Mucosal Vaccines: Innovation for Preventing Infectious Diseases [Reprint ed.] 0128119241, 9780128119242

Table of contents :
Cover
Mucosal Vaccines: Innovation for Preventing
Infectious Diseases
Copyright
Dedication
List of Contributors
Preface
Part I: Introduction
1 Historical Perspectives on Mucosal Vaccines
I Introduction
II Existing Mucosal Vaccines
III Strategies for Enhancing Mucosal Vaccines
IV Oral Tolerance
V Concluding Remarks and Future Perspectives
References
Part II: Principles of Mucosal Vaccine
2 Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immuni...
I Introduction
II Evolutionary Requirement for Mucosal Lymphoid Organs
III Gut-Associated Lymphoid Tissue
A Cryptopatches
B Isolated Lymphoid Follicles
C Peyer’s Patches, Cecal Patches, and Colonic Patches
IV Development of Gut-Associated Lymphoid Tissue
V M Cell Differentiation in Gut-Associated Lymphoid Tissue
VI Lymphoid Tissues of the Respiratory Tract
A Nasopharyngeal-Associated Lymphoid Tissue
B Bronchus-Associated Lymphoid Tissue
VII Concluding Remarks
References
3 Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells
I Introduction
II Basic Components of the Mucosal Barrier and Lymphoid Tissues
III Antigen Uptake Across the Villus Epithelium
A Paracellular and Transcellular Transport Across Villus Epithelial Cells
B Myeloid Cell Uptake of Antigens and Bacteria in the Lamina Propria
C Summary and Future Perspectives
References
4 Protective Activities of Mucosal Antibodies
I Introduction
II Properties of Antibodies of Various Ig Isotypes in External Secretions
III Origin of Antibodies in External Secretions
IV Protective Effect of Mucosal Antibodies
V The Role of IgG in Mucosal Immunity
VI Mechanisms of Protection Mediated by Mucosal IgA Antibodies
A Inhibition of Antigen Absorption
B Inhibition of Bacterial Adherence
C Polyreactivity
D Ig Glycan–Dependent Reactivity With Microorganisms
E Neutralization of Biologically Active Antigens
F Interactions of Mucosal Ig With Innate Antimicrobial Components of Mucosal Defense
VII Concluding Remarks
References
Further Reading
5 Mucosal Immunity for Inflammation: Regulation of Gut-Specific Lymphocyte Migration by Integrins
I Introduction
II Molecular Mechanisms for the Recruitment of Circulating Lymphocytes to Tissues
III Integrin Deactivation as a Regulatory Mechanism for Efficient Cell Migration
IV Interstitial Migration
V Immunological Synapse
VI Imprinting of Homing Specificity by Dendritic Cells
VII Integrin α4β7–MAdCAM-1 Interactions
VIII Therapeutic Integrin Inhibition for Inflammatory Bowel Diseases
IX Iatrogenic and Genetic Immune-Deficiencies Involving Integrins
X Therapeutic Integrin Inhibition for HIV Infection
References
6 Innate Immunity at Mucosal Surfaces
I Introduction
II Innate Mucosal Barriers in the Gut
A Structures of the Mucosal Barriers
B Innate Barrier Dysfunction and Disease Pathogenesis
III Innate Immune Regulation in the Gut
A Pattern Recognition Receptors
B Associations Between Individual TLRs and Intestinal Inflammation
C Association Between CLRs and Intestinal Inflammation
D Association Between RLRs and Intestinal Inflammation
E Association Between NLRs and Intestinal Inflammation
IV Concluding Remarks
References
7 Induction and Regulation of Mucosal Memory B Cell Responses
I Introduction
II Mucosal Vaccine Induction of Memory B Cells
III The Inductive Site for Gut Memory B Cell Responses Is the Peyer’s Patches
IV A Model System to Study Mucosal Memory B Cell Development
V The Germinal Center Reaction in Peripheral Lymph Nodes and Peyer’s Patches
VI Respiratory Tract Infections and Mucosal Memory B Cells
VII Mucosal Memory B Cells and Homing Markers
VIII Are Mucosal Memory B Cells Sessile or Recirculating?
IX Memory B Cells Show Poor Clonal Relatedness to Long-Lived Plasma Cells Following Oral Priming Immunizations
X Considerations for Mucosal Subcomponent Vaccine Development
XI Concluding Remarks
References
8 Induction and Regulation of Mucosal Memory T Cell Responses
I Introduction
II Mucosal Compartmentalization That Causes Inflammatory Responses in the Airway
A Preference of Tissue-Resident Memory T Cells for Mucosal Tissues
B Presence of Heterogenic Memory Th2 Cells in Different Mucosal Organs
C Epithelial Cytokines as Key Mediators for Mucosal Immune Responses: IL-25, IL-33, and TSLP
D Innate and Adaptive Immunity Involvement in IL-33-Induced Inflammation at Mucosal Sites
III Mucosal Inflammation and Inducible Bronchus-Associated Lymphoid Tissue
A Induction of Inducible Bronchus-Associated Lymphoid Tissue During Inflammation at Mucosal Tissue Sites
B Maintenance of Memory T Cells Within the Inducible Bronchus-Associated Lymphoid Tissue in the Mucosal Site
IV Model of Disease Induction by Tpath Cells
V Concluding Remarks
References
9 Influence of Commensal Microbiota and Metabolite for Mucosal Immunity
I Introduction
II Microbiota
III Mucosal Immunity
A Gut Microbiota and Mucosal Immunity
1 Gut Mucosal Surface
2 Cell-Mediated Immunity
3 Humoral Immunity
B Microbiota and Extraintestinal Immunity
1 Oral Immunity
2 Respiratory Tract Immunity
3 Other Mucosal Immune Sites
IV Skin Immunity
V Microbiota and Vaccines
VI Concluding Remarks
Financial Support
References
Part III: Mucosal Modulations for Induction of Effective Immunity
10 Innate Immunity-Based Mucosal Modulators and Adjuvants
I Introduction
II Innate Immune System Activators as Adjuvants for Mucosally Administered Subunit Vaccines
III Cytokines as Mucosal Vaccine Adjuvants
A Interferon Alpha
B Interleukin-1 Family
IV Nanoemulsions as Mucosal Vaccine Adjuvants
V Mast-Cell-Activating Compounds With Adjuvant Activity
A Compound 48/80
B Other Mast Cell Activators With Mucosal Adjuvant Activity
VI Cationic Polyethyleneimine Used as a Mucosal Vaccine Adjuvant
VII Mucosal Adjuvant Activity of Toll-Like Receptor Ligands
A CpG Mucosal Adjuvant and Modulator Activity
B Monophosphoryl Lipid A Mucosal Adjuvant and Modulator Activity
VIII Concluding Remarks and Future Perspective
References
11 Toxin-Based Modulators for Regulation of Mucosal Immune Responses
I Introduction
II Toxins Used For Modulation of Immune Responses
III Toxin-Derivative Adjuvants for Mucosal Vaccines
A ADP-Ribosylation of Defective Mutants of Cholera Toxin and Labile Toxin
B Other Cholera Toxin and Labile Toxin Derivatives
C Derivatives of Other Toxin Adjuvant
D Delivery Systems for Toxin-Based Adjuvant
IV Innate Mechanisms Regulated by Toxin-Based Adjuvants for Induction of Mucosal Immunity
V Induction of Tolerance by Toxin-Based Adjuvants
VI Concluding Remarks and Future Perspectives
References
12 Influence of Dietary Components and Commensal Bacteria on the Control of Mucosal Immunity
I Introduction
II Vitamins
A Vitamin A
B Vitamin B Complex
1 Vitamin B1
2 Vitamin B3
3 Vitamin B9
C Vitamin D
III Lipids
IV Commensal Bacteria and Their Metabolites
V Concluding Remarks
References
13 Mast Cells for the Control of Mucosal Immunity
I Introduction
II Exocytosis of Biologically Active Modulators
III Peripheral Location
IV Multipronged Activation at Infection Sites
A Direct Recognition of Pathogens or Their Products
B Indirect Recognition of Pathogens
C Activation by Endogenous Danger Signals
V Initiation of Local Innate Immune Responses
A Direct Bactericidal Activity
B Proteolytic Degradation of Toxins
C Induction of Immune Cell Trafficking to Sites of Infection
VI Contributions to Adaptive Immune Responses
A Enhancing Influx of Immune Cells Into Draining Lymph Nodes
B Antigen Presentation
C Effectors of Adaptive Immunity
D Adjuvant Activity of Mast Cell Activators and Products
VII Dysregulated or Impaired Mast Cell Activity During Infection
VIII Concluding Remarks and Future Perspectives
References
14 Innate Lymphoid Cells for the Control of Mucosal Immunity
I Introduction
II Innate Lymphoid Cell Development and Tissue Heterogeneity
III Organizing and Initiating Innate Lymphoid Cell–Dependent Barrier Immunity
A Cellular Regulation of Mucosal ILCs
B. Enteric Nervous System Regulates Mucosal Innate Lymphoid Cells
C Microbial and Metabolic Regulation of Mucosal Innate Lymphoid Cells
IV Mucosal Innate Lymphoid Cells in Pathogen Defense
V Innate Lymphoid Cells in Mucosal Tissue Repair
VI Innate Lymphoid Cells in Allergy, Autoimmunity, and Persistent Inflammation
VII Cross-Regulation of Innate and Adaptive Immunity by Mucosal Innate Lymphoid Cells
VIII Concluding Remarks
References
15 Mucosal Regulatory System for Balanced Immunity in the Gut
I Introduction
II Induction of Intestinal Immune Tolerance by CD103+ Dendritic Cells
III Regulation of Immune Homeostasis by Intestinal Resident CX3CR1high Macrophages
IV Roles of Human Intestinal Myeloid Cells in the Maintenance of Gut Homeostasis and Inflammatory Bowel Disease
V Concluding Remarks
References
16 Regulation of Mucosal Immunity in the Genital Tract: Balancing Reproduction and Protective Immunity
I Introduction
II Immunology of the Female Genital Tract
A Innate Immune Cells in the Upper Female Genital Tract
B Adaptive Immune Cells in the Upper Female Genital Tract
C Innate Immune Cells in the Lower Female Genital Tract
D Adaptive Immune Cells in the Lower Female Genital Tract
E Soluble Mediators in the Female Genital Tract
F Toll-Like Receptor Expression in the Female Genital Tract
G Hormone Effects on Genital Tract Infections
H Contraceptive-Driven Susceptibility to Sexually Transmitted Diseases in the Female Genital Tract
I Hormone Receptors and Signaling
III Immunology of the Male Reproductive Tract
IV Chlamydia
A Pathogenesis of C. trachomatis Genital Tract Infections
B Natural Immunity and Vaccines
C Protective Immunity Against Chlamydia
D Choice of Antigen
E Adjuvant Selection and Route of Immunization
V Chlamydia as a Gastrointestinal Commensal: The Elephant in the Room?
VI Genital Herpes
A HSV-2 Vaccine Development
B Future Perspectives
VII Concluding Remarks and Future Perspectives
VIII Key Points
References
17 Mucosal Regulatory System for Balanced Ocular Immunity
I Introduction
II Organization of the Ocular Surface Mucosa
III Immunology of the Ocular Surface Mucosa
IV Targets and Strategies for Vaccine Development
A Common Ocular Pathogens
B Strategies for Ocular Vaccine Development
References
Further Reading
18 Mucosal Regulatory System for the Balanced Immunity in the Middle Ear and Nasopharynx
I Introduction
II Innate and Acquired Immunity of Middle Ear and Nasopharynx
A Distribution of Toll-Like Receptors in Human Epithelial Cells in the Middle Ear and Changes That Result From the Ensuing ...
B Distribution of Toll-Like Receptors in Human Epithelial Cells in Nasopharyngeal Mucosae and Its Modification of Type I Al...
III Immunomodulation of Middle Ear and Nasopharyngeal Mucosae and Its Clinical Impact
IV Innovative Immunotherapy for Attenuating Nasal Symptoms of Cedar Pollinosis via the Mucosal Route With Transgenic Rice S...
V Concluding Remarks and Future Perspectives
References
Part IV: Current and New Approaches for Mucosal Vaccine Delivery
19 Current and New Approaches for Mucosal Vaccine Delivery
I Introduction
II Nano/Microscale Carriers as Promising Delivery Tools for Vaccines
III Mucosal Vaccine Delivery: Past, Present and Future
A Oral Vaccine Delivery
1 Advantages and Limitations of Oral Vaccines
2 Oral Vaccine Delivery Systems
a Emulsions and Micelles
IV Liposomes
V Immunostimulating Complexes
VI Virus-Like Particles
VII Polymeric Particle-Based Oral Delivery
VIII Oral Delivery of Vaccines Using Food Materials
A Tablets and Capsules
B Nasal Vaccine Delivery
1 Advantages and Limitations of Nasal Vaccines
2 Nasal Vaccine Delivery Systems
a Nanoemulsions
IX Liposomes
X Chitosan
XI Starch Nanoparticles
XII Polymer Nanoparticles
XIII Nanogels
XIV Concluding Remarks and Future Perspectives
References
Further Reading
20 Plant-Based Mucosal Vaccine Delivery Systems
I Introduction
II Transgenic Technologies for Vaccine Production in Plants
A Biolistic Method for Stable Transformation
B Agrobacterium-Mediated Transformation
C MucoRice System
III Plant-Based Vaccines for the Prevention and Control of Infectious Diseases
A Enterotoxigenic Escherichia coli
B Norovirus
C Hepatitis B Virus
D Rabies
E Influenza Virus
F Diarrhea Caused by Vibrio cholerae
IV Concluding Remarks
References
21 Plant-Based Mucosal Immunotherapy: Challenges for Commercialization
I INTRODUCTION
II Advances in Developing Plant-Based Mucosal Immunotherapy Products: A Quarter of a Century Later
III A Lack of Recent Human Clinical Trials Testing the Safety and/or Efficacy of Plant-Based Mucosal Immunotherapy
IV Cost of Production for Plant-Based Mucosal Immunotherapeutics: Is This a Realistic Advantage?
V Infrastructure and Protocols for Plant-Based Mucosal Immunotherapeutic Manufacturing
VI Regulatory Approval for Plant-Based Mucosal Immunotherapy
VII Safety of Plant-Made Biologics and Plant-Specific Glycosylation
VIII Mucosal Tolerance Therapy Using Plant-Made Proteins
IX Global Contamination From Food-Crop-Made Vaccines or Tolerogens
X Safety of Consumable Formulations From Food-Crop-Made Vaccines or Tolerogens
XI Separating Expression From Finishing the Product Geographically and Over Extended Periods of Time Using Seed-Based Platforms
XII Plant-Based Mucosal Immunotherapy: Challenges for Commercialization
References
22 Attenuated Salmonella for Oral Immunization
I Introduction
II Approaches for Attenuation
A Serial passage
B Deletion mutants
C Mutations in Salmonellapathogenicity islands
D Vectoring guest antigens
E Antigen delivery —location
F Regulated delayed attenuation
G Regulated delayed antigen synthesis
H Regulated delayed vaccine lysis
I Strategies for reducing lipid A toxicity
J Sugar-inducible acid resistance
K Modification of fimbriae
III Vaccines against nontyphoidal Salmonella
IV Concluding remarks
References
23 Recombinant Bacillus Calmette-Guérin for Mucosal Immunity
I Introduction
II Oral Immunization
A Mycobacterium tuberculosis
B HIV
III Intravesical Immunotherapy
A Bladder Cancer
IV Stimulation of Pulmonary Immune Responses
A Tuberculosis
B Coccidiosis, Schistosoma, and Borrelia
References
Further Reading
24 Recombinant Adenovirus Vectors as Mucosal Vaccines
I Introduction
II Adenoviruses
III Immune Responses to Adenoviruses
IV Types of Adenovirus Vectors
V Construction, Purification, and Titration of Adenovirus Vectors
VI Quality Control of Adenovirus Vectors
VII Thermostability of Adenovirus Vectors
VIII Immunogenicity of Adenovirus Vectors
IX The Mucosal Immune System
X Mucosal Vaccines
XI Adenovirus Vectors as Oral Vaccines
XII Adenovirus Vectors as Intranasal Vaccines
XIII Immunizations Through the Rectal or Genital Mucosa
XIV Use of Adjuvants for Mucosal Adenovirus Vector Vaccines
XV Concluding Remarks and Future Perspectives
References
25 Mucosal Approaches for Systemic Immunity to Anthrax, Brucellosis, and Plague
I Introduction
II Origins of Vaccination
III Anthrax
A The Disease and Historical Perspective
B Development of First Anthrax Vaccines
C Live Vaccines for Anthrax
IV Brucellosis
A Etiological Agents and Disease
B Protection to Brucella Infections Is Th1 Cell-Dependent
C Current Vaccines
D Mucosal Vaccination Approaches for Brucellosis
V Plague
A The Disease and Historical Perspective
B Antibody-Dependence for Immune Protection to Plague
C Th1- and Th17-Mediated Immunity Against Plague
D Attempt to Develop Plague Mucosal Vaccine
References
26 Nanodelivery Vehicles for Mucosal Vaccines
I Introduction
II Characteristics of the Nasal Immune System
A Structure and Function of Nasopharyngeal-Associated Lymphoid Tissue
B Mechanism for the Induction of Antigen-Specific Nasal Immune Responses
C Lymphocyte Imprinting and Homing Mechanisms in the Nasal Immune System
III Drug-Delivery Systems for Nasal Vaccines
IV cCHP Nanogel as a Drug-Delivery System for Nasal Vaccines
V Development of a Nanogel-Based Nasal Vaccine Against Pneumonia
VI Application of cCHP Nasal Vaccines Against Noninfectious Diseases
VII Concluding Remarks and Future Perspectives
Abbreviations
References
27 Effectiveness of Sublingual Immunization: Innovation for Preventing Infectious Diseases
I Introduction
II Localization of Antigen-Presenting Cells in the Sublingual Mucosa
III Role of Draining Lymph Nodes in Sublingual Vaccination
IV Mechanism for Induction of CD4+ T Cell Activation Following Sublingual Vaccination
V Sublingual Vaccination Induces Both Systemic and Mucosal Antibody Responses
VI Sublingual Administration Is Useful for Induction of Antibody Against Viral Infection
VII Sublingual Administration Does Not Redirect Antigens to the Central Nervous System
VIII Sublingual Vaccination Induces T and B Cell Activation in Female Mouse Genital Tissues
IX Concluding Remarks and Future Perspectives
References
28 M Cell-Targeted Vaccines
I Introduction
II M Cell Differentiation
III Candidate Molecules for M Cell Targeting and Their Ligands
A α(1,2)-Linked Fucose
B GP2
C PrPC
D Integrins
E Uromodulin (Umod)
F C5aR
G Claudin 4
H Secretory IgA (SIgA) Receptors
I α2-3-Linked Sialic Acid
IV Contribution of M Cells for the Development of Oral Tolerance
V Enhancement of M Cell Number and Function
VI Concluding Remarks and Future Perspectives
References
Part V: Mucosal Vaccines for Bacterial Diseases
29 Induction of Local and Systemic Immunity by Salmonella Typhi in Humans
I Introduction
II Current Vaccines and Models to Study Immunogenicity to Salmonella Typhi
III Live Attenuated Oral Vaccine and Human Challenge Model of Typhoid Fever
IV Systemic T Cell Immunity Induced by Oral Salmonella Typhi
V Humoral and Systemic B Cell Immunity Induced by Oral Salmonella Typhi
VI Changes in Innate and Mucosal-Associate Invariant T Cells Induced by Oral Salmonella Typhi
VII Gut-Homing Memory T Cells: A Window Into Mucosal Immunity
VIII Accumulation and Retention of Gut-Homing Memory T Cells in the Mucosa
IX Mucosal Immunity to Salmonella Typhi
X Relationship Between Systemic and Mucosal Immunity to Salmonella Typhi
XI Concluding Remarks
References
30 Oral Shigella Vaccines
I Introduction
II Shigella Infection: Burden of Disease and Vulnerable Groups
III Shigella Pathogenesis and Virulence Factors
IV Naturally Acquired Immunity Against Shigella
V Immune Responses to Shigella in Children
VI Animal Models of Mucosal Shigella Infection
VII History of Oral Shigella Vaccine Candidates
VIII Immunity Induced by Oral Shigella Vaccines
IX Multivalent Oral Shigella Vaccines
A Shigella–Typhoid
B Escherichia coli–Shigella
C Shigella–ETEC
X Parenteral Shigella Vaccine Candidates
XI An Ideal Oral Shigella Vaccine: Features and Implementation
XII Concluding Remarks
References
31 Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control
I Introduction
II Susceptibility and Innate Immunity in Cholera
III Oral Cholera Vaccines
IV Adaptive Mucosal Immune Responses in Cholera
A Mucosal IgA Antibody and Antibody-Secreting Cell Responses
B Intestine-Derived Gut-Homing Circulating Antibody-Secreting Cells
C Intestinal Mucosal T Cells
V Systemic Antibody and Memory B Cell Responses
A Vibriocidal Antibody Responses
B Serum Antibody Responses to Defined Antigens
1 IgA Responses
2 IgG Responses
3 Immunoglobulin M
C Memory B Cell Responses in Cholera
VI Modifiers of Immune Responses
VII Public Health Use of Oral Cholera Vaccines
VIII WHO Recommendations and a “Global RoadMap to End Cholera by 2030”
IX Concluding Remarks and Future Perspectives
References
32 Oral Vaccines for Enterotoxigenic Escherichia coli
I Introduction
II Enterotoxigenic Escherichia coli Vaccine Candidates
A Fimbrial Antigens and Heat-Labile Enterotoxin Are Major Protective Antigens
1 Studies in Animals
2 Studies in Humans
B Structure of the Major Putative Protective Enterotoxigenic Escherichia coli Antigens: Colonization Factor Antigens and En...
1 Colonization Factors
2 Heat-Labile Enterotoxin (LT)
3 ST Heat-Stable enterotoxin
4 O Antigens
III Evaluation of Optimal Administration Routes of an Enterotoxigenic Escherichia coli Vaccine
A Passive Protection Trials in Humans to Get Proof of Concept
B Candidate Vaccines in Preclinical Phase
1 Recombinant Attenuated Bacteria
2 Purified Antigens
C Multiepitope Protein Antigens
IV Oral Mucosal Adjuvants
V Methods for Assessing Mucosal Immune Responses Against Enterotoxigenic Escherichia coli Candidate Vaccines in Humans
VI Candidate Vaccines in Clinical Development
A ACE527
B Tip Adhesins
C LT patch
D Oral Inactivated Whole Cell Enterotoxigenic Escherichia coli Vaccines
1 rCTB-CF Enterotoxigenic Escherichia coli Vaccine
2 Multivalent ETEC vaccine(ETVAX)
VII Concluding Remarks
A Preclinical Vaccine Candidates
B Vaccine Candidates in Clinical Development
References
33 A Future for a Vaccine Against the Cancer-Inducing Bacterium Helicobacter pylori?
I Introduction
II The Therapeutic Tool Box of Helicobacter pylori Infections
III The Unheeded Role of Epithelial Cells in the Execution of Mucosal Defense
IV Vaccines as Stimulators of Mucosal Immunity
V Natural Immunity Against Helicobacter pylori
VI The Failure of Past Vaccination Attempts
VII Unmasking Helicobacter pylori’s Immune Evasion Strategy
VIII Concluding Remarks and Future Perspectives
References
34 Mucosal Vaccines for Streptococcus pneumoniae
I Introduction
II Host Immune Responses to Pneumococci During Nasopharyngeal Colonization
A The Role of Innate Immunity Against Pneumococci
B The Role of Acquired Immunity Against Pneumococci
III Immunization Against Streptococcus pneumoniae at Mucosal Surfaces
IV Current and Future Status of Human Mucosal Vaccine Trials
V Concluding Remarks
References
35 Development of a Mucosal TB Vaccine Using Human Parainfluenza Type 2 Virus
I Introduction
II Mucosal Immune Responses in TB Infection
III Vaccine Delivery Systems for Induction of Mucosal Immunity
IV Novel Vaccine Candidate, rHPIV2, in TB Protection
V Protective Effects of an rHPIV2 Vaccine in Mice With TB
VI Possibilities of rHPIV2 as a Next-Generation Vaccine Candidate
VII Future Study Using the HPIV2 Vaccine
Abbreviations
References
36 Sexually Transmitted Infections and the Urgent Need for Vaccines: A Review of Four Major Bacterial STI Pathogens
I Introduction
II Chlamydia
A Microbiology
B Clinical Manifestations
C Epidemiology
D Current Treatment Options
E Immune Responses Associated With Pathology
F Vaccine-Related Research
G Preclinical Vaccine Studies and Vaccine Trials in Progress
H Challenges to the Development of a Vaccine
III Gonorrhea
A Microbiology
B Clinical Manifestations
C Epidemiology
D Current Treatment Options
E Immune Responses Associated With Pathology
F Vaccine-Related Research
G Future Vaccine Implications
IV Syphilis
A Microbiology
B Epidemiology
C Clinical Manifestations
D Current Treatment Options
E Immune Responses Associated With Pathology
F Vaccine-Related Research
G Conclusions
V Mycoplasma
A Microbiology
B Epidemiology
C Clinical Manifestations
D Current Treatment Options
E Immune Responses Associated With Pathology
F Vaccination Research
G Conclusions
VI Concluding Remarks
References
37 Mucosal Vaccines for Oral Disease
I Introduction
II Mucosal Vaccines for Caries Prevention
III Mucosal Vaccines for Periodontal Disease
IV Protein Based Mucosal Vaccine
V DNA-Based Vaccine
VI Nasal Administration of Periodontal Vaccine
VII Sublingual Vaccine for Periodontal Diseases
VIII Concluding Remarks
References
Part VI: Mucosal Vaccines for Viral Diseases
38 Vaccination Against Respiratory Syncytial Virus
I Introduction
II Global Impact and Clinical Disease
III Correlates of Protection
IV Maternal Immunization to Protect Vulnerable Infants
V RSV Immunity and Vaccination in Infants and Young Children
VI RSV Immunity and Vaccination in Older Adults
VII Concluding Remarks
References
39 Nasal Influenza Vaccines
I Introduction
II Humoral Immune Responses to Influenza Virus Infection
III Development of the Nasal Influenza Vaccines
A Live Attenuated Influenza Vaccines
B Intranasal Inactivated Influenza Vaccines
References
40 The Role of Innate Immunity in Regulating Rotavirus Replication, Pathogenesis, and Host Range Restriction and the Implic...
I Introduction
II Host Innate Immune Sensors and Rotavirus Infection
A Cytoplasmic Sensors
B Membrane-Associated Sensors
C Other Sensors
III Host Innate Responses to Rotavirus and Their Effects on Viral Replication
IV Regulation of the Interferon Induction Pathway by Rotavirus
A Regulation of the Interferon Signaling Pathway by Rotavirus
B Regulation of STAT1 by Rotavirus
C Degradation of Different Types of Interferon Receptors
D STAT1 Sequestration in the Cytoplasm
E Regulation of IRF7 and IRF9
F Rotavirus Regulation of Other Effector Antiviral Factors
V Taking Advantage of Rotavirus Host Range Restriction to Reliably Attenuate Live Rotavirus Vaccine Candidates
References
41 Development of Oral Rotavirus and Norovirus Vaccines
I Introduction
II Rotavirus Vaccine Development
A Rotavirus Disease
B Rotavirus Classification
C Initial Vaccine Efforts Using Live Animal Rotavirus
D RotaShield, the First Licensed Rotavirus Vaccine
E RotaTeq and Rotarix, the Second Generation of Licensed Rotavirus Vaccines
F Other Nationally Licensed Rotavirus Vaccines
G Remaining Obstacles to Rotavirus Vaccination and Future Directions
III Norovirus Vaccine Development
A Norovirus Disease
B Norovirus Structure
C Norovirus Classification
D Challenges in Norovirus Vaccine Development
E Virus-Like Particle-Based Norovirus Vaccines
F Alternative Norovirus Vaccines in the Pipeline
G Future Directions in Norovirus Vaccine Development
IV Concluding Remarks
References
42 Mucosal Vaccines Against HIV/SIV Infection
I Introduction
II Mucosal Vaccines Inducing HIV-Specific Antibody Responses
A Characterization of Anti-HIV Neutralizing Antibodies to Design a Mucosal HIV Vaccine
B Passive Anti-HIV Antibody Administration as a Model of Mucosal Vaccines
C Antibody-Related Correlates in HIV Vaccine Clinical Trials
D Analysis of Mucosal Tissues and Ab Effector Function for Vaccine Design
III Mucosal Vaccines Inducing HIV-Specific T Cell Responses
A Mucosal HIV Infection in the Acute Phase Toward Systemic Infection
B Viral Vectors for Mucosal Anti-HIV T Cell Responses
C Mucosal T Cell Responses Effective Against HIV Infection
References
43 Mucosal Vaccines for Genital Herpes
I Introduction
II HSV-1 and HSV-2 Virus Life Cycle
III Pathogenesis of Genital Herpes
IV Symptoms of Genital Herpes
V Immune Protective Mechanisms Against HSV Infections
A Innate Immune Response to HSV Infections
B Protective Memory Responses Against Genital Herpes Infection
1 Antibody-Mediated Protection
2 T Cell-Mediated Protection
VI Vaccine Approaches Against Genital Herpes
A Past Prophylactic Vaccine Trials
B Mucosal Vaccines Against HSV-2
1 Intravaginal HSV-2 Vaccines
2 Intranasal Vaccines
VII Future Strategies for HSV-2 Vaccine
VIII Concluding Remarks and Future Perspectives
References
44 Maternal Vaccination for Protection Against Maternal and Infant Bacterial and Viral Pathogens
I Introduction
II Diphtheria, Pertussis, and Tetanus
III Influenza Virus
IV Measles, Mumps, and Rubella
V The Next Frontier of Maternal Vaccines
A Respiratory Syncytial Virus
B Human Immunodeficiency Virus
C Human Cytomegalovirus
D Herpes Simplex Virus
E Zika Virus
F Group B Streptococcus
VI Concluding Remarks
VII Summary of Key Points
Funding
References
Part VII: New and Novel Approaches for Mucosal Vaccine Development
45 Systems Biological Approaches for Mucosal Vaccine Development
I Introduction
II Systems Vaccinology
A Proof of Concept: Studies With the Yellow Fever Vaccine YF-17D
B Extending Systems Vaccinology to Other Vaccines
C Are Molecular Signatures of Immunogenicity Versus Efficacy the Same?
D Beyond Blood: Systems Analysis of Gene Signatures in Lymphoid and Nonlymphoid Tissues
E From Data to Knowledge to Understanding
III Systems Biology of Vaccines Against Mucosal Infections
Challenge 1: Discovering Correlates of Protection
1 Controlled Human Infection Models
2 Can Signatures in the Blood Predict Mucosal Immunity?
Challenge 2: Discovering Fundamental Immunological Mechanisms of Mucosal Immunity
1 How Can Vaccines and Adjuvants Imprint Mucosal Homing of Antigen-Specific T and B Cells?
2 How do Antibodies Protect Against Infection at Mucosal Sites?
3 What Roles Do T Cells Play in Protection Against Infection at Mucosal Sites?
Challenge 3: How Can the Durability of Mucosal Responses Be Enhanced?
Challenge 4: Understanding the Basis of Population Differences in the Efficacy of Mucosal Vaccines
IV Concluding Remarks
References
46 Harnessing γδ T Cells as Natural Immune Modulators
I Introduction
II γδ T Cell Surface Receptors
III Similarities of γδ T Cells to Myeloid and Macrophage Cells
IV γδ T Cell-Mediated Cytotoxicity
V γδ T Cell Cytokine Production
VI Role of γδ  T Cells in Infectious Diseases
VII Therapeutic Potential for Manipulation of γδ T Cells
VIII Plant Polyphenols for the Activation of γδ T Cells
A Plant Polysaccharides as γδ T Cell-Targeted Immunomodulator
B Microbial Products for the Regulation of γδ T Cells
IX Concluding Remarks
Abbreviations
References
47 Mucosal Vaccines for Aged: Challenges and Struggles in Immunosenescence
I Introduction
II Age-Associated Changes in the Gastrointestinal Tract Immune System
III Potential Mechanisms in Gut Aging: Roles of M Cells
IV Involvement of Mucosal CD4+ T Cells in Gut Aging
V The Intestinal Microbiota Potentially Shapes Mucosal Immunosenescence
VI Rejuvenation of Gut Immunity by Mesenchymal Stem Cell Transfer
VII Nasopharygeal-Associated Lymphoid Tissue Versus Gut-Associated Lymphoid Tissue: Similarities and Gaps
VIII Distinct Aging Process of Nasopharygeal-Associated Lymphoid Tissue Function
IX Mucosal Vaccines and Therapies Fight for Immunosenescence
X A Dentritic Cell-Targeting Mucosal Vaccines for Aged
XI Next Generation of Potent Mucosal Vaccines for the Elderly
References
Part VIII: Can Mucosal Vaccines be Applied to Other Infectious and Noninfectious Diseases?
48 Mucosal Vaccine Development for Veterinary and Aquatic Diseases
I INTRODUCTION
A Considerations for Mucosal Veterinary Vaccine
1 Mass Delivery
2 Differentiation of Infected From Vaccinated Animals Vaccines
3 Economics and Trade
II Exploration of Commercial and Experimental Mucosal Veterinary Vaccines
A Mucosal Vaccines for Livestock
1 Suidae
a Porcine transmissible gastroenteriditis and porcine epidemic diarrhea virus
2 Caprinae and Ovidae
a Brucella ovis
3 Bovinae
a Bovine herpesvirus 1
b Hemorrhagic septicemia
c Bovine viral diarrhea virus
4 Avian Species
A Avian influenza
B Companion Animals
1 Leporidae
a Rabbit hemmorrhagic disease
C Wildlife
1 Koala
a Chlamydia pecorum
2 Prairie Dogs
a Plague
3 Multispecies
a Rabies
D Mucosal Aquatic Vaccines
1 Immersion Vaccines
2 Oral and Intranasal Vaccines
3 Injected Vaccine
E Immunocontraceptive Vaccines
1 Efficacy, Safety, and Economic Feasibility of Immunocontraceptive Vaccines
F Mucosal Vaccines to Improve Fertility
III Veterinary Vaccines and One Health
IV Concluding Remarks
References
49 Mucosal Vaccine for Malaria
I Introduction
II Malaria’s Effect on the Gastrointestinal System
III Gastrointestinal system’s effect on malaria: a role for gut microbiata?
IV Mucosal Vaccines Against Malaria
V Concluding Remarks
References
50 Mucosal Vaccine for Parasitic Infections
I Introduction
II Protozoan Infections
A Amoebiasis
B Giardiasis
C Cryptosporidiosis
D Toxoplasmosis
III Helminth Infections
A Schistosomiasis (Trematodiasis)
B Cysticercosis (Cestodiasis)
C Echinococcosis (Cestodiasis)
D Trichinellosis (Nematodiasis)
IV Concluding Remarks
References
51 Mucosal Vaccines for Allergy and Tolerance
I Introduction
II Immunotherapy
A Hypoallergenic Antigens
B Anti-IgE
C Adjuvants and Delivery
D DNA Vaccines
III Microbiota
IV Concluding Remarks
References
52 Novel Strategies for Targeting the Control of Mucosal Inflammation
I Introduction
II For Every Action, There Is an Equal and Opposite Reaction
III Key Features of the Host Response in Homeostasis
IV Microbial Communities, Microbial Pathogenesis, and Dysbiosis
V The Concept of a Homeostatic Scar and Immunological Dysfunction
VI The Impact of Immunization on Homeostasis
A Passive Immunization
B Active Immunization
VII Future Perspectives
VIII Concluding Remarks
References
Index
Back Cover

Citation preview

MUCOSAL VACCINES

MUCOSAL VACCINES Innovation for Preventing Infectious Diseases SECOND EDITION Edited by

Hiroshi Kiyono The University of Tokyo, Tokyo, Japan; Chiba University, Chiba, Japan; University of California San Diego, CA, United States

David W. Pascual The University of Florida, FL, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-811924-2 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Wolff Acquisition Editor: Linda Versteeg-buschman Editorial Project Manager: Gabriela Capille Production Project Manager: Poulouse Joseph Cover Designer: Mark Rogers Cover credit: Dr. Yosuke Kurashima, Chiba University and the University of Tokyo, Japan Typeset by MPS Limited, Chennai, India

Dedication This book is dedicated to Prof. Jerry R. McGhee and Prof. Pearay L. Ogra for their pioneering scientific contributions in the area of Mucosal Immunology and Mucosal Vaccine. Together with HK, they contributed their expertise as the editorial team of 1st edition of “Mucosal Vaccines” published in 1996. Without the institutional memory of the original book, we could not edit the current version of Mucosal Vaccines: Innovation for Preventing Infectious Diseases. Finally, the book is also dedicated to our families, Momoyo and Erika Kiyono; Virginia, Michelle, Monica, Luke, and Michael A., Sr., Pascual for their support and understanding during the preparation of this book.

List of Contributors

Soman N. Abraham Departments of Pathology, Immunology and Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States; Program in Emerging Infectious Diseases, Duke-National University of Singapore, Singapore, Singapore; Department of Immunology, Duke University School of Medicine, Durham, NC, United States David Artis Jill Roberts Institute for Research in IBD, Weill Cornell Medicine, New York, NY, United States Zayed Attia Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, United States Tatsuhiko Azegami International Research and The Development Center for Mucosal Vaccines, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Health Center, Keio University, Tokyo, Japan Lorne A. Babiuk Department of Agricultural Food & Nutritional Science, University of Alberta, Edmonton, AB, Canada Eileen M. Barry Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, United States Kenneth W. Beagley Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia Jayaum S. Booth Departments of Medicine and Pediatrics, Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, United States Kenneth L. Bost Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, United States; SoyMeds Inc, Davidson, NC, United States

Prosper N. Boyaka Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, United States Emily R. Bryan Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia Nils Carlin Scandinavian Biopharma, Solna, Sweden Daniel J.J. Carr Departments of Ophthalmology, and Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States Hae Woong Choi Department of Pathology, Duke University School of Medicine, Durham, NC, United States Hiutung Chu Department of Pathology, Center for Veterinary Sciences and Comparative Medicine, Chiba University-UCSD Center for Mucosal Immunology, Allergy and Vaccine Development (CU-UCSD cMAV), University of California San Diego, La Jolla, CA, United States; The Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, UC Davis, Davis, CA, United States; Department of Immunology, Chiba University, Chiba, Japan John D. Clemens International Centre for Diarrhoeal Disease Research (icddr,b), Dhaka, Bangladesh Cevayir Coban Laboratory of Malaria Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Osaka, Japan; Division of Malaria Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo (IMSUT), Tokyo, Japan

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LIST OF CONTRIBUTORS

Estelle Cormet-Boyaka Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, United States Michelle C. Crank Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States Toni Darville Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Steven C. Derrick Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States Siyuan Ding Departments of Medicine and Microbiology and Immunology, Stanford University, School of Medicine, Stanford, CA, United States Ioannis Drygiannakis Department of Pathology, Center for Veterinary Sciences and Comparative Medicine; Chiba University-UCSD Center for Mucosal Immunology, Allergy and Vaccine Development (CU-UCSD cMAV), University of California San Diego, La Jolla, CA, United States Kristel L. Emmer Gene Therapy & Vaccines Program, University of Pennsylvania School of Medicine, Philadelphia, PA, United States; Wistar Institute Vaccine Center, Philadelphia, PA, United States Peter B. Ernst Department of Pathology, Center for Veterinary Sciences and Comparative Medicine; Chiba University-UCSD Center for Mucosal Immunology, Allergy and Vaccine Development (CU-UCSD cMAV), University of California San Diego, La Jolla, CA, United States; The Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, UC Davis, Davis, CA, United States; Department of Immunology, Chiba University, Chiba, Japan Hildegund C.J. Ertl Wistar Institute Vaccine Center, Philadelphia, PA, United States Kohtaro Fujihashi International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Department of Pediatric Dentistry, The Institute of Oral Health Research,

The School of Dentistry, The University of Alabama at Birmingham, Birmingham, AL, United States Kosuke Fujimoto Department of Immunology and Genomics, Osaka City University Graduate School of Medicine, Osaka, Japan; Division of Innate Immune Regulation, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Volker Gerdts Vaccine and Infectious Disease Organization (VIDO) – International Vaccine Centre (InterVac), University of Saskatchewan, Saskatoon, SK, Canada; Veterinary Microbiology, Western College of Veterinary Medicine (WCVM), University of Saskatchewan, Saskatoon, SK, Canada Barney S. Graham Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States Katrina R. Grau Department of Molecular Genetics & Microbiology, Emerging Pathogens Institute, Center for Inflammation and Mucosal Immunology, University of Florida, Gainesville, FL, United States Harry B. Greenberg Departments of Medicine and Microbiology and Immunology, Stanford University, Stanford, CA, United States Hideki Hasegawa Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan Tomomi Hashizume-Takizawa Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Chiba, Japan Jodi F. Hedges Department of Microbiology and Immunology, Montana State University, Bozeman, MT, United States Danica K. Hickey Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia Kiyoshi Hirahara Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan

LIST OF CONTRIBUTORS

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Jan Holmgren Department of Micriobiology & Immunology, University of Gothenburg, Gothenburg, Sweden

Hideyuki Kawauchi Department of Otorhinolaryngology, Faculty of Medicine, Shimane University, Izumo City, Japan

Adam Huys Department of Molecular Genetics & Microbiology, Emerging Pathogens Institute, Center for Inflammation and Mucosal Immunology, University of Florida, Gainesville, FL, United States

Hisako Kayama Department of Microbiology and Immunology, Graduate School of Medicine; WPI Immunology Frontier Research Center, Osaka University, Suita, Japan; Core Research for Evolutional Science and Technology, Japan Agency for Medical Research and Development, Tokyo, Japan

Hiroshi Ishii AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan Akiko Iwasaki Department of Immunobiology, Yale University School of Medicine, New Haven, CT, United States; Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT, United States; Howard Hughes Medical Institute, Chevy Chase, MD, United States Yanlong Jiang College of Animal Science & Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agriculture University, Changchun, China Christian Jobin Department of Medicine, Department of Infectious Diseases and Immunology, Department of Anatomy and Cell Physiology, University of Florida, Gainesville, FL, United States Brandi T. Johnson-Weaver Department of Pathology, Duke University School of Medicine, Durham, NC, United States Mark A. Jutila Department of Microbiology and Immunology, Montana State University, Bozeman, MT, United States Stephanie M. Karst Department of Molecular Genetics & Microbiology, Emerging Pathogens Institute, Center for Inflammation and Mucosal Immunology, University of Florida, Gainesville, FL, United States Hirotomo Kato Division of Medical Zoology, Department of Infection and Immunity, Jichi Medical University, Tochigi, Japan Eiji Kawamoto Department of Disaster and Emergency Medicine, Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, Tsu, Japan

Brian L. Kelsall Mucosal Immunobiology Section, Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States Andrea M. Kemter Department of Pathology and Committee on Immunology, The University of Chicago, Chicago, IL, United States Eunsoo Kim Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, United States Hiroshi Kiyono Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Mucosal Immunology and Allergy Therapeutics, Graduate School of Medicine, Chiba University, Chiba, Japan; Division of Gastroenterology, Department of Medicine, (CU-UCSD cMAV) Center for Mucosal Immunology, Allergy and Vaccines, University of California, San Diego, CA, United States Ryoki Kobayashi Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Chiba, Japan Avinash Kollipara Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Qingke Kong Department of Infectious Diseases & Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States

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LIST OF CONTRIBUTORS

Jun Kunisawa Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan; International Research and Development Center for Mucosal Vaccines, The Institute of Medical Sciences, The University of Tokyo, Tokyo, Japan; Department of Microbiology and Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan; Graduate School of Medicine, Graduate School of Pharmaceutical Sciences and Graduate School of Dentistry, Osaka University, Osaka, Japan Tomoko Kurita-Ochiai Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Chiba, Japan Mi-Na Kweon Mucosal Immunology Laboratory, Department of Convergence Medicine, University of Ulsan College of Medicine/Asan Medical Center, Seoul, Republic of Korea De’Ashia Lee Department of Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Michelle Sue Jann Lee Laboratory of Malaria Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Osaka, Japan Randy S. Longman Jill Roberts Institute for Research in IBD, Weill Cornell Medicine, New York, NY, United States Nils Lycke Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden Jesse Mangold Duke Human Vaccine Institute, Durham, NC, United States David R. Martinez Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, United States; Duke Human Vaccine Institute, Durham, NC, United States

Jiri Mestecky Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, United States; Laboratory of Cellular and Molecular Immunology, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic Maeva Metz Jill Roberts Institute for Research in IBD, Weill Cornell Medicine, New York, NY, United States Thomas F. Meyer Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany Micaela L. Montgomery Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States Kaitlyn M. Morabito Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States Pau Morey Instituto Universitario de Investigacio´n en Ciencias de la Salud (IUNICS), Universidad de las Islas Baleares, Palma de Mallorca, Spain Peter Mulvey Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australia Cathryn R. Nagler Department of Pathology and Committee on Immunology, The University of Chicago, Chicago, IL, United States Rika Nakahashi-Ouchida Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Toshinori Nakayama Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan

Tetsuro Matano AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan; The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Pearay L. Ogra Division of Infectious Diseases, Department of Pediatrics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, United States

Larry S. McDaniel Department of Microbiology & Immunology, University of Mississippi Medical Center, Jackson, MS, United States

Ji Eun Oh Department of Immunobiology, Yale University School of Medicine, New Haven, CT, United States

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Eun Jeong Park Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, Tsu, Japan

Michael W. Russell Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY, United States

David W. Pascual Department of Infectious Diseases & Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States

Shintaro Sato Mucosal Vaccine Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

Marcela F. Pasetti Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, United States

Adrish Sen Departments of Medicine and Microbiology and Immunology, Stanford University, Stanford, CA, United States

Sallie R. Permar Department of Molecular Genetics & Microbiology and Department of Pediatrics, Duke University Medical Center, Durham, NC, United States; Duke Human Vaccine Institute, Durham, NC, United States

Motomu Shimaoka Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, Tsu, Japan

Kenneth J. Piller The Center for Biomedical Engineering & Science, University of North Carolina at Charlotte, Charlotte, NC, United States; SoyMeds Inc, Davidson, NC, United States Jillian L. Pope Department of Medicine, University of Florida, Gainesville, FL, United States Bali Pulendran Department of Pathology, Department of Microbiology & Immunology, Institute for Immunity, Transplantation and Infection, Stanford University, Stanford, CA, United States

Aaron Silva-Sanchez Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, United States Herman F. Staats Department of Pathology, Duke Human Vaccine Institute, Department of Immunology and Department of Medicine, Duke University School of Medicine, Durham, NC, United States

Firdausi Qadri International Centre for Diarrhoeal Disease Research (icddr,b), Dhaka, Bangladesh

Hidehiko Suzuki Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan

Troy D. Randall Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, United States

Ann-Mari Svennerholm Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

Joon Haeng Rhee Clinical Vaccine R&D Center and Department of Microbiology, Chonnam National University, Gwangju, South Korea

Edwin Swiatlo Section of Infectious Diseases, Southeast Louisiana VA Medical Center, New Orleans, LA, United States

Kenneth L. Roland The Biodesign Institute, Arizona State University, Tempe, AZ, United States

Marcelo B. Sztein Center for Vaccine Development and Global Health, Department of Medicine, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, United States

Derek J. Royer Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States Tracy J. Ruckwardt Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States

Kiyoshi Takeda Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center, Osaka University, Suita, Japan; Core Research for Evolutional Science and Technology, Japan Agency for Medical Research and Development, Tokyo, Japan

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LIST OF CONTRIBUTORS

Franklin R. Toapanta Center for Vaccine Development and Global Health, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, United States Sarah Tomkovich Department of Medicine, University of Florida, Gainesville, FL, United States Logan Trim Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia Yusuke Tsujimura Laboratory of Immunoregulation and Vaccine Research, Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)Tsukuba, Japan Satoshi Uematsu Department of Immunology and Genomics, Osaka City University Graduate School of Medicine, Osaka, Japan; Division of Innate Immune Regulation, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Malabi M. Venkatesan Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD, United States

Heather L. Wilson Vaccine and Infectious Disease Organization (VIDO) – International Vaccine Centre (InterVac), Veterinary Microbiology, Western College of Veterinary Medicine (WCVM), School of Public Health, Vaccinology & Immunotherapeutics Program, University of Saskatchewan, Saskatoon, SK, Canada Hiroyuki Yamamoto AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan Masafumi Yamamoto Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Chiba, Japan Yasuhiro Yasutomi Laboratory of Immunoregulation and Vaccine Research, Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)Tsukuba, Japan; Department of Immunoregulation, Mie University Graduate School of Medicine, Tsu, Japan Yoshikazu Yuki Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Preface The importance of herd immunity against infectious diseases has never been more relevant than it is now, as we witness attrition in compliance with childhood vaccinations. Vaccination programs implemented since the 1960s have proven effective in minimizing the toll of childhood diseases on the populace. Although reports of higher numbers of measles outbreaks were noted in the late 1980s to early 1990s, most cases since then have arisen in unvaccinated close-knit communities or as a result of travel to infected areas. The public’s increased antivaccination sentiment, coupled with the current lack of firsthand knowledge of the severity of these diseases, has led to a generational complacency about childhood diseases. Before vaccination became common, these infectious diseases led to the demise of diverse populations on all continents except Antarctica. Vaccination is a tool to elicit and maintain immune memory of these diseases and promote herd immunity. Compliance with childhood vaccinations is imperative to protect the populace against these lifethreatening infectious diseases, which are readily preventable. Relying on natural immunity is not an option for protection against certain infectious diseases. There is no natural immunity to plague, which wrought havoc in the 14th through 17th centuries, reducing Europe’s population by as much as 30%. The concept of immunity dates back to the 5th century BCE, when plague survivors were refractive to reinfection and could assist infected individuals. The concept of inducing active protection emerged from variolation practices using smallpox pustules to

infect naı¨ve individuals via scarification of the skin. Such treatments were commonly practiced in Europe and even in the American colonies before the Revolutionary War. In fact, this technology as well as nasal application originated in China, where it was found to be effective in protecting against smallpox. Adapting related or attenuated strains was initiated by Dr. Edward Jenner, who more than 220 years ago noticed that the lesion patterns on milkmaids’ hands were similar to the lesions observed on smallpox-infected patients. This visual cue led to the concept of applying variolae vaccinae, meaning “smallpox of the cow,” a practice that became known as “vaccination” to prevent smallpox. The work of Pasteur and others further developed this theory, leading to vaccination against livestock diseases, such as chicken cholera and anthrax. In addition to the importance of childhood vaccinations for the control of life-threatening infectious diseases, the public needs to be aware of how the increase in life expectancy has led to an aging society. The global population of individuals older than 60 years of age is expected to reach over 2 billion by 2050. There are approximately 125 million people above 80 years old worldwide, and this number is expected to increase dramatically to well over 400 million by 2050. As one might expect, the severity of infectious diseases is generally worse in the elderly than in younger adults because immunosenescence weakens memory immune responses. Infectious diseases in the elderly often lead to long-lived aftereffects that diminish quality of life, causing individuals to become infirm, lose normal daily life activities,

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and/or lose their independence. The size of the aging population presents a major challenge to our societies and to everyone associated with public health systems. An obvious and logical intercession is the prevention of infectious diseases in the elderly to ensure healthy aging and maintain a good quality of life. Nobody can reasonably dispute the huge success of childhood vaccination in creating a healthier society. At same time, the world needs to recognize the importance of vaccination programs for the elderly to sustain lifelong protective immunity. Mucosal Vaccines offers a timely reinforcement of the concept of vaccinating via mucosal surfaces to best induce protective immunity against mucosal diseases and the pathogens that cross the mucosa to infect the host. We have enlisted outstanding scientists and clinicians from various disciplines of mucosal immunology to address the development of better mucosal vaccines. This volume is divided into eight sections, each addressing a broad area of interest. We begin with the Introduction providing a historical analysis on mucosal vaccines. The chapters in Part II, “Principles of Mucosal Vaccine,” survey the different mucosal anatomical structures, the principal cellular and molecular players involved, how these interact to provide vast coverage by the mucosal immune system to provide innate and adaptive immune protection, and the impact of microbiota on maintaining immune homeostasis. In Part III “Mucosal Modulations for Induction of Effective Immunity,” the chapters examine the role of mucosal adjuvants and mucosal players to amplify host responses to infection, as well as the role of various mucosal regulatory mechanisms to dampen and control inflammation in these mucosal tissues. In Part IV, “Current and New Approaches for Mucosal Vaccine Delivery,” delineates approaches to varied mucosal vaccination routes and recombinant

live or subunit delivery platforms that target specific tissues and infectious diseases and immunological disorders. The chapters in Part V, “Mucosal Vaccines for Bacterial Diseases,” focus on how mucosal vaccines protect against various bacterial oral, gastrointestinal, pulmonary, and sexually transmitted diseases. Part VI, “Mucosal Vaccines for Viral Diseases,” reviews the status of current vaccines against various viral pathogens that infect the respiratory, gastrointestinal, and genital tracts and the role of breast milk antibodies protecting against viral diseases. In Part VII, “New and Novel Approaches for Mucosal Vaccine Development,” the chapters consider systems biological approaches and chemical libraries to design better vaccines and adjuvants for the general population and also consider the effects of aging on immune function. The last section, “Can Mucosal Vaccines Be Applied to Other Infectious and Noninfectious Diseases?,” delves into the possibility of adapting mucosal vaccination strategies for parasitic, blood-borne, veterinary, and aquatic pathogen-based diseases and to treat allergies and control inflammation. This book will benefit basic scientists, clinicians, and students who have an interest in adapting mucosal immunity to develop vaccines against infectious diseases and immunological disorders. This resource will aid anyone who is considering the various attributes and limitations of the different mucosal tissues, and we hope that it will inspire new approaches to produce better vaccines. We thank Ms. Gabriela Capille and Mr. Poulouse Joseph of Elsevier for their tireless efforts to make publication of this book possible. We also appreciate Dr. Yosuke Kurashima who contributed the design of cover. Hiroshi Kiyono and David W. Pascual

C H A P T E R

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Historical Perspectives on Mucosal Vaccines Michael W. Russell1 and Pearay L. Ogra2 1

2

Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY, United States Division of Infectious Diseases, Department of Pediatrics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, United States

I. INTRODUCTION

of the mucosal surfaces, where most infections occur or gain entry to the body. Furthermore, as recent studies have amply shown, the mucosal immune system has an integral role in interacting with the microbiota to establish a state of mutual coexistence, which is essential for health. Quantitative data clearly show that the great majority of immune cells are located within or adjacent to the mucosal epithelia of the orogastrointestinal, respiratory, and genital tracts and that the production of mucosal immunoglobulins, predominantly consisting of secretory IgA (SIgA), greatly exceeds the production of circulating immunoglobulins of all isotypes [1] (Chapter 4, Protective Activities of Mucosal Antibodies). The first historical efforts to induce prophylactic immunity appear to have used a mucosal route of immunization. In 10th century China, ground-up scabs of healed smallpox lesions were administered intranasally to generate

The science of immunology originated in large part to explain the findings of the pioneering vaccinologists such as Edward Jenner and Louis Pasteur, who demonstrated that infectious diseases could be prevented by the prior administration, usually by injection, of killed or attenuated pathogenic agents or their detoxified products. This had the unfortunate effect of instilling in the minds of medical practitioners and scientists the concept that immunity is elicited by systemic inoculation and consists of the induction of blood-borne (circulating) antibodies and immune cells. Indeed, the word “vaccine” is invariably associated, by both the general public and the media, with an image of the hypodermic syringe. Yet it should now be abundantly clear, at least to contemporary immunologists, that the primary function of the vertebrate immune system, including that of humans, is the protection

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00001-8

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© 2020 Elsevier Inc. All rights reserved.

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1. HISTORICAL PERSPECTIVES ON MUCOSAL VACCINES

immunity to this dread disease. The practice spread to India and thence to Turkey, but along the way it became changed to cutaneous inoculation, which was eventually adopted in Europe as “variolation” until it was supplanted by Jenner’s vaccination with cowpox. Paul Ehrlich in the late 19th century demonstrated that oral immunization of mice and other animals with two plant toxins, ricin and abrin, could induce protection against subcutaneous challenge with up to 800 times the lethal dose of these toxins [2,3]. He further showed that oral immunization protected the eyes against necrosis caused by the ocular application of ricin or abrin. In a similar historical perspective, ingestion of the leaves of poison ivy and related plants (Toxicodendron sp.) to modify the severity of reactions to skin exposure was reported to be an age-old practice in the United States [4 6]. In his classic monograph, Alexandre Besredka [7] observed: “The idea of local immunity as we conceive it, that is, an immunity without the obligatory participation of antibodies, has barely made its appearance. This conception already rests upon a large number of facts. Many of the phenomena, which cannot be explained by the accepted theories, are cleared up in the light of this new conception. As a result of these researches, applications to vaccination and vaccinotherapy have followed, and are now being employed in daily practice.” He thereby proposed the framework for modern concepts of mucosal immunity, based on his own studies and those of other contemporary investigators, including Shiga, Dumas, Chvostek, and Metchnikoff [8]. Besredka’s classic studies of immunity to Salmonella Typhi and Shigella dysenteriae had earlier demonstrated the induction of intestinal antibodies by oral immunization, which accounted for protection against these infections [9]. All of these remarkable clinical observations preceded by decades the discovery of IgA

[10] as well as the identification in human external secretions of SIgA as a unique immunoglobulin containing secretory component (SC [11,12]), the discovery of the J chain [13,14], and the characterization of the common mucosal immune system (CMIS [15]) and its mucosa-associated lymphoid tissues (MALT [16,17]) in many mammalian species (Chapter 2, Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance, Chapter 4, Protective Activities of Mucosal Antibodies and Chapter 5, Mucosal Immunity for Inflammation: Regulation of Gut-Specific Lymphocyte Migration by Integrins). These basic discoveries include the predominance of polymeric IgA-secreting plasma cells in mucosal tissues [18], their origin in mucosal inductive sites such as intestinal Peyer’s patches and trafficking to effector sites such as the lamina propria [19], the transepithelial transport of polymeric IgA into secretions by means of the polymeric Ig receptor that becomes the SC [20,21], and the function of the M cells in the follicle-associated epithelia in taking up antigens into the MALT [22], where they are processed by antigen-presenting cells for presentation to T cells and thence to stimulate B cells to switch to IgA production. The components of mucosal immunity have been expanded to include the mucosal epithelial cells themselves, intraepithelial lymphocytes, regulatory T cells, numerous cytokines, various antimicrobial proteins, and neuropeptides; all of this basic information on the mucosal immune system is extensively reviewed in the fourth edition of Mucosal Immunology [23]. Recent additions to the mucosal armamentarium include innate lymphoid cells [24] and mucosa-associated invariant T cells [25]. Arguably, one of the most important recent developments concerns the role of the microbiome in human health [26] and its effect on the development and daily operation of the mucosal immune system [27] (Chapter 9,

I. INTRODUCTION

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II. EXISTING MUCOSAL VACCINES

Influence of Commensal Microbiota and Metabolite for Mucosal Immunity). Recent findings have revealed the involvement of the microbiome in a variety of human health conditions, including inflammatory bowel disease, diabetes, obesity, allergies, asthma, and autoimmune disease [28]. It is also becoming clear that the microbiome has a considerable impact on the effectiveness of mucosal vaccination [29]. This chapter will briefly highlight the general principles of mucosal vaccines and vaccination in a historical context. Further details on

specific aspects of various mucosal vaccines will be discussed in subsequent chapters.

II. EXISTING MUCOSAL VACCINES Historically, a large variety of antigens have been used in experimental mucosal immunization studies (Table 1.1). Depending on the nature of the antigens and the experimental objectives, various outcomes were recorded. Pasteur, Roux, and Chamberland

TABLE 1.1 Selected List of Bacterial and Food Antigens Used in Mucosal Immunization Studies in Humans and Animals Antigen

Results and comments

References

Pasteurella multocida (chicken cholera)

Oral immunization; protection induced

[30]

Vibrio cholerae

Oral immunization, moderate protection

[31,32]

Mycobacterium tuberculosis

Serum antibodies induced by oral immunization

[33]

Yersinia pestis, Corynebacterium diphtheria

[34,35]

Shigella dysenteriae

Limited protection

[7,9]

Salmonella Typhi

Oral immunization preferable to systemic

[36]

Streptococcus pneumoniae

Protection achieved by nasal immunization

[37]

Protection achieved by oral immunization

[38]

Abrin, ricin

Oral immunization results in systemic and mucosal protection

[2,3]

Cow’s milk and whey

Prevention of anaphylaxis by feeding

[39]

Cow’s milk, ox blood, egg white, zein, oats

Decrease in systemic reactivity after prolonged but not short ingestion of these antigens

[40]

Dinitrochlorobenzene

Inhibition of systemic (skin) reactivity after hapten feeding; inability to suppress skin sensitivity by oral immunization in previously sensitized animals

[41]

Poison ivy

Oral ingestion results in decreased skin reactivity in a few studies; [42] discouraged for lack of efficacy

Horse serum and meat

Sensitization for anaphylaxis

[43]

Proteins from rice, corn, and oat flour

Precipitins in serum

[44]

Reproduced with modifications from Mestecky J, Bienenstock J, Lamm ME, Strober W, Cebra JJ, Mayer L, et al. Historical perspectives of mucosal immunity. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015b. p. xxxi lvii [45] with permission.

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experimented with chicken cholera, which is caused by what is now known as Pasteurella multocida, and achieved protection against infection by including the bacteria in the feed [30]. Over the following years, many others were able to induce immunity with varying degrees of success against Vibrio cholerae, Salmonella Typhi, Yersinia pestis, Corynebacterium diphtheriae, S. dysenteriae, and Mycobacterium tuberculosis in animals or in some cases in humans by oral administration of the bacteria [7,9,31,33 36,46,47]. At first, it was thought that protection was mediated by serum antibodies, but as was noted above, Besredka among others challenged that view and proposed the presence of antibodies in intestinal fluids. It is noteworthy that the bacillus Calmette Gue´rin (BCG) vaccine against tuberculosis was first developed for oral administration [33], although it is now given percutaneously or intradermally. Intranasal

immunization was also used to protect rabbits against Streptococcus pneumoniae [37]. Interestingly, these authors were unable to detect serum antibody by the method then in use (complement fixation), but we now know that this test would not have detected IgA (which had not been discovered at that time), and they did not examine nasal secretions. Several mucosally administered vaccines have already been developed for human use (Table 1.2). The oral polio vaccine developed by Albert Sabin [49] has been in use for several decades and has been instrumental in achieving the almost complete eradication of poliomyelitis from most countries worldwide. This vaccine consists of three attenuated serotypes of the poliovirus and appears to work by colonization of the gut tissues to maintain longterm stimulation of immune responses that result in circulating as well as intestinal virusneutralizing antibodies. Vivotif, an oral vaccine

TABLE 1.2 Existing Mucosal Vaccines for Human Application Infection

Vaccine

Route

Comments

Poliomyelitis

Oral polio vaccine (Sabin)

Oral

Three attenuated serotypes

Salmonella Typhi

Ty21a (Vivotif)

Oral

Live attenuated Salmonella Typhi

Cholera

Dukoral

Oral

Killed V. cholerae plus CTB

Shanchol, Orc-Vax

Oral

Killed V. cholerae

CVD103-HgR

Oral

Live attenuated V. cholerae

HA plus LT

Intranasal

Withdrawn

Flumist

Intranasal

Attenuated virus

Rotashield

Oral

Withdrawn

Rotarix

Oral

Attenuated virus, monovalent

RotaTeq

Oral

Attenuated virus, pentavalent

Adenovirus

Oral

Live adenovirus types 4 and 7

Influenza

Rotavirus

Adenovirus

Military use only CTB, Cholera toxin B subunit; HA, hemagglutinin; LT, E. coli heat-labile enterotoxin. Reproduced with modifications from Russell MW, Mestecky J. Mucosal vaccines: an overview. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015. p. 1039 1046 [48] with permission.

I. INTRODUCTION

II. EXISTING MUCOSAL VACCINES

against typhoid fever, consists of Salmonella enterica Typhi bacteria that were chemically mutagenized to render them avirulent [50]. The resulting Ty21a strain contains numerous mutations, including galE, which is involved in lipopolysaccharide synthesis, and it lacks the Vi polysaccharide capsule. However, it accomplishes a satisfactory level of protection lasting for up to 7 years [51], and it is still in use. The first oral cholera vaccine, Dukoral, which was developed in Sweden [52], consists of recombinant cholera toxin (CT) B subunit (CTB) plus inactivated V. cholerae bacteria and has demonstrated overall protective efficacy up to 85% in the first year, though efficacy declines over 3 years [51]. Other oral cholera vaccines consist of inactivated cholera vibrios (Shanchol, Orc-Vax) or live V. cholerae cells attenuated by genetic engineering to delete most of the toxin A subunit (CVD 103-HgR, Orochol, Mutacol), but are generally less effective. On the other hand, field evidence indicates that these oral vaccines have a substantial effect in diminishing the community burden of cholera infection by reducing transmission and enhancing herd immunity [51] (Chapter 22, Attenuated Salmonella for Oral Immunization, Chapter 29, Induction of Local and Systemic Immunity by Salmonella Typhi in Humans and Chapter 31, Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control). Mucosal vaccines have also been developed against viral infections, including influenza (Flumist), an attenuated virus that is given intranasally. Another intranasal influenza vaccine that consisted of the viral hemagglutinin plus a low dose of the labile toxin from Escherichia coli as adjuvant was withdrawn after concerns that a cluster of cases of Bell’s palsy occurred among some recipients [53]. Oral vaccines against rotavirus also have a checkered history. The first, Rotashield, was withdrawn because a small number of infants developed intestinal intussusception after receiving it. It was replaced by Rotatrix (monovalent)

7

and RotaTeq (pentavalent), attenuated viral vaccines that appear to avoid this serious adverse event. In this context it is noteworthy that rotavirus vaccines have been found less effective in Africa and Asia, and the composition of the intestinal microbiome has been found to be markedly different in responders and nonresponders [54]. Finally, an oral live adenovirus vaccine based on adenovirus types 4 and 7 has been developed to protect against these upper respiratory tract infections, but it is available only for military use. A larger number of mucosal vaccines have been developed for veterinary use in farm livestock or recreational animals, and some of these have been in use for many years (reviewed in Ref. [55]) (Chapter 48, Mucosal Vaccine Development for Veterinary and Aquatic Diseases). Administration of oral vaccines in the feed affords an easy means of immunizing large numbers of farm animals; for other vaccines, including some intended for pet animals that are treated on an individual basis, intranasal delivery is used. The control of rabies in wildlife populations has also been targeted by means of baited vaccines containing vaccinia virus-vectored rabies antigen, intended for consumption by species such as raccoons, foxes, and coyotes. The success of these vaccines illustrates the potential for further development of these approaches but at the same time reveals challenges that need to be overcome. Ensuing chapters will discuss the progress that has been made. Some advantages and disadvantages of mucosal vaccines and the various routes of administration are summarized in Table 1.3. Only two routes, oral (enteric) and intranasal, have actually been used for human application, but potential clearly exists for other routes, as has been demonstrated in various experimental animal models. In particular, the sublingual route [56] may avoid some complications of intranasal immunization, in which the possibility exists for uptake of adjuvants such as CT

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TABLE 1.3 Routes, Advantages, and Disadvantages of Mucosal Vaccines Route

In use?

Advantages

Disadvantages

Oral (enteric)

Yes

Most convenient

Destruction by gastric acid and digestive enzymes

CMIS dissemination Applicable to GI, also oral, genital (?) infections Needs delivery system and/or adjuvant Intranasal

Yes

Simple

May need spray device

CMIS dissemination

Risk of retrograde neural uptake in olfactory nerve (some adjuvants and antigens)

Applicable to URT, also oral, LRT, genital infections Sublingual

Rectal

Noa

a

No

Simple

None known

Applicable to URT, also oral, LRT, genital (?) infections

Mechanisms of dissemination uncertain

Avoids complications of intranasal

Target tissues uncertain

Applicable to lower GI, also genital infections

Low acceptability Limited to lower GI, genital tracts

Intravaginal

a

No

Applicable to female genital tract

Only applicable to female

a

Experimental use only, mainly in animals (few in humans). URT, Upper respiratory tract; LRT, lower respiratory tract; GI, gastrointestinal. Reproduced with modifications from Russell MW, Mestecky J. Mucosal vaccines: an overview. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015. p. 1039 1046, with permission.

and related enterotoxins that bind to ganglioside receptors and their retrograde migration into the olfactory bulb [57] (Chapter 27, Effectiveness of Sublingual Immunization: Innovation for Preventing Infectious Diseases). The rectal and vaginal routes have been shown to work in various experimental animal models; whether they can become applicable to humans is partly a matter of cultural acceptance. Different routes of administration may find utility in targeting different mucosal tracts, as it is now clear that the “common” mucosal immune system is not uniform but compartmentalized through the differential distribution of homing receptors and their addressins and of chemokines and their receptors that direct lymphocytes stimulated in different tissues to

migrate to particular effector sites. An often overlooked advantage of mucosal vaccines, especially when administered intranasally, is that they induce systemic immune responses manifested by circulating IgG antibodies as well as mucosal SIgA antibodies. The converse is rarely the case: Parenteral immunization usually does not induce effective SIgA responses in secretions.

III. STRATEGIES FOR ENHANCING MUCOSAL VACCINES The mucosal immune system, by virtue of its presence at the interface with the outside environment, must discriminate between antigenic

I. INTRODUCTION

III. STRATEGIES FOR ENHANCING MUCOSAL VACCINES

material of different nature. In the gastrointestinal tract the great majority of foreign antigens consist of food. Although most of this is digested to subimmunogenic elements—amino acids, peptides, sugars, and short glycans— even a minute fraction can represent an immunologically significant amount. For example, it has been estimated that up to about 0.001% of ingested protein can be absorbed into the circulation in relatively intact form [58,59]. It is clearly undesirable for food materials to elicit immune responses when they pose no threat; however, adverse reactions such as food allergies can cause debilitating effects in some individuals. To a large extent the mucosal immune system tolerates these materials by mounting regulatory or suppressor responses or ignores them altogether, as they are not presented concomitantly with signals to the innate pathogen-recognition receptors found in antigen-presenting cells (Chapter 51, Mucosal Vaccines for Allergy and Tolerance). The respiratory tract must also contend with inhaled dust particles, including plant pollens, which can induce quite severe allergic reactions in some unfortunate individuals yet, in most cases, are tolerated. The female reproductive tract has the important role of admitting allogeneic sperm and accommodating the implantation of a semiallogenic fetus, and immune responses to these can result in reproductive failure. The ways in which the fetal tissues are accepted at the maternofetal interface within the placenta have been well studied [60] but are beyond the scope of this chapter. At the next level is the huge microbiota, thought to comprise in humans some 1014 microbial cells representing thousands of species, many of which have not been cultured in vitro or even adequately described and are known only through metagenomic sequencing (Chapter 9, Influence of Commensal Microbiota and Metabolite for Mucosal Immunity). Not only are most commensals nonthreatening, but many play an essential role in health

9

by contributing essential micronutrients, displacing potentially pathogenic species, and stimulating the normal development of the immune system. It has long been known that animals raised in a germ-free environment have severely stunted immune systems that are partially restored when a normal microbiota is established. It has become clear that certain bacterial species, among them the so-called segmented filamentous bacterium [61], Bacteroides fragilis, and certain species of Clostridium, have specific effects in stimulating the development of some T cell lineages ([62 64]; reviewed in Ref. [65]). Conversely, the suppression of the normal microbiota by treatment with broadspectrum antibiotics can lead to colonization by pathogens such as Clostridium difficile. The microbiota again must be tolerated yet kept in place, as some species can become pathogenic if allowed to overgrow, invade, or gain access to tracts where they are not normally present. For example, Neisseria meningitidis and S. pneumoniae can be carried as commensals in the human nasopharynx and become pathogenic only if they invade or descend into the lungs. Likewise, urinary tract infections can be caused by commensal types of E. coli that are normally present in the colon. Immune responses in the form of SIgA antibodies can usually be detected in intestinal and other secretions to commensal organisms, and studies have indicated that these responses continue as long as the stimulus remains present but can be displaced by other responses if the original stimulus (i.e., the inducing organism) is eliminated [66]. An intriguing consideration is that the so-called pathogen-associated molecular patterns, which are considered necessary to engage the innate pattern-recognition receptors in antigen-presenting cells and thereby induce strong responses, occur also in nonpathogenic members of the microbiota, which generally do not induce the same level of responses. This suggests that pathogens exert an additional stimulus, perhaps as a consequence of the

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1. HISTORICAL PERSPECTIVES ON MUCOSAL VACCINES

damage they inflict on the host tissues or because they invade into the host cells. At the top level, as originally observed by Besredka and his contemporaries, the mucosal immune system can mount powerful responses to overt pathogens, and protective immunity to these infections can be achieved by such responses. In developing mucosal vaccines, therefore, it becomes necessary to mimic the kind of stimulus generated by these pathogens or their products in order to induce effective responses. For many years, mucosal immune responses were induced experimentally by putting antigens such as soluble model proteins or killed microorganisms in the drinking water or administering them by gastric intubation. In many cases, responses were recorded in terms of SIgA antibodies in saliva, milk, feces, or intestinal or respiratory lavages, thereby demonstrating the operation of the CMIS. Indeed, the first demonstration of the CMIS in humans was achieved by this approach; after drinking inactivated suspensions of the oral bacterium Streptococcus mutans, most subjects responded by developing salivary IgA antibodies [67]. A subsequent paper further showed that subjects immunized with S. mutans cells given in enteric capsules developed IgA antibody-secreting cells in the circulation 7 10 days after immunization, providing evidence supporting the trafficking of IgA antibody-producing cells from the inductive sites (in this case, presumably the Peyer’s patches) to the effector sites such as the salivary glands [68]. However, responses declined over several weeks, and no evidence was seen for heightened responses to repeated immunization, indicative of an anamnestic response. At the time, it was unclear whether memory could be established and effectively recalled within the mucosal immune system. However, studies using the much more potent mucosal immunogen CT or CTB subunit showed that memory could be induced and rapidly recalled at least in the intestine [69,70] (Chapter 7, Induction and Regulation of

Mucosal Memory B Cell Responses). Meanwhile, CT was shown to be not only a potent immunogen, but also an effective mucosal adjuvant when mixed with other antigens and given orally [71,72] (Chapter 11, ToxinBased Modulators for Regulation of Mucosal Immune Responses). Furthermore, coupling of antigens to CTB, either chemically or by genetic engineering, was shown to promote their mucosal immunogenicity [73 75]. Controversy arose, however, over the relative adjuvant properties of CT and CTB, because commercially available preparations of biochemically purified CTB always contained small amounts of intact CT that were sufficient to give CTB its adjuvant effect [76]. While some studies showed that the adjuvant effect of CT depended upon its toxic enzyme activity [77], others showed that recombinant CTB devoid of toxic activity could still serve as a mucosal adjuvant [78]. It seems likely that the intrinsic properties of the nominal antigen as well as route of administration contributed to the divergent findings. A huge literature has since arisen demonstrating the mucosal adjuvant properties of CT and the related labile enterotoxins of E. coli in a variety of experimental models and potential applications (reviewed in Ref. [79]). Considerable efforts have been made to separate the adjuvanticity of these enterotoxins from their toxicity and develop them for human use, and while some success has been reported, for example, by mutation of E. coli heat-labile enterotoxin to delete key residues required for toxicity [80], none has yet been developed for human use. Several other strategies have been evaluated to enhance the immune response to mucosally administered vaccines (Table 1.4). It was found early on that microparticulate antigens were often more immunogenic than soluble antigens when delivered orally, and uptake of such particles by the M cells of the Peyer’s patches was demonstrated (Chapter 28, M CellTargeted Vaccines). Therefore efforts were

I. INTRODUCTION

III. STRATEGIES FOR ENHANCING MUCOSAL VACCINES

TABLE 1.4 Strategies for Enhancing Mucosal Vaccines Principle

Examples

Mucosal adjuvants

Cholera toxin (CT), E. coli labile toxins (LT), lectins

Antigen coupled to carriers

CTB, etc., chemical or genetic constructs

Microparticles (etc.)

Biodegradable polymers (e.g., PLG), liposomes, etc.

Retention at surfaces

Adhesive polymer matrices

Live bacterial vectors

Attenuated Salmonella, etc., BCG, commensals (lactobacilli, oral streptococci)

Live viral vectors

Vaccinia, adenovirus, canarypox, poliovirus replicons

Edible plant vectors

Potatoes, tomatoes, corn, rice

CTB, Cholera toxin B subunit; PLG, poly(lactate-co-glycolate); BCG, bacillus Calmette Gue´rin.

made to incorporate antigens in microparticles composed of biocompatible and biodegradable polymers, such as poly(lactate-co-glycolate), from which resorbable sutures are made, to deliver them into the Peyer’s patches, where the antigens would be released upon hydrolysis of the polymers. These formulations have the additional desirable property of protecting the antigens from digestion by the gastrointestinal fluids. In addition, liposomes and a variety of other microparticles including cochleates, immunostimulating complexes based on Quil A saponins, and other proprietary formulations have been evaluated with varying degrees of success [81]. Since Peyer’s patches were shown to be a route of entry for enteric pathogens [82] and genetic engineering made it possible to express foreign antigens in genetically “crippled” pathogens, the concept of delivering heterologous vaccines in live bacterial vectors became feasible. This was first demonstrated by using Salmonella carrying deletions in virulence genes and expressing antigens from S. mutans [83]. Since then, a large variety of bacterial vectors

11

for cloned antigens have been proposed, including avirulent Salmonella and other enteric bacteria, the BCG strain of mycobacteria, and commensals such as lactobacilli or oral streptococci [84] (Chapter 22, Attenuated Salmonella for Oral Immunization and Chapter 23, Recombinant Bacillus Calmette-Guerin for Mucosal Immunity). Advantages include the possibility of creating multivalent vaccines by cloning numerous different antigens in the vector. Limitations include low levels of expression of the cloned antigens and usually a strong immune response against the vector itself that limits its continued use. Experimental success has been reported especially for Salmonella, which invades and delivers the cloned antigens into the Peyer’s patches, but achieving a balance between delivery of sufficient antigen and lack of pathogenicity is key. Commensal vectors should be a potentially safer alternative [85], but given that the mucosal immune system generally mounts only limited responses to commensals, it is open to question whether they can effectively serve to induce responses against vaccine antigens cloned in them. Live viral vectors have also been proposed, including vaccinia, adenoviruses, poliovirus replicons, or pseudoviruses [86] (Chapter 24, Recombinant Adenovirus Vectors as Mucosal Vaccines and Chapter 42, Mucosal Vaccines Against HIV/SIV Infection). Being much smaller than bacteria, viruses cannot usually carry large amounts of cloned antigen genes, although this is less of a problem for large DNA viruses such as vaccinia. Adenoviruses have been used as vectors in the search for an effective vaccine against HIV, but again the problem can be that responses to the vector, including preexisting immunity, may interfere with the induction of the desired response to the delivered antigen. One such candidate vaccine had to be withdrawn from clinical trial when it became apparent that vaccinated subjects were rendered more susceptible to HIV than the control subjects were.

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1. HISTORICAL PERSPECTIVES ON MUCOSAL VACCINES

Finally, the ability to clone antigens from pathogens into plants raises the intriguing possibility of creating edible mucosal vaccines. Potatoes, tomatoes, corn, rice, and other food plants have been proposed, and some degree of success has been reported [87]. For example, potatoes have been engineered to express the hepatitis B viral antigen, and consumption of these leads to the development of neutralizing antibodies [88]. Advantages include the potential to grow large quantities of vaccine material at low cost. However, levels of expression of the desired antigen are often low, and it can be difficult to ensure reproducible delivery of the required vaccine dose. Cooking or other processing of the food plant might easily destroy the vaccine antigen, but a more serious concern is that the mucosal immune system has evolved to make only weak responses to food antigens, so disguising a vaccine as food is likely to be unproductive. The inclusion of an immunostimulating agent or adjuvant in the vaccine construct might overcome this limitation but could have the unfortunate consequence of inducing a response to the food material itself. On the other hand, plant-grown antigen material could be used as a feedstock for producing and purifying large quantities of vaccine antigen, though this might not necessarily be administered mucosally. It is interesting that plants (tobacco) have been used to engineer and produce humanized monoclonal SIgA antibody against S. mutans, for passive immunization against dental caries [89]. While this particular product met with only limited success in clinical trial [90], considerable potential exists for exploiting the principle for manufacturing other products for passive protection of mucosal surfaces (Chapter 20, Plant-Based Mucosal Vaccine Delivery Systems and Chapter 21, Plant-Based Mucosal Immunotherapy: Challenges for Commercialization). Subsequent chapters in this volume will elaborate on the more recent use of these and other strategies for developing mucosal vaccines.

IV. ORAL TOLERANCE A frequently raised concern in connection with mucosal, especially oral, vaccination is whether counterproductive tolerance might be induced instead of the desired positive immune response. Much of this concern, however, is based on a misunderstanding of the phenomenon of oral tolerance, what it consists of, and the conditions required for its induction. Oral tolerance was first described by Besredka [39] and Wells and Osborne [40], who showed that ingestion by animals of proteins such as cow’s milk, egg white, or antigens derived from corn or oats could lead to a state of unresponsiveness when the same materials were subsequently given by injection. Later, Chase [41] reported that skin sensitization with the hapten 2,4-dinitrochlorobenzene was abrogated by prior ingestion of the hapten. A number of other studies since then have amply confirmed these findings: that the first administration of antigens by the oral (enteric) route suppresses the development of immune responses when the same antigens are subsequently given parenterally. This is the phenomenon correctly described as “oral tolerance.” Because it has also been demonstrated by prior intranasal [91] or even intravaginal administration [92], the concept has been expanded as “mucosal tolerance.” Unfortunately, the phenomenon has often been confused with the lack of response to otherwise immunogenic materials when these are given mucosally. However, as we noted above, the mucosal immune system usually does not respond to bland antigens, such as food materials, that do not pose a threat or cause damage to the mucosal surfaces. Lack of immune responsiveness can occur for many reasons, including the absence of costimulatory signals such as are provided by microbe-associated molecular patterns, which are necessary to trigger the patternrecognition receptors on antigen-presenting cells. True oral tolerance is most readily

I. INTRODUCTION

V. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

induced by soluble rather than particulate antigens and in young animals. Moreover, it can co-occur with a mucosal (SIgA) antibody response, described as “split tolerance” [93]. Oral tolerance is more readily displayed in the T cell compartment than in B cells, as revealed by the suppression of delayed-type hypersensitivity more than of antibody responses [94]. Mucosal tolerance is now known to be mediated by regulatory T cells and the cytokines TGF-β and IL-10, which are also implicated in switching B cells to produce IgA in the mucosal inductive sites. An important point is that mucosal exposure must precede systemic exposure to the antigen. Prior sensitization to the antigen abrogates the induction of tolerance by mucosal exposure; thus it is extremely difficult to reverse by mucosal application of an antigen an immune response that has already been induced. Most human populations are naturally exposed from birth to a variety of microbial antigens in the environment and therefore make subliminal if not overt responses to them. Thus it is difficult to identify adults who have not had some degree of exposure to a given antigen and are therefore susceptible to the induction of mucosal tolerance as opposed to an active response. It can therefore be argued that the potential induction of oral (mucosal) tolerance by mucosal vaccination is an overstated problem [94]. Conversely, the intentional induction of mucosal tolerance by oral immunization has been investigated as a potential therapy for adverse immune reactions, such as atopic allergies and autoimmune diseases, or even to suppress allograft rejection (Chapter 51, Mucosal Vaccines for Allergy and Tolerance). If this could be accomplished, it would be of enormous medical benefit. For the most part, however, success has been extremely limited. As was noted above, once an immune response has been induced, it is very difficult to reverse or modify it. However, an exception

13

is provided by the finding that ingestion of some antigens chemically conjugated to pure recombinant CTB can substantially reverse previously induced immune responses against the antigen [95]. The presence of even small amounts of intact CT prevented this generation of tolerance. Potential applications of this strategy include autoimmune encephalitis [96], diabetes [97], arthritis [98], uveitis [99], and atopic allergy [100], as shown in experimental animal models. However, none has yet been developed for human application.

V. CONCLUDING REMARKS AND FUTURE PERSPECTIVES The science of mucosal immunology has made enormous strides in the past 20 years or so, especially with respect to cellular aspects such as the diversity of immune cells present in mucosal tissues, including recently discovered innate lymphoid cells, invariant T cells, regulatory T cells, and the role of antigenpresenting cells of different functional types in the induction of different modes of response. These findings have added immeasurably to understanding the complexity of mucosal immunity, which for a long time was dominated by a focus on the role of SIgA as the primary mediator of mucosal immune protection. In addition, numerous humoral factors of innate defense, such as various antimicrobial proteins produced by epithelial cells, have been shown to have a significant role in guarding the mucosal epithelia against microbial attack. Of crucial importance is that protection of the mucosae must be accomplished without inflicting inflammatory damage on the mucosal epithelium itself, which in most areas consists of a single layer of cells. This requires balancing the responses against the nature of the threats represented by different antigenic challenges, which range from essentially harmless food components, through the complex microbiota

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1. HISTORICAL PERSPECTIVES ON MUCOSAL VACCINES

of commensals that are necessary to health, to outright pathogens. Dysregulation of these responses can lead to chronic inflammatory conditions such as inflammatory bowel disease and possibly to systemic manifestations, including autoimmune disease. The challenge in mucosal vaccine development, therefore, is to devise strategies for eliciting the kind of effective immune responses induced by pathogens and their products but without inflicting pathological damage and to establish immune memory that can be rapidly recalled to forestall pathogens at the portals of entry, the mucosal epithelia. While that goal is essentially the same as that for conventional parenteral vaccine development, achieving it requires attention to the subtle complexities of immune responsiveness at the mucosae and to the mechanisms of defense that must discriminate between pathogens, commensals, and food materials while preserving the integrity of the mucosal barrier.

References [1] Pabst R, Russell MW, Brandtzaeg P. Tissue distribution of lymphocytes and plasma cells and the role of the gut. Trends Immunol 2008;29:206 8. [2] Ehrlich P. Experimentelle untersuchungen u¨ber ¨ ber ricin. Deutsche Med Wochenschr immunita¨t I. U 1891;17:976 9. [3] Ehrlich P. Experimentelle untersuchungen u¨ber ¨ ber abrin Deutsche Med Wochenschr immunita¨t II. U 1891;17:1218 19. [4] Duncan CH. Autotherapy in ivy poisoning. NY Med J 1916;104:901. [5] French, J.M. Treatment of ivy poisoning. Clin Med 1916;August:753 755. [6] Shelmire B. Cutaneous and systemic reactions observed during oral poison ivy therapy. J Allergy 1941;12:252 71. [7] Besredka A. Local immunization. Baltimore, MD: Williams & Wilkins; 1927. p. 7. [8] Metchnikoff E, Besredka A. Ann. Instit. Pasteur 1911; March:210. [9] Besredka A. De la vaccination contre les e´tats typhoides par la voie buccale. Ann Inst Pasteur 1919;33:882 903.

[10] Heremans JF, Heremans MT, Schultze HE. Isolation and description of a few properties of the beta 2A-globulin of human serum. Clin Chim Acta 1959;4:96 102. ˚ . Comparative immunological studies of the [11] Hanson LA immune globulins of human milk and of blood serum. Int Arch Allergy Appl Immunol 1961;18:241 67. [12] Tomasi TB, Tan EM, Solomon A, Prendergast RA. Characteristics of an immune system common to certain external secretions. J Exp Med 1965;121:101 24. [13] Koshland ME. The coming of age of the immunoglobulin J chain. Annu Rev Immunol 1985;3:425 53. [14] Mestecky J, Zikan J, Butler WT. Immunoglobulin M and secretory immunoglobulin A: presence of a common polypeptide chain different from light chains. Science 1971;171:1163 5. [15] Mestecky J. The common mucosal immune system and current strategies for induction of immune response in external secretions. J Clin Immunol 1987;7:265 76. [16] McDermott MR, Bienenstock J. Evidence for a common mucosal immunologic system I. Migration of B immunoblasts into intestinal, respiratory, and genital tissues. J Immunol 1979;122:1892 8. [17] Brandtzaeg P, Kiyono H, Pabst R, Russell MW. Terminology: nomenclature of mucosa-associated lymphoid tissue. Mucosal Immunol 2008;1:31 7. [18] Brandtzaeg P. The mucosal B cell system. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/ Elsevier; 2015. p. 623 81. [19] Craig SW, Cebra JJ. Peyer’s patches: an enriched source of precursors for IgA-producing immunocytes in rabbit. J Exp Med 1971;134:188 200. [20] Crago SS, Kulhavy R, Prince SJ, Mestecky J. Secretory component on epithelial cells is a surface receptor for polymeric immunoglobulins. J Exp Med 1978;147:1832 7. [21] Brandtzaeg P. Polymeric IgA is complexed with secretory component (SC) on the surface of human intestinal epithelial cells. Scand J Immunol 1978;8:39. [22] Bockman DE, Cooper MD. Pinocytosis by epithelium associated with lymphoid follicles in the bursa of Fabricius, appendix, and Peyer’s patches. An electron microscopic study. Am J Anat 1973;136:455 77. [23] Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/ Elsevier; 2015. [24] Artis D, Spits H. The biology of innate lymphoid cells. Nature 2015;517:293 301. [25] Wong EB, Ndung’u T, Kasprowicz VO. The role of mucosal-associated invariant T cells in infectious diseases. Immunology 2017;150:45 54.

I. INTRODUCTION

REFERENCES

[26] Foster KR, Schluter J, Coyte KZ, Rakoff-Nahoum S. The evolution of the host microbiome as an ecosystem on a leash. Nature 2017;548:43 51. [27] Ogra PL. Role of mucosal microbiome in the development and function of mucosal immune system. Biotascope 2016;20 7. [28] Clemente JC, Ursell LK, Parfrey LW, Knight R. The impact of the gut microbiota on human health: an integrative view. Cell 2012;148:1258 70. [29] Valdez Y, Brown EM, Finlay BB. Influence of the microbiota on vaccine effectiveness. Trends Immunol 2014;35:526 37. [30] Pasteur L. De l’attenuation du virus du chole´ra des poules. Compt Rend Acad Sci 1880;91:673 80. [31] Klemperer G. Untersuchungen u¨ber ku¨nstlichen Impfschutz gegen Choleraintoxication. Berliner Klin Wochenschr 1892;29:789 93. [32] Metchnikoff E. In: Mitchell PC, editor. The nature of man. New York: G.P. Putman; 1903. p. 72 3. [33] Calmette A. Les vaccinations microbiennes par voie buccale. Ann Inst Pasteur 1923;37:900 20. [34] Dserzgowdky SK. Ueber die aktive Immunisierung des Menschen gegen Diphtherie. Zeitschr Immunita¨tsforsh Exp Therap 1910;3:602. [35] Enlows EMA. Vaccination by mouth against bacillary dysentery. Publ Health Report 1925;40:639 49. [36] Vaillant L. Note sur l’emploi du vaccine bilie´ de Besredka par la voie buccale dans quelques foyers e´pide´miques de fie´vre typhoı¨de. Ann Inst Pasteur 1922;36:149 56. [37] Bull CG, McKee CM. Respiratory immunity in rabbits. VII. Resistance to intranasal infection in the absence of demonstrable antibodies. Am J Hyg 1929;9:490 9. [38] Ross V. Oral immunization against pneumococcus. Use of bile salt dissolved organisms, etc., time of appearance of immunity and dosage. J Exp Med 1930;51:585 607. [39] Besredka A. De l’anaphylaxie. De l’anaphylaxie lactique. Ann Inst Pasteur 1909;23:166 76. [40] Wells HG, Osborne JB. The biological reactions of vegetable proteins. J Infect Dis 1911;8:66 124. [41] Chase MW. Inhibition of experimental drug allergy by prior feeding of the sensitizing agent. Proc Soc Exp Biol Med 1946;61:257 9. [42] Stevens FA. Council on pharmacy and chemistry. Report of the council: status of poison ivy extracts. J Am Med Assoc 1945;127:912 21. [43] Rosenau MJ, Anderson JF. Further studies on hypersusceptibility and immunity. J Med Res 1907;16:381 418. [44] Magus W. Weitere Ergebnisse der Serum-Diagnostik fu¨r die theoretische und angewandte Botanik. Ber Deut Bot Gesell 1906;26a:532 9.

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[45] Mestecky J, Bienenstock J, Lamm ME, Strober W, Cebra JJ, Mayer L, et al. Historical perspectives of mucosal immunity. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015. p. xxxi lvii. [46] Combiesco D, Magheru A, Calab G. Vaccination preventive contre la dysente´rie par la voie digestive, chez le lapin. Compt Rend Seanc Soc Biol Filial 1923;88:904 6. [47] Gay FP. Local resistance and local immunity to bacteria. Physiol Rev 1924;4:191 214. [48] Russell MW, Mestecky J. Mucosal vaccines: an overview. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/ Elsevier; 2015. p. 1039 46. [49] Ogra PL, Karzon DT, Righthand F, MacGillivray M. Immunoglobulin response in serum and secretions after immunization with live and inactivated poliovaccine and natural infection. N Engl J Med 1968;279:893 900. [50] Germanier R, Furer E. Isolation and characterization of galE mutant Ty21a of Salmonella typhi: a candidate strain for a live oral typhoid vaccine. J Infect Dis 1975;141:553 8. [51] Holmgren J, Levine MM. Vaccines against bacterial enteric infections. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015. p. 1041 82. [52] Clemens JD, Sack DA, Harris JR, Van Loon F, Chakraborty J, Ahmed F, et al. Field trial of oral cholera vaccines in Bangladesh: results from three-year follow-up. Lancet 1990;335:270 3. [53] Mutsch M, Zhou W, Rhodes P, Bopp M, Chen RT, Linder T, et al. Use of the inactivated intranasal influenza vaccine and the risk of Bell’s palsy in Switzerland. N Engl J Med 2004;350:896 903. [54] Harris VC, Armah G, Fuentes S, Korpela KE, Parashar U, Victor JC, et al. Significant correlation between the infant gut microbiome and rotavirus vaccine response in rural Ghana. J Infect Dis 2017;215:34 41. [55] Hodgins DC, Chattha K, Vlasova A, Parren˜o V, Corbeil LB, Renukaradhya GJ, et al. Mucosal veterinary vaccines: comparative vaccinology. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/ Elsevier; 2015. p. 1337 61. [56] Czerkinsky C, Cuburu N, Kweon MN, Anjuere F, Holmgren J. Sublingual vaccination. Hum Vacc 2011;7:110 14.

I. INTRODUCTION

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1. HISTORICAL PERSPECTIVES ON MUCOSAL VACCINES

[57] van Ginkel FW, Jackson RJ, Yuki Y, McGhee JR. Cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues. J Immunol 2000;165:4778 82. [58] Brown TA, Russell MW, Mestecky J. Elimination of intestinally absorbed antigen into the bile by IgA. J Immunol 1984;132:780 2. [59] Husby S, Jensenius JC, Svehag S-E. Passage of undegraded dietary antigen into the blood of healthy adults. Quantification, estimation of size distribution, and relation of uptake to levels of specific antibodies. Scand J Immunol 1985;22:83 92. [60] Shivhare SB, Bulmer JN, Lash GF. Immunity at the maternal-fetal interface. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015. p. 2231 50. [61] Schnupf P, Gaboriau-Routhiau V, Gros M, Friedman R, Moya-Nilges M, Nigro G, et al. Growth and host interaction of mouse segmented filamentous bacteria in vitro. Nature 2015;520:99 103. [62] Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H, et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 2013;500:232 6. [63] Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 2009;139:485 98. [64] Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 2005;122:107 18. [65] Round JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 2009;9:313 23. [66] Hapfelmeier S, Lawson MA, Slack E, Kirundi JK, Stoel M, Heikenwalder M, et al. Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses. Science 2010;328:1705 9. [67] Mestecky J, McGhee JR, Arnold RR, Michalek SM, Prince SJ, Babb JL. Selective induction of an immune response in human external secretions by ingestion of bacterial antigen. J Clin Invest 1978;61:731 7. [68] Czerkinsky C, Prince SJ, Michalek SM, Jackson S, Russell MW, Moldoveanu Z, et al. IgA antibodyproducing cells in peripheral blood after antigen ingestion: evidence for a common mucosal immune system in humans. Proc Natl Acad Sci USA 1987;84:2449 53. [69] Lycke N, Holmgren J. Intestinal mucosal memory and presence of memory cells in lamina propria and Peyer’s patches in mice 2 years after oral immunization with cholera toxin. Scand J Immunol 1986;23:611 16. [70] Lycke N, Holmgren J. Long-term cholera antitoxin memory in the gut can be triggered to antibody

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

formation associated with protection within hours of an oral challenge immunization. Scand J Immunol 1987;25:407 12. Elson CO, Ealding W. Cholera toxin feeding did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated protein antigen. J Immunol 1984;133:2892 7. Lycke N, Holmgren J. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. Immunology 1986;59:301 8. Czerkinsky C, Russell MW, Lycke N, Lindblad M, Holmgren J. Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary glands and extramucosal tissues. Infect Immun 1989;57:1072 7. Hajishengallis G, Hollingshead SK, Koga T, Russell MW. Mucosal immunization with a bacterial protein antigen genetically coupled to cholera toxin A2/B subunits. J Immunol 1995;154:4322 32. Russell MW, Wu H-Y. Distribution, persistence, and recall of serum and salivary antibody responses to peroral immunization with protein antigen I/II of Streptococcus mutans coupled to the cholera toxin B subunit. Infect Immun 1991;59:4061 70. Wilson AD, Clarke CJ, Stokes CR. Whole cholera toxin and B subunit act synergistically as an adjuvant for the mucosal immune response of mice to keyhole limpet haemocyanin. Scand J Immunol 1990;31:443 51. Lycke N, Tsuji T, Holmgren J. The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity. Eur J Immunol 1992;22:2277 81. Wu H-Y, Russell MW. Induction of mucosal and systemic immune responses by intranasal immunization using recombinant cholera toxin B subunit as an adjuvant. Vaccine 1998;16:286 92. Freytag LC, Clements JD. Mucosal adjuvants: new developments and challenges. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015. p. 1183 99. Pizza M, Giuliani MM, Fontana MR, Monaci E, Douce G, Dougan G, et al. Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants. Vaccine 2001;19:2534 41. McEntee C, Lavelle EC, O’Hagan DT. Antigen delivery systems I: nonliving microparticles, liposomes, and immune-stimulating complexes (ISCOMs. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/ Elsevier; 2015. p. 1211 31.

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REFERENCES

[82] Carter PB, Collins FM. The route of enteric infection in normal mice. J Exp Med 1974;139:1189 203. [83] Curtiss R, Goldschmidt RM, Fletchall NB, Kelly SM. Avirulent Salmonella typhimurium Δcya Δcrp oral vaccine strains expressing a streptococcal colonization and virulence antigen. Vaccine 1988;6:155 60. [84] Curtiss R. Antigen delivery system II: development of live attenuated bacterial vectors. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015. p. 1233 69. [85] Wells JM, Robinson K, Chamberlain LM, Schofield KM, Le Page RWF. Lactic acid bacteria as vaccine delivery vehicles. Antonie Van Leeuwenhoek 1996;70:317 30. [86] Rosenthal KL, Jeyanathan M, Xing J. Filling the immunological gap: recombinant viral vectors for mucosal vaccines. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015. p. 1291 306. [87] Mason HS, Thueneman E, Kiyono H, Kessans S, Matoba N, Mor T. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Academic Press/Elsevier; 2015. p. 1271 89. [88] Thanavala Y, Yang YF, Lyons P, Mason HS, Arntzen C. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc Natl Acad Sci USA 1995;92:3358 61. [89] Ma JK-C, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, et al. Generation and assembly of secretory antibodies in plants. Science 1995;268:716 19. [90] Weintraub JA, Hilton JF, White JM, Hoover CI, Wycoff KL, Yu L, et al. Clinical trial of a plantderived antibody on recolonization of mutans streptococci. Caries Res 2005;39:241 50. [91] Waldo FB, van den Wall Bake AWL, Mestecky J, Husby S. Suppression of the immune response by

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

nasal immunization. Clin Immunol Immunopath 1994;72:30 4. Black CA, Rohan LC, Cost M, Watkins SC, Draviam R, Alber S, et al. Vaginal mucosa serves as an inductive site for tolerance. J Immunol 2000;165:5077 83. Challacombe SJ, Tomasi TB. Systemic tolerance and secretory immunity after oral immunization. J Exp Med 1980;152:1459 72. Mestecky J, Russell MW, Elson CO. Perspectives on mucosal vaccines: is oral tolerance a barrier? J Immunol 2007;179:5633 8. Sun JB, Holmgren J, Czerkinsky C. Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proc Natl Acad Sci USA 1994;91:10795 9. Sun JB, Rask C, Olsson T, Holmgren J, Czerkinsky C. Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit. Proc Natl Acad Sci USA 1996;93:7196 201. Bergerot I, Ploix C, Petersen J, Moulin V, Rask C, Fabien N, et al. A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes. Proc Natl Acad Sci USA 1997;94:4610 14. Tarkowski A, Sun JB, Holmdahl R, Holmgren J, Czerkinsky C. Treatment of experimental autoimmune arthritis by nasal administration of a type II collagen-cholera toxoid conjugate vaccine. Arthr Rheum 1999;42:1628 34. Phipps PA, Stanford MR, Sun JB, Xiao BG, Holmgren J, Shinnick T, et al. Prevention of mucosally induced uveitis with a HSP60-derived peptide linked to cholera toxin B subunit. Eur J Immunol 2003;33:224 32. Rask C, Holmgren J, Fredriksson M, Lindblad M, Nordstro¨m I, Sun JB, et al. Prolonged oral treatment with low doses of allergen conjugated to cholera toxin B subunit suppresses immunoglobulin E antibody responses in sensitized mice. Clin Exp Allergy 2000;30:1024 32.

I. INTRODUCTION

C H A P T E R

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Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance Aaron Silva-Sanchez and Troy D. Randall Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, United States

I. INTRODUCTION

responses that might impair the physiological function of the host organ. Therefore mucosal lymphoid organs must have robust mechanisms to fine-tune immune responses to the types of antigen they encounter. Immune responses to mucosal antigens are initiated in a variety of secondary lymphoid organs (SLOs) that collect antigens from mucosal surfaces, recruit naı¨ve B and T cells from the blood, and utilize networks of fibroblastic stromal cells to organize encounters among various cell types and generate immune responses that are appropriate for the type of antigen and the tissue where the antigens are encountered. In the gut, the inductive tissues, gut-associated lymphoid tissues (GALT), include the Peyer’s patches, cecal patches, colonic patches, isolated lymphoid follicles (ILFs), and cryptopatches, which, along with the gut-draining mesenteric

The digestive and respiratory tracts are continuously exposed to antigens, commensals, and pathogens present in the lumen that can sometimes cross the mucosal epithelium, a barrier that is necessary for nutrient acquisition in the gut and for gas exchange in the respiratory tract. However, the exposure of the mucosal surface to exogenous materials also makes these tissues ports of entry for potentially pathogenic organisms. As a result, the immune system devotes enormous resources to the defense of mucosal surfaces. Although this defense needs to effectively prevent microbes from invading the underlying tissue, it must critically discriminate between innocuous antigens, commensal organisms, and pathogens in order to prevent inappropriate or inflammatory immune

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00002-X

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© 2020 Elsevier Inc. All rights reserved.

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lymph nodes (MLNs), are inductive sites for adaptive immune responses to gut-derived antigens. In the respiratory tract, immune-inductive tissues include the nasopharyngeal-associated lymphoid tissue (NALT) and inducible bronchus-associated lymphoid tissue (iBALT), which, along with the cervical and mediastinal lymph nodes (LNs), generate adaptive immune responses to antigens encountered in the upper and lower respiratory tracts, respectively [1]. Mucosal lymphoid tissues and systemic lymphoid organs share basic architectural features that are required for the immune-inductive functions of these organs. For example, each lymphoid organ has mechanisms for recruiting leukocytes from the blood, mechanisms for spatially arranging lymphocytes and myeloid cells to maximize the efficiency of cellular interactions, mechanisms for sampling antigens from the surrounding tissue or from the luminal surface of the mucosal epithelium, and, finally, mechanisms for sending activated effector lymphocytes back into the periphery or to distal portions of the mucosal surface, where they eliminate pathogens or reside as memory cells (Chapter 3: Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells) and Chapter 5: Mucosal Immunity for Inflammation: Regulation of Gut-Specific Lymphocyte Migration by Integrins). Although these activities are generally similar in each type of mucosal lymphoid tissue, the mechanistic details that control each of these functions are often different, depending on the host organ (lung or gut), the location of the mucosal tissue in that organ, and the types of antigens that are encountered. Although most mucosal lymphoid tissues are true SLOs and form at predictable sites during embryogenesis, independently of antigen or microbial signals, other mucosal lymphoid tissues form after birth at sites of infection or inflammation and require microbial or inflammatory signals for their development. These tissues are often referred to as ectopic or tertiary lymphoid organs (TLOs) [2]. Despite their

different developmental origins, SLOs and TLOs share many overlapping developmental, architectural, and functional features. Nevertheless, the mechanistic details governing the immune functions of these organs are often different, in part owing to the stimuli that triggered their development and to the host organ in which they reside. Given that mucosal lymphoid organs can be involved in either pathogenesis, symbiosis or immune protection in a variety of clinical conditions and experimental disease models, it is essential to understand the mechanistic details controlling the development and function of each type of mucosal lymphoid organ in order to intervene in local mucosal immune responses. In this chapter, we will discuss the specific mechanisms that control the structure, development, and function of various mucosal lymphoid tissues in the respiratory and digestive tracts.

II. EVOLUTIONARY REQUIREMENT FOR MUCOSAL LYMPHOID ORGANS The oldest known receptor-based, adaptive immune systems are found in jawless fish (lamprey and hagfish) and cartilaginous fish (shark) [3,4]. The immune systems of these organisms feature two distinct cell types that use genetic recombination to generate diverse repertoires of antigen receptors. For example, lampreys have one cell type that expresses variable lymphocyte receptors (VLR)-A/C and another cell type that expresses VLR-B [5,6]. These cell types are functionally analogous to the T and B cells that are found in higher vertebrates, and they express diverse repertoires of recombinant T cell antigen receptors (TCRs) and B cell antigen receptors (BCRs) [3,7]. Interestingly, all vertebrates with adaptive immune systems also have lymphoid aggregates along the gut tube (Fig. 2.1). For example, the gut lamina propria of jawless fish contains lymphoid aggregates that fulfill both

II. PRINCIPLES OF MUCOSAL VACCINE

II. EVOLUTIONARY REQUIREMENT FOR MUCOSAL LYMPHOID ORGANS

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FIGURE 2.1 Evolution of organized lymphoid tissues. All classes of vertebrates possess some form of adaptive immune system using recombinant antigen receptors. These animals also form aggregates of immune cells along mucosal tissues. A higher degree of lymphocyte organization appears in the mucosa of bony fish and amphibians, in which T cell and B cell areas are segregated by fibroblastic stromal cells. However, specialized follicular dendritic cells within the B cell follicles are found only in birds and mammals. The presence of encapsulated lymph nodes is restricted to mammals.

hematopoietic and immune response functions, including the development of VLR-expressing cells as well as the proliferation and differentiation of those cells after antigenic encounter [8 10]. Similarly, the GALT of rabbits facilitates the diversification of the primary BCR repertoire and promotes antigen-dependent immune responses [11,12]. However, in other

vertebrates, the generation of the primary lymphocyte repertoire is physically separated from the immune response to environmental antigens. Nevertheless, the GALT in all vertebrates is involved in immune responses to the luminal contents of the digestive tract. Why should adaptive immune systems and complex lymphoid tissues coevolve? The

II. PRINCIPLES OF MUCOSAL VACCINE

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likeliest explanation is that, although a widely diverse repertoire of antigen receptors allows the recognition of a broad array of antigens, the frequency of any particular receptor that recognizes any particular antigen is very low, thereby reducing the probability that those few antigen-specific lymphocytes will encounter their cognate antigens. Given that mucosal tissues are continuously exposed to the external environment and that the digestive tract in particular is filled with a myriad of microorganisms, it makes sense that vertebrates evolved clusters of antigen-receptor-bearing lymphocytes directly beneath the mucosal epithelium, where they would be in close contact with potential antigens. Although all vertebrates have mucosaassociated lymphoid aggregates, the highly organized lymphoid architecture observed in mammalian lymphoid tissues is a more recent evolutionary development [13]. For example, bony fish lack an obvious segregation of B and T cells in the lymphocyte clusters associated with the gut, gills, and nasopharynx [14 16], whereas amphibians and reptiles separate B and T cell areas in the spleen but not in the GALT [17]. In contrast, mucosal lymphoid tissues in birds and mammals are highly organized [13,14], with separated B and T cell areas, specialized fibroblastic stromal cells, strategically placed phagocytic and antigen-presenting cells, and well-demarcated germinal centers. Why is lymphoid organization important? The evolution of specialized antigen-presenting cells, B cells and T cells, all of which need to interact for successful immune responses, requires mechanisms for these cells to find each other and additional mechanisms to support their proliferation and survival during immune responses [14]. In this regard, the development of specialized stromal cells, including fibroblastic reticular cells (FRCs) in the T cell area and follicular dendritic cells (FDCs) in the B cell area, is likely a key innovation. These cell types provide the scaffolding of lymphoid organs

and help to spatially organize lymphocytes and antigen-presenting cells. For example, FRCs express the chemokine CCL19, which attracts T cells and activated dendritic cells (DCs) into the T cell zone [18,19], whereas FDCs express the chemokines CXCL12 and CXCL13, which attract B cells and T follicular helper (Tfh) cells and direct their spatial positioning in the B cell follicle and germinal center [20 23]. Although B and T cell separation occurs in amphibians and reptiles, identifiable stromal cells such as FDCs are observed only in birds and mammals [14] (Fig. 2.1), which probably explains why these species (and not others) are able to support germinal center responses and generate high-affinity antibodies [24,25]. Thus the mechanisms that control the differentiation of stromal cell elements are critical for the proper organization and function of lymphoid organs. Not surprisingly, the genetic mechanisms that control lymphoid organ development and organization have coevolved with changes in immune architecture and function. For example, the development and organization of lymphoid tissues are regulated, in part, by the activities of tumor necrosis factor (TNF) superfamily members, TNFα, lymphotoxin-α (LT-α), and LT-β, and their receptors [26 28]. These genes are functionally expressed in lymphoid organs of higher vertebrates and sarcopterygian fish (lungfish and coelacanth) but not in teleost fish [16], an observation that correlates with lymphoid organization in each of these species. Interestingly, despite being evolutionarily advanced vertebrates, avian species appear to have lost the gene cluster encoding TNF, LT-α, and LT-β, as well as other TNF family members [29 31], which perhaps explains their inability to develop LNs [14]. However, avian species still develop highly organized mucosal lymphoid tissues, including cecal tonsil, esophageal tonsil, and Peyer’s patches [32 35], suggesting that additional or alternative TNF family members contribute to the development and organization of these tissues.

II. PRINCIPLES OF MUCOSAL VACCINE

III. GUT-ASSOCIATED LYMPHOID TISSUE

In fact, a variety of TNF superfamily members and their ligands contribute in various ways to the development, organization, and maintenance of various systemic and mucosal lymphoid organs. These cytokines are important for the differentiation of FDCs [22,36] and FRCs [37 39], for the expression of lymphocyte-attracting chemokines [27,40,41], for the development and maintenance of high endothelial venules (HEVs) [42 44], for the survival of some DCs [45 47], for the activation of lymphocytes [48], and for the differentiation of antigen-transporting microfold (M) cells [49,50]. Importantly, the various TNF superfamily members and their receptors often have overlapping functions. As a result, different TNF superfamily ligand receptor pairs are often used for similar purposes in different mucosal lymphoid tissues, depending on the cell types involved and the timing of the interactions. In the following sections, we will highlight which TNF superfamily members are important for the various features and functions of mucosal lymphoid tissues.

III. GUT-ASSOCIATED LYMPHOID TISSUE The mucosal epithelium along the digestive tract is exposed to a vast array of foreign antigens derived from the diet as well as the community of microorganisms (the microbiota) that colonizes the gut. The gut microbiota is essential for the digestion of food and helps to catabolize nutrients that otherwise would be impossible for the host [51]. However, if not properly contained, the microbiota can breach the mucosal barrier and cause local or systemic infections that may lead to chronic or acute inflammation or even death [52]. Thus although commensal colonization is clearly beneficial to the host, noninflammatory immune control is required to prevent commensal encroachment into host tissues and simultaneously maintain

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microbial homeostasis at mucosal surfaces. To accomplish this goal, the digestive tract is lined with mucosa-associated lymphoid tissues (MALTs) that support adaptive immune responses to antigens derived from food and from the microbiota itself. These tissues include the cryptopatches, ILFs, Peyer’s patches, cecal patches, colonic patches, MLNs, and even ectopic lymphoid tissues that can develop anywhere between the stomach and the colon.

A. Cryptopatches Cryptopatches are the smallest and most numerous lymphoid aggregates in the mammalian gut, numbering around 30,000 in humans and around 1500 in mice [53]. Their distribution is not homogenous, with a higher frequency in the ileum and colon compared to the duodenum and jejunum. As their name suggests, cryptopatches are found beneath the crypts at the base of the intestinal villi; they consist of a small cluster (fewer than 1000 cells) of c-kit1, IL-7R1 lymphocytes, and CD11c1CX3CR11 myeloid cells, but almost no B cells, T cells, or identifiable stromal cell networks [54] (Fig. 2.2). Initially, the c-kit1IL-7R1 cells were thought to be precursors of intestinal epithelial lymphocytes, and cryptopatches were thought to serve as sites of extrathymic T cell development [55]. However, more recent data show that the c-kit1IL-7R1 cells in cryptopatches are actually innate lymphoid cells (ILC3 cells) [56,57], which depend on the transcription factor RORγt and produce cytokines such as IL-17, IL-22, and GM-CSF [58,59] (Chapter 14: Innate Lymphoid Cells for the Control of Mucosal Immunity). The expression of the chemokine receptors CCR6 and CXCR3 divide intestinal ILC3 cells into two populations. Those that express CXCR3 coexpress the transcription factors RORγt and T-bet and are located throughout the intestinal lamina propria [60 62], whereas those that express CCR6 also express NKp46 and c-kit and are the adult counterparts of fetal lymphoid

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FIGURE 2.2 Organization and formation of cryptopatches. Cryptopatches (CPs) are located beneath the crypts at the base of the villi along the small and large intestines. The cryptopatches contain CD11c1CX3CR11 myeloid cells and RORγt-dependent ILC3 cells that express either CCR6 or CXCR3. ILC3 cells in CPs are dependent on metabolites such as aromatic hydrocarbons and retinoic acid. Under homeostatic conditions, the CCR61 ILC3s function as lymphoid tissue inducer cells. Upon sensing of microbial products, CX3CR11 myeloid cells in the CPs produce IL-23, which induces ILC3 cells to secrete IL-17 and IL-22, thereby promoting granulocyte recruitment as well as epithelial cell proliferation and repair.

tissue inducer (LTi) cells [63 66]. Importantly, the CCR61 ILC3 cells reside in cryptopatches [54] and ILFs [67], where they control intestinal epithelial homeostasis and, following infection, promote intestinal inflammation. Given the lack of B and T cells in cryptopatches, these structures are probably not primary inductive sites for adaptive immune responses. Nevertheless, interactions between ILC3 cells and CD11c1CX3CR11 myeloid cells in cryptopatches are an important component of intestinal immunity and homeostasis. For example, in response to microbial signals, the CX3CR11 myeloid cells in cryptopatches produce IL-23, which triggers the activation of ILC3 cells and their expression of cytokines such as IL-17 and IL-22 [68] (Fig. 2.2). These cytokines contribute to antimicrobial defense in the intestine by promoting the expression of antimicrobial molecules [69], triggering the recruitment of granulocytes [70], and enhancing epithelial proliferation and repair [71,72]. Upon the appropriate stimulation, CX3CR11 myeloid cells produce additional cytokines, such as IL-1β and TL1A, which also trigger ILC3 cell activation and the expression of IL-17 and IL-22 [73].

ILC3 cells are also potent producers of GM-CSF under both steady-state and inflammatory conditions [74 76]. Under homeostatic conditions, ILC3-derived GM-CSF helps to maintain immune tolerance by promoting the development of DCs that promote regulatory T cell (Treg) differentiation [75]. Conversely, the inflammatory activation of CX3CR11 myeloid cells leads to IL-23-driven activation and GM-CSF production by ILC3 cells, which in turn promotes colitis [76,77]. Moreover, GM-CSF feeds back on ILC3 cells, leading to their mobilization from cryptopatches and inflammation throughout the gut [76]. In fact, a variety of microbial signals trigger CX3CR11 myeloid cells to express inflammatory cytokines, including IL-1β, IL-23, and TL1A, that activate ILC3 cells to produce GM-CSF [74,75]. Thus the interactions between the microbiota, CX3CR11 myeloid cells, and ILC3 cells are important for intestinal homeostasis and inflammation. In addition to inflammatory cytokines, microbe-derived metabolites are also essential for the formation and activation of ILC3 cells. For example, the production and maintenance of ILC3 cells depend on vitamin-A-derived

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retinoic acid [78], which promotes expression of the transcription factor RORγt in developing ILC3 cells [79]. Similarly, the aryl hydrocarbon receptor, AHR, is expressed by ILC3 cells and senses soluble aromatic hydrocarbons that are produced by commensal bacteria or derived from the diet [80]. The AHR is essential for ILC3, for the development of ILC3 cells, and for their production of cytokines such as IL-22 [57]. ILC3 cells also sense oxysterols through the G-protein-coupled receptor, GPR183 [81], which is expressed by CCR6-expressing ILC3 cells in the cryptopatches and ILFs. Oxysterol compounds are constitutively produced by fibroblastic stromal cells [81], which recruit ILC3 cells to the cryptopatches and ILFs under steady-state conditions. Moreover, oxysterols are produced at higher levels during inflammatory responses, leading to local ILC3 activation and promoting inflammatory responses. Thus a variety of environmental triggers activate ILC3

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cells in cryptopatches and help to maintain barrier function during homeostasis and trigger inflammatory responses following infection.

B. Isolated Lymphoid Follicles Unlike the primitive structure of cryptopatches, the lymphoid architecture of ILFs is much more complex and, in many ways, resembles that of Peyer’s patches, the classic MALT in the gut. ILFs, which are found throughout the small and large intestines of mice [53], consist of a single B cell follicle that often contains a germinal center and a network of CD21/35-expressing fibroblastic stromal cells, the FDCs. The B cell follicle of ILFs is positioned beneath a specialized dome epithelium containing M cells that transport antigen from the intestinal lumen to the leukocytes beneath [67] (Fig. 2.3). A small number of T cells, primarily CD41 cells, are present in the

FIGURE 2.3 Organization and formation of isolated lymphoid follicles (ILFs). The transformation from cryptopatch to ILF entails the recruitment of lymphocytes, the development of specialized epithelial M cells, and the formation of FDC networks. Microbial products activate the intestinal epithelial cells to produce CCL20 and recruit CCR61 lymphocytes, including ILC3 cells, which in turn express membrane-bound LTαβ that reinforces M cell and FDC differentiation. Once formed, ILFs are maintained by constitutive expression of LTαβ on B cells, which interact with M cells and FDCs. Unlike the more complex Peyer’s patches, ILFs consist mostly of B cells, forming a single B cell follicle positioned beneath a subepithelial dome formed by dendritic cells and a few CD41 T cells.

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B cell follicle but do not form a discrete T cell zone. Dendritic cells are found beneath the dome and surrounding the B cell follicle [82]. Like cryptopatches, ILFs contain a significant fraction of CCR6-expressing ILC3 cells [83]. Although the structures of cryptopatches and ILFs are distinct, they are actually part of a spectrum of intestinal lymphoid tissues that ranges from nascent cryptopatches to fully developed ILFs [84,85], with cryptopatches vastly outnumbering ILFs. Interestingly, although cryptopatches appear only after birth, their development does not require signals from the microbiota [54] (Fig. 2.3). In contrast, the transition of cryptopatches to ILFs requires microbial exposure, which activates intestinal epithelial cells via innate sensing mechanisms, including peptidoglycan recognition by NOD1 in epithelial cells [86]. As a result, starting around 2 weeks after birth, some cryptopatches begin to recruit B cells and develop into ILFs [84,85]. The ability of lymphoid tissues to recruit lymphocytes is dependent on specialized vascular structures, known as HEVs, which display a variety of adhesion molecules and chemokines that allow circulating lymphocytes to adhere to the vascular wall and migrate into the tissue [44]. Although cryptopatches lack fully developed HEVs, MAdCAM-1-expressing HEVs are formed as cryptopatches become ILFs [87], thereby allowing the recruitment of α4β7-expressing B and T cells. Although the blockade of α4β7 has no effect on the numbers or cellularity of cryptopatches, it significantly impairs their ability to transition into ILFs [87]. Interactions between the chemokine receptor CCR6 and its ligand, CCL20, are also important for the formation and architecture of cryptopatches and ILFs [83] (Fig. 2.3). For example, mature ILFs have a dome epithelium with M cells that express CCL20 [86], which recruits specialized DCs, B and T cells [83,86], which cluster beneath the M cell-containing dome epithelium, where they await antigens that are

transported across the epithelium. CCL20 expression by epithelial cells is also important for the development of ILFs, because it attracts CCR61 ILC3 cells, which express lymphotoxin and other activating ligands that promote the differentiation of M cells and the maturation of fibroblastic stromal cells that support the lymphoid structure of the ILF [83]. Epithelial cells also produce IL-7, which expands ILC3 cells and promotes their expression of lymphotoxin. In fact, cryptopatches and ILFs fail to develop in the absence of IL-7, primarily owing to the absence of ILC3 cells. Whereas the cryptopatches primarily support innate immune responses due to a paucity of B and T cells, ILFs are important sites for the induction of adaptive immune responses, particularly the differentiation of IgA-producing B cells responding to the gut microbiota or food antigens [88,89]. Interestingly, ILFs are also an important source of T-cell-independent antimicrobial IgA [89], with no direct relation to the immune responses occurring in Peyer’s patches [90]. Recent work has broadened our understanding of the role of ILFs, and a general consensus is that they act in a tolerogenic manner to control intestinal immune responses by generating both IgA-secreting plasma cells and regulatory T cells [91,92], both of which are involved in the noninflammatory containment of commensal organisms. Conversely, the absence of ILFs leads to poor IgA production and a dramatic expansion of the intestinal microbiota [88]. Thus ILFs are powerhouses of IgA production in response to microbial antigens [90].

C. Peyer’s Patches, Cecal Patches, and Colonic Patches The Peyer’s patches, colonic patches, and cecal patches are among the largest MALT in the gut, and they are found in the submucosa on the antimesenteric side of the small

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intestine, colon, and cecum, respectively (Fig. 2.4). Structurally, the various intestinal patches are very similar to one another, differing mostly in their location and number, although the fine details of the cecal and colonic patches have not been thoroughly studied. It is important to remember, however, that the density and composition of the gut microbiota vary dramatically from the stomach to the

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colon [93] and will certainly affect the development, activity, and functions of the various lymphoid organs in different locations. Like ILFs, Peyer’s, cecal, and colonic patches have a prominent dome epithelium or follicleassociated epithelium that consists of enterocytes as well as antigen-transporting M cells [1,94,95]. The M cells are large epithelial cells that lack microvilli on the luminal side and

FIGURE 2.4

Peyer’s patches (PP) and large intestine patches. Large, multifollicular lymphoid organs found in the submucosa of the small and large intestines are termed Peyer’s patches (small intestine), cecal patches (cecum), or colonic patches (colon). (A) Peyer’s patches are found beneath a specialized follicle-associated epithelial layer that contains M cells. The region immediately below the epithelium is called the subepithelial dome and contains SIRPa1XCR12 dendritic cells that present antigen and help B cells switch to IgA. Monocyte-derived CX3CR11 macrophages in the dome region also contribute to the clearance and processing of lumen-derived antigens. B cell follicles separated by T-cell-rich interfollicular areas are found below the dome region. (B) Frozen sections of Peyer’s patches showing B cell follicles (red), interfollicular T cell areas (blue), and the follicular dendritic cell network in B cell follicles (green). (C) White arrows indicate the dome region that contains CD11c1 cells (green). Between B cell follicles (Fo), fibroblastic reticular cells (blue) surround HEVs (red).

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have a deep invagination of the basolateral membrane (also known as the pocket) [49,96], which harbors a variety of DCs, macrophages, and lymphocytes that sample and respond to the transcytosed antigens. M cells are highly pinocytic and mediate the transcytosis of antigens from the luminal side to the basal side of the epithelium [49,96,97]. The area immediately below the epithelium, called the subepithelial dome, is where immune cells, mainly DCs and macrophages, can first encounter antigens transported from the lumen of the intestine (Chapter 3: Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells) and Chapter 28: M Cell-Targeted Vaccines). Like ILFs, Peyer’s patches have large B cell follicles, often with germinal centers, extending beneath the subepithelial dome toward the muscularis layer. Unlike ILFs however, which have a single B cell follicle and essentially no T cell zone, Peyer’s patches have numerous follicles underneath their domes. Moreover, the B cell follicles of Peyer’s patches are separated by interfollicular regions, which are functionally analogous to the T cell zones of LNs and contain both DCs and T cells [98]. Thus the Peyer’s patches and other intestinal patches are structurally more complex than ILFs. A variety of DCs reside in the Peyer’s patches under homeostatic and inflammatory conditions [99 101]. Dendritic cells in regional LNs are broadly divided into those that migrated from peripheral tissues though afferent lymphatics (migratory DCs) and those that developed in the LN itself (resident DCs) [102]. In addition, both the migratory and resident DCs can be divided into those that express XCR1 (cDC1 cells) and those that express SIRPα (cDC2 cells) [103]. Since Peyer’s patches lack afferent lymphatics, they lack migratory DCs (by definition) but have other DC subsets that perform similar functions. For example, the CD11b1 DCs in the subepithelial dome region (SIRPα1 cDC2 cells) lack CCR7, but upon activation, they turn on CCR7 and

migrate to the interfollicular region [100,104], where they present antigen to T cells. The other migratory DCs found in the intestine are the cDC1 CD1031 DCs [105,106], which migrate through afferent lymphatics to MLNs, where they cross-present antigens to CD81 T cells [107,108]. The CD1031 DCs also efficiently promote the expression of the gut-homing receptors, CCR9 and α4β7 integrin, on responding T cells [109,110]. Importantly, under steady-state conditions, CD1031 DCs can trigger naive T cells to differentiate into Foxp3-expressing regulatory T cells through a mechanism that requires TGF-β and retinoic acid signaling [111 113]. Although CD1031 DCs are found in Peyer’s patches [114], these cells are most likely resident CD8α1 DCs found in the interfollicular region. The interfollicular region also has CD8α2CD11b2 (double negative) resident DCs near the base of the dome area near the follicle [115]. These double negative DCs are most likely cDC2 cells, since they express SIRPα [115] but are distinct from the CD11b1 DCs in the dome region. Additional antigen-presenting cells found in the Peyer’s patches, but not in conventional LNs, express lysozyme and are known as either Lyso-DCs or Lyso-macrophages, the latter of which can be further divided on their expression of the apoptotic cell receptor TIM4. All three of these cell types express the chemokine receptor CX3CR1 [115], a feature identifying them as monocyte-derived cells. Lyso-DCs that express CD11b and Lyso-Macs that express CD4 but not TIM4 are located in the dome region, whereas CD41 Tim41 lyso-Macs are located in the interfollicular region [115]. As it does in other SLOs, the fibroblastic stromal architecture of Peyer’s patches acts as a scaffolding that supports the structure of the tissue and produces a variety of factors that promote the survival of lymphocytes and regulate their position. For example, the FDC networks of Peyer’s patches express, or at least display, the chemokine CXCL13, which attracts and organizes B cells and Tfh cells in germinal

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centers [21,116]. In fact, nearly every follicle in Peyer’s patches has a germinal center filled with rapidly proliferating, antigen-responsive B cells, most of which have switched to IgA [104]. The FDCs in Peyer’s patches express typical markers such as CD21/35 and FDCM1, as well as the Fcα/μR, which is important for restraining germinal centers and the production of serum IgA [117]. Peyer’s patches also have dense networks of FRCs, which primarily define the T cell zone (the interfollicular area in Peyer’s patches) [19,116], where they express CCL19, the highaffinity ligand for CCR7, which attracts naı¨ve T cells and activated DCs. The FRCs also facilitate T cell trafficking within the interfollicular region [19,21] and help to coordinate interactions between antigen-presenting DCs and naı¨ve T cells. Importantly, FRCs in the interfollicular areas express IL-7 [118,119], a cytokine necessary for the homeostatic maintenance of naı¨ve T cells. Although FRCs are primarily associated with the T cell zones of lymphoid organs, they also extend into the B cell follicles, where they express IL-7 and BAFF [120], which maintains the viability of B cells. Finally, FRCs in Peyer’s patches modulate the activity of ILC1 cells through the transpresentation of IL-15 [121]. Thus FRCs are essential for the placement, movement, and survival of a variety of hematopoietic cell types. In addition to the chemokines made by fibroblastic stromal cells, chemokines made by epithelial cells in the dome are also important for cellular positioning in Peyer’s patches [100]. For example, dome epithelial cells express CCL20 and CCL9 [100], which attract subsets of immature CCR6- and CCR1-expressing DCs to the subepithelial dome of Peyer’s patches [100]. CCL20 is also important for attracting B cells to the dome, where they interact with CD11b1 DCs and isotype-switch to IgA [88,122]. Another chemokine, CXCL16, is also expressed by the dome epithelium of Peyer’s patches, ILFs, and cecal patches [123], whereas its

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receptor, CXCR6, is observed on subpopulations of activated and memory CD41 and CD81 T cells in the dome region. Thus the chemokines expressed by the dome epithelial cells are critical for the placement and juxtaposition of antigen-presenting cells and subsets of activated and memory lymphocytes beneath the antigen-transporting dome epithelium. Although M cells nonspecifically sample luminal antigens by pinocytosis, they also acquire antigens by receptor mediated endocytosis [124]. In fact, M cells express a variety of receptor-like molecules that help them sample a wide array of microbial pathogens and antigens. Some of these receptors have been identified by comparing gene expression profiles of M cells with those of enterocytes [125]. For example, the GPI-anchored glycoprotein 2 (GP2) is expressed on the luminal surface of M cells and acts as a receptor for type I piliated bacteria, including Escherichia coli and Salmonella typhimurium [126]. Similarly, a homolog of GP2, the urinary protein uromodulin, is also expressed on M cells [127,128], where it also binds type I piliated E. coli. Another GPIanchored protein, the cellular prion protein (PrPc), is also abundantly expressed on the luminal surface of M cells [125,129]. PrPc interacts with Hsp60 of Brucella abortus and helps its internalization into M cells [130]. Other M-cellexpressed proteins with potential antigensampling activity include ANXA5, which binds the lipid A domain of LPS and facilitates the uptake of Gram-negative bacteria [131], and the peptidoglycan recognition protein (PGLRP)-1, which binds to bacterial peptidoglycans [132] (Chapter 28: M Cell-Targeted Vaccines). Although M cells use their receptors to sample antigens and microorganisms in the gut, the reverse is also true, and some pathogens have evolved mechanisms to use M cells as a point of entry. For example, Yersinia enterocolitica, Listeria monocytogenes, S. typhimurium, human immunodeficiency virus, influenza virus, polio virus, and reovirus all use M cells and the

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receptors they express as ports of entry [133 135]. Moreover, the enterotoxin of Clostridium perfringens (CPE) binds to members of the Claudin tight-junction protein family, one of which, Claudin 4, is expressed in the cytoplasm of M cells, where it facilitates endocytosis [136]. In fact, peptides from the c-terminal domain of CPE binds to Claudin 4 can be used to target vaccine antigens to M cells [137]. Similarly, a peptide from the outer membrane protein H (OmpH β1α1) of Y. enterocolitica binds to C5aR on the luminal surface of M cells, and it can be used to enhance antigen uptake and mucosal vaccine responsiveness [138]. Thus the natural antigen-sampling activity of M cells can be exploited to enhance mucosal vaccination and tolerance. Of course, one of the main functions of MALT in the gut, including ILFs, Peyer’s patches, cecal patches, and colonic patches, is the production of IgA in response to antigens and commensal organisms in the gut lumen [74,95,104,139]. The process by which naı¨ve B cells become activated and switch to IgA is very dependent on the structural architecture and cell types in GALT. In particular, activated B cells use the chemokine receptor CCR6 to migrate to the dome region of Peyer’s patches, where they interact with DCs that prompt B cells to undergo isotype-switching to IgA [88]. Switching to IgA requires B cells to encounter active TGF-β, which is converted from the latent form by integrin αvβ8-expressing DCs in the dome epithelium [99]. Isotype-switching to IgA also requires ILC3 cells [56,80], which provide lymphotoxin-β receptor (LTβR)-dependent signals to DCs and stromal cells. A similar process likely occurs in ILFs, although much of the IgA production in ILFs occurs independently of T cells [89].

IV. DEVELOPMENT OF GUTASSOCIATED LYMPHOID TISSUE The development of the GALT in many ways is similar to the development of conventional

LNs. Both types of lymphoid organs develop according to a developmentally programmed series of cellular interactions that occurs independently of exogenous antigen or inflammation [1]. This process begins during fetal development and requires interactions between lymphoid tissue inducer (LTi) cells of hematopoietic origin [63] and lymphoid tissue organizer (LTo) cells of mesenchymal origin [140,141]. LTi cells are a subset of ILC3 cells that express the transcription factor RORγt [66] and produce cytokines such as TNF, LT-α, LT-β, IL-17, and IL-22 [59], all of which are involved in some aspect of lymphoid organ development or maturation. The development of lymphoid organs and GALT in particular has been previous reviewed [1,53], and we will summarize some of the most pertinent aspects here. In Peyer’s patches, the first step in development occurs around day 12.5 of embryogenesis and involves the activation of lymphoid tissue initiator (LTin) cells [142]. LTin cells are defined as CD451CD11c1c-kit1 cells that express the LTβR and the tyrosine kinase receptor, RET [140]. LTin cells are activated by the recognition of the RET ligands, leading to LTin accumulation around VCAM1 LTo cells, which become activated and express CXCL13, thereby prompting the recruitment and clustering of CXCR5-expressing LTi cells [143]. The reciprocal interactions between LTαβexpressing LTi cells and LTβR-expressing LTo cells reinforce the expression of CXCL13 and leads to the development of the Peyer’s patch anlagen. Once the Peyer’s patch anlagen is established, the developing stromal compartment recruits additional leukocytes via the induced expression of cytokines (IL-7, VEGF-C, and RANK ligand) [27], chemokines (CXCL12, CXCL13, CCL19, and CCL21), and adhesion molecules (VCAM-1 and ICAM-1) [144]. Taken together, these molecules help to recruit leukocytes and lymphocytes, maintain their survival, and promote their spatial positioning.

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The organogenesis of Peyer’s patches strictly requires the function of LTi cells [66]. As a result, mice lacking the transcription factors RORγt and Id2 lack LTi cells; consequently, they do not develop LNs or Peyer’s patches [142]. Similarly, mice lacking either CXCL13 or its receptor, CXCR5, fail to develop LNs or Peyer’s patches, owing to the inability of LTi cells to cluster in the lymphoid anlagen [23]. Moreover, mice lacking LT-α, LT-β, or LTβR also fail to form LNs or Peyer’s patches, owing to an inability of LTi cells to activate LTo cells and promote their eventual differentiation into mature lymphoid stroma [145,146]. Importantly, TNFR1 and LTβR are coupled to both the canonical and noncanonical NFκB signaling pathways [147], both of which are important in Peyer’s patch development [148,149]. The final steps of Peyer’s patch development occur after birth when T and B cells begin to populate their corresponding niches. At this time, LTi cells downregulate the expression of CXCR5 and upregulate the expression of CXCR4 [116], thereby changing their migration away from the B cell follicle and toward the interfollicular region. This process is coordinated by TGF-β and is necessary for the differentiation of FRCs [150]. Once Peyer’s patches are fully developed and populated, leukocytes help to maintain stromal cell differentiation; B cells are particularly important for the maintenance of FDCs [151], whereas DCs are important for the maintenance of FRCs [152]. Finally, the interactions between the microbiota and epithelial cells are arguably as important as the interactions between leukocytes and stromal cells for the maintenance of Peyer’s patches [153], particularly for the differentiation of M cells [154], the expression of epithelial chemokines, and the organization of the dome region [129]. Although the mechanisms controlling the development of Peyer’s patches are well defined, those controlling the development of colonic patches and cecal patches have not been

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studied in depth; the assumption is that the development of these organs will be similar to that of Peyer’s patches. However, the muscularis layer is different in the small intestine and colon, and the density of commensal organisms in the colon greatly exceeds the density of those in the small intestine [53]. Thus the mechanisms that control the development, maturation, or function of these organs may be different as well. In this regard, there are notable differences in the organogenesis and regulation of ILFs in the small intestine and colon [94]. For example, although the development and maturation of ILFs in the small intestine require microbial colonization, the ILFs in the colon develop independently of microbial colonization and signaling through CCR6, RANK, and CXCL13 [94]. In contrast, colonic ILFs have specific requirements that the small intestine ILFs do not. For example, colonic ILFs require MyD88dependent signals for maturation [94], whereas small intestine ILFs do not. Similarly, colonic ILFs require IL-23 signaling [155] but are suppressed by IL-25 signaling. These observations suggest that the molecular mediators of ILF maturation differ between the small and large intestines, with colonic ILFs more dependent on microbial detection and small intestine ILFs more dependent on danger signals. The mechanisms controlling the development of cryptopatches are different from those controlling the development of Peyer’s patches or LNs [53], in part because cryptopatches lack B and T cell domains. In fact, the cryptopatches in adults are in some ways analogous to the Peyer’s patch anlagen in developing embryos. However, instead of primary interactions between CXCR5-expressing LTi cells and CXCL13-expressing LTo cells, the formation of cryptopatches is dependent on interactions between CCR6-expressing ILC3 cells and CCL20-expressing epithelial cells [83], which also produce IL-7 and thereby help to maintain ILC3 cells [54]. Moreover, like the development

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of most lymphoid tissues, the formation of cryptopatches is entirely dependent on RORγt [87], which is essential for ILC3 differentiation. Interestingly, although the development of cryptopatches requires lymphotoxin signaling, it uses only the canonical pathway and does not require RelB [156]. The maturation of cryptopatches into ILFs often begins with microbial signals to the epithelium, which increases expression of CCL20 and IL-7 [86], leading to the activation of ILC3 cells [83]. Alternatively, ILC3 cells can be activated directly by metabolic products that engage the AHR [57] or by IL-23 generated from activated myeloid cells [155]. Once ILC3 cells are activated, they express LTαβ and trigger the differentiation of LTβR-expressing stromal cell precursors, which upregulate CXCL13, CCL19, and CCL21 as well as VCAM1 and ICAM-1 for the recruitment and retention of lymphocytes [157]. Further accumulation of B cells, recruitment of T cells, and differentiation of follicle-associated epithelium (FAE) mark the maturation of an ILF. The cytokine RANKL (receptor activator of NF-KB ligand) and its receptor (RANK) are crucial for the maturation of ILFs [158] but not for the formation of CPs, primarily because of the role of RANK signaling in the differentiation of M cells, which will be discussed in the next section.

V. M CELL DIFFERENTIATION IN GUT-ASSOCIATED LYMPHOID TISSUE The development of M cells in the dome epithelium is a specific feature of mucosal lymphoid tissues and, not surprisingly, involves reciprocal interactions between various cell types and signaling through receptors in the TNFR family. For example, M cell differentiation requires signaling through the TNFR family member RANK, which is expressed on epithelial stem cells [159]. RANK signaling

activates TRAF6 and NF-KB in epithelial cells [160] and triggers the expression of the transcription factor Spi-B, which is required for the final maturation and accumulation of M cells in the dome epithelium [160]. Interestingly, the systemic administration of RANKL induces the ectopic differentiation of enterocytes into M cells throughout the villous epithelium [161], suggesting that any enterocyte is capable of differentiating into M cells but that the normal availability of RANK ligand is spatially restricted to the epithelium overlaying lymphoid clusters or FAE. In fact, RANK ligand is primarily expressed by subepithelial stromal cells in areas of lymphoid tissue (e.g., Peyer’s patches) [162]. Interestingly, stromal cells in developing LNs express RANK ligand following interactions with lymphotoxin-expressing LTi cells [27]. In turn, RANK ligand maintains the survival of LTi cells and increases their expression of lymphotoxin, generating a positive feedback loop. Given that lymphotoxin is important for the differentiation of the M cell-containing FAE [49] and that RORγt-expressing ILC3 cells are necessary for the differentiation of M cellcontaining ILFs [89], it makes sense that the ILC3 cells in GALTs participate in M cell differentiation. The expression of CCL20 in the intestinal epithelium is rapidly induced by RANK ligand stimulation and enhanced by TNFR and LTβR stimulation [163]. Other signals triggered by enteroinvasive bacteria, including S. typhimurium, Salmonella enteritidis, and L. monocytogenes, also induce CCL20 expression by intestinal epithelial cells [164]. The pathogen-mediated expression of CCL20 and β-defensin-2 by epithelial cells may defend against bacterial infections by attracting CCR6-expressing Th17 cells [165,166]. However, pathogens may also exploit this mechanism by stimulating the differentiation of enterocytes into M cells and thereby facilitating their translocation from the gut lumen.

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B cells and DCs are also involved in M cell maturation; however, neither of these cell types expresses RANK ligand. Moreover, B-cell-derived TNF and lymphotoxin are each dispensable for the development of the FAE and M cells [163]. Nevertheless, the expression of CCR6 on B cells and DCs is required for their attraction to the dome epithelium [99] and for the final maturation of GP2expressing M cells [159]. In fact, the basolateral pocket of M cells in Peyer’s patches usually contains at least one B lymphocyte [167]. Moreover, adding B cells to cultures of Caco-2 intestinal epithelial cells promotes their differentiation into M cells [168,169], and mice that lack B lymphocytes appear to lack M cell transcytosis function [170]. The factors provided by B cells (or other hematopoietic cells) that stimulate M cell differentiation include signaling between B cells and M cells via CD137L/CD137 [50] or macrophage migration inhibitory factor [171].

VI. LYMPHOID TISSUES OF THE RESPIRATORY TRACT The mucosal lining the upper and lower respiratory tracts is constantly exposed to inhaled antigens and has endogenous microbial communities [172], although the density of the respiratory microbiota is dramatically less than that in the gut, and the species that compose these communities are also different [173]. As a result, MALTs that trigger local immune responses are present in the upper respiratory tract and nasal passages as well as in the lower respiratory tract and lung. Like the MALT of the gut (or GALTs), the mucosal tissues of the respiratory tract can generate local immune responses to pathogens and inflammatory antigens and help to maintain homeostatic symbiosis with the local microbial communities.

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A. Nasopharyngeal-Associated Lymphoid Tissue The upper respiratory tract has a variety of mucosal lymphoid organs whose location and structure differ greatly among the various mammalian species. For example, humans lack constitutively organized lymphoid tissues in the nasal passages but often have diffuse lymphoid tissues (likely ectopic lymphoid aggregates) [174]. Instead, humans have welldeveloped mucosal lymphoid tissues in the nasopharynx (adenoids) and the oropharynx (tonsils), which together are termed Waldeyer’s ring [175]. Although sheep, goats, pigs, dogs, and cats lack detectable lymphoid tissues in the nose [175], horses have nasal structures similar to ILFs [175,176], and rabbits have nasalassociated mucosal tissues as well as a variety of ILF-like structures in the nasal passages [177]. By contrast, rodents develop constitutively organized lymphoid tissues in the nasal cavity that are termed nasal-associated lymphoid tissue (NALT) [178]. In mice, NALT is found on the dorsal side of the soft palate at the bottom of the nasal passages [179] (Fig. 2.5). In cross section, NALT appears as two bell-shaped structures located on the ventral region of the nasal passages above the hard palate [175,179]. NALT extends along the length of the nasal passages and consists of multiple B cell follicles arranged like a string of beads separated by interfollicular areas [175,179]. Like Peyer’s patches, the interfollicular areas contain numerous T cells and DCs that are arranged on a stromal network of CCL19-expressing FRCs, whereas the B cell follicles and germinal centers are organized around stromal networks of CXCL13-expressing FDCs [175,180]. Similar to MALT in the gut, NALT has a dome epithelium that expresses the chemokines CCL20 and CCL9 [181,182]. However, unlike Peyer’s patches, it does not express CXCL16 [123]. M cells with short microvilli and

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FIGURE 2.5 Nasopharyngeal-associated lymphoid tissue (NALT). A cross section of a decalcified mouse nose shows NALT areas (arrows) on either side of the nasal passage (NP), above the oral cavity (OC).

microfold morphology are easily identified in the dome epithelium of NALT as well as turbinate epithelium of nasal cavity [96,183] and efficiently acquire luminal antigens and particulates [184], which they transcytose across the epithelium. Not surprisingly, NALT M cells express markers, including GP2, Tnfaip2, and Spi-B [184], which are also expressed in M cells of the intestinal tract. CCR6-expressing DCs and B cells fill the dome region under the epithelium and are closely associated with M cells [185]. As in Peyer’s patches, interactions between CCR6-expressing leukocytes and M cells are important for the maintenance of NALT structure and function [182]. The HEVs of NALT are found in the interfollicular regions or at the T cell B cell boundary [186]. Interestingly, NALT HEVs express peripheral node addressin (PNAd) [179,187] alone or mostly associated with mucosal addressin cell adhesion molecule (MAdCAM) [179], a difference that seems to be dependent on the molecular details of lymphotoxin signaling [188]. Hence MAdCAM expression on the HEVs is expressed only in conjunction with PNAd [179]. HEVs in NALT also express the chemokine CCL21 [181], which facilitates lymphocyte trafficking from the blood. Like other mucosal lymphoid organs, NALT lacks afferent lymphatic vessels. However,

efferent lymphatic vessels are found at the outside edge of the interfollicular T cell areas of NALT, where they collect effector cells that were primed and expanded in NALT [180]. Interestingly, the lymphatic vasculature of NALT connects with the lymphatic vasculature draining the ocular region that contains another mucosal lymphoid tissue, the lacrimal-ductassociated lymphoid tissue (LDALT) [180,189]. Thus ocular and nasal lymphatics seem to converge on their way to downstream cervical LNs. The ontogeny of NALT is unique among mucosal lymphoid organs, perhaps because of its position in the nasal passages. Although the development of NALT resembles that of ILFs in the sense that it occurs after birth in response to antigenic or microbial encounter [190], there are important differences in terms of the cell types and molecular pathways required for its development. For example, LTi cells seem dispensable for NALT formation [191] as well as for LDALT formation [189], since mice that are deficient in the transcription factor RORγt (and therefore deficient in ILC3 cells, including LTi cells [191,192]) still have apparently normal NALT structures. Similarly, mice lacking IL-7 (which fail to support LTi cells as well as most lymphocytes) still have rudimentary NALT structures. In contrast, mice lacking the

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transcription factor Id2 (which lack all ILC cells [142]) completely fail to develop NALT [191]. Thus it seems that an ILC population other than ILC3 cells or LTi cells is required for NALT development. In this regard, ILC2 cells are more prominently represented in the respiratory tract than are ILC3 cells [1,193]. The requirement of lymphotoxin signaling is also different in NALT than it is in most lymphoid tissues. For example, unlike the development of Peyer’s patches and essentially all other lymphoid organs, the development of NALT still occurs in the absence of LT-α [192,194,195]. Although NALT remains in LT-α-deficient mice, its structure is abnormal, with poorly defined B and T cell domains, a lack of FDCs, and reduced expression of chemokines (CXCL13, CCL19, CCL21, and CCL20) [194], which normally maintain the lymphoid architecture of NALT. This result is likely due to the fact that LTi cells are not involved in NALT development and that LT-α is the primary mediator of LTi cell activity in lymphoid organ development. NALT development also occurs normally mice lacking CXCL13 [181], which is required for the development of most lymphoid organs because LTi cells use CXCR5 to find and interact with LTo cells. Again, however, the organization of NALT is impaired in the absence of CXCL13 or CXCR5, as FDCs fail to develop and stromal cells are unable to organize a B cell follicle [192,194]. However, increasing antigenic encounter leads to the further organization of NALT, even in the absence of CXCR5 or LT-α [181,194,195], suggesting that the structural maturation of NALT can use alternative pathways, such as CD40-dependent signaling [194], which may substitute for LT-α signaling by engaging the noncanonical NFκB pathway [196]. The differentiation of M cells and the formation of a dome region are also important for the proper structure and function of NALT. As in Peyer’s patches, the differentiation of NALT M

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cells requires interactions between RANK, expressed on epithelial cells, and its ligand, expressed by stromal cells in the subepithelial dome region [162,197]. As in the intestine, the administration of RANK ligand to the nasal passages activates RelB and Spi-B in epithelial cell progenitors [184], ultimately leading to the differentiation of additional GP21Tnfaip21 epithelial M cells in the nasal passages [127,160,184,197]. Interestingly, unlike the development of M cells in the intestine, which are derived from proliferating precursors in the crypts, the M cells in NALT appear to differentiate in the absence of proliferation [184]. Interactions with lymphocytes are also an important part of M cell maturation in NALT, as the morphology of M cells and the basolateral pocket are lost when lymphocytes are depleted [198]. The interactions between leukocytes and M cells utilizes CCR6 [182,185], as the number of M cells is reduced in the NALT of CCR6-deficient mice. Moreover, the antigentransporting ability of the M cells remaining in CCR6-deficient mice is also impaired [182], leading to poor IgA responses to nasally administered antigens. Although interactions with B cells are not required for the initial development of M cells, B cells trigger the TNFR family member CD137 (commonly known as 4-1BB) on M cells by its ligand on B cells [50], which maintains M cell morphology, promotes the formation of the basolateral pocket, and facilitates their ability to transport antigens and particulates [50]. The antigen-transporting activity of M cells in NALT also allows pathogen entry. For example, pathogens such as mouse mammary tumor virus (MMTV) [199], group A streptococcus [200,201], and Burkholderia pseudomallei [202] all target NALT as a portal of entry. Similarly, molecules expressed on the surface of NALT M cells can be targeted to enhance the uptake and response to vaccine antigens. For example, the Claudin-4-targeting peptide of CPE promotes the uptake of nanoparticles through NALT M

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cells and enhances local IgA responses [137]. Moreover, M-cell-binding lectins, including Griffonia simplicifolia I isolectin B4 (GSI-B4) [203], Dolichos biflorus agglutinin (DBA) [203], and Ulex europaeus agglutinin (UEA) [203], and M-cell-binding proteins such as reovirus protein σ1 [204] promote antigen uptake by M cells in NALT and, when used in combination with adjuvants such as cholera toxin [205] or heatlabile toxin IIa-B5 [206], elicit potent IgA responses in the nasal passages. As a mucosal lymphoid organ, it might seem that NALT would preferentially generate IgA responses following vaccination. However, mucosal lymphoid tissues in the respiratory and digestive tracts are not functionally identical [207]. However, immunization or infection of the nasal passages typically elicits as much IgG as IgA [208]. These responses are clearly biased by the type of adjuvant involved, as immunization with cholera toxin typically leads to IgA responses [205,209], whereas proteincontaining microparticles primarily elicit IgG responses [210]. Infection with common respiratory viruses, such as influenza [211] and reovirus [212], elicits both IgG- and IgA-secreting cells in NALT. Nevertheless, B cells responding to influenza more often switch to IgA in NALT than in mediastinal LNs, lung, or spleen [213]. Intriguingly, IgA memory B cells generated in NALT accumulate more somatic mutations than do IgG memory cells [214], suggesting a selective entry into the memory compartment, perhaps by interactions with the CCR6expressing DCs in the subepithelial dome region (Chapter 7: Induction and Regulation of Mucosal Memory B Cell Responses).

B. Bronchus-Associated Lymphoid Tissue The lower respiratory tract includes the trachea, bronchi, bronchioles, and alveoli, which are continuously exposed to inhaled antigens and pathogens, including common respiratory viruses such as influenza virus, respiratory

syncytial virus, and rhinovirus. Therefore the lower respiratory tract has to be protected by robust immunological mechanisms. However, the lung is also the primary site of gas exchange, which is most efficient when the distance between the respiratory epithelium of the alveoli and the vascular endothelium of the capillaries is minimized. As a result, the delicate structures of the lower lung are susceptible to damage by inflammatory responses, implying that immune responses in the lung must be appropriately tempered to provide protection against pathogens without causing undue inflammation and damage. Given the exposure of the lung to inhaled antigens and pathogens, one might expect that the lower respiratory tract would be filled with mucosal lymphoid tissues along the bronchi and bronchioles. In fact, some species have mucosal lymphoid structures, termed BALT, along the respiratory tract [215 217]. However, the lungs of healthy humans have relatively few BALT-like areas [218,219], and the lungs of mice from clean animal facilities completely lack such structures [220]. Nevertheless, humans with chronic lung diseases such as chronic obstructive pulmonary disease [221 223], hypersensitivity pneumonitis [224], and even pulmonary complications of rheumatoid arthritis [225 227] have many well-developed BALT-like areas that seem to react with local antigens. Moreover, mice also develop BALT-like structures in their lungs following pulmonary infection or inflammation [228] (Fig. 2.6). These findings suggest that the BALT-like areas in humans and mice are not true SLOs that develop during embryogenesis but instead are ectopic or tertiary lymphoid tissues that develop in response to local inflammation [228,229]. Interestingly, some mammals, such as pigs and goats, develop BALT during gestation [215,230 233], apparently independent of exposure to antigen or microbes, suggesting that in these species, BALT is a true SLO. Therefore to distinguish between BALT formed according to a developmental program

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FIGURE 2.6

Inducible bronchus-associated lymphoid tissue (iBALT). (A) The formation of iBALT requires pulmonary inflammation and the recruitment and activation of neutrophils, macrophages, and dendritic cells. Inflammatory cytokines such as IL-17A, IL-1α, and IL-23 promote the production of CCL19 and CXCL13 by fibroblastic cells, which recruit CCR71 T cells and CXCR51 B cells. Activated lymphocytes provide LTαβ, which promotes the differentiation of fibroblast and epithelial cells into lymphocyte-organizing stromal cells. The structure of iBALT resembles that of other lymphoid organs in which segregated B cell and T cell areas are located near epithelia and are supported by specialized stromal populations. (B) H&E staining of lung sections showing iBALT. (C) Immunofluorescence staining of B2201 B cells (blue) and CD41 T cells (red). The follicle is highlighted (dashed line). (D) CD41 T cells (red) and CD21/351 FDC network (green). B, Bronchi; b, blood vessel.

and the lung-associated lymphoid tissues that are inducible and are formed after inflammation, we coined the term “inducible BALT” (iBALT) [225,234,235]. As the name suggests, iBALT forms along the bronchi and bronchioles in the lung, often at branch points of the bronchial tree and usually next to or even surrounding a pulmonary artery [228,234,236 238]. In fact, the structure of iBALT often fills the perivascular space and, depending on how the lung is sectioned, may appear to be exclusively perivascular [237,239]. BALT-like lymphoid aggregates in the lung

have a spectrum of phenotypes, beginning with a small, loosely organized cluster of B cells that lacks FDCs and ending with a dense lymphoid accumulation that includes multiple germinalcenter-containing B cell follicles arranged on networks of CD21-expressing FDCs and separated by interfollicular T cell areas with networks of podoplanin-expressing FRCs [229,240,241]. In some cases, iBALT areas can be found with extensive FDC networks and germinal-center-containing B cell follicles but no discernible T cell areas [229,241]. In other cases, however, iBALT areas can be observed

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with dense clusters of T cells and podoplaninexpressing FRCs but no B cell follicles [229,241]. These different structures are undoubtedly distinct at a functional level; however, the underlying reasons why different types of structures are formed remain unclear. Although BALT areas in some species seem to have a specialized dome epithelium with M cells [96,242], the iBALT observed in mice and humans typically lacks this feature [1], consistent with the idea that iBALT is an ectopic lymphoid tissue rather than a truly MALT of the airway. However, the absence of M cells in the iBALT of mice and humans raises the question of how these tissues acquire antigen. In this regard, it is clear that areas of iBALT have associated lymphatic vessels around the B cell follicle at the edge of the T cell area [243,244]. In fact, the inflammatory processes that trigger iBALT formation also trigger the growth of new lymphatic vessels [245]. It seems likely that some of these vessels are efferent lymphatic vessels that carry effector cells away from iBALT and toward the downstream draining mediastinal LN. However, some of these vessels may also be afferent lymphatics that carry antigen and DCs from distal portions of the lung into iBALT. In fact, nasally administered DCs rapidly migrate into the T cell areas of iBALT, most probably via afferent lymphatic vessels [43,246,247]. Well-developed areas of BALT also have HEVs in the T cell area next to the T cell B cell boundary [248,249]. Consistent with the idea that iBALT is an ectopic rather than a classical MALT, the HEVs in iBALT express CCL21 and PNAd but not MAdCAM-1 [248,249], suggesting that α4β7-expressing mucosal-homing lymphocytes are not preferentially recruited to iBALT. Together with the lack of a dome epithelium, these observations reinforce the conclusion that iBALT is an ectopic rather than a mucosal lymphoid organ. The lymphoid chemokines CXCL13, CCL19, and CCL21 are expressed by fibroblastic

stromal cells in iBALT and are important for its structure and function. For example, although iBALT develops in CXCL13-deficient mice and even has separated B and T cell areas [241], it lacks FDCs and fails to form true B cell follicles or germinal centers. In contrast, the iBALT that forms in plt/plt mice (lacking both CCL19 and CCL21 [240,250]), has B cell follicles with FDCs but fails to develop T cell areas and cannot form germinal centers [250], probably owing to the failure of Tfh differentiation. Mice lacking all three chemokines fail to form any iBALT at all [250]. Unlike Peyer’s patches and ILFs, CCR6 is not important for the formation or function of iBALT [1], consistent with the lack of an M cell-containing dome epithelium (or FAE) in this tissue. Numerous inflammatory or infectious stimuli can trigger the formation of iBALT via a variety of pathways [228]. However, many of the core mechanisms involved in SLO development are also involved in the formation of iBALT. In general, iBALT is formed most easily during the neonatal period in both mice and humans [219,240], perhaps owing to increased frequencies of ILCs or decreased frequencies of Tregs [240,251,252]. In fact, CCR7-deficient mice spontaneously develop iBALT, owing to the inability of Tregs to suppress pulmonary inflammation [251,253,254]. Conversely, the reconstitution of CCR7-deficient mice with WT Tregs prevents the formation of iBALT [251]. These observations are likely pertinent for the development of iBALT and other ectopic lymphoid tissues in autoimmune patients, who often have defects in Treg function. Although MALT in the intestine (or GALT), including cryptopatches, ILFs, Peyer’s patches, cecal patches, and colonic patches, requires ILC3 cells, these cells are not required for iBALT development, as iBALT forms easily in mice lacking the transcription factor RORγt [240]. This observation implies that other cell types such as ILC2 cells, which are common in neonatal lungs [255], may play important roles

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in iBALT formation. Despite the nonessential role of ILC3 cells, both IL-17 and IL-22 are important for the formation of iBALT in response to the inert inflammatory molecules, LPS [240]. These cytokines are provided by γδ T cells and follicular-homing Th17 cells during iBALT development [240]. The role of these cytokines is somewhat surprising, given that iBALT forms independently of RORγt, which is normally required for the differentiation of ILC3 cells, γδ T cells, and Th17 cells. Thus it seems that there are redundant mechanisms for iBALT formation when IL-17 is limiting. The formation of iBALT in response to IL-17 involves neutrophil accumulation via the expression of the inflammatory chemokines, CXCL9, CXCL10, and CXCL11 [252,256]. Neutrophils and other granulocytes cause damage, in part because of the release of proteases [257 259], which trigger the expression of homeostatic and inflammatory chemokines and lead to iBALT formation [259]. Similarly, IL-17 directly promotes the expression of the homeostatic chemokines CXCL13 and CCL19 [240,260,261], which are important for the recruitment of B and T cells and for the formation of iBALT. Although IL-17 promotes the expression of these chemokines under inflammatory conditions, lymphotoxin signaling maintains the expression of these chemokines under homeostatic conditions [240,241,261]. Thus once iBALT is formed, it no longer requires IL-17 but instead requires lymphotoxin-expressing lymphocytes and DCs to maintain stromal architecture, chemokine expression, and HEV differentiation. Importantly, IL-17 is required for iBALT formation only under some conditions, most notably bacterial infections or exposure to bacterial products [228]. However, under other conditions, such as intranasal infection with modified vaccinia virus Ankara (MVA), iBALT forms independently of IL-17 [241]. As might be expected, iBALT formation in response to bacterial infection is dependent on TLR signaling through MyD88 and Trif [241], whereas

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iBALT formation in response to viral infection is independent of these pathways. Nevertheless, all of the inflammatory pathways eventually converge on the differentiation and maintenance of stromal cells via lymphotoxin and the expression of homeostatic chemokines. Other types of inflammatory stimuli trigger iBALT formation via other mechanisms. For example, pulmonary exposure to alum or silica particles trigger dying macrophages to release IL-1α, which acts as an alarmin that promotes the recruitment of eosinophils, macrophages, neutrophils, DCs, and T cells to the lungs and ultimately leads to iBALT formation [262]. In another example, pulmonary infection with Pneumocystis murina elicits the expression of IL-25 and IL-17, which indirectly trigger the expression of CXCL13 by lung fibroblasts via IL-13 and IL-6 [261]. Interestingly, the IL-25 and IL-17 receptor complexes share a common subunit, the IL-17Rα [263,264]. Once past the inflammatory stage, the mechanisms that maintain iBALT parallel those of most lymphoid tissues. For example, the loss of DCs leads to the shrinkage of iBALT areas [247,265], perhaps because DCs are required for the maintenance of HEVs [266 268] and, without them, lymphocytes fail to be recruited and the structures fall apart. Fibroblastic stromal cells and lymphatic endothelial cells also contribute to the maintenance of iBALT, in part by expression of homeostatic chemokines that maintain the B and T cell areas [43] but also by expression of cytokines such as IL-7 and BAFF [120] that promote the survival of naı¨ve and memory T and B cells. Many of these homeostatic mechanisms require lymphotoxin or TNF signaling between leukocytes and stromal cells [269], similar to what occurs in other lymphoid organs. The areas of iBALT clearly function like other lymphoid organs and can independently support primary and secondary B and T cell responses to pulmonary antigens [1,2,270,271]. This functional capacity is apparent in mice

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lacking conventional LNs and spleen [2,234]. However, the initial activation of T cells can also be observed in iBALT following the intranasal administration of antigen-loaded DCs [247]. Moreover, the formation of germinal centers in iBALT is a clear sign that antigenspecific B cells are being primed locally [265]. Furthermore, antigen-specific plasmablasts accumulate at the border between the B cell follicle and the T cell area [265], suggesting that they were generated locally. Interestingly, immune responses triggered in BALT do not seem to bias the antibody class switch toward a particular isotype, such as IgA [265]. This observation might be explained by the lack of a dome epithelium (or FAE) and specialized DCs that facilitate the activation of TGF-β and promote IgA switching. Again, this observation is consistent with the idea that iBALT is an ectopic lymphoid tissue rather than a MALT. Even though iBALT is not a constitutive SLO in the lung, its presence has a strong impact on the success of pulmonary immune responses. In the context of pulmonary infection, the presence of iBALT is uniformly beneficial and usually leads to faster clearance of the pathogen with less pulmonary and systemic pathology [228]. For example, immune responses against pulmonary infection with influenza virus, SARS corona virus, Coxiella burnetii, Francisella tularensis, and Mycobacterium tuberculosis are all more effective when iBALT is present in the lung [235,265,272 274]. In part, this success can be attributed to an accelerated B cell and antibody response [234,273,275]. Given the location of antigen-specific germinal center B cells and plasmablasts, this faster B cell response may be simply due to proximity to antigen. Alternatively, the local differentiation of T cells may be biased toward Tfh cells rather than effector cells. Given the unique functional attributes of iBALT, it may be desirable to develop vaccines that transiently induce iBALT for the initiation of antigen-specific humoral and/or cellular immunity against respiratory pathogens.

In contrast to its beneficial role in responding to pulmonary infection, the presence of iBALT in inflammatory lung diseases may be detrimental to the host, because enhanced local immune responses to autoantigens or environmental antigens would likely exacerbate inflammation and tissue damage. For example, areas of iBALT are found in patients with hypersensitivity pneumonitis [224], rheumatic lung disease [225,227], chronic obstructive pulmonary disease [223,276,277], and even idiopathic pulmonary fibrosis [254,278]. These diseases are associated with chronic inflammation and continual or repeated exposure to antigens. Moreover antigen-specific B and T cells are found in the iBALT areas [235], suggesting that they are contributing to disease pathogenesis. Despite the link between chronic lung disease and the formation of iBALT, it is not clear whether iBALT is exacerbating disease or is actually attenuating inflammation by sequestering antigens and effector cells. Finally, despite the fact that iBALT is an ectopic lymphoid tissue, it is clearly a critical component of the pulmonary immune system and has the ability to fine-tune local immune responses and either ameliorate or exacerbate pulmonary pathology. Given that iBALT is formed in response to environmental exposures, particularly during early life, it seems likely that the type and frequency of these exposures may set the tone of pulmonary immunity for many years and perhaps the life span of the individual [218,279]. Therefore it is essential that we understand the mechanisms controlling the development and function of iBALT in order to harness its infection-preventing properties during vaccination and to restrain its pathological activities in inflammatory diseases.

VII. CONCLUDING REMARKS In summary, the mucosal lymphoid tissues of the intestinal and respiratory tracts share

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REFERENCES

many developmental, architectural, and functional features that allow them to sample mucosal antigens and generate appropriate immune responses. However, the mechanistic details that regulate how each of these tissues functions are often slightly different, in part owing to the location in which they reside, the number and type of microbial communities to which they are exposed, and the cell types involved in their function. As a result, vaccines that target particular mucosal sites or immunemodulating therapies that are directed toward particular organs must account for these differences in order to be effective.

References [1] Randall TD, Mebius RE. The development and function of mucosal lymphoid tissues: a balancing act with micro-organisms. Mucosal Immunol 2014;7:455 66. [2] Carragher DM, Rangel-Moreno J, Randall TD. Ectopic lymphoid tissues and local immunity. Semin Immunol 2008;20:26 42. [3] Kasahara M, Sutoh Y. Two forms of adaptive immunity in vertebrates: similarities and differences. Adv Immunol 2014;122:59 90. [4] Danilova N. The evolution of adaptive immunity. Adv Exp Med Biol 2012;738:218 35. [5] Das S, Li J, Hirano M, Sutoh Y, Herrin BR, Cooper MD. Evolution of two prototypic T cell lineages. Cell Immunol 2015;296:87 94. [6] Alder MN, Herrin BR, Sadlonova A, Stockard CR, Grizzle WE, Gartland LA, et al. Antibody responses of variable lymphocyte receptors in the lamprey. Nat Immunol 2008;9:319 27. [7] Kishishita N, Nagawa F. Evolution of adaptive immunity: implications of a third lymphocyte lineage in lampreys. Bioessays 2014;36:244 50. [8] Matsunaga T, Rahman A. In search of the origin of the thymus: the thymus and GALT may be evolutionarily related. Scand J Immunol 2001;53:1 6. [9] Shields JW. The functional evolution of GALT: a review. Lymphology 2000;33:47 57. [10] Finke D, Meier D. Molecular networks orchestrating GALT development. Curr Top Microbiol Immunol 2006;308:19 57. [11] Pospisil R, Mage RG. Rabbit appendix: a site of development and selection of the B cell repertoire. Curr Top Microbiol Immunol 1998;229:59 70.

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[12] Vajdy M, Sethupathi P, Knight KL. Dependence of antibody somatic diversification on gut-associated lymphoid tissue in rabbits. J Immunol 1998;160:2725 9. [13] Boehm T, Hess I, Swann JB. Evolution of lymphoid tissues. Trends Immunol 2012;33:315 21. [14] Neely HR, Flajnik MF. Emergence and evolution of secondary lymphoid organs. Annu Rev Cell Dev Biol 2016;32:693 711. [15] Salinas I. The mucosal immune system of teleost fish. Biology (Basel) 2015;4:525 39. [16] Tacchi L, Larragoite ET, Munoz P, Amemiya CT, Salinas I. African lungfish reveal the evolutionary origins of organized mucosal lymphoid tissue in vertebrates. Curr Biol 2015;25:2417 24. [17] Colombo BM, Scalvenzi T, Benlamara S, Pollet N. Microbiota and mucosal immunity in amphibians. Front Immunol 2015;6:111. [18] Link A, Vogt TK, Favre S, Britschgi MR, Acha-Orbea H, Hinz B, et al. Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nat Immunol 2007;8:1255 65. [19] Comerford I, Harata-Lee Y, Bunting MD, Gregor C, Kara EE, McColl SR. A myriad of functions and complex regulation of the CCR7/CCL19/CCL21 chemokine axis in the adaptive immune system. Cytokine Growth Factor Rev 2013;24:269 83. [20] Li L, Choi YS. Follicular dendritic cell-signaling molecules required for proliferation and differentiation of GC-B cells. Semin Immunol 2002;14:259 66. [21] Rodda LB, Bannard O, Ludewig B, Nagasawa T, Cyster JG. Phenotypic and morphological properties of germinal center dark zone Cxcl12-expressing reticular cells. J Immunol 2015;195:4781 91. [22] Aguzzi A, Kranich J, Krautler NJ. Follicular dendritic cells: origin, phenotype, and function in health and disease. Trends Immunol 2014;35:105 13. [23] Ansel KM, Ngo VN, Hyman PL, Luther SA, Forster R, Sedgwick JD, et al. A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 2000;406:309 14. [24] Mebius RE, Kraal G. Structure and function of the spleen. Nat Rev Immunol 2005;5:606 16. [25] Yasuda M, Taura Y, Yokomizo Y, Ekino S. A comparative study of germinal center: fowls and mammals. Comp Immunol Microbiol Infect Dis 1998;21:179 89. [26] Ruddle NH. Lymphoid neo-organogenesis: lymphotoxin’s role in inflammation and development. Immunol Res 1999;19:119 25. [27] Vondenhoff MF, Greuter M, Goverse G, Elewaut D, Dewint P, Ware CF, et al. LTbetaR signaling induces cytokine expression and up-regulates lymphangiogenic factors in lymph node anlagen. J Immunol 2009;182:5439 45.

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44

2. ANATOMICAL UNIQUENESS OF THE MUCOSAL IMMUNE SYSTEM

[28] Drayton DL, Liao S, Mounzer RH, Ruddle NH. Lymphoid organ development: from ontogeny to neogenesis. Nat Immunol 2006;7:344 53. [29] Kaiser P, Poh TY, Rothwell L, Avery S, Balu S, Pathania US, et al. A genomic analysis of chicken cytokines and chemokines. J Interferon Cytokine Res 2005;25:467 84. [30] Magor KE, Miranzo Navarro D, Barber MR, Petkau K, Fleming-Canepa X, Blyth GA, et al. Defense genes missing from the flight division. Dev Comp Immunol 2013;41:377 88. [31] Dalloul RA, Long JA, Zimin AV, Aslam L, Beal K, Blomberg Le A, et al. Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo): genome assembly and analysis. PLoS Biol 2010;8. Available from: http://dx.doi.org/10.1371/journal. pbio.1000475. [32] Bienenstock J, Befus D. Gut- and bronchus-associated lymphoid tissue. Am J Anat 1984;170:437 45. [33] Olah I, Nagy N, Magyar A, Palya V. Esophageal tonsil: a novel gut-associated lymphoid organ. Poult Sci 2003;82:767 70. [34] Casteleyn C, Doom M, Lambrechts E, Van den Broeck W, Simoens P, Cornillie P. Locations of gut-associated lymphoid tissue in the 3-month-old chicken: a review. Avian Pathol 2010;39:143 50. [35] Gomez Del Moral M, Fonfria J, Varas A, Jimenez E, Moreno J, Zapata AG. Appearance and development of lymphoid cells in the chicken (Gallus gallus) caecal tonsil. Anat Rec 1998;250:182 9. [36] Jarjour M, Jorquera A, Mondor I, Wienert S, Narang P, Coles MC, et al. Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells. J Exp Med 2014;211:1109 22. [37] Bajenoff M, Germain RN. B-cell follicle development remodels the conduit system and allows soluble antigen delivery to follicular dendritic cells. Blood 2009;114:4989 97. [38] Lu TT, Browning JL. Role of the lymphotoxin/LIGHT system in the development and maintenance of reticular networks and vasculature in lymphoid tissues. Front Immunol 2014;5:47. [39] Zhao L, Chen J, Liu L, Gao J, Guo B, Zhu B. Essential role of TNF-alpha in development of spleen fibroblastic reticular cells. Cell Immunol 2015;293:130 6. [40] Ngo VN, Korner H, Gunn MD, Schmidt KN, Riminton DS, Cooper MD, et al. Lymphotoxin alpha/beta and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen. J Exp Med 1999;189:403 12. [41] Yu P, Wang Y, Chin RK, Martinez-Pomares L, Gordon S, Kosco-Vibois MH, et al. B cells control the migration of a subset of dendritic cells into B cell follicles via

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

CXC chemokine ligand 13 in a lymphotoxindependent fashion. J Immunol 2002;168:5117 23. Onder L, Danuser R, Scandella E, Firner S, Chai Q, Hehlgans T, et al. Endothelial cell-specific lymphotoxin-beta receptor signaling is critical for lymph node and high endothelial venule formation. J Exp Med 2013;210:465 73. Stranford S, Ruddle NH. Follicular dendritic cells, conduits, lymphatic vessels, and high endothelial venules in tertiary lymphoid organs: parallels with lymph node stroma. Front Immunol 2012;3:350. Ager A. High endothelial venules and other blood vessels: critical regulators of lymphoid organ development and function. Front Immunol 2017;8:45. Josien R, Wong BR, Li HL, Steinman RM, Choi Y. TRANCE, a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendritic cells. J Immunol 1999;162:2562 8. Baldwin HM, Ito-Ihara T, Isaacs JD, Hilkens CM. Tumour necrosis factor alpha blockade impairs dendritic cell survival and function in rheumatoid arthritis. Ann Rheum Dis 2010;69:1200 7. Thorne A, Tomic S, Pavlovic B, Mihajlovic D, Dzopalic T, Colic M. Tumor necrosis factor-alpha promotes survival and phenotypic maturation of poly(I:C)-treated dendritic cells but impairs their Th1 and Th17 polarizing capability. Cytotherapy 2015;17:633 46. Fu YX, Huang G, Wang Y, Chaplin DD. Lymphotoxinalpha-dependent spleen microenvironment supports the generation of memory B cells and is required for their subsequent antigen-induced activation. J Immunol 2000;164:2508 14. Debard N, Sierro F, Browning J, Kraehenbuhl JP. Effect of mature lymphocytes and lymphotoxin on the development of the follicle-associated epithelium and M cells in mouse Peyer’s patches. Gastroenterology 2001;120:1173 82. Hsieh EH, Fernandez X, Wang J, Hamer M, Calvillo S, Croft M, et al. CD137 is required for M cell functional maturation but not lineage commitment. Am J Pathol 2010;177:666 76. Brestoff JR, Artis D. Commensal bacteria at the interface of host metabolism and the immune system. Nat Immunol 2013;14:676 84. Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature 2011;474:298 306. Agace WW, McCoy KD. Regionalized development and maintenance of the intestinal adaptive immune landscape. Immunity 2017;46:532 48. Kanamori Y, Ishimaru K, Nanno M, Maki K, Ikuta K, Nariuchi H, et al. Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

c-kit 1 IL-7R 1 Thy1 1 lympho-hemopoietic progenitors develop. J Exp Med 1996;184:1449 59. Lundqvist C, Baranov V, Hammarstrom S, Athlin L, Hammarstrom ML. Intra-epithelial lymphocytes. Evidence for regional specialization and extrathymic T cell maturation in the human gut epithelium. Int Immunol 1995;7:1473 87. Kiss EA, Vonarbourg C, Kopfmann S, Hobeika E, Finke D, Esser C, et al. Natural aryl hydrocarbon receptor ligands control organogenesis of intestinal lymphoid follicles. Science 2011;334:1561 5. Lee JS, Cella M, McDonald KG, Garlanda C, Kennedy GD, Nukaya M, et al. AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch. Nat Immunol 2011;13:144 51. Nagasawa M, Spits H, Ros XR. Innate lymphoid cells (ILCs): cytokine hubs regulating immunity and tissue homeostasis. Cold Spring Harb Perspect Biol 2018;10. Zhong C, Zheng M, Zhu J. Lymphoid tissue inducer-A divergent member of the ILC family. Cytokine Growth Factor Rev 2018;. Available from: http://dx.doi.org/ 10.1101/cshperspect.a028563. Luci C, Reynders A, Ivanov II, Cognet C, Chiche L, Chasson L, et al. Influence of the transcription factor RORgammat on the development of NKp46 1 cell populations in gut and skin. Nat Immunol 2009;10:75 82. Sanos SL, Bui VL, Mortha A, Oberle K, Heners C, Johner C, et al. RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46 1 cells. Nat Immunol 2009;10:83 91. Satoh-Takayama N, Vosshenrich CA, Lesjean-Pottier S, Sawa S, Lochner M, Rattis F, et al. Microbial flora drives interleukin 22 production in intestinal NKp46 1 cells that provide innate mucosal immune defense. Immunity 2008;29:958 70. Mebius RE, Rennert P, Weissman IL. Developing lymph nodes collect CD4 1 CD3- LTbeta 1 cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells. Immunity 1997;7:493 504. Sawa S, Cherrier M, Lochner M, Satoh-Takayama N, Fehling HJ, Langa F, et al. Lineage relationship analysis of RORgammat 1 innate lymphoid cells. Science 2010;330:665 9. Eberl G, Littman DR. Thymic origin of intestinal alphabeta T cells revealed by fate mapping of RORgammat 1 cells. Science 2004;305:248 51. Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol 2004;5:64 73.

45

[67] Hamada H, Hiroi T, Nishiyama Y, Takahashi H, Masunaga Y, Hachimura S, et al. Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J Immunol 2002;168:57 64. [68] Savage AK, Liang HE, Locksley RM. The development of steady-state activation hubs between adult LTi ILC3s and primed macrophages in small intestine. J Immunol 2017;199:1912 22. [69] Liang SC, Tan XY, Luxenberg DP, Karim R, DunussiJoannopoulos K, Collins M, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 2006;203:2271 9. [70] Aden K, Rehman A, Falk-Paulsen M, Secher T, Kuiper J, Tran F, et al. Epithelial IL-23R signaling licenses protective IL-22 responses in intestinal inflammation. Cell Rep 2016;16:2208 18. [71] Li Z, Hodgkinson T, Gothard EJ, Boroumand S, Lamb R, Cummins I, et al. Epidermal Notch1 recruits RORgamma(1) group 3 innate lymphoid cells to orchestrate normal skin repair. Nat Commun 2016;7:11394. [72] Eyerich S, Wagener J, Wenzel V, Scarponi C, Pennino D, Albanesi C, et al. IL-22 and TNF-alpha represent a key cytokine combination for epidermal integrity during infection with Candida albicans. Eur J Immunol 2011;41:1894 901. [73] Longman RS, Diehl GE, Victorio DA, Huh JR, Galan C, Miraldi ER, et al. CX(3)CR1(1) mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22. J Exp Med 2014;211:1571 83. [74] Magri G, Miyajima M, Bascones S, Mortha A, Puga I, Cassis L, et al. Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells. Nat Immunol 2014;15:354 64. [75] Mortha A, Chudnovskiy A, Hashimoto D, Bogunovic M, Spencer SP, Belkaid Y, et al. Microbiota-dependent crosstalk between macrophages and ILC3 promotes intestinal homeostasis. Science 2014;343:1249288. [76] Pearson C, Thornton EE, McKenzie B, Schaupp AL, Huskens N, Griseri T, et al. ILC3 GM-CSF production and mobilisation orchestrate acute intestinal inflammation. eLife 2016;5:e10066. [77] Eken A, Singh AK, Treuting PM, Oukka M. IL-23R 1 innate lymphoid cells induce colitis via interleukin-22dependent mechanism. Mucosal Immunol 2014;7:143 54. [78] Goverse G, Labao-Almeida C, Ferreira M, Molenaar R, Wahlen S, Konijn T, et al. Vitamin A controls the presence of RORgamma 1 innate lymphoid cells and lymphoid tissue in the small intestine. J Immunol 2016;196:5148 55.

II. PRINCIPLES OF MUCOSAL VACCINE

46

2. ANATOMICAL UNIQUENESS OF THE MUCOSAL IMMUNE SYSTEM

[79] van de Pavert SA, Ferreira M, Domingues RG, Ribeiro H, Molenaar R, Moreira-Santos L, et al. Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity. Nature 2014;508:123 7. [80] Kiss EA, Diefenbach A. Role of the aryl hydrocarbon receptor in controlling maintenance and functional programs of RORgammat(1) innate lymphoid cells and intraepithelial lymphocytes. Front Immunol 2012;3:124. [81] Emgard J, Kammoun H, Garcia-Cassani B, Chesne J, Parigi SM, Jacob JM, et al. Oxysterol sensing through the receptor GPR183 promotes the lymphoid-tissue-inducing function of innate lymphoid cells and colonic inflammation. Immunity 2018;48 120 32 e128. [82] McDonald KG, McDonough JS, Dieckgraefe BK, Newberry RD. Dendritic cells produce CXCL13 and participate in the development of murine small intestine lymphoid tissues. Am J Pathol 2010;176:2367 77. [83] Lugering A, Ross M, Sieker M, Heidemann J, Williams IR, Domschke W, et al. CCR6 identifies lymphoid tissue inducer cells within cryptopatches. Clin Exp Immunol 2010;160:440 9. [84] Lorenz RG, Chaplin DD, McDonald KG, McDonough JS, Newberry RD. Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxinsufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function. J Immunol 2003;170:5475 82. [85] Pabst O, Herbrand H, Friedrichsen M, Velaga S, Dorsch M, Berhardt G, et al. Adaptation of solitary intestinal lymphoid tissue in response to microbiota and chemokine receptor CCR7 signaling. J Immunol 2006;177:6824 32. [86] Bouskra D, Brezillon C, Berard M, Werts C, Varona R, Boneca IG, et al. Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 2008;456:507 10. [87] Wang C, McDonough JS, McDonald KG, Huang C, Newberry RD. Alpha4beta7/MAdCAM-1 interactions play an essential role in transitioning cryptopatches into isolated lymphoid follicles and a nonessential role in cryptopatch formation. J Immunol 2008;181:4052 61. [88] Lin YL, Ip PP, Liao F. CCR6 deficiency impairs IgA production and dysregulates antimicrobial peptide production, altering the intestinal flora. Front Immunol 2017;8:805. [89] Tsuji M, Suzuki K, Kitamura H, Maruya M, Kinoshita K, Ivanov II, et al. Requirement for lymphoid tissueinducer cells in isolated follicle formation and T cellindependent immunoglobulin A generation in the gut. Immunity 2008;29:261 71.

[90] Knoop KA, Newberry RD. Isolated lymphoid follicles are dynamic reservoirs for the induction of intestinal IgA. Front Immunol 2012;3:84. [91] Russler-Germain EV, Rengarajan S, Hsieh CS. Antigen-specific regulatory T-cell responses to intestinal microbiota. Mucosal Immunol 2017;10:1375 86. [92] Wichner K, Fischer A, Winter S, Tetzlaff S, Heimesaat MM, Bereswill S, et al. Transition from an autoimmuneprone state to fatal autoimmune disease in CCR7 and RORgammat double-deficient mice is dependent on gut microbiota. J Autoimmun 2013;47:58 72. [93] Pere-Vedrenne C, Flahou B, Loke MF, Menard A, Vadivelu J. Other Helicobacters, gastric and gut microbiota. Helicobacter 2017;22(Suppl. 1). Available from: http://dx.doi.org/10.1111/hel.12407. [94] Baptista AP, Olivier BJ, Goverse G, Greuter M, Knippenberg M, Kusser K, et al. Colonic patch and colonic SILT development are independent and differentially regulated events. Mucosal Immunol 2013;6:511 21. [95] Masahata K, Umemoto E, Kayama H, Kotani M, Nakamura S, Kurakawa T, et al. Generation of colonic IgA-secreting cells in the caecal patch. Nat Commun 2014;5:3704. [96] Wang M, Gao Z, Zhang Z, Pan L, Zhang Y. Roles of M cells in infection and mucosal vaccines. Hum Vaccin Immunother 2014;10:3544 51. [97] Brayden DJ, Baird AW. A distinctive electrophysiological signature from the Peyer’s patches of rabbit intestine. Br J Pharmacol 1994;113:593 9. [98] Pabst O, Herbrand H, Worbs T, Friedrichsen M, Yan S, Hoffmann MW, et al. Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes. Eur J Immunol 2005;35:98 107. [99] Reboldi A, Arnon TI, Rodda LB, Atakilit A, Sheppard D, Cyster JG. IgA production requires B cell interaction with subepithelial dendritic cells in Peyer’s patches. Science 2016;352:aaf4822. [100] Zhao X, Sato A, Dela Cruz CS, Linehan M, Luegering A, Kucharzik T, et al. CCL9 is secreted by the follicleassociated epithelium and recruits dome region Peyer’s patch CD11b 1 dendritic cells. J Immunol 2003;171:2797 803. [101] Da Silva C, Wagner C, Bonnardel J, Gorvel JP, Lelouard H. The Peyer’s patch mononuclear phagocyte system at steady state and during infection. Front Immunol 2017;8:1254. [102] Austyn JM. Dendritic cells in the immune systemhistory, lineages, tissues, tolerance, and immunity. Microbiol Spectr 2016;4. [103] Guilliams M, Dutertre CA, Scott CL, McGovern N, Sichien D, Chakarov S, et al. Unsupervised high-

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[104]

[105]

[106]

[107]

[108]

[109]

[110]

[111]

[112]

[113]

[114]

[115]

dimensional analysis aligns dendritic cells across tissues and species. Immunity 2016;45:669 84. Reboldi A, Cyster JG. Peyer’s patches: organizing Bcell responses at the intestinal frontier. Immunol Rev 2016;271:230 45. Luda KM, Joeris T, Persson EK, Rivollier A, Demiri M, Sitnik KM, et al. IRF8 transcription-factordependent classical dendritic cells are essential for intestinal T cell homeostasis. Immunity 2016;44:860 74. Ohta T, Sugiyama M, Hemmi H, Yamazaki C, Okura S, Sasaki I, et al. Crucial roles of XCR1-expressing dendritic cells and the XCR1-XCL1 chemokine axis in intestinal immune homeostasis. Sci Rep 2016;6:23505. Bekiaris V, Persson EK, Agace WW. Intestinal dendritic cells in the regulation of mucosal immunity. Immunol Rev 2014;260:86 101. Persson EK, Scott CL, Mowat AM, Agace WW. Dendritic cell subsets in the intestinal lamina propria: ontogeny and function. Eur J Immunol 2013;43:3098 107. Mora JR, Bono MR, Manjunath N, Weninger W, Cavanagh LL, Rosemblatt M, et al. Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature 2003;424:88 93. Johansson-Lindbom B, Agace WW. Generation of gut-homing T cells and their localization to the small intestinal mucosa. Immunol Rev 2007;215:226 42. Paidassi H, Acharya M, Zhang A, Mukhopadhyay S, Kwon M, Chow C, et al. Preferential expression of integrin alphavbeta8 promotes generation of regulatory T cells by mouse CD103 1 dendritic cells. Gastroenterology 2011;141:1813 20. Boucard-Jourdin M, Kugler D, Endale Ahanda ML, This S, De Calisto J, Zhang A, et al. Beta8 integrin expression and activation of TGF-beta by intestinal dendritic cells are determined by both tissue microenvironment and cell lineage. J Immunol 2016;197:1968 78. Worthington JJ, Czajkowska BI, Melton AC, Travis MA. Intestinal dendritic cells specialize to activate transforming growth factor-beta and induce Foxp3 1 regulatory T cells via integrin alphavbeta8. Gastroenterology 2011;141:1802 12. Johansson-Lindbom B, Svensson M, Pabst O, Palmqvist C, Marquez G, Forster R, et al. Functional specialization of gut CD103 1 dendritic cells in the regulation of tissue-selective T cell homing. J Exp Med 2005;202:1063 73. Bonnardel J, Da Silva C, Wagner C, Bonifay R, Chasson L, Masse M, et al. Distribution, location, and transcriptional profile of Peyer’s patch conventional DC subsets at steady state and under TLR7 ligand stimulation. Mucosal Immunol 2017;10:1412 30.

47

[116] Okada T, Ngo VN, Ekland EH, Forster R, Lipp M, Littman DR, et al. Chemokine requirements for B cell entry to lymph nodes and Peyer’s patches. J Exp Med 2002;196:65 75. [117] Kurita N, Honda S, Shibuya A. Increased serum IgA in Fcalpha/muR-deficient mice on the (129 x C57BL/ 6) F1 genetic background. Mol Immunol 2015;63:367 72. [118] Onder L, Narang P, Scandella E, Chai Q, Iolyeva M, Hoorweg K, et al. IL-7-producing stromal cells are critical for lymph node remodeling. Blood 2012;120:4675 83. [119] Hara T, Shitara S, Imai K, Miyachi H, Kitano S, Yao H, et al. Identification of IL-7-producing cells in primary and secondary lymphoid organs using IL-7GFP knock-in mice. J Immunol 2012;189:1577 84. [120] Cremasco V, Woodruff MC, Onder L, Cupovic J, Nieves-Bonilla JM, Schildberg FA, et al. B cell homeostasis and follicle confines are governed by fibroblastic reticular cells. Nat Immunol 2014;15:973 81. [121] Gil-Cruz C, Perez-Shibayama C, Onder L, Chai Q, Cupovic J, Cheng HW, et al. Fibroblastic reticular cells regulate intestinal inflammation via IL-15mediated control of group 1 ILCs. Nat Immunol 2016;17:1388 96. [122] McDonald KG, Wheeler LW, McDole JR, Joerger S, Gustafsson JK, Kulkarni DH, et al. CCR6 promotes steady-state mononuclear phagocyte association with the intestinal epithelium, imprinting and immune surveillance. Immunology 2017;152:613 27. [123] Hase K, Murakami T, Takatsu H, Shimaoka T, Iimura M, Hamura K, et al. The membrane-bound chemokine CXCL16 expressed on follicle-associated epithelium and M cells mediates lympho-epithelial interaction in GALT. J Immunol 2006;176:43 51. [124] Mantis NJ, Cheung MC, Chintalacharuvu KR, Rey J, Corthesy B, Neutra MR. Selective adherence of IgA to murine Peyer’s patch M cells: evidence for a novel IgA receptor. J Immunol 2002;169:1844 51. [125] Nakato G, Fukuda S, Hase K, Goitsuka R, Cooper MD, Ohno H. New approach for m-cell-specific molecules screening by comprehensive transcriptome analysis. DNA Res 2009;16:227 35. [126] Hase K, Kawano K, Nochi T, Pontes GS, Fukuda S, Ebisawa M, et al. Uptake through glycoprotein 2 of FimH(1) bacteria by M cells initiates mucosal immune response. Nature 2009;462:226 30. [127] Sato S, Kaneto S, Shibata N, Takahashi Y, Okura H, Yuki Y, et al. Transcription factor Spi-B-dependent and -independent pathways for the development of Peyer’s patch M cells. Mucosal Immunol 2013;6:838 46. [128] Yanagihara S, Kanaya T, Fukuda S, Nakato G, Hanazato M, Wu XR, et al. Uromodulin-SlpA binding

II. PRINCIPLES OF MUCOSAL VACCINE

48

[129] [130]

[131]

[132]

[133]

[134]

[135]

[136]

[137]

[138]

[139]

[140]

[141]

2. ANATOMICAL UNIQUENESS OF THE MUCOSAL IMMUNE SYSTEM

dictates Lactobacillus acidophilus uptake by intestinal epithelial M cells. Int Immunol 2017;29:357 63. Ohno H. Intestinal M cells. J Biochem 2016;159:151 60. Nakato G, Hase K, Suzuki M, Kimura M, Ato M, Hanazato M, et al. Cutting edge: Brucella abortus exploits a cellular prion protein on intestinal M cells as an invasive receptor. J Immunol 2012;189:1540 4. Wood MB, Rios D, Williams IR. TNF-alpha augments RANKL-dependent intestinal M cell differentiation in enteroid cultures. Am J Physiol Cell Physiol 2016;311: C498 507. Lo D, Tynan W, Dickerson J, Mendy J, Chang HW, Scharf M, et al. Peptidoglycan recognition protein expression in mouse Peyer’s Patch follicle associated epithelium suggests functional specialization. Cell Immunol 2003;224:8 16. Sansonetti PJ, Phalipon A. M cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process. Semin Immunol 1999;11:193 203. Clark MA, Hirst BH, Jepson MA. M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer’s patch M cells. Infect Immun 1998;66:1237 43. Jensen VB, Harty JT, Jones BD. Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer’s patches. Infect Immun 1998;66:3758 66. Matsumura T, Sugawara Y, Yutani M, Amatsu S, Yagita H, Kohda T, et al. Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity. Nat Commun 2015;6:6255. Rajapaksa TE, Stover-Hamer M, Fernandez X, Eckelhoefer HA, Lo DD. Claudin 4-targeted protein incorporated into PLGA nanoparticles can mediate M cell targeted delivery. J Control Release 2010;142:196 205. Kim SH, Jung DI, Yang IY, Kim J, Lee KY, Nochi T, et al. M cells expressing the complement C5a receptor are efficient targets for mucosal vaccine delivery. Eur J Immunol 2011;41:3219 29. Tsuji M, Suzuki K, Kinoshita K, Fagarasan S. Dynamic interactions between bacteria and immune cells leading to intestinal IgA synthesis. Semin Immunol 2008;20:59 66. Veiga-Fernandes H, Coles MC, Foster KE, Patel A, Williams A, Natarajan D, et al. Tyrosine kinase receptor RET is a key regulator of Peyer’s patch organogenesis. Nature 2007;446:547 51. White A, Carragher D, Parnell S, Msaki A, Perkins N, Lane P, et al. Lymphotoxin a-dependent and

[142]

[143]

[144]

[145]

[146]

[147]

[148]

[149]

[150]

[151]

[152]

-independent signals regulate stromal organizer cell homeostasis during lymph node organogenesis. Blood 2007;110:1950 9. Cherrier M, Sawa S, Eberl G. Notch, Id2, and RORgammat sequentially orchestrate the fetal development of lymphoid tissue inducer cells. J Exp Med 2012;209:729 40. Patel A, Harker N, Moreira-Santos L, Ferreira M, Alden K, Timmis J, et al. Differential RET signaling pathways drive development of the enteric lymphoid and nervous systems. Sci Signal 2012;5:ra55. Adachi S, Yoshida H, Kataoka H, Nishikawa S. Three distinctive steps in Peyer’s patch formation of murine embryo. Int Immunol 1997;9:507 14. Rennert PD, Browning JL, Mebius R, Mackay F, Hochman PS. Surface lymphotoxin alpha/beta complex is required for the development of peripheral lymphoid organs. J Exp Med 1996;184:1999 2006. De Togni P, Goellner J, Ruddle NH, Streeter PR, Fick A, Mariathasan S, et al. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science 1994;264:703 7. Matsushima A, Kaisho T, Rennert PD, Nakano H, Kurosawa K, Uchida D, et al. Essential role of nuclear factor (NF)-kappaB-inducing kinase and inhibitor of kappaB (IkappaB) kinase alpha in NF-kappaB activation through lymphotoxin beta receptor, but not through tumor necrosis factor receptor I. J Exp Med 2001;193:631 6. Piao JH, Yoshida H, Yeh WC, Doi T, Xue X, Yagita H, et al. TNF receptor-associated factor 2-dependent canonical pathway is crucial for the development of Peyer’s patches. J Immunol 2007;178:2272 7. Futterer A, Mink K, Luz A, Kosco-Vilbois MH, Pfeffer K. The lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 1998;9:59 70. Nakagawa R, Togawa A, Nagasawa T, Nishikawa S. Peyer’s patch inducer cells play a leading role in the formation of B and T cell zone architecture. J Immunol 2013;190:3309 18. Endres R, Alimzhanov MB, Plitz T, Futterer A, KoscoVilbois MH, Nedospasov SA, et al. Mature follicular dendritic cell networks depend on expression of lymphotoxin beta receptor by radioresistant stromal cells and of lymphotoxin beta and tumor necrosis factor by B cells. J Exp Med 1999;189:159 68. Saito Y, Respatika D, Komori S, Washio K, Nishimura T, Kotani T, et al. SIRPalpha(1) dendritic cells regulate homeostasis of fibroblastic reticular cells via TNF receptor ligands in the adult spleen. Proc Natl Acad Sci USA 2017;114:E10151 60.

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[153] Eberl G, Lochner M. The development of intestinal lymphoid tissues at the interface of self and microbiota. Mucosal Immunol 2009;2:478 85. [154] Yamanaka T, Helgeland L, Farstad IN, Fukushima H, Midtvedt T, Brandtzaeg P. Microbial colonization drives lymphocyte accumulation and differentiation in the follicle-associated epithelium of Peyer’s patches. J Immunol 2003;170:816 22. [155] Donaldson DS, Bradford BM, Artis D, Mabbott NA. Reciprocal regulation of lymphoid tissue development in the large intestine by IL-25 and IL-23. Mucosal Immunol 2015;8:582 95. [156] Taylor RT, Lugering A, Newell KA, Williams IR. Intestinal cryptopatch formation in mice requires lymphotoxin alpha and the lymphotoxin beta receptor. J Immunol 2004;173:7183 9. [157] Honda K, Nakano H, Yoshida H, Nishikawa S, Rennert P, Ikuta K, et al. Molecular basis for hematopoietic/mesenchymal interaction during initiation of Peyer’s patch organogenesis. J Exp Med 2001;193:621 30. [158] Knoop KA, Butler BR, Kumar N, Newberry RD, Williams IR. Distinct developmental requirements for isolated lymphoid follicle formation in the small and large intestine: RANKL is essential only in the small intestine. Am J Pathol 2011;179:1861 71. [159] Kimura S, Yamakami-Kimura M, Obata Y, Hase K, Kitamura H, Ohno H, et al. Visualization of the entire differentiation process of murine M cells: suppression of their maturation in cecal patches. Mucosal Immunol 2015;8:650 60. [160] Kanaya T, Hase K, Takahashi D, Fukuda S, Hoshino K, Sasaki I, et al. The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells. Nat Immunol 2012;13:729 36. [161] Donaldson DS, Sehgal A, Rios D, Williams IR, Mabbott NA. Increased abundance of M cells in the gut epithelium dramatically enhances oral prion disease susceptibility. PLoS Pathog 2016;12:e1006075. [162] Knoop KA, Kumar N, Butler BR, Sakthivel SK, Taylor RT, Nochi T, et al. RANKL is necessary and sufficient to initiate development of antigensampling M cells in the intestinal epithelium. J Immunol 2009;183:5738 47. [163] Sehgal A, Kobayashi A, Donaldson DS, Mabbott NA. c-Rel is dispensable for the differentiation and functional maturation of M cells in the follicle-associated epithelium. Immunobiology 2017;222:316 26. [164] Regan T, Nally K, Carmody R, Houston A, Shanahan F, Macsharry J, et al. Identification of TLR10 as a key mediator of the inflammatory response to Listeria monocytogenes in intestinal epithelial cells and macrophages. J Immunol 2013;191:6084 92.

49

[165] Ghannam S, Dejou C, Pedretti N, Giot JP, Dorgham K, Boukhaddaoui H, et al. CCL20 and beta-defensin-2 induce arrest of human Th17 cells on inflamed endothelium in vitro under flow conditions. J Immunol 2011;186:1411 20. [166] Hirata T, Osuga Y, Takamura M, Kodama A, Hirota Y, Koga K, et al. Recruitment of CCR6-expressing Th17 cells by CCL 20 secreted from IL-1 beta-, TNFalpha-, and IL-17A-stimulated endometriotic stromal cells. Endocrinology 2010;151:5468 76. [167] Ermak TH, Bhagat HR, Pappo J. Lymphocyte compartments in antigen-sampling regions of rabbit mucosal lymphoid organs. Am J Trop Med Hyg 1994;50:14 28. [168] Kerne´is S, Bogdanova A, Kraehenbuhl JP, Pringault E. Conversion by Peyer’s patch lymphocytes of human enterocytes into M cells that transport bacteria. Science 1997;277:949 52. [169] Araujo F, Sarmento B. Towards the characterization of an in vitro triple co-culture intestine cell model for permeability studies. Int J Pharm 2013;458:128 34. [170] Kobayashi A, Donaldson DS, Erridge C, Kanaya T, Williams IR, Ohno H, et al. The functional maturation of M cells is dramatically reduced in the Peyer’s patches of aged mice. Mucosal Immunol 2013;6:1027 37. [171] Man AL, Lodi F, Bertelli E, Regoli M, Pin C, Mulholland F, et al. Macrophage migration inhibitory factor plays a role in the regulation of microfold (M) cell-mediated transport in the gut. J Immunol 2008;181:5673 80. [172] Wang J, Li F, Tian Z. Role of microbiota on lung homeostasis and diseases. Sci China Life Sci 2017;60:1407 15. [173] Tipton L, Ghedin E, Morris A. The lung mycobiome in the next-generation sequencing era. Virulence 2017;8:334 41. [174] Sepahi A, Salinas I. The evolution of nasal immune systems in vertebrates. Mol Immunol 2016;69:131 8. [175] Pabst R. Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)Structure, function and species differences. Vaccine 2015;33:4406 13. [176] Mair TS, Batten EH, Stokes CR, Bourne FJ. The histological features of the immune system of the equine respiratory tract. J Comp Pathol 1987;97:575 86. [177] Casteleyn C, Broos AM, Simoens P, Van den Broeck W. NALT (nasal cavity-associated lymphoid tissue) in the rabbit. Vet Immunol Immunopathol 2010;133:212 18. [178] Bienenstock J, McDermott MR. Bronchus- and nasalassociated lymphoid tissues. Immunol Rev 2005;206:22 31.

II. PRINCIPLES OF MUCOSAL VACCINE

50

2. ANATOMICAL UNIQUENESS OF THE MUCOSAL IMMUNE SYSTEM

[179] Csencsits KL, Jutila MA, Pascual DW. Nasalassociated lymphoid tissue: phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site. J Immunol 1999;163:1382 9. [180] Lohrberg M, Pabst R, Wilting J. Co-localization of lymphoid aggregates and lymphatic networks in nose- (NALT) and lacrimal duct-associated lymphoid tissue (LDALT) of mice. BMC Immunol 2018;19:5. [181] Rangel-Moreno J, Moyron-Quiroz J, Kusser K, Hartson L, Nakano H, Randall TD. Role of CXC chemokine ligand 13, CC chemokine ligand (CCL) 19, and CCL21 in the organization and function of nasal-associated lymphoid tissue. J Immunol 2005;175:4904 13. [182] Date Y, Ebisawa M, Fukuda S, Shima H, Obata Y, Takahashi D, et al. NALT M cells are important for immune induction for the common mucosal immune system. Int Immunol 2017;29:471 8. [183] Kim DY, Sato A, Fukuyama S, Sagara H, Nagatake T, Kong IG, et al. The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens. J Immunol 2011;186:4253 62. [184] Mutoh M, Kimura S, Takahashi-Iwanaga H, Hisamoto M, Iwanaga T, Iida J. RANKL regulates differentiation of microfold cells in mouse nasopharynxassociated lymphoid tissue (NALT). Cell Tissue Res 2016;364:175 84. [185] Fukuyama Y, Tokuhara D, Sekine S, Aso K, Kataoka K, Davydova J, et al. Potential roles of CCR5(1) CCR6 (1) dendritic cells induced by nasal ovalbumin plus Flt3 ligand expressing adenovirus for mucosal IgA responses. PLoS One 2013;8:e60453. [186] Ying X, Chan K, Shenoy P, Hill M, Ruddle NH. Lymphotoxin plays a crucial role in the development and function of nasal-associated lymphoid tissue through regulation of chemokines and peripheral node addressin. Am J Pathol 2005;166:135 46. [187] Ohmichi Y, Hirakawa J, Imai Y, Fukuda M, Kawashima H. Essential role of peripheral node addressin in lymphocyte homing to nasal-associated lymphoid tissues and allergic immune responses. J Exp Med 2011;208:1015 25. [188] Browning JL, Allaire N, Ngam-Ek A, Notidis E, Hunt J, Perrin S, et al. Lymphotoxin-beta receptor signaling is required for the homeostatic control of HEV differentiation and function. Immunity 2005;23:539 50. [189] Nagatake T, Fukuyama S, Kim DY, Goda K, Igarashi O, Sato S, et al. Id2-, RORgammat-, and LTbetaRindependent initiation of lymphoid organogenesis in ocular immunity. J Exp Med 2009;206:2351 64.

[190] Kunisawa J, Fukuyama S, Kiyono H. Mucosaassociated lymphoid tissues in the aerodigestive tract: their shared and divergent traits and their importance to the orchestration of the mucosal immune system. Curr Mol Med 2005;5:557 72. [191] Fukuyama S, Hiroi T, Yokota Y, Rennert PD, Yanagita M, Kinoshita N, et al. Initiation of NALT organogenesis is independent of the IL-7R, LTbetaR, and NIK signaling pathways but requires the Id2 gene and CD3(-) CD4(1)CD45(1) cells. Immunity 2002;17:31 40. [192] Rangel-Moreno J, Carragher D, Randall TD. Role of lymphotoxin and homeostatic chemokines in the development and function of local lymphoid tissues in the respiratory tract. Immunologia 2007;26:13 28. [193] Nagatake T, Fukuyama S, Sato S, Okura H, Tachibana M, Taniuchi I, et al. Central role of core binding factor beta2 in mucosa-associated lymphoid tissue organogenesis in mouse. PLoS One 2015;10:e0127460. [194] Krege J, Seth S, Hardtke S, Davalos-Misslitz AC, Forster R. Antigen-dependent rescue of noseassociated lymphoid tissue (NALT) development independent of LTbetaR and CXCR5 signaling. Eur J Immunol 2009;39:2765 78. [195] Harmsen A, Kusser K, Hartson L, Tighe M, Sunshine MJ, Sedgwick JD, et al. Cutting edge: organogenesis of nasal-associated lymphoid tissue (NALT) occurs independently of lymphotoxin-α (LT α) and retinoic acid receptor-related orphan receptor-γ, but the organization of NALT is LT α dependent. J Immunol 2002;168:986 90. [196] Hostager BS, Bishop GA. CD40-mediated activation of the NF-kappaB2 pathway. Front Immunol 2013;4:376. [197] de Lau W, Kujala P, Schneeberger K, Middendorp S, Li VS, Barker N, et al. Peyer’s patch M cells derived from Lgr5(1) stem cells require SpiB and are induced by RankL in cultured “miniguts”. Mol Cell Biol 2012;32:3639 47. [198] Ermak TH, Steger HJ, Strober S, Owen RL. M cells and granular mononuclear cells in Peyer’s patch domes of mice depleted of their lymphocytes by total lymphoid irradiation. Am J Pathol 1989;134:529 37. [199] Velin D, Fotopoulos G, Luthi F, Kraehenbuhl JP. The nasal-associated lymphoid tissue of adult mice acts as an entry site for the mouse mammary tumor retrovirus. J Exp Med 1997;185:1871 6. [200] Park HS, Cleary PP. Active and passive intranasal immunizations with streptococcal surface protein C5a peptidase prevent infection of murine nasal mucosaassociated lymphoid tissue, a functional homologue of human tonsils. Infect Immun 2005;73:7878 86. [201] Park HS, Costalonga M, Reinhardt RL, Dombek PE, Jenkins MK, Cleary PP. Primary induction of CD4T

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[202]

[203]

[204]

[205]

[206]

[207]

[208]

[209]

[210]

[211]

[212]

[213]

cell responses in nasal associated lymphoid tissue during group A streptococcal infection. Eur J Immunol 2004;34:2843 53. Owen SJ, Batzloff M, Chehrehasa F, Meedeniya A, Casart Y, Logue CA, et al. Nasal-associated lymphoid tissue and olfactory epithelium as portals of entry for Burkholderia pseudomallei in murine melioidosis. J Infect Dis 2009;199:1761 70. Takata S, Ohtani O, Watanabe Y. Lectin binding patterns in rat nasal-associated lymphoid tissue (NALT) and the influence of various types of lectin on particle uptake in NALT. Arch Histol Cytol 2000;63:305 12. Wu Y, Wang X, Csencsits KL, Haddad A, Walters N, Pascual DW. M cell-targeted DNA vaccination. Proc Natl Acad Sci USA 2001;98:9318 23. Shukla A, Katare OP, Singh B, Vyas SP. M-cell targeted delivery of recombinant hepatitis B surface antigen using cholera toxin B subunit conjugated bilosomes. Int J Pharm 2010;385:47 52. Yuki Y, Kiyono H. New generation of mucosal adjuvants for the induction of protective immunity. Rev Med Virol 2003;13:293 310. Kiyono H, Fukuyama S. NALT- versus Peyer’s-patchmediated mucosal immunity. Nat Rev Immunol 2004;4:699 710. Kataoka K, Fujihashi K, Oma K, Fukuyama Y, Hollingshead SK, Sekine S, et al. The nasal dendritic cell-targeting Flt3 ligand as a safe adjuvant elicits effective protection against fatal pneumococcal pneumonia. Infect Immun 2011;79:2819 28. Matsuo K, Yoshikawa T, Asanuma H, Iwasaki T, Hagiwara Y, Chen Z, et al. Induction of innate immunity by nasal influenza vaccine administered in combination with an adjuvant (cholera toxin). Vaccine 2000;18:2713 22. Heritage PL, Underdown BJ, Brook MA, McDermott MR. Oral administration of polymer-grafted starch microparticles activates gut-associated lymphocytes and primes mice for a subsequent systemic antigen challenge. Vaccine 1998;16:2010 17. Tamura S, Iwasaki T, Thompson AH, Asanuma H, Chen Z, Suzuki Y, et al. Antibody-forming cells in the nasal-associated lymphoid tissue during primary influenza virus infection. J Gen Virol 1998;79(Pt 2):291 9. Zuercher AW, Coffin SE, Thurnheer MC, Fundova P, Cebra JJ. Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses. J Immunol 2002;168:1796 803. Boyden AW, Legge KL, Waldschmidt TJ. Pulmonary infection with influenza A virus induces site-specific germinal center and T follicular helper cell responses. PLoS One 2012;7:e40733.

51

[214] Shimoda M, Nakamura T, Takahashi Y, Asanuma H, Tamura S, Kurata T, et al. Isotype-specific selection of high affinity memory B cells in nasal-associated lymphoid tissue. J Exp Med 2001;194:1597 607. [215] Barman NN, Bhattacharyya R, Upadhyaya TN, Baishya G. Development of bronchus-associated lymphoid tissue in goats. Lung 1996;174:127 31. [216] Watt NJ, MacIntyre N, Collie D, Sargan D, McConnell I. Phenotypic analysis of lymphocyte populations in the lungs and regional lymphoid tissue of sheep naturally infected with maedi visna virus. Clin Exp Immunol 1992;90:204 8. [217] Delventhal S, Hensel A, Petzoldt K, Pabst R. Effects of microbial stimulation on the number, size and activity of bronchus-associated lymphoid tissue (BALT) structures in the pig. Int J Exp Pathol 1992;73:351 7. [218] Delventhal S, Brandis A, Ostertag H, Pabst R. Low incidence of bronchus-associated lymphoid tissue (BALT) in chronically inflamed human lungs. Virchows Arch B Cell Pathol Incl Mol Pathol 1992;62:271 4. [219] Gould SJ, Isaacson PG. Bronchus-associated lymphoid tissue (BALT) in human fetal and infant lung. J Pathol 1993;169:229 34. [220] Kolopp-Sarda MN, Bene MC, Massin N, Moulin JJ, Faure GC. Immunohistological analysis of macrophages, B-cells, and T-cells in the mouse lung. Anat Rec 1994;239:150 7. [221] Johnson JD, Houchens DP, Kluwe WM, Craig DK, Fisher GL. Effects of mainstream and environmental tobacco smoke on the immune system in animals and humans: a review. Crit Rev Toxicol 1990;20:369 95. [222] Meuwissen HJ, Hussain M. Bronchus-associated lymphoid tissue in human lung: correlation of hyperplasia with chronic pulmonary disease. Clin Immunol Immunopathol 1982;23:548 61. [223] Tschernig T, Pabst R. Bronchus-associated lymphoid tissue (BALT) is not present in the normal adult lung but in different diseases. Pathobiology 2000;68:1 8. [224] Suda T, Chida K, Hayakawa H, Imokawa S, Iwata M, Nakamura H, et al. Development of bronchusassociated lymphoid tissue in chronic hypersensitivity pneumonitis. Chest 1999;115:357 63. [225] Rangel-Moreno J, Hartson L, Navarro C, Gaxiola M, Selman M, Randall TD. Inducible bronchusassociated lymphoid tissue (iBALT) in patients with pulmonary complications of rheumatoid arthritis. J Clin Invest 2006;116:3183 94. [226] Sato A, Hayakawa H, Uchiyama H, Chida K. Cellular distribution of bronchus-associated lymphoid tissue in rheumatoid arthritis. Am J Respir Crit Care Med 1996;154:1903 7.

II. PRINCIPLES OF MUCOSAL VACCINE

52

2. ANATOMICAL UNIQUENESS OF THE MUCOSAL IMMUNE SYSTEM

[227] Shilling RA, Williams JW, Perera J, Berry E, Wu Q, Cummings OW, et al. Autoreactive T and B cells induce the development of bronchus-associated lymphoid tissue in the lung. Am J Respir Cell Mol Biol 2013;48:406 14. [228] Hwang JY, Randall TD, Silva-Sanchez A. Inducible bronchus-associated lymphoid tissue: taming inflammation in the lung. Front Immunol 2016;7:258. [229] Fleige H, Forster R. Induction and analysis of bronchus-associated lymphoid tissue. Methods Mol Biol 2017;1559:185 98. [230] Pabst R, Binns RM. The immune system of the respiratory tract in pigs. Vet Immunol Immunopathol 1994;43:151 6. [231] Vranckx K, Maes D, Marchioro SB, Villarreal I, Chiers K, Pasmans F, et al. Vaccination reduces macrophage infiltration in bronchus-associated lymphoid tissue in pigs infected with a highly virulent Mycoplasma hyopneumoniae strain. BMC Vet Res 2012;8:24. [232] Zamri-Saad M, Effendy AW. The effects of dexamethasone on the response of bronchus-associated lymphoid tissue to intranasal administration of formalin-killed Pasteurella haemolytica A2 in goats. Vet Res Commun 1999;23:467 73. [233] Effendy AW, Zamri-Saad M, Maswati MA, Ismail MS, Jamil SM. Stimulation of the bronchus-associated lymphoid tissue of goats and its effect on in vitro colonization by Pasteurella haemolytica. Vet Res Commun 1998;22:147 53. [234] Moyron-Quiroz JE, Rangel-Moreno J, Kusser K, Hartson L, Sprague F, Goodrich S, et al. Role of inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity. Nat Med 2004;10:927 34. [235] Wiley JA, Richert LE, Swain SD, Harmsen A, Barnard DL, Randall TD, et al. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses. PLoS One 2009;4:e7142. [236] Plesch BE, Gamelkoorn GJ, van de Ende M. Development of bronchus associated lymphoid tissue (BALT) in the rat, with special reference to T- and B-cells. Dev Comp Immunol 1983;7:179 88. [237] Gadaleanu V. Ovalbumin-induced immunomorphologic reaction of rat lung with emphasis on bronchusassociated lymphoid tissue (BALT). Morphol Embryol (Bucur) 1982;28:65 9. [238] Gregson RL, Davey MJ, Prentice DE. The response of rat bronchus-associated lymphoid tissue to local antigenic challenge. Br J Exp Pathol 1979;60:471 82. [239] van der Brugge-Gamelkoorn GJ, Plesch BE, Sminia T, Langevoort HL. Specific antibody-forming cells in bronchus-associated lymphoid tissue (BALT) and lung of the rat after intratracheal challenge with

[240]

[241]

[242]

[243]

[244]

[245]

[246]

[247]

[248]

[249]

[250]

horseradish peroxidase. Virchows Arch B Cell Pathol Incl Mol Pathol 1985;49:269 76. Rangel-Moreno J, Carragher DM, de la Luz GarciaHernandez M, Hwang JY, Kusser K, Hartson L, et al. The development of inducible bronchus-associated lymphoid tissue depends on IL-17. Nat Immunol 2011;12:639 46. Fleige H, Ravens S, Moschovakis GL, Bolter J, Willenzon S, Sutter G, et al. IL-17-induced CXCL12 recruits B cells and induces follicle formation in BALT in the absence of differentiated FDCs. J Exp Med 2014;211:643 51. Gebert A, Hach G. Vimentin antibodies stain membranous epithelial cells in the rabbit bronchusassociated lymphoid tissue (BALT). Histochemistry 1992;98:271 3. Kawamata N, Xu B, Nishijima H, Aoyama K, Kusumoto M, Takeuchi T, et al. Expression of endothelia and lymphocyte adhesion molecules in bronchus-associated lymphoid tissue (BALT) in adult human lung. Respir Res 2009;10:97. Shinoda K, Hirahara K, Iinuma T, Ichikawa T, Suzuki AS, Sugaya K, et al. Thy1 1 IL-7 1 lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation. Proc Natl Acad Sci USA 2016;113:E2842 2851. Baluk P, Adams A, Phillips K, Feng J, Hong YK, Brown MB, et al. Preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation. Am J Pathol 2014;184:1577 92. Muniz LR, Pacer ME, Lira SA, Furtado GC. A critical role for dendritic cells in the formation of lymphatic vessels within tertiary lymphoid structures. J Immunol 2011;187:828 34. Halle S, Dujardin HC, Bakocevic N, Fleige H, Danzer H, Willenzon S, et al. Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells. J Exp Med 2009;206:2593 601. Otsuki Y, Ito Y, Magari S. Lymphocyte subpopulations in high endothelial venules and lymphatic capillaries of bronchus-associated lymphoid tissue (BALT) in the rat. Am J Anat 1989;184:139 46. Xu B, Wagner N, Pham LN, Magno V, Shan Z, Butcher EC, et al. Lymphocyte homing to bronchusassociated lymphoid tissue (BALT) is mediated by L-selectin/PNAd, alpha4beta1 integrin/VCAM-1, and LFA-1 adhesion pathways. J Exp Med 2003;197:1255 67. Rangel-Moreno J, Moyron-Quiroz JE, Hartson L, Kusser K, Randall TD. Pulmonary expression of CXC chemokine ligand 13, CC chemokine ligand 19, and CC chemokine ligand 21 is essential for local

II. PRINCIPLES OF MUCOSAL VACCINE

53

REFERENCES

[251]

[252]

[253]

[254]

[255]

[256]

[257]

[258]

[259]

[260]

[261]

[262]

immunity to influenza. Proc Natl Acad Sci USA 2007;104:10577 82. Kocks JR, Davalos-Misslitz AC, Hintzen G, Ohl L, Forster R. Regulatory T cells interfere with the development of bronchus-associated lymphoid tissue. J Exp Med 2007;204:723 34. Foo SY, Zhang V, Lalwani A, Lynch JP, Zhuang A, Lam CE, et al. Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation. J Immunol 2015;194:4567 76. Kocks JR, Adler H, Danzer H, Hoffmann K, Jonigk D, Lehmann U, et al. Chemokine receptor CCR7 contributes to a rapid and efficient clearance of lytic murine gamma-herpes virus 68 from the lung, whereas bronchus-associated lymphoid tissue harbors virus during latency. J Immunol 2009;182:6861 9. Trujillo G, Hartigan AJ, Hogaboam CM. T regulatory cells and attenuated bleomycin-induced fibrosis in lungs of CCR7-/- mice. Fibrogenesis Tissue Repair 2010;3:18. de Kleer IM, Kool M, de Bruijn MJ, Willart M, van Moorleghem J, Schuijs MJ, et al. Perinatal activation of the interleukin-33 pathway promotes type 2 immunity in the developing lung. Immunity 2016;45:1285 98. Das A, Kole L, Wang L, Barrios R, Moorthy B, Jaiswal AK. BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2. Free Radic Biol Med 2006;40:1843 56. Bashir A, Shah NN, Hazari YM, Habib M, Bashir S, Hilal N, et al. Novel variants of SERPIN1A gene: interplay between alpha1-antitrypsin deficiency and chronic obstructive pulmonary disease. Respir Med 2016;117:139 49. Polverino E, Rosales-Mayor E, Dale GE, Dembowsky K, Torres A. The role of neutrophil elastase inhibitors in lung diseases. Chest 2017;152:249 62. Solleti SK, Srisuma S, Bhattacharya S, Rangel-Moreno J, Bijli KM, Randall TD, et al. Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation. FASEB J 2016;30:2615 26. Jones GW, Jones SA. Ectopic lymphoid follicles: inducible centres for generating antigen-specific immune responses within tissues. Immunology 2016;147:141 51. Eddens T, Elsegeiny W, Garcia-Hernadez ML, Castillo P, Trevejo-Nunez G, Serody K, et al. Pneumocystis-driven inducible bronchus-associated lymphoid tissue formation requires Th2 and Th17 immunity. Cell Rep 2017;18:3078 90. Kuroda E, Ozasa K, Temizoz B, Ohata K, Koo CX, Kanuma T, et al. Inhaled fine particles induce alveolar

[263] [264]

[265]

[266]

[267]

[268]

[269]

[270]

[271]

[272]

[273]

[274]

macrophage death and interleukin-1alpha release to promote inducible bronchus-associated lymphoid tissue formation. Immunity 2016;45:1299 310. Gaffen SL. Structure and signalling in the IL-17 receptor family. Nat Rev Immunol 2009;9:556 67. Ho AW, Gaffen SL. IL-17RC: a partner in IL-17 signaling and beyond. Semin Immunopathol 2010;32:33 42. GeurtsvanKessel CH, Willart MA, Bergen IM, van Rijt LS, Muskens F, Elewaut D, et al. Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus-infected mice. J Exp Med 2009;206:2339 49. Martinet L, Filleron T, Le Guellec S, Rochaix P, Garrido I, Girard JP. High endothelial venule blood vessels for tumor-infiltrating lymphocytes are associated with lymphotoxin beta-producing dendritic cells in human breast cancer. J Immunol 2013;191:2001 8. Martinet L, Girard JP. Regulation of tumor-associated high-endothelial venules by dendritic cells: a new opportunity to promote lymphocyte infiltration into breast cancer? Oncoimmunology 2013;2:e26470. Moussion C, Girard JP. Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules. Nature 2011;479:542 6. Matsumoto M. Role of TNF ligand and receptor family in the lymphoid organogenesis defined by gene targeting. J Med Invest 1999;46:141 50. Moyron-Quiroz JE, Rangel-Moreno J, Hartson L, Kusser K, Tighe MP, Klonowski KD, et al. Persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs. Immunity 2006;25:643 54. Chiavolini D, Rangel-Moreno J, Berg G, Christian K, Oliveira-Nascimento L, Weir S, et al. Bronchusassociated lymphoid tissue (BALT) and survival in a vaccine mouse model of tularemia. PLoS One 2010;5: e11156. Khader SA, Guglani L, Rangel-Moreno J, Gopal R, Junecko BA, Fountain JJ, et al. IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung. J Immunol 2011;187:5402 7. Richert LE, Harmsen AL, Rynda-Apple A, Wiley JA, Servid AE, Douglas T, et al. Inducible bronchusassociated lymphoid tissue (iBALT) synergizes with local lymph nodes during antiviral CD4 1 T cell responses. Lymphat Res Biol 2013;11:196 202. Saif LJ. Mucosal immunity: an overview and studies of enteric and respiratory coronavirus infections in a swine model of enteric disease. Vet Immunol Immunopathol 1996;54:163 9.

II. PRINCIPLES OF MUCOSAL VACCINE

54

2. ANATOMICAL UNIQUENESS OF THE MUCOSAL IMMUNE SYSTEM

[275] Foo SY, Phipps S. Regulation of inducible BALT formation and contribution to immunity and pathology. Mucosal Immunol 2010;3:537 44. [276] John-Schuster G, Hager K, Conlon TM, Irmler M, Beckers J, Eickelberg O, et al. Cigarette smokeinduced iBALT mediates macrophage activation in a B cell-dependent manner in COPD. Am J Physiol Lung Cell Mol Physiol 2014;307:L692 706. [277] McDonough JE, Yuan R, Suzuki M, Seyednejad N, Elliott WM, Sanchez PG, et al. Small-airway

obstruction and emphysema in chronic obstructive pulmonary disease. N Engl J Med 2011;365:1567 75. [278] Jupelli M, Shimada K, Chiba N, Slepenkin A, Alsabeh R, Jones HD, et al. Chlamydia pneumoniae infection in mice induces chronic lung inflammation, iBALT formation, and fibrosis. PLoS One 2013;8:e77447. [279] Richmond I, Pritchard GE, Ashcroft T, Avery A, Corris PA, Walters EH. Bronchus associated lymphoid tissue (BALT) in human lung: its distribution in smokers and non-smokers. Thorax 1993;48:1130 4.

II. PRINCIPLES OF MUCOSAL VACCINE

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Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells Brian L. Kelsall Mucosal Immunobiology Section, Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States

I. INTRODUCTION

immune defense mechanisms that eliminate organisms that breach the surface and prevent their spread to distant sites. Furthermore, active immune mechanisms exist to suppress potentially harmful responses to enviromental and food antigens, as well as certain symbiotic bacteria. Thus the principal challenge for the development of vaccines against mucosal pathogens is the formulation of strategies for effective uptake of vaccine antigens across this protective mucosal barrier that also result in appropriate adaptive immunity that blocks pathogen entry and/or clears invading organisms. In this chapter, we review mechanisms that are known to be involved in the uptake of dietary and microbial antigens, with the perspective of developing new strategies for vaccine delivery, and we highlight areas that need further investigation. We will first briefly discuss the components of the mucosal immune system and the intestinal barrier. We

Mucosal tissues, including those of the the nasophayrngeal, pulmonary, and gastrointestinal tracts, represent the largest surface area of the human body that is exposed to the external environment. Mucosal tissues are major sites for the the entry of nutrients, microbes, and environmental antigens contained in the food and water that we ingest and the air that we breath. They also contain vast collections of immune cells, more than all other nonmucosal tissues combined, which together with nonimmune cells and tissues have the complex tasks of combating infections while preventing harmful immune responses to environmental antigens and symbiotic microbiota [1 4]. This is accomplished by the formation of a barrier to penetration by pathogens as well as the contact of symbiotic bacteria with the epithelium, together with appropriate innate and adaptive

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00003-1

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will then divide our discussion into sections addressing antigen uptake across organized lymphoid tissues, such as Peyer’s patches and isolated lymphoid follicles in the gut, and across the nonlymphoid tissue-associated epithelia. Finally, we will discuss a number of past and current attempts to exploit some of these uptake mechanisms for vaccination. Because of limited data on many mucosal tissues, we will focus our comments on the gastrointestinal tract.

II. BASIC COMPONENTS OF THE MUCOSAL BARRIER AND LYMPHOID TISSUES Mucosal tissues have been traditionally divided into three major components: the mucosa-associated lymphoid tissues (MALT), such as Peyer’s patches; the nonlymphoid tissue-associated tissues, such as the absorptive intestinal villus; and the draining lymph nodes [3]. Tissues lacking MALT, such as the oral and vaginal mucosa, are called type 1 mucosal tissues, and are covered by stratified squamous epithelial cells. Mucosal tissues that have MALT are called type 2 mucosal tissues and are covered by columnar epithelial cells, goblet cells, M cells, tuft cells, other enteroendocrine cells, and stem cells [5]. Draining lymph nodes are present in both type 1 and type 2 mucosal tissues. They are the only major site for the induction of adaptive immune responses for type 1 tissues, whereas somewhat different immune responses are generated in MALT and draining lymph nodes for type 2 tissues [5]. Type 2 mucosal barriers differ from more secure type 1 mucosal barriers, as in type 2 mucosal tissues, there is a need to adsorb nutrients and fluids but exclude the vast majority of exogenous antigens and bacteria [5,6]. At the same time, selective uptake of bacterial and exogenous antigens as well as bacterial metabolites continuously occurs to allow for the

development and maintenance of immune tissues and for the generation of factors that in turn protect against pathogen invasion and maintain homeostasis with our commensal bacteria. Central to mucosal barrier function is a single-cell layer of epithelial cells that separates the mucosal lumen from the underlying lamina propria (LP). In the intestine, epithelial cells develop from stem cells in the crypts into five main epithelial cell types: columnar enterocytes, goblet cells, enteroendocrine cells, tuft cells, and Paneth cells [7]. Furthermore, specialized epithelial cells, called microfold (M) cells, develop over MALT and in certain conditions within the non-MALT-associated epithelium that have unique capacities to take up large macromolecules, viruses, and bacteria [8] (Chapter 28: M Cell-Targeted Vaccines). Epithelial cells form regulated adhesions between each other via a complex interaction of proteins that include tight junctions (claudins, occludins, junctional adhesion molecule A, and tricellulin), adherens junctions (E-cadherins), and desmosomes (see Ref. [9]). In addition to the barrier formed by epithelial cells and their junctional connections, mucus is produced in very high quantities, particularly in the intestine, and is essential for controlling antigen uptake. Mucin glycoproteins comprise three primary families: secreted gel-forming mucins, cell surface mucins, and secreted non-gel-forming mucins [10]. Each mucosal tissue has unique members of these families; however, all mucosal surfaces are covered in a layer of highly viscous mucus made up of secreted gel-forming mucus produced by cells in the epithelium (e.g., goblet cells) or in submucous glands. This layer forms a highly impervious layer that blocks uptake by trapping of microbiota, provides lubrication, and acts as a matrix for antimicrobial factors, including antimicrobial proteins and peptides, immunoglobulins, and enzymes [10,11]. Cell surface membrane anchored mucins form the

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glycocalyx, a dense layer extending from the surface toward the lumen that interacts with secreted mucins to form the mucus barrier [10]. This glycocalyx varies in depth between mucosal tissues and locations; for example, the microvillus tip is thick in comparison to the lateral surface, and the surface over M cells is much less than that over adjacent enterocytes [12,13]. Furthermore, glycosylation patterns of mucin glycoproteins vary between tissues, cell types, developmental stage, host genetics, and environmental factors such as the presence of inflammation, based on the expression of different glycosyl transferases [10,14,15]. Terminal glycosylation residues define histo-bloodgroup antigens, such as A, B, H, and Lewis antigens, as well as secretor status, which can determine susceptibility to specific pathogens, such as Norwalk virus and Helicobacter pylori [14,16,17]. Importantly, in the intestine, surface and gel-forming mucin glycoproteins both turn over quickly (over hours, not days) and are both expressed constitutively and induced by a variety of environmental factors, including inflammatory cytokines such as interleukin 1 beta (IL-1β), IL-4, IL-9, IL-13, interferons, tumor necrosis factor alpha (TNFα), nitric oxide, microbial metabolites including shortchain fatty acids, neutrophil elastase, and direct activation by both Gram-positive and Gram-negative bacterial products including lipopolysaccharide [10,14,18]. Owing to a variety of factors, the tertiary structure of interacting mucin glycoproteins is also a factor in excluding bacteria and other antigens from contact with epithelial surfaces [10,15]. This has been described for the layers of MUC2, the major secreted mucin glycoprotein in the intestine, where it forms tentlike structures resulting in a very impenetrable discrete barrier at the epithelial surface of the colon but not the small intestine, where a more continuous but progressively more hydrated and less viscous layer

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is formed from the epithelial cell surface to the lumen [15]. The glycosylation of epithelial cells, especially α(1,2)-fucose expressed on the apical side of epithelial cells, is one of the primary interfaces for the host microbe interaction [19 21]. Epithelial α(1,2)-fucosylation is mediated by fucosyltransferase 2 (FUT2), and dysregulation of FUT2 gene expression has been shown to associate with various human disorders, including infection (e.g., reovirus) and chronic inflammatory diseases (e.g., inflammatory bowel diseases) [22]. Emerging evidence suggests that mucosal innate and acquired immune cells, especially type 3 innate lymphoid cells (ILC3s) and CD41 T cells, are critical modulators of the FUT2-mediated epithelial glycosylation system [22]. In response to commensal bacteria and pathogenic bacteria, IL-22producing ILC3s induce FUT2 for epithelial α(1,2)-fucosylation [19]. On the other hand, IL-10-producing T cells, a family of regulatory T cells, are engaged in the downregulation of epithelial α(1,2)-fucosylation [23]. The mucosal innate and acquired immune system cooperatively orchestrate FUT2-mediated gut epithelial α(1,2)-fucosylation for the creation of a physiologically and immunologically important homeostatic environment in the intestine. It will be interesting to further examine whether the epithelial glycosylation system is involved in the sampling of luminal antigens. In addition to forming a physical barrier to the entry of bacteria and antigens, the mucus layer also acts as a matrix for antimicrobial enzymes, such as lysozyme and lactoferrin, antimicrobial peptides and proteins, such as defensins, cathelicidins, and regenerating islet-derived proteins 3 (Reg3) beta and gamma, made by Paneth and other epithelial cell populations; and IgA, which through its hydrophilic Fc tail strongly interacts with mucin side chains to trap antigens and microbes in the mucus coat, allowing clearance by peristaltic or mucociliary activity [24,25].

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Limitations of antigen penetration through the mucus coat are important to considerations for the design of antigens and vectors to use for oral vaccination. In this regard, the thin mucus coat over M cells has been considered a particularly useful site for vaccine targeting (see Chapter 28: M Cell-Targeted Vaccines). Furthermore, despite these formidable defenses of the mucus layer that exclude the majority of commensal microbes, certain commensal bacteria and symbionts, and all pathogens have evolved the ability to penetrate and/ or actually thrive in the mucus layers. These include commensal bacteria that express flagella to help them migrate through mucus, produce mucin-degrading enzymes, or use mucin glycoproteins as energy sources, including those that actually take advantage of changes induced in cell surface mucins during inflammation, such as Escherichia coli strains that sense and metabolize inflammationinduced fucose generated from fucosylated mucins by intestinal microbiota [21,26,27]. In addition, certain bacteria such as recently shown for Bacteroides fragilis actually depend on specific IgA for their colonization and survival in the mucus layer in the intestine [28]. Commensals that have evolved mechanisms to penetrate mucus and live in close proximity to the epithelium or in direct contact with it, such as Clostridium spp., and segmented filamentous bacteria, respectively, are those that have the most effects on steady-state numbers of effector and regulatory T cells in the intestinal LP. This is thought to be due to epithelial cell effects of bacterial metabolites, responses to the contact per se, other mechanisms resulting in subsequent effects on underlying LP dendritic cells (DCs) or other cells via secreted cytokines, and other factors [29 31] (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity). In addition to mucus degradation and the use of flagella, true pathogens may also produce enzymes that cleave IgA and disable

epithelial cell tight junctions and use pathogenic secretion systems that allow bacteria to inject bacterial mediators into cells to affect epithelial cell apoptosis and turnover, allowing them to enter the host, often in conjunction with epithelial cell destruction and disruption of the intestinal barrier but also, in some cases, preservation of epithelial cell integrity when it is an advantage for the microbe [32].

III. ANTIGEN UPTAKE ACROSS THE VILLUS EPITHELIUM Although most dietary proteins are digested by proteases in the stomach and upper small intestine, immunologically intact molecules are present throughout the intestine, either as nondegraded food antigens or as microbial products, and despite the presence of the formidable barrier mentioned above, multiple pathways by which these substances and microbes from the intestinal lumen can traverse the intestinal epithelium have been identified (see Ref. [33]). These include paracellular transport (“leak”) across epithelial cell junctions, active endocytosis and transcellular transport across epithelial cells, in particular goblet cells, transport of antibody-bound antigens by Fc receptors, and active uptake by populations of macrophages and DCs that extend dendrites or migrate into the epithelial layers [9,34 38]. In addition, pathogens have developed sophisticated mechanisms to invade and destroy epithelial barriers to allow for invasion and spread to systemic tissues [6,39 41].

A. Paracellular and Transcellular Transport Across Villus Epithelial Cells Epithelial cohesion and polarity are maintained by the apical junctional complex, which is composed of tight and adherens junctions, and by the subjacent desmosomes. Transport

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of molecules across these adhesions is largely controlled at the level of tight junctions, which are rate limiting for the uptake of luminal molecules, owing to their small pore size compared to deeper adhesions [9,34,36]. Recent studies using live confocal imaging after oral gavage of fluorescent 10-kD dextran to normal mice have highlighted that paracellular leakage of molecules in the small intestine does occur and is transported to the draining lymphatics but was found not to be avidly taken up by underlying DCs in the LP [35]. Epithelial cell junctions are altered by inflammatory signals, including the inflammatory cytokines interferon gamma (IFNγ), TNF-α, IL-1β, and IL-6, as well as by direct activation of NFκB by bacterial products, as seen with Listeria monocytogenes [39]. Activation of NFκB by bacterial products via interactions with heat-shock proteins, toll-like receptors (TLRs), or NOD2 or by inflammatory cytokines released by epithelial cells or other inflammatory cells results in activation of myosin light-chain kinase and subsequent redistribution or internalization of junctional proteins claudin, occludins, and E-cadherin, which can increase permeability. Increased permeability can facilitate uptake of bacterial pathogens through exposure of ligands such as E-cadherins [39] and likely results in enhanced uptake of soluble food and bacterial products as well. In addition to paracellular “leakage” of bacterial and dietary antigens across junctions between columnar epithelial cells, which is clearly enhanced by inflammatory compounds and bacteria, transcellular transport of antigens and bacteria across epithelial cells can also occur. This was first indicated in studies showing limited uptake of horseradish peroxidase by intestinal epithelial cells in rats by active endocytosis [42]. In addition, certain intestinal pathogens such as adherent-invasive E. coli can induce micropinocytosis of junctional proteins together with adherent bacteria for transepithelial cell transport into the LP (see Ref. [40]).

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Furthermore, it was recently shown that small nanoparticles (in the range of 20 40 nm) and bacterial debris may gain access to the LP through an active phagocytic process in epithelial cells in noninfected mice, whereas larger nanoparticles were preferentially taken up across the follicle-associated epithelium over Peyer’s patches [43]. The role of this direct transcellular uptake pathway across villus epithelial has received little attention in recent studies, as it has been considered primarily a mechanism of nutrient adsorption [37]. In contrast, recent studies have suggested a significant role for constitutive transport of antigens from the intestinal lumen to the LP that occurs by endocytosis and transport across intestinal goblet cells. While previously identified, this pathway has recently gained significant attention, as it was shown that it can result in adaptive immune responses, including the induction of oral tolerance (see review Ref. [37]). These so-called goblet-cell-associated passages (GAPs) were initially identified by microscopy following intraluminal administration of fluorescent dextran, bovine serum albumin, and ovalbumin in both the small intestine [35] and the colon [44]. GAPs are also associated with goblet cells at other mucosal surfaces and have been shown to be present in the human jejunum, but they have been studied primarily in the mouse intestine. GAPs were shown to be columns of antigen that are actively passed to underlying CD1031 DCs [35]. GAPs are largely present in the small intestine and the distal but not proximal colon, and they develop in these different regions at different stages of life. GAPs are induced by acetylcholine acting on goblet-cell-expressed muscarinic acetylcholine receptor 4, which also induces compound but not primary mucus granule exocytosis, and are inhibited by epidermal growth factor receptor (EGFR) signaling [45]. In the murine small intestine, GAPs are formed just before weaning at day 18 of life, are present through adulthood, and are not

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dependent on the presence of commensal bacteria. Interestingly, Salmonella typhimurium infection resulted in both GAP-dependent bacterial uptake early in infection and the inhibition of GAP formation at later time points secondary to IL-1β signaling, resulting in less dissemination of bacteria and dietary antigens to the mesenteric lymph nodes (MLN), as did elimination of goblet cells, suggesting that in addition to dietary antigens, intestinal pathogens may use GAPs for bacterial entry [38]. GAPs are expressed in the proximal colon for only a short window, from day 10 of life to day 21, near weaning, and are inhibited by intestinal bacteria signaling through TLRs and EGFR pathways. Following treatment with antibiotics, proximal colon GAPs were not inhibited and allowed for pathogenic immune responses to commensal bacteria and pathogens, resulting in intestinal inflammation [38,44,46]. In the distal colon, GAPs are present post weaning and are not inhibited by commensal bacteria. It has been speculated that the suppression of GAPs after weaning in the proximal colon, where the mucus layer may not be as protective, is critical for limiting harmful immune responses to intestinal bacteria, but exposure during the critical period of GAP formation during weaning may be harmful. The distal colon may be protected from exposure throughout life, owing to a thicker mucus coat; however, the functions of persistent GAPs at this site are not yet known [37]. Additional transcellular transport mechanisms have been demonstrated for antibody-bound antigens or immune complexes. For these bound antigens, transcellular transport across epithelial cells can occur by Fc-receptor-mediated mechanisms [47,48]. The polymeric immunoglobulin receptor (PIgR) is well known for its role in binding and transporting polymeric IgA and immune complexes from the LP to the luminal surface. This transport is unidirectional, and IgA is secreted in large amounts into the intestinal lumen. However, specific IgA can also be

directed to viral-containing endosomes of infected epithelial cells, resulting in viral neuralization. Transport by PIgR is also highly regulated, as its expression is strongly induced by cytokines such as IFNγ, IL-4, and TNF-α, pathogenic stimuli that activate NFkB via TLR signaling, and hormones including estrogens, androgens, glucocorticoids, and prolactin [47]. In contrast to PIgR, the neonatal Fc receptor (FcRn) is an MHC I-like molecule associated with β2 microglobulin that acts to transport immunoglobulin G (IgG) and associated immune complexes in a bidirectional manner (see Ref. [48]). In rodents, FcRn is expressed on the epithelium of the intestine only in neonates before weaning (thus its name), but in humans, it is continually expressed into adulthood, being present at higher levels in the proximal small intestine, where the acidic environment facilitates transport to more basic basolateral epithelial surfaces [49]. The regulation of FcRn expression is not well understood; however, both corticosteroids and thyroxin have been shown to suppress expression in the neonatal rat [50]. FcRn facilitates transport of IgG and immune complexes from maternal milk to an infant’s bloodstream across the villus epithelium and to transport IgG or immune complexes into the intestinal lumen; the former is shown to be helpful for the control of C. rodentium infection [51]. It is also expressed by the lung and mammary gland epithelium, acting to transport IgG transport into maternal milk and pulmonary secretions, respectively. In addition, it is expressed highly in the placenta, facilitating IgG transfer from mother to infant, and has a role in preserving the half-life of circulating IgG in mammals, possibly via recycling trough nondegrading endosomes. The role of FcRn in antigen uptake in the intestine has been explored in mice with genetically induced constitutive expression of human FcRn on intestinal epithelial cells [51 53]. In such mice, oral administration of IgG antigen immune complexes was shown to be

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transported to underlying DCs, that migrate to LNs, where they cross-present the associated antigen (e.g., ovalbumin) to induce CD81 T cell responses. Furthermore, IgG antigen immune complexes present in the milk of systemically immunized mother mice can be transferred to neonatal mice via FcRn to induce CD41 FoxP31 regulatory T cells that can prevent subsequent allergic responses to the complexed antigen when weaned mice are subject to allergic sensitization protocols [54,55]. This effect was also dependent on expression of FcRn on CD11c1 cells, including DCs and macrophages [56]. Because FcRn also promotes cross-presentation of antigen IgG complexes by DCs to CD81 T cells [57] and FcRn is expressed highly by macrophages, it may facilitate one response over another depending on the capturing cell type or additional cosignals, including those from additional FcRs on DCs or local macrophages [58]. Suffice it say that immunization of mothers may provide a novel method to prevent allergies in their offspring. Additionally, FcRn contributes to CD41 and CD81 T cell activation in response to vaccination with IgGopsonized and inactivated Francisella tularensis [59]. Furthermore, FcRn-mediated protection from infection in response to IgG-complexed microbial antigens also extends to viral infections, including herpes simplex virus and HIV [60,61]. Finally, specialized antigen-sampling M cells have been described to be present, not only in gut-associated lymphoid tissues but also on intestinal epithelial cells [62], which can be induced by systemic administration of receptor activator of nuclear factor kappa-B ligand (RANKL/TNFSF11), a factor that is important for their differentiation [63]. Importantly, these villous M cells were shown to bind bacteria [62] and RANKL administration induced M cell differentiation on all small intestinal villi, which were able to avidly take up Salmonella enterica serovar Typhimurium and Yesinia enterocolitica as well as 200-nm fluorescent microbeads [63].

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Furthermore, villous M cells may be induced by infection with pathogenic bacteria [64] or stimulation with bacterial toxin, such as cholera toxin [65].

B. Myeloid Cell Uptake of Antigens and Bacteria in the Lamina Propria Multiple populations of macrophages and DCs are present in the intestinal LP that are derived from unique bone marrow precursors and have unique surface markers, anatomical locations, and functions (see Ref. [66]). Both macrophages and DCs have been implicated in direct antigen uptake across the intestinal epithelium [67 71]. In the mouse, circulating Ly6Chi monocytes continuously migrate to the LP, where they differentiate into mature macrophages marked by the progressive loss of Ly6C and acquisition of CD11c, MHC II, CX3CR1, F4/80, CD64 (FcγR1), and MerTK, dependent on local factors including TGFβ, IL-10, and CSF2 (GMCSF) [66]. Furthermore, mature macrophages can be distinguished by their level of expression of CD11c (CD11chi and CD11clo) [72]. CX3CR1hi CD641 macrophages are highly represented in the LP, where they account for 70% 80% of MHCIIhi CD11c1 cells in the small and large intestines [72,73]. They are long-lived (14 30 days), are highly vacuolated and phagocytic, express high levels of lysozyme and cathepsins, are poorly capable of presenting soluble antigens to CD41 and CD81 T cells, and express low levels of inflammatory cytokines but higher levels of anti-inflammatory factors such as IL-10 and IL1RA when stimulated in vitro [72]. In contrast to monocyte-derived cells, circulating pre-DCs give rise to three primary CD64 2 DC populations that depend on unique transcription factors for their differentitation and can be distinguished by their expression of CD103, CD11b, XCR1, and SIRPα (see Ref. [74]). In mice, CD1031 CD11b1 (SIRPα1, XCR12)

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and CD1031 CD11b2 (SIRPα2, XCR11) DCs account for the majority (85 90%) of DCs, while CD1032 CD11b1 cells account for 10% 15% of DCs in the small intestine LP. CD1031 CD11b1 cells are more prevalent than CD1031 CD11b2 in the small intestine, while the opposite is true for the colon [75]. These populations have also been shown to be present in other species, including humans, the definitions of which depend on the expression of XCR1 and SIRPa, which have emerged as more reliable markers than CD11b and CD8 across species. All three DC populations have high turnover rates (7 14 days), are poorly phagocytic, express low levels of digestive enzymes, have a high capacity to stimulate naı¨ve T cells, and express different levels of cytokines and factors that influence their differential ability to drive specific T cell responses [66,72,76 79]. In in vitro differentiation assays and in some in vivo models, CD1031 CD11b1 cells can drive the de novo differention of Th17 cells, CD1031 CD11b2 DCs are highly capable of cross-presenting anntigens to CD81 T cells, and CD1032 CD11b1 cells have been shown to preferentially drive Th1 cell differentiation, while all three have the capcity to induce Treg cell differentiaion ex vivo. However, CD1031 DCs likely play a primary role in vivo because they are the predominant DCs that migrate to the MLNs, at least under steady-state conditions [66,79]. CD1031 DCs and CX3CR11 CD641 macrophages can be found in different locations in the LP, and recent studies have elucidated possible unique mechanisms of antigen uptake by these cell types. CX3CR11 macrophages are associated with the epithelium and can extend protrusions directly into the lumen for bacterial and antigen sampling. In now classic studies, Rescigno and colleagues demonstrated, using an in vitro cell system, that bone-marrowderived DCs (likely more akin to macrophages in vivo) can extend dendrites between Caco-2 epithelial cells in monolayers to sample antigens in a well-orchestrated process that

involves the coordinated expression of the tight-junction proteins occludin, claudin-1, and junctional adhesion molecule (JAM) by the DCs that allow for extended dendrites to form tightjunction-like connections with epithelial cells that prevent loss of barrier function [71]. The authors then showed that the process occurs in vivo in isolated small intestinal loops, in which probing transepithelial dendrites (TEDs) were found in contact with intestinal bacteria [71]. In addition, studies by others showed that TEDs were present in the proximal jejunum and distal ilium, the latter induced in response to pathogenic and nonpathogenic S. typhimurium infection and by orally administered TLR-2, -4, and -9 ligands, and were capable of capturing both invasive and noninvasive S. typhimurium and nonpathogenic E. coli [67,69]. TED induction was dependent on MyD88 signaling on nonhematopoietic cells, were generated without the disruption of barrier function, and were absent in mice deficient for CX3CR1 or CXCL1 that also displayed defects in the internalization of noninvasive bacteria [67,80], suggesting that epithelial activation by bacteria drives TED formation, possibly by the elaboration of CXCL1. These findings were consistent with the observation that depletion of CD11c1 cells reduced bacterial colonization in the colonic LP after oral administration of noninvasive S. typhimurium [81]. Interestingly, TEDs were not found in the colon, suggesting a unique mechanism of small bowel antigen uptake. Furthermore, CX3CR11 cells in the small intestine were shown in one study to be superior to CD1031 DCs in the uptake of soluble antigens, with active accumulation of orally administered ovalbumin into vacuoles in CX3CR11 cells in the LP [69]. Whether these cells represent true macrophages or CD1032 CD11b1 CX3CR1int DCs is not yet clear. In addition, their true function in soluble antigen uptake is also not yet clear, as mice deficient in CX3CR1 have shown normal induction of oral

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tolerance to soluble antigens in some studies [82,83] but not others [84]. Finally, CX3CR11 phagocytes were found to be essential in sampling of noninvasive S. typhimurium [81] and have been shown to be capable of taking up soluble antigens in the absence of TEDs [85], suggesting that these cells may capture soluble antigens transported to the LP by other routes (e.g., across goblet cells or via transcellular or paracellular transport across epithelial cells). In contrast to CX3CR11 cells, CD1031 LP DCs have been found more deeply within the LP or more superficially in the epithelium [73,85], and similar to intestinal macrophages, LP CD1031 CD11b1 DCs capture soluble antigens and bacteria given orally but also have the capacity to migrate to the MLN, where they activate T cells [85]. Furthermore, CD1031 DCs were also found to carry bacteria as well as fragments of intestinal epithelial cells, possibly in the form of apoptotic bodies, to the MLN [73,86], suggesting that luminal antigens taken up by or bound to epithelial cells may be carried to the MLN together with epithelial cell fragments by CD1031 DCs. How LP CD1031 DCs acquire bacterial, soluble, and epithelial-associated antigens has been addressed in several studies, suggesting that this can occur by several mechanisms. First, in mice expressing both CX3CR1-egfp and CD11cyfp, small numbers of highly motile CX3CR12 CD11c1 CD1031 DCs were found to migrate into the epithelium above the basement membrane where they moved between epithelial cells, before returning to the LP [68]. Within the epithelium, these cells captured soluble Alexa 594-labled ovalbumin, but not as efficiently as CX3CR11 cells in the LP. Furthermore, CD1031 DCs in the LP actively migrated into the epithelium following Salmonella exposure, which depended on MyD88 and/or TRIF signaling, where they sent TEDs into the lumen to capture invasive and noninvasive S. typhimurium. Furthermore, similar to CX3CR11 cells, CD1031 DCs expressed the tight-junction proteins Claudin 4 and ZO-2, which may allow

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for tight-junction formation with epithelial cells [68]. Second, CD11c1 CD1031 CX3CR12 DCs were found to directly capture antigens transported by GAPs. Antigen capture by CD1031 DCs occurred throughout the length of the small intestine, was limited to small molecules and not intact bacteria or particulates, and appeared to be a major pathway for the uptake of soluble proteins, as LP CD1031 DCs from mice fed antigens without goblet cells were not able to stimulate T cells ex vivo and soluble antigen uptake by TEDs was not observed in these studies [35]. These data are also consistent with studies showing that uptake of labeled ovalbumin by CD1031 LP DCs is intact in CX3CR1-deficient mice lacking TEDs [85]. Third, it was recently shown that CX3CR11 cells in the small intestine, in particular in the duodenum, acquired ovalbumin in ligated intestinal loops and was able to transfer ovalbumin to CD1031 cells that was dependent on connexin-43, a major component of the gap junctions that were formed between the CX3CR11 cells and CD1031 DC. Furthermore, oral tolerance to soluble ovalbumin was ablated in connexin-43- or CX3CR1-deficient mice [84].

C. Summary and Future Perspectives In summary, antigen uptake across the absorptive epithelium is normally controlled by multiple mechanisms, including the presence of an extensive mucus layer and tight junctions that form between epithelial cells, largely function to limit systemic antigen exposure, and protect against microbial invasion. For this reason, efforts to develop new oral vaccine formulations have largely focused on antigen or attenuated pathogen delivery to gut-associated lymphoid tissues (e.g., Peyer’s patches), where these barriers are less of an issue, or to alternative sites such as the airway mucosa and nasopharynx-associated lymphoid tissues. Indeed, the poor uptake of nonliving vaccines

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has severely limited this approach to a myriad of soluble and microparticle vaccines that require huge doses of antigens owing to inefficient uptake, although new plant-based approaches can overcome this obstacle by providing sufficient and inexpensive amounts of immunogenic antigens together with adjuvants (Chapter 20: Plant-Based Mucosal Vaccine Delivery Systems). Regardless of these limitations, both soluble and microbial antigens as well as microbes are selectively and actively sampled across the vast epithelial surfaces not associated with organized lymphoid tissues under steady-state conditions by the mechanisms described in this chapter and likely play a role in immunity to some current vaccines as well as offering new opportunities for future vaccine development. In particular, FcRn targeting has been demonstrated to be a viable strategy for vaccine delivery (see Refs. [87,88]). FcRn is expressed not only by intestinal and lung epithelial cells [89], but also by DCs, monocytes, macrophages, and B cells [90,91], where it functions in antigen uptake, trafficking to endosomes, and presentation via MHC I (in cooperation with FcRγ) and MHC II pathways to CD81 and CD41 T cells, respectively, thus mediating a coordinated T

cell response [57,60,87]. This strategy was used to develop vaccines against HSV-2 and HIV, using HSV glycoprotein D or HIV-gag Fc-fusion proteins, administered vaginally or nasally, respectively [60,92]. Whether this strategy will work for orally administered vaccines has not yet been shown; however, as was mentioned above, antigens have been delivered across intestinal and lung epithelial cells following oral or nasal administration of immune complexes, resulting in mucosal and systemic immune responses. An additional challenge will be to focus these responses toward effective immunity and away from the induction of tolerance. An additional opportunity for vaccine development may be the targeting of GAPs for antigen uptake. Goblet cells are capable of transporting both soluble antigens and pathogens such as S. typhimurium [38] and L. monocytogenes [93], suggesting that targeting goblet cells with specific antigens or with modified proteins or microparticles may be exploited for mucosal vaccine delivery. This would, however, would entail a more detailed knowledge of mechanisms of antigen uptake, including the role of E-cadherin that has been implicated in Listeria

FIGURE 3.1 Mechanisms of antigen transport by the cells of the villus epithelium. Shown are the various mechanisms that have been described together with the antigens and microbes that access the pathways that are discussed in the text, as well as factors that have been shown to influence their regulation. See text for further details.

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uptake [93] as well as overcoming the problems with antigen dose due to mucus trapping and degradation. In this regard, it may theoretically be possible to engineer nonpathogenic microbes or even soluble proteins with bacterial ligands

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from these pathogens to improve goblet cell targeting. It should also be mentioned that studies showing effectiveness of nanoparticle or plant-based vaccines, such as those using

FIGURE 3.2 Antigen uptake by populations of mucosal macrophages and dendritic cells. Three main populations of dendritic cells (DCs) and two populations of macrophages (MPs) are present in the intestinal LP that differentiate from different blood precursors, pre-DCs, and monocytes, as dictated by specific transcription factors (see Ref. [67]). Macrophage dendrites extend across the intestinal epithelium to capture bacteria and soluble antigens that are transferred to CD1031 DCs, the latter by mechanisms involving connexin-43. IgG immune complexes transfer antigens to CD1031 DCs, which is dependent on FcRn and FcγR expression by the DCs (see Ref. [87]). CD1031 DCs can be recruited into the intestinal epithelium, where they can capture bacteria and return to the LP. Soluble antigens may be also be captured by macrophages in the absence of TEDs and possibly from goblet cells (although these may also include CD1032CX3CR1intCD11b1 DCs), together with CD1031 DCs. CD1031 DCs are the primary cells carrying antigens to the MLN, and these are mainly the CD11b1 population. However three DC populations have been shown to be present in the LP and migrate to the MLN, where they may have unique capacities to influence T cell responses (see Ref. [66]). The cells responsible for antigen uptake from villous M cells in not clear, nor is it know which MP populations are primarily responsible for soluble of bacterial antigen uptake. It is clearer, however, that both populations of MPs are highly phagocytic, and LP MPs of one population and/or the other can transfer both bacteria and antigens to CD1031 DCs, which then carry these to the MLN. Under certain conditions, such as those induced by microbial dysbiosis or during experimental intestinal inflammation, MPs can also migrate to the MLNs, although the consequences of antigen presentation by these cells is less clear. The antigens and bacteria shown in the figure are some of those that have been studied experimentally and discussed in this chapter. See legend of Fig. 3.1 for details.

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recombinant rice [94 96], targeting M cells, in fact, be partially mediated or enhanced by epithelial cell uptake as precise sites of targeting for many of these approaches has never been proven, and M cells capable of transporting microorganisms has been demonstrated in the nonfollicular epithelium [64,65]. In addition, studies mentioned above have shown that particularly small nanoparticles (20 40 nm) can be phagocytosed and transported across intestinal epithelial cells, while larger particles targeted Peyer’s patch M cells [43], suggesting the possibility of using different-sized nanoparticles to deliver antigens to both Peyer’s patches and nonepithelial cells for enhanced vaccine efficacy. Finally, the ability to target DCs and macrophage antigen uptake mechanisms for improved mucosal vaccine efficacy should be considered. However, to date, little information is available addressing this possibility. One possibility is this regard may be to provide stimuli, such as TLR-2-ligands, that enhance transepithelial cell dendrite formation [67] to improve uptake of nonpathogenic bacterial-based vaccines. However, the consequences of this strategy are not yet clear, as the downstream effects of microbe macrophage and DC interactions by this uptake mechanism still need to be examined, and it requires further study. Furthermore, it would be of interest to identify the specific subtypes of cells and the expression of receptors responsible for antigen and pathogen uptake by TEDs and migratory DCs, as well as TLRs and other pattern-recognition receptors, to inform rational approaches to targeting these cell types for possible vaccine development (Figs. 3.1 and 3.2).

Acknowledgments This work was funded by the Intramural Research program of the NIAID, NIH, Bethesda, MD, United States.

References [1] Hill DA, Artis D. Intestinal bacteria and the regulation of immune cell homeostasis. Annu Rev Immunol 2010;28:623 67. [2] Kabat AM, Srinivasan N, Maloy KJ. Modulation of immune development and function by intestinal microbiota. Trends Immunol 2014;35(11):507 17. [3] Mayer L. Mucosal immunity. Immunol Rev 2005;206:5. [4] Pabst O, Mowat AM. Oral tolerance to food protein. Mucosal Immunol 2012;5(3):232 9. [5] Iwasaki A. Mucosal dendritic cells. Annu Rev Immunol 2007;25:381 418. [6] France MM, Turner JR. The mucosal barrier at a glance. J Cell Sci 2017;130(2):307 14. [7] Allaire JM, et al. The intestinal epithelium: central coordinator of mucosal immunity. Trends Immunol 2018;39(9):677 96. [8] Ohno H. Intestinal M cells. J Biochem 2016;159 (2):151 60. [9] Buckley A, Turner JR. Cell biology of tight junction barrier regulation and mucosal disease. Cold Spring Harb Perspect Biol 2018;10:1. [10] Linden SK, et al. Mucins in the mucosal barrier to infection. Mucosal Immunol 2008;1(3):183 97. [11] Pelaseyed T, et al. The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system. Immunol Rev 2014;260(1):8 20. [12] Owen RL. Sequential uptake of horseradish peroxidase by lymphoid follicle epithelium of Peyer’s patches in the normal unobstructed mouse intestine: an ultrastructural study. Gastroenterology 1977;72(3):440 51. [13] Nakamura Y, Kimura S, Hase K. M cell-dependent antigen uptake on follicle-associated epithelium for mucosal immune surveillance. Inflamm Regen 2018;38:15. [14] McGuckin MA, et al. Mucin dynamics and enteric pathogens. Nat Rev Microbiol 2011;9(4):265 78. [15] Johansson ME, Hansson GC. Immunological aspects of intestinal mucus and mucins. Nat Rev Immunol 2016;16(10):639 49. [16] Heggelund JE, et al. Histo-blood group antigens as mediators of infections. Curr Opin Struct Biol 2017;44:190 200. [17] Brandao de Mattos CC, de Mattos LC. Histo-blood group carbohydrates as facilitators for infection by Helicobacter pylori. Infect Genet Evol 2017;53:167 74. [18] Birchenough GM, et al. New developments in goblet cell mucus secretion and function. Mucosal Immunol 2015;8(4):712 19. [19] Goto Y, et al. Innate lymphoid cells regulate intestinal epithelial cell glycosylation. Science 2014;345 (6202):1254009.

II. PRINCIPLES OF MUCOSAL VACCINE

67

REFERENCES

[20] Pham TA, et al. Epithelial IL-22RA1-mediated fucosylation promotes intestinal colonization resistance to an opportunistic pathogen. Cell Host Microbe 2014;16 (4):504 16. [21] Pickard JM, et al. Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness. Nature 2014;514(7524):638 41. [22] Goto Y, Uematsu S, Kiyono H. Epithelial glycosylation in gut homeostasis and inflammation. Nat Immunol 2016;17(11):1244 51. [23] Goto Y, et al. IL-10-producing CD4(1) T cells negatively regulate fucosylation of epithelial cells in the gut. Sci Rep 2015;5:15918. [24] Mantis NJ, Rol N, Corthesy B. Secretory IgA’s complex roles in immunity and mucosal homeostasis in the gut. Mucosal Immunol 2011;4(6):603 11. [25] Dupont A, et al. Antimicrobial peptides and the enteric mucus layer act in concert to protect the intestinal mucosa. Gut Microbes 2014;5(6):761 5. [26] Pacheco AR, et al. Fucose sensing regulates bacterial intestinal colonization. Nature 2012;492(7427):113 17. [27] Pickard JM, Chervonsky AV. Intestinal fucose as a mediator of host-microbe symbiosis. J Immunol 2015;194(12):5588 93. [28] Donaldson GP, et al. Gut microbiota utilize immunoglobulin A for mucosal colonization. Science 2018;360 (6390):795 800. [29] Atarashi K, et al. Th17 cell induction by adhesion of microbes to intestinal epithelial cells. Cell 2015;163 (2):367 80. [30] Nagano Y, Itoh K, Honda K. The induction of Treg cells by gut-indigenous Clostridium. Curr Opin Immunol 2012;24(4):392 7. [31] Honda K, Littman DR. The microbiota in adaptive immune homeostasis and disease. Nature 2016;535 (7610):75 84. [32] Ashida H, et al. Bacteria and host interactions in the gut epithelial barrier. Nat Chem Biol 2012;8(1):36 45. [33] Schulz O, Pabst O. Antigen sampling in the small intestine. Trends Immunol 2013;34(4):155 61. [34] Adson A, et al. Quantitative approaches to delineate paracellular diffusion in cultured epithelial cell monolayers. J Pharm Sci 1994;83(11):1529 36. [35] McDole JR, et al. Goblet cells deliver luminal antigen to CD103 1 dendritic cells in the small intestine. Nature 2012;483(7389):345 9. [36] Menard S, Cerf-Bensussan N, Heyman M. Multiple facets of intestinal permeability and epithelial handling of dietary antigens. Mucosal Immunol 2010;3(3):247 59. [37] Knoop KA, Newberry RD. Goblet cells: multifaceted players in immunity at mucosal surfaces. Mucosal Immunol 2018;. [38] Kulkarni DH, et al. Goblet cell associated antigen passages are inhibited during Salmonella typhimur/cium

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49] [50]

[51]

[52]

[53]

infection to prevent pathogen dissemination and limit responses to dietary antigens. Mucosal Immunol 2018;11(4):1103 13. Drolia R, et al. Listeria adhesion protein induces intestinal epithelial barrier dysfunction for bacterial translocation. Cell Host Microbe 2018;23(4):470 84 e7. Shawki A, McCole DF. Mechanisms of intestinal epithelial barrier dysfunction by adherent-invasive Escherichia coli. Cell Mol Gastroenterol Hepatol 2017;3 (1):41 50. Pinaud L, Sansonetti PJ, Phalipon A. Host cell targeting by enteropathogenic bacteria T3SS effectors. Trends Microbiol 2018;26(4):266 83. Walker WA, et al. Macromolecular absorption. Mechanism of horseradish peroxidase uptake and transport in adult and neonatal rat intestine. J Cell Biol 1972;54(2):195 205. Howe SE, et al. The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine. PLoS One 2014;9(1):e86656. Knoop KA, et al. Antibiotics promote inflammation through the translocation of native commensal colonic bacteria. Gut 2016;65(7):1100 9. Knoop KA, et al. Microbial antigen encounter during a preweaning interval is critical for tolerance to gut bacteria. Sci Immunol 2017;2(18) eaao1314. Knoop KA, et al. Antibiotics promote the sampling of luminal antigens and bacteria via colonic goblet cell associated antigen passages. Gut Microbes 2017;8 (4):400 11. Johansen FE, Kaetzel CS. Regulation of the polymeric immunoglobulin receptor and IgA transport: new advances in environmental factors that stimulate pIgR expression and its role in mucosal immunity. Mucosal Immunol 2011;4(6):598 602. Pyzik M, et al. FcRn: the architect behind the immune and nonimmune functions of IgG and albumin. J Immunol 2015;194(10):4595 603. Rodewald R, Kraehenbuhl JP. Receptor-mediated transport of IgG. J Cell Biol 1984;99(1 Pt 2):159s 64s. Martin MG, Wu SV, Walsh JH. Hormonal control of intestinal Fc receptor gene expression and immunoglobulin transport in suckling rats. J Clin Invest 1993;91(6):2844 9. Yoshida M, et al. Neonatal Fc receptor for IgG regulates mucosal immune responses to luminal bacteria. J Clin Invest 2006;116(8):2142 51. Yoshida M, et al. Human neonatal Fc receptor mediates transport of IgG into luminal secretions for delivery of antigens to mucosal dendritic cells. Immunity 2004;20(6):769 83. Ohshaki A, et al. Maternal IgG immune complexes induce food allergen-specific tolerance in offspring. J Exp Med 2018;215(1):91 113.

II. PRINCIPLES OF MUCOSAL VACCINE

68

3. MUCOSAL ANTIGEN SAMPLING ACROSS THE VILLUS EPITHELIUM BY EPITHELIAL AND MYELOID CELLS

[54] Mosconi E, et al. Breast milk immune complexes are potent inducers of oral tolerance in neonates and prevent asthma development. Mucosal Immunol 2010;3(5):461 74. [55] Nakata K, et al. The transfer of maternal antigenspecific IgG regulates the development of allergic airway inflammation early in life in an FcRndependent manner. Biochem Biophys Res Commun 2010;395(2):238 43. [56] Ohsaki A, et al. Maternal IgG immune complexes induce food allergen-specific tolerance in offspring. J Exp Med 2018;215(1):91 113. [57] Baker K, et al. Neonatal Fc receptor for IgG (FcRn) regulates cross-presentation of IgG immune complexes by CD8-CD11b 1 dendritic cells. Proc Natl Acad Sci U S A 2011;108(24):9927 32. [58] Lambrecht BN. FcRn is mother’s milk to allergen tolerance. J Exp Med 2018;215(1):1 3. [59] Rawool DB, et al. Utilization of Fc receptors as a mucosal vaccine strategy against an intracellular bacterium, Francisella tularensis. J Immunol 2008;180 (8):5548 57. [60] Liu X, et al. The neonatal FcR-mediated presentation of immune-complexed antigen is associated with endosomal and phagosomal pH and antigen stability in macrophages and dendritic cells. J Immunol 2011;186 (8):4674 86. [61] Lu L, et al. A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection. J Virol 2011;85 (20):10542 53. [62] Jang MH, et al. Intestinal villous M cells: an antigen entry site in the mucosal epithelium. Proc Natl Acad Sci U S A 2004;101(16):6110 15. [63] Knoop KA, et al. RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium. J Immunol 2009;183 (9):5738 47. [64] Valpotic H, et al. Increased number of intestinal villous M cells in levamisole -pretreated weaned pigs experimentally infected with F4ac(1) enterotoxigenic Escherichia coli strain. Eur J Histochem 2010;54(2):e18. [65] Wang J, et al. Convergent and divergent development among M cell lineages in mouse mucosal epithelium. J Immunol 2011;187(10):5277 85. [66] Joeris T, et al. Diversity and functions of intestinal mononuclear phagocytes. Mucosal Immunol 2017;10 (4):845 64. [67] Chieppa M, et al. Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J Exp Med 2006;203 (13):2841 52.

[68] Farache J, et al. Luminal bacteria recruit CD103 1 dendritic cells into the intestinal epithelium to sample bacterial antigens for presentation. Immunity 2013;38 (3):581 95. [69] Niess JH, et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 2005;307(5707):254 8. [70] Rescigno M, et al. Dendritic cells shuttle microbes across gut epithelial monolayers. Immunobiology 2001;204(5):572 81. [71] Rescigno M, et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2001;2(4):361 7. [72] Rivollier A, et al. Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon. J Exp Med 2012;209(1):139 55. [73] Bogunovic M, et al. Origin of the lamina propria dendritic cell network. Immunity 2009;31(3):513 25. [74] Bekiaris V, Persson EK, Agace WW. Intestinal dendritic cells in the regulation of mucosal immunity. Immunol Rev 2014;260(1):86 101. [75] Mowat AM, Agace WW. Regional specialization within the intestinal immune system. Nat Rev Immunol 2014;14(10):667 85. [76] Mayer JU, et al. Different populations of CD11b(1) dendritic cells drive Th2 responses in the small intestine and colon. Nat Commun 2017;8:15820. [77] Cerovic V, et al. Intestinal CD103(-) dendritic cells migrate in lymph and prime effector T cells. Mucosal Immunol 2013;6(1):104 13. [78] Cerovic V, et al. Lymph-borne CD8alpha 1 dendritic cells are uniquely able to cross-prime CD8 1 T cells with antigen acquired from intestinal epithelial cells. Mucosal Immunol 2015;8(1):38 48. [79] Scott CL, et al. CCR2(1)CD103(-) intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells. Mucosal Immunol 2015;8(2):327 39. [80] Kim KW, et al. In vivo structure/function and expression analysis of the CX3C chemokine fractalkine. Blood 2011;118(22):e156 67. [81] Hapfelmeier S, et al. Microbe sampling by mucosal dendritic cells is a discrete, MyD88-independent step in DeltainvG S. Typhimurium colitis. J Exp Med 2008;205(2):437 50. [82] Vallon-Eberhard A, et al. Transepithelial pathogen uptake into the small intestinal lamina propria. J Immunol 2006;176(4):2465 9. [83] Worbs T, et al. Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells. J Exp Med 2006;203(3):519 27.

II. PRINCIPLES OF MUCOSAL VACCINE

69

REFERENCES

[84] Mazzini E, et al. Oral tolerance can be established via gap junction transfer of fed antigens from CX3CR1(1) macrophages to CD103(1) dendritic cells. Immunity 2014;40(2):248 61. [85] Schulz O, et al. Intestinal CD103 1 , but not CX3CR1 1 , antigen sampling cells migrate in lymph and serve classical dendritic cell functions. J Exp Med 2009;206(13):3101 14. [86] Huang FP, et al. A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J Exp Med 2000;191(3):435 44. [87] Baker K, et al. The role of FcRn in antigen presentation. Front Immunol 2014;5:408. [88] Sockolosky JT, Szoka FC. The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy. Adv Drug Deliv Rev 2015;91:109 24. [89] Spiekermann GM, et al. Receptor-mediated immunoglobulin G transport across mucosal barriers in adult life: functional expression of FcRn in the mammalian lung. J Exp Med 2002;196(3):303 10. [90] Montoyo HP, et al. Conditional deletion of the MHC class I-related receptor FcRn reveals the sites of IgG

[91]

[92]

[93]

[94]

[95]

[96]

homeostasis in mice. Proc Natl Acad Sci U S A 2009;106(8):2788 93. Zhu X, et al. MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells. J Immunol 2001;166(5):3266 76. Li Z, et al. Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection. Proc Natl Acad Sci U S A 2011;108(11):4388 93. Nikitas G, et al. Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin. J Exp Med 2011;208 (11):2263 77. Takaiwa F, et al. Rice seed for delivery of vaccines to gut mucosal immune tissues. Plant Biotechnol J 2015;13(8):1041 55. Yuki Y, et al. Oral MucoRice expressing double-mutant cholera toxin A and B subunits induces toxin-specific neutralising immunity. Vaccine 2009;27(43):5982 8. Azegami T, Yuki Y, Kiyono H. Challenges in mucosal vaccines for the control of infectious diseases. Int Immunol 2014;26(9):517 28.

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C H A P T E R

4

Protective Activities of Mucosal Antibodies Jiri Mestecky1,2 1

Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, United States 2 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic

I. INTRODUCTION

IgM, IgG, and IgA isotypes, owing to the marked differences in their circulatory half-life and distribution in various body fluids. In humans, the daily production of IgA exceeds the combined production of Ig of all isotypes [2,3]. Serum IgA levels are lower than those of IgG because of a significantly shorter half-life in the circulation (4 6 days for IgA vs 21 days for IgG) and the high level of local IgA production in mucosal tissue with an effective receptor-mediated transepithelial transport into external secretions [4,5]. Furthermore, the great variability in measured levels of Igs in external secretions depends on the methods of collection; assays used for measurement; hormonal status, especially for secretions of the female genital tract; local inflammation; and presence of proteolytic enzymes of endogenous or exogenous origin, which may degrade secretory Igs [1].

Humoral and cellular factors of the innate immune system provide broad-spectrum protection of mucosal surfaces. The adaptive immune system, which is capable of responding to specific antigenic challenges, is represented by T cells and B cells with their terminal differentiation into antibody-secreting cells and the selective transport of antibodies into the mucosal fluids. Antibodies in human and other vertebrate external secretions are represented by immunoglobulins (Ig) of various isotypes. In humans, IgA is the dominant isotype in all external secretions with the exception of those of the genitourinary tract, in which IgG represents the dominant isotype [1]. The levels of Ig, irrespective of their isotype, are generally much lower in individual secretions than in sera. However, this does not reflect the biosynthetic rates of

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4. PROTECTIVE ACTIVITIES OF MUCOSAL ANTIBODIES

II. PROPERTIES OF ANTIBODIES OF VARIOUS IG ISOTYPES IN EXTERNAL SECRETIONS Although Igs of all isotypes are present in external secretions, their relative proportions, polypeptide chain structures, and biological properties are different from those present in plasma (Table 4.1). The dominant Ig isotype of external secretions, secretory IgA (SIgA), is present in the form of polymers (p), specifically dimers and tetramers, in contrast to the monomeric (m) serum IgA, which consists of two heavy (alpha) and two light chains covalently linked [4]. The structure, distribution of IgA subclasses, spectrum of antigen specificities, cellular origins, and different maturation patterns of IgA in the systemic versus mucosal compartments convincingly demonstrate their mutual independence [2] and must be considered in vaccination strategies aimed at the selective induction of desired humoral responses. SIgA in its dominant polymeric (p) form comprises two molecules of mIgA mutually linked through their Fc regions with participation of one molecule of low-molecular-mass

joining (J) chain and one large-glycoprotein secretory component (SC), the extracellular part of the polymeric Ig receptor (pIgR) covalently linked to the alpha chains and acquired during the epithelial transport of pIgA [5]. Tetrameric SIgA contains four covalently associated molecules of mIgA, one J chain, and one SC. These structural differences between mIgA in serum and pIgA in secretions confer significant functional advantages on pIgA with respect to epithelial transport, enormous increase in the avidity of dimers and tetramers over mIgA for reactivity with antigens, and resistance to proteolysis mediated by SC association [6,7] (see later). IgM present in external secretions differs from its serum counterpart polypeptide structure by the presence of SC acquired during the transepithelial transport common to pIgA and IgM [5]. In the early phase of an immune response after systemic immunization, a small fraction of serum IgM occurs in the form of hexamers lacking the J chain [8]. Owing to the presence of an additional pair of antigen-binding sites, the avidity of such IgM is further increased, and the ability of IgM to activate complement is enhanced [8]. However,

TABLE 4.1 Isotypes and Biological Activities of Mucosal Immunoglobulins Isotype Properties

Presence

Biological activity

SIgA

Dimeric and tetrameric forms IgA1 $ IgA2

Dominant Ig in human external secretions except for the genitourinary tract

Inhibition of Ag uptake Inhibition of bacterial adherence to epithelial cells Neutralization of biologically active antigens (toxins, enzymes, viruses)

SIgM

Pentameric molecules with SC attached

SIgM . SIgA in secretions of IgAdeficient individuals

Functionally analogous to SIgA

IgG

Monomeric form, IgG subclass distribution differs in various body fluids

Dominant Ig isotype in the Neutralization of biologically active antigens in genitourinary tract transported by tissues may activate complement resulting in FcRn on epithelial cells the impairment of mucosal barriers

IgD

Monomeric form

IgD1 plasma cells abundant in the Antibody activity against selected bacteria lacrimal glands and upper colonizing or infecting the respiratory tract respiratory tract mucosae

IgE

Monomeric form

Trace amounts in external secretions

Type I hypersensitivity possibly antiparasitic immunity

II. PRINCIPLES OF MUCOSAL VACCINE

IV. PROTECTIVE EFFECT OF MUCOSAL ANTIBODIES

hexametric IgM is unlikely to be transported into external secretions because of the absence of J chain, which is strictly required for the reactivity of pIgs with the pIgR and therefore their transepithelial transport [9]. IgG present in plasma and external secretions is structurally identical except for slight differences in the distribution of IgG subclasses [1]. IgG is transported by the FcRn receptor expressed on various types of epithelial cells [5]. In contrast to pIgR, which as the sacrificial receptor remains bound to its pIg ligand, FcRn is a recyclable receptor and is therefore not in external secretions linked with IgG [5]. IgG is dominant in external secretions of the genitourinary tract and the lower respiratory tract [1,5,10] (Chapter 16: Regulation of Mucosal Immunity in the Genital Tract: Balancing Reproduction and Protective Immunity). Because of the mostly circulatory origin of IgG, systemic vaccination is an effective route for the induction of protective immune responses in these secretions [11]. IgD and IgE are present in external secretions in trace amounts. Interestingly, IgDproducing plasma cells are frequently present in the human lacrimal gland and nasal mucosa [9], but the IgD receptor mediated transport mechanism has not been identified. The specific antibody activity of IgD is apparently restricted to the pathogens of the upper respiratory tract [12]. IgD responses to mucosally or systemically administered vaccines in humans have not been explored.

III. ORIGIN OF ANTIBODIES IN EXTERNAL SECRETIONS Although the presence of antibodies in external secretions was reported in early studies (for a historical review, see Ref. [13]), their origin and routes of effective vaccination were initiated in parallel with advances and applications of novel immunochemical and immunohistochemical methods. The original suggestion that SIgA is generated from serum-derived or

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locally produced mIgA, polymerized within epithelial cells with SC participation, was not confirmed by immunochemical analyses of human and rabbit SIgA with respect to the presence of allotypic IgA determinants in heterozygous rabbits or association of pIgA molecules with either κ or λ light chains [5]. Furthermore, the virtual absence of monoclonal plasma-derived pIgA in saliva of patients with pIgA myeloma [14] and trace amounts of intravenously injected radioactively labeled pIgA in the intestinal secretions [15] clearly indicated that for an effective transport, pIgA must be produced locally by plasma cells adjacent to epithelial cells. The same conclusions were reached in studies of SIgA origin performed in mice [16]. External secretions did not contain detectable levels of systemically injected pIgA of murine origin. The only exception was the bile, which contained high levels of pIgA, owing to the fact that in mice, rats, and rabbits, pIgR is also expressed on hepatocytes [5,17], which efficiently transport pIgA from the circulation into the bile and ultimately into the intestinal secretions. However, this is not the case for human hepatocytes, which do not express pIgR [5,18]. Therefore the passive systemic immunization of humans with pIgA of desired specificity is unlikely to provide SIgAmediated protection of mucosal surfaces. Obviously, the majority, if not all, of SIgA in secretions is produced locally by large numbers of plasma cells adjacent to pIgR-expressing epithelial cells. Importantly, the effectiveness of pIgR-mediated transport is regulated by cytokines (e.g., IL-4, IFNγ), vitamin A, a variety of microbial products (e.g., butyrate, lipopolysaccharide), and hormones (e.g., estrogens, androgens) in the female genital tract [5].

IV. PROTECTIVE EFFECT OF MUCOSAL ANTIBODIES The protective effect of mucosal antibodies has been evaluated in experimental models

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involving animals actively immunized with an antigen or passively immunized with preformed antibodies by the mucosal or systemic routes and subsequent mucosal challenge with corresponding antigens. In humans, the protective effect of mucosal antibodies is evaluated by the incidence of a given mucosal infection in the immunized population. In early studies, many investigators provided direct evidence that antibodies induced in feces of guinea pigs systemically or orally immunized with Vibrio cholerae were protected from mucosal challenge (for a historical review, see Ref. [13]) (Chapter 1: Historical Perspectives on Mucosal Vaccines). Numerous subsequent studies comprehensively reviewed elsewhere [7,19] convincingly demonstrated that mucosal antibodies induced actively by various immunization routes protect experimental animals from viral (e.g., influenza and herpes viruses, simian immune deficiency virus) or bacterial (e.g., pneumococcus, V. cholerae, Escherichia coli) infections [7,20,21]. The determination of Ig isotypes and duration of protective humoral responses in external secretions indicated that variable levels of SIgA and IgG are induced in secretions depending on the route of mucosal immunization or combination of systemic and mucosal sites and the use of suitable mucosal adjuvants [22]. Passive immunization with polyclonal or monoclonal antibodies of various Ig isotypes, precisely defined antigen specificities, and characterized physicochemical properties provided direct evidence of mucosal protection against microbial challenge [7,23 28]. The protective role of polyclonal antibodies against mucosal challenge with E. coli was demonstrated in a unique model of germfree, colostrum- and milk-deprived newborn piglets, which in the absence of transplacental acquisition lack any antibodies in plasma or secretions [29 31]. Consequently, such animals succumb to infection with E. coli. However, animals that received serum, milk, or isolated Igs by the oral route survived the infection, irrespective of the Ig source [31]. Passive administration of antibodies of IgA or IgG isotypes given by the

oral, intranasal, vaginal, or respiratory tract route protected experimental animals (mice, rats, rabbits, dogs, sheep, monkeys) against challenge with a variety of viral and bacterial pathogens [27]. Particularly relevant are studies of passively administered monoclonal antibodies or IgA-producing cell lines (backpack tumors) in murine models of protection against viral and bacterial pathogens [7,21,24,32] including the influenza, Sendai, respiratory syncytial, rota, reo, and simian immunodeficiency viruses and Streptococcus pyogenes, Streptococcus pneumoniae, V. cholerae, Salmonella typhimurium, Shigella flexneri, and others. Importantly, monoclonal antibodies to a single but relevant antigenic determinant were protective depending on the mechanisms involved, such as inhibition of adherence or neutralization (see later). This is not the case for polyclonal antibodies induced by active immunization, which are usually present in all major Ig isotypes and display the Ig isotype-dependent variability of specificities to antigenic determinants, duration of immune responses, and effector mechanisms as described later. Furthermore, the results of experiments performed in animals may not have validity in humans, owing to the marked differences in Igs with respect to their structure, sensitivity to proteases, epithelial and hepatic transport, catabolism, and antigen specificity due to the species-specific generation of antibody diversity and effector mechanisms [33].

V. THE ROLE OF IGG IN MUCOSAL IMMUNITY In humans, external secretions of genitourinary and lower respiratory tract IgG is the dominant Ig isotype (Table 4.1). Thus human semen, cervicovaginal fluid collected at individual stages of the menstrual cycle, and urine contain slightly higher levels of IgG than of IgA [1,34 36]. Although IgG-producing cells are numerous in the uterine endocervix [37], most IgG is derived from the circulation. This is an

II. PRINCIPLES OF MUCOSAL VACCINE

VI. MECHANISMS OF PROTECTION MEDIATED BY MUCOSAL IGA ANTIBODIES

important point with respect to the immunization routes that induce IgG antibodies in external secretions. Systemic immunization of females and males with several vaccines induced corresponding antibodies in the cervicovaginal fluid [38] and in semen [36]. Systemic immunization of rhesus macaques with antigens of SIV-induced IgG antibodies in female genital tract secretions [21]. Furthermore, the majority of currently available vaccines against microorganisms that enter the body through mucosal surfaces, particularly of the respiratory tract, are administered by the systemic route [11]. These include vaccines against influenza, polio, papilloma, and rubella viruses as well as Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae, and S. pneumoniae [11]. Although in general, systemic immunization does not effectively induce mucosal SIgA responses, the presence of relatively high levels of IgG antibodies in external secretions of the respiratory tract provides sufficient levels of specific humoral immunity to some mucosal pathogens (e.g., pneumococcus and influenza virus) (Chapter 34: Mucosal Vaccines for Streptococcus pneumoniae and Chapter 39: Nasal Influenza Vaccines). Injectable vaccines against other mucosal infections of the gastrointestinal tract (e.g., V. cholerae, S. typhi, Shigellae), respiratory tract (e.g., respiratory syncytial virus), and genital tract (e.g., human immunodeficiency virus [SIV]) have been evaluated or are in the developmental stage [11,21]. The available data on the above-described systemically administered vaccines with significant protection encourage such efforts, albeit with applicability restricted to mucosal surfaces that have relatively high levels of IgG. Passive immunization acquired by the ingestion of milk may provide immunity in animals in which protection is acquired after birth, owing to the absence of prenatal transplacental Ig transport. In these species (e.g., pigs, horses, cattle, sheep, and goats), the dominant isotype in milk is IgG, which is absorbed by the receptor-mediated pathway from the intestinal tract for a few days

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after birth to provide protection against intestinal microorganisms [39].

VI. MECHANISMS OF PROTECTION MEDIATED BY MUCOSAL IGA ANTIBODIES The biological outcome of the interaction of an antigen with antibody depends not only on its specificity but also on the isotype and molecular form. On this point, IgA differs from other Ig isotypes. There are two IgA subclasses in humans (15 subclasses in lagomorphs) and various molecular forms: monomers, dimers, and tetramers with characteristic distributions in body fluids [4,40]. This structural heterogeneity influences the biological and functional activity of individual forms of IgA molecules. Furthermore, IgA exhibits remarkable intrinsic resistance to endogenous and exogenous proteases, a characteristic that is enhanced by association with SC [41] and allows SIgA to function in adverse environments. In contrast to other Ig isotypes, IgA in its nonaggregated and fully glycosylated form displays strong antiinflammatory activity that is advantageous in mucosal tissues, especially in the intestine, which is rich in food- and microbiotaassociated antigens [7,42] (Fig. 4.1).

A. Inhibition of Antigen Absorption Despite the existence of the mucosal barrier, which limits the penetration of environmental antigens, minute amounts of, for example, food antigens (e.g., cow milk proteins) are absorbed in an undigested form and are detectable in the circulation in the form of immune complexes [43 45]. In IgA-deficient individuals, there is an increased absorption of such antigens, as detected by higher levels of circulating immune complexes and increased incidence of allergic diseases and autoimmune diseases [43,44]. SIgA-mediated inhibition of antigen and

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3.

Inhibition of bacterial adherence – Specific antibody-dependent – Glycan-mediated

4.

Enhancement of activities of innate humoral factors (e.g., mucin, lactoferrin, peroxidase system)

5.

Intracellular neutralization

6.

Elimination of immune complexes formed within epithelialcells

1

2

3

– Inhibition of complement activation

8.

– Elimination of pIgA-containing immune complexes

Mucosal surface 4

5

6

Antiinflammatory activity 7.

FIGURE 4.1 The roles of IgA in protection at mucosal surfaces.

Antigen

7

C

carcinogen penetration has been demonstrated in several experiments, emphasizing the importance of IgA and its antiinflammatory potential [42,46 48]. The cell types involved in the mucosal antigen uptake include enterocytes, M cells found on Peyer’s patches and bronchusassociated lymphoepithelial tissue [49], and dendritic cells with intraepithelial processes involved in the internalization and antigen presentation [50] (Chapter 3: Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells and Chapter 28: M Cell-Targeted Vaccines). Recent results suggest that the immune complexes formed in the intestinal lumen that contain SIgA are taken up by M cells that exhibit the corresponding IgA lectin receptor, designated as Dectin-1 [51]. Therefore SIgA may actually potentiate rather than inhibit immune responses. This finding should be viewed with respect to the absence of IgA in M cells [9], and the presence of these immune complexes should also be considered as potential factors participating not only in the enhancement of humoral immune responses but also in the induction of mucosal tolerance. Induction of IgA antibodies to environmental allergens and interference of IgA with IgEmediated allergic reactions have been a matter of attention for several decades [52]. This possibility is supported by a higher incidence of hypersensitivity reactions in IgA-deficient

Tissue

Inhibition of uptake of soluble or particulate antigens Neutralization of biologically active antigens

8

Mucosal

1. 2.

Epithelium

Lamina propria

individuals [43]. Oral immunization with allergens from birch or grass pollens has been explored to demonstrate the beneficial effect of the induction of allergen-specific SIgA responses in the respiratory tract and in parallel cell-mediated mucosal tolerance [52].

B. Inhibition of Bacterial Adherence Mucosal surfaces are colonized by enormous numbers of highly heterogeneous microorganisms, particularly in the large intestine [53]; it has been estimated that 99.9% of the 1013 bacteria in our microbiome is present in the large intestine [54]. Epithelial cells display on their surface receptors, mostly of glycan structures, that interact with microbial ligands [55,56]. Importantly, humoral factors of innate immunity such as mucins or adaptive immunity, Igs and SIgA in particular, interfere with the epithelial receptor-bacterial ligand interactions through specific antibody activity, polyreactivity, and glycan-dependent interactions [56 58]. Bacteria present on mucosal membranes are coated in vivo with IgA antibodies [56,59 61] that limit their adherence to epithelia and subsequent tissue penetration. This binding is dependent on the specific antibody activity and polyreactivity of IgA antibodies as demonstrated by the inhibition of bacterial adherence to epithelial cells [55] and the protective effect

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of monoclonal IgA antibodies specific for microbial antigens [24] as described earlier. These antibodies are induced by vaccination with well-defined antigens and display protective activity against corresponding pathogens. Their specificity, magnitude, and distribution in various secretions are influenced by the site of immunization at the mucosal inductive sites and the use of adjuvants, which play an important role [20]. These antibodies participate in neutralization of virulence factors, such as cholera toxin (CT), and inhibition of attachment to epithelial receptors [7] (Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control). A novel mechanism involved in protection of the intestine has been demonstrated in a murine model using high-avidity IgA antibodies [62]. It should be stressed that the high avidity of antibodies is related to the number of antigenbinding sites, which in pIgA is four per dimer and eight per tetramer; this increased number of antigen-binding sites, as compared to bivalent mIgA or IgG, enormously enhances the binding of pIg to antigens despite their low affinity. The protective effect of high-avidity IgA antibodies against bacterial infection with S. typhimurium is mediated by enchaining of growing bacteria, which facilitates their elimination [62]. IgA is involved in the process of formation of clumps by cross-linking enchains of daughter bacteria, thus preventing their separation after division with accelerated clearance from the gut lumen [62].

C. Polyreactivity Reactivity of secretory antibodies has been considered with respect to the protection of mucosal surfaces in a number of studies. Polyreactive antibodies, also called natural, antibodies, interact with a broad spectrum of structurally diverse antigens, including DNA, autoantigens, and antigens of microbial origin derived from unrelated viruses and bacteria (e.g., influenza virus or HIV and mucosal

77

bacteria with the possible involvement of common glycan structures on microorganisms) [63 68]. These antibodies may be present in all three major isotypes, are of low affinity, and are induced even in the absence of stimulating antigen [64]. Furthermore, it appears that their induction is not T cell dependent and is associated in mice with the cells of B1 phenotype [69 71]. In contrast to the high-avidity IgA antibodies induced by vaccination or infection by pathogens leading to their elimination, lowavidity polyreactive antibodies may participate in interactions with commensal microbiota and thus promote their long-term residence [72]. The proportion of high-avidity, highly antigenspecific versus polyreactive low-avidity antibodies appears to be different in mice and humans; the majority of intestinal IgA- or IgGproducing cells in humans is antigen-specific [73], in contrast to mice [64]. The precise structural basis of antibody polyreactivity remains unknown. Apparently, the complementaritydetermining region 3 of the variable domain of heavy chains plays the most important role, owing to its flexibility, changes in conformational reconfiguration allowing antigen accommodation or selective glycosylation within variable regions of heavy chains, resulting in altered specificity [66,74 76]. Interestingly, Ig glycosylation outside of the antigen-binding domain alters the neutralizing activity of HIV1-specific antibodies [77].

D. Ig Glycan Dependent Reactivity With Microorganisms In addition to the reactivity of antibodies with microorganisms based on the specificity of antigen-binding site, Ig-associated glycans of heavy chains of IgA and perhaps also IgM and SC are also involved in broadly based interactions with microorganisms as components of innate immunity [7,56,78 85]. Although SIgA from human milk agglutinated and inhibited the adherence of E. coli with type I fimbriae to colonic epithelial cells that expressed a

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corresponding receptor (e.g., mannose-dependent) several IgA2 myeloma proteins displayed the same activity independently of specific antibodies [79]. This activity was inhibited by the addition of mannose (Man). Detailed analyses of glycan moieties with the determination of complete glycan primary structures revealed that Man-rich N-linked chains were abundantly present in IgA2 myeloma proteins. Thus Mandependent agglutination and the inhibition of epithelial adherence by Man-rich N-linked chains on IgA indicate that they act as decoy receptors. Other in vitro studies confirmed this concept and extended analogous results to other bacterial species, including Helicobacter pylori, S. pneumoniae, Clostridium difficile, and S. flexneri, and some viruses such as influenza, respiratory syncytial, rotaviruses, and HIV (for a review, see Ref. [56]). In addition to N- and Olinked glycans on IgA1 and IgA2, SC covalently bound in SIgA is also heavily glycosylated with seven N-linked glycans [85,86]. In vitro studies indicated that SC also exhibits through its glycan moiety innate-like properties [87 89] and inhibits the binding of pathogens and toxin A from C. difficile to human epithelial cell lines (for a review, see Ref. [90]). Importantly, an in vivo study in a mouse model of respiratory tract infection by S. flexneri indicated that the protective ability of specific pIgA was enhanced by association with SC [91]. In addition to the inhibition of microbial adherence to epithelial cells, most recent data indicate that the IgA glycan-dependent interaction with bacteria alters the expression of genes involved in metabolism [92]. Thus heavily glycosylated IgA altered the expression of polysaccharide utilization loci, including the mucusassociated functional factor, which facilitate symbiosis with other bacteria and participate in colonic homeostasis. Thus the specific antibody, polyreactivity, and glycan-mediated interactions enforce the protective ability of SIgA.

E. Neutralization of Biologically Active Antigens Protective effects of mucosal antibodies against biologically active antigens, including enzymes, toxins, and viruses, require the presence of neutralizing antibodies. Glycosyltransferases from Streptococcus mutans or Streptococcus sobrinus have been used as components of experimental vaccines for the prevention of dental caries [93] (Chapter 37: Mucosal Vaccines for Oral Disease). Induction of neutralizing antibodies in nasal secretions to neuraminidase of the influenza virus is a desirable goal of current systemically or intranasally administered virus-inactivated or live-attenuated vaccines, respectively [94]. Neutralizing antibodies to several toxins of microbial or plant origin have been induced in external secretions by various routes of immunization. Immune responses to CT or its B subunit enterotoxins from E. coli and C. difficile toxin have been extensively studied, as is described in detail in Chapter 30, Oral Shigella Vaccines and Chapter 31, Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control. The plantderived toxins ricin and abrin were used in early studies, and Paul Ehrlich was the first investigator to demonstrate the protective effect of antibodies induced by mucosal immunization (for a historical review, see Ref. [13]). Neutralization of viruses, including influenza, respiratory syncytial, rota, reo, herpes simplex, papilloma, and HIV, by serum or secretory antibodies is a goal of currently used or developing vaccines [7,95 99]. The Ig isotype of antibodies is important, and therefore immunization routes and strategies must consider the differences in individual target tissues and secretions [20]. Systemically administered vaccines against influenza and papilloma viruses or HIV induce immune responses predominantly of the IgG isotype, which are also found at levels higher than those of IgA in secretions

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of the human lower respiratory tract, semen, and cervicovaginal fluids (Table 4.1). Dimeric and tetrameric forms of SIgA display functional advantages in virus neutralization in comparison to mIgA or IgG. Owing to the presence of four or eight antigen-binding sites in dimeric and tetrameric SIgA, the bonus effect of multivalency significantly enhances virus neutralization by at least one order of magnitude, despite the low affinity of individual antigen-binding sites [21,100 102]. Furthermore, pIgA is internalized by epithelial cells that express pIgR, and virus neutralization is exhibited within the infected cell [7,103 107]. In addition, immune complexes formed in the lamina propria containing pIgA with corresponding antigens can be subsequently internalized with pIgR participation by epithelial cells and reexported by the same pathway as free pIgA into external secretions [106]. The in vitro effectiveness of intracellular neutralization has been convincingly demonstrated by using several epithelial cell lines and viruses (Sendai, influenza, measles, rotaviruses, and HIV) [106]. Apparently, the transcytotic pathways of pIgA and viral assembly within the infected epithelial cells intercept intracellular virus and result in neutralization. It is assumed that vesicles containing pIgA pIgR complexes and viral envelope antigens fuse during their transcellular journey. There are, however, several questions concerning the in vivo applicability of this attractive mechanism of protection that must be considered. The epithelial cells (enterocytes) in the intestine are generated from stem cells and subsequently differentiate into phenotypically and functionally distinct cell types that change their mission during the pathway from the crypts to the surface. This is important for the transport of pIgA because the expression of pIgR is restricted to the enterocytes in intestinal crypts rather than those on surfaces of villi [108]. Consequently, only viruses that infect cells in the crypts would be sensitive to intracellular neutralization. Furthermore, if the epithelial

79

cells contain virus-specific pIgA, it is highly probable that free, transcytosed SIgA will be also present in the gut lumen and will exhibit neutralizing activity. Therefore the temporal sequence of events must be considered. Finally, pIgA present in human sera in small quantities is not taken up and transported into external secretions as described earlier [14,15]. Only pIgA locally produced by plasma cells adjacent to epithelia is bound to pIgR and transported. Consequently, pIgA-producing cells specific for a given antigen would have to be present in a high density to achieve the generalized protection of the vast surface area of intestinal mucosa. The majority of viruses that cause intestinal infections enter the mucosae through the M cells rather than regular pIgR-expressing enterocytes [49]. As a matter of fact, SIgA virus complexes are taken up by M cells to induce humoral immune responses as described earlier [7]. A novel concept of intracellular neutralization emerged from studies of the interaction of adenovirus with the human cervical cancer HeLa cell line [109 112]. A virus coated with nonneutralizing antibodies is still recognized by the corresponding virus receptor expressed on the permissive targeted cell type. Obviously, the bound antibody does not interfere and prevent the virus binding to the cellular receptor. Upon the receptor-mediated internalization of the virus, the Fc region of nonneutralizing antibody is recognized by the newly identified cytosolic receptor, tripartite motif-containing protein 21 (TRIM21), which directs the internalized virus for destruction by the proteasome. This receptor interacts with the Fc regions of IgG, IgM. and IgA of both subclasses and exhibits binding affinity exceeding that of any other known Fc receptor. Although this mechanism of intracellular virus neutralization has been demonstrated for nonenveloped adenovirus, it is likely that other viruses (e.g., poliovirus, rhinovirus, HIV) will also be neutralized by this pathway. Furthermore, it will be important to

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determine whether SC that covers the Fc region of SIgA prevents the interaction with TRIM21.

explored to generate relevant mucosal immune responses at the sites of pathogen entry.

F. Interactions of Mucosal Ig With Innate Antimicrobial Components of Mucosal Defense

References

The protective activity of mucosal antibodies can be further enhanced through interaction with humoral factors of innate immunity [7]. Thus lactoferrin, a glycoprotein found in external secretions, which may form covalently linked complexes with SIgA, focuses its antibacterial activity because of such association. In the same vein, the antibacterial activity of the lactoperoxidase system, comprising the enzyme, H2O2, and SCN 2 , may be enhanced by IgA proteins. In addition, mucin present in the majority of external secretions forms complexes with SIgA, which enhance the entrapment of microorganisms. Binding of IgG and IgA to mucus has gained recent attention in association with HIV infection [113 115].

VII. CONCLUDING REMARKS Mucosal antibodies represented in the majority of human external secretions by SIgA in its polymeric form with associated functional advantages display protective activity by several complementary mechanisms. These include the antigen-dependent specificity generated by various routes of mucosal vaccination or infection, polyreactivity of “natural” antibodies, and Ig glycan dependent interactions with the ultimate goal of preventing pathogenic penetration through the enormous surface area of mucosal membranes. Based on the immunological differences among individual compartments of the mucosal immune system, for example, the intestinal versus genitourinary tract, immunization strategies, including immunization sites and the use of acceptable adjuvants, should be further

[1] Jackson S, Moldoveanu Z, Mestecky J. Appendix I: Collection and processing of human mucosal secretions. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 2345 54. [2] Conley ME, Delacroix DL. Intravascular and mucosal immunoglobulin A: two separate but related systems of immune defense? Ann Intern Med 1987;106:892 9. [3] Mestecky J, Russell MW, Jackson S, et al. The human IgA system: a reassessment. Clin Immunol Immunopathol 1986;40:105 14. [4] Woof JM, Mestecky J. Mucosal immunoglobulins [chapter 17] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/ Academic Press; 2015. p. 287 324. [5] Baker K, Blumberg RS, Kaetzel CS. Immunoglobulin transport and immunoglobulin receptors [chapter 19] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 349 408. [6] Kilian M, Mestecky J, Russell MW. Defense mechanisms involving Fc-dependent functions of immunoglobulin A and their subversion by bacterial immunoglobulin A proteases. Microbiol Rev 1988;52:296 303. [7] Russell MW, Kilian M, Mantis NJ, Corthesy B. Biological activities of IgA [chapter 21] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 429 54. [8] Randall TD, King LB, Corley RB. The biological effects of IgM hexamer formation. Eur J Immunol 1990;20:1971 9. [9] Brandtzaeg P. The mucosal B cell system [chapter 31] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, et al., editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 623 69. [10] Mestecky J, Raska M, Novak J, et al. Antibodymediated protection and the mucosal immune system of the genital tract: relevance to vaccine design. J Reprod Immunol 2010;85:81 5. [11] Underdown BJ, Strober W. Parenteral immunization and protection from mucosal infection [chapter 70] In: Mestecky J, Strober W, Russell MW, Kelsall BL,

II. PRINCIPLES OF MUCOSAL VACCINE

81

REFERENCES

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21] [22]

[23]

[24]

Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 1391 403. Chorny A, Cerutti A. Regulation and function of mucosal IgA and IgD [chapter 32] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 683 700. Mestecky J, McGhee JR, Bienenstock J, et al. Historical aspects of mucosal immunology. In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed Amsterdam: Elsevier/Academic Press; 2015. p. XXXI VII. Kubagawa H, Bertoli LF, Barton JC, et al. Analysis of paraprotein transport into the saliva by using antiidiotype antibodies. J Immunol 1987;138:435 9. Jonard PP, Rambaud JC, Vaerman JP, Galian A, Delacroix DL. Secretion of immunoglobulins and plasma proteins from the jejunal mucosa. Transport rate and origin of polymeric immunoglobulin A. J Clin Invest 1984;74:525 35. Russell MW, Brown TA, Mestecky J. Preferential transport of IgA and IgA-immune complexes to bile compared with other external secretions. Mol Immunol 1982;19:677 82. Jackson GDF, Lemaitre-Coelho I, Vaerman J-P. Rapid disappearance from serum of intravenously injected rat myeloma IgA and its secretion into bile. Eur J Immunol 1978;8:123 6. Tomana M, Kulhavy R, Mestecky J. Receptor-mediated binding and uptake of immunoglobulin A by human liver. Gastroenterology 1988;94:762 70. Mestecky J, Russell MW, Elson CO. Intestinal IgA: novel views on its function in the defence of the largest mucosal surface. Gut 1999;44:2 5. Boyaka PN, McGhee JR, Czerkinsky C, et al. Mucosal vaccines: an overview [chapter 47] In: Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, Mayer L, editors. Mucosal immunology. 3rd ed. Amsterdam: Elsevier/Academic Press; 2005. p. 855 74. Mestecky J, Tomaras G. HIV-1/SIV humoral responses in external secretions. Curr Immunol Rev 2019;15:49 62. Freytag LC, Clements DM. Mucosal adjuvants: new developments and challenges [chapter 61] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 1183 99. Veazey RS, Shattock RJ, Pope M, et al. Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1 gp120. Nat Med 2003;9:343 6. Kraehenbuhl J-P, Neutra MR. Monoclonal secretory IgA for protection of the intestinal mucosa against viral and bacterial pathogens. In: Ogra PL, Mestecky J,

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

Lamm ME, Strober W, McGhee JR, Bienenstock J, editors. Handbook of mucosal immunology. San Diego, CA: Academic Press; 1994. p. 403 10. Mascola JR. Passive transfer studies to elucidate the role of antibody-mediated protection against HIV-1. Vaccine 2002;20:1922 5. Michetti P, Mahan MJ, Slauch JM, et al. Monoclonal secretory immunoglobulin A protects mice against oral challenge with the invasive pathogen Salmonella typhimurium. Infect Immun 1992;60:1786 92. Renegar KB. Passive immunization: systemic and mucosal. In: 3rd ed. Mestecky J, Bienenstock J, Lamm ME, McGhee JR, Strober W, editors. Mucosal immunology, vol. 2. Amsterdam: Elsevier/Academic Press; 2005. p. 267 89. Sholukh AM, Watkins JD, Hemant KV, et al. Defensein-depth by mucosally administered anti-HIV-1 dimeric IgA2 and systemic IgG1 mAbs: complete protection of rhesus monkeys from mucosal SHIV-1 challenge. Vaccine 2015;33:2086 95. Tlaskalova H, Rejnek J, Travnicek J, et al. The effect of antibodies present in the intestinal tract of germfree piglets on the infection caused by the intravenous administration of the pathogenic strain Escherichia coli 055. Folia Microbiol 1970;15:372 6. Miler I, Cerna J, Travnicek J, et al. The role of immune pig colostrum, serum and immunoglobulins IgG, IgM, and IgA, in local intestinal immunity against enterotoxic strain in Escherichia coli O55 in germfree piglets. Folia Microbiol 1975;20:433 8. Rejnek J, Travnicek J, Kostka J, et al. Study of the effect of antibodies in the intestinal tract of germ-free baby pigs. Folia Microbiol 1968;13:36 42. Yoo EM, Chintalacharuvu KR, Morrison SL. Recombinant IgA antibodies [chapter 15] In: Kaetzel CS, editor. Mucosal immune defense: immunoglobulin A. New York: Springer Science Business Media, LLC; 2007. p. 390 415. Kaetzel CS, Russell MW. Phylogeny and comparative physiology of mucosal immunoglobulins [chapter 18] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 325 47. Franklin RD, Kutteh WH. Characterization of immunoglobulins and cytokines in human cervical mucus: influence of exogenous and endogenous hormones. J Reprod Immunol 1999;42:93 106. Kutteh WH, Moldoveanu Z, Mestecky J. Mucosal immunity in the female reproductive tract: correlation of immunoglobulins, cytokines and reproductive hormones in human cervical mucus around the time of ovulation. AIDS Res Human Retroviruses 1998;14: S51 5. Moldoveanu Z, Huang W-Q, Kulhavy R, Pate MS, Mestecky J. Human male genital tract secretions: both

II. PRINCIPLES OF MUCOSAL VACCINE

82

[37]

[38]

[39]

[40]

[41] [42]

[43]

[44]

[45]

[46]

[47] [48]

[49]

[50]

4. PROTECTIVE ACTIVITIES OF MUCOSAL ANTIBODIES

mucosal and systemic immune compartments contribute to the humoral immunity. J Immunol 2005;175:4127 36. Crowley-Nowick PA, Bell M, Edwards RP, et al. Normal uterine cervix: characterization of isolated lymphocyte phenotypes and immunoglobulin secretion. Am J Reprod Immunol 1995;34:241 7. Bouvet JP, Belec L, Pires R, Pillot J. Immunoglobulin G antibodies in human vaginal secretions after parenteral vaccination. Infect Immun 1994;62:3957 61. Tlaskalova-Hogenova H, Kverka M, Verdu EF, et al. Gnotobiology and the study of complex interactions between the intestinal microbiota, probiotics, and the host [chapter 8] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed Amsterdam: Elsevier/ Academic Press; 2015. p. 109 33. Mestecky J, Russell MW. IgA subclasses. In: Shakib F, editor. Basic and clinical aspects of IgG subclasses. S. Karger, Basel. Monogr. Allergy, vol. 19; 1986.p. 277 301. Corthesy B. Roundtrip ticket for secretory IgA: role in mucosal homeostasis? J Immunol 2007;178:27 32. Russell MW, Sibley DA, Nikolova EB, Tomana M, Mestecky J. IgA antibody as a non-inflammatory regulator of immunity. Biochem Soc Trans 1997;25:466 70. Cunningham-Rundles C. Immunodeficiency and mucosal immunity [chapter 64] In: Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, et al., editors. Mucosal immunology. 3rd ed. Amsterdam: Elsevier/Academic Press; 2005. p. 1145 57. Cunningham-Rundles C, Brandeis WE, Good RA, et al. Milk precipitins, circulating immune complexes, and IgA deficiency. Proc Natl Acad Sci USA 1978;75:3387 98. Husby SJ, Jensenius C, Svehag S-E. Passage of undegraded dietary antigens into the blood of healthy adults: quantification, estimation of size distribution and relation of uptake to levels of specific antibodies. Scand J Immunol 1985;22:83 92. Walker WA, Isselbacher KJ, Bloch KJ. Intestinal uptake of macromolecules: effect of oral immunization. Science 1972;177:608 10. Brandtzaeg P, Tolo K. Mucosal penetrability enhanced by serum-derived antibodies. Nature 1977;266:262 3. Silbart LK, Keren DF, et al. Reduction of intestinal carcinogen absorption by carcinogen-specific secretory immunity. Science 1989;243:1462 4. Williams IR, Owen RL. M cells: specialized antigen sampling cells in the follicle-associated epithelium [chapter 21] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/ Academic Press; 2015. p. 429 54. Lambrecht BN, Iwasaki A, Kelsall BL. Mucosal dendritic cells: origins, subsets, and biology [chapter 25] In: Mestecky J, Strober W, Russell MW, Kelsall BL,

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 489 541. Rochereau N, Drocourt D, Perouzel E, et al. Dectin-1 is essential for reverse transcytosis of glycosylated SIgAantigen complexes by intestinal M cells. PLoS Biol 2013;11:e1001658. Mestecky J, Russell MW, Kilian M. The potential role of IgA-mediated mucosal immunity in the prevention of hypersensitivity reactions in the respiratory tract. In: Johansson SGO, editor. Progress in allergy and clinical immunology, 3. Seattle: Hogrefe & Huber Publ.; 1995. Jacobs J, Braun J. The mucosal microbiome: imprinting the immune system of the intestinal tract [chapter 5] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. 2015. p. 63 77. Savage DC. Mucosal microbiota. In: Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, editors. Mucosal immunology. San Diego, CA: Academic Press; 1998. p. 19 30. Abraham SN, Bishop BL, Sharon N, Ofek I. Adhesion of bacteria to mucosal surfaces [chapter 3] In: Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, Mayer L, editors. Mucosal immunology. 3rd ed. Amsterdam: Elsevier/Academic Press; 2005. p. 35 48. Mestecky J, Russell MW. Specific antibody activity, glycan heterogeneity and polyreactivity contribute to the protective activity of S-IgA at mucosal surfaces. Immunol Lett 2009;124:57 62. Kaetzel CS. Cooperativity among secretory IgA, the polymeric immunoglobulin receptor, and the gut microbiota promotes host-microbial mutualism. Immunol Lett 2014;162:10 21. Backhed F, Ley RE, Sonnenburg JL, et al. Hostbacterial mutualism in the human intestine. Science 2005;307:1915 20. van der Waaij LA, Limburg PC, Mesander G, et al. In vivo IgA coating of anaerobic bacteria in human faeces. Gut 1996;38:348 54. Macpherson AJ, Geuking MB, McCoy KD. Immune responses that adapt the intestinal mucosal to commensal intestinal bacteria. Immunology 2005;115:153 62. Shroff KE, Meslin K, Cebra JJ. Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut. Infect Immun 1995;63:3904 13. Moore K, Diard M, Sellin ME, et al. High-avidity IgA protects the intestine by enchaining growing bacteria. Nature 2017;544:498 505. Wijburg OLC, Uren TK, Simpfendorfer K, et al. Innate secretory antibodies protect against natural Salmonella typhimurium infection. J Exp Med 2006;203:21 6.

II. PRINCIPLES OF MUCOSAL VACCINE

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REFERENCES

[64] Bunker JJ, Erickson SA, Flynn TM, et al. Natural polyreactive IgA antibodies coat the intestinal microbiota. Science 2017;358. Available from: https://doi.org/ 10.1126/scuebce,asb6619. [65] Fransen F, Zagato E, Mazzini E, et al. BALB/c and C57BL/6 mice differ in polyreactive IgA abundance, which impacts the generation of antigen-specific IgA and microbiota diversity. Immunity 2015;43: 527 40. [66] Mouquet H, Nussenzweig MC. Polyreactive antibodies in adaptive immune responses to viruses. Cell Mol Life Sci 2012;69:1435 45. [67] Quan CP, Berneman A, Pires R, et al. Natural polyreactive secretory immunoglobulin A autoantibodies as a possible barrier to infection in humans. Infect Immun 1997;65:3997 4004. [68] Rollenske T, Szijarto V, Lukasiewicz J, et al. Crossspecificity of protective human antibodies against Klebsiella pneumoniae LPS O-antigen. Nat Immunol 2018;19:617 24. [69] Bos NA, Cebra JJ, Kroese FG. B-1 cells and the intestinal microflora. Curr Top Microbiol Immunol 2000;252:211 20. [70] Fagarasan S, Kawamoto S, Kanagawa O, Suzuki K. Adaptive immune regulation in the gut: T celldependent and T cell-independent IgA synthesis. Annu Rev Immunol 2010;28:243 73. [71] Macpherson AJ, Gatto D, Sainsbury E, et al. A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 2000;288:2222 6. [72] Peterson DA, McNulty NP, Guruge JL, et al. IgA response to symbiotic bacteria as a mediator of gut homeostasis. Cell Host Microbe 2007;2:329 39. [73] Benckert J, Schmolka N, Kreschel C, et al. The majority of intestinal IgA 1 and IgG 1 plasmablasts in the human gut are antigen-specific. J Clin Invest 2011;121:1946 55. [74] Dunn-Walters D, Boursier L, Spencer J. Effect of somatic hypermutation on potential N-glycosylation sties in human immunoglobulin heavy chain variable regions. Mol Immunol 2000;37:107 13. [75] Fernandez C, Alarcon-Riquelme ME, Abedi-Valugerdi M, et al. Polyreactive binding of antibodies generated by polyclonal B cell activation. I. Polyreactivity could be caused by differential glycosylation of immunoglobulins. Scand J Immunol 1997;45:231 9. [76] Fernandez C, Alarcon-Riquelme ME, Sverremark E. Polyreactive binding of antibodies generated by polyclonal B cell activation. II. Crossreactive and monospecific antibodies can be generated from an identical Ig rearrangement by differential glycosylation. Scand J Immunol 1997;45:240 7. [77] Miranda LR, Duval M, Doherty H, Seaman MS, Posner MR, Cavacini LA. The neutralization properties of a

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

HIV-1-specific antibody are markedly altered by glycosylation events outside the antigen-binding domain. J Immunol 2007;178:7132 8. Walz A, Odenbreit S, Mahdavi J, et al. Identification and characterization of binding properties off Helicobacter pylori by glycoconjugate arrays. Glycobiology 2005;15:700 8. Wold AE, Mestecky J, Tomana M, et al. Secretory immunoglobulin A carries oligosaccharide receptors for Escherichia coli type 1 fimbrial lectin. Infect Immun 1990;58:3073 7. Anthony BF, Concepcion NF, Puentes SM, et al. Nonimmune binding of human immunoglobulin A to type II group B streptococci. Infect Immun 1990;58: 1789 95. Firon N, Ofek I, Sharon N. Carbohydrate specificity of the surface lectins of Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium. Carbohydr Res 1983;120:235 49. Hooper LV, Gordon JI. Glycans as legislators of hostmicrobial interactions: spanning the spectrum from symbiosis to pathogenicity. Glycobiology 2001;11:1R 10R. Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol 2007;25:21 50. Mathias A, Corthesy B. Recognition of intestinal grampositive bacteria by hybridoma- and colostrumderived secretory immunoglobulin A is mediated by carbohydrates. J Biol Chem 2011;286:17239 47. Royle L, Roos A, Harvey DJ, et al. Secretory IgA N-and O-glycans provide a link between the innate and adaptive immune systems. J Biol Chem 2003;278:20140 53. Mizoguchi A, Mizuochi T, Kobata A. Structures of the carbohydrate moieties of secretory component purified from human milk. J Biol Chem 1978;89:110 18. Davin JC, Senterre J, Mahieu PR. The high lectinbinding capacity of human secretory IgA protects nonspecifically mucosae against environmental antigens. Biol Neonate 1991;59:121 5. Giugliano LG, Ribeiro ST, Vainstein MH, et al. Free secretory component and lactoferrin of human milk inhibit the adhesion of enterotoxigenic Escherichia coli. J Med Microbiol 1995;42:3 9. Perrier C, Sprenger N, Corthesy B. Glycans on secretory component participate in innate protection against mucosal pathogens. J Biol Chem 2006;281:14280 7. Phalipon AJ, Corthesy B. Novel functions for mucosal SIgA. In: Kaetzel CS, editor. Mucosal immune defense: immunoglobulin A. New York: Springer Science Business Media, LLC; 2007. p. 183 202. Phalipon A, Cardona A, Kraehenbuhl JP, et al. Secretory component: a new role in secretory IgAmediated immune exclusion in vivo. Immunity 2002;17:107 15.

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84

4. PROTECTIVE ACTIVITIES OF MUCOSAL ANTIBODIES

[92] Nakajima A, Vogelzang A, Maruya M, et al. IgA regulates the composition and metabolic function of gut microbiota by promoting symbiosis between bacteria. J Exp Med 2018. Available from: https://doi.org/ 10.1084/jem.20180427. [93] Taubman MA, Smith DJ. Mucosal vaccines for dental disease [chapter 69] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. 2015. p. 1363 89. [94] Broadbent AJ, Boonnak K, Subbarao K. Respiratory virus vaccines [chapter 59] In: Mestecky J, Strober W, Russell MW, Kelsall BL, Cheroutre H, Lambrecht BN, editors. Mucosal immunology. 4th ed. Amsterdam: Elsevier/Academic Press; 2015. p. 1129 70. [95] Bomsel M, Tudor D, Drillet AS, et al. Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV-1 challenges. Immunity 2011;34: 269 80. [96] Alexander R, Mestecky J. Neutralizing antibodies in mucosal secretions: IgG or IgA? Curr HIV Res 2007;5:588 93 PMID: 18045115. [97] Burton DR, Hessell AJ, Keele BF, et al. Limited or no protection by weakly or nonneutralizing antibodies against vaginal SHIV-1 challenge of macaques compared with a strongly neutralizing antibody. Proc Natl Acad Sci USA 2011;108:11181 6. [98] Castilla J, Sola I, Enjuanes L. Interference of coronavirus infection by expression of immunoglobulin G (IgG) or IgA virus-neutralizing antibodies. J Virol 1997;71:5251 8. [99] Baba TW, Liska V, Hofmann-Lehmann R, et al. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med 2000;6:200 6. [100] Taylor HP, Dimmock NJ. Mechanism of neutralization of influenza virus by secretory IgA is different from that of monomeric IgA or IgG. J Exp Med 1985;161:198 209. [101] Armstrong SJ, Dimmock NJ. Neutralization of influenza virus by low concentrations of hemagglutininspecific polymeric immunoglobulin A inhibits viral fusion activity, but activation of the ribonucleoprotein is also inhibited. J Virol 1992;66:3823 32. [102] Renegar KB, Jackson GDF, Mestecky J. In vitro comparison of the biologic activities of monoclonal monomeric IgA, polymeric IgA and secretory IgA. J Immunol 1998;160:1219 23. [103] Wright A, Lamm ME, Huang YT. Excretion of human immunodeficiency virus type 1 through-polarized epithelium by immunoglobulin A. J Virol 2008;82:11526 35.

[104] Huang YT, Wright A, Gao X, Kulick L, Yan H, Lamm M. Intraepithelial cell neutralization of HIV-1 replication. J Immunol 2005;174:4828 35. [105] Kaetzel CS, Robinson JK, Chintalacharuvu KR, et al. The polymeric immunoglobulin receptor (secretory component) mediates transport of immune complexes across epithelial cells: a local defense function for IgA. Proc Natl Acad Sci USA 1991;88:8796 800. [106] Lamm ME. Protection of mucosal epithelia by IgA: intracellular neutralization and excretion of antigens. In: Kaetzel CS, editor. Mucosal immune defense: immunoglobulin A. New York: Springer Science Business Media, LLC; 2007. p. 173 82. [107] Mazanec MB, Nedrud JG, Kaetzel CS, et al. A threetiered view of the role of IgA in mucosal defense. Immunol Today 1993;14:430 5. [108] Bjerke K, Brandtzaeg P. Lack of relation between expression of HLA-DR and secretory component (SC) in follicle-associated epithelium of human Peyer’s patches. Clin Exp Immunol 1988;7:502 7. [109] Vaysburd M, Watkinson RE, Cooper H, et al. Intracellular antibody receptor TRIM21 prevents fatal viral infection. Proc Natl Acad Sci USA 2013;110:12397 401. [110] Bidgood SR, Tam JCH, McEwan WA, Mallery DL, James LC. Translocalized IgA mediates neutralization and stimulates innate immunity inside infected cells. Proc Natl Acad Sci USA 2014;111:13463 8. [111] Mallery DL, McEwan WA, Bidgood SR, Towers GJ, Johnson CM, James LC. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21). Proc Natl Acad Sci USA 2010;107:19985 90. [112] McEwan WA, Mallery DL, Rhodes DA, Trowsdale J, James LC. Intracellular antibody-mediated immunity and the role of TRIM21. BioEssays 2011;33:803 9. [113] Fahrbach KM, Malykhina O, Stieh DJ, Hope TJ. Differential binding of IgG and IgA to mucus of the female reproductive tract. PLoS One 2013;8:e76176. [114] Gunn B, Schneider J, Shansab M, et al. Enhanced binding of antibodies generated during chronic HIV infection to mucus component MUC16. Mucosal Immunol 2016;9:1549 58. [115] Shukair SA, Allen SA, Cianci GC, et al. Human cervicovaginal mucus contains an activity that hinders HIV-1 movement. Mucosal Immunol 2013;6:427 34.

Further Reading Landsverk OJB, Snir O, Bartolome R, et al. Antibodysecreting plasma cells persist for decades in human intestine. J Exp Med 2017;214:309 17.

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Mucosal Immunity for Inflammation: Regulation of Gut-Specific Lymphocyte Migration by Integrins Eun Jeong Park1, Eiji Kawamoto1,2 and Motomu Shimaoka1 1

Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, Tsu, Japan 2Department of Disaster and Emergency Medicine, Mie University Graduate School of Medicine, Tsu, Japan

I. INTRODUCTION

receptors. A better understanding of the molecular mechanisms by which lymphocyte homing to the gut is regulated would advance the development of novel approaches, not only to augment the induction of mucosal immunity elicited by vaccines such as that for rotavirus infections, but also to abrogate excessive immune responses that lead to inflammation, as seen in the inflammatory bowel diseases (IBD). This chapter seeks to describe the underlying molecular mechanisms that control gutdirected T cell homing by cell adhesion molecule integrins and their implication and relevance to mucosal inflammation and therapeutics. The key players enabling gut-specific lymphocyte homing include the integrin α4β7 and its major ligand, MAdCAM-1 [4]. The adhesiveness of integrins is regulated not only by cell surface

The migration patterns of lymphocyte circulation in tissues throughout the body is not random but rather highly regulated to optimize the efficacy of antigen-specific T cells to encounter pathogens containing cognate antigens [1 3]. Lymphocyte subsets activated in gut-associated lymphoreticular tissues (GALT) are programmed to travel back to gut compartments such as the lamina propria regions. By contrast, lymphocyte subsets activated in the peripheral lymph nodes preferentially recirculate through the skin tissues. The mechanisms that enable effector/memory T cells to home back to the specific tissue compartment, where T cells are activated by specific antigens, comprise the regulated expression of specific pairs of cell adhesion molecules and chemokine

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expression levels and the extent to which integrins are laterally associated (referred to as avidity upregulation), but also by enhancements to ligand-binding strength via single integrin molecules (referred to as affinity upregulation) [5,6]. This chapter addresses three areas: the molecular and structural mechanisms by which the activity and conformations of integrins are dynamically regulated in migrating lymphocytes; the cellular and molecular mechanisms by which the cell surface expression of α4β7 integrin is upregulated on gut-homing lymphocytes upon activation in the GALT; and how integrin antagonists, such as natalizumab and vedolizumab, have been developed in clinical trials to target IBD as well as multiple sclerosis [7 9].

II. MOLECULAR MECHANISMS FOR THE RECRUITMENT OF CIRCULATING LYMPHOCYTES TO TISSUES Recruitment of circulating T cells to secondary lymphoid organs is regulated by a series of collaborations between cell adhesion molecules and chemokines [1 3]. Entry of T cells into secondary lymphoid tissues occurs through specialized endothelial cells known as high endothelial venules (HEVs). Flowing T cells start to interact with vascular endothelial cells through L-selectin expressed on naı¨ve lymphocytes (Fig. 5.1A). Peripheral node addressin (PNAd) is the major endothelial L-selectin ligand expressed on HEVs. The interaction of

FIGURE 5.1 Distinct modes of lymphocyte migration in different microenvironments. (A) Intravascular migration involves a series of adhesive interactions with endothelial cells under shear stress. (B) The transendothelial migration step allows lymphocytes to breach the vascular endothelial barrier and enter the interstitial space. (C) Rapid interstitial migration is usually made possible by an amoeboid-like cell migration mechanisms. (D) The immunological synapse is formed at the stable contact between T cells and DCs. pSMAC, peripheral supramolecular activation cluster; cSMAC, central supramolecular activation cluster.

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III. INTEGRIN DEACTIVATION AS A REGULATORY MECHANISM FOR EFFICIENT CELL MIGRATION

L-selectin with its ligands, including PNAd, is kinetically characterized by a very fast on-rate and a very fast off-rate, thereby enabling rapid cycles of adhesion and detachment that make possible the transient adhesive interactions seen in tethering and subsequent rolling interactions [10]. Selectin-mediated rolling interactions allow lymphocytes to slow down and travel along the surface of endothelial cells [11]. Consequently, lymphocytes are placed in close proximity to the surface of endothelial cells, where chemokines are displayed. P-selectin ligand PSGL-1 (P-selectin glycan ligand-1) expressed on lymphocytes binds to P-selectin upregulated on inflamed endothelial cells. Ligation of PSGL-1 and possibly L-selectin triggers signals to lymphocytes, thereby eliciting intracellular signaling cascades leading to the priming of integrin activation [12]. The priming of integrin activation induces an intermediateaffinity state that is strong enough to slow the rolling velocity. However, primed integrins are not competent enough to support cell arrest and shear-resistant stable adhesion. Subsequent chemokine activation, along with mechanical stress by shear flow, is required to convert the primed intermediate-affinity state to the high-affinity state, thereby supporting shearresistant firmly adhesive interactions with endothelial integrin ligands [13]. Rolling lymphocytes eventually encounter chemokines on HEVs that signals through G-protein-coupled chemokine-receptors in order to activate integrins to the ligand-competent high-affinity state. Firmly adherent lymphocytes undergo diapedesis through substantial dynamic cytoskeletal rearrangement that is induced as a result of integrin signaling and chemokine signaling, thereby migrating or crawling on the surface of endothelial cells to transmigration sites across the endothelial monolayer and into the tissue [14] (Fig. 5.1B). Two distinctive routes of breaching the endothelial cell barrier by lymphocytes have been found, namely, paracellular

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transendothelial migration (TEM) and transcellular TEM routes [15]. In the former, lymphocytes travel in between adjacent endothelial cells by breaching cell cell junctions. In the latter route, lymphocytes travel through individual endothelial cells by squeezing into a “hole” formed in the endothelial cell body in response to contact with adherent migrating lymphocytes. Although the transcellular TEM is rarely seen in in vitro experiments, in in vivo settings, this TEM route is as common as the paracellular route and might actually be the predominant route at sites of inflammation [16,17].

III. INTEGRIN DEACTIVATION AS A REGULATORY MECHANISM FOR EFFICIENT CELL MIGRATION The most prominent feature of integrins is that the ability to bind ligand is dynamically regulated by bidirectional transmembrane signals, termed inside-out and outside-in signaling [6,18,19]. Integrins on resting lymphocytes usually remain inactive and are thereby unable to bind ligand (Fig. 5.2A). Only upon cellular activation by chemokines and/or TCRligation do integrins start to undergo global conformational changes, thereby converting to a primed intermediate-affinity state. This conformational conversion from the low- to the intermediate-affinity state is achieved by an association between the integrin cytoplasmic domain and talin, an intracellular adaptor protein that connects the integrin to the actin cytoskeleton (Fig. 5.2B). The mere association of talin to the integrin does not elicit a conformational driving force strong enough to induce a high-affinity fully activated state [13]. The actin cytoskeleton is indirectly connected to the integrin β cytoplasmic domain via talin. There is a constant retrograde actin flow running underneath the plasma membrane of T cells at the contact region with antigen-presenting

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FIGURE 5.2 Conformational regulation of integrin activation. (A) Integrins are usually in an inactive low-affinity state in resting cells, thus adopting a bent conformation, in which the ligand-binding headpiece is folded back to the leg-piece. This latent conformation is stabilized by associations of the α and β cytoplasmic domains. (B) Binding of talin induces the dissociation of the integrin cytoplasmic domains, thereby triggering the conversion to the extend conformation via a switchblade-like opening of the headpiece and leg-piece interface. (C) The tensile force applied to the β cytoplasmic domain via talin and the actin cytoskeleton converts the closed configuration to the open headpiece conformation, thereby stabilizing the ligand-bound high-affinity state.

cells. Actin flow leads to an application of tensile forces to integrins (e.g., LFA-1), which are anchored at the proximal cytoplasmic end to the actin (via talin) and at the distal extracellular end to ICAM-1 immobilized on the opposing antigen-presenting cells [20]. Tensile force is the mechanotransduction driver that converts the intermediate-affinity state to the high-affinity state, thereby supporting the stable tight adhesive interaction with ICAM-1 (Fig. 5.2C). In fact, this tensile force, which is otherwise disruptive to adhesive bonds (termed “slip bonds”), paradoxically strengthens the LFA-1 and ICAM-1

interaction (termed “catch bond”) [21,22]. This catch-bond-type mechanism, driven by tensile forces via actin linkage, may play an important role in converting chemokine-primed intermediate-affinity integrins to shear-driven high-affinity integrins during abrupt lymphocyte arrests to HEVs in lymphocyte homing [20,23]. The force-driven conformational upregulation of integrin activity in a catchbond-type manner constitutes a critical component of the mechanotransduction machinery that enables cells to probe the mechanical properties of their extracellular microenvironments [24].

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Efficient lymphocyte migration on the substrate, such as that on the endothelial surface, requires a balanced cycle of integrin activation and deactivation [25,26]. At the leading edge of a migrating lymphocyte, integrins are activated, thereby mediating cell adhesion to ligands on the substrates [23]. By contrast, at the trailing edge of the migrating lymphocyte, integrins are deactivated, thereby facilitating cell detachment from the substrates [27]. Parts of detached unliganded integrins are internalized and transported back to the plasma membrane at the leading edge [28]. A simple dissociation of talin from the integrin cytoplasmic domain is likely the major mechanism of integrin deactivation. Talin is thought to dissociate as stimulatory signal inputs downregulate. For example, chemokine receptor-mediated signal inputs are downregulated by receptor internalization, thereby resulting in the reduction of cell surface expression levels of the chemokine receptor [29]. This downregulation of the cell surface chemokine receptor is a physiologic mechanism to facilitate integrin deactivation and cell detachment. Genetic mutations at the C-terminal region critical for internalization of CXCR4 cause the aberrant integrin deactivation is the primary pathogenesis driving WHIM (wart, hypogammaglobulinemia, infections, and myelokathexis) syndrome, a rare autosomal immunodeficiency disorder [30]. In WHIM patients, the physiologic mechanism to downregulate CXCR4 signaling is perturbed. Thus chemokine signaling remains aberrantly persistent, thereby disabling deactivation of integrin-mediated cell adhesion. Downregulation of CXCR4 signaling and subsequent downregulation of integrin α4β1-mediated cell adhesion are important for neutrophil egress from the bone marrow to the circulation. In WHIM syndrome, neutrophil egress is impaired, thereby inducing excessive retention of neutrophils in the bone marrow, manifested as myelokathexis, a Greek term that implies retention in the bone marrow.

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In addition to the passive dissociation of talin from the integrin cytoplasmic domain, alternative mechanisms for integrin deactivation have been discovered, including several integrin inactivators that interfere with talin and the integrin cytoplasmic domain (e.g., ICAP1, Dok1, and SHARPIN) [31]. For example, ICAP1 (integrin cytoplasmic domainassociated protein 1), like talin, is able to bind to the β integrin cytoplasmic domain. However, unlike talin, ICAP1 fails to destabilize the α β integrin association, thereby disabling the triggering of integrin activation [32]. Thus ICAP1 interferers with talin binding and inhibits talinmediated integrin activation. Integrin inactivators such as ICAP1 are thought to participate in maintaining the latent form of inactive integrins on resting cells. In addition to integrin inactivators, integrins themselves contain intramolecular regulatory structures that negatively control conformational activation [33]. Studies have described knock-in mice in which one of the intramolecular regulatory structures were disabled [25,26,34,35]. Knock-in lymphocytes exhibited delayed cell migration on the substrate as well as across HEVs, owing to impaired detachment of the trailing edge, thereby revealing the extremely elongated tail. Consequently, tissuespecific homing was suppressed. Because integrins at the trailing edge failed to dissociate from the substrates, integrins along with parts of the plasma membrane were pulled away from the cell tail and left behind, deposited on the substrate as cell debris. The integrin-containing cellular debris deposited on the substrates have been observed not only in an artificial context, in which the mutation to disable the intrinsic integrin regulatory structures was engineered in knock-in mice [25,26,34,35], but also in a physiologic context, in which native neutrophils migrated to the site of an influenza virus infection in the lung [36]. Interestingly, this neutrophil debris contained integrins as well as chemokine

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CXCL12 and was deposited along the migrating paths of neutrophils, thereby creating a pathway (or trail) that followed the route to the site of the viral infection in the lung. Chemokine CXCL12 incorporated into such a pathway attracts effector lymphocytes expressing CXCR4 and guides them exactly where they need to go in order to reach the sites of infection by following after neutrophils. Thus the neutrophil chemokine pathway serves as an important mechanism facilitating the seamless transition from the acute inflammatory phase (by neutrophils) to the chronic inflammatory phase (by effector lymphocytes). Formation of the neutrophil pathways that express chemokines is a good example of physiologic mechanisms in which incomplete deactivation of integrin adhesiveness is exploited by nature.

IV. INTERSTITIAL MIGRATION After completing TEM, lymphocytes migrate within the interstitial space of the lymphoid tissue. In contrast to the intravascular space, where lymphocytes are constantly exposed to shear stress and migrate along the endothelial cells in a two-dimensional (2D) migration manner, the interstitial space constitutes a completely different microenvironment in which lymphocytes undergo three-dimensional (3D) migration through extracellular matrix (ECM) mesh structures under less shear stress [37,38] (Fig. 5.1C). Modes of migration are very different in the 2D and 3D microenvironments. In the former, lymphocytes primarily utilize integrins to produce the traction force that drives movement on the 2D substrate, such as occurs on the surface of endothelial cells. However, in the 3D migration mode, cells undergo an amoeboid-like movement in which cytoskeletal contractions push and squeeze through the ECM mesh structures. Integrins appear to be dispensable in the 3D amoeboid-like cell migration, as immune cells

[e.g., dendritic cells (DCs), which are engineered to lack all integrins] move in the interstitial space of lymph nodes. In fact, they do so as efficiently as wild-type cells, despite the presence of stromal ICAM-1 and VCAM-1 expressed by fibroblastic reticular cells and which constitute the meshlike network structures of the lymph node interstitial space [39]. This suggests that integrin activity is maintained in the inactive low-affinity state during interstitial 3D migration. A similar observation was made in a previous report, in which lymphocyte migration in the interstitial space was studied in the presence of two components: (1) lymphocytes engineered to detect subsets of integrins and (2) the pharmacological inhibition of other lymphocyte integrins [40]. Although integrins appear to be dispensable in lymphocyte interstitial 3D migration under normal conditions, another previous report that examined inflamed tissues in which stromal VCAM-1 was upregulated has shown that integrin VLA-4 plays an important role in the efficient migration of interstitial lymphocytes [41].

V. IMMUNOLOGICAL SYNAPSE While migrating in the interstitial spaces of lymph nodes, T cells make a series of apparently random short contacts with many different DCs as a way of probing for a cognate antigen-expressing DC [42]. Eventually, some T cells form a stable contact with the cognate antigen-expressing DC, thereby initiating productive T cell activation. This stable contact is made possible by the formation of the immunological synapse, the supramolecular structure formed between the T cell and the DC contact interface [43] (Fig. 5.1D). The dynamic adhesive interaction of integrin LFA-1 on T cells with ICAM-1 on the DC drives the initial contact and subsequent maturation of the immunological synapse [44]. In the mature immunological synapse, the LFA-1 ICAM-1 complex aligns

II. PRINCIPLES OF MUCOSAL VACCINE

VI. IMPRINTING OF HOMING SPECIFICITY BY DENDRITIC CELLS

with the outer edge of the synapse, thereby creating the peripheral SMAC (pSMAC). In contrast, the TCR MHC complex sits at the center of the matured immunological synapse. Formation of the immunological synapse not only serves to sustain the complex of signaling molecules required for T cell activation, but also creates the junction enabling the exchange of mediators secreted to the synaptic gap in the central SMAC (cSMAC) between T cells and the DC. Those mediators include cytokines and exosomes [44] as well as—in the case of CD8 T cells—cytotoxic granules containing granzymes [45]. The TCR MHC complex, along with costimulatory and checkpoint-regulating molecules (CD28, CTLA-4, PD-1), is concentrated at the cSMAC, thereby determining the levels of productive T cell activation [46,47]. Integrin LFA-1 complexed with ICAM-1 (located at the pSMAC) also functions as an additional costimulation molecule to modulate TCR signaling [48]. Interestingly, a dynamic cytoskeletal rearrangement at the immunological synapse not only determines the spatial organization of LFA1 at the pSMAC, but also provides mechanotransduction regulation to the conformational enhancement of binding affinity to ICAM-1 [20].

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VI. IMPRINTING OF HOMING SPECIFICITY BY DENDRITIC CELLS Strengths and durations of cell surface receptor ligand interactions at the immunological synapse, including TCR and costimulation molecules, are integrated into the signals to permit productive T cell activation and proliferation. However, those signals are not sufficient for determining the homing specificity of activated T cells. Early studies have shown that T cells activated with mesenteric lymph node DCs acquired the propensity to home to the intestine by upregulating integrin α4β7 and the chemokine receptor CCR9 [3]. By contrast, T cells activated with DCs from peripheral lymph nodes acquired the propensity to home to the skin by engaging CLA (cutaneous leukocyteassociated antigen) and CCR4 or CCR10 [1,49]. Thus it is the distinct natures of the DCs from different tissue origins that imprint the tissue homing specificity of activated lymphocytes (Fig. 5.3). The major breakthrough into the understanding of the underlying molecular mechanisms by which gut DCs specifically induce a gut-homing phenotype of activated lymphocytes comes from the following experimental

FIGURE 5.3 Imprinting of gut-homing T cells by mucosal DCs. (Right) T cell activation by DCs at the mesenteric lymph nodes and Peyer’s patches, which involves retinoic acid. This induces the upregulation of integrin α4β7 and chemokine receptor CCR9, thereby imprinting a gut-homing capability. (Left) By contrast, T cell activation by DCs at the peripheral lymph nodes leads to the acquisition of a default skin-homing capability.

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findings [50]. The ability to upregulate integrin α4β7 and CCR9 is recapitulated by T cell activation in the presence of the vitamin A metabolite, all-trans retinoic acid (RA). Vitamin A acquired from diet is subjected to a series of metabolic reactions and is thereby converted to RA. The final and rate-limiting step is catalyzed by the cytosolic retinal dehydrogenase (RALDH) enzyme family, which contains at least four enzymes: RALDH1, RALDH2, RALDH3, and RALDH4 [3]. Expression of RALDH is restricted to specific tissues and cell types, thereby enabling the unique distribution of RA to certain microenvironments. Mesenteric lymph node DCs, but not peripheral lymph node DCs, express multiple isoforms of RALDH such as RALDH1, RALDH2, and RALDH3 [3]. Intestinal epithelial cells, but not skin epithelial cells, also express RALDH1. Thus although precursors of RA such as retinal may be ubiquitously expressed in many tissue and cell types, the biologically active RA synthesized from RA precursors is highly concentrated in the GALT, including mesenteric lymph nodes. This makes the GALT a special microenvironment that imprints the gut-homing phenotype onto lymphocytes [51] (Fig. 5.3).

VII. INTEGRIN α4β7 MADCAM-1 INTERACTIONS MAdCAM-1 is the principal and strongest binding ligand for integrin α4β7 [4,52]. Whereas integrin α4β7 binds weakly to α4β1 integrin ligand VCAM-1 and ECM protein fibronectin, its interaction with MAdCAM-1 is one of the most biologically relevant in mediating gut-directed lymphocyte migration. Like ICAM-1 and VCAM-1, MAdCAM-1 is an immunoglobulin superfamily (IgSF) transmembrane molecule that contains three IgSF domains

[53]. Two N-terminal domains adopt a typical IgSF fold that contains the α4β7 binding site. By contrast, the third IgSF domain shows a structural similarity to the Cα domain of IgA1 and is separated from the second domain via a long serine threonine-rich mucin-like domain highly glycosylated to form an L-selectin binding site. Patterns and levels of glycosylation are critical to the functioning of MAdCAM-1. Only properly glycosylated MAdCAM-1 expressed in HEVs serves as ligands for both integrin α4β7 and L-selectin. In adult healthy mice, the expression of MAdCAM-1 is restricted to mucosal HEVs, thereby establishing a molecular basis to support gut-specific lymphocyte homing [54]. The expression of MAdCAM-1 in the HEVs of the GALT is driven by the transcription factor NKx2.3. This has been shown by knockout mice lacking NKx2.3, which exhibited impaired expression of MAdCAM-1 as well as defects in lymphocyte homing [55]. Proinflammatory cytokines, such as TNF, augment the expression of MAdCAM-1 in cultured vascular endothelial cells [56]. In vivo, the presence of inflammation upregulates MAdCAM-1 expression in the gut, as seen in IBD mice models as well as in IBD patient samples [56]. The interaction of integrin α4β7 with MAdCAM-1 mediates the migration of activated pathogenic lymphocytes to inflamed gut tissues, thereby playing an important role in the pathogenesis of IBD. Under certain pathologic conditions, the extragut expression of MAdCAM-1 has been reported [57]. For example, hepatic inflammation, such as is observed in chronic hepatitis, has been shown to induce MAdCAM-1 expression in hepatic endothelial cells. This hepatic expression of MAdCAM-1 suggests not only the possibility that gutimprinted lymphocytes contribute to the pathogenesis and progression of chronic hepatitis, but also the potential link between IBD and IBD-associated hepatic complications such as primary sclerosing cholangitis [57].

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VIII. THERAPEUTIC INTEGRIN INHIBITION FOR INFLAMMATORY BOWEL DISEASES

VIII. THERAPEUTIC INTEGRIN INHIBITION FOR INFLAMMATORY BOWEL DISEASES The interaction of integrin α4β7 with MAdCAM-1 is critically important not only in physiologic lymphocyte homing to the gut, but also in lymphocyte migration to the inflamed gut as seen in IBD [58]. The key preclinical experiment that confirmed integrin α4β7 as a therapeutic target for IBD was performed in spontaneously occurring chronic colitis in cotton-top tamarin monkeys [59]. The mouse monoclonal antibody clone ACT-1, which is specific to human integrin α4β7, has similarly been shown to block the monkey integrin α4β7. The ability of ACT-1 to recognize only the α4β7 integrin heterodimer complex but not the single α4 or β7 chain strongly suggested that ACT-1 binds to the epitope at the interface of the α4 and β7 association [60]. ACT-1 was demonstrated to ameliorate colitis in these cotton-top tamarins. Subsequent to the preclinical success in treating chronic colitis in monkeys [59], ACT-1 was fully humanized to the therapeutic antibody vedolizumab (Entyvio), which retained the binding specificity to human integrin α4β7 [61]. After successful completion of phase 3 clinical trials, vedolizumab was approved in the United States in 2014 and in the European Union in 2015 for the treatment of ulcerative colitis and Crohn’s disease [58,62]. The successful FDA approval of vedolizumab for treating ulcerative colitis and Crohn’s disease represents a major breakthrough in recent years [62]. However, we should not forget the rise and fall of natalizumab, an earlier humanized monoclonal antibody specific to the integrin α4 subunit [63]. The integrin α4 subunit associates with the integrin β1 and β7 subunits, thereby forming functional α4β1 and α4β7 integrin heterodimers, respectively. Unlike integrin α4β7, integrin α4β1 binds to an endothelial ligand VCAM-1 and an ECM ligand

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fibronectin. The expression of VCAM-1 is upregulated on inflamed endothelial cells, and fibronectin is deposited on inflamed endothelial cells. It is of great clinical significance to reveal that the binding of integrin α4β1 with VCAM-1 and fibronectin on brain microvascular endothelial cells plays a critical role in regulating the migration of autoreactive lymphocytes to the inflamed brain [64]. The key preclinical experiments were performed in experimental autoimmune encephalomyelitis (EAE), an established mouse model for multiple sclerosis. This demonstrated how the inhibitory antibody to integrin α4 prevented the infiltration of leukocytes into the brain and alleviated neurological motor defects. While the antibody to integrin α4 subunit can block both integrins α4β1 and α4β7, accumulating evidence drawn from animal studies strongly suggested that inhibition of the integrin α4β1 was predominantly responsible for ameliorating leukocyte accumulation within the inflamed brain [64]. Following the success of the pivotal preclinical studies using the EAE model, natalizumab, a fully humanized mAb to the human integrin α4 subunit, was developed, with the goal of carrying out clinical trials for the treatment of multiple sclerosis [7,64]. After the first small randomized, double-blind, placebo-controlled trial involving 72 multiple sclerosis patients, which showed some clinical efficacy, natalizumab successfully passed a series of phase 1 to phase 3 clinical trials, including two randomized, multicenter, placebo-controlled, doubleblind trials: AFFIRM (Natalizumab Safety and Efficacy in Relapsing and Remitting Multiple Sclerosis) [65] and SENTINEL (Safety and Efficacy of Natalizumab in Combination with Interferon Beta-1a in Patients with Relapsing Remitting Multiple Sclerosis) [66]. The AFFIRM study, which examined 942 multiple sclerosis patients for more than 2 years, showed that natalizumab treatment significantly reduced rates of clinical relapse as well as longer relapse

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or disease-free periods compared to placebo [65]. The SENTINEL study, which involved 1171 multiple sclerosis patients for over 2 years, demonstrated that natalizumab combined with interferon β1a significantly reduced both the risk of disability progression and the rate of clinical relapse compare with the interferon β1a monotherapy [66]. Natalizumabs’s remarkable therapeutic effects in the phase 3 clinical trials, such as the AFFIRM and SENTINEL studies, convinced the FDA to approve natalizumab in 2004 for the accelerated First Track Program for the treatment of multiple sclerosis [7,64]. Following the clinical trials in multiple sclerosis patients, natalizumab was clinically investigated for its therapeutic efficacy in Crohn’s disease, since it can block not only integrin α4β1, but also integrin α4β7 [67]. Indeed, before the ACT-1 was tested, another preclinical study had treated the cotton-top tamarin IBD model with this antibody to integrin α4 subunit, though not to integrin α4β7, and it had been shown to alleviate colitis [68]. After partial success in clinical trials, including the Efficacy of Natalizumab as Active Crohn’s Therapy (ENACT-1) induction trial, which included 905 Crohn’s disease patients, and the Evaluation of Natalizumab as Continuous Therapy (ENACT2) maintenance trial, which included 339 patients who had responded in the ENACT-1 trial, natalizumab was approved in 2008 by the FDA for the treatment of Crohn’s disease [67].

IX. IATROGENIC AND GENETIC IMMUNE-DEFICIENCIES INVOLVING INTEGRINS Antiinflammation is a double-edged sword; treatment with potent antiinflammatory drugs often carries the risk of excessive antiinflammation, which could induce iatrogenic immune suppression [68,69]. Natalizumab is not an exception. The first major setback in the use of natalizumab involved a few multiple sclerosis

and Crohn’s disease patients who developed progressive multifocal leukoencephalopathy (PML) [7,68]. PML is caused by reactivation of a latent John Cunningham (JC) virus infection during immunosuppression. The major clinical and pathological manifestations of PML include severe damage to the brain elicited by viral replication, inflammation, and cell death, thereby inducing often fatal encephalitis. Four months after its accelerated approval, when these fatal PML cases were revealed, natalizumab, whose US trade name is Tysabri, was voluntarily withdrawn from the market [7,63]. The FDA performed a comprehensive review of all multiple sclerosis and Crohn’s disease patients treated with natalizumab during clinical trials. Given its remarkable clinical efficacy to alleviate clinical severity in relapsing multiple sclerosis, which had not been achieved by any other treatments, the FDA needed to carefully weigh the balance between the associated risk of fatal PML and natalizumab’s beneficial effects [8]. Although natalizumab was reapproved by the FDA for the treatment of relapsing multiple sclerosis, its indication is strictly restricted to those patients who have had a suboptimal response to standard treatment. A comprehensive review of all patients, including those in postmarketing surveillance, found the presence of positive-serum anti-JC virus antibody titers. The presence of these antibody titers serves as a robust biomarker and constitutes an important component of the enhanced patient safety and pharmacovigilance program known as TOUCH (Tysabri Outreach Unified Commitment to Health), a prescription program in the United States [8]. In hindsight, the induction of iatrogenic immune suppression by lymphocyte integrin antagonists was not surprising, given the critical importance of integrins to lymphocytes in immune competence for host defense. The latter is illustrated by subtypes of a rare genetic disorder known as leukocyte adhesion deficiency (LAD), which constitutes a primary

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X. THERAPEUTIC INTEGRIN INHIBITION FOR HIV INFECTION

immune defect [70]. LAD types 1, 2, and 3 are all characterized by recurrent and often fatal infections due to impairments in many important cell adhesion-related immune functions. LAD-1 is caused by a genetic defect in the β2 integrin gene, resulting in the loss of all leukocyte integrin family members such as αLβ2 (LFA-1), αMβ2 (Mac-1), αXβ2, and αDβ2. LAD-2 is caused by a genetic defect in the GDP fucose transporter critical for the generation of carbohydrate selectin ligand sialyl Lewis X (SLex). LAD-3 is caused by a genetic defect in kindlin-3, which constitutes the critical signaling pathway to integrin activation [71]. In LAD-3, unlike LAD-1, the expression of integrins on such lymphocytes as αLβ2, α4β1, and probably α4β7 are unaffected. However, their binding to cognate integrin ligands is severely impaired [72]. Specifically, chemokine and other agonistic activation failed to enhance the adhesiveness of those integrins [73]. Although the role of α4 integrins in the pathogenesis of inflammation in the brain and gut are well documented, the role of α4 integrin in the maintenance of viral immunity, including that to JC virus, remained among the least understood by biomedical researchers until the revelations of the PML cases in human trials of integrin α4 therapeutic inhibition. Excluding patients who are at high risk (e.g., positive-serum JC virus titer, prior or current immunosuppressive therapy, and duration of natalizumab treatment) is an effective and conservative approach to ensure that natalizumab can continue to be used for the treatment of multiple sclerosis and Crohn’s disease [8]. Natalizumab not only validated the α4 integrins as the therapeutic target for alleviating inflammation in the central nervous system and gut, but also revealed the iatrogenic immune defects causing PML and how to identify and exclude patients at high risk. Building on the FDA approval of natalizumab, firategrast, an orally active smallmolecule antagonist to integrin α4, has undergone clinical trials in multiple sclerosis patients

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that thus far are showing therapeutic effectiveness with no occurrence of PML [74]. In addition to a conservative approach for excluding patients at high risk, a more creative question to address in the development of therapeutic applications of anti-cell adhesion molecules was whether or not the highly selective inhibition of only integrin α4β7, while not affecting integrin α4β1, might mitigate the risk of PML. It has been thought that integrin α4β1, but not α4β7, is responsible for immune surveillance of the central nervous system [8]. This has led to the hypothesis that an antibody that blocks only integrin α4β7 could greatly reduce the risk of PML while also retaining the therapeutic potential for Crohn’s disease. This led to the clinical development and FDA approval of vedolizumab, which is highly selective to integrin α4β7, for the treatment of Crohn’s disease and ulcerative colitis [75].

X. THERAPEUTIC INTEGRIN INHIBITION FOR HIV INFECTION Migration and retention of pathogen-specific T cells are an integral part of mucosal immunity for protecting the host from infections. In this context, boosting the activity of integrins to bind to cellular ligands is supposed to be beneficial for augmenting the host defense to eliminate pathogens [76,77]. However, another compounding factor is that some virus proteins function as integrin ligands, thereby exploiting cell adhesion molecules as entry receptors to cells for establishing infections [78,79]. One recently revealed significant example of integrin-mediated virus host interactions is the binding of integrin α4β7 to the HIV envelope protein gp120 [80]. The latter is known to bind to its primary cellular receptor CD4 as well as to its coreceptors CCR5 and CXCR4. Integrin α4β7 has been shown to laterally associate with CD4 on the cell surface of T cells [81]. Like the authentic ligand MAdCAM-1, binding

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of gp120 to integrin α4β7 transmits outside-in signals to activate lymphocytes, potentially facilitating virus propagation and dissemination of mucosally infected viruses to systemic sites. Given the possibility that the interaction of integrin α4β7 with the HIV envelope protein gp120 could promote and sustain viral infection, the effects of integrin α4β7 inhibition by vedolizumab on viral infection have been studied in rhesus macaque monkeys challenged with SIV, an established HIV infection model that is highly relevant to humans. Monkeys infected with SIV were treated with antiretroviral therapy (ART) combined with the simianized vedolizumab, resulting in the suppression of circulating SIV to undetectable levels [82]. Remarkably, even after stopping the ART and vedolizumab, SIV remained undetectable, thereby supporting the possibility that the virus was completely eradicated. It has been shown that the exploitation of immune cell trafficking by viruses can play a pivotal role in the pathogenesis of sustained infection. Furthermore, the trafficking pathway hijacked by a virus can be expected to serve as a promising therapeutic target. The therapeutic efficacy of vedolizumab in combination with ART for the treatment of HIV infection is currently being investigated in clinical trials.

References [1] Sigmundsdottir H, Butcher EC. Environmental cues, dendritic cells and the programming of tissue-selective lymphocyte trafficking. Nat Immunol 2008;9:981 7. [2] Lian J, Luster AD. Chemokine-guided cell positioning in the lymph node orchestrates the generation of adaptive immune responses. Curr Opin Cell Biol 2015;36:1 6. [3] Mora JR, Iwata M, von Andrian UH. Vitamin effects on the immune system: vitamins A and D take centre stage. Nat Rev Immunol 2008;8:685 98. [4] Berlin C, Bargatze RF, Campbell JJ, von Andrian UH, Szabo MC, Hasslen SR, et al. alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 1995;80:413 22.

[5] Carman CV, Springer TA. Integrin avidity regulation: are changes in affinity and conformation underemphasized? Curr Opin Cell Biol 2003;15:547 56. [6] Shimaoka M, Takagi J, Springer TA. Conformational regulation of integrin structure and function. Annu Rev Biophys Biomol Struct 2002;31:485 516. [7] Kawamoto E, Nakahashi S, Okamoto T, Imai H, Shimaoka M. Anti-integrin therapy for multiple sclerosis. Autoimmune Dis 2012;2012:357101. [8] Stu¨ve O, Marra CM, Jerome KR, Cook L, Cravens PD, Cepok S, et al. Immune surveillance in multiple sclerosis patients treated with natalizumab. Ann Neurol 2006;59:743 7. [9] Bryant RV, Sandborn WJ, Travis SP. Introducing vedolizumab to clinical practice: who, when, and how? J Crohns Colitis 2015;9:356 66. [10] Marshall BT, Long M, Piper JW, Yago T, McEver RP, Zhu C. Direct observation of catch bonds involving cell-adhesion molecules. Nature 2003;423:190 3. [11] Puri KD, Finger EB, Springer TA. The faster kinetics of L-selectin than of E-selectin and P-selectin rolling at comparable binding strength. J Immunol 1997;158:405 13. [12] Zarbock A, Abram CL, Hundt M, Altman A, Lowell CA, Ley K. PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcR gamma to induce slow leukocyte rolling. J Exp Med 2008;205:2339 47. [13] Comrie WA, Babich A, Burkhardt JK. F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse. J Cell Biol 2015;208:475 91. [14] Shulman Z, Shinder V, Klein E, Grabovsky V, Yeger O, Geron E, et al. Lymphocyte crawling and transendothelial migration require chemokine triggering of highaffinity LFA-1 integrin. Immunity 2009;30:384 96. [15] Carman CV, Springer TA. Trans-cellular migration: cell-cell contacts get intimate. Curr Opin Cell Biol 2008;20:533 40. [16] Barzilai S, Yadav SK, Morrell S, Roncato F, Klein E, Stoler-Barak L, et al. Leukocytes breach endothelial barriers by insertion of nuclear lobes and disassembly of endothelial actin filaments. Cell Rep 2017;18:685 99. [17] Nieminen M, Henttinen T, Merinen M, MarttilaIchihara F, Eriksson JE, Jalkanen S. Vimentin function in lymphocyte adhesion and transcellular migration. Nat Cell Biol 2006;8:156 62. [18] Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end game. Nat Rev Mol Cell Biol 2010;11:288 300. [19] Moser M, Legate KR, Zent R, Fa¨ssler R. The tail of integrins, talin, and kindlins. Science 2009;324:895 9. [20] Comrie WA, Li S, Boyle S, Burkhardt JK. The dendritic cell cytoskeleton promotes T cell adhesion and

II. PRINCIPLES OF MUCOSAL VACCINE

97

REFERENCES

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

activation by constraining ICAM-1 mobility. J Cell Biol 2015;208:457 73. Thomas W. Catch bonds in adhesion. Annu Rev Biomed Eng 2008;10:39 57. Astrof NS, Salas A, Shimaoka M, Chen J, Springer TA. Importance of force linkage in mechanochemistry of adhesion receptors. Biochemistry 2006;45:15020 8. Nordenfelt P, Elliott HL, Springer TA. Coordinated integrin activation by actin-dependent force during Tcell migration. Nat Commun 2016;7:13119. Feigelson SW, Pasvolsky R, Cemerski S, Shulman Z, Grabovsky V, Ilani T, et al. Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state. J Immunol 2010;185:7394 404. Semmrich M, Smith A, Feterowski C, Beer S, Engelhardt B, Busch DH, et al. Importance of integrin LFA-1 deactivation for the generation of immune responses. J Exp Med 2005;201:1987 98. Park EJ, Mora JR, Carman CV, Chen J, Sasaki Y, Cheng G, et al. Aberrant activation of integrin alpha4beta7 suppresses lymphocyte migration to the gut. J Clin Invest 2007;117:2526 38. Morin NA, Oakes PW, Hyun YM, Lee D, Chin YE, Chin EY, et al. Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration. J Exp Med 2008;205:195 205. Stanley P, Tooze S, Hogg N. A role for Rap2 in recycling the extended conformation of LFA-1 during T cell migration. Biol Open 2012;1:1161 8. Dagan-Berger M, Feniger-Barish R, Avniel S, Wald H, Galun E, Grabovsky V, et al. Role of CXCR3 carboxyl terminus and third intracellular loop in receptormediated migration, adhesion and internalization in response to CXCL11. Blood 2006;107:3821 31. Liu Q, Chen H, Ojode T, Gao X, Anaya-O’Brien S, Turner NA, et al. WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4. Blood 2012;120:181 9. Bouvard D, Pouwels J, De Franceschi N, Ivaska J. Integrin inactivators: balancing cellular functions in vitro and in vivo. Nat Rev Mol Cell Biol 2013;14:430 42. Liu W, Draheim KM, Zhang R, Calderwood DA, Boggon TJ. Mechanism for KRIT1 release of ICAP1mediated suppression of integrin activation. Mol Cell 2013;49:719 29. Park EJ, Yuki Y, Kiyono H, Shimaoka M. Structural basis of blocking integrin activation and deactivation for anti-inflammation. J Biomed Sci 2015;22:51. Imai Y, Park EJ, Peer D, Peixoto A, Cheng G, von Andrian UH, et al. Genetic perturbation of the putative

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

cytoplasmic membrane-proximal salt bridge aberrantly activates alpha(4) integrins. Blood 2008;112:5007 15. Park EJ, Peixoto A, Imai Y, Goodarzi A, Cheng G, Carman CV, et al. Distinct roles for LFA-1 affinity regulation during T-cell adhesion, diapedesis, and interstitial migration in lymph nodes. Blood 2010;115:1572 81. Lim K, Hyun YM, Lambert-Emo K, Capece T, Bae S, Miller R, et al. Neutrophil trails guide influenzaspecific CD81 T cells in the airways. Science 2015;349: aaa4352. Weninger W, Biro M, Jain R. Leukocyte migration in the interstitial space of non-lymphoid organs. Nat Rev Immunol 2014;14:232 46. Paluch EK, Aspalter IM, Sixt M. Focal adhesionindependent cell migration. Annu Rev Cell Dev Biol 2016;32:469 90. La¨mmermann T, Bader BL, Monkley SJ, Worbs T, Wedlich-So¨ldner R, Hirsch K, et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 2008;453:51 5. Woolf E, Grigorova I, Sagiv A, Grabovsky V, Feigelson SW, Shulman Z, et al. Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces. Nat Immunol 2007;8:1076 85. Overstreet MG, Gaylo A, Angermann BR, Hughson A, Hyun YM, Lambert K, et al. Inflammation-induced interstitial migration of effector CD41 T cells is dependent on integrin αV. Nat Immunol 2013;14:949 58. Moreau HD, Lemaıˆtre F, Terriac E, Azar G, Piel M, Lennon-Dumenil AM, et al. Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo. Immunity 2012;37:351 63. Moreau HD, Bousso P. In vivo imaging of T cell immunological synapses and kinapses in lymph nodes. Methods Mol Biol 2017;1584:559 68. Dustin ML, Choudhuri K. Signaling and polarized communication across the T cell immunological synapse. Annu Rev Cell Dev Biol 2016;32:303 25. Clark RH, Stinchcombe JC, Day A, Blott E, Booth S, Bossi G, et al. Adaptor protein 3-dependent microtubule-mediated movement of lytic granules to the immunological synapse. Nat Immunol 2003;4:1111 20. Pentcheva-Hoang T, Chen L, Pardoll DM, Allison JP. Programmed death-1 concentration at the immunological synapse is determined by ligand affinity and availability. Proc Natl Acad Sci U S A 2007;104:17765 70. Reichardt P, Dornbach B, Gunzer M. The molecular makeup and function of regulatory and effector synapses. Immunol Rev 2007;218:165 77. Ni HT, Deeths MJ, Li W, Mueller DL, Mescher MF. Signaling pathways activated by leukocyte function-

II. PRINCIPLES OF MUCOSAL VACCINE

98

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

5. MUCOSAL IMMUNITY FOR INFLAMMATION

associated Ag-1-dependent costimulation. J Immunol 1999;162:5183 9. Sigmundsdottir H, Pan J, Debes GF, Alt C, Habtezion A, Soler D, et al. DCs metabolize sunlight-induced vitamin D3 to ’program’ T cell attraction to the epidermal chemokine CCL27. Nat Immunol 2007;8:285 93. Mora JR, Iwata M, Eksteen B, Song SY, Junt T, Senman B, et al. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science 2006;314:1157 60. Erkelens MN, Mebius RE. Retinoic acid and immune homeostasis: a balancing act. Trends Immunol 2017;38:168 80. Gorfu G, Rivera-Nieves J, Ley K. Role of beta7 integrins in intestinal lymphocyte homing and retention. Curr Mol Med 2009;9:836 50. Briskin MJ, Rott L, Butcher EC. Structural requirements for mucosal vascular addressin binding to its lymphocyte receptor alpha 4 beta 7. Common themes among integrin-Ig family interactions. J Immunol 1996;156:719 26. Mebius RE, Schadee-Eestermans IL, Weissman IL. MAdCAM-1 dependent colonization of developing lymph nodes involves a unique subset of CD4 1 CD3hematolymphoid cells. Cell Adhes Commun 1998;6:97 103. Pabst O, Fo¨rster R, Lipp M, Engel H, Arnold HH. NKX2.3 is required for MAdCAM-1 expression and homing of lymphocytes in spleen and mucosaassociated lymphoid tissue. EMBO J 2000;19:2015 23. Arihiro S, Ohtani H, Suzuki M, Murata M, Ejima C, Oki M, et al. Differential expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in ulcerative colitis and Crohn’s disease. Pathol Int 2002;52:367 74. Grant AJ, Lalor PF, Salmi M, Jalkanen S, Adams DH. Homing of mucosal lymphocytes to the liver in the pathogenesis of hepatic complications of inflammatory bowel disease. Lancet 2002;359:150 7. Danese S, Pane´s J. Development of drugs to target interactions between leukocytes and endothelial cells and treatment algorithms for inflammatory bowel diseases. Gastroenterology 2014;147:981 9. Hesterberg PE, Winsor-Hines D, Briskin MJ, SolerFerran D, Merrill C, Mackay CR, et al. Rapid resolution of chronic colitis in the cotton-top tamarin with an antibody to a gut-homing integrin alpha 4 beta 7. Gastroenterology 1996;111:1373 80. Schweighoffer T, Tanaka Y, Tidswell M, Erle DJ, Horgan KJ, Luce GE, et al. Selective expression of integrin alpha 4 beta 7 on a subset of human CD4 1 memory T cells with Hallmarks of gut-trophism. J Immunol 1993;151:717 29.

[61] Wyant T, Estevam J, Yang L, Rosario M. Development and validation of receptor occupancy pharmacodynamic assays used in the clinical development of the monoclonal antibody vedolizumab. Cytometry B Clin Cytom 2016;90:168 76. [62] Danese S, Vuitton L, Peyrin-Biroulet L. Biologic agents for IBD: practical insights. Nat Rev Gastroenterol Hepatol 2015;12:537 45. [63] Poulakos M, Machin JD, Pauly J, Grace Y. Vedolizumab: a new opponent in the battle against Crohn’s disease and ulcerative colitis. J Pharm Pract 2016;29:503 15. [64] Rice GP, Hartung HP, Calabresi PA. Anti-alpha4 integrin therapy for multiple sclerosis: mechanisms and rationale. Neurology 2005;64:1336 42. [65] Miller DH, Soon D, Fernando KT, MacManus DG, Barker GJ, Yousry TA, et al. MRI outcomes in a placebo-controlled trial of natalizumab in relapsing MS. Neurology 2007;68:1390 401. [66] Rudick RA, Stuart WH, Calabresi PA, Confavreux C, Galetta SL, Radue EW, et al. Natalizumab plus interferon beta-1a for relapsing multiple sclerosis. N Engl J Med 2006;354:911 23. [67] Sandborn WJ, Colombel JF, Enns R, Feagan BG, Hanauer SB, Lawrance IC, et al. IEoNaACsTE-T, group EoNaCTE-T. Natalizumab induction and maintenance therapy for Crohn’s disease. N Engl J Med 2005;353:1912 25. [68] Podolsky DK, Lobb R, King N, Benjamin CD, Pepinsky B, Sehgal P, et al. Attenuation of colitis in the cottontop tamarin by anti-alpha 4 integrin monoclonal antibody. J Clin Invest 1993;92:372 80. [69] DePry JL, Reed KB, Cook-Norris RH, Brewer JD. Iatrogenic immunosuppression and cutaneous malignancy. Clin Dermatol 2011;29:602 13. [70] Schmidt S, Moser M, Sperandio M. The molecular basis of leukocyte recruitment and its deficiencies. Mol Immunol 2013;55:49 58. [71] Ye F, Snider AK, Ginsberg MH. Talin and kindlin: the one-two punch in integrin activation. Front Med 2014;8:6 16. [72] Moretti FA, Moser M, Lyck R, Abadier M, Ruppert R, Engelhardt B, et al. Kindlin-3 regulates integrin activation and adhesion reinforcement of effector T cells. Proc Natl Acad Sci U S A 2013;110:17005 10. [73] Malinin NL, Zhang L, Choi J, Ciocea A, Razorenova O, Ma YQ, et al. A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans. Nat Med 2009;15:313 18. [74] Miller DH, Weber T, Grove R, Wardell C, Horrigan J, Graff O, et al. Firategrast for relapsing remitting multiple sclerosis: a phase 2, randomised, double-blind, placebo-controlled trial. Lancet Neurol 2012;11:131 9.

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[75] Verstockt B, Ferrante M, Vermeire S, Van Assche G. New treatment options for inflammatory bowel diseases. J Gastroenterol 2018;53:585 90. [76] Mwanza-Lisulo M, Kelly P. Potential for use of retinoic acid as an oral vaccine adjuvant. Philos Trans R Soc Lond B Biol Sci 2015;370. [77] Belyakov IM, Berzofsky JA. Immunobiology of mucosal HIV infection and the basis for development of a new generation of mucosal AIDS vaccines. Immunity 2004;20:247 53. [78] LaFoya B, Munroe JA, Miyamoto A, Detweiler MA, Crow JJ, Gazdik T, et al. Beyond the matrix: the many non-ECM ligands for integrins. Int J Mol Sci 2018;19. [79] Stewart PL, Nemerow GR. Cell integrins: commonly used receptors for diverse viral pathogens. Trends Microbiol 2007;15:500 7.

99

[80] Arthos J, Cicala C, Martinelli E, Macleod K, Van Ryk D, Wei D, et al. HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nat Immunol 2008;9:301 9. [81] Cicala C, Martinelli E, McNally JP, Goode DJ, Gopaul R, Hiatt J, et al. The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1. Proc Natl Acad Sci U S A 2009;106: 20877 82. [82] Byrareddy SN, Arthos J, Cicala C, Villinger F, Ortiz KT, Little D, et al. Sustained virologic control in SIV 1 macaques after antiretroviral and α4β7 antibody therapy. Science 2016;354:197 202.

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Innate Immunity at Mucosal Surfaces Kosuke Fujimoto1,2 and Satoshi Uematsu1,2 1

Department of Immunology and Genomics, Osaka City University Graduate School of Medicine, Osaka, Japan 2Division of Innate Immune Regulation, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

I. INTRODUCTION The mucosal immune system, which comprises the gastrointestinal tract, the respiratory tract, and the urogenital tract, protects the internal surfaces of the body from damage. Among these tracts, the gastrointestinal tract, which consists of the oral cavity, esophagus, stomach, and intestines, is unique because its lumen is constantly exposed to food particles and pathogenic microorganisms. The large surface area (almost 400 m2) of the intestine is, in fact, much larger than the surface area of the skin or lungs in humans [1,2]. Hosts have developed a multidefense system for impeding pathogen invasion [3]. For instance, the intestinal epithelium and its mucus layer play pivotal roles in the frontline defenses of the mucosal surfaces, and as such they are regarded as constituents of the innate immune system [4,5]. A wide variety of epithelial cells, including absorptive epithelial cells, endocrine cells, goblet cells, tuft cells, Paneth cells, stem cells, transit-amplifying cells, and M cells, make up a thin layer of the epithelium and create a physiological barrier against external antigens (Fig. 6.1) [6] (Chapter 3: Mucosal Antigen Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00006-7

Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells and Chapter 28: M Cell-Targeted Vaccines). Innate immunity also protects the gastrointestinal mucosa from damage by effectively recognizing and eliminating pathogens from hosts [7 9]. Many cell types, such as dendritic cells, macrophages, and epithelial cells, play important roles in the innate immune response initiated by the germline-encoded pattern recognition receptors (PRRs), which recognize specific patterns in the microbial components expressed by microorganisms [10]. Recently, communication between the innate immune system and the intestine via disturbed intestinal function has been shown to be involved in a multitude of diseases [9,11], such as rheumatoid arthritis [12 15], ankylosing spondylitis [16,17], type 1 diabetes [18,19], inflammatory bowel diseases (IBDs) [20 26], allergic diseases [27], nonalcoholic fatty liver disease [28], carcinogenesis [29,30], obesity [31 33], and atherosclerosis [34 37] (Fig. 6.2). This indicates that innate immune responses provide a solid causal link between the disease-associated alteration of the intestinal environment known as dysbiosis and the pathophysiological functions of the host.

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FIGURE 6.1 Intestinal epithelial cells. Diagrammatic representation of intestinal epithelial cells in the gut. There are many different types of intestinal epithelial cells: absorptive epithelial cells, endocrine cells, goblet cells, tuft cells, Paneth cells, stem cells, transit-amplifying cells, and M cells. These cells play crucial roles in regulating intestinal homeostasis.

FIGURE 6.2 Relationships between intestinal mucosal dysfunction and human diseases. Alterations in innate mucosal immunity, such as disruption of the intestinal barrier system and innate immune function in the gut, contribute to the induction and/or exacerbation of many inflammatory disorders, including rheumatoid arthritis, ankylosing spondylitis, type 1 diabetes, inflammatory bowel diseases, allergic diseases, nonalcoholic fatty liver disease, carcinogenesis, obesity, and atherosclerosis (Chapter 52: Mucosal Immunity for Inflammation).

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In addition, several lines of evidence have shown that dysfunctional PRRs are involved in the induction of intestinal inflammation [5,38]. This chapter describes the current knowledge on intestinal mucosal immunity, with special focus placed on the innate immune responses occurring at the mucosal surfaces of the gut.

II. INNATE MUCOSAL BARRIERS IN THE GUT A. Structures of the Mucosal Barriers There are two main types of mucosal barriers in the small and large intestines: physical barriers and chemical barriers [5]. They separate a plethora of foreign antigens in the form of commensal microorganisms, pathogenic microbes, and food antigens on the luminal side and superbly regulate gut homeostasis. Several types of intestinal epithelial cells, such as goblet cells, Paneth cells, and absorptive epithelial cells, are present in the two barriers. Physical barriers are represented mostly by cell cell junctions and the mucus layers produced by intestinal secretary cells. The biophysical features of these barriers are central to protecting the mucosal surfaces from various intestinal components. Cell cell junctions such as tight junctions are critically important to the maintenance of the intestinal barrier system [39 41]. The huge diversity of microbiota and food antigens present in the intestines makes effective regulation of junctional integrity and paracellular permeability essential for maintaining innate immune mucosal homeostasis. Dysfunctional intestinal permeability has been shown to induce various intestinal inflammation conditions [see 1,2]. It should be noted that the mucus layers in the large intestine differ from those in the small intestine. To be more precise, in the small intestine, a variety of antimicrobial peptides (AMPs) such as defensins, which are major contributors to the chemical

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barriers in this organ, are crucially important for the segregation of commensal bacteria and intestinal epithelial cells [42,43]. In contrast, the mucus layers of the large intestine are thick (10 700 μm). They are divided into an inner firm mucus layer and an outer loose mucus layer and are organized in this fashion by gelforming MUC2 (Fig. 6.3). The sterile inner mucus layer acts as an efficient physical barrier against bacteria [44]. Conversely, huge numbers of commensal microbiota exist in the outer mucus layer [44,45], suggesting that the outer mucus layer in the large intestine forms a unique microbial niche. In fact, the number of goblet cells in the large intestine is much higher than that in the small intestine; hence the mucus layer in the large intestine is thought to be fundamentally thick. This thick mucus layer is able to establish a concentration gradient, resulting in retention of AMPs and immunoglobulins. Germ-free animals have decreased numbers of goblet cells and reduced goblet cell thecae sizes. However, under germ-free conditions, goblet cells are constantly produced despite being unnecessary for protection against enteropathogens [46]. These findings suggest that intestinal microorganisms are only partially essential for the induction of goblet cells. Chemical barriers include various types of AMPs that mainly segregate intestinal epithelial cells from intestinal microorganisms [47,48]. As antimicrobial molecules, AMPs such as the defensins produced by Paneth cells in the small intestine are gene-encoded natural antibiotics that regulate the intestinal microbiota [49,50]. Defensins are active against Gram-negative and Gram-positive bacteria, as well as against fungi, viruses, and protozoa [4,51], and are thought to be key factors in innate mucosal immunity in the intestine. The 17 defensins found at various bodily sites in humans fall into two major categories: α-defensins and β-defensins [52]. Panethcell-related dysbiosis has revealed the role played by defensins in the host mucosal immune system [53]. Indeed, studies of several mouse

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FIGURE 6.3 Distinct intestinal barriers in the small and large intestines. Diagrammatic representation of the secreted mucosal barrier in (A) the small intestine and (B) the large intestine. Specialized secretory cells such as goblet cells and Paneth cells are shown. In the small intestine, chemical barriers represented by the AMPs produced by Paneth cells are central factors for the segregation of intestinal microorganisms and intestinal epithelial cells. In the large intestine, however, the enormous number of intestinal microorganisms and intestinal epithelial cells are separated by an inner mucus layer, which is a special feature of the intestinal mucus of this organ.

strains with genetically modified α-defensin production by their Paneth cells showed that the gut microbiome composition in the small intestine is influenced by the α-defensins secreted by Paneth cells [53,54]. In HD5 transgenic mice, which express human Paneth cell defensin (DEFA5) in their own Paneth cells, the ratio of Bacteroides to Firmicutes was higher than that in their wildtype littermate controls [55]. In contrast, in matrix metalloproteinase-7-deficient mice, which lack mature α-defensin in their Paneth cells, the ratio of Bacteroides to Firmicutes decreased in comparison with that of their wild-type littermate controls [55]. These findings indicate that Paneth cell defensins can regulate the commensal microbiome and may be involved in the

pathophysiology of dysbiosis related-diseases such as IBDs. However, interestingly, there are no Paneth cells in the large intestine, and the level of antimicrobial peptides is lower than that in the small intestine [56]. A recent study has clearly shown that Ly6/Plaur domain-containing 8 (Lypd8) is essential for segregating the intestinal bacteria and intestinal epithelial cells in the large intestine [57]. Lypd8 can bind to flagellated bacteria (e.g., Escherichia, Proteus, and Helicobacter) and inhibit bacterial invasion of colonic epithelial cells [57]. Lypd8-deficient mice also have an absence of bacterial free space [57], suggesting that Lypd8 regulates intestinal homeostasis by suppressing bacterial invasion of the colonic mucosa.

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B. Innate Barrier Dysfunction and Disease Pathogenesis The innate mucosal barrier system is indispensible for the regulation of gut homeostasis. As well as disruption of the epithelial barrier, access of luminal contents to the lamina propria and the abnormal immune response that follows are key factors for the progression of intestinal diseases. A variety of intestinal diseases, including IBDs [58 60], Clostridioidesdifficile (C. difficile)-induced colitis [61], enteropathogenic Escherichia coli (EPEC) infection [62], and graftversus-host disease (GVHD) [63], are associated with a dysfunctional mucosal barrier in the gut. Recent genome-wide association studies have identified more than 200 susceptible loci for the IBDs represented by Crohn’s disease (CD) and ulcerative colitis (UC) [64,65]. Interestingly, these genetic analyses have strongly linked an increased intestinal permeability with such diseases. For example, a frameshift insertion at position 3020 of the gene encoding the cytoplasmic sensor nucleotidebinding oligomerization domain-containing 2 (NOD2) protein induces barrier defects in patients with CD [66]. MUC19 is one of the gelforming mucins expressed in epithelial cells along with MUC2, MUC5AC, MUC5B, and MUC6 [67]. Genetic variants of MUC19 in patients with IBD cause quantitative changes in mucus production or structural changes in the glycoprotein core, resulting in mucosal barrier dysfunction [68]. In addition to being associated with UC, polymorphism in the RING finger protein 186, which is a member of the RING finger protein family, induces increased intestinal permeability and an increased risk of intestinal inflammation with enhanced endoplasmic reticulum stress in colonic epithelial cells [69]. Furthermore, nonsense polymorphisms in the gene encoding fucosyltransferase 2 (FUT2) are correlated with various pathophysiological conditions in humans [3]. Loss-of-

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function mutations in FUT2 are associated with microbiome changes and an increased risk of developing IBDs [70,71]. Antibiotic use has increased the number of infectious diseases that are curable. However, the extensive use of antibiotics has caused an increase in the number of infections with C. difficile, which is a spore-forming, toxinproducing, Gram-positive anaerobic bacterium that normally colonizes healthy individuals. C. difficile produces toxins A and B, which are major virulence factors for C. difficile-induced colitis [72 74]. Release of these toxins at disease onset disrupts actomyosin and induces glycotransferase toxin expression in this bacterium. Subsequent inactivation of RHO or RAC GTPases causes dysfunctional tight junctions and disrupts the epithelial integrity of the gut [61,75]. In developing countries, EPEC infections are among the leading causes of childhood mortality [76]. Both the architecture and the barrier function of intestinal tight junctions are altered by EPEC, and the apical basal polarity of intestinal cells is also perturbed by such infections [77]. The type III secretion system, which is a special protein-export apparatus in Gramnegative bacteria, is involved in this intestinal barrier defect [78]. GVHD develops after transplantation with bone marrow or hematopoietic stem cells. Recently, it has been found that intestinal barrier dysfunction plays a role in the pathogenesis of GVHD [79 81]. For example, altered expression and localization of occludin, which is a tight junction molecule, contribute to increased intestinal permeability and disruption of tight junctions [82,83]. Structural epithelial alterations occur when increased claudin-2 expression occurs [84] and epithelial cells undergo apoptosis [85]. Pertinently, the resulting intestinal barrier loss is thought to be associated with alterations in the gut microbiota in GVHD as well as in IBDs [86 91].

