How to Make a Vaccine: An Essential Guide for COVID-19 and Beyond 9780226792651

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H OW T O M A K E A VAC C I N E

HOW TO MAKE A VACCINE AN ESSENTIAL GUIDE FOR COVID-­19 & BEYOND

JOHN RHODES The University of Chicago Press CHICAGO AND LONDON

The University of Chicago Press, Chicago 60637 The University of Chicago Press, Ltd., London © 2021 by John Rhodes All rights reserved. No part of this book may be used or reproduced in any manner whatsoever without written permission, except in the case of brief quotations in critical articles and reviews. For more information, contact the University of Chicago Press, 1427 E. 60th St., Chicago, IL 60637. Published 2021 Printed in the United States of America 30 29 28 27 26 25 24 23 22 21    1 2 3 4 5 ISBN-­13: 978-­0-­226-­79251-­4 (paper) ISBN-­13: 978-­0-­226-­79265-­1 (e-­book) DOI: https://doi.org/10.7208/chicago/9780226792651.001.0001 Library of Congress Cataloging-in-Publication Data Names: Rhodes, John, 1947– author. Title: How to make a vaccine : an essential guide for COVID-19 and beyond / John Rhodes. Description: Chicago : University of Chicago Press, 2021. | Includes bibliographical references and index. Identifiers: LCCN 2020053338 | ISBN 9780226792514 (paperback) | ISBN 9780226792651 (ebook) Subjects: LCSH: COVID-19 (Disease) | Vaccines. Classification: LCC RA644.C67 R48 2021 | DDC 614.5/92414—dc23 LC record available at https://lccn.loc.gov/2020053338  This paper meets the requirements of ANSI/NISO Z39.48–­1992 (Permanence of Paper).

To my students Gao Xiaoming, Zheng Biao, and Chen Huaqing and senior visitor Liu Dong Shen, with gratitude

CONTENTS

Preface, ix

1.

Understand the Virus

2.

Explore the Immune System

14

3.

Discover a Vaccine

35

4.

Develop Vaccines

53

5.

Evaluate the Contenders

66

6.

Don’t Count on the Magic Bullet

93

7.

Overcome the Hurdles

108

8.

Embrace Many Solutions

124

1

Epilogue, 129   Acknowledgments, 133 Appendix: COVID-­19 Vaccines and Vaccine Candidates, 137 Further Reading, 141   Index, 163

PRE FACE

The year 2021 marks the 300th anniversary of immunization. It is also the year when my colleagues, immunologists around the world, began to deliver vaccines for global use against a new virus, the cause of the most serious pandemic infection in living memory. I doubt that anyone working hard on vaccines to combat the COVID-­19 pandemic celebrated this anniversary. But history had gifted them with the resources necessary to be effective. They didn’t need any new discoveries to build a vaccine. They already understood the complex workings of the human immune system. They had the tools to develop vaccines, and manufacturing techniques stood ready for large-­scale production of the vaccines they would develop. And yet readiness is not a word many would use to describe 2020. The possibility of combating the spreading pandemic with as yet undiscovered vaccines understandably made many anxious. The desire to help calm this public uncertainty was one reason I decided to write this book. Though in 2020 I shared the public’s concern regarding the loss of life as COVID-­19 spread, I felt optimistic that we would soon have a vaccine (in fact, vaccines) available for global use. And I hope this book will leave you optimistic about vaccine development to stop future pandemics. Remember, our readiness—­in terms of tools, technologies, and knowledge—­will continue to improve. The other r eason I was keen to pen this book is much sim-

pler: vaccines are fascinating. As an immunologist, I have had a lifelong passion for the science of immunology, the history

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of vaccination, and the often-­surprising relationship between the two. From the global perspective, immunization has greatly reduced the burden of infectious diseases and, as the most successful biomedical measure preventing sickness, it continues to do so around the world. The 20th century saw the complete eradication of smallpox, one of the deadliest diseases known to humankind, through vaccination. In the 21st century, the global campaign to put an end to polio through vaccination remains tantalizingly close to success, and in 2015 researchers introduced the first vaccine against a parasitic disease, malaria, for use in Africa, where it substantially relieves the burden of disease on health-­care services. I believe that vaccines to bring an end to the COVID-­19 pandemic crown this story of success. This does not mean that we will eliminate the coronavirus causing COVID-­19, but vaccination will stop this virus from becoming a burden for our health systems and an interruption to our ways of life. The global emergency that began in 2020 brings home to all of us the importance of a concerted international effort in combating pandemic disease. The sharing of scientific information is an essential part of this, and soon after the uncertainty surrounding the earliest events, a policy of open and collaborative exchange was established and has been sustained ever since to a degree never seen before. This contrasts starkly with the events of history. Pandemics can start anywhere, and though we strive to analyze and explain the situations that give rise to them, we can never be entirely sure where epidemics will have their beginnings. Toward the end of the First World War, in the spring of 1918, soldiers were living in very close quarters in large forts and camps across the United States, training to join the war in Europe. One of the larger communities was Camp Funston, near Fort Riley in northeast Kansas, whose roll included more than 25,000 men. The winter of 1917–­18 was harsh in the Midwest, and conditions at the camp were far from ideal. The region suffered dust storms that blew across the open

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plain, endured by the soldiers and the several thousand mules and horses in their service. Smoke and ashes from bonfires of horse manure thickened the pall, which grew so heavy it sometimes blotted out the sun. Shortly before breakfast on Monday, March 11, a day or so after a particularly bad dust storm, a company cook named Albert Mitchell reported to the infirmary complaining of a sore throat, fever, and muscle aches. A medic recommended bed rest. By noon, 107 soldiers had fallen sick. By the end of the week, 500. Other outbreaks followed, including in New Jersey, South Carolina, and Colorado. In April and May, more than 500 prisoners at San Quentin in California were suffering the same condition. This spreading tide was influenza, and it gradually engulfed North America to become the worst pandemic of contagious disease in modern human history. No one is certain how the flu arrived at Funston, but some experts point to Haskill County in southwestern Kansas, the home of many recruits. Haskill County was at the time a rural community, where farmers raised hogs and poultry alongside their families in soddies (simple farmsteads built of prairie sod)—­ideal conditions for transmission of pig and bird viruses to humans. In January 1918, a local doctor became so concerned about the number of flu cases in his area, he reported the outbreak to the national authorities. The weight of evidence therefore favors Kansas as the origin of what was to become the Spanish flu (also called La Grippe, or La Pesadilla, meaning “nightmare”). The naming of epidemic diseases has often been haphazard and not at all useful for our understanding. By April 1918, nearly 200,000 US soldiers had crossed the Atlantic, carrying the infection with them. By May, the illness was established on two continents, and the infection spread through Europe into Spain. There, for the first time, newspapers widely reported the large number of people falling sick. Because Spain was a neutral country, sensi-

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tivities around reporting sickness in combat troops did not exist. This led to the name we still use today. Other authorities point to Étaples-­sur-­Mer in northern France as a possible starting point for Spanish flu. There, at a staging camp for British troops, a mysterious respiratory infection appeared during the winter of 1915–­16, and this may have presaged the 1918 flu. Whatever its origin, the first wave of Spanish flu, which mostly affected older and compromised individuals, was highly infectious but not nearly as serious as the contagion would become. Initially it had been called the “three-­day fever”—­the sickness started with a cough and headache, followed by intense chills and a fever that could quickly rise. It could take a month before survivors felt completely well again. In late August, returning troops carried an outbreak centered on the port of Brest back across the Atlantic to New England. In September 1918, soldiers at an army base near Boston, Massachusetts, suddenly began to die. The virus of the second wave was far more lethal than the first. Instead of targeting the old and sick, the second wave of Spanish flu proved fatal for the young and healthy. Almost half of the deaths were among 20-­to 40-­year-­olds, which some experts attribute to their stronger immune response, which prompted massive inflammation in the lungs. In the month of October 1918 alone, 195,000 died of influenza in the United States. No vaccine was available or on the horizon. The tools and technologies that benefit today’s vaccinologists were a lifetime away, and the global tragedy did not trigger vaccine development. The influenza pandemic of 1918–­19 is estimated to have killed 50 million people worldwide, spreading even to the Arctic and remote Pacific islands, making it one of the deadliest natural disasters in human history. Most deaths worldwide occurred in a 16-­ week period, from mid-­September to mid-­December 1918. Some 500 million people (27 percent of the global population) are estimated to have suffered infection. But the first flu vaccines would

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not be developed until 1941. By 1945 United States troops fighting World War II were being immunized against influenza. As an immunologist with a long career in both academic and

industrial research, I’ve had a lifelong fascination for the science we now call vaccinology. One of its most remarkable features is just how much researchers achieved before we had gained any detailed understanding of the workings of the immune system. As you will see in this book, virologist Jonas Salk introduced the first effective vaccine against polio in 1954, a time when virtually nothing was known about the cells of the human immune system. The same was still true when virus expert Albert Sabin developed a second vaccine in 1959. And in the next decade, still without a deep understanding of the mechanisms of the immune system, virologist John Enders discovered a measles vaccine in 1963, and Maurice Hilleman an improved form in 1968. These important breakthroughs in vaccine development related instead to techniques for growing viruses in tissue culture. This book will show just how much researchers have transformed the relationship between the science of immunology and the discovery of vaccines since then. It will also trace the development of vaccines from the first exploratory investigations, through vaccine manufacture, to the large-­scale clinical testing of vaccines to establish their safety and effectiveness. Once regulatory agencies have approved vaccines, the nature of the vaccine endeavor changes character again. There are many hurdles still to overcome, from the logistics of vaccine delivery and equitable distribution around the globe to the acceptability of vaccines for those uncertain about the safety and desirability of immunization. This issue has been present for 300 years—­since the very first organized trials of immunization to prevent smallpox in 1721. Despite our celebration of 2021 as an anniversary, immunization has even older origins in traditional practices in African and

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Asian cultures. When Chinese Emperor Fulin died of smallpox in 1661, his third son, K’ang-­hsi, succeeded him. In the late 1600s, K’ang-­hsi wrote about the value of immunization in a letter to his children: “The method of inoculation having been brought to light during my reign, I had it used upon you, my sons and daughters . . . and you all passed through the smallpox in the happiest possible manner. . . . In the beginning, when I had it tested on one or two people, some older person taxed me with extravagance, and spoke very strongly against inoculation. The courage which I summoned up to insist on its practice has saved the lives and health of millions of men. This is an extremely important thing, of which I am very proud.” When I tr ained as a young immunologist in the Laboratory

of Clinical Investigation at the National Institute of Allergy and Infectious Diseases, and afterward pursued research at the University of Cambridge, I saw myself as an academic scientist, exploring the fundamental mysteries of pure science. But later in my career, at the Wellcome Foundation, Glaxo Wellcome, and Glaxo Smith Kline, I learned, with some difficulty, to navigate the occasionally problematic path between fundamental and applied research, balancing the natural impulse of all scientists to share their work, through publications and conference communications, with the need to patent and protect intellectual property before judiciously allowing it into the public domain. For me, perhaps the most important compensatory privilege in leading this double life has been the chance to supervise research students. This book is therefore dedicated to my students, now long established in their own scientific careers in China. It is they who will teach us how to make the vaccines of the future.

CHAPTER ONE

Understand the Virus A poem by Yuen Ren Chao tells the story of a poet visiting a live market. There, just as he had hoped, the poet finds 10 lions. He shoots them with his trusty arrows and takes them home to his stone den. But when he tries to eat them, he finds them turned to stone. What can this mean? Yuen wrote the poem in 1931 in classical Chinese. When pronounced in modern Mandarin, however, the entire story is a single syllable—­shi—­repeated many times over with different tones. For me, there are parallels to Yuen’s poem in the race to find a vaccine. We begin with a single word, vaccine, but in the endlessly inventive minds of vaccinologists rushing to produce an effective weapon against COVID-­19, this small term had a hundred different meanings depending on the context, tone, color, and inflection they have chosen to employ. There are no lions for sale in the live markets of Guangzhou, a major trading center on the Pearl River in the far southeast of China. Once called Canton, the historic gateway for Western travelers to Imperial China, it is also an ancient center of learning. Because the region is blessed with a year-­round growing season, Guangzhou is known as the City of Flowers, and the fertile river valleys of Guangdong Province provide three crops of rice each year. The region is rich in wildlife. Tigers, rhinoceroses, leopards, wolves, and bears once roamed the Guangdong hills, but now these are rare or lost forever because of deforestation and the illegal trade in traditional medicines. Instead, the smaller crea-

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tures rule: bats, squirrels, mice, and rats thrive in the farmlands; mongoose-­like mammals called palm civets and small-­clawed otters represent the carnivores. Sometime around the middle of 2002, a farmer in the Shunde district of Foshan County, just across the river from Guangzhou, encountered a wild civet at a market or in the countryside and contracted a viral infection. He suffered fever and inflamed lungs and had difficulty breathing. Such events are not uncommon—­the farmer’s illness was a zoonosis, an infection contracted by a person from an animal. But in this case, as the virus grew, it changed sufficiently to allow for transmission between one person and the next—­the first ominous step in a zoonotic epidemic. By mid-­November, this particular epidemic, severe acute respiratory syndrome (SARS), had firmly taken hold. On January 31, Zhou Zuofen, a fishmonger, was admitted to the Sun Yat-­sen Memorial Hospital in Guangzhou, where his infection passed to 30 doctors and nurses. The virus soon spread to nearby hospitals, and on February 10, with 305 cases (including 105 health-­care workers) and five deaths, the Chinese Government notified the World Health Organization (WHO). On February 21, Liu Jianlun, a health-­care worker treating patients in that same hospital, traveled to Hong Kong to celebrate a family wedding and checked in at the Metropole Hotel. Despite feeling ill, he visited his family and toured the city with his wife. The next morning his illness worsened, and he walked to nearby Kwong Wah Hospital to seek treatment, urging staff to place him in isolation. He died in the intensive care unit on March 4. His infection had already spread to more than 20 Metropole guests. Among them was Kwan Sui-­Chu, an elderly visitor from Toronto, Canada. She returned home on February 23, and there her son, Tse Chi Kwai, became infected. Kwan Sui-­Chu died at home on March 5, but the disease had already gained a foothold.

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Tse Chi Kwai was admitted to Scarborough Grace Hospital, where the infection quickly spread to other patients. Canada eventually reported 251 cases and 43 deaths—­a mortality rate approaching 1 in 6. These individuals caught up in a tragedy provide a personal picture of how a deadly virus spreads around the globe. Eventually, this virus would infect more than 8,000 people in 29 countries, killing 774 before the epidemic was contained. Early in 2003, with worldwide cases running at 3,400 and deaths standing at 143, Canada became the Western nation most severely impacted by the SARS epidemic. In response, scientists at the British Columbia Cancer Agency Genome Sciences Centre in Vancouver switched from their normal task, studying genetic changes in cancer, to sequencing the virus’s genome. It took just six days to sequence the 30,000 “letters” of the virus’s genetic code, and the data placed the new virus firmly in the coronavirus family—­a group of viruses known for causing mild infections of the upper respiratory system. From this, the deadly new virus received its name: SARS coronavirus, conveniently shortened to SARS-­CoV. It would be one of several deadly epidemics caused by zoonotic viruses to emerge early in the 21st century. The scale of these outbreaks, however, would be dwarfed by the events that unfolded in 2020. As each zoonotic epidemic emerges, I take a close interest. In part this interest began with a very personal experience of a viral contagion. When I was 22 years old, just starting my career in science and researching for my PhD in immunology at University College London, something dreadful happened. A mysterious infectious agent, silently confined to a life in animals, somehow succeeded in making the leap into humans—­two humans to be precise, me and Ted, the lab technician who looked after our laboratory guinea pigs.

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When Ted first became ill, I stepped up to look after the animals in his absence. After two weeks, there was still no word on when Ted would return. I didn’t mind continuing to care for the animals, but the pleasure of their company as I cleaned their cages, fed, and watered them was marred by a new concern. The guinea pigs had seemed normal enough when they arrived from a reputable supplier. Now some of them looked sick. It wasn’t long before something else started troubling me. I had developed sores inside my mouth. The nearest thing I could liken them to were cold sores, but my sores seemed deeper, almost cavernous, and more aggressive. Each one cleared, slowly, but a new one would replace it, just a little farther down my throat. Somehow the sores felt malevolent. Then they disappeared. My throat recovered, and I felt tired but relieved. Just a week later another symptom took their place. I had a dull ache at the top of my esophagus. It felt swollen and constricted, and I found it difficult to swallow. I began to feel delicate and strangely vulnerable, and I realized with alarm what was happening—­something, I didn’t know what, was making its way inside me. I was young and fit, and such dark and morbid imaginings weren’t like me at all. Then suddenly the ache went away, and I felt better. I was sure this would be an end to it and that at last I was back to normal. One week later, I felt something in my chest. It was a kind of flutter—­like a small bird trapped beneath my ribs, trying to escape—­soft, painless, gone in a trice. The next day I felt another —­stronger and more distinct, as if my heart had missed a beat. I didn’t feel ill or tired, but I felt something else—­a cold terror seeping down my spine. As the missed heartbeats began to string themselves together in longer and longer chains, I would pray for my heart to start beating again. I managed to get back to the student health doctor for a third visit. This time he sent me straight to Coppett’s Wood Hospital, London’s specialist isolation

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unit, tucked away in the remote suburbs north of Muswell Hill. The infection almost put an end to me, and to Ted, but happily, one year later, we’d both fully recovered, though still troubled by a few missed heartbeats now and then. The cause of our illness was a guinea pig coxsackievirus targeting the heart. Ted and I had each contracted the virus directly from the infected guinea pigs. Thankfully for us, and those we love, this virus cannot be transmitted person to person. This life-­threatening infection, suffered right at the start of my career, endowed me with three things—­an enduring zest for life, an uncanny ability to control my heart rate just by thinking about it, and a lifelong interest in zoonotic viruses. When SARS emerged in 2002, I was eager to understand the full picture. It took Chinese scientists almost 15 years of work, but eventu-

ally their efforts came to fruition. Zheng-­Li Shi and her colleagues at the Wuhan Institute of Virology succeeded in tracing the primary cause of the SARS outbreak. By sequencing the genetic material of viruses in the mongoose-­like masked palm civets sold in Guangdong’s live markets, virologists had already confirmed these creatures as the likely source of the zoonotic viral strain, meaning the strain that made the jump from civets to humans. Subsequent testing in other animals revealed large numbers of SARS-­related coronaviruses in horseshoe bats, tiny mammals abundant across Asia. This suggested that the deadly strain had originated in bats and later passed through civets before reaching humans. But a crucial gene—­the gene for the key protein that allows the virus to latch on to and infect cells—­was different in the human and the known bat versions of the virus, leaving room for doubt. Shi’s long search for a better match eventually led as far as Yunnan Province in Southwest China—­a land of snow-­capped mountains and steep valleys with the richest flora in the whole of China and an equally diverse human population. There, in a