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III. INNATE IMMUNE REGULATION IN THE GUT A. Pattern Recognition Receptors The immune system is divided into two major branches: the innate immune system and the adaptive immune system. After pathogenic microorganisms invade beyond the structural barriers in the host, the first line of defense against pathogens is innate immunity, and the second line is adaptive immunity. A key function of innate immunity is to recognize invading pathogens and quickly mount defensive responses against them. For a long time, innate immunity has been considered to be nonspecific in its actions, its main functions being to digest pathogens and to present antigens to the cells involved in adaptive immunity. However, recent studies have shown that innate immunity is not necessarily nonspecific but is specific enough to distinguish between self and pathogens using evolutionarily preserved receptors, such as toll-like receptors (TLRs) [10,92 94]. Germline-encoded PRRs can sense microorganisms. Currently, four different PRR family classes have been identified: TLRs, C-type lectin receptors (CLRs), retinoic-acid-inducible gene-I-like receptors (RLRs), and NOD-like receptors (NLRs) [95]. PRRs recognize the highly conserved pathogen-associated molecular patterns (PAMPs) expressed by microorganisms but not by mammalian host cells. After PAMP recognition, PRRs immediately induce a series of signaling systems that execute the first line of host defensive responses. PRR signaling also introduces the maturation of particular immune cells, such as dendritic cells (DCs), which subsequently leads to activation of antigen-specific acquired immunity. The intestinal barrier system uses many different mechanisms to protect mucosal surfaces from pathogens. For example, intestinal epithelial cells express PRRs and can recognize intestinal microorganisms through secretion of

AMPs and cytokines [96]. A deficiency of PRRrelated genes in the intestinal epithelial cells of mice has revealed the crucial role they play in mucosal immune homeostasis [97,98], indicating that the function of PRRs in the mucosal barrier is a fundamental aspect of innate mucosal immunity.

B. Associations Between Individual TLRs and Intestinal Inflammation TLRs were identified as the first PRRs. To date, the mammalian TLR family comprises 13 members, and 10 and 12 functional TLRs have been characterized in humans (TLR1 TLR10) and mice (TLR1 TLR9, TLR11 TLR13), respectively. Each TLR recognizes distinct PAMPs derived from bacteria, viruses, mycobacteria, fungi, and parasites (Table 6.1). In common with TABLE 6.1 TLR Ligands TLR family Ligands TLR1

Triacyl lipopeptides

TLR2

Peptidoglycan, lipoprotein, lipopeptides, lipoteichoic acid, zymosan, glycolipids, GPI anchored

TLR3

dsRNA, Poly(I:C)

TLR4

LPS, endogenous ligands (HSPS, fibronectin, hyaluronic acid)

TLR5

Flagellin

TLR6

Diacyl lipopeptides

TLR7

ssRNA, imiquimod

TLR8

ssRNA, imiquimod

TLR9

CpG DNA, hemozin

TLR10

Unknown

TLR11

Profilin, flagellin

TLR12

Profilin

TLR13

Bacterial 23S ribosomal RNA

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other organs, intestinal TLRs play an essential role in the induction of host defenses (Chapter 10: Innate Immunity based Mucosal Modulator and Adjuvant). However, their distribution and effects on the gut are noteworthy because of the presence of commensal microflora in this organ (Chapter 9: Influence of Commensal Miro-biota and metabolite for mucosal immunity). TLR1, which is functionally associated with TLR2 [99], is involved in the recognition of triacyl lipopeptides derived from bacteria [100]. It has been reported that none of the singlenucleotide polymorphisms (SNPs) in TLR1 are associated with a susceptibility to IBDs; however, a nonsynonymous variant in TLR1 (R80T) is linked to the pancolitis of UC [101]. The involvement of TLR2 in the recognition of components from a variety of microbial pathogens, such as peptidoglycan, lipopeptides, lipoteichoic acid, zymosan, and glycolipids, has been established [10]. TLR2 cooperates with TLR1 and TLR6, resulting in the ability to detect various microbial components. TLR2 constantly senses commensal microorganisms and regulates intestinal inflammation by controlling intestinal barrier functions [102 104]. In the inflamed colonic mucosa of IBD patients, TLR2 levels were higher than those in the controls in one study [105]. Additionally, polymorphisms in TLR2 are associated with the risk of developing IBDs [106]. TLR3 is involved in recognizing the dsRNA produced by viruses during their replication. TLR3 is constitutively expressed on the intestinal epithelial cells of healthy individuals [107]. However, in active CD, TLR3 expression was found to be highly downregulated in intestinal epithelial cells [107]. TLR3 is a potential mediator of CCL20 production, the levels of which can be used to predict the risk of IBD. In fact, TLR3 silencing is believed to have an effect on intestinal inflammation [108]. TLR3 also plays a critical role in the pathogenesis of radiationinduced intestinal inflammation and the

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subsequent gastrointestinal syndrome associated with p53-dependent crypt cell death [109], indicating that targeting it may offer a therapeutic strategy for reducing intestinal inflammation. TLR4 mainly recognizes the lipid A component of lipopolysaccharide (LPS) from Gramnegative bacteria. LPS is a crucial mediator of the AMPs produced by intestinal epithelial cells and Paneth cells [42]. Several lines of evidence show that TLR4 is involved in the pathogenesis of IBDs. TLR4 expression is increased in both CD and UC [107]. TLR4-deficient mice and Myd88-deficient mice are highly susceptible to dextran sodium sulfate (DSS)-induced intestinal inflammation [38]. SNPs in TLR4, such as A299G in IBD patients, are associated with IBDs, and they carry an increased risk of infection occurring with Gram-negative bacteria [110 113]. The T399I polymorphism in TLR4 has also been reported in UC patients [114]. TLR4 rs4986790A . G and rs4986791C . T genetic polymorphisms are also correlated with an increased risk of IBDs [115]. TLR5, which recognizes bacterial flagellin, is expressed on the basolateral surface of intestinal epithelial cells, indicating that it performs a crucial role in detecting invasive flagellated bacteria at the mucosal surface [116,117]. When exposed to flagellin, interleukin 8 (IL-8) was found to be produced by human intestinal epithelial cell lines, and this resulted in the induction of neutrophil and immature DC migration [118]. Intestinal epithelial cell TLR5 also maintains the intestinal microbiome by preventing intestinal inflammation [119]. However, in the intestinal lamina propria, TLR5 is expressed mainly on CD11c1 DCs [120]. Low-density cells in the small intestinal lamina propria can be divided into four subsets based on their CD11c and CD11b expression patterns: CD11chiCD11blo DCs, CD11chiCD11bhi DCs, CD11cintCD11bint macrophages, and CD11cintCD11bhi eosinophils [121,122]. Among them, the CD11chiCD11bhi DCs, which have also been characterized as

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FIGURE 6.4 The role of TLR5 in the small intestine. TLR5 is expressed on the basolateral surface of intestinal epithelial cells. TLR5 is highly expressed by CD11chiCD11bhi lamina propria DCs in the small intestine. These specialized DCs recognize invasive flagellated bacteria through TLR5 and induce the expansion of IgA1 plasma cells, Th1 cells, and Th17 cells after TLR5 stimulation.

CD1031 conventional DCs, specifically express TLR5, and contribute not only to the differentiation of naı¨ve B cells into immunoglobulin A positive (IgA1) plasma cells, but also to the differentiation of antigen-specific T helper type 1 (Th1) and Th17 cells in response to flagellin (Fig. 6.4) [121,122]. Interestingly, the polymorphisms in TLR5 are correlated with UC in North Indian patients, in whom they have been found to induce decreased levels of inflammatory cytokines [123]. Functional SNPs in TLR5 have also been seen to be involved in the response to anti-tumor necrosis factor therapy in IBD patients [124]. In common with TLR1, TLR6 has been shown to functionally cooperate with TLR2. TLR6 recognizes the diacyl lipopeptides derived from bacteria [125]. TLR6 levels in the inflamed colon were higher than those in the noninflamed colon in one study [126]. TLR6 controls Th1 and Th17 responses in gastrointestinal-associated lymphoid tissue, and TLR6-deficient mice are protected against DSS-induced intestinal inflammation [126].

TLR7 is another important innate immune response regulator. It recognizes singlestranded (ss) RNA and synthetic compounds such as imiquimod, a medicinal immune response modifier. Imiquimod administration can ameliorate DSS-induced intestinal inflammation [127]. TLR7 stimulation induces type I interferon and antimicrobial molecules, indicating a potential role for TLR7 as an agonistic therapy for IBDs. TLR8, the gene for which is located on the X chromosome, is involved in recognizing ssRNA and imidazoquinolines in humans. However, mouse TLR8 is known to be nonfunctional [10]. TLR8 expressed on regulatory T cells plays an important role in regulating immune responses to cancer [128]. Furthermore, TLR8 haplotypes are correlated with IBDs in females, where they have also been shown to be predisposing and protective factors for these diseases [129]. TLR9 mediates the recognition of bacterial DNA containing unmethylated CpG motifs (otherwise known as CpG DNA) [130]. Expression of TLR9 on the apical side of

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intestinal epithelial cells contributes to the maintenance of colonic homeostasis when activated by bacterial DNA. TLR9 has a protective function against intestinal inflammation via the induction of type I interferon [131]. Increased levels of it in memory B cells from patients with IBD are correlated with disease severity [132]. Administration of a Western-style diet altered the small intestinal mucosa in TLR9 knockout mice but not in TLR2-, TLR4-, or NOD2deficient mice, indicating that TLR9 signaling is crucial for regulating the small intestinal mucosa [133]. Despite thousands of reports about TLRs in the scientific literature, a role for TLR10 remains elusive. Very recently, it has been shown that TLR10 is expressed on human B cells and is involved in B cell activity [134]. TLR10 can also regulate the differentiation of primary human monocytes into DCs [135]. However, the association between TLR10 and intestinal inflammation remains unclear. TLR11 recognizes flagellin from Salmonella typhimurium [136]. Both TLR11 and TLR12 are receptors for profilin derived from Toxoplasma gondii [137]. TLR13 is associated with the recognition of bacterial 23S ribosomal RNA [137,138]. However, there are no reports that TLR11, TLR12, and TLR13 are associated with the pathogenesis of intestinal inflammation.

C. Association Between CLRs and Intestinal Inflammation CLRs are an emerging family of receptors that recognize various carbohydrate structures, such as mannose, fucose, sialic acid, and β-glucan [139]. CLRs, such as dectin-1, dectin-2, DC-specific intercellular adhesion molecule-3grabbing nonintegrin, and CLR-specific intracellular adhesion molecule-3-grabbing nonintegrin homolog-related 3 (SIGNR3), are expressed by myeloid cells, in particular by macrophages and dendritic cells [140]. CLR recognition of microorganisms on antigen-presenting cells leads to

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their internalization and subsequent antigen processing and presentation [141]. Previous studies have shown that CLRs are responsible for regulating intestinal inflammation. Dectin-1deficient mice are susceptible to DSS-induced intestinal inflammation with alteration of their commensal fungi [26]. In addition, a polymorphism in the human dectin-1 gene (CLEC7A) is associated with a severe form of UC [26]. Mice that are deficient in SIGNR1 are resistant to experimental colitis [142], and SIGNR3 is an important regulator of mucosal immunity against commensal fungi in the colon [143]. Macrophage-restricted C-type lectin and the dendritic cell immunoreceptor also contribute to the pathogenesis of DSS-induced intestinal inflammation [140].

D. Association Between RLRs and Intestinal Inflammation RLRs are cytoplasmic viral RNA receptors that recognize viral double-stranded (ds) RNA and trigger a signal that induces the innate immune response, whereas TLRs recognize only viral components at the cell surface or in endosomes [144 147]. Retinoic-acid-inducible gene I (RIG-I) and melanoma differentiationassociated gene 5 (MDA5) encode RLRs, and each protein encodes a caspase activation domain and a recruitment domain [148]. Because RIG-I recognizes comparatively short dsRNA and MDA5 recognizes long dsRNA, different RNA viruses are recognized by each of them [149 152]. While RIG-I is essential for immune responses against Sendai virus, influenza A virus, vesicular stomatitis virus, and hepatitis C virus, MDA5 is involved in the detection of encephalomyocarditis virus, poliovirus, and picornaviruses [144,147,153 155]. However, both RIG-I and MDA5 recognize West Nile virus and Japanese encephalitis virus [144,147]. RIG-I-deficient mice are highly susceptible to DSS-induced intestinal inflammation and have Peyer’s patches of decreased size

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and number, abnormal activation of peripheral T cells, and downregulated Gai2, which is one of a number of candidate genes for IBD [156]. In addition, RIG-I was found to be predominantly expressed on the apical side of intestinal epithelial cells, and RIG-I expression was downregulated in the intestinal tissues of patients with CD [157].

E. Association Between NLRs and Intestinal Inflammation That NLRs recognize cytoplasmic pathogens indicates that they are essential sensors for immune responses against intracellular bacteria. NLRs are multidomain proteins, and structurally and functionally, they represent the most diverse family among the PRRs [158]. Recently, it has been shown that NLR function is crucial for autophagy, antigen presentation, and embryonic development as well as for pattern recognition [159,160]. Among the NLRs, NOD1 and NOD2, which are regarded as noninflamasomeforming NLRs, are well characterized members of the NLR family. NOD2 dysfunction is strongly linked with susceptibility to CD, but NOD2 normally plays a pivotal role in regulating intestinal homeostasis [161]. Polymorphisms in NOD2, such as those encoding R702W, G908R, and L1007insC, are widely known to be genetic risk factors for CD [22,162,163], but the common NOD2 variants found in Western individuals are not correlated with predisposition to CD in the Japanese [164]. Nevertheless, it is obvious that NOD2 is involved in the pathogenesis of CD. However, interestingly, NOD2deficient mice and NOD1-deficient mice do not develop spontaneous intestinal inflammation [165,166]. These findings indicate that more scientific and clinical knowledge is needed to determine the pathophysiological role of NOD2 in CD. In contrast, NLRP3, which is one of the inflammasome-forming NLRs, is also a wellknown NLR. In humans, polymorphisms in NLRP3 are correlated with increased

susceptibility to CD [167]. NLRP3-deficient mice show severe intestinal inflammation in experimental colitis models, with dysregulation of IL1β and/or IL-18 production [168,169].

IV. CONCLUDING REMARKS The mucosal surface is the main bodily interface between the host and external antigens such as microorganisms. In the intestines, the barrier system plays a fundamental role in mucosal protection from antigens. In this organ, the monolayer epithelial cells form physical and chemical barriers consisting of nonstructural components such as the AMPs produced by Paneth cells and the mucus produced by goblet cells. Both barriers provide protection against enteropathogenic microorganisms. Intestinal epithelial cell dysfunction leads to disruption of the mucosal barrier in the gut and subsequent induction of intestinal disorders. On the other hand, a series of cells relevant to innate immunity constitutively express gene-encoded PRRs, including TLRs, CLRs, RLRs, and NLRs. When the mucosal surface is exposed to pathogens, the innate immune responses mediated by the PRRs provide key functions that regulate mucosal homeostasis. To date, a large number of molecules involved in mucosal innate immunity have been identified, and defective innate immune functioning in the gut is thought to contribute to intestinal inflammation. However, the need to elucidate the mechanisms of pathophysiology in human disorders with the goal of developing new therapeutic agents remains because a large number of patients with intractable diseases have been waiting for them.

References [1] Helander HF, Fandriks L. Surface area of the digestive tract - revisited. Scand J Gastroenterol 2014;49:681 9. [2] Hasleton PS. The internal surface area of the adult human lung. J Anat 1972;112:391 400.

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REFERENCES

[3] Goto Y, Uematsu S, Kiyono H. Epithelial glycosylation in gut homeostasis and inflammation. Nat Immunol 2016;17:1244 51. [4] Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710 20. [5] Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol 2014;14:141 53. [6] Marchiando AM, Graham WV, Turner JR. Epithelial barriers in homeostasis and disease. Annu Rev Pathol 2010;5:119 44. [7] Perez-Lopez A, Behnsen J, Nuccio SP, Raffatellu M. Mucosal immunity to pathogenic intestinal bacteria. Nat Rev Immunol 2016;16:135 48. [8] Uematsu S, Fujimoto K. The innate immune system in the intestine. Microbiol Immunol 2010;54:645 57. [9] Thaiss CA, Zmora N, Levy M, Elinav E. The microbiome and innate immunity. Nature 2016;535:65 74. [10] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783 801. [11] Gilbert JA, et al. Microbiome-wide association studies link dynamic microbial consortia to disease. Nature 2016;535:94 103. [12] Scher JU, et al. Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. Elife 2013;2:e01202. [13] Phillips R. Rheumatoid arthritis: microbiome reflects status of RA and response to therapy. Nat Rev Rheumatol 2015;11:502. [14] Scher JU, et al. The lung microbiota in early rheumatoid arthritis and autoimmunity. Microbiome 2016;4:60. [15] Maeda Y, et al. Dysbiosis contributes to arthritis development via activation of autoreactive T cells in the intestine. Arthritis Rheumatol 2016;68:2646 61. [16] Costello ME, Robinson PC, Benham H, Brown MA. The intestinal microbiome in human disease and how it relates to arthritis and spondyloarthritis. Best Pract Res Clin Rheumatol 2015;29:202 12. [17] Gill T, Asquith M, Rosenbaum JT, Colbert RA. The intestinal microbiome in spondyloarthritis. Curr Opin Rheumatol 2015;27:319 25. [18] Kostic AD, et al. The dynamics of the human infant gut microbiome in development and in progression toward type 1 diabetes. Cell Host Microbe 2015;17:260 73. [19] Wen L, et al. Innate immunity and intestinal microbiota in the development of Type 1 diabetes. Nature 2008;455:1109 13. [20] Carvalho FA, et al. Transient inability to manage proteobacteria promotes chronic gut inflammation in TLR5-deficient mice. Cell Host Microbe 2012;12:139 52. [21] Rigottier-Gois L. Dysbiosis in inflammatory bowel diseases: the oxygen hypothesis. ISME J 2013;7:1256 61.

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[22] Hugot JP, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 2001;411:599 603. [23] Ogura Y, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 2001;411:603 6. [24] Hampe J, et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet 2007;39:207 11. [25] Rioux JD, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet 2007;39:596 604. [26] Iliev ID, et al. Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science 2012;336:1314 17. [27] Nowak-Wegrzyn A, Szajewska H, Lack G. Food allergy and the gut. Nat Rev Gastroenterol Hepatol 2016;. [28] Marchesi JR, et al. The gut microbiota and host health: a new clinical frontier. Gut 2016;65:330 9. [29] Irrazabal T, Belcheva A, Girardin SE, Martin A, Philpott DJ. The multifaceted role of the intestinal microbiota in colon cancer. Mol Cell 2014;54:309 20. [30] Grivennikov SI, et al. Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth. Nature 2012;491:254 8. [31] Turnbaugh PJ, et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006;444:1027 31. [32] Le Chatelier E, et al. Richness of human gut microbiome correlates with metabolic markers. Nature 2013;500:541 6. [33] Sonnenburg JL, Backhed F. Diet-microbiota interactions as moderators of human metabolism. Nature 2016;535:56 64. [34] Koren O, et al. Human oral, gut, and plaque microbiota in patients with atherosclerosis. Proc Natl Acad Sci U S A 2011;108(Suppl 1):4592 8. [35] Wang Z, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 2011;472:57 63. [36] Wang Z, et al. Non-lethal inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis. Cell 2015;163:1585 95. [37] Jin C, Henao-Mejia J, Flavell RA. Innate immune receptors: key regulators of metabolic disease progression. Cell Metab 2013;17:873 82. [38] Fukata M, et al. Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis. Am J Physiol Gastrointest Liver Physiol 2005;288: G1055 1065.

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112

6. INNATE IMMUNITY AT MUCOSAL SURFACES

[39] Garrett WS, Gordon JI, Glimcher LH. Homeostasis and inflammation in the intestine. Cell 2010;140:859 70. [40] Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature 2011;474:298 306. [41] Tamura A, Tsukita S. Paracellular barrier and channel functions of TJ claudins in organizing biological systems: advances in the field of barriology revealed in knockout mice. Semin Cell Dev Biol 2014;36:177 85. [42] Ayabe T, et al. Secretion of microbicidal alphadefensins by intestinal Paneth cells in response to bacteria. Nat Immunol 2000;1:113 18. [43] Vaishnava S, et al. The antibacterial lectin RegIIIγ promotes the spatial segregation of microbiota and host in the intestine. Science 2011;334:255 8. [44] Johansson ME, et al. The inner of the two Muc2 mucindependent mucus layers in colon is devoid of bacteria. Proc Natl Acad Sci U S A 2008;105:15064 9. [45] Hansson GC, Johansson ME. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Gut Microbes 2010;1:51 4. [46] Kim JJ, Khan WI. Goblet cells and mucins: role in innate defense in enteric infections. Pathogens 2013;2:55 70. [47] Zasloff M. Antimicrobial peptides in health and disease. N Engl J Med 2002;347:1199 200. [48] Lievin-Le Moal V, Servin AL. The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota. Clin Microbiol Rev 2006;19:315 37. [49] Boman HG. Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 1995;13:61 92. [50] Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389 95. [51] Lehrer RI, Lichtenstein AK, Ganz T. Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Annu Rev Immunol 1993;11:105 28. [52] Jarczak J, et al. Defensins: natural component of human innate immunity. Hum Immunol 2013;74: 1069 79. [53] Salzman NH, Underwood MA, Bevins CL. Paneth cells, defensins, and the commensal microbiota: a hypothesis on intimate interplay at the intestinal mucosa. Semin Immunol 2007;19:70 83. [54] Mastroianni JR, et al. Alternative luminal activation mechanisms for paneth cell alpha-defensins. J Biol Chem 2012;287:11205 12. [55] Salzman NH. Paneth cell defensins and the regulation of the microbiome: detente at mucosal surfaces. Gut Microbes 2010;1:401 6. [56] Cunliffe RN, Mahida YR. Expression and regulation of antimicrobial peptides in the gastrointestinal tract. J Leukoc Biol 2004;75:49 58.

[57] Okumura R, et al. Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia. Nature 2016;532:117 21. [58] Heller F, et al. Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution. Gastroenterology 2005;129:550 64. [59] Blair SA, Kane SV, Clayburgh DR, Turner JR. Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease. Lab Invest 2006;86:191 201. [60] Zeissig S, et al. Changes in expression and distribution of claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn’s disease. Gut 2007;56:61 72. [61] Nusrat A, et al. Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins. Infect Immun 2001;69:1329 36. [62] McNamara BP, et al. Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. J Clin Invest 2001;107:621 9. [63] Brown GR, et al. Tumor necrosis factor inhibitor ameliorates murine intestinal graft-versus-host disease. Gastroenterology 1999;116:593 601. [64] Jostins L, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012;491:119 24. [65] Liu JZ, et al. Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight shared genetic risk across populations. Nat Genet 2015;47:979 86. [66] Angelow S, Yu AS. Structure-function studies of claudin extracellular domains by cysteine-scanning mutagenesis. J Biol Chem 2009;284:29205 17. [67] Linden SK, Sutton P, Karlsson NG, Korolik V, McGuckin MA. Mucins in the mucosal barrier to infection. Mucosal Immunol 2008;1:183 97. [68] Rivas MA, et al. Deep resequencing of GWAS loci identifies independent rare variants associated with inflammatory bowel disease. Nat Genet 2011;43: 1066 73. [69] Fujimoto K, et al. Regulation of intestinal homeostasis by the ulcerative colitis-associated gene RNF186. Mucosal Immunol 2017;10:446 59. [70] Rausch P, et al. Colonic mucosa-associated microbiota is influenced by an interaction of Crohn disease and FUT2 (Secretor) genotype. Proc Natl Acad Sci U S A 2011;108:19030 5. [71] Miyoshi J, et al. Ectopic expression of blood type antigens in inflamed mucosa with higher incidence of FUT2 secretor status in colonic Crohn’s disease. J Gastroenterol 2011;46:1056 63.

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[72] Lyras D, et al. Toxin B is essential for virulence of Clostridium difficile. Nature 2009;458:1176 9. [73] Kuehne SA, et al. The role of toxin A and toxin B in Clostridium difficile infection. Nature 2010;467:711 13. [74] Carter GP, et al. Defining the roles of TcdA and TcdB in localized gastrointestinal disease, systemic organ damage, and the host response during Clostridium difficile infections. MBio 2015;6:e00551. [75] Abt MC, McKenney PT, Pamer EG. Clostridium difficile colitis: pathogenesis and host defence. Nat Rev Microbiol 2016;14:609 20. [76] Pearson JS, Giogha C, Wong Fok Lung T, Hartland EL. The genetics of enteropathogenic Escherichia coli virulence. Annu Rev Genet 2016;50:493 513. [77] Glotfelty LG, et al. Enteropathogenic E. coli effectors EspG1/G2 disrupt microtubules, contribute to tight junction perturbation and inhibit restoration. Cell Microbiol 2014;16:1767 83. [78] Jarvis KG, et al. Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc Natl Acad Sci U S A 1995;92:7996 8000. [79] Hill GR, Ferrara JL. The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation. Blood 2000;95:2754 9. [80] Penack O, Holler E, van den Brink MR. Graft-versushost disease: regulation by microbe-associated molecules and innate immune receptors. Blood 2010;115:1865 72. [81] Nalle SC, Turner JR. Endothelial and epithelial barriers in graft-versus-host disease. Adv Exp Med Biol 2012;763:105 31. [82] Noth R, et al. Increased intestinal permeability and tight junction disruption by altered expression and localization of occludin in a murine graft versus host disease model. BMC Gastroenterol 2011;11:109. [83] Odenwald MA, Turner JR. The intestinal epithelial barrier: a therapeutic target? Nat Rev Gastroenterol Hepatol 2017;14:9 21. [84] Nalle SC, et al. Recipient NK cell inactivation and intestinal barrier loss are required for MHC-matched graft-versus-host disease. Sci Transl Med 2014;6 243ra287. [85] Washington K, Jagasia M. Pathology of graft-versushost disease in the gastrointestinal tract. Hum Pathol 2009;40:909 17. [86] Heimesaat MM, et al. MyD88/TLR9 mediated immunopathology and gut microbiota dynamics in a novel murine model of intestinal graft-versus-host disease. Gut 2010;59:1079 87.

113

[87] Jenq RR, et al. Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation. J Exp Med 2012;209:903 11. [88] Eriguchi Y, et al. Graft-versus-host disease disrupts intestinal microbial ecology by inhibiting Paneth cell production of alpha-defensins. Blood 2012;120:223 31. [89] Heidegger S, van den Brink MR, Haas T, Poeck H. The role of pattern-recognition receptors in graftversus-host disease and graft-versus-leukemia after allogeneic stem cell transplantation. Front Immunol 2014;5:337. [90] Schwab L, et al. Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance graft-versus-host disease via tissue damage. Nat Med 2014;20:648 54. [91] Nalle SC, Turner JR. Intestinal barrier loss as a critical pathogenic link between inflammatory bowel disease and graft-versus-host disease. Mucosal Immunol 2015;8:720 30. [92] Beutler B, et al. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu Rev Immunol 2006;24:353 89. [93] Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell 2010;140:805 20. [94] Takeda K, Akira S. Toll-like receptors. Curr Protoc Immunol 2015;109 14 12 11-10. [95] Akira S. Innate immunity and adjuvants. Philos Trans R Soc Lond B Biol Sci 2011;366:2748 55. [96] Abraham C, Medzhitov R. Interactions between the host innate immune system and microbes in inflammatory bowel disease. Gastroenterology 2011;140:1729 37. [97] Nenci A, et al. Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 2007;446:557 61. [98] Zaph C, et al. Epithelial-cell-intrinsic IKK-beta expression regulates intestinal immune homeostasis. Nature 2007;446:552 6. [99] Wyllie DH, et al. Evidence for an accessory protein function for Toll-like receptor 1 in anti-bacterial responses. J Immunol 2000;165:7125 32. [100] Takeuchi O, et al. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 2002;169:10 14. [101] Pierik M, et al. Toll-like receptor-1, -2, and -6 polymorphisms influence disease extension in inflammatory bowel diseases. Inflamm Bowel Dis 2006;12:1 8. [102] Cario E, Podolsky DK. Toll-like receptor signaling and its relevance to intestinal inflammation. Ann N Y Acad Sci 2006;1072:332 8. [103] Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 controls mucosal inflammation by regulating epithelial barrier function. Gastroenterology 2007;132: 1359 74.

II. PRINCIPLES OF MUCOSAL VACCINE

114

6. INNATE IMMUNITY AT MUCOSAL SURFACES

[104] Cario E. Barrier-protective function of intestinal epithelial Toll-like receptor 2. Mucosal Immunol 2008;1 (Suppl 1):S62 66. [105] Szebeni B, et al. Increased expression of Toll-like receptor (TLR) 2 and TLR4 in the colonic mucosa of children with inflammatory bowel disease. Clin Exp Immunol 2008;151:34 41. [106] Bank S, et al. Polymorphisms in the inflammatory pathway genes TLR2, TLR4, TLR9, LY96, NFKBIA, NFKB1, TNFA, TNFRSF1A, IL6R, IL10, IL23R, PTPN22, and PPARG are associated with susceptibility of inflammatory bowel disease in a Danish cohort. PLoS One 2014;9:e98815. [107] Cario E, Podolsky DK. Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun 2000;68:7010 17. [108] Skovdahl HK, et al. Expression of CCL20 and its corresponding receptor CCR6 is enhanced in active inflammatory bowel disease, and TLR3 mediates CCL20 expression in colonic epithelial cells. PLoS One 2015;10:e0141710. [109] Takemura N, et al. Blockade of TLR3 protects mice from lethal radiation-induced gastrointestinal syndrome. Nat Commun 2014;5:3492. [110] Arbour NC, et al. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 2000;25:187 91. [111] Lorenz E, Patel DD, Hartung T, Schwartz DA. Tolllike receptor 4 (TLR4)-deficient murine macrophage cell line as an in vitro assay system to show TLR4independent signaling of Bacteroides fragilis lipopolysaccharide. Infect Immun 2002;70:4892 6. [112] Franchimont D, et al. Deficient host-bacteria interactions in inflammatory bowel disease? The Toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn’s disease and ulcerative colitis. Gut 2004;53:987 92. [113] Cheng Y, et al. Association between TLR2 and TLR4 gene polymorphisms and the susceptibility to inflammatory bowel disease: a meta-analysis. PLoS One 2015;10:e0126803. [114] Mohammadi M, Zahedi MJ, Nikpoor AR, Baneshi MR, Hayatbakhsh MM. Interleukin-17 serum levels and TLR4 polymorphisms in ulcerative colitis. Iran J Immunol 2013;10:83 92. [115] Ao R, Wang Y, Zhnag DR, Du YQ. Role of TLR4 rs4986790A . G and rs4986791C . T Polymorphisms in the risk of inflammatory bowel disease. Gastroenterol Res Pract 2015;2015:141070. [116] Hayashi F, et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001;410:1099 103.

[117] Gewirtz AT, Navas TA, Lyons S, Godowski PJ, Madara JL. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol 2001;167:1882 5. [118] Sierro F, et al. Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells. Proc Natl Acad Sci U S A 2001;98:13722 7. [119] Chassaing B, Ley RE, Gewirtz AT. Intestinal epithelial cell toll-like receptor 5 regulates the intestinal microbiota to prevent low-grade inflammation and metabolic syndrome in mice. Gastroenterology 2014;147 1363-1377 e1317. [120] Uematsu S, et al. Detection of pathogenic intestinal bacteria by Toll-like receptor 5 on intestinal CD11c 1 lamina propria cells. Nat Immunol 2006;7:868 74. [121] Uematsu S, et al. Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5. Nat Immunol 2008;9: 769 76. [122] Fujimoto K, et al. A new subset of CD103 1 CD8alpha 1 dendritic cells in the small intestine expresses TLR3, TLR7, and TLR9 and induces Th1 response and CTL activity. J Immunol 2011;186:6287 95. [123] Meena NK, Ahuja V, Meena K, Paul J. Association of TLR5 gene polymorphisms in ulcerative colitis patients of north India and their role in cytokine homeostasis. PLoS One 2015;10:e0120697. [124] Bank S, et al. Genetically determined high activity of IL-12 and IL-18 in ulcerative colitis and TLR5 in Crohns disease were associated with non-response to anti-TNF therapy. Pharmacogenomics J 2017;. [125] Takeuchi O, et al. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol 2001;13: 933 40. [126] Morgan ME, et al. Toll-like receptor 6 stimulation promotes T-helper 1 and 17 responses in gastrointestinalassociated lymphoid tissue and modulates murine experimental colitis. Mucosal Immunol 2014;7:1266 77. [127] Sainathan SK, et al. Toll-like receptor-7 ligand Imiquimod induces type I interferon and antimicrobial peptides to ameliorate dextran sodium sulfateinduced acute colitis. Inflamm Bowel Dis 2012;18: 955 67. [128] Peng G, et al. Toll-like receptor 8-mediated reversal of CD4 1 regulatory T cell function. Science 2005;309: 1380 4. [129] Saruta M, et al. High-frequency haplotypes in the X chromosome locus TLR8 are associated with both CD and UC in females. Inflamm Bowel Dis 2009;15: 321 7.

II. PRINCIPLES OF MUCOSAL VACCINE

115

REFERENCES

[130] Hemmi H, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000;408:740 5. [131] Stenson WF. The role of toll-like receptor 9 in the intestine. Curr Opin Gastroenterol 2005;21:360 2. [132] Berkowitz D, Peri R, Lavy A, Kessel A. Increased Toll-like receptor 9 expression by B cells from inflammatory bowel disease patients. Hum Immunol 2013;74:1519 23. [133] Sardi C, et al. Three months of Western diet induces small intestinal mucosa alteration in TLR KO mice. Microsc Res Tech 2017;80:563 9. [134] Hess NJ, Jiang S, Li X, Guan Y, Tapping RI. TLR10 is a B cell intrinsic suppressor of adaptive immune responses. J Immunol 2017;198:699 707. [135] Hess NJ, Felicelli C, Grage J, Tapping RI. TLR10 suppresses the activation and differentiation of monocytes with effects on DC-mediated adaptive immune responses. J Leukoc Biol 2017;101:1245 52. [136] Mathur R, et al. A mouse model of Salmonella typhi infection. Cell 2012;151:590 602. [137] Koblansky AA, et al. Recognition of profilin by Tolllike receptor 12 is critical for host resistance to Toxoplasma gondii. Immunity 2013;38:119 30. [138] Oldenburg M, et al. TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification. Science 2012;337:1111 15. [139] Geijtenbeek TB, Gringhuis SI. Signalling through Ctype lectin receptors: shaping immune responses. Nat Rev Immunol 2009;9:465 79. [140] Hutter J, et al. Role of the C-type lectin receptors MCL and DCIR in experimental colitis. PLoS One 2014;9:e103281. [141] Geijtenbeek TB, Gringhuis SI. C-type lectin receptors in the control of T helper cell differentiation. Nat Rev Immunol 2016;16:433 48. [142] Saunders SP, et al. C-type lectin SIGN-R1 has a role in experimental colitis and responsiveness to lipopolysaccharide. J Immunol 2010;184:2627 37. [143] Eriksson M, et al. The C-type lectin receptor SIGNR3 binds to fungi present in commensal microbiota and influences immune regulation in experimental colitis. Front Immunol 2013;4:196. [144] Kato H, et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 2006;441:101 5. [145] Kawai T, Akira S. Innate immune recognition of viral infection. Nat Immunol 2006;7:131 7. [146] Takeuchi O, Akira S. MDA5/RIG-I and virus recognition. Curr Opin Immunol 2008;20:17 22. [147] Loo YM, Gale Jr. M. Immune signaling by RIG-I-like receptors. Immunity 2011;34:680 92. [148] Yoneyama M, et al. Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2

[149] [150]

[151]

[152]

[153] [154]

[155]

[156]

[157]

[158]

[159]

[160]

[161]

[162]

[163]

[164]

in antiviral innate immunity. J Immunol 2005;175:2851 8. Hornung V, et al. 5’-Triphosphate RNA is the ligand for RIG-I. Science 2006;314:994 7. Kato H, et al. Length-dependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-I and melanoma differentiationassociated gene 5. J Exp Med 2008;205:1601 10. Goubau D, et al. Antiviral immunity via RIG-Imediated recognition of RNA bearing 5’-diphosphates. Nature 2014;514:372 5. Oshiumi H, Kouwaki T, Seya T. Accessory factors of cytoplasmic viral RNA sensors required for antiviral innate immune response. Front Immunol 2016;7:200. Kato H, et al. Cell type-specific involvement of RIG-I in antiviral response. Immunity 2005;23:19 28. Foy E, et al. Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling. Proc Natl Acad Sci U S A 2005;102:2986 91. Chang TH, Liao CL, Lin YL. Flavivirus induces interferon-beta gene expression through a pathway involving RIG-I-dependent IRF-3 and PI3Kdependent NF-kappaB activation. Microbes Infect 2006;8:157 71. Wang Y, et al. Rig-I2/2 mice develop colitis associated with downregulation of Gαi2. Cell Res 2007;17:858 68. Funke B, et al. Selective downregulation of retinoic acid-inducible gene I within the intestinal epithelial compartment in Crohn’s disease. Inflamm Bowel Dis 2011;17:1943 54. Meunier E, Broz P. Evolutionary convergence and divergence in NLR function and structure. Trends Immunol 2017;. Lange C, et al. Defining the origins of the NOD-like receptor system at the base of animal evolution. Mol Biol Evol 2011;28:1687 702. Duenez-Guzman EA, Haig D. The evolution of reproduction-related NLRP genes. J Mol Evol 2014;78: 194 201. Caruso R, Warner N, Inohara N, Nunez G. NOD1 and NOD2: signaling, host defense, and inflammatory disease. Immunity 2014;41:898 908. Chamaillard M, et al. Gene-environment interaction modulated by allelic heterogeneity in inflammatory diseases. Proc Natl Acad Sci U S A 2003;100:3455 60. Hugot JP, et al. Prevalence of CARD15/NOD2 mutations in Caucasian healthy people. Am J Gastroenterol 2007;102:1259 67. Inoue N, et al. Lack of common NOD2 variants in Japanese patients with Crohn’s disease. Gastroenterology 2002;123:86 91.

II. PRINCIPLES OF MUCOSAL VACCINE

116

6. INNATE IMMUNITY AT MUCOSAL SURFACES

[165] Pauleau AL, Murray PJ. Role of nod2 in the response of macrophages to toll-like receptor agonists. Mol Cell Biol 2003;23:7531 9. [166] Kobayashi KS, et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 2005;307:731 4. [167] Villani AC, et al. Common variants in the NLRP3 region contribute to Crohn’s disease susceptibility. Nat Genet 2009;41:71 6.

[168] Zaki MH, et al. The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis. Immunity 2010;32: 379 91. [169] Allen IC, et al. The NLRP3 inflammasome functions as a negative regulator of tumorigenesis during colitis-associated cancer. J Exp Med 2010;207: 1045 56.

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Induction and Regulation of Mucosal Memory B Cell Responses Nils Lycke Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden

I. INTRODUCTION Although critical for successful mucosal vaccination, our understanding of how and where immunological memory is induced and maintained is still incomplete [1]. Despite the fact that mucosal vaccines have been used for decades and found effective at protecting against enteric as well as lung infections, detailed knowledge about mucosal memory B and T cells and how these are regulated is largely lacking. Two examples of excellent long-term memory B cell development come from the fields of oral cholera and live attenuated rotavirus vaccines [2 4]. Both these vaccines stimulate strong IgA memory, although protection against the former infection has been considered short-lived compared to what is normally observed with injectable vaccines [5]. This also applies to memory development against certain viruses such as respiratory syncytial virus, in which immunity appears short-lived and recurrent infections are common [6 8] (Chapter 38). Acellular pertussis vaccination has also been found to stimulate relatively poor mucosal

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00007-9

memory B cell responses [9]. This can most likely be ascribed to the nature of the microorganism involved or the features of the vaccine, being live attenuated (e.g., the rotavirus vaccines) or killed (e.g., the oral cholera vaccines), rather than to the route of vaccine administration. Alternatively, there could be genetic limitations, as have recently been described for naı¨ve B cells with germline-encoded high-avidity B cell receptors (BCRs) that fail to develop into stable memory B cell clones [10]. A major question is whether the short-term increase in antibody titers correctly reflects the size and function of the memory B cell pool following vaccination. Several experimental studies have attested that this is not the case. For example, immunizations using the same antigen (e.g., NP-chicken gamma globulin) but different adjuvants (e.g., alum, monophosphoryl lipid A, or CTA1-DD) were found to stimulate comparable short-term serum antibody levels, while after 3 months, the serum antibody levels declined rapidly in two of the adjuvanted groups (e.g., alum and monophosphoryl lipid A), which also responded less to a booster

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immunization [11]. Thus the adjuvant formulation may play a central role in vaccine stimulation of memory [12] (Chapter 10: Innate Immunity-Based Mucosal Modulators and Adjuvants and Chapter 11: Toxin-Based Modulators for Regulation of Mucosal Immune Responses). Today, we still lack biomarkers and reliable correlates of memory B cell development that could be used to improve mucosal vaccine efficacy [13]. Of course, we could propose identifying the frequency of memory B cells that arise after vaccination using specific markers (e.g., CD73, CD80, CD27) as a proxy of memory development [14,15]. However, we do not know much about mucosal memory B cell reactivation requirements and functions; hence cell numbers may not correctly reflect the protective potential of the memory B cell pool. Besides, there is still debate about the definition of a memory B cell, and it is likely that these cells are not a homogenous population but rather are quite heterogenous and differ in several parameters [15]. Nevertheless, the classic definition would be that these cells have encountered antigen, and subsequent to a number of cell divisions, a rewired memory B cell clone returns to a quiescent stage in the tissues, where it resides until reactivated by antigen [16]. Memory B cells are also likely recirculating cells, but this has been incompletely studied. Most important, the memory B cells differ from the naı¨ve B cells in several aspects, such as their requirements for activation, their most often higher affinity for the antigen, and the gene transcriptional profile that they carry to develop a more rapid and mature response [17,18]. Experimental models have been instrumental in the study of immunological memory, but conflicting views as to the efficiency by which memory development occurs at mucosal membranes is also observed in these models [19]. For example, colonization of germ-free mice with a commensal auxotrophic Escherichia coli

strain was reported to fail in stimulating memory B cells, but in other mouse models, bacterial colonization of the intestine could effectively drive memory B cell development [20,21]. Now there is emerging evidence to indicate that different bacterial species of the microbiota indeed have greatly different ability to elicit memory B cell responses [22 24]. Clearly, a better understanding of which microbial properties or features of a vaccine formulation that stimulate memory would be instrumental for improving vaccine efficacy. In oral cholera vaccinated individuals, a significant recall response to the vaccine can be elicited even after 5 years, which is when specific IgA plasma cells appear to be absent in the intestine [4] (Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control). Indeed, most studies support that mucosal sites have the ability to develop and sustain memory B cells for a considerable period of time [19,25]. Another example is natural cholera infection, which has been found to prime long-term memory B cells [1,26]. Also providing evidence of a strong ability to maintain memory is the fact that an unbiased analysis of IgA V-region gene sequences in conventionally reared mice as well as in humans revealed a progressive accumulation of somatic hypermutations (SHMs) with age, supporting the concept of memory B cell development [27,28]. In addition, selective depletion of gut IgA plasma cells did not change the repertoire or clonality of the IgA plasma cells that reappeared in the gut lamina propria (LP) a few weeks later. Thus the unique gene sequences in the gut IgA plasma cell repertoire tend to remain stable in adulthood and can be taken to reflect the stability of mucosal memory B cell clones. Whether these gut mucosal memory B cell clones are specific not only for T-cell-dependent (TD) antigens but also for T-cell-independent (TI) IgA B cell responses against, for example, the commensal flora, has been incompletely investigated.

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III. THE INDUCTIVE SITE FOR GUT MEMORY B CELL RESPONSES IS THE PEYER’S PATCHES

However, it appears likely that this is the case, given the complete dominance of mutated IgA V gene sequences in adult mice [27]. Whereas much of our knowledge about the induction and regulation of mucosal memory B cell responses emanates from studies of the gutassociated lymphoid tissues (GALT), the present chapter will focus mostly on the gastrointestinal tract, although some aspects of memory B cell development at other mucosal sites, such as the lung and respiratory tract, will also be discussed.

II. MUCOSAL VACCINE INDUCTION OF MEMORY B CELLS The list of vaccines delivered by the mucosal route is short; hence only limited information can be gained from clinical studies with regard to memory B cells other than that they exist and can be found, for example, in peripheral blood [1,29]. Some recent studies have attempted to investigate their homing properties and location to lymph nodes [30,31]. Following oral cholera or enterotoxigenic E. coli vaccination, memory B cell responses were detected in peripheral blood with phenotypic markers (e.g., CD27, IgA, CCR9, CCR10, CLA, integrin β7) for homing to the mucosal membranes [30] (Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control and Chapter 32: Oral Vaccines for Enterotoxigenic Escherichia coli). Following mucosal immunization memory B cells can remain IgM1 or may isotype-switch before appearing as quiescent B cells in the lymphoid tissues [e.g., Peyer’s patches (PPs), mesenteric lymph nodes (MLNs), or the spleen] [32]. Currently, there is much interest in the IgM1 memory B cells that, when reactivated, either secrete IgM or undergo class-switch recombination (CSR) to IgG subclasses or IgA [15,17,32,33]. In fact, in the mouse model, oral immunization has been found to generate a dominance of IgM memory B cells in the MLNs

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as well as in the spleen and systemic secondary lymph nodes, while PPs are dominated by IgA1 memory B cells [32]. After a booster immunization 1 year later, the great majority of IgM1 memory B cells undergo isotypeswitching to IgA or IgG subclasses in mucosaassociated (e.g., PPs) and systemic secondary lymphoid (e.g., peripheral lymph node) tissues, respectively [11].

III. THE INDUCTIVE SITE FOR GUT MEMORY B CELL RESPONSES IS THE PEYER’S PATCHES From studies of systemic immunizations, we have learned that memory B cells are generated predominantly in the germinal centers (GCs) that are formed in the spleen or draining lymph nodes within 10 days following immunization [15,34,35]. Although memory B cells can also develop in the absence of a GC reaction, these cells usually carry only a few mutations and are not expected to contribute to protection against most types of infection [34 36]. However, there have been some examples of GC-independent memory IgG immunity that contributed to protection, as has been found with Salmonella infection, for example [37,38]. The GC reaction in systemic secondary lymphoid tissues has been dissected and characterized in great detail, and a significant understanding of the regulatory requirements exists today, but whether this knowledge applies to the inductive sites for mucosal immune responses has only recently been addressed [19,32,36]. The best-analyzed tissues are the PPs, which constitute the most important sites for stimulating gut memory B cell responses [39] (Fig. 7.1) (Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance). MLNs, although central to the draining lymph from the small and large intestine, are less strongly associated with gut IgA B

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Peyer’s patch

Gut lumen

Mucus layer Epithelial cells

Germinal center

Follicular dendritic cell

TFH cell B cell

Long-lived plasma cells

Timeline Memory B cells IgM IgG

cell responses and host only a few GC reactions following oral immunization, which may, in fact, represent responses initiated in the PPs with migrating activated B cells [40,41]. The extent to which GC reactions are critical for gut mucosal B cell memory development is not completely understood [1]. Whereas experimental studies using oral immunizations with cholera toxin (CT), a well-known TD antigen and mucosal adjuvant, have shown strong dependence on GC formations [42], oral rotavirus immunizations stimulated long-term IgA memory that protected against infection but was reported to be TD- and GC-independent [42,43] (Chapters 40 and 41). A GC-independent pathway for memory B cell development has been described, but unlike GC-dependent memory B cells, these cells exhibit few IgH V gene mutations [27,42]. As was mentioned above, the fact that there is an accumulation of mutated IgA V genes in the gut with age would argue against GC-independent memory B cell development at the gut mucosal site and probably also at other mucosal sites [25,28]. On the other

CD80 CD73 PD-L2 IgA

FIGURE 7.1 The formation of mucosal memory B cells is only beginning to be unraveled. Whereas the systemic induction of memory B cells is known in some detail, the formation of mucosal memory B cells has only recently been investigated. The GC reaction is central to mucosal memory B cells, and when naı¨ve B cells are stimulated by antigen in PPs, they generate a GC, which is the site for memory B cell development. Memory B cells undergo a selection process that appears to be faster than that of long-lived plasma cells, and memory B cell clones are therefore poorly related to the long-lived plasma cells.

CD38

hand, it has been observed in CD40-deficient mice that gut B cells are reactive against TI antigens of the bacterial commensal flora [42]. Therefore, to accommodate the finding of accumulating SHMs in IgA gene sequences with age in wild-type mice with experiments in CD40deficient mice (showing near normal levels of IgA plasma cells), we have hypothesized that TI antigen-activated B cells also undergo SHMs in GC in PPs [19]. Given that GC formation requires the involvement of CD41 T cells, B cells activated by TI antigen would have to use already existing GCs in PPs to undergo SHMs. Indeed, PPs are known to consistently host GC reactions; hence this scenario is entirely feasible, provided that cognate MHC class II-dependent T B cell interactions are not required but rather only CD40L-CD40 interactions are critical for SHMs in these reutilized GCs [41]. Thus under normal conditions, B cells that are activated by TI antigen also could undergo SHMs in GCs in PPs, making these responses indirectly dependent on CD4 T cells. In fact, GCs in PPs have been found to host multiple B cell clones, and

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FIGURE 7.2 The B1-8high B cell transfer model for detailed studies of gut B cells. The NP-CT model system using

adoptive transfer of B1-8high NP-specific GFP1 λ1B cells into naı¨ve recipient C57Bl/6 mice. The animals are then immunized orally with NP-CT, and the distribution of responding CD191 GFP1 B cells (green) is monitored by flow cytometry (PP B cells) and imaging of tissue sections from Peyer’s patches and small intestinal lamina propria (LP). The responding B cell clones eventually are found as IgA plasma cells localized to the LP of the intestine (lower left panel). Tissue sections were labeled with specific antibodies for B cells (B220), germinal center (GC) B cells (GL71), and subepithelial dome (SED) B cells (CCR61). Responding GFP1 B cells are found primarily in the GC but also in the SED.

these polyclonal GC sites could allow for both TD- and TI-antigen-stimulated IgA B cell responses [14,44,45].

IV. A MODEL SYSTEM TO STUDY MUCOSAL MEMORY B CELL DEVELOPMENT To allow for a detailed study of regulatory requirements for mucosal memory B cell development, we have used an adoptive transfer model with B1-8high/GFP1 NP-specific B cells and using oral immunization with the hapten

(4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to CT [46,47]. This has allowed us to undertake detailed studies of the induction, location, and regulatory requirements for mucosal memory B cell responses (Fig. 7.2). A particular asset to the NP system is that we know which genes are used for recognition of NP, so we can study the acquisition of SHMs in the NP-specific IgA sequences in the VH186.2 V region and identify when the B cells undergo IgA CSR. Indeed, a mutation in VH186.2 W33 to L33 in the CDR1 region implies a 10-fold enhanced affinity for the NP antigen and is a sensitive marker for productive SHMs in the

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PPs [48,49]. Following oral immunization with NP-CT, we can identify the inductive sites (e.g., PPs or MLNs) for the NP B cell response through tracing of the GFP-expressing B cells. We can also identify the formation of GC, follow the migration of activated B cells (GFP1, IgDlow, CD38low, GL71/2ICOS1, Bcl61), and identify the memory B cells (GFP1, IgDlow, CD381, CD731, CD801, PD-L21) and the longlived plasma cells (GFP1, CD1381, B220(1)) and their location (e.g., PPs, MLNs, LP, spleen, or peripheral lymph nodes), as will be discussed later in this chapter.

V. THE GERMINAL CENTER REACTION IN PERIPHERAL LYMPH NODES AND PEYER’S PATCHES A particular aspect of PPs is that they do not receive afferent lymph, as is seen in peripheral lymph nodes [50]. However, both types of tissues have efferent lymph, leaving the immune inductive site. Hence both tissues can be expected to generate memory B cells that can emigrate in the efferent lymph [41,51]. Whether the generation of memory B cells is similar or different in the GALT and peripheral lymph nodes is at present not completely clear. On the one hand, the PPs are sites for synchronizing IgA plasma cell responses in the gut LP, and this requires spreading of activated B cells to multiple PPs and the reutilization of preexisting GCs in these PPs, as has already been mentioned [46]. The strongest evidence for the existence of a synchronizing system is that clonally closely related antigen-specific B cells are found in multiple PPs and that the response following oral immunization is oligoclonal [46]. Indeed, following oral immunization, the NPspecific B cell response was oligoclonal and dominated by cells that carried the highaffinity mutation VH186.2 W33 to L33 in the CDR1 region. When NP-specific IgA sequences were analyzed, it was observed that clonally

related B cells representing three to six clones were found in multiple PPs in the same mouse, suggesting that the expansion of these clones was synchronized and was the consequence of an antigen-driven selection process. A clonal tree analysis revealed that NP-specific B cell clones were closely related not only in multiple PPs, but also in MLNs, suggesting that early after activation, B cells migrated to different PPs and MLNs, where they continued to participate in affinity selection and differentiation. Thus the memory B cell and long-lived IgA plasma cell responses can undergo synchronization to represent only the high-affinity clones selected in the GALT [14]. This implies that at an early stage of the response, antigen-specific B cells can leave the PP and migrate to reutilize already established GCs in multiple other PPs. Unpublished work in our laboratory has clearly shown that activated B cells with a GCphenotype can be found in the thoracic duct after oral immunization with NP-CT (N-Lycke, unpublished data). We propose that reutilization of preexisting GCs in PPs is a prime feature of the induction of memory IgA B cell responses in PPs and the reason why highquality SIgA antibodies are found in the gut LP upon recall activation [52].

VI. RESPIRATORY TRACT INFECTIONS AND MUCOSAL MEMORY B CELLS Human studies and evidence from experimental models provide strong support for the need to develop mucosal B cell memory to protect against respiratory tract infections [53]. As a consequence of infection, such as influenza, inducible bronchoalveolar lymphoid tissues (iBALT) develop in the lung, and these tissues host complete inductive sites for antigenspecific immune responses [54] (Chapter 2. Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the

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VII. MUCOSAL MEMORY B CELLS AND HOMING MARKERS

Induction and Regulation of Mucosal Immunity and Tolerance). The iBALT is also a site for memory B cell development as well as a site for their residency [55,56]. Whether there are resident memory B cells in the lung, similar to resident CD41 and CD81 T cells, is currently a debated question, but following influenza infection, memory B cells that are CD381, CD731, and CD801 appear in the lung [53,57]. These memory B cells are CD691 and CXCR31 and persisted in the lung for at least 5 months in mice after infection [55]. The persistence of memory B cells in the lung following influenza infection has been associated with the persistence of GCs in the lung. Indeed, it was observed that these lung GC sites were strong in promoting cross-reactive memory B cells that could target conserved regions of HA, and in this way neutralizing viral escape mutants [56]. Thus, harboring GC reactions for extended periods of time following infection appears somewhat unique to the lung. The memory B cell response in the respiratory tract is dominated by IgG antibodies, although a fair production of IgA antibodies can also be found. The IgA antibodies in the nasal compartment are considered to be a first line of defense, while IgG antibodies in the lung serve as a backup [58]. Hence the role of IgG antibodies for protection is more critical in the lung than in the gut intestine. The production of IgG B cells is a key feature of iBALT, although significant IgA B cell numbers are also formed [59]. In contrast to the gut intestine, a considerable amount of IgG antibodies in the respiratory tract are derived from serum, while the IgA production is mainly by local plasma cells in the mucosal membrane [8,58]. The memory B cells that reside in the lung in immune individuals are known to respond rapidly with local IgG and IgA antibody production to an infection, providing early protection against spreading of virus [55]. Hence memory B cell development is well described for the respiratory tract [60].

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However, it is clear that different antigens are differently effective at stimulating memory B cell responses [61]. The DTaP vaccine, for example, stimulates stronger and more longlived protection against the tetanus and diphtheria components than against the pertussis antigens [62,63]. Protection against respiratory tract infections (Bordetella pertussis, influenza virus, respiratory syncytial virus, adenovirus, and many more) is achieved more effectively through mucosal vaccination than through parenteral vaccination, and lung-resident memory CD4 T cells have been shown to have a central role for immune protection against, for example, influenza virus and several other pathogens [53,57,64 66]. Mucosal immunization has been found to promote both Th1 and Th17 cells, which are considered beneficial for protection against infection (influenza virus, B. pertussis, and many others) [65,67,68]. For example, a recent study of intranasal immunization with an acellular pertussis vaccine demonstrated strong resident memory Th17 immunity and IL-17 production as well as local IgA, which gave long-term protection [69].

VII. MUCOSAL MEMORY B CELLS AND HOMING MARKERS Mucosal memory B cells need to express homing markers to be effective at protecting against infection. For example, upon adoptive transfer to naı¨ve recipient mice, rotavirusspecific memory IgA B cells could mediate protection only if they expressed α4β7 integrin, one of the important gut-homing molecules [70]. Hence IgG memory B cells that did not carry α4β7 integrin did not protect against the infection [43,71]. Moreover, if splenic memory B cells from oral immunizations expressed only α4β7 homing markers, they failed to home to the small intestinal LP, as this also required CCR9 expression, which was acquired only in

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Effector site

Site for memory B cells Inductive site

Small intestine α4β7

Peyer’s patch

CCR9

Peyer’s patch

α4β7

IgA

CD38 (GL7–)

Large intestine

EbI2 IgA CXCR5Peri-GC CXCR4 CCR6

?

α4β7

IgA

CCR10

Skin Memory B cells CD80 CD73 PD-L2 CD38

Spleen IgA/M α4β7

CD38 (GL7–)

α4β1

CLA

CCR10

IgG

Lung/respiratory tract

CXCR4 Peri-GC CCR6 α4β1

α4β1 IgG/M

CCR3 CXCR3

CCR6

IgG CCR10

FIGURE 7.3 Homing receptors and surface markers identifying memory B cells. Memory B cells can be identified by some surface molecules that are expressed on their cell membrane. Following development in the GC, the memory B cells circulate or reside as quiescent cells in the B cell follicle of the lymph nodes and spleen. Following immunization, they acquire homing receptors that guide them to the correct tissue after reactivation, or they could acquire additional homing receptors upon reactivation, depending on the site of reactivation.

PPs and not in the spleen [32]. Therefore a combination of several homing markers rather than a single marker is needed to migrate to the preferred tissues (Chapter 5). Hence the expression patterns of homing receptors make up an intricate system to secure the tissue specificity of migrating memory B cells. Markers that are specifically associated with mucosal homing are CXCR4, CCR6, CXCR6, CCR9, CLA, and α4β7, which are differentially expressed to guide trafficking memory B cells to the site where they can exit [19]. For example, CXCR4 and CCR6 expression levels were elevated on naı¨ve compared to memory B cells, CCR9 and α4β7 were significantly elevated on mucosal IgA1 memory B cells homing to the small intestine, and CCR10 and α4β7 directed cells to the colon [72]. However, memory B cells expressing CCR10 bind to CCL28, which is also

expressed in the respiratory tract together with VCAM-1, as well as to CCL27, which is produced in the skin. Therefore expression of a single chemokine receptor, such as CCR10 or CCR9, does not fully identify the tissue-homing preference of the memory B cells [31]. Of note, retinoic acid, a vitamin A metabolite, is known to induce immune cells to express CCR9, thereby promoting homing to the small intestine [73]. Thus the local microenvironment dictates the homing preferences of the induced memory B cells. Moreover, homing profiles of nonmucosal memory B cells that, for example, traffic to the airways or skin share expression of CCR10 and α4β1, but they differ with regard to CLA, which binds to E-selectin in the skin. Hence the combination of several homing markers is needed to analyze the homing preferences of memory B cells (Fig. 7.3).

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VIII. ARE MUCOSAL MEMORY B CELLS SESSILE OR RECIRCULATING?

VIII. ARE MUCOSAL MEMORY B CELLS SESSILE OR RECIRCULATING? Whether memory B cells are recirculating cells or sessile in the secondary lymphoid tissues is a matter of debate [15]. Most memory B cells are unswitched IgM cells, as has been observed, for example, after rotavirus infections in humans [3,71]. Furthermore, a large number of IgA- and IgM-expressing cells with a memory phenotype (e.g., CD271, CD801) do circulate in human blood [31,74]. On the other hand, the anatomic localization of memory B cells has

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proven critical for their ability to participate in secondary responses to antigen exposure [15]. For example, chemokine-dependent localization of memory B cells to the marginal and perifollicular areas of the lymph node was shown to depend on CCR6 expression and was found to be important for their ability to respond to recall responses [75]. Whether this applies to mucosal tissues is not completely clear, but CCR6 has been associated with migration of antigen-specific lymphocytes to mucosal tissues [76,77]. In CCR6-deficient mice, the IgA response was reduced, and IgA memory B cell development and positioning in PPs were

FIGURE 7.4 Rare antigen-specific memory B cells respond rapidly to reactivation. The frequency of GFP1 memory B (green) cells in the tissues at 1 year following oral immunization is sparse, possibly as low as 1 NP memory B cell per 100.000 B cells (left panel). The arrows mark the memory GFP1 B cells, of which a majority reside in the B cell follicle close to the germinal center areas (GL71, red) in the Peyer’s patches (PPs). Five days after a single booster immunization with NP-CT, 1 year after priming immunizations, an impressive expansion of the memory B cell response can be observed. The PPs become loaded with GFP1 memory B cells expanding in secondary GC, and the differentiation of IgA plasma cells, which appear in the lamina propria (LP) of the intestinal villi, is very strong. As many as 15% of all IgA plasma cells in the LP were found to be GFP1 NP-specific.

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impaired, affecting, for example, protection against rotavirus infection [78,79]. Of note, CCR6 has been found to mark memory B precursor cells in both mouse and human GCs [80]. By contrast, human memory B cells uniquely express CD27, which does not apply to mouse memory B cells [81]. However, not all CD271 B cells are memory cells, as this marker also identifies newly antigenexperienced B cells [82]. Furthermore, memory B cells could also be CD272 as has been shown, for example, in systemic lupus erythematosus patients [83]. By studying our NP system, we found that 1 year following oral immunization, specific memory B cells resided in the B cell follicles, often close to GCs, in the spleen, MLNs, and GALT [32] (Fig. 7.4). Thus while the response was initiated in the PPs, the presence of clonally related memory B cells could be found both systemically and locally. These memory B cells were rare in the tissues at 1 year after immunization, but we calculated a frequency of memory B cells that was 1 per 100.000 isolated B cells. However, following a recall response, rapid expansion was observed, and from hardly detectable levels of memory B cells in the tissues, we achieved 15% of all IgA plasma cells in the gut LP that were NP-specific within 5 days of an oral challenge with NP-CT. As has been well documented for systemically generated memory B cells as well as for memory B cells in the lung, these orally induced NPspecific memory B cells expressed CD80, PDL2, and CD73 [55]. Upon adoptive transfer of the CD801 memory B cells to a naı¨ve host, we observed strong IgA and IgG responses to a single oral NP-CT challenge immunization. The memory B cells expressed α4β7 integrin, and upon reactivation in PPs, they underwent strong clonal expansion and selection in GCs with additional affinity maturation and upregulated expression of CCR9 [32]. This allowed memory B cells from PPs, but not from the spleen, to home to gut mucosal LP and give rise

to an NP-specific IgA plasma cell response. Hence mucosal memory B cells in the spleen cannot contribute to the gut LP IgA plasma cell response unless they are migrating to a site, such as a PP, where CCR9 expression is acquired [32]. On the other hand, irrespective of whether the memory B cells were isolated from PPs, MLNs, or the spleen, the progeny gave rise to a strong IgA plasma cell response in the small intestinal LP following oral immunization of recipient mice, indicating that it is the site of reexposure to antigen that determines whether the memory B cell acquires CCR9, a property that is imprinted in PPs but not in the spleen. Thus both α4β7 and CCR9 expression are required for mucosal memory B cell responses to locate to the small intestinal LP [32]. Furthermore, the isotype of the memory B cell appears not to be critical because transfer of memory B cells from the spleen, dominated by IgM1 cells, was as effective at giving rise to a gut IgA plasma cell response in the LP as transfer of cells from the PPs [84]. We found that memory B cells in the spleen exhibited enhanced expression of α4β7 only after oral but not after systemic priming, and we concluded that clear distinctions apply to the homing properties imprinted after mucosal as opposed to systemic immunizations. This is why parenteral vaccination poorly primes for a mucosal IgA response [1,5,74]. The extent to which mucosal memory B cells are different from those derived from systemic priming with respect to other features is unclear, but IgG1 memory B cells appear to depend on the T-bet transcription factor (Tbx21), whereas memory IgA1 B cells have been shown to express RORα, which was found to upregulate α4β7 [85]. Of note, rotavirus-specific memory B cells that did not express α4β7 failed to transfer gut LP IgA immunity, but they effectively transferred a systemic IgG response [43]. On the basis of our observations, we propose that the GC environment in PPs is quite unique in that it allows for expansion, selection, and

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IX. MEMORY B CELLS SHOW POOR CLONAL

differentiation of IgM1 as well as IgA1 memory B cells [41]. In addition, we know now that significant IgG CSR also occurs in the PPs upon oral priming and that long-lived IgG plasma cells in the bone marrow are clonally related to long-lived IgA plasma cells in the bone marrow and in the gut [32]. It therefore appears likely that the memory B cells acquire different homing receptors in the PPs depending on whether they express IgA or IgG isotypes. In fact, IgG2a1 memory B cells, in contrast to IgA1 memory B cells, have been shown to express CXCR3, which facilitates localization to the lung or inflammatory sites [85]. Why IgGswitched B cells do not express CCR9 is poorly known at present. Additional work is needed to better understand the complexity of how clonally related IgG and IgA memory B cells and plasma cells can populate the same or distinctly different tissues following oral immunizations.

IX. MEMORY B CELLS SHOW POOR CLONAL RELATEDNESS TO LONGLIVED PLASMA CELLS FOLLOWING ORAL PRIMING IMMUNIZATIONS Previous studies have shown that isotypeswitched IgA1 memory B cells can engage in secondary GC reactions [32]. This is further supported by studies at the single-cell level in which increased BCR diversification in switched memory B cells was achieved in secondary GC [86]. Memory B cells’ ability to reengage in GC is ascribed to the expression of CD80 and PD-L2 [15,87]. But following BCR reactivation, we observed downmodulated expression of PD-L2, while the CD80 expression was preserved [32]. Nevertheless, the importance of a secondary selective step of highavidity memory B cells upon recall antigen reactivation was clearly observed in our NP-specific system [32]. Using next-generation sequencing analysis of sorted NP-specific IgA IghV gene

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sequences in memory B cells and gut IgA plasma cells, we could demonstrate that the cells were only poorly related, but after an oral challenge immunization with NP-CT, these clones became closely related [32]. We therefore proposed a model in which plasticity of the GALT allows for the generation of memory B cells concomitant with the generation of longlived plasma cells but involving temporarily separate processes in the GC or even anatomically separate GC leading to lower-affinity maturation in memory B cells. Others have documented that a fundamental dichotomy exists between processes that drive development of memory B cells and those that drive long-lived plasma cells during a GC response [80]. Indeed, systemic immunizations have demonstrated that memory B cells leave the GC before long-lived plasma cells do and therefore have acquired fewer SHMs [17,88]. This process may not be stochastic in nature but rather may be regulated by Tfh cells and the expression of the transcriptional repressor Bach2 gene in the B cell, directing the differentiation to a memory B cell [89]. We have speculated that this allows for a broader, less mutated repertoire of mucosal IgM1 and IgA1 memory B cells that, upon reexposure to antigen, undergo strong selection and differentiation into high-affinity gut LP IgA plasma cells; that is, only strongly mutated clones are represented. It can be envisioned that a broad memory B cell repertoire is functionally important and could, for example, explain cross-protection against related pathogens, as is seen between Vibrio cholerae and enterotoxigenic E. coli infections [74] (Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control and Chapter 32: Oral Vaccines for Enterotoxigenic Escherichia coli). The transition from a clonally poorly related mucosal IgA1 memory B cell and long-lived plasma cell response to a clonally closely related gut IgA recall response supports the notion that mucosal memory B cells likely participate in secondary GC reactions in the

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PPs. It will be important to dissect the secondary GC reaction in PPs in greater detail in future studies.

X. CONSIDERATIONS FOR MUCOSAL SUBCOMPONENT VACCINE DEVELOPMENT In natural cholera infection, memory B cells develop, and upon reexposure to antigen, strong IgA1 plasma cell responses in the gut LP associated with protection against disease can be seen [26]. The goal of vaccination is to create conditions that effectively mimic such a response pattern. However, a central question is whether a mucosal vaccine can be made from subcomponents or whether it requires the whole organism. Surprisingly, a study found that while V. cholerae provides a large antigen diversity, as many as 75% of the investigated clones of circulating memory B cells reacted with CT or lipopolysaccharide, indicating that a select number of dominant antigens appear to convey immune protection [26]. Moreover, the anti-CT memory B cell clones exhibited a high degree of clonality, suggesting a very focused response. Hence specific gut IgA responses appear to be oligoclonal, and essentially all gut LP IgA plasma cells have acquired extensive SHMs, suggesting that these are GC-dependent responses. Therefore it appears that a mucosal vaccine with a strong memory-B-cell-inducing capability directed against a set of subcomponents rather than against the whole microorganism could be a winning strategy. From experimental studies, a plethora of mucosal subcomponent vaccines have been developed and tested with promising results. However, the limitation at present is whether we can identify mucosal adjuvants that are safe, effective, and, above all, potent drivers of memory B cell development [1,12]. The best-studied adjuvant systems for enhancing mucosal immune responses and stimulating mucosal memory B

cells are the enteroxins CT and E. coli heatlabile toxin, which unfortunately have been found to be too toxic to be used in clinical vaccines [90] (Chapter 11: Toxin-Based Modulators for Regulation of Mucosal Immune Responses). Several mutants or derivatives of these enterotoxins have therefore been developed, and some have been clinically tested with promising results. We have developed the mucosal CTA1-DD adjuvant, which was to be found superior to alum or CpG in stimulating memory B cells and long-lived plasma cells in the mouse model [84]. However, more efforts are needed to evaluate mucosal adjuvant formulations for how effective they are at stimulating memory B cell development. Thus we may identify effective mucosal adjuvants that allows us to design and develop the next generation of effective mucosal vaccines based on selected subcomponents.

XI. CONCLUDING REMARKS Recent progress in the study of mucosal memory B cells has been quite exceptional. This has been greatly helped by the identification of markers to be used to selectively study mucosal memory B cells [15]. It is commonly agreed that CD80, PD-L2, and CD73 in the mouse system and CD27 and CD80 in the human system are useful markers with which to identify memory B cells. These markers will allow for detailed investigations of the site of formation, persistence, regulation, and fate of memory B cell responses following mucosal vaccination. Establishing models in which the memory B cells can be traced and monitored in real time by two-photon microscopy and single-cell analysis will provide a shift in paradigm of how to understand their function and their requirements for activation. In particular, given the fast development of single-cell RNA-seq analysis, a better characterization of mucosal memory B cells will soon be available [18]. Although

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REFERENCES

many questions remain regarding mucosal memory B cells, the rapid development of new technologies provides cause for optimism about the prospects for the development of a next generation of mucosal vaccines that more effectively stimulate memory B cell responses.

References [1] Lycke N. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 2012;12(8):592 605. [2] Harris AM, et al. Antigen-specific memory B-cell responses to Vibrio cholerae O1 infection in Bangladesh. Infect Immun 2009;77(9):3850 6. [3] Narvaez CF, et al. Human rotavirus-specific IgM Memory B cells have differential cloning efficiencies and switch capacities and play a role in antiviral immunity in vivo. J Virol 2012;86(19):10829 40. [4] Quiding M, et al. Intestinal immune responses in humans. Oral cholera vaccination induces strong intestinal antibody responses and interferon-gamma production and evokes local immunological memory. J Clin Invest 1991;88(1):143 8. [5] Pasetti MF, et al. Immunology of gut mucosal vaccines. Immunol Rev 2011;239(1):125 48. [6] Falsey AR, Singh HK, Walsh EE. Serum antibody decay in adults following natural respiratory syncytial virus infection. J Med Virol 2006;78(11):1493 7. [7] Walsh EE, Falsey AR. Humoral and mucosal immunity in protection from natural respiratory syncytial virus infection in adults. J Infect Dis 2004;190(2):373 8. [8] Habibi MS, et al. Impaired antibody-mediated protection and defective IgA B-cell memory in experimental infection of adults with respiratory syncytial virus. Am J Respir Crit Care Med 2015;191(9):1040 9. [9] Witt MA, Katz PH, Witt DJ. Unexpectedly limited durability of immunity following acellular pertussis vaccination in preadolescents in a North American outbreak. Clin Infect Dis 2012;54(12):1730 5. [10] Pape KA, et al. Naive B cells with high-avidity germline-encoded antigen receptors produce persistent IgM ( 1 ) and transient IgG( 1 ) memory B cells. Immunity 2018;48(6):1135 1143 e4. [11] Bemark M, et al. A unique role of the cholera toxin A1DD adjuvant for long-term plasma and memory B cell development. J Immunol 2011;186(3):1399 410. [12] Lycke N. Is the choice of vaccine adjuvant critical for long-term memory development? Expert Rev Vaccines 2010;9(12):1357 61.

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[13] Inoue T, et al. Generation of memory B cells and their reactivation. Immunol Rev 2018;283(1):138 49. [14] Pape KA, et al. Different B cell populations mediate early and late memory during an endogenous immune response. Science 2011;331(6021):1203 7. [15] Weisel F, Shlomchik M. Memory B cells of mice and humans. Annu Rev Immunol 2017;35:255 84. [16] Anderson SM, et al. New markers for murine memory B cells that define mutated and unmutated subsets. J Exp Med 2007;204(9):2103 14. [17] Phan TG, Tangye SG. Memory B cells: total recall. Curr Opin Immunol 2017;45:132 40. [18] Nguyen A, et al. Single cell RNA sequencing of rare immune cell populations. Front Immunol 2018;9:1553. [19] Lycke NY, Bemark M. The regulation of gut mucosal IgA B-cell responses: recent developments. Mucosal Immunol 2017;10(6):1361 74. [20] Hapfelmeier S, et al. Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses. Science 2010;328(5986):1705 9. [21] Macpherson AJ, et al. IgA function in relation to the intestinal microbiota. Annu Rev Immunol 2018;36:359 81. [22] Bunker JJ, et al. Innate and adaptive humoral responses coat distinct commensal bacteria with immunoglobulin A. Immunity 2015;43(3):541 53. [23] Le Gallou S, et al. A splenic IgM memory subset with antibacterial specificities is sustained from persistent mucosal responses. J Exp Med 2018;215(8):2035 53. [24] Magri G, et al. Human secretory IgM emerges from plasma cells clonally related to gut memory B cells and targets highly diverse commensals. Immunity 2017;47 (1):118 34 e8. [25] Spencer J, Sollid LM. The human intestinal B-cell response. Mucosal Immunol 2016;9(5):1113 24. [26] Kauffman RC, et al. Single-cell analysis of the plasmablast response to Vibrio cholerae demonstrates expansion of cross-reactive memory B cells. MBio 2016;7(6). [27] Lindner C, et al. Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota. Nat Immunol 2015;16(8):880 8. [28] Lindner C, et al. Age, microbiota, and T cells shape diverse individual IgA repertoires in the intestine. J Exp Med 2012;209(2):365 77. [29] Berkowska MA, et al. Circulating human CD27-IgA 1 memory B cells recognize bacteria with polyreactive Igs. J Immunol 2015;195(4):1417 26. [30] Cardeno A, et al. Activated T follicular helper-like cells are released into blood after oral vaccination and correlate with vaccine specific mucosal B-cell memory. Sci Rep 2018;8(1):2729. [31] van Splunter M, et al. Oral cholera vaccination promotes homing of IgA( 1 ) memory B cells to the large

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130

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44] [45]

[46]

[47]

7. INDUCTION AND REGULATION OF MUCOSAL MEMORY B CELL RESPONSES

intestine and the respiratory tract. Mucosal Immunol 2018;11(4):1254 64. Bemark M, et al. Limited clonal relatedness between gut IgA plasma cells and memory B cells after oral immunization. Nat Commun 2016;7:12698. Vajdy M, Lycke N. Mucosal memory B cells retain the ability to produce IgM antibodies 2 years after oral immunization. Immunology 1995;86(3):336 42. Toyama H, et al. Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells. Immunity 2002;17(3):329 39. Taylor JJ, Jenkins MK, Pape KA. Heterogeneity in the differentiation and function of memory B cells. Trends Immunol 2012;33(12):590 7. Tarlinton D, Victora G. Editorial overview: germinal centers and memory B-cells: from here to eternity. Curr Opin Immunol 2017;45:v viii. Cunningham AF, et al. Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection. J Immunol 2007;178(10):6200 7. Inamine A, et al. Two waves of memory B-cell generation in the primary immune response. Int Immunol 2005;17(5):581 9. Gebert A, et al. Antigen transport into Peyer’s patches: increased uptake by constant numbers of M cells. Am J Pathol 2004;164(1):65 72. Hahn A, et al. Mesenteric lymph nodes are not required for an intestinal immunoglobulin A response to oral cholera toxin. Immunology 2010;129(3):427 36. Lycke NY, Bemark M. The role of Peyer’s patches in synchronizing gut IgA responses. Front Immunol 2012;3:329. Bergqvist P, et al. T cell-independent IgA class switch recombination is restricted to the GALT and occurs prior to manifest germinal center formation. J Immunol 2010;184(7):3545 53. Williams MB, et al. The memory B cell subset responsible for the secretory IgA response and protective humoral immunity to rotavirus expresses the intestinal homing receptor, alpha4beta7. J Immunol 1998;161(8):4227 35. Papa I, Vinuesa CG. Synaptic interactions in germinal centers. Front Immunol 2018;9:1858. Arakawa H, et al. Oligoclonal development of B cells bearing discrete Ig chains in chicken single germinal centers. J Immunol 1998;160(9):4232 41. Bergqvist P, et al. Re-utilization of germinal centers in multiple Peyer’s patches results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses. Mucosal Immunol 2013;6(1):122 35. Takahashi Y, et al. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl.

[48]

[49]

[50]

[51] [52]

[53] [54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

V. Affinity maturation develops in two stages of clonal selection. J Exp Med 1998;187(6):885 95. Allen D, et al. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO J 1988;7(7):1995 2001. Jacob J, et al. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells. J Exp Med 1993;178 (4):1293 307. Reboldi A, Cyster JG. Peyer’s patches: organizing Bcell responses at the intestinal frontier. Immunol Rev 2016;271(1):230 45. Victora GD, Nussenzweig MC. Germinal centers. Annu Rev Immunol 2012;30:429 57. Kau AL, et al. Functional characterization of IgAtargeted bacterial taxa from undernourished Malawian children that produce diet-dependent enteropathy. Sci Transl Med 2015;7(276) 276ra24. Allie SR, Randall TD. Pulmonary immunity to viruses. Clin Sci (Lond) 2017;131(14):1737 62. Hwang JY, Randall TD, Silva-Sanchez A. Inducible bronchus-associated lymphoid tissue: taming inflammation in the lung. Front Immunol 2016;7:258. Onodera T, et al. Memory B cells in the lung participate in protective humoral immune responses to pulmonary influenza virus reinfection. Proc Natl Acad Sci USA 2012;109(7):2485 90. Adachi Y, et al. Distinct germinal center selection at local sites shapes memory B cell response to viral escape. J Exp Med 2015;212(10):1709 23. Kumar BV, et al. Human tissue-resident memory T cells are defined by core transcriptional and functional signatures in lymphoid and mucosal sites. Cell Rep 2017;20(12):2921 34. Renegar KB, et al. Role of IgA versus IgG in the control of influenza viral infection in the murine respiratory tract. J Immunol 2004;173(3):1978 86. Moyron-Quiroz JE, et al. Role of inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity. Nat Med 2004;10(9):927 34. Boyden AW, et al. Primary and long-term B-cell responses in the upper airway and lung after influenza A virus infection. Immunol Res 2014;59(1 3):73 80. Warfel JM, Edwards KM. Pertussis vaccines and the challenge of inducing durable immunity. Curr Opin Immunol 2015;35:48 54. Amanna IJ, Carlson NE, Slifka MK. Duration of humoral immunity to common viral and vaccine antigens. N Engl J Med 2007;357(19):1903 15. Le T, et al. Immune responses and antibody decay after immunization of adolescents and adults with an

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

acellular pertussis vaccine: the APERT Study. J Infect Dis 2004;190(3):535 44. Aguilo N, et al. Pulmonary but not subcutaneous delivery of BCG vaccine confers protection to tuberculosis-susceptible mice by an interleukin 17-dependent mechanism. J Infect Dis 2016;213 (5):831 9. Raeven RH, et al. Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination. Mucosal Immunol 2018;11(3):979 93. Turner DL, et al. Lung niches for the generation and maintenance of tissue-resident memory T cells. Mucosal Immunol 2014;7(3):501 10. Ross PJ, et al. Relative contribution of Th1 and Th17 cells in adaptive immunity to Bordetella pertussis: towards the rational design of an improved acellular pertussis vaccine. PLoS Pathog 2013;9(4):e1003264. Eliasson DG, et al. M2e-tetramer-specific memory CD4 T cells are broadly protective against influenza infection. Mucosal Immunol 2018;11(1):273 89. Allen AC, et al. Sustained protective immunity against Bordetella pertussis nasal colonization by intranasal immunization with a vaccine-adjuvant combination that induces IL-17-secreting TRM cells. Mucosal Immunol 2018;11:1763 76. Lamb CA, et al. Gut-selective integrin-targeted therapies for inflammatory bowel disease. J Crohns Colitis 2018;12(Suppl. 2):S653 68. Blutt SE, et al. IgA is important for clearance and critical for protection from rotavirus infection. Mucosal Immunol 2012;5(6):712 19. Demberg T, et al. Phenotypes and distribution of mucosal memory B-cell populations in the SIV/SHIV rhesus macaque model. Clin Immunol 2014;153 (2):264 76. Bakdash G, et al. Retinoic acid primes human dendritic cells to induce gut-homing, IL-10-producing regulatory T cells. Mucosal Immunol 2015;8(2):265 78. Czerkinsky C, Holmgren J. Vaccines against enteric infections for the developing world. Philos Trans R Soc Lond B Biol Sci 2015;370:1671. Elgueta R, et al. CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen. J Immunol 2015;194(2):505 13. McDonald KG, et al. CCR6 promotes steady-state mononuclear phagocyte association with the intestinal epithelium, imprinting and immune surveillance. Immunology 2017;152(4):613 27.

131

[77] Calenda G, et al. Integrin alpha4beta7 blockade preferentially impacts CCR6( 1 ) lymphocyte subsets in blood and mucosal tissues of naive rhesus macaques. J Immunol 2018;200(2):810 20. [78] Cook DN, et al. CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue. Immunity 2000;12(5):495 503. [79] Lin YL, Ip PP, Liao F. CCR6 deficiency impairs IgA production and dysregulates antimicrobial peptide production, altering the intestinal flora. Front Immunol 2017;8:805. [80] Suan D, et al. CCR6 defines memory B cell precursors in mouse and human germinal centers, revealing lightzone location and predominant low antigen affinity. Immunity 2017;47(6):1142 53 e4. [81] Klein U, Kuppers R, Rajewsky K. Evidence for a large compartment of IgM-expressing memory B cells in humans. Blood 1997;89(4):1288 98. [82] Macallan DC, et al. B-cell kinetics in humans: rapid turnover of peripheral blood memory cells. Blood 2005;105(9):3633 40. [83] Wei C, et al. A new population of cells lacking expression of CD27 represents a notable component of the B cell memory compartment in systemic lupus erythematosus. J Immunol 2007;178(10): 6624 33. [84] Lycke N, Bemark M. Mucosal adjuvants and long-term memory development with special focus on CTA1-DD and other ADP-ribosylating toxins. Mucosal Immunol 2010;3(6):556 66. [85] Wang NS, et al. Divergent transcriptional programming of class-specific B cell memory by T-bet and RORalpha. Nat Immunol 2012;13(6):604 11. [86] McHeyzer-Williams LJ, et al. Class-switched memory B cells remodel BCRs within secondary germinal centers. Nat Immunol 2015;16(3):296 305. [87] Zuccarino-Catania GV, et al. CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype. Nat Immunol 2014;15(7):631 7. [88] Weisel FJ, et al. A temporal switch in the germinal center determines differential output of memory B and plasma cells. Immunity 2016;44(1):116 30. [89] Shinnakasu R, Kurosaki T. Regulation of memory B and plasma cell differentiation. Curr Opin Immunol 2017;45:126 31. [90] Lycke N, Lebrero-Fernandez C. ADP-ribosylating enterotoxins as vaccine adjuvants. Curr Opin Pharmacol 2018;41:42 51.

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C H A P T E R

8

Induction and Regulation of Mucosal Memory T Cell Responses Kiyoshi Hirahara and Toshinori Nakayama Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan

I. INTRODUCTION To protect the host from the continuous invasion of various harmful particles, the lung develops a unique mucosal barrier system that consists of various types of immune cells, including tissue-resident immune cells. Based on the migration pattern within the body, memory T cells can be divided into three subpopulations: central memory T (TCM) cells, effector memory T (TEM) cells, and the recently identified subpopulation tissue-resident memory T (TRM) cells [11,12]. TCM cells express CD62L and CCR7 and can respond rapidly to pathogens independently of T cell recruitment from the blood [13]. In contrast, TEM cells, which circulate in the blood, nonlymphoid tissue, and secondary lymphoid organs, exhibit a low expression of CD62L and CCR7 [13].

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00008-0

II. MUCOSAL COMPARTMENTALIZATION THAT CAUSES INFLAMMATORY RESPONSES IN THE AIRWAY A. Preference of Tissue-Resident Memory T Cells for Mucosal Tissues TRM cells reside permanently in the epithelium, particularly the mucosal tissues, being maintained independently of the lymphoid and circulating T cell populations with a high expression of CD69 and CD103 [1]. Although the concept of TRM was initially driven by CD81 TRM cells in studies of viral infectious disease [2], CD41 TRM cells were subsequently confirmed in mucosal organs such as the lungs, reproductive organs, and skin [3 5]. CD41 TRM cells are required for protective immune responses to viral infections at mucosal sites in the

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reproductive tract [6,7]. In contrast, certain lung CD41 TRM cells are involved in the pathogenesis of respiratory allergic inflammation, such as bronchial asthma [8,9]. Interestingly, visceral adipose tissue (mesenchymal, gonadal, and subcutaneous fat tissue) has recently been identified as a reservoir of CD41 TRM cells in the lung [10]. Further investigations are needed to clarify the precise mechanism underlying the induction and maintenance of CD41 TRM cells in the lung.

different mucosal organs may therefore be crucial for the pathology of chronic immune-mediated diseases (Fig. 8.1). Taken together, these findings from recent studies imply that T helper (Th) cells show far more plasticity and heterogeneity than was previously imagined. Precise assessments regarding the nature of Th cells will be indispensable in the development of new therapeutic approaches for these immune-related diseases.

B. Presence of Heterogenic Memory Th2 Cells in Different Mucosal Organs

C. Epithelial Cytokines as Key Mediators for Mucosal Immune Responses: IL-25, IL-33, and TSLP

Emerging technological developments have allowed us to investigate the genetic expression at the single-cell level among various T cell subsets [14 17]. A single-cell analysis revealed that the populations of T cell subsets are more heterogeneous than predicted. For Th2 cells, different types of effector and memory Th2 cells are involved in shaping the type 2 immune responses in vivo [18 21]. Among these subpopulations, interleukin (IL)5-producing Th2 cells are known to contribute to the pathogenicity of immune-mediated inflammatory diseases in both humans and mice because these IL-5-producing populations have the ability to induce the massive infiltration of eosinophils in the local mucosal sites [18 25]. ST2-expressing memory Th2 cells have the ability to produce a large amount of IL-5 after the stimulation of IL-33; these ST2 1 memory Th2 cells have therefore been named pathogenic Th2 (Tpath2) cells [18,26]. IL-33 confers the induction of memory Tpath2 cells from effector Th2 cells through the enhancement of the ST2 expression on T cells during chronic lung inflammation [18], whereas CCR8 1 memory Th2 cells are key pathogenic players that induce the production of high levels of IL-5 during chronic skin inflammation [19]. Another memory Th2 cell subset with a high expression of GPR15 is increased in the colon of patients with ulcerative colitis [23]. The heterogeneity of memory Th2 cells among

The mucosal barriers at the respiratory epithelial sites functions as the first line of defense against harmful pathogens and have a distinct role that initiates the sequential immune response. Damage to the mucosal barrier causes the identification of several epithelial cytokines, including IL-25, thymic stromal lymphopoietin (TSLP), and IL-33. All of these epithelial cytokines have the potential to induce type 2 immune responses via both innate and adaptive immunity at the mucosal sites. In 2005, the IL-1 family member IL-33 was identified as a ligand of ST2, an IL-1 receptor family member [27]. IL-33 is constitutively expressed on epithelial cells and other types of mesenchymal cells [28 31]. IL-33 is not secreted like a conventional cytokine because of the absence of signal sequence [32]. Indeed, IL-33 is localized in the nuclei of these epithelial cells and mesenchymal cells and is associated with chromatin by a chromatin-binding motif, functioning as a transcriptional repressor for the cellular homeostasis under steady state [33]. However, necrotic cell death or cellular activation through ATP signaling in the absence of cell death that is induced by mechanical injury or chronic exposure to various exogenous allergens or pathogens, such as tobacco smoke or inhaled irritant particles, prompts epithelial cells to release their stored IL-33 into the extracellular space [34,35].

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Lung: CD44highCD62LlowCXCR3lowCCR4highCCR8highIL-7Rα αhigh ST2high memory Th2 (Tpath2) cells ST2

(Endo Y et al. Immunity 2011) (Endo Y et al. Immunity 2015) (Nakayama T et al. Annu Rev Immunol 2017)

CRTH2highCD161highCD49dhigh Th2 cells (Wambre E et al. Sci Transl Med 2017) CD161

CRTH2

Chronic allergic inflammation

Skin: CCR8high Th2 cells

CCR8

(Islam SA et al. Nat Immunol 2011)

Chronic atopic dermatitis

Upper gastrointestinal tract: CRTH2highCD161highhPGDSpositive Th2 cells CD161

CRTH2

(Mitson-Salazar A et al. J Allergy Clin Immunol 2016)

Eosinophilic gastrointestinal disease

Colon: CD4+CD45ROhighGPR15high memory like Th2 cells GPR15

(Nguyen LP et al. Nat Immunol 2015)

Ulcerative colitis

FIGURE 8.1 Various pathogenic helper-2 T (Tpath2) cells are involved in the pathogenesis of the inflammatory diseases at the mucosal sites. Source: Modified rendition of one that was previously published from Hirahara K, Nakayama T. CD41 T-cell subsets in inflammatory diseases: beyond the Th1/Th2 paradigm. Int Immunol 2016;28:163 71.

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It is difficult to interpret experimental results using mouse models and predict the IL-33-targeting therapeutic effects in human because the route of IL-33 release differs between humans and mice, although mesenchymal cells preserve IL-33 in the nuclei at the mucosal tissues in both human and mouse. For example, human endothelial cells preserve IL-33 universally, whereas the expression of IL-33 by vascular endothelial cells is not constitutive in mice [29,36,37].

D. Innate and Adaptive Immunity Involvement in IL-33-Induced Inflammation at Mucosal Sites IL-33 activates both innate and adaptive immune cells, which are involved in type 2 immune responses, such as effector Th2 cells, some memory Th2 cells, mast cells (for further description of mast cells in mucosal immunity, see Chapter 13: Mast Cells for the Control of Mucosal Immunity), eosinophils, and ILC2s [38 41]. Consequently, IL-33 induces robust type 2 immune responses followed by eosinophilic inflammation at mucosal sites. In particular, ILC2s and CD41 T cells that express high levels of ST2 work as key cell populations during IL-33-induced inflammatory responses. A subpopulation of CD41 TRM cells that express high levels of ST2 accompanied by the high expression of CD44 and low expression of CD62L resides in the lung [42]. ILCs are another tissue-resident cell population that react promptly to various inner signals, such as cytokines, pathogens, and allergens, without antigen-specific stimulation [40,43]. Among ILCs, ILC2s express high levels of ST2 [40]. ILC2s play pivotal roles in the induction of the type 2 immune response during the neonatal period, as adaptive immunity is immature in the postnatal stage [44]. ILC2s preferentially reside in the lung and produce large amounts

of Th2 cytokines, such as IL-5 and IL-13, in response to IL-33, IL-25, and TSLP in an antigen-independent manner [45 47]. Crosstalk between ILC2 and CD41 T cells has also been reported. Mice lacking ILC2s showed impaired Th2 cell differentiation [48,49], and ILC2 modulated the Th2 cell differentiation in a cell-contact manner through MHC class ll [50]. Both ST2 1 CD41 TRM cells and ILC2s are involved in the IL-33-induced eosinophilic inflammation [42]. In a mouse model of eosinophilic lung inflammation induced by IL-33, steroid treatment induced no inhibitory effects in the infiltration of ST21CD41 TRM and ILC2s cells that produced substantial amounts of IL-5 and IL-13 [42]. IL-33 is involved in different types of human diseases, such as allergic diseases, infectious diseases, cardiovascular diseases, and metabolic diseases [36]. In asthma in particular, genome-wide association studies have repeatedly shown that both IL-33 and ST2 gene loci are significantly associated with the onset of asthma [51], and an IL-33 signal pathway polymorphism was found to be associated with the clinical phenotype of asthma in childhood [52]. Given the above findings, IL-33 is a key epithelial cytokine that directs the inflammatory diseases at the mucosal sites.

III. MUCOSAL INFLAMMATION AND INDUCIBLE BRONCHUSASSOCIATED LYMPHOID TISSUE A. Induction of Inducible Bronchus-Associated Lymphoid Tissue During Inflammation at Mucosal Tissue Sites Bronchus-associated lymphoid tissue (BALT) develops as normal mucosal lymphoid tissue in the airway in some mammalian species, such as rabbits and rats, but not in humans

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III. MUCOSAL INFLAMMATION AND INDUCIBLE BRONCHUS-ASSOCIATED LYMPHOID TISSUE

or mice [53]. In humans, chronic inflammation induced by smoking, infection, or autoimmune diseases causes the formation of BALT structures that are termed inducible BALT (iBALT) [9,54 56]. iBALT (for further description of iBALT, see Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance) does not develop in a preprogrammed way, and its development and size in the lung depend on the type and duration of antigenic exposure at mucosal sites; iBALT is, therefore, a classic example of a tertiary lymphoid tissue [57 59]. Similar ectopic lymphoid structures known as skin-associated lymphoid tissues and nasalassociated lymphoreticular tissue are observed at other mucosal sites such as the skin and upper airway, respectively [60,61]. The typical iBALT structure consists of separate T and B cell areas accompanied by the presence of specific cell populations, such as follicular dendritic cells (FDCs), resident dendritic cells (DCs), high endothelial venules, and lymphatics [55,62]. Resident DCs within iBALT may present antigens, and FDCs are needed for the development of proper B cell follicles [56]. CD11c1 DCs are involved in the maintenance of the iBALT structure [63]. Massive lymphangiogenesis also occurs around the iBALT structure during sustained inflammation [64]. iBALT participates in the pathogenesis of mucosal inflammatory diseases in a couple of ways: Antigen-presentation and T cell-priming can occur directly in iBALT in response to antigens derived from the airways; [65], and antigen-specific memory T cells can be maintained in iBALT as CD41 TRM cells, which react efficiently to secondary immune responses [9,66] Thus the formation of iBALT is closely associated with the pathology of various inflammatory diseases. For example, the formation of iBALT is observed in the lungs of patients with chronic obstructive

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pulmonary disease, and the disease severity is correlated with the amount of iBALT in the lung [67]. iBALT formation can also be observed in the lungs of patients with rheumatoid arthritis or Sjo¨gren syndrome [68]. Furthermore, the Th2 cell-associated immune response in experimental settings, such as house dust mite driven airway inflammation or ovalbumin-induced chronic allergic airway inflammation, can induce iBALT structures in mice [9,69].

B. Maintenance of Memory T Cells Within the Inducible BronchusAssociated Lymphoid Tissue in the Mucosal Site After the induction of inflammation at mucosal sites, some antigen-specific memory T cells are maintained in the iBALT as CD41 TRM cells for efficient secondary immune responses [9,66]. In the case of chronic allergic airway inflammation, the maintenance of antigen-specific memory Th2 cells is dependent on Thy1 1 IL-7-producing lymphatic endothelial cells (LECs), which are localized within iBALT structures [12]. After the induction of allergic airway inflammation, antigen-specific memory Th2 cells preferentially localize in the iBALT and are maintained in an IL-7-dependent manner. IL-7-producing LECs increase in number under inflammatory conditions, suggesting that the survival of antigen-specific memory Th2 cells within iBALT is induced by the modification of the lung microenvironment. Interestingly, an unbiased genome-wide gene expression analysis of Thy1 1 IL-7producing LECs revealed that this cell population also produces IL-33 [9]. As was discussed earlier in the chapter, IL-33 directly instructs ST2 1 memory-type Th2 cells to enhance their IL-5 production,

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thereby exacerbating eosinophilic inflammation [18,70]. Thus IL-33 stimulation by Thy1 1 IL-7-producing LECs within iBALT is critical for memory Tpath2 cells to maintain their pathogenic ability, such as producing type 2 cytokines. Taken together, these findings indicate that IL-7 supports the survival of memory Tpath2 cells and suggest that IL-33 may be responsible for those cells’ pathogenic functionality in terms of their ability to produce high amounts of IL-5 and induce eosinophilic inflammation in the airway.

We must reconsider the pathogenicity of T cell mediated inflammatory diseases beyond the classical Th1/Th2 balance disease induction model in light of our current understanding of the increased complexity and heterogeneity of Th cells. The heterogeneity of memory Th cells suggests that a specific population of Th cells directs the pathology of immune-mediated inflammatory diseases at mucosal sites. We have therefore proposed a “pathogenic Th

SLO, NLT

SLO Effector

Memory Tpath1

Th1

Tpath2

Naive CD4 + T cells

IV. MODEL OF DISEASE INDUCTION BY TPATH CELLS

Th2 Tpath2+17

Tpath17

Species

Phenotype

Involved diseases

Human

CXCR3high

Type 1 diabetes

Murine

CXCR3lowCCR4high IL-7R highST2high

Chronic airway inflammation

Human

CD45ROhighCD69high ST2highIL17RBhigh

Eosinophilic chronic rhinosinusitis

Human

CD45ROhighCRTH2high CCR4highCXCR3lowCCR6low CD27low

Alder pollen allergy

Murine

CCR8high

Atopic dermatitis

Human

CRTH2highCD161high hPGDShigh

Atopic dermatitis

Human

CRTH2highCD161high hPGDShigh

Eosinophilic gastro-intestinal disease

Human

CD45ROhigh GPR15high

Ulcerative colitis

Human

CRTH2high CCR6high

Asthma

Human

CD161high CCR6high

Asthma

Murine

IL-23Rhigh

Experimental Encephalitis

Th17 FIGURE 8.2 A schematic representation of the pathogenic Th population disease induction model. Memory Th1, Th2, and Th17 cells are generated in the secondary lymphoid organs (SLOs), while Tpath cells are generated in nonlymphoid tissues (NLTs), and Tpath cells appear to reside preferentially in these tissues [70]. Source: Credit Annual Review of Immunology.

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V. CONCLUDING REMARKS

population disease induction model” [70]. In this model, a pathogenic subpopulation of Th cells that possess distinguishing effector function(s), for example, atypical expression of cytokines, are generated in vivo and are involved in the pathogenesis (induction and persistence) of immune-mediated inflammatory diseases, regardless of the overall population balance of Th1 and Th2 subsets. For example, in type 2 immune pathologies, such as asthma, eosinophilic pneumonia, or chronic dermatitis, IL-5-producing Th2 cell subpopulations are considered to be the pathogenic populations, as IL-5-producing Th2 cells can induce the massive infiltration of eosinophils into the local inflammatory sites. Indeed, several groups, including our own, have reported that various distinct subpopulations of Th cells have been identified in mice and humans and play crucial roles in the pathogenesis of certain types of immune-mediated inflammatory diseases (Fig. 8.2). For example, Tpath2 cells are involved in chronic allergic inflammation of the airway, eosinophilic chronic rhinosinusitis, chronic atopic dermatitis, ulcerative colitis, and eosinophilic gastrointestinal diseases in both mice and humans [18 21,26]. In the case of Tpath1 cells, CXCR3high Th1 cells are known to be crucial for the pathology of type 1 diabetes [71]. Pathogenic Th17 cells are induced under conditions without TGF-β1 or with TGF-β3 [72,73]. A recent report showed that AIM (CD5L) restrains the Th17 cell pathogenicity through the regulation of lipid biosynthesis [74]. Thus our “pathogenic Th population disease induction model” is a key concept for understanding the pathogenicity of immune-mediated inflammatory diseases. In this model, rather than the balance among the Th cell subsets, a pathogenic subpopulation of Th cells that is induced under certain conditions is critical for the pathogenesis of immunemediated diseases [70]. Tpath cells, such as

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Tpath1, Tpath2, and Tpath17, may be potent specific-therapeutic targets for immunemediated intractable inflammatory diseases.

V. CONCLUDING REMARKS We have briefly reviewed the recent progress in research on the involvement of memory Th2 cells in the immune-mediated inflammatory diseases. The dysregulation of type 2 immune responses via various stimuli at mucosal sites induces many allergic inflammatory diseases, such as asthma. IL-33 plays a particularly crucial pathogenic role in directing the inflammatory diseases at mucosal sites. Tertiary lymphoid tissue, such as iBALT, is a key structure for the maintenance of memory T cells at mucosal sites. These ectopic lymphoid structures and the heterogeneity of memory Tpath cells might be involved in the induction of resistance to certain therapies that is observed in various immune-mediated inflammatory diseases. It will therefore become increasingly important to understand the precise features of Tpath cells to overcome intractable immune-mediated inflammatory diseases.

Acknowledgments We appreciate all of the members in Department of Immunology, Graduate School of Medicine, Chiba University, Japan. This work was supported by the following grants: Ministry of Education, Culture, Sports, Science and Technology (MEXT Japan) Grants-in-Aid for Scientific Research (S) 26221305, (C) 17K0876; AMED-CREST, AMED (No. JP18gm1210003); AMED-PRIME, AMED (No. JP19gm6110005); Practical Research Project for Allergic Diseases and Immunology (Research on Allergic Diseases and Immunology) from AMED (No. JP19ek0410060, JP19ek0410045), Mochida Memorial Foundation for Medical and Pharmaceutical Research, The Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care and Takeda Science Foundation.

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References [1] Mueller SN, Gebhardt T, Carbone FR, Heath WR. Memory T cell subsets, migration patterns, and tissue residence. Annu Rev Immunol 2013;31:137 61. [2] Masopust D, Vezys V, Marzo AL, Lefrancois L. Preferential localization of effector memory cells in nonlymphoid tissue. Science 2001;291:2413 17. [3] Koelle DM, Schomogyi M, Corey L. Antigen-specific T cells localize to the uterine cervix in women with genital herpes simplex virus type 2 infection. J Infect Dis 2000;182:662 70. [4] Mackay LK, Stock AT, Ma JZ, Jones CM, Kent SJ, Mueller SN, et al. Long-lived epithelial immunity by tissue-resident memory T (TRM) cells in the absence of persisting local antigen presentation. Proc Natl Acad Sci U S A 2012;109:7037 42. [5] Turner DL, Bickham KL, Thome JJ, Kim CY, D’Ovidio F, Wherry EJ, et al. Lung niches for the generation and maintenance of tissue-resident memory T cells. Mucosal Immunol 2014;7:501 10. [6] Iijima N, Iwasaki A. T cell memory. A local macrophage chemokine network sustains protective tissue-resident memory CD4 T cells. Science 2014;346:93 8. [7] Iijima N, Iwasaki A. Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help. Nature 2016;533:552 6. [8] Hondowicz BD, An D, Schenkel JM, Kim KS, Steach HR, Krishnamurty AT, et al. Interleukin-2-dependent allergen-specific tissue-resident memory cells drive asthma. Immunity 2016;44:155 66. [9] Shinoda K, Hirahara K, Iinuma T, Ichikawa T, Suzuki AS, Sugaya K, et al. Thy1 1 IL-7 1 lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation. Proc Natl Acad Sci U S A 2016;113:E2842 2851. [10] Han SJ, Glatman Zaretsky A, Andrade-Oliveira V, Collins N, Dzutsev A, Shaik J, et al. White adipose tissue is a reservoir for memory T cells and promotes protective memory responses to infection. Immunity 2017;47:1154 68 e1156. [11] Gebhardt T, Wakim LM, Eidsmo L, Reading PC, Heath WR, Carbone FR. Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus. Nat Immunol 2009;10:524 30. [12] Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999;401:708 12. [13] Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function,

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

generation, and maintenance. Annu Rev Immunol 2004;22:745 63. Arsenio J, Kakaradov B, Metz PJ, Kim SH, Yeo GW, Chang JT. Early specification of CD8 1 T lymphocyte fates during adaptive immunity revealed by single-cell gene-expression analyses. Nat Immunol 2014;15:365 72. Lonnberg T, Svensson V, James KR, Fernandez-Ruiz D, Sebina I, Montandon R, et al. Single-cell RNA-seq and computational analysis using temporal mixture modelling resolves Th1/Tfh fate bifurcation in malaria. Sci Immunol 2017;2. Available from: http://dx.doi.org/ 10.1126/sciimmunol.aal2192. Zemmour D, Zilionis R, Kiner E, Klein AM, Mathis D, Benoist C. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nat Immunol 2018;19:291 301. Zheng C, Zheng L, Yoo JK, Guo H, Zhang Y, Guo X, et al. Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell 2017;169: 1342 56 e1316. Endo Y, Hirahara K, Iinuma T, Shinoda K, Tumes DJ, Asou HK, et al. The interleukin-33-p38 kinase axis confers memory T helper 2 cell pathogenicity in the airway. Immunity 2015;42:294 308. Islam SA, Chang DS, Colvin RA, Byrne MH, McCully ML, Moser B, et al. Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5 1 T(H)2 cells. Nat Immunol 2011;12:167 77. Mitson-Salazar A, Yin Y, Wansley DL, Young M, Bolan H, Arceo S, et al. Hematopoietic prostaglandin D synthase defines a proeosinophilic pathogenic effector human TH2 cell subpopulation with enhanced function. J Allergy Clin Immunol 2016;137:907 18 e909. Wambre E, Bajzik V, DeLong JH, O’Brien K, Nguyen QA, Speake C, et al. A phenotypically and functionally distinct human TH2 cell subpopulation is associated with allergic disorders. Sci Transl Med 2017;9:1 10. Iinuma T, Okamoto Y, Yamamoto H, Inamine-Sasaki A, Ohki Y, Sakurai T, et al. Interleukin-25 and mucosal T cells in noneosinophilic and eosinophilic chronic rhinosinusitis. Ann Allergy Asthma Immunol 2015;114:289 98. Nguyen LP, Pan J, Dinh TT, Hadeiba H, O’Hara 3rd E, Ebtikar A, et al. Role and species-specific expression of colon T cell homing receptor GPR15 in colitis. Nat Immunol 2015;16:207 13. Upadhyaya B, Yin Y, Hill BJ, Douek DC, Prussin C. Hierarchical IL-5 expression defines a subpopulation of highly differentiated human Th2 cells. J Immunol 2011;187:3111 20. Wambre E, DeLong JH, James EA, LaFond RE, Robinson D, Kwok WW. Differentiation stage

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

determines pathologic and protective allergen-specific CD4 1 T-cell outcomes during specific immunotherapy. J Allergy Clin Immunol 2012;129:544 51 e541 e547. Endo Y, Iwamura C, Kuwahara M, Suzuki A, Sugaya K, Tumes DJ, et al. Eomesodermin controls interleukin-5 production in memory T helper 2 cells through inhibition of activity of the transcription factor GATA3. Immunity 2011;35:733 45. Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005;23:479 90. Kolodin D, van Panhuys N, Li C, Magnuson AM, Cipolletta D, Miller CM, et al. Antigen- and cytokinedriven accumulation of regulatory T cells in visceral adipose tissue of lean mice. Cell Metab 2015;21:543 57. Pichery M, Mirey E, Mercier P, Lefrancais E, Dujardin A, Ortega N, et al. Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain. J Immunol 2012;188:3488 95. Sanada S, Hakuno D, Higgins LJ, Schreiter ER, McKenzie AN, Lee RT. IL-33 and ST2 comprise a critical biomechanically induced and cardioprotective signaling system. J Clin Invest 2007;117:1538 49. Sponheim J, Pollheimer J, Olsen T, Balogh J, Hammarstrom C, Loos T, et al. Inflammatory bowel disease-associated interleukin-33 is preferentially expressed in ulceration-associated myofibroblasts. Am J Pathol 2010;177:2804 15. Keller M, Ruegg A, Werner S, Beer HD. Active caspase-1 is a regulator of unconventional protein secretion. Cell 2008;132:818 31. Carriere V, Roussel L, Ortega N, Lacorre DA, Americh L, Aguilar L, et al. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo. Proc Natl Acad Sci U S A 2007;104:282 7. Cayrol C, Girard JP. The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1. Proc Natl Acad Sci U S A 2009;106:9021 6. Cayrol C, Girard JP. IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy. Curr Opin Immunol 2014;31:31 7. Liew FY, Girard JP, Turnquist HR. Interleukin-33 in health and disease. Nat Rev Immunol 2016;16:676 89. Moussion C, Ortega N, Girard JP. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ‘alarmin’? PLoS One 2008;3:e3331.

141

[38] Allakhverdi Z, Smith DE, Comeau MR, Delespesse G. Cutting edge: the ST2 ligand IL-33 potently activates and drives maturation of human mast cells. J Immunol 2007;179:2051 4. [39] Klein Wolterink RG, Serafini N, van Nimwegen M, Vosshenrich CA, de Bruijn MJ, Fonseca Pereira D, et al. Essential, dose-dependent role for the transcription factor Gata3 in the development of IL-5 1 and IL-13 1 type 2 innate lymphoid cells. Proc Natl Acad Sci U S A 2013;110:10240 5. [40] Moro K, Yamada T, Tanabe M, Takeuchi T, Ikawa T, Kawamoto H, et al. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(1)Sca-1(1) lymphoid cells. Nature 2010;463:540 4. [41] Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H. A novel IL-1 family cytokine, IL-33, potently activates human eosinophils. J Allergy Clin Immunol 2008;121:1484 90. [42] Mato N, Hirahara K, Ichikawa T, Kumagai J, Nakayama M, Yamasawa H, et al. Memory-type ST2 1 CD4 1 T cells participate in the steroid-resistant pathology of eosinophilic pneumonia. Sci Rep 2017;7:6805. [43] Neill DR, Wong SH, Bellosi A, Flynn RJ, Daly M, Langford TK, et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010;464:1367 70. [44] de Kleer IM, Kool M, de Bruijn MJ, Willart M, van Moorleghem J, Schuijs MJ, et al. Perinatal activation of the interleukin-33 pathway promotes type 2 immunity in the developing lung. Immunity 2016;45:1285 98. [45] Hammad H, Lambrecht BN. Barrier epithelial cells and the control of type 2 immunity. Immunity 2015;43:29 40. [46] Mohapatra A, Van Dyken SJ, Schneider C, Nussbaum JC, Liang HE, Locksley RM. Group 2 innate lymphoid cells utilize the IRF4-IL-9 module to coordinate epithelial cell maintenance of lung homeostasis. Mucosal Immunol 2016;9:275 86. [47] Verma M, Liu S, Michalec L, Sripada A, Gorska MM, Alam R. Experimental asthma persists in IL-33 receptor knockout mice because of the emergence of thymic stromal lymphopoietin-driven IL-9(1) and IL-13(1) type 2 innate lymphoid cell subpopulations. J Allergy Clin Immunol 2017;142 793 803.e8. [48] Halim TY, Hwang YY, Scanlon ST, Zaghouani H, Garbi N, Fallon PG, et al. Group 2 innate lymphoid cells license dendritic cells to potentiate memory TH2 cell responses. Nat Immunol 2016;17:57 64. [49] Halim TY, Steer CA, Matha L, Gold MJ, MartinezGonzalez I, McNagny KM, et al. Group 2 innate lymphoid cells are critical for the initiation of adaptive T helper 2 cell-mediated allergic lung inflammation. Immunity 2014;40:425 35.

II. PRINCIPLES OF MUCOSAL VACCINE

142

8. INDUCTION AND REGULATION OF MUCOSAL MEMORY T CELL RESPONSES

[50] Oliphant CJ, Hwang YY, Walker JA, Salimi M, Wong SH, Brewer JM, et al. MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(1) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion. Immunity 2014;41:283 95. [51] Gudbjartsson DF, Bjornsdottir US, Halapi E, Helgadottir A, Sulem P, Jonsdottir GM, et al. Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction. Nat Genet 2009;41:342 7. [52] Savenije OE, Mahachie John JM, Granell R, Kerkhof M, Dijk FN, de Jongste JC, et al. Association of IL33-IL-1 receptor-like 1 (IL1RL1) pathway polymorphisms with wheezing phenotypes and asthma in childhood. J Allergy Clin Immunol 2014;134:170 7. [53] Sminia T, van der Brugge-Gamelkoorn GJ, Jeurissen SH. Structure and function of bronchus-associated lymphoid tissue (BALT). Crit Rev Immunol 1989;9:119 50. [54] Kuroda E, Ozasa K, Temizoz B, Ohata K, Koo CX, Kanuma T, et al. Inhaled fine particles induce alveolar macrophage death and interleukin-1alpha release to promote inducible bronchus-associated lymphoid tissue formation. Immunity 2016;45:1299 310. [55] Moyron-Quiroz JE, Rangel-Moreno J, Kusser K, Hartson L, Sprague F, Goodrich S, et al. Role of inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity. Nat Med 2004;10:927 34. [56] Randall TD. Bronchus-associated lymphoid tissue (BALT) structure and function. Adv Immunol 2010; 107:187 241. [57] Delventhal S, Hensel A, Petzoldt K, Pabst R. Effects of microbial stimulation on the number, size and activity of bronchus-associated lymphoid tissue (BALT) structures in the pig. Int J Exp Pathol 1992;73:351 7. [58] Hwang JY, Randall TD, Silva-Sanchez A. Inducible bronchus-associated lymphoid tissue: taming inflammation in the lung. Front Immunol 2016;7:258. [59] Tschernig T, Pabst R. Bronchus-associated lymphoid tissue (BALT) is not present in the normal adult lung but in different diseases. Pathobiology 2000;68:1 8. [60] Kiyono H, Fukuyama S. NALT- versus Peyer’s-patchmediated mucosal immunity. Nat Rev Immunol 2004;4:699 710. [61] Natsuaki Y, Egawa G, Nakamizo S, Ono S, Hanakawa S, Okada T, et al. Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin. Nat Immunol 2014;15:1064 9. [62] Carragher DM, Rangel-Moreno J, Randall TD. Ectopic lymphoid tissues and local immunity. Semin Immunol 2008;20:26 42.

[63] Shinoda K, Hirahara K, Nakayama T. Maintenance of pathogenic Th2 cells in allergic disorders. Allergol Int 2017;66:369 76. [64] Baluk P, Adams A, Phillips K, Feng J, Hong YK, Brown MB, et al. Preferential lymphatic growth in bronchusassociated lymphoid tissue in sustained lung inflammation. Am J Pathol 2014;184:1577 92. [65] Halle S, Dujardin HC, Bakocevic N, Fleige H, Danzer H, Willenzon S, et al. Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells. J Exp Med 2009;206:2593 601. [66] Moyron-Quiroz JE, Rangel-Moreno J, Hartson L, Kusser K, Tighe MP, Klonowski KD, et al. Persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs. Immunity 2006;25:643 54. [67] Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2004;350:2645 53. [68] Rangel-Moreno J, Hartson L, Navarro C, Gaxiola M, Selman M, Randall TD. Inducible bronchus-associated lymphoid tissue (iBALT) in patients with pulmonary complications of rheumatoid arthritis. J Clin Invest 2006;116:3183 94. [69] Vroman H, Bergen IM, Li BW, van Hulst JA, Lukkes M, van Uden D, et al. Development of eosinophilic inflammation is independent of B-T cell interaction in a chronic house dust mite-driven asthma model. Clin Exp Allergy 2017;47:551 64. [70] Nakayama T, Hirahara K, Onodera A, Endo Y, Hosokawa H, Shinoda K, et al. Th2 cells in health and disease. Annu Rev Immunol 2017;35:53 84. [71] Antonelli A, Ferrari SM, Corrado A, Ferrannini E, Fallahi P. CXCR3, CXCL10 and type 1 diabetes. Cytokine Growth Factor Rev 2014;25:57 65. [72] Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, et al. Generation of pathogenic T(H)17 cells in the absence of TGF-beta signalling. Nature 2010;467:967 71. [73] Lee Y, Awasthi A, Yosef N, Quintana FJ, Xiao S, Peters A, et al. Induction and molecular signature of pathogenic TH17 cells. Nat Immunol 2012;13:991 9. [74] Wang C, Yosef N, Gaublomme J, Wu C, Lee Y, Clish CB, et al. CD5L/AIM regulates lipid biosynthesis and restrains Th17 cell pathogenicity. Cell 2015;163: 1413 27.

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Influence of Commensal Microbiota and Metabolite for Mucosal Immunity Jillian L. Pope1,*, Sarah Tomkovich1,* and Christian Jobin1,2,3 1

Department of Medicine, University of Florida, Gainesville, FL, United States 2Department of Infectious Diseases and Immunology, University of Florida, Gainesville, FL, United States 3 Department of Anatomy and Cell Physiology, University of Florida, Gainesville, FL, United States

I. INTRODUCTION The conglomerate of microorganisms, or microbiota, populating human surface and cavities contributes to numerous physiological processes such as nutrition, metabolism, and immune responses [1]. These microbial communities are composed of an ensemble of microorganisms such as bacteria, viruses, archaea, and fungi that share different ecosystems distributed around the body. These microorganisms are thought to form a complex interactive network at the interkingdom and intrakingdom level that are in tune with the host’s own network, leading to the concept that a human should be viewed as a metaorganism. Most of the data supporting a role for microorganisms in host health and disease status were derived from studies on bacteria. Bacterial communities have been studied in various locations such as the skin, mouth, gastrointestinal tract,

and genital tract, and their composition in healthy vs diseased states has been investigated. For example, the composition of intestinal bacteria has been linked to different diseases, such as inflammatory bowel diseases, colorectal cancer, diabetes, and liver cirrhosis [2]. The link between bacterial composition and disease states has led to the concept that a compositional or functional equilibrium must exist among the community to maintain host homeostasis, although a clear picture of this “ideal” microbial distribution has not been identified. The communication network between bacteria and host is complex and implicates a spectrum of microbial structures (RNA, DNA, membrane components) and metabolites (short-chain fatty acids (SCFAs), extracellular ATP) activating various host cells (immune and nonimmune cells) to trigger a number of homeostatic responses. Consequently, alteration of microbial equilibrium disrupts the homeostatic

* These authors contributed equally to the manuscript.

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00009-2

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network between host and microbe in a way that could have deleterious consequences for the host if the recovery system fails to respond properly. Genetics, nutrition, infection, inflammation, and environmental factors all have the potential to affect bacterial network breakdown. Among the important signaling networks influenced by bacteria host interactions is immunity, a critical component of vaccine responsiveness. This chapter will highlight important concepts implicated in host microbe interactions with respect to immune response. The chapter will also focus on the wide impact of microbial communities on immune responses.

II. MICROBIOTA Although previous estimates of bacterial burden within humans have been revised, the fact remains that bacteria outnumber host cells two to one [3]. Microbes are distributed throughout the body, establishing habitation niches in specified regions, including mucosal cavities. While the gut microbiota is most abundant and has been studied in greatest detail, it is equally important to discuss the role and contribution of extraintestinal sites and the local influence they provide. The human colon is home to approximately 3.9 3 1013 bacteria [3], composed of more than 1000 different species, which can transcribe approximately 150 times more genes than the eukaryotic cells [4]. This is the most densely populated area in the human body, along with the oral cavity and skin, which have approximately 1012 and 1011 bacteria, respectively [5]. In addition to the number of bacteria present, the representative phyla are also distinct among various locations. In the stomach, the phyla Firmicutes and Actinobacteria predominate, with Bacteroidetes, Proteobacteria, and Fusobacteria also in attendance [6 8]. In contrast, Firmicutes and Proteobacteria are

dominant in the small intestine (SI), while Firmicutes and Bacteroidetes are dominant in the colon, which has the highest bacterial diversity of the gastrointestinal tract [9 11]. Firmicutes also predominates in the oral and lung microbiota along with Bacteroidetes and Proteobacteria [12,13]. Beyond bacteria, the roles of other microbiota members such as viruses, helminths, and fungi and their effects on health are emerging [14]. Along with distribution differences, there exist temporal differences in intestinal bacterial colonization. The majority of the neonatal microbiome is established at birth and is shaped by the method of delivery (vaginal vs cesarean) [15 17] and the source of nutrition (breast milk, which is not sterile, vs formula) [16,18 22]. Vertical transmission from the mother appears to be the primary route of colonization. However, horizontal transmission from the environment also plays a role, as the hospital at which an infant is born is linked with microbial alterations in preterm infants [23,24]. During the first 2 3 years of life, there is a steady normalization of the enteric microbiome, with remarkable interindividual variability during the first months of life to an adult-like steady state around 2 years of age [20,25 29]. The initial colonizers are facultative anaerobes such as Staphylococcus, Streptococcus, Lactobacillus, and Enterobacteriaceae. As these bacteria use up the enteric oxygen, strict anaerobes are able to colonize, including Bifidobacterium, Bacteroides, and Clostridium [18]. This heralds the slow movement toward an anaerobe-rich adult microbiota with the phyla Bacteroidetes and Firmicutes representing approximately 95% of the bacterial community. The mature microbiota of each individual is relatively stable over time but shows a high level of variability between people. The microbial members show constant minor fluctuations that return to steady state in a dynamic equilibrium, demonstrating resistance to perturbation. However, infection, the use of antibiotics, or

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substantial dietary changes can overcome this resistance and cause dysbiosis. Microbial diversity is a key factor in resilience, and decreased diversity can indicate, or perhaps initiate, pathologies related to dysbiosis, including inflammatory bowel disease, colorectal cancer, necrotizing enterocolitis, obesity, diabetes, metabolic disorders, and even neurological disorders [30]. When the microbiota is investigated beyond determining which microbes are present, it becomes clear that the microbiota is metabolically active in shaping the intestinal habitat [31]. For example, approximately 77 out of 179 nondietary metabolites detected in the mouse colon were significantly increased when intestinal bacteria were present [32]. Another 48 metabolites were decreased upon colonization, while 56 were unchanged. From this study, the authors were able to observe differences in several compound classes including amino acid derivatives, carbohydrate metabolism intermediates, coenzymes and derivatives, and central carbon metabolism intermediates. Furthermore, this study replicated well-established roles for the intestinal microbiota in SCFA synthesis [33], bile salt transformation [34], and polyamine metabolism [35]. Microbes also produce extracellular ATP [31] and indole derivatives from dietary tryptophan [36,37], both of which interact with host cells of the intestinal epithelium and immune system. These metabolites can circulate throughout the host to distant organs, extending the reach of the microbiome beyond its locale, contributing to systemic homeostasis or diseases (allergy, arthritis, autism, obesity).

III. MUCOSAL IMMUNITY The microbiota and its biosynthetic capacity in the form of metabolites play a crucial role in regulating both innate and adaptive immunity. Therefore the microbiota is able not only to affect its local environment but also to influence

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immune function at distant sites through the release of a complex network of metabolites circulating throughout the body. Microbiota manipulation through the use of gnotobiotic technology and antibiotics has provided a wealth of information about the key role of bacteria in both development and activation of mucosal immunity. Germ-free (GF) mice, devoid of microorganisms, can be populated by defined bacteria ranging from a single organism (monocolonization) to a complex community in a fashion that allows investigation of temporal dynamic changes in the immune system [38,39]. Here, we discuss the key players in mucosal immunity and how microbes participate in their regulation and maintenance.

A. Gut Microbiota and Mucosal Immunity 1. Gut Mucosal Surface A defining characteristic of mucosal sites is its intimate contact with the external environment. Thus having a physical barrier against these foreign antigens is paramount to the preservation of these regions. This physical barrier includes the mucus layer and the underlying epithelium, which undergo influences by the microbiota. The mucus layer has a twofold purpose: as the first hurdle that foreign substances must traverse and as guardian of the underlying cells within the epithelium and lamina propria. The intestinal microbiota is involved in the fortification of this defense. The thickness of the mucus layer of GF mice is significantly less compared to that of specific pathogen-free (SPF) mice [40,41]. More interestingly, changes in diet can cause shifts in the abundance of bacteria that lead to mucin degradation and decreased mucosal thickness [42]. A high-fiber diet has been demonstrated to be a valuable source of nutrients for bacteria and beneficial for the host, as the bacteria can biotransform the fiber into SCFAs that are released into the mucosa to be utilized by

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epithelial and immune cells [33,43] (see also Chapter 12: Influence of Dietary Components and Commensal Bacteria on the Control of Mucosal Immunity). However, in the absence of fiber, mice demonstrate an increase in the abundance of Akkermansia muciniphila and Bacteriodes caccae bacteria, which utilize mucin within the mucus layer and lamina propria as a nutrient source. As the architectural landscape and microbial load vary between the SI and the colon, so does the mucus barrier. The SI contains a single layer of mucus, while the colon, which carries a larger microbial burden, contains a mucus bilayer. The outermost layer is thickest and is where commensals reside; the inner layer is relatively thin and does not contain microbes [44]. The second component of the mucosal surface is the epithelium, and while vulnerable to invading pathogens, it also shields the underlying lamina propria. Kissing points between individual cells of the epithelial sheet are tightly regulated by junctional adhesion molecules that maintain paracellular permeability. GF mice have reduced gene expression of tight junction proteins (claudin-7, occludin, and tjp1), and adherens junctions proteins (b catenin and e cadherin), which has been linked to the absence of indole, a product of bacterial metabolism [45]. Clostridium sporogenes was shown to regulate epithelial permeability through indole metabolism in GF Swiss Webster mice [46]. Furthermore, indole supplementation of GF mice was sufficient to restore junctional protein expression and protect against dextran sulfate sodium induced experimental colitis [45]. These data demonstrate the regulatory control of the microbiota and its metabolites in the function and control of gut mucosal surface. 2. Cell-Mediated Immunity Aside from physical exclusion of pathogens, the intestinal epithelium contains specialized cell types that play mediatory roles in gut immunity. For example, goblet cells secrete mucin, which contributes to the composition and maintenance of the mucus barrier. In addition, goblet cells

have the ability to form goblet cell associated antigen passages, which provide a route for antigens to be processed by dendritic cells (DCs) residing in the lamina propria [47] (Fig. 9.1). While this effect was intrinsic to antigen processing within the SI, colonic goblet cells were able to exhibit this function only after antibiotic treatment or deletion of myeloid differentiation primary response gene 88 (MyD88), a microbial sensing adapter protein, suggesting an alternative regulation by commensal microbes within the colon [47]. This differential control among the SI and colon is similar to that noticed for the differences in mucosal lining, and also observed for many immune cell functions. It remains to be determined whether goblet cells possess this feature in extraintestinal mucosal sites such as the respiratory tract. Specific to the SI, Paneth cells are another differentiated cell type of the epithelium functioning in the secretion of antimicrobial peptides (AMPs) for innate defense (Chapter 6: Innate Immunity at Mucosal Surfaces). These AMPs include defensins, cathelicidins, lysozyme, and the Reg III family of proteins [48]. While there exist no developmental defects of Paneth cells between GF and SPF mice, there is a disparity in the contents and output based on microbial status. Microbiota depletion decreases AMPs’ gene expression [49] (Fig. 9.1), similar to the effect observed in microbial desensitization by Nod1 and Nod2 deletion [50]. Conventionalization of GF mice, a process by which GF mice are gavaged with SPF microbiota or cohoused with SPF mice to achieve similar bacterial levels, was sufficient to induce maximal expression of AMPs by day 4 of colonization [38]. The microfold (M) cells are also essential to innate immunity, serving as a molecular middleman between antigens and immune cells (see also Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance and Chapter 28: M CellTargeted Vaccines). As early as three decades ago,

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Outer mucus

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sIgA

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FIGURE 9.1 Intestinal microbiota regulates mucosal immunity. The intestinal microbiota influences a number of mucosal immune responses. The presence of microbiota (1) increases the production of SIgA and (2) anti-microbial peptides (AMP) release from Paneth cells. (3) Microbes influence the amount of microfold cells (M cells), which function to deliver luminal antigens to dendritic cells. (4) In lower concentrations of microbiota, goblet cells can form goblet cell associated antigen passages (GAPs), which facilitate antigen delivery. Germinal centers reside in the lymphoid follicles (5), where B cells reside and mature to produce IgA and specific antibodies under microbiota influence. The microbiota (6) increases the number of intraepithelial lymphocytes and (7) iNKT cells, which bridge between dendritic and B cells. (8) T cells undergo Th17 specification in the presence of microbiota, thus producing more IL-17 and IL-22 cytokines. (9) MHCII expression on macrophages and (10) IL-10 cytokine expression are regulated by microbes, which increases the amount of Foxp31 Treg cells. (11) Microbes increase the number of RORγt1 cells.

the microbiota was identified as an important component shaping the formation of intestinal M cells within Peyer’s patches [51] (Fig. 9.1). Generally, antigens that are coated in secretory immunoglobulin A are delivered to the M cells via transcytosis and passed along to DCs for further processing [52]. The M cells work in close contact with another major component of the gut-associated lymphoid tissue (GALT) (see also Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (DEVELOPMENTAL FOR STRUCTURES, E.G., Ibalt, GALT, NALT) for the Induction and Regulation of Mucosal Immunity & Tolerance). These are the Peyer’s patches of the SI (referred to as lymphoid follicles of the colon or colonic patches), which are reservoirs for several innate immune cell types.

It is also within these reservoirs that B cells reside and mature in pockets called germinal centers (GCs) [53,54], whose presence is also dependent upon the microbiota [55,56] (Fig. 9.1). Interestingly, along the SI, the Peyer’s patches have varied immune cell composition which can be attributed to the variegated presence of specific members of the microbiota along the intestine. For example, the Peyer’s patches of the 1 ileum contained fewer NKp46 group 3 innate lymphoid cells (ILC3s) than those of the jejunum, and this correlates with increased butyrate levels [57] within those regions (Chapter 14: Innate Lymphoid Cells for the Control of Mucosal Immunity). Most naı¨ve immune cell populations found in originator sites, such as the thymus and bone

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marrow, are similar between GF and conventionalized mice [58]. However, once immune cells enter the intestine, gut microbes can direct differentiation and specialization of these cells by regulating the expression of cell surface markers, thus influencing their function. Some of the early responders of the immune system reside in close contact with the epithelium. These cells are distinct from traditional T cells and are known as intraepithelial lymphocytes (IELs). GF mice have significantly fewer IELs [59] (Fig. 9.1). Antibiotic treatment is sufficient to reduce colonic IELs (similar to GF levels) that can be recovered by cohousing with conventionally derived mice and, more specifically, colonization with certain members of the order Bacteriodales [59]. As was mentioned previously, further specification can be regulated by surface expression of immune receptors. Signaling lymphocyte activation molecule family member 4 (SLAMF4) is a cell surface receptor expressed on IELs in the lamina propria of the SI and colon in mice. Expression of SLAMF4 on CD451 cells was higher in conventionally raised mice than in GF mice [60]. Furthermore, SLAMF4 expression was demonstrated to be under dynamic control of the microbiota through conventionalization and antibiotic experiments. SLAMF4 expression on NK cells and lymphocytes is necessary for the protection and immune response against Listeria monocytogenes and Citrobacter rodentium induced disease. DCs are key mediators of antigen-stimulated immune response, and their maturation is supported by the presence of microbes. DCs expressing CD11c were surveyed in the spleen and mesenteric lymph nodes (MLNs) to assess systemic and local levels, respectively. GF mice had a significantly lower percentage of CD11c1 DCs than did conventionalized mice [61]. When DC subtypes were further analyzed, GF mice had a significant increase in the CD8α2/ CD11b1/CD11c1 population and a decrease in the number of CD8α1/CD11b2/CD11c1 cells

when compared to SPF mice, suggesting that microbes preferentially regulate CD8α1 cells, a major player for the induction and regulation of viral immunity and tolerance in the GALT [61]. While the microbiota does not affect overall amounts of regulatory T cells (Tregs) (Foxp31/ CD41) levels, they do have local control of Treg specificity. As Tregs encounter commensal bacteria, they begin to express T cell receptors that have specificity toward certain commensals of the microbiota [58]. Colonization of GF mice with chloroform-resistant bacteria that were fed a high-fat diet led to an increased production of butyrate and enrichment of Foxp31/CD1031 Treg cells [62]. Bacteria were shown to regulate colonic interleukin 10 (IL-10) levels, which in turn controlled the amount of Foxp31 Tregs [63] (Fig. 9.1). Regulation of CD41 T cells is sensitive to the presence of specific microbiota members. Altered Schaedler flora, a minimally defined microbiota, was sufficient to increase the number of CD41/CD251/Foxp31 T cells specifically in the colonic lamina propria of GF mice [39]. Intestinal microbes also have local control in the specification of RORγt1 Tregs. While GF mice exhibit higher levels of Helios1 Tregs, they have significantly reduced amounts of RORγt1 Tregs in comparison to SPF mice. Colonization of GF mice with SPF microbiota was sufficient to increase RORγt1 expression levels (Fig. 9.1) in the colon, SI, and MLNs but did not alter expression in the spleen [64]. Antibiotic treatment also resulted in reduced RORγt1 Tregs without affecting total Helios1 cellular levels, suggesting that microbes regulate the conversion of Helios1 to RORγt1 cells but do not contribute to the overall production of thymus-derived cells. Further experimentation showed that colonization of GF mice with a mixture of seven Clostridia species increased the amount of IL-10-expressing RORγt1 cells in the colon, while SFB (segmented filamentous bacteria) monocolonization induced RORγt1 cells in the SI. Both bacteria are capable of

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producing butyrate, which was shown to be important for the peripheral education of Tregs in the intestine [64] (Chapter 15: Mucosal Regulatory System for the Balanced Immunity in the Gut). Another important T cell subtype is the Th17 cells. GF mice have significantly less expression of IL-17 in lamina propria CD41 T cells compared to SPF mice [65 67], indicative of a decreased Th17 signature. The presence of a specific bacterial community known as CFB (Cytophaga-Flavobacterium-Bacteriodes) was sufficient to induce IL-17 expression [66] (Fig. 9.1). SFB colonization of GF mice was also sufficient to induce the number of TCRβ1 CD41 cells expressing IL-17 and IL-22 cytokines (markers of Th17 signature) [67]. More interestingly, SFB was able to produce this same phenotype in conventionally raised mice from Jackson laboratories, which were shown to lack SFB bacteria and have a reduced Th17 T cell signature [67]. Within the colon, Clostridia, not SFB, have the ability to increase the number of CD81 T cells [68]. Presentation of antigens through major histocompatibility complex (MHC) proteins is another important step in antigen processing, that is, also regulated by microbes (Fig. 9.1). As early as 4 days postconventionalization, GF mice demonstrate an increase expression of key genes of the MHC Class I and II complexes (Tap1, Tap2) in both the SI and the colon. These levels were maintained throughout the experiment [38], suggesting tight control of this ratelimiting step of the adaptive immune system by the commensal microbes. The microbiota also regulates MHC Class II expression on macrophages [69], and within the SI, total MHCII expression levels were increased with conventionalization; much of the contribution was due to the presence of SFB [68]. Invariant NKT cells (iNKT) are a subset of immune cells that help mediate the antigen processing from DCs to B cells, linking the innate immune response to the adaptive

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immune response. Manipulation of iNKTs can prove beneficial for vaccines [70,71]. Interestingly, when compared to SPF mice, GF mice have a significantly higher amount of colonic iNKT cells (Fig. 9.1) while having lower numbers in the SI and MLNs [72], suggesting differential regulation between the compartments. When comparing microbiota compositions, mice with a conventional biota, rich in Bacteroidetes, produce a significantly higher amount of iNKT cells than do mice rederived to carry a “restricted biota” composed of higher abundance of Firmicutes [73]. This suggests that microbial composition is also a factor to be considered in immune cell recruitment and specification. 3. Humoral Immunity The dissemination of antibodies and other secreted factors is the defining element of humoral immunity. Among these factors are the immunoglobulin family of proteins, the most common being immunoglobulin A (IgA) (see also Chapter 4: Mucosal Lymphocyte Homing). This immunoglobulin is one of the first molecules encountered by most bacteria and viruses entering the intestine. IgA is secreted as secretory IgA (SIgA) into the mucus layer, where it participates in fortifying the barrier by binding foreign antigens that penetrate the mucus. Gnotobiotic studies highlight a dependence of IgA levels on the presence of microbes [55,74,75]. Colonization of GF mice increases the production and secretion of IgA [39,55] (Fig. 9.1) in a M-cell-dependent manner [76]. Not all microbial species are capable of inducing IgA within the intestine. While SFB can increase the number of IgA-producing cells in both the SI and the colon [68], monocolonization with commensal Bacteroides acidifaciens increases IgA levels in the colon but not in the SI [55] by augmenting the number of IgA1B2201 B cells. More recently, it has been shown that the microbial metabolite acetate participates in IgA production [74]. B cells, the

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cellular source of IgA, express a G-proteincoupled receptor, GPR43, that is, capable of sensing microbial metabolites, specifically SCFAs such as acetate. Mice deficient in GPR43 have diminished output of total IgA concomitant with a loss of IgA1 B cells. Microbiotadepleted mice supplemented with acetate showed restored IgA levels, a response that was abolished in GPR432/2 mice deficient for acetate response [74]. This regulation was localized to the intestine, as no changes were observed within the spleen. It is important to note that while there exist notable differences in the amount of IgA in GF and colonized mice, administration of antibiotics is unable to alter IgA levels, indicating that commensals are essential for the development but not the maintenance of IgA levels. While control of IgA levels is an important indicator of the role of intestinal microbes in immunity, what is even more telling is the specification of the IgA response. This metric also gives an indication of the regulatory control the microbes have over B cells function. The gut microbiota directly influences the diversity of the IgA pool [77]. To demonstrate this relationship experimentally, GF mice were colonized with either an SPF microbiota, individual commensals (E. coli Nissle, Lactobacillus GG), or a mixture of two Clostridia strains to analyze the sequences of their IgA heavy-chain regions (VH and JH). The IgA diversity correlated with the complexity of the microbiota; GF and monocolonized strains were less diverse than the SPF colonized mice. While IgA is predominant in the GALT, B cells are also capable of expressing other members of the immunoglobulin family. The ability to undergo isotype switching is an important process in the maturation of B cells. This process requires the expression of recombinant activating genes (RAG-1 and RAG-2). GF mice have been shown to have significantly lower amounts of IgA, IgM, and IgG [75] yet exhibit higher levels of serum IgE compared to

conventionalized mice [78]. When GF mice were cohoused with SPF-born mice for 7 days, an increase in Rag-1 and Rag-2 expression was observed in the bone marrow and spleen, suggesting that microbes can control systemic levels of recombination proteins in addition to the intestinal lamina propria [79]. Although Rag expression was increased at the systemic control, this was not indicative of systemic control of B cell maturation. Cohousing experiments confirmed B cell maturation as measured by the increased ratio of Igλ/Igκ in B cells of the lamina propria and not the bone marrow or the spleen. Activation of B cells is among the most coveted of vaccine-mediated immunity, and this can be measured by the production of antibodies. Measurement of antibody titers allows assessment of the fitness of vaccine-mediated immunity. GF mice produce lower amounts of antibody titers following systemic immunization compared to SPF mice that can be restored upon conventionalization [80]. This suggests that commensal microbes are crucial to the development of antibody titers and thus B cell activation. Further, the study indicates that commensal microbiota can influence the outcome of antigen-specific immune responses induced by injection-type vaccination.

B. Microbiota and Extraintestinal Immunity 1. Oral Immunity The interplay between the host’s intestinal microbiota and immune response extends to extraintestinal regions harboring their own microbiota, which together have the capacity to influence local immunity (Table 9.1). In contrast to the intestine, SFB colonization does not appear to affect Th17 levels because SFB is not part of the mouse oral microbiota, and Th17 cell levels in the gingiva are similar regardless of whether SFB colonized the intestine [115].

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TABLE 9.1 Immunomodulatory Effects of Microbes, Microbial Components, and Metabolites Immune Site

Microbiota

Effect

References

Mouth

Oral microbiota

Modulates γδT17 number and function in cervical lymph nodes, lungs, and spleen

[81]

Microbiota

Induces GAS6 expression, which controls Treg/Th17 ratio in cervical lymph nodes

[82]

Induces B cell class-switch recombination and IgD secretion

[78]

Microbiota

Promotes NALT development

[83]

Nasal Streptococcaceae, Pseudomonadaceae, Staphylococcaceae

Correlates with proinflammatory Th1 response

[84]

Nasal Corynebacteriaceae

Correlates with IL-5

[84]

Nasal Proteobacteria

Correlates with IL-1α

[85]

Intestinal microbiota

Stimulates gut DCs, which trigger ILC3 recruitment to lungs

[86]

Intestinal SFB

Induces Th17 cells and effector cytokines, which influence resistance to microbial lung infections

[87,88]

Intestinal fungi (Aspergillus amstelodami, Epicoccum nigrum, and Wallena sebi)

Promotes Th2 response, increasing lung IgG, IgE, and eosinophils during allergic airway disease

[89]

Lung Prevotella, Rothia, and Veillonella

Correlates with lung Th17 cells, Th17 cytokines, [90] and neutrophils

Nasal LPS

Promotes adaptive immune response to influenza A infection

[91]

Intestinal LPS, CpG, Poly I:C, peptidoglycan

Promotes adaptive immune response to influenza A infection

[91]

Intestinal NOD1/NOD2 ligands

Promotes antibacterial defenses of lung macrophages during infection

[92]

Intestinal helminth (Heligomosomoides polygorus)

Induces type I interferons and interferonstimulated genes in the lung and intestine

[93]

Respiratory Intestinal microbiota tract

Helminth product (antiinflammatory protein- Promotes Treg induction 2 from Ancylostoma caninum)

[94]

Intestinal Lactobacillus johnsonii

Reduces activated DCs and T cells during allergen or RSV challenge

[95]

Intranasal CpG

Increases IL-10-producing interstitial macrophages in lung

[96]

SCFAs (propionate)

Decrease eosinophils, IL-17A, IgE during allergic airway disease

[97] (Continued)

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TABLE 9.1 (Continued) Immune Site

Vagina

Eye

Skin

Microbiota

Effect

References

Desminotyrosine (microbiota-produced flavonoid)

Promotes type I interferon signaling in macrophages, reducing lung pathology associated with influenza infection

[98]

Vaginal Proteobacteria

Induces mucosal IL-33 and ILC2 recruitment, suppressing effector T cell recruitment and promoting herpes simplex virus-2 infection

[99]

Vaginal Prevotella bivia

Increases CD41 T cells and TNF-α in mucosa, promoting HIV susceptibility

[100]

Eye and intestinal microbiota

Induces IL-1B to boost immune effectors and neutrophil numbers in the eye to protect against infection

[101]

Ocular Corynebacterium mastitidis

Stimulates IL-17A production by γδ T cells, promoting resistance to eye infections

[102]

Microbiota

Activates retina-specific Th17 cells in the gut that can promote uveitis development

[103,104]

Skin microbiota (Staphylococcus epidermidis, Corynebacterium accolens, and Propionibacterium acnes)

Induces Ccl20 in hair follicles to recruit Treg cells

[105]

Skin microbiota

Controls ratio of Tregs/effector T cells

[106]

1

Skin Staphylococcus epidermidis, Rothia nasimurium, Staphylococcus aureus, Staphylococcus lentus, and Staphylococcus xylosus

Recruits IL-17A-producing CD8 T cells

[107 109]

Skin Corynebacterium bovis

Induces a Th17 response

[109]

LTA on skin

Promotes mast cell maturation and function and Treg recruitment; increases eosinophils and basophils during dermatitis

[105,110,111]

Skin SCFAs (propionate and valerate)

Induce proinflammatory cytokines in keratinocytes and antiinflammatory cytokines in monocytes and Tregs through histone deacetylase inhibition

[112,113]

Skin helminth infection (Schistosoma mansoni larvae)

Suppresses neutrophil recruitment and inflammation

[114]

Additionally, since the numbers of Th17 levels are similar between GF and SPF mice, the microbiota in general appears to have less impact on Th17 cells in the oral compartment compared to other environmental signals such

as the damage that accrues over time from mastication [115]. The influence of the oral microbiota extends to the immune cells residing in the cervical lymph nodes, which drain antigen-presenting cells from the oral and nasal

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mucosa [116]. GF and antibiotic treatment experiments demonstrated the microbiota modulated IL-17-producing γδ T cell (γδT17) numbers and function, most prominently within cervical lymph nodes but also in the lungs and spleen [81]. Additionally, γδT17 cells appeared to control the oral microbiota composition, as IL-17r2/2 mice had an altered microbiota composition, which included increased Lactobacillus and decreased Aggregatibacter [81]. Studies with GF and MyD882/2 mice revealed that growth-arrest-specific 6 (GAS6) expression, a TYRO3-AXL-MERTK (TAM) receptor family ligand in the oral epithelium and immune cells, is induced by the microbiota [82]. Additional experiments with Gas62/2 mice demonstrated that GAS6 increased Treg cells while limiting Th17 cells in the cervical lymph nodes and controlled the oral microbiota composition by limiting the expansion of anaerobic bacteria such as Prevotella, Aggregatibacter, and Helicobacter [82]. 2. Respiratory Tract Immunity The respiratory tract ranges from the nasal cavity and nasopharynx to the lung and is under constant exposure to the environment, which in turn influences the local microbiota [117]. Although much of the insight for how the local and intestinal microbiota shapes the respiratory tract immune response comes from preclinical models studying infection and allergic diseases; many of these responses involve DCs, T cell activation, and IgA induction, all of which are important targets for designing effective mucosal vaccines. Similar to the GALT, the nasal-associated lymphoid tissue (NALT) is also impacted by the intestinal microbiota. For example, comparing GF and conventionally raised WT C57BL/ 6J mice revealed that NALT-associated B cells undergo Sμ-σδ class-switch recombination to secrete IgD in an intestinal microbiota- and MyD88-dependent manner [78]. Furthermore, comparing sinus histology between GF and SPF

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C3H/Orl mice showed that GF mice have a thinner mucosa and epithelium, reduced goblet cells, and less NALT, suggesting that the local microbiota could influence sinus mucosal tissue development [83]. Disease states such as acute infections, inflammation, or allergies promote variation in the human nasal microbiome, which can in turn alter local immunity [84,85]. Characterizing the sinus microbiota of chronic rhinosinusitis patients via 16S rRNA gene sequencing demonstrated four distinct microbiota types, each dominated by a pathogen-associated bacterial family [84]. Simultaneous assessment of the host immune response with a gene microarray demonstrated that the nasal-associated microbiotas dominated by Streptococcaceae, Pseudomonadaceae, or Staphylococcaceae were associated with more proinflammatory Th1 immune responses, while a Corynebacteriaceaedominated microbiota was associated with increased IL-5 [84]. Examination of the interplay between nasal bacteria and the host immune response in pediatric asthma patients using dual RNA sequencing revealed positive correlations between Proteobacteria abundance or bacterial adhesion gene expression and host IL-1α expression [85]. Taken together, these observations indicate that nasal bacteria have differential effects on the host’s immune response in a disease state. Like the microbiota of the nasal cavity, members of both the local and intestinal microbiota have been implicated in promoting innate and adaptive immune responses within the lung. Lung immunity to Streptococcus pneumoniae infection in newborn mice is shaped by the recruitment of IL-22-producing ILC3s into the lungs upon intestinal DC recognition of gut bacteria [86]. Lung ILC3s were decreased in GF and antibiotic-treated mice, and IL-22 administration, ILC3 cell transfer, or reintroduction of intestinal bacteria restored protection against S. pneumoniae infection [86]. Similarly, broad-spectrum antibiotic treatment of adult mice increased their

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susceptibility to acute Klebsiella pneumoniae infection [92]. In mice, SFB have the capacity to modulate immunity at mucosal sites beyond the intestine, such as the lung. For example, SFB intestinal colonization was associated with resistance to Staphylococcus aureus pneumonia challenge due to increased Th17 cell effector cytokines such as IL-22 in the lung [87]. Additional evidence for the intestinal microbiota and SFB affecting lung adaptive immunity was shown in the context of a lung fungal infection in which vancomycin treatment of SFBcolonized mice, which selectively depleted Gram-positive bacteria, decreased lung Th17 cells and increased susceptibility to Aspergillus fumigatus infection [88]. Besides the bacterial members of the intestinal microbiota, fungi also appear to affect lung immunity. For example, fungal intestinal dysbiosis in mice after oral administration of antifungal drugs (decreased Candida and increased Aspergillus, Wallemia, and Epicoccum spp.) was associated with increased susceptibility to allergic airway disease [89]. Increased disease susceptibility could be replicated by oral gavage of Aspergillus amstelodami, Epicoccum nigrum, and Wallemia sebi, suggesting that these species promote the Th2 response, which increases IgG, IgE, and eosinophils in the lung during allergic airway disease [89]. In addition to the gut microbiota, bacteria in the lung may also influence immunity, as positive correlations were observed between Prevotella, Rothia, and Veillonella and Th17 cytokines, lung Th17 cells, and neutrophils collected via bronchoalveolar lavage from healthy and pulmonary disease human patients [90]. Similar to the intestine, many of the interactions between the microbiota and respiratory tract immunity are mediated by pattern recognition receptor (PRR) detection of microbial antigens. A study utilizing a mouse model of influenza A infection revealed that antibiotics targeting the respiratory tract reduced the adaptive, inflammasome-dependent immune

response to the virus [91]. Furthermore, administration of the toll-like receptor 4 (TLR4) ligand LPS via intranasal or rectal inoculation of other TLR ligands CpG (TLR9), Poly I:C (TLR3), and peptidoglycan (TLR2) could recover the immune response to influenza in the lungs of antibiotic-treated mice, suggesting that both the nasal and gut microbiota can modify the immune response to influenza [91]. In a mouse model of acute bacterial lung infection with K. pneumoniae, depleting the microbiota with antibiotics reduced the antibacterial capacity of alveolar macrophages by lowering reactive oxygen species production [92]. Gavage of Nod-like receptor (NOD1 or NOD2) but not TLR ligands (TLR2, 4, or 9) was able to restore the antibacterial defenses of lung macrophages [92]. In contrast to the microbiota members that promote innate and adaptive immune responses, other microorganisms such as helminths and certain bacteria have been shown to suppress immunity in the lung. Gavaging mice with enteric Heligomosomoides polygorus reduced inflammation and viral load in the lung after intranasal inoculation with respiratory syncytial virus (RSV) [93]. The helminth’s protective effects depended on the microbiota and were mediated by type I interferons and interferonstimulated gene induction in the duodenum and lung [93]. Many of the immunomodulatory effects of helminths relate to products they secrete and do not require the live organism to have an effect [118]. For example, in a mouse model of allergic asthma, intraperitoneal or intranasal administration of antiinflammatory protein-2 (secreted by the hookworm Ancylostoma caninum) promoted Treg induction, which reduced DC stimulation and T cell activation [94]. Accounting for potential immunosuppression from helminths could be particularly important for mucosal vaccine design, especially for vaccines that will be used in developing countries and tropical regions where helminth infections are common,

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affecting approximately one third of the world’s population [119]. Immunosuppressive effects have also been demonstrated for bacteria. For example, exposing mice to dust collected from homes with a pet dog reduced inflammation in a model of allergen-induced airway disease and was associated with an increase abundance of cecal Lactobacillus johnsonii. Administering L. johnsonii to mice undergoing allergen or RSV challenge reduced the number and proportion of activated DCs and T cells in the lung and decreased Th2 cytokines [95]. Similarly, some bacterial components also appear to have immunosuppressive effects. For example, intranasal administration of CpG, the bacterial TLR9 ligand, increased the number of IL-10producing interstitial macrophages in the lung and decreased inflammation in a house dust mite mouse model of asthma [96]. Microbial metabolites are another way in which lung immune suppression by the microbiota can occur (Table 9.1). SCFAs modulate lung immunity in addition to their established intestinal effects. For example, a high-fiber diet was associated with high circulating levels of SCFAs and decreased inflammation (eosinophils, Il-17A, IgE) in an intranasal house dust mite mouse model of allergic airway disease [97]. Additional experiments with propionate alone demonstrated that the decreased inflammation was mediated through a promotional effect on precursor DCs in the bone marrow, which upon migration to the lungs were less effective at inducing Th2 effector cells [97]. Another metabolite, desminotyrosine, a flavonoid produced by the microbiota, is able to promote type I interferon signaling in macrophages, reducing lung pathology in a mouse model of influenza [98]. Importantly, gavaging the desminotyrosine-producing bacteria Clostridium orbscindens or deaminotyrosine restored immunity to influenza infection in antibiotic-treated mice [98].

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3. Other Mucosal Immune Sites The vaginal microbiota has also been implicated in modulation of the local immune response (Table 9.1). Treating mice with oral antibiotics altered the vaginal microbiome by increasing members of the Proteobacteria phylum [99]. Microbiota depletion made the mice more susceptible to herpes simplex virus-2 infection, owing to impaired recruitment of effector CD41 and CD81 T cells as a result of mucosal IL-33 induction and group 2 ILC (ILC2) recruitment [99]. Additional experiments demonstrated that papain, a cysteine protease, promoted IL-33 secretion, suggesting that cysteine proteases produced by Proteobacteria such as Serratia and Pseudomonas indirectly suppress effector T cell recruitment [99]. The observation of the local microbiota modulating vaginal immunity extends to humans with a low Lactobacillus and high anaerobic spp. vaginal microbiome associated with more active CD41 T cells, TNF-α, and IL-17 in the mucosa and an increased risk of acquiring HIV [100]. Intravaginal inoculation of GF mice with Prevotella bivia increased CD41 T cells and TNFα in the genital mucosa, while Lactobacillus crispatus did not, demonstrating that bacteria colonizing the vaginal mucosa also have different immunomodulatory capacities. Mucosal immunity in the eye is also affected by the local and intestinal microbiota, and the eye has been explored as a potential mucosal vaccine delivery route [120,121] (Chapter 17: Mucosal Regulatory System for the Balanced Occular Immunity). SPF mice are less susceptible to Pseudomonas aeruginosa eye infection than are GF and antibiotic-treated mice, owing to increased immune effectors and neutrophil recruitment that depend on IL-1β induction by the eye and gut microbiota [101]. Corynebacterium mastitidis, a member of the mouse eye microbiota, stimulated Il-17A production by γδ T cells and promoted resistance to Candida albicans and P. aeruginosa eye infections [102]. In a genetically

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engineered mouse model of spontaneous uveitis, an autoimmune eye disease that leads to blindness, deriving mice into GF conditions or treating them with antibiotics attenuated symptoms [103,104]. Interestingly, the microbiota or microbiota-derived protein extracts activated the retina-specific Th17 cells in the gut and lead to uveitis development, indicating a role for the microbiota in eye autoimmunity [103,104].

IV. SKIN IMMUNITY Although the skin is not part of the mucosal immune system, transcutaneous vaccines remain attractive, owing to their simplicity and ease of use [122]. Since transcutaneous vaccine administration has the capacity to generate mucosal immune responses [123], it is important to consider how the local microbiota may affect the skin immune response to vaccination. Mutualism between the host’s immune system and the skin microbiota commences early in life. Comparisons between 2-week-old GF and SPF mice revealed that FoxP31 Treg accumulation within skin hair follicles is microbiotadependent and mediated by induction of the chemokine Ccl20 in hair follicles, which recruits Ccr6-expressing Treg cells [105]. In vitro experiments demonstrated that multiple bacterial strains such as Staphylococcus epidermidis, Corynebacterium accolens, and Propionibacterium acnes have the capacity to induce Ccl20 mRNA expression in human skin cells [105]. Once established, the Treg cells specific for skin bacteria antigens promote antigen-specific tolerance by suppressing inflammation in adult mice after a skin abrasion challenge [124]. Comparisons between GF and SPF mice demonstrated that the skin microbiota also influences the balance between Tregs and effector T cells in a MyD88- and IL-1-dependent manner [106]. Topical application of S. epidermidis to the skin of SPF or GF mice recruited IL-17Aproducing CD81 T cells specific to the

bacterium, a process dependent on the presence of DCs [107]. IL-17A production bolstered keratinocyte innate immune defenses by promoting S100a8/9 mRNA expression [107]. Interestingly, other skin bacteria such as Rothia nasimurium, S. aureus, Staphylococcus lentus, and Staphylococcus xylosus were also able to increase skin IL-17A1 T cells [107]. The interactions between the skin bacteria and the host are crucial for generating effective immune responses to skin infections with the protozoan Leishmania major or the fungus C. albicans [106,107]. Additional insight into how the skin microbiota affects skin immunity derives from research focused on inflammatory skin diseases such as atopic dermatitis or eczema. Skin microbiota dysbiosis in a mouse model of dermatitis due to a deficiency in Nfkbiz (a transcriptional regulator of NF-κB) along with topical application of S. xylosus increased IL-17A1 CD4 T cells [108]. An altered skin microbiota is also associated with inflammation in another mouse model of eczema dermatitis due to deficiency in the transmembrane protease, A disintegrin and metalloproteinase 17 (ADAM17) [109]. Dysbiosis in mice included increased S. aureus and Corynebacterium bovis, with S. aureus promoting Th17 inflammation through interactions with Langerhans cells and C. bovis inducing a Th2 response [109]. Similarly in human immunodeficient patients suffering from atopic dermatitis-like eczema, S. aureus abundance positively correlated with markers of disease severity, while P. acnes negatively correlated [125]. These observations suggest that interactions with the skin microbiota are carefully controlled by the host immune system (Table 9.1) and that disruption of this balance drives skin diseases, which could in turn affect the immune response to transcutaneous vaccines. Similar to the gut and other mucosal immune sites, microbial PRR ligands and metabolites direct host microbe interactions within the skin. Investigations using GF and SPF mice

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V. MICROBIOTA AND VACCINES

revealed that microbiota and, more specifically, the Gram-positive cell wall product lipotechoic acid (LTA), a TLR2 ligand, promoted mast cell maturation and function in the skin by inducing keratinocytes to secrete stem cell factor [110]. Mast cells have roles in Langerhans cell (a skin-specific DC) migration, inflammation, and cytotoxic T lymphocyte induction following transcutaneous vaccine administration [123]. LTA and the bacterial cell wall component peptidoglycan are also implicated in skin Treg cell recruitment via CCL20 induction [105]. S. aureus LTA and muramyl dipeptide (MDP), a NOD2 ligand, increased eosinophils and basophils through interactions with dermal fibroblasts in a chemically induced model of dermatitis [111]. LTA localizes to the epidermis of healthy humans, and a high LTA level correlates with severe pediatric atopic dermatitis disease, implicating LTA as an important factor for skin immune homeostasis [126,127]. In vitro culturing of bacterial metabolites with monocyte-derived DCs from atopic dermatitis or control patient blood demonstrated that skin bacterial metabolites have strain-specific immunomodulatory capacities [128]. S. aureus metabolites promoted CD41 Th2/Th22 activation, while S. epidermidis induced DC IL-10 secretion and Treg proliferation [128]. SCFAs also have immunomodulatory effects on the skin immune system, promoting proinflammatory cytokines (e.g., TNF-α) in keratinocytes and antiinflammatory cytokines (e.g., IL-10) in monocytes and Tregs through histone deacetylase inhibition [112,113]. P. acnes grown under anaerobic conditions that mimic a blocked hair follicle environment produce propionate and valerate, suggesting that skin bacteria also produce SCFAs [112]. Future investigations are needed to determine the physiological skin SCFA levels and associated bacteria and how they affect skin immunity during healthy and disease states. Along with bacteria, helminths can suppress inflammatory responses in the skin. For example, a mouse model of repeated

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Schistosoma mansoni larvae skin infection to mimic exposure in endemic areas revealed that the helminth induced IL-10-producing CD41 T cells at the infection site, which suppressed neutrophil recruitment and inflammation [114]. Interestingly, IL-10 production on the first day of infection was in response to skin bacterial antigens from the local microbiota and was subsequently induced by helminth and skin bacterial antigens [114].

V. MICROBIOTA AND VACCINES As the field of vaccine development has continued to press forward, researchers have begun to recognize the influence and potential of the microbiome as adjuvants and delivery agents of vaccines (Fig. 9.2). Several studies highlight the usage of specific commensal microbes and commonly utilized probiotics as adjuvants to increase the efficacy of vaccines (Fig. 9.2). Bacillus subtilis is a commensal microbe that was engineered to express tetanus toxin fragment C, administered sublingually, acting as an antigen carrier to induce mucosal immunity in pigs and mice [129,130]. In addition, microbial sensors appear to play a role in flu vaccination in mice. For example, TLR5deficient GF and antibiotic-treated mice showed a decrease in trivalent influenza vaccine-specific antibody-secreting cells and B cells after subcutaneous injection of vaccine. This phenotype was reversed following the transfer of GF mice to SPF housing. This study outlines the importance of host microbe interactions, with host-derived TLR5 promoting signaling of flagellated microbes [131]. In addition, IgA class-switch recombination in B cells was impaired after culture with lung DCs from either GF or antibiotic-treated mice. This phenomenon was MyD88- and TRIFdependent, suggesting that lung DCs require interaction with the microbiota to promote activation of IgA induction pathway [132]. The

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2

1 Antigen carriers

Vaccine response

3

PRR

4

Adjuvants

FIGURE 9.2 Role of microbiota in vaccines. Intestinal or local microbiota at mucosal and vaccine administration sites can enhance or restrict the efficacy of vaccines. Microbes have been utilized as a vehicle (1) for toxin delivery to the mucosal site. When combined with vaccines, microbes and/or their pattern recognition receptors (PRRs) can serve as (2) adjuvants to help facilitate activation of antigen-specific immune response. The microbiota can (3) favorably promote (4) or inhibit the response to vaccines through PRR interactions or the production of metabolites.

ability of the microbiota to increase serum, lung, and stool IgA levels post intranasal vaccination with inactive cholera toxin highlights the microbiota’s importance for mucosal vaccine studies, although further investigations are needed to tease out the responsible microbiota members [132]. In addition to the role of PRR ligands from the lung and intestinal microbiota, antigens from nasal microbiota could also be involved in antibody induction. For example, enhanced IgA production by B cells in the lung after intranasal administration of OVA and recombinant flagellin was abrogated upon antibody blockade of granulocyte-macrophage colony-stimulating factor (GM-CSF), suggesting that lung DCs respond to GM-CSF released by TLR5-activated nasal epithelial cells [133]. Furthermore, experiments with GF or mice treated intranasally with antibiotics demonstrated that the microbiota enhanced CpG and cholera toxin adjuvant activities for nasal or oral but not intraperitoneal human serum albumin vaccination [134]. Additional experiments, revealed that cholera toxin’s ability to enhance the antigen-specific IgG response was NOD2dependent and could be restored in GF mice by

administering MDP (Nod2 ligand) or colonizing the nasal cavity with Staphylococcus gallinarum, a bacteria with high NOD2 stimulatory activity in vitro [134]. Depending on the type of vaccine and the administration route, the local microbiota also has the capacity to influence vaccine efficacy (Fig. 9.2). The observation that intranasal administration of IL-1α was a potent adjuvant with influenza vaccine in mice, generating more secretory IgA upon challenge, is interesting, given the association between nasal Proteobacteria and host IL-1α [85,135]. Furthermore, positive correlations between members of the human nasal microbiome (Streptococcus infantis, Prevotella melaninogenica, and Lactobacillus helveticus) of young adults and nasal IgA response to live attenuated influenza vaccine demonstrate the capacity of different nasal bacteria to influence the IgA response to mucosal vaccines in humans [136]. Further investigations are needed to determine the mechanisms by which nasal bacteria influence the response to influenza and other mucosal vaccines. However, immunomodulatory microbes may also have detrimental effects (Fig. 9.2) in the

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FINANCIAL SUPPORT

context of vaccine response, owing to their immunosuppressive capabilities. For example, Helicobacter hepaticus, a common mouse intestinal microbiota member, induced IL-10 mRNA expression in the colon and was associated with a reduced lung immune response to a Mycobacterium tuberculosis subunit vaccine [137]. Alternatively, the intestinal microbiota may prevent immunization efficacy by outcompeting vaccine-responsive B cells with preexisting cross-reactive B cells, thus driving a nonprotective antibody response to the vaccine. This was shown for the intramuscularly administered HIV-1 envelope vaccine DNA-rAD5 [138]. Prior to vaccine or virus exposure, polyreactive antibodies against HIV envelope subunit gp41 are present in human samples, originating from terminal ileum B cells and cross-reacting with commensal bacteria as well as other environmental antigens [139,140]. When the HIV-1 vaccine was administered intramuscularly, systemic titers of the protective gp120 antibodies did increase, but nonneutralizing gp41 antibodies remained dominant and were associated with low vaccine efficacy [138]. The authors hypothesized that this indirect inhibition by the microbiota could be circumvented by early vaccination of infants, before their immature immune system has established a microbe-specific B cell repertoire [138]. In fact, neonates are able to develop broadly neutralizing antibodies against HIV-1 at least as effectively as adults [141] (Chapter 42: Mucosal Vaccines Against HIV/SIV infection). However, caution should be applied to this line of thinking, as intestinal homeostasis is a delicate balance between reactivity to and tolerance of commensal bacteria. Disrupting this equilibrium by decreasing pools of microbe-specific antibodies could potentially increase susceptibility to intestinal infections or opportunistic pathogens. For example, an abundance of Actinobacteria (in particular Bifidobacterium) in infant stool directly correlated with systemic vaccine responsiveness to oral polio virus, bacillus Calmette-Gue´rin, and tetanus toxoid but not

hepatitis B virus. Conversely, the amounts of Enterobacteriales, Pseudomondales, and Clostridiales were associated with systemic inflammation and decreased vaccine response [142].

VI. CONCLUDING REMARKS The critical role of microbiota in immune cell function and differentiation is well established, and the potential impact of these microbial communities on vaccine response requires further attention. The gut microbiota plays a crucial role in mucosal immunity development and maintenance, which is important to consider in determining the efficacy of vaccines within a specific population or even an individual. Depending on the mucosal vaccine type and administration route, both the local and intestinal microbiota should be accounted for because of their capacity to influence extraintestinal immunity. Since the immunomodulatory activities of the microbiota range from stimulatory to suppressive, it is important that future mucosal vaccine design and efficacy studies take into account both types of effects. Continuing to elucidate the microbial components and/or metabolites that modulate immunity at different administration sites could lead to new mucosal vaccines and adjuvant combinations that will maximize vaccine efficacy.

Acknowledgment The authors would like to thank Dr. Christina Ohland for helping to write part of sections II and V of this chapter.

FINANCIAL SUPPORT This research was supported by National Institutes of Health grants RO1DK073338, RO1AT08623, and R21CA195226 to C. Jobin.

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References [1] Kau AL, Ahern PP, Griffin NW, Goodman AL, Gordon JI. Human nutrition, the gut microbiome and the immune system. Nature 2011;474:327 36. [2] Pasolli E, Truong DT, Malik F, Waldron L, Segata N. Machine learning meta-analysis of large metagenomic datasets: tools and biological insights. PLoS Comput Biol 2016;12:e1004977. [3] Sender R, Fuchs S, Milo R. Revised estimates for the number of human and bacteria cells in the body. PLoS Biol 2016;14:e1002533. [4] Qin J, et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 2010;464:59 65. [5] Sender R, Fuchs S, Milo R. Revised estimates for the number of human and bacteria cells in the body. BioRxiv 2016;. Available from: https://doi.org/ 10.1101/036103. [6] Andersson AF, et al. Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS One 2008;3:e2836. [7] von Rosenvinge EC, et al. Immune status, antibiotic medication and pH are associated with changes in the stomach fluid microbiota. ISME J 2013;7:1354 66. [8] Dong Q, et al. Characterization of gastric microbiota in twins. Curr Microbiol 2017;74:224 9. [9] Ringel Y, et al. High throughput sequencing reveals distinct microbial populations within the mucosal and luminal niches in healthy individuals. Gut Microbes 2015;6:173 81. [10] Sekirov I, Russell SL, Antunes LCM, Finlay BB. Gut microbiota in health and disease. Physiol Rev 2010;90:859 904. [11] Ohland CL, Jobin C. Microbial activities and intestinal homeostasis: a delicate balance between health and disease. Cell Mol Gastroenterol Hepatol 2015;1:28 40. [12] Dewhirst FE, et al. The human oral microbiome. J Bacteriol 2010;192:5002 17. [13] Dickson RP, Erb-Downward JR, Huffnagle GB. The role of the bacterial microbiome in lung disease. Expert Rev Resp Med 2013;7:245 57. [14] Lloyd CM, Marsland BJ. Lung homeostasis: influence of age, microbes, and the immune system. Immunity 2017;46:549 61. [15] Dominguez-Bello MG, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci USA 2010;107:11971 5. [16] Martin R, et al. Early-life events, including mode of delivery and type of feeding, siblings and gender, shape the developing gut microbiota. PLoS One 2016;11:e0158498.

[17] Milani C, et al. Exploring vertical transmission of bifidobacteria from mother to child. Appl Environ Microbiol 2015;81:7078 87. [18] Davis EC, Wang M, Donovan SM. The role of early life nutrition in the establishment of gastrointestinal microbial composition and function. Gut Microbes 2017; 8:143 71. [19] Nejrup RG, Licht TR, Hellgren LI. Fatty acid composition and phospholipid types used in infant formulas modifies the establishment of human gut bacteria in germ-free mice. Sci Rep 2017;7:3975. [20] Planer JD, et al. Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice. Nature 2016;534:263 6. [21] Harmsen HJ, et al. Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 2000;30:61 7. [22] Praveen P, Jordan F, Priami C, Morine MJ. The role of breast-feeding in infant immune system: a systems perspective on the intestinal microbiome. Microbiome 2015;3:41. [23] Taft DH, et al. Intestinal microbiota of preterm infants differ over time and between hospitals. Microbiome 2014;2:36. [24] Torrazza RM, et al. Intestinal microbial ecology and environmental factors affecting necrotizing enterocolitis. PLoS One 2013;8:e83304. [25] Bergstro¨m A, et al. Establishment of intestinal microbiota during early life: a longitudinal, explorative study of a large cohort of Danish infants. Appl Environ Microbiol 2014;80:2889 900. [26] Koenig JE, et al. Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci USA 2011;108(Suppl. 1):4578 85. [27] Yatsunenko T, et al. Human gut microbiome viewed across age and geography. Nature 2012;486:222 7. [28] Palmer C, Bik EM, DiGiulio DB, Relman DA, Brown PO. Development of the human infant intestinal microbiota. PLoS Biol 2007;5:e177. [29] Avershina E, et al. Transition from infant- to adult-like gut microbiota. Environ Microbiol 2016;18:2226 36. [30] Weiss GA, Hennet T. Mechanisms and consequences of intestinal dysbiosis. Cell Mol Life Sci 2017;. Available from: https://doi.org/10.1007/s00018-017-2509-x. [31] Postler TS, Ghosh S. Understanding the holobiont: how microbial metabolites affect human health and shape the immune system. Cell Metab 2017;26:110 30. [32] Matsumoto M, et al. Impact of intestinal microbiota on intestinal luminal metabolome. Sci Rep 2012;2:233. [33] Morrison DJ, Preston T. Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes 2016;7:189 200.

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[34] Ridlon JM, Harris SC, Bhowmik S, Kang D-J, Hylemon PB. Consequences of bile salt biotransformations by intestinal bacteria. Gut Microbes 2016;7:22 39. [35] Michael AJ. Biosynthesis of polyamines and polyamine-containing molecules. Biochem J 2016; 473:2315 29. [36] Wikoff WR, et al. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proc Natl Acad Sci USA 2009;106:3698 703. [37] Zelante T, et al. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity 2013; 39:372 85. [38] El Aidy S, et al. Temporal and spatial interplay of microbiota and intestinal mucosa drive establishment of immune homeostasis in conventionalized mice. Mucosal Immunol 2012;5:567 79. [39] Geuking MB, et al. Intestinal bacterial colonization induces mutualistic regulatory T cell responses. Immunity 2011;34:794 806. [40] Perez-Mun˜oz ME, et al. Discordance between changes in the gut microbiota and pathogenicity in a mouse model of spontaneous colitis. Gut Microbes 2014; 5:286 95. [41] Petersson J, et al. Importance and regulation of the colonic mucus barrier in a mouse model of colitis. Am J Physiol Gastrointest Liver Physiol 2011;300:G327 33. [42] Desai MS, et al. A dietary fiber-deprived gut microbiota degrades the colonic mucus barrier and enhances pathogen susceptibility. Cell 2016;167:1339 1353.e21. [43] den Besten G, et al. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. J Lipid Res 2013;54:2325 40. [44] Johansson MEV, et al. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc Natl Acad Sci USA 2008;105:15064 9. [45] Shimada Y, et al. Commensal bacteria-dependent indole production enhances epithelial barrier function in the colon. PLoS One 2013;8:e80604. [46] Venkatesh M, et al. Symbiotic bacterial metabolites regulate gastrointestinal barrier function via the xenobiotic sensor PXR and Toll-like receptor 4. Immunity 2014;41:296 310. [47] Knoop KA, McDonald KG, McCrate S, McDole JR, Newberry RD. Microbial sensing by goblet cells controls immune surveillance of luminal antigens in the colon. Mucosal Immunol 2015;8:198 210. [48] Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol 2014;14:141 53. [49] Reikvam DH, et al. Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression. PLoS One 2011;6:e17996.

161

[50] Natividad JMM, et al. Commensal and probiotic bacteria influence intestinal barrier function and susceptibility to colitis in Nod1-/-; Nod2-/- mice. Inflamm Bowel Dis 2012;18:1434 46. [51] Smith MW, James PS, Tivey DR. M cell numbers increase after transfer of SPF mice to a normal animal house environment. Am J Pathol 1987;128:385 9. [52] Corr SC, Gahan CCGM, Hill C. M-cells: origin, morphology and role in mucosal immunity and microbial pathogenesis. FEMS Immunol Med Microbiol 2008;52:2 12. [53] Boursier L, Gordon JN, Thiagamoorthy S, Edgeworth JD, Spencer J. Human intestinal IgA response is generated in the organized gut-associated lymphoid tissue but not in the lamina propria. Gastroenterology 2005;128:1879 89. [54] Barone F, et al. IgA-producing plasma cells originate from germinal centers that are induced by B-cell receptor engagement in humans. Gastroenterology 2011;140:947 56. [55] Yanagibashi T, et al. IgA production in the large intestine is modulated by a different mechanism than in the small intestine: Bacteroides acidifaciens promotes IgA production in the large intestine by inducing germinal center formation and increasing the number of IgA 1 B cells. Immunobiology 2013;218:645 51. [56] Yamanaka T, et al. Microbial colonization drives lymphocyte accumulation and differentiation in the follicle-associated epithelium of Peyer’s patches. J Immunol 2003;170:816 22. [57] Kim S-H, Cho B-H, Kiyono H, Jang Y-S. Microbiotaderived butyrate suppresses group 3 innate lymphoid cells in terminal ileal Peyer’s patches. Sci Rep 2017;7:3980. [58] Lathrop SK, et al. Peripheral education of the immune system by colonic commensal microbiota. Nature 2011;478:250 4. [59] Kuhn KA, et al. Bacteroidales recruit IL-6-producing intraepithelial lymphocytes in the colon to promote barrier integrity. Mucosal Immunol 2017;. Available from: https://doi.org/10.1038/mi.2017.55. [60] Cabinian A, et al. Gut symbiotic microbes imprint intestinal immune cells with the innate receptor SLAMF4 which contributes to gut immune protection against enteric pathogens. Gut 2017;. Available from: https://doi.org/10.1136/gutjnl-2016-313214. [61] Walton KLW, He J, Kelsall BL, Sartor RB, Fisher NC. Dendritic cells in germ-free and specific pathogen-free mice have similar phenotypes and in vitro antigen presenting function. Immunol Lett 2006;102:16 24. [62] Furusawa Y, et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 2013;504:446 50.

II. PRINCIPLES OF MUCOSAL VACCINE

162

9. INFLUENCE OF COMMENSAL MICROBIOTA AND METABOLITE FOR MUCOSAL IMMUNITY

[63] Round JL, Mazmanian SK. Inducible Foxp3 1 regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci USA 2010;107:12204 9. [64] Ohnmacht C, et al. Mucosal immunology. The microbiota regulates type 2 immunity through RORγt1 T cells. Science 2015;349:989 93. [65] Atarashi K, et al. Th17 cell induction by adhesion of microbes to intestinal epithelial cells. Cell 2015;163: 367 80. [66] Ivanov II, et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 2008;4: 337 49. [67] Ivanov II, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 2009;139: 485 98. [68] Umesaki Y, Setoyama H, Matsumoto S, Imaoka A, Itoh K. Differential roles of segmented filamentous bacteria and clostridia in development of the intestinal immune system. Infect Immun 1999;67:3504 11. [69] Bain CC, et al. Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nat Immunol 2014;15:929 37. [70] Cerundolo V, Silk JD, Masri SH, Salio M. Harnessing invariant NKT cells in vaccination strategies. Nat Rev Immunol 2009;9:28 38. [71] Carren˜o LJ, Kharkwal SS, Porcelli SA. Optimizing NKT cell ligands as vaccine adjuvants. Immunotherapy 2014;6:309 20. [72] Olszak T, et al. Microbial exposure during early life has persistent effects on natural killer T cell function. Science 2012;336:489 93. [73] Wei B, et al. Commensal microbiota and CD8 1 T cells shape the formation of invariant NKT cells. J Immunol 2010;184:1218 26. [74] Wu W, et al. Microbiota metabolite short-chain fatty acid acetate promotes intestinal IgA response to microbiota which is mediated by GPR43. Mucosal Immunol 2017;10:946 56. [75] Cahenzli J, Ko¨ller Y, Wyss M, Geuking MB, McCoy KD. Intestinal microbial diversity during early-life colonization shapes long-term IgE levels. Cell Host Microbe 2013;14:559 70. [76] Rios D, et al. Antigen sampling by intestinal M cells is the principal pathway initiating mucosal IgA production to commensal enteric bacteria. Mucosal Immunol 2016;9:907 16. [77] Lindner C, et al. Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota. Nat Immunol 2015;16:880 8. [78] Choi JH, et al. IgD class switching is initiated by microbiota and limited to mucosa-associated lymphoid

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

tissue in mice. Proc Natl Acad Sci USA 2017;114: E1196 204. Wesemann DR, et al. Microbial colonization influences early B-lineage development in the gut lamina propria. Nature 2013;501:112 15. Lamouse´-Smith ES, Tzeng A, Starnbach MN. The intestinal flora is required to support antibody responses to systemic immunization in infant and germ free mice. PLoS One 2011;6:e27662. Fleming C, et al. Microbiota-activated CD103(1) DCs stemming from microbiota adaptation specifically drive γδT17 proliferation and activation. Microbiome 2017;5:46. Nassar M, et al. GAS6 is a key homeostatic immunological regulator of host-commensal interactions in the oral mucosa. Proc Natl Acad Sci USA 2017;114:E337 46. Jain R, Waldvogel-Thurlow S, Darveau R, Douglas R. Differences in the paranasal sinuses between germ-free and pathogen-free mice. Int Forum Allergy Rhinol 2016;6:631 7. Cope EK, Goldberg AN, Pletcher SD, Lynch SV. Compositionally and functionally distinct sinus microbiota in chronic rhinosinusitis patients have immunological and clinically divergent consequences. Microbiome 2017;5:53. Pe´rez-Losada M, Castro-Nallar E, Bendall ML, Freishtat RJ, Crandall KA. Dual transcriptomic profiling of host and microbiota during health and disease in pediatric asthma. PLoS One 2015;10:e0131819. Gray J, et al. Intestinal commensal bacteria mediate lung mucosal immunity and promote resistance of newborn mice to infection. Sci Transl Med 2017;9. Available from: http://dx.doi.org/10.1126/ scitranslmed.aaf9412. Gauguet S, et al. Intestinal microbiota of mice influences resistance to Staphylococcus aureus pneumonia. Infect Immun 2015;83:4003 14. McAleer JP, et al. Pulmonary th17 antifungal immunity is regulated by the gut microbiome. J Immunol 2016;197:97 107. Wheeler ML, et al. Immunological consequences of intestinal fungal dysbiosis. Cell Host Microbe 2016;19:865 73. Segal LN, et al. Enrichment of the lung microbiome with oral taxa is associated with lung inflammation of a Th17 phenotype. Nat Microbiol 2016;1:16031. Ichinohe T, et al. Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proc Natl Acad Sci USA 2011;108:5354 9. Clarke TB. Early innate immunity to bacterial infection in the lung is regulated systemically by the commensal microbiota via nod-like receptor ligands. Infect Immun 2014;82:4596 606.

II. PRINCIPLES OF MUCOSAL VACCINE

REFERENCES

[93] McFarlane AJ, et al. Enteric helminth-induced type I interferon signaling protects against pulmonary virus infection through interaction with the microbiota. J Allergy Clin Immunol 2017;. Available from: https:// doi.org/10.1016/j.jaci.2017.01.016. [94] Navarro S, et al. Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma. Sci Transl Med 2016;8:362ra143. [95] Fujimura KE, et al. House dust exposure mediates gut microbiome Lactobacillus enrichment and airway immune defense against allergens and virus infection. Proc Natl Acad Sci USA 2014;111:805 10. [96] Sabatel C, et al. Exposure to bacterial CpG DNA protects from airway allergic inflammation by expanding regulatory lung interstitial macrophages. Immunity 2017;46:457 73. [97] Trompette A, et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat Med 2014;20:159 66. [98] Steed AL, et al. The microbial metabolite desaminotyrosine protects from influenza through type I interferon. Science 2017;357:498 502. [99] Oh JE, et al. Dysbiosis-induced IL-33 contributes to impaired antiviral immunity in the genital mucosa. Proc Natl Acad Sci USA 2016;113:E762 71. [100] Gosmann C, et al. Lactobacillus-deficient cervicovaginal bacterial communities are associated with increased HIV acquisition in young South African women. Immunity 2017;46:29 37. [101] Kugadas A, et al. Impact of microbiota on resistance to ocular Pseudomonas aeruginosa-induced keratitis. PLoS Pathog 2016;12:e1005855. [102] St Leger AJ, et al. An ocular commensal protects against corneal infection by driving an interleukin-17 response from mucosal γδ T cells. Immunity 2017;47:148 158.e5. [103] Za´rate-Blade´s CR, et al. Gut microbiota as a source of a surrogate antigen that triggers autoimmunity in an immune privileged site. Gut Microbes 2017;8:59 66. [104] Horai R, et al. Microbiota-dependent activation of an autoreactive T cell receptor provokes autoimmunity in an immunologically privileged site. Immunity 2015;43:343 53. [105] Scharschmidt TC, et al. Commensal microbes and hair follicle morphogenesis coordinately drive treg migration into neonatal skin. Cell Host Microbe 2017;21:467 477.e5. [106] Naik S, et al. Compartmentalized control of skin immunity by resident commensals. Science 2012;337:1115 19. [107] Naik S, et al. Commensal-dendritic-cell interaction specifies a unique protective skin immune signature. Nature 2015;520:104 8.

163

[108] Kim Y, et al. The resident pathobiont Staphylococcus xylosus in Nfkbiz-deficient skin accelerates spontaneous skin inflammation. Sci Rep 2017;7:6348. [109] Kobayashi T, et al. Dysbiosis and Staphylococcus aureus colonization drives inflammation in atopic dermatitis. Immunity 2015;42:756 66. [110] Wang Z, et al. Skin microbiome promotes mast cell maturation by triggering stem cell factor production in keratinocytes. J Allergy Clin Immunol 2017;139: 1205 1216.e6. [111] Jiao D, et al. NOD2 and TLR2 ligands trigger the activation of basophils and eosinophils by interacting with dermal fibroblasts in atopic dermatitis-like skin inflammation. Cell Mol Immunol 2016;13:535 50. [112] Sanford JA, et al. Inhibition of HDAC8 and HDAC9 by microbial short-chain fatty acids breaks immune tolerance of the epidermis to TLR ligands. Sci Immunol 2016;1. [113] Schwarz A, Bruhs A, Schwarz T. The short-chain fatty acid sodium butyrate functions as a regulator of the skin immune system. J Invest Dermatol 2017;137: 855 64. [114] Sanin DE, Prendergast CT, Bourke CD, Mountford AP. Helminth infection and commensal microbiota drive early IL-10 production in the skin by CD4 1 T cells that are functionally suppressive. PLoS Pathog 2015;11:e1004841. [115] Dutzan N, et al. On-going mechanical damage from mastication drives homeostatic Th17 cell responses at the oral barrier. Immunity 2017;46:133 47. [116] Moingeon P. Update on immune mechanisms associated with sublingual immunotherapy: practical implications for the clinician. J Allergy Clin Immunol Pract 2013;1:228 41. [117] Man WH, de Steenhuijsen Piters WAA, Bogaert D. The microbiota of the respiratory tract: gatekeeper to respiratory health. Nat Rev Microbiol 2017;15: 259 70. [118] Girgis NM, Gundra UM, Loke P. Immune regulation during helminth infections. PLoS Pathog 2013;9: e1003250. [119] de Ruiter K, et al. Helminths, hygiene hypothesis and type 2 diabetes. Parasite Immunol 2017;39. [120] Seo KY, et al. Eye mucosa: an efficient vaccine delivery route for inducing protective immunity. J Immunol 2010;185:3610 19. [121] Barisani-Asenbauer T, et al. The ocular conjunctiva as a mucosal immunization route: a profile of the immune response to the model antigen tetanus toxoid. PLoS One 2013;8:e60682. [122] Engelke L, Winter G, Hook S, Engert J. Recent insights into cutaneous immunization: how to vaccinate via the skin. Vaccine 2015;33:4663 74.

II. PRINCIPLES OF MUCOSAL VACCINE

164

9. INFLUENCE OF COMMENSAL MICROBIOTA AND METABOLITE FOR MUCOSAL IMMUNITY

[123] Lawson LB, Clements JD, Freytag LC. Mucosal immune responses induced by transcutaneous vaccines. Curr Top Microbiol Immunol 2012;354:19 37. [124] Scharschmidt TC, et al. A wave of regulatory T cells into neonatal skin mediates tolerance to commensal microbes. Immunity 2015;43:1011 21. [125] Oh J, et al. The altered landscape of the human skin microbiome in patients with primary immunodeficiencies. Genome Res 2013;23:2103 14. [126] Wang Z, MacLeod DT, Di Nardo A. Commensal bacteria lipoteichoic acid increases skin mast cell antimicrobial activity against vaccinia viruses. J Immunol 2012;189:1551 8. [127] Travers JB, et al. Infected atopic dermatitis lesions contain pharmacologic amounts of lipoteichoic acid. J Allergy Clin Immunol 2010;125:146 152.e1. [128] Laborel-Pre´neron E, et al. Effects of the Staphylococcus aureus and Staphylococcus epidermidis secretomes isolated from the skin microbiota of atopic children on CD4 1 T cell activation. PLoS One 2015;10:e0141067. [129] Amuguni JH, et al. Sublingually administered Bacillus subtilis cells expressing tetanus toxin C fragment induce protective systemic and mucosal antibodies against tetanus toxin in mice. Vaccine 2011;29:4778 84. [130] Amuguni H, et al. Sublingual immunization with an engineered Bacillus subtilis strain expressing tetanus toxin fragment C induces systemic and mucosal immune responses in piglets. Microbes Infect 2012;14:447 56. [131] Oh JZ, et al. TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination. Immunity 2014;41:478 92. [132] Ruane D, et al. Microbiota regulate the ability of lung dendritic cells to induce IgA class-switch recombination and generate protective gastrointestinal immune responses. J Exp Med 2016;213:53 73.

[133] Cao Y, et al. Frontline science: nasal epithelial GMCSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response. J Leukoc Biol 2017;. Available from: https://doi.org/ 10.1189/jlb.3HI0816-368RR. [134] Kim D, et al. Nod2-mediated recognition of the microbiota is critical for mucosal adjuvant activity of cholera toxin. Nat Med 2016;22:524 30. [135] Kayamuro H, et al. Interleukin-1 family cytokines as mucosal vaccine adjuvants for induction of protective immunity against influenza virus. J Virol 2010;84: 12703 12. [136] Salk HM, et al. Taxa of the nasal microbiome are associated with influenza-specific IgA response to live attenuated influenza vaccine. PLoS One 2016;11: e0162803. [137] Arnold IC, et al. Helicobacter hepaticus infection in BALB/c mice abolishes subunit-vaccine-induced protection against M. tuberculosis. Vaccine 2015;33: 1808 14. [138] Williams WB, et al. HIV-1 VACCINES. Diversion of HIV-1 vaccine-induced immunity by gp41microbiota cross-reactive antibodies. Science 2015; 349:aab1253. [139] Trama AM, et al. HIV-1 envelope gp41 antibodies can originate from terminal ileum B cells that share crossreactivity with commensal bacteria. Cell Host Microbe 2014;16:215 26. [140] Campion SL, et al. Proteome-wide analysis of HIVspecific naive and memory CD4(1) T cells in unexposed blood donors. J Exp Med 2014;211:1273 80. [141] Goo L, Chohan V, Nduati R, Overbaugh J. Early development of broadly neutralizing antibodies in HIV-1-infected infants. Nat Med 2014;20:655 8. [142] Huda MN, et al. Stool microbiota and vaccine responses of infants. Pediatrics 2014;134:e362 72.

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Innate Immunity-Based Mucosal Modulators and Adjuvants Brandi T. Johnson-Weaver1, Soman N. Abraham1,2,3 and Herman F. Staats1,2,4 1

Department of Pathology, Duke University School of Medicine, Durham, NC, United States Department of Immunology, Duke University School of Medicine, Durham, NC, United States 3 Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, United States 4Department of Medicine, Duke University School of Medicine, Durham, NC, United States 2

I. INTRODUCTION Vaccines represent one of the greatest achievements of biomedical research and continue to have an impact on public health by preventing nearly 2.5 million deaths each year [1]. Most vaccines are administered by needle injections, although a few vaccines have been approved for delivery by mucosal routes. Immunization by mucosal routes, usually oral or intranasal, offers some real and potential benefits over injected vaccines. One real benefit of mucosal vaccination is that it can be achieved without needles, which may be desirable for individuals with needle phobia, and needlefree methods of immunization may also be more practical in developing countries [2 4]. A potential benefit of mucosal immunization is

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00010-9

the induction of antigen-specific immune responses not only in systemic compartments but also in mucosal tissues [5]. Since parenteral immunization rarely induces antigen-specific immune responses in mucosal tissues and delivery of vaccines by mucosal routes may induce antigen-specific immune responses at systemic and mucosal compartments [6 9], mucosal vaccination may provide protection against pathogens that initiate infection at the mucosal surfaces that is superior to protection provided by injected vaccines. Adjuvants are compounds coadministered with vaccines that enhance the induction of antigen-specific adaptive immune responses. Activation of the innate immune system is one pathway that adjuvants may use to enhance or modulate antigen-specific immunity [10,11].

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Mucosally administered vaccines often require adjuvants to activate the innate immune system, since antigen exposure by mucosal routes in the absence of adjuvants may lead to the induction of immunological tolerance [12]. While antigen-specific immunological tolerance may be desirable for the treatment of autoimmune, allergic, or antitransplant immune responses (see Chapter 51: Mucosal Vaccine for Allergy and Tolerance), immunological tolerance to infectious agents of disease would be detrimental to the host.

II. INNATE IMMUNE SYSTEM ACTIVATORS AS ADJUVANTS FOR MUCOSALLY ADMINISTERED SUBUNIT VACCINES Although live-attenuated or killed organisms are approved for use as mucosally administered vaccines, mucosal immunization with subunit immunogens may be desired, since subunit vaccines may induce the desired antigen-specific immune responses without the risks (or lack of efficacy) associated with liveattenuated or killed vaccines. However, as was mentioned above, mucosal vaccination with subunit immunogens will likely require the addition of an adjuvant to enhance the induction of antigen-specific immunity instead of antigen-specific tolerance [12,13]. Adjuvants that activate the innate immune system may provide immune-enhancing activity by a variety of different pathways, including increased local proinflammatory cytokine production by stromal cells [11,14], activation of local mast cells [14 16], and induction of localized cell death [14,16] with each pathway leading to enhanced antigen-specific adaptive immune responses due to activation and enhanced trafficking of antigen-presenting cells (APCs) and lymphocytes to the draining lymph node [11,17] (Table 10.1). While some researchers may consider cell death to be an

unacceptable activity for a vaccine adjuvant, we hypothesize that localized cell damage and cell death induced by a vaccine adjuvant may more closely mimic host responses to natural infections at a mucosal surface [30,31]. Adjuvant-induced cell damage or cell death may lead to the release of danger-associated molecular patterns (DAMPs) that enhance the induction of adaptive immunity in a manner similar to that provided by a natural infection. This chapter will discuss some of the mucosal adjuvants currently in development that modulate innate immune responses to enhance the induction of antigen-specific adaptive immunity. We are unable to discuss every unique adjuvant that has been evaluated when delivered by a mucosal route. Instead, we will focus on mucosal adjuvants with more detailed information on their ability to activate the innate immune system and those that have been evaluated in clinical studies. It is important to note that the enterotoxins cholera toxin (CT) and heat-labile toxin (LT) produced by Vibrio cholera and enterotoxigenic Escherichia coli, respectively, are related molecules [32] with a long history of use as mucosal vaccine adjuvants and are often considered the gold standard mucosal vaccine adjuvants. Derivatives of CT, such as CTA1-DD, which contains the enzymatic region of CT and two immunoglobulin-binding domains of staphylococcal protein A, also exhibit mucosal adjuvant activity [33]. The toxin-based adjuvants will not be discussed in this chapter because they are reviewed thoroughly in Chapter 11, ToxinBased Modulators for Regulation of Mucosal Immune Responses.

III. CYTOKINES AS MUCOSAL VACCINE ADJUVANTS Local induction of proinflammatory cytokine expression is one mechanism vaccine adjuvants may use to activate the innate immune system

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TABLE 10.1

Mucosal Adjuvants That Activate the Innate Immune System

Adjuvant

Induced innate immune response

References

Interferon alpha

• Activate myeloid DCs and pDCs • m Activated cells toward site of IFN-α administration • m Costimulatory molecules on dendritic cells and enhance DC function

[18 21]

Interleukin-1

• Induce ILC3 cells to produce IL-17 and IL-22 • m Enhance cytokine production, including IL-6, G-CSF, KC, MCP-1, and MIP-1β from stromal cells after mucosal delivery

[11,22]

W805EC

• • • •

[14,17,23,24]

Induce localized epithelial cell death and release caspase enzymes m Proinflammatory cytokines m Costimulatory molecules on APCs Activate TLRs

Compound 48/80 • Induce mast cell degranulation • m Dendritic cell trafficking to lymph nodes • Increase TNF production from mast cells

[15]

Polyethyleneimine • Enhance APC antigen uptake without direct APC activation • Induce DNA release

[16]

CpG

• m Costimulatory molecules on plasmacytoid DCs • Enhance IFN-α, TNF-α, and IL-6 from human pDCs and IFN-α and IFN-γ from human PBMCs • Activate cells through TLR9

[25 27]

MPL

• Activate cells through TLR4 • Induce TNF-α, IL-10 and IL-12p40 from human monocytes • Stimulates DCs to increase IFN-γ and reduce IL-4 and IL-5

[28,29]

to enhance antigen-specific adaptive immune responses. Thus, it is possible that directly applying one or more proinflammatory cytokines may exhibit adjuvant activity for mucosally administered vaccines by inducing local production of other cytokines, which in turn will enhance antigen presentation in the draining lymph nodes. Interferon alpha and interleukin-1 family members have demonstrated effective mucosal adjuvant activity and will be discussed below.

A. Interferon Alpha Interferon alpha (IFNα) is a member of the interferon cytokine family that is often produced after viral infections and helps to

mediate antiviral activities within the host [34]. IFNα is also an effective adjuvant in murine nasal influenza vaccine formulations [35,36]. Mice nasally immunized with an IFNα-adjuvanted influenza vaccine developed elevated antigen-specific serum and nasal antibodies [35,36]. Since the IFNα-adjuvanted influenza vaccine enhanced the induction of protective immune responses in mice, IFNα-adjuvanted inactivated influenza vaccines were evaluated for safety and immunogenicity in humans. In contrast to the results observed in the murine studies, IFNα did not provide adjuvant activity in humans, and subjects who received the high dose of IFNα in the nasal vaccine formulation developed severe systemic adverse reactions [35,37]. Although IFNα is an effective therapeutic approved for human use as an antiviral/

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anticancer agent [38,39] and demonstrates mucosal adjuvant activity in mouse models, additional studies are needed to further optimize vaccine conditions to better evaluate the safety and efficacy of IFNα as an effective mucosal vaccine adjuvant in humans.

B. Interleukin-1 Family The interleukin-1 (IL-1) family members include several cytokines that share a cellsignaling pathway that includes the Toll-IL-1 receptor (TIR) domain and myeloid differentiation factor 88 (MyD88) adapter molecule [22]. Members of the IL-1 family diversely affect host innate immune responses, with IL-1 family cytokines IL-1α and IL-1β associated with Th17-type responses [22], while other IL-1 family cytokines such as IL-33 are associated with Th2-type responses [22]. The mucosal adjuvant activities of several IL-1 family members were compared in a mouse model of an influenza vaccine, and IL-1, IL-18, and IL-33 were shown to be the most effective cytokines with nasal adjuvant activity [40]. Since the IL-1 family member IL-1α/β has been the focus of numerous investigations, we will limit our discussion of the mucosal adjuvant activity to IL-1α/β. There are two isoforms of IL-1, IL-1α and IL1β, and both bind the IL-1 receptor, IL-1R1 [22]. Both hematopoietic and stromal cells express IL-1α that exists in membrane-bound and soluble forms, which is released by damaged cells [41,42]. IL-1β expression is induced in cells primed by stimuli, such as DAMPs. Monocytes are the primary cell source of IL-1β [43,44]. Activation of the IL-1R1 by either IL-1α or IL-1β occurs through the TIR domain and recruits MyD88 to complete IL-1 signaling [45]. IL-1 enhances the survival and effector function of innate immune cells, including neutrophils, and activates ILC3s [22]. In addition to innate immune responses, IL-1 enhances cytokine

responses in CD41 T cells, including IFN-γ, IL4, and IL-17, and expands populations of in vitro differentiated Th1, Th2, and Th17 cells in vivo [46]. IL-1 is a potent mucosal vaccine adjuvant that enhances antigen-specific and protective immune responses. Nasal vaccination adjuvanted with IL-1α or IL-1β in C57BL/6 or BALB/c mice induced antigen-specific serum IgG and lymphocyte proliferative responses similar to those induced by CT [47]. Others have reported that mice nasally immunized with recombinant influenza virus hemagglutinin (HA) adjuvanted with IL-1α or IL-1β displayed 80% and 100% survival, respectively, after a lethal influenza virus challenge, while only 20% survival was observed in mice vaccinated with antigen alone [40]. The mucosal adjuvant activity of IL-1 has also been confirmed in rabbit models of nasal immunization. Rabbits provide a unique preclinical model to evaluate the safety and efficacy of nasal vaccine adjuvants, since the volume of the rabbit nasal cavity is similar to that of humans [48]. Rabbits immunized intranasally with IL-1β and tetanus toxoid (TT) developed measurable toxoid-specific serum IgG [49], although antibody responses induced by nasal immunization were lower than those induced by intramuscular immunization. IL-1α provided effective nasal adjuvant activity when coadministered with recombinant anthraxprotective antigen in rabbits; however, nasal immunization was less effective than intramuscular immunization for induction of anthrax lethal toxin-neutralizing antibody responses [49]. We believe that optimization of vaccine formulations [50,51] and delivery methods [49,52] is required to enhance delivery and retention of the vaccine in the upper respiratory tract to maximize the immunogenicity of nasally delivered vaccines. Immunization of the oral mucosa with IL-1adjuvanted vaccines has also been evaluated in rabbits. Delivery of IL-1 combined with

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inactivated Streptococcus sobrinus to the palatine tonsils in rabbits significantly enhanced S. sobrinus-specific IgA responses in the saliva and IgG responses in the plasma [53]. Thus, IL-1 is a potent mucosal adjuvant active by two mucosal routes (intranasal and oral) in at least two animal species (mice and rabbits). Despite the effective adjuvant activity of recombinant cytokines, including IL-1, the high cost to produce recombinant cytokines may slow their development for use in humans. The mechanism of adjuvant activity of IL-1α has been investigated by using intranasal immunization. Since nasal immunization likely results in the vaccine formulation predominantly contacting the nasal epithelial cells, one possible mechanism of action for nasally administered adjuvants could be adjuvantdependent activation of the nasal epithelium to produce proinflammatory cytokines that subsequently activate APCs to enhance the induction of an adaptive immune response. In fact, nasal delivery of IL-1α increased IL-6, G-CSF, and KC cytokines in the nasal secretions of immunized mice 6 hours after delivery [54]. Additionally, the production of proinflammatory cytokines was observed in the serum of mice 6 hours after nasal immunization of IL-1α [54]. While the IL-1α-induced production of proinflammatory cytokines after nasal immunization correlated with adjuvant activity, it was not clear what cells were responsible for the production of the proinflammatory cytokines after nasal immunization. The study used genetically engineered mice that lack the IL-1R1 and wildtype mice to generate bone marrow chimeric animals. Bone marrow chimeric mice were utilized to determine whether IL-1R1 signaling through stromal cells only or hematopoietic cells only was sufficient to provide adjuvant activity after nasal delivery of IL-1α [11]. While IL-1α exhibited nasal adjuvant activity in both groups of chimeric mice (IL-1R12/2 - WT and WT -IL-1R12/2), IL-1R1 was required on bone-marrow-derived cells to observe maximal

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adjuvant activity. However, IL-1R1 on stromal cells was required for the maximal IL-1α adjuvant-induced cytokine production, suggesting that adjuvant activation of stromal cells was sufficient to initiate host innate immune responses that provided adjuvant activity, but was not required for the nasal adjuvant activity of IL-1α [11]. Additional studies determined that CD11c1 cells must respond to IL-1 signaling for maximal adjuvant activity after nasal delivery of IL-1α [11]. The results from these studies demonstrate that both stromal cells and hematopoietic cells are sufficient to mediate the nasal adjuvant activity of IL-1α, while CD11c1 APCs must be able to be directly activated by the adjuvant (e.g., IL-1α) for maximal adjuvant activity to be observed.

IV. NANOEMULSIONS AS MUCOSAL VACCINE ADJUVANTS Nanoemulsions represent another category of innate immune-activating adjuvants for use with mucosally administered vaccines. Historically, nanoemulsions were produced by diffusing oil with water and surfactants to create particulates with a diameter that can range from 10 to 500 nm [55,56]. Many oils, including corn, soybean, olive, peanut, and castor, can be used to create nanoemulsions [57]. Common surfactants used to create nanoemulsions include polyethoxylated glycerides, poloxamers, sodium dodecyl sulfate, and polysaccharides [56,57]. Although nanoemulsions are valuable drug delivery tools, they can also be used as immunemodulating adjuvants for mucosal vaccines. W805EC is a nanoemulsion formulation with nasal adjuvant activity that contains Tween 80, ethanol, cetylpyridinium chloride, soybean oil, and water [23]. Nasal immunization with W805EC and an inactivated influenza virus enhanced the induction of antigen-specific serum IgG and IgA and mucosal IgA in the nasal washes of immunized mice [58].

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Increased serum HA-inhibiting antibodies decreased viral loads in the lungs and nasal cavity, and a reduction in morbidity after a lethal influenza challenge were observed in mice nasally immunized with a W805ECadjuvanted vaccine when compared to control animals immunized with antigen formulated in phosphate-buffered saline [58]. Nanoemulsions have also demonstrated mucosal adjuvant activity in animal species other than mice, including guinea pigs and ferrets. Nasally administered nanoemulsionadjuvanted influenza vaccines provided sterilizing immunity against challenge with influenza A/Wisconsin/67/2005 (H3N2) in ferrets [59]. Interestingly, nanoemulsion-adjuvanted influenza virus vaccines induced antibody responses that cross-reacted with other strains of influenza not present in the vaccine, possibly owing to the nanoemulsion adjuvant influencing the epitope specificity of the vaccine-induced antibodies [60]. Guinea pigs nasally immunized with hepatitis B antigens combined with nanoemulsions developed elevated antigen-specific serum IgG [61]. Similarly, mice and guinea pigs were used to determine the adjuvant activity of nanoemulsions when coadministered with recombinant protective antigen (rPA) [62]. In mice, nasal immunization with nanoemulsion-adjuvanted rPA induced serum anti-rPA IgG responses that were significantly greater than anti-rPA IgG induced by rPA adjuvanted with monophosphoryl lipid A (MPL) or CpG oligodeoxynucleotide [62]. Guinea pigs nasally immunized with nanoemulsion-adjuvanted rPA developed increased serum rPA-specific IgG, anthrax lethal toxin-neutralizing antibodies and were protected against a lethal anthrax infection [62]. Clinical trials have evaluated the safety and immunogenicity of the nanoemulsion adjuvant W805EC after nasal administration with seasonal influenza virus vaccines. Research subjects received either 4 or 10 μg of antigen alone or combined with 5%, 10%, 15%, or 20% of the nanoemulsion adjuvant. There were no

adjuvant-induced adverse events with the exception of minor acute throat irritation in subjects who received the 10 μg antigen 1 10% adjuvant formulation [63]. While the intranasally immunized subjects did not develop serum antiinfluenza antibody responses comparable to those induced by intramuscular immunization, W805EC provided effective nasal adjuvant activity based on its ability to induce serum antiinfluenza IgG responses that were significantly higher than those produced by nasal vaccines containing the influenza antigen in the absence of added adjuvant [63]. It is important to consider that the development of effective nasal vaccines for influenza may require the use of higher antigen doses [6] (vs intramuscular vaccination) or alternative vaccine formulations [50,51] to maximize the induction of serum antiinfluenza IgG responses. Alternatively, nasally administered influenza virus vaccines may be more appropriately evaluated on the basis of their ability to induce mucosal anti-influenza IgA responses [6 9]. Nanoemulsions mediate their adjuvant activity as a result of effective activation of the innate immune system. Since epithelial cells are the first cell source to encounter nanoemulsion adjuvants after mucosal delivery, their response to the nanoemulsions may initiate the proinflammatory cascade, resulting in adjuvant activity. In human epithelial cells, the nanoemulsion W805EC induces cell death by several mechanisms, including apoptosis, necrosis and pyroptosis, and increased production of caspase enzymes, including 1, 3/7, 6, 8, and 9 [24]. The release of caspase enzymes from epithelial cells may initiate a cascade, resulting in enhanced proinflammatory cytokine production that activates the innate immune system to provide adjuvant activity. For example, caspase-1 cleaves pro-IL-1β, which leads to IL-1β activation and cellular secretion [64]. Nasal delivery of nanoemulsions enhanced inflammatory cytokine production in local tissues, including IL-1, IL-5, IL-6, IP-10, TGF-β, and TSLP [14]. Epithelial cells

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engulf antigen in vitro and in vivo in the presence of nanoemulsions [65] and increase MHC II molecule expression [14]. After the nanoemulsions increase antigen uptake and apoptosis in epithelial cells, dendritic cells (DCs) then consume the apoptotic epithelial cells and increase expression of costimulatory molecules [17], which may further increase T cell activation. Mucosal delivery of nanoemulsions also increase DCs ability to traffic antigens to lymph nodes [14], where they may activate T cells to enhance adaptive immune responses. The importance of toll-like receptor (TLR) signaling in the adjuvant activity of W805EC was investigated by using mice deficient in TLR2, TLR4, and MyD88. Although MyD88, TLR2, and TLR4 were not required for W805ECinduced antigen-specific serum antibody responses, T cell responses induced by the W805EC adjuvant were altered in the absence of MyD88, TLR2, and TLR4 [23]. Nasal delivery of W805EC-adjuvanted rPA in MyD882/2, TLR22/2, or TLR42/2 mice induced lower levels of IFN-γ and higher levels of IL-5 and IL-13 compared to WT mice [23]. It is possible that W805EC utilizes TLRs and MyD88 to enhance APC maturation, since human DCs increase MHC II, CD80, and CD86 expression in response to the nanoemulsion, W805EC [23]. Costimulatory molecules found on APCs, such as CD40, CD80, and CD86, are important for maximal nasal adjuvant activity of W805EC. Nanoemulsions represent a promising mucosal vaccine adjuvant system that warrants continued evaluation in clinical studies because of their capacity to activate immune system via multiple pathways.

V. MAST-CELL-ACTIVATING COMPOUNDS WITH ADJUVANT ACTIVITY Mast cells play an important protective role in the host response to infectious agents [66 69], although they are often recognized

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only for their role in allergic reactions. For example, mast cells regulate lymph node hypertrophy during bacterial infection [69] that likely contributes to the induction of antigenspecific adaptive immunity and resolution of infection. Compound 48/80, a chemical mast cell activator, reproduces infection-induced, mast-cell-dependent lymph node hypertrophy, resulting in increased sequestration of DCs and T lymphocytes in the draining lymph node. Such evidence suggests that mast cell activators exhibit vaccine adjuvant qualities. In this section, we will briefly discuss mast-cell-activating compounds that exhibit vaccine adjuvant activity (see Chapter 13: Mast Cells for the Control of Mucosal Immunity) and their proposed mode of action when delivered by mucosal routes.

A. Compound 48/80 Compound 48/80 (C48/80) is a chemical compound that contains several polymers of pmethoxy-N-methyl phenylethylamine that can activate mast cells to degranulate [70,71] and secrete histamine [70]. Mucosal vaccine studies have demonstrated that C48/80 provided nasal adjuvant activity when coadministered with anthrax rPA and was as effective as CT for the induction of antigen-specific serum IgG and mucosal IgA [72]. Nasal immunization with poxvirus protein B5R combined with C48/80 provided enhanced survival (50%) against a lethal vaccinia virus infection, while mice immunized with B5R alone did not survive [15]. Similarly, nasal delivery of a C48/80-adjuvanted influenza virus vaccine protected 90% of mice from a lethal influenza challenge, while mice immunized with influenza alone did not survive [72]. Pediatric populations often have an immature immune system, and potent vaccine adjuvants may be required to enhance immunity in children. C48/80 induced significant antigenspecific serum IgG responses in young mice when coadministered with a pneumococcal

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vaccine [73]. As observed in influenza virus models, C48/80-adjuvanted vaccines protected against a lethal pneumococcal challenge [73]. The method of formulation of C48/80 in a vaccine (e.g., aqueous formulation, nanoparticles, dry powders, etc.) can also influence its adjuvant activity. Nasal immunization with C48/80 encapsulated into chitosan nanoparticles enhanced antigen retention and antigenspecific IgA responses in the nasal cavity of mice compared to aqueous formulations [74]. C48/80 formulation can also affect host immune responses in rabbits. Although C48/80 effectively enhanced antigen-specific IgG and anthrax toxin-neutralizing antibody responses after nasal immunization of rabbits with rPA prepared as an aqueous solution or a spraydried powder, 75% of rabbits receiving an aqueous formulation of C48/80-adjuvanted vaccines developed significant toxin-neutralizing antibodies, while 100% of animals vaccinated with the powder form developed significant toxinneutralizing antibody responses [75]. A rabbit model of a botulinum neurotoxin type A vaccine demonstrated toxin-neutralizing antibody responses after nasal vaccination with the antigen and C48/80 similar to those induced by nasal immunization with the same antigen and CT [76]. Collectively, these murine and rabbit studies demonstrate the potency of C48/80 when used as a nasal vaccine adjuvant. C48/80’s adjuvant activity requires mast cells for maximal adjuvant activity. In the presence of mast cells, C48/80 induces DC migration to the local draining lymph nodes. In the absence of mast cells, DC trafficking is reduced, and antigen-specific antibody responses induced after nasal immunization with C48/80 are decreased [15]. Additionally, reconstitution of mast-cell-deficient mice with mast cells restored the vaccine-induced antigen-specific antibody response to the level observed in wild-type mice [15]. Mast cell production of tumor necrosis factor (TNF) is required for maximal adjuvant activity induced by C48/80 [15]. Importantly, C48/80 did not exhibit any

direct TLR activation activity in vitro, and MyD88 signaling was not required for the adjuvant activity of C48/80 (supplemental data [15]). Despite the effective adjuvant activity of C48/80, it may not be a desirable adjuvant candidate for development for use in humans, owing to its chemical composition. For example, C48/80 is a “Condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde” (SIGMA, Cat. #C2313) and represents a mixture of polymer species [77]. The use of C48/80 in humans would likely require the identification of the single species that mediates the adjuvant activity (if it exists). The purification of the single active species from C48/80 or the chemical synthesis of a specific adjuvant species would likely be difficult.

B. Other Mast Cell Activators With Mucosal Adjuvant Activity Other compounds that activate mast cells have been reported to exhibit mucosal adjuvant activity. Polymyxins and colistin are clinically utilized polypeptide antibiotics that cause mast cell degranulation and histamine release (an indicator of mast cell activation) and also exhibit effective adjuvant activity after nasal immunization in mice [78]. Melittin is an antimicrobial peptide found in bee venom that activates mast cells [79], and nasal delivery of melittin combined with tetanus toxoid (TT) or diphtheria toxoid enhanced antigen-specific serum antibody responses [80]. Mast cells are also required for maximal adjuvant activity of the IL-1 family cytokine IL-18 [40]. Finally, nasal immunization with HPV antigens combined with CTA1-DD/IgG complexes induced viral neutralizing antibodies and mucosal IgA by a mechanism that involved mast cell activation [81]. CTA1-DD/IgG induces mast cell activation in connective tissue mast cells, but not mucosal mast cells through the FcγRIIIA [81]. While there may be concerns about the use of mast cell activators as vaccine adjuvants, it is

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important to keep in mind that mast cells are important innate immune system cells that contribute to the host-protective response against bacterial infection [66 69]. Additionally, mast cell activators used as vaccine adjuvants are applied locally with antigen; therefore, systemic adverse effects such as anaphylaxis have not been observed [15,75,76]. Finally, clinically used peptide antibiotics such as polymyxin B and colistin are known mast cell activators, yet they are safely used in human medicine while also providing effective nasal adjuvant activity. Additional studies are warranted to better define the mechanism of action of mast cell activator mucosal vaccine adjuvants and to evaluate their safety and efficacy in nonrodent preclinical models and in clinical studies.

VI. CATIONIC POLYETHYLENEIMINE USED AS A MUCOSAL VACCINE ADJUVANT Polyethylenimine (PEI) is a cationic polymer (linear or branched) that has a long history of use as a nucleic acid delivery agent [82 84] that also exhibits mucosal adjuvant activity. PEI was previously used as DNA delivery agent for an intranasally administered DNA vaccine [85], and was recently used as a nasal vaccine adjuvant when delivered with viral glycoprotein antigens [16]. Both linear and branched forms of PEI exhibited nasal adjuvant activity, while branched forms of PEI (both 750 and 25 kD) coadministered with HIV-1 gp140 induced antigen-specific vaginal IgA in an amount 10fold greater than that induced by CTB [16]. PEI (B25) used as a nasal vaccine adjuvant-induced higher avidity antigen-specific IgG than did CT, CTB, or CpG [16]. PEI (B25) also provided effective nasal adjuvant activity when coadministered with influenza virus HA or HSV-2 glycoprotein D, demonstrating that PEI provides effective nasal adjuvant activity with a variety of different viral glycoprotein antigens [16]. The mucosal adjuvant activity of PEI, when

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delivered nasal or sublingually, has been confirmed by other investigators [86 88]. When used as a nasal vaccine adjuvant, PEI combines with antigen to form nanocomplexes that enhance antigen uptake both in vitro and in vivo, although PEI did not directly activate APC or TLR reporter cells [16]. Because PEI exhibits dose-dependent cellular cytotoxicity [89,90], it was evaluated for its ability to induce cell death in vitro and DNA release in vivo, since cell death and release of DNA were recently described as contributing to the adjuvant activity of alum [91]. PEI causes DNA release in vivo, and nasal adjuvant activity of PEI, but not CT, was reduced by treatment with DNase, suggesting that the adjuvant activity of PEI is related to its ability to cause cell damage and DNA release in vivo [16]. Mice deficient in interferon regulatory factor 3 (IRF3) exhibit a significantly reduced antigen-specific IgG1 after nasal immunization with antigen combined with PEI (but not CT), demonstrating that IRF3 is required for maximal adjuvant activity of PEI [16]. Since PEI also exhibits mast cell degranulation activity with characteristics similar to the mast cell degranulation activity of C48/80 [92,93], it would be interesting to determine whether the mucosal adjuvant activity of PEI involves mast cell activation. The cationic nature of PEI may also contribute to its adjuvant activity, owing to its ability to enhance antigen binding to epithelial cells and antigen uptake by DC [87]. Additional safety and efficacy studies are needed to evaluate PEI as a next-generation mucosal adjuvant to determine whether it is suitable for use in humans.

VII. MUCOSAL ADJUVANT ACTIVITY OF TOLL-LIKE RECEPTOR LIGANDS TLR ligands are microbial components (or synthetic analogs) that are included in the group of molecules referred to as pathogen-associated molecular patterns (PAMPs) that activate the

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innate immune system by binding to their specific receptor on host cells. TLR ligands provide adjuvant activity to enhance adaptive immunity to coadministered antigens [94 96]. While a variety of TLR ligands, including LPS and MPL (TLR4), CpG (TLR9), rintatolimod and poly I:C (TLR3), flagellin (TLR5), Pam3CSK4 (TLR1/2), FSL-1 (TLR2/6), 3M-019 (TLR7), and Resiquimod/R848 (TLR7/8), have demonstrated mucosal adjuvant activity [8,54,97 100], we will limit our discussion to MPL and CpG. These TLR ligands are used in FDA-approved injected vaccines [54], and therefore are likely to be the TLR ligands closest to use for mucosal vaccination in humans.

A. CpG Mucosal Adjuvant and Modulator Activity CpG oligodeoxynucleotides exhibit mucosal adjuvant activity that enhances antigen-specific Th1 cell-associated responses. Nasal immunization with CpG increased Th1 cell-associated antigen-specific serum IgG2c (C57BL/6) and IgG2a (BALB/c) in mice [25]. Increased antigenspecific lymphocyte IFN-γ responses were also observed in mice nasally vaccinated with CpGadjuvanted vaccines [54]. The Th1 cell-skewing activity observed after nasal vaccination with CpG required plasmacytoid DCs (pDCs), since in their absence, IgG2a responses were reduced [101]. An increase in mucosal antibody responses was also observed in lung, nasal lavages, and feces of CpG-immunized BALB/c and C57BL/6 mice [25]. Nasal immunization of mice with a CpG-adjuvanted pertussis vaccine enhanced antigen-specific serum antibody responses and reduced lung bacterial burden after challenge with Bordetella pertussis compared to immunization with antigen alone [102]. Combined oral delivery of CpG with allergen as an immunotherapy also has been shown to diminish allergic disease. Mouse models of peanut (PN) allergen immunotherapy show reduced PN-induced hypothermia and allergy

clinical symptoms after oral PN immunotherapy formulations containing PN proteins and CpG encapsulated in nanoparticles [103]. CpG also modulates host immune responses after mucosal delivery to human subjects. For example, increased IL-1β, IL-6, and IL-8 were observed in the nasal lavage fluids 24 hours after nasal CpG delivery, and increased IL-12p70 was observed 48 hours after delivery [26]. Mucosal delivery of CpG-adjuvanted vaccines can provide measurable protection against both infectious and allergic diseases. The adjuvant activity of CpG involves activation of components of both the innate and adaptive immune systems. B cells activated by CpG increase cellular proliferation and CD40, CD80, and MHC II expression as well as their ability to stimulate T cells [27]. Mouse pDCs respond to CpG by increasing expression of the costimulatory and antigen-presenting molecules, CD40, CD80, and MHC II, which lead to enhanced T cell proliferation in a coculture that contains CpG-adjuvanted pDCs pulsed with antigen [25]. CpG stimulation of human pDCs increase IFNα, TNF-α, and IL-6 [25]. Human PBMCs cultured in the presence of CpG increase IFN-α and IFN-γ production [27]. Similarly, mouse bone marrow cells increase IFN-α, IFN-γ, IL-12p40/ p70, and IL-5 after CpG stimulation [25]. The increased costimulatory molecules and cytokines modulate naı¨ve T cells to obtain a regulatory phenotype that suppress T cell responses [27] or a Th1 phenotype, which aids in increasing IgG2a antibodies and CTL responses in mice [104]. TLR9 and pDCs are required for CpG to provide maximal nasal adjuvant activity [25]. MyD88, an adapter molecule downstream of TLR9 signaling, is also required for maximal CpG adjuvant activity [105].

B. Monophosphoryl Lipid A Mucosal Adjuvant and Modulator Activity MPL is a TLR4 ligand derived from Gramnegative bacteria lipopolysaccharide that is

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approved for use in human vaccines [106], and has been evaluated as a mucosal vaccine adjuvant in preclinical and clinical studies. The mucosal adjuvant activity of MPL was demonstrated in mice after nasal immunization using three different antigens: influenza HA, TT, and hepatitis B surface antigen (HBsAg) [107]. Doses of MPL ranging from 10 to 25 μg enhanced antigen-specific serum IgG1 and IgG2a and mucosal IgA responses after nasal immunization to all three antigens in separate vaccine experiments. Mice nasally vaccinated with HA 1 MPL exhibited a 100% survival rate compared to the 0% survival in mockvaccinated animals [107]. However, when compared to CT, CpG, and alum in an influenza virus-like particle (VLP) vaccine formulation, MPL induced significantly lower serum IgG responses compared to CpG, alum (10 μg), and CT [108]. However, mice nasally immunized with influenza VLP and MPL developed serum viral neutralization activity similar to that of mice immunized with a high dose of alum (60 μg). MPL used as a nasal adjuvant activity in rabbits provided protective immunity against an aerosol anthrax spore challenge [109,110]. Therefore, studies from mouse and rabbit models suggest that MPL effectively enhances antigen-specific immune responses and can induce protective immunity against viral and bacterial challenges. Nasal immunization of mice with a Mycobacterium tuberculosis (Mtb) lipopeptide vaccine combined with MPL enhanced the induction of protective immunity when compared to immunization with lipopeptide alone [111]. By comparison, nasal immunization with Mtb lipopeptides combined with TLR3 or TLR7/8 agonists was associated with reduced protection when compared to immunization with Mtb lipopeptides alone [111], demonstrating that some adjuvants may not enhance protective immune responses even if they augment antigen-specific adaptive immune responses. The use of MPL to enhance the induction of protective immunity against Mtb was associated with a low level of

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myeloid-derived suppressor cells (MDSCs), while TLR3 and TLR7/8 ligands (which did not provide protective immunity) increased MDSCs [111]. The impact of vaccine adjuvants on MDSCs should be closely evaluated during preclinical and clinical evaluation of mucosal vaccine adjuvants [112,113]. Clinical studies have evaluated the safety and immunogenicity of MPL-adjuvanted, nasally delivered norovirus VLP vaccines. Nasal delivery of a dry powder formulation containing norovirus VLPs adjuvanted with MPL and chitosan, as a mucoadhesive, did not induce local adverse symptoms different from those seen with the placebo control [114]. A dose-dependent increase in antigen-secreting cells and antigen-specific antibodies was observed in subjects immunized with MPLadjuvanted vaccines, but not in subjects who received adjuvants in the absence of antigen [114]. Seroconversion was obtained in 38.9% of subjects receiving a low-dose vaccine and 73.7% of those receiving the high-dose vaccine 1 month after vaccination [114]. The safety and preliminary immunogenicity observed with nasal delivery of MPL to humans with the norovirus VLP suggest that MPL is an attractive nasal adjuvant candidate for additional clinical testing. A TLR4 ligand, CRX-675, has also been evaluated as an immune modulator when delivered by the nasal route in humans [115]. CRX-675 was safe when administered by the nasal route to subjects with seasonal allergic rhinitis, and it improved symptom scores at a 100 μg dose [115]. The adjuvant activity of MPL may be associated with its ability to activate the innate immune system. MPL induces human monocytes to produce TNF-α, IL-10, and IL-12p40 in a manner that is dependent on TLR2 and TLR4 ligation [28]. Similarly, MPL can stimulate APCs (e.g., B cells, DCs, and macrophages) to enhance T cell proliferation [29]. APCs pulsed with antigen and stimulated with MPL enhanced antibody production by plasma cells. Antigen-pulsed macrophages stimulated with

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MPL enhanced IgG2a antibodies, and B cells pulsed with antigen and stimulated with MPL led to increased IgG1 and IgG2a antibodies compared to B cells with antigen alone. MPL stimulation of DCs leads to enhanced IFN-γ and reduced IL-4 and IL-5 compared to DCs without MPL stimulation, demonstrating the ability of MPL to enhance Th1-type immunity while reducing Th2 cell responses. Macrophages and B cells stimulated with MPL increased IFN-γ and IL-5 compared to unstimulated cells [29]. However, the Th cell-skewing activity of MPL may be dose-dependent, as observed with LPS [116].

VIII. CONCLUDING REMARKS AND FUTURE PERSPECTIVE Mucosal immunization represents an attractive, needle-free method of vaccination that allows for expanded vaccine coverage and enhances the induction of antigen-specific mucosal immune responses. Innate immunemodulating adjuvants will likely be needed to maximize the immunogenicity of mucosally administered vaccines, especially vaccines that utilize highly purified subunit immunogens. Various mucosal vaccine adjuvant candidates have been described in the literature that utilize a variety of mechanisms of action to activate the innate immune system, including bacterial enterotoxins, recombinant proinflammatory cytokines, mast cell activators, nanoemulsions, and TLR ligands. Results from both preclinical and clinical studies suggest that the mucosal adjuvants and immune-modulators described in this chapter provide adjuvant activity in the absence of adverse events. Since several different adjuvants provide adjuvant activity while inducing local cell death and tissue damage, the role of adjuvant-induced cytotoxicity in mucosal adjuvant activity requires additional research. A careful comparative evaluation of adjuvant candidates is warranted in preclinical vaccine testing, since effective adjuvant activity

based on enhancement of antigen-specific adaptive immune responses does not always translate to induction of enhanced protective immunity against the pathogen of interest. Adjuvants may also differentially influence the induction of MDSCs and modulate protective immunity. Evaluation of adjuvant combinations is also warranted, since combination adjuvants may provide superior adjuvant activity than individual adjuvants. With many innate immune-modulating vaccine adjuvant candidates available and more being described on a regular basis, we are optimistic that safe and effective mucosally administered subunit vaccines can be developed for human use in the near future.

References [1] Patlak, M. Vaccines: essential weapons in the fight against disease breakthroughs in bioscience [Online], p. 15. ,https://www.faseb.org/Portals/2/PDFs/opa/2015/ 10.23.15%20FASEB-BreakthroughsInBioscience-Vaccines %20-WEB.pdf.; 2015 [accessed 8.15.17]. [2] Aggerbeck H, Gizurarson S, Wantzin J, Heron I. Intranasal booster vaccination against diphtheria and tetanus in man. Vaccine 1997;15(3):307 16. [3] Tuckerman JL, Shrestha L, Collins JE, Marshall HS. Understanding motivators and barriers of hospitalbased obstetric and pediatric health care worker influenza vaccination programs in Australia. Hum Vaccin Immunother 2016;12(7):1749 56. [4] Levine MM. “IDEAL” vaccines for resource poor settings. Vaccine 2011;29(Suppl. 4):D116 25. [5] Lycke N. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 2012;12(8):592 605. [6] Atmar RL, Keitel WA, Cate TR, Munoz FM, Ruben F, Couch RB. A dose-response evaluation of inactivated influenza vaccine given intranasally and intramuscularly to healthy young adults. Vaccine 2007;25(29):5367. [7] Hoft DF, Lottenbach KR, Blazevic A, Turan A, Blevins TP, Pacatte TP, et al. Comparisons of the humoral and cellular immune responses induced by live attenuated influenza vaccine and inactivated influenza vaccine in adults. Clin Vaccine Immunol 2017;24(1):e00414-16. [8] Overton ET, Goepfert PA, Cunningham P, Carter WA, Horvath J, Young D, et al. Intranasal seasonal influenza vaccine and a TLR-3 agonist, rintatolimod, induced cross-reactive IgA antibody formation against avian

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

H5N1 and H7N9 influenza HA in humans. Vaccine 2014;32(42):5490 5. [9] Morokutti A, Muster T, Ferko B. Intranasal vaccination with a replication-deficient influenza virus induces heterosubtypic neutralising mucosal IgA antibodies in humans. Vaccine 2014;32(17):1897 900. [10] Van Maele L, Fougeron D, Janot L, Didierlaurent A, Cayet D, Tabareau J, et al. Airway structural cells regulate TLR5-mediated mucosal adjuvant activity. Mucosal Immunol 2014;7(3):489 500. [11] Thompson AL, Johnson BT, Sempowski GD, Gunn MD, Hou B, Defranco AL, et al. Maximal adjuvant activity of nasally delivered IL-1alpha requires adjuvant-responsive CD11c 1 cells and does not correlate with adjuvant-induced in vivo cytokine production. J Immunol 2012;188(6):2834 46. [12] Rezende RM, Weiner HL. History and mechanisms of oral tolerance. Semin Immunol 2017;30:3 11. [13] Pabst O, da Cunha AP, Weiner H. Mechanisms of orla tolerance to soluble P. In: 4th ed Mestecky J, Strober W, Russel M, Cheroute H, Lambrecht B, Kelsall B, editors. Mucosal Immunology, vol. 1. Boston, MA: Academic Press of Elsevier; 2015. p. 831 48. [14] Makidon PE, Belyakov IM, Blanco LP, Janczak KW, Landers J, Bielinska AU, et al. Nanoemulsion mucosal adjuvant uniquely activates cytokine production by nasal ciliated epithelium and induces dendritic cell trafficking. Eur J Immunol 2012;42(8):2073 86. [15] McLachlan JB, Shelburne CP, Hart JP, Pizzo SV, Goyal R, Brooking-Dixon R, et al. Mast cell activators: a new class of highly effective vaccine adjuvants. Nat Med 2008;14(5):536 41. [16] Wegmann F, Gartlan KH, Harandi AM, Brinckmann SA, Coccia M, Hillson WR, et al. Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens. Nat Biotechnol 2012;30(9):883 8. [17] Myc A, Kukowska-Latallo JF, Smith DM, Passmore C, Pham T, Wong P, et al. Nanoemulsion nasal adjuvant W (8)(0)5EC induces dendritic cell engulfment of antigenprimed epithelial cells. Vaccine 2013;31(7):1072 9. [18] Tovey MG, Lallemand C, Meritet JF, Maury C. Adjuvant activity of interferon alpha: mechanism(s) of action. Vaccine 2006;24(Suppl. 2):S2 46-7. [19] Rizza P, Capone I, Moretti F, Proietti E, Belardelli F. IFN-alpha as a vaccine adjuvant: recent insights into the mechanisms and perspectives for its clinical use. Expert Rev Vaccines 2011;10(4):487 98. [20] Luft T, Pang KC, Thomas E, Hertzog P, Hart DN, Trapani J, et al. IFNs enhance the terminal differentiation of dendritic cells. J Immunol 1998;161(4):1947 53. [21] Paquette RL, Hsu NC, Kiertscher SM, Park AN, Tran L, Roth MD, et al. Interferon-alpha and granulocytemacrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigenpresenting cells. J Leukoc Biol 1998;64(3):358 67.

179

[22] Garlanda C, Dinarello CA, Mantovani A. The interleukin-1 family: back to the future. Immunity 2013;39(6):1003 18. [23] Bielinska AU, Makidon PE, Janczak KW, Blanco LP, Swanson B, Smith DM, et al. Distinct pathways of humoral and cellular immunity induced with the mucosal administration of a nanoemulsion adjuvant. J Immunol 2014;192(6):2722 33. [24] Orzechowska BU, Kukowska-Latallo JF, Coulter AD, Szabo Z, Gamian A, Myc A. Nanoemulsion-based mucosal adjuvant induces apoptosis in human epithelial cells. Vaccine 2015;33(19):2289 96. [25] Maeyama J-i, Takatsuka H, Suzuki F, Kubota A, Horiguchi S, Komiya T, et al. A palindromic CpGcontaining phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated TH1 immunity. PLoS One 2014;9(2): e88846. [26] Mansson A, Bachar O, Adner M, Cardell LO. Nasal CpG oligodeoxynucleotide administration induces a local inflammatory response in nonallergic individuals. Allergy 2009;64(9):1292 300. [27] Moseman EA, Liang X, Dawson AJ, PanoskaltsisMortari A, Krieg AM, Liu YJ, et al. Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4 1 CD25 1 regulatory T cells. J Immunol 2004;173(7):4433 42. [28] Martin M, Michalek SM, Katz J. Role of innate immune factors in the adjuvant activity of monophosphoryl lipid A. Infect Immun 2003;71(5):2498 507. [29] De Becker G, Moulin V, Pajak B, Bruck C, Francotte M, Thiriart C, et al. The adjuvant monophosphoryl lipid A increases the function of antigen-presenting cells. Int Immunol 2000;12(6):807 15. [30] Noah TL, Henderson FW, Wortman IA, Devlin RB, Handy J, Koren HS, et al. Nasal cytokine production in viral acute upper respiratory infection of childhood. J Infect Dis 1995;171(3):584 92. [31] Laham FR, Trott AA, Bennett BL, Kozinetz CA, Jewell AM, Garofalo RP, et al. LDH concentration in nasalwash fluid as a biochemical predictor of bronchiolitis severity. Pediatrics 2010;125(2):e225 33. [32] Spangler BD. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev 1992;56(4):622 47. [33] Agren LC, Ekman L, Lowenadler B, Lycke NY. Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit. J Immunol 1997;158(8): 3936 46. [34] Le Page C, Genin P, Baines MG, Hiscott J. Interferon activation and innate immunity. Rev Immunogenet 2000;2(3):374 86. [35] Couch RB, Atmar RL, Cate TR, Quarles JM, Keitel WA, Arden NH, et al. Contrasting effects of type I interferon

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

180

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

10. INNATE IMMUNITY-BASED MUCOSAL MODULATORS AND ADJUVANTS

as a mucosal adjuvant for influenza vaccine in mice and humans. Vaccine 2009;27(39):5344 8. Bracci L, Canini I, Puzelli S, Sestili P, Venditti M, Spada M, et al. Type I IFN is a powerful mucosal adjuvant for a selective intranasal vaccination against influenza virus in mice and affects antigen capture at mucosal level. Vaccine 2005;23(23):2994 3004. Samo TC, Greenberg SB, Couch RB, Quarles J, Johnson PE, Hook S, et al. Efficacy and tolerance of intranasally applied recombinant leukocyte a interferon in normal volunteers. J Infect Dis 1983;148(3):535 42. Ives NJ, Suciu S, Eggermont AMM, Kirkwood J, Lorigan P, Markovic SN, et al. Adjuvant interferonalpha for the treatment of high-risk melanoma: an individual patient data meta-analysis. Eur J Cancer 2017; 82:171 83 (Oxford, England: 1990). Baumert TF, Juhling F, Ono A, Hoshida Y. Hepatitis Crelated hepatocellular carcinoma in the era of new generation antivirals. BMC Med 2017;15(1):52. Kayamuro H, Yoshioka Y, Abe Y, Arita S, Katayama K, Nomura T, et al. Interleukin-1 family cytokines as mucosal vaccine adjuvants for induction of protective immunity against influenza virus. J Virol 2010;84(24): 12703 12. Di Paolo NC, Shayakhmetov DM. Interleukin 1[alpha] and the inflammatory process. Nat Immunol 2016;17(8): 906 13. Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 2009;27:519 50. Allen JN, Herzyk DJ, Allen ED, Wewers MD. Human whole blood interleukin-1-beta production: kinetics, cell source, and comparison with TNF-alpha. J Lab Clin Med 1992;119(5):538 46. Hsi ED, Remick DG. Monocytes are the major producers of interleukin-1 beta in an ex vivo model of local cytokine production. J Interferon Cytokine Res 1995;15 (1):89 94. Dinarello CA. Interleukin-1 in the pathogenesis and treatment of inflammatory diseases. Blood 2011;117 (14):3720 32. Ben-Sasson SZ, Hu-Li J, Quiel J, Cauchetaux S, Ratner M, Shapira I, et al. IL-1 acts directly on CD4T cells to enhance their antigen-driven expansion and differentiation. Proc. Natl Acad Sci 2009;106(17):7119 24. Staats HF, Ennis Jr. FA. IL-1 is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens. J Immunol 1999;162(10):6141 7. Casteleyn C, Broos AM, Simoens P, Van den Broeck W. NALT (nasal cavity-associated lymphoid tissue) in the rabbit. Vet Immunol Immunopathol 2010;133(24):212 18.

[49] Gwinn WM, Kirwan SM, Wang SH, Ashcraft KA, Sparks NL, Doil CR, et al. Effective induction of protective systemic immunity with nasally administered vaccines adjuvanted with IL-1. Vaccine 2010;28(42): 6901 14. [50] Nordone SK, Peacock JW, Kirwan SM, Staats HF. Capric acid and hydroxypropylmethylcellulose increase the immunogenicity of nasally administered peptide vaccines. AIDS Res Hum Retroviruses 2006;22 (6):558 68. [51] Saito S, Ainai A, Suzuki T, Harada N, Ami Y, Yuki Y, et al. The effect of mucoadhesive excipient on the nasal retention time of and the antibody responses induced by an intranasal influenza vaccine. Vaccine 2016;34 (9):1201 7. [52] Bryant ML, Brown P, Gurevich N, McDougall IR. Comparison of the clearance of radiolabelled nose drops and nasal spray as mucosally delivered vaccine. Nucl Med Commun 1999;20(2):171 4. [53] Kokuryo S, Inoue H, Fukuizumi T, Tsujisawa T, Tominaga K, Fukuda J. Evaluation of interleukin 1 as a mucosal adjuvant in immunization with Streptococcus sobrinus cells by tonsillar application in rabbits. Oral Microbiol Immunol 2002;17(3):163 71. [54] Gwinn WM, Johnson BT, Kirwan SM, Sobel AE, Abraham SN, Gunn MD, et al. A comparison of nontoxin vaccine adjuvants for their ability to enhance the immunogenicity of nasally-administered anthrax recombinant protective antigen. Vaccine 2013;31(11):1480 9. [55] Mundada V, Patel M, Sawant K. Submicron emulsions and their applications in oral delivery. Crit Rev Ther Drug Carrier Syst 2016;33(3):265 308. [56] Singh Y, Meher JG, Raval K, Khan FA, Chaurasia M, Jain NK, et al. Nanoemulsion: concepts, development and applications in drug delivery. J Control Release 2017;252:28 49. [57] Cerpnjak K, Zvonar A, Gasperlin M, Vrecer F. Lipidbased systems as a promising approach for enhancing the bioavailability of poorly water-soluble drugs. Acta Pharm 2013;63(4):427 45. [58] Das SC, Hatta M, Wilker PR, Myc A, Hamouda T, Neumann G, et al. Nanoemulsion W805EC improves immune responses upon intranasal delivery of an inactivated pandemic H1N1 influenza vaccine. Vaccine 2012;30(48):6871 7. [59] Hamouda T, Sutcliffe JA, Ciotti S, Baker Jr. JR. Intranasal immunization of ferrets with commercial trivalent influenza vaccines formulated in a nanoemulsion-based adjuvant. Clin Vaccine Immunol 2011;18(7):1167 75. [60] Kallewaard NL, Corti D, Collins PJ, Neu U, McAuliffe JM, Benjamin E, et al. Structure and function analysis of an antibody recognizing all influenza A subtypes. Cell 2016;166(3):596 608.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

181

REFERENCES

[61] Makidon PE, Bielinska AU, Nigavekar SS, Janczak KW, Knowlton J, Scott AJ, et al. Pre-clinical evaluation of a novel nanoemulsion-based hepatitis B mucosal vaccine. PLoS One 2008;3(8):e2954. [62] Bielinska AU, Janczak KW, Landers JJ, Makidon P, Sower LE, Peterson JW, et al. Mucosal immunization with a novel nanoemulsion-based recombinant anthrax protective antigen vaccine protects against Bacillus anthracis spore challenge. Infect Immun 2007;75(8): 4020 9. [63] Stanberry LR, Simon JK, Johnson C, Robinson PL, Morry J, Flack MR, et al. Safety and immunogenicity of a novel nanoemulsion mucosal adjuvant W805EC combined with approved seasonal influenza antigens. Vaccine 2012;30(2):307 16. [64] Man SM, Kanneganti T-D. Converging roles of caspases in inflammasome activation, cell death and innate immunity. Nat Rev Immunol 2016;16(1):7 21. [65] Wong PT, Wang SH, Ciotti S, Makidon PE, Smith DM, Fan Y. Formulation and characterization of nanoemulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immunogenicity. Mol Pharm 2014;11(2):531 44. [66] Malaviya R, Ikeda T, Ross E, Abraham SN. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature 1996;381(6577):77 80. [67] McLachlan JB, Abraham SN. Studies of the multifaceted mast cell response to bacteria. Curr Opin Microbiol 2001;4(3):260 6. [68] Malaviya R, Abraham SN. Mast cell modulation of immune responses to bacteria. Immunol Rev 2001;179: 16 24. [69] McLachlan JB, Hart JP, Pizzo SV, Shelburne CP, Staats HF, Gunn MD, et al. Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection. Nat Immunol 2003;4(12):1199 205. [70] Mousli M, Bueb JL, Bronner C, Rouot B, Landry Y. G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides. Trends Pharmacol Sci 1990;11(9):358 62. [71] Paton WD. Compound 48/80: a potent histamine liberator. Br J Pharmacol 1951;6(3):499 508. [72] Meng S, Liu Z, Xu L, Li L, Mei S, Bao L, et al. Intranasal immunization with recombinant HA and mast cell activator C48/80 elicits protective immunity against 2009 pandemic H1N1 influenza in mice. PLoS One 2011;6(5):e19863. [73] Zeng L, Liu Y, Wang H, Liao P, Song Z, Gao S, et al. Compound 48/80 acts as a potent mucosal adjuvant for vaccination against Streptococcus pneumoniae infection in young mice. Vaccine 2015;33(8):1008 16. [74] Bento D, Staats HF, Goncalves T, Borges O. Development of a novel adjuvanted nasal vaccine: C48/80 associated with chitosan nanoparticles as a

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

path to enhance mucosal immunity. Eur J Pharm Biopharm 2015;93:149 64. Wang SH, Kirwan SM, Abraham SN, Staats HF, Hickey AJ. Stable dry powder formulation for nasal delivery of anthrax vaccine. J Pharm Sci 2012;101(1):31 47. Staats HF, Fielhauer JR, Thompson AL, Tripp AA, Sobel AE, Maddaloni M, et al. Mucosal targeting of a BoNT/A subunit vaccine adjuvanted with a mast cell activator enhances induction of BoNT/A neutralizing antibodies in rabbits. PLoS One 2011;6(1):e16532. Koibuchi Y, Ichikawa A, Nakagawa M, Tomita K. Binding of active components of compound 48/80 to rat peritoneal mast cells. Eur J Pharmacol 1985;115 (2-3):171 7. Yoshino N, Endo M, Kanno H, Matsukawa N, Tsutsumi R, Takeshita R, et al. Polymyxins as novel and safe mucosal adjuvants to induce humoral immune responses in mice. PLoS One 2013;8(4):e61643. Nishikawa H, Kitani S. Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A2-induced degranulation in mast cells. Toxicol Appl Pharmacol 2011;252(3):228 36. Bramwell VW, Somavarapu S, Outschoorn I, Alpar HO. Adjuvant action of melittin following intranasal immunisation with tetanus and diphtheria toxoids. J Drug Target 2003;11(8-10):525 30. Fang Y, Zhang T, Lidell L, Xu X, Lycke N, Xiang Z. The immune complex CTA1-DD/IgG adjuvant specifically targets connective tissue mast cells through FcgammaRIIIA and augments anti-HPV immunity after nasal immunization. Mucosal Immunol 2013;6 (6):1168 78. Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine) and its role in gene delivery. J Control Release 1999;60 (2 3):149 60. Hobel S, Aigner A. Polyethylenimines for siRNA and miRNA delivery in vivo. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2013;5(5):484 501. Pandey AP, Sawant KK. Polyethylenimine: a versatile, multifunctional non-viral vector for nucleic acid delivery. Mater Sci Eng C Mater Biol Appl 2016;68:904 18. Huang X, Xu J, Qiu C, Ren L, Liu L, Wan Y, et al. Mucosal priming with PEI/DNA complex and systemic boosting with recombinant TianTan vaccinia stimulate vigorous mucosal and systemic immune responses. Vaccine 2007;25(14):2620 9. Klein K, Mann JF, Rogers P, Shattock RJ. Polymeric penetration enhancers promote humoral immune responses to mucosal vaccines. J Control Release 2014;183:43 50. Qin T, Yin Y, Huang L, Yu Q, Yang Q. H9N2 influenza whole inactivated virus combined with polyethyleneimine strongly enhances mucosal and systemic immunity after intranasal immunization in mice. Clin Vaccine Immunol 2015;22(4):421 9.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

182

10. INNATE IMMUNITY-BASED MUCOSAL MODULATORS AND ADJUVANTS

[88] Song L, Xiong D, Song H, Wu L, Zhang M, Kang X, et al. Mucosal and systemic immune responses to influenza H7N9 antigen HA1-2 Co-delivered intranasally with flagellin or polyethyleneimine in mice and chickens. Front Immunol 2017;8:326. [89] Monnery BD, Wright M, Cavill R, Hoogenboom R, Shaunak S, Steinke JH, et al. Cytotoxicity of polycations: relationship of molecular weight and the hydrolytic theory of the mechanism of toxicity. Int J Pharm 2017;521(1-2):249 58. [90] Hunter AC. Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity. Adv Drug Deliv Rev 2006;58(14):1523 31. [91] Marichal T, Ohata K, Bedoret D, Mesnil C, Sabatel C, Kobiyama K, et al. DNA released from dying host cells mediates aluminum adjuvant activity. Nat Med 2011;17(8):996 1002. [92] Suzuki-Nishimura T, Oku N, Nango M, Uchida MK. PEI6, a new basic secretagogue in rat peritoneal mast cells: characteristics of polyethylenimine PEI6 resemble those of compound 48/80. Gen Pharmacol 1995;26 (6):1171 8. [93] Yoshino Y, Nagaya K, Sekino H, Uchida MK, SuzukiNishimura T. Comparison of histamine release induced by synthetic polycations with that by compound 48/80 from rat mast cells. Jpn J Pharmacol 1990;52(3):387 95. [94] Bergmann-Leitner ES, Leitner WW. Adjuvants in the driver’s seat: how magnitude, type, fine specificity and longevity of immune responses are driven by distinct classes of immune potentiators. Vaccines 2014;2 (2):252 96. [95] Odendall C, Kagan JC. Activation and pathogenic manipulation of the sensors of the innate immune system. Microbes Infect 2017;19(4-5):229 37. [96] Reed SG, Hsu FC, Carter D, Orr MT. The science of vaccine adjuvants: advances in TLR4 ligand adjuvants. Curr Opin Immunol 2016;41:85 90. [97] Nelson CS, Pollara J, Kunz EL, Jeffries Jr TL, Duffy R, Beck C, et al. Combined HIV-1 envelope systemic and mucosal immunization of lactating rhesus monkeys induces robust IgA-isotype B cell response in breast milk. J Virol 2016;90(10):4951 65. [98] Fouda GG, Amos JD, Wilks AB, Pollara J, Ray CA, Chand A, et al. Mucosal immunization of lactating female rhesus monkeys with a transmitted/founder HIV-1 envelope induces strong Env-specific IgA antibody responses in breast milk. J Virol 2013;87(12): 6986 99. [99] Honko AN, Sriranganathan N, Lees CJ, Mizel SB. Flagellin is an effective adjuvant for immunization against lethal respiratory challenge with Yersinia pestis. Infect Immun 2006;74(2):1113 20.

[100] Buffa V, Klein K, Fischetti L, Shattock RJ. Evaluation of TLR agonists as potential mucosal adjuvants for HIV gp140 and tetanus toxoid in mice. PLoS One 2012;7(12):e50529. [101] Iho S, Maeyama J, Suzuki F. CpG oligodeoxynucleotides as mucosal adjuvants. Hum Vaccin Immunother 2015;11(3):755 60. [102] Asokanathan C, Corbel M, Xing D. A CpG-containing oligodeoxynucleotide adjuvant for acellular pertussis vaccine improves the protective response against Bordetella pertussis. Hum Vaccin Immunother 2013;9 (2):325 31. [103] Srivastava KD, Siefert A, Fahmy TM, Caplan MJ, Li XM, Sampson HA. Investigation of peanut oral immunotherapy with CpG/peanut nanoparticles in a murine model of peanut allergy. J Allergy Clin Immunol 2016;138(2):536 43 e4. [104] Bode C, Zhao G, Steinhagen F, Kinjo T, Klinman DM. CpG DNA as a vaccine adjuvant. Expert Rev Vaccines 2011;10(4):499 511. [105] Mosaheb MM, Reiser ML, Wetzler LM. Toll-like receptor ligand-based vaccine adjuvants require intact MyD88 signaling in antigen-presenting cells for germinal center formation and antibody production. Front Immunol 2017;8:225. [106] Lawrence S, Wilby OK, Willoughby CR, Veenstra S, Deschamps M. Evaluation of the intramuscular administration of Cervarix vaccine on fertility; preand post-natal development in rats. Reprod Toxicol 2010;. [107] Baldridge JR, Yorgensen Y, Ward JR, Ulrich JT. Monophosphoryl lipid A enhances mucosal and systemic immunity to vaccine antigens following intranasal administration. Vaccine 2000;18(22):2416 25. [108] Quan FS, Ko EJ, Kwon YM, Joo KH, Compans RW, Kang SM. Mucosal adjuvants for influenza virus-like particle vaccine. Viral Immunol 2013;26(6):385 95. [109] Wimer-Mackin S, Hinchcliffe M, Petrie CR, Warwood SJ, Tino WT, Williams MS, et al. An intranasal vaccine targeting both the Bacillus anthracis toxin and bacterium provides protection against aerosol spore challenge in rabbits. Vaccine 2006;24(18):3953 63. [110] Klas SD, Petrie CR, Warwood SJ, Williams MS, Olds CL, Stenz JP, et al. A single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge. Vaccine 2008;26(43):5494 502. [111] Gupta N, Vedi S, Kunimoto DY, Agrawal B, Kumar R. Novel lipopeptides of ESAT-6 induce strong protective immunity against Mycobacterium tuberculosis: routes of immunization and TLR agonists critically impact vaccine’s efficacy. Vaccine 2016;34(46):5677 88. [112] Sui Y, Hogg A, Wang Y, Frey B, Yu H, Xia Z, et al. Vaccine-induced myeloid cell population dampens

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REFERENCES

protective immunity to SIV. J Clin Invest 2014;124(6): 2538 49. [113] Permar SR, Staats HF. Which comes first: the antigen or the adjuvant? J Clin Invest 2014;1 2. [114] El-Kamary SS, Pasetti MF, Mendelman PM, Frey SE, Bernstein DI, Treanor JJ, et al. Adjuvanted intranasal Norwalk virus-like particle vaccine elicits antibodies and antibody-secreting cells that express homing receptors for mucosal and peripheral lymphoid tissues. J Infect Dis 2010;202(11):1649 58.

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[115] Casale TB, Kessler J, Romero FA. Safety of the intranasal toll-like receptor 4 agonist CRX-675 in allergic rhinitis. Ann Allergy Asthma Immunol 2006;97(4):454 6. [116] Eisenbarth SC, Piggott DA, Huleatt JW, Visintin I, Herrick CA, Bottomly K. Lipopolysaccharideenhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 2002;196(12):1645 51.

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Toxin-Based Modulators for Regulation of Mucosal Immune Responses Eunsoo Kim, Zayed Attia, Estelle Cormet-Boyaka and Prosper N. Boyaka Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, United States

I. INTRODUCTION Most toxins used for modulation of immune responses are AB-type toxins and consist of a binding B subunit and an enzymatically active A subunit (Fig. 11.1). A large majority of toxins and toxin derivatives used to modulate immune responses to vaccines are of microbial origin. While plant toxins can also display immunomodulatory effects, they have been the objects of only a limited number of studies.

II. TOXINS USED FOR MODULATION OF IMMUNE RESPONSES Early work by Elson showed that unlike many protein antigens, the bacterial enterotoxin cholera toxin (CT) is highly immunogenic when administered by the mucosal route [1]. Further studies showed that CT and the related heatlabile toxin I (LT-I or LT) I from Escherichia coli

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00011-0

are effective adjuvants that promote mucosal immunity and enhance systemic immune responses to coadministered antigens [2 4]. The LT from E. coli is closely related to CT, and the two enterotoxins share 80% amino acid sequence homology [5]. These molecules are AB5 toxins consisting of two structurally and functionally separate A and B subunits (Fig. 11.1) [6 8]. The B subunit of CT (CTB) consists of five identical 11.6-kD peptides that bind to GM1 ganglioside, whereas the B subunit of LT-I (LTB) is more promiscuous and binds GM1 as well as asialo GM1 and GM2 [9,10]. After binding of the B subunit to epithelial cell GM1 or GM2 receptors, the A subunit reaches the cytosol and, after activation, binds to nicotinamide adenosyl diphosphate and catalyzes ADP-ribosylation of Gsα protein [11]. The latter GTP-binding protein activates adenylyl cyclase (commonly known as adenyl cyclase) with subsequent elevation of cAMP in epithelial cells followed by secretion of water and chloride ions into the intestinal lumen [12]. Knowledge about CT interaction with mammalian cells

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continues to increase, and a new receptor for CTB has been recently reported [13]. Thus the use a metabolically incorporated photocrosslinking sugar, helped reveal that CTB binds to a fucosylated glycoprotein and that it is the fucosylated structure, not the protein, that promotes the entry of CTB into epithelial cells [13]. Although CT and LT were identified as very effective mucosal adjuvants for oral, nasal, rectal, or sublingual vaccines, the toxicity of these molecules has precluded their use in human vaccines. One of the main strategies undertaken to make these molecules more suitable for use in humans consisted of developing mutants that lack the ADP ribosyltransferase activity of the native toxins. Other approaches include substitution of the B subunit by a B-cell-targeting moiety, the covalent binding of protein antigens to CTB or LTB, and expression of toxin subunits or toxin derivatives in plants (Chapter 7: Induction and Regulation of Mucosal Memory B Cell Responses and Chapter 20: Plant-based Mucosal Vaccine Delivery Systems). Bacillus anthracis edema factor (EF) is a class II adenylyl cyclase [14]. It is one component of the tripartite anthrax exotoxin, which comprises the binding subunit protective antigen (PA) and the enzymatically active EF and lethal factor of anthrax (LF) [15]. The PA subunit targets cells via the anthrax toxin receptor 1 (ATR1), which resembles the tumor endothelial marker 8 (TEM8) [16], and the related ATR2, which is similar to the capillary morphogenesis gene 2 (CMG2) [17]. After the 83-kD PA (PA83) binds to the ATR1/TEM8 or ATR2/CMG2, a protease furin cleaves a 20-kD peptide (PA20) [18], which allows the formation of a PA63 oligomer ring in the host cell membrane [19]. This PA63 ring serves to anchor the PA-binding domain of EF [20] and allows internalization of EF and its release into the cytoplasm. Once there, following binding to calmodulin, EF will exert its adenylyl cyclase activity and elevate intracellular cAMP levels [21]. The combination of B. anthracis PA and EF produces edema toxin, an AB-type toxin that induces edema.

The CyaA toxin from Bordetella pertussis (the agent of whooping cough) [22] is another class II adenylyl cyclase that has been tested in mucosal vaccines. The catalytic domain of CyaA shares similarity with that of B. anthracis EF, but CyaA binds to the cells via an integrin binding located in its C-terminus portion [23]. CyaA invades a variety of cell types through a mechanism involving receptor-independent binding to the cell surface. In addition to this nonspecific interaction, CyaA binding to, and intoxication of myeloid cells involves a high-affinity receptor identified as the αMβ2 integrin or macrophage-1 antigen (Mac-1; CD11b/CD18) [24]. T cells do not express Mac-1, and CyaA binds to these cells via the αLβ2 integrin LFA-1 (CD11a/CD18) [25]. The ExoY from Pseudomonas aeruginosa (the agent of nosocomial infections), the Yp ACT-A/ B from Yersinia pestis (the agent of plague), and the Yp ACT from Yersinia pseudotuberculis (responsible for gastrointestinal symptoms) are other class II adenylyl cyclases. However, to our knowledge, these toxins have not been tested as adjuvants for mucosal vaccines. Shiga toxins are AB5 toxins produced by Shigella dysenteriae and the shigatoxigenic serotypes of E. coli (STEC). The pentameric B subunit binds to globotriaosylceramides (Gb3 or CD77) on the host cells and allows the internalization of the enzymatic A subunit [26]. Within the cells, the A1 subunit binds to the ribosomes and disrupts protein synthesis by cleaving an adenine from the 28S RNA of the 60S subunit of the ribosomes and thus depurinates ribosomes. Two types of Shiga toxins (Stx-1 and Stx-2) are produced by E. coli strains. The E. coli Stx-1 is identical to the Stx of Shigella spp. and shares 56% sequence identity with Stx-2 [27]. In vivo studies in mice have revealed that Stx-2 is up to 400 times more toxic than Stx-1 [28,29]. The ribotoxic stress response induced by Stx in infected cells consists of proinflammatory and proapoptotic responses [30,31]. Ricin is a potent toxin found in the beans of Ricinus communis. This molecule is an AB-type toxin that impairs ribosomal function [30,31]. This ribosome-inactivating protein consists of two

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subunits covalently linked by a single disulfide bond. The B subunit of ricin (RTB) binds to terminal galactose residues on cell surface. The A subunit (RTA) is an N-glycosyl hydrolase that removes an adenine from the 28S RNA of the 60S subunit of the ribosome and thus, depurinates the ribosomes. Ingestion of ricin causes severe nausea, vomiting, diarrhea, and hemorrhage, and its inhalation results in cough and fever. The fact that Stx and ricin are targeted to the endosome after receptor-mediated endocytosis has been exploited to deliver peptide antigens for induction of CD81 cytotoxic T cells (CTLs). However, as was described above for

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CT and LT, safer derivatives will need to be developed to circumvent the adverse effects of these toxins.

III. TOXIN-DERIVATIVE ADJUVANTS FOR MUCOSAL VACCINES A. ADP-Ribosylation of Defective Mutants of Cholera Toxin and Labile Toxin Mutant toxins defective in ADP ribosyltransferase activity were generated by

FIGURE 11.1 Structure of toxins used for modulation of immune responses. The biochemical structure of native toxins and functional characteristics of the binding B subunits and the catalytic A subunits. Toxin derivatives are generated by genetic modification of the catalytic A subunit or coupling (genetic or chemical) of either the A or the B subunit with antigen(s) or molecule(s) targeting a specific receptor on host cells.

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single amino acid substitutions in the ADP-ribosylation activity site of the A subunit of CT or LT, or in the protease-sensitive loop of LT. CT mutants were constructed by substitution of serine by phenylalanine at position 61 (CT-S61F) or glutamate by lysine at position 112 (CT-E112K) in the ADP ribosyltransferase activity center of the CT gene from Vibrio cholerae 01 strain GP14 [32]. Similar substitutions in LT have been shown to completely inactivate the ADP ribosyltransferase activity and enterotoxicity [33,34]. The antigen-specific serum IgG and secretory IgA (SIgA) antibodies induced by the mutant CT after nasal immunization were comparable to those induced by wild-type holotoxin and significantly higher than those induced by CTB [32,35]. Further, like CT, the mutant CT-E112K induces Th2 responses through a preferential inhibition of Th1 CD41 T cells. Both wild-type CT and enzymatically inactive CT mutants enhanced the expression of costimulatory molecules of the B7 family and their corresponding receptors [36,37]. Mutations at other sites of the CT molecule were reported to induce nontoxic derivatives, but the adjuvant activity was also affected. For example, the CT-106S mutant CT with a partial knockout of the ADPribosylating activity exhibited an adjuvant activity lower than that of wild-type holotoxin [38]. Mutant LT molecules either with residual ADP ribosyltransferase activity (e.g., LT-72R) or totally devoid of such enzymatic activity (e.g., LT-R72 and LT-K63) can function as mucosal adjuvants when nasally administered to mice together with unrelated antigens [39,40]. When mutants of LT were tested as mucosal adjuvants, they generally induced mucosal and systemic antibody (Ab) responses comparable to those of the wild-type LT, although higher doses of LT mutants were often needed [41]. A double mutant of CT (dmCT) with two amino acid substitutions in the ADP ribosyltransferase active center (E112K) and in the COOH-terminal KDEL (E112K/KDEV or E112K/KDGL) retained mucosal adjuvant

activity [42]. Thus when nasally coadminstered with an antigen, this dmCT enhanced antigenspecific IgA Ab responses in nasal washes, fecal extracts, and saliva [42]. Similar to the double mutant of CT, a double mutant of LT-I [LT (R192G/L211A)] was developed by introducing two amino acid substitutions in its A subunit [43]. This dmLT adjuvant did not induce water secretion after oral delivery and enhanced immunity to vaccine antigens coadministered by the oral, nasal, epicutaneous, and sublingual routes [43 46]. In addition to the induction of Th2-type immune response, the adjuvant activity of CT was shown to promote Th17 responses [47,48]. The dmLT [LT(R192G/ L211A)] and a dmCT prepared by introducing the same mutations in CT were both found also to induce Th17 responses despite exhibiting only negligible cAMP-promoting activity [49].

B. Other Cholera Toxin and Labile Toxin Derivatives It has been hypothesized that the strong toxic effect of CT and LT could be largely due to their promiscuous binding to cells via their B subunits. This assumption led to the construction of a fusion protein consisting of CTA1 and two Ig-binding domains (DD) of staphylococcal protein A that binds IgG, IgE, IgA, and IgM and thus primarily targets B lymphocytes [50]. The CTA1-DD fusion protein displayed adjuvant activity when given by the nasal route and promoted both mucosal and systemic antibody responses [50]. These Ab responses were associated with mixed Th1 and Th17 responses [51,52]. It also has been established that the adjuvant activity of CTA1-DD promotes both T-cell-dependent and T-cell-independent responses and requires both the ADPribosylation and Ig-binding activities [53,54]. It was reported that a mutant of the holotoxin LK lacking binding capacity on gangliosides (LT-E112K/G33D) retains adjuvant activity and

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enhances immunity to nasally coadministered antigens [55]. The controversial notion that targeting of LTA to specific cellular receptors was not a requirement for the adjuvanticity of this toxin was further supported by a report by the same group that nasal immunization with LTA stimulates serum and mucosal antibody responses toward coadministered influenza subunit antigens and that these responses were of the same magnitude and profile as promoted by immunization with the native holotoxin or the mLT-E112K as adjuvant [56]. These intriguing findings could somewhat be explained by the recent observation that the A2 domain of LTA has cell-penetrating capabilities [57]. More specifically, it was reported that proteins fused to LTA2 that penetrate the cell membrane are internalized through membrane transport pathways and localized in the endoplasmic reticulum [57]. The same study showed that the LTA2-mediated entry of coupled protein involved the clathrin-mediated endocytosis and the macropinocytosis pathways. Type II heat-labile enterotoxins (LT-II) represent another group of adenylate cyclases produced by enteric bacteria [58,59]. Like CT and LT, these toxins are oligomeric AB-type proteins made of a catalytic A subunit with ADP ribosyltransferase activity, which is noncovalently bound to a pentameric B subunit. However, LT-II toxins bind to different cell surface gangliosides than LT: LT-IIa binds to GD1b, GD1a, and GM1; LT-IIb binds to GD1a, GT1b, GM2, and GM3; and LT-IIc binds to GM1, GM2, GM3, and GD1a [9,60]. A previous report showed that when used as adjuvant for nasal vaccines, LT-IIb promotes both systemic and mucosal immunity and balanced Th1/ Th2 immune responses [61]. More recent studies have shown that like other ADP ribosyltransferase toxins, LT-IIb as mucosal adjuvant also promotes Th17 responses [62]. Single amino acid substitution in the B subunit of LT-IIb [LT-IIb (T13I)] reduced the affinity for GD1a gangliosides and

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the toxicity of this toxin for mammalian cells [63,64]. Nonetheless, LT-IIb (T13I) retains mucosal adjuvant activity for nasally coadministered antigens [60,61]. Similar to LT-IIb (T13I), mutants of LT-IIa and LT-IIc were developed, and despite altered binding to ganglioside receptors, some of them retain mucosal adjuvant activity [59].

C. Derivatives of Other Toxin Adjuvant Several approaches have been developed over the last three decades to take advantage of adenylate cyclase for beneficial regulation of immune responses. The combination of anthrax PA plus the 1 254 N-terminal fragment of LF (LFn1 254) or EF (EFn1 254) has been used as a “molecular syringe” to introduce foreign antigens into target cells. This strategy successfully delivered short peptides [65,66] as well as proteins [66,67] coupled to LFn1 254, and this formulation was able to promote CTL responses. Mechanistic studies with PA mutants attenuated in self-assembly or translocation [66,68] showed that delivery of antigens involves the same toxin self-assembly and translocation steps that occurred during intoxication. This delivery system primed CTL responses in both BALB/c (H 2d) and C57BL/6 (H 2b) mice [65]. In addition, injection of mice with dendritic cells treated in vitro with an LFn1 254 fusion protein and PA effectively primed CTL responses [69]. Studies with the proteasome inhibitor lactacystin have shown that the LFn1 254 and PA-mediated delivery of proteins switches their processing from the cytosolic degradative pathway into the classical processing pathway for presentation by MHC class I molecules [66,70]. While the cellular delivery of LFn-coupled antigens optimally occurred in the presence of a functional PA, LFn fusion proteins were able to enter the classical MHC class I degradation pathway and prime CTL responses in the absence of PA [70,71].

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This PA-independent LFn delivery of antigens could be abrogated in the presence of the phagocytosis inhibitors cytochalasin B or brefeldin A, which inhibit exocytosis of proteins [70,72]. Finally, fluorescent antigens delivered in the absence of PA colocalized with proteosomes, which degrade proteins into peptides capable of entering the classical MHC class I pathway [71]. The observation that coinjection of anthrax EF enhances the magnitude of serum anti-PA IgG responses was the first evidence of the adjuvant activity of EdTx [73]. Nasal coapplication of B. anthracis PA together with a mutant of B. anthracis EF with reduced adenylate cyclase activity (EF-S414N) enhanced anti-PA Ab responses but also displayed mucosal adjuvant activity for coadministered unrelated antigens [74]. The adjuvant activity of this mutant as well as that of the native EF promote mixed Th1, Th2 and Th17 responses [75]. Furthermore, this EdTx adjuvant did not target CNS tissues or induce IL-1 mRNA responses in the olfactory bulb epithelium [74]. Studies addressing the relative contribution of the binding and catalytic subunits of EdTx revealed that binding of EF to PA oligomers was required for its adjuvanticity following nasal administration. Coadministration of EF with PA-dFF, a PA mutant that allows the cellular uptake of EF but fails to efficiently mediate its translocation into the cytosol, enhanced serum Ab responses, but these responses were lower than those measured after immunization with native PA. These studies also showed that immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. Thus a fully functional PA and a minimum level of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant [76]. Similar to the anthrax toxin derivative, B. pertussis has been engineered to deliver molecules coupled to its N-terminal catalytic domain into the cytosol of eukaryotic cells. The recombinant B. pertussis adenylate cyclase

could codeliver CD81 T cell and CD41 T cell epitopes efficiently and induce CTL responses [72]. The molecular syringe activity of B. pertussis adenylate cyclase required the full-length invasive molecule but did not depend upon its adenylate cyclase activity [77]. An enzymatically inactive mutant of B. pertussis Cya toxin was generated by insertion of a Leu-Gln dipeptide within the ATP-binding site [78]. Intranasal immunization with the mutant Cya enhanced serum IgG and IgA antibody responses to the coadministered antigen. Interestingly, the adjuvant activity of the mutant Cya for antibody and T cell responses was superior to that of the native toxin [79]. Furthermore, mutant Cya induced IgA responses in mucosal secretion and, when coadministered with B. pertussis pertactin as a vaccine antigen, protected mice against nasal challenge with B. pertussis [79]. It is worth noting that the adenylate cyclase toxin of B. anthracis was shown to be a potent inducer of Th17 cells [80].

D. Delivery Systems for Toxin-Based Adjuvant Toxin derivatives often display adjuvant activity and promote the desired mucosal B and T cell responses after nasal immunization. However, it is generally accepted that the reduced levels of catalytic activity of these molecules and their reduced stability in the harsh environment of the gastrointestinal tract limit their adjuvant activity for oral vaccines. To circumvent these limitations, toxin derivatives have been produced or incorporated in vaccine vectors for oral immunization. The development of “edible” vaccines has been a major advance in the field of vaccinology. Since tolerance is the natural response to ingested antigens, toxin-based adjuvants were incorporated into plant vaccines. Major progress have been made since the initial report that

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a rice-based oral vaccine expressing CTB induced CTB-specific serum IgG and mucosal IgA antibodies with neutralizing activity [81]. This MucoRice vaccine has been tested in many preclinical animal models where it induced long-standing protection against CT- or LTinduced diarrhea in mice [82], pigs [83], and nonhuman primates [84]. Oral immunization of mice with a MucoRice vaccine expressing a dmCT resulted in anti-CTB serum IgA and SIgA responses that were of the same magnitude as those achieved after oral administration of MucoRice-CTB [85]. Expression of fusion proteins consisting of CTB and irrelevant vaccine antigens in rice was shown to be an effective vaccination approach for promoting immunity against the unrelated antigen. Thus oral immunization of mice with transgenic rice expressing a consensus envelope protein domain III (cEDIII) of dengue virus fused to CTB induced cEDIII-specific serum IgG and mucosal IgA response in feces [86]. Methods for mass production of MucoRice are being developed to exploit the multiple advantages of this platform, including the extended stability (years) at room temperature, formulation without purification of vaccines, and ease of vaccine administration [87]. Probiotic and bacteria generally recognized as safe (GRAS) are increasingly gaining interest as potential live vectors for delivery of vaccines. To increase the immunogenicity of protein antigens expressed at the surface of a Lactobacillus casei, the vector was engineered to express fusion protein of these antigens and CTA1. Oral or nasal immunization of mice with such L. casei expressing CTA1 fused to flu antigens was found to promote both systemic IgG and mucosal IgA responses and to improve protection against challenge with influenza viruses [88,89]. As was described with Lactobacilli vectors expressing CTA1, mice oral immunization with L. casei vector coexpression LTAK63 and LTB together with the fimbrial adhesin FaeG (rLpPG-2-FaeG) exhibited enhanced

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anti-rLpPG-2-FaeG antibody responses and increased protection against challenge with Enterotoxigenic Escherichia coli (ETEC) [90].

IV. INNATE MECHANISMS REGULATED BY TOXIN-BASED ADJUVANTS FOR INDUCTION OF MUCOSAL IMMUNITY SIgA Ab as well as Th cell and CTL responses can be induced by pathogens triggering the organized mucosal inductive sites such as gut-associated lymphoid tissues (GALT) and nasopharyngeal-associated lymphoid tissues (NALT). Effective protection against mucosal pathogens requires prophylactic B and T cell responses that can be achieved by mucosal vaccines. Toxin-based adjuvants target a variety of immune cells to trigger a cascade of innate responses that will ultimately result in the induction of protective B and T cell responses in mucosal tissues and in the general bloodstream (Fig. 11.2). Because of the broad expression of GM1 gangliosides, CT and LT virtually bind to all mammalian cells, including epithelial cells, macrophages, dendritic cells, B cells, CD41 T cells, and CD81 T cells. In contrast, LT-IIa and LT-IIb bind to most dendritic cells and macrophages but only to a limited percentage of CD41 T cells, CD81 T cells, and B cells [91]. Although ATR1 and ATR2 are expressed in virtually all cells, EdTx cannot penetrate polarized epithelial cells via the apical membrane exposed to the lumen [92]. Toxin-based adjuvants that bind to epithelial cells and increase cAMP stimulate IL-1 and IL6 secretion by these cells. CT and CTB also induce TGF-β responses by epithelial cells [93]. Myeloid cell subsets contribute to many signals that support IgA production and mucosal homing of effector B and T cells. B cells can undergo Ig class-switch recombination (CSR) and acquire the ability to produce IgA after CD40 CD40L ligation in the presence of

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TGF-β with contribution from other cytokines, including IL-4, IL-5, IL-6, IL-10, and IL-21 [94 97]. Other costimulatory signals such as B-cell-activating factor of the TNF family (BAFF), a proliferation-inducing ligand (APRIL), retinoic acid (RA), and nitric oxide (NO) also facilitate CSR for the production of IgA [94,98,99]. BAFF and APRIL further enhance IgA responses by providing survival signals and/or inducing plasma cell differentiation and IgA secretion [99,100]. RA, a metabolite of vitamin A, plays an important role in the production of mucosal IgA, since it both acts synergistically with IL-5 and IL-6 to induce IgA secretion [101] and induces expression of gut-homing receptors (i.e., α4β7 or CCR9) on effector B and T cells [101]. CT and toxin-based adjuvants that elevate intracellular cAMP stimulate the expression of MHC class II and costimulatory molecules by macrophages [76,102 104]. These toxins also induce production of innate cytokines that will facilitate differentiation of Th1, Th2, Th17, and Treg cells. For example, CT, a powerful inducer of Th2 cells, suppresses IL-12 production by macrophages and dendritic cells [105] via inhibition of interferon regulatory factor 8 (IRF8) [106]. Despite the close similarity between CT and LT, the latter toxin more effectively induced Th1 responses necessary for generation of CTLs. To address the relative importance of A or B subunits in CD41 Th cell subset responses, chimeras of CT and LT (i.e., CT-A/ LT-B and LT-A/CT-B) were constructed by switching the B subunits of the two adjuvants [107]. Nasal immunization of mice with these chimeras showed that the B subunits of these enterotoxin adjuvants differentially regulated IL-12R expression and subsequent Th1 cell responses [107]. The predominant role played by enterotoxin B subunits for the induction of antigen-specific CD81 T cell was recently confirmed by studies with chimera of LT-IIb and LT-IIc, which showed that the differences in the CD81 T cell responses elicited by native

holotoxins were dictated by their respective B subunits [108]. The ability of the ADP ribosyl transferase and adenylate cyclase adjuvants to promote differentiation of Th17 cells is a consequence of their induction of IL-6, TGF-β, and inflammasome-dependent IL-1β secretion by myeloid cells [49,75,109,110]. It was recently suggested that induction of Th1 by CT can occur in the absence of IL-12p402/2 [48]. It remains unclear whether IL-12p35 dimers or IL-18 compensated for induction of these responses. In general, the binding of the B subunit of toxin adjuvants to their cellular receptors does not stimulate production of inflammatory cytokines and stimulates only modest expression of costimulatory molecules. As we will discuss below, mucosal delivery of antigen linked to the CTB or LTB can induce immunological tolerance. Unlike the B subunit of these enterotoxins, LT-IIa-B5 and LT-IIb-B5 activate human monocytes or mouse macrophages in a TLR2-dependent fashion [111]. LTIIa-B5 enhances production of proinflammatory cytokines and chemokines by dendritic cells and the homing capacity into lymph nodes [112] and displays adjuvant activities in vivo [113]. The stimulatory effects of LT-IIaB5 and LT-IIb-B5 have been shown to result from the fact that upon binding to GD1 gangliosides, these molecules interact with TLR2/ TLR1 and induce NF-κ B activation and cytokine response in a MyD88-dependent fashion [111,113]. Recent studies have provided new clues about the relative contribution of myeloid cell signals to the ability of toxin-based adjuvants to promote mucosal immunity. In this regard, CD81 dendritic cells, which regulate the development of CD81 T cell responses, were found to be dispensable for the adjuvant activity of CT [48]. The same authors showed that the adjuvant activity of CT was completely lost in mice with Gsα-deficient CD11c cells, demonstrating the greater role played by dendritic cells compared to

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IV. INNATE MECHANISMS REGULATED BY TOXIN-BASED ADJUVANTS FOR INDUCTION OF MUCOSAL IMMUNITY

macrophages in the adjuvant activity of CT toward primary responses [48]. Dendritic cells appear to be the main target of other toxinbased adjuvants, since after nasal delivery, antigens coupled to the B subunit of STx are preferentially targeted to those cells (myeloid CD11b dendritic cells, lymph-node-resident CD8α1 dendritic cells, and CD1031 dendritic cells) in mucosal lung-associated mediastinal lymph nodes but not to lung Ly6C1 macrophages expressing CD11c [114]. In vitro, CTBtreated bone-marrow-derived dendritic cells were primed for IgA production by B cells without the help of T cells but required cosignaling by different toll-like receptor (TLR) ligands acting via the MyD88 pathway [115]. Induction of IgA production by CTBtreated DCs could be blocked in vitro or in vivo by RA receptor antagonists, inhibitors of TGF-β signaling, or by neutralizing antiTGF-β antibodies [115]. In these studies, CTB showed no major effect on BAFF, APRIL, or NO production by dendritic cells. CTA1-DD is the only toxin adjuvant that selectively binds to B cells, owing to the presence of an immunoglobulin-binding domain of Staphylococcus aureus protein A [53], and thus allowed us to gain a better understanding of how B cells initiate adaptive immunity. Addition of CTA1-DD to a culture of human peripheral blood mononuclear cells upregulated expression of CD86 and ICAM-1 by B cells [116]. This treatment also stimulated secretion of IL-6, TNF-α, and IL-8 by purified B cells [116]. Neither of these effects was reported after exposure of cells to an enzymatically inactive CTA1-R7K-DD mutant [53,117]. It is worth noting that CTA1-DD was reported to reduce apoptosis of Ag-receptor-activated B cells via production of Bcl2 and to stimulate germinal centers formation [53]. Neutrophils have long been considered minor players in adaptive immunity. This view was initially challenged by the report that the widely used adjuvant alum acts via Gr-11 splenic

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myeloid cells expressing IL-4 to stimulate early B cell priming [118]. It has since been demonstrated that alum stimulates NLRP3 inflammasome and IL-1β secretion to recruit neutrophils and enhance antibody responses [119 121]. Neutrophil depletion in mice was shown to increase CD4 T cells and antibody responses to protein antigens after immunization with alum, indicating that neutrophils compete with antigen-presenting cells for access to antigens [122]. However, splenic neutrophils but not circulating neutrophils were identified as B cell helper neutrophils capable of stimulating marginal zone B cells and promoting T-cellindependent antibody responses through secretion of BAFF, APRIL, and IL-21 [123]. While CT and cAMP-stimulating toxins stimulate NLRP3 inflammasome and IL-1β secretion, no information was available about a potential modulatory role of neutrophils for immune responses to mucosal immunization with toxin adjuvants. While trying to understand why, unlike CT, EdTx as adjuvant failed to induce SIgA responses after immunization via the sublingual route, we found that EdTx recruited a high number of neutrophils in the sublingual tissues and cervical lymph nodes shortly (3 6 hours) after immunization [75]. Depletion of neutrophils before sublingual immunization with EdTx allowed the development of antigen-specific serum and mucosal IgA responses, further confirming that circulating neutrophils recruited in mucosal inductive sites prevented the induction of IgA responses but not IgG responses [75]. These findings suggest that tests used for preclinical evaluation of toxin-based adjuvants for induction of mucosal immunity could include assessment of their ability to recruit neutrophils. In addition to epithelial cells, CT targets intraepithelial lymphocytes (IELs). Oral administration of CT, but not CTB, induces a transient reduction of the number of CD81 T cells in intestinal villi of the jejunum [124,125]. Interestingly, the decrease in intraepithelial CD81 T cells was associated with a reduction

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in the amount of the chemokine RANTES that was produced in intestinal villi [125]. CT binds CD41 T cells but less efficiently than on CD81 T cells. After oral administration of CT, there is an increase in the number of TCRγδ IELs in the ileum [126]. It was recently shown that TCRγδ IELs stimulated with CT express

higher levels of MHC class II molecules, as well as the costimulatory molecules CD80 and CD86 [127]. In addition to these features generally associated with antigen-presenting cells, CT-stimulated TCRγδ IELs produce IL-10 and IL-17 [127] and thus can influence the profile of antigen-specific T helper cells’ response.

FIGURE 11.2 Innate responses regulated by toxin-based adjuvants for induction of mucosal immunity and tolerance. Toxin-based adjuvants target a variety of host cells, including epithelial cells, dendritic cells, macrophages, T lymphocytes, and B lymphocytes. The tropism of toxins or toxin derivatives for a given cell type is dictated by their affinity for receptors expressed on the mammalian cell. Mucosal immunity is induced after exposure to toxins or toxin derivatives, which stimulate production of proinflammatory cytokines and enhance interactions between APC and effector cells via stimulation of the expression of MHC class II costimulatory molecules. Induction of cytokines (IL-10, TGF-β) and factors (RA) that promote expression of mucosal homing receptors and production of mucosal IgA also contribute to development of mucosal immunity. Tolerance results from exposure to toxin derivatives that suppress proinflammatory responses, promote production of IL-10, and facilitate the development of regulatory T cells.

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VI. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

V. INDUCTION OF TOLERANCE BY TOXIN-BASED ADJUVANTS The immunomodulatory effects of toxinbased adjuvants can be used to suppress rather than stimulate immune responses to mucosally coadministered antigens (Fig. 11.2). More specifically, when administered at the right dose and formulation, toxin-based adjuvants can promote Foxp31 Treg, secreting IL-10 and reduce the magnitude of antibody responses against the coadministered antigens. Since toxin-based adjuvants induce more effective and lasting tolerance than mucosal administration of antigens alone, they are being investigated for the treatment of a variety of autoimmune diseases as well as allergy. Studies with CT and LT derivatives have shown that induction of tolerance requires oral, nasal, or sublingual administration of low doses of antigen coupled to the B subunit of the holotoxins or to enzymatically inactive mutants [128 132]. Dendritic cells play an important role in the induction of tolerance to antigens linked to CTB. Thus incubation with CTB antigen fusion protein can inhibit expression of costimulatory molecules (CD80, CD86, and CD40) and secretion of inflammatory cytokines by human dendritic cells while enhancing secretion of the inhibitory cytokine IL-10 [133]. Evidence that B lymphocytes regulate the induction of tolerance was provided by the report that FcγRIIB expression on B cells was required for the development of tolerance following sublingual administration of CTB conjugated to an antigen [134]. Enzymatically inactive mutants of CTA1-DD engineered to coexpress unrelated antigens are being evaluated for induction of therapeutic tolerance and prevention of autoimmune diseases. Nasal administration of such molecules protected mice from collagen-induced arthritis [135]. The tolerance-induced protection was associated with reduced levels of antigen-specific antibodies and Th1 and Th17 responses [135] and could be induced after oral therapy with an

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edible form of this molecule expressed in a plant [136] (Chapter 21: Plant-based Mucosal Immunotherapy).

VI. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Our understanding of the immunomodulatory effects of toxins has greatly improved the last couple of decades. The ability to disassociate their toxicity and their modulatory effects led to the development of safer derivatives that could be incorporated in future human vaccines. For example, a dmLT adjuvant tested for safety and adjuvanticity for an oral vaccine in a double-blind placebo-controlled phase I study was well tolerated and induced mucosal immune responses [45]. The fact that toxin subunits or toxin derivatives expressed in plant, or live recombinant microbial vector can modulate immune responses and promote mucosal immunity will certainly have a significant impact on the development of future prophylactic and therapeutic vaccines. Stability of vaccines throughout the cold chain and the need for trained health care professionals and needles are major points to consider for production and distribution of vaccines. The extensive stability at room temperature of vaccines expressed in rice and the ease of oral administration of rice power will likely boost the development of plant-based vaccines containing different toxin subunits or toxin derivatives as adjuvant. While toxin adjuvants were initially used to increase immune responses, reports that formulations containing toxin subunits or enzymatically inactive derivatives can suppress unwanted immune responses such as autoimmune or allergic responses [137] will likely increase their use for applications unrelated to protection against infectious pathogens. However, a knowledge gap still remains to be filled about host cell response to toxin subunits and derivatives. This point is especially

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important because delivery of these molecules in different mucosal sites targets different immune cells and could result in different outcomes.

Acknowledgments This work was supported by National Institutes of Health grants AI18958 and DK101323.

References

[14]

[15]

[16]

[17]

[1] Elson CO, Ealding W. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J Immunol 1984;132:2736 41. [2] Clements JD, Hartzog NM, Lyon FL. Adjuvant activity of Escherichia coli heat-labile enterotoxin and effect on the induction of oral tolerance in mice to unrelated protein antigens. Vaccine 1988;6:269 77. [3] Elson CO, Ealding W. Cholera toxin feeding did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated protein antigen. J Immunol 1984;133:2892 7. [4] Lycke N, Holmgren J. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. Immunology 1986;59:301 8. [5] Dallas WS, Falkow S. Amino acid sequence homology between cholera toxin and Escherichia coli heat-labile toxin. Nature 1980;288:499 501. [6] Spangler BD. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev 1992;56:622 47. [7] Gill DM. The arrangement of subunits in cholera toxin. Biochemistry 1976;15:1242 8. [8] Gill DM, Clements JD, Robertson DC, Finkelstein RA. Subunit number and arrangement in Escherichia coli heat-labile enterotoxin. Infect Immun 1981;33:677 82. [9] Fukuta S, Magnani JL, Twiddy EM, Holmes RK, Ginsburg V. Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heatlabile enterotoxins LTh-I, LT-IIa, and LT-IIb. Infect Immun 1988;56:1748 53. [10] Holmgren J, Lindblad M, Fredman P, Svennerholm L, Myrvold H. Comparison of receptors for cholera and Escherichia coli enterotoxins in human intestine. Gastroenterology 1985;89:27 35. [11] Gill DM, King CA. The mechanism of action of cholera toxin in pigeon erythrocyte lysates. J Biol Chem 1975;250:6424 32. [12] Field M, Rao MC, Chang EB. Intestinal electrolyte transport and diarrheal disease (1). N Engl J Med 1989;321:800 6. [13] Wands AM, Fujita A, McCombs JE, Cervin J, Dedic B, Rodriguez AC, et al. Fucosylation and protein

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

glycosylation create functional receptors for cholera toxin. eLife 2015;4:e09545. Leppla SH. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci U S A 1982;79:3162 6. Liu S, Moayeri M, Leppla SH. Anthrax lethal and edema toxins in anthrax pathogenesis. Trends Microbiol 2014;22:317 25. Bradley KA, Mogridge J, Mourez M, Collier RJ, Young JA. Identification of the cellular receptor for anthrax toxin. Nature 2001;414:225 9. Scobie HM, Rainey GJ, Bradley KA, Young JA. Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc Natl Acad Sci U S A 2003;100:5170 4. Klimpel KR, Molloy SS, Thomas G, Leppla SH. Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc Natl Acad Sci U S A 1992;89:10277 81. Milne JC, Furlong D, Hanna PC, Wall JS, Collier RJ. Anthrax protective antigen forms oligomers during intoxication of mammalian cells. J Biol Chem 1994;269:20607 12. Elliott JL, Mogridge J, Collier RJ. A quantitative study of the interactions of Bacillus anthracis edema factor and lethal factor with activated protective antigen. Biochemistry 2000;39:6706 13. Drum CL, Yan SZ, Bard J, Shen YQ, Lu D, Soelaiman S, et al. Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin. Nature 2002;415:396 402. Guiso N. Bordetella adenylate cyclase-hemolysin toxins. Toxins (Basel) 2017;9. Available from: http://dx. doi.org/10.3390/toxins9090277. Guo Q, Shen Y, Lee YS, Gibbs CS, Mrksich M, Tang WJ. Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin. EMBO J 2005;24:3190 201. Guermonprez P, Khelef N, Blouin E, Rieu P, RicciardiCastagnoli P, Guiso N, et al. The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18). J Exp Med 2001;193:1035 44. Paccani SR, Finetti F, Davi M, Patrussi L, D’Elios MM, Ladant D, et al. The Bordetella pertussis adenylate cyclase toxin binds to T cells via LFA-1 and induces its disengagement from the immune synapse. J Exp Med 2011;208:1317 30. Sandvig K, van Deurs B. Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. EMBO J 2000;19:5943 50. Melton-Celsa AR. Shiga toxin (Stx) classification, structure, and function. Microbiol Spectr 2014;2 EHEC0024-2013.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[28] Smith MJ, Teel LD, Carvalho HM, Melton-Celsa AR, O’Brien AD. Development of a hybrid Shiga holotoxoid vaccine to elicit heterologous protection against Shiga toxins types 1 and 2. Vaccine 2006;24:4122 9. [29] Tesh VL, Burris JA, Owens JW, Gordon VM, Wadolkowski EA, O’Brien AD, et al. Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice. Infect Immun 1993;61:3392 402. [30] Tesh VL. The induction of apoptosis by Shiga toxins and ricin. Curr Top Microbiol Immunol 2012;357: 137 78. [31] Jandhyala DM, Thorpe CM, Magun B. Ricin and Shiga toxins: effects on host cell signal transduction. Curr Top Microbiol Immunol 2012;357:41 65. [32] Yamamoto S, Takeda Y, Yamamoto M, Kurazono H, Imaoka K, Yamamoto M, et al. Mutants in the ADPribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity. J Exp Med 1997;185:1203 10. [33] Tsuji T, Inoue T, Miyama A, Okamoto K, Honda T, Miwatani T. A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity. J Biol Chem 1990;265:22520 5. [34] Harford S, Dykes CW, Hobden AN, Read MJ, Halliday IJ. Inactivation of the Escherichia coli heat-labile enterotoxin by in vitro mutagenesis of the A-subunit gene. Eur J Biochem 1989;183:311 16. [35] Yamamoto S, Kiyono H, Yamamoto M, Imaoka K, Fujihashi K, Van Ginkel FW, et al. A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity. Proc Natl Acad Sci U S A 1997;94:5267 72. [36] Yamamoto M, Kiyono H, Yamamoto S, Batanero E, Kweon MN, Otake S, et al. Direct effects on antigenpresenting cells and T lymphocytes explain the adjuvanticity of a nontoxic cholera toxin mutant. J Immunol 1999;162:7015 21. [37] Cong Y, Weaver CT, Elson CO. The mucosal adjuvanticity of cholera toxin involves enhancement of costimulatory activity by selective up-regulation of B7.2 expression. J Immunol 1997;159:5301 8. [38] Douce G, Fontana M, Pizza M, Rappuoli R, Dougan G. Intranasal immunogenicity and adjuvanticity of sitedirected mutant derivatives of cholera toxin. Infect Immun 1997;65:2821 8. [39] Douce G, Giuliani MM, Giannelli V, Pizza MG, Rappuoli R, Dougan G. Mucosal immunogenicity of genetically detoxified derivatives of heat labile toxin from Escherichia coli. Vaccine 1998;16:1065 73. [40] Giuliani MM, Del Giudice G, Giannelli V, Dougan G, Douce G, Rappuoli R, et al. Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. J Exp Med 1998;187:1123 32.

197

[41] Rappuoli R, Pizza M, Douce G, Dougan G. Structure and mucosal adjuvanticity of cholera and Escherichia coli heat-labile enterotoxins. Immunol Today 1999;20:493 500. [42] Hagiwara Y, Kawamura YI, Kataoka K, Rahima B, Jackson RJ, Komase K, et al. A second generation of double mutant cholera toxin adjuvants: enhanced immunity without intracellular trafficking. J Immunol 2006;177:3045 54. [43] Norton EB, Lawson LB, Freytag LC, Clements JD. Characterization of a mutant Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral adjuvant. Clin Vaccine Immunol 2011;18:546 51. [44] White JA, Blum JS, Hosken NA, Marshak JO, Duncan L, Zhu C, et al. Serum and mucosal antibody responses to inactivated polio vaccine after sublingual immunization using a thermoresponsive gel delivery system. Hum Vaccin Immunother 2014;10:3611 21. [45] Lundgren A, Bourgeois L, Carlin N, Clements J, Gustafsson B, Hartford M, et al. Safety and immunogenicity of an improved oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine administered alone and together with dmLT adjuvant in a double-blind, randomized, placebo-controlled Phase I study. Vaccine 2014;32:7077 84. [46] Novotny LA, Clements JD, Bakaletz LO. Transcutaneous immunization as preventative and therapeutic regimens to protect against experimental otitis media due to nontypeable Haemophilus influenzae. Mucosal Immunol 2011;4:456 67. [47] Datta SK, Sabet M, Nguyen KP, Valdez PA, GonzalezNavajas JM, Islam S, et al. Mucosal adjuvant activity of cholera toxin requires Th17 cells and protects against inhalation anthrax. Proc Natl Acad Sci U S A 2010;107:10638 43. [48] Mattsson J, Schon K, Ekman L, Fahlen-Yrlid L, Yrlid U, Lycke NY. Cholera toxin adjuvant promotes a balanced Th1/Th2/Th17 response independently of IL-12 and IL-17 by acting on Gsalpha in CD11b(1) DCs. Mucosal Immunol 2015;8:815 27. [49] Larena M, Holmgren J, Lebens M, Terrinoni M, Lundgren A. Cholera toxin, and the related nontoxic adjuvants mmCT and dmLT, promote human Th17 responses via cyclic AMP-protein kinase A and inflammasome-dependent IL-1 signaling. J Immunol 2015;194:3829 39. [50] Agren LC, Ekman L, Lowenadler B, Lycke NY. Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit. J Immunol 1997;158:3936 46. [51] O’Meara CP, Armitage CW, Harvie MC, Timms P, Lycke NY, Beagley KW. Immunization with a MOMPbased vaccine protects mice against a pulmonary Chlamydia challenge and identifies a disconnection

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

198

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

11. TOXIN-BASED MODULATORS FOR REGULATION OF MUCOSAL IMMUNE RESPONSES

between infection and pathology. PLoS One 2013;8: e61962. Nedrud JG, Bagheri N, Schon K, Xin W, Bergroth H, Eliasson DG, et al. Subcomponent vaccine based on CTA1-DD adjuvant with incorporated UreB class II peptides stimulates protective Helicobacter pylori immunity. PLoS One 2013;8:e83321. Agren L, Sverremark E, Ekman L, Schon K, Lowenadler B, Fernandez C, et al. The ADPribosylating CTA1-DD adjuvant enhances T celldependent and independent responses by direct action on B cells involving anti-apoptotic Bcl-2- and germinal center-promoting effects. J Immunol 2000;164:6276 86. Agren LC, Ekman L, Lowenadler B, Nedrud JG, Lycke NY. Adjuvanticity of the cholera toxin A1-based gene fusion protein, CTA1-DD, is critically dependent on the ADP-ribosyltransferase and Ig-binding activity. J Immunol 1999;162:2432 40. de Haan L, Feil IK, Verweij WR, Holtrop M, Hol WG, Agsteribbe E, et al. Mutational analysis of the role of ADP-ribosylation activity and GM1-binding activity in the adjuvant properties of the Escherichia coli heatlabile enterotoxin towards intranasally administered keyhole limpet hemocyanin. Eur J Immunol 1998;28:1243 50. De Haan L, Holtrop M, Verweij WR, Agsteribbe E, Wilschut J. Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the Escherichia coli heat-labile enterotoxin. Immunology 1999;97: 706 13. Liu D, Guo H, Zheng W, Zhang N, Wang T, Wang P, et al. Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin. Appl Microbiol Biotechnol 2016;100:5079 88. van den Akker F, Sarfaty S, Twiddy EM, Connell TD, Holmes RK, Hol WG. Crystal structure of a new heatlabile enterotoxin, LT-IIb. Structure 1996;4:665 78. Hajishengallis G, Connell TD. Type II heat-labile enterotoxins: structure, function, and immunomodulatory properties. Vet Immunol Immunopathol 2013;152: 68 77. Nawar HF, Greene CJ, Lee CH, Mandell LM, Hajishengallis G, Connell TD. LT-IIc, a new member of the type II heat-labile enterotoxin family, exhibits potent immunomodulatory properties that are different from those induced by LT-IIa or LT-IIb. Vaccine 2011;29:721 7. Nawar HF, Arce S, Russell MW, Connell TD. Mucosal adjuvant properties of mutant LT-IIa and LT-IIb enterotoxins that exhibit altered ganglioside-binding activities. Infect Immun 2005;73:1330 42.

[62] Gopal R, Rangel-Moreno J, Fallert Junecko BA, Mallon DJ, Chen K, Pociask DA, et al. Mucosal pre-exposure to Th17-inducing adjuvants exacerbates pathology after influenza infection. Am J Pathol 2014;184:55 63. [63] Connell TD. Cholera toxin, LT-I, LT-IIa and LT-IIb: the critical role of ganglioside binding in immunomodulation by type I and type II heat-labile enterotoxins. Expert Rev Vaccines 2007;6:821 34. [64] Berenson CS, Nawar HF, Yohe HC, Castle SA, Ashline DJ, Reinhold VN, et al. Mammalian cell gangliosidebinding specificities of E. coli enterotoxins LT-IIb and variant LT-IIb(T13I). Glycobiology 2010;20:41 54. [65] Doling AM, Ballard JD, Shen H, Krishna KM, Ahmed R, Collier RJ, et al. Cytotoxic T-lymphocyte epitopes fused to anthrax toxin induce protective antiviral immunity. Infect Immun 1999;67:3290 6. [66] Goletz TJ, Klimpel KR, Arora N, Leppla SH, Keith JM, Berzofsky JA. Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein. Proc Natl Acad Sci U S A 1997;94:12059 64. [67] Lu Y, Friedman R, Kushner N, Doling A, Thomas L, Touzjian N, et al. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity. Proc Natl Acad Sci U S A 2000;97:8027 32. [68] Ballard JD, Collier RJ, Starnbach MN. Anthrax toxinmediated delivery of a cytotoxic T-cell epitope in vivo. Proc Natl Acad Sci U S A 1996;93:12531 4. [69] Moriya O, Matsui M, Osorio M, Miyazawa H, Rice CM, Feinstone SM, et al. Induction of hepatitis C virusspecific cytotoxic T lymphocytes in mice by immunization with dendritic cells treated with an anthrax toxin fusion protein. Vaccine 2001;20:789 96. [70] Cao H, Agrawal D, Kushner N, Touzjian N, Essex M, Lu Y. Delivery of exogenous protein antigens to major histocompatibility complex class I pathway in cytosol. J Infect Dis 2002;185:244 51. [71] Kushner N, Zhang D, Touzjian N, Essex M, Lieberman J, Lu Y. A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen. Proc Natl Acad Sci U S A 2003;100:6652 7. [72] Schlecht G, Loucka J, Najar H, Sebo P, Leclerc C. Antigen targeting to CD11b allows efficient presentation of CD41 and CD8 1 T cell epitopes and in vivo Th1-polarized T cell priming. J Immunol 2004;173:6089 97. [73] Quesnel-Hellmann A, Cleret A, Vidal DR, Tournier JN. Evidence for adjuvanticity of anthrax edema toxin. Vaccine 2006;24:699 702. [74] Duverger A, Jackson RJ, van Ginkel FW, Fischer R, Tafaro A, Leppla SH, et al. Bacillus anthracis edema toxin acts as an adjuvant for mucosal immune

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

199

REFERENCES

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

responses to nasally administered vaccine antigens. J Immunol 2006;176:1776 83. Jee J, Bonnegarde-Bernard A, Duverger A, Iwakura Y, Cormet-Boyaka E, Martin TL, et al. Neutrophils negatively regulate induction of mucosal IgA responses after sublingual immunization. Mucosal Immunol 2015;8:735 45. Duverger A, Carre JM, Jee J, Leppla SH, CormetBoyaka E, Tang WJ, et al. Contributions of edema factor and protective antigen to the induction of protective immunity by Bacillus anthracis edema toxin as an intranasal adjuvant. J Immunol 2010;185:5943 52. Fayolle C, Sebo P, Ladant D, Ullmann A, Leclerc C. In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying viral CD8 1 T cell epitopes. J Immunol 1996;156:4697 706. Ladant D, Glaser P, Ullmann A. Insertional mutagenesis of Bordetella pertussis adenylate cyclase. J Biol Chem 1992;267:2244 50. Orr B, Douce G, Baillie S, Parton R, Coote J. Adjuvant effects of adenylate cyclase toxin of Bordetella pertussis after intranasal immunisation of mice. Vaccine 2007;25:64 71. Paccani SR, Benagiano M, Savino MT, Finetti F, Tonello F, D’Elios MM, et al. The adenylate cyclase toxin of Bacillus anthracis is a potent promoter of T(H) 17 cell development. J Allergy Clin Immunol 2011;127:1635 7. Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, et al. Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Acad Sci U S A 2007;104:10986 91. Tokuhara D, Yuki Y, Nochi T, Kodama T, Mejima M, Kurokawa S, et al. Secretory IgA-mediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine. Proc Natl Acad Sci U S A 2010;107:8794 9. Takeyama N, Yuki Y, Tokuhara D, Oroku K, Mejima M, Kurokawa S, et al. Oral rice-based vaccine induces passive and active immunity against enterotoxigenic E. coli-mediated diarrhea in pigs. Vaccine 2015;33: 5204 11. Nochi T, Yuki Y, Katakai Y, Shibata H, Tokuhara D, Mejima M, et al. A rice-based oral cholera vaccine induces macaque-specific systemic neutralizing antibodies but does not influence pre-existing intestinal immunity. J Immunol 2009;183:6538 44. Yuki Y, Tokuhara D, Nochi T, Yasuda H, Mejima M, Kurokawa S, et al. Oral MucoRice expressing doublemutant cholera toxin A and B subunits induces toxinspecific neutralising immunity. Vaccine 2009;27:5982 8. Kim MY, Kim BY, Oh SM, Reljic R, Jang YS, Yang MS. Oral immunisation of mice with transgenic rice calli

[87]

[88]

[89]

[90]

[91]

[92]

[93]

[94] [95]

[96]

[97]

[98]

expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes. Plant Mol Biol 2016;92:347 56. Kashima K, Yuki Y, Mejima M, Kurokawa S, Suzuki Y, Minakawa S, et al. Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants. Plant Cell Rep 2016;35:667 79. Chowdhury MY, Li R, Kim JH, Park ME, Kim TH, Pathinayake P, et al. Mucosal vaccination with recombinant Lactobacillus casei-displayed CTA1-conjugated consensus matrix protein-2 (sM2) induces broad protection against divergent influenza subtypes in BALB/ c mice. PLoS One 2014;9:e94051. Li R, Chowdhury MY, Kim JH, Kim TH, Pathinayake P, Koo WS, et al. Mucosally administered Lactobacillus surface-displayed influenza antigens (sM2 and HA2) with cholera toxin subunit A1 (CTA1) induce broadly protective immune responses against divergent influenza subtypes. Vet Microbiol 2015;179:250 63. Yu M, Qi R, Chen C, Yin J, Ma S, Shi W, et al. Immunogenicity of recombinant Lactobacillus caseiexpressing F4 (K88) fimbrial adhesin FaeG in conjunction with a heat-labile enterotoxin A (LTAK63) and heat-labile enterotoxin B (LTB) of enterotoxigenic Escherichia coli as an oral adjuvant in mice. J Appl Microbiol 2017;122:506 15. Arce S, Nawar HF, Russell MW, Connell TD. Differential binding of Escherichia coli enterotoxins LTIIa and LT-IIb and of cholera toxin elicits differences in apoptosis, proliferation, and activation of lymphoid cells. Infect Immun 2005;73:2718 27. Beauregard KE, Wimer-Mackin S, Collier RJ, Lencer WI. Anthrax toxin entry into polarized epithelial cells. Infect Immun 1999;67:3026 30. Baldauf KJ, Royal JM, Kouokam JC, Haribabu B, Jala VR, Yaddanapudi K, et al. Oral administration of a recombinant cholera toxin B subunit promotes mucosal healing in the colon. Mucosal Immunol 2017;10:887 900. Pabst O. New concepts in the generation and functions of IgA. Nat Rev Immunol 2012;12:821 32. Reboldi A, Cyster JG. Peyer’s patches: organizing Bcell responses at the intestinal frontier. Immunol Rev 2016;271:230 45. Cao AT, Yao S, Gong B, Nurieva RI, Elson CO, Cong Y. Interleukin (IL)-21 promotes intestinal IgA response to microbiota. Mucosal Immunol 2015;8:1072 82. Honda K, Littman DR. The microbiota in adaptive immune homeostasis and disease. Nature 2016;535:75 84. Gommerman JL, Rojas OL, Fritz JH. Re-thinking the functions of IgA(1) plasma cells. Gut Microbes 2014;5:652 62.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

200

11. TOXIN-BASED MODULATORS FOR REGULATION OF MUCOSAL IMMUNE RESPONSES

[99] Gutzeit C, Magri G, Cerutti A. Intestinal IgA production and its role in host-microbe interaction. Immunol Rev 2014;260:76 85. [100] Veldhoen M, Brucklacher-Waldert V. Dietary influences on intestinal immunity. Nat Rev Immunol 2012;12:696 708. [101] Mora JR, Iwata M, Eksteen B, Song SY, Junt T, Senman B, et al. Generation of gut-homing IgAsecreting B cells by intestinal dendritic cells. Science 2006;314:1157 60. [102] Cong Y, Oliver AO, Elson CO. Effects of cholera toxin on macrophage production of co-stimulatory cytokines. Eur J Immunol 2001;31:64 71. [103] Lavelle EC, McNeela E, Armstrong ME, Leavy O, Higgins SC, Mills KH. Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation. J Immunol 2003;171:2384 92. [104] Negri DR, Pinto D, Vendetti S, Patrizio M, Sanchez M, Riccomi A, et al. Cholera toxin and Escherichia coli heatlabile enterotoxin, but not their nontoxic counterparts, improve the antigen-presenting cell function of human B lymphocytes. Infect Immun 2009;77:1924 35. [105] Braun MC, He J, Wu CY, Kelsall BL. Cholera toxin suppresses interleukin (IL)-12 production and IL-12 receptor beta1 and beta2 chain expression. J Exp Med 1999;189:541 52. [106] la Sala A, He J, Laricchia-Robbio L, Gorini S, Iwasaki A, Braun M, et al. Cholera toxin inhibits IL-12 production and CD8alpha 1 dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function. J Exp Med 2009;206:1227 35. [107] Boyaka PN, Ohmura M, Fujihashi K, Koga T, Yamamoto M, Kweon MN, et al. Chimeras of labile toxin one and cholera toxin retain mucosal adjuvanticity and direct Th cell subsets via their B subunit. J Immunol 2003;170:454 62. [108] Hu JC, Greene CJ, King-Lyons ND, Connell TD. The divergent CD8 1 T cell adjuvant properties of LT-IIb and LT-IIc, two type II heat-labile enterotoxins, are conferred by their ganglioside-binding B subunits. PLoS One 2015;10:e0142942. [109] Brereton CF, Sutton CE, Ross PJ, Iwakura Y, Pizza M, Rappuoli R, et al. Escherichia coli heat-labile enterotoxin promotes protective Th17 responses against infection by driving innate IL-1 and IL-23 production. J Immunol 2011;186:5896 906. [110] Dunne A, Ross PJ, Pospisilova E, Masin J, Meaney A, Sutton CE, et al. Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis. J Immunol 2010;185: 1711 19.

[111] Hajishengallis G, Tapping RI, Martin MH, Nawar H, Lyle EA, Russell MW, et al. Toll-like receptor 2 mediates cellular activation by the B subunits of type II heat-labile enterotoxins. Infect Immun 2005;73: 1343 9. [112] Lee CH, Hajishengallis G, Connell TD. Dendritic cellmediated mechanisms triggered by LT-IIa-B5, a mucosal adjuvant derived from a type II heat-labile enterotoxin of Escherichia coli. J Microbiol Biotechnol 2017;27:709 17. [113] Liang S, Hosur KB, Nawar HF, Russell MW, Connell TD, Hajishengallis G. In vivo and in vitro adjuvant activities of the B subunit of type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli. Vaccine 2009;27:4302 8. [114] Sandoval F, Terme M, Nizard M, Badoual C, Bureau MF, Freyburger L, et al. Mucosal imprinting of vaccine-induced CD8(1) T cells is crucial to inhibit the growth of mucosal tumors. Sci Transl Med 2013;5 172ra120. [115] Gloudemans AK, Plantinga M, Guilliams M, Willart MA, Ozir-Fazalalikhan A, van der Ham A, et al. The mucosal adjuvant cholera toxin B instructs nonmucosal dendritic cells to promote IgA production via retinoic acid and TGF-beta. PLoS One 2013;8:e59822. [116] Eriksson A, Lycke N. The CTA1-DD vaccine adjuvant binds to human B cells and potentiates their T cell stimulating ability. Vaccine 2003;22:185 93. [117] Lycke N. ADP-ribosylating bacterial enzymes for the targeted control of mucosal tolerance and immunity. Ann N Y Acad Sci 2004;1029:193 208. [118] Jordan MB, Mills DM, Kappler J, Marrack P, Cambier JC. Promotion of B cell immune responses via an alum-induced myeloid cell population. Science 2004;304:1808 10. [119] Oleszycka E, Moran HB, Tynan GA, Hearnden CH, Coutts G, Campbell M, et al. IL-1alpha and inflammasome-independent IL-1beta promote neutrophil infiltration following alum vaccination. FEBS J 2016;283:9 24. [120] Eisenbarth SC, Colegio OR, O’Connor W, Sutterwala FS, Flavell RA. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 2008;453:1122 6. [121] Franchi L, Nunez G. The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity. Eur J Immunol 2008;38:2085 9. [122] Yang CW, Strong BS, Miller MJ, Unanue ER. Neutrophils influence the level of antigen presentation during the immune response to protein antigens in adjuvants. J Immunol 2010;185:2927 34.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

201

REFERENCES

[123] Puga I, Cols M, Barra CM, He B, Cassis L, Gentile M, et al. B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen. Nat Immunol 2011;13:170 80. [124] Elson CO, Holland SP, Dertzbaugh MT, Cuff CF, Anderson AO. Morphologic and functional alterations of mucosal T cells by cholera toxin and its B subunit. J Immunol 1995;154:1032 40. [125] Flach CF, Lange S, Jennische E, Lonnroth I, Holmgren J. Cholera toxin induces a transient depletion of CD8 1 intraepithelial lymphocytes in the rat small intestine as detected by microarray and immunohistochemistry. Infect Immun 2005;73:5595 602. [126] Penney I, Kilshaw PJ, MacDonald TT. Increased division of alpha beta TCR 1 and gamma delta TCR 1 intestinal intraepithelial lymphocytes after oral administration of cholera toxin. Immunology 1996;89:54 8. [127] Frossard CP, Asigbetse KE, Burger D, Eigenmann PA. Gut T cell receptor-gammadelta(1) intraepithelial lymphocytes are activated selectively by cholera toxin to break oral tolerance in mice. Clin Exp Immunol 2015;180:118 30. [128] Royal JM, Matoba N. Therapeutic potential of cholera toxin B subunit for the treatment of inflammatory diseases of the mucosa. Toxins (Basel) 2017;9:379. [129] Sun JB, Czerkinsky C, Holmgren J. Mucosally induced immunological tolerance, regulatory T cells and the adjuvant effect by cholera toxin B subunit. Scand J Immunol 2010;71:1 11. [130] Sun JB, Holmgren J, Czerkinsky C. Cholera toxin B subunit: an efficient transmucosal carrier-delivery

[131]

[132]

[133]

[134]

[135]

[136]

[137]

system for induction of peripheral immunological tolerance. Proc Natl Acad Sci U S A 1994;91:10795 9. Sun JB, Rask C, Olsson T, Holmgren J, Czerkinsky C. Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit. Proc Natl Acad Sci U S A 1996;93:7196 201. Bergerot I, Ploix C, Petersen J, Moulin V, Rask C, Fabien N, et al. A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes. Proc Natl Acad Sci U S A 1997;94:4610 14. Odumosu O, Nicholas D, Payne K, Langridge W. Cholera toxin B subunit linked to glutamic acid decarboxylase suppresses dendritic cell maturation and function. Vaccine 2011;29:8451 8. Sun JB, Xiang Z, Smith KG, Holmgren J. Important role for FcgammaRIIB on B lymphocytes for mucosal antigen-induced tolerance and Foxp3 1 regulatory T cells. J Immunol 2013;191:4412 22. Hasselberg A, Schon K, Tarkowski A, Lycke N. Role of CTA1R7K-COL-DD as a novel therapeutic mucosal tolerance-inducing vector for treatment of collageninduced arthritis. Arthritis Rheum 2009;60:1672 82. Hansson C, Schon K, Kalbina I, Strid A, Andersson S, Bokarewa MI, et al. Feeding transgenic plants that express a tolerogenic fusion protein effectively protects against arthritis. Plant Biotechnol J 2016;14:1106 15. Lin IP, Hsu YS, Kang SW, Hsieh MH, Wang JY. Escherichia coli heat-labile detoxified enterotoxin modulates dendritic cell function and attenuates allergic airway inflammation. PLoS One 2014;9:e90293.

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Influence of Dietary Components and Commensal Bacteria on the Control of Mucosal Immunity Hidehiko Suzuki1 and Jun Kunisawa1,2,3,4 1

Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan 2International Research and Development Center for Mucosal Vaccines, The Institute of Medical Sciences, The University of Tokyo, Tokyo, Japan 3Department of Microbiology and Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan 4Graduate School of Medicine, Graduate School of Pharmaceutical Sciences and Graduate School of Dentistry, Osaka University, Osaka, Japan

I. INTRODUCTION To maintain immunological homeostasis, intestinal tissues contain several types of immune cells, including regulatory T cells (Tregs), T helper 17 (Th17) cells, cytotoxic T cells (CTLs), γδ T cells, dendritic cells (DCs), IgA-producing plasma cells (IgA PCs), and macrophages. These cells are continuously exposed to dietary components and pathogenic and commensal microorganisms, and together, these provide a selective barrier against harmful materials and microorganisms and permit beneficial interactions to take place. Dietary components such as vitamins and metabolites

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00012-2

produced by commensal bacteria are essential for the development and maintenance of the immune system [1]. Indeed, poor or excess nutrient intake has been shown to increase the risk of infection and susceptibility to inflammatory or allergic diseases. Therefore, understanding the immunological functions of dietary components and metabolites will provide useful insights for the development of effective mucosal vaccines. In this chapter, we focus on the immunological relevance of how nutrients influence the regulation of the host immune system and discuss how this knowledge can be applied to develop improved and effective mucosal vaccines.

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II. VITAMINS Vitamins are essential nutrients for the development, maintenance, and regulation of the host immune system [1]. Vitamins are classified as either hydrophilic (vitamin B complex, vitamin C) or hydrophobic (vitamins A, D, E, and K). Since mammals, including humans, are unable to synthesize most vitamins [1], they must be obtained from the diet or be produced by commensal bacteria in the gut [2].

A. Vitamin A Vitamin A is essential for the maintenance of physiological functions such as cell differentiation and reproduction. Vitamin A is also essential for regulation of immunological homeostasis. Vitamin A-deficient mice have been shown to be highly susceptible to infection by Citrobacter rodentium and Escherichia coli and to be at high risk of developing immune diseases [3,4]. Similarly, in children, vitamin A deficiency is associated with a high susceptibility to infection by the microorganisms that cause measles, diarrhea, and respiratory diseases. [5], and vitamin A supplementation has been shown to decrease the incidence of diarrhea [6]. Vitamin A is obtained from the diet as alltrans-retinyl esters and β-carotene, which are metabolized to retinol [7]. Retinol is then metabolized to retinoic acid by alcohol dehydrogenase and retinal dehydrogenase. CD1031 DCs in mesenteric lymph nodes and Peyer’s patches express large amounts of retinal dehydrogenase, and therefore produce retinoic acid from vitamin A [8]. Immunologically, retinoic acid plays an important role in the regulation of cell trafficking by inducing T and B cells to express the guthoming molecules integrin α4β7 and chemokine receptor CCR9 [9,10]. The ligand of integrin α4β7 is mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), which is expressed by high endothelial venules (HEVs) present in the Peyer’s patches, the mesenteric lymph nodes, and the

lamina propria (for more on lymphocyte homing, see Chapter 5: Mucosal Immunity for Inflammation: Regulation of Gut-Specific Lymphocyte Migration by Integrins). The ligand of CCR9 is CCL25, which is produced by intestinal epithelial cells [11,12]. Because of this tissuespecific ligand expression, integrin α4β7 and CCR9 are key molecules in the homing of retinoicacid-activated T and B cells to the gut (Fig. 12.1). Vitamin A-deficient mice have decreased numbers of T cells and IgA PCs in the intestine [9,10]. Retinoic acid modulates the differentiation of B and T cells. Also, together with transforming growth factor beta (TGF-β) [13], retinoic acid enhances IgA class-switching, which induces interleukin (IL)-5 and -6 production for the enhancement of IgA production [10]. Furthermore, retinoic acid also controls TGF-β-driven T cell differentiation; retinoic acid-producing CD1031 DCs promote the differentiation of naı¨ve CD41 T cells into Tregs in a TGF-β-dependent manner [8]. However, retinoic acid also inhibits the differentiation of naı¨ve T cells into Th17 cells in the steady state (Fig. 12.1) [14]. These immunological functions of retinoic acid underlie a new type of vaccine strategy. In general, systemic vaccination (e.g., via the subcutaneous route) induces antigen-specific immune responses in systemic immune compartments but not in intestinal tissues. However, subcutaneous immunization together with retinoic acid induces the expression of integrin α4β7 and CCR9 on T and B cells, leading to the trafficking of antigen-specific T cells and IgA PCs to the intestine, resulting in protective immunity against intestinal pathogens (e.g., Salmonella, cholera toxin) [15,16].

B. Vitamin B Complex The vitamin B complex comprises eight vitamins: thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid

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FIGURE 12.1 Vitamin-mediated intestinal immunological responses. CD1031 dendritic cells (DCs) express retinal dehydrogenase (RALDH), which converts vitamin A to retinoic acid. Retinoic acid induces the expression of gut-homing molecules (integrin α4β7 and CCR9) by B cells and T cells. Retinoic acid promotes IgA class-switching and controls positively and negatively the differentiation of naı¨ve T cells into regulatory T cells (Tregs) and T helper 17 (Th17) cells, respectively. GPR109a vitamin B3 signaling promotes the production of interleukin (IL)-10 by DCs and the differentiation of naı¨ve T cells to Tregs. Folate receptor 4 (FR4) is expressed on Tregs, and the vitamin B9 FR4 axis is essential for their survival. The differentiation from naı¨ve B cells into IgA-producing plasma cells (IgA PCs) leads to a change in their glycolysis and vitamin B1 dependency.

(vitamin B5), pyridoxine (vitamin B6), biotin (vitamin B7), folic acid (vitamin B9), and various cobalamins (vitamin B12). Several of the vitamin B complex vitamins play roles in human energy metabolism systems. 1. Vitamin B1 Vitamin B1 is a key molecule in the production of ATP via glycolysis and the tricarboxylic acid (TCA) cycle. In the TCA cycle, vitamin B1 is an essential cofactor for the activity of 2oxoglutarate dehydrogenase and pyruvate dehydrogenase [17]. Therefore vitamin B1 deficiency disrupts the TCA cycle. To examine the immunological function of vitamin B1 in the intestine, we fed mice vitamin B1-deficient diets and found that these mice had diminished numbers of naı¨ve B cells in

their Peyer’s patches, but retained normal numbers of IgA PCs in their lamina propria compared to control mice. This suggests that the differentiation of naı¨ve B cells into IgA PCs is associated with the changes in vitamin B1 dependency (Fig. 12.1) [18]. This dependency can be explained by a change in energy metabolism; naı¨ve B cells in the Peyer’s patches preferentially and exclusively obtain ATP from the TCA cycle, whereas IgA PCs shift to glycolysis to obtain ATP (Fig. 12.1) [18]. Although it is well known that vitamin B1 deficiency causes beriberi and Wernicke Korsakoff syndrome, it can also affect the efficacy of oral vaccines. For example, mice fed a vitamin B1-deficient diet during oral immunization had decreased levels of antigen-specific IgA in their feces [18]. Together, these results

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imply that vitamin B1 deficiency increases susceptibility to infectious diseases by affecting host energy metabolism. 2. Vitamin B3 Many foods contain vitamin B3 (e.g., meat, fish, eggs). Since vitamin B3 deficiency causes pellagra, which is characterized by diarrhea and inflammation of the intestine and skin [19], vitamin B3 likely has immunological functions. It is reported that GPR109a is expressed on DCs, macrophages, and T cells and that it acts as a receptor for vitamin B3 [20]. In DCs and macrophages, GPR109a signaling induces the expression of the antiinflammatory cytokine IL-10. On T cells, GPR109a signaling promotes the differentiation of naı¨ve T cells to Tregs and IL-10-producing T cells (Fig. 12.1). Indeed, GPR109a-knockout mice have few Tregs and IL-10-producing T cells, which results in increased susceptibility to inflammatory diseases [21]. Thus, vitamin B3 is crucial for the induction of regulatory immune responses. 3. Vitamin B9 Vitamin B9 is essential for nucleotide and protein synthesis [22]; it also plays an important role in the regulation of the immune system by maintaining the survival of Tregs. Folate receptor 4, a receptor for vitamin B9, is highly expressed on Tregs [23] (Fig. 12.1). In the absence of vitamin B9, Tregs differentiate from naı¨ve T cells but do not survive, owing to decreased expression of antiapoptotic molecules such as Bcl-2 [24]. Indeed, mice fed a vitamin B9-deficient diet have decreased numbers of Tregs in the intestine and increased susceptibility to intestinal inflammation [25].

C. Vitamin D Vitamin D is an essential factor for calcium homeostasis and cell differentiation and growth [26]. Mice fed a vitamin D-deficient diet have

increased susceptibility to inflammatory diseases [27]. It is reported that patients with inflammatory bowel disease have low serum levels of vitamin D, and that vitamin D supplementation decreases the inflammatory symptoms in these patients [28]. In determining lymphocyte uptake, vitamin D receptor was found to be expressed on macrophages, DCs, and T cells. Treatment of these immune cells with 1,25-dihydroxy-vitamin D, an active form of vitamin D, results in downregulation of the expression of CD40, CD80, and CD86; reduced expression of inflammatory cytokines (e.g., IL-12); increased expression of the antiinflammatory cytokine IL-10; and enhanced differentiation of naı¨ve T cells to Tregs [29]. Thus, vitamin D is a negative regulator for host immunity.

III. LIPIDS Lipids are one of the three major essential nutrients obtained from the diet. It is well known that excessive amounts of lipid in the diet leads to the development of inflammation [30]. In addition to the quantity of dietary lipids, the composition of dietary lipids is also an important factor in the regulation of host immunity and inflammation. In general, dietary lipids are composed of long-chain saturated fatty acids and mono- or polyunsaturated fatty acids. Mammals are unable to synthesize omega-3 and omega-6 polyunsaturated fatty acids; therefore, these are referred to as essential fatty acids. To examine the effects of the fatty acid composition of dietary lipids on host immunity, mice were fed a diet containing different types of dietary lipids. We found that allergic responses in the intestine were ameliorated when mice were fed a diet containing linseed oil, which contains high concentrations of α-linolenic acid, an omega-3 polyunsaturated fatty acid [31]. In the intestine, α-linolenic acid is

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IV. COMMENSAL BACTERIA AND THEIR METABOLITES

metabolized into eicosapentaenoic acid, and our subsequent lipidomics analysis revealed that eicosapentaenoic acid is further metabolized to 17,18-epoxy eicosatetraenoic acid, which has antiallergic effects in the intestine [31]. We also found that the level of intestinal IgA was different depending on the fatty acid composition of dietary lipids [32]. For instance, mice fed a diet containing palm oil had a higher level of intestinal IgA compared with mice fed a diet containing soybean oil. Since palm oil contains a higher ratio of palmitic acids compared with soybean oil, we next fed mice diets containing soybean oil plus palmitic acid and found that these mice had increased levels of fecal IgA and numbers of IgA PCs. Palm oil and palmitic acid are reported to enhance antigen-specific intestinal IgA responses induced by oral vaccination [32]. We identified two mechanisms underlying the IgA enhancement by palmitic acid [32] (Fig. 12.2). First, palmitic acid directly stimulates IgA PCs to produce IgA. Second, palmitic acid is metabolized to sphingolipids (e.g.,

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ceramide, sphingosine, sphingosine-1-phosphate) via the action of serine palmitoyltransferase, which promotes the growth of IgA PCs in the large intestine [32]. This suggests that the high level of palmitic acid in palm oil both directly and indirectly stimulates IgA production and therefore that palmitic acid is a potential mucosal adjuvant.

IV. COMMENSAL BACTERIA AND THEIR METABOLITES It is estimated that 10 million to 100 trillion commensal bacteria from approximately 400 species colonize the human intestine [33]. Crosstalk between commensal bacteria and the host immune system has been shown to be important for the development and maintenance of host immune responses, including the production of intestinal IgA [34]. Previously, we identified a unique subset of IgA PCs in the intestine that are induced by commensal bacteria [35]. In addition, we found that PP-null mice failed to develop FIGURE 12.2 Palmitic acid stimulates IgA production. Palmitic acid, which is present at high concentrations in palm oil, directly stimulates IgA-producing plasma cells (IgA PCs) to produce IgA. Palmitic acid is also converted into sphingolipids (e.g., sphingosine, ceramide, sphingosine-1-phosphate) by several enzymes such as serine palmitoyltransferase. These sphingolipids induce proliferation of IgA PCs in the large intestine.

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these commensal bacteria-induced IgA PCs, suggesting that Peyer’s patches were necessary for the induction of these cells. Further examination of the commensal bacteria inside Peyer’s patches led to the discovery that species in the genus Alcaligenes colonize Peyer’s patches [36]. We also found that Alcaligenes species localize inside DCs, where they stimulate the production of several cytokines that enhance IgA production including IL-6, B-cell-activating factor, and a proliferation-inducing ligand [36]. Thus, Alcaligenes species appear to be a natural adjuvant stimulating IgA production in the Peyer’s patches. Probiotics are live microorganisms that confer a health benefit to the host, such as modulation of the immune system and suppression of the development of intestinal inflammation and metabolic disorders [37,38]. Probiotics have various effects on immune cells, such as cytokine and IgA production. Previously, we found that Lactobacillus pentosus strain b240 was taken up by Peyer’s patches, and that this enhanced the production of IL-6 by DCs, resulting in induction of IgA [39]. Probiotics have also been shown to support the effects of vaccines; oral

administration of Lactobacillus GG enhanced IgA immune responses induced by oral administration of a rotavirus vaccine [40]. Commensal bacteria, such as Lactobacillus species, produce short-chain fatty acids such as acetate, propionate, and butyrate from dietary fiber that act as effector molecules. Their collective concentration in the human colon is from 50 to 150 mM [41]. Short-chain fatty acids regulate host energy metabolism [42], and recent studies have revealed that they affect immune responses, including IgA production. For example, the short-chain fatty acid receptor GPR43, which is an acetate receptor, is involved in IgA production [43]. Indeed, GPR43-knockout mice show decreased intestinal IgA production, and supplementation with acetate promotes IgA production in wild-type mice but not in GPR43-knockout mice [43]. Although acetate does not directly act on B cells in the promotion of IgA production, the binding of acetate to GPR43 on DC induces the expression of retinal dehydrogenase by DCs. Moreover, activation of retinoic acid receptor signaling by acetatetreated DCs induces IgA production (Fig. 12.3), suggesting that acetate induces the production FIGURE 12.3 Commensal bacteriaderived short-chain fatty acids control intestinal immune responses. Commensal bacteria (e.g., Clostridium and Lactobacillus spp.) produce short-chain fatty acids (e.g., acetate, propionate, butyrate). The binding of acetate to GPR43 on DCs induces the expression of retinal dehydrogenase (RALDH). Activation of retinoic acid receptor (RAR) signaling by acetate-stimulated DCs promotes IgA production. Propionate binds to GPR43 on naı¨ve T cells, and the expression of Foxp3 is upregulated by inhibition of histone deacetylase (HDAC), leading to the differentiation of naı¨ve T cells to Tregs. Butyrate promotes the expression of AID-silencing microRNAs by B cells, which inhibit the differentiation of B cells to IgA PCs.

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REFERENCES

of retinoic acid by DCs via GPR43 to promote IgA production [43]. GPR43 also recognizes propionate, which promotes the induction of Tregs. Indeed, wild-type mice, but not GPR43-knockout mice, that orally consumed propionate, showed increased levels of Tregs in their colons [44]. Mechanistically, propionate inhibits histone deacetylase enhancing the expression of Foxp3 in T cells, and promoting their differentiation to Tregs [44] (Fig. 12.3). In contrast to acetate, butyrate suppresses IgA class-switching and the production of IgA. Butyrate inhibits histone deacetylase and upregulates microRNAs 155, 181b, 361, 23b, 30a, and 125b in human and mouse B cells, which inhibit class-switching and somatic hypermutation by silencing AID and Blimp-1 [45], resulting in reduced B cell differentiation and IgA production (Fig. 12.3).

V. CONCLUDING REMARKS In this chapter, we provided an overview of the immunological functions of several dietary components and commensal bacteria. Clinical and experimental evidence has revealed associations between the development of immunological diseases and nutritional deficiencies. Research is ongoing to clarify the molecular and cellular mechanisms underlying nutrientmediated or commensal bacteria-mediated immune regulation. The insights provided by this research will be essential for the development of improved vaccines.

Acknowledgment This chapter contains results obtained from our studies that were supported at least partly by grants from the Japan Agency for Medical Research and Development (AMED); the Ministry of Health, Labour and Welfare of Japan; the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry; Crossministerial Strategic Innovation Promotion Program (SIP); and the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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References [1] Suzuki H, Kunisawa J. Vitamin-mediated immune regulation in the development of inflammatory diseases. Endocr Metab Immune Disord Drug Targets 2015;15 (3):212 15. [2] Kau AL, Ahern PP, Griffin NW, Goodman AL, Gordon JI. Human nutrition, the gut microbiome and the immune system. Nature 2011;474 (7351):327 36. [3] Bhaskaram P. Micronutrient malnutrition, infection, and immunity: an overview. Nutr Rev 2002;60(5 Pt 2): S40 45. [4] McDaniel KL, Restori KH, Dodds JW, Kennett MJ, Ross AC, Cantorna MT. Vitamin A-deficient hosts become nonsymptomatic reservoirs of Escherichia colilike enteric infections. Infect Immun 2015;83 (7):2984 91. [5] West Jr. KP. Vitamin A deficiency disorders in children and women. Food Nutr Bull 2003;24(Suppl. 4): S78 90. [6] Villamor E, Fawzi WW. Effects of vitamin a supplementation on immune responses and correlation with clinical outcomes. Clin Microbiol Rev 2005;18 (3):446 64. [7] Theodosiou M, Laudet V, Schubert M. From carrot to clinic: an overview of the retinoic acid signaling pathway. Cell Mol Life Sci 2010;67(9):1423 45. [8] Coombes JL, Siddiqui KR, Arancibia-Carcamo CV, et al. A functionally specialized population of mucosal CD103 1 DCs induces Foxp3 1 regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J Exp Med 2007;204(8):1757 64. [9] Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C, Song SY. Retinoic acid imprints gut-homing specificity on T cells. Immunity 2004;21(4):527 38. [10] Mora JR, Iwata M, Eksteen B, et al. Generation of guthoming IgA-secreting B cells by intestinal dendritic cells. Science 2006;314(5802):1157 60. [11] Kunkel EJ, Campbell JJ, Haraldsen G, et al. Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity. J Exp Med 2000;192(5):761 8. [12] Wurbel MA, Philippe JM, Nguyen C, et al. The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9. Eur J Immunol 2000;30(1):262 71. [13] Watanabe K, Sugai M, Nambu Y, et al. Requirement for Runx proteins in IgA class switching acting downstream of TGF-beta 1 and retinoic acid signaling. J Immunol 2010;184(6):2785 92.

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[14] Mucida D, Park Y, Kim G, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 2007;317(5835):256 60. [15] Hammerschmidt SI, Friedrichsen M, Boelter J, et al. Retinoic acid induces homing of protective T and B cells to the gut after subcutaneous immunization in mice. J Clin Invest 2011;121(8):3051 61. [16] Tan X, Sande JL, Pufnock JS, Blattman JN, Greenberg PD. Retinoic acid as a vaccine adjuvant enhances CD8 1 T cell response and mucosal protection from viral challenge. J Virol 2011;85(16):8316 27. [17] Frank RA, Leeper FJ, Luisi BF. Structure, mechanism and catalytic duality of thiamine-dependent enzymes. Cell Mol Life Sci 2007;64(7-8):892 905. [18] Kunisawa J, Sugiura Y, Wake T, et al. Mode of bioenergetic metabolism during B cell differentiation in the intestine determines the distinct requirement for vitamin B1. Cell Rep 2015;13(1):122 31. [19] Hegyi J, Schwartz RA, Hegyi V. Pellagra: dermatitis, dementia, and diarrhea. Int J Dermatol 2004;43(1):1 5. [20] Lukasova M, Malaval C, Gille A, Kero J, Offermanns S. Nicotinic acid inhibits progression of atherosclerosis in mice through its receptor GPR109A expressed by immune cells. J Clin Invest 2011;121(3):1163 73. [21] Singh N, Gurav A, Sivaprakasam S, et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity 2014;40(1):128 39. [22] Stover PJ. Physiology of folate and vitamin B12 in health and disease. Nutr Rev 2004;62(6 Pt 2):S3 12 discussion S13. [23] Yamaguchi T, Hirota K, Nagahama K, et al. Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. Immunity 2007;27 (1):145 59. [24] Kunisawa J, Hashimoto E, Ishikawa I, Kiyono H. A pivotal role of vitamin B9 in the maintenance of regulatory T cells in vitro and in vivo. PLoS One 2012;7(2): e32094. [25] Kinoshita M, Kayama H, Kusu T, et al. Dietary folic acid promotes survival of Foxp3 1 regulatory T cells in the colon. J Immunol 2012;189(6):2869 78. [26] Holick MF, Grant WB. Vitamin D status and ill health. Lancet Diabetes Endocrinol 2014;2(4):273 4. [27] Iijima H, Shinzaki S, Takehara T. The importance of vitamins D and K for the bone health and immune function in inflammatory bowel disease. Curr Opin Clin Nutr Metab Care 2012;15(6):635 40. [28] Suibhne TN, Cox G, Healy M, O’Morain C, O’Sullivan M. Vitamin D deficiency in Crohn’s disease: prevalence, risk factors and supplement use in an outpatient setting. J Crohn’s Colitis 2012;6(2):182 8.

[29] Chang JH, Cha HR, Lee DS, Seo KY, Kweon MN. 1,25Dihydroxyvitamin D3 inhibits the differentiation and migration of T(H)17 cells to protect against experimental autoimmune encephalomyelitis. PLoS One 2010;5 (9):e12925. [30] Jin C, Flavell RA. Innate sensors of pathogen and stress: linking inflammation to obesity. J Allergy Clin Immunol 2013;132(2):287 94. [31] Kunisawa J, Arita M, Hayasaka T, et al. Dietary omega3 fatty acid exerts anti-allergic effect through the conversion to 17,18-epoxyeicosatetraenoic acid in the gut. Sci Rep 2015;5:9750. [32] Kunisawa J, Hashimoto E, Inoue A, et al. Regulation of intestinal IgA responses by dietary palmitic acid and its metabolism. J Immunol 2014;193(4):1666 71. [33] Bourlioux P, Koletzko B, Guarner F, Braesco V. The intestine and its microflora are partners for the protection of the host: report on the danone symposium “the intelligent intestine,” held in Paris, June 14, 2002. Am J Clin Nutr 2003;78(4):675 83. [34] Goto Y, Ivanov II. Intestinal epithelial cells as mediators of the commensal-host immune crosstalk. Immunol Cell Biol 2013;91(3):204 14. [35] Kunisawa J, Gohda M, Hashimoto E, et al. Microbedependent CD11b 1 IgA 1 plasma cells mediate robust early-phase intestinal IgA responses in mice. Nat Commun 1772;2013:4. [36] Obata T, Goto Y, Kunisawa J, et al. Indigenous opportunistic bacteria inhabit mammalian gut-associated lymphoid tissues and share a mucosal antibodymediated symbiosis. Proc Natl Acad Sci USA 2010;107 (16):7419 24. [37] Ganji-Arjenaki M, Rafieian-Kopaei M. Probiotics are a good choice in remission of inflammatory bowel diseases: a meta analysis and systematic review. J Cell Physiol 2018;233(3):2091 103. [38] Yoo JY, Kim SS. Probiotics and prebiotics: present status and future perspectives on metabolic disorders. Nutrients 2016;8(3):173. [39] Kotani Y, Kunisawa J, Suzuki Y, et al. Role of Lactobacillus pentosus strain b240 and the Toll-like receptor 2 axis in Peyer’s patch dendritic cell-mediated immunoglobulin A enhancement. PLoS One 2014;9(3): e91857. [40] Isolauri E, Joensuu J, Suomalainen H, Luomala M, Vesikari T. Improved immunogenicity of oral D x RRV reassortant rotavirus vaccine by Lactobacillus casei GG. Vaccine 1995;13(3):310 12. [41] Cummings JH, Pomare EW, Branch WJ, Naylor CP, Macfarlane GT. Short chain fatty acids in human large intestine, portal, hepatic and venous blood. Gut 1987;28(10):1221 7.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[42] Kasubuchi M, Hasegawa S, Hiramatsu T, Ichimura A, Kimura I. Dietary gut microbial metabolites, shortchain fatty acids, and host metabolic regulation. Nutrients 2015;7(4):2839 49. [43] Wu W, Sun M, Chen F, et al. Microbiota metabolite short-chain fatty acid acetate promotes intestinal IgA response to microbiota which is mediated by GPR43. Mucosal Immunol 2017;10(4):946 56.

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[44] Smith PM, Howitt MR, Panikov N, et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 2013;341(6145):569 73. [45] White CA, Pone EJ, Lam T, et al. Histone deacetylase inhibitors upregulate B cell microRNAs that silence AID and Blimp-1 expression for epigenetic modulation of antibody and autoantibody responses. J Immunol 2014;193(12):5933 50.

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C H A P T E R

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Mast Cells for the Control of Mucosal Immunity Hae Woong Choi1, Brandi Johnson-Weaver1, Herman F. Staats1,2,3 and Soman N. Abraham1,3,4,5 1

Department of Pathology, Duke University School of Medicine, Durham, NC, United States Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC, United States 3 Department of Immunology, Duke University Medical Center, Durham, NC, United States 4 Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States 5Program in Emerging Infectious Diseases, Duke-National University of Singapore, Singapore, Singapore 2

I. INTRODUCTION Having been overlooked by infectious diseases researchers for over a century, mast cells (MCs) are turning out to be very critical players in immune surveillance [1,2]. They possess an intrinsic capacity to recognize a broad range of infectious agents and to mobilize both innate and adaptive immune responses to these challenges. Much of this activity involves recruiting various immune cells either to the site of infection to clear pathogens or to the draining lymph nodes (DLNs), where the mobilized immune cells initiate the development of pathogenspecific immune responses. MC traits, including modulating immune cell trafficking into DLNs and remodeling secondary lymphoid

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00013-4

organs for maximal immune responses, have even encouraged studies examining the utility of MC-specific activators as adjuvants for various vaccines delivered in the skin and nasal passages [3,4] (see Chapter 10: Innate Immunity-Based Mucosal Modulators and Adjuvants). MCs have several unique properties that facilitate their prominent role in immune surveillance. These traits include their strategic location at host environment interfaces, where their expression of a wide range of receptors allows them to respond promptly to extrinsic challenge with both immediate degranulation and secretion of de novo synthesized mediators [1,2,5]. In this chapter, we will describe some of the key immunomodulatory properties of MCs

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and discuss how they are harnessed to simultaneously direct both innate and adaptive arms of the immune response when microbes attack. Since MCs are also known for their pathological contributions during chronic inflammatory diseases such as asthma and arthritis [6 8], it is not surprising that MCs sometimes aggravate the disease state during infection. This chapter will describe MCs’ contribution to beneficial and detrimental host immunity. Since the data pertaining to the mucosae are sometimes sparse, contributions of MCs at other relevant body sites have been included.

II. EXOCYTOSIS OF BIOLOGICALLY ACTIVE MODULATORS MCs are characterized by the numerous electron-dense secretory granules populating their cytosolic space. Upon activation, MCs utilize a highly specialized intracellular transport system to rapidly exteriorize granules within seconds to minutes [2,9]. MC granules contain high amounts of anionic sulfated proteoglycans such as heparin and chondroitin sulfate, but the major constituents are MC-specific proteases, which are tightly complexed with the sulfated proteoglycans by electrostatic interactions [2,10,11]. Embedded in the MC granule matrix are biogenic amines such as histamine and the neurotransmitter serotonin [11 13]. Several proinflammatory cytokines and growth factors are also contained within granules, such as tumor necrosis factor (TNF) and vascular endothelial growth factor [14,15]. When MC granules are exteriorized, they do not disassemble immediately. Instead, they remain in the tissue microenvironment, serving as slow-release devices, releasing their payload gradually until the granules disintegrate or are taken up by surrounding cells [16]. The release of particular cargo from the granule matrix varies, depending on their physicochemical attributes. For example, histamine escapes granules within

minutes [17], while TNF is retained for hours within the granule matrix [18]. MC granules are able to protect their cargo from dilution and degradation in the extracellular environment by stably retaining their conformation for hours [2,19]. Additionally, MCs sustain inflammatory reactions by releasing their cargo of soluble mediators over protracted periods. MCs are able to sustain inflammatory reactions even after the degranulation event is concluded. Interestingly, MCs do not undergo programmed cell death upon degranulation, unlike certain other immune cells, such as neutrophils [20,21]. Instead, they regranulate and, within a few hours, are able to undertake additional cycles of degranulation and regranulation as needed. Additionally, these cells are able to increase their numbers at inflammatory sites by proliferating in tissue in response to appropriate cues, despite being terminally differentiated [22,23]. The ability of MCs to rapidly exocytose their granules and associated preformed contents within seconds to a few minutes following activation is especially effective in mobilizing the immune system. Other immune cells, such as skin-resident dendritic cells (DCs) or epithelial cells, require considerably more time to mount a response, owing to the requirement for de novo synthesis of mediators [1]. In addition to the immediate activation response, MCs have a potent second phase mediator response that occurs hours after the initial stimulus, in which they initiate de novo synthesis of mediators such as cytokines and eicosanoids. Eicosanoids, which are composed of prostaglandins and related compounds such as leukotrienes, can rapidly be converted from arachidonic acid to an active form [24,25]. Once secreted, they function as proinflammatory mediators that enhance vascular permeability and cellular recruitment. MCs also secrete a wide array of chemokines and cytokines depending on the type and strength of the stimulus [9]. Potentially, MCs can follow up an immediate

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degranulation response with the synthesis of additional factors that are tailored to the stimulus, allowing for enormous plasticity in the duration, nature, and specificity of its mediator response. Thus MCs possess several physical attributes that extenuate their capacity to mediate inflammation to mount appropriate responses to pathogens at sites of infection [1,26 30]. Table 13.1 lists MC mediators released in response to various microbial pathogens and their functions. TABLE 13.1

III. PERIPHERAL LOCATION MCs originate from bone marrow hematopoietic progenitors that circulate in the blood [52] and mature upon entering tissues. These progenitors differentiate into mature cells based on the particular cytokine milieu in the microenvironment [53,54]. Consequently, MCs at different anatomical sites are phenotypically heterogeneous [55] and can be identified on the basis of protein and proteoglycan granule

MC Mediator Responses to Various Microbial Pathogens and Their Functional Roles

Species

Pathogens

MC Mediators

Functional Roles

Reference

Bacteria

Klebsiella pneumoniae

TNF-α, IL-6, tryptase

Recruitment of neutrophils

[27,31,32]

Escherichia coli

TNF-α, leukotriene

Recruitment of neutrophils/DCs/T cells

[3,33 35]

Francisella tularensis

IL-4

Inhibiting replication and killing of pathogens inside macrophage

[36,37]

Pseudomonas aeruginosa

IL-1α/β

Recruitment of neutrophils

[38,39]

Clostridium difficile

IL-8

Recruitment of neutrophils

[40,41]

Group A Streptococcus

Cathelicidin

Direct killing of pathogens

[42]

Group B Streptococcus

Chymase

Preventing adherence to host cells

[43]

Listeria monocytogenes

TNFα

Recruitment of neutrophils

[44,45]

Cytomegalovirus

CCL5

Recruitment of protective CD8 T cells

[28]

Dengue virus

TNFα, IFNα CCL5, CXCL12, CX3CL1

Recruitment of NK and NK T cells

[29]

Respiratory syncytial virus

Type I INFs, CXCL10, CCL4 Effector cell recruitment

[46]

Newcastle virus

CCL5

CD8 T cell recruitment

[47]

Chymase, Tryptase

Parasite expulsion, eosinophil recruitment

[48,49]

Strongyloides venezuelensis

IL-3

Parasite expulsion

[50]

Heligmosomoides polygyrus

IL-33

Induction of group 2 innate lymphoid cells

[51]

Virus

Parasites Trichinella spiralis

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components. For example, rodent MCs can be categorized into mucosal type, which are present in the epithelia of mucosal tissues such as the gut and lung, and connective tissue type, which are found in the submucosae and muscle layer [56,57]. Connective tissue MCs express both tryptases and chymases complexed with heparin, while mucosal MCs express only chymases, complexed with chondroitin sulfate [10,11,58]. Similarly, human MCs, are classified either as tryptase- and chymase-positive (MCTC) or as tryptase-positive (MCT), though there is less stringency in tissue-type specificity in humans than in rodents [59,60]. As was indicated above, MCs are mostly concentrated at interfaces between the host and environment, such as the skin and the gastrointestinal and respiratory tracts. Thus they are ideally situated to be among the first responders to pathogens or other extrinsic agents that breach the host epithelial barrier [1]. MCs are often strategically located proximal to blood vessels, lymphatics [18], and nerve fibers [61] where they can quickly relay information about invading pathogens by releasing mediators in the vicinity of these signaling conduits. Interestingly, a recent report suggests that perivascular MCs may even directly acquire IgE antibodies circulating in the blood by extending cellular processes across blood vessel walls [62]. This ability to sample vascular contents may also apply to detecting pathogens or pathogen-associated molecules in the circulation.

IV. MULTIPRONGED ACTIVATION AT INFECTION SITES During infection, local MCs are activated by multiple mechanisms. MC activation can occur when membrane receptors are directly engaged by pathogens or antibody receptors on MCs for engaging antibody-coated pathogens [63 66]. Since MCs also possess receptors for damage-

associated molecular patterns (DAMPs) that are released by cells under stress, they can be activated in the absence of pathogen by-products by surrounding distressed cells at inflamed sites [67,68] (Chapter 6: Innate Immunity at Mucosal Surfaces).

A. Direct Recognition of Pathogens or Their Products Various pattern recognition receptors are expressed on MC membranes that allow them to directly recognize pathogen-associated molecular patterns (PAMPs). These include tolllike receptors (TLRs), C-type lectins (e.g., dectin-1), and other surface molecules such as CD14 [69 71]. Most known TLRs are expressed by MCs, at least at the mRNA level, and they have been shown to be either constitutively expressed or upregulated based on cues from the microenvironment. TLR4 expression on human MCs derived from cord blood, for example, is induced in vitro in the presence of interferon gamma (IFNγ) or IL-4 but is not expressed constitutively [72]. Certain pathogen-associated ligands can also activate MCs by a combination of TLRs and other surface molecules, such as fungal zymosan, which is recognized by both TLR2 and dectin-1 [71], and LPS, which is recognized by TLR4 and CD14 [73]. Differential ligation of TLRs alone or in combination with other receptors can result in distinct MC exocytic outcomes. While complete degranulation may occur in some cases, partial or no degranulation occurs in others. TLR2 activation via bacterial peptidoglycan, for instance, results in both MC degranulation and cytokine production, while TLR4 ligation via LPS only results in cytokine release [74]. Additionally, simultaneous ligation of both TLRs and other activating receptors, such as the high-affinity receptor for IgE antibodies, FcεRI, can enhance the release of cytokines [69], adding a further dimension to TLR-mediated MC activation.

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V. INITIATION OF LOCAL INNATE IMMUNE RESPONSES

Remarkably, TLR signaling may influence the MC functional phenotype by downregulating FcεRI expression, altering protease composition within granules, and influencing cytokine secretion [75]. Prolonged interaction of MCs with pathogens and their associated PAMPs, which may activate MCs via TLRs, in chronically inflamed tissues could markedly shape MCdirected immune responses [47,72]. However, microbial shaping of MC behavior is not limited to inflamed tissues. Commensal microbes, such as the skin microbiome, are critical in shaping MC maturation in the skin [76]. For example, lipoteichoic acid, a component of Staphylococcus epidermidis, triggers secretion of stem cell factor (SCF) from keratinocytes in the steady state [76]. SCF secretion is suggested to be pivotal in recruiting MC progenitors from the circulation to the dermis and inducing maturation, since the numbers of mature skin MCs are limited in germ-free mice [76].

B. Indirect Recognition of Pathogens MC membranes express ample numbers of receptors for various antibody types, allowing them to bind antigen-specific immunoglobulins and become sensitized to an antigen previously encountered by the host [7,19,77]. These immobilized antibodies enable MCs to specifically recognize and vigorously react to previously encountered pathogens. The high-affinity receptor for IgE, FcεRI, is amply expressed on MCs and is responsible for protective IgEmediated responses to metazoan parasites such as roundworms [66]. However, this receptor is also implicated in the misdirected IgEmediated hypersensitivity reactions to harmless antigens [7,57]. MCs also express the FcγR family of receptors for IgG, of which FcγRI and FcγRIII are activating and FcγRIIB is inhibitory [77 81]. Cross-linking of FcγR by IgG immune complexes or IgE-bound FcεRI by multivalent antigen results in MC activation, degranulation, and cytokine secretion [77,82]. Coligation of

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FcεRI and the inhibitory FcγRIIB, however, results in the downregulation of FcεRI signaling [83]. It might be interested to consider that this MC-associated FcR family could be a novel candidate for the development of MC-targeted adjuvant or immunomodulator.

C. Activation by Endogenous Danger Signals During infection, many endogenous DAMPs that are elaborated by stressed or dying cells have potent MC-activating properties. These host-derived proinflammatory mediators include ATP, complement components, antimicrobial peptides, substance P, and endothelin-1 [84 86]. Human MCs are activated by the antimicrobial peptide LL-37 through the MAS-related G-protein-coupled receptor-X2 (MRGPRX2), found on the MC membrane [87]. Endothelin-1, which is produced during sepsis by endothelial cells, activates MCs via the ET-1 receptor [84]. Notably, components of the complement system, in particular C5a, can also activate MC, inducing their chemotaxis, degranulation, and production of cytokines through complement receptors on MCs [86].

V. INITIATION OF LOCAL INNATE IMMUNE RESPONSES MCs are pivotal modulators of the innate immune system following various toxic challenges (Fig. 13.1). In addition to directly contributing to the killing and degradation of microbes or other noxious substances, they can direct the activation and recruitment of appropriate immune cells to the site of infection to facilitate early clearance of the insult [1,3,29,31,47,51].

A. Direct Bactericidal Activity Through the extracellular release of granuleassociated antimicrobial peptides such as

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FIGURE 13.1 Immune regulatory properties of MCs during various infections. In most cases, upon perceiving bacteria, parasites, or virus, MCs evoke a vigorous response involving the release of many prestored mediators (degranulation) and the secretion of de novo synthesized mediators. Upon release of these mediators, various immune responses are initiated, including the recruitment of effector cells and the initiation of adaptive immunity, through activation of immune cells in the distal draining lymph nodes. MCs also release antimicrobial peptides that can directly kill pathogens. In view of the powerful role of MCs in mobilizing innate and adaptive immune responses, several pathogen and commensal bacteria have evolved mechanisms to inactivate MCs, resulting in a limited response from these immune cells.

cathelicidins, MCs have the capacity to kill pathogens in their vicinity [42,88]. Additionally, MCs are able to endocytose enterobacteria, such as Escherichia coli, that express type I fimbriae, by binding the fimbrial component FimH to the cell surface molecule CD48 [89]. Engulfed bacteria are then killed through

oxidative burst and acidification of vacuoles. MCs, like neutrophils, appear capable of ensnaring and killing bacteria via extracellular trap formation. MC extracellular traps [90] are composed of nuclear histones, antimicrobial peptides, and tryptases and are released in a reactive oxygen species dependent cell death

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mechanism. However, since MCs are a small population of cells at infection sites, relative to the large numbers of recruited professional phagocytes and other immune cells, it is unlikely that the bactericidal actions of MCs contribute significantly to direct bacterial killing in vivo.

B. Proteolytic Degradation of Toxins MC granules also contain large amounts of active proteases that could potentially cleave toxic peptides of endogenous and exogenous origin. In a cecal ligation and puncture rodent model of bacterial sepsis, carboxypeptidase A3 released from MC granules was shown to be protective against locally produced inflammatory peptides such as neurotensin and endothelin that contribute to mortality [30,91,92]. MCs also play an important role in the defense against insect or reptile venom-derived toxins such as Sarafotoxin, a homolog of the endogenous inflammatory peptide endothelin, through the release of degradative carbopeptidases [30,92]. This is beneficial to the host, since venom exotoxins can mimic the activity of endogenous inflammatory peptides s, such as endothelin-1, and cause severe inflammatory reactions. Chymase is also a major component of MC granules that possess chymotrypsin-like cleavage specificity, which is protective against severe group B streptococcus infections [26]. This MC product was found to reduce adherence of bacteria to host tissue by degrading fibronectin, a host extracellular matrix protein [26].

C. Induction of Immune Cell Trafficking to Sites of Infection An important MC role is to alter composition of the extracellular milieu at the infection site through the secretion of a wide variety of chemoattractant and cytokines [1]. This surge in

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the local levels of immunomodulatory agents promotes and directs leukocyte trafficking to sites of infection, where they contribute to pathogen clearance or amplify inflammation by secretion of proinflammatory cytokines. An important prestored as well as de novo produced cytokine is TNF-α, which is pivotal in the trafficking of neutrophils to sites of MC activation [31]. MC granule proteases, released upon degranulation, are also known to have chemotactic activity for leukocytes. The mouse MC protease 6 (mMCP6) induces neutrophil migration to bacterial infection sites [27]. This protease also contributes to eosinophil recruitment and cytokine production in response to parasitic infections [48]. During bacterial infections, MC-generated leukotrienes (LT), such as LTB4 and LTC4, upregulate P-selectin expression on endothelial cells, enhancing the rolling phase of the leukocyte adhesion cascade preceding neutrophil recruitment and bacterial clearance [93]. MCs also regulate cell trafficking in response to viral challenge. Respiratory syncytial virus, while only poorly infective for human MCs, is able to induce the secretion of multiple chemotactic factors for leukocytes, such as C-C motif ligand 4 (CCL4), CCL5, and C-X-C motif chemokine 10 (CXCL10) [46]. In a mouse model of dengue infection, MCs upregulate the chemokines CX3CL1, CXCL12, and CCL5, which recruit natural killer (NK) cells to the site of viral infection [29]. Similarly, MCs are pivotal in protecting mice against cytomegalovirus-induced interstitial pneumonia by recruiting CD81 T cells through the secretion of a wave of CCL5 [28]. Through the release of IL-33, MCs in the intestines have been shown to be protective against nematodes by recruiting IL-13-producing type 2 innate lymphoid cells [51]. This role of MCs to recruit appropriate pathogen-clearing immune cells to sites of bacterial, viral, and parasitic infections is arguably the most important innate immune activity mediated by these immune surveillance cells.

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VI. CONTRIBUTIONS TO ADAPTIVE IMMUNE RESPONSES MC inflammatory responses appear to contribute in many ways to developing adaptive immune responses. In addition to promoting remodeling of the DLNs, the epicenter of the adaptive immune response, MCs also serve as effector cells in collaboration with adaptive immune cells.

A. Enhancing Influx of Immune Cells Into Draining Lymph Nodes While MCs are instigating innate immune responses at microbial infection sites, they are concurrently initiating adaptive immune responses. During infection by bacteria such as E. coli, MCs recruit large numbers of DCs into infected tissues, which then proceed to migrate into DLNs [33]. This influx of DCs to the infection site is instigated by MC-derived TNF, which upregulates E-selectin on endothelial cells of neighboring blood vessels and is an essential step in the development of E. coli specific immune responses [33]. Thus MCs may be important cells that initiate host adaptive immune responses to pathogens, since DC activation and trafficking to lymph nodes constitute a critical first step in the initiation of adaptive immunity (Fig. 13.1). The DLNs are dynamic lymphoid structures that can sequester large numbers of circulating lymphocytes during infection, causing them to swell dramatically in size [3,33,94]. T cells entrapped in the nodes in this manner interact with antigen-loaded DCs that have been trafficked from sites of infection to initiate the adaptive immune response. It is now known that the hypertrophy of DLN that occurs within 24 hours of bacterial infection is mediated by MCs at the infection site [33]. How peripheral MCs regulate immune cell trafficking was a

mystery until it was discovered that a significant number of exteriorized MC granules are trafficked via the lymphatics to local DLNs. These granules release their cargo, including TNF, which induces production of the chemokine CCL21 inside the DLN, resulting in the influx of DCs and T cells [33]. In addition to TNF, MC-derived histamine and IL-6 stimulate trafficking of immune cells into the DLNs following stimulation with bacterial peptidoglycan [94]. Thus following infection at peripheral sites, MCs kick-start adaptive immunity by promoting the coordinate trafficking of professional antigen-presenting cells (APCs), such as DCs, from infection site into the DLNs and simultaneously sequestering T lymphocytes to facilitate optimal antigen presentation.

B. Antigen Presentation While not regarded as professional APCs, MCs are reported capable of directly presenting antigens to T cells and evoking responses [95]. MCs constitutively express major histocompatibility complex (MHC) class I but appear to express MHC class II molecules only at infection sites [96]. MCs also express costimulatory molecules such as OX40 ligand upon activation [97]. CD81 T cells respond to MC MHC class Idependent antigen presentation by increasing cellular proliferation and IL-2 and granzyme B production in an autoimmune state, but it is possible that modulation of CD81 T cell function could occur during infection [95]. However, the number of MCs compared to other recruited APCs at infection sites is low. MCs are more likely to play a supporting role in antigen presentation to other APCs than to directly mediate this immune function. For instance, histamine, a MC product, enhances DC antigen uptake and cross-presentation and upregulates costimulatory molecules [98]. During helminth infections, MC-derived cytokines can also prime APCs to skew immune

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responses toward Th2, [99], suggesting that MCs may have a role in shaping the nature of the adaptive immune response that the initiate.

C. Effectors of Adaptive Immunity MCs exert effector functions in collaboration with adaptive immunity. MC- and IgEassociated responses are thought to contribute to host defense against helminths and other parasites that are too large to be endocytosed [51,65,66]. MC products, such as proteases that are secreted upon activation through parasiticspecific IgE on MC surfaces are believed to contribute to killing and expulsion of parasites [66]. Additionally, MCs may serve as effectors of pathogen-specific immune responses by directing the trafficking of effector CD81 T cells to sites of viral infection through the secretion of CXCL10 and CCL4 [47].

D. Adjuvant Activity of Mast Cell Activators and Products MCs’ contribution to induction and modulation of adaptive immunity has inspired studies harnessing MC activities to improve immune responses to vaccine antigens [4]. One approach is to coadminister small-molecule MC activators as adjuvants in vaccine formulations. Since MC activators (MCA) are highly efficacious in provoking rapid mobilization of immune cells at the infection sites and DLNs, it is possible that MCA can enhance immunity at sites of vaccine administration as well. Indeed, the MCA compound 48/80 has been shown to be a powerful adjuvant when coadministered with various vaccine antigens in the skin and nasal passages [4]. Because this related topic is adequately discussed in Chapter 10, Innate Immunity Based Mucosal Modulator and Adjuvant, it will not be discussed further here. The use of synthetic MC granules is another technique to induce vaccine adjuvant activity.

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These synthetic granules are inspired by MCs and can be loaded with powerful immunomodulatory cytokines [100]. When injected at peripheral sites, synthetic granules, similar to natural MC granules, are trafficked via the lymph to the DLN and release their components [100]. The resulting cytokine burst markedly enhances the influx of immune cells into the DLNs from both the site of vaccination and blood, culminating in an enhanced immune response to the vaccine antigen. Synthetic MC granules can also be engineered to modulate adaptive immune responses [100]. MCs are thought to predominantly enhance Th2-type responses, defined by enhanced antibody production and cytokines, such as IL-4, which may aid in the production of neutralizing antibodies against pathogens [101,102]. Unfortunately, MCs are not a source of Th1associated immunity, which is characterized by cytotoxic T cell activity and cytokines, such as IL-12 and IFNγ, and is more effective against intracellular pathogens [103,104]. However, synthetic MC granules can be utilized to include different cytokines and polarize immune responses as needed to combat pathogens. Thus this technology is potentially a viable strategy to improve vaccination. MC granule-inspired particles can be readily synthesized by encapsulating specific cytokines within heparin chitosan complexes at acidic pH, forming nanoparticles between 200 and 1000 nm in diameter [100]. Indeed, such synthetic MC granules bearing TNF protected mice against a lethal influenza challenge with efficacy on a par with that of conventional alum adjuvant [100]. This strategy was also recently used to reprogram the immune response to allergens from a harmful pathological one to a benign one in mice [100]. It was found that immunizing atopic mice with a formulation comprising the allergen and IL-12-loaded synthetic granules resulted in enhanced Th1polarized immunity with minimal Th2-type activity. When these reprogrammed mice were

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challenged with the allergen, no harmful anaphylaxis was observed compared to atopic mice that were not reprogrammed and to atopic mice treated with the allergen and soluble IL-12 [100]. Thus using synthetic granules loaded with immunomodulatory cytokines to target lymph nodes in order to enhance and/or polarize immune responses is potentially a powerful strategy for modulating immunity. This approach also provides tremendous flexibility in vaccine design by tailoring the specific cytokines encapsulated to a desired immune response.

VII. DYSREGULATED OR IMPAIRED MAST CELL ACTIVITY DURING INFECTION While the responses of mucosal and skin MCs to microbial infection are generally beneficial, these responses can become harmful if pathogens induce prolonged MC activation or trigger aberrant immune responses, causing impaired bacterial clearance [105,106]. MC mediator responses can also become problematic if pathogens or their products escape peripheral defenses and enter the circulation, causing septic shock. Certain pathogens and commensal bacteria also appear to have evolved mechanisms to suppress MC secretory functions, allowing them to colonize the host with minimal inflammatory reactions [107 109]. Prolonged peripheral MCs activation can result in chronic inflammation and tissue remodeling, which is observed with asthma [110]. Staphylococcus aureus-derived peptidoglycan activates MCs and stimulate sustained proinflammatory cytokine production, while the delta-toxin causes MC degranulation [111]. These responses result in chronic skin inflammation, which is aggravated by MCs [111]. Similarly, Chlamydia pneumoniae coopts the capacity of MCs to recruit immune cells into the airspaces of the lung in order to provide an

optimal replicative environment to the pathogen [112]. Indeed, treatment of infected mice with the MC stabilizer cromolyn significantly improves bacterial burden in these mice [113]. Mechanistically, MCs may contribute to detrimental host responses by impeding the phagocytic activities of neighboring macrophages, as has been demonstrated in models of peritoneal polymicrobial infections [105]. Systemic activation of MCs lining the entire vasculature can occur when pathogens or their products overcome the mucosal or skin barrier and reach the bloodstream [29,105,106]. The simultaneous release of mediators from a vast number of MCs into blood can result in severe vascular leakage and shock. In rodent models of polymicrobial sepsis, systemic activation of MCs resulted in extensive release of IL-6 and loss of viability [32]. Similarly, humans with dengue hemorrhagic fever experience systemic MC activation, and the elevated presence of MC protease in their serum is associated with vascular leakage and higher morbidity [114]. Interestingly, blocking of MC degranulation with MC-stabilizing drugs such as ketotifen and cromolyn during severe dengue hemorrhagic fever has been markedly protective in mice studies [114] and may be a potentially beneficial therapy for humans. In view of the prominence in mobilizing innate immune responses, certain host-adapted pathogens and commensal microbes appear to have evolved distinct mechanisms to suppress MCs (Fig. 13.1) [109,115]. Salmonella typhimurium is a highly virulent gut pathogen that is capable of injecting a phosphatase SptP into MCs, completely inhibiting degranulation [109]. As a consequence of this action, neutrophil recruitment to infection sites is delayed, allowing the pathogen to gain entry into mesenteric lymph nodes from the gut [109]. MC suppression was also observed in mucosa infected with human papillomavirus (HPV). Infected squamous epithelial cells were found to recruit MCs to infection sites through the

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release of CCL2 and CCL5 chemokines [116]. Although the suppressive mechanisms remain unclear, MC arrival to the infected mucosa promoted a local microenvironment that appeared to promote a persistent HPV infection and induce precancerous lesions [116]. While MCs can contribute to infectious pathogen-induced detriment to the host, they are also implicated in suppressive immune responses to commensal microbes. The commensal bacteria Bifidobacterium and Lactobacillus, found in the skin and in the gut, have been described as MC suppressors [107,108]. Although the underlying mechanisms appear unclear, commensal microbes are thought to suppress local inflammation to prevent chronic inflammatory disorders. Indeed, MC and inflammation suppression is a property of certain commensal bacteria that partially contributes to their use as probiotics. Delivery of probiotics or their by-products to the gut has been found to quell many gut-associated inflammatory disorders [117]. Thus MC can display very divergent roles in immunity, in which acute local responses, such as at mucosal surfaces and in the skin, enhance the clearance of pathogens while sustained or systemic activation is associated with harmful sequelae.

VIII. CONCLUDING REMARKS AND FUTURE PERSPECTIVES With the rapid upsurge in emergent infectious disease frequency and infections caused by multiresistant bacteria in recent years, there is a dire need for developing nontraditional and effective therapeutic strategies to combat these infections. The recent successes of cancer immunotherapy point to utilizing host immunity as a potential strategy to counter recalcitrant infections. Following the recognition of MCs’ capacity to mediate protective and sometimes destructive inflammatory reactions, several recent studies have revealed the remarkable therapeutic potential of targeting

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MCs either before or during infection with agents that either activate or inhibit their release of bioactive mediators. Since these studies are only at the preclinical stage, there is an urgent need to advance these studies to human trials and investigate their clinical benefit.

Acknowledgements The authors’ work was supported by NIH grants R01-AI068074, R01-AI-096305 and R01-HL-112921 and HHSN272201400054C, NIH Adjuvant Discovery Program Contract.

References [1] Abraham SN, St John AL. Mast cell-orchestrated immunity to pathogens. Nat Rev Immunol 2010;10:440 52. Available from: https://doi.org/ 10.1038/nri2782. [2] Wernersson S, Pejler G. Mast cell secretory granules: armed for battle. Nat Rev Immunol 2014;14:478 94. Available from: https://doi.org/10.1038/nri3690. [3] McLachlan JB, et al. Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection. Nat Immunol 2003;4:1199 205. Available from: https://doi.org/10.1038/ni1005. [4] McLachlan JB, et al. Mast cell activators: a new class of highly effective vaccine adjuvants. Nat Med 2008;14:536 41. Available from: https://doi.org/ 10.1038/nm1757. [5] Galli SJ, Grimbaldeston M, Tsai M. Immunomodulatory mast cells: negative, as well as positive, regulators of immunity. Nat Rev Immunol 2008;8:478 86. Available from: https://doi.org/10.1038/nri2327. [6] Yu M, et al. Mast cells can promote the development of multiple features of chronic asthma in mice. J Clin Invest 2006;116:1633 41. Available from: https://doi. org/10.1172/JCI25702. [7] Bischoff SC. Role of mast cells in allergic and nonallergic immune responses: comparison of human and murine data. Nat Rev Immunol 2007;7:93 104. Available from: https://doi.org/10.1038/nri2018. [8] Nigrovic PA, Lee DM. Mast cells in inflammatory arthritis. Arthritis Res Ther 2005;7:1 11. Available from: https://doi.org/10.1186/ar1446. [9] Gaudenzio N, et al. Different activation signals induce distinct mast cell degranulation strategies. J Clin Invest 2016;126:3981 98. Available from: https://doi.org/ 10.1172/JCI85538. [10] Henningsson F, Hergeth S, Cortelius R, Abrink M, Pejler G. A role for serglycin proteoglycan in granular

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

224

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

13. MAST CELLS FOR THE CONTROL OF MUCOSAL IMMUNITY

retention and processing of mast cell secretory granule components. FEBS J 2006;273:4901 12. Available from: https://doi.org/10.1111/j.1742-4658.2006.05489.x. Abrink M, Grujic M, Pejler G. Serglycin is essential for maturation of mast cell secretory granule. J Biol Chem 2004;279:40897 905. Available from: https://doi.org/ 10.1074/jbc.M405856200. Ringvall M, et al. Serotonin and histamine storage in mast cell secretory granules is dependent on serglycin proteoglycan. J Allergy Clin Immunol 2008;121: 1020 6. Available from: https://doi.org/10.1016/j. jaci.2007.11.031. Garcia-Faroldi G, et al. Polyamines are present in mast cell secretory granules and are important for granule homeostasis. PLoS One 2010;5:e15071. Available from: https://doi.org/10.1371/journal.pone.0015071. Grutzkau A, et al. Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications for the biological significance of VEGF206. Mol Biol Cell 1998;9:875 84. Olszewski MB, Groot AJ, Dastych J, Knol EF. TNF trafficking to human mast cell granules: mature chaindependent endocytosis. J Immunol 2007;178:5701 9. Subba Rao PV, Friedman MM, Atkins FM, Metcalfe DD. Phagocytosis of mast cell granules by cultured fibroblasts. J Immunol 1983;130:341 9. Johansen T. Mechanism of histamine release from rat mast cells induced by the ionophore A23187: effects of calcium and temperature. Br J Pharmacol 1978;63: 643 9. Kunder CA, et al. Mast cell-derived particles deliver peripheral signals to remote lymph nodes. J Exp Med 2009;206:2455 67. Available from: https://doi.org/ 10.1084/jem.20090805. Palm NW, Rosenstein RK, Medzhitov R. Allergic host defences. Nature 2012;484:465 72. Available from: https://doi.org/10.1038/nature11047. Nielsen EH, Clausen J. Electron microscopic study of the regeneration in vivo of rat peritoneal mast cells after histamine secretion. Cell Tissue Res 1982;224:465 8. Fawcett DW. An experimental study of mast cell degranulation and regeneration. Anat Rec 1955;121: 29 51. Theoharides TC, et al. Mast cells and inflammation. Biochim Biophys Acta 2012;1822:21 33. Available from: https://doi.org/10.1016/j.bbadis.2010.12.014. Bischoff SC, et al. IL-4 enhances proliferation and mediator release in mature human mast cells. Proc Natl Acad Sci U S A 1999;96:8080 5. Benyon RC, Robinson C, Church MK. Differential release of histamine and eicosanoids from human skin

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

mast cells activated by IgE-dependent and nonimmunological stimuli. Br J Pharmacol 1989;97: 898 904. Levi-Schaffer F, Shalit M. Differential release of histamine and prostaglandin D2 in rat peritoneal mast cells activated with peptides. Int Arch Allergy Appl Immunol 1989;90:352 7. Gendrin C, et al. Mast cell chymase decreases the severity of group B Streptococcus infections. J Allergy Clin Immunol 2017;. Available from: https://doi.org/ 10.1016/j.jaci.2017.07.042. Thakurdas SM, et al. The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections. J Biol Chem 2007;282:20809 15. Available from: https://doi.org/10.1074/jbc.M611842200. Ebert S, et al. Mast cells expedite control of pulmonary murine cytomegalovirus infection by enhancing the recruitment of protective CD8 T cells to the lungs. PLoS Pathog 2014;10:e1004100. Available from: https://doi.org/10.1371/journal.ppat.1004100. St John AL, et al. Immune surveillance by mast cells during dengue infection promotes natural killer (NK) and NKT-cell recruitment and viral clearance. Proc Natl Acad Sci U S A 2011;108:9190 5. Available from: https://doi.org/10.1073/pnas.1105079108. Metz M, et al. Mast cells can enhance resistance to snake and honeybee venoms. Science 2006;313:526 30. Available from: https://doi.org/10.1126/science.1128877. Malaviya R, Ikeda T, Ross E, Abraham SN. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature 1996;381:77 80. Available from: https://doi.org/ 10.1038/381077a0. Sutherland RE, Olsen JS, McKinstry A, Villalta SA, Wolters PJ. Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by enhancing neutrophil killing. J Immunol 2008;181:5598 605. Shelburne CP, et al. Mast cells augment adaptive immunity by orchestrating dendritic cell trafficking through infected tissues. Cell Host Microbe 2009;6:331 42. Available from: https://doi.org/ 10.1016/j.chom.2009.09.004. Malaviya R, Abraham SN. Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis. J Leukoc Biol 2000;67:841 6. Echtenacher B, Mannel DN, Hultner L. Critical protective role of mast cells in a model of acute septic peritonitis. Nature 1996;381:75 7. Available from: https:// doi.org/10.1038/381075a0. Rodriguez AR, et al. Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis. J Immunol 2012;188:5604 11. Available from: https://doi.org/ 10.4049/jimmunol.1200039.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[37] Ketavarapu JM, et al. Mast cells inhibit intramacrophage Francisella tularensis replication via contact and secreted products including IL-4. Proc Natl Acad Sci U S A 2008;105:9313 18. Available from: https://doi. org/10.1073/pnas.0707636105. [38] Siebenhaar F, et al. Control of Pseudomonas aeruginosa skin infections in mice is mast cell-dependent. Am J Pathol 2007;170:1910 16. Available from: https://doi. org/10.2353/ajpath.2007.060770. [39] Lin TJ, Garduno R, Boudreau RT, Issekutz AC. Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta. J Immunol 2002;169:4522 30. [40] Wershil BK, Castagliuolo I, Pothoulakis C. Direct evidence of mast cell involvement in Clostridium difficile toxin A-induced enteritis in mice. Gastroenterology 1998;114:956 64. [41] Meyer GK, et al. Clostridium difficile toxins A and B directly stimulate human mast cells. Infect Immun 2007;75:3868 76. Available from: https://doi.org/ 10.1128/IAI.00195-07. [42] Di Nardo A, Yamasaki K, Dorschner RA, Lai Y, Gallo RL. Mast cell cathelicidin antimicrobial peptide prevents invasive group A Streptococcus infection of the skin. J Immunol 2008;180:7565 73. [43] Gendrin C, et al. Mast cell chymase decreases the severity of group B Streptococcus infections. J Allergy Clin Immunol 2018;142:120 9. Available from: https://doi.org/10.1016/j.jaci.2017.07.042 e126. [44] Gekara NO, Weiss S. Mast cells initiate early antiListeria host defences. Cell Microbiol 2008;10:225 36. Available from: https://doi.org/10.1111/j.14625822.2007.01033.x. [45] Dietrich N, et al. Mast cells elicit proinflammatory but not type I interferon responses upon activation of TLRs by bacteria. Proc Natl Acad Sci U S A 2010;107:8748 53. Available from: https://doi.org/ 10.1073/pnas.0912551107. [46] Al-Afif A, et al. Respiratory syncytial virus infection of primary human mast cells induces the selective production of type I interferons, CXCL10, and CCL4. J Allergy Clin Immunol 2015;136:1346 54. Available from: https://doi.org/10.1016/j.jaci.2015.01.042 e1341. [47] Orinska Z, et al. TLR3-induced activation of mast cells modulates CD8 1 T-cell recruitment. Blood 2005;106:978 87. Available from: https://doi.org/ 10.1182/blood-2004-07-2656. [48] Shin K, et al. Mouse mast cell tryptase mMCP-6 is a critical link between adaptive and innate immunity in the chronic phase of Trichinella spiralis infection. J Immunol 2008;180:4885 91.

225

[49] Knight PA, Wright SH, Lawrence CE, Paterson YY, Miller HR. Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1. J Exp Med 2000;192:1849 56. [50] Lantz CS, et al. Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature 1998;392:90 3. Available from: https://doi. org/10.1038/32190. [51] Shimokawa C, et al. Mast cells are crucial for induction of group 2 innate lymphoid cells and clearance of helminth infections. Immunity 2017;46:863 74. Available from: https://doi.org/10.1016/j.immuni.2017.04.017 e864. [52] Dahlin JS, Hallgren J. Mast cell progenitors: origin, development and migration to tissues. Mol Immunol 2015;63:9 17. Available from: https://doi.org/ 10.1016/j.molimm.2014.01.018. [53] Kitamura Y, Ito A. Mast cell-committed progenitors. Proc Natl Acad Sci U S A 2005;102:11129 30. Available from: https://doi.org/10.1073/pnas.0505073102. [54] Chen CC, Grimbaldeston MA, Tsai M, Weissman IL, Galli SJ. Identification of mast cell progenitors in adult mice. Proc Natl Acad Sci U S A 2005;102:11408 13. Available from: https://doi.org/10.1073/ pnas.0504197102. [55] Bradding P, Okayama Y, Howarth PH, Church MK, Holgate ST. Heterogeneity of human mast cells based on cytokine content. J Immunol 1995;155:297 307. [56] Enerback L. Mast cells in rat gastrointestinal mucosa. 2. Dye-binding and metachromatic properties. Acta Pathol Microbiol Scand 1966;66:303 12. [57] Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev 1997;77:1033 79. [58] Braga T, et al. Serglycin proteoglycan is required for secretory granule integrity in mucosal mast cells. Biochem J 2007;403:49 57. Available from: https:// doi.org/10.1042/BJ20061257. [59] Welle M. Development, significance, and heterogeneity of mast cells with particular regard to the mast cellspecific proteases chymase and tryptase. J Leukoc Biol 1997;61:233 45. [60] Befus AD, Pearce FL, Gauldie J, Horsewood P, Bienenstock J. Mucosal mast cells. I. Isolation and functional characteristics of rat intestinal mast cells. J Immunol 1982;128:2475 80. [61] Goetzl EJ, Chernov T, Renold F, Payan DG. Neuropeptide regulation of the expression of immediate hypersensitivity. J Immunol 1985;135:802s 5s. [62] Cheng LE, Hartmann K, Roers A, Krummel MF, Locksley RM. Perivascular mast cells dynamically probe cutaneous blood vessels to capture immunoglobulin E. Immunity 2013;38:166 75. Available from: https://doi. org/10.1016/j.immuni.2012.09.022.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

226

13. MAST CELLS FOR THE CONTROL OF MUCOSAL IMMUNITY

[63] Schwartz C, et al. Basophil-mediated protection against gastrointestinal helminths requires IgEinduced cytokine secretion. Proc Natl Acad Sci U S A 2014;111:E5169 5177. Available from: https://doi. org/10.1073/pnas.1412663111. [64] Patella V, Florio G, Petraroli A, Marone G. HIV-1 gp120 induces IL-4 and IL-13 release from human Fc epsilon RI 1 cells through interaction with the VH3 region of IgE. J Immunol 2000;164:589 95. [65] Mukai K, Tsai M, Starkl P, Marichal T, Galli SJ. IgE and mast cells in host defense against parasites and venoms. Semin Immunopathol 2016;38:581 603. Available from: https://doi.org/10.1007/s00281-016-0565-1. [66] Gurish MF, et al. IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis. J Immunol 2004;172:1139 45. [67] Enoksson M, et al. Mast cells as sensors of cell injury through IL-33 recognition. J Immunol 2011;186:2523 8. Available from: https://doi.org/10.4049/ jimmunol.1003383. [68] Lunderius-Andersson C, Enoksson M, Nilsson G. Mast cells respond to cell injury through the recognition of IL-33. Front Immunol 2012;3:82. Available from: https://doi.org/10.3389/fimmu.2012.00082. [69] Qiao H, Andrade MV, Lisboa FA, Morgan K, Beaven MA. FcepsilonR1 and toll-like receptors mediate synergistic signals to markedly augment production of inflammatory cytokines in murine mast cells. Blood 2006;107:610 18. Available from: https://doi.org/ 10.1182/blood-2005-06-2271. [70] Gondokaryono SP, et al. The extra domain A of fibronectin stimulates murine mast cells via toll-like receptor 4. J Leukoc Biol 2007;82:657 65. Available from: https://doi.org/10.1189/jlb.1206730. [71] Olynych TJ, Jakeman DL, Marshall JS. Fungal zymosan induces leukotriene production by human mast cells through a dectin-1-dependent mechanism. J Allergy Clin Immunol 2006;118:837 43. Available from: https://doi.org/10.1016/j.jaci.2006.06.008. [72] Varadaradjalou S, et al. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells. Eur J Immunol 2003;33:899 906. Available from: https:// doi.org/10.1002/eji.200323830. [73] McCurdy JD, Lin TJ, Marshall JS. Toll-like receptor 4mediated activation of murine mast cells. J Leukoc Biol 2001;70:977 84. [74] Supajatura V, et al. Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J Clin Invest 2002;109:1351 9. Available from: https://doi.org/10.1172/JCI14704. [75] Sandig H, Bulfone-Paus S. TLR signaling in mast cells: common and unique features. Front Immunol 2012;3:185. Available from: https://doi.org/10.3389/ fimmu.2012.00185.