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remote cave, scientists at last identified a single population of horseshoe bats harboring a coronavirus strain with all the genetic building blocks to match the strain infecting humans in 2002. So, SARS-­CoV is a bat coronavirus. But what exactly does this mean? Where did coronaviruses come from? And how did we know about them in the first place? American virologists Arthur Schalk and M. C. Hawn first discovered what would become known as coronaviruses in chickens on a farm in North Dakota in 1931. Schalk and Hawn, early pioneers in the study of viruses, described an aggressive bronchial infection in newborn chicks that caused gasping and listlessness. More than half of the vulnerable newborns succumbed to the disease. Six years later, virologists Fred Beaudette and Charles Hudson succeeded in isolating and transferring the virus that caused the sickness, characterizing it as chicken bronchitis virus. Two more viruses, discovered in mice in the 1940s, would also turn out to be members of the same family (that is, a group with similar characteristics, suggesting an evolutionary relationship). At this time, the study of viruses was in its infancy. The observations were largely inferential and the virus itself, unlike bacteria, which could readily be studied under a microscope, remained an invisible and intangible entity. The mystery of viruses began in 1892 when Russian biologist Dimitri Ivanovsky, working at the University of St. Petersburg, discovered that the agent causing a certain disease in tobacco plants could pass through unglazed porcelain filters (which had the smallest pores then available to science) to be collected in the culture vessel below. This was a great surprise. At the time, all living things were thought to be too large to pass through such pores. Even bacteria, the smallest known living things, were too big to pass through that filter. And unlike bacteria, this mysterious substance was not capable of growing independently on nutrient broth in a test tube. In 1898, the Dutch microbiologist Martinus Beijerinck repeated Ivanovsky’s experi-

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ment and became convinced that the filtered solution contained a new form of infectious agent that could thrive only in growing plants. He called the liquid living thing a virus, a term from the Latin word for poison, originally referring to snake venom. An Italian microbiologist, Adelchi Negri, later showed that the smallpox germ was also a filterable agent, pronouncing it a virus in 1906. But it wasn’t until 1935 that viruses were first seen as objects under an electron microscope. This feat was achieved by Wendell Stanley, a young chemist working in the Rockefeller Institute’s laboratories in Princeton, New Jersey. He succeeded in purifying the tobacco plant virus in the form of needle-­shaped crystals, which possessed the chemical properties of a protein. It was again a startling discovery. How could a virus, with its ability to infect and multiply, also be an inanimate chemical? An inert molecule? Stanley’s finding prompted fundamental philosophical questions about what constitutes life. Further research soon confirmed that the infectious substance, what we now call tobacco mosaic virus, was a combination of protein and nucleic acid. We now know that all genetic material consists of nucleic acid. At the time of Stanley’s breakthrough, however, the discovery of the genetic blueprint for all living organisms, by James Watson and Francis Crick, still lay eight years in the future. For his achievements, Stanley shared the 1946 Nobel Prize in Chemistry. Despite their early studies of chickens and mice, researchers did not discover human coronaviruses until much later. In 1960 David Tyrrell, working at the Common Cold Unit of the British Medical Research Council, succeeded in isolating a novel virus from a boy with the common cold. When this isolated virus was put in healthy volunteers’ noses, it reliably caused the common cold. We now know that coronaviruses cause around 20 percent of colds. (Another virus family, rhinoviruses, account for twice this number.) Around the same time, virologists Dorothy Hamre

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and John Procknow at the University of Chicago isolated a novel cold virus from medical students, which turned out to be the same pathogen. But when would researchers actually succeed in laying eyes on this virus? The novel cold virus was first seen under an electron microscope by Scottish virologist June Almeida working at St. Thomas Hospital in London in 1967. The daughter of a Glasgow bus driver, Almeida left school at 16. But this didn’t stop her becoming a pioneering electron microscopist with a gift for photographing viruses. It was Almeida, together with David Tyrrell, who named the germ coronavirus because of its appearance. Her new name first appeared in the journal Nature in 1968. “[The viral] particles are more or less rounded in profile . . . there is also a characteristic fringe of projections 200 angstroms long, which are rounded or petal shaped, rather than sharp or pointed. This appearance, recalling the solar corona, is shared by mouse hepatitis virus and several viruses recently recovered from man.” I was lucky to meet Almeida late in her career, toward the end of the 1980s. By then she had returned to work at the Wellcome Research Laboratories in the leafy suburbs of south London to advise on the best way to photograph a novel pathogen recently given its new and final name: HIV. At the time of this book’s writing, most readers can conjure

the image of coronavirus, common from news reports around the world. A sphere with crown-­like protrusions named for the solar corona, the aura of plasma that surrounds the Sun. But what are those spikes? And what’s inside the ball? In technical terms, coronavirus is an “enveloped, nonsegmented, positive sense RNA virus.” You may remember the role of RNA from an introductory biology course; its job is to carry the instructions in the DNA (our cellular blueprints) out of the nucleus into the cytoplasm, allowing the building of protein. The

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single strand of viral RNA in coronavirus is longer than in many other viruses, consisting of 26,000 to 32,000 bases or RNA “letters.” The viral contents are enclosed in a fatty outer layer, the envelope which gives the virus its round shape. Spikes, formed from three protein molecules bound together, protrude through the envelope. These spikes are perfectly shaped to lock on to a target molecule, called “angiotensin-­converting enzyme 2” or ACE2, on the surface of human cells. The viral target, ACE2, is not just a passive protein on the surface of cells. When blood pressure is too high, ACE2’s job is to reverse a chemical message instructing blood vessels to contract, causing vessels to dilate instead. ACE2 appears on cells in the lungs, arteries, heart, kidneys, and intestines, so the virus can in principle affect these organs. Most people infected with COVID-­19 have respiratory problems as lung tissues are the first to be exposed to the virus in large amounts. In those who recover quickly, the effects are confined to the lungs. But in individuals who fail to shake off the virus, other organs are at risk. As the virus reproduces, cells lining blood vessels (which also have ACE2 molecules) may become leaky, attracting inflammatory cells, leading to blood clots affecting heart, brain, lung, and kidney function. These effects appear only in more severe cases. Once the spike protein gets a grip on ACE2 at the surface of lung cells, the spike changes its shape to expose what’s called a “membrane fusion element,” rather like unsheathing a weapon. This change is effected by enzymes produced by the target cell. The exposed element then fuses with the membrane of the human cell it’s attacking, allowing the virus inside. Once inside, the viral RNA directs the production of copies of the original virus attacker. Viral proteins self-­assemble into particles, the new RNA is packaged, and the progeny leave the cell through the normal secretory pathway used in the export of proteins, picking up a lipid coat on the way out. Infected cells continue to export virus

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in this way until their own integrity becomes exhausted or they’re picked off by the immune system. Of course, on December 30, 2019, no one could see these secret

machinations, hidden away inside the human cell, that would freeze the life of a city of 11 million. On this date in Wuhan, Hubei Province, China, a cluster of pneumonia cases was reported to the China National Health Commission. Wuhan is a trading center 1,000 kilometers south of the capital, Beijing. It is the most populous city in Central China. Chinese health authorities set in motion an investigation to characterize and contain the disease, including isolation of those with symptoms, close monitoring of contacts, and epidemiological and clinical data collection. By January 7, Chinese scientists had isolated a novel virus from patients in Wuhan and analyzed the genetic material. The sequence they obtained revealed it as a novel coronavirus, distinct from SARS-­CoV, the cause of the outbreak 18 years before. In consequence, SARS-­CoV was renamed SARS-­CoV-­1 and the new corona virus dubbed SARS-­CoV-­2. Using this sequence (the order in which the four chemical building blocks of RNA are arranged), scientists constructed diagnostic tests for detecting the virus. The scientists provided full genome sequence information to the WHO and shared their data on the Global Initiative for Sharing All Influenza Data platform. Of the initial 41 people hospitalized with pneumonia and officially identified as having SARS-­CoV-­2 infection, two-­thirds were exposed in the Huanan Seafood Wholesale Market. Wuhan is a trading center on the Yangtze River. The Huanan market, 50,000 meters square with more than 1,000 traders, is among the largest in the region, with wild animals traded in its western zone. It’s in a newer part of town and, although it is a modern market governed by the latest regulations and routinely inspected by statutory authorities, the spirit of this and other such

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markets is ancient as can be. Picture the lanes and crowded stalls with caged wild creatures, butchered carcasses, offal from slaughter and skinning, garbage piling up, clusters of trussed, protesting poultry being swung onto the scales, stunned reptiles, starving civets, live frogs bagged for sale. Imagine the clamorous cries of mongers, shrewd shoppers sampling the wares, lightning-­speed transactions made with skill, aligned with custom, yet with all the appearance of chaos. Such are the scenes of a typical live market. On January 11, the first SARS-­CoV-­2 fatality was recorded. Five days later came the first case in Japan. On January 19, the first case in Korea occurred, two cases in Beijing had come to light, and one case was recorded to the south in Guangdong Province. By January 24, 835 cases had been reported in China with 549 in Hubei Province, and 286 from the other 31 provinces, municipalities, and special administrative regions of China. On January 23, the local government in Wuhan announced the suspension of public transportation, with closure of airports, railway stations, and highways in the city to prevent further disease transmission. The government implemented active surveillance for new cases and close monitoring of their contacts. The SARS outbreak of 2002–­2004 was fresh in the minds of presiding officials. Investigative journalists would later challenge this official version of events and provide evidence that cases were higher much earlier and that the lockdown in Wuhan came too late—­a mistake that, sadly, would be repeated around the world. Other laboratories soon confirmed the genetic sequence of the new virus. Scientists in the Department of Immunology and Microbiology at Scripps Research Institute in Southern California concluded that the virus was a natural (known as wild-­type) virus originating in horseshoe bats but evolving in an intermediate host into a form that could infect humans. In the words of infectious diseases specialist Kristian Andersen and his coauthors, “Our analyses clearly show that SARS-­CoV-­2 is not a laboratory con-

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struct or a purposefully manipulated virus.” The likely culprit as secondary host, the animal that transferred the virus to humans, according to these authors, was the pangolin. Pangolins are small, ant-­eating mammals illegally trafficked in large numbers for sale in live markets across China as a source of traditional medicines. Further evolution in humans had probably increased the virus’s ability to bind to human cells. On March 11, the WHO declared the SARS-­CoV-­2 outbreak a pandemic. Studies soon showed that much of the spread of SARS-­ CoV-­2 might be due to transmission by infected individuals with no symptoms. The highest levels of the virus in people’s noses and throats appear shortly before they exhibit symptoms, and one study, using test and trace surveillance techniques, showed that this period of maximum virus levels accounted for half of the total transmissions. It’s April 17, 2020. The sliding waters of the Yangtze River, an arched brow to the still eye of the lake below, continue on their eastward journey. This city has seen many troubles. In 1938 the Imperial Japanese Army captured it. In 1944 the city erupted in a firestorm as American forces fire-­bombed the Japanese. The cherry trees beside East Lake are a legacy of the Japanese occupation, but in Wuhan the much-­loved plum blossom reigns supreme. In 2020, no one could attend the plum blossom festival, but there was a different spectacle to impress and inspire us. On the wide embankment of the river, just below First Bridge, a striking figure takes the center of the stage and boldly leaps into the air. She’s wearing sports shoes, a white face mask, and black pajamas richly decked with gold and red adornments suggestive of twin dragons. The bridge is quiet. The river is deserted. With just her smartphone on its trusty tripod for company, she is the perfect symbol of resistance to the latest enemy of the people. Her name is Wu Xueqin, a Wuhan Sports University teacher deter-

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mined to continue her lessons to students of the martial arts, no matter what may come. In modern China, the martial arts are, at their best, elegant, intricate, precise, beautiful in their complexity, surprising in their depth, and as much about restraint, inhibition, and forbearance as about striking down an enemy. The image reminds me of the human immune system.

CHAPTER TWO

Explore the Immune System This story begins in Japan. Each spring, across the five islands, people gather to celebrate the transient beauty of sakura (cherry blossoms) with hanami—­elaborate viewing picnics held beneath the laden trees. The long-­awaited spectacle begins in Kyushu, the southernmost island, in the fine parks of the city of Kumamoto and moves slowly northward in a wave of pale pink. But this is not Kumamoto’s only claim to fame. In 1872, a local boy named Kitasato Shibasaburō arrived in Kumamoto from his village in the countryside to study at the city’s medical school. From his unlikely beginnings in a rural community, a feudal land of forests, hot springs, and volcanoes, Kitasato would become the cofounder of the science of immunology. After graduation from Kumamoto Medical School, Kitasato embarked on a research career, and in 1886, on the recommendation of the chief of the Public Health Bureau, he traveled to the West to join the world’s leading bacteriology laboratory, led by Robert Koch at the University of Berlin. Kitasato was soon put to work with a Prussian researcher, Emil von Behring, another unlikely recruit to medical research. One of 12 children from a modest household in Hansdorf, now in Poland, Behring was planning to become a schoolteacher or a parish priest when he learned that he could get a free medical education by serving in the Prussian Army. During his early career as a military surgeon, he developed an interest in disinfectants as internal treatments for wound infections. The problem was that

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disinfectants were as toxic to human tissue as they were to bacteria, and this prompted him to dream of something much more specific—­something entirely harmless to the patient but lethal to the germ. In 1889 he joined the Institute for Hygiene at the University of Berlin to become Robert Koch’s full-­time assistant. At this time, diphtheria, a bacterial infection that can lead to difficulty breathing and heart failure, was a major killer of young children. The reason for the infection’s deadly effect had just been discovered by French scientists. Instead of the bacteria causing the sickness, it was the toxin the bacteria released—­a deadly poison that attacked the tissues of the throat and heart. Behring and Kitasato decided to investigate how guinea pigs reacted to this dangerous toxin. Earlier research had shown that iodine could render the toxin harmless, so Behring and Kitasato injected guinea pigs with toxin disabled in this way and later examined the guinea pigs’ blood. They found a mysterious substance in the serum, produced in response to the injection. When added to an unaltered toxin, the invisible agent neutralized the deadly poison. They established the same result with tetanus toxin. This was a momentous discovery. The serum contained nothing less than the pathogen-­specific weapon Behring had dreamed about. In his own words: “Kitasato and I reported experiments which showed that the immunity to tetanus of experimental animals resides in the ability of the blood to render harmless the toxic products of the tetanus bacillus. The same mechanism was advanced for diphtheria immunity.” They named this miraculous substance antitoxin. Each antitoxin appeared to inactivate only the toxin used to produce it. Antitoxin B failed to combat toxin A, while antitoxin A had no effect on toxin B. Mixed with the “wrong” antitoxin, the bacterial poisons retained all their deadly potency. Antitoxins could not only tell the difference between germs and normal tissues, they could discriminate between toxins. This, one of the greatest

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moments in the history of biomedicine, was the discovery of what would later be called an antibody—­the protein actively produced by the body to neutralize, eliminate, or kill invading germs. Kitasato was destined to return to his native land, but Behring went on to use antibodies to save the lives of children with diphtheria. In 1901 he was awarded the first ever Nobel Prize in Physiology or Medicine for having “placed in the hands of the physician a victorious weapon against illness and deaths.” In line with the new picture of immunity, the fragments of germs (pathogens) stimulating the production of antibodies soon became known as antigens. But how did antibodies work, and how did the body produce them? For the first 50 years of the 20th century, chemists held the reins in immunology and came up with the suggestion that the shapes of bacteria become impressed on antibodies. These “instructive” theories held that the immune system learns the shapes of pathogens in order to fight them. In 1930, Felix Haurowitz and Friedrich Breinl, working in Prague, proposed that antibodies were newly made on the surface of the pathogen, and this gave them the right shape. Ten years later, the chemist Linus Pauling suggested more simply that the antibody folded into the right shape on the foreign structure. One year later the Australian Frank Macfarlane Burnet refined the theory and, taking account of the fact that these events might take place on cells, proposed that these cells then grew to produce numerous offspring, all making antibodies of the same shape or recognition specificity. An insurmountable problem for these instructive theories came with Watson and Crick’s discovery of DNA in 1953: all information for protein shape is encoded in the genes and so cannot be learned from subsequent encounters with microbes. The alternative was hard to believe: to cover the possibilities, the immune system must premake all the shapes for everything it might encounter in the universe of microbes. Neils Jerne, a Dan-

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ish immunologist, suggested that the body already knew how to make all possible antibody shapes and simply had to select which shapes to produce in large quantities when invading pathogens appeared. Where and how this kind of adaptation to infection took place was then tackled by Burnet. Burnet was a virologist by training, but he would eventually become a principal contributor to our understanding of how the immune system learns and remembers infections. The son of Scottish immigrants, he grew up in rural Australia in the bushlands of the coastal plain, due west of Melbourne. Here, as a boy in the small town of Traralgon, he enjoyed the wildlife of the creeks and open forest and led a rather solitary childhood. But his early experiences didn’t prevent him from becoming one of the most highly decorated and honored scientists in Australian history. Burnet proposed, in the late 1950s, that receptors (locks) fitting all shapes (keys) in the universe of microbes are present on immune cells but that each cell has only one receptor shape. An invading pathogen will “unlock” a cell with a receptor of the corresponding shape. This unlocking spurs the rapid multiplication of that cell, the progeny of which produce antibodies of the same shape. Think of the receptors on the individual immune cells as a cell-­bound version of an antibody. Antibodies of precisely the same shape or “specificity” as the parent receptor are released by the daughters of that particular cell into bodily fluids. The antibodies then neutralize the pathogens matching the type that unlocked the parent cell. This theory of immunity, which Burnet dubbed “clonal selection,” would later be refined but has essentially stood the test of time. At this moment in history, the precise chemical structure of antibodies remained a tantalizing mystery. Just how did antibodies seize hold of pathogens and neutralize them? At one level, antibodies had been defined in quite simple terms, but the picture remained very limited.

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The first breakthrough in understanding depended, rather surprisingly, on a small fruit tree native to South America. Because of its sweet and creamy fruit, the papaya tree has been cultivated in tropical regions all around the globe, but the indigenous peoples of South America also use the unripe fruit as a tenderizer for red meat in traditional cooking. The secret is a plant enzyme, papain, which snips through proteins, breaking them down into smaller portions. The English scientist Rodney Robert Porter used this enzyme to great effect, and papain would prove crucial to understanding the shape of antibodies. Porter, the son of a railway clerk, was born in 1917 in Newton-­ le-­Willows, a small market town between Liverpool and Manchester in northwest England. He went to nearby Liverpool University, where he studied biochemistry, graduating in 1939 and registering to study for a PhD. The war effort interrupted this path when he joined the Royal Army Service Corps. Porter couldn’t resume his studies until 1946, when he managed to win a place at Cambridge. His supervisor was the biochemist Frederick Sanger, one of only four people to win the Nobel Prize twice. When Porter joined his laboratory, Sanger was busy pioneering the sequencing of important proteins, with insulin as his primary target. But the young Porter was interested in a very different molecule. A new book had just arrived in England—­The Specificity of Serological Reactions, by Karl Landsteiner (discoverer of blood groups and polio virus)—­and it was all about antibodies and how they recognize pathogens. In it, a great conundrum confronted Porter. From early studies, the structure of antibodies appeared highly uniform—­and yet they could recognize a vast range of different pathogens and toxins. How was this possible? Studies of horse antibodies used to treat diphtheria (and others to treat tetanus) had already yielded clues. It was clear that the whole antibody molecule was not needed to recognize pathogens. Armed with this knowledge, Porter became keen to isolate the

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small, “important” bit that made the difference. The only thing he could find to split an antibody without destroying its recognition function was papain (already used by others to study protein structure). Porter’s work was interrupted for a while, but seven years later he found himself at the National Institute of Medical Research in London, and by this time much purer preparations of papain had become available. He soon found that pure papain consistently split the antibody molecules into three parts. Two of these portions could still bind the antigen, and it gradually became clear that these two portions were identical. The third portion did not bind the antigen at all, and to Porter’s great surprise, this fragment could also form crystals, transforming in the cold into diamond-­ shaped plates. These findings were a great step forward, but it would take the work of an American scientist, Gerald Edelman, to put the next pieces of the puzzle in place. The prize would be a complete picture of the antibody molecule. Gerald Edelman was born in 1929 in New York City and studied medicine at the University of Pennsylvania School of Medicine. In 1957, after service in the US Army Medical Corps, he joined the Rockefeller Institute under the guidance of Henry Kunkel. Kunkel was already well known for discovering that the antibodies made in large amounts by patients with cancerous immune cells (myeloma proteins) are just the same as normal antibodies and therefore provide a ready supply of material for study. Edelman, armed with these human myeloma proteins, began by assuming that antibodies, like most biologically active proteins, might be composed of proteins held together by cross-­links of some kind. Breaking these links using chemicals, he found that antibodies are composed of four chains. When he tested them individually, none of these chains retained the ability to recognize antigens. This was again a major breakthrough—­the antibody binding site was composed of different chains acting together.

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Edelman then used various molecular sizing techniques to show that antibodies are composed of two pairs of chains, one short and the other long. He sensibly named the short pair “light” and the long pair “heavy,” publishing his findings in the Journal of the American Chemical Society in June 1959. Imagine Porter’s excitement when he learned about the four-­ chain composition of human antibodies. Now he simply needed to put this structure together with his own three-­part model and a picture of an antibody would emerge. The experiments, as he noted in his Nobel Lecture of 1972, were now “easy.” By this time he was working at St. Mary’s Hospital in London. There, aided by his assistant Elizabeth Press, he repeated Edelman’s treatment. By putting this information together with his own picture of papain products, he created a four-­chain model that explained all the findings. So, what did antibodies look like? The answer: antibodies are pleasing Y-­shaped molecules. The arms of the Y consist of the two light chains linked with the upper portion of the two heavy chains. The tail of the Y consists of just the lower portion of the two heavy chains. This structure remains the picture of an antibody that we have today. Edelman meanwhile repeated Porter’s studies and happily arrived at the same conclusions. But if all antibodies have the same overall structure, where does that amazing spectrum of recognition capability reside? The answer lies in the arms of the Y. In the 1970s, studies of the arms would reveal hot spots of variability between different antibodies. And within these variable hot spots are regions of even greater diversity. These come together in the intact molecule to form the highly specific pockets in which antigens fit—­again, like keys in a lock. This gradually emerging picture was a source of great satisfaction to both Porter and Edelman. In 1972, the two scientists jointly received the Nobel Prize in Physiology or Medicine for their achievements.

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Though scientists now understood the structure of antibod-

ies, they hadn’t yet discovered exactly how the body creates these virus-­fighting proteins. As described earlier, when immune cells with receptors of the right shape meet antigens (from viruses or other pathogens) of the matching shape, the cells unlock, increase their numbers, and produce antibodies of the same shape, which then neutralize the antigens. But this description glosses over a few important parts of the process. An early clue that antibodies weren’t the whole picture came in the aftermath of the Second World War. At this time, hospitals struggled to cope with combatants’ burns, and skin grafting became a vital, cutting-­edge procedure. The British scientist Peter Medawar showed that the human body frequently treated skin grafts as invaders and rejected them. He noted that the inflammation surrounding rejected grafts contained many lymphocytes, a particular type of immune cell. In 1957, he began suggesting that cellular immunity, mediated by a particular type of lymphocyte acting directly, cell-­to-­cell, against foreign tissue, might be important in tissue rejection, rather than the antibodies produced by immune cells. This implied that there were two different types of lymphocyte working through very different mechanisms. Later, the special class of lymphocyte that rejected the skin grafts would be named T-­cells, to distinguish them from B-­cells, lymphocytes that produce antibodies. At the same time, James Gowans, an Oxford researcher, was studying the behavior of lymphocytes, showing how these cells endlessly circulate between the vessels and nodes of the lymphatic system and the blood. It was to be the dawn of a new age—­an understanding of what we now know as cellular, or cell-­mediated, immunity. In the early 1970s, as studies moved from animals into the test tube, several investigators found that pure lymphocyte populations could not respond to antigens. The body also needed a third type of immune cell to produce an immune response—­a sticky

IMMUNE CELL TASKS

Cell

Task 1

Task 2

Task 3

Surveillance cell

Patrol the body consuming pathogens (such as bacteria or viruses).

Detect danger signals (molecular patterns associated with pathogens). Move fragments of digested pathogens to cell surface. Release cytokines (signal proteins) to attract T‑cells.

Present pathogen fragments to T‑cells (first signal). Transmit second signal to T‑cells to activate immune response.

Helper T‑cell

Migrate between lymph nodes, blood, and sites of infection. Respond to cytokines from surveillance cells.

Recognize pathogen fragments presented by surveillance cells. Detect activation signal and rapidly multiply.

Release cytokines to attract other T‑ and B‑cells. Activate B‑cells.

Killer T‑cell

Migrate between lymph nodes, blood, and sites of infection. Respond to cytokines from surveillance cells.

Recognize pathogen fragments presented by surveillance cells. Detect activation signal and rapidly multiply in response.

Identify matching pathogen fragments on the surfaces of infected tissue cells. Kill infected cells.

B‑cell

Recognize invading pathogens and digest them. Present pathogen fragments to helper T‑cells.

Receive activating signal from helper T‑cells and rapidly multiply in response.

Produce daughter cells, which release antibodies.

Regulatory T‑cell

Recognize pathogen fragments presented by surveillance cells and B‑cells.

Produce cytokines to orchestrate and regulate the immune response.

Stop the immune response after perceived success defeating the pathogen.

Memory B‑cell

Persist long-­term, ready to respond to previously encountered pathogens.

Ingest pathogens and present pathogen fragments to corresponding memory T‑cells.

Produce daughter cells, which release antibodies in an amplified secondary immune response.

Memory T‑cell

Persist long-­term, ready to respond to previously encountered pathogens.

Recognize pathogen fragments presented by corresponding memory B-­cells.

Detect activation signal and rapidly multiply to mount a potent secondary response.

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cell they found clinging to the glass surfaces of test tubes and petri dishes. Gradually, scientists discovered the way this cell works. Such cells are sticky because they are forever trying to consume things. Their job is to patrol the body as the first line of immune defense, an ancient system appearing in evolution long before the adaptive immune system. These surveillance cells quickly engulf and digest any invading pathogens. They then transport pathogen fragments to their surface and at the same time release signaling molecules called cytokines to attract T-­cells. A key molecule mobilizing immune defenses at this stage is a cytokine called interferon, and some pathogens seek to evade immunity by dampening its production. When the T-­cells arrive, they inspect the surface of the surveillance cell, looking for antigens in the form of fragments of pathogens. Those T-­cells whose receptors fit the viral fragments then begin to divide, growing quickly and greatly increasing their numbers. This growing army of lymphocytes, all with the right-­shaped receptors to recognize the virus, produce a range of daughter cells. Some (helper T-­cells) collaborate with B-­cells, promoting their rapid growth and thereby greatly amplifying the production of antibodies. Still others (killer T-­cells) recognize virus-­infected cells and kill them directly. The fourth class of lymphocyte (regulatory T-­cells) produces chemical messengers that orchestrate the immune response and close down the operation once it’s been successful. Crucially, when it is all over, antibodies remain to fight off any subsequent encounter with the virus. It’s important to remember a crucial difference between how antibodies function and how cell-­mediated immunity works. Antibodies can bind to viruses directly (in the same sort of way as the spike protein of coronavirus binds to ACE2). This means they can recognize the native shapes of viruses. But in cell-­mediated immunity, T-­cells see only small fragments of viruses—­remnants of the viruses that were digested by surveillance cells. But T-­cells

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can then identify the same tell-­tale fragments on the surfaces of virus-­infected cells, after which they kill the host cell and, consequently, the virus. But how do T-­cells know what to look for, and what exactly do they see? The answer would be key to understanding the skin graft rejections that began this whole area of research. Scientists took steps toward answering this question at the John Curtin School of Medicine in Canberra, Australia, in 1973, when a young Australian scientist, Peter Doherty, was joined by a junior visitor from Switzerland, Rolf Zinkernagel. Pursuing their mutual interests, the two immunologists began studying a virus that causes meningitis in various inbred strains of mice. Such mouse strains differ in the genes controlling immune responses in a highly consistent way. In the test tube, T-­cells reliably killed virus-­ infected mouse cells, but how did the T-­cells recognize the virus? They soon observed that virus-­infected cells were killed only if the infected cells and the killer T-­cells were from the same strain of genetically identical mice and therefore shared the same genes. Although specific for the virus, T-­cells could not, for some reason, see the virus on a “foreign” cell whose genes encoded subtly different cell-­surface molecules. This suggested the T-­cell might be seeing not just the virus but the virus plus a normal healthy molecule on the target cell. If this healthy molecule was different in each inbred strain of mouse, then that would explain the findings. Gradually it dawned on them that the T-­cells were indeed identifying not a virus alone but a molecular complex of the virus plus a specialized molecule on the target cell. The beauty of this discovery lay in the fact that these specialized molecules were already well known to science. They were the so-­called transplantation antigens, different in each mouse strain. And these are the cell-­surface molecules that cause skin grafts to be rejected. Transplantation antigens are special because this is where the immune system looks for foreign invaders. The job of these cell-­surface molecules, which are different in every individual, is

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to provide a molecular “window” routinely inspected by T-­cells looking for pathogens. Doherty and Zinkernagel published their findings in the journal Nature in 1974. In their own words, T-­cells recognize “a little bit of transplantation antigen, and a little bit of virus.” We now call transplantation antigens MHC molecules (standing for “major histocompatibility complex”), and they are structures of some beauty, pleasingly equipped with a cleft or groove in which the pathogen fragment sits, ready for inspection by T-­cell sensors. In other words, the pathogen fragment is like a hot dog and the MHC molecule is the bun. Although the structure of the T-­cell receptor was not known in the 1970s, the great mystery of T-­cell recognition (and the biological reason behind surgical graft rejection) had been solved in the suburbs of Canberra, Australia. Twenty-­two years later, in 1996, Zinkernagel and Doherty were awarded the Nobel Prize in Physiology or Medicine for their discovery. In the early spring of 1973, fully recovered from the zoonotic

virus infection, a PhD in my pocket, and newly wed, I arrived in the United States at the National Institute of Allergy and Infectious Diseases, on the outskirts of Washington, DC. I was about to join a select group of young US talent in biomedical research who had been excused from service in the Vietnam War and, though not in fact afraid of the draft, had dubbed themselves the Yellow Berets. Initially, the idea of joining these scientists was daunting, but I felt blessed with a new self-­confidence acquired, somehow, mid-­ Atlantic. Stateside I felt brash, warm, eager to engage. My host was Charles Kirkpatrick, an immunologist who stood astride the academic and clinical arenas. My sponsor was Alan Rosenthal, a man right in the middle of a seminal discovery. The clinical head of the whole thing was Sheldon Wolff. Among the charming, fresh-­faced company were Charles Dinarello and Anthony “Tony” Fauci. Dinarello, with his mentor Sheldon Wolff, would go on to discover

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the prime mediator of clinical fever. Fauci would take the helm of the institute, guiding it through major storms and remaining in the thick of it for 36 years and counting. The Vietnam War had ended, Watergate had yet to kick off, and Washington was endlessly exciting for my wide-­eyed wife and me. President Nixon had visited China with the outstretched hand of friendship and returned home with two giant pandas. We went to see them at the National Zoo. Ling-­Ling, the female, and Hsing-­Hsing, her mate, seemed to have every comfort they could wish for. Later we visited the Lincoln Memorial and stood alone in awe, listening to the silence, bathed in marble light, striking the pose of statues, one foot set forward, gazing up, striving to do justice to the seated giant and the famous words. A short bus ride later we touched a fragment of the surface of the moon and posed enthusiastically (behind a cardboard cutout) as astronauts on the Apollo mission. Soon, around the Tidal Basin, the cherry trees (a gift from Japan in 1912) would blossom, filling the air with delicate petals touched with pink, softening the marble of the Jefferson Memorial, setting it adrift on waves of falling flowers. Alan Rosenthal was working with his colleague Ethan Shevach on two strains of guinea pigs with small differences in the genes encoding their respective MHC molecules. These animals had been purposely inbred to subtly differentiate their genetic profiles. Rosenthal and Shevach used the surveillance cells of one strain to present antigens to the T-­cells of the same strain and also to the slightly mismatched strain. What they found was thoroughly intriguing: it was only when the presenting and responding cells were from the same guinea pig strain that the T-­cells were able to respond and proliferate. T-­cells could not “see” antigens presented on the “wrong” surveillance cells. This strongly suggested that MHC molecules on the surface of cells were key in the intimate cellular interaction during which T-­cells “saw” antigens. They could also prevent the response to the “right” antigen-­

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presenting cells using antibodies that masked the MHC molecules. MHC from the wrong guinea pig strain was too mismatched to present antigens but not so greatly mismatched as to provoke a graft-­rejection type of response. These findings mirrored those of Doherty and Zinkernagel in a rather different experimental system. And they foretold the truth about the nature of T-­cell recognition. Whether they were killing virus-­infected cells or being switched on in the first stages of an immune response, T-­cells saw antigens not on their own but lodged in the framing window of MHC molecules. But where do T-­cells come from? Cell-­mediated immunity depends on the thymus, a small pinkish gray organ situated just behind the sternum, in between the lungs. This seemingly insignificant gland is active only in children. By the time children reach age 15, the thymus has discharged its huge responsibilities. Slowly it starts to shrink—­its complex architecture gradually yielding to fat. Once adults reach 70 years of age, its once vital structure has been completely obliterated. But during early life this small bi-­ lobed organ is packed with lymphocytes and is responsible for generating a T-­cell army with a full repertoire of functions that will last a lifetime. Between 1964 and 2000, one immunologist in particular was ever present at the forefront of the effort to understand where T-­cells come from. His name was Jacques Miller. Miller was born in France in 1931 as Jacques Meunier, but his family soon moved to China, where his father was a manager at the Franco-­Chinese Bank in Shanghai. When the Japanese invaded part of the city, Jacques’s father worked undercover for the resistance movement. As the situation deteriorated, British authorities in an adjacent zone hastily arranged a passport for M. Meunier under the name of Miller, and he set about organizing his family’s escape. In 1943, they just managed to board the last cargo ship out of Shanghai, arriving, at last, in Sydney, Australia.

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In their newly adopted country, Jacques began his school studies at St. Aloysius’ College in Sydney, while his younger sister attended the convent school nearby. After failing his first exams (he was still learning English), Jacques’s school career began its upward path. He was determined to study medicine—­determined to understand why his older sister had died from tuberculosis. He won a place at the University of Sydney and eventually gained a fellowship to visit England. In London, he was sent to work at an outpost of the Chester Beatty Institute in rural Buckinghamshire, where he examined a kind of leukemia in mice caused by a virus. The leukemia began in the thymus. In order to dissect the process, young Miller removed the thymus in newborn mice to see if this prevented disease. What he saw was unrelated to leukemia. Without a thymus, the mice could not develop immune functions. Most strikingly, they could not reject foreign skin grafts. The astonishing implication was that, far from being a vestigial relic, the thymus is essential for the development of lymphocytes—­ the cells shown by Peter Medawar to be responsible for the rejection of skin grafts. This was a hugely controversial claim. Nevertheless, in 1961, the junior researcher managed to publish his observations in the British journal The Lancet. In 1965, armed with a PhD, Miller returned to Australia at the invitation of the distinguished Australian immunologist Gus Nossal, the new director of the Walter and Eliza Hall Institute in Melbourne. And it was here, with his first PhD student, Graham Mitchell, that he made a breakthrough that changed the course of immunology. (At this time, many other immunologists around the globe were on the point of reaching the same conclusions.) By injecting genetically marked lymphocytes from the thymus together with genetically marked lymphocytes taken from the bone marrow, Miller and Mitchell were able to restore the full range of immune responses—­antibody and cell-­mediated func-

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tions—­in mice without a thymus. Both thymic and bone marrow cells were essential for this restoration. In this way Miller was the last person in history to discover the function of a major organ. His pioneering work showed that the thymus populates the body with T-­cells, while B-­cells are produced in the bone marrow. But the extraordinary details of just how the thymus works emerged only gradually in the years ahead. First, we must look at the amazing work of a Japanese scientist. Li k e K ita sato, t h e cofounder of immunology, Susumu

Tonegawa was born in the countryside of southern Japan. Later he was sent to school in Tokyo, and there he won a place at the Imperial Kyoto University on his second attempt (he failed chemistry the first time round). The year was 1959 and, because the postwar defense treaty between Japan and the United States was about to expire, the university was a hotbed of debate about the future. This convinced him to remain an academic, and with the help of his professors, he succeeded in joining a graduate program in molecular biology at the University of San Diego in Southern California, eventually gaining his PhD. In 1971, with his US visa running out, Tonegawa migrated to the Basel Institute in Switzerland, a lab full of young immunologists. Here the fledgling molecular biologist had little choice but to take an interest in the immune system. His progress was extraordinary—­between 1974 and 1981, he succeeded in resolving the long debate about how the immune system creates all the shapes it needs to fit the universe of pathogens. He began by looking at immune cells from mice (which happily are very similar to human immune cells). The first thing he noticed was that the genes coding for antibodies in mouse embryos were farther apart from one another than on the chromosomes of adult mice. Somehow, in the time taken for embryonic cells to mature into antibody-­producing cells, the genes had

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been rearranged. A central tenet of the genetic code is the faithful transmission of characteristics unchanged over many generations. This means that DNA normally resists all change. But not in the developing immune system. Investigating the genetic material in greater detail, Tonegawa found that rather than existing as whole genes, the genes coding for antibodies are made up of strings of units, like beads on a thread. As each immune cell is created during embryonic growth, these units are randomly shuffled—­a sort of intensive cut and paste—­to eventually form the mature antibody gene. This random shuffling generates a huge number of possible combinations from a relatively small number of genes—­like the large number of possible sequences in a shuffled pack of cards (you can arrange 10 playing cards in more than three and a half million ways). New bits of DNA are also added randomly, and these genetic recombinations produce many millions of new receptor components. Still more diversity is generated when the microbe-­binding bits of antibody molecules (antibody receptors) are randomly assembled from different component parts. Some estimates suggest these processes generate more than 10 billion (1010) configurations. In other words, DNA mutations and recombinations, occurring during fetal development, generate the huge diversity of antibodies. With these remarkable discoveries, Tonegawa opened a window to an elegant and beautiful solution to the problem of countering infectious disease. His discovery gives us the opportunity to marvel at the evolution of immunity. Tonegawa likened the process to the commercial production of cars. An automobile company produces different models for different customers’ tastes not by creating new pieces for each model but by taking the same parts and assembling them in different configurations. For this achievement Tonegawa became, in 1987, the first Japa-

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nese scientist to receive the Nobel Prize for Physiology or Medicine (though perhaps Kitasato ought to have been honored in this way 80 years before). We can now return to Jacques Miller and the role of the thymus.