[76] Wang Z, et al. Skin microbiome promotes mast cell maturation by triggering stem cell factor production in keratinocytes. J Allergy Clin Immunol 2017;139:1205 16. Available from: https://doi.org/ 10.1016/j.jaci.2016.09.019 e1206. [77] Malbec O, Daeron M. The mast cell IgG receptors and their roles in tissue inflammation. Immunol Rev 2007;217:206 21. Available from: https://doi.org/ 10.1111/j.1600-065X.2007.00510.x. [78] Woolhiser MR, Okayama Y, Gilfillan AM, Metcalfe DD. IgG-dependent activation of human mast cells following up-regulation of FcgammaRI by IFN-gamma. Eur J Immunol 2001;31:3298 307. Available from: https://doi.org/10.1002/1521-4141(200111) 31:113.0.CO;2-U. [79] Zhao W, et al. Fc gamma RIIa, not Fc gamma RIIb, is constitutively and functionally expressed on skinderived human mast cells. J Immunol 2006;177:694 701. [80] Kepley CL, et al. Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigeninduced secretion and involves SHIP-Grb2-Dok complexes. J Biol Chem 2004;279:35139 49. Available from: https://doi.org/10.1074/jbc.M404318200. [81] Katz HR, Lobell RB. Expression and function of Fc gamma R in mouse mast cells. Int Arch Allergy Immunol 1995;107:76 8. Available from: https://doi. org/10.1159/000236936. [82] Malbec O, et al. Peritoneal cell-derived mast cells: an in vitro model of mature serosal-type mouse mast cells. J Immunol 2007;178:6465 75. [83] Malbec O, et al. Fc epsilon receptor I-associated lyndependent phosphorylation of Fc gamma receptor IIB during negative regulation of mast cell activation. J Immunol 1998;160:1647 58. [84] Maurer M, et al. Mast cells promote homeostasis by limiting endothelin-1-induced toxicity. Nature 2004;432:512 16. Available from: https://doi.org/ 10.1038/nature03085. [85] Johnson D, Krenger W. Interactions of mast cells with the nervous system--recent advances. Neurochem Res 1992;17:939 51. [86] Nilsson G, et al. C3a and C5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway. J Immunol 1996;157:1693 8. [87] Subramanian H, Gupta K, Guo Q, Price R, Ali H. Masrelated gene X2 (MrgX2) is a novel G protein-coupled receptor for the antimicrobial peptide LL-37 in human mast cells: resistance to receptor phosphorylation, desensitization, and internalization. J Biol Chem 2011;286:44739 49. Available from: https://doi.org/ 10.1074/jbc.M111.277152. [88] Di Nardo A, Vitiello A, Gallo RL. Cutting edge: mast cell antimicrobial activity is mediated by expression of

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

227

REFERENCES

cathelicidin antimicrobial peptide. J Immunol 2003;170:2274 8. [89] Malaviya R, Gao Z, Thankavel K, van der Merwe PA, Abraham SN. The mast cell tumor necrosis factor alpha response to FimH-expressing Escherichia coli is mediated by the glycosylphosphatidylinositolanchored molecule CD48. Proc Natl Acad Sci U S A 1999;96:8110 15. [90] Mollerherm H, von Kockritz-Blickwede M, BranitzkiHeinemann K. Antimicrobial activity of mast cells: role and relevance of extracellular DNA traps. Front Immunol 2016;7:265. Available from: https://doi. org/10.3389/fimmu.2016.00265. [91] Caughey GH. Mast cell proteases as protective and inflammatory mediators. Adv Exp Med Biol 2011;716:212 34. Available from: https://doi.org/ 10.1007/978-1-4419-9533-9_12. [92] Schneider LA, Schlenner SM, Feyerabend TB, Wunderlin M, Rodewald HR. Molecular mechanism of mast cell mediated innate defense against endothelin and snake venom sarafotoxin. J Exp Med 2007;204:2629 39. Available from: https://doi.org/ 10.1084/jem.20071262. [93] Datta YH, et al. Peptido-leukotrienes are potent agonists of von Willebrand factor secretion and P-selectin surface expression in human umbilical vein endothelial cells. Circulation 1995;92:3304 11. [94] Jawdat DM, Rowden G, Marshall JS. Mast cells have a pivotal role in TNF-independent lymph node hypertrophy and the mobilization of Langerhans cells in response to bacterial peptidoglycan. J Immunol 2006;177:1755 62. [95] Stelekati E, et al. Mast cell-mediated antigen presentation regulates CD8 1 T cell effector functions. Immunity 2009;31:665 76. Available from: https:// doi.org/10.1016/j.immuni.2009.08.022. [96] Gong J, et al. The antigen presentation function of bone marrow-derived mast cells is spatiotemporally restricted to a subset expressing high levels of cell surface FcepsilonRI and MHC II. BMC Immunol 2010;11:34. Available from: https://doi.org/10.1186/ 1471-2172-11-34. [97] Piconese S, et al. Mast cells counteract regulatory Tcell suppression through interleukin-6 and OX40/ OX40L axis toward Th17-cell differentiation. Blood 2009;114:2639 48. Available from: https://doi.org/ 10.1182/blood-2009-05-220004. [98] Amaral MM, et al. Histamine improves antigen uptake and cross-presentation by dendritic cells. J Immunol 2007;179:3425 33. [99] Ierna MX, Scales HE, Saunders KL, Lawrence CE. Mast cell production of IL-4 and TNF may be required for protective and pathological responses in

[100]

[101]

[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

gastrointestinal helminth infection. Mucosal Immunol 2008;1:147 55. Available from: https://doi.org/ 10.1038/mi.2007.16. St John AL, Chan CY, Staats HF, Leong KW, Abraham SN. Synthetic mast-cell granules as adjuvants to promote and polarize immunity in lymph nodes. Nat Mater 2012;11:250 7. Available from: https://doi.org/10.1038/nmat3222. Aoki I, et al. Contribution of mast cells to the T helper 2 response induced by simultaneous subcutaneous and oral immunization. Immunology 1999;98:519 24. Sherman MA, Secor VH, Lee SK, Lopez RD, Brown MA. STAT6-independent production of IL-4 by mast cells. Eur J Immunol 1999;29:1235 42. Available from: https://doi.org/10.1002/(SICI)1521-4141 (199904)29:04 , 1235::AID-IMMU1235 . 3.0.CO;2-0. Wasiuk A, et al. Mast cells impair the development of protective anti-tumor immunity. Cancer Immunol Immunother 2012;61:2273 82. Available from: https://doi.org/10.1007/s00262-012-1276-7. Mazzoni A, Siraganian RP, Leifer CA, Segal DM. Dendritic cell modulation by mast cells controls the Th1/Th2 balance in responding T cells. J Immunol 2006;177:3577 81. Dahdah A, et al. Mast cells aggravate sepsis by inhibiting peritoneal macrophage phagocytosis. J Clin Invest 2014;124:4577 89. Available from: https://doi. org/10.1172/JCI75212. Seeley EJ, Sutherland RE, Kim SS, Wolters PJ. Systemic mast cell degranulation increases mortality during polymicrobial septic peritonitis in mice. J Leukoc Biol 2011;90:591 7. Available from: https:// doi.org/10.1189/jlb.0910531. Harata G, et al. Bifidobacterium suppresses IgEmediated degranulation of rat basophilic leukemia (RBL-2H3) cells. Microbiol Immunol 2010;54:54 7. Available from: https://doi.org/10.1111/j.13480421.2009.00185.x. Forsythe P, Wang B, Khambati I, Kunze WA. Systemic effects of ingested Lactobacillus rhamnosus: inhibition of mast cell membrane potassium (IKCa) current and degranulation. PLoS One 2012;7:e41234. Available from: https://doi.org/10.1371/journal. pone.0041234. Choi HW, et al. Salmonella typhimurium impedes innate immunity with a mast-cell-suppressing protein tyrosine phosphatase, SptP. Immunity 2013;39:1108 20. Available from: https://doi.org/ 10.1016/j.immuni.2013.11.009. Brightling CE, et al. Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med 2002;346:1699 705. Available from: https://doi.org/ 10.1056/NEJMoa012705.

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13. MAST CELLS FOR THE CONTROL OF MUCOSAL IMMUNITY

[111] Nakamura Y, et al. Staphylococcus delta-toxin induces allergic skin disease by activating mast cells. Nature 2013;503:397 401. Available from: https:// doi.org/10.1038/nature12655. [112] Chiba N, et al. Mast cells play an important role in Chlamydia pneumoniae lung infection by facilitating immune cell recruitment into the airway. J Immunol 2015;194:3840 51. Available from: https://doi.org/ 10.4049/jimmunol.1402685. [113] Finn DF, Walsh JJ. Twenty-first century mast cell stabilizers. Br J Pharmacol 2013;170:23 37. Available from: https://doi.org/10.1111/bph.12138. [114] St John AL, Rathore AP, Raghavan B, Ng ML, Abraham SN. Contributions of mast cells and vasoactive products, leukotrienes and chymase, to dengue

virus-induced vascular leakage. eLife 2013;2:e00481. Available from: https://doi.org/10.7554/eLife.00481. [115] Melendez AJ, et al. Inhibition of Fc epsilon RI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes. Nat Med 2007;13: 1375 81. Available from: https://doi.org/10.1038/ nm1654. [116] Bergot AS, et al. HPV16-E7 expression in squamous epithelium creates a local immune suppressive environment via CCL2- and CCL5- mediated recruitment of mast cells. PLoS Pathog 2014;10:e1004466. Available from: https://doi.org/10.1371/journal.ppat.1004466. [117] Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell 2014;157:121 41. Available from: https://doi.org/10.1016/j.cell.2014.03.011.

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Innate Lymphoid Cells for the Control of Mucosal Immunity Randy S. Longman, Maeva Metz and David Artis Jill Roberts Institute for Research in IBD, Weill Cornell Medicine, New York, NY, United States

I. INTRODUCTION The discovery of natural killer (NK) cells and lymphoid tissue inducer (LTi) cells marked the conceptual emergence of a group of innate lymphoid cells (ILCs), which, in contrast to adaptive lymphocytes, develop independently of somatic recombination and lack rearranged antigen receptors [1 3]. Although the characterization of prototypical conventional NK cells (cNKs) and LTi cells occurred decades earlier, advances in flow cytometry enabled the identification of alternative ILC sources of type 1 (interferon gamma), type 2 (interleukin 5 (IL-5) and IL-13), and type 3 (IL-17 and IL-22) cytokines [4,5]. These ILCs share the expression of IL-7 receptor α (CD127) and common gamma chain (γc) or IL-2 receptor subunit gamma (IL2RG) consistent with their identification as ILCs, but are developmentally and functionally distinct from cytotoxic cNK cells. Genetic, developmental, and functional studies identified subsets of these noncytotoxic ILCs as innate counterparts of conventional helper T cells, now termed ILC1, ILC2, and ILC3 [1,4]. In

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00014-6

contrast to T cells, ILCs reside predominantly in mucosal tissue, where they orchestrate critical functions of mucosal immunity in health and disease. In addition to extensive recent reviews on ILCs [2,6,7], this chapter will focus on the functional contribution and regulation of mucosal ILCs to barrier immunity.

II. INNATE LYMPHOID CELL DEVELOPMENT AND TISSUE HETEROGENEITY Similar to adaptive immune cells, ILCs develop in the fetal liver and adult bone marrow from common lymphoid progenitor (CLP) cells [8 10]. Although somatic recombination required for antigen-specific receptor generation is not required, ILC development through various precursor populations is under tight transcriptional control [7]. All ILC subsets differentiate from a CLP in a process that requires inhibitor of DNA binding 2 (ID2) and nuclear factor interleukin 3 (NFIL3) (Fig. 14.1) [11 13]. Mechanistically, mesenchyme-derived IL-7

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FIGURE 14.1 ILC development and heterogeneity at mucosal surfaces. All ILCs develop from a common lymphoid progenitor (CLP). Common innate lymphoid progenitor (CILP) cells differentiate into NK cell precursors (NKP) and common helper innate lymphoid precursors (CHILP). CHILPs give rise to all helper ILC subsets, as well as lymphoid tissue inducer (LTi) cells. Development and cytokine production are tightly regulated by sequential transcription factor expression and/or exposure to the indicated stimuli. Source: Illustration by Maeva Metz. III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

II. INNATE LYMPHOID CELL DEVELOPMENT AND TISSUE HETEROGENEITY

signaling induces NFIL3, which subsequently triggers Id2 and ILC lineage development [14]. However, NFIL3-deficient mice do not fully replicate the ILC deficiency seen ID2-deficient mice, suggesting a potential role for alternative or redundant pathways in driving Id2 [14,15]. Additional transcriptional factors, including TOX [16,17], TCF7 [18,19], and GATA-3 [20 22], are required to generate cNK precursors as well as common helper innate lymphoid precursors (CHILPs). CHILPs gives rise to all key subsets of the mucosal helper ILCs [6,9]. Notch-dependent pathways in adult bone marrow allow for the upregulation of α4β7 and CXCR6, which enables intestinal homing of conventional CCR61 LTi cells [8,23]. Promyelocytic leukemia zinc finger (PLZF)dependent signaling is required for development of an ILC precursor (ILCP) from a CHILP that is capable of differentiating into a conventional ILC1, ILC2, and CCR6 2 ILC3 precursors in the tissue [9]. While conventional analysis has identified phenotypically and functionally distinct ILC subsets, unbiased characterization of tissue ILC subsets with next-generation sequencing technologies revealed additional cellular clusters that reflect either additional ILC subsets or an unrecognized plasticity between the subsets [24,25]. In humans, ILC precursors exist in the circulating blood and secondary lymphoid tissue [26,27]. In particular, circulating CD1171 ILC precursors can differentiate into all ILC subsets, including cNK. Although these CD1171 ILC precursors do not express transcriptional signatures of ILC differentiation, they have epigenetic modifications that may facilitate rapid differentiation in response to tissue-specific signals [28]. ILC1s are functionally characterized by their production of type 1 cytokines interferon gamma (IFNγ) and TNFα in response to IL-12 stimulation [1]. This subset reflects a heterogeneous group of ILCs that includes cNK cells, CD1271 NKp461 ILC1s, and CD1272 NKp441 intraepithelial ILC1s. While cNK cells require

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T-box transcription factors Eomesodermin (EOMES) and IL-15 for development, ID2 [29] and T-BET [30] are required for terminal maturation and not lineage specification. In contrast, CD1271 ILC1s require T-BET, ID2, and GATA3 for development and maintenance [10]. Despite these differences from cNK cells in transcriptional regulation of development, CD1271 ILC1s upregulate IL-2Rβ (CD122) and downregulate CD127 in maintenance and differentiation, revealing an underlying shared requirement for IL-15. Intraepithelial ILC1s are a smaller group of ILC1s in the mucosa that express CD103. They develop independently of IL-15 but require both EOMES and T-BET [31]. Functionally, both cNK cells and CD1031 ILC1s can additionally express granzyme and perforin, while promyelocytic leukemia zinc finger (PZLF)-dependent ILC1s produce TNFα [10]. ILC2s are abundant in both lung and gut mucosal tissues as well as white adipose depots in mice and humans [32]. Functionally, they are responsive to epithelial signals and secrete effectors molecules to help protect and restore the barrier. ILC2s express receptors for epithelial mucosa derived signals, including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the tissue chemoattractant prostaglandin D2 receptor (CRTh2). In response to stimulation, ILC2s produce type 2 effector cytokines (IL-4, IL-5, IL-9, IL-13) and the epidermal growth factor (EGF)-like molecule amphiregulin [32]. GATA-3 is required for the development and maintenance of these cells in both humans and mice [22,33]. ILC3s are most abundant in the gut mucosal tissue and functionally play a critical role in gut lymphoid organogenesis, tissue repair, and inflammation [34]. All ILC3s require the transcription factor RORγt, but substantial heterogeneity exists in ILC3 subsets. LTis were the first group of ILC3s described and play a primary role in lymphoid organogenesis in the gut [35,36]. Through the expression of α4β7, LTi

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cells home to intestine and interact with stromal cells to direct the formation of Peyer’s patches and isolated lymphoid follicles (ILFs). This process of lymphogenesis requires lymphotoxin-α/β as well as intercellular adhesion molecule/vascular cell adhesion molecule dependent recruitment [37]. Phenotypically, LTi cells express CCR6 with or without CD4 surface expression. In addition to LTi cells, CCR6 2 ILC3s exist throughout the skin and intestinal lamina propria, including a subset of lymphocytes expressing NKp46 (termed NCR1 ILC3s) (Fig. 14.1) [38 40]. Both CCR61 and CCR6 2 ILC3s can secrete effector cytokines IL-22 and IL-17 in response to dendritic cell (DC) and macrophage-derived IL-1β, IL-6, IL23, and TNF-like ligand 1A (TL1A) [6,41]. In contrast to LTi cells, both NCR1 and NCR2 CCR62 ILC3s require PLZF for development [9]. The majority of CCR62 ILC3s express TBET and require microbiota and Ahr for development [42]. T-BET expression in ILC3s can downregulate RORγt and lead to an inflammatory phenotype associated with IFNγ production [39,43]. These ex-RORγt1 ILCs reveal the heterogeneity and potential plasticity in the ILC subsets.

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A. Cellular Regulation of Mucosal ILCs Localization to the mucosal tissue is a key characteristic of ILC immunity. Although all adult ILCs develop from bone marrow precursors, noncytolytic ILCs localize primarily to mucosal tissues, whereas cNK cells localize to secondary lymphoid organs [34]. Recruitment of ILC precursors is tightly regulated by α4β7

expression and interaction with mucosal vascular addressin cell adhesion molecule 1 (MadCAM-1). At steady state or during inflammatory or infectious conditions, tissue ILCs rarely repopulate from a hematogenous source [44]. Instead, parabiosis experiments have revealed evidence for the local proliferation of tissue-resident ILCs, whose function is dictated by the local microenvironment and availability of IL-2 [45]. In situ, these tissue ILCs reciprocally shape intestinal immunity and the gut microbiota. Membrane-bound LTα1β2 is required for T-cell-independent IgA, while soluble LTα3 is required for T cell dependent IgA, which subsequently alters the composition of the intestinal microbiome [46]. Tissue localization of ILCs enables them to respond quickly to barrier insults. Conventional NK cell responses are triggered by ligands that are expressed (or not expressed) by nonhealthy self [47]. For example, the activating receptor natural killer group 2D (NKG2D) recognizes induced-self protein ligands expressed on stressed epithelial cells. The activating receptor NKp44 is expressed on both ILC1s and NCR1 ILC3s and triggers increased proinflammatory cytokines [48], but an essential function of these receptors on noncytotoxic ILCs is not clear. Inhibitory Ly49 family receptors play a critical role in balancing the activation of cNK cells [47]. ILC1s, by contrast, are devoid of inhibitory receptors that recognize major histocompatibility complex class I (MHCI) but also lack cytotoxic capacity, which may obviate the need for this tight regulation [10]. In lieu of other innate receptors expressed by cNK, ILCs have evolved a tightly regulated dialogue with epithelial cell signals to facilitate a rapid response. In the lung, epithelial cell derived TSLP, IL-25, and IL-33 contribute to the ILC2 inflammatory response to environmental triggers, including allergens, viral infection, and helminth challenge [49]. In the intestine, chemosensory epithelial cells called

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tuft cells are a critical source of IL-25. Subsequent secretion of IL-13 by ILC2s induces tuft cell hyperplasia and links ILC2 activation with epithelial cell derived signals in a regulatory circuit [50 52]. Commensal-dependent epithelial cell derived IL-25 can simultaneously act on DCs to suppress ILC3 effector function at steady state [53]. During inflammation, reduction in IL-25 relieves tonic inhibition of IL-22 production. ILC3 production of IL-22, in turn, feeds back on the epithelium to promote mucosal healing. In particular, Lgr51 stem cells express the IL-22R and mediate a STAT3-dependent regulation of epithelial regeneration [54]. A critical feature of innate immunity includes rapid recognition of pathogenassociated molecular patterns (PAMPs) to initiate immune responses. Human tonsillar ILCs can express toll-like receptors (TLRs), including TLR1, TLR2, TLR5, TLR6, TLR7, and TLR9 [55] (Chapter 6: Innate Immunity at Mucosal Surfaces). Stimulation of human, but not mouse, ILCs with TLR2 ligands triggers proliferation and IL-22 cytokine production (Crellin et al., 2010). In addition to direct recognition of PAMPs by human ILCs, both human and mouse mucosal ILCs rely on a complex network of DCs and mononoculear phagocytes (MNPs) to sense PAMPs. Stimulation with intravenous TLR5 ligand flagellin [56] or TLR7 agonist R848 [57] can induce conventional, migratory CD1031 DC production of IL-23, which subsequently induces ILC3 production of IL-22 in secondary lymph organs [58,59]. In the intestine, CX3CR11 MNPs maintain regulated access to luminal microbiota and PAMPs through transepithelial dendrites [60,61]. PAMP-stimulated MNPs can rapidly regulate mucosal ILC3 effector cytokine secretion through the production of IL-23, IL-1β, and TL1A [41]. MNPs and ILC3s spatially colocalize in tissue to promote this circuitry [41]. Tissueresident CX3CR11 MNPs express CXCL16 and enable colocalization with CXCR61 NKp461 ILC3s in the lamina propria [62]. Spatial colocalization in the mucosa also enables ILC2s to

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respond rapidly. In the lung and the skin, basophils can trigger an ILC2 response in an IL-4dependent manner following allergen exposure [63,64]. In addition, the production of prostaglandin D2 (PGD2) by mast cells that colocalize with ILC2s in lung tissue can stimulate an IL-13 response [65]. These cellular circuits enable rapid and selective regulation of mucosal ILC function.

B. Enteric Nervous System Regulates Mucosal Innate Lymphoid Cells The enteric nervous system has recently been recognized as a key regulator of mucosal ILC function. The receptor tyrosine kinase (RET), which is required for mammalian enteric nervous system formation, is highly expressed by LTi cells and required for Peyer’s patch formation [66] (Chapters 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance). Enteric glial cell ILC3 interactions via RET ligands remain active in the adult mouse. Detection of PAMPs by glial-cell-intrinsic MYD88 is required to regulate ILC3-intrinsic RET-dependent production of IL-22, and ILC3-specific deletion of Ret results in an increased sensitivity to chemical and infectious colitis [67]. The connection with the enteric nervous system also regulates ILC2 function. Simultaneous reports revealed ILC2-specific expression of the neuromedin U (NMU) receptor and the colocalization of ILC2s with NMU expressing cholinergic neurons in the lung and gut mucosal tissue [68 70]. NMU enhances ILC2 proliferation and cytokine production of IL-5, IL-9, and IL-13 in synergy with IL-25 [68 70]. Thus the NMU receptor is required in vivo for eosinophil recruitment and accelerated helminth expulsion. In addition, the β2-adrenergic receptor (β2AR) is highly expressed on small intestinal ILC2s and, in contrast to cholinergic signals,

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neuronal adrenergic signals act to repress ILC2 proliferation and function [71]. In the lung and gut, β2AR agonists negatively regulate ILC2dependent responses to helminth infection [71]. Together, these findings indicate an evolutionarily conserved group of molecular mechanisms through which the neuronal system and ILCs interact to maintain mucosal tissue homeostasis.

C. Microbial and Metabolic Regulation of Mucosal Innate Lymphoid Cells Spatial localization of ILCs at the mucosal barrier affords a unique opportunity for the microbiota to shape their development and function. While LTi cells derived from fetal liver seed the intestine and develop even in the absence of commensal microbiota [53], NCR1 ILC3s are reduced in frequency and number [40] and function [38]. Specific commensals that reside within the intestinal lymphoid tissues [72] or that tightly adhere to the epithelial mucosa can promote the accumulation and function of ILC3s in the intestinal lamina propria [73 75]. Although phenotypically normal ILC2s develop under germ-free conditions [76], the proportion of ILC2s appears higher, owing to a decrease in other microbe-dependent populations [77]. The impact of the microbiota is also seen at the transcriptional and epigenetic levels. Antibiotic treatment diminishes lineagespecific H3K4me2 methylation marks of Tcf7, cd93, and Gjb2 loci, and ILC1s and ILC2s acquire transcriptional elements associated with ILC3s [24]. Consistent with functional reports showing the microbiome dependency of ILC1 and ILC3 plasticity [39,78], antibiotic treatment results in a reduction in Il17a across all subsets, particularly in Tbx21 expressing clusters of ILC3s [24] (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity). In addition to regulation by the microbiota, mucosal ILCs can be affected by exposure to dietary and environmental metabolites. Caloric intake can trigger circadian rhythm dependent

vasoactive intestinal peptide (VIP) release, which acts on ILC2s (through VIP receptor type 2) to regulate IL-5- and IL-13-dependent eosinophil recruitment [79]. A delay in eosinophil response to allergic challenge during periods of reduced caloric intake or malnutrition highlights the importance of dietary signals in mucosal ILC2 function. Vitamin A deficiency occurring during chronic malnutrition attempts to limit this impairment of mucosal immunity during nutritional deficiency. In particular, vitamin A deficiency results in an increase in mucosal ILC2s and enhanced production of IL13 [80]. This response allows the host to sustain barrier immunity to helminth infection in the face of malnutrition. Transcriptional profiling of ILC2s identified RARγ and PPARγ, which may mediate the direct sensing of vitamin A metabolites [25]. Additional genes (including Dgat2, Mc5r, and Alox5) reveal the potential importance of lipid sensing and fatty acid metabolism in ILC2s to fuel the barrier response when dietary nutrients are limited [81]. In contrast to states of deficiency, arginase 1 (Arg1) plays an ILC2 cell-intrinsic role in arginine catabolism and aerobic glycolysis, serving as a metabolic checkpoint in the development of type 2 airway inflammation [82]. Collectively, these dietary and environmental metabolic cues serve to both enhance and restrain the function of ILC2s in mucosal tissue. The metabolic environment similarly shapes the development and function of mucosal ILC3s. Dietary retinoids are key regulators of prenatal ILCs. Deficiency in retinoic acid (RA) signaling impairs secondary lymphoid organ generation in the gut and lung and can alter the ability to clear mucosal viral infection [83] (Chapter 12: Influence of Dietary Components and Commensal Bacteria on the Control of Mucosal Immunity). In addition to RA, metabolism of dietary tryptophan by commensals to yield aryl hydrocarbon receptor (Ahr) ligand indo-3-carbinol in pregnant dams is sufficient to increase mucosal NCR1 ILC3s and protect against bacterial translocation in the progeny [84].

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IV. MUCOSAL INNATE LYMPHOID CELLS IN PATHOGEN DEFENSE

Next-generation sequencing platforms have identified ILC subset-specific intracellular metabolism that may couple unique metabolic profiles with functional activities of ILC subsets. These include mTOR and Notch signaling in ILC1s, sphingolipid and amino acid metabolism in ILC2s, and carbohydrate metabolism in ILC3s [24]. In particular, hexokinase (Hk2 and Hk3), enolase-1, and phosphofructokinase expression in ILC3 may reflect a critical dependence on active glycolysis. In addition, although Arg1 is expressed in both ILC2s and ILC3s [24], ILC2s have an additional peak upstream of the transcriptional start site that may reflect regulated transcriptional control of subset-specific metabolism. Additional studies are needed to define epigenetic and post-translational changes that may also contribute to this functional regulation.

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IV. MUCOSAL INNATE LYMPHOID CELLS IN PATHOGEN DEFENSE As primary producers of mucosal IL-22, ILC3s play a critical role in the response to attaching and effacing gut bacteria [6,38,85]. IL22 acts primarily on epithelial cells to stimulate antimicrobial peptides (REG3G, REG3B), lipocalin, and mucus production, which reinforce barrier protection. In addition to this direct antimicrobial effect, IL-22 induces epithelial fucosyltransferase 2 (Fut2) [86 88] (Fig. 14.2). FUT2 allows for fucosylation of oligosaccharides at the mucosal border and provides a key nutrient for establishing a healthy and diverse microbiome. Mice that lack fucosyltransferase have abnormal barrier function that renders the host more susceptible to enteric bacterial

FIGURE 14.2 Organizing and initiating ILC-dependent barrier immunity. Microbial triggers at the mucosal barrier and local tissue factors regulate effector function of mucosal ILCs. Spatial colocalization of ILCs with dendritic cells, macrophages, and mononuclear phagocytes (MNPs) facilitates the integration of these signals and regulates effector functions. Specialized epithelial cells such as tuft cells and enteric neurons also stimulate barrier immunity and tissue repair functions. Source: Illustration by Maeva Metz. III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

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infections. ILC3 production of lymphotoxin α1β2 regulates the production of IL-22 during infection and helps to restore the structure of colonic patches and ILFs [89]. LTβR signaling on DCs promotes this positive feedback loop on ILC3 production of IL-22 and pathogen defense [90]. Adherent commensal microbiota (including segmented filamentous bacteria and adherent-invasive Escherichia coli) can also induce ILC3 production of IL-22 and effector cytokines in the absence of disease [73,75], and both offer protection from chemical dextran sodium sulfate induced or infectious Citrobacter colitis [91]. Mucosal release of prostaglandin E2, signaling through EP4 on ILC3s, promotes gut IL-22 and blocks lipopolysaccharide translocation, leading to systemic inflammation [92]. Collectively, these responses protect the mucosal barrier from invasive Gram-negative bacterial infections. Intestinal ILC3 production of IL-22 also plays an important role in protecting against mucosal viral infection. Rotavirus infection is the most common diarrheal disease in children. Epithelial cell production of IL-1α and TLR5 activation of DCs following rotavirus infection is required for induction of IL-22-producing ILC3s [93] (Fig. 14.2). In addition, cooperation of IL-22 with IFNλ is required for optimal STAT1 activation and viral clearance [94]. While ILC3s play an effector role primarily at the intestinal barrier, protective effects are also seen at other mucosal sites. Commensaldependent upregulation of the lung homing CC chemokine receptor 4 (CCR4) allows for NCR1 ILC3 influx into the lung shortly after birth and protects against intratracheal challenge with Streptococcus pneumoniae [95]. In addition to IL-22, ILC3 production of IL-17 reinforces barrier immunity in oropharyngeal infections. Experiments in recombinationactivating gene (RAG)-deficient mice revealed a key role for CCR61 ILC3 production of IL-17 and IL-22 in oropharyngeal barrier defense [96]. Similarly, in humans, biallelic disruption

of RORC gene is associated with increased susceptibility to infection with mucosal pathogens Candida and Mycobacterium [97]. A notable limitation in assigning the role for ILCs in mucosal immunity is that the majority of data supporting the essential role for ILCs derives from T- and B-cell-deficient mice. Although ILC3s are major early producers of IL-22, T cell derived IL-22 and B cells contribute significantly to pathogen resistance later in infection [98]. Apparent redundancy in these cellular responses may reflect quantitative or temporal differences in exposure. Selective targeting of NCR1 ILC3s during Citrobacter infection revealed that this subset is dispensable for response in the presence of T cells [99,100]. However, this is consistent with data in RAGdeficient mice showing a critical role for CD41 ILC3s and may suggest qualitative differences in the contribution of ILC3 subsets depending on the kinetics and dose of infection. As robust producers of type 2 cytokines IL-4, IL-5, and IL-13, ILC2s play an essential role in mediating helminth immunity in the lung and gut. IL-4 and IL-13 can support antimicrobial peptide production by goblet cells, including RELMβ, to limit parasite infection [101]. In response to epithelial cell derived cytokines IL-25 and IL-33, ILC2s support B1 cell proliferation and goblet cell hyperplasia to further curtail helminth infection [102 104]. ILC2 immunity is sufficient to clear intestinal infections of both Nippostrongylus brasiliensis and Trichuris muris even in the absence of adaptive immunity. Closer analysis of prototypical Th1dependent infections has revealed a central role for ILC1s in pathogen control at the mucosal barrier as well. In response to the IFNγ-dependent Toxoplasma gondii infection, ILC1s were found to be the main producers of IFNγ and TNFα in response to oral challenge [10]. Transfer of CHILP-derived ILC1s blocked infection of alymphoid (RAG-, IL2RG-deficient) mice and restored recruitment of inflammatory

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monocytes. Similarly, ILC1s mediated T-BET and IFNγ-dependent protection from Clostridium difficile infection in synergy with an ILC3 response [105]. These data reveal an essential contribution of ILC1s to the mucosal immune response dependent on conventional Th1 cytokines.

V. INNATE LYMPHOID CELLS IN MUCOSAL TISSUE REPAIR In addition to their response in pathogen clearance, ILC2s play an equally important role in limiting disease by initiating tissue repair. In influenza infection of the lung, for example, ILC2s are not responsible for innate control of viral infection, but they are essential for restoring airway integrity [76]. This function is mediated by IL-33, but is independent of IL-13 and IL-22. In this capacity, IL-33 triggers ILC2 production of EGF family member amphiregulin, which can act directly on the lung epithelium to stimulate regeneration. This repair process similarly plays a critical role following intestinal inflammation and links mucosal ILCs with a central EGF pathway critical in supporting intestinal epithelial cell renewal [105]. Following helminth infection, lung ILC2s are the main producers of IL-9, which acts in an autocrine fashion to regulate IL-5, IL-13, and amphiregulin and to promote tissue repair in the recovery phase of the infection [107]. ILC3s also play a role in tissue repair following injury. The disruption of lymphoid tissue architecture following mucosal infection can be restored by ILC3 expression of LTα1β2 [108]. Similarly, in response to radiation-induced thymic damage, ILC3 production of IL-22 orchestrates thymic tissue repair [109]. This tissue repair function plays an essential role in restoring immune competence following infection or injury. ILC3s also play a local role in tissue repair following lung or intestinal mucosal damage. Production of IL-22 by ILC3s is

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required following viral- or chemical-induced lung damage [110]. In addition, following ablation therapy prior to stem cell transplantation, radio-resistant ILC3s are activated by IL-23 and produce IL-22 to limit intestinal epithelial cell apoptosis and preserve barrier function [111]. ILC3 production of IL-22 acts on the Lgr51 intestinal stem cells at the base of the crypts to stimulate epithelial regeneration. ILC3s therefore cooperate with the intestinal epithelial stem cell niche to stimulate proliferation and preserve barrier function through a STAT3dependent mechanism [54]. This restorative function of ILC3-derived IL-22 is similarly critical in chemical and infectious colitis [41,112]. In humans with acute inflammatory bowel disease, increased ILC3-derived IL-22 may also support epithelial regeneration [41]. Similarly, the reduction in mucosal ILC3 following alloreactive T cell transfer induced graft-versus-host disease (GVHD) correlates with increased mucosal damage [111]. Consistent with this finding, studies of patients following hematopoietic stem cell transplantation revealed an inverse correlation between circulating ILC3s and the severity of GVHD [113], revealing a potential role for circulating ILC3s as a biomarker of mucosal barrier protection.

VI. INNATE LYMPHOID CELLS IN ALLERGY, AUTOIMMUNITY, AND PERSISTENT INFLAMMATION Allergic and autoimmune inflammatory disease is commonly manifested at mucosal surfaces (Chapter 51: Mucosal Vaccines for Allergy and Tolerance and Chapter 52: Novel Strategies for Targeting the Control of Mucosal Inflammation). ILC2s are recognized as central drivers of Th2 immunity at these sites, contributing to asthma and pulmonary fibrosis [49]. As primary producers of IL-5, they recruit eosinophils to the lung, which are the main effectors of allergic inflammatory disease. In addition,

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ILC2 production of IL-13 enhances airway smooth muscle contraction and mucus production contributing to respiratory spasm and mucus plugging symptomatically associated with allergic asthma. Finally, IL-13 polarizes alternatively activate macrophages allowing for increase collagen deposition [114,115]. Despite the role for ILC3s acutely in tissue protection, local microenvironment cues can alter the effector functions of these cells in situ. In lung tissue, for example, ILC3s expand in response to NLRP3-dependent IL-1β production by macrophages and induce ILC3-derived IL-17 causing airway hyperresponsiveness [116]. Similarly in the gut, chronic models of colitis in mice can support proinflammatory ILCs that express both IL17A and IFNγ [78,117]. Transcriptional plasticity may underlie this regulation of effector function. A subset of inflammatory ILC3s can lose expression of RORγt and upregulate T-BET and IFNγ, producing ex-ILC3 ILC1s [39,43]. In humans, despite the initial increase in ILC3 production of IL-22 in acute colitis [41], chronic colitis may reflect a transition from tissue repair ILC3 to inflammatory ex-RORγt1 ILC1s [118,119]. In addition to the direct impact of IL-22, downstream effectors may promote a chronic inflammatory cascade that needs to be tightly regulated. Following T. gondii infection, IL-22 triggers epithelial cell production of IL-18, which subsequently augments Th1 tissue activity and feedback on ILC3s to sustain IL-22 in an inflammatory loop [120]. This inflammatory loop may support broad cross-reactive adaptive immunity generated during the primary infection [121]. Furthermore, the proliferative effects of IL-22 on the intestinal crypt can ultimately lead to hyperproliferation and cancer during persistent inflammation [122]. Tight regulation of IL-22 is therefore essential in managing this response. In addition to removing the trigger, DC-derived IL-22-binding protein (IL-22BP) binds directly to IL-22 and reduces the risk for chronic inflammatory effects of this cytokine [123].

VII. CROSS-REGULATION OF INNATE AND ADAPTIVE IMMUNITY BY MUCOSAL INNATE LYMPHOID CELLS Although many experimental studies have used RAG-deficient animals to evaluate the sufficiency of ILCs in disease models, the overall function of mucosal ILCs must be considered within the complex and sometimes redundant immune cell landscape at barrier surfaces. The presence of T cell immunity, particularly CD41 T cell immunity, provides a critical function at the mucosal barrier and may limit ILC3 effector functions [124,125]. In a reciprocal fashion, mucosal ILCs regulate innate and adaptive immunity in the tissue [34]. In addition to their role as the major source for IL-5 and IL-13, enabling eosinophil recruitment to the tissue, mucosal ILC2s can promote Th2 immunity [126]. One mechanism allowing for this support in allergic asthma is via IL-13elicited DC migration to lung-draining lymph nodes, which subsequently enables Th2 priming [127]. ILC2s are also recognized to express MHCII antigen presentation machinery, which enables them to engage and stimulate antigenspecific CD41 T cells [128,129]. ILC2 expression of costimulatory molecules, including OX40L and ICOSL, can additionally regulate Th2 cell responses and airway hyperreactivity [130,131]. The expression of MHCII is higher on intestinal lymph node and Peyer’s-patch-derived ILC2s compared with lung- and peritoneal-derived ILC2s, allowing for mucosal tissue specificity of this regulation. Adaptive immunity can also cross-regulate the mucosal ILC response. Adaptive immune responses are required for prolonged ILC2 expansion and clearance of helminth infection [103]. Helper CD41 T cells can directly regulate type 2 cytokines including IL-4 [130], IL-5, and IL-13 [128] through antigen-specific T-cell production of IL-2. Furthermore, T cells are

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VIII. CONCLUDING REMARKS

required for optimal papain-induced IL-9 that is also supported by IL-2 [81]. IL-33 stimulation of ILC2s augments this cross-regulation by increasing IL-2R (CD25) expression by ILC2s. Collectively, these findings reveal an ILC2 T cell positive feedback loop that enables ILC2 proliferation and IL-13 production [129]. This feedback loop may help to sensitize ILC2s and/ or sequester IL-2 away from other cellular sources in the mucosa. NK cells and ILC1s can also modulate adaptive immunity, albeit independently of MHCII expression [132,133]. Salivary NK cells express TNF-related apoptosis-inducing ligand and eliminate activated CD41 T cell during chronic murine cytomegalovirus (MCMV) infection [132]. Although this function enables chronicity of MCMV infection, it constrains viral-induced autoimmunity associated with a Sjo¨gren-like syndrome. In addition to limiting the T cell response, expression of OX40 ligand by activated NK cells can costimulate T cell receptor (TCR)-induced proliferation and IFNγ production of human autologous CD41 T cells [133]. Finally, ILC3s also play an important role in regulating adaptive and innate immunity in an immunocompetent host. LTi-derived soluble LTα3 regulates T cell dependent production of IgA by controlling T cell homing to the gut. This cross-regulation affects IgA-dependent immunity and shapes the gut microbiome [46]. In addition, effector cytokines produced by ILC3s such as IL-22 can act on the epithelium to regulate tissue-specific licensing of Th1 (via IL18) [120] or Th17 cells in the intestine (via serum amyloid A) [75]. ILC3s can similarly shape the local response by interacting with tissue macrophages. Expression of CXCL16 by MNPs enables colocalization with CXCR61 NKp461 ILC3s within the lamina propria [62]. This colocalization is reinforced by IL-1β- and TL1A-mediated induction of GM-CSF [41,134], which further enables the recruitment of MNPs and the generation of regulatory T cells to luminal antigens [134].

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Similar to ILC2s, a subsets of ILC3s express MHCII and are able to process and present antigens [135]. ILC3s in the spleen can express conventional costimulatory molecules [136], but conventional B7 costimulatory molecules CD80 and CD86 are not seen in MHCII1 ILC3s in the intestine even under inflammatory conditions. Alternative costimulatory molecules are expressed on ILC3s, including OX40L and CD30L, which can regulate the maintenance of CD41 memory Th cells [137]. In the absence of costimulatory molecules, intestinal MHCII1 ILC3s can engage antigen-specific T cells and induce tolerance by sequestering IL-2 [135]. Deletion of MHCII in ILC3s causes the expansion of commensal specific proinflammatory colonic CD41 T cell responses and the development of spontaneous colitis [135]. Consistent with this integral role for MHCII1 ILC3s in maintaining mucosal T cell tolerance, intestinal ILC3s from patients with Crohn’s disease have lower levels of MHCII, and this correlates with increased frequency of Th17 and commensal bacteria-specific IgG [138].

VIII. CONCLUDING REMARKS This chapter highlights the remarkable pace of research in defining the development and function of ILCs at mucosal barrier surfaces. While conventional NK and LTi cells were described decades ago, the formal discovery of the entire ILC family has emerged over the last ten years. Recent discoveries outlined here provide significant new insight into their critical roles in mucosal immunity, inflammation, metabolic homeostasis, and tissue repair. The mechanisms through which ILCs interact with and regulate adaptive immunity, the epithelial barrier, and the nervous system offer an exciting new framework for understanding immunity at mucosal surfaces. This framework identifies essential aspects of ILC mediated mucosal immunity that will serve as key targets

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in the development of novel strategies for mucosal vaccine development.

References [1] Spits H, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, et al. Innate lymphoid cells--a proposal for uniform nomenclature. Nat Rev Immunol 2013;13:145 9. [2] Eberl G, Colonna M, Di Santo JP, McKenzie AN. Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology. Science 2015;348:aaa6566. [3] Artis D, Spits H. The biology of innate lymphoid cells. Nature 2015;517:293 301. [4] Sonnenberg GF, Mjosberg J, Spits H, Artis D. Snapshot: innate lymphoid cells. Immunity 2013;39 622-622.e621. [5] Vivier E, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, et al. Innate lymphoid cells: 10 years on. Cell 2018;174:1054 66. [6] Sonnenberg GF, Artis D. Innate lymphoid cells in the initiation, regulation and resolution of inflammation. Nat Med 2015;21:698 708. [7] Diefenbach A, Colonna M, Koyasu S. Development, differentiation, and diversity of innate lymphoid cells. Immunity 2014;41:354 65. [8] Cherrier M, Sawa S, Eberl G. Notch, Id2, and RORgammat sequentially orchestrate the fetal development of lymphoid tissue inducer cells. J Exp Med 2012;209:729 40. [9] Constantinides MG, McDonald BD, Verhoef PA, Bendelac A. A committed precursor to innate lymphoid cells. Nature 2014;508:397 401. [10] Klose CSN, Flach M, Mohle L, Rogell L, Hoyler T, Ebert K, et al. Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages. Cell 2014;157:340 56. [11] Seillet C, Rankin LC, Groom JR, Mielke LA, Tellier J, Chopin M, et al. Nfil3 is required for the development of all innate lymphoid cell subsets. J Exp Med 2014;211:1733 40. [12] Yu X, Wang Y, Deng M, Li Y, Ruhn KA, Zhang CC, et al. The basic leucine zipper transcription factor NFIL3 directs the development of a common innate lymphoid cell precursor. Elife 2014;3:e04406. [13] Geiger TL, Abt MC, Gasteiger G, Firth MA, O’Connor MH, Geary CD, et al. Nfil3 is crucial for development of innate lymphoid cells and host protection against intestinal pathogens. J Exp Med 2014;211:1723 31. [14] Xu W, Domingues RG, Fonseca-Pereira D, Ferreira M, Ribeiro H, Lopez-Lastra S, et al. NFIL3 orchestrates the emergence of common helper innate lymphoid cell precursors. Cell Rep 2015;10:2043 54.

[15] Cortez VS, Fuchs A, Cella M, Gilfillan S, Colonna M. Cutting edge: salivary gland NK cells develop independently of Nfil3 in steady-state. J Immunol 2014;192:4487 91. [16] Seehus CR, Aliahmad P, de la Torre B, Iliev ID, Spurka L, Funari VA, et al. The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor. Nat Immunol 2015;16:599 608. [17] Aliahmad P, de la Torre B, Kaye J. Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages. Nat Immunol 2010;11:945 52. [18] Yang Q, Monticelli LA, Saenz SA, Chi AW, Sonnenberg GF, Tang J, et al. T cell factor 1 is required for group 2 innate lymphoid cell generation. Immunity 2013;38:694 704. [19] Mielke LA, Groom JR, Rankin LC, Seillet C, Masson F, Putoczki T, et al. TCF-1 controls ILC2 and NKp46 1 RORgammat 1 innate lymphocyte differentiation and protection in intestinal inflammation. J Immunol 2013;191:4383 91. [20] Serafini N, Klein Wolterink RG, Satoh-Takayama N, Xu W, Vosshenrich CA, Hendriks RW, et al. Gata3 drives development of RORgammat 1 group 3 innate lymphoid cells. J Exp Med 2014;211:199 208. [21] Yagi R, Zhong C, Northrup DL, Yu F, Bouladoux N, Spencer S, et al. The transcription factor GATA3 is critical for the development of all IL-7Ralpha-expressing innate lymphoid cells. Immunity 2014;40:378 88. [22] Hoyler T, Klose CS, Souabni A, Turqueti-Neves A, Pfeifer D, Rawlins EL, et al. The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells. Immunity 2012;37:634 48. [23] Possot C, Schmutz S, Chea S, Boucontet L, Louise A, Cumano A, et al. Notch signaling is necessary for adult, but not fetal, development of RORgammat( 1 ) innate lymphoid cells. Nat Immunol 2011;12:949 58. [24] Gury-BenAri M, Thaiss CA, Serafini N, Winter DR, Giladi A, Lara-Astiaso D, et al. The spectrum and regulatory landscape of intestinal innate lymphoid cells are shaped by the microbiome. Cell 2016;166:1231 1246. e1213. [25] Robinette ML, Fuchs A, Cortez VS, Lee JS, Wang Y, Durum SK, et al. Transcriptional programs define molecular characteristics of innate lymphoid cell classes and subsets. Nat Immunol 2015;16:306 17. [26] Scoville SD, Mundy-Bosse BL, Zhang MH, Chen L, Zhang X, Keller KA, et al. A progenitor cell expressing transcription factor RORgammat generates all human innate lymphoid cell subsets. Immunity 2016;44:1140 50. [27] Montaldo E, Teixeira-Alves LG, Glatzer T, Durek P, Stervbo U, Hamann W, et al. Human RORgammat( 1 )

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39]

[40]

CD34( 1 ) cells are lineage-specified progenitors of group 3 RORgammat( 1 ) innate lymphoid cells. Immunity 2014;41:988 1000. Lim AI, Li Y, Lopez-Lastra S, Stadhouders R, Paul F, Casrouge A, et al. Systemic human ILC precursors provide a substrate for tissue ILC differentiation. Cell 2017;168:1086 1100.e1010. Boos MD, Yokota Y, Eberl G, Kee BL. Mature natural killer cell and lymphoid tissue-inducing cell development requires Id2-mediated suppression of E protein activity. J Exp Med 2007;204:1119 30. Townsend MJ, Weinmann AS, Matsuda JL, Salomon R, Farnham PJ, Biron CA, et al. T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells. Immunity 2004;20:477 94. Fuchs A, Vermi W, Lee JS, Lonardi S, Gilfillan S, Newberry RD, et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15responsive IFN-gamma-producing cells. Immunity 2013;38:769 81. Kim BS, Artis D. Group 2 innate lymphoid cells in health and disease. Cold Spring Harb Perspect Biol 2015;7:a016337. Mjosberg J, Bernink J, Golebski K, Karrich JJ, Peters CP, Blom B, et al. The transcription factor GATA3 is essential for the function of human type 2 innate lymphoid cells. Immunity 2012;37:649 59. Klose CS, Artis D. Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis. Nat Immunol 2016;17:765 74. Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol 2004;5:64 73. Mebius RE. Organogenesis of lymphoid tissues. Nat Rev Immunol 2003;3:292 303. van de Pavert SA, Mebius RE. New insights into the development of lymphoid tissues. Nat Rev Immunol 2010;10:664 74. Satoh-Takayama N, Vosshenrich CA, Lesjean-Pottier S, Sawa S, Lochner M, Rattis F, et al. Microbial flora drives interleukin 22 production in intestinal NKp46 1 cells that provide innate mucosal immune defense. Immunity 2008;29:958 70. Klose CS, Kiss EA, Schwierzeck V, Ebert K, Hoyler T, d’Hargues Y, et al. A T-bet gradient controls the fate and function of CCR6-RORgammat 1 innate lymphoid cells. Nature 2013;494:261 5. Sanos SL, Bui VL, Mortha A, Oberle K, Heners C, Johner C, et al. RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46 1 cells. Nat Immunol 2009;10:83 91.

241

[41] Longman RS, Diehl GE, Victorio DA, Huh JR, Galan C, Miraldi ER, et al. CX(3)CR1( 1 ) mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22. J Exp Med 2014;211: 1571 83. [42] Lee JS, Cella M, McDonald KG, Garlanda C, Kennedy GD, Nukaya M, et al. AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch. Nat Immunol 2011;13:144 51. [43] Vonarbourg C, Mortha A, Bui VL, Hernandez PP, Kiss EA, Hoyler T, et al. Regulated expression of nuclear receptor RORgammat confers distinct functional fates to NK cell receptor-expressing RORgammat( 1 ) innate lymphocytes. Immunity 2010;33:736 51. [44] Fan X, Rudensky AY. Hallmarks of tissue-resident lymphocytes. Cell 2016;164:1198 211. [45] Gasteiger G, Fan X, Dikiy S, Lee SY, Rudensky AY. Tissue residency of innate lymphoid cells in lymphoid and nonlymphoid organs. Science 2015;350:981 5. [46] Kruglov AA, Grivennikov SI, Kuprash DV, Winsauer C, Prepens S, Seleznik GM, et al. Nonredundant function of soluble LTalpha3 produced by innate lymphoid cells in intestinal homeostasis. Science 2013;342: 1243 6. [47] Diefenbach A, Raulet DH. Innate immune recognition by stimulatory immunoreceptors. Curr Opin Immunol 2003;15:37 44. [48] Glatzer T, Killig M, Meisig J, Ommert I, LuetkeEversloh M, Babic M, et al. RORgammat( 1 ) innate lymphoid cells acquire a proinflammatory program upon engagement of the activating receptor NKp44. Immunity 2013;38:1223 35. [49] Monticelli LA, Sonnenberg GF, Artis D. Innate lymphoid cells: critical regulators of allergic inflammation and tissue repair in the lung. Curr Opin Immunol 2012;24:284 9. [50] von Moltke J, Ji M, Liang HE, Locksley RM. Tuft-cellderived IL-25 regulates an intestinal ILC2-epithelial response circuit. Nature 2016;529:221 5. [51] Gerbe F, Sidot E, Smyth DJ, Ohmoto M, Matsumoto I, Dardalhon V, et al. Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites. Nature 2016;529:226 30. [52] Howitt MR, Lavoie S, Michaud M, Blum AM, Tran SV, Weinstock JV, et al. Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut. Science 2016;351:1329 33. [53] Sawa S, Lochner M, Satoh-Takayama N, Dulauroy S, Berard M, Kleinschek M, et al. RORgammat 1 innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota. Nat Immunol 2011;12:320 6.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

242

14. INNATE LYMPHOID CELLS FOR THE CONTROL OF MUCOSAL IMMUNITY

[54] Lindemans CA, Calafiore M, Mertelsmann AM, O’Connor MH, Dudakov JA, Jenq RR, et al. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration. Nature 2015;528:560 4. [55] Crellin NK, Trifari S, Kaplan CD, Satoh-Takayama N, Di Santo JP, Spits H. Regulation of cytokine secretion in human CD127( 1 ) LTi-like innate lymphoid cells by Toll-like receptor 2. Immunity 2010;33(5):752 64. [56] Kinnebrew MA, Buffie CG, Diehl GE, Zenewicz LA, Leiner I, Hohl TM, et al. Interleukin 23 production by intestinal CD103( 1 )CD11b( 1 ) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense. Immunity 2012;36:276 87. [57] Abt MC, Buffie CG, Susac B, Becattini S, Carter RA, Leiner I, et al. TLR-7 activation enhances IL-22mediated colonization resistance against vancomycinresistant enterococcus. Sci Transl Med 2016;8:327ra325. [58] Satpathy AT, Briseno CG, Lee JS, Ng D, Manieri NA, Kc W, et al. Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-andeffacing bacterial pathogens. Nat Immunol 2013;14:937 48. [59] Mackley EC, Houston S, Marriott CL, Halford EE, Lucas B, Cerovic V, et al. CCR7-dependent trafficking of RORgamma( 1 ) ILCs creates a unique microenvironment within mucosal draining lymph nodes. Nat Commun 2015;6:5862. [60] Chieppa M, Rescigno M, Huang AY, Germain RN. Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J Exp Med 2006;203:2841 52. [61] Niess JH, Brand S, Gu X, Landsman L, Jung S, McCormick BA, et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 2005;307:254 8. [62] Satoh-Takayama N, Serafini N, Verrier T, Rekiki A, Renauld JC, Frankel G, et al. The chemokine receptor CXCR6 controls the functional topography of interleukin-22 producing intestinal innate lymphoid cells. Immunity 2014;41:776 88. [63] Kim BS, Siracusa MC, Saenz SA, Noti M, Monticelli LA, Sonnenberg GF, et al. TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation. Sci Transl Med 2013;5:170ra116. [64] Motomura Y, Morita H, Moro K, Nakae S, Artis D, Endo TA, et al. Basophil-derived interleukin-4 controls the function of natural helper cells, a member of ILC2s, in lung inflammation. Immunity 2014;40:758 71. [65] Barnig C, Cernadas M, Dutile S, Liu X, Perrella MA, Kazani S, et al. Lipoxin A4 regulates natural killer cell and type 2 innate lymphoid cell activation in asthma. Sci Transl Med 2013;5:174ra126.

[66] Veiga-Fernandes H, Coles MC, Foster KE, Patel A, Williams A, Natarajan D, et al. Tyrosine kinase receptor RET is a key regulator of Peyer’s patch organogenesis. Nature 2007;446:547 51. [67] Ibiza S, Garcia-Cassani B, Ribeiro H, Carvalho T, Almeida L, Marques R, et al. Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence. Nature 2016;535:440 3. [68] Cardoso V, Chesne J, Ribeiro H, Garcia-Cassani B, Carvalho T, Bouchery T, et al. Neuronal regulation of type 2 innate lymphoid cells via neuromedin U. Nature 2017;549:277 81. [69] Wallrapp A, Riesenfeld SJ, Burkett PR, Abdulnour RE, Nyman J, Dionne D, et al. The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation. Nature 2017;549:351 6. [70] Klose CSN, Mahlakoiv T, Moeller JB, Rankin LC, Flamar AL, Kabata H, et al. The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation. Nature 2017;549:282 6. [71] Moriyama S, Brestoff JR, Flamar AL, Moeller JB, Klose CSN, Rankin LC, et al. beta2-adrenergic receptor-mediated negative regulation of group 2 innate lymphoid cell responses. Science 2018;359:1056 61. [72] Fung TC, Bessman NJ, Hepworth MR, Kumar N, Shibata N, Kobuley D, et al. Lymphoid-tissue-resident commensal bacteria promote members of the IL-10 cytokine family to establish mutualism. Immunity 2016;44:634 46. [73] Viladomiu M, Kivolowitz C, Abdulhamid A, Dogan B, Victorio D, Castellanos JG, et al. IgA-coated E. coli enriched in Crohn’s disease spondyloarthritis promote TH17-dependent inflammation. Sci Transl Med 2017;9: eaaf9655. [74] Atarashi K, Tanoue T, Ando M, Kamada N, Nagano Y, Narushima S, et al. Th17 cell induction by adhesion of microbes to intestinal epithelial cells. Cell 2015;163:367 80. [75] Sano T, Huang W, Hall JA, Yang Y, Chen A, Gavzy SJ, et al. An IL-23R/IL-22 circuit regulates epithelial serum amyloid A to promote local effector Th17 responses. Cell 2016;164:324. [76] Monticelli LA, Sonnenberg GF, Abt MC, Alenghat T, Ziegler CG, Doering TA, et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nat Immunol 2011;12:1045 54. [77] Kernbauer E, Ding Y, Cadwell K. An enteric virus can replace the beneficial function of commensal bacteria. Nature 2014;516:94 8. [78] Buonocore S, Ahern PP, Uhlig HH, Ivanov II, Littman DR, Maloy KJ, et al. Innate lymphoid cells drive

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

243

REFERENCES

interleukin-23-dependent innate intestinal pathology. Nature 2010;464:1371 5. [79] Nussbaum JC, Van Dyken SJ, von Moltke J, Cheng LE, Mohapatra A, Molofsky AB, Thornton EE, Krummel MF, Chawla A, Liang HE, Locksley RM. Type 2 innate lymphoid cells control eosinophil homeostasis. Nature 2013;502(7470):245 8. [80] Spencer SP, Wilhelm C, Yang Q, Hall JA, Bouladoux N, Boyd A, et al. Adaptation of innate lymphoid cells to a micronutrient deficiency promotes type 2 barrier immunity. Science 2014;343:432 7. [81] Wilhelm C, Harrison OJ, Schmitt V, Pelletier M, Spencer SP, Urban JF, et al. Critical role of fatty acid metabolism in ILC2-mediated barrier protection during malnutrition and helminth infection. J Exp Med 2016;213:1409 18. [82] Monticelli LA, Buck MD, Flamar AL, Saenz SA, Tait Wojno ED, Yudanin NA, et al. Arginase 1 is an innate lymphoid-cell-intrinsic metabolic checkpoint controlling type 2 inflammation. Nat Immunol 2016;17:656 65. [83] van de Pavert SA, Ferreira M, Domingues RG, Ribeiro H, Molenaar R, Moreira-Santos L, et al. Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity. Nature 2014;508:123 7. [84] Gomez de Aguero M, Ganal-Vonarburg SC, Fuhrer T, Rupp S, Uchimura Y, Li H, et al. The maternal microbiota drives early postnatal innate immune development. Science 2016;351:1296 302. [85] Zheng Y, Valdez PA, Danilenko DM, Hu Y, Sa SM, Gong Q, et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med 2008;14:282 9. [86] Goto Y, Obata T, Kunisawa J, Sato S, Ivanov II, Lamichhane A, et al. Innate lymphoid cells regulate intestinal epithelial cell glycosylation. Science 2014;345:1254009. [87] Pham TA, Clare S, Goulding D, Arasteh JM, Stares MD, Browne HP, et al. Epithelial IL-22RA1-mediated fucosylation promotes intestinal colonization resistance to an opportunistic pathogen. Cell Host Microbe 2014;16:504 16. [88] Pickard JM, Maurice CF, Kinnebrew MA, Abt MC, Schenten D, Golovkina TV, et al. Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness. Nature 2014;514:638 41. [89] Ota N, Wong K, Valdez PA, Zheng Y, Crellin NK, Diehl L, et al. IL-22 bridges the lymphotoxin pathway with the maintenance of colonic lymphoid structures during infection with Citrobacter rodentium. Nat Immunol 2011;12:941 8. [90] Tumanov AV, Koroleva EP, Guo X, Wang Y, Kruglov A, Nedospasov S, et al. Lymphotoxin controls the IL-

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

22 protection pathway in gut innate lymphoid cells during mucosal pathogen challenge. Cell Host Microbe 2011;10:44 53. Guo X, Liang Y, Zhang Y, Lasorella A, Kee BL, Fu YX. Innate lymphoid cells control early colonization resistance against intestinal pathogens through ID2dependent regulation of the microbiota. Immunity 2015;42:731 43. Duffin R, O’Connor RA, Crittenden S, Forster T, Yu C, Zheng X, et al. Prostaglandin E(2) constrains systemic inflammation through an innate lymphoid cellIL-22 axis. Science 2016;351:1333 8. Zhang B, Chassaing B, Shi Z, Uchiyama R, Zhang Z, Denning TL, et al. Viral infection. Prevention and cure of rotavirus infection via TLR5/NLRC4-mediated production of IL-22 and IL-18. Science 2014;346:861 5. Hernandez PP, Mahlakoiv T, Yang I, Schwierzeck V, Nguyen N, Guendel F, et al. Interferon-lambda and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection. Nat Immunol 2015;16:698 707. Gray J, Oehrle K, Worthen G, Alenghat T, Whitsett J, Deshmukh H. Intestinal commensal bacteria mediate lung mucosal immunity and promote resistance of newborn mice to infection. Sci Transl Med 2017;9: eaaf9412. Gladiator A, Wangler N, Trautwein-Weidner K, LeibundGut-Landmann S. Cutting edge: IL-17secreting innate lymphoid cells are essential for host defense against fungal infection. J Immunol 2013;190:521 5. Okada S, Markle JG, Deenick EK, Mele F, Averbuch D, Lagos M, et al. IMMUNODEFICIENCIES. Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations. Science 2015;349:606 13. Basu R, O’Quinn DB, Silberger DJ, Schoeb TR, Fouser L, Ouyang W, et al. Th22 cells are an important source of IL-22 for host protection against enteropathogenic bacteria. Immunity 2012;37:1061 75. Song C, Lee JS, Gilfillan S, Robinette ML, Newberry RD, Stappenbeck TS, et al. Unique and redundant functions of NKp46 1 ILC3s in models of intestinal inflammation. J Exp Med 2015;212:1869 82. Rankin LC, Girard-Madoux MJ, Seillet C, Mielke LA, Kerdiles Y, Fenis A, et al. Complementarity and redundancy of IL-22-producing innate lymphoid cells. Nat Immunol 2016;17:179 86. Artis D, Wang ML, Keilbaugh SA, He W, Brenes M, Swain GP, et al. RELMbeta/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract. Proc Natl Acad Sci USA 2004;101:13596 600.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

244

14. INNATE LYMPHOID CELLS FOR THE CONTROL OF MUCOSAL IMMUNITY

[102] Moro K, Yamada T, Tanabe M, Takeuchi T, Ikawa T, Kawamoto H, et al. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit( 1 )Sca-1( 1 ) lymphoid cells. Nature 2010;463:540 4. [103] Neill DR, Wong SH, Bellosi A, Flynn RJ, Daly M, Langford TK, et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010;464:1367 70. [104] Price AE, Liang HE, Sullivan BM, Reinhardt RL, Eisley CJ, Erle DJ, et al. Systemically dispersed innate IL-13-expressing cells in type 2 immunity. Proc Natl Acad Sci USA 2010;107:11489 94. [105] Abt MC, Lewis BB, Caballero S, Xiong H, Carter RA, Susac B, et al. Innate immune defenses mediated by two ILC subsets are critical for protection against acute clostridium difficile infection. Cell Host Microbe 2015;18:27 37. [106] Monticelli LA, Osborne LC, Noti M, Tran SV, Zaiss DM, Artis D. IL-33 promotes an innate immune pathway of intestinal tissue protection dependent on amphiregulin-EGFR interactions. Proc Natl Acad Sci USA 2015;112:10762 7. [107] Turner JE, Morrison PJ, Wilhelm C, Wilson M, Ahlfors H, Renauld JC, et al. IL-9-mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation. J Exp Med 2013;210:2951 65. [108] Scandella E, Bolinger B, Lattmann E, Miller S, Favre S, Littman DR, et al. Restoration of lymphoid organ integrity through the interaction of lymphoid tissueinducer cells with stroma of the T cell zone. Nat Immunol 2008;9:667 75. [109] Dudakov JA, Hanash AM, Jenq RR, Young LF, Ghosh A, Singer NV, et al. Interleukin-22 drives endogenous thymic regeneration in mice. Science 2012;336:91 5. [110] Sonnenberg GF, Nair MG, Kirn TJ, Zaph C, Fouser LA, Artis D. Pathological versus protective functions of IL-22 in airway inflammation are regulated by IL17A. J Exp Med 2010;207:1293 305. [111] Hanash AM, Dudakov JA, Hua G, O’Connor MH, Young LF, Singer NV, et al. Interleukin-22 protects intestinal stem cells from immune-mediated tissue damage and regulates sensitivity to graft versus host disease. Immunity 2012;37:339 50. [112] Sonnenberg GF, Monticelli LA, Elloso MM, Fouser LA, Artis D. CD4( 1 ) lymphoid tissue-inducer cells promote innate immunity in the gut. Immunity 2011;34:122 34. [113] Munneke JM, Bjorklund AT, Mjosberg JM, GarmingLegert K, Bernink JH, Blom B, et al. Activated innate lymphoid cells are associated with a reduced susceptibility to graft-versus-host disease. Blood 2014;124: 812 21.

[114] Chang YJ, Kim HY, Albacker LA, Baumgarth N, McKenzie AN, Smith DE, et al. Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity. Nat Immunol 2011;12:631 8. [115] Hams E, Armstrong ME, Barlow JL, Saunders SP, Schwartz C, Cooke G, et al. IL-25 and type 2 innate lymphoid cells induce pulmonary fibrosis. Proc Natl Acad Sci USA 2014;111:367 72. [116] Kim HY, Lee HJ, Chang YJ, Pichavant M, Shore SA, Fitzgerald KA, et al. Interleukin-17-producing innate lymphoid cells and the NLRP3 inflammasome facilitate obesity-associated airway hyperreactivity. Nat Med 2014;20:54 61. [117] Garrett WS, Lord GM, Punit S, Lugo-Villarino G, Mazmanian SK, Ito S, et al. Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system. Cell 2007;131:33 45. [118] Geremia A, Arancibia-Carcamo CV, Fleming MP, Rust N, Singh B, Mortensen NJ, et al. IL-23responsive innate lymphoid cells are increased in inflammatory bowel disease. J Exp Med 2011;208: 1127 33. [119] Bernink JH, Peters CP, Munneke M, te Velde AA, Meijer SL, Weijer K, et al. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nat Immunol 2013;14:221 9. [120] Munoz M, Eidenschenk C, Ota N, Wong K, Lohmann U, Kuhl AA, et al. Interleukin-22 induces interleukin18 expression from epithelial cells during intestinal infection. Immunity 2015;42:321 31. [121] Hand TW, Dos Santos LM, Bouladoux N, Molloy MJ, Pagan AJ, Pepper M, et al. Acute gastrointestinal infection induces long-lived microbiota-specific T cell responses. Science 2012;337:1553 6. [122] Kirchberger S, Royston DJ, Boulard O, Thornton E, Franchini F, Szabady RL, et al. Innate lymphoid cells sustain colon cancer through production of interleukin-22 in a mouse model. J Exp Med 2013;210:917 31. [123] Huber S, Gagliani N, Zenewicz LA, Huber FJ, Bosurgi L, Hu B, et al. IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine. Nature 2012;491:259 63. [124] Korn LL, Thomas HL, Hubbeling HG, Spencer SP, Sinha R, Simkins HM, et al. Conventional CD4 1 T cells regulate IL-22-producing intestinal innate lymphoid cells. Mucosal Immunol 2014;7:1045 57. [125] Mao K, Baptista AP, Tamoutounour S, Zhuang L, Bouladoux N, Martins AJ, et al. Innate and adaptive lymphocytes sequentially shape the gut microbiota and lipid metabolism. Nature 2018;554:255 9.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

245

REFERENCES

[126] Gold MJ, Antignano F, Halim TY, Hirota JA, Blanchet MR, Zaph C, et al. Group 2 innate lymphoid cells facilitate sensitization to local, but not systemic, TH2inducing allergen exposures. J Allergy Clin Immunol 2014;133:1142 8. [127] Halim TY, Steer CA, Matha L, Gold MJ, MartinezGonzalez I, McNagny KM, et al. Group 2 innate lymphoid cells are critical for the initiation of adaptive T helper 2 cell-mediated allergic lung inflammation. Immunity 2014;40:425 35. [128] Mirchandani AS, Besnard AG, Yip E, Scott C, Bain CC, Cerovic V, et al. Type 2 innate lymphoid cells drive CD4 1 Th2 cell responses. J Immunol 2014;192:2442 8. [129] Oliphant CJ, Hwang YY, Walker JA, Salimi M, Wong SH, Brewer JM, et al. MHCII-mediated dialog between group 2 innate lymphoid cells and CD4( 1 ) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion. Immunity 2014;41:283 95. [130] Drake LY, Iijima K, Kita H. Group 2 innate lymphoid cells and CD4 1 T cells cooperate to mediate type 2 immune response in mice. Allergy 2014;69:1300 7. [131] Maazi H, Patel N, Sankaranarayanan I, Suzuki Y, Rigas D, Soroosh P, et al. ICOS:ICOS-ligand interaction is required for type 2 innate lymphoid cell function, homeostasis, and induction of airway hyperreactivity. Immunity 2015;42:538 51. [132] Schuster IS, Wikstrom ME, Brizard G, Coudert JD, Estcourt MJ, Manzur M, et al. TRAIL 1 NK cells

[133]

[134]

[135]

[136]

[137]

[138]

control CD4 1 T cell responses during chronic viral infection to limit autoimmunity. Immunity 2014;41:646 56. Zingoni A, Sornasse T, Cocks BG, Tanaka Y, Santoni A, Lanier LL. Cross-talk between activated human NK cells and CD4 1 T cells via OX40-OX40 ligand interactions. J Immunol 2004;173:3716 24. Mortha A, Chudnovskiy A, Hashimoto D, Bogunovic M, Spencer SP, Belkaid Y, et al. Microbiotadependent crosstalk between macrophages and ILC3 promotes intestinal homeostasis. Science 2014;343:1249288. Hepworth MR, Monticelli LA, Fung TC, Ziegler CG, Grunberg S, Sinha R, et al. Innate lymphoid cells regulate CD4 1 T-cell responses to intestinal commensal bacteria. Nature 2013;498:113 17. von Burg N, Chappaz S, Baerenwaldt A, Horvath E, Bose Dasgupta S, Ashok D, et al. Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses. Proc Natl Acad Sci USA 2014;111:12835 40. Withers DR, Gaspal FM, Mackley EC, Marriott CL, Ross EA, Desanti GE, et al. Cutting edge: lymphoid tissue inducer cells maintain memory CD4 T cells within secondary lymphoid tissue. J Immunol 2012;189:2094 8. Hepworth MR, Fung TC, Masur SH, Kelsen JR, McConnell FM, Dubrot J, et al. Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4( 1 ) T cells. Science 2015;348:1031 5.

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Mucosal Regulatory System for Balanced Immunity in the Gut Hisako Kayama1,2,3 and Kiyoshi Takeda1,2,3 1

Department of Microbiology and Immunology, Graduate School of Medicine, Osaka University, Suita, Japan 2WPI Immunology Frontier Research Center, Osaka University, Suita, Japan 3Core Research for Evolutional Science and Technology, Japan Agency for Medical Research and Development, Tokyo, Japan

I. INTRODUCTION In the intestinal mucosa, the immune system is balanced between activating proinflammatory pathways for host defense against invading pathogens and remaining unresponsive to symbiotic microorganisms and dietary antigens. Because constitutive or excessive activation of immune responses causes intestinal tissue destruction, various immune cells contribute to the induction of tolerance for the maintenance of gut homeostasis as well as driving proinflammatory responses to pathogens. The two major forms of inflammatory bowel disease (IBD) are ulcerative colitis (UC) and Crohn’s disease (CD), both of which are characterized by chronic relapsing-remitting courses [1]. Excessive adaptive immune responses such as T helper (Th)1 and Th17 cell responses are implicated in the pathogenesis of IBD [2]. In the intestinal mucosa, antiinflammatory cytokine

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00015-8

interleukin-10 (IL-10)-producing CD41 T cells such as Foxp31 regulatory cells (Tregs) and Foxp32 type 1 regulatory T (Tr1) cells comprise the major regulatory cells in the adaptive immune system [3]. IL-10 produced by Foxp31 Tregs and Tr1 cells is thought to regulate the activity of innate myeloid cells in the intestine [4] and thereby prevent intestinal inflammation. In addition to IL-10, Foxp31 Tregs inhibit inflammatory responses by producing antiinflammatory cytokine transforming growth factor beta (TGF-β) as well as by expressing CTLA-4, LAG-3, CD39, and Nrp-1 [5]. During chronic inflammation, regulatory B cells (Breg) develop and suppress inflammatory responses through the production of IL-10 and TGF-β [6]. CD1d expression on Bregs is required for the production of IL-10. Accordingly, Bregs lacking Cd1d are unable to suppress the exacerbation of intestinal inflammation [7]. The transfer of IL10-producing Bregs induced by IL-33 had a

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therapeutic effect on colitis in Il102/2 mice [8]. These studies demonstrate that regulatory cells in the adaptive immune system are required for sustained intestinal immune balance and thereby repress enteric inflammation. Because innate myeloid cells can shape adaptive immunity through antigen presentation and cytokine production via the activation of pattern recognition receptors, such as tolllike receptors (TLRs) [9 12], the dysregulation of the innate immune system is linked to the inadequate activation of adaptive immunity and thus is implicated in the pathogenesis of IBD. Recent studies have identified a variety of innate myeloid cell subsets that modulate intestinal immune responses in the murine gut [13]. Among these subsets, some innate myeloid

cells promote intestinal inflammation by activating colitogenic effector T cells through the upregulation of gut-homing receptor α4β7 [14] and the production of proinflammatory cytokines such as IL-6 and IL-23, both of which induce Th17 responses [15]. Accordingly, patients with CD have abnormal intestinal macrophages with IL-23-induced interferon gamma (IFN-γ) production and enhanced antigen-presenting activity [16,17]. These findings indicate that intestinal innate immunity might be tightly controlled to maintain gut homeostasis and that its dysregulation might be associated with the pathogenesis of IBD. This chapter focuses on the role of murine (Fig. 15.1) and human (Fig. 15.2) intestinal innate myeloid cells in the organization of

FIGURE 15.1 Maintenance of immunological tolerance by murine intestinal innate myeloid cells. (A, B) CD1031 DCs

induce Foxp31 Tregs through the production of TGF-β and retinoic acid (RA) (A) or indoleamine 2,3-dioxygenase (IDO) (B). (C) CD1031 CD11b2 DCs activated by probiotic Bifidobacterium breve induce IL-10-producing Tr1 cells through the production of IL-10 and IL-27. (D) Proinflammatory cytokine production, such as IL-12 and IL-23, by intestinal CX3CR1high macrophages is tightly regulated by an IL-10/Stat3 axis-dependent mechanism. (E) Intestinal CX3CR1high macrophages in the large intestinal lamina propria inhibit CD41 T cell proliferative responses through the high expression of adhesion molecules such as ICAM-1 and VCAM-1 and lower expression of CD80/CD86. (F) IL-10 produced by intestinal CX3CR1high macrophages maintains Foxp31 Tregs.

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FIGURE 15.2

Regulation of gut homeostasis and ulcerative colitis by human intestinal innate myeloid cells. (A) In the colon of healthy individuals, CD1031 DCs induce the FOXP31 Tregs. (B) Colonic CD1031 DCs from patients with ulcerative colitis (UC) decrease FOXP31 Treg-inducing activity but markedly increase the ability to promote development of Th1, Th2, and Th17 cells with higher expressions of IL6, IL23A, IL12p35, and TNF. (C) CD141 CD163high CD160high cells constitutively produce IL-10 and inhibit effector CD41 T cell proliferation. (D) In UC patients, the number of CD141 CD163high CD160high cells is markedly decreased. In addition, CD163high CD160high cells from UC patients do not suppress effector CD41 T cell proliferative responses.

mucosal regulatory systems for maintenance of the gut homeostasis.

II. INDUCTION OF INTESTINAL IMMUNE TOLERANCE BY CD1031 DENDRITIC CELLS Mononuclear phagocytes in the murine intestine have been characterized into a variety of subsets with distinct abilities to either promote or inhibit T cell responses for the maintenance of gut homeostasis [18,19]. In particular, murine intestinal CD1031 dendritic cells (DCs) and CX3CR11 CD11b1 cells have been well characterized. Among CD1031 DCs, CD1031 CD11c1 DCs promote the proliferation of CD41

and CD81 T cells [20], induce cytotoxic T lymphocytes [21], regulate antimicrobial peptide expression in epithelial cells by inducing IL-22 production by innate lymphoid cells through flagellin-dependent IL-23 production [22], and induce Th1 and Th17 cell differentiation [23,24]. In addition to mucosal defense, CD1031 DCs contribute to intestinal tolerance by inducing Foxp31 Tregs through the production of TGF-β and retinoic acid, which is a metabolite of vitamin A [25] (Fig. 15.1A). In addition, CD1031 DCs promote the differentiation of Foxp31 Tregs through expression of indoleamine 2,3dioxygenase (IDO), which is a tryptophan catabolism enzyme [26] (Fig. 15.1B). Accordingly, the inhibition of IDO disrupted oral tolerance and exacerbated intestinal

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inflammation [26]. In the colon, CD1031 CD11b2 DCs activated by probiotic Bifidobacterium breve via the TLR2/MyD88 pathway produce IL-10 and IL-27 and thereby induce IL-10-producing Tr1 cells [27] (Fig. 15.1C). In addition, oral B. breve administration improved T-cell-mediated colitis in an IL-10-dependent manner. Thus CD1031 DCs play multiple roles in the induction of immune tolerance as well as effector responses against pathogens in the gut.

III. REGULATION OF IMMUNE HOMEOSTASIS BY INTESTINAL RESIDENT CX3CR1HIGH MACROPHAGES Intestinal CX3CR11 CD11b1 cells are heterogeneous and can be further divided into CX3CR1high CD11b1 macrophages and intermediate (int) CX3CR1 CD11b1 DCs [28,29]. CX3CR1int CD11b1 DCs promote inflammatory responses by inducing the differentiation of Th17 cells [30,31]. CX3CR1high CD11b1 cells are intestinal resident macrophages [32]. CX3CR1high macrophages in the intestinal lamina propria of neonates arise from embryonic precursors in the yolk sac [33], whereas both CX3CR1int DCs and CX3CR1high macrophages in the intestine of adult mice are derived from blood Ly6Chigh CCR21 monocytes [34 36]. A recent study showed that miR-223 deficiency resulted in a decreased number of intestinal CX3CR1high macrophages and severe dextran sodium sulfate-induced colitis [37], suggesting that the appropriate differentiation and function of intestinal CX3CR1high macrophages are required for sustained gut homeostasis. Intestinal CX3CR1high macrophages are nonmigratory cells even under inflammatory conditions [23,38]. A previous study showed that the migration of intestinal CX3CR1high macrophages from the lamina propria to mesenteric lymph nodes was suppressed by a commensal

bacteria-dependent mechanism and inhibited the activation of T cell responses and IgA production [39]. Intestinal CX3CR1high macrophages are crucial for host defense against invading pathogens based on their high phagocytic activity [35]. To maintain immune tolerance in the gut, intestinal CX3CR1high macrophages mediate their noninflammatory features via IL-10 receptor signaling-dependent mechanisms [4,40] (Fig. 15.1D). A previous study revealed that the myeloid lineage cellspecific deletion of Stat3, a downstream transcription factor of the IL-10 signaling pathway, resulted in the spontaneous development of colitis with the hyperproduction of proinflammatory cytokines such as IL-12, IL-6, and tumor necrosis factor alpha (TNF-α) by innate myeloid cells [41]. In addition, large intestinal CX3CR1high macrophages from Il102/2 mice showed the increased production of proinflammatory cytokines, including IL-12 and IL-23 [34], suggesting that the expression of a subset of proinflammatory cytokines that mediate the induction/activation of colitogenic Th1 and Th17 cells in intestinal CX3CR1high macrophages might be tightly regulated by an IL-10/ Stat3 axis-dependent mechanism. Furthermore, CX3CR1high macrophages in the large intestinal lamina propria acquire their activity to inhibit CD41 T cell proliferative responses through the IL-10/Stat3 signaling pathway and thereby prevent intestinal inflammation [29]. Large intestinal CX3CR1high macrophages preferentially interacted with effector CD41 T cells through the high expression of adhesion molecules such as ICAM-1 and VCAM-1. However, these cells did not activate CD41 T cells because their expression of CD80/CD86 was severely suppressed via IL-10/Stat3 signaling [29]. In comparison to other tissue-resident macrophages, intestinal CX3CR1high macrophages produce a large amount of IL-10 constitutively, which maintained Foxp31 Tregs [42]. In addition, autocrine IL-10 signals from intestinal CX3CR1high macrophages suppressed

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IV

ROLES OF HUMAN INTESTINAL MYELOID CELLS

colitogenic IL-23 production [43]. Thus murine intestinal CX3CR1high CD11b1 cells are unique tissue-resident macrophages with regulatory properties. Small intestinal CX3CR1high macrophages develop by a Notch signaling-dependent mechanism [44]. CX3CR1high macrophages residing in the small intestine possess another unique property allowing them to extend their dendrites directly into the intestinal lumen. The transepithelial sampling of microbes by CX3CR1high macrophages in the small intestine is dependent on CX3CR1 expression. Accordingly, in CX3CR1-deficient mice, the sampling of entero-invasive pathogens by small intestinal innate myeloid cells was impaired, and their susceptibility to Salmonella enterica serovar Typhimurium was enhanced [45]. In addition to CX3CR1, TLR/MyD88 signaling pathways in intestinal epithelial cells are required for dendrite extension by CX3CR1high macrophages [46]. Recently, a study showed that CX3CR1high macrophages contributed to the induction of Foxp31 Treg cell-mediated tolerance to a food antigen by taking up soluble fed antigen and transferring it to CD1031 DCs via a gap-junction-mediated mechanism [47]. Thus small intestinal CX3CR1high macrophages contribute to sustained immunological tolerance through the sampling of luminal antigens.

IV. ROLES OF HUMAN INTESTINAL MYELOID CELLS IN THE MAINTENANCE OF GUT HOMEOSTASIS AND INFLAMMATORY BOWEL DISEASE Similar to CD1031 DCs in the murine intestine, human intestinal CD1031 DCs induce the expression of gut-homing receptors, such as CCR9 and integrin α4β7, on T cells [20]. In the human small intestine, CD1031 Sirpα2 DCs, equivalent to murine CD1031 CD11b2 DCs, evoke Th17 cell differentiation [48]. In addition,

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CD1031 Sirpα1 DCs, equivalent to murine CD1031 CD11b1 DCs, induce FOXP31 Tregs as well as Th17 cells [48]. In the colon, CD1031 DCs from healthy individuals induced the conversion of naı¨ve T cells into FOXP31 Tregs but not into Th1 or Th17 cells [49] (Fig. 15.2A). In contrast, colonic CD1031 DCs from UC patients showed decreased in FOXP31 Treg-inducing activity but exhibited a marked increase in the development of Th1, Th2, and Th17 cells, with higher expression of proinflammatory cytokine genes IL6, IL23A, IL12p35, and TNF (Fig. 15.2B). In the human intestinal mucosa, CD141 macrophages are increased in CD patients and produce greater amounts of inflammatory cytokines, such as IL-6, IL-23, and TNF-α, in response to commensal bacteria [16]. In addition, CD141 macrophage-derived IL-23 facilitates colitogenic IL-17 and IFN-γ-producing CD41 T cell differentiation [16,17,50,51]. Human intestinal HLA-DRhigh CD141 innate myeloid cells are further characterized as CD141 CD163low cells and CD141 CD163high cells. CD141 CD163low cells induce Th17 cell differentiation by producing Th17-related cytokines, such as IL-6, IL-23p19, TNF-α, and IL-1β, through TLR2, TLR4, and TLR5 signaling pathways [52]. In CD patients, the activity of CD141 CD163low cells to induce Th17 cells is increased. CD141 CD163high cells can be subdivided into two subsets, CD141 CD163high CD160high and CD141 CD163high CD160low cells, based on the expression levels of CD160 [53]. Similar to murine intestinal CX3CR1high macrophages, CD141 CD163high CD160high cells constitutively produce high amounts of IL-10 and exert high phagocytic activity. In addition, CD141 CD163high CD160high cells inhibit effector CD41 T cell proliferation in a FOXP31 Tregindependent manner (Fig. 15.2C). In the colon of UC patients, the number of CD141 CD163high CD160high cells is markedly decreased. In this context, CD163high CD160high cells show higher CD80/CD86 expression with

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decreased IL10RB expression. Furthermore, CD163high CD160high cells from UC patients do not suppress effector CD41 T cell proliferative responses (Fig. 15.2D). Thus several regulatory innate myeloid cells, including CD1031 DCs and CD141 CD163high CD160high cells, are crucial for balanced immunity in the human and murine intestine.

V. CONCLUDING REMARKS In the intestinal mucosa, appropriate effector T cell responses play an important role in protection against invading pathogens in the steady state, whereas their excessive activation leads to intestinal inflammation. Recent advances have demonstrated the key roles of intestinal innate immune cells in immunological tolerance through the regulation of the adaptive immune system to maintain gut homeostasis. Further human studies to characterize the innate and adaptive immune systems responsible for either the maintenance of gut homeostasis or the pathogenesis of intestinal inflammation will advance diagnostic and therapeutic approaches for IBD.

[5] [6] [7]

[8]

[9] [10]

[11]

[12]

[13] [14]

[15]

Acknowledgments We thank C. Hidaka for secretarial assistance and J.L. Croxford, PhD, from Edanz Group (www.edanzediting. com/ac) for editing a draft of this manuscript.

[16]

References

[17]

[1] Baumgart DC, Sandborn WJ. Crohn’s disease. Lancet 2012;380:1590 605. [2] Rovedatti L, Kudo T, Biancheri P, Sarra M, Knowles CH, Rampton DS, et al. Differential regulation of interleukin 17 and interferon gamma production in inflammatory bowel disease. Gut 2009;58:1629 36. [3] Barnes MJ, Powrie F. Regulatory T cells reinforce intestinal homeostasis. Immunity 2009;31:401 11. [4] Zigmond E, Bernshtein B, Friedlander G, Walker CR, Yona S, Kim KW, et al. Macrophage-restricted interleukin-10 receptor deficiency, but not IL-10

[18] [19]

[20]

deficiency, causes severe spontaneous colitis. Immunity 2014;40:720 33. Shevach EM. Mechanisms of Foxp3 1 T regulatory cell-mediated suppression. Immunity 2009;30:636 45. Mizoguchi A, Bhan AK. A case for regulatory B cells. J Immunol 2006;176:705 10. Mizoguchi A, Mizoguchi E, Takedatsu H, Blumberg RS, Bhan AK. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity 2002;16:219 30. Sattler S, Ling GS, Xu D, Hussaarts L, Romaine A, Zhao H, et al. IL-10-producing regulatory B cells induced by IL-33 (Breg(IL-33)) effectively attenuate mucosal inflammatory responses in the gut. J Autoimmun 2014;50:107 22. Mills KH. TLR-dependent T cell activation in autoimmunity. Nat Rev Immunol 2011;11:807 22. Gaffen SL, Jain R, Garg AV, Cua DJ. The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing. Nat Rev Immunol 2014;14:585 600. Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3:133 46. Hunter CA. New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions. Nat Rev Immunol 2005;5:521 31. Garrett WS, Gordon JI, Glimcher LH. Homeostasis and inflammation in the intestine. Cell 2010;140:859 70. Do JS, Visperas A, Freeman ML, Iwakura Y, Oukka M, Min B. Colitogenic effector T cells: roles of gut-homing integrin, gut antigen specificity and gammadelta T cells. Immunol Cell Biol 2014;92:90 8. Siddiqui KR, Laffont S, Powrie F. E-cadherin marks a subset of inflammatory dendritic cells that promote T cell-mediated colitis. Immunity 2010;32:557 67. Kamada N, Hisamatsu T, Okamoto S, Chinen H, Kobayashi T, Sato T, et al. Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-gamma axis. J Clin Invest 2008;118:2269 80. Kamada N, Hisamatsu T, Honda H, Kobayashi T, Chinen H, Kitazume MT, et al. Human CD14 1 macrophages in intestinal lamina propria exhibit potent antigen-presenting ability. J Immunol 2009; 183:1724 31. Coombes JL, Powrie F. Dendritic cells in intestinal immune regulation. Nat Rev Immunol 2008;8:435 46. Varol C, Zigmond E, Jung S. Securing the immune tightrope: mononuclear phagocytes in the intestinal lamina propria. Nat Rev Immunol 2010;10:415 26. Jaensson E, Uronen-Hansson H, Pabst O, Eksteen B, Tian J, Coombes JL, et al. Small intestinal CD103 1 dendritic cells display unique functional properties

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

that are conserved between mice and humans. J Exp Med 2008;205:2139 49. Fujimoto K, Karuppuchamy T, Takemura N, Shimohigoshi M, Machida T, Haseda Y, et al. A new subset of CD103 1 CD8alpha 1 dendritic cells in the small intestine expresses TLR3, TLR7, and TLR9 and induces Th1 response and CTL activity. J Immunol 2011;186:6287 95. Kinnebrew MA, Buffie CG, Diehl GE, Zenewicz LA, Leiner I, Hohl TM, et al. Interleukin 23 production by intestinal CD103(1)CD11b(1) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense. Immunity 2012;36:276 87. Schulz O, Jaensson E, Persson EK, Liu X, Worbs T, Agace WW, et al. Intestinal CD103 1 , but not CX3CR1 1 , antigen sampling cells migrate in lymph and serve classical dendritic cell functions. J Exp Med 2009;206:3101 14. Persson EK, Uronen-Hansson H, Semmrich M, Rivollier A, Hagerbrand K, Marsal J, et al. IRF4 transcription-factor-dependent CD103(1)CD11b(1) dendritic cells drive mucosal T helper 17 cell differentiation. Immunity 2013;38:958 69. Coombes JL, Siddiqui KR, Arancibia-Carcamo CV, Hall J, Sun CM, Belkaid Y, et al. A functionally specialized population of mucosal CD103 1 DCs induces Foxp3 1 regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J Exp Med 2007;204:1757 64. Matteoli G, Mazzini E, Iliev ID, Mileti E, Fallarino F, Puccetti P, et al. Gut CD103 1 dendritic cells express indoleamine 2,3-dioxygenase which influences T regulatory/T effector cell balance and oral tolerance induction. Gut 2010;59:595 604. Jeon SG, Kayama H, Ueda Y, Takahashi T, Asahara T, Tsuji H, et al. Probiotic Bifidobacterium breve induces IL10-producing Tr1 cells in the colon. PLoS Pathog 2012;8:e1002714. Varol C, Vallon-Eberhard A, Elinav E, Aychek T, Shapira Y, Luche H, et al. Intestinal lamina propria dendritic cell subsets have different origin and functions. Immunity 2009;31:502 12. Kayama H, Ueda Y, Sawa Y, Jeon SG, Ma JS, Okumura R, et al. Intestinal CX3C chemokine receptor 1(high) (CX3CR1(high)) myeloid cells prevent T-celldependent colitis. Proc Natl Acad Sci U S A 2012;109:5010 15. Atarashi K, Nishimura J, Shima T, Umesaki Y, Yamamoto M, Onoue M, et al. ATP drives lamina propria T(H)17 cell differentiation. Nature 2008;455:808 12. Denning TL, Wang YC, Patel SR, Williams IR, Pulendran B. Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17-producing T cell responses. Nat Immunol 2007;8:1086 94.

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[32] Davies LC, Jenkins SJ, Allen JE, Taylor PR. Tissueresident macrophages. Nat Immunol 2013;14:986 95. [33] Bain CC, Bravo-Blas A, Scott CL, Gomez Perdiguero E, Geissmann F, Henri S, et al. Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nat Immunol 2014;15:929 37. [34] Rivollier A, He J, Kole A, Valatas V, Kelsall BL. Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon. J Exp Med 2012;209:139 55. [35] Bain CC, Scott CL, Uronen-Hansson H, Gudjonsson S, Jansson O, Grip O, et al. Resident and proinflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunol 2013;6:498 510. [36] Zigmond E, Varol C, Farache J, Elmaliah E, Satpathy AT, Friedlander G, et al. Ly6C hi monocytes in the inflamed colon give rise to proinflammatory effector cells and migratory antigen-presenting cells. Immunity 2012;37:1076 90. [37] Zhou H, Xiao J, Wu N, Liu C, Xu J, Liu F, et al. MicroRNA-223 regulates the differentiation and function of intestinal dendritic cells and macrophages by targeting C/EBPbeta. Cell Rep 2015;13:1149 60. [38] Niess JH, Brand S, Gu X, Landsman L, Jung S, McCormick BA, et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 2005;307:254 8. [39] Diehl GE, Longman RS, Zhang JX, Breart B, Galan C, Cuesta A, et al. Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells. Nature 2013;494:116 20. [40] Shouval DS, Biswas A, Goettel JA, McCann K, Conaway E, Redhu NS, et al. Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function. Immunity 2014;40:706 19. [41] Kobayashi M, Kweon MN, Kuwata H, Schreiber RD, Kiyono H, Takeda K, et al. Toll-like receptordependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice. J Clin Invest 2003;111:1297 308. [42] Murai M, Turovskaya O, Kim G, Madan R, Karp CL, Cheroutre H, et al. Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis. Nat Immunol 2009;10:1178 84. [43] Krause P, Morris V, Greenbaum JA, Park Y, Bjoerheden U, Mikulski Z, et al. IL-10-producing intestinal macrophages prevent excessive antibacterial innate immunity by limiting IL-23 synthesis. Nat Commun 2015;6:7055.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

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[44] Ishifune C, Maruyama S, Sasaki Y, Yagita H, Hozumi K, Tomita T, et al. Differentiation of CD11c 1 CX3CR1 1 cells in the small intestine requires Notch signaling. Proc Natl Acad Sci U S A 2014;111:5986 91. [45] Evason K, Huang C, Yamben I, Covey DF, Kornfeld K. Anticonvulsant medications extend worm life-span. Science 2005;307:258 62. [46] Chieppa M, Rescigno M, Huang AY, Germain RN. Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J Exp Med 2006;203:2841 52. [47] Mazzini E, Massimiliano L, Penna G, Rescigno M. Oral tolerance can be established via gap junction transfer of fed antigens from CX3CR1(1) macrophages to CD103(1) dendritic cells. Immunity 2014;40:248 61. [48] Watchmaker PB, Lahl K, Lee M, Baumjohann D, Morton J, Kim SJ, et al. Comparative transcriptional and functional profiling defines conserved programs of intestinal DC differentiation in humans and mice. Nat Immunol 2014;15:98 108.

[49] Matsuno H, Kayama H, Nishimura J, Sekido Y, Osawa H, Barman S, et al. CD103 1 dendritic cell function is altered in the Colons of Patients with Ulcerative Colitis. Inflamm Bowel Dis 2017;23:1524 34. [50] Lee YK, Turner H, Maynard CL, Oliver JR, Chen D, Elson CO, et al. Late developmental plasticity in the T helper 17 lineage. Immunity 2009;30:92 107. [51] Ahern PP, Schiering C, Buonocore S, McGeachy MJ, Cua DJ, Maloy KJ, et al. Interleukin-23 drives intestinal inflammation through direct activity on T cells. Immunity 2010;33:279 88. [52] Ogino T, Nishimura J, Barman S, Kayama H, Uematsu S, Okuzaki D, et al. Increased Th17-inducing activity of CD14 1 CD163 low myeloid cells in intestinal lamina propria of patients with Crohn’s disease. Gastroenterology 2013;145:1380 91 e1381. [53] Barman S, Kayama H, Okuzaki D, Ogino T, Osawa H, Matsuno H, et al. Identification of a human intestinal myeloid cell subset that regulates gut homeostasis. Int Immunol 2016;28:533 45.

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Regulation of Mucosal Immunity in the Genital Tract: Balancing Reproduction and Protective Immunity Danica K. Hickey1, Peter Mulvey2, Emily R. Bryan1, Logan Trim1 and Kenneth W. Beagley1 1

Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia 2Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australia

I. INTRODUCTION According to the World Health Organization (https://www.who.int/news-room/fact-sheets/ detail/sexually-transmitted-infections-(stis)), in 2016 more than 1 million sexually transmitted infections (STIs) were acquired every day. Each year, there are an estimated 215 million new infections with one of three bacterial STIs— chlamydia (131 million), gonorrhea (78 million), and syphilis (5.6 million)—in addition to 143 million infections with the sexually transmitted protozoan parasite Trichomonas vaginalis. Furthermore, more than 290 million women have a human papillomavirus (HPV) infection, and more than 500 million people have genital herpes simplex virus (HSV) infection. Some of these infections are asymptomatic, meaning

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00016-X

that the numbers may be an underestimate. All STIs are spread by sexual contact, but many can also be transmitted from mother to child during pregnancy and childbirth, causing adverse reproductive outcomes, including low birth weight, sepsis, pneumonia, conjunctivitis, neurological problems, and stillbirth. For example, 900,000 pregnant women were infected with syphilis, resulting in 350,00 adverse outcomes [1]. Increasing rates of syphilis in pregnant women have been reported in both low-income countries [1] and first-world countries such as the United States [2]. Many STIs, particularly genital herpes, have been associated with an increased risk of infection with human immunodeficiency virus (HIV), and in 2017, there were 37 million people living with HIV/acquired immunodeficiency

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syndrome (AIDS) worldwide with 1.8 million new infections that year (UNAIDS, https:// www.hiv.gov/hiv-basics/overview/data-andtrends/global-statistics). Chlamydia, gonorrhea, and syphilis can be treated with antibiotics, although in many parts of the world, multidrug-resistant gonococcal strains may make gonorrhea an untreatable condition [3] (https://www.cdc.gov/std/gonorrhea/arg/ default.htm) (Chapter 36: Mucosal Vaccines for Bacterial Sexually Transmitted Diseases). Despite available treatments, the incidence of each of these infections continues to increase. To date, the only vaccines approved to target STIs in humans are the HPV vaccines Gardasil and Cervarix and the vaccine against hepatitis B, a virus that can also be sexually transmitted. Gonococcal infections fail to elicit immunity against reinfection, and natural chlamydial infections elicit only short-lived immunity, that is, serovar-specific immunity. Repeat syphilis infections have also been reported [4], suggesting that natural immunity is incomplete. While most sexually transmitted pathogens have evolved multiple mechanisms to evade the host immune system, there are constraints on immunity in both the female and male genital tracts that may also contribute to the increasing incidence of infections and the difficulty facing vaccine development. Immunity in the female genital tract (FGT) must be regulated to permit fertilization, implantation, and the development of a semiallogeneic fetus, meaning that immune responses to paternal antigens must be prevented or suppressed [5]. This suppression of FGT immunity is due mainly to sex hormone regulation of immunity in the FGT, which may also increase susceptibility to infection [6]. In the testes, sperm production begins at puberty, long after tolerance to self has developed; thus autoimmune responses to sperm antigens must also be prevented or suppressed. Prevention of antisperm responses are maintained by physical separation of the testicular sperm from the immune system by the blood:

testis barrier and by the action of specialized macrophages and other immune cells that maintain a suppressive environment, both of which could increase susceptibility to infection with STIs and allow chronic or latent infections to persist. This is supported by our findings of chronic chlamydial infection of the testes in male mice (Ref. [7] and personal observations) and the detection of Zika virus in mouse testes and human semen (reviewed in Ref. [8]). In this chapter, we review the immune system of the female and male genital tracts, highlighting the unique features that balance the reproductive and immune protective functions of these tissues, and describe progress in the development of vaccines to target the major STIs chlamydia and genital herpes.

II. IMMUNOLOGY OF THE FEMALE GENITAL TRACT The FGT is a unique immunological site that must prevent infection by sexually transmitted organisms while maintaining tolerance against allogeneic sperm and permitting fertilization and implantation [9]. Over the course of the menstrual cycle, physiological changes that facilitate ovulation, fertilization, and implantation are locally controlled by endogenous sex steroid hormones produced by the ovaries, including estradiol and progesterone [10]. Cyclic hormone concentrations are characterized by increasing estradiol levels during the follicular phase (days 0 14), with a peak estradiol level measured prior to ovulation at midcycle, followed by increasing progesterone levels postovulation and throughout the secretory/luteal phase (days 14 28) [10]. It is well recognized that mucosal immune responses in the FGT are also regulated by changes in hormones over the menstrual cycle [11 13]. Furthermore, the susceptibility of reproductive tissue to infection and the subsequent shedding of pathogenic organisms are believed to be

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cyclically regulated, with immune dampening observed at ovulation essentially creating a window of vulnerability to infection [14]. Protective immunity versus susceptibility to infection is a balance of hormonally controlled mechanisms, including tissue barrier physiology, soluble mediators, and cellular responses [9]. The local mucosal immune system of the FGT comprises both innate and adaptive immune cells and soluble mediators distributed throughout the entire tract with trafficking and activation changes over the menstrual cycle [9]. The FGT is compartmentalized into the lower FGT, comprising the vagina and ectocervix, and the upper FGT, comprising the endocervix, endometrium, fallopian tubes, and ovaries. During the menstrual cycle, hormone-driven tissue proliferation in the endometrium is required to facilitate implantation [10]. In the absence of pregnancy, decidualization of the endometrium leads to shedding and menses. The essential physiological role of the endometrium in reproduction results in the endometrium being highly responsive to sex steroid hormones compared to other FGT sites [10]. Thus hormones drive unique immunological changes in the endometrium over the menstrual cycle, and this is the most studied tissue site in the FGT. Using flow cytometric analysis, Givan et al. [15] quantitated the distribution of leukocytes in the upper and lower FGT. Overall, a higher proportion of immune cells are localized within the upper FGT compared to the lower FGT, and cell numbers primarily in the upper FGT are under cyclic hormone control [15].

A. Innate Immune Cells in the Upper Female Genital Tract Endometrial-specific natural killer (NK) CD32CD56bright CD162 cells (also known as glandular lymphocytes) are present in the FGT depending on the stage of the menstrual cycle [16,17]. During the proliferative phase, low NK

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numbers are found in the stroma. However, a large recruitment of NK cells throughout the stroma, with accumulation around endometrial glands and blood vessels, occurs during the secretory phase, representing 70% of all leukocytes in the endometrium [17,18]. Macrophage numbers are also reported to increase and accumulate around endometrial glands late in the secretory phase, with numbers peaking during menses [18,19]. Both M1 and M2 macrophage phenotypes are present in the endometrium and are associated with bacterial clearance and inflammation and with postmenses angiogenesis and repair, respectively [20,21]. Likewise, neutrophil numbers increase during the secretory phase and peak during the menses [22].

B. Adaptive Immune Cells in the Upper Female Genital Tract In contrast with innate cells, CD31 T lymphocyte numbers are highest during the proliferative phase before decreasing over the cycle and are lowest during the secretory phase [15]. T lymphocytes are found in the endometrium as basal lymphoid aggregates but also scattered throughout stromal layers and epithelium. Interestingly, unlike peripheral blood with a CD4:CD8 ratio of 2:1, CD81 cytotoxic lymphocytes are more commonly present in the FGT [23]. Furthermore, this ratio is modulated over the normal menstrual cycle. A study by Mettler et al. [23] investigating endometrial CD41 and CD81 T lymphocytes reported that during the early proliferative phase, at the peak of lymphocyte numbers in the FGT, the ratio of CD41 to CD81 lymphocytes was 1:1. As total lymphocytes numbers decrease through the proliferative phase, a shift to a predominant CD81 distribution is observed, with a CD41 to CD81 ratio of 1:2 at time of ovulation. Following ovulation, a further drop in total and predominantly CD41 lymphocyte numbers occurs over the secretory phase, resulting in a ratio of CD41 to CD81 of 1:4 at the end of the menstrual cycle [23].

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The high abundance of endometrial CD81 lymphocytes is associated with the formation of lymphoid aggregates from midcycle and during the secretory phase, containing almost exclusively CD81 T lymphocytes surrounding a B lymphocyte core [24]. The cytolytic activity of endometrial T cells is present during the proliferative phase but not the secretory phase, suggesting another function of CD81 cells in lymphoid aggregates in the second half of the menstrual cycle [22], potentially preventing rejection of the semiallogeneic fetus. The inhibition of cytolytic activity correlates with high progesterone and estradiol levels, and no such cycle-associated changes in endometrial cytolytic T cell capacity is observed in postmenopausal women [22,25]. The main role of the FGT is to sustain reproduction, as such immune regulation of responses against the antigenically distinct fetus and placenta is essential. Specialized populations of CD41 T helper cells known as regulatory T cells (Tregs) are potent suppressors of the inflammatory immune response. In women, endometrial Tregs populations expand over the proliferative phase of the menstrual cycle [26]. Studies in mice show that forkhead box P3 (FoxP3) expression is lowest at diestrus, increasing across the cycle to peak at metestrus in nonpregnant mice; during pregnancy, FoxP3 expression is further increased. [27]. The recruitment and retention of Treg cells were associated with cycle-associated changes in the expression of multiple chemokine genes, including CCL3, CCL4, CCL22, and CX3CL1. The data are consistent with a role of Treg cells in preparing the uterus for implantation and maintaining tolerance to the semiallogeneic fetus during pregnancy. Consistent with endometrial T lymphocyte distribution, the number of fallopian tube CD31 T cells peaks in the proliferative phase and then decreases following ovulation to low levels in the secretory phase [15]. The FGT contains a number of unique T cell populations. Murine studies have shown that unique

populations of nonrecirculating tissue-resident memory cells (TRMs), both CD81 and CD41, which are phenotypically distinct from both circulating central memory and effector memory T cells are localized within the uterine, cervical, and vaginal mucosa [28]. In the upper FGT, there is limited understanding of the role of TRMs. However, in the lower FGT, TRMs are likely to be important for controlling STIs and preventing the ascent of microorganisms to the upper FGT [29]. B lymphocyte populations in the FGT are present consistently but in relatively low numbers compared to CD31 T lymphocytes. In endometrial tissue, B cells are predominantly associated with lymphoid aggregates, forming a celluar core surrounded by CD81 lymphocytes [24]. Lymphoid aggreagates increase in size during the secretory phase, with increasing cell numbers derived from recruitment rather than local proliferation [24]. B cells are also localized within endocervical tissues, two-third of subepithelial lymphocytes being polymeric immunoglobulin A (IgA) producing plasma cells [30]. Both local production of immunoglobulin in the endometrium and endocervix and serum-derived immunoglobulin contribute to the immunoglobulin content of the cervical mucus (CM). Immunoglobulin G (IgG) and IgA are actively transported into upper FGT secretions via the neonatal Fc receptor (FcRn) and the polymeric immunoglubulin receptor (pIgR), respectively (Chapter 4: Protective Activities of Mucosal Antibodies). FcRnmediated transport of IgG is bidirectional, potentially enhancing infection of sexually transmitted pathogens [31], while pIgRmediated transport of IgA is unidirectional, from the lamina propria to the FGT lumen. Upper FGT epithelium express both these immunoglobulin transport molecules. In vitro, estradiol treatment has been shown to upregulate the surface expression and polymeric IgA binding by secretory component, a part of the pIgR receptor on human glandular endometrial

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cells [32]. Using a mouse model, Wang et al. [33] showed that treatment of ovariectomized mice with estradiol rescues pIgR-mediated IgA transport into the FGT [33]. This suggests that estradiol directly regulates transport of IgA to the lumen in the FGT. In contrast to pIgR, FcRn-mediated IgG transport is regulated by pH. Little is known about menstrual cycle hormone regulation of FcRn expression. Overall, little is known about cyclic regulation of immunoglobulin concentrations in the upper FGT. However, in the CM, levels of both IgG and IgA peak prior to ovulation and are lowest during the secretory phase [34]. It is unclear whether this reduction is due to decreased local production, decreased serum exudation, dilution of total mucus, or a combination of the three [35]. Fahrbach et al. [36] found that both IgA and IgG present in CM bind by protein protein interactions rather than by passive association. Furthermore, in the CM, IgG accumulates densely on the periphery, while IgA is spread throughout the local CM network [36]. CM is formed from the secretion of mucins, including Muc1, Muc5AC, Muc5B, Muc6, and Muc16 by endometrial and cervical columnar epithelial cells and cervical crypt goblet cells. As CM moves down the FGT, it mixes with vaginal mucus and secretions, producing cervicovaginal mucus (CVM), which is less viscous than CM (Section E, Soluble Mediators in the Female Genital Tract). The cervix represents a transition zone in which the lower FGT multilayered squamous epithelium of the ectocervix transitions to the single layer of columnar epithelium of the ectocervix and the rest of the upper FGT. The transition zone contains high numbers of CD41 and CD81 T lymphocytes as well as macrophages and granulocytes [37] and is a highly active immunological site and the primary infection site for many sexually transmitted pathogens. While the total proportion of leukocytes across the two sites is consistent, specific cells type numbers differ. For example, ectocervical tissue

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contains increased numbers of both T (CD41 and CD81) and B lymphocytes when compared to the endocervix [38]. Consistent with the upper FGT, CD81 T lymphocytes predominate over CD41 T lymphocytes. In both the ectocervix and the endocervix, CD41 and CD81 T lymphocytes are present as intraepithelial lymphocytes (IEL), with CD81 IELs expressing the TIA1 cytotoxic granule-associated RNA binding protein (TIA1), a marker of cytotoxic potential, suggesting increased cytotoxic function aimed at stopping infections ascending to the upper FGT during the second half of the menstrual cycle [37]. Furthermore, in the cervix, a subset of T helper 17 (Th17) CD41 IEL have been identified as HIV infection targets due to the coexpression of HIV coreceptor CCR5 and the mucosal integrin α4β7 integrin [35,39]. In addition to classical αβ T helper lymphocytes, γδ T cells are present within epithelial barriers throughout the FGT [40,41]. These cells show innate characteristics with direct antimicrobial and cytotoxic activity without priming and production of a number of cytokines consistent with an activated phenotype. In the endocervix, the majority of γδ T cells lack expression of both CD4 and CD8 (CD42 CD82) and are defined as γδ type 1 subset cells [40]. The endocervix also contains a small number of the γδ type 2 subset that express CD4 and the chemokine receptor CCR5. Similar to Th17 CD4 cells, type 2 γδ cells may be targets for HIV infection; a loss of endocervical γδ T cells has been observed in women with HIV infection [40]. While the regulation of γδ T cell numbers by the menstrual cycle is unclear, it has been shown that changes in the local microbiome and inflammatory profile during bacterial vaginosis is associated with a drop in γδ type 1 cells in the cervix [41]. In contrast to a decrease in γδ type 1, during bacterial vaginosis an influx of γδ type 2 cells (CD41 CCR51) enters the cervix, presumably from the periphery [41]. This suggests that the cervix contains a number of cellular targets for HIV transmission in the FGT. A study by Alcaide et al. suggests that women with

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abnormal vaginal flora have decreased cervical γδ type 1 and increased γδ type 2 T cells, which increases the likelihood of bacterial vaginosis and HIV acquisition [41]. While lymphocytes are predominant in the ectocervix, NK cells, DCs, and macrophages are equally distributed across the ectocervix and the endocervix. Interestingly, while most cervical CD141 cells are conventional macrophages (CD11c2), one third are CD11c1 DCs that coexpress DC-SIGN, which may facilitate HIV infection through DC-SIGN-mediated viral transfer [37] to T cells [38,42].

C. Innate Immune Cells in the Lower Female Genital Tract In contrast to the upper FGT, the lower FGT is lined with multiple layers of stratified squamous epithelial cells. These stratified squamous epithelial cells overlay basal epithelial cells that, through tight junctions, form a physical barrier. Immune cells in the lower FGT remain relatively constant over the menstrual cycle in both the cervix and the vagina and constitute a lower proportion of cells compared to the upper FGT [15,37]. While overall there are lower numbers of immune cells in the lower FGT, a large population of NK cells has been reported. Vaginal NK cells differ from upper FGT NK cells in the expression of CD16, reflecting a circulating NK phenotype with a primary role in microbial control [43].

D. Adaptive Immune Cells in the Lower Female Genital Tract Similar to the upper FGT, CD81 cell populations are more prominent than CD41 T cell populations in the lower FGT. T lymphocytes are found localized near the basal epithelium at the stromal cell interphase and as IELs [37]. In contrast to cervical tissue, the cytotoxic activity of lower FGT CD81 T lymphocytes is low throughout the reproductive cycle, with no

expression of TIA1, a marker of cytotoxic potential, observed. This suggests a regulatory role rather than a cytotoxic role for these cells in the vagina [37]. Interestingly, in vaginal tissue, CD81 cells predominate in the IEL population, with few CD41 T cells present within the epithelium [37]. IELs in the lower FGT may include populations of γδ T cells and TRMs [38]. In response to infection, such as HSV, murine vaginal CD81 TRM cells rapidly release IFNγ, inducing local immune activation, inflammation, and the recruitment of circulating B and T cells into vaginal tissue [44,45]. While CD81 cells have been studied in greater detail in controlling infection in the vaginal tract, the function of CD81 TRMs is critically regulated by local CD41 TRMs [46]. Humoral responses in the LGT are are characterized by the local production and exudation of immunoglobulins from serum. While B cells are present only in small numbers in the vagina, secreted antibodies present in CVM, collected via cervicovaginal lavage (CVL), may be derived from resident B plasma cells in the cervix, both the endocervix and ectocervix [47]. In contrast to other mucosal sites where secretory IgA (SIgA) is the predominant isotype, immunoglobulin present in CVL samples collected from normal cycling women contain a greater proportion of IgG than SIgA [48,49]. While both IgG and SIgA are present in CM, little SIgA is present in the lower FGT within CVM. This decrease in SIgA in CVM compared to CM is associated with reduced protein binding of SIgA to CMV compared to IgG, which maintains a stable IgG mucus interaction [36]. Studies of immunoglobulin concentrations in CVLs from women and rhesus macaque monkneys reported that IgG and SIgA concentrations varied with the stages of the menstrual cycle, with a drop in immunoglobulins around the time of ovulation at midcycle, which coincides with the frequency of IgA- and IgG-secreting cells peaking 3 5 days before ovulation, followed by a rapid decline [13,38,48,49].

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E. Soluble Mediators in the Female Genital Tract In addition to the presence of immunoglobulins, mucus provides a physical barrier rich in antimicrobial molecules, which protect against disease through direct killing or inhibition of potential pathogens. CVM is a combination of upper FGT CM migrated down through the cervix into the vagina mixed with locally produced mucins from ectocervical and vaginal epithelial cells, forming a hydrophobic mucus rich in both immunoglobulins and antimicrobial peptides (AMPs). The viscosity and complement of bioactive molecules in CVM are regulated by the menstrual cycle [50]. At ovulation, following expression of high levels of estradiol, CVM is less viscous with a higher liquid content and higher pH (favorable for spermatozoa) compared to the high-progesterone luteal phase, when CVM is highly viscous with a low pH (impenetrable by spermatozoa) [51]. During the luteal phase, trapped microbes are destroyed by the presence of AMPs and lysozymes entrapped within the CMV [52]. Throughout the FGT, soluble antimicrobial immune molecules include cytokines, chemokines, proteases, and enzymes, which regulate direct microbial killing, tissue homeostasis, immunomodulation, and antiinflammatory pathways (see the review in Ref. [53]). Therefore tight regulation of soluble immune mediators is required for both reproduction and maintaining protection against STIs. Cyclic changes in cytokines and AMPs, such as secretory leukocyte protease inhibitor (SLPI) and human beta defensins, have been observed in CVLs [13]. Analysis of CVLs observed that while there is a consistent concentration of some cytokines, AMPs, and transforming growth factor beta (TGF-β) over the menstrual cycle, a midcycle decrease in IL-6, IL-8, SLPI, and human beta defensin 2 (HBD-2) occurs and in some cases decreases over the secretory phase [11,12]. Furthermore, a study looking at

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HBD-5 reported that increasing HBD-5 concentration from the late proliferative phase peaks in the secretory phase [54]. This suggests that cytokines and AMPs in the lower FGT are regulated over the menstrual cycle in a molecule-specific manner. At ovulation and during the secretory phase following ovulation, an increase in HNP1-3, HD-5, HBD-1, HBD-3, SLPI, and elafin is observed [54 58]. This expression may support microbial protection during a period of immune suppression in the upper FGT. Site-specific and molecule-specific differential regulation of cytokines, chemokines, and AMPs observed in women is supported by observed murine estrous cycle changes in the upper and lower genital tracts. Hickey et al. reported that most vaginal cytokines are present at a consistent level throughout the estrous cycle. However, both IL-1β and CXCL1 concentrations peaked with declining estradiol at diestrus and estrus [59]. In contrast, a significant inhibition of uterine cytokines and chemokines occurs at diestrus immediately following ovulation in the mouse in vivo [59]. Furthermore, while uterine cytokines are inhibited at midcycle, the production of AMPs in both the uterine and vaginal compartments, while influenced by hormones, is altered over the estrous cycle in an AMP-specific manner [59]. While the cyclic changes in specific AMPs may overlap to broadly protect against STI susceptibility, specific molecules are more effective at killing specific microbial pathogens, and cyclic changes in concentrations may drive susceptibility to some infections. For example, the known anti-HIV AMPs SLPI, elafin, and HBD-2 [6,60 64] are inhibited in CVLs at midcycle [65] which coincides with an increase in the number of HIV-1 target cells and may drive an increased susceptibility to HIV. This suggests that combined menstrual cycle regulation of cellular and soluble immunity in the FGT drives specific pathogen susceptibility.

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F. Toll-Like Receptor Expression in the Female Genital Tract The innate immune system is rapidly activated following the binding of conserved microbial products know as pathogenassociated molecular patterns by host pattern recognition receptors such as toll-like receptors (TLRs) [66] (Chapter 6: Innate Immunity at Mucosal Surfaces). In the FGT, TLRs are expressed by immune and epithelial cells and play a significant role in preventing STIs [67,68]. Expression of TLRs by epithelial cells shows that while TLR1, TLR2, TLR3, TLR5, and TLR6 are observed throughout the upper and lower FGT, TLR4 has expression restricted to the endocervix, endometrium, and fallopian tubes [67], although a study by Pivarcsi et al. [69] did report the presence of low levels of TLR4 in the vagina. In addition to membranebound TLR4, a soluble form was identified that is associated with endocervical glands [67,70]. This soluble form of TLR4 may neutralize lipopolysaccharide in the lower FGT, potentially preventing induction of inflammation by the normal vaginal microbiota. The absence or weak expression or soluble forms of TLR4 in the vagina and ectocervix but its presence in endocervical glands suggests a role in the maintenance of immunological tolerance in the upper FGT. In the endometrium, expression of all TLRs, 1 10, are observed; furthermore, endometrial TLR expression is regulated by the menstrual cycle [71]. While constitutive expression is observed in the FGT, TLR2, TLR3, TLR4, TLR5, TLR6, TLR9, and TLR10 mRNA in the endometrial tissue is reduced during menses and the proliferative phase [71,72]. In the murine FRT, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR9, and TLR13 are expressed predominantly in vaginal tissue, and TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR9, and TLR12 are expressed predominantly in uterine tissues [59]. During the murine estrous cycle, overall uterine TLR expression peaks at estrus

after ovulation before a significant reduction at diestrus. In contrast, vaginal TLRs over the estrous cycle are expressed differentially in a TLR-specific manner [59].

G. Hormone Effects on Genital Tract Infections Several animal models of genital tract infection have shown the importance of hormones in the establishment and progression of infection and disease. Exogenous hormone treatment is required to establish chlamydial infections in rodent models, with progesterone pretreatment to induce diestrus necessary to establish a Chlamydia muridarum infection in both mice [73] and rats [74]. Guinea pigs, however, can be infected with Chlamydophila caviae throughout their cycle without the need for hormone pretreatment. However, guinea pigs infected at day 11 of their cycle had significantly more oviduct inflammation [75], and pretreatment of guinea pigs with estradiol resulted in infections of longer duration and greater intensity and increased ascending infection causing endometritis and salpingitis [76]. Progesterone also increases the susceptibility of mice to experimental HSV infection [77], while guinea pigs can be infected with the same HSV strains throughout their cycle. Conversely, infection of female mice with Neisseria gonorrhoeae requires pretreatment of mice with estradiol [78]. These studies show that hormone treatment not only is important in establishing infection but also regulates both the magnitude and duration of infection and infection-induced pathology in animal models. While understanding the direct role of estradiol and progesterone in chlamydia infections in women is difficult, in vitro studies suggest a critical need for hormones for the establishment of infection and also the progression of a persistent state. Estradiol pretreatment of HeLa cells prior to infection with Chlamydia trachomatis

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serovars K or L1 enhanced elementary body (EB) attachment and inclusion body development [79]. Furthermore, Amirshahi et al. showed that in vitro treatment with both estradiol and progesterone resulted in altered gene expression associated with chlamydial persistence [80]. It is currently unclear whether hormones regulate establishment of infection directly or whether the major effects of hormones on infection are indirect, via modulation of the host immune responses, leading to increased susceptibility.

H. Contraceptive-Driven Susceptibility to Sexually Transmitted Diseases in the Female Genital Tract In women, exogenous hormones have been shown to regulate immunity and, potentially, susceptibility to infection in vivo. A large number of epidemiological studies have reported that the use of steroid contraceptives such as depot medroxyprogesterone acetate (DMPA) or combined oral contraceptives (COCs) may enhanced the risk of women acquiring a STI. The most recent systematic reviews of oral contraceptive use and the risk of non-HIV STIs by Deese et al. [81] and McCarthy et al. [82] report that the current evidence of hormone contraceptive driven susceptibility to HPV, chlamydia, gonorrhea, and syphilis infections is unclear. Owing to the variable types of contraceptives with varying hormone compositions currently in use by women, more detailed studies are required to examine each hormonal contraception type with STI susceptibility. For example, of the hormone contraceptives used by women, DPMA specifically has been associated with increased susceptibility to HIV infections [83]. A systematic review conducted by Polis et al. showed that women using the DPMA-type hormone contraceptive are 40% more likely to acquire HIV infection than are women not using hormonal contraceptive [84].

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Interestingly, the current data suggest that DMPA is associated with reduced risk of T. vaginalis infection [82] but an increased susceptibility to HSV-2 (see reviews by Deese et al. and McCarthy et al.). While the drivers of infection susceptibility are unclear, Fichorova et al showed that DMPA and COC use alters cervical cytokine profiles during infection (non-HIV STIs) in a pathogen- and contraceptive-specific manner [85]. This study also reported that an increased proportion of women using hormone contraceptives had asymptomatic infections. This suggests that hormonal contraceptive use not only enhances the risk of acquisition and transmission of sexually infections (STIs) but may also drive asymptomatic infections that could increase the risk of chronic infection and reproductive tract disease.

I. Hormone Receptors and Signaling Steroid action of estradiol and progesterone occurs through a number of mechanisms. “Classical” activation occurs through hormone binding and activation of nuclear receptors, including estrogen receptors (ERα and ERβ subtypes) and progesterone receptors (PR-A and PR-B), resulting in the direct gene regulation [86,87]. “Nonclassical” responses are driven by diverse signaling pathways and second messenger systems independently of transcriptional or classical genomic regulation, driving a rapid hormone effect on tissues [88]. ERs and PRs are expressed in many cell types in the FGT, including immune and tissue cells (reviewed in Ref. [89]). During the menstrual cycle, similar to concentrations of sex hormones, the expression of both ER and PR are altered in the endometrium. Immunohistochemical staining of human endometrial tissue demonstrated strong expression of both receptors in endometrial cells during the proliferative phase, which decreased significantly in the secretory phase [90]. In addition to estrogen and progesterone, steroid receptors bind and are activated by a large number of

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other molecules, including selective estrogen receptor modulators (SERMs) and selective progesterone receptor modulators (SPRMs) [91]. Treatment with SERMs such as tamoxifen, which act directly through ERs, for treatment of ER-positive breast cancer has been shown to have proliferative effects on endometrial tissues in patients and may cause adverse genital tract outcomes [92]. In murine studies, specific ERα targeting SERMs regulated the production of uterine chemokines CCL20 and CXCL1 in vivo and in vitro by uterine epithelial cells and directly opposed the effects of estradiol [93]. Studies with freshly isolated human vaginal epithelial cells showed that SERM treatment had no effect on the production of HBD-2 and elafin [94], although production of both were suppressed by estradiol. Understanding the positive and negative physiological and immunological effects of hormone receptor activation following ligand binding and subsequent effects on downstream signaling will require further study and must be considered in our understanding of endocrine regulation driving immune protection against STIs. Fig. 16.1 summarizes the influence of sex hormones on immunity in the FGT throughout the estrous cycle.

III. IMMUNOLOGY OF THE MALE REPRODUCTIVE TRACT STIs affect both women and men, and male infections represent the reservoir of infection in the community. The immune system of the male reproductive tract is poorly studied, despite the fact that vaccine-induced protection against the spread of STIs is likely to be more effective if immunity is induced in both sexes. We have included a review of what is known about immunity in the male reproductive tract. As the site of initial exposure for STIs and UTIs, the penile urethra has a plethora of innate and adaptive immune response mechanisms.

As with all mucosal body sites, mucus production by epithelium creates a protective layer. The penile epithelium produces membraneassociated mucins (MUC1, MUC3, MUC4, MUC13, MUC15, MUC17, and MUC20), and one gel-forming mucin (MUC5AC) is produced by urethral glands [95]. The mucus layer forms a barrier that helps to prevent invasion of larger pathogens, including bacteria, but is potentially ineffective against viruses. Antibody-mediated agglutination of pathogens may assist in preventing invasion of pathogens through the small pores of the mucin matrix [96,97]. The secreted mucus also contains AMPs, although these are constitutively expressed at most mucosal sites by innate immune and epithelial cells, such as lysozyme, lactoferrin, serine leukocyte protease inhibitor, and defensins (human β-defensins 1, 2, and 3 and α-defensins 5 and 6 are largely expressed by epithelial cells; neutrophil α-defensins 1, 2, 3, and 4 are mostly secreted by granulocytes) [98 100]. Further innate protection in the urethra occurs with the expression of many TLRs, including TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9, which are, however, quite sparse in the upper reproductive tract [101]. TLR2, TLR3, and TLR4 are relatively rarely expressed in the urethra, predominantly on lymphocytes residing in the lamina propria, whereas TLR1, TLR5, and TL7 are more frequently expressed on immune cells, and TLR9 is expressed on mucosal epithelial cells [101]. Cells morphologically categorized as macrophages can be dispersed throughout the epithelium (TLR11, TLR71, TLR91), lamina propria (TLR11), and fossa navicularis (TLR11), depending on the health of the tissue [102]. TLR51 lymphocytes can be found in the same sites. Other pattern recognition receptors, including NOD-like receptors, have not yet been characterized in the penile tissue and urethra. Activation of innate defenses can lead to production of type 1 interferons (IFNα and IFNβ) and type 2 interferons (IFNγ) predominantly by

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FIGURE 16.1 Summary of hormonal, physiological, cellular and immunological changes of the endometrium during the menstrual cycle in women. E2, 17b estradiol; ER, estrogen receptor; HBD, human beta defensing; P4, progesterone; PR, progesterone receptor; NK, natural killer cell; SLPI, secretory leukocyte protease inhibitor; TLR, toll-like receptor; TRM, tissue-resident memory cell.

immune cells and by epithelial cells coexpressing TLR9 [102,103]. Interferons and other cytokines (e.g., IL-17, IL-22, TNF-α, and IL-2) and chemokines and receptors (e.g., CCR3, CCR6,

CCR9, and CCR10) contribute to recruitment of effector cells to extend protection. Penile innate immune cells capable of expressing these include CD161, CD561(dim), NKp441 NK cells,

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macrophages, and dendritic cells (DC) [103]. These cells display diversity in surface marker expression and abundance depending on their location; for example, the urethra houses abundant macrophages, while DCs appear to be absent, yet the reverse is true for the meatus and fossa, which normally contain abundant DCs but few macrophages. Adaptive immunity includes both T and B lymphocytes. CD41 and CD81 T cells commonly expressing the mucosal-associated antigen αEβ7 and the memory markers CD45RO/ RA and CCR7 are present in varying levels in all penile tissues [103,104]. A small proportion of T cells are also CD691, CD62L1, and CD1031 TRMs. CD191, CD21-positive and -negative, and CD27-positive and -negative B lymphocytes are similarly ubiquitous, indicating the presence of naı¨ve, resting, and activated memory cells [103]. CD191, CD381, and CD1381 plasma cells are also present and can secrete IgA and IgG [103]. A similar distribution of immune cell subsets occurs in the accessory organs (seminal vesicles, bulbourethral gland, and prostate) and the vas deferens, with continuing compartment-specific differences in cell density. CD81 T cells dominate the T cell phenotypes. Macrophages are common in the stroma of the tissues. These cells are most likely to be the origin of the leukocytes (most commonly macrophages, neutrophils, and lymphocytes) that can occur naturally in semen. In support of this is the fact that vasectomy drastically reduces the number of leukocytes present in semen [105,106]. The major difference in these tissues progressing to the upper reproductive tract is secretion of antiinflammatory and regulatory cytokines and androgenic steroids, which function to limit sperm-specific antibody production and subsequent contribution to autoimmune infertility. The requirement of suppressing autoimmunity to sperm increases in the epididymis, the site of sperm maturation and storage [107]. The epididymis becomes compartmentalized

into the interstitial and epithelial tubules for sperm storage. Epithelial cell junctions create a blood epididymis barrier, which provides protection for sperm but is comparatively leaky compared to the blood testis barrier [108,109]. Immune cells are still relatively common in both the epithelium and interstitial compartments of the epididymis. Most information for epididymal and testicular immunobiology is extrapolated from animal models, as obtaining these tissues from healthy humans is difficult. However, MHC class II restricted CD41 T cells appear commonly in the interstitium, while CD81 T cells are more frequent in the epithelium, with some differences in density between the caput, corpus, and cauda epididymis [110]. Macrophages and DC-like cells are most frequently observed within both compartments, although other leukocytes are present, which predominantly clear apoptotic sperm from tubules [111]. Marked differences occur within epididymal macrophages, where the interstitial cells express MHC class II but the epithelial macrophages do not. This truncated antigen presentation and proinflammatory regulatory ability intensify in moving from the epididymis into the testes [112]. Testicular macrophages constitute the majority of testicular immune cells [113]. A proportion of DCs, mast cells, eosinophils, NK, and lymphocytes (predominantly MHC class I restricted CD81 and cytotoxic and CD41 regulatory T cells) recruited from circulation also exist [114]. The macrophages are predominantly of an M2, CD1631 phenotype and are the major contributor to the immune suppressive or immune-privileged environment within the testes that is required for spermatogenesis [115]. Murine testicular macrophages have been fairly well characterized; these are generally the resident F4/801/CD2061/Ym11/Mrc11/ 1 Arg1 /CD1631/MHCII1 cells [114]. These cells originate during embryogenesis and have selfrenewing capacity [114,116]. There is also a

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smaller population of testicular macrophages that most likely originate from the circulating population that are CD1632/CD861 [117]. Testicular macrophages retain their phagocytic ability; however, they have reduced expression of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α to prevent inflammatory cell recruitment and immunopathology within the testis [112,117]. Because testicular macrophages are also responsible for protecting the testis from infection, this becomes a difficult balance between effective clearance of infection and avoiding immunopathology [117]. Testicular macrophages contribute heavily to the immune-privileged status of the testis. They create the tolerance for immunogenic germcell-derived antigens that would otherwise elicit an autoimmune response against the germ cells from which sperm develop [118]. This occurs through the suppressive effects and truncated inflammatory abilities of the testicular macrophages. This process works in concert with the tight junctions between intratubular Sertoli cells, which provide the physical blood testis barrier to shield the germ cell antigens more effectively.

IV. CHLAMYDIA C. trachomatis is an atypical intracellular Gram-negative bacterium with a biphasic life cycle, consisting of an infectious metabolically inert extracellular EB and the replicating, noninfectious intracellular reticulate body (RB) [119]. Uptake of EBs begins with electrostatic binding to heparin sulfate proteoglycans on target epithelial cells [120], followed by clathrinmediated uptake and remodeling of the host cell actin cytoskeleton [121,122]. Following uptake, the EB forms a parasitophorous vacuole, the inclusion, which is considered nonfusogenic [123], isolating the Chlamydia from host endocytic and lysosomal pathways. This inclusion can fuse with host cell vesicle-trafficking

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pathways allowing the dividing Chlamydia to hijack and sequester host cell lipids and other metabolites required for EB RB transition and RB division [124,125]. Replication of the RB occurs by binary fission within the inclusion, yielding 100 900 progeny, depending on the serovars or species of Chlamydia. Following transition of RB to EB, release of newly formed chlamydial EB occurs by either lysis of the host cell or extrusion of packaged chlamydial EB in host-membrane-bound extrusions [126]. Chlamydiae can also enter a persistent state when exposed to stressors such as antibiotics, nutritional deprivation, and exposure to inflammatory cytokines such as IFNγ [127,128]. Persistence is characterized by large, irregular EBs termed aberrant bodies (ABs), which are noninfectious but remain viable within the host cell [127,128]. Upon removal of the stressors, ABs can revert to RBs, and the replicative cycle resumes. This ability to maintain viability in the face of multiple stressors may complicate the development of vaccines or the effectiveness of antibiotic treatments. C. trachomatis is the most common bacterial STI worldwide, with 130 million new infections each year [129]. It has been the most reported STI in the United States for the past 20 years, with twice as many females infected as males [130]. Up to 70% of infections in females and 50% in males are asymptomatic, indicating that infection data are probably underestimates [131]. Despite effective antibiotic treatment (azithromycin or doxycycline), infection rates have been consistently increased for the past 30 years, and the consensus view is that only an effective vaccine will reverse this trend [132]. C. trachomatis serovars D K and the more invasive serovars L1 L3 responsible for lymphogranuloma venereum are the cause of genital tract infections [133 138]. Serovars A C are the cause of ocular infections leading to trachoma, the most common cause of infectious blindness worldwide. Genital infections in women cause pelvic inflammatory disease

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(PID) and tubal blockage leading to infertility and ectopic pregnancy [139]. Infections in males are less well studied but have been linked to epididymitis, prostatitis, orchitis, and decreased fertility due to increased sperm DNA damage and reduced motility and viability [140].

A. Pathogenesis of C. trachomatis Genital Tract Infections The major health costs associated with chlamydial infections are due to damaging tissue pathology associated with infection of the FGT. Infection of the endocervical epithelium induces the secretion of CXCL1, CXCL16, and the inflammatory cytokines IL1, IL6, IL8, GMCSF, and TNF-α [141], inducing recruitment of neutrophils and NK cells, which secrete IFNα, IFNβ, IFNγ, and IL-12, which are necessary to polarize the adaptive immune response to a protective Th1/Th17 immune response [142,143] but at the same time drive pathology in the murine FRT [144,145]. Chlamydial infection of mouse oviducts leads to rapid recruitment of neutrophils, and neutrophil numbers directly correlate with oviduct occlusion [146,147]. Furthermore, depletion of neutrophils or inhibition of neutrophil chemotaxins (IL8/MIP-2, CXCR2) reduces the severity of immunopathology [147]. Production of matrix metalloproteinases (MMPs) by neutrophils has been implicated in tissue damage in the mouse oviduct (MMP9) [148]; in human tissues, MMP2 and MMP9 can be produced by neutrophils, epithelial cells, and stromal cells [149]. Signaling through TLR2 has also been shown to be important for tissue pathology in mouse models; TLR2-deficient animals show significantly reduced tissue damage [150]. Using human fallopian tube explants, Hvid et al. [151] showed that infection directly causes tissue destruction of the ciliated epithelial cells due to excess IL1α and IL8 production, and this

cellular hypothesis [152] of pathology posits that FGT tissue damage is due to the continuous production of inflammatory cytokines (IL1, IL6, IL8, and TNF-α) by the infected epithelial cells alone, even in the absence of recruited leukocytes. Recruited leukocytes would further exacerbate damage. Further studies in mouse models have also implicated TNF-α-producing CD81 T cells as having a major role in oviduct destruction [153]. OT-1 mice, which cannot develop chlamydia-specific CD81 T cells, do not develop hydrosalpinx following infection. However, adoptive transfer of wild-type CD8 cells prior to infection restored infectioninduced oviduct pathology [154], confirming the role of pathogenic CD81 T cells, at least in the mouse model. Furthermore, monkey [155] and guinea pig [156] models of chlamydia infection indicate that both CD41 and CD81 T cells rapidly infiltrate the oviducts on repeat infections and play a role in tissue fibrosis and scarring. Thus in terms of pathogenesis, chlamydia infections, which are apparently benign, inasmuch as 70% of infections are asymptomatic in women [131], activate multiple immune mechanisms that ultimately lead to scarring, fibrosis, and obstruction of the fallopian tubes and oviducts. In summary, inflammation results from sustained overproduction of multiple mediators by infected nonimmune host epithelial cells. Molecules such as IL-1α, TNF-α, and MMPs directly damage the mucosal epithelium, while various cytokines and chemokines recruit inflammatory immune cells. Neutrophils play a major role in mouse infection models, recruited by ongoing secretion of chemokines by infected epithelium. CD81 T cells have been implicated in pathology in mouse, guinea pig, and monkey models, but their role in human disease has not yet been defined. In guinea pigs and monkeys, immunity elicited by the initial infection is not able to prevent reinfection, and repeat infections exacerbate tissue pathology similarly to what is observed in human infections [157,158].

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B. Natural Immunity and Vaccines Infection-induced immunity is only serovarspecific and transient [159], and repeat and chronic infections are common [160], although epidemiological studies show a decreased prevalence of infection and bacterial load with increasing age despite continuous exposure [158,161]. Prospective studies have also shown that women who spontaneously clear infections between testing and treatment are less likely to become reinfected [162], indicating that immunity does develop in some women. In mouse models, infection with either wild-type or plasmid-deficient C. muridarum (Cmu) strains protects against reinfection [73,163], as determined by shedding of Cmu in vaginal swabs, but inflammatory pathology may be further increased by repeat infections. In guinea pigs [164] and nonhuman primates (NHPs) [165], primary infection induces partial immunity as measured by shorter duration and magnitude of repeat infection but does not offer protection against upper genital tract pathology. These observations of limited immunity induced by primary infection are promising for development of a human vaccine. The first human chlamydia vaccines were trialed in the 1960s for trachoma and used live or formalin-fixed bacteria (reviewed in Refs. [166,167]). Some protection against trachoma was demonstrated, although protection was only partial and short lived (1 2 years). Studies in NHPs with the same vaccines showed some protection when high doses were used, but at low doses, enhanced ocular inflammation was observed on challenge with heterologous serovars [168,169]. Studies of a live trachoma vaccine in Gambian children also raised concerns regarding exacerbation of disease in vaccine recipients upon rechallenge [170], although reanalysis of the data using current trachoma grading systems suggested that these concerns were unfounded [171,172]. However, the original concerns focused most subsequent vaccine

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research around subunit rather than whole cell vaccines, and they highlight the need to test potential vaccine antigens to ensure that their use would not increase inflammatory tissue damage in sensitized hosts. Since these early studies, vaccine development has involved (1) identification of protective immune responses, (2) choice of antigen, (3) adjuvant selection, and (4) defining the optimal route of immunization (Chapter 36: Mucosal Vaccines for Bacterial Sexually Transmitted Diseases).

C. Protective Immunity Against Chlamydia The consensus in the field is that IFNγ-producing CD41 Th1 cells are essential for longterm protective immunity. Nude mice develop a chronic chlamydia infection, and mice deficient in MHC class II, CD4, IL-12, IFNγ, and the IFNγ receptor [173 177] all have enhanced susceptibility to infection. Transfer of chlamydia-specific Th2 cells does not protect mice against infection [178], and Th2 responses correlate with increased pathology during human ocular infection [179]. Some studies also suggest that polyfunctional CD41 T cells, secreting IFNγ plus TNF-α and/or IL-17, may also be important in protection [180,181], although the requirement for TNF-α and IL-17 production is debated, as both have been implicated in pathology. Data showing that HIV-infected patients with low CD41 T cell counts are at higher risk of genital chlamydia infections support the importance of CD41 T cells [182]. The protective role of IFNγ appears to differ in humans and mice, induction of indoleamine 2,3-dioxygenase and subsequent tryptophan starvation being the protective mechanism in humans, whereas in mice, IFNγ-mediated induction of p47 GTPases [183] and possibly inducible nitric oxide synthase and nitric oxide [184,185] provides protection. Mouse models show that CD81 T cells are not required for protective

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immunity, although chlamydia-specific CD81 T cell clones can contribute to protection through production of IFNγ [73]. Studies by Murthy et al. suggest that CD81 T cells actually contribute to pathology and tissue damage via production of TNF-α [153] and that upregulation of PD-L1 in the reproductive tract may inhibit CD8 T cell expansion as a means of preventing pathology [186]. The role of antibodies in protection is not clear. B-cell-deficient mice clear a primary chlamydia infection as effectively as wild-type controls [187] but are more susceptible to reinfection [188]. Immune wild-type mice depleted of CD41 or CD81 T cells can clear a secondary infection, but B-cell-deficient mice depleted of CD4 cells are unable to clear a secondary infection [189], leading Farris et al. to conclude that both CD41 T cells and antibody are required for optimal protection with antibody likely acting to enhance Th1 CD4 T cell development [190]. This hypothesis is supported by studies with FcR-deficient mice, which are less able to clear a secondary infection compared to wild-type control mice, and the determination that FcR2/2 antigenpresenting cells (APCs) are less efficient at inducing protective Th1 cells than are APCs with an intact FcR [191]. Antibody transfer studies have shown that monoclonal antibodies specific for the chlamydial mitochondrial outer membrane protein (MOMP) can protect mice against infection [192]. While many studies have demonstrated that antibodies generated by vaccination or infection can neutralize chlamydial infection in vitro, most of these studies are performed at neutral pH [193]. In the acidic environment of the FGT, IgG antibodies directed at MOMP, which neutralize infection at pH 7, actually enhance infection at a pH of 6 6.5, representative of the in vivo environment in the FGT [31]. The enhanced infectivity depends on the expression of the neonatal Fc receptor (FcRn), which is normally expressed in the FGT. Because IgG is the major isotype in

both female and male genital tract secretions, a potential infection-enhancing function of antibodies should be considered in choosing vaccine antigens. In terms of what type of immune response potential vaccines should try to elicit, the consensus is that a CD41 Th1 response would provide optimum protection against infection. In recent studies, TRMs have emerged as major players in protective immunity at mucosal sites [194]. These cells reside long term in mucosal epithelial tissues (many months in the mouse), including the FGT, and respond rapidly to local pathogen challenge, independent of recruitment of circulating memory T cells [194]. These cells have been studied mostly in HSV infection models, in which parenteral priming “pull” was used to elicit circulating memory cells, which were then “pulled” into the murine FGT by topical chemokine application [195], protecting mice against lethal HSV infection. Stary et al. [196] showed that mucosal immunization with UV-inactivated C. trachomatis complexed with charge-switching synthetic adjuvant particles (cSAPs) elicited long-lived protection in conventional and humanized mice and that protection required both a local TRM T cell response and recruited circulating memory T cells. In mice that have recovered from a Cmu infection and are immune to reinfection, CD41 T lymphocyte clusters persist in FGT tissues long after the infection has resolved [197], and we also identified CD1031 and CD81 TRMs in these same tissues [181]. Lymphoid follicles containing activated lymphocytes and plasma cells were also identified in cervical and endometrial samples from women with C. trachomatis infections [198], and these have recently been termed memory lymphocyte clusters. While knowledge of human TRMs and memory lymphocyte clusters is sparse, it can be concluded that a successful vaccine will likely require the induction of a local CD41 Th1 tissue-resident response in genital tissues for optimum protection [199].

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D. Choice of Antigen Historically, surface-expressed antigens, particularly MOMP, have been the most studied, particularly in the mouse model. It has been suggested that only natural MOMP in its native configuration is effective at eliciting protective immunity [200], but producing such a product in sufficient quantities for a human vaccine is certainly impractical. However, other studies have demonstrated that recombinant MOMP can be used to induce protective immunity in mice [181,201,202]. With the recognition that CD41 T cell responses are required for protection, bioinformatic and various highthroughput approaches have been used to identify class II-restricted antigens and peptides that will activate a Th1 response. Barker et al. [203] used in silico T cell epitope prediction algorithms to identify promiscuous T cell epitopes conserved across C. trachomatis and Chlamydia pneumoniae. Karunakaran et al. [204] pulsed DCs with live Chlamydia and identified peptides that were loaded into class II MHC molecules, identifying five antigens that elicited partial protection in the mouse infection model [205]. Finco et al. [206] selected 120 Chlamydia proteins and assessed their immunogenicity. Proteins were screened against sera from C. trachomatis-infected patients for antibodyinducing antigens and were used to stimulate splenocytes from infected mice to identify antigens that elicited a CD41, IFNγ response. Seven antigens were identified that elicited partial protection in the mouse model either alone or in combination [206]. Other groups have used proteomic approaches where proteomic libraries of Escherichia coli expressed chlamydial proteins were fed to APCs which were then used to stimulate human T cells from patients to identify IFNγ-secreting CD41 and CD81 T cells [207,208]. This approach identified eight CD4 and eighteen CD8 antigens associated with clearance or resistance to infection [207]. Many of these approaches have identified novel

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antigens and often did not identify surface exposed antigens such as MOMP and polymorphic membrane proteins (Pmps) that have been shown to be protective in the mouse model [208]. Future work will require development of a pipeline to evaluate these novel antigens in animal models (mouse, guinea pig, NHP) for selection of the best candidates to proceed to human studies. Although new antigens will undoubtedly be required for an effective vaccine, a novel formulation containing multiple MOMP VD4 regions from three C. trachomatis serovars has been shown to be protective in mice [209], with protection dependent on both CD4 T cells and antibodies. The only chlamydial vaccine currently in human trials is based on this approach (https:// clinicaltrials.gov/ct2/show/NCT02787109).

E. Adjuvant Selection and Route of Immunization Only a limited number of vaccine adjuvants have been approved for human use. These include alum, ASO4 (alum plus monophosphoryl lipid A), the squalene-based adjuvants AS03 and MF59, and liposome formulations [210]. The ISCOMATRIX platform has been used in a number of clinical trials in human cancer vaccines, HPV vaccines, and influenza vaccines [211] and has been shown to have a good safety profile. Many novel adjuvants have been used in preclinical and early-phase clinical studies [212], including TLR agonists (such as resiquimod, CpG), particulate systems such as virus-like particles, various nanoparticle formulations (PEG-PLGA nanoparticles), nanoemulsion adjuvants (NanoVax), polysaccharide/inulin-based adjuvants (Advax), plant carbohydrates (saponin), lipid-based adjuvants (Liporale), adjuvants based on bacterial toxins (LKT63 and CTA1-DD), and combination adjuvants such as DDA-DTB and TriAdj (VIDOIntervac) [213]. Other replicating delivery systems based on adenoviruses [214], vaccinia

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virus (modified vaccinia virus Ankara, MVA) [215], HPV, and fowl pox-based vectors [216] provide alternative vaccine platforms to traditional adjuvants. DNA vaccines have also shown promise in animal trials, although this potential has yet to be realized in human studies [217] (Chapter 10: Innate Immunity based Mucosal Modulators and Adjuvants, Chapter 11: Toxin-based Modulators for Regulation of Mucosal Immune Responses). The key to a successful chlamydia vaccine will be the identification of the best combination of antigen, adjuvant/delivery system, and delivery route that elicits a CD41 Th1 response, both tissue resident and circulating, that is, long lasting and protects against multiple serovars of C. trachomatis. Intranasal immunization of mice with MOMP combined with ISCOMATRIX, CTA1-DD, cholera toxin plus CpG, NanoVax, Advax, and VIDO TriAdj [144,145,181,218] all elicited partial protection against Cmu vaginal infection, and ISCOMATRIX, NanoVax, and VIDO TriAdj immunized mice also showed a significant reduction in oviduct pathology [218]. Other studies have used intranasal immunization with MOMP-containing vault nanocapsules [219], MOMP/IncA/CPAF plus IL-12, or CpG as an adjuvant [220,221], type III secretion proteins plus CpG adjuvant [222], or intranasal infection with heterologous C. pneumoniae [223] or homologous Cmu [224] to demonstrate partial protection against infection and FGT pathology in the mouse model of infection, further indicating that the intranasal immunization route is effective at generating immunity in the FGT. Intranasal immunization with MOMP plus cholera toxin or ISCOMATRIX also reduced the duration of infection and testicular chlamydial burden in male mice challenged into the penile meatus with Cmu [181,225,226]. Systemic vaccination (subcutaneous and intramuscular) has also been used to induce partial protection against infection (reduced bacterial shedding) and, in some cases, reduced pathology (hydrosalpinx). Adjuvants used in

these studies included CAF01, alum, CpG, AbISCO-100, DDA/TDB, ASO1B, and CpG-IFA combined with various subunit antigens [180,224,227 229]. Partial protection was also induced by transcutaneous immunization of mice with MOMP plus cholera toxin CpG adjuvant [201] and oral immunization using MOMP plus Liporale [230]. A common feature of most of the mouse studies described is that sterilizing immunity was not achieved, with most reporting partial protection against infection and/or pathology (hydrosalpinx). Studies of Cmu respiratory infection have also demonstrated that immunization with MOMP, combined with different adjuvants can protect against either immunopathology or infection but not both [231]. It should also be noted that most of these studies did not investigate whether TRMs were present in the FGT after vaccination. This raises important questions about what endpoints should be targeted with a human chlamydia vaccine, as not all experimental vaccines protected against both infection and pathology. Interestingly, Johnson et al. [232] recently identified a unique population of CD41 T cells secreting IFNγ and IL-13, isolated from immune T cells stimulated with immune B cells as APCs. Clones derived from these cells had a transcriptome representative of TRMs and when adoptively transferred to naı¨ve mice prevented the development of oviduct pathology without accelerating chlamydial clearance. If sterilizing immunity cannot be achieved by a human vaccine, should the primary endpoint be prevention of oviduct/fallopian tube pathology, which is the cause of PID, infertility, and ectopic pregnancy, the most costly health outcomes associated with a chlamydial infection? Such an outcome could be achieved by either preventing ascending infection from the cervix or targeting the immune mechanisms responsible for oviduct scarring and occlusion. Recent studies by Stary et al. [196] have provided hope that a vaccine that both reduces infection and prevents pathology may be achievable.

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VI. GENITAL HERPES

Mucosal vaccination (intrauterine or intranasal) of mice with UV-inactivated C. trachomatis combined with charge-switching synthetic adjuvant particles containing the TLR7/8 agonist resiquimod protected mice against both infection and hydrosalpinx [196]. The success of this vaccine depended on the targeting of antigen to CD1032 immunogenic DCs rather than tolerogenic CD1031 DCs and the subsequent generation of both tissue-resident CD41 cells and circulating mucosal trophic memory CD41 T cells. It will be interesting to see whether the same approach is able to protect guinea pigs and NHPs. While most chlamydial vaccine research has involved vaccination only of females, we have shown that intranasal vaccination of male mice can provide partial protection as measured by reduced testicular, epididymal, and prostatic chlamydial burden. Protection required both antigen-specific CD41 T cells and local IgA [225,226]. Importantly, our studies have also shown that immunization of both female and male mice, even though sterilizing immunity was not achieved in either, resulted in almost complete protection of females [181], suggesting that immunity in both sexes may synergize to provide complete protection against sexual transmission, a finding not predicted in previous modeling studies [233]. Table 16.1 summarizes the preclinical chlamydial vaccine trials described in this chapter.

V. CHLAMYDIA AS A GASTROINTESTINAL COMMENSAL: THE ELEPHANT IN THE ROOM? Investigators working with nonhuman chlamydial infections have recognized for more than a century that chlamydial species persist in the gastrointestinal (GI) tract of animals and birds, potentially for life (reviewed in Ref. [234]). Rectal chlamydia has been demonstrated

in women attending STI clinics [235 238], and often there was no correlation between selfreported anal sex and the presence of rectal chlamydia [238]. Studies in animal models suggest that chlamydia may persist in the GI tract even in the presence of a host immune response that clears a genital tract infection [234] and furthermore that GI chlamydia may be resistant to antibiotic treatment [239,240], meaning that a GI reservoir may persist, giving rise to reinfection via autoinoculation. If rectal chlamydial carriage is common in humans, this could have important implications for both antibiotic therapy and vaccine development.

VI. GENITAL HERPES HSV-2 is a STI that causes genital herpes. Genital herpes is a global issue; an estimated 417 million people were living with the infection in 2012 (https://www.who.int/newsroom/fact-sheets/detail/herpes-simplex-virus) [241]. The prevalence of HSV-2 infection was estimated to be highest in Africa (31.5%), followed by the Americas (14.45%) and Australia (12%) [242]. More women than men are infected with HSV-2; in 2012, it was estimated that 267 million women and 150 million men were living with a genital herpes infection [241]. Symptoms of herpes include painful blisters and ulcers at the site of infection. Herpes infections are most contagious when symptoms are present but can still be transmitted in the absence of symptoms [243]. Infection with HSV-2 increases the risk of acquiring and transmitting HIV infection threefold [244]. HSV-2 is among the most common infections in people living with HIV, occurring in 60% 90% of HIV-infected individuals [241,245]. HSV-2 symptoms are more severe in presentation and more frequent in HIVinfected individuals [246]. In advanced HIV disease, HSV-2 may lead to more serious

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

TABLE 16.1 Chlamydia Vaccine Trials Animal model Mouse genital infection (female)

Chlamydial organism

Infectious dose

BALB/c

C. muridarum Nigg

1 3 10 IFU Ovarian bursa

CpG1 alum

i.n.1 s.c.

Unaffected Unaffected

BALB/c

C. muridarum Nigg

5 3 104 IFU Intravaginal rMOMP

CpG1 CT

t.c.

Protected

Not reported

[201]

BALB/c

C. muridarum weiss

50, 100 or 500 IFU

Intravaginal rMOMP

ISCOMATRIX i.n.

Protected

Protected

[181]

BALB/c

C. muridarum

1.5 3 103 IFU

Intravaginal PmpG, PmpE, PmpF, Aasf, MOMP, RpIF, TC0825, or TC0420

DDA-MPL

s.c.

Protected

Not reported

[204]

C57BL/6

C. muridarum Nigg

1.5 3 103 IFU

Intravaginal PmpG-125 500, RplF, PmpE/F-225 575, or MOMP

Pulsed BMDCs

i.v.

Protected

Not reported

[205]

C57BL/6

C. trachomatis D

5 3 105 IFU Intravaginal CT823 (htrA) or CT144

AbISCO-100

s.c.

Protected

Not reported

[207]

B6C3F1

C. trachomatis D

4-8 3 105 IFU

CAF01

i.n.1 s.c.

Protected

Protected

[209]

B6C3F1

C. trachomatis F

1 3 106 IFU Intravaginal

CAF01

i.n.1 s.c.

Protected

Protected

BALB/c

C. muridarum weiss

5 3 104 IFU Intravaginal rMOMP

CpG1 Ct

i.n. or t.c.

Protected

Protected

[144]

BALB/c

C. muridarum weiss

500 IFU

CTA1-DD

i.n.

Unaffected Protected

[145]

CpG1 Ct

i.n.

Protected

CTA1-DD

t.c.

Unaffected Unaffected

CpG1 CT

t.c.

Protected

Strain

Infection route

5

Antigen

Adjuvant

native MOMP

CpG1 Montanide

Intravaginal Hirep-1

Intravaginal rMOMP

C. muridarum weiss

500 IFU

C57BL/6

C. muridarum

1.5 3 105 IFU

BALB/c

C. muridarum

1 3 105 IFU Intravaginal rMOMP

BALB/c

Intravaginal rMOMP1 PmpG1 TC05001 TC0873 rMOMP1 PmpG1 TC05001 TC0873 Intravaginal MOMP-mINT

Mode of delivery

Infection

Pathology

References

i.n.1 s.c.

Protected

Protected

[200]

Unaffected

Protected

CTA1-DD

s.l.

Unaffected Protected

CpG1 CT

s.l.

Protected

Protected

IMX

i.n.

Protected

Protected

VIDO

s.c.

Protected

Unaffected

Vault nanoparticles

i.n.

Protected

Not reported

[219]

[220]

i.n.

Protected

Protected

rCPAF

IL-12

i.n.

Protected

Protected

rIncA

i.n.

Protected

Protected

rCPAF1 rMOMP

i.n.

Protected

Protected

rCPAF1 rIncA

i.n.

Protected

Protected

[218]

BALB/c

C. muridarum

5 3 104 IFU Intravaginal rCPAF1 rCPAF rMOMP1 rIncA

CpG

i.n.

Protected

Protected

rCPAF

CpG

i.p.

Protected

Protected

CpG

i.n.

Protected

Protected

[222]

i.n.

Protected

Protected

[223]

s.c.

Unaffected Not reported

Intravaginal CT443

s.c.

Protected

Not reported

Intravaginal CTH1

s.c.

Protected

Not reported

C. muridarum Nigg

1 3 10 IFU Intravaginal CTH1

s.c.

Protected

Not reported

C. muridarum Nigg

1.5 3 103 IFU

C57BL/6

C. muridarum Nigg

1 3 105 IFU Intravaginal BD584

C57BL/6

C. muridarum Nigg

5 3 104 IFU Intravaginal C. pneumoniae AR39 (1x10^6 IFU)

C3H/HeN and CB6F1 mice

C. trachomatis D

1 3 107

C57BL/6

Intravaginal CT521

CAF01

5

Intravaginal PmpG

LN CpG

s.c.

Unaffected Not reported

Intravaginal PmpG

CpG

s.c.

Unaffected Not reported

Intravaginal MOMP

LN CpG

s.c.

Unaffected Not reported

Intravaginal PmpG

AbISCO-100

Intravaginal PmpF

Protected

Unaffected Not reported

[224]

[180]

Not reported

Intravaginal MOMP

s.c.

Unaffected Not reported

Intravaginal PmpG1 PmpF1 MOMP

s.c.

Protected

Not reported

s.c.

Protected

Not reported

Intravaginal PmpF

s.c.

Protected

Not reported

Intravaginal MOMP

s.c.

Protected

Not reported

Intravaginal PmpG1 PmpF1 MOMP

s.c.

Protected

Not reported

DDA/TDB

s.c.

Protected

Not reported

CpG1 CFA

i.m.

Protected

Protected

[227]

AS01B

Base of tail

Protected

Not reported

[228]

Intravaginal PmpG

Balb/c

C. muridarum Nigg

1.5 3 10 IFU

Balb/c

C. muridarum Nigg

1 3 104 IFU Intravaginal Tarp

C57BL/6, C3H and BALB/c

C. trachomatis K 5 3 105 IFU Intravaginal UV-inactivated C. trachomatis E Ebs

3

s.c. s.c.

[221]

Intravaginal PmpG1 PmpF1 MOMP

DDA/TDB

(Continued)

TABLE 16.1 (Continued) Animal model

Strain

Chlamydial organism

BALB/c

C. trachomatis K 1 3 10 IFU Intrauterine

C57/NL6J

BALB/c

Not reported

Infectious dose

Infection route

6

Antigen UV-inactivated C. trachomatis E Ebs

Mode of delivery

Infection

Pathology

i.m.

Protected

Protected

pmpDpd

i.m.

Protected

Protected

CT622

i.m.

Protected

Protected

MOMP F

i.m.

Protected

Protected

CT875

i.m.

Protected

Unaffected

swib

i.m.

Protected

Unaffected

pmpHpd

i.m.

Protected

Protected

CT089

i.m.

Protected

Unaffected

CT772

i.m.

Protected

Unaffected

CT858

i.m.

Protected

Protected

rpoB

i.m.

Protected

Protected

CT322

i.m.

Protected

Protected

Lpda

i.m.

Protected

Protected

CT8751 MOMP F

i.m.

Protected

Protected

C. muridarum Nigg

1.5 3 10 IFU

C. muridarum

5 3 10 IFU Intravaginal rMOMP

5

Adjuvant

Intravaginal Native MOMP

CAF01

s.c.

Protected

Unaffected

Native MOMP

Alhydrogel

s.c.

Protected

Unaffected

rMOMP

CAF01

s.c.

Protected

Unaffected

4

CpG1 CT

i.g.

Protected

Unaffected

rMOMP

Lipid C

i.g.

Protected

Unaffected

rMOMP

Lipid C1 CpG i.g. 1 Ct

Protected

Unaffected

cSAP

i.u.

Protected

Not reported

Not reported

C. trachomatis L2

1 3 106 IFU Intrauterine

UV-Ct

C. trachomatis L2

1 3 106 IFU Intrauterine

UV-Ct

i.n.

Protected

C. trachomatis L2

1 3 106 IFU Intrauterine

UV-Ct

s.c.

Unaffected Not reported

C. muridarum

Not reported

i.u.

Protected

Intravaginal UC-Cm

Protected

References

[229]

[230]

[196]

Mouse respiratory infection

BALB/c

BALB.c

C. muridarum Nigg II

1 3 103 IFU Intranasal

C. murdarum Nigg

rMOMP

CPG1 CT

i.n.

Protected

Protected

rMOMP

CPG1 CT

t.c.

Protected

Protected

1 3 103 IFU Intranasal

TC0106 1 TC0210 1 TC0313 1 TC0741

LKT63 1 CpG

i.m.

Protected

Not reported

1 3 103 IFU Intranasal

TC0106 1 TC0431 1 TC0551 1 TC0890

LKT63 1 CpG

i.m.

Protected

Not reported

PmpG-125 500, RplF, PmpE/F-225 575, or MOMP

Pulsed BMDCs

i.v.

Protected

Protected

[205]

rMOMP

CTA1-DD

Unaffected

[231]

C57BL/6

C. muridarum Nigg

2 3 103 IFU Intranasal

BALB/c

C. muridarum

1 3 103 IFU Intranasal

i.n.

Protected

rMOMP

t.c.

Unaffected Protected

rMOMP

s.l.

Unaffected Unaffected

rMOMP

CpG1 CT

rMOMP rMOMP Mouse genital infection (male)

C57BL/6, BALB/c

C. muridarum

1 3 105 IFU Intrapenile

rMOMP

Note: i.n., intranasal; s.c., subcutaneous; t.c., transcutaneous; s.l., sublingual; i.g., intragastric; i.p., intraperitoneal; i.u., intrauterine; 1.v., intravenous.

CT

i.n.

Protected

Unaffected

t.c.

Protected

Protected

s.l.

Protected

Protected

i.n.

Protection

Not reported

[202]

[206]

[181,225,226]

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complications, including meningoencephalitis and disseminated infection [247]. Herpes treatments include antivirals such as acyclovir, famciclovir, and valacyclovir [248]. These can help to reduce the severity and frequency of symptoms but cannot cure the infection, owing to the lifelong latent infection that occurs in neurons of the nerve ganglia. Prevention of infection by abstaining from sexual activity and consistent use of condoms may help to reduce the risk of spreading genital herpes [249]. However, condoms provide only partial protection, as HSV can be found in areas not covered by the condom [249]. The lack of effective treatments to prevent or cure HSV-2 infection has led to additional research to develop more effective prevention methods, such as vaccines. The World Health Organization has been working to accelerate research to develop new strategies for prevention and control of genital HSV-2 infection [250]. Despite several promising vaccine candidates entering into clinical trials over the last decade, an effective vaccine to prevent genital HSV-2 infection is still missing [251,252].

A. HSV-2 Vaccine Development Two approaches are being pursued for HSV-2 vaccine development: prophylactic and therapeutic vaccines [252 256]. Prophylactic vaccines would provide protective immunity against genital HSV-2 infection prior to exposure, preventing the spread of infection. Therapeutic vaccines aim to reduce genital lesions and viral shedding in HSV-2-seropositive individuals. Female mice can be infected vaginally with HSV-2 to closely mimic human genital HSV disease [257]. Female mice must be infected during the progesterone-dominant phase of the mouse estrous cycle or be pretreated with medroxyprogesterone [258]. The virus is introduced directly into the vagina, where it infects epithelial cells and causes ulcers to appear within 2 days of

infection [257]. The virus rapidly accesses the dorsal root ganglia (DRG) to establish latency and progresses to lethal encephalitis approximately 7 days after infection [259]. Vaccine studies in mouse models examine the protective effect of prophylactic vaccines. Early prophylactic vaccine studies intravaginally inoculated mice with thymidine kinase (TK) deficient HSV-2 [257], causing primary genital lesions without neurological illness [258]. TKHSV-2infected mice were immune to lethal secondary wild-type HSV-2 challenge with original studies, suggesting IgG antibody-mediated protection against HSV-2 challenge [260]. However, later studies using B cell and T cell knockout mice showed that protection was mediated by a combined antibody and T cell immune response [261,262]. HSV-2 infected B cell knockout mice had significantly higher viral loads in the genital epithelium and more severe infection outcomes compared to wild-type mice [261]. Likewise, HSV-2 infected CD4 knockout mice had significantly higher viral loads in the genital epithelium and DRG compared to wild-type mice [262,263]. The increased viral susceptibility was attributed to reduced CD81 T cell infiltration into the FRT during primary HSV-2 infection [262]. Most recent studies have indicated that establishment of CD41 and CD81 TRMs in the FGT following genital TK- HSV-2 infection are important for protection against secondary wildtype HSV-2 challenge [195]. When TRMs are present in the genital tract before wild-type HSV-2 challenge, mortality significantly decreases, and disease outcome and HSV-2 infiltration are significantly lower [195]. These studies concluded that optimal protection against HSV-2 infection of the DRG requires antibodyand antigen-specific T cells in the FRT. The pursuit of prophylactic HSV-2 vaccines has led to the testing of live, inactivated, replication-defective, subunit, peptide, live vector, and DNA vaccines with varying degrees of success. Live attenuated vaccines, which are nonpathogenic or low-pathogenic mutant

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

VI. GENITAL HERPES

strains, can induce effective protection against genital HSV-2 challenge [264]. ICP0-mutant virus subcutaneously injected into mice before lethal intravaginal challenge increased survival rates by 99.1% compared to unimmunized controls [264]. When compared to an HSV glycoprotein D (gD2) subunit vaccine, HSV-2 ICP0-immunized mice shed approximately125fold less wild-type HSV-2, resulting in 10 100 times greater protection than their gD2immunized counterparts. Protection was attributed to significantly higher IgG responses in ICP0-immunized mice compared to gD2immunized controls [264]. Another attractive live attenuated HSV-2 candidate is HSV-2 gD27, which was mutated to prevent infection of neuronal cells, including the ganglia of mice [265]. Intramuscular immunization of mice with HSV-2 gD27 before lethal intravaginal challenge with wild-type HSV-2 led to 100% survival [265]. HSV-2 gD27-immunized mice had significantly lower viral titers in the vagina and increased serum antibody compared to unimmunized controls [265]. Neither live vaccine was administered mucosally, suggesting that protective mucosal immunity may not require mucosal vaccination. Live vector vaccines, such as vaccinia virus or adenovirus, which express HSV-2 antigens, can also elicit strong immune responses against HSV-2 [266]. Needle-free injection of mice with modified vaccinia virus vector expressing HSV2 gpD significantly enhanced serum IgG and IFNγ compared to unimmunized controls [266]. Similar serum responses were recorded in mice that were intramuscularly immunized with adenovirus-expressing HSV-2 gpD [267]. Immunized mice showed a higher survival rate (75%) compared to unimmunized controls (0%) and had significantly lower disease scores following intravaginal wild-type HSV-2 challenge [267]. These studies suggested live attenuated and live vector vaccines are promising candidates to protect against genital HSV-2 infection. However, in these studies, immunogenicity of

279

each vaccine was determined only in the blood and not in the FGT, so data on mucosal immunity elicited by the vaccines were not available [265,267]. In contrast, Sato et al. investigated the mucosal immune response to intranasal or intraperitoneal administration of live TK- HSV2 in mice [268]. Intranasal immunization led to development of effector CD41 and CD81 T cells in the FGT of mice whereas intraperitoneal immunization elicited only a weak mucosal immune response [268]. Consequently, intranasally immunized mice had significantly better viral clearance and survival following lethal intravaginal challenge with wild-type HSV-2. Further investigation through adoptive transfer experiments concluded that protection was achieved through mucosal immunity, not systemic immune responses [268]. Thus Sato et al. provide clear evidence that mucosal immunization may be better at eliciting mucosal immunity compared to systemic immunization [268]. However, live vaccines also pose the potential risk of reversion to the wild-type phenotype. DNA vaccines do not carry risk of reversion and commonly utilize DNA encoding the HSV2 gpD. Intramuscular immunization of mice with gpD plasmid DNA mixed with Vaxfectin induced high titers of serum IgG [269]. Following lethal intravaginal HSV-2 challenge, immunized mice had significantly lower virus copies in the DRG and an improved survival rate (80%) compared to controls immunized with Vaxfectin alone (0%) [269]. The DNA vaccine was improved by using gpD and gpB CTL epitope plasmid DNA, which was intramuscularly administered to naı¨ve mice. The vaccine induced strong serum IgG and Th1 immune responses, leading to a 90% survival rate following lethal intravaginal HSV-2 challenge [269]. While this study clearly shows DNA vaccine efficacy against HSV-2 challenge in mice, the potential of DNA vaccines has yet to be realized in humans [217]. Subunit vaccines offer strong protein immunogenicity and utilize one or more antigenic

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16. REGULATION OF MUCOSAL IMMUNITY IN THE GENITAL TRACT

HSV glycoproteins usually mixed with an adjuvant to elicit protective immune responses in mice [270]. The most extensively tested subunit vaccine has been recombinant glycoprotein D (gpD) [270], which has high amino acid identity (88% 99%) across different HSV strains and is a major target for neutralizing antibodies and CD41 and CD81 T cell responses [270,271]. Subcutaneous immunization of mice with gpD/alum significantly reduced mortality compared to unimmunized controls [272]. Immunized mice had antibodies in serum that neutralized HSV-2 in vitro [272]. The success that subunit vaccines have achieved in preventing HSV-2 infection has been replicated in therapeutic vaccine guinea pig studies [273]. Subunit vaccines have been used in guinea pig studies to determine whether vaccination can prevent reactivation of genital herpes (therapeutic vaccine) [273]. Much like humans, guinea pigs infected with genital herpes have periodic reactivation of HSV-2 from the ganglia into the vaginal vault, leading to genital lesions. Subcutaneous injection of HSV-2-infected guinea pigs with gpD mixed with AS04 prevented recurrent lesions in 90% of immunized guinea pigs up to 63 days following immunization [273]. However, when AS04 was replaced with alum, only 36% of guinea pigs were protected from recurrent lesions despite both vaccines generating strong HSV-2-specific sera antibodies following immunization [273]. The Vaxfectin-gD2 and Vaxfectin-gD2/ UL46/UL47 vaccines were also evaluated in guinea pigs and were effective as both prophylactic and therapeutic vaccines in the guinea pig model of genital herpes [254]. The HSV-2 plasmid DNA vaccines were administered intramuscularly three times at 2-week intervals. Both vaccines significantly reduced viral titer in the vaginal vault and viral infiltration into the DRG compared to controls immunized with Vaxfectin alone [254]. The Vaxfectin-gD2/ UL46/UL47 vaccine was the most effective vaccine, protecting 93% of guinea pigs from HSV-2

DRG infection [254]. Protection was associated with increased vaccine-specific antibodies in the sera compared to Vaxfectin-immunized controls. Immunization of HSV-2-infected guinea pigs with Vaxfectin-gD2/UL46/UL47 significantly reduced mean lesion days compared to Vaxfectin-immunized controls [254]. The promising results of the Vaxfectin vaccine and its safety as a DNA vaccine warranted further investigation into developing Vaxfectin for clinical trials. These studies also highlight that vaccines that contain multiple antigens are likely to induce better protection.A DNA vaccine encoding two HSV proteins formulated with Vaxfectin was tested in phase 1 2 clinical trials. In HSV-2-positive individuals, plasmid DNA vaccine containing genes encoding one or two HSV-2 proteins plus Vaxfectin was administered intramuscularly (https://clinicaltrials. gov/ct2/show/NCT02030301). The bivalent vaccine reduced shedding by 19% from baseline, compared to a 45% reduction in the placebo-immunized group. The bivalent vaccine also reduced lesion development by 51%, although the placebo group had a reduction of 46% compared to baseline. The monovalent vaccine increased lesion development by 3% when compared to the placebo-immunized controls. Both vaccines generated high levels of HSV-2-specific T cell and B cell immune responses in immunized individuals. Clearly, protective immune correlates against genital HSV-2 infection that are defined in animal models may not apply to humans. The most recent therapeutic vaccine candidate against genital HSV-2 infection was GEN003, a subunit vaccine containing a deletion mutant of gD2 and a portion of infected cell protein 4 (ICP4) with Matrix-M2 adjuvant (Genocea) [274]. In a phase 1 2a study, participants who were given the most effective GEN-003 vaccine had a 50% decrease in viral shedding and a 65% decrease in days with genital lesions, persisting 12 months post vaccination. Vaccine-specific T cell and antibody

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

VI. GENITAL HERPES

responses in the blood remained elevated for 12 months. These immunogenicity results provide the first evidence that the GEN-003 vaccine stimulates sustained cell-mediated and antibody responses against vaccine antigens. A follow-up phase 2 GEN-003 clinical trial comparing the effectiveness of different vaccine doses in reducing genital shedding and lesion development from baseline did not demonstrate significant efficacy [251]. The most effective vaccine doses (60 μg/50 μg and 60 μg/75 μg) reduced shedding and lesion development by 50% compared to baseline measurements. Doses comprising 100 μg of antigen and 100 μg of adjuvant reduced viral shedding by only 33%, suggesting that the vaccine response is not dose dependent [251]. Furthermore, protection was short-lived, with no protection evident at 1 year post vaccination. Given the moderate degree of efficacy seen in HSV-2-positive individuals, GEN-003 seems unlikely to be an effective therapeutic vaccine, and further development has been discontinued. In the past two decades, two prophylactic vaccines for the prevention of genital herpes have been tested for efficacy and safety in Phase III clinical trials. The subunit vaccines were made of gpB and gpD plus MF59 adjuvant (Chiron) [275] or gpD plus alum/MPL adjuvant (HerpeVac, GSK) [276]. High antibody titers were induced in individuals injected with either vaccine, while HerpeVac also elicited systemic Th1 responses. Despite the high antibody titers, the Chiron vaccine showed no efficacy for the prevention of disease or infection with HSV-2 [275]. Also, no difference was observed between the control and vaccinated groups in the duration to first episode or in recurrence of the disease [275]. The largest clinical trial of a HSV subunit vaccine, HerpeVac, immunized HSV-1- and/or HSV-2seronegative women with gpD2 combined with alum/MPL adjuvant [276]. Significant efficacy was not achieved, efficacy across the two

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studies being only 40% in terms of acquisition of genital herpes disease, although efficacy was higher (74%) in couples in which female partners were seronegative for both HSV-1 and HSV-2. The vaccine was not efficacious in men [276]. Vaccination elicited HSV-neutralizing antibodies and gpD-specific lymphoproliferation and IFNγ in serum and PBMC, respectively. HSV-2 protection was associated with significantly higher gD2 antibody titers in sera 7 months post immunization compared to those infected with HSV-1. The immune correlate does suggest that immunity can be achieved against HSV-1, although it is unclear whether this is simply a mechanistic correlate. Table 16.2 summarizes recent and current HSV-2 vaccines in clinical trials. The reasons for these vaccine failures are unclear. Neutralizing antibodies measured in the sera waned after boosting with HerpeVac but did not correlate with increased risk of HSV-2 infection [276]. Likewise, no differences were observed in the cellular immune level of participants who were infected with HSV-2 and those who remained HSV-2 free [275]. As in most animal studies, the immune response was measured only in the blood; no measures of mucosal immunity were examined. HSV is a mucosal pathogen, infecting the mucosa of the FGT and then the local nerve endings that allow virus to become latent in the DRG. Thus measuring antibody and cell-mediated immune responses in blood is unlikely to represent local mucosal immunity at the infection site. Although this will be difficult, correlates of protection at the site of infection need to be defined rather than looking at systemic immunity elicited by vaccines. The natural course of infection may provide clues to this. Over time, infection recurrences become less frequent, and viral shedding is reduced. This coincides with the accumulation of HSV-specific memory CD81 T cells in the genital skin close to the nerve endings [246,280]. Thus a successful vaccine may require the establishment of this local TRM

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282 TABLE 16.2 Vaccine candidate

16. REGULATION OF MUCOSAL IMMUNITY IN THE GENITAL TRACT

Clinical Genital Herpes Vaccines Company

Vaccine constitution

Developmental stage

References

GEN-003

Genocea

gD2 and Matrix-M2

Failed phase 2

[251,252]

VCL-HB01

Vical

gD2 1 / 2 UL46 and Vaxfectin DNA vaccine

Phase 2b planned

[254,256]

HerpeVac

GlaxoSmithKline

gD2 and AS04

Failed phase 3

[277]

NE-HSV-2

BlueWillow biologics

Nanoemulsion with gB2 and gD2

Phase 1 planned

[278]

SUBUNIT

LIVE ATTENUATED HSV529

Sanofi pasteur

Replication-defective HSV-2, UL5, UL29 deletion

Phase 1 ongoing

[255]

RVX201

Rational vaccines

HSV-2 ICP0- mutant

Phase 2 planned

[264]

HerpV

Agenus

Peptide vaccine1 QS-21 Stimulon

Phase 2 finished

[253]

COR-1

Admedus

gD2 codon DNA vaccine

Phase 2 planned

[279]

PEPTIDE/DNA

population (TRM, see below) at the site of infection and reactivation (Chapter 43: Mucosal Vaccines for Genital Herpes).

B. Future Perspectives Evidence suggests that protective immunity elicited by HSV-2 vaccines in animal models may not be protective in humans. Hence most clinical trials of vaccines in humans have failed because correlates of protection in humans are currently unknown. Furthermore, most of these vaccines have been delivered by intramuscular injection, which may not effectively target immunity to the FRT. Intravaginal immunization of mice with an attenuated TK- HSV-2 strain prevents disease and death caused by lethal challenge with wildtype HSV-2 [257]. This immunization strategy elicits CD8 T cell responses in the vaginal epithelium and stroma and increases virus-specific IgG in the vagina, which together confer robust immunity [195]. Although intravaginal

immunization is a useful tool for studying HSV2-generated immune responses in animal models, the vaccine is not translatable to humans. Mucosal immunization strategies, such as oral and intranasal, take advantage of the common mucosal immune system to elicit an immune response in the FGT [281,282]. Intranasal immunization can lead to HSV-2-specific IgA and IgG in the genital tract, increased numbers of T cells in the tissue, and improved protection against lethal HSV-2 challenge, compared to controls immunized through other routes [282,283]. Intranasal immunization of mice with an IgG Fc fragment conjugated to gD2, which is transported across the mucosal membrane barrier in the lungs and genital tract, leads to a strong systemic immune response, high titers of gD2specific IgG in the genital tract, and protection against HSV-2 vaginal challenge [283]. Thus modifying the route of immunization, away from injected vaccines to mucosal delivery, may enhance HSV-2 vaccine efficacy.

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VI. GENITAL HERPES

The only vaccine method to date that has reproduced similar levels of HSV-2 antigenspecific IgG and TRMs in the murine FGT was a prime-and-pull method [195]. Through parenteral (subcutaneous) immunization, a pool of circulating antigen-specific memory T cells was established. The robust systemic cellular immune response was then redirected to the FGT through the vaginal application of the chemoattractants CXCL9/10. Subsequently, the vaccine elicited HSV-specific CD8 TRMs, which were retained in the vagina for 12 weeks post pull [195]. Immunized mice challenged genitally with a lethal dose of HSV-2 were fully protected from death, lost significantly less weight, and developed significantly less severe symptoms than the Prime-only controls. A significant reduction of HSV-2 in the ganglia was achieved in prime and pull immunized mice compared to Prime-only controls. Similar results have been found by using oral priming of mice with live attenuated HSV in Liporale followed by a vaginal application of DNFB to recruit CD81 T cells to the vaginal epithelium. Mice were protected against wild-type HSV challenge and pathology development (US Patent 62/481,226). The same oral immunization and vaginal pull also reduced reactivation of HSV and associated pathology in a guinea pig model, suggesting that the prime-and-pull approach is also applicable for therapeutic vaccines (personal observation). Effective control of HSV in the FGT mucosae will depend on recruitment of CD81 and CD41 TRMs into the FGT and their long-term retention, together with a local antibody response, and should be the aim of future vaccine efforts. Such a local immune response that can be rapidly mobilized will be essential for prophylactic vaccines, for which the time between initial infection of the epithelia and establishment of latent infection, when virions enter the innervating sensory neuron axons, may be as short as 12 hours. For a therapeutic vaccine to limit the magnitude and duration of reactivation at the mucosal

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epithelia will also require a local response spatially close to the nerve endings. While progress toward an effective vaccine for genital herpes (prophylactic or therapeutic) has been disappointing, the recent FDA approval of two vaccines targeting herpes zoster (HZ), also known as shingles, caused by reactivation of another herpes virus, the varicella zoster virus (VZV), may provide hope. Zostavax (Merck) is a live attenuated vaccine that provides partial protection against HZ (51% overall efficacy); protection declines over a period of 5 8 years. More recently, Shingrix (GSK), a recombinant subunit vaccine (VZV gE) combined with the AS01B adjuvant system, was approved for use and has demonstrated an efficacy of 90% against HZ following two immunizations 2 6 months apart. Importantly, protection showed minimal decline with age (reviewed in Refs. [284,285]). Adverse events were more common with the subunit vaccine, mainly at the injection site, although protection was superior. These findings are important in that both a live attenuated vaccine and, perhaps surprisingly, a vaccine based on a single viral protein protected against reactivation of a latent herpes virus infection. With regard to shingles, the initial immune response boosted by either the live attenuated vaccine (Zostavax) or the subunit vaccine (Shingrix) was elicited by a natural infection (chickenpox), a scenario that would be the same for a therapeutic HSV-2 vaccine, although the target organ of shingles, the skin or innervated dermatome, is unlikely to be affected by sex hormones. Restimulating memory T and B cells, activated by a natural infection, is an easier task than stimulating naı¨ve lymphocytes (required for a prophylactic vaccine), as memory cells are likely to be more abundant and do not require costimulation for activation [286]. Additionally, because the target population for the VZV vaccines is people older than 60 years of age, the initiating chickenpox infection may have occurred more than 50 years previously, and VZV-specific

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cell-mediated immunity is known to decline progressively with age, beginning in the fourth decade of life [287]. This immunosenescence is unlikely to be a major problem for a genital herpes therapeutic vaccine, as the target population for such a vaccine is likely to be much younger and the time between the natural infection and vaccination is likely to be considerably shorter. Immune correlates of protection were not established for either HZ vaccine, although a rise in VSV antibodies was a good predictor of protection elicited by the live attenuated HSV vaccine. It would be interesting to know whether either vaccine elicited CD8 TRMs in the skin, as has been shown in animal models of HSV skin infections, because the human cutaneous surface contains approximately 20 billion T cells, almost twice the number present in the circulation [288].

VII. CONCLUDING REMARKS AND FUTURE PERSPECTIVES More than 30 different bacteria, viruses, and parasites are transmitted through sexual contact (https://www.who.int/news-room/fact-sheets/ detail/sexually-transmitted-infections-(stis)). Despite available curative treatments for some bacterial STIs, some viral infections (HSV, HPV, and HIV) are incurable, although disease can be reduced or ameliorated by antivirals. Across the globe, the number of STIs continues to increase in what has been termed “the hidden epidemic” by the Institute of Medicine, and effective vaccines for STIs represents a major unmet need. All of the major pathogens are extremely well adapted to survival in the genital tract and have evolved multiple mechanisms to avoid the host immune system. For example, there are multiple serovars of C. trachomatis, which is an intracellular pathogen that can also enter a quiescent persistent form to avoid stresses imposed by the host immune system. HSV

migrates rapidly from the genital epithelium to the DRG and establishes a lifelong latent infection in the nervous system, then reactivates periodically. Syphilis also establishes a longlived persistent latent infection, aided by a paucity of proteins expressed on the treponemal surface, antigenic variation in the Treponema pallidum Tpr protein family, and residence in the immunoprotected niche of the central nervous system [289]. N. gonorrhoeae is also poorly immunogenic [290], and infection fails to elicit protective immunity, meaning that repeat infections are frequent [291]. Increased antibiotic resistance in N. gonorrhoeae represents a major impediment to controlling and treating infections [292]. Hence the problem for vaccine developers is to find ways to induce protective immunity that is better than that elicited by a natural infection and can overcome the multiple immune evasion mechanisms developed by these incredibly successful pathogens. Both female and male genital tissues, the niche occupied by STIs, constrain the host immune response in order to facilitate reproductive functions. These constraints may also represent a barrier to the development of successful vaccines. When multiple immune mechanisms are suppressed in the FGT to facilitate fertilization and implantation, a window of opportunity may open for pathogens to establish infection. Male seminal plasma also contains multiple immunosuppressive molecules such as prostaglandins, complement inhibitors, and TGF-β [293], which may further suppress immunity in females, at least temporarily. Owing to suppression of immunity at certain times in the menstrual cycle, the challenge may be developing vaccines that induce a robust and sustained immune response that can mediate protection even during these windows of immune suppression. It should be noted, however, that most data on hormone regulation of FGT immunity come from animal models, and data on hormone influences on human responses are scarce, although cycle-associated changes in vaccine-

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VII. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

induced immunity have been documented in women vaccinated by the intranasal, vaginal, and intramuscular routes [294,295]. Although most vaccine research has targeted females, males likely represent the main reservoir of STIs in the community. The isolation of spermatogenesis from the immune system by the blood testis barrier and the immunosuppressive environment of the testes potentially contribute to the establishment of chronic infections in the MRT contributing to this reservoir. The immune-privileged nature of the testes could also reduce the effectiveness of local mucosal vaccine-induced immunity in males, something that may not be reflected in the serum and peripheral blood responses of vaccinees, the parameters usually evaluated in human trials. Males certainly mount an immune response to chlamydial infections, and in mouse studies, we showed that antibodies in male ejaculate can enhance infectivity and pathology in female mice [31]. Antibodies isolated from human seminal plasma can also enhance chlamydial infections in vitro via an FcRn- and pH-dependent mechanism (personal observation). The potential of vaccine-mediated antibody to enhance infection should be considered in future vaccine development. Correlates of protection for various sexually transmitted pathogens have been evaluated in animal models, and Th1 IFNγ-secreting cells have been identified as key mediators of protection against chlamydia and a combination of T-cell- and antibody-mediated immunity required for prevention of HSV infections. Studies of human HSV vaccine-induced immunity has suggested that correlates of protection identified in mice and guinea pigs may not always be the protective responses required for protection against human HSV infections, although in the case of the human HPV vaccines (Gardasil and Cervarix), HPV-specific antibody has been demonstrated as a correlate of protection [296]. Animal studies have also emphasized the importance of local mucosal

285

immunity for protection, and the concept of tissue-resident memory being essential for protection is gaining traction. Local memory responses are difficult to measure in humans, and most clinical trials of vaccines still rely on responses that are measurable in blood. Developing methods to measure local immunity in the human female and male genital tracts remains a major problem for vaccine developers. Optimal targeting of immunity to the genital tract may require delivery of vaccines by routes other than conventional intramuscular (IM) injection. Since Ogra first demonstrated local antibody responses in the FGT to the oral polio vaccine in 1973 [297], many animal studies have confirmed the effectiveness of mucosal, particularly intranasal, vaccination as a means of targeting the genital tract. The majority of human vaccine trials still use IM vaccine delivery, and most adjuvants currently licensed for human use cannot be used for mucosal delivery. However, this may be changing. The novel mucosal adjuvant NanoVax (BlueWillow Biologics), a novel oil-in-water nanoemulsion adjuvant, has been shown to be safe and effective in human trials [298] following intranasal delivery and has been shown in animal studies to protect mice and guinea pigs against chlamydia and HSV infections, respectively (http://www.bluewillow.com/vaccine-pipeline/). Conventional systemic vaccination may also be effective if combined with local immune stimulation at the target tissue site of infection. Such prime-and-pull approaches [195] have yet to be tried in humans but may represent a way forward for some STI vaccines, although methods to measure the recruitment of immune effector cells into the genital mucosae will be required to enable these studies. However, for pathogens against which antibody is the main protective mechanism, conventional IM delivery is still effective. The new nine-valent HPV vaccine is highly efficacious against the HPV types that cause 90% of cervical cancer [299]

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and represents a major success story. Recent findings from a New Zealand study provide hope for success of a gonorrhea vaccine in that reduced rates of gonorrhea diagnosis were seen in recipients of a meningococcal B vaccine (MeNZB) [300]. There is an 80% 90% genetic homology between N. meningitidis and N. gonorrhoeae, and the demonstration of cross-reactive protection bodes well for the development of a gonorrhea vaccine. While most STI vaccine research has focused on females, the HPV vaccines are now given to both females and males in some countries. With regard to HPV, how to best prevent transmission of a STI at a population level and the downstream consequences of infection in individuals (cervical, anal, penile, and oropharyngeal cancers) have been debated [301,302]. When vaccines do become available for other STIs (chlamydia, syphilis, gonorrhea, HSV), modeling and cost benefit studies will be required to determine how best to establish herd immunity for each infection in both highand low-income settings. Our own studies have shown that induction of partial immunity in both female and male mice protects female mice against chlamydial oviduct disease [181], demonstrating that, in this model at least, vaccination of both genders may be beneficial at a population level.

VIII. KEY POINTS New STI vaccines are needed to address the increasing global burden of STIs. Increasing antibiotic resistance in gonorrhea and the increased incidence of syphilis is some settings highlight the importance of vaccines for STIs. The correlates of protection in humans need to be determined for each major STI. Do we need tissue-resident immune responses for protection against STIs?

Evaluation of the safety of vaccines will be essential to ensure that inflammation is not exacerbated by vaccination. It is necessary to determine whether vaccineinduced immunity is affected by sex hormones in the FGT and the immunosuppressed environment of the MGT. The best antigen/adjuvant combination and immunization route(s) to target protective immunity to the female and male genital tracts need to be determined. It is necessary to determine how best to use a vaccine to maximize herd immunity.

References [1] Newman L, et al. Global estimates of syphilis in pregnancy and associated adverse outcomes: analysis of multinational antenatal surveillance data. PLoS Med 2013;10(2):e1001396. [2] Aslam MV, et al. Increasing Syphilis diagnoses among females giving birth in US hospitals, 2010-2014. Sex Transm Dis 2019;46(3):147 52. [3] Blomquist PB, et al. Is gonorrhea becoming untreatable? Future Microbiol 2014;9(2):189 201. [4] Kenyon C, et al. Syphilis reinfections pose problems for syphilis diagnosis in Antwerp, Belgium - 1992 to 2012. Euro Surveill 2014;19(45):20958. [5] Mor G, Cardenas I. The immune system in pregnancy: a unique complexity. Am J Reprod Immunol 2010;63 (6):425 33. [6] Wira CR, et al. Innate immunity in the human female reproductive tract: endocrine regulation of endogenous antimicrobial protection against HIV and other sexually transmitted infections. Am J Reprod Immunol 2011;65(3):196 211. [7] Sobinoff AP, et al. Chlamydia muridarum infectioninduced destruction of male germ cells and sertoli cells is partially prevented by Chlamydia major outer membrane protein-specific immune CD4 cells. Biol Reprod 2015;92(1):27. [8] Stassen L, et al. Zika virus in the male reproductive tract. Viruses 2018;10(4). [9] Wira CR, et al. Innate and adaptive immunity in female genital tract: cellular responses and interactions. Immunol Rev 2005;206:306 35. [10] Jones RE, Lopez KH. The female reproductive system. Human reproductive biology. Academic Press; 2013. p. 53.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[11] Valore EV, et al. Antimicrobial components of vaginal fluid. Am J Obstet Gynecol 2002;187(3):561 8. [12] Macneill C, et al. Cyclic changes in the level of the innate immune molecule, surfactant protein-a, and cytokines in vaginal fluid. Am J Reprod Immunol 2012;68(3):244 50. [13] Keller MJ, et al. PRO 2000 elicits a decline in genital tract immune mediators without compromising intrinsic antimicrobial activity. AIDS 2007;21(4): 467 76. [14] Wira CR, Fahey JV. A new strategy to understand how HIV infects women: identification of a window of vulnerability during the menstrual cycle. AIDS 2008;22 (15):1909 17. [15] Givan AL, et al. Flow cytometric analysis of leukocytes in the human female reproductive tract: comparison of fallopian tube, uterus, cervix, and vagina. Am J Reprod Immunol 1997;38(5):350 9. [16] King AE, et al. Expression of secretory leukocyte protease inhibitor and elafin in human fallopian tube and in an in-vitro model of Chlamydia trachomatis infection. Hum Reprod 2009;24(3):679 86. [17] Lee JY, Lee M, Lee SK. Role of endometrial immune cells in implantation. Clin Exp Reprod Med 2011;38 (3):119 25. [18] King A. Uterine leukocytes and decidualization. Hum Reprod Update 2000;6(1):28 36. [19] Salamonsen LA, Zhang J, Brasted M. Leukocyte networks and human endometrial remodelling. J Reprod Immunol 2002;57(1-2):95 108. [20] Jensen AL, et al. A subset of human uterine endometrial macrophages is alternatively activated. Am J Reprod Immunol 2012;68(5):374 86. [21] Gordon S, Pluddemann A, Martinez Estrada F. Macrophage heterogeneity in tissues: phenotypic diversity and functions. Immunol Rev 2014;262 (1):36 55. [22] White HD, et al. CD3 1 CD8 1 CTL activity within the human female reproductive tract: influence of stage of the menstrual cycle and menopause. J Immunol 1997;158(6):3017 27. [23] Mettler L, et al. Lymphocyte subsets in the endometrium of patients with endometriosis throughout the menstrual cycle. Am J Reprod Immunol 1996;36 (6):342 8. [24] Yeaman GR, et al. Unique CD8 1 T cell-rich lymphoid aggregates in human uterine endometrium. J Leukocyte Biol 1997;61(4):427 35. [25] White HD, et al. Mucosal immunity in the human female reproductive tract: cytotoxic T lymphocyte function in the cervix and vagina of premenopausal and postmenopausal women. Am J Reprod Immunol 1997;37(1):30 8.

287

[26] Arruvito L, et al. Expansion of CD4 1 CD25 1 and FOXP3 1 regulatory T cells during the follicular phase of the menstrual cycle: implications for human reproduction. J Reprod Immunol 2007;178(4):2572 8. [27] Kallikourdis M, Betz AG. Periodic accumulation of regulatory T cells in the uterus: preparation for the implantation of a semi-allogeneic fetus? PLoS One 2007;2(4):e382. [28] Steinert EM, et al. Quantifying memory CD8 T cells reveals regionalization of immunosurveillance. Cell 2015;161(4):737 49. [29] Topham DJ, Reilly EC. Tissue-resident memory CD8 (1) T cells: from phenotype to function. Front Immunol 2018;9:515. [30] Kutteh WH, et al. Secretory immune system of the female reproductive tract: I. Immunoglobulin and secretory component-containing cells. Obstet Gynecol 1988;71(1):56 60. [31] Armitage CW, et al. Divergent outcomes following transcytosis of IgG targeting intracellular and extracellular chlamydial antigens. Immunol Cell Biol 2014;92 (5):417 26. [32] Menge AC, Mestecky J. Surface expression of secretory component and HLA class II DR antigen on glandular epithelial cells from human endometrium and two endometrial adenocarcinoma cell lines. J Clin Immunol 1993;13(4):259 64. [33] Wang Y, et al. Transport of anti-sperm monoclonal IgA and IgG into murine male and female genital tracts from blood. Effect of sex hormones. J Immunol 1996;156(3):1014 19. [34] Kutteh WH, et al. Variations in immunoglobulins and IgA subclasses of human uterine cervical secretions around the time of ovulation. Clin Exp Immunol 1996;104(3):538 42. [35] Cicala C, et al. The integrin α4β7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1. Proc Natl Acad Sci U S A 2009;106(49):20877 82. [36] Fahrbach KM, et al. Differential binding of IgG and IgA to mucus of the female reproductive tract. PLoS One 2013;8(10):e76176. [37] Pudney J, Quayle AJ, Anderson DJ. Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone. Biol Reprod 2005;73 (6):1253 63. [38] Trifonova RT, Lieberman J, van Baarle D. Distribution of immune cells in the human cervix and implications for HIV transmission. Am J Reprod Immunol 2014;71 (3):252 64. [39] McKinnon LR, et al. Characterization of a human cervical CD4 1 T cell subset coexpressing multiple

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

288

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

16. REGULATION OF MUCOSAL IMMUNITY IN THE GENITAL TRACT

markers of HIV susceptibility. J Immunol 2011;187 (11):6032 42. Strbo N, et al. Loss of intra-epithelial endocervical gamma delta (GD) 1 T cells in HIV-infected women. Am J Reprod Immunol 2016;75(2):134 45. Alcaide ML, et al. Bacterial vaginosis is associated with loss of gamma delta T cells in the female reproductive tract in women in the Miami Women Interagency HIV Study (WIHS): a cross sectional study. PLoS One 2016;11(4):e0153045. Geijtenbeek TB, et al. DC-SIGN, a dendritic cellspecific HIV-1-binding protein that enhances transinfection of T cells. Cell 2000;100(5):587 97. Mselle TF, et al. Unique characteristics of NK cells throughout the human female reproductive tract. Clin Immunol 2007;124(1):69 76. Schenkel JM, et al. Sensing and alarm function of resident memory CD8(1) T cells. Nat Immunol 2013;14 (5):509 13. Schenkel JM, et al. T cell memory. Resident memory CD8T cells trigger protective innate and adaptive immune responses. Science 2014;346(6205):98 101. Iijima N, Iwasaki A. T cell memory. A local macrophage chemokine network sustains protective tissueresident memory CD4T cells. Science 2014;346 (6205):93 8. Nguyen PV, et al. Innate and adaptive immune responses in male and female reproductive tracts in homeostasis and following HIV infection. Cell Mol Immunol 2014;11(5):410 27. Wright PF. Inductive/effector mechanisms for humoral immunity at mucosal sites. Am J Reprod Immunol 2011;65(3):248 52. Russell MW, Mestecky J. Humoral immune responses to microbial infections in the genital tract. Microbes Infect 2002;4(6):667 77. Lopez-Gatius F, et al. Rheological properties of the anterior vaginal fluid from superovulated dairy heifers at estrus. Theriogenology 1993;40(1):167 80. Kirkman-Brown JC, Smith DJ. Sperm motility: is viscosity fundamental to progress? Mol Hum Reprod 2011;17(8):539 44. Wessels JM, et al. The relationship between sex hormones, the vaginal microbiome and immunity in HIV1 susceptibility in women. Dis Model Mech 2018;11(9). Yarbrough VL, Winkle S, Herbst-Kralovetz MM. Antimicrobial peptides in the female reproductive tract: a critical component of the mucosal immune barrier with physiological and clinical implications. Hum Reprod Update 2015;21(3):353 77. Quayle AJ, et al. Gene expression, immunolocalization, and secretion of human defensin-5 in human female reproductive tract. Am J Pathol 1998;152(5):1247 58.

[55] King AE, Critchley HO, Kelly RW. Presence of secretory leukocyte protease inhibitor in human endometrium and first trimester decidua suggests an antibacterial protective role. Mol Hum Reprod 2000;6 (2):191 6. [56] Fleming DC, et al. Hormonal contraception can suppress natural antimicrobial gene transcription in human endometrium. Fertil Steril 2003;79(4):856 63. [57] King AE, et al. Differential expression of the natural antimicrobials, beta-defensins 3 and 4, in human endometrium. J Reprod Immunol 2003;59(1):1 16. [58] Shust GF, et al. Female genital tract secretions inhibit herpes simplex virus infection: correlation with soluble mucosal immune mediators and impact of hormonal contraception. Am J Reprod Immunol 2010;63 (2):110 19. [59] Hickey DK, Fahey JV, Wira CR. Mouse estrous cycle regulation of vaginal versus uterine cytokines, chemokines, α-/β-defensins and TLRs. Innate Immun 2013;19 (2):121 31. [60] Wira CR, Veronese F. Mucosal immunity in the male and female reproductive tract and prevention of HIV transmission. Am J Reprod Immunol 2011;65 (3):182 5. [61] Aboud L, et al. The role of serpin and cystatin antiproteases in mucosal innate immunity and their defense against HIV. Am J Reprod Immunol 2014;71(1):12 23. [62] Ghosh M, et al. Anti-HIV activity in cervical-vaginal secretions from HIV-positive and -negative women correlate with innate antimicrobial levels and IgG antibodies. PLoS One 2010;5(6):e11366. [63] McNeely TB, et al. Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro. J Clin Invest 1995;96(1):456 64. [64] Lee-Huang S, et al. Structural and functional modeling of human lysozyme reveals a unique nonapeptide, HL9, with anti-HIV activity. Biochemistry 2005;44 (12):4648 55. [65] Keller MJ, et al. Longitudinal assessment of systemic and genital tract inflammatory markers and endogenous genital tract E. coli inhibitory activity in HIVinfected and uninfected women. Am J Reprod Immunol 2016;75(6):631 42. [66] Brubaker SW, et al. Innate immune pattern recognition: a cell biological perspective. Annu Rev Immunol 2015;33:257 90. [67] Fazeli A, Bruce C, Anumba DO. Characterization of toll-like receptors in the female reproductive tract in humans. Hum Reprod 2005;20(5):1372 8. [68] Aflatoonian R, Fazeli A. Toll-like receptors in female reproductive tract and their menstrual cycle dependent expression. J Reprod Immunol 2008;77(1):7 13.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[69] Pivarcsi A, et al. Microbial compounds induce the expression of pro-inflammatory cytokines, chemokines and human beta-defensin-2 in vaginal epithelial cells. Microbes Infect 2005;7(9-10):1117 27. [70] Pioli PA, et al. Differential expression of toll-like receptors 2 and 4 in tissues of the human female reproductive tract. Infect Immun 2004;72(10):5799 806. [71] Aflatoonian R, et al. Menstrual cycle-dependent changes of toll-like receptors in endometrium. Hum Reprod 2007;22(2):586 93. [72] Jorgenson RL, et al. Human endometrial epithelial cells cyclically express toll-like receptor 3 (TLR3) and exhibit TLR3-dependent responses to dsRNA. Hum Immunol 2005;66(5):469 82. [73] O’Meara CP, Andrew DW, Beagley KW. The mouse model of Chlamydia genital tract infection: a review of infection, disease, immunity and vaccine development. Curr Mol Med 2014;14(3):396 421. [74] Kaushic C, et al. Chlamydia trachomatis infection in the female reproductive tract of the rat: influence of progesterone on infectivity and immune response. Infect Immun 1998;66(3):893 8. [75] Rank RG, Sanders MM, Kidd AT. Influence of the estrous cycle on the development of upper genital tract pathology as a result of chlamydial infection in the guinea pig model of pelvic inflammatory disease. Am J Pathol 1993;142(4):1291 6. [76] Rank RG, et al. Effect of estradiol on chlamydial genital infection of female guinea pigs. Infection Immunity 1982;38(2):699 705. [77] Kaushic C, et al. Progesterone increases susceptibility and decreases immune responses to genital herpes infection. J Virol 2003;77(8):4558 65. [78] Jerse AE. Experimental gonococcal genital tract infection and opacity protein expression in estradiol-treated mice. Infect Immun 1999;67(11):5699 708. [79] Bose SK, Goswami PC. Enhancement of adherence and growth of Chlamydia trachomatis by estrogen treatment of HeLa cells. Infect Immun 1986;53(3):646 50. [80] Amirshahi A, et al. Modulation of the Chlamydia trachomatis in vitro transcriptome response by the sex hormones estradiol and progesterone. BMC Microbiol 2011;11:150. [81] Deese J, et al. Contraceptive use and the risk of sexually transmitted infection: systematic review and current perspectives. Open Access J Contracept 2018;9:91 112. [82] McCarthy KJ, et al. Hormonal contraceptives and the acquisition of sexually transmitted infections: an updated systematic review. Sex Transm Dis 2019;. [83] Hapgood JP, Kaushic C, Hel Z. Hormonal contraception and HIV-1 acquisition: biological mechanisms. Endocr Rev 2018;39(1):36 78.

289

[84] Polis CB, et al. An updated systematic review of epidemiological evidence on hormonal contraceptive methods and HIV acquisition in women. AIDS 2016;30 (17):2665 83. [85] Fichorova RN, et al. The contribution of cervicovaginal infections to the immunomodulatory effects of hormonal contraception. MBio 2015;6(5). p. e00221-15. [86] Tsai MJ, O’Malley BW. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 1994;63:451 86. [87] McKenna NJ, Lanz RB, O’Malley BW. Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 1999;20(3):321 44. [88] McDevitt MA, et al. New insights into the classical and non-classical actions of estrogen: evidence from estrogen receptor knock-out and knock-in mice. Mol Cell Endocrinol 2008;290(1-2):24 30. [89] Fahey JV, et al. New approaches to making the microenvironment of the female reproductive tract hostile to HIV. Am J Reprod Immunol 2011;65(3):334 43. [90] Mertens HJ, et al. Androgen, estrogen and progesterone receptor expression in the human uterus during the menstrual cycle. Eur J Obstet Gynecol Reprod Biol 2001;98(1):58 65. [91] Martinkovich S, et al. Selective estrogen receptor modulators: tissue specificity and clinical utility. Clin Interv Aging 2014;9:1437 52. [92] Polin SA, Ascher SM. The effect of tamoxifen on the genital tract. Cancer Imaging 2008;8:135 45. [93] Hickey DK, Fahey JV, Wira CR. Estrogen receptor alpha antagonists mediate changes in CCL20 and CXCL1 secretions in the murine female reproductive tract. Am J Reprod Immunol 2013;69(2):159 67. [94] Patel MV, et al. Innate immunity in the vagina (part I): estradiol inhibits HBD2 and elafin secretion by human vaginal epithelial cells. Am J Reprod Immunol 2013;69 (5):463 74. [95] Russo CL, et al. Mucin gene expression in human male urogenital tract epithelia. Hum Reprod 2006;21(11): 2783 93. [96] Nelson AL, et al. Capsule enhances pneumococcal colonization by limiting mucus-mediated clearance. Infect Immun 2007;75(1):83 90. [97] Roche AM, et al. Antibody blocks acquisition of bacterial colonization through agglutination. Mucosal Immunol 2015;8(1):176 85. [98] Porter E, Yang H, Yavagal S, Preza GC, Murillo O, Lima H, et al. Distinct defensin profiles in Neisseria gonorrhoeae and Chlamydia trachomatis urethritis reveal novel epithelial cell-neutrophil interactions. Infect Immun 2005;73(8):4823 33. [99] Quayle AJ, Porter EM, Nussbaum AA, Wang YM, Brabec C, Yip KP, et al. Gene expression,

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

290

16. REGULATION OF MUCOSAL IMMUNITY IN THE GENITAL TRACT

immunolocalization, and secretion of human defensin5 in human female reproductive tract. Am J Pathol 1998;152(5):1247 58. [100] Klotman ME, Rapista A, Teleshova N, Micsenyi A, Jarvis GA, Lu W, Porter E, Chang TL. Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission. J Immunol 2008;180(9):6176 85. [101] Pudney J, Anderson DJ. Expression of toll-like receptors in genital tract tissues from normal and HIV-infected men. Am J Reprod Immunol 2011;65(1):28 43. [102] Pudney J, Anderson D. Innate and acquired immunity in the human penile urethra. J Reprod Immunol 2011;88(2):219 27. [103] Sennepin A, et al. The human penis is a genuine immunological effector site. Front Immunol 2017;8: 1732. [104] Pudney J, Anderson DJ. Immunobiology of the human penile urethra. Am J Pathol 1995;147(1): 155 65. [105] Hedger MP, Hales DB. Immunophysiology of the male reproductive tract. In: Neill JD, editor. Physiology of reproduction. Academic Press; 2006. p. 1195 286. [106] Hedger M. The immunophysiology of male reproduction, in Knobil and Neill’s Physiology of ReproductionIn: Plant T, Zeleznik A, editors. Elsevier; 2015. p. 805 92. [107] Arroteia KF, et al. The epididymis: function and its role in fertilization and infertility. In: Pereira LV, editor. Embryology - updates and highlights on classic topics. Rijeka, Croatia: InTech; 2012. p. 41 66. [108] Hinton BT, et al. The epididymis as protector of maturing spermatozoa. Reprod Fertil Dev 1995;7 (4):731 45. [109] Mital P, Hinton BT, Dufour JM. The blood-testis and blood-epididymis barriers are more than just their tight junctions. Biol Reprod 2011;84(5):851 8. [110] Meinhardt A, Hedger MP. Immunological, paracrine and endocrine aspects of testicular immune privilege. Mol Cell Endocrinol 2011;335(1):60 8. [111] Da Silva N, et al. A dense network of dendritic cells populates the murine epididymis. Reproduction 2011;141(5):653 63. [112] Hedger MP. Immunophysiology and pathology of inflammation in the testis and epididymis. J Androl 2011;32(6):625 40. [113] Goluza T, et al. Macrophages and Leydig cells in testicular biopsies of azoospermic men. Biomed Res Int 2014;2014:828697. [114] Winnall WR, Hedger MP. Phenotypic and functional heterogeneity of the testicular macrophage population: a new regulatory model. J Reprod Immunol 2013;97(2):147 58.

[115] Winnall WR, Muir JA, Hedger MP. Rat resident testicular macrophages have an alternatively activated phenotype and constitutively produce interleukin-10 in vitro. J Leukoc Biol 2011;90(1):133 43. [116] DeFalco T, et al. Yolk-sac-derived macrophages regulate fetal testis vascularization and morphogenesis. Proc Natl Acad Sci U S A 2014;111(23):E2384 93. [117] Bhushan S, et al. Uropathogenic E. coli induce different immune response in testicular and peritoneal macrophages: implications for testicular immune privilege. PLoS One 2011;6(12):e28452. [118] Fijak M, et al. Identification of immunodominant autoantigens in rat autoimmune orchitis. J Pathol 2005;207(2):127 38. [119] Beagley KW, Timms P. Chlamydia trachomatis infection: incidence, health costs and prospects for vaccine development. J Reproductive Immunol 2000;48(1):47 68. [120] Su H, et al. A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells. Proceedings of the National Academy of Sciences of the United States of America 1996;93(20):11143 8. [121] Hodinka RL, et al. Ultrastructural study of endocytosis of Chlamydia trachomatis by McCoy cells. Infect Immun 1988;56(6):1456 63. [122] Majeed M, Kihlstrom E. Mobilization of F-actin and clathrin during redistribution of Chlamydia trachomatis to an intracellular site in eucaryotic cells. Infect Immun 1991;59(12):4465 72. [123] Fields KA, Hackstadt T. The chlamydial inclusion: escape from the endocytic pathway. Annu Rev Cell Dev Biol 2002;18:221 45. [124] Hackstadt T, Scidmore MA, Rockey DD. Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion. Proc Natl Acad Sci U S A 1995;92 (11):4877 81. [125] Stephens RS, et al. Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 1998;282(5389):754 9. [126] Hybiske K, Stephens RS. Mechanisms of host cell exit by the intracellular bacterium Chlamydia. Proc Natl Acad Sci U S A 2007;104(27):11430 5. [127] Hogan RJ, et al. Chlamydial persistence: beyond the biphasic paradigm. Infect Immun 2004;72(4):1843 55. [128] Panzetta ME, Valdivia RH, Saka HA. Chlamydia persistence: a survival strategy to evade antimicrobial effects in-vitro and in-vivo. Front Microbiol 2018;9:3101. [129] WHO, Sexually Transmitted Infections (STIs). Fact SheetsAugust 2016. 2016. [130] Prevention, C.C.f.D.C.a. 2016 Sexually Transmitted Diseases Surveillance. 2016. Available from: ,https:// www.cdc.gov/std/stats16/chlamydia.htm..

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[131] Torrone E, et al. Prevalence of Chlamydia trachomatis genital infection among persons aged 14-39 years— United States, 2007-2012. MMWR Morb Mortal Wkly Rep 2014;63(38):834 8. [132] Brunham RC, Rappuoli R. Chlamydia trachomatis control requires a vaccine. Vaccine 2013;31 (15):1892 7. [133] Lister NA, et al. Chlamydia trachomatis serovars causing urogenital infections in women in Melbourne, Australia. J Clin Microbiol 2005;43(5):2546 7. [134] Mossman D, et al. Genotyping of urogenital Chlamydia trachomatis in Regional New South Wales, Australia. Sex Transm Dis 2008;35(6):614 16. [135] Papadogeorgakis H, et al. Chlamydia trachomatis serovar distribution and Neisseria gonorrhoeae coinfection in male patients with urethritis in Greece. J Clin Microbiol 2010;48(6):2231 4. [136] Jurstrand M, et al. Characterization of Chlamydia trachomatis omp1 genotypes among sexually transmitted disease patients in Sweden. J Clin Microbiol 2001;39 (11):3915 19. [137] Spaargaren J, et al. Analysis of Chlamydia trachomatis serovar distribution changes in the Netherlands (1986 2002). Sex Transm Infect 2004;80(2):151 2. [138] Stoner BP, Cohen SE. Lymphogranuloma venereum 2015: clinical presentation, diagnosis, and treatment. Clin Infect Dis 2015;61(Suppl. 8):S865 73. [139] Kavanagh K, et al. Estimation of the risk of tubal factor infertility associated with genital chlamydial infection in women: a statistical modelling study. Int J Epidemiol 2013;42(2):493 503. [140] Redgrove KA, McLaughlin EA. The role of the immune response in Chlamydia trachomatis infection of the male genital tract: a double-edged sword. Front Immunol 2014;5:534. [141] Rasmussen SJ, et al. Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in chlamydial pathogenesis. J Clin Invest 1997;99 (1):77 87. [142] Johnson RM. Murine oviduct epithelial cell cytokine responses to Chlamydia muridarum infection include interleukin-12-p70 secretion. Infect Immun 2004;72 (7):3951 60. [143] Maxion HK, Kelly KA. Chemokine expression patterns differ within anatomically distinct regions of the genital tract during Chlamydia trachomatis infection. Infect Immun 2002;70(3):1538 46. [144] Andrew DW, et al. The duration of Chlamydia muridarum genital tract infection and associated chronic pathological changes are reduced in IL-17 knockout mice but protection is not increased further by immunization. PLoS One 2013;8(9):e76664.

291

[145] O’Meara CP, et al. Immunity against a Chlamydia infection and disease may be determined by a balance of IL-17 signaling. Immunol Cell Biol 2014;92(3):287 97. [146] Lee HY, et al. A role for CXC chemokine receptor-2 in the pathogenesis of urogenital Chlamydia muridarum infection in mice. FEMS Immunol Med Microbiol 2010;60(1):49 56. [147] Lee HY, et al. A link between neutrophils and chronic disease manifestations of Chlamydia muridarum urogenital infection of mice. FEMS Immunol Med Microbiol 2010;59(1):108 16. [148] Imtiaz MT, et al. A role for matrix metalloproteinase9 in pathogenesis of urogenital Chlamydia muridarum infection in mice. Microbes Infect 2007;9(14-15): 1561 6. [149] Ault KA, et al. Chlamydia trachomatis enhances the expression of matrix metalloproteinases in an in vitro model of the human fallopian tube infection. Am J Obstet Gynecol 2002;187(5):1377 83. [150] Darville T, et al. Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection. J Immunol 2003;171(11):6187 97. [151] Hvid M, et al. Interleukin-1 is the initiator of Fallopian tube destruction during Chlamydia trachomatis infection. Cell Microbiol 2007;9(12):2795 803. [152] Stephens RS. The cellular paradigm of chlamydial pathogenesis. Trends Microbiol 2003;11(1):44 51. [153] Murthy AK, et al. Tumor necrosis factor alpha production from CD8 1 T cells mediates oviduct pathological sequelae following primary genital Chlamydia muridarum infection. Infect Immun 2011;79 (7):2928 35. [154] Manam S, Nicholson BJ, Murthy AK. OT-1 mice display minimal upper genital tract pathology following primary intravaginal Chlamydia muridarum infection. Pathog Dis 2013;67(3):221 4. [155] Van Voorhis WC, et al. Repeated Chlamydia trachomatis infection of Macaca nemestrina fallopian tubes produces a Th1-like cytokine response associated with fibrosis and scarring. Infect Immun 1997;65(6):2175 82. [156] Rank RG, Bowlin AK, Kelly KA. Characterization of lymphocyte response in the female genital tract during ascending Chlamydial genital infection in the guinea pig model. Infect Immun 2000;68(9):5293 8. [157] Westrom L, et al. Pelvic inflammatory disease and fertility. A cohort study of 1,844 women with laparoscopically verified disease and 657 control women with normal laparoscopic results. Sex Transm Dis 1992;19 (4):185 92. [158] Osser S, Persson K, Liedholm P. Tubal infertility and silent chlamydial salpingitis. Hum Reprod 1989;4 (3):280 4.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

292

16. REGULATION OF MUCOSAL IMMUNITY IN THE GENITAL TRACT

[159] Rank RG, Whittum-Hudson JA. Protective immunity to chlamydial genital infection: evidence from animal studies. J Infect Dis 2010;201(Suppl. 2): S168 77. [160] Molano M, et al. The natural course of Chlamydia trachomatis infection in asymptomatic Colombian women: a 5-year follow-up study. J Infect Dis 2005;191(6):907 16. [161] Blythe MJ, et al. Recurrent genitourinary chlamydial infections in sexually active female adolescents. J Pediatr 1992;121(3):487 93. [162] Geisler WM, et al. Spontaneous resolution of genital Chlamydia trachomatis infection in women and protection from reinfection. J Infect Dis 2013;207(12): 1850 6. [163] O’Connell CM, et al. Plasmid-deficient Chlamydia muridarum fail to induce immune pathology and protect against oviduct disease. J Immunol 2007;179 (6):4027 34. [164] Rank RG, et al. Role of cell-mediated immunity in the resolution of secondary chlamydial genital infection in guinea pigs infected with the agent of guinea pig inclusion conjunctivitis. Infect Immun 1989;57 (3):706 10. [165] Qu Y, et al. Comparable genital tract infection, pathology, and immunity in rhesus macaques inoculated with wild-type or plasmid-deficient Chlamydia trachomatis serovar D. Infect Immun 2015;83 (10):4056 67. [166] Grayston JT. Immunisation against trachoma. Pan American Health Organization Scientific Publication 1965;147:549. [167] Grayston JT, Wang SP. The potential for vaccine against infection of the genital tract with Chlamydia trachomatis. Sex Transm Dis 1978;5(2):73 7. [168] Wang SP, Grayston JT, Alexander ER. Trachoma vaccine studies in monkeys. Am J Ophthalmol 1967;63 (5):1615 30. p. Suppl. [169] Grayston JT, et al. Protective studies in monkeys with trivalent and monovalent trachoma vaccines. Trachoma and related disorders cause by Chlamydial Agents. Amsterdam and New York: ExcerptaMedica; 1971. p. 377 85. [170] Sowa S, et al. Trachoma vaccine field trials in The Gambia. J Hyg (Lond) 1969;67(4):699 717. [171] Bailey R, Burton M, Mabey D. Trachoma vaccine trials in the Gambia. In: Schachter J, Byrne GI, Chernesky M, editors. Proceedings of the thirteenth international symposium on human chlamydial infections. Pacific Grove, CA, USA; 2014. p. 485 8. [172] Derrick T, et al. Trachoma and ocular chlamydial infection in the era of genomics. Mediators Inflamm 2015;2015:791847.

[173] Morrison RP, Feilzer K, Tumas DB. Gene knockout mice establish a primary protective role for major histocompatibility complex class II-restricted responses in Chlamydia trachomatis genital tract infection. Infect Immun 1995;63(12):4661 8. [174] Morrison SG, et al. Immunity to murine Chlamydia trachomatis genital tract reinfection involves B cells and CD4(1) T cells but not CD8(1) T cells. Infect Immun 2000;68(12):6979 87. [175] Perry LL, Feilzer K, Caldwell HD. Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-gamma-dependent and -independent pathways. J Immunol 1997;158(7):3344 52. [176] Wang S, et al. IFN-gamma knockout mice show Th2associated delayed-type hypersensitivity and the inflammatory cells fail to localize and control chlamydial infection. Eur J Immunol 1999;29 (11):3782 92. [177] Johansson M, et al. Genital tract infection with Chlamydia trachomatis fails to induce protective immunity in gamma interferon receptor-deficient mice despite a strong local immunoglobulin A response. Infect Immun 1997;65(3):1032 44. [178] Hawkins RA, Rank RG, Kelly KA. A Chlamydia trachomatis-specific Th2 clone does not provide protection against a genital infection and displays reduced trafficking to the infected genital mucosa. Infect Immun 2002;70(9):5132 9. [179] Holland MJ, et al. T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring. Clin Exp Immunol 1996;105(3):429 35. [180] Yu H, et al. Chlamydia muridarum T-cell antigens formulated with the adjuvant DDA/TDB induce immunity against infection that correlates with a high frequency of gamma interferon (IFN-gamma)/tumor necrosis factor alpha and IFN-gamma/interleukin-17 double-positive CD4 1 T cells. Infect Immun 2010;78 (5):2272 82. [181] O’Meara CP, et al. Induction of partial immunity in both males and females is sufficient to protect females against sexual transmission of Chlamydia. Mucosal Immunol 2016;9(4):1076 88. [182] Plummer FA, et al. Cofactors in male-female sexual transmission of human immunodeficiency virus type 1. J Infect Dis 1991;163(2):233 9. [183] Nelson DE, et al. Chlamydial IFN-γ immune evasion is linked to host infection tropism. Proc Natl Acad Sci U S A 2005;102(30):10658 63. [184] Igietseme JU. The molecular mechanism of T-cell control of Chlamydia in mice: role of nitric oxide. Immunology 1996;87(1):1 8.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[185] Zhang Y, et al. IL-17A synergizes with IFN-γ to upregulate iNOS and NO production and inhibit chlamydial growth. PLoS One 2012;7(6):e39214. [186] Fankhauser SC, Starnbach MN. PD-L1 limits the mucosal CD8 1 T cell response to Chlamydia trachomatis. J Immunol 2014;192(3):1079 90. [187] Ramsey KH, Soderberg LS, Rank RG. Resolution of chlamydial genital infection in B-cell-deficient mice and immunity to reinfection. Infect Immun 1988;56 (5):1320 5. [188] Su H, et al. Chlamydia trachomatis genital tract infection of antibody-deficient gene knockout mice. Infect Immun 1997;65(6):1993 9. [189] Morrison SG, Morrison RP. Resolution of secondary Chlamydia trachomatis genital tract infection in immune mice with depletion of both CD4 1 and CD8 1 T cells. Infect Immun 2001;69(4):2643 9. [190] Farris CM, Morrison SG, Morrison RP. CD4 1 T cells and antibody are required for optimal major outer membrane protein vaccine-induced immunity to Chlamydia muridarum genital infection. Infect Immun 2010;78(10):4374 83. [191] Moore T, et al. Fc receptor regulation of protective immunity against Chlamydia trachomatis. Immunology 2002;105(2):213 21. [192] Zhang YX, et al. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis. J Immunol 1987;138(2):575 81. [193] Byrne GI, et al. Workshop on in vitro neutralization of Chlamydia trachomatis: summary of proceedings. Journal of Infectious Diseases 1993;168(2):415 20. [194] Mueller SN, Mackay LK. Tissue-resident memory T cells: local specialists in immune defence. Nat Rev Immunol 2016;16(2):79 89. [195] Shin H, Iwasaki A. A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature 2012;491(7424):463 7. [196] Stary G, et al. VACCINES. A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells. Science 2015;348 (6241):aaa8205. [197] Morrison SG, Morrison RP. In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection. Infect Immun 2000;68(5):2870 9. [198] Kiviat NB, et al. Endometrial histopathology in patients with culture-proved upper genital tract infection and laparoscopically diagnosed acute salpingitis. Am J Surg Pathol 1990;14(2):167 75. [199] Johnson RM, Brunham RC. Tissue-resident T cells as the central paradigm of chlamydia immunity. Infect Immun 2016;84(4):868 73.

293

[200] Pal S, Peterson EM, de la Maza LM. Vaccination with the Chlamydia trachomatis major outer membrane protein can elicit an immune response as protective as that resulting from inoculation with live bacteria. Infect Immun 2005;73(12):8153 60. [201] Berry LJ, et al. Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection. Infect Immun 2004;72(2):1019 28. [202] Skelding KA, et al. Comparison of intranasal and transcutaneous immunization for induction of protective immunity against Chlamydia muridarum respiratory tract infection. Vaccine 2006;24 (3):355 66. [203] Barker CJ, et al. In silico identification and in vivo analysis of a novel T-cell antigen from Chlamydia, NrdB. Vaccine 2008;26(10):1285 96. [204] Karunakaran KP, et al. Using MHC molecules to define a Chlamydia T cell vaccine. Methods Mol Biol 2016;1403:419 32. [205] Yu H, et al. Novel Chlamydia muridarum T cell antigens induce protective immunity against lung and genital tract infection in murine models. J Immunol 2009;182(3):1602 8. [206] Finco O, et al. Approach to discover T- and B-cell antigens of intracellular pathogens applied to the design of Chlamydia trachomatis vaccines. Proc Natl Acad Sci U S A 2011;108(24):9969 74. [207] Picard MD, et al. High-throughput proteomic screening identifies Chlamydia trachomatis antigens that are capable of eliciting T cell and antibody responses that provide protection against vaginal challenge. Vaccine 2012;30(29):4387 93. [208] Grubaugh D, Flechtner JB, Higgins DE. Proteins as T cell antigens: methods for high-throughput identification. Vaccine 2013;31(37):3805 10. [209] Olsen AW, et al. Protection against Chlamydia trachomatis infection and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of the major outer membrane protein. J Infect Dis 2015;212(6):978 89. [210] Petrovsky N. Comparative safety of vaccine adjuvants: a summary of current evidence and future needs. Drug Saf 2015;38(11):1059 74. [211] McKenzie A, Watt M, Gittleson C. ISCOMATRIXTM vaccines: safety in human clinical studies. Hum Vaccin 2010;6(3). [212] O’Hagan DT, Fox CB. Are we entering a new age for human vaccine adjuvants? Expert Rev Vaccines 2015;14(7):909 11. [213] Mutwiri G, et al. Combination adjuvants: the next generation of adjuvants? Expert Rev Vaccines 2011;10 (1):95 107.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

294

16. REGULATION OF MUCOSAL IMMUNITY IN THE GENITAL TRACT

[214] Fougeroux C, Holst PJ. Future prospects for the development of cost-effective adenovirus vaccines. Int J Mol Sci 2017;18(4). [215] Altenburg AF, et al. Modified vaccinia virus ankara (MVA) as production platform for vaccines against influenza and other viral respiratory diseases. Viruses 2014;6(7):2735 61. [216] Townsend DG, et al. Recombinant fowlpox virus vector-based vaccines: expression kinetics, dissemination and safety profile following intranasal delivery. J Gen Virol 2017;98(3):496 505. [217] Li L, Saade F, Petrovsky N. The future of human DNA vaccines. J Biotechnol 2012;162(2-3):171 82. [218] O’Meara CP, et al. Multistage vaccines containing outer membrane, type III secretion system and inclusion membrane proteins protects against a Chlamydia genital tract infection and pathology. Vaccine 2017;35(31):3883 8. [219] Champion CI, et al. A vault nanoparticle vaccine induces protective mucosal immunity. PLoS One 2009;4(4):e5409. [220] Li W, et al. Induction of cross-serovar protection against genital chlamydial infection by a targeted multisubunit vaccination approach. Clin Vaccine Immunol 2007;14(12):1537 44. [221] Cong Y, et al. Intranasal immunization with chlamydial protease-like activity factor and CpG deoxynucleotides enhances protective immunity against genital Chlamydia muridarum infection. Vaccine 2007;25 (19):3773 80. [222] Bulir DC, et al. Immunization with chlamydial type III secretion antigens reduces vaginal shedding and prevents fallopian tube pathology following live C. muridarum challenge. Vaccine 2016;34(34):3979 85. [223] Manam S, et al. Intranasal vaccination with Chlamydia pneumoniae induces cross-species immunity against genital Chlamydia muridarum challenge in mice. PLoS One 2013;8(5):e64917. [224] Olsen AW, et al. Protection against Chlamydia promoted by a subunit vaccine (CTH1) compared with a primary intranasal infection in a mouse genital challenge model. PLoS One 2010;5(5):e10768. [225] Cunningham KA, et al. Poly-immunoglobulin receptor-mediated transport of IgA into the male genital tract is important for clearance of Chlamydia muridarum infection. Am J Reprod Immunol 2008;60 (5):405 14. [226] Cunningham KA, et al. CD4 1 T cells reduce the tissue burden of Chlamydia muridarum in male BALB/c mice. Vaccine 2010;28(31):4861 3. [227] Wang J, et al. A chlamydial type III-secreted effector protein (Tarp) is predominantly recognized by antibodies from humans infected with Chlamydia trachomatis and induces protective immunity against upper

[228]

[229]

[230]

[231]

[232]

[233]

[234]

[235]

[236]

[237]

[238]

[239]

genital tract pathologies in mice. Vaccine 2009;27 (22):2967 80. Coler RN, et al. Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis. FEMS Immunol Med Microbiol 2009;55 (2):258 70. Hansen J, et al. Liposome delivery of Chlamydia muridarum major outer membrane protein primes a Th1 response that protects against genital chlamydial infection in a mouse model. J Infect Dis 2008;198 (5):758 67. Hickey DK, Aldwell FE, Beagley KW. Oral immunization with a novel lipid-based adjuvant protects against genital Chlamydia infection. Vaccine 2010;28 (7):1668 72. O’Meara CP, et al. Immunization with a MOMPbased vaccine protects mice against a pulmonary Chlamydia challenge and identifies a disconnection between infection and pathology. PLoS One 2013;8(4): e61962. Johnson RM, et al. B cell presentation of Chlamydia antigen selects out protective CD4gamma13T cells: implications for genital tract tissue-resident memory lymphocyte clusters. Infect Immun 2018;86(2). Gray RT, et al. Modeling the impact of potential vaccines on epidemics of sexually transmitted Chlamydia trachomatis infection. J Infect Dis 2009;199 (11):1680 8. Rank RG, Yeruva L. Hidden in plain sight: chlamydial gastrointestinal infection and its relevance to persistence in human genital infection. Infect Immun 2014;82(4):1362 71. Gratrix J, et al. Evidence for increased Chlamydia case finding after the introduction of rectal screening among women attending 2 Canadian sexually transmitted infection clinics. Clin Infect Dis 2015;60 (3):398 404. Barry PM, et al. Results of a program to test women for rectal chlamydia and gonorrhea. Obstet Gynecol 2010;115(4):753 9. Hunte T, Alcaide M, Castro J. Rectal infections with chlamydia and gonorrhoea in women attending a multiethnic sexually transmitted diseases urban clinic. Int J Std AIDS 2010;21(12):819 22. Chan PA, et al. Extragenital infections caused by Chlamydia trachomatis and Neisseria gonorrhoeae: a review of the literature. Infect Dis Obstet Gynecol 2016;2016:5758387. Yeruva L, et al. Differential susceptibilities to azithromycin treatment of chlamydial infection in the gastrointestinal tract and cervix. Antimicrob Agents Chemother 2013;57(12):6290 4.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[240] Whittington WL, et al. Determinants of persistent and recurrent Chlamydia trachomatis infection in young women: results of a multicenter cohort study. Sex Transm Dis 2001;28(2):117 23. [241] Looker KJ, et al. Global estimates of prevalent and incident herpes simplex virus type 2 infections in 2012. PLoS One 2015;10(1). p. e114989. [242] Cunningham AL, et al. Prevalence of infection with herpes simplex virus types 1 and 2 in Australia: a nationwide population based survey. Sex Transm Infect 2006;82(2):164 8. [243] Tronstein E, et al. Genital shedding of herpes simplex virus among symptomatic and asymptomatic persons with HSV-2 infection. Jama 2011;305(14):1441 9. [244] Schacker T, et al. Frequency of symptomatic and asymptomatic herpes simplex virus type 2 reactivations among human immunodeficiency virus-infected men. J Infect Dis 1998;178(6):1616 22. [245] Freeman EE, et al. Proportion of new HIV infections attributable to herpes simplex 2 increases over time: simulations of the changing role of sexually transmitted infections in sub-Saharan African HIV epidemics. Sex Transm Infect 2007;83(Suppl. 1):i17 24. [246] Posavad CM, et al. Severe genital herpes infections in HIV-infected individuals with impaired herpes simplex virus-specific CD8 1 cytotoxic T lymphocyte responses. Proc Natl Acad Sci U S A 1997;94 (19):10289 94. [247] Johnson RT. The pathogenesis of herpes virus encephalitis. I. Virus pathways to the nervous system of suckling mice demonstrated by fluorescent antibody staining. J Exp Med 1964;119:343 56. [248] Australian Medical Handbook, 2019. In: Buckley N, Calabretto H, Del Mar C, Dowden J, Rossi S, Jane Smith W, Somogyi A, Tonkin A, editors. Adelaide. Australian Medicines Handbook Pty Ltd; 2019. Available from: https://amhonline.amh.net.au/. [249] Magaret AS, et al. Effect of condom use on per-act HSV-2 transmission risk in HIV-1, HSV-2-discordant couples. Clin Infect Dis 2016;62(4):456 61. [250] Organisation WH. Herpes simplex virus. Geneva: World Health Organisation; 2017. [251] Van Wagoner N, et al. Effects of different doses of GEN-003, a therapeutic vaccine for genital herpes simplex virus-2, on viral shedding and lesions: results of a randomized placebo-controlled trial. J Infectious Dis 2018;218(12):1890 9. [252] Bernstein DI, et al. Therapeutic vaccine for genital herpes simplex virus-2 infection: findings from a randomized trial. J Infectious Dis 2017;215(6):856 64. [253] Wald A, et al. Safety and immunogenicity of long HSV-2 peptides complexed with rhHsc70 in HSV-2 seropositive persons. Vaccine 2011;29(47):8520 9.

295

[254] Veselenak RL, et al. A Vaxfectin((R))-adjuvanted HSV-2 plasmid DNA vaccine is effective for prophylactic and therapeutic use in the guinea pig model of genital herpes. Vaccine 2012;30(49):7046 51. [255] Bernard M-C, et al. Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs. PLoS One 2015;10(4):e0121518. [256] Vical, Vical reports phase 2 trial of HSV-2 therapeutic vaccine did not meet primary endpoint. 2018. [257] McDermott MR, et al. Immunity in the female genital tract after intravaginal vaccination of mice with an attenuated strain of herpes simplex virus type 2. J Virol 1984;51(3):747 53. [258] Parr MB, et al. A mouse model for studies of mucosal immunity to vaginal infection by herpes simplex virus type 2. Lab Invest 1994;70(3):369 80. [259] Wakim LM, et al. CD8(1) T-cell attenuation of cutaneous herpes simplex virus infection reduces the average viral copy number of the ensuing latent infection. Immunol Cell Biol 2008;86(8):666 75. [260] Parr EL, Parr MB. Immunoglobulin G is the main protective antibody in mouse vaginal secretions after vaginal immunization with attenuated herpes simplex virus type 2. J Virol 1997;71(11):8109 15. [261] Parr MB, Parr EL. Immunity to vaginal herpes simplex virus-2 infection in B-cell knockout mice. Immunology 2000;101(1):126 31. [262] Nakanishi Y, et al. CD8(1) T lymphocyte mobilization to virus-infected tissue requires CD4(1) T-cell help. Nature 2009;462(7272):510 13. [263] Morrison LA. Replication-defective virus vaccineinduced protection of mice from genital herpes simplex virus 2 requires CD4 T cells. Virology 2008;376 (1):205 10. [264] Halford WP, et al. A live-attenuated HSV-2 ICP0 virus elicits 10 to 100 times greater protection against genital herpes than a glycoprotein D subunit vaccine. PLoS One 2011;6(3):e17748. [265] Wang K, et al. A herpes simplex virus 2 (HSV-2) gD mutant impaired for neural tropism is superior to an HSV-2 gD subunit vaccine to protect animals from challenge with HSV-2. J Virol 2015;90 (1):562 74. [266] Meseda CA, Stout Rr Fau - Weir JP, Weir JP. Evaluation of a needle-free delivery platform for prime-boost immunization with DNA and modified vaccinia virus ankara vectors expressing herpes simplex virus 2 glycoprotein D. Viral Immunol 2006;19 (2):250 9. [267] Awasthi S, et al. A dual-modality herpes simplex virus 2 vaccine for preventing genital herpes by using glycoprotein C and D subunit antigens to induce

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

296

[268]

[269]

[270]

[271]

[272]

[273]

[274]

[275]

[276]

[277] [278] [279]

[280]

[281]

16. REGULATION OF MUCOSAL IMMUNITY IN THE GENITAL TRACT

potent antibody responses and adenovirus vectors containing capsid and tegument proteins as T cell immunogens. J Virol 2015;89(16):8497 509. Sato A, et al. Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease. J Virol 2014;88(23):13699 708. Shlapobersky M, et al. Vaxfectin-adjuvanted plasmid DNA vaccine improves protection and immunogenicity in a murine model of genital herpes infection. J Gen Virol 2012;93(6):1305 15. Whitbeck JC, et al. Repertoire of epitopes recognized by serum IgG from humans vaccinated with herpes simplex virus 2 glycoprotein D. J Virol 2014;88 (14):7786 95. Long D, et al. Disulfide bond structure of glycoprotein D of herpes simplex virus types 1 and 2. J Virol 1992;66(11):6668 85. Landolfi V, et al. Baculovirus-expressed herpes simplex virus type 2 glycoprotein D is immunogenic and protective against lethal HSV challenge. Vaccine 1993;11(4):407 14. Bourne N, et al. Herpes simplex virus (HSV) type 2 glycoprotein D subunit vaccines and protection against genital HSV-1 or HSV-2 disease in guinea pigs. J Infect Dis 2003;187(4):542 9. Flechtner JB, et al. Immune responses elicited by the GEN-003 candidate HSV-2 therapeutic vaccine in a randomized controlled dose-ranging phase 1/2a trial. Vaccine 2016;34(44):5314 20. Corey L, et al. Recombinant glycoprotein vaccine for the prevention of genital HSV-2 infection: two randomized controlled trials. Chiron HSV Vaccine Study Group. JAMA 1999;282(4):331 40. Stanberry LR, et al. Glycoprotein-D-adjuvant vaccine to prevent genital herpes. N Engl J Med 2002;347 (21):1652 61. Belshe RB, et al. Efficacy results of a trial of a herpes simplex vaccine. N Engl J Med 2012;366(1):34 43. Biologics, B. HSV-2 Vaccine. 2018. http://www.bluewillow.com/vaccine-pipeline/hsv-2-vaccine/. Dutton JL, et al. An escalating dose study to assess the safety, tolerability and immunogenicity of a Herpes Simplex Virus DNA vaccine, COR-1. Human Vacc Immunother 2016;12(12):3079 88. Zhu J, et al. Virus-specific CD8 1 T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation. J Exp Med 2007;204 (3):595 603. Gallichan WS, Rosenthal KL. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J Exp Med 1996;184 (5):1879 90.

[282] Morrison LA, Da Costa XJ, Knipe DM. Influence of mucosal and parenteral immunization with a replication-defective mutant of HSV-2 on immune responses and protection from genital challenge. Virology 1998;243(1):178 87. [283] Ye L, et al. Efficient mucosal vaccination mediated by the neonatal Fc receptor. Nat Biotechnol 2011;29 (2):158 63. [284] Cunningham AL, Levin MJ. Herpes zoster vaccines. J Infect Dis 2018;218(Suppl. 2):S127 33. [285] Tricco AC, et al. Efficacy, effectiveness, and safety of herpes zoster vaccines in adults aged 50 and older: systematic review and network meta-analysis. BMJ 2018;363:k4029. [286] Sandgren KJ, Bertram K, Cunningham AL. Understanding natural herpes simplex virus immunity to inform next-generation vaccine design. Clin Transl Immunol 2016;5(7):e94. [287] Weinberg A, et al. Influence of age and nature of primary infection on varicella-zoster virus-specific cellmediated immune responses. J Infect Dis 2010;201 (7):1024 30. [288] Clark RA. Skin-resident T cells: the ups and downs of on site immunity. J Invest Dermatol 2010;130 (2):362 70. [289] Cameron CE, Lukehart SA. Current status of syphilis vaccine development: need, challenges, prospects. Vaccine 2014;32(14):1602 9. [290] Quillin SJ, Seifert HS. Neisseria gonorrhoeae host adaptation and pathogenesis. Nat Rev Microbiol 2018;16 (4):226 40. [291] Vincent LR, Jerse AE. Biological feasibility and importance of a gonorrhea vaccine for global public health. Vaccine 2018;. [292] Bolan GA, Sparling PF, Wasserheit JN. The emerging threat of untreatable gonococcal infection. N Engl J Med 2012;366(6):485 7. [293] Kelly RW. Immunosuppressive mechanisms in semen: implications for contraception. Hum Reprod 1995;10(7):1686 93. [294] Johansson EL, et al. Antibodies and antibodysecreting cells in the female genital tract after vaginal or intranasal immunization with cholera toxin B subunit or conjugates. Infection Immunity 1998;66 (2):514 20. [295] Nardelli-Haefliger D, et al. Specific antibody levels at the cervix during the menstrual cycle of women vaccinated with human papillomavirus 16 virus-like particles. J Natl Cancer Inst 2003;95(15):1128 37. [296] Pinto LA, et al. Immunogenicity of HPV prophylactic vaccines: serology assays and their use in HPV vaccine evaluation and development. Vaccine 2018;36(32 Pt A):4792 9.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[297] Ogra PL, Ogra SS. Local antibody response to poliovaccine in the human female genital tract. J Immunol 1973;110(5):1307 11. [298] Stanberry LR, et al. Safety and immunogenicity of a novel nanoemulsion mucosal adjuvant W805EC combined with approved seasonal influenza antigens. Vaccine 2012;30(2):307 16. [299] Joura EA, et al. A 9-valent HPV vaccine against infection and intraepithelial neoplasia in women. N Engl J Med 2015;372(8):711 23.

297

[300] Petousis-Harris H, et al. Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study. Lancet 2017;390(10102): 1603 10. [301] Hibbitts S. Should boys receive the human papillomavirus vaccine? Yes. Bmj 2009;339:b4928. [302] Human papillomavirus vaccines: WHO position paper, May 2017-Recommendations. Vaccine, 2017;35 (43):5753 5.

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C H A P T E R

17

Mucosal Regulatory System for Balanced Ocular Immunity Derek J. Royer1, Micaela L. Montgomery2 and Daniel J.J. Carr1,2 1

Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States 2Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States

I. INTRODUCTION Limiting inflammation at the ocular surface is paramount for maintenance of normal visual acuity. As vaccine development for ocular pathogens moves forward, it will be important to carefully assess and characterize the impact of vaccination on immune responses and pathology in the ocular surface mucosa. The local environment fashions a potent immunoregulatory theater composed of innate molecules, cells, and organisms. In addition, a continuous mechanical flushing of the ocular surface to maintain structural integrity and clarity and to prevent microbial pathogen colonization and replication on or within resident cells. Simultaneously, autocrine and neuroendocrine mechanisms are in place to dampen the adaptive immune response to preserve the visual axis. Therefore, the development of a vaccine to pathogens that infect the eye must

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00017-1

not only consider the efficacy of the vaccine, but also the principal effector cells (B and/or T lymphocytes) to target for activation, the contribution of the memory cell subsets (e.g., resident memory vs central memory T cells) in resistance to infection, and the preservation of the visual axis. The latter topic is often forgotten, but is critical in the overall vaccine development scheme. The ocular surface is continually exposed to the external environment, yet the default regulation of this mucosal site involves immune privilege to maintain normal vision [1,2]. Many regulatory mechanisms work in unison to create an immunological balance to dampen inflammatory responses to environmental antigens and to limit microbial growth on the ocular surface mucosa to preserve vision. However, the ocular surface mucosa remains vulnerable to a variety of pathogens that can break tolerance and provoke substantial ocular

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immunopathology [3]. The rival immunological mechanisms governing privilege and pathology in the eye present a distinct conundrum for vaccine development: Can we judiciously amplify the immune response in the ocular mucosa without provoking visual morbidity? Even short-lived inflammatory events in the eye can have deleterious visual outcomes. This chapter will review the organization and immunology of the ocular surface mucosa, common ocular pathogens, and current strategies guiding ocular vaccine development for two of the leading infectious causes of blindness worldwide: Chlamydia trachomatis and herpes simplex virus type 1 (HSV-1). While the ocular mucosa is now a common delivery route for vaccines against diseases of veterinary and agricultural importance [4,5], this chapter will focus on human pathogens and their impact on vision.

II. ORGANIZATION OF THE OCULAR SURFACE MUCOSA Eye-associated lymphoid tissue (EALT) has been suggested as a cohesive parlance for the ocular mucosal immune system. The EALT is an amalgamation of mucosal tissues, including conjunctiva-associated lymphoid tissue (CALT), tear-associated lymphoid tissue (TALT) encompassing the lacrimal apparatus and tear ducts, and divisions of the nasopharyngeal-associated lymphoid tissue (NALT) into which tears drain [6]. These mucosal aspects of the ocular surface and adnexa are organized anatomically and functionally to support and protect the avascular cornea. Fig. 17.1 details these structures. The cornea serves as the primary refractive component of the visual axis. Structurally, the vertebrate cornea is composed of three principal layers: the external epithelium, the central

FIGURE 17.1 Ocular mucosal tissues and glands in humans and mice. (A) Corneal epithelium. (B) Bowman’s layer (human only). (C) Corneal stroma. (D) Descemet’s membrane (human only). (E) Corneal endothelium. (F) Relative anatomic location of cornea. (G) Palpebral conjunctiva lining the inside of the eyelid. (H) Corneal limbus. (I) Bulbar conjunctiva covering the sclera (white of the eye). (J) Intraorbital lacrimal gland (mouse). (K) Extraorbital lacrimal gland (mouse). (L) Harderian gland (mouse only). (M) Meibomian glands (mouse). (N) Lacrimal gland (human). (O) Meibomian glands (human). (P) Nasolacrimal duct.

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stroma, and an endothelial monolayer critical for fluid homeostasis [7]. The epithelium is a tight-junction-rich protective barrier covered with a carbohydrate glycocalyx that serves to limit microbial adhesion [8 10]. The stroma comprises the majority of corneal thickness and consists of interspersed keratocytes within a dense extracellular matrix of collagen fibril lamellae optimized for optical transparency and durability [11,12]. Glycosaminoglycans are abundant in the cornea and help to maintain the structural integrity, plasticity, and transparency of the layers [13]. Although the healthy cornea is avascular, it is surrounded by blood and lymphatic vessels in the limbus—the junction between the cornea and sclera. In contrast, the cornea contains the body’s highest density of sensory nerve fibers, bar none. Corneal innervation is supplied from the ophthalmic branches of the trigeminal ganglia (cranial nerve V), which provide sensitivity to pressure, dryness, and pain and ultimately regulate blinking, wound healing, and tear production [14,15]. Alterations in corneal structure, hydration, vascularity, or sensation can have severe and often irreversible impacts on visual acuity. One of the primary roles of the EALT involves lubrication and protection of the ocular surface by maintaining a healthy tear film. The tear film is an approximately 3-μm layer containing many components produced by the EALT, including the aqueous fraction, mucins, lipids, neuropeptides, growth factors, antimicrobial peptides, and immunoglobulins [16]. The conjunctiva is a vascularized mucosal membrane that lines the inside of the eyelids (palpebral conjunctiva) and covers the sclera of the external eye (bulbar conjunctiva). The conjunctiva is composed predominantly of epithelial cells and goblet cells. Goblet cells produce gellike mucins that form a protective glycocalyx covering the ocular surface epithelia [10,17]. Lacrimal glands are multilobed exocrine structures that secrete the aqueous layer of the tear film in addition to other products [16,18].

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Although rodents are indispensable for preclinical research, they have accessory Harderian glands not found in primates that support the ocular surface [19]. Fig. 17.1 highlights differences in the lacrimal apparatus between humans and mice. Meibomian glands line the eyelid margins and secrete lipids into the tear film to limit evaporation [20]. The microbiota is an integral component of mucosal tissues [21]. The systemic physiological effects of the microbiome are well appreciated, even in immune-privileged tissues sequestered deep in the eye [22]. The ocular surface is considered to be a paucibacterial site compared to periocular skin and other mucosae, which have greater microbial density and diversity [23]. The status of the ocular surface microbiome remains controversial, as microbes may be only transient colonizers [24]. However, recent metagenomic studies indicate that the ocular surface harbors greater microbial diversity than previously reported, with a profile distinct from that of the periocular skin. Moreover, elevations in Propionibacterium acnes, Bacillus spp., and certain Klebsiella spp. have been noted in patients with dry eye disease [25]. Whether the bacteria are commensal or transient, the ocular surface microbiome contributes to immune regulation and IgA secretion by the EALT [23,26].

III. IMMUNOLOGY OF THE OCULAR SURFACE MUCOSA Immune responses at the ocular surface are balanced by immune privilege during homeostasis to maintain normal vision. In addition to supporting the tear film, the EALT is an immunoregulatory tissue that contains an abundance of innate effectors, resident antigen-presenting cells (APCs), and lymphocytes. Accordingly, the EALT is essential for immune surveillance and defense at the ocular surface. Ocular immune privilege can be lost during infection

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or may result from other inflammatory conditions. Loss of tolerance is often concomitant with the onset of immune-driven tissue pathology. Contemporary reviews provide a broad overview of the regulatory mechanisms of immunity in the cornea [27] and ocular surface mucosa [28]. Here, we briefly underscore aspects relevant to vaccine design and their role in inducing immunopathology in the ocular surface mucosa. The eyelids and tear film make up the first line of defense for the ocular surface. Autonomic blink reflexes distribute the tear film evenly across the ocular surface and wash foreign particles, pollutants, bacteria, and allergens away from the EALT. Tear film contains an array of antimicrobial factors with bactericidal and bacteriostatic properties. Tears also contain serum components, including complement proteins, secretory IgA (SIgA), and neutrophils (overnight) [29 31]. The glycocalyx covering the ocular surface epithelium provides an additional barrier to microbial invasion [17]. However, corneal denervation, allergy, dry eye disease, ocular surface trauma or surgery, and contact lens use can compromise these intrinsic protective assets [10,14,18,32]. The ocular surface epithelium is more than a protective barrier, as it has great inflammatory potential. Ocular surface epithelia express an armament of innate sensors to detect and respond to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) with cytokines and other inflammatory mediators [33,34]. Accordingly, the ocular surface epithelium is a major contributor to leukocyte recruitment during pathological states, such as dry eye disease or infection [28]. Regulatory mechanisms keep unwarranted inflammatory signaling at bay in the ocular surface epithelium to prevent immunopathology during normal conditions [28]. Maintenance of immune privilege in the EALT involves active mechanisms that limit inflammation and suppress immune cell

activation. Within the cornea, these mechanisms include conditions that favor the induction of Foxp31 regulatory T cells, tissue expression of FasL and PD-L1 to suppress effector T cell proliferation and survival, and restriction of proangiogenic factors to maintain angiogenic privilege. Moreover, the lack of blood and lymphatic vessels in the healthy cornea limit leukocyte infiltration and lymphatic drainage during homeostasis [2,3,27,35]. Cellular immune responses to infection in the cornea and conjunctiva occur in the submandibular and preauricular lymph nodes, respectively. Lymphoid follicles within the EALT also undergo hyperplasia during conjunctivitis [36]. Thus, the tolerogenic mechanisms that are active in the EALT affect both local and distal adaptive immune responses. The antigen-sampling system in the cornea and conjunctiva involves tissue-resident APCs that include dendritic cells, macrophages, and Langerhans cells. These APCs are normally tolerogenic upon migration to the regional draining lymph nodes or local lymphoid follicles. However, they upregulate costimulatory molecules in response to proinflammatory cytokines in the tissue microenvironment and galvanize effector T cell responses during infection [28,36]. In addition, mast cells are selectively inducible surrogate APCs that reside in the lacrimal gland, limbus, and conjunctiva [37,38]. Although mast cells are the primary initiators of IgE-mediated allergic conjunctivitis, they also function as innate sentinels to activate and fine-tune mucosal immune responses [39,40]. Moreover, recent evidence indicates a probable neuroimmune feedback loop in which substance P abolishes corneal immune privilege by activating dendritic cells in response to corneal nerve damage [2,41,42]. Humoral immunity in the EALT is also subject to regulation. One mechanism involves restricted biodistribution of immunoglobulin in the EALT. The peripheral blood vasculature is the primary source of IgG and serum proteins in the ocular surface mucosa, but IgG and other

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large macromolecules diffuse slowly in avascular corneas. As a result, the healthy cornea contains lower concentrations of IgG and complement proteins than vascularized tissues do, and the tear film harbors even less [43,44]. IgM and IgA are restricted to the corneal periphery and apical surface epithelium, respectively. In contrast, SIgA is present in the tear film and is produced locally by antibodysecreting cells in the EALT [45]. However, corneal inflammation enables a drastic influx of IgG into the cornea [46,47]. One important mechanism underlying this effect involves increased expression of the neonatal Fc receptor (FcRn) in the cornea following infection or injury [47]. The function of FcRn as an enhancer of IgG transcytosis is unique to mucosal barriers, as FcRn functions as an IgG efflux pump in the retina and central nervous system [48 50]. Other important regulatory mechanisms of antibody function have yet to be elucidated in the ocular mucosa, including expression profiles of activating or inhibitory Fcγ receptors or the effects of Fc/Fab glycosylation patterns on antibody effector function [51 53]. Inflammation is a dilemma for the eye. While the ocular surface mucosa is sufficiently armed to respond to infection, it is balanced with protective regulatory mechanisms to limit excessive inflammation. Although host immunity mediates pathogen clearance in the ocular surface mucosa and restricts systemic dissemination, it often comes at a cost to visual acuity. Disruption of ocular immune privilege is the first step in the pathogenesis of a variety of blinding sequelae, including corneal opacification, denervation, scarring, and neovascularization. Sustained activation of innate and adaptive defenses in the EALT during (and after) infection initiates such pathologies. However, it is apparent that disruption of immune privilege alone is not sufficient for ocular pathology, given the high success rate of HLA-discordant corneal transplants [3,27].

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Multiple mechanisms contribute to the loss of visual acuity. Pathogen-induced lesions increase light scatter in the cornea, and microbial proteases can further disrupt extracellular matrix organization [54]. Infections can also lead to long-term ocular pathologies after the resolution of the primary infection, such as corneal neovascularization, which is the growth of blood and/or lymphatic vessels into the cornea [34]. Many proangiogenic factors, including proinflammatory cytokines, growth factors, and matrix metalloproteinases, are upregulated in the cornea as a result of infection or tissue repair programs and contribute to corneal neovascularization [55]. Similarly, inflammatory cytokines and growth factors can transform stromal keratocytes into activated fibrotic phenotypes that contribute to corneal scarring [56]. Damage to the corneal endothelium drives edematous opacification [7]. In addition, dysfunction in corneal nerve sensation is associated with a variety of bacterial, viral, and fungal infections; sensation loss is a risk factor for other ocular surface sequelae [14]. Microbial infection of the lacrimal gland (dacryoadenitis) may contribute to dry eye disease indirectly through inflammatory tissue damage [16,18]. All of these processes alter visual acuity and can result in temporary or permanent corneal vision loss. Insidious and recurrent infections are particularly notorious for eliciting corneal pathologies. In the next section, we will review common pathogens of the EALT and consider how the host immune response to infections associated with vision loss can inform approaches for ocular vaccine development.

IV. TARGETS AND STRATEGIES FOR VACCINE DEVELOPMENT A. Common Ocular Pathogens The risk of vaccine-associated immunopathology remains a universal concern in the field of ocular vaccine development. This concern is

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TABLE 17.1

Common and Vaccine-Preventablea Infections of the Ocular Mucosa

Location

Presentation

Associated pathogens

Eyelid

Blepharitis

Viruses

Herpes simplex virus (HSV-1), varicella-zoster virus (VZV)a

Hordeolum

Bacteria

Staphylococcus epidermidis, Staphylococcus aureus, Propionibacterium acnes

Dacryoadenitis

Viruses

Mumpsa, measlesa, Epstein-Barr virus (EBV), HSV-1

Bacteria

S. aureus, Streptococcus pneumoniae, Haemophilus influenzaea, Neisseria gonorrhoeae

Viruses

Adenovirus, HSV-1, rubellaa, measlesa, EBV, influenza A

Bacteria

SS. aureus, S. pneumoniae, H. influenzaea,

Lacrimal Apparatus Conjunctiva

Conjunctivitis

N. gonorrhoeae/Neisseria meningitidisa, Bartonella henselae

Cornea

a

Trachoma

Bacteria

Chlamydia trachomatis

Keratitis

Viruses

HSV-1, VZVa, adenovirus, EBV

Bacteria

Pseudomonas aeruginosa, S. pneumoniae, S. aureus, S. epidermidis, Moraxella species,

Fungi

Fusarium, Aspergillus, Candida

Parasites

Acanthamoeba, Onchocerca volvulus (and mutualistic Wolbachia)

Vaccine available.

rational, as infection of the ocular surface mucosa is an important cause of corneal blindness worldwide [57]. Moreover, predisposing factors influence infections of the ocular surface mucosa such as trauma, contact lens wear, topical medication use, and systemic disease [58,59]. Table 17.1 lists many common infections of the ocular mucosal tissues as well as others that have been diminished by vaccination [57,60]. Other infections of clinical importance are excluded, owing to their rarity, limited geographic distribution, or indirect association with ocular findings. However, C. trachomatis, Onchocerca volvulus, and HSV-1 are important pathogens for consideration in vaccine research given their status as the three leading infectious causes of corneal blindness [61,62]. No licensed vaccines currently exist for these three pathogens. However, optimism about the future of vaccines for ocular pathogens is warranted, as recent advances in basic research and clinical trials are promising [63,64].

The pathogenesis of onchocerciasis (river blindness) depends upon a complex ecological life cycle involving the filarial nematode O. volvulus; its vector, the black fly; and the bacterium Wolbachia, an endosymbiotic parasite of Onchocerca. The Onchocerca life cycle has been reviewed elsewhere [65]; here, the important point is that the death of immature larvae migrating through the skin (or ocular mucosae) releases Wolbachia. Corneal immunopathology is driven by TLR signaling in response to Wolbachia [65]. The widespread prevention of onchocerciasis through control of the black fly vector and annual treatment of infected populations is a modern public health triumph. However, it is estimated that there are still 12 40 cases of visual impairment or blindness per 10,000 people in areas of endemic infection [61,66]. Accordingly, the Onchocerciasis Vaccine for Africa (TOVA) initiative has been created to develop additional avenues of disease elimination [67]. Protective mechanisms of

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IV. TARGETS AND STRATEGIES FOR VACCINE DEVELOPMENT

an effective vaccine for river blindness would likely facilitate pathogen clearance prior to the onset of ocular disease. Thus the risk of vaccine-associated ocular immunopathology is limited for prophylactic management of onchocerciasis. Ocular immunopathology is of much greater concern in the realm of vaccine development for C. trachomatis and HSV-1. Recurrent ocular reactivation of HSV-1 from neurons in the trigeminal ganglia can lead to blinding keratitis involving corneal opacification, neovascularization, scarring, and sensation loss [62]. Repeated ocular infection with C. trachomatis leads to trachoma, a progressive inflammatory disease of the palpebral conjunctiva that can eventually result in eyelash inversion (trichiasis) and mechanical corneal damage. Public health interventions to eliminate trachoma involve the “SAFE” strategy (surgery to treat eyelid inversion, antibiotics, facial cleanliness, and environmental improvement to reduce transmission) [68]. The pathogenesis of HSV-1 and C. trachomatis in the onset of ocular disease have been reviewed elsewhere [62,69]. They have several

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common features. First, both are intracellular pathogens that exhibit tropisms for the ocular and genital mucosae. In the eye, blinding pathology ensues as a result of the immune response to recurrent infection over time, as shown in Fig. 17.2 for trachoma. In addition, several decades of targeted research has failed to generate a licensed vaccine for either pathogen [70,71]. Moreover, some experts question whether sterilizing immunity can be achieved through vaccination, as both pathogens establish persistent and recurrent infections in immunocompetent individuals, albeit through very different mechanisms [72 74]. The pathology induced by ocular infection can be corrected clinically by corneal transplantation, but the risk of recurrent disease and subsequent allograft rejection is high [75,76]. Fig. 17.3 depicts a recurrent HSV-1 lesion in the epithelium of a corneal graft.

B. Strategies for Ocular Vaccine Development The classical correlates of protection elicited by vaccination (cell-mediated and humoral FIGURE 17.2 Pathogenesis of trachoma. (A) Timeline of trachoma pathogenesis following repeated ocular Chlamydia trachoma infection during childhood. (B) Follicular inflammatory foci in the palpebral conjunctiva associated with repeated infection. (C) Trachomatous scarring of the palpebral conjunctiva. (D) Advanced trachoma with diffuse corneal opacity. Source: Graphic (A) was obtained from Hunter’s Tropical Medicine and Emerging Infectious Diseases, 9th Ed., Ch. 35, Trachoma and Inclusion Conjunctivitis by Taylor and Matthew. http://www. sciencedirect.com/science/article/pii/B9781416043904000357; Photographs (B D) were obtained from The Lancet Vol. 384, Issue 9960, 13 19, December 2014, page 2143. http://www.sciencedirect.com/science/article/pii/ S0140673613621820#fig1.

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FIGURE 17.3 Herpetic corneal lesion in a patient with previous corneal graft. Dendritic ulcers in the corneal epithelium are a pathognomonic sign of HSV-1 infection in the corneal epithelium. Fluorescein was used here as a contrast agent to accentuate the lesion. Source: Photograph was obtained from Hunter’s Tropical Medicine and Emerging Infectious Diseases, 9th Ed., Ch. 9, Ophthalmological Diseases by Taylor and Turner. http:// www.sciencedirect.com/science/article/pii/B9781416043 904000096.

immunity) are useful for clinical diagnostics and licensure requirements, yet the correlates of protection paradigm is limited in explaining specific mechanisms of immunological protection within mucosal microenvironments [77]. Leveraging the unique physiology of the ocular mucosal immune system in the pursuit of vaccine development is therefore a worthy goal. Controversy surrounds the best approaches for ocular vaccines, mechanisms of protection, and associated risks. Accordingly, vaccine development with respect to the ocular surface mucosa falls into two broad categories: mucosal vaccine delivery for ocular and/or systemic mucosal protection and parenteral vaccination for protection against ocular pathogens. Pearls from clinical practice and research provide insights into the benefits, risks, and mechanisms of vaccine-mediated protection with each approach in the eye. These insights will help answer the central question in ocular vaccine research: Can we judiciously amplify the immune response to ocular pathogens without provoking visual morbidity? The rationale supporting local immunization in the eye is multifactorial. Recent trends in vaccine development have centered on tailoring vaccines to mimic the immune response to a pathogen at its natural point of entry.

Inducing immune responses in local mucosal sites facilitates establishment of pathogenspecific tissue resident memory T cells (TRM) and local IgA production [78,79]. Indeed, ocular vaccination has been verified in the agricultural industry as a beneficial needle-free immunization alternative that facilitates mucosal protection [4,5]. However, the long-term safety of topical ocular vaccines and adjuvants has not been investigated with respect to toxicity, impacts on immune privilege, or visual acuity. Although the EALT is an immune-privileged site, experimental evidence indicates that topical immunization elicits humoral immunity in the ocular mucosa [80,81]. In mice, an inactivated influenza eye drop vaccine adjuvanted with the TLR3 agonist poly(I:C) induces protective titers of IgG and IgA in both the tear film and serum [82]. Similarly, topical tetanus toxoid elicits IgG and IgA in tears and serum of mice [5]. Direct ocular vaccination using a HSV glycoprotein subunit adjuvanted with CpG DNA in rabbits induces virus-neutralizing IgG and IgA in the tear film and serum [83]. Intranasal immunization is another proven route of SIgA induction in the tear film, and it may be more efficacious in certain instances [84]. Collectively, this suggests that that crosstalk exists between the EALT and the NALT

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REFERENCES

that supports coinduction of mucosal immune responses. However, TRM induction associated with ocular vaccination and the contributions of TRM to protection or pathology upon challenge have not been systematically investigated. Although mucosal immunization may elicit greater IgA responses than parenteral vaccination at sites of pathogen entry, IgA is not protective against every mucosal pathogen. This is especially true of HSV-1, as IgA titers are not predictive of prophylactic protection or viral reactivation [85 88]. Likewise, decades of research has yielded insufficient evidence to suggest that IgA offers adequate protection against C. trachomatis infection in the ocular (or genital) mucosa [71]. On the other hand, mucosal vaccination may help recruit ΤRM for protection against Chlamydia. This could be experimentally determined by using hybrid prime-and-pull immunization strategies involving parenteral vaccination with mucosal delivery of cytokines to establish TRM in mucosal sites [89]. How the unique immunological functions of the privileged ocular surface mucosa affect the establishment and retention of TRM is unknown. However, such approaches must proceed cautiously in light of the prevailing role of T cells in various ocular immunopathologies [90]. Finally, an important immunological concept relevant to vaccine development is that the immunological mechanisms undergirding prophylactic protection and resolution of natural infection may differ [77]. Classic parenteral vaccination is the other principal option for eliciting ocular protection [91,92]. Many parenteral vaccines currently licensed for use have drastically limited the occurrence of infections associated with the EALT, including measles, mumps, and varicella-zoster virus [93]. Not all routes of parenteral vaccination are equal; however, recent data using a live-attenuated HSV-1 vaccine show that subcutaneous vaccination is superior to intramuscular vaccination in terms of

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antibody titer [47]. Evidence from clinical trials and basic research using parenteral HSV vaccines show that antibody is the major correlate of protection against HSV-1 infection and disease [87,94]. Moreover, intramuscular immunization against C. trachomatis has been shown to elicit pathogen-specific antibodies in the tear film [95]. Additional factors in mucosal defense that apply to both local and parenteral vaccination strategies for ocular protection must also be considered. Vaccine composition is a crucial component for effective vaccines, as the antigenic breadth of the immunogen governs the repertoire of the resulting immune response [96]. In addition, the host inflammatory program induced by vaccination has a significant impact on the longevity of humoral protection by regulating B cell responses [97,98]. Effector functions of IgG subclasses differ between rodents and humans. This is also true of the corresponding Fcγ receptor affinities and intracellular activation/inhibition signaling properties [51]. For example, parenteral vaccination against Onchocerca in mice leads to FcγRdependent inflammatory corneal pathology upon challenge [43]. Thus mechanisms of protection elucidated from vaccine studies in rodents need to be interpreted cautiously in being extrapolated to the human immune system. As a final point, it is time to challenge the dogma that T cells respond to intracellular pathogens and immunoglobulin patrols the extracellular space, as evidence for protective functions of intracellular IgG is accumulating [99,100].

References [1] Forrester JV, Xu H, Lambe T, Cornall R. Immune privilege or privileged immunity? Mucosal Immunol 2008;1:372 81. Available from: https://doi.org/ 10.1038/mi.2008.27. [2] Reyes NJ, O’Koren EG, Saban DR. New insights into mononuclear phagocyte biology from the visual system. Nat Rev Immunol 2017;17:322 32. Available from: https://doi.org/10.1038/nri.2017.13.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

308

17. MUCOSAL REGULATORY SYSTEM FOR BALANCED OCULAR IMMUNITY

[3] Niederkorn JY. Cornea: window to ocular immunology. Curr Immunol Rev 2011;7:328 35. Available from: https://doi.org/10.2174/157339511796196593. [4] van Ginkel FW, Gulley SL, Lammers A, Hoerr FJ, Gurjar R, Toro H. Conjunctiva-associated lymphoid tissue in avian mucosal immunity. Dev Comp Immunol 2012;36:289 97. Available from: https://doi. org/10.1016/j.dci.2011.04.012. [5] Barisani-Asenbauer T, Inic-Kanada A, Belij S, Marinkovic E, Stojicevic I, Montanaro J, et al. The ocular conjunctiva as a mucosal immunization route: a profile of the immune response to the model antigen tetanus toxoid. PLoS One 2013;8:e60682. Available from: https://doi.org/10.1371/journal.pone.0060682. [6] Knop E, Knop N. The role of eye-associated lymphoid tissue in corneal immune protection. J Anat 2005;206:271 85. Available from: https://doi.org/ 10.1111/j.1469-7580.2005.00394.x. [7] Diecke FPJ, Ma L, Iserovich P, Fischbarg J. Corneal endothelium transports fluid in the absence of net solute transport. Biochim Biophys Acta (BBA) Biomembr 2007;1768:2043 8. Available from: https:// doi.org/10.1016/j.bbamem.2007.05.020. [8] Yi X, Wang Y, Yu FS. Corneal epithelial tight junctions and their response to lipopolysaccharide challenge. Invest Ophthalmol Vis Sci 2000;41:4093 100. [9] Govindarajan B, Gipson IK. Membrane-tethered mucins have multiple functions on the ocular surface. Exp Eye Res 2010;90:655 63. Available from: https:// doi.org/10.1016/j.exer.2010.02.014. [10] Mantelli F, Mauris J, Argu¨eso P. The ocular surface epithelial barrier and other mechanisms of mucosal protection: from allergy to infectious diseases. Curr Opin Allergy Clin Immunol 2013;13:563 8. Available from: https://doi.org/10.1097/ACI.0b013e3283645899. [11] Morishige N, Petroll WM, Nishida T, Kenney MC, Jester JV. Noninvasive corneal stromal collagen imaging using two-photon-generated secondharmonic signals. J Cataract Refract Surg 2006;32:1784 91. Available from: https://doi.org/ 10.1016/j.jcrs.2006.08.027. [12] Chen S, Mienaltowski MJ, Birk DE. Regulation of corneal stroma extracellular matrix assembly. Exp Eye Res 2015;133:69 80. Available from: https://doi.org/ 10.1016/j.exer.2014.08.001. [13] Pacella E, Pacella F, De Paolis G, Parisella FR, Turchetti P, Anello G, et al. Glycosaminoglycans in the human cornea: age-related changes. Ophthalmol Eye Dis 2015;7:1 5. Available from: https://doi.org/10.4137/ OED.S17204. [14] Shaheen BS, Bakir M, Jain S. Corneal nerves in health and disease. Surv Ophthalmol 2014;59:263 85. Available from: https://doi.org/10.1016/j.survophthal.2013.09.002.

[15] Stepp MA, Tadvalkar G, Hakh R, Pal-Ghosh S. Corneal epithelial cells function as surrogate Schwann cells for their sensory nerves. Glia 2017;65:851 63. Available from: https://doi.org/10.1002/glia.23102. [16] Zoukhri D. Effect of inflammation on lacrimal gland function. Exp Eye Res 2006;82:885 98. Available from: https://doi.org/10.1016/j.exer.2005.10.018. [17] Barbosa F, Xiao Y, Bian F, Coursey T, Ko B, Clevers H, et al. Goblet cells contribute to ocular surface immune tolerance—implications for dry eye disease. Int J Mol Sci 2017;18:978. Available from: https://doi.org/ 10.3390/ijms18050978. [18] Conrady CD, Joos ZP, Patel BCK. Review: the lacrimal gland and its role in dry eye. J Ophthalmol 2016;2016:1 11. Available from: https://doi.org/ 10.1155/2016/7542929. [19] Chieffi G, Baccari GC, Di Matteo L, d’Istria M, Minucci S, Varriale B. Cell biology of the harderian gland. Int Rev Cytol 1996;168:1 80. [20] Butovich IA. On the lipid composition of human meibum and tears: comparative analysis of nonpolar lipids. Investig Opthalmol Vis Sci 2008;49:3779. Available from: https://doi.org/10.1167/iovs.081889. [21] McHardy IH, Goudarzi M, Tong M, Ruegger PM, Schwager E, Weger JR, et al. Integrative analysis of the microbiome and metabolome of the human intestinal mucosal surface reveals exquisite inter-relationships. Microbiome 2013;1:17. Available from: https://doi. org/10.1186/2049-2618-1-17. [22] Horai R, Za´rate-Blade´s CR, Dillenburg-Pilla P, Chen J, Kielczewski JL, Silver PB, et al. Microbiota-dependent activation of an autoreactive T cell receptor provokes autoimmunity in an immunologically privileged site. Immunity 2015;43:343 53. Available from: https:// doi.org/10.1016/j.immuni.2015.07.014. [23] Doan T, Akileswaran L, Andersen D, Johnson B, Ko N, Shrestha A, et al. Paucibacterial microbiome and resident DNA virome of the healthy conjunctiva. Invest Ophthalmol Vis Sci 2016;57:5116 26. Available from: https://doi.org/10.1167/iovs.16-19803. [24] Willcox MDP. Characterization of the normal microbiota of the ocular surface. Exp Eye Res 2013;117:99 105. Available from: https://doi.org/ 10.1016/j.exer.2013.06.003. [25] Graham JE, Moore JE, Jiru X, Moore JE, Goodall EA, Dooley JSG, et al. Ocular pathogen or commensal: a PCR-based study of surface bacterial flora in normal and dry eyes. Investig Opthalmol Vis Sci 2007;48:5616. Available from: https://doi.org/10.1167/iovs.07-0588. [26] Kugadas A, Gadjeva M. Impact of microbiome on ocular health. Ocul Surf 2016;14:342 9. Available from: https://doi.org/10.1016/j.jtos.2016.04.004.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

[27] Taylor AW. Ocular immune privilege and transplantation. Front Immunol 2016;7:37. Available from: https://doi.org/10.3389/fimmu.2016.00037. [28] Galletti JG, Guzma´n M, Giordano MN. Mucosal immune tolerance at the ocular surface in health and disease. Immunology 2017;150:397 407. Available from: https://doi.org/10.1111/imm.12716. [29] McDermott AM. Antimicrobial compounds in tears. Exp Eye Res 2013;117:53 61. Available from: https:// doi.org/10.1016/j.exer.2013.07.014. [30] Tan KO, Sack RA, Holden BA, Swarbrick HA. Temporal sequence of changes in tear film composition during sleep. Curr Eye Res 1993;12:1001 7. [31] Bora NS, Jha P, Bora PS. The role of complement in ocular pathology. Semin Immunopathol 2008;30:85 95. Available from: https://doi.org/10.1007/s00281-0080110-y. [32] Evans DJ, Fleiszig SMJ. Microbial keratitis: could contact lens material affect disease pathogenesis? Eye Contact Lens 2013;39:73 8. Available from: https:// doi.org/10.1097/ICL.0b013e318275b473. [33] Ueta M, Kinoshita S. Ocular surface inflammation is regulated by innate immunity. Prog Retin Eye Res 2012;31:551 75. Available from: https://doi.org/ 10.1016/j.preteyeres.2012.05.003. [34] Bryant-Hudson K, Conrady CD, Carr DJJ. Type I interferon and lymphangiogenesis in the HSV-1 infected cornea are they beneficial to the host? Prog Retin Eye Res 2013;36:281 91. Available from: https://doi. org/10.1016/j.preteyeres.2013.06.003. [35] Foulsham W, Marmalidou A, Amouzegar A, Coco G, Chen Y, Dana R. Review: the function of regulatory T cells at the ocular surface. Ocul Surf 2017;15:652 9. Available from: https://doi.org/ 10.1016/j.jtos.2017.05.013. [36] Chodosh J, Kennedy RC. The conjunctival lymphoid follicle in mucosal immunology. DNA Cell Biol 2002;21:421 33. Available from: https://doi.org/ 10.1089/10445490260099719. [37] Kambayashi T, Laufer TM. Atypical MHC class IIexpressing antigen-presenting cells: can anything replace a dendritic cell? Nat Rev Immunol 2014;14:719 30. Available from: https://doi.org/10.1038/nri3754. [38] Royer DJ, Zheng M, Conrady CD, Carr DJJ. Granulocytes in ocular HSV-1 infection: opposing roles of mast cells and neutrophils. Invest Ophthalmol Vis Sci 2015;56:3763 75. Available from: https://doi.org/ 10.1167/iovs.15-16900. [39] Ackerman S, Smith LM, Gomes PJ. Ocular itch associated with allergic conjunctivitis: latest evidence and clinical management. Ther Adv Chronic Dis 2016;7:52 67. Available from: https://doi.org/ 10.1177/2040622315612745.

309

[40] Frossi B, Mion F, Tripodo C, Colombo MP, Pucillo CE. Rheostatic functions of mast cells in the control of innate and adaptive immune responses. Trends Immunol 2017;38:648 56. Available from: https://doi. org/10.1016/j.it.2017.04.001. [41] Paunicka KJ, Mellon J, Robertson D, Petroll M, Brown JR, Niederkorn JY. Severing corneal nerves in one eye induces sympathetic loss of immune privilege and promotes rejection of future corneal allografts placed in either eye. Am J Transplant 2015;15:1490 501. Available from: https://doi.org/10.1111/ajt.13240. [42] Mo J, Neelam S, Mellon J, Brown JR, Niederkorn JY. Effect of corneal nerve ablation on immune tolerance induced by corneal allografts, oral immunization, or anterior chamber injection of antigens. Investig Opthalmol Vis Sci 2017;58:137. Available from: https://doi.org/10.1167/iovs.16-20601. [43] Hall LR, Diaconu E, Pearlman E. A dominant role for Fc receptors in antibody-dependent corneal inflammation. J Immunol 2001;167:919 25. Available from: https://doi.org/10.4049/jimmunol.167.2.919. [44] Osusky R, Morell A, Imbach P, Lerch PG. Diffusion of immunoglobulins into rabbit cornea after subconjunctival injection: experimental demonstration and mathematical model. Graefes Arch Clin Exp Ophthalmol 1993;231:122 8. Available from: https://doi.org/ 10.1007/BF00920226. [45] Hazlett LD, Berk RS. Kinetics of immunoglobulin appearance at the ocular surface. Reg Immunol 1989;2:294 9. [46] Preston MJ, Kernacki KA, Berk JM, Hazlett LD, Berk RS. Kinetics of serum, tear, and corneal antibody responses in resistant and susceptible mice intracorneally infected with Pseudomonas aeruginosa. Infect Immun 1992;60:885 91. [47] Royer DJ, Carr MM, Gurung HR, Halford WP, Carr DJJ. The neonatal fc receptor and complement fixation facilitate prophylactic vaccine-mediated humoral protection against viral infection in the ocular mucosa. J Immunol 2017;199:1898 911. Available from: https:// doi.org/10.4049/jimmunol.1700316. [48] Bai Y, Ye L, Tesar DB, Song H, Zhao D, Bjorkman PJ, et al. Intracellular neutralization of viral infection in polarized epithelial cells by neonatal Fc receptor (FcRn)-mediated IgG transport. Proc Natl Acad Sci 2011;108:18406 11. Available from: https://doi.org/ 10.1073/pnas.1115348108. [49] Kim H, Robinson SB, Csaky KG. FcRn receptormediated pharmacokinetics of therapeutic IgG in the eye. Mol Vis 2009;15:2803 12. [50] Pyzik M, Rath T, Lencer WI, Baker K, Blumberg RS. FcRn: the architect behind the immune and nonimmune functions of IgG and albumin. J Immunol

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

310

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60] [61]

[62]

17. MUCOSAL REGULATORY SYSTEM FOR BALANCED OCULAR IMMUNITY

2015;194:4595 603. Available from: https://doi.org/ 10.4049/jimmunol.1403014. Bruhns P. Properties of mouse and human IgG receptors and their contribution to disease models. Blood 2012;119:5640 9. Available from: https://doi.org/ 10.1182/blood-2012-01-380121. Huhn C, Selman MHJ, Ruhaak LR, Deelder AM, Wuhrer M. IgG glycosylation analysis. Proteomics 2009;9:882 913. Available from: https://doi.org/ 10.1002/pmic.200800715. van de Bovenkamp FS, Hafkenscheid L, Rispens T, Rombouts Y. The emerging importance of IgG fab glycosylation in immunity. J Immunol 2016;196:1435 41. Available from: https://doi.org/ 10.4049/jimmunol.1502136. Sharma S, Garg P, Rao G, Gopinathan U. Review of epidemiological features, microbiological diagnosis and treatment outcome of microbial keratitis: experience of over a decade. Indian J Ophthalmol 2009;57:273. Available from: https://doi.org/10.4103/ 0301-4738.53051. Gurung HR, Carr MM, Bryant K, Chucair-Elliott AJ, Carr DJ. Fibroblast growth factor-2 drives and maintains progressive corneal neovascularization following HSV-1 infection. Mucosal Immunol 2018;11:172 85. Available from: https://doi.org/10.1038/mi.2017.26. Wilson SE, Mohan RR, Mohan RR, Ambro´sio R, Hong J, Lee J. The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Prog Retin Eye Res 2001;20:625 37. Available from: https://doi.org/ 10.1016/S1350-9462(01)00008-8. Tan SZ, Walkden A, Au L, Fullwood C, Hamilton A, Qamruddin A, et al. Twelve-year analysis of microbial keratitis trends at a UK tertiary hospital. Eye 2017;31:1229 36. Available from: https://doi.org/ 10.1038/eye.2017.55. Green M, Apel A, Stapleton F. Risk factors and causative organisms in microbial keratitis. Cornea 2008;27:22 7. Available from: https://doi.org/ 10.1097/ICO.0b013e318156caf2. Keay L, Edwards K, Naduvilath T, Taylor HR, Snibson GR, Forde K, et al. Microbial keratitis. Ophthalmology 2006;113:109 16. Available from: https://doi.org/ 10.1016/j.ophtha.2005.08.013. Armstrong RA. The microbiology of the eye. Ophthalmic Physiol 2000;20:429 41. Whitcher JP, Srinivasan M, Upadhyay MP. Corneal blindness: a global perspective. Bull World Health Organ 2001;79:214 21. Farooq AV, Shukla D. Herpes simplex epithelial and stromal keratitis: an epidemiologic update. Surv Ophthalmol 2012;57:448 62. Available from: https:// doi.org/10.1016/j.survophthal.2012.01.005.

[63] Gottlieb SL, Deal CD, Giersing B, Rees H, Bolan G, Johnston C, et al. The global roadmap for advancing development of vaccines against sexually transmitted infections: update and next steps. Vaccine 2016;34:2939 47. Available from: https://doi.org/ 10.1016/j.vaccine.2016.03.111. [64] de la Maza LM, Zhong G, Brunham RC. Update on Chlamydia trachomatis vaccinology. Clin Vaccine Immunol 2017;24. Available from: https://doi.org/ 10.1128/CVI.00543-16 e00543 16. [65] Tamarozzi F, Halliday A, Gentil K, Hoerauf A, Pearlman E, Taylor MJ. Onchocerciasis: the role of wolbachia bacterial endosymbionts in parasite biology, disease pathogenesis, and treatment. Clin Microbiol Rev 2011;24:459 68. Available from: https://doi.org/ 10.1128/CMR.00057-10. [66] Kim YE, Stolk WA, Tanner M, Tediosi F. Modelling the health and economic impacts of the elimination of river blindness (onchocerciasis) in Africa. BMJ Glob Health 2017;2:e000158. Available from: https://doi.org/ 10.1136/bmjgh-2016-000158. [67] Hotez PJ, Bottazzi ME, Zhan B, Makepeace BL, Klei TR, Abraham D, et al. The onchocerciasis vaccine for Africa—TOVA—initiative. PLoS Negl Trop Dis 2015;9: e0003422. Available from: https://doi.org/10.1371/ journal.pntd.0003422. [68] Kuper H, Solomon AW, Buchan J, Zondervan M, Foster A, Mabey D. A critical review of the SAFE strategy for the prevention of blinding trachoma. Lancet Infect Dis 2003;3:372 81. Available from: https://doi. org/10.1016/S1473-3099(03)00659-5. [69] Hu VH, Holland MJ, Burton MJ. Trachoma: protective and pathogenic ocular immune responses to Chlamydia trachomatis. PLoS Negl Trop Dis 2013;7:e2020. Available from: https://doi.org/10.1371/journal. pntd.0002020. [70] Royer DJ, Cohen AW, Carr DJ. The current state of vaccine development for ocular HSV-1 infection. Expert Rev Ophthalmol 2015;10:113 26. Available from: https://doi.org/10.1586/17469899.2015.1004315. [71] Mabey DCW, Hu V, Bailey RL, Burton MJ, Holland MJ. Towards a safe and effective chlamydial vaccine: lessons from the eye. Vaccine 2014;32:1572 8. Available from: https://doi.org/10.1016/j.vaccine.2013.10.016. [72] Whitley RJ, Roizman B. Herpes simplex viruses: is a vaccine tenable? J Clin Invest 2002;110:145 51. Available from: https://doi.org/10.1172/JCI16126. [73] Morrison RP. New insights into a persistent problem— chlamydial infections. J Clin Invest 2003;111:1647 9. Available from: https://doi.org/10.1172/JCI200318770. [74] Liang S, Bulir D, Kaushic C, Mahony J. Considerations for the rational design of a Chlamydia vaccine. Hum Vaccin Immunother 2017;13:831 5. Available from: https://doi.org/10.1080/21645515.2016.1252886.

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

311

REFERENCES

[75] Al-Fawaz A, Wagoner MD, King Khaled Eye Specialist Hospital Corneal Transplant Study Group. Penetrating keratoplasty for trachomatous corneal scarring. Cornea 2008;27:129 32. Available from: https://doi.org/10.1097/ICO.0b013e318158b49e. [76] Kuffova L, Knickelbein JE, Yu T, Medina C, Amescua G, Rowe AM, et al. High-risk corneal graft rejection in the setting of previous corneal herpes simplex virus (HSV)-1 infection. Investig Opthalmol Vis Sci 2016;57:1578. Available from: https://doi. org/10.1167/iovs.15-17894. [77] Plotkin SA. Correlates of protection induced by vaccination. Clin Vaccine Immunol 2010;17:1055 65. Available from: https://doi.org/10.1128/CVI.00131-10. [78] Boyaka PN. Inducing mucosal IgA: a challenge for vaccine adjuvants and delivery systems. J Immunol 2017;199:9 16. Available from: https://doi.org/ 10.4049/jimmunol.1601775. [79] Belyakov IM, Ahlers JD. What role does the route of immunization play in the generation of protective immunity against mucosal pathogens? J Immunol 2009;183:6883 92. Available from: https://doi.org/ 10.4049/jimmunol.0901466. [80] Seo KY, Han SJ, Cha H-R, Seo S-U, Song J-H, Chung S-H, et al. Eye mucosa: an efficient vaccine delivery route for inducing protective immunity. J Immunol 2010;185:3610 19. Available from: https://doi.org/ 10.4049/jimmunol.1000680. [81] Nesburn AB, Bettahi I, Zhang X, Zhu X, Chamberlain W, Afifi RE, et al. Topical/mucosal delivery of subunit vaccines that stimulate the ocular mucosal immune system. Ocul Surf 2006;4:178 87. [82] Kim E-D, Han SJ, Byun Y-H, Yoon SC, Choi KS, Seong BL, et al. Inactivated eyedrop influenza vaccine adjuvanted with poly(I:C) is safe and effective for inducing protective systemic and mucosal immunity. PLoS One 2015;10:e0137608. Available from: https://doi. org/10.1371/journal.pone.0137608. [83] Nesburn AB, Ramos TV, Zhu X, Asgarzadeh H, Nguyen V, BenMohamed L. Local and systemic B cell and Th1 responses induced following ocular mucosal delivery of multiple epitopes of herpes simplex virus type 1 glycoprotein D together with cytosine phosphate guanine adjuvant. Vaccine 2005;23:873 83. Available from: https://doi.org/ 10.1016/j.vaccine.2004.08.019. [84] Farid M, Agrawal A, Fremgen D, Tao J, Chuyi H, Nesburn AB, et al. Age-related defects in ocular and nasal mucosal immune system and the immunopathology of dry eye disease. Ocul Immunol Inflamm 2014;1 21. Available from: https://doi.org/10.3109/ 09273948.2014.986581. [85] Friedman MG, Kimmel N. Herpes simplex virusspecific serum immunoglobulin a: detection in patients

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

with primary or recurrent herpes infections and in healthy adults. Infect Immun 1982;37:374 7. Ashley RL, Crisostomo FM, Doss M, Sekulovich RE, Burke RL, Shaughnessy M, et al. Cervical antibody responses to a herpes simplex virus type 2 glycoprotein subunit vaccine. J Infect Dis 1998;178:1 7. Belshe RB, Heineman TC, Bernstein DI, Bellamy AR, Ewell M, van der Most R, et al. Correlate of immune protection against HSV-1 genital disease in vaccinated women. J Infect Dis 2014;209:828 36. Available from: https://doi.org/10.1093/infdis/jit651. Petro C, Gonza´lez PA, Cheshenko N, Jandl T, Khajoueinejad N, Be´nard A, et al. Herpes simplex type 2 virus deleted in glycoprotein D protects against vaginal, skin and neural disease. eLife 2015;4:e06054. Available from: https://doi.org/10.7554/eLife.06054. Shin H, Iwasaki A. A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature 2012;491:463 7. Available from: https:// doi.org/10.1038/nature11522. Larsen IV, Clausius H, Kolb AW, Brandt CR. Both CD8 1 and CD4 1 T cells contribute to corneal clouding and viral clearance following vaccinia virus infection in C57BL/6 mice. J Virol 2016;90:6557 72. Available from: https://doi.org/10.1128/JVI.00570-16. Herzog C. Influence of parenteral administration routes and additional factors on vaccine safety and immunogenicity: a review of recent literature. Expert Rev Vaccines 2014;13:399 415. Available from: https://doi.org/10.1586/14760584.2014.883285. Clements JD, Freytag LC. Parenteral vaccination can be an effective means of inducing protective mucosal responses. Clin Vaccine Immunol 2016;23:438 41. Available from: https://doi.org/10.1128/CVI.00214-16. Yawn BP, Wollan PC, St Sauver JL, Butterfield LC. Herpes zoster eye complications: rates and trends. Mayo Clin Proc 2013;88:562 70. Available from: https://doi.org/10.1016/j.mayocp.2013.03.014. Royer DJ, Gurung HR, Jinkins JK, Geltz JJ, Wu JL, Halford WP, et al. A highly efficacious herpes simplex virus 1 vaccine blocks viral pathogenesis and prevents corneal immunopathology via humoral immunity. J Virol 2016;90:5514 29. Available from: https://doi. org/10.1128/JVI.00517-16. Badamchi-Zadeh A, McKay PF, Holland MJ, Paes W, Brzozowski A, Lacey C, et al. Intramuscular immunisation with chlamydial proteins induces Chlamydia trachomatis specific ocular antibodies. PLoS One 2015;10: e0141209. Available from: https://doi.org/10.1371/ journal.pone.0141209. Halford WP. Antigenic breadth: a missing ingredient in HSV-2 subunit vaccines? Expert Rev Vaccines 2014;13:691 710. Available from: https://doi.org/ 10.1586/14760584.2014.910121.

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[97] Lewis GK, DeVico AL, Gallo RC. Antibody persistence and T-cell balance: two key factors confronting HIV vaccine development. Proc Natl Acad Sci U S A 2014;111:15614 21. Available from: https://doi.org/ 10.1073/pnas.1413550111. [98] Amanna IJ, Slifka MK. Mechanisms that determine plasma cell lifespan and the duration of humoral immunity. Immunol Rev 2010;236:125 38. Available from: https://doi.org/10.1111/j.1600-065X.2010.00912.x. [99] Casadevall A. Antibody-mediated immunity against intracellular pathogens: two-dimensional thinking comes full circle. Infect Immun 2003;71:4225 8. Available from: https://doi.org/10.1128/IAI.71.8.42254228.2003.

[100] Mallery DL, McEwan WA, Bidgood SR, Towers GJ, Johnson CM, James LC. Antibodies mediate intracellular immunity through tripartite motifcontaining 21 (TRIM21). Proc Natl Acad Sci 2010;107:19985 90. Available from: https://doi. org/10.1073/pnas.1014074107.

Further Reading Hattori T, Takahashi H, Dana R. Novel insights into the immunoregulatory function and localization of dendritic cells. Cornea 2016;35(Suppl. 1):S49 54. Available from: https://doi.org/10.1097/ICO.0000000000001005.

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Mucosal Regulatory System for the Balanced Immunity in the Middle Ear and Nasopharynx Mucosal Immunity of Middle Ear and Nasopharynx Hideyuki Kawauchi Department of Otorhinolaryngology, Faculty of Medicine, Shimane University, Izumo City, Japan

I. INTRODUCTION Bacteria and their components, such as lipopolysaccharide (LPS) or teichoic acid (TA), induce middle ear or nasopharyngeal inflammation, which can become a so-called vicious circle in the auditory tube and tympanic cavity and the paranasal sinus. Innate and adaptive immune cells recognize microbes via various innate and antigen-specific receptors, respectively. The former includes toll-like receptors (TLRs) expressed on dendritic cells, macrophages, endothelial cells, and γδ T cells. Once the ostium blockade occurs, mucosal swelling and paranasal sinus inflammation can persist. The vicious circle in the middle ear cleft or paranasal sinus had been proposed and

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00018-3

classically explained by the complement pathway. A revised consideration now takes into account the interactions between microbial products and TLRs present on resident epithelial cells and/or recruited inflammatory cells. These various TLR-expressing cells can secrete different inflammatory cytokines and/or chemokines in response to the continuous stimulation by molecules with pathogen-associated molecular patterns (PAMPs) [1 4]. In this chapter, innate and adaptive immunity of the middle ear and nasopharynx are discussed. We discuss the clinical impact of mucosal regulatory system to modify inflammatory diseases such as otitis media and allergic rhinitis, using a number of our experimental studies as examples.

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II. INNATE AND ACQUIRED IMMUNITY OF MIDDLE EAR AND NASOPHARYNX A. Distribution of Toll-Like Receptors in Human Epithelial Cells in the Middle Ear and Changes That Result From the Ensuing Middle Ear Inflammatory Response to Microbial Infection At least ten different TLRs have been detected on the epithelial cell surface in the human middle ear. TLRs contain an N-terminal extracellular leucine-rich repeat domain and a cytoplasmic toll/IL-1 receptor domain. TLR molecules provide protection against infection by recognizing infectious agents through their invariant PAMPs, resulting in the mobilization of appropriate immune defenses [5,6]. The activation of most TLRs results in downstream activation of the mitogen-activated protein kinase or the nuclear factor kappa B (NF-κB)-dependent cell signaling cascades, thus leading to further activation of immune responses [7]. In otitis media, recognition of nontypable Haemophilus influenzae (NTHi) is associated with several PAMPs acting as TLR ligands. NTHi cell surface peptidoglycans and the associated proteins, such as outer membrane protein P6, serve as TLR2 ligands [8]. Another notable example is lipooligosaccharide, which serves as a ligand for TLR2 and TLR4 [9]. Not surprisingly, polymorphisms in the gene encoding for TLR4 have been associated with recurrent acute otitis media [10]. When infected with NTHi, TLR4knockout mice exhibit a depressed mucosal immune response compared to wild-type mice [11]. These TLR4-knockout mice display inferior immune responses with regard to mucosal IgA, systemic IgG, and T helper 1 (Th1) cells [11]. NTHi and its various immunogenic molecules have been shown not only to directly activate

TLR, but also to upregulate TLR2 gene expression in middle ear epithelial cell lines [12]. Patients with chronic middle ear disease, such as chronic secretory otitis media (CSOM), show lower mRNA levels for TLR4, TLR5, and TLR7 than the control group [13]. A recent report has further confirmed these findings, demonstrating lower mRNA and protein levels for TLR2, TLR4, and TLR5 in the middle ear mucosa of CSOM patients [14]. The downregulation of TLR expression during otitis media can lead to inefficient host defense in the middle ear. This can cause repeated infections and persistent inflammation, eventually leading to recurrent, persistent chronic middle ear diseases.

B. Distribution of Toll-Like Receptors in Human Epithelial Cells in Nasopharyngeal Mucosae and Its Modification of Type I Allergic Inflammation in Nasal Mucosae In our recent studies, we examined the role of TLRs of upper respiratory tract mucosal epithelial cells in chemokine (interleukin-8, IL-8) and cytokine (interleukin-15, IL-15) induction and intracellular signaling pathway and modification of inflammatory response by antiinflammatory agents. Northern blot analysis and reverse transcription polymerase chain reaction (RT-PCR) were done to determine the TLR distribution by cultured human nasal epithelial cells (HNECs). Both TLR4 and TLR 9 mRNA expression were undetectable. Respiratory epithelial cells constitutively expressed mRNA for TLR2, TLR 3, and TLR6 but not for TLR4 and TLR9 (Fig. 18.1). Northern blot analysis revealed IL-15-specific mRNA being strongly expressed after lipoprotein stimulation. In contrast, it was not found following TLR4 agonist stimulation. Lipoprotein significantly induced IL-8 production by both A549 cells and HNECs, whereas

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II. INNATE AND ACQUIRED IMMUNITY OF MIDDLE EAR AND NASOPHARYNX

FIGURE 18.1 Expression of TLR mRNA by macrophages and nasal epithelial cells. Expression of TLR mRNA by A549 cells, HNEC-1, and HNEC-2 was examined by RT-PCR. Both A549 cells and HNECs showed TLR2 and TLR3 mRNA expression; however, HNECs expressed no TLR4 mRNA. A549 cells and HNEC1 expressed TLR6 mRNA, but A549 cells and HNECs expressed no TLR9 mRNA. TLR1, TLR5, TLR7, TLR8, and TLR10 mRNAs were also not expressed by A549 cells and HNECs. U937, macrophage lineage cells used as controls, expressed markedly high levels of TLR2, TLR3, TLR4, TLR6, and TLR9 mRNA. RT-PCR analysis of beta-actin expression confirmed the quality of all RNA preparations used for RT-PCR. No band was detected in the non-RT sample by PCR.

stimulation of these cells with lipid A resulted in no induction of IL-8 production (Fig. 18.2). The IL-15 concentration in the supernatants of CCL185 cells was also upregulated after lipoprotein stimulation in a dose-dependent manner. Lipoprotein induced IL-15 and IL-8 production by respiratory epithelial cells, which are strictly dependent upon TLR2 stimulation.

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Lipoprotein-induced IL-15 production by respiratory epithelial cells was abolished upon NF-κB inhibition. Exposure to airborne endotoxin in infancy may protect against asthma by promoting enhanced Th1-type response and tolerance to allergens. On the other hand, later in life, exposure to airborne endotoxin adversely affects patients with asthma. Mast cells, the key player for eliciting allergic rhinitis, produce Th2 cytokines subsequent to in vitro LPS stimulation via TLR4, but in vivo studies remain to be performed [15 17]. LPS inhalation exacerbates allergic airway inflammation by activating mast cells via TLR4 and promoting Th2 responses. To investigate the effect of LPS on eliciting murine allergic rhinitis, an experimental model was tested. At the elicitation phase, ovalbumin (OVA) was administered nasally for 7 consecutive days without or with LPS, and on the final challenge, sneezing rates were measured along with nasal tissue analysis and Th2 cytokine. As a result (Fig. 18.3), LPS aggravated the induction of type 1 allergic reaction [18]. Furthermore, a significant difference in sneezing rates between C3H/HeN mice challenged with OVA alone and those challenged with OVA plus LPS was found, but this difference was not detected in TLR4mutant C3H/HeJ mice. Eosinophil infiltration was more prominent in C3H/HeN mice challenged with OVA and LPS in comparison to that in mice challenged with OVA alone. By Western blot analysis, IL-5, IL-10, and IL-13 expression was detected in both groups, but IL-5 expression was upregulated in mice challenged with OVA plus LPS. However, there was no significant difference in eosinophil infiltration and Th2 cytokine expression between C3H/HeJ mice challenged with OVA alone and OVA plus LPS. Collectively, these data suggest that LPS aggravates nasal symptoms, upregulating Th2 cytokine production of mast cells in a TLR4dependent fashion.

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FIGURE 18.2 IL-8 production from A549, HNEC-1, and HNEC-2 cells activated with lipoprotein or lipid A. IL-8-producing activities in A549 cells and HNECs after stimulation with lipoprotein (as a TLR2 ligand) and lipid A (as a TLR4 ligand) were examined. Lipoprotein significantly induced IL-8 production in both A549 cells and HNECs, whereas stimulation of these cells with lipid A resulted in no induction of IL-8 production.

FIGURE 18.3 Effect of intranasal LPS administration at eliciting phase of allergic rhinitis in murine model. At the elicitation phase, ovalbumin (OVA) antigens alone or with LPS were introduced intranasally for 7 consecutive days. On the final challenge, sneezing rates were counted in a 10-min period. A significant difference in sneezing rates between C3H/ HeN mice challenged with OVA alone and those challenged with OVA and LPS was found.

III. IMMUNOMODULATION OF MIDDLE EAR AND NASOPHARYNGEAL MUCOSAE AND ITS CLINICAL IMPACT The pathogenesis of acute otitis media or otitis media with effusion (OME) was extensively elucidated in the 1980s and 1990s. Bacterial or

viral infections and their products in the middle ear were found to be responsible for triggering middle ear acute otitis media or OME [19 22]. Suppression of immune-mediated OME by mucosa-derived suppressor T cells was first reported by Ueyama et al. [23], suggesting a mucosal regulatory system via antigen-specific

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IV. INNOVATIVE IMMUNOTHERAPY FOR ATTENUATING NASAL SYMPTOMS

immunomodulation. In their study, the effects of mucosa-derived suppressor T cells in mice were investigated upon induction of IgGmediated OME, since antigen antibody reactions in the tympanic cavity are pathogenic mechanisms of OME. Splenic T suppressor cells from orally OVA-dosed C3H/HeN female mice were transferred intravenously to syngeneic mice. The recipients were then immunized intraperitoneally with OVA and later challenged with OVA into the tympanic cavity. Nine of ten control mice, which received splenic T cells from saline-fed mice, developed OME, while OME was seen in only one of ten recipients receiving splenic T suppressor cells from OVA-fed mice. The results showed that IgG-mediated OME can be suppressed to a certain extent by the induction of antigen-specific, mucosa-derived T suppressor cells. Allergic rhinitis is well elucidated as a type 1 allergy of the nasal mucosa. Sublingual immunotherapy (SLIT) has been recently employed as a painless and effective therapeutic treatment modality for allergic rhinitis. So far, its mechanism of action has been elucidated by using immune serum and lymphocytes in an antigenspecific fashion [24 26]. Because of the limitations in sampling human tissues, there is still controversy among many reports between clinical efficacy and experimental results. To ascertain the mechanism of action, further studies in promising rodents and nonhuman primates need to be pursued. To this end, we successfully developed an effective murine model for SLIT for allergic rhinitis. Mice were treated with OVA sublingually followed by intraperitoneal sensitization and nasal challenge [27 28]. Sublingually treated mice showed significantly decreased OVA-specific IgE responses as well as suppressed Th2 cell immune responses. Nasal symptoms and eosinophil recruitment into the nasal mucosa were significantly diminished in sublingually treated mice. Sublingual OVA administration did not alter the frequency of CD41CD251 regulatory T cells (Tregs), but led

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to upregulation of Foxp3- and IL-10-specific mRNAs in the Tregs of the cervical lymph nodes (CLNs), which strongly suppressed Th2-type cytokine production from CD41CD252 effector T cells in vitro. Furthermore, sublingual administration of plasmids, encoding the lymphoid chemokines CCL19 and CCL21-Ser DNA together with OVA, suppressed allergic responses. These results suggest that IL-10-expressing CD41CD251Foxp31 Tregs in the CLNs are involved in the suppression of allergic responses and that CCL19/CCL21 may contribute to it in mice that received SLIT.

IV. INNOVATIVE IMMUNOTHERAPY FOR ATTENUATING NASAL SYMPTOMS OF CEDAR POLLINOSIS VIA THE MUCOSAL ROUTE WITH TRANSGENIC RICE SEEDS CONTAINING HYPOALLERGENIC CRYJ1 AND CRYJ2 T CELL EPITOPES Allergen-specific subcutaneous immunotherapy with Japanese cedar pollen extract has been employed to desensitize patients with cedar pollinosis. SLIT for allergy to Japanese cedar pollen is allowed in Japan and has been determined to be a clinically safe and effective method of treatment. However, its mechanism of action remains to be determined, and the question of adverse consequences of SLIT needs to be further studied. We examined the effect of sublingual administration of protein bodies (PB) of transgenic (Tg) rice seeds (see further description of plant-based vaccines and immunotherapeutics in Chapters 20: Plant-Based Mucosal Vaccine Delivery Systems and Chapter 21: Plant-Based Mucosal Immunotherapy: Challenges for Commercialization) expressing hypoallergenic whole T cell epitopes of Cryj1 and Cryj2 (PB Tg rice) in a murine model of cedar pollinosis [29]. BALB/c mice were

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sublingually dosed with PB Tg rice, followed by an intraperitoneal sensitization and nasal challenge of cedar pollen extract. For sublingual treatment, 20 or 100 mg of PB Tg rice powder was introduced on the sublingual mucosae of anesthetized mice every 4 days for 3 weeks. For an induction of a murine model of cedar pollinosis, mice were systemically sensitized by intraperitoneal injections of 100 μg of Cryj antigens with 5 mg of alum once a week for 3 weeks and then were intranasally challenged with 1 mg of Cryj for 14 days. Clinical symptoms were evaluated by counting the number of sneezes and scratches in 2 minutes at the final intranasal challenge of Cryj antigens. For an analysis of cytokine production from CLNs or spleen cells, mononuclear cells were cultured in vitro, and cytokine levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Gene expression was analyzed by a quantitative real-time RT-PCR. The number of

sneezes after the final intranasal challenge in sublingually treated mice with 20 and 100 mg of PB Tg rice powder were significantly diminished when compared to mice that had been given no sublingual treatment (Fig. 18.4). Nasal scratching also decreased among sublingually treated mice. Corresponding histopathological findings showed that the number of eosinophils infiltrating into the nasal mucosa decreased and that damage to the epithelium was less in the sublingually treated mice (Fig. 18.5). Cryjspecific cytokine productions by cultured spleen cells were evaluated by an ELISA. IL-13 levels in the splenic culture supernatants were significantly reduced subsequent to sublingual treatment, but no difference in IL-5 production was observed. CLN IL-13 and IL-5 production diminished significantly in sublingually treated mice with concomitant increase in interferon gamma (IFNγ) production (Fig. 18.6). The results from testing this murine model of cedar

FIGURE 18.4 The effect of sublingual administration of protein body fraction from Tg rice in a murine model of cedar pollinosis. BALB/c mice were sublingually administered PB Tg rice followed by an intraperitoneal sensitization and nasal challenge of cedar pollen extract. For sublingual treatment, 20 or 100 mg of PB Tg rice powder was introduced on the sublingual mucosae of anesthetized mice every 4 days for 3 weeks. For an induction of a murine model of cedar pollinosis, mice were systemically sensitized by intraperitoneal injections of 100 μg of Cryj antigens with 5 mg of alum once a week for 3 weeks and then were intranasally challenged with 1 mg of Cryj for 14 days. Clinical symptoms were evaluated by counting the number of sneezes and scratches in 2 min at the final intranasal challenge of Cryj antigens. Sublingual immunotherapy with 20 and 100 mg of Tg rice seed powder containing protein bodies significantly attenuated the number of scratches and sneezes. III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

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V. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

FIGURE 18.5 Number of eosinophils infiltrating into the nasal mucosa in a murine model of cedar pollinosis. Sublingual immunotherapy with 20 and 100 mg of Tg rice seed powder containing protein bodies significantly decreased the number of eosinophils infiltrating into nasal mucosa.

IL-13

IL-5

(pg/mL)

600

IFN-γγ

(pg/mL)

(pg/mL)

90

90

** 400

60

60

*

* 200

30

30

** 0

**

0 Control sl. 20 mg

sl. 100 mg

0 Control sl. 20 mg

sl. 100 mg

Control sl. 20 mg

sl. 100 mg

* **

pollinosis showed its suitability in recapitulating nasal symptoms and examining the effect of SLIT. Sublingually treated mice with PB Tg rice powder showed significantly decreased nasal symptoms and suppressed Th2-type immune responses in the nasal mucosa and draining CLNs. These results suggest that sublingual administration with PB-Tg rice expressing whole T cell epitopes of Cryj1 and Cryj2 suppress cedar pollinosis in this murine model.

V. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Notably, TLRs expressed on epithelial cells, mast cells, and macrophages residing in the

FIGURE 18.6 Cryj-specific cytokine production of T cells from CLN cells in murine model of cedar pollinosis. Cryj-specific cytokine production by cultured CLN cells was evaluated by an ELISA. IL-13 and IL-5 production were reduced in sublingually treated mice, while interferon gamma (IFNγ) production increased.

P < .05 P < .01

upper respiratory tract mucosae have an important role in the pathogenesis of persistent inflammation in the nasopharyngeal cavity and middle ear cleft. Persistent inflammation of the paranasal sinus or middle ear might be explained by such an interaction between bacterial products and TLRs on residing on epithelial cells and/or recruited inflammatory cells. Innate immune cells can nonspecifically remove nasopharyngeal or middle ear pathogens recognized by the TLRs expressed on epithelial cells and/or recruited inflammatory cells into the paranasal sinus or middle ear. To this end, our results may lead us to new therapeutic strategies that may include immunotherapies, TLR antagonists, signal transduction inhibitors, or

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antisense therapy to downregulate the stagnant inflammation in nasopharyngeal or tubotympanal mucosae. I have no conflict of interest with anyone as regard this manuscript preparation.

[13]

[14]

References [1] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783 801. [2] Medzhitov R, Preston-Hurlburt P, Janeway Jr. CA. A human homologue of the Drosophila toll protein signals activation of adaptive immunity. Nature 1997;388: 394 7. [3] Verstak B, Hertzog P, Mansell A. Toll-like receptor signaling and the clinical benefits that lie within. Inflamm Res 2007;56:1 10. [4] Akira S. Mammalian toll-like receptors. Curr Opin Immunol 2003;15:5 11. ´ [5] Szczepanski M, Szyfter W, Jenek R, et al. Toll-like receptors 2, 3 and 4 (TLR-2, TLR-3 and TLR-4) are expressed in the microenvironment of human acquired cholesteatoma. Eur Arch Otorhinolaryngol 2006;263 (7):603 7. [6] McClure R, Massari P. TLR-dependent human mucosal epithelial cell responses to microbial pathogens. Front Immunol 2014;5:386. [7] Lee HY, Takeshita T, Shimada J, et al. Induction of beta defensin 2 by NTHi requires TLR2 mediated MyD88 and IRAK-TRAF6-p38MAPK signaling pathway in human middle ear epithelial cells. BMC Infect Dis 2008;8:87. [8] Moon SK, Woo JI, Lee HY, et al. Toll-like receptor 2-dependent NF-kappa-B activation is involved in nontypeable Haemophilus influenzae-induced monocyte chemotactic protein 1 upregulation in the spiral ligament fibrocytes of the inner ear. Infect Immun 2007;75 (7):3361 72. [9] Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 2011;34:637 50. [10] Kumar S, Ingle H, Prasad DV, Kumar H. Recognition of bacterial infection by innate immune sensors. Crit Rev Microbiol 2013;39:229 46. [11] Song DH, Lee JO. Sensing of microbial molecular patterns by toll-like receptors. Immunol Rev 2012;250:216 29. [12] Chen R, Lim JH, Jono H, Gu XX, Kim YS, Basbaum CB, et al. Nontypeable Haemophilus influenzae lipoprotein P6 induces MUC5AC mucin transcription via TLR2TAK1-dependent p38 MAPK-AP1 and IKKbeta-

[15] [16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

IkappaBalpha- NF-kappaB signaling pathways. Biochem Biophys Res Commun 2004;324:1087 94. DeMaria TF, Apicella MA, Nichols WA, Leake ER. Evaluation of the virulence of nontypeable Haemophilus influenzae lipooligosaccharide htrB and rfaD mutants in the chinchilla model of otitis media. Infect Immun 1997;65:4431 5. Emonts M, Veenhoven RH, Wiertsema SP, HouwingDuistermaat JJ, Walraven V, de Groot R, et al. Genetic polymorphisms in Immuno response genes TNFA, IL6, IL10, and TLR4 are associated with recurrent acute otitis media. Pediatrics 2007;120:814 23. Reed CE, Milton DK. J Allergy Clin Immunol 2001;108 (2):157 66. Murakami D, Yamada H, Yajima T, Masuda A, Komune S, Yoshikai Y. Lipopolysaccharide inhalation exacerbates allergic airway inflammation by activating mast cells and promoting Th2 responses. Clin Exp Allergy 2007;37(3):339 47. Yamashita M, Nakayama T. Regulation of allergic airway inflammation through toll-like receptor 4 mediated modification of mast cell function. J Pharmacol Sci 2008;106(3):332 5. Kawauchi H, Goda K, Tongu M, et al. Short review on sublingual immunotherapy for patients with allergic rhinitis: from bench to bedside. Adv Otorhinolaryngol 2011;72:103 6. Ichimiya I, Kawauchi H, Mogi G. Analysis of immunocompetent cells in the middle ear mucosa. Arch Otolaryngol Head Neck Surg 1990;116(3):324 30. Kodama S, Hirano T, Suenaga S, Abe N, Suzuki M. Eustachian tube possesses immunological characteristics as a mucosal effector site and responds to P6 outer membrane protein of nontypeable Haemophilus influenzae. Vaccine 2006;24(7):1016 27. Kodama S, Suenaga S, Hirano T, Suzuki M, Mogi G. Induction of specific immunoglobulin A and Th2 immune responses to P6 outer membrane protein of nontypeable Haemophilus influenzae in middle ear mucosa by intranasal immunization. Infect Immun 2000;68(4):2294 300. Heikkinen T, Ruuskanen O, Waris M, Ziegler T, Arola M, Halonen P. Influenza vaccination in the prevention of acute otitis media in children. Am J Dis Child 1991;145(4):445 8. Ueyama S, Kawauchi H, Mogi G. Suppression of immune-mediated OME by mucosa-derived suppressor T cells. Arch Otolaryngol Head Neck Surg 1988;114 (8):878 82. Fujimura T, Yonekura S, Taniguchi Y, et al. The induced regulatory T cell level, defined as the proportion of IL-10 Foxp31 cells among CD251CD41 leukocytes, is a potential therapeutic biomarker for

III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

REFERENCES

sublingual immunotherapy: a preliminary report. Int Arch Allergy Immunol 2010;153(4):378 87. [25] Shamji MH, Durham SR. Mechanisms of immunotherapy to aeroallergens. Clin Exp Allergy 2011;41(9):1235 46. [26] Yamanaka KI, Yuta A, Kakeda M, et al. Induction of IL-10-producing regulatory T cells with TCR diversity by epitopespecific immunotherapy in pollinosis. J Allergy Clin Immunol 2009;124(4):842 5. [27] Yamada T, Tongu M, Goda K, Aoi N, Morikura I. Sublingual immunotherapy induces regulatory function of IL-10-expressing CD41CD251Foxp31 T cells of

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cervical lymph nodes in murine allergic rhinitismodel. J Allergy 2012;. Available from: https://doi.org/ 10.1155/2012/490905 Article ID 490905, 11 pages. [28] Qu Y, Tongu M, et al. Sublingual immunotherapy induces regulatory function of IL-10 expressing CD41 CD251 Foxp31 T cells of cervical lymph nodes and actually attenuates nasal symptoms upon allergen exposure in murine allergic rhinitis model. JJIAO 2013;31(2):49 50. [29] Takagi H, Takaiwa F. Production of rice seed-based allergy vaccines. Vaccine Design 2016;713 21.

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Current and New Approaches for Mucosal Vaccine Delivery Joon Haeng Rhee Clinical Vaccine R&D Center and Department of Microbiology, Chonnam National University, South Korea

I. INTRODUCTION Mucosal surfaces are the interface between the host’s internal milieu and the external environment, and they have dual functions, serving as physical barriers to foreign bodies and pathogenic microbes and providing the foundation for crucial survival functions such as uptake of air and nutrients, reproduction, and perception of signals. The protection of mucosal surfaces is ensured by the specialized mucosal-associated lymphoid tissues (MALTs). Mucosal vaccines, in contrast to parenteral vaccines, generally induce more efficacious protective immune reactions by inducing secretory IgA responses and cell-mediated immunity in mucosal tissues and portals of entries of mucosal pathogens. For food components and inert materials in breathing air, the MALT should remain tolerant so as not to cause unnecessary inflammatory responses. Despite the many advantages of mucosal vaccines, there are only limited numbers of licensed mucosal vaccines. Almost all licensed mucosal vaccines are composed of

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00019-5

whole components of pathogens, either live or dead. There is no successful subunit mucosal vaccine so far. Live attenuated or whole cell (WC) killed vaccines are not formulated with any specific adjuvant or delivery system. In those vaccines, pathogen-associated molecular patterns (PAMPs) play the role of built-in adjuvants, and cell corpuscles serve as delivery systems for protective antigens. There could be many reasons for the sluggish progress of development of mucosal vaccines. Concerns about safety are the most prominent reason. Mucosal surfaces are continuously exposed to environmental and food antigens and allergens, and inflammatory immune responses against mucosal vaccine antigens would result in sustained pathologic inflammation. In the case of nasal vaccination, the nasal cavity is separated from the central nervous system by a thin partition, and olfactory nerves are directly projected from the brain to cavity. The scarcity of optimal delivery systems is another reason for the slow progress. In the mucosal environment, there are many physicochemical conditions that

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would interfere with proper stimulation of immune cells by antigens and adjuvants. In the case of oral administration, to be taken up microfold (M) cells in the distal jejunum and ileum, antigens should be able to accommodate very low pH in the stomach and a sudden alkaline surge in the duodenum, and they should be able to resist proteolytic attacks of digestive enzymes. In contrast to the oral route, antigens delivered intranasally do not experience that dramatic fluctuation in pH, but they should be able to survive profuse mucosal secretions, mucociliary clearance, and the relative inefficiency of antigen uptake by antigen-presenting cells (APCs). For more efficient delivery of mucosal vaccines, many new delivery systems TABLE 19.1

based on nanotechnology and biomaterials have been studied, but very few of them have been approved for clinical use. More vigorous clinically oriented research is needed [1,2].

II. NANO/MICROSCALE CARRIERS AS PROMISING DELIVERY TOOLS FOR VACCINES In vaccine formulations currently approved or under clinical trials, nanoscale (,1000 nm) carriers are already in use [3]. Current nanotechnology and nanocarriers on the market or in the literature are summarized in Table 19.1 and Fig. 19.1. They include virus-like particles

Current Nanotechnology and Nanocarriers Used for Vaccine Delivery

Technology or Nanocarrier

Example (Antigens or Carriers)

Reference

Virus vector

Adenovirus, vaccinia virus Ankara (MVA), canary pox, yellow fever virus, pox virus, vesicular stomatitis virus, measles virus

Humphreys and Sebastian (2018)

Virus-like particles (VLPs) and virosomes

Hepatitis A/B/E virus, human papillomavirus (HPV), influenza, human immunodeficiency virus (HIV), norovirus, respiratory syncytial virus (RSV), SARS-CoV

[3], Fuenmayor, Godia et al. (2017)

Emulsions

MF59, AS03, AS02, Montanide, GLA-SE

[4], O’Hagan and Fox (2015)

Immunostimulating complexes

ISCOM

[5]

Monophosphoryl lipid A

AS04, AS02, DETOX, Melacine

[3]

Calcium phosphate nanoparticles

CaP, anthrax, hepatitis B virus, influenza

He et al. (2000)

Polymeric nanoparticles

PLG, PLA, PLGA, chitosan

Cordeiro et al. (2015)

Liposomes

Hepatitis virus B/C, RSV, influenza, Burkholderia, Candida, malaria, leishmaniasis

De Serrano and Burkhart (2017)

Proteosomes

Neisseria meningitides, Shigella, Haemophilus influenzae type b (Hib), Streptococcus pneumonia, influenza

Fries et al. (2001), [3], Burt et al. (2011)

Cholesterol-bearing pullulan nanoparticles

Cholesteryl group-bearing pullulan (CHP) nanogel

[6]

Self-assembled peptides

Self-assembled peptide nanostructures with adjuvanticity as well as antigenicity

[7]

SCOMATRIX

Herpes simplex virus

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FIGURE 19.1

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Different micro/nanocarriers that could be applied to the development of mucosal vaccine delivery

systems.

(VLPs), emulsions, liposomes, immunostimulating complexes (ISCOMs), polymeric and nondegradable nanoparticles (NPs), and nanogels [8]. Some of the NPs are able to enter APCs by diverse pathways, thereby differentially modulating downstream immune responses. Moreover, the nano-based delivery systems are also able to carry antigens and specific adjuvants such as TLR ligands simultaneously in the same carriers; carriers by themselves sometimes exert adjuvant activities. The nanoscale vaccine carrier systems generally constitute three key components: an antigen, against which adaptive immune responses are induced; an adjuvant, to potentiate the interaction between innate and adaptive immune systems in reacting to the antigen(s); and a delivery or targeting system to ensure that the antigen(s) and adjuvant(s) are delivered together to the right location at right time [9].

In this context, many effective mucosal delivery systems using the nano/microscale carriers have been very actively researched up to clinical trial levels in recent years. Viral-vectored vaccines and live or killed virus vaccines by themselves are already nanocarriers with built-in PAMP adjuvants. VLPs and virosomes behave similarly to viruses in stimulating immune responses and carrying antigens in nanoscales. Some adjuvant formulations are already composed of nanoscale structures. Formulation of adjuvants with vaccine antigens became inevitable in modern vaccine development to enhance the immunogenicity of highly purified antigens that have insufficient immunostimulatory capabilities. While early adjuvants (e.g., aluminum, oil-in-water emulsions) were used empirically, rapidly increasing knowledge of how the immune system interacts with pathogens allowed better understanding

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of the role of adjuvants and how the formulation of modern vaccines can be better tailored for the desired clinical benefit [4]. Of interest, currently licensed oil-in-water emulsion adjuvants such as MF59, AS02, and AS03 comprise nanoscale structures in the formulation. Squalene is popularly incorporated in oil-inwater emulsions because of its physical and immunostimulatory properties [10]. MF59 (Novartis Vaccines & Diagnostics), AS03 (GSK Biologics), and glucopyranosyl lipid adjuvantstable emulsion (GLA-SE) (infectious disease research institute (IDRI)) have a squalene content of around 2% 4% (w/w) with additional emulsifying agents [11]. MF59 was the first oilin-water emulsion on the market produced by microfluidization and contains sorbitan trioleate and polysorbate-80 (PS80) as surfactants. MF59 has a particle size of around 160 nm [12]. AS03 contains α-tocopherol and PS80 as a surfactant. AS03 has a particle size of about 150 nm [13]. GLA-SE (IDRI) consists of a squalene emulsion in combination with GLA, which is a synthetic form of monophosphoryl lipid A (MPLA) and a potent immunopotentiator. GLA-SE has a particle size of about 100 nm [14]. It has been suggested that emulsions with particle sizes ranging from 100 to 200 nm are efficiently taken up by dendritic cells (DCs) and hence effectively stimulate immune responses against coadministered vaccine antigens [15]. TLR ligands and immunostimulatory agents such as QS21 are formulated into oil-water emulsion or liposomes to make adjuvants for vaccines against diverse infectious agents and tumor immunotherapy. ISCOMs are approximately 40-nm cage-like particles produced by combining protein antigens, cholesterol, phospholipid, and the saponin adjuvant Quil A [5]. ISCOM is composed of matrix serving traps for protein antigens. Typically, membrane antigens containing hydrophobic domains are well trapped in ISCOM through apolar interactions [16]. Liposomes are spherical carriers composed of one or more phospholipid membranes with

aqueous core. Thanks to the structure, liposomes provide a wide range of options for vaccine formulation design. Proteins, peptides, DNA, RNA, and adjuvant components can be readily encapsulated inside the aqueous core, embedded within the lipid layer, or attached to the surface by adsorption, hydrophobic anchor insertion, or covalent fusion. Liposomes by themselves are considered to be nontoxic and biodegradable when mainly phospholipids are used, since they are normal components of mammalian cell membranes and lipids are relatively nonimmunogenic. Liposomes could be designed and manufactured to have the desired physicochemical characteristics optimal for inducing desired immune responses against vaccine antigens: vesicle size, lamellarity (number of lipid layers), surface charge, bilayer fluidity, and incorporation of immunostimulatory components [16a]. Polymeric NPs are have also been robustly studied to deliver vaccine antigens and adjuvants. Polymeric NPs are submicron-sized colloidal systems of natural or synthetic polymers used as delivery carriers of chemical drugs, proteins, peptides, and nucleic acids, owing to their high bioavailability, controlled release, biodegradable and biocompatible properties, and low toxic profiles [17]. Compared with liposomes, polymeric NPs can more easily incorporate both hydrophilic and hydrophobic biomolecules and have better storage stability. The most commonly studied polymers are poly(D,L-lactide-co-glycolide) (PLG), polylactide (PLA), and poly(D,L-lactide-co-glycolide) (PLGA) [18,19]. These biodegradable, biocompatible polymers are well characterized and have been approved by the U.S. Food and Drug Administration (FDA) for use in humans because of their excellent safety profiles. They have been extensively studied for the formulation of vaccine antigens (proteins, peptides, DNA, etc.) [20]. In these formulations, antigens can be either entrapped or adsorbed to the surface of the particles and are protected

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from proteolytic degradation conferring longer half-lives in vivo. By not-so-difficult additional engineering, these particles can be regulated to degrade or to release cargos (adjuvant and/or antigens) over a wide range of rates. Additionally, polymeric particles more easily pass M cells and reach to APCs in the MALT after surviving harsh physicochemical conditions in many mucosal compartments [3,18]. PLG polymers have been evaluated for drug delivery since the early 1980s and have been used widely for pharmaceutical and medical device applications with excellent safety profiles in humans [21]. Generally, PLG forms microparticles rather than NPs. PLG microparticles received attention for vaccine delivery by the World Health Organization (WHO) Special Program for Vaccine Development from the late 1980s [22]. Triggered by this program, many antigens, such as tetanus toxoid, hepatitis B antigen, and diphtheria toxoid, were formulated with PLG microparticles and evaluated in comparison with aluminum salt adjuvants [21]. Although some promising results with PLG-based vaccines in small animal models were reported, challenges concerning antigen stability and insufficient immune responses compared with alum-adjuvanted vaccines prevented the use of PLG microparticles in commercial vaccines [23]. One more disadvantage of PLG polymer-based vaccines is their inefficiency in translocating to lymph nodes where APCs present antigens to T lymphocytes. The particle size can influence transport to specific location and cell types in the draining lymph nodes [24,25]. NPs (20 200 nm) drained to the lymph nodes and localized in the lymph-node-resident DCs and macrophages, whereas larger particles (500 2000 nm) were mostly associated with DCs at the injection site. PLG particles would be inefficient in presenting antigens to DCs in draining lymph nodes. In this regard, PLG microparticles have handicaps to being more widely applied to the development of vaccine delivery systems.

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PLA is a linear aliphatic polyester composed of lactic acid building blocks that are naturally occurring organic acids derived from sugarcane and cornstarch [9]. PLA’s physical properties can be tuned through combining racemic mixtures of these enantiomers: poly-L-lactide and poly-DL-lactide semicrystalline and amorphous polymers, respectively [26]. Thanks to their good safety profiles, PLA-based products have been approved by the FDA and the European Medicines Agency (EMA) for multiple biomedical applications. This polymer can be easily chemically modified with different ligands to improve their specificity to targeted cells [27]. In vivo, PLA is hydrolyzed into α-hydroxy acid, which is easily metabolized in the body via the Krebs cycle [28]. PLA polymers can be fabricated into both microparticles and NPs [27]. Despite its physicochemical and pharmaceutical advantages, PLA has been less intensively applied than other polymers, such as its copolymer PLGA, in clinical stage vaccine developments. Although a considerable number of reports have shown the usefulness of PLA polymer NPs as versatile vaccine carriers, the scaling-up of these laboratory methods to industrial production has faced hurdles, which are mostly related to particle size and size distribution. For further development of PLA polymers as clinical-grade vaccine delivery systems, these practical problems need to be solved in advance. Biodegradable nano/microparticles of PLGA and PLGA-based polymers are more widely explored as carriers for controlled delivery of macromolecular therapeutics such as proteins, peptides, vaccines, genes, antigens, and growth factors. PLGA is one of the most successfully developed biodegradable polymers. Among the different polymers developed to formulate polymeric NPs, PLGA has won strong attention, owing to its attractive properties: biodegradability and biocompatibility, FDA and EMA approval in drug delivery systems for parenteral administration, well-described

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formulations and methods of production adapted to various types of drugs such as hydrophilic or hydrophobic small molecules or macromolecules, protection of the drug from degradation, the possibility of sustained release, the possibility of modifying surface properties to provide stealthiness and/or better interaction with biological materials, and the possibility of targeting NPs to specific organs or cells [29]. The PLGA-based carriers’ cargo release characteristics could be relatively well controlled by modulating encapsulation, particle size, formulation additives, molecular mass, ratio of lactide to glycolide moieties in PLGA, and surface morphology [30]. The most widely used PLGA, with a monomer composition of 50:50, has the fastest biodegradation rate; it completely occurs in approximately 50 60 days. The polyglycolide acid is more hydrophilic than polylactide, owing to the absence of a methyl side group [31]. A higher glycolic acid percentage causes more water uptake and consequently faster degradation of PLGA polymers. PLGA is hydrolyzed into the original monomers lactic acid and glycolic acid, which are by-products of various metabolic pathways and are not associated with any significant toxicity except lowered pH. Next to release characteristics, various other physical traits of PLGA particles can be manipulated, including particle size, size distribution, zeta potential, polydispersity index, encapsulation efficiency, and cargo loading [32]. Among a myriad of choices in nano/microcarrier polymers, PLGA has more advantages other than those listed above: PLGA particles can be administered via diverse routes; PLGA particle formulation may dampen toxicities of vaccine components; PLGA particles could protect the antigen from degradation and allow controlled release; PLGA particles could be made to target APCs and increase cross-presentation of the antigen; and PLGA particles allow concomitant delivery of multiple vaccine components with dose sparing. While many properties of PLGA

polymers are favorable and controllable as vaccine delivery tools, there are drawbacks as well. PLGA has a negative effect on the stability of encapsulated protein antigens during preparation and storage, primarily owing to the acid-catalyzed nature of its degradation. Its hydrolysis leads to the accumulation of lactic and glycolic acids in the microenvironment, which will denature encapsulated protein antigens and consequently compromise immunogenicity [30]. In addition, processing conditions used in manufacturing PLGA carriers have negative effects on protein secondary structures [33]. To overcome problems associated with protein degradation, many efforts have been made to optimize the manufacturing process and to add excipients that would protect the protein antigens being encapsulated. Protein antigens adsorbed to PLGA particles are relatively more protected from those physicochemical insults. Adsorbed antigen would offer improved stability and activity over encapsulated antigen by avoiding exposure to organic solvents used during formulation and acidic pH conditions caused by degradation of the polymer. But this may result in premature high burst release of the antigen before uptake by APCs, which can lead to deficient immune responses [34]. Natural polymers are attractive vaccine delivery vehicles, owing to their low toxicity and biocompatibility. Synthetic polymers such as PLGA or PLA are also reported to be safe and biocompatible. Their degradation products affect the microenvironment by lowering pH which may be detrimental to functions of APCs and compromise immunogenicity of vaccine antigens. On the other hand, natural polymers are generally composed of biological components, making them physiologically resorbable with few to no adverse effects [35]. The major drawback of natural polymers as vaccine delivery systems is reproducibility, which should be overcome by further technological researches. There are two major groups of

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natural polymers that are used to manufacture particulate carriers: peptide/proteins and polyelectrolytes including alginate, chitosan, and dextran [36]. Chitosan and chitosan derivatives are cationic polymers, which, owing to their structure, have excellent mucoadhesive and absorption-promoting properties. Chitosan is manufactured by alkaline deacetylation of chitin (e.g., derived commercially from exoskeleton of crustaceans or fungi) and is a linear copolymer of β1-4 linked monomers of D-glucosamine and N-acetyl-D-glucosamine [36]. It is biodegradable and biocompatible. The pKa of the primary amine group of chitosan is approximately 6.5, and the nascent polymer at neural pH carries no charge; hence chitosan is insoluble in water. This solubility characteristic should prevent nascent chitosan from being able to deliver antigens that are soluble and stable at neutral pH. Structural modifications have been made to chitosan to produce derivatives that are soluble at neutral pH yet retain the positive charge and unique properties of nascent chitosan. Because chemical modifications make it possible to substitute both amine and hydroxyl functional groups of chitosan, various chitosan derivatives have been produced by introducing hydrophilic groups such as hydroxyalkyl, carboxyalkyl, succinyl, thiol, and sulfate or by grafting solubility enhancer polymers such as polyethylene glycol and poloxamer [37]. Of all the water-soluble derivatives, N-trimethyl and carboxymethyl derivatives of chitosan have been studied most extensively, owing to their relative ease of synthesis, ampholytic character, and ample application possibilities. Soluble N-trimethyl chitosan has both mucoadhesive properties and excellent absorption-enhancing effects even at neutral pH because of its cationic charge at neutral pH [38]. N-trimethyl chitosan is rather widely applied to the development of mucosal vaccine delivery systems, since it is mucoadhesive and has penetration-enhancing ability through the paracellular route even at neutral

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pH. Chitosan particles can be fabricated to successfully deliver both adjuvant and antigen to DCs in the tissue or draining lymph nodes [39]. Adjuvants and antigens could be either incorporated inside or adsorbed on the surface of chitosan microparticles or NPs on purpose. In addition to being a carrier, chitosan can be used to coat other polymer particles to enhance their immunogenicity, bioadhesiveness, and surface adsorption potential [36]. N-trimethyl chitosan is freely soluble over a wide pH range as compared to other chitosan derivatives and bears positive charges, independently of the environmental pH. Methylcarboxy chitosan is a polyampholytic polymer that is able to form viscoelastic gels in aqueous environments or with anionic macromolecules at neutral pH values. On the basis of these characteristics, the complexation of two chitosan derivatives without using any cross-linker could generate a vaccine delivery carrier that has high loading efficiency and can maintain integrity of a protein antigen [40]. Alginate is a linear, anionic polysaccharide found in the cell walls of brown algae. It has a high affinity for water and forms an inert and highly aqueous environment within the particle, which limits its ability to carry hydrophobic vaccine antigens and adjuvants. It is also biocompatible, biodegradable, and easily eliminated from the body [36,41]. As with chitosan particles, adsorption of adjuvant onto the surface of microspheres allows differential release of the antigen and adjuvant for temporally controlled stimulation of immune cells [42]. Alginate itself, besides its role as a vaccine carrier, has immune-stimulatory activities though stimulating NF-κB signaling pathway [43]. Dextran is a polysaccharide composed of repeating branched glucose molecules. Its most commonly used form is dextran sulfate [44], which is biocompatible and hydrophilic and decomposes into natural byproducts. Anionic dextran sulfate is often fabricated with cationic poly-L-arginine to make layer-by-layer antigen-adjuvant carriers

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[45]. Because this type of particle is assembled layer by layer, multiple antigens and adjuvants can be incorporated in multiple layers to maximize targeting and activation of immune cells. Self-assembled peptides have been reported to be useful candidates for future vaccine delivery systems [7]. Peptide molecules can be rationally designed to self-assemble into specific nanoarchitectures in response to changes in their assembly environment, including pH, temperature, ionic strength, and interactions between host and guest molecules. They could be manufactured in the forms of nanomicelles, nanovesicles, nanofibers, nanotubes, nanoribbons, and hydrogels and would have a diverse range of mechanical and physicochemical properties [46]. Peptide delivery systems may have potential advantages over liposome or NPs, since they can be composed of amphiphilic molecules with high loading efficiency, low antigen leakage, biodegradability, and high permeability to biomembranes of target cells. These molecules can be designed for cellspecific targeting by including adhesion ligands, receptor recognition ligands, or peptide-based antigens in their design, often in a multivalent display [7]. These molecules can also act as intracellular transporters and respond to changes in the physiological environment. Generally, self-assembling peptides are nonimmunogenic, serving as built-in adjuvants for fused antigenic peptides [47]. The adjuvant activity is closely related to nanostructures, since the mutation of key amino acid residues in the self-assembling domain demolishes the immunogenicity of the self-assembled peptide vaccines [48]. The adjuvanticity in a nanofiber self-assembled peptide vaccine was reported to be T-cell- and MyD88-dependent, but specific interactions with TLR2 and TLR5 as well as NALP3 were not noted, suggesting a novel immunomodulating mechanism. Although peptide nanofiber vaccines are more efficiently taken up by DCs and subsequently activate them, these vaccines do not cause

reactogenicity and nonspecific inflammatory reactions at the administration site [49]. Nanogels became more prominent recently as a vaccine delivery system. The term “nanogel” defines refers to nanoscale particles (,100 nm in diameter) composed of physically or chemically cross-linked bifunctional networks having good swelling capacity in aqueous environments [50]. Nanogels have a high cargo loading capacity, biocompatibility, and biodegradability. Cationic nanogels are adhesive to epithelial cell surface and serve as artificial chaperones protecting antigens from aggregation and denaturation. Loaded antigens are subsequently released in native forms and captured by appropriate APCs nearby [51]. The surfaces of nanogels are relatively easy to modify by specific ligands, enabling targeted delivery to specific cells or tissues. Nanogel vaccine formulations can be delivered via a wide range of routes, such as parenteral, oral, nasal, pulmonary, or ocular administration [52]. Nanogels can be formulated by various polysaccharides such as chitosan, mannan, hyaluronic acid, dextrin, cycloamylose, pullulan, and enzymatically synthesized glucogen [53]. In recent years, pullulan has played a critical role in the development of nanogel systems for vaccine and drug delivery [54]. Pullulan is an aqueous polysaccharide synthesized by the yeast-like fungus Aureobasidium pullulans. It consists of hundreds of repeated units of a maltotriose trimer. Pullulan is widely used in diverse biomedical industries because it is easily modified by rather simple chemical reactions that are nontoxic, nonmutagenic, noncarcinogenic, and, most important, nonimmunogenic [55,56]. Pullulan hydrophobized by cholesterol becomes amphiphilic and forms self-aggregates [57]. The cholesterol-bearing pullulan (CHP) can form complex NPs with various protein antigens. CHP can self-assemble in water into the NPs and encapsulate protein antigens in the internal space through hydrophobic interactions. The complex NP protects internal protein

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antigens against physicochemical or enzymatic degradation, serves as an ideal delivery vehicle, and releases payloads in a controlled fashion [58]. The most valuable characteristic of CHP nanogels is its artificial molecular chaperon activity [59]. Protein antigens are captured in denatured form in the CHP nanogel under reversible denaturation temperature or in the presence of reversible denaturation reagents [60]. In the nanomatrix, the nanogel protects denatured protein antigen as an artificial molecular chaperone and helps in proper refolding after release [61]. Another advantage of CHP is its targeting ability to APCs. The CHP nanogels and protein antigens could form colloidally stable NPs 50 nm in diameter, which is a relevant size allowing effective uptake by epithelial cells and APCs [59]. Moreover, CHP nanogels can be modified to have cationic charge (cCHP) by adding amine groups to the CHP nanogels [62]. The cCHP nanogels could be well formulated with protein antigens and effectively carry vaccine antigen to the negatively charged nasal epithelium after intranasal administration [51]. The positive charge of cCHP nanogel provides more efficient adhesion to negatively charged nasal mucus and epithelia, leading to higher level and more sustained delivery of antigens to DCs inhabiting underneath mucosa. More important, the cCHP vaccine formulation could induce significantly higher immune responses without adjuvant addition, while the cCHP nanogel itself could not activate immature DCs, suggesting no biologically active adjuvant-like activity [51,63]. The reason why cCHP, having no direct stimulatory effect on innate immune cells, could significantly enhance the immunogenicity of cargo antigens was thought to be the improved antigen residence time in the nasal cavity, which leads to better antigen transport to the nasal DCs. The 40-nm cCHP nanogels carrying Clostridium botulinum type A neurotoxin heavychain C-terminus (BoHc/A) were bound by nasal epithelial cells and subsequently

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endocytosed. The BoHc/A antigen was separated from the nanogel by protein exchange and sustainably released by exocytosis, which was subsequently taken up by CD11c1 DCs in the mucosa [51]. Epithelial cells served as a reservoir for the cargo antigen, while no overt cytotoxicity was observed. Neither the cCHP nanogel nor cargo BoHc/A antigen was taken up by the olfactory bulb or brain tissue, suggesting that the cCHP nanogel system is safe for nasal administration (Chapter 26: Nanodelivery for Mucosal Vaccines).

III. MUCOSAL VACCINE DELIVERY: PAST, PRESENT AND FUTURE A. Oral Vaccine Delivery Gastrointestinal (GI) infection is a significant global health challenge, especially in developing countries. Most GI infections are spread via the fecal oral route, primarily through contaminated water and food due to poor sanitation and social infrastructure. An efficacious vaccination policy is the most economical way of solving GI infection problems from a public health perspective. The oral vaccination is generally the best way to induce secretory immunoglobulin A (SIgA) in the GI tract and IgG antibody responses in the systemic compartment. In fact, the only oral vaccine that has been widely used globally in infants and children in a national immunization program is the oral polio vaccine (OPV) developed by Albert Sabin in the 1950s. Since Sabin’s OPV vaccination, several oral vaccines against rotavirus, Salmonella Typhi, and Vibrio cholerae have been licensed and marketed [64]. Those vaccines are made of live attenuated organism or killed microbial cells. There is as yet no licensed subunit oral vaccine on the market. There have been continuous efforts to develop oral vaccines because of the advantages of oral vaccination. As was noted during the 2010 Haitian

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cholera epidemic, oral vaccination is a faster way of containing circulating infections and prevention of further outbreaks [65]. After the September 11, 2001, terrorist attacks, the threat of biological warfare became highlighted worldwide. The potential bioterrorism agents are likely to be disseminated by either aerosol or in food or water supplies targeting the wide mucosal surfaces in the respiratory or GI tracts, respectively. Considering that the bioterrorism agents invade from the GI mucosa, oral vaccines, inducing protective immune responses at the route of entry, have generated the most interest as a frontline tool in biodefense [66]. 1. Advantages and Limitations of Oral Vaccines Oral vaccination has several advantages, such as better patient compliance, mass immunization capability, easy administration or selfdelivery, simplified production and storage, lower production cost, and no needleassociated risks such as injuries and carryover infections (Table 19.2) [67]. The most important virtues of oral vaccination are its needle-free painless administration and that there is no need for trained personnel for administration. Two major mucosal vaccination routes, oral and intranasal, are compared in Table 19.3. The most widely used oral vaccine is Sabin’s OPV, which contributed enormously to the eradication of poliomyelitis worldwide. But recently, despite its efficacy, OPV has been replaced by injectable poliovirus vaccine (IPV) in developed countries. OPV is likely to be replaced by IPV globally over several coming years. Live poliovirus was discovered in the stool of OPV vaccines, possibly spreading infectious material in the environment [68]. Another serious concern about OPV is the rare event of reversion to virulent strains in vivo, which could cause a serous iatrogenic vaccine-associated paralytic poliomyelitis [69]. The same concerns apply to other mucosal vaccines using live attenuated organisms. But generally, oral vaccines are regarded as a better choice than injectable

parenteral vaccines from production, economic, and regulatory perspectives [70]. Oral vaccines have better compliance and fewer adverse reactions. Oral vaccines are better for large-scale production and mass vaccination campaigns in developing countries, since no needles are required and self-administration is possible. Thermostabilization technologies would enable successful cold-chain-free vaccination of killed as well as live attenuated formulations in resourcepoor settings such as developing countries [71]. Thanks to the many virtues of oral vaccines and the success of OPV, research into oral vaccines has rather a long history, but only a very limited number of oral vaccine products have become available. Many oral vaccines that proved to be efficacious in preclinical studies have failed in clinical trials. Live-attenuated vaccines against rotavirus and S. Typhi have been successfully introduced into the market. In the case of cholera, two types of oral cholera vaccines (OCVs) are currently available: WC-rBS, which are killed WC monovalent (O1) vaccines with a recombinant B subunit (rBS) of cholera toxin (CT) (Dukoral), and killed modified WC bivalent (O1 and O139) vaccines without the B subunit (Shanchol, Euvichol, and mORCVAX) (WHO cholera vaccine position paper—August 2017 at www.who.int) (Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control). The three WC vaccines are based on the same cholera strains and dosage. Although the WC killed cholera vaccines listed proved to be efficacious in multiple clinical trials, many other prototype killed and subunit vaccines could not be put on the market because of suboptimal immunogenicity. Oral vaccines should have strong immune-stimulatory adjuvants and optimal delivery strategies to drive effective innate and adaptive immune responses against vaccine antigens [64]. To achieve optimal immunogenicity, Dukoral has a huge number of killed V. cholerae cells (1.25 3 1011 CFU) and 1 mg of rBS. The three bivalent WC vaccines contain 2100 LPS ELISA unit of killed cells. Vaxchora,

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TABLE 19.2 Delivery System

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Common Oral Delivery Systems and Their Advantages and Disadvantages

Application

Advantages

Disadvantages

Live attenuated, whole cell killed, proteins, peptides, conjugates

Inexpensive buffer, flexible, easy administration

Often requires the use of bicarbonate salts to neutralize gastric acid, dilution of formulation, lack of clean water in poor regions

Emulsions Whole cell killed, proteins, peptides, conjugates

Potential for different formulations to facilitate specific immunomodulation, for example, Th1 (water-in-oil) and Th2 (oil-in-water)

Does not protect vaccine from GI tract harsh environment, efficacy by the oral route uncertain, no licensed oral vaccine yet

Virus-like particles

Nonreplicating, high uptake, selfassembling, can conjugate additional molecules for targeted tissue/cell specificity and immune modulation (adjuvant)

Expensive and difficult to scale up, requires purification and often formulation with additional adjuvants, no licensed oral vaccine yet

Liposomes Proteins, DNA, peptides

Ease of surface modification, can accommodate a wide variety of antigen types, controlled release

Poor antigen loading efficiency, low stability, nonspecific interactions, toxicity of cationic liposomes, degradation by bile salts and lipases in the small intestine

ISCOMs

Proteins, peptides

Intrinsic adjuvant capabilities, ease of antigen loading and surface modification, efficient induction of CTLs

Difficulty of loading hydrophobic antigens

Synthetic and natural particles

Proteins, peptides, conjugates

Highly adaptable, can protect contents from both environmental and physiological effects, controlled release, modifiable surface chemistry, carrier size can be engineered

Low loading efficiency, manufacturing process may degrade antigens, surface antigen exposed to proteolysis, can become trapped in mucus, difficulty to scale up

Pills and capsules

Live attenuated, whole cell killed, proteins, peptides, conjugates other delivery systems

Highly adaptable, can protect contents from both environmental and physiological challenges, controlled release, two or more carriers could be coformulated, easy administration

Formulation process may damage components, loading complications

Solution

Plasmid DNA, proteins, peptides, conjugates

a newly licensed single-dose OCV, contains 2.0 3 108 to approximately 2.0 3 109 CFU of live attenuated CVD 103-HgR cells [72 74]. The reason why oral vaccines manufactured with live attenuated organisms or high-dose WC killed bacteria are on the market reflects the poor delivery efficiency and the consequent higher requirement of built-in adjuvants (PAMPs).

2. Oral Vaccine Delivery Systems A. EMULSIONS AND MICELLES

Emulsions such as MF59 and AS03 are extensively tested for many vaccines and immunization routes. However, in the literature, it is rather rare to find successful test results for oral vaccines against infectious diseases employing

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TABLE 19.3 Vaccine Route

Comparison of the Intranasal Vaccination Route With the Oral and Parenteral Routes

Advantages

Disadvantages

Intranasal Needle free, noninvasive No cross-contamination Amenable for mass vaccination Self-administration possible Increased compliance of vaccines Cost-efficient logistics Large absorption surface area Reduced risk of anaphylactic shock due to slow absorption Stimulates both mucosal and systemic responses Responses at distant mucosal sites through common mucosal system activation Avoidance of first-pass metabolism No antigen degradation in the stomach

Short nasal residence time Quick clearance by mucociliary action Need for adjuvants/delivery systems for subunit vaccines Requires higher antigen dosage than parenteral vaccination Limitations for live vaccines (e.g., certain age and risk groups) Shedding of live vaccine organisms Administration problems in vaccines with nasal obstruction

Oral

Inaccurate dosage/low bioavailability Need for adjuvants/delivery systems for subunit vaccines Requires higher antigen dosage than parenteral vaccines Site-restricted mucosal responses Gastric degradation First-pass metabolism of antigens Shedding of live vaccine organisms Administration problem in vaccines with accelerated enteric transit (e.g., diarrhea) or small children

Needle free, noninvasive No cross-contamination Amenable for mass vaccination Self-administration Increased compliance of vaccines Cost-efficient logistics Stimulates both mucosal and systemic responses Permissive to low antigen purity

Parenteral Accurate dosage Fast absorption rate No shedding Strong systemic immune responses No gastric degradation

an oil-in-water or water-in-oil emulsion formulation or adjuvant system. A successful oral tumor vaccine study showed that an antigen complex (melanoma antigen MAGE1, heat shock protein 70, and staphylococcal enterotoxin A) incorporated in a nanoemulsion with a small size of 15 25 nm induced efficacious protective immune responses comparable to those of subcutaneous administration [75,76]. Vaccine antigen can be delivered inside the core or attached on the outside to the shell of micelles, depending on the electrochemical properties of

Invasive Poor acceptance by vaccines Medical trained personnel required Reduced patient’s compliance Expensive logistics No stimulation of mucosal responses Poor protection against mucosal infection/colonization

the vaccine formulation [77]. Oral immunization of PLA-PEG-PLA and PLGA-PEG-PLGA copolymer micelles loaded with DNA encoding HCV multiple epitope antigen could induce satisfactory immune responses [78]. The copolymers showed an innate adjuvant activity and caused no significant adverse reactions [78]. Micelles can be synthesized as nanocarriers and engineered to penetrate mucus and be taken up by mucosal APCs [79]. However, micelles may have a propensity to dissociate when diluted, leading to a loss of loaded antigen.

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IV. LIPOSOMES While most clinical trials and delivery system developments employing liposomes have focused on the parenteral routes, there are still continued efforts to develop liposome-based oral delivery tools [80]. Conventional liposomes are vulnerable to acidic gastric juice and are easily digested by pancreatic lipase [81]. Also, intestinal bile salts can destroy the phospholipid membrane integrity and lyse the liposomes, resulting in the premature release of vaccine antigens [82]. To tackle these problems, researchers have investigated different lipid moieties such as archaeal lipids or bile salts in the liposomal membrane [83,84]. An important determinant of the adjuvanticity of liposomes is the surface charge. Positively charged (cationic) liposomes have been demonstrated to possess the strongest adjuvanticity compared to neutral and negatively charged liposomes [85]. Mucoadhesion is promoted by the cationic surface charge that would have a stronger interaction with negatively charged GI mucus. Cationic liposomes also better adhere to negatively charged membranes of M cells and enterocytes, limiting flushing by peristalsis and providing a better chance to be internalized [86]. However, the greater toxicity of cationic than anionic liposomes is of concern. Recently, a study reported that cationic liposomes induce necrosis to release damageassociated molecular patterns and cause inflammation in vivo [87]. To extend the clinical applications of liposomes as carriers of oral vaccines by improving stability and sustainability in GI tract mucosa, the surface modification of liposomes has been rigorously investigated [88 90].

V. IMMUNOSTIMULATING COMPLEXES The addition of cholesterol and Quil A saponin to liposomes results in the generation

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of self-assembling pentagonal dodecahedrons that have intrinsic adjuvant properties and thus earned the name ISCOMs. Two types of ISCOMs exist. The first, “classical” type was manufactured to entrap protein antigens, making them act as both a vaccine antigen delivery and an adjuvant system. The second type, referred as ISCOM matrices, contains no entrapped antigen and serves as a codelivered adjuvant, which is later formulated with vaccine antigens. Early ISCOM formulations could not be evaluated in humans and were limited to veterinary use, owing to the reactogenicity of Quil A. Quil A was later replaced with QS21, and ISCOMs could be then given clinical trials. QS21 is more purified in nature and shows an improved safety profile while remaining active as an adjuvant [16]. While ISCOMs were widely tried with very diverse antigens for parenteral and intranasal vaccination studies, trials with oral vaccines have been relatively scarce [91,92]. One major challenge in working with ISCOMs is the difficulty associated with antigen incorporation. In this context, many trials used ISCOM matrices purely as adjuvants and formulated with vaccine antigens rather than incorporating them, which elicited immune responses as beneficial as those elicited by classical ISCOMs [93]. The use of unmodified ISCOMs as adjuvants would significantly simplify production; however, the benefit of antigen encapsulation, which is an important key to success for the oral route, might be lost. For this reason, ISCOM matrix adjuvants in combination with enteric vaccine antigens are used for boosting through intranasal or parenteral routes after oral priming with antigens only or antigens with other adjuvants [92,94 96]. To use ISCOMs for the induction of efficacious immune responses in the GI tract, the immunization protocol employing intelligent oral or injectable prime-boost regimens and incorporating additional coatings or adjuvants should be tried.

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VI. VIRUS-LIKE PARTICLES VLPs imitate the three-dimensional conformation of real virus and mimic infection by authentic viruses. Moreover, VLPs can be engineered to express additional antigens and target epitopes in repeated array. VLPs have been studied as oral vaccines against virus or tumor antigens [97 104]. Those VLP oral vaccines induced humoral and cellular immune responses in both systemic and mucosal compartments. Significant antigen-specific SIgA responses were also observed. VLPs could also be expressed in plant tissue successfully [103,105,106] and could be purified from plant tissue at lower cost. On the other hand, rather crude freeze-dried plant tissue containing VLPs could be directly administered orally to induce protective immune responses in animals, which suggests the possible development of human edible vaccines using VLPs expressed in plant tissues such as tomato and potato [103,107 110].

VII. POLYMERIC PARTICLE-BASED ORAL DELIVERY Synthetic particles can serve as the most versatile oral delivery tools in which vaccine antigens and adjuvant could be loaded by encapsulation, conjugation, or adsorption. With advances in polymer material chemistry and formulation technology, different types of natural and synthetic NPs are being actively tested for possible use for effective oral vaccine delivery. These recent technologies enable overcoming previous barriers in oral vaccination and allow better targeting of antigens and adjuvants to the desired tissue location and cells. Particles can be engineered to release antigens and adjuvants upon degradation, swelling, and diffusion from the polymer, or change in electrostatic interactions. The production of particles in defined sizes, architectures, and chemical

properties would enable oral delivery, which is the most difficult vaccination route in terms of targeted delivery, thanks to the major development in nanotechnology and biomaterial science. Depending upon the polymer choices, the delivery systems by themselves provide adjuvant activity along with biocompatibility and biodegradability. The most extensively studied biodegradable polymers for the development of oral vaccine nanocarriers are PLA, PLGA, β-glucans, alginate, and chitosan [8,111]. In the case of oral delivery, the greater surface area of NPs, owing to their small size, would allow increased absorption across the intestinal epithelium, which will furnish reduced dosage and administration volume [67]. Hydrophilic NPs are generally transported through enterocytes, whereas hydrophobic polymeric NPs are better transported through M cells [30,99,112,113]. Orally administered PLA NPs (200 250 nm) reached the Peyer’s patches (PPs) through a three-step process. Most particles are first entrapped in the mucus. Then crossing of the epithelial barrier takes place exclusively through M cells, leading to an accumulation in PPs. Finally, the NPs interact with underlying B cells and DCs in the PP tissue. All three steps can occur within 15 minutes. Furthermore, DCs engulfing NPs were induced of TLR expression [114]. To be licensed and clinically used for mucosal vaccine delivery, nanocarriers should be able to protect the payload from degradation, to penetrate the mucus barriers, and to control the release of both antigens and adjuvants at targeted sites. In principle, these properties could be tuned by altering their particle size, surface chemistry, and three-dimensional architecture [79]. It is widely accepted that particulate antigens are more efficiently trafficked across the mucosa and delivered to mucosal APCs than are soluble antigens [67]. Uptake of particles in the GI tract occurs primarily via M cells. Despite some controversies, particles with a diameter smaller than 1 μm are thought to have a better chance of being taken up by M cells [115].

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To meet requisites for delivering diverse vaccine antigens and adjuvants to a specific mucosal target, PLGA surface characteristics have been extensively modified by coating with ionic surfactants or polymers such as polyethylene glycol (PEG), sodium dodecyl sulfate, aminodextran, chitosan, polyethylene imine, poly-Llysine, protamine, or cetyltrimethylammonium [30,116 118]. The surface chemistry of PLGA particles can also be altered to increase diffusion through mucus and uptake by M cells through oral delivery [119,120]. Other methods, such as coating antigen/adjuvant-loaded PLGA NPs with methacrylate-based polymer Eudragit FS30D, produced gastric-acid-resistant microparticles ($10 μm) that released payloads from the terminal ileum, where the pH level reaches 7.0 or higher [121]. The pH-sensitive polymers derived from methyl methacrylate, methacrylate, methacrylic acid, acrylate, and/or dimethacrylate have been blended with PLGA for the protection of payloads in the NP against enzymatic attacks in the stomach and small intestine [122]. The antigen encapsulated by PLGA NPs sequentially coated with phase-transitional shielding layer, poly[(methyl methacrylate)-co(methyl acrylate)-co-(methacrylic acid)] poly(D, L-lactide-co-glycolide) (PMMMA-PLGA), was found to protect antigens in the GI tract and achieve targeted vaccination in the large intestine [123]. Hydroxypropyl methylcellulose phthalate (HPMCP), another enteric coating agent, was shown to make acid-resistant B200nm NPs with PLGA carrying Helicobacter pylori antigen effectively induce Th1/Th17 protective immune responses [124]. To induce enhanced protective immune responses against antigens delivered by PLGA carriers, PLGA particles are functionalized by M-cell-targeting ligands in combination with stabilizing agents. Incorporation of M-celltargeting lectins such as UEA or LTA into PLGA NPs would enhance antigen-specific immune responses [125,126]. Arginine-glycine-aspartate (RGD) ligand binding to β1 integrin can also

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target M cells. PEGylated PLGA NPs grafted with RGD or RGD peptidomimetic ligand showed significantly increased uptake by M cells and enhanced specific IgG responses [113,127]. Claudin 4, one a member of the integral membrane protein family expressed primarily in tight junctions, also serves as a target for developing M-cell-binding nanocarriers [128]. In one study, an antigen-loaded porous PLGA microparticle was successfully coated with water-soluble chitosan conjugated with an M-cell-targeting peptide (CKS9). The resulting microparticles effectively reached PPs through M cell transcytosis to induce balanced Th1/Th2protective immune responses [129,130]. The pHsensitive polymeric delivery systems employing hydroxypropyl methylcellulose phthalate (HPMCP) could be attuned by adding thiol groups to be selectively released under ileal pH condition. By formulating M-cell-homing peptide (CKSTHPLSC) conjugated BmpB antigen with attuned HPMCP, delivery to PPs and subsequent adaptive immune responses could be significantly enhanced [131,132]. Recent research into oral vaccine delivery of NPs has been directed toward the incorporation of mucoadhesive polymers. The mucoadhesive polymers prolong retention time of the particles in the mucus by steric or adhesive interactions. Coating of nonbioadhesive nanospheres with poly(butadiene-maleic anhydrideco-L-DOPA) increased the particle uptake by 10-fold in the small intestine [133]. Conjugation of immunostimulatory ligands to bioadhesive polymers should induce longer-lasting mucosal and systemic immune responses against entrapped antigens. Bioadhesive poly(anhydride) NPs (300 400 nm) coated with mannose or Salmonella flagellin induced more potent and balanced Th1/Th2 immune responses compared with noncoated particles [134]. Chitosan and its derivatives have been tried for the oral delivery of protein and DNA vaccines [135 139]. The limited solubility of chitosan at alkaline and neutral pH has been

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circumvented by fabrication of chitosan by graft copolymerization with acyl, alkyl, monomeric, and polymeric moieties. Modifications through quarterization, thiolation, acylation, and grafting resulted in copolymers with higher mucoadhesion strength, increased hydrophobic interactions (advantageous in hydrophobic antigen entrapment), and increased solubility in alkaline pH, higher solubility, and controlled/ extended release profiles, which consequently confer wider application of chitosan derivatives for oral vaccine delivery [140,141]. Chitosan and its derivatives are mucoadhesive and have the ability to stimulate immune cells either by directly interacting with the M cells or by opening the tight junctions between the epithelial cells [142]. Because of the advantages mentioned above, chitosan has been applied to the manufacture of orally deliverable NPs or coating of micro/nanocarriers made of other synthetic or natural biopolymers [143,144]. Alginate has been used to make oral vaccine carriers utilizing its acid resistance and immunostimulatory properties [145]. In order to overcome chitosan’s instability in low-pH environments, cationic chitosan NPs can be coated with acid-resistant alginate to make composite carriers [144,146]. Alginate encapsulation of chitosan NPs entrapping protein antigens was proved to protect payload protein antigens and DNA from acidic attack in the stomach after oral administration. Owing to its acid resistance property, alginate is also used to encapsulate bacterial cells to develop oral vaccines. A single oral dose of alginateencapsulated BCG elicited effective long-lasting mucosal and systemic immune responses [147]. Cold-chain-free OCV could be developed by encapsulating heat-inactivated bacterial cells with alginate [148]. Oral vaccines against Edwardsiella, Brucella, and Aeromonas infections were also developed by encapsulation with alginate [149 152]. Glucan particles are porous 2- to 4-micron cell wall shells manufactured by treating

baker’s yeast (Saccharomyces cerevisiae) with a series of alkaline, acid, and solvent extractions [153]. By in situ layer-by-layer synthesis through electrostatic interactions, DNA could be encapsulated at high density [154]. Protein antigens could be encapsulated in glucan particles through hydration and lyophilization and could induce significant intestinal SIgA and Th1/Th17 cellular responses to encapsulated antigens following oral vaccination [155]. Glucan microparticles target enterocytes and M cells for uptake and activate them to secrete and express cytokines and β-glucan receptors [154 156]. β-Glucans, the major component of glucan microparticles, are fungal PAMPs, signaling through receptors such as dectin-1 and complement receptor 3 expressed on DCs, monocytes, and neutrophils [153,157]. Although the efficacy of glucan particles as an oral vaccine carrier was well proved with model antigens such as OVA or BSA, the application to pathogenic microorganisms has been relatively rare [158]. One reason for the limited use of glucan particles is that their manufacture is currently limited to liquid formulations, which require cold-chain storage and therefore are not optimal for the use in poorer regions [79].

VIII. ORAL DELIVERY OF VACCINES USING FOOD MATERIALS Plant-based oral vaccines have advantages over the traditional vaccines in cost, safety, and scalability. Since 1990, researchers have manufactured edible plant-based vaccines in carrot, soybean, tomato, rice, potato, and tobacco against microbial pathogen antigens such as the heat-labile toxin B subunit (LTB) of enterotoxigenic Escherichia coli, cholera toxin B subunit (CTB), and antigens from Yersinia pestis and viruses, such as hepatitis B virus, rotavirus, and Norwalk virus [159]. Many conventional vaccines are not widely distributed in developing

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countries where those vaccines are urgently needed, because of high production costs and the requirement of better infrastructure. One more problem standing in the way of wider distribution of desperately needed vaccines is that the conventional cell fermentation systems for producing recombinant protein vaccine antigens are often expensive and are not easily scalable [160]. Another emerging infectious disease field is One Health, dealing with zoonotic diseases spreading in both animals and humans. A solution to this may be the use of plants or plant cells as bioreactors. Molecular farming has become well established for the production of vaccines, and many proofs of principle and important proofs of efficacy are accumulating continuously [161]. MucoRice should be one of the most innovative approaches for oral vaccine delivery using edible rice as a carrier (Chapter 20: Plant-Based Mucosal Vaccine Delivery Systems). Rice seeds have stability and resistance to digestion in the stomach, making MucoRice an attractive oral vaccine delivery system. In 2007, it was first reported that cholera toxin B subunit (CTB) could be expressed in the rice seed. As much as 30 μg of CTB per seed was stored in the storage organelle protein body. When orally ingested, rice seeds expressing CTB induced CTB-specific serum IgG and mucosal IgA antibodies with neutralizing activity, while no rice storage-specific immune response was noted. When expressed in rice, CTB was protected from pepsin digestion in vitro [162]. Rice-expressed CTB also remained stable and thus maintained immunogenicity at room temperature for more than 3 years, and it provided more than 6 months of protection against CT- or LT-induced diarrhea after primary immunization [163]. These results show that the MucoRice vaccine could be stockpiled longer at room temperature and could be widely used for oral vaccination without cold-chain management. Rice-based oral vaccine developments are under way against many infectious diseases and noninfectious diseases such as allergy, autoimmunity, and Alzheimer’s disease [159].

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A. Tablets and Capsules The most widely used form of whole bacterial cell vaccines for cholera and typhoid fever was liquid suspension. Because of the lack of shelf stability, the liquid format is unsuitable for storage and distribution in developing countries. In this regard, a stable solid dosage vaccine platform is required for those vaccines. Formulation in tablets or capsules would provide more stability and ease of handling. In comparison to microparticles and NPs, capsules are significantly larger in size and could serve reservoir for multiple vaccine/ adjuvant formulations [79]. While no subunit or WC killed oral vaccine is currently delivered by capsule or tablet, the live attenuated Salmonella vaccine Vivotif is routinely delivered in an enteric-coated format [164]. Capsules could be manufactured in appropriate physical sizes (the average size of capsules and tablets ranges from 5 to 20 mm) suitable for administration to target populations. With enteric coatings, tablets and capsules could be protected from gastric acid and endowed with controlled release properties, which will provide facilitated delivery to discrete locations in the intestine. In principle, capsules allow the incorporation of many previously introduced delivery technologies in one primary delivery format. Recently, a tablet-based oral avian influenza vaccine was shown to elicit strong antiviral antibody and IFNγ T-cell responses. This approach utilized a nonreplicating adenovirus type 5 vector expressing avian flu hemagglutinin antigens together with a dsRNA TLR3 agonist [165].

B. Nasal Vaccine Delivery Among the choices of mucosal routes for vaccine administration, nasal delivery has been the most widely employed for innovative research because of the ease of approach and less harsh physicochemical conditions in the nasal cavity compared with the GI tract.

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Furthermore, nasal vaccines could be administered without professional training. There are at least three nasal vaccines licensed worldwide. The FDA approved FluMist, a live attenuated trivalent/quadrivalent influenza vaccine. Fluenz is an EMA-approved quadrivalent influenza vaccine. These two intranasal vaccines are manufactured by the same company, MedImmune. The Serum Institute of India licensed the monovalent NasoVac against pandemic A/California/7/2009 H1N1 influenza (Chapter 39: Nasal Influenza Vaccines). Besides influenza vaccines, the nasal route has been widely studied for development of many prophylactic and therapeutic vaccines against other infectious diseases, such as allergy, cancer, Alzheimer’s disease, and lifestyle-related diseases [166 169]. The human nasal cavity is an attractive route of mucosal immunization, having a total surface area of 150 cm2 with a volume of 15 20 mL [52,170,171]. The nasal cavity is divided into five anatomical and functional regions: the nasal vestibule, the atrium, the respiratory region, the olfactory region, and the nasopharynx [171]. The respiratory region is where nasal delivery of drugs and vaccines occurs, since it is the most permeable region, having a large surface area and a rich vascular bed [172]. The respiratory region is covered by a pseudostratified epithelium composed of columnar cells interspersed with goblet cells, which are interconnected by tight junctions (zonae occludens). The tight junctions are relatively resistant to paracellular passages of particulate materials in the breathed air [173]. The respiratory region is where mucus production actively takes place. The mucus layer in the nasal tract is relatively thinner (5 μm) than other mucosal surfaces. The nasal cavity is equipped with nasopharyngeal-associated lymphoid tissue (NALT), which is highly similar to Peyer’s patches in the ileum [174]. NALT is also covered with M cells that have active antigen sampling capacity [175]. Intraepithelial DCs project dendrites toward mucosal lumen and sample antigens. Particulate antigens are preferentially

sampled by M cells, and small soluble antigens have access to epithelium, where they are captured by intraepithelial DCs [173] (Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance). The mucociliary clearance mechanism should have negative effects on nasal vaccination. The rapid turnover of mucus (10 15 minutes) and fast mucus flow (B5 mm/min) in the nasal cavity limit the length of residence of administered vaccine. Continuous outward movement of cilia on the epithelial apical surface accelerates the clearance of mucus-trapped substances. To make matters worse, nasal enzymes and local pH negatively affect the stability of nasally administered vaccine antigens [170]. This could be why only live attenuated influenza vaccines proved effective in clinical trials and were approved by the FDA and EMA. A live influenza virus should be able to survive in the nasal mucosa and be harnessed with built-in adjuvants. An inactivated split influenza vaccine was also tested for nasal delivery but proved ineffective without coformulation with appropriate mucosal adjuvants [176 178]. To achieve equivalent antibody responses without adjuvant, an inactivated split antigen should be given at least three times more, or an inactivated whole virus should have been immunized [179 181]. Given that even an inactivated virus antigen requires potent mucosal adjuvants to achieve optimal immune responses in the systemic and mucosal compartments, protein antigens should employ even stronger mucosal adjuvants to be effective by nasal vaccination. Many mucosal adjuvants are suggested as formulation partners of nasal vaccine antigens [182,183]. CT and related E. coli heat-labile toxin (LT) and their mutant derivatives are the most widely tried mucosal adjuvant in preclinical studies [167]. Although those enterotoxins served as potent adjuvants for nasal vaccination of diverse antigens in animal studies, they

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have seldom been adopted for the development of human nasal vaccines. The use of enterotoxins as nasal vaccine adjuvants has a very serious failure history. The subunit influenza nasal vaccine Nasalflu Berna adjuvanted with E. coli LT had been significantly connected with Bell’s palsy with an odd ratio of 84 in an epidemiological study in Switzerland. The vaccine was consequently withdrawn from the market [184]. The adverse effect could be related with the capacity of LTB and CTB subunit to bind to GM1 ganglioside expressed on neuronal cell and retrograde translocation toward the brain [185,186]. Since the nasal cavity and brain are separated by a thin anatomical structure and are directly connected by the olfactory nerve, binding of any vaccine component to the olfactory nerve should contribute neurotoxicity. In this context, any nasal vaccine, adjuvant, or delivery system must clear the safety concern to be introduced to the market. Given the versatility and ease of nasal vaccination, numerous research groups are studying safe nasal adjuvants that could replace CT and LT. PAMPs are most widely studied as alternative nasal adjuvants. Recently published literature shows that ligands of TLR2, TLR5, and TLR9, STING agonist, and Flt3 ligand could be used as effective and safe nasal adjuvants [177,187 190]. Vaccine antigens should remain sufficiently stable in the nasal mucosa and should be able to reach to antigen-capturing cells surviving the mucociliary clearance mechanism. To overcome those hurdles, micro/nanocarriers for nasal vaccine delivery have been actively researched. To increase the residence time at mucosal surfaces, several strategies have been developed to increase adhesiveness of antigen delivery systems to the nasal mucus [191,192]. However, the mucus is not a static barrier; it is continuously secreted and cleared from the nasal cavity by the cilia beating on columnar epithelial cells. Mucoadhesion ability of delivery carrier would cause earlier removal of vaccine antigens when mucociliary clearance mechanism is intact. To cope with this problem,

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nasal vaccine carriers should cross the mucus layer rapidly and deliver antigens to M cells and DCs rather than strongly adhering to it [193 195]. Strategies that prevent vaccine carrier mucus interactions and hence allow for free diffusion by mucopenetration should be more effective in inducing efficacious immune responses [196]. However, many reports claimed that mucoadhesive NPs effectively enhanced the efficacy of mucosal vaccines [192]. One study showed that mucoadhesive NPs disrupted the protective human mucus barrier by altering its microstructure [197]. The disrupted microstructure resulted in a 20% increase in mucus mesh pore size. This disrupted mucus mesh would allow increased passage of other NPs to reach to the epithelium. 1. Advantages and Limitations of Nasal Vaccines The comparative advantages and disadvantages of intranasal vaccination are summarized in Table 19.3. The most outstanding advantage is the ease of administration, while the safety issue is the most essential problem to be resolved. 2. Nasal Vaccine Delivery Systems A. NANOEMULSIONS

Nanoemulsions, owing to their stability, small droplet size, and optimal solubilization properties, have great potential in nasal drug delivery. Furthermore, they may act as an active adjuvant in nasal vaccine formulations. Despite the promising results of in vitro and animal studies, the application of nanoemulsions for nasal delivery in humans appears to be hindered mainly by the lack of detailed toxicology studies and the lack of extensive clinical trials [198]. A cationic nanoemulsion formulation could have facilitated cellular uptake of model antigen ovalbumin in the nasal epithelial cell line [199]. The intranasal vaccination of HIV gp120 immunogen formulated in oil-inwater nanoemulsions induced robust serum

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anti-gp120 IgG and Th1-polarized systemic cellular immune responses [200].

IX. LIPOSOMES A large number of studies have investigated the potential of liposomes as a delivery system for nasal vaccination. Those liposome formulation vaccines targeted diverse pathogens: viruses (influenza, human immunodeficiency virus [HIV], lymphocytic choriomeningitis virus, hepatitis B virus, respiratory syncytial virus, Newcastle disease virus) and bacteria (Mycobacterium tuberculosis, V. cholerae, Pseudomonas aeruginosa, Y. pestis, Actinobacillus pleuropneumoniae) [201]. Peptides, proteins, and DNA can be successfully carried by liposomes having neutral, negative, and positive charges. Cationic liposomes were shown to interact more efficiently with epithelial cells and DCs [202,203]. Induction of cell-mediated immunity is another important feature of liposomemediated adjuvanticity [204]. Intranasal administration of DNA vaccine formulated with cationic liposomes, together with IL-12- and/or GM-CSF-expressing plasmids, resulted in both high levels of HIV-1-neutralizing antibodies in feces and serum and high levels of HIV-specific CTL responses [205]. Using this characteristic of the liposomal delivery system, a successful antitumor cellular immune response could be induced by intranasal immunization of DCtargeting liposomes carrying a tumor antigen [206]. Similarly, intranasal immunization with liposome-encapsulated plasmid DNA encoding influenza virus hemagglutinin elicited both mucosal cell-mediated and humoral (IgA and IgG) immune responses [207].

X. CHITOSAN Intranasal chitosan solution formulations were reported to enhance protective immune

responses against many antigens, including diphtheria, pertussis, and influenza [208 210]. Chitosan solutions seem to induce balanced Th1 and Th2 responses with neutralizing antibodies [211]. Whole influenza virus formulated with trimethylated chitosan showed much closer interaction with the epithelial surface, with the potential to generate enhanced uptake and induction of immune responses with minimal local toxicity in terms of ciliary beat frequency in the nasal cavity [212]. Chitosan dry power in salt form enables a thermally stable vaccine formulation that does not require cold chains. Chitosan power formulations were shown to outperform solutions in eliciting humoral responses against diphtheria, anthrax, and norovirus [213]. Chitosan microparticles and NPs are being robustly studied for the intranasal delivery of vaccines. Chitosan particles are basically mucoadhesive and able to deliver adjuvants and antigen cargos to efficiently promote humoral and cellular immune responses. Protein and peptide antigen-loaded chitosan particles are taken up by APCs in the administration site and eventually trafficked to draining lymph nodes, where T-cell activation occurs [39]. To be used for better intranasal delivery, chitosan should be chemically modified for better solubility, stability, mucoadhesiveness, safety, and resilience against degradation [214]. Chitosan itself shows strong adhesion to mucosal surfaces, providing a longer retention time at the nasal mucosa, and disrupts the tight junctions between nasal epithelial cells, which leads to enhanced paracellular transport of antigens [167]. The paracellular transport of the vaccine formulation into the nasal mucosa would lead to enhanced antigen uptake and presentation by APCs, with consequently augmented adaptive immunity [215]. This was demonstrated in a subunit influenza vaccine study showing protection against a heterologous viral challenge [216]. Ntrimethyl chitosan nasal vaccine formulation

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was superior to PLGA NPs in inducing nasal IgA responses [217]. Improved mucosal (IgA) and humoral (IgG) antibody responses are generally observed in mice as well as in other animal models such as rat and rabbit [218]. The cationic chitosan enhanced Th1 and Th17 responses as well as DC maturation through type I interferon induction by the cGAS-STING pathway, suggesting the involvement of multiple immune components [219]. Modified chitosan particles were generated to improve delivery efficiency and targeting. PEGylation improved water solubility and stability of conjugated antigens [37]. Chitosan coated poly-(ε-caprolactone) NPs induced enhanced mucosal immune responses against coformulated influenza antigen [220]. Protein antigen-loaded Pluronic F-127/chitosan microparticles showed improved antigen release and induced higher antigen-specific secretory IgA responses after intranasal vaccination [221]. OVA-loaded trimethyl chitosan hyaluronic acid NPs demonstrated superior immunogenicity after intranasal immunization [222]. Mannosylation of chitosan particles enhanced macrophage targeting and antigen-specific secretory IgA responses in mucosal secretions after intranasal immunization [223]. Chitosan application to intranasal vaccination has already reached clinical trial stages (phases 2 and 3) in the form of NPs [224] and antigen-conjugates [225]. Norovirus VLP formulated with MPLA was administered intranasally twice to healthy volunteers, inducing specific IgA responses in 70% of vaccinated individuals [224]. A clinical study testing intranasal vaccination of Neisseria meningitidis serogroup C polysaccharide (MCP)-CRM197 conjugate antigen mixed with chitosan showed specific IgA responses in nasal washes and balanced IgG1/IgG2 responses in serum [225]. Despite several clinical studies with promising results, no chitosan-based product for intranasal vaccination has yet reached the market.

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XI. STARCH NANOPARTICLES Influenza viral antigens encapsulated within bioadhesive starch and propylacrylic acid mixtures induced significant systemic antigen-specific IgG responses but not mucosal IgA after intranasal delivery of the influenza vaccine in rabbit [226]. Inactivated influenza antigens in positively charged NPs have been tested in a phase 1 clinical study. Significant mucosal IgA antibodies were produced in individuals who received two-dose nasal immunizations [227]. A cationic maltodextrin NP (cationic surface with an anionic lipid core) showed longer nasal residence time after nasal administration than liposomes and PLGA NPs [228].

XII. POLYMER NANOPARTICLES The PLGA NPs are the most used synthetic polymer for nasal vaccine delivery studies. Cationic modification of PLGA enhanced residence time in the mucosa and resulted in better immune responses with higher serum antibody and SIgA levels [217]. The surface modification of PLGA carriers with chitosan can increase mucoadhesion through a change of zeta potential from negative to positive without affecting particle size and dispersion. Moreover, the clearance rate in the nasal cavity was reduced, resulting in enhanced systemic and mucosal antibody responses [229]. Many antigens encapsulated in PLGA nano/microparticles were immunized through the nasal route to show enhanced immune responses in both systemic and mucosal immune compartments; they were ovalbumin, bovine serum albumin, bovine parainfluenza virus, bovine syncytial virus, HBsAg, malaria, swine fever virus, Y. pestis, and Streptococcus equi [230]. Targeting efficiency to M cells could also be achieved by functionalization of the particle surface [19].

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XIII. NANOGELS Although many types of nanogels were tested as vaccine delivery systems, the cholesteryl group-bearing pullulan (CHP) is the most extensively studied one for mucosal vaccine delivery [6]. The cationic CHP (cCHP) nanogel binds better to epithelial cells and is subsequently taken up with high efficiency into the cells. The cCHP nanogel itself did not activate DCs and did not have biologically active adjuvant activity. However, vigorous neutralizing serum IgG and SIgA responses were noted without coadministration of mucosal adjuvants. The cCHP nanogel was suggested as a universal protein-based antigen delivery vehicle for adjuvant-free intranasal vaccinations [51] (Chapter 26: Nanodelivery Vehicles for Mucosal Vaccines). To further potentiate immune responses against cargo antigens, cytokines or adjuvant could be coencapsulated in the CHP nanogel. TNFα encapsulated in the nanogel acted as a vaccine adjuvant for a nasal influenza vaccine [231]. In an antiobesity vaccine study, cCHP was engineered to carry the adjuvant cyclic di-GMP along with self-origin antigen ghrelin peptide hormone conjugated to a carrier protein PspA [166,169]. The cCHP-based nasal vaccines were successfully tested for use against several infectious diseases and lifestyle-related diseases: influenza, Streptococcus pneumoniae, C. botulinum, obesity, and hypertension [51,63,166,168,169,232]. Another very promising virtue of the cCHP nanogel nasal delivery system is safety. It was shown reiteratively that protein antigens carried by the nanogel did not accumulate in the olfactory bulb and brain, thus excluding the risk of neurotoxicity or brain damage [51,232]. When all the physicochemical, biological, and immunological characteristics are considered, the cCHP nanogel platform seems to be the most promising nasal delivery system to be translated into more aggressive clinical applications.

XIV. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Although mucosal vaccination has many advantages, a very limited number of mucosal vaccines have been licensed. The most widely tested vaccination routes are oral and intranasal. Currently licensed oral and intranasal vaccines are composed predominantly of WC killed or live attenuated microorganisms, where cell bodies serve as delivery systems and whose cell components act as built-in adjuvants. Future mucosal vaccines should be made with more purified antigen components, which will require safe and efficacious adjuvants and delivery systems. Recent developments in biomaterials and nanotechnology have enabled many innovative mucosal vaccine trials. For oral vaccination, the vaccine delivery system should be able to stably carry antigens and adjuvants and resist the harsh physicochemical conditions in the stomach and intestinal tract. Besides many nano/microcarrier tools generated by using natural and chemical materials, the development of oral vaccine delivery systems using food materials should be more robustly researched to expand vaccine coverage of GI infections in developing countries. For intranasal vaccination, the vaccine delivery system should survive the very active mucociliary clearance mechanisms and provide safety, given the anatomical location of the nasal cavity, which is separated from the central nervous system by a thin barrier. Future mucosal vaccine carriers, regardless of administration routes, should share common characteristics. They should maintain stability in given environments, be mucoadhesive, and have targeting ability to specific tissues and cells.

References [1] Ventola CL. Progress in nanomedicine: approved and investigational nanodrugs. P T 2017;42(12):742 55. [2] Bobo D, Robinson KJ, Islam J, Thurecht KJ, Corrie SR. Nanoparticle-based medicines: a review of FDA-approved materials and clinical trials to date. Pharm Res 2016;33 (10):2373 87.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[3] Peek LJ, Middaugh CR, Berkland C. Nanotechnology in vaccine delivery. Adv Drug Deliv Rev 2008;60 (8):915 28. [4] Di Pasquale A, Preiss S, Tavares Da Silva F, Garcon N. Vaccine adjuvants: from 1920 to 2015 and beyond. Vaccines (Basel) 2015;3(2):320 43. [5] Garcia A, Lema D. An updated review of ISCOMSTM and ISCOMATRIXTM vaccines. Curr Pharm Des 2016;22(41):6294 9. [6] Nakahashi-Ouchida R, Yuki Y, Kiyono H. Development of a nanogel-based nasal vaccine as a novel antigen delivery system. Expert Rev Vaccines 2017;16(12):1231 40. [7] Eskandari S, Guerin T, Toth I, Stephenson RJ. Recent advances in self-assembled peptides: implications for targeted drug delivery and vaccine engineering. Adv Drug Deliv Rev 2017;110 111:169 87. [8] Gregory AE, Titball R, Williamson D. Vaccine delivery using nanoparticles. Front Cell Infect Microbiol 2013;3:13. [9] Peres C, Matos AI, Conniot J, Sainz V, Zupancic E, Silva JM, et al. Poly(lactic acid)-based particulate systems are promising tools for immune modulation. Acta Biomater 2017;48:41 57. [10] Fox CB. Squalene emulsions for parenteral vaccine and drug delivery. Molecules 2009;14(9):3286 312. [11] Fox CB, Haensler J. An update on safety and immunogenicity of vaccines containing emulsion-based adjuvants. Expert Rev Vaccines 2013;12(7):747 58. [12] Schultze V, D’Agosto V, Wack A, Novicki D, Zorn J, Hennig R. Safety of MF59 adjuvant. Vaccine 2008;26 (26):3209 22. [13] Garcon N, Vaughn DW, Didierlaurent AM. Development and evaluation of AS03, an adjuvant system containing alpha-tocopherol and squalene in an oil-in-water emulsion. Expert Rev Vaccines 2012;11 (3):349 66. [14] Misquith A, Fung HW, Dowling QM, Guderian JA, Vedvick TS, Fox CB. In vitro evaluation of TLR4 agonist activity: formulation effects. Colloids Surf B Biointerfaces 2014;113:312 19. [15] Ott G, Barchfeld GL, Chernoff D, Radhakrishnan R, van Hoogevest P, Van Nest G. MF59. Design and evaluation of a safe and potent adjuvant for human vaccines. Pharm Biotechnol 1995;6:277 96. [16] Cox JC, Sjolander A, Barr IG. ISCOMs and other saponin based adjuvants. Adv Drug Deliv Rev 1998;32 (3):247 71. [16a] De Serrano LO, Burkhart DJ. Liposomal vaccine formulations as prophylactic agents: design considerations for modern vaccines. J Nanobiotechnol 2017;15 (1):83.

347

[17] Mishra B, Patel BB, Tiwari S. Colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery. Nanomedicine 2010;6(1):9 24. [18] Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev 2003;55(3):329 47. [19] Allahyari M, Mohit E. Peptide/protein vaccine delivery system based on PLGA particles. Hum Vaccin Immunother 2016;12(3):806 28. [20] Wendorf J, Singh M, Chesko J, Kazzaz J, Soewanan E, Ugozzoli M, et al. A practical approach to the use of nanoparticles for vaccine delivery. J Pharm Sci 2006;95 (12):2738 50. [21] Jain S, O’Hagan DT, Singh M. The long-term potential of biodegradable poly(lactide-co-glycolide) microparticles as the next-generation vaccine adjuvant. Expert Rev Vaccines 2011;10(12):1731 42. [22] Johansen P, Men Y, Merkle HP, Gander B. Revisiting PLA/PLGA microspheres: an analysis of their potential in parenteral vaccination. Eur J Pharm Biopharm 2000;50(1):129 46. [23] Crotts G, Park TG. Protein delivery from poly(lactic-coglycolic acid) biodegradable microspheres: release kinetics and stability issues. J Microencapsul 1998;15 (6):699 713. [24] Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol 2008;38(5):1404 13. [25] Bachmann MF, Jennings GT. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev Immunol 2010;10(11):787 96. [26] Lasprilla AJ, Martinez GA, Lunelli BH, Jardini AL, Filho RM. Poly-lactic acid synthesis for application in biomedical devices a review. Biotechnol Adv 2012;30(1):321 8. [27] Lee BK, Yun Y, Park K. PLA micro- and nano-particles. Adv Drug Deliv Rev 2016;107:176 91. [28] Athanasiou KA, Niederauer GG, Agrawal CM. Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers. Biomaterials 1996;17(2):93 102. [29] Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Preat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release 2012;161 (2):505 22. [30] Mundargi RC, Babu VR, Rangaswamy V, Patel P, Aminabhavi TM. Nano/micro technologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and its derivatives. J Control Release 2008;125(3):193 209.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

348

19. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

[31] Giteau A, Venier-Julienne MC, Aubert-Pouessel A, Benoit JP. How to achieve sustained and complete protein release from PLGA-based microparticles? Int J Pharm 2008;350(1 2):14 26. [32] Mohammadi-Samani S, Taghipour B. PLGA micro and nanoparticles in delivery of peptides and proteins; problems and approaches. Pharm Dev Technol 2015;20 (4):385 93. [33] Johansen P, Men Y, Audran R, Corradin G, Merkle HP, Gander B. Improving stability and release kinetics of microencapsulated tetanus toxoid by co-encapsulation of additives. Pharm Res 1998;15(7):1103 10. [34] Silva AL, Rosalia RA, Sazak A, Carstens MG, Ossendorp F, Oostendorp J, et al. Optimization of encapsulation of a synthetic long peptide in PLGA nanoparticles: low-burst release is crucial for efficient CD8(1) T cell activation. Eur J Pharm Biopharm 2013;83(3):338 45. [35] Dang JM, Leong KW. Natural polymers for gene delivery and tissue engineering. Adv Drug Deliv Rev 2006;58(4):487 99. [36] Leleux J, Roy K. Micro and nanoparticle-based delivery systems for vaccine immunotherapy: an immunological and materials perspective. Adv Healthc Mater 2013;2(1):72 94. [37] Singh B, Maharjan S, Cho KH, Cui L, Park IK, Choi YJ, et al. Chitosan-based particulate systems for the delivery of mucosal vaccines against infectious diseases. Int J Biol Macromol 2018;110:54 64. [38] Thanou M, Florea BI, Langemeyer MW, Verhoef JC, Junginger HE. N-trimethylated chitosan chloride (TMC) improves the intestinal permeation of the peptide drug buserelin in vitro (Caco-2 cells) and in vivo (rats). Pharm Res 2000;17(1):27 31. [39] Chua BY, Al Kobaisi M, Zeng W, Mainwaring D, Jackson DC. Chitosan microparticles and nanoparticles as biocompatible delivery vehicles for peptide and protein-based immunocontraceptive vaccines. Mol Pharm 2012;9(1):81 90. [40] Sayin B, Somavarapu S, Li XW, Sesardic D, Senel S, Alpar OH. TMC-MCC (N-trimethyl chitosan-mono-Ncarboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines. Eur J Pharm Sci 2009;38(4):362 9. [41] Wee S, Gombotz WR. Protein release from alginate matrices. Adv Drug Deliv Rev 1998;31(3):267 85. [42] Hori Y, Winans AM, Irvine DJ. Modular injectable matrices based on alginate solution/microsphere mixtures that gel in situ and co-deliver immunomodulatory factors. Acta Biomater 2009;5(4):969 82. [43] Yang D, Jones KS. Effect of alginate on innate immune activation of macrophages. J Biomed Mater Res A 2009;90(2):411 18.

[44] McCarthy RE, Arnold LW, Babcock GF. Dextran sulphate: an adjuvant for cell-mediated immune responses. Immunology 1977;32(6):963 74. [45] De Geest BG, Willart MA, Lambrecht BN, Pollard C, Vervaet C, Remon JP, et al. Surface-engineered polyelectrolyte multilayer capsules: synthetic vaccines mimicking microbial structure and function. Angew Chem Int Ed Engl 2012;51(16):3862 6. [46] Rad-Malekshahi M, Lempsink L, Amidi M, Hennink WE, Mastrobattista E. Biomedical applications of selfassembling peptides. Bioconjug Chem 2016;27(1):3 18. [47] Rudra JS, Tian YF, Jung JP, Collier JH. A selfassembling peptide acting as an immune adjuvant. Proc Natl Acad Sci U S A 2010;107(2):622 7. [48] Rudra JS, Sun T, Bird KC, Daniels MD, Gasiorowski JZ, Chong AS, et al. Modulating adaptive immune responses to peptide self-assemblies. ACS Nano 2012;6 (2):1557 64. [49] Chen JJ, Pompano RR, Santiago FW, Maillat L, Sciammas R, Sun T, et al. The use of self-adjuvanting nanofiber vaccines to elicit high-affinity B cell responses to peptide antigens without inflammation. Biomaterials 2013;34(34):8776 85. [50] Zarekar NS, Lingayat VJ, Pande VV. Nanogel as a novel platform for smart drug delivery system. Nanosci Nanotechnol Res 2017;4(1):25 31. [51] Nochi T, Yuki Y, Takahashi H, Sawada S, Mejima M, Kohda T, et al. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines. Nat Mater 2010;9(7):572 8. [52] Salatin S, Barar J, Barzegar-Jalali M, Adibkia K, Milani MA, Jelvehgari M. Hydrogel nanoparticles and nanocomposites for nasal drug/vaccine delivery. Arch Pharm Res 2016;39(9):1181 92. [53] Zhang T, Yang R, Yang S, Guan J, Zhang D, Ma Y, et al. Research progress of self-assembled nanogel and hybrid hydrogel systems based on pullulan derivatives. Drug Deliv 2018;25(1):278 92. [54] Sasaki Y, Akiyoshi K. Nanogel engineering for new nanobiomaterials: from chaperoning engineering to biomedical applications. Chem Rec 2010;10(6):366 76. [55] Kimoto T, Shibuya T, Shiobara S. Safety studies of a novel starch, pullulan: chronic toxicity in rats and bacterial mutagenicity. Food Chem Toxicol 1997;35 (3 4):323 9. [56] Ferreira SA, Coutinho PJG, Gama FM. Synthesis and characterization of self-assembled nanogels made of pullulan. Materials (Basel) 2011;4(4):601 20. [57] Akiyoshi K, Deguchi S, Moriguchi N, Yamaguchi S, Sunamoto J. Self-aggregates of hydrophobized polysaccharides in water. Formation and characteristics of nanoparticles. Macromolecules 1993;26(12):3062 8.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[58] Shimizu T, Kishida T, Hasegawa U, Ueda Y, Imanishi J, Yamagishi H, et al. Nanogel DDS enables sustained release of IL-12 for tumor immunotherapy. Biochem Biophys Res Commun 2008;367(2):330 5. [59] Yuki Y, Nochi T, Kong IG, Takahashi H, Sawada S, Akiyoshi K, et al. Nanogel-based antigen-delivery system for nasal vaccines. Biotechnol Genet Eng Rev 2013;29:61 72. [60] Asayama W, Sawada S, Taguchi H, Akiyoshi K. Comparison of refolding activities between nanogel artificial chaperone and GroEL systems. Int J Biol Macromol 2008;42(3):241 6. [61] Nomura Y, Ikeda M, Yamaguchi N, Aoyama Y, Akiyoshi K. Protein refolding assisted by selfassembled nanogels as novel artificial molecular chaperone. FEBS Lett 2003;553(3):271 6. [62] Ayame H, Morimoto N, Akiyoshi K. Self-assembled cationic nanogels for intracellular protein delivery. Bioconjug Chem 2008;19(4):882 90. [63] Kong IG, Sato A, Yuki Y, Nochi T, Takahashi H, Sawada S, et al. Nanogel-based PspA intranasal vaccine prevents invasive disease and nasal colonization by Streptococcus pneumoniae. Infect Immun 2013;81 (5):1625 34. [64] Lycke N. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 2012;12(8):592 605. [65] Date KA, Vicari A, Hyde TB, Mintz E, DanovaroHolliday MC, Henry A, et al. Considerations for oral cholera vaccine use during outbreak after earthquake in Haiti, 2010 2011. Emerg Infect Dis 2011;17(11):2105 12. [66] Mantis NJ, Morici LA, Roy CJ. Mucosal vaccines for biodefense. Curr Top Microbiol Immunol 2012;354:181 95. [67] Marasini N, Skwarczynski M, Toth I. Oral delivery of nanoparticle-based vaccines. Expert Rev Vaccines 2014;13(11):1361 76. [68] Modlin J, Wenger J. Achieving and maintaining polio eradication new strategies. N Engl J Med 2014;371 (16):1476 9. [69] Strebel PM, Sutter RW, Cochi SL, Biellik RJ, Brink EW, Kew OM, et al. Epidemiology of poliomyelitis in the United States one decade after the last reported case of indigenous wild virus-associated disease. Clin Infect Dis 1992;14(2):568 79. [70] Levine MM. Immunogenicity and efficacy of oral vaccines in developing countries: lessons from a live cholera vaccine. BMC Biol 2010;8:129. [71] Alcock R, Cottingham MG, Rollier CS, Furze J, De Costa SD, Hanlon M, et al. Long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass. Sci Transl Med 2010;2(19):19ra12.

349

[72] Levine MM, Chen WH, Kaper JB, Lock M, Danzig L, Gurwith M. PaxVax CVD 103-HgR single-dose live oral cholera vaccine. Expert Rev Vaccines 2017;16 (3):197 213. [73] Sow SO, Tapia MD, Chen WH, Haidara FC, Kotloff KL, Pasetti MF, et al. A randomized, placebo-controlled, double-blind Phase 2 trial comparing the reactogenicity and immunogenicity of a single $ 2x108 colony forming units [cfu] standard-dose versus a $ 2x109 cfu high-dose of CVD 103-HgR live attenuated oral cholera vaccine, with Shanchol inactivated oral vaccine as an open label immunologic comparator. Clin Vaccine Immunol 2017;. Available from: https://doi.org/ 10.1128/CVI.00265-17. [74] Cabrera A, Lepage JE, Sullivan KM, Seed SM. Vaxchora: a single-dose oral cholera vaccine. Ann Pharmacother 2017;51(7):584 9. [75] Ge W, Hu PZ, Huang Y, Wang XM, Zhang XM, Sun YJ, et al. The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following different administration routes. Oncol Rep 2009;22(4):915 20. [76] Ge W, Li Y, Li ZS, Zhang SH, Sun YJ, Hu PZ, et al. The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following peroral administration route. Cancer Immunol Immunother 2009;58(2):201 8. [77] Kataoka K, Harada A, Nagasaki Y. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv Drug Deliv Rev 2001;47(1):113 31. [78] Yang Y, Kuang Y, Liu Y, Li W, Jiang Z, Xiao L, et al. Immunogenicity of multiple-epitope antigen gene of HCV carried by novel biodegradable polymers. Comp Immunol Microbiol Infect Dis 2011;34(1):65 72. [79] Davitt CJ, Lavelle EC. Delivery strategies to enhance oral vaccination against enteric infections. Adv Drug Deliv Rev 2015;91:52 69. [80] Silva AC, Santos D, Ferreira D, Lopes CM. Lipid-based nanocarriers as an alternative for oral delivery of poorly water-soluble drugs: peroral and mucosal routes. Curr Med Chem 2012;19(26):4495 510. [81] Zho F, Neutra MR. Antigen delivery to mucosaassociated lymphoid tissues using liposomes as a carrier. Biosci Rep 2002;22(2):355 69. [82] Parmentier J, Thomas N, Mullertz A, Fricker G, Rades T. Exploring the fate of liposomes in the intestine by dynamic in vitro lipolysis. Int J Pharm 2012;437 (1 2):253 63. [83] Chauhan MK, Sahoo PK, Rawat AS, Singh A, Bamrara A, Sharma D. Bilosomes: a novel approach to meet the challenges in oral immunization. Recent Pat Drug Deliv Formul 2015;9(3):201 12.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

350

19. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

[84] Jacobsen AC, Jensen SM, Fricker G, Brandl M, Treusch AH. Archaeal lipids in oral delivery of therapeutic peptides. Eur J Pharm Sci 2017;108:101 10. [85] Nakanishi T, Kunisawa J, Hayashi A, Tsutsumi Y, Kubo K, Nakagawa S, et al. Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins. J Control Release 1999;61(1 2):233 40. [86] Christensen D, Korsholm KS, Rosenkrands I, Lindenstrom T, Andersen P, Agger EM. Cationic liposomes as vaccine adjuvants. Expert Rev Vaccines 2007;6(5):785 96. [87] Wei X, Shao B, He Z, Ye T, Luo M, Sang Y, et al. Cationic nanocarriers induce cell necrosis through impairment of Na(1)/K(1)-ATPase and cause subsequent inflammatory response. Cell Res 2015;25 (2):237 53. [88] Nguyen TX, Huang L, Gauthier M, Yang G, Wang Q. Recent advances in liposome surface modification for oral drug delivery. Nanomedicine (Lond) 2016;11 (9):1169 85. [89] Venkatesan N, Vyas SP. Polysaccharide coated liposomes for oral immunization development and characterization. Int J Pharm 2000;203(1 2):169 77. [90] Chen H, Torchilin V, Langer R. Lectin-bearing polymerized liposomes as potential oral vaccine carriers. Pharm Res 1996;13(9):1378 83. [91] Souza M, Costantini V, Azevedo MS, Saif LJ. A human norovirus-like particle vaccine adjuvanted with ISCOM or mLT induces cytokine and antibody responses and protection to the homologous GII.4 human norovirus in a gnotobiotic pig disease model. Vaccine 2007;25(50):8448 59. [92] Aguila A, Donachie AM, Peyre M, McSharry CP, Sesardic D, Mowat AM. Induction of protective and mucosal immunity against diphtheria by a immune stimulating complex (ISCOMS) based vaccine. Vaccine 2006;24(24):5201 10. [93] Eliasson DG, Helgeby A, Schon K, Nygren C, ElBakkouri K, Fiers W, et al. A novel non-toxic combined CTA1-DD and ISCOMS adjuvant vector for effective mucosal immunization against influenza virus. Vaccine 2011;29(23):3951 61. [94] Lobaina Y, Palenzuela D, Pichardo D, Muzio V, Guillen G, Aguilar JC. Immunological characterization of two hepatitis B core antigen variants and their immunoenhancing effect on co-delivered hepatitis B surface antigen. Mol Immunol 2005;42(3):289 94. [95] Nguyen TV, Yuan L, Azevedo MS, Jeong KI, Gonzalez AM, Iosef C, et al. High titers of circulating maternal antibodies suppress effector and memory B-cell responses induced by an attenuated rotavirus priming and rotavirus-like particle-immunostimulating complex

[96]

[97]

[98] [99]

[100]

[101]

[102]

[103]

[104]

[105]

[106] [107]

boosting vaccine regimen. Clin Vaccine Immunol 2006;13(4):475 85. Azevedo MS, Gonzalez AM, Yuan L, Jeong KI, Iosef C, Van Nguyen T, et al. An oral versus intranasal prime/boost regimen using attenuated human rotavirus or VP2 and VP6 virus-like particles with immunostimulating complexes influences protection and antibody-secreting cell responses to rotavirus in a neonatal gnotobiotic pig model. Clin Vaccine Immunol 2010;17(3):420 8. Jariyapong P, Xing L, van Houten NE, Li TC, Weerachatyanukul W, Hsieh B, et al. Chimeric hepatitis E virus-like particle as a carrier for oral-delivery. Vaccine 2013;31(2):417 24. Kuate Defo Z, Lee B. New approaches in oral rotavirus vaccines. Crit Rev Microbiol 2016;42(3):495 505. Nerome K, Matsuda S, Maegawa K, Sugita S, Kuroda K, Kawasaki K, et al. Quantitative analysis of the yield of avian H7 influenza virus haemagglutinin protein produced in silkworm pupae with the use of the codon-optimized DNA: a possible oral vaccine. Vaccine 2017;35(5):738 46. Shuttleworth G, Eckery DC, Awram P. Oral and intraperitoneal immunization with rotavirus 2/6 virus-like particles stimulates a systemic and mucosal immune response in mice. Arch Virol 2005;150(2):341 9. Tacket CO, Sztein MB, Losonsky GA, Wasserman SS, Estes MK. Humoral, mucosal, and cellular immune responses to oral Norwalk virus-like particles in volunteers. Clin Immunol 2003;108(3):241 7. Zhai Y, Zhong Z, Zariffard M, Spear GT, Qiao L. Bovine papillomavirus-like particles presenting conserved epitopes from membrane-proximal external region of HIV-1 gp41 induced mucosal and systemic antibodies. Vaccine 2013;31(46):5422 9. Czyz M, Dembczynski R, Marecik R, Wojas-Turek J, Milczarek M, Pajtasz-Piasecka E, et al. Freeze-drying of plant tissue containing HBV surface antigen for the oral vaccine against hepatitis B. Biomed Res Int 2014;2014:485689. Huang Y, Fayad R, Smock A, Ullrich AM, Qiao L. Induction of mucosal and systemic immune responses against human carcinoembryonic antigen by an oral vaccine. Cancer Res 2005;65(15):6990 9. Scotti N, Rybicki EP. Virus-like particles produced in plants as potential vaccines. Expert Rev Vaccines 2013;12(2):211 24. Chen Q, Lai H. Plant-derived virus-like particles as vaccines. Hum Vaccin Immunother 2013;9(1):26 49. Santi L, Batchelor L, Huang Z, Hjelm B, Kilbourne J, Arntzen CJ, et al. An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles. Vaccine 2008;26(15):1846 54.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[108] Warzecha H, Mason HS, Lane C, Tryggvesson A, Rybicki E, Williamson AL, et al. Oral immunogenicity of human papillomavirus-like particles expressed in potato. J Virol 2003;77(16):8702 11. [109] Zhang X, Buehner NA, Hutson AM, Estes MK, Mason HS. Tomato is a highly effective vehicle for expression and oral immunization with Norwalk virus capsid protein. Plant Biotechnol J 2006;4(4):419 32. [110] Huang Z, Elkin G, Maloney BJ, Beuhner N, Arntzen CJ, Thanavala Y, et al. Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses. Vaccine 2005;23(15):1851 8. [111] des Rieux A, Fievez V, Garinot M, Schneider YJ, Preat V. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J Control Release 2006;116(1):1 27. [112] Wang T, Zou M, Jiang H, Ji Z, Gao P, Cheng G. Synthesis of a novel kind of carbon nanoparticle with large mesopores and macropores and its application as an oral vaccine adjuvant. Eur J Pharm Sci 2011;44 (5):653 9. [113] Garinot M, Fievez V, Pourcelle V, Stoffelbach F, des Rieux A, Plapied L, et al. PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination. J Control Release 2007;120(3):195 204. [114] Primard C, Rochereau N, Luciani E, Genin C, Delair T, Paul S, et al. Traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to subepithelial immune competent cells. Biomaterials 2010;31(23):6060 8. [115] Brayden DJ, Baird AW. Microparticle vaccine approaches to stimulate mucosal immunisation. Microbes Infect 2001;3(10):867 76. [116] Wischke C, Borchert HH, Zimmermann J, Siebenbrodt I, Lorenzen DR. Stable cationic microparticles for enhanced model antigen delivery to dendritic cells. J Control Release 2006;114(3):359 68. [117] Castellanos IJ, Crespo R, Griebenow K. Poly(ethylene glycol) as stabilizer and emulsifying agent: a novel stabilization approach preventing aggregation and inactivation of proteins upon encapsulation in bioerodible polyester microspheres. J Control Release 2003;88(1):135 45. [118] Fischer S, Uetz-von Allmen E, Waeckerle-Men Y, Groettrup M, Merkle HP, Gander B. The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles. Biomaterials 2007;28(6):994 1004. [119] Jung T, Kamm W, Breitenbach A, Kaiserling E, Xiao JX, Kissel T. Biodegradable nanoparticles for oral delivery of peptides: is there a role for polymers to affect mucosal uptake? Eur J Pharm Biopharm 2000;50(1):147 60.

351

[120] Jain RA. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 2000;21(23): 2475 90. [121] Zhu Q, Talton J, Zhang G, Cunningham T, Wang Z, Waters RC, et al. Large intestine-targeted, nanoparticle-releasing oral vaccine to control genitorectal viral infection. Nat Med 2012;18(8):1291 6. [122] Reyes-Ortega F, Rodriguez G, Aguilar MR, Lord M, Whitelock J, Stenzel MH, et al. Encapsulation of low molecular weight heparin (bemiparin) into polymeric nanoparticles obtained from cationic block copolymers: properties and cell activity. J Mater Chem B 2013;1(6):850 60. [123] Zhang L, Zeng ZZ, Hu CH, Bellis SL, Yang WD, Su YT, et al. Controlled and targeted release of antigens by intelligent shell for improving applicability of oral vaccines. Biomaterials 2016;77:307 19. [124] Tan Z, Liu W, Liu H, Li C, Zhang Y, Meng X, et al. Oral Helicobacter pylori vaccine-encapsulated acidresistant HP55/PLGA nanoparticles promote immune protection. Eur J Pharm Biopharm 2017;111:33 43. [125] Mishra N, Tiwari S, Vaidya B, Agrawal GP, Vyas SP. Lectin anchored PLGA nanoparticles for oral mucosal immunization against hepatitis B. J Drug Target 2011;19(1):67 78. [126] Du L, Yu Z, Pang F, Xu X, Mao A, Yuan W, et al. Targeted delivery of GP5 antigen of PRRSV to M cells enhances the antigen-specific systemic and mucosal immune responses. Front Cell Infect Microbiol 2018;8:7. [127] Fievez V, Plapied L, des Rieux A, Pourcelle V, Freichels H, Wascotte V, et al. Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination. Eur J Pharm Biopharm 2009;73(1):16 24. [128] Peter Y, Goodenough D. Claudins. Curr Biol 2004;14 (8):R293 4. [129] Jiang T, Singh B, Li HS, Kim YK, Kang SK, Nah JW, et al. Targeted oral delivery of BmpB vaccine using porous PLGA microparticles coated with M cell homing peptide-coupled chitosan. Biomaterials 2014;35 (7):2365 73. [130] Yoo MK, Kang SK, Choi JH, Park IK, Na HS, Lee HC, et al. Targeted delivery of chitosan nanoparticles to Peyer’s patch using M cell-homing peptide selected by phage display technique. Biomaterials 2010;31 (30):7738 47. [131] Singh B, Maharjan S, Jiang T, Kang SK, Choi YJ, Cho CS. Attuning hydroxypropyl methylcellulose phthalate to oral delivery vehicle for effective and selective delivery of protein vaccine in ileum. Biomaterials 2015;59:144 59.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

352

19. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

[132] Singh B, Maharjan S, Jiang T, Kang SK, Choi YJ, Cho CS. Combinatorial approach of antigen delivery using M cell-homing peptide and mucoadhesive vehicle to enhance the efficacy of oral vaccine. Mol Pharm 2015;12(11):3816 28. [133] Reineke J, Cho DY, Dingle YL, Cheifetz P, Laulicht B, Lavin D, et al. Can bioadhesive nanoparticles allow for more effective particle uptake from the small intestine? J Control Release 2013;170 (3):477 84. [134] Salman HH, Irache JM, Gamazo C. Immunoadjuvant capacity of flagellin and mannosamine-coated poly (anhydride) nanoparticles in oral vaccination. Vaccine 2009;27(35):4784 90. [135] Abkar M, Fasihi-Ramandi M, Kooshki H, Lotfi AS. Intraperitoneal immunization with urease loaded N-trimethyl chitosan nanoparticles elicits high protection against Brucella melitensis and Brucella abortus infections. Immunol Lett 2018;199:53 60. [136] Kole S, Kumari R, Anand D, Kumar S, Sharma R, Tripathi G, et al. Nanoconjugation of bicistronic DNA vaccine against Edwardsiella tarda using chitosan nanoparticles: evaluation of its protective efficacy and immune modulatory effects in Labeo rohita vaccinated by different delivery routes. Vaccine 2018;36 (16):2155 65. [137] Pathinayake PS, Gayan Chathuranga WA, Lee HC, Chowdhury MYE, Sung MH, Lee JS, et al. Inactivated enterovirus 71 with poly-gamma-glutamic acid/ chitosan nano particles (PC NPs) induces high cellular and humoral immune responses in BALB/c mice. Arch Virol 2018;163(8):2073 83. [138] Walke S, Srivastava G, Routaray CB, Dhavale D, Pai K, Doshi J, et al. Preparation and characterization of microencapsulated DwPT trivalent vaccine using water soluble chitosan and its in-vitro and in-vivo immunological properties. Int J Biol Macromol 2018;107(Pt B):2044 56. [139] Xu B, Zhang W, Chen Y, Xu Y, Wang B, Zong L. Eudragit(R) L100-coated mannosylated chitosan nanoparticles for oral protein vaccine delivery. Int J Biol Macromol 2018;113:534 42. [140] Bhavsar C, Momin M, Gharat S, Omri A. Functionalized and graft copolymers of chitosan and its pharmaceutical applications. Expert Opin Drug Deliv 2017;14(10):1189 204. [141] Islam MA, Firdous J, Choi YJ, Yun CH, Cho CS. Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review. Int J Nanomedicine 2012;7:6077 93. [142] Mi FL, Wu YY, Lin YH, Sonaje K, Ho YC, Chen CT, et al. Oral delivery of peptide drugs using nanoparticles self-assembled by poly(gamma-glutamic acid)

[143]

[144]

[145]

[146]

[147]

[148]

[149]

[150]

[151]

[152]

and a chitosan derivative functionalized by trimethylation. Bioconjug Chem 2008;19(6):1248 55. Nomura D, Saito M, Takahashi Y, Takahashi Y, Takakura Y, Nishikawa M. Development of orallydeliverable DNA hydrogel by microemulsification and chitosan coating. Int J Pharm 2018;547 (1 2):556 62. Onuigbo E, Iseghohimhen J, Chah K, Gyang M, Attama A. Chitosan/alginate microparticles for the oral delivery of fowl typhoid vaccine: innate and acquired immunity. Vaccine 2018;36(33):4973 8. Oliveira CR, Rezende CM, Silva MR, Pego AP, Borges O, Goes AM. A new strategy based on SmRho protein loaded chitosan nanoparticles as a candidate oral vaccine against schistosomiasis. PLoS Negl Trop Dis 2012;6(11):e1894. Shukla A, Mishra V, Bhoop BS, Katare OP. Alginate coated chitosan microparticles mediated oral delivery of diphtheria toxoid. Part A. Systematic optimization, development and characterization. Int J Pharm 2015;495(1):220 33. Hosseini M, Dobakhti F, Pakzad SR, Ajdary S. Immunization with single oral dose of alginateencapsulated BCG elicits effective and long-lasting mucosal immune responses. Scand J Immunol 2015;82(6):489 97. Pastor M, Esquisabel A, Talavera A, Ano G, Fernandez S, Cedre B, et al. An approach to a cold chain free oral cholera vaccine: in vitro and in vivo characterization of Vibrio cholerae gastro-resistant microparticles. Int J Pharm 2013;448(1):247 58. Arenas-Gamboa AM, Ficht TA, Davis DS, Elzer PH, Kahl-McDonagh M, Wong-Gonzalez A, et al. Oral vaccination with microencapsuled strain 19 vaccine confers enhanced protection against Brucella abortus strain 2308 challenge in red deer (Cervus elaphus elaphus). J Wildl Dis 2009;45(4):1021 9. Sun H, Pan H, Yang Z, Shi M. The immune response and protective efficacy of vaccination with oral microparticle Aeromonas sobria vaccine in mice. Int Immunopharmacol 2007;7(9):1259 64. Sun Y, Liu CS, Sun L. Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain. Vaccine 2010;28(40):6603 8. Clark S, Cross ML, Smith A, Court P, Vipond J, Nadian A, et al. Assessment of different formulations of oral Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine in rodent models for immunogenicity and protection against aerosol challenge with M. bovis. Vaccine 2008;26(46):5791 7.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

353

REFERENCES

[153] De Smet R, Allais L, Cuvelier CA. Recent advances in oral vaccine development: yeast-derived beta-glucan particles. Hum Vaccin Immunother 2014;10 (5):1309 18. [154] Soto ER, Ostroff GR. Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery. Bioconjug Chem 2008;19(4):840 8. [155] De Smet R, Demoor T, Verschuere S, Dullaers M, Ostroff GR, Leclercq G, et al. beta-Glucan microparticles are good candidates for mucosal antigen delivery in oral vaccination. J Control Release 2013;172 (3):671 8. [156] Huang H, Ostroff GR, Lee CK, Wang JP, Specht CA, Levitz SM. Distinct patterns of dendritic cell cytokine release stimulated by fungal beta-glucans and toll-like receptor agonists. Infect Immun 2009;77(5):1774 81. [157] Brown GD, Gordon S. Immune recognition. A new receptor for beta-glucans. Nature 2001;413(6851):36 7. [158] Soares E, Jesus S, Borges O. Oral hepatitis B vaccine: chitosan or glucan based delivery systems for efficient HBsAg immunization following subcutaneous priming. Int J Pharm 2018;535(1 2):261 71. [159] Azegami T, Itoh H, Kiyono H, Yuki Y. Novel transgenic rice-based vaccines. Arch Immunol Ther Exp (Warsz) 2015;63(2):87 99. [160] Sohrab SS, Suhail M, Kamal MA, Husen A, Azhar EI. Recent development and future prospects of plantbased vaccines. Curr Drug Metab 2017;18(9):831 41. [161] Rybicki EP. Plant-made vaccines and reagents for the One Health initiative. Hum Vaccin Immunother 2017;13(12):2912 17. [162] Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, et al. Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Acad Sci U S A 2007;104 (26):10986 91. [163] Tokuhara D, Yuki Y, Nochi T, Kodama T, Mejima M, Kurokawa S, et al. Secretory IgA-mediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine. Proc Natl Acad Sci U S A 2010;107(19):8794 9. [164] Simanjuntak CH, Paleologo FP, Punjabi NH, Darmowigoto R, Soeprawoto, Totosudirjo H, et al. Oral immunisation against typhoid fever in Indonesia with Ty21a vaccine. Lancet 1991;338(8774):1055 9. [165] Peters W, Brandl JR, Lindbloom JD, Martinez CJ, Scallan CD, Trager GR, et al. Oral administration of an adenovirus vector encoding both an avian influenza A hemagglutinin and a TLR3 ligand induces antigen specific granzyme B and IFN-gamma T cell responses in humans. Vaccine 2013;31(13):1752 8. [166] Azegami T, Yuki Y, Hayashi K, Hishikawa A, Sawada SI, Ishige K, et al. Intranasal vaccination against

[167]

[168]

[169]

[170]

[171]

[172] [173]

[174]

[175]

[176]

[177]

[178]

[179]

angiotensin II type 1 receptor and pneumococcal surface protein A attenuates hypertension and pneumococcal infection in rodents. J Hypertens 2017;36 (2):387 94. Riese P, Sakthivel P, Trittel S, Guzman CA. Intranasal formulations: promising strategy to deliver vaccines. Expert Opin Drug Deliv 2014;11(10):1619 34. Azegami T, Yuki Y, Nakahashi R, Itoh H, Kiyono H. Nanogel-based nasal vaccines for infectious and lifestyle-related diseases. Mol Immunol 2018;98: 19 24. Azegami T, Yuki Y, Sawada S, Mejima M, Ishige K, Akiyoshi K, et al. Nanogel-based nasal ghrelin vaccine prevents obesity. Mucosal Immunol 2017;10 (5):1351 60. Pires A, Fortuna A, Alves G, Falcao A. Intranasal drug delivery: how, why and what for? J Pharm Pharm Sci 2009;12(3):288 311. Dahl R, Mygind N. Anatomy, physiology and function of the nasal cavities in health and disease. Adv Drug Deliv Rev 1998;29(1 2):3 12. Illum L. Nasal drug delivery: new developments and strategies. Drug Discov Today 2002;7(23):1184 9. Illum L. Nanoparticulate systems for nasal delivery of drugs: a real improvement over simple systems? J Pharm Sci 2007;96(3):473 83. Heurtault B, Gentine P, Thomann JS, Baehr C, Frisch B, Pons F. Design of a liposomal candidate vaccine against Pseudomonas aeruginosa and its evaluation in triggering systemic and lung mucosal immunity. Pharm Res 2009;26(2):276 85. Date Y, Ebisawa M, Fukuda S, Shima H, Obata Y, Takahashi D, et al. NALT M cells are important for immune induction for the common mucosal immune system. Int Immunol 2017;29(10):471 8. Petersson P, Hedenskog M, Alves D, Brytting M, Schroder U, Linde A, et al. The Eurocine L3 adjuvants with subunit influenza antigens induce protective immunity in mice after intranasal vaccination. Vaccine 2010;28(39):6491 7. Hong SH, Byun YH, Nguyen CT, Kim SY, Seong BL, Park S, et al. Intranasal administration of a flagellinadjuvanted inactivated influenza vaccine enhances mucosal immune responses to protect mice against lethal infection. Vaccine 2012;30(2):466 74. Ichinohe T, Watanabe I, Ito S, Fujii H, Moriyama M, Tamura S, et al. Synthetic double-stranded RNA poly (I:C) combined with mucosal vaccine protects against influenza virus infection. J Virol 2005;79(5):2910 19. Ainai A, Suzuki T, Tamura SI, Hasegawa H. Intranasal administration of whole inactivated influenza virus vaccine as a promising influenza vaccine candidate. Viral Immunol 2017;30(6):451 62.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

354

19. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

[180] Ainai A, Tamura S, Suzuki T, Ito R, Asanuma H, Tanimoto T, et al. Characterization of neutralizing antibodies in adults after intranasal vaccination with an inactivated influenza vaccine. J Med Virol 2012;84 (2):336 44. [181] Ainai A, Tamura S, Suzuki T, van Riet E, Ito R, Odagiri T, et al. Intranasal vaccination with an inactivated whole influenza virus vaccine induces strong antibody responses in serum and nasal mucus of healthy adults. Hum Vaccin Immunother 2013;9(9):1962 70. [182] Aoshi T. Modes of action for mucosal vaccine adjuvants. Viral Immunol 2017;30(6):463 70. [183] Rhee JH, Lee SE, Kim SY. Mucosal vaccine adjuvants update. Clin Exp Vaccine Res 2012;1(1):50 63. [184] Mutsch M, Zhou W, Rhodes P, Bopp M, Chen RT, Linder T, et al. Use of the inactivated intranasal influenza vaccine and the risk of Bell’s palsy in Switzerland. N Engl J Med 2004;350(9):896 903. [185] Connell TD. Cholera toxin, LT-I, LT-IIa and LT-IIb: the critical role of ganglioside binding in immunomodulation by type I and type II heat-labile enterotoxins. Expert Rev Vaccines 2007;6(5):821 34. [186] Fukuyama Y, Okada K, Yamaguchi M, Kiyono H, Mori K, Yuki Y. Nasal administration of cholera toxin as a mucosal adjuvant damages the olfactory system in mice. PLoS One 2015;10(9):e0139368. [187] Lin YL, Chow YH, Huang LM, Hsieh SM, Cheng PY, Hu KC, et al. A CpG-adjuvanted intranasal enterovirus 71 vaccine elicits mucosal and systemic immune responses and protects human SCARB2-transgenic mice against lethal challenge. Sci Rep 2018;8(1):10713. [188] Nguyen CT, Kim SY, Kim MS, Lee SE, Rhee JH. Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice. Vaccine 2011;29(34):5731 9. [189] Fukuyama Y, Ikeda Y, Ohori J, Sugita G, Aso K, Fujihashi K, et al. A molecular mucosal adjuvant to enhance immunity against pneumococcal infection in the elderly. Immune Netw 2015;15(1):9 15. [190] Allen AC, Wilk MM, Misiak A, Borkner L, Murphy D, Mills KHG. Sustained protective immunity against Bordetella pertussis nasal colonization by intranasal immunization with a vaccine-adjuvant combination that induces IL-17-secreting TRM cells. Mucosal Immunol. 2018;11(6):1763 76. [191] Ugwoke MI, Agu RU, Verbeke N, Kinget R. Nasal mucoadhesive drug delivery: background, applications, trends and future perspectives. Adv Drug Deliv Rev 2005;57(11):1640 65. [192] Baudner BC, O’Hagan DT. Bioadhesive delivery systems for mucosal vaccine delivery. J Drug Target 2010;18(10):752 70.

[193] Lai SK, Wang YY, Hanes J. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv Drug Deliv Rev 2009;61(2):158 71. [194] Witten J, Samad T, Ribbeck K. Selective permeability of mucus barriers. Curr Opin Biotechnol 2018;52:124 33. [195] Cone RA. Barrier properties of mucus. Adv Drug Deliv Rev 2009;61(2):75 85. [196] Schneider CS, Xu Q, Boylan NJ, Chisholm J, Tang BC, Schuster BS, et al. Nanoparticles that do not adhere to mucus provide uniform and long-lasting drug delivery to airways following inhalation. Sci Adv 2017;3 (4):e1601556. [197] Wang YY, Lai SK, So C, Schneider C, Cone R, Hanes J. Mucoadhesive nanoparticles may disrupt the protective human mucus barrier by altering its microstructure. PLoS One 2011;6(6):e21547. [198] Comfort C, Garrastazu G, Pozzoli M, Sonvico F. Opportunities and challenges for the nasal administration of nanoemulsions. Curr Top Med Chem 2015;15(4):356 68. [199] Wong PT, Wang SH, Ciotti S, Makidon PE, Smith DM, Fan Y, et al. Formulation and characterization of nanoemulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immunogenicity. Mol Pharm 2014;11(2):531 44. [200] Bielinska AU, Janczak KW, Landers JJ, Markovitz DM, Montefiori DC, Baker Jr. JR. Nasal immunization with a recombinant HIV gp120 and nanoemulsion adjuvant produces Th1 polarized responses and neutralizing antibodies to primary HIV type 1 isolates. AIDS Res Hum Retroviruses 2008;24(2):271 81. [201] Heurtault B, Frisch B, Pons F. Liposomes as delivery systems for nasal vaccination: strategies and outcomes. Expert Opin Drug Deliv 2010;7(7):829 44. [202] Foged C, Arigita C, Sundblad A, Jiskoot W, Storm G, Frokjaer S. Interaction of dendritic cells with antigencontaining liposomes: effect of bilayer composition. Vaccine 2004;22(15 16):1903 13. [203] Ingvarsson PT, Rasmussen IS, Viaene M, Irlik PJ, Nielsen HM, Foged C. The surface charge of liposomal adjuvants is decisive for their interactions with the Calu-3 and A549 airway epithelial cell culture models. Eur J Pharm Biopharm 2014;87(3):480 8. [204] Lopes LM, Chain BM. Liposome-mediated delivery stimulates a class I-restricted cytotoxic T cell response to soluble antigen. Eur J Immunol 1992;22(1):287 90. [205] Okada E, Sasaki S, Ishii N, Aoki I, Yasuda T, Nishioka K, et al. Intranasal immunization of a DNA vaccine with IL-12- and granulocyte-macrophage colonystimulating factor (GM-CSF)-expressing plasmids in liposomes induces strong mucosal and cell-mediated immune responses against HIV-1 antigens. J Immunol 1997;159(7):3638 47.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

355

REFERENCES

[206] Jiang PL, Lin HJ, Wang HW, Tsai WY, Lin SF, Chien MY, et al. Galactosylated liposome as a dendritic cell-targeted mucosal vaccine for inducing protective anti-tumor immunity. Acta Biomater 2015;11: 356 67. [207] Wang D, Christopher ME, Nagata LP, Zabielski MA, Li H, Wong JP, et al. Intranasal immunization with liposome-encapsulated plasmid DNA encoding influenza virus hemagglutinin elicits mucosal, cellular and humoral immune responses. J Clin Virol 2004;31 (Suppl. 1):S99 106. [208] McNeela EA, O’Connor D, Jabbal-Gill I, Illum L, Davis SS, Pizza M, et al. A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM(197)) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery. Vaccine 2000;19 (9 10):1188 98. [209] Hagenaars N, Verheul RJ, Mooren I, de Jong PH, Mastrobattista E, Glansbeek HL, et al. Relationship between structure and adjuvanticity of N,N,N-trimethyl chitosan (TMC) structural variants in a nasal influenza vaccine. J Control Release 2009;140 (2):126 33. [210] Jabbal-Gill I, Fisher AN, Rappuoli R, Davis SS, Illum L. Stimulation of mucosal and systemic antibody responses against Bordetella pertussis filamentous haemagglutinin and recombinant pertussis toxin after nasal administration with chitosan in mice. Vaccine 1998;16(20):2039 46. [211] McNeela EA, Jabbal-Gill I, Illum L, Pizza M, Rappuoli R, Podda A, et al. Intranasal immunization with genetically detoxified diphtheria toxin induces T cell responses in humans: enhancement of Th2 responses and toxin-neutralizing antibodies by formulation with chitosan. Vaccine 2004;22(8):909 14. [212] Hagenaars N, Mania M, de Jong P, Que I, Nieuwland R, Slutter B, et al. Role of trimethylated chitosan (TMC) in nasal residence time, local distribution and toxicity of an intranasal influenza vaccine. J Control Release 2010;144(1):17 24. [213] Jabbal-Gill I, Watts P, Smith A. Chitosan-based delivery systems for mucosal vaccines. Expert Opin Drug Deliv 2012;9(9):1051 67. [214] Zhang J, Xia W, Liu P, Cheng Q, Tahirou T, Gu W, et al. Chitosan modification and pharmaceutical/biomedical applications. Mar Drugs 2010;8(7):1962 87. [215] Dodane V, Amin Khan M, Merwin JR. Effect of chitosan on epithelial permeability and structure. Int J Pharm 1999;182(1):21 32. [216] Vicente S, Peleteiro M, Diaz-Freitas B, Sanchez A, Gonzalez-Fernandez A, Alonso MJ. Co-delivery of viral proteins and a TLR7 agonist from polysaccharide

[217]

[218] [219]

[220]

[221]

[222]

[223]

[224]

[225]

[226]

nanocapsules: a needle-free vaccination strategy. J Control Release 2013;172(3):773 81. Slutter B, Bal S, Keijzer C, Mallants R, Hagenaars N, Que I, et al. Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen. Vaccine 2010;28(38):6282 91. Bernocchi B, Carpentier R, Betbeder D. Nasal nanovaccines. Int J Pharm 2017;530(1 2):128 38. Carroll EC, Jin L, Mori A, Munoz-Wolf N, Oleszycka E, Moran HBT, et al. The vaccine adjuvant chitosan promotes cellular immunity via DNA sensor cGASSTING-dependent induction of type I interferons. Immunity 2016;44(3):597 608. Gupta NK, Tomar P, Sharma V, Dixit VK. Development and characterization of chitosan coated poly-(varepsilon-caprolactone) nanoparticulate system for effective immunization against influenza. Vaccine 2011;29(48):9026 37. Kang ML, Jiang HL, Kang SG, Guo DD, Lee DY, Cho CS, et al. Pluronic F127 enhances the effect as an adjuvant of chitosan microspheres in the intranasal delivery of Bordetella bronchiseptica antigens containing dermonecrotoxin. Vaccine 2007;25 (23):4602 10. Verheul RJ, Slutter B, Bal SM, Bouwstra JA, Jiskoot W, Hennink WE. Covalently stabilized trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination. J Control Release 2011;156 (1):46 52. Jiang HL, Kang ML, Quan JS, Kang SG, Akaike T, Yoo HS, et al. The potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization. Biomaterials 2008;29(12):1931 9. Atmar RL, Bernstein DI, Harro CD, Al-Ibrahim MS, Chen WH, Ferreira J, et al. Norovirus vaccine against experimental human Norwalk Virus illness. N Engl J Med 2011;365(23):2178 87. Huo Z, Sinha R, McNeela EA, Borrow R, Giemza R, Cosgrove C, et al. Induction of protective serum meningococcal bactericidal and diphtherianeutralizing antibodies and mucosal immunoglobulin A in volunteers by nasal insufflations of the Neisseria meningitidis serogroup C polysaccharide-CRM197 conjugate vaccine mixed with chitosan. Infect Immun 2005;73(12):8256 65. Coucke D, Schotsaert M, Libert C, Pringels E, Vervaet C, Foreman P, et al. Spray-dried powders of starch and crosslinked poly(acrylic acid) as carriers for nasal delivery of inactivated influenza vaccine. Vaccine 2009;27(8):1279 86.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

356

19. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

[227] von Hoegen P. Synthetic biomimetic supra molecular Biovector (SMBV) particles for nasal vaccine delivery. Adv Drug Deliv Rev 2001;51(1 3):113 25. [228] Le MQ, Carpentier R, Lantier I, Ducournau C, Dimier-Poisson I, Betbeder D. Residence time and uptake of porous and cationic maltodextrin-based nanoparticles in the nasal mucosa: comparison with anionic and cationic nanoparticles. Int J Pharm 2018;550(1 2):316 24. [229] Jaganathan KS, Vyas SP. Strong systemic and mucosal immune responses to surface-modified PLGA microspheres containing recombinant hepatitis B antigen administered intranasally. Vaccine 2006;24(19):4201 11. [230] Wang S, Liu H, Zhang X, Qian F. Intranasal and oral vaccination with protein-based antigens: advantages, challenges and formulation strategies. Protein Cell 2015;6(7):480 503. [231] Nagatomo D, Taniai M, Ariyasu H, Taniguchi M, Aga M, Ariyasu T, et al. Cholesteryl pullulan encapsulated TNF-alpha nanoparticles are an effective mucosal vaccine adjuvant against influenza virus. Biomed Res Int 2015;2015:471468. [232] Nakahashi-Ouchida R, Yuki Y, Kiyono H. Cationic pullulan nanogel as a safe and effective nasal vaccine delivery system for respiratory infectious diseases. Hum Vaccin Immunother 2018;1 5.

Further Reading Brandhonneur N, Loizel C, Chevanne F, Wakeley P, Jestin A, Le Potier MF, et al. Mucosal or systemic administration of rE2 glycoprotein antigen loaded PLGA microspheres. Int J Pharm 2009;373(1 2):16 23.

Jia Y, Krishnan L, Omri A. Nasal and pulmonary vaccine delivery using particulate carriers. Expert Opin Drug Deliv 2015;12(6):993 1008. Lewis DJ, Huo Z, Barnett S, Kromann I, Giemza R, Galiza E, et al. Transient facial nerve paralysis (Bell’s palsy) following intranasal delivery of a genetically detoxified mutant of Escherichia coli heat labile toxin. PLoS One 2009;4(9):e6999. Mansoor F, Earley B, Cassidy JP, Markey B, Foster C, Doherty S, et al. Intranasal delivery of nanoparticles encapsulating BPI3V proteins induces an early humoral immune response in mice. Res Vet Sci 2014;96 (3):551 7. Mills KH. Induction, function and regulation of IL-17producing T cells. Eur J Immunol 2008;38(10):2636 49. Pan L, Zhang Z, Lv J, Zhou P, Hu W, Fang Y, et al. Induction of mucosal immune responses and protection of cattle against direct-contact challenge by intranasal delivery with foot-and-mouth disease virus antigen mediated by nanoparticles. Int J Nanomedicine 2014;9:5603 18. Tamura S, Yamanaka A, Shimohara M, Tomita T, Komase K, Tsuda Y, et al. Synergistic action of cholera toxin B subunit (and Escherichia coli heat-labile toxin B subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine. Vaccine 1994;12 (5):419 26. Therien HM, Shahum E. Importance of physical association between antigen and liposomes in liposomes adjuvanticity. Immunol Lett 1989;22(4):253 8. van Ginkel FW, Jackson RJ, Yuki Y, McGhee JR. Cutting edge: The mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues. J Immunol 2000;165 (9):4778 82.

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Plant-Based Mucosal Vaccine Delivery Systems Tatsuhiko Azegami1, Yoshikazu Yuki1 and Hiroshi Kiyono2,3,4 1

International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 2Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 3 Mucosal Immunology and Allergy Therapeutics, Graduate School of Medicine, Chiba University, Chiba, Japan 4Division of Gastroenterology, Department of Medicine, (CU-UCSD cMAV) Center for Mucosal Immunology, Allergy and Vaccines, University of California, San Diego, CA, United States

I. INTRODUCTION The discovery of the mechanism of gene transfer between Agrobacterium tumefaciens and plants [1] advanced plant genetic engineering. In 1990, Curtiss and Cardineau showed the use of this transfer for vaccines, expressing the Streptococcus mutans surface protein antigen A, a virulent antigen associated with dental caries, in tobacco leaves (Curtiss R and Cardineau C, 1990, WIPO Patent: WO90/02484). Since then, researchers have tried to develop plantbased vaccines (PbVs) as a platform for the realization of “edible vaccines” because of their possible practical advantages over traditional injectable vaccines, notably convenient administration, safety, low cost, and long storage life (Table 20.1).

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00020-1

Cost-related hurdles, including the production, storage, transportation, and administration of vaccines by medical staff, need to be overcome for expanding mass immunization programs in developing countries, where two thirds of deaths are due to infectious diseases [2], and vaccines are most effective for disease control [3]. PbVs can substantially reduce the cost of production of vaccine antigens, and thus permit affordable mass immunization in low-income countries. Costs of raw materials (the antigens) produced in plants can be up to 99.9% lower than the costs of those produced in animal cells, and 99% lower than the cost of bacterial or yeast cell production; such cost reductions are estimated to reduce the vaccine price by 31% [4]. In addition, antigens

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TABLE 20.1 Advantages and Disadvantages of Plant-Based Vaccines ADVANTAGES Safety

Low risk of contamination with pathogenic microorganisms during vaccine production

Cost

Low cost of production No requirement for purification

Storage

No need for cold chain

Delivery

Resistant to gastrointestinal digestion

Scaling-up

Rapid scaling-up of production

DISADVANTAGES Allergy

Risk of allergenic potential of plant-derived proteins

Immune response

Risk of induction of oral tolerance after oral administration

produced in plants can save the costs of antigen purification and cold-chain storage; for example, cereals (e.g., corn and rice) have been tested for the preparation of cold-chain-free vaccines [5 7]. Among grain-based candidates, MucoRice, a rice-based vaccine production and oral delivery system, has been developed and advanced to the clinical trial stage [8]. MucoRice can be administered orally after pulverization of rice grains without any other purification and can be stored at room temperature for several years [6,9] (Chapter 19: Current and New Approaches for Mucosal Vaccine Delivery). In addition, the production of antigens in plants prevents the risk of contamination by pathogens, which can occur during the preparation of vaccines from whole pathogens or derived subunits produced in Escherichia coli, yeast, or mammalian cell culture systems [10], as in previous viral vaccines (e.g., SV40 in polio vaccines, bacteriophages in measles and polio vaccines, and porcine circovirus in rotavirus vaccines) [11]. PbVs can eliminate the possibility of such contamination because the plantbased transgene expression system requires neither pathogens nor primary cell cultures. In contrast, antigens produced in plants are sometimes modified posttranslationally (e.g.,

N-glycosylation), acquiring the potential to induce allergic responses [8]. However, recent advances in glycol engineering in plants enable the optimization of glycan structures to improve safety and to control posttranslational modification of glycoproteins [12]. PbVs have the further advantage of rapid scaling up, enabling the rapid creation of vaccines for pandemic and endemic situations. A PbV-targeting hemagglutinin (HA) of influenza viruses is a promising approach for controlling seasonal influenza, because antigenic variation in influenza viruses caused by mutations in HA can cause recurrent seasonal outbreaks and pandemics. For example, 50 90 mg of purified vaccine antigen can be obtained from 1 kg of tobacco leaves, and this system can be easily scaled up under Good Manufacturing Practice (GMP) conditions [13]. Orally administered PbVs should effectively induce antigen-specific mucosal and systemic immune responses and simultaneously overcome the possibility of immunological hyporesponsiveness (i.e., oral tolerance). Although the induction of oral tolerance to orally delivered vaccines is a potential concern, evidence of oral tolerance to PbVs has not been documented [14]. In general, orally

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II. TRANSGENIC TECHNOLOGIES FOR VACCINE PRODUCTION IN PLANTS

administered antigens should be taken up from the lumen of the digestive tract by microfold (M) cells in the follicle-associated epithelium of gut-associated lymphoreticular tissue (GALT) for the induction of antigenspecific mucosal immune responses [15,16] (see related discussions in Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance, Chapter 3: Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells and Chapter 28: M Cell-Targeted Vaccines). When M cell-specific monoclonal antibody (NKM 16-2-4) is used as a carrier for M cell-targeted oral vaccine, the conjugated vaccine antigens are effectively taken up from M cells and induce antigenspecific serum IgG and mucosal IgA antibodies [17]. Rice seeds expressing cholera toxin B subunit (CTB) are protected from digestive enzymes when orally administered, and the inserted CTB is effectively delivered to and taken up by mucosal M cells [6]. This rice-based oral CTB vaccine induced a protective immune response against cholera infection in mice [6,7]. Another M celltargeting PbV candidate, transgenic rice calluses expressing a gene encoding the cEDIII envelope protein of dengue virus fused to the Co1 M cell-targeting peptide ligand, delivered the cEDIII Co1 fusion protein to M cells in Peyer’s patches when orally administered [18]. This chapter introduces and discusses genetic engineering technologies applied to PbVs and summarizes progress in the development of PbVs against human infectious diseases.

II. TRANSGENIC TECHNOLOGIES FOR VACCINE PRODUCTION IN PLANTS Various plants can express potential antigenic foreign proteins via stable or transient genetic transformation (Fig. 20.1) [8,19 21]. In stable transformation, genes are integrated into the nuclear genome or a plasmid genome

FIGURE 20.1

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Plant-based vaccine expression system.

by biolistic transformation or A. tumefaciensmediated transformation, and the resulting transgenic plants can genomically express antigenic proteins [8]. In transient transformation, simple tissue infiltration with A. tumefaciens (agroinfiltration) or viral vectors can deliver antigenic proteins within a week for short-term expression [8].

A. Biolistic Method for Stable Transformation Biolistic transformation enables the direct introduction of DNA or RNA into plant cells. In this method, a foreign DNA or RNA construct is coated onto gold or tungsten particles. The particles are released from a gene gun by high-pressure helium gas and directly penetrate the host cell wall [22]. For example, biolistic bombardment with a gene encoding an antigen of Bacillus anthracis, which causes the deadly disease anthrax, into chloroplasts of tobacco-produced transgenic tobacco leaves [23] in which the antigen constituted up to 14.2% of total soluble proteins. Mice subcutaneously immunized with this antigen were completely protected against a lethal challenge of anthrax toxin [23]. The advantages of the biolistic method are that a variety of plants can be transformed, no vector is required, and the protocol is simple [24]. The disadvantages

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include that DNA integrates randomly into plant cells (e.g., messy integration pattern, uncontrolled cellular target), the gene gun is expensive, and the plant may be subjected to severe tissue damage [24].

B. Agrobacterium-Mediated Transformation A. tumefaciens is a Gram-negative bacterium that naturally infects many different plants and transfers its genes (via transfer DNA:T-DNA) into the plant genome [1]. Introduced T-DNA is transcribed in the plant cells. A gene of interest can be inserted into the tumor-inducing (Ti) plasmid of the T-DNA. The Ti plasmid includes genes encoding plant hormones such as auxins and cytokinins, which support tumor induction in plants. These genes are deleted in order to produce vaccine antigens in plants [8]. For vaccine production, a T-DNA vector incorporating a gene of interest is transformed into A. tumefaciens. Infection of plants by the transformed A. tumefaciens results in the integration of the gene of interest into the host plant genomic DNA [1]. After the establishment of a

transgenic line, stable production of the target protein is obtained. The advantages of Agrobacterium-mediated transformation include minimal equipment costs, the insertion of a single copy or low copy number of the transgene, the transfer of a large segment of foreign DNA, and the high quality and fertility of transgenic plants [25]. In addition, Agrobacterium is used to transiently and rapidly introduce recombinant genes into leaves in a method generally called agroinfiltration [26]. In the popular syringe agroinfiltration method, A. tumefaciens harboring a gene of interest is suspended in infiltration buffer, and the suspension is injected into nicks in leaves with a needleless syringe [27]. Syringe infiltration is a simple and easy procedure without the need for any specialized equipment, and allows rapid and high-level transient expression of vaccine antigens.

C. MucoRice System As an example of a transgenic expression system, we describe the MucoRice protocol (Fig. 20.2). First, a gene for an antigen is FIGURE 20.2 MucoRice production system. Each batch of MucoRiceCTB was produced in a full-closed type hydroponics system according to GMP. The workflow inside the facility is shown, from germinating seeds from the seed bank to processing the drug substance from harvested rice. The final formulation of the vaccine product for phase I study is performed in a GMP-compliant facility.

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III. PLANT-BASED VACCINES FOR THE PREVENTION AND CONTROL OF INFECTIOUS DISEASES

optimized for translation into rice and inserted into a plasmid vector. The resultant T-DNA plasmid vector contains an antigen overexpression cassette (vaccine cassette) with the sequence encoding the antigen under the control of the rice 13-kD prolamin promoter, an RNAi cassette including an antisense sequence specific to endogenous rice storage proteins to suppress major storage proteins (13-kD prolamin and glutelin) under the control of the ubiquitin promoter, and an antibiotic resistance gene [6]. This plasmid is transformed into A. tumefaciens by electroporation. For the induction of callus (a mass of unorganized parenchymal cells), rice seeds are incubated on a medium containing an auxin. The rice callus is coincubated with transformed A. tumefaciens on a medium including acetosyringone, which enhances the virulence of A. tumefaciens [28]. The infected callus is then incubated with antibiotics for the selection of transformed tissues. Selected callus is incubated and grown until shoot formation. Then shoots are transferred to the growing medium, and the rice plants are raised in pots until the harvest of rice grains. The plant line with the highest levels of antigen accumulated in the rice seed is selected and advanced to the next generation by self-crossing to obtain homozygous lines [29].

III. PLANT-BASED VACCINES FOR THE PREVENTION AND CONTROL OF INFECTIOUS DISEASES A few PbVs against infectious diseases have reached the clinical trial stage (Table 20.2) [8]. These vaccines are administered via the mucosal (oral) or systemic (intramuscular) route and target gastrointestinal and other infectious diseases.

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A. Enterotoxigenic Escherichia coli According to the Global Burden of Disease Study 2015, enterotoxigenic E. coli (ETEC) infection causes an estimated 741,000 deaths worldwide every year [45]. ETEC produces various enterotoxins, including heat-labile enterotoxin (LT) and heat-stable enterotoxin. LT is composed of an enzymatically active A subunit and a nonactive pentameric B subunit (LT-B) [46]. LT-B binds irreversibly to GM1 ganglioside, which is abundant in the intestinal brush border membrane, and the A subunit activates adenylate cyclase and subsequently increases intracellular cyclic AMP, resulting in fluid secretion in the intestine [47]. Oral administration of recombinant subunit vaccine consisting of an epitope of LT-B induces anti-LT-B immune responses and neutralizes the toxicity of LT in humans [48]. Therefore LT-B, which is an enzymatically inactive (i.e., nontoxic) subunit of LT, is a promising candidate for a vaccine antigen against ETEC infection (Chapter 32: Oral Vaccines for Enterotoxigenic Escherichia coli). Tacket et al. produced LT-B as a potential vaccine antigen in potato and corn [30,31]. Their transgenic potato produced 3.7 15.7 µg/g of LT-B [30]. When volunteers ate 50 or 100 g of transgenic raw potato containing 0.4 1.1 mg (mean: 0.75 mg) of LT-B on days 0, 7, and 21, 91% developed serum anti-LT IgG antibodies, 55% developed serum anti-LT IgA antibodies, and 50% developed fecal anti-LT secretory IgA (SIgA) antibodies [30]. In addition, serum antibodies in 73% of volunteers showed LTneutralizing activity in vitro [30]. The transgenic raw potato was generally well tolerated; 18% of volunteers reported nausea, and 36% reported loose stools, but there was no significant difference in adverse events between vaccinees and placebo controls [30]. LT-B expressed in corn seeds by Agrobacterium-mediated stable transformation

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Plant-Based Vaccines Against Human Infectious Diseases in Clinical Trials Antigen

Plant

Expression System

Vaccine Clinical Administration Trial References

Escherichia coli Heat-labile enterotoxin B subunit Potato

Plant transformation vector

Oral

Phase I

[30]

Escherichia coli Heat-labile enterotoxin B subunit Corn

Agrobacterium Oral stable transformation

Phase I

[31]

Norovirus

Capsid protein

Potato

Plant transformation vector

Oral

Phase I

[32]

Hepatitis B virus

HBs antigen

Lettuce

Agrobacterium Oral stable transformation

Phase I

[33]

Hepatitis B virus

HBs antigen

Potato

Agrobacterium Oral stable transformation

Phase I

[34]

Rabies virus

Chimeric peptide of glycoprotein Spinach plant transformation and nucleoprotein vector

Oral

Phase I

[35]

Influenza virus

Hemagglutinin (A/H5N1)

Tobacco Agrobacterium infiltration (transient)

Intramuscular

Phase I

[36]

Influenza virus

Hemagglutinin (A/H1N1)

Tobacco Agrobacterium infiltration (transient)

Intramuscular

Phase I

[37]

Influenza virus

Hemagglutinin (A/H1N1)

Tobacco Agrobacterium infiltration (transient)

Intramuscular

Phase I

[38]

Influenza virus

Hemagglutinin (H5 or H1)—VLP Tobacco Agrobacterium infiltration (transient)

Intramuscular

Phases I and II

[39 41]

Influenza virus

Hemagglutinin (A/H1N1, A/ H3N2, B/Bris, B/Wis)—VLP

Tobacco Agrobacterium infiltration (transient)

Intramuscular

Phases I and II

[42]

Phase I

[6,7,9,43,44]

Vibrio cholerae Cholera toxin B subunit

Rice

was more stable and homogeneous than that expressed in potato [31]. Volunteers who ate 2.1 g of transgenic corn-germ meal containing 1 mg of LT-B tolerated it well; 11% of vaccinees developed mild diarrhea, and 22% experienced mild cramps, but no significant difference in adverse events between vaccinees and placebo controls was found [31]. Of

Agrobacterium Oral stable transformation

volunteers who ingested transgenic corn containing 1 mg of LT-B on days 0, 7, and 21, 78% developed serum anti-LT IgG, 44% developed serum anti-LT IgA, and 44% developed fecal SIgA. These findings show that orally administered potato and corn containing LT-B induce antigen-specific antibody responses in humans.

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B. Norovirus Norovirus is a leading cause of acute gastroenteritis (with an estimated prevalence of 18%) across all age groups [49]. Although a strategy for the prevention of norovirus infection is needed, there are currently no available vaccines and only a few candidates [50]. The plant-based norovirus VP1 vaccine was produced in potato by using a binary vector containing VP1 expression cassettes and an antibiotic resistance gene cassette [32]. Peeled, raw transgenic potatoes contained 215 751 µg of VP1 per 150-g dose. Oral doses of transgenic potato on days 0, 7, and 21 or on days 0 and 21 increased antigen-specific IgA-secreting cells in the blood of vaccinees [32]. Serum anti-VP1 IgG antibodies were detected in some volunteers even before vaccination because of the high prevalence of norovirus infection. However, 20% of vaccinees newly developed serum antiVP1 IgG, and 30% developed fecal anti-VP1 IgA [32]. Although this vaccine is an attractive strategy for the prevention of norovirusinduced gastroenteritis, problems such as its weak immune response remain to be resolved.

C. Hepatitis B Virus Hepatitis B Virus (HBV) infection causes liver inflammation and can result in liver cirrhosis, hepatocellular carcinoma, and even death. According to the World Health Organization, 257 million people are infected with HBV, and 887,000 people die from complications each year [51]. Although HBV is commonly spread through perinatal transmission or exposure to infected blood, mucosal exposure to infected blood and body fluids can also spread it [51]. Therefore, the development of a vaccine that induces HBV-specific systemic and mucosal immune responses is sought to reduce its spread. To develop an HBV vaccine, Kupusta et al. produced HBV surface antigen (HBsAg) in

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transgenic lettuce (Lactuca sativa) using Agrobacterium-mediated stable transformation and fed it to human healthy volunteers [33]. Raw transgenic lettuce leaves containing 0.1 0.5 µg of HBsAg per 100 g of fresh tissue were administered for oral consumption in two doses 2 months apart (200 1 150 g) to three volunteers, who neither had previous HBV vaccination nor HBV infection [33]. After the second vaccination, HBsAg-specific serum IgG titers above 10 IU/L (protective titer) were induced in two of the vaccinees without any obvious side effects [33]. However, contrary to expectation, it did not induce HBsAg-specific serum IgA [33]. A transgenic potato approach was also tested [34,52,53]. Agrobacterium-mediated stable transformation produced 8.5 6 2.1 µg of HBsAg per 1 g of tuber. In 63% of healthy volunteers given three doses of 100 110 g of peeled raw potato (on days 0, 14, and 28) and in 53% of subjects given two doses (on days 0 and 28), serum anti-HBs IgG titers more than doubled [34]. The levels remained high for at least 6 weeks after vaccination [34]. These trials indicate that oral doses of lettuce- and potato-based HBs vaccines can induce systemic, not mucosal immune responses, against HBsAg.

D. Rabies Rabies is a vaccine-preventable viral disease, yet tens of thousands of cases occur annually in more than 150 countries [54]. The rabies virus infects peripheral motor neurons, which can often result in death upon reaching the central nervous system [54]. An available vaccine for the prevention and treatment of rabies consists of inactivated whole rabies virus, inducing not only antigen-specific serum IgG, but also Th1type cytokine responses [55]. Rabies virus is an RNA virus that encodes nucleoprotein, glycoprotein, phosphoprotein, matrix protein, and

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polymerase [54]. The nucleoprotein plays an important role in viral RNA transcription and replication; the glycoprotein mediates viral entry into host cells through the acetylcholine receptor [54,56,57]. Both nucleoprotein and glycoprotein are antigenic, and potential candidates for vaccine antigens [58,59]. A chimeric peptide containing rabies virus glycoprotein and nucleoprotein was produced in spinach (Spinacia oleracea) by transient expression using tobacco mosaic virus [35]. The transgenic spinach contained 84 µg of chimeric rabies peptide in 20 g of raw leaves. Three oral doses of 20 g of leaves at 2-week intervals induced rabies virus-specific serum IgGs, but did not elicit virus-neutralizing antibodies, in three of five vaccinees [35]. Although vaccination with transgenic spinach alone did not induce virus-neutralizing antibodies, a combination of transgenic spinach (three doses of 150 g of raw leaves on days 0, 14, and 28) and a commercially available rabies vaccine (a single intramuscular injection on day 35) did so in three of nine other vaccinees. This result suggests that a prime-boost vaccination strategy using plant-based and conventional vaccines may enhance immunity against rabies virus, and that the PbV can be used in combination with the current systemic vaccine for priming or boosting immunization.

E. Influenza Virus Seasonal influenza is an acute respiratory infection caused by the influenza virus. There are four types of influenza virus: types A, B, C, and D. Human influenza A and B viruses cause seasonal endemic outbreaks [60], which cause an estimated 291,243 645,832 deaths annually [60]. Since influenza viruses continuously undergo genetic changes in the HA and neuraminidase (NA) genes, called antigenic drift, annual vaccine updates are recommended [61]. Although it takes at least 6 months to produce

large quantities of licensed influenza vaccines through conventional cultures in eggs, transgenic tobacco containing the HA antigen can be harvested a week after agroinfiltration [39]. Hence, a rapid, scalable, low-cost transient expression system will aid in vaccination against seasonal influenza. Chichester et al. reported a phase I clinical trial designed to evaluate the safety and immunogenicity of an HA-based vaccine produced in tobacco leaves. HAI-05, a recombinant subunit HA antigen from the A/Indonesia/05/2005 (H5N1) strain of influenza A virus, was expressed as a vaccine antigen in Nicotiana benthamiana using a tobacco mosaic virus-based transient expression system. Harvested transgenic tobacco leaves were homogenized in buffer and purified to produce HAI-05 antigen [36]. Two doses of HAI-05 vaccine (15, 45, or 90 µg) were injected intramuscularly with 0.3% alum adjuvant into subjects 3 weeks apart. Other volunteers were injected intramuscularly with HAI-05 vaccine (90 µg) without adjuvant. The tobacco HAI-05 vaccine caused no serious adverse events and was well tolerated. Some of the immunized subjects, with or without adjuvant, produced serum HA-inhibiting or NA-inhibiting antibodies [36]. The authors concluded that the HAI-05 vaccine did not require adjuvant for the induction of antibody responses. Jul-Larsen et al. also developed an HA-based influenza vaccine expressed in tobacco leaves [37]. They produced recombinant HA antigen from the A/California/04/2009 (H1N1) strain of influenza A virus in N. benthamiana by agroinfiltration. At 14 days after intramuscular immunization with the HA vaccine together with oil-in-water adjuvant (AS03), 15 of 16 vaccinees produced HA inhibition (HI) titers of $ 40, which is enough to protect against infection with influenza virus [37]. Cummings et al. produced another HA vaccine from the A/California/04/2009 (H1N1) strain in N. benthamiana by agroinfiltration [38].

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III. PLANT-BASED VACCINES FOR THE PREVENTION AND CONTROL OF INFECTIOUS DISEASES

This vaccine was injected intramuscularly into subjects twice at 15, 45, or 90 µg, with or without 0.3% alum adjuvant, 3 weeks apart. HI titer ($40) and seroconversion showed dose responses, with the highest rates of HI titer (100%) and seroconversion (78%) at 90 µg without adjuvant. Contrary to expectation, alum adjuvant did not enhance the HA-specific antibody responses. These results indicate that HA purified from tobacco leaves and administered without adjuvant has potent immunogenicity capable of stimulating antigen-specific humoral immunity. A plant-made virus-like particle (VLP) vaccine bearing HA of influenza virus is another potential candidate vaccine because of favorable immunological characteristics of VLPs due to their size, repeating surface geometry, and potential to induce both innate and adaptive immune responses [62]. The H1-VLP and H5VLP vaccines contained HA of A/California/ 07/2009 (H1N1) or A/Indonesia/05/2005 (H5N1) anchored in the plasma membrane of N. benthamiana cells; the 135-nm-diameter particles consisted of a lipid bilayer studded with HA trimer [39 41]. In a phase I clinical trial, healthy volunteers received a single dose of H1-VLP vaccine at 5, 13, or 28 µg without adjuvant; the HI titer dose-independently reached $ 40 in 75.0% 88.9% of subjects without any serious adverse events or allergic symptoms [39,40]. In contrast, two doses of H5-VLP vaccine at 5, 10, or 20 µg with alum adjuvant induced HI titers of $ 40 in only 16.7%, 25%, and 50%, respectively, of the vaccinees [39,40]. Interestingly, in addition to the induction of antibody responses, both vaccines elicited antigen-specific protective polyfunctional CD41 T cell responses, but only the H1-VLP vaccine induced protective polyfunctional CD81 T cell responses, possibly because only H5-VLP vaccination used alum adjuvant [41]. To protect against different influenza viruses, Pillet et al. developed a plant-based quadrivalent VLP vaccine [42]. They

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produced HA sequences of A/California/07/ 2009 (H1N1), A/Victoria/361/11 (H3N2), B/ Brisbane/60/08 (B/Bris, Victoria lineage) and B/Wisconsin/1/10 (B/Wis, Yamagata lineage) in N. benthamiana by transient expression by agroinfiltration and combined them into a quadrivalent product [42]. Intramuscular injection of the vaccine (one dose of 3, 9, or 15 µg of VLP per strain) induced high HI titers against the four homologous strains with a small dose response, and elicited cross-reactive HI antibody and CD41 T cell responses [42]. These clinical trials evaluating the safety and efficacy of tobacco-derived influenza vaccines suggest that a tobacco-based flu vaccine may be an alternative to the bioreactor system for the production of antigen because of its safety and potential for the induction of antibody responses. In addition, because protein expression in agroinfiltration-induced transgenic tobacco provides a rapid, large-scale production of vaccine antigens, a rapid preparation of tobacco-based flu vaccine will be attractive for the control of pandemic and endemic influenza infection caused by regular mutations in HA.

F. Diarrhea Caused by Vibrio cholerae Cholera is a rapidly dehydrating diarrheal disease caused by infection with Vibrio cholerae. Its annual incidence is estimated to be 2.0 cases per 1000 people in high-risk countries [63]. Serogroups O1 and O139 cause outbreaks worldwide [64]. Vaxchora, which is a live, attenuated, single-dose oral vaccine, is the only vaccine approved by the US Food and Drug Administration for prophylaxis against V. cholera infection [64]. Oral administration of Vaxchora is 90% effective against V. cholera O1 challenge at 10 days after vaccination, and 80% effective at 90 days, but it does not protect against O139 or other non-O1 serogroups [64]. Therefore, it is necessary to develop a new vaccine that covers other serogroups.

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One potential target as a universal cholera vaccine is cholera toxin (CT) enterotoxin. After V. cholerae colonizes the gut, it produces CT when it reaches the small intestine and causes severe diarrhea [65]. CT consists of an enzymatically active dimeric A subunit (CTA) and five identical nontoxic B subunits (CTB) [66]. CTB binds to GM1 ganglioside on the intestinal surface and creates a membrane pore ring for the entrance of CTA into the endosome to initiate the ADP-ribosylate G-protein-mediated adenylate cyclase cascade, leading to rapid fluid loss [67,68]. Thus, CTB is an important CT-binding and entrance molecule with good immunogenicity [66] (Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control). In countries where cholera is endemic (mostly developing countries in Asia and Africa), 1.4 billion people are at risk for the disease, and an estimated 2.8 million cases occur annually [63]. Mass immunization against cholera in these countries at an affordable cost is needed to reduce this global burden. MucoRice (Section II.C) offers a promising strategy [6]. An Agrobacterium-transformed rice seed expressing CTB, known as MucoRice-CTB, was created for the control of V. cholerae enterotoxin induced diarrhea [6,7]. The CTB protein is accumulated in rice protein bodies, which protect it from digestive enzymes [6]. CTB (in 2 3 µm of protein bodies) is taken up by antigen-sampling M cells in the follicle-associated epithelium of Peyer’s patches of the GALT for the initiation of antigen-specific immune responses [6]. CTB in rice seeds is stable at room temperature for more than 3 years without impacting its immunogenicity [7]. In addition to its other advantages, MucoRice-CTB induces antigen-specific protective immune responses in both the systemic and mucosal compartments. Oral administration of MucoRice-CTB to mice induced CT-neutralizing serum IgG and fecal IgA and protected mice from CT-induced diarrhea [6,7]. When cynomolgus macaques

were orally immunized with 667 mg of MucoRice-CTB containing 1 mg of CTB, CTneutralizing serum IgG antibodies were induced and maintained for at least 6 months [69]. Interestingly, MucoRice-CTB may also protect against ETEC infection, because crossprotective immunity against LT is sometimes induced by CT vaccination, owing to the close similarity between the CTB and LT-B subunits [70]. In fact, oral immunization with MucoRice-CTB induced SIgA-mediated protective immunity against LT-induced diarrhea in mice and pigs [7,71]. We recently developed a second-generation MucoRice-CTB, with molecular uniformity and without plant-derived modifications, by overexpressing CTB(N4Q) with a single amino acid substitution at position 4 to remove glycosylation. This MucoRice-CTB was produced at a high level (2.35 mg of CTB/g) in rice seeds by using a CTB(N4Q) overexpression system together with RNAi to suppress the production of prolamin and glutelin B [29,43]. It shows good immunogenicity, and it induced toxin-specific neutralizing SIgA-mediated protective immunity in mice and macaques [43]. In addition, we established an antibiotic-selectionmarker-free MucoRice-CTB for human use by using the Agrobacterium cotransformation system [44]. By performing whole-genome resequencing of this MucoRice-CTB line 51A, we identified and confirmed the location and structure of the transgenes, whereby CTB overexpression and RNAi cassettes were inserted into chromosomes 3 and 12, respectively, as a full tandem structure and a single halved structure [44]. For clinical trials, marker-free MucoRiceCTB line 51A as a seed stock was cultured in a closed MucoRice hydroponics factory at the Institute of Medical Science, University of Tokyo (IMSUT), Japan, which was approved as a GMP factory by the Minister for Health, Labor and Welfare of Japan in 2014 [9] (Fig. 20.2). Because MucoRice-CTB line 51A is considered a genetically modified organism

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REFERENCES

(GMO) and is used as a human oral vaccine, the most essential criterion of the enclosed GMP facility is that the MucoRice is contained within this factory to prevent its release or its exposure to the outside environment. The yield of MucoRice-CTB rice was estimated to be 300 500 g/m2, and the concentration of recombinant CTB protein reached 3 µg/mg in seeds [9] (Fig. 20.2). After the development of the specification test method for MucoRice-CTB, a powder formulation, and the completion of the Good Laboratory Practice safety test, upon receiving approval by the IMSUT Institutional Review Board, we applied to Pharmaceutical and Medical Devices Agency (PMDA) in Japan for permission to investigate this new drug. A double-blind, physician-initiated, phase I clinical trial (UMIN 000018001) was conducted with dose escalation cohorts of 20 healthy subjects. In each cohort, 10 subjects were randomly assigned to the drug arm or a placebo (wildtype rice powder). The MucoRice-CTB or the placebo was administered orally in four doses of 1, 3, or 6 g at 2-week intervals. The Clinical Trial Evaluation Independent Committee found no serious adverse events during the study. Although no events predominantly occurred for the MucoRice-CTB group, the adverse events with high incidence were mostly of low severity. Furthermore, the immunogenicity of MucoRiceCTB was confirmed in humans, and the antigen-specific immune responses were dosedependent (manuscript in revision). These findings suggest that MucoRice-CTB is a safe and immunogenic oral vaccine in humans.

IV. CONCLUDING REMARKS A PbV is a potential candidate for the creation of new mucosal vaccines as well as vaccine production system. Various plants are used as hosts for the stable or transient expression of the transgenes. Because of their low costs of

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production, purification, and storage, these plants are suitable for the development of PbVs against various infectious diseases that occur frequently in low-income countries. The rapid scaling-up of PbVs will enable a substantial contribution to mass vaccination against endemic infectious diseases. Although no PbV has yet been licensed anywhere for clinical use, the advantages of such vaccines will cause them to be approved for human vaccines in the near future.

References [1] Schell J, Van Montagu M. The Ti-plasmid of Agrobacterium tumefaciens, a natural vector for the introduction of nif genes in plants? Basic Life Sci 1977;9:159 79. [2] Dye C. After 2015: infectious diseases in a new era of health and development. Philos Trans R Soc Lond B Biol Sci 2014;369:20130426. [3] Ozawa S, Clark S, Portnoy A, Grewal S, Stack ML, Sinha A, et al. Estimated economic impact of vaccinations in 73 low- and middle-income countries, 2001 2020. Bull World Health Organ 2017;95:629 38. [4] Rybicki EP. Plant-produced vaccines: promise and reality. Drug Discov Today 2009;14:16 24. [5] Hayden CA, Egelkrout EM, Moscoso AM, Enrique C, Keener TK, Jimenez-Flores R, et al. Production of highly concentrated, heat-stable hepatitis B surface antigen in maize. Plant Biotechnol J 2012;10:979 84. [6] Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, et al. Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Acad Sci USA 2007;104:10986 91. [7] Tokuhara D, Yuki Y, Nochi T, Kodama T, Mejima M, Kurokawa S, et al. Secretory IgA-mediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine. Proc Natl Acad Sci USA 2010;107:8794 9. [8] Takeyama N, Kiyono H, Yuki Y. Plant-based vaccines for animals and humans: recent advances in technology and clinical trials. Ther Adv Vaccines 2015;3:139 54. [9] Kashima K, Yuki Y, Mejima M, Kurokawa S, Suzuki Y, Minakawa S, et al. Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants. Plant Cell Rep 2016;35:667 79. [10] Minor P. Considerations for setting the specifications of vaccines. Expert Rev Vaccines 2012;11:579 85.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

368

20. PLANT-BASED MUCOSAL VACCINE DELIVERY SYSTEMS

[11] Petricciani J, Sheets R, Griffiths E, Knezevic I. Adventitious agents in viral vaccines: lessons learned from 4 case studies. Biol J. Int. Assoc. Biol. Standardization 2014;42:223 36. [12] Kim HS, Jeon JH, Lee KJ, Ko K. N-glycosylation modification of plant-derived virus-like particles: an application in vaccines. Biomed Res Int 2014;2014:249519. [13] Shoji Y, Chichester JA, Jones M, Manceva SD, Damon E, Mett V, et al. Plant-based rapid production of recombinant subunit hemagglutinin vaccines targeting H1N1 and H5N1 influenza. Hum Vaccin 2011;7(Suppl):41 50. [14] Streatfield SJ. Plant-based vaccines for animal health. Rev Sci Tech 2005;24:189 99. [15] Azegami T, Yuki Y, Kiyono H. Challenges in mucosal vaccines for the control of infectious diseases. Int Immunol 2014;26:517 28. [16] Ohno H. Intestinal M cells. J Biochem 2016;159:151 60. [17] Nochi T, Yuki Y, Matsumura A, Mejima M, Terahara K, Kim DY, et al. A novel M cell-specific carbohydratetargeted mucosal vaccine effectively induces antigenspecific immune responses. J Exp Med 2007;204:2789 96. [18] Kim TG, Kim MY, Huy NX, Kim SH, Yang MS. M celltargeting ligand and consensus dengue virus envelope protein domain III fusion protein production in transgenic rice calli. Mol Biotechnol 2013;54:880 7. [19] Gleba YY, Tuse´ D, Giritch A. Plant viral vectors for delivery by Agrobacterium. Curr Top Microbiol Immunol 2014;375:155 92. [20] Hefferon K. Plant virus expression vector development: new perspectives. Biomed Res Int 2014;2014:785382. [21] Verma D, Daniell H. Chloroplast vector systems for biotechnology applications. Plant Physiol 2007;145:1129 43. [22] Kikkert JR, Vidal JR, Reisch BI. Stable transformation of plant cells by particle bombardment/biolistics. Methods Mol Biol 2005;286:61 78. [23] Koya V, Moayeri M, Leppla SH, Daniell H. Plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect Immun 2005;73:8266 74. [24] Baltes NJ, Gil-Humanes J, Voytas DF. Genome engineering and agriculture: opportunities and challenges. Prog Mol Biol Transl Sci 2017;149:1 26. [25] Penney CA, Thomas DR, Deen SS, Walmsley AM. Plant-made vaccines in support of the millennium development goals. Plant Cell Rep 2011;30:789 98. [26] Janssen BJ, Gardner RC. Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol 1990;14:61 72. [27] Chen Q, Lai H, Hurtado J, Stahnke J, Leuzinger K, Dent M. Agroinfiltration as an effective and scalable strategy of gene delivery for production of pharmaceutical proteins. Adv Tech Biol Med 2013;1.

[28] Stachel SE, Messens E, Montagu MV, Zambryski P. Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 1985;318:624 9. [29] Kurokawa S, Nakamura R, Mejima M, Kozuka-Hata H, Kuroda M, Takeyama N, et al. J Proteome Res 2013;12:3372 82. [30] Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM, Arntzen CJ. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat Med 1998;4:607 69. [31] Tacket CO, Pasetti MF, Edelman R, Howard JA, Streatfield S. Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn. Vaccine 2004;22:4385 9. [32] Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM, Arntzen CJ. Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes. J Infect Dis 2000;182:302 5. [33] Kapusta J, Modelska A, Figlerowicz M, Pniewski T, Letellier M, Lisowa O, et al. A plant-derived edible vaccine against hepatitis B virus. FASEB J 1999;13:1796 9. [34] Thanavala Y, Mahoney M, Pal S, Scott A, Richter L, Natarajan N, et al. Immunogenicity in humans of an edible vaccine for hepatitis B. Proc Natl Acad Sci USA 2005;102:3378 82. [35] Yusibov V, Hooper DC, Spitsin SV, Fleysh N, Kean RB, Mikheeva T, et al. Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine. Vaccine 2002;20:3155 64. [36] Chichester JA, Jones RM, Green BJ, Stow M, Miao F, Moonsammy G, et al. Safety and immunogenicity of a plant-produced recombinant hemagglutinin-based influenza vaccine (HAI-05) derived from A/Indonesia/ 05/2005 (H5N1) influenza virus: a phase 1 randomized, double-blind, placebo-controlled, dose-escalation study in healthy adults. Viruses 2012;4:3227 44. [37] Jul-Larsen A, Madhun AS, Brokstad KA, Montomoli E, Yusibov V, Cox RJ. The human potential of a recombinant pandemic influenza vaccine produced in tobacco plants. Human Vaccines Immunother 2012;8:653 61. [38] Cummings JF, Guerrero ML, Moon JE, Waterman P, Nielsen RK, Jefferson S, et al. Safety and immunogenicity of a plant-produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza A (H1N1)pdm09 virus: a phase 1 dose-escalation study in healthy adults. Vaccine 2014;32:2251 9. [39] Landry N, Ward BJ, Trepanier S, Montomoli E, Dargis M, Lapini G, et al. Preclinical and clinical development of plant-made virus-like particle vaccine against avian H5N1 influenza. PLoS One 2010;5:e15559.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[40] Ward BJ, Landry N, Trepanier S, Mercier G, Dargis M, Couture M, et al. Human antibody response to Nglycans present on plant-made influenza virus-like particle (VLP) vaccines. Vaccine 2014;32:6098 106. [41] Landry N, Pillet S, Favre D, Poulin JF, Trepanier S, Yassine-Diab B, et al. Influenza virus-like particle vaccines made in Nicotiana benthamiana elicit durable, poly-functional and cross-reactive T cell responses to influenza HA antigens. Clin Immunol 2014;154:164 77. [42] Pillet S, Aubin E, Trepanier S, Bussiere D, Dargis M, Poulin JF, et al. A plant-derived quadrivalent virus like particle influenza vaccine induces cross-reactive antibody and T cell response in healthy adults. Clin Immunol 2016;168:72 87. [43] Yuki Y, Mejima M, Kurokawa S, Hiroiwa T, Takahashi Y, Tokuhara D, et al. Induction of toxin-specific neutralizing immunity by molecularly uniform ricebased oral cholera toxin B subunit vaccine without plant-associated sugar modification. Plant Biotechnol J 2013;11:799 808. [44] Mejima M, Kashima K, Kuroda M, Takeyama N, Kurokawa S, Fukuyama Y, et al. Determination of genomic location and structure of transgenes in marker-free rice-based cholera vaccine by using whole genome resequencing approach. Plant Cell Tiss Organ Cult 2015;120:35 48. [45] GBD 2015 Mortality and Causes of Death Collaborators. Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980 2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet 2016;388:1459 544. [46] Fleckenstein J, Sheikh A, Qadri F. Novel antigens for enterotoxigenic Escherichia coli vaccines. Expert Rev Vaccines 2014;13:631 9. [47] Moss J, Richardson SH. Activation of adenylate cyclase by heat-labile Escherichia coli enterotoxin. Evidence for ADP-ribosyltransferase activity similar to that of choleragen. J Clin Invest 1978;62:281 5. [48] Klipstein FA, Engert RF, Houghten RA. Immunisation of volunteers with a synthetic peptide vaccine for enterotoxigenic Escherichia coli. Lancet 1986;1:471 2. [49] Ahmed SM, Hall AJ, Robinson AE, Verhoef L, Premkumar P, Parashar UD, et al. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis. Lancet Infect Dis 2014;14:725 30. [50] Lucero Y, Vidal R, O’Ryan GM. Norovirus vaccines under development. Vaccine 2017;36(36):5435 41. [51] World Health Organization. Hepatitis B. Fact sheet 5 July 2017. Available from: ,http://www.who.int/ news-room/fact-sheets/detail/hepatitis-b..

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[52] Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS, Thanavala Y. Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc Natl Acad Sci USA 2001;98:11539 44. [53] Richter LJ, Thanavala Y, Arntzen CJ, Mason HS. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotechnol 2000;18:1167 71. [54] Fooks AR, Cliquet F, Finke S, Freuling C, Hemachudha T, Mani RS, et al. Rabies. Nat Rev Dis Primers 2017;3:17091. [55] Moore SM, Wilkerson MJ, Davis RD, Wyatt CR, Briggs DJ. Detection of cellular immunity to rabies antigens in human vaccinees. J Clin Immunol 2006;26:533 45. [56] Lentz TL, Burrage TG, Smith AL, Crick J, Tignor GH. Is the acetylcholine receptor a rabies virus receptor? Science 1982;215:182 4. [57] Lentz TL, Wilson PT, Hawrot E, Speicher DW. Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins. Science 1984;226:847 8. [58] Fu ZF, Dietzschold B, Schumacher CL, Wunner WH, Ertl HC, Koprowski H. Rabies virus nucleoprotein expressed in and purified from insect cells is efficacious as a vaccine. Proc Natl Acad Sci USA 1991;88:2001 5. [59] Benmansour A, Leblois H, Coulon P, Tuffereau C, Gaudin Y, Flamand A, et al. Antigenicity of rabies virus glycoprotein. J Virol 1991;65:4198 203. [60] Iuliano AD, Roguski KM, Chang HH, Muscatello DJ, Palekar R, Tempia S, et al. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet 2018;391:1285 300. [61] Paules CI, Sullivan SG, Subbarao K, Fauci AS. Chasing seasonal influenza - the need for a universal influenza vaccine. N Engl J Med 2018;378:7 9. [62] Mohsen MO, Zha L, Cabral-Miranda G, Bachmann MF. Major findings and recent advances in virus-like particle (VLP)-based vaccines. Semin Immunol 2017;34:123 32. [63] Ali M, Lopez AL, You YA, Kim YE, Sah B, Maskery B, et al. The global burden of cholera. Bull World Health Organ 2012;90:209 218A. [64] Mosley 2nd JF, Smith LL, Brantley P, Locke D, Como M. Vaxchora: the first FDA-approved cholera vaccination in the United States. P&T Peer-Rev J Form Manage 2017;42:638 40. [65] Harris JB, LaRocque RC, Qadri F, Ryan ET, Calderwood SB. Cholera. Lancet 2012;379:2466 76. [66] King CA, Van Heyningen WE. Deactivation of cholera toxin by a sialidase-resistant monosialosylganglioside. J Infect Dis 1973;127:639 47.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

370

20. PLANT-BASED MUCOSAL VACCINE DELIVERY SYSTEMS

[67] Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature 1981;292:413 17. [68] Kimberg DV, Field M, Johnson J, Henderson A, Gershon E. Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins. J Clin Invest 1971;50:1218 30. [69] Nochi T, Yuki Y, Katakai Y, Shibata H, Tokuhara D, Mejima M, et al. A rice-based oral cholera vaccine induces macaque-specific systemic neutralizing antibodies but does not influence pre-existing intestinal immunity. J Immunol 2009;183:6538 44.

[70] Peltola H, Siitonen A, Kyronseppa H, Simula I, Mattila L, Oksanen P, et al. Prevention of travellers’ diarrhoea by oral B-subunit/whole-cell cholera vaccine. Lancet 1991;338:1285 9. [71] Takeyama N, Yuki Y, Tokuhara D, Oroku K, Mejima M, Kurokawa S, et al. Oral rice-based vaccine induces passive and active immunity against enterotoxigenic E. coli-mediated diarrhea in pigs. Vaccine 2015;33:5204 11.

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Plant-Based Mucosal Immunotherapy: Challenges for Commercialization Kenneth L. Bost1,2 and Kenneth J. Piller1,2 1

Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, United States 2SoyMeds Inc, Davidson, NC, United States

I. INTRODUCTION The technologies that have been used in preclinical studies to produce plant-based immunotherapies are diverse. While tobacco is the most common plant used for transformation [1 5], algae [6], potato [7], corn [8], rice [9], moss [10], papaya [11], soy [12,13], and other plant genera [14] have been successfully transformed to express potential immunotherapeutic proteins. In addition to the type of plant used, there are numerous methods for transforming particular genera. Stable nuclear transformants using Agrobacterium binary vectors were some of the earliest examples of this transkingdom DNA transfer [15]. Later, transformation of plastid DNA (e.g., chloroplasts) using biolistic bombardment was used to express proteins in plant

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00021-3

organelles without the necessity of altering chromosomal DNA [16]. From a commercial point of view, transient expression using biolistic bombardment, Agrobacterium infiltration, or virus vectors has been the most popular method used to date [17]. Plant cell culture systems have also been used to generate commercial products [18]. Therefore an important commercial decision in designing plant-based mucosal immunotherapeutics is the selection of the plant and the transformation method, since each has advantages and disadvantages [1,14,19,20]. In light of the numerous reviews summarizing preclinical data using animal models that were referenced above, the present work will focus on the challenges for achieving the ultimate goal in plant-made mucosal immunotherapy: commercial products.

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II. ADVANCES IN DEVELOPING PLANT-BASED MUCOSAL IMMUNOTHERAPY PRODUCTS: A QUARTER OF A CENTURY LATER The hepatitis B surface antigen was expressed in transgenic tobacco leaves in 1992 [21], suggesting the possibility that edible vaccines were feasible [19,22 25] pending further development of this technology. However, the enthusiasm for edible vaccines was soon tempered by some legitimate and some perceived hurdles for commercialization of such products [26,27]. Nonetheless, during the past 26 years, numerous review articles [1,16,20,28,29] have summarized a wealth of basic research showing the expression of numerous antigens and/or tolerogens in a diversity of plant expression systems, as well as highlighting their efficacy in preclinical animal models. Despite this abundance of academic and industry R&D for over a quarter of a century, few products have been evaluated in clinical trials using human or animal subjects, and even fewer have received regulatory approval for commercial use. One notable example of such regulatory approval occurred in 2006 when Dow Agrosciences, Inc. was successful in obtaining a US Department of Agriculture (USDA) permit for a tobacco-derived Newcastle disease virus protein subunit vaccine for use in chickens [30]. Despite USDA approval and the continuing need for preventive measures targeting this avian virus infection [31], the vaccine was never marketed by Dow Agrosciences, Inc. To date, the company has marketed no additional follow-on vaccines using this technology. After so much time and effort by so many [1,16,20,28,29], the lack of commercially available mucosal immunotherapies derived from genetically modified plants is surprising. The translation of preclinical research efforts into approved, marketed plantderived pharmaceuticals remains the most significant current challenge for the field.

III. A LACK OF RECENT HUMAN CLINICAL TRIALS TESTING THE SAFETY AND/OR EFFICACY OF PLANT-BASED MUCOSAL IMMUNOTHERAPY Despite the absence of any marketed plantderived pharmaceuticals and/or agriceuticals [32], some clinical trial information regarding the safety and efficacy of plant-based immunotherapies is available. In 1998, 11 human volunteers consumed raw transgenic potatoes expressing the beta subunit of Escherichia coli heat-labile toxin [7]. The treatment was generally well tolerated and antibodies against the vaccine developed in each volunteer. A second example of a Phase I clinical trial was reported in 2005 evaluating the safety and immunogenicity of ingested transgenic potatoes expressing the hepatitis B surface antigen [33]. The majority of vaccinated human volunteers demonstrated increases in antibody responses against this viral antigen, with approximately 40% classed as nonresponders. Additional Phase I clinical trials in human subjects exposed to oral antigens expressed in plants have been previously reviewed [14,34]. Though limited in number, each of these Phase I clinical trials showed no major safety concerns, and each produced some detectable level of an immune response against the vaccine antigen following oral administration. Despite the apparent lack of significant adverse events during these Phase I trials, no follow-up Phase II clinical trials have been reported for these mucosal vaccine candidates [14]. Further, for more than a decade, there has been a lack of human clinical trials for plantderived mucosal immunotherapeutics, which seems surprising. Since efficacy in human subjects is the gold standard, the lack of clinical data is a significant gap in our knowledge in attempting to evaluate the ability of plantbased vaccines and tolerogens to function as mucosal therapeutics.

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IV. COST OF PRODUCTION FOR PLANT-BASED MUCOSAL IMMUNOTHERAPEUTICS: IS THIS A REALISTIC ADVANTAGE?

IV. COST OF PRODUCTION FOR PLANT-BASED MUCOSAL IMMUNOTHERAPEUTICS: IS THIS A REALISTIC ADVANTAGE? The current lack of commercial success for plant-based mucosal immunotherapy suggests market challenges for this technology. Early enthusiasm for edible vaccines [19,22 25] has not translated into products for routine human and animal use. Review articles continue to tout the potential for plant-based expression systems, a low cost of production being one advantage that is commonly stated [16,35]. However, it is not clear whether a low cost of production is an actual advantage or merely one that is perceived. When this advantage is stated, quantification of such savings is rarely provided. Further, when such estimations have been attempted [36], they have included assumptions that may or may not be holistic. One difficulty in estimating cost savings for producing plant-derived immunotherapies stems, in part, from the diversity of transformation technologies that might be used. As was noted above, stable versus transient protein expressions or whole plant versus plant cell cultures would vary significantly in their production costs. It has been estimated that production using GMP-compliant greenhouses would be three to five times more expensive than an open-field system [37]. Further, costs associated with bioreactor facilities for plant cell culture could be 30 80 times greater, which approaches the current costs for conventional recombinant protein expression platforms [37]. Since the proposed use of open-field systems is an antiquated concept, given present-day technologies (see below), and since plant cell culture facilities are costly, any substantial savings during production would likely be limited to greenhouse-based facilities. The lack of commercially available products prevents a clear understanding of any potential costs savings for the different production

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platforms. If there were current data for the cost of goods for numerous manufactured plant-derived immunotherapies, then it might be possible to more accurately quantify any cost savings in comparison to conventional vaccine production technologies [38,39]. Unfortunately, information regarding any potential cost savings is mostly theoretical at this point [35,36]. However, there is a recent example of a plant-derived influenza subunit vaccine made by Caliber Biotherapeutics resulting from the DARPA commissioned Blue Angel Project [40]. A candidate hemagglutinin protein was transiently expressed in tobacco leaves using vacuum-facilitated infiltration of Agrobacterium expressing the flu protein construct. Following growth of the transformed tobacco plants, leaves were harvested, and the vaccine candidate was extracted and purified. This success allowed calculation of the production costs to be between $0.10 and $0.12 for the anticipated 50-µg vaccine dose on an annualized cost basis [40]. While there is no direct cost comparison for this subunit vaccine candidate with conventional recombinant protein expression systems [38], the cost of goods for manufacturing Chinese Hamster Ovary (CHO) cell-derived monoclonal antibodies is approximately $100 g21 with projections to be less in coming years with technological advances and efficiencies [41]. While it might seem obvious that it would be cheaper to grow transgenic plants than to culture transformed CHO cells in large containers, the actual expression of a particular recombinant protein represents only a fraction of the total cost of goods. Harvesting, concentrating, purifying, finishing and quality control represent the bulk of expense for manufacturing therapeutic recombinant proteins, whether it is a vaccine [38,39] or another protein [41]. While some plant-derived proteins may be cheaper to express, the cost of goods for plant-derived immunotherapeutics will depend largely on the costs of commercial-scale harvesting,

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concentrating, purifying, finishing, and quality control of the final product. Stated simply, the bulk costs associated with a commercial, plantbased immunotherapy will depend on the final product [37], not merely on how it was expressed. Further, the cost of goods will likely be a fraction of the retail price for plant-based immunotherapies. Additional costs (e.g., marketing) and corporate revenues will factor into the final wholesale and/or retail price. Companies will profit from their commercial products regardless of the expression platform. While cost savings in the manufacturing process might attract a company to utilize plantbased technologies, whether any savings in production are passed on to the consumer will depend largely on what the market will bear. Again, since no commercially available plantderived mucosal immunotherapeutics are currently being marketed, it is difficult to assess any savings in retail costs for the consumer. While not a vaccine, ELELYSO (taliglucerase alfa) is an example of an FDA-approved plant-made therapeutic protein that is currently being marketed by Protalix for treatment of Gaucher’s disease [42]. This recombinant protein, glucocerebrosidase, is expressed in a carrot cell culture system and purified to treat patients deficient in this enzyme. The current retail price for a 1-year treatment regimen for an adult patient is more than $300,000. While this biologic does have orphan drug status, it does illustrate how expression costs are a small fraction of the retail cost, which is determined in large part by what the market will bear. In summary, reviews of plant-based protein expression systems continue to tout cost savings as a significant advantage for this expression platform [16,35]. However, without an actual commercial example demonstrating such savings, it is difficult to imagine a scenario in which plant-made immunotherapeutics will provide the consumer with mucosal vaccines and tolerogens that cost significantly less than current vaccines.

V. INFRASTRUCTURE AND PROTOCOLS FOR PLANT-BASED MUCOSAL IMMUNOTHERAPEUTIC MANUFACTURING Despite the lack of marketed products for plant-based mucosal immunotherapeutics, a number of commercial facilities have been constructed that are capable of expressing therapeutic proteins from plants using Good Manufacturing Practices (GMP). For example, from 2007 to 2009, the Defense Advanced Research Projects Agency (DARPA) contributed to the infrastructure of four facilities designed to manufacture plant proteins [40]. These included facilities at Fraunhofer CMB in Newark, Delaware; Kentucky BioProcessing in Owensboro, Kentucky; Medicago in Durham, North Carolina; and Caliber Biotherapeutics in Bryan, Texas. One goal of this DARPA funding was to validate the manufacturing of a tobaccoderived influenza hemagglutinin protein as a candidate subunit vaccine in several different locations under time restraints [40]. Following the successful completion of this project, these sponsored facilities continue to function as high-capacity manufacturing facilities, effectively increasing the infrastructure for plantmade pharmaceuticals. Other examples of GMP-compliant commercial facilities capable of manufacturing plant-derived proteins have been reviewed [14,40]. Further, the logistics and facility requirements for manufacturing plantmade proteins have been developed and recently discussed [2,40,43 47]. Stated simply, protocols and facilities currently exist for commercial production of plant-derived proteins. Therefore the manufacturing hurdles for producing edible vaccines that existed in the early 2000s [26,27] are no longer as prohibitive. The lack of commercially available plant-made mucosal immunotherapeutics cannot be explained by inadequate infrastructure or underdeveloped technologies.

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Similarly to the developing infrastructure for manufacturing plant-made proteins, approved products and candidate therapies currently in clinical trials have provided examples of regulatory approvals that might be followed. As was mentioned earlier, USDA regulatory approval of a tobacco-derived Newcastle disease virus protein subunit vaccine for use in chickens occurred more than a decade ago [30]. There have also been several notable plant-made therapeutics approved for use in patients, including ELELYSO made in carrot cells by Protalix [18]. While no plantmade vaccines are currently being marketed, several candidates have passed regulatory scrutiny for use in human clinical trials. For example, clinical trials for a virus-like particle influenza vaccine made in tobacco by Medicago [3], a subunit influenza vaccine made in tobacco by Fraunhofer [48], and a conjugate vaccine for the targeted treatment of B cell follicular lymphoma made in tobacco by Icon Genetics [4] have been completed. It should be noted that each of these human clinical trial examples used a vaccine formulation given parenterally and not mucosally. With the exception of some therapies targeting allergies (see Chapter 51: Mucosal Vaccine for Parasitic Infections), no current, ongoing human clinical trials using plant-made mucosal vaccines could be found. The successful approval of plant-made biologics for commercial use or for ongoing human clinical trials demonstrates successful examples of navigating the regulatory requirements for such products. The regulatory landscape for plant-made animal vaccines has also been discussed [49]. The lack of commercially available plant-made mucosal immunotherapeutics cannot be explained solely by an onerous regulatory approval process.

Early on, there were many reasons to suspect that purified, plant-made proteins would be safe for use in humans and animals [19,22 25]. The absence of contamination by human or animal pathogens when using plant expression systems remains an obvious advantage. Further, since humans and domestic animals are exposed to normal plant-derived proteins in their diets, it was logical to assume that plant-expressed transgenic proteins would be well tolerated, despite any posttranslational modifications. Definitive evidence for the safety of plant-made proteins came from the postmarketing surveillance for a commercial product [50] as well as recent clinical trials data for potential vaccine candidates [3,4,48,51]. In the presence of an abundance of safety data for purified, plant-made protein vaccines following parenteral immunization, it is unclear why there continues to be the assertion that plant-specific glycosylation remains a significant consideration for adverse events following human treatments [52 54]. Enumerable glycosylated proteins are produced by the approximately 200 crop plants that humans typically consume [55], with glycosylation of these proteins having commonalities [56]. Given that only a very small number of the multitude of consumed glycosylated plant proteins cause any significant disease (e.g., food allergy [57]), it is clear that glycosylation by itself does not render a plant protein allergenic. It has been suggested that some unique combination of a particular “allergic” amino acid sequence combined with a particular posttranslational modification (such as glycosylation) might account for a percentage of the allergic epitopes that some patients react to [58]. The likelihood of a particular recombinant protein expressed in transgenic plants having such a

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unique “allergic” combination seems remote. Safety profiles in patients have supported this likelihood [3,4,48,51]. In fact, there is at least one example in which the presence of plant glycosylation actually reduced the allergenicity of a recombinant protein when compared to its yeast-derived counterpart [59]. Further, recent articles provide some convincing evidence to support the notion that N-linked glycosylation of transgenic plant proteins are at least as safe as their CHO-derived counterparts [50] or do not induce detectable allergic reactions [60]. Taken together, this direct evidence strongly supports the safety of glycosylated proteins that are made in plants. The presence of cross-reactive carbohydrate determinants (CCDs) in diagnostic assays to define patient reactivity to allergens has been purported to support the notion of the dangers of plant glycosylation [61]. While CCDs are problematic for diagnostic assays as false positives, there are compelling arguments to suggest that most of this cross-reactivity is clinically insignificant [61]. In the absence of some compelling demonstration that a significant number of transgenic plant proteins can cause adverse events in an unacceptable number of patients owing to glycosylation, the assertion that this posttranslational modification is problematic seems unfounded.

VIII. MUCOSAL TOLERANCE THERAPY USING PLANT-MADE PROTEINS Edible vaccines dominated the discussion for plant-made proteins early on, with published results using preclinical animal models [19,22 25]. However, there were attempts to use plant-based expression systems for manufacturing products to induce oral tolerance in models of autoimmunity [62]. As with plant-made vaccines, more than 20 years have elapsed with no oral tolerance products

targeting autoimmune diseases progressing through Phase III clinical trials. This statement is true for plant-made proteins as well as for candidate tolerogens manufactured by conventional methods [63 65]. Despite encouraging results in animal models of autoimmunity, the notion that oral tolerance therapy does not translate to human disease has persisted based on the lack of efficacy in a very limited number of Phase I clinical trials [63 65]. The apparent discrepancy between large number of animal studies and a few human trials has been used to suggest that the human mucosal barrier to vaccination and tolerance induction may be a result of differences that are not wholly appreciated [66]. Perhaps the best evidence for the ability of mucosal-delivered antigens to alter clinical outcomes in human patients comes from recent trials focused on food allergies [57,67 69]. Completed and/or ongoing human Phase III clinical trials have clearly demonstrated the ability to modulate the allergic response in patients once treated with allergens at mucosal surfaces. Whether current treatment modalities will result in significant disease reduction but not necessarily a cure for allergies is not clear [68]. Technically, some of these clinical trials are being performed with plant-made proteins, even though some antigens are naturally occurring (e.g., peanut proteins) and not recombinant proteins made in transgenic plants. Regardless, these clinical trials clearly demonstrate the efficacy of mucosal therapies to modulate antigen-specific human immune responses. As our understanding of antigenspecific allergen therapies advances, perhaps this knowledge can be applied to modulating autoimmune diseases. Early intervention for affecting outcomes in allergic patients seems an especially promising approach [70]. The notion that it will be easier to positively affect patient outcomes if the disease is treated in its early stages or prior to its development is a tenet of prophylactic

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vaccination [71]. Success with this early intervention strategy may provide some insight into the lack of efficacy for oral tolerance in human clinical trials for autoimmune diseases. The ability to treat patients prior to an established disease state or the ability to prophylactically treat individuals who have a high probability of developing a particular autoimmunity seems an attractive strategy. Unlike some viral pathogens (e.g., influenza virus) of which antigenically distinct strains can evolve rapidly, there is no such selective pressure for autoimmune antigens or for allergens to change their epitopes. Stated simply, autoimmune antigens or allergens do not evolve rapidly. Once an effective tolerogenic regimen has been identified, it can be used in perpetuity. While the rapidity of plant-made proteins using transient expression in tobacco may be required for producing vaccines for rapidly evolving pathogens [17], such technology is not required for antigens used in tolerance therapy. For such applications, stably transduced seeds might simplify the downstream manufacturing process, since the production process can be discontinuous, as will be discussed later.

IX. GLOBAL CONTAMINATION FROM FOOD-CROP-MADE VACCINES OR TOLEROGENS For an immunotherapy to be easily “edible,” it seemed logical to transform food crops that could be consumed [1,72,73]. Unfortunately, there have been suggestions that such a strategy would threaten the global contamination of food crops [27]. The Prodigene fiasco [74] in 2002 supports such a notion. Many current commercial efforts to express therapeutics in plants avoid food crops altogether and focus on transient tobacco transformations [2,3] or plant cell culture [46]. Stated simply, the plantmade protein industry is mostly limited to

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technologies that require extensive protein purification during the manufacturing process. This limitation certainly removes the fear of contaminating food crops. However, if one examines the current advances in using food crops as bioreactors, there are many advantages over tobacco and plant cell cultures. Further, the fear of contamination seen in the Prodigene era [74] is currently not a well-founded argument. For example, using transgenic soybean seeds to manufacture therapeutic proteins [12,36] differs from transforming value-added traits into soy plants [72,73]. One significant difference is the goal of expressing high levels of a particular recombinant therapeutic protein. In fact, recent technological advances have increased the yield of transgenic protein per seed to a point at which there is little reason for growth of transformed soybean plants in open fields to manufacture large amounts of recombinant protein. Production of kilogram amounts of recombinant protein within secure greenhouses is theoretically feasible [36] and currently practical (unpublished results). At the range of 10 25 µg per dose of a subunit vaccine (e.g., influenza and hepatitis B), this level of production represents hundreds of millions of doses per greenhouse acre. As technological advances occur, efficiencies can only be expected to increase. Since GMP-compliant growth of transgenic soy would require the same level of containment as those recombinant protein manufacturing facilities currently using GMO bacteria, GMO yeasts, GMO viruses, and the like, the risks of accidental release into the environment would be similar. In other words, there is no reason to believe that seed containment will be any more difficult than microbial containment. Further, one advantage of transgenic seed expression is the ease of such containment. Equipment to reduce seeds to powder at a commercial scale are common, and such a processing step can easily be performed in the growth facility following seed harvest. Because powder is nongerminal, this material

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can then be packaged and shipped without fear of growth outside the production facility.

X. SAFETY OF CONSUMABLE FORMULATIONS FROM FOOD-CROP-MADE VACCINES OR TOLEROGENS While it may seem obvious that a consumable formulation made from an edible transgenic plant expressing a vaccine would be safe and therefore advantageous, this possibility was also suggested to be a potential disadvantage based on several unlikely speculations. Specifically, there was a concern about possibly mounting an immune response against normal plant proteins present in a plant-derived vaccine formulation [26,27]. Conversely, it was suggested that a vaccine given within the context of other normal plant proteins might result in tolerance to the vaccine, since tolerance is the default response to most plant proteins [26,27]. The logic for these early concerns remains unclear. Mechanisms required for instantly breaking tolerance to normal plant proteins after a lifetime of exposure or mechanisms required for immediately inducing tolerance to a foreign vaccine candidate are difficult to imagine. Previous Phase I clinical trials showed evidence of immune responses against the particular vaccine candidate, not tolerance [14,34]. Further, when transgenic foodstuffs or formulations were given, no detectable antibody responses against normal plant proteins were detected, and these administrations were well tolerated [14,34]. These results are consistent with data from most animal models [1,9,16,20,28,29]. While more clinical trial data would be useful, it seems that the obvious observation will be the correct one: A consumable formulation made from an edible transgenic plant expressing a vaccine is likely to cause few adverse effects. Another argument against formulating food-crop-made therapeutics is the notion that regulatory agencies would never approve such complex protein mixtures for human therapies

[26]. Currently, there is little reason to support such a notion. Examples of clinical trials using edible vaccines made from food crops have been reviewed [14,34], and the candidate vaccines in each of these clinical trials required regulatory approval prior to use. When formulations made directly from seed crops are considered, soybeans again can be used to illustrate regulatory approval and safety. A seedbased formulation containing a concentration of the Bowman-Birk protease inhibitor was produced in the early 1990s for use in human clinical trials [75]. This concentrate was easy to produce and contained a precise dose of the inhibitor along with other normal soybean proteins. Since its production and characterization, several Phase I and Phase IIa/b clinical trials have been performed [76,77], and the safety of this soy formulation has been evaluated [78]. While this soy concentrate was made from normal (i.e., nontransgenic) seeds, these studies demonstrate the likelihood of such formulations gaining regulatory approval, as well as their safety once given orally to patients even when transgenic seeds are being formulated. While formulating edible formulations from food crops for oral therapy is intuitively attractive, purification of recombinant proteins from transgenic food crops can also be performed if a mucosal delivery method (e.g., intranasal) requires removal of extraneous proteins. However, regardless of the process, transgenic seed crops have advantages that other food crop based therapies do not.

XI. SEPARATING EXPRESSION FROM FINISHING THE PRODUCT GEOGRAPHICALLY AND OVER EXTENDED PERIODS OF TIME USING SEED-BASED PLATFORMS Conventional recombinant protein expression platforms usually employ a continuous production timeline [79]. Once culturing of transformed bacteria, yeast, or mammalian cells

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begins, the process of growing, harvesting, concentrating, purifying, and finishing the biologic proceeds on a continuous schedule. This often results in production, purification, and finishing facilities being housed in the same structure or in very close proximity [80]. If any step fails GMP, the entire process repeats from the beginning. This is also true for plant cell culture platforms [46]. While it is possible at certain steps to lyophilize or freeze materials and continue at a later time [16], such processes add to the time and expense of production. Seed-based platforms for protein expression have the advantage of a discontinuous manufacturing process. It is possible to stably transform plant lines that target expression of a particular recombinant protein to the seed. Once seeds have been harvested, a simple grinding of these transgenic seeds to nongerminable powder allows shipping to geographically distant facilities for extended storage [81]. We have stored powder from transgenic soybeans in dry conditions at room temperature for times approaching one decade with minimal or no detectable degradation of particular recombinant proteins (unpublished data). When needed, aliquots of transgenic powder can be removed for formulating soy-based therapies or for purification of the recombinant protein from soy. Theoretically, seeds can be harvested in one geographic location, powders sent to storage facilities elsewhere, and finishing of the product completed at some future date. The ability to segregate the manufacturing process geographically and over an extended time period is unique to such plantmade protein expression platforms when compared to current processes [80] (Chapter 20: PlantBased Mucosal Vaccine Delivery Systems).

XII. PLANT-BASED MUCOSAL IMMUNOTHERAPY: CHALLENGES FOR COMMERCIALIZATION In conclusion, the present-day challenges for developing commercially viable mucosal

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immunotherapies using plant-based platforms are little different from the challenges faced by any mucosal vaccinologist [82]. Specifically, the efficacy of mucosal immunotherapy depends upon the delivery of an antigenic formulation that will either stimulate a vaccine response or induce tolerance, as required, with acceptable adverse effects to the host. This can be a challenging task, as some immunotherapies require delivery systems to transverse mucosal barriers or require adjuvants to augment the desired response. Targeting of antigens to particular mucosal cell types may also be required to increase bioavailability and therefore efficacy. As previously reviewed [82], these challenges can be significant ones and are considerations for all mucosal immunotherapies, including plant-based ones. If these challenges are the same, why use plants as an expression platform? As we discussed in this chapter, there are some significant differences in comparison to current recombinant protein expression systems. For example, plant-based expression platforms begin the manufacturing process with no animal pathogens or animal toxins (e.g., prions) being present. With previous exposure to normal dietary and environmental plant proteins over a person’s lifetime, it is logical to assume that any posttranslational modifications made to immunotherapeutics in these expression systems will be well tolerated. In addition to these safety considerations, each platform using plant-made proteins has its own advantages. For example, transient expression of influenza vaccine candidates in tobacco has proven speed and flexibility in its manufacturing processes [17]. Disadvantages include the relatively high leaf biomass required for initial processing and a continuous manufacturing process, since the protein must be purified. Seed-based expression platforms are often stable transformations that require time and effort to establish such lines [8,9,36]. However, these platforms produce relatively high protein to biomass (i.e., seed) ratios, as

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well as having the possibility of a discontinuous manufacturing process and direct formulation into consumable therapies that may not require purification of the protein. Nonseed food crop transformations can also be formulated into consumables [16] but can have relatively low protein to biomass (e.g., leaf or fruit) ratios and continuous manufacturing unless lyophilization or freezing is added to the process. Finally, plant cell culture systems [46] have demonstrated the speed of transformation and production. These manufacturing platforms are not dissimilar in their logistics from conventional cell culture based systems, requiring continuous processing and purification of the protein. This diversity of advantages over conventional recombinant protein expression platforms requires some thought in considering which plant-based system might be the most advantageous for any particular mucosal immunotherapy application. Each of these plant-based technologies also has its own history of intellectual property rights, which must be considered [83 88]. Despite the current lack of commercially marketed plant-based immunotherapeutics, it is likely that this will not be the case for very long [3,14]. Many of the perceived hurdles for such products voiced more than a decade ago [26,27] are not well founded or have already been addressed, as discussed previously. The forthcoming commercial successes of plantbased immunotherapeutics should provide additional incentives for follow-on products, as well as the evolution of products for modulating mucosal immune responses.

References [1] Abiri R, et al. A critical review of the concept of transgenic plants: insights into pharmaceutical biotechnology and molecular farming. Curr Issues Mol Biol 2016;18:21 42.

[2] Ma JK, et al. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants. Plant Biotechnol J 2015;13(8):1106 20. [3] Pillet S, et al. A plant-derived quadrivalent virus like particle influenza vaccine induces cross-reactive antibody and T cell response in healthy adults. Clin Immunol 2016;168:72 87. [4] Tuse D, et al. Clinical safety and immunogenicity of tumor-targeted, plant-made Id-KLH conjugate vaccines for follicular lymphoma. Biomed Res Int 2015;2015:648143. [5] Zahin M, et al. Scalable production of HPV16 L1 protein and VLPs from tobacco leaves. PLoS One 2016;11 (8):e0160995. [6] Specht EA, Mayfield SP. Algae-based oral recombinant vaccines. Front Microbiol 2014;5. [7] Tacket CO, et al. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat Med 1998;4(5):607 9. [8] Rosales-Mendoza S, et al. Corn-based vaccines: current status and prospects. Planta 2017;245(5):875 88. [9] Azegami T, et al. Novel transgenic rice-based vaccines. Arch Immunol Ther Exp (Warsz) 2015;63(2):87 99. [10] Reski R, Parsons J, Decker EL. Moss-made pharmaceuticals: from bench to bedside. Plant Biotechnol J 2015;13(8):1191 8. [11] Fragoso G, et al. Transgenic papaya: a useful platform for oral vaccines. Planta 2017;245(5):1037 48. [12] Hudson LC, et al. Soybean seeds: a practical host for the production of functional subunit vaccines. Biomed Res Int 2014;2014:340804. [13] Piller KJ, et al. Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean. Planta 2005;222(1):6 18. [14] Takeyama N, Kiyono H, Yuki Y. Plant-based vaccines for animals and humans: recent advances in technology and clinical trials. Ther Adv Vaccines 2015;3(5-6): 139 54. [15] Tzfira T, Citovsky V. Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol 2006;17(2):147 54. [16] Daniell H, Chan HT, Pasoreck EK. Vaccination via chloroplast genetics: affordable protein drugs for the prevention and treatment of inherited or infectious human diseases. Annu Rev Genet 2016;50:595 618. [17] Canto T. Transient expression systems in plants: potentialities and constraints. Adv Exp Med Biol 2016;896:287 301. [18] Grabowski GA, Golembo M, Shaaltiel Y. Taliglucerase alfa: an enzyme replacement therapy using plant cell expression technology. Mol Genet Metab 2014;112 (1):1 8.

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REFERENCES

[19] Giddings G, et al. Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 2000;18(11): 1151 5. [20] Lomonossoff GP, D’Aoust MA. Plant-produced biopharmaceuticals: a case of technical developments driving clinical deployment. Science 2016;353 (6305):1237 40. [21] Mason HS, Lam DM, Arntzen CJ. Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci U S A 1992;89(24):11745 9. [22] Langridge WH. Edible vaccines. Sci Am 2000;283 (3):66 71. [23] Mason HS, et al. Edible plant vaccines: applications for prophylactic and therapeutic molecular medicine. Trends Mol Med 2002;8(7):324 9. [24] Mor TS, Gomez-Lim MA, Palmer KE. Perspective: edible vaccines--a concept coming of age. Trends Microbiol 1998;6(11):449 53. [25] Walmsley AM, Arntzen CJ. Plants for delivery of edible vaccines. Curr Opin Biotechnol 2000;11(2):126 9. [26] Bonetta L. Edible vaccines: not quite ready for prime time. Nat Med 2002;8(2):94. [27] Vermij P. Edible vaccines not ready for main course. Nat Med 2004;10(9):881. [28] Marsian J, Lomonossoff GP. Molecular pharming VLPs made in plants. Curr Opin Biotechnol 2016; 37:201 6. [29] Merlin M, Pezzotti M, Avesani L. Edible plants for oral delivery of biopharmaceuticals. Br J Clin Pharmacol 2017;83(1):71 81. [30] Vermij P. USDA approves the first plant-based vaccine. Nat Biotechnol 2006;24(3):234. [31] Kapczynski DR, Afonso CL, Miller PJ. Immune responses of poultry to Newcastle disease virus. Dev Comp Immunol 2013;41(3):447 53. [32] Goldberg R. The business of agriceuticals. Nat Biotechnol 1999;17:Bv5 6. [33] Thanavala Y, et al. Immunogenicity in humans of an edible vaccine for hepatitis B. Proc Natl Acad Sci U S A 2005;102(9):3378 82. [34] Streatfield SJ. Mucosal immunization using recombinant plant-based oral vaccines. Methods 2006;38 (2):150 7. [35] Waheed MT, et al. Need of cost-effective vaccines in developing countries: what plant biotechnology can offer? Springerplus 2016;5:65. [36] Bost K, Piller K. Protein expression systems: why soybean seeds? In: Sudaric A, editor. Soybean: molecular aspects of breeding. Croatia: InTech; 2011. p. 3 18. [37] Hefferon KL. Plant-derived pharmaceuticals: principles and applications for developing countries. CABI biotechnology series. Boston, MA: CABI; 2014. x, 170 pages.

381

[38] Preiss S, et al. Vaccine provision: delivering sustained & widespread use. Vaccine 2016;34(52):6665 71. [39] Wen EP, Ellis RJ, Pujar NS. Vaccine development and manufacturing. Wiley series in biotechnology and bioengineering. Hoboken, NJ: Wiley, a John Wiley & Sons, Inc. Publication; 2015. xii, 440 pages. [40] Holtz BR, et al. Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals. Plant Biotechnol J 2015;13(8):1180 90. [41] Kelley B. Industrialization of mAb production technology: the bioprocessing industry at a crossroads. MAbs 2009;1(5):443 52. [42] Mor TS. Molecular pharming’s foot in the FDA’s door: Protalix’s trailblazing story. Biotechnol Lett 2015;37 (11):2147 50. [43] Buyel JF, Twyman RM, Fischer R. Very-large-scale production of antibodies in plants: the biologization of manufacturing. Biotechnol Adv 2017;35(4):458 65. [44] Kashima K, et al. Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants. Plant Cell Rep 2016;35(3):667 79. [45] Lai H, Chen Q. Bioprocessing of plant-derived viruslike particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations. Plant Cell Rep 2012;31(3):573 84. [46] Tekoah Y, et al. Large-scale production of pharmaceutical proteins in plant cell culture-the Protalix experience. Plant Biotechnol J 2015;13(8):1199 208. [47] Tuse D, Tu T, McDonald KA. Manufacturing economics of plant-made biologics: case studies in therapeutic and industrial enzymes. Biomed Res Int 2014;2014: 256135. [48] Cummings JF, et al. Safety and immunogenicity of a plant-produced recombinant monomer hemagglutininbased influenza vaccine derived from influenza A (H1N1)pdm09 virus: a Phase 1 dose-escalation study in healthy adults. Vaccine 2014;32(19):2251 9. [49] MacDonald J, et al. Bringing plant-based veterinary vaccines to market: managing regulatory and commercial hurdles. Biotechnol Adv 2015;33(8):1572 81. [50] Shaaltiel Y, Tekoah Y. Plant specific N-glycans do not have proven adverse effects in humans. Nat Biotechnol 2016;34(7):706 8. [51] Chichester JA, et al. Safety and immunogenicity of a plant-produced recombinant hemagglutinin-based influenza vaccine (HAI-05) derived from A/Indonesia/ 05/2005 (H5N1) influenza virus: a phase 1 randomized, double-blind, placebo-controlled, dose-escalation study in healthy adults. Viruses 2012;4(11):3227 44. [52] Chen Q. Glycoengineering of plants yields glycoproteins with polysialylation and other defined N-glycoforms. Proc Natl Acad Sci U S A 2016;113(34):9404 6.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

382

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[53] Piron R, et al. Using GlycoDelete to produce proteins lacking plant-specific N-glycan modification in seeds. Nat Biotechnol 2015;33(11):1135 7. [54] Sheshukova EV, Komarova TV, Dorokhov YL. Plant factories for the production of monoclonal antibodies. Biochemistry (Mosc) 2016;81(10):1118 35. [55] Warren J. The nature of crops: how we came to eat the plants we do. Boston, MA: CAB International; 2015. [56] Strasser R. Plant protein glycosylation. Glycobiology 2016;26(9):926 39. [57] Kattan J. The prevalence and natural history of food allergy. Curr Allergy Asthma Rep 2016;16(7):47. [58] Huby RD, Dearman RJ, Kimber I. Why are some proteins allergens? Toxicol Sci 2000;55(2):235 46. [59] Zafred D, et al. Crystal structure and immunologic characterization of the major grass pollen allergen Phl p 4. J Allergy Clin Immunol 2013;132(3):696 703 e10. [60] Ward BJ, et al. Human antibody response to N-glycans present on plant-made influenza virus-like particle (VLP) vaccines. Vaccine 2014;32(46):6098 106. [61] Altmann F. The role of protein glycosylation in allergy. Int Arch Allergy Immunol 2007;142(2):99 115. [62] Ma SW, et al. Transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance. Nat Med 1997;3(7):793 6. [63] Ilan Y. Oral tolerance: can we make it work? Hum Immunol 2009;70(10):768 76. [64] Meyer T, Ullrich R, Zeitz M. Oral tolerance induction in humans. Exp Mol Pathol 2012;93(3):449 54. [65] Weiner HL, et al. Oral tolerance. Immunol Rev 2011;241(1):241 59. [66] Mestecky J, Russell MW, Elson CO. Perspectives on mucosal vaccines: is mucosal tolerance a barrier? J Immunol 2007;179(9):5633 8. [67] Hamad A, Burks WA. Emerging approaches to food desensitization in children. Curr Allergy Asthma Rep 2017;17(5):32. [68] MacGinnite A. Update on potential therapies for IgEmediated food allergy. Curr Allergy Asthma Rep 2017;17(1):4. [69] Yu W, Freeland DM, Nadeau KC. Food allergy: immune mechanisms, diagnosis and immunotherapy. Nat Rev Immunol 2016;16(12):751 65. [70] Greenhawt MJ, Fleischer DM. Primary prevention of food allergy. Curr Allergy Asthma Rep 2017;17(4):26. [71] Fine PE. Herd immunity: history, theory, practice. Epidemiol Rev 1993;15(2):265 302. [72] McGloughlin MN. Modifying agricultural crops for improved nutrition. N Biotechnol 2010;27(5):494 504.

[73] Parisi C, Tillie P, Rodriguez-Cerezo E. The global pipeline of GM crops out to 2020. Nat Biotechnol 2016;34(1):31 6. [74] Fox JL. Puzzling industry response to ProdiGene fiasco. Nat Biotechnol 2003;21(1):3 4. [75] Kennedy AR, et al. Preparation and production of a cancer chemopreventive agent, Bowman-Birk inhibitor concentrate. Nutr Cancer 1993;19(3):281 302. [76] Armstrong WB, et al. Clinical modulation of oral leukoplakia and protease activity by Bowman-Birk inhibitor concentrate in a phase IIa chemoprevention trial. Clin Cancer Res 2000;6(12):4684 91. [77] Armstrong WB, et al. Bowman birk inhibitor concentrate and oral leukoplakia: a randomized phase IIb trial. Cancer Prev Res (Phila) 2013;6(5):410 18. [78] Lin LL, et al. Phase I randomized double-blind placebo-controlled single-dose safety studies of BowmanBirk inhibitor concentrate. Oncol Lett 2014;7(4):1151 8. [79] Wells EA, Robinson AS. Cellular engineering for therapeutic protein production: product quality, host modification, and process improvement. Biotechnol J 2017;12(1). [80] Behme S. Manufacturing of pharmaceutical proteins: from technology to economy. Weinheim: Wiley-VCH; 2009. xiv, 390 p. [81] Oakes JL, Bost KL, Piller KJ. Stability of a soybean seed-derived vaccine antigen following long-term storage, processing and transport in the absence of a cold chain. J Sci Food Agric 2009;89(13):2191 9. [82] Lamichhane A, Azegamia T, Kiyonoa H. The mucosal immune system for vaccine development. Vaccine 2014;32(49):6711 23. [83] Chi-Ham CL, et al. An intellectual property sharing initiative in agricultural biotechnology: development of broadly accessible technologies for plant transformation. Plant Biotechnol J 2012;10(5):501 10. [84] Chilton MD. Adding diversity to plant transformation. Nat Biotechnol 2005;23(3):309 10. [85] Jefferson DJ, et al. The emergence of agbiogenerics. Nat Biotechnol 2015;33(8):819 23. [86] Jefferson OA, et al. The ownership question of plant gene and genome intellectual properties. Nat Biotechnol 2015;33(11):1138 43. [87] Kowalski SP, et al. Transgenic crops, biotechnology and ownership rights: what scientists need to know. Plant J 2002;31(4):407 21. [88] Miralpeix B, et al. Strategic patent analysis in plant biotechnology: terpenoid indole alkaloid metabolic engineering as a case study. Plant Biotechnol J 2014;12 (2):117 34.

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Attenuated Salmonella for Oral Immunization Kenneth L. Roland1, Qingke Kong2 and Yanlong Jiang3 1

The Biodesign Institute, Arizona State University, Tempe, AZ, United States 2Department of Infectious Diseases & Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States 3College of Animal Sciences & Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agriculture University, Changchun, China

I. INTRODUCTION Salmonella enterica is a mucosal pathogen that interacts with the host immune system, making it an attractive target for use as an antigen delivery vector. Infection with wild-type Salmonella typically generates a robust immune response that leads to lifelong immunity. Recombinant Salmonella strains expressing heterologous genes can be orally administered to elicit an immune response against the pathogen from which the heterologous gene was derived. While strains of other bacteria, such as Escherichia coli, Listeria, Shigella, and Vibrio, have been and continue to be evaluated as oral vaccines, the invasive nature of Salmonella and its propensity to home to host immune cells make it adept at eliciting B and T cell memory responses with the greatest potential to elicit long-lasting mucosal, humoral, and cellular immunity (Chapter 29: Induction of Local and Systemic Immunity by Salmonella Typhi in Humans, Chapter 30: Oral Shigella Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00022-5

Vaccines and Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control). Pathogenic S. enterica are introduced into the human body via the gastrointestinal tract by ingestion of contaminated food. The ingested cells must then pass through and survive the low-pH environment of the stomach before reaching the small intestines. The environment in the human gut is characterized by high osmolarity, the presence of antimicrobial peptides such as defensins, short-chain fatty acids, and resident microflora. In the ileum, Salmonella makes its way through the mucus layer that coats the intestinal epithelium, where it adheres and invades enterocytes or the follicle associated epithelium (FAE) that overlays the Peyer’s patches (PPs). The PPs consist of B cell-rich follicles, T cells, macrophages, and dendritic cells (DCs) that constitute a major component of the gut-associated lymphoid tissue (GALT). One hallmark of the FAE is the presence of microfold

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(M) cells. M cells specialize in the transcytosis of intact luminal material such as soluble proteins, bacteria, and viruses (Chapter 28: M CellTargeted Vaccines). After invasion into the PPs, Salmonella rapidly encounters DCs and is phagocytized [1,2]. These Salmonella-containing DCs may interact directly with B cells within the PPs, resulting in IgA switching and production of intestinal IgA [3]. In addition, T cell priming by the Salmonella-containing DCs begins in the PPs and continues in the deeper immunological tissues (e.g., the spleen) [4,5], resulting in activation of B cells and CD41 and CD81 T cells, leading to production of a systemic cell-mediated and humoral immune response [5
7]. Salmonella enterica Typhimurium initially stimulates a strong proinflammatory immune response that assists with effector cell recruitment and DC maturation. During invasion of PPs and intestinal epithelial cells, the host immune system is exposed to numerous pathogen-associated molecular patterns (PAMPs) produced by S. Typhimurium, including flagella, lipopolysaccharide (LPS), and bacterial DNA [8
11]. These PAMPs are recognized by their cognate Toll-like receptors (TLRs), TLR5, TLR4, and TLR9, respectively. The interactions with TLRs result in the secretion of IL-8 and the proinflammatory cytokines IL-1β, IL-6, TNFα, and IFNγ [12
15]. The production of these cytokines recruits and activates neutrophils, monocytes, and DCs [16]. Colonization of PPs by Salmonella is required to facilitate a strong mucosal IgA response [17]. When salmonellae are phagocytosed by a macrophage, their fate is not necessarily death. While most bacterial cells are killed by macrophages, Salmonella is equipped to survive the encounter because of the genes present in Salmonella pathogenicity island 2 (SPI-2), allowing it to grow within the macrophage in a specialized structure called the Salmonellacontaining vacuole. The Salmonella may be subsequently transported by the macrophage to deeper tissues such as spleen or liver, where it interacts further with lymphoid cells. These properties of Salmonella, including its ability to

stimulate mucosal, humoral, and cellular immunity, when adequately attenuated, make it an attractive antigen delivery vaccine vector.

II. APPROACHES FOR ATTENUATION A. Serial passage In the early studies, attenuated Salmonella vaccines were developed by in vitro serial passage or by random mutagenesis with chemical mutagens. The random mutagenesis method was widely used to develop both agricultural vaccines, such as Salmonella enterica Gallinarum 9R for fowl typhoid [18], and human vaccines, including the Salmonella Typhi strain Ty21a for typhoid fever [19]. Both strains are still in use today. While these methods were in use for many decades, the approach is not without drawbacks. One major problem is an incomplete understanding of the basis of attenuation. For example, two easily distinguished phenotypes of Ty21a are its lack of the Vi capsule, present in nearly all wild-type isolates, and its requirement for exogenous galactose in order to produce O-antigen due to a mutation in the galE gene. For many years, these two defects were assumed to constitute the sole basis of attenuation. However, this assumption was proven to be incorrect in a 1988 study in which a derivative of S. typhi Ty2, the parent of Ty21a, carrying a defined deletion in galE and a spontaneous mutation resulting in lack of Vi was found to retain virulence in human volunteers [20]. Subsequent DNA sequence analysis of the Ty21a genome showed that a number of other mutations are present that could affect attenuation [21]. It is likely that the combined impact of mutations generated during random mutagenesis resulted in a general reduction of the strain’s ability to grow and survive in vivo that are responsible for its attenuation. Other drawbacks include low immunogenicity and risk of reversion to virulence. The Ty21a

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vaccine must be given orally in three or four doses, though its genetic structure and attenuation phenotype appear to be quite stable [21]. The S. Gallinarum 9R vaccine must be injected rather than being given orally, and there are ongoing concerns about its potential virulence in some breeds of chickens [22].

B. Deletion mutants With the development of techniques to produce deletions in specific target genes, Salmonella-defined deletion mutants were constructed, providing better control over the genetic composition of vaccine strains. Salmonella mutants deleted for genes in the biosynthetic pathway for aromatic amino acid synthesis were first described as potential vaccines in the 1980s [23]. These mutants affect the shikimate pathway and are unable to synthesize aromatic compounds, including aromatic amino acids and certain vitamins. Salmonella mutants with deletions in aroA, aroC, and/or aroD are immunogenic and have been extensively studied as vaccine candidates [23]. An aroA S. Typhimurium mutant is available commercially for use in poultry [24]. aro mutants are attenuated, largely owing to their inability to replicate in host tissues where aromatic amino acids are limiting. In addition, aroA and aroD mutants exhibit cell wall defects resulting in greater sensitivity to serum and other components of the innate immune system [25]. In a detailed study of a S. Typhimurium aroA mutant, the mutation was found to have pleiotropic effects, including increased sensitivity to complement and phagocytic uptake, reduced motility, and altered expression of several virulence genes, and when delivered intravenously to BALB/c mice, they elicit increased levels of TNF-α [26]. Deletion of certain regulatory genes also attenuates. S. Typhimurium mutants with deletions in cya and/or crp and phoPQ were shown to be attenuated and immunogenic in mice [27,28]. Crp, in conjunction with adenylate cyclase, encoded by cya, regulates expression of

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a number of genes and operons required for transport and catabolism of sugars, as well as a variety of virulence factors, including fimbriae, flagella, and outer membrane proteins. PhoP is a DNA-binding protein that regulates many Salmonella genes in response to magnesium and pH, sensed by the membrane protein PhoQ [29]. PhoPQ constitutes a master regulator of Salmonella virulence, including survival in macrophages [29]. A ΔphoPQ S. typhi mutant was safe in humans [30], while a Δcya Δcrp S. typhi mutant was highly reactogenic [31]. These results highlight the need for caution in translating mouse safety data obtained with S. Typhimurium into S. Typhi strains for humans. Fur (ferric uptake regulator) acts as a repressor of many genes whose products are involved in iron, zinc, and manganese acquisition and uptake [32,33]. In Salmonella, Fur also modulates expression of genes involved in surviving acid shock, adaptation to low pH [34,35], and oxidative stress resistance [36,37]. In addition, Fur plays a role in regulation of the Salmonella pathogenicity island 1 (SPI-1) genes (e.g., hilA and hilD) necessary for invasion [38
40]. An Salmonellaenterica Enteritidis Δfur strain is attenuated, and immunization of mice with this strain results in a decreased bacterial load in systemic organs after challenge with the wildtype strain [41]. An S. Gallinarum Δfur mutant is safe and immunogenic in chickens, eliciting a protective immune response against challenge with virulent S. Gallinarum [42]. A Δfur mutation was employed to improve the safety of a S. Typhimurium ΔssaV mutant (discussed in the next section). Introduction of a Δfur into a ΔssaV strain improved its safety profile in immunocompromised mice without compromising immunogenicity [43].

C. Mutations in Salmonellapathogenicity islands Salmonella carry genomic “islands,” generally with a GC content lower than the rest of

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the chromosome, that have been acquired by horizontal gene transfer. Many of these regions facilitate pathogenesis and are known as pathogenicity islands [44]. Although the number of pathogenicity islands and their specific gene content can vary by serovar, all Salmonella serovars that infect warm-blooded animals carry Salmonella pathogenicity island 1 (SPI-1) and SPI-2 [45]. Both SPI-1 and SPI-2 encode a type 3 secretion system (T3SS), a specialized secretion apparatus that secretes virulence proteins across host cell membranes directly into host cells [46]. The current paradigm is that SPI-1 facilitates invasion of host epithelial cells, and SPI-2 is important for survival in macrophages [47]. The ssaV gene encodes a component of the SPI-2 T3SS and strains lacking ssaV are unable to secrete SPI-2 effector proteins and are unable to survive in macrophages [48]. The ΔssaV ΔaroC S. Typhi strain M01ZH09 is safe and immunogenic when administered orally to human volunteers [49,50]. At the highest dose, all 15 vaccinees were positive for anti-Salmonella IgA antibody-secreting cells (ASCs). By day 28, 75% of the volunteers produced anti-Salmonella serum IgG responses that were at least fourfold above background, suggesting that this strain is a promising typhoid vaccine candidate [50]. Strain M01ZH09 was used to deliver the B subunit of heat-labile toxin (LTB) from E. coli in a clinical trial [51]. The eltB gene, encoding the LTB subunit, was inserted into the chromosome of M01ZH09 and expressed from the in vivoinducible ssaG promoter. At the highest dose, 92% of vaccinees produced anti-Salmonella IgA ASCs, and 63% produced anti-LTB IgA ASCs after one or two doses. When combined with ELISA data measuring serum IgG responses, 67% of the vaccinees demonstrated immune responses against LTB, and 95% of the test subjects produced anti-Salmonella immune responses. To date, these are the most promising results for a Salmonella-vectored vaccine in humans (Chapter 32: Oral Vaccines for Enterotoxigenic Escherichia coli).

D. Vectoring guest antigens As was mentioned in the above example, one method for modifying attenuated Salmonella for antigen delivery is to express a foreign gene from the bacterial chromosome. However, a more common approach is to express foreign genes from multicopy plasmids. While expression from the chromosome ensures that the antigen gene will not be lost from the population, the amount of protein produced from a single copy of the gene is often not adequate to promote a protective immune response. Increasing the gene dosage within the vaccine strains results in higher levels of protein production, thereby increasing the subsequent immune response. To be suitable for antigen delivery by Salmonella, the plasmid vector must carry a promoter to transcribe the guest antigen gene, an origin of plasmid replication, a selectable marker, and a means to maintain the plasmid in the vaccine cell population. The use of plasmids necessitates methods to select for and maintain their presence in the cell. In most nonvaccine applications, a geneencoding antibiotic resistance is used. When the plasmid is first introduced into the bacterium, plasmid-containing cells are selected on media containing the antibiotic whose resistance is encoded by the plasmid. Plasmidbearing cells are then grown in the presence of antibiotics to maintain the plasmid in the population. This approach is not practical for vaccine applications, as antibiotic resistance genes cannot be used. There are a variety of novel methods to select for and maintain plasmids in vaccine strains (for a review, see Ref. [52]). Here, we will discuss only the AsdA-balanced lethal plasmid stabilization system [53], as it is widely used. The asdA gene encodes aspartate semialdehyde dehydrogenase, an enzyme required for the synthesis of arginine, lysine, threonine, and methionine. One intermediate in the lysine

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biosynthetic pathway is diaminopimelic acid (DAP), an essential component of the peptidoglycan layer in the bacterial cell wall. An asdA Salmonella mutant cannot grow, even on rich media, unless the growth medium is supplemented with DAP. In the absence of DAP, asdA mutants undergo lysis [54]. Inclusion of a copy of asdA gene on the plasmid carrying a heterologous gene of interest permits selection of plasmid-bearing ΔasdA strains by simply plating on any rich medium. The plasmid is maintained in the population because any cell that loses the plasmid will lyse. This system has proven to be convenient for the selection and maintenance of plasmids in vaccine strains designed to deliver a wide variety of protein antigens.

E. Antigen delivery —location The final location of the antigen in the Salmonella cell can have a huge impact on immunogenicity. Typically, the protein products of antigen genes are retained in the cytoplasm of the Salmonella vaccine. The importance of antigen location was investigated in a study in which two Δcrp S. Typhimurium mutant strains producing the Streptococcus pneumoniae protein PspA were compared. In one strain, the PspA was retained in the cytoplasm. In the other strain, the PspA was fused to a type 2 secretion signal, resulting in secretion of PspA into the periplasmic space and into the growth medium [55]. The two strains produced equivalent levels of PspA. When used to immunize mice, the strain that secreted PspA elicited significantly greater serum anti-PspA IgG responses than the strain in which PspA was retained in the cytoplasm [56]. This strain also elicited strong anti-PspA mucosal IgA responses in immunized mice [55]. A direct comparison of strains in which the early secretory antigen 6 (ESAT-6) from Mycobacterium tuberculosis was localized either in the cytoplasm, on the bacterial cell surface, or secreted was conducted by using a ΔaroA

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S. Typhimurium strain [57]. As in the previous study, all strains produced similar levels of the antigen, varying only in where the antigen was localized. Compared to the cytoplasmic construct, surface-expressed and secreted constructs elicited significantly greater numbers of splenic ESAT-61 T helper 1 (Th1) cells. No IFNγ1 splenic T cells could be detected by using an ESAT-6 MHC II tetramer from mice immunized with the cytoplasmic construct; by contrast, the levels in mice immunized with the other two constructs achieved 3%
30% of total splenic ESAT-61 CD41 T cells. The impact on serum IgG responses was not evaluated for the cytoplasmic construct, but the strains that secreted ESAT-6 to the cell surface or into the extracellular environment elicited high titers of anti-ESAT-6 IgG. In this regard, the two strains were similar, although on day 21, the IgG titers in mice immunized with the surface-expressed strain were significantly lower than mice receiving the secreted construct. However, on days 35 and 42, there was no significant difference in the titers between groups. These results provide further evidence that antigen location is an important consideration in designing Salmonella-vectored vaccines. Technologies for enhancing the immunogenicity of Salmonella vaccines. Traditionally attenuated Salmonella vaccines rely on mutations designed to weaken the strain, making it less virulent. One caveat of this approach is that the resulting strains exhibit a reduced capacity to survive host defenses and interact with host immune cells, leading to reduced immunogenicity. To overcome these issues and enhance the ability of Salmonella to survive and replicate in target immune tissues, Roy Curtiss and colleagues have described a variety of novel approaches for vaccine development, including regulated delayed attenuation, regulated delayed antigen synthesis, and regulated delayed lysis (for a detailed review of these approaches, see Ref. [58]). In this scenario, the vaccine strains are not burdened by debilitating mutations or

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excessive heterologous antigen synthesis at the time of administration and during the initial stages of infection. Once the strain reaches target immune tissues, the attenuation phenotype is expressed, and heterologous antigen synthesis begins.

F. Regulated delayed attenuation Vaccine strains with regulated delayed attenuation display wild-type characteristics when grown in media containing appropriate sugars, providing them with a full complement of virulence factors required to survive transit through the gastrointestinal tract and to carry out the initial stages of infection. Once inside host tissues, expression of specific virulence genes or attributes shuts off, resulting in a fully attenuated strain. Several methods have been used to construct strains with the regulated delayed attenuation phenotype. For example, deletion of the pmi, encoding 6-phosphomannose isomerase, or galE, encoding uridine diphosphategalactose-4-epimerase, results in strains that are dependent on exogenous mannose or galactose, respectively, for the synthesis of O-antigen. O-antigen is a cell surface carbohydrate polymer that serves to protect Salmonella from the action of complement [59], and is important for penetration of the mucus that overlays the intestinal epithelium [60]. Strains are grown with the appropriate sugar prior to administration, resulting in full-length O-antigen. Free mannose and free galactose are not present in host tissues, and the cells gradually lose their O-antigen as they divide. However, pmi deletion mutants of S. Typhimurium are only partially attenuated in mice [61], and Salmonella Typhi galE mutants remain virulent for humans [20]. Thus, pmi and galE mutations cannot be used as the sole basis of attenuation but may serve as secondary mutations to make the cell more susceptible to host defenses. The regulation of virulence genes can be modified by replacing the native promoter

with a sugar-inducible promoter. The arabinose-regulated PBAD promoter, along with the gene encoding the arabinosesensitive transcriptional activator AraC, has been frequently used for this purpose. In one study, the araC PBAD promoter was used to drive expression of individual Salmonella virulence genes, including crp, phoP, rpoS, and/or fur [62]. When the vaccine is grown prior to administration, arabinose is added to the culture medium, and the arabinose-regulated virulence gene(s) is expressed. Thus upon immunization, the strain is producing its full complement of virulence factors. After immunization, when the vaccine strain reaches host tissues where free arabinose is not present, virulence gene expression ceases, and their protein products are lost by dilution as the bacteria divide. When administered orally, strains carrying arabinose-regulated crp, phoP, or rpoS genes were highly attenuated, while arabinose-regulated fur mutants were partially attenuated [62]. The safety and immunogenicity of vaccine strains designed by using this approach have been demonstrated for S. Typhimurium in mice and for S. Gallinarum in chickens [63,64]. However, since key virulence genes are expressed in response to sugars, arabinose in particular, it is possible that host diet could affect the virulence of these strains. While this question has not been specifically addressed for S. Typhimurium in mouse studies, it was investigated in chickens using a S. Gallinarum vaccine. Rhode Island red chicks were immunized with a highly virulent S. Gallinarum strain attenuated by replacement of the crp promoter with araC PBAD [64], a construct essentially identical to the one used in the S. Typhimurium studies. Note that the parent S. Gallinarum strain has an oral LD50 of less than 1 3 106 CFU [42]. Two groups of chicks were orally inoculated at 4 and 18 days of age with the vaccine strain. After the first inoculation, one group of birds was supplied with water

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containing 0.2% arabinose, and the other group was given water with no arabinose. All chickens survived, indicating that dietary arabinose intake does not affect the virulence of strains attenuated in this manner. The immunized birds in both groups were equally protected against challenge with the parent S. Gallinarum strain at 4 weeks of age.

G. Regulated delayed antigen synthesis The strongest case for developing Salmonella vaccines is its utility as an antigen delivery vector. Typically, genes encoding one or more antigens derived from a pathogen of interest are introduced into attenuated Salmonella on a plasmid. The choice of promoters to drive antigen gene(s) expression is an important parameter influencing the efficacy of the vaccine. The strong, constitutive Ptrc promoter can drive high levels of antigen synthesis in cells grown in vitro. In mouse studies, this promoter was shown to be a good, but not ideal, choice for eliciting optimal immune responses [65]. The suboptimal results using the Ptrc promoter are likely to be related to the fact that expression is unregulated in Salmonella. Unregulated heterologous antigen synthesis consumes cellular resources, reducing the ability of the vaccine strain to grow and to cope with host defenses. To overcome this problem, the heterologous antigen gene is placed under transcriptional control of a promoter that is active only in vivo. For example, the PssaG or PpagC promoters are turned on in macrophages [66,67], while the PnirB promoter is expressed under anaerobic conditions [68]. As an alternative approach, a method to regulate heterologous gene expression from the Ptrc promoter in Salmonella was developed. The Ptrc promoter carries a binding site for the transcriptional repressor LacI. The lacI gene is not native to Salmonella, so an arabinose-regulated lacI gene was introduced into the S. Typhimurium chromosome. This results in

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expression of lacI when the strain is grown in the presence of arabinose. When the LacI repressor is synthesized, it binds to Ptrc, thereby reducing or eliminating transcription of the heterologous passenger gene [69]. When arabinose is absent (as in host tissues), lacI is no longer transcribed. The LacI concentration in the cell decreases by dilution as the cell divides, allowing transcription from Ptrc to proceed. Synthesis of heterologous antigen increases, reaching maximum levels after approximately nine cell divisions due to dilution of LacI [69]. One potential drawback of this technology is that overproduction of LacI can reduce Salmonella virulence, which may reduce the immunogenicity of vaccine strains [70]. Despite this, the presence of an arabinose-inducible lacI gene was found to enhance the immunogenicity of a vaccine strain carrying a Ptrc-driven heterologous antigen gene, but reduces immunogenicity in strains carrying other, in vivoinducible promoters, such as PssaG, that do not bind LacI [71]. Thus, it is likely that the presence of the Ptrc promoter on a multicopy plasmid titrates the LacI so that its negative effect on virulence and immunogenicity is minimized. Despite its potential drawbacks, this system may be more flexible than in vivoregulated promoters, since the regulated Ptrc promoter system is compatible with a wider variety of attenuation strategies [71]. For example, promoters that rely on PhoP for activation, such as PpagC and PssaG, will not function properly in a ΔphoP background. Live attenuated Salmonella vaccine strains for antigen delivery have been constructed that utilize both regulated delayed attenuation and regulated delayed antigen synthesis. S. Typhimurium χ9558 includes arabinoseregulated crp and fur genes in addition to a Δpmi mutation [72]. This strain also carries an arabinose-regulated lacI gene to control heterologous antigen gene expression and a ΔasdA mutation to allow use of the AsdA-based

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FIGURE 22.1

Overview of the regulated delayed lysis system. (A) Arabinose regulation of wild-type araBAD. When arabinose is bound to AraC, it binds to the araI1 and araI2 sites. AraC and the cAMP receptor protein (CRP) activate transcription of from the PBAD promoter, driving expression of araBAD and araC. (B) In the absence of arabinose, AraC binds to the araO2 and araI1 regions, forming a DNA loop, repressing the transcription of all four genes. (C) The RASV lysis system is composed of plasmid and chromosomal components. Synthesis of MurA, C2, and LacI from chromosome and of MurA and AsdA from the plasmid occurs when arabinose is present. In the absence of arabinose, LacI is not made, and antigen genes are transcribed from Ptrc. murA and asdA are no longer expressed, leading to elimination of MurA and AsdA by dilution as the cell divides, eventually resulting in cell lysis.

balanced-lethal plasmid maintenance system. Immunization of mice with strain χ9558 carrying an antigen from S. pneumoniae resulted in a greater level of protection against lethal S. pneumoniae challenge than a traditionally attenuated S. Typhimurium strain producing the same antigen [72]. Strain χ9558 derivatives carrying Yersinia antigens have also been shown to protect mice against lethal challenge with Yersinia pestis [73]. Several S. Typhi vaccine candidates with genotypes similar to χ9558 were used to deliver PspA in a clinical trial. The strains were safe but only weakly immunogenic [74]. However, it should be noted that each of these S. Typhi strains carried 10 or more mutations, which may have resulted in over attenuation.

H. Regulated delayed vaccine lysis Another innovative technology is the design of vaccine strains that undergo regulated delayed vaccine lysis [75]. This system was developed to address a number of goals, including biocontainment, release of nonsecreted protein antigens, and DNA vaccine delivery. The basic components of this system are outlined in Fig. 22.1. These vaccine strains feature a ΔasdA deletion mutation and arabinose-controlled expression murA. The MurA gene product, like AsdA, is required for synthesis of the peptidoglycan component of the bacterial cell wall. The murA gene is essential, and its absence cannot be corrected by adding a nutrient to the growth medium, so a

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conditional lethal mutation was constructed by placing the gene under transcriptional control of araC PBAD. The plasmid component of this system carries arabinose-controlled asdA and murA genes, along with antigen genes of interest. The Ptrc promoter drives antigen gene expression for delivery of protein antigens, and is regulated by an arabinose-regulated lacI gene present in the chromosome. S. Typhimurium lysis strains designed by using this technology have been used to deliver proteins from Gram-positive pathogens and from influenza A virus. In the first chapter describing this system, mice were orally immunized with lysis strains producing the S. pneumoniae protein PspA. Immunized mice produced both humoral and mucosal responses against PspA and Salmonella proteins [75]. Humoral responses against Salmonella antigens were strongly Th1, while a mixed Th1/Th2 response was elicited against PspA. In another study, a lysis strain carrying a plasmid encoding a woodchuck hepatitis virus-like particle (VLP) modified to produce the influenza A M2e protein was used to orally or intranasally immunize mice twice at a 3-week interval [76]. The strain elicited both humoral IgG and mucosal IgA responses against M2e. Intranasal immunization resulted in a more rapid antibody response, but by 6 weeks, the two groups showed similar levels of IgG in the serum. These responses were greater than those in a control group that received the VLP-M2e delivered by a nonlysis Δcya Δcrp S. Typhimurium strain. Five days after being challenged with influenza virus, immunized mice had lower titers of virus in their lungs compared to nonimmunized controls, suggesting mucosal protection, although the difference was not statistically significant. Immunized mice also showed a significant increase in weight gain and increased survival compare to Salmonellaonly controls. A similar strain, further modified with a ΔsifA deletion to allow escape from the endosome prior to lysis [77], was used to orally

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deliver the influenza A NP protein in three or four doses, eliciting a strong Th1-type response [78]. The vaccine elicited protection against weight loss and led to increased survival after influenza challenge. A lysis strain was used to deliver Clostridium perfringens antigens to chickens. C. perfringens causes necrotic enteritis in chickens, a disease characterized in part by intestinal lesions. Chickens were orally immunized with two doses of a Salmonella lysis strain producing two relevant antigens, alpha toxoid and NetB toxoid from a plasmid [79]. Overall, serum responses were low, with maximum IgY, the chicken equivalent of IgG, titers about four-fold greater than those in controls. However, the vaccine elicited strong mucosal responses, with maximum intestinal IgA, IgM, and IgY titers against both antigens that were 30- to 60-fold greater than those in controls. After challenge, immunized birds exhibited fewer and milder lesions than Salmonella-only controls. Protection was not as great when a nonlysis strain delivering the same antigens was used as the immunogen (Jiang, Roland, and Curtiss, unpublished). A Salmonella lysis strain is also useful for delivering DNA vaccines. In this context, the plasmid component of the lysis system is modified to include a eukaryotic promoter and nuclear targeting sequences. A number of additional mutations were included in the vaccine strain to facilitate efficient DNA delivery, including ΔsifA [80]. The system was used to deliver a gene encoding the influenza hemagglutinin antigen (HA). Results from the study showed that the lysis DNA delivery system was effective, eliciting high serum IgG titers against HA and protection against challenge with influenza virus. Mucosal responses were not reported. Taken together, results from the studies described in this section suggest that lysis strains are superior to nonlysis strains for eliciting protective mucosal responses, at

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least against the pathogens tested. Thus far, this approach has not been tested in a clinical trial, so how useful and applicable it is for human vaccines remains an open question. One concern about this system relates to its potential to trigger sepsis due to release of lipid A during lysis. While symptoms of sepsis have not been reported in animal systems, this remains a potential obstacle that must be addressed before this system can be approved for human trials. In the next section, I will discuss strategies for lowering the toxicity of lipid A by altering its structure in vivo.

I. Strategies for reducing lipid A toxicity Lipid A, also known as endotoxin, is a component of the complex molecule called LPS which forms the outer monolayer of the outer membrane of Salmonella. LPS is composed of lipid A, core sugars, and the highly immunogenic, multimeric O-antigen polysaccharide. Salmonella lipid A is itself a complex molecule consisting of a β(1
6)-linked glucosamine disaccharide, with phosphate groups at the 1 and 40 positions (Fig. 22.2). The disaccharide is typically hexa-acylated with 12- to 16-carbon acyl groups, although the exact number and length of the acyl groups can vary depending on the environment. Lipid A is responsible for the toxic effects of LPS (endotoxin), which include fever and sepsis [81]. Lipid A containing two phosphate groups, located at the 1 and 40 positions in the molecule, and six acyl chains that are 12
14 carbons in length activates proinflammatory responses through the TLR4MD2-CD14 pathway, while lipid A variants with fewer acyl chains, such as tetra- or pentaacylated lipid A species, have significantly diminished immunostimulatory activity [82,83]. A monophosphoryl lipid A derivative of E. coli lipid A (MPL), in which the 1-phosphate and several acyl groups are

FIGURE 22.2 Diagram of Salmonella lipid A. The figure illustrates the basic structure of lipid A, including glucosamines, acyl groups, and phosphate groups. Carbons comprising the glucosamine moieties are numbered. Depending on conditions, the phosphate groups may be modified with 4-amino-4 deoxy-L-arabinose or phosphoethanolamine moieties, and additional acyl groups or modified acyl groups may be present.

chemically removed, is much less toxic while retaining its ability to bind to TLR4 and is a potent mucosal adjuvant [84]. Kong and associates have taken a unique approach to enhancing Salmonella vaccine safety by modifying the lipid A in living cells to the less toxic, 40 -monophosphoryl form while retaining its adjuvant properties [85]. To achieve this, the Francisella tularensis lpxE gene, encoding an inner membrane phosphatase, was introduced into the Salmonella chromosome to facilitate removal of the 1-phosphate group from lipid A. Constitutive expression of lpxE from the chromosome, along with deletion of several Salmonella genes involved in lipid A modification, resulted in a strain that produced lipid A that was essentially 100% dephosphorylated at the 1 position. The resulting 40 -monophosphorylated lipid A

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was purified and used to stimulate the human monocytic cell line MM6, inducing significantly lower levels of IL-6 than lipid A isolated from wild-type S. Typhimurium [85]. In a rabbit ligated loop assay, the S. Typhimurium strain producing 40 -monophosphorylated lipid A did not elicit a detectable inflammatory response, while its wild-type parent was highly inflammatory, as characterized by flattening of epithelial villi, substantial PMN infiltration, and significant tissue destruction. An attenuated S. Typhimurium strain modified to produce 40 -monophosphorylated lipid A retained immunogenicity and was able to deliver the pneumococcal protein PspA, eliciting antiPspA serum IgG and mucosal IgA, resulting in significant protection against challenge with S. pneumoniae. This seems to be a promising approach for overcoming potential issues with the delayed lysis technology. However, as yet, there are no reports of lysis strains engineered to produce modified lipid A. Vaccine strains with lipid A with the 1 and 40 phosphates removed have been explored. However, this modification resulted in lower immunogenicity [86]. Lipid A toxicity can also be reduced by modification of the acyl groups attached to the glucosamine component of lipid A [87]. Deletion of the waaN (msbB) gene results in pentaacylated lipid A missing a myristoyl group [88]. This modification results in a loss of virulence and decreased endotoxicity linked to a reduced ability of the penta-acylated lipid A to induce TNF-α in human monocytes [89]. A waaN mutant of S. Typhimurium strains with modified acyl groups has been investigated for use as an antigen delivery vector [90]. Introduction of a ΔwaaN mutation into an attenuated ΔpabA ΔpabB mutant enhanced mucosal IgA responses to a vectored antigen, PspA, although anti-PspA serum IgG responses were delayed. This is a promising alternative approach to 1-dephosphorylation for detoxifying lipid A in lysis strains.

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J. Sugar-inducible acid resistance Attenuated S. Typhi strains are the preferred Salmonella vector for human vaccines. However, S. Typhi is more sensitive to the low pH environment of the human stomach than other enteric pathogens such as S. Typhimurium, E. coli or Shigella [91]. Many attenuated S. Typhi strains exhibit a further increase in sensitivity to low pH. To survive the low gastric pH in humans, oral Salmonella vaccines are typically given with an agent designed to increase the gastric pH, such as bicarbonate, or they are incorporated into an enteric-coated capsule. While this approach is helpful, it precludes the Salmonella vaccine from sensing an important environmental signal—low pH—signifying its entry into a host and, in the case of encapsulation, reduces overall efficacy. Increasing the acid resistance of S. Typhi vaccines would preclude the need to bypass stomach acidity, enhancing its ability to reach the host mucosa and initiate invasion of local tissues in the gut, leading to enhanced immunogenicity. One caveat of this approach is that fewer attenuated S. Typhi cells may be required to elicit a protective immune response, thus lowering the effective vaccine dose and perhaps also the frequency of vaccination. Enteric bacteria have evolved a number of different strategies to survive low-pH environments, including amino acid decarboxylase systems. Several groups have taken advantage of these existing genes and have engineered S. Typhi vaccine strains to express them from sugar-inducible promoters such as araC PBAD. In my laboratory, we modified the native S. Typhi acid resistance genes adiA and adiC, encoding arginine decarboxylase and the arginine
agmatine antiporter, such that their expression is driven by a sugarinducible promoter [92]. Addition of the appropriate sugar results in expression of adiA and adiC and a concomitant increase in acid resistance. Our results demonstrated that

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this system significantly increases in survival for ΔaroD and ΔphoPQ S. Typhi strains at both pH 3.0 and pH 2.5 [92] and in a low gastric pH mouse model [93]. We also examined the use of the glutamate decarboxylase (GAD) system from E. coli. This system allows E. coli to survive at a lower pH than S. Typhi [94]. The GAD system is composed of two homologous decarboxylases (GadA and GadB) and a glutamate/ γ
aminobutyric acid antiporter (GadC) [95]. GadA and GadB are biochemically indistinguishable, and only GadB is strictly required for survival at pH 2.5 in E. coli [96]. However, both are required for survival at pH 2 [96,97]. In E. coli, this system maintains an internal pH between 4 and 5 [98]. We introduced an arabinose-inducible glutamate decarboxylase system (gadBC) into S. Typhi guaBA, phoPQ, and fur mutants. The presence of gadBC enhanced survival to acid shock in vitro and survival during passage through the gastrointestinal tract in a low gastric pH mouse model (Brenneman and Roland, unpublished). Dennis Kopecko’s group introduced the gadA gadBC system from Shigella flexneri into the licensed typhoid vaccine strain Ty21a under transcriptional control of araC PBAD [99]. The inclusion of this system resulted in significant increases in survival at pH 3.0 and pH 2.5 compared to Ty21a without this system. The impact of this approach on immunogenicity has not yet been tested in a clinical trial.

K. Modification of fimbriae Bacterial fimbriae are extracellular structures whose primary function is to present adhesins that enable the bacterium to bind to surfaces. In addition to adherence to surfaces, fimbriae are involved in other functions, including conjugation, biofilm formation, and evasion of host phagocytes. Although fimbriae have been used

as vaccine antigens, the role of fimbriae in vaccine design to influence host immune responses has been an understudied area. However, there have been a few reports suggesting that host immune responses can be influenced by the fimbrial composition of the vaccine strain. When the E. coli CFA/I fimbriae were produced in an S. Typhimurium vaccine, there was a shift in the subsequent early immune response from Th1- to Th2-type, compared with a strain that does not produce CFA/I [100]. This was due to altered interaction with host macrophages, resulting in a reduction in the early proinflammatory cytokine responses [101]. In another study, four S. Typhimurium fimbrial operons—agf, saf, sti, and stc—were identified as being expressed in the mouse spleen. In the context of an attenuated S. Typhimurium vaccine strain delivering PspA, constitutive expression of either sti, saf, or stc significantly enhanced protection against challenge with S. pneumoniae [42], suggesting that this approach has promise for vectored vaccines. We have been investigating the influence of modifying the S. Typhi fimbrial profile as a means to improve immunogenicity. The S. Typhi stg fimbrial operon facilitates adherence to enterocytes [102], while the S. Typhimurium lpf fimbrial operon facilitates adherence to M cells, leading to efficient colonization of PPs with subsequent stimulation of innate immune responses [103]. We have found that deleting the stg fimbrial operon and introducing lpf in wildtype S. Typhi resulted in increased adherence and invasion of M cells, with subsequent increases in release of the proinflammatory cytokine interleukin-8 [104]. More important, these modifications had a profound effect on the adherence and invasion of M cells by the licensed vaccine strain Ty21a (Tafoya, Roland unpublished), suggesting that this approach may significantly enhance immune responses to this vaccine.

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III. VACCINES AGAINST NONTYPHOIDAL SALMONELLA Bacteremia due to invasive nontyphoidal Salmonella (iNTS) is an emerging disease in parts of sub-Saharan Africa. The primary serovars associated with this disease are S. Typhimurium and S. Enteritidis, although other serovars may also be involved. This situation has driven efforts to develop live attenuated vaccines to prevent infections by iNTS. Prior to these efforts, only two attenuated S. Typhimurium vaccines were tested in humans: the ΔaroC ΔssaV mutant WT05 [49] and a ΔphoPQ mutant expressing the urease gene from Helicobacter pylori [105]. Both strains were safe and immunogenic, although the WT05 strain was shed in feces for up to 22 days. However, these early results indicate that NTS strains may be suitable as human vaccines. iNTS isolates were used to construct S. Typhimurium and S. Enteritidis strains carrying ΔguaBA ΔclpP or ΔguaBA ΔclpX mutations as human vaccines. Preclinical studies evaluating these strains are underway [106] (Chapter 29: Mucosal Vaccines for Salmonella typhi Infection).

IV. CONCLUDING REMARKS Salmonella has unique characteristics that make it an ideal antigen delivery vector. As our understanding of Salmonella pathogenesis has improved, so has our ability to harness Salmonella’s usefulness. From putting “Salmonella on a string” to induce attenuation and/or lysis after infection to advancing safety by modifying its lipid A and enhancing immunogenicity by inactivating immunosuppressive genes, we are finally poised to make human Salmonella vaccines a reality. Thus, the technology for constructing Salmonella vaccines is reaching its maturity. The lack of informative animal models for S. Typhi leaves human trials as the best option

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for testing S. Typhi vaccines. As of 2013, there were only 13 clinical trials testing Salmonella vectoring antigens. Advanced tissue culture models are being developed, but it is unclear how well these will mimic the complicated interplay between Salmonella and host. What is needed now are more clinical studies to test the innovative ideas outlined in this chapter and lead the way to achieving the great potential of Salmonella vaccines.

References [1] Sundquist M, Wick MJ. TNF-a-dependent and -independent maturation of dendritic cells and recruited CD11c(int)CD11b1 cells during oral Salmonella infection. J Immunol 2005;175(5):3287
98. [2] Hopkins SA, Niedergang F, Corthesy-Theulaz IE, Kraehenbuhl JP. A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer’s patch dendritic cells. Cell Microbiol 2000;2 (1):59
68. [3] Reboldi A, Arnon TI, Rodda LB, Atakilit A, Sheppard D, Cyster JG. IgA production requires B cell interaction with subepithelial dendritic cells in Peyer’s patches. Science 2016;352(6287):aaf4822. [4] Johansson C, Wick MJ. Liver dendritic cells present bacterial antigens and produce cytokines upon Salmonella encounter. J Immunol 2004;172(4):2496
503. [5] Yrlid U, Svensson M, Hakansson A, Chambers BJ, Ljunggren HG, Wick MJ. In vivo activation of dendritic cells and T cells during Salmonella enterica serovar Typhimurium infection. Infect Immun 2001;69 (9):5726
35. [6] Salazar-Gonzalez RM, Niess JH, Zammit DJ, Ravindran R, Srinivasan A, Maxwell JR, et al. CCR6-mediated dendritic cell activation of pathogen-specific T cells in Peyer’s patches. Immunity 2006;24(5):623
32. [7] McSorley SJ, Asch S, Costalonga M, Reinhardt RL, Jenkins MK. Tracking Salmonella-specific CD4 T cells in vivo reveals a local mucosal response to a disseminated infection. Immunity 2002;16(3):365
77. [8] Trinchieri G, Sher A. Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol 2007;7(3):179
90. [9] Feuillet V, Medjane S, Mondor I, Demaria O, Pagni PP, Galan JE, et al. Involvement of Toll-like receptor 5 in the recognition of flagellated bacteria. Proc Natl Acad Sci U S A 2006;103(33):12487
92.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

396

22. ATTENUATED SALMONELLA FOR ORAL IMMUNIZATION

[10] Carpenter S, O’Neill LA. How important are Toll-like receptors for antimicrobial responses? Cell Microbiol 2007;9(8):1891
901. [11] de Jong HK, Parry CM, van der Poll T, Wiersinga WJ. Host-pathogen interaction in invasive Salmonellosis. PLoS Pathog 2012;8(10):e1002933. [12] Raffatellu M, Chessa D, Wilson RP, Tukel C, Akcelik M, Baumler AJ. Capsule-mediated immune evasion: a new hypothesis explaining aspects of typhoid fever pathogenesis. Infect Immun 2006;74(1):19
27. [13] Fournier B, Williams IR, Gewirtz AT, Neish AS. Tolllike receptor 5-dependent regulation of inflammation in systemic Salmonella enterica serovar Typhimurium infection. Infect Immun 2009;77(9):4121
9. [14] Klimpel GR, Asuncion M, Haithcoat J, Niesel DW. Cholera toxin and Salmonella typhimurium induce different cytokine profiles in the gastrointestinal tract. Infect Immun 1995;63(3):1134
7. [15] Kupz A, Guarda G, Gebhardt T, Sander LE, Short KR, Diavatopoulos DA, et al. NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8(1) T cells. Nat Immunol 2012;13 (2):162
9. [16] Rydstro¨m A, Wick MJ. Monocyte recruitment, activation, and function in the gut-associated lymphoid tissue during oral Salmonella infection. J Immunol 2007;178(9):5789
801. [17] Martinoli C, Chiavelli A, Rescigno M. Entry route of Salmonella typhimurium directs the type of induced immune response. Immunity 2007;27 (6):975
84. [18] Smith HW. The use of live vaccines in experimental Salmonella gallinarum infection in chickens with observations on their interference effect. J Hyg (Lond) 1956;54(3):419
32. [19] Germanier R, Furer E. Isolation and characterization of Gal E mutant Ty 21a of Salmonella typhi: a candidate strain for a live, oral typhoid vaccine. J Infect Dis 1975;131(5):553
8. [20] Hone DM, Attridge SR, Forrest B, Morona R, Daniels D, LaBrooy JT, et al. A galE via (Vi antigen-negative) mutant of Salmonella typhi Ty2 retains virulence in humans. Infect Immun 1988;56(5):1326
33. [21] Kopecko DJ, Sieber H, Ures JA, Furer A, Schlup J, Knof U, et al. Genetic stability of vaccine strain Salmonella Typhi Ty21a over 25 years. Int J Med Microbiol 2009;299(4):233
46. [22] Kwon HJ, Cho SH. Pathogenicity of SG 9R, a rough vaccine strain against fowl typhoid. Vaccine 2011;29 (6):1311
18. [23] Hoiseth SK, Stocker BA. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 1981;291(5812):238
9.

[24] Alderton MR, Fahey KJ, Coloe PJ. Humoral responses and salmonellosis protection in chickens given a vitamin-dependent Salmonella typhimurium mutant. Avian Dis 1991;35(3):435
42. [25] Sebkova A, Karasova D, Crhanova M, Budinska E, Rychlik I. aro Mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity. J Bacteriol 2008;190(9):3155
60. [26] Felgner S, Frahm M, Kocijancic D, Rohde M, Eckweiler D, Bielecka A, et al. aroA-Deficient Salmonella enterica serovar typhimurium is more than a metabolically attenuated mutant. MBio 2016;7:5. [27] Curtiss 3rd R, Kelly SM. Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect Immun 1987;55(12):3035
43. [28] Miller SI, Loomis WP, Alpuche-Aranda C, Behlau I, Hohmann E. The PhoP virulence regulon and live oral Salmonella vaccines. Vaccine 1993;11(2):122
5. [29] Groisman EA, Mouslim C. Sensing by bacterial regulatory systems in host and non-host environments. Nat Rev Microbiol 2006;4(9):705
9. [30] Hohmann EL, Oletta CA, Killeen KP, Miller SI. phoP/ phoQ-deleted Salmonella typhi (Ty800) is a safe and immunogenic single-dose typhoid fever vaccine in volunteers. J Infect Dis 1996;173(6):1408
14. [31] Tacket CO, Hone DM, Curtiss III R, Kelly SM, Losonsky G, Guers L, et al. Comparison of the safety and immunogenicity of DaroC DaroD and Dcya Dcrp Salmonella typhi strains in adult volunteers. Infect Immun 1992;60(2):536
41. [32] Troxell B, Hassan HM. Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria. Front Cell Infect Microbiol 2013;3:59. [33] Garrido ME, Bosch M, Medina R, Llagostera M, Perez de Rozas AM, Badiola I, et al. The high-affinity zincuptake system znuACB is under control of the ironuptake regulator (fur) gene in the animal pathogen Pasteurella multocida. FEMS Microbiol Lett 2003;221 (1):31
7. [34] Hall HK, Foster JW. The role of fur in the acid tolerance response of SalmonellaTyphimurium is physiologically and genetically separable from its role in iron acquisition. J Bacteriol 1996;178(19):5683
91. [35] Baik HS, Bearson S, Dunbar S, Foster JW. The acid tolerance response of Salmonella typhimurium provides protection against organic acids. Microbiology 1996;142(Pt 11):3195
200. [36] Leclerc JM, Dozois CM, Daigle F. Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells. Microbiology 2013;159(Pt 3):591
602.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

397

REFERENCES

[37] Velayudhan J, Castor M, Richardson A, Main-Hester KL, Fang FC. The role of ferritins in the physiology of Salmonella enterica sv. Typhimurium: a unique role for ferritin B in iron-sulphur cluster repair and virulence. Mol Microbiol 2007;63(5):1495
507. [38] Teixido L, Carrasco B, Alonso JC, Barbe J, Campoy S. Fur activates the expression of Salmonella enterica pathogenicity island 1 by directly interacting with the hilD operator in vivo and in vitro. PloS One 2011;6(5): e19711. [39] Troxell B, Sikes ML, Fink RC, Vazquez-Torres A, Jones-Carson J, Hassan HM. Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Typhimurium. J Bacteriol 2011;193(2):497
505. [40] Ellermeier JR, Slauch JM. Fur regulates expression of the Salmonella pathogenicity island 1 type III secretion system through HilD. J Bacteriol 2008;190 (2):476
86. [41] Karasova D, Sebkova A, Vrbas V, Havlickova H, Sisak F, Rychlik I. Comparative analysis of Salmonella enterica serovar Enteritidis mutants with a vaccine potential. Vaccine 2009;27(38):5265
70. [42] Laniewski P, Mitra A, Karaca K, Khan A, Prasad R, Curtiss 3rd R, et al. Evaluation of protective efficacy of live attenuated Salmonella enterica serovar Gallinarum vaccine strains against fowl typhoid in chickens. Clin Vaccine Immunol 2014;21(9):1267
76. [43] Vishwakarma V, Pati NB, Chandel HS, Sahoo SS, Saha B, Suar M. Evaluation of Salmonella enterica serovar Typhimurium TTSS-2 deficient fur mutant as safe liveattenuated vaccine candidate for immunocompromised mice. PloS One 2012;7(12):e52043. [44] Marcus SL, Brumell JH, Pfeifer CG, Finlay BB. Salmonella pathogenicity islands: big virulence in small packages. Microbes Infect 2000;2(2):145
56. [45] Figueira R, Holden DW. Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors. Microbiology 2012;158(Pt 5):1147
61. [46] Notti RQ, Stebbins CE. The structure and function of type III secretion systems. Microbiol Spectr 2016;4(1). [47] Buckner MM, Croxen MA, Arena ET, Finlay BB. A comprehensive study of the contribution of Salmonella enterica serovar Typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models. Virulence 2011;2(3):208
16. [48] Hansen-Wester I, Stecher B, Hensel M. Type III secretion of Salmonella enterica serovar Typhimurium translocated effectors and SseFG. Infect Immun 2002;70 (3):1403
9. [49] Hindle Z, Chatfield SN, Phillimore J, Bentley M, Johnson J, Cosgrove CA, et al. Characterization of

[50]

[51]

[52] [53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

Salmonella enterica derivatives harboring defined aroC and Salmonella pathogenicity island 2 type III secretion system (ssaV) mutations by immunization of healthy volunteers. Infect Immun 2002;70(7):3457
67. Kirkpatrick BD, McKenzie R, O’Neill JP, Larsson CJ, Bourgeois AL, Shimko J, et al. Evaluation of Salmonella enterica serovar Typhi (Ty2 aroC-ssaV-) M01ZH09, with a defined mutation in the Salmonella pathogenicity island 2, as a live, oral typhoid vaccine in human volunteers. Vaccine 2006;24(2):116
23. Khan S, Chatfield S, Stratford R, Bedwell J, Bentley M, Sulsh S, et al. Ability of SPI2 mutant of S. typhi to effectively induce antibody responses to the mucosal antigen enterotoxigenic E. coli heat labile toxin B subunit after oral delivery to humans. Vaccine 2007;25 (21):4175
82. Roland KL, Brenneman KE. Salmonella as a vaccine delivery vehicle. Expert Rev Vaccines 2013;12(9):1033
45. Galan JE, Nakayama K, Curtiss 3rd R. Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains. Gene 1990;94 (1):29
35. Loessner H, Endmann A, Rohde M, Curtiss 3rd R, Weiss S. Differential effect of auxotrophies on the release of macromolecules by Salmonella enterica vaccine strains. FEMS Microbiol Lett 2006;265(1):81
8. Kang HY, Srinivasan J, Curtiss 3rd R. Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar Typhimurium vaccine. Infect Immun 2002;70 (4):1739
49. Kang HY, Curtiss 3rd R. Immune responses dependent on antigen location in recombinant attenuated Salmonella typhimurium vaccines following oral immunization. FEMS Immunol Med Microbiol 2003;37 (2
3):99
104. Sevastsyanovich YR, Withers DR, Marriott CL, Morris FC, Wells TJ, Browning DF, et al. Antigen localization influences the magnitude and kinetics of endogenous adaptive immune response to recombinant Salmonella vaccines. Infect Immun 2017;85(12). Available from: http://dx.doi.org/10.1128/IAI.00593-17. Wang S, Kong Q, Curtiss 3rd R. New technologies in developing recombinant attenuated Salmonella vaccine vectors. Microb Pathog 2012;58:17
28. Murray GL, Attridge SR, Morona R. Altering the length of the lipopolysaccharide O antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with macrophages and complement. J Bacteriol 2006;188(7):2735
9. McCormick BA, Stocker BA, Laux DC, Cohen PS. Roles of motility, chemotaxis, and penetration through

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

398

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

22. ATTENUATED SALMONELLA FOR ORAL IMMUNIZATION

and growth in intestinal mucus in the ability of an avirulent strain of Salmonella typhimurium to colonize the large intestine of streptomycin-treated mice. Infect Immun 1988;56(9):2209
17. Collins LV, Attridge S, Hackett J. Mutations at rfc or pmi attenuate Salmonella typhimurium virulence for mice. Infect Immun 1991;59(3):1079
85. Curtiss 3rd R, Wanda SY, Gunn BM, Zhang X, Tinge SA, Ananthnarayan V, et al. Salmonella strains with regulated delayed attenuation in vivo. Infect Immun 2009;77(3):1071
82. Mitra A, Laniewski P, Curtiss 3rd R, Roland KL, Live Oral A. Fowl Typhoid vaccine with reversible Oantigen production. Avian Dis 2015;59(1):52
6. Mitra A, Loh A, Gonzales A, Laniewski P, Willingham C, Curtiss III R, et al. Safety and protective efficacy of live attenuated Salmonella Gallinarum mutants in Rhode Island Red chickens. Vaccine 2013;31(7):1094
9. Dunstan SJ, Simmons CP, Strugnell RA. Use of in vivoregulated promoters to deliver antigens from attenuated Salmonella enterica var. Typhimurium. Infect Immun 1999;67(10):5133
41. Hohmann EL, Oletta CA, Loomis WP, Miller SI. Macrophage-inducible expression of a model antigen in Salmonella typhimurium enhances immunogenicity. Proc Natl Acad Sci U S A 1995;92(7):2904
8. McKelvie ND, Stratford R, Wu T, Bellaby T, Aldred E, Hughes NJ, et al. Expression of heterologous antigens in SalmonellaTyphimurium vaccine vectors using the in vivo-inducible, SPI-2 promoter, ssaG. Vaccine 2004;22 (25-26):3243
55. Chatfield SN, Charles IG, Makoff AJ, Oxer MD, Dougan G, Pickard D, et al. Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus vaccine. Biotechnology (N Y) 1992;10(8):888
92. Wang S, Li Y, Scarpellini G, Kong W, Shi H, Baek CH, et al. Salmonella vaccine vectors displaying delayed antigen synthesis in vivo to enhance immunogenicity. Infect Immun 2010;78(9):3969
80. Eswarappa SM, Karnam G, Nagarajan AG, Chakraborty S, Chakravortty D. lac repressor is an antivirulence factor of Salmonella enterica: its role in the evolution of virulence in Salmonella. Plos One 2009;4 (6):e5789. Wang S, Li Y, Shi H, Sun W, Roland KL, Curtiss 3rd R. Comparison of a regulated delayed antigen synthesis system with in vivo-inducible promoters for antigen delivery by live attenuated Salmonella vaccines. Infect Immun 2011;79(2):937
49. Li Y, Wang S, Scarpellini G, Gunn B, Xin W, Wanda SY, et al. Evaluation of new generation Salmonella

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81] [82]

[83]

[84]

enterica serovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA. Proc Natl Acad Sci U S A 2009;106 (2):593
8. Branger CG, Sun W, Torres-Escobar A, Perry R, Roland KL, Fetherston J, et al. Evaluation of Psn, HmuR and a modified LcrV protein delivered to mice by live attenuated Salmonella as a vaccine against bubonic and pneumonic Yersinia pestis challenge. Vaccine 2010;29(2):274
82. Frey SE, Lottenbach KR, Hill H, Blevins TP, Yu Y, Zhang Y, et al. A Phase I, dose-escalation trial in adults of three recombinant attenuated Salmonella Typhi vaccine vectors producing Streptococcus pneumoniae surface protein antigen PspA. Vaccine 2013;31 (42):4874
80. Kong W, Wanda SY, Zhang X, Bollen W, Tinge SA, Roland KL, et al. Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment. Proc Natl Acad Sci U S A 2008;105(27):9361
6. Ameiss K, Ashraf S, Kong W, Pekosz A, Wu WH, Milich D, et al. Delivery of woodchuck hepatitis viruslike particle presented influenza M2e by recombinant attenuated Salmonella displaying a delayed lysis phenotype. Vaccine 2010;28(41):6704
13. Brumell JH, Goosney DL, Finlay BB. SifA, a type III secreted effector of Salmonella typhimurium, directs Salmonella-induced filament (Sif) formation along microtubules. Traffic 2002;3(6):407
15. Ashraf S, Kong W, Wang S, Yang J, Curtiss 3rd R. Protective cellular responses elicited by vaccination with influenza nucleoprotein delivered by a live recombinant attenuated Salmonella vaccine. Vaccine 2011;29(23):3990
4002. Jiang Y, Mo H, Willingham C, Wang S, Park JY, Kong W, et al. Protection against necrotic enteritis in broiler chickens by regulated delayed lysis Salmonella vaccines. Avian Dis 2015;59(4):475
85. Kong W, Brovold M, Koeneman BA, Clark-Curtiss J, Curtiss 3rd R. Turning self-destructing Salmonella into a universal DNA vaccine delivery platform. Proc Natl Acad Sci U S A 2012;109(47):19414
19. Parrillo JE. Pathogenetic mechanisms of septic shock. N Engl J Med 1993;328(20):1471
7. Miller SI, Ernst RK, Bader MW. LPS, TLR4 and infectious disease diversity. Nat Rev Microbiol 2005;3 (1):36
46. Brandenburg K, Wiese A. Endotoxins: relationships between structure, function, and activity. Curr Top Med Chem 2004;4(11):1127
46. Freytag LC, Clements JD. Mucosal adjuvants. Vaccine 2005;23(15):1804
13.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[85] Kong Q, Six DA, Roland KL, Liu Q, Gu L, Reynolds CM, et al. Salmonella synthesizing 1dephosphorylated [corrected] lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity. J Immunol 2011;187(1):412
23. [86] Kong Q, Six DA, Liu Q, Gu L, Wang S, Alamuri P, et al. Phosphate groups of lipid A are essential for Salmonella enterica serovar Typhimurium virulence and affect innate and adaptive immunity. Infect Immun 2012;80(9):3215
24. [87] Wang X, Quinn PJ, Yan A. Kdo2 -lipid A: structural diversity and impact on immunopharmacology. Biol Rev Camb Philos Soc 2015;90(2):408
27. [88] Tran AX, Lester ME, Stead CM, Raetz CR, Maskell DJ, McGrath SC, et al. Resistance to the antimicrobial peptide polymyxin requires myristoylation of Escherichia coli and Salmonella typhimurium lipid A. J Biol Chem 2005;280(31):28186
94. [89] Low KB, Ittensohn M, Le T, Platt J, Sodi S, Amoss M, et al. Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumortargeting in vivo. Nat Biotechnol 1999;17(1):37
41. [90] Kong Q, Six DA, Liu Q, Gu L, Roland KL, Raetz CR, et al. Palmitoylation state impacts induction of innate and acquired immunity by the Salmonella enterica serovar typhimurium msbB mutant. Infect Immun 2011;79(12):5027
38. [91] Verdu EF, Fraser R, Armstrong D, Blum AL. Effects of omeprazole and lansoprazole on 24-hour intragastric pH in Helicobacter pylori-positive volunteers. Scand J Gastroenterol 1994;29(12):1065
9. [92] Brenneman KE, Willingham C, Kong W, Curtiss 3rd R, Roland KL. Low-pH rescue of acid-sensitive Salmonella enterica serovar Typhi strains by a rhamnose-regulated arginine decarboxylase system. J Bacteriol 2013;195(13):3062
72. [93] Brenneman KE, Willingham C, Kilbourne JA, Curtiss 3rd R, Roland KL. A low gastric pH mouse model to evaluate live attenuated bacterial vaccines. PloS One 2014;9(1):e87411. [94] Hone DM, Harris AM, Levine MM. Adaptive acid tolerance response by Salmonella typhi and candidate live oral typhoid vaccine strains. Vaccine 1994;12 (10):895
8. [95] Smith DK, Kassam T, Singh B, Elliott JF. Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci. J Bacteriol 1992;174 (18):5820
6.

399

[96] De Biase D, Tramonti A, Bossa F, Visca P. The response to stationary-phase stress conditions in Escherichia coli: role and regulation of the glutamic acid decarboxylase system. Mol Microbiol 1999;32 (6):1198
211. [97] De Biase D, Tramonti A, John RA, Bossa F. Isolation, overexpression, and biochemical characterization of the two isoforms of glutamic acid decarboxylase from Escherichia coli. Protein Expr Purif 1996;8(4):430
8. [98] Richard H, Foster JW. Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential. J Bacteriol 2004;186(18):6032
41. [99] Dharmasena MN, Feuille CM, Starke CE, Bhagwat AA, Stibitz S, Kopecko DJ. Development of an acidresistant Salmonella Typhi Ty21a attenuated vector for improved oral vaccine delivery. PloS One 2016;11(9): e0163511. [100] Pascual DW, Hone DM, Hall S, van Ginkel FW, Yamamoto M, Walters N, et al. Expression of recombinant enterotoxigenic colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response. Infect Immun 1999;67:6249
56. [101] Pascual DW, Trunkle T, Sura J. Fimbriated Salmonella enterica serovar typhimurium abates initial inflammatory responses by macrophages. Infect Immun 2002;70(8):4273
81. [102] Forest C, Faucher SP, Poirier K, Houle S, Dozois CM, Daigle F. Contribution of the stg fimbrial operon of Salmonella enterica serovar Typhi during interaction with human cells. Infect Immun 2007;75(11):5264
71. [103] Ba¨umler AJ, Tsolis RM, Heffron F. The lpf fimbrial operon mediates adhesion of Salmonella typhimurium to murine Peyer’s patches. Proc Natl Acad Sci U S A 1996;93(1):279
83. [104] Gonzales AM, Wilde S, Roland KL. New insights into the roles of Lpf and Stg fimbriae in Salmonella interactions with enterocytes and M cells. Infect Immun 2017. [105] Angelakopoulos H, Hohmann EL. Pilot study of phoP/phoQ-deleted Salmonella enterica serovar Typhimurium expressing Helicobacter pylori urease in adult volunteers. Infect Immun 2000;68(4):2135
41. [106] Tennant SM, MacLennan CA, Simon R, Martin LB, Khan MI. Nontyphoidal Salmonella disease: current status of vaccine research and development. Vaccine 2016;34(26):2907
10.

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Recombinant Bacillus Calmette-Gue´rin for Mucosal Immunity Steven C. Derrick Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States

I. INTRODUCTION With the 100th anniversary of its introduction approaching, more people (an estimated 3 billion) have been immunized with bacillus Calmette-Gue´rin (BCG) than with any other vaccine, and BCG continues to be the most widely administered vaccine globally with around 91% of children immunized worldwide in 2014. While parenteral immunization with BCG protects against disseminated forms of Mycobacterium tuberculosisdisease (TB) in children (e.g., TB meningitis and military TB), protection wanes by early adulthood and cannot be boosted with additional BCG immunizations. Furthermore, protection against pulmonary TB has been shown to be highly variable in several controlled clinical studies, ranging from 0% to 80%, and for reasons that are not understood, protection is low or nonexistent in some of the areas in which TB is most endemic, which partially explains why it is still an immense global public health problem, notwithstanding extensive coverage with an

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00023-7

available vaccine. These shortcomings may be at least partially linked to the standard parenteral route of immunization, and mucosal delivery is a feasible alternative that could improve efficacy. In fact, BCG was initially administered orally when it was released in the early 1920s. Additionally, mycobacterial cell wall components make BCG an excellent adjuvant capable of stimulating both T helper 1 (Th1) and T helper 2 (Th2) cell immune responses when delivered either mucosally or parenterally. Moreover, BCG is amenable to recombinant DNA technology, which can be exploited to further augment the immunogenicity of the bacillus by engineering strains that overexpress antigens, relevant cytokines, or stimulatory molecules. Because of these and other properties, recombinant BCG (rBCG) has been evaluated clinically and experimentally as a platform for immunization against a variety of diseases, including viral, bacterial, and parasitic infections and cancer. BCG’s long history of use, relatively low cost of manufacture, stability, and adjuvant properties that stimulate durable

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humoral and cell-mediated immune responses following parenteral or mucosal delivery (pathogen-tailored delivery) are some of the reasons many investigators have turned to BCG as an expression vector for inducing antigen-specific immune responses.

II. ORAL IMMUNIZATION Oral vaccinations do not require needles and syringes; therefore no special training is required for their administration. However, special challenges present in the gastrointestinal (GI) mucosa should be considered in contemplating development of an oral vaccine. The vaccine must be able to resist inactivation or degradation in the harsh environment of the GI tract such as acidic pH, proteolytic enzymes, and a mucus barrier. Despite such obstacles, orally administered vaccines have been approved in the United States for polio (no longer distributed in the United States), adenovirus disease (military vaccine), cholera, rotavirus disease, and typhoid [1]. The effectiveness of oral vaccination is partially dependent on the interactions of orally applied antigens with the gut-associated lymphoid tissue (GALT) (Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (DEVELOPMENTAL FOR STRUCTURES, E.G., Ibalt, GALT, NALT) for the Induction and Regulation of Mucosal Immunity & Tolerance). Representing the greatest assemblage of lymphoid tissues in the body, GALT consists of the organized lymphoid structure with unique epithelium (or follicle-associated epithelium, FAE) such as Peyer’s patches connected with mesenteric lymph nodes (Fig. 23.1) [2]. The epithelium contains microfold (M) cells that sample the intestinal luminal antigens and transport them across the mucosa to antigenpresenting cells (APCs) in Peyer’s patches (Chapter 3: Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid

FIGURE 23.1

Peyer’s patches are a major inductive site for orally-administered vaccine. Anatomy of the gastrointestinal immune system. There are two main components in the gut-associated lymphoid tissue (GALT): inductive and effector sites. Inductive tissues include the action of Peyer’s patches, lymphoid follicles (within lymph nodes), and antigen presenting cells (APCs). Meanwhile, effector sites comprise the laminapropria and the surface epithelium. Upon entering to the intestinal lumen, antigens are transported across the intestinal epithelium barrier by sampling M cells, transcytosed and delivered to APCs (i.e. DCs). Activated DCs travel and prime CD41 T cells in germinal centers (GCs), present in the Peyer’s Patches (PPs) and mesenteric lymph nodes, needed to initiate an immune response. Primed CD41 T cells then activate B cells, which undergo isotype switching, thus generating IgA1 B cells. These B cells then leave the PPs using the lymphatic system to enter circulation and reach effector sites in the lamina propria, mature, and become IgA producing-plasma B cells. Source: From Vela Ramirez JE, Sharpe LA, Peppas NA. Current state and challenges in developing oral vaccines. Adv Drug Deliv Rev 2017;114:116 131.

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Cells and Chapter 28: M Cell-Targeted Vaccines). APCs can then present antigens to specific naı¨ve T cells that become either memory cells or effector cells, which can traffic to other mucosal sites or remain as tissue-resident memory (RM) T cells (Chapter 5: Mucosal Immunity for Inflammation: Regulation of Gut-Specific Lymphocyte Migration by Integrins). Thus vaccine antigens administered orally must have the ability to interact with M cells in order to cross the mucus layer and epithelium to induce an immune response. A study by Fujimura showed via electron microscopy that bacillus Calmette-Gue´rin (BCG) was capable of binding to M cells in the GI tract of rabbits reporting that microfolds of the M cells appeared to stretch toward the bacillus to capture them [3]. BCG was subsequently found in macrophages located below the epithelium.

A. Mycobacterium tuberculosis Currently, immunizations with BCG are routinely administered intradermally; however, the vaccine was initially administered orally [4]. Developed by Albert Calmette and Camille Gue´rin as a vaccine against TB, BCG is an attenuated strain of Mycobacterium bovis. After 231 passages over a period of 13 years, the bacterium was deemed sufficiently attenuated, and it was used for the first time in humans in 1921 [5]. Between 1921 and 1924, 300 children received three oral doses of BCG, and between 1924 and 1928, 114,000 children were immunized via this route [4]. The vaccine was considered safe with evidence for efficacy; however, subcutaneous immunizations became popular after 1927, since this route, unlike the oral route, consistently led to purified protein derivative skin test conversion, which was erroneously considered an indicator of protective immunity. Routine oral immunizations, however, continued in Brazil until the 1970s [6]. Although T cells and cytokines, such as IFNγ and TFN-α, have been shown to be absolutely necessary for protection against Mycobacterium

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tuberculosis [7], a complete picture of what constitutes a protective immune response remains ill defined (Chapter 35: Development of a Mucosal TB Vaccine Using Human Parainfluenza Type 2 Virus). The fact that individuals who acquire human immunodeficiency virus (HIV) are highly susceptible to TB diseases relative to HIVuninfected people has provided compelling evidence for the necessity of CD41 T cells for controlling TB infection [8]. Moreover, individuals with congenital deficiencies and individuals taking immunosuppressive medications that suppress the production of or response to cytokines such as IFNγ, TNF-α, or IL-12 are also highly susceptible to TB. BCG does induce both T helper 1 (Th1) and T helper 2 (Th2) cell immune responses [9]. The involvement of the humoral response has been less clear. In one study using mice lacking B cells, the animals did not appear to be more susceptible to M. tuberculosis infection, having comparable organ bacterial burdens with less severe lung pathology than B-cell-competent C57BL/6 mice [10]. Recently, cynomolgus macaque monkeys were examined for susceptibility to an aerosol infection with M. tuberculosis following treatment with rituximab, an antibody specific for CD20, to deplete the animals of B cells [11]. After a low dose (4 8 CFUs) intrabronchial instillation of M. tuberculosis, no difference was observed in overall pathology and clinical outcome between the treated and untreated animals. Other animal studies, however, have yielded opposing results, supporting a role for B cells in protection against TB, as reviewed by Achkar et al. [12]. Interestingly, B cells may play a role in generating an optimum BCG immune response. Different investigators have presented evidence suggesting that B cells may help to prevent neutrophilia at the site of BCG intradermal (ID) immunization in mice [13,14]. Neutrophilia seems to adversely affect BCG efficacy in B-celldeficient mice, but after treatment with sera from M. tuberculosis infected mice, neutrophilia was hindered, suggesting that humoral immunity may contribute to BCG protection by performing

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a primarily immunomodulatory function [13]. The importance of B cells and secretory IgA (SIgA) in BCG-mediated mucosal immunity is not currently clear, although BCG efficiently induces SIgA production when delivered mucosally, as will be discussed later [15]. Hence, given the ambiguous role of humoral immunity in the context of mucosal BCG vaccination against TB, the emphasis in this chapter will be primarily on T cell immune responses when considering immunity to TB. Animal studies have shown that BCG delivered orally provides protection against TB comparable to that provided by the parenteral route. In guinea pigs, oral administration of BCG Danish 1331 or Moreau (Rio de Janeiro) formulated in lipid was as protective as subcutaneous (SC) delivery of BCG, but protection was absent when BCG was delivered alone, presumably owing to the lipid’s preservation of the bacillus in the GI tract [16]. Lipid formulations of BCG have also been shown to protect mice, cattle, badgers, white-tailed deer, and possums following oral delivery [17]. In a study conducted by Ancelet et al., the persistence of TB-specific T cells and memory T cells was compared between mice fed BCG formulated in lipid versus those given a parenteral immunization [18]. Antigen-85B (Ag85B)-specific T cells were elevated similarly and persisted in both vaccinated groups over 30 weeks of observation in the spleen relative to naı¨ve mice. In lungs and spleens, effector, effector memory, and central memory CD41 T cells were elevated similarly in both vaccinated groups up to 30-week postimmunization. Moreover, the frequency of multifunctional CD41 T cells producing multiple cytokines was significantly elevated at the 30week time point in the lungs of mice immunized with BCG orally relative to naı¨ve controls and mice immunized subcutaneously [18]. Another study, using guinea pigs immunized orally with a recombinant BCG (rBCG) strain secreting a fusion of Antigen-85B (Ag85B) and Esat-6 M. tuberculosis antigens (formulated in sodium bicarbonate) and then boosted with the fusion

protein intranasally (6 weeks apart, formulated in adjuvant), demonstrated that the oral nasal immunization route was as effective as the parenteral route in a survival study. However, the rBCG prime, fusion protein boost regimen was not more protective than priming orally or subcutaneously with wtBCG [19]. In this study, postimmunization Ag85B and Esat-6 IFNγ responses and postchallenge Esat-6 IFNγ responses in mice were significantly lower in animals receiving an oral BCG immunization relative to the subcutaneous route. The magnitude of the IFNγ response, as is now recognized, is not a correlate of protection for TB [20], as these results demonstrate, and suggest that targeting two different mucosal sites for heterologous prime-boost immunizations or engineering BCG to overexpress TB antigens may not have an additive effect. Together, these animal studies demonstrate that an oral BCG delivery can provide protection equivalent to that of the conventional parenteral route and appears to induce comparable cytokine responses and numbers of antigen-specific T cells. A study by Hoft and colleagues compared systemic and mucosal immune responses in human subjects immunized with BCG Danish 1331 orally, intradermally, or by both routes [21]. Subjects receiving oral BCG immunizations had consumed sodium bicarbonate just prior to BCG ingestion. Systemic responses were determined by measuring peripheral blood mononuclear cell (PBMC) proliferation and performing IFNγ ELISPOT assays following incubation with BCG or M. tuberculosis antigens. Mucosal responses were examined by measuring lipoarabinomannan (LAM)-specific SIgA in tears and nasal washes as well as M. tuberculosis-specific T cell responses (via ELISPOT and flow cytometry) from bronchoalveolar lavage (BAL) fluid. The authors found that BCG delivered intradermally induced a strong systemic Th1type response, whereas oral administration with or without ID BCG vaccination induced a strong mucosal response. Significantly higher frequencies of M. tuberculosis-specific T cells were observed in the lungs (BAL-derived), which

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II. ORAL IMMUNIZATION

expressed CXCR3 and α4β1 (homing molecules for lymphocyte trafficking to the lungs) in subjects receiving BCG orally, an obvious advantage over ID BCG immunization. Detection of SIgA in nasal washes and tears and T cell responses in the lung following an oral BCG immunization indicates that mediators of BCG-induced mucosal immune responses can be trafficked to other mucosal sites. Importantly, optimal activation of both systemic and humoral responses was achieved via both immunization routes, which would be the most desirable attribute of a TB vaccine. Transcriptome profiles of CD41 T cells were compared between the two immunization routes by Illumine BeadArray transcriptomal analysis. Interestingly, gene expression profiles were very different between the two immunization routes with only 2 3 genes upregulated in both groups out of 100 genes analyzed, suggesting that the different immunization routes induce very different gene pathways. Further studies will be needed to define the specific gene pathways that contribute to the protective immune response (Table 23.1).

B. HIV The HIV vaccine community has investigated the feasibility of using BCG or attenuated M. tuberculosis strains to express viral antigens to generate protective immunity. The involvement TABLE 23.1

of both humoral and cytotoxic T cell responses in immunity to HIV in addition to the option for mucosal delivery has made BCG an appealing vector for antigen expression. Mucosal administration is especially relevant for immunization against HIV, since the GI, vaginal, and anal mucosa are the principal sites of infection [22]. Moreover, given that HIV replication occurs mainly in GALT from which HIV disseminates and remains a key reservoir of HIV replication, BCG is a rational choice for HIV antigen expression in the GI tract [22] (Chapter 42: Mucosal Vaccines Against HIV/SIV Infection). Several studies have investigated the immunogenicity of attenuated, recombinant mycobacterial simian immunodeficiency virus (SIV) vaccines. Jensen et al. described a dual-purpose TB/SIV vaccine consisting of two live, attenuated M. tuberculosis replication-deficient auxotroph strains with deletions in the panCD, leuCD, and secA2 genes. These strains were engineered to express the full-length SIV Gag (mc26435) or Env (mc26439) genes [23]. The authors immunized infant rhesus macaques orally with both auxotroph strains followed by an intramuscular boost immunization with a modified vaccinia ankara (MVA) virus construct expressing the SIV Gag, Pol, and Env genes. The animals were then challenged orally with SIV to model mother-to-child HIV transmission via breast feeding. This vaccine regimen did not prevent infection with SIV;

Summary of Clinical or Preclinical BCG Mucosal Vaccine Studies

BCG Strain

Model

Route

Formulation

Efficacy

Moreau & Danish 1331

Guinea pig

Oral

Lipid

Equivalent to BCG SC

Moreau

Guinea pig

Oral

None

None

?

Mice, cattle, badgers, possums, deer

Oral

Lipid

1

rBCG (Ag85B-Esat-6)

Guinea pig

Oral 1 subunit IN boost

NaBicarb

Equivalent to BCG SC

Danish 1331

Clinical

Oral or ID

NaBicarb

ID: Strong systemic Th1 response Oral: Strong mucosal response

IN, intranasal; ID, intradermal; rBCG, recombinant BCG; SC, subcutaneous.

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however, a subset of immunized animals had significantly lower peak viremia, which was associated with increased prechallenge antigenspecific IgA levels in the saliva and intestines and higher Env-specific IgG levels in plasma with elevated avidity. These animals also experienced significantly less CD41 T cell loss than controls after SIV infection. While mycobacteria-specific T cell responses were detected in immunized macaques [24], vaccineinduced protection against a M. tuberculosis challenge has yet to be reported. Importantly, the mc26435 auxotroph was shown to be safe following oral immunization of infant macaques (1week old) with or without prior SIV infection [25]. The authors were unable to recover viable mycobacteria from relevant tissues (lungs, spleen, lymph nodes, skin), and the animals exhibited normal lung histopathology with no clinical symptoms, demonstrating this live vaccine to be highly attenuated. Since wtBCG is contraindicated for immunization of HIV infected infants, vaccination with attenuated M. tuberculosis strains represents a potential alternative. Recombinant BCG vaccines given orally have also induced relevant antiviral immune responses. In papers preceding the aforementioned studies, a rBCG strain was constructed expressing three different segments of the SIV Env gene and administered orally to guinea pigs. The vaccine was reported to induce IgA production in the GI tract [26]. In a separate study, these authors immunized Balb/c mice with three separate rBCG strains expressing either the full-length SIV Nef gene, a portion of the Env gene, or the Gag gene [27]. The mice were immunized orally, intranasally, rectally, and aerogenically with a mixture of all three strains. All the different immunization routes induced local antigen-specific IgA responses, specific IgG responses in the sera, and specific cytotoxic responses in the spleen, indicating that both Th1 and Th2 cell responses were induced locally and systemically by mucosal immunization with rBCG. A paper by Hiroi et al. described a rBCG capable of secreting the

HIV V3 domain (rBCG-V3J1) [28]. When delivered either orally, intranasally, or parenterally (subcutaneously) to mice, equivalent antigenspecific IgG responses were detected in the sera up to a year postimmunization. These authors reported that intranasal immunization induced antigen-specific IgG in the sera that neutralized HIV in vitro. Whether the immune responses described in all of these papers induced by the different mucosally delivered rBCG constructs were sufficient to provide protection has not yet been reported. Importantly, Ami et al. investigated a SIV vaccine consisting of rBCG expressing the SIV gag gene as an ID prime immunization in macaques followed by a boost (parenteral) with a replication-deficient vaccinia virus expressing the same antigen administered intravenously (IV) [29]. Following intrarectal challenge with SIV, the prime-boosted animals were protected up to 1-year postchallenge, whereas each vaccine component administered alone was not protective. Protection was assessed by measuring plasma viral RNA, CD41 T cell counts, and survival. During the 1-year observation period, two of three immunized animals exhibited undetectable viremia (,500 RNA copies/ mL), and all the animals survived with no weight loss, while nonimmunized controls exhibited relatively high viremia, and two of three animals succumbed to the infection. Protection was associated with elevated gag-specific IFNγ PBMC responses. Andrieu and colleagues were also able to achieve protection in macaques against an intravenous or intrarectal SIV challenge by immunizing the animals via the vaginal or oral route with inactive SIV particles (iSIV) formulated with either BCG, Lactobacillus plantarum, or L. rhamnosus as adjuvants through the vaginal or oral route [30,31]. Four of six macaques immunized intravaginally with iSIV/BCG had no detectable viral RNA in the plasma at 60day postintravenous challenge, and the remaining two animals had significantly lower plasma viral RNA concentrations (almost 3 log10 less)

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than nonimmunized controls. Three of the four protected macaques were administered repeated intravenous challenges with SIV at 2 and 8 months following the initial intravenous challenge and also an intrarectal challenge 21 and 27 months following the initial immunization. In each case, after an initial low-level spike in viral RNA plasma levels, viral loads dropped to undetectable levels. When a separate group of animals was immunized orally (followed by a sodium bicarbonate chase) and then challenged intrarectally, the monkeys exhibited sterile protection (N 5 4) with no plasma viral RNA detected. The authors also examined the presence of provirus from PBMCs. Of the four of six immunized animals protected from an intravenous challenge, PBMCs chronically harbored the provirus, while PBMCs from 8 of 11 animals protected from an intrarectal challenge had no detectable SIV cellular DNA. Unexpectedly, no anti-SIV antibody or specific IFNγ producing T cells were detected in the protected animals. Sterile protection was also achieved for oral immunizations with iSIV/L. plantarum or iSIV/L. rhamnosus vaccine formulations followed with an intrarectal challenge, but monkeys immunized with each vaccine component alone were not protected [31]. Surprisingly, the mechanism of protection may have been through the induction of noncytolytic, CD81 regulatory T cells that suppressed CD41 T cell activation. The authors proposed that suppressing activation resulted in suppression of viral replication at the level of viral DNA integration in CD41 T cells from the animals immunized and challenged mucosally, since coculture of CD41 and CD81 T cells from immunized animals, but not from nonprotected (nonimmunized or immunized with BCG alone) animals, suppressed both viral replication and CD41 T cell activation ex vivo [31]. Furthermore, CD81 T cell depletion of immunized animals previously infected via the IV route (and thus harboring provirus) experienced a spike in viremia, which then declined to undetectable

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levels, corresponding to recovery of the CD81 T cell population. In contrast, animals immunized orally and previously challenged intrarectally (sterilely protected with no provirus) did not experience a spike in viremia upon depletion of their CD81 T cells [31]. Although the authors did not utilize a rBCG for these studies but simply mixed wtBCG with attenuated virus, it is reasonable to speculate that designing a rBCG expressing CD8 T-cell-specific HIV epitopes might result in similar protection in humans following a mucosal immunization and challenge.

III. INTRAVESICAL IMMUNOTHERAPY A. Bladder Cancer Intravesical BCG immunotherapy has been the treatment of choice for nonmuscle invasive bladder cancer since the 1970s. Following transurethral resection of the tumor, the efficacy of BCG therapy for prevention of recurrence is 50% 75%, depending of the type of cancer being treated. For greatest efficacy, therapy should continue for 1 3 years [25,26]. The immune responses necessary for BCG efficacy against bladder cancer are becoming better defined, and innate and adaptive responses are equally important. Upon intravesical instillation of BCG, the following proinflammatory cytokines are found in the urine: IL-1, IL-2, IL6, IL-8, IL-12, IL-18, TNF-α, and IFNγ [32]. BCG binds the bladder mucosa, possibly via attachment to fibronectin, which could be blocked in vitro with antifibronectin antibody [33]. Binding leads to internalization of BCG by cancer cells, which are induced to produce nitric oxide and cytokines, including IL-6, IL-8, IL-10, TNF-α, IFNγ, IL-6, and GM-CSF, resulting in the accumulation of APCs, lymphocytes, and natural killer (NK) cells [32,34,35]. These activated cells in turn amplify the proinflammatory

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cytokine response, which leads to the formation of a BCG granuloma in the bladder wall [29]. In addition to dendritic cells (DCs) and macrophages, mouse bladder tumor cells have also been shown to present BCG antigens to CD41 T cells via MHCII [35]. Moreover, tissue biopsies of BCG-treated but not untreated patients showed that superficial bladder carcinoma cells expressed ICAM1, which could render these cells susceptible to killing by cytotoxic lymphocytes [36]. In vitro studies have implicated the involvement of different populations of cytotoxic cells in tumor elimination, including NK cells, BCG-activated killer cells, macrophage-activated killer cells, lymphokine-activated killer cells, and cytotoxic T cells [34,35]. Neutrophils may also play an important role in BCG therapy for bladder cancer, as neutrophils are a major source of TNF-related apoptosis-inducing ligand (TRAIL), which is also found in the urine of BCG-treated bladder cancer patients [37,38]. TRAIL induces apoptosis in cancer cells but not normal cells, and elevated levels in the urine are associated with superior BCG responsiveness [37]. Moreover, IFNα primed neutrophils are stimulated to secrete elevated levels of TRAIL relative to unprimed neutrophils [39]. Administering recombinant IFNα (rIFNα) with BCG has been shown to be beneficial to patients in whom initial BCG therapy was not effective (see below). Additionally, as described below, depletion of neutrophils using a specific antibody significantly reduced BCG effectiveness in a mouse bladder cancer model [40]. Given that BCG immunotherapy against bladder cancer has a failure rate of up to 40%, many investigators have sought to improve efficacy by engineering BCG strains to express different immunomodulators. One promising study, which has served as proof of principle, administered BCG combined with rIFNαβ to patients in whom conventional BCG therapy had failed [41]. At 24-month follow-up, 53% of patients were disease free, and the treatment was tolerated as well as BCG alone following the initial

doses. However, subsequent maintenance cycle treatments were not as well tolerated, and only 46% of patients completed all three treatment cycles. Nevertheless, these results have prompted additional studies using rBCG strains. To improve the efficacy of BCG against bladder cancer, several in vitro and mouse protection studies have been conducted using unique rBCG constructs. An in vitro study showed that human PBMCs stimulated with rBCG expressing IFNα exhibited enhanced cytotoxicity to human bladder cancer cells relative to PBMCs stimulated with control BCG. This enhanced cytotoxicity was determined to be mediated by NK and CD81 T cells [42]. Likewise, macrophages stimulated with a rBCG strain secreting IL-2 or IL-18 exhibited enhanced killing of a bladder cancer cell line [43]. Using a murine bladder cancer model, intravesical instillation of a rBCG strain secreting IFNγ extended survival significantly longer than control BCG [44]. In a similar study, using an orthotopic MB49 bladder cancer mouse model, Sun et al. treated mice with rBCG expressing human IFNα2b, wtBCG, or wtBCG mixed with hIFNα2b [45]. Both the rBCG and BCG1hIFNα2b treatments enhanced survival significantly longer than was seen in naı¨ve and BCG control groups. Furthermore, the expression or addition of hIFNα2b rendered BCG significantly more cytotoxic to MB49 cells than control BCG in vitro [45]. Another rBCG strain expressing genetically detoxified pertussis toxin (S1PT) was tested for efficacy using the MB49 bladder cancer mouse model [46]. The rBCG-S1PT group survived significantly longer than control groups (naı¨ve and control BCG) with a significant reduction in bladder weight and increased in vivo induction of TNF-α and IL-10 relative to control BCG. Interestingly, Takeuchi et al. investigated the efficacy of a rBCG strain expressing a fusion of IL-15 and antigen-85B, an immunodominant mycobacterial antigen [40]. This strain was also shown to enhance survival of treated mice using the bladder cancer model described above

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relative to untreated or BCG controls. Protection was associated with elevated numbers of neutrophils and elevated MIP-1α and MIP-2 expression in the bladder. In mice depleted of neutrophils or TCRγδ T cells, protection mediated by control BCG or the rBCG was either significantly reduced or abolished, suggesting that these cells are necessary for BCG-mediated protection against bladder cancer [40]. Given the reported success using bladder cancer models, immunotherapeutic studies using rBCG are being initiated in humans. A BCG strain (VPM1002) expressing listeriolysin O (from Listeria monocytogenes), which disrupts the phagosomal membrane under acidic conditions, and a deletion of the urease C gene (to help maintain a phagosomal acidic pH) has been shown to have superior protection against TB in animal models [47]. Enhanced immunogenicity was attributed to augmented phagolysosomal fusion, apoptosis, autophagy, and antigen presentation via MHCI molecules as antigens leak from the phagosome into the cytosol and/or by antigen cross-presentation from phagocytosis of infected, apoptotic cells by APC’s [47]. Phase 1 and phase 2 clinical studies have been completed in adults and newborns following parenteral immunization. The VPM1002 strain has been tested in a phase 1 study for immunotherapy in patients with bladder cancer (nonmuscle invasive). A phase 2 study in bladder cancer patients is currently ongoing in Switzerland and Germany and is scheduled to be completed in 2022 [47].

IV. STIMULATION OF PULMONARY IMMUNE RESPONSES A. Tuberculosis Given that TB arises from an infection in the lung, induction of a protective immune response in the lung may most effectively stimulate appropriate local immune responses to

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protect against pulmonary TB. Antigens delivered intranasally are taken up by M cells of the nasopharyngeal-associated lymphoid tissue (NALT) and then transported across the FAE to underlying APC’s and lymphoid tissue consisting of high endothelial venules and germinal centers with T and B cells (Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance). Thus BCG delivered intranasally can enter the NALT associated with the upper and lower respiratory tracts and can also be swallowed and enter the GALT. Subsequently, innate and adaptive immune responses develop and, following exposure to M. tuberculosis, antigen-specific, memory T cells can traffic to the lung upon infection or even take up residence in the lung and respond relatively quickly [48,49]. As discussed below, the induction of a pulmonary immune response that can proceed rapidly post M. tuberculosis infection may be important for improving protection over that achieved via conventional, parenteral BCG immunization. We and others have shown that BCG delivered intranasally provides superior protection in mice relative to the subcutaneous route [48 51]. In our experience, intranasal (IN) BCG immunization significantly augmented pulmonary protection 2- and 4-month postvaccination at the 1- and 3-month postchallenge time points with up to 1.0 log10 CFU reduction relative to the SC route and 2.0 log10 better than nonimmunized control C57BL/6 mice [49]. However, this enhanced efficacy was absent at later postvaccination time points (8 and 10 months). Enhanced protection in the spleen, however, was more durable. At early postvaccination time points up to 1.65 and 2.8 log10, CFU reduction in the IN group relative to the SC route and nonimmunized mice, respectively, was observed. At 10month postvaccination, however, the animals receiving BCG IN were protected 1.0 and 1.9 log10 CFU better in the spleen than mice

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receiving BCG SC and nonimmunized mice, respectively. The improved protection was associated with significantly higher frequencies of mycobacteria-specific CD41 and CD81 T cells in the lungs and spleens. Consistent with our results, Aguilo et al. showed that DBA/2 mice were protected significantly better with IN BCG immunization relative to the SC route [50]. Mice receiving IN BCG vaccination were protected 2.0 log10 better in the lungs relative to mice immunized SC following an intranasal challenge with H37Rv and survived significantly longer. The enhanced protection was partially mediated by IL-17A, since in vivo neutralization with antiIL17A antibody significantly reduced protective efficacy afforded by the IN delivery route. Additionally, Griffiths et al. demonstrated that enhanced IL-17 in the lungs following IN administration with BCG in mice could be further augmented by formulating BCG in cholera toxin (CT) [51]. BCG delivered IN with CT protected Balb/c mice significantly better than BCG IN without CT or BCG ID. Protection was further augmented when mice immunized with BCG1CT were boosted 10 weeks later with MVA expressing Ag85A. This elevated response was partially mediated by IL-17, since in vivo treatment with antiIL-17 neutralizing antibody abolished the enhanced protection. Importantly, the effectiveness of BCG delivered via aerosol has also been evaluated in primate models. The protective efficacy of aerosol delivery of BCG was compared with intracutaneous immunization in monkeys (Macaca mulatta) by Barclay et al. in the 1970s following an aerosol M. tuberculosis challenge [52]. After the infection, tuberculous mycobacteria were isolated from 8 of 10 control animals and animals immunized intracutaneously, whereas M. tuberculosis CFUs were isolated from 2 of 10 animals receiving an aerosol BCG immunization. Similarly, Verreck et al. using rhesus macaques showed that delivery of BCG via bronchoscope reduced lung CFUs by 0.5 log10 relative to BCG delivered ID and nonimmunized animals after

an intratracheal challenge with M. tuberculosis [53]. The simultaneous delivery of BCG intradermally and mucosally did not improve protection achieved by immunization via bronchoscopy alone. BCG administered intradermally alone, however, did not protect in this study. To examine whether an attenuated M. tuberculosis mutant induced enhanced protective immune responses, Kaushal et al. compared the protective efficacy of a M. tuberculosis sigH deletion mutant versus BCG delivered by aerosol to rhesus macaques [54]. The mutant was shown to be attenuated and could not be isolated from the lungs 8-week post-aerosol immunization. Furthermore, immunization with the mutant was found to be more protective compared to nonimmunized monkeys and BCG-immunized animals, with significant improvements in lung pathology, control of mycobacterial growth in the lungs, and extension in survival following an aerosol challenge with virulent M. tuberculosis (CDC1551). The superior protection was associated with significantly higher frequencies of postvaccination CD41 and CD81 central memory, CD691, CXCR31, and CXCR51 T cells recovered from BAL [54]. Recent imaging studies have emphasized the need to induce early localized immune responses in order to adequately control M. tuberculosis infection. PET CT scans of the lungs of M. tuberculosis infected macaques have provided evidence that early postinfection pulmonary immune responses may be important for preventing TB disease [55]. Imaging data revealed that animals with active TB disease exhibited elevated numbers of pulmonary granulomas at 3-week postinfection relative to those that developed a latent infection. Furthermore, the number of granulomas in those with active disease increased over time, whereas lesion numbers in those with latent infection remained static. Thus immunological events occurring within 3-week postinfection appear to be important for determining long-term control of the infection. It is now

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recognized that in humans, induction of a critical Th1-type response is delayed 2 3-week postinfection with M. tuberculosis [55]. Among the reasons for this delay is the suppression of the immune response by the bacterium. The immunoevasive properties of M. tuberculosis, among which is the ability to delay APC migration to the lymph nodes from the lungs, are an integral component of its success as a pathogen [56]. Interestingly, recruitment of DC migration to the lung could be accelerated by the delivery of FimH, a TLR4 ligand, intranasally. The intranasal delivery of FimH accelerated Th1 cell immunity via earlier (less than 2 weeks) induction of TNF-α and IL-12 in the lungs and enhanced DC migration to the lungs and antigen presentation, which was associated with earlier control of M. tuberculosis infection [56]. Griffiths et al. adoptively transferred M. tuberculosis antigen-pulsed, stimulated DCs into the lungs of BCG-immunized mice at the time of TB challenge, which resulted in accelerated CD41 T cell activation and superior protection relative to naı¨ve and BCG SC controls supporting the hypothesis that early activation of T cells may overcome the delayed protective immune response characterized by BCG SC immunization [57]. Other work showed that BCG delivered intratracheally dramatically alters the cellular composition of the lung [58]. While the cellular composition of bronchoalveolar lavage fluid (BALF) from naı¨ve mouse lungs or from BCG SC-immunized mice 60-day postvaccination was primarily alveolar macrophages, the composition of the lungs of mice receiving BCG intratracheally was primarily DC’s. Furthermore, BALF from mice immunized intratracheally or intranasally was populated with significantly higher frequencies of effector memory (EM) and resident memory (RM) CD41 and CD81 T cells than lungs from mice immunized with BCG SC, and adoptive transfer of these cells were protective against a TB challenge. These observations form the basis

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of the model illustrated in Fig. 23.2, which show that the formation of TRM in draining lymph nodes following pulmonary immunization with BCG is likely critical to early protective immune responses, since these cells are trafficked back to the lung and respond relatively quickly following infection with TB [59]. Horvath et al. showed that infection with nonvirulent M. tuberculosis strains and parenteral immunization with BCG are also characterized by a delay in the induction of Th1 cell immunity [60]. These authors found that parenteral immunization with BCG was associated with the recruitment of T cells to the lung interstitium, but recruitment of airway luminal T cells (ALT) was delayed 10-day post M. tuberculosis challenge, which coincided with the absence of protection for the first 2 weeks [60]. When ALT cells were adoptively transferred into mice immunized parenterally (SC) with BCG, the mice were protected significantly better than BCG SC and naı¨ve controls at 2-weeks postchallenge. Furthermore, ALT cells could be recruited to the lungs by intranasal delivery of M. tuberculosis culture filtrate proteins (CFP). Intranasal delivery of CFP to mice immunized with BCG via the SC route prior to M. tuberculosis infection augmented protection at 1- and 4weeks postchallenge; thus the limitations of parenterally administered BCG could be potentially overcome by mucosal immunization. IN delivery of BCG establishes a pool of memory and effector T cells in the lymph nodes adjacent to the lungs, and ALT cells in the lungs that could induce a Th1-type response postinfection with greater rapidity than can be achieved by infection with the pathogen or by parenteral BCG immunization [60]. Furthermore, Th1 cell responses might be augmented further by mucosal immunization with rBCG strains that express TLR ligands such as FimH and/or strains that overexpress mycobacterial antigens. Future work with animal models will be required to determine whether such rBCG

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FIGURE 23.2 Mucosal vaccination confers increased protection by inducing tissue RM T cells in the lungs. (A) Infection by Mtb. (1) Lung dendritic cell (DC) activation and migration is inhibited by Mtb infection, resulting in delayed T cell responses. (2) Owing to inhibition of DC activity, antigens are presented to naı¨ve T cells (To) by bystander DCs in the draining lymph node (LN) rather than by the infected DC. (3) Proliferation and differentiation of antigen-specific T central memory (TCM) and T effector memory (TEM) cells. TEM (KLGR1) home to the site of infection and produce cytokines. Less differentiated memory T cells (KLGR12) enter the circulation, where they travel to secondary lymphoid organs to establish populations of TCM or, in the presence of inflammation, differentiate into TRM in tissues. (4) IFNγ produced by T cells activates alveolar macrophages (AMs), inducing Mtb growth inhibition. (B) Mucosal vaccination. (1) Antigens administered by aerosol or intranasal vaccination are taken up by DCs and carried to the draining LN. (2) DCs prime antigen-specific T cells. (3) T cells home to the lung or circulate to secondary lymphoid organs. (4) In the presence of the correct tissue signals, less differentiated KLGR1 2 T cells differentiate into TRM cells expressing CD69 and CD103 and remain in the lung. (5) Upon Mtb infection, preexisting TRM cells are activated and produce cytokines such as IFNγ, which induces Mtb growth inhibition in AMs. Source: From Gengenbacher M, Nieuwenhuizen NE, Kaufmann SHE. BCG old workhorse, new skills. Curr Opin Immunol 2017;47:8 16.

strains delivered mucosally would augment BCG protection at early time points. As of this writing, very few studies have been published addressing this question, notwithstanding many reports of significant improvement in BCG protection with intranasal delivery of

mycobacterial antigens formulated in adjuvant or expressed from plasmid or viral vectors. Examples of these include an Ag85B, Esat-6 fusion protein formulated in detoxified Escherichia coli heat-labile enterotoxin (LT) delivered IN as a BCG boost immunization

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(in mice); [61] an adenovirus expressing Ag85A immunized intrabronchially and concomitantly with BCG (SC) in cattle [62]; and an IN DNA vaccine prime immunization expressing a fusion of Ag85A, Esat-6, and IL-21 followed by a BCG boost vaccination (SC) in mice [63]. These studies show that the potential exists of improving protective immunity beyond IN delivery of wtBCG using rBCG strains overexpressing certain antigens, cytokines, and/or innate immune receptor agonists such as FimH. Collectively, the limited data from the literature that do address the potential utility of rBCG intranasal immunization are encouraging. Given the substantial delay in the establishment of both innate and adaptive pulmonary immune responses post M. tuberculosis infection, intranasal delivery of BCG represents a relatively simple measure to improve protective immunity. Results from animal models have shown a potential advantage of the mucosal route over the conventional, parenteral route in that BCG immunization induces a population of local immune cells (e.g., RM T cells) that may respond more rapidly and potentially lead to earlier control of M. tuberculosis infection, as has been shown in animal models and illustrated in Fig. 23.2 [48,49]. Moreover, given the routine manner with which BCG can now be manipulated to overexpress relevant antigens and/or immunomodulators, the possibility exists for creating even more immunogenic and protective rBCG strains for mucosal immunization alone or as part of prime-boost regimens as supported by animal studies.

B. Coccidiosis, Schistosoma, and Borrelia Recombinant BCG strains have also been constructed as mucosal vaccines against other pathogens, including protozoa, flatworms, and spirochetes. Wang et al. constructed a rBCG strain expressing rhomboid, an Eimeria tenella antigen, and chicken IL-2 (separate BCG

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constructs). When it was delivered intranasally or subcutaneously to chickens, the host for E. tenella infections, the IN route protected the animals from coccidiosis cacal lesions and reduced oocyst shedding better than the SC route [64]. Kremer et al. designed rBCG expressing the glutathione S-transferase (GST) of Schistosoma mansoni [65]. Antibody responses against this antigen are thought to be associated with protection, and following expression in BCG, GST exhibited enzymatic activity. After intraperitoneal (IP), IN, or SC immunizations of Balb/c mice, antiGST humoral responses were effective at neutralizing enzymatic activity, and the antibody responses were relatively high for at least 1 year. Protection against Borrelia burgdorferi, the causative agent of Lyme disease, has been demonstrated in Balb/c mice following IN immunization with rBCG expressing the B. burgdorferi outer surface protein A [66]. Protective durable IgG and IgA responses were generated that persisted for more than a year. Both IP and IN routes induced antibody responses that inhibited growth of the bacterium in vitro, and following an ID challenge of mice with B. burgdorferi, no spirochetes were detected in different tissues of animals receiving either IN or IP vaccinations (N 5 7). In contrast, spirochetes were detected in all nonimmunized controls and animals immunized with wtBCG by either route. These studies, including the aforementioned discussions on HIV and TB, highlight the adaptability of rBCG for immunization against a variety of infectious diseases. The option of parenteral or mucosal administration and the relative ease of manipulation to express foreign antigens with or without immunomodulators provide vaccinologists with the ability to generate pathogen-specific rBCG strains. Moreover, customizing rBCG strains to express tumor antigens and/or immunomodulators may give future oncologists additional options for cancer therapy. Such BCG strains have shown promise in bladder cancer preclinical studies (Table 23.2).

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TABLE 23.2 Chapter

Summary of Clinical or Preclinical rBCG or BCG-Formulated Mucosal Vaccine Studies Discussed in this

Disease Vaccine

Route P

P

B

Model

Efficacy

Mice

Equivalent to BCG SC or BCG-IN prime

TB

rBCG(Esat-6-Ag85B) , Esat- O , IN 6-Ag85BB

TB

BCGΔSigH

IN, Aer, Br

Mice, Macaque

Increased survival vs nonimmunized and wtBCG controls

TB

BCG1 Ag85B-Esat-6

IN

Mice

Improved protection relative to BCG alone

TB

BCG SC, Ad85A-Br

SC, Br

Cattle

Improved protection relative to BCG alone

Mice

Improved protection relative to BCG alone

TB

DNA (Ag85A, Esat-6, IL-21)p

P

IN , SCB

BCG-SCB HIV

mc26435(Gag)1 mc26439 (Env)P

OP, IMB Macaque

Lower peak viremia; elevated CD41 T cell counts

MVA(Gag, Pol, Env)B HIV

rBCG(Nef)1 rBCG(Env)1 rBCG(Gag)

O, Ir, Aer

Mice

? The rBCG strains immunized via the different routes induced specific IgG and IgA responses

HIV

rBCG(V3J1)

O, IN, SC

Mice

? In vitro neutralizing IgG responses by all the different routes

HIV

rBCG(Gag)

IDP, SCB

Macaque

No viremia detected in prime-boosted animals up to 1 year

HIV

iSIV 1 BCG

IVa or O

Macaque

Sterile protection via the oral route

BC

BCG1 rIFNα/β

Ives

Clinical

53% of patients were disease free at 24-month posttreatment following unsuccessful conventional BCG therapy

BC

rBCG(IFNγ)

Ives

Mice

Increased survival relative to wtBCG

BC

rBCG(IFNα2b) 6 rIFNα2b

Ives

Mice

Increased survival relative to wtBCG and untreated mice

BC

rBCG(S1PT) (pertussis toxin)

Ives

Mice

Increased survival relative to wtBCG and untreated mice

BC

rBCG(IL-15-Ag85B)

Ives

Mice

Increased survival relative to wtBCG and untreated mice

Coc

rBCG(rhomboid-IL-2)

IN or SC

Chickens

IN immunization superior to SC

Sch

rBCG(GST)

IP, IN, SC

Mice

? Induced neutralizing antibody responses via the different immunization routes

LD

rBCG(protein A)

IN, IP

Mice

No postchallenge spirochetes detected by either route

Aer, aerosol; B, boost; BC, bladder cancer; Br, intrabronchial; Coc, coccidiosis; ID, intradermal; IM, intramuscular; IN, intranasal; IP, intraperitoneal; Ir, intrarectal; Iva, intravaginal; Ives, intravesical; LD, Lyme disease; O, oral; P, prime; SC, subcutaneous; Sch, Schistosoma; ?, unknown efficacy. ( ) Indicates antigens expressed from a rBCG strain. 1 Indicates admixture.

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REFERENCES

References [1] https://www.cdc.gov/vaccines/terms/usvaccines. html. [2] Vela Ramirez JE, Sharpe LA, Peppas NA. Current state and challenges in developing oral vaccines. Adv Drug Deliv Rev 2017;114:116 31. [3] Fujimura Y. Functional morphology of microfold cells (M cells) in Peyer’s patches phagocytosis and transport of BCG by M cells into rabbit Peyer’s patches. Gastroenterol Jpn 1986;21(4):325 35. [4] Benevolo-de-Andrade TC, Monteiro-Maia R, Cosgrove C, Castello-Branco LR. BCG Moreau Rio de Janeiro: an oral vaccine against tuberculosis review. Mem Inst Oswaldo Cruz 2005;100(5):459 65. [5] Lucas S, Mihaescu T. History of BCG vaccine. Maedica (Buchar) 2013;8(1):53 8. [6] Monteiro-Maia R, Pinho RT. Oral bacillus CalmetteGuerin vaccine against tuberculosis: why not? Mem Inst Oswaldo Cruz 2014;109(6):838 45. [7] Cadena AM, Flynn JL, Fortune SM. The importance of first impresions: early events in Mycobacterium tuberculosis infection influence outcome. mBio 2016;7(2) e00342-16. [8] Diedrich CR, Flynn JL. HIV-1/Mycobacterium tuberculosis coinfection immunology: how does HIV-1 exacerbate tuberculosis? Infect Immun 2011;79(4):1407 17. [9] Lalor MK, Smith SG, Floyd S, Gorak-Stolinska P, Weir RE, Blitz R, et al. Complex cytokine profiles induced by BCG vaccination in UK infants. Vaccine 2010;28 (6):1635 41. [10] Bosio CM, Gardner D, Elkins KL. Infection of B celldeficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lung pathology. J Immunol 2000;164:6417 25. [11] Phuah J, Wong EA, Gideon HP, Maiello P, Coleman MT, Hendricks MR, et al. Effects of B cell depletion on early Mycobacterium tuberculosis infection in Cynomolgus Macaques. Infect Immun 2016;84(5):1301 11. [12] Achkar JM, Chan J, Casadevall A. B cells and antibodies in the defense against Mycobacterium tuberculosis infection. Immunol Rev 2015;264:167 81. [13] Kozakiewicz L, Chen Y, Xu J, Wang Y, DunussiJoannopoulos K, Ou Q, et al. B cells regulate neutrophilia during Mycobacterium tuberculosis infection and BCG vaccination by modulating the interleukin-17 response. PLoS ONE 2013;9(7):e1003472. [14] Kondratieva TK, Rubakova EI, Linge IA, Evstifeev VV, Majorov KB, Apt AS. B cells delay neutrophil migration toward the site of stimulus: tardiness critical for effective bacillus Calmette-Gue´rin vaccination against tuberculosis infection in mice. J Immunol 2010;184:1227 34.

415

[15] Hosseini M, Dobakhti F, Pakzad SR, Ajdary S. Immunization with single oral dose of alginateencapsulated BCG elicits effective and long-lasting mucosal immune responses. Scand J Immunol 2015;82 (6):489 97. [16] Clark SO, Kelly DL, Badell E, Castello-Branco LR, Aldwell F, Winter N, et al. Oral delivery of BCG Moreau Rio de Janeiro gives equivalent protection against tuberculosis but with reduced pathology compared to parenteral BCG Danish vaccination. Vaccine 2010;28(43):7109 16. [17] Gormley E, Nı´ Bhuachalla D, O’Keeffe J, Murphy D, Aldwell FE, Fitzsimons T, et al. Oral vaccination of Free-Living Badgers (Meles meles) with Bacille Calmette Gue´rin (BCG) vaccine confers protection against tuberculosis. PLoS ONE 2017;12(1):eo168851. [18] Ancelet LR, Aldwell FE, Rich FJ, Kirman JR. Oral vaccination with lipid-formulated BCG induces a longlived, multifunctional CD4(1) T cell memory immune response. PLoS ONE 2012;7(9):e45888. [19] Badell E, Nicolle F, Clark S, Majlessi L, Boudou F, Martino A, et al. Protection against tuberculosis induced by oral prime with Mycobacterium bovis BCG and intranasal subunit boost based on the vaccine candidate Ag85B-ESAT-6 does not correlate with circulating IFN-gamma producing T-cells. Vaccine 2009;27 (1):28 37. [20] Sakai S, Kauffman KD, Sallin MA, Sharpe AH, Young HA, Ganusov VV, et al. CD4 T cell-derived IFN-γ plays a minimal role in control of pulmonary Mycobacterium tuberculosis infection and must be actively repressed by PD-1 to prevent lethal disease. PLoS Pathog 2016;12(5): e1005667. [21] Hoft DF, Xia M, Zhang GL, Blazevic A, Tennant J, Kaplan C, et al. PO and ID BCG vaccination in humans induce distinct mucosal and systemic immune responses and CD4 1 T cell transcriptomal molecular signatures. Mucosal Immunol 2017; epub ahead of print. [22] Chapman R, Chege G, Shephard E, Stutz H, Williamson AL, et al. Recombinant Mycobacterium bovis BCG as an HIV vaccine vector. Curr HIV Res 2010;8 (4):282 98. [23] Jensen K, Nabi R, Van Rompay KK, Robichaux S, Lifson JD, Piatak Jr M, et al. Vaccine-elicited mucosal and systemic antibody responses are associated with reduced simian immunodeficiency viremia in infant rhesus macaques. J Virol 2016;90(16):7285 302. [24] Jensen K, Pena MG, Wilson RL, Ranganathan UD, Jacobs Jr WR, Fennelly G, et al. A neonatal oral Mycobacterium tuberculosis-SIV prime/intramuscular MVA-SIV boost combination vaccine induces both SIV and Mtb-specific immune responses in infant macaques. Trials Vaccinol 2013;2:53 63.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

416

´ RIN FOR MUCOSAL IMMUNITY 23. RECOMBINANT BACILLUS CALMETTE-GUE

[25] Jensen K, Ranganathan UD, Van Rompay KK, Canfield DR, Khan I, Ravindran RA, et al. A recombinant attenuated Mycobacterium tuberculosis vaccine strain is safe in immunosuppressed simian immunodeficiency virus-infected infant macaques. Clin Vaccine Immunol 2012;19(8):1170 81. [26] Lim EM, Lagranderie M, Le Grand R, Rauzier J, Gheorghiu M, Gicquel B, et al. Recombinant Mycobacterium bovis BCG producing the N-terminal half of SIVmac251 Env antigen induces neutralizing antibodies and cytotoxic T lymphocyte responses in mice and guinea pigs. AIDS Res Hum Retroviruses 1997;13(18):1573 81. [27] Lagranderie M, Winter N, Balazuc AM, Gicquel B, Gheorghiu M. A cocktail of Mycobacterium bovis BCG recombinants expressing the SIV Nef, Env, and Gag antigens induces antibody and cytotoxic responses in mice vaccinated by different mucosal routes. AIDS Res Hum Reroviruses 1998;14(18):1625 33. [28] Hiroi T, Goto H, Someya K, Yanagita M, Honda M, Yamanaka N, et al. HIV mucosal vaccine: nasal immunization with rBCG-V3J1 induces a long term V3J1 peptide-specific neutralizing immunity in Th1- and Th2-deficient conditions. J Immunol 2001;167:5862 7. [29] Ami Y, Izumi Y, Matsuo K, Someya K, Kanekiyo M, Horibata S, et al. Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus CalmetteGue´rin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity. J Virol 2005;79(20):12871 9. [30] Andrieu JM, Chen S, Lai C, Guo W, Lu W, Mucosal SIV. Vaccines comprising inactivated virus particles and bacterial adjuvants induce CD8(1) T-regulatory cells that suppress SIV-positive CD4(1) T-cell activation and prevent SIV infection in the macaque model. Front Immunol 2014;5:297. [31] Lu W, Chen S, Lai C, Guo W, Fu L, Andrieu JM. Induction of CD8 1 regulatory T cells protects macaques against SIV challenge. Cell Rep 2012;2(6):1736 46. [32] Zheng YQ, Naguib YW, Dong Y, Shi YC, Bou S, Cui Z. Applications of bacillus Calmette-Guerin and recombinant bacillus Calmette-Guerin in vaccine development and tumor immunotherapy. Expert Rev Vaccines 2015;14(9):1255 75. [33] Zhao W, Schorey JS, Bong-Mastek M, Ritchey J, Brown EJ, Ratliff TL. Role of a bacillus Calmette-Gue´rin fibronectin attachment protein in BCG-induced antitumor activity. Int J Cancer 2000;86(1):83 6. [34] Luo Y, Henning J, O’Donnell MA. Th1 cytokinesecreting recombinant Mycobacterium bovis bacillus Calmette-Gue´rin and prospective use in immunotherapy of bladder cancer. Clin Dev Immunol 2011;2011: 728930.

[35] Bevers RFM, Kurth K-H, Schamhart DHJ. Role of urothelial cells in BCG immunotherapy for superficial bladder cancer. Br J Cancer 2004;91:607 12. [36] Jackson AM, Alexandroff AB, McIntyre M, Esuvaranathan K, James K, Chisholm GD, et al. Induction of ICAM 1 expression on bladder tumours by BCG immunotherapy. J Clin Pathol 1994;47(4):309 12. [37] Ludwig AT, Moore JM, Luo Y, Chen X, Saltsgaver NA, O’Donnell MA, et al. Tumor necrosis factor-related apoptosis-inducing ligand: a novel mechanism for Bacillus Calmette-Gue´rin-induced antitumor activity. Cancer 2004;64(10):3386 90. [38] Kemp TJ, Ludwig AT, Earel JK, Moore JM, Vanoosten RL, Moses B. Neutrophil stimulation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in the release of functional soluble TRAIL/ Apo-2L. Blood 2005;106(10):3474 82. [39] Simons MP, Moore JM, Kemp TJ, Griffith TS. Identification of the mycobacterial subcomponents involved in the release of tumor necrosis factor-related apoptosis-inducing ligand from human neutrophils. Infect Immun 2007;75(3):1265 71. [40] Takeuchi A, Eto M, Tatsugami K, Shiota M, Yamada H, Kamiryo Y, et al. Antitumor activity of recombinant bacille Calmette-Guerin secreting interleukin-15Ag85B fusion protein against bladder cancer. Int Immunopharmacol 2016;35:327 31. [41] O’Donnell MA, Krohn J, DeWolf WC. Salvage intravesical therapy with interferon-alpha 2b plus low dose bacillus Calmette-Guerin is effective in patients with superficial bladder cancer in whom bacillus CalmetteGuerin alone previously failed. J Urol 2001;166:1300 5. [42] Liu W, O’Donnell MA, Chen X, Han R, Luo Y. Recombinant bacillus Calmette-Gue´rin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro. Cancer Immunol Immunother 2009;58 (10):1647 55. [43] Luo Y, Yamada H, Evanoff DP, Chen X. Role of Th1stimulating cytokines in bacillus Calmette-Gue´rin (BCG)-induced macrophage cytotoxicity against mouse bladder cancer MBT-2 cells. Clin Exp Immunol 2006;146(1):181 8. [44] Arnold J, de Boer EC, O’Donnell MA, Bohle A, Brandau S. Immunotherapy of experimental bladder cancer with recombinant BCG expressing interferongamma. J Immunother 2004;27(2):116 23. [45] Sun E, Fan X, Wang L, Lei M, Zhou X, Liu C, et al. Recombinant hIFN-α2b-BCG inhibits tumor growth in a mouse model of bladder cancer. Oncol Rep 2015;34:183 94. [46] Andrade PM, Chade DC, Borra RC, Nacimento IP, Villanova FE, Leite LC, et al. The therapeutic potential

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

FURTHER READING

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

of recombinant BCG expressing the antigen S1PT in the intravesical treatment of bladder cancer. Urol Oncol 2010;28(5):520 5. Nieuwenhuizen NE, Kulkarni PS, Shaligram U, Cotton MF, Rentsch CA, Eisele B, et al. The recombinant bacilli Calmette-Guerin vaccine VPM1002: ready for clinical efficacy testing. Front Immunol 2017;8:1147. Chen L, Wang J, Zganiacz A, Xing Z. Single intranasal mucosal Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis. Infect Immun 2004;72(1):238 46. Derrick SC, Kolibab K, Yang A, Morris SL. Intranasal administration of Mycobacterium bovis BCG induces superior protection against aerosol infection with Mycobacterium tuberculosis in mice. Clin Vaccine Immunol 2014;21(10):1443 51. Aguilo N, Alvarez-Arguedas S, Uranga S, Marinova D, Monzon M, Badiola J, et al. Pulmonary but not subcutaneous delivery of BCG vaccine confers protection to tuberculosis-susceptible mice by an interleukin 17dependent mechanism. J Infect Dis 2016;213:831 9. Griffiths KL, Stylianou E, Poyntz HC, Betts GJ, Fletcher HA, McShane H. Cholera toxin enhances vaccineinduced protection against Mycobacterium tuberculosis challenge in mice. PLoS ONE 2013;8(10):e78312. Barclay WR, Busey WM, Dalgard DW, Good RC, Janicki BW, Kasik JE. Protection of monkeys against airborne tuberculosis by aerosol vaccination with bacillus Calmette-Guerin. Am Rev Respir Dis 1973;107 (3):351 8. Verrek F, Tchilian EZ, Vervenne R, Sombroek CC, Kondova I, Eissen OA, et al. Variable BCG efficacy in rhesus populations: pulmonary BCG provides protection where standard intra-dermal vaccination fails. Tuberculosis 2017;104:46 57. Kaushal D, Foreman TW, Gautam US, Alvarez X, Adekambi T, Moreno-Rangel J, et al. Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis. Nat Commun 2015;6:8533. Coleman MT, Maiello P, Tomko J, Frye LJ, Fillmore D, Janssen C, et al. Early changes by (18)fluorodeoxyglucose positron emission tomography coregistered with computed tomography predict outcome after Mycobacterium tuberculosis infection in cynomolgus macaques. Infect Immun 2014;82(6):2400 4. Lai R, Jeyanathan M, Shaler CR, Damjanovic D, Khera A, Horvath C, et al. Restoration of innate immune activation accelerates Th1-cell priming and protection following pulmonary mycobacterial infection. Eur J Immunol 2014;44(5):1375 86.

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[57] Griffiths KL, Ahmed M, Das S, Gopal R, Horne W, Connell TD, et al. Targeting dendritic cells to accelerate T-cell activation overcomes a bottleneck in tuberculosis vaccine efficacy. Nat Commun 2016;7:13894. [58] Perfomo C, Zedler U, Kuhl AA, Lozza L, Saikali P, Sander LE, et al. Mucosal BCG vaccination induces protective lung-resident memory T cell populations against tuberculosis. mBio 2016;7(6) e01686-16. [59] Gengenbacher M, Nieuwenhuizen NE, Kaufmann SHE. BCG old workhorse, new skills. Curr Opin Immunol 2017;47:8 16. [60] Horvath CN, Shaler CR, Jeyanathan M, Zganiacz A, Xing Z. Mechanisms of delayed anti-tuberculosis protection in the lung of parenteral BCG-vaccinated hosts: a critical role of airway luminal T cells. Mucosal Immuno 2012;5(4):420 31. [61] Dietrich J, Andersen C, Rappuoli R, Doherty TM, Jensen CG, Andersen P. Mucosal administration of Ag85BESAT-6 protects against infection with Mycobacterium tuberculosis and boosts prior bacillus Calmette-Guerin immunity. J Immunol 2006;177:6353 60. [62] Dean GS, Clifford D, Whelan AO, Tchilian EZ, Beverley PC, Salguero F, et al. Protection induced by simultaneous subcutaneous and endobronchial vaccination with BCG/BCG and BCG/adenovirus expressing antigen 85A against Mycobacterium bovis in cattle. PLoS ONE 2015;10(11):e0142270. [63] Dou J, Wang Y, Yu F, Yang H, Wang J, He X, et al. Protection against Mycobacterium tuberculosis challenge in mice by DNA vaccine Ag85A-ESAT-6-IL-21 priming and BCG boosting. Int J Immunogenet 2012;39(2):183 90. [64] Wang Q, Chen L, Li J, Zheng J, Cai N, Gong P, et al. A novel recombinant BCG vaccine encoding Eimeria tenella rhomboid and chicken IL-2 induces protective immunity against coccidiosis. Korean J Parasitol 2014;52(3):251 6. [65] Kremer L, Riveau G, Baulard A, Capron A, Locht C. Neutrolizaing antibody responses elicited in mice immunized with recominanat bacillus CalmetteGuerin producing the Schistosoma mansoni glutathione S-transferase. J Immunol 1996;156(11):4309 17. [66] Langermann S, Palaszynski S, Sadzlene A, Stover CK, Koenig S. Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi. Nature 1994;372:552 5.

Further Reading Bo¨hle A, Gerdes J, Ulmer AJ, Hofstetter AG, Flad HD. Effects of local bacillus Calmette-Guerin therapy in patients with bladder carcinoma on immunocompetent cells of the bladder wall. J Urol 1990;144(1):53 8.

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C H A P T E R

24

Recombinant Adenovirus Vectors as Mucosal Vaccines Kristel L. Emmer1,2 and Hildegund C.J. Ertl2 1

Gene Therapy & Vaccines Program, University of Pennsylvania School of Medicine, Philadelphia, PA, United States 2Wistar Institute Vaccine Center, Philadelphia, PA, United States

I. INTRODUCTION Vaccines have saved millions of lives through the prevention of infectious diseases. Traditionally, most vaccines have been created by attenuation or inactivation of viral or bacterial pathogens. Advances in molecular virology have led to the development of new vaccine prototypes based on recombinant viral vectors that encode or express antigens from heterologous pathogens. Numerous viruses, including RNA and DNA viruses, have been modified to allow for their use as antigen-delivery vehicles [1
8]. Of those, vectors based on adenoviruses have consistently shown induction of potent and sustained B and T cell responses to inserted foreign antigens [9
12]. Replication-defective adenovirus vectors mostly based on human serotype 5 (HAdV5) were initially generated for the treatment of genetic diseases such as cystic fibrosis, hemophilia, and muscular dystrophy [13
15]. The recombinant adenoviruses had excellent transduction rates upon application through various

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00024-9

routes. Nevertheless, expression of the therapeutic protein was transient, owing to robust immune responses directed toward both the adenoviral proteins and the transgene products causing rapid elimination of the infected cells [16,17]. Although the high immunogenicity of adenovirus vectors caused insurmountable problems for their use in permanent gene replacement protocols, it invited their use as vaccine carriers. Vaccine vectors have been generated from multiple adenovirus serotypes derived from different species. The vectors’ ability to replicate has been genetically altered. The foreign antigen has been expressed as a transgene, or epitopes thereof have been incorporated into the vectors’ capsid [18,19]. Methods for mass production, quality control, purification, and storage at ambient temperatures have been developed and are being refined [20
25]. Adenovirus vectors have been tested extensively in experimental animals as well as in human volunteers. In most human trials, vectors were given intramuscularly, but mucosal routes have been explored as well.

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© 2020 Elsevier Inc. All rights reserved.

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Mucosal immunization has a number of advantages over systemic immunization. The relative ease of mucosal administration facilitates mass vaccination campaigns. It may reduce the cost of vaccination and decrease the risk of side effects due to inadvertent syringe or vial contamination. Since mucosal surfaces are the most common portal of entry for pathogens, vaccine-induced responses of the mucosal immune system, which is unique from the systemic immune system, may be better suited for prophylaxis. This review gives a brief overview about adenoviruses and adenovirus vectors and then discusses the use of adenovirus vectors for mucosal immunization, addressing available results, feasibility, and potential advantages as well as disadvantages.

II. ADENOVIRUSES Adenoviruses are nonenveloped viruses with an icosahedral capsid and a doublestranded DNA that ranges in size from 26 to 46 kb [26]. A number of early (E) and late (L) transcription units encode between 23 and 46 proteins. Human adenoviruses have the five early transcription units—E1A, E1B, E2, E3, and E4—which produce polypeptides that are required for viral transcription and replication of the viral DNA or serve to suppress the hosts’ immune responses. E1, E2, and E4 are essential, and their deletion cripples the ability of adenovirus to replicate. E3, which encodes polypeptides that block major histocompatibility class (MHC) I-restricted antigen presentation and inhibit various pathways of apoptosis, is nonessential for viral replication. The five late transcription units L1
L5 encode the three major capsid proteins, that is, hexon, fiber, and penton base, as well as a number of minor capsid components that stabilize the capsid or participate in its assembly.

Adenoviruses are species-specific and infect a wide range of hosts, including mammals, birds, and amphibians. Adenoviruses are divided into five genera. The genus Mastadenovirus includes all of the human adenoviruses, which are subdivided into seven species, A
G. Each species is further separated into serotypes, which are distinguished by their reactivity with virus-neutralizing antibodies directed mainly to the hypervariable loops of the major coat protein hexon. Neutralizing antibodies can also be directed against fiber, although this response is less pronounced. To date, 57 distinct serotypes of human adenoviruses have been identified. Adenoviruses cause upper respiratory infections, conjunctivitis, tonsillitis, or otitis. Human serotypes 40 and 41 cause gastroenteritis. Infections are usually self-limiting, but can be fatal in immunocompromised individuals or occasionally in otherwise healthy humans. Adenovirus vaccines are not available to the general public; however, vaccines to serotypes 4 and 7 are given to U.S. military recruits. Adenoviruses persist even though their genome does not integrate into the host cell genome. The level of persistence seems to depend on the serotype and the host species. While most humans stop shedding adenovirus within a few weeks after infection, adenoviruses can readily be isolated from the feces of most nonhuman primates [27]. Even after cessation of shedding, the virus continues to persist episomally at low levels in T cells, presumably for the lifetime of the individual [28]. The continued presence of adenovirus in turn maintains an effector-like T cell response to its antigens [29,30].

III. IMMUNE RESPONSES TO ADENOVIRUSES Adenoviruses induce vigorous innate and adaptive immune responses. Innate responses

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III. IMMUNE RESPONSES TO ADENOVIRUSES

are triggered by a number of pathogenassociated molecular patterns (PAMPs) on adenoviruses interacting with host cell pathogenrecognition receptors (PRRs), which are located on the cell surface, in endosomes, and in the cytosol. Adenoviruses bind to cellular receptors such as the coxsackievirus and adenovirus receptor (CAR) or integrins, used by most human serotypes, and are then are taken up by endocytosis [31]. Cytokines such as interleukin 8 (IL-8) or tumor necrosis factor alpha (TNF-α) facilitate entry by increasing the density of CAR and integrins on the cell surface [32]. Species B adenoviruses bind to CD46 or desmoglein-2 and are taken up by pinocytosis [33]. On the cell surface, adenoviruses interact with toll-like receptor 2 (TLR-2), triggering a proinflammatory response [34]. Adenoviruses that form a complex with coagulation factor X can also interact with TLR-4, another cell surface PRR [35]. CD46-binding adenoviruses have been reported to interact with TLR-9 [36], a sensor for unmethylated CpG motifs in double-stranded DNA [36]. Within the cytosol, the adenoviral genome binds to additional sets of DNA sensors such as NLRs, which are the core proteins of inflammasomes, gammainterferon-inducible protein 16 (IFI16), DEADbox helicase 41 (DDX41), and protein cyclic GMP-AMP synthase (cGAS); the latter initiates the stimulator of interferon genes (STING) pathway [37]. The adenovirus genome encodes two short viral RNAs that are produced with the help of host cell polymerases and interact with RIG-I, a cellular double-stranded RNA sensor. These different PAMP
PRR interactions trigger a robust proinflammatory response to adenoviruses, which is also observed with adenoviral vectors [38]. The strong innate responses facilitate induction of adaptive responses by providing a proinflammatory milieu rich in cytokines and activated professional antigen-presenting cells. B cells are induced to different late gene products and produce both nonneutralizing and

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neutralizing antibodies. Nonneutralizing antibodies directed to different adenovirus proteins cross-react between serotypes. Neutralizing antibodies, directed against hexon or fiber, are serotype-specific. The prevalence of neutralizing antibodies to adenoviruses differs depending on serotypes and geographic location [39
41]. In the United States, there is a clear distinction between common serotypes to which a large proportion of the adult population carries neutralizing antibodies, best exemplified by adenovirus of human serotype 5 (HAdV5), and so-called rate serotypes to which fewer than 10% of human adults show serological evidence of preexisting immunity. This distinction is blurred in less developed countries, where neutralizing antibodies to serotypes that are rare in the United States tend to be far more common, as best exemplified by HAdV26 (Table 24.1). Preexisting neutralizing antibodies induced by natural infection lessen the uptake of adenoviral vectors. This in turn diminishes the amount of transgene that is synthesized, and thereby reduces immune responses to the vaccine antigen [42
44]. Neutralizing antibodies to simian origin adenoviruses, which are phylogenetically clustered with human adenoviruses in the genus Mastadenovirus, are rare in humans [39,45]. Vectors based on nonhuman primate serotypes have been developed as vaccine carriers to circumvent preexisting immunity to human serotypes and therefore avoid dampening of transgene product-specific immune responses. Adenoviruses induce both CD41 and CD81 T cell responses to E and L gene products. These T cell responses cross-react between multiple serotypes [46]. Owing to the low-level persistence of adenoviruses, ratios of T cells to adenoviral antigens are fairly high in humans. On average, 1%
2% of CD41 and 3%
5% of CD81 T cells in human blood respond to adenovirus antigens. T cells produce different effector functions, including interferon gamma (IFNγ), TNF-α, and lytic enzymes [46].

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TABLE 24.1

Prevalence of Adenovirus Neutralizing Antibodies in Humans (Percentage of Population)

Serotypes

United States (%)

Europe (%)

South America (%)

Africa (%)

Asia (%)

HAdV5

40
70

60

80

80
90

70
90

HAdV6

50

50

50

70
80

70
80

HAdV26

, 10

90

50

90

20
50

HAdV35

, 10

HAdV36

, 10

HAdV41

, 10

SAdV23

, 10

SAdV24

, 10

IV. TYPES OF ADENOVIRUS VECTORS Replication-competent adenovirus (RCA) vectors that are deleted only in E3 have been constructed. Such vectors may not meet the safety requirements for human preventive vaccines; however, they have been used for immunization of wildlife against rabies [47,48]. Most adenovirus vectors are deleted in E1, which is essential for viral replication, as it encodes polypeptides that initiate transcription of viral genes. E1 deletions thus render adenoviruses replication-defective. E1-deleted adenovirus vectors can be grown in cell lines such as HEK 293 cells or PerC6 cells that transcomplement E1. For many serotypes, including simian adenoviruses (SAdV), the deleted E1 domain can be transcomplemented by E1 of HAdV5; for others, packaging cell lines carrying the serotype-specific E1 have to be developed. Many adenovirus vectors are deleted in both E1 and E3 to increase the permitted size of the transgene’s expression cassette that is generally inserted into E1. For expression of toxic glycoproteins, partial E3 deletions that maintain sequences encoding antiapoptotic peptides have been explored [49]. Additional deletions, such as deletion of E4, have been used to

17
20 50

50

50 50

20

5
30

5
12

5
10

13

dampen immune responses against antigens of adenovirus, and again these genes have to be provided in trans during virus propagation [50]. Some vectors based on rare or nonhuman primate serotypes are further modified to replace part of the E4 domain with that of HAdV5 to increase vector yields during production [51]. Vectors that express two expression cassettes in forward or reverse orientation within E1 and E3 have been developed [52]. Expression of the transgene product is in general driven by a potent ubiquitously active promoter such as the early promoter of cytomegalovirus combined with an enhancer and introns to optimize protein production. Instead of using adenovirus vectors as genedelivery vehicles, other researchers have modified the viral hexon by incorporating short, linear B cell epitopes from a different virus [18,19,53]. Such vectors, which display the epitopes in a repetitive fashion on the virus surface, promote the induction of B cell responses and could be combined with second, longer sequences incorporated within an expression cassette into E1. Adenovirus vectors have been generated from multiple different human and nonhuman serotypes. Although many of the characteristics of such vectors are similar, there are also

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VI. QUALITY CONTROL OF ADENOVIRUS VECTORS

notable differences in their performance as vaccines, as discussed below.

V. CONSTRUCTION, PURIFICATION, AND TITRATION OF ADENOVIRUS VECTORS Initially, adenovirus vectors were constructed by homologous recombination. This rather cumbersome procedure has been replaced by using viral molecular clones in which the adenovirus sequences with appropriate deletions and suitable rare restriction enzymes sites flanking the deletions are cloned into a plasmid vector or a bacterial artificial chromosome for propagation in bacteria [54,55]. By using standard cloning procedures, the transgene expression cassette is first cloned into a pShuttle vector and, from there, is inserted into the deleted E1 domain of the viral molecular clone. The recombinant viral molecular clones are linearized by a restriction enzyme targeting a site just upstream of the 50 inverted terminal repeat of the adenovirus genome and transfected into packaging cells. The recombinant virus, which in general forms plaques on the packaging cell monolayers within 7
14 days, is then serially expanded. Once a sufficiently large batch has been generated, the adenovirus vector is released by freeze-thawing or by treatment with detergents. The vector is then cleared by low-speed centrifugation followed by cesium chloride gradient purification. Alternative purification methods that avoid centrifugations are available; these include clearance by filtration and purification by chromatography [23
25,56]. The viral particle content of a vector preparation is determined by its absorbency at 260 nm. In clinical trials, adenovirus vectors are dosed according to their viral particle content, as this parameter directly determines the vector’s toxicity. Immunogenicity of the vectors, on the other hand, depends on the

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number of virus particles that are able to infect cells and to transcribe the transgene product. In general, the numbers of infectious virus particles are measured by plaque assays or by an endpoint dilution assay for cytopathic effects. Plaque formation is dependent on several viral factors and is not reliable for all adenovirus serotypes. Alternative assays based on amplification of the adenovirus vectors transcripts or on staining for hexon with an antibody to a conserved domain of adenovirus hexon have been developed and validated.

VI. QUALITY CONTROL OF ADENOVIRUS VECTORS The numbers of release assays that are required for adenovirus vectors used in clinical trials is extensive and beyond the scope of this article. For preclinical experiments, vector batches are checked for RCA, which can emerge during the creation and propagation of E1-deleted replication-defective adenoviruses as a result of recombination between overlapping viral sequences in the packaging cells and the vectors [57]. Depending on the level of RCA in vector preparations, it can have a significant impact on vector performance, host immune responses, and toxicological profiles for in vivo experiments. Vectors should be tested for sterility and endotoxin content. The genetic integrity and stability of vectors should be assessed by restriction enzyme digest of purified viral DNA from early passaged virus and virus that has been propagated sequentially for 12
15 passages in comparison to the viral molecular clone. Although most adenovirus vectors are genetically stable, deletion of the transgene or part of the backbone can happen and may necessitate rerescue of the virus or even reconstruction of the viral molecular clone.

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VII. THERMOSTABILITY OF ADENOVIRUS VECTORS Adenovirus vectors kept in suitable buffers are stable if stored at temperatures below 65 C. Upon storage at room temperature, they rapidly lose infectivity. Cold chains are difficult to maintain in less developed countries, and methods have been developed to increase the thermostability of adenoviral vectors [58,59]. These methods rely on resuspension of vectors in solutions with a high sugar content, such as mannitol and sucrose or trehalose and sucrose, followed by spray-drying or dry-coating onto membranes. Dried vectors in sugar formulations are stable for weeks at room temperature.

VIII. IMMUNOGENICITY OF ADENOVIRUS VECTORS E1-deleted adenovirus vectors induce potent transgene product-specific T and B cell responses after a single dose. Upon systemic immunization, CD41 T cell responses are biased toward T helper 1 (Th1) cell responses that support induction of cellular immunity [58,59]. Immune responses are sustained, presumably owing to low-level vector persistence in a transcriptionally active form within lymphatic tissues [29,30]. Despite continuing T cell stimulation, adenovirus vector-induced T cells do not differentiate toward exhaustion, but rather maintain populations of effector and effector memory cells. Immune responses can be further increased by booster immunizations using an adenovirus vector from a different serotype or an unrelated vaccine prototype [60
62]. Although all adenovirus vectors tested to date have shown excellent immunogenicity, there are some clear differences. Innate responses to some of the chimpanzee adenovirus vectors derived from species E were shown to be more pronounced than those to HAdV5 [63]. Another study described that vectors

based on CD46-binding adenoviruses of species B elicit higher levels of proinflammatory cytokines compared to HAdV5 vectors, a species C member [64]. Vectors based on species C viruses tend to induce more potent adaptive immune responses than those from species E viruses, which in turn, are more immunogenic compared to B virus vectors [65]. This may, in part, relate to the hierarchy of proinflammatory responses which, when overly induced, may dampen adaptive responses [66].

IX. THE MUCOSAL IMMUNE SYSTEM Mucosal surfaces that cover the gastrointestinal, urogenital, and respiratory tracts as well as the eyes, middle ears, and exocrine gland ducts are the most common portals of entry for pathogens. In addition, foreign innocuous antigens from food or commensal bacteria constantly bombard mucosal surfaces. The mucosal immune system must therefore distinguish between harmless antigens and those derived from pathogens, a challenge that is not faced by the systemic immune system. The mucosal immune system is thus distinct yet interconnected with the systemic immune system [67]. The mucosal immune system is divided into inductive sites, which are the organized mucosaassociated lymphoid tissues, and effector sites, which are lymphocytes dispersed within mucosal membranes. Inductive sites such as Peyer’s patches in the small intestine also include lymph nodes draining the mucosal surfaces, such as mesenteric lymph nodes within the peritoneal cavity or the Waldeyer’s tonsillar ring of the nasopharynx and oropharynx. Inductive sites contain B cell follicles with follicular dendritic cells, which are surrounded by T cells. A single layer of epithelial cells separates the lymphoid tissue from the mucosal surface. Within the gut, this area contains microfold (M) cells, which take up antigen from the lumen and then transfer it to antigen-presenting cells. Other

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X. MUCOSAL VACCINES

mechanisms allow mucosal dendritic cells to take up antigen. Soluble antigen crosses epithelial barriers. Larger particulate antigen is transferred across epithelial cells through transcellular routes by enterocytes and potentially by macrophages and CX3CR11 myeloid cells. MHC class II positive enterocytes release partially degraded antigen in the form of exosomes. The antigen is then taken up by dendritic cells within the lamina propria and transported to inductive sites. Mucosal dendritic cells are distinct from those present in the central immune system, presumably owing to differences in their microenvironment, which, for example, within the gut is rich in retinoid acids (RAs). RAs prime dendritic cells to induce IL-10 and transforming growth factor beta (TGF-β), resulting in the generation of regulatory T cells (Tregs) [68], which play a crucial role in maintaining tolerance to harmless antigens (Chapter 3: Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells. Chapter 28: M Cell-Targeted Vaccines). Effector sites such as the lamina propria and the interstitial epithelium of the gut mucosa contain intraepithelial lymphocytes (IELs) that originate either directly from the thymus (tIELs) or from the periphery (pIELs). tIELs are largely T cell receptor (TcR)γδ1CD8αα1 or TcRα/β1CD8αα1). They migrate from the thymus to the gut shortly after birth, while pIELs, which are TcRαβ1CD81 or TcRαβ1CD41, are induced in peripheral lymphatic tissues and then migrate to the mucosa. Within the mucosa, pIELs adapt and progressively increase expression of CD8αα homodimers [69]. Mucosal B cells belong both to the conventional B2 subset and the B1 subsets; the latter may arise from peritoneal B1 cells. Mucosal B cells mainly produce secretory immunoglobulin A (SIgA), which is resistant to degradation, and thus is able to survive the harsh environment of the gastrointestinal tract. Class-switch recombination of mucosal B cells is facilitated by local follicular Th cells, which originate from Th17 cells and, through

the secretion of IL-21 and TGF-β, promote class switching to IgA [70]. Class switching can also be achieved without T cell help through direct interaction of the B cells’ TLRs with PAMPs on gut bacteria [71] or by microbiota metabolites [72]. The common mucosal immune system is partially interconnected in that induction of a response at one site leads to effector responses at distant sites. Nevertheless, responses are compartmentalized in that oral immunization through the gastrointestinal tract favors local responses; intranasal immunization favors responses within the oral cavity, the airways, and the female reproductive tract; and intrarectal immunization induces the strongest responses within the rectum and the genital tract but not within the oral cavity. Vaginal immunization induces a weak and mainly local response, reflecting the lack of local inductive sites in the vaginal mucosa. The mucosal immune system is shaped and regulated by the microbiome and vice versa: The composition of the microbiome is molded by the mucosal immune system [73] (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity). The microbiome varies between individuals, in part owing to differences in diets, and may thus influence the outcome of mucosal immune responses to pathogens or vaccines. This clearly has to be investigated in more depth, as geographic differences in the microbiome may make it difficult to predict global mucosal vaccine efficacy based on clinical trials, which are typically conducted in a few selected areas.

X. MUCOSAL VACCINES Most commercially available vaccines are administered by a systemic route such as intramuscular, subcutaneous, or intradermal injections. Only six vaccines used in humans are given to mucosal surfaces. Most are live attenuated vaccines such as those for poliovirus,

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

426

24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

rotavirus (see Chapter 40: The Role of Innate Immunity in Regulating Rotavirus Replication, Pathogenesis, and Host Range Restriction and the Implications for Live Rotaviral Vaccine Development and Chapter 41: Development of Oral Rotavirus and Norovirus Vaccines), Salmonella enterica serovar Typhi (see Chapter 29: Induction of Local and Systemic Immunity by Salmonella Typhi in Humans), Vibrio cholerae (see Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control), adenovirus serotypes 4 and 7 given orally, and a coldadapted influenza vaccine given intranasally (see Chapter 39: Nasal Influenza Vaccines). Several rabies vaccines based on a recombinant vaccinia virus vector, a recombinant replicationcompetent HAdV5 vector expressing both rabies virus glycoproteins as their transgene product, or attenuated rabies viruses have been used for oral immunization of wildlife. With the exception of the rabies and adenovirus vaccines, mucosal vaccines are given through their natural routes of infection, which avoids the need to protect the vaccine from digestion during passage through the gastrointestinal tract or to facilitate uptake through the mucosa. Oral rabies vaccines virus do not pass into the intestinal tract, but rather induce a response locally within the oral cavity. The vaccines for adenovirus serotypes 4 and 7 induce immune responses within the intestine. They are given in tablets with an inner capsule containing dried virus and an outer layer containing microcrystalline cellulose, magnesium stearate, and anhydrous lactose and an enteric coating consisting of cellulose acetate phthalate, alcohol, acetone, and castor oil, which protects the virus until it has passed through the upper digestive tract [74]. Immunity induced by live attenuated vaccines given systemically, such as the yellow fever vaccine, is very long lasting [75]. Less is known about the longevity of local immune responses induced by mucosal vaccinations. Available evidence obtained upon oral application of poliovirus vaccines suggests that responses may

wane after a fairly short period (approximately 1 year) [76]. Age may also affect the effectiveness of mucosal vaccines, presumably in part owing to the delayed development of the mucosal immune system. The killed oral cholera vaccine, for example, induces a CD41 T cell response in older children, but is relatively ineffective in children under 6 years of age [77]. A study conducted with the pentavalent rotavirus vaccine in Nicaragua reported good protection during the first year after vaccination but a decline in prevention after that [78]. In contrast, an inactivated whole cell oral cholera vaccine or an attenuated S. Typhi live oral vaccine resulted in sustained protective immunity for a 3-year or a 7-year period, respectively [79].

XI. ADENOVIRUS VECTORS AS ORAL VACCINES Oral immunization is the method of choice for vaccine delivery. It is less expansive, it does not require the extensive purification needed for systemic vaccines, mass vaccination programs can be conducted rapidly and with ease, and it would be safe for replication-defective adenovirus vectors. Replicating adenoviruses would be less suited for use in humans, as the viruses would be shed and then pose risks of fecal transmission to others. As an additional benefit, preexisting neutralizing antibodies to adenovirus, which dampen transgene product-specific immune responses upon systemic immunization, fail to affect those induced by oral immunization. Most adenovirus serotypes infect through the mucosal membranes of the airways, the naso-oropharynx, or the conjunctiva. Only two serotypes, HAdV40 and HAdV41, belonging to species F, are enteric viruses. Nevertheless, some humans and nearly all great apes shed multiple serotypes of infectious adenoviruses, indicating that these viruses survive within the intestinal tract [28,80]. By the same token, DNA from most adenovirus species has been isolated from intestinal tissues of primates [81].

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

TABLE 24.2 Oral Adenovirus Vector Vaccines

Vector (s)

Regimen

Species

Transgene product

Vaccine target

Immune responses/protection/side effects

Interference by vectorspecific NAs

References

REPLICATION-COMPETENT HAdV7, Prime-boost HAdV4

Chimpanzee, Hepatitis B virus dogs (HBV)

Surface antigen Antibody responses, partial protection (sAg)

n.t.

[82,83]

HAdV7, Prime-boost HAdV4

Humans

HBV

sAg

No response

n.t.

[84]

CAdV2

1 dose

Raccoons

Rabies virus

Glycoprotein (G)

Multifocal necrotizng bronchiolitis

n.t.

[85]

HAdV5

1 dose

Mice

Human Gag immunodeficiency virus (HIV)-1

B and T cells

n.t.

[86]

CAdV2

1 dose

Cats

Rabies virus

G

Poorly immunogenic per os, protective after systemic immunization

n.t.

[87]

HAdV5

1 dose

Raccoons, skunks

Rabies virus

G

Protective levels of serum antibdy titers

n.t.

[88]

CAdV2

1 dose

Raccoons, skunks

Rabies virus

G

Neutralizing antibodies, protection against challenge

n.t.

[89]

HAdV4

Enteric-coated capsules, 3 doses 6 parental H5N1 vaccine boost

Humans

Influenza virus

Hemagglutinin (HA)

Cellular responses, low seroconversion rates after oral immunization, increase after parental boost, oral priming increases antibody avidity

Yes at lower vaccine doses

[90,91]

REPLICATION-DEFECTIVE HAdV5

1 dose

Mice, cotton rats

Measles virus

HA, fusion (F) protein

Antibodies, protection

n.t.

[92]

HAdV5

1 dose

Mice

Measles virus

Nucleocapsid protein (NC)

Antibodies, splenic CD81 T cells in spleens

n.t.

[93]

HAdV5, 1 dose SAdV25

Mice

Rabies virus

G

Antibodies in serum, vaginal lavage, feces, protection, biodistribution mainly to oral cavity and airways

no

[94,95]

HAdV5, 1 dose SAdV25

Neonatal mice

Rabies virus

G

Antibodies in serum, vaginal lavage, feces, protection

n.t.

[96] (Continued)

TABLE 24.2 (Continued)

Vector (s)

Regimen

Species

Vaccine target

Transgene product

Immune responses/protection/side effects 1

Interference by vectorspecific NAs

References

SAdV23

1 dose, prime-boost

Mice

HIV-1

Gag

CD8 T cells, protection against surrogate challenge

n.t.

[97]

SADv?

1 dose

Mice

HIV-1

Gag

Intestinal CD81 T cells induced by IM but not oral immunization

n.t.

[98]

HAdV5

1 dose

Mice

Ebola virus

G

Systemic and mucosal T and B cell responses, protection

n.t.

[99]

HAdV5

1 enteric-coated dose Macaques

HIV-1

Gag, Env epitopes

T cell responses

n.t.

[100]

HAdV5

1 dose

Mice

respiratory syncytial virus (RSV)

G

No response after oral immunization

n.t.

[101]

HAdV41 HAdV5 boost

Mice

HIV-1

Env

Intestinal CD81 T cell response after oral n.t. immunization and HAdV5 boost, more potent after ileal priming

[102]

HAdV5

1 dose, iliac immunization, HAdV5 i.m. boost

Mice

HIV-1

Env

Intestinal CD81 T cell responses after iliac immunization, increase after boost

n.t.

[103]

HAdV5

1 dose, sublingual

Mice

HIV-1

Gag

Systemic and mucosal CD81 T cells

no

[104]

HAdV5

1 dose, adjuvant

Mice, ferrets

Influenza virus

HA

Antibodies, protection

no

[105]

HAdV5

1 dose

Mice

C. botulinum

Heavy-chain C- Antibodies, protection fragment

n.t.

[106]

HAdV5

1 or 2 doses, encapsidated, adjuvant

Humans

Avian influenza virus

HA

CD81 T cell responses

n.t.

[107]

HAdV5

1 or 2 doses, encapsidated, adjuvant

Humans

Influenza virus

HA

Increases in neutralizing antibody titers

No

[108]

HAdV5

1 or 2 doses, radiocontrolled capsules, adjuvant

Humans

Influenza virus

HA

Mucosal and serum B cell responses, higher upon vaccine release in the ilium than the jejunum

n.t.

[109]

n.t., not tested.

XI. ADENOVIRUS VECTORS AS ORAL VACCINES

Oral vaccine studies have been conducted with both replication-competent and replicationdefective adenovirus vectors derived from different human and nonhuman serotypes [82
101] (Table 24.2). Although poorly immunogenic in cats [86], RCA vectors, based on a canine serotype expressing the rabies virus glycoproteins, were shown to induce neutralizing antibodies and protect against challenge in raccoons and skunks [87,88]. Vectors based on human serotype 4 or 7 induced immune responses, albeit low ones, to hepatitis B surface antigen (HBsAg) in chimpanzees [82]. A clinical trial was conducted with the replication-competent HAdV7-HBsAg vector [84]. The vaccine was given to three human volunteers in the form of enteric-coated tablets. The vaccine was well tolerated and was shed for up to 2 weeks. Two out of three vaccinees showed increases in antibody titers to HAdV7, and none of them developed antibodies to hepatitis B virus. Another clinical trial was conducted using the live HAdV4 vector expressing the hemagglutinin of the poorly immunogenic H5N1 virus [90,91]. Each volunteer received three doses of vector in enteric-coated capsules. Some were then boosted parentally with an inactivated H5N1 vaccine. The oral HAdV4 vaccine-induced cellular immune responses, but only marginal neutralized antibody titers. Nevertheless, upon a systemic boost with a traditional vaccine, titers and avidity of influenza virus-specific antibodies were markedly higher in HAdV4-immunized individuals than in volunteers who had received only the systemic vaccine, indicating that the oral vaccine had primed the B cell compartment. Several preclinical studies report on oral immunizations with replication-defective adenovirus vectors. Oral immunization with HAdV5 or a SAdV of species E expressing the highly immunogenic rabies virus glycoprotein was shown to induce antibody responses to the transgene product in mice [94,95]. Using a different transgene in another SAdV serotype for oral immunization of mice resulted in genital CD81 T cell responses [97]. Additional studies

429

showed that T cell responses within the gut or the female genital tract were more robust upon systemic delivery than mucosal vaccine delivery [98]. Biodistribution studies in mice showed that, upon oral immunization, the adenovirus vectors were mainly recovered from cells of the oral cavity and the airways but not from the intestine [94]. Another group used a HAdV5 vector backbone for gastric immunization of mice to a measles virus antigen and reported induction of sustained humoral and cellular responses, in most, but not all mice [93]. Direct surgical inoculation of HAdV5 vectors into the intestine resulted in potent immune responses in experimental animals that were not achieved upon oral immunization [103]. Our attempt to induce antibody responses failed in nonhuman primates following oral immunization with high doses of SAdV vectors that were found to be efficacious in orally immunized mice (unpublished data). Oral immunization with nonenteric adenovirus vectors, when applied in a liquid form rather than within enteric-coated capsules, induced immune responses locally within the oral cavity and the airways rather than within the intestinal tract as a consequence of the vectors being labile to the stomach’s low pH and to gastric and pancreatic enzymes [103]. This is further supported by a study that reported that in mice, an E1-deleted HAdV5 vector induces higher immune responses upon sublingual than oral application [104]. While oral adenovirus vector immunization of mice has repeatedly been reported to induce transgene productspecific immune responses, oral immunization of nonhuman primates using the same vectors failed. This could reflect anatomic differences within the oral cavity. Primates have tonsils and adenoids guarding the oral cavity and the pharynx, while most rodents lack these organized structures, but instead have more diffuse nasal-associated lymphoid tissues that may more readily be infected with adenovirus vectors. Chewing habits differ between species. Raccoons and skunks chew their food carefully,

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

430

24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

allowing for their oral vaccination with recombinant vaccinia or adenovirus vectors to rabies, while dogs swallow big chunks of food and do not respond as well to oral immunization with the same vaccine virus vectors. If the duration of contact between the recombinant vaccine vector and the mucosa of the oral cavity indeed affects effectiveness of oral immunization, it would be unsuited for small children, as they may or may not follow instructions. Vectors based on nonenteric adenoviruses could be encapsulated similarly to the live HAdV4 and HAdV7 vaccines used by the U.S. military. To this end, an enteric polymethacrylate formulation for coating hydroxy-propylmethyl-cellulose capsules containing lyophilized HAdV5 expressing HIV-1 gag and a string of Env epitopes was used [100]. Vectors were thermolabile, and infectivity dropped within a week when tablets were stored at 4 C. Animals fed with these capsules developed transient SIgA responses, and responses in saliva were detected in only two thirds of animals. Cellular responses were weak but could be boosted by an adjuvanted Env peptide cocktail. Additional clinical trials tested the immunogenicity of an HAdV5 influenza vaccine given within enteric-coated tablets. The HAdV5 vectors expressed the virus hemagglutinin together with a molecular double-stranded RNA hairpin as a TLR adjuvant. The vaccine was well tolerated, and volunteers were shown to develop transgene product-specific CD81 T cells and antibodies. Immune responses were not affected by preexisting antibodies to the vaccine carrier [107,108]. In a follow-up trial, humans were given the HAdV5 vector within radio-controlled capsules that allowed for vaccine release within the ilium or the jejunum [109]. Systemic and mucosal immune responses were induced at either site, but rates of vaccine responders were higher when the vaccine was released within the ilium. These results are very promising and invite further trials. One of the attractive features of adenovirus vectors is that once their production has been

optimized, they are expected to be highly cost effective, with a single dose within the $1 range [110]. Encapsulation would increase the cost, which could be offset in part by reduced production cost due to less extensive purification and savings during vaccine delivery. Methods to ensure thermostability would have to be modified to allow for their use in encapsidated adenovirus vectors. Alternatively, one could develop vectors based on enteric adenovirus serotypes of species F. E1-deleted vectors based on HAdV41 have been constructed. The vector, which was genetically unstable [111], was shown upon oral immunization to induce a low immune response in mice that could be boosted with a HAdV5 vector expressing the same transgene and given systemically [102]. This line of research should be pursued but would first necessitate the development of genetically stable vectors.

XII. ADENOVIRUS VECTORS AS INTRANASAL VACCINES Adenovirus vectors transduce the endothelial cells covering the airways, although transduction is inefficient, as the major adenovirus receptor CAR is expressed on the basolateral rather than the apical surface of airway epithelial cells [112]. Nevertheless, a number of preclinical studies explored intranasal immunizations with adenovirus vectors derived from different serotypes [113
152] (Table 24.3). Initial studies used RCA vectors based on human, canine, or bovine serotypes expressing a variety of foreign viral antigens and reported induction of mucosal and systemic antibody responses, including IgA responses in mice, rats, dogs, cats, and nonhuman primates [86,113
122]. In mice, replication-competent HAdV5 vectors were shown to induce mucosal memory CD81 T cells [122]. Responses were blunted in the presence of preexisting immunity to the vaccine carrier [120].

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

TABLE 24.3 Vector(s)

Intranasal Adenovirus Vector Vaccines

Regimen

Species

Vaccine target

Transgene product

Immune responses/ protection

Interference by preexisting NAs

References

n.t.

[113]

REPLICATION-COMPETENT VECTORS HAdV4, 7, 5

Vectors given sequentially

Chimpanzee

HIV-1

Gag, Env

Serum, salivary, nasal, rectal, vaginal secretions, proliferative T cell responses

HAdV5

1 dose

Mice

Herpes simplex virus (HSV)-2

gB

IgA and IgG responses n.t. serum, vaginal secretions, transient systemic and sustained mucosal CD81 T cells, protection

[114,115]

HAdV5

1 dose

Mice

Simian Gag immunodeficiency virus (SIV)

n.t. Sustained systemic antibody and CD81 T cells

[86]

HAdV5

1 dose

Cotton rats, mice

Bovine herpes gD virus type (BHV) 1

IgA in lungs and nasal wash, systemic T cells, protection

n.t.

[116,117]

HAdV5

2 doses

Cattle

Foot and mouth disease virus

Precursor polypeptide 1

No protection

n.t.

[118]

BAdV3

1 dose

Cotton rats

Viral diarrhea virus

gE2

Systemic and mucosal IgA n.t. and IgG

[119]

CAdV2

1 dose

Canine puppies

Canine distemper virus

HA, F

Protection in CAdV2 seronegative but not seropositive puppies

Yes

[120]

CAdV2

1 dose

Mice

Rabies virus

Glycoprotein (G)

Serum neutralizing antibody titers, protection

n.t.

[121]

HAdV5

Listeria vector prime and protein boost

Macaques

SIV

Gag

T cells to Gag in blood and n.t. rectal mucosa, partial protection

[122]

REPLICATION-DEFECTIVE HAdV5

1 dose

Mice

Clostridium tetani

Tetanus toxin C fragment

Protection

n.t.

[123]

HAdV5

1 dose

Calves

BHV 1

gD or gC

Protection after second vaccine dose

n.t.

[124] (Continued)

TABLE 24.3

(Continued) Interference by preexisting NAs

References

n.t.

[125]

Gag, Pol, Env

Mucosal IgAs, Yes transduction of the central nervous system (CNS)

[126]

EGFP

Biodistribution study: transduction of the olfactory bulb but not other areas of the CNS

n.t.

[127]

Rabies virus

G

IgA in serum, genital secretions, and feces

n.t.

[128]

Mice

Rabies virus

G

Systemic and genital antibody responses, impaired B cell response in HAdV5-per-immune animals without DNA prime

Yes

[129]

1 dose

Neonatal mice

Rabies virus

G

Protection

n.t.

[96]

SAdV23

1 dose

Mice

HIV-1

Gag

Genital CD81 T cells

n.t.

[130]

HAdV5

1 dose

Neonatal mice

Rotavirus

VP4

intestinal IgG and IgA, immunization of dams protects offspring

n.t.

[131]

HAdV5

1 dose

Mice

MCMV

gH

Antibody responses in sera, bronchoalveolar lavage, fecal suspensions and vaginal washings; protection

n.t.

[132]

HAdV5

After BCG prime

Mice, pigs

Mycobacterium tuberculosis

Ag85a

Increased protection

n.t.

[133,134]

Vaccine target

Transgene product

Immune responses/ protection

Vector(s)

Regimen

Species

HAdV5

1 dose

Mice

Murine cytomegalovirus (MCMV)

gB

Antibodies in serum, bronchoalveolar lavage, fecal suspensions and vaginal washings, protection

HAdV5

1 dose

Mice

HIV-1

HAdV5

1 dose

Mice

HAdV5

1 dose

Mice

HAdV5

DNA vaccine prime

HAdV5,

HAdV5

1 dose

Mice

Norovirus

Capsid protein

IgG, IgA, IGM in sera, n.t. feces, intestines, lungs and lung lavage, systemic T cells

[135]

HAdV5

1 dose

Mice

RSV

G

Mucosal IgA and serum IgG responses, systemic CD81 T cell responses, protection

n.t.

[101,136]

HAdV5

1 dose

Mice

Clostridium botulinum

Heavy-chain C-fragment

Mucosal IgA and IgG, protection

No

[137]

HAdV5

1 dose

Mice

Streptococcus pneumoniae

sAg A, detoxified pneumolysin

Serum IgG, protection

n.t.

[138]

HAdV5

1 dose

Mice

Mycobacterium tuberculosis

Ag85a TB0:4

T cells in spleen and lung lumen, protection

n.t.

[139]

HAdV5, HAdV35

1 dose, aerosols

Mice, ferrets

Various

Various, including influenza virus HA

T cells in blood, BAL, protection against influenza virus challenge

n.t.

[140]

HAdV5

1 dose

Guinea pigs

Ebola virus

G

Protective immunity

no

[141]

HAdV5

1 dose

Mice

Hepatitis C virus (HCV)

CE1
E2

High antibody and low T cell responses, protection against surrogate challenge

n.t.

[142]

HAdV5

Protein boost

Mice

Chlamydia

CPAF

Strong antibody and weak n.t. T cell responses after the prime, protection after the boost

[143]

HAdV5

1 dose

Pigs

Influenza virus

HA

n.t. Full protection against homologous challenge, partial protection against heterologous challenge, vaccine-induced enhanced respiratory disease following heterologous challenge

[144]

(Continued)

TABLE 24.3

(Continued) Interference by preexisting NAs

References

Strong T and B cell responses systemically and within the airways, protection

n.t.

[145]

HA

Stronger neutralizing antibody responses than after IM immunization, protection

No

[146]

RSV

G, F

Weak serum antibody responses, IgG and IgA in BAL, protection

n.t.

[147]

Mice

Influenza virus

HA and ectodomain of M2

Serum antibody responses, protection against heterotypic challenge

n.t.

[148]

Rabbits

Bacillus anthracis

Protective Ag83

Protection after 2 doses

n.t.

[149]

Boosted with MVA or Human PanAd3 adults

RSV

F, NC, M

No increase and serum antibody titer and marginal increases in T cell frequencies in blood after IN prime

n.t.

[150]

HAdV5

2 doses

Macaques

Yersinia pestis

ycsF, caf1, and lcrV

Protection

No

[151]

HAdV5

1 dose

Mice

Pneumonia virus

NP, M

Weak systemic antibody and CD41 T cell responses, potent CD81 T cell responses, protection

n.t.

[152]

Vector(s)

Regimen

Species

PanAd3

1 dose

Mice

Influenza virus

Nucleoprotein (NP)
matrix protein (M) fusion protein

HAdV26, 28, 48

1 dose

Mice

Influenza virus

HAdV5

Protein boost

African green monkeys

HAdV5

1 dose

HAdV5

1 or 2 doses

PanAd3

n.t., not tested.

Vaccine target

Transgene product

Immune responses/ protection

XII. ADENOVIRUS VECTORS AS INTRANASAL VACCINES

Other researchers used replication-defective adenovirus vectors based on human or simian serotypes expressing viral or bacterial antigens in mice, guinea pigs, pigs, or nonhuman primates. The adenovirus vectors were used as single vaccine modalities or combined with DNA or, in the case of a vaccine to Mycobacterium tuberculosis, BCG primes or as booster immunizations with protein or a poxvirus vector. Most studies reported induction of antibody and T cells within the airways and protection against a subsequent challenge. Intranasal immunization elicited genital and, in some studies, intestinal antibody responses. Protective immune responses could be induced in neonatal mice [96], and responses induced in female mice were transferred and shown to be protective in their offspring [131]. A comparison of vaccines based on species C and D of adenovirus showed that vaccines from species D were less effective than those from species C upon systemic immunization but elicited equal transgene product-specific immune responses when given to the airways [146]. Some studies reported that preexisting neutralizing antibodies to the vaccine carrier had no effect on the immunogenicity of adenovirus vector vaccine given intranasally [137,142,146,151]; others disagreed [126,129]. Biodistribution studies in mice showed that adenovirus vectors upon intranasal application to mice localize to the olfactory bulb [126], but fail to disseminate to other regions of the central nervous system [127]. A clinical trial was conducted in healthy human adults with a vector based on a chimpanzee adenovirus called PanAd3 expressing an artificial fusion antigen of respiratory syncytial virus (RSV) [150]. The human volunteers had preexisting immunity to RSV due to natural infections during childhood. Vectors were given as a prime intramuscularly or intranasally. Humans were then boosted intramuscularly by a second dose of the same vector or a modified vaccinia Ankara vector. The vaccines were well tolerated, although several individuals reported a sore throat after intranasal immunization. RSV-specific neutralizing

435

antibodies in serum increased upon systemic but not intranasal prime. Circulating T cell responses increased in some individuals after the prime given through either route, but the responses were more robust and common after systemic immunization. Mucosal responses were not assessed, and it is thus not possible to draw firm conclusions about the effectiveness of intranasal immunization to elicit or boost responses within the airways. Nevertheless, lack of a robust systemic recall response upon intranasal immunization is worrisome and may predict that this route of vaccine delivery, although highly efficient in mice, is not suitable for immunization of humans. In nonhuman primates, intranasal immunization with a replication-defective HAdV5 vector expressing RSV antigens elicited a modest systemic antibody response against RSV’s fusion protein in all animals, but only a fraction responded against the viral glycoprotein [147]. Responses were more pronounced in animals that were primed with the vector given systemically. Mucosal responses were assessed after booster immunizations. Results indicate that the intranasal prime was superior at promoting responses in the airways, and it induced the highest degree of protection against intranasal RSV challenge. Similar results were obtained with a HAdV5 vaccine to Yersinia pestis, for which intranasal priming followed by a protein boost resulted in complete protection against challenge [151]. Neither of the nonhuman primate studies assessed protection after a single intranasal immunization, but rather boosted responses with a second vaccine prototype given systemically. It is thus currently impossible to conclude that intranasal adenovirus vector immunization as a single-dose vaccine modality is suited for use in primates. The apparent lack of a strong systemic immune response upon intranasal immunization of primates contrasts results in rodents and may again reflect anatomic differences of the oro-pharyngeal immune system. This may be overcome by retargeting the adenovirus vector

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

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to receptors that are more accessible than CAR within the airways. The sigma 1 protein of reovirus serotype T3 binds sialic acid and junctional adhesion molecule 1. It forms a trimer, and its structure is related to that of adenovirus fiber [153]. Adenovirus vectors with a chimeric fiber, in which the reovirus sigma I protein was fused to the C-terminus of fiber, resulted paradoxically in strongly reduced transduction rates within the airways, but improved transgene productspecific T cell responses upon intranasal immunization [154]. Further modifications of the fusion protein by increasing the length of the fusion protein resulted in improved transduction rates in the airways of mice, presumably by making the receptor-targeting domain more accessible [155]. In mice, modified vectors were shown to induce higher antibody responses in serum and in genital secretions compared to vectors with wild-type fiber. This line of research should be explored further, although its potential success would hinge not only on improved transduction rates and superior T and B cell responses, but also on genetic stability of modified vectors and vector growth characteristics, which will dictate vector yields. Overall, intranasal immunization with adenovirus vectors have been shown to prime a mucosal immune response, which, in order to become protective, may require a systemic booster immunization. Safety concerns due to transfer of the vectors into the olfactory bulb or other parts of the central nervous system may have to be addressed for some serotypes in more detail in species, such as nonhuman primates, that are more predictable for outcome in humans (For the use of adenovirus vector for HIV vaccine, see Chapter 42: Mucosal Vaccines Against HIV/SIV Infection).

XIII. IMMUNIZATIONS THROUGH THE RECTAL OR GENITAL MUCOSA Adenovirus vectors have been tested for induction of immune responses upon their

application to the genital, rectal, or colorectal mucosa of experimental animals [156
159]. We view these routes of application, which may yield basic knowledge about the mucosal immune system, as too intrusive and overall impractical for use in humans.

XIV. USE OF ADJUVANTS FOR MUCOSAL ADENOVIRUS VECTOR VACCINES Adenovirus vectors could be formulated with traditional adjuvants, or they could be designed to encode an inflammatory sequence such as the TLR-binding sequence explored in clinical trials for the oral HAdV5 vector for influenza virus [103,104]. Others demonstrated that the formulations of HAdV5 vectors in α-galactosylceramide [160], Escherichia coli heat-labile enterotoxin [161], a synthetic TLR-4 agonist [162], chitooligosaccharide-based nanoparticles with mannosylated polyethyleneiminetriethyleneglycol [163], or Ftl3 [164] further enhance mucosal transgene product-specific immune responses. We would like to caution that adenovirus vectors already induce very potent innate immune responses that cause dose-limiting toxicity, which may worsen upon the addition of adjuvants.

XV. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Adenovirus vectors induce potent and sustained transgene product-specific T and B cell responses and are thus highly suited as vaccine carriers. As systemic vaccines, they are expected to be cost-effective and to provide lasting immunity after a single dose. Will they be as cheap and efficacious as mucosal vaccines? Intranasal immunization, routinely used in humans for the attenuated influenza vaccine, has yielded disappointing results in primates, which may reflect that CAR, the receptor used

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REFERENCES

by most adenoviruses, is not expressed on the apical surface on the mucosal endothelial layer. Further studies are needed to assess whether retargeting of vectors or the use of CD46binding vectors would improve vaccine uptake into the airways and whether such vectors would meet the requirements of vaccines for mass vaccination: genetic stability, high yields during production, and efficacy in single-dose regimens. In addition, transduction of the olfactory bulb raises safety concerns that need to be addressed in more detail. The use of adenovirus vector as oral vaccines, which in many ways are more practical for mass vaccination campaigns in developing countries, appears more feasible. Results obtained in clinical trials thus far are promising. Methods to encapsidate adenovirus vectors have been developed for the live HAdV4 and HAdV7 vaccines. They may have to be adjusted to ensure thermostability of vectors and delivery of the vaccines to the ileum.

References [1] Volz A, Sutter G. Modified vaccinia virus ankara: history, value in basic research, and current perspectives for vaccine development. Adv Virus Res 2017;97:187
243. [2] Kaplan C. Vaccinia virus: a suitable vehicle for recombinant vaccines? Arch Virol 1989;106(1
2):127
39. [3] Falzarano D, Geisbert TW, Feldmann H. Progress in filovirus vaccine development: evaluating the potential for clinical use. Expert Rev Vaccines 2011;10(1):63
77. [4] Geisbert TW, Feldmann H. Recombinant vesicular stomatitis virus-based vaccines against Ebola and Marburg virus infections. J Infect Dis 2011;204(Suppl. 3):S1075
81. [5] McKenna PM, McGettigan JP, Pomerantz RJ, Dietzschold B, Schnell MJ. Recombinant rhabdoviruses as potential vaccines for HIV-1 and other diseases. Curr HIV Res 2003;1(2):229
37. [6] Nogales A, Martı´nez-Sobrido L. Reverse genetics approaches for the development of influenza vaccines. Int J Mol Sci 2016;18(1). [7] Takimoto T, Hurwitz JL, Zhan X, Krishnamurthy S, Prouser C, Brown B, et al. Recombinant Sendai virus as a novel vaccine candidate for respiratory syncytial virus. Viral Immunol 2005;18(2):255
66.

437

[8] Michalik M, Djahanshiri B, Leo JC, Linke D. Reverse vaccinology: the pathway from genomes and epitope predictions to tailored recombinant vaccines. Methods Mol Biol Clifton NJ 2016;1403:87
106. [9] Lasaro MO, Ertl HCJ. New insights on adenovirus as vaccine vectors. Mol Ther J Am Soc Gene Ther 2009;17 (8):1333
9. [10] Gilbert SC, Warimwe GM. Rapid development of vaccines against emerging pathogens: The replicationdeficient simian adenovirus platform technology. Vaccine 2017;35(35 Pt A):4461
4. [11] Fougeroux C, Holst PJ. Future prospects for the development of cost-effective adenovirus vaccines. Int J Mol Sci 2017;18(4):686. [12] Shiver JW, Emini EA. Recent advances in the development of HIV-1 vaccines using replication-incompetent adenovirus vectors. Annu Rev Med 2004;55:355
72. [13] Wilson JM. Adenoviruses as gene-delivery vehicles. N Engl J Med 1996;334(18):1185
7. [14] Kung SH, Hagstrom JN, Cass D, Tai SJ, Lin HF, Stafford DW, et al. Human factor IX corrects the bleeding diathesis of mice with hemophilia B. Blood 1998;91 (3):784
90. [15] Cao B, Mytinger JR, Huard J. Adenovirus mediated gene transfer to skeletal muscle. Microsc Res Tech 2002;58(1):45
51. [16] Yang Y, Ertl HC, Wilson JM. MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity 1994;1(5):433
42. [17] Bessis N, GarciaCozar FJ, Boissier M-C. Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther 2004;11 (Suppl. 1):S10
17. [18] Zhou D, Wu T-L, Emmer KL, Kurupati R, Tuyishime S, Li Y, et al. Hexon-modified recombinant E1-deleted adenovirus vectors as dual specificity vaccine carriers for influenza virus. Mol Ther J Am Soc Gene Ther 2013;21(3):696
706. [19] Worgall S, Krause A, Rivara M, Hee K-K, Vintayen EV, Hackett NR, et al. Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid. J Clin Invest 2005;115(5):1281
9. [20] Altaras NE, Aunins JG, Evans RK, Kamen A, Konz JO, Wolf JJ. Production and formulation of adenovirus vectors. Adv Biochem Eng Biotechnol 2005;99:193
260. [21] Gardner TA, Ko SC, Yang L, Cadwell JJ, Chung LW, Kao C. Serum-free recombinant production of adenovirus using a hollow fiber capillary system. Biotechniques 2001;30(2):422
7. [22] Kamen A, Henry O. Development and optimization of an adenovirus production process. J Gene Med 2004;6 (Suppl. 1):S184
92.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

438

24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

[23] Burova E, Ioffe E. Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications. Gene Ther 2005;12 (Suppl. 1):S5
17. [24] Eglon MN, Duffy AM, O’Brien T, Strappe PM. Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography. J Gene Med 2009;11(11):978
89. [25] Konz JO, Lee AL, Lewis JA, Sagar SL. Development of a purification process for adenovirus: controlling virus aggregation to improve the clearance of host cell DNA. Biotechnol Prog 2005;21(2):466
72. [26] Doerfler, W., Bo¨hm, P., editors. Adenoviruses: model and vectors in virus-host interactions. Springer; 2003 [cited 2017 July 5]. Available from: http://www. springer.com/us/book/9783540001546. [27] Roy S, Vandenberghe LH, Kryazhimskiy S, Grant R, Calcedo R, Yuan X, et al. Isolation and characterization of adenoviruses persistently shed from the gastrointestinal tract of non-human primates. PLoS Pathog 2009;5 (7):e1000503. [28] Horvath J, Palkonyay L, Weber J. Group C adenovirus DNA sequences in human lymphoid cells. J Virol 1986;59(1):189
92. [29] Tatsis N, Fitzgerald JC, Reyes-Sandoval A, HarrisMcCoy KC, Hensley SE, Zhou D, et al. Adenoviral vectors persist in vivo and maintain activated CD8 1 T cells: implications for their use as vaccines. Blood 2007;110(6):1916
23. [30] Finn JD, Bassett J, Millar JB, Grinshtein N, Yang TC, Parsons R, et al. Persistence of transgene expression influences CD8 1 T-cell expansion and maintenance following immunization with recombinant adenovirus. J Virol 2009;83(23):12027
36. [31] Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997;275(5304):1320
3. [32] Lu¨tschg V, Boucke K, Hemmi S, Greber UF. Chemotactic antiviral cytokines promote infectious apical entry of human adenovirus into polarized epithelial cells. Nat Commun 2011;2:391. [33] Gaggar A, Shayakhmetov DM, Lieber A. CD46 is a cellular receptor for group B adenoviruses. Nat Med 2003;9(11):1408
12. [34] Appledorn DM, Patial S, McBride A, Godbehere S, Van Rooijen N, Parameswaran N, et al. Adenovirus vector-induced innate inflammatory mediators, MAPK signaling, as well as adaptive immune responses are dependent upon both TLR2 and TLR9 in vivo. J Immunol 2008;181(3):2134
44. [35] Doronin K, Flatt JW, Di Paolo NC, Khare R, Kalyuzhniy O, Acchione M, et al. Coagulation factor X

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

activates innate immunity to human species C adenovirus. Science 2012;338(6108):795
8. Iacobelli-Martinez M, Nemerow GR. Preferential activation of Toll-like receptor nine by CD46-utilizing adenoviruses. J Virol 2007;81(3):1305
12. Ferguson BJ, Mansur DS, Peters NE, Ren H, Smith GL. DNA-PK is a DNA sensor for IRF-3-dependent innate immunity. eLife 2012;1:e00047. Hendrickx R, Stichling N, Koelen J, Kuryk L, Lipiec A, Greber UF. Innate immunity to adenovirus. Hum Gene Ther 2014;25(4):265
84. Chen H, Xiang ZQ, Li Y, Kurupati RK, Jia B, Bian A, et al. Adenovirus-based vaccines: comparison of vectors from three species of adenoviridae. J Virol 2010;84 (20):10522
32. Barouch DH, Kik SV, Weverling GJ, Dilan R, King SL, Maxfield LF, et al. International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations. Vaccine 2011;29 (32):5203
9. Mast TC, Kierstead L, Gupta SB, Nikas AA, Kallas EG, Novitsky V, et al. International epidemiology of human pre-existing adenovirus (Ad) type-5, type-6, type-26 and type-36 neutralizing antibodies: correlates of high Ad5 titers and implications for potential HIV vaccine trials. Vaccine 2010;28(4):950
7. Small JC, Haut LH, Bian A, Ertl HCJ. The effect of adenovirus-specific antibodies on adenoviral vectorinduced, transgene product-specific T cell responses. J Leukoc Biol 2014;96(5):821
31. Pandey A, Singh N, Vemula SV, Coue¨til L, Katz JM, Donis R, et al. Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine. PLoS One 2012;7(3):e33428. Casimiro DR, Chen L, Fu T-M, Evans RK, Caulfield MJ, Davies M-E, et al. Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene. J Virol 2003;77(11):6305
13. Rux JJ, Kuser PR, Burnett RM. Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods. J Virol 2003;77 (17):9553
66. Hutnick NA, Carnathan D, Demers K, Makedonas G, Ertl HCJ, Betts MR. Adenovirus-specific human T cells are pervasive, polyfunctional, and cross-reactive. Vaccine 2010;28(8):1932
41. Brown LJ, Rosatte RC, Fehlner-Gardiner C, Ellison JA, Jackson FR, Bachmann P, et al. Oral vaccination and protection of striped skunks (Mephitis mephitis)

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

439

REFERENCES

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

against rabies using ONRABs. Vaccine 2014;32 (29):3675
9. Slate D, Chipman RB, Algeo TP, Mills SA, Nelson KM, Croson CK, et al. Safety and immunogenicity of Ontario Rabies Vaccine Bait (ONRAB) in the first us field trial in raccoons (Procyon lotor). J Wildl Dis 2014;50(3):582
95. Haut LH, Gill AL, Kurupati RK, Bian A, Li Y, GilesDavis W, et al. A Partial E3 Deletion in ReplicationDefective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products. Hum Gene Ther Methods 2016;27(5):187
96. Koup RA, Lamoreaux L, Zarkowsky D, Bailer RT, King CR, Gall JGD, et al. Replication-defective adenovirus vectors with multiple deletions do not induce measurable vector-specific T cells in human trials. J Virol 2009;83(12):6318
22. Morris SJ, Sebastian S, Spencer AJ, Gilbert SC. Simian adenoviruses as vaccine vectors. Future Virol 2016;11 (9):649
59. Small JC, Kurupati RK, Zhou X, Bian A, Chi E, Li Y, et al. Construction and characterization of E1- and E3deleted adenovirus vectors expressing two antigens from two separate expression cassettes. Hum Gene Ther 2014;25(4):328
38. Matthews QL, Fatima A, Tang Y, Perry BA, Tsuruta Y, Komarova S, et al. HIV antigen incorporation within adenovirus hexon hypervariable 2 for a novel HIV vaccine approach. PLoS One 2010;5(7):e11815. Zhou D, Zhou X, Bian A, Li H, Chen H, Small JC, et al. An efficient method of directly cloning chimpanzee adenovirus as a vaccine vector. Nat Protoc 2010;5 (11):1775
85. Ruzsics Z, Lemnitzer F, Thirion C. Engineering adenovirus genome by bacterial artificial chromosome (BACj) technology. Methods Mol Biol Clifton NJ 2014;1089:143
58. Nestola P, Silva RJS, Peixoto C, Alves PM, Carrondo MJT, Mota JPB. Robust design of adenovirus purification by two-column, simulated moving-bed, sizeexclusion chromatography. J Biotechnol 2015;213: 109
19. Ishii-Watabe A, Uchida E, Iwata A, Nagata R, Satoh K, Fan K, et al. Detection of replication-competent adenoviruses spiked into recombinant adenovirus vector products by infectivity PCR. Mol Ther J Am Soc Gene Ther 2003;8(6):1009
16. Afkhami S, LeClair DA, Haddadi S, Lai R, Toniolo SP, Ertl HC, et al. Spray dried human and chimpanzee adenoviral-vectored vaccines are thermally stable and immunogenic in vivo. Vaccine 2017;35(22):2916
24. Pearson FE, McNeilly CL, Crichton ML, Primiero CA, Yukiko SR, Fernando GJP, et al. Dry-coated live viral

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

vector vaccines delivered by nanopatch microprojections retain long-term thermostability and induce transgene-specific T cell responses in mice. PLoS One 2013;8(7):e67888. Pinto AR, Fitzgerald JC, Giles-Davis W, Gao GP, Wilson JM, Ertl HCJ. Induction of CD8 1 T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers. J Immunol 2003;171(12):6774
9. Mensah VA, Gueye A, Ndiaye M, Edwards NJ, Wright D, Anagnostou NA, et al. Safety, immunogenicity and fficacy of prime-boost vaccination with ChAd63 and MVA ncoding ME-TRAP against Plasmodium falciparum infection in adults in Senegal. PLoS One 2016;11 (12):e0167951. Rollier CS, Verschoor EJ, Verstrepen BE, Drexhage J AR, Paranhos-Baccala G, Liljestro¨m P, et al. T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates. Gene Ther 2016;23 (10):753
9. Hensley SE, Giles-Davis W, McCoy KC, Weninger W, Ertl HCJ. Dendritic cell maturation, but not CD8 1 T cell induction, is dependent on type I IFN signaling during vaccination with adenovirus vectors. J Immunol 2005;175(9):6032
41. Tatsis N, Blejer A, Lasaro MO, Hensley SE, Cun A, Tesema L, et al. A CD46-binding chimpanzee adenovirus vector as a vaccine carrier. Mol Ther J Am Soc Gene Ther 2007;15(3):608
17. Colloca S, Barnes E, Folgori A, Ammendola V, Capone S, Cirillo A, et al. Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species. Sci Transl Med 2012;4(115):115ra2. Hensley SE, Cun AS, Giles-Davis W, Li Y, Xiang Z, Lasaro MO, et al. Type I interferon inhibits antibody responses induced by a chimpanzee adenovirus vector. Mol Ther J Am Soc Gene Ther 2007;15(2):393
403. Society for Mucosal Immunology, P. Smith, T. MacDonald, R. Blumberg. Principles of Mucosal Immunology [Internet]. 1st ed. Garland Science; 2012 [cited 2017 July 5]. 512 p. Available from: http:// www.garlandscience.com/product/isbn/ 9780815344438. Benson MJ, Pino-Lagos K, Rosemblatt M, Noelle RJ. All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of co-stimulation. J Exp Med 2007;204 (8):1765
74. Sheridan BS, Lefranc¸ois L. Intraepithelial lymphocytes: to serve and protect. Curr Gastroenterol Rep 2010;12 (6):513
21.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

440

24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

[70] Hirota K, Turner J-E, Villa M, Duarte JH, Demengeot J, Steinmetz OM, et al. Plasticity of Th17 cells in Peyer’s patches is responsible for the induction of T celldependent IgA responses. Nat Immunol 2013;14 (4):372
9. [71] Bergqvist P, Stensson A, Lycke NY, Bemark M. T cellindependent IgA class switch recombination is restricted to the GALT and occurs prior to manifest germinal center formation. J Immunol 2010;184 (7):3545
53. [72] Wu W, Sun M, Chen F, Cao AT, Liu H, Zhao Y, et al. Microbiota metabolite short-chain fatty acid acetate promotes intestinal IgA response to microbiota which is mediated by GPR43. Mucosal Immunol 2017;10 (4):946
56. [73] Kato LM, Kawamoto S, Maruya M, Fagarasan S. The role of the adaptive immune system in regulation of gut microbiota. Immunol Rev 2014;260(1):67
75. [74] Gray GC, Erdman DD. Adenovirus vaccines. Plotkin’s Vaccines. 7th ed Elsevier Health Sciences; 2017. p. 121
33. [75] Gotuzzo E, Yactayo S, Co´rdova E. Efficacy and duration of immunity after yellow fever vaccination: systematic review on the need for a booster every 10 years. Am J Trop Med Hyg 2013;89(3):434
44. [76] Grassly NC, Jafari H, Bahl S, Sethi R, Deshpande JM, Wolff C, et al. Waning intestinal immunity after vaccination with oral poliovirus vaccines in India. J Infect Dis 2012;205(10):1554
61. [77] Arifuzzaman M, Rashu R, Leung DT, Hosen MI, Bhuiyan TR, Bhuiyan MS, et al. Antigen-specific memory T cell responses after vaccination with an oral killed cholera vaccine in Bangladeshi children and comparison to responses in patients with naturally acquired cholera. Clin Vaccine Immunol CVI 2012;19 (8):1304
11. [78] Patel M, Pedreira C, De Oliveira LH, Uman˜a J, Tate J, Lopman B, et al. Duration of protection of pentavalent rotavirus vaccination in Nicaragua. Pediatrics 2012;130 (2):e365
72. [79] Sur D, Kanungo S, Sah B, Manna B, Ali M, Paisley AM, et al. Efficacy of a low-cost, inactivated whole-cell oral cholera vaccine: results from 3 years of follow-up of a randomized, controlled trial. PLoS Negl Trop Dis 2011;5(10):e1289. [80] Fox JP, Brandt CD, Wassermann FE, Hall CE, Spigland I, Kogon A, et al. The virus watch program: a continuing surveillance of viral infections in metropolitan New York families. VI. Observations of adenovirus infections: virus excretion patterns, antibody response, efficiency of surveillance, patterns of infections, and relation to illness. Am J Epidemiol 1969;89(1):25
50. [81] Roy S, Calcedo R, Medina-Jaszek A, Keough M, Peng H, Wilson JM. Adenoviruses in lymphocytes of the

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

human gastro-intestinal tract. PLoS One 2011;6(9): e24859. Lubeck MD, Davis AR, Chengalvala M, Natuk RJ, Morin JE, Molnar-Kimber K, et al. Immunogenicity and efficacy testing in chimpanzees of an oral hepatitis B vaccine based on live recombinant adenovirus. Proc Natl Acad Sci U S A 1989;86(17):6763
7. Chengalvala MV, Bhat BM, Bhat R, Lubeck MD, Mizutani S, Davis AR, et al. Immunogenicity of high expression adenovirus-hepatitis B virus recombinant vaccines in dogs. J Gen Virol 1994;75(Pt 1):125
31. Tacket CO, Losonsky G, Lubeck MD, Davis AR, Mizutani S, Horwith G, et al. Initial safety and immunogenicity studies of an oral recombinant adenohepatitis B vaccine. Vaccine 1992;10(10):673
6. Hamir AN, Raju N, Rupprecht CE. Experimental oral administration of canine adenovirus (type 2) to raccoons (Procyon lotor). Vet Pathol 1992;29(6):509
13. Flanagan B, Pringle CR, Leppard KN. A recombinant human adenovirus expressing the simian immunodeficiency virus Gag antigen can induce long-lived immune responses in mice. J Gen Virol 1997;78 (Pt 5):991
7. Hu RL, Liu Y, Zhang SF, Zhang F, Fooks AR. Experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies. Vaccine 2007;25(29):5301
7. Rosatte RC, Donovan D, Davies JC, Allan M, Bachmann P, Stevenson B, et al. Aerial distribution of ONRAB baits as a tactic to control rabies in raccoons and striped skunks in Ontario, Canada. J Wildl Dis 2009;45(2):363
74. Henderson H, Jackson F, Bean K, Panasuk B, Niezgoda M, Slate D, et al. Oral immunization of raccoons and skunks with a canine adenovirus recombinant rabies vaccine. Vaccine 2009;27(51):7194
7. Gurwith M, Lock M, Taylor EM, Ishioka G, Alexander J, Mayall T, et al. Safety and immunogenicity of an oral, replicating adenovirus serotype 4 vector vaccine for H5N1 influenza: a randomised, double-blind, placebo-controlled, phase 1 study. Lancet Infect Dis 2013;13(3):238
50. Khurana S, Coyle EM, Manischewitz J, King LR, Ishioka G, Alexander J, et al. Oral priming with replicating adenovirus serotype 4 followed by subunit H5N1 vaccine boost promotes antibody affinity maturation and expands H5N1 cross-clade neutralization. PLoS One 2015;10(1):e0115476. Fooks AR, Jeevarajah D, Lee J, Warnes A, Niewiesk S, ter Meulen V, et al. Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins:

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

protective immune responses in rodents. J Gen Virol 1998;79(Pt 5):1027
31. [93] Sharpe S, Fooks A, Lee J, Hayes K, Clegg C, Cranage M. Single oral immunization with replication deficient recombinant adenovirus elicits long-lived transgene-specific cellular and humoral immune responses. Virology 2002;293(2):210
16. [94] Xiang ZQ, Gao GP, Reyes-Sandoval A, Li Y, Wilson JM, Ertl HCJ. Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier. J Virol 2003;77(20):10780
9. [95] Zhou D, Cun A, Li Y, Xiang Z, Ertl HCJ. A chimpanzee-origin adenovirus vector expressing the rabies virus glycoprotein as an oral vaccine against inhalation infection with rabies virus. Mol Ther J Am Soc Gene Ther 2006;14(5):662
72. [96] Xiang Z, Li Y, Gao G, Wilson JM, Ertl HCJ. Mucosally delivered E1-deleted adenoviral vaccine carriers induce transgene product-specific antibody responses in neonatal mice. J Immunol 2003;171(8):4287
93. [97] Pinto AR, Fitzgerald JC, Gao GP, Wilson JM, Ertl HCJ. Induction of CD8 1 T cells to an HIV-1 antigen upon oral immunization of mice with a simian E1-deleted adenoviral vector. Vaccine 2004;22 (5
6):697
703. [98] Lin SW, Cun AS, Harris-McCoy K, Ertl HC. Intramuscular rather than oral administration of replication-defective adenoviral vaccine vector induces specific CD8 1 T cell responses in the gut. Vaccine 2007;25(12):2187
93. [99] Patel A, Zhang Y, Croyle M, Tran K, Gray M, Strong J, et al. Mucosal delivery of adenovirus-based vaccine protects against Ebola virus infection in mice. J Infect Dis 2007;196(Suppl. 2):S413
20. [100] Mercier GT, Nehete PN, Passeri MF, Nehete BN, Weaver EA, Templeton NS, et al. Oral immunization of rhesus macaques with adenoviral HIV vaccines using enteric-coated capsules. Vaccine 2007;25 (52):8687
701. [101] Yu J-R, Kim S, Lee J-B, Chang J. Single intranasal immunization with recombinant adenovirus-based vaccine induces protective immunity against respiratory syncytial virus infection. J Virol 2008;82 (5):2350
7. [102] Ko S-Y, Cheng C, Kong W-P, Wang L, Kanekiyo M, Einfeld D, et al. Enhanced induction of intestinal cellular immunity by oral priming with enteric adenovirus 41 vectors. J Virol 2009;83(2):748
56. [103] Wang L, Cheng C, Ko S-Y, Kong W-P, Kanekiyo M, Einfeld D, et al. Delivery of human immunodeficiency virus vaccine vectors to the intestine induces enhanced mucosal cellular immunity. J Virol 2009;83 (14):7166
75.

441

[104] Appledorn DM, Aldhamen YA, Godbehere S, Seregin SS, Amalfitano A. Sublingual administration of an adenovirus serotype 5 (Ad5)-based vaccine confirms Toll-like receptor agonist activity in the oral cavity and elicits improved mucosal and systemic cellmediated responses against HIV antigens despite preexisting Ad5 immunity. Clin Vaccine Immunol CVI 2011;18(1):150
60. [105] Scallan CD, Tingley DW, Lindbloom JD, Toomey JS, Tucker SN. An adenovirus-based vaccine with a double-stranded RNA adjuvant protects mice and ferrets against H5N1 avian influenza in oral delivery models. Clin Vaccine Immunol CVI 2013;20 (1):85
94. [106] Chen S, Xu Q, Zeng M. Oral vaccination with an adenovirus-vectored vaccine protects against botulism. Vaccine 2013;31(7):1009
11. [107] Peters W, Brandl JR, Lindbloom JD, Martinez CJ, Scallan CD, Trager GR, et al. Oral administration of an adenovirus vector encoding both an avian influenza A hemagglutinin and a TLR3 ligand induces antigen specific granzyme B and IFN-γ T cell responses in humans. Vaccine 2013;31(13):1752
8. [108] Liebowitz D, Lindbloom JD, Brandl JR, Garg SJ, Tucker SN. High titre neutralising antibodies to influenza after oral tablet immunisation: a phase 1, randomised, placebo-controlled trial. Lancet Infect Dis 2015;15(9):1041
8. [109] Kim L, Martinez CJ, Hodgson KA, Trager GR, Brandl JR, Sandefer EP, et al. Systemic and mucosal immune responses following oral adenoviral delivery of influenza vaccine to the human intestine by radio controlled capsule. Sci Rep 2016;6:37295. [110] Vellinga J, Smith JP, Lipiec A, Majhen D, Lemckert A, van Ooij M, et al. Challenges in manufacturing adenoviral vectors for global vaccine product deployment. Hum Gene Ther 2014;25(4):318
27. [111] Lemiale F, Haddada H, Nabel GJ, Brough DE, King CR, Gall JGD. Novel adenovirus vaccine vectors based on the enteric-tropic serotype 41. Vaccine 2007;25(11):2074
84. [112] Zabner J, Freimuth P, Puga A, Fabrega A, Welsh MJ. Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection. J Clin Invest 1997;100(5):1144
9. [113] Natuk RJ, Davis AR, Chanda PK, Lubeck MD, Chengalvala M, Murthy SC, et al. Adenovirus vectored vaccines. Dev Biol Stand 1994;82:71
7. [114] Gallichan WS, Rosenthal KL. Specific secretory immune responses in the female genital tract following intranasal immunization with a recombinant adenovirus expressing glycoprotein B of herpes simplex virus. Vaccine 1995;13(16):1589
95.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

442

24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

[115] Gallichan WS, Rosenthal KL. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J Exp Med 1996;184 (5):1879
90. [116] Papp Z, Babiuk LA, Baca-Estrada ME. Induction of immunity in the respiratory tract and protection from bovine herpesvirus type 1 infection by different routes of immunization with recombinant adenovirus. Viral Immunol 1998;11(2):79
91. [117] Papp Z, Babiuk LA, Baca-Estrada ME. Antigenspecific cytokine and antibody isotype profiles induced by mucosal and systemic immunization with recombinant adenoviruses. Viral Immunol 1999;12 (2):107
16. [118] Sanz-Parra A, Va´zquez B, Sobrino F, Cox SJ, Ley V, Salt JS. Evidence of partial protection against footand-mouth disease in cattle immunized with a recombinant adenovirus vector expressing the precursor polypeptide (P1) of foot-and-mouth disease virus capsid proteins. J Gen Virol 1999;80(Pt 3):671
9. [119] Baxi MK, Deregt D, Robertson J, Babiuk LA, Schlapp T, Tikoo SK. Recombinant bovine adenovirus type 3 expressing bovine viral diarrhea virus glycoprotein E2 induces an immune response in cotton rats. Virology 2000;278(1):234
43. [120] Fischer L, Tronel JP, Pardo-David C, Tanner P, Colombet G, Minke J, et al. Vaccination of puppies born to immune dams with a canine adenovirusbased vaccine protects against a canine distemper virus challenge. Vaccine 2002;20(29
30):3485
97. [121] Li J, Faber M, Papaneri A, Faber M-L, McGettigan JP, Schnell MJ, et al. A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice. Virology 2006;356(1
2):147
54. [122] Lakhashe SK, Velu V, Sciaranghella G, Siddappa NB, Dipasquale JM, Hemashettar G, et al. Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges. Vaccine 2011;29(34):5611
22. [123] Shi Z, Zeng M, Yang G, Siegel F, Cain LJ, van Kampen KR, et al. Protection against tetanus by needle-free inoculation of adenovirus-vectored nasal and epicutaneous vaccines. J Virol 2001;75(23):11474
82. [124] Gogev S, Vanderheijden N, Lemaire M, Schynts F, D’Offay J, Deprez I, et al. Induction of protective immunity to bovine herpesvirus type 1 in cattle by intranasal administration of replication-defective human adenovirus type 5 expressing glycoprotein gC or gD. Vaccine 2002;20(9
10):1451
65. [125] Shanley JD, Wu CA. Mucosal immunization with a replication-deficient adenovirus vector expressing

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[136]

murine cytomegalovirus glycoprotein B induces mucosal and systemic immunity. Vaccine 2003;21 (19
20):2632
42. Lemiale F, Kong W, Akyu¨rek LM, Ling X, Huang Y, Chakrabarti BK, et al. Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodeficiency virus vaccine and localization in the central nervous system. J Virol 2003;77 (18):10078
87. Damjanovic D, Zhang X, Mu J, Medina MF, Xing Z. Organ distribution of transgene expression following intranasal mucosal delivery of recombinant replication-defective adenovirus gene transfer vector. Genet Vaccines Ther 2008;6:5. Xiang Z, Ertl HC. Induction of mucosal immunity with a replication-defective adenoviral recombinant. Vaccine 1999;17(15
16):2003
8. Xiang ZQ, Pasquini S, Ertl HC. Induction of genital immunity by DNA priming and intranasal booster immunization with a replication-defective adenoviral recombinant. J Immunol 1999;162(11):6716
23. de Souza APD, Haut LH, Silva R, Ferreira SI, Zanetti CR, Ertl HCJ, et al. Genital CD8 1 T cell response to HIV-1 gag in mice immunized by mucosal routes with a recombinant simian adenovirus. Vaccine 2007;25(1):109
16. Liu X, Yang T, Sun QM, Sun MS. Efficient intranasal immunization of newborn mice with recombinant adenovirus expressing rotavirus protein VP4 against oral rotavirus infection. Acta Virol 2005;49(1):17
22. Shanley JD, Wu CA. Intranasal immunization with a replication-deficient adenovirus vector expressing glycoprotein H of murine cytomegalovirus induces mucosal and systemic immunity. Vaccine 2005;23 (8):996
1003. Santosuosso M, McCormick S, Zhang X, Zganiacz A, Xing Z. Intranasal boosting with an adenovirusvectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis. Infect Immun 2006;74(8):4634
43. Xing Z, McFarland CT, Sallenave J-M, Izzo A, Wang J, McMurray DN. Intranasal mucosal boosting with an adenovirus-vectored vaccine markedly enhances the protection of BCG-primed guinea pigs against pulmonary tuberculosis. PLoS One 2009;4(6):e5856. Guo L, Wang J, Zhou H, Si H, Wang M, Song J, et al. Intranasal administration of a recombinant adenovirus expressing the norovirus capsid protein stimulates specific humoral, mucosal, and cellular immune responses in mice. Vaccine 2008;26(4):460
8. Fu Y, He J, Zheng X, Wu Q, Zhang M, Wang X, et al. Intranasal immunization with a replication-deficient

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[137]

[138]

[139]

[140]

[141]

[142]

[143]

[144]

[145]

adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice. Biochem Biophys Res Commun 2009;381(4):528
32. Xu Q, Pichichero ME, Simpson LL, Elias M, Smith LA, Zeng M. An adenoviral vector-based mucosal vaccine is effective in protection against botulism. Gene Ther 2009;16(3):367
75. Are´valo MT, Xu Q, Paton JC, Hollingshead SK, Pichichero ME, Briles DE, et al. Mucosal vaccination with a multicomponent adenovirus-vectored vaccine protects against Streptococcus pneumoniae infection in the lung. FEMS Immunol Med Microbiol 2009;55 (3):346
51. Mu J, Jeyanathan M, Small C-L, Zhang X, Roediger E, Feng X, et al. Immunization with a bivalent adenovirus-vectored tuberculosis vaccine provides markedly improved protection over its monovalent counterpart against pulmonary tuberculosis. Mol Ther J Am Soc Gene Ther 2009;17(6):1093
100. Song K, Bolton DL, Wei C-J, Wilson RL, Camp JV, Bao S, et al. Genetic immunization in the lung induces potent local and systemic immune responses. Proc Natl Acad Sci U S A 2010;107(51):22213
18. Richardson JS, Abou MC, Tran KN, Kumar A, Sahai BM, Kobinger GP. Impact of systemic or mucosal immunity to adenovirus on Ad-based Ebola virus vaccine efficacy in guinea pigs. J Infect Dis 2011;204 (Suppl. 3):S1032
42. Guan J, Wen B, Deng Y, Zhang K, Chen H, Wu X, et al. Effect of route of delivery on heterologous protection against HCV induced by an adenovirus vector carrying HCV structural genes. Virol J 2011;8:506. Brown THT, David J, Acosta-Ramirez E, Moore JM, Lee S, Zhong G, et al. Comparison of immune responses and protective efficacy of intranasal primeboost immunization regimens using adenovirusbased and CpG/HH2 adjuvanted-subunit vaccines against genital Chlamydia muridarum infection. Vaccine 2012;30(2):350
60. Braucher DR, Henningson JN, Loving CL, Vincent AL, Kim E, Steitz J, et al. Intranasal vaccination with replication-defective adenovirus type 5 encoding influenza virus hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs. Clin Vaccine Immunol CVI 2012;19(11):1722
9. Vitelli A, Quirion MR, Lo C-Y, Misplon JA, Grabowska AK, Pierantoni A, et al. Vaccination to conserved influenza antigens in mice using a novel Simian adenovirus vector, PanAd3, derived from the bonobo Pan paniscus. PLoS One 2013;8(3): e55435.

443

[146] Weaver EA, Barry MA. Low seroprevalent species D adenovirus vectors as influenza vaccines. PLoS One 2013;8(8):e73313. [147] Eyles JE, Johnson JE, Megati S, Roopchand V, Cockle PJ, Weeratna R, et al. Nonreplicating vaccines can protect african green monkeys from the memphis 37 strain of respiratory syncytial virus. J Infect Dis 2013;208(2):319
29. [148] Kim EH, Park H-J, Han G-Y, Song M-K, Pereboev A, Hong JS, et al. Intranasal adenovirus-vectored vaccine for induction of long-lasting humoral immunitymediated broad protection against influenza in mice. J Virol 2014;88(17):9693
703. [149] Krishnan V, Andersen BH, Shoemaker C, Sivko GS, Tordoff KP, Stark GV, et al. Efficacy and immunogenicity of single-dose AdVAV intranasal anthrax vaccine compared to anthrax vaccine absorbed in an aerosolized spore rabbit challenge model. Clin Vaccine Immunol CVI 2015;22(4):430
9. [150] Green CA, Scarselli E, Sande CJ, Thompson AJ, de Lara CM, Taylor KS, et al. Chimpanzee adenovirusand MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults. Sci Transl Med 2015;7(300):300ra126. [151] Sha J, Kirtley ML, Klages C, Erova TE, Telepnev M, Ponnusamy D, et al. A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates. Clin Vaccine Immunol CVI 2016;23(7):586
600. [152] Maunder HE, Taylor G, Leppard KN, Easton AJ. Intranasal immunisation with recombinant adenovirus vaccines protects against a lethal challenge with pneumonia virus of mice. Vaccine 2015;33 (48):6641
9. [153] Forrest JC, Campbell JA, Schelling P, Stehle T, Dermody TS. Structure-function analysis of reovirus binding to junctional adhesion molecule 1. Implications for the mechanism of reovirus attachment. J Biol Chem 2003;278(48):48434
44. [154] Mercier GT, Campbell JA, Chappell JD, Stehle T, Dermody TS, Barry MA. A chimeric adenovirus vector encoding reovirus attachment protein sigma1 targets cells expressing junctional adhesion molecule 1. Proc Natl Acad Sci U S A 2004;101(16):6188
93. [155] Weaver EA, Camacho ZT, Hillestad ML, Crosby CM, Turner MA, Guenzel AJ, et al. Mucosal vaccination by adenoviruses displaying reovirus sigma 1. Virology 2015;482:60
6. [156] Patterson LJ, Kuate S, Daltabuit-Test M, Li Q, Xiao P, McKinnon K, et al. Replicating adenovirus-simian immunodeficiency virus (SIV) vectors efficiently prime SIV-specific systemic and mucosal immune

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

444

[157]

[158]

[159]

[160]

24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

responses by targeting myeloid dendritic cells and persisting in rectal macrophages, regardless of immunization route. Clin Vaccine Immunol CVI 2012;19 (5):629
37. Zhu Q, Thomson CW, Rosenthal KL, McDermott MR, Collins SM, Gauldie J. Immunization with adenovirus at the large intestinal mucosa as an effective vaccination strategy against sexually transmitted viral infection. Mucosal Immunol 2008;1(1):78
88. Ji Z, Xie Z, Wang Q, Zhang Z, Gong T, Sun X. A Prime-Boost Strategy Combining Intravaginal and Intramuscular Administration of Homologous Adenovirus to Enhance Immune Response Against HIV-1 in Mice. Hum Gene Ther 2016;27(3):219
29. Zhang X, Dervillez X, Chentoufi AA, Badakhshan T, Bettahi I, Benmohamed L. Targeting the genital tract mucosa with a lipopeptide/recombinant adenovirus prime/boost vaccine induces potent and long-lasting CD8 1 T cell immunity against herpes: importance of MyD88. J Immunol 2012;189(9):4496
509. Singh S, Nehete PN, Yang G, He H, Nehete B, Hanley PW, et al. Enhancement of Mucosal Immunogenicity of Viral Vectored Vaccines by the NKT Cell Agonist

[161]

[162]

[163]

[164]

Alpha-Galactosylceramide as Adjuvant. Vaccines 2014;2(4):686
706. Alejo DM, Moraes MP, Liao X, Dias CC, Tulman ER, Diaz-San Segundo F, et al. An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine. Vaccine 2013;31(18):2302
9. Jiang Y, Li M, Zhang Z, Gong T, Sun X. Enhancement of nasal HIV vaccination with adenoviral vector-based nanocomplexes using mucoadhesive and DC-targeting adjuvants. Pharm Res 2014;31 (10):2748
61. Agirre M, Zarate J, Ojeda E, Puras G, Rojas LA, Alemany R, et al. Delivery of an adenovirus vector plasmid by ultrapure oligochitosan based polyplexes. Int J Pharm 2015;479(2):312
19. Sekine S, Kataoka K, Fukuyama Y, Adachi Y, Davydova J, Yamamoto M, et al. A novel adenovirus expressing Flt3 ligand enhances mucosal immunity by inducing mature nasopharyngeal-associated lymphoreticular tissue dendritic cell migration. J Immunol 2008;180(12):8126
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Mucosal Approaches for Systemic Immunity to Anthrax, Brucellosis, and Plague David W. Pascual Department of Infectious Diseases & Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States

I. INTRODUCTION Vaccination is a highly effective means to achieve protection from infectious diseases. While most vaccines are given parenterally, mucosal vaccines are adept in conferring protection against mucosal disease and systemic sequelae. Such advantage was observed with the oral Sabin polio vaccine, which maintained the ability to replicate in the gut [1] despite the attenuation of neurovirulence [2]. This in vivo infection and replication capacity drives the stimulation of local mucosal immunity sufficiently to neutralize wild-type virus infection [3,4]; hence such immunity prevents systemic poliovirus dissemination to the central nervous system [4]. As occurs in a natural infection, some live vaccines disseminate into the host systemic tissues, resulting in vaccination of

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00025-0

both mucosal and systemic compartments. This particular feature of how mucosal vaccines can combat systemic infections will be discussed in this chapter. Customarily, parenteral vaccines are used to protect against systemic infections; however, mucosal vaccination strategies should be considered, since these have been shown to be as effective and can offer greater protection status in the systemic compartment as well [4]. Utilization of mucosally derived, live vaccines for anthrax, brucellosis, and plague will be emphasized in this chapter. Each of these diseases has a mucosal component that contributes to disease onset or pathogenesis. These bacteria typically produce a systemic disease, and the host’s ability to neutralize or destroy the challenging pathogen is key to protection [5 10]. We will discuss here how adapting mucosal vaccine approaches can

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effectively confer protective immunity against both systemic and mucosal attributes of disease.

II. ORIGINS OF VACCINATION The concept of immunity was described first in Athens as early as the 5th century BCE, when survivors of a plague epidemic were observed to be refractive to reinfection [11]. In 10th century China, dried pustules taken from individuals with mild smallpox were applied to scarified skin or were wafted into the nostrils of uninfected individuals, in perhaps the first form of nasal vaccination [11]. Either method resulted in milder disease symptoms and protection against subsequent smallpox infections [11,12]. This skin scarification method, referred to as variolation, was a common practice in 17th century Africa and was later adopted in 18th century Europe to protect children against smallpox [11,13]. However, this technique became unpopular, owing to the variability of the source of inoculum and inconsistency in protection [14]. During this period, smallpox was lethal for as much as 20% of the population, prompting a desperate search for a method of safe and consistent protection [14]. In 1796, Edward Jenner observed that milkmaids who acquired cowpox exhibited skin lesions similar to those caused by smallpox, and he astutely recognized that these women were resistant to smallpox. As described in his 1798 publication [15], Jenner demonstrated that variolation using cowpox pustules from cows protected individuals against smallpox. Furthermore, inoculum from pustules on cowpox-variolated individuals provided the same effect, showing that cowpox can be transferred from person to person, thus bypassing the need for an infected cow. In his publication, Jenner used the term “variolae vaccinae,” meaning “smallpox of the cow,” which led to the term “vaccination” to describe the act of becoming actively immune to disease [14] (See Chapter 1: Historical Perspectives on Mucosal Vaccines).

III. ANTHRAX A. The Disease and Historical Perspective The Gram-positive, spore-forming Bacillus anthracis, the causative agent for anthrax [5], was identified in 1876 by Robert Koch [16] as filamentous rods capable of cultivation on artificial medium. When injected into rabbits, the bacilli could cause disease and be reisolated from the rabbit’s blood. His work with B. anthracis preceded the development of Koch’s postulates, universally used to identify a new contagion: the infectious agent is present in all diseased animals; it can be isolated and propagated to a pure culture; it can cause disease when reintroduced into a naı¨ve, susceptible host; and it can be reisolated from the new host and show identity to the initial isolate [17]. In the 19th century, anthrax, or “splenic fever” [18], presumably afflicted livestock and, rarely, the livestock handlers [19]. In 95% of human infections, anthrax presents as cutaneous lesions of finite duration [20]. If left untreated, cutaneous anthrax, also known as “wool sorter’s disease,” is lethal in 10% 40% of infected individuals. Mucosal exposures (e.g., oral or inhalational) have an even higher mortality rate [21]. Oral exposure to anthrax in raw or undercooked meat can result in infection of the oropharyngeal and gastrointestinal tracts. Inhalation of bacilli or spores can cause anthrax to disseminate to other tissues via the bloodstream [19,21]. Early intervention with antibiotics is essential to reduce levels of secreted toxins, which mediate tissue destruction [19,21]. Inhalation of B. anthracis spores is commonly fatal. Germination of spores occurs irregularly, making it difficult to determine when the disease will emerge [19]. While the exact human LD50 dose is unclear, data extrapolated from nonhuman primate studies estimate the LD50 to be 5 3 104 spores [22]. In the 2001 US anthrax terror attack, anthrax spores caused death in 5 of 11 individuals exposed to aerosolized

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III. ANTHRAX

spores after opening spore-laden letters, with an additional 7 confirmed cases of cutaneous anthrax [19,23]. The concentration of anthrax contained in the envelopes was estimated to be 1 trillion spores per gram of powder. All of the exposed individuals, numbering 625, were administered a 60-day course of antibiotics [24]. The lethal effects of anthrax are due to the production of two plasmid (pX01)-encoded exotoxins termed lethal toxin and edema toxin [5,19,25]. Replicating B. anthracis secretes three proteins responsible for anthrax’s destructive toxins: protective antigen (PA), lethal factor (LF), and edema factor (EF) [19,25]. PA combines with LF to form lethal toxin, or alternatively, PA combines with EF to form edema toxin. PA is responsible for binding to host cells via one of two anthrax receptors, ANTXR1 and ANTXR2 [5,25]. Following furin-mediated cleavage, PA polymerizes into heptamers or octamers to form a channel within the host cell membrane to facilitate the translocation of PAbound LF or EF into the host cell. Lethal toxin, a metalloprotease that acts upon cardiomyocytes and vascular smooth muscle cells, causes nonhemorrhagic hypotension [25]. Lethal toxin activates the inflammasome, resulting in increases in caspase-1, interleukin 1 (IL-1), and IL-18 production [5]. Lethal toxin also inhibits neutrophil chemotaxis, is cytotoxic for macrophages and dendritic cells, and primarily targets the liver [5]. Edema toxin increases intracellular cyclic adenosine monophosphate levels, inhibits neutrophil function, and causes edema. B. anthracis also exhibits an alternative defense mechanism. Encoded within a second virulence plasmid (pX02), a poly-γ-D-glutamic acid capsule is produced to subvert immune detection, prevent phagocytosis, and enable dissemination [5,6].

B. Development of First Anthrax Vaccines The vegetative B. anthracis bacilli were first described by Koch in 1876 as long, filamentous

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chains [16]. In the same year, Cohn published his observation that exposure to a temperature as high as 100 C was insufficient to kill the infectivity of the bacilli. Importantly, he noted the presence of small, dense bodies [26]. These dense bodies, the spores, were resistant to the heat [20,27]. These findings along with others formed the foundation for acceptance of the “germ theory” that live pathogens cause disease [20]. Such concurrent findings led efforts emphasizing methods to attenuate virulent strains. In 1878, Burdet-Sanderson and Greenfield found that culturing B. anthracis at a lower temperature resulted in diminished virulence with the retention of the ability to induce immunity [13]. In contrast, a year before Pasteur’s famous exhibition of his anthrax vaccine, Toussaint showed that blood from B. anthracis-infected animals that was heated to 55 C contained only dead bacteria and that, when transferred to dogs and sheep, these killed bacteria behaved as an immunogen, inducing protective immunity against repetitive challenges with virulent B. anthracis [13,20]. In 1881, Pasteur sought to prove that an attenuated vaccine for anthrax could protect livestock against virulent challenge [13,20,28]. In Pouillyle-Fort, France, he vaccinated sheep and other livestock with a heat-treated (42 C 43 C) B. anthracis culture, which unknowingly may have led to loss of the virulence plasmids [29]. He gave a booster dose 12 days later with B. anthracis culture treated with potassium bichromate, an antiseptic originally employed by Joseph Lister to treat surgical wounds [20]. Two weeks later, both vaccinated and unvaccinated animals were challenged with virulent B. anthracis, and only the vaccinated animals survived; naı¨ve animals succumbed to the virulent pathogen [13,20,28]. The public success of this trial led to a cattle vaccination campaign that reduced disease incidence in France to only 0.3% by 1894 [28]. This work and Pasteur’s success in attenuating Pasteurella multocida, the agent responsible for fowl cholera, led to the

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development of other live, attenuated vaccines for preventing or treating diseases [11,13,20,28].

C. Live Vaccines for Anthrax Currently, conventional vaccines for anthrax using cell culture filtrates containing PA and varying amounts of LF and/or EF require multiple parenteral administrations [29,30] to achieve protective antibody levels [30]. Early vaccines were live, attenuated [20,30], but subsequent anthrax vaccines relied on spores. The live spore vaccine is the basis for the attenuated Sterne vaccine currently used for immunizing livestock against anthrax [29,31]. Discovered in 1937 [31], the B. anthracis Sterne strain lacks the pX02 virulence plasmid and thus cannot produce the capsule, making it less exotoxic. Another spore vaccine, the ST1 strain, developed in the former Soviet Union for use in livestock [29,32], was later adapted for human use in provinces where anthrax remained endemic [32]. The spore vaccine, when later tested in humans by the aerosol route [33], was found to be relatively more effective than vaccination by subcutaneous injection or scarification, as determined by skin test-positive reactions [32]. Oral vaccination with spores has also been tested [34]. Stimulation of neutralizing anti-PA antibodies that protected against subcutaneous challenge was observed in guinea pigs that were fed spores from a strain that overexpresses a mutant PA that is defective in translocating LF or EF [34]. In an effort to develop a subunit anthrax vaccine, recombinant PA produced by attenuated Salmonella vaccine vectors was given via the oral route and tested for efficacy against spore challenge. Initial attempts found that PA was poorly expressed in Salmonella, resulting in minimal protection, although upon in vivo selection, an isolated clone proved more effective than the parental strain and showed improved protection in one third of the orally

vaccinated mice [35]. Stabilizing PA expression in Salmonella was effective in eliciting elevated anti-PA antibody titers in intravenously vaccinated mice, but poor titers were still obtained following oral vaccination [36]. As a result, the intravenously vaccinated mice showed protection against parenteral lethal spore challenge, while the orally administered vaccine failed to confer protection [36]. Other researchers successfully expressed intact PA in Salmonella and showed an efficacy of 83% against an aerosolized spore challenge following oral vaccination [37]. To facilitate eventual testing in humans, the emphasis shifted to developing an S. Typhibased vaccine. The FDA-approved S. Typhi Ty21a vaccine strain, when modified to express PA and administered nasally or parenterally at 2-week intervals, conferred complete protection against aerosolized anthrax spore challenge in mice [38]. To study the influence of age and the presence of maternal antibodies, 7-day-old neonates were nasally primed with Ty21a expressing PA and boosted intramuscularly with PA on alum [39]. Priming either intramuscularly with PA on alum or nasally with PA-expressing Ty21a, followed by PA on alum boost for both methods, proved effective against a nasal spore challenge. Because of Ty21a’s safety in children, this vaccine could be used in human populations at high risk for anthrax [39] (See Chapter 29: Induction of Local and Systemic Immunity by Salmonella Typhi in Humans).

IV. BRUCELLOSIS A. Etiological Agents and Disease Brucella is a highly homogenous genus of 10 12 Gram-negative species; B. melitensis, B. abortus, and B. suis are the ones responsible for human disease [40 42]. In 1886, these Gramnegative rods were first identified by David Bruce in patients hospitalized with Malta fever [43]. He named these small cocciobacilli Micrococcus melitensis [44]. Immunity to any one

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IV. BRUCELLOSIS

of these species protects against heterologous Brucella species, since the genus has more than 94% DNA homology [42,45,46]. Thus any effective brucellosis vaccine would likely crossprotect against other Brucella species. Brucellosis, the most common zoonotic disease worldwide, is now listed as the third most neglected zoonotic disease [47]. Brucellosis has a global distribution, including the Mediterranean rim, the Middle East, Central Asia, South America, and the US states bordering Mexico [48 50]. In some areas, prevalence exceeds 500 per 1 3 106 inhabitants [48,51], and in endemic regions, cases often go underreported by more than 26-fold [46,51]. The high incidence of Brucella infections is attributed to the sustained prevalence of brucellosis in infected livestock [52], which is the source of unpasteurized milk consumed in various dairy products [7,50]. Oral exposure is the primary route of infection in humans worldwide [53], although aerosol exposure from Brucellainfected livestock can occur [54]. While deadly in livestock by inducing abortion [7,53,55,56], brucellosis is seldom (in fewer than 0.5% of cases) life-threatening in humans, instead producing a debilitating, systemic disease [49 52] caused by either B. melitensis or B. abortus [57,58]. Brucellosis in humans presents with flu-like symptoms, fever, chills, malaise, headaches, hepatomegaly, and splenomegaly [50 52,54]. Interestingly, intestinal manifestations are rare [59,60]. Chronic brucellosis manifests as a relapsing, undulant fever, malaise, chronic fatigue, and positive Brucella blood cultures [52,54,59], which can lead to endocarditis and arthritis [61 65]. Brucella is sensitive to antibiotics, but despite a prolonged twoantibiotic regimen [54,59], sequelae persist in approximately 16% of the infected individuals [59], and 50% of them remain bacteremic [59]. The absence of brucellae or pathology in the intestinal tract [53,66] suggests other mucosal sites, such as the oropharyngeal tissues, as the site in which human infections occur [66 71].

Currently, no FDA-approved human brucellosis exists.

vaccine

for

B. Protection to Brucella Infections Is Th1 Cell-Dependent Macrophages are believed to be the principal cells targeted by Brucella [72]. Elimination of brucellae from the intracellular compartment depends upon whether macrophages have been previously activated, particularly by interferon gamma (IFNγ) [73 75] and tumor necrosis factor alpha (TNF-α) [58,76]. IFNγ is essential in resolving Brucella infection of macrophages [77,78] in an IL-12-dependent [79] and TNF-α-dependent fashion [80], implicating cellular immunity as important for protection to brucellosis.

C. Current Vaccines Four conventional live vaccines for livestock are available: B. abortus strain 19 (S19) for cattle, B. abortus RB51 for cattle, B. melitensis Rev. 1 for sheep and goats, and B. suis strain 2 (S2) for pigs [8]. Because no subunit vaccine has effectively protected animals against Brucellainduced abortions, only live vaccines will be discussed further here. Strain 19 (S19) Vaccine. The cattle S19 vaccine, developed from a spontaneously attenuated mutant discovered in 1923 [81], has a 703-bp deletion of the erythritol catabolic genes [82]. Vaccination with S19 is highly effective in protecting cattle against B. abortus-induced abortion [83] with little impact on normal calf parturitions [84]. S19 can also act therapeutically to reduce infections in Brucella-infected herds [85]. Despite these positive attributes, S19 is estimated to have only 70% efficacy in cattle [86]. The S19 vaccine is normally given to female calves at 3 6 months of age as a single subcutaneous dose of 5 8 3 1010 colonyforming units (CFUs). Adult cattle receive reduced doses ranging from 3 3 108 to 3 3 109

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CFUs or two doses of 5 3 109 CFUs via the conjunctival route [87]. The adult vaccination schedule allows protection without a persistent antibody response, reducing the risks of abortion and excretion into milk [87]. S19 was successfully used in humans during the 1950s in the former Soviet Union to significantly reduce the incidence of B. melitensis infections in livestock workers [88]. However, subsequent studies performed in the United States showed that this vaccine and the attenuated goat vaccine, B. melitensis Rev. 1 strain, are reactogenic in humans [89] even at a reduced dose [90]. RB51 Vaccine. The B. abortus RB51 vaccine was derived from a spontaneous rough mutant that was selected after repeated passages of wild-type B. abortus 2308 selected on rifampinand penicillin-containing media [91]. The mutation resulted in an interruption of the enzyme wboA glycosyltransferase, which contributes to O-Ag biosynthesis [92]. Lacking its O-Ag (LPS), RB51 allows for serological diagnosis of B. abortus-infected animals, since RB51 does not elicit a positive response [93]. Hence RB51 allows vaccinated animals to be distinguished from naturally infected animals. RB51 vaccination of cattle is efficacious in preventing Brucella-induced abortion and fetal infection and is relatively safe compared to S19 [94]. Vaccine efficacy can vary with age. Older animals vaccinated at greater than 5 6 months of age showed no abortions when they were B. abortus challenged as mature pregnant cows (2 3 years of age), whereas heifer calves vaccinated at 3 months of age showed efficacy reduced to 60% when challenged as pregnant adults [85,95]. Although RB51 efficacy is similar to S19 [94], vaccination does not preclude surveillance. RB51 has since replaced S19 vaccine for the brucellosis eradication program in the United States and in other countries that implement a test-and-slaughter policy. RB51 is best suited for countries with effective veterinary services, well-controlled herds, and low disease prevalence [96].

Rev. 1 vaccine. Rev. 1, one of the first vaccines developed for B. melitensis, was originally derived from an avirulent streptomycinresistant strain, but an isolated revertant that regained its sensitivity to streptomycin became the vaccine strain [97]. This live vaccine has an effective dose of 1 3 109 CFUs and is normally given to sheep and goats, either as a subcutaneous injection or via the conjunctiva [85]. Rev. 1 confers protection against B. melitensis-induced abortion. Small ruminants are typically vaccinated when not pregnant to avoid vaccine-induced abortion [8,85]. Rev. 1 is a smooth vaccine, making it difficult to distinguish vaccinated animals from naturally infected animals, but this vaccine has the advantage of crossprotecting against other Brucella species [8] (see Chapter 48).

D. Mucosal Vaccination Approaches for Brucellosis Independent of the route of infection, Brucella infections cause systemic disease [98,99]. While oral exposures are the most common route of infection for both animals and humans, Brucella is unable to sustain an infection of the gut [60,100]. Instead, infections arise in the lymphoid tissues that drain the nasooropharyngeal lymph nodes [101] as a result of animals sniffing or licking Brucella-infected aborted fetuses and/or infected placental tissues [102,103]. This evidence points to a mucosal route of first contact. Although Brucella infections primarily occur following mucosal exposures [50,51,53,58], few studies have considered the impact of oral or nasal vaccination for protection against brucellosis. Oral RB51 vaccination conferred little protection against intraperitoneal challenge [104,105] and only approximately 2 logs of protection against oral B. abortus challenge [104]. Oral vaccination with irradiated RB51 or Brucella neotomae, a pathogen of wood rats and

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possibly humans [106], reduced brucellae dissemination following virulent intraperitoneal challenge with B. abortus [107]. Since none of the studies found improved efficacy by these approaches, different vaccine candidates are needed to achieve protection. To this end, various research groups have generated new attenuated mutants for oral vaccination. The ΔpurEK B. melitensis 16M (WR201) mutant delivered orally provided potent protection against nasal challenge with virulent B. melitensis 16M, as evidenced by deterring colonization of the lungs and reducing dissemination into systemic tissues [108]. Furthermore, the ΔznuA B. melitensis mutant conferred exquisite protection against nasal challenge with virulent B. melitensis 16M [75]. In fact, mice orally vaccinated with this mutant showed nearly complete protection against dissemination in an IFNγdependent fashion [75]. ΔznuA B. melitensis administered orally to IFNγ2/2 mice was unable to confer complete protection, although these mice showed some resistance to colonization by wild-type B. melitensis in an IL-17dependent fashion [75]. Neutralization of IL-17 in immunocompetent mice had no significant impact upon protection [75]. These collective findings support the notion that protection at the site of infection can be achieved with an appropriate mucosal vaccination, given the proper dose and regimen [75,104,105,107,108]. Unlike oral vaccinations, nasal administration, as was done with RB51, yielded poor protection against pulmonary B. abortus challenge [109]. However, coadministration of TLR agonists with nasal RB51 did improve protection [110]. In contrast, a single nasal dose of ΔznuA B. melitensis potently protected mice against pulmonary B. melitensis challenge, wherein more than half of the mice had no detectable brucellae in their lungs or spleens [111]. As with the ones orally vaccinated, protection was IFNγ-dependent, and the reduced protection in nasally vaccinated IFNγ2/2 mice was abrogated upon IL-17 neutralization [111].

Interestingly, nasal vaccination with ΔznuA B. melitensis elicited an effector memory CD81 T cell population in the lungs, wherein cells were positive for IFNγ, TNF-α, and granzyme B. The relevance of these CD81 T cells was affirmed when, in their absence, protection abated [111]. These findings confirm that different brucellosis vaccines and vaccination strategies that can induce a potent cell-mediated immunity need to be considered in order to maximize protection against brucellosis. Vaccinations in cattle are customarily delivered subcutaneously, and only a few studies have examined mucosal vaccination methods. S19 vaccination delivered orally proved to be as effective as the subcutaneous route in protecting pregnant heifers from Brucella-induced abortion [112,113]. In addition, oral RB51 provided cattle with protection equivalent to subcutaneous vaccination of cattle against abortion and brucellae colonization [114]. Developed in the early 1950s, the B. suis strain 2 (S2) vaccine was isolated from an aborted B. suis-infected fetal pig and was passed repeatedly to attenuate it [115,116]. Since 1971, S2 has been used in China as an oral vaccine for swine, administered via drinking water [115]. S2 is highly effective in curbing B. suis and other Brucella infections in swine [115,116] and cross-protects against other Brucella species [115 117].

V. PLAGUE A. The Disease and Historical Perspective Yersinia pestis is a Gram-negative bacterium [118], typically transmitted to humans via a bite from a flea that had previously fed on an infected rodent [119]. Y. pestis is the etiologic agent for plague, referred to as “Black Death” during the time from the 14th through 17th centuries when approximately 30% of Europe’s population succumbed to this disease

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[119,120]. The most common presentation of plague is a bubo, an enlargement of a regional lymph node that drains the site of the flea bite. If left untreated, yersiniae within these buboes can disseminate, causing a bacteremia that could ultimately lead to a fatal septicemia [119,121]. Pneumonic plague results from inhalational infection of the lungs with Y. pestis. While bubonic plague is transmitted via an insect bite, pneumonic plague is typically transmitted via aerosols from infected humans or animals [119,121]. Pneumonic plague is the most virulent form, with death occurring within 24 hours of infection [121]. Yersiniae disseminate in the victim via infected macrophages, where they proliferate and produce virulence factors in response to drops in Ca11 concentrations and pH within macrophages. In turn, this facilitates the survival of Y. pestis [118,119]. Upon apoptosis, the virulent bacteria are released, causing bacteremia, and without antibiotic intervention, subjects usually succumb to septicemia. Sporadic outbreaks of pneumonic plague have occurred in the United States and other countries [121 124]. Plague remains endemic in prairie dogs in the western United States [125]. In 1894, Alexandre Yersin first described Y. pestis bacilli as faintly Gram-negative bacteria, cultured them from buboes present on plague cadavers during an outbreak in Hong Kong, and named them Bacillus pestis [120,126,127]. B. pestis was later named Pasteurella pestis and finally Yersinia pestis [120]. Guinea pigs that Yersin inoculated with some bubo fluid all died within a few days [126]. The bacilli isolated from the guinea pigs resembled the initial inoculum, being poorly Gram-negative with “rounded ends,” confirming B. pestis [126 128]. Yersin found that rats were the source of the infective bacilli, since the rats’ lymph nodes also contained the bacteria [120,128]. Later, Paul-Louis Simond discovered fleas on infected rats to be the source of transmission [128,129]. Yersin also showed that horse antiserum to plague could be

used to treat plague-infected patients [120,130] demonstrating that antibodies are protective.

B. Antibody-Dependence for Immune Protection to Plague Various vaccines have been developed for plague. A whole-cell-inactivated vaccine, Cutter vaccine, is simply a formaldehyde-killed Y. pestis 195/P strain. It was previously licensed in the United States and is believed to stimulate protection against the F1 capsular protein [131]. Another killed whole cell vaccine, called KWC, was a heat-killed suspension of Y. pestis that required multiple boosters to maintain protection. The KWC vaccine was proven effective for prevention of bubonic plague in US soldiers in the Vietnam War but was ineffective against pneumonic plague [132,133]. Because of the limitations of killed vaccines, a subunit plague vaccine was pursued. Both the F1 capsule and V antigens were found to confer high levels of protection [134]. F1 antigen is a capsular protein of 15.5 17.5 kDa encoded by the caf1 operon on the virulence pFra plasmid or the pMT1 plasmid for the Kurdistan Iran man (KIM) strains [119]. The F1 antigen is antiphagocytic [119,134], and expressed only at 37 C [131]. Sera from F1 antigen-immunized human volunteers can passively protect mice against plague [135], as can anti-F1 antigen mAbs [136]. In addition to F1 capsule, immunization with recombinant Virulence (V) antigen is also protective [134]. V antigen, encoded by the LCR virulence plasmid, is a 35- to 37-kDa protein [118,119], which is secreted by Y. pestis under low Ca11 conditions. V antigen is believed to be important for regulating delivery of Yops into host cells [118]. V antigen’s mode of action may be suppression of both IFNγ and TNF-α production, thereby limiting inflammation [137] via IL-10 [138] and suppression of neutrophil chemotaxis [139]. Immunization with V-Ag alone [140] or in combination with F1

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antigen also protects against plague challenge [141]. Soluble F1 and V antigens make up the active components of RypVax [142]. Likewise, immunization with an F1-V antigen fusion protein confers complete protection against F11 or F12 pneumonic plague [143], and the F1-V has been developed into a vaccine [142]. To abate V antigen’s antiinflammatory properties, a shortened version capable of maintaining protection against pneumonic plague in mice and macaques was developed [144].

C. Th1- and Th17-Mediated Immunity Against Plague Interest in live, attenuated Y. pestis-based vaccines has diminished, largely owing to the unpredictability of the attenuation and the tendency of the earlier attenuated vaccines, such as EV76, to produce a nonfatal disease in humans while, in some instances, causing a fatal disease in animals and nonhuman primates [132,145]. Development of a suitable live vaccine has been hindered by Y. pestis’ genomic instability that results in the loss of various genes [142]. Another caveat, not extensively considered, has been the contributions of T cell immunity for protection against Y. pestis. Mice that were primed, then boosted with the virulent Y. pestis KIM5 showed protection against pneumonic plague, but at the cost of considerable attrition from primary infection [146]. To generate a genetically defined mutant, efforts focused on the replacement of the Y. pestis tetra-acylated lipid A that lacks TLR4 stimulatory activity. To enable TLR4 engagement, the KIM61 (pCD1Ap) was modified by overexpressing the Escherichia coli lpxL to produce hexaacylated lipid A, which in turn made the strain immunogenic and more attenuated [147]. An additional attenuation was done by regulating its crp expression, resulting in a strain conferring 80% protection against pneumonic KIM61 (pCD1Ap) challenge [147].

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Concern about genetic reversion diminished enthusiasm for genetically manipulated virulent strains for vaccine development. While the combination of F1 and V antigens showed promise as a potential vaccine strategy, concerns remained regarding how to confer protection against F1-negative Y. pestis strains and to variants of V antigen [145,148,149]. To allay these concerns, alternative Yersinia species were considered. Yersinia pseudotuberculosis and its genetic cousin, Y. pestis, are believed to have diverged about 6500 years ago [150]. They share more than 95% genomic identity [151], rendering similar off-target antigens that may contribute to cross-protection [142]. Y. pseudotuberculosis has the advantage of producing only a nonfatal, mild gastroenteritis. One Y. pseudotuberculosis strain was identified that lacked the superantigens YPM, the pathogenicity island, and a type IV pilus [152]. Immunization with two doses of this less virulent strain protected 88% of mice against a lethal bubonic Y. pestis challenge [152]. Another approach developed a defined, avirulent mutant, ΔHPI ΔpsaA ΔyopK Y. pseudotuberculosis V674 strain, which was subsequently modified with the caf operon for expressing the Y. pestis F1 antigen [153]. Oral vaccination with this F1 antigen-modified V674 strain was highly efficacious against pneumonic F11 and F12 Y. pestis challenges, owing to the stimulation of IFNγ and, to a lesser degree, IL-17 [153]. Chromosomal insertion of the caf operon into the V674 strain resulted in a highly efficient vaccine strain, which conferred complete protection against both pneumonic and bubonic plague challenges in mice that were orally vaccinated once [154]. Elevated levels of IFNγ and IL-17 were also induced in these mice following vaccination [154]. W. Sun et al. used two approaches to attenuate Y. pseudotuberculosis as a live vaccine for plague [155,156]. The first approach replaced Y. pseudotuberculosis msbB with an E. coli acyltransferase homolog to produce a TLR4 agonistic hexaacylated lipid A. This resulted in making the

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recombinant Y. pseudotuberculosis strain immunogenic, and for additional attenuation, the mutant was modified for regulated expression of its crp [155]. To enhance immunogenicity, a fusion of YopE1 138 and V antigen was secreted, using a type III secretion apparatus, and these collective modifications stimulated immunity via elevated IFNγ and IL-17, which resulted in efficacy [155] similar to that of their attenuated Y. pestis strain [147]. Their second approach developed a ΔyopJ ΔyopK Y. pseudotuberculosis strain with chromosomal expression of the caf operon and achieved 90% protection against pneumonic KIM61 (pCD1Ap) challenge [156].

D. Attempt to Develop Plague Mucosal Vaccine Heterologous vaccine vectors have also been adapted for oral and nasal plague vaccinations. The first attempts to express the caf operon in an attenuated Salmonella enterica serovar Typhimurium led to suboptimal expression and/or immunity [157,158]. For V antigen, multiple doses were required for protection [159]. An attenuated S. Typhimurium vector containing two separate plasmids, one for the caf operon with its endogenous promoter, and another, a chimeric promoter for V antigen expression, demonstrated improved and stable expression of F1 and V antigens [160]. Mice orally vaccinated with this S. Typhimurium strain showed 88% protection against lethal, 1000 LD50 bubonic and 100 LD50 pneumonic plague challenges [160]. In a separate study, mice vaccinated with an S. Typhimurium bearing chromosomal F1 antigen and plasmid-based expression of V antigen and Psn (the outer membrane receptor for the siderophore yersiniabactin) conferred 100% protection against a bubonic challenge (B570 LD50) and 60% protection against a pneumonic plague challenge (50 LD50) [161]. A more relevant vector for human vaccines, an attenuated S. Typhi expressing F1 antigen, resulted in modest protection against a bubonic challenge [162]. Other

researchers tested plasmid-based expression of F1 antigen and chromosomal expression of V antigen in different attenuated S. Typhi strains [163]. Mice were nasally immunized twice with attenuated S. Typhi strains bearing both F1 and V antigens and boosted once with V antigen on alum. Five weeks after the boost, all mice that were vaccinated with constructs bearing both F1 and V antigens were completely protected against 37 and 177 LD50 pneumonic challenges [163]. Hence these studies demonstrated the benefits and limitations of using related attenuated Y. pseudotuberculosis strains as well as heterologous vaccine vectors as mucosal plague vaccines (See Chapter 22).

Acknowledgment This work was supported in part by US Public Health grants R01 AI-123244 and R01 AI-125516.

References [1] Sabin AB. Oral poliovirus vaccine. History of its development and prospects for eradication of poliomyelitis. JAMA 1965;194:872 6. [2] Almond JW. The attenuation of poliovirus neurovirulence. Annu Rev Microbiol 1987;41:153 80. [3] Hird TR, Grassly NC. Systematic review of mucosal immunity induced by oral and inactivated poliovirus vaccines against virus shedding following oral poliovirus challenge. PLoS Pathog 2012;8:e1002599. [4] Pasetti MF, Simon JK, Sztein MB, Levine MM. Immunology of gut mucosal vaccines. Immunol Rev 2011;239:125 48. [5] Moayeri M, Leppla SH, Vrentas C, Pomerantsev AP, Liu S. Anthrax pathogenesis. Annu Rev Microbiol 2015;69:185 208. [6] Williamson ED, Dyson EH. Anthrax prophylaxis: recent advances and future directions. Front Microbiol 2015;6:1009. [7] Byndloss MX, Tsolis RM. Brucella spp. Virulence factors and immunity. Annu Rev Anim Biosci. 2016;4:111 27. [8] Yang X, Skyberg JA, Cao L, Thornburg T, Clapp B, Pascual DW. Progress in Brucella vaccine development. Front Biol. (Beijing) 2013;8:60 77. [9] Viboud GI, Bliska JB. Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis. Annu Rev Microbiol 2005;59:69 89.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[10] Sun W, Curtiss R. Rational considerations about development of live attenuated Yersinia pestis vaccines. Curr Pharm Biotechnol 2013;14:878 86. [11] De Gregorio E, Rappuoli R. From empiricism to rational design: a personal perspective of the evolution of vaccine development. Nat Rev Immunol 2014;14: 505 14. [12] Needham J. China and the origins of immunology. East Horiz 1980;19:6 12. [13] Lombard M, Pastoret PP, Moulin AM. A brief history of vaccines and vaccination. Rev Sci Tech 2007;26: 29 48. [14] Baxby D. Edward Jenner’s Inquiry; a bicentenary analysis. Vaccine 1999;17:301 7. [15] Jenner E. An inquiry into the causes and effects of the variolae vaccinae. London: Sampson Low; 1798. [16] Koch R. Die aetiologie der milzbrand-krankheit, begrundet auf die entwicklungsgeschichte des Bacillus antracis. Beitr Biol Pflanz 1876;2:277 310. [17] Mu¨nch R. Robert Koch. Microbes Infect 2003;5:69 74. [18] Pasteur L. Remarks on anthracic vaccination as a prophylactic of splenic fever. Br Med J 1882;1:489. [19] Inglesby TV, O’Toole T, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Anthrax as a biological weapon, 2002: updated recommendations for management. JAMA 2002;287:2236 52. [20] Smith KA. Louis Pasteur, the father of immunology? Front Immunol 2012;3:68. [21] Welkos S, Bozue J, Twenhafel N, Cote C. Animal models for the pathogenesis, treatment, and prevention of infection by Bacillus anthracis. Microbiol Spectr 2015;3 TBS-0001-2012. [22] Gutting BW, Rukhin A, Mackie RS, Marchette D, Thran B. Evaluation of inhaled versus deposited dose using the exponential dose-response model for inhalational anthrax in nonhuman primate, rabbit, and guinea pig. Risk Anal 2015;35:811 27. [23] Fennelly KP, Davidow AL, Miller SL, Connell N, Ellner JJ. Airborne infection with Bacillus anthracis from mills to mail. Emerg Infect Dis 2004;10:996 1002. [24] Hsu VP, Lukacs SL, Handzel T, Hayslett J, Harper S, Hales T, et al. Opening a Bacillus anthraciscontaining envelope, Capitol Hill, Washington, D.C.: the public health response. Emerg Infect Dis 2002;8:1039 43. [25] Friebe S, van der Goot FG, Bu¨rgi J. The ins and outs of anthrax toxin. Toxins (Basel) 2016;8:69. [26] Cohn F. Untersuchungen uber bakterien. Beitr Biol Pflanz 1876;2:249 76. [27] Gould GW. History of science spores. J Appl Microbiol 2006;101:507 13. [28] Berche P. Louis Pasteur, from crystals of life to vaccination. Clin Microbiol Infect 2012;18(Suppl. 5):1 6.

455

[29] Hambleton P, Carman JA, Melling J. Anthrax: the disease in relation to vaccines. Vaccine 1984;2:125 32. [30] Chitlaru T, Altboum Z, Reuveny S, Shafferman A. Progress and novel strategies in vaccine development and treatment of anthrax. Immunol Rev 2011;239: 221 36. [31] Sterne M. The effects of different carbon dioxide concentrations on the growth of virulent anthrax strains. Pathogenicity and immunity tests on guinea-pigs and sheep with anthrax variants derived from virulent strains. Onderstepoort J Vet Sci 1937;9:49 67. [32] Shlyakhov EN, Rubinstein E. Human live anthrax vaccine in the former USSR. Vaccine 1994;12:727 30. [33] Aleksandrov NI, Gefen NE, Garin NS, Gapochko lEG, Sergev VM, Smirnov MS, et al. Experience in massive aerogenic vaccination against anthrax. Voen Med Zh 1959;8:27 32 (in Russian). [34] Aloni-Grinstein R, Gat O, Altboum Z, Velan B, Cohen S, Shafferman A. Oral spore vaccine based on live attenuated nontoxinogenic Bacillus anthracis expressing recombinant mutant protective antigen. Infect Immun 2005;73:4043 53. [35] Coulson NM, Fulop M, Titball RW. Bacillus anthracis protective antigen, expressed in Salmonella typhimurium SL 3261, affords protection against anthrax spore challenge. Vaccine 1994;12:1395 401. [36] Garmory HS, Titball RW, Griffin KF, Hahn U, Bo¨hm R, Beyer W. Salmonella enterica serovar Typhimurium expressing a chromosomally integrated copy of the Bacillus anthracis protective antigen gene protects mice against an anthrax spore challenge. Infect Immun 2003;71:3831 6. [37] Stokes MG, Titball RW, Neeson BN, Galen JE, Walker NJ, Stagg AJ, et al. Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis. Infect Immun 2007;75:1827 34. [38] Osorio M, Wu Y, Singh S, Merkel TJ, Bhattacharyya S, Blake MS, et al. Anthrax protective antigen delivered by Salmonella enterica serovar Typhi Ty21a protects mice from a lethal anthrax spore challenge. Infect Immun 2009;77:1475 82. [39] Ramirez K, Ditamo Y, Galen JE, Baillie LW, Pasetti MF. Mucosal priming of newborn mice with S. Typhi Ty21a expressing anthrax protective antigen (PA) followed by parenteral PA-boost induces B and T cellmediated immunity that protects against infection bypassing maternal antibodies. Vaccine 2010;28: 6065 75. [40] Guzma´n-Verri C, Gonza´lez-Barrientos R, Herna´ndezMora G, Morales JA, Baquero-Calvo E, Chaves-Olarte E, et al. Brucella ceti and brucellosis in cetaceans. Front Cell Infect Microbiol 2012;2:3.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

456

25. MUCOSAL APPROACHES FOR SYSTEMIC IMMUNITY TO ANTHRAX, BRUCELLOSIS, AND PLAGUE

[41] Olsen SC, Palmer MV. Advancement of knowledge of Brucella over the past 50 years. Vet Pathol 2014;51: 1076 89. [42] Whatmore AM. Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infect Genet Evol 2009;9:1168 84. [43] Bruce D. Observations on Malta Fever. Br Med J 1889;1:1101 5. [44] Bruce D. Note on the discovery of a micro organism in Malta Fever. Practitioner 1887;39:161 70. [45] Wattam AR, Williams KP, Snyder EE, Almeida Jr NF, Shukla M, Dickerman AW, et al. Analysis of ten Brucella genomes reveals evidence for horizontal gene transfer despite a preferred intracellular lifestyle. J Bacteriol 2009;191:3569 79. [46] Van der Henst C, de Barsy M, Zorreguieta A, Letesson JJ, De Bolle X. The Brucella pathogens are polarized bacteria. Microbes Infect 2013;15:998 1004. [47] Mableson HE, Okello A, Picozzi K, Welburn SC. Neglected zoonotic diseases-the long and winding road to advocacy. PLoS Negl Trop Dis 2014;8: e2800. [48] Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV. The new global map of human brucellosis. Lancet Infect Dis 2006;6:91 9. [49] Corbel MJ. Brucellosis in humans and animals. Geneva: WHO; 2006. p. 1 102. [50] Pappas G, Akritidis N, Bosilkovski M, Tsianos E. Brucellosis. N Engl J Med 2005;352:2325 36. [51] Franco MP, Mulder M, Gilman RH, Smits HL. Human brucellosis. Lancet Infect Dis 2007;7:775 86. [52] Young EJ. Human brucellosis. Rev Infect Dis 1983;5: 821 42. [53] Godfroid J, Scholz HC, Barbier T, Nicolas C, Wattiau P, Fretin D, et al. Brucellosis at the animal/ecosystem/ human interface at the beginning of the 21st century. Prev Vet Med 2011;102:118 31. [54] Corbel M. Brucellosis: an overview. Emerg Infect Dis 1997;3:213 21. [55] Bang B. Infectious abortion in cattle. J Comp Pathol Ther 1906;19:191 202. [56] Poester FP, Samartino LE, Santos RL. Pathogenesis and pathobiology of brucellosis in livestock. Rev Sci Tech 2013;32:105 15. [57] Godfroid J, DeBolle X, Roop RM, O’Callaghan D, Tsolis RM, Baldwin C, et al. The quest for a true One Health perspective of brucellosis. Rev Sci Tech Off Int Epiz 2014;33:521 38. [58] Tsolis RM, Young GM, Solnick JV, Ba¨umler AJ. From bench to bedside: stealth of enteroinvasive pathogens. Nat Rev Microbiol 2008;6:883 92. [59] Ariza J, Corredoira J, Pallares R, Viladrich PF, Rufi G, Pujol M, et al. Characteristics of and risk factors for




[60]

[61]

[62] [63]

[64]

[65]

[66]

[67] [68] [69]

[70]

[71]

[72] [73]

[74]

relapse of brucellosis in humans. Clin Infect Dis 1995;20:1241 9. Ablin J, Mevorach D, Eliakim R. Brucellosis and the gastrointestinal tract. The odd couple. J Clin Gastroenterol 1997;24:25 9. Reguera JM, Alarco´n A, Miralles F, Pacho´n J, Jua´rez C, Colmenero JD. Brucella endocarditis: clinical, diagnostic, and therapeutic approach. Eur J Clin Microbiol Infect Dis 2003;22:647 50. Rajapakse CN. Bacterial infections: osteoarticular brucellosis. Baillieres Clin Rheumatol 1995;9:161 77. Shen MW. Diagnostic and therapeutic challenges of childhood brucellosis in a nonendemic country. Pediatrics 2008;121:e1178 83. Skyberg JA, Thornburg T, Kochetkova I, Layton W, Callis G, Rollins MF, et al. IFN-γ-deficient mice develop IL-1-dependent cutaneous and musculoskeletal inflammation during experimental brucellosis. J Leukoc Biol 2012;92:375 87. Lacey CA, Keleher LL, Mitchell WJ, Brown CR, Skyberg JA. CXCR2 mediates Brucella-induced arthritis in interferon γ-deficient mice. J Infect Dis 2016;214:151 60. Ron-Roma´n J, Ron-Garrido L, Abatih E, Celi-Erazo M, Vizcaı´no-Ordo´n˜ez L, Calva-Pacheco J, et al. Human brucellosis in northwest Ecuador: typifying Brucella spp., seroprevalence, and associated risk factors. Vector Borne Zoonotic Dis 2014;14:124 33. Carpenter CM, Boak RA. The isolation of Brucella abortus from tonsils. JAMA 1932;99:296 8. Poelma LJ, Pickens EM. Brucella abortus in human tonsils. J Bact 1932;23:112 13. Suraud V, Olivier M, Bodier CC, Guilloteau LA. Differential expression of homing receptors and vascular addressins in tonsils and draining lymph nodes: effect of Brucella infection in sheep. Vet Immunol Immunopathol 2007;115:239 50. Zachou K, Papamichalis PA, Dalekos GN. Severe pharyngitis in stockbreeders: an unusual presentation of brucellosis. Occup Med (Lond) 2008;58:305 7. von Bargen K, Gagnaire A, Arce-Gorvel V, de Bovis B, Baudimont F, Chasson L, et al. Cervical lymph nodes as a selective niche for Brucella during oral infections. PLoS One 2015;10:e121790. Celli J. The changing nature of the Brucella-containing vacuole. Cell Microbiol 2015;17:951 8. Jiang X, Baldwin CL. Effects of cytokines on intracellular growth of Brucella abortus. Infect Immun 1993;61: 124 34. Rodriguez-Zapata M, Salmeron I, Manzano L, Salmeron OJ, Prieto A, Alvarez-Mon M. Defective interferon-gamma production by T-lymphocytes from patients with acute brucellosis. Eur J Clin Invest 1996;26:136 40.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[75] Clapp B, Skyberg JA, Yang X, Thornburg T, Walters N, Pascual DW. Protective live oral brucellosis vaccines stimulate Th1 and Th17 cell responses. Infect Immun 2011;79:4165 74. [76] Macedo GC, Magnani DM, Carvalho NB, BrunaRomero O, Gazzinelli RT, Oliveira SC. Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection. J Immunol 2008;180: 1080 7. [77] Eze MO, Yuan L, Crawford RM, Paranavitana CM, Hadfield TL, Bhattacharjee AK, et al. Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro. Infect Immun 2000;68:257 63. [78] Skyberg JA, Thornburg T, Rollins M, Huarte E, Jutila MA, Pascual DW. Murine and bovine γδ T cells enhance innate immunity against Brucella abortus infections. PLoS One 2011;6:e21978. [79] Zhan Y, Cheers C. Endogenous interleukin-12 is involved in resistance to Brucella abortus infection. Infect Immun 1995;63:1387 90. [80] Zhan Y, Cheers C. Control of IL-12 and IFN-γ production in response to live or dead bacteria by TNF and other factors. J Immunol 1998;161:1447 53. [81] Buck JM. Studies of vaccination during calfhood to prevent bovine infectious abortion. J Agric Res 1930;41:667 89. [82] Sangari FJ, Garcı´a-Lobo JM, Agu¨ero J. The Brucella abortus vaccine strain B19 carries a deletion in the erythritol catabolic genes. FEMS Microbiol Lett 1994;121:337 42. [83] Confer AW, Hall SM, Faulkner CB, Espe BH, Deyoe BL, Morton RJ, et al. Effects of challenge dose on the clinical and immune responses of cattle vaccinated with reduced doses of Brucella abortus strain 19. Vet Microbiol 1985;10:561 75. [84] Wright AE. Report of the co-operative bovine brucellosis work in the United States. Proc US Livestock San Assoc 1942;47:149 54. [85] Olsen SC, Stoffregen WS. Essential role of vaccines in brucellosis control and eradication programs for livestock. Expert Rev Vaccines 2005;4:915 28. [86] Lubroth J, Rweyemamu MM, Viljoen G, Diallo A, Dungu B, Amanfu W. Veterinary vaccines and their use in developing countries. Rev Sci Tech 2007;26: 179 201. [87] World Animal Health Organization (OIE). Manual of diagnostic tests and vaccines for terrestrial animals. World Animal Health Organization; 2014. [88] Vershilova PA. The use of live vaccine for vaccination of human beings against brucellosis in the USSR. Bull World Health Organ 1961;24:85 9.