This organ populates the body with T-­cells, but is there some kind of filter for that huge array of specificities that randomly shuffled genes produce? Remember that the starting population of immature T-­cells in the fetal thymus possesses a vast range of randomly generated receptors which may or may not bind to pathogens, and which may or may not recognize MHC molecules. What happens is this. During early development, the vast, new random family of receptors in the thymus is screened to weed out those that recognize the self—­the natural healthy profiles of the body. In other words, immune cells that could attack the normal tissues and organs of the body are eliminated at this early stage. Among these are cells that bind too readily to MHC molecules. Cells that bind too weakly to MHC molecules are also useless, and these too are eliminated. It’s a case of the Goldilocks principle. Some developing T-­cells bind too strongly to MHC. Others bind too weakly. Only the ones in between are “just right.” And these are the only cells that survive the rigorous thymic selection process in the developing fetus. All the rest are killed by a mechanism called programmed cell death. The result is a tailor-­made population of mature T-­cells ready to recognize pathogens (in fragmented form) when they are bound in the groove of MHC molecules on the surface of antigen-­presenting surveillance cells. I have long been fascinated by this crucial collaborative interaction between two very different cells that triggers the immune response. And the most exciting phase of my career was devoted to this subject. Sometime in the late summer of 1988, at the Wellcome Research Laboratories in southeast London, I was studying

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the interactions between antigen-­presenting cells and T-­cells in tissue culture, gently modifying the surfaces of both cells with subtle chemical treatments and measuring immune responses (the proliferation of T-­cells). My assistant, Barbara Pearce, was extremely good at these meticulous, timed manipulations. It was already well established that one very powerful way to stimulate T-­cells was to artificially create chemical bonds bridging surveillance cells and T-­cells. This caused all T-­cells to proliferate (in the absence of an antigen). But what our studies now showed was that the formation of such chemical bonds between molecules on surveillance cell and T-­cell surfaces was a natural and essential process in the response of T-­cells to a foreign antigen. These reversible bonds, which connect nitrogen to carbon, are called Schiff bases. Over the next three years, we were able to publish four more major studies consolidating this finding, and we also began to look at ways to exploit this natural phenomenon. We succeeded in producing an experimental injectable drug which greatly amplifies immune responses to a range of vaccines in mice. We published these findings in Science in June 1992. I hoped this experimental substance would be developed and commercialized as a new product to enhance immune responses. A rou n d this point, in the mid-­1980s, scientists generally

agreed that T-­cells saw antigens bound to MHC molecules, and this was the primary signal initiating an immune response. But something more was needed. Additional crucial signals were required to work together with the primary signal to switch the T-­cell on. This has been characterized as the second signal or the costimulatory signal, and it brings us to the last piece of the immune system puzzle: danger signals. During the last decades of the 20th century, the techniques of genetic engineering arrived, and it became easy to produce

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viral and bacterial proteins in the test tube. Researchers planned to make suitable combinations to produce synthetic (or subunit) vaccines, with all the right fragments to promote antibody production but without the risks of live infection or killed microbes. However, attempts to provoke immune responses with these purified proteins were doomed to failure. Immunologists had long known of the need for what they call adjuvants (from the Latin adjuvare, which means “to help”). These include chemical agents and poorly defined mixes of microbial components—­discovered by chance and working mysteriously—­which pivotally enhance the immune response. Adjuvants have always been used by researchers to help provoke immune responses, but they were rarely mentioned outside the “recipe” section of research reports. In 1989, a much-­admired researcher, Charles Janeway of Yale University, reminded his fellow immunologists of these “dirty little secrets” and suggested that in addition to foreignness, a good immune response needs some indication that the foreign thing is truly dangerous. He was right. The foreign pathogen fragment bound in the MHC molecule provides the primary signal to trigger the immune response, but something else is also needed. A crucial second signal is required, or the immune system does not respond. Adjuvants work because they contribute to, and amplify, this natural second signal. Like the primary signal, this second signal also originates from surveillance cells. Surveillance cells can sense the general features of dangerous pathogens—­molecular patterns widely present in pathogens but not found in human cells. These “flags” prompt the surveillance cell to send the all-­important second signal to T-­cells. In the years that followed, Janeway and other researchers characterized these pathogen-­associated molecular patterns. They also discovered the families of receptors on surveillance cells whose job is to detect them. The ancient, innate immune system

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and the “intelligent” adaptive immune system, which recognizes and remembers pathogens, work hand in hand. This knowledge remains crucial in the design of vaccines. The more danger signals delivered by a vaccine, the more effective the vaccine will be.

CHAPTER THREE

Discover a Vaccine On August 9, 1721, the first well-­documented trial of deliberate immunization to prevent infectious disease began in London. This was not vaccination as we’ve come to know it. But the basic principles were just the same as in modern vaccination—­using a pathogen in a weakened form to protect against a virulent infection. The sample size was small; just six participating subjects assembled to receive the inoculation. But somebody has to go first. The team of observers was large and illustrious. At least 25 members of the College of Physicians and assorted fellows of the Royal Society, the leaders of the capital’s medical and scientific establishment, were in attendance, privileged to witness the moment in history. As the first subject steadied himself, preparing to submit to the surgeon’s knife, the audience leaned forward in attentive silence. It has now become customary on these occasions to celebrate the prime volunteer in the first trial of any new vaccine. Such was the case on April 26, 1954, when Randy Kerr, a pupil at Franklin Sherman Elementary School in McLean, Virginia, was photographed bravely and cheerfully receiving the first shot of Jonas Salk’s polio vaccine. Thus it was a six-­year-­old schoolboy who launched the inoculation that would end infantile paralysis in the developed world. London met the 1721 launch with an equivalent Georgian fanfare. The event was widely reported in the press, and a host of celebrities endorsed the proceedings, but what were the chances of this vaccine protecting the volunteers and saving their lives, at

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least for a time? Strangely, the odds were better than they would ever be again, and to go unvaccinated would mean certain death. The names of the participants did not pass into history. Each one of them was a keen volunteer. And each one of them was a convicted prisoner in Newgate Prison, condemned to hang on the gallows. They knew that by agreeing to participate they would earn their freedom. This historic vaccine trial—­the Royal Experiment—­followed the death of the grandchild of King George I from smallpox. The clearest account we have of the proceedings is by Sir Hans Sloane, the Royal Physician: “Caroline . . . then princess of Wales, to secure her other children, and for the common good, begged the lives of six condemned prisoners who had not had the smallpox in order to try the experiment of inoculation upon them.” Scottish surgeon Charles Maitland conducted the experiment. For five prisoners, he made small incisions on the right arm and leg of each subject and inserted material taken from the characteristic skin rash of a smallpox patient. For the sixth, Maitland administered the preparation through her nose. Three days later, he examined the inoculation sites and decided they were not as inflamed as he’d hoped. Anxious to succeed, he obtained fresh smallpox material and repeated the insertion. All six recovered quickly, received pardon from the king, and walked free from Newgate Prison on September 6. At least one of them was later exposed to smallpox and shown to be immune. According to Hans Sloane’s account: “Dr. Steigertahl, physician to the late king, and I joined our purses to pay one of those, who had it by inoculation at Newgate [a girl of 19], who was sent to Hertford, where the disease in the natural way was epidemical and very mortal and where this person nursed, and lay in bed with one, who had it [a 10-­year-­old boy], without receiving any new infection.” This was the practice of variolation (variola is another name

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for smallpox, from the Latin varius, meaning “spotted”), the forerunner to vaccination. Chinese, Turkish, and African practitioners had used variolation for centuries before aristocrat Lady Mary Wortley Montague brought the practice to England. The customary variolation practice involved inserting a small amount of dried matter from the rash of a patient with a mild case of smallpox into the skin of a healthy recipient. This typically produced a localized infection and a minor sickness, followed by good recovery and many years of full protection. In the same year as the Royal Experiment in London, the practice of variolation was introduced in America. During a serious outbreak of smallpox in Boston in 1721, the Puritan minister Cotton Mather wrote to all the doctors of the town appealing for a trial of the practice of variolation. Only one, Dr. Zabdiel Boylston, responded. Boylston, a highly respected practitioner, tried the procedure on his only son and two slaves: a man and a boy. All three recovered in about a week. Boylston went on to inoculate 242 people during the Boston epidemic. But how did Mather know about the practice? In 1706 Mather had purchased a slave newly transported from Africa. He named him Onesimus (after a biblical slave) and questioned him about his origins. Among the traditional customs that Onesimus confided was variolation, widely practiced in his homeland to prevent smallpox. In this way, the ancient wisdom of deliberate immunization to prevent disease was carried to the New World by a man whose name we do not know. In the seven years following Princess Caroline’s experiment, 897 variolations were recorded in Britain, America, and Hanover, and 17 of the recipients—­2 percent—­died of the resulting disease. This means that anyone contemplating variolation had to face a 1 in 50 chance of death. There was another problem with this strategy. Any one in close proximity to the variolated person during the period of their mild illness was in danger of infection by the

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natural route (usually through inhalation) and of suffering serious disease. As time went on, this risk gradually declined, through refinements in technique, to around a 1 in 1,000 chance of death. When we consider the value of variolation from our modern standpoint, it’s hard to grasp the realities of life in the 18th century. For modern vaccines given to healthy people, our concerns focus on extremely small risks. Live polio vaccine, for example, has an average chance of causing paralysis of approximately 1 in 2.7 million administrations. But throughout the 1700s, smallpox killed an estimated 400,000 Europeans every year. The mortality rate for smallpox was extremely high: 1 to 3 patients in every 10 died of the disease. Disfigurement was the expected outcome in survivors, and smallpox blinded many of its victims. In 1729, since the majority of children were likely to get smallpox, variolation made very good sense. Descriptions of individual variolations are rare, but one late 18th-­century account survives in the writings of the Reverend Thomas Dudley Fosbroke of Gloucestershire: “The boy [an orphan] was bled to ascertain whether his blood was fine; was purged repeatedly, till he became emaciated and feeble. . . . After this barbarism of human-­veterinary practice he was removed to one of the inoculation stables and haltered up with others. . . . By good fortune [he] escaped with a mild exhibition of the disease.” We also know the identity of this particular orphan. His name was Edward Jenner, and he would become famous for his own contribution to the development of vaccines. As a country doctor in the small town of Berkeley, Glouces-

tershire, Jenner knew all about variolation, and he practiced the refined version on the patients in his care. But he also knew of the local belief that milkmaids who suffered cowpox infection (a mild disorder causing a localized rash) were protected from smallpox.

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Other medical men had also taken an interest in the local folklore. In 1765 Jenner’s great friend and colleague, John Fewster, wrote a paper for the London Medical Society entitled “Cow Pox and Its Ability to Prevent Smallpox,” and he discussed the subject with Jenner at meetings of their local medical society. But, for whatever reason, no one pursued the proposition any further. But at last on May 14, 1796, Jenner performed his now well-­ known experiment. Many paintings and engravings commemorate the famous scene. Jenner sits upright, supporting the extended arm of James Phipps, age 8, the son of his gardener. Applying his needle to the upper surface of James’s arm, he concentrates on the shallow delivery of the vaccine. Behind Jenner stands the milkmaid, Sarah Nelmes, holding a cloth to her hand where Jenner has withdrawn the fluid from a cowpox blister. In nine days or so, James will have a slight fever lasting a couple of days. On July 1, some seven weeks later, Jenner variolated his small patient with smallpox material by the procedure he (and every other dutiful practitioner) routinely employed. He then watched anxiously for the result. To Jenner’s great delight, Phipps showed only a very small reaction of the kind seen in subjects already protected against smallpox. In great excitement, Jenner wrote to his friend James Gardner: I have at length accomplish’d what I have been so long waiting for, the passing of the vaccine Virus from one human being to another by the ordinary mode of inoculation. A boy named James Phipps was inoculated on the arm from a pustule on the hand of a young woman who was infected by her Masters Cows. Having never seen the disease but in its casual way before, that is, when communicated from the Cow to the hand of the milker, I was astonish’d at the close resemblance of the Pustules in some of their stages to the variolous Pustules. But

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now listen to the most delightful part of my Story. The boy has since been inoculated for the smallpox which as I ventured to predict produce’d no effect.

The very word vaccine is derived from the Latin word vacca, meaning “cow,” and modern vaccines follow from Edward Jenner’s discovery of the smallpox vaccine. Still I hesitate to give Jenner exclusive title as the originator of vaccination with cowpox. For one, he wasn’t actually the first. Long before Jenner’s historic experiment, a Gloucestershire farmer decided to act on the same folk belief. This man, Benjamin Jesty, resolved to protect his family when a local outbreak threatened his village in 1774. Jesty searched out a suitable cow and, with a darning needle, transferred pustular material from the cow’s udder to the arms of his loved ones. His two sons had mild reactions and quickly recovered, but his wife, Elizabeth, was less fortunate. Her arm became swollen and highly inflamed, and for a time her condition gave cause for grave concern. (She probably had a secondary infection.) Happily, Elizabeth too eventually recovered. But Jesty never tested the success of his intervention and did not document the experience. There is no doubt that Edward Jenner richly deserves his title as the father of vaccination. But I still feel bound to make the point that variolations are in some sense closer to today’s practices. The kinship between cowpox and smallpox is extremely rare, and most modern vaccines consist of weakened, killed, fragmented, or otherwise altered versions of the pathogen they protect against. And this, for me, makes variolation the first (highly imperfect) vaccine. Jenner’s discovery was a radical leap forward. His realization that one mysterious infectious entity, from a different species, could protect against another, quite different disease was an insight without precedent. The leap he took depended on his

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insights about the nature of infection. Without knowing anything about pathogens, he was able to visualize their characteristics, grasping that different pathogens might share certain features. Convincing the world at large to adopt an “unnatural” practice was just as big a challenge, but with the publication in 1798 of his book An Inquiry into the Causes and Effects of the Variolae vaccinae, Jenner would ultimately triumph. And for all his life thereafter Jenner would reign as vaccine advocate-­in-­chief to all nations, or, as he preferred to call himself, “Vaccine Clerk to the world.” Jenner’s smallpox vaccine was a live virus, and although it was administered by an unnatural route, it infected cells and grew in the body, providing natural danger signals and ensuring a strong immune response. A single dose was therefore sufficient to provide protection for many years. With the benefit of hard-­won knowledge (unimaginable for Jenner but detailed in chapter 2), we can now describe what happened when Jenner injected young Phipps. The cowpox virus entered ordinary tissue cells around the injection site and hijacked the normal cellular machinery to reproduce itself. In the course of this, viral fragments ended up in MHC molecules, flagging the cells as infected. At the same time, specialized surveillance cells of the immune system took up the cowpox virus, digested it, and presented fragments, bound to MHC, to the small proportion of immune lymphocytes whose receptors could recognize them. The surveillance cells, stimulated by danger signals on the proliferating virus, also sent the crucial second message: an alarm signal, kicking the virus-­specific T-­cells into action. The T-­cells multiplied; some of their progeny became killer T-­cells, directly eliminating virus-­infected cells. Others recruited B-­cells, which also multiplied and produced antibodies to neutralize the virus. In this way the growth of cowpox was limited to the local site of injection, though James felt feverish because of circulating cytokines—­the chemical messengers regu-

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lating the immune response. Regulatory T-­cells closed down the response when all the cowpox virus was cleared. Crucially, memory T-­cells and antibodies remained long-­ term. When, seven weeks later, Jenner injected James with smallpox, the immune system was already primed to antigens shared by cowpox and smallpox. Existing antibodies and memory T-­cells were ready to attack the virus at once, and the immune system quickly contained the smallpox invasion. In the long-­term, neutralizing antibodies continued to circulate so that James was protected against any subsequent infection with the dread disease. Today’s vaccines must be manufactured to extremely high standards in dedicated facilities for large-­s cale distribution around the globe. But, for Jenner’s smallpox vaccine, standards were very different. One of the means of transport, used over long distances, was quite extraordinary. The vaccine was transferred between humans, arm to arm. Ships’ crews, willing passengers, and orphaned children procured for the purpose and offered a fresh chance in the New World served as links in this vital human chain. Vaccine transfers were made serially and judiciously to conserve fresh material. When a vaccine voyage, commissioned by Charles IV of Spain, arrived in Caracas, Venezuela, just one boy still had fresh pustules to transfer infection to a native recipient. But one boy was enough for vaccination to be launched in the South American continent, nearly three centuries after Spanish adventurers had inadvertently carried smallpox there. Vaccines come in different classes. Jenner’s vaccine represents a highly popular category: the live virus class. The vaccines used to protect children against measles, mumps, and rubella are live virus vaccines. But there are others, detailed in chapter 5, including killed or inactivated vaccines, toxoid vaccines, subunit vaccines, and, more recently, nucleic acid vaccines. In the 19th century, as an understanding of pathogens began to grow, Louis Pasteur introduced vaccines for cholera, anthrax,

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and rabies. All of these were in the whole pathogen category. The cholera bacterium was weakened by repeatedly transferring bacteria among tissue-­culture flasks; the anthrax bacterium, by chemical treatment; and rabies, by storage in rabbit spinal cord tissue (the only way a virus could be stored at this time). Exploiting Pasteur’s principles of weakening and inactivation of pathogens, vaccines against typhoid, shigella, and whooping cough were introduced early in the 20th century. So too were vaccines protecting against diphtheria and tetanus, but these used a different principle. As discussed in chapter 2, because toxins caused these two diseases, the protein toxin (free of bacteria) could be pretreated with antibodies or chemicals to render it harmless. Both procedures neutralized the toxin but, because its shape and native structure remained unchanged, a good immune response could be induced safely. But with nonliving vaccines, several doses are required, and the immune response is nothing like as vigorous as the reaction to a living pathogen. At the heart of every vaccine lies an ingenious trick: convincing the immune system to produce antibodies in the absence of a genuine and dangerous infection; and this is more difficult with a nonliving vaccine. Fortunately, just at the right moment, help for this problem came to hand. In 1926, Alexander Glenny, working at the Wellcome Research Laboratories, was purifying diphtheria toxin. Although he had a passion for order, organization, and tidiness, Glenny also had an eye for happy accidents. He was using aluminum salts to purify the toxin, and when he injected preparations still “contaminated” with this purifying agent, he saw a huge improvement in the antibody response. This material, alum, was the first adjuvant, discussed briefly in chapter 2—­the first enhancing agent added to a vaccine to amplify the immune response. Not only was it the first, it was by far the most successful. It is still widely used today in the vaccines we give to children.