457

[89] Spink WW, Hall 3rd JW, Finstad J, Mallet E. Immunization with viable Brucella organisms. Results of a safety test in humans. Bull World Health Organ 1962;26:409 19. [90] Pappagianis D, Elberg SS, Crouch D. Immunization against Brucella infections. Effects of graded doses of viable attenuated Brucella melitensis in humans. Am J Epidemiol 1966;84:21 31. [91] Schurig GG, Roop 2nd RM, Bagchi T, Boyle S, Buhrman D, Sriranganathan N. Biological properties of RB51; a stable rough strain of Brucella abortus. Vet Microbiol 1991;28:171 88. [92] Vemulapalli R, McQuiston JR, Schurig GG, Sriranganathan N, Halling SM, Boyle SM. Identification of an IS711 element interrupting the wboA gene of Brucella abortus vaccine strain RB51 and a PCR assay to distinguish strain RB51 from other Brucella species and strains. Clin Diagn Lab Immunol 1999;6:760 4. [93] Stevens MG, Hennager SG, Olsen SC, Cheville NF. Serologic responses in diagnostic tests for brucellosis in cattle vaccinated with Brucella abortus 19 or RB51. J Clin Microbiol 1994;32:1065 6. [94] Olsen SC. Immune responses and efficacy after administration of a commercial Brucella abortus strain RB51 vaccine to cattle. Vet Ther 2000;1:183 91. [95] Cheville NF, Olsen SC, Jensen AE, Stevens MG, Palmer MV, Florance AM. Effects of age at vaccination on efficacy of Brucella abortus strain RB51 to protect cattle against brucellosis. Am J Vet Res 1996;57:1153 6. [96] Moriyo´n I, Grillo´ MJ, Monreal D, Gonza´lez D, Marı´n C, Lo´pez-Gon˜i I, et al. Rough vaccines in animal brucellosis: structural and genetic basis and present status. Vet Res 2004;35:1 38. [97] Herzberg M, Elberg SS. Immunization against Brucella infection. III. Response of mice and guinea pigs to injection of viable and nonviable suspensions of a streptomycin-dependent mutant of Brucella melitensis. J Bacteriol 1955;69:432 5. [98] Atluri VL, Xavier MN, de Jong MF, den Hartigh AB, Tsolis RM. Interactions of the human pathogenic Brucella species with their hosts. Annu Rev Microbiol 2011;65:523 41. [99] de Figueiredo P, Ficht TA, Rice-Ficht A, Rossetti CA, Adams LG. Pathogenesis and immunobiology of brucellosis: review of Brucella-host interactions. Am J Pathol 2015;185:1505 17. [100] Delpino MV, Marchesini MI, Estein SM, Comerci DJ, Cassataro J, Fossati CA, et al. A bile salt hydrolase of Brucella abortus contributes to the establishment of a successful infection through the oral route in mice. Infect Immun 2007;75:299 305.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

458

25. MUCOSAL APPROACHES FOR SYSTEMIC IMMUNITY TO ANTHRAX, BRUCELLOSIS, AND PLAGUE

[101] Meador VP, Warner DP, Deyoe BL. Distribution of Brucella abortus organisms in calves after conjunctival exposure. Am J Vet Res 1988;49:2015 17. [102] Samartino LE, Enright FM. Pathogenesis of abortion of bovine brucellosis. Comp Immunol Microbiol Infect Dis 1993;16:95 101. [103] Schumaker B. Risks of Brucella abortus spillover in the Greater Yellowstone area. Rev Sci Tech 2013;32:71 7. [104] Pasquali P, Rosanna A, Pistoia C, Petrucci P, Ciuchini F. Brucella abortus RB51 induces protection in mice orally infected with the virulent strain B. abortus 2308. Infect Immun 2003;71:2326 30. [105] Pasquevich KA, Garcı´a Samartino C, Coria LM, Estein SM, Zwerdling A, Iban˜ez AE, et al. The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising selfadjuvanting vaccine against systemic and oral acquired brucellosis. J Immunol 2010;184:5200 12. [106] Sua´rez-Esquivel M, Ruiz-Villalobos N, Jime´nez-Rojas C, Barquero-Calvo E, Chaco´n-Dı´az C, Vı´quez-Ruiz E, et al. Brucella neotomae infection in humans, Costa Rica. Emerg Infect Dis 2017;23:997 1000. [107] Dabral N, Moreno-Lafont M, Sriranganathan N, Vemulapalli R. Oral immunization of mice with gamma-irradiated Brucella neotomae induces protection against intraperitoneal and intranasal challenge with virulent B. abortus 2308. PLoS One 2014;9:e107180. [108] Izadjoo MJ, Bhattacharjee AK, Paranavitana CM, Hadfield TL, Hoover DL. Oral vaccination with Brucella melitensis WR201 protects mice against intranasal challenge with virulent Brucella melitensis 16M. Infect Immun 2004;72:4031 9. [109] Surendran N, Sriranganathan N, Lawler H, Boyle SM, Hiltbold EM, Heid B, et al. Efficacy of vaccination strategies against intranasal challenge with Brucella abortus in BALB/c mice. Vaccine 2011;29:2749 55. [110] Surendran N, Sriranganathan N, Boyle SM, Hiltbold EM, Tenpenny N, Walker M, et al. Protection to respiratory challenge of Brucella abortus strain 2308 in the lung. Vaccine 2013;31:4103 10. [111] Clapp B, Yang X, Thornburg T, Walters N, Pascual DW. Nasal vaccination stimulates CD81 T cells for potent protection against mucosal Brucella melitensis challenge. Immunol Cell Biol 2016;94:496 508. [112] Nicoletti P, Milward FW. Protection by oral administration of Brucella abortus strain 19 against an oral challenge exposure with a pathogenic strain of Brucella. Am J Vet Res 1983;44:1641 3. [113] Nicoletti P. Vaccination of cattle with Brucella abortus strain 19 administered by differing routes and doses. Vaccine 1984;2:133 5.

[114] Elzer PH, Enright FM, Colby L, Hagius SD, Walker JV, Fatemi MB, et al. Protection against infection and abortion induced by virulent challenge exposure after oral vaccination of cattle with Brucella abortus strain RB51. Am J Vet Res 1998;59:1575 8. [115] Xin X. Orally administrable brucellosis vaccine: Brucella suis strain 2 vaccine. Vaccine 1986;4:212 16. [116] Zhu L, Feng Y, Zhang G, Jiang H, Zhang Z, Wang N, et al. Brucella suis strain 2 vaccine is safe and protective against heterologous Brucella spp. infections. Vaccine 2016;34:395 400. [117] Bosseray N, Plommet M. Brucella suis S2, Brucella melitensis Rev. 1 and Brucella abortus S19 living vaccines: residual virulence and immunity induced against three Brucella species challenge strains in mice. Vaccine 1990;8:462 8. [118] McNally A, Thomson NR, Reuter S, Wren BW. ‘Add, stir and reduce’: Yersinia spp. as model bacteria for pathogen evolution. Nat Rev Microbiol 2016;14: 177 90. [119] Perry RD, Fetherston JD. Yersinia pestis Etiologic agent of plague. Clin Microbiol Rev 1997;10:35 66. [120] Butler T. Plague history: Yersin’s discovery of the causative bacterium in 1894 enabled, in the subsequent century, scientific progress in understanding the disease and the development of treatments and vaccines. Clin Microbiol Infect 2014;20:202 9. [121] Koirala J. Plague: disease, management, and recognition of act of terrorism. Infect Dis Clin North Am 2006;20:273 87. [122] Runfola JK, House J, Miller L, Colton L, Hite D, Hawley A, et al. Outbreak of human pneumonic plague with dog-to-human and possible human-tohuman transmission Colorado, June-July 2014. MMWR Morb Mortal Wkly Rep 2015;64:429 34. [123] Ramasindrazana B, Andrianaivoarimanana V, Rakotondramanga JM, Birdsell DN, Ratsitorahina M, Rajerison M. Pneumonic plague transmission, Moramanga, Madagascar, 2015. Emerg Infect Dis 2017;23:521 4. [124] Donaires LF, Ce´spedes M, Valencia P, Salas JC, Luna ME, Castan˜eda A, et al. Primary pneumonic plague with nosocomial transmission in La Libertad, Peru 2010. Rev Peru Med Exp Salud Publica 2010;27:326 36. [125] Richgels KL, Russell RE, Bron GM, Rocke TE. Evaluation of Yersinia pestis transmission pathways for sylvatic plague in prairie dog populations in the western U.S. Ecohealth 2016;13:415 27. [126] Yersin A. Le peste bubonique a` Hong Kong. Ann Inst Pasteur 1894;8:662 7. [127] Hawgood BJ. Alexandre Yersin (1863-1943): discoverer of the plague bacillus, explorer and agronomist. J Med Biogr 2008;16:167 72.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[128] Gross L. How the plague bacillus and its transmission through fleas were discovered: reminiscences from my years at the Pasteur Institute in Paris. Proc Natl Acad Sci USA 1995;92:7609 11. [129] Simond P-L. La propagation de la peste. Ann Inst Pasteur 1898;12:625 87. [130] Yersin A. Sur la peste bubonique (serotherapie). Ann Inst Pasteur 1897;11:81 93. [131] Friedlander AM, Welkos SL, Worsham PL, Andrews GP, Heath DG, Anderson Jr GW, et al. Relationship between virulence and immunity as revealed in recent studies of the F1 capsule of Yersinia pestis. Clin Infect Dis 1995;21(Suppl. 2):S178 81. [132] Meyer KF. Effectiveness of live or killed plague vaccines in man. Bull World Health Organ 1970;42: 653 66. [133] Speck RS, Wolchow H. Studies on the experimental epidemiology of respiratory infections. VIII. Experimental pneumonic plague in Maeaeus rhesus. J Infect Dis 1957;100:58 69. [134] Williamson ED, Oyston PC. Protecting against plague: towards a next-generation vaccine. Clin Exp Immunol 2013;172:1 8. [135] Meyer KF, Foster LE. Measurement of protective serum antibodies in human volunteers inoculated with plague prophylatics. Stanford Med Bull 1948;6: 75 9. [136] Anderson Jr GW, Worsham PL, Bolt CR, Andrews GP, Welkos SL, Friedlander AM, et al. Protection of mice from fatal bubonic and pneumonic plague by passive immunization with monoclonal antibodies against the F1 protein of Yersinia pestis. Am J Trop Med Hyg 1997;56:471 3. [137] Nakajima R, Motin VL, Brubaker RR. Suppression of cytokines in mice by protein A-V antigen fusion peptide and restoration of synthesis by active immunization. Infect Immun 1995;63:3021 9. [138] Sing A, Rost D, Tvardovskaia N, Roggenkamp A, Wiedemann A, Kirschning CJ, et al. Yersinia V-antigen exploits toll-like receptor 2 and CD14 for interleukin 10-mediated immunosuppression. J Exp Med 2002;196:1017 24. [139] Welkos S, Friedlander A, McDowell D, Weeks J, Tobery S. V antigen of Yersinia pestis inhibits neutrophil chemotaxis. Microb Pathog 1998;24:185 96. [140] Leary SE, Williamson ED, Griffin KF, Russell P, Eley SM, Titball RW. Active immunisation with V-antigen from Yersinia pestis protects against plague. Infect Immun 1995;63:2854 8. [141] Williamson ED, Vesey PM, Gillhespy KJ, Eley SM, Green M, Titball RW. An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model. Clin Exp Immunol 1999;116:107 14.

459

[142] Demeure CE, Derbise A, Carniel E. Oral vaccination against plague using Yersinia pseudotuberculosis. Chem Biol Interact 2017;267:89 95. [143] Heath DG, Anderson Jr GW, Mauro JM, Welkos SL, Andrews GP, Adamovicz J, et al. Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine. Vaccine 1998;16:1131 7. [144] Cornelius CA, Quenee LE, Overheim KA, Koster F, Brasel TL, Elli D, et al. Immunization with recombinant V10 protects cynomolgus macaques from lethal pneumonic plague. Infect Immun 2008;76:5588 97. [145] Verma SK, Tuteja U. Plague vaccine development: current research and future trends. Front Immunol 2016;7:602. [146] Philipovskiy AV, Smiley ST. Vaccination with live Yersinia pestis primes CD4 and CD8 T cells that synergistically protect against lethal pulmonary Y. pestis infection. Infect Immun 2007;75:878 85. [147] Sun W, Six D, Kuang X, Roland KL, Raetz CR, Curtiss 3rd R. A live attenuated strain of Yersinia pestis KIM as a vaccine against plague. Vaccine 2011;29:2986 98. [148] Feodorova VA, Corbel MJ. Prospects for new plague vaccines. Expert Rev Vaccines 2009;8:1721 38. [149] Anisimov AP, Dentovskaya SV, Panfertsev EA, Svetoch TE, Kopylov Pkh, Segelke BW, et al. Amino acid and structural variability of Yersinia pestis LcrV protein. Infect Genet Evol 2010;10:137 45. [150] Achtman M, Morelli G, Zhu P, Wirth T, Diehl I, Kusecek B, et al. Microevolution and history of the plague bacillus, Yersinia pestis. Proc Natl Acad Sci USA 2004;101:17837 42. [151] Chain PS, Carniel E, Larimer FW, Lamerdin J, Stoutland PO, Regala WM, et al. Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. Proc Natl Acad Sci USA 2004;101:13826 31. [152] Blisnick T, Ave P, Huerre M, Carniel E, Demeure CE. Oral vaccination against bubonic plague using a live avirulent Yersinia pseudotuberculosis strain. Infect Immun 2008;76:3808 16. [153] Derbise A, Cerda` Marı´n A, Ave P, Blisnick T, Huerre M, Carniel E, et al. An encapsulated Yersinia pseudotuberculosis is a highly efficient vaccine against pneumonic plague. PLoS Negl Trop Dis 2012;6:e1528. [154] Derbise A, Hanada Y, Khalife´ M, Carniel E, Demeure CE. Complete protection against pneumonic and bubonic plague after a single oral vaccination. PLoS Negl Trop Dis 2015;9:e0004162. [155] Sun W, Sanapala S, Henderson JC, Sam S, Olinzock J, Trent MS, et al. LcrV delivered via type III secretion system of live attenuated Yersinia pseudotuberculosis

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

460

[156]

[157]

[158]

[159]

25. MUCOSAL APPROACHES FOR SYSTEMIC IMMUNITY TO ANTHRAX, BRUCELLOSIS, AND PLAGUE

enhances immunogenicity against pneumonic plague. Infect Immun 2014;82:4390 404. Sun W, Sanapala S, Rahav H, Curtiss 3rd R. Oral administration of a recombinant attenuated Yersinia pseudotuberculosis strain elicits protective immunity against plague. Vaccine 2015;33:6727 35. Oyston PC, Williamson ED, Leary SE, Eley SM, Griffin KF, Titball RW. Immunization with live recombinant Salmonella typhimurium aroA producing F1 antigen protects against plague. Infect Immun 1995;63:563 8. Titball RW, Howells AM, Oyston PC, Williamson ED. Expression of the Yersinia pestis capsular antigen (F1 antigen) on the surface of an aroA mutant of Salmonella typhimurium induces high levels of protection against plague. Infect Immun 1997;65:1926 30. Garmory HS, Griffin KF, Brown KA, Titball RW. Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague. Vaccine 2003;21:3051 7.

[160] Yang X, Hinnebusch BJ, Trunkle T, Bosio CM, Suo Z, Tighe M, et al. Oral vaccination with Salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague. J Immunol 2007;178:1059 67. [161] Sanapala S, Rahav H, Patel H, Sun W, Curtiss R. Multiple antigens of Yersinia pestis delivered by live recombinant attenuated Salmonella vaccine strains elicit protective immunity against plague. Vaccine 2016;34:2410 16. [162] Morton M, Garmory HS, Perkins SD, O’Dowd AM, Griffin KF, Turner AK, et al. A Salmonella enterica serovar Typhi vaccine expressing Yersinia pestis F1 antigen on its surface provides protection against plague in mice. Vaccine 2004;22:2524 32. [163] Galen JE, Wang JY, Carrasco JA, Lloyd SA, MelladoSanchez G, Diaz-McNair J, et al. A bivalent typhoid live vector vaccine expressing both chromosome- and plasmid-encoded Yersinia pestis antigens fully protects against murine lethal pulmonary plague infection. Infect Immun 2015;83:161 72.

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Nanodelivery Vehicles for Mucosal Vaccines Rika Nakahashi-Ouchida1,2, Yoshikazu Yuki1,2 and Hiroshi Kiyono1,2,3,4 1

Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, The Institute of Medical Science, University of Tokyo, Tokyo, Japan 2International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, University of Tokyo, Tokyo, Japan 3Mucosal Immunology and Allergy Therapeutics, Graduate School of Medicine, Chiba University, Chiba, Japan 4 Division of Gastroenterology, Department of Medicine, (CU-UCSD cMAV) Center for Mucosal Immunology, Allergy and Vaccines, University of California, San Diego, CA, United States

I. INTRODUCTION Infectious agents such as viruses and bacteria generally invade the host via the mucosal surfaces of the respiratory and gastrointestinal tracts. To protect the host from invasion and to prevent the spread of pathogens, the induction of neutralizing antibody-based humoral immunity and/or cell-mediated immunity against pathogens at the mucosal surface is a logical strategy [1]. Oral and nasal vaccines that can effectively induce antigen-specific immune responses at mucosal surfaces and in systemic immune compartments [2,3] and mucosal vaccines that target various infectious diseases are currently under development [4]. The nasal cavity is considered to be a particularly attractive site for antigen deposition because the Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00026-2

nasal mucosa has the capacity to facilitate an active antigen uptake, processing, and presenting system in the form of microfold (M) cells and dendritic cells (DCs), and because it also has the nasopharyngeal-associated lymphoid tissue (NALT) for the induction of antigenspecific immune responses in respiratory tissues [5,6]. Furthermore, the nasal cavity is enriched with blood vessels, so antigens are rapidly transported into the circulation and are ultimately deposited in peripheral lymph nodes for initiation of antigen-specific systemic immune responses [7]. In addition to the induction of antigen-specific immune responses in the upper and lower respiratory tracts, nasal vaccination has been shown to effectively induce antigen-specific immune responses at distal mucosal surfaces, particularly the

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reproductive tract [8] (Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance and Chapter 18: Mucosal Regulatory System for the Balanced Immunity in the Middle Ear and Nasopharynx). Although the nasal cavity is an attractive location for mucosal vaccination, there are physical and chemical protective barriers that must be overcome for successful nasal vaccination. For example, mucociliary clearance is an innate defense mechanism that uses ciliary movement and a mucous layer to exclude inhaled microbes and foreign substances, including vaccine antigens, from the nasal cavity [9]. Moreover, epithelial cells provide a physical barrier in the form of tight junctions between cells and a chemical barrier in the form of antimicrobial peptide secretion. In addition to these barriers, metabolic enzymes secreted by nasal glands, mucus produced by goblet cells, and transudate from plasma are present in the nasal cavity. Thus, there is a high possibility that vaccine antigens delivered to the nasal cavity will be decomposed or excluded before an antigen-specific immune response is evoked [10]. Because of these protective barriers in the nasal cavity, the accessibility to and adsorption of nasally administered vaccine antigen by the mucosal immune system means that nasal vaccination is less effective than other types of vaccination (e.g., subcutaneous or intramuscular injection). Consequently, large amounts of antigen are often required for administration, and all the while only a weak antigen-specific immune response is induced [11]. To date, this issue has been addressed by the use of various combinations of live vectors, inactivated antigens, artificial-antigen-delivery vehicles, and sometimes mucosal adjuvants [12 14]. An inactivated intranasal influenza vaccine (Nasalflu) containing Escherichia coli heat-labile toxin as an adjuvant was approved for use in Switzerland during the 2001 02 influenza season [15]. Although the vaccine was found to

induce protective immunity, several cases of facial nerve palsy (Bell’s palsy) were reported during postmarketing surveillance, and the vaccine was withdrawn as a result of safety concerns [15]. This incident clearly highlighted the potential for nasal vaccines to affect the central nervous system via the olfactory nerve and raised the bar for nasal vaccine development. Thus, effective and safe nasal vaccines should be able to (1) overcome various physical and chemical barriers at the nasal mucosa for efficient delivery of vaccine antigen, (2) induce an effective antigen-specific immune response with or without a mucosal adjuvant, and (3) have a suitable safety profile and not influence the central nervous system.

II. CHARACTERISTICS OF THE NASAL IMMUNE SYSTEM A. Structure and Function of Nasopharyngeal-Associated Lymphoid Tissue The mucosal immune system can be subdivided into inductive and effector sites (Fig. 26.1). The inductive sites are collectively referred to as mucosa-associated lymphoid tissue (MALT), and this is where antigensampling M cells are located and follicleassociated epithelium and immunocompetent cells such as T and B lymphocytes, DCs, and macrophages accumulate in high numbers [8]. In rodents, the nasal immune system contains NALT, which is located basally on both sides of the rodent nasal cavity, and is responsible for the initiation of antigen-specific immune responses [16]. In humans, the paired palatine tonsils and the unpaired nasopharyngeal tonsils (adenoids), which are two components of Waldeyer’s ring, are thought to be functionally related to the NALT in rodent [17]. The NALT is covered with follicle-associated epithelium, which contains M cells, which specialize in the uptake transcytosis of antigens from the

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FIGURE 26.1 The mucosal immune system. Antigens are taken up by microfold (M) cells and then captured by antigenpresenting cells such as dendritic cells. Antigen-primed T and B lymphocytes express homing or polymeric molecules and chemokine receptors and migrate to the effector site through the thoracic ducts and blood circulation. IgA1 B cells differentiate into plasma cells by IgA-enhancing cytokines such as IL-6 and IL-10 secreted from Th2 cells. Dimeric IgA secreted from plasma cells is transported to the mucosal surface as secretory secretory IgA (SIgA) through the polymeric Ig receptor (pIgR) expressed on the basal membrane of epithelial cells.

mucosal lumen to antigen-presenting cells (e.g., DCs) for processing and presentation to T and B lymphocytes. There is also a special subset of M cells, called respiratory M cells, that exist as a monolayer on top of the single layer of epithelium that covers the lateral surfaces of the nasal turbinates [18]. Respiratory M cells resemble classical M cells with their depressed surface and irregular microvilli, but unlike classical M cells, they do not have a pocket on their basolateral side [18]. Like classical M cells, respiratory

M cells can take up soluble protein or bacterial antigens and therefore are an antigen-sampling pathway for the nasal immune system.

B. Mechanism for the Induction of Antigen-Specific Nasal Immune Responses After antigens have been captured by M cells, these are transported via transcytosis to antigen-presenting cells such as DCs located

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beneath the epithelial layer or follicleassociated epithelium (Fig. 26.1). DCs process these antigens and present the peptide together with major histocompatibility complex II to CD41 T cells located at induction sites. Antigen-primed CD41 T cells secret cytokines such as transforming growth factor beta and interleukin 5 (IL-5) and promote isotype class switching in B cells from immunoglobulin M to A [19 21]. Eventually, the antigen-specific T cells and IgA1 B cells migrate to effector sites via the thoracic ducts, blood circulation, and lymphocyte homing system (discussed in the next section) [22]. IgA1 B cells then terminally differentiate into plasma cells that secrete dimeric or polymeric IgA in the presence of IL6 and IL-10 produced by T helper cells [23]. At effector sites, dimeric or polymeric IgA binds to polymeric immunoglobulin receptor, a transmembrane protein expressed on the basement membrane of nasal epithelial cells, and then forms secretory IgA (SIgA), which is secreted into the lumen [24]. SIgA promotes the clearance of pathogens via direct binding and neutralizing activity to prevent pathogen invasion and proliferation at mucosal surfaces. Thus, SIgA plays a central role in the mucosal immune system as a first line of defense [25,26] (Chapter 4: Protective Activities of Mucosal Antibodies).

C. Lymphocyte Imprinting and Homing Mechanisms in the Nasal Immune System The lymphocyte homing system interconnects the inductive and effector sites of the mucosal immune system [27]. In the case of the nasal immune system, antigen-stimulated lymphocytes express α4β1 integrin, which is a receptor for vascular cell adhesion molecule-1 and C-C motif chemokine receptor 10, which is a receptor for C-C motif chemokine ligand 28 [28 30]. Because vascular cell adhesion molecule-1 and C-C motif chemokine ligand 28 are predominantly expressed by vascular

endothelial cells and epithelial cells in mucosal tissues, α4β1- and C-C motif chemokine receptor 10-positive lymphocytes preferentially migrate from the NALT to the respiratory tract, salivary glands, mammary glands, lacrimal glands, and genitourinary tract via the thoracic ducts and blood circulation after antigen stimulation and acquiring these imprinting molecules [31]. It has been shown that the thymic stromal lymphopoietin thymic stromal lymphopoietin receptor signaling cascade is important for the induction of antigen-specific IgA production following nasal administration of antigens [32]. This signaling cascade has been shown to enhance IgA production via the induction of IL-6 production in mucosal DCs after nasal immunization of antigen together with cholera toxin adjuvant, suggesting that adjuvants that promote activation of the thymic stromal lymphopoietin-mediated signaling may be useful for effective mucosal vaccine development.

III. DRUG-DELIVERY SYSTEMS FOR NASAL VACCINES The use of various nanoparticle-based delivery systems for nasal vaccines has been examined (Table 26.1). The chemical and physical properties of nanomaterials can be controlled via surface modification, which makes it easy to encapsulate vaccine antigens and/or adjuvants within the nanomaterials or make vaccine antigen nanomaterial complexes via noncovalent binding or covalent interactions [51]. Liposomes have been examined for their suitability as carriers for mucosal vaccines. Since liposomes are amphipathic, antigens and adjuvants can be encapsulated within the aqueous space regardless of their hydrophilicity and hydrophobicity [52]. To maximize vaccine immunogenicity, the physicochemical properties of liposomes, such as their size, lipid composition, and structure, can be adjusted on the

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III. DRUG-DELIVERY SYSTEMS FOR NASAL VACCINES

TABLE 26.1

Studies Using Nanoparticles for Nasal Vaccines

Delivery system

Materials

Antigens

Reference

Lipid nanoparticles

Liposome

Ovalbumin

[33]

Multivalent group A streptococcal M protein

[34]

Formaldehyde-killed whole cell of Yersinia pestis

[35]

Ovalbumin

[36]

Ovalbumin

[37]

X8P

Influenza A

[38]

W205EC

Bacillus anthracis

[39]

Polylactic acid (PLA)

Tetanus toxoid

[40]

Poly(lactic-glycolic acid) (PLGA)

Enterotoxigenic Escherichia coli colonization factor CS6

[41]

Chitosan PLGA

Tetanus toxoid

[42]

Chitosan

Influenza subunit

[43]

RSV M2 protein expressing plasmid

[44]

VP1 protein of Coxsackie virus B3

[45]

Glycol chitosan PLGA

Hepatitis B surface antigen

[46]

PLA polyethylene glycol

Tetanus toxoid

[47]

cCHP

Botulinum type A neurotoxin fragment

[48]

Pneumococcal surface protein A

[49]

Pneumococcal surface protein A

[50]

Emulsion

Polymeric nanoparticles

W805EC

basis of the specific characteristics of the vaccine antigen [52]. Several studies have utilized liposomes for the development of nasal vaccines [33 35]. For example, nasal immunization with a liposome-formulated Yersinia pestis formaldehyde-killed whole cell vaccine significantly enhanced not only the antigen-specific immune response in systemic immune compartments, but also the amount of IgA and IgG in mucosal secretions in the lung and nasal cavity and consequently protected mice from intranasal lethal challenge with Y. pestis when compared with mice immunized with the formaldehyde-killed whole-cell vaccine alone [35] (Chapter 19: Current and New Approaches for Mucosal Vaccine Delivery).

Nanomaterial emulsions (nanoemulsions), which are water-in-oil formulations stabilized by small amounts of surfactant, can also be used to deliver vaccines because they can trap antigens inside their core [53]. For instance, nasal administration of the nanoemulsion W805EC, which is composed of cetylpyridinium chloride, Tween 80, ethanol, and soybean oil, promoted engulfment and antigen presentation by DCs and evoked an immune response to the model antigen ovalbumin in mice [36,37]. Other nanoemulsions have been examined for the development of influenza or Bacillus anthracis vaccines [38,39]. In the case of influenza, the nanoemulsion X8P, which is composed of tributyl phosphate, Triton X-100, and soybean oil in

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water, was mixed with inactivated influenza A to create a vaccine. Nasal vaccination with this mixture protected mice from death or viral pneumonitis after lethal challenge with a congenic strain of influenza virus [38]. Similarly, the nanoemulsion W205EC, which is composed of cetylpyridinium chloride, Tween 20, and ethanol in water with hot-pressed soybean oil, has been used as an adjuvant in a mixture with B. anthracis antigen rPA [39]. Nasal immunization with one or two doses of the mixture elicited an antigen-specific systemic IgG response as well as bronchial IgA and IgG responses and T helper 1 (Th1)-polarized cytokine secretion by splenocytes in mice. High titers of toxin-neutralizing serum IgG obtained in both mice and guinea pigs were found to protect the guinea pigs from intradermal or intranasal lethal challenge with B. anthracis [39]. Of the different nanoparticles examined to date, polymers are the best-studied carriers, with polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) being the most-studied synthetic polymers [54]. With these two polymers, it is possible to control the rate of release of vaccine antigen by changing the composition ratio and molecular weight of PLA and glycolic acid in the formulation [40,55]. Furthermore, these synthetic polymers are superior in biocompatibility and biodegradability and have high practicability, and they have been approved by the US Food and Drug Administration for use in nanomedicine formulations [56]. Tetanus toxoid has been adsorbed onto PLA, and E. coli colonization factor CS6 has been encapsulated in PLGA microspheres [40,41]. Nasal administration of PLA-adsorbed tetanus toxoid to guinea pigs enhanced the antigenspecific immune response compared to those administered free antigen [40]. Similarly, nasal administration of CS6 encapsulated in PLGA led to greater fecal IgG and IgA immune responses compared to mice that were administered noncapsulated antigen [41]. New formulations using PLA or PLGA to polymerize with polyethylene glycol (PEG) or chitosan are being

tested to improve the stability of vaccine formulations [42]. It has been reported that nasal administration of tetanus toxoid-encapsulated in PLA PEG nanoparticles (PLA polymerized with PEG) generates high, long-lasting tetanus toxoid-specific immunity compared to tetanus toxoid-encapsulated in PLA alone [42]. Chitosan is a well-studied natural polymer [57]. It has mucoadhesive properties and can loosen the tight junctions between epithelial cells, so it is a potentially useful molecule for promoting antigen uptake at the mucosal surface [58]. To take advantage of these characteristics of chitosan, nanoparticles containing N-trimethyl chitosan and monovalent influenza A subunit H3N2 have been developed as a prototype chitosan-based nasal influenza vaccine. Nasal immunization with this vaccine resulted in potent inhibition of hemagglutination that was dependent on the induction of antigenspecific IgG and SIgA responses [43]. Similarly, a plasmid DNA encoding the cytotoxic T lymphocyte epitope derived from respiratory syncytial virus (RSV) M2 protein has been combined with chitosan to develop a vaccine against RSV [44]. Nasal immunization with the chitosan DNA RSV vaccine induced a virusspecific cytotoxic T lymphocyte response in mice, and a significant reduction in virus titer was observed in the lung after RSV challenge compared with the results in a nonimmunized control group [44]. As a vaccine against Coxsackie virus B3 infection, which is the cause of acute and chronic myocarditis, nasal immunization of chitosan-DNA expressing VP1, a major structural protein of Coxsackie virus B3, induced antigen-specific serum IgG and mucosal SIgA responses and resulted in protection against intraperitoneal injection of a lethal dose of Coxsackie virus B3 in mice [45]. Furthermore, an immunogenicity study in mice revealed that nasal administration with hepatitis B virus antigen in combination with glycol chitosan PLGA polymers was more effective at inducing systemic and mucosal immune responses when compared to the antigen encapsulated with

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V. DEVELOPMENT OF A NANOGEL-BASED NASAL VACCINE AGAINST PNEUMONIA

PLGA or chitosan PLGA polymers [46]. Glycol chitosan improves vaccine retention because this polymer remains on the mucous membrane owing to its better mucoadhesiveness arising from its positive charge, which leads to sustained antigen release. Furthermore, the effectiveness of hydrophilic PEG PLA particles as nasal transporters has been demonstrated by using tetanus toxoid as a model antigen [47]. In the presence of lysozyme, PLA nanoparticles immediately aggregate, whereas PEG PLA particles remain in their soluble form. Tetanus toxoid-specific antibody titers induced following nasal administration of PEG PLA tetanus toxoid are significantly higher and more prolonged than those following nasal administration of PLA tetanus toxoid [47]. It is thought that the PEG coating stabilizes the PLA particles in the mucosal fluid and facilitates the transport of the antigen to the nasal epithelium, where it elicits a long-lasting antigen-specific antibody immune response [47]. Cross-linked hydrogel vaccine antigen complexes can also be used to deliver vaccines to mucosal surfaces [48]. For instance, cationic formulations of cholesteryl-bearing pullulan (cCHP) nanogel can attach to negatively charged nasal mucosal epithelium because it has a positive charge [48]. As a result, sustained antigen release from the encapsulated vaccine and subsequent antigen uptake elicit antigen-specific immune responses both in systemic (e.g., IgG) and mucosal (e.g., IgA) compartments. The use of cCHP nanogel in nasal vaccines against infectious and noninfectious diseases is discussed in the remaining sections of this chapter.

IV. CCHP NANOGEL AS A DRUGDELIVERY SYSTEM FOR NASAL VACCINES cCHP nanogel is composed of cholesterolbound pullulan molecules that associate via

467

hydrophobic interactions to create a spherical structure (diameter, approx. 40 nm) [59 61]. Vaccine antigens can be easily incorporated into and released from the internal space of the cCHP nanogel sphere as a natural form owing to the artificial chaperone function of CHP nanogel [59]. To enhance attachment of CHP nanogel to the negatively charged mammalian nasal mucosal surface [62], cCHP nanogel in which an amino group has also been introduced to the pullulan molecules has been developed (Fig. 26.2) [59]. It has been shown that cCHP nanogel containing the C-terminus fragment of heavy chain of botulinum neurotoxin type A (BoHc) attaches strongly to the nasal mucosal epithelium after nasal administration (Fig. 26.3) [48]. After attachment, the BoHc antigen is gradually released from the cCHP nanogel and taken up by nasal epithelial cells and M cells for at least 12 hours after administration [48]. In addition, it has been shown that approximately 40% of nasal DCs take up the BoHc antigen after nasal administration of cCHP BoHc nanogel compared with only 2% after nasal administration of BoHc antigen alone, resulting in greater BoHc-specific serum IgG and SIgA responses and protection from lethal challenge with neurotoxin [48]. With respect to safety, the antigen itself does not migrate to the olfactory bulb after nasal administration, having no effect on the olfactory nerve [48,63]. These data suggest that cCHP nanogels are a safe, adjuvant-free means of delivering vaccine antigens to the nasal cavity and can induce effective antigen-specific immune responses in both systemic and mucosal compartments.

V. DEVELOPMENT OF A NANOGEL-BASED NASAL VACCINE AGAINST PNEUMONIA To verify the suitability of using cCHP nanogel to deliver vaccine antigen to the nasal cavity, a cCHP nanogel-based nasal vaccine

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FIGURE 26.2

Schematic illustration of cCHP nanogel with artificial chaperone function. cCHP consists of a cholesterylgroup-bearing pullulan (CHP) introduced cationic amino group. cCHP nanogel can encapsulate proteins in the interior space through hydrophobic interactions and are effectively retained in the nasal mucosa, which is negatively charged following intranasal vaccination.

against respiratory infection has been developed. Streptococcus pneumoniae is a pathogen that causes bacterial pneumonia mainly in infants and the elderly and can lead to death as a result of severe upper respiratory tract infection [64]. Two intramuscular injection types of vaccines against pneumonia have been developed: pneumococcal polysaccharide vaccine (PPSV) and pneumococcal polysaccharide conjugate vaccine

(PCV) [65]. PPSV contains capsular polysaccharides purified from various serotypes of S. pneumoniae and induces antigen-specific responses in a T-cell-independent manner [66]. PCV is a conjugate vaccine containing capsular polysaccharides, a carrier protein (Corynebacterium diphtheriae mutant 197, a nontoxic mutant of diphtheria toxin), and an aluminum phosphate adjuvant that induces a T cell-dependent immune

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469

FIGURE 26.3 Uptake of cCHP antigen complex from the paranasal sinuses in the nasal mucosa. The BoHc/A antigens are gradually released from the cCHP nanogel and taken up by nasal epithelial cells and M cells in NALT for at least 12 h after administration. The NKM 16-2-4 is an M-cell-specific monoclonal antibody.

response [67]. Although these vaccines have potent immunogenicity against multiple major serotypes that cause bacterial pneumonia (23 serotypes in PPSV, 13 serotypes in PCV), their efficacy is lost once serotype replacement occurs [68,69]. Therefore, a vaccine effective against all serotypes of S. pneumoniae is required.

To address this issue, pneumococcal surface protein A (PspA), which is expressed by all serotypes of S. pneumoniae, has been examined as a promising candidate antigen for next-generation pneumococcal vaccines that can induce crossreactive immune responses against different serotypes [70,71]. Indeed, nasal administration of a

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PspA Vibrio vulnificus-derived flagellin fusion protein induced antigen-specific IgG and IgA responses both in serum and at mucosal surfaces, and provided protective immunity against lethal challenge with S. pneumoniae in mice [72]. In addition, nasal immunization using chitosan DNA nanoparticles expressing PspA suppressed pneumococcal colony formation in the nasal cavity of mice [73]. Also, a unique antigen-delivery method targeting Claudin 4, a major cell adhesion molecule in the tight junctions of NALT epithelial cells, has been developed [74]. For instance, a PspA C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) fusion protein has been shown to bind to Claudin 4 after nasal immunization and induces the production of PspA-specific IgG in serum and SIgA in nasal and bronchoalveolar lavage fluids [74]. As a result, mice nasally vaccinated with PspA C-CPE are protected from pneumococcal respiratory infection. In addition, nasal administration of PspA together with Flt3 ligand expression plasmid and CpG oligodeoxynucleotide as mucosal adjuvants induced PspAspecific SIgA in 2-year-old or pregnant mice, resulting in inhibition of bacterial colonization both in aged mice and in offspring of the vaccinated group [75]. Therefore, PspA is a promising new candidate vaccine antigen for the vaccination of children and elderly people [70,71]. Based on the advantages of PspA for the generation of protective immunity against all serotypes of S. pneumoniae, cCHP PspA nanogel has been developed, and its safety and efficacy have been investigated both in mice and in nonhuman primates [49,50]. When cCHP PspA nanogel was intranasally administered to mice three times at 1-week intervals, antigen-specific IgG was significantly increased both in serum and in bronchial fluid [49]. Antigen-specific SIgA in nasal fluid was also elevated. In contrast, administration of PspA antigen alone failed to induce an antigenspecific antibody response [49]. These results indicate that PspA antigen is efficiently

delivered to nasal epithelium and taken up by DCs in the nasal cavity for the initiation of an antigen-specific immune response. Furthermore, bacterial growth after S. pneumoniae infection was suppressed both in the lung and in the nasal cavity of the mice vaccinated with cCHP PspA nanogel, but not in the mice that received PspA alone [49]. Consequently, the cCHP PspA vaccinated mice survived after lethal challenge with S. pneumoniae [49]. The protective immunity elicited by the cCHP PspA nanogel was a humoral immune response, and was accompanied by the production of both Th2- and Th17type cytokines by antigen-specific CD41 T cells, which are associated with protective immunity against S. pneumoniae [49]. To examine the clinical applicability the cCHP-based pneumococcal vaccine in humans, the safety and immunogenicity of cCHP PspA were analyzed in cynomolgus macaques (Macaca fascicularis) [50]. To confirm the deposition and fate of vaccine antigen in the nasal cavity, olfactory bulbs, and central nervous system, a positron emission tomography study combined with magnetic resonance imaging was performed after nasal administration of 18Flabeled PspA in the macaques. cCHP 18F-PspA nanogel was retained at the nasal epithelium for as long as 6 hours after administration, whereas 18 F-PspA antigen alone had been eliminated from the nasal cavity by 3 hours after administration [50]. Furthermore, no nanogel-delivered antigen was found in the olfactory bulb or brain, even at 6 hours after nasal immunization, suggesting that the cCHP PspA nanogel nasal vaccine does not have neurological side effects. When the cCHP PspA nanogel was nasally administered to cynomolgus macaques five times at 2-week intervals, PspA-specific IgG was markedly elevated in serum and then gradually diminished over a period of 8 months [50]. Similarly, PspA-specific SIgA was elevated in nasal washes as well as bronchoalveolar lavage fluids, and it then decreased in the same manner as did PspA-specific IgG in the serum. After

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VII. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

these macaques received a nasal administration of cCHP PspA nanogel at 9 months after the final immunization, PspA-specific IgG and SIgA were rapidly boosted to the levels achieved with the primary responses [50]. This suggests that the cCHP PspA nanogel vaccine effectively induces antigen-specific immunological memory. In addition, mice that received PspA-specific antibodies transferred from the macaques exhibited protection against lethal challenge with S. pneumoniae via the production of neutralizing antibodies [50]. Together, these results show that the cCHP PspA nanogel is a promising nasal vaccine candidate for the prevention and control of diseases caused by S. pneumoniae.

VI. APPLICATION OF CCHP NASAL VACCINES AGAINST NONINFECTIOUS DISEASES Recently, cCHP nasal vaccines against obesity and hypertension have been developed, confirming that cCHP vaccines are effective against not only infectious diseases, but also lifestyle-related diseases [76,77]. In the antiobesity vaccine, a ghrelin PspA fusion protein was used, where ghrelin is the vaccine antigen and PspA is the carrier protein. Ghrelin is a peptide hormone produced in the stomach that promotes secretion of growth hormone from the pituitary gland, which stimulates the hypothalamus to enhance appetite [76]. When the ghrelin PspA fusion protein was encapsulated in a cationic nanogel mixed with cyclic guanosine diphosphate as an adjuvant and administered to diet-induced obese mice, both ghrelin-specific IgG in the serum and energy consumption were increased. As a result, both the amount of visceral fat and the body weight were decreased in treated mice compared with untreated mice [76]. A hypertension vaccine has also been developed. Angiotensin II receptor type 1 (AT1R)

471

promotes the functional and structural integrity of the arterial wall and contributes to vascular homeostasis, but it also causes an increase in blood pressure [77]. When an AT1R PspA conjugate was encapsulated in a nanogel mixed with cyclic guanosine diphosphate and administered to hypertensive rats, both AT1R- and PspA-specific IgG levels were elevated in the serum, the onset of hypertension was attenuated, and the rats were protected from lethal challenge with S. pneumoniae [77]. Since hypertension is reported to increase the mortality risk from pneumonia, a single nasal vaccine against both diseases would be of great value. From the results of these two studies, cCHPbased nasal vaccines are expected to be a major part of the next generation of therapies for the treatment of lifestyle-related diseases because of their noninvasiveness and long period of efficacy.

VII. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Nanomaterials are promising tools for improving the immunogenicity of vaccines and effectively delivering antigens and/or adjuvants to mucosal surfaces. It is possible to increase the efficiency of antigen uptake into mucosal tissues, including MALT, by modifying the size and surface properties of these nanomaterials. cCHP nanogel is a vaccine-delivery system that is close to being ready for use in the clinical setting. After nasal administration, cCHP nanogel has excellent safety and efficacy profiles with respect to the induction of protective immunity in both the systemic and mucosal immune compartments. Although cCHP nanogel itself has no biological adjuvant activity, it delivers vaccine antigens to the nasal epithelium and follicle-associated epithelium of the NALT and to DCs in the nasal cavity; therefore, it is capable of inducing antigen-specific

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immune responses in both systemic and mucosal immune compartments even in the absence of an adjuvant. As a result, it induces humoral immune responses providing protection against infection. cCHP nanogel-based nasal vaccines are showing promise as the next generation of vaccines to prevent infectious diseases. However, before these vaccines can be used in the clinical setting, manufacturing techniques and guidelines for their preparation must first be developed through collaboration among industry, academia, and government.

ABBREVIATIONS AT1R BoHc cCHP C-CPE DC Ig IL MALT NALT PCV PEG PLA PLGA PPSV PspA RSV SIgA

angiotensin II receptor type 1 Botulinum type A neurotoxin fragment cationic formulation of cholesterylgroup-bearing pullulan C-terminal fragment of Clostridium perfringens enterotoxin dendritic cell immunoglobulin interleukin mucosa-associated lymphoid tissue nasopharynx-associated lymphoid tissue pneumococcal polysaccharide conjugate vaccine polyethylene glycol polylactic acid poly(lactic-co-glycolic acid) pneumococcal polysaccharide vaccine pneumococcal surface protein A respiratory syncytial virus secretory IgA

References [1] Kiyono H, Kunisawa J, McGhee JR, Mestecky J. The mucosal immune system. In: Paul WE, editor. Fundamental immunology. Philadelphia: Lippincott Williams & Wilkins; 2008. p. 983 1030.

[2] Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, et al. Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Acad Sci USA 2007;104(26):10986 91. [3] Azegami T, Yuki Y, Kiyono H. Challenges in mucosal vaccines for the control of infectious diseases. Int Immunol 2014;26(9):517 28. [4] Ogra PL. Mucosal immunity: some historical perspective on host-pathogen interactions and implications for mucosal vaccines. Immunol Cell Biol 2003;81:23 33. [5] Nochi T, Yuki Y, Matsumura A, Mejima M, Terahara K, Kim DY, et al. A novel M cell-specific carbohydratetargeted mucosal vaccine effectively induces antigenspecific immune responses. J Exp Med 2007;204 (12):2789 96. [6] Kiyono H, Fukuyama S. NALT- versus Peyer’s-patchmediated mucosal immunity. Nat Rev Immunol 2004;4 (9):699 710. [7] Dkhar LK, Bartley J, White D, Seyfoddin A. Intranasal drug delivery devices and interventions associated with post-operative endoscopic sinus surgery. Pharm Dev Technol 2018;23(3):282 94. [8] Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med 2005;11:45 53. [9] Jones N. The nose and paranasal sinuses physiology and anatomy. Adv Drug Deliv Rev 2001;51(1 3):5 19. [10] Oliveria P, Fortuna A, Alves G, Falcao A. Drugmetabolizing enzymes and efflux transporters in nasal epithelium: influence on the bioavailability of intranasally administered drugs. Curr Drug Metab 2016;17 (7):628 47. [11] Haan L, Verweij WR, Holtrop M, Brands R, van Scharrenburg GJ, Palache AM, et al. Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity. Vaccine 2001;19:2898 907. [12] Riese P, Sakthivel P, Trittel S, Guzma´n CA. Intranasal formulations: promising strategy to deliver vaccines. Expert Opin Drug Deliv 2014;11(10):1619 34. [13] Srivastava A, Gowda DV, Madhunapantula SV, Shinde CG, Iyer M. Mucosal vaccines: a paradigm shift in the development of mucosal adjuvants and delivery vehicles. APMIS 2015;123(4):275 88. [14] Aoshi T. Modes of action for mucosal vaccine adjuvants. Viral Immunol 2017;30(6):463 70. [15] Mutsch M, Zhou W, Rhodes P, Bopp M, Chen RT, Linder T, et al. Use of the inactivated intranasal influenza vaccine and the risk of Bell’s palsy in Switzerland. N Engl J Med 2004;350(9):896 903. [16] Kuper CF, Koornstra PJ, Hameleers DM, Biewenga J, Spit BJ, Duijvestijn AM, et al. The role of nasopharyngeal lymphoid tissue. Immunol Today 1992;13:219 24.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[17] Rerry M, Whyte A. Immunology of the tonsils. Immunol Today 1998;19:414 20. [18] Kim DY, Sato A, Fukuyama S, Sagara H, Nagatake T, Kong IG, et al. The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens. J Immunol 2011;186(7):4253 62. [19] Sonoda E, Matsumoto R, Hitoshi Y, Ishii T, Sugimoto M, Araki S, et al. Transforming growth factor beta induces IgA production and acts additively with interleukin 5 for IgA production. J Exp Med 1989;170: 1415 20. [20] Coffman RL, Lebman DA, Shrader B. Transforming growth factor beta specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes. J Exp Med 1989;170(3):1039 44. [21] Horikawa K, Takatsu K. Interleukin-5 regulates genes involved in B-cell terminal maturation. Immunology 2006;118:497 508. [22] Neutra MR, Kozlowski PA. Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 2006;6 (2):148 58. [23] McGhee JR, Fujihashi K, Beagley KW, Kiyono H. Role of interleukin-6 in human and mouse mucosal IgA plasma cell responses. Immunol Res 1991;10(3 4): 418 22. [24] Kaetzel CS. The polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces. Immunol Rev 2005;206:83 99. [25] Williams RC, Gibbons RJ. Inhibition of bacterial adherence by secretory immunoglobulin A: a mechanism of antigen disposal. Science 1972;177:697 9. [26] Taylor HP, Dimmock NJ. Mechanism of neutralization of influenza virus by secretory IgA is different from that of monomeric IgA or IgG. J Exp Med 1985;161: 198 209. [27] Brandtzaeg P. Mucosal immunity: induction, dissemination, and effector functions. Scand J Immunol 2009;70(6):505 15. [28] Bentley AM, Durham SR, Robinson DS, Menz G, Storz C, Cromwell O, et al. Expression of endothelial and leukocyte adhesion molecules intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 in the bronchial mucosa in steady-state and allergen-induced asthma. J Allergy Clin Immunol 1993;92(6):857 68. [29] Campbell JJ, Brightling CE, Symon FA, Qin S, Murphy KE, Hodge M, et al. Expression of chemokine receptors by lung T cells from normal and asthmatic subjects. J Immunol 2001;166(4):2842 8. [30] Brinkman CC, Peske JD, Engelhard VH. Peripheral tissue homing receptor control of naı¨ve, effector, and memory CD8 T cell localization in lymphoid and nonlymphoid tissues. Front Immunol 2013;4:241.

473

[31] Brandtzaeg P, Johansen FE. Mucosal B cells: phenotypic characteristics, transcriptional regulation, and homing properties. Immunol Rev 2005;206:32 63. [32] Joo S, Fukuyama Y, Park EJ, Yuki Y, Kurashima Y, Ouchida R, et al. Critical role of TSLP-responsive mucosal dendritic cells in the induction of nasal antigen-specific IgA response. Mucosal Immunol 2017;10(4):901 11. [33] Patel GB, Ponce A, Zhou H, Chen W. Structural characterization of archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) formulations prepared by different protocols and their efficacy upon intranasal immunization of mice. J Liposome Res 2008;18 (2):127 43. [34] Hall MA, Stroop SD, Hu MC, Walls MA, Reddish MA, Burt DS, et al. Intranasal immunization with multivalent group A streptococcal vaccines protect mice against intranasal challenge infections. Infect Immun 2004;72(5):2507 12. [35] Baca-Estrada ME, Foldvari MM, Snider MM, Harding KK, Kournikakis BB, Babiuk LA, et al. Intranasal immunization with liposome-formulated Yersinia pestis vaccine enhances mucosal immune responses. Vaccine 2000;18(21):2203 11. [36] Makidon PE, Nigavekar SS, Bielinska AU, Mank N, Shetty AM, Suman J, et al. Characterization of stability and nasal delivery systems for immunization with nanoemulsion-based vaccines. J Aerosol Med Pulm Drug Deliv 2010;23(2):77 89. [37] Myc A, Kukowska-Latallo JF, Smith DM, Passmore C, Pham T, Wong P, et al. Nanoemulsion nasal adjuvant W805EC induces dendritic cell engulfment of antigenprimed epithelial cells. Vaccine 2013;31(7):1072 9. [38] Myc A, Kukowska-Latallo JF, Bielinska AU, Cao P, Myc PP, Janczak K, et al. Development of immune response that protects mice from viral pneumonitis after a single intranasal immunization with influenza A virus and nanoemulsion. Vaccine 2003;21(25 26):3801 14. [39] Bielinska AU, Janczak KW, Landers JJ, Makidon P, Sower LE, Peterson JW, et al. Mucosal immunization with a novel nanoemulsion-based recombinant anthrax protective antigen vaccine protects against Bacillus anthracis spore challenge. Infect Immun 2007;75 (8):4020 9. [40] Almeida AJ, Alpar HO, Brown MR. Immune response to nasal delivery of antigenically intact tetanus toxoid associated with poly (l-lactic acid) microspheres in rats, rabbits and guinea-pigs. J Pharm Pharmacol 1993;45:198 203. [41] Byrd W, Cassels FJ. Intranasal immunization of BALB/c mice with enterotoxigenic Escherichia coli colonization factor CS6 encapsulated in biodegradable poly(DL-lactideco-glycolide) microspheres. Vaccine 2006;24(9):1359 66.

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474

26. NANODELIVERY VEHICLES FOR MUCOSAL VACCINES

[42] Vila A, Sa´nchez A, Tobı´o M, Calvo P, Alonso MJ. Design of biodegradable particles for protein delivery. J Control Release 2002;78(1 3):15 24. [43] Amidi M, Romeijn SG, Verhoef JC, Junginger HE, Bungener L, Huckriede A, et al. N-trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model. Vaccine 2007;25(1):144 53. [44] Iqbal M, Lin W, Jabbal-Gill I, Davis SS, Steward MW, Illum L. Nasal delivery of chitosan-DNA plasmid expressing epitopes of respiratory syncytial virus (RSV) induces protective CTL responses in BALB/c mice. Vaccine 2003;21(13 14):1478 85. [45] Xu W, Shen Y, Jiang Z, Wang Y, Chu Y, Xiong S. Intranasal delivery of chitosan-DNA vaccine generates mucosal SIgA and anti-CVB3 protection. Vaccine 2004;22(27 28):3603 12. [46] Pawar D, Mangal S, Goswami R, Jaganathan KS. Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity. Eur J Pharm Biopharm 2013;85(3 Pt A):550 9. [47] Vila A, Sa´nchez A, Evora C, Soriano I, Vila Jato JL, Alonso MJ. PEG-PLA nanoparticles as carriers for nasal vaccine delivery. J Aerosol Med 2004;17 (2):174 85. [48] Nochi T, Yuki Y, Takahashi H, Sawada S, Mejima M, Kohda T, et al. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines. Nat Mater 2010;9(7):572 8. [49] Kong IG, Sato A, Yuki Y, Nochi T, Takahashi H, Sawada S, et al. Nanogel-based PspA intranasal vaccine prevents invasive disease and nasal colonization by Streptococcus pneumoniae. Infect Immun 2013;81 (5):1625 34. [50] Fukuyama Y, Yuki Y, Katakai Y, Harada N, Takahashi H, Takeda S, et al. Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNAassociated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques. Mucosal Immunol 2015;8(5):1144 53. [51] Peek LJ, Middaugh CR, Berkland C. Nanotechnology in vaccine delivery. Adv Drug Deliv Rev 2008;60 (8):915 28. [52] Baca-Estrada ME, Foldvari M, Babiuk SL, Babiuk LA. Vaccine delivery: lipid-based delivery systems. J Biotechnol 2000;83:91 104. [53] Kim MG, Park JY, Shon Y, Kim G, Shim G, Oh YK. Nanotechnology and vaccine development. Asian J Pharm Sci 2014;9:227 35.

[54] Ko¨ping-Ho¨gga˚rd M, Sa´nchez A, Alonso MJ. Nanoparticles as carriers for nasal vaccine delivery. Expert Rev Vaccines 2005;4(2):185 96. [55] Akagi T, Baba M, Akashi M. Biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine. Adv Polym Sci 2012;247:31 64. [56] Bobo D, Robinson KJ, Islam J, Thurecht KJ, Corrie SR. Nanoparticle-based medicines: a review of FDAapproved materials and clinical trials to date. Pharm Res 2016;33(10):2373 87. [57] Kang ML, Cho CS, Yoo HS. Application of chitosan microspheres for nasal delivery of vaccines. Biotechnol Adv 2009;27(6):857 65. [58] Dodane V, Khan MA, Merwin JR. Effect of chitosan on epithelial permeability and structure. Int J Pharm 1999;182:21 32. [59] Ayame H, Morimoto N, Akiyoshi K. Self-assembled cationic nanogels for intracellular protein delivery. Bioconjug Chem 2008;19(4):882 90. [60] Sasaki Y, Akiyoshi K. Nanogel engineering for new nanobiomaterials: from chaperoning engineering to biomedical applications. Chem Rec 2010;10(6):366 76. [61] Nakahashi-Ouchida R, Yuki Y, Kiyono H. Development of a nanogel-based nasal vaccine as a novel antigen delivery system. Expert Rev Vaccines 2017;16(12):1231 40. [62] Baldwin AL, Wu NZ, Stein DL. Endothelial surface charge of intestinal mucosal capillaries and its modulation by dextran. Microvasc Res 1991;42(2):160 78. [63] Yuki Y, Nochi T, Harada N, Katakai Y, Shibata H, Mejima M, et al. In vivo molecular imaging analysis of a nasal vaccine that induces protective immunity against botulism in nonhuman primates. J Immunol 2010;185(9):5436 43. [64] Cilloniz C, Amaro R, Torres A. Pneumococcal vaccination. Curr Opin Infect Dis 2016;29(2):187 96. [65] Dinleyici EC. Current status of pneumococcal vaccines: lessons to be learned and new insights. Expert Rev Vaccines 2010;9(9):1017 22. [66] Haas KM, Blevins MW, High KP, Pang B, Swords WE, Yammani RD. Aging promotes B-1b cell responses to native, but not protein-conjugated, pneumococcal polysaccharides: implications for vaccine protection in older adults. J Infect Dis 2014;209(1):87 97. [67] Laferriere C. The immunogenicity of pneumococcal polysaccharides in infants and children: a metaregression. Vaccine 2011;29(40):6838 47. [68] Richter SS, Heilmann KP, Dohrn CL, Riahi F, Diekema DJ, Doern GV. Pneumococcal serotypes before and after introduction of conjugate vaccines, United States, 1999-2011(1). Emerg Infect Dis 2013;19(7):1074 83.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

475

REFERENCES

[69] Weinberger DM, Malley R, Lipsitch M. Serotype replacement in disease after pneumococcal vaccination. Lancet 2011;378(9807):1962 73. [70] Berry AM, Yother J, Briles DE, Hansman D, Paton JC. Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae. Infect Immun 1989;57(7):2037 42. [71] McDaniel LS, Yother J, Vijayakumar M, McGarry L, Guild WR, Briles DE. Use of insertional inactivation to facilitate studies of biological properties of pneumococcal surface protein A (PspA). J Exp Med 1987;165 (2):381 94. [72] Nguyen CT, Kim SY, Kim MS, Lee SE, Rhee JH. Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice. Vaccine 2011;29(34):5731 9. [73] Xu J, Dai W, Wang Z, Chen B, Li Z, Fan X. Intranasal vaccination with chitosan-DNA nanoparticles expressing pneumococcal surface antigen a protects mice against nasopharyngeal colonization by

[74]

[75]

[76]

[77]

Streptococcus pneumoniae. Clin Vaccine Immunol 2011;18(1):75 81. Suzuki H, Watari A, Hashimoto E, Yonemitsu M, Kiyono H, Yagi K, et al. C-Terminal Clostridium perfringens enterotoxin-mediated antigen delivery for nasal pneumococcal vaccine. PLoS One 2015;10(5):e0126352. Fukuyama Y, King JD, Kataoka K, Kobayashi R, Gilbert RS, Hollingshead SK, et al. A combination of Flt3 ligand cDNA and CpG oligodeoxynucleotide as nasal adjuvant elicits protective secretory-IgA immunity to Streptococcus pneumoniae in aged mice. J Immunol 2011;186(4):2454 61. Azegami T, Yuki Y, Sawada S, Mejima M, Ishige K, Akiyoshi K, et al. Nanogel-based nasal ghrelin vaccine prevents obesity. Mucosal Immunol 2017;10(5): 1351 60. Azegami T, Yuki Y, Hayashi K, Hishikawa A, Sawada SI, Ishige K, et al. Intranasal vaccination against angiotensin II type 1 receptor and pneumococcal surface protein A attenuates hypertension and pneumococcal infection in rodents. J Hypertens 2018;36(2):387 94.

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C H A P T E R

27

Effectiveness of Sublingual Immunization: Innovation for Preventing Infectious Diseases Mi-Na Kweon Mucosal Immunology Laboratory, Department of Convergence Medicine, University of Ulsan College of Medicine/Asan Medical Center, Seoul, Republic of Korea

I. INTRODUCTION As the main entry site of most environmental pathogens, mucosal surfaces such as those of the respiratory, gastrointestinal, and genital tracts act as the first line of defense against infectious diseases [1]. Many recent studies have focused on developing mucosal vaccines capable of effectively inducing both mucosal and systemic immune responses, thereby resulting in two layers of host protection [2]. Mucosal vaccination, in contrast to parenteral vaccination, is of particular interest, since it can elicit immune responses, mainly secretory immunoglobulin A (SIgA) antibodies, which are located at the portal of entry of most infectious pathogens [3,4] (Chapter 4: Protective Activities of Mucosal Antibodies). Further, since the route of vaccine administration has a significant effect on the outcome of immune responses, much effort has focused on the development of novel mucosal vaccine delivery routes [5 10]. Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00027-4

Mucosal and skin surfaces, both boundaries with the exterior environment, are covered with special epithelial layers that act as barriers against exogenous challenges by pathogens and soluble antigens. Functionally independent of the systemic immune apparatus, the mucosal immune system has developed its own highly organized immunological tissues [11]. These tissues maintain homeostasis in the vast mucosa by mounting specialized antiinflammatory immune defenses, such as the production of SIgA antibodies and the induction of tolerance against innocuous soluble substances as well as commensal bacteria [12]. Moreover, when administered with appropriate adjuvants, mucosal vaccination can induce not only protective antigen-specific SIgA antibodies and cytotoxic lymphocyte (CTL) responses against pathogens invading via a mucosal surface, but also immunoglobulin G (IgG) responses in the systemic compartment [13]. Furthermore, owing to the migration of IgA antibody-secreting cells

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© 2020 Elsevier Inc. All rights reserved.

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(ASCs), local mucosal immunization leads to antigen-specific IgA production at distant mucosal sites [14]. In human studies, the strongest responses were elicited in mucosal tissues that were directly exposed to antigen and in the adjacent mucosa, respectively [15]. Oral vaccination can induce strong IgA responses in the small intestine, proximal colon, and mammary glands but is relatively less efficient in the respiratory and reproductive tracts [6]. In contrast, intranasal (i.n.) immunization induces SIgA antibodies in the respiratory system and reproductive tract but is less effective for gut immune responses [16]. However, i.n. administration of certain vaccine antigens requires special delivery devices (nebulizers), which have raised safety concerns. In a clinical trial in Switzerland, i.n. administration of inactivated influenza vaccine with heat-labile enterotoxin from enterotoxigenic Escherichia coli (LT) as mucosal adjuvant proved capable of eliciting brisk levels of systemic and mucosal immunity, but some study participants developed Bell’s palsy [17]. Murine studies have demonstrated that i.n. administration of native cholera toxin (nCT) can redirect coadministered vaccine antigen into the central nervous system (CNS), for example, in the olfactory nerves and epithelium, olfactory bulbs, and brain [18,19]. Possibly, facial nerve fibers adsorb the adjuvant, which is followed by retrograde transport and neuronal damage. To date, only one nasal vaccine against seasonal influenza virus has been licensed, and only for use in a specific age group. Oral mucosae, including buccal (cheek lining), sublingual (underside of the tongue), and gingival mucosae, have recently received much attention as novel delivery sites for therapeutic drugs because they do not subject proteins and/or peptides to the degradation associated with gastrointestinal administration. Among oral mucosal routes, the sublingual (s.l.) route is commonly used for immunotherapeutic treatment of allergies because it quickly absorbs

antigen, allowing direct entry into the bloodstream without passing through the intestine or liver and thereby eliciting allergen-specific tolerance [20 23]. No cases of anaphylactic shock were observed in recent human studies of s.l. administered immunotherapy targeting allergies [24 26]. On the basis of these findings, it has been assumed that the s.l. route might be promising for delivery of vaccines targeting infectious diseases.

II. LOCALIZATION OF ANTIGENPRESENTING CELLS IN THE SUBLINGUAL MUCOSA Histologically, the murine s.l. mucosa shows a superficial keratinized pluristratified epithelium overlying a thin lamina propria (LP) with capillary vessels, scattered mononuclear cells, and numerous fibroblasts. Beneath the epithelial layer, the LP contains mononuclear cells devoid of organized lymphoid structures. In an immunohistochemical study (Fig. 27.1), MHC class II1 and/or CD11b1 cells were detected in both the epithelium and LP of mice at steady state; the numbers were much increased within 2 hours after nCT administration via the s.l. mucosa. CD11c1 and Langerin1 cells were also found, although the expression intensities were moderate [27]. To trace the antigen uptake pattern in s.l. mucosa, mice were administered FITC-ovalbumin (OVA) alone or with nCT by the s.l. route in a time-dependent manner [28]. The FITC-conjugated OVA adhered to the s.l. mucosa for 2 hours after s.l. administration without nCT and with nCT; most FITC-OVA disappeared within 4 hours of administration. The histological traits of the s.l. mucosa resemble those of other mucosal tissues (e.g., skin and buccal and vaginal mucosa): They possess stratified epithelium, lack mucosa-associated lymphoid tissues, and show ability to adhere to and penetrate antigen with or without mucosal adjuvant.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

IV. MECHANISM FOR INDUCTION OF CD4 1 T CELL ACTIVATION FOLLOWING SUBLINGUAL VACCINATION

479

FIGURE 27.1 Morphology of the s.l. mucosa and in vivo antigen uptake patterns following sublingual immunization. Small pieces of s.l. mucosal tissue of naı¨ve BALB/c mice were fixed with paraffin, cut longitudinally (length of the tongue), and stained with H&E (A). Sublingual mucosa was isolated at 0 h (B) or 2 h following s.l. immunization with OVA plus nCT (C, D). Tissue sections were stained with FITC-conjugated anti-MHC class II and PE-conjugated anti-CD11b mAbs. Naı¨ve BALB/c mice were immunized with FITC-OVA (E) and FITC-OVA plus nCT (F) by the s.l. route, and the s.l. mucosa was harvested 2 h later. DAPI was used for counterstaining.

III. ROLE OF DRAINING LYMPH NODES IN SUBLINGUAL VACCINATION It is important to identify the site of primary antigen presentation after vaccination. By day 3 after s.l. administration of OVA plus nCT [28], adoptively transferred OVA-specific CD41 T cells isolated from DO11.10 mice had proliferated mainly in the cervical and mediastinal lymph nodes (LNs) and spleen of recipient BALB/c mice. After s.l. vaccination of mice treated with FTY 720, which prevents the egress of T cells from secondary lymphoid organs [29], proliferating OVA-specific CD41 T cells were found only in the cervical LNs, not in the mediastinal LNs or spleen [28]. The cervical LN is the primary draining LN for priming of CD41 T cells after s.l. vaccination, and s.l. administration of

protein antigen with mucosal adjuvant enhances the antigen-specific CD41 T cell activation that initially takes place in cervical LNs.

IV. MECHANISM FOR INDUCTION OF CD41 T CELL ACTIVATION FOLLOWING SUBLINGUAL VACCINATION After identifying the site of primary antigen presentation, my group assessed mRNA expression levels of chemokines and chemokine receptors in s.l. mucosa and cervical LN in a time-dependent manner. CCL19 (ligand of CCR7) and CCL27 and CCL28 (ligands of CCR10) in the s.l. mucosa were substantially expressed at steady state [28]. mRNA expression levels of CCL19 were higher at 0.5 and 2 hours but were

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considerably lower by 6 hours after s.l. administration with nCT. Sublingual mucosa possessed mature professional antigen-presenting cells (APCs) and had unique chemokine expression patterns at both steady state and after nCT administration. mRNA expression levels of CCL21 in cervical LN were approximately 30 times higher 24 hours after s.l. vaccination with nCT than at steady state and other time points. In addition, predominant enhanced levels of CCL21 in cervical LN following s.l. administration were also confirmed by immunohistochemical analysis of protein levels [28]. Thus both CCL19 and CCL21 may play substantial roles in immune responses in the s.l. mucosa (Fig. 27.2). In order to identify crucial APCs involved in ferrying and presentation of antigen in draining LNs after s.l. vaccination, several selective APC

depletion systems were used (i.e., langerinDTR, CD11c-DTR, and macrophage depletion). The CD11c1 dendritic cell (DC)-depleted mice completely lacked OVA-specific CD41 T cell proliferation in both the cervical and mediastinal LNs, whereas both langerin-depleted and macrophage-depleted mice had similar levels of CD41 T cell proliferation and differed only from recipient wild-type C57BL/6 mice [28]. This analysis revealed that the majority of T cell priming in the cervical LNs was mediated by the CD11c1 DCs, even though the T cell priming of langerin-DTR and clodronate-treated mice was lower than in wild-type mice. Of note, OVA-specific CD41 T cell proliferation was completely abrogated in CCR72/2 mice but not in CCR62/2 mice [28]. Thus antigenspecific CD41 T cell proliferation induced by

FIGURE 27.2 Depiction of antigen delivery and antigen presentation by sublingual vaccination. Epithelial cells in the s.l. mucosa express high levels of mRNA for CCL19, CCL27, and CCL28 at steady-state conditions. In the s.l. mucosa, Langerhans cells are within the stratified epithelial layer and submucosal CD8α2 DCs are beneath the layer. DCs migrate into cervical LN, which highly express CCL19 and CCL21 following s.l. vaccination with antigen plus nCT as mucosal adjuvant. Migratory CD8α2 DCs derived from s.l. submucosa may have a role in acquiring antigen and in sharing it with resident CD8α1 DCs. Finally, both CD8α2 and CD8α1 DCs act as predominant APCs to induce CD41 T cell activation in cervical lymph nodes. IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

VI. SUBLINGUAL ADMINISTRATION IS USEFUL FOR INDUCTION OF ANTIBODY AGAINST VIRAL INFECTION

s.l. vaccination must be tightly regulated by CD11c1 DCs in a CCR7-CCL19/21-dependent manner (Fig. 27.2).

V. SUBLINGUAL VACCINATION INDUCES BOTH SYSTEMIC AND MUCOSAL ANTIBODY RESPONSES Groups of mice were immunized s.l. twice with OVA (10, 50, 200 μg) plus nCT (2 μg) as mucosal adjuvant and antigen-specific antibody levels were determined in the serum and mucosal compartments (saliva, nasal wash, and bronchial alveolar lavage samples) [27]. Antigenspecific IgG antibodies in sera were readily detected in mice immunized twice with 10 μg of OVA plus nCT and were comparable to those of mice vaccinated i.n. Increased doses (50 or 200 μg) of OVA antigen by s.l. or i.n. routes resulted in more significant and/or consistent serum IgG antibodies. Importantly, s.l. administration with OVA plus nCT elicited high levels of OVAspecific IgA antibodies in the mucosal secretions and were of same magnitude as those induced via the i.n. route [27]. Systemic and mucosal antibody responses against CT were identical.

VI. SUBLINGUAL ADMINISTRATION IS USEFUL FOR INDUCTION OF ANTIBODY AGAINST VIRAL INFECTION Mice were immunized s.l. twice at 2-week intervals with formalin-inactivated A/PR/8 virus plus mCTA/LTB, a subunit of mutant CT E112K with the pentameric B subunit of a LT as a nontoxic adjuvant [16] to determine the efficacy of s.l. influenza virus vaccination (Chapter 11: Toxinbased Modulators for Regulation of Mucosal Immune Responses). Mouse groups receiving inactivated A/PR/8 virus either alone or together with mCTA/LTB by the s.l. route had higher levels of A/PR/8-specific IgG and IgA antibodies in serum, bronchial alveolar lavage (BAL) fluid,

481

and nasal wash than the control mice vaccinated with PBS [30]. The serum IgG1 and IgG2a antibody response profiles of immunized mice were parallel and not partial to IgG1 or IgG2a antibody responses. However, no significant levels of IgE antibodies were elicited by s.l. vaccination of inactivated A/PR/8 virus with or without mCTA/ LTB. These finding suggest that s.l. vaccination could avoid the danger of anaphylactic shock and/or allergic reactions provoked by IgE antibodies. To further ascertain the levels of A/PR/8specific IgA antibodies in mucosal compartments, BAL fluid, nasal wash samples, saliva, and fecal extract were collected 1 week after the final vaccination. Mice vaccinated s.l. with inactivated A/ PR/8 alone or together with mCTA/LTB showed significantly higher levels of A/PR/8-specific IgA antibodies in mucosal secretions than did the PBS-vaccinated animals [30]. In addition, s.l. vaccination with formalin-inactivated A/PR/8 virus plus mCTA/LTB elicited more A/PR/8-specific IgG ASCs in the spleen and lung than did PBS or inactivated A/PR/8 virus alone. High numbers of A/PR/8-specific IgA ASCs were detected in nasal passages, submandibular glands, and small and large intestines of mice vaccinated s.l. with formalin-inactivated A/PR/8 virus plus mCTA/ LTB. Mice given mCTA/LTB adjuvant plus inactivated whole virus antigen s.l. had 100% survival against lethal i.n. challenge of influenza virus. Consistent with these findings, viral titers in the BAL fluid of mice vaccinated s.l. with inactivated A/PR/8 virus plus mucosal adjuvant showed complete clearance of A/PR/8 virus following i. n. challenge [30]. Previous studies showed that human papillomavirus (HPV)-like particles (VLPs) are potent immunogens for both parenteral and mucosal immunization [31,32]. To determine the effectiveness of the s.l. route, mice were immunized s.l. with HPV16 L1 VLP or PBS and challenged by the genital route with HPV16 pseudovirions carrying a luciferase reporter gene [33]. The results showed that s.l. immunization with HPV16 VLP was more protective against HPV16 genital infection than was PBS treatment. Vaccination s.l. with HPV16 L1 VLP conferred

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complete protection against HPV infection, since no luminescence activity was detected above the background levels measured in the negative control mice that remained unchallenged with HPV16 luciferase pseudovirions [33]. Similarly, mice immunized with HPV16 VLP with nCT showed complete protection, as did mice immunized intramuscularly with HPV16 VLP plus alum. Overall, the s.l. immunization route elicited neutralizing antibodies in the cervicovaginal region, which may account for the complete protection.

VII. SUBLINGUAL ADMINISTRATION DOES NOT REDIRECT ANTIGENS TO THE CENTRAL NERVOUS SYSTEM Trafficking of vaccine antigens and adjuvants into the CNS is crucial for development of a novel mucosal delivery route. When acridium-labeled A/PR/8 virus was administered i.n. without adjuvant, viral vaccine antigen accumulated in the olfactory bulbs and brain within 24 hours; however, labeled A/PR/ 8 virus was undetectable in either area following s.l. administration [30]. In addition, viral RNA was strongly expressed in both lung and olfactory bulb tissues of mice infected i.n. with the A/PR/8 virus, but the A/PR/8 virus gene was not detected in the lungs, olfactory bulbs, and brains of mice infected s.l [30]. Thus inactivated and live A/PR/8 virus can be transported into CNS tissues after i.n. administration but not after s.l. administration.

VIII. SUBLINGUAL VACCINATION INDUCES T AND B CELL ACTIVATION IN FEMALE MOUSE GENITAL TISSUES The potential of s.l. immunization to induce remote antibody responses and CTL in the

mouse female genital tract was examined (Chapter 16: Regulation of Mucosal Immunity in the Genital Tract: Balancing Reproduction and Protective Immunity). We found that s.l. immunization induces large numbers of OVA-specific and CT-specific ASCs in the genital tract [33]. These responses were dominated by IgA ASCs and were comparable to or higher than those seen after i.n. and intravaginal immunization. Furthermore, in vivo specific cytotoxic activity was readily detected in the genital tracts of mice immunized s.l. with OVA and nCT, and these responses were comparable to those seen after intravaginal immunization and somewhat lower than those seen after i.n. immunization [33]. These data indicate that s.l. immunization can induce antigen-specific antibody-secreting B cells and effector CTL responses in genital tissues.

IX. CONCLUDING REMARKS AND FUTURE PERSPECTIVES The results described suggest that the s.l. delivery route may be highly effective and safe. It allows protective immune responses by nonreplicating antigens or inactivated and live virus and enhances systemic IgG and mucosal IgA antibodies and CTL responses. Many issues regarding s.l. administration remain to be resolved, including the development of mucosal adjuvants and improved formulations that would enable enhanced efficacy and lowered dose. An important observation derived from recent studies is that the s.l. route may offer a safer alternative to nasal delivery of vaccines. Other findings strongly suggest that the s.l. delivery route could be a more effective avenue than traditional approaches for vaccination against both respiratory and genital viral infections. Previous study indicate that s.l. administration is convenient for delivery of drugs and low-molecular-weight molecules to the bloodstream, avoiding the enterohepatic circulation and immediate destruction of ingested

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IX. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

molecules by gastric acid or partial first-pass effects of hepatic metabolism [34]. At present, the s.l. route is commonly used for human allergenspecific immunotherapies [21,26,35]. It seems likely that different immune modulators modify APCs in the s.l. mucosa and set the T cells (e.g., Th2/Th1 vs Treg immune responses) in the draining LN for tolerance or immunity. Recent findings of the simultaneous induction of TABLE 27.1

secretory antibodies and CTL in mucosal compartments after s.l. immunization with a nonreplicating antigen or inactivated and live virus provide a foundation for further evaluation of this alternative form of vaccination against pathogens, as summarized in Table 27.1 [51]. Controlled clinical trials will be necessary to determine the safety and efficacy of this novel mucosal route for administration of vaccines

Results of Sublingual Vaccination With Various Antigens and Adjuvants

Antigen

Adjuvant

Protection

Reference

Influenza A/PR/8

2

1/ 2

[30]

deltaNS1 influenza A

2

1

[36]

RSV sFsyn

HDAd

1

[37]

SARS-S protein (spike)

rAd

n.d.

[38]

HIV Env (envelope glycoprotein)

rAd5

n.d

[39]

Ebola ZGP (Zaire glycoprotein)

rAd5

1

[40]

Tetanus toxin fragment C (TTFC)

Bacillus subtilis mLT

1

[41]

Influenza WIV (β-propiolactone-inactivated)

2

n.d

[42]

Influenza WIV (formalin-inactivated)

mCTA-LT (5 μg)

1

[30]

Influenza HA subunit

LTK63 (5 μg)

n.d

[43]

Influenza A virosome

c-di-GMP (7.5 μg)

n.d

[44]

Influenza 3M2eC protein

CT (2 μg)

1

[45]

HPV16L1 VLP

CT (2 μg)

1

[33]

HPV16L1 VLP

CTB (10 μg)

n.d

[46]

HIV-1 Pol

CTB

n.d

[47]

CT (1 μg) HIV-1 gp41

CT (1 μg)

n.d

[47]

Salmonella proteins (SPP)

CT (1 μg)

1

[48]

CpG (10 μg) Helicobacter pylori lysate

CT (10 μg)

1

[49]

Ovalbumin (OVA)

CT (2 μg)

n.d

[27]

Ovalbumin (OVA)

Ad2F (25 μg)

n.d

[50]

CT (2 μg) n.d., not determined.

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27. EFFECTIVENESS OF SUBLINGUAL IMMUNIZATION: INNOVATION FOR PREVENTING INFECTIOUS DISEASES

originally formulated for delivery by other routes [52]. The development of mucoadhesive formulations with enhanced permeabilizing properties to facilitate contact of vaccine antigens with the s.l. epithelium will be a major milestone for the eventual use of s.l. vaccines in humans.

References [1] McGhee JR, Kiyono H. The mucosal immune system. In: Paul WE, editor. Fundamental Immunology. Philadelphia: Lippincott Williams & Wilkins; 2004. p. 965 1020. [2] Boyaka PN, McGhee JR, Czerkinsky C, Mestecky J. Mucosal vaccines: an overwiew. In: Mestecky J, Bienenstock J, Lamm ME, mayer L, McGhee JR, Strober W, editors. Mucosal Immunology. 3rd ed Amsterdam: Elsevier/Academic Press; 2007. p. p855 874. [3] Brandtzaeg P. Induction of secretory immunity and memory at mucosal surfaces. Vaccine 2007;25:5467 84. [4] Russell MW, Kilian M. Biological activities of IgA. In: Mestecky J, Bienenstock J, Lamm ME, mayer L, McGhee JR, Strober W), editors. Mucosal Immunology. 3rd ed Amsterdam: Elsevier/Academic Press; 2007. p. p267 289. [5] Nair PN, Schroeder HE. Local immune response to repeated topical antigen application in the simian labial mucosa. Infect Immun 1983;41:399 409. [6] Quiding M, Nordstrom I, Kilander A, Andersson G, Hanson LA, et al. Intestinal immune responses in humans. Oral cholera vaccination induces strong intestinal antibody responses and interferon-gamma production and evokes local immunological memory. J Clin Invest 1991;88:143 8. [7] Kozlowski PA, Cu-Uvin S, Neutra MR, Flanigan TP. Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women. Infect Immun 1997;65:1387 94. [8] Jertborn M, Nordstrom I, Kilander A, Czerkinsky C, Holmgren J. Local and systemic immune responses to rectal administration of recombinant cholera toxin B subunit in humans. Infect Immun 2001;69:4125 8. [9] Johansson EL, Wassen L, Holmgren J, Jertborn M, Rudin A. Nasal and vaginal vaccinations have differential effects on antibody responses in vaginal and cervical secretions in humans. Infect Immun 2001;69:7481 6. [10] Glenn GM, Taylor TD, Li X, Frankel S, Montemarano A, Alving CR. Transcutaneous immunization: a human vaccine delivery strategy using a patch. Nat Med 2000;6:1403 6.

[11] Brandtzaeg P, Pabst R. Let’s go mucosal: communication on slippery ground. Trends Immunol 2004;25:570 7. [12] Suzuki K, Fagarasan S. How host-bacterial interactions lead to IgA synthesis in the gut. Trends Immunol 2008;29:523 31. [13] Yuki Y, Kiyono H. New generation of mucosal adjuvants for the induction of protective immunity. Rev Med Virol 2003;13:293 310. [14] Kunkel EJ, Butcher EC. Plasma-cell homing. Nat Rev Immunol 2003;3:822 9. [15] Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med 2005;11:S45 53. [16] Kweon MN, Yamamoto M, Watanabe F, Tamura S, Van Ginkel FW, et al. A nontoxic chimeric enterotoxin adjuvant induces protective immunity in both mucosal and systemic compartments with reduced IgE antibodies. J Infect Dis 2002;186:1261 9. [17] Mutsch M, Zhou W, Rhodes P, Bopp M, Chen RT, et al. Use of the inactivated intranasal influenza vaccine and the risk of Bell’s Palsy in Switzerland. N Engl J Med 2004;350:896 903. [18] van Ginkel FW, Jackson RJ, Yuki Y, McGhee JR. Cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues. J Immunol 2000;165:4778 82. [19] van Ginkel FW, Jackson RJ, Yoshino N, Hagiwara Y, Metzger DJ, et al. Enterotoxin-based mucosal adjuvants alter antigen trafficking and induce inflammatory responses in the nasal tract. Infect Immun 2005;73:6892 902. [20] Kildsgaard J, Brimnes J, Jacobi H, Lund K. Sublingual immunotherapy in sensitized mice. Ann Allergy Asthma Immunol 2007;98:366 72. [21] Brimnes J, Kildsgaard J, Jacobi H, Lund K. Sublingual immunotherapy reduces allergic symptoms in a mouse model of rhinitis. Clin Exp Allergy 2007;37:488 97. [22] Noonan PK, Benet LZ. Incomplete and delayed bioavailability of sublingual nitroglycerin. Am J Cardiol 1985;55:184 7. [23] Cleary JF. Pharmacokinetic and pharmacodynamic issues in the treatment of breakthrough pain. Semin Oncol 1997;24:S16 13-19. [24] Agostinis F, Tellarini L, Canonica GW, Falagiani P, Passalacqua G. Safety of sublingual immunotherapy with a monomeric allergoid in very young children. Allergy 2005;60:133. [25] Grosclaude M, Bouillot P, Alt R, Leynadier F, Scheinmann P, et al. Safety of various dosage regimens during induction of sublingual immunotherapy. A preliminary study. Int Arch Allergy Immunol 2002;129:248 53. [26] Olaguibel JM, Alvarez Puebla MJ. Efficacy of sublingual allergen vaccination for respiratory allergy in

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

485

REFERENCES

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

children. Conclusions from one meta-analysis. J Investig Allergol Clin Immunol 2005;15:9 16. Cuburu N, Kweon MN, Song JH, Hervouet C, Luci C, et al. Sublingual immunization induces broad-based systemic and mucosal immune responses in mice. Vaccine 2007;25:8598 610. Song JH, Kim JI, Kwon HJ, Shim DH, Cuburu N, et al. CCR7-CCL19/CCL21 regulated dendritic cells are responsible for the effectiveness of sublingual vaccination. J Immunol 2009;183:7851 9. Fujii R, Kanai T, Nemoto Y, Makita S, Oshima S, et al. FTY720 suppresses CD41CD44highCD62L2 effector memory T cell-mediated colitis. Am J Physiol Gastrointest Liver Physiol 2006;291:G267 274. Song JH, Nguyen HH, Cuburu N, Horimoto T, Ko SY, et al. Sublingual vaccination with influenza virus protects mice against lethal viral infection. Proc Natl Acad Sci USA 2008;105:1644 9. Bowman EP, Kuklin NA, Youngman KR, Lazarus NH, Kunkel EJ, et al. The intestinal chemokine thymusexpressed chemokine (CCL25) attracts IgA antibodysecreting cells. J Exp Med 2002;195:269 75. Balmelli C, Roden R, Potts A, Schiller J, De Grandi P, et al. Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions. J Virol 1998;72:8220 9. Cuburu N, Kweon MN, Hervouet C, Cha HR, Pang YY, et al. Sublingual immunization with nonreplicating antigens induces antibody-forming cells and cytotoxic T cells in the female genital tract mucosa and protects against genital papillomavirus infection. J Immunol 2009;183:7851 9. Senel S, Kremer M, Nagy K, Squier C. Delivery of bioactive peptides and proteins across oral (buccal) mucosa. Curr Pharm Biotechnol 2001;2:175 86. Moingeon P, Batard T, Fadel R, Frati F, Sieber J, et al. Immune mechanisms of allergen-specific sublingual immunotherapy. Allergy 2006;61:151 65. Park HJ, Ferko B, Byun YH, Song JH, Han GY, et al. Sublingual immunization with a live attenuated influenza a virus lacking the nonstructural protein 1 induces broad protective immunity in mice. PLoS One 2012;7:e39921. Fu YH, Jiao YY, He JS, Giang GY, Zhang W, et al. Sublingual administration of a helper-dependent adenoviral vector expressing the codon-optimized soluble fusion glycoprotein of human respiratory syncytial virus elicits protective immunity in mice. Antiviral Res 2014;105:72 9. Shim BS, Stadler K, Nguyen HH, Yun CH, Kim DW, et al. Sublingual immunization with recombinant adenovirus encoding SARS-CoV spike protein induces

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

systemic and mucosal immunity without redirection of the virus to the brain. Virol J 2012;9:215. Domm W, Brooks L, Chung HL, Feng C, Bowers WJ, et al. Robust antigen-specific humoral immune responses to sublingually delivered adenoviral vectors encoding HIV-1 Env: association with mucoadhesion and efficient penetration of the sublingual barrier. Vaccine 2011;29:7080 9. Choi JH, Schafer SC, Zhang L, Kobinger GP, Juelich T, et al. A single sublingual dose of an adenovirus-based vaccine protects against lethal Ebola challenge in mice and guinea pigs. Mol Pharm 2012;9:156 67. Amuguni H, Lee S, Kerstein K, Brown D, Belitsky B, et al. Sublingual immunization with an engineered Bacillus subtilis strain expressing tetanus toxin fragment C induces systemic and mucosal immune responses in piglets. Microbes Infect 2012;14:447 56. Murugappan S, Patil HP, Frijlink HW, Huckriede A, Hinrichs WL. Simplifying influenza vaccination during pandemics: sublingual priming and intramuscular boosting of immune responses with heterologous whole inactivated influenza vaccine. AAPS J 2014;16:342 9. Gallorini S, Taccone M, Bonci A, Nardelli F, Casini D, et al. Sublingual immunization with a subunit influenza vaccine elicits comparable systemic immune response as intramuscular immunization, but also induces local IgA and Th17 responses. Vaccine 2014;32:2382 8. Pedersen GK, Ebensen T, Gjeraker IH, Svindland S, Bredholt G, et al. Evaluation of the sublingual route for administration of influenza H5N1 virosomes in combination with the bacterial second messenger c-di-GMP. PLoS One 2011;6:e26973. Shim BS, Choi YK, Yun CH, Lee EG, Jeon YS, et al. Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza. PLoS One 2011;6:e27953. Cho HJ, Kim JY, Lee Y, Kim JM, Kim YB, et al. Enhanced humoral and cellular immune responses after sublingual immunization against human papillomavirus 16 L1 protein with adjuvants. Vaccine 2010;28:2598 606. Hervouet C, Luci C, Cuburu N, Cremel M, Bekri S, et al. Sublingual immunization with an HIV subunit vaccine induces antibodies and cytotoxic T cells in the mouse female genital tract. Vaccine 2010;28:5582 90. Huang CF, Wu TC, Wu CC, Lee CC, Lo WT, et al. Sublingual vaccination with sonicated Salmonella proteins and mucosal adjuvant induces mucosal and systemic immunity and protects mice from lethal enteritis. APMIS 2011;119:468 78. Raghavan S, Ostberg AK, Flach CF, Ekman A, Blomquist M, et al. Sublingual immunization protects

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

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against Helicobacter pylori infection and induces T and B cell responses in the stomach. Infect Immun 2010;78:4251 60. [50] Jun S, Clapp B, Zlotkowska D, Hoyt T, Holderness K, et al. Sublingual immunization with adenovirus F protein-based vaccines stimulates protective immunity against botulinum neurotoxin A intoxication. Int Immunol 2012;24:117 28.

[51] Kraan H, Vrieling H, Czerkinsky C, Jiskoot W, Kersten G, et al. Buccal and sublingual vaccine delivery. J Control Release 2014;190:580 92. [52] Razafindratsita A, Saint-Lu N, Mascarell L, Berjont N, Bardon T, et al. Improvement of sublingual immunotherapy efficacy with a mucoadhesive allergen formulation. J Allergy Clin Immunol 2007;120:278 85.

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C H A P T E R

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M Cell-Targeted Vaccines Shintaro Sato1 and David W. Pascual2 1

Mucosal Vaccine Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 2Department of Infectious Diseases & Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States

I. INTRODUCTION Discovered in the 1970s, microfold (M) cells are a subtype of intestinal epithelial cells. Electron microscopy studies revealed their close proximity to lymphocytes, implying a relevance for antigen (Ag) uptake [1,2]. M cells are found in the follicle-associated epithelium (FAE), which covers the luminal side of the mucosal-associated lymphoid tissue (MALT), such as Peyer’s patches (PPs) in the intestine (Fig. 28.1) [2,3]. The microvilli on the luminal surface of M cells are short and sparse in comparison to villi present on the surrounding columnar epithelial cells. M cells have a pocket structure that encloses antigen-presenting cells (APCs), including dendritic cells, macrophages, and lymphocytes [4]. Furthermore, their production and expression of mucus and digestive enzymes are remarkably low [4]. Because of the proximity of M cell-bearing Ags to APCs, it is thought to convey macromolecules from the M cell luminal surface to APCs. In fact, some pathogens enter the host via PP M cells, exploiting this Ag-sampling process for infection. Members of the enterobacteria family [5], such Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00028-6

as Salmonella, Escherichia coli, Vibrio cholerae, and Shigella, and viruses such as reovirus types 1 and 3 [6 8], murine norovirus [9], rotaviruses [10], poliovirus [11], and HIV [12,13], and prions [14] infect via the use of the M cell entry system. Consequently, vaccination with M celltargeted Ags is expected to be much more effective than using soluble Ags alone, allowing a much lower dose to be effective for the initiation of antigen-specific immune response in the inductive sites of aerodigestive tracts, including nasopharyngeal-associated lymphoid tissue (NALT) and gut-associated lymphoid tissues (GALT). Molecules expressed on M cells’ luminal surface are indispensable for M cell targeting, and efforts are under way to help identify new targets to enable M cell vaccination (Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance). In addition to their morphological appearance and their short microvilli as evidenced by electron microscopy, M cells have a modified α(1,2)-linked fucose capable of binding the lectin Ulex europaeus agglutinin-1 (UEA-1) [15].

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α(1,2)-linked fucose Short and sparse microvilli

Lower expression of mucus and digestive enzymes

M cell

FIGURE 28.1

Characterization of M cells

(see text).

FAE

Pocket structure at basal side DC Mϕ L

In contrast, wheat germ agglutinin (WGA) lectin binds intestinal epithelial cells, not M cells [16]; hence, WGA-negative UEA-1-positive intestinal epithelial cells are originally designated as M cells. When their distribution was analyzed by WGA and UEA-1 staining, M cells were found to be lower in abundance, limiting biological analysis of isolated M cells. With the availability of UEA-1 lectin and a monoclonal antibody (mAb) directed to α(1,2)-linked fucose on M cells, cell-sorted M cells were evaluated for their gene expression patterns in comparison to other epithelial cells [17]. The study identified M-cell-specific genes, one of which was the glycoprotein 2 (GP2) present on its luminal surface [17]. GP2 is now recognized as a surface marker for mature M cells [17,18]. In addition to the “traditional” M cells, sporadic M-like cells are found outside of the FAE; these are the intestinal villus M cells [19] and respiratory M cells [20]. Although both villus and respiratory M cells show short and irregular microvilli, like PP and NALT M cells, they have less chance to be in contact with antigenpresenting cells [21]. However, these cells are also considered to contribute to antigen uptake because oral or nasal immunization to GALTor NALT-deficient mice could induce antigenspecific responses, even though organized PP and NALT M cells do not exist in these mice [19,20].

II. M CELL DIFFERENTIATION The transcription factors that regulate differentiation into M cells are unclear, unlike those regulating other intestinal epithelial cells, such as absorptive epithelial cells, enteroendocrine cells, goblet cells, and Paneth cells [22]. Receptor activator of nuclear factor kappa-B (RANK) is expressed on the basal membrane side of intestinal epithelial cells, but in mucosal tissues, its ligand, RANK ligand (RANKL), has been detected only in the subepithelial dome, which is directly beneath the FAE in PPs [23]. Since the number of PP M cells is markedly reduced in RANKL-deficient mice, RANKL plays a critical role in inducing M cell differentiation [23]. These RANKL-expressing M cell inducer cells belong to Podoplanin1 CD312 mesenchymal cell populations [24]. Therefore, Twist2-Cre or Col6a1-Cre Rankl-floxed mice that lack the inducer cell pathway also show severe reduction of M cells [24,25], similar to intestinal epithelial-cell-specific RANK-deficient mice [26]. Conversely, systemic administration of recombinant RANKL led to the formation of cells associated with PPs as well as diffuse and dense M cell expression on intestinal villi outside the FAE [23]. RANKL stimulation has been shown to induce M cell differentiation in in vitro cultured human primary intestinal epithelial cells [27]. This evidence shows that the

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II. M CELL DIFFERENTIATION

RANK RANKL signaling pathway is essential for the development of M cells. In the search for transcription factors that mediate gene expression by intestinal epithelial cells under the influence of the RANKL stimulation, Spi-B, a member of the ETS family, was identified as such a candidate [28]. Spib is found to be a gene specifically expressed in M cells [17], and Spi-B-deficient mice are devoid of GP21 mature M cells [28 30]. A transcription factor Spi-B may be a master regulator of M cells, since it was found to be important for M cell differentiation and responsible for the expression of several M-cell-associated genes, including GP2 [28,30]. In fact, Spi-B-deficient mice were incapable of the uptake of fluorescently labeled beads and not susceptible to oral infection with Salmonella, implicating Spi-B’s role in M cell differentiation [28,30]. However, Spi-B deficiency did not prevent PP colonization by the endogenous intratissue commensal bacteria Alcaligenes, although the level tended to be lower in Spi-B2/2 mice when compared to colonization of wild-type mice [30]. Epithelial organoids cultured in the presence of RANKL enabled Spi-B-dependent differentiation of FAE into M cells, and intestinal epithelial organoids derived from Spi-B2/2 mice were unable to induce M cell differentiation

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[29]. Hence, these studies demonstrate the importance of Spi-B and RANKL for M cell differentiation, although there remains a Spi-Bindependent pathway, since some M cells do remain in Spi-B2/2 mice [30]. Mature M cells are denoted as GP2high Tnfaip21, whereas immature M cells are denoted as GP2low Tnfaip21 [31]. It has been reported that forced expression of Spi-B in intestinal epithelial organoids alone failed to induce M cell differentiation. This observation suggests that as-yet-unidentified molecule induced by the RANKL stimulus, most likely a transcription factor, may be necessary for cooperative effects with Spi-B to initiate differentiation into M cell [29]. The fact that RelB, a member of the nuclear factor-κB (NF-κB) family, is essential for the RANKL-induced differentiation of osteoclasts suggests that RelB activation may also be relevant for M cell maturation [31]. Exogenous RANKL treatment of Peyer’s patchless aly/aly mice failed to induce M cell maturation. This was further supported in a recent study showing the association of NF-κB with M cell maturation and differentiation. The canonical (RelA) as well as noncanonical (RelB) NF-κB activation via TRAF6 were important for M cell development (Fig. 28.2) [32]. FIGURE 28.2 Differentiation of M cells. RANKL stimulation triggers M cell differentiation by sequential activation of several transcription factors via TRAF6. Other as-yet-unidentified transcription factor(s) induced by RANKL stimulus might involve in this signaling. Source: This is modified figure of original one by Kanaya T, Sakakibara S, Jinnohara T, Hachisuka M, Tachibana N, Hidano S, et al. Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-kappaB signaling. J Exp Med 2018;215(2):501 519 [32].

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III. CANDIDATE MOLECULES FOR M CELL TARGETING AND THEIR LIGANDS Given that pathogens have hijacked M cells as a route of infection [6 13] and luminal Ags are sampled via M cells [3,5], it is logical to target M cells for effective mucosal vaccination. Several in vivo studies conducted in small experimental animals and nonhuman primates have described the potency for M cell-targeted vaccination for infectious diseases and tolerance. Promising and/or interesting luminal M cell ligands and their applicability as targeting molecules are discussed below.

A. α(1,2)-Linked Fucose As was mentioned above, α(1,2)-linked fucose, which is recognized by UEA-1, has long been known to detect M cells [15]. In fact, a couple of studies have tested the use of UEA-1 for M cell targeting for vaccination [33,34]. In a DNA vaccine for HIV-1, UEA-1 proved effective in mucosal targeting of the nasal mucosa, eliciting serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) anti-gp120 binding Abs, as well as increased CTL responses [34]. In an effort to develop an M cell-specific mAb, UEA-11 PP epithelial cells were cell-sorted to immunize rats and successfully developed the anti-M-cell mAb, NKM16-2-4 clone [35]. This anti-M-cell mAb recognizes a specific glycosylation site of α(1,2)-linked fucose in the same way as UEA-1 does, but it also appears to recognize complexes with other sugar chains, since UEA-1 does not completely inhibit NKM16-2-4 binding. When this mAb is conjugated to botulinum toxin heavy-chain C-terminus (Hc) and is administered to mice, it elicits an immune response even at doses that stimulated no effect with the Hc alone. In addition, NKM16-2-4-conjugated Ags elicited greater Ab responses than did UEA-1-conjugated Ags [35]. However, since

α(1,2)-linked fucose is expressed by other luminal, absorptive epithelial cells in the ileum and large intestine, such as goblet and Paneth cells, as well as enteric bacteria [36], the UEA-1 and NKM16-2-4 mAb may be delivering vaccine antigens to M cells as well as other epithelial cells.

B. GP2 The mature M cell marker GP2 is a receptor for FimH, a structural protein for type I pili from Salmonella and E. coli, as well as being a receptor for botulinum neurotoxin complex [18,37]. GP2 facilitates the uptake of these microorganisms and toxins by M cells. Thus FimH and the nontoxic hemagglutinin from botulinum neurotoxin complex are potential M-cell-targeting molecules. A recent study developed an aptamer with a strong affinity for murine GP2 using the systematic evolution of ligands by exponential enrichment (SELEX) technique [38]. Additionally, this group tested a streptavidin (SA)-conjugated anti-GP2 Ab that, when mixed with a biotinylated Ag, created M celltargeted immunogens [39]. Importantly, oral administration of biotinylated ovalbumin (OVA) conjugated with anti-GP2-SA efficiently induced OVA-specific fecal IgA, independently of the presence or absence of cholera toxin as a mucosal adjuvant. The other group used GP2 to screen a library of 12amino-acid phage peptides and identified one peptide with M cell tropism that binds strongly to GP2 [40]. Subsequently, a vaccine conjugate made from the C-terminus of this peptide fused to a reporter protein enhanced green fluorescent protein (EGFP). Oral administration of this conjugate into mice without any adjuvant induced EGFP-specific serum IgG and fecal IgA Abs. Thus, this peptide is a potential targeting molecule. Further elucidation is needed to determine the peptide’s adjuvant properties.

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III. CANDIDATE MOLECULES FOR M CELL TARGETING AND THEIR LIGANDS

The presence of GP2 in other tissues may diminish the feasibility of using GP2 ligands for potential M cell-targeted vaccination. GP2 is also expressed on pancreatic zymogen granule membranes. A portion of GP2 is secreted, and a large amount of GP2 is present in pancreatic fluid [41]. A potential unexpected consequence is that GP2-targeted oral vaccines may be bound by GP2 in pancreatic secretions flowing into lumen of intestinal tract; thus, it is important to determine the pharmacokinetics of their transport.

C. PrPC The bursa of Fabricius, a lymphoid organ found only in birds, is an important tissue for B cell maturation [1,42]. The internal surface of the bursa of Fabricius contains many folds and large numbers of lymphoid follicles. This surface is covered with specialized epithelial cells that can take up luminal Ags. Thus, the luminal surface of the bursa of Fabricius contains cells that resemble M cells [1]. Comprehensive transcriptome analysis identified 28 molecules expressed by both bursa of Fabricius epithelial cells and the mouse PP FAE [43]. The cellular prion protein (PrPC) was among these molecules showing M cell specificity, and it was demonstrated that anti-PrPC Abs is specifically bound by GP21 M cells in the murine FAE [43]. In subsequent analyses, it was shown that Brucella abortus Hsp60 bound to macrophageexpressed PrPC. Consequently, M cell PrPC contributes to effective uptake of B. abortus via Hsp60 on B. abortus [44]. These results suggest that targeting M cell PrPC with Hsp60 or antiPrPC Abs may be a suitable vaccine antigen delivery vehicle for oral vaccination.

D. Integrins β1 integrin is generally expressed on the basal membrane of intestinal epithelial cells

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[45]. However, it was shown that β1 integrin was present only on the M cell luminal surface [46]. In fact, Yersinia spp. effectively invade through M cells via invasin, an outer-membrane protein capable of binding to β1 integrin [47,48]. Although fibronectin is an endogenous ligand to α5β1 integrin, the Arg-Gly-Asp (RGD) peptide is sufficient for binding by this integrin. When poly(D,L-lactide-co-glycolide) particles, which have been shown to be safe vaccine delivery vehicles in humans, are conjugated to RGD peptide or low-molecular-weight mimetics, they can selectively deliver OVA as Ag to M cells [49 51]. Recently, allograft inflammatory factor 1 (Aif1), a protein that binds calcium ions, was demonstrated to be important for the activation of β1 integrin on the M cell luminal surface via calcium-ion-dependent activation of small G-protein [52]. Aif1-mediated small G-protein activation is also thought to be important for actin remodeling on the M cell luminal surface [52]. The finding that Aif1 activation may be crucial for M cell uptake has significant implications for drug delivery systems.

E. Uromodulin (Umod) Umod (also known as Tamm-Horsfall protein) shows .50% amino acid homology with GP2, and is strongly expressed in the kidneys [53]. Umod is a GPI-anchor protein and is expressed on the luminal side of the urinary tract, including the bladder. Soluble Umod contributes to the recognition and excretion of FimH1 type 1-fimbriated E. coli but not P-fimbriated E. coli, thus reducing uropathogenic E. coli from colonizing the mammalian urinary tract [54,55]. Umod mRNA is expressed by M cells, not other epithelial cells, and expression is regulated by Spi-B, since Spi-B2/2 mice show lessened Umod expression [30]. Umod also acts as a scaffold receptor for Lactobacillus

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acidophilus via its outer protein SlpA [56]. Hence, Umod is another candidate as an M cell-targeting molecule. However, so far, no studies have used Umod as the M cell-targeted vaccine delivery molecule.

F. C5aR An M-cell-targeting peptide, Co1, was selected from a phage display library against M-like cells prepared by the coculture system of Caco-2 and Raji cells [57]. Fusion of Co1 with EGFP showed binding to mouse PP M cells and induced serum IgG and mucosal IgA antiEGFP Abs even in the absence of adjuvant [57]. Subsequent studies determined the target of Co1 is C5aR [58], found on PP M cells’ luminal surface. The Co1 peptide shares partial homology to Yersinia enterocolitica outer-membrane protein, OmpH, which contributes to efficient uptake of Yersinia [59]. Dengue virus envelope domain III (EDIII) fused to either Co1 or OmpH proved effective as M cell-targeted vaccines [59]. Notably, Co1 also exhibited adjuvant activity but may be linked to the nature of vaccine Ag used. Additional studies are needed to discern Co1’s adjuvant activity and to validate its safety for the development of novel M-celltargeted delivery vehicles with self-build in adjuvant.

G. Claudin 4 Comparison of the expression of Claudin 2, 3, 4 to occludin located between the villus epithelium and FAE revealed that only Claudin 4 was expressed strongly in the FAE of PPs [60]. Examining total gene expression analysis, Claudin 4 was more highly expressed in Caco-2 cells differentiated into M-like cells as well as by human FAE [61]. Like GP2, Claudin 4 is expressed in M cells of NALT [62]. Owing to its strong affinity, Claudin 4 initially was

described as a receptor for Clostridium perfringens enterotoxin (CPE) [63]. When vaccine Ags (e.g., hemagglutinin and PspA) are fused to CPE C-terminus and administered either orally [64] or nasally [65], they elicit significantly stronger Ag-specific immune responses than when these vaccine Ags are administered alone without the CPE C-terminus. These findings indicate that the Claudin 4-targeted delivery of vaccine antigen may be another attractive strategy for the development of oral and nasal vaccines.

H. Secretory IgA (SIgA) Receptors SIgA is polymeric IgA produced in the mucosal secretions of the eye, oral cavity, intestinal tract, and respiratory tract. SIgA Abs are formed and transported to the mucosal lumen via the polymeric immunoglobulin receptor (pIgR), which is expressed on the basolateral surface of the epithelium. Upon binding of dimeric or polymeric forms of IgA with pIgR, the IgA pIgR complexes traverse the epithelium to the luminal surface, where IgA Ab is released as SIgA. The SIgA Ag complexes can also be taken up from the luminal surface via M cells present on mucosa-associated lymphoid tissues (e.g., GALT and NALT) [66], suggesting an alternative function to IgA-mediated Ag uptake. The uptake of these complexes was believed to occur via pinocytosis, but it was reported that dectin-1 functions as a receptor for these complexes in M-like cells derived from Caco-2 cells, resulting in reverse transcytosis [67]. However, gene expression analysis of mouse PP M cells did not show dectin-1 to be an M cell-specific gene, and other researchers could not confirm this finding [52]. Further, in vivo studies are needed to discern how SIgA Ag complexes are sampled by M cells and how they contribute to the initiation of antigen-specific immunity or tolerance.

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IV. CONTRIBUTION OF M CELLS FOR THE DEVELOPMENT OF ORAL TOLERANCE

I. α2-3-Linked Sialic Acid Types 1 and 3 reoviruses adhere to PP M cells via their minor outer capsid hemagglutinin, protein σ1 [8]. Protein σ1 binding is facilitated by both a C-terminal domain [68,69] and a sialic-acid-binding domain [68]. The former is believed to interact with host junction adhesion molecule-1 (JAM-1), particularly for type 3 reoviruses [70], and is commonly found in tight junctions between endothelial and epithelial cells as well as on mouse L cells [70]. The sialicacid-binding domain in type 1 reoviruses adheres to α2,3-linked sialic acids on M cells [6], and that for type 3 reoviruses binds to 5-Nacetylneuraminic acid [68]. Because of its M cell tropism, reovirus protein σ1 was tested for its ability to deliver nasal DNA vaccines when modified with poly-L-lysine for NALT M cell targeting [71]. Nasal vaccination with protein σ1-DNA complexes resulted in stimulation of systemic IgG and mucosal IgA Abs and increased CTL responses to a reporter gene and gp120 [71,72]. α2,3-linked sialic acids also bind specifically to Siglec-F, an endogenous lectin, which is present on murine PP M cells [73]. In fact, when used in the intestinal loop assay, Siglec-F bound to the apical surface of mouse M cells [73]. Taken together with the results obtained by UEA-1 and NKM16-2-4 above, unique glycosylated molecule(s) produced by M cells could be considered as the M cellspecific targets for vaccine antigen delivery.

IV. CONTRIBUTION OF M CELLS FOR THE DEVELOPMENT OF ORAL TOLERANCE Tolerance is the active induction of peripheral unresponsiveness to a specific Ag by exposure to multiple doses of Ag (low-dose tolerance or T cell anergy) or to a high dose of Ag (high-dose tolerance or clonal deletion) [74].

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For the mucosal immune system, one of important regulatory arms is the creation of quiescent immunological condition, known as oral tolerance, to maintain unresponsiveness under the harsh environment present in the gastrointestinal tract [75]. PPs are thought to be required for the induction of tolerance based upon the findings that treatment of female mice with soluble lymphotoxin-β receptor-Ig (LTβR-Ig) during gestation impaired PP formation. When PPdeficient and intact mice were fed with large doses of OVA for the induction of oral tolerance, the former mice were unresponsive to OVA tolerance, while the result in the latter mice was OVA-specific oral tolerance [76]. Hence PP-null mice or mice without M cells would be expected to be unable to produce tolerance. The exact role of the M cell for the induction of oral tolerance needs to be examined by the M-cell-specific deficient condition (e.g., Spi-B2/2 or RANKL2/2 mice). To circumvent the failures of traditional oral tolerance methods, employing M cell ligands might represent a feasible method for the stimulation of tolerance. As was previously mentioned, reovirus protein σ1 can effectively target M cells [6 8]. A single nasal or oral administration of low-dose of the OVA protein σ1 fusion construct induced potent oral tolerance via the stimulation of regulatory CD41 T cells (Tregs), producing transforming growth factor beta (TGF-β) and interleukin 10 (IL-10), and suppressing proinflammatory cytokine production [77,78]. Both serum and mucosal anti-OVA Abs were suppressed, and since no anti-protein-σ1 Abs were induced, protein σ1 can be adapted for inducing tolerance against any number of Ags (e.g., self-protein, autoantigen, and allergen). Adoptively transferred Tregs from OVA protein-σ1-tolerized mice conferred tolerance in naı¨ve recipients [77]. The OVA protein σ1 complex could not induce tolerance in IL-102/2 mice [77]. These findings suggest that protein σ1 is a promising M cell-targeting

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molecule for the induction of Treg-mediated oral tolerance via PPs and NALT. Since the OVA protein σ1 fusion worked so effectively, further studies were conducted to test its application to suppress autoimmune diseases. The first study [79] tested a modification of OVA protein σ1 fusion expressing the encephalitogenic proteolipid protein (PLP130151) to tolerize against experimental autoimmune encephalomyelitis (EAE), which causes a remitting disease in SJL mice. Tolerance was induced by using this tolerogen-suppressing proinflammatory cytokine responses, resulting in the stimulation of elevated inhibitory cytokines TGF-β, IL-10, and IL-28 production by Foxp31 CD41 Tregs for protection against EAE. Th2 cells were also elicited, but their protective potency was not as great as the induced Tregs. After adoptive transfer to EAE mice, Treg recipients showed better protection than did Th2 cell recipients. To further test its therapeutic potential, protein σ1 fusion with myelin oligodendrocyte glycoprotein29-146 (MOG) was constructed (MOG pσ1) [80]. When tested in MOG35 55-induced EAE mice, which produce a monophasic disease, mice orally tolerized with the MOG pσ1, but not with MOG protein alone, showed complete protection against EAE in an IL-10-dependent fashion. Anti-MOG Abs were suppressed in the protected mice by oral administration with MOG pσ1. The MOG pσ1 could also reverse disease in mice exhibiting peak disease symptoms, and within 24 hours of treatment, EAE was abated. Subsequent studies with MOG pσ1 showed that in addition to induced Tregs, regulatory NK cells [81] and regulatory B cells [82] are also induced, and are important for conferring protection against EAE. Such findings show the feasibility of inducing oral tolerance to effectively tolerize and treat autoimmune diseases, and possibly allergies, when using low and few doses of tolerogens that target M cells [77 82].

V. ENHANCEMENT OF M CELL NUMBER AND FUNCTION From the standpoint of preventive medicine, vaccination is important for the most vulnerable, including infants with naı¨ve immunity and the elderly with reduced immunity for inducing protective immunity against infectious agents (see Chapter 47: Mucosal Vaccines for Aged: Challenges and Struggles in Immunosenescence). Experiments on geriatric mice indicate that the number of PP M cells decreases with age [83]. Except for rodents, there is limited research on aging and its impact upon PP function in humans. Human PPs from senior adults have reduced follicles [84], which may be tied to reduced PP performance as the inductive tissue with aging. For this reason, strategies may need to consider methods to transiently increase the function of PPs, including number of M cells or temporarily strengthen antigen uptake, especially in the elderly. Salmonella spp. uses the type III secretion system to induce membrane ruffling in M cells and intestinal epithelial cells so that the bacteria can pass through the epithelial layer [85,86]. The number of M cells in the FAE is known to increase as a result of Salmonella spp. infection in a SopB-dependent fashion [87]. SopB is an effector protein for the Salmonella spp. type III secretion system. SopB inserts into the cell, triggering Wnt/β-catenin signaling by phosphorylating host Akt, causing an epithelial mesenchymaltransition-like event and inducing the production of RANKL. The resulting RANKL regulates autocrine signaling and drives differentiation of FAE-enterocyte to M cells. The use of SopB or a low-molecular-weight mimetic with similar effects to target M cells or the FAE may transiently improve the reduced number of M cells in elderly mice. Furthermore, transiently activated Aif1 (see Section III, Subsection D above) improves uptake efficiency, even with few

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M cells. Thus a substance that can exert this action may be effective at M cell targeting and ferrying a vaccine Ag.

VI. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Improved and modernized experimental methods have led to rapid progress in the characterization of M cell differentiation and have defined specific surface and functional molecules expressed by M cells. Research is needed to determine whether these findings apply to humans and to take advantage of M celltargeted mucosal vaccines for infectious and autoimmune diseases. Various molecules with M cell tropism have been described, including large molecules, such as Abs specific for M cell molecules, and smaller molecules, such as peptides, which often can be easily produced. Furthermore, since intestinal epithelial stem cell marker Lgr5 has been identified [88], human biopsy samples and human induced pluripotent stem-cell-derived primary cultures of intestinal epithelial cells have been successfully used to study stem cell differentiation into human M cells upon addition of RANKL [27,89,90]. These new experimental methods will result in the molecular and cellular understanding of human M cells, which will lead to the development of human M cell-targeted mucosal vaccines in the near future.

References [1] Bockman DE, Cooper MD. Pinocytosis by epithelium associated with lymphoid follicles in the bursa of Fabricius, appendix, and Peyer’s patches. An electron microscopic study. Am J Anat 1973;136(4):455 77. [2] Owen RL, Jones AL. Epithelial cell specialization within human Peyer’s patches: an ultrastructural study of intestinal lymphoid follicles. Gastroenterology 1974;66(2):189 203. [3] Schulz O, Pabst O. Antigen sampling in the small intestine. Trends Immunol 2013;34(4):155 61.

495

[4] Owen RL, Apple RT, Bhalla DK. Morphometric and cytochemical analysis of lysosomes in rat Peyer’s patch follicle epithelium: their reduction in volume fraction and acid phosphatase content in M cells compared to adjacent enterocytes. Anat Rec 1986;216(4):521 7. [5] Mabbott NA, Donaldson DS, Ohno H, Williams IR, Mahajan A. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunol. 2013;6(4):666 77. [6] Helander A, Silvey KJ, Mantis NJ, Hutchings AB, Chandran K, Lucas WT, et al. The viral sigma1 protein and glycoconjugates containing alpha2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces. J Virol 2003;77(14):7964 77. [7] Wolf JL, Kauffman RS, Finberg R, Dambrauskas R, Fields BN, Trier JS. Determinants of reovirus interaction with the intestinal M cells and absorptive cells of murine intestine. Gastroenterology 1983;85(2):291 300. [8] Wolf JL, Rubin DH, Finberg R, Kauffman RS, Sharpe AH, Trier JS, et al. Intestinal M cells: a pathway for entry of reovirus into the host. Science 1981;212 (4493):471 2. [9] Gonzalez-Hernandez MB, Liu T, Payne HC, StencelBaerenwald JE, Ikizler M, Yagita H, et al. Efficient norovirus and reovirus replication in the mouse intestine requires microfold (M) cells. J Virol 2014;88 (12):6934 43. [10] Dharakul T, Riepenhoff-Talty M, Albini B, Ogra PL. Distribution of rotavirus antigen in intestinal lymphoid tissues: potential role in development of the mucosal immune response to rotavirus. Clin Exp Immunol 1988;74(1):14 19. [11] Sicinski P, Rowinski J, Warchol JB, Jarzabek Z, Gut W, Szczygiel B, et al. Poliovirus type 1 enters the human host through intestinal M cells. Gastroenterology 1990;98(1):56 8. [12] Amerongen HM, Weltzin R, Farnet CM, Michetti P, Haseltine WA, Neutra MR. Transepithelial transport of HIV-1 by intestinal M cells: a mechanism for transmission of AIDS. J Acquir Immune Defic Syndr 1991;4 (8):760 5. [13] Owen RL. M cells as portals of entry for HIV. Pathobiol J Immunopathol Mol Cell Biol 1998;66 (3 4):141 4. [14] Donaldson DS, Kobayashi A, Ohno H, Yagita H, Williams IR, Mabbott NA. M cell-depletion blocks oral prion disease pathogenesis. Mucosal Immunol 2012;5 (2):216 25. [15] Sharma R, Schumacher U, Adam E. Lectin histochemistry reveals the appearance of M-cells in Peyer’s patches of SCID mice after syngeneic normal bone marrow transplantation. J Histochem Cytochem 1998;46(2):143 8.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

496

28. M CELL-TARGETED VACCINES

[16] Clark MA, Jepson MA, Simmons NL, Booth TA, Hirst BH. Differential expression of lectin-binding sites defines mouse intestinal M-cells. J Histochem Cytochem 1993;41(11):1679 87. [17] Terahara K, Yoshida M, Igarashi O, Nochi T, Pontes GS, Hase K, et al. Comprehensive gene expression profiling of Peyer’s patch M cells, villous M-like cells, and intestinal epithelial cells. J Immunol 2008;180(12):7840 6. [18] Hase K, Kawano K, Nochi T, Pontes GS, Fukuda S, Ebisawa M, et al. Uptake through glycoprotein 2 of FimH(1) bacteria by M cells initiates mucosal immune response. Nature 2009;462(7270):226 30. [19] Jang MH, Kweon MN, Iwatani K, Yamamoto M, Terahara K, Sasakawa C, et al. Intestinal villous M cells: an antigen entry site in the mucosal epithelium. Proc Natl Acad Sci U S A 2004;101(16):6110 15. [20] Kim DY, Sato A, Fukuyama S, Sagara H, Nagatake T, Kong IG, et al. The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens. J Immunol 2011;186(7):4253 62. [21] Wang J, Gusti V, Saraswati A, Lo DD. Convergent and divergent development among M cell lineages in mouse mucosal epithelium. J Immunol 2011;187 (10):5277 85. [22] Gerbe F, van Es JH, Makrini L, Brulin B, Mellitzer G, Robine S, et al. Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium. J Cell Biol 2011;192(5):767 80. [23] Knoop KA, Kumar N, Butler BR, Sakthivel SK, Taylor RT, Nochi T, et al. RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium. J Immunol 2009;183 (9):5738 47. [24] Nagashima K, Sawa S, Nitta T, Tsutsumi M, Okamura T, Penninger JM, et al. Identification of subepithelial mesenchymal cells that induce IgA and diversify gut microbiota. Nat Immunol 2017;18(6):675 82. [25] Nagashima K, Sawa S, Nitta T, Prados A, Koliaraki V, Kollias G, et al. Targeted deletion of RANKL in M cell inducer cells by the Col6a1-Cre driver. Biochem Biophys Res Commun 2017;493(1):437 43. [26] Rios D, Wood MB, Li J, Chassaing B, Gewirtz AT, Williams IR. Antigen sampling by intestinal M cells is the principal pathway initiating mucosal IgA production to commensal enteric bacteria. Mucosal Immunol 2016;9(4):907 16. [27] Rouch JD, Scott A, Lei NY, Solorzano-Vargas RS, Wang J, Hanson EM, et al. Development of Functional Microfold (M) Cells from Intestinal Stem Cells in Primary Human Enteroids. PLoS One 2016;11(1): e0148216. [28] Kanaya T, Hase K, Takahashi D, Fukuda S, Hoshino K, Sasaki I, et al. The Ets transcription factor Spi-B is

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

essential for the differentiation of intestinal microfold cells. Nat Immunol 2012;13(8):729 36. de Lau W, Kujala P, Schneeberger K, Middendorp S, Li VS, Barker N, et al. Peyer’s patch M cells derived from Lgr5(1) stem cells require SpiB and are induced by RankL in cultured “miniguts”. Mol Cell Biol 2012;32 (18):3639 47. Sato S, Kaneto S, Shibata N, Takahashi Y, Okura H, Yuki Y, et al. Transcription factor Spi-B-dependent and -independent pathways for the development of Peyer’s patch M cells. Mucosal Immunol 2013;6(4):838 46. Kimura S, Yamakami-Kimura M, Obata Y, Hase K, Kitamura H, Ohno H, et al. Visualization of the entire differentiation process of murine M cells: suppression of their maturation in cecal patches. Mucosal Immunol 2015;8(3):650 60. Kanaya T, Sakakibara S, Jinnohara T, Hachisuka M, Tachibana N, Hidano S, et al. Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-kappaB signaling. J Exp Med 2018;215(2):501 19. Manocha M, Pal PC, Chitralekha KT, Thomas BE, Tripathi V, Gupta SD, et al. Enhanced mucosal and systemic immune response with intranasal immunization of mice with HIV peptides entrapped in PLG microparticles in combination with Ulex europaeus-I lectin as M cell target. Vaccine 2005;23(48 49):5599 617. Wang X, Kochetkova I, Haddad A, Hoyt T, Hone DM, Pascual DW. Transgene vaccination using Ulex europaeus agglutinin I (UEA-1) for targeted mucosal immunization against HIV-1 envelope. Vaccine 2005;23 (29):3836 42. Nochi T, Yuki Y, Matsumura A, Mejima M, Terahara K, Kim DY, et al. A novel M cell-specific carbohydratetargeted mucosal vaccine effectively induces antigenspecific immune responses. J Exp Med 2007;204 (12):2789 96. Goto Y, Obata T, Kunisawa J, Sato S, Ivanov II, Lamichhane A, et al. Innate lymphoid cells regulate intestinal epithelial cell glycosylation. Science 2014;345 (6202):1254009. Matsumura T, Sugawara Y, Yutani M, Amatsu S, Yagita H, Kohda T, et al. Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity. Nat Commun 2015;6:6255. Hanazato M, Nakato G, Nishikawa F, Hase K, Nishikawa S, Ohno H. Selection of an aptamer against mouse GP2 by SELEX. Cell Struct Funct 2014;39 (1):23 9. Shima H, Watanabe T, Fukuda S, Fukuoka S, Ohara O, Ohno H. A novel mucosal vaccine targeting Peyer’s patch M cells induces protective antigen-specific IgA responses. Int Immunol 2014;26(11):619 25.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

REFERENCES

[40] Khan IU, Huang J, Liu R, Wang J, Xie J, Zhu N. Phage display-derived ligand for mucosal transcytotic receptor GP-2 promotes antigen delivery to M cells and induces antigen-specific immune response. SLAS Discov 2017;22(7):879 86. [41] Hoops TC, Rindler MJ. Isolation of the cDNA encoding glycoprotein-2 (GP-2), the major zymogen granule membrane protein. Homology to uromodulin/TammHorsfall protein. J Biol Chem 1991;266(7):4257 63. [42] Cooper MD, Raymond DA, Peterson RD, South MA, Good RA. The functions of the thymus system and the bursa system in the chicken. J Exp Med 1966;123 (1):75 102. [43] Nakato G, Fukuda S, Hase K, Goitsuka R, Cooper MD, Ohno H. New approach for m-cell-specific molecules screening by comprehensive transcriptome analysis. DNA Res 2009;16(4):227 35. [44] Nakato G, Hase K, Suzuki M, Kimura M, Ato M, Hanazato M, et al. Cutting Edge: Brucella abortus exploits a cellular prion protein on intestinal M cells as an invasive receptor. J Immunol 2012;189(4):1540 4. [45] Beaulieu JF. Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine. J Cell Sci 1992;102(Pt 3):427 36. [46] Clark MA, Hirst BH, Jepson MA. M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer’s patch M cells. Infect Immun 1998;66(3):1237 43. [47] Grutzkau A, Hanski C, Hahn H, Riecken EO. Involvement of M cells in the bacterial invasion of Peyer’s patches: a common mechanism shared by Yersinia enterocolitica and other enteroinvasive bacteria. Gut 1990;31(9):1011 15. [48] Isberg RR, Leong JM. Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 1990;60(5):861 71. [49] Fievez V, Plapied L, des Rieux A, Pourcelle V, Freichels H, Wascotte V, et al. Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination. Eur J Pharm Biopharm 2009;73(1):16 24. [50] Garinot M, Fievez V, Pourcelle V, Stoffelbach F, des Rieux A, Plapied L, et al. PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination. J Control Release 2007;120(3):195 204. [51] Gullberg E, Keita AV, Salim SY, Andersson M, Caldwell KD, Soderholm JD, et al. Identification of cell adhesion molecules in the human follicle-associated epithelium that improve nanoparticle uptake into the Peyer’s patches. J Pharmacol Exp Ther 2006;319(2):632 9. [52] Kishikawa S, Sato S, Kaneto S, Uchino S, Kohsaka S, Nakamura S, et al. Allograft inflammatory factor 1 is a regulator of transcytosis in M cells. Nat Commun 2017;8:14509.

497

[53] Fukuoka S, Freedman SD, Yu H, Sukhatme VP, Scheele GA. GP-2/THP gene family encodes selfbinding glycosylphosphatidylinositol-anchored proteins in apical secretory compartments of pancreas and kidney. Proc Natl Acad Sci U S A. 1992;89 (4):1189 93. [54] Bates JM, Raffi HM, Prasadan K, Mascarenhas R, Laszik Z, Maeda N, et al. Tamm-Horsfall protein knockout mice are more prone to urinary tract infection: rapid communication. Kidney Int 2004;65(3):791 7. [55] Mo L, Zhu XH, Huang HY, Shapiro E, Hasty DL, Wu XR. Ablation of the Tamm-Horsfall protein gene increases susceptibility of mice to bladder colonization by type 1-fimbriated Escherichia coli. Am J Physiol Renal Physiol 2004;286(4):F795 802. [56] Yanagihara S, Kanaya T, Fukuda S, Nakato G, Hanazato M, Wu XR, et al. Uromodulin-SlpA binding dictates Lactobacillus acidophilus uptake by intestinal epithelial M cells. Int Immunol 2017;29(8):357 63. [57] Kim SH, Seo KW, Kim J, Lee KY, Jang YS. The M celltargeting ligand promotes antigen delivery and induces antigen-specific immune responses in mucosal vaccination. J Immunol 2010;185(10):5787 95. [58] Kim SH, Jung DI, Yang IY, Kim J, Lee KY, Nochi T, et al. M cells expressing the complement C5a receptor are efficient targets for mucosal vaccine delivery. Eur J Immunol 2011;41(11):3219 29. [59] Kim SH, Jung DI, Yang IY, Jang SH, Kim J, Truong TT, et al. Application of an M-cell-targeting ligand for oral vaccination induces efficient systemic and mucosal immune responses against a viral antigen. Int Immunol 2013;25(11):623 32. [60] Tamagawa H, Takahashi I, Furuse M, YoshitakeKitano Y, Tsukita S, Ito T, et al. Characteristics of claudin expression in follicle-associated epithelium of Peyer’s patches: preferential localization of claudin-4 at the apex of the dome region. Lab Investigat J Tech Methods Pathol 2003;83(7):1045 53. [61] Lo D. Cell culture modeling of specialized tissue: identification of genes expressed specifically by follicleassociated epithelium of Peyer’s patch by expression profiling of Caco-2/Raji co-cultures. Int Immunol 2004;16(1):91 9. [62] Kakutani H, Kondoh M, Fukasaka M, Suzuki H, Hamakubo T, Yagi K. Mucosal vaccination using claudin-4-targeting. Biomaterials 2010;31(20):5463 71. [63] Katahira J, Inoue N, Horiguchi Y, Matsuda M, Sugimoto N. Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin. J Cell Biol 1997;136(6):1239 47. [64] Lo DD, Ling J, Eckelhoefer AH. M cell targeting by a Claudin 4 targeting peptide can enhance mucosal IgA responses. BMC Biotechnol 2012;12:7.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

498

28. M CELL-TARGETED VACCINES

[65] Suzuki H, Watari A, Hashimoto E, Yonemitsu M, Kiyono H, Yagi K, et al. C-terminal clostridium perfringens enterotoxin-mediated antigen delivery for nasal pneumococcal vaccine. PLoS One 2015;10(5):e0126352. [66] Rey J, Garin N, Spertini F, Corthesy B. Targeting of secretory IgA to Peyer’s patch dendritic and T cells after transport by intestinal M cells. J Immunol 2004;172(5):3026 33. [67] Rochereau N, Drocourt D, Perouzel E, Pavot V, Redelinghuys P, Brown GD, et al. Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells. PLoS Biol 2013;11(9): e1001658. [68] Barton ES, Chappell JD, Connolly JL, Forrest JC, Dermody TS. Reovirus receptors and apoptosis. Virology 2001;290(2):173 80. [69] Turner DL, Duncan R, Lee PW. Site-directed mutagenesis of the C-terminal portion of reovirus protein sigma 1: evidence for a conformation-dependent receptor binding domain. Virology 1992;186(1):219 27. [70] Barton ES, Forrest JC, Connolly JL, Chappell JD, Liu Y, Schnell FJ, et al. Junction adhesion molecule is a receptor for reovirus. Cell. 2001;104(3):441 51. [71] Wu Y, Wang X, Csencsits KL, Haddad A, Walters N, Pascual DW. M cell-targeted DNA vaccination. Proc Natl Acad Sci U S A. 2001;98(16):9318 23. [72] Wang X, Hone DM, Haddad A, Shata MT, Pascual DW. M cell DNA vaccination for CTL immunity to HIV. J Immunol 2003;171(9):4717 25. [73] Gicheva N, Macauley MS, Arlian BM, Paulson JC, Kawasaki N. Siglec-F is a novel intestinal M cell marker. Biochem Biophys Res Commun 2016;479 (1):1 4. [74] Bilate AM, Lafaille JJ. Induced CD4 1 Foxp3 1 regulatory T cells in immune tolerance. Annu Rev Immunol 2012;30:733 58. [75] Weiner HL, Friedman A, Miller A, Khoury SJ, alSabbagh A, Santos L, et al. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu Rev Immunol 1994;12:809 37. [76] Fujihashi K, Dohi T, Rennert PD, Yamamoto M, Koga T, Kiyono H, et al. Peyer’s patches are required for oral tolerance to proteins. Proc Natl Acad Sci U S A 2001;98 (6):3310 15. [77] Rynda A, Maddaloni M, Mierzejewska D, OchoaRepa´raz J, Maslanka T, Crist K, et al. Low-dose tolerance is mediated by the microfold cell ligand, reovirus protein sigma1. J Immunol 2008;180(8):5187 200. [78] Suzuki H, Sekine S, Kataoka K, Pascual DW, Maddaloni M, Kobayashi R, et al. Ovalbumin-protein sigma 1 M-cell targeting facilitates oral tolerance

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

with reduction of antigen-specific CD4 1 T cells. Gastroenterology 2008;135(3):917 25. Rynda A, Maddaloni M, Ochoa-Repa´raz J, Callis G, Pascual DW. IL-28 supplants requirement for T(reg) cells in protein sigma1-mediated protection against murine experimental autoimmune encephalomyelitis (EAE). PLoS One 2010;5(1):e8720. Rynda-Apple A, Huarte E, Maddaloni M, Callis G, Skyberg JA, Pascual DW. Active immunization using a single dose immunotherapeutic abates established EAE via IL-10 and regulatory T cells. Eur J Immunol 2011;41(2):313 23. Huarte E, Rynda-Apple A, Riccardi C, Skyberg JA, Golden S, Rollins MF, et al. Tolerogen-induced interferon-producing killer dendritic cells (IKDCs) protect against EAE. J Autoimmun 2011;37(4):328 41. Huarte E, Jun S, Rynda-Apple A, Golden S, Jackiw L, Hoffman C, et al. Regulatory T cell dysfunction acquiesces to BTLA 1 regulatory B cells subsequent to oral intervention in experimental autoimmune encephalomyelitis. J Immunol 2016;196(12):5036 46. Kobayashi A, Donaldson DS, Erridge C, Kanaya T, Williams IR, Ohno H, et al. The functional maturation of M cells is dramatically reduced in the Peyer’s patches of aged mice. Mucosal Immunol 2013;6(5):1027 37. Kato T, Owen R. Structure and function of intestinal mucosal epithelium. In: Mestecky JB, Lamm ME, Mayer L, McGhee JR, Strober W, editors. Mucosal Immunology. Burlington, MA: Elsevier-Academic Press, Inc; 2005. p. 131 51. Dramsi S, Cossart P. Intracellular pathogens and the actin cytoskeleton. Annu Rev Cell Dev Biol 1998;14: 137 66. Reis RS, Horn F. Enteropathogenic Escherichia coli, Samonella, Shigella and Yersinia: cellular aspects of hostbacteria interactions in enteric diseases. Gut Pathog 2010;2(1):8. Tahoun A, Mahajan S, Paxton E, Malterer G, Donaldson DS, Wang D, et al. Salmonella transforms follicle-associated epithelial cells into M cells to promote intestinal invasion. Cell Host Microbe 2012;12 (5):645 56. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009;459(7244):262 5. Spence JR, Mayhew CN, Rankin SA, Kuhar MF, Vallance JE, Tolle K, et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 2011;470(7332):105 9. Wang X, Yamamoto Y, Wilson LH, Zhang T, Howitt BE, Farrow MA, et al. Cloning and variation of ground state intestinal stem cells. Nature 2015;522(7555):173 8.

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Induction of Local and Systemic Immunity by Salmonella Typhi in Humans Franklin R. Toapanta1,2, Jayaum S. Booth1,2,3 and Marcelo B. Sztein1,2,3 1

Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, United States 2Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, United States 3Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, United States

I. INTRODUCTION Salmonella enterica serovar Typhi (S. Typhi), the etiological agent of typhoid fever, is a highly invasive bacterium that rapidly and efficiently infects the intestinal mucosa of humans, it’s only natural host [1,2]. Although the disease burden varies between countries [3], it is estimated that 11.9 20.6 million people are infected annually and approximately 130,000 223,000 people worldwide die of the disease [4 6]. The spread of the disease, which occurs by the fecal oral route, depends on several factors, such as personal hygiene, quality of the sanitation system, socioeconomic status, and the emergence of drug-resistant strains in epidemic and endemic regions [2,3,7,8]. Outbreaks are most likely to occur in developing countries as the result of poor sanitation systems. However, typhoid fever also occurs in industrialized countries, mostly among known risk groups, such as microbiology Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00029-8

lab workers, military personnel, and travelers to endemic regions [2]. The emergence of multidrug-resistant [9] and extensively drugresistant strains, including the H58 S. Typhi strain [10] has spurred renewed attention to the disease. In addition, the potential use of S. Typhi as a bioterrorism agent, given its potential to cause high morbidity and mortality and its ability to contaminate food and water supplies, has added a new sense of urgency for the development of more effective typhoid vaccines.

II. CURRENT VACCINES AND MODELS TO STUDY IMMUNOGENICITY TO SALMONELLA TYPHI None of the currently licensed typhoid vaccines is ideal. Ty21a (the only licensed live attenuated oral vaccine) generates only modest

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immunogenicity and requires three to four spaced doses to confer acceptable protection [11 13]. Of importance, this vaccine is not licensed for children younger than 6 years of age, a particularly susceptible age group [14,15]. The purified Vi capsular polysaccharide vaccine is well tolerated but moderately immunogenic [2,16]. Of note, Vi vaccines are not immunogenic in children younger than 2 years of age. Recently, the World Health Organization prequalified and recommended the introduction of a Vi tetanus toxoid (Vi TT) conjugate vaccine into areas with a high incidence of typhoid fever [17,18]. This recommendation was due to the ability of Vi TT to induce immunity even in children younger than 2 years of age [18]. The development of better vaccines, particularly broad-spectrum vaccines that are also effective against other enteric Salmonella infections (e.g., the rapid dissemination of S. Paratyphi A in Southeast Asia, for which no vaccines are available) [19 24], is urgently needed. However, efforts to develop novel vaccines have been hampered by an incomplete understanding of the immune factors responsible for protection (correlates of protection, or CoP), particularly at the mucosal (gut) level. Animal models do not recapitulate important aspects of the pathogenesis and immunogenicity induced by S. Typhi in humans. Therefore human studies are necessary to uncover critical aspects of the immunity induced by this microorganism. The licensed live attenuated Ty21a typhoid vaccine is remarkable in that it induces long-term immunity lasting 5 7 years [11]. This makes Ty21a immunization an excellent human model to use in studying the mechanisms underlying protective immunity to enteric bacterial infections in humans. Studies with Ty21a have begun to uncover the role that memory T (TM) cells play in the long-term immunity elicited at the systemic level and, more recently, the gut mucosa (local) level [25 28]. Moreover, in recent years, the reestablishment of the human challenge

model of typhoid fever in Oxford, United Kingdom (UK) [29], has provided a unique opportunity to start uncovering responses that might represent mechanistic or nonmechanistic immunological CoP [30].

III. LIVE ATTENUATED ORAL VACCINE AND HUMAN CHALLENGE MODEL OF TYPHOID FEVER During the 1960s and 1970s, several induced human infection (challenge) studies with S. Typhi were conducted at the University of Maryland [31]. Importantly, these challenge studies reproduced fatefully the illness reported in naturally occurring typhoid fever [32,33]. The University of Maryland studies allowed evaluation of multiple aspects of the pathogen host interaction and permitted a direct evaluation of the efficacy of typhoid vaccines in preventing clinical illness. For example, these studies demonstrated the importance of the Vi capsule for virulence, since the attack rate of Vi-expressing strains (Quailes, Ty2, Zermatt) was almost double than that of Vinegative strains (Ty2W, O-901): 51% vs 26%, respectively [34]. These trials also explored the relationship between different infectious doses with attack rates and incubation periods to develop clinical illness. For this, oral doses of 103 109 of S. Typhi (Quailes, suspended in milk) were tested. The attack rate correlated directly with the challenge dose but inversely with the incubation period [34,35]. Since S. Typhi challenge replicated natural typhoid fever, these studies were ideal for evaluating vaccine candidates (parenteral and oral). Several oral vaccine candidates were tested [36,37], including a successful vaccine trial involving the live attenuated vaccine strain Ty21a (derived from Ty2) [38]. The protective efficacy of Ty21a grown in galactose (5 8 doses; 3 10 3 1010 CFU) after challenge

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(105 CFU of S. Typhi; 5 9 weeks after immunization) was 87%. Similar protective efficacy was later observed in a field trial in Egypt [39]. Subsequent field trials showed that the overall protective efficacy of Ty21a was 67% 80% and lasted up to 7 years. Ty21a became the first licensed live oral vaccine for the prevention of typhoid infection [40 42] and remains an example of the usefulness of human challenge models for rapid and controlled testing of vaccine candidates. While the human challenge studies performed at the University of Maryland provided various insights into the prevention and protection to this pathogen, the in-depth study of immune responses was limited, owing to the technical limitation of the time. The program in the United States was cancelled in 1974; therefore, the recent reestablishment of a new human challenge model for S. Typhi by the Oxford Group in the UK [43] has provided new opportunities to study in detail the immunity to this pathogen. In the subsequent sections, we summarize some of the most important new immunological insights provided by this human challenge model. Additionally, where relevant, we include information on the immunity derived from human studies involving oral immunization with Ty21a.

IV. SYSTEMIC T CELL IMMUNITY INDUCED BY ORAL SALMONELLA TYPHI In the Oxford human challenge study, 40 volunteers were exposed to either 103 (low dose) or 104 (high dose) CFU of S. Typhi (Quailes strain) in sodium bicarbonate solution. These doses resulted in attack rates of 55% (11 of 20 subjects) and 65% (13 of 20 subjects), respectively [43]. Typhoid disease (TD) was diagnosed as a temperature of 38 C or higher sustained over 12 or more hours or bacteremia. Challenged volunteers were catalogued as

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those who developed TD and those who did not (NoTD). These studies revealed for the first time that higher multifunctional (MF) S. Typhispecific CD81 baseline responses were associated with protection against typhoid fever and delayed onset of disease when volunteers were challenged with a low-dose inoculum (103 CFU) [44]. Furthermore, after challenge, development of TD was accompanied by a reduction of circulating S. Typhi-specific CD81 T effector memory (TEM) cells with gut-homing potential (integrin α4β71), suggesting that these cells were migrating to the site(s) of infection. In contrast, protection against disease was associated with low or no changes in circulating S. Typhi-specific TEM cells. Importantly, although the presence of higher levels of CD81 TEM cells was associated with increased time to disease onset in the high-dose challenge (104 CFU), recipients were not protected from eventually succumbing to disease [45]. Of note, protection against disease in lowand high-dose challenges was associated with low or no changes in circulating S. Typhi-specific TEM cells [44,45]. These studies have also provided the first evidence that prechallenge upregulation of the gut-homing molecule integrin α4β7 by regulatory T (Treg) cells, followed by a significant downregulation postchallenge consistent with Treg cells homing to the gut, was associated with the development of typhoid fever, suggesting that Treg cells play an important role in determining clinical outcome [46].

V. HUMORAL AND SYSTEMIC B CELL IMMUNITY INDUCED BY ORAL SALMONELLA TYPHI The role of antibodies in protection from typhoid fever remains unclear. The Oxford human challenge study showed that relatively high baseline levels of S. Typhi LPS specific antibody in nonexposed/nonvaccinated study participants did not correlate with subsequent risk of infection [43]. In a subsequent study,

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robust anti-LPS and antiflagella titers induced by two independent S. Typhi live attenuated vaccines were unable to provide protection against S. Typhi challenge [47]. Additionally, protection in this model was not correlated with the functional ability of antibodies to mediate serum bactericidal activity, although there was some decrease in severity associated with increased antibody titer [48]. Antibodies are produced by B cells; however, to date, little or no information regarding the changes in B cell subtypes induced by S. Typhi infection is available. The Oxford human challenge study allowed evaluation of various B cell subsets. In volunteers who received 104 CFU of S. Typhi and developed disease (days 9 14 postchallenge), a reduction in the frequency of B cells was noted. Plasmablasts, which are early antibody producers, increased expression of integrin α4β7 during the disease period, suggesting that these cells were readying to migrate to the gut. At later time points, expression of activation molecules (CD40 and CD21) was also detected. Most of the changes in the other B cell subsets were focalized in the disease period (e.g., reduction of CD21 expression by classswitched CD271 and CD272 B cells) and evident only in the volunteers who developed disease [49]. Therefore profound changes in various B cell subsets occur, mostly in TD volunteers, following challenge with S. Typhi

VI. CHANGES IN INNATE AND MUCOSAL-ASSOCIATE INVARIANT T CELLS INDUCED BY ORAL SALMONELLA TYPHI One of the first lines of defense against pathogens is the innate immune system, which consists of many cell types, including macrophages and dendritic cells (DCs). Circulating monocytes, derived from bone marrow precursors, show great plasticity and are able to differentiate into various types of macrophages

and DCs as they migrate to tissues or when exposed to the appropriate cytokine environment [50 52]. Limited information regarding the changes induced by S. Typhi infection in human monocytes and DCs is available. Specimens from volunteers challenged with 104 CFU of S. Typhi revealed that monocytes and DCs of TD volunteers upregulated expression of CD38 and CD40 during disease days (days 9 14 postchallenge) [53]. Interestingly, integrin α4β7 was upregulated on monocytes but not on DCs, suggesting preferential migration of only one of these cell types to the gut. Of particular note, monocytes and DCs from NoTD volunteers showed increased binding to S. Typhi 1 day postchallenge, and upon stimulation with S. Typhi-LPS-QDot micelles, monocytes showed phosphorylation of p38MAPK, NFkB, and Erk1/2. In contrast, monocytes from TD volunteers showed only a moderate increase in S. Typhi binding 48 and 96 hours postdisease diagnosis (days 9 16 postchallenge) and only Erk1/2 phosphorylation [53]. Therefore S. Typhi infection on volunteers who developed disease induced radical changes on monocytes and DCs, and early responses in monocytes seem to be associated with reduced susceptibility to disease. Mucosal-associate invariant T (MAIT) cells are an innate effector-memory-like T cell population that display a TCR Vα7.21CD1611 phenotype and are restricted by the nonclassical MHC-related molecule 1 (MR1) [54,55]. MAIT cells are important in immunity at the mucosal sites and appear to play a particular role in immunity to gut pathogens [56]. CD81 MAIT cells can be activated and kill host cells that have been exposed to S. Typhi; these responses depend on bacterial load [56]. Specimens from the Oxford study showed that TD volunteers exhibited a sharp decline in circulating MAIT cells during the development of typhoid fever. Interestingly, MAIT cells from volunteers who received a low dose (103 CFU) of S. Typhi and developed disease

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had higher levels of CD38 coexpressing CCR9, CCR6, and Ki67 during the development of typhoid fever than did volunteers who received 104 CFU and developed disease [57]. Similarly, MAIT cells from low-dose (103 CFU) TD volunteers showed a stronger upregulation of CCR9 during disease days than did cells from high-dose (104 CFU) TD volunteers. The changes were not evident in volunteers who did not develop disease, regardless of the bacterial dose they received [57]. In sum, the Oxford challenge studies allowed more detailed evaluation of the changes that S. Typhi induce at the cellular level in various human immune cells. These changes are mainly identified in volunteers who developed disease, and most of the changes are clustered during the acute phase of typhoid fever. Importantly, expression of homing molecules such as integrin α4β7 and/or CCR9 during the acute phase suggests that a large percentage of these cells are actively migrating to the gut, which is the initial port of infection. Therefore, the study of local responses is of particular importance. To start addressing the local responses to Salmonella, oral immunization with Ty21a has been used as an initial model.

VII. GUT-HOMING MEMORY T CELLS: A WINDOW INTO MUCOSAL IMMUNITY Leukocyte homing and trafficking to peripheral tissues during infection or homeostasis requires an array of chemokines and activation of their cognate receptors. The key step in this intricate process is the adherence of lymphocytes expressing surface-homing receptors to their cognate 7-transmembrane domain Gprotein couple receptors on endothelial and/or epithelial cells [58 60]. Remarkably, the pattern of homing receptors of S. Typhi-specific plasmablasts has been shown to differ significantly following immunization with either the

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Vi (parenteral) or Ty21a (oral) vaccines [61,62]. While both vaccines elicited similar number of S. Typhi-specific plasmablasts, Vi induced mainly the expression of systemic-homing receptors, while Ty21a induced largely the expression of gut-homing receptors such as integrin α4β7 [61,62]. Furthermore, antigenspecific IgA2 plasmablasts were found to express more frequently integrin α4β7 than IgA1 following immunization with Ty21a [63]. Interestingly, cross-reacting LPS-specific plasmablasts producing IgG and IgA have been shown to express mostly CD27 and integrin α4β7, with a significant proportion coexpressing CD62L (secondary lymphoid tissue homing) [64]. Concerning effector memory T cells, these cells home selectively to the lamina propria of the small intestine mainly by expressing integrin α4β7 and CCR9 [65 67]. Induction of specific memory CD41 and CD81 T cells with guthoming potential following immunization with the oral typhoid vaccine Ty21a has been well documented [68 70]. Initial reports in Ty21a vaccinees showed that sorted integrin β7-expressing memory T cells (CD45RA2 β7hi) produced more IFNγ (approximately 10-fold) than CD45RA2 β72 or CD45RA2 β7int T cells [68]. The gut-homing potential and induction of IFNγ production were further delineated in central (TCM, CD45RO1CD62L1) and effector (TEM, CD45RO1CD62L2) memory T populations from volunteers orally immunized with the S. Typhi vaccine candidate strain CVD 909 [70]. Interestingly, this study also showed that the homing potential of CD41 and CD81 TM subsets was distinct, since CD41 TCM, but not CD81 TCM cells, expressed predominantly integrin α4β7 [70]. In addition, another study reported that S. Typhi-specific CD81 T cells coexpressed high levels of integrin α4β7, intermediate levels of CCR9, and low levels of CD103 [69]. Thus oral typhoid immunization (Ty21a or CVD 909) induces circulating S. Typhi-specific TM cells to upregulate homing

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markers, allowing for their trafficking to the gut and other lymphoid tissues. Furthermore, these S. Typhi-responsive T cells expressing integrin α4β7 were also shown to be multifunctional, producing multiple cytokines and/or expressing CD107a (a marker of cytotoxic activity), and cross-reactive to S. Paratyphi A and S. Paratyphi B [71,72]. All these observations were made in peripheral blood specimens from orally immunized volunteers. Recent studies in the human challenge model (Oxford studies) demonstrated that wild-type S. Typhi infection elicits S. Typhiresponsive T effector cells with gut-homing potential which were associated with disease protection [44,45]. In these studies, following S. Typhi challenge, TD volunteers showed a reduction in total integrin α4β71 CD81 TEM as well as in both integrin α4β72 and α4β71 S. Typhi-specific TEM cells. However, 48 hours after TD diagnosis, the proportion of MF S. Typhi-specific integrin α4β71 TEM in TD subjects was higher than in integrin α4β72 cells [45]. Taken together, these observations highlight the association of disease protection and delayed time to diagnosis to strong MF CD81 TEM responses with gut-homing potential as well as to other lymphoid tissues. These observations complement and expand our previous findings showing that oral immunization against S. Typhi elicited integrin α4β71 CD81 effector T cells [69,70,73], which most likely migrate to the gut to reduce S. Typhi replication during the incubation phase and thereby delay disease onset. Although all the earlier observations about antigen-specific cells were made in the systemic compartment by examining their potential to home to the gut and other tissues, they nevertheless provide a window into the local (gut) responses to S. Typhi. However, it is becoming increasing clear that the generation and characteristics of mucosal immunity at the site of infection are distinct from systemic immunity. Therefore it is crucial to determine the

characteristic and mechanisms defining mucosal immunity to S. Typhi. We will next describe recent advances in research on S. Typhi immunity at the site of infection in the gut mucosa.

VIII. ACCUMULATION AND RETENTION OF GUT-HOMING MEMORY T CELLS IN THE MUCOSA Novel studies following oral Ty21a immunization have been performed to understand the accumulation and potential retention of S. Typhi-responsive cells expressing homing markers in human duodenal lamina propria mononuclear cells (LPMCs) (integrin β7) [28], terminal ileum (TI) LPMC (α4β7, CCR9, and CCR6) [27], and their peripheral blood counterparts. Interestingly, while there were significant decreases of integrin α4β7 and CCR9 expression in peripheral blood, expression of integrin α4β7, CCR9, and CCR6 homing molecules on TI LPMC CD81 T cells exhibited no significant differences following Ty21a immunization [27]. However, integrin α4β71CCR91CD81 LPMC T cells accumulated in significantly higher numbers in the TI mucosa [27]. These data suggest that specific CD81 TM are recruited and retained at the TI mucosa. In the duodenum, integrin β7 expression [a component of both integrin α4β7 and αεβ7 (CD103)] among LPMC CD41 T cell subsets was significantly higher than that in PBMC [28]. Furthermore, Ty21aresponsive and influenza-virus-responsive CD81 T cells obtained from duodenal biopsies expressed higher levels of integrin β7 than did total CD81 T cells [28]. Remarkably, CCR91 T cells have been shown to home to the small intestine but not to the colon and play an important role in the generation of antigenspecific lymphocytes [66]. Thus S. Typhiresponsive cells expressing homing molecules, particularly integrin α4β71CCR91 T cells, seem to be recruited and compartmentalized to the mid-gut region (Fig. 29.1). These cells may play

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FIGURE 29.1

Mucosal and systemic human immunity to Salmonella Typhi. Immunity to S. Typhi is extremely complex. This diagram presents a summary of our current knowledge of mucosal and systemic immunity obtained from orally immunized volunteers and S. Typhi-challenged volunteers. It involves multiple antigen-presenting cells (e.g., macrophages, dendritic cells, B cells) and effector cells (e.g., various effector and regulatory T cell subsets, B cells, NK cells, and MAIT cells). APC, Antigen-presenting cells; ASC, antibody-secreting cells; BCR, B cell receptor; CD4, CD41 T cells; CD8, CD81 T cells; DC, dendritic cells; HLA, human leukocyte antigen; HLA-I, HLA class I; HLA-II, HLA class II; Ig, immunoglobulin; MΦ, macrophages; MAIT, mucosal-associated invariant T cells; MR1, HLA-I nonclassical (b) molecule MR1; NK, natural killer cells; PMN, polymorphonuclear cells, neutrophils; TCM, central memory T cells; TCR, T cell receptor; TEM, effector memory T cells; TEMRA, effector-memory-expressing CD45RA; TM, memory T cells; Treg, regulatory T cells.

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a crucial role in the generation of S. Typhispecific cells in the human small intestine.

IX. MUCOSAL IMMUNITY TO SALMONELLA TYPHI Ingested enteric pathogens (e.g., S. Typhi) actively invade the host mucosal surfaces via epithelial cells and antigen-sampling M cells, which are specialized epithelial cells covering the Peyer’s patches. Consequently, S. Typhi translocates to the submucosa and encounters intestinal lymphoid tissues before entering the draining mesenteric lymph nodes [2,74]. Subsequently, S. Typhi disseminates to the liver, spleen, and other secondary lymphoid tissues, causing systemic illness and typhoid fever [2,74]. The most serious complication of typhoid fever is intestinal perforation, which occurs primarily (approximately 78% of cases) in the TI [75]. At the perforation site, acute and chronic inflammatory cells, particularly macrophages (CD681) and T (CD31) cells, have been observed [76]. Other pathological changes that have been described include hypertrophy, necrosis, and ulceration of the intestinal and mesenteric lymphatic tissue [76 78]. Duodenal and appendiceal (fewer than 2% of cases) perforations have rarely been reported [79]. While S. Typhi can potentially invade any site harboring M cells (e.g., duodenum, jejunum) along the intestine, these clinical observations suggest that the human TI is the favored intestinal active invasion site for S. Typhi [74,75]. Although the human gastrointestinal tract constitutes a major reservoir of total body lymphocytes (approximately 60%) and represents an area of high antigenic exposure, our understanding of the intestinal mucosal immunity is sparse, particularly with respect to the events after oral immunization. This gap in knowledge is hindering the rational development of new oral vaccines. Recently, following Ty21a immunization, cell-mediated immunity (CMI) was

meticulously characterized in the human intestinal mucosa (duodenum, colon [28], and TI [27]). Direct evidence revealed that oral Ty21a immunization elicited significant S. Typhispecific LPMC CD81 T cell responses (IFNγ, TNF-α, IL-17A, and CD107a) in biopsies obtained from the TI of healthy volunteers [27]. Additionally, all the major CD81 TM subsets (TEM, TCM, and TEMRA) were induced in the TI mucosa. Of note, each of these subsets displayed unique response profiles (specific cytokines production and/or cytotoxicity) following Ty21a immunization [27]. These data suggest that local mechanisms present in the mucosa strongly activate distinct CD81 TM subsets following oral vaccination. Likewise, following Ty21a immunization, human duodenum CD41 and CD81 T cells showed robust responses to Ty21a killed bacteria and heterologous influenza virus, while human colon T cells were unresponsive to either antigen [28]. Taken together, these data indicate that human intestinal responses to oral Ty21a immunization are compartmentalized to the embryological mid-gut. However, the magnitude and characteristics of S. Typhi-responsive cells were different between TI, duodenum and colon. Based on the frequencies of S. Typhiresponsive CD81 T cells from both studies, LPMC CD81 T responses seem to be stronger in the TI than duodenum and colon. In addition, TI LPMC CD81 TM responses were chiefly MF, while duodenal CD81 T responses were largely attributed to single cytokine-producing (S) cells [27,28]. Thus, oral Ty21a immunization elicited TI LPMC CD81 TM cells that differentiated predominantly into MF Tc1 (IFNγ and TNFα), S and MF Tc17 (IL-17A), and S and MF cytotoxic (CD107a1) CD81 T effector cells. These effector sets are well suited to protect against intracellular pathogens. In contrast, the duodenal CD81 T cell S. Typhi-responsive phenotype was mostly S Tc1 (IFNγ, TNF-α, or IL-2) CD81 T effector cells. The differences between these tissues might be attributed to various

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XI. CONCLUDING REMARKS

factors, including microbiota composition and distribution of lymphoid structures (e.g., Peyer’s patches, isolated lymphoid follicles, M cells) between the colon, duodenum, and TI [80,81] as well as the type of antigenic stimulation used in the studies. Altogether, these data suggest that oral Ty21a immunization elicits distinct site-specific mucosal immune responses along the intestine (Fig. 29.1). Evaluation of mucosal CMI at distinct sites of infection along the intestine might help in the identification of functional CoP and offer new insights in the mechanisms operating locally.

X. RELATIONSHIP BETWEEN SYSTEMIC AND MUCOSAL IMMUNITY TO SALMONELLA TYPHI Most human studies have sampled peripheral blood from which relationships to the site of infection (e.g., mucosal sites) have been inferred. However, recent findings have indicated that immune responses at the site of infection (mucosal) are distinct from those in peripheral blood [27,82 85]. We have previously reported that TI CD81 TM cells respond differently from their systemic counterparts [27]. For example, the frequency of S. Typhiresponsive CD81 TEM is higher in magnitude in the intestinal mucosa than in PBMC, which might be due to the constant exposure to the microbiome. Furthermore, S. Typhi-specific responses in the mucosa can have different characteristics. For instance, TI cytotoxic (CD107a1) CD81 TEM responses shift from CD107a1 MF in unvaccinated to predominantly S in Ty21a-vaccinated volunteers [27]. These S. Typhi-responsive CD81 T effector phenotypes are also elicited in peripheral blood, but at a lower magnitude and exclusively as MF cells [27]. Importantly, the generation of both mucosal and systemic T cell effectors effectively provides protective immunity against enteric

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diseases. However, information about the relationship between the generation of immune responses between the two compartments is scarce. Recent evidence following Ty21a immunization indicates that there is a significant positive correlation between the TI mucosa and peripheral blood exclusively within the MF CD81 TEM responses (CD107a, IFNγ, TNF-α, IL-17A, and MIP1β) [27]. These data argue that MF CD81 TEM S. Typhi-responsive cells might shuttle between the mucosa and periphery and possibly accumulate and/or proliferate locally, as evidenced by the qualitatively stronger MF responses observed in LPMC. This finding further supports a recent report showing that MF S. Typhi-specific CD81 T cell responses correlated with protection against typhoid fever and delayed disease onset in humans challenged with wild-type S. Typhi [44]. In sum, the generation of MF S. Typhi-specific responses at the site of infection could be a major determinant in protection against TD. Thus, the development of a highly efficacious oral Salmonella vaccine should prioritize the induction of mucosal MF CD81 TEM effectors (Fig. 29.1). In addition, these studies provide novel insights to advance the development of oral vaccines for enteric pathogens other than S. Typhi.

XI. CONCLUDING REMARKS The re-established human challenge model of typhoid fever has shown that infection with S. Typhi induces profound changes in multiple branches of the immune system. Also, since most of the changes in the volunteers who developed TD were focalized during disease days, the results suggest a complex and synchronized response. Importantly, at the systemic level, two notable features associated with protection from disease were revealed: One of these features is pre-existing MF CD81 TM cell immunity (induced either by previous exposure to this bacterium or a related

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microorganism or due to cross-reactive epitopes in the gut microbiota) with gut-homing capacity which was associated with protection from disease (low-dose challenge) or delayed disease onset (high-dose challenge). Second, enhanced affinity of monocytes for S. Typhi at early time points after challenge and activation of signaling pathways in these cells was also associated with protection from disease. Conversely, one feature suggesting susceptibility to disease development involved a prechallenge upregulation of the gut-homing molecule integrin α4β7 by Treg cells. At the intestinal level, studies with Ty21a uncovered the presence of S. Typhi-responsive CD81 TEM cells in the TI and duodenum. Moreover, these cells appear to be shuttling between the gut mucosa and peripheral blood, providing a link between local and systemic responses (Fig. 29.1). The presence of S. Typhi-responsive CD81 TEM cells in the intestine further supports a protective role for CD81 TEM against S. Typhi. Finally, despite all the progress that has been made, major questions remain to be explored. For example, do cell populations identified at the systemic level after challenge (e.g., MAIT cells, B cells, DCs, monocytes, and macrophages) behave similarly at the intestinal level (Fig. 29.1)? What are the roles of other immune cells, such as follicular helper cells, natural killer (NK) cells, NK-T cells, different subsets of monocytes and macrophages, and DCs, at the systemic and local levels? Future studies will be able to shed light on these questions by emphasizing the investigation of local responses and their correlation with responses observed systemically. The human challenge model and the Ty21a immunization model remain our best tools to explore these questions and guide the development of more efficient Salmonella vaccine candidates.

Acknowledgment This work was funded by the National Institute of Allergy and Infectious Diseases (NIAID), NIH, DHHS grants R01-

AI036525, U19-AI082655 [Cooperative Center for Human Immunology (CCHI)] and U19-AI109776 [Center of Excellence for Translational Research (CETR)].

References [1] Levine MM. Typhoid vaccines ready for implementation. N Engl J Med 2009;361(4):403 5. [2] Levine MM. Typhoid fever vaccines. In: Plotkin SA, Orenstein WA, Offit PA, Edwards KM, editors. Plokin’s Vaccines. 7th ed. Philadelphia: Elsevier, Inc; 2018. p. 1114 44. [3] John J, Van Aart CJ, Grassly NC. The burden of typhoid and paratyphoid in India: systematic review and meta-analysis. PLoS Negl Trop Dis 2016;10(4): e0004616. [4] Mogasale V, Maskery B, Ochiai RL, Lee JS, Mogasale VV, Ramani E, et al. Burden of typhoid fever in lowincome and middle-income countries: a systematic, literature-based update with risk-factor adjustment. Lancet Global Health 2014;2(10):e570 80. [5] Crump JA. Updating and refining estimates of typhoid fever burden for public health action. Lancet Global Health 2014;2(10):e551 3. [6] Mogasale V, Mogasale VV, Ramani E, Lee JS, Park JY, Lee KS, et al. Revisiting typhoid fever surveillance in low and middle income countries: lessons from systematic literature review of population-based longitudinal studies. BMC Infect Dis 2016;16:35. [7] Harish BN, Menezes GA. Antimicrobial resistance in typhoidal salmonellae. Indian J Med Microbiol 2011;29 (3):223 9. [8] Paterson GK, Maskell DJ. Recent advances in the field of Salmonella Typhi vaccines. Hum Vaccin 2010;6 (5):379 84. [9] Mitchell DH. Ciprofloxacin-resistant Salmonella typhi: an emerging problem. Med J Aust 1997;167(3):172. [10] Klemm EJ, Shakoor S, Page AJ, Qamar FN, Judge K, Saeed DK, et al. Emergence of an extensively drugresistant Salmonella enterica Serovar Typhi clone harboring a promiscuous plasmid encoding resistance to fluoroquinolones and third-generation cephalosporins. mBio 2018;9:1. [11] Levine MM, Ferreccio C, Abrego P, Martin OS, Ortiz E, Cryz S. Duration of efficacy of Ty21a, attenuated Salmonella typhi live oral vaccine. Vaccine 1999;17 (Suppl. 2):S22 7. [12] Black RE, Levine MM, Ferreccio C, Clements ML, Lanata C, Rooney J, et al. Efficacy of one or two doses of Ty21a Salmonella typhi vaccine in enteric-coated capsules in a controlled field trial. Vaccine 1990;8:81 4. [13] Ferreccio C, Levine MM, Rodriguez H, Contreras R. Comparative efficacy of two, three, or four doses of Ty21a live oral typhoid vaccine in enteric-coated

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REFERENCES

[14] [15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

capsules: a field trial in an endemic area. J Infect Dis 1989;159:766 9. Wain J, Hendriksen RS, Mikoleit ML, Keddy KH, Ochiai RL. Typhoid fever. Lancet 2015;385(9973):1136 45. Levine MP, Sztein MB, Pasetti MF. Salmonella enterica Serovar Typhi (typhoid) vaccines. In: WHO, editor. Immunization, vaccines and biologicals. WHO; 2011. http://apps.who.int/iris/bitstream/handle/10665/ 44752/9789241502610_eng.pdf;jsessionid 5 B7BCCD 5D3658080E77798D156E7161D6?sequence 5 1. Tacket CO, Ferreccio C, Robbins JB, Tsai CM, Schulz D, Cadoz M, et al. Safety and immunogenicity of two Salmonella typhi Vi capsular polysaccharide vaccines. J Infect Dis 1986;154(2):342 5. Jin C, Gibani MM, Moore M, Juel HB, Jones E, Meiring J, et al. Efficacy and immunogenicity of a Vi-tetanus toxoid conjugate vaccine in the prevention of typhoid fever using a controlled human infection model of Salmonella Typhi: a randomised controlled, phase 2b trial. Lancet 2017;390(10111):2472 80. World Health Organization. Typhoid Vaccines: WHO position paper, March 2018 Recommendations. Vaccine; 2018. Levine MM. Typhoid fever vaccines. In: Plotkin SA, Mortimer EA, editors. Vaccines. Philadelphia: W.B. Saunders Company; 1994. p. 597 633. Sahastrabuddhe S, Carbis R, Wierzba TF, Ochiai RL. Increasing rates of Salmonella Paratyphi A and the current status of its vaccine development. Expert Rev Vaccines 2013;12(9):1021 31. Maskey AP, Day JN, Phung QT, Thwaites GE, Campbell JI, Zimmerman M, et al. Salmonella enterica serovar Paratyphi A and S. enterica serovar Typhi cause indistinguishable clinical syndromes in Kathmandu, Nepal. Clin Infect Dis 2006;42(9):1247 53. Nasstrom E, Vu Thieu NT, Dongol S, Karkey A, Voong Vinh P, Ha Thanh T, et al. Salmonella Typhi and Salmonella Paratyphi A elaborate distinct systemic metabolite signatures during enteric fever. eLife 2014;3. Karkey A, Thompson CN, Tran Vu Thieu N, Dongol S, Le Thi Phuong T, Voong Vinh P, et al. Differential epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu, Nepal: a matched case control investigation in a highly endemic enteric fever setting. PLoS Negl Trop Dis 2013;7(8):e2391. Kuijpers LMF, Phe T, Veng CH, Lim K, Ieng S, Kham C, et al. The clinical and microbiological characteristics of enteric fever in Cambodia, 2008 2015. PLoS Negl Trop Dis 2017;11(9):e0005964. Salerno-Goncalves R, Pasetti MF, Sztein MB. Characterization of CD8(1) effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine. J Immunol 2002;169(4):2196 203.

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[26] Salerno-Goncalves R, Wyant TL, Pasetti MF, Fernandez-Vina M, Tacket CO, Levine MM, et al. Concomitant induction of CD4 1 and CD8 1 T cell responses in volunteers immunized with Salmonella enterica serovar typhi strain CVD 908-htrA. J Immunol 2003;170(5):2734 41. [27] Booth JS, Patil SA, Ghazi L, Barnes R, Fraser CM, Fasano A, et al. Systemic and terminal ileum mucosal immunity elicited by oral immunization with the Ty21a typhoid vaccine in humans. Cell Mol Gastroenterol Hepatol 2017;4(3):419 37. [28] Pennington SH, Thompson AL, Wright AK, Ferreira DM, Jambo KC, Wright AD, et al. Oral typhoid vaccination with live-attenuated Salmonella Typhi strain Ty21a generates Ty21a-responsive and heterologous influenza virus-responsive CD4 1 and CD8 1 T cells at the human intestinal mucosa. J Infect Dis 2016;213 (11):1809 19. [29] Waddington CS, Darton TC, Jones C, Haworth K, Peters A, John T, et al. An outpatient, ambulantdesign, controlled human infection model using escalating doses of Salmonella Typhi challenge delivered in sodium bicarbonate solution. Clin Infect Dis 2014;58 (9):1230 40. [30] Plotkin SA, Gilbert PB. Nomenclature for immune correlates of protection after vaccination. Clin Infect Dis 2012;54(11):1615 17. [31] Waddington CS, Darton TC, Woodward WE, Angus B, Levine MM, Pollard AJ. Advancing the management and control of typhoid fever: a review of the historical role of human challenge studies. J Infect 2014;68(5):405 18. [32] Hornick RB, Woodward TE. Appraisal of typhoid vaccine in experimentally infected human subjects. Trans Am Clin Climatol Assoc 1967;78:70 8. [33] Greisman SE, Woodward TE, Hornick RB, Snyder MJ, Carozza Jr. FA. Typhoid fever: a study of pathogenesis and physiologic abnormalities. Trans Am Clin Climatol Assoc 1961;73:146 61. [34] Hornick RB, Greisman SE, Woodward TE, DuPont HL, Dawkins AT, Snyder MJ. Typhoid fever: pathogenesis and immunologic control. N Engl J Med 1970;283 (13):686 91. [35] Levine MM, Tacket CO, Sztein MB. Host-Salmonella interaction: human trials. Microbes Infect 2001;3 (14 15):1271 9. [36] Dupont HL, Hornick RB, Snyder MJ, Dawkins AT, Heiner GG, Woodward TE. Studies of immunity in typhoid fever. Protection induced by killed oral antigens or by primary infection. Bull World Health Organ 1971;44(5):667 72. [37] Levine MM, DuPont HL, Hornick RB, Snyder MJ, Woodward W, Gilman RH, et al. Attenuated, streptomycin-dependent Salmonella typhi oral vaccine: potential deleterious effects of lyophilization. J Infect Dis 1976;133(4):424 9.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

512

29. INDUCTION OF LOCAL AND SYSTEMIC IMMUNITY BY SALMONELLA TYPHI IN HUMANS

[38] Gilman RH, Hornick RB, Woodward WE, DuPont HL, Snyder MJ, Levine MM, et al. Evaluation of a UDPglucose-4-epimeraseless mutant of Salmonella typhi as a live oral vaccine. J Infect Dis 1977;136(6):717 23. [39] Wahdan MH, Serie C, Cerisier Y, Sallam S, Germanier R. A controlled field trial of live Salmonella typhi strain Ty 21a oral vaccine against typhoid: three-year results. J Infect Dis 1982;145(3):292 5. [40] Levine MM, Ferreccio C, Black RE, Germanier R. Large-scale field trial of Ty21a live oral typhoid vaccine in enteric-coated capsule formulation. Lancet 1987;1(8541):1049 52. [41] Ferreccio C, Levine MM, Rodriguez H, Contreras R. Comparative efficacy of two, three, or four doses of TY21a live oral typhoid vaccine in enteric-coated capsules: a field trial in an endemic area. J Infect Dis 1989;159(4):766 9. [42] Begier EM, Burwen DR, Haber P, Ball R, Vaccine Adverse Event Reporting System Working G. Postmarketing safety surveillance for typhoid fever vaccines from the Vaccine Adverse Event Reporting System, July 1990 through June 2002. Clin Infect Dis 2004;38(6):771 9. [43] Waddington CS, Darton TC, Jones C, Haworth K, Peters A, John T, et al. An outpatient, ambulantdesign, controlled human infection model using escalating doses of Salmonella typhi challenge delivered in sodium bicarbonate solution. Clin Infect Dis 2014;58 (9):1230 40. [44] Fresnay S, McArthur MA, Magder L, Darton TC, Jones C, Waddington CS, et al. Salmonella Typhi-specific multifunctional CD8 1 T cells play a dominant role in protection from typhoid fever in humans. J Transl Med 2016;14(1):62. [45] Fresnay S, McArthur MA, Magder LS, Darton TC, Jones C, Waddington CS, et al. Importance of Salmonella typhi-responsive CD8 1 T cell immunity in a human typhoid fever challenge model. Front Immunol 2017;8:208. [46] McArthur MA, Fresnay S, Magder LS, Darton TC, Jones C, Waddington CS, et al. Activation of Salmonella typhi-specific regulatory T cells in typhoid disease in a wild-type S. Typhi challenge model. PLoS Pathog 2015;11(5):e1004914. [47] Darton TC, Jones C, Blohmke CJ, Waddington CS, Zhou L, Peters A, et al. Using a human challenge model of infection to measure vaccine efficacy: a randomised, controlled trial comparing the typhoid vaccines M01ZH09 with placebo and Ty21a. PLoS Negl Trop Dis 2016;10(8):e0004926. [48] Juel HB, Thomaides-Brears HB, Darton TC, Jones C, Jones E, Shrestha S, et al. Salmonella typhi bactericidal antibodies reduce disease severity but do not protect against typhoid fever in a controlled human infection model. Front Immunol 2017;8:1916.

[49] Toapanta FR, Bernal PJ, Fresnay S, Magder LS, Darton TC, Jones C, et al. Oral challenge with wild-type Salmonella typhi induces distinct changes in B cell subsets in individuals who develop typhoid disease. PLoS Negl Trop Dis 2016;10(6):e0004766. [50] van Furth R, Cohn ZA. The origin and kinetics of mononuclear phagocytes. J Exp Med 1968;128(3):415 35. [51] Peters JH, Ruppert J, Gieseler RK, Najar HM, Xu H. Differentiation of human monocytes into CD14 negative accessory cells: do dendritic cells derive from the monocytic lineage? Pathobiol: J Immunopathol Mol Cell Biol 1991;59(3):122 6. [52] Romani N, Gruner S, Brang D, Kampgen E, Lenz A, Trockenbacher B, et al. Proliferating dendritic cell progenitors in human blood. J Exp Med 1994;180 (1):83 93. [53] Toapanta FR, Bernal PJ, Fresnay S, Darton TC, Jones C, Waddington CS, et al. Oral wild-type Salmonella typhi challenge induces activation of circulating monocytes and dendritic cells in individuals who develop typhoid disease. PLoS Negl Trop Dis 2015;9(6):e0003837. [54] Dusseaux M, Martin E, Serriari N, Peguillet I, Premel V, Louis D, et al. Human MAIT cells are xenobioticresistant, tissue-targeted, CD161hi IL-17-secreting T cells. Blood 2011;117(4):1250 9. [55] Treiner E. Mucosal-associated invariant T cells in inflammatory bowel diseases: bystanders, defenders, or offenders? Front Immunol 2015;6:27. [56] Salerno-Goncalves R, Rezwan T, Sztein MB. B cells modulate mucosal associated invariant T cell immune responses. Front Immunol 2014;4:511. [57] Salerno-Goncalves R, Luo D, Fresnay S, Magder L, Darton TC, Jones C, et al. Challenge of humans with wild-type Salmonella enterica Serovar Typhi elicits changes in the activation and homing characteristics of mucosal-associated invariant T cells. Front Immunol 2017;8:398. [58] Kantele A, Zivny J, Hakkinen M, Elson CO, Mestecky J. Differential homing commitments of antigen-specific T cells after oral or parenteral immunization in humans. J Immunol 1999;162(9):5173 7. [59] Svensson M, Marsal J, Ericsson A, Carramolino L, Broden T, Marquez G, et al. CCL25 mediates the localization of recently activated CD8alphabeta(1) lymphocytes to the small-intestinal mucosa. J Clin Invest 2002;110(8):1113 21. [60] Brandtzaeg P, Farstad IN, Haraldsen G. Regional specialization in the mucosal immune system: primed cells do not always home along the same track. Immunol Today 1999;20(6):267 77. [61] Kantele A, Pakkanen SH, Karttunen R, Kantele JM. Head-to-head comparison of humoral immune responses to Vi capsular polysaccharide and Salmonella Typhi Ty21a typhoid vaccines--a randomized trial. PLoS One 2013;8(4):e60583.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

513

REFERENCES

[62] Pakkanen SH, Kantele JM, Savolainen LE, Rombo L, Kantele A. Specific and cross-reactive immune response to oral Salmonella Typhi Ty21a and parenteral Vi capsular polysaccharide typhoid vaccines administered concomitantly. Vaccine 2015;33(3):451 8. [63] Pakkanen SH, Kantele JM, Moldoveanu Z, Hedges S, Hakkinen M, Mestecky J, et al. Expression of homing receptors on IgA1 and IgA2 plasmablasts in blood reflects differential distribution of IgA1 and IgA2 in various body fluids. Clin Vaccine immunol: CVI 2010;17 (3):393 401. [64] Wahid R, Simon R, Zafar SJ, Levine MM, Sztein MB. Live oral typhoid vaccine Ty21a induces cross-reactive humoral immune responses against Salmonella enterica serovar Paratyphi A and S. Paratyphi B in humans. Clin Vaccine Immunol: CVI 2012;19(6):825 34. [65] Butcher EC, Williams M, Youngman K, Rott L, Briskin M. Lymphocyte trafficking and regional immunity. Adv Immunol 1999;72:209 53. [66] Kunkel EJ, Campbell JJ, Haraldsen G, Pan J, Boisvert J, Roberts AI, et al. Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity. J Exp Med 2000;192(5):761 8. [67] Hamann A, Andrew DP, Jablonski-Westrich D, Holzmann B, Butcher EC. Role of alpha 4-integrins in lymphocyte homing to mucosal tissues in vivo. J Immunol 1994;152(7):3282 93. [68] Lundin BS, Johansson C, Svennerholm AM. Oral immunization with a Salmonella enterica serovar typhi vaccine induces specific circulating mucosa-homing CD4(1) and CD8(1) T cells in humans. Infect Immun 2002;70(10):5622 7. [69] Salerno-Goncalves R, Wahid R, Sztein MB. Immunization of volunteers with Salmonella enterica serovar Typhi strain Ty21a elicits the oligoclonal expansion of CD8 1 T cells with predominant Vbeta repertoires. Infect Immun 2005;73(6):3521 30. [70] Wahid R, Salerno-Goncalves R, Tacket CO, Levine MM, Sztein MB. Generation of specific effector and memory T cells with gut- and secondary lymphoid tissue- homing potential by oral attenuated CVD 909 typhoid vaccine in humans. Mucosal Immunol 2008;1(5):389 98. [71] Wahid R, Fresnay S, Levine MM, Sztein MB. Immunization with Ty21a live oral typhoid vaccine elicits crossreactive multifunctional CD8 1 T-cell responses against Salmonella enterica serovar Typhi, S. Paratyphi A, and S. Paratyphi B in humans. Mucosal Immunol 2015;8(6):1349 59. [72] Wahid R, Fresnay S, Levine MM, Sztein MB. Crossreactive multifunctional CD4 1 T cell responses against

[73]

[74] [75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

Salmonella enterica serovars Typhi, Paratyphi A and Paratyphi B in humans following immunization with live oral typhoid vaccine Ty21a. Clin Immunol 2016;173:87 95. Sztein MB. Cell-mediated immunity and antibody responses elicited by attenuated Salmonella enterica Serovar Typhi strains used as live oral vaccines in humans. Clin Infect Dis 2007;45(Suppl. 1):S15 19. Parry CM, Hien TT, Dougan G, White NJ, Farrar JJ. Typhoid fever. N Engl J Med 2002;347(22):1770 82. Ukwenya AY, Ahmed A, Garba ES. Progress in management of typhoid perforation. Ann Afr Med 2011;10 (4):259 65. Nguyen QC, Everest P, Tran TK, House D, Murch S, Parry C, et al. A clinical, microbiological, and pathological study of intestinal perforation associated with typhoid fever. Clin Infect Dis 2004;39 (1):61 7. Mukawi TJ. Histopathological study of typhoid perforation of the small intestines. Southeast Asian J Trop Med Public Health 1978;9(2):252 5. Everest P, Wain J, Roberts M, Rook G, Dougan G. The molecular mechanisms of severe typhoid fever. Trends Microbiol 2001;9(7):316 20. Golakai VK, Makunike R. Perforation of terminal ileum and appendix in typhoid enteritis: report of two cases. East Afr Med J 1997;74(12):796 9. Mowat AM, Agace WW. Regional specialization within the intestinal immune system. Nat Rev Immunol 2014;14(10):667 85. Ohland CL, Jobin C. Microbial activities and intestinal homeostasis: a delicate balance between health and disease. Cell Mol Gastroenterol Hepatol 2015;1(1):28 40. Jozwik A, Habibi MS, Paras A, Zhu J, Guvenel A, Dhariwal J, et al. RSV-specific airway resident memory CD8 1 T cells and differential disease severity after experimental human infection. Nat Commun 2015;6:10224. Yang OO, Ibarrondo FJ, Price C, Hultin LE, Elliott J, Hultin PM, et al. Differential blood and mucosal immune responses against an HIV-1 vaccine administered via inguinal or deltoid injection. PLoS One 2014;9 (2):e88621. Booth JS, Toapanta FR, Salerno-Goncalves R, Patil S, Kader HA, Safta AM, et al. Characterization and functional properties of gastric tissue-resident memory T cells from children, adults, and the elderly. Front Immunol 2014;5:294. Booth JS, Salerno-Goncalves R, Blanchard TG, Patil SA, Kader HA, Safta AM, et al. Mucosal-associated invariant T cells in the human gastric mucosa and blood: role in Helicobacter pylori infection. Front Immunol 2015;6:466.

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Oral Shigella Vaccines Marcela F. Pasetti1, Malabi M. Venkatesan2 and Eileen M. Barry1 1

Center for Vaccine Development and Global Health, University of Maryland School of Medicine; Baltimore, MD, United States 2Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD, United States

I. INTRODUCTION Shigella species cause severe diarrheal and dysenteric illness in humans, both endemically and as epidemics. Clinical symptoms and signs of disease include fever, intestinal pain, moderate to severe watery diarrhea, and fecal depositions with blood and mucus (dysentery). If left untreated, infection can result in uncontrolled intestinal inflammation, dehydration, and death. Shigella-induced diarrhea is seen primarily in young children living in impoverished areas of the world [1,2] and in all other age groups during outbreaks [3 5]. There are four pathogenic species of Shigella (S. sonnei, S. flexneri, S. boydii, and S. dysenteriae), which include several serotypes and subtypes based on differences in the O-polysaccharide antigen. S. dysenteriae 1, S. sonnei, and all serotypes and subtypes of S. flexneri are associated with the highest morbidity rates. While therapy with antibiotics to which Shigella species are sensitive can significantly lessen the severity and duration of illness and can limit shedding [6], Shigella species are

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00030-4

notorious for rapidly developing antibiotic resistance [7,8]. The high transmissibility of this organism, the clinical severity of bacillary dysentery, and the emergence of multiple antibiotic-resistant strains have impelled interest in immunological control of shigellosis by means of well-tolerated, practical, and effective vaccines. Unfortunately, no approved vaccine is currently available. A number of promising candidates have been produced, the most advanced being orally administered whole cell inactivated [9,10] and live attenuated Shigella strains [11 13] as well as parenterally administered subunit candidates such as O-specific polysaccharide-protein conjugates [14 16], subcellular complexes containing lipopolysaccharide (LPS) and invasion plasmid antigens (Ipas) [17], and outer membrane particles [18]. These vaccine candidates have been tested in human clinical studies with a wide range of outcomes (reviewed in Refs. [19 22,177]). Oral administration of live attenuated strains has been a leading immunization strategy, and is based on modeling natural Shigella infection (Fig. 30.1), which is known to confer protective

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FIGURE 30.1

Steps involved in oral Shigella infection and vaccination. (A) The normal colon in steady state. The intestinal epithelial cell barrier separates the lumen content from the highly regulated internal compartment. Commensal organisms help to maintain gut homeostasis. Peyer’s patches alongside the small and large intestine and colon are immune inductive sites. Antigens translocate the epithelial barrier through microfold (M) cells, and are delivered to underlying phagocytes antigen-presenting cells (macrophages and dendritic cells). Peyer’s patches contain B and T cells, which can be activated by luminal antigens (directly or through antigen-presenting cells, respectively). (B) Orally delivered Shigella antigens and (attenuated) vaccine organisms translocate through M cells and are delivered to adjacent phagocytic cells. Live vaccines may induce a controlled replication in the colonic epithelium, resulting in stronger immune stimulation than killed organisms. Infection of macrophages leads to cell death (apoptosis, pyroptosis, and necrosis). Released antigens alone or captured by antigen-presenting cells stimulate B and T cells. (C) During acute infection, Shigella invades and disrupts the rectal and colonic mucosa (blood dissemination is rare except in malnourished children). The organism infects the epithelial cells from the basolateral side and spreads intracellularly and cell-to-cell across the epithelium. Upon invasion, epithelial cells produce inflammatory molecules, particularly IL-8. Polymorphonuclear cells (neutrophils) rapidly accumulate, resulting in inflammation, disruption of the epithelium, and dysentery (blood and mucus in stool).

immunity [23 25]. Likewise, inactivated whole cell organisms have been pursued as candidates for oral vaccination. Both concepts have been evaluated in multiple human clinical trials involving different populations, including field sites. Immunization via the oral route is practical and offers the opportunity to induce local as well as systemic protective immunity as it occurs during natural exposure. This chapter reviews the progress of Shigella vaccine development, focusing on clinically advanced oral vaccine candidates. It provides an update on disease burden, animal models

used in vaccine studies, and human immune responses to Shigella. Gaps in knowledge and the future of oral vaccines to prevent Shigellainduced diarrhea are discussed.

II. SHIGELLA INFECTION: BURDEN OF DISEASE AND VULNERABLE GROUPS The greatest burden of shigellosis is borne by children 1 5 years of age living in poor countries that lack adequate sanitation and access to

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III. SHIGELLA PATHOGENESIS AND VIRULENCE FACTORS

clean drinking water. The Global Enteric Multicenter Study identified Shigella as one of the top attributable agents of moderate to severe diarrhea (MSD) among children 12 23 months old living in sub-Saharan Africa and South Asia (exceeded only by rotavirus), and the most prevalent etiology of MSD among children 24 59 months of age [1,26]. Similarly, Shigella has been associated with the highest severity of diarrheal disease during the second year of life, with an increasing trend, in the Malnutrition and Enteric Disease birth cohort study [27]. Significant morbidity and mortality have also been reported among children older than 5 years of age and adolescents [28], and these estimates are likely underestimates [26,29]. Even if not life threatening, repeated episodes of diarrhea during childhood impair physical and cognitive development, resulting in long-term disability and reduced life expectancy [30]. Owing to the mode of transmission (fecal oral and person-to-person) and the low infectious dose, the risk of contracting shigellosis is heightened in overcrowded conditions. S. flexneri is the major cause of endemic diarrhea in developing countries, while S. sonnei is the most common cause of shigellosis in industrialized and transitional countries [31]. S. dysenteriae type 1, which, unlike the other serotypes, produces Shiga toxin, is responsible for largescale outbreaks among displaced populations and those living in refugee settlements [32]; these major epidemics (or pandemics) are associated with high attack rates and case fatality and usually involve antibiotic-resistant strains [7]. Shigella outbreaks have also been reported in confined groups, such as patients in hospitals [33] and children in school and day care centers [34]. Shigella is an opportunistic pathogen in immunocompromised individuals and those infected with HIV [35]. Travelers to regions where Shigella is endemic and military personnel are vulnerable to diarrhea caused primarily by Shigella and enterotoxigenic Escherichia coli (ETEC) [36].

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III. SHIGELLA PATHOGENESIS AND VIRULENCE FACTORS The defining feature of Shigella pathogenesis is its ability to invade, replicate, and spread within the host mucosal epithelium [37]. Acquired via contaminated food or water or from human-to-human contact, Shigella traverses the human intestinal epithelial cell barrier via microfold (M) cells and accesses underlying macrophages and other immune cells within the lymphoid follicles (Fig. 30.1). Instead of being killed by macrophages, internalized Shigella causes a caspase-1-mediated cell death, originally called apoptosis [38] and now referred to as pyroptosis [39,40], and the secretion of proinflammatory cytokines. Following release from macrophages, Shigella invades adjacent intestinal epithelial cells from the basolateral side, where they proceed to replicate intracellularly and spread cell to cell in a lateral fashion, using host cell actin for propulsion. The ensuing cell death and inflammation cause an influx of polymorphonuclear granulocytes, a hallmark of Shigella infection and the clinical symptoms of diarrhea and dysentery. The molecular mechanisms underlying these processes have been dissected in elegant detail and are reviewed elsewhere [41,42]. While chromosomally encoded factors play a role in pathogenesis, the large virulence plasmid that is present in all strains of pathogenic Shigella encodes key factors essential for invasion: a type 3 secretion system (encoded by the mxi-spa loci) that enables the secretion of several effector proteins such as the Ipas, as well as other virulence factors (including IcsA or VirG) required for pathogenicity [43,44]. The recent completion of a large number of Shigella genome sequences has revealed the variable presence of genes encoding other potential virulence elements in a strain-dependent manner, including pathogenicity islands [45]. In addition to the presence of virulence loci, Shigella genomes have lost or inactivated several genes

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that are present in the closely related E. coli. The loss of these genes, termed black holes or antivirulence loci, confer pathogenic properties to the Shigella strain [46,47]. The understanding of pathogenic mechanisms and identification of virulence factors has guided Shigella vaccine development strategies by providing targets for gene inactivation to produce live attenuated candidates (i.e., virG [48] and guaBA [49]), and by uncovering protective antigens (e.g., IpaB and IpaD [50]).

IV. NATURALLY ACQUIRED IMMUNITY AGAINST SHIGELLA Natural infection leads to protective immunity against shigellosis that is serotype-specific and targets the O-polysaccharide. It is known from epidemiological and seroepidemiological studies that older individuals living in endemic regions and repeatedly exposed to the organism have a lower risk of infection, and that this trend coincides with a gradual (age-dependent) increase in protective immunity [51 53]. It was further documented in experimental challenge studies in nonhuman primates [54] and humans [25,55] that an initial clinical infection with Shigella results in short-lived, serotypespecific protection. Both children and adults living in areas where Shigella is endemic develop serum antibodies against Shigella LPS, Ipas, and other bacterial antigens, such as IcsA [53,56 59]. Antibody levels increase during convalescence and with age as a result of subsequent infections [53]. No firm immune correlates of protection against shigellosis have been established. However, seroepidemiological studies have demonstrated a strong association between reduced incidence of shigellosis and preexisting serum IgG against LPS of the infecting serotype [60,61]. Furthermore, the efficacy of conjugate vaccines has been predicated on the presence of high levels of serum IgG against

the O-antigen based on their association with vaccine-induced protection [15,62]. Elevated IpaB and VirG-specific serum IgG antibodies and IpaB-specific B memory cells have also been associated with reduced disease in adult human volunteers experimentally challenged with virulent S. flexneri 2a [63,64]. Serum antibodies produced during natural Shigella infection promote complementmediated killing [65 67] and opsonophagocytic activity [66,68]. The magnitude of serum antibodies with bactericidal (SBA) and opsonophagocytic killing activity (OPKA) prior to infection was found to be associated with clinical protection in a controlled Shigella human challenge study [63]. Antibodies specific for the Shigella O-antigen have bactericidal activity [69]. Whether other antigens induce and/or are targets of functional antibody activity remains to be investigated. In addition to systemic immunity, oral exposure to Shigella results in strong local immune responses. Mucosally primed LPS and Ipaspecific antibody secreting cells (ASCs) can be detected in peripheral blood 7 10 days after exposure [70]. These cells bear intestinal homing markers (CCR9 and α4β7) [70], and have the capacity to migrate to intestinal (e.g., colon) effector sites for production of local antibodies. The frequency of circulating LPS IgA ASCs is also considered a proxy of protective immunity [71,48]. More recently, the measurement of antibodies in lymphocyte supernatant (ALS), representing antigen-specific immunoglobulins produced ex vivo by mucosally primed ASCs, has been proposed as a practical method to determine Shigella-induced mucosal immunity [72]. Expansion of IgA- (IgA2 . IgA1) and IgG2producing cells has been reported in rectal mucosal biopsies from Shigella-infected patients, which persisted long after the clinical symptoms had resolved [73]. The expression of the secretory component of IgA transport was also increased in mucosal infected tissue. The presence of IgG21 cells in the mucosa of acute

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V. IMMUNE RESPONSES TO SHIGELLA IN CHILDREN

phase patients has been linked with the early increase in LPS-specific IgG serum antibodies following infection [73]. LPS- and Ipa-specific IgG and IgA have also been found in the stool of infected individuals as early as 3 5 days after onset [58], and an increase in fecal IgA is prominent in patients with severe disease [74]. A severe mucosal inflammation (associated with a complex cellular reaction) is typically seen in adults with acute shigellosis, which persists through convalescence [75,76]. This process has been well documented through histopathological changes [73] and increased production of proinflammatory, T helper 1 (Th1)- and Th2-type cytokines (IL-1, IL-4, IL-6, IL-8, IL-10, TNF-α, IFNγ, GM-CSF, and TGF-β) in rectal biopsies [77]. Interestingly, the receptors for some of these cytokines were downregulated during acute stage of disease, possibly aiming to control bacteria-induced cell damage [77 79]. Elevated levels of proinflammatory cytokines (IL-1β, IL1ra, TNF-α, IL-6, IL-8, and GM-CSF) in stool during acute Shigella infection correlated with severity of disease [79]. Some contend that this acute inflammation, through the production of immunosuppressive cytokines and mucosal cell death, may interfere with the development of adaptive immunity [80,81]. The presence of intraepithelial lymphocytes, infiltration of CD41 and CD81 T cells in the lamina propria, and expanded mucosal T cell repertoires have also been described in patients with bacillary dysentery [82]. IFNγ tissue deposition and levels of IFNγ receptor expression in stool were increased in convalescent as compared to acutely infected patients, suggesting a role for IFNγ in clearing infection [79]. The inflammation associated with Shigella infection is also reflected systemically with increased leukocytes and T cell counts. Activated and memory CD41 and CD81 T cells, as well as NK cells, have been found in peripheral blood of Shigella-infected patients [83]. In the early stages of infection, T cells expressing a

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gut-specific homing/activation marker (CD103) detected in circulation suggest local T cell activation and homing [83]. Shigella can infect human T cells and B cells [81,84,85], although it is not clear how these processes directly relate to or influence adaptive immunity. The contribution of T cells in controlling Shigella infection, particularly resident memory cells, requires further investigation. Imminent clinical studies of new and improved vaccine candidates, particularly studies in endemic areas and in target groups, as well as controlled human infection models will provide opportunities to further dissect innate and adaptive immunity to Shigella and define the elusive immunological correlates of protection.

V. IMMUNE RESPONSES TO SHIGELLA IN CHILDREN Knowledge of Shigella pathogenicity and immunity derives primarily from infected adults. The host pathogen interaction and immunological priming in younger individuals are less known. Both children and adults with acute shigellosis exhibit mucosal innate immune cell activation. However, lower levels of stool superoxide dismutase and persistent production of lactoferrin have been reported in children, which may contribute to chronic inflammation and worsen tissue damage [86]. Similarly, Shigella-infected children purportedly mount an immune response that is delayed and of lower magnitude compared with that of adults. They produce primarily IgG1 antibodies in response to LPS, while IgG2 is the predominant adult response. At younger ages (3 5 years), children fail to produce IgG3, a subclass that is important for Fc-mediated functional activity [53]. Delayed responses in the production of stool IgA, TNF-α, and IFNγ by peripheral blood cells upon in vitro antigen stimulation were also reported [53]. Identifying the effector responses necessary for protection

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30. ORAL SHIGELLA VACCINES

of children against shigellosis and establishing reliable methods to determine such responses have a high priority for this highly vulnerable group.

VI. ANIMAL MODELS OF MUCOSAL SHIGELLA INFECTION There is no animal model that fully recapitulates the disease caused by Shigella in humans. While this has curtailed advances in vaccine development, several animal models that provide useful insight into aspects of human Shigella infection have been used in the evaluation of vaccine candidates. Infection of nonhuman primates with Shigella results in diarrhea and disease similar to that found in humans. This model has revealed important aspects of Shigella pathogenesis and has been used to assess a variety of vaccine candidates [87,88]. A weakness of this nonhuman primate model is the requirement for an infectious dose that is more than 100,000 times higher than that required for human infection. In addition, its high cost and the requirement for special facilities and expertise make this model impractical and prevent widespread use. Guinea pigs have been used extensively to assess the safety, immunogenicity, and protective capacity of Shigella vaccine candidates. Immunization of guinea pigs by intranasal or intraocular routes followed by ocular challenge with wild-type organisms in the Sereny test has been used to evaluate the protective capacity of live and subunit vaccines [49,89,90]. The lack of inflammation and keratoconjunctivitis in the Sereny reaction has served as a test for the safety of live attenuated vaccine candidates [91]. In addition, vaccine-induced systemic and mucosal antibodies can be quantified in serum and tears. More recently, a guinea pig rectocolitis model has been developed wherein intrarectal inoculation with virulent Shigella was shown to produce diarrhea, dysentery, and pathological

features similar to those found in human disease [92,93]. This model is appealing for its relevant disease characteristics, and is currently being applied to vaccine development. The mouse pulmonary model (pulmonary pneumonia model) has been widely used in vaccine evaluation [50,94 97]. Mice can be immunized by the intranasal route, and vaccines can be examined for their capacity to elicit a wide array of immune responses and protection against a lethal pulmonary challenge. In this model, the pulmonary tract acts as a mucosal surrogate for natural infection in the gastrointestinal tract. An important observation derived from mouse studies is the thymic independence of adaptive immunity to Shigella [98], which supports a major role for humoral immunity, particularly antibodies other than IgA [99], in protection against infection. The mouse model has been adapted to include vaccination of infant mice to investigate immune responses and protection at very early life stages and passively transferred maternal immunity [100,101]. Other murine models involve oral challenge of newborn mice that results in colonic invasion and cellular destruction [102] and intraperitoneal infection of adult mice [103]. These models have not been widely used and are constrained by the limitations in animal age and incomplete characterization, respectively. Rabbits were also used extensively in the past to study Shigella pathogenesis, especially invasion in the rabbit ileal loop model [104,105]. This model has not been adapted for evaluation of vaccine candidates.

VII. HISTORY OF ORAL SHIGELLA VACCINE CANDIDATES Early oral Shigella vaccine candidates consisting of single or combined (multiple serotypes) heat-killed, formalin-inactivated, or UVirradiated strains administered parenterally (subcutaneously) at doses from 1.5 3 109 to 6.3 3 109 CFU (three immunizations at weekly

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VIII. IMMUNITY INDUCED BY ORAL SHIGELLA VACCINES

intervals) proved ineffective against naturally occurring Shigella infections [106,107]. The same vaccines (at lower doses) were administered subcutaneously to infants and children living in areas of high endemicity, but with no evidence of efficacy [106]. The failure of this initial approach propelled studies of oral vaccination. An acetone-killed and dried Shigella vaccine fed to monkeys in multiple doses also failed to mount protective immunity [108]. The lack of protection in individuals who received killed vaccines prompted clinical trials with live, but noninvasive vaccine candidates (Table 30.1). These included the opaque colonial variants of S. flexneri 2a strain 2457T (referred to as 2457O), the streptomycindependent strains, which provided 90% protection in field trials in Yugoslavia, and the T-32 Istrati strain (Vadizen), which was safe and afforded protection for 6 months [23,110 113]. The need for multiple high doses, which makes it particularly difficult for use in children, discouraged further development of these candidates. More recently, a refined formalin-killed Shigella vaccine has been evaluated orally at high doses with some success [129]. The recognition of Shigella O-antigen mediated serotype-specific protection and of the role of the large virulence plasmid in pathogenesis led to a series of Salmonella Typhi and E. coli Shigella hybrid vaccine candidates (listed in Table 30.1 and described below). These hybrid strains were tested in multiple doses in humans, found to be reactogenic, and to yield inconsistent efficacy rates [71,114,115]. More recently, Shigella-based live oral vaccine candidates were designed that contained specific mutations either in metabolic genes such as aroD or guaBA, in specific virulence-associated genes such as virG(icsA), senA, senB, or in a combination of both (Tables 30.1 and 30.2). The rationale for this approach was to develop a vaccine that would recreate the steps of natural infection: reach the colon, translocate across the intestinal barrier, possibly undergo limited spreading

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(intercellularly and intracellularly) minimizing inflammation, and activate a local and systemic adaptive immune response. Table 30.1 provides a comprehensive list of oral Shigella vaccines that have been tested in human clinical studies. Most of these candidates have proven to be safe, immunogenic, and in some cases protective against challenge. Some of them are progressing through different stages of clinical development.

VIII. IMMUNITY INDUCED BY ORAL SHIGELLA VACCINES O-antigens and Ipas are considered the major immune-stimulating Shigella antigens [148]. Antibody and B cell responses reported in humans immunized orally with whole cell vaccine candidates include: LPS- and Ipaspecific serum IgG and IgA; LPS- and Ipaspecific fecal IgA; LPS- and Ipa-specific IgG and IgA ASC and ALS, Shigella-specific BM cells bearing the α4β71 gut homing marker and serum functional antibodies (bactericidal and opsonophagocytic killing). T cell responses include the production of cytokines, mainly IFNγ, by antigen-stimulated peripheral blood mononuclear cells and by CD4 6 and α4β7 6 CD81 T effector memory cells in live vaccine recipients. Immunogenicity data from historic and recent oral Shigella human vaccine studies are summarized in Tables 30.1 and 30.2. Most of the information that is available pertains to humoral immunity, expected to be a major contributor to protection: IgA (against LPS and other antigens) by interfering with bacterial invasion in the gut and IgG by facilitating complement-mediated killing as well as phagocytic (e.g., macrophage, neutrophil) uptake and killing. In contrast to the wide array of responses induced by whole cell vaccines, the efficacy of O-polysaccharide conjugate vaccines relies primarily on the production of high levels of LPS-specific serum IgG. These antibodies exhibit bactericidal activity [16].

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TABLE 30.1 History of Oral Shigella Vaccines Vaccine

Mutation

Dose

2457O

Spontaneous noninvasive mutant, IS1 insertion in virF

High

Escherichia coli Shigella hybrid, X16

Invasive, incorporates High section of E. coli from xylrha into Shigella flexneri 2a, cadA 1 , Sereny 1

Number of doses

Safety

Multiple oral doses, reverts to virulence

Reactogenic, caused dysentery in 34% of volunteers

Single dose

Avirulent in monkeys; virulent for one investigator

Immunogenicity/ efficacy Comments

Reference

Protection

[23]

Caused inflammation but did not persist in lumen of starved guinea pigs

[109]

Streptomycin- Noninvasive, incapable 5 3 1010 CFU dependent of growing in the absence (SmD) of streptomycin

Multiple doses Diarrhea and vomiting in Protectiona in field trials 15% 35% volunteers, in some trials reversion to strep independence, low reactogenicity

Unstable phenotype; [23,110,111] inconsistent efficacy

S. flexneri 2a strain T32ISTRATI (Vadizen)

Multiple doses Safe, mild adverse reaction at 1011 CFU

Approximately 80% protectionb

Spontaneous loss of ipa-mxi-spa, invA, and virG loci

[112,113]

Two or three doses of freshly harvested vaccine weekly, preceded by bicarbonate

Safe

Only 10% of volunteers showed increased serum antibodies to 2a O-antigen, no protection

Shed for 2 weeksc

[114]

One study at USAMRIID, three doses given in skim milk after bicarbonate

Safe

Overall inconsistent protection; lot-tolot variationd

[25]

Two or three doses

High dose caused diarrhea in 30% of volunteers, lower doses were safe

Little protection at lower doses

[115]

Noninvasive, developed 2 3 1011 CFU in Romania in 1961 after repeated passage on agar of S. flexneri 2a strain

PGA1-42-1-15 Noninvasive, E. coli 08 with his and met markers conjugally transferred from an S. flexneri 2a Hfr donor, expresses S. flexneri 2a O-antigen

5076-IC: Ty21a, galE-, Vi-, with Shigella sonnei VP

3 3 1010 CFU, challenge 8 weeks after last dose with 104 or 102 CFU of 2457T

Ty21a is a chemically 107 1010 CFU induced S. Typhi galE mutant, 5076-1C is Ty21a with S. sonnei virulent plasmid carrying Oantigen

E. coli-Shigella Invasive, E. coli K12 with 109 CFU and hybrid, His, Pro, Arg from S. 107 CFU Ecsf2a-1 flexneri 2a and VP from S. flexneri 5 no fluid secretion in ligated rabbit intestine

E. coli-Shigella Invasive, EcSf2a-1 with hybrid, aroD mutation and VP EcSf2a-2 from S. flexneri 5

109 and 1010 CFU

Three or four doses

Safe, reactogenic at 1010 CFU due to some bacterial spreading, tiny plaques seen in plaque assays

Immunogenic, protection seen against dysentery and diarrhea, not fever

Requires exogenous PABA (precursor of folic acid) not present in mammalian cells

[71]

S. flexneri Y strain, Sfl124

2 3 109 CFU

Multiple doses Safe in adult Swedish and Vietnamese volunteers, also given to Vietnamese children 9 14 years at 107 109 CFU

Vaccine was shed in adults and children; immunogenice

Lyophilized, made by National Bacteriology Lab, Stockholm; no diarrhea in monkeys given 1011 CFU

[22,116 118]

Three times, on days 0, 2, and 4, tested at four dosage levels

At the highest dose, four of nine subjects were sick. At 108 CFU, three of nine subjects had mild constitutional symptoms

Immunogenic with systemic and mucosal responsesf

Tested in adult Swedes

[22,119]

Invasive, parent strain Sfl1 is moderately virulent, Sfl124 has a 1.4kb deletion in the aroD, makes small plaques

S. flexneri 2a Invasive, parent strain 105 109 CFU strain, Sfl1070 more virulent 2457T with aroD deletion

S. flexneri 2a strain CVD1203

Invasive, parent strain 2457T, deletion in aroA and virG(icsA)

106, 108, 109 Two doses, CFU, 108 CFU day 0 and day 14

AEs at the two highest doses

Serum and mucosal Ab responsesg

85% 90% shed the vaccine

[120]

CVD1204

Invasive, 918-bp deletion in the guaBA operon of 2457T

107 109 CFU

See under CVD1208

Anti-LPS IgA ASC; LPS serum IgG and IgA; fecal IgA in all subjects at 109 CFU

86% shedding at 109 CFU; mean peak excretion 1.1x105 CFU/g stool

[12]

CVD1205

CVD1204 with ΔvirG

CVD1207

Invasive, CVD1205 with loss of senA, setAB, Sereny negative

106 1010 CFU Single dose

At the two highest doses, 1 of 12 and 1 of 6 subjects had diarrhea and emesis, no fever or dysentery

65% 100% shedders also had LPS ASC, modest serum Ab

100% shedding at the two highest doses for 1 3 days

[121]

CVD1208

CVD1204 with loss of senA and setAB

107 109 CFU

1 of 21 subjects receiving CVD1208 had mild fever and only at the highest dose in contrast to 8 of 23 who received CVD 1204

Anti-LPS IgA ASC; LPS serum IgG and IgA; fecal IgA at 109 CFU of CVD1208

86% shedding at 109 CFU; mean peak excretion 1.9x104 CFU/g stool

[12]

Single dose

Single dose

(Continued)

TABLE 30.1 (Continued) Vaccine

Mutation

Number of doses

Dose

Safety

Immunogenicity/ efficacy Comments

81% and 19% of subjects had mild and moderate symptoms, respectively; no dose response in frequency or intensity of symptoms

Dose levels of $ 105 CFU induced LPS-IgA ASCs, few subjects had serum antibody

Three subjects shed for 1 day, no immune response to StxB

[122,123]

Protection after challengeh

Proof-of-concept study for loss of virG-based attenuation

[48,124]

Shigella dysenteriae 1 strain SC599

Invasive, parent strain 1617, lacks icsA, ent, fep, and stxA

10

S. flexneri 2a strain SC602

Invasive, parent strain 2457T, lacks virG(icsA) and iuc, plaque and Sereny negative

102 108 CFU, Single dose, 1 of 12 subjects at 104 safe at 104 CFU had mild diarrhea, ciprofloxacin CFU given on day 8 no fever or SAE, 7 of these subjects were challenged with 1000 CFU of 2457T 2 months later

2

8

10 CFU

Single dose; ciprofloxacin given on day 4

Reference

S. sonnei Invasive, lacks virG(icsA), 103 106 CFU strain, WRSS1 more recently shown to lack ent and fep as part of a 82-kb chromosomal deletion

Single dose

22% had mild transient fever, 50% 100% of the vaccinees shed, safe at 103 104 CFU

Highly immunogenici

Challenge in Thai [11,125 127] adults after immunization with a single dose of 104 CFU, B40% protection

S. dysenteriae 1 strain WRSd1

Single dose

20% had mild transient diarrhea, nine of 40 subjects shed for 1 2 days

Immunogenicj

Overall poor shedding, likely owing to loss of fnr

Lacks virG(icsA), stxAB, and fnr

103 107 CFU

[11,128]

a Developed in Yugoslavia for S. flexneri 1, 2a, 3a, 4 and S. sonnei, up to 90% protection in field trials for more than 6 months but less than 1 year; a single oral booster dose after 1 year maintained protection for an additional year. US trials conducted in the early 1970s, also in institutionalized children, showed marginal protection. b Multiple doses in 12 separate field trials in Romania (60,000 volunteers) and China (5000 volunteers), produced and packaged by the Cantacuzino Institute in Bucharest as a suspension of 1011 CFU/mL in a synthetic preserving medium. This preparation is stable at 4 C for 60 days. c Type II serotype is linked to met, rfb loci; 3,4 group antigen linked to His. d USAMRIID study: Only 2 of 10 volunteers had antibody response to S. sonnei. In a second study, 40 vaccinees and 38 controls were fed three different lots of vaccines; two lots protected against dysentery. The third lot made at Swiss serum institute; three doses were given on days 0, 3, 7, and vaccine recipients were challenged 1 month later with 500 CFU of S. sonnei 53G. IgA ASC were measured for the first time. The vaccine did not protect. e One or three doses on days 0, 2, 4, in Swedish adults, booster dose 9 10 months later to those receiving three doses, single dose in Swedish children 9 14 years, given 30 mL 4% bicarb-orange juice mixed with vaccine (pH 7.4), children not allowed to have food and beverage for 2 h pre- and post-vaccination. Two of 21 adult Swedes had diarrhea and fever for a single day at 107 CFU dose. No shedding was detected by culture; 9 of 10 children had shedding documented by immune-magnetic beads and PCR, at 108 CFU dose, 2 of 10 shed by culture, by PCR 9 of 10 shed, at 109 CFU, 3 of 10 shed by culture, by PCR, 10 of 10 were positive. ASC responses in children in 1 and 5 of 10 subjects at 108 and 109 CFU dose, respectively; SIgA were detected in stools. f Vigorous IgA ASC response to LPS, 990 per 106 PBMCs; 78% 90% had fecal IgA response; 15 of 128 volunteers had serum antibody responses to LPS, less to Ipa. g After a single dose of the CVD1203 vaccine, 70%, 46%, and 64% of the subjects showed a serum TNF-α response in groups 1, 2, and 3, respectively. However, fecal TNF-α response was seen in 0%, 27%, and 18% of the subjects, which is a lower responder rate than for serum levels. More volunteers responded with ASCs and SIgA than with serum antibody responses. h 7 of 12 subjects who received 104 CFU had LPS IgA ASC responses, and 4 had fourfold or greater increase in LPS serum IgG and IgA; 7 vaccinated subjects were challenged 2 months later; 4 were protected against fever, moderate to severe diarrhea, dysentery, and shigellosis, while the remaining 3 subjects had mild fever and diarrhea after challenge. i Geometric mean of LPS-specific IgA ASC were 99, 39, 278, and 233 per 106 PBMC at the four doses tested, 70% had a greater than fourfold increase in LPS serum IgA and IgG. j 24 of 40 subjects (60%) had a positive IgA ASC response, and 8 of 40 subjects had .40 ASC, but this was not dose related; 10 of 32 subjects had a fourfold increase in serum IgA to LPS, 5 of these subjects also had serum IgG response.

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IX. MULTIVALENT ORAL SHIGELLA VACCINES

TABLE 30.2

Current oral Shigella Vaccine Candidates

Vaccine

Developer

Phase of study

Whole cell killed vaccine

PATH

Live attenuated VirG derivatives

WRAIR, Silver Spring, MD

GuaBA series

CVD, University of Maryland Phase 1

ShigETEC

EveliQure Biotechnologies GmbH, Vienna, Austria

Preclinical Deletion of rfbF; lacks O-antigen; carries heatstable ST toxoid from ETEC

[139,140]

Ty21a with Shigella LPS

Combivax, Rockville, MD Protein Potential

Preclinical Ty21a with Shigella O-antigen operon integrated onto the Ty21a chromosome

[141 144]

Comments

References

Phase 1

Formalin-killed S. flexneri 2a (Sf2aWC) and S. sonnei (SsWC)

[9,10,130]

Phase 1

S. sonnei WRSs2/WRSs3 with loss of virG, senA, [131 135] senB, and msbB2; safe and immunogenic up to 106 CFU; other serotypes available S. flexneri 2a strain CVD1208S with loss of guaBA and setAB, senA, and senB; safe and immunogenic at 109 CFU dose

Truncated Shigella International Vaccine Preclinical O-Ag polymerase is disrupted; Δdwzy LPS exposing Institute, Seoul, Korea, PATH mutation retains one unit of O-antigen IcsP, PSSP1 Heat-inactivated hexavalent Shigella vaccine

National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India

Preclinical Shigella dysenteriae 1, S. flexneri 2a, 3a, 6, S. boydii 4, and S. sonnei

SC599 series with virG, ent/fep, and stxA genes

Institute Pasteur, Paris, France

Phase 2

IX. MULTIVALENT ORAL SHIGELLA VACCINES A. Shigella Typhoid An oral live, attenuated vaccine (Ty21a) is available for prevention of typhoid fever caused by Salmonella enterica serovar Typhi. A bivalent vaccine based on S. sonnei form I O-polysaccharide expression by Ty21a was produced in the early 1980s [149]. This hybrid vaccine was well tolerated and conferred significant protection against diarrhea and dysentery in humans [150]. However, the O-antigen expression was unstable and resulted in lot-to-lot variation. Using the same concept, a new acid-resistant Ty21a expressing S. sonnei O-antigen has been developed in which the

S. dysenteriae 1, live attenuated

[136 138]

[145]

[146,147]

[122,123]

O-antigen is stably expressed from the Ty21a chromosome. This vaccine protected mice (90% survival) from lethal S. sonnei infection [141] but remains to be tested in humans. Similar hybrid vaccines were produced based on Ty21a expression of S. flexneri 2a or 3a [151,152] and S. dysenteriae O-antigens [142], all of which prevented Shigella infection in mice. These strains were further manipulated to remove antibiotic resistance plasmids and ensure genetic stability for future evaluation in humans.

B. Escherichia coli Shigella A similar strategy was employed to create E. coli Shigella hybrid vaccines. The use of classical genetic techniques allowed Formal,

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30. ORAL SHIGELLA VACCINES

Kopecko, and colleagues at the Walter Reed Army Institute of Research (WRAIR) to develop vaccines that utilized an E. coli backbone into which the genes encoding invasive properties of Shigella as well as genes encoding S. flexneri 2a group and type-specific antigens were mobilized. The resultant vaccine, EcSf2a-1, was tested in volunteers and was found to be well tolerated at doses up to 5 3 107, but induced fever and diarrhea in some volunteers at higher doses of 1 3 109 CFU, at which the vaccine was more immunogenic. Vaccination resulted in 20% efficacy against wild-type challenge [115]. The reactogenicity of this strain prompted investigators to create the second-generation EcSf2a-2 vaccine strain, which harbored an aroD mutation. This strain was extensively tested in volunteers, in whom it was found to be well tolerated and to confer 48% efficacy in a challenge study. However, in two subsequent challenge studies, no significant protection was observed [71]. This vaccine was tested under military field conditions in the Israeli Defense Force, but a lack of S. flexneri 2a disease in controls precluded efficacy calculations. Advancement of this candidate was discouraged, as more precise molecular techniques were becoming popular for the engineering of specifically targeted Shigella-based oral vaccines (Table 30.2).

C. Shigella ETEC Live attenuated Shigella vaccine strains have been engineered for the expression and delivery of heterologous antigens. The concept of a multivalent Shigella ETEC vaccine has been advanced by investigators at the University of Maryland Center for Vaccine Development (CVD) and WRAIR under the premise that a vaccine that targets both pathogens would be a practical and valuable public health intervention. ETEC causes disease by attaching to the gastrointestinal tract via fimbriae and secreting

heat-labile (LT) and/or heat-stable (ST) toxins that result in watery diarrhea. The hybrid vaccines consist of Shigella expressing critical ETEC virulence factors (fimbriae) as well as antigens to induce LT neutralizing antibodies. Individual Shigella strains expressing one or two ETEC antigens elicited antibody responses against the Shigella live vector as well as each ETEC antigen when delivered mucosally to guinea pigs [153 156]. The co-administration to guinea pigs of S. flexneri, S. sonnei, and S. dysenteriae attenuated strains, each expressing one or two ETEC antigens, induced serum and mucosal antibody responses to each of the three Shigella strains and each of the five ETEC antigens. [154]. These studies support the concept of a multivalent vaccine candidate that could be given orally to prevent infection by enteric pathogens.

X. PARENTERAL SHIGELLA VACCINE CANDIDATES The conjugate vaccine developed by John Robbins’ group at the National Institutes of Health is currently the most advanced of the subunit Shigella vaccine candidates (Table 30.3). It has been evaluated in Phase III trials in Israeli soldiers, children, and toddlers younger than 3 years of age [15]. The vaccine is made up of the O-specific polysaccharide covalently attached to succinylated mutant Pseudomonas aeruginosa exotoxin A (rEPAsucc) or native or succinylated Corynebacterium diphtheria toxin mutant (CRM9 or CRM9succ) [168]. These candidates have been tested in several clinical trials in Israel and have been shown to provide up to 70% protection when given intramuscularly to adults and young children 3 4 years old, with evidence that a critical level of LPS-specific IgG prevents disease [15,168,170]. In children younger than 3 years of age, the efficacy of S. sonnei rEPA conjugate vaccine candidate fell to 28%, emphasizing the importance of understanding what

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X. PARENTERAL SHIGELLA VACCINE CANDIDATES

TABLE 30.3

Current Parenteral Shigella Vaccines Phase of study

Vaccine

Developer

Comments

Chemical conjugates of O-polysaccharide with detoxified proteins

John Robbins lab, NICHD, NIH, USA

Escherichia coli expressing recombinant glycoconjugate

Limmatech Biologics-now Phase 2b 30% protection against shigellosis part of GlaxoSmithKline completed 50% protection against more severe disease (GSK) or dysentery

[16,157]

GMMA, generalized modules of membrane antigens

GSK Vaccines Institute for Phase 2b Global Health, Siena, Italy

Genetically derived outer membrane particles without LPS

[122,158]

Artificial Invaplex

WRAIR, USA

Phase 1

Mixture of purified LPS, IpaB, and IpaC; safe and immunogenic given intranasally; each dose given 3 times on days 0, 14, and 28; highest dose tested B500 μg of protein with B1 mg of LPS

[159 161]

Synthetic glycoconjugate as haptens for a conjugate vaccine

Institute Pasteur, Paris, France

Phase 1

Immunogenic; serum IgG to O-polysaccharide

[162,163]

IpaB-D fusion

Kansas State University, USA

Preclinical Protective in mice

rIpaB-GroEL

DIPAS (Defense Institute of Physiology and Allied Sciences), Delhi, India

Preclinical Organization is a component of the Defense Research and Development Organization in India

[164,165]

OMV encapsulated in nanoparticles

University of Navarra, Spain

Preclinical Protective against pulmonary infection in mice immunized orally or intranasally

[166]

34-kDa OMP

NICED, Kolkata, India

Preclinical Protective against pulmonary infection in mice immunized intranasally

[167]

Phase 3 Conjugates of O-polysaccharide with CRM trials or Pseudomonas aeruginosa exotoxin protein completed A (rEPA). In Israeli field trials, 60% 70% protection in adults, ,30% protection in children # 3 years of age

References [15,168,169]

[94]

Manufacturing issues; Phase 1 not initiated

constitutes protective immunity in this group, which is the main target for vaccination. These results bring to mind that the efficacy of parenteral immunization with O-conjugate vaccines was originally reported in immunologically primed (not naı¨ve) individuals. A bioconjugate S. flexneri 2a vaccine candidate Flexyn2a, developed by in vivo conjugation of the Opolysaccharide with the carrier protein within

E. coli [171] has been tested for safety, immunogenicity, and efficacy in adult human volunteers; it induced O-polysaccharide specific serum IgG, SBA titers and greater than 40% efficacy [16,172]. An artificial Invaplex subunit vaccine candidate containing purified S. flexneri 2a LPS mixed with rIpaB and rIpaC was recently tested in a phase 1 trial in adults, who received increasing dosage levels

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30. ORAL SHIGELLA VACCINES

intranasally, and it was shown to be safe and immunogenic [159].

XI. AN IDEAL ORAL SHIGELLA VACCINE: FEATURES AND IMPLEMENTATION Oral vaccines offer an opportunity to induce local immunity that helps to contain the enteropathogens at the site of infection [173]. The success of oral polio and rotavirus immunization demonstrates the effectiveness of this approach in preventing childhood disease. Furthermore, the existence of two licensed oral cholera vaccines—Dukoral, which consists of inactivated whole cell Vibrio cholerae with recombinant cholera toxin B subunit, and the live attenuated CVD 103-HgR (Vaxchora)—provides a formidable precedent for effective oral vaccination to prevent enteric diarrheal illness. An ideal oral Shigella vaccine is expected to be: • Well tolerated and able to induce robust and long-lasting protective immunity, particularly in infants and young children living in endemic areas. Oral vaccines can potentially elicit effective mucosal immunity comparable to or greater than that provided by natural infection but in a safe manner. • Broadly protective across multiple serotypes. The serotype-specific protection could be overcome by the combination of multiple strains; a vaccine that includes S. sonnei and S. flexneri serotypes 2a, 3a, and 6 is expected to prevent the majority of Shigella-induced diarrhea in endemic regions [174]. Whole organisms will likely induce good responses to Ipas and other bacterial proteins, strengthening immunity. • Practical and easy to administer. An oral vaccine (amenable for self- or parental delivery) would facilitate logistics and implementation, particularly in field settings. • Stable, easy to manufacture, and affordable.

Subunit Shigella antigens given orally or sublingually have shown modest efficacy in animal models [175]. Inactivated whole cell vaccines have been shown to be safe and immunogenic in adults yet remain to be tested for clinical efficacy. Live attenuated vaccines have been able to induce protective immunity in adults and children and might be closer to meeting the desired requirements. An inherent difficulty of this approach is attaining the right balance between attenuation and immunogenicity. The increased understanding of pathogenesis and refined methods for vaccine engineering as well as options for vaccine formulation mitigate this challenge. Another concern associated with the use of oral vaccines has been their traditionally lower immunogenicity in less developed countries and endemic areas as compared to industrialized regions. Reasons proposed to explain this discrepancy include the interference of maternal antibodies or other components in breast milk, small intestinal overgrowth, the microbiome, genetic factors, coinfection with other bacterial or helminths, environmental inflammation, malnutrition, and micronutrient deficiency. These and other issues, as well as the prospects for effective oral vaccination against shigellosis, have been discussed elsewhere [148,176 178]. Clinical studies in endemic regions and in the intended population (i.e., infants, toddlers, and young children) will be necessary to ascertain the effectiveness of oral and other Shigella vaccine candidates.

XII. CONCLUDING REMARKS The success of licensed oral vaccines against enteric pathogens, such as the typhoid vaccine Ty21a and Vaxchora, a live cholera vaccine, supports and encourages efforts to produce an effective oral Shigella vaccine. Live attenuated organisms represent the most advanced concept (from the clinical standpoint) for oral immunization to prevent shigellosis, offering the

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

advantages of simple manufacturing and practical implementation for young children living in impoverished countries, who represent the key target population. Leading candidates have been well tolerated and immunogenic in multiple studies conducted in human adults. While effective oral immunization in endemic countries is more challenging, a better understanding of Shigella pathogenesis and host immunity, refined molecular engineering, new adjuvants, improved formulations (i.e., multistrain combinations), and optimized schedules are expected to overcome existing barriers and contribute to successful outcomes. The increasing appreciation of Shigella as one of the most significant pathogens causing long-term childhood morbidity and death has invigorated efforts from public health and regulatory authorities, scientists, industry, and funding agencies to accelerate Shigella vaccine development efforts. Their coordinated strength and commitment has created a momentum for the realization of this muchneeded prophylactic product.

Acknowledgments We dedicate this chapter to the memory of our friend Lillian Van de Verg (1948 2016) who championed the development of Shigella vaccines. The authors thank Shannon Heine and Ed Oaks for assisting with the preparation and critical reading of this manuscript, and Alyssa Cunningham for the artistic representation of Fig. 30.1. This work was supported, in part, by NIH/NIAID awards R01AI117734 and R01125841 to MFP and R01AI132257 to EMB. The views expressed here are the private views of the authors and do not reflect the official views of WRAIR, the Department of the Army, or the Department of Defense.

References [1] Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 2013;382(9888):209 22.

529

[2] Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 2012;380(9859):2095 128. [3] Nygren BL, Schilling KA, Blanton EM, Silk BJ, Cole DJ, Mintz ED. Foodborne outbreaks of shigellosis in the USA, 1998 2008. Epidemiol Infect 2013;141(2):233 41. [4] Sjolund Karlsson M, Bowen A, Reporter R, Folster JP, Grass JE, Howie RL, et al. Outbreak of infections caused by Shigella sonnei with reduced susceptibility to azithromycin in the United States. Antimicrob Agents Chemother 2013;57(3):1559 60. [5] Kozyreva VK, Jospin G, Greninger AL, Watt JP, Eisen JA, Chaturvedi V. Recent outbreaks of shigellosis in California caused by two distinct populations of Shigella sonnei with either increased virulence or fluoroquinolone resistance. mSphere 2016;1(6) e00344 16. [6] Salam MA, Bennish ML. Antimicrobial therapy for shigellosis. Rev Infect Dis 1991;13(Suppl. 4):S332 41. [7] Chung The H, Rabaa MA, Pham Thanh D, De Lappe N, Cormican M, Valcanis M, et al. South Asia as a reservoir for the global spread of ciprofloxacin-resistant Shigella sonnei: a cross-sectional study. PLoS Med 2016;13(8):e1002055. [8] Klontz EH, Das SK, Ahmed D, Ahmed S, Chisti MJ, Malek MA, et al. Long-term comparison of antibiotic resistance in Vibrio cholerae O1 and Shigella species between urban and rural Bangladesh. Clin Infect Dis 2014;58(9):e133 6. [9] Chakraborty S, Harro C, DeNearing B, Bream J, Bauers N, Dally L, et al. Evaluation of the safety, tolerability, and immunogenicity of an oral, inactivated whole-cell Shigella flexneri 2a vaccine in healthy adult subjects. Clin Vaccine Immunol 2016;23(4):315 25. [10] McKenzie R, Walker RI, Nabors GS, Van de Verg LL, Carpenter C, Gomes G, et al. Safety and immunogenicity of an oral, inactivated, whole-cell vaccine for Shigella sonnei: preclinical studies and a Phase I trial. Vaccine 2006;24(18):3735 45. [11] Kotloff KL, Taylor DN, Sztein MB, Wasserman SS, Losonsky GA, Nataro JP, et al. Phase I evaluation of ΔvirG Shigella sonnei live, attenuated, oral vaccine strain WRSS1 in healthy adults. Infect Immun 2002;70(4):2016 21. [12] Kotloff KL, Pasetti MF, Barry EM, Nataro JP, Wasserman SS, Sztein MB, et al. Deletion in the Shigella enterotoxin genes further attenuates Shigella flexneri 2a bearing guanine auxotrophy in a phase 1 trial of CVD 1204 and CVD 1208. J Infect Dis 2004;190(10):1745 54. [13] Rahman KM, Arifeen SE, Zaman K, Rahman M, Raqib R, Yunus M, et al. Safety, dose, immunogenicity, and transmissibility of an oral live attenuated Shigella flexneri 2a vaccine candidate (SC602) among healthy adults and school children in Matlab, Bangladesh. Vaccine 2011;29(6):1347 54.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

530

30. ORAL SHIGELLA VACCINES

[14] Cohen D, Ashkenazi S, Green M, Lerman Y, Slepon R, Robin G, et al. Safety and immunogenicity of investigational Shigella conjugate vaccines in Israeli volunteers. Infect Immun 1996;64(10):4074 7. [15] Passwell JH, Ashkenzi S, Banet-Levi Y, Ramon-Saraf R, Farzam N, Lerner-Geva L, et al. Age-related efficacy of Shigella O-specific polysaccharide conjugates in 1-4year-old Israeli children. Vaccine 2010;28(10):2231 5. [16] Riddle MS, Kaminski RW, Di Paolo C, Porter CK, Gutierrez RL, Clarkson KA, et al. Safety and immunogenicity of a candidate bioconjugate vaccine against Shigella flexneri 2a administered to healthy adults: a single-blind, randomized Phase I study. Clin Vaccine Immunol 2016;23(12):908 17. [17] Riddle MS, Kaminski RW, Williams C, Porter C, Baqar S, Kordis A, et al. Safety and immunogenicity of an intranasal Shigella flexneri 2a Invaplex50 vaccine. Vaccine 2011;29(40):7009 19. [18] Obiero CW, Ndiaye AGW, Scire AS, Kaunyangi BM, Marchetti E, Gone AM, et al. A Phase 2a randomized study to evaluate the safety and immunogenicity of the 1790GAHB generalized modules for membrane antigen vaccine against Shigella sonnei administered intramuscularly to adults from a shigellosis-endemic country. Front Immunol 2017;8:1884. [19] Kaminski RW, Oaks EV. Inactivated and subunit vaccines to prevent shigellosis. Expert Rev Vaccines 2009; 8(12):1693 704. [20] Barry EM, Pasetti MF, Sztein MB, Fasano A, Kotloff KL, Levine MM. Progress and pitfalls in Shigella vaccine research. Nat Rev Gastroenterol Hepatol 2013;10(4): 245 55. [21] Levine MM, Kotloff KL, Barry EM, Pasetti MF, Sztein MB. Clinical trials of Shigella vaccines: two steps forward and one step back on a long, hard road. Nat Rev Microbiol 2007;5(7):540 53. [22] Venkatesan MM, Ranallo RT. Live-attenuated Shigella vaccines. Expert Rev Vaccines 2006;5(5):669 86. [23] DuPont HL, Hornick RB, Snyder MJ, Libonati JP, Formal SB, Gangarosa EJ. Immunity in shigellosis. I. Response of man to attenuated strains of Shigella. J Infect Dis 1972;125(1):5 11. [24] Ferreccio C, Prado V, Ojeda A, Cayyazo M, Abrego P, Guers L, et al. Epidemiologic patterns of acute diarrhea and endemic Shigella infections in children in a poor periurban setting in Santiago, Chile. Am J Epidemiol 1991;134(6):614 27. [25] Herrington DA, Van de Verg L, Formal SB, Hale TL, Tall BD, Cryz SJ, et al. Studies in volunteers to evaluate candidate Shigella vaccines: further experience with a bivalent Salmonella typhi-Shigella sonnei vaccine and protection conferred by previous Shigella sonnei disease. Vaccine 1990;8(4):353 7. [26] Liu J, Platts-Mills JA, Juma J, Kabir F, Nkeze J, Okoi C, et al. Use of quantitative molecular diagnostic methods

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study. Lancet 2016;388(10051): 1291 301. Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, Haque R, Havt A, et al. Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED). Lancet Glob Health 2015;3(9): e564 75. Lamberti LM, Bourgeois AL, Fischer Walker CL, Black RE, Sack D. Estimating diarrheal illness and deaths attributable to Shigellae and enterotoxigenic Escherichia coli among older children, adolescents, and adults in South Asia and Africa. PLoS Negl Trop Dis 2014;8(2): e2705. Lindsay B, Ochieng JB, Ikumapayi UN, Toure A, Ahmed D, Li S, et al. Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in lowincome countries. J Clin Microbiol 2013;51(6):1740 6. Murray CJ, Vos T, Lozano R, Naghavi M, Flaxman AD, Michaud C, et al. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 2012;380(9859):2197 223. Kotloff KL. The burden and etiology of diarrheal illness in developing countries. Pediatr Clin North Am 2017;64(4):799 814. Kerneis S, Guerin PJ, von Seidlein L, Legros D, Grais RF. A look back at an ongoing problem: Shigella dysenteriae type 1 epidemics in refugee settings in Central Africa (1993 1995). PLoS One 2009;4(2):e4494. Centers for Disease C. Hospital-associated outbreak of Shigella dysenteriae type 2 Maryland. MMWR Morb Mortal Wkly Rep 1983;32(19):250 2. Arvelo W, Hinkle CJ, Nguyen TA, Weiser T, Steinmuller N, Khan F, et al. Transmission risk factors and treatment of pediatric shigellosis during a large daycare center-associated outbreak of multidrug resistant Shigella sonnei: implications for the management of shigellosis outbreaks among children. Pediatr Infect Dis J 2009;28(11):976 80. Aragon TJ, Vugia DJ, Shallow S, Samuel MC, Reingold A, Angulo FJ, et al. Case-control study of shigellosis in San Francisco: the role of sexual transmission and HIV infection. Clin Infect Dis 2007;44(3):327 34. Shah N, DuPont HL, Ramsey DJ. Global etiology of travelers’ diarrhea: systematic review from 1973 to the present. Am J Trop Med Hyg 2009;80(4):609 14. Labrec EH, Schneider H, Magnani TJ, Formal SB. Epithelial cell penetration as an essential step in the pathogenesis of bacillary dysentery. J Bacteriol 1964;88(5): 1503 18. Zychlinsky A, Prevost MC, Sansonetti PJ. Shigella flexneri induces apoptosis in infected macrophages. Nature 1992;358(6382):167 9.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

531

REFERENCES

[39] Suzuki T, Nakanishi K, Tsutsui H, Iwai H, Akira S, Inohara N, et al. A novel caspase-1/toll-like receptor 4independent pathway of cell death induced by cytosolic Shigella in infected macrophages. J Biol Chem 2005;280(14):14042 50. [40] Hilbi H, Moss JE, Hersh D, Chen Y, Arondel J, Banerjee S, et al. Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB. J Biol Chem 1998;273(49):32895 900. [41] Mattock E, Blocker AJ. How do the virulence factors of Shigella work together to cause disease? Front Cell Infect Microbiol 2017;7:64. [42] Carayol N, Tran Van, Nhieu G. The inside story of Shigella invasion of intestinal epithelial cells. Cold Spring Harb Perspect Med 2013;3(10):a016717. [43] Sansonetti PJ, Kopecko DJ, Formal SB. Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence. Infect Immun 1981;34(1):75 83. [44] Parsot C, Menard R, Gounon P, Sansonetti PJ. Enhanced secretion through the Shigella flexneri MxiSpa translocon leads to assembly of extracellular proteins into macromolecular structures. Mol Microbiol 1995;16(2):291 300. [45] Sahl JW, Caporaso JG, Rasko DA, Keim P. The largescale blast score ratio (LS-BSR) pipeline: a method to rapidly compare genetic content between bacterial genomes. PeerJ 2014;2:e332. [46] Day Jr. WA, Fernandez RE, Maurelli AT. Pathoadaptive mutations that enhance virulence: genetic organization of the cadA regions of Shigella spp. Infect Immun 2001; 69(12):7471 80. [47] Maurelli AT. Black holes, antivirulence genes, and gene inactivation in the evolution of bacterial pathogens. FEMS Microbiol Lett 2007;267(1):1 8. [48] Coster TS, Hoge CW, VanDeVerg LL, Hartman AB, Oaks EV, Venkatesan MM, et al. Vaccination against shigellosis with attenuated Shigella flexneri 2a strain SC602. Infect Immun 1999;67(7):3437 43. [49] Noriega FR, Losonsky G, Lauderbaugh C, Liao FM, Wang JY, Levine MM. Engineered ΔguaBA and ΔvirG Shigella flexneri 2a strain CVD 1205: construction, safety, immunogenicity, and potential efficacy as a mucosal vaccine. Infect Immun 1996;64(8):3055 61. [50] Martinez-Becerra FJ, Kissmann JM, Diaz-McNair J, Choudhari SP, Quick AM, Mellado-Sanchez G, et al. Broadly protective Shigella vaccine based on type III secretion apparatus proteins. Infect Immun 2012;80(3): 1222 31. [51] Van de Verg LL, Herrington DA, Boslego J, Lindberg AA, Levine MM. Age-specific prevalence of serum antibodies to the invasion plasmid and lipopolysaccharide antigens of Shigella species in Chilean and North American populations. J Infect Dis 1992;166(1):158 61. [52] Cohen D, Green MS, Block C, Slepon R, Lerman Y. Natural immunity to shigellosis in two groups with

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

different previous risks of exposure to Shigella is only partly expressed by serum antibodies to lipopolysaccharide [letter]. J Infect Dis 1992;165(4):785 7. Raqib R, Qadri F, Sarker P, Mia SM, Sansonnetti PJ, Albert MJ, et al. Delayed and reduced adaptive humoral immune responses in children with shigellosis compared with in adults. Scand J Immunol 2002;55 (4):414 23. Formal SB, Oaks EV, Olsen RE, Wingfield-Eggleston M, Snoy PJ, Cogan JP. Effect of prior infection with virulent Shigella flexneri 2a on the resistance of monkeys to subsequent infection with Shigella sonnei. J Infect Dis 1991;164(3):533 7. Kotloff KL, Nataro JP, Losonsky GA, Wasserman SS, Hale TL, Taylor DN, et al. A modified Shigella volunteer challenge model in which the inoculum is administered with bicarbonate buffer: clinical experience and implications for Shigella infectivity. Vaccine 1995;13 (16):1488 94. Cam PD, Pal T, Lindberg AA. Immune response against lipopolysaccharide and invasion plasmidcoded antigens of shigellae in Vietnamese and Swedish dysenteric patients. J Clin Microbiol 1993;31 (2):454 7. Cohen D, Block C, Green MS, Lowell G, Ofek I. Immunoglobulin M, A, and G antibody response to lipopolysaccharide O antigen in symptomatic and asymptomatic Shigella infections. J Clin Microbiol 1989; 27(1):162 7. Islam D, Wretlind B, Ryd M, Lindberg AA, Christensson B. Immunoglobulin subclass distribution and dynamics of Shigella- specific antibody responses in serum and stool samples in shigellosis. Infect Immun 1995;63(5):2054 61. Oberhelman RA, Kopecko DJ, Salazar-Lindo E, Gotuzzo E, Buysse JM, Venkatesan MM, et al. Prospective study of systemic and mucosal immune responses in dysenteric patients to specific Shigella invasion plasmid antigens and lipopolysaccharides. Infect Immun 1991;59(7):2341 50. Cohen D, Green MS, Block C, Rouach T, Ofek I. Serum antibodies to lipopolysaccharide and natural immunity to shigellosis in an Israeli military population. J Infect Dis 1988;157(5):1068 71. Robin G, Cohen D, Orr N, Markus I, Slepon R, Ashkenazi S, et al. Characterization and quantitative analysis of serum IgG class and subclass response to Shigella sonnei and Shigella flexneri 2a lipopolysaccharide following natural Shigella infection. J Infect Dis 1997;175(5):1128 33. Cohen D, Ashkenazi S, Green MS, Gdalevich M, Robin G, Slepon R, et al. Double-blind vaccine-controlled randomised efficacy trial of an investigational Shigella sonnei conjugate vaccine in young adults. Lancet 1997;349 (9046):155 9.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

532

30. ORAL SHIGELLA VACCINES

[63] Shimanovich AA, Buskirk AD, Heine SJ, Blackwelder WC, Wahid R, Kotloff KL, et al. Functional and antigen-specific serum antibody levels as correlates of protection against shigellosis in a controlled human challenge study. Clin Vaccine Immunol 2017;24(2): e00412 16. [64] Wahid R, Simon JK, Picking WL, Kotloff KL, Levine MM, Sztein MB. Shigella antigen-specific B memory cells are associated with decreased disease severity in subjects challenged with wild-type Shigella flexneri 2a. Clin Immunol 2013;148(1):35 43. [65] Reed WP, Albright EL. Serum factors responsible for killing of Shigella. Immunology 1974;26(1):205 15. [66] Sayem MA, Ahmad SM, Rekha RS, Sarker P, Agerberth B, Talukder KA, et al. Differential host immune responses to epidemic and endemic strains of Shigella dysenteriae type I. J Health Popul Nutr 2011;29(5):429 37. [67] Okamura N, Nakaya R, Suzuki K, Kondo S, Hisatsune K, Imagawa Y, et al. Differences among Shigella spp. in susceptibility to the bactericidal activity of human serum. J Gen Microbiol 1988;134(7):2057 65. [68] Lowell GH, MacDermott RP, Summers PL, Reeder AA, Bertovich MJ, Formal SB. Antibody-dependent cellmediated antibacterial activity: K lymphocytes, monocytes, and granulocytes are effective against Shigella. J Immunol 1980;125:2778 84. [69] Lin J, Smith MA, Benjamin Jr. WH, Kaminski RW, Wenzel H, Nahm MH. Monoclonal antibodies to Shigella lipopolysaccharide are useful for vaccine production. Clin Vaccine Immunol 2016;23(8):681 8. [70] Sinha A, Dey A, Saletti G, Samanta P, Chakraborty PS, Bhattacharya MK, et al. Circulating gut-homing (α4 β71) plasmablast responses against Shigella surface protein antigens among hospitalized patients with diarrhea. Clin Vaccine Immunol 2016;23(7):610 17. [71] Kotloff KL, Losonsky GA, Nataro JP, Wasserman SS, Hale TL, Taylor DN, et al. Evaluation of the safety, immunogenicity, and efficacy in healthy adults of four doses of live oral hybrid Escherichia coli-Shigella flexneri 2a vaccine strain EcSf2a-2. Vaccine 1995;13(5):495 502. [72] Feller AJ, McKenzie R, Taylor DN, Woods CC, Grahek SL, Islam D, et al. Comparative evaluation of the antibody in lymphocyte supernatant (ALS) and enzymelinked immunospot (ELISPOT) assays for measuring mucosal immune responses to Shigella antigens. Vaccine 2011;29(47):8487 9. [73] Islam D, Veress B, Bardhan PK, Lindberg AA, Christensson B. Quantitative assessment of IgG and IgA subclass producing cells in rectal mucosa during shigellosis. J Clin Pathol 1997;50(6):513 20. [74] Islam D, Wretlind B, Hammarstrom L, Christensson B, Lindberg AA. Semiquantitative estimation of Shigella antigen-specific antibodies: correlation with disease severity during shigellosis. APMIS 1996;104(7-8):563 74.

[75] Raqib R, Lindberg AA, Wretlind B, Bardhan PK, Andersson U, Andersson J. Persistence of local cytokine production in shigellosis in acute and convalescent stages. Inf Immun 1995;63(1):289 96. [76] Islam D, Veress B, Bardhan PK, Lindberg AA, Christensson B. In situ characterization of inflammatory responses in the rectal mucosae of patients with shigellosis. Infect Immun 1997;65(2):739 49. [77] Raqib R, Lindberg AA, Bjork L, Bardhan PK, Wretlind B, Andersson U, et al. Down-regulation of gamma interferon, tumor necrosis factor type I, interleukin 1 (IL-1) type I, IL-3, IL-4, and transforming growth factor beta type I receptors at the local site during the acute phase of Shigella infection. Inf Immun 1995;63(8): 3079 87. [78] Raqib R, Ljungdahl A, Lindberg AA, Andersson U, Andersson J. Local entrapment of interferon gamma in the recovery from Shigella dysenteriae type 1 infection. Gut 1996;38(3):328 36. [79] Raqib R, Wretlind B, Andersson J, Lindberg AA. Cytokine secretion in acute shigellosis is correlated to disease activity and directed more to stool than to plasma. J Infect Dis 1995;171(2):376 84. [80] Islam MM, Azad AK, Bardhan PK, Raqib R, Islam D. Pathology of shigellosis and its complications. Histopathology 1994;24(1):65 71. [81] Nothelfer K, Arena ET, Pinaud L, Neunlist M, Mozeleski B, Belotserkovsky I, et al. B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection. J Exp Med 2014;211(6):1215 29. [82] Raqib R, Reinholt FP, Bardhan PK, Karnell A, Lindberg AA. Immunopathological patterns in the rectal mucosa of patients with shigellosis: expression of HLA-DR antigens and T-lymphocyte subsets. APMIS 1994;102(5):371 80. [83] Islam D, Bardhan PK, Lindberg AA, Christensson B. Shigella infection induces cellular activation of T and B cells and distinct species-related changes in peripheral blood lymphocyte subsets during the course of the disease. Infect Immun 1995;63(8):2941 9. [84] Pinaud L, Samassa F, Porat Z, Ferrari ML, Belotserkovsky I, Parsot C, et al. Injection of T3SS effectors not resulting in invasion is the main targeting mechanism of Shigella toward human lymphocytes. Proc Natl Acad Sci USA 2017;114(37):9954 9. [85] Salgado-Pabon W, Konradt C, Sansonetti PJ, Phalipon A. New insights into the crosstalk between Shigella and T lymphocytes. Trends Microbiol 2014;22(4):192 8. [86] Raqib R, Mia SM, Qadri F, Alam TI, Alam NH, Chowdhury AK, et al. Innate immune responses in children and adults with Shigellosis. Infect Immun 2000;68(6):3620 9. [87] Gregory M, Kaminski RW, Lugo-Roman LA, Galvez Carrillo H, Tilley DH, Baldeviano C, et al.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

Development of an Aotus nancymaae model for Shigella Vaccine immunogenicity and efficacy studies. Infect Immun 2014;82(5):2027 36. [88] Oaks EV, Hale TL, Formal SB. Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp. Infect Immun 1986;53(1):57 63. [89] Hartman AB, Powell CJ, Schultz CL, Oaks EV, Eckels KH. Small-animal model to measure efficacy and immunogenicity of Shigella vaccine strains. Infect Immun 1991;59(11):4075 83. [90] Hartman AB, Van de Verg LL, Collins Jr. HH, Tang DB, Bendiuk NO, Taylor DN, et al. Local immune response and protection in the guinea pig keratoconjunctivitis model following immunization with Shigella vaccines. Infect Immun 1994;62(2):412 20. [91] Sereny B. Experimental shigella keratoconjunctivitis; a preliminary report. Acta Microbiol Acad Sci Hung 1955;2(3):293 6. [92] Shim DH, Suzuki T, Chang SY, Park SM, Sansonetti PJ, Sasakawa C, et al. New animal model of shigellosis in the Guinea pig: its usefulness for protective efficacy studies. J Immunol 2007;178(4):2476 82. [93] Arena ET, Campbell-Valois FX, Tinevez JY, Nigro G, Sachse M, Moya-Nilges M, et al. Bioimage analysis of Shigella infection reveals targeting of colonic crypts. Proc Natl Acad Sci USA 2015;112(25):E3282 90. [94] Martinez-Becerra FJ, Chen X, Dickenson NE, Choudhari SP, Harrison K, Clements JD, et al. Characterization of a novel fusion protein from IpaB and IpaD of Shigella spp. and its potential as a panShigella vaccine. Infect Immun 2013;81(12):4470 7. [95] Heine SJ, Diaz-McNair J, Martinez-Becerra FJ, Choudhari SP, Clements JD, Picking WL, et al. Evaluation of immunogenicity and protective efficacy of orally delivered Shigella type III secretion system proteins IpaB and IpaD. Vaccine 2013;31(28):2919 29. [96] Mallett CP, Van DeVerg L, Collins HH, Hale TL. Evaluation of Shigella vaccine safety and efficacy in an intranasally challenged mouse model. Vaccine 1993; 11(2):190 6. [97] Phalipon A, Sansonetti P. Live attenuated Shigella flexneri mutants as vaccine candidates against shigellosis and vectors for antigen delivery. Biologicals 1995;23(2): 125 34. [98] Way SS, Borczuk AC, Goldberg MB. Thymic independence of adaptive immunity to the intracellular pathogen Shigella flexneri serotype 2a. Infect Immun 1999; 67(8):3970 9. [99] Way SS, Borczuk AC, Goldberg MB. Adaptive immune response to Shigella flexneri 2a cydC in immunocompetent mice and mice lacking immunoglobulin A. Infect Immun 1999;67(4):2001 4.

533

[100] Fernandez MI, Thuizat A, Pedron T, Neutra M, Phalipon A, Sansonetti PJ. A newborn mouse model for the study of intestinal pathogenesis of shigellosis. Cell Microbiol 2003;5(7):481 91. [101] Mitra S, Barman S, Nag D, Sinha R, Saha DR, Koley H. Outer membrane vesicles of Shigella boydii type 4 induce passive immunity in neonatal mice. FEMS Immunol Med Microbiol 2012;66(2):240 50. [102] Singer M, Sansonetti PJ. IL-8 is a key chemokine regulating neutrophil recruitment in a new mouse model of Shigella-induced colitis. J Immunol 2004;173(6): 4197 206. [103] Yang JY, Lee SN, Chang SY, Ko HJ, Ryu S, Kweon MN. A mouse model of shigellosis by intraperitoneal infection. J Infect Dis 2014;209(2):203 15. [104] Wassef JS, Keren DF, Mailloux JL. Role of M cells in initial antigen uptake and in ulcer formation in the rabbit intestinal loop model of shigellosis. Infect Immun 1989;57(3):858 63. [105] Marteyn B, West NP, Browning DF, Cole JA, Shaw JG, Palm F, et al. Modulation of Shigella virulence in response to available oxygen in vivo. Nature 2010;465 (7296):355 8. [106] Hardy AV, Decapito T, Halbert SP. Studies of the acute diarrheal diseases; immunization in shigellosis. Public Health Rep 1948;63(21):685 8. [107] Higgins AR, Floyd TM, Kader MA. Studies in shigellosis. III. A controlled evaluation of a monovalent Shigella vaccine in a highly endemic environment. Am J Trop Med Hyg 1955;4:281 8. [108] Formal SB, Labrec EH, Palmer A, Falkow S. Protection of monkeys against experimental shigellosis with attenuated vaccines. J Bacteriol 1965;90(1):63 8. [109] Formal SB, Labrec EH, Kent TH, Falkow S. Abortive intestinal infection with an Escherichia coli-Shigella flexneri hybrid strain. J Bacteriol 1965;89:1374 82. [110] Levine MM, Gangarosa EJ, Werner M, Morris GK. Shigellosis in custodial institutions. 3. Prospective clinical and bacteriologic surveillance of children vaccinated with oral attenuated shigella vaccines. J Pediatr 1974;84(6):803 6. [111] Mel DM, Terzin AL, Vuksic L. Studies on vaccination against bacillary dysentery. 3. Effective oral immunization against Shigella flexneri 2a in a field trial. Bull World Health Organ 1965;32(5):647 55. [112] Meitert T, Pencu E, Ciudin L, Tonciu M. Vaccine strain S. flexneri T32-Istrati. Studies in animals and in volunteers. Antidysentery immunoprophylaxis and immunotherapy by live vaccine Vadizen (S. flexneri T32-Istrati). Arch Roum Pathol Exp Microbiol 1984; 43(3 4):251 78. [113] Venkatesan M, Fernandez-Prada C, Buysse JM, Formal SB, Hale TL. Virulence phenotype and genetic

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

534

[114]

[115]

[116]

[117]

[118]

[119]

[120]

[121]

[122]

[123]

[124]

30. ORAL SHIGELLA VACCINES

characteristics of the T32-ISTRATI Shigella flexneri 2a vaccine strain. Vaccine 1991;9(5):358 63. Levine MM, Woodward WE, Formal SB, Gemski Jr. P, DuPont HL, Hornick RB, et al. Studies with a new generation of oral attenuated shigella vaccine: Escherichia coli bearing surface antigens of Shigella flexneri. J Infect Dis 1977;136(4):577 82. Kotloff KL, Herrington DA, Hale TL, Newland JW, Van De Verg L, Cogan JP, et al. Safety, immunogenicity, and efficacy in monkeys and humans of invasive Escherichia coli K-12 hybrid vaccine candidates expressing Shigella flexneri 2a somatic antigen. Infect Immun 1992;60(6):2218 24. Li A, Cam PD, Islam D, Minh NB, Huan PT, Rong ZC, et al. Immune responses in Vietnamese children after a single dose of the auxotrophic, live Shigella flexneri Y vaccine strain SFL124. J Infect 1994;28(1):11 23. Li A, Karnell A, Huan PT, Cam PD, Minh NB, Tram LN, et al. Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in adult Vietnamese volunteers. Vaccine 1993;11(2):180 9. Li A, Pal T, Forsum U, Lindberg AA. Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in volunteers. Vaccine 1992;10(6):395 404. Karnell A, Li A, Zhao CR, Karlsson K, Nguyen BM, Lindberg AA. Safety and immunogenicity study of the auxotrophic Shigella flexneri 2a vaccine SFL1070 with a deleted aroD gene in adult Swedish volunteers. Vaccine 1995;13(1):88 99. Kotloff KL, Noriega F, Losonsky GA, Sztein MB, Wasserman SS, Nataro JP, et al. Safety, immunogenicity, and transmissibility in humans of CVD 1203, a live oral Shigella flexneri 2a vaccine candidate attenuated by deletions in aroA and virG. Infect Immun 1996;64(11):4542 8. Kotloff KL, Noriega FR, Samandari T, Sztein MB, Losonsky GA, Nataro JP, et al. Shigella flexneri 2a Strain CVD 1207, with Specific Deletions in virG, sen, set, and guaBA, Is Highly Attenuated in Humans. Infect Immun 2000;68(3):1034 9. Launay O, Sadorge C, Jolly N, Poirier B, Bechet S, van der Vliet D, et al. Safety and immunogenicity of SC599, an oral live attenuated Shigella dysenteriae type-1 vaccine in healthy volunteers: results of a Phase 2, randomized, double-blind placebo-controlled trial. Vaccine 2009;27(8):1184 91. Sadorge C, Ndiaye A, Beveridge N, Frazer S, Giemza R, Jolly N, et al. Phase 1 clinical trial of live attenuated Shigella dysenteriae type-1 ΔicsA Δent Δfep ΔstxA: HgR oral vaccine SC599 in healthy human adult volunteers. Vaccine 2008;26(7):978 87. Katz DE, Coster TS, Wolf MK, Trespalacios FC, Cohen D, Robins G, et al. Two studies evaluating the

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers. Infect Immun 2004;72(2):923 30. Hartman AB, Venkatesan MM. Construction of a stable attenuated Shigella sonnei ΔvirG vaccine strain, WRSS1, and protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model. Infect Immun 1998;66(9):4572 6. Orr N, Katz DE, Atsmon J, Radu P, Yavzori M, Halperin T, et al. Community-based safety, immunogenicity, and transmissibility study of the Shigella sonnei WRSS1 vaccine in Israeli volunteers. Infect Immun 2005;73(12):8027 32. Pitisuttithum P, Islam D, Chamnanchanunt S, Ruamsap N, Khantapura P, Kaewkungwal J, et al. Clinical trial of an oral live Shigella sonnei vaccine candidate, WRSS1, in Thai adults. Clin Vaccine Immunol 2016;23(7):564 75. McKenzie R, Venkatesan MM, Wolf MK, Islam D, Grahek S, Jones AM, et al. Safety and immunogenicity of WRSd1, a live attenuated Shigella dysenteriae type 1 vaccine candidate. Vaccine 2008;26(26):3291 6. Walker RI. An assessment of enterotoxigenic Escherichia coli and Shigella vaccine candidates for infants and children. Vaccine 2015;33(8):954 65. Kaminski RW, Wu M, Turbyfill KR, Clarkson K, Tai B, Bourgeois AL, et al. Development and preclinical evaluation of a trivalent, formalin-inactivated Shigella whole-cell vaccine. Clin Vaccine Immunol 2014;21(3): 366 82. Ranallo RT, Thakkar S, Chen Q, Venkatesan MM. Immunogenicity and characterization of WRSF2G11: a second generation live attenuated Shigella flexneri 2a vaccine strain. Vaccine 2007;25(12):2269 78. Barnoy S, Jeong KI, Helm RF, Suvarnapunya AE, Ranallo RT, Tzipori S, et al. Characterization of WRSs2 and WRSs3, new second-generation virG (icsA)-based Shigella sonnei vaccine candidates with the potential for reduced reactogenicity. Vaccine 2010;28(6):1642 54. Barnoy S, Baqar S, Kaminski RW, Collins T, Nemelka K, Hale TL, et al. Shigella sonnei vaccine candidates WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine candidate, in a primate model of infection. Vaccine 2011;29(37):6371 8. Bedford L, Fonseka S, Boren T, Ranallo RT, Suvarnapunya AE, Lee JE, et al. Further characterization of Shigella sonnei live vaccine candidates WRSs2 and WRSs3-plasmid composition, invasion assays and Sereny reactions. Gut Microbes 2011;2(4):244 51. Frenck RW, Kaminski R, Suvarnapunya AE, Barnoy S, McNeal M, El-Khorazaty J, et al., editors. A Phase I

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

[136]

[137]

[138]

[139]

[140]

[141]

[142]

[143]

[144]

[145]

[146]

study of two live oral Shigella sonnei vaccine candidates WRSs2 and WRSs3. In: 9th International conference on Vaccines for Enteric Diseases (VED) 9-11 Oct 2017, Albufeira, Portugal; 2017. Toapanta FR, Bernal PJ, Kotloff KL, Levine MM, Sztein MB. T cell mediated immunity induced by the live-attenuated Shigella flexneri 2a vaccine candidate CVD 1208S in humans. J Transl Med 2018;16(1):61. Kotloff KL, Simon JK, Pasetti MF, Sztein MB, Wooden SL, Livio S, et al. Safety and immunogenicity of CVD 1208S, a live, oral ΔguaBA Δsen Δset Shigella flexneri 2a vaccine grown on animal-free media. Hum Vaccin 2007;3(6):268 75. Toapanta FR, Simon JK, Barry EM, Pasetti MF, Levine MM, Kotloff KL, et al. Gut-homing conventional plasmablasts and CD27(-) plasmablasts elicited after a short time of exposure to an oral live-attenuated shigella vaccine candidate in humans. Front Immunol 2014;5:374. Tennant SM, Steele AD, Pasetti MF. Highlights of the 8th international conference on vaccines for enteric diseases: the scottish encounter to defeat diarrheal diseases. Clin Vaccine Immunol 2016;23(4):272 81. Nagy G, Henicks T, Szija´rto´ V, Nagy E, inventors; Eveliqure Biotechnologies Gmbh, assignee. A novel live attenuated Shigella vaccine. 2014. Wu Y, Chakravarty S, Li M, Wai TT, Hoffman SL, Sim BK. Development of a live attenuated bivalent oral vaccine against Shigella sonnei shigellosis and typhoid fever. J Infect Dis 2017;215(2):259 68. Xu DQ, Cisar JO, Osorio M, Wai TT, Kopecko DJ. Core-linked LPS expression of Shigella dysenteriae serotype 1 O-antigen in live Salmonella Typhi vaccine vector Ty21a: preclinical evidence of immunogenicity and protection. Vaccine 2007;25(33):6167 75. Dharmasena MN, Hanisch BW, Wai TT, Kopecko DJ. Stable expression of Shigella sonnei form I Opolysaccharide genes recombineered into the chromosome of live Salmonella oral vaccine vector Ty21a. Int J Med Microbiol 2013;303(3):105 13. Dharmasena MN, Osorio M, Filipova S, Marsh C, Stibitz S, Kopecko DJ. Stable expression of Shigella dysenteriae serotype 1 O-antigen genes integrated into the chromosome of live Salmonella oral vaccine vector Ty21a. Pathog Dis 2016;74:ftw098. Kim J-O. Development of a broad spectrum vaccine against shigellosis. In: 8th International conference on Vaccines for Enteric Diseases (VED) 8-10 July 2015, Edinburgh, UK; 2015. Nag D, Sinha R, Mitra S, Barman S, Takeda Y, Shinoda S, et al. Heat killed multi-serotype Shigella immunogens induced humoral immunity and protection against heterologous challenge in rabbit model. Immunobiology 2015;220(11):1275 83.

535

[147] Barman S, Koley H, Ramamurthy T, Chakrabarti MK, Shinoda S, Nair GB, et al. Protective immunity by oral immunization with heat-killed Shigella strains in a guinea pig colitis model. Microbiol Immunol 2013;57 (11):762 71. [148] Walker RI. Considerations for development of whole cell bacterial vaccines to prevent diarrheal diseases in children in developing countries. Vaccine 2005;23(26): 3369 85. [149] Formal SB, Baron LS, Kopecko DJ, Washington O, Powell C, Life CA. Construction of a potential bivalent vaccine strain: introduction of Shigella sonnei form I antigen genes into the galE Salmonella typhi Ty21a typhoid vaccine strain. Infect Immun 1981;34(3): 746 50. [150] Black RE, Levine MM, Clements ML, Losonsky G, Herrington D, Berman S, et al. Prevention of shigellosis by a Salmonella Typhi-Shigella sonnei bivalent vaccine. J Infect Dis 1987;155:1260 5. [151] Baron LS, Kopecko DJ, Formal SB, Seid R, Guerry P, Powell C. Introduction of Shigella flexneri 2a type and group antigen genes into oral typhoid vaccine strain Salmonella typhi Ty21a. Infect Immun 1987;55(11): 2797 801. [152] Dharmasena MN, Osorio M, Takeda K, Stibitz S, Kopecko DJ. Stable chromosomal expression of Shigella flexneri 2a and 3a O-antigens in the live Salmonella oral vaccine vector Ty21a. Clin Vaccine Immunol 2017;24. [153] Altboum Z, Barry EM, Losonsky G, Galen JE, Levine MM. Attenuated Shigella flexneri 2a Delta guaBA strain CVD 1204 expressing enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 fimbriae as a live mucosal vaccine against Shigella and ETEC infection. Infect Immun 2001;69(5):3150 8. [154] Barry EM, Wang J, Wu T, Davis T, Levine MM. Immunogenicity of multivalent Shigella-ETEC candidate vaccine strains in a guinea pig model. Vaccine 2006;24(18):3727 34. [155] Noriega FR, Losonsky G, Wang JY, Formal SB, Levine MM. Further characterization of ΔaroA ΔvirG Shigella flexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for delivering antigens of enterotoxigenic Escherichia coli. Infect Immun 1996;64(1):23 7. [156] Ranallo RT, Fonseka CP, Cassels F, Srinivasan J, Venkatesan MM. Construction and characterization of bivalent Shigella flexneri 2a vaccine strains SC608 (pCFAI) and SC608(pCFAI/LTB) that express antigens from enterotoxigenic Escherichia coli. Infect Immun 2005;73(1):258 67. [157] Ka¨mpf MM, Braun M, Sirena D, Ihssen J, Tho¨ny-Meyer L, Ren Q. In vivo production of a novel glycoconjugate

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

536

[158]

[159]

[160]

[161]

[162]

[163]

[164]

[165]

[166]

[167]

30. ORAL SHIGELLA VACCINES

vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation. Microb Cell Fact 2015;14(1):12. Citiulo F, Colucci A, Giannelli C, Sollai L, Pisoni I, Moriel D, et al. Generation and development of 4-valent GMMA-based Shigella vaccine. In: 9th International Conference on Vaccines for Enteric Diseases (VED), 911 Oct 2017, Albufeira, Portugal; 2017. Clarkson KA DC, Turbyfill KR, Porter C, Gutierrez R, Riddle MS, Lee T, et al. A phase I open label, dose escalating study of Shigella flexneri 2a artificial invaplex administered intranasally to healthy adult volunteers. In: 9th International Conference on Vaccines for Enteric Diseases (VED) 9-11 Oct 2017, Albufeira, Portugal; 2017. Turbyfill KR, Kaminski RW, Oaks EV. Immunogenicity and efficacy of highly purified invasin complex vaccine from Shigella flexneri 2a. Vaccine 2008;26(10):1353 64. Turbyfill KR, Clarkson KA, Vortherms AR, Oaks EV, Kaminski RW. Assembly, biochemical characterization, immunogenicity, adjuvanticity, and efficacy of Shigella artificial invaplex. mSphere 2018;3(2). Belot F, Guerreiro C, Baleux F, Mulard LA. Synthesis of two linear PADRE conjugates bearing a deca- or pentadecasaccharide B epitope as potential synthetic vaccines against Shigella flexneri serotype 2a infection. Chemistry 2005;11(5):1625 35. Cohen D, Atsmon J, Artaud C, Meron-Sudai S, Gougeon M-L, Bialik A, et al. A phase I dose escalation study to assess the safety and immunogenicity of the SF2a-TT15 conjugate vaccine against S. flexneri 2a in healthy adult volunteers (preliminary results). In: 9th International Conference on Vaccines for Enteric Diseases (VED), 9-11 Oct 2017, Albufeira, Portugal; 2017. Chitradevi STS, Kaur G, Sivaramakrishna U, Singh D, Bansal A. Development of recombinant vaccine candidate molecule against Shigella infection. Vaccine 2016;34(44):5376 83. Chitradevi STS, Kaur G, Uppalapati S, Yadav A, Singh D, Bansal A. Co-administration of rIpaB domain of Shigella with rGroEL of S. Typhi enhances the immune responses and protective efficacy against Shigella infection. Cell Mol Immunol 2015;12(6):757 67. Camacho AI, Irache JM, de Souza J, Sanchez-Gomez S, Gamazo C. Nanoparticle-based vaccine for mucosal protection against Shigella flexneri in mice. Vaccine 2013;31(32):3288 94. Pore D, Chakrabarti MK. Outer membrane protein A (OmpA) from Shigella flexneri 2a: a promising subunit vaccine candidate. Vaccine 2013;31(36):3644 50.

[168] Passwell JH, Harlev E, Ashkenazi S, Chu C, Miron D, Ramon R, et al. Safety and immunogenicity of improved Shigella O-specific polysaccharide-protein conjugate vaccines in adults in Israel. Infect Immun 2001;69(3):1351 7. [169] Pozsgay V, Chu C, Pannell L, Wolfe J, Robbins JB, Schneerson R. Protein conjugates of synthetic saccharides elicit higher levels of serum IgG lipopolysaccharide antibodies in mice than do those of the O-specific polysaccharide from Shigella dysenteriae type 1. Proc Natl Acad Sci USA 1999;96(9):5194 7. [170] Passwell JH, Ashkenazi S, Harlev E, Miron D, Ramon R, Farzam N, et al. Safety and immunogenicity of Shigella sonnei-CRM9 and Shigella flexneri type 2arEPAsucc conjugate vaccines in one- to four-year-old children. Pediatr Infect Dis J 2003;22(8):701 6. [171] Ravenscroft N, Haeuptle MA, Kowarik M, Fernandez FS, Carranza P, Brunner A, et al. Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli. Glycobiology 2016;26(1):51 62. [172] Talaat KR AC, Bourgeois AL, Kaminski RW, Dreyer A, Porter CK, Chakraborty S, et al. Flexyn 2a, a candidate bioconjugate vaccine against Shigella flexneri 2a induces protective immune response in a controlled human infection model. In: 9th International Conference on Vaccines for Enteric Diseases (VED), 911 Oct 2017, Albufeira, Portugal; 2017. [173] Levine MM, Sztein MB. Vaccine development strategies for improving immunization: the role of modern immunology. Nat Immunol 2004;5(5):460 4. [174] Livio S, Strockbine NA, Panchalingam S, Tennant SM, Barry EM, Marohn ME, et al. Shigella isolates from the global enteric multicenter study inform vaccine development. Clin Infect Dis 2014;59:933 41. [175] Lee S, Picking WL, Tzipori S. The immune response of two microbial antigens delivered intradermally, sublingually, or the combination thereof. Microbes Infect 2014;16(9):796 803. [176] Pasetti MF, Simon JK, Sztein MB, Levine MM. Immunology of gut mucosal vaccines. Immunol Rev 2011;239(1):125 48. [177] Mani S, Wierzba T, Walker RI. Status of vaccine research and development for Shigella. Vaccine 2016; 34(26):2887 94. [178] Walker RI, Wierzba TF, Mani S, Bourgeois AL. Vaccines against Shigella and enterotoxigenic Escherichia coli: a summary of the 2016 VASE Conference. Vaccine 2017;35(49 Pt A):6775 82.

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Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control Firdausi Qadri1, John D. Clemens1 and Jan Holmgren2,* 1

International Centre for Diarrhoeal Disease Research (icddr,b), Dhaka, Bangladesh 2Department of Micriobiology & Immunology, University of Gothenburg, Gothenburg, Sweden

I. INTRODUCTION Cholera, the severe, often life-threatening watery diarrheal disease caused by enteric infection with Vibrio cholerae bacteria producing cholera toxin, remains an important global health problem, having a major impact on health and development in many countries of Africa and Southeast Asia and, since 2010, on a large scale also in Hispaniola. Since the 1970s, V. cholerae of serogroup O1, predominantly of the El Tor biotype and comprising the Ogawa and Inaba serotypes, has been the causative agent of at least 98% all cholera cases in the world; a small percentage of cholera was in the 1990s transiently and restricted to Southeast Asia caused by El Tor V. cholerae of the then new serogroup O139. First discovered in the 1990s and, since the early 2000s, completely replacing the previous O1

pandemic strains worldwide, new variants of O1 El Tor strains have emerged that carry the classical biotype cholera toxin-encoding (CTX) prophage [1]. These altered strains may have originated through lateral gene transfer and recombination events, possibly from environmental V. cholerae O141 strains carrying the classical CTX prophage, and appear to give rise to a more severe disease than the original seventh pandemic El Tor strain. They may also have a selective advantage, explaining their rapid complete takeover worldwide. Cholera is a largely water- and food-borne infection that is spread by the fecal oral route. Endemic cholera is intimately related to poverty and poor environmental conditions associated with the use of contaminated drinking water and poor food hygiene, sanitation, and waste management. Natural disasters, political

*Corresponding author.

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00031-6

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conflicts, and climate change are additional identified risk factors for endemic cholera and cholera outbreaks. Altogether, recent estimates by WHO suggest that there are approximately 2.86 million cholera cases leading to 95,000 deaths annually in endemic countries, with Africa accounting for 60% of the cases and 68% of the deaths [2,3]. However, the true global burden of cholera remains largely unknown, since probably only 5% 10% of the cholera cases are officially reported, owing to lack of epidemiological surveillance and laboratory facilities and to social, political, and economic restrictions against reporting [2,3]. Cholera affects all age groups, and in countries where cholera is endemic, most individuals above age 5 years show serological evidence of previous exposure to V. cholerae O1 [4]. In cholera-endemic areas, both adults and children are at risk; however, children under 5 years of age have the highest incidence and lethality of disease, mainly owing to the relative lack of acquired immunity in this age group compared to older individuals [5,6]. By contrast, in areas exposed to cholera for the first time, as in Haiti in 2010, infection and disease tend to affect all age groups to a similar extent [7,8]. Importantly, cholera is a vaccine-preventable disease. Convalescents from clinical cholera are usually well protected against new cholera disease for at least the next 3 years [9]. There is also an impressive body of evidence demonstrating effective immune protection at both individual and community levels from the use of oral cholera vaccines (OCVs) [7,10,11]. OCVs of proven efficacy have been available since the 1990s, yet it has taken a long time for OCVs to be accepted as an important public health tool for the control of cholera (with the notable exception of Vietnam, which has used OCV for control of cholera in the Mekong delta since 1998). However, after the WHO in 2010 recommended the use of OCV in control programs for both endemic cholera and cholera outbreaks and in 2013 recommended building

an OCV stockpile for use in outbreaks and emergencies, more than 10 million doses were used in 2017 alone, and more than 15 million doses were requested from the stockpile in the first half of 2018 [12,13]. Indeed, OCV is now a mainstay (in combination with improved water quality, sanitation, and hygiene, or WaSH) for the global control of cholera as outlined in the program “Ending Cholera—A Global Roadmap to 2030,” launched in November 2017 by the WHO and 50 other partner organizations [2]. Of special relevance for this volume, the detailed immunological studies that have been undertaken with OCVs (especially the first licensed OCV containing CTB in addition to killed cholera Vibrios) have also provided important overall knowledge about mucosal immunity in humans. Thus much of our current knowledge about the localization of mucosal immune responses after different routes of immunization, the links between mucosal inductive and expression sites in humans, and the properties and impressive longevity of mucosal B cell immunological memory has come from studies using OCVs and/or CTB [14]. For this reason, in this chapter, after a brief treatise on susceptibility and innate immunity in cholera, we describe, the composition and protective efficacy of the different licensed OCVs with a focus on the current WHOprequalified vaccines, the nature of adaptive immunity in cholera including the mechanisms of immune protection and mucosal and systemic immune responses and immunological memory to infection or immunization, various factors that have been found to modify the immune response, and the current plans and activities with regard to public health use of OCVs in the global efforts to “end cholera by 2030.”

II. SUSCEPTIBILITY AND INNATE IMMUNITY IN CHOLERA Susceptibility to infection with V. cholerae depends on both innate host factors and

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II. SUSCEPTIBILITY AND INNATE IMMUNITY IN CHOLERA

acquired immunity induced by previous infection or vaccination. Stomach acidity and ABO blood groups are the most studied innate immune factors. In Bangladesh, low gastric acid level has been associated with more severe cholera disease [15]. Case control studies have found that individuals with blood group O are at increased risk of hospitalization due to both V. cholerae O1 and V. cholerae O139 [16], and it was proposed that cholera might have been a selection factor leading to a uniquely low prevalence of blood group O in Bangladesh and West Bengal [17]. It was later found that the correlation between blood group O and disease severity in Bangladesh was restricted to cholera caused by the El Tor biotype [18]. The innate immune response is involved in the initial defense against many pathogens, both by exerting antiinfectious functions of its own and by triggering the adaptive immune response. Innate immune responses may be especially important in young children for controlling infections, since they, especially infants, have limited past exposures to antigens and therefore less ability compared to older children and adults to rely on rapidly deployed anamnestic immune responses (Chapter 6: Innate Immunity at Mucosal Surfaces). Recent studies have shown that the innate immune response is upregulated in cholera [19 21]. Thus studies of patients with severe acute cholera have demonstrated that blood and stool levels of innate immune response mediators, including leukotriene B4, lactoferrin, myeloperoxidase, and nitric oxide, are elevated in the initial phase of infection in both children and adults compared with age-group-matched healthy controls [21,22]. Whole genome microarray screening of duodenal biopsies from adults acutely infected with V. cholerae O1 has shown that the majority of upregulated genes encode for proteins that are part of the innate response [19]. Histopathological studies of duodenal biopsies demonstrate that neutrophils infiltrate the mucosa, followed by an increase in

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degranulated mast cells and eosinophils during convalescence [20,23]. Levels of other mediators of the innate response, including cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β), as well as bactericidal proteins, including lactoferrin, myeloperoxidase, and defensins, are also elevated [20,24]. Furthermore, it has been suggested that the long palate, lung, and nasal epithelium clone 1 (LPUNC1) protein expressed in Paneth cells, which was identified as the most upregulated gene in duodenal biopsies from patients with acute cholera [11], also plays a role in modulating the innate response to V. cholerae lipopolysaccharide (LPS) [25]. Studies of duodenal biopsies in children with cholera have not yet been performed. However, rectal biopsies from children with acute cholera show findings similar to those in adults, including an increase in neutrophils at onset of disease followed by an increase in mast cells at early convalescence [20]. These findings are associated with elevated expressions of myeloperoxidase, lactoferrin, and nitric oxide by immunohistochemistry, all of which persist up to 30 days longer than is seen in adults [21]. The stimulation of the innate immune system in response to cholera infection and oral vaccination is a prerequisite for effective induction of a cholera-specific adaptive mucosal immune response in the intestine. While additional bacterial factors may be involved, experimental work has identified V. cholerae LPS and cholera toxin or CTB not only as the most important protective antigens, but also as the predominant stimulators of innate immune pathways promoting the adaptive immune response to infection or vaccination. Several of the innate immunity proteins that were enhanced in intestinal biopsies from patients with acute cholera disease are under control of the also upregulated cytokine IL-1β [11], which has been shown to be a strong adjuvant for promoting mucosal IgA immune responses [26,27]

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and to be strongly increased in both human intestinal epithelial and myeloid and lymphoid cells by in vitro exposure to cholera toxin as well as LPS [19,26] In particular, these bacterial antigens have been shown to have potent adjuvant-stimulating activity on antigenpresenting cells involving several innate immune pathways that promote antigen presentation and effective T cell activation, including the promotion of T helper 17 (Th17) cells, which have been found to be of particular importance to support mucosal IgA formation, and mucosal germinal center formation, which is critical for effective mucosal IgA B/plasma cell and IgA memory responses [28,29] (Chapter 4: Protective Activities of Mucosal Antibodies and Chapter 7: Induction and Regulation of Mucosal Memory B Cell Responses).

III. ORAL CHOLERA VACCINES The current situation for cholera vaccination and the properties of cholera vaccines were recently described in a WHO position paper [30]. There are three WHO-prequalified OCVs to date, all based on inactivated whole cell cholera Vibrios: the Swedish oral whole cell/recombinantly produced CTB vaccine (WC/rCTB) Dukoral (Valneva, Sweden) and the whole-cellonly vaccines Shanchol (Sanofi/Shanta Biotechnics, India) and Euvichol (Eubiologics, South Korea). The compositions of these vaccines are shown in Table 31.1. Dukoral, which was licensed in the early 1990s, consists of 1 3 1011 heat- or formalinkilled V. cholerae O1 classical and El Tor Inaba and Ogawa bacteria, together with 1 mg rCTB. Large, placebo-controlled field trials in Bangladesh in the 1980s and in Peru during the 1990s as well as a case control effectiveness study in Mozambique in 2004 have demonstrated 78% 90% protection after two doses in the first 6 months, gradually decreasing thereafter, with clear efficacy demonstrable for at

least 2 years [31 34]. Like the other inactivated whole cell OCVs, Dukoral requires at least two doses for optimal efficacy, and since the B subunit component is acid labile, the vaccine is administered with a bicarbonate buffer solution. Dukoral is internationally licensed for individuals above 2 years of age, but its current price is considered too high for routine public use in developing countries. Since Dukoral through its B subunit component also gives significant short-term protection against diarrhea caused by enterotoxigenic Escherichia coli (ETEC) producing cholera toxin-like heat-labile enterotoxin, it is largely used as a traveler’s vaccine in industrialized countries [35,36]. Following technology transfer from Swedish scientists, the government of Vietnam developed a killed whole cell cholera vaccine (WCV) without the B subunit. This vaccine, OrcVax, which contains both V. cholerae O1 (the same strains and inactivation methods as in the Dukoral OCV) and a formalin-killed O139 strain, was shown to provide 66% protection after 8 10 months and 50% protection for up to 5 years when administered in two doses [37]. OrcVax, which has been licensed only in Vietnam, has been used since 1998 by the public health system in high-risk areas and during emergencies, such as floods, with more than 15 million doses having been distributed to date. While having lesser short-term efficacy than Dukoral, this and similar inactivated WCVs have advantages for use in developing countries: They do not require a buffer, making them easier to administer, and they are easier to produce and at lower cost. However, certain manufacturing issues (including the exchange of one of the Dukoral O1 strains with a strain with greater risk for residual cholera toxin) and a lack of a WHO-accepted national regulatory agency (NRA) in Vietnam precluded international licensing and WHO prequalification of this vaccine. With the goal of developing a vaccine that could be prequalified by the WHO for

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TABLE 31.1 Composition (Active Ingredients) in the Currently WHO-Prequalified Inactivated Oral Cholera Vaccines Dukoral, Shanchol, and Euvichol Dukoral

Shanchol, Euvichol

Quantity per dose

Vibrio cholerae O1 Inaba classical strain Cairo 48

Same as in Dukoral

300 Elisa units (EU) of lipopolysaccharide (LPS)a (B2.5 3 1010 bacteria)

Same as in Dukoral

300 EU of LPS (B2.5 3 1010 bacteria)

Same as in Dukoral

300 EU of LPS (B2.5 3 1010 bacteria)

Same as in Dukoral

600 EU of LPS (B5 3 1010 bacteria)

V. cholerae O139 strain 4260B

600 EU of LPS (B5 3 1010 bacteria)

Heat inactivated V. cholerae O1 Ogawa classical strain Cairo 50 Heat inactivated V. cholerae O1 Ogawa classical strain Cairo 50 Formaldehyde inactivated V. cholerae O1 Inaba E1 Tor strain Phil 6973 Formaldehyde inactivated

Formaldehyde inactivated Cholera toxin B subunit (rCTB)

1 mg

a

Determined for the individual bulk components before mixing by an inhibition ELISA method (Holmgren and Svennerholm,1973, Infect Immun 7, 759 763; and used as a potency test by manufacturers of current WHO-prequalified OCVs) in which serial dilutions of the inactivated bacteria are incubated with LPS-specific monoclonal or absorbed polyclonal antibody and the 50% inhibition titer is determined in a subsequent LPS-specific ELISA and compared with the inhibition titer of a reference inactivated V. cholerae O1 or O139 bacterial preparation.

international use to control both endemic and epidemic cholera, the International Vaccine Institute, working with the Vietnamese producer, modified the production methods and reformulated the O1 vaccine composition back to that of Dukoral while retaining the O139 component, thus making the modified OrcVax (mOrcVax) OCV compliant with WHO manufacturing standards. Importantly, the production of this modified vaccine was also transferred to a manufacturer in India with a WHO approved-NRA. The Indian OCV named Shanchol, with the composition of the reformulated Vietnam WCV, was licensed in 2009 after a large pivotal placebo-controlled efficacy trial in age group 1 year and above in Kolkata. In this trial, the vaccine afforded over 60%

protection over a 5-year observation period, and Shanchol was WHO prequalified in 2011 [35,38]. The manufacturing technology has further been transferred to a producer in South Korea, and the vaccine Euvichol, which has the same composition as Shanchol, was also prequalified by WHO in 2015 after being demonstrated to be noninferior to Shanchol with regard to safety and vibriocidal antibody immunogenicity [39]. Another OCV, Oravacs (Shanghai United Cell Biotechnology, Shanghai, China), is licensed only in China and the Philippines. It closely resembles Dukoral, containing the same composition of killed V. cholerae O1 inactivated bacteria as well as rCTB, and comes in a dry formulation, three-dose, enteric-coated capsule formulation,

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thus avoiding the need for administration with a buffer. At least two more inactivated whole cell OCVs are on a trajectory to licensing: Cholvax (Incepta, Bangladesh), which is a locally produced, inexpensive version of Shanchol and Euvichol and is primarily intended for national use in Bangladesh, and Hillchol (MSB-Wellcome Trust Hilleman Laboratories, India and University of Gothenburg, Sweden), which is an inexpensively manufactured single-strain, formaldehyde-inactivated V. cholerae O1 El Tor Hikojima (combined Inaba and Ogawa) serotype WCV, that is, aiming at international licensure and WHO prequalification. A limitation of the killed OCVs, both Dukoral and the bivalent reformulated WCVs, is the need for a two-dose regimen. Much effort has been made to develop live attenuated OCVs for single-dose administration, and several new candidate vaccines are currently in different stages of development. The first such vaccine, CVD 103HgR (Orochol, Berna, Switzerland), which was derived from the classical Inaba 569B strain by deletion of the cholera toxin A subunit, was shown to give a high degree of protection against cholera in challenge studies in the United States, which provided the basis for its licensure in several industrialized countries for use in travelers to cholera-endemic settings [40]. A large field trial in Indonesia, however, failed to demonstrate protective efficacy, and production of the vaccine was halted in 2004 [35,41]. However, after being produced by a different manufacturer and renamed VAXCHORA, the CVD 103HgR vaccine was licensed in 2016 for use by US travelers based on the significant (approximately 65%) protection seen in North American volunteers challenged with V. cholerae O1 El Tor up to 3 months after vaccination [41b]. A recent large phase III trial in Bangladesh has further addressed the question of whether a single-dose inactivated whole cell OCV could give satisfactory protection in a high-endemicity population with, at least in older individuals,

frequent previous natural exposure to V. cholerae. It was found that while a single dose of the Shanchol OCV gave 63% protection against severe cholera for at least 2 years in subjects who were 5 years of age and older, it did not afford any protection in children below age 5 years, probably owing to the relatively naı¨ve immunological status of young children. A twodose regimen is required for an effective immune response in this age group [42]. Transfer of the killed whole-cell-only OCV production technology to high quality, emerging producers is now taking place at increased pace. Production by these emerging producers offers the prospect of an adequate supply of low-cost vaccine, thereby addressing key supply issues for the use of this vaccine in the control of endemic cholera.

IV. ADAPTIVE MUCOSAL IMMUNE RESPONSES IN CHOLERA The most studied correlate of adaptive immunity to V. cholerae is serum vibriocidal antibody titer. Seroepidemiological studies have shown that in cholera-endemic areas, vibriocidal antibodies, which are largely immunoglobulin M (IgM) antibodies against the bacterial cell wall LPS, increase with age and that the risk of disease is inversely proportional to the vibriocidal antibody titer [4]. However, vibriocidal antibodies in both unvaccinated and vaccinated individuals are only a surrogate marker for the intestinal mucosal immune status. For instance, parenteral vaccines confer only limited and short-lived protection, even though they induce extremely high vibriocidal antibody titers. Instead, there is strong evidence indicating that immune protection in cholera, both that mediating recovery from ongoing infection and disease and that preventing cholera infection and disease after effective immunization, depends on the stimulation of a mucosal

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immune response in the intestine and is mediated largely by mucosal secretory IgA (SIgA) antibacterial and/or antitoxin antibodies [18,19,25] (Chapter 4: Protective Activities of Mucosal Antibodies).

A. Mucosal IgA Antibody and AntibodySecreting Cell Responses V. cholerae is a noninvasive enteric pathogen, and consistent with this, antigen-specific immune responses at the intestinal surface, primarily SIgA antibody to V. cholerae LPS and to cholera toxin/CTB antigens and corresponding mucosal IgA B cell memory responses, have been found to play a major role in protective immunity [7,43,44]. Experimental as well as clinical epidemiological findings support that both antibacterial (mainly anti-O1 LPS) and antitoxin (mainly anti-CTB) mediate effective immune protection against cholera [24 26]. In animal models, antibacterial and antitoxic antibodies capable of preventing bacterial colonization and the binding and action of CT in the small intestine were found to effectively protect against experimentally induced V. cholerae infection and disease. SIgA is the predominant immunoglobulin at the mucosal surface [24,26]. Both antibacterial and antitoxic immunity were found to depend mainly, if not exclusively, on locally produced SIgA mucosal antibodies of the SIgA type directed mainly against LPS and CTB, respectively [45,46]. It was further noted that in the intestine, such antibacterial and antitoxic antibodies produce a synergistic cooperative effect in protection against disease [42,47]. Similarly, epidemiological studies have found a strong correlation in breast-fed Bangladeshi infants and young children between the specific SIgA anti-LPS and anti-CTB titers in the ingested breast milk and reduction in cholera incidence, including evidence of synergistic protection against cholera through the combined SIgA titers against LPS and CTB [48].

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In adults, both antitoxin and anti-LPS SIgA responses are detected in intestinal fluid, breast milk, and saliva after both cholera infection and vaccination [49]. The intestinal lavage method, in which participating volunteers drink an isotonic solution until they discharge a watery stool, which is collected and used for measurements of total SIgA and cholera-specific SIgA antibody levels by ELISA methods, has contributed much of the current information about the intestinal mucosal immune response in humans after cholera infection or oral vaccination. The results obtained with this method directly guided the development of the first licensed OCV (Dukoral) [50]. As illustrated in Fig. 31.1, it was shown that two oral immunizations with a mixture of 0.5 mg or, even better, 2.5 mg CTB and 5 3 1010 heat-killed whole cell cholera Vibrios could elicit anti-CTB as well as anti-LPS IgA antibody responses of magnitudes similar to those of the responses in convalescents from severe cholera disease; a single dose was significantly less immunogenic, as were two parenteral doses [29]. Subsequent studies confirmed that two oral doses with the chosen combination of 1 mg CTB and 1 3 1011 inactivated cholera Vibrios, now also including formalininactivated classical and El Tor bacteria, elicited strong intestinal mucosal SIgA responses in both cholera-endemic (Bangladesh) and nonendemic (Sweden) populations and also demonstrated that the interval between the two doses could be varied between 1 and 6 weeks without affecting the mucosal immunogenicity, while a 3-day interval was less immunogenic [51]. Anamnestic SIgA responses also likely contribute to protection. Thus while significant immune protection lasts for at least 3 years after either a clinical cholera episode or effective OCV immunization, the acute SIgA anti-LPS and anti-CTB responses peak within the first 7 30 days and have largely vanished after 6 9 months [33]. In contrast, the intestinal mucosal immunological memory is of very long duration, as is shown by rapid strong SIgA

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IgA Anti-LPS

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FIGURE 31.1 Intestinal lavage geometric mean IgA antibody responses in Bangladeshi cholera patients (Dis) and healthy adults receiving two peroral (PO) immunizations with an inactivated Vibrio cholerae O1 whole cell (WCB5 3 1010 bacteria) 1 B subunit vaccine (CTB, either 0.5 or 2.5 mg) or, for comparison, two intramuscular (IM) immunizations with a reduced dose of the WC 1 CTB vaccine at days 0 and 28; the cholera patients were followed for 28 days and then given a single PO0.5 (WC 1 O.5 mg CTB) immunization. Source: Adapted from Svennerholm AM, Jertborn M, Gothefors L, Karim AM, Sack DA, Holmgren J. Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a combined B subunit-whole cell vaccine. J Infect Dis 1984;149:884 93.

responses induced by single-dose OCV boosting in Swedish volunteers immunized as long as 10 14 years after an initial two-dose OCV immunization (Fig. 31.2) [52,53]. An important prerequisite for a protective function would be that an anamnestic response to the pathogen would be rapid enough to avert the infection before it has caused disease. Consistent with this, a rapid rise in intestinal lavage antitoxin and anti-LPS IgA was seen by Day 3 (which was the earliest time point examined) in adults receiving a dose of an oral B subunit/killed whole cell OCV either 1 month after primary immunization or after an episode of clinical cholera (Fig. 31.1) [50]. Such rapid anamnestic immune responses would appear to be particularly important for the duration of protective immunity in cholera. Several studies have also examined the antibody-secreting cell (ASC) response in

human intestinal mucosa after cholera infection or oral vaccination by isolating mononuclear cells (MNCs) from duodenal mucosal biopsies and then assaying the numbers of antigen-specific ASCs in relation to total MNCs or isotype-matched immunoglobulinsecreting cells by the ELISPOT method. A pivotal study by Quiding et al. [54] examined the kinetics of the duodenal mucosal ASC response in Swedish volunteers after oral immunizations with the WCV/rCTB Dukoral prototype vaccine demonstrating a significant IgA, immunoglobulin G (IgG), and IgM ASC response 7 days after a first immunization, which, for the IgA ASCs, was increased fivefold by a second dose; after 5 months, the duodenal ASC levels had returned almost to prevaccination background levels but were rapidly reelicited to maximal levels upon a single booster immunization at that time

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V2

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FIGURE 31.2 Long-lasting ( . 10 years) immunological memory for intestinal IgA antibody responses shown in Swedish human adult volunteers after oral cholera vaccination. Volunteers received two oral immunizations at a 2-week interval (V1 and V2) with Dukoral whole cell 1 B subunit OCV (left panel), and other volunteers who had received a similar two-dose immunization with the same vaccine 6 months to 14 years earlier received a single oral booster dose (right panel). Peripheral blood mononuclear cells (MNCs) were isolated at indicated time points and were cultured for 5 days, whereupon the culture supernatants were collected and analyzed by ELISA for CTB-subunit-specific IgA antibody titers. These intestine-derived antibody-in-lymphocyte-supernatant ALS titers, which closely correlate with antigen-specific IgA antibody-secreting cell numbers in MNCs from the same time points, show that a single booster immunization given as late as up to 14 years after primary two-dose immunization elicits a strong anamnestic IgA response and that the peak response occurs earlier (Day 4 and 5) after the second or late booster dose than after the first dose. Source: Adapted from Leach S, Lundgren A, Svennerholm AM. Different kinetics of circulating antibody-secreting cell responses after primary and booster oral immunizations: a tool for assessing immunological memory. Vaccine 2013;31:3035 8.

(Fig. 31.3A). Similarly, and important for the development of the bivalent OCVs containing formalin-killed V. cholerae O139 bacteria in addition to the inactivated O1 strains, such a bivalent O1/O1391rCTB prototype OCV developed in Sweden, after proving safe and inducing good intestinal lavage IgA antibody responses in Swedish volunteers to both the O1 and O139 components [55], was shown in immunized Bangladeshi adults to induce IgA ASC responses in duodenal biopsies as well as IgA antibody responses in intestinal lavage and fecal extracts to all three antigens (CTB, O1 LPS, and O139 LPS) [56]. The CTB and

anti-O1 intestinal responses were as strong as those induced by the comparator Dukoral (O1 WC/rCTB) vaccine, supporting that the O139 vaccine component not only was immunogenic but also did not interfere with the anti-O1 or anti-CTB responses [55,56]. In adult cholera patients in Bangladesh, ASC responses to both CTB and O1 LPS were maximal at 30 days after onset of disease and, in an apparent difference from orally immunized Swedish adults, remained elevated above the levels in healthy controls for 180 days [57]; the longer duration in the endemic population might reflect renewed subclinical natural exposure

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FIGURE 31.3 Kinetics of geometric mean (A) intestinal mucosal IgA, IgG, and IgM antiCTB antibody-secreting cell IgA IgG IgM (ASC) responses, (B) intestinal 150 mucosal interferon-gammasecreting T cell response, and (C) peripheral blood IgA, IgG, and IgM anti-CTB ASC responses in 100 Swedish human adult volunteers before and 7 days after each dose of an initial two-dose oral immu50 nization at a 2-week interval with cholera toxin B subunit (CTB)/whole cell Dukoral OCV 0 and then again 5 months later before and after a single booster Pre d+7 d+21 5 months d+7 immunization with the same Boost 1st 2nd vaccine. Responses were analyzed by ELISPOT methods in mononuclear cells (MNCs) iso(B) (C) Duodenal T cell response Peripheral blood ASC responses lated from duodenal biopsies (A 700 15 and B) or peripheral blood (C). Source: Adapted from Quiding M, 600 Nordstrom I, Kilander A, Andersson G, Hanson LA, 500 Holmgren J, et al. Intestinal 10 immune responses in humans. Oral 400 IgA cholera vaccination induces strong intestinal antibody responses and IgG 300 interferon-gamma production and IgM evokes local immunological memory. 5 200 J Clin Invest 1991;88:143 8.

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maintaining or renewing the primed mucosal IgA ASC response.

B. Intestine-Derived Gut-Homing Circulating Antibody-Secreting Cells Following antigen presentation in the gut mucosa, both intestinal B and T lymphocytes expressing the gut-homing α4β7 integrin on their

d+21

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surface transiently migrate in the peripheral circulation before rehoming to the gut for final differentiation into mainly IgA plasma cells for the B cells [58] (Chapter 5: Mucosal Immunity for Inflammation: Regulation of Gut-Specific Lymphocyte Migration by Integrins). Such B plasmablast ASCs are detectable in blood even before antigen-specific SIgA in intestinal secretion is detectable, peaking at 7 10 days after a primary oral immunization [54,55,59] and at

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4 5 days after booster immunization [53]. Circulating ASCs to LPS and CTB have been demonstrated in adults infected with either V. cholerae O1 or O139 [60]. When blood is collected at the appropriate time after oral cholera vaccination or infection, such intestine-derived, gut-homing antigen-specific ASCs account for a high proportion of the total IgA-secreting cells in the peripheral blood; thus among flow cytometrically sorted populations of gut-homing plasmablasts, almost 50% of the cells recognized either CTB or V. cholerae O1 LPS [61]. Young Bangladeshi children infected with V. cholerae O1 Ogawa mounted ASC responses to CTB and LPS that peaked by Day 7 after onset of illness and were comparable in magnitude to those seen in older children and adults [62]. Since enumeration of intestine-derived ASC responses in peripheral blood is logistically and technically much easier than corresponding enumeration of ASCs from intestinal mucosal biopsies and can be adapted to small blood volumes that can be collected from even young children, this approach has emerged as a major tool for assaying the intestinal mucosal immunogenicity of a broad range of new enteric vaccines [59,63,64]. The latter is even more and increasingly true for the ALS (antibodies-in-lymphocyte supernatants) method, in which peripheral blood MNCs are cultivated for 4 5 days and the cell supernatants are then collected, stored by freezing, and then analyzed for specific antibody levels by ELISA methods [65 68] (see also Chapter 32: Oral Vaccines for Enterotoxigenic Escherichia coli). While showing excellent agreement with ASC determinations for blood samples collected at the same time, a marked practical advantage of the ALS method is that, unlike determinations of ASCs, which have to be done on freshly isolated MNCs, the ALS specimens can be stored to allow side-by-side testing of prevaccination and postvaccination samples. However, it should be noted that although both the ASC and ALS methods are highly useful for comparing immune responses to oral vaccines on a

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group basis, they provide only crude proxy estimates of the intestinal ASC response on an individual basis.

C. Intestinal Mucosal T Cells Although V. cholerae is a noninvasive mucosal pathogen, and pathogen-specific effector mucosal defense against V. cholerae is thought to be largely B cell mediated as discussed previously, helper T cells likely play an important role in the development of B cell immunity directed against protein antigens, as seen in the involvement of Th17 cells [69] and the chemokine receptor CXCR5 [70] in the generation of mucosal immune responses against CT. Early work with Swedish volunteers showed that two oral immunizations with the CTB/O1 whole cell Dukoral prototype vaccine in addition to inducing a strong IgA ASC response elicited a significant interferon-gamma (IFNγ) secreting T cell response in duodenal mucosal biopsies (Fig. 31.3A) [54]. Bangladeshi adults recovering from cholera were found to have increased frequencies of gut-homing, CD41 expressing, effector, and central memory T cells that peaked at Day 7 [71 73]. Furthermore, cytokines associated with a Th17 response are detectable in duodenal biopsy lamina propria samples during the acute stage of cholera [72]. There is limited information on cellular immune responses in children with cholera. One study assessed cellular responses in infants aged 10 18 months given two doses of WC-rBS vaccine [74]. In these children, stimulation of postvaccination lymphocytes ex vivo with a modified CTB resulted in increased CD41 blast formation and IFNγ production compared with responses elicited by using prevaccination lymphocytes; however, in contrast with what occurs in adults, stimulation with a cholera membrane preparation did not produce any increases in children. Further studies of T cell responses in children with natural infection are needed.

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V. SYSTEMIC ANTIBODY AND MEMORY B CELL RESPONSES Although, as discussed previously, protective immunity in cholera is mainly, if not exclusively, mediated by mucosal antibodies produced locally in the intestine, the majority of studies of immune responses to cholera infection or vaccination in both adults and children have largely assessed serum vibriocidal and other systemic antibody responses.

A. Vibriocidal Antibody Responses The serum vibriocidal antibody response is the most studied immunological marker of cholera infection and also of immune responses to OCVs. The antibodies are bactericidal in the presence of complement and are mostly of the IgM isotype and directed against LPS [75 77]. Titers increase after both symptomatic and asymptomatic infection [78], but levels wane quickly and fall to baseline levels within 9 12 months [79,80]. In endemic settings, the percentage of a population with detectable vibriocidal titers increases with increasing age, and 40% 80% of individuals have detectable vibriocidal antibodies by 15 years of age [4,81]. Although higher titers have been associated with protection against V. cholerae O1 infection and disease [4,81], there is no threshold at which protection is complete [82]. A study of patients infected with V. cholerae O1 showed that children 12 years of age and younger had, in general, lower acute-phase (Day 2) vibriocidal antibody titers but developed a stronger vibriocidal response to infection than those older than 12 years of age [83]. Similarly, a study of children hospitalized for V. cholerae O1 Ogawa infection showed that while young children (2 5 years of age) had significantly lower acute-phase titers than adults, the young children had higher fold increases in vibriocidal antibodies compared with adults, resulting in comparable peak titers

during convalescence [62]. It appears that young children are able to mount vibriocidal responses following wild-type cholera infection that are comparable to those induced in older children and adults. The few studies with age-group-specific comparisons of vibriocidal responses after oral vaccination have shown much heterogeneity in results depending on the type of vaccine and criteria of response [57 60,63]. Interlaboratory variability also limits comparison across studies, although fold increase in response to infection or vaccination can be used as the important outcome. Overall, studies of immunogenicity as measured by fold change or responder frequency of vibriocidal titer rises show that young children have immune responses to vaccination comparable to those of older children and adults. A study of WC/BS OCV, a predecessor of the current killed, whole cell OCV Dukoral, in Bangladesh showed that, in general, children aged 2 5 years had responder frequencies and fold changes similar to those of older children and adults [84,85], a response that was comparable whether or not a recombinant B subunit was included in the vaccine. Notably, young children, but not adults, had incremental increases in responder frequency with subsequent vaccine doses. These findings were also seen in a study of the inactivated whole cell Shanchol OCV, in which young children (both 2 5 years and 12 23 months of age) had responder rates similar to those of adults [86]. The live attenuated El Tor Inaba V. cholerae O1 OCV (Peru-15) also elicited comparable vibriocidal response rates in young children aged 2 5 years and infants aged 9 24 months as adults, although infants achieved the lowest rates [87]. Still, as will be discussed below, with all tested OCVs, the protective efficacy in young children is less and of shorter duration than that in adults and older children, underlining the fact that the vibriocidal antibody response after immunization is a very imperfect measure of immune protection.

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Akin to what is seen following natural infection, studies of both live and killed formulations of cholera vaccines have demonstrated lower seroconversion rates [88 90] and lower fold increases [91] in children with higher baseline or acute-phase titers, largely reflecting a higher baseline/acute-phase level in endemic zones, where exposure to V. cholerae is often repetitive.

B. Serum Antibody Responses to Defined Antigens 1. IgA Responses Serum IgA levels increase progressively in childhood, from 1% of adult levels in the newborn to 20% at 1 year, 50% at 5 years, and 75% by 16 years of age [92]. There is evidence that serum IgA antibodies to specific antigens, mainly LPS and CT/CTB, may better reflect immune protection after disease and OCV immunization than the vibriocidal antibody response. In a study in Bangladesh among household contacts, including children, of cholera patients observed for 21 days after identification of the index case, specific serum IgA levels against V. cholerae LPS and CT antigens were associated with protection against subsequent V. cholerae O1 infection during follow-up [93]. In children infected with V. cholerae O1 Ogawa, levels of serum IgA against LPS and CTB rise, with peak levels at Day 7, similar to the levels achieved in older children and adults [62]. By Day 30 of convalescence, levels of antigen-specific IgA are still above baseline levels, although adults maintain LPS IgA levels that are significantly higher than those of younger children. In a study that included both O1 and O139 infections, patients older than 12 years of age had higher levels of homologous anti-LPS IgA in serum than those 12 years of age and younger at both acute and convalescent phases of infection, whereas younger patients had higher serum CTB IgA responses, as they also had higher mucosal anti-CTB IgA levels in

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feces [83]. This may be due to younger children having more recent exposures to ETEC [94], a common childhood pathogen whose heat-labile enterotoxin is immunologically cross-reactive with CT [95]. Most studies utilizing serum IgA responses after vaccination have focused on the antitoxin response. Approximately 80% of children in Bangladesh aged 2 5 years achieved a more than or equal to twofold increase in anti-CT IgA after two doses of a WC-rBS vaccine given 2 weeks apart [96], and a similar responder frequency rate was found in children younger than 2 years of age given a WC-rBS vaccine in a subsequent study [97]. Immunity against cholera is serogroup specific, as previous infection with V. cholerae O1 does not provide protection against O139 and vice versa [98]. Few studies have examined serum IgA responses to LPS following vaccination, although the available data suggest that young children may have lower anti-LPS responses than adults. Thus in Bangladeshi children given Peru-15, a live attenuated Inaba vaccine, 54% of 2- to 5-year-old children achieved a twofold or greater increase in IgA anti-LPS, while only 34% of those younger than 2 years of age achieved such increases [87]; both results were lower than the responses seen in adults (88%) receiving the same vaccination in an earlier study [99]. Studies of the killed Shanchol WC vaccine, also among Bangladeshi subjects, showed that while young children (2 5 years and 12 23 months) achieved comparable frequencies of a twofold or greater response, the peak geometric mean titer decreased with age (LPS IgA against O1 Inaba, geometric mean titer of 171 in adults, 37 in those aged 2 5 years and 13 in those aged 12 23 months) [86]. LPS is a T-cell-independent antigen, and despite the similar responder rates, the lower magnitude of the absolute antibody response may be a reflection of poorer humoral responses to T-cell-independent antigens in very young children [100].

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2. IgG Responses Significant systemic IgG responses to V. cholerae antigens are detected following both natural infection and vaccination. In adults, elevations in serum IgG to CTB are detectable for at least 270 days following both natural infection [80] and two doses of vaccination with Dukoral WC-rBS vaccine [101]. However, studies of household contacts of cholera patients have not found levels of plasma antigen-specific IgG on exposure to be predictive of protection against cholera [80]. Children with V. cholerae infection also mount significant increases in plasma IgG titers against CTB and LPS by Day 7 of infection [62,83]. Notably, baseline levels of plasma CTB IgG are significantly lower in adults than in children, likely reflecting more recent exposure to ETEC in children. During V. cholerae O1 Ogawa infection, all age groups achieve similar magnitudes of CTB- and LPS-specific plasma IgG at convalescence [62], while a study of patients infected with V. cholerae O1 or O139 showed that adults mounted higher LPS IgG levels at convalescence, while children mounted higher CTB IgG levels [83]. In adults, systemic antibody responses in multiple subclasses of IgG have been demonstrated against both CT and LPS [102]. Such subclass studies have not been performed in children with cholera. Antitoxin IgG responses have been demonstrated in children receiving OCV. In studies using WC-BS, a predecessor to the Dukoral WCrBS vaccine, children aged 2 5 years achieved a 2.6-fold rise in CT IgG compared with those receiving placebo, while those older than 15 years had a 4.7-fold rise [85]. A more recent study has shown that approximately 55% of Bangladeshi children aged 2 5 years receiving two doses of WC-rBS achieved a twofold or greater increase in serum IgG antibody to CTB [96]. Similar to other serum antibody responses in cholera, including vibriocidal antibody responses, the IgG anti-LPS or anti-CT responses to infection or immunization do not protect, nor do the specific levels of these

antibodies in contrast to vibriocidal antibodies (which are largely of IgM isotype) correlate with protection [85]. 3. Immunoglobulin M Antigen-specific IgM is the first antibody isotype to rise in the serum after exposure to an antigen and plays an important role in subsequent affinity maturation and isotype switching, giving way to other antibody isotypes such as high-affinity IgG [103]. In adults with V. cholerae O1 infection, IgM responses against LPS are elevated by Day 7 after onset of illness and remain persistently elevated above baseline for at least 30 days, the last period examined [104]. This finding is not unexpected, as the vibriocidal antibody is composed mostly of IgM directed against LPS [77]. Levels of IgM to CTB do not appear to change with cholera infection. Similarly, adults vaccinated with Peru-15 also produced low levels of serum IgM to CTB [99]. No studies are available characterizing IgM responses in children with cholera. Such investigations are needed, given the importance of IgM antibody in humoral immunity against T-cell-independent antigens such as LPS [105] and its role in long-term immunity involving memory B cells (MBCs) [104].

C. Memory B Cell Responses in Cholera MBCs are found in the circulation after natural infection and vaccination and are thought to play a critical role in maintaining long-term protective immunity by facilitating rapid anamnestic antibody responses upon reexposure to antigen [106]. Crotty et al. [107] developed a practical ELISPOT-based method for determining MBCs in peripheral blood by stimulating MNCs in culture with a cocktail of B cell mitogens and then determining antigen-specific ASCs. When this method was used in hospitalized cholera patients, both IgA and IgG MBCs against V. cholerae antigens were detectable by Day 30 after infection [80]. In fact, IgG MBC responses to

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VI. MODIFIERS OF IMMUNE RESPONSES

T-cell-dependent antigens CTB and TcpA (a major pilus colonization factor of V. cholerae) persisted for up to 1 year, longer than any other known marker of cholera immunity [80]. Despite the lack of increase in systemic IgM responses against CTB, IgM MBCs against LPS and CTB have also been described up to 30 days following acute infection with V. cholerae O1 [104]. Younger children with V. cholerae O1 Ogawa infection mount CTB- and LPS-specific MBC responses by Day 30 after infection comparable to those of older children and adults [62], and there is a trend for younger children to mount higher levels of CTB IgG MBC responses than older children, likely reflecting more recent exposure to the cross-reactive heat-labile toxin antigen of ETEC. Evaluation of the MBC responses in children with longer follow-up is needed. In Bangladeshi adults receiving WC-rBS vaccine, MBC responses to CTB and LPS were significantly shorter in duration and lower in magnitude than those in adults recovering from cholera [101]. However, using a different B-cell-stimulating cocktail, it was found that a two-dose oral immunization with the rBS-WC Dukoral vaccine induced a strong vaccinespecific peripheral blood IgA BMC response in Swedish adult volunteers that persisted for at least 9 months after vaccination [80a]. The evaluation of MBC in children receiving cholera vaccination remains to be reported, and comparisons between vaccinated children and adults may uncover the role that MBCs play in determining the duration of vaccine protection. Of note also is that the B memory cells (BMCs) that have been studied to date have not been examined for the presence of gut-homing markers such as α4β7. Further, BMCs may exist that are not detected in blood, either because they are present in other sites, such as gutassociated germinal centers, or even if present in the blood, because they may not be detected with the usual methods. Further examinations of these aspects are needed, since, as was mentioned above, there is functional evidence of B

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cell mucosal memory lasting for more than 10 years in Swedish volunteers after vaccination (Fig. 31.3A) [53] (Chapter 7: Induction and Regulation of Mucosal Memory B Cell Responses).

VI. MODIFIERS OF IMMUNE RESPONSES A number of host factors modify disease severity and immune responses following infection and vaccination, including coinfections, blood group, genetic polymorphisms, and nutritional and micronutrient deficiencies [108]. In cholera-endemic regions, concomitant intestinal infection with parasites and bacteria is common, especially in children. More than one third of children aged 10 years and younger presenting with acute V. cholerae infection to a hospital in India had concomitant parasitic infection [86,109]. The impact of helminthic coinfection on blunting the mucosal immune response to cholera infection and vaccination has been demonstrated in both adults and children [110,111], and there is evidence that parasite-associated alterations in cell-mediated immunity are responsible for differences in mucosal responses. In patients presenting with severe cholera in Bangladesh, helminth coinfection was associated with decreased fecal and serum IgA responses to the T-dependent antigen CTB but not to the T-independent antigen LPS [111]. Additionally, Ecuadorean children aged 13 17 years infected with Ascaris lumbricoides had a diminished T helper 1 (Th1) cytokine response to vaccination with live attenuated OCV CVD 103HgR compared with noninfected US controls; this diminished response was partially reversed in a group treated with albendazole [110]. Coinfection with ETEC, an enteric bacterial pathogen commonly coendemic with V. cholerae, also appears to alter the immune response to V. cholerae. In Bangladeshi adults and children

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hospitalized for diarrhea, those infected with both ETEC and V. cholerae (13% of patients) produced higher vibriocidal and higher antibody levels to CTB and LPS than did those infected with V. cholerae alone [112]. Several studies have begun to address the role that genetics may play in the host immune response to cholera. As described previously, it has long been known that patients with blood group O have increased severity of cholera [16,17]. In a study of household contacts of index cholera patients in Bangladesh, familial segregation of susceptibility within households independent of blood group was observed, suggesting possible additional genetic contributions to cholera susceptibility and severity [111]. A family-based candidate gene association study in Dhaka, Bangladesh, identified a variant in the promoter region of long palate, lung, and nasal epithelium clone 1 (LPLUNC-1) gene to be associated with cholera disease [113]. Further exploration of genetic factors and their role in susceptibility to infection may uncover additional factors of importance affecting host immunity during cholera. Malnutrition and micronutrient deficiency can also negatively affect adaptive immune responses in cholera. Studies in animal models have demonstrated that protein deprivation results in impaired mucosal antitoxin responses [114] and severely diminished antibody responses to cholera vaccination [115]. In humans, malnutrition and intestinal infections are integrally related [116], and in Bangladesh, malnutrition is associated with up to half of deaths due to diarrhea in children under 5 years of age [117]. Low weight-for-age Z scores are associated with an almost 10-fold increased risk of mortality from diarrhea [118], and in hospitalized patients with V. cholerae infection, individuals with protein calorie malnutrition have increased stool losses and prolonged diarrhea [119]. Vitamin A, or retinol, and its metabolite, retinoic acid, are associated with host defense

against infectious diseases, possibly through their effects on CD41 T cell function [120]. Retinoic acid is fundamentally important for the mucosal immune defense. The small intestine CD1031 dendritic cells have increased ability to generate retinoic acid, which specifically underlies their ability to induce the gut-homing receptors CC chemokine receptor 9 and α4β7 on mucosal T and B cells and to enhance T regulatory and IgA plasma cell differentiation [121]. Consistent with this, vitamin-A-deficient rats immunized with CTB-containing OCV had an impaired intestinal mucosal antibody response to CTB compared to immunized normal rats [122,123]. Vitamin A is also known to influence the composition of microbiota [121]. Vitamin A supplementation has been associated with reductions in mortality and morbidity, including incidence of diarrhea, when given to nonhospitalized young children in developing countries [124]. In patients hospitalized for cholera, retinol deficiency is more common in children younger than 12 years of age than in older patients [83] and is associated with an increased risk of V. cholerae O1 infection and symptomatic disease [111]. However, vitamin A supplementation of oral cholera vaccination in young children produced only small increases in vibriocidal antibody responses [125] and had no effect on antitoxin antibody responses [96,125]. Zinc deficiency in children is associated with an increased risk of diarrhea, and supplementation of children with zinc in developing countries is associated with reductions in both the incidence and severity of diarrhea [126]. In children with V. cholerae infection, zinc supplementation decreases the duration of diarrhea and stool output [127], although zinc deficiency was not associated with increased susceptibility to infection in household contacts [111]. By contrast to vitamin A supplementation, zinc supplementation in children has a differential effect on the immunogenicity of killed OCV in that it increases the vibriocidal antibody

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VII. PUBLIC HEALTH USE OF ORAL CHOLERA VACCINES

response [97,125] and IFNγ production by CD41 T cells [74] but lowered the antitoxin responses [96]. In ETEC, a related acute toxigenic diarrheal infection, zinc supplementation resulted in an increase of complement C3 levels and in phagocytic functional activities [128].

VII. PUBLIC HEALTH USE OF ORAL CHOLERA VACCINES As described in Section III of this chapter, effective OCVs prequalified by WHO for purchase in the UN system have been available since the early 1990s. Considering the great need for effective control of cholera in the world, it is remarkable that it has taken so long for OCVs to be accepted as a valuable public health tool in the control of cholera. For many years, concerns were expressed in parts of the WHO and the water-sanitation-hygiene (WaSH) community that using OCV might have a negative impact on the implementation of oral rehydration treatment, WaSH activities, and other established cholera control interventions. One concern was that people might feel safe by being protected by the vaccine and thus would be less compliant to WaSH practices. Another concern was that access to OCV would take away the pressure on communities and governments to make needed infrastructural investments toward sustainable access to safe water and adequate sanitation. However, as has since long been argued by us and others [129,130] and as is now increasingly being adopted, there are good reasons to believe that the opposite is true: WaSH interventions and cholera vaccination should work well and probably even synergistically together, with OCV improving the effectiveness of WaSH and vice versa. Immunization will decrease the proportion of susceptible individuals in the community and reduce environmental contamination, thus helping to stop transmission of the disease and improving the effectiveness of

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WaSH interventions. Conversely, WaSH could make cholera vaccination more effective by reducing the median inoculum size. A common fallacy in discussions about the use of OCVs has been that their protective efficacy must be very high to be useful in controlling cholera. Overlooked is the fact that in addition to their specific vaccine efficacy, OCVs provide strong herd protection resulting from the ability of immunization to reduce person-toperson transmission of cholera, thus providing protection of nonvaccinees who reside in vaccinated neighborhoods as well as enhanced protection of vaccinees [131 133]. Indeed, available evidence indicates that with vaccineinduced herd protection, the available OCVs even at less than 50% vaccination coverage levels of targeted populations may result in virtually complete control of cholera despite their moderate, approximately 60% 70% levels of direct vaccine efficacy [134]. There is a strong need for the use of OCVs, both in settings where cholera is highly endemic and in places where outbreaks are likely to occur because of conflict, war, or natural disasters that result in large numbers of displaced people living in high-risk, cholera-prone settings. Often, but not always (e.g., the large Haiti epidemic in 2010), cholera outbreaks occur in regions and populations where cholera is already endemic. It has been reported that the majority of refugee camps in choleraendemic regions experience cholera within the first 2 years of their existence. Under conditions of conflict, war, or natural disasters, normally acceptable water and sanitation systems often collapse, and displaced populations then have no access to safe water or sanitation, making them very prone to cholera. The large cholera epidemics in Haiti, Yemen [135,136], and other areas facing humanitarian crises in recent years demonstrate the importance of large OCV campaigns globally. In this context, the successful use of OCVs in the large-scale preemptive vaccination of displaced Rohingya nationals from

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Myanmar into Bangladesh is an example in which a large epidemic may have been averted by quick response on the part of the WHO and the government [137]. With regard to the public health use of OCVs, it is important to distinguish endemic from epidemic cholera, especially when the latter occurs in a population that has not previously been exposed to cholera [138]. There are several implications of these two epidemiological forms of cholera for the use of OCVs. First, whereas regular and periodic immunization, for example, at 3-year intervals, makes epidemiological sense for the control of endemic cholera, such a strategy is not rational for epidemic cholera, which instead requires either selective, preemptive vaccination of populations at high risk of an epidemic or reactive vaccination shortly after onset of the epidemic. Second, populations experiencing epidemic cholera often have limited background natural immunity to cholera, whereas the recurrent nature of endemic cholera means that background immunity is common in affected populations. Therefore vaccines for epidemic cholera must be effective in immunologically naı¨ve individuals, whereas those for endemic cholera must work in the face of background immunity. Third, owing to these different patterns of background immunity, cholera is a greater problem in children than adults in endemic settings, whereas it tends to affect all age groups relatively equally in epidemics. Thus targeting young children and adolescents may be rational for controlling endemic cholera with vaccines, whereas a focus on all age groups will be necessary for vaccinating against epidemics. Finally, early onset of protection, preferably after the first dose of vaccine, is of great importance to the impact of using a vaccine in epidemic situations, whereas long-term duration of protection is less critical. The opposite applies for vaccines used in the control of endemic cholera.

VIII. WHO RECOMMENDATIONS AND A “GLOBAL ROADMAP TO END CHOLERA BY 2030” As early as in 1999 and again in 2002, there were recommendations from the WHO to consider the use of OCV together with standard water, hygiene, and sanitation methods for prevention of cholera in populations at high risk of cholera epidemics and to set up a vaccine stockpile for such use. However, it was not until 2010, after WHO recommendations for use of OCV were strengthened and extended to use of OCV in the control of both epidemic and endemic cholera, that member countries were encouraged to take action in their implementation. In 2013, the WHO established the Global OCV stockpile and received long-term support from Gavi, the Vaccine Alliance, for use of OCV in epidemic and endemic settings. Since then, the targeted use of the vaccine in endemic, epidemic, and humanitarian settings has increased dramatically under the guidance of the renovated Global Task Force on Cholera Control (GTFCC), which was renovated in 2014. In line with the latest WHO position paper on cholera vaccines from 2017 [30], many countries are now integrating the use of OCV within their cholera control programs. As of May 2018, over 25 million doses had been administered through mass vaccination campaigns in 19 countries since the stockpile was created in 2013. In October 2017, the GTFCC of the WHO, with support and commitment from more than 50 other signatory organizations and governments, launched a renewed strategy, “Ending Cholera—A Global Roadmap to 2030,” for cholera control to support countries in the implementation of cholera control plans, based on multisectoral interventions targeting cholera hotspots. It emphasizes that OCV is an important and, until recently, largely unused tool for both short-term and long-term cholera

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IX. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

prevention and control and presents a proposed multisectoral approach to use OCVs together with WaSH (clean water, improved sanitation and hygiene, improved surveillance and access to treatment) and other interventions for the control of both epidemic and endemic cholera [2]. By implementing the strategy between now and 2030, the more than 50 GTFCC partners have committed to supporting countries to reduce global cholera deaths by 90%. It is envisioned that with the commitment of cholera-affected countries, technical partners, and donors, as many as 20 countries could eliminate disease transmission by 2030.

IX. CONCLUDING REMARKS AND FUTURE PERSPECTIVES The development of OCVs is a good example of how basic research can generate new knowledge that can be translated into a life-saving medical product. At the same time, the OCV story also illustrates how long and tedious the process from product to public health use often is, especially for medical products targeted for use against diseases for which there is no or minimal commercial market in higher-income countries. Cholera is a typical disease of poverty, mainly affecting the poorest populations in low-income or at best middle-income countries. Had it not been for the fact that the first effective OCV, Dukoral, through its B subunit component, in addition to protecting against cholera also gives significant short-term protection against ETEC (traveler’s diarrhea) and thus could be marketed commercially as a traveler’s vaccine, the development of licensed effective OCVs would almost certainly have been delayed by many years. Even so, given the undiminished global cholera problem over the past 30 years and the availability of increasingly affordable OCVs with consistent protective effectiveness against

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both endemic cholera and cholera outbreaks in more than 15 large field trials in Asia, Africa, and Latin America, it is for most cholera experts frustrating that it has taken so long for OCVs to be used as an important public health tool in the control of cholera. As was discussed in section VII of this chapter, there were for many years concerns among parts of the WHO and the ORS and WaSH communities that providing OCV might have a negative impact on the implementation of oral rehydration treatment, WaSH activities, and other traditional cholera control interventions. It is only recently that it has become more fully understood that the real situation is rather the opposite: that OCV will likely increase the effectiveness of nonvaccine interventions and may likely synergize with WaSH-type interventions. There are now great hopes that the new insights and the new global commitment embodied in “Ending Cholera—A Global Roadmap to 2030” by the WHO and many partners will be a real game changer in the fight against cholera. Still, there are many challenges before its goals to reduce cholera deaths by 90% and eliminate cholera transmission in at least 20 countries will become a reality. Its success will require a sustained political will at all levels, adequate sustained financing for the whole period, a motivated global health community, and an effective research and development (R&D) program. For the R&D program as much as for the overall global roadmap, a multisectoral approach will be needed. Focusing on the OCVs, there is a need for much operational research to define and evaluate the best ways of using existing OCVs together with other tools in different settings. There is also a need for continued development of further improved OCVs. This includes, for example, development and evaluation of variants of the current types of OCVs with improvements in reduced cost of manufacturing or administration, heat-stable

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dry formulation capsule or tablet vaccines that would not need any cold chain at any stage, and OCVs with further improved immunogenicity and efficacy in young children, possibly requiring coadministration with an effective mucosal adjuvant. All these developments are in progress, but while their development is being supported, it is important not to let the search for the ideal be the enemy of the good but instead to let them proceed in parallel with the development of further improved tools maximize and optimize the use of the already existing effective OCVs and other tools in the public health fight against cholera.

References [1] Nair GB, Qadri F, Holmgren J, Svennerholm AM, Safa A, Bhuiyan NA, et al. Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh. J Clin Microbiol 2006;44:4211 13. [2] WHO Global Task Force for Cholera Control. Ending cholera A global road map to 2030; 2017. ,https:// www.who.int/cholera/publications/global-roadmap. pdf.. [3] Ali M, Nelson AR, Lopez AL, Sack DA. Updated global burden of cholera in endemic countries. PLoS Negl Trop Dis 2015;9:e0003832. [4] Mosley WH, Benenson AS, Barui R. A serological survey for cholera antibodies in rural east Pakistan. 2. A comparison of antibody titres in the immunized and control population of a cholera-vaccine field-trial area and the relation of antibody titre to cholera case rate. Bull World Health Organ 1968;38:335 46. [5] Deen JL, von Seidlein L, Sur D, Agtini M, Lucas ME, Lopez AL, et al. The high burden of cholera in children: comparison of incidence from endemic areas in Asia and Africa. PLoS Negl Trop Dis 2008;2:e173. [6] Legros D, McCormick M, Mugero C, Skinnider M, Bek’Obita DD, Okware SI. Epidemiology of cholera outbreak in Kampala, Uganda. East Afr Med J 2000;77: 347 9. [7] Clemens JD, Nair GB, Ahmed T, Qadri F, Holmgren J. Cholera. Lancet 2017;390:1539 49. [8] Piarroux R, Barrais R, Faucher B, Haus R, Piarroux M, Gaudart J, et al. Understanding the cholera epidemic, Haiti. Emerg Infect Dis 2011;17:1161 8.

[9] Ali M, Emch M, Park JK, Yunus M, Clemens J. Natural cholera infection-derived immunity in an endemic setting. J Infect Dis 2011;204:912 18. [10] Bi Q, Ferreras E, Pezzoli L, Legros D, Ivers LC, Date K, et al. Protection against cholera from killed whole-cell oral cholera vaccines: a systematic review and metaanalysis. Lancet Infect Dis 2017;17:1080 8. [11] Schwerdtle P, Onekon CK, Recoche K. A quantitative systematic review and meta-analysis of the effectiveness of oral cholera vaccine as a reactive measure in cholera outbreaks. Prehosp Disaster Med 2018;33:2 6. [12] Organization WH. Cholera vaccine: WHO position paper. Weekly Epidemiological Record/Health Section of Secretariat of the League of Nations 2010;85:117 28. [13] Organization WH. Meeting of the Strategic Advisory Group Experts and Immunization: Conclusions and Recommendations. Weekly Epidemiological Record/ Health Section of Secretariat of the League of Nations 2017;92:301 20. [14] Czerkinsky C, Holmgren J. Mucosal delivery routes for optimal immunization: targeting immunity to the right tissues. Curr Top Microbiol Immunol 2012;354:1 18. [15] Evans CA, Gilman RH, Rabbani GH, Salazar G, Ali A. Gastric acid secretion and enteric infection in Bangladesh. Trans R Soc Trop Med Hyg 1997;91:681 5. [16] Harris JB, Khan AI, LaRocque RC, Dorer DJ, Chowdhury F, Faruque AS, et al. Blood group, immunity, and risk of infection with Vibrio cholerae in an area of endemicity. Infect Immun 2005;73:7422 7. [17] Glass RI, Holmgren J, Haley CE, Khan MR, Svennerholm AM, Stoll BJ, et al. Predisposition for cholera of individuals with O blood group. Possible evolutionary significance. Am J Epidemiol 1985;121:791 6. [18] Clemens JD, Sack DA, Harris JR, Chakraborty J, Khan MR, Huda S, et al. ABO blood groups and cholera: new observations on specificity of risk and modification of vaccine efficacy. J Infect Dis 1989;159:770 3. [19] Flach CF, Qadri F, Bhuiyan TR, Alam NH, Jennische E, Lonnroth I, et al. Broad up-regulation of innate defense factors during acute cholera. Infect Immun 2007;75: 2343 50. [20] Qadri F, Bhuiyan TR, Dutta KK, Raqib R, Alam MS, Alam NH, et al. Acute dehydrating disease caused by Vibrio cholerae serogroups O1 and O139 induce increases in innate cells and inflammatory mediators at the mucosal surface of the gut. Gut 2004;53:62 9. [21] Qadri F, Raqib R, Ahmed F, Rahman T, Wenneras C, Das SK, et al. Increased levels of inflammatory mediators in children and adults infected with Vibrio cholerae O1 and O139. Clin Diagn Lab Immunol 2002;9:221 9. [22] Rabbani GH, Islam S, Chowdhury AK, Mitra AK, Miller MJ, Fuchs G. Increased nitrite and nitrate

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30] [31]

[32]

[33]

[34]

[35]

concentrations in sera and urine of patients with cholera or shigellosis. Am J Gastroenterol 2001;96:467 72. Mathan MM, Chandy G, Mathan VI. Ultrastructural changes in the upper small intestinal mucosa in patients with cholera. Gastroenterology 1995;109: 422 30. Shirin T, Rahman A, Danielsson A, Uddin T, Bhuyian TR, Sheikh A, et al. Antimicrobial peptides in the duodenum at the acute and convalescent stages in patients with diarrhea due to Vibrio cholerae O1 or enterotoxigenic Escherichia coli infection. Microbes Infect 2011;13: 1111 20. Shin OS, Uddin T, Citorik R, Wang JP, Della Pelle P, Kradin RL, et al. LPLUNC1 modulates innate immune responses to Vibrio cholerae. J Infect Dis 2011;204: 1349 57. Royaee AR, Mendis C, Das R, Jett M, Yang DC. Cholera toxin induced gene expression alterations. Mol Immunol 2006;43:702 9. Staats HF, Ennis Jr. FA. IL-1 is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens. J Immunol 1999; 162:6141 7. Larena M, Holmgren J, Lebens M, Terrinoni M, Lundgren A. Cholera toxin, and the related nontoxic adjuvants mmCT and dmLT, promote human Th17 responses via cyclic AMP-protein kinase A and inflammasome-dependent IL-1 signaling. J Immunol 2015;194:3829 39. Sun JB, Holmgren J, Larena M, Terrinoni M, Fang Y, Bresnick AR, et al. Deficiency in calcium-binding protein S100A4 impairs the adjuvant action of cholera toxin. Front Immunol 2017;8:1119. World Health Organization’s Cholera Position Paper. World Health Organization; 2017. Clemens JD, Sack DA, Harris JR, Chakraborty J, Khan MR, Stanton BF, et al. Field trial of oral cholera vaccines in Bangladesh. Lancet 1986;2:124 7. Clemens JD, Sack DA, Harris JR, Van Loon F, Chakraborty J, Ahmed F, et al. Field trial of oral cholera vaccines in Bangladesh: results from three-year follow-up. Lancet 1990;335:270 3. Lucas ME, Deen JL, von Seidlein L, Wang XY, Ampuero J, Puri M, et al. Effectiveness of mass oral cholera vaccination in Beira, Mozambique. N Engl J Med 2005;352:757 67. Sanchez JL, Vasquez B, Begue RE, Meza R, Castellares G, Cabezas C, et al. Protective efficacy of oral wholecell/recombinant-B-subunit cholera vaccine in Peruvian military recruits. Lancet 1994;344:1273 6. Clemens J, Shin S, Sur D, Nair GB, Holmgren J. Newgeneration vaccines against cholera. Nat Rev Gastroenterol Hepatol 2011;8:701 10.

557

[36] Holmgren J, Svennerholm AM. Vaccines against mucosal infections. Curr Opin Immunol 2012;24:343 53. [37] Thiem VD, Deen JL, von Seidlein L, Canh DG, Anh DD, Park JK, et al. Long-term effectiveness against cholera of oral killed whole-cell vaccine produced in Vietnam. Vaccine 2006;24:4297 303. [38] Bhattacharya SK, Sur D, Ali M, Kanungo S, You YA, Manna B, et al. 5 year efficacy of a bivalent killed whole-cell oral cholera vaccine in Kolkata, India: a cluster-randomised, double-blind, placebo-controlled trial. Lancet Infect Dis, 13. 2013. p. 1050 6. [39] Odevall L, Hong D, Digilio L, Sahastrabuddhe S, Mogasale V, Baik Y, et al. The Euvichol story Development and licensure of a safe, effective and affordable oral cholera vaccine through global public private partnerships. Vaccine 2018;36:6606 14. [40] Tacket CO, Cohen MB, Wasserman SS, Losonsky G, Livio S, Kotloff K, et al. Randomized, double-blind, placebo-controlled, multicentered trial of the efficacy of a single dose of live oral cholera vaccine CVD 103HgR in preventing cholera following challenge with Vibrio cholerae O1 El tor inaba three months after vaccination. Infect Immun 1999;67:6341 5. [41] a.Richie EE, Punjabi NH, Sidharta YY, Peetosutan KK, Sukandar MM, Wasserman SS, et al. Efficacy trial of single-dose live oral cholera vaccine CVD 103-HgR in North Jakarta, Indonesia, a cholera-endemic area. Vaccine 2000;18:2399 410.b. https://www.paxvaxconnect.com/vaxchora. [42] Svennerholm AM, Holmgren J. Synergistic protective effect in rabbits of immunization with Vibrio cholerae lipopolysaccharide and toxin/toxoid. Infect Immun 1976;13:735 40. [43] Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature 1981;292:413 17. [44] Holmgren J, Svennerholm AM. Cholera and the immune response. Prog Allergy 1983;33:106 19. [45] Lange S, Holmgren J. Protective antitoxic cholera immunity in mice: influence of route and number of immunizations and mode of action of protective antibodies. Acta Pathol Microbiol Scand C 1978;86C:145 52. [46] Svennerholm A, Lange S, Holmgren J. Correlation between intestinal synthesis of specific immunoglobulin A and protection against experimental cholera in mice. Infect Immun 1978;21:1 6. [47] Holmgren J, Svennerholm AM, Lonnroth I, FallPersson M, Markman B, Lundbeck H. Development of improved cholera vaccine based on subunit toxoid. Nature 1977;269:602 4. [48] Glass RI, Svennerholm AM, Stoll BJ, Khan MR, Hossain KM, Huq MI, et al. Protection against cholera in breast-fed children by antibodies in breast milk. N Engl J Med 1983;308:1389 92.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

558

31. CHOLERA IMMUNITY AND DEVELOPMENT AND USE OF ORAL CHOLERA VACCINES FOR DISEASE CONTROL

[49] Jertborn M, Svennerholm AM, Holmgren J. Saliva, breast milk, and serum antibody responses as indirect measures of intestinal immunity after oral cholera vaccination or natural disease. J Clin Microbiol 1986;24: 203 9. [50] Svennerholm AM, Jertborn M, Gothefors L, Karim AM, Sack DA, Holmgren J. Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a combined B subunit-whole cell vaccine. J Infect Dis 1984;149:884 93. [51] Jertborn M, Svennerholm AM, Holmgren J. Evaluation of different immunization schedules for oral cholera B subunit-whole cell vaccine in Swedish volunteers. Vaccine 1993;11:1007 12. [52] Jertborn M, Svennerholm AM, Holmgren J. Immunological memory after immunization with oral cholera B subunit whole-cell vaccine in Swedish volunteers. Vaccine 1994;12:1078 82. [53] Leach S, Lundgren A, Svennerholm AM. Different kinetics of circulating antibody-secreting cell responses after primary and booster oral immunizations: a tool for assessing immunological memory. Vaccine 2013;31: 3035 8. [54] Quiding M, Nordstrom I, Kilander A, Andersson G, Hanson LA, Holmgren J, et al. Intestinal immune responses in humans. Oral cholera vaccination induces strong intestinal antibody responses and interferongamma production and evokes local immunological memory. J Clin Invest 1991;88:143 8. [55] Jertborn M, Svennerholm AM, Holmgren J. Intestinal and systemic immune responses in humans after oral immunization with a bivalent B subunit-O1/O139 whole cell cholera vaccine. Vaccine 1996;14:1459 65. [56] Shamsuzzaman S, Ahmed T, Mannoor K, Begum YA, Bardhan PK, Sack RB, et al. Robust gut associated vaccine-specific antibody-secreting cell responses are detected at the mucosal surface of Bangladeshi subjects after immunization with an oral killed bivalent V. cholerae O1/O139 whole cell cholera vaccine: comparison with other mucosal and systemic responses. Vaccine 2009;27:1386 92. [57] Uddin T, Harris JB, Bhuiyan TR, Shirin T, Uddin MI, Khan AI, et al. Mucosal immunologic responses in cholera patients in Bangladesh. Clin Vaccine Immunol 2011;18:506 12. [58] Habtezion A, Nguyen LP, Hadeiba H, Butcher EC. Leukocyte trafficking to the small intestine and colon. Gastroenterology 2016;150:340 54. [59] Czerkinsky C, Prince SJ, Michalek SM, Jackson S, Russell MW, Moldoveanu Z, et al. IgA antibodyproducing cells in peripheral blood after antigen ingestion: evidence for a common mucosal immune system in humans. Proc Natl Acad Sci USA 1987;84:2449 53.

[60] Qadri F, Wenneras C, Albert MJ, Hossain J, Mannoor K, Begum YA, et al. Comparison of immune responses in patients infected with Vibrio cholerae O139 and O1. Infect Immun 1997;65:3571 6. [61] Rahman A, Rashu R, Bhuiyan TR, Chowdhury F, Khan AI, Islam K, et al. Antibody-secreting cell responses after Vibrio cholerae O1 infection and oral cholera vaccination in adults in Bangladesh. Clin Vaccine Immunol 2013;20:1592 8. [62] Leung DT, Rahman MA, Mohasin M, Riyadh MA, Patel SM, Alam MM, et al. Comparison of memory B cell, antibody-secreting cell, and plasma antibody responses in young children, older children, and adults with infection caused by Vibrio cholerae O1 El Tor Ogawa in Bangladesh. Clin Vaccine Immunol 2011;18:1317 25. [63] Czerkinsky C, Svennerholm AM, Quiding M, Jonsson R, Holmgren J. Antibody-producing cells in peripheral blood and salivary glands after oral cholera vaccination of humans. Infect Immun 1991;59:996 1001. [64] Saletti G, Cuburu N, Yang JS, Dey A, Czerkinsky C. Enzyme-linked immunospot assays for direct ex vivo measurement of vaccine-induced human humoral immune responses in blood. Nat Protoc 2013;8:1073 87. [65] Carpenter CM, Hall ER, Randall R, McKenzie R, Cassels F, Diaz N, et al. Comparison of the antibody in lymphocyte supernatant (ALS) and ELISPOT assays for detection of mucosal immune responses to antigens of enterotoxigenic Escherichia coli in challenged and vaccinated volunteers. Vaccine 2006;24:3709 18. [66] Chang HS, Sack DA. Development of a novel in vitro assay (ALS assay) for evaluation of vaccine-induced antibody secretion from circulating mucosal lymphocytes. Clin Diagn Lab Immunol 2001;8:482 8. [67] Lundgren A, Bourgeois L, Carlin N, Clements J, Gustafsson B, Hartford M, et al. Safety and immunogenicity of an improved oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine administered alone and together with dmLT adjuvant in a double-blind, randomized, placebo-controlled Phase I study. Vaccine 2014;32:7077 84. [68] Qadri F, Ryan ET, Faruque AS, Ahmed F, Khan AI, Islam MM, et al. Antigen-specific immunoglobulin A antibodies secreted from circulating B cells are an effective marker for recent local immune responses in patients with cholera: comparison to antibody-secreting cell responses and other immunological markers. Infect Immun 2003;71:4808 14. [69] Datta SK, Sabet M, Nguyen KP, Valdez PA, GonzalezNavajas JM, Islam S, et al. Mucosal adjuvant activity of cholera toxin requires Th17 cells and protects against inhalation anthrax. Proc Natl Acad Sci USA 2010;107: 10638 43.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

559

REFERENCES

[70] Velaga S, Herbrand H, Friedrichsen M, Jiong T, Dorsch M, Hoffmann MW, et al. Chemokine receptor CXCR5 supports solitary intestinal lymphoid tissue formation, B cell homing, and induction of intestinal IgA responses. J Immunol 2009;182:2610 19. [71] Bhuiyan TR, Lundin SB, Khan AI, Lundgren A, Harris JB, Calderwood SB, et al. Cholera caused by Vibrio cholerae O1 induces T-cell responses in the circulation. Infect Immun 2009;77:1888 93. [72] Kuchta A, Rahman T, Sennott EL, Bhuyian TR, Uddin T, Rashu R, et al. Vibrio cholerae O1 infection induces proinflammatory CD4 1 T-cell responses in blood and intestinal mucosa of infected humans. Clin Vaccine Immunol 2011;18:1371 7. [73] Weil AA, Arifuzzaman M, Bhuiyan TR, LaRocque RC, Harris AM, Kendall EA, et al. Memory T-cell responses to Vibrio cholerae O1 infection. Infect Immun 2009;77: 5090 6. [74] Ahmed T, Arifuzzaman M, Lebens M, Qadri F, Lundgren A. CD4 1 T-cell responses to an oral inactivated cholera vaccine in young children in a cholera endemic country and the enhancing effect of zinc supplementation. Vaccine 2009;28:422 9. [75] Losonsky GA, Yunyongying J, Lim V, Reymann M, Lim YL, Wasserman SS, et al. Factors influencing secondary vibriocidal immune responses: relevance for understanding immunity to cholera. Infect Immun 1996;64:10 15. [76] Majumdar AS, Ghose AC. Evaluation of the biological properties of different classes of human antibodies in relation to cholera. Infect Immun 1981;32:9 14. [77] Neoh SH, Rowley D. The antigens of Vibrio cholerae involved in the vibriocidal action of antibody and complement. J Infect Dis 1970;121:505 13. [78] Mosley WH, Ahmad S, Benenson AS, Ahmed A. The relationship of vibriocidal antibody titre to susceptibility to cholera in family contacts of cholera patients. Bull World Health Organ 1968;38:777 85. [79] Clements ML, Levine MM, Young CR, Black RE, Lim YL, Robins-Browne RM, et al. Magnitude, kinetics, and duration of vibriocidal antibody responses in North Americans after ingestion of Vibrio cholerae. J Infect Dis 1982;145:465 73. [80] Harris AM, Bhuiyan MS, Chowdhury F, Khan AI, Hossain A, Kendall EA, et al. Antigen-specific memory B-cell responses to Vibrio cholerae O1 infection in Bangladesh. Infect Immun 2009;77:3850 6. [81] Glass RI, Svennerholm AM, Khan MR, Huda S, Huq MI, Holmgren J. Seroepidemiological studies of El Tor cholera in Bangladesh: association of serum antibody levels with protection. J Infect Dis 1985;151:236 42. [82] Saha D, LaRocque RC, Khan AI, Harris JB, Begum YA, Akramuzzaman SM, et al. Incomplete correlation of

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

serum vibriocidal antibody titer with protection from Vibrio cholerae infection in urban Bangladesh. J Infect Dis 2004;189:2318 22. Chowdhury F, Khan AI, Harris JB, LaRocque RC, Chowdhury MI, Ryan ET, et al. A comparison of clinical and immunologic features in children and older patients hospitalized with severe cholera in Bangladesh. Pediatr Infect Dis J 2008;27:986 92. Clemens JD, Stanton BF, Chakraborty J, Sack DA, Khan MR, Huda S, et al. B subunit-whole cell and whole cell-only oral vaccines against cholera: studies on reactogenicity and immunogenicity. J Infect Dis 1987;155:79 85. Sack DA, Clemens JD, Huda S, Harris JR, Khan MR, Chakraborty J, et al. Antibody responses after immunization with killed oral cholera vaccines during the 1985 vaccine field trial in Bangladesh. J Infect Dis 1991; 164:407 11. Saha A, Chowdhury MI, Khanam F, Bhuiyan MS, Chowdhury F, Khan AI, et al. Safety and immunogenicity study of a killed bivalent (O1 and O139) wholecell oral cholera vaccine Shanchol, in Bangladeshi adults and children as young as 1 year of age. Vaccine 2011;29:8285 92. Qadri F, Chowdhury MI, Faruque SM, Salam MA, Ahmed T, Begum YA, et al. Peru-15, a live attenuated oral cholera vaccine, is safe and immunogenic in Bangladeshi toddlers and infants. Vaccine 2007;25: 231 8. Kanungo S, Paisley A, Lopez AL, Bhattacharya M, Manna B, Kim DR, et al. Immune responses following one and two doses of the reformulated, bivalent, killed, whole-cell, oral cholera vaccine among adults and children in Kolkata, India: a randomized, placebocontrolled trial. Vaccine., 27. 2009. p. 6887 93. Su-Arehawaratana P, Singharaj P, Taylor DN, Hoge C, Trofa A, Kuvanont K, et al. Safety and immunogenicity of different immunization regimens of CVD 103-HgR live oral cholera vaccine in soldiers and civilians in Thailand. J Infect Dis 1992;165:1042 8. Taylor DN, Cardenas V, Perez J, Puga R, Svennerholm AM. Safety, immunogenicity, and lot stability of the whole cell/recombinant B subunit (WC/rCTB) cholera vaccine in Peruvian adults and children. Am J Trop Med Hyg 1999;61:869 73. Mahalanabis D, Lopez AL, Sur D, Deen J, Manna B, Kanungo S, et al. A randomized, placebo-controlled trial of the bivalent killed, whole-cell, oral cholera vaccine in adults and children in a cholera endemic area in Kolkata, India. PLoS One 2008;3:e2323. Stiehm ER, Fudenberg HH. Serum levels of immune globulins in health and disease: a survey. Pediatrics 1966;37:715 27.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

560

31. CHOLERA IMMUNITY AND DEVELOPMENT AND USE OF ORAL CHOLERA VACCINES FOR DISEASE CONTROL

[93] Harris JB, LaRocque RC, Chowdhury F, Khan AI, Logvinenko T, Faruque AS, et al. Susceptibility to Vibrio cholerae infection in a cohort of household contacts of patients with cholera in Bangladesh. PLoS Negl Trop Dis 2008;2:e221. [94] Chowdhury F, Rahman MA, Begum YA, Khan AI, Faruque AS, Saha NC, et al. Impact of rapid urbanization on the rates of infection by Vibrio cholerae O1 and enterotoxigenic Escherichia coli in Dhaka, Bangladesh. PLoS Negl Trop Dis 2011;5:e999. [95] Smith NW, Sack RB. Immunologic cross-reactions of enterotoxins from Escherichia coli and Vibrio cholerae. J Infect Dis 1973;127:164 70. [96] Qadri F, Ahmed T, Wahed MA, Ahmed F, Bhuiyan NA, Rahman AS, et al. Suppressive effect of zinc on antibody response to cholera toxin in children given the killed, B subunit-whole cell, oral cholera vaccine. Vaccine 2004;22:416 21. [97] Ahmed T, Svennerholm AM, Al Tarique A, Sultana GN, Qadri F. Enhanced immunogenicity of an oral inactivated cholera vaccine in infants in Bangladesh obtained by zinc supplementation and by temporary withholding breast-feeding. Vaccine 2009;27:1433 9. [98] Qadri F, Mohi G, Hossain J, Azim T, Khan AM, Salam MA, et al. Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1. Clin Diagn Lab Immunol 1995;2:685 8. [99] Qadri F, Chowdhury MI, Faruque SM, Salam MA, Ahmed T, Begum YA, et al. Randomized, controlled study of the safety and immunogenicity of Peru-15, a live attenuated oral vaccine candidate for cholera, in adult volunteers in Bangladesh. J Infect Dis 2005;192: 573 9. [100] Clutterbuck EA, Oh S, Hamaluba M, Westcar S, Beverley PC, Pollard AJ. Serotype-specific and age-dependent generation of pneumococcal polysaccharide-specific memory B-cell and antibody responses to immunization with a pneumococcal conjugate vaccine. Clin Vaccine Immunol 2008;15:182 93. [101] Alam MM, Riyadh MA, Fatema K, Rahman MA, Akhtar N, Ahmed T, et al. Antigen-specific memory Bcell responses in Bangladeshi adults after one- or twodose oral killed cholera vaccination and comparison with responses in patients with naturally acquired cholera. Clin Vaccine Immunol 2011;18:844 50. [102] Qadri F, Ahmed F, Karim MM, Wenneras C, Begum YA, Abdus Salam M, et al. Lipopolysaccharide- and cholera toxin-specific subclass distribution of B-cell responses in cholera. Clin Diagn Lab Immunol 1999;6: 812 18. [103] Boes M. Role of natural and immune IgM antibodies in immune responses. Mol Immunol 2000;37:1141 9.

[104] Kendall EA, Tarique AA, Hossain A, Alam MM, Arifuzzaman M, Akhtar N, et al. Development of immunoglobulin M memory to both a T-cellindependent and a T-cell-dependent antigen following infection with Vibrio cholerae O1 in Bangladesh. Infect Immun 2010;78:253 9. [105] Mond JJ, Vos Q, Lees A, Snapper CM. T cell independent antigens. Curr Opin Immunol 1995;7:349 54. [106] Kelly DF, Pollard AJ, Moxon ER. Immunological memory: the role of B cells in long-term protection against invasive bacterial pathogens. JAMA 2005;294: 3019 23. [107] Crotty S, Aubert RD, Glidewell J, Ahmed R. Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system. J Immunol Methods 2004;286:111 22. [108] Levine MM. Immunogenicity and efficacy of oral vaccines in developing countries: lessons from a live cholera vaccine. BMC Biol 2010;8:129. [109] Saha DR, Rajendran K, Ramamurthy T, Nandy RK, Bhattacharya SK. Intestinal parasitism and Vibrio cholerae infection among diarrhoeal patients in Kolkata, India. Epidemiol Infect 2008;136:661 4. [110] Cooper PJ, Chico ME, Losonsky G, Sandoval C, Espinel I, Sridhara R, et al. Albendazole treatment of children with ascariasis enhances the vibriocidal antibody response to the live attenuated oral cholera vaccine CVD 103-HgR. J Infect Dis 2000;182:1199 206. [111] Harris JB, Podolsky MJ, Bhuiyan TR, Chowdhury F, Khan AI, Larocque RC, et al. Immunologic responses to Vibrio cholerae in patients co-infected with intestinal parasites in Bangladesh. PLoS Negl Trop Dis 2009;3: e403. [112] Chowdhury F, Begum YA, Alam MM, Khan AI, Ahmed T, Bhuiyan MS, et al. Concomitant enterotoxigenic Escherichia coli infection induces increased immune responses to Vibrio cholerae O1 antigens in patients with cholera in Bangladesh. Infect Immun 2010;78:2117 24. [113] Larocque RC, Sabeti P, Duggal P, Chowdhury F, Khan AI, Lebrun LM, et al. A variant in long palate, lung and nasal epithelium clone 1 is associated with cholera in a Bangladeshi population. Genes Immun 2009;10:267 72. [114] Barry WS, Pierce NF. Protein deprivation causes reversible impariment of mucosal immune response to cholera toxoid/toxin in rat gut. Nature 1979;281:64 5. [115] Flo J, Roux ME, Massouh E. Deficient induction of the immune response to oral immunization with cholera toxin in malnourished rats during suckling. Infect Immun 1994;62:4948 54. [116] Guerrant RL, Oria RB, Moore SR, Oria MO, Lima AA. Malnutrition as an enteric infectious disease with

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

[117]

[118]

[119]

[120]

[121]

[122]

[123]

[124]

[125]

[126]

long-term effects on child development. Nutr Rev 2008;66:487 505. Baqui AH, Black RE, Arifeen SE, Hill K, Mitra SN, al Sabir A. Causes of childhood deaths in Bangladesh: results of a nationwide verbal autopsy study. Bull World Health Organ 1998;76:161 71. Black RE, Allen LH, Bhutta ZA, Caulfield LE, de Onis M, Ezzati M, et al. Maternal and child undernutrition: global and regional exposures and health consequences. Lancet 2008;371:243 60. Palmer DL, Koster FT, Alam AK, Islam MR. Nutritional status: a determinant of severity of diarrhea in patients with cholera. J Infect Dis 1976;134:8 14. Pino-Lagos K, Guo Y, Brown C, Alexander MP, Elgueta R, Bennett KA, et al. A retinoic aciddependent checkpoint in the development of CD4 1 T cell-mediated immunity. J Exp Med 2011;208: 1767 75. Sirisinha S. The pleiotropic role of vitamin A in regulating mucosal immunity. Asian Pac J Allergy Immunol 2015;33:71 89. Cassani B, Villablanca EJ, De Calisto J, Wang S, Mora JR. Vitamin A and immune regulation: role of retinoic acid in gut-associated dendritic cell education, immune protection and tolerance. Mol Aspects Med 2012;33: 63 76. Wiedermann U, Hanson LA, Holmgren J, Kahu H, Dahlgren UI. Impaired mucosal antibody response to cholera toxin in vitamin A-deficient rats immunized with oral cholera vaccine. Infect Immun 1993;61: 3952 7. Mayo-Wilson E, Imdad A, Herzer K, Yakoob MY, Bhutta ZA. Vitamin A supplements for preventing mortality, illness, and blindness in children aged under 5: systematic review and meta-analysis. BMJ 2011;343:d5094. Albert MJ, Qadri F, Wahed MA, Ahmed T, Rahman AS, Ahmed F, et al. Supplementation with zinc, but not vitamin A, improves seroconversion to vibriocidal antibody in children given an oral cholera vaccine. J Infect Dis 2003;187:909 13. Aggarwal R, Sentz J, Miller MA. Role of zinc administration in prevention of childhood diarrhea and respiratory illnesses: a meta-analysis. Pediatrics 2007;119: 1120 30.

561

[127] Roy SK, Hossain MJ, Khatun W, Chakraborty B, Chowdhury S, Begum A, et al. Zinc supplementation in children with cholera in Bangladesh: randomised controlled trial. BMJ 2008;336:266 8. [128] Sheikh A, Shamsuzzaman S, Ahmad SM, Nasrin D, Nahar S, Alam MM, et al. Zinc influences innate immune responses in children with enterotoxigenic Escherichia coli-induced diarrhea. J Nutr 2010;140: 1049 56. [129] Bhattacharya S, Black R, Bourgeois L, Clemens J, Cravioto A, Deen JL, et al. Public health. The cholera crisis in Africa. Science 2009;324:885. [130] Clemens J, Holmgren J. Urgent need of cholera vaccines in public health-control programs. Future Microbiol 2009;4:381 5. [131] Ali M, Emch M, von Seidlein L, Yunus M, Sack DA, Rao M, et al. Herd immunity conferred by killed oral cholera vaccines in Bangladesh: a reanalysis. Lancet 2005;366:44 9. [132] Ali M, Sur D, You YA, Kanungo S, Sah B, Manna B, et al. Herd protection by a bivalent killed whole-cell oral cholera vaccine in the slums of Kolkata, India. Clin Infect Dis 2013;56:1123 31. [133] Khatib AM, Ali M, von Seidlein L, Kim DR, Hashim R, Reyburn R, et al. Effectiveness of an oral cholera vaccine in Zanzibar: findings from a mass vaccination campaign and observational cohort study. Lancet Infect Dis 2012;12:837 44. [134] Longini Jr. IM, Nizam A, Ali M, Yunus M, Shenvi N, Clemens JD. Controlling endemic cholera with oral vaccines. PLoS Med 2007;4:e336. [135] Estupinan-Day S, Lafontant C, Acuna MC. Integrating oral health into Haiti’s National Health Plan: from disaster relief to sustainable development. Rev Panam Salud Publica 2011;30:484 9. [136] Qadri F, Islam T, Clemens JD. Cholera in Yemen an old foe rearing its ugly head. N Engl J Med 2017;377: 2005 7. [137] Qadri F, Azad AK, Flora MS, Khan AI, Islam MT, Nair GB, et al. Emergency deployment of oral cholera vaccine for the Rohingya in Bangladesh. Lancet 2018; 391:1877 9. [138] Clemens J, Holmgren J. When, how, and where can oral cholera vaccines be used to interrupt cholera outbreaks? Curr Top Microbiol Immunol 2014;379:231 58.

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Oral Vaccines for Enterotoxigenic Escherichia coli Nils Carlin1 and Ann-Mari Svennerholm2 1

Scandinavian Biopharma, Solna, Sweden 2Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

I. INTRODUCTION Intense efforts are in progress in many laboratories and the vaccine industry to develop an effective vaccine against the most common bacterial cause of diarrhea, that is, enterotoxigenic Escherichia coli (ETEC), in children in developing countries and in travelers to these areas. ETEC colonizes the intestinal mucosa by different specific colonization factors (CFs) and possibly additional conserved antigens located on the surface of the bacteria. Following colonization, the bacteria produce a heat-labile enterotoxin (LT) and/or a heat-stable toxin (ST) that cause watery diarrhea [1,2]. The lack of a licensed human ETEC vaccine so far is partly due to the fact that ETEC causing disease in humans is very heterogeneous with more than 70 different O antigens, at least 25 different CFs, and production of two different toxins alone or in combination [1 3]. However, epidemiological studies have confirmed that only a few ETEC lineages predominate worldwide

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00032-8

and over time and that only a few CFs (CFA/I, CS1-CS6, CS14, CS17, and CS7) are found on a majority of clinical ETEC isolates [1,4]. There is strong evidence that it will be possible to develop an effective ETEC vaccine. Both ETEC infection and immunization with candidate vaccines have been shown to induce protection against reinfection with related ETEC strains in animal models as well as in humans and to induce mucosal immune responses against CFs and LT, which are considered important for protection [2,5,6]. An effective ETEC vaccine, particularly for use in ETEC-endemic countries, not only should induce acute, effective immune responses against the most prevalent types of ETEC, but also should give rise to long-lasting immunological memory that can be boosted by repeated vaccination given several years after the initial vaccination. In this chapter, we describe the identification of putative protective ETEC antigens and recombinant strains and suitable administration routes for the development of promising

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ETEC vaccine candidates as well as clinical trials of such vaccines in challenge models and in ETEC-endemic areas.

II. ENTEROTOXIGENIC ESCHERICHIA COLI VACCINE CANDIDATES A. Fimbrial Antigens and Heat-Labile Enterotoxin Are Major Protective Antigens 1. Studies in Animals In early preclinical studies, we showed that antibodies against LT and different CFs provided significant passive protection against challenge with ETEC expressing LT and homologous CFs and that these antibody specificities cooperated synergistically for protection in rabbit ileal loops [7]. In subsequent studies in a nonligated rabbit model, we demonstrated that an initial oral infection with CF-positive ETEC protected against rechallenge with heterologous, virulent ETEC strains expressing the same CF as the infecting strain [8]. 2. Studies in Humans Levine and coworkers showed that human volunteers fed with a single dose of a nontoxigenic E. coli strain expressing the CFs CS1 1 CS3 were protected against challenge with a heterologous ETEC strain producing ST and LT and expressing TABLE 32.1

CS1 1 CS3, indicating that the CFs of the infecting strain were protective [9] (Table 32.1). Further clinical support for the protective capacity of ETEC CFs are results from a birth cohort study in Bangladesh showing that children with an initial ETEC infection were protected against reinfection with ETEC expressing homologous CFs during their first 2 years of life [6]. Clinical evidence for the protective efficacy of anti-LT antibodies comes from two different studies with Dukoral, an oral cholera vaccine containing whole cell inactivated Vibrio cholerae bacteria and recombinant cholera toxin B subunit (rCTB) (Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control). The B subunit of E. coli LT (LTB) has more than 85% homology with CTB; hence anti-CTB antibodies cross-react with LT [10,13]. In an endemic setting, Clemens and coworkers [10] described that the CTBcontaining cholera vaccine not only protected against cholera, but also conferred crossprotection (approximately 60%) against LT-onlyand LT/ST-expressing E. coli; similar results were found in a study of Finnish travelers to Morocco [11] (Table 32.1). The first evidence for the potential of a killed whole cell ETEC vaccine was the work of Evans and coworkers, who showed protection in a human challenge study using colicin E2 killed E. coli bacteria as an oral immunogen followed by challenge with the same live strain and later also a strain with heterologous O antigen [14,15].

Proof of Concept Clinical Trials

Vaccine/antibodies

Type of protection

Efficacy

Readout

Live attenuated E1392-75-2A CS1 1 CS3 LT2ST2 O6:H16

Challenge, virulent CS1 1 CS3 LT1, ST1 O139:H28

PE: 75%

CFs are protective antigens in [9] live attenuated vaccines

Dukoral, whole cell cholera vaccine with 1 mg of rCTB

Field trials, Bangladesh (endemic) and Morocco (travelers)

PE:  60% in Antibodies to LT toxin B both trials subunit are protective

[10,11]

Bovine colostrum raised against CFA/E tip adhesion

Passive protection against challenge with virulent H10407

PE: 63%

[12]

Tip adhesin is a protective component in CFA/I

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References

III. EVALUATION OF OPTIMAL ADMINISTRATION ROUTES OF AN ENTEROTOXIGENIC ESCHERICHIA COLI VACCINE

B. Structure of the Major Putative Protective Enterotoxigenic Escherichia coli Antigens: Colonization Factor Antigens and Enterotoxins 1. Colonization Factors Most ETEC CFs are plasmid encoded; the operons are usually composed of genes encoding one or two structural subunits, a chaperone, and an usher. The vast majority of CFs involved in human disease are of the α-fimbrial clade, also known as the CFA/I family [16,17]. Structurally the fimbriae are built by the alternate chaperone/usher pathway, and the fimbrial shaft is built up by multiple copies of the major subunit, which in its distal end carries a tip adhesin [18]. Members of the CFA/I family include CFA/I, CS1, CS2, CS4, CS5, CS14, CS17, CS19, and PFC071. CS3 and CS6 fimbriae commonly expressed by clinical ETEC isolates belong to the γ3 family. CS6 assembles into nonfimbrial structures that are thought to be made up of fibrillae that are too thin to be resolved by electron microscopy; the two subunits that build up the CS6 structure are in a 1:1 ratio, thereby differing from most other chaperone/ usher fimbrial structures [19] The CS3 fibrillae assemble in structures that can be seen in electron microscopy [18]. 2. Heat-Labile Enterotoxin (LT) The LT of ETEC is built up by the classical 5B 1 1A subunit structure, in which the five B subunits forms a pentameric structure that can bind to its cellular receptor, the GM1 ganglioside, whereas the enzymatically active A subunit, which is noncovalently associated with the pentameric B subunit, exhibits the toxicity [13,20]. Induction of intestinal fluid secretion occurs after a series of events involving both changes of toxin structure and activation of intracellular signaling pathways, which lead to excretion of fluid and electrolytes [21]. The B subunit is highly immunogenic, and anti-LTB

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antibodies are protective; data are emerging that the A subunit may also induce protective antibodies [22]. 3. ST Heat-Stable enterotoxin E. coli ST exists in two forms: the human STh and the porcine STp. The molecule consists, in its mature form of 18 (STp) or 19 (STh) amino acids tightly bound by three internal cysteine bridges [23,24]. The molecule itself is too small to evoke an immune response and is also highly enterotoxic. Toxicity is exerted by binding to an extracellular domain of guanylyl cyclase C (GCC) on the apical surface of intestinal epithelial cells [25]. This in turn activates the intracellular part of GC-C, which converts GTP to the intracellular messenger cGMP, starting a cascade that ultimately leading to diarrhea. 4. O Antigens The carbohydrate moiety of the smooth lipopolysaccharide (LPS) molecule mediating the serotype is linked to the lipid A molecule, anchoring the LPS to the outer membrane of Gram-negative bacteria [26]. LPS or O antigens have been shown to mediate protection in numerous Gram-negative infections. The plethora of different E. coli serotypes involved in ETEC infections, since more 70 different O groups have been identified in clinical ETEC isolates [27], makes the development of a vaccine based on O antigens with meaningful coverage impossible.

III. EVALUATION OF OPTIMAL ADMINISTRATION ROUTES OF AN ENTEROTOXIGENIC ESCHERICHIA COLI VACCINE Since both the ETEC bacteria and the toxins they produce are confined to the small intestinal mucosa during infection, locally produced antibodies in the intestine seem to be of prime

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importance for protection [28]. Such antibodies are optimally produced by repeated oral antigen administration [29]. However, in mucosally primed individuals, for example, those natural primed with enteric pathogens, a parenteral vaccine may also elicit mucosal immune responses [30]. Other routes that have been suggested to stimulate intestinal immunity are the transcutaneous route, which has been reported to induce mucosal immune responses [31], or the sublingual route, which has shown promise in animals [32], although no successful example has yet been described for humans. Intranasal immunization with LT or LTB [33] and numerous other antigens and vaccines or vaccine candidates has been reported to induce mucosal immune responses in the intestine of mice. However, repeated intranasal administration of comparatively high doses of CTB failed to induce a significant antibody-secreting cell (ASC) response locally in the stomach and intestine, as tested in gastric and duodenal biopsies from vaccinated human volunteers [34]. Hence most efforts to date are focused on the development of oral ETEC vaccines or oral feeding of putative protective ETEC antibodies.

A. Passive Protection Trials in Humans to Get Proof of Concept Oral feeding with specific colostrum antibodies to healthy volunteers, followed by challenge with a well-defined inoculum of a virulent challenge strain, has been used in different ETEC challenge experiments [35]. Usually, the volunteers ingest gram quantities of antibodies together with an antacid, starting 2 7 days before and during challenge, and continue this regimen during the evaluation phase. Recently, Savarino et al. showed that antibodies raised against CFA/I as well as the recombinant tip adhesin CfaE of CFA/I fimbriae were protective against challenge with a virulent ETEC CFA/I-positive strain [12]. The value of

orally transferred antibodies as an alternative to active immunization remains to be shown. Ingestion of relatively large amounts of antibodies three times per day together with an antacid could increase the risk for infection with other enteric pathogens. Enteric coating of the antibodies, eliminating the need for a neutralizing buffer, might be an alternative, but conflicting results have been seen in passive protection trials [36,37].

B. Candidate Vaccines in Preclinical Phase A number of different antigens and microbial candidate vaccines, such as attenuated strains or recombinant bacteria, have been suggested as candidate vaccines on the basis of encouraging preclinical studies. Some of those are described below (Table 32.2). 1. Recombinant Attenuated Bacteria A live vaccine candidate consisting of GuaBA attenuated Shigella strains expressing recombinant CF antigens and LTB has been developed at the Center for Vaccine Development, University of Maryland. The aim is to produce an oral vaccine with dual protection against both Shigella and ETEC. A vaccine consisting of Shigella flexneri 2a (CVD1208) expressing CFA/I and CS3, Shigella sonnei expressing CS4 and LTK63, and Shigella dysenteriae type 1 expressing CS2 has been tested in guinea pigs. Antibody responses against all antigens were shown [40], but no evidence of protection against ETEC has been reported. A company Protein Potential in Rockville, Maryland, is developing an oral live attenuated vaccine based on the Salmonella typhi Ty21a strain, licensed as a typhoid vaccine in many countries [61]. The company has, by chromosomal integration, included the genes for the University of Kansas MEFA proteins (see below

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III. EVALUATION OF OPTIMAL ADMINISTRATION ROUTES OF AN ENTEROTOXIGENIC ESCHERICHIA COLI VACCINE

TABLE 32.2

567

Vaccine Candidates in Development

Type of studies

Preclinical

Live attenuated whole cell vaccines

Phase 1

CHIM

ACE527 [38]

ACE527 [39]

Phase Phase 2 2b

Attenuated Shigella strains expressing CFAs and LTB [40] Salmonella typhi Ty21a expressing MEFA antigens [41] Inactivated whole cell vaccine

ETVAX [42,43]

Subunit vaccine candidates

CfaE/CfaE-LTB chimera 1 mLT (transcutaneous vs intradermal) [46]

ETVAX ETVAX [44] [45] CfaE mLT ID [46]

dmLT [47] ST analogs linked to LTB [48] Yghj (SslE) [49 51] EtpA [52 55] EatA [56] FliC [53,57] EaeH [57,58] LTA [22] MEFA [59,60]

in the discussion of multiepitope protein antigens) in what is called Ty21a-ETEC-MEFA [41] 2. Purified Antigens Several proteins/antigens to be used in subunit vaccines for parenteral and/or mucosal administration have been proposed: 1. ST. The prospect of having a vaccine based on the LT and ST toxins has the appealing prospect of covering all ETEC isolates irrespective of CFs or O antigens. Considerable work has been done to mutate the ST molecule to abolish the toxicity or to produce atoxic recombinant or synthetic ST

molecules [62,63] and to make the molecule immunogenic by coupling it with a larger immunogenic carrier protein (mutated LT or LTB; CFA/I or BSA [64], reviewed in Ref. [48]). The close similarity of both STh and STp to human guanylin and uroguanylin has raised concerns that anti-ST antibodies could react with guanylin, resulting in autoimmunity [25,65,66]. The induction of such potential cross-reacting antibodies will in all likelihood require an extensive safety investigation if a clinically viable ST toxoid is developed. Furthermore, the number of ST molecules produced in comparison to LT and the molar ratio of secretory

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2.

3.

4.

5.

6.

32. ORAL VACCINES FOR ENTEROTOXIGENIC ESCHERICHIA COLI

immunoglobulin A (SIgA) molecules present in the human gut have raised the question of whether it is possible for enough local SIgA antibodies to be produced in the gut to neutralize the ST toxin that is produced [67]. Yghj or SslE (secreted and surface associated lipoprotein) is a cell-surface-associated and secreted lipoprotein. However, SslE has been shown to be present in enteropathogenic, septicemic, and commensal strains of E. coli [49 51], casting some doubts on whether it is a credible vaccine candidate [49]. The EtpA adhesin was identified by Fleckenstein and coworkers together with the EatA serine protease by transposon mutagenesis of the large 92-kilobase virulence plasmid of the ETEC H10407 strain [52]. EtpA has been shown to mediate adhesion between flagellae and the epithelial cells in the gut mucosa [53], to be immunogenic [54], and to induce protection when given intranasally in a mouse model of ETEC infection [55]. EatA is an autotransporter and can modulate adherence by digesting EtpA, but it is also a mucinase [56]. Mice vaccinated intranasally with recombinant EatA combined with LT demonstrated significantly reduced colonization of the small intestine when challenged with live ETEC bacteria. The flagellin (fliC) of E. coli has also been implicated as a putative vaccine antigen [53] either alone or in conjunction with EtpA. The flagellin is present in both commensal and pathogenic E. coli [57]. The EaeH gene was originally identified in an ETEC strain by subtractive hybridization with a laboratory strain of E. coli. The gene is upregulated when in contact with epithelial cells. The gene product is a surfaceexpressed protein; however, it is barely detectable if not cocultured with epithelial cells or supernatants from epithelial cells. Under permissive conditions, it promotes

binding to epithelial cells in vitro and plays an active part in the attachment of ETEC to the gut mucosa in a mouse model [58]. EaeH is present in different E. coli pathotypes, including uropathogenic E. coli, but also in commensal strains [57], which might have implications for the potential as a vaccine antigen. 7. LTA. Also the A subunit of E. coli LT has been suggested as a protective antigen by Norton et al. [22]. They investigated the presence of anti-A subunit antibodies in sera from challenged volunteers and patients with natural infection. High titers against both the A and B subunits of LT were found. Antisera raised against recombinant A and B subunits were shown to neutralize toxin in an in vitro cAMP assay. These data have been suggested by the investigators to support inclusion of an A subunit component to increase the antitoxin response in a putative oral ETEC vaccine.

C. Multiepitope Protein Antigens Several groups have used bioinformatics to link epitopes of fimbrial adhesins together with epitopes of toxins and/or other surface exposed proteins for use as parenteral vaccines [59,68]. Zeinalzadeh et al. used bioinformatics to design a multiepitope fusion antigen (MEFA) consisting of epitopes of CfaB (major subunit in CFA/I fimbriae), heat-stable toxoid, CssA, CssB (structural genes for CS6), and LTB subunit, thus covering CFA/I-, CS6-, LT-, and ST-producing strains. The chimeric protein was shown to be immunogenic when given parentally to mice; antisera induced by the protein conferred protection against ST fluid secretion in the suckling mouse model and also protected against intraperitoneal challenge with virulent ETEC [60]. The most advanced MEFA vaccine candidate, consisting of a protein expressing seven

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V METHODS FOR ASSESSING MUCOSAL IMMUNE

CFs and both toxins, has been developed by Zhang and Sack [59]. Mice intraperitoneally immunized with this protein developed antibodies against all the CFs and neutralized both toxins. A remaining challenge for this concept and other subunit vaccine candidates that are immunogenic when given parenterally to mice is to establish a suitable administration route in humans, resulting in potent local secretory immune response in the small intestine.

IV. ORAL MUCOSAL ADJUVANTS Several different oral mucosal adjuvants have been tested in animal models, among them cholera toxin (CT) [69], E. coli LT [33], and chitosan [70] (Chapter 11: Toxin-Based Modulators for Regulation of Mucosal Immune Responses). Both CT and LT are powerful toxins and also strong mucosal adjuvants [71]. In mouse models, CT and LT have been used as adjuvants in high doses (15 25 μg), well beyond what would cause moderate to severe diarrhea in humans [72,73]. Chitosan has been tested in humans for oral drug delivery but not specifically as an oral adjuvant [74]. Mutants of both CT and LT have been developed by several groups in efforts to reduce or eliminate the toxic part while retaining the adjuvant property of the molecules [21,75], The side effects of these adjuvants when given intranasally to humans, in particular transient Bell’s palsy, seem to have stopped development of several of them [76]. Clements et al. first developed a single mutant of the LT molecule mLT (LT(R192G)) [77]. The mutation inactivates a trypsin-sensitive site in the A subunit that separates the A1 from the A2 unit, effectively stopping the nicking of the protein. The mLT was shown to have a favorable safety profile and retained adjuvanticity in animal models as well as when tested alone in a clinical trial [78]. Later, it was discovered that mLT

569

retained some reactogenicity when given orally together with a whole cell Helicobacter vaccine [79], which led the investigators to develop a double mutant of LT, dmLT LT(R192G/L211A) [21]. The second mutation, L211A, is located at a putative pepsin sensitive site in the A2 domain. The dmLT has been shown to be safe both in animal models and when given orally to humans in doses up to 100 μg [47]. dmLT has now also been used successfully as an adjuvant with two different oral whole cell ETEC vaccine candidates, ACE527 [39] and ETVAX [42].

V. METHODS FOR ASSESSING MUCOSAL IMMUNE RESPONSES AGAINST ENTEROTOXIGENIC ESCHERICHIA COLI CANDIDATE VACCINES IN HUMANS Since both the bacteria and the toxins they produce are confined to the small intestine during infection, determination of mucosal immune responses, particularly in the intestine, have been considered of prime importance in assessing immune response against ETEC candidate vaccines. The gold standard to assess intestinal immune responses is to determine SIgA responses in intestinal lavage fluid [29,80]. However, since this method is very laborious and time consuming, we have searched for alternative approaches to determine responses that may reflect mucosal immune responses locally in the gut. Using the oral cholera vaccine Dukoral or oral ETEC candidate vaccines as model vaccines, we have shown very good agreement between the immune responses they induce in intestinal lavages and in fecal extracts as well as with circulating ASC responses determined either by the ELISPOT or the antibody in lymphocyte secretion (ALS) method [42,80]. Determination of mucosal immune responses in saliva (to be published) or in serum [42] has been less sensitive, particularly in individuals

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not primed by natural infection, to reflect intestinal immune responses induced by these oral vaccines. Methods that may be or have been used to determine immune responses against ETEC candidate vaccines are summarized in Table 32.3.

TABLE 32.3 Methods and Parameters Assessed for Determination of Mucosal Immune Responses Against ETEC Vaccines Parameters Assessed

Methods

Samples

Quantities and isotypes of specific antibodies

ELISA/ Intestinal electrochemiluminescence lavages Fecal extracts Saliva Biopsy extracts Antibodies in lymphocyte secretion (ALS) supernatants

Function of specific antibodies

Toxin neutralization assays Avidity assays Inhibition of binding, etc.

Serum/ plasma ALS samples

Antibodysecreting cells

ELISPOT ALS

Peripheral blood mononuclear cell (PBMCs) Cells extracted from intestinal biopsies

Immunological Polyclonal stimulation memory assays 1 ELISPOT Kinetic analysis of ALS/ serum responses

PBMCs

Phenotypes of B cells

Whole blood/ PBMCs Cells extracted from biopsies

FACS Immunomagnetic cell sorting

VI. CANDIDATE VACCINES IN CLINICAL DEVELOPMENT A. ACE527 The ETEC vaccine candidates presently in clinical trials span over quite a large variety of formulations, from recombinant whole cells (attenuated or killed), and protein antigens such as fimbrial subunits or toxoids. Also the route of administration varies, from oral to parenteral to skin administration. The most advanced live attenuated ETEC vaccine in clinical trials is the ACE527 vaccine, developed by Darsley et al. [38]. This vaccine consists of three recombinant strains of ETEC attenuated by aroC, omp F, and Omp C mutations. The three different strains express the colonization factors CFA/I, CS1, CS2, CS3, CS5, CS6, and LTB. The vaccine has been shown to induce mucosal immune responses against all CFs when administered orally to adult humans [38]. In a recent controlled human infection model (CHIM), the vaccine was given orally on three occasions with or without 25 μg of dmLT adjuvant. When challenged with virulent ETEC bacteria 6 months later, the group that had been given vaccine 1 dmLT was significantly protected (protective efficacy: 65.9%; P 5 .01), whereas the group given ACE527 alone was not protected (protective efficacy: 20.5%; P 5 .3) [39]. The clinical development of this vaccine is currently not being pursued.

B. Tip Adhesins The US Naval Medical Research Center (NMRC) has constructed a CFaE tip protein adhesin of CFA/I as a prototype vaccine candidate [81]. When given intradermally and transcutaneously together with the LT-based adjuvant mLT, this protein gave rise to significant serum and ASC (IgG/IgA) antibody responses to CFA/I in a majority of the volunteers; intradermal administration was also

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VI. CANDIDATE VACCINES IN CLINICAL DEVELOPMENT

shown to induce high hemagglutination inhibition titers. However, when tested in a clinical phase 2b trial encompassing three different cohorts, including 84 adult American volunteers (41 vaccinees and 43 placebo recipients), intradermal administration of three doses of the tip vaccine induced highly variable protection against ETEC challenge in the different cohorts [46]. A passive protection study [12] using bovine milk containing high titers of anti-CFaE antibodies has provided some promising results. The success of using tip protein adhesins as immunogens will depend on the extent to which protective antibodies against a majority of prevalent CFs can be raised and whether intestinal immune responses can be induced by such proteins administered parenterally or orally.

C. LT patch The LT toxin is a major protective antigen. Previous studies have shown that transcutaneous immunization could elicit high anti-LT antibody responses, both systemic and mucosal, of both IgA and IgG subclasses comparable to those obtained after challenge with an LTpositive ETEC strain or after oral immunization with a CTB-containing cholera vaccine [31]. Despite promising preliminary results in a small phase 2 trial, in which an overall protection rate of 75% against all-cause moderate to severe diarrhea was recorded in European travelers to Guatemala or Mexico [82], the vaccine did not meet its primary objectives in a subsequent phase 2 trial in India [83] or in a larger phase 3 trial [84] in European travelers to Guatemala or Mexico. In both the latter trials, two doses of LT (37.5 μg) was delivered by a patch on the upper arm 2 3 weeks apart to adult human volunteers before travel to the target area 7 30 days later. Immunogenicity data indicated that there was a robust vaccine take; 84% 90% of the participants seroconverted with IgG and IgA antibodies

571

against LT in serum [83,84], respectively. In the phase 2 trial conducted in India, no protection was recorded against any ETEC. In the phase 3 trial done in travelers to Guatemala or Mexico, there was a tendency (not statistically significant) of protection against LT-only strains but not against LT/ST or ST-only strains. On the basis of these results, development of this vaccine was halted.

D. Oral Inactivated Whole Cell Enterotoxigenic Escherichia coli Vaccines 1. rCTB-CF Enterotoxigenic Escherichia coli Vaccine We have previously developed an oral ETEC vaccine consisting of a combination of recombinantly produced CTB (rCTB) and formalininactivated ETEC bacteria expressing major CFs (as reviewed by Svennerholm and Lundgren [28]). Extensive clinical evaluation of this first-generation ETEC vaccine (rCTB-CF) showed that it was safe and induced significant mucosal immune responses in all age groups except in children 6 17 months of age in endemic areas. In the youngest children, a full adult dose was associated with an increased frequency of vomiting; however, a quarter of a full dose of vaccine was safe and immunogenic [85]. The rCTB-CF vaccine provided significant protective efficacy (77%, P 5 .039) against nonmild ETEC disease in American travelers to Mexico and Guatemala [86] but not significant protection (protective efficacy: 20%) against ETEC diarrhea in Egyptian infants with mostly mild disease [87]. 2. Multivalent ETEC vaccine(ETVAX) Based on the experience with the rCTB-CF ETEC vaccine, we have developed a modified second-generation oral inactivated ETEC vaccine with the aim of improving the immunogenicity without increasing the dosage of bacteria, allowing administration of reduced doses to

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infants and young children. The new vaccine consists of recombinant E. coli strains overexpressing the most prevalent ETEC CFs in combination with a CTB/LTB hybrid protein (LCTBA), which induces stronger anti-LT responses than CTB does in mice and humans [69,88] (N. Carlin, unpublished). This multivalent ETEC vaccine (ETVAX) contains four different inactivated E. coli strains expressing substantially higher levels of CFA/I, CS3, CS5, and CS6 than the first-generation vaccine, plus LCTBA. In addition, we have evaluated the possibility of furthering enhance the immunogenicity of the vaccine by coadministration with the mucosal dmLT adjuvant [21], which significantly improved both the anti-CF and anti-LT responses following oral immunization with ETVAX in mice [69] (Fig. 32.1). The vaccine 6 dmLT adjuvant is administered orally as a drink in a glass of bicarbonate solution for human trial. We have recently tested the safety and immunogenicity of ETVAX in large phase 1 trial in adult Swedish volunteers [42]. These

studies have shown that two oral doses of the vaccine were safe and well tolerated and capable of inducing strong mucosal immune responses against the key vaccine antigens, that is, the CFs and LTB, in a majority of vaccinees. In these and subsequent clinical trials of ETVAX, mucosal immune responses have been determined by analysis of intestine-derived ASC responses in ALS and as SIgA/total SIgA antibody responses in fecal extracts. Addition of dmLT adjuvant in a dose of 10 μg enhanced the antigen-specific immune response against the CFs present in the lowest amounts in the vaccine, supporting the dose-sparing effect of the adjuvant previously observed in animal studies [69]. In continued studies in Swedish volunteers, we showed that the vaccine induced mucosal immune responses not only against the vaccine CFs but also against related CFs of the CFA/I group and against CS7 that cross-reacts with CS5 [89]. In a subsequent trial, we showed that Swedish adults who 1 2 years earlier had been given two doses of ETVAX responded considerably earlier

FIGURE 32.1 ETVAX contains four recombinant Escherichia coli strains overexpressing the most common colonization factors and a toxoid, LCTBA, together with the dmLT adjuvant. All these colonization facors have been shown to be protective in animal studies [8].

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VII. CONCLUDING REMARKS

and with higher mucosal immune responses to a single booster dose of ETVAX than did previously unimmunized matched control subjects [43]. Furthermore, the avidity of the immune responses was also higher after the late booster than after the two initial vaccinations [89]. Based on these promising results a large phase 1 2 trial (495 subjects) was initiated in descending age groups in Bangladesh. Groups of adults, 2- to 5year-old children, children aged 1 2 years, and infants aged 6 11 months were given different doses of vaccine alone or together with dmLT adjuvant or placebo (buffer only) 2 weeks apart, and blood and feces were collected from all subjects before the first dose and then 7, 19, and 28 days (feces only) after onset of vaccine or placebo administration [44]. Analyses of ALS responses in the different age groups revealed that 100% of all vaccinated adults responded to all the five key vaccine antigens, whereas only a few placebo recipients responded to a single antigen [90]. Responses in the Bangladeshi adults were higher and more frequent than responses previously recorded in adult Swedes. High and frequent ALS responses similar to those in the Bangladeshi adults were recorded in 2- to 5-yearold Bangladeshi children [91] ALS responses in 1- to 2-year-old children were also observed against the key vaccine antigens in a majority of the volunteers. Mucosal immune responses in infants analyzed from ALS and/or fecal specimens showed significant immune response against all vaccine antigens in a majority of the volunteers. A study is in progress to evaluate the safety, immunogenicity, and protective efficacy of ETVAX in Finnish travelers to Benin in Africa [45,92]. Eight hundred adult volunteers participating in a double-blind phase 2b study are receiving either two doses of ETVAX or placebo (buffer only). All participants are analyzed for immune responses in serum as well as for possible episodes of ETEC diarrhea during a 12-day stay in Africa and the first 6 days after their return to Finland. The study was

573

planned to have the last volunteer included in February 2019. Studies are also in planning to evaluate ETVAX for protection against moderate to severe ETEC diarrhea in young children in a highly ETEC-endemic area in Africa.

VII. CONCLUDING REMARKS A. Preclinical Vaccine Candidates The field of oral vaccines against ETEC is evolving rapidly. Promising new noncanonical antigens have been proposed on the basis of new knowledge acquired through whole genome sequencing and reversed vaccinology [59,93,94]. The concept of using MEFA candidates is an approach considered to complement administration of already established antigens. The renewed interest in ST as a candidate immunogen has led to a new generation of ST analogs that holds some promise based on studies in animal models [25]. At the same time, numerous ST analogs coupled to carrier proteins have been studied as potential candidate vaccine components without identifying a safe and protective ST toxoid for more than 30 years [67]. Thus new formulations of nontoxic ST conjugates will have to be tested in the clinic before their value can be assessed. The refinement and diversification of CHIM models for evaluation of protective efficacies of candidate vaccines and formulations may help in clinical development of ETEC vaccines.

B. Vaccine Candidates in Clinical Development The tip adhesin strategy pioneered by Savarino et al. is interesting from a scientific point of view and has shown promising results in passive protection studies [12]. The ACE527 candidate represents the most advanced live attenuated vaccine [38]. When given alone in a

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CHIM model, it did not provide protection, but when it was supplemented with the dmLT adjuvant, significant protection was recorded [39]; however, further development of this vaccine has been halted [95]. The ETVAX vaccine candidate comprising a whole cell component together with an LTB-CTB toxoid and the dmLT adjuvant is the most advanced vaccine candidate [42,91,92,95]. Production is taking place on a commercial scale, and the vaccine has been shown to be highly immunogenic both in Western adults and in adults and young children in the developing world. The efficacy of the vaccine in adult European travelers to Africa is being tested in a phase 2b trial. The development landscape for ETEC vaccines is promising at present with several attractive candidates in preclinical, early, and late phase clinical trials, giving hope that one or more of these vaccines will reach licensure within the next 3 5 years.

[6]

[7]

[8]

[9]

[10]

[11]

[12]

References [1] Isidean SD, Riddle MS, Savarino SJ, et al. A systematic review of ETEC epidemiology focusing on colonization factor and toxin expression. Vaccine 2011;29:6167 78. Available from: http://www.sciencedirect.com/science/article/pii/S0264410X11009583. [2] Qadri F, Svennerholm A-M, Faruque ASG, et al. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev 2005;18: 465 83. Available from: http://cmr.asm.org/cgi/content/abstract/18/3/465. [3] Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol 1996;4:444 52. [4] von Mentzer A, Connor TR, Wieler LH, et al. Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution. Nat Genet 2014;46:1321 6. Available from: https://doi.org/ 10.1038/ng.3145. [5] Ahren CM, Svennerholm AM. Experimental enterotoxin-induced Escherichia coli diarrhea and protection induced by previous infection with bacteria of the same adhesin or enterotoxin type. Infect Immun

[13]

[14]

[15]

[16]

1985;50:255 61. Available from: http://iai.asm.org/ cgi/content/abstract/50/1/255. Qadri F, Saha A, Ahmed T, et al. Disease burden due to enterotoxigenic Escherichia coli in the first 2 years of life in an urban community in Bangladesh. Infect Immun 2007;75:3961 8. Available from: http://iai. asm.org/cgi/content/abstract/75/8/3961. Svennerholm AM, Ahren C. Immune protection against enterotoxinogenic E. coli: search for synergy between antibodies to enterotoxin and somatic antigens. Acta Pathol Microbiol Immunol Scand C 1982;90:1 6. Svennerholm AM. From cholera to enterotoxigenic Escherichia coli (ETEC) vaccine development. Indian J Med Res 2011;2011(133):188 96. Levine MM, Morris JG, Losonsky G, et al. Fimbriae (Pili) adhesins as vaccines. In: Lark DL, editor. Protein-Carbohydrate Interact Biol Syst Mol Biol Microb Pathog (FEMS Symp). London: Academic Press; 1986. p. 143 5. Clemens JD, Sack DA, Harris JR, et al. Cross-protection by B subunit-whole cell cholera vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial. J Infect Dis 1988;158:372 7. Peltola H, Siitonen A, Kyronseppa H, et al. Prevention of travellers’ diarrhoea by oral B-subunit/whole-cell cholera vaccine. Lancet 1991;338:1285 9. Savarino SJ, McKenzie R, Tribble DR, et al. Prophylactic efficacy of hyperimmune bovine colostral antiadhesin antibodies against enterotoxigenic Escherichia coli diarrhea: a randomized, double-blind, placebo-controlled, phase 1 trial. J Infect Dis 2017;216:7 13. Available from: https://doi.org/10.1093/infdis/jix144. Spangler BD. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev 1992;56:622 47. Available from: http:// mmbr.asm.org/cgi/content/abstract/56/4/622. Evans DG, Evans Jr. DJ, Opekun AR, et al. Nonreplicating oral whole cell vaccine protective against enterotoxigenic Escherichia coli (ETEC) diarrhea: stimulation of anti-CFA (CFA/I) and anti-enterotoxin (antiLT) intestinal IgA and protection against challenge with ETEC belonging to heterologous. FEMS Microbiol Immunol 1988;1:117 25. Evans Doyle JJ, Evans DG, Opekun AR, et al. Immunoprotective oral whole cell vaccine for enterotoxigenic Escherichia coli diarrhea prepared by in situ destruction of chromosomal and plasmid DNA with colicin E2. FEMS Microbiol Immunol 1988;1:9 18. Available from: https://doi.org/10.1111/j.1574-6968. 1988.tb02485.x. Vipin Madhavan TP, Sakellaris H. Chapter Five Colonization factors of enterotoxigenic Escherichia coli.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

575

REFERENCES

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

Adv appl Microbiol. Boston: Academic Press; 2015. p. 155 97. Anantha RP, McVeigh AL, Lee LH, et al. Evolutionary and functional relationships of colonization factor antigen I and other class 5 adhesive fimbriae of enterotoxigenic Escherichia coli. Infect Immun 2004;72:7190 201. Available from: http://iai.asm.org/cgi/content/ abstract/72/12/7190. Nuccio S-P, Ba¨umler AJ. Evolution of the chaperone/ usher assembly pathway: fimbrial classification goes greek. Microbiol Mol Biol Rev 2007;71:551 75. Available from: http://www.ncbi.nlm.nih.gov/pmc/ articles/PMC2168650/. Roy SP, Rahman MM, Yu XD, et al. Crystal structure of enterotoxigenic Escherichia coli colonization factor CS6 reveals a novel type of functional assembly. Mol Microbiol 2012;86:1100 15. Available from: https:// doi.org/10.1111/mmi.12044. Sa´nchez J, Holmgren J. Cholera toxin structure, gene regulation and pathophysiological and immunological aspects. Cell Mol Life Sci 2008;65:1347 60. Available from: https://doi.org/10.1007/s00018-008-7496-5. Clements JD, Norton EB. The Mucosal Vaccine Adjuvant LT(R192G/L211A) or dmLT. Papasian CJ, editor. mSphere [Internet] 2018;3(4). Available from: Papasian CJ, editor . Available from: http://msphere. asm.org/content/3/4/e00215-18.abstract. Norton EB, Branco LM, Clements JD. Evaluating the A-subunit of the heat-labile toxin (LT) as an immunogen and a protective antigen against enterotoxigenic Escherichia coli (ETEC). PLoS One 2015;10:e0136302. Yoshimura S, Ikemura H, Watanabe H, et al. Essential structure for full enterotoxigenic activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli. FEBS Lett 1985;181:138 42. Available from: https://doi.org/10.1016/0014-5793(85)81129-7. Ozaki H, Sato T, Kubota H, et al. Molecular structure of the toxin domain of heat-stable enterotoxin produced by a pathogenic strain of Escherichia coli. A putative binding site for a binding protein on rat intestinal epithelial cell membranes. J Biol Chem 1991;266: 5934 41. Available from: http://www.jbc.org/content/266/9/5934.abstract. Zegeye ED, Govasli ML, Sommerfelt H, et al. Development of an enterotoxigenic Escherichia coli vaccine based on the heat-stable toxin. Hum Vaccin Immunother 2018;1 10. Available from: https://doi. org/10.1080/21645515.2018.1496768. Whitfield C, Trent MS. Biosynthesis and export of bacterial lipopolysaccharides. Annu Rev Biochem 2014;83: 99 128. Available from: https://doi.org/10.1146/ annurev-biochem-060713-035600. Wolf MK. Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

toxins of enterotoxigenic Escherichia coli. Clin Microbiol Rev 1997;10:569 84. Available from: http://cmr.asm. org/cgi/content/abstract/10/4/569. Svennerholm A-M, Lundgren A. Recent progress toward an enterotoxigenic Escherichia coli vaccine. Expert Rev Vaccines 2012;11:495 507. Available from: https://doi.org/10.1586/erv.12.12. Svennerholm A-M, Jertborn M, Gothefors L, et al. Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a combined B subunit-whole cell vaccine. J Infect Dis 1984; 149:884 93. Available from: http://libsta28.lib.cam.ac. uk:2163/stable/30131477. ˚ , et al. Svennerholm A-M, Holmgren J, Hanson L-A Boosting of secretory IgA antibody responses in man by parenteral cholera vaccination. Scand J Immunol 1977;6:1345 9. Available from: https://doi.org/ 10.1111/j.1365-3083.1977.tb00376.x. Glenn GM, Villar CP, Flyer DC, et al. Safety and immunogenicity of an enterotoxigenic Escherichia coli vaccine patch containing heat-labile toxin: use of skin pretreatment to disrupt the stratum corneum. Infect Immun 2007;75:2163 70. Available from: http://iai.asm.org/ cgi/content/abstract/75/5/2163. ¨ stberg AK, Flach C-F, et al. Sublingual Raghavan S, O immunization protects against Helicobacter pylori infection and induces T and B cell responses in the stomach. Infect Immun 2010;78:4251 60. Available from: http:// www.ncbi.nlm.nih.gov/pmc/articles/PMC2950356/. Haan L, de, Holtrop M, Verweij WR, et al. Mucosal immunogenicity of the Escherichia coli heat-labile enterotoxin: role of the A subunit. Vaccine 1996;14: 260 6. Available from: http://www.sciencedirect. com/science/article/pii/0264410X9500235S. Johansson E-L, Bergquist C, Edebo A, et al. Comparison of different routes of vaccination for eliciting antibody responses in the human stomach. Vaccine 2004;22:984 90. Available from: http://www.sciencedirect.com/science/article/pii/S0264410X0300642X. Tacket CO, Losonsky G, Link H, et al. Protection by milk immunoglobulin concentrate against oral challenge with enterotoxigenic Escherichia coli. N Engl J Med 1988;318:1240 3. Tacket CO, Losonsky G, Livio S, et al. Lack of prophylactic efficacy of an enteric-coated bovine hyperimmune milk product against enterotoxigenic Escherichia coli challenge administered during a standard meal. J Infect Dis 1999;180:2056 9. Otto W, Najnigier B, Stelmasiak T, et al. Randomized control trials using a tablet formulation of hyperimmune bovine colostrum to prevent diarrhea caused by enterotoxigenic Escherichia coli in volunteers. Scand J Gastroenterol 2011;46:862 8. Available from: http:// www.ncbi.nlm.nih.gov/pmc/articles/PMC3154584/.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

576

32. ORAL VACCINES FOR ENTEROTOXIGENIC ESCHERICHIA COLI

[38] Darsley MJ, Chakraborty S, DeNearing B, et al. The oral, live attenuated enterotoxigenic Escherichia coli vaccine ACE527 reduces the incidence and severity of diarrhea in a human challenge model of diarrheal disease. Clin Vaccine Immunol 2012;19:1921 31. Available from: http://cvi.asm.org/content/19/12/1921.abstract. [39] Harro C, Louis Bourgeois A, Sack D, Walker R, DeNearing B, Brubaker J, et al. Live attenuated enterotoxigenic Escherichia coli (ETEC) vaccine with dmLT adjuvant protects human volunteers against virulent experimental ETEC challenge. Vaccine [Internet] 2019. Available from: http://www.sciencedirect.com/ science/article/pii/S0264410X19302208. [40] Barry EM, Wang J, Wu T, et al. Immunogenicity of multivalent Shigella-ETEC candidate vaccine strains in a guinea pig model. Vaccine 2006;24:3727 34. Available from: http://www.sciencedirect.com/science/article/B6TD44GP6WDX-1/2/195fae62c200ca258b10c11d19417e22. [41] Wu Y, Wai TT, Jackson JM, et al. Development and characterization of Salmonella enterica serovar Typhi Ty21a Vaccine platform the promise and insight for vaccines against Shigellosis, ETEC diarrhea, typhoid fever, and non-typhoidal Salmonellosis. Albufeira: Meetings Management; 2017. [42] Lundgren A, Bourgeois L, Carlin N, et al. Safety and immunogenicity of an improved oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine administered alone and together with dmLT adjuvant in a double-blind, randomized, placebo-controlled Phase I study. Vaccine 2014;32:7077 84. [43] Lundgren A, Jertborn M, Svennerholm A-M. Induction of long term mucosal immunological memory in humans by an oral inactivated multivalent enterotoxigenic Escherichia coli vaccine. Vaccine 2016;34:3132 40. Available from: http://www.sciencedirect.com/science/article/pii/S0264410X16302092. [44] Qadri F, Chowdhury MI, Bhuiyan TR, et al. A phase I/ II trial of the oral inactivated ETEC vaccine (ETVAX; OEV 122) in descending age groups in Bangladesh. Albufeira: Meetings Management; 2017. [45] Kantele A. OEV123 A clincial trial on ETVAX, an oral vaccine against enterotoxigenic Escherichia coli diarrhea. Albufeira: Meetings Management; 2017. [46] Harro C, Gutierrez RL, Talaat KR, et al. Protective efficacy of an enterotoxigenic E. coli fimbrial tip adhesin vaccine given with LTR192G by intradermal vaccination against experimental challenge with CFA/I-ETEC in adult volunteers. In: 50th US-Japan Coop. Med. Sci. Progr. Jt. Panel Conf. Cholera Other Bact. Enteric Infect. Bethesda, MD; 2016. p. 126 30. [47] El-Kamary SS, Cohen MB, Bourgeois AL, et al. Safety and immunogenicity of a single oral dose of recombinant double mutant heat-labile toxin derived from

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

enterotoxigenic Escherichia coli. Clin Vaccine Immunol 2013;20:1764 70. Available from: http://cvi.asm.org/ content/20/11/1764.abstract. Taxt A, Aasland R, Sommerfelt H, et al. Heat-stable enterotoxin of enterotoxigenic Escherichia coli as a vaccine target. Infect Immun 2010;78:1824 31. Available from: http://iai.asm.org/cgi/content/abstract/78/5/1824. Tapader R, Bose D, Basu P, et al. Role in proinflammatory response of YghJ, a secreted metalloprotease from neonatal septicemic Escherichia coli. Int J Med Microbiol 2016;306:554 65. Available from: http://www.sciencedirect.com/science/article/pii/S1438422116301382. Tapader R, Bose D, Pal A. YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop. Microb Pathog 2017;105:96 9. Available from: http://www.sciencedirect.com/science/article/pii/S0882401016307999. DeCanio MS, Landick R, Haft RJF. The non-pathogenic Escherichia coli strain W secretes SslE via the virulenceassociated type II secretion system beta. BMC Microbiol 2013;13:130. Available from: http://www. ncbi.nlm.nih.gov/pmc/articles/PMC3707838/. Fleckenstein JM, Roy K, Fischer JF, et al. Identification of a two-partner secretion locus of enterotoxigenic Escherichia coli. Infect Immun 2006;74:2245 58. Available from: http://iai.asm.org/cgi/content/ abstract/74/4/2245. Roy K, Hilliard GM, Hamilton DJ, et al. Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells. Nature 2008;457:594 8. Available from: https://doi.org/10.1038/nature07568. Roy K, Bartels S, Qadri F, et al. Enterotoxigenic Escherichia coli elicits immune responses to multiple surface proteins. Infect Immun 2010;78:3027 35. Available from: http://iai.asm.org/cgi/content/abstract/78/7/3027. Roy K, Hamilton D, Ostmann MM, et al. Vaccination with EtpA glycoprotein or flagellin protects against colonization with enterotoxigenic Escherichia coli in a murine model. Vaccine 2009;27:4601 8. Available from: http://www.sciencedirect.com/science/article/ B6TD4-4WH0G02-7/2/ b90f8e42dca7b28faa6d44b7591a4302. Kumar P, Luo Q, Vickers TJ, et al. EatA, an immunogenic protective antigen of enterotoxigenic Escherichia coli, degrades intestinal mucin. Infect Immun 2014;82: 500 8. Available from: http://iai.asm.org/content/ 82/2/500.abstract. Fleckenstein JM, Sheikh A, Qadri F. Novel antigens for enterotoxigenic Escherichia coli (ETEC) vaccines. Expert Rev Vaccines 2014;13:631 9. Available from: http:// www.ncbi.nlm.nih.gov/pmc/articles/PMC4199203/. Sheikh A, Luo Q, Roy K, et al. Contribution of the highly conserved EaeH surface protein to

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

577

REFERENCES

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

enterotoxigenic Escherichia coli pathogenesis. Payne SM, editor. Infect Immun 2014;82:3657 66. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC4187836/. Zhang W, Sack DA. Current progress in developing subunit vaccines against enterotoxigenic Escherichia coli-associated diarrhea. Clin Vaccine Immunol 2015; 22:983 91. Available from: http://cvi.asm.org/content/22/9/983.abstract. Zeinalzadeh N, Salmanian AH, Goujani G, et al. A chimeric protein of CFA/I, CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli. Microbiol Immunol 2017;61: 272 9. Available from: https://doi.org/10.1111/ 1348-0421.12491. Amicizia D, Arata L, Zangrillo F, et al. Overview of the impact of typhoid and paratyphoid fever. Utility of Ty21a vaccine (Vivotifs). J Prev Med Hyg 2017;58: E1 8. Available from: http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC5432773/. Yamasaki S, Hidaka Y, Ito H, et al. Structural requirements for the spatial structure and toxicity of heatstable enterotoxin (STh) of enterotoxigenic Escherichia coli. Bull Chem Soc Jpn 1988;61:1701 6. Available from: http://www.journal.csj.jp/doi/10.1246/ bcsj.61.1701. Taxt AM, Diaz Y, Aasland R, et al. Towards rational design of a toxoid vaccine against the heat-stable toxin of Escherichia coli. Infect Immun 2016;84:1239 49. Available from: http://iai.asm.org/content/84/4/1239.abstract. Sanchez J, Svennerholm A-M, Holmgren J. Genetic fusion of a non-toxic heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit. FEBS Lett 1988;241:110 14. Available from: http://doi. wiley.com/10.1016/0014-5793%2888%2981041-X. Taxt AM, Diaz Y, Bacle A, et al. Characterization of immunological cross-reactivity between enterotoxigenic Escherichia coli heat-stable toxin and human guanylin and uroguanylin. Infect Immun 2014;82:2913 22. Available from: http://iai.asm.org/content/82/7/ 2913.abstract. Diaz Y, Larsen MA., Zegeye ED, et al. Both the STh and the STp variant of the heat-stable toxin of enterotoxigenic Escherichia coli can elicit antibodies that cross-react with guanylin and/or uroguanylin. In: 9th Int. Conf. vaccines enteric Dis. Albufeira, Portugal; 2017. p. B117. Holmgren J, Levine MM. Chapter 56 Vaccines against bacterial enteric infections. Mucosal Immunol 2015;1047 82. Available from: http://www.sciencedirect.com/science/article/pii/B9780124158474000562. Zeinalzadeh N, Salmanian AH, Ahangari G, et al. Design and characterization of a chimeric multiepitope construct containing CfaB, heat-stable toxoid, CssA,

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

CssB, and heat-labile toxin subunit B of enterotoxigenic Escherichia coli: a bioinformatic approach. Biotechnol Appl Biochem 2014;61:517 27. Holmgren J, Bourgeois L, Carlin N, et al. Development and preclinical evaluation of safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli bacteria overexpressing colonization factors CFA/I, CS3, CS5 and CS6 combined with a hybrid LT/ CT B subunit antigen, administered al. Vaccine 2013; 31:2457 64. Borges O, Tavares J, de Sousa A, et al. Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles. Eur J Pharm Sci 2007;32:278 90. Available from: http:// www.sciencedirect.com/science/article/pii/ S0928098707003284. Simmons CP, Ghaem-Magami M, Petrovska L, et al. Immunomodulation using bacterial enterotoxins. Scand J Immunol, 53. 2001. p. 218 26. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1046/ j.1365-3083.2001.00884.x. Levine MM, Kaper JB, Black RE, et al. New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development. Microbiol Rev 1983;47:510 50. Michetti P, Kreiss C, Kotloff KL, et al. Oral immunization with urease and Escherichia coli heat-labile enterotoxin is safe and immunogenic in Helicobacter pylori infected adults. Gastroenterology 1999;116: 804 12. Available from: http://www.sciencedirect. com/science/article/pii/S0016508599700636. Bowman K, Leong KW. Chitosan nanoparticles for oral drug and gene delivery. Int J Nanomedicine 2006;1: 117 28. Available from: http://www.ncbi.nlm.nih. gov/pmc/articles/PMC2426784/. Pizza M, Giuliani MM, Fontana MR, et al. LTK63 and LTR72, two mucosal adjuvants ready for clinical trials. Int J Med Microbiol 2000;290:455 61. Available from: http://www.sciencedirect.com/science/article/pii/ S1438422100800648. Lewis DJM, Huo Z, Barnett S, et al. Transient facial nerve paralysis (Bell’s Palsy) following intranasal delivery of a genetically detoxified mutant of Escherichia coli heat labile toxin. PLoS One 2009;4:e6999. Available from: http://doi.org/10.1371%2Fjournal.pone.0006999. Dickinson BL, Clements JD. Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADPribosyltransferase activity. Infect Immun 1995;63: 1617 23. Available from: http://iai.asm.org/cgi/content/abstract/63/5/1617. Oplinger ML, Bakar S, Trofa AF, et al. Safety and immunogenicity in volunteers of a new candidate mucosal adjuvant. LT(R192G) Programs Abstr 37th Intersci Conf

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

578

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

32. ORAL VACCINES FOR ENTEROTOXIGENIC ESCHERICHIA COLI

Antimicrob Agents Chemother. Washington, DC: American Society for Microbiology; 1997. Kotloff KL, Sztein MB, Wasserman SS, et al. Safety and immunogenicity of oral inactivated whole-cell Helicobacter pylori vaccine with adjuvant among volunteers with or without subclinical infection. Infect Immun 2001;69:3581 90. Available from: http://iai. asm.org/cgi/content/abstract/69/6/3581. Ahre´n C, Jertborn M, Svennerholm A-M. Intestinal immune responses to an inactivated oral enterotoxigenic Escherichia coli vaccine and associated immunoglobulin A responses in blood. Infect Immun 1998;66: 3311 16. Available from: http://www.ncbi.nlm.nih. gov/pmc/articles/PMC108347/. Gutierrez R, Riddle M, Porter C, et al. Phase I Cinical evaluation of Adhesin-based ETEC. Vaccines 7th Int Vaccines Enteric Dis Conf. Bangkok: Meetings Management; 2013. Frech SA, DuPont HL, Bourgeois AL, et al. Use of a patch containing heat-labile toxin from Escherichia coli against travellers’ diarrhoea: a phase II, randomised, double-blind, placebo-controlled field trial. Lancet 2008;371:2019 25. Available from: http://www.sciencedirect.com/science/article/B6T1B-4SRC3VB-1/2/ d3e0048424db74cb4f6dec7a713d2447. Steffen R, Cramer JP, Burchard G, et al. Efficacy of a travelers’ diarrhea vaccine system in travelers to India. J Travel Med 2013;20:374 9. Available from: https:// doi.org/10.1111/jtm.12064. Behrens RH, Cramer JP, Jelinek T, et al. Efficacy and safety of a patch vaccine containing heat-labile toxin from Escherichia coli against travellers’ diarrhoea: a phase 3, randomised, double-blind, placebo-controlled field trial in travellers from Europe to Mexico and Guatemala. Lancet Infect Dis 2014;14:197 204. Qadri F, Ahmed T, Ahmed F, et al. Reduced doses of oral killed enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine is safe and immunogenic in Bangladeshi infants 6 17 months of age: dosing studies in different age groups. Vaccine 2006;24:1726 33. Available from: http://www.sciencedirect.com/science/article/pii/S0264410X05010212. Sack DA, Shimko J, Torres O, et al. Randomised, doubleblind, safety and efficacy of a killed oral vaccine for enterotoxigenic E. coli diarrhoea of travellers to Guatemala and Mexico. Vaccine 2007;25:4392 400. Available from: http://www.sciencedirect.com/science/article/B6TD4-4NDMRFF-1/2/41837e38fad0d 38cd018c55e3a295223. Walker RI, Steele D, Aguado T. Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E. coli (ETEC) disease. Vaccine 2007;25:2545 66. Available from: http://www.

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

sciencedirect.com/science/article/B6TD4-4MM24RH-1/ 2/35d272df72f37411000b16419059ef52. Tobias J, Svennerholm A-M. Strategies to overexpress enterotoxigenic Escherichia coli (ETEC) colonization factors for the construction of oral whole-cell inactivated ETEC vaccine candidates. Appl Microbiol Biotechnol 2012;93:2291 3000. Available from: https://doi.org/ 10.1007/s00253-012-3930-6. Leach S, Lundgren A, Carlin N, et al. Cross-reactivity and avidity of antibody responses induced in humans by the oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX. Vaccine 2017; 35:3966 73. Available from: http://www.sciencedirect.com/science/article/pii/S0264410X17307831. Akhtar M, Chowdhury MI, Bhuiyan TR, Kaim J, Ahmed T, Rafique TA, et al. Evaluation of the safety and immunogenicity of the oral inactivated multivalent enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi adults in a double-blind, randomized, placebo-controlled Phase I trial using electrochemiluminescence and ELISA a. Vaccine [Internet] 2018. Available from: http://www.sciencedirect.com/science/article/pii/S0264410X18315639. Svennerholm A-M, Lundgren A, Akhtar M, et al. Mucosal immune responses to an oral inactivated ETEC vaccine (ETVAX) among descending age groups in Bangladesh. In: PATH, editor. 2nd Vaccines against Shigella ETEC Conf. June 12 to 14, Hyatt Regency, Mexico City; 2018. p. 96. Carlin NIA, Svennerholm A-M, Kantele A. A Phase 2b clinical trial of ETVAX, an oral whole-cell inactivated vaccine against enterotoxigenic Escherichia coli, in Finnish travelers to Benin. In: 2nd Vaccines against Shigella ETEC Conf. June 12 to 14, Hyatt Regency, Mexico City: PATH; 2018. p. 104. Chakraborty S, Randall A, Vickers TJ, et al. Human experimental challenge with enterotoxigenic Escherichia coli elicits immune responses to canonical and novel antigens relevant to vaccine development. J Infect Dis 2018;218:1436 46. Available from: https:// doi.org/10.1093/infdis/jiy312. Nesta B, Valeri M, Spagnuolo A, et al. SslE elicits functional antibodies that impair in vitro mucinase activity and in vivo colonization by both intestinal and extraintestinal Escherichia coli strains. Sperandio V, editor. PLoS Pathog 2014;10:e1004124. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC4014459/. Hosangadi D, Smith PG, Kaslow DC, et al. WHO consultation on ETEC and Shigella burden of disease, Geneva, 6 7th April 2017: Meeting report. Vaccine 2018;S0264 74. Available from: http://www.sciencedirect.com/science/article/pii/S0264410X17313762.

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A Future for a Vaccine Against the Cancer-Inducing Bacterium Helicobacter pylori? Thomas F. Meyer1 and Pau Morey2 1

2

Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany Instituto Universitario de Investigacio´n en Ciencias de la Salud (IUNICS), Universidad de las Islas Baleares, Palma de Mallorca, Spain

I. INTRODUCTION The human stomach provides an essentially hostile environment for infectious agents, yet the Gram-negative bacterium Helicobacter pylori has adapted to colonize the gastric mucosa in early childhood and persist for life [1,2]. These adaptations, forged during more than 100,000 years of coevolution with H. pylori’s human host [3,4], include unique mechanisms geared toward withstanding harsh acidic conditions and evading immune surveillance [5]. Indeed, such is the degree of bacterial specialization that H. pylori’s habitat is restricted almost exclusively to the human gastric mucosa, embedded in the luminal mucus layer in close contact with epithelial cells [6 8]. It is estimated that 4.4 billion people were colonized with H. pylori in 2015; this comprises more than 50% of the world population, with a prevalence ranging

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00033-X

from approximately 20% in some developed countries in Western Europe, Oceania, and North America to more than 80% particularly, yet not exclusively, in subdeveloped and emerging countries [9]. Most colonized individuals develop marginal inflammation, remaining largely asymptomatic [10]. In about 20% of cases, however, H. pylori leads to severe complications such as chronic active gastritis or peptic ulcer disease [6]. Barry Marshall and Robert Warren were awarded the Nobel Prize in Physiology and Medicine in 2005 for the discovery of this association [11]. Moreover, chronic colonization with H. pylori is strongly connected with the development of mucosa-associated lymphoid tissue (MALT) lymphoma [12] and gastric adenocarcinoma, which has a high mortality rate. H. pylori is the only bacterium classified as a class 1 (definite) causative agent of cancer by the World Health

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Organization (WHO) and is therefore considered a serious threat to human health [10]. Being a chronically infectious agent, H. pylori has evolved effective immunosuppressive strategies [5]. These are mediated by the VacA and GGT immunomodulators, which interfere with the maturation of T cells [5,13,14]. Indeed, several vaccine trials have successfully compensated for this deficit by inducing substantial H. pylori-specific immune responses in both mice and humans [15]. Surprisingly, though, the vaccines failed to confer complete protection against infection [16,17]. In this chapter, we review recent findings that provide an astounding explanation for the failure of past vaccination attempts. We also illuminate scenarios through which future approaches could lead to better results, also taking into account the possible risks of vaccination.

II. THE THERAPEUTIC TOOL BOX OF HELICOBACTER PYLORI INFECTIONS H. pylori is an enlightening example in an array of discouraging experiences with chronically persistent pathogens that have escaped conventional vaccination approaches. Pathogenic bacteria rely on a large repertoire of tools to suppress immune activation [18]. Besides the exhibition of structural features, such as antigenic mimicry of self-epitopes, they pursue direct interference with immune activation pathways. The result of such failure may, for example, involve an ineffective generation of pathogen-specific immune cells, an unfavorable balance of effector versus regulatory cells, and a failure in generating specific antibodies. In the case of H. pylori, immune suppression is conferred by at least two secreted factors, VacA and GGT, which effectively downregulate Th1 and Th17 T cell activation [5,13,14]. However, by applying appropriate vaccination

protocols, the deficiency of immune activation could be overcome [19], thus in principle mounting robust protection lines. In fact, successful rational vaccine design strategies have been developed against numerous pathogens through assembling efficacious antigen cocktails in combination with highly potent adjuvants [20]. In this way, appropriate branches of the immune system could be activated, directing immunity toward desired organ sites [21]. Despite great success in preventing various pathogenic infections [22], surprisingly, this strategy appears to fail for H. pylori, as further addressed below. Also for this reason, current treatment of H. pylori infections still relies on antibiotic therapy. A combination of two antibiotics is usually given together with a proton pump inhibitor [23]. However, bacterial resistance is steadily increasing, to such an extent that the WHO has now given high priority to the development of new therapies against clarithromycin-resistant H. pylori [24]. Further problems associated with antibiotic treatment are linked to low patient compliance, the occurrence of reinfections, and the uncertainty about whether treatment is indicated for asymptomatic patients [25]. In this light, a vaccine, whether prophylactic or therapeutic, would obviously be highly desirable. It would theoretically solve the problems associated with current antibiotic treatment and constitute a milestone for the prevention of peptic ulcer and gastric cancer. Much effort has therefore been invested in developing vaccines in preclinical and clinical settings, but so far, none of them have provided more than scarce protection [15,25 27]. Even in mouse studies, vaccination approaches have mostly achieved only partial reduction of pathogen colonization rather than sterile immunity [28 31]. Apart from this, critical voices have been raised indicating potential risks. For example, vaccination against H. pylori may lead to an exaggerated yet nonproductive inflammatory

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III. THE UNHEEDED ROLE OF EPITHELIAL CELLS IN THE EXECUTION OF MUCOSAL DEFENSE

response, particularly if subsequent eradication is incomplete. Thus, failure to achieve sterile immunity could increase the risk of associated gastric cancer [32 34]. Moreover, boosting T helper responses might diminish the immuneregulatory effects associated with H. pylori infections, which have been considered to be partially beneficial in preventing autoaggressive diseases, such as certain forms of asthma and colitis [5]. Attention has also been drawn to the potential positive effects of H. pylori in reducing the risk of Barrett’s disease and esophageal cancer [35,36], possibly in connection with the decreased gastric acid production that occurs under the influence of H. pylori infection [35,36]. Thus any vaccination program aimed against H. pylori must take these various considerations into account.

III. THE UNHEEDED ROLE OF EPITHELIAL CELLS IN THE EXECUTION OF MUCOSAL DEFENSE The mucosal surfaces constitute the interface between an organism and the environment, forming the key barrier for preventing infiltration of surrounding microbiota and infectious agents to the internal tissues. This barrier consists of either stratified or columnar epithelium. The latter type, making up the gastrointestinal surfaces from the gastroesophageal junction to the end of the colon, consists of a single layer of polarized epithelial cells covered by a protective mucus film on the luminal side and supported by stroma and the MALT on the basal side [37,38]. Owing to the innumerable invaginations, referred to as glands and crypts, as well as the intestinal villi, the gastrointestinal tract displays the largest of these mucosal surfaces. As the digestive channel for passing the essentially nonsterile cargo, the gastrointestinal tract has adapted to keep potentially harmful

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microorganisms at bay while tolerating colonization by commensal microbes. However, pathogenic bacteria of the mucosa are often equipped with an array of virulence factors capable of disturbing the existing equilibrium and penetrating the mucosal barrier [39]. To cope with such challenges, the mucosa possesses intricate surveillance and alert systems that are poised for a fine-tuned response, depending on the vicinity and aggressiveness of potential invaders (Fig. 33.1, left). Accordingly, propagation in the lumen does not provoke substantial responses, while epithelial cell attachment or even cell and tissue invasion induces progressively greater responses, potentially culminating in sepsis, typically associated with an overwhelming inflammatory burst. In a naı¨ve situation, these defense mechanisms will act in the absence of adaptive immunity, relying solely on the action of the immediately available and autonomous surveillance and defense systems of the epithelium [40]. This can, for example, involve the release of antimicrobial molecules and the mobilization of phagocytic and innate epithelial cells to the site of infection. If clearance of the initial assault is delayed, the early innate response may be advanced via the adaptive branches of the immune surveillance (Fig. 33.1). These subsequent mechanisms typically involve antigen-presenting cells (APCs) and CD41 T cells, which, depending on the nature of the aggression and the type of microorganism, mount an appropriate response. CD41 T cells are key players in coordinating the clearance of an infection by stimulating B cells to produce specific antibodies, activating cytotoxic immune cells and also boosting the activity of the innate defense mechanisms (Fig. 33.1, right) [21]. Therefore a correct barrier function of the mucosal surfaces requires tight coordination among many different cell types. Because the epithelial lining of the gastrointestinal tract is permanently exposed to microorganisms, to maintain its integrity it

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FIGURE 33.1 Pathogen sensing and response to infection on the mucosal surfaces. Epithelial cells respond with gradual intensity to an infection. (Left) The tighter bacteria interact with mucosal cells (e.g., distant location, epithelial cell adherence, cellular invasion, subepithelial penetration, and entering blood vessels and sepsis), the stronger the proinflammatory response will be. (Right) The chemokine signals elicited will serve to activate and recruit innate and adaptive immune cells to the site of infection. Inflammatory cytokines released from recruited immune cells will activate epithelial defenses, allowing for the translocation of secretory immunoglobulin A (SIgA) as well as the extravasation of professional phagocytes, for example, neutrophils and macrophages (Mϕ).

undergoes constant renewal, facilitated by stem cells located deep in the base of the glands or crypts in the stomach and the intestine, respectively [38,41,42]. Stem cells differentiate into different, specialized epithelial subtypes with distinct cellular functions, such as nutrient absorption and the production and secretion of mucus, hormones, or acid [38,42]. Remarkably, the role of epithelial cells in the defense against mucosal pathogens goes far beyond physical containment. Together with the hematopoietic immune surveillance, epithelial cells are key players in the maintenance and homeostasis of the barrier function. Their location at the border between the external environment and the underlying immune cells allows the epithelial compartment to act as a coordinating hub of mucosal immunity [37]. Epithelial cells display both intracellular and extracellular innate pattern recognition receptors to detect the presence of microbes [37,38,43]. Epithelial sensing of potentially harmful microorganisms triggers the first wave of the innate immune responses, leading to the

secretion of chemotactic cytokines that recruit professional immune cells to the site of infection (Chapter 6: Innate Immunity at Mucosal Surfaces). Immune effector function and microbial killing are often associated with “professional” immune cells. However, the epithelium also participates actively in the antimicrobial defense by secreting bactericidal compounds along with mucins [44]. Antimicrobial peptides (AMPs) are the paradigm of innate epithelial endogenous antibiotics. These small molecules may be produced in millimolar concentrations and are active against Gram-positive and Gram-negative bacteria [45]. AMPs are often positively charged, amphipathic molecules, which allows them to specifically bind the negatively charged bacterial surface and destabilize the plasma membrane [45]. They kill or inactivate bacteria by inducing leakage of cytoplasmic content, binding to intracellular targets and/or delocalization of membrane proteins [45 47]. This powerful way of clearance— fast, specific to bacteria, and cost-effective in energetic terms—is widespread in both

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vertebrates and invertebrates and provides effective protection in animal species that lack an immune system [48]. In humans, several families of these peptides have been described (e.g., cathelicidins, histatins), but the defensin family is the most prominent in terms of expressed genes [49]. Epithelial cells express only β-defensins, except for intestinal Paneth cells, which also express α-defensins 5 and 6 [49]. Expression of epithelial AMPs can be either constitutive or inducible. As a frontline defense mechanism, bacterial sensing by epithelial cells induces immediate AMP expression via a cell-autonomous mechanism [44,50 52]. The hematopoietic system can also boost the production of AMPs via cytokine stimulation of epithelial cells. Interleukin 1 beta (IL-1β) or tumor necrosis factor alpha produced by innate immune cells induces expression of hBD2 and other AMPs [52,53]. Likewise, cytokines from the adaptive immune response, such as interferon gamma (IFNγ), IL17, and IL-22, can engage epithelial cells to produce antimicrobial compounds [44,53 55]. Therefore apart from the immediate autonomous response of epithelial cells, AMPs can be released by the epithelium in a second wave of attack based on instructions from the underlying professional immune cells. In other words, AMPs, a primitive yet effective defense mechanism, are also utilized in the sophisticated adaptive immune response of vertebrates as a powerful weapon to fight infections. Remarkably, epithelial molecules other than AMPs can also contribute in a decisive manner to pathogen clearance in response to inflammation: C-type lectins (e.g., RegIIIα, RegIIIβ, RegIIIγ) can agglutinate bacteria or even form pores in bacterial membranes [56] and are crucial for protection against attaching and effacing pathogens such as Citrobacter rodentium [57]. Protease inhibitors (e.g., elafin, SLPI), hydrolases attacking the bacterial envelope (e.g., lysozyme or phospholipase A), and chemokines (e.g., CXCL9) have also been connected to bacterial killing [58]. Other defensive

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factors induced upon infection can indirectly target bacterial growth, such as lactoferrin or lipocalin 2, which sequester extracellular iron or bacterial siderophores, respectively [59]. Most AMPs and bactericidal factors are retained and accumulate in the protective mucus layer [60,61], preventing bacterial contact with the host cells and therefore uncontrolled inflammation [62]. There do not seem to be fundamental differences between the principal tissue organization and protective functions along the gastrointestinal tract. Yet while the healthy stomach is usually a rather sterile compartment and therefore shows little infiltration of hematopoietic cells [41], the increasing density of the microbiota toward the colon is counterbalanced by increasing numbers of innate lymphoid cells as well as T and B cell populations in the tissue. Upon infection with H. pylori, however, this condition is somewhat mirrored in the stomach mucosa, which then also exhibits immune cell infiltration, including neutrophil recruitment in the case of chronic active gastritis [63]. The epithelial lining can thus be viewed as a central element in orchestrating the equilibrium between the immune system and the adjacent microbiome. If the balance shifts, epithelial cells can enroll potent immune responses to effectively fight infections both autonomously and upon instruction by the professional immune compartment.

IV. VACCINES AS STIMULATORS OF MUCOSAL IMMUNITY The ability to actively vaccinate signifies a huge breakthrough in our fight against infections. Most vaccines rely on the induction of a pathogen-specific adaptive immune response. This occurs by activation of T cells through the presentation of one or more antigens via MHC molecules on the surface of APCs (see Fig. 33.2). Typically, it is the professional

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FIGURE 33.2 Mechanisms of immune suppression and immune escape by Helicobacter pylori. To allow for lifelong colonization in the human gastric mucosa H. pylori elicits several immune-regulatory factors. Two of those, VacA and GGT, lead to a suppression of T cell activation, dampening antimicrobial Th1/Th17 responses. Two additional mechanisms act at the epithelial effector site, preventing the synthesis of bactericidal AMPs. One mechanism is CagA-dependent and thus counteracts the increased inflammatory potential of type 4 secretion system positive strains. The other mechanism applies for all H. pylori isolates and leads to the destruction of lipid raft-dependent receptors, including the IFNγ and IL-22 receptors.

phagocytes that present antigens via class II MHC molecules to induce CD41 T cells and, farther downstream, lead to the activation of B cells and the production of pathogen-specific neutralizing antibodies [21]. However, besides their role in activating such specific B cells, increasing evidence points to a wider role for T cells and associated effector molecules in mucosal immune surveillance [21]. Targeting mucosal pathogens through vaccination is thought to require particular approaches, as the mucosal immune mechanisms differ from the systemic responses. A hallmark of adaptive mucosal immunity is the production of secretory immunoglobulin A (SIgA) [40] (Chapter 4: Protective Activities of Mucosal Antibodies). SIgA is accumulated in the mucus layer and contributes to barrier

function by controlling the overgrowth of commensal pathogens, opsonizing luminal bacteria to facilitate phagocytosis, or neutralizing toxins and pathogens [64]. The significance of SIgA activity has been mainly connected to immune exclusion, limiting the access of potentially harmful microorganisms to the epithelial layer [65]. Strikingly, though, individuals who experience an IgA deficiency do not overly suffer from recurrent infections [66]. It is plausible then to speculate that in some cases, SIgA may a have a regulatory function in mucosal immunity rather than in the decisive clearance of pathogens [66,67]. Keeping commensal microbiota and potentially associated pathogens in balance is likely an important aspect of IgA function and might involve polyreactive lowaffinity antibodies that differ from systemic immune responses, which usually exhibit high specificity and affinity [68] (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity). Finally, IgM class antibodies produced by plasma cells located in the lamina propria can also be transported to the gut lumen and help to keep pathogens and toxins at bay [69]. Effective immune protection against different mucosal pathogens likely requires distinct vaccination regimens. Depending on the type of pathogenic challenge, innate immune cells, such as APCs, instruct naı¨ve CD41 T cells to proliferate and to differentiate into a particular T helper cell subset. Each subset activates distinct effector functions, capable of conducting pathogen control and clearance. Since every pathogen may be more or less susceptible to a specific set of effectors, an appropriate immune response depends very much on both the induction of the best-suited T helper cell subset and the correct functioning of the downstream effector mechanisms (Fig. 33.1) [70]. The main aim of a vaccine is to induce and boost the appropriate immune response for a given pathogen. Ironically, the vast majority of existing vaccines have been discovered empirically,

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ignoring inductor and effector mechanisms involved in the acquired protection [21]. Recently acquired basic knowledge about the variety of mechanisms ruling the host pathogen interplay could drive the design of new strategies in a potentially more efficient way.

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concerned with explaining the underlying mechanisms leading to such a striking inflammatory response and have only recently led to an unexpected yet conclusive insight: A small carbohydrate precursor molecule of lipopolysaccharide (LPS) synthesis, adenosine diphosphate (ADP) heptose, is released into host cells in a T4SS-dependent manner to target a novel innate pattern recognition system [74,75]. Binding of ADP-heptose to the ALPK1 kinase

H. pylori’s ability to persist for life obviously means that the immune system fails to elicit responses that are appropriate and sufficient to overcome the infection. In this light, it seems puzzling that the infection is limited to about 50% of the population and not everybody is infected. Does not everyone come into contact with this pathogen, or do some people withstand infection by an unknown natural mechanism? Support for the latter notion derives from the observation that a subgroup of human volunteers deliberately infected with H. pylori exhibited spontaneous clearance irrespective of a vaccination attempt [16]. While this provides hope for the existence of early clearance mechanisms capable of preventing establishment of H. pylori persistence, it may depend on the predisposition of the individual. In contrast, immune mechanisms seen during persistency of H. pylori seem to be rather ineffective in clearing the infection, although a balance between pathogen and host may be achieved, allowing for the majority of infections to proceed asymptomatically. Yet almost all H. pylori infections are connected with at least a mild stage of inflammation [6]. Only occasionally, this inflammatory condition expands to cause an active chronic gastritis as the first stage of a sequence of even more severe pathologies [6]. The more aggressive forms of the infection appear to depend on the presence of H. pylori’s cagPAI encoded type 4 secretion system (T4SS) [71 73]. Numerous studies have been

FIGURE 33.3 Pathogen recognition and early proinflammatory response to infection with Helicobacter pylori. Highly pathogenic traits of H. pylori characterized by a cagPAI type 4 secretion system (T4SS) are readily recognized by human epithelial cells via their release of ADPheptose. ALPK1 serves as a pattern recognition receptor for ADP-heptose, leading to vivid activation of NF-κB and expression of proinflammatory downstream targets.

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[43] thus leads to the activation of TIFA [76] followed by strong NF-κB activation and IL-8 secretion in epithelial cells (Fig. 33.3). Secreted IL-8 results in massive phagocyte and neutrophil infiltration [77], generating a proinflammatory milieu, which in turn leads to a Th1 and Th17 biased adaptive immune response upon activation of T helper cells [5]. Numerous studies have described the predominant differentiation of naı¨ve CD41 T cells to the Th1 lineage [78 80]. In animal models, Th1-related IFNγ contributes to a higher protection against H. pylori by the natural host response [32,81]. Th17 and Th22 subsets have also been linked to the response to H. pylori. Infected IL-23 KO mice showed a lower inflammation score associated with decreased IL-17 levels and a higher bacterial load compared to wild-type mice [82]. IL-23 has also been described to induce Th22 differentiation and IL-22 production, contributing to gastritis progression in H. pylori-infected mice as well as patients [83,84]. Finally, abundant iNOSproducing plasma cells have been shown to infiltrate the H. pylori-infected mucosa, some of which produce pathogen-specific antibodies [85]. While this highly inflamed environment is thought to keep the infection at balance, it is also held responsible for driving H. pylori-associated pathology, being incapable of clearing the infection.

VI. THE FAILURE OF PAST VACCINATION ATTEMPTS Since the discovery of H. pylori approximately 35 years ago [11], numerous vaccination studies have sought to confer protection against this pathogen, with moderate success in animal models and complete failure to eradicate infection in humans, regardless of the antigen, adjuvants, or route of administration (extensively reviewed in 15, 25, 27). The only report of successful vaccination has been in children;

however, the lack of reported details and the lack of follow-up data since the original publication have prevented a full evaluation of the vaccine’s success and suitability for large-scale application [86]. At present, therefore, research in this field seems to have arrived at a dead end, and most pharmaceutical companies no longer pursue development of an H. pylori vaccine [26]. Nevertheless, despite the lack of success, preclinical and clinical vaccine trials together with basic research on H. pylori-driven inflammation have provided various types of insight that could help to generate new approaches for clearing this bacterium. Preclinical vaccination studies have started in the early 1990s and initially generated encouraging results in immunized mice challenged with Helicobacter felis; protective immune responses and even bacterial clearance were achieved [87]. Subsequent infection models for H. pylori itself, however, yielded only partial protection [88,89]. While these initial vaccines were administered orally and based on urease as antigen, either in combination with cholera toxin as adjuvant or expressed in recombinant attenuated Salmonella typhimurium carrier strains, many other variations were explored in both animals and humans, also involving other pathogen antigens [90]. Despite the lack of achievement of sterilizing immunity, insight from animal models has helped to pinpoint the tentatively best approaches to reach protection. In most cases, different degrees of protection were associated with T-cell-mediated responses, involving Th1 and/or Th17 subsets in particular [28 31], while antibody responses seemed to be dispensable [91 93]. Clinical trials performed in humans rendered fewer but similar conclusions [16,94]. H. pylori antigens had been chosen on the basis of different criteria, such as natural immunogenicity, protein abundance, surface exposition, and structural conservation within the H. pylori population, and these antigens included urease, Hp281, CagA, Hsp60, and VacA. Apart from

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these, certain antigen targets may have particular potential, as their inhibition via vaccination are considered to prevent immune suppression (GGT) or at least dampen inflammation by reducing the pathogen’s tissue-invasive potential by targeting related factors such as HtrA [27,95]. Several studies demonstrated significant responses to vaccine candidates, although they ultimately failed to confer full protection or clearance [15]. It is therefore believed that the main obstacle to obtaining a sterilizing vaccine is not primarily linked to the antigenic composition of the vaccine but rather lies in the vast capacity of the bacteria to evade immunity and that only new, creative solutions will be able to overcome this obstacle.

VII. UNMASKING HELICOBACTER PYLORI’S IMMUNE EVASION STRATEGY The ability to so effectively escape the powerful immune response of the host must be based in elaborate mechanisms employed by the pathogen [17]. As mentioned above, H. pylori has been shown to suppress T cell activation [5]. This is due to the bacterial factors VacA and GGT, which together promote immune tolerance by preventing activation and proliferation of CD41 T cells (Fig. 33.2, left) while inducing differentiation toward a Treg subset [13,14]. In consequence, differentiation to Th1 and Th17 lineages is reduced and their effector functions are suppressed by Treg cells [5,13,14,96]. These discoveries suggested that overcoming immune tolerance could be the last hurdle to obtaining a sterilizing vaccine, as Treg induction appeared to be the main bacterial mechanism to evade the protective natural immune response. Efforts were made to target GGT in order to counteract immune suppression [96]. Nonetheless, any new vaccine candidates have failed to eliminate infection despite a robust enhancement of cellmediated immunity [17], suggesting that the

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link between CD41 T cells and relevant effector functions at the mucosal sites against H. pylori must be blocked [97]. Until recently, the role of the epithelium in the defense against H. pylori received little attention. This shortfall is somewhat surprising in light of the predominant location of H. pylori at the luminal side of the epithelium [8,98] with only little penetration of bacteria into the submucosal layers except in the case of progressive inflammatory damage and ulcer formation [6,95,99 101]. Thus the epithelium obviously must have a major role not only in pathogen sensing but also as the main effector side of the infection (Fig. 33.1). Not surprisingly, recent studies aimed at elucidating the modulation of epithelial defense mechanisms by H. pylori have led to some astounding observations. Epithelial AMPs appear to play a relevant role as immune effectors against H. pylori. Unlike intestinal commensal bacteria, H. pylori requires intimate contact with epithelial cells and therefore needs to overcome the antimicrobial epithelial factors retained in the deep mucus layers, which ensure a spatial separation from the epithelium [62]. Epithelial cells have the potential to secrete antibacterial factors, active against H. pylori [102]. Surprisingly though, such bactericidal factors, including hBD3, are not detected in biopsies from infected patients [50,103]. Rather, the infected mucosa exhibits human β-defensins 1 and 2 (hBD1 and hBD2) and elafin, which are only moderately effective against H. pylori [50,103], probably owing to particular modifications in the lipid A part of H. pylori LPS [104]. Despite continuous bacterial stimulation of epithelial cells and a favorable proinflammatory cytokine milieu, H. pylori actively shuts down expression of hBD3 in different ways. The first mechanism was shown to be dependent on the active translocation of CagA protein to infected cells via the T4SS [50]. While upon contact of H. pylori with host epithelial cells an immediate upregulation of hBD3 has

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FIGURE 33.4 Initial activation and subsequent downregulation of human β-defensin 3 (hBD3) by Helicobacter pylori. Early in the course of infection, H. pylori triggers an antibacterial response involving the epidermal growth factor receptor (EGFR). Subsequent release of human hBD3 leads to the partial killing of H. pylori. However, surviving bacteria translocate CagA, which upon phosphorylation activates SHP2 to cause inactivation of EGFR and downregulation of hBD3, supporting H. pylori survival.

been observed in vitro, that is, via activation of EGFR and likely additional receptors, this led to the killing of some bacteria on the surface of epithelial cells, as evidenced by the appearance of coccoid forms of the pathogen [50,105]. However, some bacteria still managed to translocate CagA into host cells, which upon phosphorylation caused an activation of SHP2 phosphatase [106], followed by dephosphorylation of the EGF receptor. Consequently, blockage of the EGF pathway causes downregulation of hBD3 and thus an increased survival of CagA translocationcompetent bacteria on the epithelial cell surface (Fig. 33.4). While hBD3 indeed seems to be among the most effective defensins against H. pylori, it is downregulated in the infected stomach [103]. Thus it has been postulated that this mechanisms serves to ensure the persistence of highly pathogenic, that is, cagPAI type 4 secretion system positive H. pylori. However, this would not explain the ability of cagPAI negative H. pylori strains to persist or the lack of induction of hBD3 expression under such a proinflammatory cytokine milieu.

Most important, a postulated second, highly effective escape mechanism has recently been described [81]. It is based on the bacterial cholesterol-α-glucosyltransferase (CGT), which mediates cholesterol depletion from the surface of host epithelial cells [34,107]. Although it was obvious from this initial study that cholesterol and CGT play a prominent role in the regulation of the interferon-driven response against H. pylori, the precise function of CGT remained obscure for a long time. Only recently, it became clear that CGT activity blocks the activation of JAK/STAT signaling in human gastric primary epithelial cells treated with IFNγ [81]. This could be explained by the finding that receptors for IFNγ require functional lipid rafts to transduce signaling [108]. Accordingly, epithelial cells infected with H. pylori become unresponsive to important T-cell-derived inflammatory cytokines such as IFNγ or IL-22 (Fig. 33.5). The response to other cytokines, such as IFNβ, or IL-6, is also silenced by this mechanism. One result of JAK/STAT blockage is the loss of hBD3 synthesis in IFNγ or IL-22 treated epithelial cells, explaining why

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FIGURE 33.5 Blockage of innate defense mechanisms by Helicobacter pylori via cholesterol glucosylation and extraction from the site of infection. Upon glucosylation, host cell cholesterol becomes incorporated in the bacterial membrane by the action of CGT. However, gastric epithelial cells respond to inflammatory cytokines, such as IFNγ and IL-22, via respective receptors (e.g., IFNGR and IL22R), whose subunits require lipid rafts for assembly. Under normal conditions, stimulation with IFNγ or IL-22 produced by T cells triggers JAK/STAT pathway activation, leading to STAT1 or STAT3 phosphorylation. Once activated, STAT1 and STAT3 are translocated to the nucleus to promote expression of genes involved in inflammation and defense. Infection by H. pylori results in CGT-mediated depletion of host cell cholesterol, disruption of lipid rafts, and disassembly of IFNGR and IL22R subunits, thereby preventing JAK/STAT signaling.

H. pylori, whether cagPAI positive or negative, is able to persist in the stomach [81]. The recent development of advanced, highly polarized epithelial culture models based on primary cells has for the first time enabled infections to be observed for several days [102]. Under these conditions, H. pylori forms localized foci of infection where JAK/STAT signaling is silenced, while noninfected areas of the same culture show a robust activation of STAT1 or STAT3 upon cytokine treatment [81]. These data suggest that H. pylori generates

epithelial microniches that are protected from inflammatory stimuli. This notion is consistent with recent observations indicating that superinfections with H. pylori take place only at gastric sites already colonized by H. pylori [109]. Together, these findings shed critical light on our current vaccination strategies against H. pylori in that they suggest that even the strongest induction of an immune response would fail to mobilize crucial epithelial immune effectors, such as hBD3, that are blocked as a result of CGT activity (Fig. 33.6).

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FIGURE 33.6 Overview of Helicobacter pylori mechanisms affecting inflammation and defense on the gastric mucosal surfaces. One of the very early events of H. pylori infection is the induction of a proinflammatory response, owing to the release of ADP-heptose. To combat the resulting epithelial defense mechanisms, H. pylori factors interfere with host immune mechanisms. Both VacA and GGT act to prevent proper T cell activation at the immune induction site. The translocated CagA protein counteracts the increased inflammatory response elicited by its T4SS. However, by modifying and extracting host cell cholesterol, CGT prevents the activation of infected epithelial cells in more general terms, as it blocks the inflammatory response and associated epithelial cells, generating a protected niche for lifelong mucosal colonization by H. pylori. While the dampened T cell activation theoretically could be overcome by appropriate vaccination strategies, vaccination does not seem to rescue the epithelial shutoff at the site of infection, thus explaining the failure of current vaccination programs.

VIII. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Although vaccination approaches have been highly successful against numerous important pathogens [110], some pathogens are persistent by nature and have resisted truly effective immunization, despite the enormous efforts that have been made in some of these cases, such as malaria, tuberculosis, HIV, and now H. pylori as well. The multilayered immune evasion strategy of H. pylori poses an enormous challenge for the design of a sterilizing vaccine. Recent investigations, however, have provided important clues to help in understanding the failure of past vaccination attempts. As a case in point, H. pylori blocks host immunity at the two principal stages: the induction of effective

T cell responses and the execution of the immune defense, for example, by the release of powerful effector molecules from the mucosal surfaces (Fig. 33.2). While the first blockage could be circumvented by effective immunization, this could cause adverse effects, resulting in an increased inflammatory burden with little effect of H. pylori clearance. It is also doubtful whether the second blockage can be overcome by simple vaccination. This observation provides a compelling paradigm rationalizing the limits of traditional vaccination strategies that, so far, have rested on the predominant search for steadily improved antigens and adjuvant regimens. Given the lessons learned from H. pylori, it appears possible that other pathogens that still resist effective vaccination rely on related

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REFERENCES

blockage mechanisms at the site of immune effector function. The considerable list of vaccine failures thus urges for rational investigation of the underlying reasons. Interestingly, a critical role of cholesterol downregulation has also been implicated in Leishmania parasite survival in the infected host, hinting at potentially related processes [111]. Another principal escape mechanism, capable of paralyzing an otherwise functional immune effector program, involves vigorous antigenic variation, as seen for the gonococcus, African trypanosomes or nontypable Haemophilus influenzae [112 114]. No robust vaccine has been found thus far to overcome effective blockage at the immune execution site. The fact that past H. pylori vaccination attempts have already generated a robust T cell response suggests that immunization, but not protection, can be achieved in infected individuals [15,25 27]. This has stimulated researchers not only to search for better antigens, vaccine formulations, or delivery routes, but also to specifically inhibit immune interference mechanisms via vaccination. Obvious antigen targets of H. pylori would include the immunerepressive factors VacA, GGT, and CagA. However, even for these immune-relevant targets, no protection has been reported. Also, the immune escape factor CGT presented here could be such a target. However, it might be difficult to achieve full enzymatic inhibition of the protein in this way in order to prevent cholesterol modification and its depletion from infected cells. Finally, for solutions focused on preventing the disease outcome of H. pylori infections rather than the pathogen’s complete elimination, one might consider antigen targets that are assumed to be involved in increased inflammation and pathology of the infection, such as the CagA protein and HtrA, which has recently been implicated in mucosal penetration [95]. However, while such strategies appear to be plausible in theory, to achieve functional inhibition of these factors by

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immunization, one might begin to consider the suitability of combination approaches involving both pharmacological inhibition of immune escape factors and vaccination. Moreover, particularly in therapeutic vaccination settings, reversal of the immunosuppressive features of H. pylori in the stomach may amplify the risks related to inflammationinduced damage. Particularly, vaccination that fails to achieve complete pathogen clearance could result in increased inflammation and a worsening of the H. pylori-related pathology, especially if the vaccine targets the immune suppressors VacA or GGT. Likewise, the inflammatory responses associated with a sterilizing vaccine could aid cancer initiation or progression, as many of the protective cytokines (e.g., IL-17, IL-22, IFNγ) have been associated with increased epithelial proliferation, the appearance of preneoplastic lesions, and tumorigenesis [32,115 117]. Rigorous risk assessment of any future vaccine or combination approaches therefore remains mandatory. Nonetheless, considering the immense burden of gastric cancer that continues to exist, it seems of utmost importance, on the basis of the recent scientific advancements, to pursue novel concepts and design innovative strategies to ban this pathogen’s deleterious outcomes.

References [1] Monack DM. Helicobacter and salmonella persistent infection strategies. Cold Spring Harb Perspect Med 2013;3:a010348. [2] Yucel O. Prevention of Helicobacter pylori infection in childhood. World J Gastroenterol 2014;20:10348 54. [3] Linz B, Balloux F, Moodley Y, Manica A, Liu H, Roumagnac P, et al. An African origin for the intimate association between humans and Helicobacter pylori. Nature 2007;445:915 18. [4] Smet A, Yahara K, Rossi M, Tay A, Backert S, Armin E, et al. Macroevolution of gastric Helicobacter species unveils interspecies admixture and time of divergence. ISME J 2018;12:2518 31.

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33. A FUTURE FOR A VACCINE AGAINST THE CANCER-INDUCING BACTERIUM HELICOBACTER PYLORI?

[5] Salama NR, Hartung ML, Mu¨ller A. Life in the human stomach: persistence strategies of the bacterial pathogen Helicobacter pylori. Nat Rev Microbiol 2013;11:385 99. [6] Bauer B, Meyer TF. The human gastric pathogen Helicobacter pylori and its association with gastric cancer and ulcer disease. Ulcers 2011;2011:1 23. [7] Johnson KS, Ottemann KM. Colonization, localization, and inflammation: the roles of H. pylori chemotaxis in vivo. Curr Opin Microbiol 2018;41:51 7. [8] Schreiber S, Konradt M, Groll C, Scheid P, Hanauer G, Werling HO, et al. The spatial orientation of Helicobacter pylori in the gastric mucus. Proc Natl Acad Sci USA 2004;101:5024 9. [9] Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. Global prevalence of Helicobacter pylori infection: systematic review and meta-analysis. Gastroenterology 2017;153:420 9. [10] Hunt RH, Camilleri M, Crowe SE, El-Omar EM, Fox JG, Kuipers EJ, et al. The stomach in health and disease. Gut 2015;64:1650 68. [11] Warren JR, Marshall B. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983;321:1273 5. [12] Mazzucchelli L, Blaser A, Kappeler A, Scharli P, Laissue JA, Baggiolini M, et al. BCA-1 is highly expressed in Helicobacter pylori-induced mucosaassociated lymphoid tissue and gastric lymphoma. J Clin Invest 1999;104:R49 54. [13] Gebert B, Fischer W, Weiss E, Hoffmann R, Haas R. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. Science 2003;301:1099 102. [14] Oertli M, Noben M, Engler DB, Semper RP, Reuter S, Maxeiner J, et al. Helicobacter pylori γ-glutamyl transpeptidase and vacuolating cytotoxin promote gastric persistence and immune tolerance. Proc Natl Acad Sci USA 2013;110:3047 52. [15] Czinn SJ, Blanchard T. Vaccinating against Helicobacter pylori infection. Nat Rev Gastroenterol Hepatol 2011;8:133 40. [16] Aebischer T, Bumann D, Epple HJ, Metzger W, Schneider T, Cherepnev G, et al. Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines. Gut 2008;57:1065 72. [17] Malfertheiner P, Selgrad M, Wex T, Romi B, Borgogni E, Spensieri F, et al. Efficacy, immunogenicity, and safety of a parenteral vaccine against Helicobacter pylori in healthy volunteers challenged with a Cagpositive strain: a randomised, placebo-controlled phase 1/2 study. Lancet Gastroenterol Hepatol 2018;3:698 707.

[18] Van Avondt K, van Sorge NM, Meyaard L. Bacterial immune evasion through manipulation of host inhibitory immune signaling. PLoS Pathog 2015;11:e1004644. [19] Malfertheiner P, Schultze V, Rosenkranz B, Kaufmann SH, Ulrichs T, Novicki D, et al. Safety and immunogenicity of an intramuscular Helicobacter pylori vaccine in noninfected volunteers: a phase I study. Gastroenterology 2008;135:787 95. [20] De Gregorio E, Rappuoli R. From empiricism to rational design: a personal perspective of the evolution of vaccine development. Nat Rev Immunol 2014;14: 505 14. [21] Pulendran B, Ahmed R. Immunological mechanisms of vaccination. Nat Immunol 2011;12:509 17. [22] Finco O, Rappuoli R. Designing vaccines for the twenty-first century society. Front Immunol 2014;5:12. [23] O’Morain NR, Dore MP, O’Connor AJP, Gisbert JP, O’Morain CA. Treatment of Helicobacter pylori infection in 2018. Helicobacter 2018;23(Suppl. 1):e12519. [24] Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 2018;18:318 27. [25] Del Giudice G, Malfertheiner P, Rappuoli R. Development of vaccines against Helicobacter pylori. Expert Rev Vaccines 2009;8:1037 49. [26] Sutton P, Boag JM. Status of vaccine research and development for Helicobacter pylori. Vaccine 2018;. Available from: https://doi.org/10.1016/j.vaccine.2018.01.001 [Epub ahead of print]. [27] Sutton P, Chionh YT. Why can’t we make an effective vaccine against Helicobacter pylori? Expert Rev Vaccines 2013;12:433 41. [28] Akhiani AA, Pappo J, Kabok Z, Schon K, Gao W, Franze´n LE, et al. Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells. J Immunol 2002;169:6977 84. [29] Flach CF, Svensson N, Blomquist M, Ekman A, Raghavan S, Holmgren J. A truncated form of HpaA is a promising antigen for use in a vaccine against Helicobacter pylori. Vaccine 2011;29:1235 41. [30] Hitzler I, Oertli M, Becher B, Agger EM, Mu¨ller A. Dendritic cells prevent rather than promote immunity conferred by a helicobacter vaccine using a mycobacterial adjuvant. Gastroenterology 2011;141:186 196.e1. [31] Sawai N, Kita M, Kodama T, Tanahashi T, Yamaoka Y, Tagawa Y, et al. Role of gamma interferon in Helicobacter pylori-induced gastric inflammatory responses in a mouse model. Infect Immun 1999;67:279 85.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

[32] Sayi A, Kohler E, Hitzler I, Arnold I, Schwendener R, Rehrauer H, et al. The CD4 1 T cell-mediated IFNgamma response to Helicobacter infection is essential for clearance and determines gastric cancer risk. J Immunol 2009;182:7085 101. [33] Sutton P, Danon SJ, Walker M, Thompson LJ, Wilson J, Kosaka T, et al. Post-immunisation gastritis and Helicobacter infection in the mouse: a long term study. Gut 2001;49:467 73. [34] Wunder C, Churin Y, Winau F, Warnecke D, Vieth M, Lindner B, et al. Cholesterol glucosylation promotes immune evasion by Helicobacter pylori. Nat Med 2006;12:1030 8. [35] Fischbach LA, Nordenstedt H, Kramer JR, Gandhi S, Dick-Onuoha S, Lewis A, et al. The association between Barrett’s esophagus and Helicobacter pylori infection: a meta-analysis. Helicobacter 2012;17:163 75. [36] Smolka AJ, Schubert ML. Helicobacter pylori-induced changes in gastric acid secretion and upper gastrointestinal disease. Curr Top Microbiol Immunol 2017;400:227 52. [37] Allaire JM, Crowley SM, Law HT, Chang SY, Ko HJ, Vallance BA. The intestinal epithelium: central coordinator of mucosal immunity. Trends Immunol 2018;39:677 96. [38] Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol 2014;14:141 53. [39] Ahluwalia B, Magnusson MK, Ohman L. Mucosal immune system of the gastrointestinal tract: maintaining balance between the good and the bad. Scand J Gastroenterol 2017;52:1185 93. [40] Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt’s essential immunology. 13th ed. John Wiley & Sons; 2017. [41] Sigal M, Logan CY, Kapalczynska M, Mollenkopf HJ, Berger H, Wiedenmann B, et al. Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis. Nature 2017;548:451 5. [42] Willet SG, Mills JC. Stomach organ and cell lineage differentiation: from embryogenesis to adult homeostasis. Cell Mol Gastroenterol Hepatol 2016;2:546 59. [43] Zhou P, She Y, Dong N, Li P, He H, Borio A, et al. Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose. Nature 2018;561:122 6. [44] Ostaff MJ, Stange EF, Wehkamp J. Antimicrobial peptides and gut microbiota in homeostasis and pathology. EMBO Mol Med 2013;5:1465 83. [45] Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 2005;3:238 50.

593

[46] Gee ML, Burton M, Grevis-James A, Hossain MA, McArthur S, Palombo EA, et al. Imaging the action of antimicrobial peptides on living bacterial cells. Sci Rep 2013;3:1557. [47] Wenzel M, Chiriac AI, Otto A, Zweytick D, May C, Schumacher C, et al. Small cationic antimicrobial peptides delocalize peripheral membrane proteins. Proc Natl Acad Sci 2014;111:E1409 18. [48] Zasloff M. Antimicrobial peptides of multicellular organisms, 2002. https://doi.org/10.1038/415389a. [49] Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710 20. [50] Bauer B, Pang E, Holland C, Kessler M, Bartfeld S, Meyer TF. The Helicobacter pylori virulence effector CagA abrogates human b-defensin 3 expression via inactivation of EGFR signaling. Cell Host Microbe 2012;11:576 86. [51] Boughan PK, Argent RH, Body-malapel M, Park J-H, Ewings KE, Bowie AG, et al. Nucleotide-binding oligomerization domain-1 and epidermal growth factor receptor. J Biol Chem 2006;281:11637 48. [52] Harder J, Meyer-Hoffert U, Teran LM, Schwichtenberg L, Bartels J, Maune S, et al. Mucoid Pseudomonas aeruginosa, TNF-alpha, and IL-1beta, but not IL-6, induce human beta-defensin-2 in respiratory epithelia. Am J Respir Cell Mol Biol 2000;22:714 21. [53] Kolls JK, McCray PB, Chan YR. Cytokine-mediated regulation of antimicrobial proteins. Nat Rev Immunol 2008;8:829 35. [54] Joly S, Organ CC, Johnson GK, McCray PB, Guthmiller JM. Correlation between beta-defensin expression and induction profiles in gingival keratinocytes. Mol Immunol 2005;42:1073 84. [55] Wolk K, Kunz S, Witte E, Friedrich M, Asadullah K, Sabat R. IL-22 increases the innate immunity of tissues. Immunity 2004;21:241 54. [56] Mukherjee S, Zheng H, Derebe MG, Callenberg KM, Partch CL, Rollins D, et al. Antibacterial membrane attack by a pore-forming intestinal C-type lectin. Nature 2014;505:103 7. [57] Zheng Y, Valdez PA, Danilenko DM, Hu Y, Sa SM, Gong Q, et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med 2008;14:282 9. [58] Chung LK, Raffatellu M. G.I. pros: antimicrobial defense in the gastrointestinal tract. Semin Cell Dev Biol 2018;. Available from: https://doi.org/10.1016/ j.semcdb.2018.02.001. [59] Wilson BR, Bogdan AR, Miyazawa M, Hashimoto K, Tsuji Y, Magill SS, et al. Siderophores in iron metabolism: from mechanism to therapy potential. Trends Mol Med 2016;22:1077 90.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

594

33. A FUTURE FOR A VACCINE AGAINST THE CANCER-INDUCING BACTERIUM HELICOBACTER PYLORI?

[60] Antoni L, Nuding S, Weller D, Gersemann M, Ott G, Wehkamp J, et al. Human colonic mucus is a reservoir for antimicrobial peptides. J Crohn’s Colitis 2013;. Available from: https://doi.org/10.1016/j. crohns.2013.05.006. [61] Meyer-Hoffert U, Hornef MW, Henriques-Normark B, Axelsson LG, Midtvedt T, Pu¨tsep K, et al. Secreted enteric antimicrobial activity localises to the mucus surface layer. Gut 2008;57:764 71. [62] Vaishnava S, Yamamoto M, Severson KM, Ruhn KA, Yu X, Koren O, et al. The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine. Science 2011;334:255 8. [63] Correa P, Piazuelo MB. The gastric precancerous cascade. J Dig Dis 2012;13:2 9. [64] Boyaka PN. Inducing mucosal IgA: a challenge for vaccine adjuvants and delivery systems. J Immunol 2017;199:9 16. [65] Corthe´sy B. Multi-faceted functions of secretory IgA at mucosal surfaces. Front Immunol 2013;4:185. [66] Gommerman JL, Rojas OL, Fritz JH. Re-thinking the functions of IgA 1 plasma cells. Gut Microbes 2014;5:652 62. [67] Bunker JJ, Erickson SA, Flynn TM, Henry C, Koval JC, Meisel M, et al. Natural polyreactive IgA antibodies coat the intestinal microbiota. Science 2017;358. [68] Bunker JJ, Bendelac A. IgA responses to microbiota. Immunity 2018;49:211 24. [69] Janeway CA, Travers P, Walport M, Shlomchik MJ. Immunobiology: the immune system in health and disease. New York: Garland Science; 2005. [70] O’Shea JJ, Paul WE. Mechanisms underlying lineage commitment and plasticity of helper CD4 1 T cells. Science 2010;327:1098 102. [71] Blaser MJ, Perez-Perez GI, Kleanthous H, Cover TL, Peek RM, Chyou P, et al. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res 1995;55:2111 15. [72] Covacci A, Telford JL, Del Giudice G, Parsonnet J, Rappuoli R. Helicobacter pylori virulence and genetic geography. Science 1999;284:1328 33. [73] Parsonnet J, Friedman G, Orentreich N, Vogelman H. Risk for gastric cancer in people with CagA positive or CagA negative Helicobacter pylori infection. Gut 1997;40:297 301. [74] Pfannkuch L, Hurwitz R, Traulsen J, Kosma P, Schmid M, Meyer T. ADP heptose, a novel pathogen associated molecular pattern associated with Helicobacter pylori type 4 secretion. bioRxiv, 2018. [75] Zimmermann S, Pfannkuch L, Al-Zeer MA, Bartfeld S, Koch M, Liu J, et al. ALPK1- and TIFA-dependent

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

innate immune response triggered by the Helicobacter pylori Type IV Secretion System. Cell Rep 2017;20:2384 95. Gaudet RG, Sintsova A, Buckwalter CM, Leung N, Cochrane A, Li J, et al. INNATE IMMUNITY. Cytosolic detection of the bacterial metabolite HBP activates TIFA-dependent innate immunity. Science 2015;348:1251 5. Eck M, Schmausser B, Scheller K, Toksoy A, Kraus M, Menzel T, et al. CXC chemokines Gro(alpha)/IL-8 and IP-10/MIG in Helicobacter pylori gastritis. Clin Exp Immunol 2000;122:192 9. Bagheri N, Salimzadeh L, Shirzad H. The role of T helper 1-cell response in Helicobacter pylori-infection. Microb Pathog 2018;123:1 8. Itoh T, Wakatsuki Y, Yoshida M, Usui T, Matsunaga Y, Kaneko S, et al. The vast majority of gastric T cells are polarized to produce T helper 1 type cytokines upon antigenic stimulation despite the absence of Helicobacter pylori infection. J Gastroenterol 1999;34:560 70. Smythies LE, Waites KB, Lindsey JR, Harris PR, Ghiara P, Smith PD. Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-gamma, gene-deficient mice. J Immunol 2000;165:1022 9. Morey P, Pfannkuch L, Pang E, Boccellato F, Sigal M, Imai-Matsushima A, et al. Helicobacter pylori depletes cholesterol in gastric glands to prevent interferon gamma signaling and escape the inflammatory response. Gastroenterology 2018;154:1391 1404.e9. Horvath DJ, Washington MK, Cope VA, Algood HMS. IL-23 contributes to control of chronic Helicobacter pylori infection and the development of T helper responses in a mouse model. Front Immunol 2012;3:56. Dixon BREA, Radin JN, Piazuelo MB, Contreras DC, Algood HMS, Noto J, et al. IL-17a and IL-22 induce expression of antimicrobials in gastrointestinal epithelial cells and may contribute to epithelial cell defense against Helicobacter pylori. PLoS One 2016;11:e0148514. Zhuang Y, Cheng P, Liu XF, Peng LS, Li BS, Wang TT, et al. A pro-inflammatory role for Th22 cells in Helicobacter pylori-associated gastritis. Gut 2015;64:1368 78. Neumann L, Mueller M, Moos V, Heller F, Meyer TF, Loddenkemper C, et al. Mucosal inducible NO synthase-producing IgA 1 plasma cells in Helicobacter pylori-infected patients. J Immunol 2016;197:1801 8. Zeng M, Mao XH, Li JX, Tong WD, Wang B, Zhang YJ, et al. Efficacy, safety, and immunogenicity of an oral recombinant Helicobacter pylori vaccine in children in China: a randomised, double-blind, placebocontrolled, phase 3 trial. Lancet 2015;386:1457 64.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

[87] Michetti P, Corthesy-Theulaz I, Davin C, Haas R, Vaney AC, Heitz M, et al. Immunization of BALB/c mice against Helicobacter felis infection with Helicobacter pylori urease. Gastroenterology 1994;107:1002 11. [88] Corthesy-Theulaz I, Porta N, Glauser M, Saraga E, Vaney AC, Haas R, et al. Oral immunization with Helicobacter pylori urease B subunit as a treatment against Helicobacter infection in mice. Gastroenterology 1995;109:115 21. [89] Gomez-Duarte OG, Lucas B, Yan ZX, Panthel K, Haas R, Meyer TF. Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B. Vaccine 1998;16:460 71. [90] Sutton P, Doidge C. Helicobacter pylori vaccines spiral into the new millennium. Dig Liver Dis 2003;35: 675 87. [91] Ermak TH, Giannasca PJ, Nichols R, Myers GA, Nedrud J, Weltzin R, et al. Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses. J Exp Med 1998;188:2277 88. [92] Hitzler I, Kohler E, Engler DB, Yazgan AS, Mu¨ller A. The role of Th cell subsets in the control of Helicobacter infections and in T cell-driven gastric immunopathology. Front Immunol 2012;3:142. [93] Sutton P, Wilson J, Kosaka T, Wolowczuk I, Lee A. Therapeutic immunization against Helicobacter pylori infection in the absence of antibodies. Immunol Cell Biol 2000;78:28 30. [94] Metzger WG, Mansouri E, Kronawitter M, Diescher S, Soerensen M, Hurwitz R, et al. Impact of vectorpriming on the immunogenicity of a live recombinant Salmonella enterica serovar typhi Ty21a vaccine expressing urease A and B from Helicobacter pylori in human volunteers. Vaccine 2004;22:2273 7. [95] Tegtmeyer N, Wessler S, Necchi V, Rohde M, Harrer A, Rau TT, et al. Helicobacter pylori employs a unique basolateral type IV secretion mechanism for CagA delivery. Cell Host Microbe 2017;22:552 560.e5. [96] Wu¨stner S, Anderl F, Wanisch A, Sachs C, Steiger K, Nerlich A, et al. Helicobacter pylori gamma-glutamyl transferase contributes to colonization and differential recruitment of T cells during persistence. Sci Rep 2017;7:13636. [97] Aebischer T, Meyer TF, Andersen LP. Inflammation, immunity, and vaccines for Helicobacter. Helicobacter 2010;15(Suppl. 1):21 8. [98] Earle KA, Billings G, Sigal M, Lichtman JS, Hansson GC, Elias JE, et al. Quantitative imaging of gut microbiota spatial organization. Cell Host Microbe 2015;18:478 88.

595

[99] Howitt MR, Lee JY, Lertsethtakarn P, Vogelmann R, Joubert L-M, Ottemann KM, et al. ChePep controls Helicobacter pylori Infection of the gastric glands and chemotaxis in the Epsilonproteobacteria. mBio 2011;2 e00098-11. [100] Keilberg D, Zavros Y, Shepherd B, Salama NR, Ottemann KM. Spatial and temporal shifts in bacterial biogeography and gland occupation during the development of a chronic infection. mBio 2016;7: e01705 16. [101] Sigal M, Rothenberg ME, Logan CY, Lee JY, Honaker RW, Cooper RL, et al. Helicobacter pylori activates and expands Lgr5(1) stem cells through direct colonization of the gastric glands. Gastroenterology 2015;148 1392-404.e21. [102] Boccellato F, Woelffling S, Imai-Matsushima A, Sanchez G, Goosmann C, Schmid M, et al. Polarised epithelial monolayers of the gastric mucosa reveal insights into mucosal homeostasis and defence against infection. Gut 2018;. Available from: https://doi.org/ 10.1136/gutjnl-2017-314540:gutjnl-2017-314540. [103] Nuding S, Gersemann M, Hosaka Y, Konietzny S, Schaefer C, Beisner J, et al. Gastric antimicrobial peptides fail to eradicate Helicobacter pylori infection due to selective induction and resistance. PLoS One 2013;8:e73867. [104] Cullen TW, Giles DK, Wolf LN, Ecobichon C, Boneca IG, Trent MS. Helicobacter pylori versus the host: remodeling of the bacterial outer membrane is required for survival in the gastric mucosa. PLoS Pathog 2011;7 e1002454-e1002454. [105] Kusters JG, Gerrits MM, Van Strijp JA, Vandenbroucke-Grauls CM. Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infect Immun 1997;65:3672 9. [106] Higashi H, Tsutsumi R, Muto S, Sugiyama T, Azuma T, Asaka M, et al. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science 2002;295:683 6. [107] Lebrun A-H, Wunder C, Hildebrand J, Churin Y, Za¨hringer U, Lindner B, et al. Cloning of a cholesterol-alpha-glucosyltransferase from Helicobacter pylori. J Biol Chem 2006;281:27765 72. [108] Blouin CM, Lamaze C. Interferon gamma receptor: the beginning of the journey. Front Immunol 2013;4:267. [109] Salama NR, Gonzalez-Valencia G, Deatherage B, Aviles-Jimenez F, Atherton JC, Graham DY, et al. Genetic analysis of Helicobacter pylori strain populations colonizing the stomach at different times postinfection. J Bacteriol 2007;189:3834 45. [110] Bloom DE, Black S, Salisbury D, Rappuoli R. Antimicrobial resistance and the role of vaccines. Proc Natl Acad Sci USA 2018;115:12868 71.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

596

33. A FUTURE FOR A VACCINE AGAINST THE CANCER-INDUCING BACTERIUM HELICOBACTER PYLORI?

[111] Ghosh J, Bose M, Roy S, Bhattacharyya SN. Leishmania donovani targets Dicer1 to downregulate miR-122, lower serum cholesterol, and facilitate murine liver infection. Cell Host Microbe 2013;13:277 88. [112] Garmendia J, Marti-Lliteras P, Moleres J, Puig C, Bengoechea JA. Genotypic and phenotypic diversity of the noncapsulated Haemophilus influenzae: adaptation and pathogenesis in the human airways. Int Microbiol 2012;15:159 72. [113] Horn D. Antigenic variation in African trypanosomes. Mol Biochem Parasitol 2014;195:123 9. [114] Meyer TF, van Putten JP. Genetic mechanisms and biological implications of phase variation in

pathogenic neisseriae. Clin Microbiol Rev 1989;2 (Suppl.):S139 45. [115] Bockerstett KA, DiPaolo RJ. Regulation of gastric carcinogenesis by inflammatory cytokines. Cell Mol Gastroenterol Hepatol 2017;4:47 53. [116] Syu L-J, El-Zaatari M, Eaton KA, Liu Z, Tetarbe M, Keeley TM, et al. Transgenic expression of interferon-γ in mouse stomach leads to inflammation, metaplasia, and dysplasia. Am J Pathol 2012;181: 2114 25. [117] West NR, McCuaig S, Franchini F, Powrie F. Emerging cytokine networks in colorectal cancer. Nat Rev Immunol 2015;15:615 19.

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Mucosal Vaccines for Streptococcus pneumoniae Edwin Swiatlo1 and Larry S. McDaniel2 1

Section of Infectious Diseases, Southeast Louisiana VA Medical Center, New Orleans, LA, United States 2Department of Microbiology & Immunology, University of Mississippi Medical Center, Jackson, MS, United States

I. INTRODUCTION Infections of the lower respiratory tract are collectively one of the most common causes of death globally, and Streptococcus pneumoniae (pneumococcus) is among the most common bacterial etiologies of these infections [1]. In addition to lower respiratory infections such as pneumonia, pneumococci are common causes of bacteremia and sepsis, meningitis, empyema, and upper respiratory infections such as sinusitis and otitis media [2]. There is no question that pneumococcal infections exact a large toll in terms of morbidity and mortality worldwide. The pneumococcus is a Gram-positive bacterium that grows as diplococci or short chains (Fig. 34.1) and expresses a large variety of surface structures that are associated with virulence and host immune responses [1]. The extracellular polysaccharide capsule is widely considered the most dominant and crucial virulence factor of pneumococci and is the antigen component of currently available vaccines [3]. Serum antibodies to capsule are regarded as a Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00034-1

surrogate marker of protection; however, these antibodies are variably effective at preventing both respiratory tract infections and invasive disease. Also, there are nearly 100 antigenically distinct capsule types, and new serotypes continued to be identified [3]. Consequently, capsule-based vaccines are perpetually fraught with incomplete coverage and the continuous shifting of serotype prevalence in at-risk populations [4,5]. The pneumococcal capsule has received a great deal of study and development, but there are other molecules, both surface-associated and cytoplasmic, that are involved in pneumococcal virulence and can induce robust antigenspecific immune responses (Fig. 34.2). These proteins can be organized into groups based on their mechanism of attachment to the cell surface. Pneumococci express lipoproteins anchored in the cell membrane, and proteins that are attached to the cell wall peptidoglycan. A unique feature of pneumococci is a family of surface proteins that bind phosphocholine in the teichoic acids of the cell wall and membrane

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FIGURE 34.1 Gram stain of sputum showing Grampositive cocci in short chains and pairs characteristic of pneumococcus.

[6]. These choline-binding proteins have been studied for their role in immune evasion and their ability to induce protective responses both systemically and at mucosal surfaces [7]. Representatives from these classes of proteins have been studied for their immunogenicity and will be discussed in the following sections. Pneumococci normally inhabit the mucosal epithelium of the nasopharynx (NP) and oropharynx (OP) of humans. As commensal organisms of the upper respiratory tract, pneumococcal colonization causes no detectable symptoms in the great majority of people [8]. Pneumococcal colonization is most prevalent in children younger than 5 years of age and in adults over 65 years of age, as well as those with certain underlying conditions or risks, such as smokers and individuals infected with HIV [9]. Carriage of pneumococci is transient, as strains and serotypes persist for weeks to months but are eventually cleared by both adaptive and

FIGURE 34.2 Schematic representation of the pneumococcal cell surface showing major macromolecular constituents.

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innate immune responses. The exact host bacteria interactions that control and subsequently eliminate colonization are not fully known, but host responses such as anti-capsule antibody, anti-surface-protein antibodies, and T helper 17 (Th17) type immune responses have all been implicated [10,11]. Although pneumococci live a relatively benign commensal lifestyle, they can translocate out from the pharynx and cause a variety of respiratory and systemic diseases [8]. The most common manifestations of pneumococcal disease in adults are pneumonia, bronchitis, and sinusitis. Children can demonstrate all these as well as otitis media, an infection of the middle ear that is uncommon in adults. In all age groups, infection of respiratory mucosal surfaces can progress to invasion of the blood, and in a small fraction of these,

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invasion of the subarachnoid space resulting in meningitis [12]. The full complement of host and pathogen factors that allow pneumococci to persist at mucosal surfaces and, in some cases, escape cellular and humoral defenses is not understood. Undoubtedly, the outcome of host pathogen interactions is the result of a multitude of bacterial and human factors that control host immune responses and the physiology of both bacteria and their human environment. Pneumococci are transmitted person-toperson by respiratory droplets or fomites contaminated with respiratory secretions (Fig. 34.3). There is no known environmental or animal reservoir outside of humans. This ecology suggests that pneumococcal disease could potentially be eradicated in humans if the transmission chain could be effectively broken.

FIGURE 34.3 Pneumococci are primarily commensal inhabitants of the upper airways and spread person-to-person through droplets or fomites. Under conditions not completely understood pneumococci can translocate to lungs and distal sites via the bloodstream.

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The biggest impediment to this goal is the large number of antigenically distinct capsular serotypes expressed by pneumococci. Capsular polysaccharide is the largest macromolecule on the pneumococcal surface and is the overwhelmingly dominant epitope in immune interactions [3]. For these reasons, capsular polysaccharide is the antigen used in all currently available pneumococcal vaccines [13]. Conjugation of polysaccharide to a peptide improves the immunogenicity over purified polysaccharide alone, and has resulted in a large decrease in invasive disease as well as carriage for vaccine types in both children and adults [14]. Nonetheless, the limited coverage of capsule-based vaccines and consequent emergence of nonvaccine types and nonencapsulated strains are deficits, in current preventive efforts that will be very difficult to overcome in the face of continued reliance on capsule-based vaccines [15]. Conserved proteins that are invariant among pneumococcal capsule types have been intensively studied as protective immunogens [16]. Many of these proteins are effective immunogens when given systemically or topically on mucosal surfaces. The following sections will discuss immune responses at the mucosal surface in response to pneumococcal adherence, and will highlight the extensive work that has been done to characterize immune responses to pneumococcal antigens at mucosal surfaces. Protection against colonization and carriage in the upper airways would prevent the first essential step in pneumococcal pathogenesis, and is the ideal for all vaccines against bacterial pathogens of the respiratory tract. For the purposes of this chapter, the section will be limited to discussion of the upper respiratory tract, including the NP and OP down through the trachea. This is where the human respiratory system has a prototypical mucosal epithelial architecture and immune system [17]. Furthermore, mucosal vaccines against pneumococcal infection are designed for delivery to

the upper respiratory mucosa. The lower respiratory tract, particularly the alveoli, is an important site of pneumococcal infection. However, the histology of the alveoli is significantly different from that of a mucosal surface, and is more aligned functionally with systemic immune responses.

II. HOST IMMUNE RESPONSES TO PNEUMOCOCCI DURING NASOPHARYNGEAL COLONIZATION The distinction between pneumococcal carriage and infection is somewhat blurred, as pneumococcal strains, as defined by capsular type, are cleared by the host after weeks to months of ostensibly asymptomatic carriage in the NP [18]. This suggests that pneumococci are not merely resting inertly on mucosal epithelial cells but are engaging with the host immune system and triggering both innate and adaptive immune responses that will ultimately result in their elimination. The development of a murine model of NP carriage has provided a readily available and flexible system to study both host and pathogen factors that drive the dynamics of bacterial carriage, invasion, and elimination at the mucosal surface [19].

A. The Role of Innate Immunity Against Pneumococci Nonspecific soluble effectors of the immune system, such as complement, C-reactive protein, mannose-binding lectin, and naturally occurring antibodies, are all present on mucosal surfaces to a greater or lesser extent, and serve as effective first defenses at the mucosal surface to contain and potentially eliminate microbes, including pneumococci [20]. These humoral effectors are not generated in response to previous exposure, and are critical for protection

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against a wide variety of pathogens across kingdoms until antigen-specific adaptive responses can be deployed. Bacteria arriving at mucosal surfaces quickly encounter pattern-recognition receptors on and within cells of the immune system and epithelial cells of the mucosa. These include wellcharacterized receptors such as toll-like receptors (TLRs) 1, 2, 6, and 9; NOD-like receptors (NLRs); mannose receptors; C-reactive protein; and serum amyloid P [21]. These receptors recognize common structural and sequence motifs present within macromolecules of most microbes (see also Chapter 7: Induction and Regulation of Mucosal Memory B Cell Responses). These interactions do not require previous sensitization, and are among the earliest innate immune responses. TLRs are a highly conserved family of proteins that recognize a broad variety of molecular patterns, and initiate intracellular signaling cascades that ultimately result in activation of transcription factors NFκB or IRF3/IRF7 and myriad inflammatory responses [22]. Lipoteichoic acids from pneumococci bind and activate TLR2 [23], and pneumococcal lipoproteins, when considered as a broad family, have been shown to engage TLR2 as well [24]. Unmethylated CpG DNA derived from endosomal DNA from internalized pneumococci interacts with TLR9, and comparative studies suggested that TLR9 deficiency results in greater susceptibility to infection than mice deficient in TLR2, TLR4, TLR6, or interleukin 1R (IL-1R) [25]. Interestingly, uptake of pneumococci by TLR9-deficient phagocytes was reduced, an unexpected finding since TLR9 is an intracellular receptor that has not been detected on cell surfaces to this point [25]. Pneumolysin, a multifunctional pneumococcal toxin, has been reported to activate TLR4, a receptor classically associated with responses to lipopolysaccharide [26]. NLRs and a subgroup that form inflammasomes (NLRPs) are also associated with innate responses to pneumococcal cellular components

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in the cytoplasm. NOD2 sensing of intracellularly digested peptidoglycan results in CCL2 expression by macrophages and recruitment of additional macrophages, which culminates in the clearance of pneumococci from the NP of mice [27]. The NLRP3 component of inflammasomes is necessary for IL-1β and IL-18 production in response to pneumolysin, and is crucial for controlling invasive infection [28]. It is not known whether inflammasomes are involved at mucosal surfaces, but it would not be unreasonable to assume that they have some role in innate immune responses to pneumococci at the mucosa, similar to other members of the NLR family. Interferons (IFNs) classified as type I (α/β) are classically associated with antiviral responses and effects. However, the role of type I IFNs in innate immunity is rapidly broadening to other pathogens, including bacteria. Pneumococcal colonization of the upper airways elicits IFN production, and subsequent expression of IFN-responsive genes that generate anti-bacterial effector mechanisms of epithelial cells and macrophages [29]. Paradoxically, the effect of type I IFNs during influenza coinfection may be to promote, rather than inhibit, pneumococcal colonization by a mechanism that remains unknown [30]. A somewhat opposing observation has been offered in which live attenuated influenza vaccine, but not pneumococcal conjugate vaccine, decreases pneumococcal density and duration of NP carriage in mice [31]. The mechanisms by which pneumococci induce IFN responses are not clear, but they are clearly distinct from viral mechanisms. Some investigators have suggested that pneumococci can induce IFN without invasion or uptake by cells [32]; others have described an intracellular mechanism dependent on the cytosolic receptor NOD2 [30]. The subcellular components of pneumococci that drive IFN responses are still unclear, but at least one report suggests that pneumolysindependent release of bacterial DNA can induce

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a robust type I IFN response in a mouse NP carriage model [32]. In addition to the pattern-recognition receptors expressed by many immune and epithelial cells, a significant subset of T lymphocytes express T cell receptors of limited diversity and may be considered a type of innate immune response system. These “atypical” or “nonconventional” T cells that are best characterized include mucosa-associated invariant T (MAIT) cells, γδ T cells, and natural killer T (NKT) cells [33]. MAIT cells have never been linked with pneumococcal infection or protection, and it is unknown whether pneumococci can produce the riboflavin metabolites that are the primary ligands for MAIT receptors. In mice, γδ T cells are part of the early response in lungs to pneumococcal pneumonia, but any role in carriage in the upper airway remains unknown [34,35]. The same is true for NKT cells; mice that lack NKT cells are more susceptible to invasive pneumococcal infection, possibly by reduced recruitment of neutrophils related to deficient NKT-derived MIP-2 [36]. Despite this observation in a murine model of pneumococcal pneumonia, no role has yet been ascribed to NKT cells specifically in the NP during pneumococcal colonization.

B. The Role of Acquired Immunity Against Pneumococci While interaction with the innate immune system occurs rapidly and leads to a variety of cellular and humoral inflammatory responses that can reduce or possibly eliminate NP carriage, adaptive immune responses are traditionally thought to correlate with elimination of pneumococcal carriage [18]. This dogma is derived primarily from the strong evidence that antibodies to pneumococcal capsule are sufficient to protect against invasive infection [37]. Historically, the appearance of serum antibodies to capsular polysaccharide has also been correlated with

elimination of, and protection against, NP carriage. This may occur either by natural exposure or by immunization with currently available vaccines [11,38,39]. Colonization of the upper airway also elicits secretory IgA (SIgA) in the OP and likely contributes, to some degree, to serotypespecific protection against carriage [40]. The dogma classically associated with serum and SIgA is now being reassessed. In experimental human carriage, serum anti-capsule antibodies do not correlate with the duration or intensity of carriage and in a murine model of pneumococcal carriage, may not be related to pneumococcalspecific antibodies at all [41,42]. Serotype-independent protection against carriage has been proposed as the principal mechanism by which pneumococcal carriage declines after 2 years of age and into early and middle adulthood [43]. Protein antigens have been intensely studied for their role in mucosal immunity to pneumococci, and are at the advancing front in development of novel pneumococcal vaccines. Immunization with unencapsulated, live attenuated pneumococci conferred protection against carriage that was dependent on antibodies [44]. An experimental NP infection of humans found a strong correlation between pneumococcal surface protein A (PspA) and protection against carriage. Subjects with preexisting PspA antibodies had less intensity and duration of carriage compared with subjects who had low or nonexistent preinfection antibody [41]. There appears to be a strong correlation between pneumococcal protein antibodies and age as well [45]. Since pneumococcal carriage declines with age until late in life, this correlation provides evidence of the protective capacity of pneumococcal protein antibodies. Another study in humans of experimental pneumococcal carriage looked at preinfection and postinfection serum IgG to 27 proteins. Only 5 of 27 preselected proteins induced significant antibody responses, and three of the five were uncharacterized proteins. Neither of the other two proteins were PspA [46].

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Although the role of protein antigens in protection against pneumococcal carriage remains somewhat ambiguous, in the animal and human studies that did show protection against carriage with protein antigens described above, the antibodies were also protective against invasive disease. These associations have provided impetus for further work to develop protein-based pneumococcal vaccines for both carriage and invasive disease. Despite the strong historical association between antibodies and pneumococcal carriage, there is recent emerging evidence for adaptive cellular immunity in pneumococcal colonization. Mice that have been immunized with inactivated, unencapsulated whole pneumococci are protected against NP colonization, and this protection is dependent on CD41 T cells and not functional B cells and antibody [47,48]. The search for mechanisms by which cellular immune responses affect NP colonization by pneumococci led to the identification of CD41 T cells producing IL-17A, a signature cytokine for Th17 cells. Th17 cells are important in controlling NP carriage in mouse models, probably by promoting chemotaxis of neutrophils and macrophages via IL-17A [49,50]. An association between Th17 cells and pneumococcal carriage in humans is more difficult to study directly; however, evidence is accumulating, although it is somewhat circumstantial. Peripheral blood mononuclear cells (PBMCs) from adults and children in Sweden and Bangladesh produced IL-17A after stimulation with whole cell pneumococcal antigen [51]. A study of NP-associated lymphoid tissue and tonsillar lymphoid cells in children was designed to look at pneumococcal carriage and T cell subsets. This work showed a strong relationship between Th17 and regulatory T cells (Treg) and carriage, with carriage being associated with a greater proportion of Treg cells. Conversely, protection against carriage correlated with a higher number of Th17 cells [52]. Cellular responses to heat-killed pneumococci

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were examined in children prone to otitis media and nonprone children. Infection-prone children had significantly fewer Th17 PBMCs after stimulation than did nonprone children, a difference that could be eliminated by addition of IL-6, IL-1β, IL-23, or transforming growth factor beta [53]. Invasive pneumococcal infections develop almost exclusively after NP carriage has been established [2]. Early animal studies were designed to primarily study invasive pneumococcal infections, primarily bloodstream infection, and they led to fairly effective, polysaccharide-based vaccines directed against pneumococcal capsule [54]. These capsulebased vaccines have some critical weaknesses that preclude their effectiveness on a global scale. Foremost among these is that capsulebased vaccines can never be completely comprehensive, as there are now nearly 100 distinct serotypes of pneumococcal capsule that have been described [54]. Strongly immunogenic, serotype-independent vaccines delivered to the mucosal surface to prevent colonization are a top public health priority and the focus of expanding research efforts.

III. IMMUNIZATION AGAINST STREPTOCOCCUS PNEUMONIAE AT MUCOSAL SURFACES In the 1920s, classic experiments by Oswald Avery and Frederick Griffith established polysaccharide capsule as the immunodominant antigen of pneumococci responsible for serotype diversity. From that time until the present, commercial vaccine efforts have focused on the capsule and how to increase the coverage and immunogenicity of polysaccharide vaccines. Two types of pneumococcal vaccines are in use today: a 23-valent purified polysaccharide mixture and a polysaccharide protein conjugate vaccine that currently contains up to 13 capsule types [54]. The purified polysaccharide vaccine

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has never been shown to reduce or affect carriage. Conversely, the conjugate vaccine has been shown to reduce NP carriage of serotypes included in the vaccine [55,56]. As was previously mentioned, polysaccharide-based vaccines can never be fully comprehensive, so the search for effective, mucosal, serotypeindependent pneumococcal antigens has been launched. The development of a reproducible murine model of pneumococcal carriage has made possible an accelerated pathway to mucosal vaccine development. Very quickly after the establishment of a mouse model of pneumococcal carriage, the same investigators reported that intranasal immunization with PspA and the B subunit of cholera toxin (CTB) could reduce colonization, pneumonia, and sepsis [57]. A followup report showed similar efficacy with a nontoxic, mutated cholera holotoxin [58]. A second surface protein, pneumococcal surface adhesion A (PsaA), was subsequently found to be immunogenic when delivered to the mucosal surface of the NP. In the same report, the combination of PspA and PsaA was more protective than either protein alone [59]. Although antigenspecific responses are typically desired and considered surrogates of protection, nonspecific inflammatory responses may be just as effective in the case of pneumococcal NP carriage. Immune responses generated by CTB alone are sufficient to reduce pneumococcal carriage in a mouse model [60]. This “adjuvant-alone” effect was also seen in a mucosal immunization study with PspA and killed whole cell pertussis as adjuvant [61]. Perhaps head-to-head comparisons between adjuvant antigen and adjuvant alone should be considered to assess exactly what additional benefits specific immune responses have in the immediate postimmunization period. Adjuvants alone will not confer antigen-specific immune memory and protection against rechallenge, but may prime the adaptive immune system by nonspecifically

increasing processing and presentation of antigens by early innate responses. Cholera toxin and variants thereof carry safety concerns in humans, and less potentially toxic yet immunogenic adjuvants for mucosal vaccines are a research priority. To address these specific problems, a cholesterol-containing pullulan nanogel was developed and used in a mucosal vaccine containing PspA [62]. Nasal immunization with this preparation protected mice against carriage, pneumonia, and sepsis. A vast array of immune responses were queried that demonstrated that the vaccine produced high levels of PspA-specific serum IgG, nasopharyngeal and pulmonary IgA, and nasal and systemic Th17 responses. A similar study demonstrating the immunogenicity of PspA in nanogel was performed in cynomolgus macaques; however, no nasal or systemic challenge was done following immunization [63]. This same nanogel vehicle was used with cyclic di-GMP as an adjuvant to immunize spontaneously hypertensive rats via the NP mucosa with a fusion protein consisting of a peptide fragment of angiotensin I receptor and PspA. Immunized animals produced both angiotensin receptorand PspA-specific antibodies, and rats developed limited, less severe blood pressure elevation than did controls. Immune sera from immunized rats passively protected mice from otherwise fatal pneumococcal sepsis [64]. Unmethylated DNA has also been shown to be an effective adjuvant when combined with PspA to protect against NP carriage. In this same study, phosphorylcholine (ChoP), a component of teichoic and lipoteichoic acids of all pneumococci, was combined with keyhole limpet hemocyanin to induce anti-ChoP antibodies and subsequent mucosal protection against colonization [65]. It is known that naturally occurring anti-ChoP antibodies contribute to innate resistance to pneumococcal infections in mice and likely in humans as well [66]. Because ChoP moieties are widely distributed in

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phospholipids, this immunization strategy bears careful scrutiny. A novel antigen carrier consisting of bacteria-like particles (BLP), generated by hot acid inactivation of Lactococcus lactis, was used to deliver a mutated recombinant pneumolysin toxoid to the NP of mice [67]. Serum IgG and IgA from bronchoalveolar lavage were measured 2 weeks following the last of a series of three immunizations. Animals immunized with BLPs and pneumolysin toxoid generated 10fold more IgG and IgA than did animals that received toxoid alone. In this case, challenge or protection experiments were not done in immunized animals. Peptidoglycan from Lactobacillus rhamnosus has been shown to have salutary immunomodulatory effects at the respiratory mucosal surface. Immunization with both intact Lactobacillus cells and purified peptidoglycan protected mice from immunemediated lung injury following respiratory syncytial virus infection followed by pneumococcal superinfection [68]. A similar observation was reported with Corynebacterium pseudodiphtheriticum [69]. It is likely that other bacterial taxa, or subcellular components thereof, that inhabit mucosal surfaces will be studied as vehicles and adjuvants to deliver pneumococcal antigens to the upper respiratory tract. Although protein antigens were initially used as mucosal vaccines for pneumococcal carriage and infection, pneumococcal whole cell preparations were soon thereafter studied as mucosal vaccines. Heat-killed, whole cell vaccines administered with cholera holotoxin [70] or CTB [71] protected against NP carriage after challenge with multiple capsular serotypes of pneumococci. This work was extended by immunization with attenuated encapsulated strains of serotypes 4 or 6A and challenge with wild-type parent strain 5 weeks following a two-dose series of intranasal immunizations. In this case, challenge was intranasal, and animals were followed for carriage and sepsis.

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Complete protection against sepsis and a significant reduction in bacterial burden in the NP were observed in immunized mice, with concomitant increases in serum IgG and nasal IgA reactive with wild-type pneumococcal cells [44]. An attenuated mutant of a serotype 2 strain that is unable to autolyse protected against NP colonization with heterologous serotypes as well as nonencapsulated and nontypable strains [72]. In contrast, other work has shown that NP colonization with live bacteria or immunization with killed pneumococci is not protective against pneumonia, and only delivery of antigens to the lungs can protect against pneumococcal pneumonia with heterologous capsule types [73]. Pneumococcal infections are most prevalent at the extremes of age, and age-specific vaccines, particularly those targeted to a senescent immune system, may potentially improve immunogenicity of vaccines studied primarily in children and young, healthy adults. A step in this direction was undertaken in aged (2-yearold) mice using nucleic acid adjuvants. A plasmid containing cDNA for the Flt3 ligand, when combined with a nonspecific CpG-containing oligodeoxynucleotide, enhances PspA-specific SIgA as well as specific cellular immune responses [74]. As the field of mucosal immunity to pneumococci has rapidly embraced cellular immunity, particularly IL-17A-secreting CD41 T cells, vaccine development has also shifted to surrogates of protection other than immunoglobulins. The first published data in this direction examined CD41 T lymphocytes collected from mice immunized intranasally with a whole cell pneumococcal vaccine. An expression library containing more than 90% of predicted open reading frames of a type 4 pneumococcal strain was added to these CD41 T cells, and secreted IL-17A was measured as a marker of activated Th17 cells. The 17 antigens that strongly activated CD41 T lymphocytes in this manner

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were predominantly proteins that had unknown function and were mostly uncharacterized [75]. Several of these were lipoproteins that induced Th17-type cytokines by engaging TLR2 [76]. These findings stand in stark contrast to the long tradition of capsule and wellcharacterized surface proteins, which have been studied intensively for their ability to induce robust antibody responses.

IV. CURRENT AND FUTURE STATUS OF HUMAN MUCOSAL VACCINE TRIALS Mucosal vaccine trials for pneumococcal carriage and invasive disease in humans have lagged behind animal work, despite compelling evidence that many strategies are worth pursuing. There are effective pneumococcal vaccines for at-risk groups, and low-risk individuals who are not candidates for the current vaccines have a very low incidence of carriage and invasive disease. This makes trials for new pneumococcal vaccines very large and prolonged, with the associated expense. Surrogate markers of protection, at least for carriage, are now more nebulous as compared with immunoglobulin levels and invasive disease used in early vaccine trials [77]. Of course, there are safety and ethical concerns about deliberately infecting humans with pneumococci as part of vaccine trials to assess resistance to NP carriage. But research with human subjects has now progressed to the point at which strictly controlled human infections can be done, and immune responses can be measured. While not designed to test a vaccine, experimental human carriage experiments have measured both mucosal and systemic immunity [41,78]. Although not strictly a vaccine trial, the first human infection experiment that looked at the protective effect of carriage on rechallenge showed that humans deliberately colonized with a serotype 6B strain

were protected from subsequent colonization with the same strain [46]. The first properly controlled human vaccine trial with postimmunization mucosal challenge was reported 2 years later [79]. This protocol involved immunization with a 13-valent polysaccharide-conjugate vaccine, and challenge at some undefined time postvaccination with a serotype 6B strain. Immunized subjects were less frequently colonized and had less bacterial density than controls. Although a defined vaccine study with mucosal challenge, this was not a demonstration of mucosal immunization, and no immunological data were collected in this study. A similar approach was used with a ten-valent conjugate vaccine in children; it found that vaccinated children were not protected against carriage, although pneumococcal cells from vaccinated children were less metabolically active than pneumococci from unvaccinated carriers, as measured by 16S DNA:mRNA ratios [80].

V. CONCLUDING REMARKS Many respiratory pathogens exist primarily as commensal members of the upper respiratory microbiota and cause disease only under specific circumstances related to both host and pathogen characteristics. The pneumococcus is the archetype of this lifestyle, and since it was first identified as a pathogen, vaccine efforts have targeted the most common and serious manifestations of infection: pneumonia and bacteremia. As we continue to develop our understanding of mucosal immunity, vaccine efforts have turned to target the necessary first step in pneumococcal infection: colonization of the NP. Reduction of carriage not only will protect immunized persons, but also will protect nonimmunized persons by reducing transmission overall in the population. Lest we forget, though, pneumococci are first and foremost commensal organisms that occupy a niche in respiratory microbial communities of a significant number of people. We still

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REFERENCES

do not have a clear idea of what impact selectively removing pneumococci will have on microbial communities. Will nonvaccine strains fill the void? Will other potential respiratory pathogens find a hospitable landscape? A convergence of mucosal immunology with microbiota and microbiome research will be necessary to move forward and reduce the pneumococcal disease burden with no unintended negative consequences.

[12]

[13]

[14]

[15] [16]

Acknowledgment The authors would like to thank Lance Keller for his invaluable assistance with illustrations and Jessica Bradshaw and Haley Pipkins for their thoughtful review of the manuscript.

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[18]

References [1] Kadioglu A, et al. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008;288 301. [2] Bogaert D, De Groot R, Hermans PW. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect Dis 2004;4(3):144 54. [3] Geno KA, et al. Pneumococcal capsules and their types: past, present, and future. Clin Microbiol Rev 2015;28(3): 871 99. [4] Pilishvili T, et al. Sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine. J Infect Dis 2010;201(1):32 41. [5] Pollard AJ, Perrett KP, Beverley PC. Maintaining protection against invasive bacteria with proteinpolysaccharide conjugate vaccines. Nat Rev Immunol 2009;9(3):213 20. [6] Swiatlo E, McDaniel L, Briles D. The Pneumococcus. In: Tuomanen E, et al., editors. The Pneumococcus. Washington, DC: ASM Press; 2004. p. 49 60. [7] Hakenbeck R, et al. Versatility of choline metabolism and choline-binding proteins in Streptococcus pneumoniae and commensal streptococci. FEMS Microbiol Rev 2009;33(3):572 86. [8] Gillespie SH, Balakrishnan I. Pathogenesis of pneumococcal infection. J Med Microbiol 2000;49(12):1057 67. [9] Siegel SJ, Weiser JN. Mechanisms of bacterial colonization of the respiratory tract. Ann Rev Microbiol 2015; 69:425 44. [10] Esposito S, et al. Pneumococcal colonization in older adults. Immun Ageing 2016;13(2):1 10. [11] Pennington SH, et al. Polysaccharide-specific memory B cells predict protection against experimental human

[19]

[20]

[21] [22] [23]

[24]

[25]

[26]

[27]

pneumococcal carriage. Am J Respir Crit Care Med 2016;194(12):1523 31. Koedel U, Scheld WM, Pfister HW. Pathogenesis and pathophysiology of pneumococcal meningitis. Lancet Infect Dis 2002;2:721 36. Feldman C, Anderson R. Review: current and new generation pneumococcal vaccines. J Infect 2014;69(4): 309 25. Whitney CG, et al. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine. N Engl J Med 2003;348(18):1737 46. Moffitt KL, Malley R. Next generation pneumococcal vaccines. Curr Opin Immunol 2011;23:407 13. Miyaji EN, et al. Serotype-independent pneumococcal vaccines. Cell Mol Life Sci 2013;70(18):3303 26. Lloyd CM, Marsland BJ. Lung homeostasis: influence of age, microbes, and the immune system. Immunity 2017;18:549 61. Mackenzie GA, et al. Epidemiology of nasopharyngeal carriage of respiratory bacterial pathogens in children and adults: cross-sectional surveys in a population with high rates of pneumococcal disease. BMC Infect Dis 2010;10. Available from: https://doi.org/10.1186/ 1471-2334-10-304. Wu HY, Virolainen A, et al. Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice. Microb Pathog 1997;23(3):127 37. Koppe U, Suttorp N, Opitz B. Recognition of Streptococcus pneumoniae by the innate immune system. Cell Microbiol 2012;14(4):460 6. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124(4):783 801. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4(7):499 511. Schroder NW, et al. Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved. J Biol Chem 2003;278(18):15587 94. Tomlinson G, et al. TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins. J Immunol 2014;193(7):3736 45. Albiger B, et al. Toll-like receptor 9 acts at an early stage in host defence against pneumococcal infection. Cell Microbiol 2007;9(3):633 44. Malley R, et al. Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection. Proc Natl Acad Sci USA 2003;100(4):1966 71. Davis KM, Nakamura S, Weiser JN. Nod2 sensing of lysozyme-digested peptidoglycan promotes macrophage recruitment and clearance of S. pneumoniae colonization in mice. J Clin Invest 2011;121(9):3666 76.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

608

34. MUCOSAL VACCINES FOR STREPTOCOCCUS PNEUMONIAE

[28] Witzenrath M, et al. The NLRP3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia. J Immunol 2011;187(1):434 40. [29] Joyce EA, Popper SJ, Falkow S. Streptococcus pneumoniae nasopharyngeal colonization induces type I interferons and interferon-induced gene expression. BMC Genomics 2009;10:404 10. [30] Nakamura S, Davis KM, Weiser JN. Synergistic stimulation of type I interferons during influenza virus coinfection promotes Streptococcus pneumoniae colonization in mice. J Clin Invest 2011;121(9):3657 65. [31] Mina MJ, Klugman KP, McCullers JA. Live attenuated influenza vaccine, but not pneumococcal conjugate vaccine, protects against increased density and duration of pneumococcal carriage after influenza infection in pneumococcal colonized mice. J Infect Dis 2013;208 (8):1281 5. [32] Parker D, et al. Streptococcus pneumoniae DNA initiates type I interferon signaling in the respiratory tract. mBio 2011;2(3). Available from: https://doi.org/ 10.1128/mBio.00016-11 e00016-11. [33] Ivanov S, Paget C, Trottein F. Role of non-conventional T lymphocytes in respiratory infections: the case of the pneumococcus. PLoS Pathog 2014;10(10). Available from: https://doi.org/10.1371/journal.ppat.1004300 e1004300. [34] Kirby AC, et al. Evidence for the involvement of lungspecific γ δ T cell subsets in local responses to Streptococcus pneumoniae infection. Eur J Immunol 2007; 37(12):3404 13. [35] Nakasone C, et al. Accumulation of γ/δ T cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection. Microb Infect 2007;9(3):251 8. [36] Kawakami K, et al. Critical role of Valpha14 1 natural killer T cells in the innate phase of host protection against Streptococcus pneumoniae infection. Eur J Immunol 2003;33(12):3322 30. [37] Falkenhorst G, et al. Effectiveness of the 23-valent pneumococcal polysaccharide vaccine (ppv23) against pneumococcal disease in the elderly: systematic review and meta-analysis. PLoS One 2017;12(1) e0169368. [38] Ghaffar F, Friedland IR, McCracken Jr. GH. Dynamics of nasopharyngeal colonization by Streptococcus pneumoniae. Pediatr Infect Dis J 1999;18(7):638 46. [39] Khan MN, Pichichero ME. The host immune dynamics of pneumococcal colonization: implications for novel vaccine development. Hum Vaccin Immunother 2014; 10(12):3688 99. [40] Simell B, Kilpi TM, Kayhty H. Pneumococcal carriage and otitis media induce salivary antibodies to pneumococcal capsular polysaccharides in children. J Infect Dis 2002;186(8):1106 14.

[41] McCool TL, et al. The immune response to pneumococcal proteins during experimental human carriage. J Exp Med 2002;195(3):359 65. [42] McCool TL, Weiser JN. Limited role of antibody in clearance of Streptococcus pneumoniae in a murine model of colonization. Infect Immun 2004;72(10):5807 13. [43] Cobey S, Lipsitch M. Niche and neutral effects of acquired immunity permit coexistence of pneumococcal serotypes. Science 2012;335(6074):1376 80. [44] Roche AM, King SJ, Weiser JN. Live attenuated Streptococcus pneumoniae strains induce serotypeindependent mucosal and systemic protection in mice. Infect Immun 2007;75(5):2469 75. [45] Azarian T, et al. Association of pneumococcal protein antigen serology with age and antigenic profile of colonizing isolates. J Infect Dis 2017;215(5):713 22. [46] Ferreira DM, et al. Controlled human infection and rechallenge with Streptococcus pneumoniae reveals the protective efficacy of carriage in healthy adults. Am J Respir Crit Care Med 2013;187(8):855 64. [47] Malley R, et al. CD41 T cells mediate antibodyindependent acquired immunity to pneumococcal colonization. Proc Natl Acad Sci USA 2005;102(13): 4848 53. [48] van Rossum AM, Lysenko ES, Weiser JN. Host and bacterial factors contributing to the clearance of colonization by Streptococcus pneumoniae in a murine model. Infect Immun 2005;73(11):7718 26. [49] Lu YJ, et al. Interleukin-17A mediates acquired immunity to pneumococcal colonization. PLoS Pathog 2008;4 (9):e1000159. Available from: https://doi.org/ 10.1371/journal.ppat.1000159. [50] Zhang Z, Clarke TB, Weiser JN. Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice. J Clin Invest 2009;119(7):1899 909. [51] Lundgren A, et al. Characterization of Th17 responses to Streptococcus pneumoniae in humans: comparisons between adults and children in a developed and a developing country. Vaccine 2012;30(26): 3897 907. [52] Mubarak A, et al. A dynamic relationship between mucosal T helper type 17 and regulatory T-cell populations in nasopharynx evolves with age and associates with the clearance of pneumococcal carriage in humans. Clin Microbiol Infect 2016;22(8) 736 e1-7. [53] Basha S, et al. Reduced T-helper 17 responses to Streptococcus pneumoniae in infection-prone children can be rescued by addition of innate cytokines. J Infect Dis 2017;215(8):1321 30. [54] Poolman JT. Pneumococcal vaccine development. Exp Rev Vaccines 2004;3(5):597 604. [55] Esposito S, Principi N. Impacts of the 13-valent pneumococcal conjugate vaccine in children. J Immunol Res 2015;2015:1 6.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

609

REFERENCES

[56] van Hoek AJ, et al. Pneumococcal carriage in children and adults two years after introduction of the thirteen valent pneumococcal conjugate vaccine in England. Vaccine 2014;32(34):4349 55. [57] Wu HY, Nahm MH, et al. Intranasal immunization of mice with PspA (pneumococcal surface protein A) can prevent intranasal carriage, pulmonary infection, and sepsis with Streptococcus pneumoniae. J Infect Dis 1997; 175(4):839 46. [58] Yamamoto M, et al. A nontoxic adjuvant for mucosal immunity to pneumococcal surface protein A. J Immunol 1998;161(8):4115 21. [59] Briles DE, et al. Intranasal immunization of mice with a mixture of the pneumococcal proteins PsaA and PspA is highly protective against nasopharyngeal carriage of Streptococcus pneumoniae. Infect Immun 2000;68 (2):796 800. [60] Kuipers K, et al. Antigen-independent restriction of pneumococcal density by mucosal adjuvant cholera toxin subunit B. J Infect Dis 2016;214(10):1588 96. [61] Tostes RO, et al. Protection elicited by nasal immunization with recombinant pneumococcal surface protein A (rPspA) adjuvanted with whole-cell pertussis vaccine (wP) against co-colonization of mice with Streptococcus pneumoniae. PLoS One 2017;12(1):e0170157. Available from: https://doi.org/10.1371/journal.pone.0170157. [62] Kong IG, et al. Nanogel-based PspA intranasal vaccine prevents invasive disease and nasal colonization by Streptococcus pneumoniae. Infect Immun 2013;81(5): 1625 34. [63] Fukuyama Y, et al. Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNAassociated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques. Mucosal Immunol 2015;8(5):1144 53. [64] Azegami T, et al. Intranasal vaccination against angiotensin II type 1 receptor and pneumococcal surface protein A attenuates hypertension and pneumococcal infection in rodents. J Hypertens 2018;36(2):387 94. [65] Kataoka K, et al. Dendritic cell-targeting DNA-based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae. Microbiol Immunol 2017;61(6): 195 205. [66] Briles DE, Forman C, Crain M. Mouse antibody to phosphocholine can protect mice from infection with mouse-virulent human isolates of Streptococcus pneumoniae. Infect Immun 1992;60(5):1957 62. [67] Lu J, et al. Systemic and mucosal immune responses elicited by intranasal immunization with a pneumococcal bacterium-like particle-based vaccine displaying pneumolysin mutant Plym2. Immunol Lett 2017;187:41 6. [68] Clua P, et al. Peptidoglycan from immunobiotic Lactobacillus rhamnosus improves resistance of infant

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77] [78]

[79]

[80]

mice to respiratory syncytial viral infection and secondary pneumococcal pneumonia. Front Immunol 2017;8(948):1 15. Kanmani P, et al. Respiratory commensal bacteria Corynebacterium pseudodiphtheriticum improves resistance of infant mice to respiratory syncytial virus and Streptococcus pneumoniae superinfection. Front Microbiol 2017;8:1613. Malley R, et al. Intranasal immunization with killed unencapsulated whole cells prevents colonization and invasive disease by capsulated pneumococci. Infect Immun 2001;69(8):4870 3. Malley R, et al. Multiserotype protection of mice against pneumococcal colonization of the nasopharynx and middle ear by killed nonencapsulated cells given intranasally with a nontoxic adjuvant. Infect Immun 2004;72(7):4290 2. Kim GL, et al. Pneumococcal pep27 mutant immunization stimulates cytokine secretion and confers longterm immunity with a wide range of protection, including against non-typeable strains. Vaccine 2016; 34(51):6481 92. Wang Y, et al. Cross-protective mucosal immunity mediated by memory Th17 cells against Streptococcus pneumoniae lung infection. Mucosal Immunol 2017;10 (1):250 9. Fukuyama Y, et al. A combination of Flt3 ligand cDNA and CpG oligodeoxynucleotide as nasal adjuvant elicits protective secretory-IgA immunity to Streptococcus pneumoniae in aged mice. J Immunol 2011;186(4): 2454 61. Moffitt KL, et al. T(H)17-based vaccine design for prevention of Streptococcus pneumoniae colonization. Cell Host Microbe 2011;9(2):158 65. Moffitt K, et al. Toll-like receptor 2-dependent protection against pneumococcal carriage by immunization with lipidated pneumococcal proteins. Infect Immun 2014;82(5):2079 86. Plotkin SA. Vaccines: correlates of vaccine-induced immunity. Clin Infect Dis 2008;47(3):401 9. Wright AK, et al. Human nasal challenge with Streptococcus pneumoniae is immunising in the absence of carriage. PLoS Pathog 2012;8(4):e1002622. Available from: https://doi.org/10.1371/journal.ppat.1002622. Collins AM, et al. First human challenge testing of a pneumococcal vaccine. Double-blind randomized controlled trial. Am J Respir Crit Care Med 2015;192(7): 853 8. Andrade DC, et al. 10-valent pneumococcal conjugate vaccine (PCV10) decreases metabolic activity but not nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae. Vaccine 2017;35(33): 4105 11.

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Development of a Mucosal TB Vaccine Using Human Parainfluenza Type 2 Virus Yusuke Tsujimura1 and Yasuhiro Yasutomi1,2 1

Laboratory of Immunoregulation and Vaccine Research, Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)Tsukuba, Japan 2Department of Immunoregulation, Mie University Graduate School of Medicine, Tsu, Japan

I. INTRODUCTION Most people infected with Mycobacterium tuberculosis (Mtb) never develop disease, indicating that the host
pathogen balance can be tipped in favor of the host, leading to protective immunity [1]. A better understanding of mycobacterial immunity and the life cycle of Mtb would facilitate a rational design of new successful vaccines and the prediction of vaccine targets (Fig. 35.1). Mtb has invented complex mechanisms to survive in the intracellular niche, in which it counteracts or evades the numerous defense mechanisms. The mechanisms for survival of Mtb include (1) reprogramming of macrophages after primary infection or phagocytosis to prevent its own destruction; (2) initiating the formation of well-organized granulomas that serve as both habitat and containment for Mtb to minimize cross-talk between the pathogen and the host immune system; (3) shutting down its own central metabolism,

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00035-3

terminating replication and thereby transitioning into a stage of dormancy, rendering it extremely resistant to host defense and drug treatment; and (4) reactivating TB disease through the failure of T cells to maintain protective immunity (Fig. 35.1) [2]. Vaccines have had a substantial global impact on morbidity and mortality of a variety of bacterial and viral infections. Nevertheless, there are no licensed vaccines that are protective against human immunodeficiency virus (HIV), malaria, or pulmonary tuberculosis (TB) infections [3]. For these pathogens, the T cell-mediated immune responses profoundly contribute to the control of infection and prevention of or delay in the onset of disease. Type 1 T helper (Th1) cells produce interferon gamma (IFNγ) to activate infected macrophages and promote the formation of granulomas around infected macrophages. CD81 T cells and unconventional T cells also produce IFNγ and participate in protective

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FIGURE 35.1 Success of Mtb infection and targets for TB vaccines [1
4]. Upon inhalation of droplets containing Mtb, the pathogen reaches lung airways and is phagocytosed by alveolar macrophages. The infected host cell induces a localized proinflammatory response that attracts mononuclear cells and T lymphocytes to build up a granuloma, the hallmark tissue reaction of TB. Healthy individuals can control the pathogen at this stage, but they remain latently infected and thus are at lifelong risk of reactivation. Granuloma maturation (solid, necrotic, and caseous) occurs at different rates and typically culminates in the coexistence of all lesion forms during active TB. The caseating granuloma loses solidity because of decay of its center into a structureless accumulation of host cell debris, the caseum. The number of Mtb bacteria increases, and the bacteria are released into airways and coughed out as a contagious aerosol.

responses against bacterial growth. Th17 cells produce interleukin 17 (IL-17) to promote the mobilization of immunocompetent cells and contribute to the granuloma formation [4]. On the other hand, Th2 cells and regulatory T cells (Tregs) interfere with these protective proinflammatory immune responses [5]. The onset of adaptive immunity in Mtb infection is delayed about 14 days in mice and up to 6 weeks in humans [6]. At this point, distinct T cell subsets and B cells migrate to the site of infection and execute their different effector functions [7]. After onset of adaptive immunity, 90%
97% of infected individuals develop sustained infection without clinical

symptoms, termed latent TB infection (LTBI) [4]. LTBI was initially considered a static phase, but it is now known that this stage is hallmarked by the presence of granulomas in various stages (caseous, noncaseous, and fibrotic) and an ongoing balance between anti-mycobacterial activity and regulatory mechanisms to minimize immunopathology [8]. Mtb is an intracellular pathogen that is mucosally transmitted. Mucosal and cellular immunity have, therefore, been suggested to be pivotal in protection against Mtb infection. A vector that can be delivered via a mucosal route and elicit potent antigen (Ag)-specific immune responses may be an ideal candidate as an

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anti-TB vaccine. Recently, we evaluated the effectiveness of intranasal administration of Ag85B-expressed human parainfluenza type 2 virus (rHPIV2-Ag85B) as a novel mucosal TB vaccine. rHPIV2 vectors have gained increasing attention in anti-TB vaccine development owing to their intrinsic adjuvant activity, excellent safety, and high levels of Ag release as well as their suitability for intranasal mucosal delivery. In addition, rHPIV2 vectors are highly effective in eliciting robust cellular immunity in experimental animal models [9], indicating that they are promising antigen delivery vectors for the development of a TB vaccine.

II. MUCOSAL IMMUNE RESPONSES IN TB INFECTION The immune response can greatly alter the proportions and absolute numbers of actively replicating Mtb pathogens in infected individuals with concomitant changes in TB disease risks. Because the infection is largely intracellular during paucibacillary LTBI and early reactivation disease, T cell responses are critically important for protective immunity. Conventional CD41 and CD81 T cell responses play key roles in immunity to Mtb and are targeted by current vaccine and immunodiagnostic strategies [10,11]. Meanwhile, cells of the innate immune system use several receptor systems to recognize pathogens and act as the first line of defense against infection. Certain innate cell subsets, such as natural killer (NK) cells, type 1 innate lymphoid cells (ILC1) [12], and invariant natural killer T (iNKT) cells [13] respond rapidly following detection of Mtb-infected cells and can modulate other cell types [14]. Thus, by being an early producer of IFNγ and suppressing intracellular bacterial growth, these cells function as an important part of the early immune response against Mtb. In addition, γδ T cells and ILC3s produce IL-17, which facilitates optimal activation of myeloid cells and efficient recall

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responses [15
19]. During this process, loosely aggregated innate granulomas are already formed [20]. While innate lymphocytes serve important roles in host resistance to different infections, it remains controversial whether ILC1s and ILC3s contribute to immunity against Mtb infection [19]. The predominant lipid component of the M. tuberculosis cell wall is mycolic acid, which was the first CD1-restricted lipid to be identified [21], making it the prototypical microbial lipid Ag. Mycolic acid is a key virulence factor, protecting Mtb from dehydration, exposure to drugs, and the hostile environment of the macrophage phagolysosome [22
24]. Group 1 CD1-restricted T cells specific to Mtb lipid have been detected in patients with active or latent TB infection, and they have been shown to confer protection against Mtb in human group 1 CD1 transgenic mice [13]. Innate-like MR1 (nonclassical MHC-1b)-restricted mucosalassociated invariant T (MAIT) cells have also been shown to be important players in mycobacterial immunity by potentially acting as early sentinels to Mtb infection [25,26]. Increasing evidence shows that nonconventional CD81 T cells restricted by MHC-Ib molecules can recognize distinct microbial Ags and contribute to protection against Mtb infection [27,28]. Cellular immunity to Mtb requires a coordinated response between the innate and adaptive arms of the immune system, resulting in a type 1 cytokine response, which is associated with control of infection. Mtb has an incredible capacity to adapt in vivo to a variety of stressful conditions. Every responses from all cells induced on the mucosal surface will be considered major targets for vaccine development because they can recognize intracellular Mtb, the major pathogen reservoir during LTBI. In most vaccine studies for infections requiring Th1 cell responses, the proportion of IFNγ-producing cells is assessed as the primary immune correlate of protection [29]. Although IFNγ is clearly necessary [30],

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using it as a single immune parameter may not always be sufficient to predict protection [31]. Tumor necrosis factor (TNF) is another effector cytokine that can mediate control of intracellular infections. Indeed, IFNγ and TNF synergize in their capacity to mediate killing of pathogens [32]. IL-2 has little direct effector function but strongly enhances the expansion of CD41 and CD81 T cells, leading to a more efficient effector response. Some studies showed that mucosal immunization with Mtb Ag induces a population of lung-resident Th17 cells. IL-17 produced by Th17 cells mediates protection in the host by inducing formation of lymphoid

follicles in the lung to initiate T cell localization near infected macrophages for Mtb control. More recent evidence points toward a role of B cells in modulating immune responses to Mtb infection. Mucosal antibody responses also could protect against initial infection and transmission [33]. Evidence for other correlates of protective immunity arose with the discovery of polyfunctional CD41 T cells that simultaneously produce multiple cytokines (usually TNF, IFNγ, IL-17, and IL-2). The contribution of these cells to mycobacterial immunity has been discussed in detail elsewhere [34], but it is important to

FIGURE 35.2 Mucosal immune responses to TB infections. Mucosal immune responses are important for protection against TB infection. Responses from all cells induced on the mucosal surface have crucial roles in the regulation of TB pathogenesis. Responses of CD41 effector T cells (Th1, Th17, and polyfunctional T cells) [10,11,34
36]; effector CD81 T cells (cytotoxic T lymphocyte (CTL), tissue resident memory T (TRM) cells, polyfunctional T cells) [10,11,37]; NK cells [12]; γδ T cells [14
17]; mucosal-associated invariant T (MAIT) cells [25,26]; HLA-E-restricted CD8 T cells [27,28]; ILCs [12,19]; CD1restricted T cells [20,21]; CD1d-restricted iNKT cells [13,24]; and SIgA/IgG antibodies [33] are potentially protective against LTBI reactivation, which could reduce both TB disease and TB transmission. Effector CD41 Th2 cells [11], Tregs [5], T cell exhaustion [38], M2 macrophages [39], polymorphonuclear (PMN) cells [40], and myeloid-derived suppressor cells [41] interfere with the above protective immune responses.

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stress that polyfunctional T cells do not always correlate with protective immunity [35], and their importance should be carefully evaluated. Thus, polyfunctional T cells may be a part of the solution [36], but may not contribute as significantly as was initially thought to protect against TB. Although the role of CD81 T cells in TB is less clear compared with that of CD41 T cells, CD81 T cells can also contribute to optimal immunity and protection against Mtb by their production of proinflammatory cytokines [37]. Internal influences may also confound protective immunity. These include suppression by cytokines (IL-10, TGF-β) from Tregs. Tregs can recognize Mtb-derived Ags and can potently restrict protective immune responses during TB [5]. Th17 cells secreting IL-17, IL-21, and IL-22 and Tregs secreting IL-10 and TGF-β have been implicated in coordinating and balancing the cellular immune responses. T cell exhaustion [38], alternatively activated macrophages [39] unable to kill intracellular Mtb, and type I IFN-induced polymorphonuclear neutrophils can negatively regulate protective immunity in the lungs [40]. Myeloid-derived suppressor cells (MDSCs) are involved in increased TB disease severity with induction of hypoxic necrotic granulomas [41] (Fig. 35.2). Together, the cells of the adaptive immune system orchestrate the immune response in an attempt to establish Mtb-induced immunity. Understanding how distinct populations of cytokine-producing cells are optimized for effector function and determining how they demonstrate a correlate of protection represent crucial steps in developing T cell vaccines to Mtb [42].

III. VACCINE DELIVERY SYSTEMS FOR INDUCTION OF MUCOSAL IMMUNITY Many infectious diseases, such as TB, initially establish infection in mucosal tissues. Therefore, the best defense against these predominantly

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mucosal pathogens is mucosal vaccines that are capable of inducing both systemic immunity and mucosal immunity. However, the mucosal immune system is unique and is different from systemic immune responses [43,44]. Conventional injectable vaccines are often ineffective in eliciting mucosal immune responses in the desired target mucosal tissue. Viral vectors show promise for delivering subunit vaccines. Since Mtb normally enters the host via mucosal surfaces of the lungs, the best defense against Mtb is a mucosal vaccine capable of inducing both systemic immunity and mucosal immunity. Many viral vectors have been tested as recombinant viral vaccines eliciting suitable Ag-specific immune responses, yet many were found ineffective, such as vaccinia virus Ankara adenovirus, Sendai virus, and cytomegalovirus. These viral vectors have also been evaluated in several clinical trials for TB and HIV vaccines [45
47]. Experience in the HIV vaccine field has emphasized the importance of avoiding anti-vector immune responses in developing a vectored vaccine [48]. Often, immune responses to the vaccine vectors prevent or lessen the induction of desired immunity to the recombinant Mtb Ags. From these findings, elimination of the immunogenicity of a vaccine vector is critical for a recombinant viral vaccine. As TB vaccines, recombinant vaccinia virus and adenovirus, which are immunogenic viruses and carry Mtb epitopes, have not been shown to be effective against TB infection, when used solely to vaccinate against it. Instead, these two recombinant TB vaccines were utilized for booster immunizations in BCG-primed animals [49,50]. Such an approach adopting heterologous prime-boost strategies diminished immune responses to the viral vectors, indicating their potential when used in prime-boost vaccination strategies for TB prophylaxis [51,52]. In this same vein, to attenuate a virus, what is commonly done is to make it a replication-defective virus to prevent its in vivo replication. Such a strategy was applied to HPIV2 to make it

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FIGURE 35.3 Schematic illustration of rHPIV2-Ag85B construct. (A) Construction of recombinant (r)HPIV2-Ag85B by reverse genetics [53]. (B) Schematic diagram of rHPIV2 and description of its 6 polypeptides [54].

a vaccine vector. With technological advances in reverse genetics, this virus was made replication-incompetent by elimination of some key viral genes [53] (Fig. 35.3A).

IV. NOVEL VACCINE CANDIDATE, RHPIV2, IN TB PROTECTION Human parainfluenza type 2 virus (HPIV2) is a member of the genus Rubulavirus of the family paramyxoviridae, and it possesses a single-stranded, nonsegmented, and negativestranded RNA genome that encodes seven viral proteins from six genes arranged in the order 30
N-P/V-M-F-HN-L-50 . The viral nucleocapsid protein (N), the phosphoprotein (P), and the large polymerase (L) protein direct transcription and replication [53]. The fusion (F) and hemagglutinin-neuraminidase (HN) transmembrane glycoproteins are the major protective Ags that induce neutralizing

antibodies, and the matrix protein (M) supports virion morphogenesis (Fig. 35.3B). The V protein is expressed from unedited P/V mRNA, and its reported function is to inhibit production of type I IFN, which plays a pivotal role in host innate immunity [54]. Furthermore, HPIV’s tropism is for the respiratory tract, where it efficiently infects, but it does not spread far beyond there, which is an important safety attribute. This virus does not have a DNA phase during its life cycle and can avoid genetic modifications. HPIVbased vectors have been shown to be effective in inducing local immunity and systemic immunity against passenger Ags. Thus, HPIVs have several advantages as vaccine vectors. As an application of reverse genetics to produce the replication-incompetent virus to ensure safety to the vaccinated host, target gene products that would interfere with the vector’s safety are eliminated. Moreover, this approach also enables the development of a vaccine bearing the desired recombinant Ags.

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V. PROTECTIVE EFFECTS OF AN RHPIV2 VACCINE IN MICE WITH TB We assessed the effectiveness of a novel mucosal TB vaccine using human rHPIV2 as a vaccine vector in BALB/c mice [9]. Replicationincompetent rHPIV2 expressing Ag85B

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(rHPIV2-Ag85B) was constructed by using reverse genetics. The construction of replicationcompetent rHPIV2-Ag85B is depicted in Fig. 35.4A. Unlike other viral vaccine vectors, the immune responses against Ag85B induced by rHPIV2-Ag85B immunization was found to be enhanced over the viral vector (Fig. 35.4B and C). The rHPIV2 vector showed

FIGURE 35.4 Expression of Ag85B and its immunogenicity when delivered by nonreplicating rHPIV2 into mice [9]. (A) Construction of M-protein-depleted-rHPIV2 (ΔM-rHPIV2)-Ag85B. (B) Expression of Ag85B (left) and NP (right) genes in BEAS-2B cells infected with ΔM-rHPIV2 or ΔM-rHPIV2-Ag85B at each time point was determined by real-time PCR. (C) Mice were immunized twice with ΔM-rHPIV2 or ΔM-rHPIV2-Ag85B at a 2-week interval by intranasal inoculation (N 5 5 per group). Spleen, peripheral lymph node (pLN), and bronchoalveolar lavage (BAL) cells were collected from immunized mice 2 weeks after the final immunization and were examined by IFNγ ELISPOT assay. These isolated cells were stimulated in vitro with syngeneic splenic lymphocytes infected with ΔM-rHPIV2 or ΔM-rHPIV2-Ag85B for 24 hours. Error bars represent standard deviations. Statistically significant differences are indicated by asterisks (*P , .05 compared to the group stimulated with ΔM-rHPIV2).

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weaker immunogenicity; however, intranasal immunization with rHPIV2-Ag85B induced Mtb-specific immune responses, and the vaccinated mice showed a substantial reduction in the number of CFUs by Mtb in lungs and spleens compared with mice subjected to conventional BCG vaccination (Fig. 35.5). Importantly, rHPIV2-Ag85B in itself alone was protective, not requiring a heterologous prime boost to be effective. These findings provide further evidence for the possibility of rHPIV2Ag85B as a novel TB vaccine.

VI. POSSIBILITIES OF RHPIV2 AS A NEXT-GENERATION VACCINE CANDIDATE Viral vectors are promising vaccine delivery platforms for eliciting Ag-specific immune responses [55,56]. Preexisting anti-vector antibodies, however, constitute an obstacle for their application in humans [57]. Although antibodies against HPIV2 are known to cross-react with Sendai virus, a Sendai virus vector is considered to be effective for human use when

FIGURE 35.5 Repeated immunization with rHPIV2-Ag85B results in protection against TB [9]. (A) Groups of mice were vaccinated using this schedule. (B) Inhibition of bacterial growth in the lungs and spleen following immunization with rHPIV2-Ag85B. Groups of mice were immunized four times with rHPIV2-Ag85B or BCG followed by Mtb infection. The numbers of Mtb CFUs in the lungs and spleen were determined by a colony enumeration assay. The bacterial load is represented as the mean log10 CFUs per organ. Error bars represent standard deviations. Statistically significant differences are indicated by asterisks (*P , .05, **P , .005).

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given nasally [58]. Additionally, a Sendai virus vector was not affected by antibodies against Sendai virus for induction of T cell responses, especially when it was administered intranasally [59]. Furthermore, multiple administrations with rHPIV2-Ag85B showed better prophylaxis than did mice immunized twice with rHPIV2-Ag85B [9]. From these findings, intranasal administration of the HPIV2 vector is considered to have a clinical benefit for human use. The HPIV2 vector has an additional advantage over other viral vectors. Aside from the relatively small insertion size for the Ag85B gene (978 bp), the potency of the induced cellular immune responses against Ag85B is thought to depend on the rHPIV2’s expression mechanisms. This is related to the frequency with which viral RNA polymerase reinitiates the

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next mRNA, which occurs at gene junctions. Consequently, this being an imperfect system results in a gradient of mRNA abundance that decreases according to distance from the genome 30 end [60]. Insertion of the Ag85B gene into the 30 end proximal to the first locus between the leader sequence and the NP gene results in the highest level of gene expression. As a result, Ag85B is transcribed earlier and more abundantly than other viral products (Fig. 35.4A and B). This property of rHPIV2Ag85B leads to the elicitation of stronger Ag85B-specific immune responses than vectorspecific responses in this system (Figs. 35.4C and 35.5B), although recombinant virus vaccine immunization usually induces overwhelming viral-specific immunity compared to the passenger Ag [61,62]. We also demonstrated that intranasal administration of the rHPIV2 vector

FIGURE 35.6 Schematic illustration of the proposed effects of rHPIV2-Ag85B and/or rAg85B in lung inflammation. This summarized view is based on the cited literature [9,67].

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in mice had no adverse effects and provided sufficient immunogenicity for optimal protective immunity against Mtb. These results suggest that intranasal administration of rHPIV2-Ag85B allows for repeated use, minimizing anti-vector immunity, an attribute making rHPIV2 vectors suitable for clinical testing. Since rHPIV2 vectors work effectively in the respiratory tract and Mtb is a respiratory pathogen, these features may contribute to the observed enhanced protection against Mtb infection. Indeed, a number of recombinant TB vaccines have been developed and evaluated for respiratory mucosal immunization [63,64]. Aside from the importance of adaptive immunity for protection to Mtb infections, induction of innate immunity is also crucial for vaccines to elicit potent Ag-specific immune responses. Ligands to host pattern-recognition receptors have been studied as potential targets as adjuvants. dsRNA is a dominant activator of innate immunity because viral dsRNA is recognized by TLR3, RIG-I, and MDA5 [65,66]. As a result, it was demonstrated that the rHPIV2 vector had a potent adjuvant activity residing within the dsRNA recognized by host RIG-I receptor, enhancing not only local innate immunity, but also systemic adaptive immunity. In using rHPIV2-Ag85B for vaccination, it is possible that no additional adjuvant is required because of the adjuvant properties inherent in this vaccine vector. Moreover, recombinant Ag85B was found to be involved in tissue homeostasis, suppressing allergic responses via the stimulation of IFNγ, IL-17, and IL-22 [67]. Additional research is needed to better understand the function of various Mtb vaccine epitopes [68]. In summary, our studies suggest that intranasal administration of rHPIV2-Ag85B induces both mucosal and systemic immunity to Mtb. This vaccine has the advantage of providing significant protection against TB without any additional heterologous vaccine and without requiring an exogenous adjuvant (Fig. 35.6).

VII. FUTURE STUDY USING THE HPIV2 VACCINE One of the ultimate goals for vaccine development is the containment of Mtb so that latent infection is sustained and TB disease reactivation is prevented or at least delayed. From a number of reports, frequency, phenotype, quality, and persistence of memory T cells are thought to contribute to successful vaccination outcomes [69]. It will be necessary to evaluate the detailed mechanisms induced by the HPIV2 vaccine using a TB model of the cynomolgus monkey, which is the only animal that closely recapitulates human TB pathogenesis. Further studies will contribute to the ultimate goal of establishing a new vaccine strategy for the prevention of Mtb infections.

ABBREVIATIONS BAL BCG rHPIV2 Mtb TB Ags LTBI ILC NKT IFN IL Treg MHC Ig TGF HIV dsRNA TLR RIG-I MDA5

bronchoalveolar lavage Mycobacterium bovis bacillus Calmette-Gue´rin recombinant human parainfluenza type 2 virus Mycobacterium tuberculosis tuberculosis antigens latent TB infection innate lymphoid cell invariant natural killer T interferon interleukin regulatory T cell major histocompatibility complex immunoglobulin transforming growth factor human immunodeficiency virus double-stranded RNA toll-like receptor retinoic acid-inducible gene-I melanoma differentiation-associated gene 5.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

References [1] Dye C, Williams BG. The population dynamics and control of tuberculosis. Science 2010;328(5980):856
61. [2] Gengenbacher M, Kaufmann SHE. Mycobacterium tuberculosis: success through dormancy. FEMS Microbiol Rev 2012;36(3):514
32. [3] Seder RA, Hill AV. Vaccines against intracellular infections requiring cellular immunity. Nature 2000;406 (6797):793
8. [4] Mack U, Migliori GB, Sester M, Rieder HL, Ehlers S, Goletti D, et al. LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement. Eur Respir J 2009;33 (5):956
73. [5] Shafiani S, Tucker-Heard G, Kariyone A, Takatsu K, Urdahl KB. Pathogen-specific regulatory T cells delay the arrival of effector T cells in the lung during early tuberculosis. J Exp Med 2010;207(7):1409
20. [6] Grosset J. Mycobacterium tuberculosis in the extracellular compartment: an underestimated adversary. Antimicrob Agents Chemother 2003;47(3):833
6. [7] Reece ST, Kaufmann SHE. Floating between the poles of pathology and protection: can we pin down the granuloma in tuberculosis? Curr Opin Microbiol 2012;15(1):63
70. [8] Halle S, Dujardin HC, Bakocevic N, Fleige H, Danzer H, Willenzon S, et al. Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells. J Exp Med 2009;206(12):2593
601. [9] Watanabe K, Matsubara A, Kawano M, Mizuno S, Okamura T, Tsujimura Y, et al. Recombinant Ag85B vaccine by taking advantage of characteristics of human parainfluenza type 2 virus vector showed Mycobacteria-specific immune responses by intranasal immunization. Vaccine 2014;32(15):1727
35. [10] Kaufmann SH, Baumann S, Nasser Eddine A. Exploiting immunology and molecular genetics for rational vaccine design against tuberculosis. Int J Tuberc Lung Dis 2006;10(10):1068
79. [11] Prezzemolo T, Guggino G, La Manna MP, Di Liberto D, Dieli F, et al. Functional signatures of human CD4 and CD8 T cell responses to Mycobacterium tuberculosis. Front Immunol 2014;5:180. [12] Spits H, Bernink JH, Lanier L. NK cells and type 1 innate lymphoid cells: partners in host defense. Nat Immunol 2016;17(7):758
64. [13] Chackerian A, Alt J, Perera V, Behar SM. Activation of NKT cells protects mice from tuberculosis. Infect Immun 2002;70(11):6302
9. [14] Bendelac A, Bonneville M, Kearney JF. Autoreactivity by design: innate B and T lymphocytes. Nat Rev Immunol 2001;1(3):177
86.

621

[15] Yao S, Huang D, Chen CY, Halliday L, Zeng G, Wang RC, et al. Differentiation, distribution and gammadelta T cell-driven regulation of IL-22-producing T cells in tuberculosis. PLoS Pathog 2010;6(2):e1000789. [16] Li B, Rossman MD, Imir T, Oner-Eyuboglu AF, Lee CW, Biancaniello R, et al. Disease-specific changes in gammadelta T cell repertoire and function in patients with pulmonary tuberculosis. J Immunol 1996;157 (9):4222
9. [17] Lockhart E, Green AM, Flynn JL. IL-17 production is dominated by gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis infection. J Immunol 2006;177(7):4662
9. [18] Sutton CE, Lalor SJ, Sweeney CM, Brereton CF, Lavelle EC, Mills KH. Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity. Immunity 2009;31 (2):331
41. [19] Klose CS, Artis D. Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis. Nat Immunol 2016;17(7):765
74. [20] Shaler CR, Horvath CN, Jeyanathan M, Xing Z. Within the Enemy’s Camp: contribution of the granuloma to the dissemination, persistence and transmission of Mycobacterium tuberculosis. Front Immunol 2013;4:30. [21] Beckman EM, PRecognitiorcelli SA, Morita CT, Behar SM, Furlong ST, Brenner MB. Recognition of a lipid antigen by CD1-restricted alpha beta 1 T cells. Nature 1994;372(6507):691
4. [22] Montamat-Sicotte DJ, Millington KA, Willcox CR, Hingley-Wilson S, Hackforth S, Innes J, et al. A mycolic acid-specific CD1-restricted T cell population contributes to acute and memory immune responses in human tuberculosis infection. J Clin Invest 2011;121 (6):2493
503. [23] Takayama K, Wang C, Besra GS. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol Rev 2005;18(1):81
101. [24] Dubnau E, Chan J, Raynaud C, Mohan VP, Laneelle MA, Yu K, et al. Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol Microbiol 2000;36(3):630
7. [25] Harriff MJ, Karamooz E, Burr A, Grant WF, Canfield ET, Sorensen ML, et al. Endosomal MR1 trafficking plays a key role in presentation of Mycobacterium tuberculosis ligands to MAIT cells. PLoS Pathog 2016;12(3):e1005524. [26] Greene JM, Dash P, Roy S, McMurtrey C, Awad W, Reed JS, et al. MR1 restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates. Mucosal Immunol 2017;10(3):802
13. [27] Bian Y, Shang S, Siddiqui S, Zhao J, Joosten SA, Ottenhoff THM, et al. MHC Ib molecule Qa-1 presents Mycobacterium tuberculosis peptide antigens to CD8 1

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

622

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

35. DEVELOPMENT OF A MUCOSAL TB VACCINE USING HUMAN PARAINFLUENZA TYPE 2 VIRUS

T cells and contributes to protection against infection. PLoS Pathog 2017;13(5):e1006384. Lewinsohn DM, Briden AL, Reed SG, Grabstein KH, Alderson MR. Mycobacterium tuberculosis-reactive CD8 1 T lymphocytes: the relative contribution of classical versus nonclassical HLA restriction. J Immunol 2000;165(2):925
30. Reece WH, Pinder M, Gothard PK, Milligan P, Bojang K, Doherty T, et al. A CD4(1) T-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural Plasmodium falciparum infection and disease. Nat Med 2004;10 (4):406
10. Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J Exp Med 1993;178(6):2249
54. Oliveira MR, Tafuri WL, Afonso LC, Oliveira MA, Nicoli JR, Vieira EC, et al. Germ-free mice produce high levels of interferon-gamma in response to infection with Leishmania major but fail to heal lesions. Parasitology 2005;131(Pt 4):477
88. Liew FY, Li Y, Millott S. Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishmania major through the induction of nitric oxide. J Immunol 1990;145(12):4306
10. Zimmermann N, Thormann V, Hu B, Kohler AB, ImaiMatsushima A, Locht C, et al. Human isotypedependent inhibitory antibody responses against Mycobacterium tuberculosis. EMBO Mol Med 2016;8 (11):1325
39. Bhatt K, Verma S, Ellner JJ, Salgame P. Quest for correlates of protection against tuberculosis. Clin Vaccine Immunol 2015;22(3):258
66. Hawkridge T, Scriba TJ, Gelderbloem S, Smit E, Tameris M, Moyo S, et al. Safety and immunogenicity of a new tuberculosis vaccine, MVA85A, in healthy adults in South Africa. J Infect Dis 2008;198(4):544
52. Qiu Z, Zhang M, Zhu Y, Zheng F, Lu P, Liu H, et al. Multifunctional CD4 T cell responses in patients with active tuberculosis. Sci Rep 2012;2:216. Grotzke JE, Lewinsohn DM. Role of CD8 1 T lymphocytes in control of Mycobacterium tuberculosis infection. Microbes Infect 2005;7(4):776
88. Jayaraman P, Jacques MK, Zhu C, Steblenko KM, Stowell BL, Madi A, et al. TIM3 mediates T cell exhaustion during Mycobacterium tuberculosis infection. PLoS Pathog. 2016;12(3):e1005490. Venkatasubramanian S, Tripathi D, Tucker T, Paidipally P, Cheekatla S, Welch E, et al. Tissue factor expression by myeloid cells contributes to protective immune response against Mycobacterium tuberculosis infection. Eur J Immunol 2016;46(2):464
79.

[40] Mishra BB, Rathinam VA, Martens GW, Martinot AJ, Kornfeld H, Fitzgerald KA, et al. Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent processing of IL1beta. Nat Immunol 2013;14(1):52
60. [41] Domingo-Gonzalez R, Das S, Griffiths KL, Ahmed M, Bambouskova M, Gopal R, et al. Interleukin-17 limits hypoxia-inducible factor 1a and development of hypoxic granulomas during tuberculosis. JCI Insight 2017;2(19):e92973. [42] Lindenstrom T, Agger EM, Korsholm KS, Darrah PA, Aagaard C, Seder RA, et al. Tuberculosis subunit vaccination provides long-term protective immunity characterized by multifunctional CD4 memory T cells. J Immunol 2009;182(12):8047
55. [43] Zinselmeyer BH, Dempster J, Gurney AM, Wokosin D, Miller M, Ho H, et al. In situ characterization of CD41 T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance. J Exp Med 2005;201(11):1815
23. [44] Dwivedy A, Aich P. Importance of innate mucosal immunity and the promises it holds. Int J Gen Med 2011;4:299
311. [45] McShane H, Brookes R, Gilbert SC, Hill AVS. Enhanced immunogenicity of CD41 T-cell responses and protective efficacy of a DNA-modified vaccinia virus ankara prime-boost vaccination regimen for murine tuberculosis Petri WA, ed. Inf Immun 2001;69 (2):681
6. [46] Radoˇsevi´c K, Wieland CW, Rodriguez A, Weverling GJ, Mintardjo R, Gillissen G, et al. Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 Tcell epitope mapping and role of gamma interferon. Inf Immun 2007;75(8):4105
15. [47] Munier CM, Andersen CR, Kelleher AD. HIV vaccines: progress to date. Drugs 2011;71(4):387
414. [48] Cheng C, Wang L, Gall JGD, Nason M, Schwartz RM, McElrath MJ, et al. Decreased pre-existing Ad5 capsid and Ad35 neutralizing antibodies increase HIV-1 infection risk in the step trial independent of vaccination Kremer EJ, ed. PLoS One 2012;7(4):e33969. [49] Abel B, Tameris M, Mansoor N, Gelderbloem S, Hughes J, Abrahams D, et al. The novel tuberculosis vaccine, AERAS-402, induces robust and polyfunctional CD4 1 and CD8 1 T cells in adults. Am J Respir Crit Care Med 2010;181(12):1407
17. [50] McShane H, Pathan AA, Sander CR, Keating SM, Gilbert SC, Huygen K, et al. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat Med 2004;10 (11):1240
4.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

[51] Rahman S, Magalhaes I, Rahman J, Ahmed RK, Sizemore DR, Scanga CA, et al. Prime-boost vaccination with rBCG/rAd35 enhances CD8(1) cytolytic T-cell responses in lesions from Mycobacterium tuberculosisinfected primates. Mol Med 2012;18:647
58. [52] Pathan AA, Minassian AM, Sander CR, Rowland R, Porter DW, Poulton ID, et al. Effect of vaccine dose on the safety and immunogenicity of a candidate TB vaccine, MVA85A, in BCG vaccinated UK adults. Vaccine 2012;30(38):5616
24. [53] Kawano M, Kaito M, Kozuka Y, Komada H, Noda N, Nanba K, et al. Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain. Virology 2001;284:99
112. [54] Schaap-Nutt A, Higgins C, Amaro-Carambot E, Nolan SM, D’Angelo C, Murphy BR, et al. Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates. J Virol 2011;85(8):4007
19. [55] Draper SJ, Heeney JL. Viruses as vaccine vectors for infectious diseases and cancer. Nat Rev Microbiol 2010;8(1):62
73. [56] Clark KR, Johnson PR. Gene delivery of vaccines for infectious disease. Curr Opin Mol Ther 2001;3(4):375
84. [57] Priddy FH, Brown D, Kublin J, Monahan K, Wright DP, Lalezari J, et al. Safety and immunogenicity of a replication-incompetent adenovirus type 5 HIV-1 clade B gag/pol/nef vaccine in healthy adults. Clin Infect Dis 2008;46(11):1769
81. [58] Hara H, Hironaka T, Inoue M, Iida A, Shu T, Hasegawa M, et al. Prevalence of specific neutralizing antibodies against Sendai virus in populations from different geographic areas: implications for AIDS vaccine development using Sendai virus vectors. Hum Vaccin 2011;7(6):639
45. [59] Moriya C, Horiba S, Kurihara K, Kamada T, Takahara Y, Inoue M, et al. Intranasal Sendai viral vector vaccination is more immunogenic than intramuscular under pre-existing anti-vector antibodies. Vaccine 2011;29 (47):8557
63.

623

[60] Tokusumi T, Iida A, Hirata T, Kato A, Nagai Y, Hasegawa M. Recombinant Sendai viruses expressing different levels of a foreign reporter gene. Virus Res 2002;86(1
2):33
8. [61] Sakurai H, Kawabata K, Sakurai F, Nakagawa S, Mizuguchi H. Innate immune response induced by gene delivery vectors. Int J Pharm 2008;354(1
2):9
15. [62] Chen D, Murphy B, Sung R, Bromberg JS. Adaptive and innate immune responses to gene transfer vectors: role of cytokines and chemokines in vector function. Gene Ther 2003;10(11):991
8. [63] Dietrich J, Andersen C, Rappuoli R, Doherty TM, Jensen CG, Andersen P. Mucosal administration of Ag85B-ESAT-6 protects against infection with Mycobacterium tuberculosis and boosts prior bacillus Calmette-Guerin immunity. J Immunol 2006;177 (9):6353
60. [64] Ballester M, Nembrini C, Dhar N, de Titta A, de Piano C, Pasquier M, et al. Nanoparticle conjugation and pulmonary delivery enhance the protective efficacy of Ag85B and CpG against tuberculosis. Vaccine 2011;29 (40):6959
66. [65] Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3. Nature 2001;413 (6857):732
8. [66] Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 2006;441(7089):101
5. [67] Tsujimura Y, Inada H, Yoneda M, Fujita T, Matsuo K, Yasutomi Y. Effects of mycobacteria major secretion protein, Ag85B, on allergic inflammation in the lung. Hoshino Y, ed PLoS One 2014;9(9):e106807. [68] Sreejit G, Ahmed A, Parveen N, Jha V, Valluri VL, Ghosh S, et al. The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (β2M) affecting antigen presentation function of macrophage Lewinsohn DM, ed. PLoS Pathog 2014;10(10):e1004446. [69] Kaufmann SH. Future vaccination strategies against tuberculosis: thinking outside the box. Immunity 2010;33(4):567
77.

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Sexually Transmitted Infections and the Urgent Need for Vaccines: A Review of Four Major Bacterial STI Pathogens Avinash Kollipara1, De’Ashia Lee2 and Toni Darville1 1

Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Department of Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

2

I. INTRODUCTION Over 1 million sexually transmitted infections (STIs) are acquired every day worldwide. Every year, there are an estimated 357 million new infections with one of four STIs: chlamydia (131 million), gonorrhea (78 million), syphilis (5.6 million), and trichomoniasis (143 million). The 2017 surveillance report released by the Centers for Disease Control and Prevention (CDC) marked the third consecutive year in which overall rates for these pathogens increased [1]. STIs cause serious consequences beyond the immediate impact of infection itself. Viral and bacterial STIs can increase the risk of HIV acquisition [2]. Mother-to-child transmission can result in neonatal conjunctivitis, pneumonia, sepsis, low birth weight, stillbirth, and death [3]. Over 900,000 pregnant women were infected with syphilis in 2012, resulting in

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00036-5

approximately 350,000 adverse birth outcomes, including stillbirth [4]. STIs are spread predominantly by sexual contact through vaginal, anal, or oral sex. Some STIs can also be spread through nonsexual means such as via blood. An individual can have a STI without obvious symptoms of disease, which contributes to high prevalence. Symptoms of STIs include vaginal discharge in women, urethral discharge in men, painful urination, genital ulcers, and abdominal or pelvic pain in both sexes [5]. Currently, safe and highly efficacious vaccines are available for only two viral STIs: hepatitis B [6] and human papillomavirus (HPV) [7]. These vaccines have led to huge advances in the area of STI prevention. The hepatitis B vaccine is included as part of infant immunization programs in 93% of countries and has already prevented an estimated 1.3 million deaths from

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chronic liver disease and cancer [8]. HPV vaccine is available as part of routine immunization programs in over 65 countries, most of them high- and middle-income countries, and has been forecasted to prevent the deaths of more than 4 million women over the next decade if 70% immunization coverage can be achieved [9]. Progress for vaccine development related to four leading bacterial STIs—chlamydia, gonorrhea, syphilis, and the emerging pathogen Mycoplasma genitalium [10]—varies according to pathogen, the most progress being found for chlamydia. In this chapter, we review these pathogens with respect to their microbiology, clinical manifestations, epidemiology, pathologic and protective immune responses, and vaccine development.

II. CHLAMYDIA

mucous discharge or mucopurulent cervicitis and dysuria in women and urethritis in men [11]. Despite initiating local inflammation, C. trachomatis infection remains subclinical in 70% 90% of women and 30% 50% of men [13]. For women, a common anxiety with a diagnosis of Chlamydia is the effect on fertility. Ascension of C. trachomatis to the endometrium of the uterus and to the fallopian tubes can lead to pelvic pain or pelvic inflammatory disease (PID), which can increase the risk for ectopic pregnancy, tubal factor infertility, and chronic pelvic pain [11]. Chlamydia infection has also been linked to other adverse pregnancy outcomes such as chorioamnionitis, placentitis, premature rupture of membranes, and preterm birth. It can also cause conjunctivitis and pneumonitis in newborns. In men, Chlamydia has been associated with urethritis, epididymitis, orchitis, and prostatitis [14], but the connection to male infertility is less clear.

A. Microbiology Chlamydia trachomatis is an obligate intracellular, Gram-negative bacterium that infects human ocular, genital tract, and respiratory epithelium [11]. The developmental cycle of C. trachomatis is approximately 48 72 hours and starts with an elementary body (EB), a small infectious particle that attaches to and enters host epithelial cells, where the EB transforms into a reticulate body (RB), which replicates in a membrane-bound vacuole called an inclusion. After 24 48 hours, RBs transform back to EBs, which exit the cell by lysis of the inclusion and the host cell or via extrusion of the inclusion leaving the host cell intact [12].

B. Clinical Manifestations Chlamydia infects the single-cell columnar epithelium of the endocervix of women and the urethra of men. At the mucosal site, inflammation is characterized by erythema, edema, and

C. Epidemiology C. trachomatis is the most common bacterial STI and results in substantial morbidity and economic costs worldwide. The World Health Organization (WHO) [15] reported that in 2012, 131 million new cases of chlamydia occurred among adults and adolescents, with a global incidence rate of 38 per 100 females and 33 per 1000 males [16]. The annual surveillance report released by the CDC showed that during 2015 16, the rates of Chlamydia increased by 4.7% from previous years [1].

D. Current Treatment Options The WHO recommends either azithromycin or doxycycline as oral treatment options, and these drugs are equally effective in eradicating infection [16]. Unexpectedly, widespread screening and treatment efforts have been accompanied

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II. CHLAMYDIA

by rising reinfection rates, which are attributed to a reduced duration of infection arresting the development of herd immunity. Thus a vaccine is essential for control efforts [17].

E. Immune Responses Associated With Pathology Inflammatory mediators are capable of inducing tissue destruction during chlamydial infection [18]. Animal models of trachoma and of female genital infection reveal a direct correlation between neutrophil influx and development of tissue damage [19 24]. In addition, human transcriptional profiling and genetic studies have determined an association of enhanced innate proinflammatory responses with trachomatous scarring [24 26]. Finally, there is in vitro evidence for interleukin 1 (IL-1), a cytokine released by neutrophils and monocytes, to cause direct oviduct cell damage [27]. Since the innate inflammatory response is induced by the interaction of pathogenassociated molecular patterns (PAMPs) with pathogen-recognition receptors on innate inflammatory cells and host epithelial cells, it should not be surprising that increased bacterial burden leads to enhanced inflammation and disease [20,28,29] (Fig. 36.1). The mouse model of genital infection revealed that repeated infections that were abbreviated by antibiotic treatment led to protection from oviduct disease that was associated with a significant reduction in frequency of neutrophils and an increase in the frequency of T cells infiltrating the genital tract upon challenge [30]. Furthermore, a single infection with a plasmiddeficient strain of C. muridarum protects mice from oviduct disease upon challenge with the fully virulent parental strain [31]. This protection is again associated with reduced neutrophil influx and an anamnestic T cell response [30]. Thus avoidance of chlamydial-induced neutrophil influx and neutrophil activation

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appears essential for disease prevention. A vaccine that promotes adaptive T cell responses that are innocuous for the tissue but potently chlamydiacidal should protect from disease by avoidance of PAMP-induced tissue-damaging innate immune responses. Human epidemiological studies demonstrate an increased risk of disease with recurrent infections [32,33]. However, the contribution of pathological effects of the primary infection versus subsequent infections is unknown, and each successful infection would induce an element of tissue-damaging innate responses. Interferon gamma (IFNγ) and IL-12 mediate protective T helper 1 (Th1) cell responses [34], while T helper 2 (Th2) cell responses have been shown to be nonprotective [35]. CD8 T cells have been shown to play a role in pathogenesis in the macaque and mouse models of genital tract infection, possibly through the production of tumor necrosis factor alpha (TNF-α) [36,37]. Currently, there is no evidence for the role of B cells in tissue pathology during chlamydial infection. Recent technological advances in immune profiling using animal models and human clinical samples provide an opportunity to discern specific components of the immune response that contribute to pathology and provide insight for safe vaccination strategies.

F. Vaccine-Related Research The critical role of T cells in chlamydial immunity was first demonstrated 30 years ago with the observation that athymic nude mice developed a chronic C. muridarum infection [38]. T cells are detected at the site of infection in mice and humans; antigen-presenting cells can prime T cells in the lymph nodes, where they migrate to inductive sites within the genital tract to clonally expand in response to chlamydial infection. These inductive sites contain CD41 T cells that form perivascular lymphoid clusters [39,40]. CD41 T cells that produce IFNγ mediate

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36. SEXUALLY TRANSMITTED INFECTIONS AND THE URGENT NEED FOR VACCINES

FIGURE 36.1

Innate immunopathological mechanisms of Chlamydia infection inside a host cell. Chlamydia infection is not “silent” as it activates membrane-bound (TLR2, denoted by the yellow rhombus) as well as intracellular pathogenrecognition receptors (NOD, NLRP, caspase 1/11), cytosolic DNA sensors (cGAS, STING), and DAMP pathways (IL-1α, IL1β), leading to neutrophil infiltration thereby causing inflammation.

protection from C. muridarum and C. trachomatis. Mice deficient in MHC class II [41], CD4 [42], IL-12 [34], IFNγ [43], or the IFNγ receptor [44] have an enhanced susceptibility to infection. Th2-type responses correlate with disease progression and pathology during human ocular infection [35]. Transfer of chlamydial-specific Th2 cell clones fails to protect mice from genital infection [45]. These studies suggest that induction of antigen-specific Th1 cell responses should be a goal of vaccination.

IFNγ-mediated control of in vivo infection is not fully understood, but IFNγ controls in vitro growth of C. trachomatis in human cells by inducing the production of indoleamine-2,3dioxygenase (IDO) [46]. IDO leads to tryptophan degradation and lethality to C. trachomatis by starvation of the essential amino acid, but the bacterium can be rescued through the addition of indole [47]. Additional effector mechanisms include the activation of phagocytic macrophages [48] and CD41 T cell cytotoxicity

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II. CHLAMYDIA

[49,50]. Evidence suggests that a Th1 cell response that generates long-term, sterilizing mucosal immunity should be the optimal goal of immunization. Tissue-resident memory T (TRM) cells have emerged as an important subset of memory T cells in tissue-specific immune responses [51]. These cells reside in epithelial barrier tissues that interface with the environment, such as the gut, lungs, skin, and reproductive tract, and can provide rapid, effective immunity against previously encountered pathogens. TRM cells are able to respond to a pathogen challenge independent of recruited, circulating systemic T cells [52]. The female genital tract mucosa is an important barrier to pathogenic microorganisms. Mouse studies illustrate that HSV infection and vaccination generate an accumulation of CD41 TRM cells in the vaginal mucosa that are maintained by a local chemokine gradient and mount a rapid, anamnestic response upon antigenic challenge [53,54]. Mucosal immunization, for example, via the nasal route, with ultraviolet-light-inactivated [55] C. trachomatis complexed with chargeswitching synthetic adjuvant particles (cSAPs) incorporating the TLR7 agonist resiquimod elicited long-lived protection against chlamydial infection in conventional and humanized mice [56]. Vaccination generated mucosal and systemic T cell responses, but optimal clearance required TRM induction in the uterine mucosa. Mucosal CD41 Th1 cells will likely be instrumental to Chlamydia vaccine success, as the intensity of mucosal CD41 Th1 cellular responses is an important correlate of immunity [57] (Fig. 36.2) (Chapter 16: Regulation of Mucosal Immunity in the Genital Tract: Balancing Reproduction and Protective Immunity). Mouse models demonstrate that CD81 T cells are not needed for infection clearance; however, antigen-specific CD81 T cell clones can home to the genital tract and enhance clearance through their production of IFNγ [58,59]. Evidence suggests that upregulation of PD-L1

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in the genital tract following infection may impair CD81 T cell expansion via PD-1 ligation, hampering development of CD8 memory responses [60]. This could be a mechanism to avoid cell-mediated genital tract pathology, as CD81 T cells can play a role in tissue damage via production of TNF-α [36]. The role of B cells and antibody in the context of antichlamydial immunity is not completely understood [61]. Mice lacking B cells do not demonstrate an altered course of primary genital infection with C. muridarum [62] but are more susceptible to reinfection [63]. Immune wildtype mice depleted of CD41 or CD81 T cells clear a secondary challenge, but B-cell-deficient mice are unable to resolve secondary infection after CD41 T cell depletion [42,64]. Passive transfer of immune serum to naı¨ve mice does not provide protection, but antigen-experienced mice with primed CD41 T cells and immune serum are afforded optimal protection [65]. Additionally, B-cell-deficient mice have a reduced capacity to prime CD41 T cells, leading to bacterial dissemination [66]. Studies utilizing B-cell-deficient mice are limited, owing to the inherent reduction of a significant antigenpresenting cell population and cytokine source, less efficient memory CD41 T cell initiation, possible disruption of lymphoid architecture and subcapsular sinus macrophages, and enhanced chlamydial dissemination [61]. Although early human studies suggested that Chlamydia-specific antibodies might play a role in C. trachomatis immunity based on in vitro neutralization assays [67 69], epidemiological studies indicate that high antibody titers are associated with infertility and increased exposure to chlamydial infection and do not correlate with infection resolution or control of ascending infection [70,71]. However, antibodies specific for chlamydial outer-membrane proteins have been shown to correlate with protection [72,73]. Their protective effects are likely due to their ability to enhance Th1 cell activation and cellular effector responses [74].

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FIGURE 36.2 Protective immune responses involved in clearing Chlamydia infection in the genital tract. Chlamydia enters via the vagina and infects the cervical epithelial cells. This figure illustrates both the immune and nonimmune conditions in the genital tract. On the left side, the immune system in naı¨ve individuals encounters Chlamydia for the first time, and if not controlled, it ascends to infect the endometrium and fallopian tubes, causing pelvic inflammatory disease, scarring, and occlusion, which can lead to infertility or ectopic pregnancy. On the right side, Chlamydia enters an immune individual (e.g., due to vaccination or natural immunity), and the immune system is primed and recognizes Chlamydia directly as soon as it is encountered. Antibodies (SIgA and IgG) and T cells secreting IFNγ react in an orchestrated fashion to clear the infection before it ascends to the upper genital tract.

G. Preclinical Vaccine Studies and Vaccine Trials in Progress The first phase 1 trials of chlamydial vaccine candidates are under way, and scientific advances hold promise for additional candidates to enter clinical evaluation in the coming years [75]. Current strategies hinge on a variety of different platforms and are supported by academic, government, and corporate institutions. A major focus is development of vaccines prepared with the major outer-membrane protein (MOMP) of C. trachomatis [76]. MOMP vaccination utilizing cationic liposomes (CAF01) induced robust antibody responses, type 1 immunity, and partial protection from infection in minipigs and significant protection from genital tract disease in mice [77,78]. A second MOMP formulation prepared with a novel oilin-water nanoemulsion (Nanostat) and delivered nasally purportedly decreased oviduct

pathology in mice by 80% [79]. Protection was associated with high levels of serum and vaginal antibodies and robust IFNγ responses. An immunoproteomics approach identified Chlamydia polymorphic membrane proteins (PMPs) preferentially loading MHC Class II, and vaccination with three MOMP and four PMP alleles emulsified with DDA/MPL adjuvant significantly reduced bacterial shedding in a transcervical C. trachomatis mouse model [80]. Current investigation is centered on development of an outer-membrane protein based vaccine for phase 1 testing. The ability to generate vaccine-induced TRM cells in the mouse genital mucosa is a major advancement in the field [56]. Mucosal immunization with ultraviolet-light-inactivated C. trachomatis complexed with novel, chargeswitching synthetic adjuvant particles (cSAPs) incorporating the TLR7 agonist resiquimod conferred significant protection against

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chlamydial infection in mice [55]. Uterine vaccination induced mucosal resident and systemic T cell responses that induced optimal chlamydial clearance compared to intranasal and intramuscular vaccine delivery. Another major advancement is the use of high-throughput technology for determination of T-cell-specific epitopes. Examination of T cell IFNγ responses in a cohort of 141 subjects led to identification of eight CD4 antigens and 18 CD8 antigens associated with clearance or resistance to infection [81]. Another group assessed 120 Chlamydia proteins and identified seven novel antigens that conferred partial protection in mice [82]. Recent analysis demonstrated chlamydial proteins recognized by highly exposed women that limit or resist genital tract infection [83]. These proteins were primarily involved in protein synthesis, central metabolism, and type III secretion. Ongoing research is focused on in vitro screening of PBMC responses from previously infected subjects to chlamydial proteins. These efforts may help to identify protective antigens broadly expressed by human leukocyte antigen (HLA) haplotypes to better guide an effective vaccine strategy. A Vaxonella platform for chlamydia immunization is being investigated for immunogenicity and efficacy in animal models [75]. The oral delivery system utilizes an attenuated Salmonella enterica vector that has passed phase 2 trials as the Typhella vaccine and allows for insertion of chlamydial antigenic gene sequences. Salmonella acts as an immunostimulator, bypassing the necessity of additional adjuvants. The vector is constructed with technology designed to generate stable attenuation and is formulated to exclude toxic bile salts during ingestion for optimal delivery [84,85]. Finally, work related to vaccine development for C. trachomatis ocular infection might shed light on vaccine development for the genital tract. Ocular inoculation of macaques with attenuated, plasmid-deficient C. trachomatis ocular serovar A elicited partial protection against a

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virulent strain in a subset of cynomolgus macaques that appeared to correlate with MHC Class II haplotype [86]. Interestingly, depletion of CD81 T cell responses in vaccinated macaques abrogated the protective response [87]. This strategy is currently in preclinical development with the National Institute of Allergy and Infectious Diseases (NIAID). Murine genital inoculation with plasmid-deficient C. muridarum conferred protection against upper genital tract pathology [31]. These results were not replicated in the macaque model of genital infection; however, pathology was minimal in macaques inoculated with wild-type C. trachomatis genital serovar D [29]. This illustrates the need for delineating protective immune mechanisms and optimal vaccine formulations in ocular versus genital tract infections [88].

H. Challenges to the Development of a Vaccine Critical advances have been made in the field of chlamydial immunology that includes identification of the basic correlates of protective immunity and capacity of vaccination to induce resident-memory T cells. However, there are many challenges and questions that need to be addressed regarding the adaptive response to infection, in order to develop an efficacious chlamydial vaccine. Use of the mouse model is limited because T-cell-mediated clearance operates through different mechanisms compared to humans. The mechanism by which C. trachomatis induces genital tract pathology in humans is also unclear, and we lack understanding of why some patients remain asymptomatic with no pathology and others develop PID. Additionally, there is a basic requirement to characterize the correlates of immunity that allow for chronic infection versus spontaneous clearance. Exacerbating this problem is the paucity of data reflecting protective responses in humans, and the macaque model for vaccine testing has demonstrated more success for

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trachoma than for genital infection. Furthermore, identification of protective human antigens is in its infancy [81,82]. To enhance and guide the proper immune response to vaccination, current mucosal adjuvants will require further testing, and new adjuvants may need to be developed (Chapter 10: Innate Immunity-Based Mucosal Modulators and Adjuvants, Chapter 11: Toxin-Based Modulators for Regulation of Mucosal Immune Responses, and Chapter 46: Harnessing γδ T Cells as Natural Immune Modulators). A mucosal vaccine may not be necessary if systemic immunity is capable of preventing upper genital tract infection and pathology. Sterilizing immunity is the ultimate goal; however, a partially protective vaccine that could be boosted by a live infection may be more pragmatic, particularly if it is able to prevent disease, prevent transmission, and seed the genital mucosa with TRM. Ideally, a chlamydial vaccine would be combined with other vaccinations delivered during childhood or adolescence to enhance vaccine uptake, improve marketability, and avoid multiple immunizations. Economic analysis suggests a vaccine that provides partial immunity would be cost-effective compared to current screening and treatment strategies [89]. A partially protective vaccine would reduce the prevalence of genital infection [90], and vaccination of both sexes could synergize to impart sterilizing immunity against sexual transmission [91]. Current research must continue to focus on identifying correlates of protective immunity versus pathogenic responses and delineate adjuvants and antigens that can enhance protective T cell responses.

III. GONORRHEA A. Microbiology Neisseria gonorrhoeae is a Gram-negative, obligate, fastidious, diplococcus bacteria. Like all

Gram-negative bacteria, N. gonorrhoeae possesses a cell envelope composed of an inner cytoplasmic membrane, a middle layer of peptidoglycan, and an outer membrane [92]. The outer membrane contains lipooligosaccharide (LOS; also called endotoxin), phospholipid, and a variety of proteins that contribute to cell adherence, tissue invasion, and resistance to host defenses. N. gonorrhoeae has many dynamic polymeric protein filaments called type IV pili, which allow the bacteria to adhere to and move along surfaces [93]. N. gonorrhoeae is a naturally competent bacteria, and its type IV pilus is involved in DNA exchange, specifically proteins Pil Q and Pil T [94]. This allows for N. gonorrhoeae to acquire new genes. This is especially dangerous in the clinical setting because it has led to a rise in antibiotic-resistant strains of bacteria [95]. Additionally, N. gonorrhoeae has surface proteins, called Opa proteins, that bind to receptors on immune cells and suppress the immune response. As a result the host is unable to develop immunological memory. N. gonorrhoeae is also able to evade the immune response through antigenic variation; the bacteria alter antigenic determinants of Opa proteins and Type IV pili that adorn its surface [96,97]. The many permutations of the surface proteins make it more difficult for immune cells to recognize and kill the bacteria.

B. Clinical Manifestations Gonorrhea is manifested by a spectrum of clinical presentations from asymptomatic carriage to localized mucosal inflammation and, rarely, disseminated infection. As many as 80% of females with urogenital gonorrhea are asymptomatic. By contrast, men are asymptomatic only 50% of the time [98]. Male gonococcal urethritis is characterized by a purulent discharge and painful urination. Untreated, it may resolve spontaneously in several weeks or may

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III. GONORRHEA

be complicated by epididymitis, a common cause of male infertility in some regions [99]. In women, gonococcal infection can cause symptomatic urethritis, cervicitis, and PID. Complications of infertility, ectopic pregnancy, and chronic pelvic pain are frequent and debilitating [100,101]. Disseminated gonoccocal infections are rare (0.5% 3%) and include gonococcal arthritis-dermatitis syndrome, suppurative arthritis, and infrequently endocarditis, meningitis, or other localized infections [100]. Newborns exposed to infected mothers during birth may develop eye infection (ophthalmic neonatorum) and/or, rarely, disseminated infection [102].

C. Epidemiology Gonorrhea is a major global public health problem, and the WHO estimated that in 2008, there were 78.3 million new cases in adults worldwide [103]. In the United States, the CDC reports gonorrhea as the second most common notifiable bacterial infection [104]. In 2016, approximately 468,500 cases of gonorrhea were reported in the United States, at a rate of 145.8 gonorrhea cases per 100,000 population [104]. During 2015 16, the rate of reported gonorrhea cases increased 18.5%, and it had increased 48.6% since the historic low in 2009 [104].

D. Current Treatment Options In the absence of a vaccine, effective antibiotic therapy of gonorrhea is critical to cure infected persons and to reduce the spread of infection; however, gonorrhea is on the rise worldwide, and strains that are resistant to many antibiotics are emerging [105 107]. The current CDC-recommended treatment includes dual therapy consisting of intramuscular ceftriaxone and oral azithromycin [108,109]. Recently, a strain that was resistant to this dual regimen was isolated from an infected

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individual [110]. Currently, third-generation cephalosporins remain the last line of antibiotics for empiric monotherapy [102]. Owing to the rising incidence of gonococcal infections and the rapid emergence of antibiotic resistance, there is an urgent need for newer antibiotics and a prophylactic vaccine.

E. Immune Responses Associated With Pathology The only natural host of N. gonorrhoeae is the human. During initial infection, gonoccoci can induce pus-filled exudates that consist of neutrophils, epithelial cells, and intracellular and extracellular bacteria. N. gonorrhoeae has evolved to evade several host immune defenses. Gonococci are equipped to defend against host cationic antimicrobial proteins and evade complement [102]. In experimentally infected men, elevated proinflammatory cytokines and chemokines have been detected [111]. Mouse models have shown that gonococci selectively induce Th17 cells, which leads to the recruitment of neutrophils that are active in host defense [112]. However, gonococci possess mechanisms to resist killing by neutrophils [102]. The adaptive immune response to gonorrhea is ineffective, and no memory develops, leading to repeated infections being common. Serum and local antibody responses in women and men infected with gonorrhea are modest, unsustained, and not protective [113 115]. The lack of a protective response following gonococcal infection has led to investigation of gonococcal immunosuppressive pathways [116 120]. In mice, gonococci suppress the development of Th1- and Th2-type adaptive immune responses by mechanisms dependent on TGF-β and IL-10 as well as type 1 regulatory T cells [121 123]. This immunosuppression occurs alongside induction of IL-17, resulting in strong inflammatory responses but no adaptive immune response. Humans with gonorrhea have elevated serum IL-17 and IL-23 compared to healthy

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controls [124]. Human monocyte-derived dendritic cells (DCs) secrete IL-23 and IL-10 upon stimulation with N. gonorrhoeae in vitro [112,121]. Other mechanisms of immunosuppression include induction of apoptosis in antigenpresenting cells and inhibition of DC-induced proliferation of T cells [117,120]. Gonococcal Opa proteins that bind CEACAM1 were reported to downregulate proliferation of activated CD41 T cells and also B cells [116,125].

F. Vaccine-Related Research Progress on gonorrhea vaccines has been hindered for decades for many reasons. First, correlates of protection in humans have not been identified. Second, early vaccine efforts failed, owing to the highly antigenically variable surface of N. gonorrhoeae, and a small animal model for identifying protective responses and for testing of antigens and immunization routes was only recently developed [100]. Only two antigens, killed whole cells and purified pilin, have been tested in clinical trials, which occurred over 30 years ago and were unsuccessful [114]. This has discouraged research, funding, and commercial interest in gonorrhea vaccines. Advances in microbial pathogenesis, immunology, and molecular epidemiology combined with new infection models and the powerful new tools of genomics, proteomics, and glycomics have renewed and intensified research on gonorrhea vaccine development. Knowledge of the specific immune mechanisms that protect against N. gonorrhoeae infection is severely lacking. The lack of evidence that natural infection induces resistance to reinfection seriously limits the ability to define the types of immune responses that an effective vaccine must induce. Conventional thinking suggests that antibody-mediated immunity rather than cell-mediated immunity would be a key mediator for protection; however, this has not been experimentally proven. In addition, to

the extent that N. gonorrhoeae reside intracellularly and thereby escape antibody-mediated defenses, T-cell-mediated immunity could have a role that merits exploration. In one report, repeatedly infected women in Nairobi, Kenya, showed partial strain-specific immunity; however, this finding was not replicated in a study of less exposed subjects in the United States [126,127]. Antibodies against the reductionmodifiable protein (Rmp) block the bactericidal activity of PorB or LOS-specific antibodies, and the relative proportion of bactericidal and blocking antibodies have been proposed to correlate with immunity or disease [128]. Lacking are studies on the effect of high-titer bactericidal antibody, which natural infection does not induce, or cellular immunity in protecting against human infection. A successful vaccine must protect against all antigenic types, and novel approaches to address this challenge are needed. In addition, if the mechanisms by which N. gonorrhoeae evades the host immune response can be identified, vaccines might be designed to inhibit or sidestep these mechanisms and allow an effective protective immune response to develop. The relative contributions of Th17-cell-driven innate responses and Th1/Th2-cell-driven adaptive responses to protective immunity remain to be elucidated. N. gonorrhoeae-induced immunosuppression in mice can be reversed by treatment with blocking antibodies against TGF-β and IL-10, which permit the development of Th1-type- and Th2-type-dependent responses with circulating and vaginal anti-N. gonorrhoeae antibodies, immunological memory, and protective immunity against reinfection [122,129]. However, neutralization of TGF-β also interferes with Th17 cell responses [129]. Alternatively, intravaginal administration of IL-12 incorporated in sustained-release microspheres enhanced Th1-cell-dependent adaptive immune responses, including generation of anti-N. gonorrhoeae antibodies, accelerated clearance of infection, and protection against reinfection, but

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IV. SYPHILIS

without interfering with the induction of Th17type responses [130]. Systematic testing of antigens, immunization routes, and adjuvants is greatly facilitated by animal models, but the human specificity of N. gonorrhoeae poses a challenge for animal modeling [129]. Experimental urethral infection of male volunteers has been used to define the innate and humoral responses to infection and reinfection and the importance of selected virulence factors [111,131 133]. This wellcharacterized model provides a system for testing of vaccine candidates [132]. However, the human challenge model can assess only immunoprotection against early stages of male urethral infection and might not identify vaccine candidates that would be effective in women or prevent complicated infections or disseminated gonoccocal infections. Chimpanzees are less subject to N. gonorrhoeae host restrictions than other laboratory animals. Male chimpanzees develop N. gonorrhoeae urethritis that is similar to that observed in humans, and natural transmission of gonorrhea from a male chimpanzee to two females was documented. Immunization of chimpanzees with a whole cell vaccine resulted in increased resistance to infection [114]. Chimpanzees are no longer available for gonorrhea research, but the insights gained from these experiments should not be ignored.

G. Future Vaccine Implications The futile search for a gonorrhea vaccine dates back more than a century. All the candidates that made it to clinical trials failed to provide any protection against the pathogen. Hope has recently come from a group B meningococcal vaccine. A recent retrospective case control study demonstrated that exposure to this vaccine MeNZB was associated with reduced rates of gonorrhea; [134]. MeNZB’s more broadly protective successor, 4CMenB (marketed as Bexsero by GSK Vaccines) has three genetically

modified meningococcal proteins. Two of these proteins are shared with N. gonorrhoeae. Hence GSK plans to explore the potential of 4CMenB to protect against gonorrhea [135].

IV. SYPHILIS A. Microbiology Treponema pallidum is a Gram-negative, obligate, intracellular, microaerophilic spirochete that causes syphilis [136,137]. T. pallidum is very small, and requires dark-field microscopy for visualization [136]. It is rapidly inactivated by mild heat, cold, desiccation, and most disinfectants [138]. T. pallidum is incapable of fixing carbon to form its own organic compounds, so it has very limited metabolic capabilities. Because T. pallidum relies heavily on its host for nutrients, it has not been successfully cultured in vitro. Unlike most Gramnegative bacteria, T. pallidum lacks lipopolysaccharide (LPS) and type III secretion system homologs. Although treponemes possess both outer and cytoplasmic membranes, they differ considerably in structure from enteric Gramnegative bacteria. The organism has an outer membrane containing an extremely low density of surface-exposed transmembrane proteins. Because of the fragility of its outer membrane, genetic manipulation of T. pallidum has been very difficult [138]. The genome of T. pallidum does not reveal any obvious classical virulence factors, which makes it difficult to determine factors that contribute to pathogenesis [136]. The only known natural host of T. pallidum is the human [137,139]. Rabbits can be infected and are used to study syphilis because they develop lesions consistent with both primary and secondary syphilis [140,141].

B. Epidemiology In the United States, syphilis has been a nationally notifiable disease since 1944. The WHO

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estimates that worldwide in 2012, there were 18 million prevalent cases of syphilis in adolescents and adults aged 15 49 years and 5.6 million new cases [103]. The number of cases of primary and secondary syphilis has increased since 2001, and this trend mostly continued through 2015 [142]. In 2015, the national rate increased by 19% to 7.5 cases per 100,000 population, which is the highest rate reported since 1994 [143]. During 2014 15, primary and secondary syphilis rates increased among men and women in every region of the country and in every racial and ethnic group except American Indians and Alaska Natives [143]. The rise in the rate of reported syphilis cases is attributable primarily to increased cases among men who have sex with men [142]. The overall rate of reported congenital syphilis has also increased, with 12.4 cases per 100,000 live births reported in 2015 [142].

C. Clinical Manifestations Syphilis is a chronic disease that is divided into stages (primary, secondary, latent, and tertiary), with different signs and symptoms associated with each stage [144 146]. Infection is initiated when T. pallidum penetrates dermal microabrasions or intact mucous membranes, resulting in a single chancre or multiple chancres at the site of inoculation, usually the genitalia [136]. Chancres are usually firm, round, and painless and last for 3 6 weeks. The chancre usually disappears whether or not treatment is received. Because the chancre is painless and may be located in anatomical sites that may not be noticeable to the patient, diagnosis is often delayed until secondary or other late disease manifestations become apparent. Manifestations of secondary syphilis usually occur within 3 months of initial infection. The secondary stage of syphilis causes a range of diverse symptoms, including fever, swollen lymph nodes, and rash; this is the stage when most people present for treatment. Left

untreated or inadequately treated, syphilis can progress to tertiary disease or latency [147]. Latent syphilis commonly refers to the period during which a person is infected but has no symptoms. Early latency is defined as the first 4 years of infection, when patients can experience recurrent secondary manifestations. Late latent syphilis is defined as asymptomatic infection of longer than 4 years or unknown duration. While sexual transmission of late latency syphilis is unlikely, congenital transmission may still occur. Latent syphilis can end when antibiotics are administered or when manifestations of tertiary syphilis occur [138]. Tertiary syphilis describes patients with late syphilis who have symptomatic manifestations involving the cardiovascular system or gummatous disease. Gummatous disease involves nodular lesions, known as gummas, that most commonly affect the skin and bones; however, these lesions can also occur in the liver, heart, brain, stomach, and upper respiratory tract. Unlike chancres, gummas do not resolve spontaneously but can be treated with appropriate antibiotic therapy [144,146].

D. Current Treatment Options In 1943, benzathine penicillin G was determined to be an effective cure for primary syphilis [148]. To date, STI control efforts for all stages of syphilis are mainly dependent on the timely diagnosis and prompt treatment of infected individuals and their contacts with penicillin. Antibiotic resistance to penicillin is thus far not a concern in syphilis treatment [136]. However, resistance to second-line therapy such as macrolide-based antibiotics has emerged [149]. Because there is no vaccine to prevent syphilis, timely diagnosis and treatment of infected individuals and their sexual partners are key to syphilis control programs, which should also include sex education and promotion of condom use to prevent infection.

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E. Immune Responses Associated With Pathology Two key mechanisms are essential to T. pallidum survival [150]. First, it is highly invasive. An example of T. pallidum’s invasive capabilities is that it can cross the placental barrier to cause congenital syphilis. It can also disseminate widely via the bloodstream and lymphatics to infect and cause damage to many major organs [151,152]. This is possible because T. pallidum can attach to and penetrates intact membranes and endothelial cell monolayers [153,154]. Second, T. pallidum has the capacity to evade the immune response and persist for extended periods of time. Many of the virulence mechanisms of T. pallidum are unknown. The bacteria lack surface exposed proteins such as LPS and other known virulence factors [155]. This allows the bacteria to escape immune detection [156]. In rabbits, protection from reinfection is dependent on the induction of a Th1 cell response, the production of IFNγ, opsonic antibodies, and macrophages [157,158]. Immunoglobulin M (IgM) antibodies are usually the first to develop after establishment of bacterial infection, followed shortly by immunoglobulin G (IgG). In the intratesticular model of syphilis infection in rabbits, anti-T. pallidum IgM and IgG are detectable as early as 6 days post infection [156,159,160]. The antibodies are able to neutralize the treponemes [161]. Although antibodies delay lesion formation, they do not protect against infection or kill the bacteria [136,161,162]. Previous research has revealed that primary and secondary syphilis lesions are primarily infiltrated with macrophages that clear the treponemes. Additionally, there is a Th1 cellular response, and IFNγproducing CD41 and CD81 T cells attract and activate macrophages that clear opsonized bacteria [150,157,158,163 165]. Targeting the invasive nature of T. pallidum and understanding disease pathogenesis are crucial to the development of vaccines against this pathogen.

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F. Vaccine-Related Research Little is known about immune protection against infection with T. pallidum in humans. The fact that people can become infected over and over again suggests against naturally acquired immunity. While several animals can be infected with T. pallidum, only a few develop clinical disease [150]. This has significantly hindered basic and clinical research. Nevertheless, sterile protection against challenge with a homologous T. pallidum strain has been achieved in rabbits [166]. In the early 1970s, rabbits given 60 intravenous injections of γ-irradiated T. pallidum over 37 weeks became immune to infection on subsequent intradermal challenge that lasted for at least 1 year after final immunization [166]. This demonstrated that protection against syphilis is possible, but the vaccination regimen that was used is impractical for humans [150]. The increasing prevalence of syphilis, despite the continued sensitivity of T. pallidum to treatment with penicillin, highlights the need for syphilis vaccine development. The technical challenges associated with T. pallidum experimentation, including its fragility, genetic complexity, and unusual ultrastructure, combined with the fact that few basic researchers study T. pallidum, has hindered the field of T. pallidum vaccine research [136,150]. Current efforts focus on reverse vaccinology and targeted functional studies to identify antigens that are important in host pathogen interactions and disease pathogenesis [167,168]. Sequencing circulating syphilis strains provides additional information on potential crossprotection across selected targets [169]. The challenge now is to generate the right combination of these potential vaccine targets, with appropriate adjuvants, to develop a viable syphilis vaccine candidate [170].

G. Conclusions T. pallidum is an obligate human pathogen that can cause chronic debilitating disease, and

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its prevalence is increasing. Although syphilis can be effectively treated with penicillin, there is no evidence of protective immunity after infection resolution in humans. A useful rabbit model for syphilis infection has enabled insights into correlates of disease protection. Extending vaccine studies in rabbits, in addition to investing in basic research, is needed to propel the field of syphilis vaccine research forward.

V. MYCOPLASMA A. Microbiology M. genitalium is a tiny, cell-wall-less, facultative anaerobic bacterium. It was first isolated from urethral specimens of two men with nongonococcal urethritis (NGU) [171]. It has a unique differentiated terminal structure to attach to tissue cells and to erythrocytes. It has the smallest known genome of any free-living bacteria [172]. It has taken some time for M. genitalium to gain attention as a sexually transmitted pathogen. The CDC recently identified it as an emerging STI pathogen [173].

B. Epidemiology The prevalence of M. genitalium in the general population is reported to be between 1.1% and 3.3%. This is higher than the prevalence of N. gonorrhoeae at 0.4% but lower than C. trachomatis at 4.2% and T. vaginalis at 2.3% [173,174]. Several studies have shown statistically significant increased rates of infection among sexually active women, with the rate and risk of infection increasing with two or more sexual partners. The prevalence of M. genitalium increases by 10% with each additional sexual partner [175].

C. Clinical Manifestations Although Chlamydia and Neisseria are wellknown causative agents of male urethritis, M.

genitalium has stood out as another cause. M. genitalium has also been implicated in balanitis and posthitis, inflammation of the foreskin [176]. In men who have sex with men, M. genitalium may colonize the rectum. A recent study revealed that 71.4% of 1778 men had positive anorectal swabs for M. genitalium [177]. The incidence of M. genitalium is similar to that of C. trachomatis in high-risk women. The organism has been associated with mucopurulent cervicitis [178], endometritis [179], PID [178], preterm labor [180], and infertility [181].

D. Current Treatment Options The current treatment guidelines for empiric treatment of NGU are oral doxycycline or azithromycin. This is problematic, since NGU may also be caused by M. genitalium and doxycycline is ineffective in 17% 90% [181]. In addition, azithromycin has become progressively less effective [182]. Resistance to macrolides has been reported as high as 30% 40% in certain populations [183]. Extended azithromycin dosing schedules have been suggested to combat the development of resistant strains [184]. Since M. genitalium lacks a cell wall, it is inherently resistant to antibiotics targeting cell wall synthesis. The fluoroquinolone moxifloxacin has shown significant efficacy, but this drug is costly.

E. Immune Responses Associated With Pathology Research investigating the immune responses associated with pathology has not progressed much in humans. Iverson et al. have detected M. genitalium reactive cervicovaginal immunoglobulin A (IgA) and IgG antibodies in infected women [185]. Wood and colleagues have developed a macaque model of M. genitalium by inoculating the cervix and salpingeal pockets generated by transplanting autologous fallopian tube tissue subcutaneously. Humoral and

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VI. CONCLUDING REMARKS

cervicovaginal antibodies reacting to the major adhesion protein MgpB were induced post inoculation and persisted throughout the infection [186]. McGowin and colleagues have demonstrated that human vaginal and cervical epithelial cells are immunologically responsive to M. genitalium, secreting proinflammatory cytokines, including IL-6 and IL-8, upon exposure to live bacteria [187]. The same group has also shown the intracellular localization of the bacterium within vaginal and cervical epithelial cells, which allows it to avoid immune attack [188]. M. genitalium has also been reported to establish long-term infection of human endocervical cells that resulted in chronic inflammatory cytokine secretion. Despite persistent cytokine elaboration, no host cell cytotoxicity was observed except with high loads of M. genitalium, suggesting that persistent infection occurs with minimal damage to the epithelium in the absence of immune cells [189].

F. Vaccination Research When compared to Chlamydia and Neisseria vaccine research programs, M. genitalium has not drawn the attention of infectious diseases researchers. A single study has screened human sera for immunogenic proteins and found the attachment protein MgPa to be an immunodominant protein [190].

G. Conclusions M. genitalium is now increasingly recognized as a STI that has been associated with cervicitis, PID, and infertility in women and urethritis in men. It is also exhibiting alarming capabilities of developing antimicrobial resistance, and the widespread use of azithromycin as front-line treatment for Chlamydia and, more recently, for gonorrhea appears to be driving even higher rates of resistance [191,192]. Owing to antibiotic

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resistance patterns, alternatives to existing antibiotics as well as development of a prophylactic vaccine must be investigated.

VI. CONCLUDING REMARKS With an alarming rise in incidence of STIs for the last three consecutive years, it is obvious that antibiotic treatment is not sufficient for their control. Hence the need for a vaccine for each of the pathogens discussed above becomes necessary. In this chapter, we have summarized the status of vaccine development for each of the pathogens in Table 36.1. Chlamydial vaccine development has been enhanced by the availability of animal models such as mouse, guinea pig, pigs, and nonhuman primates. All the animal models have demonstrated that induction of Th1-type immune responses are key for a vaccine to protect against genital tract infection. However, CD81 T cells were surprisingly shown to play a role in protection induced by live attenuated C. trachomatis vaccine for trachoma [87]. The first phase 1 clinical trials of a chlamydial recombinant vaccine candidate (CTH522) formulated with CAF01 adjuvant system in healthy women aged 18 45 years has been completed and monitored until day 168, and the examination of its protective efficacy is under way [193]. Several other vaccine candidates, such as MOMP-nanoemulsion adjuvant (NanoBio) by Beagley et al., QUT, Australia (unpublished) [79], MOMP plus Pmp [80], cSAP TLR7 agonist with UV-killed Chlamydia [56], and a synthetic salmonella vector containing chlamydial antigens, are in their preclinical phase [85]. The recent emergence of antibiotic-resistant cases of gonorrhea has reengaged biotech companies to pursue development of gonorrhea vaccines. Clinical trials are being conducted by GSK using successful meningococcal vaccines (4CMenB and rLP2086) that share many of the antigens of gonorrhea [135]. Syphilis vaccine research has been hindered, owing to the lack of

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

TABLE 36.1 Overview of the Vaccine Candidates at Different Stages of Development for Chlamydia trachomatis, Neisseria gonorrhea, Treponema pallidum, and Mycoplasma genitalium

Key accine candidates

Vaccineinduced immune Preclinical phase responses vaccines

Pathogen

Animal models

Chlamydia trachomatis

Mouse (Mus musculus)

MOMP, CPAF, Pmp [194]

Th1

(1) Intranasal MOMP nanoemulsion [NanoBio Corporation] [79]; (2) MOMP1 Pmp [Panprovincial Vaccine Enterprise Inc. (PREVENT) and British Columbia CDC] [80]; (3) cSAP TLR7 agonist with UV-killed Chlamydia [Selecta Biosciences] [56]; (4) Vaxonella platform (Salmonella vector) [Prokarium] [85]

Guinea pig (Cavia porcellus) [195]

MOMP [196]

Th1

Not available

Nonhuman primates (Cynomolgus rhesus, pigtailed macaque, and rhesus macaque) [197]

MOMP, Live attenuated trachoma vaccine

Tc (CD81 T cell) [87]

Live attenuated plasmid-deficient trachoma vaccine [NIH/ NIAID] [86]

Pig (Suis domesticus) [198]

MOMP [78], Pmp [199]

Th1

Not available

Neisseria gonorrhoeae

Mouse (M. musculus) [102]

NGO1985, NGO2121, MetQ [200]

Th1

(1) Outer-membrane vesicle (OMV) vaccine along with Quil-A adjuvant; (2) rrPorBVRP vaccine using a subcutaneous prime and footpad boost regime; (3) MAP1—peptide mimetic of 2C7 lipooligosaccharide epitope through intraperitoneal route [201]

Treponema pallidum

Rabbit (Oryctolagus cuniculus) [140]

Endoflagella, 4D, GpD, TmpB, Tp92, TpN15, TprF, TprI, TprK [136]

Antibody- Recombinant Tp0751 mediated along with TiterMax protection Gold adjuvant through subcutaneous route [170]

Not available

Not available

Mycoplasma genitalium

Mouse (M. musculus) [202]

MgPa [190]

Not available

Not available

Not available

Not available

Clinical phase vaccines Status Phase 1 human, double- Ongoing blind, parallel, randomized and placebo controlled clinical trial of the safety of SSI’s adjuvanted chlamydia vaccine CTH522 in healthy women aged 18 45 years. Sponsored by Statens Serum Institut, Denmark [193] https://clinicaltrials. gov/ct2/show/record/ NCT02787109

Since 1970, three clinical 3 failed; trials failed: whole cell, 2 pilus, and Por vaccine ongoing formulations. Ongoing trials are in progress using 4CMenB and rLP2086. Both of them consists of NadA, FHbp, and NHBA antigens [135]

REFERENCES

animal models except for rabbits. Although the rabbit model has demonstrated that antibodies play a protective role, there is only one vaccine candidate in preclinical phase [170], and there are none in clinical trials. The recently emerging STI pathogen M. genitalium needs a lot of attention from researchers; no vaccine candidates against this pathogen exist in either the preclinical or clinical phase.

References [1] Prevention, C.f.D.C.a., Sexually transmitted disease surveillance report, 2016, 2017. [2] Ward H, Ro¨nn M. Contribution of sexually transmitted infections to the sexual transmission of HIV. Curr Opin HIV AIDS 2010;5(4):305 10. [3] World Health Organization, Sexually transmitted infections, Department of Reproductive Health and Research, 2014. [4] Newman L, et al. Global estimates of syphilis in pregnancy and associated adverse outcomes: analysis of multinational antenatal surveillance data. PLoS Med 2013;10(2):e1001396. [5] Chernesky MA, Patrick D. Syndromes associated with sexually transmitted infections. Can J Infect Dis Med Microbiol 2005;16(1):13 14. [6] Van Damme P. Long-term protection after hepatitis B vaccine. J Infect Dis 2016;214(1):1 3. [7] Garland SM, et al. Impact and effectiveness of the quadrivalent human papillomavirus vaccine: a systematic review of 10 years of real-world experience. Clin Infect Dis 2016;63(4):519 27. [8] Sarah Schillie CV, Reingold A, Harris A, Haber P, Ward JW, Nelson NP. Prevention of Hepatitis B virus infection in the United States: recommendations of the advisory committee on immunization practices. MMWR Recomm Rep 2018;67(RR-1):1 31. [9] Lee LY, Garland SM. Human papillomavirus vaccination: the population impact. F1000 Res 2017;6:866. [10] Murray GL, Bradshaw CS, Bissessor M, Danielewski J, Garland SM, Jensen JS, et al. Increasing macrolide and fluoroquinolone resistance in Mycoplasma genitalium. Emerg Infect Dis 2017;23(5):809 12. [11] Darville T. Chlamydia trachomatis. Red book: 2012 report of the committee on infectious diseases. twenty ninth ed Elk Grove Village, IL: American Academy of Pediatrics; 2012. [12] Hybiske K, Stephens RS. Mechanisms of host cell exit by the intracellular bacterium Chlamydia. Proc Natl Acad Sci 2007;104(27):11430 5.

641

[13] Peipert JF. Clinical practice. Genital chlamydial infections. N Engl J Med 2003;349(25):2424 30. [14] Brookings C, Goldmeier D, Sadeghi-Nejad H. Sexually transmitted infections. In: Mulhall JP, Hsiao W, editors. Men’s sexual health and fertility: a clinician’s guide. New York, NY: Springer; 2014. p. 67 87. [15] WHO, Sexually transmitted infections (STIs). Fact Sheet, 2016. [16] Organization WH. WHO guidelines for the treatment of Chlamydia trachomatis. 2016;44. [17] Brunham RC, Rappuoli R. Chlamydia trachomatis control requires a vaccine. Vaccine 2013;31(15):1892 7. [18] Brunham RC, Rey-Ladino J. Immunology of Chlamydia infection: implications for a Chlamydia trachomatis vaccine. Nat Rev Immunol 2005;5(2):149 61. [19] Imtiaz MT, et al. A role for matrix metalloproteinase-9 in pathogenesis of urogenital Chlamydia muridarum infection in mice. Microbes. Infect. 2007;9(14 15):1565 6. [20] Darville T, et al. Mouse strain-dependent variation in the course and outcome of chlamydial genital tract infection is associated with differences in host response. Infect Immun 1997;65(8):3065 73. [21] Lacy HM, et al. Essential role for neutrophils in pathogenesis and adaptive immunity in Chlamydia caviae ocular infections. Infect Immun 2011;79(5):1889 97. [22] Prantner D, et al. Critical role for interleukin-1beta (IL1beta) during Chlamydia muridarum genital infection and bacterial replication-independent secretion of IL1beta in mouse macrophages. Infect Immun 2009;77(12): 5334 46. [23] Ramsey KH, et al. Role for inducible nitric oxide synthase in protection from chronic Chlamydia trachomatis urogenital disease in mice and its regulation by oxygen free radicals. Infect Immun 2001;69(12):7374 9. [24] Shah AA, et al. Histopathologic changes related to fibrotic oviduct occlusion after genital tract infection of mice with Chlamydia muridarum. Sex Transm Dis 2005; 32(1):49 56. [25] Burton MJ, et al. Cytokine and fibrogenic gene expression in the conjunctivas of subjects from a Gambian community where trachoma is endemic. Infect Immun 2004;72(12):7352 6. [26] Hu VH, et al. Innate immune responses and modified extracellular matrix regulation characterize bacterial infection and cellular/connective tissue changes in scarring trachoma. Infect Immun 2012;80(1):121 30. [27] Hvid M, et al. Interleukin-1 is the initiator of Fallopian tube destruction during Chlamydia trachomatis infection. Cell Microbiol 2007;9(12):2795 803. [28] Zhang H, et al. Lack of long-lasting hydrosalpinx in A/J mice correlates with rapid but transient chlamydial ascension and neutrophil recruitment in the oviduct following intravaginal inoculation with Chlamydia muridarum. Infect Immun 2014;82(7):2688 96.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

642

36. SEXUALLY TRANSMITTED INFECTIONS AND THE URGENT NEED FOR VACCINES

[29] Qu Y, et al. Comparable genital tract infection, pathology, and immunity in rhesus macaques inoculated with wild-type or plasmid-deficient chlamydia trachomatis serovar D. Infect Immun 2015;83(10):4056 67. [30] Riley MM, et al. The recall response induced by genital challenge with Chlamydia muridarum protects the oviduct from pathology but not from reinfection. Infect Immun 2012;80(6):2194 203. [31] O’Connell CM, et al. Plasmid-deficient Chlamydia muridarum fail to induce immune pathology and protect against oviduct disease. J Immunol 2007;179(6):4027 34. [32] Kimani J, et al. Risk factors for Chlamydia trachomatis pelvic inflammatory disease among sex workers in Nairobi, Kenya. J Infect Dis 1996;173(6):1437 44. [33] Bakken IJ, Skjeldestad FE, Nordbo SA. Chlamydia trachomatis infections increase the risk for ectopic pregnancy: a population-based, nested case-control study. Sex Transm Dis 2007;34(3):166 9. [34] Perry LL, Feilzer K, Caldwell HD. Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-gamma-dependent and -independent pathways. J Immunol 1997;158(7):3344 52. [35] Holland MJ, et al. T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring. Clin Exp Immunol 1996;105(3):429 35. [36] Murthy AK, et al. Tumor necrosis factor alpha production from CD81 T cells mediates oviduct pathological sequelae following primary genital Chlamydia muridarum infection. Infect Immun 2011;79(7):2928 35. [37] Van Voorhis WC, et al. Repeated Chlamydia trachomatis infection of Macaca nemestrina fallopian tubes produces a Th1-like cytokine response associated with fibrosis and scarring. Infect Immun 1997;65(6):2175 82. [38] Rank RG, Soderberg LS, Barron AL. Chronic chlamydial genital infection in congenitally athymic nude mice. Infect Immun 1985;48(3):847 9. [39] Morrison SG, Morrison RP. In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection. Infect Immun 2000;68(5):2870 9. [40] Kiviat NB, et al. Endometrial histopathology in patients with culture-proved upper genital tract infection and laparoscopically diagnosed acute salpingitis. Am J Surg Pathol 1990;14(2):167 75. [41] Morrison RP, Feilzer K, Tumas DB. Gene knockout mice establish a primary protective role for major histocompatibility complex class II-restricted responses in Chlamydia trachomatis genital tract infection. Infect Immun 1995;63(12):4661 8. [42] Morrison SG, et al. Immunity to murine Chlamydia trachomatis genital tract reinfection involves B cells and

[43]

[44]

[45]

[46]

[47]

[48]

[49] [50]

[51] [52] [53]

[54]

[55]

[56]

[57]

CD4(1) T cells but not CD8(1) T cells. Infect Immun 2000;68(12):6979 87. Wang S, et al. IFN-gamma knockout mice show Th2associated delayed-type hypersensitivity and the inflammatory cells fail to localize and control chlamydial infection. Eur J Immunol 1999;29(11):3782 92. Johansson M, et al. Genital tract infection with Chlamydia trachomatis fails to induce protective immunity in gamma interferon receptor-deficient mice despite a strong local immunoglobulin A response. Infect Immun 1997;65(3):1032 44. Hawkins RA, Rank RG, Kelly KA. A Chlamydia trachomatis-specific Th2 clone does not provide protection against a genital infection and displays reduced trafficking to the infected genital mucosa. Infect Immun 2002;70(9):5132 9. Beatty WL, et al. Tryptophan depletion as a mechanism of gamma interferon-mediated chlamydial persistence. Infect Immun 1994;62(9):3705 11. Nelson DE, et al. Chlamydial IFN-gamma immune evasion is linked to host infection tropism. Proc Natl Acad Sci U S A 2005;102(30):10658 63. Bancroft GJ, et al. A T cell-independent mechanism of macrophage activation by interferon-gamma. J Immunol 1987;139(4):1104 7. Cheroutre H, Husain MM. CD4 CTL: living up to the challenge. Semin Immunol 2013;25(4):273 81. Johnson RM, Kerr MS, Slaven JE. Plac8-dependent and inducible NO synthase-dependent mechanisms clear Chlamydia muridarum infections from the genital tract. J Immunol 2012;188(4):1896 904. Shin H, Iwasaki A. Tissue-resident memory T cells. Immunol Rev 2013;255(1):165 81. Clark RA. Resident memory T cells in human health and disease. Sci Transl Med 2015;7(269):269rv1. Iijima N, Iwasaki A. T cell memory. A local macrophage chemokine network sustains protective tissueresident memory CD4 T cells. Science 2014;346(6205): 93 8. Shin H, Iwasaki A. A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature 2012;491(7424):463 7. Nagarajan UM, et al. Significant role of IL-1 signaling, but limited role of inflammasome activation, in oviduct pathology during Chlamydia muridarum genital infection. J Immunol 2012;188(6):2866 75. Stary G, et al. VACCINES. A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells. Science 2015;348(6241):aaa8205. Igietseme JU, Rank RG. Susceptibility to reinfection after a primary chlamydial genital infection is associated with a decrease of antigen-specific T cells in the genital tract. Infect Immun 1991;59(4):1346 51.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

643

REFERENCES

[58] Igietseme JU, et al. Role for CD81 T cells in antichlamydial immunity defined by Chlamydia-specific T-lymphocyte clones. Infect Immun 1994;62(11):5195 7. [59] Wizel B, et al. Role of CD8(1)T cells in the host response to Chlamydia. Microbes Infect 2008;10(14-15): 1420 30. [60] Fankhauser SC, Starnbach MN. PD-L1 limits the mucosal CD81 T cell response to Chlamydia trachomatis. J Immunol 2014;192(3):1079 90. [61] Li LX, McSorley SJ. A re-evaluation of the role of B cells in protective immunity to Chlamydia infection. Immunol Lett 2015;164(2):88 93. [62] Ramsey KH, Soderberg LS, Rank RG. Resolution of chlamydial genital infection in B-cell-deficient mice and immunity to reinfection. Infect Immun 1988;56(5): 1320 5. [63] Su H, et al. Chlamydia trachomatis genital tract infection of antibody-deficient gene knockout mice. Infect Immun 1997;65(6):1993 9. [64] Morrison SG, Morrison RP. Resolution of secondary Chlamydia trachomatis genital tract infection in immune mice with depletion of both CD41 and CD81 T cells. Infect Immun 2001;69(4):2643 9. [65] Morrison SG, Morrison RP. A predominant role for antibody in acquired immunity to chlamydial genital tract reinfection. J Immunol 2005;175(11):7536 42. [66] Li LX, McSorley SJ. B cells enhance antigen-specific CD4 T cell priming and prevent bacteria dissemination following Chlamydia muridarum genital tract infection. PLoS Pathog 2013;9(10):e1003707. [67] Barenfanger J, MacDonald AB. The role of immunoglobulin in the neutralization of trachoma infectivity. J Immunol 1974;113(5):1607 17. [68] Jawetz E, et al. Experimental inclusion conjunctivitis in man: measurements of infectivity and resistance. JAMA 1965;194(6):620 32. [69] Byrne GI, et al. Workshop on in vitro neutralization of Chlamydia trachomatis: summary of proceedings. J Infect Dis 1993;168(2):415 20. [70] Punnonen R, et al. Chlamydial serology in infertile women by immunofluorescence. Fertil Steril 1979;31 (6):656 9. [71] Russell AN, et al. Analysis of factors driving incident and ascending infection and the role of serum antibody in chlamydia trachomatis genital tract infection. J Infect Dis 2015;. [72] Zhang YX, et al. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis. J Immunol 1987;138(2): 575 81. [73] Zhong G, Berry J, Brunham RC. Antibody recognition of a neutralization epitope on the major outer

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

membrane protein of Chlamydia trachomatis. Infect Immun 1994;62(5):1576 83. Brady LJ. Antibody-mediated immunomodulation: a strategy to improve host responses against microbial antigens. Infect Immun 2005;73(2):671 8. Poston TB, Gottlieb SL, Darville T. Status of vaccine research and development of vaccines for Chlamydia trachomatis infection. Vaccine 2017;. Liang S, et al. Considerations for the rational design of a Chlamydia vaccine. Hum Vaccin Immunother 2017; 13(4):831 5. Olsen AW, et al. Protection against Chlamydia trachomatis infection and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of the major outer membrane protein. J Infect Dis 2015;212(6):978 89. Boje S, et al. A multi-subunit Chlamydia vaccine inducing neutralizing antibodies and strong IFNgamma(1) CMI responses protects against a genital infection in minipigs. Immunol Cell Biol 2016;94(2): 185 95. NanoBio Corporation. Nanobio’s Chlamydia Vaccine Improves Clearance of Bacteria, Prevents Pelvic Inflammatory Disease in Mice. 2015; Available from: ,http://www.nanobio.com/chlamydia-vaccineupdate/.. Karunakaran KP, et al. Outer membrane proteins preferentially load MHC class II peptides: implications for a Chlamydia trachomatis T cell vaccine. Vaccine 2015;33 (18):2159 66. Picard MD, et al. Resolution of Chlamydia trachomatis infection is associated with a distinct T cell response profile. Clin Vaccine Immunol 2015;22(11):1206 18. Finco O, et al. Approach to discover T- and B-cell antigens of intracellular pathogens applied to the design of Chlamydia trachomatis vaccines. Proc Natl Acad Sci U S A 2011;108(24):9969 74. Russell AN, Zheng X, O’Connell CM, Wiesenfeld HC, Hillier SL, Taylor BD, et al. Identification of Chlamydia trachomatis antigens recognized by T cells from highly exposed women who limit or resist genital tract infection. J Infect Dis 2016;. Edwards AD, Slater NK. Protection of live bacteria from bile acid toxicity using bile acid adsorbing resins. Vaccine 2009;27(29):3897 903. Garmory HS, et al. Antibiotic-free plasmid stabilization by operator-repressor titration for vaccine delivery by using live Salmonella enterica Serovar typhimurium. Infect Immun 2005;73(4):2005 11. Kari L, et al. A live-attenuated chlamydial vaccine protects against trachoma in nonhuman primates. J Exp Med 2011;208(11):2217 23.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

644

36. SEXUALLY TRANSMITTED INFECTIONS AND THE URGENT NEED FOR VACCINES

[87] Olivares-Zavaleta N, et al. CD81 T cells define an unexpected role in live-attenuated vaccine protective immunity against Chlamydia trachomatis infection in macaques. J Immunol 2014;192(10):4648 54. [88] Mabey DC, et al. Towards a safe and effective chlamydial vaccine: lessons from the eye. Vaccine 2014;32(14): 1572 8. [89] Owusu-Edusei Jr. K, et al. Cost-effectiveness of Chlamydia vaccination programs for young women. Emerg Infect Dis 2015;21(6):960 8. [90] Gray RT, et al. Modeling the impact of potential vaccines on epidemics of sexually transmitted Chlamydia trachomatis infection. J Infect Dis 2009;199(11):1680 8. [91] O’Meara CP, et al. Induction of partial immunity in both males and females is sufficient to protect females against sexual transmission of Chlamydia. Mucosal Immunol 2015;9(4):1076 88. [92] Hill SA, Masters TL, Wachter J. Gonorrhea - an evolving disease of the new millennium. Microb Cell 2016; 3(9):371 89. [93] Skerker JM, Berg HC. Direct observation of extension and retraction of type IV pili. Proc Natl Acad Sci U S A 2001;98(12):6901 4. [94] Obergfell KP, Seifert HS. Mobile DNA in the pathogenic Neisseria. Microbiol Spectr 2015;3(3). [95] Aas FE, et al. Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression. Mol Microbiol 2002;46(3):749 60. [96] Stern A, et al. Opacity genes in Neisseria gonorrhoeae: control of phase and antigenic variation. Cell 1986;47 (1):61 71. [97] Cahoon LA, Seifert HS. Focusing homologous recombination: pilin antigenic variation in the pathogenic Neisseria. Mol Microbiol 2011;81(5):1136 43. [98] Detels R, et al. The incidence and correlates of symptomatic and asymptomatic Chlamydia trachomatis and Neisseria gonorrhoeae infections in selected populations in five countries. Sex Transm Dis 2011;38(6):503 9. [99] Campbell MF. The surgical pathology of epididymitis. Ann Surg 1928;88(1):98 111. [100] Jerse AE, Bash MC, Russell MW. Vaccines against gonorrhea: current status and future challenges. Vaccine 2014;32(14):1579 87. [101] Westrom LV. Sexually transmitted diseases and infertility. Sex Transm Dis 1994;21(Suppl. 2):S32 7. [102] Rice PA, et al. Neisseria gonorrhoeae: drug resistance, mouse models, and vaccine development. Annu Rev Microbiol 2017;71:665 86. [103] Newman L, et al. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One 2015;10(12):e0143304.

[104] Prevention, C.f.D.C.a. Gonorrhea statistics, 2016. Available from: https://www.cdc.gov/std/stats16/ gonorrhea.htm. [105] Ohnishi M, et al. Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhea?: detailed characterization of the first strain with high-level resistance to ceftriaxone. Antimicrob Agents Chemother 2011;55 (7):3538 45. [106] Unemo M, et al. High-level cefixime- and ceftriaxoneresistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure. Antimicrob Agents Chemother 2012;56(3):1273 80. [107] Unemo M, Shafer WM. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future. Clin Microbiol Rev 2014;27(3): 587 613. [108] Bolan GA, Sparling PF, Wasserheit JN. The emerging threat of untreatable gonococcal infection. N Engl J Med 2012;366(6):485 7. [109] Bloomfield P. Update on emerging infections: news from the Centers for Disease Control and Prevention. Update to CDC’s Sexually Transmitted Diseases Treatment Guidelines, 2006: fluoroquinolones no longer recommended for treatment of gonococcal infections. Ann Emerg Med 2007;50(3):232 5. [110] Fifer H, et al. Failure of dual antimicrobial therapy in treatment of gonorrhea. N Engl J Med 2016;374(25): 2504 6. [111] Ramsey KH, et al. Inflammatory cytokine response to experimental human infection with Neisseria gonorrhoeae. Ann N Y Acad Sci 1994;730:322 5. [112] Feinen B, et al. Critical role of Th17 responses in a murine model of Neisseria gonorrhoeae genital infection. Mucosal Immunol 2010;3(3):312 21. [113] Hedges SR, et al. Cytokine and antibody responses in women infected with Neisseria gonorrhoeae: effects of concomitant infections. J Infect Dis 1998;178(3): 742 51. [114] Zhu W, et al. Vaccines for gonorrhea: can we rise to the challenge? Front Microbiol 2011;2:124. [115] Hedges SR, et al. Limited local and systemic antibody responses to Neisseria gonorrhoeae during uncomplicated genital infections. Infect Immun 1999;67(8): 3937 46. [116] Boulton IC, Gray-Owen SD. Neisserial binding to CEACAM1 arrests the activation and proliferation of CD41 T lymphocytes. Nat Immunol 2002;3(3): 229 36. [117] Duncan JA, et al. Neisseria gonorrhoeae activates the proteinase cathepsin B to mediate the signaling activities of the NLRP3 and ASC-containing inflammasome. J Immunol 2009;182(10):6460 9.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

645

REFERENCES

[118] Normark S, Albiger B, Jonsson AB. Gonococci cause immunosuppression by engaging a coinhibitory receptor on T lymphocytes. Nat Immunol 2002;3(3): 210 11. [119] Pantelic M, et al. Retinoic acid treated HL60 cells express CEACAM1 (CD66a) and phagocytose Neisseria gonorrhoeae. FEMS Immunol Med Microbiol 2004;42(2):261 6. [120] Zhu W, et al. Neisseria gonorrhoeae suppresses dendritic cell-induced, antigen-dependent CD4 T cell proliferation. PLoS One 2012;7(7):e41260. [121] Liu Y, et al. Neisseria gonorrhoeae selectively suppresses the development of Th1 and Th2 cells, and enhances Th17 cell responses, through TGF-betadependent mechanisms. Mucosal Immunol 2012;5(3): 320 31. [122] Liu Y, Liu W, Russell MW. Suppression of host adaptive immune responses by Neisseria gonorrhoeae: role of interleukin 10 and type 1 regulatory T cells. Mucosal Immunol 2014;7(1):165 76. [123] Imarai M, et al. Regulatory T cells are locally induced during intravaginal infection of mice with Neisseria gonorrhoeae. Infect Immun 2008;76(12):5456 65. [124] Gagliardi MC, et al. Circulating levels of interleukin17A and interleukin-23 are increased in patients with gonococcal infection. FEMS Immunol Med Microbiol 2011;61(1):129 32. [125] Pantelic M, et al. Neisseria gonorrhoeae kills carcinoembryonic antigen-related cellular adhesion molecule 1 (CD66a)-expressing human B cells and inhibits antibody production. Infect Immun 2005;73 (7):4171 9. [126] Plummer FA, et al. Epidemiologic evidence for the development of serovar-specific immunity after gonococcal infection. J Clin Invest 1989;83(5):1472 6. [127] Fox KK, et al. Longitudinal evaluation of serovarspecific immunity to Neisseria gonorrhoeae. Am J Epidemiol 1999;149(4):353 8. [128] Rice PA, et al. Immunoglobulin G antibodies directed against protein III block killing of serum-resistant Neisseria gonorrhoeae by immune serum. J Exp Med 1986;164(5):1735 48. [129] Arko RJ. Animal models for pathogenic Neisseria species. Clin Microbiol Rev 1989;2(Suppl):S56 9. [130] Liu Y, Egilmez NK, Russell MW. Enhancement of adaptive immunity to Neisseria gonorrhoeae by local intravaginal administration of microencapsulated interleukin 12. J Infect Dis 2013;208(11):1821 9. [131] Cohen MS, Cannon JG. Human experimentation with Neisseria gonorrhoeae: progress and goals. J Infect Dis 1999;179(Suppl 2):S375 9. [132] Hobbs MM, et al. Experimental gonococcal infection in male volunteers: cumulative experience with

[133]

[134]

[135] [136] [137]

[138]

[139]

[140]

[141]

[142]

[143]

[144]

[145]

[146]

Neisseria gonorrhoeae strains FA1090 and MS11mkC. Front Microbiol 2011;2:123. Schmidt KA, et al. Experimental gonococcal urethritis and reinfection with homologous gonococci in male volunteers. Sex Transm Dis 2001;28(10):555 64. Petousis-Harris H, et al. Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study. Lancet 2017;390(10102):1603 10. Abbasi J. New hope for a gonorrhea vaccine. JAMA 2017;318(10):894 5. Lafond RE, Lukehart SA. Biological basis for syphilis. Clin Microbiol Rev 2006;19(1):29 49. Norris SJ. Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group. Microbiol Rev 1993;57 (3):750 79. Radolf JD. Treponema, in medical microbiology, Baron S., Editor. The University of Texas Medical Branch, Galveston, TX; 1996. Norris SJ, Edmondson DG. Factors affecting the multiplication and subculture of Treponema pallidum subsp. pallidum in a tissue culture system. Infect Immun 1986;53(3):534 9. Sell S, et al. Host response to Treponema pallidum in intradermally-infected rabbits: evidence for persistence of infection at local and distant sites. J Invest Dermatol 1980;75(6):470 5. Baker-Zander S, Sell S. A histopathologic and immunologic study of the course of syphilis in the experimentally infected rabbit. Demonstration of longlasting cellular immunity. Am J Pathol 1980;101(2): 387 414. Division of STD Prevention, N.C.f.H.A., Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention Syphilis. 2015 Sexually Transmitted Disease Surveillance 2015 October 17, 2016; Available from: https://www.cdc. gov/std/stats15/syphilis.htm. Patton ME, et al. Primary and secondary syphilis— United States, 2005 2013. MMWR Morb Mortal Wkly Rep 2014;63(18):402 6. Clark EG, Danbolt N. The Oslo study of the natural course of untreated syphilis: an epidemiologic investigation based on re-study of Boeck-Bruusgaard material. Med Clin North Am 1964;48(3):613 23. Rosahn PD, Black-Schaffer B. Studies in syphilis I review of the incidence of syphilis in autopsies on adults. Arch Intern Med 1943;72(1):78 90. Rockwell DH, Moore MB, Yobs AR. Tuskegee study of untreated syphilis. Arch Intern Med 1964;114(6): 792.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

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36. SEXUALLY TRANSMITTED INFECTIONS AND THE URGENT NEED FOR VACCINES

[147] Primary and secondary syphilis—United States, 1997. JAMA 1998;280(14):1218 1219. (Reprinted from MMWR 1998;47:493 97). [148] Mahoney JF, Arnold RC, Harris A. Penicillin treatment of early syphilis-A preliminary report. Am J Public Health Nations Health 1943;33(12):1387 91. [149] Marra CM, et al. Antibiotic selection may contribute to increases in macrolide-resistant Treponema pallidum. J Infect Dis 2006;194(12):1771 3. [150] Cameron CE, Lukehart SA. Current status of syphilis vaccine development: need, challenges, prospects. Vaccine 2014;32(14):1602 9. [151] Raiziss GW, Severac M. Rapidity with which Spirochaeta pallida invades the blood stream. Arch Derm Syphilol 1937;35(6):1101 9. [152] Cumberland MC, Turner TB. The rate of multiplication of Treponema pallidum in normal and immune rabbits. Am J Syph Gonorrhea Vener Dis 1949;33(3): 201 12. [153] Riviere GR, Thomas DD, Cobb CM. In vitro model of Treponema pallidum invasiveness. Infect Immun 1989; 57(8):2267 71. [154] Thomas DD, et al. Treponema-pallidum invades intercellular-junctions of endothelial-cell monolayers. Proc Natl Acad Sci USA 1988;85(10):3608 12. [155] Fraser CM, et al. Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science 1998;281(5375):375 88. [156] Salazar JC, Hazlett KR, Radolf JD. The immune response to infection with Treponema pallidum, the stealth pathogen. Microbes Infect 2002;4(11):1133 40. [157] Baker-Zander SA, Lukehart SA. Macrophagemediated killing of opsonized Treponema pallidum. J Infect Dis 1992;165(1):69 74. [158] Lukehart SA, Baker-Zander SA, Sell S. Characterization of lymphocyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens. J Immunol 1980;124(1):454 60. [159] Lukehart SA, Baker-Zander SA, Sell S. Characterization of the humoral immune response of the rabbit to antigens of Treponema pallidum after experimental infection and therapy. Sex Transm Dis 1986;13(1):9 15. [160] Muller F, Oelerich S. Treponema-specific and antilipoidal 19S(IgM) antibodies in penicillin-treated and untreated rabbits after infection with Treponema pallidum. Br J Vener Dis 1981;57(1):15 19. [161] Bishop NH, Miller JN. Humoral immunity in experimental syphilis. I. The demonstration of resistance conferred by passive immunization. J Immunol 1976; 117(1):191 6. [162] Weiser RS, et al. Immunity to syphilis: passive transfer in rabbits using serial doses of immune serum. Infect Immun 1976;13(5):1402 7.

[163] Van Voorhis WC, et al. Primary and secondary syphilis lesions contain mRNA for Th1 cytokines. J Infect Dis 1996;173(2):491 5. [164] Salazar JC, et al. Treponema pallidum elicits innate and adaptive cellular immune responses in skin and blood during secondary syphilis: a flow-cytometric analysis. J Infect Dis 2007;195(6):879 87. [165] Stary G, et al. Host defense mechanisms in secondary syphilitic lesions: a role for IFN-gamma-/IL-17-producing CD81 T cells? Am J Pathol 2010;177(5):2421 32. [166] Miller JN. Immunity in experimental syphilis. VI. Successful vaccination of rabbits with Treponema pallidum, Nichols strain, attenuated by γ-irradiation. J Immunol 1973;110(5):1206 15. [167] Houston S, et al. Conservation of the host-interacting proteins Tp0750 and pallilysin among treponemes and restriction of proteolytic capacity to Treponema pallidum. Infect Immun 2015;83(11):4204 16. [168] Ke W, et al. Treponema pallidum subsp. pallidum TP0136 protein is heterogeneous among isolates and binds cellular and plasma fibronectin via its NH2terminal end. PLoS Negl Trop Dis 2015;9(3):e0003662. [169] Cejkova D, et al. A retrospective study on genetic heterogeneity within treponema strains: subpopulations are genetically distinct in a limited number of positions. PLoS Negl Trop Dis 2015;9(10). [170] Lithgow KV, et al. A defined syphilis vaccine candidate inhibits dissemination of Treponema pallidum subspecies pallidum. Nat Commun 2017;8:14273. [171] Tully J, et al. A newly discovered mycoplasma in the human urogenital tract. Lancet 1981;317(8233):1288 91. [172] Fraser CM, et al. The minimal gene complement of Mycoplasma genitalium. Science 1995;270(5235):397 403. [173] Bolan KAWaGA. Sexually transmitted diseases treatment guidelines, in morbidity and mortality weekly report, 2015. [174] Taylor-Robinson D, Jensen JS. Mycoplasma genitalium: from Chrysalis to multicolored butterfly. Clin Microbiol Rev 2011;24(3):498 514. [175] Manhart LE, et al. Mycoplasma genitalium among young adults in the United States: an emerging sexually transmitted infection. Am J Public Health 2007;97(6): 1118 25. [176] Horner PJ, Taylor-Robinson D. Association of Mycoplasma genitalium with balanoposthitis in men with non-gonococcal urethritis. Sex Transm Infect 2011;87(1):38 40. [177] Reinton N, et al. Anatomic distribution of Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium infections in men who have sex with men. Sex Health 2013;10(3):199 203. [178] Manhart LE, et al. Mucopurulent cervicitis and Mycoplasma genitalium. J Infect Dis 2003;187(4):650 7.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

REFERENCES

[179] Jurstrand M, et al. A serological study of the role of Mycoplasma genitalium in pelvic inflammatory disease and ectopic pregnancy. Sex Transm Infect 2007;83(4):319 23. [180] Hitti J, et al. Correlates of cervical Mycoplasma genitalium and risk of preterm birth among Peruvian women. Sex Transm Dis 2010;37(2):81 5. [181] Manhart LE, et al. Efficacy of antimicrobial therapy for Mycoplasma genitalium infections. Clin Infect Dis 2015;61(Suppl 8):S802 17. [182] Lau A, et al. The efficacy of azithromycin for the treatment of genital Mycoplasma genitalium: a systematic review and meta-analysis. Clin Infect Dis 2015;61(9): 1389 99. [183] Nijhuis RH, et al. High levels of macrolide resistanceassociated mutations in Mycoplasma genitalium warrant antibiotic susceptibility-guided treatment. J Antimicrob Chemother 2015;70(9):2515 18. [184] Manhart LE. Diagnostic and resistance testing for Mycoplasma genitalium: what will it take? Clin Infect Dis 2014;59(1):31 3. [185] Iverson-Cabral SL, Manhart LE, Totten PA. Detection of Mycoplasma genitalium-reactive cervicovaginal antibodies among infected women. Clin Vaccine Immunol 2011;18(10):1783 6. [186] Wood GE, et al. Persistence, immune response, and antigenic variation of Mycoplasma genitalium in an experimentally infected pig-tailed macaque (Macaca nemestrina). Infect Immun 2013;81(8):2938 51. [187] McGowin CL, et al. Mycoplasma genitalium-encoded MG309 activates NF-kappaB via Toll-like receptors 2 and 6 to elicit proinflammatory cytokine secretion from human genital epithelial cells. Infect Immun 2009;77(3):1175 81. [188] McGowin CL, Popov VL, Pyles RB. Intracellular Mycoplasma genitalium infection of human vaginal and cervical epithelial cells elicits distinct patterns of inflammatory cytokine secretion and provides a possible survival niche against macrophage-mediated killing. BMC Microbiol 2009;9:139. [189] McGowin CL, et al. Persistent Mycoplasma genitalium infection of human endocervical epithelial cells elicits chronic inflammatory cytokine secretion. Infect Immun 2012;80(11):3842 9. [190] Svenstrup HF, et al. Identification and characterization of immunogenic proteins of Mycoplasma genitalium. Clin Vaccine Immunol 2006;13(8):913 22.

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[191] Jensen JS, Bradshaw C. Management of Mycoplasma genitalium infections can we hit a moving target? BMC Infect Dis 2015;15:343. [192] Unemo M, Jensen JS. Antimicrobial-resistant sexually transmitted infections: gonorrhoea and Mycoplasma genitalium. Nat Rev Urol 2017;14(3): 139 52. [193] Institute SS. Safety of Chlamydia Vaccine CTH522 in Healthy Women Aged 18 to 45 Years. NIH US National Library of Medicine, ClinicalTrials.gov, 2017. doi: https://clinicaltrials.gov/ct2/show/record/NCT02787109. [194] Farris CM, Morrison RP. Vaccination against Chlamydia genital infection utilizing the murine C. muridarum model. Infect Immun 2011;79(3):986 96. [195] Rank RG, et al. Characterization of chlamydial genital infection resulting from sexual transmission from male to female guinea pigs and determination of infectious dose. Infect Immun 2003;71(11):6148 54. [196] Andrew DW, et al. Partial protection against chlamydial reproductive tract infection by a recombinant major outer membrane protein/CpG/cholera toxin intranasal vaccine in the guinea pig Chlamydia caviae model. J Reprod Immunol 2011;91(1-2):9 16. [197] Bell JD, et al. Nonhuman primate models used to study pelvic inflammatory disease caused by Chlamydia trachomatis. Infect Dis Obstet Gynecol 2011; 2011:7. [198] Kaser T, et al. Chlamydia suis and Chlamydia trachomatis induce multifunctional CD4 T cells in pigs. Vaccine 2017;35(1):91 100. [199] Schautteet K, et al. Validation of the Chlamydia trachomatis genital challenge pig model for testing recombinant protein vaccines. J Med Microbiol 2011;60: 117 27. [200] Baarda BI, et al. Deciphering function of new gonococcal vaccine antigens using phenotypic microarrays. J Bacteriol 2017;199 e00037 e00017. [201] Wetzler LM, et al. Summary and recommendations from the National Institute of Allergy and Infectious Diseases (NIAID) Workshop “Gonorrhea Vaccines: the Way Forward”. Clin Vaccine Immunol 2016;23(8): 656 63. [202] McGowin CL, Spagnuolo RA, Pyles RB. Mycoplasma genitalium rapidly disseminates to the upper reproductive tracts and knees of female mice following vaginal inoculation. Infect Immun 2010;78(2): 726 36.

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Mucosal Vaccines for Oral Disease Tomoko Kurita-Ochiai, Tomomi Hashizume-Takizawa, Ryoki Kobayashi and Masafumi Yamamoto Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Chiba, Japan

I. INTRODUCTION It has been well established that while the mucosal and systemic immune systems coexist, the mucosal system acts separately from the systemic immune system [1]. Therefore, a systemic immune response induced by parenteral immunization does not result in significant mucosal immunity. However, mucosal immunization can and often does result in protective mucosal immunity as evidenced in external secretions as well as immunity in systemic compartment [1]. In many ways, the oral cavity is an important and characteristic compartment of the mucosal immune system because, unlike other mucosal compartments, its local immune responses are operated by both mucosal and systemic arms of immunity. Local immune responses emanating from the salivary gland are part of the mucosal immune system, while immune responses emanating from the crevicular fluid are part of the systemic immune system because these are derived from tissue fluids in blood capillaries [2]. Thus, although the main antibody isotype of the oral cavity is

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00037-7

secretory immunoglobulin A (SIgA), it is clear that systemic-derived immunoglobulin G (IgG) in crevicular fluid is also biologically active within the oral cavity. Hence, effective protection against oral infections such as caries and periodontal disease requires both mucosal and systemic antibody responses. If an effective vaccine for the oral cavity is to be designed, careful consideration must be given to the various immune responses and antigen-delivery systems. Because of the risk of diseases associated with reuse and improper disposal of needles, needle-free delivery has become a global priority. Nasal administration of vaccines has been widely adopted for mucosal immunization because it delivers antigen directly to IgAinductive sites termed nasopharyngealassociated lymphoid tissues (NALT) without the influence of enzymes and acids in the gastrointestinal tract. Sublingual mucosae are also attractive vaccine delivery sites that may have advantages over other routes because of their anatomy and physiology [3]. Furthermore, these forms of immunization are capable of

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inducing both mucosal and systemic immune responses, which result in two layers of host protection against infectious diseases [4,5].

II. MUCOSAL VACCINES FOR CARIES PREVENTION Dental caries result from a chronic infectious disease caused by the formation of biofilm on tooth surfaces. Among the oral bacteria, mutans streptococci are considered to be causative agents of dental caries [6]. Streptococcus mutans produces a cell-surface protein antigen (Ag) with a molecular mass of 190 kDa, referred to as the surface protein antigen of S. mutans serotype c (PAc) [7] (also termed antigen I/II, protein B, or P1 [8]). Because PAc has been presented as a key virulence factor for tooth adherence by S. mutans [9], blocking or inhibiting this cell surface adhesin by antigen-specific SIgA in saliva is a logical approach for the prevention of the initial colonization of teeth by S. mutans. In this regard, it has been shown that nasal or oral immunization with PAc chemically conjugated with B subunit of cholera toxin (CT-B), which contain trace amounts of the holotoxin, elicits significant Ag-specific salivary IgA antibody (Ab) responses [9,10]. Furthermore, a fusion protein made with the saliva-binding region (SBR) of PAc and CT-B induced high titers of anti-PAc salivary IgA Abs when given orally or nasally [11]. Thus mucosally administered PAc plus CT or its subunit appears to be an effective form of mucosal vaccine for induction of PAc-specific salivary IgA Ab responses. An enterotoxin adjuvant (e.g., CT) that causes clinical manifestations of cholera is unsuitable for use in humans. To eliminate the risk of unfavorable clinical manifestations while retaining the adjuvanticity of CT, two nontoxic mutants of CT (mCTs), S61F and E112K, were generated. The mutants harbor single amino acid substitutions in the ADP ribosyltransferase active center of

the toxin that render them enzymatically inactive. However, these mCTs still supported Ag-specific immune responses when administered nasally [12,13]. Furthermore, nasal administration of PAc and mCT E112K was potentially an effective mucosal vaccine against dental caries because it reduced the colonization of S. mutans in the oral cavity [14]. As with native CT, mCT E112K induces adjuvant responses in part via upregulation of B7-2 on antigen-presenting cells (APCs), which is independent of ADP-ribosyltransferase activity [15]. Furthermore, attenuated Salmonella strains have been used as vaccine vectors for delivery of recombinantly expressed vaccine Ags [16]. When the Salmonella vaccine strain expressing SBR or SBR linked to A2/B subunits of CT was used to immunize mice nasally or orally, elevated levels of anti-SBR Ab responses were induced in saliva and serum. These coincided with significant protection against S. mutans infection in the oral cavity in mice [16]. By using bacteria as a carrier in this way, persistence of the Ab can be expected if it can fix and proliferate in the intestinal tract. So far, different forms of the PAc protein have been tested in experimental systems. These include the PAc recombinant or synthetic peptide [17], protein carbohydrate conjugate [18], or DNA-based active vaccines [19]. An anticaries DNA vaccine (pGJA-P/VAX) nasally administered with recombinant flagellin protein derived from Salmonella as a mucosal adjuvant also enhanced the salivary IgA Ab response, inhibited S. mutans colonization on tooth surfaces, and endowed better protection with significant fewer caries lesions [20]. But the relatively weak immunogenicity of DNA vaccines and the large dosage of plasmid DNA (100 μg per rat) needed for immunization remain significant challenges [20]. To improve the immunogenicity of vaccines, the pathogen-associated molecule pattern (PAMP) bacterial flagellin can be used as an effective adjuvant because it is a known potent immune activator [21]. Flagellin, a protein

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II. MUCOSAL VACCINES FOR CARIES PREVENTION

subunit of the flagellum, can be recognized by the cell surface toll-like receptor (TLR) 5 and NOD-like receptors NLRC4 and NAIP5 [22,23]. As a PAMP, flagellin has been demonstrated to be an effective mucosal adjuvant and has aided different vaccines types inducing elevated levels of local IgA Ab responses [24,25]. Furthermore, a fusion protein of the A-P fragment of PAc from S. mutans in combination with flagellin from Escherichia coli could induce a high level of systemic and mucosal immune responses by nasal immunization and confer robust protection against dental caries [26 28]. S. mutans also possesses phosphatebinding protein (PstS), a component of ATPbinding cassette transporters, which are involved in bioadhesion of the bacteria [29]. When mice were sublingually immunized with the recombinant form of PstS plus heatlabile toxin from E. coli, systemic and mucosal Ab responses were induced and partially inhibited oral colonization of S. mutans after challenge [30]. Like S. mutans, another major pathogen that causes dental caries is Streptococcus sobrinus [31]. Both bacteria produce water-soluble and water-insoluble glucans from sucrose through the action of glucosyltransferases [6,31]. Synthesis of the water-insoluble glucan is necessary for the accumulation of these bacteria on the tooth surface leading to the induction of dental caries [32,33]. A previous study has shown that suppression of sucrose-dependent and -independent adhesion of both S. sobrinus and S. mutans in vitro occurred when specific Abs were induced by a fusion protein consisting of the alanine-rich region of surface protein antigen (PAg) and glucosyltransferase GTF-I produced by S. sobrinus [34]. Furthermore, immunization with a plasmid encoding PAc of S. mutans and the GTF glucan-binding domain or the catalytic regions of S. sobrinus GTF-I induced protective Ab responses against the oral infection with S. mutans and S. sobrinus [35,36].

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The development of a nasal GTF-I vaccine for human use may be a significant milestone in the quest for an effective anticaries vaccine. In assessing the efficacy of nasally administered GTF-I, GTF-I alone induced significant antigenspecific Ab responses in both serum and saliva samples, while GTF-I plus CpG dinucleotides (CpG-ODN) as an adjuvant further increased the levels of salivary Ab responses [37]. The GTF-I-specific IgG Abs induced by the nasal vaccine significantly diminished biofilm formation by S. sobrinus. Furthermore, mice given GTF-I alone or GTF-I plus CpG-ODN were significantly protected against the development of dental caries caused by oral infection with S. sobrinus. These results suggest that nasal immunization with GTF-I may be an effective vaccination regimen for the induction of protective immune responses against S. sobrinus as well as S. mutans infection. To date, protein molecules and cell-surface pathogenic factors specific to S. mutans and S. sobrinus have been studied as vaccine targets (Fig. 37.1). These mucosal vaccine candidates

FIGURE 37.1

Relevant cell surface proteins of Streptococcus mutans and Streptococcus sobrinus targeted for vaccine development against caries. S. mutans and S. sobrinus produce both cell-associated and secreted glucosyltransferases, GTF-B and GTF-I, respectively. These GTFs synthesize water-insoluble glucans (WIGs). S. mutans and S. sobrinus express similar protein antigens (Ags) on their surface, which regulate adhesion of the bacteria to acquired pellicle. S. mutans expresses PAc (AgI/II), including the saliva-binding region (SBR), and S. sobrinus expresses PAg. S. mutans expresses an antigenic phosphate-binding protein that is a component of ATP-binding cassette transporters.

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FIGURE 37.2 Mucosal vaccine strategy for dental caries prevention. Vaccination against dental caries pathogens via nasal or oral routes induces Ag-specific secretory IgA (SIgA) antibodies (Abs) in saliva and Agspecific IgG Abs derived from gingival crevicular fluid in the oral cavity inhibits adhesion of caries bacteria to the tooth surface and plaque formation.

are expected to reduce caries formation through Ag-specific SIgA in saliva and Agspecific IgG production in gingival crevicular fluids (Fig. 37.2). It is also necessary to develop multivalent vaccines that can induce Abs simultaneously against multiple pathogenic factors. In addition, since dental caries occurs on various tooth surfaces, such as the smooth surface, pit fissure groove, and root surface, and the mechanism of its development is complicated, alternative vaccine approaches for the control of caries need to be considered. For example, the development of an Ab drug administration system that delivers and sustains neutralizing Abs against caries bacteria in biofilms could be an attractive strategy for the control of oral diseases in the future.

III. MUCOSAL VACCINES FOR PERIODONTAL DISEASE Oral health is also threatened by chronic periodontitis that destroys periodontal tissues and eventually leads to tooth loss [38]. Moreover, periodontal diseases have been linked to a number of systemic diseases, such as cardiovascular diseases, diabetes, rheumatoid arthritis, and Alzheimer’s disease [39 41], suggesting that prevention of periodontitis might be relevant for both oral and systemic health. A major pathogen that causes chronic periodontitis is the Gram-negative anaerobe,

Porphyromonas gingivalis. The colonization of gingival tissues by P. gingivalis is considered to be the first step in the pathogenic process of periodontal disease that results in gingival tissue destruction [42,43]. Recent evidence suggests that this bacterium contributes to periodontitis by functioning as a keystone pathogen [44,45]. Molecules such as fimbriae, hemagglutinins, aggregation factors, and lipopolysaccharides responsible for colonization have been identified previously as virulence factors [42,43].

IV. PROTEIN BASED MUCOSAL VACCINE An outer-membrane protein (OMP) with a molecular mass of 40 kDa produced by P. gingivalis (40K-OMP) is a key virulence factor involved in the coaggregation activity of P. gingivalis [46]. Furthermore, this OMP has been previously shown to be a hemin-binding protein [47]. The 40K-OMP resides both on the cell surface and in extracellular vesicles and is found in many strains of P. gingivalis [46,48 50]. Previous studies have demonstrated that specific IgG Abs induced by nasal or transcutaneous administration of 40K-OMP with CT inhibited coaggregation and hemagglutinin activity by P. gingivalis in mice and rats [51 53]. Nasal immunization with 40K-OMP plus CT also induced Ab responses that provided protective immunity

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V. DNA-BASED VACCINE

against P. gingivalis infection in aged mice [54]. These studies suggest that the induction of 40KOMP-specific Abs in the oral mucosa is a logical approach for the prevention of P. gingivalis infection. Further studies have demonstrated that nasal administration of 40K-OMP plus nontoxic chimeric enterotoxin adjuvant or an oral vaccine containing 40K-OMP plus unmethylated CpGODN elicited 40K-OMP-specific saliva SIgA and serum IgG Abs that reduced alveolar bone loss caused by oral infection with P. gingivalis [55,56]. Furthermore, when apolipoprotein-E-deficient mice, which were spontaneously hyperlipidemic and were nasally immunized with 40K-OMP plus CT before P. gingivalis infection, atherosclerotic plaque accumulation in the aortic sinus was significantly reduced compared to nonimmunized mice [57]. These studies indicate that 40K-OMP may be an effective vaccine Ag for the prevention of P. gingivalis-mediated periodontal disease and atherosclerosis. Furthermore, nasal immunization with P. gingivalis outer-membrane vesicle and TLR3 agonist, poly(I:C), also enhanced specific Ab and bacterial clearance [58,59]. Another important vaccine antigen candidate against P. gingivalis is fimbriae. Fimbriae, specifically the major subunit protein fimbrillin (FimA), are one of the adhesive agents of P. gingivalis. A dysfunction in fimbriae leads to a loss of bacterial adhesive properties, resulting in reduced invasion and decreased periodontal bone decay in experimental animals [60 62]. Previous results have indicated that oral or nasal immunization of P. gingivalis fimbriae with CT induced fimbriae-specific serum IgG and salivary IgA, which resulted in the inhibition of bacterial attachment to epithelial cells [63,64]. Further, nasal immunization of mice with fimbriae plus CT-B elicited Ab responses in serum and saliva and inhibited P. gingivalismediated alveolar bone loss [65]. Oral delivery of P. gingivalis FimA epitopes via Streptococcus gordonii vectors also resulted in the induction of FimA-specific serum IgG and salivary IgA Ab

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responses. These immune responses were also protective against subsequent P. gingivalisinduced alveolar bone loss [66]. Therefore, mucosal vaccination with fimbriae can be an effective means of preventing P. gingivalismediated periodontitis.

V. DNA-BASED VACCINE DNA vaccination against P. gingivalis has also been investigated. When mice were immunized nasally with a plasmid expressing fimA and IL-15 genes, anti-FimA Ab responses were elicited in systemic and mucosal compartments [67]. DNA plasmids may offer many potential advantages over protein-based vaccines because of greater chemical stability, relatively ease of purification, correct and native conformation of the expressed protein, and the possibility for creation of a polyvalent vaccine against several kinds of pathogens within one plasmid vector [68,69]. The rates of integrationinduced mutation with DNA plasmids in animal models were found to be much lower than the rates of spontaneous mutation for a mammalian genome [70,71]. Thus, DNA vaccines, in addition to producing immunogens, are thought to be much safer. Dendritic cells (DCs) are considered to be the most potent APCs that are pivotal for the initiation and regulation of antigen-specific immune responses. DCs are strategically located at potential sites of pathogen entry, such as peripheral epithelial and mucosal inductive sites. Upon encountering pathogens, DCs capture and process antigens, undergo maturation as defined by upregulation of major histocompatibility complex (MHC) class II molecules and costimulatory molecules such as CD40, CD80, and CD86 and migrate to lymph nodes, where they present antigens primarily as MHC peptide complexes to naı¨ve T lymphocytes and initiate T cell-mediated immune responses [72]. Cytotoxic T-lymphocyte-associated antigen 4

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(CTLA4) is a membrane-bound molecule located mainly on activated T cells. Its extracellular V-domain is considered to be involved in mediating binding to the B7 molecule on DCs [73]. Using the interaction between CTLA4 and B7, specific antigens can be targeted to DCs by fusion to CTLA4. DNA vaccines with additional targeting components have also been under investigation. For example, the immunogenicity of a DCtargeted FimA DNA vaccine against P. gingivalis was evaluated in mice. The targeted DNA plasmid pCTLA4 FimA, which encoded the signal peptide and extracellular regions of mouse CTLA4, the hinge and Fc regions of human Igγ1, and FimA of P. gingivalis, was constructed [74]. The DC-targeted DNA construct pCTLA4 FimA enhanced both systemic and mucosal immunity following nasal immunization. A DNA-based immunization strategy may be an effective way to attenuate periodontitis induced by P. gingivalis.

VI. NASAL ADMINISTRATION OF PERIODONTAL VACCINE Hemagglutinin protein has also been considered as a candidate for periodontal vaccination. Hemagglutinin protein is expressed on the cell surface of P. gingivalis and regulates bacterial adhesion to host cells. It also agglutinates and causes hemolysis. Multiple hemagglutinin genes have been cloned from P. gingivalis by functional screening [75,76]. Previous studies have shown that oral vaccination of mice with an attenuated vaccine strain of Salmonella expressing hemagglutinin B (HagB) from P. gingivalis induced HagB-specific humoral immunity [75,77]. Nasal immunization with HagB protein mixed with the quillaja saponin semisynthetic analog GPI-0100 potentiated mucosal and systemic responses to recombinant HagB from P. gingivalis [78]. Hemagglutinin A (Ht6agA) is also thought to possess a functional domain and

thus to be a potential candidate for periodontal vaccination [79]. Maltose-binding protein (MBP) is a highaffinity maltose/maltodextrin-binding protein and a periplasmic receptor for the capture and transport of maltodextrins from the periplasmic space in Gram-negative bacteria [80]. MBP was used as a chaperone component in various vaccines and was shown to enhance antigen-specific immune responses [81,82]. Additionally, MBP was recently reported to act as an adjuvant that elicits innate immunity through TLR4 [83]. Although hagA was originally easy to aggregate as an inclusion body [84], even the minimal antigenic region of the 25-kDa protein, the fusion form of the 25k-hagA-MBP protein aided in improving its solubility. Hence, this form was studied further. Nasal administration of 25khagA-MBP induced high levels of 25k-hagAspecific serum IgG and IgA and salivary IgA Ab responses that were similar to those induced by 25k-hagA plus an established mucosal adjuvant, CT [85]. Furthermore, these Abs persisted for at least 1 year. The induction of Ab responses was associated with elevated numbers of activated CD11c1 CD81 DCs in both mucosal and systemic lymphoid tissues. Thus, increased proportions of CD11c1 CD81 DCs with upregulated expression of MHC II, CD40, CD80, and CD86 molecules in NALT, cervical lymph nodes, and the spleen were noted in mice given 25k-hagA-MBP. Furthermore, 25k-hagA-MBP induced CD41 T cells producing predominantly T helper 2 (Th2) cytokines [interleukin 4 (IL-4) and IL-5], as well as IgG1 and IgG2b responses. Importantly, mice given 25k-hagA-MBP were significantly protected against alveolar bone loss caused by oral infection with P. gingivalis, even 1 year after immunization. These studies demonstrate that MBP is an effective adjuvant for nasal immunization and that, when used as a fusion partner for 25k-hagA, it facilitates the development of a long-term protective Ab response. Bacterial flagellin was also used as an excellent mucosal adjuvant in periodontal disease prevention [86].

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VII. SUBLINGUAL VACCINE FOR PERIODONTAL DISEASES

Gingipain, a cysteine protease produced by P. gingivalis, has also been considered a major virulence factor [87,88]. The Hgp44 domain polypeptide of Arg gingipain A has been tested as a mucosal antigen [89]. Mice administered a mixture of Hgp44 and flagellin via the nasal route exhibited significant reduction in alveolar bone loss induced by live P. gingivalis infection. Nasal administration has been widely used for mucosal immunization because Ags are not subject to the digestive degradation usually caused by oral administration. However, several studies have reported that nasally administered Ags, such as CT and adenovirus vectors, diffuse through the perineural space as a result of retrograde passage through the olfactory epithelium [90,91]. A clinical study also suggested a strong association between nasal influenza vaccine and Bell’s palsy [92]. These findings raise concerns with regard to nasal administration and the potential threat posed by vaccine trafficking in neural tissues, including the central nervous system. Interestingly, when 25khagA-MBP or CT was given nasally to enable examination of their presence in neuronal tissues, the amounts of 25k-hagA-MBP were significantly lower than those of CT. Recent advances in the development of safe and effective nasal delivery vehicle system are leading to overcome the safety issue of nasal route of vaccination (see also Chapter 26: Nanodelivery Vehicles for Mucosal Vaccines).

VII. SUBLINGUAL VACCINE FOR PERIODONTAL DISEASES Sublingual administration is another convenient method to deliver drugs and lowmolecular-weight molecules to the bloodstream via a mucosal surface. It also avoids enterohepatic circulation and the partial first-pass effects of hepatic metabolism, as well as the immediate destruction of ingested molecules by gastric

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acids. Furthermore, recent studies have demonstrated that sublingual immunization (SLI) with an antigenic macromolecule plus CT as an adjuvant or inactivated influenza virus induced Agspecific immune responses in both mucosal and systemic compartments [93,94]. This indicated that the sublingual route is useful for delivery of vaccines targeting infectious diseases. FMS-like tyrosine kinase 3 (Flt3) ligand (Flt3L), a type 1 transmembrane protein, binds the fetal liver kinase 2 (Flk2)/Flt3 receptor. Flt3L has multiple roles in early hematopoiesis and B lymphopoiesis [95]. Interestingly, daily administration of recombinant Flt3L into mice induces Ag-specific immune responses that are comparable to those supported by the CT adjuvant [96]. Furthermore, nasal delivery of a DNA plasmid encoding Flt3L (pFL) with a protein Ag resulted in the induction of Ab responses in both mucosal and systemic sites [97]. Sublingual delivery of pFL plus 40k-OMP also elicited high titers of 40k-OMP-specific serum IgG and IgA, and salivary IgA Ab responses [98]. Induction of Ab responses was associated with elevated numbers of activated CD11C1 CD11b1 and CD11C1 CD8a1 DCs in the submandibular lymph nodes and spleen. Furthermore, pFL as a mucosal adjuvant induced CD41 T cells producing Th1- (IFNγ) and Th2-type (IL-4 and IL-5) cytokines. Importantly, SLI with 40k-OMP plus pFL provided significant protection against oral infection with P. gingivalis. SLI with the fusion protein 25k-hagA-MBP also augmented the activity of IFNγ-producing Th1- and IL-4producing Th2-type cells for the induction of serum IgG and IgA, and mucosal IgA Ab responses in comparison to 25k-hagA alone [99]. Furthermore, 25k-hagA-MBP-specific immune responses provided protective immunity against alveolar bone loss after P. gingivalis infection. These results suggest that SLI with 40k-OMP plus pFL or 25k-hagA-MBP may be a candidate for an efficient and safe vaccine against periodontal infection.

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Several epidemiologic studies have revealed that the host immune reaction against persistent infectious pathogens such as P. gingivalis may promote the development of atherosclerosis [100]. In particular, immune activation by the pathogen-derived heat shock protein (HSP) GroEL may result in an autoimmune response followed by atherosclerosis via the structural similarity or “molecular mimicry” of host HSP60 and GroEL. Previous studies have shown that HSP60 is selectively located in atherosclerotic lesions rather than nonatherosclerotic areas of the arterial wall [101]. In addition, a significant correlation was observed between anti-HSP Ab levels and the severity of atherosclerosis. High titers of anti-HSP60 Abs have been found in patients with carotid atherosclerosis, coronary disease, and stroke [102]. HSP of a major periodontal pathogen, such as P. gingivalis (GroEL), was also suggested to be a key molecule linking periodontitis, an infectious disease, with atherosclerosis, an autoimmune disease [103]. Clonal analysis of the T cells clearly demonstrated the presence of both human HSP60- and P. gingivalis GroEL-reactive T cell populations in the peripheral circulation of atherosclerosis patients [104]. Conversely, P. gingivalis GroEL immunization was reported to significantly reduce the levels of alveolar bone loss induced by multiple periodontopathic bacteria in an animal model [105]. Moreover, antiP. gingivalis GroEL serum showed cross-species recognition and exerted an opsonophagocytic effect on multiple periodontopathic bacteria [106]. Therefore, sublingual vaccination of atherosclerosis-related autoantigens is an effective method of attenuating autoimmune diseases by inducing an unresponsive state of tolerance [107]. SLI with rGroEL induced significant rGroELspecific serum IgG responses [108]. Antigenspecific cells isolated from the spleen produced significantly high levels of IL-10 and IFNγ after antigen restimulation in vitro. Flow cytometric analysis indicated that the frequencies of both

IL-101 and IFNγ1 CD41 Foxp31 cells increased significantly in the submandibular glands (SMGs). Furthermore, SLI with rGroEL significantly reduced atherosclerosis lesion formation in the aortic sinus and decreased serum Creactive protein, monocyte chemoattractant protein-1, and oxidized low-density lipoprotein levels. These findings suggest that SLI with rGroEL is associated with the increase of IFNγ1 or IL-101 Foxp31 cells in SMGs and a systemic humoral response, which could be an effective strategy for the prevention of naturally occurring or P. gingivalis-accelerated atherosclerosis. To date, pathogenic factors such as OMPs, fimbriae, gingipain, and hemagglutinin proteins, which P. gingivalis as a keystone pathogen possesses, have been studied as targets of mucosal vaccines. These vaccines are expected to reduce periodontal disease through Agspecific SIgA in saliva and Ag-specific IgG production in gingival crevicular fluid and to contribute to the prevention of systemic diseases derived from periodontal disease (Fig. 37.3).

VIII. CONCLUDING REMARKS To prevent oral infections such as periodontal disease and caries, vaccines that use many bacterial and/or viral components or adjuvant candidates to initially activate innate immunity leading to potent antigen-specific acquired immunity have been examined, and provided useful information for strategic mucosal vaccine development that can be adapted to prevent other infectious diseases. Since oral infectious diseases are caused by mixed infections and are associated with various systemic inflammatory diseases, the future development of safe, effective, and inexpensive vaccines targeting inflammatory markers caused by oral infections can also be taken into consideration. Further, oral fluids represent both mucosal and systemic arms of the immune system, one might consider the use of mucosally induced

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FIGURE 37.3

Suppression of periodontal disease by mucosal immunity. Porphyromonas gingivalis is an important pathogen in periodontal disease. Accordingly, intranasal or sublingual immunization of the pathogenic agent of P. gingivalis with various adjuvants can induce an Ag-specific SIgA response from saliva and an Ag-specific IgG response from gingival crevicular fluid that is derived from serum. These antibodies could inhibit bone resorption by P. gingivalis infection and suppress inflammation and arteriosclerosis caused by infection.

SIgA and serum IgG Abs for the control of infectious and inflammatory disease associated with the rest of digestive and pharyngeal tracts.

Acknowledgment This study was supported by Grants-in-Aid for Scientific Research (18592270, 19390537, 19791624, 22390398, and 26463145) from the Japan Society for the Promotion of Science, by an “Academic Frontier” Project (2007 11) and the “Strategic Research Base Development” Program (Japan [MEXT], 2010 14 [S1001024]) for Private Universities matching fund subsidy from the Ministry of Education, Cultures, Sports, Science and Technology, and by the Nihon University Multidisciplinary Research Grant (07-094 and 07-095; 14-019 and 15-016).

References [1] Kunisawa J, Nochi T, Kiyono H. Immunological commonalities and distinctions between airway and digestive immunity. Trends Immunol 2008;29:505 13. [2] Challacombe SJ, Shirlaw PJ. Immunity of diseases of the oral cavity. San Diego, CA: Academic Press; 1999. [3] Kraan H, Vrieling H, Czerkinsky C, Jiskoot W, Kersten G, Amorij JP. Buccal and sublingual vaccine delivery. J Control Release 2014;190:580 92. [4] Holmgren J, Czerkinsky C, Eriksson K, Mharandi A. Mucosal immunisation and adjuvants: a brief overview of recent advances and challenges. Vaccine 2003;21(Suppl. 2):S89 95. [5] Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med 2005;11:S45 53. [6] Hamada S, Slade HD. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev 1980;44:331 84.

[7] Okahashi N, Sasakawa C, Yoshikawa M, Hamada S, Koga T. Cloning of a surface protein antigen gene from serotype c Streptococcus mutans. Mol Microbiol 1989;3: 221 8. [8] Hajishengallis G, Michalek SM. Current status of a mucosal vaccine against dental caries. Oral Microbiol Immunol 1999;14:1 20. [9] Czerkinsky C, Russell MW, Lycke N, Lindblad M, Holmgren J. Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary glands and extramucosal tissues. Infect Immun 1989;57:1072 7. [10] Takahashi I, Okahashi N, Matsushita K, Tokuda M, Kanamoto T, Munekata E, et al. Immunogenicity and protective effect against oral colonization by Streptococcus mutans of synthetic peptides of a streptococcal surface protein antigen. J Immunol 1991;146:332 6. [11] Hajishengallis G, Hollingshead SK, Koga T, Russell MW. Mucosal immunization with a bacterial protein antigen genetically coupled to cholera toxin A2/B subunits. J Immunol 1995;154:4322 32. [12] Yamamoto S, Takeda Y, Yamamoto M, Kurazono H, Imaoka K, Yamamoto M, et al. Mutants in the ADPribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity. J Exp Med 1997;185: 1203 10. [13] Yamamoto M, Briles DE, Yamamoto S, Ohmura M, Kiyono H, McGhee JR. A nontoxic adjuvant for mucosal immunity to pneumococcal surface protein A. J Immunol 1998;161:4115 21. [14] Saito M, Otake S, Ohmura M, Hirasawa M, Takada K, Mega J, et al. Protective immunity to Streptococcus mutans induced by nasal vaccination with surface protein antigen and mutant cholera toxin adjuvant. J Infect Dis 2001;183:823 6. [15] Yamamoto M, Kiyono H, Yamamoto S, Batanero E, Kweon MN, Otake S, et al. Direct effects on antigenpresenting cells and T lymphocytes explain the

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

658

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

37. MUCOSAL VACCINES FOR ORAL DISEASE

adjuvanticity of a nontoxic cholera toxin mutant. J Immunol 1999;162:7015 21. Huang Y, Hajishengallis G, Michalek SM. Induction of protective immunity against Streptococcus mutans colonization after mucosal immunization with attenuated Salmonella enterica serovar typhimurium expressing an S. mutans adhesin under the control of in vivo-inducible nirB promoter. Infect Immun 2001;69:2154 61. Smith DJ, King WF, Rivero J, Taubman MA. Immunological and protective effects of diepitopic subunit dental caries vaccines. Infect Immun 2005;73: 2797 804. Wachsmann D, Klein JP, Scholler M, Ogier J, Ackermans F, Frank RM. Serum and salivary antibody responses in rats orally immunized with Streptococcus mutans carbohydrate protein conjugate associated with liposomes. Infect Immun 1986;52:408 13. Xu QA, Yu F, Fan MW, Bian Z, Chen Z, Peng B, et al. Protective efficacy of a targeted anti-caries DNA plasmid against cariogenic bacteria infections. Vaccine 2007;25:1191 5. Shi W, Li YH, Liu F, Yang JY, Zhou DH, Chen YQ, et al. Flagellin enhances saliva IgA response and protection of anti-caries DNA vaccine. J Dent Res 2012;91: 249 54. Mizel SB, Bates JT. Flagellin as an adjuvant: cellular mechanisms and potential. J Immunol 2010;185: 5677 82. Lightfield KL, Persson J, Brubaker SW, Witte CE, von Moltke J, Dunipace EA, et al. Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin. Nat Immunol 2008;9:1171 8. Zhao Y, Yang J, Shi J, Gong YN, Lu Q, Xu H, et al. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 2011;477: 596 600. Honko AN, Sriranganathan N, Lees CJ, Mizel SB. Flagellin is an effective adjuvant for immunization against lethal respiratory challenge with Yersinia pestis. Infect Immun 2006;74:1113 20. Campodonico VL, Llosa NJ, Bentancor LV, MairaLitran T, Pier GB. Efficacy of a conjugate vaccine containing polymannuronic acid and flagellin against experimental Pseudomonas aeruginosa lung infection in mice. Infect Immun 2011;79:3455 64. Sun Y, Shi W, Yang JY, Zhou DH, Chen YQ, Zhang Y, et al. Flagellin-PAc fusion protein is a high-efficacy anti-caries mucosal vaccine. J Dent Res 2012;91:941 7. Bao R, Yang JY, Sun Y, Zhou DH, Yang Y, Li YM, et al. Flagellin-PAc fusion protein inhibits progression of established caries. J Dent Res 2015;94:955 60. Yang J, Sun Y, Bao R, Zhou D, Yang Y, Cao Y, et al. Second-generation flagellin-rPAc fusion protein, KFD2-rPAc, shows high protective efficacy against

[29]

[30]

[31] [32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

dental caries with low potential side effects. Sci Rep 2017;7:11191. Luz DE, Nepomuceno RS, Spira B, Ferreira RC. The Pst system of Streptococcus mutans is important for phosphate transport and adhesion to abiotic surfaces. Mol Oral Microbiol 2012;27:172 81. Ferreira EL, Batista MT, Cavalcante RC, Pegos VR, Passos HM, Silva DA, et al. Sublingual immunization with the phosphate-binding-protein (PstS) reduces oral colonization by Streptococcus mutans. Mol Oral Microbiol 2016;31:410 22. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50:353 80. Yamashita Y, Bowen WH, Burne RA, Kuramitsu HK. Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model. Infect Immun 1993;61:3811 17. Koga T, Asakawa H, Okahashi N, Hamada S. Sucrosedependent cell adherence and cariogenicity of serotype c Streptococcus mutans. J Gen Microbiol 1986;132: 2873 83. Kawato T, Yamashita Y, Katono T, Kimura A, Maeno M. Effects of antibodies against a fusion protein consisting of parts of cell surface protein antigen and glucosyltransferase of Streptococcus sobrinus on cell adhesion of mutans streptococci. Oral Microbiol Immunol 2008;23:14 20. Sun J, Yang X, Xu QA, Bian Z, Chen Z, Fan M. Protective efficacy of two new anti-caries DNA vaccines. Vaccine 2009;27:7459 66. Niu Y, Sun J, Fan M, Xu QA, Guo J, Jia R, et al. Construction of a new fusion anti-caries DNA vaccine. J Dent Res 2009;88:455 60. Watanabe K, Hashizume T, Kurita-Ochiai T, Akimoto Y, Yamamoto M. Nasal administration of glucosyltransferase-I of Streptococcus sobrinus without adjuvant induces protective immunity. J Vaccines Vaccin 2010;1. Cutler CW, Kalmar JR, Genco CA. Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis. Trends Microbiol 1995;3:45 51. Li X, Kolltveit KM, Tronstad L, Olsen I. Systemic diseases caused by oral infection. Clin Microbiol Rev 2000;13:547 58. Wu Z, Nakanishi H. Connection between periodontitis and Alzheimer’s disease: possible roles of microglia and leptomeningeal cells. J Pharmacol Sci 2014;126:8 13. Hajishengallis G. Periodontitis: from microbial immune subversion to systemic inflammation. Nat Rev Immunol 2015;15:30 44. Holt SC, Kesavalu L, Walker S, Genco CA. Virulence factors of Porphyromonas gingivalis. Periodontol 2000 1999;20:168 238. Maiden MF, Carman RJ, Curtis MA, Gillett IR, Griffiths GS, Sterne JA, et al. Detection of high-risk

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

659

REFERENCES

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

groups and individuals for periodontal diseases: laboratory markers based on the microbiological analysis of subgingival plaque. J Clin Periodontol 1990;17: 1 13. Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA, et al. Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 2011;10:497 506. Darveau RP, Hajishengallis G, Curtis MA. Porphyromonas gingivalis as a potential community activist for disease. J Dent Res 2012;91:816 20. Hiratsuka K, Abiko Y, Hayakawa M, Ito T, Sasahara H, Takiguchi H. Role of Porphyromonas gingivalis 40-kDa outer membrane protein in the aggregation of P. gingivalis vesicles and Actinomyces viscosus. Arch Oral Biol 1992;37:717 24. Shibata Y, Hiratsuka K, Hayakawa M, Shiroza T, Takiguchi H, Nagatsuka Y, et al. A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis. Biochem Biophys Res Commun 2003;300: 351 6. Abiko Y, Ogura N, Matsuda U, Yanagi K, Takiguchi H. A human monoclonal antibody which inhibits the coaggregation activity of Porphyromonas gingivalis. Infect Immun 1997;65:3966 9. Hamajima S, Maruyama M, Hijiya T, Hatta H, Abiko Y. Egg yolk-derived immunoglobulin (IgY) against Porphyromonas gingivalis 40-kDa outer membrane protein inhibits coaggregation activity. Arch Oral Biol 2007;52:697 704. Saito S, Hiratsuka K, Hayakawa M, Takiguchi H, Abiko Y. Inhibition of a Porphyromonas gingivalis colonizing factor between Actinomyces viscosus ATCC 19246 by monoclonal antibodies against recombinant 40-kDa outer-membrane protein. Gen Pharmacol 1997; 28:675 80. Namikoshi J, Otake S, Maeba S, Hayakawa M, Abiko Y, Yamamoto M. Specific antibodies induced by nasally administered 40-kDa outer membrane protein of Porphyromonas gingivalis inhibits coaggregation activity of P. gingivalis. Vaccine 2003;22:250 6. Maeba S, Otake S, Namikoshi J, Shibata Y, Hayakawa M, Abiko Y, et al. Transcutaneous immunization with a 40-kDa outer membrane protein of Porphyromonas gingivalis induces specific antibodies which inhibit coaggregation by P. gingivalis. Vaccine 2005;23:2513 21. Koizumi Y, Kurita-Ochiai T, Yamamoto M. Transcutaneous immunization with an outer membrane protein of Porphyromonas gingivalis without adjuvant elicits marked antibody responses. Oral Microbiol Immunol 2008;23:131 8. Cai Y, Kurita-Ochiai T, Kobayashi R, Hashizume T, Yamamoto M. Nasal immunization with the 40-kDa outer membrane protein of Porphyromonas gingivalis

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

plus cholera toxin induces protective immunity in aged mice. J Oral Sci 2013;55:107 14. Momoi F, Hashizume T, Kurita-Ochiai T, Yuki Y, Kiyono H, Yamamoto M. Nasal vaccination with the 40-kilodalton outer membrane protein of Porphyromonas gingivalis and a nontoxic chimeric enterotoxin adjuvant induces long-term protective immunity with reduced levels of immunoglobulin E antibodies. Infect Immun 2008;76:2777 84. Liu C, Hashizume T, Kurita-Ochiai T, Fujihashi K, Yamamoto M. Oral immunization with Porphyromonas gingivalis outer membrane protein and CpGoligodeoxynucleotides elicits T helper 1 and 2 cytokines for enhanced protective immunity. Mol Oral Microbiol 2010;25:178 89. Koizumi Y, Kurita-Ochiai T, Oguchi S, Yamamoto M. Nasal immunization with Porphyromonas gingivalis outer membrane protein decreases P. gingivalisinduced atherosclerosis and inflammation in spontaneously hyperlipidemic mice. Infect Immun 2008;76: 2958 65. Nakao R, Hasegawa H, Ochiai K, Takashiba S, Ainai A, Ohnishi M, et al. Outer membrane vesicles of Porphyromonas gingivalis elicit a mucosal immune response. PLoS One 2011;6:e26163. Nakao R, Hasegawa H, Dongying B, Ohnishi M, Senpuku H. Assessment of outer membrane vesicles of periodontopathic bacterium Porphyromonas gingivalis as possible mucosal immunogen. Vaccine 2016;34: 4626 34. Evans RT, Klausen B, Sojar HT, Bedi GS, Sfintescu C, Ramamurthy NS, et al. Immunization with Porphyromonas (Bacteroides) gingivalis fimbriae protects against periodontal destruction. Infect Immun 1992;60: 2926 35. Njoroge T, Genco RJ, Sojar HT, Hamada N, Genco CA. A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells. Infect Immun 1997;65:1980 4. Weinberg A, Belton CM, Park Y, Lamont RJ. Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells. Infect Immun 1997;65:313 16. Yanagita M, Hiroi T, Kitagaki N, Hamada S, Ito HO, Shimauchi H, et al. Nasopharyngeal-associated lymphoreticular tissue (NALT) immunity: fimbriaespecific Th1 and Th2 cell-regulated IgA responses for the inhibition of bacterial attachment to epithelial cells and subsequent inflammatory cytokine production. J Immunol 1999;162:3559 65. Nagasawa T, Aramaki M, Takamatsu N, Koseki T, Kobayashi H, Ishikawa I. Oral administration of Porphyromonas gingivalis fimbriae with cholera toxin induces anti-fimbriae serum IgG, IgM, IgA and salivary IgA antibodies. J Periodontal Res 1999;34:169 74. Takahashi Y, Kumada H, Hamada N, Haishima Y, Ozono S, Isaka M, et al. Induction of immune

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

660

[66]

[67]

[68] [69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

37. MUCOSAL VACCINES FOR ORAL DISEASE

responses and prevention of alveolar bone loss by intranasal administration of mice with Porphyromonas gingivalis fimbriae and recombinant cholera toxin B subunit. Oral Microbiol Immunol 2007;22:374 80. Sharma A, Honma K, Evans RT, Hruby DE, Genco RJ. Oral immunization with recombinant Streptococcus gordonii expressing Porphyromonas gingivalis FimA domains. Infect Immun 2001;69:2928 34. Guo H, Wang X, Jiang G, Yang P. Construction of a sIgA-enhancing anti-Porphyromonas gingivalis FimA vaccine and nasal immunization in mice. Immunol Lett 2006;107:71 5. Kowalczyk DW, Ertl HC. Immune responses to DNA vaccines. Cell Mol Life Sci 1999;55:751 70. Conry RM, Curiel DT, Strong TV, Moore SE, Allen KO, Barlow DL, et al. Safety and immunogenicity of a DNA vaccine encoding carcinoembryonic antigen and hepatitis B surface antigen in colorectal carcinoma patients. Clin Cancer Res 2002;8:2782 7. Nichols WW, Ledwith BJ, Manam SV, Troilo PJ. Potential DNA vaccine integration into host cell genome. Ann NY Acad Sci 1995;772:30 9. Martin T, Parker SE, Hedstrom R, Le T, Hoffman SL, Norman J, et al. Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum Gene Ther 1999;10:759 68. Lakey RL, Morgan TG, Rowan AD, Isaacs JD, Cawston TE, Hilkens CM. A novel paradigm for dendritic cells as effectors of cartilage destruction. Rheumatology (Oxford) 2009;48:502 7. Parsons KR, Young JR, Collins BA, Howard CJ. Cattle CTLA-4, CD28 and chicken CD28 bind CD86: MYPPPY is not conserved in cattle CD28. Immunogenetics 1996; 43:388 91. Yu F, Xu QA, Chen W. A targeted fimA DNA vaccine prevents alveolar bone loss in mice after intra-nasal administration. J Clin Periodontol 2011;38:334 40. Progulske-Fox A, Tumwasorn S, Lepine G, Whitlock J, Savett D, Ferretti JJ, et al. The cloning, expression and sequence analysis of a second Porphyromonas gingivalis gene that codes for a protein involved in hemagglutination. Oral Microbiol Immunol 1995;10:311 18. Lepine G, Progulske-Fox A. Duplication and differential expression of hemagglutinin genes in Porphyromonas gingivalis. Oral Microbiol Immunol 1996;11:65 78. Dusek DM, Progulske-Fox A, Brown TA. Systemic and mucosal immune responses in mice orally immunized with avirulent Salmonella typhimurium expressing a cloned Porphyromonas gingivalis hemagglutinin. Infect Immun 1994;62:1652 7. Zhang P, Yang QB, Marciani DJ, Martin M, Clements JD, Michalek SM, et al. Effectiveness of the quillaja saponin semi-synthetic analog GPI-0100 in potentiating mucosal and systemic responses to recombinant

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

HagB from Porphyromonas gingivalis. Vaccine 2003;21: 4459 71. Kozarov E, Miyashita N, Burks J, Cerveny K, Brown TA, McArthur WP, et al. Expression and immunogenicity of hemagglutinin A from Porphyromonas gingivalis in an avirulent Salmonella enterica serovar typhimurium vaccine strain. Infect Immun 2000;68:732 9. Boos W, Shuman H. Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol Mol Biol Rev 1998;62:204 29. Seong SY, Huh MS, Jang WJ, Park SG, Kim JG, Woo SG, et al. Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56. Infect Immun 1997;65:1541 5. Kushwaha A, Rao PP, Suresh RP, Chauhan VS. Immunogenicity of recombinant fragments of Plasmodium falciparum acidic basic repeat antigen produced in Escherichia coli. Parasite Immunol 2001;23: 435 44. Fernandez S, Palmer DR, Simmons M, Sun P, Bisbing J, McClain S, et al. Potential role for Toll-like receptor 4 in mediating Escherichia coli maltose-binding protein activation of dendritic cells. Infect Immun 2007;75: 1359 63. Fox JD, Kapust RB, Waugh DS. Single amino acid substitutions on the surface of Escherichia coli maltosebinding protein can have a profound impact on the solubility of fusion proteins. Protein Sci 2001;10: 622 30. Du Y, Hashizume T, Kurita-Ochiai T, Yuzawa S, Abiko Y, Yamamoto M. Nasal immunization with a fusion protein consisting of the hemagglutinin A antigenic region and the maltose-binding protein elicits CD11c (1) CD8(1) dendritic cells for induced long-term protective immunity. Infect Immun 2011;79:895 904. Hong SH, Byun YH, Nguyen CT, Kim SY, Seong BL, Park S, et al. Intranasal administration of a flagellinadjuvanted inactivated influenza vaccine enhances mucosal immune responses to protect mice against lethal infection. Vaccine 2012;30:466 74. Curtis MA, Kuramitsu HK, Lantz M, Macrina FL, Nakayama K, Potempa J, et al. Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis. J Periodontal Res 1999;34:464 72. Nakayama K. Porphyromonas gingivalis cell-induced hemagglutination and platelet aggregation. Periodontol 2000 2010;54:45 52. Puth S, Hong SH, Park MJ, Lee HH, Lee YS, Jeong K, et al. Mucosal immunization with a flagellin-adjuvanted Hgp44 vaccine enhances protective immune responses in a murine Porphyromonas gingivalis infection model. Hum Vaccin Immunother 2017;1 10.

V. MUCOSAL VACCINES FOR BACTERIAL DISEASES

661

REFERENCES

[90] van Ginkel FW, Jackson RJ, Yuki Y, McGhee JR. Cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues. J Immunol 2000;165:4778 82. [91] Lemiale F, Kong WP, Akyurek LM, Ling X, Huang Y, Chakrabarti BK, et al. Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodeficiency virus vaccine and localization in the central nervous system. J Virol 2003;77:10078 87. [92] Mutsch M, Zhou W, Rhodes P, Bopp M, Chen RT, Linder T, et al. Use of the inactivated intranasal influenza vaccine and the risk of Bell’s palsy in Switzerland. N Engl J Med 2004;350:896 903. [93] Cuburu N, Kweon MN, Song JH, Hervouet C, Luci C, Sun JB, et al. Sublingual immunization induces broad-based systemic and mucosal immune responses in mice. Vaccine 2007;25:8598 610. [94] Song JH, Kim JI, Kwon HJ, Shim DH, Parajuli N, Cuburu N, et al. CCR7-CCL19/CCL21-regulated dendritic cells are responsible for effectiveness of sublingual vaccination. J Immunol 2009;182:6851 60. [95] Hunte BE, Hudak S, Campbell D, Xu Y, Rennick D. flk2/flt3 ligand is a potent cofactor for the growth of primitive B cell progenitors. J Immunol 1996;156: 489 96. [96] Pulendran B, Smith JL, Caspary G, Brasel K, Pettit D, Maraskovsky E, et al. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc Natl Acad Sci USA 1999;96:1036 41. [97] Kataoka K, McGhee JR, Kobayashi R, Fujihashi K, Shizukuishi S, Fujihashi K. Nasal Flt3 ligand cDNA elicits CD11c 1 CD8 1 dendritic cells for enhanced mucosal immunity. J Immunol 2004;172:3612 19. [98] Zhang T, Hashizume T, Kurita-Ochiai T, Yamamoto M. Sublingual vaccination with outer membrane protein of Porphyromonas gingivalis and Flt3 ligand elicits protective immunity in the oral cavity. Biochem Biophys Res Commun 2009;390:937 41. [99] Yuzawa S, Kurita-Ochiai T, Hashizume T, Kobayashi R, Abiko Y, Yamamoto M. Sublingual vaccination with fusion protein consisting of the functional domain of hemagglutinin A of Porphyromonas

[100]

[101]

[102]

[103]

[104]

[105]

[106]

[107]

[108]

gingivalis and Escherichia coli maltose-binding protein elicits protective immunity in the oral cavity. FEMS Immunol Med Microbiol 2012;64:265 72. Taniguchi A, Nishimura F, Murayama Y, Nagasaka S, Fukushima M, Sakai M, et al. Porphyromonas gingivalis infection is associated with carotid atherosclerosis in non-obese Japanese type 2 diabetic patients. Metabolism 2003;52:142 5. Kol A, Sukhova GK, Lichtman AH, Libby P. Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-alpha and matrix metalloproteinase expression. Circulation 1998;98:300 7. Mandal K, Jahangiri M, Xu Q. Autoimmunity to heat shock proteins in atherosclerosis. Autoimmun Rev 2004;3:31 7. Rajaiah R, Moudgil KD. Heat-shock proteins can promote as well as regulate autoimmunity. Autoimmun Rev 2009;8:388 93. Yamazaki K, Ohsawa Y, Itoh H, Ueki K, Tabeta K, Oda T, et al. T-cell clonality to Porphyromonas gingivalis and human heat shock protein 60s in patients with atherosclerosis and periodontitis. Oral Microbiol Immunol 2004;19:160 7. Lee JY, Yi NN, Kim US, Choi JS, Kim SJ, Choi JI. Porphyromonas gingivalis heat shock protein vaccine reduces the alveolar bone loss induced by multiple periodontopathogenic bacteria. J Periodontal Res 2006;41:10 14. Choi JI, Choi KS, Yi NN, Kim US, Choi JS, Kim SJ. Recognition and phagocytosis of multiple periodontopathogenic bacteria by anti-Porphyromonas gingivalis heat-shock protein 60 antisera. Oral Microbiol Immunol 2005;20:51 5. Calder CJ, Nicholson LB, Dick AD. Mechanisms for inducing nasal mucosal tolerance in experimental autoimmune uveoretinitis. Methods 2006;38:69 76. Hagiwara M, Kurita-Ochiai T, Kobayashi R, Hashizume-Takizawa T, Yamazaki K, Yamamoto M. Sublingual vaccine with GroEL attenuates atherosclerosis. J Dent Res 2014;93:382 7.

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Vaccination Against Respiratory Syncytial Virus Tracy J. Ruckwardt, Michelle C. Crank, Kaitlyn M. Morabito and Barney S. Graham Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

I. INTRODUCTION Respiratory syncytial virus (RSV) is an enveloped, negative-sense, nonsegmented RNA virus with a 15-kb genome. RSV has 10 genes that encode 11 recognized proteins. The RNAbinding nucleoprotein (N), phosphoprotein (P), polymerase (L), and transcription processivity factor (M2-1) make up the ribonucleocapsid. The lipid bilayer envelope is supported by the matrix protein (M) and displays the glycoprotein (G), the fusion protein (F), and small hydrophobic ion channel (SH) protein on the surface. The virus also encodes M2-2, which mediates the transition between transcription and replication, and two nonstructural proteins, NS1 and NS2 [1]. RSV was a member of Paramyxoviridae family prior to reclassification in 2016 into the Orthopneumovirus genus of the Pneumoviridae family [2]. Compared to influenza virus, another common cause of respiratory tract infections, the

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00038-9

diversity of RSV is limited. There are two antigenic subtypes of RSV, A and B, which comprise a single serotype [3]. Most of the diversity is accounted for by G protein, which has less than 50% amino acid sequence identity between subtypes. The F glycoprotein is more conserved, with 89% amino acid identity between subtypes [4]. While F diversity is limited, even small amino acid differences can affect the ability of monoclonal antibodies to neutralize individual viral isolates [5]. Several RSV proteins have been found to interfere with innate and adaptive immune responses [6]. NS1 and NS2 inhibit the induction of type I and III interferon (IFN) by binding to proteins in the IFN pathway (IRF3, MAVS, RIG-I), mediating degradation of other proteins (IKKε, TRAF3, and STAT2) in the IFN pathway, and inhibiting apoptosis of RSV-infected cells [7 11]. The G protein exists in two forms. Membrane-anchored G protein mediates attachment between the virus and cell and is

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key for in vivo infection. The secreted form of the G protein can act as an immune decoy to absorb antibody and reduce neutralizing activity. Additionally, secreted G protein modulates the immune response by altering ERK1 and ERK2 signaling in dendritic cells and by inhibiting activation of NF-κB and the response to TLR2, TLR4, and TLR9 agonists; it has also been shown to induce a Th2 CD41 lymphocyte response that leads to eosinophilia in BALB/c mice [12 16]. The G protein contains a CX3C chemokine-like motif and can bind the fractalkine receptor to affect recruitment of lymphocytes into the respiratory tract [17]. These mechanisms of immune evasion are among many strategies that RSV uses to counteract host defenses and remain a successful human pathogen [6]. Humans are the only natural hosts for RSV, and infection is restricted to the superficial epithelial cells in the airway. Virus both enters and buds from the apical surface of polarized cells and can spread by inducing cell-to-cell fusion, factors that limit the ability of the immune system to fight infection. Viral particles can be filamentous or spherical in shape and can grow to high titers in the nose and upper airway of infected individuals [18]. RSV is a highly contagious pathogen for which there is no licensed vaccine, resulting in yearly epidemics of cocirculating subtype A and B strains that not only impart a substantial financial burden but also contribute to significant morbidity and mortality in susceptible populations.

II. GLOBAL IMPACT AND CLINICAL DISEASE RSV spreads via large droplets or contact with contaminated objects and has an incubation period of 3 5 days. It replicates in the nasopharynx, and if lower respiratory symptoms develop, they appear 1 3 days after the onset of upper airway symptoms. In affected

mucosal tissue, polymorphonuclear cells invade first, with lymphocytes, plasma cells, and macrophages forming peribronchiolar infiltrates. Significant edema and mucus production accompany necrosis of mucosal cells and inflammatory cell infiltrate to narrow or obstruct bronchioles and alveoli [19]. This can lead to collapse or hyperinflation of distal airways as well as the clinical symptom of wheezing [20]. Severe acute lower respiratory tract infections (ALRI) due to RSV infection have been associated with chronic wheezing or asthma later in childhood in multiple epidemiological studies, although the mechanism of this effect is still being debated [21 24]. Clinical disease from RSV ranges from mild upper respiratory symptoms in healthy children and young adults to sometimes deadly lower airway disease in infants, the elderly, and individuals with comorbid heart or lung disease. In most individuals, symptoms include rhinorrhea, cough, decreased appetite, pharyngitis, fatigue, and sometimes otitis media or fever; these symptoms are consistent with other common upper respiratory viral infections. This syndrome lasts an average of 10 days in previously healthy children and adults, and about one quarter of individuals develop lower respiratory tract disease after 3 days [25]. However, in certain populations, including those at the extremes of age and those with comorbid medical conditions, RSV disease can be much more severe. RSV is a ubiquitous viral infection affecting all age groups around the globe. In temperate climates, RSV outbreaks occur seasonally during colder months (October May in the United States) [26], and annual incidence varies. However, about 90% of children have experienced at least one RSV infection by the age of 24 months [27]. Worldwide, RSV is the leading cause of ALRI in children [28]. The number of episodes of RSV ALRI in children under 5 years of age was estimated to be 33.1 million in 2015, and those were predicted to result in 3.2

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million hospitalizations and 60,000 199,000 inhospital deaths [29,30]. Infants under 3 months of age have the highest rate of hospitalization in the United States, and very preterm infants are three times more likely to be hospitalized than infants born at term [31,32]. An estimated 10% 20% of children under 5 years of age in the United States, the majority of whom have no known risk factor for RSV disease, receive medical care for RSV-related illness, which includes 500,000 emergency room visits and 1.5 million primary care visits in addition to the hospitalizations mentioned [7,33]. In children under 6 months of age or those with chronic heart, lung, or immune defects, involvement of the small airways, or bronchiolitis, can cause airway obstruction, wheezing, and pneumonia. The youngest infants with lower airway disease may present with only lethargy, apnea, or hypoxia. Both the size of premature infants’ airways and the comorbid presence of bronchopulmonary dysplasia may explain their predisposition to severe disease, and it is for this population that the only licensed therapeutic for RSV, palivizumab (a recombinant monoclonal antibody specific for mature F protein of RSV; see below) is indicated. These high rates of hospitalizations and deaths make infants, particularly those born preterm, an important population for prevention of RSV infection. At the other extreme of age, RSV causes significant morbidity and mortality in the elderly, particularly those living in long-term care facilities in the United States [34]. In older adults or those with comorbidities, RSV can cause pneumonia or hypoxia that requires mechanical ventilation. Attack rates in congregate care populations are 5% 10%, complicated by significant rates of pneumonia (10% 20%) and death (2% 5%) [35]. More recent studies estimate mortality from RSV infection among US adults over the age of 65 years to be 10,000 per year [36]. RSV was found to be the cause of 7% of total cases and 12.5% of hospitalized cases of

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moderate to severe influenza-like illness in adults over 65 not living in group care, suggesting that RSV infection necessitates hospitalization at a rate comparable to influenza [25,37]. Thus older adults are another key population that would benefit from an RSV vaccine. The natural epidemiology of RSV infection has led to the identification of several target populations for vaccination, each with its own unique risk factors and safety considerations. Direct vaccination of infants prior to their primary infection and of elderly adults with underlying comorbidities would protect the highest-risk groups, but these populations present several challenges for achieving safe and effective immunity. Alternatively, vaccinating young children, who readily transmit disease between and within households, or of pregnant women, who may provide passive protection to their infants, could effectively reduce transmission and disease in groups at the greatest risk. Gaining a better understanding of the epidemiology of infection and correlates of protection in ongoing vaccine efficacy trials in differentaged cohorts will be a critical asset [38,39].

III. CORRELATES OF PROTECTION A significant obstacle to RSV vaccine efforts is the lack of a well-defined correlate of protection [39]. Binding and neutralizing antibodies in maternal, cord, and infant sera have been correlated with protection from infection and/or severe disease in several studies of natural infection, but there has been no consensus on a protective threshold. Additionally, passive transfer of polyclonal, high-titer RSV immunoglobulin has proven an effective strategy to protect highrisk infants from severe disease [40]. Antibodies at the site of infection may play a deterministic role, and either nasal IgA or IgG has been correlated with protection from infection in several studies [41 43]. IgA is the predominant immunoglobulin in the upper respiratory tract,

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exceeding the concentration of IgG by 2.5:1 [44]. IgA is transported from the basolateral to the apical surface of respiratory epithelial cells by the polymeric immunoglobulin receptor, which is cleaved to a secretory component that stabilizes dimeric or polymeric IgA in the mucosal lumen (Chapter 4: Protective Activities of Mucosal Antibodies). Conversely, IgG transudates between alveolar capillaries and alveolar epithelium to dominate and most efficiently protect the lower respiratory tract [45]. Thus IgG access to the upper respiratory tract is limited, and it will be difficult to achieve sufficient levels of neutralizing antibody in the upper airway to completely protect against infection following systemic vaccination. RSV neutralizing antibodies target the F and G surface glycoproteins. The diversity and immunomodulatory properties of the G protein, in addition to the larger number of neutralization-sensitive antigenic sites on the F protein, make F the more commonly selected antigen for candidate antibody-eliciting vaccines. There is proof of concept for this approach as passive prophylaxis with the monoclonal antibody palivizumab, which targets the F protein, has demonstrated efficacy for protecting high-risk infants from severe disease and has been used clinically for this purpose for 20 years. Motavizumab, which is a higher-potency derivative of palivizumab, has shown greater than 80% efficacy in full-term Native American infants [46]. The functional pretriggered form of the RSV F protein is metastable, resulting in the display of both prefusion (pre-F) and postfusion (postF) forms on viral particles [47]. High-resolution structures of pre-F and post-F have aided in the definition of antigenic sites on each conformation, and approximately 50% of the protein surface is shared between pre-F and post-F [48 51]. Sites unique to pre-F, designated antigenic sites Ø and V, are targeted by more potent neutralizing antibodies than sites II and IV that are on the shared surfaces of pre-F and

post-F [52,53]. While site III antibody contact residues are present on both pre-F and post-F surfaces, access to this site is obscured in the post-F conformation. Antibodies that bind to site III have been shown to cross-react with human metapneumovirus [54,55]. Interestingly, the infant immune repertoire is apt to respond to antigenic site III, as potent antibodies targeting this site can be generated with little to no somatic hypermutation [56]. Site I antibodies tend to bind post-F preferentially over pre-F and have weak or no neutralizing activity [53]. The majority of neutralizing antibody in normal adult sera targets the pre-F conformation [52]. Similarly, pre-F targeting antibodies from infected infants demonstrate better neutralization than antibodies targeting G or post-F [57]. Given the potency of pre-F targeting antibodies, it is not surprising that mutations designed to stabilize the F protein in the pre-F conformation have resulted in an immunogen (DS-Cav1) that elicits significantly improved serum neutralizing activity and protection compared to wild-type F or post-F proteins across multiple delivery platforms [48]. Pre-F specific antibodies have been identified that are 10 100 times more potent than palivizumab [53]. The superior potency of neutralizing antibodies to pre-Fspecific antigenic sites may explain the failure to demonstrate efficacy in two recent phase 2b and phase 3 clinical trials that used recombinant protein in the post-F conformation [58,59]. Altogether, these data suggest that stabilization of the F protein in its prefusion conformation may improve the effectiveness of subunit vaccines. Our understanding of correlates is limited in part by a lack of permissive animal models that recapitulate human infection and disease. RSV was shown to cause illness in the first experimental human challenge studies shortly after it was identified as a human pathogen [60,61]. Human challenge studies offer the advantage of knowing the time of infection and in many ways have corroborated studies of natural infection.

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IV. MATERNAL IMMUNIZATION TO PROTECT VULNERABLE INFANTS

Symptoms of disease in experimentally infected adults correlated with viral titer as previously described for RSV-infected infants [62,63]. Nasal IgA was also highly correlated with protection from infection, yet even after experimental infection, individuals were unlikely to reach nasal IgA levels that had been anticipated to be protective against reinfection [42]. This highlights the difficulty of conferring protection in the nose where immune access is limited. Limitations in local immunity may be exaggerated during RSV infection; in contrast to natural influenza virus infection, RSV failed to induce virus-specific IgA memory B cells [42]. This is an area that needs additional work with optimized antigens to better define the role of mucosal IgA responses in RSV immunity. As was previously reported following natural infection in adults and children [64,65], RSV-specific antibody is poorly maintained after experimental infection and can wane to preinfection levels as soon as 6 months post infection [42]. CD81 T cells are known to contribute to RSV immunopathology in animal models [66], but disease is prolonged in children with T cell immunodeficiency [67] and CD81 T cell responses coincide with convalescence in infected infants and experimentally infected adults [68,69]. Most often, studies of RSVspecific T cell responses are confined to peripheral blood where critical populations such as lung-tissue-resident memory T cells (TRM) cannot be measured. Serial bronchoscopy has been used in human challenge experiments to measure CD81 TRM, which were found to accumulate in the airway even through convalescence and have a distinct phenotype from CD81 T cells in the blood of the same subjects. While numbers of preexisting CD81 TRM in the airway did not predict infection, they correlated with reduced disease in individuals who were infected, indicating that T cells may be a crucial second line of defense when antibodies are unable to prevent infection [69]. Care should be taken in interpreting correlates of protection

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from either infection or severe disease gleaned from studies in healthy adults, as they may not be extrapolated to individuals at the extremes of age, in whom RSV has the largest impact, owing to the unique susceptibility factors associated with severe disease in these populations. Many candidates have emerged in the RSV vaccine landscape. These include live attenuated viruses, particle- and subunit-based vaccines, and gene-based vectors. Current approaches have been recently reviewed [59,70], and a list of preclinical and clinical trial candidates is curated and updated regularly by PATH (https://vaccineresources.org/details. php?i 5 1562). This is a rapidly changing list. In addition to defining useful immunological correlates and metrics to evaluate the success of these various approaches, consistent definitions of clinical endpoints should be generated to facilitate comparisons between trials [71,72]. A brief review of the history of vaccination and considerations for selecting candidates for the major target populations will be discussed below. The majority of candidates in the pipeline involve parenteral administration, with only a few intranasal candidates aiming for direct induction of immune responses at the mucosal site of infection.

IV. MATERNAL IMMUNIZATION TO PROTECT VULNERABLE INFANTS Little is known about the clinical impact of RSV infection in pregnant women, but their decreased immunity and increased susceptibility to other viral pathogens may portend an increased susceptibility to more severe outcomes following infection. While vaccinating pregnant women may have some potential to benefit them directly, the primary goal is to bolster neutralizing antibody titers and thus FcRnmediated transport of protective antibodies in

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utero [73]. This strategy is relatively new for RSV but has proven effective for influenza, pertussis, and several other pathogens for which maternal vaccination is used to provide infants with partial protection while they are too young to be effectively immunized [74,75]. Only IgG is capable of FcRn-mediated antenatal transcytosis across the placenta, a process that can result in protective antibody levels in infants that exceed those of their mothers prior to a full-term birth. Protective effects of maternally derived IgG may be limited in the upper airway, but passive transudation into the lung could protect from lower airway disease, and additional protection at the mucosal level may be conferred by IgA and IgG antibody in breast milk [73]. Higher levels of passively transferred maternal RSV-specific IgG have been shown to result in a lower risk of severe RSV disease, yet maternal RSV-specific antibody levels are variable and typically provide protection for only the first few months of life [76 78]. A 0.5-log increase in antibody titers in infants is estimated to extend protection by 19 days, suggesting that maternal immunization could be a feasible approach to protecting infants against severe disease [76]. Because adults have preexisting immunity to RSV, a single injection has the potential to boost maternal antibodies to extend protection of infants through 6 months of age or beyond, when active immunization approaches are more feasible (Chapter 44: Maternal Vaccination for Protection Against Maternal and Infant Bacterial and Viral Pathogens). Vaccines for pregnant women must meet high standards for safety and tolerability. Preexisting immunity and the major goal of enhancing systemic antibody for transfer precludes the use of intranasal vaccines in this population, and current strategies are focused on subunit and particle-based approaches. The candidate that is most advanced in clinical testing is a RSV post-F protein presented as a rosette that is now in phase III testing in pregnant women.

V. RSV IMMUNITY AND VACCINATION IN INFANTS AND YOUNG CHILDREN About half of all hospitalizations for severe RSV disease occur in infants under 6 months of age, so this age group has the most to gain from direct vaccination. This period of high susceptibility for infants is due to the waning of protective maternal antibody and infants’ relatively small airway size and lung capacity. However, infancy is also the most difficult age for vaccination because of lack of preexisting immunity, immunologic immaturity, and, specifically for RSV, safety concerns based on the historical formalin-inactivated vaccine-enhanced disease in young infants. Infants are forced to rely heavily on their innate defenses, which prove limited during early life [79,80]. Infants are biased toward tolerogenic and Th2 types of responses and are known to have limited capacity for somatic hypermutation to optimize antibody affinity [81]. For these reasons, it is likely that the majority of vaccines will be initially targeted to infants older than 6 months of age, in whom immunity has sufficiently matured to generate higher affinity responses with less potential for adverse events. Formalin and heat inactivation of RSV, which had been a previously successful strategy for other viral vaccines for infants, had the unfortunate consequence of enhancing disease following natural RSV infection. Trials conducted with these early vaccines served as a warning against using protein-based or inactivated viruses for this target population [82]. The enhanced respiratory disease associated with immunization with formalin-inactivated RSV (FI-RSV) has long been attributed to the induction of Th2-biased immune responses and granulocyte infiltration [83 85]. The elicitation of high titers of binding antibody with low neutralization capacity was associated with complement fixation and the deposition of immune complexes in the lung [86]. The inactivation

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process to render RSV noninfectious (72 hours at 36 C in 0.025% formalin) altered the antigenic properties of the F protein, resulting in the display of post-F epitopes without preserving the unique, neutralization-sensitive epitopes found only on the functional pre-F conformation [87]. In addition, FI-RSV failed to elicit CD81 T cells, another hallmark of vaccination with antigens that are not processed in the cytoplasm [86]. The copious amount of work done to understand the failings of FI-RSV vaccination in infants has served as a guideline for the clinical development of future vaccines. For the RSV-naı¨ve infant population, liveattenuated approaches have been extensively tested in young children and have a proven safety profile. Live-attenuated vaccines (LAV) are administered intranasally and replicate in the upper respiratory tract. Thus they elicit immunity directly at the site of infection but spread to the lower airway is limited by attenuation of replication and by the presence of maternal antibodies. Most akin to natural infection, live-attenuated RSV offers intrinsic adjuvanting signals that can direct antiviral immunity and have shown no disease enhancement over multiple clinical trials [88]. Iterative modifications have been necessary to ensure an acceptable balance between attenuation and immunogenicity and to prevent viral reversions that could potentially restore pathogenicity. The newest generation of LAV are recombinant derivatives of wild-type RSV. One approach is tailored to avoid type 1 IFN inhibition via ΔNS2 deletions, thereby attenuating replication and improving safety. Another approach is designed to increase transcription and antigen production while limiting replication using ΔM2-2 deletions, which achieves greater immunogenicity despite lower titers of vaccine virus [88]. RSV LAVs are likely to display a combination of pre-F and post-F proteins because retention of the active pre-F protein is critical for infection and replication, effectively ensuring the display of antigenic sites found

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exclusively on pre-F. Some LAV approaches have selectively focused on strains that have a less unstable version of F that maintains a relatively high level of pre-F [89,90]. Alternatively, chimeric parainfluenza LAV vectors are being designed to express RSV G or stabilized pre-F proteins and are being tested in preclinical and early phase trials and may offer the advantage of bivalent protection [59,70,91,92]. While ideal for antigen-naı¨ve populations, LAV are unable to significantly boost immunity in antigenexperienced populations where mucosal immunity prevents replication of the attenuated vaccine [93]. In addition to direct vaccination or boosting maternal antibody levels to facilitate thirdtrimester antenatal transfer of protective antibody, disease outcomes in very young infants may be improved by delivering high-potency, half-life-extended antibodies directed at pre-F exclusive sites. One such antibody, MEDI8897, is currently under clinical evaluation in preterm infants. This antibody has the potential to be accessible to all newborns and is given as a single birth dose [94,95]. While these strategies may protect infants during the critical period of severe RSV disease, the impact of passive antibodies on the generation of de novo immune responses in infants is unknown. Although infants under 6 months of age are more likely to experience severe disease, RSV is responsible for significant morbidity and mortality in children between 6 months and 5 years of age [28]. Young children infected with RSV tend to have high viral titers and extended shedding, and epidemiological studies have implicated them as a common initial source of infection for both infants and the elderly [96,97]. Therefore vaccination of children between 6 months and 5 years of age will directly benefit them and could help to mitigate disease at the extremes of age through decreased transmission. This approach of targeting transmitters with better immunological capacity could be even more effective for

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protecting the elderly than direct vaccination of older adults and would require a smaller number of doses if RSV follows the model that has been shown for influenza [97,98]. Liveattenuated and gene-based approaches that induce Th1 type immunity and antiviral CD81 T cell responses in addition to neutralizing antibodies in young children who may still be RSV naı¨ve are favored methods. After priming by these approaches or by natural infection, protein-based vaccines could be used to boost functional antibody responses. Many of these methods are currently being tested in clinical trials that may afford broad protection across multiple susceptible age groups.

VI. RSV IMMUNITY AND VACCINATION IN OLDER ADULTS The symptoms of RSV infection in older adults are not as easily discriminated from other respiratory infections as they are in infected infants. Most often, infection in older adults is mild to moderate in severity and not wellreported or confirmed by testing. It is estimated that 3% 7% of adults are infected annually [25]. Many anatomical, immunological, and lung functional changes are known to occur with age that predispose older adults to respiratory disease [99]. Chronic obstructive pulmonary disease and other comorbidities contribute to increased susceptibility to RSV disease and may be compounded by immunosenescence and a reduction of functional RSV-specific T cells associated with aging [25]. Owing to these obstacles, vaccine approaches for the elderly may need to involve higher dosages, stronger adjuvants, and potentially repeated administration (Chapter 47: Mucosal Vaccines for Aged: Challenges and Struggles in Immunosenescence). As shown in studies of healthy, young adults, serum and nasal antibody to RSV has an inverse correlation with the risk of becoming

infected, and higher serum neutralizing antibody is associated with less severe infection in the elderly. Despite their seemingly numerous immune limitations, older adults do not have significantly lower baseline RSV antibody titers than younger adults and are just as likely, if not more likely, to have a fourfold rise in antibody titer after natural infection [100]. These findings suggest that an RSV vaccine could boost neutralizing antibody titers, but other factor such as diminished CD81 T cell responses and comorbidities may also play a role in the increased susceptibility of the elderly to RSV infection. The number of variables and range of clinical presentation in the elderly make it increasingly difficult to fully define risk factors and precise correlates of protection for this atrisk population, and a better vaccine may be needed to achieve greater understanding. All adults have preexisting RSV immunity and are not candidates for LAV, owing to overattenuation in antigen-experienced individuals [93]. Thus most current vaccine approaches for the elderly are protein-based, and the recent failures of post-F vaccines in this populations highlight the importance of using pre-F or alternative target antigens to elicit optimal protective antibody responses [59]. Other candidates are gene-based, offering the attractive feature of cytoplasmic generation of antigen and simultaneous boosting of preexisting CD81 T cell responses, which may be a key correlate of protection for this population.

VII. CONCLUDING REMARKS Our improved understanding of the epidemiology and global impact of RSV disease combined with a better understanding of pathogenesis and protein structure has reinvigorated vaccine development, resulting in a large number of candidate vaccines in preclinical and clinical testing. Owing to the specific needs and characteristics of each vulnerable

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REFERENCES

population, different approaches may be required to promote the development of protective antibody and T cell responses in blood or in mucosal secretions and tissues. Vaccines are needed to protect at-risk populations from the immediate illness from acute infection and to mitigate the longer-term consequences of severe disease, including altered lung development and airway reactivity in infants and secondary infections in older adults. Active vaccination strategies have progressed into larger phase 2 and phase 3 clinical trials with efficacy endpoints for infants and older adults, and there is a robust pipeline of products in clinical and preclinical development. Passive approaches to bolster neutralizing activity in infants by direct administration of highly potent monoclonal antibody or by boosting maternal antibody transfer have also progressed to advanced clinical trials. Data obtained from these trials will allow us to further define the immune correlates of protection for each at-risk population with the hope of yielding licensed RSV vaccine products within the next several years.

References [1] Collins PL, Fearns R, Graham BS. Respiratory syncytial virus: virology, reverse genetics, and pathogenesis of disease. Curr Top Microbiol Immunol 2013;372:3 38. [2] Rima B, et al. ICTV virus taxonomy profile: pneumoviridae. J Gen Virol 2017;98(12):2912 13. [3] Collins PL, Graham BS. Viral and host factors in human respiratory syncytial virus pathogenesis. J Virol 2008;82 (5):2040 55. [4] Melero JA, Moore ML. Influence of respiratory syncytial virus strain differences on pathogenesis and immunity. Curr Top Microbiol Immunol 2013;372:59 82. [5] Tian D, et al. Structural basis of respiratory syncytial virus subtype-dependent neutralization by an antibody targeting the fusion glycoprotein. Nat Commun 2017;8(1):1877. [6] Canedo-Marroquin G, et al. Modulation of host immunity by human respiratory syncytial virus virulence factors: a synergic inhibition of both innate and adaptive immunity. Front Cell Infect Microbiol 2017;7:367.

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[7] Collins PL. Human respiratory syncytial virus. Reference module in biomedical sciences. Elsevier; 2014. [8] Boyapalle S, et al. Respiratory syncytial virus NS1 protein colocalizes with mitochondrial antiviral signaling protein MAVS following infection. PLoS One 2012;7(2): e29386. [9] Ling Z, Tran KC, Teng MN. Human respiratory syncytial virus nonstructural protein NS2 antagonizes the activation of beta interferon transcription by interacting with RIG-I. J Virol 2009;83(8):3734 42. [10] Swedan S, Musiyenko A, Barik S. Respiratory syncytial virus nonstructural proteins decrease levels of multiple members of the cellular interferon pathways. J Virol 2009;83(19):9682 93. [11] Bitko V, et al. Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NFkappaB-dependent, interferon-independent mechanism and facilitate virus growth. J Virol 2007;81(4):1786 95. [12] Arnold R, et al. Respiratory syncytial virus deficient in soluble G protein induced an increased proinflammatory response in human lung epithelial cells. Virology 2004;330(2):384 97. [13] Johnson TR, et al. Priming with secreted glycoprotein G of respiratory syncytial virus (RSV) augments interleukin-5 production and tissue eosinophilia after RSV challenge. J Virol 1998;72(4):2871 80. [14] Johnson TR, McLellan JS, Graham BS. Respiratory syncytial virus glycoprotein G interacts with DC-SIGN and L-SIGN to activate ERK1 and ERK2. J Virol 2012; 86(3):1339 47. [15] Openshaw PJ, Clarke SL, Record FM. Pulmonary eosinophilic response to respiratory syncytial virus infection in mice sensitized to the major surface glycoprotein G. Int Immunol 1992;4(4):493 500. [16] Polack FP, et al. A role for immune complexes in enhanced respiratory syncytial virus disease. J Exp Med 2002;196(6):859 65. [17] Tripp RA, et al. CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein. Nat Immunol 2001;2(8):732 8. [18] Kim YI, et al. Relating plaque morphology to respiratory syncytial virus subgroup, viral load, and disease severity in children. Pediatr Res 2015;78(4):380 8. [19] Johnson JE, et al. The histopathology of fatal untreated human respiratory syncytial virus infection. Mod Pathol 2007;20(1):108 19. [20] Knipe DM, Howley PM. Fields virology. 6th ed. Philadelphia, PA: Wolters Kluwer/Lippincott Williams & Wilkins Health; 2013. 2 volumes. [21] Gern JE, et al. Relationships among specific viral pathogens, virus-induced interleukin-8, and respiratory symptoms in infancy. Pediatr Allergy Immunol 2002;13(6):386 93.

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38. VACCINATION AGAINST RESPIRATORY SYNCYTIAL VIRUS

[22] Koponen P, et al. Preschool asthma after bronchiolitis in infancy. Eur Respir J 2012;39(1):76 80. [23] Rossi GA, Colin AA. Infantile respiratory syncytial virus and human rhinovirus infections: respective role in inception and persistence of wheezing. Eur Respir J 2015;45(3):774 89. [24] Stein RT, et al. Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet 1999;354(9178):541 5. [25] Walsh EE, Falsey AR. Respiratory syncytial virus infection in adult populations. Infect Disord Drug Targets 2012;12(2):98 102. [26] Rose EB, et al. Respiratory syncytial virus seasonality United States, 2014-2017. MMWR Morb Mortal Wkly Rep 2018;67(2):71 6. [27] Glezen WP, et al. Risk of primary infection and reinfection with respiratory syncytial virus. Am J Dis Child 1986;140(6):543 6. [28] Nair H, et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 2010;375(9725):1545 55. [29] Shi T, et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study. Lancet 2017;390(10098):946 58. [30] Hall CB. Respiratory syncytial virus in young children. Lancet 2010;375(9725):1500 2. [31] Hall CB, et al. Respiratory syncytial virus-associated hospitalizations among children less than 24 months of age. Pediatrics 2013;132(2):e341 8. [32] Stockman LJ, et al. Respiratory syncytial virusassociated hospitalizations among infants and young children in the United States, 1997-2006. Pediatr Infect Dis J 2012;31(1):5 9. [33] Hall CB, et al. The burden of respiratory syncytial virus infection in young children. N Engl J Med 2009;360(6): 588 98. [34] Falsey AR, et al. Respiratory syncytial virus infection in elderly and high-risk adults. N Engl J Med 2005;352(17): 1749 59. [35] Falsey AR, Walsh EE. Respiratory syncytial virus infection in adults. Clin Microbiol Rev 2000;13(3):371 84. [36] Thompson WW, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 2003;289(2):179 86. [37] Falsey AR, et al. Respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness. J Infect Dis 2014;209 (12):1873 81. [38] Drysdale SB, et al. RSV vaccine use--the missing data. Expert Rev Vaccines 2016;15(2):149 52. [39] Kulkarni PS, et al. Establishing correlates of protection for vaccine development: considerations for the

[40]

[41] [42]

[43]

[44] [45] [46]

[47]

[48]

[49]

[50]

[51] [52]

[53]

[54]

[55]

[56]

respiratory syncytial virus vaccine field. Viral Immunol 2018;. Piedra PA. Clinical experience with respiratory syncytial virus vaccines. Pediatr Infect Dis J 2003;22(Suppl. 2): S94 9. Falsey AR. Respiratory syncytial virus infection in adults. Semin Respir Crit Care Med 2007;28(2):171 81. Habibi MS, et al. Impaired antibody-mediated protection and defective IgA B-cell memory in experimental infection of adults with respiratory syncytial virus. Am J Respir Crit Care Med 2015;191(9):1040 9. Vissers M, et al. Mucosal IgG levels correlate better with respiratory syncytial virus load and inflammation than plasma IgG levels. Clin Vaccine Immunol 2015;23(3): 243 5. Twigg 3rd HL. Humoral immune defense (antibodies): recent advances. Proc Am Thorac Soc 2005;2(5):417 21. Allie SR, Randall TD. Pulmonary immunity to viruses. Clin Sci (Lond) 2017;131(14):1737 62. O’Brien KL, et al. Efficacy of motavizumab for the prevention of respiratory syncytial virus disease in healthy Native American infants: a phase 3 randomised double-blind placebo-controlled trial. Lancet Infect Dis 2015;15(12):1398 408. Liljeroos L, et al. Architecture of respiratory syncytial virus revealed by electron cryotomography. Proc Natl Acad Sci USA 2013;110(27):11133 8. McLellan JS, et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 2013;342(6158):592 8. McLellan JS, et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 2013;340(6136):1113 17. McLellan JS, et al. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. J Virol 2011;85(15):7788 96. Graham BS. Vaccine development for respiratory syncytial virus. Curr Opin Virol 2017;23:107 12. Ngwuta JO, et al. Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera. Sci Transl Med 2015;7(309):309ra162. Gilman MS, et al. Rapid profiling of RSV antibody repertoires from the memory B cells of naturally infected adult donors. Sci Immunol 2016;1(6). Corti D, et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 2013;501(7467):439 43. Wen X, et al. Structural basis for antibody crossneutralization of respiratory syncytial virus and human metapneumovirus. Nat Microbiol 2017;2:16272. Goodwin E, et al. Infants Infected with Respiratory Syncytial Virus Generate Potent Neutralizing Antibodies that Lack Somatic Hypermutation. Immunity 2018;48(2): 339 49 e5.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

675

REFERENCES

[57] Capella C, et al. Prefusion F, postfusion F, G antibodies, and disease severity in infants and young children with acute respiratory syncytial virus infection. J Infect Dis 2017;216(11):1398 406. [58] Falloon J, et al. An adjuvanted, postfusion f proteinbased vaccine did not prevent respiratory syncytial virus illness in older adults. J Infect Dis 2017;216(11):1362 70. [59] Villafana T, et al. Passive and active immunization against respiratory syncytial virus for the young and old. Expert Rev Vaccines 2017;16(7):1 13. [60] Chanock RM, et al. Respiratory syncytial virus. I. Virus recovery and other observations during 1960 outbreak of bronchiolitis, pneumonia, and minor respiratory diseases in children. JAMA 1961;176:647 53. [61] Johnson KM, et al. Respiratory syncytial virus. IV. Correlation of virus shedding, serologic response, and illness in adult volunteers. JAMA 1961;176:663 7. [62] DeVincenzo JP, et al. Viral load drives disease in humans experimentally infected with respiratory syncytial virus. Am J Respir Crit Care Med 2010;182(10):1305 14. [63] DeVincenzo JP, El Saleeby CM, Bush AJ. Respiratory syncytial virus load predicts disease severity in previously healthy infants. J Infect Dis 2005;191(11): 1861 8. [64] Falsey AR, Singh HK, Walsh EE. Serum antibody decay in adults following natural respiratory syncytial virus infection. J Med Virol 2006;78(11):1493 7. [65] Sande CJ, et al. Kinetics of the neutralizing antibody response to respiratory syncytial virus infections in a birth cohort. J Med Virol 2013;85(11):2020 5. [66] Graham BS, et al. Role of T lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice. J Clin Invest 1991;88(3):1026 33. [67] Hall CB, et al. Respiratory syncytial viral infection in children with compromised immune function. N Engl J Med 1986;315(2):77 81. [68] Heidema J, et al. CD81 T cell responses in bronchoalveolar lavage fluid and peripheral blood mononuclear cells of infants with severe primary respiratory syncytial virus infections. J Immunol 2007;179(12): 8410 17. [69] Jozwik A, et al. RSV-specific airway resident memory CD81 T cells and differential disease severity after experimental human infection. Nat Commun 2015;6: 10224. [70] Esposito S, Pietro GD. Respiratory syncytial virus vaccines: an update on those in the immediate pipeline. Future Microbiol 2016;11:1479 90. [71] Karron RA, Zar HJ. Determining the outcomes of interventions to prevent respiratory syncytial virus disease in children: what to measure? Lancet Respir Med 2018; 6(1):65 74. [72] Modjarrad K, et al. WHO consultation on Respiratory Syncytial Virus Vaccine Development Report from a

[73]

[74]

[75] [76]

[77]

[78]

[79] [80]

[81] [82]

[83]

[84]

[85]

[86]

[87]

[88]

World Health Organization Meeting held on 23-24 March 2015. Vaccine 2016;34(2):190 7. Saso A, Kampmann B. Vaccination against respiratory syncytial virus in pregnancy: a suitable tool to combat global infant morbidity and mortality? Lancet Infect Dis 2016;16(8):e153 63. Fouda GG, et al. The Impact of IgG transplacental transfer on early life immunity. Immunohorizons 2018;2(1):14 25. Kachikis A, Eckert LO, Englund J. Who’s the target: mother or baby? Viral Immunol 2018;. Munoz FM. Respiratory syncytial virus in infants: is maternal vaccination a realistic strategy? Curr Opin Infect Dis 2015;28(3):221 4. Nyiro JU, et al. Defining the vaccination window for respiratory syncytial virus (RSV) using age-seroprevalence data for children in Kilifi, Kenya. PLoS One 2017;12(5): e0177803. Ochola R, et al. The level and duration of RSV-specific maternal IgG in infants in Kilifi Kenya. PLoS One 2009; 4(12):e8088. Lambert L, Culley FJ. Innate immunity to respiratory infection in early life. Front Immunol 2017;8:1570. Ruckwardt TJ, Morabito KM, Graham BS. Determinants of early life immune responses to RSV infection. Curr Opin Virol 2016;16:151 7. Saso A, Kampmann B. Vaccine responses in newborns. Semin Immunopathol 2017;39(6):627 42. Kim HW, et al. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol 1969;89(4): 422 34. Connors M, et al. Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4 (IL-4) and IL-10. J Virol 1994;68(8):5321 5. Graham BS, et al. Priming immunization determines T helper cytokine mRNA expression patterns in lungs of mice challenged with respiratory syncytial virus. J Immunol 1993;151(4):2032 40. Prince GA, et al. Vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with Lot 100 or a newly prepared reference vaccine. J Gen Virol 2001;82(Pt 12):2881 8. Acosta PL, Caballero MT, Polack FP. Brief history and characterization of enhanced respiratory syncytial virus disease. Clin Vaccine Immunol 2015;23(3): 189 95. Killikelly AM, Kanekiyo M, Graham BS. Pre-fusion F is absent on the surface of formalin-inactivated respiratory syncytial virus. Sci Rep 2016;6:34108. Karron RA, Buchholz UJ, Collins PL. Live-attenuated respiratory syncytial virus vaccines. Curr Top Microbiol Immunol 2013;372:259 84.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

676

38. VACCINATION AGAINST RESPIRATORY SYNCYTIAL VIRUS

[89] Rostad CA, et al. Enhancing the thermostability and immunogenicity of a respiratory syncytial virus (RSV) live-attenuated vaccine by incorporating unique RSV line19f protein residues. J Virol 2018;92 (6). [90] Stobart CC, et al. A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation. Nat Commun 2016;7:13916. [91] Liang B, et al. Improved prefusion stability, optimized codon usage, and augmented virion packaging enhance the immunogenicity of respiratory syncytial virus fusion protein in a vectored-vaccine candidate. J Virol 2017;91(15). [92] Liu X, et al. Attenuated human parainfluenza virus type 1 expressing the respiratory syncytial virus (RSV) fusion (F) glycoprotein from an added gene: effects of prefusion stabilization and packaging of RSV F. J Virol 2017;91(22). [93] Gonzalez IM, et al. Evaluation of the live attenuated cpts 248/404 RSV vaccine in combination with a subunit RSV vaccine (PFP-2) in healthy young and older adults. Vaccine 2000;18(17):1763 72. [94] Domachowske JB, et al. Safety, tolerability, and pharmacokinetics of MEDI8897, an extended half-life

[95]

[96]

[97]

[98]

[99]

[100]

single-dose respiratory syncytial virus prefusion Ftargeting monoclonal antibody administered as a single dose to healthy preterm infants. Pediatr Infect Dis J 2018;. Zhu Q, et al. A highly potent extended half-life antibody as a potential RSV vaccine surrogate for all infants. Sci Transl Med 2017;9(388). Munywoki PK, et al. The source of respiratory syncytial virus infection in infants: a household cohort study in rural Kenya. J Infect Dis 2014;209(11): 1685 92. Poletti P, et al. Evaluating vaccination strategies for reducing infant respiratory syncytial virus infection in low-income settings. BMC Med 2015;13:49. Yamin D, et al. Vaccination strategies against respiratory syncytial virus. Proc Natl Acad Sci USA 2016;113 (46):13239 44. Sharma G, Goodwin J. Effect of aging on respiratory system physiology and immunology. Clin Interv Aging 2006;1(3):253 60. Malloy AM, Falsey AR, Ruckwardt TJ. Consequences of immature and senescent immune responses for infection with respiratory syncytial virus. Curr Top Microbiol Immunol 2013;372:211 31.

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Nasal Influenza Vaccines Hideki Hasegawa Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan

I. INTRODUCTION Influenza is the result of an influenza virus infection of the upper respiratory system. Epidemics and pandemics of influenza are caused by the frequent mutation in the virus genome and occasional exchange of genetic segments between virus strains. Vaccines for influenza have already been developed and used worldwide. However, most vaccines are now administered intramuscularly or subcutaneously, and little is known about the role of local mucosal responses that lead prevention of infection. Local mucosal immunity, including immunoglobulin A (IgA) responses, are important in the prevention of infection. IgA responses in mice and humans are also involved in cross-protection. Studies designed to improve vaccine efficacy by changing the route of vaccine administration have been conducted. In contrast to the current injectable vaccines, nasal vaccines are unique in that its administration route mimics natural influenza virus infection. Nasal vaccines are expected to confer protection against a broad spectrum of influenza virus strains in vaccine recipients by inducing the mucosal immune

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00039-0

response represented by secretory IgA (SIgA) antibodies. In this chapter, the history of nasal influenza vaccine research and development, as well as clues to overcome remaining issues that impede more efficacious and next-generation influenza vaccine development, will be introduced and discussed.

II. HUMORAL IMMUNE RESPONSES TO INFLUENZA VIRUS INFECTION The influenza viruses target the epithelial cells of the upper respiratory tract. The respiratory tract mucous membrane is the battlefront in the defense against influenza virus infection. The upper respiratory epithelium is covered by mucus, viscous liquid, and mucous membranes maintained in a moist state. Immunity, which prevents the invasion of the pathogens to the mucous membrane, exerts its impact in a viscous liquid covering the mucous membrane. As an acquired immunity, SIgA antibody distributed on a mucous membrane binds to the virus before the infection and inhibits the infection to the epithelial cells [1]. Moreover, SIgA

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antibodies on the mucosal surface abolish the infectivity of the virus [2]. Unlike serum IgG antibodies, SIgA antibody does not activate complement and is not considered to be inflammatory [3]. It would be advantageous to elicit influenza virus-specific SIgA antibodies subsequent to vaccination to protect against viral infection. In addition to the prevention of infection, SIgA antibodies are cross-protective against variant influenza viruses. Crossprotection is beneficial for prevention of infection by drifted influenza viruses. Influenza viruses are known to undergo changes in their antigenicity every year due to selective pressures causing an antigenic drift or mutation(s). The impact of vaccination is expected to be greatest when the antigenicity of the vaccine is identical or similar to the circulating virus strain; however, it is less effective when there is a mismatch, though SIgA antibodies induced in the upper respiratory tract can cross-protect against drifted viruses. The current injectable split vaccine is intended to reduce disease severity, not to prevent infection. The injectable influenza vaccine induces virusspecific serum IgG antibodies that can neutralize influenza viruses, but these IgG antibodies are less potent on mucosal surfaces, and cannot induce virus-specific SIgA antibodies. Therefore, a vaccine capable of preventing infection and providing cross-protection is desired.

III. DEVELOPMENT OF THE NASAL INFLUENZA VACCINES To have the greatest impact to minimize influenza virus epidemics, the ideal influenza vaccine is expected to induce mucosal immunity represented by SIgA antibodies in the upper respiratory tract. The induction of optimal mucosal immunity may require administration of the vaccine via a mucosal route, such as intranasal administration. In addition to the

vaccine antigen, a suitable mucosal adjuvant is needed to activate antigen-presenting cells. Animal experiments using mice and nonhuman primates revealed that vaccines administered via the nasal route resulted in the secretion of vaccine-specific IgA antibodies, which is not induced by an injectable vaccine. These SIgA antibodies work just before the virus attaches to the mucosal surface epithelial cells to prevent infection; moreover, they work widely to provide cross-protection against variant viruses [4,5]. So far, two methods have been applied to a human in clinical trials to induce mucosal immunology by nasal influenza vaccine. One is live attenuated influenza vaccines (LAIVs); the other is intranasal inactivated influenza vaccines (IIIVs)

A. Live Attenuated Influenza Vaccines It has been known that the humoral immunity induced by the natural infection is more protective and shows better cross-protection compared to immunity induced with injectable vaccines. The idea of using live attenuated influenza viruses as a vaccine candidate started in the very early in influenza vaccine development. In 1937, Smorodintseff et al. infected human volunteers with a live influenza virus that had been serially passaged and maintained in mice and ferrets for attenuation. As a result, a majority of the infected individuals were protected from the virus infection, and the remaining individuals developed only mild symptoms. The results indicated that the lessvirulent live virus attenuated by serial passages in laboratory animals can be used as a means of prophylaxis in humans [6]. The virus attenuating method by growth at low temperatures was introduced in the 1960s [7]. These attenuated viruses, called “coldadapted influenza viruses,” can replicate only at low temperature and reduce the clinical

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III. DEVELOPMENT OF THE NASAL INFLUENZA VACCINES

symptoms in humans. These cold-adapted influenza viruses were licensed in the United States in 2003 and have been used to create LAIVs [8,9]. The vaccine strains of coldadapted influenza viruses are created by genetic reassortment by the classical method or by reverse genetics to change the surface glycoproteins (hemagglutinin (HA) and neuraminidase (NA)) to circulating viral strains [8,10,11]. Field studies have shown that LAIVs induce high levels of SIgA antibodies, though they induce relatively lower levels of serum hemagglutination inhibition (HI) antibodies in comparison to parenteral vaccines [12,13]. It has also been reported that LAIVs showed broadspectrum immunity against influenza viruses [14]. However, studies have also shown that induction of systemic and local immune responses cannot necessarily be obtained in people of all ages. The systemic and mucosal immune responses to LAIVs are greatest in children, moderate in adults, and low in elderly individuals for LAIVs replicate efficiently in individuals who do not have preexisting immunity against influenza viruses [13]. FluMist, which was targeted at individuals aged 5 49 years, was approved by the US Food and Drug Administration as a trivalent LAIV in 2003. In 2007, the target was expanded to include children between 2 and 4 years of age [15].

B. Intranasal Inactivated Influenza Vaccines IIIVs has also been studied for a long time. The immune responses in humans by intranasal administration of inactivated viruses were first examined in the 1940s. At that time, little was known about the mucosal antibodies, so only the serological response was measured. Therefore, it was concluded that subcutaneous vaccination could induce higher antibody responses compared to intranasal administration. The mucosal administration was regarded

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as having no advantage over subcutaneous injection [16,17]. Clinical studies to examine the effects of intranasal inactivated vaccines were conducted to determine whether it can induce protective immunity by stimulating mucosal antibodies. The intranasal administration of split or subunit vaccines was determined not to be as effective as had been expected. It required a large amount of antigen and repeated multiple administrations to induce mucosal immunity. To circumvent multiple vaccinations or elevated doses, the inclusion of an adjuvant may prove beneficial to enhance IIV effectiveness. The first IIIV, Nasalflu (Berna Biotech Ltd., Switzerland), was licensed in 2000 in Switzerland after large-scale clinical trials [18,19]. Nasalflu contained Escherichia coli heatlabile toxin as a mucosal adjuvant [20]. This vaccine is a virosomal subunit vaccine, prepared by incorporating hemagglutinin and neuraminidase in the membranes of liposomes. Shortly after approval, there were several reported cases of Bell’s palsy, a type of facial paralysis among Nasalflu recipients. A matched case control study suggested a strong epidemiological association between Nasalflu administration and Bell’s palsy cases [21]. Thus, this vaccine is no longer in clinical use. The failure of Nasalflu elucidated the necessity of the development of a safe and effective mucosal adjuvants for IIIVs, and various candidates have been tested in animal models [22 30]. However, to date, there is no licensed mucosal adjuvant for safe and effective intranasal vaccination. Methods other than intranasal split and subunit vaccines, which require mucosal adjuvants, have been tried. Inactivated whole virus vaccines have also been tested for intranasal administration. Clinical studies have shown that immunization with trivalent inactivated whole virus vaccine via the intranasal route once or twice enhanced production of both mucosal SIgA and serum IgG antibodies in

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humans without the use of additional mucosal adjuvants [31 33]. Safety and immunogenicity of intranasal whole inactivated virus vaccines were also observed in the elderly population [31]. The immunogenicity of inactivated whole virus vaccine is not affected by the host’s preexisting immunity against influenza viruses. Animal studies showed that the intranasal inactivated whole virus vaccine could induce a broad spectrum of immune responses against influenza viruses [34,35]. In this case, viral genomic RNA may work as a built-in adjuvant that is recognized by toll-like receptor 7 (TLR-7) [36 38]. It has also been revealed that intranasal administration of whole inactivated viruses could induce high levels of specific serum IgG antibodies as well as mucosal SIgA antibodies. In a human clinical study, two intranasal doses of whole inactivated virus vaccine could induce sufficient serum HI antibody responses that met the European Medicines Agency’s criteria for the licensure of influenza vaccines [39]. SIgA antibodies play a central role in the prevention of influenza virus infection in the upper respiratory systems. Analysis of mucosal antibodies in humans in response to inactivated whole virus influenza vaccine revealed the importance of the structure of SIgA antibodies in its function [40]. It has been known that SIgA exists mostly in the form of dimers, in which two monomeric IgA molecules are covalently linked by a J chain. However, it has been also known that there are high-molecular-weight

multimeric forms of SIgA, such as trimers and tetramers. The existence of this multimeric SIgA was reported in a paper published as early as 1965; however, the detailed structure and functions of this multimeric SIgA have long remained unclear [41]. Suzuki et al. purified SIgA antibodies from human nasal wash samples collected from the recipients of intranasal inactivated whole virus vaccine recipients and conducted direct observation by atomic force microscopy of high-molecular-weight compounds within purified SIgA. As a result, it was visually confirmed that SIgAs are present at the human nasal mucosa in various quaternary structures, including the form of multimers (Fig. 39.1). Further, in the study, the relationship between the various quaternary structures of human SIgA and its virus-neutralizing potency was examined. Multimeric SIgAs exhibited higher virus-neutralizing activity compared to monomers and dimers against both homologous and heterologous virus strains to the vaccine virus strain [42]. Taken together, multimeric SIgAs exhibit higher virus neutralization activity against viruses, and the cross-protection and reactivity against variant influenza virus strains observed in IIIV-administered humans and mice may in part owe to multimeric SIgA induction. Human studies have contributed to obtaining a basic understanding of the characteristics of SIgA that are related to the mechanism of action of FIGURE 39.1 Atomic force microscopy (AFM) revealed the quaternary molecular structures of nasal IgA. AFM images of serum monomeric IgA, nasal dimeric IgA, and nasal tetrameric IgA [42].

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REFERENCES

IIIVs. IIIVs are expected to be licensed in the near future to take advantage of mucosal immunity against influenza virus infection.

References [1] Renegar KB, Small Jr. PA, Boykins LG, Wright PF. Role of IgA versus IgG in the control of influenza viral infection in the murine respiratory tract. J Immunol 2004;173:1978 86. [2] Brandtzaeg P. Induction of secretory immunity and memory at mucosal surfaces. Vaccine 2007;25:5467 84. [3] Yel L. Selective IgA deficiency. J Clin Immunol 2010;30: 10 16. [4] Ichinohe T, Kawaguchi A, Tamura S, Takahashi H, Sawa H, Ninomiya A, et al. Intranasal immunization with H5N1 vaccine plus Poly I:Poly C12U, a Toll-like receptor agonist, protects mice against homologous and heterologous virus challenge. Microbes Infect 2007;9 (11):1333 40. [5] Ichinohe T, Tamura S, Kawaguchi A, Ninomiya A, Imai M, Itamura S, et al. Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine. J Infect Dis 2007;196(9):1313 20. [6] Smorodintseff AAT, Drobyshevskaya MD, Korovin AA, Osetroff AI. Investigation on volunteers infected with the influenza virus. Am J Med Sci 1937;194: 159 70. [7] Maassab HF. Biologic and immunologic characteristics of cold-adapted influenza virus. J Immunol 1969;102(3): 728 32. [8] Maassab HF, Bryant ML. The development of live attenuated cold-adapted influenza virus vaccine for humans. Rev Med Virol 1999;9(4):237 44. [9] Ambrose CS, Levin MJ, Belshe RB. The relative efficacy of trivalent live attenuated and inactivated influenza vaccines in children and adults. Influenza Other Respir Viruses 2011;5(2):67 75. [10] Aleksandrova GI. Use of the genetic recombination method for obtaining vaccinal strains of the influenza virus. Vopr Virusol 1977;4:387 95. [11] Kendal AP. Cold-adapted live attenuated influenza vaccines developed in Russia: can they contribute to meeting the needs for influenza control in other countries? Eur J Epidemiol 1997;13(5):591 609. [12] Clements ML, Murphy BR. Development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza A virus vaccine. J Clin Microbiol 1986;23(1):66 72.

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[13] Beyer WE, Palache AM, de Jong JC, Osterhaus AD. Cold-adapted live influenza vaccine versus inactivated vaccine: systemic vaccine reactions, local and systemic antibody response, and vaccine efficacy. A metaanalysis. Vaccine 2002;20(9 10):1340 53. [14] Hoft DF, Babusis E, Worku S, et al. Live and inactivated influenza vaccines induce similar humoral responses, but only live vaccines induce diverse T-cell responses in young children. J Infect Dis 2011;204(6):845 53. [15] Centers for Disease Control and Prevention (CDC). Expansion of use of live attenuated influenza vaccine (FluMists) to children aged 2-4 years and other FluMist changes for the 2007-08 influenza season. MMWR 2007;56(46):1217 19. [16] Henle W, Henle G, et al. Experimental exposure of human subjects to viruses of influenza. J Immunol 1946;52:145 65. [17] Quilligan Jr. JJ, Francis Jr. T. Serological response to intranasal administration of inactive influenza virus in children. J Clin Invest 1947;26(6):1079 87. [18] de B, Zanasi A, Ragusa S, Gluck R, Herzog C. An open-label comparison of the immunogenicity and tolerability of intranasal and intramuscular formulations of virosomal influenza vaccine in healthy adults. Clin Ther 2002;24(1):100 11. [19] Gluck U, Gebbers JO, Gluck R. Phase 1 evaluation of intranasal virosomal influenza vaccine with and without Escherichia coli heat-labile toxin in adult volunteers. J Virol 1999;73(9):7780 6. [20] Michetti P, Kreiss C, Kotloff KL, et al. Oral immunization with urease and Escherichia coli heat-labile enterotoxin is safe and immunogenic in Helicobacter pyloriinfected adults. Gastroenterology 1999;116(4):804 12. [21] Mutsch M, Zhou W, Rhodes P, et al. Use of the inactivated intranasal influenza vaccine and the risk of Bell’s palsy in Switzerland. N Engl J Med 2004;350(9):896 903. [22] Tamura S, Ito Y, Asanuma H, et al. Cross-protection against influenza virus infection afforded by trivalent inactivated vaccines inoculated intranasally with cholera toxin B subunit. J Immunol 1992;149(3):981 8. [23] Tamura S, Samegai Y, Kurata H, Nagamine T, Aizawa C, Kurata T. Protection against influenza virus infection by vaccine inoculated intranasally with cholera toxin B subunit. Vaccine 1988;6(5):409 13. [24] Tamura S, Yamanaka A, Shimohara M, et al. Synergistic action of cholera toxin B subunit (and Escherichia coli heat-labile toxin B subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine. Vaccine 1994;12(5):419 26. [25] Ichinohe T, Watanabe I, Ito S, et al. Synthetic doublestranded RNA poly(I:C) combined with mucosal vaccine protects against influenza virus infection. J Virol 2005;79(5):2910 19.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

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[26] Ichinohe T, Ainai A, Ami Y, et al. Intranasal administration of adjuvant-combined vaccine protects monkeys from challenge with the highly pathogenic influenza A H5N1 virus. J Med Virol 2010;82(10):1754 61. [27] Ichinohe T, Ainai A, Nakamura T, et al. Induction of cross-protective immunity against influenza A virus H5N1 by an intranasal vaccine with extracts of mushroom mycelia. J Med Virol 2010;82(1):128 37. [28] Ichinohe T, Watanabe I, Tao E, et al. Protection against influenza virus infection by intranasal vaccine with surf clam microparticles (SMP) as an adjuvant. J Med Virol 2006;78(7):954 63. [29] Hasegawa H, Ichinohe T, Strong P, et al. Protection against influenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant. J Med Virol 2005;75(1):130 6. [30] Ainai A, Ichinohe T, Tamura S, et al. Zymosan enhances the mucosal adjuvant activity of poly(I:C) in a nasal influenza vaccine. J Med Virol 2010;82(3):476 84. [31] Muszkat M, Yehuda AB, Schein MH, et al. Local and systemic immune response in community-dwelling elderly after intranasal or intramuscular immunization with inactivated influenza vaccine. J Med Virol 2000; 61(1):100 6. [32] Greenbaum E, Furst A, Kiderman A, et al. Mucosal [SIgA] and serum [IgG] immunologic responses in the community after a single intra-nasal immunization with a new inactivated trivalent influenza vaccine. Vaccine 2002;20(7 8):1232 9. [33] Greenbaum E, Engelhard D, Levy R, Schlezinger M, Morag A, Zakay-Rones Z. Mucosal (SIgA) and serum (IgG) immunologic responses in young adults following intranasal administration of one or two doses of inactivated, trivalent anti-influenza vaccine. Vaccine 2004;22(20):2566 77. [34] Takada A, Matsushita S, Ninomiya A, Kawaoka Y, Kida H. Intranasal immunization with formalin-inactivated

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

virus vaccine induces a broad spectrum of heterosubtypic immunity against influenza A virus infection in mice. Vaccine 2003;21(23):3212 18. Tamura S, Hasegawa H, Kurata T. Estimation of the effective doses of nasal-inactivated influenza vaccine in humans from mouse-model experiments. Jpn J Infect Dis 2010;63(1):8 15. Lund JM, Alexopoulou L, Sato A, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA 2004;101(15):5598 603. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7mediated recognition of single-stranded RNA. Science 2004;303(5663):1529 31. Kang SM, Guo L, Yao Q, Skountzou I, Compans RW. Intranasal immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens. J Virol 2004;78(18):9624 32. Ainai A, Tamura S, Suzuki T, et al. Intranasal vaccination with an inactivated whole influenza virus vaccine induces strong antibody responses in serum and nasal mucus of healthy adults. Human Vaccines Immunother 2013;9(9):1962 70. Suzuki T, Ainai A, Hasegawa H. Functional and structural characteristics of secretory IgA antibodies elicited by mucosal vaccines against influenza virus. Vaccine 2017;35(39):5297 302. Tomasi Jr. TB, Tan EB, Solomon A, Prendergast RA. Characteristics of an Immune System Common to Certain External Secretions. The Journal of experimental medicine 1965;121:101 24. Suzuki T, Kawaguchi A, Ainai A, et al. Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus. Proc Natl Acad Sci USA 2015;112(25):7809 14.

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The Role of Innate Immunity in Regulating Rotavirus Replication, Pathogenesis, and Host Range Restriction and the Implications for Live Rotaviral Vaccine Development Adrish Sen, Siyuan Ding and Harry B. Greenberg Departments of Medicine and Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, United States

I. INTRODUCTION Rotaviruses (RVs) remain one of the two most important viral causes of gastroenteritis despite the availability of several safe and effective live attenuated vaccines [1,2]. Rotavirus infection has its biggest health impact on children under the age of 3 years, in whom it still accounts for approximately 200,000 deaths annually, almost entirely in less-developed countries [2]. RVs can infect many cells of the nonimmune host, but the overwhelming bulk of viral replication occurs in the mature villus tip cells of the small intestine [3]. In this review, we focus on the regulation of rotavirus replication by the host innate immune system, the host-restricted

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00041-9

nature of the innate immune response to specific rotavirus strains, and the practical utility of these host range barriers in the development of safe and effective RV vaccines.

II. HOST INNATE IMMUNE SENSORS AND ROTAVIRUS INFECTION A. Cytoplasmic Sensors Infection with RV results in the immediate activation of a conserved cellular innate immune signaling pathway that involves multiple pattern recognition receptors (PRRs) recognizing

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discrete RV-encoded pathogen-associated molecular patterns (PAMPs). A primary purpose of this diverse host-signaling system is to induce different types of interferons (IFNs) and a set of virus-induced stress genes (vISGs) through two principal transcriptional factors: nuclear factor-κB (NF-κB) and IFN regulatory factor 3 (IRF3) [4,5]. The induced IFNs and vISGs then function to restrict RV replication and pathogen-induced cell injury [6]. Of note, RVs, like virtually all other viral pathogens, have evolved a set of countermeasures to inhibit the host innate immune response, and these countermeasures are most pronounced during homologous RV infection (RV infection with a strain routinely isolated from that specific host species) [7]. Interestingly, RV strains that differ in their ability to regulate the secretion of IFNs similarly induce this early recognition pathway, as indicated by the transcriptional upregulation of IFNs and several vISGs [8]. Based on the collective evidence, initial RV transcription engages the two related PRRs RIG-I and MDA-5 (members of the family of RIG-I-like receptors, or RLRs) [8,9], which then trigger activation of the mitochondrial antiviral-signaling protein (MAVS). These receptors are likely to be stimulated by early RV transcriptional by-products such as exposed 50 -phosphate groups, incompletely methylated 50 -cap structures, and local dsRNA structures such as panhandle loops in viral transcripts [10]. In addition to inducing the secretion of different IFNs, RLR responses to RV are likely to orchestrate other host responses. Rotavirus activation of MDA-5 results in apoptosis, which occurs mostly in the pancreas of RV-infected mice, indicating that such PRRdependent consequences can occur in a cell or organ type-specific fashion [11] (Chapter 6: Innate Immunity at Mucosal Surfaces). In addition to RIG-I- and MDA-5-dependent host responses to RV RNA, other sensors are also recruited by the innate immune machinery to trigger early anti-RV responses. Among these is a third member of the RLR family: LGP2, which seems to exert a proviral effect on RV

replication [9] and whose activation during RV infection may represent a viral strategy to dampen this pathway. Yet another player in the innate recognition of RV is the dsRNAdependent protein kinase PKR, which is essential for RV-infected cells to secrete IFN [8]. The molecular basis for PKR’s role during RV infection is not well understood, but given the importance of PKR in antiviral signaling in general and its inhibition by a majority of viruses, PKR is likely to be important for RV pathogenicity.

B. Membrane-Associated Sensors Distinct from the cytosolic receptors discussed above, RV recognition also involves the toll-like receptors (TLRs), a class of viral receptors that function in the context of cellular membranes, including surface and endosomal membranous components. This aspect of innate RV recognition possibly reflects the RV entry process that exploits endosomal vesicle transport to gain access into host cells. So far, TLR3, TLR7, TLR2, and TLR5 have all been implicated as potential players in the innate RV detection cascade [1217]. The ability of TLR3 to recognize and regulate RV and thus perpetuate an antiviral effect has been tied to TLR3’s agedependent expression in the intestine [18]. Since RV typically causes severe disease and replicates in the intestine of infants and young children (in many mammalian species), coincidental with lower levels of TLR3 expression in infants [18], one possible implication is that age-restricted RV intestinal replication is partly due to enhanced TLR3 signaling with age in this mucosal compartment. Other TLRs play specialized roles in discrete types of cells during RV infection. The RVencoded enterotoxin NSP4 may function as a PAMP and in macrophages triggers inflammatory cytokine secretion by a TLR2-dependent pathway [14]. During RV infection in human enteroid cultures [19] and in different species of mammals [2025], different types of IFNs are

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secreted, and as will be discuss below, antiviral actions of these IFNs are actively countered in a host-range-specific manner by pathogenic RVs. Of the IFNs, type I IFN is mostly expressed in the intestinal hematopoietic cell compartment rather than in the epithelium where RV primarily replicates [26]. Studies to date have implicated TLRdependent signaling in dendritic cells in the type I IFN secretion process during RV infection [16,27,28]. Infection of plasmacytoid dendritic cells with RV, which are a major source of type I IFN secretion during viral infections, leads to endosome-dependent (and possibly TLR7-mediated) type I IFN secretion that is triggered by viral genomic dsRNA (or, potentially, a RV structural protein) [13,16,27,28]. A central role for TLR-dependent defense against RV is also indicated by the finding that the absence of MyD88, an essential convergent adaptor in signaling from the different TLRs, results in increased RV infectivity, severity of diarrhea, and impaired humoral immunity [12]. In addition, RV is susceptible to the antiviral effects of TLR5, since activation of this receptor by bacterial flagellin prevents or cures RV infection by a process that involves the secretion of IL 22 [17,29,30].

C. Other Sensors Inflammasomes are cytosolic multiprotein complexes that remain quiescent at resting state [31]. Upon activation, apoptosis-associated speck-like protein containing CARD protein, named ASC (encoded by PYCARD) and caspase-1 (encoded by CASP1), oligomerize and mediate the proteolytic processing of proinflammatory cytokines such as pro-IL-1β and pro-IL-18 and the pore-forming protein gasdermin D, ultimately leading to a lytic form of cell death known as pyroptosis [32]. These events not only contribute to the host defense against bacterial and other microbial infections, but also regulate the homeostasis of the immune system and the development of various inflammatory diseases and cancer [33].

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Although it is known that AIM2 and IFI16 inflammasomes recognize DNA viruses and that NLRP3 inflammasome responds to general stress and breach of plasma/endosomal membrane integrity [34,35], how inflammasomes control RNA virus replication is less well understood. In addition, whether noncanonical inflammasomes operate in cell types other than myeloid cells is largely unknown. Recently, we found that oral infection of suckling mice with murine RV-induced robust activation of CASP1 in the small intestinal tissue, indicating a potential role for inflammasomes in RV pathogenesis [36]. Of note, in contrast to other NOD-like receptors, including NLRP3, NLRP6, NLRC4, and NAIPs, targeted deletion of NLRP9b in intestinal epithelial cells (IECs) of suckling mice led to an increase in diarrhea, RV shedding in the feces, and intraintestinal viral replication compared to wild-type pups, highlighting a crucial role of NLRP9b in controlling RV replication. Mechanistically, we found that during RV infection, DExH-box helicase 9 (DHX9) binds to viral RNA PAMP and interacts with NLRP9 to activate the downstream signaling pathway (Fig. 40.1). Furthermore, primary mouse intestinal enteroids generated from DHX9- or NLRP9deficient mice produced less IL-18 and underwent less pyroptosis compared to wildtype enteroids upon RV infection, confirming a role for DHX9 in the activation of the NLRP9b inflammasome during RV infection [36]. Identification of the DHX9-NLRP9b-ASCCASP1 cascade as a novel RV-sensing pathway opened up new research directions. Are there other inflammasome sensors of RV or other enteric viruses? How do different RNAbinding proteins (DHX9, RIG-I, MDA-5, etc.) coordinate in the cytoplasm? What is the physiological relevance of theses sensors in the human intestine? Addressing these fundamental issues will provide new insights into the biological functions of host innate immune recognition during acute RV infection and, more generally, in overall human enteric health

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FIGURE 40.1 Rotavirus sensing by the NLRP9b inflammasome. Within the IECs, RV RNA is sensed by DHX9 and the NLRP9 inflammasome and activates the downstream ASC-CASP1 complex, which activates IL-18 secretion, GSDMD cleavage, and pyroptosis.

and disease. In addition, answering these basic questions is likely to inform more practical considerations, such as the development of better therapeutics and preventive strategies for enteric infectious diseases.

III. HOST INNATE RESPONSES TO ROTAVIRUS AND THEIR EFFECTS ON VIRAL REPLICATION The concept of host range restriction (HRR) is central to many aspects of RV replication and disease, including the development of several

of the currently available safe and effective RV vaccines [37]. RVs are distinguished by being highly pathogenic and infectious to their homologous host species (i.e., the species of host normally infected by the particular RV strain and the species in which that RV strain spreads efficiently) [38]. RVs are also subject to very severe species-specific restriction of replication and transmission in heterologous host species [39]. These fundamental properties of RVs are not only of great importance for viral pathogenicity. They also form the basis for several licensed live attenuated orally administered RV vaccines (which are attenuated by virtue of their HRR). In traditional continuous cell line culture systems, most RV strains efficiently block the induction of type I IFN and have evolved to target several different host factors that regulate the IFN pathway [40]. This multipronged subversion of the IFN response is accomplished primarily by the versatile RV nonstructural NSP1 protein, the product of RV gene 5 (see below). Most IFN-sensitive RV strains encode forms of NSP1 that exhibit defective IFN inhibition and therefore elicit enhanced IFN secretion [4048]. Although such strains are still viable infectious agents, their ability to replicate is substantially hampered. In addition, IFN sensitivity of RVs encoding full-length “functional” NSP1 proteins also occurs in specific cell lines, possibly reflecting NSP1’s inability to target host innate factors across different species [49]. Enteric infection of suckling (i.e., 3- to 5-dayold) mice with a homologous murine RV compared to a heterologous simian RV strain reveals a substantial (B45 log) host restriction of the simian RV replication in the intestine [7,26,50,51]. This host restriction phenotype is substantially reduced (down to 1 log or less) in mice lacking and three IFN receptors (IFNRs) or STAT1, a key transcription factor relaying antiviral signals from different IFNRs [7,26,50,51]. The replication phenotype strongly cosegregates with the genetic origin of the

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murine or simian RV NSP1 encoding gene 5 segments. The suckling mouse thus presents a highly tractable model in which IFN-specific effects on RV replication can be studied within the biologically relevant framework of intestinal RV replication in a natural host and in a host-range-restricted manner. Ectopic parenteral injection of purified IFN types I, II, or III in many species, including mice, results in the activation of the key downstream transcription factor STAT1 in small IECs (the predominant site of RV replication) [52]. Multiple lines of evidence indicate important roles for IFN types I, II, and III in restricting RV replication in the gut and in cell culture [7,26,50,51,53]. In mouse embryonic fibroblasts lacking both types I and II IFNRs, the replication of several nonmurine RV strains is substantially enhanced (by four to five orders of magnitude). In suckling mice lacking the types I, II, and III IFNRs (either singly or in combination) significant enhancement of simian RV intestinal replication occurs demonstrating the sensitivity of heterologous nonmurine RVs to different IFN types in the mouse gut [7,26,50,51]. In contrast, replication of the homologous murine RV is quite resistant to these IFNs (B1 log or less replication gain). The collective evidence thus highlights the important role of different types of IFNs (and their inhibition by the RV NSP1 protein) in RV pathogenicity and attenuation [7,26,50,51,53]. Deciphering the mechanisms underlying these interactions is key for rational RV strain attenuation and designing improved third-generation live attenuated RV vaccines.

IV. REGULATION OF THE INTERFERON INDUCTION PATHWAY BY ROTAVIRUS In a manner analogous to that of other RNA viruses, RV-induced IFN activation is dependent on an intact RNA sensing pathway [8].

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Postrecognition of viral RNA by the cytoplasmic sensors RIG-I and/or MDA-5, epithelial cells activate the MAVS, a mitochondriaresident adaptor protein that is alternatively known as IFN-β promoter stimulator 1 (IPS-1), CARD adapter inducing IFN beta (Cardif), or virus-induced signaling adapter (VISA) [5457]. MAVS serves as a central hub in the IFN induction pathway by activating further downstream kinases including TANK-binding kinase 1 (TBK1) and inhibitor of kappa light polypeptide gene enhancer in B cells, kinase epsilon (IKK-ε) that phosphorylate IRF3 and NF-κB, respectively [58]. These molecules translocate into the nucleus upon phosphorylation and function as transcription factors, which ultimately leads to the expression of different IFNs and the activation of IFNstimulated response elements (ISREs). In addition to IRF3, IRF7 has been characterized as an important transcription factor for type I IFN induction in immune cells, in particular dendritic cells [59]. Similar to IRF3, IRF7 undergoes phosphorylation and subsequent translocation into the nucleus in response to RV infection and activates IFN expression by functioning as transcription factors. To block such an important pathway, the RV-encoded NSP1 protein efficiently degrades both IRF3 and IRF7 in a virus-strain-dependent manner [42,60]. This process takes place first through the recognition of IRF3 using an ELLIS motif localized at the C-terminal end of NSP1 present on the NSP1 molecule derived from simian, murine, and some other nonhuman RV strains [61]. The NSP1IRF3 interaction subsequently results in a rapid and efficient degradation of IRF3 at the proteasome and suppression of IFN production in RV-infected cells (Fig. 40.2). Besides the IRF family members, NF-κB has been shown to be another key arm of the host innate immune response downstream of MAVS in many virus-infected cells [56]. NF-κB signaling is robustly activated by virus infection as well as proinflammatory cytokines, including

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FIGURE 40.2 Rotavirus regulation of the IFN induction pathway. RV RNA PAMP activates the RNA sensing pathway, which is antagonized by RV-encoded proteins at multiple steps. In unpublished studies, VP3 has been shown to directly induce the proteasomal degradation of MAVS. In addition, NSP1 from different RV strains degrades either IRF3 or β-TrCP, the latter of which is mediated by the host cullin 3E3 ligase complex. Together, these RV proteins work in concert to dampen IFN induction in RV-infected cells.

IL-1β and TNF-α, the latter of which has recently been shown to be directly antiviral against RV [62]. For RV infection of HT-29 cells, the secretion of IL-8 is dependent on the NF-κB activation [63]. In a suckling mouse model, other chemokines such as CCL3, CCL5, CXCL10, and GM-CSF were also upregulated following RV infection [64], although whether these canonical NF-κB cytokines are activated through MAVS or TLRs remains unknown. Similar to IRF3, β-TrCP, a critical protein essential for degrading cellular NF-κB inhibitor IκB, is also targeted by NSP1 for proteasomal degradation [45]. In the case of β-TrCP, the binding domain within NSP1 maps to a C-terminal DSGIS motif in human and porcine RV strains [65]. Importantly, this is the same region as the

ELLIS motif responsible for IRF3 binding mentioned above. This interesting dichotomy of NSP1substrate interaction may stem from the distinct contribution of IRF3 versus β-TrCP in IFN induction in different human and animal RV species [45]. In contrast to the previous speculation of NSP1 as a viral E3 ligase due to the presence of an N-terminal RING finger domain, we recently discovered an interesting codestruction mechanism, in which NSP1 localizes to the Golgi apparatus and hijacks the host cullin 3RING box protein 1 E3 ligase complex to induce the proteasomal degradation of both β-TrCP and NSP1 itself [44]. Chemical blockade or siRNA knockdown of cullin-3 components impaired NSP1’s ability to degrade β-TrCP, leading to a significant increase in the levels of β-TrCP and reduced RV replication (Fig. 40.2). Interestingly, the cullin complex did not appear to be required for NSP1-mediated IRF3 degradation, suggesting an alternative mechanism of action at work. More recent unpublished data from our lab indicate that in addition to IRFs and β-TrCP, MAVS itself is also subject to RV inhibition. MAVS levels were significantly reduced during RV infection, and this process is mediated, surprisingly, by the RNA methyl- and guanylyltransferase VP3 protein (Fig. 40.2). By localizing to the mitochondria and binding to MAVS through an N-terminal domain, VP3 induced efficiently proteasomal degradation of MAVS in a host-species- and virus-strain-specific manner. MAVS inhibition by VP3 is another striking manifestation of RV’s ability to subvert host innate immune signaling. This is the first example of MAVS degradation by an enteric virus, and it would be of interest to further test other enteric RNA viruses such as norovirus.

A. Regulation of the Interferon Signaling Pathway by Rotavirus The antiviral IFN response to RV infection follows a biphasic pattern consisting of an

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initial IFN induction phase followed by a ligand-mediated (and IFN receptor-relayed) amplification phase [7,8]. As was discussed above, RVs are adept at inhibiting IFN induction and the RV NSP1 protein functions to degrade the essential factors β-TrCP and/or IRF3 during IFN induction in a RV strainspecific manner [66]. Interestingly, in spite of viral antagonism of IFN induction, infection with RV leads to the transcriptional induction and secretion of different IFN types in both cell culture and in vivo [8,19,26,49,52,67]. At least two factors contribute to the failure of RV to completely suppress the induction of IFN secretion. First, synthesis of the IFN antagonist NSP1 occurs only after viral entry, uncoating of the virion, RV transcription, and translation. During this initial infection process, several byproducts of viral transcription are generated that act as potent triggers of the IFN induction pathway [8,10]. Second, RV entry into different types of cells may not always result in productive infection. For example, RV exposure to primary human pDCs results in two distinct populations of cells that differ in their level of viral infectivity [16]. Dendritic cells that are not productively infected nevertheless exhibit robust activation of the IFN induction response [16,27]. Given the remarkable efficiency of IFN secretion in this cell type, they are a likely source of substantial IFN secretion from a nonepithelial cellular compartment where RV does not replicate efficiently. In suckling mice, infection with RV leads to significant induction of different types of IFNs, of which the type I IFNs are induced primarily in intestinal immune cells rather than being derived from IECs, where viral infectivity is maximal [26]. The secretion of IFNs from different cell types poses a unique challenge to successful viral propagation and spread to uninfected bystander IECs. This is because IFN binding to its cognate cell surface receptor activates a positive feedback loop that amplifies the expression of IFNs as well as more than 300 different

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IFN-stimulated genes [68]. This IFN release then efficiently amplifies the expression of antiviral proteins targeting a variety of viral replication steps in uninfected bystander cells. Each of the three major IFN types (I, II, and III) that are found to be induced in the intestine following RV infection is capable of mediating phosphorylation of the key convergent transcription factor STAT1 (at Y701, an event that is critical for unlocking the transcriptional program resulting in an antiviral state) (Fig. 40.3). Each of these IFN types is biologically relevant in the context of modulating RV infection and spread [7,52]. Several lines of evidence indicate that RV employs potent countermeasures to subvert the antiviral state mediated by secreted IFNs during initial infection [7,19,26,52,67]. In cell culture, addition of purified exogenous IFNs after RV adsorption does not significantly hamper viral replication; instead, IFN treatment of cells prior to RV infection is required to achieve efficient RV replication restriction [69]. In the RV suckling mouse model, infection with a homologous murine RV and infection with a heterologous simian RV strain result in comparable levels of induction of different IFNs from the intestine [7,26]. However, as was noted above, the presence of IFNs during infection has a negligible effect on murine RV replication (B1-log restriction in titer) but has a potent effect on heterologous simian RV replication (4- to 5-log restriction in titer) [26,50]. These observations suggest that in order to replicate successfully, homologous RVs have evolved strategies to induce resistance to the actions of different secreted IFN types in cells prior to their actual infection (bystander cells). Classical reassortment genetic studies of the IFN-mediated replication phenotype of murine and simian RVs implicated a constellation of RV genes (encoding the VP3, NSP1, NSP2, and NSP3 viral proteins) in determining the resistance to IFN signaling [51]. Of these, NSP1 is a necessary and the major determinant of efficient intestinal RV replication in an IFN-dependent fashion.

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FIGURE 40.3

RV regulation of the IFN amplification pathway. Following infection, RV NSP1 efficiently blocks exogenous IFN-directed phosphorylation of STAT1 at Y701, shown here for the IFNAR1 (1). In the virus-infected cell, RV also mediates the lysosomal degradation of receptors for types I, II, and III IFN by an unknown process (2). Along with IRF3 and IRF5, the viral NSP1 protein also proteasomally degrades IRF9 and IRF7 in the infected cells, which are required for optimal IFN amplification responses (3). In addition, RV can inhibit the nuclear translocation of STAT1-pY701 by an unknown mechanism (4). Remarkably, in addition to these viral effects in infected cells, RV also potently inhibits STAT1 phosphorylation in uninfected bystander cells in response to different types of IFNs (5). The viral and host factors underlying this bystander inhibition of IFNs are unknown.

B. Regulation of STAT1 by Rotavirus Direct evidence for RV subversion of the antiviral state mediated by exogenous IFNs comes from the finding that RV-infected HT-29 cells (a human IEC colonic cancer cell derived line) are able to efficiently block STAT1-Y701 phosphorylation in response to exogenously added purified IFNs I and II [52,67]. Using single-cell analytic techniques, IFN-mediated STAT1 inhibition is found to occur within RVinfected cells. Remarkably, STAT1 responses to exogenous IFN ligand are also potently

inhibited in RV uninfected bystander cells, which do not express any detectable viral antigen [67] (Fig. 40.3). Although initially described for a porcine RV strain SB1-A, this bystander inhibitory effect has now been observed in vitro with several other RV strains, albeit with lower efficiency (Sen and Greenberg, unpublished observations). The ability of RV to block IFNdependent signaling has also been observed in vivo. Suckling mice infected with murine RV are able to significantly suppress IEC STAT1Y701 phosphorylation and subsequent transcription that occurs in response to parenterally

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administered purified IFNs I or III [52]. Although IFNA4, IFNA5, and IFNA5 transcripts are induced in the intestines of murine RV-infected mice, transcriptional analysis of isolated mature villous enterocytes revealed that within this compartment (where RV replication predominantly occurs), both infected and bystander cells fail to amplify the type I IFN genes [26]. In the villous epithelium, RV bystander cells also do not express elevated levels of transcripts encoding IRF7, which is upregulated in response to stimulation of cells with secreted IFNs and is critical for the optimal expression of several antiviral genes.

C. Degradation of Different Types of Interferon Receptors The effectors in RV-infected cells that mediate STAT1 inhibition in bystander cells and the rotaviral factors responsible have not been identified. In contrast, RV inhibition of IFNdirected STAT1 activation in RV-infected cells is well characterized [26,52,67]. Recent findings demonstrate that at the single-cell level, RV infection results in the efficient depletion of type I, II, and III IFNRs within RV (VP6 antigen1) infected cells [52]. Such RV-mediated IFNR degradation is unlikely to be directed by secreted IFNs (i.e., by a ligand-dependent pathway) and despite prolonged infection of cells is restricted exclusively to the subset of cells expressing VP6. The depletion of IFNRs in RV-infected cells occurs from 6 to 8 hours postinfection (hpi) onwards by a lysosomalproteasomal pathway of protein degradation and is not observed in the RV (VP 6 antigen) uninfected bystander cells, which are nevertheless highly refractory to IFN-directed STAT1 activation (Fig. 40.3). The relevance of RV-mediated IFNR degradation is shown in vivo by the significant decrease in intestinal type I and II IFNR protein expression in the small intestine following murine RV infection [52].

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Degradation of different types of IFNRs by RV represents an ingenious strategy to ensure that any autocrine IFN antiviral amplification is inhibited, thus allowing viral replication and cell to cell spread to proceed efficiently [52]. Interestingly, these findings indicate the likelihood that RV targets a common hostsignaling pathway that is responsible for the expression of all three IFNRs. Continuing to unravel the mechanisms by which RV also inhibits the response to different IFNs in bystander cells is important for several reasons. First, since the level of RV replication substantially determines host pathogenicity, such information will enable more rational attenuation of homologous and heterologous RV strains and their use as candidate thirdgeneration live vaccines. Second, for several diseases (including sepsis and systemic lupus erythematosus), an excessive IFN response is undesirable and implicated as a causative and/or exacerbation trigger of disease. In these situations, discovering novel therapeutic modalities that can dampen IFN signaling is potentially valuable.

D. STAT1 Sequestration in the Cytoplasm Other RV strategies have also been identified that are directed at disrupting STAT1 signaling during infection. The ability of RV to perturb STAT1 signaling was first reported by Holloway and colleagues [70,71], who observed that as early as 6 hpi, several RV strains inhibited the nuclear translocation of phosphorylated STAT1-Y701 in response to exogenous IFN stimulation (Fig. 40.3). Although viral factors responsible for this inhibitory effect downstream of STAT1 activation have not yet been identified, it is possible that redundancy in RV inhibition of the STAT1 pathway exists, perhaps indicative of the vital role of inhibiting IFN signaling in enabling RV replication.

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E. Regulation of IRF7 and IRF9 The ability of the RV NSP1 protein to target IRFs for proteasomal degradation extends to IRF7 and IRF9, two additional factors that are critical for the optimal amplification of IFNdependent antiviral responses [40,42]. Whereas early induction of different IFNs and antiviral transcripts is mediated primarily by IRF3, IFNmediated signaling results in an increase in IRF7 expression, which subsequently orchestrates the amplification of IFNs and of ISGs. The IFN-mediated effects on transcription of several genes (including IRF7) require the assembly of a transcriptional complex ISGF3, which includes STAT1 and IRF9. The role of IRF7 and IRF9 degradation in IFN-dependent responses during RV infection has not yet been well studied. Nevertheless, the degradation of IRF7 and IRF9 by NSP1 is likely to be an additional weapon in the RV arsenal that can be used to halt an efficient IFN amplification response (Fig. 40.3).

F. Rotavirus Regulation of Other Effector Antiviral Factors In addition to the IFN induction and amplification pathway, RV is equipped with the ability to block further downstream effector antiviral proteins. One such example is ribonuclease L (RNaseL), a key enzyme in the IFN-inducible 20 -50 -oligoadenylate synthetase (OAS)-RNaseL pathway responsible for potent RNA degradation, including both host and viral RNA molecules [72]. The RV RNA capping enzyme VP3 encodes a C-terminal phosphodiesterase (PDE) domain that was recently shown to induce the degradation of 20 ,50 -oligoadenylate, the second messenger responsible for RNaseL activation and dimerization [73]. The RV VP3 PDE domain functionally replaced the comparable domain in the murine coronavirus ns2 protein and inhibited RNaseL activity. A more recent study suggests that another VP3-independent,

yet-to-be identified, mechanism also exists and contributes to RV inhibition of RNaseL [74]. However, the actual physiological roles of RNaseL in modulating RV replication and of VP3 in antagonizing RNaseL in vivo remain to be demonstrated.

V. TAKING ADVANTAGE OF ROTAVIRUS HOST RANGE RESTRICTION TO RELIABLY ATTENUATE LIVE ROTAVIRUS VACCINE CANDIDATES There are currently two time-honored and demonstrably successful approaches to developing safe and effective human viral vaccines [75]. In the first case, a wide variety of current viral vaccines rely on the parenteral administration of replication-incompetent inactivated whole virus, the parenteral administration of a viral protein(s) component, or the administration a molecularly produced virus-like particle (VLP). All of these entities are selected because immunity to the individual protein, inactivated whole virus, or VLP induces protective immunity to the host and, at the same time, is both safe and well tolerated. There are numerous highly successful examples of this category of viral vaccine (e.g., the inactivated polio vaccine, the various preparations of inactivated influenza hemagglutinin-based vaccines, the human papilloma VLP vaccine, the hepatitis B virus vaccine, and the recently licensed herpes zoster gE protein-based vaccine). In all cases, these vaccines are administered parenterally. Several are administered with adjuvants of one kind or another to enhance immune responses, and in all cases, they appear to function primarily by stimulating systemic immunity, with the primary effector function generally mediated by systemic antibodies. Of note, none of these types of vaccines are directed at a predominantly enteric pathogen, although an investigation of a potential parenterally administered

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V. TAKING ADVANTAGE OF ROTAVIRUS HOST RANGE RESTRICTION

norovirus VLP vaccine is currently under way [76], and there are plans to study the utility of a heat-inactivated RV virion-based vaccine in humans [77]. The greatest advantages of the inactivated/recombinant protein-based vaccines are their general safety and the fact that they can be produced even when the actual pathogen cannot be readily propagated. The general disadvantage of such vaccines is that they are almost always less effective at stimulating T-cell-based immune responses, they are not very efficient at stimulating mucosal immune responses, that highly effective mucosal immune adjuvants are not yet readily available, and in some cases, immune responses to these inactivated vaccine preparations tend to diminish over time more rapidly than do responses to several live viral vaccines. Other potential disadvantages of inactivated vaccines become apparent when the antigen or antigens required to induce protective immunity are difficult to synthesize artificially or when immunity is most potent when it is directed at multiple antigens that are correctly folded only on the actual or recombinant multiprotein viral particle. Of note, RVs have at least two separate proteins (VP4 and VP7) that are targets of protective antibodies, and VP4 is cleaved by enteric trypsin into two separate protein components: VP8* and VP5* [78]. Both VP8* and VP5* are individually targeted by protective antibody responses. VP7 is correctly folded into its proper antigenic trimeric form only within the context of the RV virion, and a similar issue likely is true for VP5*. On the other hand, the RV VP8* protein can be relatively simply and accurately synthesized in several prokaryote and eukaryote systems, and because of this feature, it is currently being examined as a potential inactivated vaccine to be administered parenterally [79] (Chapter 41: Development of Oral Rotavirus & Norovirus Vaccines). The second highly successful approach to human viral vaccine development has been the production of live attenuated viral vaccines

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that actually infect susceptible people but are attenuated to such a degree that their level of reactogenicity and pathogenicity is acceptable, while they reliably generate protective immunity. As with inactivated vaccines, a number of highly effective replication-competent viral vaccines are currently available (e.g., oral Sabin polio vaccine, measles vaccine, rubella vaccine, yellow fever vaccine, smallpox vaccine, live attenuated influenza vaccine, and, of course, several licensed RV vaccines, such as RotaTeq, Rotarix, and Rotavac) [75]. Most but not all of the live attenuated viral vaccines are administered by parenteral injection; however, oral polio, rotavirus, and live attenuated influenza vaccine are all administered to a mucosal surface (the GI tract and the respiratory tract, respectively). The general thinking is that live attenuated viral vaccines more closely mimic the type and level of immunity induced by natural infection than parenterally administered inactivated vaccines do. If natural infection is an effective preventative of severe secondary infection, then reproducing it without undue reactogenicity can be desirable. This feature is present when natural immunity is operative primarily at a mucosal surface, as is the case for RVs. Natural RV infection efficiently protects against severe reinfection, and for this reason, a variety of experimental and licensed live attenuated RV vaccines have been developed or proposed. The key issue to overcome in developing a live viral vaccine is to discover a method that reliably attenuates viral pathogenicity while retaining the ability of the viral infection to induce protective immunity. Traditionally, such modification has been accomplished by multiple passage of virulent viral strains in cell culture with the hope that such multiple passages will lead to the acquisition of sufficient mutations in the viral genome (acquired to enhance cell culture replication) and that these cell culture adaptations will attenuate viral pathogenicity in the target host. This strategy is

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time honored and has been used to develop multiple vaccines (e.g., live polio, measles, mumps, rubella, yellow fever, and some RV vaccines). While this approach is frequently successful, there is no way to determine how many passages are needed to eliminate residual virulence while retaining immunogenicity, so it is often tedious and always an inexact approach. In addition, concerns about reversion to virulence are always present. That said, this approach was used successful to develop a highly effective human RV vaccine (R1, Rotarix), which consist of a single human RV strain that was repeated passed in several cell culture systems and, over the passage history, became attenuated in people [80]. While the genomic sequence of both the virulent wildtype parental Rotarix strain and the eventual vaccine strain are known, the exact genomic mutations responsible for attenuation are unclear. What has been established, however, is that, given the very substantial and decade-long safety record, this human RV vaccine strain has sufficient attenuating mutations to ensure a high degree of genetic stability in humans. Of note, the R1 Rotarix vaccine represents a single G and [P] serotype yet reliably induces protective immunity to virtually all frequently circulating human RV strains. This finding strongly supports the conclusion that serotype-specific immunity is not a major determinant of immunity to severe RV disease in humans [81]. The other strategy that has proven highly successful for the reproducible attenuation of the RVs used in live attenuated RV vaccines has been to take advantage of the HRR (see above) of heterologous (nonhuman origin) RVs as vaccine candidates for humans [82]. Several currently licensed RV vaccines (e.g., the R5 vaccines RotaTeq and Rotasil and the R1 vaccine Rotavac) consist of either natural or experimentally selected reassortants between animal RVs (in these cases, all bovine RVs) and human RVs. Both R5 vaccines are pentavalent constructs derived experimentally on the basis of

bovine RV genomes but containing individual VP7- or VP4s-encoding genes derived from various human serotypes. RotaTeq is broadly licensed around the world, while Rotasil is currently licensed only in India [83]. Rotavac is, interestingly, a naturally occurring reassortant RV derived from a human RV and a bovine RV. It contains only a bovine VP4, with all other ten RV gene segments derived from a human RV strain. This virus was originally isolated from a neonatal nursery where asymptomatic RV infection was endemic [84]. Finally, of relevance to this review, an entirely lamb origin RV strain is currently licensed for RV prevention in China. This vaccine is also highly attenuated, presumably because of the HRR of a lamb origin RV in humans. While this vaccine seems safe, the data on its efficacy are limited [85]. The key point here is that all these animal virus origin based RV vaccines are highly attenuated in humans, and this attenuation, as was discussed above, is most likely based on their host range replication restriction in humans. This HRR is primarily due to the inability of heterologous RV to efficiently suppress the human intestinal innate immune response, primarily the human IFN response, owing to the presence of heterologous NSP1s in the vaccine candidates. The active human IFN response to these heterologous RV vaccines suppresses their replication sufficiently to restrict pathogenicity and reactogenicity but not so much that the generation of effective RV immunity is suppressed. However, in the case of the Indian Rotavac vaccine, attenuation might be based on the heterologous bovine origin VP4, which might be expected to reduce RV binding to human IECs. The big question for the future is whether, with the advent of a tractable reverse genetic system and our improved understanding of the genetic determinants of HRR, we will be able to better fine-tune the replication competence and immunogenicity potential of RV vaccine candidates to further enhance their efficacy.