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The story of the two main classes of vaccine—­the live attenuated vaccine and the killed vaccine—­is best illustrated by the discovery of polio vaccines in the middle of the 20th century: the classic vaccine race. In the first half of the 20th century, polio was a hugely feared disease, and there was no shortage of funds to develop a vaccine. The US National Foundation for Infantile Paralysis, later called the March of Dimes, founded by President Roosevelt, himself a victim of the disease, would become the largest voluntary health organization ever seen in America. The year 1952 would prove to be the worst for polio in the whole of US history—­nearly 60,000 children were infected, and by the year’s end more than 20,000 had been paralyzed and 3,000 had died. The fear of polio haunted every summer season. In August 1953, the Cold War between the Soviet Union and the West deepened when the Soviet premier, Georgi Malenkov, announced that the Soviets had succeeded in producing the hydrogen bomb. A nationwide survey reported that the two greatest fears of ordinary Americans were nuclear war and the scourge of polio. We now know that only a tiny proportion of those infected with polio virus have the paralytic disease. The virus targets the digestive tract. Confined to the gut and circulating in the blood, polio virus causes few or no problems. But in around 1 in 1,000 cases, for reasons still not understood, the virus invades the spinal cord, where it damages motor nerves, causing paralysis of muscles in the limbs. When brain stem nerves are also damaged, breathing, swallowing, and bladder and bowel function become compromised. The more invasive the disease, the more likely the patient will die. The world of polio research at this time was dominated by a handful of elite researchers, and with the benefit of the new technique of growing virus in tissue-­culture cells, the experts believed that a live virus vaccine was the only way forward. The problem

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was that no weakened polio viral strain existed, and no one knew how long it would take to develop one. Since 1948, the National Foundation for Infantile Paralysis funded a young researcher named Jonas Salk at the Pittsburgh School of Medicine to investigate how many subtypes of polio virus existed. By 1951 he’d found there were three, and, from his background working on killed influenza vaccines, he was convinced a killed vaccine could work for polio. To this end he grew the three viral types in tissue-­culture cells then killed them with a sure-­ fire chemical treatment—­formaldehyde. This chemical had long been in use for making toxoid vaccines and was also key in killed vaccines against cholera, typhoid, plague, and whooping cough. Because the virus was killed, Salk knew that immunity might require three doses, and he anguished over which adjuvant he should use to strengthen the immune response. Researchers had conducted two initial tests in two institutions close to Pittsburgh. Approval by parents and the authorities charged with the care of these vulnerable children was based on their being most likely to benefit from a successful vaccine. The results in terms of antibody responses were highly encouraging and presiding officials at the National Foundation for Infantile Paralysis made the historic decision to proceed. The trial commenced in the spring of 1954, and the researchers vaccinated half a million children by the end of June (in time for the start of polio season). On April 12, 1955, Thomas Francis, trial director at the University of Michigan’s School of Public Health, was ready to announce the results. Salk’s vaccine was successful: the series of three injections was 80 to 90 percent effective in preventing paralytic polio. The press went wild. The public celebrated. And Jonas Salk became a hero. Meanwhile, his rival Albert Sabin, a leading light in the elite world of polio research, continued to pursue his goal. As early as the winter of 1954, Sabin was testing his live “attenuated” vaccine

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on prisoners at Chillicothe Federal Penitentiary in Ohio, close to his laboratory at Cincinnati Children’s Hospital. The results were highly encouraging. The vaccine was given by mouth; but how was this vaccine made? The answer: by rational empiricism, informally known as “guts and guesses.” Attenuating (weakening) a virus means transferring the dangerous virus many times into fresh cultures of tissue cells, allowing random genetic changes to accumulate. In this way the virus gradually loses its pathogenic potency to become harmless when injected into animals. Before the discovery of genetic manipulation late in the 20th century, researchers developed all live virus (and bacterial) vaccines this way. Bacillus Calmette Guérin (a bovine tuberculosis bacterium isolated in 1908) was transferred in tissue culture every three weeks throughout the First World War before it was introduced as a vaccine against tuberculosis in 1921. In the 1960s, vaccines for measles, mumps, and rubella were produced in long-­term tissue culture using the same empirical methodology. The latter were largely pioneered by the great vaccinologist Maurice Hilleman. In 1961 the Salk vaccine was working well: polio cases dropped below 1,000. But perceptions of its effectiveness were influenced by small resurgences of infection, and some harder to reach populations remained unvaccinated. Would a new vaccine, easily swallowed and without injections, revive the vaccination rate? Sabin lobbied hard. As a result, in September 1961, the Department of Health, Education, and Welfare licensed the Sabin vaccine as the replacement for the Salk killed vaccine. Large-­scale vaccination began in 1962, and polio incidence continued to fall. In 1979, the last indigenous case of polio was recorded in the United States. Today the campaign to eradicate polio remains tantalizingly close to success. What can we learn from the merits of the two polio vaccine approaches? When Salk’s killed vaccine is given in a series of

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three injections into muscle, the immune system’s surveillance cells take it up, digest it, and (using MHC) present the fragments to T-­cells, which then grow in number and prompt the production of antibodies that neutralize polio virus. But with Sabin’s single oral dose, because the virus infects cells and multiplies, both antibody and cell-­mediated immune responses are activated. The result is killer T-­cells, able to eliminate the virus hiding inside cells. And the antibodies that are produced exist in the blood and body fluids but also the lining of the digestive tract. Antibodies and memory T-­cells then remain long-­term to deal with any subsequent encounter with polio. The downside of the Sabin vaccine is that on very rare occasions it causes paralysis in the same way as the natural virus. During the 1970s, a number of major advances changed the way that vaccines are discovered. One of these was the revolutionary new field of recombinant DNA technology (genetic engineering), which allowed scientists to produce synthetic proteins in large amounts for use in vaccines. A natural trait of bacteria is the key tool for creating these vaccines. Unlike human (and animal) cells, in which the nucleus protects the genes, bacterial chromosomes are simply in with all the rest of the cellular machinery. What’s more, some genetic material is not in chromosomes at all but floating freely as small autonomous circles of self-­replicating DNA called plasmids. Bacteria can pass plasmids between them in order to share genes—­for example, the ones to resist antibiotics. Plasmids have become the workhorse of genetic engineering. Also vital are the “scissors” used by genetic engineers. These take the form of what are called “restriction enzymes,” which cut DNA at highly specific sites. In this way a gene can be cut out of a plasmid and an edited piece of DNA, encoding a protein of interest, can be inserted in its place. The engineered plasmid can then be grown in bacteria in the test tube or in large tanks known as fermentation vessels—­a technology that began in the brew-

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ing industry. But how can you be certain that the bacteria you’re growing contain the new plasmid? One ingenious answer is to place a selection marker in the plasmid—­this might be a gene coding for resistance to an antibiotic. Simply by growing the bacteria in the presence of the antibiotic, only those bacteria containing the engineered plasmid can survive and grow. When the protein of interest is a key viral or bacterial antigen, then this technology provides the means to manufacture a nonliving protein vaccine. In 1986 Maurice Hilleman, William Rutter, and Pablo Valenzuela at Chiron, a biotech company in San Francisco, produced the world’s first such genetically engineered or “recombinant protein” vaccine to protect against the chronic viral infection known as hepatitis B. Cutting out sequences of DNA, inserting new sequences, and otherwise editing the genetic material of pathogens also allows genetic engineers to change viruses in a highly targeted, fully defined way. This means that living viruses can be rendered harmless for use as live attenuated vaccines to protect against the disease that the natural or “wild-­type” virus caused. In addition to making proteins and weakened viruses for vaccines, genetic engineering has also led to the development of viral vectors. Vector here simply means carrier (from the Latin vehere, “to carry”). In 1953 scientists isolated a new virus from the tonsils and adenoids of a patient suffering a common cold. They gave this new virus the name adenovirus. Because it could readily multiply in tissue-­culture cells, adenovirus was soon shown to constitute a large family of related strains. In 1960, David Tyrell, the scientist who first characterized human coronaviruses, produced a live attenuated vaccine by transferring a virus many times in pig kidney cells until the virus became innocuous. In 1971 the US Army adopted such a vaccine, given orally in tablet form, to protect recruits. This practice, maintained over several decades, firmly established the safety of adenoviral vaccines in large populations. The advent of genetic engineering allowed scientists to cre-

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ate new versions of adenovirus. These lacked a gene essential for replication and therefore provided safe, nonreplicating viral vectors for delivering new vaccines. Because they can’t multiply in the body, such vectors are entirely harmless, but because they remain as whole pathogens with all components intact, they still infect cells and look dangerous to the immune system—­leading to stronger, more sustained immune responses. But how can you grow a vaccine virus which cannot reproduce itself? Viruses are grown in continuously dividing tissue-­culture cells (such as human embryonic kidney cells), and the missing adenovirus replication gene can be inserted into the genetic material of these incubator cells. In this way, all the molecules necessary for viral replication can be provided to the virus. But, after harvesting, the purified virus, without its own replication gene, is once more unable to multiply. Such viral vectors are similarly important as vehicles for gene therapy, and this has driven much of the research. But when the need arises, viral vectors can quickly be pressed into service. When, for example, the zoonotic virus Ebola threatened the world in 2014, Chinese scientists began working on a vaccine using nonreplicating adenovirus as a vehicle. Into it, they inserted the Ebola gene coding for the all-­important surface spike that the Ebola virus uses to lock on to human cells. Testing soon showed that it worked. It stimulated the production of good levels of neutralizing antibodies in human volunteers, and there were no safety issues. As we will see, this was to bode well when the next zoonotic virus came along. In September 2012, health officials in Saudi Arabia reported an outbreak of severe respiratory illness with fever, cough, and shortness of breath. Working backward, they eventually succeeded in tracing the first known case: a patient in Jordan reported in April 2012. The disease was soon named Middle East Respiratory Syndrome or MERS. All cases of MERS would eventually be linked

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to travel or residence in countries in or near the Arabian Peninsula. The largest known outbreak of MERS outside the peninsula occurred in the Republic of Korea in 2015, carried there by a returning traveler. Studies of the virus soon showed it to be a member of the coronavirus family; and the likely natural host species is a bat. Human MERS infections, however, are likely to have been contracted from camels. Fortunately, this virus requires close contact in order to pass from one person to the next. Its spread appears to be uncommon outside hospitals, so risk to the global population is deemed low. Because of the high rate of mortality in MERS infection (about one-­third of those diagnosed die from the disease), work on vaccines soon commenced in several centers. One of the most promising candidates, tested in Oxford, England, by Sarah Gilbert and her colleagues, exploits a chimpanzee adenoviral vector. Not all adenoviral vectors are equal, and Gilbert selected this one, ChAdOx1, engineered by Adrian Hill and his colleagues at the Jenner Institute, because pre-­existing antibodies to this virus were found to be at low levels in blood samples from the healthy human population. Because adenovirus is a common infection in humans, antibodies against it exist naturally and could prevent the vaccine doing its job—­that is, activating the immune system to create a long-­lasting immune response. It’s therefore important to choose an adenovirus vector that doesn’t face pre-­existing antibodies in vaccine recipients. In 2018, this ChAdOx1 vaccine against MERS was shown to be safe and well tolerated in human volunteers, and vaccination with a single dose produced both neutralizing antibodies to the MERS virus and killer T-­cell–­mediated immunity. Other viral carrier approaches include a vector called vesicular stomatitis virus (VSV). Researchers have used several targeted genetic modifications to weaken this animal virus (one ingenious method knocks out a gene enabling the virus to resist the immune

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response, making it easy for the immune system to control the virus). But this VSV vector differs from the adenovirus vaccines in that it does replicate in the vaccine recipient. Canadian researchers at the Public Health Agency of Canada’s National Microbiology Laboratory initially engineered this vaccine. They removed the native VSV surface protein gene and inserted the key Ebola surface protein gene in its place. In late 2014, when the Ebola outbreak in western Africa was at its peak, the pharmaceutical giant Merck acquired the vaccine, and in December 2019, this vaccine was approved for use in the nations of West Africa. A single dose proved to be effective in preventing Ebola infection in vaccinated individuals, and Merck continues to supply the vaccine for use in Africa. In 1990, in a laboratory in Madison, Wisconsin, something very

surprising took place. Working in the Department of Pediatrics at the University of Wisconsin, Jon A. Wolff and his colleagues took the bold step of injecting engineered RNA and DNA plasmids directly into the muscle tissue of mice. This was the first step in the effort to produce nucleic acid (or genetic) vaccines. It seems Wolff and his associates first used naked DNA in this way as a comparison with DNA packaged in protective parcels already known to be effective. Imagine their surprise when the naked material succeeded on its own. Or perhaps they were expecting to be lucky. It certainly paid off. Without any special delivery vehicle, the “naked” nucleic acids directed the production of their encoded proteins. This opened the door to the development of both DNA and RNA vaccines. Two years later, Stephen Johnston at the Department of Medicine, University of Texas, Dallas, repeated the observations but with an impressive addition—­researchers placed plasmid DNA onto tiny particles of gold delivered by a futuristic-­sounding “gene gun.” This produced an immune response: the mice made antibod-

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ies to human growth hormone—­the protein coded in the DNA. Just one year after that, Jeffery Ulmer and his colleagues, again studying mice, demonstrated protection against flu infection by a naked DNA vaccine encoding a key flu protein. This simple vaccine produced killer T-­cell–­mediated immune responses. And within five years the first DNA immunization study in humans succeeded in stimulating a good killer T-­cell response to a protein from the malarial parasite. Stephen L. Hoffman and his colleagues at the US Navy Research Facility in Bethesda, Maryland, conducted this research and published it in 1998. In the same year, buoyed by impetus and excitement, the pharmaceutical giant Glaxo Wellcome (now GSK), whose research center is in Stevenage Hertfordshire, signed a deal with a small company in Madison, Wisconsin, named PowderJect, part of an enterprise originally spun out from Oxford University. The task was to develop the vaccines of the 21st century administered by the gene gun. This alliance would take me on frequent visits to Madison. Such was the dizzying start to the story of nucleic acid vaccination. But, as the years passed, progress slowed, and disappointment set in. The problem was lack of potency. Human nucleic acid vaccines failed to make it to the clinic. What was needed for nucleic acid vaccines to complete the journey? The answer was a global challenge beginning in 2020—­an imperative need not seen before in the modern world.

CHAPTER FOUR

Develop Vaccines The question of when a vaccine will become available is common at the outset of a pandemic. But the answer is inevitably complex. There are six stages in the development of a new vaccine, and with today’s advanced state of knowledge, the first stage—­ exploratory research and discovery—­is often the shortest. This was never truer than in the race for a coronavirus vaccine. Many of the front-­r unners began with an existing vaccine technology, already tried and trusted while ending past epidemics. Researchers call these existing technologies “vaccine platforms” or “plug and play” because they can be used to launch new vaccines with relatively small changes. In other words, researchers can plug new vaccine elements into the platform, safe in the knowledge that the novel product will perform. But a long process of clinical development still lies before them. When I was a young research fellow in the Department of Pathology at the University of Cambridge in the late 1970s, I saw myself very much as an academic, pursuing a hugely privileged adventure in the realms of pure science; even though my chosen subject, immunology, is fundamentally about defense against disease. I saw myself working at the edge of understanding, relentlessly pushing the boundaries of science. Beyond this lay a dark, impenetrable mystery—­the border of the map, the limits of the known, the quickly darkening margin of the yet to be explored surrounding the circle of light.

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What is it like in that dark hinterland beyond the boundaries of knowledge? A forest laced with unmapped tracks and paths? A vital, undiscovered web, woven in shadow and silence? This perfect state of ignorance has something poignant about it. An innocence like that of childhood, and harder to escape. How do you move beyond it? That was the challenge I set myself. And that was how I styled my early endeavors. I’m not sure my sponsors saw things quite that way. They understood the value of fundamental research, but their money came from ordinary people, keen to see practical, applicable medical advances: my work was financed by the British Cancer Research Campaign, now Cancer Research UK. But in 1982, just home from a brief working visit to the University of Texas Medical Branch in Galveston, and with our second daughter newly arrived (it wasn’t the best planned chapter in our lives), I took a job at the Wellcome Research Laboratories. My brief, as a staff scientist, was to investigate new ways to make vaccines more powerful. Here I very slowly, sometimes painfully, learned a new reality: in order to come to fruition, biomedical discoveries need to be developed, and this is a lengthy, massively expensive journey, fraught with pitfalls, dogged with failures, and full of frustrations. This brings us to the six stages in the development of vaccines.

Stage One: Exploratory

This is the research-­intensive phase of vaccine discovery, designed to identify the right components of the pathogen to be included in the vaccine. In the case of coronavirus this is relatively straightforward: the spikes on the ball stand out, although some research teams have chosen much larger elements, including inactivated whole virus. Once researchers have identified an antigen such as the spike protein, they must determine the best way to administer

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it. This means a form of delivery promoting a strong and effective immune response without adverse effects. Because vaccines are destined to be used in large populations, safety is a principal consideration. Numerous well-­established delivery platforms are available for use with new vaccines. We should remember, however, that some of the most interesting of these have never been tested in large populations.