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REFERENCES

References [1] Platts-Mills JA, et al. Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED). Lancet Global Health 2015;3:e564575. [2] Tate JE, et al. Global, regional, and national estimates of rotavirus mortality in children ,5 years of age, 20002013. Clin Infect Dis 2016;62(Suppl. 2):S96105. [3] Estes MK, Greenberg HB. Rotaviruses. In: Knipe DM, Howley PM, et al., editors. Fields virology. 6th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2013. p. 1347401. [4] Arnold MM, et al. The battle between rotavirus and its host for control of the interferon signaling pathway. PLoS Pathog 2013;9:e1003064. [5] Brennan K, Bowie AG. Activation of host pattern recognition receptors by viruses. Curr Opin Microbiol 2010;13:5037. [6] Li MM, et al. Totranslate, or not totranslate: viral and host mRNA regulation by interferon-stimulated genes. Trends Cell Biol, 25. 2015. p. 3209. [7] Lin JD, et al. Distinct roles of type I and type III interferons in intestinal immunity to homologous and heterologous rotavirus infections. PLoS Pathog 2016;12:e1005600. [8] Sen A, et al. The early interferon response to rotavirus is regulated by PKR and depends on MAVS/IPS-1, RIG-I, MDA-5, and IRF3. J Virol 2011;85:371732. [9] Broquet AH, et al. RIG-I/MDA5/MAVS are required to signal a protective IFN response in rotavirusinfected intestinal epithelium. J Immunol 2011;186: 161826. [10] Uzri D, Greenberg HB. Characterization of rotavirus RNAs that activate innate immune signaling through the RIG-I-like receptors. PLoS One 2013;8:e69825. [11] Dou Y, et al. The innate immune receptor MDA5 limits rotavirus infection but promotes cell death and pancreatic inflammation. EMBO J 2017;36:274257. [12] Uchiyama R, et al. MyD88-mediated TLR signaling protects against acute rotavirus infection while inflammasome cytokines direct Ab response. Innate Immun 2015;21:41628. [13] Pane JA, et al. Rotavirus activates lymphocytes from non-obese diabetic mice by triggering toll-like receptor 7 signaling and interferon production in plasmacytoid dendritic cells. PLoS Pathog 2014;10:e1003998. [14] Ge Y, et al. Rotavirus NSP4 triggers secretion of proinflammatory cytokines from macrophages via toll-like receptor 2. J Virol 2013;87:111607. [15] Liu F, et al. Porcine small intestinal epithelial cell line (IPEC-J2) of rotavirus infection as a new model for the study of innate immune responses to rotaviruses and probiotics. Viral Immunol 2010;23:13549. [16] Deal EM, et al. Rotavirus structural proteins and dsRNA are required for the human primary

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25] [26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

plasmacytoid dendritic cell IFNalpha response. PLoS Pathog 2010;6:e1000931. Zhang B, et al. Viral infection. Prevention and cure of rotavirus infection via TLR5/NLRC4-mediated production of IL-22 and IL-18. Science 2014;346:8615. Pott J, et al. Age-dependent TLR3 expression of the intestinal epithelium contributes to rotavirus susceptibility. PLoS Pathog 2012;8:e1002670. Saxena K, et al. A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection. Proc Natl Acad Sci USA 2017;114:E5709. Jaimes MC, et al. Frequencies of virus-specific CD4(1) and CD8(1) T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults. J Virol 2002;76:47419. Chaplin PJ, et al. The cloning of cattle interferon-A subtypes isolated from the gut epithelium of rotavirusinfected calves. Immunogenetics 1996;44:1435. De Boissieu D, et al. Rotavirus induces alphainterferon release in children with gastroenteritis. J Pediatr Gastroenterol Nutr 1993;16:2932. Lecce JG, et al. Treatment of rotavirus infection in neonate and weanling pigs using natural human interferon alpha. Mol Biother 1990;2:21116. Vanden Broecke C, et al. Interferon response in colostrum-deprived newborn calves infected with bovine rotavirus: its possible role in the control of the pathogenicity. Ann Rech Vet 1984;15:2934. La Bonnardiere C, et al. Interferon activity in rotavirus infected newborn calves. Ann Rech Vet 1981;12:8591. Sen A, et al. Innate immune response to homologous rotavirus infection in the small intestinal villous epithelium at single-cell resolution. Proc Natl Acad Sci USA 2012;109:2066772. Deal EM, et al. Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses. J Clin Invest 2013;123:246474. Douagi I, et al. Role of interferon regulatory factor 3 in type I interferon responses in rotavirus-infected dendritic cells and fibroblasts. J Virol 2007;81:275868. Vijay-Kumar M, et al. Flagellin treatment protects against chemicals, bacteria, viruses, and radiation. J Immunol 2008;180:82805. Hernandez PP, et al. Interferon-lambda and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection. Nat Immunol 2015;16:698707. Schroder K, Tschopp J. The inflammasomes. Cell 2010;140:82132. Liu X, et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 2016;535:1538. Kanneganti TD. Inflammatory bowel disease and the NLRP3 inflammasome. N Engl J Med 2017;377:6946.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

696

40. THE ROLE OF INNATE IMMUNITY IN REGULATING ROTAVIRUS REPLICATION

[34] Rathinam VA, et al. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat Immunol 2010;11:395402. [35] Munoz-Planillo R, et al. K(1) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 2013;38:114253. [36] Zhu S, et al. Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells. Nature 2017;546: 66770. [37] Burnett E, et al. Global impact of rotavirus vaccination on childhood hospitalizations and mortality from diarrhea. J Infect Dis 2017;215:166672. [38] Feng N, et al. Roles of VP4 and NSP1 in determining the distinctive replication capacities of simian rotavirus RRV and bovine rotavirus UK in the mouse biliary tract. J Virol 2011;85:268694. [39] Crawford SE, et al. Rotavirus infection. Nat Rev Dis Primers 2017;3:17083. [40] Arnold MM, Patton JT. Diversity of interferon antagonist activities mediated by NSP1 proteins of different rotavirus strains. J Virol 2011;85:19709. [41] Arnold MM. The rotavirus interferon antagonist NSP1: many targets, many questions. J Virol 2016;90:521215. [42] Arnold MM, et al. Rotavirus NSP1 mediates degradation of interferon regulatory factors through targeting of the dimerization domain. J Virol 2013;87:981321. [43] Barro M, Patton JT. Rotavirus NSP1 inhibits expression of type I interferon by antagonizing the function of interferon regulatory factors IRF3, IRF5, and IRF7. J Virol 2007;81:447381. [44] Ding S, et al. Comparative proteomics reveals strainspecific beta-TrCP degradation via rotavirus NSP1 hijacking a host cullin-3-Rbx1 complex. PLoS Pathog 2016;12:e1005929. [45] Graff JW, et al. Rotavirus NSP1 inhibits NFkappaB activation by inducing proteasome-dependent degradation of beta-TrCP: a novel mechanism of IFN antagonism. PLoS Pathog 2009;5:e1000280. [46] Graff JW, et al. Zinc-binding domain of rotavirus NSP1 is required for proteasome-dependent degradation of IRF3 and autoregulatory NSP1 stability. J Gen Virol 2007;88:61320. [47] Graff JW, et al. Interferon regulatory factor 3 is a cellular partner of rotavirus NSP1. J Virol 2002;76:954550. [48] Di Fiore IJ, et al. NSP1 of human rotaviruses commonly inhibits NF-kappaB signalling by inducing beta-TrCP degradation. J Gen Virol 2015;96:176876. [49] Sen A, et al. IRF3 inhibition by rotavirus NSP1 is host cell and virus strain dependent but independent of NSP1 proteasomal degradation. J Virol 2009;83: 1032235. [50] Feng N, et al. Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice. J Virol 2008;82:757890.

[51] Feng N, et al. Permissive replication of homologous murine rotavirus in the mouse intestine is primarily regulated by VP4 and NSP1. J Virol 2013;87:830716. [52] Sen A, et al. Rotavirus degrades multiple type interferon receptors to inhibit IFN signaling and protects against mortality from endotoxin in suckling mice. J Virol 2017. Available from: https://doi.org/10.1128/ JVI.01394-17. [53] Feng N, et al. Variation in antagonism of the interferon response to rotavirus NSP1 results in differential infectivity in mouse embryonic fibroblasts. J Virol 2009;83: 698794. [54] Kawai T, et al. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat Immunol 2005;6:9818. [55] Meylan E, et al. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 2005;437:116772. [56] Seth RB, et al. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 2005;122:66982. [57] Xu LG, et al. VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol Cell 2005;19: 72740. [58] Liu S, et al. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science 2015;347:aaa2630. [59] Honda K, et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 2005;434:7727. [60] Barro M, Patton JT. Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3. Proc Natl Acad Sci USA 2005;102:411419. [61] Zhao B, et al. Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins. Proc Natl Acad Sci USA 2016;113:E34033412. [62] Hakim MS, et al. TNF-alpha exerts potent antirotavirus effects via the activation of classical NFkappaB pathway. Virus Res 2018;253:2837. [63] Sheth R, et al. Rotavirus stimulates IL-8 secretion from cultured epithelial cells. Virology 1996;221:2519. [64] Xu J, et al. Rotavirus and coxsackievirus infection activated different profiles of toll-like receptors and chemokines in intestinal epithelial cells. Inflamm Res 2009;58: 58592. [65] Morelli M, et al. Putative E3 ubiquitin ligase of human rotavirus inhibits NF-kappaB activation by using molecular mimicry to target beta-TrCP. mBio 2015;6. [66] Morelli M, et al. Silencing the alarms: innate immune antagonism by rotavirus NSP1 and VP3. Virology 2015;479480:7584. [67] Sen A, et al. Rotavirus NSP1 protein inhibits interferon-mediated STAT1 activation. J Virol 2014;88: 4153.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

697

REFERENCES

[68] Hoffmann HH, et al. Interferons and viruses: an evolutionary arms race of molecular interactions. Trends Immunol 2015;36:12438. [69] Bass DM. Interferon gamma and interleukin 1, but not interferon alfa, inhibit rotavirus entry into human intestinal cell lines. Gastroenterology 1997;113:819. [70] Holloway G, et al. Rotavirus inhibits IFN-induced STAT nuclear translocation by a mechanism that acts after STAT binding to importin-alpha. J Gen Virol 2014;95:172333. [71] Holloway G, et al. Rotavirus antagonizes cellular antiviral responses by inhibiting the nuclear accumulation of STAT1, STAT2, and NF-kappaB. J Virol 2009; 83:494251. [72] Silverman RH. Viral encounters with 2’,5’-oligoadenylate synthetase and RNase L during the interferon antiviral response. J Virol 2007;81:127209. [73] Zhang R, et al. Homologous 2’,5’-phosphodiesterases from disparate RNA viruses antagonize antiviral innate immunity. Proc Natl Acad Sci USA 2013;110: 1311419. [74] Sanchez-Tacuba L, et al. Rotavirus controls activation of the 20 -50 -oligoadenylate synthetase/RNase L pathway using at least two distinct mechanisms. J Virol 2015;89:1214553. [75] Plotkin SA, et al. Plotkin’s vaccines. Elsevier; 2018. [76] Riddle MS, Walker RI. Status of vaccine research and development for norovirus. Vaccine 2016;34:28959. [77] Jiang B, et al. Does a monovalent inactivated human rotavirus vaccine induce heterotypic immunity?

[78]

[79]

[80]

[81] [82]

[83]

[84]

[85]

Evidence from animal studies. Hum Vaccin Immunother 2013;9:16347. Nair N, et al. VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans. Sci Transl Med 2017;9. Fix AD, et al. Safety and immunogenicity of a parenterally administered rotavirus VP8 subunit vaccine in healthy adults. Vaccine 2015;33:376672. Vesikari T, et al. Efficacy of human rotavirus vaccine against rotavirus gastroenteritis during the first 2 years of life in European infants: randomised, double-blind controlled study. Lancet 2007;370:175763. Santosham M, Steele D. Rotavirus vaccines - a new hope. N Engl J Med 2017;376:11702. Kapikian AZ, et al. Jennerian and modified Jennerian approach to vaccination against rotavirus diarrhea using a quadrivalent rhesus rotavirus (RRV) and human-RRV reassortant vaccine. Arch Virol Suppl 1996;12:16375. Vesikari T, et al. Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med 2006;354:2333. Bhandari N, et al. Efficacy of a monovalent humanbovine (116E) rotavirus vaccine in Indian infants: a randomised, double-blind, placebo-controlled trial. Lancet 2014;383:213643. Zhen SS, et al. Effectiveness of the live attenuated rotavirus vaccine produced by a domestic manufacturer in China studied using a population-based case-control design. Emerg Microbes Infect 2015;4:e64.

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C H A P T E R

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Development of Oral Rotavirus and Norovirus Vaccines Adam Huys, Katrina R. Grau and Stephanie M. Karst Department of Molecular Genetics & Microbiology, Emerging Pathogens Institute, Center for Inflammation and Mucosal Immunology, University of Florida, Gainesville, FL, United States

I. INTRODUCTION

II. ROTAVIRUS VACCINE DEVELOPMENT

Pathogenic enteric viruses are one of the main causes of acute gastroenteritis and are implicated in a huge disease burden worldwide. Rotaviruses (RVs) and noroviruses (NoVs) are the two most common causes of acute viral gastroenteritis, RV being a common cause of pediatric diarrheal disease and NoVs afflicting people of all ages. In this chapter, we will summarize the state of the field in developing effective vaccines against these two families of viruses. A global RV vaccination program is in place, so we will present a history of vaccine development and then discuss ongoing concerns with efficacy in developing nations. NoVs have been more difficult to study than RVs and pose unique obstacles in terms of vaccine development, so we will focus our discussion on the current state of this effort and cautionary notes for the future.

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00040-7

A. Rotavirus Disease RVs are a major cause of severe gastroenteritis in infants and very young children. They are so ubiquitous in the environment that nearly every child has been infected with a RV by the age of 5 years. Prior to implementation of vaccine programs, RVs were the main cause of acute childhood diarrhea in children younger than 5 years of age worldwide [1 3]. Projection models estimated an astounding 138 million cases of RV disease per year, associated with 25 million clinic visits, 2 million hospitalizations, and nearly 500,000 deaths in children under 5 years of age [4]. The mortality rate of RV infections is much higher in developing countries than in developed ones; over half of fatal infections occur in India, Nigeria, Pakistan, Ethiopia,

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41. DEVELOPMENT OF ORAL ROTAVIRUS AND NOROVIRUS VACCINES

FIGURE 41.1 Rotavirus vaccine development timeline. Colored bars indicate the type of RV used for the vaccine formulation. Blue: attenuated animal strain; red: reassortant viruses; green: attenuated human strain. & Never licensed, owing to poor efficacy in developing countries. ☨Licensed but withdrawn, owing to correlation with intussusception. *Internationally prequalified vaccines. ☥ Licensed for use only in the country of origin.

and the Democratic Republic of Congo [5]. Therefore, developing effective RV vaccines is of high importance, and huge progress has been made in the past four decades (Fig. 41.1).

B. Rotavirus Classification RVs are segmented double-stranded RNA viruses constituting a genus in the Reoviridae family. There are nine RV serogroups, referred to as groups A I, with group A being the major cause of disease in people [6]. Group A RVs are segregated into serotypes based on neutralizing epitopes within one of two structural proteins. The G serotypes are defined by the glycoprotein VP7, and the P serotypes are defined by the protease-sensitive VP4 protein. To date, 27G genotypes and 37P genotypes have been described [6]. Because of the segmented nature of the viral genome, the genes encoding VP4 and VP7 can reassort.

C. Initial Vaccine Efforts Using Live Animal Rotavirus The earliest RV vaccine candidates developed in the early 1980s were live attenuated animal RV strains adapted by serial tissue passaging and administered to infants orally. RIT 4237, a serotype G6 RV, is a bovine RV that was sequentially passaged on primary fetal bovine cells and then a human leukemic cell line [7]. Initial trials

demonstrated that the vaccine was well tolerated and provided protection from developing disease [8]. However, efficacy in developing countries was significantly lower than that in developed nations [9,10]. Similar to RIT 4237, a minimally passaged bovine RV referred to as WC-3 displayed very promising results in clinical trials conducted in the United States [11,12], but subsequent testing in developing countries showed minimal protection [12]. Because of the poor performance of RIT 4237 and WC-3 in developing countries, further development of these bovine RV-based vaccines was abandoned. Clinical trials of a rhesus macaque RV-based vaccine candidate, RRV-1, demonstrated convincing serotype-specific protection against G3 viruses, but there were conflicting results in terms of its ability to induce heterologous protection to other RV serotypes [13 17]. Moreover, RRV-1 vaccination was associated with a febrile response in a majority of vaccinated infants and watery stools in a minority of recipients [15,17]. Efforts to test RRV-1 further were halted because of adverse outcomes and the potential serotype-specific nature of its immune response.

D. RotaShield, the First Licensed Rotavirus Vaccine Unlike the earliest RV vaccine candidates, which comprised a single attenuated virus, the first licensed vaccine was a tetravalent live

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II. ROTAVIRUS VACCINE DEVELOPMENT

attenuated vaccine called RotaShield, or RRVTV, manufactured by Wyeth. It contained the parental RRV-1 strain used in earlier trials, which encodes serotype G3 specificity in addition to three reassortant RRV-1 viruses containing the VP7 gene segment from human RV strains with serotype G1, G2, and G4 specificity, respectively [18]. RotaShield reduced severe disease by approximately 80% in three independent clinical trials conducted in the United States, Finland, and Venezuela in which infants were immunized orally at 2, 4, and 6 months of age [19 21]. These trials also demonstrated that the vaccine was well tolerated, the only adverse event reported being a mild febrile response after the first dose of the vaccine compared to placebo controls. Of note, there was no significant association with intussusception in any clinical trial of RotaShield [19 22]. On the basis of the success of these trials, RotaShield was licensed in the United States on August 31, 1998, and administration in a nationwide immunization plan was recommended. Unfortunately, within a year, it became apparent that RotaShield was associated with a small (1 2 cases per 10,000 vaccine recipients), but significant number of cases of intussusception, a form of bowel obstruction. Postlicensure studies supported a causal association [23]. The scientific driver of the development of RotaShield, Dr. Albert Kapikian at the NIH, argued that the benefits of the vaccine far outweighed this risk, especially in impoverished parts of the world, where RV fatalities were common. His view was grounded in supporting data considering that hospitalizations due to RV-induced gastroenteritis would be far higher than hospitalizations attributable to RotaShield-induced intussusception even in the United States [24,25]. Yet, the public perception of the dangers of the vaccine was widespread, leading the manufacturing companies to voluntarily withdraw RotaShield from the market, and the Centers for Disease Control and Prevention (CDC) to recommend suspending its use in 1999.

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E. RotaTeq and Rotarix, the Second Generation of Licensed Rotavirus Vaccines RotaShield clinical trials likely failed to identify a link between the vaccine and intussusception because the frequency of this adverse outcome was so low. Accordingly, new RV vaccine candidates were tested in very large subject pools. In the early 2000s, two vaccine candidates, RotaTeq and Rotarix, underwent large-scale testing involving more than 60,000 infants each. and were licensed in the United States in 2006 and 2008, respectively. RotaTeq, manufactured by Merck, is a pentavalent vaccine composed of reassortant viruses between the bovine WC-3 vaccine strain and human RV strains [26]. Four reassortants express the VP4 protein from WC-3 and different VP7 proteins from human RV strains (serotype G1, G2, G3, or G4), while the fifth virus expresses a human RV VP4 protein and the VP7 protein from WC-3 (serotype G6). A large clinical trial was carried out from 2001 to 2004 in which more than 70,000 participants from 11 countries were immunized with RotaTeq and evaluated for safety and efficacy. This trial demonstrated that RotaTeq was well tolerated with no increase in intussusception compared to the placebo group. The trial also demonstrated that RotaTeq vaccination led to a 95% decrease in severe disease when administered in three doses separated by 4 10 weeks beginning when the infant was 6 12 weeks old [27]. RotaTeq was licensed for use in the United States and Canada in 2006. Unfortunately, vaccine efficacy is substantially lower in developing countries than in developed nations. For example, while RotaTeq provided over 90% protection from severe disease in Finnish children [28], it provided only 39% and 48% protection in sub-Saharan Africa and Asia, respectively [29,30]. Rotarix, manufactured by GlaxoSmithKline, is composed of a single live attenuated serotype

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G1 human RV. The vaccine strain was originally isolated from a naturally infected infant in Cincinnati, and was subsequently passaged in African green monkey kidney cells, cloned, and minimally passaged in Vero cells [31,32]. In a phase 3 clinical trial conducted in 11 Latin American countries and Finland, 63,225 infants were administered Rotarix in two doses 4 weeks apart between 6 and 24 weeks of age. The vaccine was well tolerated, was not associated with intussusception, and resulted in a vaccine efficacy of 85% against severe disease [33]. Of note, considering that Rotarix is a monovalent vaccine, the large clinical trial and subsequent trials have demonstrated that Rotarix can provide cross-protection against other RV serotypes [34 36]. In 2004, Mexico and the Dominican Republic were the first countries to license Rotarix. It was subsequently licensed in the United States in 2008, and is currently licensed for use in more than 120 countries [37]. Although Rotarix is highly effective at reducing severe disease, its efficacy and its ability to provide protection for more than a year is diminished in developing countries compared to developed countries [38 41]. Since 2009, the World Health Organization has recommended that all countries include a RV vaccine in their national immunization programs. As of December 2016, 82 countries do so [42]. Current recommendations are that the first dose of RotaTeq or Rotarix be administered between 6 and 15 weeks of age and that all doses be given by 32 weeks of age. Specifically, RotaTeq is given in three doses at 2, 4, and 6 months of age, while Rotarix is given in two doses at 2 and 4 months of age. Importantly, both RotaTeq and Rotarix have recently been implicated in associations with intussusception, though at even lower rates (1 6 cases per 100,000 vaccine recipients) than with RotaShield. In response to these reports, the US Food and Drug Administration initiated a study of the vaccines in their Post-Licensure Rapid Immunization Safety Monitoring (PRISM)

program, which found a significant increase in the risk of intussusception after the first dose of RotaTeq, but no increased risk with Rotarix [43]. The Vaccine Safety Datalink project, a collaboration between the CDC and large managed care organizations, also examined this question but attained conflicting data compared to the PRISM study, since there appeared to be increased risk associated with Rotarix, but not with RotaTeq [44]. Regardless, it is widely agreed among policymakers and health providers that the protection provided by RV vaccines against potentially severe gastroenteritis far outweighs the extremely low risk of intussusception.

F. Other Nationally Licensed Rotavirus Vaccines While RotaTeq and Rotarix are the only internationally prequalified RV vaccines, several countries have developed novel RV vaccines for their own national use [42]. None of these have been tested in the large-scale type of trials used for RotaTeq and Rotarix. Nonetheless, some have been administered widely, and data are available to support their effectiveness. For example, the Lanzhou lamb RV (LLR) vaccine, created at the Lanzhou Institute of Biological Products in China, is an attenuated live serotype G10 RV that was originally isolated from a lamb and passaged on primary calf kidney cells [45]. LLR was licensed in China in 2000, and is recommended to be given at 2 3 months of age and then again at 3 5 years of age [46]. Although prelicensure phase 3 clinical trials were not performed, millions of Chinese children have received LLR, and retrospective analyses suggest a vaccine efficacy against severe disease of 35% 70%, depending on the study [46,47]. Rotavac, also referred to as 116E, is a natural reassortant serotype G6 human RV containing a bovine VP4 segment. Interestingly, the virus was isolated in India from infected

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infants who mounted a strong immune response to the virus, but failed to develop symptoms, suggesting its use as a vaccine candidate [48]. Sponsored by the US National Institute of Allergy and Infectious Diseases (NIAID), the Indian company Bharat Biotech carried out clinical trials in India, and found Rotavac to be safe and to confer 55% protection against severe disease [49,50]. Excitingly, this efficacy is comparable to RotaTeq and Rotarix protection in developing countries. Rotavac was licensed in India in 2014, and included in that country’s national vaccine strategy in 2015. Bharat Biotech is currently seeking prequalification of Rotavac by the World Health Organization. The Indian government also recently licensed Rotasiil, or BRV-PV, which is a vaccine composed of five reassortant UK-bovine RV strains, each encoding a distinct human VP7 protein from G1, G2, G3, G4, and G9 serotypes [51]. This vaccine was developed as a partnership between the Serum Institute of India Limited and the US NIAID. Phase 3 clinical trials conducted in Nigeria and India demonstrated that Rotasiil provides protection comparable to that of RotaTeq and Rotarix in similar demographics [52,53]. A major advantage of Rotasiil over other RV vaccines is that it is heat-stable, so it may become the preferred formulation in lowincome countries, where access to refrigeration and power can be limited. While the licensed Rotasiil formulation is lyophilized, a liquid formulation has recently been demonstrated to be safe and well tolerated in adults [54]. Finally, Rotavin-M1 is a live attenuated serotype G1 RV vaccine that was developed and licensed in Vietnam in 2012 by the Center for Research and Production of Vaccines and Biologicals. Rotavin-M1 was isolated from a 6month old Vietnamese girl and serially passaged in cell culture [55]. In clinical trials, it has been deemed safe and immunogenic, resulting in seroconversion rates comparable to those of Rotarix [56].

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G. Remaining Obstacles to Rotavirus Vaccination and Future Directions A major goal of ongoing research in RV vaccinology is to gain a better understanding of the reduced efficacy of oral vaccines in developing nations [57]. While the complete answer is likely multifactorial, there has been progress in addressing this key gap in knowledge. First, several studies have uncovered a correlation between malnutrition and suboptimal RV vaccine efficacy or increased risk of RV disease [58,59]. Studies in a gnotobiotic piglet model provide compelling evidence that vitamin A deficiency impairs RV vaccine efficacy [60 62]. Second, a strong correlation was observed between the intestinal microbiome of infants and their RV vaccine response in a cohort in rural Ghana, with vaccine responders having microbiomes more similar to those of Dutch infants than to those of nonresponders [63]. Third, coinfections can have a marked effect on RV vaccine response. For example, enterovirus quantity was associated with RV vaccine failure in a cohort in urban Bangladesh [64]. Finally, numerous studies of infants in developing countries have observed a negative association between maternal RV-specific immunoglobulin G levels before infant vaccination and the seroconversion of the infant after RV vaccination [57]. In addition to the vaccines discussed in this chapter, there are a number of novel RV vaccines and vaccine strategies in various stages of development that may prove to be superior to existing formulations in providing protection to infants in developing countries [42]. For example, a vaccine is being developed in Australia that is given to infants at birth with the hope of eliciting an immune response prior to the complete development of the intestinal microbiota. There is also a parenteral subunit RV vaccine in clinical trials in the United States that could overcome specific deficiencies in mucosal immunity in malnourished children. In

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conclusion, the use of oral live attenuated RV strains, either multivalent human animal reassortant formulations or individual human virus formulations, have been highly effective at preventing severe disease in children in the industrialized world, and have been modestly effective at protecting children in developing countries. Many research groups are working on refinements of existing vaccines and development of novel vaccine strategies and formulations, offering hope that protection in developing countries can be enhanced to levels achieved in the industrialized world.

III. NOROVIRUS VACCINE DEVELOPMENT

encoding a polyprotein that gets processed into six mature nonstructural proteins, ORF2 encoding the major capsid protein VP1, and ORF3 encoding the minor structural protein VP2. The NoV capsid is composed of 90 dimers of the VP1 protein that assemble into icosahedral particles, with a small number of VP2 molecules residing inside the virion [74,75]. VP1 has a conserved shell domain (S), which forms the internal core protecting the RNA genome, and a protruding domain (P), which forms archlike protrusions extending away from the core; the P domain is further subdivided into P1, which forms the stalk of the arches, and P2, which is a hypervariable domain located at the tips of the arches [75].

A. Norovirus Disease

C. Norovirus Classification

Recent studies of the global burden of NoVattributable disease indicate that they cause an estimated 699 million cases of gastroenteritis and 219,000 fatalities annually [65,66]. In fact, NoVs are now recognized as the leading global cause of severe childhood diarrhea [65,67,68]. While NoVs are ubiquitous in that they infect all age groups and display similar disease incidence within high-, middle-, and low-income settings, the young, elderly, and immunocompromised are at higher risk of developing more severe and prolonged infections [65,69 73]. In 2016, NoV infections resulted in an estimated global loss of more than $4 billion in direct health system costs and $60.3 billion in societal costs [66]. Given the clinical and economic burdens of NoVs, development of vaccines against these pathogens is of utmost importance.

NoVs comprise a genus within the larger Caliciviridae family. NoVs are not currently segregated into serotypes because of the lack of a cell culture system, precluding the ability to test for antibody neutralization. Instead, they are segregated into seven genogroups and further subdivided into more than 40 genotypes based on genomic sequence similarity [76]. Viruses in genogroups I (GI), GII, and GIV are responsible for disease in humans. GII strains cause more than 90% of NoV disease in the United States, with GII genotype 4 (GII.4) variants causing 50% 80% of disease depending on the year [77]. Importantly, GII.4 variants emerged in a sequential fashion to cause six pandemics over the past two decades (1995 96, 2002, 2004, 2007 08, 2009 12, and 2012) interspersed with quiescent periods [78], similar to the epochal evolution of influenza viruses. Since 2014, a novel GII.17 strain has circulated widely [79]. GI strains are the second most common cause of human disease, accounting for 11% of cases [77]. GIV strains are most often associated with sporadic cases of gastroenteritis, although several recent studies

B. Norovirus Structure Human NoVs are nonenveloped singlestranded, positive-sense RNA viruses. Their genomes are 7.4 7.7 kb organized into three open reading frames (ORF), with ORF1

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have reported a high frequency of genetically variable GIV strains in environmental samples [80,81]. Because Norwalk virus, the first human NoV to be discovered, was a GI.1 virus and has been the most extensively studied NoV to date, it was the target of early human NoV vaccine development efforts. However, more recent efforts have shifted to GII strains because of their association with a majority of human disease.

D. Challenges in Norovirus Vaccine Development There are currently no licensed NoV vaccines. This is in large part due to the lack of a robust tissue culture system. The inability to grow and passage high-titer stocks of human NoV in vitro precludes the generation of live attenuated NoV strains through cell culture adaptation, the strategy used to develop most of our currently licensed viral vaccines, including those targeting RV, as described above. In addition to difficulties in propagating human NoVs, there are a number of other significant challenges to NoV vaccine design. First, the high degree of genetic and antigenic diversity among NoV strains may necessitate the development of multivalent vaccines. There is conflicting evidence for the existence of genogroup-specific and genotypespecific cross-protection among human NoV strains; the collective available data suggest a substantial degree of virus strain-specific variability, as reviewed recently [82]. Moreover, the rapid evolution of NoV through mutation and recombination is thought to facilitate the emergence of novel immune-escape variants that may not be targeted by preexisting vaccineinduced immunity [83]. Thus, an effective NoV immunization program may require frequent reformulation, similar to the influenza virus program. Finally, data suggest that the immune response to natural NoV infection wanes quickly [84,85], and there is conflicting evidence as to

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whether the presence of virus-specific antibodies directly correlates with protection from infection, as recently reviewed [82]. One feature of human NoV infection that is critical to consider in studies of protective immunity and vaccine efficacy is the genetic basis of host susceptibility to human NoV infection. Most human NoV strains require specific histo-blood group antigens (HBGAs) for infection. Consequently, a certain proportion of the population will be resistant to any given NoV strain. The inclusion of genetically resistant individuals in prior studies of protective immunity may have markedly confounded results and contributed to the often conflicting results in the field. Overall, correlates of protective immunity and factors regulating NoV vaccine efficacy remain poorly understood. In spite of these barriers, there have been extensive efforts to develop NoV vaccines, and results from small-scale clinical trials (summarized below) have been promising.

E. Virus-Like Particle-Based Norovirus Vaccines Because of the inability to generate live attenuated human NoVs in vitro, researchers have instead focused efforts on nonreplicating protein-based vaccines. A major advance in the field was the discovery that the VP1 capsid protein self-assembles into particles that are structurally and antigenically similar to native virions [86]. These virus-like particles (VLPs) were first shown to induce a virus-specific antibody response in mice [87,88]. Phase 1 clinical trials then determined that oral or intranasal administration of GI.1 VLPs was safe and immunogenic [89 91], setting the stage for an efficacy trial in 2009 10 sponsored by LigoCyte Pharmaceuticals (later acquired by Takeda Pharmaceuticals) [92]. Specifically, two doses of GI.1 VLPs plus adjuvant were administered intranasally to 38 healthy adult recipients with a 3-week interval between doses; another 39

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individuals received placebo. Three weeks following the boost, all participants were challenged orally with GI.1 live virus. There was a 47% reduction in gastroenteritis and a 35% reduction in the severity of disease in the vaccinated subjects compared to placebo controls. There was also a correlation between protection and the level of prechallenge antibody capable of blocking virus binding to HBGA. Several limitations of this trial are important to consider. First and most important, the challenge was performed 6 weeks after the first immunization, so these results do not indicate whether VLPs will elicit a long-term immune response. Related to this, the placebo group did not receive adjuvant, so it is possible that nonspecific adjuvant effects contributed to protection. Second, the small number of subjects in each group limits the power of these findings. Finally, only adults were included in this trial. Because more than 90% of adults are seropositive to NoV, the issue of variable levels of preexisting immunity among volunteers must be taken into consideration. Nonetheless, these studies were quite encouraging in terms of the prospect that NoV VLPs can protect people from live virus challenge. Next, a bivalent VLP vaccine containing a GI.1 construct and a consensus GII.4 construct engineered from sequences of three genetically distinct GII.4 strains was tested in preclinical and clinical trials. This formulation elicited an antibody response in rabbits and people following intramuscular inoculation [93,94]. In 2012 13, an efficacy study was carried out in which healthy adults were immunized intramuscularly with the bivalent vaccine formulation plus adjuvant twice 4 weeks apart (N 5 50) or placebo (N 5 48) and then were challenged with a GII.4 virus by day 42 postimmunization [95]. While the vaccine was immunogenic, indicated by an increase in virus-specific serum antibody levels, there was no significant reduction in the rate of infection or the incidence of gastroenteritis in the vaccinated group.

However, a trend toward less severe disease was noted in the vaccinated cohort. A concern with the experimental design of this study is that only 62.5% of subjects in the placebo group became infected and only 37.5% developed disease symptoms, reducing the power of the study. Furthermore, the same limitations that were described above for the GI.1 efficacy trial apply to this trial (i.e., the short-term nature of the study, the lack of an adjuvant-only control group, and the confounding factor of variable preexisting immunity in adult subjects). This bivalent VLP vaccine, sponsored by Takeda Pharmaceuticals, is currently being tested in a phase 2b field efficacy trial in a military setting and in phase 1 trials in children and elderly subjects [82].

F. Alternative Norovirus Vaccines in the Pipeline Another bivalent NoV VLP vaccine with GI.3 and GII.4 constructs is being tested in combination with the RV VP6 protein. This RV structural protein forms the intermediate layer of the triple-layered RV particle and can assemble into nanotubes when expressed in vitro [96,97]. When the NoV VLPs and RV VP6 protein were administered together into mice intramuscularly, humoral responses were generated to both NoV and RV antigens; in fact, the RV VP6 antigen appeared to boost the antibody response to NoV [98,99]. There are non-VLP NoV constructs being tested as vaccine candidates as well, all of which are VP1 protein-based approaches. First, a recombinant adenovirus vector engineered to express a GII.4 VP1 protein has been shown to be immunogenic in mice following intranasal inoculation, and the immunogenicity was enhanced when boosted with a NoV VLP immunization [100,101]. Second, the VP1 P domain will assemble into subviral particles referred to as P particles, and these have

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recently received attention as a vaccine candidate because they are immunogenic and easily produced in Escherichia coli and yeast [102,103]. Intranasal immunization with P particles in gnotobiotic piglets has proven comparably effective to VLPs in reducing the incidence of disease [104].

G. Future Directions in Norovirus Vaccine Development While NoV VLP vaccines have generated promising results in small-scale clinical trials, many open questions pertaining to the success of these formulations on a population level remain. Will they provide long-term protection? Will they provide protection to the complement of genetically diverse NoV strains circulating at any given time as well as newly emergent strains that episodically cause pandemics? Will they be effective in adults who have complex and varied preexisting immune histories, or should efforts be tailored to infant immunizations? The large-scale phase 2b clinical trials being carried out by Takeda Pharmaceuticals should provide insight into a number of these questions. Moreover, there is renewed hope in the field for the development of live attenuated NoV vaccines: After almost 50 years of failed attempts, two in vitro propagation systems for human NoV are now available. The first, developed by the Karst lab, utilizes a human B cell line. The second, developed by the Estes group, uses human intestinal enteroids derived from intestinal stem cells [105,106]. Although both systems support human NoV replication, neither results in robust amplification of the virus, so refinement of these systems is highly warranted and is being pursued by multiple research groups. Once the field has access to a robust propagation system, true antibody neutralization assays can be performed, and live attenuated vaccine constructs will become a possibility.

IV. CONCLUDING REMARKS Since the widespread inclusion of RV vaccines in national immunization programs, the incident of RV-associated deaths has been estimated to be halved. Although this is a remarkable achievement, the vast majority of deaths occur in developing countries, where the efficacy of current vaccines is not as robust. This fact underlines the importance of continued research and refinement of RV vaccines. The success of RV vaccines lends encouragement and importance to the development of a vaccine against NoV, which has overtaken RV as the leading cause of severe childhood diarrhea. Although these efforts are in their infancy, there have been encouraging results associated with NoV vaccines, lending hope that these two prevalent diseases will 1 day be a memory of the past, similar to poliovirus infections.

References [1] Matson DO, Estes MK. Impact of rotavirus infection at a large pediatric hospital. J Infect Dis 1990;162 598 604. [2] O’Ryan M, Pe´rez-Schael I, Mamani N, Pen˜a A, Salinas B, Gonza´lez G, et al. Rotavirus-associated medical visits and hospitalizations in South America: a prospective study at three large sentinel hospitals. Pediatr Infect Dis J 2001;20:685 93. [3] Rodriguez WJ, Kim HW, Brandt CD, Bise B, Kapikian AZ, Chanock RM, et al. Rotavirus gastroenteritis in the Washington, DC, area: incidence of cases resulting in admission to the hospital. Am J Dis Child 1980;1960 (134):777 9. [4] Parashar UD, Hummelman EG, Bresee JS, Miller MA, Glass RI. Global illness and deaths caused by rotavirus disease in children. Emerg Infect Dis 2003;9:565 72. Available from: https://doi.org/10.3201/eid0905.020562. [5] Tate JE, Burton AH, Boschi-Pinto C, Steele AD, Duque J, Parashar UD. 2008 estimate of worldwide rotavirusassociated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and metaanalysis. Lancet Infect Dis 2012;12:136 41. Available from: https://doi.org/10.1016/S1473-3099(11)70253-5.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

708

41. DEVELOPMENT OF ORAL ROTAVIRUS AND NOROVIRUS VACCINES

[6] Luchs A, Timenetsky M do CST. Group A rotavirus gastroenteritis: post-vaccine era, genotypes and zoonotic transmission. Einstein 2016;14:278 87. Available from: https://doi.org/10.1590/S167945082016RB3582. [7] Delem A, Lobmann M, Zygraich N. A bovine rotavirus developed as a candidate vaccine for use in humans. J Biol Stand 1984;12:443 5. [8] Vesikari T, Isolauri E, D’Hondt E, Delem A, Andre´ FE, Zissis G. Protection of infants against rotavirus diarrhoea by RIT 4237 attenuated bovine rotavirus strain vaccine. Lancet Lond Engl 1984;1:977 81. [9] Hanlon P, Hanlon L, Marsh V, Byass P, Shenton F, Hassan-King M, et al. Trial of an attenuated bovine rotavirus vaccine (RIT 4237) in Gambian infants. Lancet Lond Engl 1987;1:1342 5. [10] Lanata CF, Black RE, del Aguila R, Gil A, Verastegui H, Gerna G, et al. Protection of Peruvian children against rotavirus diarrhea of specific serotypes by one, two, or three doses of the RIT 4237 attenuated bovine rotavirus vaccine. J Infect Dis 1989;159:452 9. [11] Clark HF, Furukawa T, Bell LM, Offit PA, Perrella PA, Plotkin SA. Immune response of infants and children to low-passage bovine rotavirus (strain WC3). Am J Dis Child 1986;1960(140):350 6. [12] Clark HF, Horian FE, Bell LM, Modesto K, Gouvea V, Plotkin SA. Protective effect of WC3 vaccine against rotavirus diarrhea in infants during a predominantly serotype 1 rotavirus Season. J Infect Dis 1988;158: 570 87. Available from: https://doi.org/10.1093/ infdis/158.3.570. [13] Vesikari T, Rautanen T, Varis T, Beards GM, Kapikian AZ. Rhesus rotavirus candidate vaccine. Clinical trial in children vaccinated between 2 and 5 months of age. Am J Dis Child 1990;1960(144):285 9. [14] Flores J, Gonzalez M, Perez M, Cunto W, Perez-Schael I, Garcia D, et al. Protection against severe rotavirus diarrhoea by rhesus rotavirus vaccine in venezuelan infants. Lancet 1987;882 4. Available from: https:// doi.org/10.1016/S0140-6736(87)92858-3 Originally published as 1(8538) 329. [15] Gothefors L, Wadell G, Juto P, Taniguchi K, Kapikian AZ, Glass RI. Prolonged efficacy of rhesus rotavirus vaccine in swedish children. J Infect Dis 1989;159: 753 7. Available from: https://doi.org/10.1093/ infdis/159.4.753. [16] Stuker G, Oshiro LS, Schmidt NJ. Antigenic comparisons of two new rotaviruses from rhesus monkeys. J Clin Microbiol 1980;11:202 3. [17] Vesikari T, Kapikian AZ, Delem A, Zissis G. A comparative trial of rhesus monkey (RRV-1) and bovine (RIT 4237) oral rotavirus vaccines in young children. J Infect Dis 1986;153:832 9.

[18] Kapikian AZ, Hoshino Y, Chanock RM, Pe´rez-Schael I. Efficacy of a quadrivalent rhesus rotavirus-based human rotavirus vaccine aimed at preventing severe rotavirus diarrhea in infants and young children. J Infect Dis 1996;174(Suppl. 1):S65 72. [19] Joensuu J, Koskenniemi E, Pang XL, Vesikari T. Randomised placebo-controlled trial of rhesus-human reassortant rotavirus vaccine for prevention of severe rotavirus gastroenteritis. Lancet Lond Engl 1997;350: 1205 9. Available from: https://doi.org/10.1016/ S0140-6736(97)05118-0. [20] Pe´rez-Schael I, Guntin˜as MJ, Pe´rez M, Pagone V, Rojas AM, Gonza´lez R, et al. Efficacy of the rhesus rotavirusbased quadrivalent vaccine in infants and young children in Venezuela. N Engl J Med 1997;337:1181 7. Available from: https://doi.org/10.1056/NEJM19971 0233371701. [21] Rennels MB, Glass RI, Dennehy PH, Bernstein DI, Pichichero ME, Zito ET, et al. Safety and efficacy of high-dose rhesus-human reassortant rotavirus vaccines--report of the National Multicenter Trial. United States Rotavirus Vaccine Efficacy Group. Pediatrics 1996;97:7 13. [22] Santosham M, Moulton LH, Reid R, Croll J, Weatherholt R, Ward R, et al. Efficacy and safety of high-dose rhesus-human reassortant rotavirus vaccine in Native American populations. J Pediatr 1997;131: 632 8. [23] Murphy TV, Gargiullo PM, Massoudi MS, Nelson DB, Jumaan AO, Okoro CA, et al. Intussusception among infants given an oral rotavirus vaccine. N Engl J Med 2001;344:564 72. Available from: https://doi.org/ 10.1056/NEJM200102223440804. [24] Glass RI, Kilgore PE, Holman RC, Jin S, Smith JC, Woods PA, et al. The epidemiology of rotavirus diarrhea in the United States: surveillance and estimates of disease burden. J Infect Dis 1996;174(Suppl. 1):S5 11. Available from: https://doi.org/10.1093/infdis/174. Supplement_1.S5. [25] Parashar UD, Holman RC, Cummings KC, Staggs NW, Curns AT, Zimmerman CM, et al. Trends in intussusception-associated hospitalizations and deaths among US infants. Pediatrics 2000;106:1413 21. [26] Heaton PM, Goveia MG, Miller JM, Offit P, Clark HF. Development of a pentavalent rotavirus vaccine against prevalent serotypes of rotavirus gastroenteritis. J Infect Dis 2005;192(Suppl. 1):S17 21. Available from: https://doi.org/10.1086/431500. [27] Vesikari T, Matson DO, Dennehy P, Van Damme P, Santosham M, Rodriguez Z, et al. Safety and efficacy of a pentavalent human bovine (WC3) reassortant rotavirus vaccine. N Engl J Med 2006;354:23 33. Available from: https://doi.org/10.1056/NEJMoa052664.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

REFERENCES

[28] Vesikari T, Karvonen A, Ferrante SA, Ciarlet M. Efficacy of the pentavalent rotavirus vaccine, RotaTeqs , in Finnish infants up to 3 years of age: the Finnish Extension Study. Eur J Pediatr 2010;169: 1379 86. Available from: https://doi.org/10.1007/ s00431-010-1242-3. [29] Zaman K, Dang DA, Victor JC, Shin S, Yunus M, Dallas MJ, et al. Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: a randomised, doubleblind, placebo-controlled trial. Lancet Lond Engl 2010;376:615 23. Available from: https://doi.org/ 10.1016/S0140-6736(10)60755-6. [30] Armah GE, Sow SO, Breiman RF, Dallas MJ, Tapia MD, Feikin DR, et al. Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub-Saharan Africa: a randomised, double-blind, placebo-controlled trial. Lancet Lond Engl 2010;376:606 14. Available from: https://doi.org/10.1016/S0140-6736(10)60889-6. [31] Bernstein DI, Smith VE, Sherwood JR, Schiff GM, Sander DS, DeFeudis D, et al. Safety and immunogenicity of live, attenuated human rotavirus vaccine 8912. Vaccine 1998;16:381 7. Available from: https:// doi.org/10.1016/S0264-410X(97)00210-7. [32] Vesikari T, Karvonen A, Korhonen T, Espo M, Lebacq E, Forster J, et al. Safety and immunogenicity of RIX4414 live attenuated human rotavirus vaccine in adults, toddlers and previously uninfected infants. Vaccine 2004;22:2836 42. Available from: https://doi. org/10.1016/j.vaccine.2004.01.044. [33] Ruiz-Palacios GM, Pe´rez-Schael I, Vela´zquez FR, Abate H, Breuer T, Clemens SC, et al. Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med 2006;354:11 22. Available from: https://doi.org/10.1056/NEJMoa052434. [34] Cortese MM, Immergluck LC, Held M, Jain S, Chan T, Grizas AP, et al. Effectiveness of monovalent and pentavalent rotavirus vaccine. Pediatrics 2013;132:e25 33. Available from: https://doi.org/10.1542/peds.2012-3804. [35] Steele AD, Neuzil KM, Cunliffe NA, Madhi SA, Bos P, Ngwira B, et al. Human rotavirus vaccine Rotarixt provides protection against diverse circulating rotavirus strains in African infants: a randomized controlled trial. BMC Infect Dis 2012;12:213. Available from: https://doi.org/10.1186/1471-2334-12-213. [36] Vesikari T, Karvonen A, Prymula R, Schuster V, Tejedor JC, Cohen R, et al. Efficacy of human rotavirus vaccine against rotavirus gastroenteritis during the first 2 years of life in European infants: randomised, double-blind controlled study. Lancet Lond Engl 2007;370:1757 63. Available from: https://doi.org/ 10.1016/S0140-6736(07)61744-9.

709

[37] O’Ryan M, Lucero Y, Linhares AC. Rotarixs : vaccine performance 6 years postlicensure. Expert Rev Vaccines 2011;10:1645 59. Available from: https:// doi.org/10.1586/erv.11.152. [38] Cunliffe NA, Witte D, Ngwira BM, Todd S, Bostock NJ, Turner AM, et al. Efficacy of human rotavirus vaccine against severe gastroenteritis in Malawian children in the first two years of life: a randomized, double-blind, placebo controlled trial. Vaccine Rotavirus Vaccines Children Dev Countries 2012;30: A36 43. Available from: https://doi.org/10.1016/j. vaccine.2011.09.120. [39] Kawamura N, Tokoeda Y, Oshima M, Okahata H, Tsutsumi H, van Doorn LJ, et al. Efficacy, safety and immunogenicity of RIX4414 in Japanese infants during the first two years of life. Vaccine 2011;29:6335 41. Available from: https://doi.org/10.1016/j.vaccine. 2011.05.017. [40] Madhi SA, Kirsten M, Louw C, Bos P, Aspinall S, Bouckenooghe A, et al. Efficacy and immunogenicity of two or three dose rotavirus-vaccine regimen in South African children over two consecutive rotavirus-seasons: a randomized, double-blind, placebo-controlled trial. Vaccine Rotavirus Vaccines Children Dev Countries 2012;30:A44 51. Available from: https://doi. org/10.1016/j.vaccine.2011.08.080. [41] O’Ryan M, Giaquinto C, Benninghoff B. Human rotavirus vaccine (Rotarix): focus on effectiveness and impact 6 years after first introduction in Africa. Expert Rev Vaccines 2015;14:1099 112. Available from: https://doi.org/10.1586/14760584.2015.1059282. [42] Deen J, Lopez AL, Kanungo S, Wang X-Y, Anh DD, Tapia M, et al. Improving rotavirus vaccine coverage: can newer-generation and locally produced vaccines help? Hum Vaccines Immunother 2018;14:495 9. Available from: https://doi.org/10.1080/21645515. 2017.1403705. [43] Yih WK, Lieu TA, Kulldorff M, Martin D, McMahillWalraven CN, Platt R, et al. Intussusception risk after rotavirus vaccination in U.S. infants. N Engl J Med 2014;370:503 12. Available from: https://doi.org/ 10.1056/NEJMoa1303164. [44] Weintraub ES, Baggs J, Duffy J, Vellozzi C, Belongia EA, Irving S, et al. Risk of intussusception after monovalent rotavirus vaccination. N Engl J Med 2014;370: 513 19. Available from: https://doi.org/10.1056/ NEJMoa1311738. [45] Chang J-T, Li X, Liu H-J, Yu L. Ovine rotavirus strain LLR-85-based bovine rotavirus candidate vaccines: construction, characterization and immunogenicity evaluation. Vet Microbiol 2010;146:35 43. Available from: https://doi.org/10.1016/j.vetmic. 2010.04.016.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

710

41. DEVELOPMENT OF ORAL ROTAVIRUS AND NOROVIRUS VACCINES

[46] Fu C, Wang M, Liang J, He T, Wang D, Xu J. Effectiveness of Lanzhou lamb rotavirus vaccine against rotavirus gastroenteritis requiring hospitalization: a matched casecontrol study. Vaccine 2007;25:8756 61. Available from: https://doi.org/10.1016/j.vaccine.2007.10.036. [47] Zhen S, Li Y, Wang S, Zhang X-J, Hao Z, Chen Y, et al. Effectiveness of the live attenuated rotavirus vaccine produced by a domestic manufacturer in China studied using a population-based case control design. Emerg Microbes Infect 2015;4:e64. Available from: https://doi.org/10.1038/emi.2015.64. [48] Bhan MK, Lew JF, Sazawal S, Das BK, Gentsch JR, Glass RI. Protection conferred by neonatal rotavirus infection against subsequent rotavirus diarrhea. J Infect Dis 1993;168:282 7. [49] Bhandari N, Rongsen-Chandola T, Bavdekar A, John J, Antony K, Taneja S, et al. Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian infants: a randomised, double-blind, placebo-controlled trial. Lancet 2014;383:2136 43. Available from: https:// doi.org/10.1016/S0140-6736(13)62630-6. [50] Bhandari N, Sharma P, Glass RI, Ray P, Greenberg H, Taneja S, et al. Safety and immunogenicity of two live attenuated human rotavirus vaccine candidates, 116E and I321, in infants: results of a randomised controlled trial. Vaccine 2006;24:5817 23. Available from: https://doi.org/10.1016/j.vaccine.2006.05.001. [51] Zade JK, Kulkarni PS, Desai SA, Sabale RN, Naik SP, Dhere RM. Bovine rotavirus pentavalent vaccine development in India. Vaccine Rotavirus India Epidemiol Vaccines 2014;32:A124 8. Available from: https://doi.org/10.1016/j.vaccine.2014.03.003. [52] Isanaka S, Guindo O, Langendorf C, Matar Seck A, Plikaytis BD, Sayinzoga-Makombe N, et al. Efficacy of a low-cost, heat-stable oral rotavirus vaccine in Niger. N Engl J Med 2017;376:1121 30. Available from: https://doi.org/10.1056/NEJMoa1609462. [53] Naik SP, Zade JK, Sabale RN, Pisal SS, Menon R, Bankar SG, et al. Stability of heat stable, live attenuated Rotavirus vaccine (ROTASIILs ). Vaccine 2017;35: 2962 9. Available from: https://doi.org/10.1016/j. vaccine.2017.04.025. [54] Anil K, Desai S, Bhamare C, Dharmadhikari A, Madhusudhan RL, Patel J, et al. Safety and tolerability of a liquid bovine rotavirus pentavalent vaccine (LBRVPV) in adults. Vaccine 2018;36:1542 4. Available from: https://doi.org/10.1016/j.vaccine.2018.02.024. [55] Le LT, Nguyen TV, Nguyen PM, Huong NT, Huong NT, Huong NTM, et al. Development and characterization of candidate rotavirus vaccine strains derived from children with diarrhoea in Vietnam. Vaccine Rotavirus Asia Dis Burden Genotypes Vaccine Introduction 2009;27:F130 8. Available from: https:// doi.org/10.1016/j.vaccine.2009.08.086.

[56] Dang DA, Nguyen VT, Vu DT, Nguyen THA, Nguyen DM, Yuhuan W, et al. A dose-escalation safety and immunogenicity study of a new live attenuated human rotavirus vaccine (Rotavin-M1) in Vietnamese children. Vaccine 2012;30(Suppl. 1):A114 121. Available from: https://doi.org/10.1016/j.vaccine.2011.07.118. [57] Desselberger U. Differences of rotavirus vaccine effectiveness by country: likely causes and contributing factors. Pathogens 2017;6:65. Available from: https://doi. org/10.3390/pathogens6040065. [58] Colgate ER, Haque R, Dickson DM, Carmolli MP, Mychaleckyj JC, Nayak U, et al. Delayed dosing of oral rotavirus vaccine demonstrates decreased risk of rotavirus gastroenteritis associated with serum zinc: a randomized controlled trial. Clin Infect Dis Off Publ Infect Dis Soc Am 2016;63:634 41. Available from: https:// doi.org/10.1093/cid/ciw346. [59] Gruber JF, Hille DA, Liu GF, Kaplan SS, Nelson M, Goveia MG, et al. Heterogeneity of rotavirus vaccine efficacy among infants in developing countries. Pediatr Infect Dis J 2017;36:72 8. Available from: https://doi.org/10.1097/INF.0000000000001362. [60] Vlasova AN, Chattha KS, Kandasamy S, Siegismund CS, Saif LJ. Prenatally acquired vitamin A deficiency alters innate immune responses to human rotavirus in a gnotobiotic pig model. J Immunol 2013;190:4742 53. Available from: https://doi.org/10.4049/jimmunol.1203575. [61] Chattha KS, Kandasamy S, Vlasova AN, Saif LJ. Vitamin A deficiency impairs adaptive B and T cell responses to a prototype monovalent attenuated human rotavirus vaccine and virulent human rotavirus challenge in a gnotobiotic piglet model. PLoS One 2013;8: e82966. Available from: https://doi.org/10.1371/journal.pone.0082966. [62] Kandasamy S, Chattha KS, Vlasova AN, Saif LJ. Prenatal vitamin A deficiency impairs adaptive immune responses to pentavalent rotavirus vaccine (RotaTeqs ) in a neonatal gnotobiotic pig model. Vaccine 2014;32:816 24. Available from: https://doi. org/10.1016/j.vaccine.2013.12.039. [63] Harris VC, Armah G, Fuentes S, Korpela KE, Parashar U, Victor JC, et al. Significant correlation between the infant gut microbiome and rotavirus vaccine response in rural ghana. J Infect Dis 2017;215:34 41. Available from: https://doi.org/10.1093/infdis/jiw518. [64] Taniuchi M, Platts-Mills JA, Begum S, Uddin MJ, Sobuz SU, Liu J, et al. Impact of enterovirus and other enteric pathogens on oral polio and rotavirus vaccine performance in Bangladeshi infants. Vaccine 2016;34: 3068 75. Available from: https://doi.org/10.1016/j. vaccine.2016.04.080. [65] Ahmed SM, Hall AJ, Robinson AE, Verhoef L, Premkumar P, Parashar UD, et al. Global prevalence of norovirus in cases of gastroenteritis: a systematic

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

REFERENCES

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

review and meta-analysis. Lancet Infect Dis 2014;14: 725 30. Available from: https://doi.org/10.1016/ S1473-3099(14)70767-4. Bartsch SM, Lopman BA, Ozawa S, Hall AJ, Lee BY. Global economic burden of norovirus gastroenteritis. PLoS One 2016;11:e0151219. Available from: https:// doi.org/10.1371/journal.pone.0151219. Koo HL, Neill FH, Estes MK, Munoz FM, Cameron A, Dupont HL, et al. Noroviruses: the most common pediatric viral enteric pathogen at a large university hospital after introduction of rotavirus vaccination. J Pediatr Infect Dis Soc 2013;2:57 60. Available from: https://doi.org/10.1093/jpids/pis070. Payne DC, Vinje´ J, Szilagyi PG, Edwards KM, Staat MA, Weinberg GA, et al. Norovirus and medically attended gastroenteritis in U.S. children. N Engl J Med 2013;368:1121 30. Available from: https://doi.org/ 10.1056/NEJMsa1206589. Bok K, Green KY. Norovirus gastroenteritis in immunocompromised patients. N Engl J Med 2012;367: 2126 32. Available from: https://doi.org/10.1056/ NEJMra1207742. Kaufman SS, Green KY, Korba BE. Treatment of norovirus infections: moving antivirals from the bench to the bedside. Antiviral Res 2014;105:80 91. Available from: https://doi.org/10.1016/j.antiviral.2014.02.012. Murata T, Katsushima N, Mizuta K, Muraki Y, Hongo S, Matsuzaki Y. Prolonged norovirus shedding in infants ,or 5 6 months of age with gastroenteritis. Pediatr Infect Dis J 2007;26:46 9. Available from: https://doi.org/10.1097/01.inf.0000247102.04997.e0. Robilotti E, Deresinski S, Pinsky BA. Norovirus. Clin Microbiol Rev 2015;28:134 64. Available from: https://doi.org/10.1128/CMR.00075-14. van Asten L, van den Wijngaard C, van Pelt W, van de Kassteele J, Meijer A, van der Hoek W, et al. Mortality attributable to 9 common infections: significant effect of influenza A, respiratory syncytial virus, influenza B, norovirus, and parainfluenza in elderly persons. J Infect Dis 2012;206:628 39. Available from: https:// doi.org/10.1093/infdis/jis415. Glass PJ, White LJ, Ball JM, Leparc-Goffart I, Hardy ME, Estes MK. Norwalk virus open reading frame 3 encodes a minor structural protein. J Virol 2000;74:6581 91. Available from: https://doi.org/ 10.1128/JVI.74.14.6581-6591.2000. Prasad BV, Rothnagel R, Jiang X, Estes MK. Threedimensional structure of baculovirus-expressed Norwalk virus capsids. J Virol 1994;68:5117 25. Kroneman A, Vega E, Vennema H, Vinje´ J, White PA, Hansman G, et al. Proposal for a unified norovirus nomenclature and genotyping. Arch Virol 2013;158: 2059 68. Available from: https://doi.org/10.1007/ s00705-013-1708-5.

711

[77] Vega E, Barclay L, Gregoricus N, Shirley SH, Lee D, Vinje´ J. Genotypic and epidemiologic trends of norovirus outbreaks in the United States, 2009 to 2013. J Clin Microbiol 2014;52:147 55. Available from: https://doi.org/10.1128/JCM.02680-13. [78] Bull RA, White PA. Mechanisms of GII.4 norovirus evolution. Trends Microbiol 2011;19:233 40. Available from: https://doi.org/10.1016/j.tim.2011.01.002. [79] de Graaf M, van Beek J, Vennema H, Podkolzin A, Hewitt J, Bucardo F, et al. Emergence of a novel GII.17 norovirus end of the GII.4 era? Euro Surveill 2015;20:21178. Available from: https://doi.org/10.2807/ 1560-7917.ES2015.20.26.21178. [80] Kitajima M, Rachmadi AT, Iker BC, Haramoto E, Gerba CP. Genetically distinct genogroup IV norovirus strains identified in wastewater. Arch Virol 2016;161:3521 5. Available from: https://doi.org/ 10.1007/s00705-016-3036-z. [81] La Rosa G, Pourshaban M, Iaconelli M, Muscillo M. Detection of genogroup IV noroviruses in environmental and clinical samples and partial sequencing through rapid amplification of cDNA ends. Arch Virol 2008;153:2077 83. Available from: https://doi.org/ 10.1007/s00705-008-0241-4. [82] Cortes-Penfield NW, Ramani S, Estes MK, Atmar RL. Prospects and challenges in the development of a norovirus vaccine. Clin Ther 2017;39:1537 49. Available from: https://doi.org/10.1016/j.clinthera. 2017.07.002. [83] Debbink K, Lindesmith LC, Donaldson EF, Baric RS. Norovirus immunity and the great escape. PLoS Pathog 2012;8:e1002921. Available from: https://doi. org/10.1371/journal.ppat.1002921. [84] Johnson PC, Mathewson JJ, DuPont HL, Greenberg HB. Multiple-challenge study of host susceptibility to Norwalk gastroenteritis in US adults. J Infect Dis 1990;161:18 21. [85] Parrino TA, Schreiber DS, Trier JS, Kapikian AZ, Blacklow NR. Clinical immunity in acute gastroenteritis caused by Norwalk agent. N Engl J Med 1977;297: 86 9. [86] Jiang X, Wang M, Graham DY, Estes MK. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein. J Virol 1992;66:6527 32. [87] Guerrero RA, Ball JM, Krater SS, Pacheco SE, Clements JD, Estes MK. Recombinant Norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses. J Virol 2001;75:9713 22. Available from: https://doi. org/10.1128/JVI.75.20.9713-9722.2001. [88] Ball JM, Hardy ME, Atmar RL, Conner ME, Estes MK. Oral immunization with recombinant Norwalk viruslike particles induces a systemic and mucosal immune response in mice. J Virol 1998;72:1345 53.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

712

41. DEVELOPMENT OF ORAL ROTAVIRUS AND NOROVIRUS VACCINES

[89] El-Kamary SS, Pasetti MF, Mendelman PM, Frey SE, Bernstein DI, Treanor JJ, et al. Adjuvanted intranasal Norwalk virus-like particle vaccine elicits antibodies and antibody-secreting cells that express homing receptors for mucosal and peripheral lymphoid tissues. J Infect Dis 2010;202:1649 58. Available from: https://doi.org/10.1086/657087. [90] Ball JM, Graham DY, Opekun AR, Gilger MA, Guerrero RA, Estes MK. Recombinant Norwalk virus like particles given orally to volunteers: phase I study. Gastroenterology 1999;117:40 8. Available from: https://doi.org/10.1016/S0016-5085(99)70548-2. [91] Ramirez K, Wahid R, Richardson C, Bargatze RF, ElKamary SS, Sztein MB, et al. Intranasal vaccination with an adjuvanted Norwalk virus-like particle vaccine elicits antigen-specific B memory responses in human adult volunteers. Clin Immunol 2012;144: 98 108. Available from: https://doi.org/10.1016/j. clim.2012.05.006. [92] Atmar RL, Bernstein DI, Harro CD, Al-Ibrahim MS, Chen WH, Ferreira J, et al. Norovirus vaccine against experimental human Norwalk virus illness. N Engl J Med 2011;365:2178 87. Available from: https://doi. org/10.1056/NEJMoa1101245. [93] Parra GI, Bok K, Taylor R, Haynes JR, Sosnovtsev SV, Richardson C, et al. Immunogenicity and specificity of norovirus Consensus GII.4 virus-like particles in monovalent and bivalent vaccine formulations. Vaccine 2012;30:3580 6. Available from: https://doi. org/10.1016/j.vaccine.2012.03.050. [94] Treanor JJ, Atmar RL, Frey SE, Gormley R, Chen WH, Ferreira J, et al. A novel intramuscular bivalent norovirus virus-like particle vaccine candidate—reactogenicity, safety, and immunogenicity in a phase 1 trial in healthy adults. J Infect Dis 2014;210:1763 71. Available from: https://doi.org/10.1093/infdis/jiu337. [95] Bernstein DI, Atmar RL, Lyon GM, Treanor JJ, Chen WH, Jiang X, et al. Norovirus vaccine against experimental human GII.4 virus illness: a challenge study in healthy adults. J Infect Dis 2015;211:870 8. Available from: https://doi.org/10.1093/infdis/jiu497. [96] Bugli F, Caprettini V, Cacaci M, Martini C, Paroni Sterbini F, Torelli R, et al. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein. Int J Nanomed 2014;9:2727 39. Available from: https:// doi.org/10.2147/IJN.S60014. [97] Estes MK, Crawford SE, Penaranda ME, Petrie BL, Burns JW, Chan WK, et al. Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system. J Virol 1987;61:1488 94.

[98] Blazevic V, Lappalainen S, Nurminen K, Huhti L, Vesikari T. Norovirus VLPs and rotavirus VP6 protein as combined vaccine for childhood gastroenteritis. Vaccine 2011;29:8126 33. Available from: https://doi. org/10.1016/j.vaccine.2011.08.026. [99] Tamminen K, Lappalainen S, Huhti L, Vesikari T, Blazevic V. Trivalent combination vaccine induces broad heterologous immune responses to norovirus and rotavirus in mice. PLoS One 2013;8:e70409. Available from: https://doi.org/10.1371/journal. pone.0070409. [100] Guo L, Wang J, Zhou H, Si H, Wang M, Song J, et al. Intranasal administration of a recombinant adenovirus expressing the norovirus capsid protein stimulates specific humoral, mucosal, and cellular immune responses in mice. Vaccine 2008;26:460 8. Available from: https://doi.org/10.1016/j.vaccine.2007.11.039. [101] Guo L, Zhou H, Wang M, Song J, Han B, Shu Y, et al. A recombinant adenovirus prime-virus-like particle boost regimen elicits effective and specific immunities against norovirus in mice. Vaccine 2009;27:5233 8. Available from: https://doi.org/10.1016/j.vaccine. 2009.06.065. [102] Tan M, Fang P, Chachiyo T, Xia M, Huang P, Fang Z, et al. Noroviral P particle: structure, function and applications in virus-host interaction. Virology 2008;382:115 23. Available from: https://doi.org/ 10.1016/j.virol.2008.08.047. [103] Tan M, Hegde RS, Jiang X. The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors. J Virol 2004;78:6233 42. Available from: https://doi.org/10.1128/JVI.78.12. 6233-6242.2004. [104] Kocher J, Bui T, Giri-Rachman E, Wen K, Li G, Yang X, et al. Intranasal P particle vaccine provided partial cross-variant protection against human GII.4 norovirus diarrhea in gnotobiotic pigs. J Virol 2014;88:9728 43. Available from: https://doi.org/ 10.1128/JVI.01249-14. [105] Ettayebi K, Crawford SE, Murakami K, Broughman JR, Karandikar U, Tenge VR, et al. Replication of human noroviruses in stem cell derived human enteroids. Science 2016;353:1387 93. Available from: https://doi.org/10.1126/science.aaf5211. [106] Jones MK, Watanabe M, Zhu S, Graves CL, Keyes LR, Grau KR, et al. Enteric bacteria promote human and mouse norovirus infection of B cells. Science 2014;346:755 9. Available from: https://doi.org/ 10.1126/science.1257147.

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Mucosal Vaccines Against HIV/SIV Infection Hiroyuki Yamamoto1, Hiroshi Ishii1 and Tetsuro Matano1,2 1

AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan 2The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

I. INTRODUCTION This chapter describes the current progress of studies for the development of mucosal HIV vaccines that will induce effective antibody and T cell responses. In the final part of the chapter, we discuss the possible synergistic efficacy of antibody and T cell responses against HIV infection.

II. MUCOSAL VACCINES INDUCING HIV-SPECIFIC ANTIBODY RESPONSES A. Characterization of Anti-HIV Neutralizing Antibodies to Design a Mucosal HIV Vaccine It is now widely recognized that HIV is highly resistant to neutralizing antibodies (NAbs), and it appears practically difficult to design a NAb-inducing vaccine by conventional strategies [1]. Due to the heavy glycosylation of the target envelope (Env) protein [2] and

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00042-0

its intrinsic morphology [3], the conserved antigenic sites of HIV Env are very difficult for normal anti-Env Abs to access. Anti-HIV NAbs often require to highly mutate by undergoing extensive B cell receptor (BCR) affinity maturation, often to build looped protrusions, mainly composed of complementarity-determining region 3 (CDR3) of the immunoglobulin G (IgG) heavy chain, to reach functionally conserved and cryptic Env epitopes such as the CD4-binding site or other regions [4]. Among such anti-HIV NAbs, a broadly neutralizing HIV antibody (bNAb) against a panel of HIV strains is considered to be the key component to be induced. Recent technical advances in single B cell/antibody isolation [5] have resulted in the identification and characterization of a vast set of bNAbs targeting several critical regions of HIV Env [6]. The important question of an antibody/NAbbased vaccine is where, anatomically, to induce these antibody titers. The two major compartments where these Abs are needed to segregate include the systemic compartment (peripheral

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blood) and mucosal compartment. Considerable interest has been taken in developing a mucosal antibody-based HIV vaccine, owing to the sexually transmitted nature of the virus [7]. In the past three decades, attempts have been made through passive immunization studies, characterization of potent monoclonal anti-HIV bNAbs, and analysis of clinical trials. Extended from these, current studies also aim for highresolution in situ analysis of infected mucosal tissues.

B. Passive Anti-HIV Antibody Administration as a Model of Mucosal Vaccines Because it is immensely difficult to build even a prototype regimen of an antibody-based HIV vaccine, efforts have been made to define the potency of anti-HIV antibodies by passive immunization studies in animal models. To this end, several important factors need to be focused to aim for a mucosal vaccine. First, assessment of antibody-mediated virus protection inevitably requires a sufficient level of viral detection sensitivity after virus challenge, which was achieved in the late 1990s by the establishment of plasma viral RNA quantitation for routine monitoring of in vivo viral replication [8]. Following this step, it was first demonstrated that administration of high-dose HIV-specific polyclonal and monoclonal NAbs can provide sufficient protection against intravenous challenge of chimeric CXCR4-tropic SHIVs (simian-human immunodeficiency viruses) in macaques [9]. The second important aspect is the virus challenge route. Modifying the challenge route of the virus to a mucosal one (e.g., oral, intrarectal, or intravaginal challenge) in macaque passive immunization models has set grounds for adequate estimation of mucosal titers required for the SHIV/SIV protection. A body of work has characterized the efficacy of passively administered anti-HIV polyclonal NAbs

and bNAbs in these mucosal CCR5-tropic SHIV challenge models, and it was found that protection against mucosally challenged virus may not require titers as high as those required against intravenous challenge, providing optimism for development of a mucosal vaccine [10 13]. Another factor related to this is the specificity of the antibody. A recent study has discovered that the biological half-life of bNAbs passively infused by a single administration determines the longevity of sterile protection against repeated intrarectal low-dose SHIV challenges [14]. These results together suggest the importance of attaining sufficient NAb titers at the mucosal interface of infection.

C. Antibody-Related Correlates in HIV Vaccine Clinical Trials Antibodies are composed of five subtypes: IgM, IgG, IgA, IgE, and IgD. IgG is the most predominant effector of systemic Ab titers, whereas IgA dimerizes and often provides bivalent protection on mucosal surfaces. In typical acute viral infections such as influenza virus infection, antiviral IgA antibodies can be the major mucosal effectors for viral blockade [15]. Therefore, it would be straightforward to expect that protective correlates in Ab-based HIV vaccine trials may derive certain protective IgA-related factors by immune correlates analysis. Uniquely differing from this expectation, the RV144 trial in Thailand of a poxvirus vector prime-protein boost vaccination eliciting HIV V1/V2-specific Ab responses as protection correlates [16] showed that a skewing to virus-specific IgG responses was protective, whereas skewing to IgA responses correlated more negatively with protection [17]. One functional in vitro study on these two subtypes later showed that binding of anti-Env IgA impedes the binding of anti-Env IgG, and the resulting cellular immunemediated effector function, providing a potential explanation of their discordant correlations [18]. These results collectively indicate that mucosal

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II. MUCOSAL VACCINES INDUCING HIV-SPECIFIC ANTIBODY RESPONSES

Ab-based HIV vaccines may need to employ mechanisms distinct from conventional vaccines to afford protection. Among these, an important candidate to focus on is the Ab Fc (constant region)-mediated effector function. In one of the passive immunization studies, abrogation of Fc receptor binding in bNAb b12 resulted in impairment of sterile SHIV protection, which provided a rationale for taking Fc effector functions into consideration [19]. Furthermore, a genotype subgroup analysis on the RV144 trial has found that the rate of protection afforded differed strikingly when participants were classified by their Fc gamma receptor polymorphisms [20], which further implicated the potential involvement of Ab effector functions in this trial. A meta-analysis performed in another study found that Fc-receptor-related profiles of anti-HIV antibodies, such as subclasses, differed among vaccine trials, which could also withhold unknown links with the outcomes [21]. Collectively, clinical trials have indicated that Ab-related protective correlates may indeed exist in an HIV vaccine, posing fundamental questions for the precise mechanism of protection against mucosal HIV transmission.

D. Analysis of Mucosal Tissues and Ab Effector Function for Vaccine Design To develop a robust Ab-based mucosal HIV vaccine, it is critical to technically enrich analysis of the mucosal site of infection. The most important step here is in situ visualization of SHIV/SIV infection in the mucosa. One practical approach for establishing such models is the use of female nonhuman primates that have been vaginally challenged with SIV or SHIV. Here, the goal is to characterize viral dynamics and infection against the very first encountered target cells, requiring sophisticated techniques for probing. Visualization of SIV infection on cells in the female reproductive tract has precisely mapped the distribution of infected cells in the mucosa, providing morphological clues to attain protective HIV Ab titers [22]. In handling mucosal

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tissues for such analysis, it is important to take into account the influence of the menstrual cycle and related mucosal changes. Studies have addressed this by monitoring the change of virus target interactions by topical administration of progestins in SIV-infected female rhesus macaques [23]. Furthermore, recent tissue culture analysis of male penile and foreskin epithelia has depicted how female-to-male transmission occurs [24]. These reports together provide important platform knowledge for designing mucosal HIV vaccines. Further analysis sheds light on the potential effector mechanisms of HIV Abs in the mucosal sites. A recent study has indicated a correlation of the Fc region glycosylation profiles of antiHIV Abs with its binding to MUC16, the most abundant type of mucin in the reproductive mucosa, suggesting that the resultant stabilization profile of anti-HIV Abs can directly modify the patterns of in situ mucosal infection as well as phagocytosis and/or virion transfer across mucosal sites [25]. This finding may provide potential explanation for the protective correlates of IgG instead of IgA responses in the RV144 trial; that is, Fc effector functions of certain IgG subclasses might have more predominant influence than the bivalent antiviral activity of IgA dimers. Thus the Ab-related environmental milieu within the mucosa could affect mucosal HIV infection, and its optimization for HIV protection may need unexpected patterns of effector function modulation (Fig. 42.1). Current studies analyzing residual viral genomes in homogenized tissues of passively NAb-immunized macaques by ultrasensitive polymerase chain reaction methods have suggested that evaluation of mucosa-associated virological parameters is critical for defining HIV protection [26,27]. Finally, a recent work revisiting protective correlates of live-attenuated nef-deleted SIV vaccines has indicated that mucosal oligomeric gp41-specific IgG induced by nef-deleted SIV infection could be a protective factor in blockade of wild-type SIV challenge [28]. Another report

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FIGURE 42.1 Modes of Ab-based protection against mucosal HIV infection. Anti-HIV Abs, including neutralizing Abs (NAbs), can exert a vast array of protective mechanisms against mucosal HIV infection, which should be taken into account for rational design of an Ab-based HIV vaccine.

has found robust Ab affinity maturation evoked by live-attenuated nef-deleted SIV vaccines [29], implying the potential contribution of mucosal anti-HIV Ab responses to HIV protection. Altogether, we need to take a vast array of factors into consideration for development of a vaccine inducing effective mucosal anti-HIV Abs.

III. MUCOSAL VACCINES INDUCING HIV-SPECIFIC T CELL RESPONSES A. Mucosal HIV Infection in the Acute Phase Toward Systemic Infection Sexual transmission of HIV occurs via the intrarectal or intravaginal route, followed by massive acute depletion of HIV-targeted

memory CD41 T cells in the gut mucosal tissues [30 32]. Involvement of dendritic cells (DCs) in mucosal HIV transmission has been indicated (Fig. 42.2). Langerhans-cell-like DC subsets and plasmacytoid DCs in the mucosal epithelium that express HIV receptors, CD4 and CCR5, and are susceptible to HIV infection can be the first targets for HIV infection [33,34]. HIV replication in DCs is not efficient, but HIV transmission from HIV-infected DCs to CD41 T cells could trigger efficient acute HIV proliferation. Alternatively, HIV captured by mucosal DCs via C-type lectin such as DC-SIGN is transmitted to CD41 T cells, facilitating efficient acute HIV proliferation [35,36]. Such DC-mediated HIV transmission results in massive HIV replication and severe depletion of memory CCR51 CD41 T cells in gut mucosal lymphoid tissues in the acute

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FIGURE 42.2 Modes of T-cellbased control of mucosal HIV replication.

phase [30 32]. This rapid CD41 T cell depletion in gut mucosal tissues impairs adaptive immune response and damages mucosal epithelium barrier, leading to chronic immune activation with persistent HIV replication in HIV-infected individuals [37]. Mucosal HIVspecific T cell responses may be the front line against acute mucosal HIV replication and rapid HIV dissemination [38]. Indeed, a previous report has indicated higher levels of mucosal T cells with higher functionality in HIV elite controllers [39]. Induction of effective mucosal T cell responses is thus considered to be important for the control of acute mucosal HIV replication and systemic HIV spread as well as the protection of HIV transmission (Fig. 42.1). Mucosal T cell responses have been analyzed in macaque AIDS models of SIV or SHIV infection. In particular, the SIV infection model played a central role in demonstrating massive depletion of memory CD41 T cells by virus infection in gut mucosal tissues in the acute phase of infection [30 32]. Repeated intrarectal low-dose SIV/SHIV challenges have been used for evaluation of vaccine efficacy against mucosal transmission [40].

Live attenuated nef-deleted SIV vaccines, although not applicable to clinical use, to safety concerns, have been intensively examined because they can confer protection against wildtype pathogenic SIV challenge [41]. In macaques infected with live attenuated nef-deleted SIV, control of nef-deleted SIV replication is not complete but partial, resulting in induction of persistent T cell responses in mucosal tissues. These T cell responses have been indicated to contribute to consistent protection from mucosal wild-type SIV challenge [42], implying the rationale for development of a vaccine inducing effective mucosal anti-HIV T cell responses.

B. Viral Vectors for Mucosal Anti-HIV T Cell Responses Optimization of vaccine delivery and immunogen is crucial for the development of an effective HIV vaccine. Recent studies have indicated the potential of several viral vectors to induce mucosal viral antigen-specific T cell responses as vaccine-delivery tools. Adenovirus (Ad) vectors have been widely used for induction of HIV/SIV antigen-specific

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T cell response. Clinical trials using recombinant Ad serotype 5 (Ad5) vectors failed to show efficacy and indicated the large inhibitory effect of preexisting anti-Ad5 Abs on vaccine immunogenicity [43,44]. Other serotype-derived Ad vectors that are less affected by preexisting antivector Abs in humans have been developed for efficient HIV-specific T cell responses [45,46]. Intramuscular Ad5/Ad26 vector vaccination has been shown to induce efficient T cell responses in intestine, vagina, and lung mucosal tissues as well as in peripheral lymphocytes in macaques [47,48]. Induction of T cell responses in mucosal tissues by intramuscular Ad26 vector vaccination has also been confirmed in a clinical trial [49]. Macaques orally immunized with aerosolized Ad5 vectors expressing SIV antigens showed efficient induction of SIVspecific T cell responses in the bronchoalveolar lavage and ameliorated CD41 T cell depletion in the intestine after intrarectal SIV challenge, compared to intramuscularly immunized animals [50]. Intramuscular and intrarectal immunization with Ad5 and chimpanzee Ad63 (ChAd63) vectors expressing SIV antigens has been indicated to reduce acquisition risk by repeated intrarectal low-dose SIV challenges in macaques [51] (Chapter 24: Recombinant Adenovirus Vectors as Mucosal Vaccines). Recombinant pox viruses such as modified vaccinia virus Ankara (MVA) and canarypox virus (CNPV) have been used as vaccinedelivery tools to induce mucosal T cell responses. Macaques intranasally immunized with DNAs coding full SIV genome, interleukin 2 (IL-2) and IL-15, and recombinant MVAs expressing SIV Gag, Pol, and Env showed efficient SIV-specific T cell induction in rectal mucosal tissues and slower AIDS progression than in intramuscularly immunized macaques [52]. The potential of intramuscular recombinant CNPV vector immunization to induce mucosal T cell responses was indicated in a clinical trial [16]. We have developed a vaccine using recombinant Sendai virus (SeV) vectors and have

shown the potential of this vector to efficiently induce antigen-specific T cell responses in macaques [53]. A phase 1 clinical trial has confirmed the safety and immunogenicity of the replication-competent SeV vector expressing HIV Gag [54]. Intranasal administration with recombinant SeV vectors efficiently induced T cell responses not only systemically, but also in the tonsil and local secondary lymphoid tissues proximal the nasal mucosa in macaques [55]. We have also detected T cell responses at the intestinal mucosa after intranasal SeV administration (unpublished data). These results indicate the potential of SeV vectors to induce mucosal T cell responses. The potential of rhesus cytomegalovirus (RhCMV) vectors to induce effective T cell responses against SIV challenge has been indicated in rhesus macaques [56]. Half of the animals vaccinated with recombinant RhCMV vectors were protected from mucosal pathogenic SIV challenge. Analysis implicated effector memory T cell responses induced by persistent RhCMV replication in this effective SIV protection. It has been reported that these SIV-specific CD81 T cells induced by RhCMV vectors target not canonical MHC-I-restricted epitopes but MHC-II or MHC-E-restricted epitopes [57].

C. Mucosal T Cell Responses Effective Against HIV Infection CD81 T cell responses are crucial for the control of HIV replication [58,59]. Individual viral antigen-specific CD81 T cells show different efficacy against virus replication, and optimization of vaccine immunogens is critical for induction of effective anti-HIV CD81 T cell responses. Dominant induction of ineffective T cell responses could inhibit induction of effective CD81 T cell responses. Cumulative studies have implicated CD81 T cell responses targeting HIV Gag and Vif in HIV control [53,60 62]. Currently, the HIVconsv

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REFERENCES

immunogen, consisting of multiple conserved regions of the HIV proteome [63], and the HTI immunogen, consisting of HIV Gag/Pol/Vif/ Nef-derived regions that were relatively conserved and targeted predominantly by individuals with reduced viral loads [64], have been proposed as optimized immunogen candidates for induction of effective CD81 T cell responses. CD41 T cell responses are crucial for effective CD81 T cell induction [65], but HIV/ SIV-specific CD41 T cells can be preferential targets for HIV infection [66]. Our recent study analyzing vaccine efficacy against intravenous SIV challenge in macaques has indicated that virus-specific CD107a2 CD41 T cell induction by vaccination did not lead to efficient CD41 T cell responses following infection, but rather accelerated viral replication in the acute phase [67]. It is therefore speculated that induction of mucosal HIV-specific CD41 T cells by vaccination may enhance acute HIV replication after mucosal HIV transmission. This suggests the benefit of avoiding virus-specific CD41 T cell induction in HIV vaccine design. CD81 T induction with the help of vector antigenspecific CD41 T cell responses by vaccination can be an effective strategy [68]. We have previously shown that passive polyclonal NAb immunization at day 7 after SIV infection results in induction of effective T cell responses, leading to long-term SIV control in rhesus macaques [69 71]. Analysis has indicated Ab-mediated enhancement of antigen uptake by DCs, followed by induction of Gagspecific polyfunctional CD41 T cell responses and increased in vitro viral suppressive activity in CD81 cells, suggesting synergism between Abs and T cells for the control of mucosal HIV/ SIV replication. This has been confirmed in a macaque model of SHIV infection by another group [72], indicating a possible benefit of the combination of Ab and T cell induction in HIV vaccine design.

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References [1] Burton DR, Ahmed R, Barouch DH, Butera ST, Crotty S, Godzik A, et al. A blueprint for HIV vaccine discovery. Cell Host Microbe 2012;12(4):396 407. [2] Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, et al. Antibody neutralization and escape by HIV-1. Nature 2003;422(6929):307 12. [3] Stewart-Jones GB, Soto C, Lemmin T, Chuang GY, Druz A, Kong R, et al. Trimeric HIV-1-Env structures define glycan shields from clades A, B, and G. Cell 2016;165(4):813 26. [4] Zhou T, Georgiev I, Wu X, Yang ZY, Dai K, Finzi A, et al. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science 2010;329(5993): 811 17. [5] Wrammert J, Smith K, Miller J, Langley WA, Kokko K, Larsen C, et al. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 2008;453(7195):667 71. [6] Kwong PD, Mascola JR, Nabel GJ. Broadly neutralizing antibodies and the search for an HIV-1 vaccine: the end of the beginning. Nat Rev Immunol 2013;13(9): 693 701. [7] Keele BF, Giorgi EE, Salazar-Gonzalez JF, Decker JM, Pham KT, Salazar MG, et al. Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci U S A 2008;105(21):7552 7. [8] Shibata R, Maldarelli F, Siemon C, Matano T, Parta M, Miller G, et al. Infection and pathogenicity of chimeric simian-human immunodeficiency viruses in macaques: determinants of high virus loads and CD4 cell killing. J Infect Dis 1997;176(2):362 73. [9] Shibata R, Igarashi T, Haigwood N, Buckler-White A, Ogert R, Ross W, et al. Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nat Med 1999;5(2):204 10. [10] Mascola JR, Stiegler G, VanCott TC, Katinger H, Carpenter CB, Hanson CE, et al. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med 2000;6(2):207 10. [11] Parren PW, Marx PA, Hessell AJ, Luckay A, Harouse J, Cheng-Mayer C, et al. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/ human immunodeficiency virus at serum levels giving complete neutralization in vitro. J Virol 2001;75(17): 8340 7. [12] Hessell AJ, Poignard P, Hunter M, Hangartner L, Tehrani DM, Bleeker WK, et al. Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. Nat Med 2009;15(8):951 4.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

720

42. MUCOSAL VACCINES AGAINST HIV/SIV INFECTION

[13] Shingai M, Donau OK, Plishka RJ, Buckler-White A, Mascola JR, Nabel GJ, et al. Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques. J Exp Med 2014;211(10):2061 74. [14] Gautam R, Nishimura Y, Pegu A, Nason MC, Klein F, Gazumyan A, et al. A single injection of anti-HIV-1 antibodies protects against repeated SHIV challenges. Nature 2016;533(7601):105 9. [15] Suzuki T, Kawaguchi A, Ainai A, Tamura S, Ito R, Multihartina P, et al. Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus. Proc Natl Acad Sci U S A 2015;112(25): 7809 14. [16] Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, et al. MOPH-TAVEG Investigators. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 2009;361(23):2209 20. [17] Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, Alam SM, et al. Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med 2012;366(14):1275 86. [18] Tomaras GD, Ferrari G, Shen X, Alam SM, Liao HX, Pollara J, et al. Vaccine-induced plasma IgA specific for the C1 region of the HIV-1 envelope blocks binding and effector function of IgG. Proc Natl Acad Sci U S A 2013;110(22):9019 24. [19] Hessell AJ, Hangartner L, Hunter M, Havenith CE, Beurskens FJ, Bakker JM, et al. Fc receptor but not complement binding is important in antibody protection against HIV. Nature 2007;449(7158):101 4. [20] Li SS, Gilbert PB, Tomaras GD, Kijak G, Ferrari G, Thomas R, et al. FCGR2C polymorphisms associate with HIV-1 vaccine protection in RV144 trial. J Clin Invest 2014;124(9):3879 90. [21] Chung AW, Ghebremichael M, Robinson H, Brown E, Choi I, Lane S, et al. Polyfunctional Fc-effector profiles mediated by IgG subclass selection distinguish RV144 and VAX003 vaccines. Sci Transl Med 2014;6(228): 228ra38. [22] Stieh DJ, Maric D, Kelley ZL, Anderson MR, Hattaway HZ, Beilfuss BA, et al. Vaginal challenge with an SIVbased dual reporter system reveals that infection can occur throughout the upper and lower female reproductive tract. PLoS Pathog 2014;10(10):e1004440. [23] Carias AM, Allen SA, Fought AJ, Kotnik Halavaty K, Anderson MR, Jimenez ML, et al. vIncreases in Endogenous or Exogenous Progestins Promote VirusTarget Cell Interactions within the Non-human Primate Female Reproductive Tract. PLoS Pathog 2016;12(9):e1005885. [24] Dinh MH, Anderson MR, McRaven MD, Cianci GC, McCoombe SG, Kelley ZL, et al. Visualization of HIV-1 interactions with penile and foreskin epithelia: clues

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

for female-to-male HIV transmission. PLoS Pathog 2015;11(3):e1004729. Gunn B, Schneider J, Shansab M, Bastian AR, Fahrbach K, Smith 4th A, et al. Enhanced binding of antibodies generated during chronic HIV infection to mucus component MUC16. Mucosal Immunol 2016;9(6):1549 58. Hessell AJ, Jaworski JP, Epson E, Matsuda K, Pandey S, Kahl C, et al. Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques. Nat Med 2016;22(4): 362 8. Liu J, Ghneim K, Sok D, Bosche WJ, Li Y, Chipriano E, et al. Antibody-mediated protection against SHIV challenge includes systemic clearance of distal virus. Science 2016;353(6303):1045 9. Voss JE, Macauley MS, Rogers KA, Villinger F, Duan L, Shang L, et al. Reproducing SIVΔnef vaccine correlates of protection: trimeric gp41 antibody concentrated at mucosal front lines. AIDS 2016;30(16): 2427 38. Adnan S, Reeves RK, Gillis J, Wong FE, Yu Y, Camp JV, et al. Persistent low-level replication of SIVΔnef drives maturation of antibody and CD8 T cell responses to induce protective immunity against vaginal SIV infection. PLoS Pathog 2016;12(12):e1006104. Veazey RS, DeMaria M, Chalifoux LV, Shvetz DE, Pauley DR, Knight HL, et al. Gastrointestinal tract as a major site of CD4 1 T cell depletion and viral replication in SIV infection. Science 1998;280:427 31. Brenchley JM, Schacker TW, Ruff LE, Price DA, Taylor JH, Beilman GJ, et al. CD4 1 T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 2004;200:749 59. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M. Massive infection and loss of memory CD4 1 T cells in multiple tissues during acute SIV infection. Nature 2005;434:1093 7. Kawamura T, Gulden FO, Sugaya M, McNamara DT, Borris DL, Lederman MM, et al. R5 HIV productively infects Langerhans cells, and infection levels are regulated by compound CCR5 polymorphisms. Proc Natl Acad Sci U S A 2003;100:8401 6. Smed-So¨rensen A, Lore´ K, Vasudevan J, Louder MK, Andersson J, Mascola JR, et al. Differential susceptibility to human immunodeficiency virus type 1 infection of myeloid and plasmacytoid dendritic cells. J Virol 2005;79:8861 9. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 2000;100:587 97. Yu HJ, Reuter MA, McDonald D. HIV traffics through a specialized, surface-accessible intracellular compartment during trans-infection of T cells by mature dendritic cells. PLoS Pathog 2008;4:e1000134.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

REFERENCES

[37] Shan L, Siliciano RF. Unraveling the relationship between microbial translocation and systemic immune activation in HIV infection. J Clin Invest 2014;124: 2368 71. [38] Whitney JB, Hill AL, Sanisetty S, Penaloza-MacMaster P, Liu J, Shetty M, et al. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys. Nature 2014;512:74 7. [39] Ferre AL, Hunt PW, Critchfield JW, Young DH, Morris MM, Garcia JC, et al. Mucosal immune responses to HIV-1 in elite controllers: a potential correlate of immune control. Blood 2009;113:3978 89. [40] Martins MA, Wilson NA, Piaskowski SM, Weisgrau KL, Furlott JR, Bonaldo MC, et al. Vaccination with gag, vif, and nef gene fragments affords partial control of viral replication after mucosal challenge with SIVmac239. J Virol 2014;88:7493 516. [41] Daniel MD, Kirchhoff F, Czajak SC, Sehgal PK, Desrosiers RC. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 1992;258:1938 41. [42] Genesca` M, Skinner PJ, Bost KM, Lu D, Wang Y, Rourke TL, et al. Protective attenuated lentivirus immunization induces SIV-specific T cells in the genital tract of rhesus monkeys. Mucosal Immunol 2008;1: 219 28. [43] Buchbinder SP, Mehrotra DV, Duerr A, Fitzgerald DW, Mogg R, Li D, et al. Step Study Protocol Team. Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebocontrolled, test-of-concept trial. Lancet 2008;372: 1881 93. [44] Hammer SM, Sobieszczyk ME, Janes H, Karuna ST, Mulligan MJ, Grove D, et al. HVTN 505 Study Team. Efficacy trial of a DNA/rAd5 HIV-1 preventive vaccine. N Engl J Med 2013;369:2083 92. [45] Liu J, O’Brien KL, Lynch DM, Simmons NL, La Porte A, Riggs AM, et al. Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 2009;457:87 91. [46] Keefer MC, Gilmour J, Hayes P, Gill D, Kopycinski J, Cheeseman H, et al. A Phase I double blind, placebocontrolled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults. PLoS One 2012;7:e41936. [47] Baig J, Levy DB, McKay PF, Schmitz JE, Santra S, Subbramanian RA, et al. Elicitation of simian immunodeficiency virus-specific cytotoxic T lymphocytes in mucosal compartments of rhesus monkeys by systemic vaccination. J Virol 2002;76:11484 90. [48] Li H, Liu J, Carville A, Mansfield KG, Lynch D, Barouch DH. Durable mucosal simian immunodeficiency virus-specific effector memory T lymphocyte responses elicited by recombinant adenovirus vectors in rhesus monkeys. J Virol 2011;85:11007 15.

721

[49] Baden LR, Liu J, Li H, Johnson JA, Walsh SR, Kleinjan JA, et al. Induction of HIV-1-specific mucosal immune responses following intramuscular recombinant adenovirus serotype 26 HIV-1 vaccination of humans. J Infect Dis 2015;211:518 28. [50] Bolton DL, Song K, Wilson RL, Kozlowski PA, Tomaras GD, Keele BF, et al. Comparison of systemic and mucosal vaccination: impact on intravenous and rectal SIV challenge. Mucosal Immunol 2012;5:41 52. [51] Xu H, Andersson AM, Ragonnaud E, Boilesen D, Tolver A, Jensen BAH, et al. Mucosal vaccination with heterologous viral vectored vaccine targeting subdominant SIV accessory antigens strongly inhibits early viral replication. EBioMedicine 2017;18:204 15. [52] Manrique M, Kozlowski PA, Wang SW, Wilson RL, Micewicz E, Montefiori DC, et al. Nasal DNA-MVA SIV vaccination provides more significant protection from progression to AIDS than a similar intramuscular vaccination. Mucosal Immunol 2009;2:536 50. [53] Matano T, Kobayashi M, Igarashi H, Takeda A, Nakamura H, Kano M, et al. Cytotoxic T lymphocytebased control of simian immunodeficiency virus replication in a preclinical AIDS vaccine trial. J Exp Med 2004;199:1709 18. [54] Nyombayire J, Anzala O, Gazzard B, Karita E, Bergin P, Hayes P, et al. First-in-human evaluation of the safety and immunogenicity of an intranasally administered replication-competent Sendai virus-vectored HIV type 1 Gag vaccine: induction of potent T-cell or antibody responses in prime-boost regimens. J Infect Dis 2017;215:95 104. [55] Takeda A, Igarashi H, Nakamura H, Kano M, Iida A, Hirata T, et al. Protective efficacy of an AIDS vaccine, a single DNA-prime followed by a single booster with a recombinant replication-defective Sendai virus vector, in a macaque AIDS model. J Virol 2003;77:9710 15. [56] Hansen SG, Ford JC, Lewis MS, Ventura AB, Hughes CM, Coyne-Johnson L, et al. Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine. Nature 2011;473:523 7. [57] Hansen SG, Sacha JB, Hughes CM, Ford JC, Burwitz BJ, Scholz I, et al. Cytomegalovirus vectors violate CD8 1 T cell epitope recognition paradigms. Science 2013;340:1237874. [58] Koup RA, Safrit JT, Cao Y, Andrews CA, Mcleod G, Borkowsky W, et al. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol 1994;68:4650 5. [59] Matano T, Shibata R, Siemon C, Connors M, Lane HC, Martin MA. Administration of an anti-CD8 monoclonal antibody interferes with the clearance of chimeric simian/human immunodeficiency virus during primary infections of rhesus macaques. J Virol 1998;72: 164 9.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

722

42. MUCOSAL VACCINES AGAINST HIV/SIV INFECTION

[60] Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, et al. CD8 1 T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med 2007;13:46 53. [61] Goulder PJ, Watkins DI. Impact of MHC class I diversity on immune control of immunodeficiency virus replication. Nat Rev Immunol 2008;8:619 30. [62] Iwamoto N, Takahashi N, Seki S, Nomura T, Yamamoto H, Inoue M, et al. Control of simian immunodeficiency virus replication by vaccine-induced Gag- and Vifspecific CD8 1 T cells. J Virol 2014;88:425 33. [63] Borthwick N, Ahmed T, Ondondo B, Hayes P, Rose A, Ebrahimsa U, et al. Vaccine-elicited human T cells recognizing conserved protein regions inhibit HIV-1. Mol Ther 2014;22:464 75. [64] Mothe B, Hu X, Llano A, Rosati M, Olvera A, Kulkarni V, et al. A human immune data-informed vaccine concept elicits strong and broad T-cell specificities associated with HIV-1 control in mice and macaques. J Transl Med 2015;13:60. [65] Swain SL, McKinstry KK, Strutt TM. Expanding roles for CD41 T cells in immunity to viruses. Nat Rev Immunol 2012;12:136 48. [66] Douek DC, Brenchley JM, Betts MR, Ambrozak DR, Hill BJ, Okamoto Y, et al. HIV preferentially infects HIV-specific CD4 1 T cells. Nature 2002;417:95 8.

[67] Terahara K, Ishii H, Nomura T, Takahashi N, Takeda A, Shiino T, et al. Vaccine-induced CD107a1 CD41 T cells are resistant to depletion following AIDS virus infection. J Virol 2014;88:14232 40. [68] Tsukamoto T, Takeda A, Yamamoto T, Yamamoto H, Kawada M, Matano T. Impact of cytotoxic-Tlymphocyte memory induction without virus-specific CD4 1 T-cell help on control of a simian immunodeficiency virus challenge in rhesus macaques. J Virol 2009;83:9339 46. [69] Yamamoto H, Kawada M, Takeda A, Igarashi H, Matano T. Post-infection immunodeficiency virus control by neutralizing antibodies. PLoS One 2007;2:e540. [70] Yamamoto T, Iwamoto N, Yamamoto H, Tsukamoto T, Kuwano T, Takeda A, et al. Polyfunctional CD4 1 Tcell induction in neutralizing antibody-triggered control of simian immunodeficiency virus infection. J Virol 2009;83:5514 24. [71] Iseda S, Takahashi N, Poplimont H, Nomura T, Seki S, Nakane T, et al. Biphasic CD8 1 T-cell defense in simian immunodeficiency virus control by acute-phase passive neutralizing antibody immunization. J Virol 2016;90:6276 90. [72] Nishimura Y, Martin MA. Of mice, macaques, and men: broadly neutralizing antibody immunotherapy for HIV-1. Cell Host Microbe 2017;22(2):207 16.

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Mucosal Vaccines for Genital Herpes Ji Eun Oh1 and Akiko Iwasaki1,2,3 1

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, United States 2 Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT, United States 3Howard Hughes Medical Institute, Chevy Chase, MD, United States

I. INTRODUCTION Genital herpes is one of the most common sexually transmitted viral infections. Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are the causative agents of genital herpes. Over 500 million people are infected with herpes virus worldwide, with 23 million new infections are reported each year [1]. In the United States, 16% of people aged 14 49 years are seropositive for HSV-2, and the seroprevalence in women (21%) is estimated to be twice as high as that in men (12%) [2]. In some regions of sub-Saharan Africa, over 80% of people are seropositive for HSV-2 [3]. Historically, HSV-1 has been associated with oral herpes, while HSV-2 has been considered to be the predominant cause of genital herpes. However, in many Western nations, HSV-1 recently surpassed HSV-2 as the leading cause of genital herpes infections [4,5] (Fig. 43.1). Furthermore, the World Health Organization estimates that, in addition to the 417 million people with genital HSV-2 infections, there are approximately

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00043-2

140 million people living with genital HSV-1 infections worldwide [6,7] (Fig. 43.1). As a matter of public health, transmission of genital herpes is difficult to prevent because over 80% of seropositive people are asymptomatic yet capable of transmitting the virus to their uninfected partners [2]. Although symptoms of genital herpes can be alleviated by antiviral drugs such as acyclovir, these drugs are not sufficient to control asymptomatic shedding and potential transmission [8]. Genital herpes is also a significant risk factor for HIV-1 transmission, with a 3.1- and 2.7-fold higher risk of HIV-1 acquisition among HSV-2 seropositive women and men, respectively [9]. The estimate of total lifetime direct medical cost of genital herpes in the United States is $540.7 million, third only to the costs of HIV and HPV among eight major sexually transmitted infections [10]. Thus development of prophylactic vaccines to prevent acquisition of HSV, or therapeutic vaccines designed to reduce viral burden, severity of disease, and transmission remains an important goal. Although several

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FIGURE 43.1 Regional estimates of the prevalence of genital herpes caused by either HSV-1 or HSV-2 among 15- to 49-year-olds in 2012 by sex. Both HSV-1 and HSV-2 contribute to genital herpes. In Africa and Southeast Asia, the vast majority of cases of genital herpes are caused by HSV-2, whereas in the Americas, Europe, and the Western Pacific region, HSV-1 is becoming a dominant cause of genital herpes, particularly in males. Source: Graphed from data reported in Looker KJ, Magaret AS, May MT, Turner KM, Vickerman P, Gottlieb SL, et al. Global and regional estimates of prevalent and incident herpes simplex virus type 1 infections in 2012. PLoS One 2015;10:e0140765; Looker KJ, Magaret AS, Turner KM, Vickerman P, Gottlieb SL, Newman LM. Global estimates of prevalent and incident herpes simplex virus type 2 infections in 2012. PLoS One 2015;10:e114989.

vaccines have been tested in clinical trials, none of the candidates have met the goal of reducing herpes acquisition and disease.

II. HSV-1 AND HSV-2 VIRUS LIFE CYCLE Both HSV-1 and HSV-2 are members of the herpesvirus family, with a large double-stranded DNA genome (B153 kb) encoding approximately 80 genes. They are enveloped within host membranes containing viral glycoproteins. Inside the envelope, the viral genomic DNA is encapsidated. The viral capsid is surrounded by the tegument, which contains many molecules necessary to promote infection once the viral envelope fuses with the host membrane. Virus

entry into host cells involves multiple steps [11,12] (Fig. 43.2). First, viral envelope glycoproteins C (gC) and B (gB) bind to heparan sulfate on the host cell membrane. Next, glycoprotein D (gD) binds to one of the viral entry receptors on the host cell membrane, namely, nectin-1, nectin2, herpesvirus entry mediator (HVEM), or 3-O sulfated heparan sulfate. Finally, gB along with gH/gL binds to the paired immunoglobulin-like type 2 receptor alpha (PILRα), forming an entry pore for the viral capsid. After viral fusion with the plasma membrane, the de-enveloped capsid travels to the host cell nucleus and releases viral genomic DNA. Inside the nucleus, viral DNA replicates using the host machinery. HSV viral DNA is then transcribed, and viral proteins are generated for the synthesis of new virions. Simultaneously, the viral genome is transcribed

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III. PATHOGENESIS OF GENITAL HERPES

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FIGURE 43.2 HSV entry receptors and ligands. HSV uses its surface glycoproteins, gB, gC, gD, gH, and gL, to mediate entry into host cells. gB and gC mediate the initial attachment of virus particles to heparan sulfate on host cell surface proteoglycans. gB binding to paired immunoglobulin-like type 2 receptor alpha (PILRα) and gD binding to herpesvirus entry mediator (HVEM), nectin-1, nectin-2, or 3-O-sulphotransferase-modified heparan sulfate trigger membrane fusion, which is mediated by gB and the gH gL heterodimer, and releases the viral nucleocapsid into the host cell cytoplasm. Viral gene ˇ ´ JR, Spear PG, transcription occurs after the release of viral DNA into the cell nucleus. Source: Figure and legend are from Sedy Ware CF. Cross-regulation between herpesviruses and the TNF superfamily members. Nat Rev Immunol 2008;8:861 73, used with permission.

to produce a wide array of proteins that are needed for evading the immune responses as well as generating more virus. HSV genomic DNA becomes encapsidated, while tegument proteins assemble around the capsid and are reenveloped and released from the cells. These new virions go on to infect neighboring epithelial cells.

III. PATHOGENESIS OF GENITAL HERPES HSV-1 and HSV-2 enter the human host by first infecting the epithelial cells of the genital mucosa. Both viruses replicate actively in the keratinocytes—a process known as lytic replication—resulting in the killing of infected cells and causing damage to the tissue. Some of the viruses released from the epithelial cells go on to infect the sensory neurons innervating the

genital tissue. Inside neurons, the viral capsid travels up the axon by retrograde transport from the axon termini to the dorsal root ganglia (DRG) [13]. HSV-1 and HSV-2 enter a period of latency in neurons, where they shut down viral protein synthesis. How latency is initiated remains unclear. During latency, the latencyassociated transcript (LAT) is expressed while viral protein coding genes are repressed, and LAT actively promotes viral latency and host cell survival through several different mechanisms [14]. When an infected person is under stress or exposed to sunlight, HSV reactivates within the sensory neurons and travels back to the mucosa, where it reinfects epithelial cells and undergoes lytic replication. This leads to symptoms such as inflammation and ulceration. However, viral shedding in the vaginal mucosa can occur in the absence of lesions or symptoms [15,16]. Mathematical modeling suggests that

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frequent HSV-2 shedding episodes in humans can be explained by nearly constant release of small numbers of viruses from neurons [17].

IV. SYMPTOMS OF GENITAL HERPES The most common symptom of genital herpes is inflammation and ulceration in the skin around the genital area during recurrent outbreaks. The transmission of virus from mother to neonate during birth could cause serious complications, leading to fatal encephalitis and long-term neurological sequelae in the newborn child [18,19]. In addition to these well-documented sequelae, genital herpes also leads to dysfunction of organs that are connected by the DRG neurons. For example, in the mouse model of genital herpes, the virus travels from the DRG to the spinal cord and reaches the enteric neurons residing in the colonic muscularis layer [20]. Lytic infection of enteric neurons causes dismotility and toxic megacolon. In humans, chronic constipation and urinary retention are symptoms associated with genital herpes [21,22].

V. IMMUNE PROTECTIVE MECHANISMS AGAINST HSV INFECTIONS In order to develop an effective vaccine against HSV-1 or HSV-2, it is essential to understand the nature of the immune responses that confer protection against these viruses [23]. This section will focus on how innate and adaptive immune responses achieve such protection.

female reproductive tract is covered by a noncornified stratified squamous epithelial layer, or type II mucosa [24]. The lining of the lower reproductive tract is covered with a mucus layer, which contains antimicrobial peptides, commensal bacteria, and natural antibodies. Once HSV-2 penetrates this barrier and infects epithelial cells, both the epithelial cells and nearby sentinel cells of the genital mucosa recognize the virus through pattern recognition receptors (PRRs). This initiates immune responses against the virus. Both HSV-1 and HSV-2 are recognized by various cell surface and cytosolic PRRs. Toll-like receptor 9 (TLR9), expressed in various leukocytes, recognizes viral double-stranded DNA, while TLR2 senses viral glycoprotein. cGMP cAMP synthase (cGAS) is a cytosolic DNA sensor that activates stimulator of interferon genes (STING) in response to HSV infection [25]. Signaling from these sensors activates transcription factors, which induces expression of antiviral mediators such as type I and type III interferons (IFNs). Type I IFNs limit viral replication in the epithelial cells in an autocrine and paracrine manner. HSV infection also recruits various immune cells such as neutrophils, monocytes, natural killer (NK) cells, and dendritic cells (DCs). DCs initiate the priming of adaptive immune responses by taking up HSV-2 antigens and priming T cells in the draining lymph nodes. Once activated, CD4 and CD8 T cells migrate to the infection sites and help to control viral infection by producing IFNγ or by directly killing infected cells. Once antigenspecific T and B cells have been induced during a primary infection with HSV-1 or HSV-2, these effector cells mediate clearance of the virus and of infected cells in the genital mucosa.

A. Innate Immune Response to HSV Infections

B. Protective Memory Responses Against Genital Herpes Infection

Multiple lines of defense protect mucosal surfaces against viral infections. The lower

In designing vaccine approaches for blocking primary genital herpes infection, it is

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V. IMMUNE PROTECTIVE MECHANISMS AGAINST HSV INFECTIONS

important to consider the nature of the adaptive immune memory response capable of conferring antiviral protection (Fig. 43.3). 1. Antibody-Mediated Protection B cells mediate protection by secreting virusspecific immunoglobulins (Ig) or antibodies. Two types of antibodies, neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs), are protective against HSV-1 and HSV-2. nAbs inhibit virus attachment or entry into host cells by blocking receptor binding or cellular uptake. The isotype of nAbs present in mucosal secretions is dependent on the ability of the antibody to cross the epithelial barrier and reach the lumen. Vaginal epithelia do not express the polymeric Ig receptors (pIgR) and are therefore incapable of transporting dimeric IgA. However, they can transport IgG into the vaginal lumen, albeit somewhat ineffectively, via their expression of the neonatal Fc receptor (FcRn) (Fig. 43.3). nnAbs cannot block virus entry into host cells. However, they mediate antiviral effector functions by engaging NK cells’ FcγR to induce antibody-dependent cellular cytotoxicity (ADCC). FcγR (CD16)-expressing NK cells can bind to the Fc portion of nnAbs, themselves bound to surface antigens of infected cells, and mediate ADCC. nnAbs also engage the FcγR of phagocytes to induce antibody-dependent cellular phagocytosis (ADCP). Phagocytic FcγR mediates the nnAbdependent direct uptake and destruction of pathogens through ADCP. Finally, nnAbs activate complement and destroy pathogens [26]. Several studies using the mouse model of genital herpes have shown that anti-HSV-2 antibodies can protect against HSV-2 infection. Passive transfer of serum immunoglobulin from immunized mice helps to protect against wildtype (WT) HSV-2 in naı¨ve B-cell-deficient mice [27]. But the development of a vaccine that focuses on generating systemic antibodies has failed, most likely owing to the very high concentration of IgG required for preventing HSV-2

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infection in the genital mucosa. The minimum concentration of anti-HSV-2 IgG2a monoclonal antibodies (mAb III-174) required for complete protection against vaginal HSV-2 was reported to be around 50 μg/mL [28]. Even in mice immunized with an attenuated strain of HSV-2 (thymidine kinase-defective HSV-2, or TKHSV-2) and boosted with WT HSV-2, the concentration of HSV-2 specific IgG in the vagina is 2.6 μg/mL, more than an order of magnitude lower than the required concentration for protection [29]. Moreover, systemic IgGs are insufficient to protect against vaginal HSV-2 infection despite the expression of FcRn, capable of transporting IgG into the vaginal lumen, on vaginal epithelia cells [29]. Thus for a vaccine to be protective, it has to generate very high and stable levels of virus-specific antibody within the vaginal lumen. If protection of the host at the genital mucosa is unattainable, the second level of protection conferred by a vaccine is at the level of neuronal infection. A recent study showed that HSV2 specific antibodies protect against viral spread between DRG neurons [30] (Fig. 43.3). Antibodies alone are insufficient to confer protection, but virus-specific CD4 T cells enable antibody access through the blood brain barrier. These observations suggest that antibodybased vaccines must simultaneously generate CD4 T cell responses that potentiate antibody effector functions by granting access to neuronal tissues [31]. 2. T Cell-Mediated Protection Memory T cells confer protection against genital infection with HSV-1 and HSV-2. Unlike B cells, which secrete antibodies into circulation, T cells have to be at the site of infection to block viral replication. There are two ways in which T cells can reach the site of HSV infection in the vaginal mucosa. First, circulating memory T cells can enter the site of infection in response to inflammatory signals. Second, T cells can reside in the vaginal tissue

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CD4 T cell Lymph node

CD8 T cell B cell

HSV

Macrophage DC IgG

CD8 TRM

FcRn

Vaginal epithelia

Anterograde transport

IFN-γ

Dorsal root ganglia

Retrograde transport

CD4 TRM Blood vessel

Memory lymphocyte cluster

Spinal cord

FIGURE 43.3 Protective memory responses against genital HSV infection. Immunization induces effector T and B cells in the lymph nodes, which migrate to the genital mucosa. Local establishment of humoral and cellular memory responses is critical for protection against HSV infection. Virusspecific IgG can be transported into the vaginal lumen via FcRn, which can bind to incoming HSV to neutralize infection. Memory lymphocyte clusters provide robust protection against HSV challenge. These clusters are made up of macrophages, DCs, and CD4 and CD8 TRM cells. CD4 TRM cells secrete IFNγ to block viral replication. CD8 TRM cells residing in the epithelial layer and the submucosa carry out immune surveillance and enable viral control by secreting IFNγ upon stimulation by DCs. HSV-2-specific antibodies also protect against viral spread inside the DRG and the spinal cord. The access of these antibodies to the neural tissues depends on virus-specific CD4 T cells. CD8 TRM cells in the DRG can also block reactivation of the latent virus.

VI. VACCINE APPROACHES AGAINST GENITAL HERPES

before encountering the virus. Such tissueresident memory T (TRM) cells can be established by the vaccine strategy known as prime and pull [32]. Circulating memory T cells include central memory T (TCM) cells, which survey secondary lymphoid organs, and effector memory T (TEM) cells, which are capable of entering peripheral tissues [33]. However, the lower female reproductive tract restricts to TEM entry at steady state [34]. In response to signals provided by inflammatory chemokines, TEM cells gain access to the vaginal tissue. But it takes time for TEM cells to proliferate, migrate, and carry out effector functions at the site of infection. Therefore vaccine strategies based solely on TCM cells are unlikely to successfully prevent HSV infection and disease. In contrast, TEM cells established within the vaginal tissue, also known as TRM cells, are able to recognize pathogens and initiate protective responses more rapidly and effectively upon local infection. These cells directly control pathogens by rapidly producing cytokines and killing infected cells. TRM cells also amplify immune responses by recruiting more inflammatory cells and TCM cells to the site of infection [35,36]. Therefore vaccine strategies that establish local TRM cells provide robust protection to the host. The evidence that both CD4 and CD8 TRM cells confer robust protection comes from both human and mouse studies. CD4 TRM cells can be established by vaginal infection with an attenuated HSV-2 strain. These CD4 TRM cells are maintained in memory lymphoid clusters, which are made up of macrophages that present viral antigens to CD4 T cells to maintain their long-term residence in the vaginal submucosa (Fig. 43.3). CD4 TRM cells mediate better protection against lethal HSV-2 vaginal challenge compared to circulating memory CD4 T cells, which provide only partial protection, as has been shown in parabiotic mouse studies [37]. However, vaccine strategies based on immunization with attenuated HSV-2 are too risky in humans. CD8 T cells persisting in sensory ganglia also suppress

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reactivation of latent virus [38] (Fig. 43.3). In humans, virus-specific CD8 TRM cells at the dermal epidermal junction, adjacent to peripheral nerve endings, provide immune surveillance and viral control upon release of virus from the neurons into the genital epithelium [39,40]. In mathematical models of HSV-2 infection, the local CD8 T cell density in the mucosa inversely correlates with HSV-2 DNA copy number and predicts whether genital lesions or asymptomatic shedding will occur [41]. Together, these studies highlight the importance of the location of antiviral memory T cells as a key factor in conferring protection against genital herpes infection (Chapter 16: Regulation of Mucosal Immunity in the Genital Tract: Balancing Reproduction and Protective Immunity).

VI. VACCINE APPROACHES AGAINST GENITAL HERPES No vaccines are currently available for preventing HSV-1 or HSV-2 infection in humans. Most previous vaccine approaches against genital herpes have focused on generating systemic IgG responses against HSV-2.

A. Past Prophylactic Vaccine Trials Most HSV vaccine trials have focused on targeting viral glycoproteins. A recombinant subunit vaccine containing major HSV-2 surface glycoproteins (gB2 and gD2) with an oil water emulsion adjuvant (Chiron) induced high titers of serum nAbs and circulating CD4 T cell responses [42]. However, the Chiron vaccine failed to prevent either disease or infection with HSV-2. Moreover, it showed no difference between the control and vaccinated groups in terms of duration of the first episode of clinical symptoms or the recurrence of disease. Another subunit vaccine containing gD2 with alum and monophosphoryl lipid A (MPLA, a

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TLR4 agonist) adjuvant underwent two phase 3 trials sponsored by GlaxoSmithKline (GSK) Biologicals. This vaccine also induced both neutralizing serum antibodies and circulating CD4 T cell responses and showed 74% vaccine efficacy in women from discordant couples who were seronegative for both HSV-1 and HSV-2 [43]. However, a follow-up study with the same vaccine in a generalized group of women found no protection against HSV-2 infection or disease, while the vaccine did prevent genital HSV-1 disease [44]. It is still unclear why these vaccines failed in clinical trials. One possible reason is that they were not able to elicit robust local immunity in the genital mucosa of the vaccinated individuals.

B. Mucosal Vaccines Against HSV-2 In contrast to parenteral vaccines, which are given via injection, mucosal vaccines are applied to the surface of the mucosal tissue. These vaccines have been highly effective in protecting humans against poliovirus (oral Sabin vaccine), influenza (nasal FluMist), and rotavirus (oral Rotarix and RotaTeq). Mucosal vaccines have obvious advantages, including the lack of needle usage, their effectiveness, and the likelihood of establishing local TRM cells at the site of inoculation. A number of preclinical studies have examined the ability of mucosal vaccine candidates to block HSV-2 infection and disease in mice and guinea pigs. 1. Intravaginal HSV-2 Vaccines Intravaginal immunization with TK- HSV-2 establishes TRM cell responses and virus-specific IgG in the vagina in mice and guinea pigs [45 48]. Mice immunized with TK- HSV-2 are protected from challenge with lethal WT HSV-2 for life. This protection requires CD4 TRM cells [37]. But intravaginal immunization requires replication-competent HSV [45,49], making this strategy unsafe as a prophylactic vaccine.

Intravaginal immunization with recombinant viral subunit protein gB plus CpG oligodeoxynucleotides (ODNs) as adjuvant can induce high levels of gB-specific IgA and IgG in vaginal secretions and in serum [50]. These mice also showed higher survival rates, lower pathology scores, and lower vaginal viral titers following genital HSV-2 challenge compared to control mice. A similar study using intravaginal immunization with recombinant gD plus CpG ODNs showed that these vaccinated mice experienced strong antigen-specific T helper 1 (Th1)-like immune responses in the genital lymph nodes and spleens as well as mucosal and systemic IgG antibodies [51]. A topical herpes vaccine in which pseudotyped human papillomavirus is used to deliver genes encoding HSV-2 gB and gD showed robust TRM cell and antibody responses leading to protection against genital HSV-2 challenge [52]. Although intravaginal immunization is a highly effective way to establish both humoral and cellular immunity, particularly TRM cells in the vagina, it is difficult to implement as a prophylactic vaccine in humans, owing to safety concerns associated with delivering infectious agents and adjuvants in young girls. 2. Intranasal Vaccines Additional studies have examined whether immunization at other mucosal surfaces, such as intranasal immunization, can elicit protective immunity against genital HSV-2 infection. Intranasal immunization with genetically disabled HSV-1 (deletion in the glycoprotein H), TK- HSV-2, and vaccinia virus expressing HSV-2 gD led to protection comparable to that elicited by intravaginal immunization against genital HSV-2 infection in mice and guinea pigs [53 55]. Antibody-mediated immunity appears to play a major role in the mechanism of protection triggered by intranasal immunization with TK- HSV-2 [54]. Intranasal, but not intraperitoneal, immunization with TK- HSV-2 provides robust protection against vaginal

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VII. FUTURE STRATEGIES FOR HSV-2 VACCINE

challenge with WT HSV-2 [30,54,56]. The mode of protection depends on CD4 T cells and antibodies but not NK cells or CD8 T cells. Intranasal immunization elicits more robust circulating memory CD4 T cells capable of migrating into the DRG. Upon recognition of viral antigens, CD4 T cells secrete IFNγ, leading to the opening of the blood neuron barrier for antibody access to the DRG [31]. This allows clearance of virus from the DRG and protection of mice from lethal disease. Intranasal immunization with replicationdefective HSV-2, 5BLacZ defective in the UL29 gene, confers protection from genital HSV-2 challenge [57]. Heterologous viral vectors have been employed as agents of mucosal vaccine against HSV-2. Intranasal immunization with recombinant adenovirus vector expressing gB or replication-defective mutant of HSV-2 can induce high levels of antigen-specific IgG and IgA in the genital tract as well as serum antibodies. In addition, this strategy induces and maintains long-lived memory cytotoxic T cells in mucosal tissues and improves protection following lethal genital HSV-2 infection. In contrast, parenteral immunization (subcutaneous or intraperitoneal) is less effective in inducing mucosal immune responses [57 60]. The combination of a parenteral prime step with DNA encoding gD followed by an intranasal boost with liposomes encapsulating gD protein also conferred protection against vaginal HSV-2 challenge [61]. This heterologous immunization regimen stimulated high titers of nAbs in the serum as well as a T helper response dependent on the DNA prime step. It also enhanced mucosal immune responses and protective immunity at the port of entry for the virus, the vaginal cavity [61]. Another study using an IgG Fc fragment conjugated to HSV-2 gD, mimicking the FcRn-mediated transport of IgG across epithelial cells, showed that intranasal immunization together with CpG induced effective mucosal and systemic

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antibodies, B and T cell immune responses, and complete protection after intravaginal virulent HSV-2 challenge [62].

VII. FUTURE STRATEGIES FOR HSV-2 VACCINE As was described above, local TRM cells play a crucial role in protecting the host against genital HSV-2 infection. However, a safe method of establishing local TRM cells has proven difficult, as effector and memory T cells do not migrate into the genital mucosa at steady state. To overcome this problem, a new vaccine strategy known as prime and pull has been developed. This strategy involves a two-step process in which T cells are first “primed” via a parenteral vaccine. Such circulating T cells are “pulled” into the vaginal mucosa through topical application of chemokines (CXCL9 and CXCL10) to the vaginal epithelium. The prime and pull strategy establishes CD8 TRM cells in the genital mucosa, and they are able to protect the host against lethal HSV-2 challenge [32]. In mice immunized with this strategy, CD8 TRM cells block viral entry into the sensory neurons in the DRG and fully protect against lethal genital HSV-2 infection. Viral immune evasion is another key issue to consider for future vaccine design against HSV. HSV-1 and HSV-2 envelope glycoproteins mediate this process. HSV-2 glycoprotein C (gC2) is an immunoevasin that inhibits complement-mediated virus neutralization and the lysis of infected cells by binding the complement component C3 [63]. In addition, gE/gI glycoproteins act as IgG Fc receptor analogs that capture nAbs via their Fc domain and render them ineffective [64]. Simultaneous immunization with immunogens targeting virus entry molecules (gD) and immunoevasins (gC and/or gE/gI) induces antibody production against all of these components, blocking the inhibitory functions of gC and gE molecules.

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Several studies using this strategy showed the improved production of nAbs and robust CD4 T cell responses. Notably, this vaccine approach protected the mouse DRG from infection and reduced recurrent vaginal shedding of HSV-2 in guinea pig [64 68]. Mucosal vaccines that take advantage of such anti-immunoevasin approaches have a better chance of eliciting protective antibody responses in the genital mucosa.

VIII. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Unlike vaccines targeting acute viral infections, vaccines against genital herpes must take into account the virus’s peculiar life cycle, alternating between lytic and latent phases. The virus must be stopped before it establishes latent infection in DRG neurons. To achieve this, an effective herpes vaccine must induce strong and prolonged local immune responses in the genital tract to prevent initial infection of the genital mucosa and DRG. A possible reason for the failure of previous vaccine approaches is the lack of robust local immunity. The concentration of nAbs required to protect against HSV-2 infection in the genital mucosa is very high, owing to the ability of the virus to evade antibody effector functions. A successful vaccine will be able to induce robust immune responses both against herpes and against the viral immune evasion strategy. Another point of consideration is that previous vaccine attempts may have failed as a result of their inability to establish local TRM cells. The presence of local TRM cells in the genital mucosa may hold the key for successful future vaccine strategies.

Acknowlegment We thank Dr. Asu Erden for editorial support.

References [1] Looker KJ, Garnett GP, Schmid GP. An estimate of the global prevalence and incidence of herpes simplex virus type 2 infection. Bull World Health Organ 2008;86:805 12 A. [2] Control, C.F.D., and Prevention. Seroprevalence of herpes simplex virus type 2 among persons aged 14 49 years--United States, 2005 2008. MMWR Morb Mortal Wkly Rep 2010;59:456. [3] Paz-Bailey G, Ramaswamy M, Hawkes SJ, Geretti AM. Herpes simplex virus type 2: epidemiology and management options in developing countries. Sex Transm Infect 2007;83:16 22. [4] Malkin JE. Epidemiology of genital herpes simplex virus infection in developed countries. Herpes 2004;11 (Suppl. 1):2A 23A. [5] Bradley H, Markowitz LE, Gibson T, McQuillan GM. Seroprevalence of herpes simplex virus types 1 and 2-United States, 1999 2010. J Infect Dis 2014;209:325 33. [6] Looker KJ, Magaret AS, May MT, Turner KM, Vickerman P, Gottlieb SL, et al. Global and regional estimates of prevalent and incident herpes simplex virus type 1 infections in 2012. PLoS One 2015;10: e0140765. [7] Looker KJ, Magaret AS, Turner KM, Vickerman P, Gottlieb SL, Newman LM. Global estimates of prevalent and incident herpes simplex virus type 2 infections in 2012. PLoS One 2015;10:e114989. [8] Johnston C, Saracino M, Kuntz S, Magaret A, Selke S, Huang ML, et al. Standard-dose and high-dose daily antiviral therapy for short episodes of genital HSV-2 reactivation: three randomised, open-label, cross-over trials. Lancet 2012;379:641 7. [9] Freeman EE, Weiss HA, Glynn JR, Cross PL, Whitworth JA, Hayes RJ. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS 2006;20:73 83. [10] Owusu-Edusei Jr. K, Chesson HW, Gift TL, Tao G, Mahajan R, Ocfemia MC, et al. The estimated direct medical cost of selected sexually transmitted infections in the United States, 2008. Sex Transm Dis 2013;40:197 201. ˇ ´ JR, Spear PG, Ware CF. Cross-regulation between [11] Sedy herpesviruses and the TNF superfamily members. Nat Rev Immunol 2008;8:861 73. [12] Spear PG. Herpes simplex virus: receptors and ligands for cell entry. Cell Microbiol 2004;6:401 10. [13] Smith G. Herpesvirus transport to the nervous system and back again. Annu Rev Microbiol 2012;66:153 76. [14] Knipe DM, Cliffe A. Chromatin control of herpes simplex virus lytic and latent infection. Nat Rev Microbiol 2008;6:211 21.

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REFERENCES

[15] Tronstein E, Johnston C, Huang ML, Selke S, Magaret A, Warren T, et al. Genital shedding of herpes simplex virus among symptomatic and asymptomatic persons with HSV-2 infection. JAMA 2011;305:1441 9. [16] Wald A, Zeh J, Selke S, Warren T, Ryncarz AJ, Ashley R, et al. Reactivation of genital herpes simplex virus type 2 infection in asymptomatic seropositive persons. N Engl J Med 2000;342:844 50. [17] Schiffer JT, Abu-Raddad L, Mark KE, Zhu J, Selke S, Magaret A, et al. Frequent release of low amounts of herpes simplex virus from neurons: results of a mathematical model. Sci Transl Med 2009;1:7ra16. [18] Brown ZA, Selke S, Zeh J, Kopelman J, Maslow A, Ashley RL, et al. The acquisition of herpes simplex virus during pregnancy. N Engl J Med 1997;337:509 15. [19] Kimberlin DW, Lin CY, Jacobs RF, Powell DA, Frenkel LM, Gruber WC, et al. Natural history of neonatal herpes simplex virus infections in the acyclovir era. Pediatrics 2001;108:223 9. [20] Khoury-Hanold W, Yordy B, Kong P, Kong Y, Ge W, Szigeti-Buck K, et al. Viral spread to enteric neurons links genital HSV-1 infection to toxic megacolon and lethality. Cell Host Microbe 2016;19:788 99. [21] Caplan LR, Kleeman FJ, Berg S. Urinary retention probably secondary to herpes genitalis. N Engl J Med 1977;297:920 1. [22] Goodell SE, Quinn TC, Mkrtichian E, Schuffler MD, Holmes KK, Corey L. Herpes simplex virus proctitis in homosexual men. Clinical, sigmoidoscopic, and histopathological features. N Engl J Med 1983;308:868 71. [23] Iwasaki A. Exploiting mucosal immunity for antiviral vaccines. Annu Rev Immunol 2016;34:575 608. [24] Iwasaki A. Mucosal dendritic cells. Annu Rev Immunol 2007;25:381 418. [25] Sun L, Wu J, Du F, Chen X, Chen ZJ. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 2013;339:786 91. [26] Gunn BM, Alter G. Modulating antibody functionality in infectious disease and vaccination. Trends Mol Med 2016;22:969 82. [27] Schiffer JT, Corey L. Rapid host immune response and viral dynamics in herpes simplex virus-2 infection. Nat Med 2013;19:280 90. [28] Whaley KJ, Zeitlin L, Barratt RA, Hoen TE, Cone RA. Passive immunization of the vagina protects mice against vaginal transmission of genital herpes infections. J Infect Dis 1994;169:647 9. [29] McDermott MR, Brais LJ, Evelegh MJ. Mucosal and systemic antiviral antibodies in mice inoculated intravaginally with herpes simplex virus type 2. J Gen Virol 1990;71(Pt 7):1497 504. [30] Iijima N, Iwasaki A. Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help. Nature 2016;533:552 6.

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[31] Iwasaki A. Immune regulation of antibody access to neuronal tissues. Trends Mol Med 2017;23:227 45. [32] Shin H, Iwasaki A. A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature 2012;491:463 7. [33] Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999;401:708 12. [34] Shin H, Iwasaki A. Tissue-resident memory T cells. Immunol Rev 2013;255:165 81. [35] Ariotti S, Hogenbirk MA, Dijkgraaf FE, Visser LL, Hoekstra ME, Song JY, et al. T cell memory. Skinresident memory CD8(1) T cells trigger a state of tissue-wide pathogen alert. Science 2014;346:101 5. [36] Schenkel JM, Fraser KA, Vezys V, Masopust D. Sensing and alarm function of resident memory CD8 (1) T cells. Nat Immunol 2013;14:509 13. [37] Iijima N, Iwasaki A. T cell memory. A local macrophage chemokine network sustains protective tissueresident memory CD4 T cells. Science 2014;346:93 8. [38] St Leger AJ, Hendricks RL. CD8 1 T cells patrol HSV1-infected trigeminal ganglia and prevent viral reactivation. J Neurovirol 2011;17:528 34. [39] Zhu J, Koelle DM, Cao J, Vazquez J, Huang ML, Hladik F, et al. Virus-specific CD8 1 T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation. J Exp Med 2007;204:595 603. [40] Zhu J, Peng T, Johnston C, Phasouk K, Kask AS, Klock A, et al. Immune surveillance by CD8alphaalpha 1 skin-resident T cells in human herpes virus infection. Nature 2013;497:494 7. [41] Schiffer JT, Abu-Raddad L, Mark KE, Zhu J, Selke S, Koelle DM, et al. Mucosal host immune response predicts the severity and duration of herpes simplex virus-2 genital tract shedding episodes. Proc Natl Acad Sci USA 2010;107:18973 8. [42] Corey L, Langenberg AG, Ashley R, Sekulovich RE, Izu AE, Douglas Jr. JM, et al. Recombinant glycoprotein vaccine for the prevention of genital HSV-2 infection: two randomized controlled trials. Chiron HSV Vaccine Study Group. JAMA 1999;282:331 40. [43] Stanberry LR, Spruance SL, Cunningham AL, Bernstein DI, Mindel A, Sacks S, et al. Glycoprotein-Dadjuvant vaccine to prevent genital herpes. N Engl J Med 2002;347:1652 61. [44] Belshe RB, Leone PA, Bernstein DI, Wald A, Levin MJ, Stapleton JT, et al. Efficacy results of a trial of a herpes simplex vaccine. N Engl J Med 2012;366:34 43. [45] McDermott MR, Smiley JR, Leslie P, Brais J, Rudzroga HE, Bienenstock J. Immunity in the female genital tract after intravaginal vaccination of mice with an attenuated strain of herpes simplex virus type 2. J Virol 1984;51:747 53.

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[46] Parr EL, Parr MB. Immunoglobulin G, plasma cells, and lymphocytes in the murine vagina after vaginal or parenteral immunization with attenuated herpes simplex virus type 2. J Virol 1998;72:5137 45. [47] Parr MB, Parr EL. Mucosal immunity to herpes simplex virus type 2 infection in the mouse vagina is impaired by in vivo depletion of T lymphocytes. J Virol 1998;72:2677 85. [48] Stanberry LR, Kit S, Myers MG. Thymidine kinasedeficient herpes simplex virus type 2 genital infection in guinea pigs. J Virol 1985;55:322 8. [49] Milligan GN, Bernstein DI. Generation of humoral immune responses against herpes simplex virus type 2 in the murine female genital tract. Virology 1995;206: 234 41. [50] Kwant A, Rosenthal KL. Intravaginal immunization with viral subunit protein plus CpG oligodeoxynucleotides induces protective immunity against HSV-2. Vaccine 2004;22:3098 104. [51] Tengvall S, Lundqvist A, Eisenberg RJ, Cohen GH, Harandi AM. Mucosal administration of CpG oligodeoxynucleotide elicits strong CC and CXC chemokine responses in the vagina and serves as a potent Th1tilting adjuvant for recombinant gD2 protein vaccination against genital herpes. J Virol 2006;80:5283 91. [52] Cuburu N, Wang K, Goodman KN, Pang YY, Thompson CD, Lowy DR, et al. Topical herpes simplex virus 2 (HSV-2) vaccination with human papillomavirus vectors expressing gB/gD ectodomains induces genital-tissue-resident memory CD8 1 T cells and reduces genital disease and viral shedding after HSV-2 challenge. J Virol 2015;89:83 96. [53] McLean CS, Ni Challanain D, Duncan I, Boursnell ME, Jennings R, Inglis SC. Induction of a protective immune response by mucosal vaccination with a DISC HSV-1 vaccine. Vaccine 1996;14:987 92. [54] Milligan GN, Dudley-McClain KL, Chu CF, Young CG. Efficacy of genital T cell responses to herpes simplex virus type 2 resulting from immunization of the nasal mucosa. Virology 2004;318:507 15. [55] Bernstein DI. Effect of route of vaccination with vaccinia virus expressing HSV-2 glycoprotein D on protection from genital HSV-2 infection. Vaccine 2000;18:1351 8. [56] Sato A, Suwanto A, Okabe M, Sato S, Nochi T, Imai T, et al. Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease. J Virol 2014;88: 13699 708. [57] Morrison LA, Da Costa XJ, Knipe DM. Influence of mucosal and parenteral immunization with a replication-defective mutant of HSV-2 on immune

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

responses and protection from genital challenge. Virology 1998;243:178 87. Gallichan WS, Johnson DC, Graham FL, Rosenthal KL. Mucosal immunity and protection after intranasal immunization with recombinant adenovirus expressing herpes simplex virus glycoprotein B. J Infect Dis 1993;168:622 9. Gallichan WS, Rosenthal KL. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J Exp Med 1996;184: 1879 90. Gallichan WS, Rosenthal KL. Long-term immunity and protection against herpes simplex virus type 2 in the murine female genital tract after mucosal but not systemic immunization. J Infect Dis 1998;177:1155 61. Tirabassi RS, Ace CI, Levchenko T, Torchilin VP, Selin LK, Nie S, et al. A mucosal vaccination approach for herpes simplex virus type 2. Vaccine 2011;29:1090 8. Ye L, Zeng R, Bai Y, Roopenian DC, Zhu X. Efficient mucosal vaccination mediated by the neonatal Fc receptor. Nat Biotechnol 2011;29:158 63. Lubinski J, Wang L, Mastellos D, Sahu A, Lambris JD, Friedman HM. In vivo role of complement-interacting domains of herpes simplex virus type 1 glycoprotein gC. J Exp Med 1999;190:1637 46. Awasthi S, Huang J, Shaw C, Friedman HM. Blocking herpes simplex virus 2 glycoprotein E immune evasion as an approach to enhance efficacy of a trivalent subunit antigen vaccine for genital herpes. J Virol 2014;88:8421 32. Awasthi S, Hook LM, Shaw CE, Pahar B, Stagray JA, Liu D, et al. An HSV-2 trivalent vaccine is immunogenic in rhesus macaques and highly efficacious in guinea pigs. PLoS Pathog 2017;13:e1006141. Awasthi S, Lubinski JM, Friedman HM. Immunization with HSV-1 glycoprotein C prevents immune evasion from complement and enhances the efficacy of an HSV1 glycoprotein D subunit vaccine. Vaccine 2009;27: 6845 53. Awasthi S, Lubinski JM, Shaw CE, Barrett SM, Cai M, Wang F, et al. Immunization with a vaccine combining herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea pigs compared to immunization with gD alone. J Virol 2011;85: 10472 86. Awasthi S, Zumbrun EE, Si H, Wang F, Shaw CE, Cai M, et al. Live attenuated herpes simplex virus 2 glycoprotein E deletion mutant as a vaccine candidate defective in neuronal spread. J Virol 2012;86:4586 98.

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Maternal Vaccination for Protection Against Maternal and Infant Bacterial and Viral Pathogens David R. Martinez1,2, Jesse Mangold2 and Sallie R. Permar1,2,3 1

Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC, United States 2Duke Human Vaccine Institute, Durham, NC, United States 3 Department of Pediatrics, Duke University Medical Center, Durham, NC, United States

I. INTRODUCTION Maternal vaccination has been a widely successful strategy to prevent both maternal and neonatal viral and bacterial infections in the first year of life. In fact, it is estimated that maternal tetanus vaccination alone has reduced yearly infant deaths worldwide from more than 750,000 in the 1980s to fewer than 50,000 cases per year by the mid-2000s [1,2]. Maternal vaccination has also been effective in combating other bacterial and viral pathogens. For example, in the 1960s, more than 60,000 congenital rubella infections occurred each year in the United States alone [3,4], but because of the widespread immunization of women of childbearing age with a universal measles-mumps-rubella (MMR) vaccine, congenital rubella virus infection has now been eliminated in the United States [5]. Despite the

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00044-4

widespread success of maternal vaccination as a means to mediate protection from mother-tochild transmission (MTCT) of neonatal pathogens, safe and effective maternal vaccines for pathogens that pose a major public health burden for both pregnant mothers and their neonates have not been developed. Relevant maternal and neonatal pathogens for which safe and effective maternal vaccines are not yet available include respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), Zika virus (ZIKV), and group B streptococcus (GBS) (Table 44.1). Given the extraordinary global public health burden of disease caused by these maternal and neonatal pathogens, there is an urgent need to gain a deeper understanding of naturally occurring maternal immune correlates of protection against these pathogens

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© 2020 Elsevier Inc. All rights reserved.

736 44. MATERNAL VACCINATION FOR PROTECTION AGAINST MATERNAL AND INFANT BACTERIAL AND VIRAL PATHOGENS TABLE 44.1 Maternal Vaccines Administered During Pregnancy and Maternal and Neonatal Pathogens for Which Maternal Vaccines Are Needed Type of vaccine/ recommendation

Vaccine

Age group recommendation

Tetanus toxoid, diphtheria, and pertussis

Inactivated

Boostrix/Adacel

10 64 years

Yes, during the last trimester

Influenza

Inactivated/protein based

Fluzone/Flucelvax

Minimum age 6 months

Yes, during the last trimester

Measles, mumps and rubella

Live attenuated

M-M-R II

Minimum age 6 months

No, contraindicated

Respiratory syncytial virus

No licensed vaccine available

Preclinical and clinical testing

n/a

n/a

Human immunodeficiency virus

No licensed vaccine available

Preclinical testing

n/a

n/a

Human cytomegalovirus

No licensed vaccine available

Preclinical testing

n/a

n/a

Herpes simplex virus

No licensed vaccine available

Preclinical testing

n/a

n/a

Zika virus

No licensed vaccine available

Preclinical and clinical testing

n/a

n/a

Group B streptococcus

No licensed vaccine available

Phase 3 efficacy testing

n/a

n/a

Maternal vaccine

and to harness our understanding of these immune correlates of protection to guide the development of novel maternal vaccination strategies that can protect against congenital and neonatal transmission. In this chapter, we outline the public health disease burden imposed by these common neonatal pathogens and highlight promising preclinical and clinical vaccination strategies against relevant maternal and neonatal pathogens. A better understanding of naturally occurring maternal immune responses and vaccine-elicited immune responses that can protect against infection could help to guide the development of rational vaccination strategies against neonatal pathogens for which safe and effective vaccines have not been developed.

Recommendation

II. DIPHTHERIA, PERTUSSIS, AND TETANUS While the burden of tetanus and diphtheria in the United States has been greatly reduced by routine vaccination, rates of pertussis have resurged in recent years, though they are now trending on a decline [6]. Infants younger than 1 year of age are especially vulnerable to pertussis infection and exhibit the highest rates of hospitalization and mortality of any age group [7,8]. Close contact exposure with infected parents accounts for 47% 60% of pertussis cases in infants [9]. Although vaccination schedules begin the tetanus toxoid, diphtheria, and pertussis (Tdap) series for infants at 2 months of age, vaccine-elicited protective responses for pertussis

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737

III. INFLUENZA VIRUS

do not strengthen until 6 months of age after at least two doses. Young infants are thus at especially elevated risk for pertussis infection. The preventive strategy of postpartum maternal immunization had limited efficacy in protecting infants from pertussis [10]. To further reduce infant disease burden from pertussis, the Centers for Disease Control and Prevention (CDC) recommended in 2012 that the Tdap vaccine be administered to all pregnant women during each pregnancy during the late second or third trimester. In the United States, vaccine administration is recommended between the gestational ages of 27 and 36 weeks, while in the United Kingdom, vaccine administration is recommended between the gestational ages of 20 and 32 weeks. Studies conducted in the United Kingdom have shown effectiveness of maternal pertussis vaccination to range from 91% to 93% [11,12]. The optimal timing of vaccine delivery is a critical question, given that passively acquired protective levels of pertussis-specific immunoglobulin G (IgG) decay to below protective thresholds in newborns within the first 2 months of life [13]. A recent study reported that infants born to pregnant women immunized with Tdap as early as 13 weeks gestational age had higher levels of passively acquired pertussis-specific IgG against both pertussis toxin and filamentous hemagglutinin compared to infants born to women immunized at more than 25 weeks gestational age [14]. The disagreement among studies about the optimal timing of gestational age at administration of pertussis vaccine warrants further investigation. Therefore detailed studies aimed at understanding determinants of maternal antibody half-lives in infants are needed to potentially improve protective levels of passively acquired pertussis-specific IgG in infants. Other open questions regarding maternal Tdap vaccination include the overall efficacy of maternal vaccination, establishing immunological correlates of protection against pertussis in the infant, and the cumulative effects of repeated doses on multiparous mothers.

III. INFLUENZA VIRUS Primarily as a result of the maternal health complications associated with influenza infection during pregnancy, maternal influenza vaccination is recommended during any trimester of pregnancy [15]. The burden of influenza among pregnant women is attributed to both seasonal and pandemic influenza. In a given year, it is estimated that 20% 30% of all pregnant women will develop an influenza-like illness and 10% of all pregnant women will have laboratory-confirmed cases of influenza [16]. In the case of influenza infection with viral strains with pandemic potential, such as the H1N1 outbreak in 2009, it was estimated that nearly 50% of all pregnant women became infected with influenza [16]. Pregnant women infected with H1N1 were at higher risk for hospitalization, intensive care unit admission related to influenza complications, and death [17]. Moreover, pregnant women infected with seasonal influenza are at higher risk of morbidity and mortality related to influenza complications compared to nonpregnant women [18]. Maternal health complications from influenza infection during pregnancy include spontaneous abortion, severe lung-associated pathologies, higher rates of respiratory illness, and cardiopulmonary complications [19,20]. Infant complications related to maternal influenza infection with highly pathogenic strains, such as the H1N1 pandemic from 2009, during pregnancy include low birth weight, preterm birth, stillbirth, and neonatal death [19,21]. Studies have shown that childhood mortality from influenza is greatest among infants up to 6 months of age, yet vaccines are not specifically licensed for use in this age range [22,23]. Thus maternal vaccination presents as a viable strategy to provide temporary protection to infants in this window of heightened vulnerability before administration of influenza vaccine according to guidelines outlined in vaccination schedules. The CDC recommends the inactivated influenza vaccine for all pregnant women during

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738 44. MATERNAL VACCINATION FOR PROTECTION AGAINST MATERNAL AND INFANT BACTERIAL AND VIRAL PATHOGENS each pregnancy during any trimester. Live attenuated influenza vaccine is contraindicated during pregnancy, owing to the theoretical risks associated with perinatal infection of the fetus. Maternal vaccination for influenza has shown great impact for both maternal and infant health outcomes. Studies have shown a 36% reduction of maternal influenza infection and a 63% decrease in infant influenza disease with administration of inactivated influenza vaccine during pregnancy [24,25]. For infants, increased birth weight and reduced rates of preterm birth and infant death are associated with maternal influenza vaccination [24,25]. Notably, maternal influenza vaccination administered in the third trimester led to a 63% reduction in laboratory-confirmed cases of influenza among infants up to 6 months of age [24]. The impact of gestational age at the time of vaccine administration on vaccine efficacy remains poorly understood and warrants further study to optimize timing for protective health outcomes in both mothers and infants. Furthermore, given the relatively low efficacy of both seasonal and pandemic influenza vaccination strategies in healthy adults, future strategies should also focus on testing the influenza vaccine efficacy for pregnant women.

IV. MEASLES, MUMPS, AND RUBELLA Measles (rubeola) is a paramyxovirus that spreads through airborne transmission and presents with symptoms of high fever, full-body rash, cough, coryza, and conjunctivitis. Common complications of measles include ear infections and diarrhea. More severe complications that may require hospitalization include pneumonia and encephalitis. Mumps is a paramyxovirus that spreads through saliva and mucus and presents with flulike symptoms and swollen salivary glands. Complications of mumps include inflammation and potential

deafness. Infection with measles or mumps during pregnancy is associated with increased risk of spontaneous abortion, preterm birth, and low birth weight [26]. Rubella is a togavirus that spreads through airborne transmission and presents with symptoms of low fever, lymphadenopathy, full-body rash, and arthralgia. Infection with rubella during pregnancy can result in transmission of the virus to the fetus, resulting in miscarriage, spontaneous abortion, or development of congenital rubella syndrome (CRS). CRS is associated with severe birth defects, including deafness, cataracts, cardiac defects, brain damage, and death [27]. Although CRS has been virtually eliminated in the United States, there have been reports of CRS imported into the United States in recent years [19]. Therefore MMR vaccination programs in the United States remain critical to public health security, as individuals infected with rubella and CRS may import the virus from around the globe [27]. MMR vaccination in healthy adults has been shown to induce long-lasting antibody responses against measles, mumps, and rubella. In fact, a study reported that even at least 20 years after of the second MMR boost, up to 95%, 74%, and 100% of 183 vaccinees were still seropositive for measles, mumps, and rubella, respectively [28]. Since MMR is a live attenuated virus vaccine, it is contraindicated during pregnancy because of theoretical risks associated with potential transmission of the virus to the fetus. Nevertheless, studies have reported that among infants born to susceptible mothers vaccinated just before or during pregnancy, 3% 5% of cord blood samples were seropositive for antirubella immunoglobulin M (IgM) antibody, yet none of the infants developed clinical symptoms of CRS [29 31]. These data support the safety of the MMR vaccine in the event of inadvertent administration during pregnancy and do not suggest that the MMR vaccine is likely to lead to adverse maternal or infant outcomes. Therefore all women of childbearing age

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V. THE NEXT FRONTIER OF MATERNAL VACCINES

should receive preconception screening for rubella-protective immunity and appropriate follow-up with MMR vaccine administration before and directly after childbirth in order to prevent infant exposure and reduce transmission risk during future pregnancies [18].

V. THE NEXT FRONTIER OF MATERNAL VACCINES A. Respiratory Syncytial Virus RSV is a paramyxovirus that causes severe disease in newborns and infants, with an estimated 33.8 million cases worldwide in infants younger than 5 years of age [32]. While therapeutic monoclonal antibody treatments for infant RSV disease, such as Palivizumab and Motavizumab, have had some efficacy in reducing hospitalization rates in high-risk infants [33], treatment costs can often be prohibitively expensive and also require multidose administration as therapeutic antibody levels wane over time. Therefore the development of a safe and effective RSV vaccine administered during pregnancy or in newborns is of critical importance. Despite several preclinical and clinical RSV vaccine concepts that have been tested, no effective RSV vaccine is available for clinical use to date [34 36]. The development of a safe and effective RSV vaccine has been hindered by a number of factors. These include the lack of RSV vaccine immunogens that can induce protective responses, the lack of small animal models that accurately recapitulate human RSV disease, an opaque understanding of potentially protective antibody responses that can block infection, and concerns about the possibility of vaccine-mediated RSV disease enhancement in infants. In the 1960s, an RSV formalin-inactivated virus vaccine tested in neonates was associated with more severe disease outcomes and higher hospitalization rates in vaccinees compared to placebo control neonates who became infected

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with RSV [37]. While it is not entirely clear why the vaccine led to higher respiratory disease outcomes in neonates, follow-up studies in cotton rats and mice suggest that formalininactivated RSV vaccine disease severity was associated with low levels of vaccine-elicited neutralizing antibodies, increased recruitment of neutrophils and macrophages to the lung, and immunopathology that is potentially modulated by inflammatory Th2 CD41 T cells in the lung [38,39]. In contrast, a purified fusion (F) protein vaccine that was tested in neonates with preexisting antibody responses found no increase in infant RSV disease severity in vaccinated infants compared to placebo controls [35]. The inconsistency in infant RSV disease enhancement that is potentially modulated by distinct RSV vaccine candidates underlines the need for more basic research and preclinical testing of RSV vaccines in relevant neonatal animal models. Recent advances in RSV vaccine research include the development of structurally optimized RSV envelope immunogens. An RSV Env protein (DSCAV-1) that contains a series of cavity-filling amino acid mutations was recently developed [40]. These cavity-filling amino acid mutations stabilize the RSV envelope into a prefusion trimer and expose the main target of neutralizing antibodies: site Ø [40]. Importantly, DSCAV-1 RSV prefusion F protein vaccination in both mice and nonhuman primate (NHP) models induced high levels of binding and neutralizing antibodies targeting site Ø [40]. As a result, a phase 1 clinical trial testing the safety and immunogenicity of DSCAV-1 in healthy adults was initiated (ClinicalTrials.gov Identifier: NCT03049488). It will be important to test whether this structurally optimized RSV Env prefusion trimer can also induce binding and neutralizing antibody responses targeting site Ø in humans and whether these antibodies can mediate protection from subsequent RSV infection. It will also be important to define the safety, immunogenicity, and efficacy of promising RSV

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740 44. MATERNAL VACCINATION FOR PROTECTION AGAINST MATERNAL AND INFANT BACTERIAL AND VIRAL PATHOGENS vaccines, such as the DSCAV-1 vaccine, in neonates, as this group is especially vulnerable to severe RSV disease in early life (Chapter 38: Vaccination Against Respiratory Syncytial Virus).

B. Human Immunodeficiency Virus HIV is a retrovirus that can cause acquired immunodeficiency syndrome (AIDS) [41]. Progression of HIV disease to AIDS can largely be prevented in patients treated with viral suppressive antiretroviral therapy (ART) [42,43]. HIV can also be vertically transmitted to the fetus during pregnancy, during delivery, or via breast feeding. In fact, it was estimated that in the year 2015, more than 150,000 pediatric infections occurred in the setting of MTCT [44]. MTCT risk of HIV has been substantially reduced by maternal and/or infant antiretroviral (ART) prophylaxis during pregnancy and the breast-feeding period [45]. The standard of clinical care ART treatment for HIV-infected women is Option B plus, which consists of triple combination ART therapy initiation in the mother upon diagnosis regardless of CD41 T cell count, and continued ART treatment for life [46]. Option B plus also recommends daily nevirapine or zidovudine treatment in infants through 6 weeks of age regardless of feeding method [46]. However, recent studies suggest that certain triple combination ART prophylaxis regimens that are administered to HIVinfected women during pregnancy can lead to higher rates of adverse maternal and fetal outcomes, including preterm birth and infant death [41]. Therefore additional immunologybased strategies that can be synergistic with ART are needed to curb the pediatric HIV epidemic. To this end, maternal HIV vaccination could be a strategy to further reduce the risk of pediatric HIV infections. A limitation in the development of a safe and effective maternal HIV vaccine field has been a lack of maternal immune correlates of protection against MTCT

of HIV. Yet important recent strides have been made in defining naturally occurring maternal immune responses that can mediate partial protection against MTCT of HIV. In a US cohort study of HIV clade B virus-infected women (N 5 248), it was recently demonstrated that maternal IgG responses specific to HIV Env variable loop 3 (V3), the ability to neutralize HIV tier 1 viruses, and CD4-binding site blocking responses were all colinear and associated with reduced MTCT risk [47]. A follow-up study on the fine specificity and function of potentially protective maternal V3-specific IgG responses in this cohort showed that maternal binding and neutralizing responses associated with reduced MTCT of HIV risk target amino acid residues within the C-terminal region of the V3 loop [48]. These studies raise the hypothesis that augmenting the level of maternal V3 loop and CD4binding site IgG-neutralizing responses through maternal vaccination could further reduce the risk of MTCT of HIV. A deeper understanding of maternal immune responses that can mediate partial protection against MTCT of HIV could help to guide the design of maternal HIV vaccination strategies by providing benchmark immune responses that a successful maternal HIV vaccine must elicit. A safety and immunogenicity phase 1 maternal HIV vaccination study tested a recombinant gp120 vaccine adjuvanted with alum in HIV-infected women without advanced HIV disease progression [49]. This maternal HIV vaccination study enrolled 17 vaccinees and 9 placebo control patients, and all the women received at least three vaccines before delivery. Importantly, maternal symptoms attributable to the HIV vaccine were mild, and there were no pregnancy adverse outcomes in either the vaccinees or the placebo control patients [49]. While there was no significant increase either in the levels of maternal V3-specific IgG binding or in neutralizing responses in vaccinees compared to placebo control women, it should be noted that women

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V. THE NEXT FRONTIER OF MATERNAL VACCINES

in both groups were HIV-infected and had had only minimal treatment, potentially explaining the limited boosting of HIV-specific immunity of this maternal HIV vaccine. This study also reported no significant differences in HIV infection rates in infants born to vaccinees in comparison to those born to placebo control women. However, it should be noted that this phase 1 vaccine trial was neither designed nor sufficiently powered to test vaccine efficacy against MTCT of HIV. This study highlights that recombinant gp120 vaccination strategies may be safely administered to HIV-infected pregnant women. Therefore, ongoing therapeutic HIV vaccine concepts undergoing phase 1 testing in HIVinfected adult volunteers should also include HIV-infected pregnant women to assess the potential role of these vaccines in reducing MTCT of HIV (Chapter 42: Mucosal Vaccines Against HIV/SIV Infection).

C. Human Cytomegalovirus Congenital HCMV is the leading infectious cause of infant brain damage and childhood permanent disabilities worldwide. Nearly 1% of all infants are born with congenital HCMV, and at least 20% of those infected infants will go on to have lifelong disabilities, most commonly hearing loss [50]. Moreover, up to a quarter of infant hearing loss is attributable to congenital HCMV infection. The annual impact of congenital HCMV in the United States alone has been estimated to be $4 billion. While antiviral treatment has recently been shown to be effective in reducing hearing loss and developmental delays associated with congenital HCMV infection in infants with symptomatic disease at birth [51], antiviral treatment had only a modest impact on disease outcome, and the impact of treatment in infants who develop disease after birth is unclear. Thus a vaccine to eliminate congenital HCMV transmission is of highest public health priority and was named a

741

top tier priority vaccine by the National Academy of Medicine over 15 years ago [52]. However, few HCMV vaccines have moved into late-phase clinical trials. The most successful clinically tested HCMV vaccine to date is the glycoprotein B vaccine, which has been tested in phase 2 studies in postpartum women [53], adolescent females [54], and renal transplant recipients [55]. It achieved approximately 50% protection against HCMV acquisition or disease in all three trials. Remarkably, this vaccine was partially effective despite the induction of only limited neutralizing antibody responses [56], raising the question of the role of nonneutralizing antibody responses in protection against HCMV. One complicating factor in HCMV vaccine development is that while primary HCMV infection of the mother poses the highest risk for congenital transmission, HCMV can be placentally transmitted in the setting of natural maternal immunity. However, the risk of HCMV transmission to the fetus after maternal reinfection in the setting of preexisting maternal immunity is considerably lower than that of primary maternal infection [57,58]. Thus a vaccine that is fully protective against congenital transmission must elicit immunity that is distinct from or improves on natural HCMV immunity. While a HCMV vaccine would be most effective against congenital HCMV infection if administered universally before the childbearing years, one possible strategy is to provide temporary passive or active immunity to women who remain without potentially protective HCMV immunity during pregnancy to ameliorate the high risk of transmission in the setting of primary maternal infection. In fact, a recent study in the NHP model of congenital HCMV transmission demonstrated that passive infusion of polyclonal anti-HCMV antibodies prior to maternal virus challenge was protective against placental virus transmission [59]. Moreover, a better understanding of maternal immune correlates of transmission risk in the

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742 44. MATERNAL VACCINATION FOR PROTECTION AGAINST MATERNAL AND INFANT BACTERIAL AND VIRAL PATHOGENS setting of preexisting immunity could direct maternal vaccine development to enhance the identified protective response during pregnancy. One such study identified HCMVneutralizing responses assessed in epithelial cells as an immune correlate of protection against congenital HCMV transmission in HIV/ HCMV coinfected women [60]. Thus, maternal vaccination for HCMV may be an effective strategy to temporarily improve on existing maternal immune responses that can block congenital HCMV transmission upon HCMV exposure.

D. Herpes Simplex Virus HSV is a widely prevalent human alpha herpesvirus that infects approximately one of every five adults in the United States [61]. In the setting of pregnancy, infection with maternal HSV-1 and HSV-2 can lead to congenital and perinatal infections. It is estimated that congenital and perinatal HSV infections occur in one out of every 3000 20,000 births [62]. Congenital and perinatal HSV infections can present with severe infant symptoms, including conjunctivitis, disseminated intravascular coagulation, seizures, and severely impaired central nervous system function, depending on the timing and severity of the infection [63]. Current strategies to reduce the risk of congenital HSV infection include the use of antiviral drugs such as acyclovir or valacyclovir during pregnancy [64]. However, likely as a result of several factors, including drug-resistant viral mutations, neonatal HSV transmission can occur even when optimal viral suppressive antiviral drug treatments are administered to pregnant women prior to delivery [65]. Therefore the failure of current prevention treatments for congenital and perinatal HSV infection underlines the need for synergistic immune-based strategies that can mediate protection against neonatal HSV infection. Such strategies could include the development of a safe and effective maternal HSV vaccine.

Neonates born to women who undergo a primary HSV infection during pregnancy are at higher risk of infection compared to neonates born to women with a recurrent infection, suggesting that preexisting maternal immune responses may play a role in reducing MTCT risk of HSV [66]. However, naturally occurring maternal immune correlates of protection against MTCT of HSV are not known. In addition, no safe and effective clinically licensed HSV vaccine exists to date. A previous phase 2 vaccine efficacy trial demonstrated that a glycoprotein D subunit vaccine adjuvanted with alum and 3-O-deacylatedmonophosphoryl lipid A was 73% efficacious in women who were seronegative for HSV-1 and HSV-2 and 74% efficacious in women who were seronegative only for HSV-2 [67]. The primary endpoint to assess vaccine efficacy in this trial was the occurrence of genital herpes in HSV-seronegative women. Because most perinatal infections result from exposure to HSV in the maternal genital tract [68], it will be important to assess whether glycoprotein D vaccines also mediate protection against perinatal HSV infections. Furthermore, it is important to note that this glycoprotein D subunit vaccine was efficacious in women who were HSV-1 and HSV-2 negative. Since the majority of congenital and perinatal HSV transmission occurs in neonates born to seronegative mothers who undergo seroconversion during pregnancy, it could also be important to examine whether similar HSV glycoprotein D subunit vaccine are efficacious against congenital and perinatal HSV transmission. In contrast, studies in mice have shown that live attenuated HSV-2 viruses with a glycoprotein D deletion induced HSVspecific IgG responses that are protective against virus challenge [69]. Protective HSVspecific IgG responses were found to have little neutralizing activity and primarily mediated antiviral activity through Fc receptors, suggesting that nonneutralizing effector functions may be important for mediating

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V. THE NEXT FRONTIER OF MATERNAL VACCINES

protection against HSV infection. Therefore ongoing vaccine safety and efficacy trials in healthy adults and pregnant women should define the antiviral function of vaccine-elicited HSV-specific IgG responses. The elucidation of the mechanism(s) of protection of vaccineelicited HSV-specific IgG responses could also be important for the design of passive immunization strategies that can mediate protection against congenital and/or perinatal HSV transmission (Chapter 43: Mucosal Vaccines for Genital Herpes).

E. Zika Virus The emergence of ZIKV in the Americas in 2015 and its association with severe microcephaly in newborns have led to an international public health crisis. The development of a safe and effective ZIKV vaccine is therefore a global health priority. ZIKV is a positively stranded RNA virus that is transmitted to humans primarily through mosquito vectors from the Aedes genus, but it can also be transmitted in utero to the fetus during pregnancy [70 72]. ZIKV infection in adults is associated with fairly mild symptoms, including fever, viremia, headaches, malaise, maculopapular rash, and arthralgia [73]. In contrast, ZIKV infection during pregnancy can have devastating effects on the fetus, including miscarriage, stillbirth, grossly abnormal brain development (i.e., microcephaly), and other forms of severe neurological impairment [74]. Preclinical vaccine concepts in animal models have shown promise and suggest that a licensed Zika vaccine may be within reach. For example, two recent studies involving mouse and NHP challenge models showed that both ZIKV premembrane and Env (prM-Env) DNA vaccine and a ZIKV heat-inactivated vaccine could induce ZIKVspecific IgG antibodies that were protective against ZIKV challenge [75,76]. ZIKV Envspecific IgG induced by these vaccines was

743

shown to mediate protection in passively immunized naı¨ve animals, suggesting that Envspecific IgG responses alone can be protective against ZIKV virus infection. A separate study involving mouse and NHP models also tested a DNA vaccine that expressed ZIKV prM-Env and elicited ZIKV Env-specific neutralizing antibodies [77]. This study also showed that the level of vaccine-elicited ZIKV-specific neutralizing antibody level was associated with the protection versus breakthrough infection after challenge. Finally, it was recently shown that both mRNA and live attenuated ZIKV vaccines can mediate protection against ZIKV-associated placental damage and congenital disease in fetal mice [78], suggesting that a ZIKV vaccine that elicits Env-specific IgG responses may mediate protection against adult and congenital ZIKV transmission. It is not yet known whether ZIKV vaccine concepts that have shown promise in mice and NHP challenge models will be directly translatable to humans, specifically in the setting of pregnancy. A recent experimental ZIKV DNA vaccine phase 1 study in healthy female and male adults showed that all subjects had seroconverted by the third vaccine dose, yet only 62% of patients developed ZIKV-specific neutralizing antibodies [79]. Interestingly, ZIKV Envspecific antibodies from postvaccinated sera from patients with and without neutralizing antibodies protected 92% of mice from lethal ZIKV challenge, while none of the mice that received baseline serum survived virus challenge [79]. This finding suggests that vaccineelicited ZIKV Env-specific IgG responses in humans are capable of blocking ZIKV transmission, and this protective effect is independent of in vitro ZIKV-specific IgG neutralizing activity. Importantly, it is not yet known whether DNA vaccines undergoing clinical development in healthy adults will be safe and effective in the setting of pregnancy. Therefore ongoing phase 1 and phase 2 vaccine clinical trials should include pregnant women who are at

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744 44. MATERNAL VACCINATION FOR PROTECTION AGAINST MATERNAL AND INFANT BACTERIAL AND VIRAL PATHOGENS risk for ZIKV infection to assess vaccine safety, immunogenicity, and efficacy in the setting of pregnancy.

F. Group B Streptococcus GBS is one of the leading causes of neonatal sepsis and meningitis [80]. In 2010, more than 390,000 neonatal deaths occurred from GBS disease worldwide [81]. Moreover, it is estimated that up to 50% of infants who survive meningitis infection suffer from disease-associated neurological impairment [80]. In the developed world, current strategies to prevent neonatal GBS infection consist primarily of intrapartum administration of antibiotic prophylaxis in infants born to GBS-positive pregnant women [82,83]. In contrast, in developing countries that have not implemented routine maternal GBS screening as part of the standard of clinical care during pregnancy, neonatal GBS disease and death rates remain high. While intrapartum antibiotic prophylaxis is generally effective at preventing early-onset GBS disease in neonates [83], there are public health concerns regarding the selection of drug-resistant strains of GBS [84]. Therefore the development of alternative prophylactic strategies that can prevent neonatal GBS disease is critically needed. Previous studies found that infant cord blood levels of maternal passively acquired GBS-specific IgG responses were associated with protection from GBS disease in infants [85]. Up to 76% of women who gave birth to infants without GBS disease had capsular polysaccharide (CPS)-specific IgG, whereas all women who gave birth to infants who developed severe GBS disease did not have CPS-specific IgG [85], suggesting that maternal passively acquired CPS-specific responses may protect infants who are at risk for GBS disease. These findings suggest that maternal IgG responses may be harnessed to prevent neonatal GBS disease. Therefore, investigating the

specificity and antibacterial function of maternal GBS-specific IgG responses could be important for guiding maternal GBS vaccination strategies that can block neonatal GBS disease. To date, several GBS CPS and conjugated CPS vaccines have been clinically tested. CPSbased GBS vaccines were shown to be immunogenic in humans [86]. However, because of the lack of induction of T-cell-dependent B cell responses induced by polysaccharide vaccines, later GBS vaccine concepts included a conjugate tetanus toxoid (TT) protein to engage T-cell-dependent B cell responses. In fact, a phase 1 vaccine trial that tested a GBS serotype III TT conjugated vaccine in pregnant women showed safety and immunogenicity with up to a fourfold increase in maternal GBS-specific IgG responses after vaccination [87]. As a result, a placebo control phase 2 vaccine trial was initiated involving pregnant women that included serotype Ia, IIb, and III TT conjugated vaccine [88]. This phase 2 placebocontrolled vaccine trial reported that immunized women have a greater than 15-fold increase in their GBS-specific IgG titers. A phase 3 vaccine efficacy trial of this GBS Ia, Ib, and III TT conjugated vaccine concept is underway [81]. The success of the completed phase 1 and 2 GBS vaccine trials in pregnant women clearly demonstrates that the development and testing of novel vaccines specific to the setting of pregnancy may be a feasible and achievable strategy to combat neonatal pathogens for which maternal vaccines are not yet available.

VI. CONCLUDING REMARKS It is clear that maternal vaccination before and during pregnancy has been a cornerstone for preventing congenital and neonatal infections. While the development and deployment of safe and effective vaccines such as Tdap and MMR have had an extraordinarily positive

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REFERENCES

impact on infant health, there still remains a great need to develop the next generation of maternal vaccines against neonatal pathogens that pose a significant public health burden. Therefore more basic, preclinical, and clinical research is needed to safely develop maternal vaccines against RSV, HIV, HCMV, HSV, ZIKV, and GBS, and to understand how vaccine immunity is modified during pregnancy. Additionally, ongoing studies should focus on defining determinants of the transplacental transfer and milk transfer of protective maternal IgG to the vulnerable fetus. Altogether, the development of passive transfer therapeutic IgG-based strategies and safe and effective maternal vaccines against these pathogens will be important to ensure that all newborns have a healthy start in life.

VII. SUMMARY OF KEY POINTS • Maternal vaccination strategies have been highly successful in combating neonatal pathogens. • Maternal vaccination strategies against tetanus, diphtheria, pertussis, and influenza have significantly reduced disease burden in both mothers and their infants. • Vaccinating women of childbearing age against measles, mumps, and rubella has eliminated congenital rubella infections in the United States. • Despite the success of maternal vaccination, there remains a need for developing new vaccines against maternal and neonatal pathogens with significant disease burden. • Relevant maternal and neonatal pathogens for which vaccines are not yet available include respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), herpes simplex virus (HSV), Zika virus (ZIKV), and group B streptococcus (GBS).

Acknowledgment We thank Stephanie Risbon for technical help with the submission of this manuscript.

FUNDING DRM is supported by an American Society of Microbiology Robert D. Watkins Graduate Research Fellowship, an NIH NIAID Ruth L. Kirschstein National Research Service Award F31 F31AI127303, and a Burroughs Wellcome Graduate Diversity Enrichment Award. SRP is supported by NIH, NIAID grants: 5R01AI106380, 1R01AI22909, and UM1AI106716. Overall support for the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Network was provided by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH) under Award Numbers UM1AI068632 (IMPAACT LOC), UM1AI068616 (IMPAACT SDMC) and UM1AI106716 (IMPAACT LC), with cofunding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute of Mental Health. The content here is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

References [1] Roper MH, Vandelaer JH, Gasse FL. Maternal and neonatal tetanus. Lancet 2007;370(9603):1947 59. [2] Liu L, Oza S, Hogan D, et al. Global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis. Lancet 2015;385(9966):430 40. [3] CDC. Elimination of rubella and congenital rubella syndrome United States, 1969-2004. MMWR Morb Mortal Wkly Rep 2005;54(11):279 82. [4] Reef SE, Redd SB, Abernathy E, Zimmerman L, Icenogle JP. The epidemiological profile of rubella and congenital rubella syndrome in the United States, 1998-2004: the evidence for absence of endemic transmission. Clin Infect Dis 2006;43(Suppl. 3):S126 132.

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746 44. MATERNAL VACCINATION FOR PROTECTION AGAINST MATERNAL AND INFANT BACTERIAL AND VIRAL PATHOGENS [5] Eurosurveillance te. Expert committee declares WHO Region of the Americas measles-free. Eurosurveillance 2016;21(39). [6] Mooi FR, Van Der Maas NA, De Melker HE. Pertussis resurgence: waning immunity and pathogen adaptationtwo sides of the same coin. Epidemiol Infect 2014;142 (4):685 94. [7] Masseria C, Martin CK, Krishnarajah G, Becker LK, Buikema A, Tan TQ. Incidence and burden of pertussis among infants less than 1 year of age. Pediatr Infect Dis J 2017;36(3):e54 61. [8] Vitek CR, Pascual FB, Baughman AL, Murphy TV. Increase in deaths from pertussis among young infants in the United States in the 1990s. Pediatr Infect Dis J 2003;22(7):628 34. [9] Edwards KM. Overview of pertussis: focus on epidemiology, sources of infection, and long term protection after infant vaccination. Pediatr Infect Dis J 2005;24(6 Suppl.):S104 108. [10] Castagnini LA, Healy CM, Rench MA, Wootton SH, Munoz FM, Baker CJ. Impact of maternal postpartum tetanus and diphtheria toxoids and acellular pertussis immunization on infant pertussis infection. Clin Infect Dis 2012;54(1):78 84. [11] Dabrera G, Amirthalingam G, Andrews N, et al. A case-control study to estimate the effectiveness of maternal pertussis vaccination in protecting newborn infants in England and Wales, 2012-2013. Clin Infect Dis 2015;60(3):333 7. [12] Amirthalingam G, Andrews N, Campbell H, et al. Effectiveness of maternal pertussis vaccination in England: an observational study. Lancet 2014;384 (9953):1521 8. [13] Healy CM, Munoz FM, Rench MA, Halasa NB, Edwards KM, Baker CJ. Prevalence of pertussis antibodies in maternal delivery, cord, and infant serum. J Infect Dis 2004;190(2):335 40. [14] Eberhardt CS, Blanchard-Rohner G, Lemaıˆtre B, et al. Pertussis antibody transfer to preterm neonates after second- versus third-trimester maternal immunization. Clin Infect Dis 2017;64(8):1129 32. [15] Practice ACoOaGCoO. ACOG committee opinion number 305, November 2004. Influenza vaccination and treatment during pregnancy. Obstet Gynecol 2004;104(5 Pt 1):1125 6. [16] Cantu J, Tita AT. Management of influenza in pregnancy. Am J Perinatol 2013;30(2):99 103. [17] Mosby LG, Rasmussen SA, Jamieson DJ. 2009 pandemic influenza A (H1N1) in pregnancy: a systematic review of the literature. Am J Obstet Gynecol 2011;205 (1):10 18. [18] Neuzil KM, Reed GW, Mitchel EF, Simonsen L, Griffin MR. Impact of influenza on acute cardiopulmonary

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

hospitalizations in pregnant women. Am J Epidemiol 1998;148(11):1094 102. Oluyomi-Obi T, Avery L, Schneider C, et al. Perinatal and maternal outcomes in critically ill obstetrics patients with pandemic H1N1 Influenza A. J Obstet Gynaecol Can 2010;32(5):443 7. Yudin MH. Risk management of seasonal influenza during pregnancy: current perspectives. Int J Women’s Health 2014;6:681 9. Pierce M, Kurinczuk JJ, Spark P, Brocklehurst P, Knight M. Perinatal outcomes after maternal 2009/ H1N1 infection: national cohort study. BMJ (Clin Res Ed.) 2011;342:d3214. Bhat N, Wright JG, Broder KR, et al. Influenzaassociated deaths among children in the United States, 2003-2004. N Engl J Med 2005;353(24):2559 67. Moriarty LF. Infants and the seasonal influenza vaccine: A global perspective on safety, effectiveness, and alternate forms of protection. Hum Vaccines Immunother 2014;10(9):2721 8. Zaman K, Roy E, Arifeen SE, et al. Effectiveness of maternal influenza immunization in mothers and infants. N Engl J Med 2008;359(15):1555 64. Ha˚berg SE, Trogstad L, Gunnes N, et al. Risk of fetal death after pandemic influenza infection or vaccination during pregnancy. N Engl J Med 2013;368 (4):333 40. Siegel M, Fuerst HT, Peress NS. Comparative fetal mortality in maternal virus diseases. A prospective study on rubella, measles, mumps, chicken pox and hepatitis. N Engl J Med 1966;274(14):768 71. Centers for Disease Control and Prevention (CDC). Three cases of congenital rubella syndrome in the postelimination era Maryland, Alabama, and Illinois, 2012. MMWR Morb Mortal Wkly Rep 2013;62 (12):226 9. Davidkin I, Jokinen S, Broman M, Leinikki P, Peltola H. Persistence of measles, mumps, and rubella antibodies in an MMR-vaccinated cohort: a 20-year followup. J Infect Dis 2008;197(7):950 6. Castillo-Solorzano C, Reef SE, Morice A, et al. Rubella vaccination of unknowingly pregnant women during mass campaigns for rubella and congenital rubella syndrome elimination, the Americas 2001-2008. J Infect Dis 2011;204(Suppl. 2):S713 717. Soares RC, Siqueira MM, Toscano CM, et al. Follow-up study of unknowingly pregnant women vaccinated against rubella in Brazil, 2001-2002. J Infect Dis 2011;204(Suppl. 2):S729 736. Hamkar R, Jalilvand S, Abdolbaghi MH, et al. Inadvertent rubella vaccination of pregnant women: evaluation of possible transplacental infection with rubella vaccine. Vaccine 2006;24(17):3558 63.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

REFERENCES

[32] Nair H, Nokes DJ, Gessner BD, et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 2010;375(9725):1545 55. [33] Geevarghese B, Simoes EA. Antibodies for prevention and treatment of respiratory syncytial virus infections in children. Antiviral Ther 2012;17(1 Pt B):201 11. [34] Higgins D, Trujillo C, Keech C. Advances in RSV vaccine research and development-a global agenda. Vaccine 2016;34(26):2870 5. [35] Groothuis JR, King SJ, Hogerman DA, Paradiso PR, Simoes EA. Safety and immunogenicity of a purified F protein respiratory syncytial virus (PFP-2) vaccine in seropositive children with bronchopulmonary dysplasia. J Infect Dis 1998;177(2):467 9. [36] Falsey AR, Walsh EE, Capellan J, et al. Comparison of the safety and immunogenicity of 2 respiratory syncytial virus (rsv) vaccines nonadjuvanted vaccine or vaccine adjuvanted with alum given concomitantly with influenza vaccine to high-risk elderly individuals. J Infect Dis 2008;198(9):1317 26. [37] Kim HW, Canchola JG, Brandt CD, et al. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol 1969;89(4):422 34. [38] Connors M, Kulkarni AB, Firestone CY, et al. Pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of CD4 1 T cells. J Virol 1992;66(12):7444 51. [39] Waris ME, Tsou C, Erdman DD, Zaki SR, Anderson LJ. Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern. J Virol 1996;70(5):2852 60. [40] McLellan JS, Chen M, Joyce MG, et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 2013;342(6158):592 8. [41] Pantaleo G, Graziosi C, Demarest JF, et al. HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature 1993;362(6418):355 8. [42] Walensky RP, Paltiel AD, Losina E, et al. The survival benefits of AIDS treatment in the United States. J Infect Dis 2006;194(1):11 19. [43] Antiretroviral Therapy Cohort Collaboration. Survival of HIV-positive patients starting antiretroviral therapy between 1996 and 2013: a collaborative analysis of cohort studies. Lancet HIV 2017;4(8): e349 56.

747

[44] UNAIDS. Preventing mother-to-child transmission of HIV; 2016. Geneva: Joint United NationsProgramme on HIV/AIDS. [45] Connor EM, Sperling RS, Gelber R, et al. Reduction of maternal-infant transmission of human immunodeficiency virus type 1 with zidovudine treatment. Pediatric AIDS Clinical Trials Group Protocol 076 Study Group. N Engl J Med 1994;331(18):1173 80. [46] (UNAIDS). JUNPoHA. Countdown to zero: global plan for the elimination of new HIV infections among children by 2015 and keeping their mothers alive, 2011-2015. UNAIDS; 2011. [47] Permar SR, Fong Y, Vandergrift N, et al. Maternal HIV-1 envelope-specific antibody responses and reduced risk of perinatal transmission. J Clin Invest 2015;125(7):2702 6. [48] Martinez DR, Vandergrift N, Douglas AO, et al. Maternal binding and neutralizing IgG responses targeting the C-terminal region of the V3 loop are predictive of reduced peripartum HIV-1 transmission risk. J Virol 2017;91(9), e02422 16. [49] Wright PF, Lambert JS, Gorse GJ, et al. Immunization with envelope MN rgp120 vaccine in human immunodeficiency virus-infected pregnant women. J Infect Dis 1999;180(4):1080 8. [50] Kenneson A, Cannon MJ. Review and meta-analysis of the epidemiology of congenital cytomegalovirus (CMV) infection. Rev Med Virol 2007;17(4):253 76. [51] Kimberlin DW, Jester PM, Sanchez PJ, et al. Valganciclovir for symptomatic congenital cytomegalovirus disease. N Engl J Med 2015;372(10):933 43. [52] Arvin AM, Fast P, Myers M, Plotkin S, Rabinovich R. National Vaccine Advisory C. Vaccine development to prevent cytomegalovirus disease: report from the National Vaccine Advisory Committee. Clin Infect Dis 2004;39(2):233 9. [53] Pass RF, Zhang C, Evans A, et al. Vaccine prevention of maternal cytomegalovirus infection. N Engl J Med 2009;360(12):1191 9. [54] Bernstein DI, Munoz FM, Callahan ST, et al. Safety and efficacy of a cytomegalovirus glycoprotein B (gB) vaccine in adolescent girls: a randomized clinical trial. Vaccine 2016;34(3):313 19. [55] Griffiths PD, Stanton A, McCarrell E, et al. Cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant in transplant recipients: a phase 2 randomised placebo-controlled trial. Lancet 2011;377(9773):1256 63. [56] Cui X, Meza BP, Adler SP, McVoy MA. Cytomegalovirus vaccines fail to induce epithelial entry neutralizing antibodies comparable to natural infection. Vaccine 2008;26(45):5760 6.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

748 44. MATERNAL VACCINATION FOR PROTECTION AGAINST MATERNAL AND INFANT BACTERIAL AND VIRAL PATHOGENS [57] Leruez-Ville M, Magny JF, Couderc S, et al. Risk factors for congenital cytomegalovirus infection following primary and nonprimary maternal infection: a prospective neonatal screening study using polymerase chain reaction in saliva. Clin Infect Dis 2017;65 (3):398 404. [58] Simonazzi G, Curti A, Cervi F, et al. Perinatal outcomes of non-primary maternal cytomegalovirus infection: a 15-year experience. Fetal Diagn Ther 2018;43:138 42. [59] Nelson CS, Cruz DV, Tran D, et al. Preexisting antibodies can protect against congenital cytomegalovirus infection in monkeys. JCI Insight 2017;2(13), e94002. [60] Bialas KM, Westreich D, Cisneros de la Rosa E, et al. Maternal antibody responses and nonprimary congenital cytomegalovirus infection of HIV-1-exposed infants. J Infect Dis 2016;214(12):1916 23. [61] Kimberlin DW, Baley J, Brady MT, et al. Guidance on management of asymptomatic neonates born to women with active genital herpes lesions. Pediatrics 2013;131(2):e635 646. [62] Brown ZA, Wald A, Morrow RA, Selke S, Zeh J, Corey L. Effect of serologic status and cesarean delivery on transmission rates of herpes simplex virus from mother to infant. JAMA 2003;289(2):203 9. [63] Berardi A, Lugli L, Rossi C, et al. Neonatal herpes simplex virus. J Matern Fetal Neonatal Med 2011;24 (Suppl. 1):88 90. [64] ACOG. Clinical management guidelines for obstetrician-gynecologists. No. 82 June 2007. Management of herpes in pregnancy. Obstet Gynecol 2007;109(6):1489 98. [65] Pinninti SG, Angara R, Feja KN, et al. Neonatal herpes disease following maternal antenatal antiviral suppressive therapy: a multicenter case series. J Pediatr 2012;161(1) 134-138.e131-133. [66] Baker DA. Consequences of herpes simplex virus in pregnancy and their prevention. Curr Opin Infect Dis 2007;20(1):73 6. [67] Stanberry LR, Spruance SL, Cunningham AL, et al. Glycoprotein-D-adjuvant vaccine to prevent genital herpes. N Engl J Med 2002;347(21):1652 61. [68] Corey L, Wald A. Maternal and neonatal HSV infections. N Engl J Med 2009;361(14):1376 85. [69] Petro C, Gonzalez PA, Cheshenko N, et al. Herpes simplex type 2 virus deleted in glycoprotein D protects against vaginal, skin and neural disease. eLife 2015;4, e06054. [70] Besnard M, Lastere S, Teissier A, Cao-Lormeau V, Musso D. Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014. Eurosurveillance 2014;19(13), 20751.

[71] Musso D, Roche C, Robin E, Nhan T, Teissier A, CaoLormeau VM. Potential sexual transmission of Zika virus. Emerg Infect Dis 2015;21(2):359 61. [72] Cunha MS, Esposito DL, Rocco IM, et al. First Complete Genome Sequence of Zika Virus (Flaviviridae, Flavivirus) from an Autochthonous Transmission in Brazil. Genome Announc 2016;4(2), e00032 16. [73] Blood GAC. Zika Virus (ZIKV). Transfus Med Hemother 2016;43(6):436 46. [74] Brasil P, Pereira Jr. JP, Moreira ME, et al. Zika virus infection in pregnant women in Rio de Janeiro. N Engl J Med 2016;375(24):2321 34. [75] Larocca RA, Abbink P, Peron JP, et al. Vaccine protection against Zika virus from Brazil. Nature 2016;536 (7617):474 8. [76] Abbink P, Larocca RA, De La Barrera RA, et al. Protective efficacy of multiple vaccine platforms against Zika virus challenge in rhesus monkeys. Science 2016;353(6304):1129 32. [77] Dowd KA, Ko SY, Morabito KM, et al. Rapid development of a DNA vaccine for Zika virus. Science 2016;354(6309):237 40. [78] Richner JM, Jagger BW, Shan C, et al. Vaccine mediated protection against Zika virus-induced congenital disease. Cell 2017;170(2) 273-283.e212. [79] Tebas P, Roberts CC, Muthumani K, et al. Safety and immunogenicity of an anti-Zika virus DNA vaccine preliminary report. N Engl J Med 2017;. [80] Dangor Z, Lala SG, Cutland CL, et al. Burden of invasive group B Streptococcus disease and early neurological sequelae in South African infants. PLoS One 2015;10(4):e0123014. [81] Madhi SA, Dangor Z, Heath PT, et al. Considerations for a phase-III trial to evaluate a group B Streptococcus polysaccharide-protein conjugate vaccine in pregnant women for the prevention of early- and late-onset invasive disease in young-infants. Vaccine 2013;31 (Suppl. 4):D52 57. [82] Schrag SJ, Verani JR. Intrapartum antibiotic prophylaxis for the prevention of perinatal group B streptococcal disease: experience in the United States and implications for a potential group B streptococcal vaccine. Vaccine 2013;31(Suppl. 4):D20 26. [83] Boyer KM, Gotoff SP. Prevention of early-onset neonatal group B streptococcal disease with selective intrapartum chemoprophylaxis. N Engl J Med 1986;314 (26):1665 9. [84] Morales WJ, Dickey SS, Bornick P, Lim DV. Change in antibiotic resistance of group B streptococcus: impact on intrapartum management. Am J Obstet Gynecol 1999;181(2):310 14.

VI. MUCOSAL VACCINES FOR VIRAL DISEASES

REFERENCES

[85] Baker CJ, Kasper DL. Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infection. N Engl J Med 1976;294 (14):753 6. [86] Baker CJ, Edwards MS, Kasper DL. Immunogenicity of polysaccharides from type III, group B Streptococcus. J Clin Invest 1978;61(4):1107 10.

749

[87] Baker CJ, Rench MA, McInnes P. Immunization of pregnant women with group B streptococcal type III capsular polysaccharide-tetanus toxoid conjugate vaccine. Vaccine 2003;21(24):3468 72. [88] Donders GG, Halperin SA, Devlieger R, et al. Maternal immunization with an investigational trivalent group B Streptococcal vaccine: a randomized controlled trial. Obstet Gynecol 2016;127(2):213 21.

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C H A P T E R

45

Systems Biological Approaches for Mucosal Vaccine Development Bali Pulendran1,2,3 1

Institute for Immunity, Transplantation and Infection, 2Department of Pathology, 3Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, United States

I. INTRODUCTION Most pathogens enter the body through a mucosal portal of entry. Therefore vaccination against mucosal infections is a major global health priority. Considerable efforts have been made toward developing vaccines that can be administered via mucosal routes, as this route of vaccination is thought to be most effective for inducing mucosal immunity [1,2]. The great efficacy of the oral polio vaccine (OPV) in controlling polio is a classic example of the success of mucosal vaccination [3]. Furthermore, mucosal vaccines have the logistical advantage of not requiring injection needles; this feature enhances vaccine compliance and lowers the risk of acquiring blood-borne infections. However, of the approximately two dozen licensed vaccines, most of which were developed against pathogens that enter the body via mucosal sites, the vast majority are administered via the parenteral route [1,2] (Fig. 45.1). Only a few licensed vaccines are administered via mucosal routes: oral vaccines against

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00045-6

cholera, typhoid, polio, and rotavirus and a nasal vaccine against influenza. Several fundamental challenges have stymied the development of mucosal vaccines. First, the harsh acidic environment of the stomach can cause degradation of proteins in oral vaccines. Second, the immune regulatory mechanisms in the intestine (e.g., regulatory T cells and macrophage and dendritic cell (DC) subsets, which induce T regulatory cells), which suppress immunity to self antigens from food and gut bacteria, pose obstacles to the induction of robust and durable immune responses through oral vaccination (Chapter 15: Mucosal Regulatory System for Balanced Immunity in the Gut). Indeed, waning intestinal immunity after oral polio [4], cholera [5], or rotavirus vaccination [6] represents a major challenge. Third, there is a paucity of understanding about the types of immune responses and the underlying immunological mechanisms that are necessary to induce durable protective immunity against mucosal infections [7]. Ironically, we lack a detailed understanding of the immunological

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Mucosal vaccines Oral polio vaccine (oral) Rotavirus (oral) Live attenuated influenza (intranasal) Cholera (oral) Typhoid (oral)

Parenteral vaccines Inactivated polio vaccine (intramuscular) Inactivated influenza (intramuscular) Typhoid (intramuscular) BCG (TB vaccine - intramuscular) Varicella (subcutaneous) Pneumococcal (intramuscular) Meningococcal (intramuscular) Diphtheria, tetanus, pertussis (intramuscular) Haemophilus (intramuscular) Human papilloma vaccine (intramuscular) MMR (measles, mumps, rubella - subcutaneous) Smallpox (multiple puncture with bifurcated needle) Anthrax (intramuscular or subcutaneous)

Protective immunity against mucosal infection

FIGURE 45.1 Licensed vaccines that induce protective immunity against mucosal infections. Most pathogens enter the body through mucosal sites. Of the approximately two dozen licensed vaccines, the vast majority are administered via the parenteral route. Only a few licensed vaccines are administered via mucosal routes. Several fundamental challenges have stymied the development of mucosal vaccines.

mechanisms by which even parenterally administered vaccines such as the BCG vaccine against tuberculosis (TB) or the inactivated polio vaccine induce protective immunity. Fourth, accurate quantitation of immune responses induced by mucosal vaccination (Chapter 23: Recombinant BCG for Mucosal Immunity Recombinant Bacillus Calmette-Gue´rin for Mucosal Immunity and Chapter 35: Development of a Mucosal TB Vaccine Using Human Parainfluenza Type 2 Virus) is lacking and poses a challenge to identifying correlates of protection. Fifth, mucosal vaccines such as the oral rotavirus vaccines have been shown to be consistently less immunogenic and efficient in infant populations in developing countries [8] (Chapter 40: The Role of Innate Immunity in Regulating Rotavirus Replication, Pathogenesis, and Host Range Restriction and the Implications for Live Rotaviral Vaccine Development and Chapter 41: Development of Oral Rotavirus and Norovirus Vaccines). It is likely that factors such as the gut microbiota, nutritional status, preexisting immunity to prior

infections, and host genetic factors affect the immunogenicity of mucosal vaccines [9] (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity). These issues underscore the lack of in-depth understanding of the molecular and cellular networks that orchestrate protective immunity induced by vaccination against mucosal infections. In recent years, researchers have begun to use vaccines as probes to explore the fundamental mechanisms driving immune responses in humans [9]. Vaccines have been described as the most costeffective public health tools in history, but to immunologists, vaccines offer an added benefit: They represent wonderful probes with which to study human immunity. Several features make them well suited for this purpose. First, they are very widely used in diverse human populations, ranging from the very young to the frail elderly, in healthy adults and in immunocompromised individuals such as transplant or HIV patients, and in geographically segregated populations with different ancestries

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I. INTRODUCTION

and microbiomes. Second, vaccines represent a plethora of different microbial stimuli such as single-stranded RNA viruses such as the oral polio or rotavirus or yellow fever vaccines, or DNA viruses such as the smallpox vaccine, or carbohydrate vaccines such as the meningococcal or pneumococcal vaccines, recombinant proteins with diverse adjuvants such as alum, M59, and the AS0 based adjuvants [10 12]. These diverse microbial stimuli stimulate the immune system through diverse pathways of innate sensing [13]. Third, vaccines allow a highly synchronized perturbation of the human immune system. We know the precise moment at which a vaccine enters the body and can follow the ensuing innate and adaptive immune responses from the earliest minutes to decades after vaccination. Finally, and perhaps most important, the recent development of the tools of systems biology has provided a means to probe the immune responses to vaccination in humans with an exquisite degree or precision [14 19]. These tools include analysis of gene expression using RNA sequencing, metabolomics, high dimensional flow cytometry approaches such as CyTOF, analysis of the epigenetic landscape of immune cells using ATAC-sequencing and EpiTOF, high throughput analysis of the antigen-specific T and B cell repertoires, as well as comprehensive profiling of the diverse array of glycan moieties and effector functions of antibodies, to analyze immune responses in the blood of vaccinated subjects [14 21] (Fig. 45.2). Such studies have helped to identify molecular signatures or molecular correlates (composed of gene expression or metabolomic signatures in blood) induced within a few days of vaccination, which correlate with the ensuing antigen-specific antibody response and T cell responses to vaccination [22 43]. Furthermore, computational approaches involving machine learning techniques have helped to define and validate signatures that can be used to predict the magnitude of the immune response in

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independent studies [22,24,33]. In addition, such an approach has recently been used to define novel correlates of protection induced by vaccination with RTS,S malaria vaccine in controlled human infection models [31,38] (Chapter 49: Mucosal Vaccine for Malaria). The data generated from such studies are being mined to obtain new biological insights into mechanisms of immunity to vaccination and are revealing unexpected insights into the immune system. Despite the promise of this so-called systems vaccinology approach, very few studies have been done to probe the immune responses to vaccination against vaccines administered via the mucosal routes. Given the lack of detailed understanding of the mechanisms driving protective immunity to mucosal vaccines, the application of systemsbased approaches provides a unique opportunity to define novel correlates of immunogenicity and/or efficacy and to delineate immune mechanisms. This is particularly relevant because, despite the development of mucosal vaccines against polio, cholera, typhoid, and rotavirus, there is little knowledge about the correlates of protection induced by the licensed mucosal vaccines—intranasal influenza, rotavirus, and typhoid—as well as for most investigational enteric vaccines [1,2]. This chapter discusses the potential application of systems vaccinology in advancing the rational design of mucosal vaccines. The first part of the chapter provides a brief overview of the emerging field of systems vaccinology, its origins, and its role in revitalizing human immunology and vaccine biology. The second part discusses the potential role that systems vaccinology approaches could play in addressing some of the challenges facing the development of mucosal vaccines. This will be discussed in the context of controlled human infection challenge models with pathogens such as influenza and typhoid, which provide a unique opportunity to deepen our understanding of mechanisms of vaccine immunity and identify novel correlates of protection.

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FIGURE 45.2 Vaccines offer a means to probe the human immune system. The toolkit of systems biology can be used to probe the behavior of genes, molecules, cells, and metabolites in response to vaccination. Source: Reproduced with permission from Nakaya HI1, Li S, Pulendran B. Systems vaccinology: learning to compute the behavior of vaccine induced immunity. Wiley Interdiscip Rev Syst Biol Med 2012;4(2):193 205.

II. SYSTEMS VACCINOLOGY Vaccination represents one of the greatest triumphs of public health, yet vaccines against disease such as HIV, TB, malaria, dengue, and many enteric diseases have been difficult to develop, despite several decades of intense effort. A major problem has been our very

limited understanding of the types of immune responses necessary for protection against such infectious diseases and the lack of strategies capable of inducing such protective immune responses. In addition, of the many vaccine concepts that have undergone preclinical testing in animal models, very few have been evaluated in phase 2 and 3 vaccine efficacy trials,

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because of the prohibitive costs of conducting such trials. In many cases, even when vaccines have been tested in phase 2 and 3 clinical trials, correlates of vaccine efficacy have been difficult to ascertain. One of the main hurdles in identifying correlates of vaccine efficacy and determining mechanisms of vaccine immunity is that, traditionally, vaccinologists have relied on the measurement of a single parameter of the concentration (e.g., binding antibody titers by enzyme-linked immunosorbent assay, or ELISA) or functional activity (e.g., neutralization titers or opsonophagocytic titers) [44]. When vaccination results in a given threshold of the magnitude or functional activity of the antibody response, this is assumed to be sufficient to confer protective immunity against infection. However, a simple measurement (e.g., hemagglutination inhibition titers for influenza)

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to assess vaccine efficacy fails to capture the complexity of the immune response against a pathogen and the multiple immune mechanisms that might synergize to mediate protection. The recent application of systems biological approaches (or systems vaccinology) to studying immune responses to vaccination has provided a new approach to testing vaccines [14 21].

A. Proof of Concept: Studies With the Yellow Fever Vaccine YF-17D The first studies to apply systems-based approaches to define molecular signatures of vaccine immunity focused on one of the most successful vaccines ever developed: the live attenuated yellow fever vaccine YF-17D [22,23] (Fig. 45.3). YF-17D consists of alive attenuated

FIGURE 45.3 Proof of concept study with the yellow fever vaccine YF-17D. Healthy adult subjects were vaccinated with YF-17D, and blood samples were collected at days 0, 3, 7, 15, and 60 90 to analyze the innate and adaptive immune responses, using the techniques of systems biology. Transcriptional profiling identified a molecular signature in the blood, induced within 3 7 days after vaccination. Machine learning techniques helped to define a signature that correlated with the vaccine-induced CD81 T cells and neutralizing antibody responses and could predict with high accuracy the responses in an independent trial. Source: Pulendran B, Li S, Nakaya HI. Systems vaccinology. Immunity 2010;33(4):516 29.

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virus, which was developed by serial passage of the pathogenic Asibi strain of yellow fever by Max Theiler at Rockefeller [14]. Vaccination with a single dose of YF-17D induces robust expansion of antigen-specific CD81 T cells and potent neutralizing antibody responses that can last more than a decade. The vaccine is highly efficacious and is considered to be one of the most successful vaccines ever made, having been administered to over 600 million people worldwide [14]. Despite its success, the immunological mechanisms by which it induces such robust and durable immune responses was poorly understood. Several years ago, we decided to use YF-17D as a model of a “gold standard” successful vaccine to deconstruct the immunological mechanisms underlying its immunogenicity. Our initial study in mice indicated that YF-17D signaled via multiple innate immune receptors (toll-like receptors (TLRs) 2, 3, 7, 8, and 9 as well as RIG-I and MDA5) and activated myeloid DCs and plasmacytoid DCs to induce a broad spectrum of innate and adaptive immune responses, including a mixed Th1/Th2 profile, CD81 T cells, and antibody responses [45]. To obtain a more global view of the innate and adaptive immune responses to vaccination, we decided to use the emerging tools of systems biology to probe the innate and adaptive responses to vaccination with YF-17D in humans. Our goals in this study were twofold. The first goal was to address a major challenge in vaccinology to prospectively determine vaccine immunity. We wished to determine whether molecular signatures of the transcriptional response to vaccination could be detected in the blood. Since YF-17D is injected subcutaneously, it was uncertain whether such signatures could be detected in the blood. We reasoned that if such signatures could indeed be detected by using machine learning approaches, it would be possible to define signatures that could be used to predict the ensuing T cell and antibody responses in an independent study of YF-17D. Our second goal

was to probe the molecular networks induced by vaccination, with a view toward discovering new biological insights about the mechanism of action of this vaccine and, more broadly, about the human immune system. In essence, our study was designed to use this vaccine as a probe to query the human immune system. Our study demonstrated that vaccination induced the expression of genes that regulate virus innate sensing and type 1 interferon production within 3 7 days of vaccination, consistent with the fact that YF-17D causes an acute viral infection [22]. Computational analyses identified a gene signature, including complement protein C1qB and eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4, also known as GCN2, an orchestrator of the integrated stress response), that correlated with the YF-17D CD81 T cell responses [22]. Furthermore, using several machine learning approaches, including discriminant analysis of integer programming, we could identify signatures that could predict with up to 90% accuracy, in an independent, blind trial done subsequently, the magnitude of the YF-17D CD81 T cell responses. A distinct signature, including B cell growth factor TNFRS17, predicted the neutralizing antibody response with up to 100% accuracy [22]. In an independent study, the group of Rafick Sekaly and colleagues identified similar transcriptional signatures induced by YF-17D in humans, including type 1 IFN and complement pathways driven by the transcription factors IRF7 and STAT1 [23]. Together, these studies highlight the utility of systems biology approaches in predicting vaccine efficacy.

B. Extending Systems Vaccinology to Other Vaccines The initial studies with YF-17D raised the question of whether systems approaches could be used to study other vaccines, particularly inactivated nonreplicating vaccines. Thus we and several other groups have extended

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the systems biology approach to study immune responses to other vaccines, including the trivalent inactivated vaccine (TIV) and live attenuated (LAIV) seasonal influenza vaccine [24,25], meningococcal vaccines [29], hepatitis B vaccine [41], shingles vaccines [39,42], malaria vaccine [27,31], Ebola vaccine [37], and HIV vaccine [28]. In our study of the seasonal influenza vaccines, we observed that immunization with LAIV induced a strong type 1 IFN antiviral signature, similar to that seen with YF-17D and other viral infections, but since the hemagglutination inhibition (HAI) response was very low, only TIV vaccines were considered in further correlation analyses. These analyses demonstrated transcriptional signatures related to the expansion of plasmablasts 7 days after vaccination, which correlated with and could be used to predict hemagglutinin titers a month later [24]. Similar findings were reported in the studies of Bucasas et al. [25] and Obermoser et al. [34]. Notably, the gene encoding TNFRSF17, TNFRSF17, which belongs to a family of molecules (BAFF, APRIL, BAFF-R, and TACI) that regulate plasma cell differentiation and antibody production and were previously shown to be the best predictor of neutralizing antibody responses to YF-17D, was shown to be one of the predictors of HAI titers. Tsang et al. [32] and Furman et al. [35] have extended this approach to search for baseline signatures capable of discriminating between high and low responders to vaccination. In our analysis of responses in young and elderly adults to inactivated influenza vaccine across five influenza seasons, we identified several transcriptional modules whose prevaccination expression was associated with an increased or diminished day 28 antibody response [33]. A key question was whether such signatures are similar across multiple seasons and in diverse populations. We therefore applied systems approaches to study immune responses in more than 400 young, elderly, and diabetic

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subjects vaccinated with the seasonal influenza vaccine across five consecutive seasons. Signatures of innate immunity and plasmablasts correlated with and predicted influenza antibody titers at 1 month after vaccination with greater than 80% accuracy across multiple seasons. Baseline signatures of lymphocyte and monocyte inflammation were positively and negatively correlated, respectively, with antibody responses at 1 month [33]. Thus these results identify shared vaccine-induced signatures across multiple seasons and in diverse populations (Chapter 39: Nasal Influenza Vaccines). An important question was whether different vaccines would induce distinct transcriptional signatures or whether they induced similar signatures and whether there was in fact a universal signature, that could be used to predict antibody responses induced by any vaccine. To address this, we used a systems-based approach to compare signatures induced by different types of vaccines (YF-17D, LAIV, TIV, and the carbohydrate meningococcal vaccine Menimmune and the conjugate meningococcal vaccine Menectra) [29]. The results indicated that although the signatures induced by different vaccines were different, they could be clustered according to the type of vaccine (i.e., viral vectors versus inactivated vaccine) and according to whether the response was a primary or a recall response. Thus recall antibody responses to inactivated vaccines, such as the seasonal influenza vaccine or diphtheria toxoid component of the conjugate meningococcal vaccine, were correlated with transcriptional signatures (or blood transcriptional modules) containing genes expressed highly in plasmablasts and other genes that orchestrate plasmablast differentiation. In contrast, the antibody response to the live attenuated yellow fever vaccine was highly correlated with blood transcriptional modules containing genes related to innate immunity and type 1 IFN responses [29]. Furthermore, antibody responses

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to the polysaccharide components of the carbohydrate or conjugate meningococcal vaccines were highly correlated with modules containing genes that orchestrate proinflammatory cytokines or activation of antigen-presenting cells. These results demonstrate that the transcriptional correlates of antibody responses to different vaccines is dependent on the type of vaccine and on whether the response was a primary or a secondary response [29].

C. Are Molecular Signatures of Immunogenicity Versus Efficacy the Same? Although most vaccines have been licensed on the basis of a single measurement of the antibody response as a correlate, in many cases antibody responses may not be the underlying mechanism or correlate of protective immunity. For example, persistent varicella-specific T cells have been shown to be indicators of protection from varicella virus infection and have been suggested as possible additional or alternative correlates of protection in children and the elderly [46,47]. Furthermore, antibody titers to influenza vaccination may be unreliable for predicting risk of influenza illness in the elderly population [48]. Therefore systems-

based approaches may offer means of identifying novel correlates of protective immunity. We and others have recently used systemsbased approaches to find correlates of protection induced by the RTS,S vaccine, an advanced malaria vaccine candidate that confers significant protection against Plasmodium falciparum infection in humans (Fig. 45.4). Little is known about the correlates and mechanisms of protection induced by the RTS,S vaccine. Our study applied a systems biology approach to analyze immune responses in subjects receiving three consecutive immunizations with RTS,S administered with the AS01 adjuvant (RRR) or in those receiving two immunizations of RTS, S/AS01 following a primary immunization with adenovirus 35 (Ad35) vector expressing circumsporozoite protein (ARR) [31]. Three weeks after the final vaccination, the vaccinees were administered a controlled human malaria challenge with Plasmodium-infected mosquitoes. Vaccination resulted in a an approximately 50% protection in both groups of vaccinees (Fig. 45.4). In the RRR group, the circumsporozoite protein (CSP)-specific binding antibody titer on the day of challenge was correlated with protection. In contrast, ARR vaccination induced lower CSP-specific antibody responses,

FIGURE 45.4 Identifying correlates of protection for the malaria RTS,S vaccine. Transcriptional profiling of signatures induced by vaccination by RTS,S vaccine could be used to identify correlates of efficacy of the RTS,S vaccine. Predictive modeling using machine learning techniques may identify signatures that classify subjects according to their infection status. Source: Nakaya HI, Pulendran B. Probiotics, antibiotics and the immune responses to vaccines. Philos Trans R Soc Lond B Biol Sci 2015;370(1671). doi:10.1098/rstb.2014.0146.

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and there was no significant correlation with protection. However, protection was associated with the magnitude of the polyfunctional CD41 T cell response 2 weeks after priming with Ad35. Transcriptional profiling revealed that in the RRR cohort, molecular signatures of B cells and plasma cells were highly correlated with antibody titers prechallenge as well with protection [31]. In contrast, in the ARR cohort, early signatures of innate immunity and DC activation were highly associated with protection. For both vaccine regimens, natural killer (NK) cell signatures were negatively correlated with and predicted protection. Molecular signatures of B cells and plasma cells detected in peripheral blood mononuclear cells (PBMCs) were highly correlated with prechallenge antibody titers and protection in the RRR cohort. These results illustrate that protective immunity against P. falciparum can be achieved via multiple immunological pathways and highlight the utility of systems-based approaches in defining molecular signatures of vaccine efficacy [31].

D. Beyond Blood: Systems Analysis of Gene Signatures in Lymphoid and Nonlymphoid Tissues The immune system comprises many lineages of cells, which are widely distributed throughout lymphoid and nonlymphoid tissues in the body. Since sampling of tissues is difficult because of the invasiveness of the collection procedures and because cell yields can be low and variable, most work to date has focused on analyzing blood samples. Peripheral blood contains cells from many different lineages and dozens of differentiation states within the immune system. Since migration represents a key feature of an immune response, many peripheral blood leukocytes represent cells en route to or from lymphoid and nonlymphoid tissues. Thus the population

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of immune cells in the peripheral blood can provide a sensitive bellwether of localized or systemic immunologic events. For this reason, efforts to date have focused on analyzing gene expression profiles from PBMCs of vaccinated individuals. However, since lymph nodes are the sites where immune responses are initiated and tissues including mucosal sites represent sites of effector function, there are now efforts to sample other tissues such as draining lymph nodes, using the technique of fine needle aspiration or biopsy. Standardization of methods to collect, process, and assay mucosal tissues has proven challenging, but many efforts are now starting to address these issues [49 51].

E. From Data to Knowledge to Understanding Systems vaccinology has yielded unprecedented volumes of omics data about immune responses induced by vaccination, but a major challenge for the field at large is learning how to extract knowledge and ultimately understanding about the mechanisms driving host immune responses. Several recent reviews have discussed this issue [9,13,16], and the reader is referred to these reviews for a detailed discussion. However, it is already clear that mining the data generated from systems vaccinology studies can yield novel insights about the immune system. For example, as was mentioned above, our recent work with the yellow fever vaccine revealed that the gene encoding the molecule GCN2, a central orchestrator of the amino acid sensing pathway in mammalian cells, was induced a few days after vaccination and was highly correlated to the ensuing CD81 T cell responses [22]. Mechanistic dissection of this using a knockout mouse model revealed an unappreciated link between GCN2 and antigen presentation by DCs to CD81 T cells and to inflammation [52,53]. Thus systems vaccinology

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revealed an unappreciated link between the ancient nutritional sensing pathway and adaptive immunity. Furthermore, our recent work with the seasonal influenza vaccine revealed that the early expression of the gene encoding TLR5, a receptor for bacterial flagellin, was highly correlated with the ensuing HAI titers. Mechanistic studies in mice revealed that TLR5 was indeed necessary for optimal induction of antibody responses to influenza vaccination and that TLR5 mediated its effects by sensing flagellin from the intestinal microbiota [54]. Thus systems vaccinology revealed a causal link between the gut microbiota and vaccine immunity. This concept is currently being tested in a human clinical trial.

III. SYSTEMS BIOLOGY OF VACCINES AGAINST MUCOSAL INFECTIONS How might systems vaccinology approaches be used to advance the development of mucosal vaccines? As indicated in Fig. 45.1, vaccines that induce protective immunity against mucosal infections fall into two categories: those that are administered parenterally and those that are administered via the mucosal route. Parenteral vaccination induces antigen-specific serum immunoglobulins and is thought to protect against (1) pathogens that infect mucosal sites (e.g., the respiratory and urogenital tracts) in which transudation of serum immunoglobulins can occur, (2) invasive pathogens that enter via a mucosal portal of entry and gain rapid systemic access via blood (e.g., HIV, influenza virus, polio virus, Streptococcus pneumoniae, Salmonella typhi), or (3) invasive pathogens that enter via a mucosal site and can cross the epithelial barrier and infect submucosal areas (e.g., Shigella, Salmonella) [1,2]. In contrast, mucosal vaccination induces in situ priming of the immune response in lymph nodes proximal

to the mucosal site of entry and the generation of plasma cells that secrete antigen-specific immunoglobulins locally. This seems critical for protection against noninvasive pathogens (e.g., cholera, ETEC) that infect mucosal tissues but that remain on the luminal side of the mucosal epithelium, at mucosal sites where transudation of serum does not occur because of lack of receptor-mediated transcytosis [1,2]. Several major challenges have plagued the development of mucosal vaccines.

Challenge 1: Discovering Correlates of Protection Only 5 of the approximately 30 licensed vaccines are mucosal vaccines [1,2,7]. Despite decades of effort, very few of the many mucosal vaccine concepts proposed and studied in mice have been tested in humans. Human efficacy trials involve several thousand subjects, last several years, and cost several hundreds of millions of dollars. The primary endpoint of such trials is an efficacy endpoint, and their trial study designs and sample collection schedules provide little information about the immune responses to vaccination and typically offer limited opportunities for a retrospective analysis of immune correlates of efficacy. For both parenterally administered and mucosal vaccines, a major challenge is that the correlates and mechanisms of their efficacy remain poorly defined. A potential solution to this problem is smaller (approximately 30 subjects), much less expensive phase-1-like (“phase 0” or experimental) trials that involves a deep immune profiling of immune responses to vaccine, using systems-based approaches. Such studies have been performed for vaccines against yellow fever and influenza, meningococcal vaccines, shingles vaccine, HIV vaccines, malaria vaccine, Ebola vaccine, and others (reviewed in Refs. [15,16]). They have provided a very detailed picture of the immune response to

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vaccination and have helped to identify early molecular signatures of immunogenicity. 1. Controlled Human Infection Models Such studies can be done in the context of controlled human infection models (e.g., typhoid [55 57], influenza [58,59], malaria [31]), in which small groups of subjects can be vaccinated and challenged with the pathogen to assess molecular correlates and mechanisms of vaccine efficacy. In this regard, as was discussed above, we and others have recently used systems-based approaches to find correlates of protection induced by the RTS,S vaccine, an advanced malaria vaccine candidate that confers significant protection against P. falciparum infection in humans [27,31,38]. Such phase 0 trials can be done for a small fraction of the cost of phase 3 efficacy trials. While they certainly do not replace the efficacy trials, they provide a deep understanding of immune responses to vaccination and help to identify putative correlates of immunogenicity and protection. 2. Can Signatures in the Blood Predict Mucosal Immunity? A particular problem in discovering correlates for mucosal immunity is that sampling blood may not necessarily provide correlates of immune responses at distal mucosal sites. Our recent studies of the efficacy of an HIV vaccine in nonhuman primates suggests that systemsbased approaches can reveal vaccine induced early molecular signatures in the blood that correlate not only with the ensuing serum antibody response, but also with antigen-specific mucosal antibody titers and protection against mucosal infection [60]. In this context, our recent work with a recombinant envelope (Env) protein against simian immune deficiency virus (SIV), adjuvanted with nanoparticle encapsulated ligands for TLRs 4 and 7/8 (MPL and R848, respectively) in nonhuman primates,

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revealed that protection against SIV infection was correlated with the prechallenge titers of Env-specific IgG antibodies in serum and vaginal secretions [60]. Transcriptional profiling of PBMCs isolated within the first few hours to days after primary vaccination revealed that adjuvanted vaccines induced a molecular signature similar to that induced by the live attenuated yellow fever viral vaccine (YF-17D). This systems approach identified early blood transcriptional signatures that correlate with Env-specific antibody responses in vaginal secretions and protection against infection [60]. These results demonstrate that transcriptional profiling can be used to identify molecular signatures that correlate with antibody titers in vaginal tissues and with protection against mucosal challenge [60] (Fig. 45.5).

Challenge 2: Discovering Fundamental Immunological Mechanisms of Mucosal Immunity Despite tremendous advances in understanding the mucosal immune system, particularly in mice, fundamental immunological questions about the cellular dynamics of the immune response to mucosal vaccination in humans remain mysterious. For example, we understand very little about how immune responses to mucosal vaccination are initiated and controlled in humans, the receptors by which the innate immune system senses mucosal vaccines, the types of DCs and macrophages that prime antigen-specific T and B cell responses to mucosal vaccines, the dynamics of the innate and adaptive immune responses, the molecular networks that drive such responses, and factors that control the durability of such responses in humans. Some of the fundamental immunological issues that need to be addressed in human vaccination studies to develop novel vaccines against mucosal infections are discussed below.

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FIGURE 45.5 Molecular signatures in blood can predict vaccine-induced mucosal immunity and vaccine efficacy against a mucosal infection. (A) A challenge in systems vaccinology is to identify early signatures in the blood that can be used to predict immune responses in mucosal tissues and efficacy against infection at mucosal sites. Rhesus macaques were vaccinated with nanoparticles containing a TLR4 ligand (MPL or monophosphoryl lipid A) and a TLR7/8 ligand (R848) mixed with soluble Envelope (Env) protein from a strain of SIV. Subsequent intravaginal challenge with SIV resulted in protected a subset of the vaccinated animals. Protection was highly correlated with the serum Env-specific IgG binding antibody titers as well as the mucosal Env-specific IgG antibody binding titers on the day of challenge. Systems analysis helped to define an early transcriptional signature (induced within a few days of vaccination), which correlated with the serum and vaginal Env-specific IgG titers several months later. In addition, we identified a signature that correlated with protection against infection 1 year later. (B) There was considerable overlap between the signature of protection and that which correlated with serum and mucosal Env-specific IgG titers. This demonstrates that systems approaches can be used to define blood signatures of mucosal vaccine immunogenicity and efficacy. Source: Kasturi SP, et al. Adjuvanting a simian immunodeficiency virus vaccine with toll-like receptor ligands encapsulated in nanoparticles induces persistent antibody responses and enhanced protection in TRIM5alpha restrictive macaques. J Virol 2017;91(4):e01844-16.

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1. How Can Vaccines and Adjuvants Imprint Mucosal Homing of Antigen-Specific T and B Cells? It has been known for decades from studies in mice that activated T cells have the propensity to migrate preferentially to the tissues in which they were initially primed [61 64]. For example, priming of T cells in the intestinal lymph nodes results in upregulation of the intestinal homing molecules α4β7 and chemokine receptor 9 (CCR9), which mediate migration of the cells to intestine [65,66]. The ligands for α4β7 and CCR9 are the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) [65] and TECK, also known as CCL25, respectively [66], which are expressed by small intestinal epithelial cells and mediate recruitment of T cells to intestinal lamina propria. Interestingly, DCs from mucosal sites can imprint T cells to migrate to the site at which they were initially primed. Thus antigen presentation and priming by intestinal DCs induce α4β7 and CCR9 on T cells [67 69]. The mechanism of this imprinting is dependent on the metabolism of vitamin A into retinoic acid (RA) [70], in a subset of intestinal CD1031 DCs [70 74]. CD1031 DCs residing within the lamina propria, Peyer’s patches, and mesenteric lymph nodes can prime naı¨ve T cells and imprint intestinal homing and gut tropism [70,75 79]. In addition to its effects on T cells, RA can also imprint intestinal homing of B cells by upregulation of α4β7 and CCR9 [77] and can also enhance production of IgA [80,81]. Similarly, a recent study in mice by Uematsu et al. has shown that CD11chi CD11bhi LPDCs induced the differentiation of naı¨ve B cells into IgA-producing plasma cells by a mechanism that is dependent on RA [82]. Interestingly, vitamin A deficiency is associated with enhanced susceptibility to almost all types of infections, with defects in both the innate and adaptive immune systems [83,84]. Finally, recent data in mice demonstrate that even lung DCs targeted by intranasal immunization are capable of

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inducing α4β7 and licensing T cells to migrate to the intestine [85]. This study suggests a coordination of immune imprinting between geographically distant mucosal sites (Chapter 5: Mucosal Immunity for Inflammation: Regulation of GutSpecific Lymphocyte Migration by Integrins). The extent to which these mechanisms exist in humans and whether they can be harnessed to develop novel mucosal vaccines are unknown. 2. How do Antibodies Protect Against Infection at Mucosal Sites? Despite the vital roles played by antibodies in protection against infection, the mechanisms by which antibodies can protect against mucosal infection are poorly understood. Typically, vaccine-induced antigen-specific antibody responses are measured by ELISA (which measures the quantity of antigen-specific binding antibodies) or by their ability to inhibit the pathogen or toxin in functional assays such as the neutralization assay (e.g., polio, HIV), hemagglutination assay (HAI for influenza), or opsonophagocytic assay (for pneumococcal bacteria) [44]. In addition, a wide range of antibody effector functions, such as antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and antibody-dependent complement deposition, have been described and could represent mechanisms of protection against mucosal infections, mediated by the Fc region of antibodies and its associated glycans [86,87]. Recent advances have used high throughput techniques to comprehensively profile the diverse range of effector functions of vaccineinduced antibodies and to assess the relevance of Fc region and the associated glycan moieties in mediating protection [87]. These diverse effector functions of antibodies are likely to be highly dependent on inflammatory signals delivered by the local innate environment. Thus the functional state of the innate immune system (e.g., DCs, macrophages, or epithelial cells) in mucosal sites may modulate the

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signals delivered by pathogen antibody complexes via Fc receptors to these cells [88]. Consistent with this, our recent studies in nonhuman primates reveal a synergy between host restriction factors and vaccine-induced immune responses in mediating protection against SIV infection [89]. Thus vaccination with nanoparticle-encapsulated TLR ligands (MPL and R848) and SIV Env and Gag proteins resulted in robust Env-specific serum and vaginal antibody titers (both IgG and IgA), which correlated with protection against an intravaginal challenge with a pathogenic SIV strain. However, there was enhanced protection in animals with TRIM5a restrictive alleles. TRIM5a is an innate restriction factor, and a role for type 1IFN in the induction of TRIM5a and other innate restriction factors such as APOBEC3 and tetherin has been documented [90]. Thus using systems-based approaches in human experimental studies or in nonhuman primate models to explore the mechanistic basis of this synergy is likely to reveal new insights. 3. What Roles Do T Cells Play in Protection Against Infection at Mucosal Sites? Most successful vaccines rely on antibodymediated protection, and since CD41 T follicular cells (Tfh) are critical for antibody production, efficient induction of Tfh is necessary for vaccine efficacy [91]. The extent to which CD81 T cells contribute to protection against mucosal infection in humans is less well understood. However, many viral vector vaccines, such as yellow fever vaccine and smallpox vaccine, induce robust expansion and differentiation of antigen-specific CD81 T cells [92,93]. Furthermore, recent work by Picker and colleagues have demonstrated that immunization of rhesus macaques with rhesus cytomegalovirus (RhCMV) vectors stimulates a high magnitude of SIV-specific effector memory T cell responses at mucosal sites of SIV infection and controls highly pathogenic SIV(MAC239) infection early after mucosal challenge in

approximately 50% of the animals. Protection correlated with the magnitude of the peak SIV-specific CD81 T cell responses in the vaccine phase [94 96]. Remarkably, CD81 T cells induced by RhCMV are restricted by either MHC-II or MHC-E but not by MHC-Ia [94,95]. The structural basis of this highly unusual recognition is not clear. MHC-E molecules (HLA-E in humans) is loaded with the highly conserved peptide VMAPRTL(V/L/I)L (VL9) encoded in the leader sequence of classical MHC-I molecules in a TAP-dependent manner [97]. The MHC-E/VL9 complex serves as a ligand for inhibitory NK cell receptors and provides an inhibitory self signal to NK cells [98]. The observation that RhCMV vectors induce an unconventional class of CD81 T cells therefore represents a potentially novel type of protective immune responses against viruses. Recent studies in mice with many different viral infections have identified tissue-resident memory T cells (TRM) that reside in nonlymphoid tissues, including mucosal sites, and respond rapidly to infection [99,100]. A key question is whether the induction of TRMs via vaccination can increase the protective efficacy against mucosal pathogens. Interestingly, a recent human study with the malaria vaccine PfSPZ demonstrated that vaccinees who were protected against controlled human infection with P. falciparum had relatively low levels of antigen-specific T cells and antibody responses in the blood. However, analysis of the immune response to PfSPZ vaccination revealed a 100-fold higher frequency of TRMs in the liver [101]. Furthermore, our recent study in nonhuman primates revealed that vaccination with a synthetic TLR7/8-ligand-adjuvanted SIV Env protein vaccine failed to induce protection against a low-dose repeated intravaginal challenge with a hard-to-neutralize SIV virus strain [102]. In contrast, immunization sequentially with three different viral vectors expressing SIV gag (in order to elicit robust expansion of Gag-specific CD81 T cells in the blood and

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TRMs in tissues) followed by booster immunizations with the TLR7/8-ligand adjuvanted Env resulted in robust Env-specific antibody responses and robust gag-specific CD81 T cells and enhanced protection in young macaques [102]. These results suggest that TRMs can synergize with antigen-specific antibodies to mediate enhanced protection against mucosal infections. Therefore learning the mechanisms by which TRMs and antibodies can synergize to induce enhanced protection will be critical in designing novel mucosal vaccines, and systems analysis of tissue biopsies in the context of vaccine studies will be informative for mucosal vaccine design.

macaques with a novel synthetic TLR7/ 8-ligand-adjuvanted vaccine containing HIV Env antigens has demonstrated remarkably long-lived bone marrow cells and persistent germinal centers and T follicular cells, as well as durable antibody responses [60]. Thus adjuvants that program the innate immune system can regulate the durability of immune responses. These results in mice and in nonhuman primates need to be tested in phase 0 experimental studies in humans, and the use of systems biological approaches is likely to yield novel insights into the molecular mechanisms that drive durable antibody responses (Chapter 42: Mucosal Vaccines Against HIV/SIV Infection).

Challenge 3: How Can the Durability of Mucosal Responses Be Enhanced?

Challenge 4: Understanding the Basis of Population Differences in the Efficacy of Mucosal Vaccines

Although many live vaccines such as smallpox vaccine and yellow fever vaccine are known to induce durable antibody responses that can last several decades, waning immunity is a major problem with many oral vaccines as well as inactivated parenteral vaccines [4,103,104]. The oral cholera vaccine Dukarol, rotavirus vaccines and inactivated parenteral vaccines such as the inactivated polio vaccine, inactivated influenza, the RTS,S malaria vaccine, and the RV144 Thai HIV vaccine trial have highlighted the problem of waning immunity [4,103,104]. Little is known about the mechanisms that regulate long-lived antibody responses and bone marrow plasma cells that maintain antibody responses. Our earlier study in mice demonstrated that immunization with nanoparticle-encapsulated ligands for TLR4 and TLR7 (MPL and R837, respectively) plus antigen (ovalbumin or influenza hemagglutinin) could induce remarkably long-lived germinal centers and durable antibody responses [105]. Our recent studies in nonhuman primates have confirmed these findings [60]. In particular, we observed that immunizing rhesus

Oral vaccines, including oral rotavirus vaccines and OPVs, are known to be less immunogenic and less effective in developing countries. Recent studies with the rotavirus vaccines Rotarix and Rotateq have shown lower efficacy in developing countries in Asia, Africa, and Latin America [106 110]. OPVs are also known to less immunogenic in developing countries than in developed countries [111 113]. Variations in each of these determinants could affect the immune response to vaccines. Indeed, it has been speculated that the diminished efficacy of oral vaccines in developing countries could be caused by a myriad of factors such as malnutrition and micronutrient deficiencies; differences in the composition of the intestinal microbiota; higher prevalence of infections such as helminthic infections, tuberculosis, and HIV [114]; or polymorphisms in genes that regulate immune responses to oral vaccines. In addition, it is possible that higher titers of maternal antibodies against enteric pathogens could interfere with viral uptake and immune responses to oral vaccines.

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Physiological systems such as the immune system are shaped by the complex interplay of three interdependent determinants: host genetics, the environment, and the microbiome. It is likely that variations in any of these determinants in populations in different parts of the world contribute substantially to the observed differences in vaccine efficacy. However, we have very little understanding of the magnitude, type, and durability of innate and adaptive responses to vaccination with oral vaccines in diverse populations. However, the evidence is conflicting, and it is now clear that the nutritional status of children influences immune responses in poorly understood and complex ways [115]. Similarly, the link between obesity and weak immunity is still being determined, but the chronic inflammation associated with obesity [116] might negatively affect vaccineinduced immunity. Therefore there is a need to critically reexamine the relationship between malnutrition or obesity and immune responses through clinical trials in which multiple parameters of innate and adaptive responses can be evaluated by cutting-edge technologies, including the tools of systems biology [15,22,23]. Systems vaccinology approaches could yield much insight and help to define the molecular networks underlying the immune response to vaccination in diverse human populations and will enable strategies to reengineer these networks to generate protective immunity. Such studies should aim to obtain an integrated high-resolution picture of the dynamic changes in the expression of genes, proteins, metabolites, and cellular composition in response to vaccination, as well as the composition of the microflora before, during, and after vaccination, in diverse populations. One study by Muyanja and colleagues compared the response to yellow fever YF-17D vaccination in adults in Entebbe, Uganda, versus Lausanne, Switzerland, and showed that YF-17D-induced CD81 T cell and B cell responses were substantially lower in the

Entebbe subjects than in the Lausanne subjects [43]. The impaired vaccine response in the Entebbe cohort was associated with reduced YF-17D replication. Interestingly, prior to vaccination, there was a higher frequency of exhausted and activated NK cells, differentiated T and B cell subsets, and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Activation of CD81 T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D-neutralizing antibody titers after vaccination [43].

IV. CONCLUDING REMARKS Vaccines represent one of the greatest lifesaving devices in history. However, the development of vaccines against global pandemics such as HIV, TB, malaria and diarrheal diseases has been stymied by a limited understanding about what types of immune responses are optimally effective for protection, and how to induce and maintain such responses. Furthermore, the evaluation of vaccine concepts in the clinic has been frustrated by the high cost and slow pace of performing phase 2 and 3 efficacy trials. For example, during the past 30 years, of the many vaccine concepts that have been proposed for an HIV vaccine, only four have been tested in efficacy trials. Systems vaccinology offers a new paradigm to evaluate the efficacy of vaccination and to decipher the mechanisms of action of vaccines. Research over the past few years has revealed the power of systems-based approaches in defining molecular signatures that predict vaccine immunogenicity and efficacy and in revealing new insights into the immunological mechanisms underlying vaccination. Systems vaccinology holds much promise in the development of mucosal vaccines and will help to define correlates of efficacy and provide rich new insights into the mechanisms driving protective immunity.

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REFERENCES

Acknowledgments I wish to acknowledge the generous support of the National Institutes of Health, the Bill and Melinda Gates Foundation, and the Soffer Endowment.

References [1] Pasetti MF, et al. Immunology of gut mucosal vaccines. Immunol Rev 2011;239(1):125 48. [2] Holmgren J, Svennerholm AM. Vaccines against mucosal infections. Curr Opin Immunol 2012;24(3):343 53. [3] Bandyopadhyay AS, et al. Polio vaccination: past, present and future. Future Microbiol 2015;10(5):791 808. [4] Grassly NC, et al. Waning intestinal immunity after vaccination with oral poliovirus vaccines in India. J Infect Dis 2012;205(10):1554 61. [5] Pasetti MF, Levine MM. Insights from natural infection-derived immunity to cholera instruct vaccine efforts. Clin Vaccine Immunol 2012;19(11):1707 11. [6] Jiang V, et al. Performance of rotavirus vaccines in developed and developing countries. Hum Vaccines 2010;6(7):532 42. [7] Holmgren J, et al. Correlates of protection for enteric vaccines. Vaccine 2017;35(26):3355 63. [8] Babji S, Kang G. Rotavirus vaccination in developing countries. Curr Opin Virol 2012;2(4):443 8. [9] Pulendran B. Systems vaccinology: probing humanity’s diverse immune systems with vaccines. Proc Natl Acad Sci USA 2014;111(34):12300 6. [10] Reed SG, Orr MT, Fox CB. Key roles of adjuvants in modern vaccines. Nat Med 2013;19(12):1597 608. [11] Del Giudice G, Rappuoli R, Didierlaurent AM. Correlates of adjuvanticity: a review on adjuvants in licensed vaccines. Semin Immunol 2018;39:14 21. [12] O’Hagan DT, et al. Towards an evidence based approach for the development of adjuvanted vaccines. Curr Opin Immunol 2017;47:93 102. [13] Pulendran B, Ahmed R. Immunological mechanisms of vaccination. Nat Immunol 2011;12(6):509 17. [14] Pulendran B. Learning immunology from the yellow fever vaccine: innate immunity to systems vaccinology. Nat Rev Immunol 2009;9(10):741 7. [15] Pulendran B, Li S, Nakaya HI. Systems vaccinology. Immunity 2010;33(4):516 29. [16] Hagan T, Pulendran B. Will systems biology deliver its promise and contribute to the development of new or improved vaccines? From data to understanding through systems biology. Cold Spring Harb Perspect Biol 2018;10(8). [17] Kuri-Cervantes L, et al. Systems biology and the quest for correlates of protection to guide the development of an HIV vaccine. Curr Opin Immunol 2016;41:91 7.

769

[18] Davis MM, Tato CM, Furman D. Systems immunology: just getting started. Nat Immunol 2017;18(7):725 32. [19] Furman D, Davis MM. New approaches to understanding the immune response to vaccination and infection. Vaccine 2015;33(40):5271 81. [20] Alter G, Ottenhoff THM, Joosten SA. Antibody glycosylation in inflammation, disease and vaccination. Semin Immunol 2018;39:102 10. [21] Gunn BM, Alter G. Modulating antibody functionality in infectious disease and vaccination. Trends Mol Med 2016;22(11):969 82. [22] Querec TD, et al. Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans. Nat Immunol 2009;10(1):116 25. [23] Gaucher D, et al. Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses. J Exp Med 2008;205(13):3119 31. [24] Nakaya HI, et al. Systems biology of vaccination for seasonal influenza in humans. Nat Immunol 2011;12 (8):786 95. [25] Bucasas KL, et al. Early patterns of gene expression correlate with the humoral immune response to influenza vaccination in humans. J Infect Dis 2011;203 (7):921 9. [26] Tan Y, et al. Gene signatures related to B-cell proliferation predict influenza vaccine-induced antibody response. Eur J Immunol 2014;44:285 95. [27] Vahey MT, et al. Expression of genes associated with immunoproteasome processing of major histocompatibility complex peptides is indicative of protection with adjuvanted RTS,S malaria vaccine. J Infect Dis 2010;201 (4):580 9. [28] Zak DE, et al. Merck Ad5/HIV induces broad innate immune activation that predicts CD8 1 T-cell responses but is attenuated by preexisting Ad5 immunity. Proc Natl Acad Sci USA 2012;109(50):E3503 12. [29] Li S, et al. Molecular signatures of antibody responses derived from a systems biology study of five human vaccines. Nat Immunol 2014;15(2):195 204. [30] Nakaya HI, et al. Systems biology of immunity to MF59-adjuvanted versus nonadjuvanted trivalent seasonal influenza vaccines in early childhood. Proc Natl Acad Sci USA 2016;113(7):1853 8. [31] Kazmin D, et al. Systems analysis of protective immune responses to RTS,S malaria vaccination in humans. Proc Natl Acad Sci USA 2017;114(9):2425 30. [32] Tsang JS, et al. Global analyses of human immune variation reveal baseline predictors of postvaccination responses. Cell 2014;157(2):499 513. [33] Nakaya HI, et al. Systems analysis of immunity to influenza vaccination across multiple years and in diverse populations reveals shared molecular signatures. Immunity 2015;43(6):1186 98.

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

770

45. SYSTEMS BIOLOGICAL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

[34] Obermoser G, et al. Systems scale interactive exploration reveals quantitative and qualitative differences in response to influenza and pneumococcal vaccines. Immunity 2013;38(4):831 44. [35] Furman D, et al. Apoptosis and other immune biomarkers predict influenza vaccine responsiveness. Mol Syst Biol 2013;9(1):659. [36] Furman D, et al. Systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination. Proc Natl Acad Sci USA 2014;111(2):869 74. [37] Rechtien A, et al. Systems vaccinology identifies an early innate immune signature as a correlate of antibody responses to the Ebola Vaccine rVSV-ZEBOV. Cell Rep 2017;20(9):2251 61. [38] van den Berg RA, et al. Predicting RTS,s vaccinemediated protection from transcriptomes in a malariachallenge clinical trial. Front Immunol 2017;8:557. [39] Qi Q, et al. Defective T memory cell differentiation after varicella zoster vaccination in older individuals. PLoS Pathog 2016;12(10):e1005892. [40] Hou J, et al. A systems vaccinology approach reveals temporal transcriptomic changes of immune responses to the yellow fever 17D vaccine. J Immunol 2017;199 (4):1476 89. [41] Fourati S, et al. Pre-vaccination inflammation and B-cell signalling predict age-related hyporesponse to hepatitis B vaccination. Nat Commun 2016;7:10369. [42] Li S, et al. Metabolic phenotypes of response to vaccination in humans. Cell 2017;169(5):862 77 e17. [43] Muyanja E, et al. Immune activation alters cellular and humoral responses to yellow fever 17D vaccine. J Clin Invest 2014;124(7):3147 58. [44] Plotkin SA. Correlates of protection induced by vaccination. Clin Vaccine Immunol 2010;17(7):1055 65. [45] Querec T, et al. Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity. J Exp Med 2006;203 (2):413 24. [46] Arvin AM. Humoral and cellular immunity to varicella-zoster virus: an overview. J Infect Dis 2008;197(Suppl. 2):S58 60. [47] Levin MJ, et al. Varicella-zoster virus-specific immune responses in elderly recipients of a herpes zoster vaccine. J Infect Dis 2008;197(6):825 35. [48] McElhaney JE, et al. T cell responses are better correlates of vaccine protection in the elderly. J Immunol 2006;176(10):6333 9. [49] Kaltsidis H, et al. Measuring human T cell responses in blood and gut samples using qualified methods suitable for evaluation of HIV vaccine candidates in clinical trials. J Immunol Methods 2011;370 (1 2):43 54.

[50] Mehra V, Musib R, Schito ML. Towards developing standardized protocols for evaluation of cellular mucosal immune responses - recommendations from a DAIDS/NIH workshop, June 15-16, 2009. Vaccine 2010;28(30):4689 94. [51] Hladik F, Hope TJ. HIV infection of the genital mucosa in women. Curr HIV/AIDS Rep 2009;6(1):20 8. [52] Ravindran R, et al. Vaccine activation of the nutrient sensor GCN2 in dendritic cells enhances antigen presentation. Science 2014;343(6168):313 17. [53] Ravindran R, et al. The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation. Nature 2016;531(7595):523 7. [54] Oh JZ, et al. TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination. Immunity 2014;41(3):478 92. [55] Jin C, et al. Efficacy and immunogenicity of a Vi-tetanus toxoid conjugate vaccine in the prevention of typhoid fever using a controlled human infection model of Salmonella Typhi: a randomised controlled, phase 2b trial. Lancet 2017;390(10111):2472 80. [56] Blohmke CJ, et al. Interferon-driven alterations of the host’s amino acid metabolism in the pathogenesis of typhoid fever. J Exp Med 2016;213(6):1061 77. [57] Salerno-Goncalves R, et al. Challenge of humans with wild-type Salmonella enterica serovar Typhi elicits changes in the activation and homing characteristics of mucosal-associated invariant T cells. Front Immunol 2017;8:398. [58] Lambkin-Williams R, et al. The human viral challenge model: accelerating the evaluation of respiratory antivirals, vaccines and novel diagnostics. Respir Res 2018;19(1):123. [59] Treanor J, Wright PF. Immune correlates of protection against influenza in the human challenge model. Dev Biol (Basel) 2003;115:97 104. [60] Kasturi SP, et al. Adjuvanting a simian immunodeficiency virus vaccine with toll-like receptor ligands encapsulated in nanoparticles induces persistent antibody responses and enhanced protection in TRIM5alpha restrictive macaques. J Virol 2017;91(4). [61] Kantele A, et al. Differential homing commitments of antigen-specific T cells after oral or parenteral immunization in humans. J Immunol 1999;162 (9):5173 7. [62] Campbell DJ, Butcher EC. Rapid acquisition of tissuespecific homing phenotypes by CD4(1) T cells activated in cutaneous or mucosal lymphoid tissues. J Exp Med 2002;195(1):135 41. [63] Cahill RN, et al. Two distinct pools of recirculating T lymphocytes: migratory characteristics of nodal and intestinal T lymphocytes. J Exp Med 1977;145 (2):420 8.

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

REFERENCES

[64] Hall JG, Hopkins J, Orlans E. Studies on the lymphocytes of sheep. III. Destination of lymph-borne immunoblasts in relation to their tissue of origin. Eur J Immunol 1977;7(1):30 7. [65] Berlin C, et al. Alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 1995;80(3):413 22. [66] Zabel BA, et al. Human G protein-coupled receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis. J Exp Med 1999;190(9):1241 56. [67] Johansson-Lindbom B, et al. Selective generation of gut tropic T cells in gut-associated lymphoid tissue (GALT): requirement for GALT dendritic cells and adjuvant. J Exp Med 2003;198(6):963 9. [68] Mora JR, et al. Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature 2003;424(6944):88 93. [69] Stagg AJ, Kamm MA, Knight SC. Intestinal dendritic cells increase T cell expression of alpha4beta7 integrin. Eur J Immunol 2002;32(5):1445 54. [70] Iwata M, et al. Retinoic acid imprints gut-homing specificity on T cells. Immunity 2004;21(4):527 38. [71] Johansson-Lindbom B, et al. Functional specialization of gut CD103 1 dendritic cells in the regulation of tissue-selective T cell homing. J Exp Med 2005;202 (8):1063 73. [72] Coombes JL, et al. A functionally specialized population of mucosal CD103 1 DCs induces Foxp3 1 regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J Exp Med 2007;204(8):1757 64. [73] Sun CM, et al. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. J Exp Med 2007;204(8):1775 85. [74] Jaensson E, et al. Small intestinal CD103 1 dendritic cells display unique functional properties that are conserved between mice and humans. J Exp Med 2008;205 (9):2139 49. [75] Elgueta R, et al. Imprinting of CCR9 on CD4 T cells requires IL-4 signaling on mesenteric lymph node dendritic cells. J Immunol 2008;180(10):6501 7. [76] Mucida D, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 2007;317(5835):256 60. [77] Mora JR, et al. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science 2006;314 (5802):1157 60. [78] Johansson-Lindbom B, Agace WW. Vitamin A helps gut T cells find their way in the dark. Nat Med 2004;10 (12):1300 1.

771

[79] Saurer L, McCullough KC, Summerfield A. In vitro induction of mucosa-type dendritic cells by all-trans retinoic acid. J Immunol 2007;179(6):3504 14. [80] Zhou L, et al. IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol 2007;8(9):967 74. [81] Pasatiempo AM, et al. The antibody response of vitamin A-deficient rats to pneumococcal polysaccharide is enhanced through coimmunization with lipopolysaccharide. J Infect Dis 1994;169(2):441 4. [82] Uematsu S, et al. Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5. Nat Immunol 2008;9 (7):769 76. [83] Bang BG, Bang FB, Foard MA. Lymphocyte depression induced in chickens on diets deficient in vitamin A and other components. Am J Pathol 1972;68 (1):147 62. [84] Stephensen CB. Vitamin A, infection, and immune function. Annu Rev Nutr 2001;21:167 92. [85] Ruane D, et al. Lung dendritic cells induce migration of protective T cells to the gastrointestinal tract. J Exp Med 2013;210(9):1871 88. [86] Tomaras GD, Plotkin SA. Complex immune correlates of protection in HIV-1 vaccine efficacy trials. Immunol Rev 2017;275(1):245 61. [87] Lu LL, et al. Beyond binding: antibody effector functions in infectious diseases. Nat Rev Immunol 2018;18 (1):46 61. [88] Bournazos S, et al. Signaling by antibodies: recent progress. Annu Rev Immunol 2017;35:285 311. [89] Letvin NL, et al. Immune and genetic correlates of vaccine protection against mucosal infection by SIV in monkeys. Sci Transl Med 2011;3(81):81ra36. [90] Altfeld M, Gale Jr. M. Innate immunity against HIV-1 infection. Nat Immunol 2015;16(6):554 62. [91] Havenar-Daughton C, Lee JH, Crotty S. Tfh cells and HIV bnAbs, an immunodominance model of the HIV neutralizing antibody generation problem. Immunol Rev 2017;275(1):49 61. [92] Akondy RS, et al. Origin and differentiation of human memory CD8 T cells after vaccination. Nature 2017;552 (7685):362 7. [93] Miller JD, et al. Human effector and memory CD8 1 T cell responses to smallpox and yellow fever vaccines. Immunity 2008;28(5):710 22. [94] Hansen SG, et al. Cytomegalovirus vectors violate CD8 1 T cell epitope recognition paradigms. Science 2013;340(6135):1237874. [95] Hansen SG, et al. Broadly targeted CD8(1) T cell responses restricted by major histocompatibility complex E. Science 2016;351(6274):714 20.

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

772

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[96] Fruh K, Picker L. CD8 1 T cell programming by cytomegalovirus vectors: applications in prophylactic and therapeutic vaccination. Curr Opin Immunol 2017;47:52 6. [97] Braud V, Jones EY, McMichael A. The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol 1997;27(5):1164 9. [98] Braud VM, et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 1998;391 (6669):795 9. [99] Rosato PC, Beura LK, Masopust D. Tissue resident memory T cells and viral immunity. Curr Opin Virol 2017;22:44 50. [100] Jameson SC, Masopust D. Understanding subset diversity in T cell memory. Immunity 2018;48 (2):214 26. [101] Epstein JE, et al. Live attenuated malaria vaccine designed to protect through hepatic CD8(1) T cell immunity. Science 2011;334(6055):475 80. [102] Petitdemange C, et al. Vaccine induction of antibodies and tissue-resident CD81T cells enhances protection against mucosal SHIV-infection in young macaques. JCI Insight 2019;4(4). [103] Gu XX, et al. Waning immunity and microbial vaccines-Workshop of the National Institute of Allergy and Infectious Diseases. Clin Vaccine Immunol 2017;24(7). [104] Plotkin SA. The importance of persistence. Clin Infect Dis 2016;63(Suppl. 4):S117 18. [105] Kasturi SP, et al. Programming the magnitude and persistence of antibody responses with innate immunity. Nature 2011;470(7335):543 7. [106] Madhi SA, et al. Effect of human rotavirus vaccine on severe diarrhea in African infants. N Engl J Med 2010;362(4):289 98.

[107] Zaman K, et al. Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: a randomised, double-blind, placebo-controlled trial. Lancet 2010;376(9741):615 23. [108] Armah GE, et al. Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub-Saharan Africa: a randomised, double-blind, placebo-controlled trial. Lancet 2010;376(9741):606 14. [109] Patel M, et al. Association between pentavalent rotavirus vaccine and severe rotavirus diarrhea among children in Nicaragua. JAMA 2009;301(21):2243 51. [110] Patel MM, et al. Effectiveness of monovalent rotavirus vaccine in Bolivia: case-control study. BMJ 2013;346: f3726. [111] John TJ. Antibody response of infants in tropics to five doses of oral polio vaccine. Br Med J 1976;1 (6013):812. [112] John TJ, Jayabal P. Oral polio vaccination of children in the tropics. I. The poor seroconversion rates and the absence of viral interference. Am J Epidemiol 1972;96(4):263 9. [113] Patriarca PA, Wright PF, John TJ. Factors affecting the immunogenicity of oral poliovirus vaccine in developing countries: review. Rev Infect Dis 1991;13 (5):926 39. [114] Praharaj I, et al. Probiotics, antibiotics and the immune responses to vaccines. Philos Trans R Soc Lond B Biol Sci 2015;370(1671). [115] Moore SE, et al. Impact of nutritional status on antibody responses to different vaccines in undernourished Gambian children. Acta Paediatr 2003;92 (2):170 6. [116] Vandanmagsar B, et al. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nat Med 2011;17(2):179 88.

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Harnessing γδ T Cells as Natural Immune Modulators Jodi F. Hedges and Mark A. Jutila Department of Microbiology and Immunology, Montana State University, Bozeman, MT, United States

I. INTRODUCTION Lymphocytes are important in both innate and adaptive immune responses. Innate lymphocytes represent a heterogeneous group of cells that include cells lacking receptors for antigen, such as the group of innate lymphoid cells, of which natural killer (NK) cells are the prototypical example. Innate lymphocytes expressing antigen receptors include B1 B cells, natural killer T (NKT) cells, and γδ T cells. While innate lymphocytes are relatively rare in circulation and in lymphoid tissues, they are found in mucosal surfaces that represent portals of entry into the body [1]. In this chapter, we will focus on some of our laboratory’s work on one of the major antigen-specific subsets of innate lymphocytes: γδ T cells. γδ T cells have important roles in both innate and adaptive immune responses, wound healing, and tissue homeostasis. There are many outstanding reviews of the biology and function of γδ T cells. A select few relevant to the topic of this chapter are listed in Table 46.1. Briefly, γδ T cells express unique T cell receptors

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00046-8

(TCRs) that recognize self and foreign antigens in the absence of the requirement for presentation by major histocompatibility complex (MHC) class I or class II molecules. This feature leads to a broad range of innate responses against pathogens, as well as recognition of stressed or tumor cells. Subsets of γδ T cells are defined by restricted TCR gene usage in addition to expression of various surface molecules and preprogrammed functional responses imprinted prior to their egress from the thymus. γδ T cells also express myriad innate receptors, such as toll-like receptors (TLRs), scavenger receptors, and lectin receptors, such as dectin-1, that can directly sense infectious agents. These receptors, along with cytokine receptors, fine-tune sensing and response of γδ T cells adapting to the tissue microenvironment. TCR stimulation leads to a variety of functional responses, such as cytolysis, cytokine production, regulatory effects, and even phagocytosis and antigen presentation, that depend on the activation of receptors and coreceptors. γδ T cells respond rapidly to external signals, leading to early cytokine responses in a

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TABLE 46.1 Recent References/Reviews for Key γδ T Cell Functions γδ T cell function Selected review article General γδ T cell

Hayday, Annu Rev Immunol 2000 [2], Chien, Annu Rev Immunol 2014 [3], Chien, Immunol Rev 2007 [4] Ciofani, Nat Rev Immunol 2010 [5], Holderness, Annu Rev Anim Biosci 2013 [6], Holderness, Crit Rev Immunol 2008 [7], Zarin, PNAS 2014 [8], Ribeiro, Frontiers Immunol 2015 [9]

γδ T cell role in cancer and cytolytic responses

Wu, Cell Mol Immunol 2017 [10], Zou, Oncotarget 2017 [11], Silva-Santos, Nat Rev Immunol 2015 [12], Rei, Cancer Res 2015 [13], Paul, Int J Cancer [14], Ramstead, J Interferon Cytokine Res [15]

γδ T cell innate and adaptive responses

Bonneville, Nat Rev Immunol 2010 [16], Jutila, Anim Health Res Rev 2007 [17], Ribeiro, Front Immunol 2015 [9]

γδ T cells in multiple species

Holderness, Annu Rev Anim Biosci 2013 [6]

Myeloid-cell-like Moser, Trends Immunol 2006 [18], Jutila, features, Anim Health Res Rev 2007 [17], Holderness, Crit Rev Immunol [7] including APC TLR expression by γδ T cells

Wesch, Cell Mol Life Sci 2011 [19], Dar, Front Immunol 2014 [20]

IL-17-producing γδ T cells

Chien, Trends Immunol., 2013 [21], Papotto, Nature Immunol. 2017 [22], Corpuz, J. Immunol., 2016 [23], McKenzie, Nature Communications, 2017 [24]

Skin/gutresident γδ T cells

Hayday, Annu Rev Immunol 2000 [2], Holderness, Crit Rev Immunol 2008 [7], Macleod, Havran, Cell Mol Life Sci 2011 [25], Nielsen, Nat Rev Immunol 2017 [1], Ebert, J Immunol 2006 [26], Sheridan, Immunity 2013 [27]

γδ T cells/ immunotherapy

Burjanadze, Br J Immunol 2007 [28], Lawand, Front Immunol 2017 [29], Mirzaei, Cancer Lett 2016 [30]

variety of disease settings. Furthermore, they are uniquely positioned at virtually all portals of entry into the body where this type of innate immune response is critical. Indeed, γδ T cells, like other innate lymphocytes, are found at all

mucosal surfaces and make up a large fraction of the intraepithelial lymphocyte population. They are also recruited to sites of inflammation, tumor growth, or other tissue insults.

II. γδ T CELL SURFACE RECEPTORS In addition to the γδ TCR, γδ T cells express a variety of non-TCR receptors that affect their function. γδ T cells express the NK C-type lectin-like receptors, such as NKG2D, which recognize cellular stress proteins resulting in cellular activation [31,32]. They also express tumor necrosis factor (TNF) receptor family molecules CD27, CD30, and CD137 [9]. CD27 is a costimulatory receptor to the TCR [33], and CD137 is also expressed on TCR-stimulated tumor-reactive γδ T cells [34]. CD28 (of the Ig superfamily) is also a γδ TCR coreceptor. The aryl hydrocarbon receptor (AhR), generally known for its role in homeostasis for mucosal T cells, is also expressed by mouse γδ T cells that produce innate interleukin 17 (IL-17) [35], as well as the mouse skin γδ T cell subset [36]. γδ T cells also express various cytokine receptors that contribute to their activation (IL-2R, IL15R, IL-23R, etc.) and-fine tune their functional responses. The expression of pathogenassociated molecular pattern receptors has been detected on γδ T cells. These include another lectin receptor, dectin-1, a receptor for fungal, plant, and bacterial-derived polysaccharides [37,38]; the TLRs [20,39]; CD36 [40]; scavenger receptors [41]; and NOD-like receptors [42]. Though not a focus of this chapter, γδ T cells also express a variety of receptors that downregulate their function. Examples include killer cell immunoglobulin-like receptor and leukocyte immunoglobulin-like receptor, B and T lymphocyte attenuator, and programmed cell death 1 receptor, which are regulatory receptors that suppress the function and/or proliferation of the cells [29].

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IV. γδ T CELL-MEDIATED CYTOTOXICITY

III. SIMILARITIES OF γδ T CELLS TO MYELOID AND MACROPHAGE CELLS γδ T cells are an ancient immune cell lineage, found in all jawed vertebrates. Phylogenetic evidence suggests that they are the progenitors of both αβ T cells and B cells [43]. They predate adaptive immunity, so it is not surprising that they retain many innate functions similar to those of monocytes and macrophages. Zebrafish γδ T cells both are phagocytic and can present antigen, in addition to their expression of CD8 [44]. We characterized transcript expression in subsets of bovine γδ T cells [45 47]. The primary outcome of these studies was the recognition of multiple transcripts similar to those found in monocyte and macrophage cells, indicating their innate function. As part of these studies, we detected B-lymphocyte-induced maturation protein 1 (BLIMP-1) transcripts in bovine γδ T cells [46]. BLIMP-1, also known as PRDI-BF1, is a key regulator in the differentiation of hematopoietic cells into myeloid or B cells [48]; therefore, its detection in a T cell subset was notable at the time. We recognized this significance and further confirmed the expression of transcripts in resting bovine γδ T cells and not αβ T cells [46]. More recent findings have further confirmed the innate function of γδ T cells in the appropriate contexts. Transcript analyses in bovine γδ T cells also suggested expression of transcripts encoding solute carrier 11A1 (SLC11A1, also denoted natural resistance-associated macrophage protein 1, or NRAMP-1) in these cells [45]. SLC11A1 is a divalent metal transporter that is thought to be expressed only in myeloid and macrophage cells; it is important in effective responses against intracellular bacterial infections [49 51]. SLC11A1 enhances signaling and activation in macrophages [52]. We defined protein expression and a similar function in activation in bovine and human γδ T cells and NK cells. Expression of SLC11A1 was strongly correlated

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to the activation and, in particular, the expression of interferon gamma (IFNγ) in these cells [53]. Thus SLC11A1 is an additional monocyte/ macrophage protein that is also expressed in γδ T cells with functional relevance. Another similarity to myeloid cells is the ability of γδ T cells in a number of species to process and present antigen. Effective antigen presentation is required for the initiation of adaptive immunity and is studied primarily in conventional antigen-presenting cells (APCs), such as dendritic cells (DCs), activated macrophages, and B cells. γδ T cells express an array of surface receptors, such as scavenger receptors, CD11b, and CD16, that facilitate uptake of particulate antigens [54,55]. Subsets also express MHC class II and necessary coreceptors for effective antigen presentation to CD41 T cells [56 58]. Antigen uptake and presentation to CD41 T cells were first shown for bovine γδ T cells [57]. It was also shown that MHC class II expression and antigen presentation is enhanced in bovine WC11 γδ T cells during viral infection [59]. Similar functions were described for porcine, human, and mouse γδ T cells [60 62]. γδ T cells in contact with bacteria can transition from cytokine-producing cells to phagocytic APCs, demonstrating their functional plasticity [63]. The phagocytic capacity of γδ T cells is augmented by opsonization [54,63]. Combined, these studies show that subsets of γδ T cells in various species can be induced to present antigens via MHC class II. Clearly, γδ T cells have a unique role in innate immunity that is similar in some respects to that of monocytes and macrophages, and further is involved in the subsequent initiation of antigendependent acquired immunity.

IV. γδ T CELL-MEDIATED CYTOTOXICITY Ligation of receptors expressed on the γδ T cell can lead to potent cytolytic responses

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against stressed, infected, and malignant cells [64 67], though γδ T cells can be permissive to growth of some tumors [15]. Ligation of TCR, in combination with other receptors such as NKG2D and cytokine receptors such as the IL23 receptor, enhances and directs cytotoxic responses along with cytokine production [68,69]. Cytotoxicity is a function of γδ T cells that is conserved across species [70 75]. For example, granzyme B, perforin, and FasL are expressed in WC11 γδ T cells from bovine peripheral blood mononuclear cells (PBMCs), with FasL expression increasing upon activation of these cells [76,77]. Perforin expression is also found in bison γδ T cells [78]. Perforin and granzyme, FasL-Fas, and the TNF-related apoptosis-inducing ligand pathway are also features of human γδ T cells [79,80]. The cytotoxic activity of γδ T cells likely plays an important role in multiple species for optimal immune responses by these cells to a subset of malignant and infected cells.

V. γδ T CELL CYTOKINE PRODUCTION Another important functional response of γδ T cells is their regulation of the tissue environment through cytokine generation. These cytokines include those that drive inflammatory responses and contribute to downstream adaptive immune responses as well as cytokines that affect epithelial cell health and tissue homeostasis. Although the number of cytokines produced by γδ T cells is large, a few, such as IL-17, IFNγ, and the tissue cytokines keratinocyte growth factor (KGF) and insulin-like growth factor (IGF), are of particular importance in the function of subsets of these cells. IL-17 and IFNγ are potent activators of cells of the myeloid lineage and contribute to downstream inflammatory responses. In mice, γδ T cells are a major source of innate IL-17 early in response to infection [81,82]. Two populations

of γδ T cells contribute to the IL-17 response. One is referred to as “natural” IL-17-producing cells, which acquire effector function prior to egress from the thymus [83]. These cells are found in mucosal tissues and are thought to be early responders to infectious insult. Another population is referred to as “induced,” and these cells rapidly acquire effector function after egress from the thymus and in response to antigen and cytokine in the periphery [84]. Some reports suggest that although human and large animal γδ T cells produce IL-17 (induced phenotype), they may not be a major early source of this cytokine in these species [10,85]. Although they are clearly protective in most instances and are thought to be important to the early innate immune response, dysregulation of IL-17 production leading to excessive IL-17 can also be pathogenic [86]. KGF and IGF are also produced by tissue γδ T cells and are important in maintaining epithelial cell health and effective wound repair responses [87 90]. Though defined as important in tissue homeostasis, these responses are also important for host defense, since health of the epithelial cell barrier contributes to protection against various pathogens and the creation of homeostatic environment for commensal microbiota.

VI. ROLE OF γδ T CELLS IN INFECTIOUS DISEASES γδ T cells have been shown to respond to and participate in host defense responses in a variety of infectious diseases, including viral, bacterial, and parasite-induced disease, many at the mucosal surface [2,91]. Recently, γδ T cells have been found to be important for protection against emerging viruses such as Chikungunya and West Nile virus [92,93]. In HIV infection, the peripheral subset of human γδ T (Vδ2) cells is severely depleted and does not completely recover, even in patients who have had successful antiretroviral treatment.

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VII. THERAPEUTIC POTENTIAL FOR MANIPULATION OF γδ T CELLS

This deficit may increase the likelihood for secondary infections and could be a critical target for new immunotherapies for HIV patients [94]. γδ T cells are clearly important in antibacterial immunity as a source of early IFNγ and IL-17 [77,82,95,96]. As human γδ T cells are preprogrammed for recognition of bacterial phosphoantigens, they are particularly important in protection from Mycobacterium and Legionella infections [97,98]. Human γδ T cells expand during Salmonella enterica serovar Typhimurium (ST) infection of the intestinal mucosa [99] and are a source of early IFNγ [100,101]. Bovine γδ T cells also respond to oral ST infection [102]. γδ T cells also play a critical role in protection against infection with Brucella sp., which are facultative intracellular bacteria [103]. This appears to be primarily through production of IFNγ, and was found in mice, cattle, and sheep [103,104]. However, our results showed no contribution of mouse γδ T cells to infection with another emerging intracellular pathogen, Coxiella burnetii (unpublished results). Following mucosal infection but not peripheral infection, mouse γδ T cells were also found to have a role in downstream memory immune responses to Listeria infection [27]. Thus, γδ T cells play an important role in response against many different bacterial infections. This suggests that their specific stimulation may contribute to protection and may potentially replace or at least reduce the need for antibiotics and could be considered as a new target for future vaccine development. γδ T cells also play protective roles in parasite infections. They respond to and are protective following initial infection with the malaria Plasmodium falciparum, owing to recognition of phosphoantigens produced by the parasite. However, upon subsequent infection, the numbers of γδ T cells drop, similar to the situation with long-term HIV infection. Nonetheless, higher numbers of functional Vδ2 T cells are correlated with greater protection from reinfection with Plasmodium and also increased

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symptoms upon infection, as they are sources of IFNγ and TNF-α [105]. Similarly, the first instance of bovine IL-17-producing cells was demonstrated and protects against a related parasite [106]. Indeed, in most instances of protection from pathogens, γδ T cells are similarly protective in humans and other animals [6]. Common features across species provide a rationale for the use of various animal models to test the role and importance of γδ T cells in disease settings of relevance to humans, which will lead to the creation of strategic platforms for γδ T-cell-targeted vaccine development.

VII. THERAPEUTIC POTENTIAL FOR MANIPULATION OF γδ T CELLS γδ T cells are characterized by a unique and specific tissue location, rapid response to external signals and insults, and the existence of preprogrammed and induced effector subsets. Combined with the ability to expand these cells in vitro and their critical roles in a variety of infectious and cancerous disease settings, γδ T cells have been the target for new immunotherapeutics [11,28 30,34,91]. In humans, both TCR and TLR agonists have been studied for their effects on enhancing γδ T cell function. Prenyl phosphates and bisphosphonates that directly or indirectly drive expansion and cytokine production in a major subset of circulating γδ T cells have been pursued for treatment of certain tumors and infections [29]. Two approaches have been used. In the first approach, γδ T cells are expanded to large numbers in vitro and then adoptively transferred to patients. In the second approach, these agonists are given directly to the patient, inducing responses in vivo. The in vivo responses of γδ T cells to these agonists are impressive, leading to significant expansion in tissues, such as the lung and production of immune cytokines [107]. Of note, though originally pursued for cancer treatments, the potential application of

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phosphoantigen stimulation of γδ T cells in infectious disease was recently demonstrated in Mycobacterium tuberculosis infection in primates [108]. The application of these therapeutic approaches to stimulate γδ T cells is limited to humans and nonhuman primates, since γδ T cell responses to the prenyl phosphates are restricted to primate cells. Other therapeutic approaches to increase γδ T cell activity have focused on other receptors, such as TLRs and scavenger receptors [19,20]. Our recent endeavor has been to expand the number of materials that enhance the activity of γδ T cells in multiple species. This was achieved by screening various natural product libraries and other sources of natural products, including nutritional supplements. They were assessed for their capacity to upregulate IL-2 receptor expression on primary γδ T cells, thereby enhancing responses to IL-2 in the absence of antigen [7,17,109 112]. Follow-up functional assays examined their cell type specificity, induced cytokine responses, and benefit in various infectious disease models [110,112]. Two classes of plant products—polyphenols and polysaccharides—and one example of a microbial product that stimulate these cells, which came from these studies, are summarized below.

VIII. PLANT POLYPHENOLS FOR THE ACTIVATION OF γδ T CELLS A class of plant polyphenol called oligomeric procyanidins (OPCs) produced by apples, grapes, and some other plants was determined to be a potent priming agent for γδ T cells. Several studies suggest that ingestion of plant and berry compounds containing polyphenols expand human γδ T cells in vivo [113 115]. Our study showed that OPCs from apple peel prime human, mouse, and bovine γδ T cells, and NK cells in some instances [109], for enhanced responses to secondary signals provided by

cytokines and antigens. Other groups also found that OPCs expand mouse γδ T cells in vivo [116] and stimulate goat γδ T cells [117]. OPCmediated γδ T cell responses increase the expression of activation markers, but the cells do not actively proliferate in the absence of a secondary signal, such as cytokine or TCR engagement [109]. OPC treatment also induces production of a restricted number of cytokines, many of which act on myeloid cells, such as colony-stimulating factors (CSFs) and chemokines such as IL-8, and various tissue growth factors [109]. One of the consequences of OPC treatment of bovine and human γδ T cells is a significant extension of the stability of CSF and chemokine transcripts [118]. The ability to extend the functional lifetime of these transcripts enables γδ T cells to more rapidly and robustly produce certain cytokines in response to secondary signals. Importantly, OPCs show bioactivity when ingested and are safe over a range of doses in all species tested [7,116,119]. Such supplements increase γδ T cells in the periphery or in tissues [119,120]. Following oral delivery of very large doses of the OPCs in mice, a significant reduction of inflammation was seen in dextran sulfate sodium (DSS)induced colitis [121]. The anti-inflammatory effects are independent of γδ T cells and require αβ T cells. Interestingly, in the absence of αβ T cells, a Rag-protein-dependent population of cells, likely γδ T cells, is responsible for a robust but noninflammatory cytokine response in OPC treated mice in the DSS model [121]. Consistent with this observation, OPC ingestion in some mice was shown to induce increased levels of G-CSF in circulation without obvious deleterious inflammation (unpublished results). Induced G-CSF is normally considered a proinflammatory response, but it can also contribute to protective immune support in certain instances. Clearly, we have much to learn about the myriad effects of ingestion of OPCs on γδ T cells and other immune cells (e.g., αβ T cells) in vivo. We expect that these potent plant

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VIII. PLANT POLYPHENOLS FOR THE ACTIVATION OF γδ T CELLS

chemicals (e.g., OPC) and their derived products may be a safe novel immunotherapeutic and immunomodulator in some settings.

A. Plant Polysaccharides as γδ T CellTargeted Immunomodulator Our study has also identified unique polysaccharides from various plants that are potent agonists for γδ T cells and other cells of the immune system. The first source of polysaccharide agonist was Funtumia elastica bark (Yamoa). Yamoa polysaccharides activate γδ T as well other immune cells, such as monocytes, and, when given in vivo, enhance protection from infection [110]. Optimal activation or priming of γδ T cells by these polysaccharides requires monocytes or macrophages in a mixed in vitro culture. Following our initial characterization of the Yamoa polysaccharides, similar activity was defined in extracts from other plants, including tansy (unpublished), juniper (unpublished), and, most recently, ac¸ai [111,122,123]. Many of the polysaccharide preparations being tested, except for those generated from ac¸ai, were positive in the limulus amebocyte lysate assay for lipopolysaccharides [124]. Ac¸ai polysaccharide responses are conserved in γδ T cells across species, including humans, cattle, and mice [111]. Monocytes and macrophages are also activated by the polysaccharides and are required for optimal responses by the γδ T cell. Instillation of ac¸ai polysaccharides into the lungs of mice induces dose-dependent IL-12 production, accumulation of myeloid cells, and activation of local DCs and macrophages [111]. It was subsequently shown that prophylactic or therapeutic nasal administration of ac¸ai polysaccharides significantly enhances host innate defense responses against the intracellular bacterial pathogens Francisella tularensis and Burkholderia pseudomallei [125]. Protection could also be achieved following

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oral delivery, although responses were more variable. Mechanism of action studies showed that ac¸ai polysaccharides enhance IFNγ expression by γδ T cells and NK cells following F. tularensis and B. pseudomallei infections. Inhibition of IFNγ blocked the protective effect of the polysaccharides [125]. Thus, the stimulation of γδ T cells, as well as other innate immune cells, by ac¸ai or similar plant agonists and subsequent type 1 T helper cell-associated responses, could have therapeutic applications in bacterial infections. Since ac¸ai is a commonly ingested dietary supplement and has shown therapeutic benefit following oral delivery [125], we examined the effects of these agonists in two additional intestinal models. Dysbiosis is a condition usually induced by antibiotic use in which the normal flora is disrupted. This state can lead to increased susceptibility to infection and colitis [126]. Mice with dysbiosis were treated with ac¸ai polysaccharides to assess whether these polysaccharides could aid in recovery from this susceptible state. When cytokine expression in mesenteric lymph nodes (MLNs) and spleen cells were measured, the feeding of ac¸ai polysaccharides induced expression of IL-12 in supernatant fluids from cultured MLN and spleen cells from the treated mice. IL-12 was also detected in the serum of the mice [127]. Expression of IFNγ was also increased in spleen cells from ac¸ai polysaccharide-fed mice, similar to the previous finding using nasal administration [125]. No adverse effects were noted in the ac¸aitreated mice. In a model of chemically induced colitis, mice that were fed ac¸ai had a reduced deleterious inflammatory response in the gut [127]. Considering that there are no adverse effects following ac¸ai ingestion, this polysaccharide could represent a safe and novel approach to stimulating γδ T cells and other innate cells, potentially to promote their innate protective and homeostatic functions at the mucosal surface.

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Our next study aimed to examine potential receptors involved in the sensing and responses to the ac¸ai polysaccharides by immune cells. Some of responses were lost in mice lacking functional TLR4 or the innate adaptor protein MyD88. However, neutrophils were still recruited into the peritoneum of these mice following intraperitoneal injection of ac¸ai [111]. The role of the β-glucan receptor dectin-1 was particularly investigated, since IL-12 is produced by immune cells following ingestion of β-glucans [128]. Our result demonstrated that ac¸ai polysaccharides contain appropriate linkages for recognition by dectin-1 using an inhibition ELISA against β-glucan [127]. Furthermore, ac¸ai polysaccharides specifically block binding of anti-dectin-1 antibodies to immune cells in a flow cytometry based assay. Thus, ac¸ai polysaccharides bind to multiple innate immune cell receptors, contributing to unique effects of innate and likely downstream adaptive immune responses. Ac¸ai polysaccharides can be considered as a new mucosal immunomodulator molecule for the regulation of antigen-specific immune response and inflammation.

B. Microbial Products for the Regulation of γδ T Cells Activation-based screening assays resulted in the detection of robust agonist activity for γδ T cells in multiple microbial extracts (unpublished results). One such agonist was determined to be amphotericin B (AmB), produced by Streptomyces nodosus. AmB is a commonly used antifungal drug that has previously been shown to stimulate innate immune cells [129 131]. AmB induces expression of cytokines in macrophages, mediated by TLR recognition [132 134]. AmB treatment of bovine PBMCs leads to increased expression of IL-2R selectively on γδ T cells, activation of bovine monocytes and NK cells, and enhanced IFNγ

from NK cells [112]. Addition of IL-2 to these cultures induces a robust, antigen-independent proliferation of the treated γδ T cells [112]. The agonist activity of AmB is not restricted to cattle, in that similar effects are seen on expression of activation markers and proliferation of γδ T cells in humans and mice as well [112]. Thus the response is highly conserved. In a separate study, AmB was shown to increase IFNγ production in mouse lung cells following in vitro infection and costimulation by avirulent C. burnetii bacteria [127]. AmB also enhances antibody responses against ovalbumin when used as an immunizing adjuvant [127]. Thus AmB has potential both to enhance innate and acquired responses to infection and to function as a vaccine adjuvant. Since bovine γδ T cells and NK cells respond to AmB at very low, nontoxic doses, our next experiment aimed to test it in an in vivo model of infectious enterocolitis. Calves were given one intravenous injection of approximately 0.029 0.031 mg/kg AmB or saline 24 hours prior to ST infection by the oral route. AmBtreated calves had lower fevers, had overall reduced morbidity, and shed less bacteria into the environment in comparison to control calves [112]. Thus AmB protected from disease severity and reduced the level of shed bacteria. The result suggested that AmB could be used as a potent immunomodulatory molecule to enhance disease resistance against ST in calves. Our efforts are continuing to assess the immune protective effects of AmB on very young calves, which are highly prone to infection. When bovine calves are less than a weekold, they have a variable colostrum status, and they experience a broad spectrum of natural scouring and respiratory maladies in their first week to 3 months of life. These symptoms are typically caused by rotavirus, coronavirus, Cryptosporidium, or a combination of virus and parasite infections. Regardless of the cause, the calves are treated with a hydration therapy. If signs of a secondary bacterial infection become

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Number of healthy assessments in first 30 days (total of 60)

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consistency, and treatments on a scale of 0 5. Health condition was assessed for all calves twice daily and was compared to calves acquired in the same 3 months in a 5-year span before and after this experiment that did not receive any treatment. In a given period, the study tallied the number of days the calves had perfect health scores (scores of 0). The calves that received one injection of AmB had improved health assessments in their first 10 days in comparison to calves that received no treatment (Fig. 46.1A). The period was then extended to the first 30 days. In this case, the untreated calves were compared to the AmB x1 and AmB x2 groups. Whereas one dose of AmB appeared to benefit in the short term (in the first 10 days), the AmB x1 treatment had no lasting effect. In contrast, calves treated with AmB x2 had longer-lasting positive benefit (Fig. 46.1B). These data suggest that minimal early doses of an innate immune stimulant could benefit the health of livestock for extended periods. This is especially important for cattle that are subject to repeated infections early in life. It also provides proof of principle that broad-spectrum

Am

apparent, antibiotics are administered. The calves were likely preexposed to a variety of pathogens; this would explain the early disease that occurs when they are housed indoors in clean facilities. With years of data on these occurrences of natural illness in our facilities, our study was directed to test whether early minimal treatments with AmB could potentially be used as a broad-spectrum prophylactic immunomodulator. A dose of 0.25 mg/kg injected intravenously as previously described [112] was used in the study. This is approximately 10-fold less than the doses given to patients for antifungal treatment and was determined to be nontoxic in calves. There were two treated groups (n 5 12 per group). One group received a single injection of AmB on the day of arrival at our facility (AmB x1). A second group received this initial dose on the day of their arrival and a second dose after 10 days (AmB x2). Thus, for the first 10 days, there were 24 calves treated with one dose of AmB. Health condition was assessed by evaluating each animal’s subjective appearance and attitude, appetite, temperature, pulse and respirations, fecal

FIGURE 46.1 Early prophylactic treatment of calves with AmB improves their health. Calves were assessed twice daily, and the perfect health assessments in the first 10 days (A) at our facility were tallied for calves that were treated once with AmB (AmB x1), or untreated. (B) Numbers of perfect health assessments in the first 30 days for calves treated with AmB once, on the day of their arrival (AmB x1), or twice, on their first and 10th days in the facility (AmB x2). Statistical analysis was by Student’s t-test. * P value , .05; ** P value , .01.

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protection against an array of mucosal pathogens can likely be achieved through prophylactic use of an innate immune stimulant.

IX. CONCLUDING REMARKS Because of their position in the body and their capacity for varied, appropriate responses depending on the environmental signals, γδ T cells are an optimal target for novel immunotherapeutic and vaccine development. Some TCR and TLR agonists that can stimulate γδ T cells have already been used extensively for new cancer treatments. Ample data suggest that the cells might also be specifically stimulated to protect from infectious and inflammatory disease. Considering the growing concerns about the use and overuse of antibiotics, it is critical that such novel approaches to counter infectious agents be pursued.

Acknowledgments We acknowledge support from the Agriculture and Food Research Initiative competitive grant no. 2014-67016-21552 and Animal Health grants of the USDA National Institute of Food and Agriculture, with partial funding through NIHNCCAM (AT0004986-01), NIH IDeA Program grant GM110732, NIH R21 AI117441, M.J. Murdock Charitable Trust and the Montana State University Agricultural Experimental Station. We acknowledge Kerri Jones for excellent animal care and Dustin Lee for database management and mining.

ABBREVIATIONS AmB APC CSF IFNγ IGF KGF MHC OPC PBMC

amphotericin B antigen presenting cell colony-stimulating factor interferon gamma insulin-like growth factor keratinocyte growth factor major histocompatibility complex oligomeric procyanidin peripheral blood mononuclear cells

ST TCR TLR TNF

Salmonella enterica serovar Typhimurium T cell receptor toll-like receptor tumor necrosis factor

References [1] Nielsen MM, Witherden DA, Havran WL. γδ T cells in homeostasis and host defence of epithelial barrier tissues. Nat Rev Immunol 2017;17:733. [2] Hayday AC. [gamma][delta] cells: a right time and a right place for a conserved third way of protection. Annu Rev Immunol 2000;18:975 1026. [3] Chien Yh, Meyer C, Bonneville M. γδ•T cells: first line of defense and beyond. Annu Rev Immunol 2014;32 (1):121 55. [4] Chien Yh, Konigshofer Y. Antigen recognition by γδ T cells. Immunol Rev 2007;215(1):46 58. [5] Ciofani M, Zu´n˜iga-Pflu¨cker JC. Determining γδ versus αβ T cell development. Nat Rev Immunol 2010;10:657. [6] Holderness J, Hedges JF, Ramstead A, Jutila MA. Comparative biology of γδ T cell function in humans, mice, and domestic animals. Annu Rev Anim Biosci 2013;1(1):99 124. [7] Holderness J, Hedges JF, Daughenbaugh KF, Kimmel E, Graff JC, Freedman B, et al. Response of γδ T cells to plant-derived tannins. Crit Rev Immunol 2008;28(5): 377 402. [8] Zarin P, Wong GW, Mohtashami M, Wiest DL, Zu´n˜iga-Pflu¨cker JC. Enforcement of γδ-lineage commitment by the pre 2 T-cell receptor in precursors with weak γδ-TCR signals. PNAS 2014;111(15):5658 63. [9] Ribeiro ST, Ribot JC, Silva-Santos B. Five layers of receptor signaling in γδ T-cell differentiation and activation. Front Immunol 2015;6:15. [10] Wu D, Wu P, Qiu F, Wei Q, Huang J. Human γδ T-cell subsets and their involvement in tumor immunity. Cell Mol Immunol 2017;14(3):245 53. [11] Zou C, Zhao P, Xiao Z, Han X, Fu F, Fu L. γδ T cells in cancer immunotherapy. Oncotarget 2017;8(5):8900 9. [12] Silva-Santos B, Serre K, Norell H. γδ T cells in cancer. Nat Rev Immunol 2015;15:683. [13] Rei M, Pennington DJ, Silva-Santos B. The emerging protumor role of γδ T lymphocytes: implications for cancer immunotherapy. Cancer Res 2015;75(5):798. [14] Paul S, Lal G. Regulatory and effector functions of gamma 2 delta (γδ) T cells and their therapeutic potential in adoptive cellular therapy for cancer. Int J Cancer 2016;139(5):976 85. [15] Ramstead AG, Jutila MA. Complex role of γδ T-cellderived cytokines and growth factors in cancer. J Interferon Cytokine Res 2012;32(12):563 9.

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

783

REFERENCES

[16] Bonneville M, O’Brien RL, Born WK. γδ T cell effector functions: a blend of innate programming and acquired plasticity. Nat Rev Immunol 2010;10:467. [17] Jutila MA, Holderness J, Graff JC, Hedges JF. Antigen Independent priming: a transitional response of bovine γδ T cells to infection. Anim Health Res Rev 2007;9(1): 47 57. [18] Moser B, Brandes M. Gammadelta T cells: an alternative type of professional APC. Trends Immunol 2006;27(3):112 18. [19] Wesch D, Peters C, Oberg HH, Pietschmann K, Kabelitz D. Modulation of γδ T cell responses by TLR ligands. Cell Mol Life Sci 2011;68(14):2357 70. [20] Dar AA, Patil RS, Chiplunkar SV. Insights into the relationship between toll like receptors and gamma delta T cell responses. Front Immunol 2014;5:366. [21] Chien Yh, Zeng X, Prinz I. The natural and the inducible: interleukin (IL)-17-producing γδ T cells. Trends Immunol 2013;34(4):151 4. [22] Papotto PH, Ribot JC, Silva-Santos B. IL-17 1 γδ T cells as kick-starters of inflammation. Nat Immunol 2017; 18:604. [23] Corpuz TM, Stolp J, Kim HO, Pinget GV, Gray DHD, Cho JH, et al. Differential responsiveness of innate-like IL-17 2 and IFN-γ-producing γδ T cells to homeostatic cytokines. J Immunol 2016;196(2):645. [24] McKenzie DR, Kara EE, Bastow CR, Tyllis TS, Fenix KA, Gregor CE, et al. IL-17-producing γδ T cells switch migratory patterns between resting and activated states. Nat Commun 2017;8:15632. [25] MacLeod AS, Havran WL. Functions of skin-resident γδ T cells. Cell Mol Life Sci 2011;68(14):2399 408. [26] Ebert LM, Meuter S, Moser B. Homing and function of human skin γδ T cells and NK cells: relevance for tumor surveillance. J Immunol 2006;176(7):4331. [27] Sheridan BS, Romagnoli PA, Pham QM, Fu HH, Alonzo F, Schubert WD, et al. γδ T cells exhibit multifunctional and protective memory in intestinal tissues. Immunity 2013;39(1):184 95. [28] Burjanadze M, Condomines M, Reme T, Quittet P, Latry P, Lugagne C, et al. In vitro expansion of gamma delta T cells with anti-myeloma cell activity by Phosphostim and IL-2 in patients with multiple myeloma. Br J Haematol 2007;139(2):206 16. [29] Lawand M, De´chanet-Merville J, Dieu-Nosjean MC. Key features of gamma-delta T-cell subsets in human diseases and their immunotherapeutic implications. Front Immunol 2017;8:761. [30] Mirzaei HR, Mirzaei H, Lee SY, Hadjati J, Till BG. Prospects for chimeric antigen receptor (CAR) γδ T cells: a potential game changer for adoptive T cell cancer immunotherapy. Cancer Lett 2016;380(2):413 23. [31] Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, et al. Activation of NK cells and T cells by NKG2D,

[32]

[33]

[34]

[35]

[36]

[37] [38] [39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

a receptor for stress-inducible MICA. Science 1999;285:727 9. Rincon-Orozco B, Kunzmann V, Wrobel P, Kabelitz D, Steinle A, Herrmann T. Activation of V gamma 9V delta 2 T cells by NKG2D. J Immunol 2005;175(4):2144 51. Ribot JC, Chaves-Ferreira M, d’Orey F, Wencker M, Gonc¸alves-Sousa N, Decalf J, et al. Cutting edge: adaptive versus innate receptor signals selectively control the pool sizes of murine IFN-γ 2 or IL-17 2 producing γδ T cells upon infection. J Immunol 2010;185(11): 6421 5. Deniger DC, Maiti SN, Mi T, Switzer KC, Ramachandran V, Hurton LV, et al. Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies. Clin Cancer Res 2014;20(22):5708 19. Martin B, Hirota K, Cua DJ, Stockinger B, Veldhoen M. Interleukin-17-producing γδ T cells selectively expand in response to pathogen products and environmental signals. Immunity 2009;31(2):321 30. Kadow S, Jux B, Zahner SP, Wingerath B, Chmill S, Clausen BE, et al. Aryl hydrocarbon receptor is critical for homeostasis of invariant γδ T cells in the murine epidermis. J Immunol 2011;187(6):3104. Brown GD, Gordon S. Fungal [beta]-glucans and mammalian immunity. Immunity 2003;19(3):311 15. Gordon S. Pattern recognition receptors: doubling up for the innate immune response. Cell 2002;111:927 30. Hedges JF, Lubick KJ, Jutila MA. γδ T cells respond directly to pathogen associated molecular patterns. J Immunol 2005;174(10):6045 53. Lubick K, Jutila MA. LTA recognition by bovine {gamma}{delta} T cells involves CD36. J Leukoc Biol 2006;79(6):1268 70. Ahn JS, Konno A, Gebe JA, Aruffo A, Hamilton MJ, Park YH, et al. Scavenger receptor cysteine-rich domains 9 and 11 of WC1 are receptors for the WC1 counter receptor. J Leukoc Biol 2002;72(2):382 90. Kerns HMM, Jutila MA, Hedges JF. The distinct response of γδ T cells to the Nod2 agonist, muramyl dipeptide. Cell Immunol 2009;257(1-2):38 43. Richards MH, Nelson JL. The evolution of vertebrate antigen receptors: a phylogenetic approach. Mol Biol Evol 2000;17:146 55. Wan F, Hu Cb, Ma Jx, Gao K, Xiang Lx, Shao Jz. Characterization of γδ T cells from zebrafish provides insights into their important role in adaptive humoral immunity. Front Immunol 2016;7:675. Hedges JF, Cockrell D, Jackiw L, Meissner N, Jutila MA. Differential mRNA expression in circulating γδ T lymphocyte subsets defines unique tissue-specific functions. J Leukoc Biol 2003;73(2):306 14. Meissner N, Radke J, Hedges JF, White M, Behnke M, Bertolino S, et al. Serial analysis of gene expression in

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

784

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

46. HARNESSING γδ T CELLS AS NATURAL IMMUNE MODULATORS

circulating γδ T cell subsets defines distinct immunoregulatory phenotypes and unexpected gene expression profiles. J Immunol 2003;170:356 64. Hedges JF, Graff JC, Jutila MA. Transcriptional profiling of gamma delta T cells. J Immunol 2003;171 (10):4959 64. Chang DH, Angelin-Duclos C, Calame K. BLIMP-1: trigger for differentiation of myeloid lineage. Nat Immunol 2000;1(IP-2):169 76. Malo D, Vogan K, Vidal S, Hu J, Cellier M, Schurr E, et al. Haplotype mapping and sequence analysis of the mouse Nramp gene predict susceptibility to infection with intracellular parasites. Genomics 1994;23(1): 51 61. Vidal S, Tremblay ML, Govoni G, Gauthier S, Sebastiani G, Malo D, et al. The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene. J Exp Med 1995;182(3):655 66. Cellier M, Shustik C, Dalton W, Rich E, Hu J, Malo D, et al. Expression of the human NRAMP1 gene in professional primary phagocytes: studies in blood cells and in HL-60 promyelocytic leukemia. J Leukoc Biol 1997;61(1):96 105. Gomez MA, Li S, Tremblay ML, Olivier M. NRAMP-1 expression modulates protein-tyrosine phosphatase activity in macrophages: impact on host cell signalling and functions. J Biol Chem 2007;282(50):36190 8. Hedges JF, Kimmel EM, Snyder DT, Jerome M, Jutila MA. SLC11A1 is expressed by innate lymphocytes and augments their activation. J Immunol 2013;190(8): 4263 73. Wu Y, Wu W, Wong WM, Ward E, Thrasher AJ, Goldblatt D, et al. Human γδ T cells: a lymphoid lineage cell capable of professional phagocytosis. J Immunol 2009;183(9):5622 9. Lahmers KK, Hedges JF, Jutila MA, Deng M, Abrahamsen MS, Brown WC. Comparative gene expression by WC1 1 {gamma}{delta} and CD4 1 {alpha}{beta} T lymphocytes, which respond to Anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell-associated genes by WC1 1 {gamma}{delta} T cells. J Leukoc Biol 2006;80(4):939 52. Taylor BC, Choi KY, Scibienski RJ, Moore PF, Stott JL. Differential expression of bovine MHC class II antigens identified by monoclonal antibodies. J Leukoc Biol 1993;53(IP-5):479 89. Collins RA, Werling D, Duggan SE, Bland AP, Parsons KR, Howard CJ. Gammadelta T cells present antigen to CD4 1 alphabeta T cells. J Leukoc Biol 1998;63: 707 14. Takamatsu HH, Denyer MS, Stirling C, Cox S, Aggarwal N, Dash P, et al. Porcine γδ T cells: possible

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

roles on the innate and adaptive immune responses following virus infection. Vet Immunol Immunopathol 2006;112(1ΓC ¸ oˆ2):49 61. Toka FN, Kenney MA, Golde WT. Rapid and transient activation of γδ T cells to IFN-γ production, NK celllike killing, and antigen processing during acute virus infection. J Immunol 2011;186(8):4853 61. Takamatsu HH, Denyer MS, Wileman TE. A subpopulation of circulating porcine γδ T cells can act as professional antigen presenting cells. Vet Immunol Immunopathol 2002;87(3):223 4. Brandes M, Willimann K, Moser B. Professional antigen-presentation function by human gammadelta T cells. Science 2005;309(5732):264 8. Cheng L, Cui Y, Shao H, Han G, Zhu L, Huang Y, et al. Mouse γδ T cells are capable of expressing MHC class II molecules, and of functioning as antigen-presenting cells. J Neuroimmunol 2008;203(1):3 11. Barisa M, Kramer AM, Majani Y, Moulding D, Saraiva L, Bajaj-Elliott M, et al. E. coli promotes human Vγ9Vδ2 T cell transition from cytokine-producing bactericidal effectors to professional phagocytic killers in a TCRdependent manner. Sci Rep 2017;7:2805. Haecker G, Wagner H. Proliferative and cytolytic responses of human gamma delta T cells display a distinct specificity pattern. Immunology 1994;81(4):564 8. Fisch P, Moris A, Rammensee HG, Handgretinger R. Inhibitory MHC class I receptors on gammadelta T cells in tumour immunity and autoimmunity. Immunol Today 2000;21:187 91. Kalyan S, Kabelitz D. Defining the nature of human γδ T cells: a biographical sketch of the highly empathetic. Cell Mol Immunol 2012;10:21. Vantourout P, Hayday A. Six-of-the-best: unique contributions of [gamma][delta] T cells to immunology. Nat Rev Immunol 2013;13(2):88 100. Moens E, Brouwer M, Dimova T, Goldman M, Willems F, Vermijlen D. IL-23R and TCR signaling drives the generation of neonatal Vgamma9Vdelta2 T cells expressing high levels of cytotoxic mediators and producing IFN-gamma and IL-17. J Leukoc Biol 2011;89(5):743 52. Von Lilienfeld-Toal M, Nattermann J, Feldmann G, Sievers E, Frank S, Strehl J, et al. Activated γδ T cells express the natural cytotoxicity receptor natural killer p44 and show cytotoxic activity against myeloma cells. Clin Exp Immunol 2006;144(3):528 33. Fox A, Meeusen E. Sheep perforin: identification and expression by gammadelta T cells from pregnant sheep uterine epithelium. Vet Immunol Immunopathol 1999;68:293 6. Phillips JH, Lanier LL. Acquisition of non-MHC restricted cytotoxic function by IL 2 activated thymocytes with an “immature” antigenic phenotype. J Immunol 1987;139(3):683.

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

REFERENCES

[72] Saito T, Pardoll DM, Fowlkes BJ, Ohno H. A murine thymocyte clone expressing γδ T cell receptor mediates natural killer-like cytolytic function and TH1-like lymphokine production. Cell Immunol 1990;131(2): 284 301. [73] Brown WC, Davis WC, Choi SH, Dobbelaere DA, Splitter GA. Functional and phenotypic characterization of WC1 1 gamma/delta T cells isolated from Babesia bovis-stimulated T cell lines. Cell Immunol 1994;153:9 27. [74] Olin MR, Batista L, Xiao Z, Dee SA, Murtaugh MP, Pijoan CC, et al. Gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus. Viral Immunol 2005;18(3):490 9. [75] Choi KD, Lillehoj HS. Role of chicken IL-2 on gammadelta T-cells and Eimeria acervulina-induced changes in intestinal IL-2 mRNA expression and gammadelta T-cells. Vet Immunol Immunopathol 2000;73:309 21. [76] Blumerman SL, Herzig CTA, Wang F, Coussens PM, Baldwin CL. Comparison of gene expression by cocultured WC1 1 γδ and CD4 1 αβ T cells exhibiting a recall response to bacterial antigen. Mol Immunol 2007;44(8):2023 35. [77] Alvarez AJ, Endsley JJ, Werling D, Mark Estes D. WC1 1 γδ T Cells Indirectly Regulate Chemokine Production During Mycobacterium bovis Infection in SCID-bo Mice. Transbound Emerg Dis 2009;56(67):275 84. [78] Nelson DD, Davis WC, Brown WC, Li H, O’Toole D, Oaks JL. CD8 1 /perforin 1 /WC1 2 γδ T cells, not CD8 1 αβ T cells, infiltrate vasculitis lesions of American bison (Bison bison) with experimental sheep-associated malignant catarrhal fever. Vet Immunol Immunopathol 2010;136(3):284 91. [79] Vincent MS, Roessner K, Lynch D, Wilson D, Cooper SM, Tschopp J, et al. Apoptosis of Fashigh CD4 1 Synovial T Cells by Borrelia-reactive Fas-ligandhigh γδ T Cells in Lyme. Arthritis J Exp Med 1996;184(6):2109. [80] Li Z, Peng H, Xu Q, Ye Z. Sensitization of human osteosarcoma cells to Vγ9Vδ2 T-cell-mediated cytotoxicity by zoledronate. J Orthop Res 2012;30(5):824 30. [81] Lockhart E, Green AM, Flynn JL. IL-17 production is dominated by {gamma}{delta} T cells rather than CD4 T cells during Mycobacterium tuberculosis infection. J Immunol 2006;177(7):4662 9. [82] Shibata K, Yamada H, Hara H, Kishihara K, Yoshikai Y. Resident V{delta}1 1 {gamma}{delta} T cells control early infiltration of neutrophils after Escherichia coli infection via IL-17 production. J Immunol 2007;178(7): 4466 72. [83] Shibata K, Yamada H, Nakamura R, Sun X, Itsumi M, Yoshikai Y. Identification of CD25 1 γδ T cells as fetal thymus-derived naturally occurring IL-17 producers. J Immunol 2008;181(9):5940.

785

[84] Zeng X, Wei YL, Huang J, Newell E, Yu H, Kidd B, et al. γδ T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen-specific interleukin17 response. Immunity 2012;37(3):524 34. [85] Deknuydt F, Scotet E, Bonneville M. Modulation of inflammation through IL-17 production by γδ T cells: mandatory in the mouse, dispensable in humans? Immunol Lett 2009;127(1):8 12. [86] Nakamizo S, Honda T, Adachi A, Nagatake T, Kunisawa J, Kitoh A, et al. High fat diet exacerbates murine psoriatic dermatitis by increasing the number of IL-17-producing γδ T cells. Sci Rep 2017;7(1):14076. [87] Chen Y, Chou K, Fuchs E, Havran WL, Boismenu R. Protection of the intestinal mucosa by intraepithelial gamma delta T cells. Proc Natl Acad Sci USA 2002;99 (22):14338 43. [88] Jameson J, Ugarte K, Chen N, Yachi P, Fuchs E, Boismenu R, et al. A role for skin gammadelta T cells in wound repair. Science 2002;296(5568):747 9. [89] Wang Y, Bai Y, Li Y, Liang G, Jiang Y, Liu Z, et al. IL15 enhances activation and IGF-1 production of dendritic epidermal T cells to promote wound healing in diabetic mice. Front Immunol 2017;8:1557. [90] Sharp LL, Jameson JM, Cauvi G, Havran WL. Dendritic epidermal T cells regulate skin homeostasis through local production of insulin-like growth factor 1. Nat Immunol 2004;6:73. [91] Zheng J, Liu Y, Lau YL, Tu W. γδ-T cells: an unpolished sword in human anti-infection immunity. Cell Mol Immunol 2012;10:50. [92] Long KM, Ferris MT, Whitmore AC, Montgomery SA, Thurlow LR, McGee CE, et al. Gamma-delta T cells play a protective role in chikungunya virus-induced disease. J Virol 2015;. [93] Wang T, Welte T. Role of natural killer and gammadelta T cells in West Nile virus infection. Viruses 2013;5(9):2298 310. [94] Pauza CD, Poonia B, Li H, Cairo C, Chaudhry S. γδ T cells in HIV disease: past, present, and future. Front Immunol 2015;5:687. [95] Vesosky B, Turner OC, Turner J, Orme IM. Gamma interferon production by bovine gamma delta T cells following stimulation with mycobacterial mycolylarabinogalactan peptidoglycan. Infect Immun 2004;72 (8):4612 18. [96] Feurle J, Espinosa E, Eckstein S, Pont F, Kunzmann V, Fournie JJ, et al. Escherichia coli produces phosphoantigens activating human gamma delta T cells. J Biol Chem 2002;277:148 54. [97] Plattner BL, Hostetter JM. Comparative gamma delta T cell immunology: a focus on mycobacterial disease in cattle. Vet Med Int 2011;2011:214384. [98] Gao Y, Williams AP. Role of innate T cells in antibacterial immunity. Front Immunol 2015;6:302.

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

786

46. HARNESSING γδ T CELLS AS NATURAL IMMUNE MODULATORS

[99] Hara T, Mizuno Y, Takaki K, Takada H, Akeda H, Aoki T, et al. Predominant activation and expansion of V gamma 9-bearing γδ T cells in vivo as well as in vitro in Salmonella infection. J Clin Invest 1992;90(1):204 10. [100] Mizuno Y, Takada H, Nomura A, Jin CH, Hattori H, Ihara K, et al. Th1 and Th1-inducing cytokines in Salmonella infection. Clin Exp Immunol 2003;131(1): 111 17. [101] Nyirenda TS, Seeley AE, Mandala WL, Drayson MT, MacLennan CA. Early interferon-γ production in human lymphocyte subsets in response to nontyphoidal Salmonella demonstrates inherent capacity in innate cells. PLoS One 2010;5(10):e13667. [102] Hedges JF, Buckner DL, Rask KM, Kerns HMM, Jackiw LO, Trunkle TC, et al. Mucosal lymphaticderived γδ T cells respond early to experimental Salmonella enterocolits by increasing expression of IL2Rα. Cell Immunol 2007;246(1):8 16. [103] Skyberg JA, Thornburg T, Rollins M, Huarte E, Jutila MA, Pascual DW. Murine and bovine γδ T cells enhance innate immunity against Brucella abortus infections. PLoS One 2011;6(7):e21978. [104] Suraud V, Jacques I, Olivier M, Guilloteau LA. Acute infection by conjunctival route with Brucella melitensis induces IgG 1 cells and IFN-γ producing cells in peripheral and mucosal lymph nodes in sheep. Microbes Infect 2008;10(12):1370 8. [105] Jagannathan P, Lutwama F, Boyle MJ, Nankya F, Farrington LA, McIntyre TI, et al. Vδ2 1 T cell response to malaria correlates with protection from infection but is attenuated with repeated exposure. Sci Rep 2017;7(1):11487. [106] Peckham RK, Brill R, Foster DS, Bowen AL, Leigh JA, Coffey TJ, et al. Two distinct populations of Bovine IL-17 1 T-cells can be induced and WC1 1 IL-17 1 γδ T-cells are effective killers of protozoan parasites. Sci Rep 2014;4:5431. [107] Chen CY, Yao S, Huang D, Wei H, Sicard H, Zeng G, et al. Phosphoantigen/IL2 expansion and differentiation of Vγ2Vδ2 T cells increase resistance to tuberculosis in nonhuman primates. PLoS Pathog 2013;9(8): e1003501. [108] Qaqish A, Huang D, Chen CY, Zhang Z, Wang R, Li S, et al. Adoptive transfer of phosphoantigen-specific γδ T cell subset attenuates Mycobacterium tuberculosis infection in nonhuman primates. J Immunol 2017;198(12): 4753. [109] Holderness J, Jackiw L, Kimmel E, Kerns HMM, Radke M, Hedges JF, et al. Select plant tannins induce IL-2Rα up-regulation and augment cell division in γδ T cells. J Immunol 2007;179(10):6468 78. [110] Graff JC, Kimmel EM, Schepetkin IA, Freedman B, Lubick KJ, Holderness J, et al. Polysaccharides

[111]

[112]

[113]

[114]

[115]

[116]

[117]

[118]

[119]

[120]

[121]

derived from Yamoat (Funtumia elastica) affect innate immunity in part by priming γδ T cells. Int Immunopharmacol 2009;9(11):1313 22. Holderness J, Schepetkin IA, Freedman B, Kirpotina LN, Quinn MT, Hedges JF, et al. Polysaccharides isolated from Acai fruit induce innate immune responses. PLoS One 2011;6(2):e17301. Hedges JF, Mitchell AM, Jones K, Kimmel E, Ramstead AG, Snyder DT, et al. Amphotericin B stimulates γδ T and NK cells, and enhances protection from Salmonella infection. Innate Immun 2015;6:598 608. Nantz MP, Rowe CA, Nieves Jr. C, Percival SS. Immunity and antioxidant capacity in humans is enhanced by consumption of a dried, encapsulated fruit and vegetable juice concentrate. J Nutr 2006;136 (10):2606 10. Percival SS, Bukowski JF, Milner J. Bioactive food components that enhance {gamma}{delta} T cell function may play a role in cancer prevention. J Nutr 2008;138(1):1 4. Nantz MP, Rowe CA, Muller C, Creasy R, Colee J, Khoo C, et al. Consumption of cranberry polyphenols enhances human gammadelta-T cell proliferation and reduces the number of symptoms associated with colds and influenza: a randomized, placebocontrolled intervention study. Nutr J 2013;12:161. Akiyama H, Sato Y, Watanabe T, Nagaoka MH, Yoshioka Y, Shoji T, et al. Dietary unripe apple polyphenol inhibits the development of food allergies in murine models. FEBS Lett 2005;579(20):4485 91. Tibe O, Pernthaner A, Sutherland I, Lesperance L, Harding DRK. Condensed tannins from Botswanan forage plants are effective priming agents of γδ T cells in ruminants. Vet Immunol Immunopathol 2012;146(3): 237 44. Daughenbaugh KF, Holderness J, Graff JC, Hedges JF, Freedman B, Graff JW, et al. Contribution of transcript stability to a conserved procyanidin-induced cytokine response in gammadelta T cells. Genes Immun 2011;12(5):378 89. Ramiro-Puig E, Pe´rez-Cano FJ, Ramos-Romero S, Pe´rez-Berezo T, Castellote C, Permanyer J, et al. Intestinal immune system of young rats influenced by cocoa-enriched diet. J Nutr Biochem 2008;19(8): 555 65. Percival SS. Grape consumption supports immunity in animals and humans. J Nutr 2009;139(9): 1801S 1805SS. Skyberg JA, Robison A, Golden S, Rollins MF, Callis G, Huarte E, et al. Apple polyphenols require T cells to ameliorate dextran sulfate sodium-induced colitis and dampen proinflammatory cytokine expression. J Leukoc Biol 2011;90(6):1043 54.

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REFERENCES

[122] Xie G, Schepetkin IA, Quinn MT. Immunomodulatory activity of acidic polysaccharides isolated from Tanacetum vulgare L. Int Immunopharmacol 2007;7(13): 1639 50. [123] Schepetkin IA, Quinn MT. Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. Int Immunopharm 2006;6(3):317 33. [124] Magalhaes PO, Lopes AM, Mazzola PG, RangelYagui C, Penna TC, Pessoa Jr. A. Methods of endotoxin removal from biological preparations: a review. J Pharm Pharm Sci 2007;10(3):388 404. [125] Skyberg JA, Rollins MF, Holderness JS, Marlenee NL, Schepetkin IA, Goodyear A, et al. Nasal Acai polysaccharides potentiate innate immunity to protect against pulmonary Francisella tularensis and Burkholderia pseudomallei infections. PLoS Pathog 2012;8(3):e1002587. [126] Chen X, Katchar K, Goldsmith JD, Nanthakumar N, Cheknis A, Gerding DN, et al. A mouse model of Clostridium difficile 2 associated disease. Gastroenterology 2008;135(6):1984 92. [127] Hedges JF, Holderness J, Jutila MA. Adjuvant materials that enhance bovine γδ T cell responses. Vet Immunol Immunopathol 2016;181(Suppl. C):30 8. [128] Volman JJ, Mensink RP, Ramakers JD, de Winther MP, Carlsen H, Blomhoff R, et al. Dietary (1 2 3),

[129]

[130]

[131]

[132]

[133]

[134]

(1 2 4)-β-D-glucans from oat activate nuclear factorκB in intestinal leukocytes and enterocytes from mice. Nutr Res 2010;30(1):40 8. Lohr KM, Snyderman R. Amphotericin B alters the affinity and functional activity of the oligopeptide chemotactic factor receptor on human polymorphonuclear leukocytes. J Immunol 1982;129(4):1594 9. Hauser WE, Remington JS. Effect of amphotericin B on natural killer cell activity in vitro. J Antimicrob Chemother 1983;11(3):257 62. Little JR, Abegg A, Plut E. The relationship between adjuvant and mitogenic effects of amphotericin methyl ester. Cell Immunol 1983;78(2):224 35. Cleary JD, Chapman SW, Nolan RL. Pharmacologic modulation of interleukin-1 expression by amphotericin B-stimulated human mononuclear cells. Antimicrob Agents Chemother 1992;36(5):977 81. Arning M, Kliche KO, Heer-Sonderhoff AH, Wehmeier A. Infusion-related toxicity of three different amphotericin B formulations and its relation to cytokine plasma levels. Mycoses 1995;38(11 12):459 65. Rogers PD, Jenkins JK, Chapman SW, Ndebele K, Chapman BA, Cleary JD. Amphotericin B activation of human genes encoding for cytokines. J Infect Dis 1998;178(6):1726 33.

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Mucosal Vaccines for Aged: Challenges and Struggles in Immunosenescence Kohtaro Fujihashi1,2 1

International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 2Department of Pediatric Dentistry, The Institute of Oral Health Research, The School of Dentistry, The University of Alabama at Birmingham, Birmingham, AL, United States

I. INTRODUCTION Immune functions are known to deteriorate with age in many species. In fact, the risk and severity of infections are higher and the susceptibility to certain types of autoimmune diseases and cancers is greater in the elderly [1,2], while responses to vaccination are diminished [1,3,4]. These studies provide evidence of dysregulation and of an overall decline in host immunity in the elderly. In systemic compartments, the ageassociated alterations of which have been studied extensively, dysfunctions occur in both B and T cells, thought the latter are considered to be more susceptible to immunosenescence [1,2,5,6]. It has been suggested that although dendritic cells (DCs) could be fully functional in aging individuals [7 10], both foreign and self antigens induce enhanced proinflammatory cytokines [11,12]. This enhancement of inflammation can

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00047-X

be detrimental; however, very old individuals with a more balanced proinflammatory and antiinflammatory phenotype may be the most fortunate [13,14]. The association of inflammation in aging has been termed “inflamm aging” [15 17]. In this regard, inflamm aging may hamper the induction of Ag-specific immune responses when active immunization is initiated, since it is essential to induce a transient inflammatory innate immune response in order to elicit subsequent acquired immunity [18]. Recent studies have shown that alteration of hematopoetic stem cells (HSCs) in bone marrow significantly influences outcomes of immune responses in the host. Thus increased frequency of the myeloid type of HSCs with decreased numbers of the lymphoid type of HSCs is seen in aged animals. This unbalanced distribution of HSC subsets, which is induced by the age-associated changes of bone marrow

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microenvironment but not caused by intrinsic changes of HSCs, most likely results in lower immune responses in the elderly [19]. The mucosal immune system plays an essential role in protecting against the majority of infectious diseases. Mucosa-associated cells and lymphoid and effector molecules (e.g., antibody, or Ab) create an integrated network for this mucosal immune systems of higher mammals. Immunoglobulin A (IgA) plays a central role in this sophisticated immune system. Along with cytokines, chemokines, and their receptors involved in IgA induction and regulation, the IgA isotype appears to function in synergy with the innate immune system, including epithelial cells, macrophages, innate lymphoid cells, and their derived cytokines and antimicrobial peptides [20,21]. In order to induce antigen (Ag)specific humoral as well as cell-mediated immune responses at these mucosal barriers, one must consider the common mucosal immune system (CMIS), which consists of functionally distinct, but highly interconnected mucosal inductive and effector tissues [20,21]. In the mammalian host, organized secondary lymphoid tissues have evolved in the upper respiratory (UR) and gastrointestinal (GI) tracts to facilitate Ag uptake, processing, and presentation for the initiation of Ag-specific immune responses. These tissues are termed nasopharygeal-associated lymphoid tissue (NALT) and gut-associated lymphoid tissue (GALT), respectively. Collectively, NALT and GALT in humans and mice comprise a mucosaassociated lymphoid tissue (MALT) network (see Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance). This MALT serves as the major mucosal inductive sites. In general, individual components of MALT are assumed to share the molecular and cellular characteristics of well-characterized Peyer’s patches (PPs). PPs are covered by a follicle-associated epithelium (FAE) interspersed with Ag-sampling microfold

(M) cells and well-organized microarchitectures, such as a subepithelium (dome) containing antigen-presenting cells (APCs) enriched in DCs and macrophages, and a B cell zone with germinal centers (GCs) and adjacent T cell areas as well as high endothelial venules (HEVs). We know that naı¨ve, recirculating B and T lymphocytes enter MALT via the HEVs [20,21]. For the initiation of Ag-specific mucosal immune responses through MALT (or PPs and NALT), the FAE M cell plays a crucial role by sampling Ags from the lumen of the gut or nasal passages (NPs) and transporting the intact form of Ag to the underlying APCs for subsequent processing and presentation of the peptide Ag. Further, APCs (e.g., PP DCs) induce necessary mucosal imprinting of the molecules CCR9 and α4β7 on Ag-specific lymphocytes [20,21]. Following this Ag presentation and activation process, Agspecific B and T cell populations then emigrate from the mucosal inductive environment via lymphatic drainage, circulate through the bloodstream, and home to mucosal effector sites, where they conduct effector functions, including the differentiation of PP-originating B cells into IgA Ab-producing plasma cells. Effector sites for mucosal immune responses include the numerous subsets of lymphoid cells in the lamina propria (LP) of the GI, UR, and reproductive tracts as well as secretory glandular tissues [20,21]. Resident in these mucosal effector sites, which are characterized by more diffuse connective tissues, are the Ag-specific CD41 type 1 T helper (Th1) cells, Th17 cells, and CD81 cytotoxic T lymphocytes (CTLs) responsible for cellmediated immunity (CMI) and CTL functions, as well as CD41 Th2 cells, IgA-committed B lymphocytes, and IgA-producing plasma cells for humoral mucosal immunity. Mucosal surfaces are protected by secretory IgA (SIgA) Abs, which is mainly produced in local effector tissues through the cellular cooperation between polymeric IgA-producing plasma cells and poly-Ig receptor expressed by columnar epithelial cells [20,21]. Since the effector sites of mucosal

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II. AGE-ASSOCIATED CHANGES IN THE GASTROINTESTINAL TRACT IMMUNE SYSTEM

surfaces play a central role as the first line of host defense, these tissues contain relatively high numbers of activated T and B cells, expressing a memory phenotype in order to be ready for an immediate immune responses to mucosally invading, undesired pathogens [20,21]. Further, regulatory T (Treg) cells and CD41 Th17 cells, which control the suppression and protection/ inflammation phases of the GALT immune system, respectively, have been identified in the intestinal LP region [20,21]. More recent evidence showed that newly identified type 3 innate lymphoid cells (ILC3) in the intestinal LP play key roles in the regulation of epithelial cell repair and glycosylation in order to assist mucosal protection [22]. Despite the achievements of extensive current studies, which provide a better understanding of aging biology and mucosal immune system, we still do not have a clear view of the age-associated alterations that occur in the sophisticated mucosal immune system, which together are termed mucosal immunosenescence. In this chapter, we will focus on the changes exhibited in both the GI and UR tracts with advanced aging and introduce potential strategies for the restoration of mucosal immunosenescence in order to describe progress toward development of effective mucosal vaccines, which are much needed in the elderly.

II. AGE-ASSOCIATED CHANGES IN THE GASTROINTESTINAL TRACT IMMUNE SYSTEM Extensive evidence for dysregulation and an overall decline in mucosal immunity in the GI tract during aging have been reported by immunological analyses [23]. Testing external secretions for the presence of SIgA Abs is the most common method for assessing mucosal immune responses. Both human and animal studies have shown that total IgA Ab levels in mucosal secretions either increased or

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remained unchanged in aging. Thus, elderly subjects had significantly higher concentrations of salivary SIgA Abs than did younger ones [24]. The same report showed that similar levels of Abs were noted in whole gut lavages of aged and young subjects [24]. Other researchers reported similar total IgA Ab responses in the serum of aged humans [25,26]. Studies in aged mice and rats also showed no reduction of total IgA Ab levels [27 30]. Similarly, our previous study showed that fecal extracts from 12- to 14month-old mice contained essentially the same levels of IgA Abs as those seen in young adult mice [31]. These results indicate that age does not impair total IgA Ab responses in external secretions. To support these findings, in vitro activated B cells from the PPs of aged mice revealed significantly higher levels of IgA Abs than those noted in identically treated PP B cells from young adult mice [32]. In contrast, when T-cell-dependent B cell mitogens were employed in the same culture system, reduced levels of Ab production were seen in the culture containing B cells from aged PPs and mesenteric lymph nodes (MLNs) [29]. These findings suggest that T cells, which are involved in the induction of Ag-specific immune responses, are more susceptible than B cells to immunosenescence in the mucosal compartment. Furthermore, it is possible that natural IgA Ab responses in aged mice could be due to increased levels of low-affinity, T cellindependent IgA Ab production. Ag-specific IgA B cell responses are known to play a central role in the induction of mucosal immunity to infectious diseases [21]. The GI tract in the elderly is particularly susceptible to infectious diseases, suggesting that Ag-specific mucosal immunity is also affected in aging [3,33]. Indeed, despite intact overall IgA Ab levels in aging, Ag-specific immunity in the elderly and in experimental animals is significantly diminished when compared with their younger counterparts. For example, intestinal lavages from aged rats given oral cholera toxin

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(CT) were shown to contain significantly lower titers of anti-CT-B IgA Abs than did those from identically immunized young rats [34]. Furthermore, the numbers of Ag-specific IgA Ab-forming cells in the intestinal LP were also reduced in aged rats [34,35] and rhesus macaques [36] given oral CT. When aged mice were orally immunized with the hemagglutinin (HA) from influenza virus along with CT as mucosal adjuvant, reduced levels of HAspecific SIgA Ab responses were noted when compared with those seen in young adult mice [37]. These results clearly indicate that Agspecific mucosal SIgA Ab responses are diminished in aged animals, especially responses associated with the GALT immune system. Of importance, our previous studies showed that 12- to 14-month-old mice given oral ovalbumin

(OVA) plus CT had reduced levels of OVAand CT-B-specific mucosal and systemic Ab responses that resembled those seen in aged (2year-old) mice given the same oral vaccine [31] (Fig. 47.1). In contrast, 12- to 14-month-old mice given OVA plus CT via the subcutaneous route revealed intact CT-B-specific Ab responses, but failed to reveal nCT adjuvanticity (essentially no OVA-specific Ab responses) [31]. On the basis of these studies, one could suggest that the systemic immune system in 12- to 14month-old mice may be in a transitional stage between a normal state and age-associated deficiency. Thus, we would conclude that ageassociated alterations may arise in the mucosal immune system of the GI tract earlier (at approximately 1 year of age) than in the systemic immune compartment.

FIGURE 47.1 Mucosal aging in GALT versus NALT. Reduced induction of Ag-specific intestinal SIgA Ab responses was noted in 1-year-old mice. Peyer’s patches (PPs) exhibited a reduced size, and lower numbers of PPs were present. Reduced numbers of naı¨ve CD41 T cells, M cells, and follicular dendritic cells were already seen in PPs of 1-year old mice. In contrast, NALT functions remained intact during aging with notable signs of mucosal immunosenescence. VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

IV. INVOLVEMENT OF MUCOSAL CD4 1 T CELLS IN GUT AGING

III. POTENTIAL MECHANISMS IN GUT AGING: ROLES OF M CELLS M cells play a central role in an Ag-sampling system that takes up luminal Ags from the gut lumen into the GALT [21]. M cells have different morphological features when compared with normal intestinal epithelial cells. In this regard, their apical sides show relatively short, irregular microvilli, and their basolateral sides form a pocket structure containing enfolds of lymphocytes and APCs. Thus, M cells can effectively transport luminal Ags from the gut lumen to underlying MALT lymphocytes [21]. Based upon this evidence, M cell-targeting strategies have been developed and successfully used to elicit mucosal immunity (see Chapter 28: M CellTargeted Vaccines). It has been shown that reovirus protein sigma one (pσ1) specifically bind to M cells [38]. In this regard, M cell-targeting DNA vaccine complexes consisting of plasmid DNA and the covalently attached reovirus pσ1 to polyL-lysine (PL) induced significant mucosal SIgA Ab responses in addition to systemic immunity [38]. Further, it has been shown that a novel M cell-specific monoclonal antibody (NKM 16-2-4) that recognizes the unique glycosylation moiety of the M cells conjugated with botulinum toxoid as a M cell-targeting mucosal vaccine provided significant protection when challenged with a lethal dose of botulinum neurotoxin [39]. Oral delivery of Ag combined with the M celltargeting peptide ligand (Co1), selected from a phage display library panning against the in vitro M cell coculture system, resulted in enhanced Ag-specific immune responses [40]. Since no in vitro M cell systems have been developed, only limited information is available about how Ag sampling actually occurs. However, glycoprotein 2 (GP2) expressed by M cells has been reported to be an M cell-specific molecule that acts as a binding receptor for FimH-expressed Escherichia coli and Salmonella spp. to elicit effective uptake and induction of specific immune responses [41,42]. Of interest, it was also reported that a transition of FAE enterocytes into M cells was

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induced by Salmonella enterica serovar Typhimurium (S. Typhimurium) type III effector protein SopB [43]. Recently, it has been shown that Spi-B, which is one of the E26 avian leukemia oncogene transformation-specific (Ets) family transcription factors, is required for the functional and structural differentiation of M cells [44 46]. M cells differentiate from leucine-rich repeat containing G-protein-coupled receptor 5-positive (Lgr51) intestinal epithelial stem cells as with all other intestinal epithelial cell lineages [44]. Receptor activator of nuclear factor-κB ligand (RANKL) signal stimulation from the subepithelial stromal cells in the FAE region [47] triggers the expression and activation of Spi-B in M cell precursors and subsequently upregulates several Spi-Btarget genes, including GP2, which is considered to be a mature M cell marker [41]. Importantly, aged mice have significantly decreased numbers of GP21, mature GALT M cells [48] (Fig. 47.1). Through an unknown mechanism, the numbers of Spi-B-positive cells are significantly reduced in the FAE region of aged mice, although the expression of RANKL and RANK and their signaling pathways are intact in aged mice. In agreement with reduced numbers of mature M cells, aged mice failed to transport latex particles into the PPs. Furthermore, T cell activation by orally delivered S. Typhimurium is markedly reduced, owing to the absence of M-cell-intrinsic Spi-B [45]. Therefore, reduced numbers of M cells may be one of the causes of impaired GI tract immunity in the elderly. Forced Spi-B activation and/or expression may be a potential target strategy for the development of effective mucosal vaccines in the elderly.

IV. INVOLVEMENT OF MUCOSAL CD41 T CELLS IN GUT AGING As was indicated above, since PPs are the major mucosal inductive tissues in the GI tract, lack of PPs can result in impaired Ag-specific SIgA Ab responses when oral CT adjuvant or

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Salmonella delivery systems are employed [49,50]. Thus, one could easily predict that this impaired Ag-specific Ab response was due to age-associated alteration in the PPs. Indeed, a substantial senescence-associated decline in numbers of lymphoid cells was found in the GALT, specifically in PPs and MLNs [29]. Further, a significant size reduction in PPs was seen in 1-year-old mice along with reduced Agspecific mucosal Ab responses [31] (Fig. 47.1). Although the ratios of CD41 and CD81 T cells and B cells were unchanged [31,51], the actual numbers of lymphocyte counts in PPs of 1year-old mice were significantly lower than those seen in young adult mice (6 8 weeks old) [31,51] (Fig. 47.1). Indeed, Ag-stimulated CD41 T cells from 1-year-old mice given oral OVA plus CT resulted in reduced Th2-type cytokine (e.g., interleukin 4) production [31]. Further, it was reported that Ag-specific T cell regulatory and helper functions in PPs were diminished by aging [51,52]. These findings clearly suggest that the development of effector T cells is influenced by senescence. Indeed, it has been shown that age-associated alterations closely parallel increases in memory type and loss of the naı¨ve T cell phenotype during aging [23]. In this regard, when the actual cell numbers of naı¨ve CD41 T cells between young adult mice (6 8 weeks old) and aging mice (12 14 months old) were compared, PPs of aging mice showed significant reductions in CD41, CD45RB1 naı¨ve T cell frequencies in addition to total cell numbers [53] (Fig. 47.1).

V. THE INTESTINAL MICROBIOTA POTENTIALLY SHAPES MUCOSAL IMMUNOSENESCENCE The density of bacteria in mammalian large intestine can reach up to 1012 bacteria per gram of intestinal content [54,55]. In this regard, 50 genera including several hundred species, which represent more genes in the gut

microflora than are seen in the human genome, were found in the human gut microbiota [56]. In order to maintain appropriate homeostatic conditions, the normal microbiota protects from potential pathogenic bacteria colonization by producing antimicrobial peptides. Further, this intestinal microbiota provides energy in the form of short-chain fatty acids and nutrients (vitamins K and B12) [55,57]. Furthermore, mucosal tissue development and the host immune system development, including SIgA Ab synthesis, were closely regulated by the intestinal microbiota [54,58,59]. For example, hypoplasia of PPs and reduced numbers of IgA plasma cells and CD41 T cells have been reported in germ-free (GF) mice [54,58,60]. When GF mice were exposed to normal mice or mice monoassociated with E. coli, these mice developed a mature mucosal immune system [61,62]. Although GF mice failed to establish tolerance to orally fed Ags, oral treatment with lipopolysaccharide converted GF mice to sensitivity to oral tolerance induction [63]. Further, IgA2 subclass switching was preferentially supported by bacterial stimulation of human intestinal epithelial cells [64]. Conversely, the absence of mucosal IgA Abs induced dysbiosis in the intestine by allowing bacterial population changes to occur. Thus, activation-induced cytidine deaminase (AID)-deficient mice, which lack an appropriate molecular environment for IgA class switching, showed aberrant expansion of segmented filamentous bacteria [65]. Further, opportunistic bacteria, largely Alcaligenes species, specifically inhabit GALT and isolated lymphoid follicles, with the associated preferential induction of Ag-specific SIgA Abs in the GI tract [66,67]. Recent studies have shown that diverse and select IgA Abs contribute to the maintenance of a diversified and balanced microbiota, which in turn facilitates the expansion of Foxp31 T cells, induction of GCs, and SIgA Ab responses in the gut through a symbiotic regulatory loop [68]. On the basis of these findings, one could predict that

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VI. REJUVENATION OF GUT IMMUNITY BY MESENCHYMAL STEM CELL TRANSFER

alterations in the intestinal microflora may lead to a dysregulation of the immune system in the GI tract as major age associated-changes occur (see Chapter 50: Mucosal Vaccine for Parasitic Infections). Indeed, it has been reported that significant changes in the intestinal microflora were noted in the elderly (over 65 years old) [69,70]. In addition, other human microbiome analyses showed that the centenarians exhibited increased inflammatory cytokine responses (inflamm aging) associated with significant changes in their microbiota when compared with those seen in young adults [71].

VI. REJUVENATION OF GUT IMMUNITY BY MESENCHYMAL STEM CELL TRANSFER Adipose tissue-derived mesenchymal stem cells (AMSCs) are attractive candidates for cell replacement therapies, since they can be obtained and expanded relatively easily. It has been shown that AMSCs can differentiate into adipocytes, chondrocytes, and osteoblasts [72]. In addition, various clinical trials have shown the regenerative capacity of AMSCs [73 75]. Previous studies suggested a therapeutic potential for AMSCs for treatment of Alzheimer’s disease [76] and periodontal disease [77]. In this regard, the potential of AMSCs to restore mucosal immunosenescence in the GI tract was investigated by adoptively transferring AMSCs into aged mice. Both OVA- and CT-B-specific SIgA Ab responses were significantly increased in aged mice (12 14 months old and over 18 months old) adoptively transferred with AMSCs when orally immunized with OVA and CT [78] (Fig. 47.2). The induction of Ag-specific SIgA Ab responses was supported by increased levels of interleukin 4 (IL-4) production in mucosal tissues of aged mice, which were achieved by pretreatment with AMSCs [78]. Of importance, Agspecific SIgA Abs in aged mice restored by AMSC transfer were functional. Thus, fecal

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extracts containing CT-B-specific SIgA Abs exhibited neutralizing activity against CT intoxication [78] (Fig. 47.2). This finding contrasts with previous studies generally showing that MSCs downregulate various immunocompetent cells. For example, MSCs inhibited both CD41 and CD81 T cell proliferation following coculture and polyclonal stimulation [79 81]. Other in vitro studies showed reduced Abs in mixed lymphocyte cultures [82] as well as reduced B cell proliferation and Ab synthesis in the presence of MSCs [83]. Finally, coculture of MSCs with splenic B cells induced regulatory B cells, producing IL-10 that ameliorated autoimmunity and aberrant Ab synthesis [84]. The major difference between these opposite studies is that one assessed AMSC functions by adoptive transfer in vivo in a mouse model instead of in vitro systems. Since adoptively transferred AMSCs were generated by serum-free medium, adoptively transferred AMSCs and their soluble products may totally differ from the in vitro studies by others and may result in upregulation of various immune competent cells. Since it is essential to induce a transient inflammatory innate immune response in order to elicit subsequent acquired immunity [18], one of the features of immunosenescence, inflamm aging, may hamper induction of Agspecific immune responses when active immunization is initiated. It has been shown that MSCs exhibited potential roles for antiinflammatory functions [85]. Thus, MSCs have been employed as therapeutic strategies for various immune disorders, including graftversus-host disease [86,87], organ transplantation [88], autoimmune diseases [89], and inflammatory bowel diseases [90,91]. Indeed, MSCs interact with T cells to reduce their proinflammatory cytokines [92,93] while increasing their production of anti-inflammatory cytokines, including IL-4 and IL-10 [94,95]. In this regard, it is possible that adoptive transfer of AMSCs into aged mice could reduce inflamm aging and facilitate the subsequent restoration

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FIGURE 47.2 Stem cell transfer to overcome GI immunity in aging. Adipose-tissue-derived mesenchymal stem cell (AMSC) transfer could reduce inflamm aging and facilitate the subsequent restoration of Ag-specific protective immunity. Further, AMSC transfer may increase the numbers of immature-type of DCs to facilitate APC function in aged mice.

of Ag-specific immune responses when mice were orally immunized with OVA and CT (Fig. 47.2). Of importance, AMSC adoptive transfer studies revealed increased numbers of IL-4-producing CD41 T cells with increased levels of OVA-induced IL-4 production by CD41 T cells in PPs [78]. Since IL-4 is an essential Th2-type cytokine for adjuvant activity of CT [96,97], these results clearly indicate that AMSCs enhanced IL-4 production in aged mice, which could also potentially downregulate inflammatory responses and simultaneously allow CT to enhance OVA-specific Ab responses (Fig. 47.2). Taken together, the AMSC transfer system would be a potent novel strategy in order to overcome mucosal immunosenescence.

VII. NASOPHARYGEALASSOCIATED LYMPHOID TISSUE VERSUS GUT-ASSOCIATED LYMPHOID TISSUE: SIMILARITIES AND GAPS It has been shown that PPs and NALT have common features; however, it is also clear that both tissues possess unique features reflecting their local microenvironments. For example, a compartmentalization occurs between the GALT and NALT immune systems for the induction of Ag-specific immune responses [21,98]. Thus oral immunization mainly elicits Ag-specific immune responses in the small intestine, in the proximal part of the large intestine, and in the mammary and salivary glands,

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VIII. DISTINCT AGING PROCESS OF NASOPHARYGEAL-ASSOCIATED LYMPHOID TISSUE FUNCTION

whereas nasal immunization induces mucosal immunity in the UR tract and the nasal and oral cavities as well as in the cervicovaginal mucosa [98]. Further, NALT and GALT organogenesis and lymphocyte trafficking are distinctly regulated [99]. For example, PPs develop during embryonic days 14 17 in an IL-7-IL-7Rα- and LTα1β2-LTβR-dependent manner, whereas NALT organogenesis occurs postnatally without involvement of either of these cytokine pathways [99,100]. In addition, PP inducer cells require both Id2 and RORγt transcripts for their development; however, NALT inducer cells require only Id2 [100]. It has been shown that activated T and B cells in GALT preferentially express α4β7 and CCR9 as gut-homing receptors, which help to guide their migration back into the intestinal LP (iLP) [20,21]. In contrast, CD62L, α4β1, and CCR10 preferentially regulate the trafficking of T and B cells from NALT into the UR tract effector tissues [99,101,102]. Finally, other investigators and our recent studies have shown that the NALT immune system represents a unique CMIS compartment that supports the induction of SIgA Ab responses in the submandibular glands and saliva [101,103]. These findings clearly show some common as well as distinct compartmentalization occurs in GI and UR tract immune systems in an otherwise framework of the CMIS.

VIII. DISTINCT AGING PROCESS OF NASOPHARYGEALASSOCIATED LYMPHOID TISSUE FUNCTION In addition to the progression during organogenesis and lymphocyte trafficking, the aging process in NALT is also distinctly regulated when compared with that of GALT. When the frequencies of naı¨ve CD41 T cells in NALT and GALT (i.e., the PPs) were compared in young adult and 1-year-old mice, reduced

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frequencies of CD41, CD45RB1 T cells were seen in aged mice [23,53]. On the other hand, the actual cell counts of naı¨ve CD41 T cells in NALT of 1-year-old mice were higher than those seen in young adult mice (Fig. 47.1). The size as well as total lymphocyte count in NALT increases approximately five- to ninefold during the aging process through the first year [23,53] (Fig. 47.1). Although the total lymphocyte count is reduced by 2 years of age, NALT contains approximately twice the number of total lymphocytes [23,53]. Thus the overall numbers of naı¨ve CD41, CD45RB1 T cells in the NALT were similar between aged and young adult mice. These results suggest that the continuous generation of this naı¨ve T cell populations in NALT plays a pivotal role in maintaining young adult mouse levels for the induction of both systemic and mucosal immune responses to nasally administered antigens in aged mice. On the basis of these findings, one could easily predict that nasal immunization of 12- to 14-month-old mice would reveal an intact mucosal immune response. In contrast to oral immunization, nasal immunization with OVA plus CT indeed effectively induced Ag-specific mucosal and systemic immune responses in 1-year-old mice [53] (Fig. 47.1). Thus equivalent levels of OVAspecific Ab responses in plasma and external secretions and Ag-specific Ab-forming cells in the nasal cavity were seen [53]. These results clearly show that both mucosal and systemic immunity occurred in 1-year-old mice following nasal immunization (Fig. 47.1). Further, 12- to 14-month-old mice given nasal tetanus toxoid vaccine were protected from tetanus intoxication [53]. These results suggest that a distinct immune aging process is occurring in NALT versus GALT that mediates Ag-specific Ab induction accounting for differences in the induction of Ag-specific mucosal SIgA and parenteral IgG Ab responses (Fig. 47.1). It is generally agreed that experimental mice should be 18 months of age or older to be

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suitable and equivalent models for evaluating immunological aging effects in order to provide useful information for the understanding of immunosenescence in humans. In this regard, when 18- to 24-month-old mice were immunized nasally with OVA and CT as adjuvant, the mice failed to undergo induction of Ag-specific SIgA Ab responses [53,104]. However, these mice underwent OVA-specific peripheral immune responses that were essentially identical to the responses seen in young adult mice [53,104]. Similarly, OVA-specific CD41 T cell proliferative as well as Th1- and Th2-type cytokine responses in spleens of 18to 24-month-old mice were comparable to those of young adult mice when CT was used as a nasal adjuvant [53]. These results further agree with the findings that mucosal immunosenescence takes place prior to systemic immune dysregulation [31], even though the process of NALT immunosenescence was

less than that seen in GALT in 18- to 24month-old mice [53]. To consider the control of infectious diseases in the elderly, one must overcome this mucosal immunosenescence and seek to develop novel immune modulators that can maintain appropriate mucosal immunity in 2-year-old mice. Further, as we described above, although the numbers of M cells in GALT were reduced in aged mice, a change in the density of mature M cells in NALT FAE with aging has not been reported [67]. Thus, it remains possible that one of the reasons for the slower process of immunosenescence in NALT of aged mice could be intact numbers of mature and functional M cells on NALT FAE. The evidence indicates another advantage of using the NALT immune system for eliciting mucosal immunity in aging. Thus, an M cell-targeting nasal delivery system would be a potent strategy for inducing mucosal immunity in the elderly (Fig. 47.3).

FIGURE 47.3 DC- and M cell-targeting nasal vaccines provide protective mucosal immunity in aging. Nasal immunization with virulence compartments plus pFL and CpG ODN as a DC-targeting adjuvant induces specific SIgA Ab immunity in aged mice (complete protection from influenza virus or Streptococcus pneumoniae challenge). In addition, nasal delivery of GP-ligand-conjugated-Ag (M cell-targeting) may potentially induce pathogen-specific immunity.

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IX. MUCOSAL VACCINES AND THERAPIES FIGHT FOR IMMUNOSENESCENCE

IX. MUCOSAL VACCINES AND THERAPIES FIGHT FOR IMMUNOSENESCENCE As we discussed, the elderly are in general much more susceptible to infections. In fact, the severity and mortality caused by the infectious pathogens invading mucosal surfaces such as influenza virus and the bacterial pathogen Streptococcus pneumoniae (pneumococcus) are sharply increased in humans of advanced age [105 110]. The highest incidence of influenza and pneumococcal diseases occurs among individuals over 65 years of age. Although vaccines to prevent two respiratory pathogens are available, they are less effective in the elderly, so a need exists to develop safe and improved vaccines [23]. Thus far, it has been shown that adjuvant systems are required in order to improve influenza vaccines in the elderly [111,112]. When MF59 was employed as adjuvant for an H5N1 vaccine, broadly crossreactive Abs and long-lived memory B cells were rapidly elicited [111]. Moreover, immune responses to inactivated 2009 H1N1 influenza vaccine in both healthy adults (18 64 years old) and older adults (more than 65 years old) were successfully enhanced by the AS03 adjuvant system (squalene, DL-α-tocopherol, and polysorbate 80; GlaxoSmithKline) [112]. In a nonhuman primate study, a cationic lipid DNA complex (CLDC) was shown to improve the efficacy of the trivalent inactivated influenza vaccine Fluzone in elderly nonhuman primates [113]. In addition to these injectable influenza vaccines, poly I:C as an adjuvant enhanced the effectiveness of an influenza-virus-like particle nasal vaccine in aged mice [114]. Despite these successful reports, one must carefully consider adjuvant selection as well as vaccine and delivery method, since mice given detergent splitinfluenza Ag [A/Uruguay716/2007 (H3N2)] plus purified monophosphoryl lipid A in liposomes via the nasal route showed transient

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weight loss, which was induced by Th17mediated immune responses [115]. As a more general approach, CpG oligodeoxynucleotide (ODN) as vaccine adjuvant has been shown to restore Ag-specific immune responses to OVA, diphtheria toxoid, hepatitis B, pneumococcal polysaccharides, amyloid beta, and tumor cells in aged mice and rats [23]. Oral immunization with OVA (considered to be a weak immunogen) plus CpG ODN induced equally increased levels of Ag-specific SIgA and IgG Ab responses in mucosally normal (3-month-old) as well as mucosally aged (18-month-old) mice [116]. These studies clearly show the potential of CpG ODN as an adjuvant to compensate for the reduced immune responses seen in aging. In addition, strategies to restore the ratio of naı¨ve to memory CD41 T cell subsets have successfully compensated for the altered immune responses in aging, since increased numbers of memory-type cells and decreased numbers of naı¨ve CD41 T cells are associated with immunosenescence [23]. In this regard, aged Fas-CD2 transgenic mice (overexpressing the Fas gene regulated by the CD2 promoter) resulted in reduced numbers of memory-type T cells and rejuvenated immune responses that resembled those of young adult mice [117]. Further, exogenous IL-2 delivery effectively restored development of effector cells from naı¨ve precursors in aged mice [118]. Similarly, mucosal IL-2 treatment reversed ageimpaired mucosal immune responses by enhancing mucosal immunity or by abrogating tolerance in aged mice [119]. Additional studies showed that keratinocyte growth factor or IL-7 treatment prevented thymic atrophy, and thus resulted in a continuous supply of naı¨ve T cells [120,121]. These studies suggest that a continuous supply of naı¨ve T cell populations is a critical factor for the maintenance of an appropriate immunological state, including the induction of Ag-specific immunity in aged mice. Both IL-2 and IL-7 are common gamma-chain cytokine receptor-related interleukins; therefore, IL-15

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treatment also restored impaired DC function in mesenteric lymph nodes of aged mice [122].

X. A DENTRITIC CELLTARGETING MUCOSAL VACCINES FOR AGED The development of effective vaccines for the elderly remains a largely unmet goal. In order to provide effective protection against influenza and S. pneumoniae in the elderly, one should strongly consider developing a new generation of vaccines that could induce pathogen-specific immunity in the respiratory tract. Although it has been shown that effective protection can be provided by pathogenspecific systemic IgG without mucosal IgA responses [123], pathogen-specific SIgA Ab responses are a necessary component for providing a first line of effective immunity against these respiratory pathogens at their entry site. Indeed, it was reported that influenza HAspecific SIgA Ab responses play a key role in protection against influenza in the UR tract. This SIgA Ab provided cross-protection against infection with a variant virus within the same subtype when compared with those of serum IgG Abs [124,125]. Further, our recent study showed that PspA-specific S-IgA, but not IgG Abs at the surface of the nasal mucosa, was essential for the prevention of S. pneumoniae carriage [126]. It has been shown that mice can survive an otherwise lethal challenge of influenza virus by the production of pathogen-specific systemic IgG without mucosal IgA responses; however, these mice became sick and showed significant weight loss [123]. Similarly, since new adjuvant systems for influenza vaccines for the elderly described above would fail to induce protective SIgA Ab responses at the UR tract mucosa, it is possible that influenza virus infection would still elicit flu symptoms and delay the recovery of elderly patients. Indeed, it is essential to

have pathogen-specific SIgA Ab responses in order to provide a first line of defense against major respiratory pathogens (i.e., influenza virus and S. pneumoniae) at their entry site [126,127]. These findings clearly suggest that Ag-specific SIgA Abs are the most important component for effective protection. However, as we have discussed thus far, Ag-specific mucosal SIgA Ab responses are diminished in aged animals and presumably in humans despite slower development of immunosenescence in the UR tract [23,128]. To explore new avenues for effective mucosal immunization strategies that can induce pathogen-specific protective SIgA Ab responses, investigators have begun to target mucosal tissues and immune cells for vaccine delivery. To this end, mucosal DC-targeting Ag delivery systems have been shown to induce Ag-specific SIgA responses [103,104,129]. The unmethylated CpG motifs are recognized by the innate immune system via toll-like receptor 9 (TLR9), expressed by B cells and plasmacytoid DCs (pDCs) [130]. Thus, CpG DNA induced the maturation and stimulation of professional pDCs as well as the subsequent Ag-specific Th1 cell and CTL responses [131,132]. Further, it has been show that CpG ODN acts as an effective adjuvant for the induction of Ag-specific immunity [133]. Indeed, CpG ODN enhanced both Ab and CMI responses to OVA in mice [134]. When viral or toxoid vaccines were given with CpG ODN, significantly increased levels of Ag-specific Ab and CTL responses were seen [135 137]. Mucosal delivery of CpG ODN plus formalininactivated influenza virus or hepatitis B virus surface antigen successfully induced Agspecific Ab responses in both external secretions and plasma of mice [136,137]. In addition, mice given nasal recombinant protective antigen (PA) of the anthrax lethal toxin plus CpG ODN exhibited high levels of PA-specific IgG2a and IgA Ab responses in both plasma and external secretions [138]. Importantly, these

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X. A DENTRITIC CELL-TARGETING MUCOSAL VACCINES FOR AGED

PA-specific Abs neutralized the lethal toxin in vitro [138]. The Flt3 ligand (FL), which binds to the fmslike tyrosine kinase receptor Flt3/Flk2, is a growth factor that dramatically increases the numbers of DCs in vivo without inducing their activation [139,140]. Treatment of mice by systemic FL injection induced marked increases in the numbers of DCs in both systemic (i.e., spleen) and mucosal lymphoid tissues (i.e., intestinal LP, PPs, and MLN) [141]. Other studies have now shown that FL treatment also favors the induction of immune responses after mucosal [142], systemic [143], or cutaneous [144] vaccine delivery. In addition, plasmid DNA encoding FL (pFL) has been systemically coadministered with plasmids encoding protein Ags or linked to the Ag itself. These studies support the use of FL as an adjuvant to induce both IgG Ab and CMI responses [145,146]. When pFL was employed as a nasal adjuvant, it induced significant expansion of mature-type CD81 DCs in NALT, which contributed to IL-4 production by CD41 T cells and enhancement of coadministered Ag-specific SIgA Ab responses [129]. Although DC-targeting adjuvants have shown promising outcomes, conflicting reports concerning functional DC subsets in aged mice have been put forth [10,147,148]. Reports suggesting impaired DC effects have included the reduced expression of CCR7 involved in cell tracking, interferon alpha (IFN-α) production after herpes simplex virus-2 infection, and IFN regulatory factor 7 (IRF-7) synthesis following CpG ODN activation [147,148]. In contrast, myeloid-type DCs were shown to exhibit intact APC functions and TLR expression in aging [10]. Nevertheless, in order to broadly stimulate potentially weakened DC functions in aging individuals and to avoid polarized Th1 (inflammatory)- or Th2 (allergic)-type immune responses in the elderly, a double adjuvant system has been developed using a combination of pFL and CpG ODN. It has been shown that aged mice given nasal OVA plus a combined

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nasal adjuvant consisting of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG ODN showed significantly increased levels of Agspecific, mucosal SIgA, and plasma IgG Ab responses [104]. It is important to note that a balanced Th1- and Th2-type cytokine response with essentially no potential inflammatory IL17 responses was induced by this double adjuvant system [104] (Fig. 47.3). In order to assess whether this double adjuvant system could successfully induce bacterial Ag-specific SIgA Ab responses in the UR tract mucosa for prevention of both S. pneumoniae carriage and infection in the elderly, aged mice were nasally immunized with pneumococcal surface protein A (PspA) plus a combination of pFL and CpG ODN. Vaccinated aged mice showed elevated levels of PspA-specific SIgA Ab responses in external secretions and plasma that were comparable to those seen in young adult mice [149] (Fig. 47.3). Significant levels of PspA-induced CD41 T cell proliferative and PspA-induced Th1- and Th2-, but not Th17type, cytokine responses were noted in NALT and cervical lymph nodes of aged mice [149]. In addition, increased numbers of mature-type CD8- or CD11b-expressing DCs were detected in mucosal tissues of aged mice as a result of the DC-targeting pFL and CpG ODN delivery [149]. Importantly, aged mice that were given PspA plus a combination of pFL and CpG ODN showed protective immunity against nasal S. pneumoniae colonization [149] (Fig. 47.3). In contrast, both aged and young adult mice that were given nasal PspA alone failed to provide sufficient protection after nasal challenge. Thus, high numbers of S. pneumoniae CFUs were seen in nasal washes (NWs) and NPs of both groups of mice when compared with mice nasally immunized with PspA plus the double adjuvant. Further, aged mice that were given PspA plus pFL or CpG ODN (single nasal adjuvant regimen) revealed high numbers of bacterial CFUs in both NWs and NPs. The numbers of S. pneumoniae CFUs were essentially the same as those

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seen in mice given PspA alone. [149]. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for the induction of necessary Ag-specific immune responses (e.g., PspA-specific SIgA and plasma IgG) for protection against S. pneumoniae in the elderly (Fig. 47.3). As we have emphasized, influenza virus is a major human respiratory pathogen in addition to S. pneumoniae and a significant cause of morbidity and death in the elderly. To this end, our study was next designed to assess whether a nasal influenza vaccine together with our double adjuvant system (pFL and CpG ODN) would enhance influenza-virus-specific immunity for the prevention of influenza virus infection in aged mice. A double adjuvant system plus A/ Puerto Rico/8/34 (PR8)-HA induced increased numbers of CD11b1 CD11c1 DCs and both CD41 Th1- and Th2-type cytokineresponses in mucosal inductive tissues and subsequently elicited PR8-HA-specific SIgA Ab responses in the UR tract of aged mice [127] (Fig. 47.3). Thus, when mice were challenged with PR8 virus via the nasal route, both aged and young adult mice that were given the double adjuvant nasal vaccine exhibited complete protection [127] (Fig. 47.3). It should be emphasized that the influenza vaccine given with the double adjuvant system induced high titers of influenzaspecific SIgA and plasma IgG Ab responses, which provided protective immunity in fully aged mice. These results support the potential use of a double adjuvant system for future human studies (Fig. 47.3).

XI. NEXT GENERATION OF POTENT MUCOSAL VACCINES FOR THE ELDERLY In addition to the oral and nasal routes, other mucosal inductive tissues such as tear duct- and conjunctiva-associated lymphoid tissues and sublingual tissue complex should be targeted as

vaccine delivery systems. To this end, it has been shown that vaccine delivery through eyedrops effectively induced Ag-specific SIgA Ab responses [150,151] (see Chapter 17: Mucosal Regulatory System for the Balanced Occular Immunity). Further, sublingual application of influenza virus vaccine successfully elicited protective mucosal immunity [152,153] (see Chapter 27: Effectiveness of Sublingual Immunization: Innovation for Preventing Infectious Diseases). New vaccine delivery systems should also be considered. It was reported that a nanometer-sized hydrogel (nanogel) consisting of a cationic cholesteryl group-bearing pullulan is an effective nasal vaccine delivery vehicle for the induction of protective immunity without coadministration of a biologically active adjuvant [154,155] (see Chapter 28: M CellTargeted Vaccines). Although these alternative mucosal immunization routes and nasal delivery vehicles have been shown to be effective for the induction of Ag-specific immune responses in both mucosal and systemic compartments, it still remains to be determined whether they are also applicable to and effective under immunosenescence situations (i.e., in individuals over 65 years of age). Conversely, it would be of great benefit to the aged population if one could use an innate adjuvant system alone, without Ag, to enhance preexisting mucosal SIgA Ab responses, since the elderly should possess pathogenspecific memory responses against past infections. Nevertheless, we still need to understand the precise cellular and molecular mechanisms for mucosal immunosenescence in order to develop novel mucosal vaccines for the elderly that can overcome their age-associated immunodeficiency.

Acknowledgment Portions of the work described in this chapter was partially supported by National Institutes of Aging (NIA) grant AG025873 (to KF) and research funding from BioMimetics Sympathy Inc. (Tokyo, JAPAN) (to KF).

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REFERENCES

References [1] Castle SC. Clinical relevance of age-related immune dysfunction. Clin Infect Dis 2000;31:578 85. [2] Miller RA. The aging immune system: primer and prospectus. Science 1996;273:70 4. [3] Powers DC. Immunological principles and emerging strategies of vaccination for the elderly. J Am Geriatr Soc 1992;40:81 94. [4] Bernstein E, Kaye D, Abrutyn E, Gross P, Dorfman M, Murasko DM. Immune response to influenza vaccination in a large healthy elderly population. Vaccine 1999;17:82 94. [5] Solana R, Pawelec G. Molecular and cellular basis of immunosenescence. Mech Ageing Dev 1998;102:115 29. [6] Globerson A, Effros RB. Ageing of lymphocytes and lymphocytes in the aged. Immunol Today 2000;21: 515 21. [7] Komatsubara S, Cinader B, Muramatsu S. Functional competence of dendritic cells of ageing C57BL/6 mice. Scand J Immunol 1986;24:517 25. [8] Pietschmann P, Hahn P, Kudlacek S, Thomas R, Peterlik M. Surface markers and transendothelial migration of dendritic cells from elderly subjects. Exp Gerontol 2000;35:213 24. [9] Steger MM, Maczek C, Grubeck-Loebenstein B. Morphologically and functionally intact dendritic cells can be derived from the peripheral blood of aged individuals. Clin Exp Immunol 1996;105:544 50. [10] Tesar BM, Walker WE, Unternaehrer J, Joshi NS, Chandele A, Haynes L, et al. Murine [corrected] myeloid dendritic cell-dependent toll-like receptor immunity is preserved with aging. Aging Cell 2006;5: 473 86. [11] Agrawal A, Agrawal S, Cao JN, Su H, Osann K, Gupta S. Altered innate immune functioning of dendritic cells in elderly humans: a role of phosphoinositide 3-kinasesignaling pathway. J Immunol 2007;178:6912 22. [12] Agrawal A, Tay J, Ton S, Agrawal S, Gupta S. Increased reactivity of dendritic cells from aged subjects to self-antigen, the human DNA. J Immunol 2009;182:1138 45. [13] Franceschi C, Capri M, Monti D, Giunta S, Olivieri F, Sevini F, et al. Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans. Mech Ageing Dev 2007;128: 92 105. [14] Van Bodegom D, May L, Meij HJ, Westendorp RG. Regulation of human life histories: the role of the inflammatory host response. Ann NY Acad Sci 2007; 1100:84 97. [15] Franceschi C, Bonafe M, Valensin S, Olivieri F, De Luca M, Ottaviani E, et al. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann NY Acad Sci 2000;908:244 54.

803

[16] Licastro F, Candore G, Lio D, Porcellini E, ColonnaRomano G, Franceschi C, et al. Innate immunity and inflammation in ageing: a key for understanding agerelated diseases. Immun Ageing 2005;2:8. [17] Shanley DP, Aw D, Manley NR, Palmer DB. An evolutionary perspective on the mechanisms of immunosenescence. Trends Immunol 2009;30:374 81. [18] Iwasaki A, Medzhitov R. Control of adaptive immunity by the innate immune system. Nat Immunol 2015;16:343 53. [19] Pang WW, Schrier SL, Weissman IL. Age-associated changes in human hematopoietic stem cells. Semin Immunol 2017;54 39 1 42. [20] Fujihashi K, Boyaka PN, McGhee JR. Host defenses at mucosal surfaces. In: Rich RT, Fleisher TA, Shearer WT, Schroeder HW, Frew AJ, Weyand CM, editors. Clinical immunology. Philadelphia, PA: Mosby Elsevier; 2013. p. 287 304. [21] Kiyono H, Kunisawa J, McGhee JR, Mestecky J. The mucosal immune system. In: Paul WE, editor. Fundamental Immunology. Philadelphia, PA: Lippincott Williams & Wilkins; 2008. p. 983 1030. [22] Goto Y, Obata T, Kunisawa J, Sato S, Ivanov II, Lamichhane A, et al. Innate lymphoid cells regulate intestinal epithelial cell glycosylation. Science 2014;345: 1254009. [23] Fujihashi K, Kiyono H. Mucosal immunosenescence: new developments and vaccines to control infectious diseases. Trends Immunol 2009;30:334 43. [24] Arranz E, O’Mahony S, Barton JR, Ferguson A. Immunosenescence and mucosal immunity: significant effects of old age on secretory IgA concentrations and intraepithelial lymphocyte counts. Gut 1992;33:882 6. [25] Ammann AJ, Schiffman G, Austrian R. The antibody responses to pneumococcal capsular polysaccharides in aged individuals. Proc. Soc. Exp. Biol. Med. 1980; 164:312 16. [26] Buckley 3rd CE, Buckley EG, Dorsey FC. Longitudinal changes in serum immunoglobulin levels in older humans. Fed. Proc. 1974;33:2036 9. [27] Ebersole JL, Smith DJ, Taubman MA. Secretory immune responses in ageing rats. I. Immunoglobulin levels. Immunology 1985;56:345 50. [28] Finkelstein MS, Tanner M, Freedman ML. Salivary and serum IgA levels in a geriatric outpatient population. J Clin Immunol 1984;4:85 91. [29] Kawanishi H, Kiely J. Immune-related alterations in aged gut-associated lymphoid tissues in mice. Dig Dis Sci 1989;34:175 84. [30] Senda S, Cheng E, Kawanishi H. Aging-associated changes in murine intestinal immunoglobulin A and M secretions. Scand J Immunol 1988;27:157 64. [31] Koga T, McGhee JR, Kato H, Kato R, Kiyono H, Fujihashi K. Evidence for early aging in the mucosal immune system. J Immunol 2000;165:5352 9.

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

804

47. MUCOSAL VACCINES FOR AGED: CHALLENGES AND STRUGGLES IN IMMUNOSENESCENCE

[32] Kawanishi H, Senda S, Ajitsu S. Aging-associated intrinsic defects in IgA production by murine Peyer’s patch B cells stimulated by autoreactive Peyer’s patch T cell hybridoma-derived B cell stimulatory factors (BSF). Mech Ageing Dev 1989;49:61 78. [33] Schmucker DL, Heyworth MF, Owen RL, Daniels CK. Impact of aging on gastrointestinal mucosal immunity. Dig Dis Sci 1996;41:1183 93. [34] Schmucker DL, Daniels CK, Wang RK, Smith K. Mucosal immune response to cholera toxin in ageing rats. I. Antibody and antibody-containing cell response. Immunology 1988;64:691 5. [35] Thoreux K, Owen RL, Schmucker DL. Intestinal lymphocyte number, migration and antibody secretion in young and old rats. Immunology 2000;101:161 7. [36] Taylor LD, Daniels CK, Schmucker DL. Ageing compromises gastrointestinal mucosal immune response in the rhesus monkey. Immunology 1992;75:614 18. [37] Enioutina EY, Visic VD, Daynes RA. Enhancement of common mucosal immunity in aged mice following their supplementation with various antioxidants. Vaccine 2000;18:2381 93. [38] Wu Y, Wang X, Csencsits KL, Haddad A, Walters N, Pascual DW. M cell-targeted DNA vaccination. Proc Natl Acad Sci USA 2001;98:9318 23. [39] Nochi T, Yuki Y, Matsumura A, Mejima M, Terahara K, Kim DY, et al. A novel M cell-specific carbohydrate-targeted mucosal vaccine effectively induces antigenspecific immune responses. J Exp Med 2007;204:2789 96. [40] Kim SH, Seo KW, Kim J, Lee KY, Jang YS. The M celltargeting ligand promotes antigen delivery and induces antigen-specific immune responses in mucosal vaccination. J Immunol 2010;185:5787 95. [41] Hase K, Kawano K, Nochi T, Pontes GS, Fukuda S, Ebisawa M, et al. Uptake through glycoprotein 2 of FimH(1) bacteria by M cells initiates mucosal immune response. Nature 2009;462:226 30. [42] Terahara K, Yoshida M, Igarashi O, Nochi T, Pontes GS, Hase K, et al. Comprehensive gene expression profiling of Peyer’s patch M cells, villous M-like cells, and intestinal epithelial cells. J Immunol 2008;180:7840 6. [43] Tahoun A, Mahajan S, Paxton E, Malterer G, Donaldson DS, Wang D, et al. Salmonella transforms follicleassociated epithelial cells into M cells to promote intestinal invasion. Cell Host Microbe 2012;12:645 56. [44] de Lau W, Kujala P, Schneeberger K, Middendorp S, Li VS, Barker N, et al. Peyer’s patch M cells derived from Lgr5(1) stem cells require SpiB and are induced by RankL in cultured “miniguts”. Mol Cell Biol 2012;32: 3639 47. [45] Kanaya T, Hase K, Takahashi D, Fukuda S, Hoshino K, Sasaki I, et al. The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells. Nat Immunol 2012;13:729 36.

[46] Sato S, Kaneto S, Shibata N, Takahashi Y, Okura H, Yuki Y, et al. Transcription factor Spi-B-dependent and -independent pathways for the development of Peyer’s patch M cells. Mucosal Immunol 2013;6:838 46. [47] Knoop KA, Kumar N, Butler BR, Sakthivel SK, Taylor RT, Nochi T, et al. RANKL is necessary and sufficient to initiate development of antigensampling M cells in the intestinal epithelium. J Immunol 2009;183:5738 47. [48] Kobayashi A, Donaldson DS, Erridge C, Kanaya T, Williams IR, Ohno H, et al. The functional maturation of M cells is dramatically reduced in the Peyer’s patches of aged mice. Mucosal Immunol 2013;6:1027 37. [49] Hashizume T, Togawa A, Nochi T, Igarashi O, Kweon MN, Kiyono H, et al. Peyer’s patches are required for intestinal immunoglobulin A responses to Salmonella spp. Infect Immun 2008;76:927 34. [50] Yamamoto M, Rennert P, McGhee JR, Kweon MN, Yamamoto S, Dohi T, et al. Alternate mucosal immune system: organized Peyer’s patches are not required for IgA responses in the gastrointestinal tract. J Immunol 2000;164:5184 91. [51] Kato H, Fujihashi K, Kato R, Dohi T, Fujihashi K, Hagiwara Y, et al. Lack of oral tolerance in aging is due to sequential loss of Peyer’s patch cell interactions. Int Immunol 2003;15:145 58. [52] Kawanishi H, Ajitsu S. Correction of antigen-specific T cell defects in aged murine gut-associated lymphoid tissues an immune intervention by combined adoptive transfer of an antigen-specific immunoregulatory CD4 T cell subset and interleukin 2 administration. Eur J Immunol 1991;21:2907 14. [53] Hagiwara Y, McGhee JR, Fujihashi K, Kobayashi R, Yoshino N, Kataoka K, et al. Protective mucosal immunity in aging is associated with functional CD41 T cells in nasopharyngeal-associated lymphoreticular tissue. J Immunol 2003;170:1754 62. [54] Macpherson AJ, McCoy KD, Johansen FE, Brandtzaeg P. The immune geography of IgA induction and function. Mucosal Immunol 2008;1:11 22. [55] Tsuji M, Suzuki K, Kinoshita K, Fagarasan S. Dynamic interactions between bacteria and immune cells leading to intestinal IgA synthesis. Semin Immunol 2008;20: 59 66. [56] Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, Toyoda A, et al. Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes. DNA Res 2007;14:169 81. [57] Tremaroli V, Backhed F. Functional interactions between the gut microbiota and host metabolism. Nature 2012;489:242 9. [58] Cebra JJ. Influences of microbiota on intestinal immune system development. Am J Clin Nutr 1999;69: 1046S 51S.

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

REFERENCES

[59] Suzuki K, Fagarasan S. How host-bacterial interactions lead to IgA synthesis in the gut. Trends Immunol. 2008;29:523 31. [60] Macpherson AJ, Harris NL. Interactions between commensal intestinal bacteria and the immune system. Nat Rev Immunol 2004;4:478 85. [61] Klaasen HL, Koopman JP, Van den Brink ME, Van Wezel HP, Beynen AC. Mono-association of mice with non-cultivable, intestinal, segmented, filamentous bacteria. Arch Microbiol 1991;156:148 51. [62] Shroff KE, Meslin K, Cebra JJ. Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut. Infect Immun 1995;63:3904 13. [63] Wannemuehler MJ, Kiyono H, Babb JL, Michalek SM, McGhee JR. Lipopolysaccharide (LPS) regulation of the immune response: LPS converts germfree mice to sensitivity to oral tolerance induction. J Immunol 1982;129:959 65. [64] He B, Xu W, Santini PA, Polydorides AD, Chiu A, Estrella J, et al. Intestinal bacteria trigger T cellindependent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity 2007;26:812 26. [65] Suzuki K, Meek B, Doi Y, Muramatsu M, Chiba T, Honjo T, et al. Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut. Proc Natl Acad Sci USA 2004;101:1981 6. [66] Obata T, Goto Y, Kunisawa J, Sato S, Sakamoto M, Setoyama H, et al. Indigenous opportunistic bacteria inhabit mammalian gut-associated lymphoid tissues and share a mucosal antibody-mediated symbiosis. Proc Natl Acad Sci USA 2010;107:7419 24. [67] Sato S, Kiyono H, Fujihashi K. Mucosal immunosenescence in the gastrointestinal tract: a mini-review. Gerontology 2015;61:336 42. [68] Kawamoto S, Maruya M, Kato LM, Suda W, Atarashi K, Doi Y, et al. Foxp3(1) T cells regulate immunoglobulin a selection and facilitate diversification of bacterial species responsible for immune homeostasis. Immunity 2014;41:152 65. [69] Claesson MJ, Cusack S, O’Sullivan O, Greene-Diniz R, de Weerd H, Flannery E, et al. Composition, variability, and temporal stability of the intestinal microbiota of the elderly. Proc Natl Acad Sci USA 2011;108(Suppl. 1):4586 91. [70] Woodmansey EJ. Intestinal bacteria and ageing. J Appl Microbiol 2007;102:1178 86. [71] Biagi E, Nylund L, Candela M, Ostan R, Bucci L, Pini E, et al. Through ageing, and beyond: gut microbiota and inflammatory status in seniors and centenarians. PLoS One 2010;5:e10667. [72] Tobita M, Orbay H, Mizuno H. Adipose-derived stem cells: current findings and future perspectives. Discov Med 2011;11:160 70.

805

[73] Garcia-Olmo D, Garcia-Arranz M, Herreros D. Expanded adipose-derived stem cells for the treatment of complex perianal fistula including Crohn’s disease. Expert Opin Biol Ther 2008;8:1417 23. [74] Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 2006;12:459 65. [75] Psaltis PJ, Zannettino AC, Worthley SG, Gronthos S. Concise review: mesenchymal stromal cells: potential for cardiovascular repair. Stem cells 2008;26:2201 10. [76] Katsuda T, Tsuchiya R, Kosaka N, Yoshioka Y, Takagaki K, Oki K, et al. Human adipose tissuederived mesenchymal stem cells secrete functional neprilysin-bound exosomes. Sci Rep 2013;3:1197. [77] Tobita M, Uysal CA, Guo X, Hyakusoku H, Mizuno H. Periodontal tissue regeneration by combined implantation of adipose tissue-derived stem cells and platelet-rich plasma in a canine model. Cytotherapy 2013;15:1517 26. [78] Aso K, Tsuruhara A, Takagaki K, Oki K, Ota M, Nose Y, et al. Adipose-derived mesenchymal stem cells restore impaired mucosal immune responses in aged mice. PLoS One 2016;11:e0148185. [79] Cuerquis J, Romieu-Mourez R, Francois M, Routy JP, Young YK, Zhao J, et al. Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-gamma and tumor necrosis factoralpha stimulation. Cytotherapy 2014;16:191 202. [80] Dorronsoro A, Ferrin I, Salcedo JM, Jakobsson E, Fernandez-Rueda J, Lang V, et al. Human mesenchymal stromal cells modulate T-cell responses through TNF-alpha-mediated activation of NF-kappaB. Eur J Immunol 2014;44:480 8. [81] Malcherek G, Jin N, Huckelhoven AG, Mani J, Wang L, Gern U, et al. Mesenchymal stromal cells inhibit proliferation of virus-specific CD8(1) T cells. Leukemia 2014;28:2388 94. [82] Comoli P, Ginevri F, Maccario R, Avanzini MA, Marconi M, Groff A, et al. Human mesenchymal stem cells inhibit antibody production induced in vitro by allostimulation. Nephrol Dial Transplant 2008;23: 1196 202. [83] Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V, Cazzanti F, et al. Human mesenchymal stem cells modulate B-cell functions. Blood 2006;107: 367 72. [84] Park MJ, Kwok SK, Lee SH, Kim EK, Park SH, Cho ML. Adipose tissue-derived mesenchymal stem cells induce expansion of interleukin-10-producing regulatory B cells and ameliorate autoimmunity in a murine model of systemic lupus erythematosus. Cell Transplant 2015;24:2367 77. [85] Ho MS, Mei SH, Stewart DJ. The immunomodulatory and therapeutic effects of mesenchymal stromal cells

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

806

47. MUCOSAL VACCINES FOR AGED: CHALLENGES AND STRUGGLES IN IMMUNOSENESCENCE

for acute lung injury and sepsis. J Cell Physiol 2015;230:2606 17. [86] Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008;371:1579 86. [87] Perez-Simon JA, Lopez-Villar O, Andreu EJ, Rifon J, Muntion S, Diez Campelo M, et al. Mesenchymal stem cells expanded in vitro with human serum for the treatment of acute and chronic graft-versus-host disease: results of a phase I/II clinical trial. Haematologica 2011;96:1072 6. [88] Casiraghi F, Perico N, Remuzzi G. Mesenchymal stromal cells to promote solid organ transplantation tolerance. Curr Opin Organ Transplant 2013;18:51 8. [89] Figueroa FE, Carrion F, Villanueva S, Khoury M. Mesenchymal stem cell treatment for autoimmune diseases: a critical review. Biol Res 2012;45:269 77. [90] Forbes GM, Sturm MJ, Leong RW, Sparrow MP, Segarajasingam D, Cummins AG, et al. A phase 2 study of allogeneic mesenchymal stromal cells for luminal Crohn’s disease refractory to biologic therapy. Clin Gastroenterol Hepatol 2014;12:64 71. [91] Knyazev OV, Parfenov AI, Shcherbakov PL, Ruchkina IN, Konoplyannikov AG. Cell therapy of refractory Crohn’s disease. Bull Exp Biol Med 2013;156:139 45. [92] Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002;99:3838 43. [93] Krampera M, Glennie S, Dyson J, Scott D, Laylor R, Simpson E, et al. Bone marrow mesenchymal stem cells inhibit the response of naı¨ve and memory antigen-specific T cells to their cognate peptide. Blood 2003;101:3722 9. [94] Kong QF, Sun B, Bai SS, Zhai DX, Wang GY, Liu YM, et al. Administration of bone marrow stromal cells ameliorates experimental autoimmune myasthenia gravis by altering the balance of Th1/Th2/Th17/Treg cell subsets through the secretion of TGF-beta. J Neuroimmunol 2009;207:83 91. [95] Prevosto C, Zancolli M, Canevali P, Zocchi MR, Poggi A. Generation of CD4 1 or CD8 1 regulatory T cells upon mesenchymal stem cell-lymphocyte interaction. Haematologica 2007;92:881 8. [96] Okahashi N, Yamamoto M, Vancott JL, Chatfield SN, Roberts M, Bluethmann H, et al. Oral immunization of interleukin-4 (IL-4) knockout mice with a recombinant Salmonella strain or cholera toxin reveals that CD41 Th2 cells producing IL-6 and IL-10 are associated with mucosal immunoglobulin A responses. Infect Immun 1996;64:1516 25.

[97] Vajdy M, Kosco-Vilbois MH, Kopf M, Kohler G, Lycke N. Impaired mucosal immune responses in interleukin 4-targeted mice. J Exp Med 1995;181:41 53. [98] Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med 2005;11:S45 53. [99] Kunisawa J, Nochi T, Kiyono H. Immunological commonalities and distinctions between airway and digestive immunity. Trends Immunol 2008;29: 505 13. [100] Fukuyama S, Hiroi T, Yokota Y, Rennert PD, Yanagita M, Kinoshita N, et al. Initiation of NALT organogenesis is independent of the IL-7R, LTβR, and NIK signaling pathways but requires the Id2 gene and CD3(-) CD4(1)CD45(1) cells. Immunity 2002;17:31 40. [101] Csencsits KL, Walters N, Pascual DW. Cutting edge: dichotomy of homing receptor dependence by mucosal effector B cells: alpha(E) versus L-selectin. J Immunol 2001;167:2441 5. [102] Pascual DW, Riccardi C, Csencsits-Smith K. Distal IgA immunity can be sustained by αEβ71 B cells in Lselectin2/2 mice following oral immunization. Mucosal Immunol 2008;1:68 77. [103] Sekine S, Kataoka K, Fukuyama Y, Adachi Y, Davydova J, Yamamoto M, et al. A novel adenovirus expressing Flt3 ligand enhances mucosal immunity by inducing mature nasopharyngeal-associated lymphoreticular tissue dendritic cell migration. J Immunol 2008;180:8126 34. [104] Fukuiwa T, Sekine S, Kobayashi R, Suzuki H, Kataoka K, Gilbert RS, et al. A combination of Flt3 ligand cDNA and CpG ODN as nasal adjuvant elicits NALT dendritic cells for prolonged mucosal immunity. Vaccine 2008;26:4849 59. [105] Bernstein JM. Waldeyer’s ring and otitis media: the nasopharyngeal tonsil and otitis media. Int J Pediatr Otorhinolaryngol 1999;49(Suppl. 1):S127 132. [106] Mufson MA. Pneumococcal pneumonia. Curr Infect Dis Rep 1999;1:57 64. [107] Webster RG. Immunity to influenza in the elderly. Vaccine 2000;18:1686 9. [108] Thompson WW, Shay DK, Weintraub E, Brammer L, Bridges CB, Cox NJ, et al. Influenza-associated hospitalizations in the United States. JAMA 2004;292: 1333 40. [109] Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 2003;289:179 86. [110] Murray CJ, Lopez AD. Mortality by cause for eight regions of the world: Global Burden of Disease Study. Lancet 1997;349:1269 76. [111] Galli G, Hancock K, Hoschler K, DeVos J, Praus M, Bardelli M, et al. Fast rise of broadly cross-reactive

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

807

REFERENCES

[112]

[113]

[114]

[115]

[116]

[117]

[118]

[119]

[120] [121]

[122]

[123]

antibodies after boosting long-lived human memory B cells primed by an MF59 adjuvanted prepandemic vaccine. Proc Natl Acad Sci USA 2009;106:7962 7. Jackson LA, Chen WH, Stapleton JT, Dekker CL, Wald A, Brady RC, et al. Immunogenicity and safety of varying dosages of a monovalent 2009 H1N1 influenza vaccine given with and without AS03 adjuvant system in healthy adults and older persons. J Infect Dis 2012;206:811 20. Carroll TD, Matzinger SR, Barry PA, McChesney MB, Fairman J, Miller CJ. Efficacy of influenza vaccination of elderly rhesus macaques is dramatically improved by addition of a cationic lipid/DNA adjuvant. J Infect Dis 2014;209:24 33. Schneider-Ohrum K, Giles BM, Weirback HK, Williams BL, DeAlmeida DR, Ross TM. Adjuvants that stimulate TLR3 or NLPR3 pathways enhance the efficiency of influenza virus-like particle vaccines in aged mice. Vaccine 2011;29:9081 92. Maroof A, Yorgensen YM, Li Y, Evans JT. Intranasal vaccination promotes detrimental Th17-mediated immunity against influenza infection. PLoS Pathog 2014;10:e1003875. Alignani D, Maletto B, Liscovsky M, Ropolo A, Moron G, Pistoresi-Palencia MC. Orally administered OVA/CpG-ODN induces specific mucosal and systemic immune response in young and aged mice. J Leukoc Biol 2005;77:898 905. Zhou T, Edwards 3rd CK, Mountz JD. Prevention of age-related T cell apoptosis defect in CD2-fastransgenic mice. J Exp Med 1995;182:129 37. Haynes L, Linton PJ, Eaton SM, Tonkonogy SL, Swain SL. Interleukin 2, but not other common gamma chain-binding cytokines, can reverse the defect in generation of CD4 effector T cells from naive T cells of aged mice. J Exp Med 1999;190:1013 24. Fayad R, Zhang H, Quinn D, Huang Y, Qiao L. Oral administration with papillomavirus pseudovirus encoding IL-2 fully restores mucosal and systemic immune responses to vaccinations in aged mice. J Immunol 2004;173:2692 8. Henson SM, Pido-Lopez J, Aspinall R. Reversal of thymic atrophy. Exp Gerontol 2004;39:673 8. Min D, Panoskaltsis-Mortari A, Kuro OM, Hollander GA, Blazar BR, Weinberg KI. Sustained thymopoiesis and improvement in functional immunity induced by exogenous KGF administration in murine models of aging. Blood 2007;109:2529 37. Moretto MM, Lawlor EM, Khan IA. Aging mice exhibit a functional defect in mucosal dendritic cell response against an intracellular pathogen. J Immunol 2008;181: 7977 84. Harriman GR, Bogue M, Rogers P, Finegold M, Pacheco S, Bradley A, et al. Targeted deletion of the IgA constant region in mice leads to IgA deficiency

[124]

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133] [134]

[135]

[136]

[137]

with alterations in expression of other Ig isotypes. J Immunol 1999;162:2521 9. Asahi-Ozaki Y, Yoshikawa T, Iwakura Y, Suzuki Y, Tamura S, Kurata T, et al. Secretory IgA antibodies provide cross-protection against infection with different strains of influenza B virus. J Med Virol 2004;74: 328 35. Tamura S, Funato H, Hirabayashi Y, Suzuki Y, Nagamine T, Aizawa C, et al. Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules. Eur J Immunol 1991;21: 1337 44. Fukuyama Y, King JD, Kataoka K, Kobayashi R, Gilbert RS, Oishi K, et al. Secretory-IgA antibodies play an important role in the immunity to Streptococcus pneumoniae. J Immunol 2010;185:1755 62. Asanuma H, Zamri NB, Sekine S, Fukuyama Y, Tokuhara D, Gilbert RS, et al. A novel combined adjuvant for nasal delivery elicits mucosal immunity to influenza in aging. Vaccine 2012;30:803 12. Fujihashi K, McGhee JR. Mucosal immunity and tolerance in the elderly. Mech Ageing Dev 2004;125: 889 98. Kataoka K, McGhee JR, Kobayashi R, Fujihashi K, Shizukuishi S, Fujihashi K. Nasal Flt3 ligand cDNA elicits CD11c1CD81 dendritic cells for enhanced mucosal immunity. J Immunol 2004;172:3612 19. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000;408:740 5. Klinman DM, Currie D, Gursel I, Verthelyi D. Use of CpG oligodeoxynucleotides as immune adjuvants. Immunol Rev 2004;199:201 16. Wagner H. Bacterial CpG DNA activates immune cells to signal infectious danger. Adv Immunol 1999;73:329 68. Klinman DM, Barnhart KM, Conover J. CpG motifs as immune adjuvants. Vaccine 1999;17:19 25. Klinman DM. Therapeutic applications of CpGcontaining oligodeoxynucleotides. Antisense Nucleic Acid Drug Dev 1998;8:181 4. Brazolot Millan CL, Weeratna R, Krieg AM, Siegrist CA, Davis HL. CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice. Proc Natl Acad Sci USA 1998;95:15553 8. McCluskie MJ, Davis HL. CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice. J Immunol 1998;161:4463 6. Moldoveanu Z, Love-Homan L, Huang WQ, Krieg AM. CpG DNA, a novel immune enhancer for systemic and mucosal immunization with influenza virus. Vaccine 1998;16:1216 24.

VII. NEW AND NOVEL APPROACHES FOR MUCOSAL VACCINE DEVELOPMENT

808

47. MUCOSAL VACCINES FOR AGED: CHALLENGES AND STRUGGLES IN IMMUNOSENESCENCE

[138] Boyaka PN, Tafaro A, Fischer R, Leppla SH, Fujihashi K, McGhee JR. Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen. J Immunol 2003;170:5636 43. [139] Brasel K, McKenna HJ, Morrissey PJ, Charrier K, Morris AE, Lee CC, et al. Hematologic effects of flt3 ligand in vivo in mice. Blood 1996;88:2004 12. [140] Maraskovsky E, Brasel K, Teepe M, Roux ER, Lyman SD, Shortman K, et al. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J Exp Med 1996;184:1953 62. [141] Viney JL, Mowat AM, O’Malley JM, Williamson E, Fanger NA. Expanding dendritic cells in vivo enhances the induction of oral tolerance. J Immunol 1998;160:5815 25. [142] Williamson E, Westrich GM, Viney JL. Modulating dendritic cells to optimize mucosal immunization protocols. J Immunol 1999;163:3668 75. [143] Pisarev VM, Parajuli P, Mosley RL, Sublet J, Kelsey L, Sarin PS, et al. Flt3 ligand enhances the immunogenicity of a gag-based HIV-1 vaccine. Int J Immunopharmacol 2000;22:865 76. [144] Baca-Estrada ME, Ewen C, Mahony D, Babiuk LA, Wilkie D, Foldvari M. The haemopoietic growth factor, Flt3L, alters the immune response induced by transcutaneous immunization. Immunology 2002;107: 69 76. [145] Hung CF, Hsu KF, Cheng WF, Chai CY, He L, Ling M, et al. Enhancement of DNA vaccine potency by linkage of antigen gene to a gene encoding the extracellular domain of Fms-like tyrosine kinase 3-ligand. Cancer Res 2001;61:1080 8. [146] Moore AC, Kong WP, Chakrabarti BK, Nabel GJ. Effects of antigen and genetic adjuvants on immune responses to human immunodeficiency virus DNA vaccines in mice. J Virol 2002;76:243 50.

[147] Kovacs EJ, Palmer JL, Fortin CF, Fulop Jr. T, Goldstein DR, Linton PJ. Aging and innate immunity in the mouse: impact of intrinsic and extrinsic factors. Trends Immunol 2009;30:319 24. [148] Shaw AC, Joshi S, Greenwood H, Panda A, Lord JM. Aging of the innate immune system. Curr Opin Immunol 2010;22:507 13. [149] Fukuyama Y, King JD, Kataoka K, Kobayashi R, Gilbert RS, Hollingshead SK, et al. A combination of Flt3 ligand cDNA and CpG oligodeoxynucleotide as nasal adjuvant elicits protective secretory-IgA immunity to Streptococcus pneumoniae in aged mice. J Immunol 2011;186:2454 61. [150] Nagatake T, Fukuyama S, Kim DY, Goda K, Igarashi O, Sato S, et al. Id2-, RORγt-, and LTβR-independent initiation of lymphoid organogenesis in ocular immunity. J Exp Med 2009;206:2351 64. [151] Seo KY, Han SJ, Cha HR, Seo SU, Song JH, Chung SH, et al. Eye mucosa: an efficient vaccine delivery route for inducing protective immunity. J Immunol 2010; 185:3610 19. [152] Park HJ, Ferko B, Byun YH, Song JH, Han GY, Roethl E, et al. Sublingual immunization with a live attenuated influenza a virus lacking the nonstructural protein 1 induces broad protective immunity in mice. PLoS One 2012;7:e39921. [153] Song JH, Nguyen HH, Cuburu N, Horimoto T, Ko SY, Park SH, et al. Sublingual vaccination with influenza virus protects mice against lethal viral infection. Proc Natl Acad Sci USA 2008;105:1644 9. [154] Kong IG, Sato A, Yuki Y, Nochi T, Takahashi H, Sawada S, et al. Nanogel-based PspA intranasal vaccine prevents invasive disease and nasal colonization by Streptococcus pneumoniae. Infect Immun 2013;81:1625 34. [155] Nochi T, Yuki Y, Takahashi H, Sawada S, Mejima M, Kohda T, et al. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines. Nat Mater 2010;9:572 8.

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Mucosal Vaccine Development for Veterinary and Aquatic Diseases Heather L. Wilson1,2,3, Volker Gerdts1,2 and Lorne A. Babiuk4 1

Vaccine and Infectious Disease Organization (VIDO) International Vaccine Centre (InterVac), University of Saskatchewan, Saskatoon, SK, Canada 2Veterinary Microbiology, Western College of Veterinary Medicine (WCVM), University of Saskatchewan, Saskatoon, SK, Canada 3School of Public Health, Vaccinology & Immunotherapeutics Program, University of Saskatchewan, Saskatoon, SK, Canada 4Department of Agricultural Food & Nutritional Science University of Alberta, Edmonton, AB, Canada

I. INTRODUCTION Vaccines are essential for controlling disease in livestock, companion, and zoo animals and wildlife and for controlling fertility and disease in pest species. Effective mucosal vaccines have myriad advantages over parenteral vaccines that are shared across human and veterinary species. For instance, mucosal vaccines stimulate both the mucosal and systemic immune systems, meaning that mucosal vaccines can reduce pathogen colonization and shedding and thus protect the population or herd against infection. Major challenges for mucosal immunization are to generate effective immunity instead of immunological tolerance as well as overcoming interference from passively acquired maternal antibodies. Oral tolerance is a suppressive mechanism designed to prevent

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00048-1

the host immune system from overreacting to innocuous antigens such as those present in feed or commensal flora [1]. Once oral tolerance has been induced, subsequent exposure to that antigen from mucosal or systemic routes will prevent induction of a robust immune response [2]. The vast majority of mucosal vaccines use live attenuated forms of a pathogen that can replicate in the target species, avoiding induction of oral tolerance, but under very rare circumstances may revert back to virulence. Other forms of live vaccines include viruses such as replication-deficient adenovirus [3], canarypox virus carrier vaccines [4], and others that act as a vector to express genes of interest from various pathogens. These carrier viruses have no ability or limited ability to replicate in the immunized species; nor is reversion to a replication competent, highly infectious virus

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© 2020 Elsevier Inc. All rights reserved.

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possible, making them a safer vaccine choice. There are some reports that mucosal delivery of live viral vaccine to the upper respiratory tract may overcome maternal antibody interference [5]. Previous chapters provided detailed analyses of the numerous advantages of mucosal vaccines, and these will not be discussed in depth here. Instead, aspects of mucosal vaccine development that are particularly important for the veterinary field, such as mass delivery, vaccines that differentiate between infection and vaccination (which has important trade implications), and economic considerations, will be discussed. Examples of the numerous commercially available mucosal veterinary vaccines and discussion of novel experimental vaccines will be provided. Vaccine development for administration in veterinary species to protect human health will also be discussed.

A. Considerations for Mucosal Veterinary Vaccine 1. Mass Delivery Mass delivery methods for mucosal immunization, such as administration of the vaccine in drinking water or in feed, in sprays (for avian species, which are ingested at preening), in ovo (for avian species), or for immersions (for aquatic species), allow vaccination of hundreds or thousands of animals over a short period of time. Most important, mass delivery of mucosal vaccines means that each individual animal does not need to be restrained, which can be extremely stressful for animals (especially those that are not routinely handled by humans), and restraint may be potentially hazardous to the person or teams of people administering the vaccines. A major drawback to mass delivery is that a uniform dose per animal is not always given across the population. Other mucosal routes that require restraint are vaccines administered intranasally, by drenching (oral immunization by syringe), or through eyedrop (conjunctival) administration. These delivery methods have the advantage that they promote a mucosal immune

response and the dose per animal is easily controlled, but they do require animal restraint, which makes them more expensive to deliver. 2. Differentiation of Infected From Vaccinated Animals Vaccines With today’s global economy, trade in animals and in animal products such as meat, eggs, and fur occurs locally, nationally, and internationally. For trade purposes, it is advantageous to be able to distinguish animals that have been infected by disease from animals that have been vaccinated. Vaccines that allow for the immunological differentiation between animals that have been infected and those that have been vaccinated are called DIVA vaccines. DIVA vaccines often lack one or more proteins present in the wild-type microorganism, which can be determined through immune assays. The first-generation DIVA vaccines were developed when it was discovered that pigs vaccinated with the attenuated strains of Aujeszky’s disease virus did not develop antibody against select protein epitopes [6], whereas animals infected by wild-type viruses did have these protein-specific antibodies in their serum. These first-generation marker vaccines were soon improved upon by using genetically modified live vaccines that lacked selected glycoproteins [7], and enzyme-linked immune sorbent assays were developed that could determine which pigs were vaccinated because they lacked antibodies against the glycoprotein but had antibodies specific for other PrV proteins [8]. By using these strains as vaccines or genetically engineered live viral vaccines, researchers could track the success of eradication efforts; this resulted in effective eradication programs of the disease in many parts of Europe [8]. Without the option of using DIVA vaccines, producers may choose not to vaccinate so as to avoid the presence of antibodies in the herd that they cannot guarantee are due to the vaccine and not the presence of the diseasecausing agent in their herd. At the same time, vaccines made from live viruses, such as

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

II. EXPLORATION OF COMMERCIAL AND EXPERIMENTAL MUCOSAL VETERINARY VACCINES

replication-deficient adenovirus that express genes of interest from various pathogens or modified viruses with select genes deleted, are considered genetically modified organisms (GMOs). Animals vaccinated with GMOs may face regulatory hurdles that must be overcome before the products from these animals can be sold. Indeed, some countries are reluctant to license GMO vaccines. 3. Economics and Trade Livestock production is a large-scale business and mucosal vaccination will not be implemented unless the disease causes significant mortality and/or morbidity (such that production metrics are negatively affected). Further, the vaccine will be used only if it is sufficiently affordable such that its use does not significantly reduce profitability. Depending on the disease, how easily it spreads, and how well the infectious agent survives in the environment, culling of a barn rather than mass vaccination may be a more economically feasible option to clear an infection. However, from a welfare point of view, such a practice is highly problematic and often results in public outcry. Moreover, as antimicrobialresistant pathogens continue to emerge, there is increasing pressure to raise livestock with reduced levels of antibiotics. Vaccines offer the potential of controlling such infections and represent effective alternatives to antibiotics. Together, adaptation of mucosal vaccines for mass delivery to livestock, DIVA vaccination and its impact on trade considerations, and the cost of vaccine development and implementation all influence whether vaccines will be used in a veterinary setting.

II. EXPLORATION OF COMMERCIAL AND EXPERIMENTAL MUCOSAL VETERINARY VACCINES Whether an effective vaccine can be generated against an infectious agent requires

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extensive knowledge of disease pathogenesis and epidemiology across all target species. Livestock (cattle and other ruminants, pigs, avian species such as chickens and turkeys, etc.), companion animals (horses, dogs, cats, rabbits, guinea pigs, etc.), wildlife (deer, bison, koala, etc.), pest species (skunks, raccoon, mice, rats, etc.), zoo animals, and aquatic species have unique economic, social, and immunological characteristics that affect whether a mucosal vaccine will be sought for development and/ or whether existing mucosal vaccines will be used.

A. Mucosal Vaccines for Livestock The growing human population has lead to an increased need for protein from food animals and for animal by-products, which has led to a steady increase in the number of intensive livestock operations worldwide. Such operations can include hundreds or thousands of animals contained in a pasture or barn, and their close proximity to one another can facilitate spread of disease to vulnerable members within the population. Because mucosal vaccination is superior to systemic vaccination in preventing colonization and shedding of pathogens, mucosal vaccination is especially important in controlling infections in livestock. Tremendous strides have been made in veterinary mucosal vaccine development and commercial availability, many of which are listed in Table 48.1. The following sections provide examples of mucosal veterinary vaccines under development against selected pathogens using animals from the target species (rather than mice or other experimental animals where possible). 1. Suidae A. PORCINE TRANSMISSIBLE GASTROENTERIDITIS AND PORCINE EPIDEMIC DIARRHEA VIRUS

Both transmissible gastroenteritis virus and porcine epidemic diarrhea virus can cause severe

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TABLE 48.1 Commercially Available Vaccines for Livestock Species, Companion Animals, Birds, and Wildlife Against Infectious Diseases Listed in the OIE Manual of Infectious Diseases [9] (http://www.oie.int/manual-of-diagnostic-testsand-vaccines-for-terrestrial-animals/) Vaccine name

Virus, bacteria, or subunit antigen

Disease

Route

Bovilis IBR

Bovine herpesvirus 1 (BHV1)

Bovine rhinotracheitis

Intranasal

TSV-2

Infectious bovine rhinotracheitis (IBR) virus and parainfluenza3 (PI3) virus

Bovine rhinotracheitis Parainfluenza,

Intranasal

INFORCE 3

BRSV, IBR, and PI3

Bovine respiratory disease

Intranasal

NASALGEN IP

IBR, PI3

Bovine rhinotracheitis Parainfluenza

Intranasal

Once PMH IN

Mannheimia haemolytica, Pasteurella multocida

M. haemolytica-P. multocida

Intranasal

BOVILIS CORONAVIRUS

Bovine coronavirus

Bovine enteric disease

Intranasal

Calf-Guard

Bovine rotavirus and coronavirus

Bovine enteric disease

Oral

Respioval

BRSV, IBR, and PI3

Bovine respiratory disease

Intranasal

Flu Avert I.N.

Equine influenza virus type H3N8 strain (EIV A/Equine 2/Kentucky/91)

Influenza

Intranasal

PINNACLE I.N.

Streptococcus equi

Strangles

Intranasal

ProSystem TREC

Rotavirus (two modified live G serotypes 5 and 4 of serogroup A), transmissible gastroenteritis virus, colibacillosis (Escherichia coli pilus antigens K88, K99, F41, and 987P), Clostridium perfringens type C

Bovine enteric disease, transmissible gastroenteritis, colibacillosis, enterotoxemia

Two oral and one intramuscular

ENTERO VAC

Avirulent live E. coli F4 (K88)

Enteritis

Oral administration in drinking water

EDEMA VAC

Avirulent live E. coli F18 (K99) vaccines Edema

Oral administration in drinking water

COLIPROTEC F4

Avirulent live E. coli F4 (K88)

Postweaning diarrhea (PWD)

Oral

Enterisol Ileitis

Live attenuated Lawsonia intracellularis

Porcine proliferative enteropathy (ileitis)

Oral administration in drinking water

NITRO-SAL FD,

Avirulent live Salmonella enterica Cholerasuis

Salmonellosis

Oral administration in drinking water

Argus SC/ST

Avirulent live S. Cholerasuis

Salmonellosis

Oral administration in drinking water

BOVIDAE

EQUINE

SUIDAE

(Continued)

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

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II. EXPLORATION OF COMMERCIAL AND EXPERIMENTAL MUCOSAL VETERINARY VACCINES

TABLE 48.1

(Continued)

Vaccine name

Virus, bacteria, or subunit antigen

Disease

Route

Salmonella T/C vaccine

Avirulent live S. Cholerasuis

Salmonellosis

Oral administration in drinking water

MAXI/GUARD Nasal Vac

Bordetella bronchiseptica

Respiratory disease

Intranasal

Ingelvac ERY-ALC

Avirulent live Erysipelothrix rhusiopathiae

Erysipelas

Oral administration in drinking water

Suvaxyn E-ora

Avirulent live E. rhusiopathiae

Erysipelas

Oral administration in drinking water

NASAGUARD-B

Avirulent live B. bronchiseptica

Canine infectious respiratory disease (CIRD) (kennel cough)

Intranasal

VANGUARD B

Avirulent live B. bronchiseptica

Kennel cough

Intranasal

BRONCHI-SHIELD Avirulent live B. bronchiseptica ORAL

Kennel cough

Administered by syringe into the buccal cavity

Diseases caused by Bordetella bronchiseptica

Intranasal

CANINE

FELINE NOBIVAC FelineBb

Avirulent live B. bronchiseptica

WILDLIFE OR PEST ANIMALS Raboral V-RG

Live vaccinia virus vaccine encoding the rabies virus glycoprotein

Rabies

Oral in baits

ONRAB

Live adenovirus vector encoding the rabies glycoprotein

Rabies

Oral in Ultralite bait matrix

RABIGEN SAG2

Modified live attenuated rabies virus vaccine

Rabies

Oral in baits

Burse BLEN-M

Live infectious bursal disease virus (IBV)

Infectious bursal disease

Drinking water

S-706 and SVS 510

Live IBV

Infectious bursal disease

Drinking water and coarse spray

Univax-Plus

ST-12 and 51 A/C4 strains of IBV

Infectious bursal disease

Drinking water

UNIVAX-BD

Live IBV

Infectious bursal disease

In ovo, Drinking water

Bursine-2

Live IDV

Infectious bursal disease

Drinking water

CLONEVAC D-78

Field isolate of IBV

Infectious bursal disease

Coarse spray, drinking water

AVIAN

(Continued)

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48. MUCOSAL VACCINE DEVELOPMENT FOR VETERINARY AND AQUATIC DISEASES

TABLE 48.1

(Continued)

Vaccine name

Virus, bacteria, or subunit antigen

Disease

Route

HVT

FC-126 live strain of turkey herpesvirus (HVT)

Marek’s disease

In ovo

SB1

SB1 strain of chicken herpesvirus

Marek’s disease

In ovo

VAXXITEK HVT 1 IBD

Live serotype 3 HVT

Marek’s disease

In ovo

Bursal Disease Marek’s Disease Vaccine Serotype 3

ST-14 strain of live bursal disease virus, Infectious bursal disease and FC-126 strain of HVT Marek’s disease

In ovo

VECTORMUNE HVT IBD vaccine

HVT expressing an infectious bursal disease key protective antigen

Marek’s disease

In ovo

CEVAC MD HVT

Serotype 3 HVT Marek’s disease virus

Marek’s disease

In ovo

MD-Vac

Live serotype 3 virus Marek’s Disease Vaccine

Marek’s disease

In ovo

Poulvac Ovoline CVI

Live serotype 1 Marek’s disease virus

Marek’s Disease

In ovo

IB-VAC-H

Live Holland strain of IBV

Massachusetts type bronchitis

Coarse spray or drinking water

Bronchitis Vaccine, Mass Type

IBV

Massachusetts type bronchitis

Intraocular, Coarse aerosol spray, Drinking water

MILDVAC-Ma5

Live Ma5 strain of Massachusetts type bronchitis

Massachusetts type bronchitis

Beak-O-Vac or coarse spray, drinking water

NEWHATCH-C2

Live B1 type C2 strain of Newcastle disease virus

Newcastle disease

Coarse spray

Gallivac HB1 Mass

B1 strain of Newcastle disease virus, Massachusetts type IBV

Newcastle disease and Massachusetts type bronchitis

Coarse spray, drinking water

NewcastleBronchitis Vaccine

B1 strain of Newcastle disease virus and IBV of the Massachusetts and Connecticut types

Newcastle disease and Massachusetts type bronchitis

Intraocular or coarse spray

AVIPRO ND-IB POLYBANCO

Newcastle disease virus and IBV of the Massachusetts and Connecticut types

Newcastle disease and infectious bronchitis, Massachusetts and Connecticut type bronchitis

Intraocular or drinking water

COMBOVAC-30

Live clone 30 strain of Newcastle disease virus and IBV Massachusetts and Connecticut types

Newcastle disease and Massachusetts and Connecticut types bronchitis

Coarse spray, drinking water

Poulvac ST

Modified-live Salmonella enterica Enteritidis Salmonella Heidelberg, or Salmonella enterica Typhimurium

Salmonellosis

Coarse spray then drinking water (Continued)

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

II. EXPLORATION OF COMMERCIAL AND EXPERIMENTAL MUCOSAL VETERINARY VACCINES

TABLE 48.1

817

(Continued)

Vaccine name

Virus, bacteria, or subunit antigen

Disease

Route

SALMUNE

Live avirulent S. Enteritidis, S. Heidelberg, or S. Typhimurium

Salmonellosis

Coarse spray, drinking water

AVIPRO MEGAN EGG

Live avirulent S.Typhimurium

Salmonellosis

Coarse spray

M-NINEVAX-C

Live avirulent M-9 strain of P. multocida, Heddleston type 3 4 crossstrains

P. multocida, owl cholera in chickens and turkeys

Drinking water

H.E. VAC

Live apathogenic avian adenovirus

Hemorrhagic enteritis

Drinking water

ORALVAX HE

Live turkey avirulent type II avian adenovirus of pheasant origin

Hemorrhagic enteritis

Drinking water

REOGUARD L

Live 1133 strain of avian reovirus

Tenosynovitis

Drinking water

ENTEROVAX

Live avian reovirus (tenosynovitus biotype)

Reovirus induced tenosynovitis (viral arthritis)

Spray or drinking water

IMMUCOX

Live oocysts of Eimeria spp.

Coccidiosis

Oral by chicken feed

COCCIVAC-B52

Live oocysts of Eimeria acervulina, Eimeria maxima, E. maxima MFP, Eimeria mivati, and Eimeria tenella

Coccidiosis

spray cabinet administration

COCCIVAC D2

E. tenella, E. mivati, E. acervulina, E. maxima, Eimeria brunetti, and Eimeria necatrix i

Coccidiosis

Spray cabinet administration

HATCHPAK COCCI III

Live oocysts of E. maxima, E. acervulina, Coccidiosis and E. tenella

Coarse spray

INNOVAX-ILT

Live recombinant serotype 3THV with genes from laryngotracheitis virus

Fowl laryngotracheitis and Marek’s disease

In ovo

LT-IVAX

Live attenuated fowl laryngotracheitis virus

Fowl laryngotracheitis

Intraocular

LT BLEN

Live fowl laryngotracheitis virus

Fowl laryngotracheitis

Intraocular or drinking water

ART VAX

Live chemically induced mutant of Bordetella avium

B. avium rhinotracheitis (turkey coryza)

Coarse spray, drinking water

TREMOR BLEN D

Live avian encephalomyelitis virus

Avian encephalomyelitis

Drinking water

AQUAVAC-ESC

Modified live Edwardsiella ictaluri RE-33

Edwardsiellosis

Immersion

Furogen Dip

Aeromonas salmonicida bacterin

Furunculosis

Immersion

Ermogen

Formalin-inactivated Yersinia ruckeri serotype I (Hagerman strain)

Enteric redmouth disease

Immersion

AquaVac Vibrio Oral

Inactivated Vibrio anguilarum 01 and 02a Vibrosis

AQUATIC SPECIES

Oral

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diarrhea in newborn piglets. A DNA vaccine expressing S proteins from both viruses delivered by attenuated Salmonella Typhimurium was constructed as a potential vaccine. and its immunogenicity was assessed [10]. Twenty-one-dayold piglets were orally immunized with the attenuated S. Typhimurium with empty DNA vaccine or DNA vaccine expressing the S proteins at a dosage of 1.6 3 1011 CFU per piglet and then booster immunized with 2.0 3 1011 CFU after 2 weeks. Virus-neutralizing S-proteinspecific immunoglobulin G (IgG) and secretory immunoglobulin A (SIgA) as well as systemic cellular immune responses (interferon gamma, interleukin 4, and lymphocyte proliferation) was significantly higher in the vaccinated group than in the control and empty DNA vaccine cohorts. These data show that S. Typhimurium can be used to carry DNA vaccines and, when delivered orally, may promote a protective immune response. 2. Caprinae and Ovidae A. BRUCELLA OVIS

Ovine epididymitis caused by Brucella ovis infection has been reported in the Americas, European countries, Australia, New Zealand, and South Africa. This disease can lead to genital lesions and reduced fertility in rams, placentitis and abortions in ewes, and increased perinatal mortality in lambs [11]. While safer than subcutaneous vaccination, conjunctival vaccination with live Brucella melitensis Rev 1 vaccine can causes abortions, is highly virulent, and is not a DIVA vaccine; therefore it is not recommended in countries that are free from B. melitensis [12]. Alternatively, conjunctival immunization in rams using a thermoresponsive and mucoadhesive in situ gel composed of poloxamer 407 (P407) and chitosan (Ch) could effectively deliver recombinant BLS-OMP31. (BLS is part of the enzyme lumazine synthase from Brucella spp. that is both highly immunogenic

and a carrier of foreign peptides and B. ovis antigen OMP31 [13].) Serum and preputial, saliva, lacrimal, and nasal secretions showed significant antigen-specific IgG antibody, and the levels remained elevated in serum only for several months. Relative to unvaccinated rams, the rams from the vaccinated cohort showed significant induction of antigen-specific SIgA after the first and second immunization in lacrimal, preputial, or nasal secretions (but not in nasal secretions or in serum), but antibodies levels declined rapidly [14]. Further, conjunctival immunization induced a significant BLS-OMP31-specific hypersensitivity response to intradermal injections relative to the control rams, which indicates induction of cell-mediated immunity. Conjunctival administration of BLS-OMP31P407-Ch may be a promising alternative to current B. ovis immunization strategies. 3. Bovinae A. BOVINE HERPESVIRUS 1

Bovine herpesvirus 1 (BoHV-1) is responsible for infectious bovine rhinotracheitis, infectious pustular vulvovaginitis, conjunctivitis, abortion, encephalomyelitis, and mastitis in cattle. Parenteral BoHV-1 glycoprotein E deleted mutant viral DIVA vaccines used in conjunction with diagnostic testing and targeted culling of animals infected with field strains has led to eradication of this disease in some European countries [15,16]. Additional DIVA mucosal vaccines are under development. For example, a small trial showed that calves vaccinated intranasally with BoHV-1 glycoprotein E deleted mutant virus or BoHV-1 triple mutant virus (BoHV-1 tmv), which incorporates mutation for three genes, including glycoprotein E within a single virus, were protected against infectious challenge [17]. While both DIVA vaccines were protective against clinical disease, only BoHV-1 tmv-vaccinated calves generated significantly higher virus-neutralizing titers

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

II. EXPLORATION OF COMMERCIAL AND EXPERIMENTAL MUCOSAL VETERINARY VACCINES

after challenge relative to the sham controls, and they showed a more rapid cellular immune response onset and a more rapid viral clearance. Although this virus has worldwide distribution, use of marker vaccines will continue to contribute to eradication efforts. B. HEMORRHAGIC SEPTICEMIA

Water buffalo, cattle, and bison are affected by hemorrhagic septicemia (HS), which is an acute, highly fatal form of pasteurellosis. This economically important bacterial disease affects Asia, Africa, and the Middle East, and sporadic outbreaks occur in Southern Europe. An HS vaccine containing avirulent Pasteurella multocida strain B:3,4 (fallow deer strain) has been used in Myanmar to control HS in cattle and water buffaloes [18]. Earlier intranasal vaccines failed to protect against subcutaneous challenge, and the efficacy of this vaccine for primary vaccination of young buffaloes was brought into question [19,20]. However, a later study showed that an intranasal vaccine containing live gdhA-derivative P. multocida B:2 that was boosted 2 weeks later was protective against a subcutaneously administered challenge with live wild-type P. multocida [21]. Importantly, the vaccine was also effective in protecting in-contact buffalo against a virulent parental strain and has been recommended by the Food and Agriculture Organization of the United Nations as an effective vaccine in Asia.

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transported across the mucosal epithelium of the upper respiratory tract) and although IgA does cross the mucosa, dimeric IgA makes up only 10% of antibodies in colostrum, it was suggested that the upper respiratory tract may not be affected by maternal interference. To test this hypothesis, cows were vaccinated with modified live bovine viral diarrhea virus (BVDV) vaccine composed of BHV-1, BVDV-1, BVDV-2, PI-3, and BRSV antigens. Calves were shown to have high circulating maternally derived IgG antibodies serum but extremely low titers of maternally derived IgG in nasal secretions [22]. Maternally derived IgA in nasal secretions were present but at much lower levels than in serum. Calves (3 8 days old) either were not vaccinated against BVDV or received one or two (day 0 and day 35) immunizations by the intranasal route. Within 5 7 days after birth, maternally derived IgA in nasal secretions were not detected. Calfderived (i.e., endogenous) BVDV1- and BVDV2-specific IgA production was detected within 10 days after vaccination. A secondary intranasal vaccination after 5 weeks induced a strong memory antibody response with sustained IgA levels in nasal secretions. Collectively, these studies demonstrated that the mucosal immune system in newborn calves is functional and responsive to vaccination without being affected by maternal interference. 4. Avian Species

C. BOVINE VIRAL DIARRHEA VIRUS

Calves do not receive maternal antibodies in utero; instead, they receive antibodies through colostrum in the neonatal period. However, while maternal antibodies are critically required to protect the vulnerable neonate against infectious diseases, circulating maternal antibodies interfere with the neonate’s ability to develop its own immunity (referred to as maternal interference). Because colostrum is composed mainly of IgG1 (which is not

Chicken, turkey, duck, and other avian barns and houses are populated by very large numbers of birds for egg production or for production of meat. Standard laying houses are reported to hold from 100,000 to 500,000 hens, and broiler houses routinely house 20,000 birds. With these numbers, it is not surprising that the industry has actively sought vaccines that could be administered by mucosal routes rather than by parenteral routes, which generally rely on injection with needles. The majority of

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mucosal avian vaccines are live viruses administered by eyedropper into the eye (intraocular or conjunctival), orally into the drinking water, as a coarse spray whereby birds consume the vaccine during preening, and through the in ovo route. In-feed oral vaccination and spray cabinet (intranasal) routes are also used for some commercial vaccines. In ovo injection has become widely used as a means to deliver precise, uniform doses with the capacity to inject up to 60,000 eggs per hour. In ovo immunization has the added benefit that this method avoids stress to chicks, is sanitary, and has an earlier exposure time than any other immunization method. A. AVIAN INFLUENZA

Many experimental mucosal avian vaccines are under development to combat avian influenza. It was reported that an intranasally delivered bioadhesive liposome using tremella or xanthan gum and containing the experimental inactivate avian H5N3 virus as a model antigen elicited high mucosal SIgA and serum IgG in chickens [23]. Even the low pathogenic strains such as H9N2 avian influenza virus (AIV) can affect the economic success of commercial poultry industry by causing mild respiratory disease and decreased egg production. Immunizations for multiple forms of avian influenza are under way as experimental vaccines. For instance, Lactobacillus plantarum NC8 strain was engineered to express select peptides from H9N2 AIV. Both oral and intranasal vaccination of 3week-old white leghorn layer chickens succeeded in inducing immunity, but the intranasal route induced stronger immunity and showed less body weight loss, lung virus titers, and pathology after challenge with the H9N2 virus [24]. These nontraditional mucosal vaccine delivery platforms showed that they may be good choices for commercial avian influenza vaccine development.

B. Companion Animals 1. Leporidae A. RABBIT HEMMORRHAGIC DISEASE

Rabbit hemorrhagic disease (RHD) is a lethal disease of adult rabbits caused by rabbit calicivirus [25]. Oral immunization of rabbits with recombinant vaccinia virus [26] or recombinant myxoma virus [27] coding for VP60, the major structural protein of RHD virus (RHDV) induced protection against challenge with virulent RHDV. However, little horizontal transfer was achieved. Other researchers showed that oral immunization with VP60 protein expressed in transgenic potatoes generated partial protection against viral challenge [28]. Rabbits have been used to investigate whether the uterus is a suitable mucosal vaccination site. An experimental vaccine consisting of ovalbumin (OVA), recombinant truncated glycoprotein 1 from bovine herpes virus, and a fusion protein of porcine parvovirus VP2 and bacterial thioredoxin (rVP2 TrX) was formulated with poly I:C, host defense peptide and polyphosphazene as adjuvants. Surgery was performed to isolate each uterine horn, and this triple antigen triple adjuvant vaccine was injected into the lumen of the uterine horns (referred to as intrauterine immunization) [29]. Significant induction of OVA and tGD-specific serum IgG and IgA was observed over time in intrauterine-immunized animals. Uterine, lung, and vaginal tissues obtained 1 month after the single immunization showed significant OVAspecific IgG and IgA response relative to sham treatment. Significantly increased tGD-specific and rVP2-TrX antigen-specific IgG titers (but not IgA titers) were observed in lung, vagina, and uterine tissue relative to controls. The results indicate that a subunit vaccine formulated with appropriate adjuvants can trigger both systemic and mucosal immunity when administered into the uterine lumen.

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

II. EXPLORATION OF COMMERCIAL AND EXPERIMENTAL MUCOSAL VETERINARY VACCINES

C. Wildlife 1. Koala A. CHLAMYDIA PECORUM

Many wild koalas in Australia are known to have Chlamydia pecorum, which causes debilitating ocular and urogenital infections in koalas with clinical signs that include conjunctivitis and infertility. A single-dose anti-C. pecorum vaccine formulated to contain three major outer-membrane proteins (MOMPs) or polymorphic membrane proteins (PMPs) (an antigenic membrane bound surface-exposed adhesion protein that is important for attachment to the cell membrane [30]) and a 1:2:1 ratio with PCEP poly[di(sodium carboxylatoethylphenoxy)phosphazene], immune defense regulatory peptide (IDR1002), and poly I:C was tested in wild koalas. Although the vaccine was administered subcutaneously, antiMOMP IgA increased 10- to 100-fold at ocular and upper genital tract (UGT) sites in 50% and 40% of the koalas, respectively. The PmpG vaccine also triggered a 10- to 100-fold increase post vaccine IgA antibodies at the UGT or the ocular sites in 40% and 50% of koalas, respectively, which suggests that the vaccines elicited a mucosal response in at least some of the koalas. The cohort vaccinated with MOMP vaccine showed decreased chlamydia loads with no new occurrence of infection, but the other vaccination group and the control group showed increased loads with incidences of new infections, suggesting that the MOMP vaccine may be superior [31]. Further development must be undertaken to improve vaccine uptake to more members of the population, but these results suggest that a parenteral vaccine may succeed in promoting mucosal responses when properly formulated. 2. Prairie Dogs A. PLAGUE

Sylvatic plague caused by Yersinia pestis and carried in fleas can significantly affect the

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population dynamics of prairie dogs (Cynomys spp.). In turn, reduced prairie dog numbers can affect the population dynamics of ferrets, burrowing owls, and several canine and avian predators. Administration of insecticides can control the fleas and reduce transmission of Y. pestis, but there is evidence that the fleas can develop resistance [32]. A vaccine that can be used as an alternative to the use of insecticides is actively sought. The orthopoxvirus raccoonpox (RCN) was genetically modified to express two protective Y. pestis antigens (designated RCN-F1/V307) and mixed with bait for oral vaccination of prairie dogs in a lab setting. Sixty percent of prairie dogs that consumed bait containing RCN-F1/307 and were then challenged at 270 days post-vaccination survived, which was a significantly higher percentage than that in the placebo group [33]. Rates of survival were improved if two oral baits were consumed months apart. 3. Multispecies A. RABIES

Rabies virus, a member of the Rhabdoviridae family, causes neuroinvasive rabies disease in many wild animals, including bats, possums, raccoons, skunks, foxes, coyotes, groundhogs, wolves, and monkeys. It can also infect companion animals such as dogs, cats, rabbits, and horses. It is spread through saliva and can be transmitted through bites and scratches. Symptoms include fever, violent movements, uncontrolled excitement, fear of water, aggressive behavior, and death. Most human cases of rabies come from contact with an infected domestic dog [34]. Raboral V-RG is an oral vaccine composed of a live vaccinia virus encoding the rabies virus glycoprotein [35]. It is encased in a packet with fish meal and set out as bait for raccoons, foxes, coyotes, and the like; the packet can have a flavor coating to attract target species [36]. An alternative oral vaccine in Canada is ONRAB, a

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

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48. MUCOSAL VACCINE DEVELOPMENT FOR VETERINARY AND AQUATIC DISEASES

live adenovirus vector encoding the rabies glycoprotein that is administered as an oral vaccine in Ultralite bait matrix [37]. A comparative study showed that when Raboral V-RG or ONRAB was distributed by aircraft at a density of 75 baits/km2 and sera from raccoons and skunks were collected 5 7 weeks later, skunks showed no significant difference in the proportion of antibody-positive animals, regardless of the vaccine used [38]. In contrast, the proportion of antibody-positive raccoons was significantly higher in the ONRAB-baited areas than in the RABORAL V-RG-baited areas, suggesting that ONRAB may be a better choice to vaccinate more species.

D. Mucosal Aquatic Vaccines The aquaculture industry is growing faster than any other farmed animal industry in the world, with an increase of more than 10% between 2011 and 2016 to 70 million tons, and an increasingly high proportion of high-quality protein used to feed the world’s growing population comes from aquaculture. As with any farming industry, the risk of infectious diseases increases as the density of animals increases, and excellent fish health management practices, such as controlling stocking densities, maintaining adequate oxygen levels and water quality, and reducing pathogen loads, are critical to control disease. Improved understanding of mucosal immunity in farmed fish and crustaceans may lead to the development of cost-effective mucosal vaccines. It is estimated that there are 25,000 fish species in the world, and they are extremely diverse to accommodate living in warm or cold climates, in fresh or salt water, and in depths at high or low pressure [39]. Teleosts (bony fish) lack bone marrow; instead, their B lymphocytes mature within the kidney [40]. Mucosaassociated lymphoid tissue in teleost fish is composed of skin-associated lymphoid tissue,

gill-associated lymphoid tissue, and a diffuse gut-associated lymphoid tissue [40]. Antibodyproducing cells have been identified in the cutaneous dermis and mucus, which may indicate a “mucosal” immune system in fish [41]. It is not yet clear whether fish B and T cells home back to mucosal sites upon mucosal infection after immunization [42]. For instance, oral or anal immunization of carp with formalin-killed Vibrio anguillarum followed by a booster immunization by the same route resulted in slightly enhanced antigen-specific Ig titers detected in skin mucus and bile. Serum antibody titers were elevated after anal intubation but not in response to oral immunization [43]. How the route of immunization affects immunity in fish warrants further study. Fish body temperature takes on the local temperature, which can have a significant impact on the metabolism and rate of growth of fish as well as on their immune system. For instance, development of antibodies in fish adapted to low temperatures (,15 C) may require at least 4 6 weeks, whereas the time period to develop antibodies may be a few weeks in fish adapted to warmer temperatures. This time frame suggests that even if fish have a functional immune system, they may not develop immunity in a timely manner to protect them from infection. The major causative agents of infectious diseases in finfish aquaculture include bacteria, viruses, parasites, and fungi. Infectious agents can infect fish at some developmental stages and not at others. Although some fish may have a functional immune system in the larva or fry stage, others may not, which means that proper biosecurity rather than vaccination may be critical to protecting them against infection. Farmed fish that are routinely vaccinated include Atlantic salmon (Salmo salar), rainbow trout (Onchorhynchus mykiss), and Atlantic cod (Gadus morhua). With effective vaccine development, there has been a decline in the use of antibiotics along with improved health and increased growth of the fish.

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

II. EXPLORATION OF COMMERCIAL AND EXPERIMENTAL MUCOSAL VETERINARY VACCINES

Currently, there are three main forms of vaccination for aquatic species: immersion, oral delivery, and injection. 1. Immersion Vaccines For immersion immunization, gills are likely the main site of antigen entry, but uptake by the skin, lateral line, and gut have also been suggested and may, in fact, contribute to induction of mucosal immunity [44]. Immersion vaccines are effective for a number of bacterial pathogens, and they are practical, cheap, and easy to batch-administer, especially to small fish. A disadvantage to this vaccination route is that it requires large amounts of vaccine, and levels of protection and duration of immunity may vary across vaccines. An experimental vaccine for immersion of catfish 10 30 days posthatch with modified live Edwardsiella ictaluri vaccine was shown to produce a protective immune response against Enteric septicemia [45]. Other researchers showed that introducing several small lesions in the skin and then immersing the fish in a vaccine suspension containing formalin-killed Streptococcus iniae produced a protective immune response against these bacteria, and they suggest that the response was equal in effectiveness to that produced by intraperitoneal injection [46]. 2. Oral and Intranasal Vaccines Oral delivery can be accomplished with fish of any age. It is relatively cheap and non-laborintensive, it is not stressful for the fish, and it is the only option to deliver vaccine to fish in the seawater growth stage. Disadvantages include the fact that large quantities of the antigen are required, it is impossible to ensure equal distribution among the farmed animals, and the duration of immunity is generally less than that observed with injection or immersion. Oral vaccines can be made to adhere to finished feed. The challenge is to maintain antigen stability in countries with high heat and humidity as well

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as in the high-acid environment of the stomach once consumed. Bioencapsulation has been used, wherein feed was incubated in a vaccine suspension prior to feeding the fry. Different encapsulation techniques, including formulation with liposomes [47] or alginate beads [48], have been used to protect the antigens from the destructive environment in the gut. As with any animal, oral administration may lead to induction of tolerance, which may be compounded by the young age of the animal and repeated low-dose administration. Some researchers believe that oral vaccines may be more suited to act only for booster immunization to avoid induction of oral tolerance. However, studies have shown that primary oral vaccination of salmon in the seawater growth stage with an oral salmonid rickettsial septicemia vaccine formulated with a bioadhesive cationic polysaccharide protected the salmon against a lethal pathogen challenge [49]. Other researchers showed that oral vaccination with a DNA vaccine, wherein the vector expressing a gene from infectious pancreatic necrosis virus encapsulated in alginate microspheres, protected salmonid fish against infectious challenge [50]. Oral vaccination of rainbow trout with an experimental vaccine bacterin of Yersinia ruckeri O1 failed to protect against enteric redmouth disease (yersiniosis), but the same dose administered anally was protective against infectious challenge. These data suggest that the oral vaccine needs to be protected from degradation in the stomach to be effective [51]. Rainbow trout (O. mykiss) vaccinated intranasally as early as 24 days posthatch with a live attenuated infectious hematopoietic necrosis virus vaccine, killed enteric red mouth bacterin, or saline. Upon challenge with the respective pathogen 28 days later, vaccinated groups were significantly more protected than their age-matched mock control groups [52]. These data suggest that the intranasal route may be amenable for vaccine targeting if it becomes adapted for mass vaccination.

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3. Injected Vaccine Many farmed fish are vaccinated by an intraperitoneal injection, a route that is very labor intensive. This method can also be stressful for the fish and must be performed on fish of sufficient size, which means that vaccination of fry is difficult. No commercial mucosal vaccines are available against viruses that infect fish, so these vaccines are administered by injection. With the rise in aquaculture comes an increased need to protect the livestock against infectious diseases. Increased efforts to elucidate the immunology of the target species of fish and pathogenesis of the parasite, virus, or bacteria that target them will undoubtedly lead to development of new vaccines suitable for mass delivery.

E. Immunocontraceptive Vaccines Population management has different considerations, depending on whether the species of interest are wildlife, pests, companion animals, or zoo animals. Generally, for all but pest species, an ideal immunocontraceptive would be reversible, safe, long-lasting, and cost-effective. While in some species, reduction in sexual or aggressive behavior may be a beneficial side effect of contraception, some species may require such behavior to maintain the herd hierarchy. Therefore species-specific needs should be a consideration before vaccination [53]. For decades, two reliable parenteral immunocontraceptives against gonadotropin-releasing hormone (GnRH; also known as luteinizing hormone releasing hormone) or zona pellucida proteins (ZP) have been used to reversibly control fertility in many animal species. Antibodies generated against GnRH neutralize this pituitary hormone, which in turn inhibits steroidogenesis and gametogenesis in male and female mammals. Porcine zona pellucida vaccines prepared by using ZP isolated from pig ovaries or recombinant

ZP antigen (SpayVac from ImmunoVaccine Technologies, Canada) are one of the most studied immunocontraceptive vaccines in wildlife. Anti-ZP antibodies bound to sperm impede binding and penetration of the ovum, and anti-ZP antibodies interfere with follicle development in some species [54,55]. The overwhelming majority of immunocontraceptive vaccines are delivered parenterally, but some experimental research has focused on delivery by mucosal routes. Delivery of an effective mucosal vaccine (i.e., not relying on injections or darting) for wildlife or zoo animals would be ideal for administration, as it would be less stressful to the animal and present less risk to the person administering the vaccine. Experimental work in rabbits showed that rabbit ZP glycoprotein B delivered by infection with myxoma virus resulted in infertility in 25% of female rabbits [56]. Brushtail possums (Trichosurus vulpecula) were vaccinated with bacterial ghosts (BGs) expressing ZP protein introduced through oral, intranasal/conjunctival, parenteral, and intraduodenal routes. AntiZP antibodies were detected in the serum and the ovarian follicular fluid after intranasal/conjunctival immunization [57]. Intraduodenal, but not oral administration of the vaccine, elicited significant systemic immune responses, indicating that protection of BG vaccines from degradation by gastric acidity would enhance the effectiveness of orally delivered vaccines. Superovulation and artificial insemination was used to assess the effect of the immunization with BG-delivered ZP. Immunization by the nasal/conjunctival route resulted in induced antibody-mediated and cell-mediated immune responses, and significantly fewer eggs were fertilized in immunized possum females [58]. Field trials will need to be performed to determine whether mucosal immunization with BG containing possum ZP antigens is suitable for fertility control of wild possum populations.

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III. VETERINARY VACCINES AND ONE HEALTH

1. Efficacy, Safety, and Economic Feasibility of Immunocontraceptive Vaccines To be adopted for use, mucosal immunocontraceptive vaccines must be effective in the target animals and have limited or no effect on nontarget animals which may include humans who consume the meat, eggs, or milk from the targeted species. For zoo animals, such as captive African and Asian elephants, altering sex hormone levels in male or females may be advantageous in that they reduce aggression during musth season, but the alterations can also interfere with dominance hierarchy in a herd, which may not be advantageous [59,60]. Further, use of oral bait delivery systems should, if possible, be designed in such a way as to reduce consumption by unintended target species. The economic practicality of vaccine development such as costs associated with manufacturing and licensing as well as costs associated with treatment, including labor, equipment, and population dynamics, will all determine whether controlling fertility with a vaccine is an effective means to control a population. For instance, research shows that an annual control campaign using baits to sterilize female foxes would reduce the red fox population density by about 30%, but an annual campaign of poisoning would reduce fox density by about 80%. Vaccination would, of course, be the better choice when animal welfare issues are taken into consideration, although it is a less effective means to control population growth [61]. Whether a population can be controlled by immunocontraception or culling depends on the species and the availability of an effective mucosal vaccine and mucosal delivery system.

F. Mucosal Vaccines to Improve Fertility In addition to contraception, immunization against select targets may be used to improve fertility. Active immunization of cows against inhibin, a protein whose major action is negative feedback regulation of pituitary follicle-

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stimulating hormone (FSH) secretion, via the subcutaneous route neutralized endogenous inhibin levels, which resulted in increased FSH secretions during the estrous cycle. The immunized cows had a greater number of follicular waves and a greater number of follicles during the estrous cycle, which could be used as a potential source of oocytes for use in in vitro fertilization and embryo transfer programs [62]. Advances have been made with mucosally delivered inhibin vaccines. In buffalo, nasal immunization with a DNA vaccine coding for inhibin and delivered by attenuated Salmonella Cholerasuis has been shown to improve follicle development and fertility [63]. However, the buffalo in this trial underwent estrous synchronization, which would not be feasible in the wild and will have to be investigated further to establish its feasibility as a fertility vaccine. While immunization against select targets may affect fertility, care should be taken that targeting a natural protein may affect other pathways that are important for the animal’s health. Further, should ovulation rates be affected, it must be established that an increased number of dams do not suffer from complications associated with multiple offspring per parturition before it is known that the vaccine is safe to use.

III. VETERINARY VACCINES AND ONE HEALTH Veterinary vaccines can also be used to reduce food-borne illness by targeting bacteria that cause no illness or only mild illness in animals, but that can be harmful to humans upon consumption. For example, chicks orally immunized on the first day of life then boosted orally or via the intramuscular route at 6 and 16 weeks of age with a novel attenuated Salmonella Enteridis vaccine candidate, showed significantly higher plasma IgG and intestinal SIgA levels as compared to those in the control group

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[64]. The lymphocyte proliferation response and CD451 CD31 T cell number in the peripheral blood of the vaccinated groups were significantly increased. When the birds were challenged intravenously with the virulent S. enteritidis strain in the 24th week, the egg contamination rates were significantly reduced in both vaccinated groups relative to the controls, but total protection was not achieved. These results indicate that this vaccine may reduce incidences of egg contamination and therefore reduce the risk of human salmonellosis. While rare, Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) can have serious consequences in the young and in the aged human population, including hemorrhagic colitis, renal failure, and death. Cattle are widely recognized as an important reservoir of STEC O157 for human exposure, making contaminated beef a potential source of food-borne infection. Parenteral vaccination with a combination of antigens associated with type III secretion system-mediated adherence results in significantly reduced shedding in orally infected animals [65]. As yet, no mucosal vaccines have been developed that significantly reduce colonization in cattle, potentially because of poor cross-protection across STEC strains. Veterinary mucosal vaccines that protect humans from food-borne infectious diseases have tremendous One Health implications. We anticipate that the number of these vaccines will continue to grow in the future.

IV. CONCLUDING REMARKS As the examples in this chapter demonstrate, mucosal vaccines are part of routine immunization practices in veterinary medicine and have been for many years. Research is underway to further improve those vaccines, be it through the use of novel adjuvants, better delivery systems, or effective targeting to the site of uptake at mucosal surfaces. However, it is important to

note that most of these vaccines are extremely cost-effective, at pennies per dose, and are used as part of mass vaccination in poultry and fish. Thus one would hope that human vaccine manufacturers and regulators recognize the benefits and potential this technology can offer and start to develop mucosal vaccines for humans at a cost-effective price. While some vaccines are already available for mucosal administration in humans, mucosal vaccination has, unfortunately, not yet become part of routine immunization practices in humans.

References [1] Strobel S, Mowat AM. Immune responses to dietary antigens: oral tolerance. Immunol Today 1998;19: 173 81. [2] Faria AMC, Weiner HL. Oral tolerance. Immunol Rev 2005;206:232 59. [3] Zakhartchouk AN, Pyne C, Mutwiri GK, Papp Z, BacaEstrada ME, Griebel P, et al. Mucosal immunization of calves with recombinant bovine adenovirus-3: induction of protective immunity to bovine herpesvirus-1. J Gen Virol 1999;80(Pt 5):1263 9. [4] Minke JM, El-Hage CM, Tazawa P, Homer D, Lemaitre L, Cozette V, et al. Evaluation of the response to an accelerated immunisation schedule using a canarypoxvectored equine influenza vaccine, shortened interdose intervals and vaccination of young foals. Aust Vet J 2011;89(Suppl. 1):137 9. [5] Kimman TG, Westenbrink F, Straver PJ. Priming for local and systemic antibody memory responses to bovine respiratory syncytial virus: effect of amount of virus, virus replication, route of administration and maternal antibodies. Vet Immunol Immunopathol 1989;22:145 60. [6] Mettenleiter TC, Lukacs N, Rziha HJ. Pseudorabies virus avirulent strains fail to express a major glycoprotein. J Virol 1985;56:307 11. [7] Quint W, Gielkens A, Van Oirschot J, Berns A, Cuypers HT. Construction and characterization of deletion mutants of pseudorabies virus: a new generation of ‘live’ vaccines. J Gen Virol 1987;68(Pt 2):523 34. [8] van Oirschot JT, Rziha HJ, Moonen PJ, Pol JM, van Zaane D. Differentiation of serum antibodies from pigs vaccinated or infected with Aujeszky’s disease virus by a competitive enzyme immunoassay. J Gen Virol 1986;67(Pt 6):1179 82.

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

827

REFERENCES

[9] Health, O. W. O. f. A. Manual of diagnostic tests and vaccines for terrestrial animals. Paris: OIE; 2017. [10] Zhang Y, Zhang X, Liao X, Huang X, Cao S, Wen X, et al. Construction of a bivalent DNA vaccine coexpressing S genes of transmissible gastroenteritis virus and porcine epidemic diarrhea virus delivered by attenuated Salmonella Typhimurium. Virus Genes 2016;52:354 64. [11] Lawrence WE. Ovine brucellosis: a review of the disease in sheep manifested by epididymitis and abortion. Br Vet J 1961;117:435 47. [12] Jimenez de Bagues MP, Marin CM, Barberan M, Blasco JM. Responses of ewes to B. melitensis Rev1 vaccine administered by subcutaneous or conjunctival routes at different stages of pregnancy. Ann Rech Vet 1989;20:205 13. [13] Laplagne DA, Zylberman V, Ainciart N, Steward MW, Sciutto E, Fossati CA, et al. Engineering of a polymeric bacterial protein as a scaffold for the multiple display of peptides. Proteins 2004;57:820 8. [14] Diaz AG, Quinteros DA, Gutierrez SE, Rivero MA, Palma SD, Allemandi DA, et al. Immune response induced by conjunctival immunization with polymeric antigen BLSOmp31 using a thermoresponsive and mucoadhesive in situ gel as vaccine delivery system for prevention of ovine brucellosis. Vet Immunol Immunopathol 2016;178:50 6. [15] Mars MH, de Jong MC, Franken P, van Oirschot JT. Efficacy of a live glycoprotein E-negative bovine herpesvirus 1 vaccine in cattle in the field. Vaccine 2001;19:1924 30. [16] Bosch JC, De Jong MC, Franken P, Frankena K, Hage JJ, Kaashoek MJ, et al. An inactivated gE-negative marker vaccine and an experimental gD-subunit vaccine reduce the incidence of bovine herpesvirus 1 infections in the field. Vaccine 1998;16:265 71. [17] Chowdhury SI, Wei H, Weiss M, Pannhorst K, Paulsen DB. A triple gene mutant of BoHV-1 administered intranasally is significantly more efficacious than a BoHV-1 glycoprotein E-deleted virus against a virulent BoHV-1 challenge. Vaccine 2014;32:4909 15. [18] Myint A, Jones TO, Nyunt HH. Safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine. Vet Rec 2005;156:41 5. [19] Verma R, Jaiswal TN. Haemorrhagic septicaemia vaccines. Vaccine 1998;16:1184 92. [20] Myint A, Carter GR. Field use of live haemorrhagic septicaemia vaccine. Vet Rec 1990;126:648. [21] Rafidah O, Zamri-Saad M, Shahirudin S, Nasip E. Efficacy of intranasal vaccination of field buffaloes against haemorrhagic septicaemia with a live gdhA derivative Pasteurella multocida B:2. Vet Rec 2012;171:175. [22] Hill KL, Hunsaker BD, Townsend HG, van Drunen Littel-van den Hurk S, Griebel PJ. Mucosal immune

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

response in newborn Holstein calves that had maternally derived antibodies and were vaccinated with an intranasal multivalent modified-live virus vaccine. J Am Vet Med Assoc 2012;240:1231 40. Chiou C-J, Tseng L-P, Deng M-C, Jiang P-R, Tasi S-L, Chung T-W, et al. Mucoadhesive liposomes for intranasal immunization with an avian influenza virus vaccine in chickens. Biomaterials 2009;30:5862 8. Yang W-T, Yang G-L, Shi S-H, Liu Y-Y, Huang H-B, Jiang Y-L, et al. Protection of chickens against H9N2 avian influenza virus challenge with recombinant Lactobacillus plantarum expressing conserved antigens. Appl Microbiol Biotechnol 2017;101:4593 603. Ohlinger VF, Haas B, Meyers G, Weiland F, Thiel HJ. Identification and characterization of the virus causing rabbit hemorrhagic disease. J Virol 1990;64:3331 6. Bertagnoli S, Gelfi J, Petit F, Vautherot JF, Rasschaert D, Laurent S, et al. Protection of rabbits against rabbit viral haemorrhagic disease with a vaccinia-RHDV recombinant virus. Vaccine 1996;14:506 10. Barcena J, Morales M, Vazquez B, Boga JA, Parra F, Lucientes J, et al. Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus. J Virol 2000;74:1114 23. Martin-Alonso JM, Castanon S, Alonso P, Parra F, Ordas R. Oral immunization using tuber extracts from transgenic potato plants expressing rabbit hemorrhagic disease virus capsid protein. Transgenic Res 2003;12:127 30. Pasternak JA, Hamonic G, Forsberg N, Wheler C, Dyck MK, Wilson HL. Intrauterine delivery of subunit vaccines induces a systemic and mucosal immune response in rabbits. Am J Reprod Immunol 2017;78. Coler RN, Bhatia A, Maisonneuve J-F, Probst P, Barth B, Ovendale P, et al. Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis. FEMS Immunol Med Microbiol 2009;55:258 70. Desclozeaux M, Robbins A, Jelocnik M, Khan SA, Hanger J, Gerdts V, et al. Immunization of a wild koala population with a recombinant Chlamydia pecorum Major Outer Membrane Protein (MOMP) or Polymorphic Membrane Protein (PMP) based vaccine: new insights into immune response, protection and clearance. PLoS One 2017;12:e0178786. Boyer S, Miarinjara A, Elissa N. Xenopsylla cheopis (Siphonaptera: Pulicidae) susceptibility to Deltamethrin in Madagascar. PLoS One 2014;9:e111998. Rocke TE, Tripp DW, Russell RE, Abbott RC, Richgels KLD, Matchett MR, et al. Sylvatic plague vaccine partially protects prairie dogs (Cynomys spp.) in field trials. Ecohealth 2017;14:438 50.

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

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48. MUCOSAL VACCINE DEVELOPMENT FOR VETERINARY AND AQUATIC DISEASES

[34] Hampson K, Dushoff J, Cleaveland S, Haydon DT, Kaare M, Packer C, et al. Transmission dynamics and prospects for the elimination of canine rabies. PLoS Biol 2009;7:e53. [35] Pastoret PP, Brochier B. The development and use of a vaccinia-rabies recombinant oral vaccine for the control of wildlife rabies; a link between Jenner and Pasteur. Epidemiol Infect 1996;116:235 40. [36] Linhart SB, Wlodkowski JC, Kavanaugh DM, MotesKreimeyer L, Montoney AJ, Chipman RB, et al. A new flavor-coated sachet bait for delivering oral rabies vaccine to raccoons and coyotes. J Wildl Dis 2002;38:363 77. [37] Rosatte RC, Donovan D, Davies JC, Brown L, Allan M, von Zuben V, et al. High-density baiting with ONRAB rabies vaccine baits to control Arctic-variant rabies in striped skunks in Ontario, Canada. J Wildl Dis 2011;47:459 65. [38] Fehlner-Gardiner C, Rudd R, Donovan D, Slate D, Kempf L, Badcock J. Comparing ONRAB AND RABORAL V-RG oral rabies vaccine field performance in raccoons and striped skunks, New Brunswick, Canada, and Maine, USA. J Wildl Dis 2012;48:157 67. [39] Sommerset I, Krossoy B, Biering E, Frost P. Vaccines for fish in aquaculture. Expert Rev Vaccines 2005;4: 89 101. [40] Salinas I, Zhang Y-A, Sunyer JO. Mucosal immunoglobulins and B cells of teleost fish. Dev Comp Immunol 2011;35:1346 65. [41] St Louis-Cormier EA, Osterland CK, Anderson PD. Evidence for a cutaneous secretory immune system in rainbow trout (Salmo gairdneri). Dev Comp Immunol 1984;8:71 80. [42] Bernard D, Six A, Rigottier-Gois L, Messiaen S, Chilmonczyk S, Quillet E, et al. Phenotypic and functional similarity of gut intraepithelial and systemic T cells in a teleost fish. J Immunol 2006;176:3942 9. [43] Rombout JW, Blok LJ, Lamers CH, Egberts E. Immunization of carp (Cyprinus carpio) with a Vibrio anguillarum bacterin: indications for a common mucosal immune system. Dev Comp Immunol 1986;10:341 51. [44] Nakanishi T, Ototake M. Antigen uptake and immune responses after immersion vaccination. Dev Biol Stand 1997;90:59 68. [45] Klesius PH, Shoemaker CA. Development and use of modified live Edwardsiella ictaluri vaccine against Enteric septicemia of catfish. Adv Vet Med 1999;41: 523 37. [46] Nakanishi T, Kiryu I, Ototake M. Development of a new vaccine delivery method for fish: percutaneous administration by immersion with application of a multiple puncture instrument. Vaccine 2002;20:3764 9. [47] Irie T, Watarai S, Iwasaki T, Kodama H. Protection against experimental Aeromonas salmonicida infection in carp by

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

oral immunisation with bacterial antigen entrapped liposomes. Fish Shellfish Immunol 2005;18:235 42. Maurice S, Nussinovitch A, Jaffe N, Shoseyov O, Gertler A. Oral immunization of Carassius auratus with modified recombinant A-layer proteins entrapped in alginate beads. Vaccine 2004;23:450 9. Tobar JA, Jerez S, Caruffo M, Bravo C, Contreras F, Bucarey SA, et al. Oral vaccination of Atlantic salmon (Salmo salar) against salmonid rickettsial septicaemia. Vaccine 2011;29:2336 40. de las Heras AI, Rodriguez Saint-Jean S, Perez-Prieto SI. Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish. Fish Shellfish Immunol 2010;28:562 70. Villumsen KR, Neumann L, Ohtani M, Strom HK, Raida MK. Oral and anal vaccination confers full protection against enteric redmouth disease (ERM) in rainbow trout. PLoS One 2014;9:e93845. Salinas I, LaPatra SE, Erhardt EB. Nasal vaccination of young rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis and enteric red mouth disease. Dev Comp Immunol 2015;53:105 11. Doughty LS, Slater K, Zitzer H, Avent T, Thompson S. The impact of male contraception on dominance hierarchy and herd association patterns of African elephants (Loxodonta africana) in a fenced game reserve. Global Ecol Conserv 2014;2:88 96. Mahi-Brown CA, Yanagimachi R, Hoffman JC, Huang Jr. TT. Fertility control in the bitch by active immunization with porcine zonae pellucidae: use of different adjuvants and patterns of estradiol and progesterone levels in estrous cycles. Biol Reprod 1985;32:761 72. Wood DM, Liu C, Dunbar BS. Effect of alloimmunization and heteroimmunization with zonae pellucidae on fertility in rabbits. Biol Reprod 1981;25:439 50. Kerr PJ, Jackson RJ, Robinson AJ, Swan J, Silvers L, French N, et al. Infertility in female rabbits (Oryctolagus cuniculus) alloimmunized with the rabbit zona pellucida protein ZPB either as a purified recombinant protein or expressed by recombinant myxoma virus. Biol Reprod 1999;61:606 13. Cui X, Duckworth JA, Lubitz P, Molinia FC, Haller C, Lubitz W, et al. Humoral immune responses in brushtail possums (Trichosurus vulpecula) induced by bacterial ghosts expressing possum zona pellucida 3 protein. Vaccine 2010;28:4268 74. Walcher P, Cui X, Arrow JA, Scobie S, Molinia FC, Cowan PE, et al. Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula). Vaccine 2008;26: 6832 8. De Nys HM, Bertschinger HJ, Turkstra JA, Colenbrander B, Palme R, Human AM. Vaccination

VIII. CAN MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

REFERENCES

against GnRH may suppress aggressive behaviour and musth in African elephant (Loxodonta africana) bulls--a pilot study. J S Afr Vet Assoc 2010;81:8 15. [60] Lueders I, Hildebrandt TB, Gray C, Botha S, Rich P, Niemuller C. Supression of testicular function in a male Asian elephant (Elephas maximus) treated with gonadotropin-releasing hormone vaccines. J Zoo Wildl Med 2014;45:611 19. [61] McLeod SR, Saunders G. Fertility control is much less effective than lethal baiting for controlling foxes. Ecol Model 2014;273:1 10. [62] Medan MS, Takedom T, Aoyagi Y, Konishi M, Yazawa S, Watanabe G, et al. The effect of active immunization against inhibin on gonadotropin secretions and follicular dynamics during the estrous cycle in cows. J Reprod Dev 2006;52:107 13.

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[63] Liu Q, Rehman ZU, Liu JJ, Han L, Liu XR, Yang LG. Nasal immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis for improving ovarian responses and fertility in crossbred buffaloes. Reprod Domest Anim 2017;52: 189 94. [64] Nandre R, Matsuda K, Lee JH. Efficacy for a new live attenuated Salmonella Enteritidis vaccine candidate to reduce internal egg contamination. Zoonoses Public Health 2014;61:55 63. [65] McNeilly TN, Mitchell MC, Rosser T, McAteer S, Low JC, Smith DGE, et al. Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge. Vaccine 2010;28:1422 8.

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Mucosal Vaccine for Malaria Michelle Sue Jann Lee1 and Cevayir Coban1,2 1

Laboratory of Malaria Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Osaka, Japan 2Division of Malaria Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo (IMSUT), Tokyo, Japan

I. INTRODUCTION Although collective global efforts toward the eradication of malaria by insecticide-treated bed nets, indoor residual sprays, and antimalarial drugs have proven to be effective in recent years [1], it is alarming that the malaria eradication efforts might not be sustainable. The rising problems of multidrug resistance in parasites (including resistance to the frontline drug artemisinin [2]) and the lack of successful malaria vaccine candidates still put almost half of the world’s population at risk of malaria infection [3]. The RTS,S vaccine is thus far the most promising malaria vaccine candidate. In the vaccine’s name, the “R” stands for the central repeat region of circumsporozoite protein (CSP) of Plasmodium falciparum, the “T” stands for the T cell epitopes of CSP (circumsporozoite protein), and the “S” stands for hepatitis B surface antigen (HBsAg). A single fusion protein of RTS is expressed in yeast cells with free HBsAg. The RTS fusion protein and free S protein have been shown to spontaneously assemble into RTS,S particles. The RTS,S vaccine has

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00049-3

proceeded to a phase 3 clinical trial with the conferral of partial protection, but it has yet to attain sufficient efficacy [4]. The efforts to combat malaria are challenged by the complexity of Plasmodium parasite life cycle, which gives rise to distinct developmental stages that express variable antigens, as well as other survival strategies of the parasite, including antigenic polymorphism, antigenic diversion, epitope masking, and host immunosuppression [5]. Malaria infection begins with the injection of Plasmodium parasites in the form of sporozoites from the Anopheles mosquito salivary gland into the skin of the human host. Some of the sporozoites travel to the liver through the bloodstream to infect the liver hepatocytes. After exiting the cycle of liver-stage infection, the parasites are released into the bloodstream in the form of merozoites to begin the pathologic blood-stage infection. The merozoites rapidly invade the erythrocytes and differentiate into ring, trophozoite, and schizont stages by digesting the host hemoglobin for nutrient to grow and multiply. When the infected erythrocyte (iRBC) in the form of mature schizont

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ruptures, numerous merozoites are released to repeat the erythrocytic cycle by infecting other healthy erythrocytes. The erythrocytic life cycle of human Plasmodium parasites is 48 72 hours, depending on the species. Some iRBCs enter the bone marrow and differentiate into gametocytes before returning to the blood circulation to be taken up during another mosquito blood meal for transmission purposes [6]. The parasites continue to differentiate in the mosquito midgut to produce sporozoites to repeat their life cycle again. During the blood-stage infection, iRBCs are not only confined in the blood tissue but also can be sequestered in various tissues via the blood circulation and elicit a cascade of immune responses that can induce tissue-specific pathology depending on the local environment of the tissues [7 11]. Expression of adhesion molecules on iRBCs and endothelial activation mediated by inflammation assist the parasites to be sequestered and escape from immune attack while this can breach the tissue barrier integrity. It has recently been getting attention that Plasmodium parasite presence in different organs should be considered in a tissue-specific context [12]. The mucosal immune system constitutes the largest component of the body’s immune tissues, owing to the large surface area of the mucosa, which is constantly exposed to the environment and the microbiome (see also Chapter 2: Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance and Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity). The intestine is lined with epithelial layer that is folded into villi, which consist of an extensive capillary network and lymphatics for the absorption of nutrients (Chapter 3: Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells). Thus there is a possibility that Plasmodium parasites and infection-related events may disrupt the vascular endothelium in the villi and lead to the activation of immune effector

cells scattered in the intestinal epithelium and lamina propria, and this may contribute to the dysregulation of gut homeostasis. This chapter will review the recent evidence that Plasmodium parasites may have an ability to modify the gut environment and vice versa. We will also discuss possible mucosal vaccination strategies against malaria in the light of recent evidence.

II. MALARIA’S EFFECT ON THE GASTROINTESTINAL SYSTEM Gastrointestinal symptoms such as abdominal pain, nausea, vomiting, and diarrhea are common during malaria infection. Impaired intestinal function with increased permeability in gastric and intestinal mucosa and intestinal damage have been noticed in humans with P. falciparum [13,14]. Endoscopic analysis and biopsies of the gastrointestinal system (GIS) have revealed that mucosal edema, superficial bleeding, microthrombosis, gastric atrophy, and intestinal metaplasia are common and may lead to ischemic changes resulting in acute gastric symptoms with both P. falciparum and Plasmodium vivax infections [15]. Recent autopsy studies of pediatric patients who died from severe malaria have clearly confirmed these early findings that infected erythrocytes sequester themselves in the entire GIS (including the stomach, jejunum, ileum, and colon) [7,16] and may cause GIS bleeding and excretion of Plasmodium DNA in feces [17]. It seems that both severe and uncomplicated malaria infections cause GIS symptoms but with varied disease durations in which severe malaria causes longer and severe symptoms, leading to major changes in the gastrointestinal tract. The recent mouse study using a severe Plasmodium berghei cerebral malaria model has shown that the shortening of the villi, bleeding in the small intestine, and sequestration of red blood cells to blood vessels are obvious and might be causing dysbiosis [18]. The V4 16S rRNA sequencing of fecal samples from

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III. GASTROINTESTINAL SYSTEM’S EFFECT ON MALARIA: A ROLE FOR GUT MICROBIATA?

infected mice concluded that Bacteroidetes and Firmicutes (Lactobacillaceae), the major components of the mouse intestinal microbiota before infection, were reduced and led to an increase in Proteobacteria (Enterobacteriaceae) and Verrucomicrobia, which are usually less represented. There are consequences of malaria-induced changes in the GIS. Malaria and invasive intestinal pathogens such as Salmonella [19 24] or helminthes [25,26] are frequently copresent in malaria-endemic regions, increasing the disease severity [27,28]. These unwanted outcomes might be due to immune cell alterations in the GIS caused by malaria infection, in addition to sequestration of infected erythrocytes into blood vessels in the gut tissue. It has been shown that an increase in gut mastocytosis during malaria infection may cause increases in ileal and plasma histamine levels and/or cytokine alterations such as interleukin 10, which might be directly associated with increased gut permeability to invasive bacterial infections [24,27,28]. The release of toxic heme due to malaria-induced hemolysis may also cause release of immature neutrophils into circulation that lack reactive oxygen species activity [21,29], although neutrophils are initially fully competent against systemic infection with Plasmodium [30]. Therefore exhausted immature neutrophils deficient in killing invasive Salmonella infections, probably initially in the gut, may allow them to proliferate inside neutrophils, in both mice and humans (Fig. 49.1).

III. GASTROINTESTINAL SYSTEM’S EFFECT ON MALARIA: A ROLE FOR GUT MICROBIATA? Growing evidence indicates the important effects of a “healthy” gut microbiota against several diseases, including malaria [31 33] (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunit). A study using microbiome analysis from the stools of

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children and adults in Malawi by 16S rRNA gene sequencing before and during malaria season has suggested an increased gut microbial diversity with age in this malaria-endemic setting [33]. A significant association between the microbiota composition before the malaria season and the risk of P. falciparum infection was found; this association was abolished once the blood-stage infection was established. Microbiota of people with a low risk of malaria contained a significantly higher proportion of Bifidobacterium, Streptococcus, Escherichia, and Lactobacillales [33], suggesting the beneficial effects of these microbes in the gut against malaria. The following mouse study further supported the hypothesis of the role of microbiota on malaria susceptibility by using genetically similar mice from different vendors with different gut microbiomes [32]. The mice with a cecal microbiome (by V4 16S rRNA sequencing) rich in Firmicutes phylum (which includes Clostridiaceae, Erysipelotrichaceae, Lactobacillaceae, and Peptostreptococcaceae) were resistant to Plasmodium infection, while the mice with a microbiome rich in Bacteroidetes phylum (which includes Bacteroidaeae and Prevotellaceae) and Proteobacteria (which includes Sutterellaceae) phylum were susceptible to Plasmodium infections. Hence it was concluded that the gut microbiome influences the parasite burden and severity of several mouse Plasmodium infections. To directly evaluate the role of Lactobacillus and Bifidobacterium in resistance to severe malaria, the malariasusceptible mice were treated with laboratorycultured yogurt supplemented with these probiotics, and a reducing effect on parasitemia was found [32]. Therefore this study clearly suggested a role for diet in shaping the composition of gut microbiota to fight against malaria, a nongut infection, via changing the immune responses of the gut (Fig. 49.1). However, the mechanism of microbiota regulation of the host immunity against malaria infection is not fully understood. Numerous microbial metabolites have been suggested to have various

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

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49. MUCOSAL VACCINE FOR MALARIA

FIGURE 49.1 Bidirectional interaction between the gut microbiota and Plasmodium parasites. Parasite sequestration, inflammation, and resultant bleeding due to malaria infection may cause major changes in the gut microbiota composition. For example, the increase in gut mastocytosis during malaria infection may cause increase in ileal and plasma histamine levels and/or cytokine alterations. In addition, the release of toxic heme due to malaria-induced hemolysis may cause immature neutrophil release into circulation, causing exhausted immature neutrophils deficient in killing invasive Salmonella. These changes may lead to dysbiosis and promote the invasion of other intestinal pathogens. On the other hand, gut microbiota may contribute to malaria susceptibility, directly or indirectly. The specific antibodies to some gut pathobionts and/or their metabolites may cross-react with sporozoites and therefore impair transmission of the parasite from mosquitoes to mammalian host.

biological roles in metabolic signaling, leukocyte migration, inflammatory responses, expression of adhesion molecules and the immune effector functions [34,35]. It is possible that the alteration of gut microbiota composition may be controlling the microbial metabolite productions, thus directly or indirectly influencing host immunity. Therefore these possible microbial metabolites need to be further investigated in response to malaria infection. One noteworthy study by Yilmaz et al. has reported that there are immunoglobulin M

(IgM) type anti-α-gal antibodies against the gut pathobiont Escherichia coli O86:B7 in serum [36] which cross-react with α-gal-expressing Plasmodium sporozoites and therefore impair transmission of the parasite from mosquitoes to mammalian host, although this cross-reactive immunity did not affect the blood-stage parasite burden [36]. Accordingly, both E. coli O86:B7 in gut and Plasmodium sporozoites exhibit glycan α-gal on their surfaces. Therefore humans could generate anti-α-gal antibodies, both IgM and IgG types if they were infected by these

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

IV. MUCOSAL VACCINES AGAINST MALARIA

pathogens [37,38], which eventually could destroy the sporozoites via complementmediated events [36]. There was an association between anti-α gal IgM levels and protection from P. falciparum infection in Malian children and adults, suggesting the gut microbiome’s modulatory effects on Plasmodium infections in mice and humans. Notably, when α-gal is used for immunizations, it induces IgM and IgG responses and has a protective role against sporozoites after a mosquito bite, suggesting its possible candidacy for mucosal vaccinations (vaccination via oral route to produce anti-α-gal antibodies) against malaria (Fig. 49.1) (Chapter 4: Protective Activities of Mucosal Antibodies). In addition, the low-risk population in Malawi had a substantial proportion of Escherichia in the stool that might be contributing to the protection, although the expression of α-gal was not examined in that study [33]. Therefore there is a great potential to use α-gal-producing gut bacteria as probiotics to protect from malaria infection.

IV. MUCOSAL VACCINES AGAINST MALARIA Because the mucosa is the largest entry route for several pathogens into the human body, vaccines that can provide protection at these sites against pathogens are needed [39,40]. Mucosal vaccines have several advantages, such as the capability of inducing protective immunity locally and systemically with lower cost (less purity is needed, owing to mucosal administration), needle-free delivery, and thus easy mass immunization benefits, especially during pandemics [41]. Moreover, mucosal immunizations have a potential to induce good immunogenicity, owing to having the ability to target larger surface areas with higher vascularity and easy accessibility to lymphoid tissues. Developing vaccines against malaria has been a great challenge, and a systemic subunit protein vaccination strategies has mainly been

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used [42 44]. The strain-specificity and the polymorphisms of antigens, the short duration of antibodies, and lack of good adjuvants to improve immunogenicity are a few drawbacks of the current strategy of malaria vaccine development. Therefore several strategies, including mucosal immunizations, have been tried, although with questions as to whether this type of immunization is likely to be beneficial against a parasite with a systemic life in erythrocytes, not invading mucosa at all. Early studies in mice showed that intranasal immunization with recombinant Plasmodium yoelii MSP119 and oral immunization with recombinant MSP4 or PyMSP4/5 in the presence of cholera toxin subunit B could induce antibodies (with targeting multiple epitopes) and a protective immunity to blood-stage malaria infection [45,46]. Another study using TLR5 ligand flagellin-conjugated sporozoite antigens showed that intranasal immunization gave rise to antibody-mediated protection comparable to that of parenteral immunization [47,48] and antibodies to antigens for blocking the transmission of malaria in mosquitoes [49 51]. Later studies have improved the production of Plasmodium antigens in plants [52 56], bacterial outer membrane vesicles [57], or wheat-germ free system [58] to be used for mucosal vaccination against malaria. Another approach was the intranasal or oral immunization with live attenuated Salmonella expressing Plasmodium antigens [59 61], which showed promising results in mice (Table 49.1). The key question with mucosal immunization against several pathogens, including malaria, is why and how the mucosal vaccination is successful. To address this question, a recent study investigated the interaction of malaria antigen (a flagellin-modified CS construct) with nasopharynx-associated lymphoid tissue (NALT) [48]. NALT is a type of mucosaassociated lymphoid tissue without afferent lymphatics, and antigen is directly transported into it via dendritic cells (DCs) (Chapter 2:

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

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49. MUCOSAL VACCINE FOR MALARIA

Summary of Mucosal Vaccines and Interventions for Malaria

Antigen type

Antigen

Recombinant proteins (with/without cholera toxin or TLR ligands)

Plasmodium yoelii MSP119

Live attenuated Salmonella expressing Plasmodium antigens

Plant-made Plasmodium antigens

Mucosal route Intranasal and/or oral

MSP4 or PyMSP4/5

Outcome

Reference

Antibodies, protection against infection challenge, transmission blocking from mammalian host to mosquitoes

[45] [46]

PvMSP-119/PfCS with flagellin

[47,48]

Pfs25

[49]

Pys25

[50]

Pvs25

[51]

Plasmodium berghei CS

Oral and/or intranasal

Antigen-specific cell-mediated immunity and/or against infection challenge

[59]

P. berghei MSP-1

[60]

Plasmodium falciparum MSP-1

[61]

P. falciparum AMA-1 and MSP-1

Oral

High antibody titers and/or Th1 responses

[52]

PyMSP4/5

[53,54]

PvMSP-1 and PvCSP-1

[55]

P. yoelii MSP119

[56]

Bacterial outer-membrane vesicles antigens

AnAPN1 and Pfs48/45

None-Probiotics (rich in Lactobacillus and Bifidobacterium)

Whole parasite

None-Escherichia coli 086: B7 gut colonization

α-Gal on sporozoite

Intranasal

High titers and transmission blocking

[57]

Oral

Lowered parasite burden

[32]

Oral

Sterile immunity after mosquito bite

[36]

Anatomical Uniqueness of the Mucosal Immune System (GALT, NALT, iBALT) for the Induction and Regulation of Mucosal Immunity and Tolerance). The study demonstrated the influx of CD11c1 DCs into the NALT (in which DCs may transfer malarial antigen and/or present antigen to T and B cells) and the expansion of predominantly B cells and, to a lesser extent, T lymphocytes after intranasal immunization. These accumulated data provide a new platform

for the development of suitable antigens as well as adjuvant formulations targeting mucosal surfaces [62,63].

V. CONCLUDING REMARKS This chapter has reviewed the dual interaction between Plasmodium parasites and the microbiota layered throughout the GIS. In this

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REFERENCES

chapter, we have emphasized that the presence of Plasmodium parasites, although it is a systemic infection in the circulation not a mucosal infection, could affect the gut microbiome and vice versa via the modulation of immune system rather than the direct effect on parasites. The chapter has also summarized malaria vaccine trials to date that have used mucosal route of immunization. However, it should be kept in mind that many pathogens, including bacteria, viruses, and parasites, are found in resourcelimited settings with no clean water and malnutrition where the mucosal surfaces are more exposed to dangerous pathogens and may have more disruption of mucosal barriers [64]. Therefore there is a great need to address the interaction between pathogens and host mucosal immunity to develop mucosal vaccines against malaria with consideration of other coinfections and conditions in the endemic settings.

Acknowledgments We thank Malaria Immunology Lab members for their valuable inputs. The authors are supported by Grants-in-Aid for Scientific Research and the Japan Agency for Medical Research and Development AMED J-PRIDE (KAKENHI Kiban-B grant no. 16H05181 and 17fm0208021h0001, respectively).

References [1] Murray CJL, Ortblad KF, Guinovart C, et al. Global, regional, and national incidence and mortality for HIV, tuberculosis, and malaria during 1990 2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2014;384(9947):1005 70. Available from: https://doi.org/10.1016/S0140-6736(14)60844-8. [2] Blasco B, Leroy D, Fidock DA. Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic. Nat Med 2017;23(8):917 28. Available from: https://doi.org/10.1038/nm.4381. [3] World Health Organization. World Malaria Report 2015. World Health. 2015:243. doi:ISBN 978 92 4 1564403. [4] Moreno A, Joyner C. Malaria vaccine clinical trials: what’s on the horizon. Curr Opin Immunol 2015;35: 98 106. Available from: https://doi.org/10.1016/j.coi. 2015.06.008.

837

[5] Re´nia L, Goh YS. Malaria parasites: the great escape. Front Immunol 2016;7:1 14. Available from: https:// doi.org/10.3389/fimmu.2016.00463. [6] Joice R, Nilsson SK, Montgomery J, et al. Plasmodium falciparum transmission stages accumulate in the human bone marrow. Sci Transl Med 2014;6(244) 244re5. [7] Milner DA, Lee JJ, Frantzreb C, et al. Quantitative assessment of multiorgan sequestration of parasites in fatal pediatric cerebral malaria. J Infect Dis 2015;212 (8):1317 21. Available from: https://doi.org/10.1093/ infdis/jiv205. [8] Lee MSJ, Maruyama K, Fujita Y, et al. Plasmodium products persist in the bone marrow and promote chronic bone loss. Sci Immunol 2017. Available from: https://doi.org/10.1126/sciimmunol.aam8093. [9] Zhao H, Aoshi T, Kawai S, et al. Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria. Cell Host Microbe 2014;15(5). Available from: https://doi.org/10.1016/j.chom.2014.04.008. [10] Ehrich JHH, Eke FU. Malaria-induced renal damage: facts and myths. Pediatr Nephrol 2007;22(5):626 37. Available from: https://doi.org/10.1007/s00467-0060332-y. [11] Milner D, Factor R, Whitten R, et al. Pulmonary pathology in pediatric cerebral malaria. Hum Pathol 2013;44 (12):2719 26. Available from: https://doi.org/ 10.1016/j.humpath.2013.07.018. [12] Coban C, Lee MSJ, Ishii KJ. Tissue-specific immunopathology during malaria infection. Nat Rev Immunol 2018;18:266 78. Available from: https://doi. org/10.1038/nri.2017.138. [13] Molyneux ME, Looareesuwan S, Menzies IS, et al. Reduced hepatic blood flow and intestinal malabsorption in severe falciparum malaria. Am J Trop Med Hyg 1989;40(5):470 6. [14] Wilairatana P, Meddings JB, Ho M, Vannaphan S, Looareesuwan S. Increased gastrointestinal permeability in patients with Plasmodium falciparum malaria. Clin Infect Dis 1997;24(3):430 5. Available from: https:// doi.org/10.1093/clinids/24.3.430. [15] Romero A, Matos C, Gonzalez MM, Nunez N, Bermudez L, de Castro G. [Changes in gastric mucosa in acute malaria]. GEN 1993;47:123 8. [16] Seydel KB, Milner Jr. DA, Kamiza SB, Molyneux ME, Taylor TE. The distribution and intensity of parasite sequestration in comatose Malawian children. J Infect Dis 2006;194(2):208 15. Available from: https://doi. org/10.1086/505078. [17] Abkallo HM, Liu W, Hokama S, et al. DNA from preerythrocytic stage malaria parasites is detectable by PCR in the faeces and blood of hosts. Int J Parasitol 2014;44(7):467 73. Available from: https://doi.org/ 10.1016/j.ijpara.2014.03.002.

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

838

49. MUCOSAL VACCINE FOR MALARIA

[18] Taniguchi T, Miyauchi E, Nakamura S, et al. Plasmodium berghei ANKA causes intestinal malaria associated with dysbiosis. Sci Rep 2015;5:15699. Available from: https://doi.org/10.1038/srep15699. [19] Feasey NA, Dougan G, Kingsley RA, Heyderman RS, Gordon MA. Invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in Africa. Lancet 2012;379(9835):2489 99. Available from: https://doi.org/10.1016/S0140-6736(11)61752-2. [20] Mabey DCW, Brown A, Greenwood BM. Plasmodium falciparum malaria and Salmonella infections in Gambian children. J Infect Dis 1987;155(6):1319 21. Available from: https://doi.org/10.1093/infdis/155.6.1319. [21] Cunnington AJA, de Souza JB, Walther M, Riley EEM. Malaria impairs resistance to Salmonella through hemeand heme oxygenase dependent dysfunctional granulocyte mobilization. Nat Med 2011;18(1):120 7. Available from: https://doi.org/10.1038/nm.2601. [22] Scott JAG, Berkley J a, Mwangi I, et al. Relation between falciparum malaria and bacteraemia in Kenyan children: a population-based, case-control study and a longitudinal study. Lancet 2011;378 (9799):1316 23. Available from: https://doi.org/ 10.1016/S0140-6736(11)60888-X. [23] Roux CM, Butler BP, Chau JY, et al. Both hemolytic anemia and malaria parasite-specific factors increase susceptibility to Nontyphoidal Salmonella enterica serovar typhimurium infection in mice. Infect Immun 2010;78(4):1520 7. Available from: https://doi.org/ 10.1128/iai.00887-09. [24] Potts RA, Tiffany CM, Pakpour N, et al. Mast cells and histamine alter intestinal permeability during malaria parasite infection. Immunobiology 2016;221:468 74. [25] Adegnika A a, Kremsner PG. Epidemiology of malaria and helminth interaction: a review from 2001 to 2011. Curr Opin HIV AIDS 2012;7(3):221 4. Available from: https://doi.org/10.1097/COH.0b013e3283524d90. [26] Tetsutani K, Ishiwata K, Ishida H, et al. Concurrent infection with Heligmosomoides polygyrus suppresses anti-Plasmodium yoelii protection partially by induction of CD4 1 CD25 1 Foxp3 1 Treg in mice. Eur J Immunol 2009;39(10):2822 30. Available from: https://doi.org/ 10.1002/eji.200939433. [27] Lokken KL, Mooney JP, Butler BP, et al. Malaria parasite infection compromises control of concurrent systemic non-typhoidal Salmonella infection via IL-10mediated alteration of myeloid cell function. PLoS Pathog 2014;10(5). Available from: https://doi.org/ 10.1371/journal.ppat.1004049. [28] Mooney JP, Butler BP, Lokken KL, et al. The mucosal inflammatory response to non-typhoidal Salmonella in the intestine is blunted by IL-10 during concurrent malaria parasite infection. Mucosal Immunol 2014;7(6):

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

1302 11. Available from: https://doi.org/10.1038/ mi.2014.18. Cunnington AJ, Njie M, Correa S, Takem EN, Riley EM, Walther M. Prolonged neutrophil dysfunction after Plasmodium falciparum malaria is related to hemolysis and heme oxygenase-1 induction. J Immunol 2012;189(11):5336 46. Available from: https://doi. org/10.4049/jimmunol.1201028. Zhao H, Konishi A, Fujita Y, et al. Lipocalin 2 bolsters innate and adaptive immune responses to blood-stage malaria infection by reinforcing host iron metabolism. Cell Host Microbe 2012;12(5):705 16. Available from: https://doi.org/10.1016/j.chom.2012.10.010. Levy M, Kolodziejczyk AA, Thaiss CA, Elinav E. Dysbiosis and the immune system. Nat Rev Immunol 2017;17(4):219 32. Available from: https://doi.org/ 10.1038/nri.2017.7. Villarino NF, LeCleir GR, Denny JE, et al. Composition of the gut microbiota modulates the severity of malaria. Proc Natl Acad Sci 2016;113(8):2235 40. Available from: https://doi.org/10.1073/pnas.1504887113. Yooseph S, Kirkness EF, Tran TM, et al. Stool microbiota composition is associated with the prospective risk of Plasmodium falciparum infection. BMC Genomics 2015;16(1):631. Available from: https://doi.org/ 10.1186/s12864-015-1819-3. Nicholson JK, Holmes E, Kinross J, et al. Host-gut microbiota metabolic interactions. Science 2012;336 (6086):1262 8. Vinolo MAR, Rodrigues HG, Nachbar RT, Curi R. Regulation of inflammation by short chain fatty acids. Nutrients 2011;858 76. Available from: https://doi. org/10.3390/nu3100858. Yilmaz B, Portugal S, Tran TM, et al. Gut microbiota elicits a protective immune response against malaria transmission. Cell 2014;159(6):1277 89. Available from: https://doi.org/10.1016/j.cell.2014.10.053. Macher BA, Galili U. The Galalpha1,3Galbeta1, 4GlcNAc-R (alpha-Gal) epitope: a carbohydrate of unique evolution and clinical relevance. Biochim Biophys Acta 2008;1780(2):75 88. Available from: https://doi.org/10.1016/j.bbagen.2007.11.003. Ngwa CJ, Pradel G. Coming soon: probiotics-based malaria vaccines. Trends Parasitol 2015;31(1):2 4. Available from: https://doi.org/10.1016/j.pt.2014.11.006. Lycke N. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 2012;12(8):592 605. Available from: https://doi.org/ 10.1038/nri3251. Azegami T, Yuki Y, Kiyono H. Challenges in mucosal vaccines for the control of infectious diseases. Int Immunol 2014;26(9):517 28. Available from: https:// doi.org/10.1093/intimm/dxu063.

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

REFERENCES

[41] Rhee JH, Lee SE, Kim SY. Mucosal vaccine adjuvants update. Clin Exp Vaccine Res 2012;1(1):50 63. Available from: https://doi.org/10.7774/cevr.2012.1.1.50. [42] Takashima E, Morita M, Tsuboi T. Vaccine candidates for malaria: what’s new? Expert Rev Vaccines 2016;15 (1):1 3. Available from: https://doi.org/10.1586/ 14760584.2016.1112744. [43] Kaslow DC, Biernaux S. RTS,S: toward a first landmark on the Malaria Vaccine Technology Roadmap. Vaccine 2015;33(52):7425 32. Available from: https://doi.org/ 10.1016/j.vaccine.2015.09.061. [44] Birkett AJ. Status of vaccine research and development of vaccines for malaria. Vaccine 2016;34(26):2915 20. Available from: https://doi.org/10.1016/j.vaccine. 2015.12.074. [45] Hirunpetcharat C, Stanisic D, Liu XQ, et al. Intranasal immunization with yeast-expressed 19 kD carboxylterminal fragment of Plasmodium yoelii merozoite surface protein-1 (yMSP119) induces protective immunity to blood stage malaria infection in mice. Parasite Immunol 1998;20(9):413 20. Available from: https:// doi.org/10.1046/j.1365-3024.1998.00161.x. [46] Wang L, Kedzierski L, Wesselingh SL, Coppel RL. Oral immunization with a recombinant malaria protein induces conformational antibodies and protects mice against lethal malaria. Infect Immun 2003;71 (5):2356 64. Available from: https://doi.org/10.1128/ IAI.71.5.2356-2364.2003. [47] Bargieri DY, Rosa DS, Braga CJM, et al. New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin. Vaccine 2008;26(48):6132 42. Available from: https://doi.org/ 10.1016/j.vaccine.2008.08.070. [48] Nacer A, Carapau D, Mitchell R, et al. Imaging murine NALT following intranasal immunization with flagellin-modified circumsporozoite protein malaria vaccines. Mucosal Immunol 2014;7(2):304 14. Available from: https://doi.org/10.1007/s12671-0130269-8.Moving. [49] Arakawa T, Komesu A, Otsuki H, et al. Nasal immunization with a malaria transmission-blocking vaccine candidate, Pfs25, induces complete protective immunity in mice against field isolates of Plasmodium falciparum. Infect Immun 2005;73(11):7375 80. Available from: https://doi.org/10.1128/IAI.73.11.7375-7380.2005. [50] Arakawa T, Tachibana M, Miyata T, et al. Malaria ookinete surface protein-based vaccination via the intranasal route completely blocks parasite transmission in both passive and active vaccination regimens in a rodent model of malaria infection. Infect Immun 2009;77(12):5496 500. Available from: https://doi. org/10.1128/IAI.00640-09.

839

[51] Miyata T, Harakuni T, Tsuboi T, et al. Plasmodium vivax ookinete surface protein Pvs25 linked to cholera toxin B subunit induces potent transmission-blocking immunity by intranasal as well as subcutaneous immunization. Infect Immun 2010;78(9):3773 82. Available from: https://doi.org/10.1128/IAI.00306-10. [52] Davoodi-Semiromi A, Schreiber M, Nalapalli S, et al. Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery. Plant Biotechnol J 2010;8(2):223 42. Available from: https://doi.org/10.1111/j.1467-7652.2009.00479.x. [53] Wang L, Webster DE, Campbell AE, Dry IB, Wesselingh SL, Coppel RL. Immunogenicity of Plasmodium yoelii merozoite surface protein 4/5 produced in transgenic plants. Int J Parasitol 2008;38 (1):103 10. Available from: https://doi.org/10.1016/j. ijpara.2007.06.005. [54] Webster DE, Wang L, Mulcair M, et al. Production and characterization of an orally immunogenic Plasmodium antigen in plants using a virus-based expression system. Plant Biotechnol J 2009;7(9):846 55. Available from: https://doi.org/10.1111/j.1467-7652.2009.00447.x. [55] Lee C, Kim H-H, Mi Choi K, et al. Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus. Malar J 2011;10(1):106. Available from: https://doi. org/10.1186/1475-2875-10-106. [56] Ma C, Wang L, Webster DE, Campbell AE, Coppel RL. Production, characterisation and immunogenicity of a plant-made Plasmodium antigen-the 19 kDa C-terminal fragment of Plasmodium yoelii merozoite surface protein 1. Appl Microbiol Biotechnol 2012;94(1):151 61. Available from: https://doi.org/10.1007/s00253-011-3772-7. [57] Pritsch M, Ben-Khaled N, Chaloupka M, et al. Comparison of intranasal outer membrane vesicles with cholera toxin and injected MF59C.1 as adjuvants for malaria transmission blocking antigens AnAPN1 and Pfs48/45. J Immunol Res 2016;2016. Available from: https://doi.org/10.1155/2016/3576028. [58] Ntege EH, Takashima E, Morita M, Nagaoka H, Ishino T, Tsuboi T. Blood-stage malaria vaccines: post-genome strategies for the identification of novel vaccine candidates. Expert Rev Vaccines 2017;1 11. Available from: https://doi.org/10.1080/14760584.2017.1341317. [59] Sadoff JC, Ballou WR, Baron LS, et al. Oral Salmonella typhimurium vaccine expressing circumsporozoite protein protects against malaria. Science 1988;240(4850):336 8. Available from: https://doi.org/10.1126/science.3281260. [60] Toebe CS, Clements JD, Cardenas L, Jennings GJ, Wiser MF. Evaluation of immunogenicity of an oral Salmonella vaccine expressing recombinant Plasmodium berghei merozoite surface protein-1. Am J Trop Med Hyg 1997;56(2):192 9.

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

840

49. MUCOSAL VACCINE FOR MALARIA

[61] Wu S, Beier M, Sztein MB, et al. Construction and immunogenicity in mice of attenuated Salmonella typhi expressing Plasmodium falciparum merozoite surface protein 1 (MSP-1) fused to tetanus toxin fragment C. J Biotechnol 2000;83(1 2):125 35. Available from: https://doi.org/10.1016/S0168-1656(00)00306-0. [62] Uraki R, Das SC, Hatta M, et al. Hemozoin as a novel adjuvant for inactivated whole virion influenza vaccine. Vaccine 2014;32(41):5295 300. Available from: https://doi.org/10.1016/j.vaccine.2014.07.079.

[63] Lee MSJ, Igari Y, Tsukui T, Ishii KJ, Coban C. Current status of synthetic hemozoin adjuvant: a preliminary safety evaluation. Vaccine 2016;34(18):2055 61. Available from: https://doi.org/10.1016/j.vaccine. 2016.02.064. [64] Glennie SJ, Williams NA, Heyderman RS. Mucosal immunity in resource-limited setting: is the battle ground different? Trends Microbiol 2010;18(11):487 93. Available from: https://doi.org/10.1016/j.tim.2010.08.002.

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Mucosal Vaccine for Parasitic Infections Hirotomo Kato Division of Medical Zoology, Department of Infection and Immunity, Jichi Medical University, Tochigi, Japan

I. INTRODUCTION Parasites are eukaryotic organisms that live in or on another organism, called the host. They obtain nutrients from the host and, in some cases, hitchhike from one host to another to develop into the next stage and complete their life cycle. Parasites are mainly classified into three groups: protozoans, helminths, and ectoparasites. Protozoans are unicellular organisms that live in the blood or tissue or inside cells in animals. Helminths are multicellular organisms with great diversity that can mostly be recognized by the naked eye. Parasitic helminths are further classified into trematodes (flukes), cestodes (tapeworms), and nematodes (roundworms). Ectoparasites parasitize on or in the skin of animals for the entire or part of their lives. Although one quarter of people in the world are estimated to be infected with helminths [1], many parasites are harmless when they parasitize the definitive hosts. However, some parasites cause severe manifestations in humans and animals. For example, malaria caused by the protozoan parasite Plasmodium spp. is the most life-threatening infectious

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00050-X

disease in the world, and a majority of neglected tropical diseases (NTDs), which are endemic in 149 countries and affect more than one billion people worldwide, are parasitic diseases [2]. In addition to prevention, the control of parasitic diseases has been carried out mainly by antiparasitic drugs that are cheap and effective against a wide spectrum of parasites. However, acquisition of drug resistance is a serious issue in various protozoan and helminth parasites [3]; therefore alternative ways to control parasitic diseases, such as vaccines, are needed. Although some vaccines against parasitic infections have been developed for poultry and other animals, no antiparasitic vaccine is available for human use [4,5]. In fact, development of effective vaccines against parasitic diseases is quite difficult, owing to the following unique characteristics of parasites, which are quite different from other pathogens such as bacteria and viruses: (1) eukaryote, (2) large body size, (3) complex life cycle including asexual and sexual reproduction, (4) morphological and antigenic diversity and changes in each developmental stage, and (5) various evasion mechanisms that have

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evolved against host immunity. An effective antiparasite host immune response is different depending on the parasite and is largely dependent on their habitat. Generally, cell-mediated immunity represented by type 1 T helper (Th1) cells is effective against intracellular protozoa [5,6]. On the other hand, Th2-mediated immune responses are important for exclusion of gastrointestinal helminths [7 10]. The main parasite infection routes are oral through contaminated water and food, and the skin by direct penetration or by arthropod transmission. A variety of parasites invade the gastrointestinal tract and/ or parasitize the gut and consequently interact with the mucosal immune system [5 10]. This chapter focuses on the recent advances in mucosal vaccines against parasitic diseases.

II. PROTOZOAN INFECTIONS Protozoa are microscopic unicellular organisms, most of which are less than 50 μm in diameter. Compared to helminths, the life cycle and morphological changes are relatively simple; however, most protozoa still possess several developmental stages, which accompany morphological and functional alterations in order to complete their life cycles. In addition, protozoa multiply in the host and employ a variety of antigenic changes to counteract host immunity. Orally transmitted protozoa, including species that are of medical and veterinary importance, inhabit and/or invade the host intestine; therefore the mucosa is the front line for interaction between parasites and the host immune system (Fig. 50.1). In addition to cellmediated immunity, secretory immunoglobulin A (SIgA) antibodies against parasite molecules associated with attachment and invasion are considered to be important for host protection. Although live attenuated oral vaccines have been widely used against coccidiosis in poultry for many years [4], to date there are no practical mucosal vaccine against orally transmitted

protozoa for humans use. Here, recent advances in the development of mucosal vaccines are discussed, with a focus on representative medically important protozoa that inhabit and/or invade through the gastrointestinal tract.

A. Amoebiasis Amoebiasis that causes colitis, bloody diarrhea, and/or amoebic liver abscesses is a worldwide health problem. It is more common in tropical areas with poor sanitation conditions, with an estimated 40,000 74,000 deaths annually [11]. The etiological agent is Entamoeba histolytica, which is transmitted by the fecal oral route via contaminated water and food or by person-to-person contact [11]. The life cycle of E. histolytica is relatively simple, consisting of two stages: cysts and trophozoites. Cysts are the infectious stage and have environmental resistance, whereas trophozoites are the motile and invasive stage associated with the disease [11]. Ingested cysts are excysted, and trophozoites are released in the small intestine. Trophozoites adhere to colonic mucin and epithelial cells and invade by destroying intestinal tissues (Fig. 50.1A). Gal/ GalNAc lectin, which binds to galactose (Gal) and N-acetylgalactosamine (GalNAc), was identified as the major surface adhesion molecule of E. histolytica essential for the adherence of the parasite to mucins and mucosal epithelial cells of the host [12,13]. Later studies showed that inhibition of E. histolytica Gal/GalNAc lectin prevented adherence and cytolysis of target cells in vitro [14]. In addition, a correlation between the presence of fecal SIgA antibodies to E. histolytica Gal/GalNAc lectin and resistance to infection was reported in humans [15,16]. Further, Gal/GalNAc lectin was associated with the virulence of E. histolytica directly by inhibiting expression using antisense RNA [17]. Therefore, E. histolytica Gal/GalNAc lectin

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FIGURE 50.1 The gastrointestinal tract is the site of inhabitation and/or invasion for protozoa. (A) Entamoeba histolytica. Ingested cysts are excysted, and trophozoites are released in the small intestine. Trophozoites adhere to colonic mucin and epithelial cells and invade by destroying intestinal tissues, resulting in development of colitis, bloody diarrhea, and/or amoebic liver abscess. (B) Giardia intestinalis. Ingested cysts are excysted, and released trophozoites attach to the small intestinal mucosa of the host and multiply on the epithelium surface. (C) Cryptosporidium hominis and Cryptosporidium parvum. Oocysts excyst in the small intestine, and sporozoites released from oocysts invade epithelial cells of the gastrointestinal tract. After invasion, parasites multiply and develop asexually and then sexually to other stages without invading deeper layers of the intestinal mucosa. (D) Toxoplasma gondii. Sporozoites and bradyzoites released from ingested oocysts and cysts, respectively, actively penetrate intestinal epithelial cells and differentiate into tachyzoites. Tachyzoites, the proliferative stage of the parasite, disseminate throughout the host, differentiate into bradyzoites, and form persistent tissue cysts, mainly in the brain and muscles. When a woman acquires initial infection with T. gondii during or just before pregnancy, the fetus may be infected through mother-to-child transmission.

is considered to be a promising target of mucosal vaccines that could induce mucosal SIgA antibodies to inhibit trophozoite attachment and invasion to the intestinal epithelium, as well as cell-mediated protective immunity in both systemic and mucosal sites [18]. Oral and nasal administration of purified Gal/GalNAc

lectin or recombinant subunits with an adjuvant such as cholera toxin (CT) provided significant protection against E. histolytica infection in experimental animals [19 24]. Although mucosal SIgA responses to Gal/GalNAc lectin are considered to be protective against infection [25], several studies have suggested that cell-

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mediated immunity elicited by mucosal immunization is sufficient for protection from intestinal amoebiasis, since adoptive transfer studies with vaccinated T cells conferred protection [19,20]. In these studies, interferon gamma (IFNγ) and interleukin 17 (IL-17) produced by CD41 and CD81 T cells, respectively, induced by mucosal vaccination played a central role in the protection against E. histolytica infection [19,20]. On the other hand, a protective effect against E. histolytica infection and subsequent colitis was induced by nasal vaccination with Gal/GalNAc lectin plus CT in baboons, a natural host of E. histolytica, and a strong inverse correlation between the fecal SIgA antibodies to Gal/GalNAc lectin and fecal E. histolytica DNA was detected in this model [26]. These results show that intestinal SIgA antibodies are protective against E. histolytica infection, although cell-mediated immunity may also be essential for the protection.

B. Giardiasis Giardiasis caused by zoonotic Giardia intestinalis (also called G. duodenalis or G. lamblia) is one of the most common intestinal protozoan diseases in the world, with approximately 200 million individuals infected [27,28]. Giardiasis is more common in developing countries with poor sanitation, but waterborne outbreaks occasionally occur in developed countries [27,28]. Clinical manifestations caused by G. intestinalis include asymptomatic, mild, and severe symptoms such as watery diarrhea, abdominal cramps, bloating, fatigue, and weight loss [27,28]. This parasite is typically transmitted by the fecal oral route via contaminated water and food or by person-to-person contact [27,28]. Like E. histolytica, the development consists of two stages: cysts and trophozoites. Cysts are the inactive, infectious form, having a strong resistance to the environment and chlorine disinfection; trophozoites excystated in the

stomach and duodenum are proliferative and adhere to the mucus and intestinal epithelium [27,28] (Fig. 50.1B). Different from E. histolytica, G. intestinalis is an extracellular organism that does not invade mucosal tissues [27,28]. It is known that Giardia-infected patients produce a large amount of mucosal SIgA antibodies and have elevated Th1-mediated IFNγ in the sera, while immunodeficient patients develop chronic giardiasis, suggesting that mucosal SIgA and cellular immunity correlate to protection against Giardia infection [29 31]. Protective immunity against Giardia infection was studied by using T-cell-depleted/deficient mice, and CD41 T cells but not CD81 T cells were found to be essential for the clearance of Giardia from the intestine [32,33]. In addition, recent studies suggest that Th17 cells are essential for protection [34,35]. On the other hand, the role of antibodies, especially mucosal SIgA antibodies, is still controversial. B-cell-deficient mice eliminated the majority of parasites between 1 and 2 weeks after Giardia infection, suggesting that the mucosal SIgA antibody response is not essential to control acute infection [33]. However, another study using IgAdeficient mice showed that mucosal SIgA antibodies played a central role in the clearance of Giardia [36]. The discrepancy may reflect different experimental designs and infectious burdens, and mucosal SIgA antibodies may be more important in the chronic phase of infection [28]. Development of oral vaccines inducing both a mucosal SIgA and cell-mediated immunity has been attempted, and a number of studies demonstrated the protective effect of two antigens: cyst wall protein-2 (CWP2) and trophozoite-specific ɑ1-giardin [37 43]. CWP2 is an important component of the cyst wall and plays an essential role in encystation [44]. ɑ1giardin is related to annexins associated with the plasma membrane and is suggested to play a key role in the parasite host interaction [45]. In several studies, an attenuated Salmonella Typhimurium approach was applied as a DNA

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vaccine delivery system [38,39]. Other than these two antigens, variant-specific surface proteins (VSPs), major surface proteins of trophozoites, were evaluated as targets for a mucosal vaccine, and high levels of protection against Giardia infection were reported in gerbils, cats, and dogs even with antigenic variations in VSPs [46,47]. Since G. intestinalis is a zoonotic parasite, vaccination of domestic animals will reduce the risk of human infection with this protozoon.

C. Cryptosporidiosis Human cryptosporidiosis, caused by Cryptosporidium hominis and Cryptosporidium parvum, is a common cause of severe diarrhea globally in young children and immunocompromised individuals [48,49]. The natural host of C. hominis is limited to humans, whereas C. parvum is zoonotic and infects a wide range of hosts, including domestic livestock animals [48,49]. Although cryptosporidiosis is more common in developing countries with poor sanitation, waterborne outbreaks occasionally occur in developed countries [48,49]. The most common symptom of cryptosporidiosis is watery diarrhea; other symptoms include stomach pain, dehydration, nausea, vomiting, fever, and weight loss [48,49]. The infection occurs following the ingestion of oocysts from contaminated water (drinking water, swimming pools, etc.) or food [48,49]. Oocysts excyst in the small intestine, and sporozoites released from oocysts invade the epithelial cells of the gastrointestinal tract [48,49]. After invasion, parasites multiply and develop asexually and then sexually into other stages such as trophozoites, merozoites, microgamonts, and macrogamonts. Finally, infectious oocysts are developed after fertilization of macrogametes by microgametes [48,49] (Fig. 50.1C). Autoinfection occurs during the course of development. Cryptosporidium parasitizes epithelial cells but does not invade deeper

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layers of the intestinal mucosa [48,49]. Protective immunity against Cryptosporidium has been studied, and CD41 T cells, but not CD81 T cells, were shown to play an important role in the clearance of the parasite [6,48,50]. This was supported by the observation that severe cryptosporidiosis occurs in AIDS patients with reduced CD41 T cell counts [6,48,50]. As for CD41 T cell-mediated immunity, the cellular immune responses and Th1 cell-mediated cytokines, such as IFNγ and IL-12, were shown to play an essential role in the control of early phase infection [6,48,50]. In addition, Th2-type cytokines may have some supportive role against infection [6,48]. On the other hand, the role of humoral immunity, including mucosal SIgA antibodies, is not fully understood in Cryptosporidium infection, and humoral immunity alone seems insufficient to control and clear this parasite. However, several studies have reported a correlation between antibody responses and asymptomatic status, suggesting that humoral immunity may have some role in the control of Cryptosporidium infection [48,50]. Based on these findings, vaccines that can induce strong cellular immunity have been extensively studied. Nasal and oral attenuated Salmonella vaccines expressing immunogenic surface proteins of Cryptosporidium, such as Cp12, Cp15, Cp23, and Cp40, were shown to elicit strong protective cell-mediated immune responses, as well as humoral immunity, against Cryptosporidium infection at both mucosal and systemic sites [48,50 52]. Importantly, the mucosal vaccine was less effective in animals with malnutrition [52], which is valuable information, considering future vaccination programs in developing countries.

D. Toxoplasmosis Toxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan Toxoplasma

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gondii. The parasite infects a wide variety of warm-blooded animals, including humans, but felines such as domestic cats are the only definitive host in which the sexual development of T. gondii occurs [53]. Approximately one third of the world’s population is exposed to this parasite [53]. T. gondii primarily exists in four stages: oocysts, sporozoites, tachyzoites, and bradyzoites. People acquire infection typically by ingesting food and water contaminated with oocysts produced in the feces of infected cats or by eating undercooked or uncooked meat containing tissue cysts that harbor hundreds to thousands of bradyzoites [54]. When ingested, sporozoites and bradyzoites released from oocysts and cysts, respectively, actively penetrate intestinal epithelial cells and differentiate into tachyzoites (Fig. 50.1D). Tachyzoites, the proliferative form of the parasite, multiply rapidly in infected cells and eventually rupture them. The released tachyzoites disseminate throughout the host, differentiate into bradyzoites, which are the dormant stage of the parasite, and form persistent tissue cysts mainly in the brain and muscles [54]. Immunocompetent adults infected by T. gondii are usually asymptomatic, but serious manifestations can occur in fetuses and infants infected through mother-to-child transmission when a woman acquires the initial infection with T. gondii during or just before pregnancy [54]. Symptoms of congenital toxoplasmosis include stillbirth or abortion of fetuses and retinochoroiditis, hydrocephalus, convulsions, intracerebral calcification, and mental and physical disabilities in infants [54]. Since T. gondii invades through the intestinal mucosa and parasitizes intracellularly, mucosal SIgA antibodies and cell-mediated immunity are responsible for protective immunity against T. gondii infection [55,56]. A vaccine against toxoplasmosis requires three strategies: (1) prevention of infection or clinical symptoms in humans, (2) prevention of infection in livestock to eliminate the risk of transmission to humans via meat ingestion, and (3) prevention of infection in cats to avoid the spread of oocysts. Since

there is no effective vaccine for humans, present strategies focus on the prevention of transmission from animals by limiting the human exposure to tissue cysts in meats or oocysts in cats. A live attenuated vaccine against T. gondii is commercially available for sheep to reduce abortions and tissue cyst formation, and it inhibits sexual development of the parasite in cats [5,53,57]. For the development of vaccines against T. gondii, SAG1, a well-characterized immunodominant surface antigen of tachyzoites, has been extensively evaluated as an immunogen [53,56]. Oral and intranasal administration of purified or recombinant SAG1 with an adjuvant induced strong mucosal SIgA antibodies as well as cellular immune responses in immunized mice, resulting in high survival rates and a significant reduction in tissue cyst formations against subsequent challenge with a virulent strain of T. gondii [58 62]. Other T. gondii proteins such as rhoptry proteins (ROP2, ROP18) essential for parasite multiplication and host invasion, actin proteins (ACT, ADF), serine protease inhibitor-1 (PI-1), dense granule proteins (GRA4, GRA5, GRA7), and the receptor for activated C kinase 1 were protective antigens of mucosal vaccines against subsequent challenge with virulent strains [63 69]. To enhance the immunogenicity of purified and recombinant proteins, a mucosal adjuvant such as QuilA, CT, CpG oligodeoxynucleotides (ODN), or heat-labile enterotoxin is commonly coadministered. In addition, effective antigen delivery systems are developed using attenuated Salmonella, adenoviruses, and porous nanoparticles [70 76]. Interestingly, recombinant plants expressing T. gondii antigens were studied as immunogens for “edible vaccines” [77 79].

III. HELMINTH INFECTIONS Helminths are multicellular organisms with a wide variation in size, morphology, developmental cycle, and habitat in the host [1]. Their life cycles are quite complicated. Most of them

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need more than one host, such as mammals, birds, fish, insects, or mollusks, to complete their life cycle, and they grow in each developmental stage in a unique host [1]. Major infection routes include ingestion via contaminated water and food and skin penetration and transmission by hematophagous insects [1]. Different from protozoans, adult helminths do not multiply, and the low number of infections mostly do not cause serious diseases in the definitive host [1]. However, they cause damage to parasitized organs and subsequent systemic manifestations in cases with a high number of infections [1]. In addition, some helminths can produce mild to severe symptoms in the intermediate hosts and in nondefinitive hosts when infected [1]. Parasitic helminths are classified as trematodes (flukes), cestodes (tapeworms), and nematodes (roundworms), and the gastrointestinal tract is the major entry and inhabited site [1]. A number of studies have revealed that exclusion of gastrointestinal helminths is mediated by activation of Th2-type responses in addition to innate immunity [7 10]. Studies to develop effective mucosal vaccines against helminth infection via the gastrointestinal tract are under way.

A. Schistosomiasis (Trematodiasis) Causative agents of schistosomiasis do not infect through gastrointestinal tract but via skin penetration [80]. The development of vaccines against schistosomiasis is one of the most anticipated among parasitic diseases because of the severity of manifestations [80]. Schistosomiasis is an acute and chronic parasitic disease caused by parasitic blood flukes of the genus Schistosoma. It is prevalent in 78 countries with more than 200 million people infected and more than 200,000 deaths per year, mainly in tropical and subtropical areas without access to safe drinking water and adequate sanitation [80]. Five Schistosoma species are known to infect

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humans: S. mansoni, S. japonicum, S. mekongi, S. intercalatum, and S. haematobium; more than 90% of schistosomiasis infections are caused by S. mansoni and S. haematobium [81]. S. haematobium causes urinary tract complications such as hematuria, bladder polyps, ulcers, and bladder cancer; others cause hepatic and intestinal disorders including intestinal disease, hepatosplenic inflammation, liver fibrosis, and liver failure [80,81]. Adult worms residing in the vesical veins (S. haematobium) or mesenteric veins (others) cause no apparent symptoms; however, parasite eggs trapped in the bladder wall or liver and intestinal wall and proteolytic enzymes produced by eggs elicit granulomatous inflammation, resulting in a severe chronic pathology [80 82]. The egg-induced granulomas are characterized by Th2 cell-dominant responses such as eosinophil infiltration, Th2 cell expansion, and elevation of IL-4, IL-5, and IL-13 cytokines [82]. Paradoxically, Th2 cell-mediated granulomatous inflammation is reported to be protective for the surrounding host tissues against antigens and toxins released from the eggs [81,82]. Antischistosomiasis vaccines have been extensively studied, and several candidates, such as glutathione S-transferase (GST), fatty-acidbinding proteins, tetraspanin protein 2 (TSP-2), Sm-p80, and Sm14, are under evaluation in clinical trials [80,83,84]. Mucosal vaccinations of GST coadministered with an adjuvant such as CT, entrapped by microparticles, coupled to liposomes, and expressed in Bacillus, were shown to induce protective immunity, reduction in worm burden and liver egg counts, suppressed granuloma inflammation, and reduced mortality in chronically infected mice [85 89].

B. Cysticercosis (Cestodiasis) Cysticercosis is a zoonotic disease caused by Taenia solium, known as pork tapeworm, that is seen worldwide [90,91]. Infection in humans occurs following consumption of undercooked

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or uncooked pork containing tissue cysts [90,91]. The pig is the intermediate host, in which parasites grow into larvae (cysticerci) from eggs, whereas humans are the definitive host, in which parasites grow into the adult stage and produce eggs [90,91]. However, humans can be the intermediate host of this parasite, and this makes it dangerous to humans [90,91]. Adult tapeworm infections (teniosis) cause few symptoms in humans, but severe clinical symptoms occur when the larvae (cysticerci) develop in the tissues such as muscles, skin, eyes, and central nervous system in the secondary infection (autoinfection) [90,91]. When cysticerci develop in the brain, they can cause serious symptoms, called neurocysticercosis, which include severe headache, blindness, convulsions, and epileptic seizures; it can be fatal [90,91]. One of the strategies to control cysticercosis is treatment of human teniosis and cysticercosis and porcine cysticercosis combined with vaccination of pigs [90 92]. Several antigens have been reported to provide protective immunity against cysticercosis in pigs, and S3vac with three peptides and an oncospheral TSOL18 antigen were identified as promising candidates [90,91]. Salmonella-expressing TSOL18 vaccine was shown to elicit protective humoral and cellular responses when administered orally to mice. In addition, the oral vaccine was confirmed to induce high levels of antibody responses specific to TSOL18 in pigs, the intermediate host of T. solium [93]. More interestingly, transgenic plants expressing T. solium antigens such as S3vac and TSOL18 in papaya and carrot have been produced, and their immunogenicity by inducing T cell immunity, as well as antibody responses, was shown in orally administrated mice and pigs [94 96].

C. Echinococcosis (Cestodiasis) Echinococcosis is caused by the larval stages of tiny tapeworms, the genus Echinococcus [97].

Of the six species recorded, Echinococcus granulosus, which causes cystic echinococcosis, and Echinococcus multilocularis, which causes alveolar echinococcosis, are the two important causative organisms of human echinococcosis [97]. Dogs and other canids are the definitive hosts for the parasites and are infected through ingestion of hydatid cyst-harboring organs of intermediate hosts, including sheep, goat, pigs, cattle, horses, and camels for E. granulosus and rodents for E. multilocularis [97]. The adult tapeworms inhabit the intestine of definitive hosts and release eggs [97]. Humans are an accidental intermediate host for E. granulosus and E. multilocularis [97]. Humans are infected by ingestion of parasite eggs in contaminated food and water or through direct contact with infected definitive hosts [97]. After ingestion, parasite eggs hatch and release embryos in the small intestine, penetrate the mucosal membrane, and finally disseminate to the liver and other organs in which cysts develop [97]. The parasite cysts grow slowly, commonly taking 5 15 years before infected individuals present with clinical symptoms such as weight loss, abdominal pain, and signs of hepatic failure [97]. Development of a vaccine against E. granulosus and E. multilocularis infection has been extensively studied, targeting definitive hosts as well as intermediate hosts [98]. Of these, a mucosal vaccination strategy aims mainly to control the definitive host. Intranasal immunization of recombinant tetraspanin of E. multilocularis (rEm-TSP3) with CpG ODN as an adjuvant was shown to induce strong mucosal SIgA antibodies as well as Th2type immunity [99]. In addition, intranasal administration of a large glycoprotein component of protoscoleces, SRf1, with CT subunit B to dogs showed a significant reduction in parasite numbers in the gut after subsequent oral challenge with E. multilocuaris protoscoleces [100]. Recombinant Bacillus subtilis, which can colonize the gut, was applied as an antigen delivery system. Oral administration of B. subtilis displaying E. granulosus antigens (tropomyosin and

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REFERENCES

paramyosin) to dogs was shown to elicit antibody responses that recognize protoscoleces, suggesting interference of the attachment and development of parasites in the gut [101].

D. Trichinellosis (Nematodiasis) Trichinellosis, also called trichinosis, is caused by tiny roundworms, Trichinella spp., and is prevalent in most parts of the world [102]. Trichinella spp. are transmitted directly from animal to animal, including humans, through the consumption of undercooked or uncooked meat containing encapsulated larvae [102]. After ingestion, larvae are released in the small intestine and develop into adult worms [102]. After mating, adult females invade the intestinal mucosa and produce larvae [102]. The larvae migrate into muscle tissues and then encapsulate [102]. Symptoms include diarrhea and abdominal cramps in the early phase of infection and, typically, muscle pain and fever in the late phase [102]. Generally, it is reported that Th2-type responses are responsible for anti-Trichinella immunity [6]. Intranasal administration of a synthetic peptide of Trichinella spiralis with CT subunit B as an adjuvant elicited Th2-type responses as well as mucosal SIgA antibodies and protected mice from subsequent challenge with T. spiralis [103]. More recently, using attenuated Salmonella for antigen delivery, oral administration of T. spiralis paramyosin, nudix hydrolase, and cystatin-like protein was shown to induce protective mucosal SIgA antibodies and Th1/Th2-type mixed immune responses and to provide significant reduction in parasite burden after challenge with T. spiralis larvae [104 106].

IV. CONCLUDING REMARKS Although many parasites do not commonly cause severe diseases in humans, there are still

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serious and life-threatening parasitic diseases such as malaria and NTDs, especially in tropical and subtropical areas [2]. The control of parasitic diseases has relied on antiparasitic drugs for many years, and development of vaccines has been neglected. This is because complex life cycles and variations in the antigenicity of parasites make it difficult to produce effective vaccines along with the expectation of little benefit [2]. However, prevention and treatment of infected individuals are essential to control infectious diseases. Mucosal vaccination inducing both mucosal and systemic immunity is the ideal prevention strategy, since the gastrointestinal tract is the major entry and inhabited site for medically relevant parasites, as detailed in this chapter [5 10]. Mucosal vaccines against parasitic diseases that are being developed target not only humans, but also, the animals that are the source of infection. Mucosal vaccination is also studied in vector-borne parasitic diseases, although such parasites enter through the skin and not the mucosa, and their effectiveness was demonstrated in several diseases such as malaria and leishmaniasis [107,108]. Mucosal immunity represented by SIgA antibodies may not be directly associated with protective effects against such infectious diseases; however, an edible vaccine may be a breakthrough and solution for sustainable distribution of vaccines that are much needed in endemic countries. Further efforts in research and development of adjuvants and antigen delivery systems will make a success of effective and safe mucosal vaccines in the near future. In addition, efficient bioinformatics analyses of big data obtained by genome and stagespecific transcriptome analyses of parasites will lead to identification of suitable antigens and antigenic sites for mucosal vaccines.

References [1] CDC, Centers for Disease Control and Prevention. About parasites, ,https://www.cdc.gov/parasites/ about.html.; 2016.

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50. MUCOSAL VACCINE FOR PARASITIC INFECTIONS

[2] WHO, World Health Organization. Neglected tropical diseases, ,http://www.who.int/neglected_diseases/ diseases/en/.; 2018. [3] Sibley CH, Hunt SY. Drug resistance in parasites: can we stay ahead of the evolutionary curve? Trends Parasitol 2003;19(11):532 7. [4] McDonald V, Shirley MW. Past and future: vaccination against Eimeria. Parasitology 2009;136(12):1477 89. [5] Vercruysse J, Schetters TP, Knox DP, Willadsen P, Claerebout E. Control of parasitic disease using vaccines: an answer to drug resistance? Rev Sci Tech 2007;26(1):105 15. [6] Weinstock JV. Mucosal immune response to parasitic infections. In: Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, editors. Mucosal immunology. 2nd ed. Academic Press; 1999. p. 709 18. [7] Zaph C, Cooper PJ, Harris NL. Mucosal immune responses following intestinal nematode infection. Parasite Immunol 2014;36(9):439 52. [8] Cooper PJ. Mucosal immunology of geohelminth infections in humans. Mucosal Immunol 2009;2(4):288 99. [9] Grencis RK. Immunity to helminths: resistance, regulation, and susceptibility to gastrointestinal nematodes. Annu Rev Immunol 2015;33:201 25. [10] Sorobetea D, Svensson-Frej M, Grencis R. Immunity to gastrointestinal nematode infections. Mucosal Immunol 2018;. Available from: https://doi.org/10.1038/mi.2017. 113. [11] Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study 2010. Lancet 2012;380:2095 128. [12] Petri Jr. WA, Smith RD, Schlesinger PH, Murphy CF, Ravdin JI. Isolation of the galactose-binding lectin that mediates the in vitro adherence of Entamoeba histolytica. J Clin Invest 1987;80(5):1238 44. [13] Ravdin JI, Guerrant RL. Role of adherence in cytopathogenic mechanisms of Entamoeba histolytica. Study with mammalian tissue culture cells and human erythrocytes. J Clin Invest 1981;68(5):1305 13. [14] Chadee K, Petri Jr. WA, Innes DJ, Ravdin JI. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica. J Clin Invest 1987;80 (5):1245 54. [15] Haque R, Ali IM, Sack RB, Farr BM, Ramakrishnan G, Petri Jr. WA. Amebiasis and mucosal IgA antibody against the Entamoeba histolytica adherence lectin in Bangladeshi children. J Infect Dis 2001;183(12):1787 93. [16] Haque R, Duggal P, Ali IM, Hossain MB, Mondal D, Sack RB, et al. Innate and acquired resistance to amebiasis in Bangladeshi children. J Infect Dis 2002;186(4): 547 52.

[17] Ankri S, Padilla-Vaca F, Stolarsky T, Koole L, Katz U, Mirelman D. Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNac lectin complex inhibits Entamoeba histolytica virulence. Mol Microbiol 1999;33(2):327 37. [18] Haque R, Mondal D, Shu J, Roy S, Kabir M, Davis AN, et al. Correlation of interferon-gamma production by peripheral blood mononuclear cells with childhood malnutrition and susceptibility to amebiasis. Am J Trop Med Hyg 2007;76(2):340 4. [19] Guo X, Barroso L, Lyerly DM, Petri Jr. WA, Houpt ER. CD41 and CD81 T cell- and IL-17-mediated protection against Entamoeba histolytica induced by a recombinant vaccine. Vaccine 2011;29(4):772 7. [20] Guo X, Barroso L, Becker SM, Lyerly DM, Vedvick TS, Reed SG, et al. Protection against intestinal amebiasis by a recombinant vaccine is transferable by T cells and mediated by gamma interferon. Infect Immun 2009;77(9): 3909 18. [21] Houpt E, Barroso L, Lockhart L, Wright R, Cramer C, Lyerly D, et al. Prevention of intestinal amebiasis by vaccination with the Entamoeba histolytica Gal/GalNac lectin. Vaccine 2004;22(5 6):611 17. [22] Moonah SN, Jiang NM, Petri Jr. WA. Host immune response to intestinal amebiasis. PLoS Pathog 2013;9 (8):e1003489. [23] Quach J, St-Pierre J, Chadee K. The future for vaccine development against Entamoeba histolytica. Hum Vaccin Immunother 2014;10(6):1514 21. [24] Beving DE, Soong CJ, Ravdin JI. Oral immunization with a recombinant cysteine-rich section of the Entamoeba histolytica galactose-inhibitable lectin elicits an intestinal secretory immunoglobulin A response that has in vitro adherence inhibition activity. Infect Immun 1996;64(4):1473 6. [25] Carrero JC, Cervantes-Rebolledo C, Aguilar-Dı´az H, Dı´az-Gallardo MY, Laclette JP, Morales-Montor J. The role of the secretory immune response in the infection by Entamoeba histolytica. Parasite Immunol 2007;29(7): 331 8. [26] Abd Alla MD, Wolf R, White GL, Kosanke SD, Cary D, Verweij JJ, et al. Efficacy of a Gal-lectin subunit vaccine against experimental Entamoeba histolytica infection and colitis in baboons (Papio sp.). Vaccine 2012;30(20): 3068 75. [27] Certad G, Viscogliosi E, Chabe´ M, Caccio` SM. Pathogenic mechanisms of Cryptosporidium and Giardia. Trends Parasitol 2017;33(7):561 76. [28] Eckmann L. Mucosal defences against Giardia. Parasite Immunol 2003;25(5):259 70. [29] Zinneman HH, Kaplan AP. The association of giardiasis with reduced intestinal secretory immunoglobulin A. Am J Dig Dis 1972;17(9):793 7.

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

REFERENCES

[30] Babaei Z, Malihi N, Zia-Ali N, Sharifi I, Mohammadi MA, Kagnoff MF, et al. Adaptive immune response in symptomatic and asymptomatic enteric protozoal infection: evidence for a determining role of parasite genetic heterogeneity in host immunity to human giardiasis. Microbes Infect 2016;18(11):687 95. [31] Snider DP, Gordon J, McDermott MR, Underdown BJ. Chronic Giardia muris infection in anti-IgM-treated mice. I. Analysis of immunoglobulin and parasitespecific antibody in normal and immunoglobulindeficient animals. J Immunol 1985;134(6):4153 62. [32] Heyworth MF, Carlson JR, Ermak TH. Clearance of Giardia muris infection requires helper/inducer T lymphocytes. J Exp Med 1987;165(6):1743 8. [33] Singer SM, Nash TE. T-cell-dependent control of acute Giardia lamblia infections in mice. Infect Immun 2000;68(1):170 5. [34] Dann SM, Manthey CF, Le C, Miyamoto Y, Gima L, Abrahim A, et al. IL-17A promotes protective IgA responses and expression of other potential effectors against the lumen-dwelling enteric parasite Giardia. Exp Parasitol 2015;156:68 78. [35] Singer SM. Control of giardiasis by interleukin-17 in humans and mice—are the questions all answered? Clin Vaccine Immunol 2015;23(1):2 5. [36] Langford TD, Housley MP, Boes M, Chen J, Kagnoff MF, Gillin FD, et al. Central importance of immunoglobulin A in host defense against Giardia spp. Infect Immun 2002;70(1):11 18. [37] Weeratunga SK, Osman A, Hu NJ, Wang CK, Mason L, Sva¨rd S, et al. Alpha-1 giardin is an annexin with highly unusual calcium-regulated mechanisms. J Mol Biol 2012;423(2):169 81. [38] Abdul-Wahid A, Faubert G. Mucosal delivery of a transmission-blocking DNA vaccine encoding Giardia lamblia CWP2 by Salmonella typhimurium bactofection vehicle. Vaccine 2007;25(50):8372 83. [39] Feng XM, Zheng WY, Zhang HM, Shi WY, Li Y, Cui BJ, et al. Vaccination with bivalent DNA vaccine of α1Giardin and CWP2 delivered by attenuated Salmonella typhimurium reduces trophozoites and cysts in the feces of mice infected with Giardia lamblia. PLoS One 2016;11(6):e0157872. [40] Jenikova G, Hruz P, Andersson MK, Tejman-Yarden N, Ferreira PC, Andersen YS, et al. A1-giardin based live heterologous vaccine protects against Giardia lamblia infection in a murine model. Vaccine 2011;29(51): 9529 37. [41] Larocque R, Nakagaki K, Lee P, Abdul-Wahid A, Faubert GM. Oral immunization of BALB/c mice with Giardia duodenalis recombinant cyst wall protein inhibits shedding of cysts. Infect Immun 2003;71(10):5662 9. [42] Lee P, Faubert GM. Oral immunization of BALB/c mice by intragastric delivery of Streptococcus gordonii-

[43]

[44]

[45]

[46]

[47]

[48]

[49] [50]

[51]

[52]

[53]

[54]

[55]

[56]

851 expressing Giardia cyst wall protein 2 decreases cyst shedding in challenged mice. FEMS Microbiol Lett 2006;265(2):225 36. Lee P, Faubert GM. Expression of the Giardia lamblia cyst wall protein 2 in Lactococcus lactis. Microbiology 2006;152(Pt 7):1981 90. Davis-Hayman SR, Hayman JR, Nash TE. Encystationspecific regulation of the cyst wall protein 2 gene in Giardia lamblia by multiple cis-acting elements. Int J Parasitol 2003;33(10):1005 12. Weiland ME, Palm JE, Griffiths WJ, McCaffery JM, Sva¨rd SG. Characterisation of alpha-1 giardin: an immunodominant Giardia lamblia annexin with glycosaminoglycan-binding activity. Int J Parasitol 2003;33(12):1341 51. Rivero FD, Saura A, Prucca CG, Carranza PG, Torri A, Lujan HD. Disruption of antigenic variation is crucial for effective parasite vaccine. Nat Med 2010;16(5):551 7. Serradell MC, Saura A, Rupil LL, Gargantini PR, Faya MI, Furlan PJ, et al. Vaccination of domestic animals with a novel oral vaccine prevents Giardia infections, alleviates signs of giardiasis and reduces transmission to humans. NPJ Vaccines 2016;1:16018. Lemieux MW, Sonzogni-Desautels K, Ndao M. Lessons learned from protective immune responses to optimize vaccines against cryptosporidiosis. Pathogens 2017;7(1):2. Tzipori S, Widmer G. A hundred-year retrospective on cryptosporidiosis. Trends Parasitol 2008;24(4):184 9. Ludington JG, Ward HD. Systemic and mucosal immune responses to Cryptosporidium-vaccine development. Curr Trop Med Rep 2015;2(3):171 80. Benitez AJ, McNair N, Mead JR. Oral immunization with attenuated Salmonella enterica serovar Typhimurium encoding Cryptosporidium parvum Cp23 and Cp40 antigens induces a specific immune response in mice. Clin Vaccine Immunol 2009;16(9):1272 8. Roche JK, Rojo AL, Costa LB, Smeltz R, Manque P, Woehlbier U, et al. Intranasal vaccination in mice with an attenuated Salmonella enterica serovar 908htrA expressing Cp15 of Cryptosporidium: impact of malnutrition with preservation of cytokine secretion. Vaccine 2013;31(6):912 18. Verma R, Khanna P. Development of Toxoplasma gondii vaccine: a global challenge. Hum Vaccin Immunother 2013;9(2):291 3. Harker KS, Ueno N, Lodoen MB. Toxoplasma gondii dissemination: a parasite’s journey through the infected host. Parasite Immunol 2015;37(3):141 9. Cohen SB, Denkers EY. The gut mucosal immune response to Toxoplasma gondii. Parasite Immunol 2015;37(3):108 17. Jongert E, Roberts CW, Gargano N, Fo¨rster-Waldl E, Petersen E. Vaccines against Toxoplasma gondii:

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

852

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

50. MUCOSAL VACCINE FOR PARASITIC INFECTIONS

challenges and opportunities. Mem Inst Oswaldo Cruz 2009;104(2):252 66. ´ Hiszczynska-Sawicka E, Gatkowska JM, Grzybowski ´ MM, Długonska H. Veterinary vaccines against toxoplasmosis. Parasitology 2014;141(11):1365 78. Debard N, Buzoni-Gatel D, Bout D. Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection. Infect Immun 1996;64(6):2158 66. Khan IA, Ely KH, Kasper LH. A purified parasite antigen (p30) mediates CD81 T cell immunity against fatal Toxoplasma gondii infection in mice. J Immunol 1991;147(10):3501 6. Me´ve´lec MN, Mercereau-Puijalon O, Buzoni-Gatel D, Bourguin I, Charde`s T, Dubremetz JF, et al. Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route. Parasite Immunol 1998;20(4):183 95. Velge-Roussel F, Marcelo P, Lepage AC, Buzoni-Gatel D, Bout DT. Intranasal immunization with Toxoplasma gondii SAG1 induces protective cells into both NALT and GALT compartments. Infect Immun 2000;68(2):969 72. Bonenfant C, Dimier-Poisson I, Velge-Roussel F, Buzoni-Gatel D, Del Giudice G, Rappuoli R, et al. Intranasal immunization with SAG1 and nontoxic mutant heat-labile enterotoxins protects mice against Toxoplasma gondii. Infect Immun 2001;69(3):1605 12. Yin LT, Hao HX, Wang HL, Zhang JH, Meng XL, Yin GR. Intranasal immunisation with recombinant Toxoplasma gondii actin partly protects mice against toxoplasmosis. PLoS One 2013;8(12):e82765. EL-Malky MA, Al-Harthi SA, Mohamed RT, EL Bali MA, Saudy NS. Vaccination with Toxoplasma lysate antigen and CpG oligodeoxynucleotides: comparison of immune responses in intranasal versus intramuscular administrations. Parasitol Res 2014;113(6):2277 84. Igarashi M, Kano F, Tamekuni K, Machado RZ, Navarro IT, Vidotto O, et al. Toxoplasma gondii: evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice. Exp Parasitol 2008;118(3):386 92. Wang HL, Pang M, Yin LT, Zhang JH, Meng XL, Yu BF, et al. Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection. Acta Trop 2014;137:58 66. Liu Z, Yin L, Li Y, Yuan F, Zhang X, Ma J, et al. Intranasal immunization with recombinant Toxoplasma gondii actin depolymerizing factor confers protective efficacy against toxoplasmosis in mice. BMC Immunol 2016;17(1):37.

[68] Rashid I, Moire´ N, He´raut B, Dimier-Poisson I, Me´ve´lec MN. Enhancement of the protective efficacy of a ROP18 vaccine against chronic toxoplasmosis by nasal route. Med Microbiol Immunol 2017;206(1): 53 62. [69] Picchio MS, Sa´nchez VR, Arcon N, Soto AS, Perrone Sibilia M, Aldirico MLA, et al. Vaccine potential of antigen cocktails composed of recombinant Toxoplasma gondii TgPI-1, ROP2 and GRA4 proteins against chronic toxoplasmosis in C3H mice. Exp Parasitol 2018;185:62 70. [70] Wang T, Yin H, Li Y, Zhao L, Sun X, Cong H. Vaccination with recombinant adenovirus expressing multi-stage antigens of Toxoplasma gondii by the mucosal route induces higher systemic cellular and local mucosal immune responses than with other vaccination routes. Parasite 2017;24:12. [71] Cong H, Gu QM, Jiang Y, He SY, Zhou HY, Yang TT, et al. Oral immunization with a live recombinant attenuated Salmonella typhimurium protects mice against Toxoplasma gondii. Parasite Immunol 2005;27(1 2): 29 35. [72] Cong H, Yuan Q, Zhao Q, Zhao L, Yin H, Zhou H, et al. Comparative efficacy of a multi-epitope DNA vaccine via intranasal, peroral, and intramuscular delivery against lethal Toxoplasma gondii infection in mice. Parasit Vectors 2014;7:145. [73] Dimier-Poisson I, Carpentier R, N’Guyen TT, Dahmani F, Ducournau C, Betbeder D. Porous nanoparticles as delivery system of complex antigens for an effective vaccine against acute and chronic Toxoplasma gondii infection. Biomaterials 2015;50:164 75. [74] Li XZ, Wang XH, Xia LJ, Weng YB, Hernandez JA, Tu LQ, et al. Protective efficacy of recombinant canine adenovirus type-2 expressing TgROP18 (CAV-2ROP18) against acute and chronic Toxoplasma gondii infection in mice. BMC Infect Dis 2015;15:114. [75] Qu D, Wang S, Cai W, Du A. Protective effect of a DNA vaccine delivered in attenuated Salmonella typhimurium against Toxoplasma gondii infection in mice. Vaccine 2008;26(35):4541 8. [76] Qu D, Yu H, Wang S, Cai W, Du A. Induction of protective immunity by multiantigenic DNA vaccine delivered in attenuated Salmonella typhimurium against Toxoplasma gondii infection in mice. Vet Parasitol 2009;166(3 4):220 7. [77] Del L Ya´cono M, Farran I, Becher ML, Sander V, Sa´nchez VR, Martı´n V, et al. A chloroplast-derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice. Plant Biotechnol J 2012;10(9):1136 44. [78] Albarracı´n RM, Becher ML, Farran I, Sander VA, Corigliano MG, Ya´cono ML, et al. The fusion of

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

REFERENCES

Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts. Biotechnol J 2015;10(5):748 59. [79] Laguı´a-Becher M, Martı´n V, Kraemer M, Corigliano M, Yacono ML, Goldman A, et al. Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice. BMC Biotechnol 2010;10:52. [80] Beaumier CM, Gillespie PM, Hotez PJ, Bottazzi ME. New vaccines for neglected parasitic diseases and dengue. Transl Res 2013;162(3):144 55. [81] Inobaya MT, Olveda RM, Chau TN, Olveda DU, Ross AG. Prevention and control of schistosomiasis: a current perspective. Res Rep Trop Med 2014;2014(5): 65 75. [82] Hams E, Aviello G, Fallon PG. The schistosoma granuloma: friend or foe? Front Immunol 2013;4:89. [83] Siddiqui AA, Siddiqui SZ. Sm-p80-based schistosomiasis vaccine: preparation for human clinical trials. Trends Parasitol 2017;33(3):194 201. [84] Tebeje BM, Harvie M, You H, Loukas A, McManus DP. Schistosomiasis vaccines: where do we stand? Parasit Vectors 2016;9(1):528. [85] Ivanoff N, Phillips N, Schacht AM, Heydari C, Capron A, Riveau G. Mucosal vaccination against schistosomiasis using liposome-associated Sm 28 kDa glutathione S-transferase. Vaccine 1996;14(12):1123 31. [86] Lebens M, Sun JB, Sadeghi H, Ba¨ckstro¨m M, Olsson I, Mielcarek N, et al. A mucosally administered recombinant fusion protein vaccine against schistosomiasis protecting against immunopathology and infection. Vaccine 2003;21(5 6):514 20. [87] Li L, Hu X, Wu Z, Xiong S, Zhou Z, Wang X, et al. Immunogenicity of self-adjuvanticity oral vaccine candidate based on use of Bacillus subtilis spore displaying Schistosoma japonicum 26 KDa GST protein. Parasitol Res 2009;105(6):1643 51. [88] Baras B, Benoit MA, Dupre´ L, Poulain-Godefroy O, Schacht AM, Capron A, et al. Single-dose mucosal immunization with biodegradable microparticles containing a Schistosoma mansoni antigen. Infect Immun 1999;67(5):2643 8. [89] Sun JB, Stadecker MJ, Mielcarek N, Lakew M, Li BL, Hernandez HJ, et al. Nasal administration of Schistosoma mansoni egg antigen-cholera B subunit conjugate suppresses hepatic granuloma formation and reduces mortality in S. mansoni-infected mice. Scand J Immunol 2001;54(5):440 7. [90] Garcia HH, O’Neal SE, Gilman RH, Cysticercosis Working Group in Peru. Elimination of Taenia solium transmission in Peru. N Engl J Med 2016;375(12): 1196 7.

853

[91] Johansen MV, Trevisan C, Gabrie¨l S, Magnussen P, Braae UC. Are we ready for Taenia solium cysticercosis elimination in sub-Saharan Africa? Parasitology 2017;144(1):59 64. [92] Sciutto E, Fragoso G, Herna´ndez M, Rosas G, Martı´nez JJ, Fleury A, et al. Development of the S3Pvac vaccine against porcine Taenia solium cysticercosis: a historical review. J Parasitol 2013;99(4): 686 92. [93] Ding J, Zheng Y, Wang Y, Dou Y, Chen X, Zhu X, et al. Immune responses to a recombinant attenuated Salmonella typhimurium strain expressing a Taenia solium oncosphere antigen TSOL18. Comp Immunol Microbiol Infect Dis 2013;36(1):17 23. [94] Fragoso G, Herna´ndez M, Cervantes-Torres J, Ramı´rez-Aquino R, Chapula H, Villalobos N, et al. Transgenic papaya: a useful platform for oral vaccines. Planta 2017;245(5):1037 48. [95] Monreal-Escalante E, Govea-Alonso DO, Herna´ndez M, Cervantes J, Salazar-Gonza´lez JA, RomeroMaldonado A, et al. Towards the development of an oral vaccine against porcine cysticercosis: expression of the protective HP6/TSOL18 antigen in transgenic carrots cells. Planta 2016;243(3):675 85. [96] Rosales-Mendoza S, Monreal-Escalante E, Gonza´lezOrtega O, Herna´ndez M, Fragoso G, Garate T, et al. Transplastomic plants yield a multicomponent vaccine against cysticercosis. J Biotechnol 2018;266: 124 32. [97] Moro P, Schantz PM. Echinococcosis: a review. Int J Infect Dis 2009;13(2):125 33. [98] Pourseif MM, Moghaddam G, Saeedi N, Barzegari A, Dehghani J, Omidi Y. Current status and future prospective of vaccine development against Echinococcus granulosus. Biologicals 2018;51:1 11. [99] Dang Z, Yagi K, Oku Y, Kouguchi H, Kajino K, Matsumoto J, et al. A pilot study on developing mucosal vaccine against alveolar echinococcosis (AE) using recombinant tetraspanin 3: vaccine efficacy and immunology. PLoS Negl Trop Dis 2012;6(3):e1570. [100] Kouguchi H, Matsumoto J, Nakao R, Yamano K, Oku Y, Yagi K. Characterization of a surface glycoprotein from Echinococcus multilocularis and its mucosal vaccine potential in dogs. PLoS One 2013;8(7):e69821. [101] Vogt CM, Armu´a-Ferna´ndez MT, Tobler K, Hilbe M, Aguilar C, Ackermann M, et al. Oral application of recombinant Bacillus subtilis spores to dogs results in a humoral response against specific Echinococcus granulosus paramyosin and tropomyosin antigens. Infect Immun 2018;86(3):e00495 17. [102] Bruschi F. Trichinellosis in developing countries: is it neglected? J Infect Dev Ctries 2012;6(3):216 22. [103] McGuire C, Chan WC, Wakelin D. Nasal immunization with homogenate and peptide antigens induces

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

854

50. MUCOSAL VACCINE FOR PARASITIC INFECTIONS

protective immunity against Trichinella spiralis. Infect Immun 2002;70(12):7149 52. [104] Wang L, Wang X, Bi K, Sun X, Yang J, Gu Y, et al. Oral vaccination with attenuated Salmonella typhimurium-delivered TsPmy DNA vaccine elicits protective immunity against Trichinella spiralis in BALB/c mice. PLoS Negl Trop Dis 2016;10(9):e0004952. [105] Liu P, Wang ZQ, Liu RD, Jiang P, Long SR, Liu LN, et al. Oral vaccination of mice with Trichinella spiralis nudix hydrolase DNA vaccine delivered by attenuated Salmonella elicited protective immunity. Exp Parasitol 2015;153:29 38.

[106] Liu XD, Wang XL, Bai X, Liu XL, Wu XP, Zhao Y, et al. Oral administration with attenuated Salmonella encoding a Trichinella cystatin-like protein elicited host immunity. Exp Parasitol 2014;141:1 11. [107] Gregory JA, Mayfield SP. Developing inexpensive malaria vaccines from plants and algae. Appl Microbiol Biotechnol 2014;98(5):1983 90. [108] Pratti JE, Ramos TD, Pereira JC, da Fonseca-Martins AM, Maciel-Oliveira D, Oliveira-Silva G, et al. Efficacy of intranasal LaAg vaccine against Leishmania amazonensis infection in partially resistant C57Bl/6 mice. Parasit Vectors 2016;9(1):534.

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Mucosal Vaccines for Allergy and Tolerance Andrea M. Kemter and Cathryn R. Nagler Department of Pathology and Committee on Immunology, The University of Chicago, Chicago, IL, United States

I. INTRODUCTION Over the past few decades, the incidence of allergies in Western countries has increased at an alarming rate [1]. This dramatic rise has largely been attributed to environmental influences, but the exact mechanism(s) by which allergies are induced and why they are increasing in prevalence have still not been fully elucidated. Sensitization against an allergen occurs if antigen-presenting cells residing at the point of entry, usually the skin or mucosal surfaces such as the airways and intestinal tract, present the allergen to T cells in a manner that elicits a type 2 T helper (Th2) response. Antigen-specific T cells produce Th2 cytokines, including interleukin 4 (IL-4), IL-13, and IL-5, and induce an antibody class switch to allergen-specific immunoglobulin E (IgE) as well as IgG1 in activated B cells. When allergen-specific IgE binds to receptors on basophils, mast cells, and eosinophils, sensitization against the allergen occurs. Subsequent contact with allergen cross-links the surface-bound IgE, leading to the release of the molecules stored in

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00051-1

the granules of these cells. The ensuing allergic reaction can range from fairly mild symptoms, such as rhinitis, to a severe and life-threatening anaphylactic response (as reviewed by Reynolds and Finlay [2]) (Chapter 13: Mast Cells for the Control of Mucosal Immunity). For food-allergic individuals, complete avoidance of the allergens and immunotherapy are currently the only options. For conditions such as rhinitis or allergic asthma, additional treatment options exist, but most target the symptoms. Avoidance and immunotherapy have a major impact on a patient’s lifestyle, and avoidance does not protect from accidental exposure; immunotherapy, while potentially providing this protection, is fraught with side effects and, following a lengthy period of regular supervised treatments, requires continued exposure to the allergen(s). Furthermore, not all patients will become desensitized by the current regimes. Improvements to existing treatments as well as novel approaches are therefore sorely needed to combat the ongoing rise of allergic disease.

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II. IMMUNOTHERAPY As first described about a century ago, the goal of immunotherapy is to treat an allergic individual with increasing doses of allergen to desensitize the person, followed by regular treatment with a maintenance dose. There are various different delivery methods for this immunotherapy; oral and sublingual are the most commonly used, but epicutaneous delivery and subcutaneous, intramuscular, or intralymphatic injections have also been described (reviewed by Hayen et al. [3]). Regardless of the route of antigen delivery, the aim of all of these approaches is to inhibit or block the pathogenic Th2 response to the allergen. Although the exact mechanisms behind the observed outcomes of the treatments have not yet been fully elucidated, a few observations have been made. A common phenomenon is the loss of T cell reactivity to the allergen, which can be due to specific T cell deletion or induction of anergy. Furthermore, a frequent observation is the expansion of regulatory T cells (Tregs) and/or the production of IL-10, which leads to a general inhibition of immune responses against the allergen. Also, frequently observed is the induction of Th1 rather than Th2 responses, with a switch from the production of antigen-specific IgE and IgG1 to IgG2; it is thought that these, together with IgG4 antibodies, might function as competitive inhibitors and thereby block the development of an allergic reaction (as reviewed by Wood [4] and Akdis and Akdis [5]). As was mentioned above, specific immunotherapy is not effective in all patients, and even if successful, it requires long-term commitment to the therapy. Furthermore, the treatment of allergic individuals with the substances to which they are allergic poses the risk of adverse effects. Reactions to allergen dosing are common and can include anaphylaxis [4]. A number of approaches are already being used or are currently being investigated to improve the

efficacy and safety of immunotherapy; these are introduced in the following sections.

A. Hypoallergenic Antigens In nature, allergens are usually part of a complex mixture of proteins. This makes reliable dosing for treatment difficult; in addition, the unmodified allergen poses a safety threat to the patient as it still contains the IgE epitopes that induce allergic reactions. Because of these concerns, hypoallergenic derivatives of allergens are often employed. These derivatives can be made from allergen extracts, synthesized, or produced as recombinant proteins. All have in common that their IgE epitopes are masked or destroyed to avoid possible allergic reactions to the treatment, while trying to preserve the immunogenicity of the molecule, that is, make sure that it will still be recognized by T cells. IgE epitopes rely on the intact structure of the protein, so in addition to their direct alteration, manipulations disrupting proper folding would destroy the unwanted B cell epitopes while leaving peptide sequences recognized by T cells intact (as reviewed by Dall’antonia et al. [6]). A number of different options for the production of hypoallergenic antigens have been described and are summarized here. Extracts of allergenic material can be chemically modified. Formalin treatment of a rye grass pollen antigen was described almost 50 years ago to significantly reduce allergenicity as measured by histamine release by human leukocytes [7]. These so-called allergoids were still able to induce antibody production in guinea pigs, and these antibodies were able to block the histamine release induced by native allergen. In a modernday application of this approach, Allergovit [8], composed of formaldehyde-treated allergens from six grass species, has shown efficacy in clinical trials [9], and was recently found to be safe to use in accelerated updosing regimes, in which fewer injections are necessary to reach the

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maintenance dose [10]. Allergoids are also currently being used by a Dutch company, HAL Allergy [11], mainly for immunotherapy of pollen allergies [12,13], although PURETHAL mites has been shown to be effective against allergic asthma [14]. A trial for a peanut allergy therapeutic is currently underway (ClinicalTrials.gov identifier: NCT02991885). The allergoids are obtained by cross-linking of proteins in the allergen extract by glutaraldehyde treatment. Furthermore, two reports on reduction and alkylation of the peanut allergen Ara h 2 [15] and the peach allergen Pru p 3 [16] demonstrated reduced allergenicity of these molecules. Hypoallergenic derivatives of allergens can also be made as recombinant proteins. A number of different options exist to remove IgE epitopes. These manipulations include deletions of parts of the sequence encoding the allergen [17 22] and targeted mutations to remove cysteines and therefore disulfide bonds [23,24] or amino acids necessary for binding of calcium in a subclass of allergens [25 28]. The order of sequences recognized by T cells can be altered by rearranging fragments within an allergen, creating a mosaic sequence [26 31], producing hybrid molecules with sequences from a number of different allergens [29 36], or even conjoining several repeats of the same one [21,37]. Provided that the exact location and binding mechanism of IgE epitopes within an allergen are known, another option is the targeted mutation of codons encoding amino acids important for recognition of this epitope [38 41]. Most reports on hypoallergenic variants have demonstrated that the molecule produced has a reduced ability to bind to IgE and induce basophil degranulation in vitro, has reduced allergenicity in skin prick tests, and/or is capable of inducing IgG rather than IgE responses upon immunization of mice, with this IgG being able to block the binding of human IgE to the allergen. In spite of the number of hypoallergenic proteins that have been generated (see Table 51.1),

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only a few have actually been tested in clinical trials. Allergic rhinitis symptoms were reduced in clinical trials of the Bet v 1 fragments as well as trimer [42,43] and the grass pollen antigen hybrid vaccine BM32 [50]. Another trial was performed to investigate the use of the fragments as a prophylactic vaccine (ClinicalTrials. gov identifier: NCT01353924). However, these treatments were all done by subcutaneous injection. Of note is one hybrid hypoallergen, designed to target birch pollen allergy as well as birchpollen-related food allergy against celery and carrot, as this hybrid has been tested as a prophylactic treatment in mice using intranasal delivery. Impressively, it could alleviate the Th2 responses induced by sensitization with all three allergens; the authors further demonstrated that this was dependent on IL-10, transforming growth factor beta (TGF-β), and CD251 T cells, implicating Tregs as crucial mediators for tolerance induction in this system [29]. An interesting approach was taken with a potential therapy for Japanese cedar polleninduced allergic conjunctivitis, as the hypoallergens were expressed in rice, which was then fed to sensitized mice. This oral immunotherapy (OIT) proved efficient in protecting the mice from subsequent allergen challenge [51]. Another option is the use of allergen-derived peptides, selecting immunodominant sequences while avoiding the inclusion of IgE epitopes. Various methods to select these peptides have been described, including testing proliferation and activation of immune cells from allergic individuals or bioinformatics methods calculating the optimal peptides dependent on human leukocyte antigen background [52]. The use of peptides for immunotherapy has been tested successfully in several mouse models of allergies. Intranasal application of immunodominant peptides from the major house dust mite allergen Der p 1 [53], the olive pollen allergen Ole e 1 [54], or slightly longer peptides from the major bee venom allergen

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51. MUCOSAL VACCINES FOR ALLERGY AND TOLERANCE

Examples of Hypoallergenic Derivatives of Allergens Produced as Recombinant Proteins

Type

Allergen

Citation

Deletion/allergen fragment

Ole e 1 (Olive tree pollen)

[17]

Phl p 5a (grass pollen)

[18]

Phl p 6 (grass pollen)

[19]

Bet v 1 (birch pollen)

[20 22]

Par j 1 (grass pollen)

Bonura et al., 2001

Lep 2 d (house dust mite)

Olsson et al., 1998

Mutation of cysteines

Removal of Ca21 binding Cyp 1 c (fish)

Mosaic

Hybrid molecule

Clinical trial

[42,43], NCT01353924

[23,24]

Bra r 5.0101 (turnip pollen)

[25]

Phl p 7 (grass pollen)

[28]

Bet v 1 (birch pollen)

[44]

Phl p 1 (grass pollen)

[45]

Phl p 2 (grass pollen)

[46]

Phl p 12 (grass pollen)

[47]

Fel d 1 (cat)

[48]

Der p 2 (house dust mite)

[49]

Bet v 1 (birch pollen) 1 Api g 1 (celery) 1 Dau c 1 (carrot) [29] Bet v 1 (birch pollen) 1 Cor a 1.04 (hazelnut) 1 Mal d 1 (apple)

[36]

Phl p 2 1 Phl p 6 (grass pollen)

[30]

Phl p 1, 2, 5, 6 (grass pollen)

[31]

Phl p 1, 2, 5, 6 (grass pollen)

[35]

Der p 1 and 2 (house dust mite)

[32] or [33]

Par j 1 and 2 (grass pollen)

[34]

Bet v 1 (birch pollen)

[21,37]

Mutation of IgE epitopes Ara h 3 (peanut)

[50]

[42,43]

[38]

Bet v 1 (birch pollen)

[39,40]

Lol p 5 (grass pollen)

[41]

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phospholipase A2 [55] could both protect from sensitization with the allergen and alleviate the allergic reaction postsensitization by impairing or altering T cell responses. Similarly, OIT with peptides from the egg-white allergen ovomucoid [56] as well as pretreatment with peptides from the milk protein β-lactoglobulin (BLG) [57] showed efficacy against allergic sensitization; both of these treatments increased the numbers of regulatory T cells. In addition to experiments in the murine model, a number of clinical trials have been performed; however, in these, the treatment was delivered intradermally. Intradermal administration of a peptide mixture derived from the cat allergen Fel d 1, called Cat-PAD [58,59], or a mix of peptides from grass antigens [60] results in a reduction of allergic symptoms in the treatment compared to placebo groups. In the case of CatPAD, this effect persisted even 2 years after the single course of treatment, although a recent phase 3 clinical trial resulted in very high placebo response rates, making it impossible to distinguish the treatment effect [61].

B. Anti-IgE Another approach to increase the safety of allergen immunotherapy is to directly target IgE with the goal of decreasing the severity of adverse reactions. A few clinical trials have been performed with promising results. The first trials for omalizumab, a monoclonal antiIgE antibody that blocks the binding of IgE to its receptors, were published in 2001; they demonstrated its ability to alleviate allergic asthma [62 64]. Omalizumab has also been tested for treatment of other IgE-mediated diseases, including allergic rhinitis [65], and has been demonstrated to improve the safety of OIT [66]. It has also been used to speed up the initial escalation of allergen dose during OIT [67,68]. Most of the patients undergoing OIT for cow’s milk allergy using a rush oral desensitization

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protocol combined with omalizumab treatment reached the maintenance dose in the targeted time frame and showed improved tolerance of milk following the study [67]. Closer investigation of the immune responses in these children found that, while there was no induction of milk-specific Tregs, levels of milk-specific serum IgE decreased. This was accompanied by an increase in milk-specific serum IgG4 as well as an increase in the ratios of the cytokines interferon gamma (IFNγ)/IL-4 and IFNγ/IL-13 produced by the proliferating subset of peripheral blood mononuclear cells. The activation of basophils from whole blood was also inhibited in these patients [69]. However, in the trial published by Nadeau et al., no OIT group without omalizumab treatment was included, so comparisons between the two treatments cannot be made. A trial of rapid oral desensitization with and without omalizumab in peanut-allergic patients reported that patients in the omalizumab group could safely reach the set maintenance dose faster than patients in the placebo group. In addition, the treatment group reached a significantly higher maintenance dose than did patients treated with placebo without increased adverse effects [68]. Also of note is a trial investigating the safety of OIT combined with omalizumab in high-risk patients, children with high levels of peanut-specific IgE, which normally indicates increased risk for the therapy to fail. While this study did not include a placebo group and consisted of only 12 children, all of the patients could tolerate peanut well enough to reach the maintenance dose [70]. However, in some cases, the treatment with omalizumab only during the initial phase of the OIT might not be sufficient. Lafuente et al. reported on cases of egg-allergic children who had successfully been desensitized but lost their tolerance of the maintenance dose after discontinuation of omalizumab treatment; when the treatment was resumed, they were desensitized again [71]. Similar results have been observed with children allergic to cow’s milk [72].

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Studies investigating the structure of the omalizumab IgE complex and why it blocks IgE from binding its receptor [73,74] have resulted in a promising potential improvement. Pennington et al. developed an IgE-Fc fragment that, not recognized by omalizumab and not able to bind antigen, could bind to IgE receptors and thereby replace the functional IgE targeted by omalizumab. The authors showed that adding this fragment greatly improved the inhibitory effect of omalizumab on basophils [75]. Another interesting approach is the development of DARPin E2_79 and bi53_79, molecules that induce the dissociation of IgE bound to FcεRI [76,77]. In addition, several alternatives for omalizumab have been developed. An anti-IgE antibody with higher affinity (QGE031 or ligelizumab) was compared to omalizumab in a clinical trial for treatment of allergic asthma and showed improved efficacy [78]. Another antibody, quilizumab, targets membrane-bound IgE and depletes IgE1 B cells; while it has proven effective in treatment of allergic rhinitis and mild asthma [79], it failed to improve symptoms in patients whose asthma was not responsive to standard treatment [80].

C. Adjuvants and Delivery To improve the efficiency of allergen immunotherapy, a number of possibilities to support the shift toward a Th1 cell response have been explored. In general, this requires the use of an adjuvant, an additional component that boosts or alters the immune response. Many adjuvants are derived from microbial products. One such adjuvant is CpG oligodeoxynucleotides, which are short, single-stranded DNA molecules that contain unmethylated CpG motifs (cytosine followed by guanine); they are common in bacterial but not mammalian cells, and they bind to the toll-like receptor (TLR) TLR9 [81]. If mice were sensitized with peanut extract (PN), cholera toxin (CT), and CpG, they showed decreased production of PN-specific serum IgE

as well as reduced histamine release and anaphylaxis scores compared to mice that were sensitized with PN 1 CT alone. Restimulated splenocytes from mice treated with CpG secreted less IL-13 and more IFNγ compared to the PN 1 CT group, indicating a shift from a Th2- to a Th1-type response [82]. In another study, PN-sensitized mice were treated with PN-containing nanoparticles with or without CpG by oral gavage [83]. The addition of CpG to the nanoparticles led to improved efficacy, as mice showed greater protection from allergic challenge compared to the mice treated with PN-containing nanoparticles alone. This correlated with reduced plasma histamine levels as well as reduced PN-specific serum IgE and IgG1, while IgG2a levels increased. Another TLR ligand that has been used as conjugate with an allergen is the bacterial protein flagellin, which binds to TLR5. Flagellin conjugated to ovalbumin (OVA) has been used as a prophylactic vaccine in a murine model of food allergy. Both intraperitoneal injection of the conjugate and intranasal application were tested; while both approaches showed efficacy, reducing allergic symptoms as well as OVAspecific IgE and increasing OVA-specific IgG2a levels, intranasal delivery was more effective [84]. A TLR4 ligand, the lipopolysaccharide monophosphoryl lipid A (MPL), which is a derivative of an extracellular bacterial cell wall component and already used for a number of other vaccines, has also been adopted as an adjuvant for allergy vaccination. A combination of MPL and allergoids (a mix of pollen extracts modified by glutaraldehyde treatment) adsorbed to L-tyrosine to ensure slow release has been subjected to numerous safety studies and clinical trials and is commercially available as Pollinex Quattro. This vaccine is injected subcutaneously before the start of the pollen season, with just four injections of increasing doses, an enormous improvement compared to conventional immunotherapy regimes [85] (Chapter 10: Innate Immunity-Based Mucosal

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Modulator and Adjuvant and Chapter 46: Harnessing γδ T Cells as Natural Immune Modulators). In addition to the use of TLR ligands as adjuvants, allergens or their derivatives have also been coupled to particles to aid delivery and act as a depot for the antigen. This method might not be sufficient to induce protective immune responses. As was mentioned above, PN-coated nanoparticles were effective at treating a PN allergy, provided that CpG was added as an adjuvant but not on their own [83]. However, there have also been reports with more promise. In vitro studies showed that carbohydrate-based particles coated with the cat allergen Fel d 1 were better able to activate human monocytederived dendritic cells, with an increased upregulation of activation markers and secretion of proinflammatory cytokines [86]. When mice were injected subcutaneously with these particles, they were protected from subsequent sensitization to Fel d 1 [87]. Similar results were obtained in a study of the particles’ use as a therapy postsensitization [88]. Another study investigated the use of microparticles loaded with a peptide from the olive pollen allergen Ole e 1 as prophylactic treatment, applied intranasally before sensitization. These particles too led to a decreased production of allergen-specific IgE and IgG1 as well as an increase in IgG2a; in addition, treated mice achieved a much better lung histopathology score [89] (Chapter 19: Current and New Approaches for Mucosal Vaccine Delivery). In addition to these delivery options, a few groups have explored the possibility of treatment with bacteria expressing recombinant allergens. Intranasal application of Lactobacillus lactis expressing the cow’s milk allergen BLG prior to sensitization increased levels of BLGspecific IgG2a in serum as well as bronchoalveolar lavage fluid (BALF), especially if it was coadministered with another strain expressing IL-12. This was accompanied by a decrease of IL-4 and IL-5 as well as reduced eosinophil numbers in the BALF of these mice, but not a reduction in

861

BLG-specific IgE [90]. Another group compared the efficiency of intranasal versus oral administration of Lactobacillus casei expressing BLG before sensitization. Interestingly, while the intranasal treatment led to an increase in BLGspecific serum IgG1 as well as IgG2a and no alteration in IgE, the oral treatment showed the opposite result with a decrease in IgE but no significant change in IgG levels [91]. The route of oral administration was chosen in two studies by Ai et al., with L. lactis expressing either the wild-type Der p 2 [92] or a hypoallergenic mutant of it [93]. In both cases, prophylactic treatment with these bacteria significantly reduced Der p 2 specific serum IgE and increased IgG2a while not affecting IgG1 levels. Both treatments also alleviated lung inflammation after inhalation challenge with house dust mite allergen, with decreased infiltration of inflammatory cells as seen by histological analysis as well as reduced levels of IL-4 and Il-5 in the BALF. In addition, both treatments at least temporarily increased the percentage of Foxp31 CD41 T cells in the mesenteric lymph nodes, indicating a potential role for regulatory T cells. In a murine model for PN allergy, administration of live L. lactis expressing Ara h 2 by oral gavage as a prophylactic treatment before sensitization against PN induced an increased production of IgG2a antibodies and reduced the levels of PN-specific IgE [94]. Of note, after promising results in murine studies [95], a clinical trial using Ara h 1, 2, and 3 with point mutations in their IgE epitopes encapsulated in killed Escherichia coli and administered rectally to PN allergic patients resulted in a fairly high rate of adverse reactions, the severity of which appeared to correlate with the patients’ baseline levels of anti-Ara h 2 IgE [96].

D. DNA Vaccines Another option explored for treatment of allergies is the use of DNA vaccines. These

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51. MUCOSAL VACCINES FOR ALLERGY AND TOLERANCE

have clear advantages, considering that any wild-type allergen sequence can be used and made hypoallergenic simply by targeting it for direct degradation either via the lysosome or via proteasomal degradation (as reviewed by Weiss et al. [97]). Weinberger et al. investigated how targeting allergens to these different compartments might influence the efficiency of a vaccine by injecting mice with plasmids containing sequences for the birch pollen allergen Bet v 1 fused to the leader sequence from LIMP-II for lysosomal targeting or fused to ubiquitin for proteasomal degradation before sensitization with Bet v 1. Both of these constructs protected from inhalation challenge induced airway hyperresponsiveness just as well as a construct leading to secretion of the intact allergen [98]. In a mouse model for Der p 1 induced allergic rhinitis, prophylactic intranasal application of plasmids encoding Der p 1 or Der p 1 fused with ubiquitin was compared to the intramuscular injection of the latter. To increase transfection efficiency, chitosan was used as an adjuvant. All three vaccines improved symptoms of allergic rhinitis, measured by observing the frequency of sneezing and nose scratching, and reduced the number of eosinophils invading the nasal lamina propria, although the two vaccines applied intranasally proved slightly more effective. Furthermore, they induced higher levels of serum IFNγ as well as lower levels of serum IL-4. Similarly, the levels of Der p 1-specific serum IgE were lower and levels of IgG were higher in the intranasally vaccinated mice, with the ubiquitin-containing variant performing better than the plasmid encoding unaltered Der p 1 [99]. In addition, it is possible to increase the efficiency of vaccination by using vectors targeting the plasmids to tissues of interest. Yamasaki et al. used recombination-deficient adenovirus 40 as a vector targeting the vaccine to the intestinal mucosa to establish whether this increases its efficacy in a murine model of sensitization

with OVA. In contrast to Ad5, which has been demonstrated to be a poor candidate for mucosal delivery of a vaccine, Ad40 was able to transfect intestinal cells after oral gavage or intraduodenal delivery, with reporter DNA found especially in Peyer’s patches of the ileum as well as in mesenteric lymph nodes. Prophylactic vaccination with Ad40 used as vector protected mice from subsequent sensitization with ovalbumin [100]. Judging from this sampling of results, the use of DNA vaccines to combat allergies is a promising strategy. DNA vaccines targeting allergens to the lysosome are currently being investigated in clinical trials (ClinicalTrials.gov identifier: NCT03101267 against Japanese red cedar pollen and ClinicalTrials.gov identifier: NCT02851277 for a vaccine against peanut allergy), although these are applied by intradermal or intramuscular injection.

III. MICROBIOTA In recent years, the influence that our microbiome has on our immune system has gained the attention of researchers as well as the public. It therefore comes as no surprise that the microbiome is under investigation for its potential to protect individuals from—or make them susceptible to—allergies. In fact, the rapid rise in allergy prevalence has been attributed to environmental and lifestyle changes in Western societies, which affect the composition of our microbiota. Even as far back as 1989, Strachan postulated that microbial infections protect against allergy, which became known as the hygiene hypothesis [101]. An expansion of the original hypothesis suggests that a number of factors affect the development and activity of our immune systems either directly or via alterations in the composition of our commensal microbiota. These include not only the abovementioned reduction in pathogenic disease, but also in infections with enteropathogens like

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REFERENCES

helminth parasites, heavy antibiotic use (prenatal as well as postnatal), mode of birth (conventional vs cesarean section), and infant feeding (breast vs formula) as well as a general change in our diet toward high-fat, low-fiber diets (as reviewed by Feehley et al. [102]). Considering this, several approaches are being investigated to combat the rise in allergies by the alteration of the gut microbiota itself. A clinical trial investigating fecal microbiota transplantation in peanut-allergic adults is currently ongoing (ClinicalTrials.gov identifier: NCT02960074). In addition to transferring healthy microbiota, which is a challenge in itself, considering that more knowledge is needed to define what constitutes a healthy microbiome, it would probably be necessary to alter the diet of the patient to include foods that support the survival of the desired microbiota and avoid a return to the original dysbiotic state. The use of probiotics is another option that is currently being pursued, although there is little evidence for the efficacy for currently available formulations postinfancy [103]. Meanwhile, the search for the composition of a healthy microbiome is ongoing and has provided some valuable insights. Several studies have investigated the microbiota composition of infants who suffered from food allergies [104] or who would later develop food allergies [105,106] or asthma [107 109]. Although the exact composition of the microbiota differed between the studies, allergic sensitization was generally associated with dysbiosis early in life. Similarly, a study in children with milk allergy analyzed the microbiome composition in fecal samples collected at age 3 6 months of age and found an enrichment of specific groups of bacteria, namely, Firmicutes and Clostridia, in the feces of children that resolved the allergy by age 8 years [110]. Earlier work using gnotobiotic mouse models showed that Clostridia protect mice from peanut-induced allergic sensitization via induction of IL-22 [111]. Moreover, Clostridia species are among the bacteria that have the

ability to ferment fiber from our diet that we ourselves are not able to digest. The absence of dietary fiber alters the composition of the gut microbiome [112,113]; the concomitant reduction in short-chain fatty acids can ultimately contribute to the development or exacerbation of allergy [114,115]. Butyrate, in particular, is of interest for the treatment of allergy, owing to its barrier protective effects and its ability to induce Treg differentiation [114,116 119], emphasizing the critical role of a bacteria-induced barrier protective response in protection against allergic sensitization [120] (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity).

IV. CONCLUDING REMARKS While the exact mechanisms behind tolerance induction or allergy are still not fully known and treatment options for allergic individuals are currently limited, the topic has gained a lot of attention from researchers as well as industry. New aspects of the immune response involved, especially in light of the contribution of our microbiome, are being uncovered regularly, and quite a few promising approaches for novel or improved prophylactic or therapeutic treatments are under development. Considering this progress, many more options for effective treatment may soon be available to allergy patients.

References [1] Allergy UK facts sheet, ,https://www.allergyuk.org/ assets/000/001/193/Stats_for_Website_original.pdf? 1501059772. [accessed 08.03.17]. [2] Reynolds LA, Finlay BB. Early life factors that affect allergy development. Nat Rev Immunol 2017;17: 518 28. [3] Hayen SM, Kostadinova AI, Garssen J, Otten HG, Willemsen LE. Novel immunotherapy approaches to food allergy. Curr Opin Allergy Clin Immunol 2014;14: 549 56.

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864

51. MUCOSAL VACCINES FOR ALLERGY AND TOLERANCE

[4] Wood RA. Food allergen immunotherapy: current status and prospects for the future. J Allergy Clin Immunol 2016;137:973 82. [5] Akdis M, Akdis CA. Mechanisms of allergen-specific immunotherapy: multiple suppressor factors at work in immune tolerance to allergens. J Allergy Clin Immunol 2014;133:621 31. [6] Dall’antonia F, Pavkov-Keller T, Zangger K, Keller W. Structure of allergens and structure based epitope predictions. Methods 2014;66:3 21. [7] Marsh DG, Lichtenstein LM, Campbell DH. Studies on “allergoids” prepared from naturally occurring allergens. I. Assay of allergenicity and antigenicity of formalinized rye group I component. Immunology 1970;18:705 22. [8] Company mentioned: Allergopharma GmbH & Co. KG, ,https://www.allergopharma.us/home/. [accessed 08.03.17]. [9] Corrigan CJ, Kettner J, Doemer C, Cromwell O, Narkus A. Efficacy and safety of preseasonal-specific immunotherapy with an aluminium-adsorbed sixgrass pollen allergoid. Allergy 2005;60:801 7. [10] Chaker AM, Al-Kadah B, Luther U, Neumann U, Wagenmann M. An accelerated dose escalation with a grass pollen allergoid is safe and well-tolerated: a randomized open label phase II trial. Clin Transl Allergy 2015;6:4. [11] Company mentioned: HAL allergy, ,http://www. hal-allergy.com/nc/home/. [accessed 08.03.17]. [12] Ceuppens JL, Bullens D, Kleinjans H, Van der Werf J, Group PBES. Immunotherapy with a modified birch pollen extract in allergic rhinoconjunctivitis: clinical and immunological effects. Clin Exp Allergy 2009;39:1903 9. [13] Bozek A, Kolodziejczyk K, Jarzab J. Safety and efficacy of tree pollen specific immunotherapy on the ultrarush administration schedule method using purethal trees. Biomed Res Int 2014;707634. [14] Lozano J, Cruz MJ, Piquer M, Giner MT, Plaza AM. Assessing the efficacy of immunotherapy with a glutaraldehyde-modified house dust mite extract in children by monitoring changes in clinical parameters and inflammatory markers in exhaled breath. Int Arch Allergy Immunol 2014;165:140 7. [15] Starkl P, Felix F, Krishnamurthy D, Stremnitzer C, Roth-Walter F, Prickett SR, et al. An unfolded variant of the major peanut allergen Ara h 2 with decreased anaphylactic potential. Clin Exp Allergy 2012;42: 1801 12. [16] Toda M, Reese G, Gadermaier G, Schulten V, Lauer I, Egger M, et al. Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen. J Allergy Clin Immunol 2011; 128:1022 30 e1-7.

[17] Marazuela EG, Rodriguez R, Barber D, Villalba M, Batanero E. Hypoallergenic mutants of Ole e 1, the major olive pollen allergen, as candidates for allergy vaccines. Clin Exp Allergy 2006;37:251 60. [18] Wald M, Kahlert H, Weber B, Jankovic M, Keller W, Cromwell O, et al. Generation of a low immunoglobulin E-binding mutant of the timothy grass pollen major allergen Phl p 5a. Clin Exp Allergy 2007;37:441 50. [19] Vrtala S, Focke M, Kopec J, Verdino P, Hartl A, Sperr WR, et al. Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity. J Immunol 2007;179:1730 9. [20] Vrtala S, Hirtenlehner K, Vangelista L, Pastore A, Eichler HG, Sperr WR, et al. Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy. J Clin Invest 1997;99:1673 81. [21] van Hage-Hamsten M, Kronqvist M, Zetterstrom O, Johansson E, Niederberger V, Vrtala S, et al. Skin test evaluation of genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1: results obtained with a mix of two recombinant Bet v 1 fragments and recombinant Bet v 1 trimer in a Swedish population before the birch pollen season. J Allergy Clin Immunol 1999;104:969 77. [22] van Hage-Hamsten M, Johansson E, Roquet A, Peterson C, Andersson M, Greiff L, et al. Nasal challenges with recombinant derivatives of the major birch pollen allergen Bet v 1 induce fewer symptoms and lower mediator release than rBet v 1 wild-type in patients with allergic rhinitis. Clin Exp Allergy 2002;32:1448 53. [23] Bonura A, Amoroso S, Locorotondo G, Di Felice G, Tinghino R, Geraci DJ, Colombo P. Hypoallergenic variants of the Parietaria judaica major allergen Par j 1: a member of the non-specific lipid transfer protein plant family. Int Arch Allergy Immunol 2001;126:32 40. [24] Olsson S, Van Hage-Hamsten M, Whitley P. Contribution of disulphide bonds to antigenicity of Lep d 2, the major allergen of the dust mite Lepidoglyphus destructor. Mol Immunol 1998;35:1017 23. [25] Swoboda I, Bugajska-Schretter A, Linhart B, Verdino P, Keller W, Schulmeister U, et al. A recombinant hypoallergenic parvalbumin mutant for immunotherapy of IgE-mediated fish allergy. J Immunol 2007;178:6290 6. [26] Zuidmeer-Jongejan L, Huber H, Swoboda I, Rigby N, Versteeg SA, Jensen BM, et al. Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy. Int Arch Allergy Immunol 2015;166:41 51.

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REFERENCES

[27] Garmatiuk T, Swoboda I, Twardosz-Kropfmuller A, Dall’antonia F, Keller W, Singh MB, et al. Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy. Immunobiology 2013;218:1155 65. [28] Westritschnig K, Focke M, Verdino P, Goessler W, Keller W, Twardosz A, et al. Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7. J Immunol 2004;172:5684 92. [29] Hoflehner E, Hufnagl K, Schabussova I, Jasinska J, Hoffmann-Sommergruber K, Bohle B, et al. Prevention of birch pollen-related food allergy by mucosal treatment with multi-allergen-chimers in mice. PLoS One 2012;7:e39409. [30] Linhart B, Mothes-Luksch N, Vrtala S, Kneidinger M, Valent P, Valenta R. A hypoallergenic hybrid molecule with increased immunogenicity consisting of derivatives of the major grass pollen allergens, Phl p 2 and Phl p 6. Biol Chem 2008;389:925 33. [31] Linhart B, Hartl A, Jahn-Schmid B, Verdino P, Keller W, Krauth MT, et al. A hybrid molecule resembling the epitope spectrum of grass pollen for allergy vaccination. J Allergy Clin Immunol 2005;115:1010 16. [32] Asturias JA, Ibarrola I, Arilla MC, Vidal C, Ferrer A, Gamboa PM, et al. Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy. Clin Exp Allergy 2009;39:1088 98. [33] Chen KW, Blatt K, Thomas WR, Swoboda I, Valent P, Valenta R, et al. Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy. J Allergy Clin Immunol 2012;130 435 43 e4. [34] Gonzalez-Rioja R, Ibarrola I, Arilla MC, Ferrer A, Mir A, Andreu C, et al. Genetically engineered hybrid proteins from Parietaria judaica pollen for allergen-specific immunotherapy. J Allergy Clin Immunol 2007;120:602 9. [35] Focke-Tejkl M, Weber M, Niespodziana K, Neubauer A, Huber H, Henning R, et al. Development and characterization of a recombinant, hypoallergenic, peptidebased vaccine for grass pollen allergy. J Allergy Clin Immunol 2015;135 1207 1207 e1-11. [36] Hofer H, Asam C, Hauser M, Nagl B, Laimer J, Himly M, et al. Tackling Bet v 1 and associated food allergies with a single hybrid protein. J Allergy Clin Immunol 2016;140:525 33. [37] Vrtala S, Hirtenlehner K, Susani M, Hufnagl P, Binder BR, Vangelista L, et al. Genetic engineering of recombinant hypoallergenic oligomers of the major birch pollen allergen, Bet v 1: candidates for specific immunotherapy. Int Arch Allergy Immunol 1999;118:218 19. [38] Rabjohn P, West CM, Connaughton C, Sampson HA, Helm RM, Burks AW, et al. Modification of peanut

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

865 allergen Ara h 3: effects on IgE binding and T cell stimulation. Int Arch Allergy Immunol 2002;128:15 23. Ferreira F, Ebner C, Kramer B, Casari G, Briza P, Kungl AJ, et al. Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEB J 1998;12: 231 42. Holm J, Gajhede M, Ferreras M, Henriksen A, Ipsen H, Larsen JN, et al. Allergy vaccine engineering: epitope modulation of recombinant Bet v 1 reduces IgE binding but retains protein folding pattern for induction of protective blocking-antibody responses. J Immunol 2004;173:5258 67. Swoboda I, De Weerd N, Bhalla PL, Niederberger V, Sperr WR, Valent P, et al. Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgEbinding capacity: candidates for grass pollen-specific immunotherapy. Eur J Immunol 2002;32:270 80. Niederberger V, Horak F, Vrtala S, Spitzauer S, Krauth MT, Valent P, et al. Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc Natl Acad Sci USA 2004;101(Suppl. 2): 14677 82. Gafvelin G, Thunberg S, Kronqvist M, Gronlund H, Gronneberg R, Troye-Blomberg M, et al. Cytokine and antibody responses in birch-pollen-allergic patients treated with genetically modified derivatives of the major birch pollen allergen Bet v 1. Int Arch Allergy Immunol 2005;138:59 66. Campana R, Vrtala S, Maderegger B, Jertschin P, Stegfellner G, Swoboda I, et al. Hypoallergenic derivatives of the major birch pollen allergen Bet v 1 obtained by rational sequence reassembly. J Allergy Clin Immunol 2010;126:1024 31 1031 e1-8. Ball T, Linhart B, Sonneck K, Blatt K, Herrmann H, Valent P, et al. Reducing allergenicity by altering allergen fold: a mosaic protein of Phl p 1 for allergy vaccination. Allergy 2009;64:569 80. Mothes-Luksch N, Stumvoll S, Linhart B, Focke M, Krauth MT, Hauswirth A, et al. Disruption of allergenic activity of the major grass pollen allergen Phl p 2 by reassembly as a mosaic protein. J Immunol 2008; 181:4864 73. Westritschnig K, Linhart B, Focke-Tejkl M, Pavkov T, Keller W, Ball T, et al. A hypoallergenic vaccine obtained by tail-to-head restructuring of timothy grass pollen profilin, Phl p 12, for the treatment of crosssensitization to profilin. J Immunol 2007;179:7624 34. Curin M, Weber M, Thalhamer T, Swoboda I, FockeTejkl M, Blatt K, et al. Hypoallergenic derivatives of Fel d 1 obtained by rational reassembly for allergy vaccination and tolerance induction. Clin Exp Allergy 2014;44:882 94.

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

866

51. MUCOSAL VACCINES FOR ALLERGY AND TOLERANCE

[49] Chen KW, Fuchs G, Sonneck K, Gieras A, Swoboda I, Douladiris N, et al. Reduction of the in vivo allergenicity of Der p 2, the major house-dust mite allergen, by genetic engineering. Mol Immunol 2008;45:2486 98. [50] Zieglmayer P, Focke-Tejkl M, Schmutz R, Lemell P, Zieglmayer R, Weber M, et al. Mechanisms, safety and efficacy of a B cell epitope-based vaccine for immunotherapy of grass pollen allergy. EBioMedicine 2016;11: 43 57. [51] Fukuda K, Ishida W, Harada Y, Wakasa Y, Takagi H, Takaiwa F, et al. Efficacy of oral immunotherapy with a rice-based edible vaccine containing hypoallergenic Japanese cedar pollen allergens for treatment of established allergic conjunctivitis in mice. Allergol Int 2018;67:119 23. [52] Prickett SR, Rolland JM, O’Hehir RE. Immunoregulatory T cell epitope peptides: the new frontier in allergy therapy. Clin Exp Allergy 2015;45:1015 26. [53] Hoyne GF, O’Hehir RE, Wraith DC, Thomas WR, Lamb JR. Inhibition of T cell and antibody responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive and sensitized mice. J Exp Med 1993;178:1783 8. [54] Marazuela EG, Rodriguez R, Fernandez-Garcia H, Garcia MS, Villalba M, Batanero E. Intranasal immunization with a dominant T-cell epitope peptide of a major allergen of olive pollen prevents mice from sensitization to the whole allergen. Mol Immunol 2008;45: 438 45. [55] Astori M, von Garnier C, Kettner A, Dufour N, Corradin G, Spertini F. Inducing tolerance by intranasal administration of long peptides in naive and primed CBA/J mice. J Immunol 2000;165:3497 505. [56] Rupa P, Mine Y. Oral immunotherapy with immunodominant T-cell epitope peptides alleviates allergic reactions in a Balb/c mouse model of egg allergy. Allergy 2012;67:74 82. [57] Meulenbroek LA, van Esch BC, Hofman GA, den Hartog Jager CF, Nauta AJ, Willemsen LE, et al. Oral treatment with beta-lactoglobulin peptides prevents clinical symptoms in a mouse model for cow’s milk allergy. Pediatr Allergy Immunol 2013;24:656 64. [58] Patel D, Couroux P, Hickey P, Salapatek AM, Laidler P, Larche M, et al. Fel d 1-derived peptide antigen desensitization shows a persistent treatment effect 1 year after the start of dosing: a randomized, placebocontrolled study. J Allergy Clin Immunol 2013;131 1039 e1-e7. [59] Couroux P, Patel D, Armstrong K, Larche M, Hafner RP. Fel d 1-derived synthetic peptide immunoregulatory epitopes show a long-term treatment effect in cat allergic subjects. Clin Exp Allergy 2015;45: 974 81.

[60] Ellis AK, Frankish CW, O’Hehir RE, Armstrong K, Steacy L, Larche M, et al. Treatment with grass allergen peptides improves symptoms of grass polleninduced allergic rhinoconjunctivitis. J Allergy Clin Immunol 2017;1 11. [61] Circassia press release from 6/20/2016, ,http:// www.circassia.com/media/press-releases/circassiaannounces-top-line-results-from-cat-allergy-phase-iiistudy/. [accessed 08.03.17]. [62] Busse W, Corren J, Lanier BQ, Mcalary M, FowlerTaylor A, Cioppa GD, et al. Omalizumab, anti-IgE recombinant humanized monoclonal antibody, for the treatment of severe allergic asthma. J Allergy Clin Immunol 2001;108:184 90. [63] Milgrom H, Berger W, Nayak A, Gupta N, Pollard S, Mcalary M, et al. Treatment of childhood asthma with anti-immunoglobulin E antibody (omalizumab). Pediatrics 2001;108:E36. [64] Soler M, Matz J, Townley R, Buhl R, O’Brien J, Fox H, et al. The anti-IgE antibody omalizumab reduces exacerbations and steroid requirement in allergic asthmatics. Eur Respir J 2001;18:254 61. [65] Casale TB, Condemi J, Laforce C, Nayak A, Rowe M, Watrous M, et al. Effect of omalizumab on symptoms of seasonal allergic rhinitis. JAMA 2001;286:2956. [66] Wood RA, Kim JS, Lindblad R, Nadeau K, Henning AK, Dawson P, et al. A randomized, double-blind, placebocontrolled study of omalizumab combined with oral immunotherapy for the treatment of cow’s milk allergy. J Allergy Clin Immunol 2016;137:1103 10 e1-11. [67] Nadeau KC, Schneider LC, Hoyte L, Borras I, Umetsu DT. Rapid oral desensitization in combination with omalizumab therapy in patients with cow’s milk allergy. J Allergy Clin Immunol 2011;127:1622 4. [68] Macginnitie AJ, Rachid R, Gragg H, Little SV, Lakin P, Cianferoni A, et al. Omalizumab facilitates rapid oral desensitization for peanut allergy. J Allergy Clin Immunol 2017;139:873 81 e8. [69] Bedoret D, Singh AK, Shaw V, Hoyte EG, Hamilton R, Dekruyff RH, et al. Changes in antigen-specific T-cell number and function during oral desensitization in cow’s milk allergy enabled with omalizumab. Mucosal Immunol 2012;5:267 76. [70] Schneider LC, Rachid R, Lebovidge J, Blood E, Mittal M, Umetsu DT. A pilot study of amalizumab to faciliate rapid oral desensitization in high-risk peanut allergic patients. J Allergy Clin Immunol 2013;132:1368 74. [71] Lafuente I, Mazon A, Nieto M, Uixera S, Pina R, Nieto A. Possible recurrence of symptoms after discontinuation of omalizumab in anti-IgE-assisted desensitization to egg. Pediatr Allergy Immunol 2015;25:717 18. [72] Martorell-Calatayud C, Michavila-Gomez A, Martorell-Aragones A, Molini-Menchon N, Cerda-Mir

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

REFERENCES

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

JC, Felix-Toledo R, et al. Anti-IgE-assisted desensitization to egg and cow’s milk in patients refractory to conventional oral immunotherapy. Pediatr Allergy Immunol 2016;27:544 6. Wright JD, Chu HM, Huang CH, Ma C, Chang TW, Lim C. Structural and physical basis for anti-IgE therapy. Sci Rep 2015;5:11581. Davies AM, Allan EG, Keeble AH, Delgado J, Cossins BP, Mitropoulou AN, et al. Allosteric mechanism of action of the therapeutic anti-IgE antibody omalizumab. J Biol Chem 2017;292:9975 87. Pennington LF, Tarchevskaya S, Brigger D, Sathiyamoorthy K, Graham MT, Nadeau KC, et al. Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange. Nat Commun 2016;7:11610. Kim B, Eggel A, Tarchevskaya SS, Vogel M, Prinz H, Jardetzky TS. Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor. Nature 2012;491:613 17. Eggel A, Baravalle G, Hobi G, Kim B, Buschor P, Forrer P, et al. Accelerated dissociation of IgE-FcepsilonRI complexes by disruptive inhibitors actively desensitizes allergic effector cells. J Allergy Clin Immunol 2014;133:1709 19 e8. Gauvreau GM, Arm JP, Boulet LP, Leigh R, Cockcroft DW, Davis BE, et al. Efficacy and safety of multiple doses of QGE031 (ligelizumab) versus omalizumab and placebo in inhibiting allergen-induced early asthmatic responses. J Allergy Clin Immunol 2016;138:1051 9. Gauvreau GM, Harris JM, Boulet LP, Scheerens H, Fitzgerald JM, Putnam WS, et al. Targeting membraneexpressed IgE B cell receptor with an antibody to the M1 prime epitope reduces IgE production. Sci Transl Med 2014;6:243ra85. Harris JM, Maciuca R, Bradley MS, Cabanski CR, Scheerens H, Lim J, et al. A randomized trial of the efficacy and safety of quilizumab in adults with inadequately controlled allergic asthma. Respir Res 2016;17:29. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000;408:740 4. Bashir ME, Louie S, Shi HN, Nagler-Anderson C. Tolllike receptor 4 signaling by intestinal microbes influences susceptibility to food allergy. J Immunol 2004; 172:6978 87. Srivastava KD, Siefert A, Fahmy TM, Caplan MJ, Li XM, Sampson HA. Investigation of peanut oral immunotherapy with CpG/peanut nanoparticles in a murine model of peanut allergy. J Allergy Clin Immunol 2016;138:536 43 e4. Schulke S, Wolfheimer S, Gadermaier G, Wangorsch A, Siebeneicher S, Briza P, et al. Prevention of intestinal allergy in mice by rflaA:Ova is associated with

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

867 enforced antigen processing and TLR5-dependent IL10 secretion by mDC. PLoS One 2014;9:e87822. Rosewich M, Lee D, Zielen S. Pollinex Quattro: an innovative four injections immunotherapy in allergic rhinitis. Human Vaccines Immunother 2013;9:1523 31. Andersson TN, Ekman GJ, Gronlund H, Buentke E, Eriksson TL, Scheynius A, et al. A novel adjuvantallergen complex, CBP-rFel d 1, induces up-regulation of CD86 expression and enhances cytokine release by human dendritic cells in vitro. Immunology 2004;113: 253 9. Thunberg S, Neimert-Andersson T, Cheng Q, Wermeling F, Bergstrom U, Swedin L, et al. Prolonged antigen-exposure with carbohydrate particle based vaccination prevents allergic immune responses in sensitized mice. Allergy 2009;64:919 26. Neimert-Andersson T, Thunberg S, Swedin L, Wiedermann U, Jacobsson-Ekman G, Dahlen SE, et al. Carbohydrate-based particles reduce allergic inflammation in a mouse model for cat allergy. Allergy 2008; 63:518 26. Marazuela EG, Prado N, Moro E, Fernandez-Garcia H, Villalba M, Rodriguez R, et al. Intranasal vaccination with poly(lactide-co-glycolide) microparticles containing a peptide T of Ole e 1 prevents mice against sensitization. Clin Exp Allergy 2008;38:520 8. Cortes-Perez NG, Ah-Leung S, Bermudez-Humaran LG, Corthier G, Wal JM, Langella P, et al. Intranasal coadministration of live lactococci producing interleukin-12 and a major cow’s milk allergen inhibits allergic reaction in mice. Clin Vaccine Immunol 2007; 14:226 33. Hazebrouck S, Przybylski-Nicaise L, Ah-Leung S, Adel-Patient K, Corthier G, Langella P, et al. Influence of the route of administration on immunomodulatory properties of bovine beta-lactoglobulin-producing Lactobacillus casei. Vaccine 2009;27:5800 5. Ai C, Zhang Q, Ren C, Wang G, Liu X, Tian F, et al. Genetically engineered Lactococcus lactis protect against house dust mite allergy in a BALB/c mouse model. PLoS One 2014;9:e109461. Ai C, Zhang Q, Ding J, Ren C, Wang G, Liu X, et al. Suppression of dust mite allergy by mucosal delivery of a hypoallergenic derivative in a mouse model. Appl Microbiol Biotechnol 2015;99:4309 19. Ren C, Zhang Q, Wang G, Ai C, Hu M, Liu X, et al. Modulation of peanut-induced allergic immune responses by oral lactic acid bacteria-based vaccines in mice. Appl Microbiol Biotechnol 2014;98:6353 64. Li XM, Srivastava K, Grishin A, Huang CK, Schofield B, Burks W, et al. Persistent protective effect of heatkilled Escherichia coli producing “engineered,” recombinant peanut proteins in a murine model of peanut allergy. J Allergy Clin Immunol 2003;112:159 67.

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NONINFECTIOUS DISEASES?

868

51. MUCOSAL VACCINES FOR ALLERGY AND TOLERANCE

[96] Wood RA, Sicherer SH, Burks AW, Grishin A, Henning AK, Lindblad R, et al. A phase 1 study of heat/phenol-killed, E. coli-encapsulated, recombinant modified peanut proteins Ara h 1, Ara h 2, and Ara h 3 (EMP-123) for the treatment of peanut allergy. Allergy 2013;68:803 8. [97] Weiss R, Schreiblhofer S, Thalhamer J. What is the antiallergic potential of DNA vaccination? Immunotherapy 2015;7:587 90. [98] Weinberger EE, Isakovic A, Scheiblhofer S, Ramsauer C, Reiter K, Hauser-Kronberger C, et al. The influence of antigen targeting to sub-cellular compartments on the anti-allergic potential of a DNA vaccine. Vaccine 2013;31:6113 21. [99] Ou J, Shi W, Xu Y, Tao Z. Intranasal immunization with DNA vaccine coexpressing Der p 1 and ubiquitin in an allergic rhinitis mouse model. Ann Allergy Asthma Immunol 2014;113:658 65 e1. [100] Yamasaki S, Miura Y, Davydova J, Vickers SM, Yamamoto M. A single intraduodenal administration of human adenovirus 40 vaccine effectively prevents anaphylactic shock. Clin Vaccine Immunol 2013;20: 1508 16. [101] Strachan DP. Hay fever, hygiene, and household size. BMJ 1989;299:1259 60. [102] Feehley T, Stefka AT, Cao S, Nagler CR. Microbial regulation of allergic responses to food. Semin Immunopathol 2012;34:671 88. [103] Elazab N, Mendy A, Gasana J, Vieira ER, Quizon A, Forno E. Probiotic administration in early life, atopy, and asthma: a meta-analysis of clinical trials. Pediatrics 2013;132:e666 76. [104] Berni canani R, Sangwan N, Stefka AT, Nocerino R, Paparo L, Aitoro R, et al. Lactobacillus rhamnosus GGsupplemented formula expands butyrate-producing bacterial strains in food allergic infants. ISME J 2016; 10:742 50. [105] Azad MB, Konya T, Guttman DS, Field CJ, Sears MR, Hayglass KT, et al. Infant gut microbiota and food sensitization: associations in the first year of life. Clin Exp Allergy 2015;45:632 43. [106] Savage JH, Lee-Sarwar KA, Sordillo J, Bunyavanich S, Zhou Y, O’Connor G, et al. A prospective microbiome-wide association study of food sensitization and food allergy in early childhood. Allergy 2017;1:AB96. [107] Arrieta MC, Stiemsma LT, Dimitriu PA, Thorson L, Russell S, Yurist-Doutsch S, et al. Early infancy microbial and metabolic alterations affect risk of childhood asthma. Sci Transl Med 2015;7:307ra152. [108] Abrahamsson TR, Jakobsson HE, Andersson AF, Bjorksten B, Engstrand L, Jenmalm MC. Low gut

[109]

[110]

[111]

[112]

[113]

[114]

[115]

[116]

[117]

[118]

[119]

[120]

microbiota diversity in early infancy precedes asthma at school age. Clin Exp Allergy 2014;44:842 50. Fujimura KE, Sitarik AR, Havstad S, Lin DL, Levan S, Fadrosh D, et al. Neonatal gut microbiota associates with childhood multisensitized atopy and T cell differentiation. Nat Med 2016;22:1187 91. Bunyavanich S, Shen N, Grishin A, Wood R, Burks W, Dawson P, et al. Early-life gut microbiome composition and milk allergy resolution. J Allergy Clin Immunol 2016;138:1122 30. Stefka AT, Feehley T, Tripathi P, Qiu J, Mccoy K, Mazmanian SK, et al. Commensal bacteria protect against food allergen sensitization. Proc Natl Acad Sci USA 2014;111:13145 50. Goverse G, Molenaar R, Macia L, Tan J, Erkelens MN, Konijn T, et al. Diet-derived short chain fatty acids stimulate intestinal epithelial cells to induce mucosal tolerogenic dendritic cells. J Immunol 2017;198: 2172 81. Desai MS, Seekatz AM, Koropatkin NM, Kamada N, Hickey CA, Wolter M, et al. A dietary fiber-deprived gut microbiota degrades the colonic mucus barrier and enhances pathogen susceptibility. Cell 2016;167: 1339 53 e21. Tan J, Mckenzie C, Vuillermin PJ, Goverse G, Vinuesa CG, Mebius RE, et al. Dietary fiber and bacterial SCFA enhance oral tolerance and protect against food allergy through diverse cellular pathways. Cell Rep 2016;15:2809 24. Trompette A, Gollwitzer ES, Yadava K, Sichelstiel AK, Sprenger N, Ngom-Bru C, et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat Med 2014;20:159 66. Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G, Takahashi D, et al. Commensal microbe-derived butyrate induces differentiation of colonic regulatory T cells. Nature 2013;504:446 50. Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly YM, et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 2013;341:569 73. Schwarz A, Bruhs A, Schwarz T. The short-chain fatty acid sodium butyrate functions as a regulator of the skin immune system. J Invest Dermatol 2017;137: 855 64. Arpaia N, Campbell C, Fan X, Dikiy S, van der Veeken J, Deroos P, et al. Metabolites produced by commensal bacteria promote peripheral regulatory T cell generation. Nature 2013;504:451 5. Wesemann DR, Nagler CR. The microbiome, timing, and barrier function in the context of allergic disease. Immunity 2016;44:728 38.

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C H A P T E R

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Novel Strategies for Targeting the Control of Mucosal Inflammation Peter B. Ernst1,2,3,4, Ioannis Drygiannakis1,2 and Hiutung Chu1,2,3,4 1

Department of Pathology, Center for Veterinary Sciences and Comparative Medicine, Chiba University, Chiba, Japan 2Chiba University-UCSD Center for Mucosal Immunology, Allergy and Vaccine Development (CU-UCSD cMAV), University of California San Diego, La Jolla, CA, United States 3 The Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, UC Davis, Davis, CA, United States 4Department of Immunology, Chiba University, Chiba, Japan

I. INTRODUCTION The gastrointestinal tract is more than a tissue with physiological functions; it is home to trillions of microbes that modulate host immunity and metabolism. This complex ecosystem requires a delicate homeostatic balance in order for the tissue to remain healthy and functional [1,2]. The intestinal niche involves a multifaceted and dynamic mixture of host cells and molecules, microbes and their metabolites, and environmental factors that may reflect diet or contamination with toxins. Furthermore, gene expression in the host can modulate transcription in microbes [3], resulting in changes in their metabolic profile, expression of virulence factors, and local immune responses that resonate throughout the body [4 7]. These biological spheres are highly interconnected such that the microbial host interactions that begin in

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00052-3

the digestive tract affect not only gastrointestinal inflammation and risks of local cancer, but also the host’s susceptibility to conditions ranging from diabetes to obesity to various neurological conditions [8]. This interconnectivity leads to the possibility that novel approaches in mucosal immunization may alter the host microbial balance in a manner that attenuates immune-mediated diseases throughout the body.

II. FOR EVERY ACTION, THERE IS AN EQUAL AND OPPOSITE REACTION Host responses and the microbiota are highly codependent. Thus an ideal microbiome would favor immunological harmony and homeostasis. The loss of homeostasis can create

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dysbiosis of the gut microbiota that appears to be associated with an increased risk of further immunological dysfunction. One approach to test this model is to build it from individual components. For example, in germ-free conditions, the host lacks its associated microbiota and has a correspondingly diminished complement of immune and inflammatory cells in the mucosa [9]. Interestingly, while attenuated, there is a detectable “innate” B cells response including polyreactive immunoglobulin A (IgA) that reacted to multiple taxa [10]. In examples in which germ-free mice are associated with limited numbers of bacterial species, the colonizing species is often found in sites it normally does not inhabit or in unusually high numbers. Similarly, the host response in gnotobiotic mice (with very limited bacterial communities) can be proinflammatory or antiinflammatory, depending on the bacterial species [11 14]. For example, Kim et al. have shown that colonization of germ-free interleukin 10 (IL-10)2/2 mice with two species of bacteria are markedly more susceptible to colitis [12]. In germ-free IL-2-deficient mice, monoassociation with a strain of Escherichia coli caused colitis while coinfecting the mice with Bacteroides vulgatus attenuated the inflammation [14]. Upon colonization with a complex microbiota during the perinatal period, microbes communicate among each other via quorum sensing and, in so doing, alter their respective gene expression patterns to establish a balance in the richness and evenness of the microbial communities [15]. Simultaneously, the innate immune system is stimulated and transitions to antigen-specific immunity in association with the accumulation of leukocytes and various immune mediators that reflect, in part, the molecular signature of the microbes. This includes an expansion of the polyreactive IgAproducing B cells in the gut [10]. After the establishment of an equilibrium, the subsequent encounters with additional commensal microbes are associated with modest changes

in cellularity (often undetectable compared to normal). Further, treatment with antibiotics can shift the microbiome [16]. Following the end of such regimens, the microbiome can return to its original state of diversity, although the restitution can be variable and affected by multiple factors [16] (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity. The microbial communities that develop in the early part of life can differ widely from host to host, owing to numerous factors. As a result, they are associated with different rates of susceptibility to a variety of diseases. For example, in the presence of gum disease, asthma, Helicobacter pylori induced gastritis, celiac disease, and inflammatory bowel disease (IBD), the tissues remain chronically inflamed, particularly with the persistence of antigenic triggers (e.g., H. pylori) or environmental cofactors (e.g., dietary gluten). H. pylori [17] and some nematodes [18] have been implicated as antiinflammatory factors, perhaps owing to the T helper (Th) cell phenotype that develops and favors their persistence (H. pylori) (Chapter 33: A Future for a Vaccine Against the CancerInducing Bacterium Helicobacter pylori) or is associated with the infection (nematodes) (Chapter 50: Mucosal Vaccine for Parasitic Infections). It is believed that the cellular and molecular microenvironment within the mucosa determine whether inflammation will resolve, equilibrate toward homeostasis, or persist and cause susceptibility to recurrent disease. Thus we propose that the microbiota selects for an “immunobiome” and vice versa. For our purposes, the immunobiome is defined as the dynamic repertoire of T and B cells receptor rearrangements and the associated cytokine milieu that is inextricably linked to the diversity of the microbiota. Evidence for the need to completely reprogram host responses comes from studies in which ablation of immunity by chemotherapy and subsequent stem cell transplantation reprogrammed the host and

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NON-INFECTIOUS DISEASES?

IV. MICROBIAL COMMUNITIES, MICROBIAL PATHOGENESIS, AND DYSBIOSIS

restored homeostasis. This approach has been reported to be effective in multiple sclerosis [19]. However, treating inflammation alone may not completely relieve the antigenic pressures that trigger, modify, and/or perpetuate the manifestations of chronic, recurrent disease. It remains to be seen whether dramatic approaches in immunotherapy (including organ transplantation) will be improved by modifying the microbiome, perhaps through fecal microbiota transplantation (FMT), targeted microbiome editing, or mucosal immunization that alters the diversity of the microbial communities toward a composition that favors an antiinflammatory environment.

III. KEY FEATURES OF THE HOST RESPONSE IN HOMEOSTASIS The goal for mucosal immune responses is to protect the host from disease without inducing so much inflammation that physiological functions are perturbed in these delicate tissues. When this is achieved, the host is in immunological homeostasis. Some of the important immunological responses that restrain host responses are the protective regulatory cells, including anergic Th cells [20], regulatory Th (Treg) cells, and B (Breg) [21] cells. Treg cells can emerge from the thymus (tTreg), develop in the periphery (pTreg), or be induced in vitro (iTreg) [22]. Breg cells, while less well understood, likely reflect varied pathways for their induction as well as their function. Mucosal tissues provide a compelling rationale for acquired peripheral tolerance. Antigen exposure in these sites can be quite dynamic, with changes in antigen concentration that occur as the result of fluctuations in diet, seasonal variation in allergens, acquired infections, or, in extreme cases, dysbiosis. In fact, the T cell receptor repertoire of tTreg and pTreg differs [23], which supports the concept that pTreg reflect a response to acquired antigens [24].

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Therefore, the host has evolved to address the fluctuations in antigenic burden by having a population of pTreg that recognize these changes and respond by mediating acquired tolerance that stabilizes immunological homeostasis (Chapter 15: Mucosal Regulatory System for the Balanced Immunity in the Gut). One of the longest standing models of this process is the phenomenon of oral tolerance. This state was defined as a systemic hyporesponsiveness induced by the persistent oral exposure to an antigen [25]. This tolerance may not have to be complete, as illustrated by the immunological responses to some naturally occurring infections. H. pylori is typically a lifelong infection of the stomach, and its persistence is favored by the presence of Treg [26 29]. Further, animal models have suggested that the Treg induced by infection with H. pylori also attenuate allergic responses in the lung [30,31]. While the persistence of H. pylori imparts its own significant risk of disease, these studies illustrate a model of homeostasis in which organisms can coexist within the host with some mutual benefit that is attributed, in part, to Treg-mediated events that limit the level of inflammation.

IV. MICROBIAL COMMUNITIES, MICROBIAL PATHOGENESIS, AND DYSBIOSIS What is normal? The exciting studies of microbial communities to date have identified tremendous variation among individuals. In some studies, the variation reflects a loss of microbial diversity, which is often associated with an increased risk of disease or phenotype, including obesity and autism [32]. The benefit of a diverse microbiota may have emerged conceptually from studies related to the hygiene hypothesis, in which certain illnesses found in economically developed countries seem to be less frequent in people from countries with

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lower standards in sanitation and more exposure to microbes [33,34]. The mixed blessing of diversity is illustrated in studies by Hsiao et al., who have shown that Ruminococcus obeum, found in Bangladeshi children, restricts colonization of Vibrio cholerae in gnotobiotic mice through quorum sensing mechanisms that lead to the repression of several V. cholerae colonization factors [35]. While diversity that includes this species may be helpful, other studies suggest that disease is enhanced when germ-free mice colonized with microbiota from Bangladeshi donors are coinfected with the cholera-inducing organism (Ansel Hsiao, UC Riverside, personal communication). These findings indicate that a single or limited number of species have the ability to regulate the outcome of disease induced by other species but that diversity per se may not always be beneficial. If the goal is diversity, it is interesting to consider how diversity is lost. As diet can have a profound effect on the microbiome, it is not surprising that modern-day intestinal microbial communities differ from those of earlier times, as illustrated by the microbial communities in the Hadza, who sustain themselves as huntergatherers [36]. One could arbitrarily take the end of World War II as a point in time when North American diets changed dramatically. If fast food and frozen dinners gave society convenience, they also are leading candidates for the loss of microbial diversity. Importantly, from generation to generation, the diminution of diversity in a mother, also combined with an increase in cesarean sections, would necessarily lead to the loss of diversity in the offspring which is perpetuated in successive generations. This was elegantly illustrated by Sonnenburg and colleagues [37], who mimicked dietary changes by varying the relative amount of fat and fiber and comparing the diversity in successive generations of mice. However, modern-day individuals still tend to outlive their ancestors despite their limited microbial diversity, making

it less important for the perpetuation of the species even if it adversely affects quality of life. When dysbiosis is created, one or more species may be represented in unusually high numbers. This may reflect an ability of the dominant microbe to suppress the growth of other species, for example, through the effects on gene expression and quorum sensing. In addition to one microbial species regulating gene expression in another, altered host responses may contribute to this selection pressure [3,38]. As tolerance can be overcome by an increase in the concentration of an antigen, dysbiosis may enhance the immunogenicity of the microbes, leading to heightened host responses to the unusually abundant organism. This could lead to a transient or perpetual state of inflammation that may be highly dependent on host genetics. For example, the benefit of protective bacteria may be lost on some genetic backgrounds, thereby enhancing host responses that lead to altered gene expression and enhanced colonization by malevolent microbes [39]. It is tempting to speculate that mucosal immunization could be used to modify the composition of the microbiota by eliminating, or at least neutralizing, factors (microbial or host) that favor the persistence of dysbiosis (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity).

V. THE CONCEPT OF A HOMEOSTATIC SCAR AND IMMUNOLOGICAL DYSFUNCTION In the ecology of the intestine, the equilibrium that exists between host responses and microbes perpetuates homeostasis. Planer and colleagues have followed changes in fecal microbes and IgA in twins over the first years of life [40]. They have shown that the waning of maternal IgA as the infant’s endogenous IgA develops is linked to the diversity in the microbiota. The question arises as to how

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homeostasis is lost in the context of recurrent chronic inflammatory disease. This question can be approached in individuals with IBD. These diseases arise from exaggerated host responses (or immunological dysfunction) to the local microbiota in genetically susceptible hosts [41 45]. Relevant to IBD, some microbes (e.g., Bilophila wadsworthia [46]) exist as symbionts, but under abnormal circumstances, they assume a pathological role [47 49]. The pathogenic potential of these microbes, or pathobionts, is influenced by multiple factors, including innate lymphoid cells [50] and Treg [51,52]. While much remains to be learned about the mechanisms, it is likely that different degrees of activation or relative numbers of these cells and their defined phenotype will shift the composition of the host response in a manner that affects the relative numbers of microbial constituents. This could be obvious at the level of phyla but may be as subtle as changes in small numbers of species. Although the studies are just emerging, many, if not all, aspects of the host response appear to contribute in their own way to the richness and evenness of the microbial community. As was mentioned above, a misshapen microbiome can perturb host responses to enhance the risk of disease. In addition, host responses are sufficient to modify microbial gene expression in a manner that alters colonization and virulence [3,38] and further stimulates host responses toward a proinflammatory state [53]. These host responses coexist with the dysbiosis and reinforce its persistence and, in so doing, create a homeostatic scar that could perpetuate recurrent disease [54]. This would suggest that one may try to treat inflammatory diseases by targeting the inflammation, but the persistent microbiota may continue to push the host responses back to the diseased state. The premise for the approach described in this chapter is that the microbiota, particularly when in dysbiosis, represent an “infectious disease” that may be targeted through immunization.

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VI. THE IMPACT OF IMMUNIZATION ON HOMEOSTASIS The mechanisms that create homeostasis successfully are complex, owing to the number of antigens or microbes that are involved, the dynamic effects on immunological status associated with new antigenic challenges, and the number of genetic and environmental factors that influence microbial communities and host responses. While much is being learned about the impact of the microbiota on host responses, health, and disease as well as how we can prevent infectious diseases through immunization, it is relatively novel to consider that immunization could be used to ameliorate host responses and/or microbial communities to prevent or treat inflammation. Antimicrobial protection can be provided by both passive and active immunization either by targeting the microbe directly or by neutralizing a microbial toxin or metabolite that favors colonization of a problematic organism. Given the potential effect of these strategies on microbes, it is reasonable to expect that they can modulate the reciprocal interactions between the host and microbiome more broadly. Further, immunological targets that promote inflammation may provide alternative strategies with which to manipulate mucosal immunity and promote health.

A. Passive Immunization In the case of passive immunity, maternal antibodies can be transferred to the fetus across the placenta in many species but also through the breast milk of all mammals [40]. In addition to the passive transfer of protection, breast milk provides many proteins, glycans, and lipids capable of serving as substrates and carbon sources that aid in the development of intestinal physiology, the

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immune system, and the microbial communities [55,56]. As many of the components of breast milk are beneficial and not represented in formula, it is a universal recommendation to feed newborns breast milk because of the nutritional as well as the disease-sparing benefits (Chapter 44: Maternal Vaccination for Protection Against Maternal and Infant Bacterial and Viral Pathogens). Increasingly, the literature is documenting the impact that breast milk has on the microbiota and the potential benefits of a stable microbiota this provides in avoiding many diseases [55 62]. Linked to effective breast feeding are the needs for maternal health, maternal nutrition, and education. Thus the first step in using immunization to prevent inflammatory diseases is to encourage breast feeding so as to benefit from whatever positive long-term effects this source of nutrition has on the diversity and stability of the microbiome and its eventual effect on homeostasis. However, given the number of factors that affect the microbiota (diet, vaginal birth, breast feeding, local environment such as bile acids, and antibiotics [32,46,47,63 65]) as well as the numerous host genes and environmental factors that affect host responses, one might expect that breast feeding alone will not be the only approach for maintaining a microbiota that favors wellness in the individual. In addition to natural passive immunization, artificial approaches to passively immunize a host may also have benefit for managing not only diseases associated with immunodeficiency, but also chronic immunemediated diseases, including colitis [66], inflammatory demyelinating polyneuropathy, Kawasaki disease, and Guillain Barre´ syndrome [67]. IgA antibodies produced by B cells also regulate the microbiota and its impact on disease. For example, Perruzza et al. showed that T follicular helper cells responded to bacterial ATP to enhance IgA and help shape and/or maintain a beneficial microbiota [68].

Further evidence of a role for IgA in modulating a disease-causing microbiota is found in a study in which multiple antibacterial monoclonal IgA were compared and one was found to have a potential therapeutic effect in a model of colitis [69]. This clinical improvement was associated with a shift in the composition of bacterial communities. However, no direct evidence for a benefit of this shift on health was established. In another example, the passive administration of antibodies targeting proinflammatory mediators, including tumor necrosis factor alpha (TNF-α), IL-5, and IL-23, as well as molecules involved in homing to mucosal tissues (e.g., α4β7) have been utilized in diseases including eosinophilic esophagitis, IBD, and asthma [70,71]. Investigators have also targeted proinflammatory mediators such as TNF-α in the lumen of the intestine using passive immunization [72]. The notion that proinflammatory factors, such as cytokines, can be targeted through the lumen finds a precedent in studies of Salmonella in rats. In earlier work, bile was shown to secrete substantial amounts of TNF-α following infection, and when this was obstructed, disease was attenuated [73]. In another study, bovine anti-TNF was administered orally to mice in which inflammation had been induced by dextran sulfate sodium [72]. Not only did these antibodies become absorbed into the mucosa, but they also appeared to successfully target TNF, as evidenced by the improvement in disease scores. Thus targeting the microbiota or proinflammatory mediators by passive oral immunization may provide an approach to improve the outcome in chronic inflammation in disease such as IBD but potentially others as well. Cellular immunotherapy is another emerging strategy in passive immunization for altering host responses, including the suppression of unwanted host responses. In one approach, host cells from an organ transplant recipient can be harvested, stimulated against an

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alloantigen in vitro, and reinjected into the donor to provide autologous cells that inhibit transplant rejection [74]. Other approaches have stimulated autologous Treg to selfantigens to treat diseases such as type 1 diabetes [75]. While these therapies have been delivered systemically, they may yet prove to have benefit in mucosal disorders though guided trafficking to the tissue of interest.

B. Active Immunization Natural, active immunization occurs during the normal encounter with microbial organisms throughout life. Arrieta and colleagues reviewed the multiple factors that affect the microbiome early in life and persist into adulthood [76]. This has been shown to begin with the exchange of organisms from mother to fetus [76] as well as the microbial communities that emerge after vaginal or cesarean births. As that review discussed, vaginal deliveries have been shown to increase microbial diversity, which is important to establish early in life. This active immunity is the basis for homeostasis. If this microbial diversity protects against inflammatory diseases, then natural childbirth would be the ideal, if not always possible, start to life. We appreciate now that sanitation, cesarean sections, antibiotic usage, diet, and immunization are some of the key factors that shape the microbiome [76] and, subsequently, the closely linked immunological responses that are the foundation of intestinal homeostasis. If passive immunization with antibodies recognizing certain microbes can be beneficial, then actively immunizing to induce the same type of response to these targets is possible. One example that illustrates the potential of this approach is found in using “protective” bacteria to induce antiinflammatory responses. The premise is based on the assumption that a dysbiosis in diseases such as IBD continuously stimulates the proinflammatory response. Thus

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by inoculating the host with a protective species of bacteria or as a result of “immunizing” with a bacterial product that is sufficient to induce inhibitory responses, the regulatory balances could shift in favor of homeostasis. One example of this is Bacteroides fragilis. This organism produces a polysaccharide A (PSA) that is sufficient to induce Treg cell responses that can offset aberrant Th cell responses and favor a stable, homeostatic environment [13]. Subsequent studies have shown that PSA is found on outer membrane vesicles that engages TLR2 on dendritic cells and induces Treg cells that, when adoptively transferred into mice, protect against colitis that can be used to deliver the protective signal [39,52,77,78]. Further, outer membrane vesicles prepared from mutant strains of B. fragilis that lack PSA no longer confer this protective effect [79]. This example blurs the line between therapeutic benefit of probiotics and immunization, but without doubt, it illustrates how a strategy to induce a favorable host response with active immunization is a promising strategy for the control of disease. The subsequent effects on the microbiota and immunological signature of the host remain to be seen, but it would be quite interesting if such a strategy could significantly reprogram the host microbial interactions that favored disease in the first place. If the administration of protective organisms or probiotics as a strategy in active immunization for antiinflammatory effects is desirable, then one might consider the need to create space for this type of inoculation. The rationale for this is that the existing mucosal immune response may be conditioned to favor the existing community, making editing of the microbial communities more difficult. An example in which antibiotics favor microbiome editing through FMT is the approach to treat Clostridium difficile [16]. This infection can develop in patients on broad spectrum antibiotics leading to a persistent and life-threatening diarrheal disease. FMT can successfully cure the disease in almost 90%

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of the cases [80]. In this disease, “space” has already been created and indeed favors the overgrowth of C. difficile. Since antibiotics impart unwanted effects on the microbiota, other strategies, such as targeting microbial metabolic pathways, may be safer and indeed show substantial promise [81]. It remains to be determined which strategies to create space to alter the microbiome or to reprogram host responses will improve the induction of a more favorable mucosal immunity that aids in preventing or treating inflammatory diseases. Other applications of active immunization would be to eliminate pathogenic organisms, neutralize virulence factors, or target species representing a dysbiosis such that the microbial communities reequilibrate to a homeostatic environment. For example, H. pylori can establish a lifelong infection in the gastric mucosa and, from there, favor the development of gastroduodenal ulcers and gastric cancer. As host responses are believed to contribute to these disease conditions, one strategy would be to administer targeted antimicrobials or mucosal vaccines that eliminate the organism and thereby remove the inflammation that precedes disease development. An alternative strategy may be to target virulence factors that are necessary for disease. This approach requires a deeper understanding of the molecular pathogenesis of gastroduodenal disease so that an appropriate target can be identified. Some studies have suggested that H. pylori contributes to a homeostatic balance that buffers the overrepresentation of Th cell responses that may favor allergy and asthma [30,31,82]. Thus targeting disease pathogenesis and allowing the organism to persist may create a win win situation for the host.

VII. FUTURE PERSPECTIVES The concept that mucosal immunization can be used as a strategy for managing immune-

mediated diseases may be in it is infancy, but it holds promise. As with other emerging concepts, a greater understanding of the basic biology will be required before the approaches can be applied clinically. Some of the outstanding challenges include (1) determining the contributions of viruses, fungi, and bacteria on inflammation directly or through their effect on each other; (2) evaluating how much the microbiome affects immunization and vice versa; (3) assessing whether one can target dominant microbes in dysbiosis with a vaccine and then alter communities and homeostasis; and (4) comparing oral, passive immunization to systemic immunotherapy to achieve protection with fewer side effects.

VIII. CONCLUDING REMARKS To bring the discussion full circle, the introduction pointed out the importance of immunological homeostasis in health. We propose that the memory that resides in stable microbial communities and host responses makes immune-mediated diseases more resistant to one-dimensional therapeutic manipulations. As the basis of homeostasis and perturbed states becomes better defined, various approaches to mucosal immunization may provide a complementary method to correct dysbiosis and to limit overexuberant and destructive host responses. The challenges are exciting, but the complex interrelationships have to be understood, and new approaches to manipulate them must be tested.

Acknowledgments The authors greatly appreciate support from NIH, including AI AI079145 (PE) and DK110534 (HC). Support has also been provided by the Wayne and Gladys Valley Foundation and the Chiba University UC San Diego Program in Mucosal Immunology, Allergy and Vaccine Development. PE and HC hold joint appointments in the Department of Immunology, Chiba University, Chiba Japan.

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REFERENCES

References [1] Rescigno M. The intestinal epithelial barrier in the control of homeostasis and immunity. Trends Immunol 2011;32:256 64. [2] Kurashima Y, Goto Y, Kiyono H. Mucosal innate immune cells regulate both gut homeostasis and intestinal inflammation. Eur J Immunol 2013;43:3108 15. [3] Arpaia N, Godec J, Lau L, Sivick KE, McLaughlin LM, Jones MB, et al. TLR signaling is required for Salmonella typhimurium virulence. Cell 2011;144:675 88. [4] Baumler AJ, Sperandio V. Interactions between the microbiota and pathogenic bacteria in the gut. Nature 2016;535:85 93. [5] Bienenstock J, Klaenhammer TR, Walker WA, Neish A. New insights into probiotic mechanisms: a harvest from functional and metagenomic studies. Gut Microbes 2013;4:94 100. [6] Donaldson GP, Lee SM, Mazmanian SK. Gut biogeography of the bacterial microbiota. Nat Rev Microbiol 2016;14:20 32. [7] Tomkovich S, Jobin C. Microbiota and host immune responses: a love-hate relationship. Immunology 2016; 147:1 10. [8] Schroeder BO, Backhed F. Signals from the gut microbiota to distant organs in physiology and disease. Nat Med 2016;22:1079 89. [9] Round JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 2009;9:313 23. [10] Bunker JJ, Erickson SA, Flynn TM, Henry C, Koval JC, Meisel M, et al. Natural polyreactive IgA antibodies coat the intestinal microbiota. Science 2017;358. [11] Ivanov II, Frutos RL, Manel N, Yoshinaga K, Rifkin DB, Sartor RB, Finlay BB, et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 2008;4:337 49. [12] Kim SC, Tonkonogy SL, Karrasch T, Jobin C, Sartor RB. Dual-association of gnotobiotic IL-10-/- mice with 2 nonpathogenic commensal bacteria induces aggressive pancolitis. Inflamm Bowel Dis 2007;13:1457 66. [13] Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 2005; 122:107 18. [14] Waidmann M, Bechtold O, Frick JS, Lehr HA, Schubert S, Dobrindt U, et al. Bacteroides vulgatus protects against Escherichia coli-induced colitis in gnotobiotic interleukin-2-deficient mice. Gastroenterology 2003; 125:162 77. [15] Vogt SL, Pena-Diaz J, Finlay BB. Chemical communication in the gut: effects of microbiota-generated

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25] [26]

[27]

[28]

[29]

877 metabolites on gastrointestinal bacterial pathogens. Anaerobe 2015;34:106 15. Lozupone CA, Stombaugh JI, Gordon JI, Jansson JK, Knight R. Diversity, stability and resilience of the human gut microbiota. Nature 2012;489:220 30. Oertli M, Sundquist M, Hitzler I, Engler DB, Arnold IC, Reuter S, et al. DC-derived IL-18 drives Treg differentiation, murine Helicobacter pylori-specific immune tolerance, and asthma protection. J Clin Invest 2012; 122:1082 96. Elliott DE, Setiawan T, Metwali A, Blum A, Urban Jr. JF, Weinstock JV. Heligmosomoides polygyrus inhibits established colitis in IL-10-deficient mice. Eur J Immunol 2004;34:2690 8. Massey JC, Sutton IJ, Ma DDF, Moore JJ. Regenerating immunotolerance in multiple sclerosis with autologous hematopoietic stem cell transplant. Front Immunol 2018;9:410. Kalekar LA, Mueller DL. Relationship between CD4 regulatory T cells and anergy in vivo. J Immunol 2017; 198:2527 33. Palomares O, Akdis M, Martin-Fontecha M, Akdis CA. Mechanisms of immune regulation in allergic diseases: the role of regulatory T and B cells. Immunol Rev 2017; 278:219 36. Abbas AK, Benoist C, Bluestone JA, Campbell DJ, Ghosh S, Hori S, et al. Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol 2013;14:307 8. Yadav M, Stephan S, Bluestone JA. Peripherally induced tregs—role in immune homeostasis and autoimmunity. Front Immunol 2013;4:232. Kim KS, Hong SW, Han D, Yi J, Jung J, Yang BG, et al. Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine. Science 2016; 351:858 63. Pabst O, Mowat AM. Oral tolerance to food protein. Mucosal Immunol 2012;5:232 9. Salama NR, Hartung ML, Muller A. Life in the human stomach: persistence strategies of the bacterial pathogen Helicobacter pylori. Nat Rev Microbiol 2013;11:385 99. Kao JY, Zhang M, Miller MJ, Mills JC, Wang B, Liu M, et al. Helicobacter pylori immune escape is mediated by dendritic cell-induced Treg skewing and Th17 suppression in mice. Gastroenterology 2010; 138:1046 54. Harris PR, Wright SW, Serrano C, Riera F, Duarte I, Torres J, et al. Helicobacter pylori gastritis in children is associated with a regulatory T-cell response. Gastroenterology 2008;134:491 9. Rad R, Brenner L, Bauer S, Schwendy S, Layland L, da Costa CP, et al. CD25 1 /Foxp3 1 T cells regulate gastric inflammation and Helicobacter pylori colonization in vivo. Gastroenterology 2006;131:525 37.

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NON-INFECTIOUS DISEASES?

878

52. NOVEL STRATEGIES FOR TARGETING THE CONTROL OF MUCOSAL INFLAMMATION

[30] Engler DB, Reuter S, van Wijck Y, Urban S, Kyburz A, Maxeiner J, et al. Effective treatment of allergic airway inflammation with Helicobacter pylori immunomodulators requires BATF3-dependent dendritic cells and IL-10. Proc Natl Acad Sci USA 2014;111: 11810 15. [31] Arnold IC, Dehzad N, Reuter S, Martin H, Becher B, Taube C, et al. Helicobacter pylori infection prevents allergic asthma in mouse models through the induction of regulatory T cells. J Clin Invest 2011;121:3088 93. [32] Gilbert JA, Blaser MJ, Caporaso JG, Jansson JK, Lynch SV, Knight R. Current understanding of the human microbiome. Nat Med 2018;24:392 400. [33] Taghipour N, Aghdaei HA, Haghighi A, Mossafa N, Tabaei SJ, Rostami-Nejad M. Potential treatment of inflammatory bowel disease: a review of helminths therapy. Gastroenterol Hepatol Bed Bench 2014;7: 9 16. [34] Weinstock JV, Elliott DE. Helminths and the IBD hygiene hypothesis. Inflamm Bowel Dis 2009;15:128 33. [35] Hsiao A, Ahmed AM, Subramanian S, Griffin NW, Drewry LL, Petri Jr. WA, et al. Members of the human gut microbiota involved in recovery from Vibrio cholerae infection. Nature 2014;515:423 6. [36] Smits SA, Leach J, Sonnenburg ED, Gonzalez CG, Lichtman JS, Reid G, et al. Seasonal cycling in the gut microbiome of the Hadza hunter-gatherers of Tanzania. Science 2017;357:802 6. [37] Sonnenburg ED, Smits SA, Tikhonov M, Higginbottom SK, Wingreen NS, Sonnenburg JL. Diet-induced extinctions in the gut microbiota compound over generations. Nature 2016;529:212 15. [38] Barrozo RM, Cooke CL, Hansen LM, Lam AM, Gaddy JA, Johnson EM, et al. Functional plasticity in the type IV secretion system of Helicobacter pylori. PLoS Pathog 2013;9:e1003189. [39] Chu H, Khosravi A, Kusumawardhani IP, Kwon AH, Vasconcelos AC, Cunha LD, et al. Gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease. Science 2016;352:1116 20. [40] Planer JD, Peng Y, Kau AL, Blanton LV, Ndao IM, Tarr PI, et al. Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice. Nature 2016;534:263 6. [41] Sartor RB. Microbial influences in inflammatory bowel diseases. Gastroenterology 2008;134:577 94. [42] Mokry M, Middendorp S, Wiegerinck CL, Witte M, Teunissen H, Meddens CA, et al. Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium. Gastroenterology 2014;146:1040 7. [43] Shanahan F, Quigley EM. Manipulation of the microbiota for treatment of IBS and IBD-challenges and controversies. Gastroenterology 2014;146:1554 63.

[44] Kostic AD, Xavier RJ, Gevers D. The microbiome in inflammatory bowel disease: current status and the future ahead. Gastroenterology 2014;146:1489 99. [45] Goto Y, Kurashima Y, Kiyono H. The gut microbiota and inflammatory bowel disease. Curr Opin Rheumatol 2015;27:388 96. [46] Devkota S, Chang EB. Interactions between diet, bile acid metabolism, gut microbiota, and inflammatory bowel diseases. Dig Dis 2015;33:351 6. [47] Kamada N, Chen GY, Inohara N, Nunez G. Control of pathogens and pathobionts by the gut microbiota. Nat Immunol 2013;14:685 90. [48] Chow J, Mazmanian SK. A pathobiont of the microbiota balances host colonization and intestinal inflammation. Cell Host Microbe 2010;7:265 76. [49] Devkota S, Wang Y, Musch MW, Leone V, FehlnerPeach H, Nadimpalli A, et al. Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice. Nature 2012;487:104 8. [50] Goto Y, Obata T, Kunisawa J, Sato S, Ivanov II, Lamichhane A, et al. Innate lymphoid cells regulate intestinal epithelial cell glycosylation. Science 2014;345: 1254009. [51] Mazmanian SK, Round JL, Kasper DL. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 2008;453:620 5. [52] Round JL, Mazmanian SK. Inducible Foxp3 1 regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci USA 2010; 107:12204 9. [53] Palm NW, de Zoete MR, Cullen TW, Barry NA, Stefanowski J, Hao L, et al. Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease. Cell 2014;158:1000 10. [54] Fonseca DM, Hand TW, Han SJ, Gerner MY, Glatman Zaretsky A, Byrd AL, et al. Microbiota-dependent sequelae of acute infection compromise tissue-specific immunity. Cell 2015;163:354 66. [55] Aakko J, Kumar H, Rautava S, Wise A, Autran C, Bode L, et al. Human milk oligosaccharide categories define the microbiota composition in human colostrum. Benef Microbes 2017;8:563 7. [56] Milani C, Duranti S, Bottacini F, Casey E, Turroni F, Mahony J, et al. The first microbial colonizers of the human gut: composition, activities, and health implications of the infant gut microbiota. Microbiol Mol Biol Rev 2017;81. [57] Dore MP, Malaty HM, Graham DY, Fanciulli G, Delitala G, Realdi G. Risk factors associated with Helicobacter pylori infection among children in a defined geographic area. Clin Infect Dis 2002;35:240 5. [58] Ivarsson A, Hernell O, Stenlund H, Persson LA. Breast-feeding protects against celiac disease. Am J Clin Nutr 2002;75:914 21.

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NON-INFECTIOUS DISEASES?

REFERENCES

[59] Kull I, Wickman M, Lilja G, Nordvall SL, Pershagen G. Breast feeding and allergic diseases in infants—a prospective birth cohort study. Arch Dis Child 2002;87: 478 81. [60] Pozo-Rubio T, Olivares M, Nova E, De Palma G, Mujico JR, Ferrer MD, et al. Immune development and intestinal microbiota in celiac disease. Clin Dev Immunol 2012;2012:654143. [61] Bode L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology 2012;22:1147 62. [62] Kirmiz N, Robinson RC, Shah IM, Barile D, Mills DA. Milk glycans and their interaction with the infant-gut microbiota. Annu Rev Food Sci Technol 2018;9:429 50. [63] Goodrich JK, Di Rienzi SC, Poole AC, Koren O, Walters WA, Caporaso JG, et al. Conducting a microbiome study. Cell 2014;158:250 62. [64] Goodrich JK, Waters JL, Poole AC, Sutter JL, Koren O, Blekhman R, et al. Human genetics shape the gut microbiome. Cell 2014;159:789 99. [65] Rothschild D, Weissbrod O, Barkan E, Kurilshikov A, Korem T, Zeevi D, et al. Environment dominates over host genetics in shaping human gut microbiota. Nature 2018;555:210 15. [66] Okai S, Usui F, Ohta M, Mori H, Kurokawa K, Matsumoto S, et al. Intestinal IgA as a modulator of the gut microbiota. Gut Microbes 2017;8:486 92. [67] Yang L, Wu EY, Tarrant TK. Immune gamma globulin therapeutic indications in immune deficiency and autoimmunity. Curr Allergy Asthma Rep 2016;16:55. [68] Perruzza L, Gargari G, Proietti M, Fosso B, D’Erchia AM, Faliti CE, et al. T follicular helper cells promote a beneficial gut ecosystem for host metabolic homeostasis by sensing microbiota-derived extracellular ATP. Cell Rep 2017;18:2566 75. [69] Okai S, Usui F, Yokota S, Hori IY, Hasegawa M, Nakamura T, et al. High-affinity monoclonal IgA regulates gut microbiota and prevents colitis in mice. Nat Microbiol 2016;1:16103. [70] Dulai PS, Sandborn WJ. Next-generation therapeutics for inflammatory bowel disease. Curr Gastroenterol Rep 2016;18:51.

879

[71] Nhu QM, Aceves SS. Tissue remodeling in chronic eosinophilic esophageal inflammation: parallels in asthma and therapeutic perspectives. Front Med (Lausanne) 2017;4:128. [72] Bhol KC, Tracey DE, Lemos BR, Lyng GD, Erlich EC, Keane DM, et al. AVX-470: a novel oral anti-TNF antibody with therapeutic potential in inflammatory bowel disease. Inflamm Bowel Dis 2013;19:2273 81. [73] Islam AF, Moss ND, Dai Y, Smith MS, Collins AM, Jackson GD. Lipopolysaccharide-induced biliary factors enhance invasion of Salmonella enteritidis in a rat model. Infect Immun 2000;68:1 5. [74] Baker KF, Isaacs JD. Prospects for therapeutic tolerance in humans. Curr Opin Rheumatol 2014;26:219 27. [75] DuPage M, Bluestone JA. Harnessing the plasticity of CD4(1) T cells to treat immune-mediated disease. Nat Rev Immunol 2016;16:149 63. [76] Arrieta MC, Stiemsma LT, Amenyogbe N, Brown EM, Finlay B. The intestinal microbiome in early life: health and disease. Front Immunol 2014;5:427. [77] Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, et al. The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota. Science 2011;332:974 7. [78] Mazmanian SK. Capsular polysaccharides of symbiotic bacteria modulate immune responses during experimental colitis. J Pediatr Gastroenterol Nutr 2008;46 (Suppl. 1):E11 12. [79] Shen Y, Giardino Torchia ML, Lawson GW, Karp CL, Ashwell JD, Mazmanian SK. Outer membrane vesicles of a human commensal mediate immune regulation and disease protection. Cell Host Microbe 2012;12:509 20. [80] Surawicz CM, Alexander J. Treatment of refractory and recurrent Clostridium difficile infection. Nat Rev Gastroenterol Hepatol 2011;8:330 9. [81] Zhu W, Winter MG, Byndloss MX, Spiga L, Duerkop BA, Hughes ER, et al. Precision editing of the gut microbiota ameliorates colitis. Nature 2018;553:208 11. [82] Konturek PC, Rienecker H, Hahn EG, Raithel M. Helicobacter pylori as a protective factor against food allergy. Med Sci Monit 2008;14:CR452 8.

VIII. CAN MUCOSAL VACCINES BE APPLIED FOR OTHER INFECTIOUS AND NON-INFECTIOUS DISEASES?

Index

Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A A disintegrin and metalloproteinase 17 (ADAM17), 156 A proliferation-inducing ligand (APRIL), 191
192 Ab. See Antibody (Ab) Aberrant bodies (ABs), 267 Ablation therapy, 237 AB-type toxins, 185 Ac¸ai, 779 polysaccharides, 780 ACE527 vaccine, 570 Acellular pertussis vaccination, 117 Acquired immunity of middle ear and nasopharynx, 314
315 role against pneumococci, 602
603 Acquired immunodeficiency syndrome (AIDS), 255
256, 740 ACT-1, 93
94 Actinobacillus pleuropneumoniae, 344 Actinobacteria, 158
159 Activation-induced cytidine deaminase-deficient mice (AIDdeficient mice), 794
795 Active immunization, 875
876 Acute gastroenteritis, 363, 699 Acute lower respiratory tract infections (ALRI), 666 Acute shigellosis, 519 Acyclovir, 278, 723
724, 742 Ad35. See Adenovirus 35 (Ad35) Ad40. See Adenovirus 40 (Ad40) Ad5. See Adenovirus 5 (Ad5) ADAM17. See A disintegrin and metalloproteinase 17 (ADAM17) Adaptive immune cells in lower FGT, 260 in upper FGT, 257
260

Adaptive immune responses, 409, 602 contributions to, 220
222 adjuvant activity of MC activators and products, 221
222 antigen presentation, 220
221 effectors of adaptive immunity, 221 enhancing influx of immune cells into draining lymph nodes, 220 to gonorrhea, 633 Adaptive immune systems, 22, 71 Adaptive immunity, 266 cross-regulation of innate immunity and, 238
239 effectors of, 221 in Mtb infection, 612 Adaptive mucosal immune responses in cholera, 542
547 intestinal mucosal T cells, 547 intestine-derived gut-homing circulating antibody-secreting cells, 546
547 mucosal IgA antibody and antibody-secreting cell responses, 543
546 ADCC. See Antibody-dependent cellular cytotoxicity (ADCC) ADCP. See Antibody-dependent cellular phagocytosis (ADCP) Adenoids. See Unpaired nasopharyngeal tonsils Adenovirus 5 (Ad5), 862 vectors, 717
718 Adenovirus 35 (Ad35), 760
761 Adenovirus 40 (Ad40), 862 Adenovirus (Ad) vectors, 419, 422
423, 717 construction, purification, and titration, 423 immunizations through rectal/ genital mucosa, 436

881

immunogenicity, 424 as intranasal vaccines, 430
436, 431t mucosal immune system, 424
425 mucosal vaccines, 425
426 as oral vaccines, 426
430, 427t quality control, 423 thermostability, 424 use of adjuvants for mucosal adenovirus vector vaccines, 436 Adenoviruses, 11, 420 immune responses to, 420
421 neutralizing antibodies prevalence in humans, 422t Adenyl cyclase, 185
186 Adherent-invasive E. coli, 59 Adjuvants, 76
77, 167
168 adjuvant-alone effect, 604 and delivery, 860
861 imprint mucosal homing of antigenspecific T and B cells, 765 for mucosal adenovirus vector vaccines, 436 ADP-ribosyltransferase activity, 650 Aedes genus, 743 Aeromonas, 340 Affinity upregulation, 85
86 AFFIRM study, 93
94 AFM. See Atomic force microscopy (AFM) Ag-specific IgA B cell responses, 791
792 Ag-specific immune responses, 802 Ag-specific salivary IgA antibody responses (Ab responses), 650 Ag85B-expressed human parainfluenza type 2 virus (rHPIV2-Ag85B), 612
613, 616f, 791
792 Aggregatibacter, 150
153 Aged, mucosal vaccines for

882 Aged, mucosal vaccines for (Continued) age-associated changes in GI tract immune system, 791
792 dentritic cell targeting mucosal vaccines for aged, 800
802 distinct aging process of nasopharygeal associated lymphoid tissue function, 797
798 gut immunity rejuvenation by mesenchymal stem cell transfer, 795
796 intestinal microbiota potentially shapes mucosal immunosenescence, 794
795 involvement of mucosal CD41 T cells in gut aging, 793
794 mucosal vaccines and therapies for immunosenescence, 799
800 nasopharygeal-associated lymphoid tissue vs. gut-associated lymphoid tissue, 796
797 next generation of potent mucosal vaccines for elderly, 802 potential mechanisms in gut aging, 793 Agonists β2AR, 233
234 TLR3 or TLR7/8, 177 Agrobacterium, 360 A. tumefaciens, 357, 360 mediated transformation, 359 Agrobacterium-mediated transformation, 360 stable transformation, 361
363 cotransformation system, 366 infiltration, 371 Agroinfiltration, 359
360 AHR. See Aryl hydrocarbon receptor (AHR) AID-deficient mice. See Activationinduced cytidine deaminasedeficient mice (AID-deficient mice) AIDS. See Acquired immunodeficiency syndrome (AIDS) Aif1. See Allograft inflammatory factor 1 (Aif1) AIM2 inflammasomes, 685 Airway luminal T cells (ALT cells), 411 AIV. See Avian influenza virus (AIV) Akkermansia muciniphila, 145
146

INDEX

Alcaligenes species, 207
208, 489, 794
795 Alginate, 330
332, 340 Allergen immunotherapy, 176, 859
861 Allergen-derived peptides, 857
859 Allergen-specific subcutaneous immunotherapy, 317
319 Allergens, 377, 856, 860
861 Allergoids, 856
857, 860
861 Allergovit, 856
857 Allergy, 855 allergic amino acid sequence, 375
376 allergic rhinitis, 317 ILCs in, 237
238 vaccination, 860
861 Allograft inflammatory factor 1 (Aif1), 491 α-defensins, 103
104 α(1,2)-linked fucose, 490 α2-3-linked sialic acid, 493 α4β1 integrin, 464 α4β7 integrin, 85
86, 91
92, 96, 126 ALPK1 kinase, 585
586 ALRI. See Acute lower respiratory tract infections (ALRI) ALS. See Antibody in lymphocyte secretion (ALS) ALT cells. See Airway luminal T cells (ALT cells) Alveolar echinococcosis, 848
849 Alveoli, 600 Amoebiasis, 842
844 Amphotericin B (AmB), 780 AMPs. See Antimicrobial peptides (AMPs) Anamnestic SIgA responses, 543
544 Anaphylaxis, 174
175 Ancylostoma caninum (Hookworm), 154
155 Angiotensin II receptor type 1 (AT1R), 471 Animal models, 344
345, 371, 411
413, 603 of mucosal Shigella infection, 520 Anionic dextran sulfate, 330
332 Ankara adenovirus, 615
616 Anopheles mosquito salivary gland, 831
832 Anthrax development of first anthrax vaccines, 447
448

disease and historical perspective, 446
447 live vaccines, 448 toxin derivative, 190 Anthrax toxin receptor 1 (ATR1), 186 Anti-CTB, 543 Anti-IgE, 859
860 Anti-immunoevasin approaches, 731
732 Anti-LPS SIgA responses, 543 Anti-O1 LPS, 543 Anti-ZP antibodies, 824 Anti-α-gal antibodies, 834
835 Antibiotic use, 105 Antibody (Ab), 187
188, 270, 307, 727, 790
791 antibody-bound antigens, 60 antibody-coated pathogens, 216 antibody-dependence for immune protection to plague, 452
453 antibody-mediated immunity, 730
731 antibody-mediated protection, 727 antibody-producing cells, 822 protection against infection at mucosal sites, 765
766 titers, 150 Antibody in lymphocyte secretion (ALS), 518, 546
547, 569
570 Antibody-dependent cellular cytotoxicity (ADCC), 727 Antibody-dependent cellular phagocytosis (ADCP), 727 Antibody-secreting cells (ASCs), 385
386, 477
478, 518, 565
566 intestine-derived gut-homing circulating, 546
547 responses, 543
546 Anticaries vaccine, 651 Antigen (Ag), 326
327, 329, 358, 651f, 790
791 antigen-binding sites, 76
77 antigen-presenting cells localization in sublingual mucosa, 478 antigen-sampling system, 302 antigen-specific nasal immune responses, 463
464 choice of, 271 delivery, 387
388 encapsulation, 337 inhibition of antigen absorption, 75
76

883

INDEX

passages, 146 presentation, 220
221 uptake across villus epithelium, 58
66 myeloid cell uptake of antigens and bacteria in lamina propria, 61
63 paracellular and transcellular transport across villus epithelial cells, 58
61 vectoring guest, 386
387 Antigen-85B (Ag85B), 404 expression, 617f Antigen-loaded porous PLGA microparticle, 339 Antigen-presenting cells (APCs), 168, 220, 325
326, 402
403, 479
480, 487, 650, 775, 790
791 Antigenic drift, 364 Antigenic sites, 668, 671, 713 Antiinflammation, 94 Antimicrobial peptides (AMPs), 103, 146, 217
219, 235
236, 261, 582
583, 587 Antimicrobial protection, 873 Antiretroviral therapy (ART), 96, 740 Antitoxin, 543, 549 Antiviral drugs, 278, 723
724, 742 Antiviral memory T cells, 729 ANTXR1 receptor, 447 ANTXR2 receptor, 447 ANXA5, 31 APCs. See Antigen-presenting cells (APCs) Apical junctional complex, 58
59 Apoptosis, 517
518 Apoptosis-associated speck-like protein containing CARD protein (ASC), 685 APRIL. See A proliferation-inducing ligand (APRIL) Aquaculture industry, 822 Aquatic species, 812 immersion vaccines, 823 injected vaccine, 824 mucosal aquatic vaccines, 822
824 oral and intranasal vaccines, 823 Arabinose, 388 Arabinose-regulated virulence gene(s), 388 Arg
gingipain A, 654 Arginase 1 (Arg1), 234 Arginine-glycine-aspartate ligand (RGD ligand), 339, 491

ART. See Antiretroviral therapy (ART) Arthritis, 213
214 Aryl hydrocarbon receptor (AHR), 26
27, 234, 774 ASC. See Apoptosis-associated specklike protein containing CARD protein (ASC) ASCs. See Antibody-secreting cells (ASCs) asdA gene, 386
387 Aspergillus amstelodami, 154 Aspergillus fumigatus, 153
154 Asthma, 136, 213
214 AT1R. See Angiotensin II receptor type 1 (AT1R) Atlantic cod. See Gadus morhua (Atlantic cod) Atlantic salmon. See Salmo salar (Atlantic salmon) Atomic force microscopy (AFM), 680f ATR1. See Anthrax toxin receptor 1 (ATR1) Attenuated Salmonella for oral immunization approaches for attenuation antigen delivery, 387
388 deletion mutants, 385 modification of fimbriae, 394
395 mutations in Salmonella pathogenicity islands, 385
386 regulated delayed antigen synthesis, 389
390 regulated delayed attenuation, 388
389 regulated delayed lysis system, 390f regulated delayed vaccine lysis, 390
392 serial passage, 384
385 strategies for reducing lipid A toxicity, 392
393 sugar-inducible acid resistance, 393
394 vectoring guest antigens, 386
387 vaccines against nontyphoidal Salmonella, 395 Attenuated vaccine (Ty21a), 525 Attenuation strategies, 389 Atypical T cells, 602 Aureobasidium pullulans, 332
333 Autocrine, 299 Autoimmune antigens. See Allergens Autoimmunity, ILCs in, 237
238

Avian influenza virus (AIV), 820 Avian species, 819
820 Avian influenza, 820 companion animals, 820 immunocontraceptive vaccines, 824
825 mucosal aquatic vaccines, 822
824 mucosal vaccines to improve fertility, 825 wildlife, 821
822 Avidity upregulation, 85
86

B 4-1BB, 37 B cell receptors (BCRs), 22, 117, 713 B cells, 24, 35, 150, 727 activation in female mouse genital tissues, 482 B lymphocyte, 35, 258
259 B memory cells (BMCs), 551 B plasmablast ASCs, 546
547 B subunit of heat-labile toxin (LTB), 185
186, 340
341, 386 B-cell-activating factor of TNF family (BAFF), 31, 191
192 B-lymphocyte-induced maturation protein 1 (BLIMP-1), 775 Bach2 gene, 127
128 Bacillus anthracis, 186, 359
360, 446
448 Bacillus Calmette-Gue´rin (BCG), 402
403 intravesical instillation of, 407
408 lipid formulations of, 404 vaccine, 5
6 Bacillus spp., 301 B. pestis, 452 B. subtilis, 157
158 Bacteremia, 395, 451
452 Bacteria-like particles (BLP), 605 Bacteria, 313, 344, 383
384, 403, 461
462, 601 Bacterial adherence, 76
77 Bacterial fimbriae, 394 Bacterial ghosts (BGs), 824 Bacteroides, 144 B. acidifaciens, 149
150 B. caccae, 145
146 B. fragilis, 9
10, 58, 875 B. vulgatus, 870 Bacteroidetes, 144, 149 BAFF. See B-cell-activating factor of TNF family (BAFF)

884 Baker’s yeast. See Saccharomyces cerevisiae (Baker’s yeast) BAL. See Bronchoalveolar lavage (BAL) BALF. See Bronchoalveolar lavage fluid (BALF) BALT. See Bronchus-associated lymphoid tissue (BALT) BCG. See Bacillus Calmette-Gue´rin (BCG) BCG intradermal (ID), 403
404 BCG vaccination, 409
410 BCRs. See B cell receptors (BCRs) Bell’s palsy, 462 Beriberi, 205
206 Besredka’s classic studies of immunity, 4 β catenin, 146 β-defensins, 103
104 β-glucans, 340 β-lactoglobulin (BLG), 857
859 β-TrCP, 688 β2 microglobulin, 60 β2-adrenergic receptor (β2AR), 233
234 BGs. See Bacterial ghosts (BGs) Bharat biotech, 702
703 Bifidobacterium, 144, 223, 833 B. breve, 249
250 Bilophila wadsworthia, 872
873 Bioencapsulation, 823 Biolistic bombardment, 371 Biolistic method for stable transformation, 359
360 Bioterrorism agents, 333
334 Biotin, 204
205 Bisphosphonates, 777
778 Bivalent NoV VLP vaccine, 706 Bivalent VLP vaccine, 706 “Black Death”, 451
452 Bladder cancer, 407
409 BLG. See β-lactoglobulin (BLG) BLIMP-1. See B-lymphocyte-induced maturation protein 1 (BLIMP-1) Blood predict mucosal immunity, signatures in, 763 BLP. See Bacteria-like particles (BLP) BLS-OMP31-P407-Ch, 818 BMCs. See B memory cells (BMCs) bNAb against. See Broadly neutralizing HIV antibody against (bNAb against) BoHc/A. See Clostridium botulinum type A neurotoxin heavy-chain C-terminus (BoHc/A)

INDEX

BoHV-1. See Bovine herpesvirus 1 (BoHV-1) BoHV-1 triple mutant virus (BoHV-1 tmv), 818
819 Bone-marrow-derived DCs, 62 Bony fish, 24 Bordetella pertussis, 74
75, 176, 186, 190 Borrelia, 413
414 Borrelia burgdorferi, 413 Bovinae, 818
819 BoHV-1, 818
819 bovine viral diarrhea virus, 819 HS, 819 Bovine anti-TNF, 874 Bovine herpesvirus 1 (BoHV-1), 818
819 glycoprotein E deleted mutant virus, 818
819 Bovine viral diarrhea virus (BVDV), 819 Bowman-Birk protease inhibitor, 378 Bradyzoites, 845
846 Breg cells. See Regulatory B cells (Breg cells) Broadly neutralizing HIV antibody against (bNAb against), 713 Bronchoalveolar lavage (BAL), 404
405 Bronchoalveolar lavage fluid (BALF), 410
411, 481
482, 861 Bronchus-associated lymphoid tissue (BALT), 38
42, 136
137 Brucella, 340, 448
449, 777, 818 B. abortus, 31 B. melitensis Rev 1 vaccine, 818 B. ovis, 818 infections, 449 protection, 449 Brucellosis, 449 current vaccines, 449
450 etiological agents and disease, 448
449 mucosal vaccination approaches, 450
451 protection to Brucella infections, 449 Brushtail possums. See Trichosurus vulpecula (Brushtail possums) BRV-PV, 703 Bubo, 451
452 Burkholderia pseudomallei, 37
38, 779 Butyrate, 148
149, 209 BVDV. See Bovine viral diarrhea virus (BVDV)

C c-kit, 25
26 C-type lectin receptors (CLRs), 106 association between CLRs and intestinal inflammation, 109 C-type lectins, 216 C1qB protein, 758 C48/80. See Compound 48/80 (C48/ 80) C5aR, 492 Caco-2 epithelial cells, 62 CagA, 586
587 Caliciviridae, 704
705 CALT. See Conjunctiva-associated lymphoid tissue (CALT) Canarypox virus (CNPV), 718 Candida, 236 C. albicans, 155
156 Capillary morphogenesis gene 2 (CMG2), 186 Caprinae, 818 Capsid protein, 362t, 420 Capsular polysaccharide, 599
600 Capsular polysaccharide-specific IgG (CPS-specific IgG), 744 Capsules, 341 capsule-based vaccines, 597 CAR. See Coxsackievirus and adenovirus receptor (CAR) CARD adapter inducing IFN beta (Cardif), 687 Caries, 649 Carriage, 600, 602 Caspase-1 (CASP1), 447, 685 cleaving pro-IL-1β, 172
173 Cat-PAD, 857
859 Catch bond, 88 Cationic CHP (cCHP), 346 Cationic lipid
DNA complex (CLDC), 799 Cationic liposomes, 337, 344 Cationic maltodextrin NP, 345 Cationic nanogels, 332
333 Cationic polyethyleneimine used as mucosal vaccine adjuvant, 175 CCDs. See Cross-reactive carbohydrate determinants (CCDs) cCHP. See Cationic CHP (cCHP) cCHP nanogel. See Cholesteryl-bearing pullulan nanogel (cCHP nanogel) CD. See Crohn’s disease (CD) CD1031 DCs, 30, 61
62

INDEX

CD1031 LP DCs, 63 CD11c, 148 CD11c1 CD1031 CX3CR1
DCs, 63 CD11chiCD11bhi DCs, 107
108 CD11chiCD11blo DCs, 107
108 CD11cintCD11bhi eosinophils, 107
108 CD11cintCD11bint macrophages, 107
108 CD137 receptor, 774 CD141 CD163high CD160high cell, 251
252 CD141 CD163high CD160low cell, 251
252 CD141 cells, 251 CD163high CD160high cells, 251
252 CD27 receptor, 774 CD30 receptor, 774 CD41 cells, 614
615 CD41 T cells, 57, 136, 463
464, 629, 637 activation, 479
481 response, 426 CD41 TRM cells, 133
134, 136 CD8 TRM cells, 731 CD81 T cells, 192
193, 220
221, 266, 268, 613
614, 629, 637, 718
719 CDC. See Centers for Disease Control and Prevention (CDC) CDR3. See Complementaritydetermining region 3 (CDR3) Cecal patches, 21
22, 28
32 Cedar pollinosis, innovative immunotherapy for, 317
319 cEDIII. See Consensus envelope protein domain III (cEDIII) Cell cell
cell junctions, 103 culture system, 704
705 Cell-mediated immunity (CMI), 146
149, 344, 508
509, 790
791 Cellular immunotherapy, 874
875 Cellular prion protein (PrPc), 31, 491 Centers for Disease Control and Prevention (CDC), 625, 701, 737 Central memory T cells (TCM cells), 133
134, 729 Central SMAC (cSMAC), 90
91 Cervarix, 255
256 Cervical lymph nodes (CLNs), 150
153, 317 Cervical mucus (CM), 258
259

Cervicovaginal lavage (CVL), 260 Cervicovaginal mucus (CVM), 258
259, 261 Cestodes, 841
842 Cestodiasis. See Cysticercosis CFA/I family, 565, 570
571 CFB. See Cytophaga-FlavobacteriumBacteriodes (CFB) CFP. See Culture filtrate proteins (CFP) CFs. See Colonization factors (CFs) CFUs. See Colony-forming units (CFUs) cGAS. See Cyclic GMP-AMP synthase (cGAS) CGT. See Cholesterolα-glucosyltransferase (CGT) Charge-switching synthetic adjuvant particles (cSAPs), 270, 629
631 Chemical barriers, 103
104 Chemokine(s), 583, 731, 778
779 CCL5, 219 CCL19, 24, 40
41, 479
480 CCL20, 28, 31, 34 CCL21, 40 CCL27, 123
124, 479
480 CCL28, 123
124, 479
480 CCR6, 25
26, 28, 123
124 CCR61 ILC3 cells, 28 CCR7, 30
31 CCR72/2mice, 480
481 CCR9, 91
92, 123
124, 126, 765 CX3CR11 cells, 62
63 macrophages, 62 myeloid cells, 26 CX3CR1
CD11c1 CD1031 DCs, 63 CX3CR1high macrophage, 250
251 CXCL10, 219 CXCL12, 24, 89
90 CXCL13, 24, 30
31, 37, 40
41 CXCL16, 31 CXCR3, 25
26, 126
127 CXCR3high Th1 cells, 139 CXCR4, 89
90, 123
124 CXCR5, 37 CXCR6, 31 receptor-mediated signal inputs, 89 Chicken cholera, 5
6 Chikungunya virus, 776
777 Children immune responses to Shigella in, 519
520 RSV infections in, 670
672 zinc deficiency in, 552
553

885 CHILPs. See Common helper innate lymphoid precursors (CHILPs) CHIM. See Controlled human infection model (CHIM) Chimeric peptide, 364 Chinese Hamster Ovary (CHO), 373 Chitin, 330
332 Chitosan (Ch), 330
332, 339
340, 344
345, 466
467, 818 Chlamydia, 267
273 adjuvant selection and route of immunization, 271
273 C. muridarum infection, 262, 269 C. pecorum, 821 C. pneumoniae, 222 C. trachomatis, 262
263, 267, 299
300, 303
305, 626, 629 genital tract infections pathogenesis, 268 vaccine candidates, 640t challenges to development of vaccine, 631
632 choice of antigen, 271 clinical manifestations, 626 current treatment options, 626
627 epidemiology, 626 as gastrointestinal commensal, 273 immune responses associated with pathology, 627 innate immunopathological mechanisms, 628f microbiology, 626 natural immunity and vaccines, 269 preclinical vaccine studies and vaccine trials, 630
631 protective immune responses, 630f protective immunity against chlamydia, 269
270 vaccine trials, 274t vaccine-related research, 627
629 Chlamydophila caviae, 262 CHO. See Chinese Hamster Ovary (CHO) Cholera, 365, 537
538 adaptive mucosal immune responses in, 542
547 MBC responses in, 550
551 susceptibility and innate immunity, 538
540 Cholera toxin (CT), 6
7, 10, 76
77, 120
121, 168, 185
186, 334
335, 342
343, 366, 409
410, 791
792, 842
844, 860
861

886 Cholera toxin (CT) (Continued) ADP-ribosylation of defective mutants of, 187
188 derivatives, 188
189 Cholera toxin B subunit (CTB), 6
7, 10, 185
186, 340
341, 358
359, 604, 650 Cholera toxin-encoding (CTX), 537 Cholesterol, 337 Cholesterol-bearing pullulan (CHP), 332
333, 346 Cholesterol-α-glucosyltransferase (CGT), 588
589 Cholesteryl-bearing pullulan nanogel (cCHP nanogel), 332
333, 467 application of cCHP nasal vaccines against noninfectious diseases, 471 with artificial chaperone function, 468f cCHP
PspA nanogel, 470
471 as drug-delivery systems for nasal vaccines, 467 uptake, 469f Choline-binding proteins, 597
598 Cholvax, 541
542 Chondroitin sulfate, 214 ChoP. See Phosphorylcholine (ChoP) CHP. See Cholesterol-bearing pullulan (CHP) Chronic active gastritis, 579
580 Chronic inflammatory diseases, 213
214 Chronic secretory otitis media (CSOM), 314 Chymase, 219 Circulating memory T cells, 727
729 Circumsporozoite protein (CSP), 760
761, 831 Citrobacter C. rodentium, 60, 147
148, 204 infection, 236 Class switching, 425 Class-switch recombination (CSR), 119, 191
192 Classic parenteral vaccination, 307 Claudin, 58
59 claudin 4, 31
32, 339, 492 claudin-4-targeting peptide of CPE, 37
38 claudin-7, 146 CLDC. See Cationic lipid
DNA complex (CLDC)

INDEX

CLEC7A gene, 109 CLNs. See Cervical lymph nodes (CLNs) Clostridia species, 9
10, 58, 144, 148
149, 863 C. botulinum, 346 C. difficile, 9
10, 77
78, 236
237, 875
876 C. difficile-induced colitis, 105 C. orbscindens, 155 C. perfringens antigens, 391 C. sporogenes, 146 Clostridiales, 158
159 Clostridium botulinum type A neurotoxin heavy-chain Cterminus (BoHc/A), 332
333 Clostridium perfringens enterotoxin (CPE), 31
32, 469
470, 492 C-terminus, 492 CLP. See Common lymphoid progenitor (CLP) CLRs. See C-type lectin receptors (CLRs) CM. See Cervical mucus (CM) 4CMenB, 635 CMG2. See Capillary morphogenesis gene 2 (CMG2) CMI. See Cell-mediated immunity (CMI) CMIS. See Common mucosal immune system (CMIS) cNKs. See Conventional NK cells (cNKs) CNPV. See Canarypox virus (CNPV) Co1 peptide, 492 Cobalamins, 204
205 Coccidiosis, 413
414 COCs. See Combined oral contraceptives (COCs) Cold-adapted influenza viruses, 678
679 Colistin, 174
175 Colon, 145
146 Colonic patches, 21
22, 28
32 Colonization factors (CFs), 563 ETEC, 565 Colony-forming units (CFUs), 449
450 Colony-stimulating factors (CSFs), 778
779 Colostrum, 819 Columnar epithelial cells, 59 Combined oral contraceptives (COCs), 263

Commensal(s), 58 bacteria and metabolites, 207
209, 208f microbes, 217 microbiota, 150 Commercialization, 372 challenges for, 379
380 Common helper innate lymphoid precursors (CHILPs), 229
231 Common lymphoid progenitor (CLP), 229
231 Common mucosal immune system (CMIS), 4
5, 790
791 Common ocular pathogens, 303
305, 304t Companion animals, 820 Complementarity-determining region 3 (CDR3), 713 Compound 48/80 (C48/80), 173
174 Congenital HCMV, 741
742 Congenital rubella syndrome (CRS), 738 Conjugated vaccine antigens, 358
359 Conjunctiva, 301 Conjunctiva-associated lymphoid tissue (CALT), 300 Connective tissue MCs, 215
216 Consensus envelope protein domain III (cEDIII), 190
191 Conserved proteins, 600 Consumable formulations, safety of, 378 Contraceptive-driven susceptibility to sexually transmitted diseases, 263 Controlled human infection model (CHIM), 570, 763 Convalescents, 538 Conventional liposomes, 337 Conventional NK cells (cNKs), 229, 232 CoP. See Correlates of protection (CoP) Corneal immunopathology, 304
305 Corneal neovascularization, 303 Correlates of protection (CoP), 501
502, 667
669 Corticosteroids, 60 Corynebacteriaceae-dominated microbiota, 153 Corynebacterium C. accolens, 156 C. bovis, 156 C. diphtheriae, 5
6

887

INDEX

C. mastitidis, 155
156 C. pseudo-diphtheriticum, 605 Cost-related hurdles, 357 CostimulAtory molecules, 195 Coxiella burnetii, 42, 777, 780 Coxsackievirus, 420
421 B3 infection, 466
467 Coxsackievirus and adenovirus receptor (CAR), 420
421, 430, 435
436 CPE. See Clostridium perfringens enterotoxin (CPE) CpG DNA, 108
109 mucosal adjuvant and modulator activity, 176 CpG oligodeoxynucleotides (CpGODN), 651, 799
800, 860
861 CPS-specific IgG. See Capsular polysaccharide-specific IgG (CPS-specific IgG) CRM9. See Native Corynebacterium diphtheria toxin mutant (CRM9) CRM9succ. See Succinylated Corynebacterium diphtheria toxin mutant (CRM9succ) Crohn’s disease (CD), 93
94, 105, 247
248 Cross-linked hydrogel
vaccine antigen complexes, 467 Cross-reactive carbohydrate determinants (CCDs), 376 Crp, 385 CRS. See Congenital rubella syndrome (CRS) CRTh2. See Tissue chemoattractant prostaglandin D2 receptor (CRTh2) CRX-675 ligand, 177 Cryptopatches, 21
22, 25
27 Cryptosporidiosis, 845 Cryptosporidium, 780
782 C. hominis, 845 C. parvum, 845 cSAPs. See Charge-switching synthetic adjuvant particles (cSAPs) CSFs. See Colony-stimulating factors (CSFs) cSMAC. See Central SMAC (cSMAC) CSOM. See Chronic secretory otitis media (CSOM) CSP. See Circumsporozoite protein (CSP)

CSR. See Class-switch recombination (CSR) CT. See Cholera toxin (CT) CTA1-DD CTA1-DD/IgG complexes, 174
175 fusion protein, 168, 188, 193 CTB. See Cholera toxin B subunit (CTB) CTLA4. See Cytotoxic T-lymphocyteassociated antigen 4 (CTLA4) CTLs. See Cytotoxic T cells/ lymphocytes (CTLs) CTX. See Cholera toxin-encoding (CTX) Culture filtrate proteins (CFP), 411 Cutaneous anthrax, 446
447 Cutter vaccine, 452
453 CVD. See University of Maryland Center for Vaccine Development (CVD) CVD1208, 566
567 CVL. See Cervicovaginal lavage (CVL) CVM. See Cervicovaginal mucus (CVM) CWP2. See Cyst wall protein-2 (CWP2) cya, 385 CyaA toxin, 186 Cyclic GMP-AMP synthase (cGAS), 420
421, 726 Cyst wall protein-2 (CWP2), 844
845 Cysticerci, 847
848 Cysticercosis, 847
848 Cysts, 842
845 CyTOF, 753
755 Cytokines, 138
139, 214
215, 264
266, 403
404, 420
421 IFNα, 169
170 IL-1 family, 170
171 as mucosal vaccine adjuvants, 168
171 Cytometric analysis, 257 Cytophaga-FlavobacteriumBacteriodes (CFB), 149 Cytoplasmic sensors, 683
684 Cytosolic retinal dehydrogenase (RALDH), 91
92 RALDH1, RALDH2, and RALDH3, 92 Cytotoxic T cells/lymphocytes (CTLs), 186
187, 203, 477
478, 790
791 Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), 653
654 Cytotoxicity, 775
776

D Damage-associated molecular patterns (DAMPs), 168, 216, 302 DAP. See Diaminopimelic acid (DAP) DARPA. See Defense Advanced Research Projects Agency (DARPA) DARPin E2_79 and bi53_79, 860 DBA. See Dolichos biflorus agglutinin (DBA) DCs. See Dendritic cells (DCs) DEADbox helicase 41 (DDX41), 420
421 Dectin-1, 75
76, 492, 773, 780 Defense Advanced Research Projects Agency (DARPA), 374 Defensins, 103 DEFA5, 103
104 Deletion mutants, 385 Delivery systems for toxin-based adjuvant, 190
191 Δcrp S. Typhimurium mutant strains, 387 Dendritic cells (DCs), 24, 27
28, 30, 35, 58, 90, 106, 137, 146, 148, 172
173, 192
193, 195, 203, 214
215, 231
232, 249
250, 264
266, 327
328, 383
384, 407
408, 461
464, 504, 633
634, 653
654, 716
717, 726, 753
755, 775, 789, 835
836 DC-like cells, 266 homing specificity imprinting by, 91
92 targeting mucosal vaccines for aged, 800
802 Depot medroxyprogesterone acetate (DMPA), 263 Der p 1, 857
859 induced allergic rhinitis, 862 Desminotyrosine, 155 DExH-box helicase 9 (DHX9), 685 Dextran, 330
332 Dextran sodium sulfate (DSS), 107, 778
779 Dextran sulfate, 330
332 DHX9. See DExH-box helicase 9 (DHX9) Diaminopimelic acid (DAP), 386
387 Diarrhea causing by Vibrio cholerae, 365
367 Dietary lipids, 206
207 Dietary retinoids, 234

888 Differentiation of infected from vaccinated animals vaccines (DIVA vaccines), 812
813 Digestive tracts, 21 1,25-Dihydroxy-vitamin D, 206 2,4-Dinitrochlorobenzene, 12
13 Diphtheria, 736
737 Direct bactericidal activity, 217
219 Disseminated gonoccocal infections, 632
633 DIVA vaccines. See Differentiation of infected from vaccinated animals vaccines (DIVA vaccines) DLNs. See Draining lymph nodes (DLNs) dmCT. See Double mutant of CT (dmCT) dmLT adjuvant, 569
570, 573
574 DMPA. See Depot medroxyprogesterone acetate (DMPA) DNA, 328, 344 DNA-based vaccine, 653
654 vaccines, 861
862 prime immunization, 411
413 viruses, 753
755 DNA plasmid encoding FL (DNA pFL), 655 Dolichos biflorus agglutinin (DBA), 37
38 Dorsal root ganglia (DRG), 278, 725
726 Double mutant of CT (dmCT), 188 Double-stranded RNA (ds RNA), 109
110 Draining lymph nodes (DLNs), 56, 213, 220 DRG. See Dorsal root ganglia (DRG) Drug delivery, 491 cCHP nanogel as, 467 systems for nasal vaccines, 464
467 ds RNA. See Double-stranded RNA (ds RNA) DSCAV-1 (RSV Env protein), 739
740 dsRNA-dependent protein kinase PKR, 684 DSS. See Dextran sodium sulfate (DSS) DTaP vaccine, 123 Dual-purpose TB/SIV vaccine, 405
406 Dukoral, 6
7, 528, 540, 541t, 543, 548 Duodenal biopsies, 539

INDEX

Dysbiosis, 101
103, 779, 871
872 Dysentery, 515 Dysfunctional intestinal permeability, 103 Dysregulated mast cell activity during infection, 222
223

E E-cadherins, 56, 58
59, 146 E1-deleted adenovirus vectors, 424 EAE. See Experimental autoimmune encephalomyelitis (EAE) EaeH gene, 568 EALT. See Eye-associated lymphoid tissue (EALT) Early secretory antigen 6 (ESAT-6), 387 EatA, 568 EB. See Elementary body (EB) Echinococcosis, 848
849 Echinococcus, 848
849 E. granulosus, 848
849 E. multilocularis, 848
849 ECM. See Extracellular matrix (ECM) Ectoparasites, 841
842 Ectopic lymphoid organs. See Tertiary lymphoid organs (TLOs) Edema factor (EF), 186, 447 Edema toxin, 447 “Edible” vaccines development, 190
191, 357 Edwardsiella, 340 E. ictaluri vaccine, 823 EF. See Edema factor (EF) Effector memory (EM), 410
411 Effector memory T cells (TEM cells), 133
134, 729 Effectors of adaptive immunity, 221 Efficacy of Natalizumab as Active Crohn’s Therapy (ENACT-1), 94 EGF. See Epidermal growth factor (EGF) EGFP. See Enhanced green fluorescent protein (EGFP) EGFR. See Epidermal growth factor receptor (EGFR) Eicosanoids, 214
215 EIF2AK4. See Eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) Elafin, 587 Elementary body (EB), 626 ELISA. See Enzyme-linked immunosorbent assay (ELISA)

ELISPOT method, 544
546, 569
570 EM. See Effector memory (EM) EMA. See European Medicines Agency (EMA) Emulsions, 335
336 ENACT-1. See Efficacy of Natalizumab as Active Crohn’s Therapy (ENACT-1) Enantiomers, 329 Endemic cholera, 537
538 “Ending Cholera—A Global Roadmap to 2030” program, 538 Endogenous danger signals, activation by, 217 Endogenous inflammatory peptides, 219 Endometrium, 257 Endothelin-1, 217, 219 Endotoxin. See Lipid A; Lipooligosaccharide (LOS) Enhanced green fluorescent protein (EGFP), 490 Enhanced immunogenicity, 409 Entamoeba histolytica, 842
844 Enteric bacteria, 393
394 Enteric nervous system, regulation of mucosal ILCs and, 233
234 Enteric septicemia, 823 Enteric vaccine development, 546
547 Enterobacteriaceae, 144 Enterobacteriales, 158
159 Enteropathogenic Escherichia coli infection (EPEC infection), 105 Enterotoxigenic Escherichia coli infection (ETEC infection), 191, 361
362, 517, 563 Enterotoxin adjuvant, 650 CT, 128 Entyvio. See Vedolizumab Env gene, 406 Enzyme-linked immunosorbent assay (ELISA), 317
319, 756
757 Eomesodermin (EOMES), 231 EPEC infection. See Enteropathogenic Escherichia coli infection (EPEC infection) Epicoccum nigrum, 154 Epidermal growth factor (EGF), 231 Epidermal growth factor receptor (EGFR), 59
60 Epithelial cell(s), 76
77, 101, 172
173, 462 junctions, 58
59, 266

INDEX

Epithelial cytokines as key mediators for mucosal immune responses, 134
136 Epithelial molecules, 583 Epithelial α(1,2)-fucosylation, 57 Epithelium, 146, 300
301, 402
403 ER. See Estrogen receptors (ER) ESAT-6. See Early secretory antigen 6 (ESAT-6) Escherichia, 833
835 Escherichia coli, 7, 10, 31, 73
74, 118, 168, 185
186, 204, 217
219, 271, 340
341, 383, 478, 487, 650
651, 706
707, 793, 861, 870 E. coli
Shigella, 525
526 heat-labile toxin, 128, 342
343 lipid A, 392 O86:B7, 834
835 strains, 58 Estradiol, 263
264, 270, 278
282 Estrogen receptors (ER), 263
264 Estrous cycle, 261, 825 ETEC infection. See Enterotoxigenic Escherichia coli infection (ETEC infection) ETEC vaccine candidates, 564
565 in clinical development, 573
574 ACE527 vaccine, 570 LT patch, 571 oral inactivated whole cell ETEC vaccines, 571
573 tip adhesins, 570
571 in development, 567t fimbrial antigens and heat-labile enterotoxin, 564 methods for assessing mucosal immune responses against, 569
570, 570t optimal administration route evaluation, 565
569 oral mucosal adjuvants, 569 putative protective ETEC antigen structure, 565 ETEC vaccine passive protection trials in humans, 566 EtpA adhesion, 568 ETVAX. See Multivalent ETEC vaccine (ETVAX) Eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4), 758 European Medicines Agency (EMA), 329

Euvichol, 540
541, 541t EV76, 453 Excessive adaptive immune responses, 247
248 Exocytosis of biologically active modulators, 214
215 Exogenous hormones, 263 Experimental autoimmune encephalomyelitis (EAE), 93, 494 Extracellular matrix (ECM), 90 Extracellular polysaccharide capsule, 597 Extraintestinal immunity, 150
156 Eye-associated lymphoid tissue (EALT), 300
302, 306
307

F F1 antigen, 452
453 FAE. See Follicle-associated epithelium (FAE) Famciclovir, 278 Farmed fish, 822 FcRn. See Neonatal Fc receptor (FcRn) FDA. See U.S. Food and Drug Administration (FDA) FDCs. See Follicular dendritic cells (FDCs) Fecal microbiota transplantation (FMT), 870
871 Fecal SIgA, 361
362, 842
844 Fel d 1, 861 Female genital tract (FGT), 255
256 adaptive immune cells in lower, 260 in upper, 257
260 contraceptive-driven susceptibility to sexually transmitted diseases, 263 hormone effects on genital tract infections, 262
263 receptors and signaling, 263
264 immunology of, 256
264 innate immune cells in lower, 260 in upper, 257 soluble mediators in, 261 TLR expression in, 262 Female reproductive tract, 8
9 Fertility, mucosal vaccines to improve, 825 FGT. See Female genital tract (FGT)

889 FI-RSV. See Formalin-inactivated RSV (FI-RSV) Fibroblastic reticular cells (FRCs), 24 Fibronectin, 491 FimA. See Fimbrillin (FimA) Fimbriae, modification of, 394
395 Fimbrial antigens, 564 proof of concept clinical trials, 564t studies in animals, 564 studies in humans, 564 Fimbrillin (FimA), 653 Firmicutes, 144, 149 Fish body temperature, 822 FITC-conjugated OVA, 478 FL. See Flt3 ligand (FL) Flagellin (fliC), 568, 860
861 flagellin-conjugated sporozoite antigens, 835 Flexyn2a, 526
528 Flt3 ligand (FL), 801 Fluenz, 341
342 FluMist, 341
342 FMT. See Fecal microbiota transplantation (FMT) Folic acid, 204
206 Follicle-associated epithelium (FAE), 34, 383
384, 402
403, 487, 790
791 Follicle-stimulating hormone (FSH), 825 Follicular dendritic cells (FDCs), 24, 137 Food materials nasal vaccine delivery, 341
344 tablets and capsules, 341 vaccines oral delivery using, 340
344 Food-crop-made therapeutics, 378 Forkhead box P3 (FoxP3), 258 Formalin-inactivated RSV (FI-RSV), 670
671 FoxP3. See Forkhead box P3 (FoxP3) Foxp31 regulatory cells, 247
248 Francisella tularensis, 42, 60
61, 779 lpxE gene, 392
393 FRCs. See Fibroblastic reticular cells (FRCs) FSH. See Follicle-stimulating hormone (FSH) Fucosyltransferase 2 (FUT2), 57, 105, 235
236 Fungal zymosan, 216 Funtumia elastica, 779

890 Fur (ferric uptake regulator), 385 Fusion (F) glycoprotein, 665 FUT2. See Fucosyltransferase 2 (FUT2)

G G-protein activation, 491 G908R, 110 Gadus morhua (Atlantic cod), 822 Gag gene, 406 Gai2, 109
110 Galactose (Gal), 842
844 galE, 6
7 GalNAc. See N-acetylgalactosamine (GalNAc) GALT. See Gut-associated lymphoid tissues (GALT) Gamma chain (γc), 229 γδ T cells (γδT17), 150
153, 203, 773
774, 774t cytokine production, 776 γδ T cell-mediated cytotoxicity, 775
776 to myeloid and macrophage cells, 775 plant polyphenols for activation, 778
782 role in infectious diseases, 776
777 surface receptors, 774 therapeutic potential for manipulation, 777
778 GAPs. See Goblet-cell-associated passages (GAPs) Gardasil, 255
256 GAS6 expression. See Growth-arrestspecific 6 expression (GAS6 expression) Gastrointestinal (GI) tract, 101, 273, 402, 790
791, 869 age-associated changes in GI tract immune system, 791
792 Gastrointestinal commensal, Chlamydia as, 273 Gastrointestinal system (GIS), malaria’s effect on, 832
833 gB. See Glycoproteins B (gB) GBS. See Group B streptococcus (GBS) gC. See Glycoproteins C (gC) GCC. See Guanylyl cyclase C (GCC) GCN2. See Eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) GCs. See Germinal centers (GCs) gD. See Glycoprotein D (gD)

INDEX

gE/gI glycoproteins, 731
732 Gene expression profiles, 404
405 Gene signatures, systems analysis of, 761 Gene-based vaccines, 672 Generally recognized as safe (GRAS), 191 Genetic engineering technologies, 359 Genetically modified organism (GMO), 366
367, 812
813 Genital herpes, 273
284, 723
724 clinical genital herpes vaccines, 282t future perspectives, 282
284 HSV-1 virus life cycle, 724
725 HSV-2 future strategies, 731
732 vaccine development, 278
282 virus life cycle, 724
725 immune protective mechanisms against HSV infections, 726
729 pathogenesis, 725
726 symptoms, 726 vaccine approaches against, 729
731 Genital mucosa, immunizations through, 436 Genital tract, mucosal immunity regulation in Chlamydia, 267
273 as gastrointestinal commensal, 273 genital herpes, 273
284 immunology of FGT, 256
264 of male reproductive tract, 264
267 Genogroups I (GI), 704
705 Genome-wide association studies, 105 Genomics, 634 Germ theory, 447
448 Germ-free animals, 103 Germ-free mice (GF mice), 145, 794
795 Germinal centers (GCs), 119
120, 146
147, 790
791 formation in iBALT, 41
42 reaction in peripheral lymph nodes and Peyer’s patches, 122 Germline-encoded PRRs, 106 GF mice. See Germ-free mice (GF mice) GI. See Genogroups I (GI) Giardia duodenalis.. See Giardia intestinalis

Giardia intestinalis, 844
845 Giardia lamblia.. See Giardia intestinalis Giardiasis, 844
845 GLA-SE. See Glucopyranosyl lipid adjuvantstable emulsion (GLASE) Glandular lymphocytes. See Natural killer cells (NK cells), CD3
CD56bright CD16
cells GlaxoSmithKline, 701
702 GlaxoSmithKline Biologicals (GSK Biologicals), 729
730 Global contamination from food-cropmade vaccines ortolerogens, 377
378 Global impact and clinical disease, 666
667 “Global Roadmap to End Cholera by 2030”, 554
555 Global RV vaccination program, 699 Global Task Force on Cholera Control (GTFCC), 554 Glucan particles, 340 Glucopyranosyl lipid adjuvantstable emulsion (GLASE), 327
328 Glutamate decarboxylase (GAD) system, 394 Glutathione S-transferase (GST), 413, 847 Glycocalyx, 56
57 Glycoprotein 2 (GP2), 31, 487
488, 490
491 GP2high Tnfaip21, 489 Glycoprotein C (gC2), 731
732 Glycoprotein D (gD), 724
725 gD2, 278
279 Glycoprotein D (gpD), 279
280 Glycoproteins B (gB), 724
725 Glycoproteins C (gC), 724
725 Glycosaminoglycans, 300
301 Glycosylated proteins, 375
376 Glycosylation, 375
376 of epithelial cells, 57 patterns of mucin glycoproteins, 57 Glycosyltransferases, 78
79 GM-CSF. See Granulocyte-macrophage colony-stimulating factor (GMCSF) GMO. See Genetically modified organism (GMO) GMP. See Good Manufacturing Practice (GMP)

INDEX

Gnotobiotic piglet model, 703 studies, 149
150 GnRH. See Gonadotropin-releasing hormone (GnRH) Goblet cells, 56
57, 103, 146 goblet-cell-expressed muscarinic acetylcholine receptor 4, 59
60 Goblet-cell-associated passages (GAPs), 59 Gonadotropin-releasing hormone (GnRH), 824 Gonorrhea. See also Chlamydia; Mycoplasma; Syphilis clinical manifestations, 632
633 current treatment options, 633 epidemiology, 633 future vaccine implications, 635 immune responses associated with pathology, 633
634 microbiology, 632 vaccine-related research, 634
635 Good Manufacturing Practice (GMP), 358, 374 GP2. See Glycoprotein 2 (GP2) gpD. See Glycoprotein D (gpD) GPR43 receptor, 149
150, 208
209 GPR183 receptor, 26
27 Graft-versus-host disease (GVHD), 105, 237 Grain-based candidates, 357
358 Granulocyte-macrophage colonystimulating factor (GM-CSF), 26, 157
158 Granuloma, 611
613, 612f, 615 GRAS. See Generally recognized as safe (GRAS) Griffonia simplicifolia I isolectin B4 (GSIB4), 37
38 Group B streptococcus (GBS), 735
736, 744 Growth-arrest-specific 6 expression (GAS6 expression), 150
153 GSI-B4. See Griffonia simplicifolia I isolectin B4 (GSI-B4) GSK Biologicals. See GlaxoSmithKline Biologicals (GSK Biologicals) GST. See Glutathione S-transferase (GST) GTFCC. See Global Task Force on Cholera Control (GTFCC) GuaBA attenuated Shigella strains, 566
567

Guanylyl cyclase C (GCC), 565 Gummas, 636 Gut aging involvement of mucosal CD41 T cells in, 793
794 potential mechanisms in, 793 Gut homeostasis, 251
252 Gut homing lymphocytes, 85
86 memory T cells, 505
506 accumulation and retention in mucosa, 506
508 receptors, 30, 251, 505 Gut immunity, rejuvenation of, 795
796 Gut intestine, 123 Gut lamina propria of jawless fish, 22
23 Gut microbiome, 833 Gut microbiota, 144
150 cell-mediated immunity, 146
149 gut mucosal surface, 145
146 humoral immunity, 149
150 role for, 833
835 Gut-associated lymphoid tissues (GALT), 21
23, 25
32, 85, 146
147, 191, 358
359, 383
384, 402
403, 487, 790
791, 796
797. See also Lymphoid tissues of respiratory tract cryptopatches, 25
27 development, 32
34 ILFs, 27
28 M cell differentiation in, 34
35 Peyer’s patches, cecal patches, and colonic patches, 28
32 Gut-associated lymphoreticular tissues. See Gut-associated lymphoid tissues (GALT) Gut-specific lymphocyte homing, 85
86 GVHD. See Graft-versus-host disease (GVHD)

H H1N1, 364
365, 737 H3N2, 365 H5N1, 364 H9N2 avian influenza virus, 820 HA. See Hemagglutinin (HA) Haemophilus influenzae, 74
75, 590
591

891 HAL Allergy, 856
857 Hapten, 12
13 HBD-2. See Human beta defensin 2 (HBD-2) hBD1. See Human β-defensins 1 (hBD1) hBD2. See Human β-defensins 2 (hBD2) hBD3. See Human β-defensins 3 (hBD3) HBGAs. See Histo-blood group antigens (HBGAs) HBs antigen, 362t HBsAg. See Hepatitis B surface antigen (HBsAg) HBV. See Hepatitis B Virus (HBV) HCMV. See Human cytomegalovirus (HCMV) HD5 transgenic mice, 103
104 Heat shock protein (HSP), 656 Heat-labile enterotoxin (LT), 361, 411
413, 563
565 patch, 571 proof of concept clinical trials, 564t studies in animals, 564 in humans, 564 Heat-labile toxin (LT), 168, 526 Heat-stable toxin (ST), 526, 563 Helicobacter hepaticus, 158
159 Helicobacter pylori, 57, 77
78, 339, 579
580, 870
871, 876 blockage of innate defense mechanisms, 589f chronic colonization with, 579
580 epithelial cells role in execution of mucosal defense, 581
583 failure of past vaccination attempts, 586
587 future perspectives, 590
591 immune suppression and immune escape, 584f mechanisms affecting inflammation and defense, 590f natural immunity against, 585
586 pathogen recognition and early proinflammatory response, 585f pathogen sensing and response, 582f therapeutic tool box of H. pylori infections, 580
581 unmasking H. pylori’s immune evasion strategy, 587
589 vaccines as stimulators of mucosal immunity, 583
585

892 Heligomosomoides polygorus, 154
155 Helminth infections, 846
849. See also Protozoan infections cysticercosis, 847
848 echinococcosis, 848
849 schistosomiasis, 847 trichinellosis, 849 Helminths, 841
842 Hemagglutination assay, 765 Hemagglutinin (HA), 170, 358, 391
392, 678
679, 791
792 Hemagglutinin inhibition (HI), 364 Hemagglutinin-neuraminidase (HN), 616 Hematopoetic stem cells (HSCs), 789
790 Hemorrhagic septicemia (HS), 819 Heparin, 214 Hepatitis B surface antigen (HBsAg), 176
177, 363, 372, 429, 831 Hepatitis B vaccine, 625
626 Hepatitis B virus (HBV), 363 Herpes genital, 273
284, 723
724 treatments, 278 Herpes simplex virus (HSV), 255, 281
282, 742
743 HSV-1, 299
300, 723, 735
736 HSV entry receptors and ligands, 725f virus life cycle, 724
725 HSV-2, 273, 723, 735
736 future strategies for HSV-2 vaccine, 731
732 HSV entry receptors and ligands, 725f plasmid DNA vaccines, 280 vaccine development, 278
282 virus life cycle, 724
725 viral DNA, 723 Herpes zoster (HZ), 283
284 Herpesvirus entry mediator (HVEM), 724
725 Heterogenic memory Th2 cells, 134 Heterologous viral vectors, 731 HEVs. See High endothelial venules (HEVs) Hexokinase, 235 HI. See Hemagglutinin inhibition (HI) High dimensional flow cytometry approaches, 753
755 High endothelial venules (HEVs), 25, 28, 36, 86
87, 204, 790
791

INDEX

High-fiber diet, 145
146 High-resolution structures, 668 Hillchol, 541
542 Histo-blood group antigens (HBGAs), 57, 705 HIV. See Human immunodeficiency virus (HIV) HIV/SIV infection HIV/SIV-specific CD41 T cells, 719 mucosal vaccines inducing HIVspecific antibody responses, 713
716 mucosal vaccines inducing HIVspecific T cell responses, 716
719 HLA. See Human leukocyte antigen (HLA) HN. See Hemagglutininneuraminidase (HN) HNECs. See Human nasal epithelial cells (HNECs) Homeostasis impact of immunization on, 873
876 key features of host response in, 871 Homeostatic scar, 872
873 Homing markers, 123
124 Homing specificity imprinting by dendritic cells, 91
92 Homologous decarboxylases, 394 Hookworm. See Ancylostoma caninum (Hookworm) Hormone effects on genital tract infections, 262
263 Hormone receptors and signaling, 263
264 Host cell membrane, 724
725 Host-derived proinflammatory mediators, 217 Host
bacteria interactions, 598
599 Host immune responses to pneumococci, 600
603 Host
pathogen interactions, 599 Host range restriction (HRR), 686 Hp281, 586
587 HPIV2. See Human parainfluenza type 2 virus (HPIV2) HPMCP. See Hydroxypropyl methylcellulose phthalate (HPMCP) HPV. See Human papillomavirus (HPV) HPV-like particles, 481
482

HRR. See Host range restriction (HRR) HS. See Hemorrhagic septicemia (HS) HSCs. See Hematopoetic stem cells (HSCs) HSP. See Heat shock protein (HSP) Hsp60, 586
587 HSV. See Herpes simplex virus (HSV) Human adenovirus serotypes (HAdVs) HAdV4 vaccine, 429
430 HAdV5 vaccine, 419, 421 HAdV7 vaccine, 429
430 HAdV40 serotype, 426 HAdV41 serotype, 426 Human beta defensin 2 (HBD-2), 261 Human challenge model of typhoid fever, 502
503 Human colon, 144 Human cytomegalovirus (HCMV), 735
736, 741
742 Human immunodeficiency virus (HIV), 255, 403
407, 611
612, 735
736, 740
741 HIV vaccine, 405, 615
616 anti-HIV neutralizing antibodies characterization to design mucosal, 713
714 antibody-related correlates in HIV vaccine clinical trials, 714
715 HIV-1 vaccine, 158
159 replication, 409
413 therapeutic integrin inhibition for infection, 95
96 transmission, 405
406 vaccine community, 405 HIV/SIV infection mucosal vaccines inducing HIVspecific antibody responses, 713
716 T cell responses, 716
719 Human leukocyte antigen (HLA), 631 Human mast cells, 215
217 Human mucosal vaccine trials, 606 Human nasal cavity, 342 microbiome, 153 Human nasal epithelial cells (HNECs), 314
315 Human oral vaccine, 366
367 Human papillomavirus (HPV), 222
223, 255, 481
482, 625
626

INDEX

Human parainfluenza type 2 virus (HPIV2), 616 future study using HPIV2 vaccine, 620 Human virus formulations, 703
704 Human β-defensins 1 (hBD1), 587 Human β-defensins 2 (hBD2), 587 Human β-defensins 3 (hBD3), 587
588, 588f Humoral B cell immunity, 503
504 Humoral immunity, 149
150, 403
404 in EALT, 302
303 Humoral inflammatory responses, 602 HVEM. See Herpesvirus entry mediator (HVEM) Hydrophilic groups, 330
332 Hydrophilic NPs, 338 Hydrophilic vitamins, 204 Hydrophobic vitamins, 204 (4-Hydroxy-3-nitrophenyl)acetyl (NP), 121
122 Hydroxypropyl methylcellulose phthalate (HPMCP), 339 Hygiene hypothesis, 862
863 Hypertension vaccine, 471 Hypertrophy, 508 Hypoallergenic antigens, 856
859, 858t Hypoallergenic Cryj2 T cell epitopes, 317
319 HZ. See Herpes zoster (HZ)

I Iatrogenic and genetic immunedeficiencies involving integrins, 94
95 iBALT. See Inducible bronchusassociated lymphoid tissue (iBALT) IBD. See Inflammatory bowel disease (IBD) ICAP1. See Integrin cytoplasmic domain-associated protein 1 (ICAP1) ICP4. See Infected cell protein 4 (ICP4) IcsA, 518 ID. See BCG intradermal (ID) ID2. See Inhibitor of DNA binding 2 (ID2) Ideal oral Shigella vaccines, 528 IDO. See Indoleamine 2, 3-dioxygenase (IDO) IDR1002. See Immune defense regulatory peptide (IDR1002)

IDRI. See Infectious disease research institute (IDRI) IECs. See Intestinal epithelial cells (IECs) IELs. See Intraepithelial lymphocytes (IELs) IFI16 inflammasomes, 685 IFN. See Interferon (IFN) IFN receptors (IFNRs), 686
687 IFN-stimulated response elements (ISREs), 687 IFN-β promoter stimulator 1 (IPS-1), 687 IFNRs. See IFN receptors (IFNRs) IFNα primed neutrophils, 407
408 IFNγ ELISPOT assays, 404
405 Ig. See Immunoglobulins (Ig) IgA. See Immunoglobulin A (IgA) IgA PCs. See IgA-producing plasma cells (IgA PCs) IgA-producing plasma cells (IgA PCs), 203 IgD-producing plasma cells, 72
73 IgE. See Immunoglobulin E (IgE) IGF. See Insulin-like growth factor (IGF) IgG. See Immunoglobulin G (IgG) IgM. See Immunoglobulin M (IgM) IgSF. See Immunoglobulin superfamily (IgSF) IIIVs. See Intranasal inactivated influenza vaccines (IIIVs) IKK-ε. See Inhibitor of kappa light polypeptide gene enhancer in B cells, kinase epsilon (IKK-ε) IL-2 receptor subunit gamma (IL2RG), 229 IL-22-binding protein (IL-22BP), 238 IL-22BP. See IL-22-binding protein (IL22BP) IL2RG. See IL-2 receptor subunit gamma (IL2RG) ILC precursor (ILCp), 229
231 ILC1. See Type 1 innate lymphoid cells (ILC1) ILC3s. See Type 3 innate lymphoid cells (ILC3s) ILCp. See ILC precursor (ILCp) ILCs. See Innate lymphoid cells (ILCs) ILFs. See Isolated lymphoid follicles (ILFs) Imiquimod, 108 Immersion vaccines, 823

893 Immune cells, 214, 266 enhancing influx of, 220 trafficking induction to infection sites, 219 Immune complexes, 60 Immune defense regulatory peptide (IDR1002), 821 Immune escape, 584, 584f Immune homeostasis regulation, 250
251 Immune protective mechanisms against HSV infections, 726
729 innate immune response to HSV infections, 726 protective memory responses against genital herpes infection, 726
729, 728f T cell-mediated protection, 727
729 Immune regulatory mechanisms, 753
755 Immune responses, 345, 756
757 to adenoviruses, 420
421 innate mechanisms regulated by toxin-based adjuvants, 191
194 modifiers of, 551
553 to mucosal antigens, 21
22 to Shigella in children, 519
520 tolerance induction by toxin-based adjuvants, 195 toxin-derivative adjuvants for mucosal vaccines, 187
191 toxins used for modulation, 185
187, 187f Immune suppression, 580, 584, 584f Immune-privileged environment, 266
267 Immunity, 677
678 antibody-mediated, 730
731 cell-mediated, 146
149 against cholera, 549 humoral, 149
150 humoral B cell, 503
504 infection-induced, 269 respiratory tract, 153
155 systemic T cell, 503 Immunization, 605 on homeostasis, 873
876 active immunization, 875
876 passive immunization, 873
875 through rectal/genital mucosa, 436 Immunobiome, 870
871

894 Immunocontraceptive vaccines, 824
825 efficacy, safety, and economic feasibility of, 825 Immunogenicity of adenovirus vectors, 424 molecular signatures of, 760
761 of Salmonella vaccines, 387
388 Immunoglobulin A (IgA), 4
5, 149
150, 258
259, 344
345, 490, 638
639, 790
791, 870 antibodies, 874 responses, 549, 677 Immunoglobulin E (IgE), 72
73, 855 Immunoglobulin G (IgG), 60, 258
259, 344
345, 490, 637, 649, 713, 737, 813
818 antibodies, 638
639 IgG2, 519
520 IgG2a, 176 IgG2c, 176 IgG
antigen immune complexes, 60
61 in mucosal immunity, 74
75 responses, 477
478, 550 Immunoglobulin M (IgM), 542, 550, 637, 738
739, 834
835 Immunoglobulin superfamily (IgSF), 92 Immunoglobulins (Ig), 261, 727 Ig glycan
dependent reactivity with microorganisms, 77
78 Immunological dysfunction, homeostatic scar and, 872
873 Immunological homeostasis, 203 Immunological synapse, 90
91 Immunology of female genital tract, 256
264 of ocular surface mucosa, 301
303 Immunomodulatory microbes, 158
159 Immunosenescence, mucosal vaccines and therapies for, 799
800 Immunostimulating complexes (ISCOMs), 326
328, 337 matrices, 337 Immunostimulatory agents, 327
328 Immunosuppressive effects, 155 Immunotherapy, 377
378, 856
862 adjuvants and delivery, 860
861 Anti-IgE, 859
860 DNA vaccines, 861
862 hypoallergenic antigens, 856
859, 858t

INDEX

IMPAACT. See International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Impaired mast cell activity during infection, 222
223 IMSUT. See Institute of Medical Science, University of Tokyo (IMSUT) IN BCG immunization. See Intranasal BCG immunization (IN BCG immunization) In ovo immunization, 819
820 In ovo injection, 819
820 In vitro regulatory Th cells (iTreg), 871 In-feed oral vaccination, 819
820 Inactivated intranasal influenza vaccine, 462 Inactive SIV particles (iSIV), 406
407 Inclusion, 626 Indoleamine 2, 3-dioxygenase (IDO), 249
250, 628
629 Inducible bronchus-associated lymphoid tissue (iBALT), 21
22, 38
39, 39f, 41
42, 122
123, 136
138 induction during inflammation at mucosal tissue sites, 136
137 maintenance of memory T cells within, 137
138 Infected callus, 361 Infected cell protein 4 (ICP4), 280
281 Infected erythrocyte (iRBC), 831
832 Infection-induced immunity, 269 Infection-prone children, 603 Infectious agents, 461
462 Infectious disease research institute (IDRI), 327
328 Infectious diseases, 3, 340
342, 346 control of, 361
367 diarrhea caused by Vibrio cholerae, 365
367 enterotoxigenic Escherichia coli, 361
362 HBV, 363 influenza virus, 364
365 norovirus, 363 rabies, 363
364 γδ T cells role in, 776
777 prevention of, 361
367 “Inflamm aging”, 789 Inflammasomes, 75, 685 Inflammatory cytokines, 251, 267
268

diseases, 313 mediators, 627 peptides, 219 Inflammatory bowel disease (IBD), 85, 101
103, 247
248, 870
871 human intestinal myeloid cells’ role in maintenance of, 251
252 therapeutic integrin inhibition for, 93
94 Influenza, 677, 730 coinfection, 601
602 influenza A, 364 influenza B, 364 influenza C, 364 influenza D, 364 vaccines, 342 viral antigens, 345 virus, 358, 364
365, 481
482, 737
738, 802 Inhibitor of DNA binding 2 (ID2), 229
231 Inhibitor of kappa light polypeptide gene enhancer in B cells, kinase epsilon (IKK-ε), 687 Injectable poliovirus vaccine (IPV), 334 Injected vaccine, 824 iNKT cells. See Invariant natural killer T cells (iNKT cells) Innate immune cells in lower FGT, 260 in upper FGT, 257 Innate immune response, 409, 539 to HSV infections, 726 Innate immune system. See Innate immunity Innate immunity, 101
103, 262, 539
540, 620, 765
766. See also Adaptive immunity cross-regulation of adaptive immunity and, 238
239 innate immune regulation in gut, 106
110 association between CLRs and intestinal inflammation, 109 association between NLRs and intestinal inflammation, 110 association between RLRs and intestinal inflammation, 109
110 associations between individual TLRs and intestinal inflammation, 106
109 pattern recognition receptors, 106

INDEX

innate immunity-based mucosal modulators and adjuvants cationic polyethyleneimine used as mucosal vaccine adjuvant, 175 cytokines as mucosal vaccine adjuvants, 168
171 innate immune system activators as adjuvants, 168 mast-cell-activating compounds with adjuvant activity, 173
175 mucosal adjuvant activity of tolllike receptor ligands, 175
178 mucosal adjuvants, 169t nanoemulsions as mucosal vaccine adjuvants, 171
173 innate mucosal barriers in gut, 103
105 distinct intestinal barriers in small and large intestines, 104f innate barrier dysfunction and disease pathogenesis, 105 structures of mucosal barriers, 103
104 of middle ear and nasopharynx, 314
315 role against pneumococci, 600
602 intestinal epithelial cells, 102f relationships between intestinal mucosal dysfunction and human diseases, 102f Innate invariant T cells, changes in, 504
505 Innate lymphocytes, 773 Innate lymphoid cells (ILCs), 229 in allergy, autoimmunity, and persistent inflammation, 237
238 cross-regulation of innate and adaptive immunity by mucosal ILCs, 238
239 development and tissue heterogeneity, 229
232, 230f ILC1s, 231 ILC2s, 40
41, 136, 155, 231, 237 ILC3, 25
26, 146
147, 231
232, 235
237, 239 in mucosal tissue repair, 237 organizing and initiating ILCs
dependent barrier immunity, 232
235 enteric nervous system regulates mucosal ILCs, 233
234

microbial and metabolic regulation of mucosal ILCs, 234
235 mucosal ILCs in pathogen defense, 235
237 Innate mechanisms regulated by toxinbased adjuvants, 191
194 Innate myeloid cells, 248 Innovative immunotherapy for cedar pollinosis, 317
319 Institute of Medical Science, University of Tokyo (IMSUT), 366
367 Insulin-like growth factor (IGF), 776 Integrin cytoplasmic domainassociated protein 1 (ICAP1), 89 Integrin(s), 85
86, 420
421, 491 integrin deactivation as regulatory mechanism, 87
90, 88f integrin α4β7
MAdCAM-1 interactions, 92 integrin-containing cellular debris, 89
90 Intentional induction of mucosal tolerance, 13 Interferon (IFN), 264
266, 601
602, 665
666, 683
684, 726 degradation of interferon receptors types, 691 IFNα, 169
170, 801 IFNγ, 58
59, 216, 231, 248, 317
319, 421, 449, 547, 583, 611
614, 627
629, 775, 842
844, 859 induction pathway regulation by RVs, 687
692 interferon signaling pathway regulation by rotavirus, 688
689 Interferon regulatory factor (IRF) IRF3, 175, 683
684, 687 IRF-7, 687, 801 regulation, 692 IRF8, 192 IRF9 regulation, 692 Interfollicular regions, 30 Interleukin (IL) IL-1, 170
171, 447, 627 IL-1α, 41, 170 IL-1β, 26, 170, 539 IL-4, 216, 236, 855 IL-5, 204, 229, 236, 463
464, 874 IL-5-producing Th2 cells, 134 IL-7, 31

895 IL-7 receptor α, 229 IL-8, 107
108, 420
421, 778
779 IL-10, 12
13, 148
149, 156
157, 247
248, 833 IL-10-producing T cells, 57 IL-12, 779 IL-13, 236 IL-15, 231 IL-17, 41, 611
612, 774, 842
844 IL-18, 447 IL-22, 236 IL-23, 874 IL-25, 41, 134
136 IL-33, 134
138 IL-33-induced inflammation, 136 International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT), 745 Interstitial migration, 90 Intestinal barrier dysfunction, 105 system, 106 Intestinal epithelial cells (IECs), 685 Intestinal IgA, 207 Intestinal immune tolerance, 249
250 Intestinal microbiota potentially shapes mucosal immunosenescence, 794
795 Intestinal mucosal T cells, 547 Intestinal niche, 869 Intestinal resident CX3CR1high macrophages, 250
251 Intestinal tissues, 203 Intestine-derived gut-homing circulating antibody-secreting cells, 546
547 Intraepithelial lymphocytes (IELs), 147
148, 193
194, 425 Intranasal BCG immunization (IN BCG immunization), 409
410 Intranasal chitosan solution formulations, 344 Intranasal immunization, 5
6, 272, 406, 435
436, 730
731 Intranasal inactivated influenza vaccines (IIIVs), 679
681 Intranasal route of administration, 7
8 Intranasal vaccines, 730
731, 823 adenovirus vectors as, 430
436, 431t Intraperitoneal immunizations (IP immunizations), 413 Intrauterine immunization, 820 Intravaginal HSV-2 vaccines, 730

896 Intravaginal immunization, 282, 730 Intravesical BCG immunotherapy, 407
408 Intravesical immunotherapy, 407
409 bladder cancer, 407
409 iNTS. See Invasive nontyphoidal Salmonella (iNTS) Invariant natural killer T cells (iNKT cells), 149, 613 Invasion plasmid antigens (Ipas), 515, 521 Invasive nontyphoidal Salmonella (iNTS), 395 Invasive pathogens, 762 IP immunizations. See Intraperitoneal immunizations (IP immunizations) Ipas. See Invasion plasmid antigens (Ipas) IPS-1. See IFN-β promoter stimulator 1 (IPS-1) IPV. See Injectable poliovirus vaccine (IPV) ISCOMATRIX platform, 271
272 ISCOMs. See Immunostimulating complexes (ISCOMs) iSIV. See Inactive SIV particles (iSIV) Isolated lymphoid follicles (ILFs), 21
22, 27
28, 231
232 organization and formation, 27f ISREs. See IFN-stimulated response elements (ISREs) iTreg. See In vitro regulatory Th cells (iTreg)

J

JAM. See Junctional adhesion molecule (JAM) Japanese cedar pollen-induced allergic conjunctivitis, 857 JC virus infection. See John Cunningham virus infection (JC virus infection) John Cunningham virus infection (JC virus infection), 94
95 Joining chain (J chain), 4
5, 72
73 Junctional adhesion molecule (JAM), 62 JAM-1, 493

K 83-kD PA (PA83), 186 Keratinocyte growth factor (KGF), 776

INDEX

Killed whole cell vaccine (KWC vaccine), 452
453 Klebsiella spp., 301 K. pneumoniae, 153
154 Knock-in lymphocytes, 89 Knockout mouse model, 761
762 Koala, 821 Kurdistan Iran man strains (KIM strains), 452
453

L L-selectin, 86
87 L-tyrosine, 860
861 L1007insC, 110 Labile toxin (LT), 342
343 ADP-ribosylation of defective mutants, 187
188 derivatives, 188
189 LT-I, 185
186 LT-IIa-B5, 192 LT-IIb-B5, 192 LacI repressor, 389 Lacrimal glands, 301 Lacrimal-duct-associated lymphoid tissue (LDALT), 36 Lactobacillus, 144, 208
209, 223 L. casei, 191, 861 L. crispatus, 155 L. johnsonii, 155 L. lactis, 861 L. pentosus, 208 L. plantarum, 406
407 NC8 strain, 820 L. rhamnosus, 406
407, 605 Lactococcus lactis, 605 LAD. See Leukocyte adhesion deficiency (LAD) LAIVs. See Live attenuated influenza vaccines (LAIVs) LAM. See Lipoarabinomannan (LAM) Lamina propria (LP), 56, 118
119, 478, 790
791 myeloid cell uptake of antigens and bacteria in, 61
63 Lamina propria mononuclear cells (LPMCs), 506
508 Lampreys, 22 Lanzhou lamb RV vaccine (LLR vaccine), 702 Large intestine patches, 29f LAT. See Latency-associated transcript (LAT)

Latency-associated transcript (LAT), 725
726 Latent syphilis, 636 Latent TB infection (LTBI), 612 LAV. See Live-attenuated vaccines (LAV) LCTBA, 571
572 LDALT. See Lacrimal-duct-associated lymphoid tissue (LDALT) LECs. See Lymphatic endothelial cells (LECs) Legionella infections, 777 Leishmania major, 156 Leporidae, 820 Lethal factor (LF), 186, 447 Lethal toxin, 447 Leukocyte adhesion deficiency (LAD), 94
95 Leukocytes, 266, 505 Leukotrienes (LT), 214
215, 219 LF. See Lethal factor (LF) LFA-1
ICAM-1 complex, 90
91 Licensed mucosal vaccines, 753
755 Lifestyle-related diseases, 346 Ligelizumab, 860 Lipid A, 392, 392f strategies for reducing toxicity, 392
393 Lipids, 206
207 Lipoarabinomannan (LAM), 404
405 Lipooligosaccharide (LOS), 314, 632 Lipopolysaccharide (LPS), 107, 313, 383
384, 392, 515, 539, 549, 565, 635 Liposomes, 328, 337, 344, 464
465 Lipotechoic acid (LTA), 156
157, 568, 604
605 Listeria, 383 infection, 777 L. monocytogenes, 31
32, 58
59, 147
148, 409 Live attenuated influenza vaccines (LAIVs), 678
679, 758
759 Live attenuated oral vaccine, 502
503 Live attenuated vaccines, 278
279, 334
335, 425
426, 528 Live vaccines, 279, 767, 811
812 for anthrax, 448 for livestock, 449 Y. pseudotuberculosis as, 453
454 Live viral vectors, 11 Live-attenuated HSV-1 vaccine, 307

INDEX

Live-attenuated vaccines (LAV), 334
335, 670
671 Livestock mucosal vaccines for, 813
820, 814t Avian species, 819
820 Bovinae, 818
819 Caprinae and Ovidae, 818 Suidae, 813
818 production, 813 LLR vaccine. See Lanzhou lamb RV vaccine (LLR vaccine) LNs. See Lymph nodes (LNs) Local innate immune responses, initiation of, 217
219 direct bactericidal activity, 217
219 immune cell trafficking induction to infection sites, 219 proteolytic degradation of toxins, 219 Local mucosal immunity, 677 Local mucosal immunization, 477
478 Long palate, lung, and nasal epithelium clone 1 protein (LPUNC1 protein), 539, 552 LOS. See Lipooligosaccharide (LOS) LP. See Lamina propria (LP) LPMC CD81 TM responses, 508
509 LPMCs. See Lamina propria mononuclear cells (LPMCs) LPS. See Lipopolysaccharide (LPS) LPUNC1 protein. See Long palate, lung, and nasal epithelium clone 1 protein (LPUNC1 protein) LT. See Heat-labile enterotoxin (LT); Heat-labile toxin (LT); Labile toxin (LT); Leukotrienes (LT) LT-II. See Type II heat-labile enterotoxins (LT-II) LT-α. See Lymphotoxin-α (LT-α) LT-β. See Lymphotoxin-β (LT-β) LTA. See Lipotechoic acid (LTA) LTB. See B subunit of heat-labile toxin (LTB) LTBI. See Latent TB infection (LTBI) LTi cells. See Lymphoid tissue inducer cells (LTi cells) LTin cells. See Lymphoid tissue initiator cells (LTin cells) LTo cells. See Lymphoid tissue organizer cells (LTo cells) LTβR. See Lymphotoxin-β receptor (LTβR)

Lung Th17 cells, 154 Luteinizing hormone releasing hormone. See Gonadotropinreleasing hormone (GnRH) Ly6/Plaur domain-containing 8 (Lypd8), 103
104 Ly49 family receptors, 232 Lymph nodes (LNs), 21
22, 479 role in sublingual vaccination, 479 Lymphatic endothelial cells (LECs), 137 Lymphocyte, 773 homing mechanisms, 464 imprinting mechanisms, 464 subsets, 85 Lymphoid chemokines, 40 follicles of colon/colonic patches, 146
147 Lymphoid tissue inducer cells (LTi cells), 25
26, 32, 229 Lymphoid tissue initiator cells (LTin cells), 32 Lymphoid tissue organizer cells (LTo cells), 32 Lymphoid tissues gene signatures systems analysis in, 761 of respiratory tract, 35
42 bronchus-associated lymphoid tissue, 38
42 NALT, 35
38, 36f Lymphotoxin-α (LT-α), 24 Lymphotoxin-β (LT-β), 24 Lymphotoxin-β receptor (LTβR), 32 Lypd8. See Ly6/Plaur domaincontaining 8 (Lypd8) Lysis strain, 391 Salmonella, 391
392 Salmonella Typhimurium, 391 Lyso-DCs, 30 Lyso-macrophages, 30 Lytic replication process, 725
726

M

M cells. See Microfold cells (M cells) M01ZH09 strain, 386 mAb. See Monoclonal antibody (mAb) Mac-1 antigen. See Macrophage-1 antigen (Mac-1 antigen) Macaca mulatta, 410 Machine learning approaches, 757
758

897 Macrolide-based antibiotics, 636 Macrophage-1 antigen (Mac-1 antigen), 186 Macrophages, 203, 266, 408 γδ T cells to macrophage cells, 775 MAdCAM. See Mucosal addressin cell adhesion molecule (MAdCAM) MAdCAM-1. See Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) MAIT. See Mucosa-associated invariant T (MAIT) MAIT cells. See Mucosal-associate invariant T cells (MAIT cells) Major histocompatibility complex (MHC), 149, 220
221, 420, 653
654, 773 MHCI, 232 Major outer-membrane proteins (MOMPs), 821 Malaria, 611
612, 841
842 effect on GIS, 832
833 gut microbiota, role for, 833
835 mucosal vaccine against, 831, 835
836, 836t Male gonococcal urethritis, 632
633 Male reproductive tract, immunology of, 264
267 Malnutrition, 552 Maltodextrin-binding protein, 654 Maltose-binding protein (MBP), 654 MALTs. See Mucosa-associated lymphoid tissues (MALTs) Mammals, 206 Man-rich N-linked chains, 77
78 MAS-related G-protein-coupled receptor-X2 (MRGPRX2), 217 Mast cells (MCs), 156
157, 213 contributions to adaptive immune responses, 220
222 dysregulated/impaired MC activity during infection, 222
223 exocytosis of biologically active modulators, 214
215 initiation of local innate immune responses, 217
219 mast-cell-activating compounds with adjuvant activity, 173
175 C48/80, 173
174 MC activators with mucosal adjuvant activity, 174
175 MC granule-inspired particles, 221
222

898 Mast cells (MCs) (Continued) mediator responses to microbial pathogens and functional roles, 215t multipronged activation at infection sites, 216
217 peripheral location, 215
216 Mastadenovirus, 420
421 Maternal antibodies, 873
874 Maternal immunization to protect vulnerable infants, 669
670 Maternal influenza vaccination, 737
738 Maternal interference, 819 Maternal vaccination, 735
736. See also Sublingual vaccination administered during pregnancy and maternal and neonatal pathogens, 736t diphtheria, pertussis, and tetanus, 736
737 influenza virus, 737
738 measles, mumps, and rubella, 738
739 next frontier of maternal vaccines, 739
744 GBS, 744 HIV, 740
741 HSV, 742
743 human cytomegalovirus, 741
742 RSV, 739
740 ZIKV, 743
744 Matrix metalloproteinase-7-deficient mice, 103
104 Matrix metalloproteinases (MMPs), 268 MAVS. See Mitochondrial antiviralsignaling protein (MAVS) MBCs. See Memory B cells (MBCs) MBP. See Maltose-binding protein (MBP) MC activators (MCA), 221 MCA. See MC activators (MCA) MCMV. See Murine cytomegalovirus (MCMV) MCP. See Neisseria meningitidis serogroup C polysaccharide (MCP) MCs. See Mast cells (MCs) MCT. See Tryptase-positive MCs (MCT) MCTC. See Tryptase-and chymasepositive MCs (MCTC)

INDEX

MDA5. See Melanoma differentiationassociated gene 5 (MDA5) MDP. See Muramyl dipeptide (MDP) MDSCs. See Myeloid-derived suppressor cells (MDSCs) Measles, 738
739 Measles-mumps-rubella vaccine (MMR vaccine), 735
736 Mechanotransduction, 88 MEFA. See Multiepitope fusion antigen (MEFA) Melanoma differentiation-associated gene 5 (MDA5), 109
110, 683
684 Melittin, 174
175 Membrane-associated sensors, 684
685 Membrane-bound LTα1β, 232 Memory B cells (MBCs), 117
118, 550
551 considerations for mucosal subcomponent vaccine development, 128 germinal center reaction in peripheral lymph nodes and Peyer’s patches, 122 inductive site for gut MBC responses, 119
121 model system to study, 121
122 mucosal, 120f memory B cells and homing markers, 123
124 mucosal vaccine induction, 119 poor clonal relatedness to long-lived plasma cells, 127
128 respiratory tract infections and mucosal, 122
123 responses in cholera, 550
551 sessile/recirculating, 125
127 Memory T cells, 133
134, 409 gut-homing memory T cells, 505
508 maintenance of, within the iBALT, 137 Meningococcal B vaccine (MeNZB), 635 Merck. See Zostavax Mesenchymal stem cell transfer, 795
796 Mesenteric lymph node DCs, 92 Mesenteric lymph nodes (MLNs), 21
22, 59
60, 119, 148, 779, 791 Metabolic regulation of mucosal ILCs, 234
235

Methylcarboxy chitosan, 330
332 MHC. See Major histocompatibility complex (MHC) Micelles, 335
336 Microbes, 144
145 microbe-derived metabolites, 26
27 Microbial communities, 870
872 Microbial diversity, 144
145 loss of, 871
872 Microbial exposure, 28 Microbial metabolites, 155 Microbial pathogenesis, 871
872 Microbial products for γδ T cells regulation, 780
782 Microbial regulation of mucosal ILCs, 234
235 Microbial sensors, 157
158 Microbiome in human health, 4
5 Microbiota, 9
10, 144
145, 234, 301, 862
863 and extraintestinal immunity, 150
156 mucosal immune sites, 155
156 oral immunity, 150
153 respiratory tract immunity, 153
155 and vaccines, 157
159 Micrococcus melitensis, 448
449 Microfold cells (M cells), 25, 29
31, 56, 146
147, 325
326, 358
359, 383
384, 402
403, 424
425, 461
463, 487, 517
518, 790
791. See also T cell(s) candidate molecules for M celltargeted vaccines α(1,2)-linked fucose, 490 α2-3-linked sialic acid, 493 C5aR, 492 Claudin 4, 492 GP2, 490
491 integrins, 491 PrPC, 491 SIgA receptors, 492 Umod, 491
492 characterization of, 488f contribution for oral tolerance development, 493
494 differentiation, 488
489, 489f in gut-associated lymphoid tissue, 34
35 M-cell-targeting lectins, 339 number and function enhancement, 494
495

INDEX

of Peyer’s patches, 10
11 role in gut aging, 793 Micronutrient deficiency, 552 Microparticles, 861 Microparticulate antigens, 10
11 Middle ear immunomodulation and clinical impact, 316
317 innate and acquired immunity, 314
315 innovative immunotherapy for attenuating nasal symptoms of cedar pollinosis, 317
319 TLRs distribution in human epithelial cells, 314 in nasopharyngeal mucosae, 314
315 Migration, 761 Mitochondrial antiviral-signaling protein (MAVS), 683
684, 688 MLNs. See Mesenteric lymph nodes (MLNs) mMCP6. See Mouse MC protease 6 (mMCP6) MMPs. See Matrix metalloproteinases (MMPs) MMR vaccine. See Measles-mumpsrubella vaccine (MMR vaccine) MMTV. See Mouse mammary tumor virus (MMTV) MNCs. See Mononuclear cells (MNCs) MNPs. See Mononoculear phagocytes (MNPs) Moderate to severe diarrhea (MSD), 516
517 Modified OrcVax (mOrcVax), 540
541 Modified vaccinia virus Ankara virus (MVA virus), 41, 271
272, 405
406, 718 MOG. See Myelin oligodendrocyte glycoprotein (MOG) Molecular farming, 340
341 Molecular mimicry, 656 Molecular signatures of immunogenicity, 760
761 MOMPs. See Major outer-membrane proteins (MOMPs) Monoclonal antibody (mAb), 73
74, 487
488 Monomeric serum IgA, 72 Mononoculear phagocytes (MNPs), 233 Mononuclear cells (MNCs), 544
546

Monophosphoryl lipid A (MPLA), 172, 327
328, 729
730, 860
861 mucosal adjuvant and modulator activity, 176
178 mOrcVax. See Modified OrcVax (mOrcVax) Motavizumab, 668, 739 Mother-to-child transmission (MTCT), 735
736, 740, 742
743 Mouse mammary tumor virus (MMTV), 37
38 Mouse MC protease 6 (mMCP6), 219 Mouse pulmonary model, 520 MPL, 392 MPLA. See Monophosphoryl lipid A (MPLA) MRGPRX2. See MAS-related Gprotein-coupled receptor-X2 (MRGPRX2) MSD. See Moderate to severe diarrhea (MSD) Mtb. See Mycobacterium tuberculosis (Mtb) MTCT. See Mother-to-child transmission (MTCT) Mucins (MUC), 146 glycoproteins, 56
57 MUC19, 105 MUC2, 57, 105 MUC5AC, 105 MUC5B, 105 MUC6, 105 Mucoadhesion, 337 Mucociliary clearance, 342, 462 MucoRice, 340
341, 357
358, 360
361, 360f MucoRice-CTB, 366
367 protocol, 360
361 vaccine, 190
191 Mucosa accumulation and retention in, 506
508 mucosa-associated cells, 790
791 Mucosa-associated invariant T (MAIT), 602 Mucosa-associated lymphoid tissues (MALTs), 25, 32, 56, 462
463, 790
791 Mucosal addressin cell adhesion molecule (MAdCAM), 36 Mucosal adjuvant activity of toll-like receptor ligands, 175
178

899 CpG mucosal adjuvant and modulator activity, 176 monophosphoryl lipid A mucosal adjuvant and modulator activity, 176
178 Mucosal anti-HIV Abs, 715
716 T cell responses, 717 Mucosal antibodies IgG in mucosal immunity, 74
75 isotypes and biological activities of mucosal immunoglobulins, 72t mechanisms of protection mediated by mucosal IgA antibodies, 75
80 Ig glycan
dependent reactivity with microorganisms, 77
78 inhibition of antigen absorption, 75
76 inhibition of bacterial adherence, 76
77 mucosal Ig interactions with innate antimicrobial components, 80 neutralization of biologically active antigens, 78
80 polyreactivity, 77 origin in external secretions, 73 properties of antibodies of Ig isotypes in external secretions, 72
73 protective effect, 73
74 responses, 481 Mucosal aquatic vaccines, 822
824 immersion vaccines, 823 injected vaccine, 824 oral and intranasal vaccines, 823 Mucosal barrier and lymphoid tissues, basic components of, 56
58 structures of, 103
104 Mucosal CCR5-tropic SHIV challenge models, 714 Mucosal compartmentalization causing inflammatory responses in airway, 133
136 epithelial cytokines as key mediators for mucosal immune responses, 134
136 heterogenic memory Th2 cells in different mucosal organs, 134 innate and adaptive immunity involvement in IL-33-induced inflammation at mucosal sites, 136

900 Mucosal compartmentalization causing inflammatory responses in airway (Continued) TRM cells for mucosal tissues, 133
134 Mucosal CTA1-DD adjuvant, 128 Mucosal defense in H. pylori, epithelial cells role in execution of, 581
583 Mucosal delivery method, 378, 402
403 Mucosal dendritic cells, 424
425 Mucosal epithelium, 25 Mucosal HIV transmission, 716
717 Mucosal human immunity, 507f Mucosal IgA antibody cell responses, 543
546 Mucosal ILCs cross-regulation of innate and adaptive immunity by, 238
239 enteric nervous system regulates, 233
234 microbial and metabolic regulation of, 234
235 in pathogen defense, 235
237 Mucosal immunity, 4, 145
156, 445
446, 505
506. See also Innate immunity control commensal bacteria and metabolites, 207
209 lipids, 206
207 vitamins, 204
206 discovering fundamental immunological mechanisms of, 763
767 protection against infection at mucosal sites by antibodies, 765
766 T cells role in protection against infection at mucosal sites, 766
767 vaccines and adjuvants imprint mucosal homing of antigenspecific T and B cells, 765 gut microbiota and, 145
150 for inflammation distinct modes of lymphocyte migration, 86f homing specificity imprinting by dendritic cells, 91
92 iatrogenic and genetic immunedeficiencies, 94
95 immunological synapse, 90
91

INDEX

integrin deactivation as regulatory mechanism, 87
90 integrin α4β7
MAdCAM-1 interactions, 92 interstitial migration, 90 molecular mechanisms for recruitment of circulating lymphocytes, 86
87 therapeutic integrin inhibition for HIV infection, 95
96 therapeutic integrin inhibition for inflammatory bowel diseases, 93
94 innate mechanisms regulated by toxin-based adjuvants for induction, 191
194 intestinal microbiota regulating, 147f microbiota and extraintestinal immunity, 150
156 relationship with systemic immunity, 509 responses in TB infection, 613
615, 614f to Salmonella Typhi, 508
509 signatures in blood predicting, 763 sites, 155
156 vaccine delivery systems for induction, 615
616 vaccines as stimulators of, 583
585 Mucosal immunity system, 101, 402
403, 424
425, 477
478, 790
791, 832 elements of, 3
5 Mucosal immunization, 167, 282, 307, 420, 630
631 Mucosal infections, systems biology of vaccines against, 762
768 Mucosal inflammation, 136
138, 519 novel strategies for targeting control, 869 future challenges, 876 homeostatic scar and immunological dysfunction, 872
873 host response features in homeostasis, 871 impact of immunization on homeostasis, 873
876 microbial communities, microbial pathogenesis, and dysbiosis, 871
872 Mucosal lymphoid organs, 22

evolution of organized lymphoid tissues, 23f evolutionary requirement for, 22
25 tissues, 22, 24, 801 Mucosal regulatory system for balanced immunity in gut human intestinal myeloid cells’ role, 251
252 immune homeostasis regulation, 250
251 intestinal immune tolerance induction by CD1031 dendritic cells, 249
250 for balanced ocular immunity immunology of ocular surface mucosa, 301
303 ocular mucosal tissues and glands in humans and mice, 300f organization of ocular surface mucosa, 300
301 targets and strategies for vaccine development, 303
307 Mucosal responses, 435 durability of, 767 Mucosal subcomponent vaccine development, considerations for, 128 Mucosal surfaces, 76
77, 477 Mucosal TB vaccine future study using HPIV2 vaccine, 620 mucosal immune responses in TB infection, 613
615, 614f rHPIV2 possibilities as next-generation vaccine candidate, 618
620 in TB protection, 616 vaccine protective effects in mice with TB, 617
618 targets for, 612f vaccine delivery systems for induction of mucosal immunity, 615
616 Mucosal tissue, 55
56, 232, 871 repair, ILCs in, 237 Mucosal tolerance, 12
13 therapy using plant-made proteins, 376
377 Mucosal vaccination, 167, 477 approaches for brucellosis, 450
451 Mucosal vaccines, 117, 325
326, 425
426, 445
446, 477, 604, 650
652, 753
755

INDEX

for caries prevention, 650
652 relevant cell surface proteins, 651f strategy for dental caries prevention, 652f efficacy of, 767
768 historical perspectives on bacterial and food antigens, 5t elements of mucosal immune system, 3
5 existing, 5
8, 6t oral tolerance, 12
13 routes, advantages, and disadvantages, 8t strategies for enhancing, 8
12, 11t against HSV-2, 730
731 intranasal vaccines, 730
731 intravaginal HSV-2 vaccines, 730 for immunosenescence, 799
800 inducing HIV-specific antibody responses, 713
716 analysis of mucosal tissues and Ab effector function, 715
716 antibody-related correlates in HIV vaccine clinical trials, 714
715 characterization of anti-HIV neutralizing antibodies, 713
714 passive anti-HIV antibody administration, 714 inducing HIV-specific T cell responses, 716
719 mucosal HIV infection in acute phase toward systemic infection, 716
717 mucosal T cell responses effective against HIV infection, 718
719 viral vectors for mucosal anti-HIV T cell responses, 717
718 induction of memory B cells, 119 for periodontal disease, 652 protein based, 652
653 strategy for dental caries prevention, 652f studies, 173
174 toxin-derivative adjuvants for ADP-ribosylation, 187
188 cholera toxin and labile toxin derivatives, 188
189 delivery systems for toxin-based adjuvant, 190
191 derivatives of other toxin adjuvant, 189
190 Mucosal vaccine delivery, 333
336

chitosan, 344
345 current nanotechnology and nanocarriers for vaccine delivery, 326t immunostimulating complexes, 337 liposomes, 337, 344 nano/microscale carriers as promising delivery tools, 326
333, 327f nanogels, 346 oral vaccine delivery, 333
336 using food materials, 340
344 polymer nanoparticles, 345 polymeric particle-based oral delivery, 338
340 starch nanoparticles, 345 virus-like particles, 338 Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), 204, 232, 765 ligand, 85
86 MAdCAM-1-expressing HEVs, 28 Mucosal veterinary vaccines, 811
813 DIVA vaccines, 812
813 economics and trade, 813 exploration of commercial and experimental, 813
825 mucosal vaccines for livestock, 813
820 mass delivery, 812 veterinary vaccines and One Health, 825
826 Mucosal-associate invariant T cells (MAIT cells), 325
326, 487, 504
505, 613, 614f changes in, 504
505 Mucosally administered vaccines, 6
7, 167
168 Mucus layer, 145
146 Multiepitope fusion antigen (MEFA), 568
569 Multiepitope protein antigens in ETEC vaccine, 568
569 Multimeric SIgAs, 680
681 Multiple sclerosis, 85
86, 93 Multipronged activation at infection sites, 216
217 activation by endogenous danger signals, 217 direct recognition of pathogens/ products, 216
217

901 indirect recognition of pathogens, 217 Multivalent ETEC vaccine (ETVAX), 571
573, 572f Multivalent oral Shigella vaccines, 525
526 E. coli
Shigella, 525
526 Shigella
ETEC, 526 Shigella
Typhoid, 525 Mumps, 738
739 murA gene, 390
391 Muramyl dipeptide (MDP), 156
157 Murine bladder cancer model, 408
409 Murine cytomegalovirus (MCMV), 239 Murine genital inoculation, 631
632 Murine model of NP carriage, 600, 604 Murine testicular macrophages, 266
267 MVA virus. See Modified vaccinia virus Ankara virus (MVA virus) Mycobacterial antigens, 411
413 Mycobacterium, 236, 777 Mycobacterium bovis, 403 Mycobacterium tuberculosis (Mtb), 5
6, 42, 158
159, 177, 344, 403
405, 435, 611
613, 777
778 clinical/preclinical BCG mucosal vaccine studies, 405t, 414t infection, 612f Mycoplasma. See also Chlamydia; Gonorrhea; Syphilis clinical manifestations, 638 current treatment options, 638 epidemiology, 638 immune responses associated with pathology, 638
639 microbiology, 638 vaccination research, 639 Mycoplasma genitalium, 638
639 vaccine candidates, 640t MyD88. See Myeloid differentiation factor 88 (MyD88) Myelin oligodendrocyte glycoprotein (MOG), 494 Myeloid cell, 191
192 γδ T cells to, 775 uptake of antigens and bacteria in lamina propria, 61
63 Myeloid differentiation factor 88 (MyD88), 146, 170 Myeloid-derived suppressor cells (MDSCs), 177, 615

902 N

N-acetylgalactosamine (GalNAc), 842
844 N-linked glycosylation, 375
376 N-trimethyl chitosan, 330
332 nasal vaccine formulation, 344
345 NA genes. See Neuraminidase genes (NA genes) NAbs. See Neutralizing antibodies (NAbs) Naı¨ve immune cell populations, 147
148 NALT. See Nasopharyngeal-associated lymphoid tissue (NALT) Nano-based delivery systems, 326
327 Nano/microscale carriers, 326
333, 327f Nanoemulsions. See Nanomaterial emulsions (nanoemulsions) Nanogels, 332
333, 346 nanogel-based nasal vaccine development against pneumonia, 467
471 vehicle, 604 Nanomaterial emulsions (nanoemulsions), 343
344, 465
466 as mucosal vaccine adjuvants, 171
173 Nanometer-sized hydrogel, 802 Nanoparticles (NPs), 326
327 Nasal administration of periodontal vaccine, 654
655 Nasal cavity, 325
326, 461
462 Nasal immune system, 463f lymphocyte imprinting and homing mechanisms, 464 mechanism for induction of antigenspecific nasal immune responses, 463
464 nasopharyngeal-associated lymphoid tissue structure and function, 462
463 Nasal immunization, 170, 464
465, 604, 653 Nasal influenza vaccines, 677 development, 678
681 IIIVs, 679
681 LAIVs, 678
679 humoral immune responses to influenza virus infection, 677
678

INDEX

Nasal passages (NPs), 790
791 Nasal vaccination, 325
326, 465
466 Nasal vaccines, 461
462. See also Oral vaccine(s) application of cCHP nasal vaccines against noninfectious diseases, 471 delivery, 341
344 advantages and limitations, 343 nanoemulsions, 343
344 drug-delivery systems for, 464
467 nanogel-based nasal vaccine development against pneumonia, 467
471 studies using nanoparticles for, 465t Nasal washes (NWs), 801
802 Nasalflu, 462 Nasalflu Berna, 342
343 Nasopharyngeal-associated lymphoid tissue (NALT), 21
22, 35
38, 36f, 153, 191, 300, 342, 409, 461
463, 487, 649
650, 790
791, 796
797, 835
836 tissue function, 797
798 Nasopharynx (NP), 598
599 immunomodulation and clinical impact, 316
317 innate and acquired immunity of, 314
315 toll-like receptors distribution in human epithelial cells, 314 innovative immunotherapy for attenuating nasal symptoms of cedar pollinosis, 317
319 Natalizumab, 85
86, 93
95 National Institute of Allergy and Infectious Diseases (NIAID), 631
632 National Institutes of Health (NIH), 745 National regulatory agency (NRA), 540 Native cholera toxin (nCT), 478 Native Corynebacterium diphtheria toxin mutant (CRM9), 526
528 Natural antibodies. See Polyreactive antibodies Natural cholera infection, 118
119 Natural immunity against H. pylori, 585
586 and vaccines, 269 Natural infection, 518

Natural killer cells (NK cells), 219, 229, 257, 407
408, 613, 726, 760
761, 773 C-type lectin-like receptors, 774 CD3
CD56bright CD16
cells, 257 NKp46, 25
26 Natural killer group 2D (NKG2D), 232 Natural killer T cells (NKT cells), 602, 773 Natural polymers, 330
332 Natural resistance-associated macrophage protein 1 (NRAMP-1), 775 Naturally acquired immunity against Shigella, 518
519 nCT. See Native cholera toxin (nCT) Nebulizers, 478 Necrosis, 508 Nef-deleted SIV vaccines, 715
716 Neglected tropical diseases (NTDs), 841
842 Neisseria gonorrhoeae, 262, 632
633 vaccine candidates, 640t Neisseria meningitidis, 9
10, 74
75, 345 Neisseria meningitidis serogroup C polysaccharide (MCP), 345 Nematodes, 841
842 Nematodiasis. See Trichinellosis Neonatal Fc receptor (FcRn), 60, 72
73, 258
259, 270, 302
303, 727 Neonatal pathogen, 735
736, 736t Neuraminidase genes (NA genes), 364, 678
679 Neurocysticercosis, 847
848 Neuroendocrine, 299 Neuromedin U (NMU), 233
234 Neutralization assay, 765 of biologically active antigens, 78
80 Neutralizing antibodies (NAbs), 421, 713, 727 Neutrophil(s), 154, 193, 217
219, 268, 780 debris, 89
90 depletion, 193 Neutrophilia, 403
404 Next-generation sequencing platforms, 235 NF-κB. See Nuclear factor kappa B (NF-κB) NFIL3. See Nuclear factor interleukin 3 (NFIL3)

INDEX

Nfkbiz, 156 NGU. See Non-gonococcal urethritis (NGU) NHP models. See Nonhuman primate models (NHP models) Niacin, 204
206 deficiency, 206 NIAID. See National Institute of Allergy and Infectious Diseases (NIAID); US National Institute of Allergy and Infectious Diseases (NIAID) Nicotiana benthamiana, 364 NIH. See National Institutes of Health (NIH) Nippostrongylus brasiliensis, 236 Nitric oxide (NO), 191
192 NK cells. See Natural killer cells (NK cells) NKG2D. See Natural killer group 2D (NKG2D) NKT cells. See Natural killer T cells (NKT cells) NLRPs, 601 NLRP3 component, 110, 601 NLRP9b inflammasome, 685, 686f NLRs. See NOD-like receptors (NLRs) NMRC. See US Naval Medical Research Center (NMRC) NMU. See Neuromedin U (NMU) nnAbs. See Non-neutralizing antibodies (nnAbs) NO. See Nitric oxide (NO) NOD-like receptors (NLRs), 106, 154, 420
421, 601 between NLRs and intestinal inflammation, 110 NOD1. See Nucleotide-binding oligomerization domaincontaining 1 (NOD1) Non-gonococcal urethritis (NGU), 638 Non-neutralizing antibodies (nnAbs), 727 Nonconventional T cells, 602 Nongerminable powder, 379 Nonhuman primate models (NHP models), 269, 739
740 Noninfectious diseases, 340
341 application of cCHP nasal vaccines against, 471 Noninvasive pathogens, 762 Nonlymphoid tissues, gene signatures systems analysis in, 761

Nonneutralizing antibodies, 421 Nontoxic mutants of CT (Nontoxic mCTs), 650 Nontypable Haemophilus influenzae (NTHi), 314 Nontyphoidal Salmonella, vaccines against, 395 Noroviruses (NoVs), 363, 699, 707 capsid, 704 vaccine development, 699 alternative Novs. vaccines in pipeline, 706
707 challenges in, 705 classification, 704
705 disease, 704 structure, 704 virus-like particle-based Novs. vaccines, 705
706 VLP vaccines, 707 Northern blot analysis, 314
315 Norwalk virus, 57 NoVs. See Noroviruses (NoVs) NP. 4-Hydroxy-3-nitrophenyl)acetyl (NP);. See Nasopharynx (NP) NPs. See Nanoparticles (NPs); Nasal passages (NPs) NRA. See National regulatory agency (NRA) NRAMP-1. See Natural resistanceassociated macrophage protein 1 (NRAMP-1) NTDs. See Neglected tropical diseases (NTDs) NTHi. See Nontypable Haemophilus influenzae (NTHi) Nuclear factor interleukin 3 (NFIL3), 229
231 Nuclear factor kappa B (NF-κB), 314, 683
684, 687
688 Nucleotide-binding oligomerization domain-containing 1 (NOD1), 110 Nucleotide-binding oligomerization domain-containing 2 (NOD2), 58
59, 105, 110 NWs. See Nasal washes (NWs)

O 2457O, 521 O-antigens, 388, 521, 525, 565 O-polysaccharide, 518 O-specific polysaccharide-protein conjugates, 515

903 OAS. See 20 -50 -Oligoadenylate synthetase (OAS) Occludins, 58
59, 105, 146 Ocular immunopathology, 305 Ocular pathogens, 299 Ocular surface epithelium, 302 mucosa immunology, 301
303 organization, 300
301 Ocular vaccine development, strategies for, 305
307 OCVs. See Oral cholera vaccines (OCVs) ODNs. See Oligodeoxynucleotides (ODNs) Oil-in-water emulsion adjuvants, 327
328 OIT. See Oral immunotherapy (OIT) Ole e 1, 861 20 -50 -Oligoadenylate synthetase (OAS), 692 Oligodeoxynucleotides (ODNs), 730, 799
800, 845
846 Oligomeric procyanidins (OPCs), 778
779 Omalizumab, 859 Omalizumab
IgE complex, 860 OME. See Otitis media with effusion (OME) OMP. See Outer-membrane protein (OMP) OmpH, 492 OmpH β1α1. See Outer membrane protein H (OmpH β1α1) Onchocerca life cycle, 304
305 O. volvulus, 303
304 Onchocerciasis, 304
305 Onchorhynchus mykiss (Rainbow trout), 822
823 One Health, 340
341, 825
826 ONRAB, 821
822 OP. See Oropharynx (OP) OPCs. See Oligomeric procyanidins (OPCs) Open reading frames (ORF), 704 Opsonophagocytic killing activity (OPKA), 518 Optimal administration route evaluation of ETEC vaccine, 565
569

904 Optimal administration route evaluation of ETEC vaccine (Continued) candidate vaccines in preclinical phase, 566
568, 573 multiepitope protein antigens, 568
569 passive protection trials in humans, 566 OPV. See Oral polio vaccine (OPV) Oral BCG immunization, 404
405 Oral cholera vaccines (OCVs), 334
335, 538, 540
544 composition in WHO-prequalified inactivated, 541t modifiers of immune responses, 551
553 perspectives and future challenges, 555
556 public health use, 553
554 systemic antibody and memory B cell responses, 548
551 WHO recommendations, 554
555 Oral disease DNA-based vaccine, 653
654 mucosal vaccines for caries prevention, 650
652 for periodontal disease, 652 protein based, 652
653 nasal administration of periodontal vaccine, 654
655 sublingual vaccine for periodontal diseases, 655
656 Oral exposure, 449 Oral immunity, 150
153 Oral immunization, 119, 402
407, 426, 429
430 attenuated Salmonella for approaches for attenuation, 384
395 vaccines against nontyphoidal Salmonella, 395 HIV, 405
407 mycobacterium tuberculosis, 403
405 Peyer’s patches, 402f Oral immunotherapy (OIT), 857, 859 Oral Inactivated Whole Cell ETEC vaccines, 571
573 Oral mucosae, 478 Oral mucosal adjuvants, 569 Oral polio vaccine (OPV), 6
7, 333
334, 753
755

INDEX

Oral RB51 vaccination, 450
451 Oral route of administration, 7
8 Oral Sabin polio vaccine, 445
446 Oral Shigella vaccines candidates, 525t history, 520
521, 522t ideal, 528 immunity induced by, 521
524 infection and vaccination, 515
516, 516f multivalent oral Shigella vaccines, 525
526 parenteral Shigella vaccine candidates, 526
528, 527t Oral tolerance, 12
13, 493, 811
812, 871 M cell contribution for, 493
494 Oral vaccination, 333
334, 402, 478 Oral vaccine(s), 335t, 461
462, 823. See also Nasal vaccines adenovirus vectors as, 426
430, 427t advantages and limitations, 334
335 comparison of intranasal vaccination route with oral and parenteral routes, 336t delivery, 333
336 emulsions and micelles, 335
336 using food materials, 340
344 nanocarriers, 338 Oravacs, 541
542 OrcVax, 540 Oreign DNA, 359
360 ORF. See Open reading frames (ORF) Orochol, 542 Oropharynx (OP), 598
599 Orthopneumovirus genus, 665 Otitis media with effusion (OME), 316 Outer membrane protein H (OmpH β1α1), 31
32 Outer-membrane protein (OMP), 652 Ovalbumin (OVA), 315, 478, 490, 791
792, 820, 860
861 OVA
protein σ1 fusion, 494 Ovidae, 818 Ovine epididymitis, 818 Oxysterols, 26
27

P P-selectin glycan ligand-1 (PSGL-1), 86
87 P407. See Poloxamer 407 (P407) PA. See Protective antigen (PA) PA20. See 20-kD Peptide (PA20)

PA83. See 83-kD PA (PA83) PAg. See Protein antigens (PAg) Paired immunoglobulin-like type 2 receptor alpha (PILRα), 724
725 Paired palatine tonsils, 462
463 Palivizumab, 739 Palm oil, 207 Palmitic acid, 207, 207f PAMPs. See Pathogen-associated molecular patterns (PAMPs) PanAd3, 435 Paneth cells, 146 paneth-cell-related dysbiosis, 103
104 Pantothenic acid, 204
205 Papain, 155 Paracellular TEM, 87 Paracellular transport across villus epithelial cells, 58
61 Paramyxoviridae family, 665 Parasites, 841
842 Parasitic helminths, 841
842 Parasitic infections helminth infections, 846
849 mucosal vaccine for, 841
842 protozoan infections, 842
846 Parental RRV-1 strain, 700
701 Parenteral immunization, 167, 375
376, 731 Shigella vaccine candidates, 526
528, 527t vaccines, 445
446, 762 Passive immunization, 873
875 Past prophylactic vaccine trials, 729
730 Pasteurella multocida, 5
6, 447
448 Pasteurella multocida strain B:3, 4, 819 Pathogen-associated molecular patterns (PAMPs), 106, 175
176, 216, 233, 302, 313, 325
326, 383
384, 420
421, 627, 650
651, 683
684 Pathogenic Th2 cells (Tpath2 cells), 134, 135f, 138
139 Pathogenic/pathogen(s), 600, 762 bacteria of mucosa, 581 direct recognition of, 216
217 enteric viruses, 699 indirect recognition, 217 pathogen-associated molecular patterns, 9
10 pathogen-induced lesions, 303

INDEX

S. enterica, 383
384 Th population disease induction model, 138
139, 138f Pattern recognition receptors (PRRs), 101
103, 106, 154, 420
421, 602, 683
684, 726 PB. See Protein bodies (PB) PBMCs. See Peripheral blood mononuclear cells (PBMCs) PbVs. See Plant-based vaccines (PbVs) PCEP. See Poly[di(sodium carboxylatoethylphenoxy) phosphazene] (PCEP) PCV. See Polysaccharide conjugate vaccine (PCV) pDCs. See Plasmacytoid DCs (pDCs) PDE. See Phosphodiesterase (PDE) Peanut (PN), 176 extract, 860
861 PN-coated nanoparticles, 861 Pediatric populations, 173
174 PEG. See Polyethylene glycol (PEG) PEI. See Polyethylenimine (PEI) Pellagra, 206 Pelvic inflammatory disease (PID), 267
268, 626 Peptic ulcer disease, 579
580 20-kD Peptide (PA20), 186 Peptide(s), 328, 344 delivery systems, 332 molecules, 332 nanofiber vaccines, 332 Peptidoglycan, 605 Peptidoglycan recognition protein-1 (PGLRP-1), 31 Perforin expression, 775
776 Periodontal disease, 649, 652 Peripheral blood mononuclear cells (PBMCs), 404
405, 603, 760
761, 775
776 Peripheral lymph nodes, germinal center reaction in, 122 Peripheral node addressin (PNAd), 36, 86
87 Peripheral SMAC (pSMAC), 90
91 Periphery intraepithelial lymphocytes (pIELs), 425 Periphery regulatory Th cells (pTreg), 871 Persistent inflammation, ILCs in, 237
238 Pertussis, 736
737

Peyer’s patches (PPs), 10
11, 21
22, 28
32, 29f, 119, 338, 383
384, 424
425, 487, 508, 790
791 germinal center reaction in, 122 organogenesis of, 33 pFL. See Plasmid DNA encoding FL (pFL) PfSPZ vaccination, 766
767 PGD2. See Prostaglandin D2 (PGD2) PGLRP-1. See Peptidoglycan recognition protein-1 (PGLRP-1) Pharmaceutical and Medical Devices Agency (PMDA), 366
367 PhoP, 385, 389 PhoPQ, 385 Phosphodiesterase (PDE), 692 Phosphorylcholine (ChoP), 604
605 Physical barriers, 103 PIs. See Protease inhibitors (PIs) PID. See Pelvic inflammatory disease (PID) pIELs. See Periphery intraepithelial lymphocytes (pIELs) PIgR. See Polymeric immunoglobulin receptor (PIgR) PILRα. See Paired immunoglobulinlike type 2 receptor alpha (PILRα) PL. See Poly-L-lysine (PL) PLA. See Polylactide (PLA) Plague, 821 antibody-dependence for immune protection, 452
453 development of plague mucosal vaccine, 454 disease and historical perspective, 451
452 Th1-and Th17-mediated immunity against, 453
454 Plant cell culture systems, 379
380 Plant pollens, 8
9 Plant polyphenols for γδ T cells activation, 778
782 microbial products for γδ T cells regulation, 780
782 plant polysaccharides as γδ T celltargeted immunomodulator, 779
780 Plant polysaccharides as γδ T celltargeted immunomodulator, 779
780 Plant-based mucosal immunotherapeutics, 371

905 manufacturing, 374 Plant-based mucosal immunotherapy, 371 advances in product development, 372 challenges for commercialization, 379
380 cost of production for, 373
374 extended periods of time using seed-based platforms, 378
379 global contamination from foodcrop-made vaccines ortolerogens, 377
378 infrastructure and protocols for, 374 lack of recent human clinical trials testing safety and/or efficacy, 372 mucosal tolerance therapy using plant-made proteins, 376
377 regulatory approval for, 375 safety of consumable formulations, 378 of plant-made biologics, 375
376 of plant-specific glycosylation, 375
376 separating expression from finishing product geographically, 378
379 Plant-based mucosal vaccine delivery systems, 357
359 transgenic technologies for vaccine production in plants, 359
361 Plant-based norovirus VP1 vaccine, 363 Plant-based vaccines (PbVs), 357 advantages, 358t control of infectious diseases, 361
367 disadvantages, 358t expression system, 359f against human infectious diseases in clinical trials, 362t oral vaccines, 340
341 prevention of infectious diseases, 361
367 Plant-derived immunotherapies, 373 Plant-grown antigen material, 12 Plant-made animal vaccines, 375 Plant-made biologics, safety of, 375
376 Plant-made protein industry, 377
378 Plaque formation, 423

906 Plasmacytoid DCs (pDCs), 176, 800
801 Plasmid DNA encoding FL (pFL), 801 Plasmids, 386 Plasmodium spp., 841
842 P. berghei cerebral malaria model, 832
833 P. falciparum, 760
761, 763, 777, 831
835 P. vivax, 832 P. yoelii MSP119, 835 sporozoites, 834
835 PLG. See Poly(D,L-lactide-co-glycolide) (PLG) PLGA. See Poly(D,L-lactide-coglycolide) (PLG) PLP. See Proteolipid protein (PLP) PMDA. See Pharmaceutical and Medical Devices Agency (PMDA) PML. See Progressive multifocal leukoencephalopathy (PML) PMMMA-PLGA. See Poly[(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid)]
poly(D,L-lactide-coglycolide) (PMMMA-PLGA) PMPs. See Polymorphic membrane proteins (PMPs) PN. See Peanut (PN) PNAd. See Peripheral node addressin (PNAd) Pneumococcal capsule, 597
598 carriage, 602 colonization, 598
599 infections, 605 strains, 600 vaccines, 606 Pneumococcal polysaccharide vaccine (PPSV), 467
469 Pneumococcal surface adhesion A (PsaA), 604 Pneumococcal surface protein A (PspA), 469
470, 602, 801
802 Pneumococci/pneumococcus, 597
600, 599f host immune responses to, 600
603 acquired immunity role against pneumococci, 602
603 innate immunity role against pneumococci, 600
602 subcellular components of, 601
602

INDEX

Pneumocystis murina, 41 Pneumolysin, 601, 605 Pneumonia, nanogel-based nasal vaccine development against, 467
471 Pneumonic plague, 451
452 Pocket, 29
30 Poliovirus, 730 Pollinex Quattro, 860
861 Poloxamer 407 (P407), 818 Poly-L-lysine (PL), 791
792 Poly(D,L-lactide-co-glycolide) (PLG), 328
330, 466 microparticles, 329 NPs, 345 polymers, 329 Poly(lactate-co-glycolate), 10
11 Poly[(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid)]
poly(D,L-lactide-coglycolide) (PMMMA-PLGA), 339 Poly[di(sodium carboxylatoethylphenoxy) phosphazene] (PCEP), 821 Polyclonal antibodies, 73
74 Polyethylene glycol (PEG), 339, 466 PEG
PLA particles, 467 Polyethylenimine (PEI), 175 Polylactic acid. See Polylactide (PLA) Polylactide (PLA), 328
329, 466 Polymer nanoparticles, 345 Polymeric immunoglobulin receptor (PIgR), 60, 72
73, 258
259, 492, 727 Polymeric NPs, 328
329 Polymeric particle-based oral delivery, 338
340 Polymorphic membrane proteins (PMPs), 271, 630, 821 Polymyxin B, 174
175 Polymyxins, 174
175 Polyreactive antibodies, 77 Polyreactivity, 77 Polysaccharide A (PSA), 875 Polysaccharide conjugate vaccine (PCV), 467
469 Polysaccharide(s), 332
333 polysaccharide-based vaccines, 603
604 Polysorbate-80 (PS80), 327
328 Porcine epidemic diarrhea virus, 813
818

Porcine transmissible gastroenteriditis virus, 813
818 Porcine zona pellucida vaccines, 824 Pork tapeworm. See Taenia solium (Pork tapeworm) Porphyromonas gingivalis, 652
653 P. gingivalis-accelerated atherosclerosis, 656 Post-Licensure Rapid Immunization Safety Monitoring program (PRISM program), 702 Postpartum maternal immunization, 737 PPs. See Peyer’s patches (PPs) PPSV. See Pneumococcal polysaccharide vaccine (PPSV) Prairie dogs, 821 PRDI-BF1. See B-lymphocyte-induced maturation protein 1 (BLIMP-1) Preclinical vaccine candidates of ETEC, 566
568, 573 Pregnancy antiviral drugs, 742 HIV during, 740 HSV infection during, 742
743 infection with rubella during, 738 influenza infection during, 737 maternal vaccines administered during, 736t seroconversion during, 742
743 Premembrane and Env DNA vaccine (prM-Env DNA vaccine), 743 Prenyl phosphates, 777
778 Prevotella, 150
154 P. bivia, 155 Primed integrins, 86
87 PRISM program. See Post-Licensure Rapid Immunization Safety Monitoring program (PRISM program) prM-Env DNA vaccine. See Premembrane and Env DNA vaccine (prM-Env DNA vaccine) Probiotics, 191, 208, 863 Progesterone, 262, 278
282 receptors, 263
264 Progressive multifocal leukoencephalopathy (PML), 94 Proinflammatory cytokines, 248, 250
251, 266
267, 407
408 Projection models, 699
700 Prophylactic vaccines, 723
724

INDEX

Propionibacterium acnes, 156, 301 Prostaglandin D2 (PGD2), 233 Protease inhibitors (PIs), 583 PI-1, 845
846 Protective antigen (PA), 186, 447
448, 800
801 Protective immunity against Chlamydia, 269
270 Protective memory responses against genital herpes infection, 726
729 antibody-mediated protection, 727 Protein antigens (PAg), 332
333, 602, 605, 651 Protein bodies (PB), 317
319 Proteins, 328, 344 Proteobacteria, 144 Proteolipid protein (PLP), 494 Proteolytic degradation of toxins, 219 Protozoan infections, 842
846. See also Helminth infections amoebiasis, 842
844 cryptosporidiosis, 845 gastrointestinal tract, 843f giardiasis, 844
845 toxoplasmosis, 845
846 Protozoans, 841
842 Protruding domain (P domain), 704 PrPc. See Cellular prion protein (PrPc) PRRs. See Pattern recognition receptors (PRRs) PS80. See Polysorbate-80 (PS80) PSA. See Polysaccharide A (PSA) PsaA. See Pneumococcal surface adhesion A (PsaA) Pseudomonadaceae, 153 Pseudomonas, 155 P. aeruginosa, 155
156, 186, 344 Pseudomondales, 158
159 PSGL-1. See P-selectin glycan ligand-1 (PSGL-1) pSMAC. See Peripheral SMAC (pSMAC) PspA. See Pneumococcal surface protein A (PspA) PstS (Phosphate-binding protein), 650
651 pTreg. See Periphery regulatory Th cells (pTreg) Public health use in OCVs, 553
554 Pullulan, 332
333 Pulmonary immune responses borrelia, 413
414

coccidiosis, 413
414 schistosoma, 413
414 stimulation of, 409
414 tuberculosis, 409
413 Pulmonary pneumonia model, 520 PURETHAL mites, 856
857 Purified antigens, 567
568 Putative protective ETEC antigen colonization factors, 565 heat-labile enterotoxin, 565 O antigens, 565 ST heat-stable enterotoxin, 565 structure, 565 Pyridoxine, 204
205 Pyroptosis, 517
518, 685

Q QGE031, 860 Quil A saponins, 10
11, 337

R

R&D program. See Research and development program (R&D program) R1 Rotarix vaccine, 693
694 R1 vaccine, 694 R5 vaccines, 694 R702W, 110 RA. See Retinoic acid (RA) Rabbit hemorrhagic disease (RHD), 820 Rabbit hemorrhagic disease virus (RHDV), 820 Rabies, 363
364, 821
822 treatment of, 363
364 Raboral V-RG, 821
822 Raccoonpox (RCN), 821 RAG. See Recombinant activating genes (RAG) RAG-deficient mice. See Recombination-activating gene-deficient mice (RAGdeficient mice) rAg85B effects, 619f Rainbow trout. See Onchorhynchus mykiss (Rainbow trout) RANK. See Receptor activator of nuclear factor kappa-B (RANK) RANKL. See Receptor activator of NFKB ligand (RANKL) RANKL/TNFSF11. See Receptor activator of nuclear factor kappa-B ligand/TNFSF11 (RANKL/TNFSF11)

907 RAs. See Retinoid acids (RAs) Rational vaccination strategies, 735
736 RB. See Reticulate body (RB) RB51 vaccine, 450 rBCG. See Recombinant Bacillus Calmette-Gue´rin (rBCG) rBS. See Recombinant B subunit (rBS) RCA vectors. See Replicationcompetent adenovirus vectors (RCA vectors) RCN. See Raccoonpox (RCN) rCTB. See Recombinantly produced CTB (rCTB) rCTB-CF ETEC vaccine, 571 Receptor activator of NF-KB ligand (RANKL), 34, 488
489, 494
495, 793 Receptor activator of nuclear factor kappa-B (RANK), 34, 488
489 Receptor activator of nuclear factor kappa-B ligand/TNFSF11 (RANKL/TNFSF11), 61 Recombinant activating genes (RAG), 150 RAG-1 and RAG-2, 150 Recombinant attenuated bacteria, 566
567 Recombinant B subunit (rBS), 334
335 Recombinant Bacillus Calmette-Gue´rin (rBCG) intravesical immunotherapy, 407
409 oral immunization, 402
407 stimulation of pulmonary immune responses, 409
414 strain, 406 vaccines, 406 Recombinant Bacillus subtilis, 848
849 Recombinant gpD, 279
280 Recombinant MOMP, 271 Recombinant protective antigen (rPA), 172 Recombinant Salmonella strains, 383 Recombinantly produced CTB (rCTB), 571 Recombination-activating genedeficient mice (RAG-deficient mice), 236 Recombination-deficient adenovirus 40, 862 Rectal mucosa, immunizations through, 436

908 Reduction-modifiable protein (Rmp), 634 Regenerating islet-derived proteins 3 (Reg3), 57 Regulated delayed antigen synthesis, 389
390 Regulated delayed attenuation, 388
389 Regulated delayed vaccine lysis, 390
392 Regulatory approval, 372, 375, 378 Regulatory B cells (Breg cells), 247
248, 871 Regulatory T cells (Treg cells), 148
149, 203, 258, 317, 603, 611
612, 790
791, 871 differentiation, 26 Reoviridae, 700 Reovirus protein σ1, 37
38 OVA
protein σ1 fusion, 493
494 Replication-competent adenovirus vectors (RCA vectors), 422
423 Replication-defective adenovirus vectors, 419 Research and development program (R&D program), 555
556 Resident DCs, 137 Resident memory (RM), 410
411 Resident memory T cells (TRM), 306 Respiratory M cells, 462
463 Respiratory syncytial virus (RSV), 154
155, 435, 466
467, 665
667, 735
736, 739
740 immunity and vaccination in infants and young children, 670
672 in older adults, 672 immunopathology, 669 Respiratory syncytial virus, 117, 219 Respiratory tract(s), 21 immunity, 153
155 infections, 122
123 lymphoid tissues of, 35
42 mucous membrane, 677
678 Restricted biota, 149 RET ligands, 233 Reticulate body (RB), 267, 626 Retinoic acid (RA), 91
92, 191
192, 204, 234, 765 Retinoic-acid-inducible gene I (RIG-I), 109
110 Retinoic-acid-inducible gene-I-like receptors (RLRs), 106

INDEX

Retinoid acids (RAs), 424
425 Retinol, 204, 552 Rev.1 vaccine, 450 Reverse transcription
polymerase chain reaction (RT-PCR), 314
315 RGD ligand. See Arginine-glycineaspartate ligand (RGD ligand) Rhamnosus vaccine, 406
407 RhCMV. See Rhesus cytomegalovirus (RhCMV) RHD. See Rabbit hemorrhagic disease (RHD) RHDV. See Rabbit hemorrhagic disease virus (RHDV) Rhesus cytomegalovirus (RhCMV), 718, 766 Rhesus macaques, 410 Rhoptry proteins, 845
846 rHPIV2 possibilities as next-generation vaccine candidate, 618
620 in TB protection, 616 vaccine protective effects in mice with TB, 617
618 rHPIV2-Ag85B. See Ag85B-expressed human parainfluenza type 2 virus (rHPIV2-Ag85B) Riboflavin, 204
205 metabolites, 602 Ribonuclease L (RNaseL), 692 Rice-based oral CTB vaccine, 358
359 Ricin, 186
187 Ricinus communis, 186
187 RIG-I. See Retinoic-acid-inducible gene I (RIG-I) RIG-I-like receptors (RLRs), 683
684 RING finger protein 186, 105 RIT 4237, 700 Rival immunological mechanisms, 299
300 RLRs. See Retinoic-acid-inducible gene-I-like receptors (RLRs); RIG-I-like receptors (RLRs) RM. See Resident memory (RM) Rmp. See Reduction-modifiable protein (Rmp) RNA, 328 metabolomics RNA sequencing, 753
755 RNaseL. See Ribonuclease L (RNaseL) RORγt, 25
26, 33
34, 40
41 RORγt1 cells, 148
149 Rotarix, 701
702, 767

RotaShield, 7 Rotasil, 694 RotaTeq, 7, 694, 767 Rotatrix, 7 Rotavac, 694, 702
703 Rotavin-M1, 703 Rotavirus disease, 699
700 Rotaviruses (RVs), 683, 699, 730 classification, 700 host innate immune sensors and rotavirus infection, 683
686 host innate responses to rotavirus and effects on viral replication, 686
687 host range restriction to reliably attenuate live rotavirus vaccine candidates, 692
694 infection, 236 regulation of effector antiviral factors, 692 of interferon induction pathway by, 687
692 vaccine development, 702
703 initial vaccine efforts using live animal RVs, 700 obstacles to, 703
704 RotaShield, 700
701 Rotateq and Rotarix, 701
702 timeline, 700f Rothia, 154 Rothia nasimurium, 156 rPA. See Recombinant protective antigen (rPA) RRV-1 vaccination, 700 viruses, 700
701 RRVTV. See RotaShield RSV. See Respiratory syncytial virus (RSV) RT-PCR. See Reverse transcription
polymerase chain reaction (RT-PCR) RTS, S vaccine, 831 Rubella, 738
739 Rubeola. See Measles Ruminococcus obeum, 871
872 RVs. See Rotaviruses (RVs)

S

S. Typhi. See Salmonella enterica serovar Typhi (S. Typhi) S1PT. See Strain expressing genetically detoxified pertussis toxin (S1PT)

INDEX

SA. See Streptavidin (SA) Saccharomyces cerevisiae (Baker’s yeast), 340 SAdV. See Simian adenoviruses (SAdV) SAFE strategy. See Surgery to treat eyelid inversion, antibiotics, facial cleanliness, and environmental improvement to reduce transmission strategy (SAFE strategy) SAG1 protein, 845
846 Saliva-binding region (SBR), 650, 651f Salmo salar (Atlantic salmon), 822 Salmonella enterica serovar Typhi (S. Typhi), 501 changes in innate and mucosalassociate invariant T cells, 504
505 gut-homing memory T cells, 505
506 human challenge model of typhoid fever, 502
503 humoral and systemic B cell immunity, 503
504 live attenuated oral vaccine, 502
503 mucosal and systemic human immunity, 507f mucosal immunity to, 508
509 relationship between systemic and mucosal immunity, 509 systemic T cell immunity, 503 vaccines and models to study immunogenicity, 501
502 Salmonella pathogenicity island 1 genes (SPI-1 genes), 385
386 Salmonella pathogenicity island 2 (SPI2), 384
386 Salmonella spp., 11, 487, 494
495, 650, 762, 777, 793, 833 mutations in Salmonella pathogenicity islands, 385
386 S. enterica, 251, 383, 777, 793 S. enterica Enteritidis Δfur strain, 385 S. enterica Gallinarum 9R, 384 S. enterica serovar Typhimurium, 61 Typhi bacteria, 6
7 Typhimurium, 383
384 S. lysis strain, 391
392 S. typhi, 4
6, 333
334, 762

S. Typhi-responsive cells, 508
509 Ty21a strain, 384, 566
567 S. typhimurium, 31
32, 73
74, 76
77, 109, 222
223 infection, 59
60, 62
63 lysis strains, 391 Salmonella-containing DCs, 383
384 Salmonella-expressing TSOL18 vaccine, 847
848 Sarafotoxin, 219 SBA activity. See Serum antibodies with bactericidal activity (SBA activity) SBR. See Saliva-binding region (SBR) SC. See Secretory component (SC) SC delivery. See Subcutaneous delivery (SC delivery) SCF. See Stem cell factor (SCF) SCFAs. See Short-chain fatty acids (SCFAs) Schistosoma, 413
414, 847 S. haematobium, 847 S. intercalatum, 847 S. japonicum, 847 S. mansoni, 156
157, 847 S. mekongi, 847 Schistosomiasis, 847 Second-generation EcSf2a-2 vaccine strain, 525
526 Secondary lymphoid organs (SLOs), 21
22 Secreted and surface associated lipoprotein (SslE), 568 Secretory component (SC), 72
73 Secretory immunoglobulin A (SIgA), 3
5, 12
13, 72
73, 149
150, 187
188, 260, 302, 333
334, 361, 403
404, 425, 463
464, 542
543, 567
568, 584, 602, 649, 790
791, 813
818, 842 antibodies, 191, 477
478, 677
679, 791 receptors, 492 Seed-based platforms, 379 Segmented filamentous bacteria (SFB), 148
149 Segmented filamentous bacterium, 9
10 Selective estrogen receptor modulators (SERMs), 263
264 Selective progesterone receptor modulators (SPRMs), 263
264

909 SELEX. See Systematic evolution of ligands by exponential enrichment (SELEX) Self-assembled peptides, 332 Sendai virus, 618
619 Sendai virus vectors (SeV), 718 Sensors, 685
686 SENTINEL study, 93
94 Serial passage, 384
385 SERMs. See Selective estrogen receptor modulators (SERMs) Seroconversion, 177 Serotype(s), 598
599 G6 RV, 700 serotype 2 strain, 605 serotype-independent vaccines, 603 Serratia, 155 Serum anti-capsule antibodies, 602 Serum antibodies with bactericidal activity (SBA activity), 518 Serum antibody, 597 IgA responses, 549 IgG responses, 550 IgM, 550 responses to defined antigens, 549
550 Serum IgA, 605 Serum IgG, 605 SeV. See Sendai virus vectors (SeV) Sexual transmission of HIV, 716
717 Sexually transmitted infections (STIs), 255, 263
264, 625 bacterial, 626 Chlamydia, 626
632 gonorrhea, 632
635 Mycoplasma, 638
639 syphilis, 635
638 Sexually transmitted viral infections, 723 SFB. See Segmented filamentous bacteria (SFB) Shanchol, 540
541, 541t, 548 Shell domain (S domain), 704 Shiga toxin-producing Escherichia coli O157:H7 (STEC O157), 826 Shiga toxins, 186 Shigatoxigenic serotypes of E. coli (STEC), 186 Shigella, 383, 487, 515, 762 immune responses in children, 519
520 infection, 516
517

910 Shigella (Continued) naturally acquired immunity against, 518
519 outbreaks, 517 pathogenesis and virulence factors, 517
518 S. dysenteriae, 4
6, 186 type 1, 517, 566
567 S. flexneri, 73
74, 77
78, 517 Shigella flexneri 2a, 566
567 S. sonnei, 517, 566
567 Shigella-induced diarrhea, 515 Shigella
ETEC, 526 Shigella
Typhoid, 525 Shingles. See Herpes zoster (HZ) SHMs. See Somatic hypermutations (SHMs) Short-chain fatty acids (SCFAs), 143
144, 155, 208
209 SIgA. See Secretory immunoglobulin A (SIgA) Signaling lymphocyte activation molecule family member 4 (SLAMF4), 147
148 Signatures in blood predict mucosal immunity, 763 SIGNR3. See Specific intracellular adhesion molecule-3-grabbing nonintegrin homolog-related 3 (SIGNR3) Simian adenoviruses (SAdV), 422 Simian immunodeficiency virus (SIV), 405
406, 714, 763 SIV-specific T cell responses, 717
718 Single-dose anti-C. pecorum vaccine, 821 Single-nucleotide polymorphisms (SNPs), 107 Single-stranded RNA viruses (ss RNA viruses), 108, 753
755 SIRPa, 61
62 SIV. See Simian immunodeficiency virus (SIV) Skin immunity, 156
157. See also Mucosal immunity Skin-resident DCs, 214
215 SLAMF4. See Signaling lymphocyte activation molecule family member 4 (SLAMF4) SLC11A1. See Solute carrier 11A1 (SLC11A1) SLI. See Sublingual immunization (SLI)

INDEX

Slip bonds, 88 SLIT. See Sublingual immunotherapy (SLIT) SLOs. See Secondary lymphoid organs (SLOs) Small hydrophobic ion channel (SH) protein, 665 Smallpox, 446 SMGs. See Submandibular glands (SMGs) SNPs. See Single-nucleotide polymorphisms (SNPs) Soluble antimicrobial immune molecules, 261 Soluble mediators in FGT, 261 Soluble proteins, 383
384 Solute carrier 11A1 (SLC11A1), 775 Somatic hypermutations (SHMs), 118
119 SopB, 494
495 Sorbitan trioleate, 327
328 Specific intracellular adhesion molecule-3-grabbing nonintegrin homolog-related 3 (SIGNR3), 109 Specific pathogen-free mice (SPF mice), 145
146 SPF mice. See Specific pathogen-free mice (SPF mice) SPI-1 genes. See Salmonella pathogenicity island 1 genes (SPI-1 genes) SPI-2. See Salmonella pathogenicity island 2 (SPI-2) Spi-B, 34, 489, 793 Spinach. See Spinacia oleracea (Spinach) Spinacia oleracea (Spinach), 364 Spirochetes, 413 Spleen cells, 779 “Splenic fever”. See Anthrax Split tolerance, 12
13 Sporadic M-like cells, 488 Spray cabinet routes, 819
820 SPRMs. See Selective progesterone receptor modulators (SPRMs) Squalene, 327
328 ss RNA viruses. See Single-stranded RNA viruses (ss RNA viruses) SslE. See Secreted and surface associated lipoprotein (SslE) ST. See Heat-stable toxin (ST) ST heat-stable enterotoxin, 565 ST2-expressing memory Th2 cells, 134

Stable nuclear transformants, 371 Staphylococcaceae, 153 Staphylococcus, 144 S. aureus, 153
154, 156 LTA, 156
157 protein A, 193 S. aureus-derived peptidoglycan activating MCs, 222 S. epidermidis, 156, 217 S. lentus, 156 S. xylosus, 156 Starch nanoparticles, 345 STAT1 regulation by rotavirus, 690
691 sequestration in cytoplasm, 691 STEC. See Shigatoxigenic serotypes of E. coli (STEC) STEC O157. See Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) Stem cell factor (SCF), 217 Sterile inner mucus layer, 103 Steroid contraceptives, 263 Stimulator of interferon genes (STING genes), 420
421, 726 STIs. See Sexually transmitted infections (STIs) Strain 19 (S19) vaccine, 449
450 Strain expressing genetically detoxified pertussis toxin (S1PT), 408
409 Streptavidin (SA), 490 Streptococcaceae, 153 Streptococcus, 144, 833 S. equi, 345 S. iniae, 823 S. mutans, 10
12, 78
79, 357, 650 S. pneumoniae, 5
6, 9
10, 73
75, 77
78, 153
154, 236, 346, 467
469, 597, 762, 799
800, 802 current and future status of human mucosal vaccine trials, 606 host immune responses to pneumococci, 600
603 immunization against, 603
606 pneumococcal cell surface, 598f S. pyogenes, 73
74 S. sobrinus, 78
79, 170
171, 651 Streptomyces nodosus, 780 Stromal cells, 34, 41 Subcutaneous delivery (SC delivery), 404

911

INDEX

Subepithelial dome, 29
30 Sublingual immunization (SLI), 655 antigen-presenting cells localization in sublingual mucosa, 478 s. l. mucosa and in vivo antigen uptake patterns, 479f Sublingual immunotherapy (SLIT), 317 Sublingual route of administration, 7
8 Sublingual vaccination antigen delivery and antigen presentation, 480f draining lymph node role in, 479 inducing both systemic and mucosal antibody responses, 481 inducing T and B cell activation in female mouse genital tissues, 482 mechanism for induction of CD41 T cell activation, 479
481 sublingual administration, 482 for induction of antibody against viral infection, 481
482 Sublingual vaccine for periodontal diseases, 655
656 Submandibular glands (SMGs), 656 Succinylated Corynebacterium diphtheria toxin mutant (CRM9succ), 526
528 Succinylated mutant Pseudomonas aeruginosa exotoxin A (rEPAsucc), 526
528 Sugar-inducible acid resistance, 393
394 Suidae, 813
818 porcine epidemic diarrhea virus, 813
818 porcine transmissible gastroenteriditis virus, 813
818 Surface protein antigen of S. Mutans serotype c (Surface PAc), 650 Surgery to treat eyelid inversion, antibiotics, facial cleanliness, and environmental improvement to reduce transmission strategy (SAFE strategy), 305 Sylvatic plague, 821 Synthetic MC granules, 221 Synthetic particles, 338 Synthetic polymers, 330
332 Syphilis. See also Chlamydia; Gonorrhea; Mycoplasma

clinical manifestations, 636 current treatment options, 636 epidemiology, 635
636 immune responses associated with pathology, 637 microbiology, 635 vaccine-related research, 637 Syringe infiltration, 360 Systematic evolution of ligands by exponential enrichment (SELEX), 490 Systemic antibody and memory B cell responses, 548
551 MBC responses in cholera, 550
551 serum antibody responses to defined antigens, 549
550 vibriocidal antibody responses, 548
549 responses, 481 Systemic B cell immunity, 503
504 Systemic human immunity, 507f Systemic immunity relationship with mucosal immunity, 509 Systemic lymphoid organs, 22 Systemic T cell immunity, 503 Systemic vaccination, 72
73, 272 Systems biological approaches for mucosal vaccine development discovering correlates of protection, 762
763 controlled human infection models, 763 signatures in blood predict mucosal immunity, 763 discovering fundamental immunological mechanisms of mucosal immunity, 763
767 durability of mucosal responses, 767 licensed vaccines, 754f population differences in efficacy of mucosal vaccines, 767
768 systems biology of vaccines against mucosal infections, 762
768 systems vaccinology, 756
762 from data to knowledge, 761
762 extending systems vaccinology to other vaccines, 758
760 molecular signatures of immunogenicity vs. efficacy, 760
761

studies with yellow fever vaccine YF-17D, 757
758 systems analysis of gene signatures, 761 vaccines, 756f

T T cell receptors (TCRs), 773 antigen receptors, 22 TCR
MHC complex, 90
91 T cell(s), 90
91, 220, 403
404 activation in female mouse genital tissues, 482 responses, 713 role in protection against infection at mucosal sites, 766
767 T cell-mediated protection, 727
729 T follicular helper cells (Tfh cells), 24, 766, 874 T helper cells (Th cells), 134, 870
871 T helper 1 cells (Th1 cells), 107
108, 123, 247
248, 314, 402
404, 611
612, 790
791, 841
842 cell responses, 424, 627 cell-dependent, 449 Th1-like immune responses, 730 Th1-mediated immunity against plague, 453
454 T helper 2 cells (Th2 cells), 611
612 response, 154, 402
404, 627, 855 Th2-mediated immune responses, 841
842 T helper 17 cells (Th17 cells), 107
108, 123, 149, 192, 203, 239, 259
260, 539
540, 633 cytokines, 154 responses, 247
248 Th17-mediated immunity against plague, 453
454 T-bet, 25
26 T-cell-dependent antigens (TD antigens), 118
119 T-cell-independent IgA B cell responses (TI IgA B cell responses), 118
119 T-DNA. See Transfer DNA (T-DNA) T3SS. See Type 3 secretion system (T3SS) T4SS. See Type 4 secretion system (T4SS) TA. See Teichoic acid (TA) Tablet(s), 341 tablet-based oral avian influenza vaccine, 341

912 Tachyzoites, 845
846 Taenia solium (Pork tapeworm), 847
848 TALT. See Tear-associated lymphoid tissue (TALT) TAM. See TYRO3-AXL-MERTK (TAM) Tamm-Horsfall protein. See Uromodulin (Umod) Tamoxifen, 263
264 TANK-binding kinase 1 (TBK1), 687 TB. See Tuberculosis (TB) TBK1. See TANK-binding kinase 1 (TBK1) Tbx21 transcription factor, 126 TCA cycle. See Tricarboxylic acid cycle (TCA cycle) TCM cells. See Central memory T cells (TCM cells) TCRs. See T cell receptors (TCRs) TD. See Typhoid disease (TD) TD antigens. See T-cell-dependent antigens (TD antigens) Tdap series. See Tetanus toxoid, diphtheria, and pertussis series (Tdap series) Tear film, 301
302 Tear-associated lymphoid tissue (TALT), 300 TECK, 765 TEDs. See Transepithelial dendrites (TEDs) Teichoic acid (TA), 313 Teleosts, 822 TEM. See Transendothelial migration (TEM) TEM cells. See Effector memory T cells (TEM cells) TEM8. See Tumor endothelial marker 8 (TEM8) Tensile force, 88 Terminal ileum (TI), 506
508 Tertiary lymphoid organs (TLOs), 22 Tertiary syphilis, 636 Testicular macrophages, 266
267 Tetanus, 736
737 Tetanus toxoid (TT), 170, 174
175, 466, 744 Tetanus toxoid, diphtheria, and pertussis series (Tdap series), 736
737 Tetrameric SIgA, 72
73 Tetraspanin protein 2 (TSP-2), 847 Tfh cells. See T follicular helper cells (Tfh cells)

INDEX

TGF-β. See Transforming growth factor beta (TGF-β) Th cells. See T helper cells (Th cells) Th1 cells. See T helper 1 cells (Th1 cells) Th2 cells. See T helper 2 cells (Th2 cells) Th17 cells. See T helper 17 cells (Th17 cells) The Onchocerciasis Vaccine for Africa (TOVA), 304
305 Therapeutic integrin inhibition for HIV infection, 95
96 for inflammatory bowel diseases, 93
94 Therapeutic tool box of H. pylori infections, 580
581 Thermostability of adenovirus vectors, 424 Thermostabilization technologies, 334 Thiamine, 204
206 Three-dimensional migration (3D migration), 90 Thymic stromal lymphopoietin (TSLP), 134
136, 231 Thymidine kinase (TK), 278 Thymus intraepithelial lymphocytes (tIELs), 425 Thymus regulatory Th cells (tTreg cells), 871 Thyroxin, 60 TI. See Terminal ileum (TI) TI IgA B cell responses. See T-cellindependent IgA B cell responses (TI IgA B cell responses) Ti plasmid. See Tumor-inducing plasmid (Ti plasmid) tIELs. See Thymus intraepithelial lymphocytes (tIELs) Tight junctions, 58
59, 103 Tip adhesins, 570
571 TIR domain. See Toll-IL-1 receptor domain (TIR domain) Tissue chemoattractant prostaglandin D2 receptor (CRTh2), 231 Tissue heterogeneity, ILCs development and, 229
232 Tissue homeostasis, 776 Tissue-resident memory T cells (TRM cells), 133
134, 258, 629, 727
729, 766
767 for mucosal tissues, 133
134

TIV. See Trivalent inactivated vaccine (TIV) tjp1, 146 TK. See Thymidine kinase (TK) TL1A. See TNF-like ligand 1A (TL1A) TLOs. See Tertiary lymphoid organs (TLOs) TLRs. See Toll-like receptors (TLRs) TNF. See Tumor necrosis factor (TNF) TNF-like ligand 1A (TL1A), 26, 231
232 TNF-related apoptosis-inducing ligand (TRAIL), 407
408 TNF-α. See Tumor necrosis factor alpha (TNF-α) TNFRSF17 gene, 758
759 Tobacco, 371 against microbial pathogen antigens, 340
341 Tolerance, 493, 857, 859 induction by toxin-based adjuvants, 195 Tolerogen, 372, 374 Toll-IL-1 receptor domain (TIR domain), 170 Toll-like receptors (TLRs), 58
59, 106
107, 173, 192
193, 216, 233, 248, 262, 313, 383
384, 601, 650
651, 684, 757
758, 860
861 associations between individual TLRs and intestinal inflammation, 106
109 distribution in human epithelial cells, 314 in nasopharyngeal mucosae, 314
315 expression in FGT, 262 ligands, 106t, 175
176, 327
328, 411 TLR1, 107 TLR2, 107, 420
421 TLR3, 107 TLR4, 107, 154 TLR5, 107
108, 108f, 761
762 TLR6, 108 TLR7, 108, 679
680 TLR8, 108 TLR9, 108
109, 601, 726, 800
801 TLR10, 109 TLR11, 109 TLR12, 109 TOUCH. See Tysabri Outreach Unified Commitment to Health (TOUCH)

913

INDEX

TOVA. See The Onchocerciasis Vaccine for Africa (TOVA) Toxicodendron sp., 3
4 Toxin(s) proteolytic degradation of, 219 toxin-based adjuvant delivery systems for, 190
191 innate mechanisms regulating by, 191
194 tolerance induction by, 195 toxin-derivative adjuvants for mucosal vaccines, 187
191 used for immune responses modulation, 185
187, 187f Toxoplasma gondii, 109, 845
846 infection, 236
238 Toxoplasmosis, 845
846 TPATH cells, model of disease induction by, 138
139 Tpath2 cells. See Pathogenic Th2 cells (Tpath2 cells) Tr1 cells. See Type 1 regulatory T cells (Tr1 cells) TRAIL. See TNF-related apoptosisinducing ligand (TRAIL) Transcellular EM, 87 transport across villus epithelial cells, 58
61 Transcriptional plasticity, 238 Transcriptome profiles of CD41 T cells, 404
405 Transendothelial migration (TEM), 87 Transepithelial dendrites (TEDs), 62 Transfer DNA (T-DNA), 360 Transforming growth factor beta (TGF-β), 12
13, 204, 247
248, 261, 857 Transgenic potato approach, 363 Transgenic rice (Tg) calluses, 358
359 seeds, 317
319 Transgenic spinach, 364 Transgenic technologies, 359
361 Agrobacterium-mediated transformation, 360 biolistic method for stable transformation, 359
360 MucoRice system, 360
361, 360f Transmembrane glycoproteins, 616 Traveler’s diarrhea, 555 Treg cells. See Regulatory T cells (Treg cells)

Trematodes, 841
842 Trematodiasis. See Schistosomiasis Treponema pallidum, 635, 637
638 vaccine candidates, 640t Tricarboxylic acid cycle (TCA cycle), 205 Trichinella spp., 849 Trichinellosis, 849 Trichinosis. See Trichinellosis Trichomonas vaginalis, 255 Trichosurus vulpecula (Brushtail possums), 824 Trichuris muris, 236 Tripartite motif-containing protein 21 (TRIM21), 79
80 Tripartite motif-containing protein 5a (TRIM5a), 765
766 Trivalent inactivated vaccine (TIV), 758 TRM. See Resident memory T cells (TRM) TRM cells. See Tissue-resident memory T cells (TRM cells) Trophozoites, 842
845 Tryptase-and chymase-positive MCs (MCTC), 215
216 Tryptase-positive MCs (MCT), 215
216 TSLP. See Thymic stromal lymphopoietin (TSLP) TSP-2. See Tetraspanin protein 2 (TSP-2) TT. See Tetanus toxoid (TT) tTreg cells. See Thymus regulatory Th cells (tTreg cells) Tuberculosis (TB), 402
403, 409
413, 611
612, 753
755 antigens, 404 infection mucosal immune responses in, 613
615, 614f rHPIV2 in TB protection, 616 Tuft cells, 232 Tumor endothelial marker 8 (TEM8), 186 Tumor necrosis factor (TNF), 24
25, 174, 214, 613
614, 774 Tumor necrosis factor alpha (TNF-α), 57, 219, 250
251, 420
421, 449, 539, 627, 874 Tumor-inducing plasmid (Ti plasmid), 360 Two-dimensional migration (2D migration), 90

Ty21a strain, 6
7 Type 1 innate lymphoid cells (ILC1), 613 Type 1 interferons, 264
266, 601
602, 726 Type 1 mucosal tissues, 56 Type 1 regulatory T cells (Tr1 cells), 247
248 Type 2 immune pathologies, 138
139 Type 2 interferons, 264
266 Type 2 mucosal tissues, 56 Type 3 innate lymphoid cells (ILC3s), 57, 790
791 Type 3 secretion system (T3SS), 105, 385
386 Type 4 secretion system (T4SS), 585
586 Type II heat-labile enterotoxins (LT-II), 189 Typhoid disease (TD), 503 Typhoid fever, 501
503 TYRO3-AXL-MERTK (TAM), 150
153 Tysabri Outreach Unified Commitment to Health (TOUCH), 94

U U.S. Food and Drug Administration (FDA), 328
329 UC. See Ulcerative colitis (UC) UEA. See Ulex europaeus agglutinin (UEA) UGT. See Upper genital tract (UGT) UK-bovine RV strains, 703 Ulceration, 508 Ulcerative colitis (UC), 93, 105, 247
248 Ulex europaeus agglutinin (UEA), 37
38 UEA-1, 487
488 Umod. See Uromodulin (Umod) University of Maryland Center for Vaccine Development (CVD), 526 Unpaired nasopharyngeal tonsils, 462
463 Upper airway colonization, 602 Upper genital tract (UGT), 821 Upper respiratory infections (UR infections), 597, 790
791 Urease, 586
587 urease C gene, 409 Uromodulin (Umod), 31, 491
492

914 US Department of Agriculture (USDA), 372 US National Institute of Allergy and Infectious Diseases (NIAID), 702
703 US Naval Medical Research Center (NMRC), 570
571 USDA. See US Department of Agriculture (USDA)

V

V antigen. See Virulence antigen (V antigen) V protein, 616 VacA, 586
587 Vaccination(s), 445
446, 756
758 in cattle, 451 correlates of protection, 667
669 global impact and clinical disease, 666
667 maternal immunization to protect vulnerable infants, 669
670 against mucosal infections, 753
755 origins, 446 RSV immunity and in infants and young children, 670
672 in older adults, 672 Vaccine Safety Datalink project, 702 Vaccine(s), 157
159, 167, 372
373, 403, 753
755, 759
760, 811
812 antigens, 328
329, 343 antigen-specific T and B cells, 765 approaches against genital herpes, 729
731 mucosal vaccines against HSV-2, 730
731 past prophylactic vaccine trials, 729
730 composition, 307 delivery systems for induction of mucosal immunity, 615
616 efficacy, 701, 705 production, 360 strain, 701
702 targets and strategies for vaccine development common ocular pathogens, 303
305, 304t pathogenesis of trachoma, 300f strategies for ocular vaccine development, 305
307 vectors, 419

INDEX

Vaginal deliveries, 875 Vaginal microbiota, 155 Valacyclovir, 278, 742 Variable lymphocyte receptors (VLR), 22 Variant-specific surface proteins (VSPs), 844
845 Varicella zoster virus (VZV), 283
284 Variolae vaccinae, 446 Variolation, 446 Vascular endothelial growth factor, 214 Vasoactive intestinal peptide (VIP), 234 Vaxchora, 334
335, 365, 528, 542 Vaxfectin-gD2 vaccines, 280 Vaxfectin-gD2/UL46/UL47 vaccines, 280 Vaxonella platform for chlamydia immunization, 631 VCAM-1, 93 Vectoring guest antigens, 386
387 Vedolizumab, 85
86, 93, 95 Veillonella, 154 Veterinary vaccines and One Health, 825
826 Vi polysaccharide capsule, 6
7 Vibrio, 383 V. anguillarum, 822 V. cholerae, 5
6, 73
74, 128, 168, 187
188, 333
334, 344, 487, 528, 537, 543, 546
547, 871
872 Vibriocidal antibody responses, 548
549 Vicious circle, 313 Villous M cells, 61 VIP. See Vasoactive intestinal peptide (VIP) Viral immune evasion, 731
732 Viral particles, 666, 668 Viral pathogens, 377 Viral replication, host innate responses to rotavirus and effects on, 686
687 Viral vectors, 359, 615
616, 618
619 Viral-vectored vaccines, 327
328 Virulence antigen (V antigen), 452
453 Virus-induced signaling adapter (VISA), 687 Virus-induced stress genes (vISGs), 683
684

Virus-like particles (VLPs), 176
177, 326
327, 338, 365, 391, 692
693, 705
706 Virus(es), 344, 383
384, 461
462, 487 neutralization, 78
79 VISA. See Virus-induced signaling adapter (VISA) Visceral adipose tissue, 133
134 vISGs. See Virus-induced stress genes (vISGs) Vitamin B1. See Thiamine Vitamin B2. See Riboflavin Vitamin B3. See Niacin Vitamin B5. See Pantothenic acid Vitamin B6. See Pyridoxine Vitamin B7. See Biotin Vitamin B9. See Folic acid Vitamin B12. See Cobalamins Vitamins, 204
206 vitamin A, 204, 552 deficiency, 204, 234, 765 vitamin B complex, 204
206 vitamin D, 206 Vivotif, 6
7, 341 VLA-4, 90 VLPs. See Virus-like particles (VLPs) VLR. See Variable lymphocyte receptors (VLR) VP1 protein, 704 VSPs. See Variant-specific surface proteins (VSPs) VZV. See Varicella zoster virus (VZV)

W W205EC nanoemulsion, 465
466 W805EC nanoemulsion, 171
172, 465
466 Waldeyer’s ring, 462
463 Wallemia sebi, 154 Walter Reed Army Institute of Research (WRAIR), 525
526 Wart, hypogammaglobulinemia, infections, and myelokathexis (WHIM), 89 WaSH community. See Watersanitation-hygiene community (WaSH community) Water-insoluble glucans (WIGs), 651f Water-sanitation-hygiene community (WaSH community), 553 wboA glycosyltransferase, 450 WC. See Whole cell (WC)

915

INDEX

WC/rCTB. See Whole cell/ recombinantly produced CTB vaccine (WC/rCTB) WCrBS, 334
335 WCV. See Whole cell cholera vaccine (WCV) Wernicke
Korsakoff syndrome, 205
206 West Nile virus, 776
777 Wheat germ agglutinin (WGA), 487
488 WHIM. See Wart, hypogammaglobulinemia, infections, and myelokathexis (WHIM) WHO. See World Health Organization (WHO) Whole cell (WC), 325
326 WC-3, 700 WC/BS OCV, 548 Whole cell cholera vaccine (WCV), 540
542 WCV/rCTB Dukoral prototype vaccine, 544
546 Whole cell/recombinantly produced CTB vaccine (WC/rCTB), 540

WIGs. See Water-insoluble glucans (WIGs) Wild-type allergen sequence, 861
862 Wild-type HSV-2 (WT HSV-2), 727 Wildlife, 821
822 Wolbachia, 304
305 Wool sorter’s disease. See Cutaneous anthrax World Health Organization (WHO), 255, 329, 501
502, 579
580, 626 recommendations for OCVs, 554
555 WRAIR. See Walter Reed Army Institute of Research (WRAIR) WT HSV-2. See Wild-type HSV-2 (WT HSV-2)

X X8P nanoemulsion, 465
466 XCR1 expression, 61
62

Y Yamoa polysaccharides, 779 Yellow fever YF-17D vaccination, 768

studies with YF-17D, 757
758 extending systems vaccinology to other vaccines, 758
760 molecular signatures of immunogenicity vs. efficacy, 760
761 Yersinia Y. enterocolitica, 31
32 Y. enterocolitica, 61 Y. pestis, 5
6, 340
341, 344
345, 435, 451
452, 464
465, 821 Y. pseudotuberculis, 186 Y. pseudotuberculosis, 453
454 Y. ruckeri O1, 823 Yghj protein, 568

Z

Zebrafish γδ T cells, 775 Zika virus (ZIKV), 735
736, 743
744 Zinc deficiency in children, 552
553 Zona pellucida proteins (ZP), 824 Zostavax, 283
284