Stage Two: Preclinical

During this phase, researchers use tissue-­culture techniques and animal studies to predict whether the candidate vaccine will produce immunity. The predominant tests measure the levels of antibodies able to neutralize or disable the virus, the induction of T-­cell responses, and adverse or unwanted events such as excessive inflammation. At this time, researchers also conduct a limited number of efficacy tests. These usually employ small numbers of primates such as rhesus macaques, a type of monkey. The animals receive the candidate vaccine and then, a month or so later, researchers infect them with a dose of live virus. The levels of infection they develop are then compared with those seen in unvaccinated animals also infected with live virus. Particular sensitivities surround the use of primates in such studies, hence the small sample size; but it is important to use a species as close as possible to humans. For example, researchers testing a COVID-­19 vaccine for effectiveness in May 2020 used nine macaques. In all animal studies, researchers employ the principle of the three Rs: replacement of laboratory animals, where possible, with alternative techniques such as using tissue culture; the reduction of animals to the minimum required to obtain the information; and the refinement of experiments to minimize stress and discomfort and improve animal welfare. At this crucial stage in vaccine discovery, researchers fre-

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quently invest great hopes in their contenders and advocate for their favorite projects; yet many vaccine candidates do not pass this hurdle. If candidate vaccines fail to produce a strong immune response, or if they cause harmful effects, the candidate does not move on to the next stage of development. And failure is not always because of poor performance. Can this project compete for development resources with others in the pipeline? Are forecasts of commercial returns on the project large enough? Does this project fit with our current strategy and complement our established portfolio of medicines? Before embarking on clinical trials, the agency or company developing the vaccine submits an Investigational New Drug application to a regulatory body such as the US Food and Drug Administration (FDA) or the European Medicines Agency (EMA). This summarizes everything known about the vaccine, including the safety and efficacy data on any delivery technology. This might include the use of lipid parcels for injection or pressure devices that fire the vaccine into the skin. The institution that will host the clinical trial establishes a review board for approval of the application. The FDA aims to approve or request modifications to an application within 30 days. This waiting period is another difficult and anxious time for project advocates and champions. Once the appropriate administrative body has approved the proposal, there are still many challenges ahead. The vaccine must succeed in three phases of clinical trials.

Stage Three: Phase I Trials

In phase I trials, researchers administer the candidate vaccine to a small group (fewer than 150 people) with the goal of determining whether the candidate vaccine is safe. This constitutes the primary endpoint of the study. But at this early stage, researchers also use every opportunity to determine potential efficacy. For example,

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for an early COVID-­19 front-­r unner called Ad5-­nCov2, the phase I study in China measured the amount of antibody to SARS-­CoV-­2 spike protein, the ability of these antibodies to neutralize or disable the virus, and the strength of killer T-­cell–­mediated immunity.

Stage Four: Phase II Trials

Phase II trials, which include hundreds of human test subjects, aim to establish efficacy and also deliver more information about safety, the quality of the immune response that the vaccine induces, the immunization schedule, and the optimal dose. The number of dosing occasions, the interval between them, and the dose level may well be different for each vaccine, while different populations may require particular dosing schedules and formulations. Flu vaccination in the elderly is an example, where a stronger adjuvant is needed.

Stage Five: Phase III Trials

Phase III trials, which can include tens of thousands of test subjects, continue to measure the safety (rare side effects do not usually appear in smaller groups) and likely effectiveness of the candidate vaccine in the population at large. Such trials are financed by global pharmaceutical companies to establish efficacy in the markets they are targeting. Developing-­world vaccines (a malaria vaccine, for example) may be trialed in developing nations by joint initiatives between commercial and charitable organizations. The unifying principle is to establish effectiveness in the population in need of the vaccine. The ultimate test of vaccine effectiveness depends on the incidence of infections (acquired by chance) in the vaccinated population compared with unvaccinated individuals. To achieve this, vaccine trials are best conducted in regions where infection rates are high.

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Stage Six: Regulatory Review and Approval

If a vaccine passes through all three phases of clinical trials, the vaccine developer submits a Biologics License Application to the regulatory authorities. Such certification systems are administered by the FDA, the EMA, and other key organizations such as the WHO. Major pharmaceutical companies provide the infrastructure, personnel, and physical plants necessary to create large quantities of vaccines for distribution around the globe. In the case of engineered viral vectors, this involves large-­scale fermentation tanks for producing the vaccine in suitable cell lines developed for this purpose. As discussed earlier, such cell lines provide the missing gene for nonreplicating viral vectors, allowing them to reproduce but only while they are in the cells used for manufacture. Harvesting the vaccine returns the viral vector to its original nonreplicating state. Even at this late stage, disaster may strike at any time. In fact, one of the two worst tragedies in the history of vaccination, the Cutter Incident, took place in the manufacturing phase. Early on Sunday, April 24, 1955, in the midst of the historic first wave of immunization with the Salk polio vaccine, a little girl in Idaho came down with symptoms resembling polio. Remember that the Salk vaccine used dead viruses to immunize recipients, so the appearance of these symptoms was especially surprising. She had received the Salk vaccine six days earlier. Officials assumed that, as with many tragic cases in the great field trial, she had received the vaccine too late, or it hadn’t sufficiently protected her. She died three days later. In the days and weeks that followed, the death toll of newly vaccinated children and the number of cases of paralysis mounted. As officials and health-­care experts scrambled to get a grip on the catastrophe, it suddenly became clear that the problem vaccine came from a single manufacturer—­

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Cutter Laboratories in Berkeley, California. Even as this information came to light, all polio vaccinations throughout the United States were suspended. Investigations would ultimately show that the problem lay in the fermentation tanks holding the virus undergoing chemical inactivation. Inadequate mixing had allowed clumps to form, and the chemical used to kill the virus could not penetrate these clumps. Filters used to remove such clumps had also performed inadequately. Estimates indicate that at least 10 children died and more than 200 were paralyzed by the defective vaccine. The Cutter Incident transformed safety standards in the manufacture of vaccines that came afterward, and later in the 20th century, parents drove further improvements. In the case of live vaccines in the COVID-­19 race, every possible measure is in place to make sure such accidents are impossible. Once a vaccine is out, it’s still not the end of the story for vac-

cine makers, or the regulatory authorities who hold them to account. Stakeholders must put in place procedures that allow them to track whether a vaccine is performing as expected. Activities include phase IV trials, which are optional studies that researchers can conduct following the release of a vaccine. Such trials establish a wider picture of safety and side effects in particular subpopulations (diabetics, for example). Other post-­licensing systems include the Vaccine Adverse Event Reporting System and the Vaccine Safety Datalink. These systems allow administrative and approval bodies to monitor the performance, safety, and effectiveness of an approved vaccine in large populations. These steps require the skills and input of numerous stakeholders, from lab researchers to policymakers to medical professionals. With such a long and daunting road to success, how do small companies or inventive academic teams ever begin the journey? One key starting point is intellectual property. Such groups often

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begin by securing patents on their new discoveries. The aspirant technology or vaccine candidate may then progress in a small spin-­off company seeded by a university or research institution. These operations, often housed in dedicated incubator sites, receive funding from venture capital providers keen to invest in early-­stage, high-­risk enterprises. Investors receive tax breaks to encourage the flow of funds. These small companies may take their project as far as “proof of concept” in small phase II clinical trials before partnering with a large pharmaceutical company. But the dream scenario for such small teams is to be bought by a global company wishing to acquire new assets with fewer risks. This pattern operates throughout Europe and North America. In other nations (China, for example), government-­sponsored research also plays a major part. Charitable organizations such as the Gates Foundation increasingly play a vital international role, particularly in developing-­world vaccines. I have a huge respect for the guardians of high standards in the development of vaccines—­those who work daily to the stringent rules of best practice. I admire the meticulous, the dispassionate, the painstaking and consistent, who safeguard the passage of projects in their care and scrupulously maintain clear records in prescribed formats. The whole field is full of virtuous language: due diligence, good practice, quality assurance, quality control, data integrity, working to spec, certification, sign-­off—­each trusted signature witnessed, date stamped, microfilmed, and filed. Above all, I value the efforts of those who generate the policy, enforcing the regulations they have carefully formulated, policing the standards, and penalizing all who transgress. But what I most love is the boundary between mystery and discovery, as described in the previous chapters: Kitasato and Behring seeing deadly toxins made harmless; Macfarlane Burnet imagining lymphocyte clones; Porter glimpsing the true shape of antibody; Doherty and Zinkernagel identifying what the T-­cell

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sees; Miller removing the thymus and pondering the resulting loss of immunity; Tonegawa conceiving of shuffled genes; and Janeway intuiting the importance of danger. Back in 1992, my own studies were about to take a very different turn. My colleagues and I had discovered a new adjuvant that worked by promoting the formation of covalent bonds between the surface of antigen-­presenting surveillance cells and T-­cells. These dynamic bridges, known as Schiff bases, form naturally between antigen-­presenting cells and T-­cells during the induction of the immune response. They take the form of a double bond linking nitrogen to carbon on specialized cell-­surface proteins. Artificially increasing their number proved to be a potent way to amplify immune responses. We had filed patents. We’d published our findings in Science. And we were desperately hoping this agent might be developed as a revolutionary product. Sadly, it was never to be. Our agent was an active mixture of two enzymes—­a difficult product to scale up and a tricky candidate to manufacture and assure consistency and quality control. But more importantly, there was no strategic will to develop a new adjuvant within the company. Other potential products were much more important, fitting our employer’s portfolio and promising immensely greater earnings. We hadn’t a hope. Never mind. This is the usual experience in drug discovery: most projects fail in the early stages. And we’d already moved on to something fascinating. We’d begun testing small organic agents—­simple molecules called aldehydes—­which naturally form Schiff bases on the surface of immune cells. What effect would these have on T-­cell responses to antigen-­presenting surveillance cells? We were generally expecting interference with the natural chemical event between cells. But to our great surprise, at the right concentrations, such molecules potently enhanced T-­cell responses. We realized at once that, as well as stabilizing the interaction between presenting cell and T-­cell, Schiff base–­

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formation also initiates a biochemical signal, urging T-­cells to proliferate. Something quite extraordinary was just about to happen. In another part of the park (Wellcome Research Laboratories sprawls through the grounds of a grand country mansion), drug developers were testing a new treatment for sickle cell anemia—­a debilitating disorder caused by a mutation in hemoglobin. But in their phase I studies in normal volunteers, they were encountering a problem. The immune systems of some recipients were going into hyperdrive. Lymph nodes were enlarging—­a sign of infection. Activated immune cells were appearing in the blood. What was going on? To find out, the drug researchers consulted immunologists outside the company. External consultants were baffled. But then we heard about it. Just how was this drug meant to work? The answer was quite stunning. Researchers had designed the investigational drug, a small organic molecule given by mouth and circulating throughout the body, to modify hemoglobin. Hemoglobin is what gives your red blood cells the ability to carry oxygen necessary for your tissues to survive. Those with sickle cell anemia do not have enough healthy red blood cells to deliver oxygen properly. The drug was meant to work by forming Schiff bases on sickle-­cell hemoglobin, giving it the ability to carry oxygen. This chemical linkage was the same as the one we had studied between antigen-­presenting cells and T-­cells. And we knew that such a chemical event produced a potent signal that switched on T-­cells, amplifying their responses to foreign antigens. No wonder the new Schiff base–­forming drug was exaggerating immune responses to the natural pathogens we encounter daily. I began a campaign to convince my colleagues that this compound could become a new immunopotentiatory drug: a small molecule given by mouth and circulating through the body to increase the responsiveness of the immune system. Such a drug could be used to treat chronic viral infections and boost the immune system in cancer patients.

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I quickly gained their close attention. The compound, known as tucaresol, was repurposed as an oral drug to enhance immune responses through an unprecedented mechanism of action. We published our findings and their therapeutic implications in Nature in September 1995. Three new development projects were launched in Europe and the United States, targeting HIV, hepatitis B, and malignant melanoma—­all diseases where the immune system needs kicking into action. A new wave of therapeutic possibilities was gathering fast. Could the drug also be given alongside vaccines to act as a super adjuvant? Surely, success beyond the bounds of dreams lay just around the corner. As well as raising hopes and generating excitement, any gen-

uinely new products inevitably pose problems for development scientists, and this was also true for vaccines for COVID-­19. The principal examples were the nucleic acid candidates, especially RNA vaccines. Yet by and large, the fully established phases of vaccine development are well prepared to incorporate new entities. After all, now-­common technologies such as recombinant protein vaccines and viral vector vaccines were once exotic newcomers. Some learning may be necessary when it comes to making RNA vaccines at large scale, but it should be readily achievable. To make large quantities of DNA, you have to grow it as plasmids, which, as we saw in chapter 3, are autonomous units of genetic material containing all the necessary elements to make the protein they encode. These plasmids are grown in bacteria. The usual choice for growing plasmids is Escherichia coli (E. coli), a type of bacteria common in human and animal intestines and necessary for normal health. E. coli can grow easily in large-­scale fermentation vessels, and researchers can then harvest the DNA of interest. Because the safety of nucleic acid vaccines is crucial, the FDA, EMA, and WHO have released a new set of regulatory guidelines to encompass nucleic acid vaccine production.

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One vaccine production challenge for DNA vaccines is that, immediately postharvest, plasmid DNA is all but lost among the total DNA in the production system. The vital DNA is only 1–­2 percent of the total, the rest being part of bacterial chromosomes. Since all DNA is made of the same building blocks, separation of the precious plasmid can be difficult. Purification depends on the fact that plasmid DNA has a particular shape and size. Researchers use well-­established fractionation techniques to target this shape and size to purify the sample. These may be thought of as sieving, filtering, and related biochemical separation processes. Because the amount of plasmid DNA in the production system is small, the production of large amounts is also a challenge. “Large scale” refers to amounts of DNA greater than 1 gram. Since at least 1 milligram is needed as the starting point for each vaccine dose, the large-­scale vessels originally used in vaccine development make only a few thousand doses per run. Fortunately “naked” nucleic acids do not provoke an antibody response when they’re injected, so once injected, the plasmid can get on with producing the protein that will induce antibodies. But as well as removing all irrelevant bacterial DNA, other components must also be eliminated. These include proteins, glycoproteins (sugar-­protein complexes), lipoproteins (fat-­protein complexes) and lipopolysaccharides (fat-­sugar complexes). You’ll remember that the business of the immune system is to respond to bacterial and viral danger flags, and since bacterial fat-­sugar complexes constitute a potent danger signal, purity is essential to prevent the immune system overreacting to produce a dangerous inflammatory response. In the last two decades, manufacturing experts have addressed these problems. In 1994, it took two months of production to produce 1 gram of plasmid DNA for the first human trial of a malaria vaccine. Today research laboratories can produce this amount in a single day, while relatively small pilot plants can produce

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100-­gram batches. In addition to improving the productivity and purity of plasmid from bacterial fermentation systems, researchers are also developing new, fully synthetic ways to make DNA in large quantities. These depend on fully automated DNA synthesizers, originally developed by the Human Genome Project to sequence all human genes. In the same way, researchers are already producing RNA vaccines using machinery that does away with the need for cell lines and fermentation vessels. At the other end of the scale, biochemical refinements continue to be made to the plasmid itself. When Jon Wolff and his colleagues, the Madison researchers who pioneered nucleic acid vaccination, first injected plasmid DNA and RNA into mouse muscle in 1990, they were relying on the fact that the plasmids would use the machinery of the mouse cells to orchestrate the production of the foreign protein. This foreign protein then stimulated the mouse’s immune system and antibodies were produced. But certain fundamental elements are also supplied by the plasmid itself. They included a DNA sequence instructing the cellular machinery where to start reading the DNA. Another key element is the promotor. This component—­ the engine driving production of protein—­is placed just before the sequence encoding the protein to be synthesized, and it signals where to start transcribing RNA. The RNA subsequently dictates the synthesis of the protein itself. Also present in the plasmid are sequences once needed during manufacture, such as “selection markers” flagging the plasmid’s location. The point here is that researchers have refined, improved, and optimized all these molecular elements over the past 20 years, which gave nucleic acid vaccines the best possible chance of success.

CHAPTER FIVE

Evaluate the Contenders Let’s rerun, for a moment, the alarming calendar of events at the start of the COVID-­19 pandemic. On December 30, 2019, Chinese health officials report a cluster of pneumonia cases in the city of Wuhan in Central China to the National Health Commission. Chinese authorities set in motion an investigation to understand and contain the disease. By January 7, Chinese scientists have isolated a mystery virus and analyzed the genetic material, naming the deadly pathogen SARS-­CoV-­2. On January 11, we learn of the first recorded death, and on January 23, the local government in Wuhan locks down the city. By January 24, China reports 835 cases, while in Korea and Japan, cases are on the rise. A little over six weeks later, on March 11, with 116,558 cases worldwide and more than 4,000 dead, the WHO declares the SARS-­CoV-­2 outbreak a global emergency. The pandemical disease caused by the virus is named COVID-­19. By this date, the United States has confirmed more than 1,000 cases across 38 states, the United Kingdom has reported six deaths, and Italy, the worst hit country in Europe, reports 631 people have died of the disease. Imagine yourself a vaccinologist facing the task of finding a solution. The threat to humanity is ancient, but you must urgently pursue the best possible strategy, exploiting the hard-­won knowledge of the past 200 years. What kind of resources are available? And where could you possibly begin? The answer is less bewildering than you might imagine.

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Assuming you want to avoid risks and be confident of success with proven technologies, you have six choices. This is because six classes of established vaccines already exist and are highly amenable for adaptation as novel vaccines to combat new threats, whatever the pathogen might be. On the other hand, if you are ambitious enough to consider something new—­something potentially revolutionary in terms of production and versatility, you have two additional options, poised, after decades of laboratory development, to enter the clinical arena. Let’s review the options.

Inactivated Whole-­V irus Vaccines

We have used this class of vaccines since the late 19th century and most famously in the mid-­20th century, when the inactivated vaccine developed by Jonas Salk in 1954 initiated a long journey to the final eradication of a dread disease—­polio—­across the developed world. But this approach is not in any way outdated or obsolescent in combating even the most formidable infectious threat. China is championing this approach today. A traditional Chinese story tells of the miracle-­working doctor Wang Shi’er: “The difference between a miracle worker and a [merely] famous doctor is like the difference between heaven and earth. When you get sick, . . . when you are about to die at any moment and other people don’t know what to do, he does. What he comes up with isn’t a tried-­and-­trusted remedy either; it is a flash of inspiration.” Dr. Wang Shi’er goes on to remove a metal splinter from a blacksmith’s eye with a novelty magnet. But scientists based in China’s capital, Beijing, may well save lives miraculously with precisely the opposite approach: these are the vaccine experts who have opted for the most old-­fashioned, tried-­ and-­trusted vaccine you could possibly wish for. There is no virtue per se in producing a high-­tech vaccine for the sake of it. The value comes from additional efficacy, speed

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of manufacture, improved safety profile, or some other tangible benefit of advanced technology. If the vaccine is safe and effective, then traditional is as good as anything, and more likely to be safe in large human populations. The Beijing scientists, led by Gao Qiang, set about collecting viruses washed from the lungs of COVID-­19 patients across a range of nationalities in order to produce a representative mix of viral variations. They then selected one common strain and reserved another 10 strains for subsequent viral challenge studies in animals. The group purified the chosen virus strain then grew it in a continuously proliferating cell line called Vero. This valuable cell line began its life on March 27, 1962, in the hands of scientists at Chiba University near Tokyo in Japan. Japanese investigators derived the line from cells taken from the kidneys of African green monkeys. The international language, Esperanto, was in vogue at the time, and the name they chose comes from an abbreviation of verda reno, which means “green kidney.” In Esperanto, vero also means “truth.” The Vero cell line is the one most widely accepted by regulatory authorities and has been used for over 30 years for the production of polio and other virus vaccines. This makes it an excellent choice for producing new vaccines against emerging pathogens. The SARS-­CoV-­2 virus was then grown in Vero cells in large (50 liter) culture vessels and subjected to further investigations. These showed that the virus, including the gene for the spike protein (the part of the coronavirus that binds ACE2) was stable in this cellular environment, allowing researchers to proceed. The next step was to kill the virus. In the history of vaccines, the conventional way to kill vaccine viruses has been with formaldehyde, which “pickles” the virus, retaining its native shape, helping to ensure that the immune system can identify it. In this case the scientists used a chemical called β-­propiolactone, which is more targeted than formaldehyde. It has been used to kill influenza viruses, and although scientists do not completely understand how it kills

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viruses, they believe it poisons the proteins whose function is to get the virus inside human cells. After killing the virus, the researchers checked to make sure its shape wasn’t damaged. Happily, using an electron microscope, the team of scientists saw discrete, intact, oval shapes “embellished with crown-­like spikes,” confirming that the virus’s shape was unaltered. As with almost all nonliving vaccines, an enhancing agent, or adjuvant, is necessary to provoke a strong immune response, and the Beijing researchers chose the tried-­and-­trusted adjuvant alum, widely used in childhood vaccines. Repeat injections are also needed with killed vaccines. Mice and rats were immunized twice at two dose levels and antibodies to SARS-­Cov-­2 spike protein and another protein, from the core of the virus, were measured. Antibodies to the spike protein dominated the response—­ just what is needed in a COVID-­19 vaccine. The levels of these antibodies in vaccinated mice were 10 times higher than levels seen in patients recovered from COVID-­19—­an encouraging picture, though the comparison should be viewed with caution. The antibodies produced in response to the vaccine also neutralized the full range of viral subtypes—­another promising indication. Researchers then moved on to a protection model in macaques. Animals received three injections of the killed vaccine and then received the COVID-­19 virus directly into the lungs. Seven days after being infected, all four animals in the high-­dose group had no detectable virus in the respiratory tract. In great contrast, animals in the control group had pneumonia and high levels of virus in the lungs. The Beijing team published their findings in Science on May 6, 2020. Meanwhile, their clinical collaborators had received regulatory permission to proceed with a combined phase I/II clinical trial (a randomized, double-­blind, placebo-­controlled study). By April 17, the first group of volunteers had received the first dose of vaccine. Volunteers were then given two doses, 14 days apart

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in one group and 28 days apart in a second group, and two dosage levels were employed. The study included 144 healthy adults, ages 18 to 59. Two-­thirds of the volunteers were vaccinated with killed vaccine while one-­third received placebo. The trial was conducted in Jiangsu Province near Shanghai. Eight other COVID-­19 vaccine candidates in the inactivated whole-­virus class began development in 2020.

Protein Subunit Vaccines

The second class of vaccines available to researchers, protein subunit vaccines, is also well established. Like the first class of inactivated whole-­virus vaccines, this class is also nonliving. In 2020 it constituted the largest category in the COVID-­19 vaccine race. Like all nonliving candidates, protein subunit vaccines require additional ploys, packaging, and enhancements to make the immune system sit up and take notice. But there are plenty of proprietary tricks to try—­as evident from the more than 40 vaccines in this class. Strategies used to catch the immune system’s attention include proteins wrapped up in intriguing (at least for an immune system) parcels and proteins mixed with adjuvants or linked to danger signals. All these contenders focused on the spike protein in its various guises. The leading example in the race for a COVID-­19 vaccine employed a recombinant version of the SARS-­CoV-­2 spike protein produced in an interesting way, together with a potent new adjuvant. The spike protein for COVID-­19 vaccines is made by conventional genetic engineering techniques, but in this example, researchers chose an insect virus known as baculovirus for its manufacture. Baculoviruses are a distinctly odd choice to use for making proteins. In their natural environment, they infect insect larvae and caterpillars, often killing their host and then dispersing to new larvae in a special package. But this lifecycle can be

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exploited to insert foreign genes for expression at very high levels. All down the West Coast of the United States, little green caterpillars blight crops. The caterpillars become the silver Y, a cryptic nocturnal moth with a pleasing Y shape etched in silver on either wing. The moths in turn suffer from baculovirus, which spreads between them in minute polyhedral packages that might conjure memories from your first geometry class. When this virus is grown in the lab, the large gene encoding the polyhedrin protein is redundant and can be safely removed without affecting viral growth. This leaves a large space for new genes to be inserted. But more importantly, the “promoter” for the deleted polyhedrin gene (the engine driving RNA copying and the synthesis of proteins) remains in place—­and it’s a very powerful promoter. Any gene downstream of it gets expressed in large quantities, hence the interest from genetic engineers. Not surprisingly, you have to grow the insect disease baculovirus in insect cells; one favorite cell line comes from embryonic tissue of the velvetbean moth. As cells for use in large-­scale culture vessels, insect cell lines are cheap and have very good safety features. Not long after the emergence of SARS, several groups of scientists constructed baculovirus expressions systems containing SARS-­CoV-­1 genes. When researchers injected these intact baculoviruses into mice, the animals produced robust antibody responses to the full-­length SARS-­CoV-­1 proteins. In this way, baculovirus systems proved useful for making coronavirus antigens in their native conformation in the bodies of experimental animals. The COVID-­19 vaccine candidates employed a different approach. It consisted of just the purified SARS-­CoV-­2 spike protein in its native conformation, formulated in a nonliving vaccine with the obligatory adjuvant—­or in this case, a cocktail of adjuvant components. Adjuvants were once the witchcraft in classical immunology—­ the “dirty little secrets” hidden away in the recipe sections of sci-

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entific publications, never mentioned in the lofty discussion until Charles Janeway (who introduced the concept of danger, as we saw in chapter 2) called them out in 1989. But where did adjuvants begin? In the early 20th century, Gaston Ramon, the French veterinarian who pioneered the chemical inactivation of bacterial toxins, was trying to improve the yield of horse antibodies used to treat children with diphtheria. He noticed that if an abscess happened to develop at an injection site, the horse produced a lot more antibodies. This prompted him to inject the horses with toxin combined with starch, breadcrumbs, and tapioca. This ingenious use of these otherwise innocuous agents produced sufficient irritation to provoke sterile abscesses, which increased the antibody yield. Developing this theme in 1942, Hungarian-­born immunologist Jules Freund and his coworker Katherine McDermott immunized guinea pigs with horse serum emulsified in mineral oil that also contained killed, dried mycobacteria tuberculosis. They found this combination of bacterial constituents with an oil that persisted at the injection site and provoked inflammation gave a very powerful kick to the immune system. Freund’s adjuvant, even without the mycobacteria, is too toxic for use in vaccines, but luckily another strand of activity had already produced a noninflammatory adjuvant suitable for human use. This began with the work of Alexander Glenny who, in 1926, discovered the adjuvant alum, as we saw in chapter 3. It seems that alum, the only licensed adjuvant throughout the 20th century, works in three ways. As a mineral gel, alum binds protein antigens attracted to it by electrical charge, and the injected gel then creates a persistent, slow-­release depot of antigen, prolonging stimulation of the immune system. The gel is also readily ingested by surveillance cells of the innate immune system. Alum also activates a complement—­a set of blood proteins important in innate first-­line immune defenses. All three mechanisms promote

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a strong antibody response by prolonging the immune system’s exposure to antigens and increasing their uptake by immune cells. In the 21st century, the list of acceptable adjuvants at last began to grow, and advances were based on refinements in biological oils used to make emulsions, the addition of danger signals, the use of virus-­shaped packages, and the exploitation of a substance from a small South American tree. If vaccines look like pathogens, surveillance cells naturally take an interest. In this case, researchers create a virus look-­alike by wrapping antigens in fatty bio-­membranes, essentially the same as those of intact viruses. This approach—­tricking the immune system by delivering proteins in doppelganger costumes—­was licensed in the 1990s and is used in vaccines against hepatitis and flu. Another tactic is to use danger signals as adjuvants. Bacterial components called “lipopolysaccharides” from pathogens such as salmonella are very potent danger signals, too dangerous to use in vaccines. But removing part of the complex danger molecule detoxifies it without dampening the strong alarm signal it provokes. This agent, known as monophosphoryl lipid A (MPL for short), is used in vaccines against hepatitis and human papilloma virus (HPV). Water-­in-­oil emulsions and oil-­in-­water emulsions, which attract the interest of surveillance cells, have also proved effective and nontoxic, and several were licensed for human use in the 21st century. An emulsion consists of a fine dispersion of minute droplets of one liquid in another liquid in which it is not soluble or miscible (“mixable”). The oils—­from marine biological or vegetable sources—­cluster molecules together, making them more easily identifiable by the surveillance cells. Vitamin E has also been found to be a useful addition. This research is largely empirical, based on testing potential combinations and assessing how well they work.

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This brings us to the small South American tree. The soapbark tree is an attractive evergreen in the family Quillajaceae, native to central Chile and used by native peoples as a soap and decongestant. It’s also a foaming agent widely used in the soft drinks trade. A bark extract, saponin, is a powerful adjuvant, but too toxic for medical use. A modified version known as QS21, formulated in various emulsions, can, however, be safely employed in vaccines. The innovative use of adjuvants in combination has been pivotal in the development of the world’s first vaccine against a parasitic infection. This new vaccine protects against malaria and is now licensed for use in Africa. Malaria, an ancient disease spread by mosquitoes, has come to live in equilibrium with the human immune system, so in order to do better than natural immunity, a four-­pronged adjuvant approach is needed. The vaccine, trade name Mosquirix, is packaged in liposomes, boosted by the danger signal MPL, enhanced by the soapbark tree adjuvant QS21, and pivotally assisted by the presence of the hepatitis B surface antigen, which self-­assembles into virus-­like particles. It can reduce episodes of malarial sickness by up to 50 percent in children. This partial reduction has the power to ease substantially the enormous burden that malaria places on hospitals and health services across Africa. But for SARS-­CoV-­2 protein vaccines, scientists don’t have to go to these lengths. For the leading COVID-­19 protein subunit vaccine, researchers used a simpler adjuvant. It’s called AS03, and it contains squalene (a marine bio-­oil), vitamin E, and an emulsifying molecule called polysorbate 80. This produces an oil-­ in-­water emulsion. This adjuvant has already proved successful for a pandemic influenza vaccine, and its clinical trials with the COVID-­19 protein subunit candidate commenced in the second half of 2020. Several small companies developing their own protein subunit candidate vaccines also gained access to this same adjuvant through licensing arrangements.

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The 40-­plus candidates in the protein subunit COVID-­19 vaccine class include many intriguing variations on the central theme, and one of them exemplifies an interesting niche approach. A Romanian company began by looking at the MHC molecules most frequently found in the Eastern European population. They then artificially synthesized protein fragments (peptides) from SARS-­CoV-­2 that most readily bind to these particular MHC subtypes, ready for recognition by immune cells. In this way they created a vaccine tailor-­made for Eastern European recipients. Though many focused on the spike protein that gives the coronavirus its name, a California biotech company took the view that other parts of the virus were likely to promote cell-mediated immunity and so chose a protein from the viral core whose job is to maintain the viral RNA in its correct shape. Evidence from patients recovered from Ebola suggested that this core protein would be a crucial target for killer T-­cells. Other approaches to protein subunit vaccines include the use of computer-­generated predictions of which bits of the virus will be recognized by T-­cells. Researchers then genetically engineer these fragments while also adding a short sequence designed to help the antigens load into MHC molecules, ready for T-­cell recognition. We’re now at the very smart end of COVID-­19 vaccine enterprises.

Live Attenuated Vaccines

Leaving behind the nonliving vaccine categories, we now consider the third major class of vaccines researchers turn to when confronted with emerging infections. This is the class known as live attenuated vaccines. Members of this class are at the forefront of highly successful established vaccines, including those we give to infants. The category includes vaccines for measles, mumps,

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and rubella (German measles) and the Sabin polio vaccine, currently deployed in the global campaign to eradicate polio. In the 20th century, pioneering vaccinologists exploited the accumulations of random mutations in tissue culture to derive weakened, nonpathogenic progeny, as detailed in chapter 3. Researchers now use precise targeted genetic manipulations to weaken viruses, leaving nothing to chance. For COVID-­19, two live attenuated vaccine candidates, codeveloped in India and Australia, make up this select class. To make these COVID-­19 viruses harmless, researchers have modified the genetic sequences encoding the viruses’ protein building blocks. Specifically, they targeted instructions, called codons, in virus RNA used to make essential viral proteins. In what’s called “codon deoptimization,” the researchers forced their modified version of the virus to use inefficient instructions to build itself, rendering it harmless. But the virus maintains the same appearance (to the human immune system) and still provokes both antibody and cellular immune responses effective against the natural virus.

Nonreplicating Viral Vector Vaccines

Instead of using viruses with inefficient reproduction, the fourth class of vaccines uses viruses that cannot reproduce at all. As we saw in chapter 3, these vaccines consist of a harmless carrier virus containing an inserted gene encoding a key antigen from the pathogenic virus. Nonreplicating vectors do not grow in the body, but they do infect cells, and the pathogenic antigens they carry present the full range of their danger signals to the innate immune system. This is key to the success of these vaccines. In March 2020, in Tianjin in North China, researchers submitted such a vaccine against SARS-­CoV-­2 for registration in a phase I clinical trial. Tianjin is a bustling port—­the place where the ancient Chi-

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nese Empire met the sea—­and a city of 13 million people. Tianjin means “Ford of Heaven,” named for the crossing of Tianjin’s Gu River by Zhu Di on his way to become the third emperor of the Ming Dynasty in 1402. This vaccine was one of the very first to reach clinical trials, just 10 short weeks after the sequencing of the SARS-­CoV-­2 virus. By the end of March 2020, the live vaccine was in phase I clinical trials in two approved hospitals in Wuhan—­the Wuhan Rest Center of the Chinese People’s Armed Police Force and Tongji Hospital, part of Tongji Medical College at the Huazhong University of Science and Technology. The trial included 180 healthy people between 18 and 60 years of age. Like all phase I trials, no placebo or mock vaccine was included for comparison, and the trial was not “blinded”—­both doctors and recipients knew what was being administered. The study began by giving the lowest vaccine dose to 36 individuals. Once this was shown to be safe (seven days later), a second group of 36 volunteers received a higher dose. Researchers then repeated the process with the third group, who received the highest dose of all—­ this being the dose predicted to be effective with similar vaccines in previous studies. Not surprisingly, the volunteers had to pass certain tests. They had to be negative for antibodies to coronavirus, meaning they hadn’t already been exposed to the disease and developed an immunity. They had to have no previous exposure to the related virus, SARS-­CoV-­1. And they needed healthy lungs. The primary purpose of this study was to establish safety, but the exciting part for the scientists running the trial was measuring neutralizing antibodies made against the virus. They made this measurement 14 days, 28 days, 3 months, and 6 months after the injection. It wasn’t too long before the first results were in hand. With no significant adverse effects to worry about, and with good levels of neutralizing antibody observed, the study proceeded to phase II on April 12, 2020, making it an early leader in the race. This time

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500 healthy volunteers were tested in the same way. But how did Chinese scientists have a vaccine against a virus new to science in their hands so quickly? To understand the answer, we have to go to West Africa on the day after Christmas in 2013 when an 18-­month-­old boy in Meliandou, a tiny forest-­edge community in Guinea, developed a fatal illness characterized by fever, bleeding, and vomiting. This too was a zoonotic virus, and this too came from bats. But these bats were not microbats like the horseshoe bats of China. They were hammer-­headed fruit bats, one of the largest bat species on the planet. And the virus they harbored was Ebola. The outbreak lasted from March 2014 to June 2016, afflicting the people of Guinea, Sierra Leone, and Liberia, with cases reported in Nigeria, Mali, Europe, and the United States. By the end of the epidemic 28,616 people had been infected and 11,310 people had died. In chapter 3, we saw how live viral vectors provide a platform ready-­made to receive new genetic elements coding for the key proteins that viruses use to enter human cells. And we saw how in 2014, as the Ebola outbreak threatened the globe, Chinese scientists began working on a vaccine using nonreplicating adenovirus as their vector, inserting the Ebola gene coding for the surface spike that Ebola virus uses to lock on to human cells. By the end of 2014, researchers had established that the vaccine was safe and produced an immune response, but the vaccine was never needed to contain Ebola. Faced with the new coronavirus epidemic in January 2020, the same team took their proprietary viral vector platform out of the freezer and inserted the SARS-­CoV-­2 gene coding for the spike protein. The nonreplicating viral vector the Tianjin scientists opted to use is an adenovirus, discussed in chapter 3. Adenoviruses are cold viruses, and they’re classified according to small differences in the antigens they express. These differences are defined by antibodies, also called antisera, and the different viral strains are

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therefore called serotypes. The Tianjin adenovirus used in the COVID-­19 vaccine race is adenovirus serotype 5 (Ad5). Given the speed at which such vaccines can be developed, it’s not surprising that other COVID-­19 vaccines use similar approaches. In chapter 3, we saw how scientists at the Jenner Institute in Oxford established an adenoviral vector as part of a vaccine technology platform, and how this was used to produce a vaccine against MERS in 2018. This vector, a chimpanzee adenovirus, was swiftly repurposed in 2020 to make a SARS-­CoV-­2 vaccine. Why use a chimpanzee adenovirus instead of a human one in a vaccine? Remember, anti-­adenovirus antibodies are common in the human population and such pre-­existing antibodies can impede the viral vector before it can do its job, which is to induce an immune response. Human immune systems have not met chimpanzee viral vectors, so this improves the chance the body will make a fresh (and stronger) immune response. So scientists select vectors for which pre-­existing antibody levels are low to ensure vaccine potency. Another reason is that it’s possible to patent new strains of adenovirus which carry this advantage. Such intellectual property claims then underpin the commercial development of new vaccines. Like the Tianjin vaccine, the gene encoded the SARS-­CoV-­2 spike protein was incorporated into the Oxford vector known as ChAdOx1. These studies were led by Sarah Gilbert, whom we met in chapter 3—­a vaccinologist well used to challenging diseases such as malaria and, more recently, influenza. Preclinical studies with the Oxford candidate in mice showed good antibody and T-­cell responses to the vaccine, known as ChAdOx1 nCoV-­19. And in a protection study in rhesus macaques, ChAdOx1 nCoV-­19 successfully prevented pneumonia in animals challenged with SARS-­CoV-­2. A human trial of this successful vaccine got underway in April 2020. In line with the urgent need to deliver a vaccine for

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COVID-­19, the trial combined phase I and phase II features in a single study. To this end, the trial was blinded—­no one knew whether they were receiving the SARS-­CoV-­2 vaccine or an irrelevant vaccine (against meningitis), which produces the same side effects (slight fever, painful injection site, and headache). Researchers measured neutralizing antibodies and killer T-­cell–­ mediated immune responses in 1,102 participants. The study used a single dose of vaccine, but a subgroup of volunteers received two doses of the vaccine given four weeks apart. The ambitious study also looked at disease prevention by following vaccinated and mock vaccinated volunteers to see how many would contract COVID-­19 by chance. Phase III trials of the Oxford vaccine began in July by recruiting more than 10,000 subjects. Older adults were the focus of additional phase II studies. On July 20, the Oxford COVID Vaccine Trial Group published their phase I/II data in human volunteers in The Lancet. Their adenovirus vector vaccine (ChAdOx1 nCoV-­19) produced good neutralizing antibodies in all subjects given a single dose (by at least one of the virus neutralization assays used). Levels of antibody were further elevated in all subjects given a second immunization 28 days later. This boosting effect was reassuring, showing that antibodies to the adenovirus vehicle created by the immune system upon receiving the first dose did not compromise a second dose. Strong T-­cell responses, important in limiting COVID-­19 infections, were also produced by the first injection. These were not boosted by the second, consistent with previous experience with such vaccines. Phase III studies began in July 2020, and participants in the phase II study continued to be monitored to measure incidence of COVID-­19 infections after natural exposure. The successes of the Jenner Institute and the Oxford vaccine group did not come out of nowhere, and one of the weapons in their well-­established arsenal is fittingly linked with Edward Jenner himself. In the centuries-­long battle to rid the world of

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smallpox, a goal that Jenner himself held dear, the original cowpox virus, so faithfully transmitted arm to arm, arm to cow, cow to water buffalo, and back to arm again, was somehow lost, or rather substituted with a closely related poxvirus called vaccinia. Nobody knows where vaccinia came from. But vaccinia, used from early in the 20th century, was the agent that finally put an end to smallpox. By transferring vaccinia virus several hundred times over into fresh cultures of chick embryo cells, Anton Mayr, a veterinarian working at the University of Munich in Germany, succeeded in producing a modified vaccinia virus—­a variant unable to grow in humans. This virus, called modified Vaccinia Ankara (MVA), was found to have lost around 10 percent of its genome, leaving it highly weakened and therefore unable to replicate in mammalian cells. In 2012, responding to the emergence of MERS (MERS-­CoV), Oxford scientists tried out a more heavyweight approach to producing a potent vaccine. This involved two vaccine doses with very different viral vectors administered four weeks apart in laboratory animals. The first was delivered in the familiar adenovirus vector (ChAdOx1), and the second in MVA. Both contained the gene encoding MERS-­CoV spike protein, but the immune system sees this key antigen in the two very different contexts provided by components of the viral vector. This recruits many more T-­cells to the defending army (even though neither vector proliferates in the body). Though no research group is using this method at the time of this writing to combat COVID-­19, in principle the strategy is waiting in the wings. Another race contender in the live virus class also had this strategy in mind. For many years a Belgian pharmaceutical company had been tackling viruses such as Ebola, Zika, respiratory syncytial virus, and HIV, and when in 2014 the need for an Ebola vaccine suddenly became urgent, they accelerated their research. The vaccine they developed consists of an adenoviral vector called

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Ad26, a serotype less common than Ad5 and therefore not likely to meet with pre-­existing Ad26 antibodies in vaccine recipients. From this established position they entered the SARS-­CoV-­2 vaccine race in March 2020, aiming to yield safety data in early 2021. Collaborations with staff in the US Department of Health and Human Services and with scientists at the Beth Israel Deaconess Medical Center (part of Harvard Medical School) facilitated this effort. Like the Oxford group, the Belgian scientists also had what’s called a “prime-­boost platform” in their portfolio, based on adding a second immunization with MVA containing Ebola genes. In general terms, prime-­boost vaccines may give the same viral antigens in both doses. Or they may give different antigens such as a spike protein in the priming dose and viral core antigens in the boosting dose. This strategy was first used in earnest in the campaign to find a vaccine to protect against HIV. But SARS-­CoV-­2 is very different from HIV. Unlike with HIV, the majority of those infected with COVID-­19 make a successful immune response. The well-­defined nature of that immunity, targeting the viral spike protein, means that a successful vaccine is a highly realistic prospect. In 2020, the nonreplicating viral vector vaccine class included more than 20 COVID-­19 vaccines. One of these stood out for its credentials in terms of international collaboration. A small biotech enterprise based in the United States worked with a Wuhan biotech firm to exploit the US company’s MVA vaccine platform—­a fifth generation vaccinia vector optimized for good expression and stability of the SARS-­CoV-­2 gene. Like all MVA vehicles, this vector is produced in chick cells but cannot grow in human cells. Experience with Ebola, Marburg, Lassa, and Zika vaccines lent this effort a potential extra edge, and additional inserted genes to make proteins self-­assemble into virus-­like particles (empty of viral genes) were included to make the vaccine look dangerous to the immune system.

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On August 11, 2020, Russia announced that it had successfully developed a COVID-­19 vaccine at the Gamaleya Research Institute of Epidemiology and Microbiology in Moscow. A surprise aspect of this news was that Russian authorities had already approved the vaccine for general use, an aspect provoking international criticism because no one had yet conducted large-­scale phase III safety and efficacy testing. Commentators suggested that vaccinated human subjects were deliberately exposed to virus in a clinical setting in order to demonstrate the vaccine’s effectiveness. Reports indicated that the vaccine employed nonreplicating adenovirus vectors delivering the gene encoding SARS-­CoV-­2 spike protein—­the same approach taken by the Belgian COVID-­19 vaccine group and Chinese scientists based in Tianjin.

Replicating Viral Vector Vaccines

We can now look at the fifth class of vaccine technologies readily adaptable to meet the challenge of emergent infectious diseases—­ the replicating viral vector vaccines. These differ from the previous class in that once they are injected and have entered human cells, they undergo several rounds of replication, infecting more cells and providing sustained stimulation of the immune system. One of these replicating viral vector vaccines, which entered the COVID-­19 vaccine race in April 2020, employs an attenuated VSV vector, already proven as a newly registered Ebola vaccine, as we saw in chapter 3. The sponsors of this candidate have also acquired an attenuated measles virus (used in the measles vaccine) engineered to deliver SARS-­CoV-­2 spike protein to the immune system of recipients. This technology, developed by scientists at the renowned Institut Pasteur in Paris, allows the genes of other viruses to be inserted into the measles vaccine. Because this vector replicates inside the cells of vaccine recipients, it also provides an extended stimulus to the immune response. In all there were

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16 replicating viral vector vaccines in the COVID-­19 candidate vaccine race in 2020.

Virus-­L ike Particle Vaccines

The sixth class of established vaccines that researchers can turn to in the hunt for new vaccines against emergent diseases consists of the virus-­like particle (VLP) vaccines. VLPs are composed of multiple copies of structural proteins found in infectious virus particles that spontaneously assemble into particles resembling the virus but are empty and contain no genetic material. Their properties make them attractive to surveillance cells of the immune system. However, because these are nonliving vaccines, they still require adjuvants to make them work. One principal example of an established vaccine exploiting this technology is the HPV vaccine protecting against cervical cancer and genital warts. The danger signal MPL is incorporated into this vaccine. Because they resemble intact viral particles presented with a potent adjuvant, these harmless protein structures provoke a strong protective antibody response. Children who receive the HPV vaccine before age 15 need only two vaccine doses to be fully protected against HPV infection. In 2020 there were nine VLP vaccines in the COVID-­19 candidate vaccine landscape. This completes the list of the six vaccine classes with a proven

track record in clinical use. But for the ambitious and those eager to explore new territory in the search for vaccines against new global threats, two additional groundbreaking vaccine classes stood ready for use. Collectively they are known as nucleic acid vaccines. As described in chapter 4, for these vaccines, antigens of choice are encoded in engineered genetic material (either DNA or RNA). When injected, the nucleic acid spontaneously orchestrates the synthetic machinery of recipient cells to produce

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the encoded viral (or other) proteins. The foreign proteins then stimulate an immune response. The success of immunization with RNA—the first COVID-19 vaccines to be approved in Europe and the United States—stands as a historic achievement.

DNA Vaccines

Investigators have examined many ingenious delivery techniques for DNA vaccines. A US biotech company in Philadelphia has produced an optimized DNA vaccine encoding the SARS-­CoV-­2 spike protein designed to be injected into the protective middle layer of the skin known as the dermis. A proprietary handheld electrical device is then used to deliver electrical pulses at the site of injection which briefly open small pores in cell membranes, allowing the plasmids to enter dermal cells before the pores close again. The company initiated their phase I clinical trials in healthy volunteers in Kansas City and Philadelphia in 2020. Swedish scientists at the prestigious Karolinska Institute explored a similar “electroporation” technique. Another DNA candidate vaccine is delivered to the middle layer of the skin with a needle-­free device. The PharmaJet technology, widely used in developing-­world vaccinations, sends a narrow, precise fluid stream to just the right depth in the dermis. Because this skin layer is a barrier, called a “sentinel layer,” against infectious pathogens, it is well supplied with surveillance cells of the immune system. At least 11 DNA-­ based vaccines entered the COVID-­19 vaccine race in 2020.

RNA Vaccines

But the successful class of nucleic acid vaccine started the race with twice this number of candidates. This is the category delivering vaccines in the form of RNA. On March 16, 2020, the same day that Chinese scientists in Tianjin launched their clinical trial

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of a COVID-­19 viral vector vaccine, scientists based in Boston commenced the first clinical trial of their RNA vaccine. This Boston company (along with others) pioneered the development of RNA vaccines aimed at COVID-­19, and the National Institute of Allergy and Infectious Diseases sponsored their trial. The excitement surrounding such vaccines is understandable. RNA has several theoretical advantages over the live vector and the nonliving protein-­based vaccines. The first is safety: mRNA or messenger RNA, is noninfectious, eliminating the risk of unwanted invasiveness, and it does not integrate with the genetic material of the human cell. The second is that mRNA, unlike DNA, interacts directly with the machinery of cells without requiring intermediate steps. The third is that once it has done its job, vaccine mRNA is naturally broken down by “housekeeping” enzymes that maintain the normal functioning of cells. RNA vaccines are also attractive because they are easy to produce. mRNA can be manufactured rapidly and inexpensively at large scale, and it provides great flexibility in the construction of vaccines for different diseases. Why is it, then, that RNA vaccines have been in the research arena since Jon Wolff ’s pioneering experiment in 1990, but took until 2020 to reach clinical trials? The same question could be posed for DNA vaccines. Before their use to combat COVID-19, nucleic acid vaccines found a small place in agricultural and veterinary applications. One nucleic acid vaccine has been licensed to protect horses against West Nile fever. And in 2016, the EMA granted a license for Clynav, a nucleic acid vaccine which protects farmed salmon against salmon pancreas disease, a viral infection. The fish vaccine has been in the pipeline for many years, and when I, as leader of Glaxo Wellcome’s first research project on nucleic acid vaccination, raised farmed salmon as an example of success, I invariably found the heads of industry gazing over my shoulder.

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The mother of nucleic acid vaccination is Margaret Liu. Liu’s parents emigrated from China as young adults in pursuit of educational opportunity and, after the early death of her father, Liu grew up in a small town in the mountains of Colorado. Hers was one of three Chinese families in the region. Turning down offers from Princeton and Yale, she went to Colorado College, closer to home, and almost opted for a career as a concert pianist before deciding on Harvard Medical School. Later she worked at Merck under the renowned vaccinologist Maurice Hilleman. As a research immunologist, she championed nucleic acid vaccine candidates, demonstrating success in mice and rats with DNA vaccines against influenza, tuberculosis, HPV, and HIV. But when studies moved into humans, Liu and other pioneers met with disappointment. In clinical settings the nucleic acid platform was dogged by lack of potency. At first people thought that humans were just too big, but this idea could hardly be defended once nucleic acid vaccination had proven successful in horses. So what was the problem? Things suddenly began to look brighter for RNA vaccines in 2005 with the publication of a paper by medical researcher Drew Weissman and colleagues at the University of Pennsylvania. This provided a promising way around the difficulties for mRNA vaccines in humans. Success hinged on chemically modifying the building blocks of mRNA in relatively subtle ways. The problem with unmodified viral RNA is that RNA constitutes a potent danger signal for the innate immune system. Let’s return for a moment to Charles Janeway and the importance of danger for the immune system. By great good fortune, just as Janeway was predicting the existence of danger sensors on surveillance cells of the innate immune system, the prototype for all danger sensors was discovered independently in Germany. Scientists working on how genes control the development of animals made the advance in 1988. And when Christiane Nüsslein-­Volhard, the

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German scientist who made the discovery, realized what she’d done, she exclaimed, “Toll!,” which means “amazing,” “great,” “cool,” “Das is ja toll!” In 1995, Nüsslein-­Volhard was awarded the Nobel Prize in Physiology or Medicine for her pioneering studies of the genes controlling development. From there afterward, the receptors detecting danger signals in the immune system became known as Toll-­like receptors. Three of these Toll-­like danger receptors in humans recognize viral RNA, triggering them to send alarm signals to the adaptive immune system. They do the same with RNA in vaccines. Double-­stranded RNA (a contaminant of single-­stranded mRNA vaccines) is a particularly potent danger signal sensed by Toll-­ like receptors. This can be eliminated by careful purification. But single-­stranded RNA—­the stuff of RNA vaccines—­is also a danger signal when it appears in the “wrong” place, as when injected as a vaccine. On the face of it, this might seem like a good thing. RNA vaccines carry a built-­in adjuvant to switch on the immune system. But it’s not as simple as that. The sensors responding to such danger signals could disrupt the balance between killer T-­cell responses and antibody responses, making the immune response to the vaccine less effective. Moreover, some elements of the defensive response to danger are designed to inhibit the function of the offending RNA, preventing its translation into protein and destroying it completely. Unmodified RNA vaccines that resemble danger signals are therefore at risk of being eliminated before they can do their job. The way around these problems, insightfully suggested in Drew Weissman’s publication in 2005, was to add small chemical groups to the building blocks of vaccine RNA. Without these groups, three kinds of Toll-­like receptors could sense the RNA. But with the groups added, no Toll-­like sensors could sense the modified RNA, allowing RNA vaccines to be effective. Comparing

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RNA from infectious pathogens and RNA from the human cellular machinery further explained what was going on: chemical modifications were converting the appearance of vaccine RNA from a pathogen-­associated profile to a profile resembling normal human RNA. This put control back in the hands of the vaccine designers. Because vaccine RNA is manufactured from DNA by a cell-­free biochemical procedure, this makes it easy to use modified building blocks in the synthetic process, allowing scientists to avoid unwanted effects on the immune system. The vaccine designers can then choose whether or not to add defined adjuvants to their modified RNA vaccines. The other problem RNA vaccine companies contend with is more straightforward. RNA is more fragile than DNA when injected into humans. It tends to get broken down. Is there a way to protect it and, at the same time, slip it into cells, especially the surveillance cells of the innate immune system? To do this, Boston-­based RNA vaccine scientists chose the nanoparticle system—­the smallest packaging available, which slips easily through cell membranes. This was the Boston group’s trial that began in Seattle on March 16, 2020—­a phase I study in healthy men and women ages 18 years and older. The purpose was to establish the safety of the vaccine, to assess any adverse effects, and to establish how well it produced a strong immune response. Three US sites recruited 155 subjects into groups that received one of five dosing levels. The vaccine was given as a single intramuscular injection, with a second injection 29 days later. Results showed that the vaccine performed well and there were no significant safety concerns. In phase II, researchers recruited around 600 participants in two groups, below age 55 and over age 55, at 10 sites across eight states. On July 14, 2020, the company behind this leading RNA vaccine published their phase I results in The New England Journal of Medicine. Given as two doses, 28 days apart, their vaccine, mRNA-­

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1273, produced good neutralizing antibodies to SARS-­CoV-­2 in all subjects, equivalent to antibody levels seen in patients recovering from natural infection. Helper T-­cell responses also occurred. And with no concerns about adverse effects, the vaccine progressed to phase III studies and approval in December 2020. Scientists at Imperial College London have developed one notable RNA vaccine with an ingenious innovation. Once it’s been injected in very small quantities, this vaccine automatically amplifies itself. Consider a million doses from just one liter of material—­this gives an idea of what can be achieved. The team at Imperial, led by Robin Shattock, used RNA encoding the stable, full-­length spike protein of SARS-­CoV-­2. They also opted for a familiar package—­the nanoparticles favored by the Boston team. But how does it work? The secret is an extra sequence of RNA encoding an enzyme called RNA polymerase, also known as replicase. Once inside the cells of the vaccine recipient, this enzyme drives the synthesis of more and more RNA encoding spike protein, and this in turn produces more and more spike protein to stimulate the immune response. Self-­amplifying RNA vaccines of this kind are derived from alphaviruses: positive-­strand, nonsegmented RNA viruses (like SARS-­CoV-­2 itself ), often transmitted between mammals by mosquitoes. The alphavirus genome is conveniently divided into two sections: one encodes proteins for the replicase enzyme, and the other for the structural alphavirus protein. But the RNA sequence encoding alphavirus structural protein can readily be replaced by an RNA sequence encoding the vaccine antigen of choice. Scientists at Imperial College developed this system with flu antigens before the emergence of SARS-­CoV-­2. Preclinical studies of COVID-­19 vaccine showed strong immune responses larger than those seen in recovering COVID-­19 patients. German scientists have developed four different mRNA formats as candidate COVID-­19 vaccines. The purpose of four can-

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didates is to compare slightly different chemical modifications to the building blocks of RNA and also to test two different forms of the spike protein. The two spike protein versions in these vaccines consist of full-­length spike protein and a smaller version focused just on the receptor-­binding domain—­the crucial region of the spike protein that fits the shape of human ACE2. The German panel of RNA vaccines also includes a self-­amplifying RNA vaccine like that produced at Imperial College London. Testing of these vaccines initially involved 200 healthy subjects between the ages of 18 and 55. A range of doses was tested in these volunteers in order to establish the optimal dose for further studies. Researchers measured immune responses, and subjects with a higher risk of COVID-­19 infection were included in the second part of the study. The German Biotech company that developed these vaccines also linked up with a Chinese company in Shanghai, giving the Chinese access to these RNA vaccines. These, then, are the eight classes of vaccine in the COVID-­19

vaccine landscape, and they represent the full spectrum of vaccine resources now available to confront all future emergent infectious diseases. On May 15, 2020, the development of some of the leading vaccines detailed here received an even greater impetus when the US government announced a new initiative—­Operation Warp Speed, a program to fast-­track funding, facilitation, and support for selected COVID-­19 vaccines. Among them was the RNA vaccine produced by scientists in Boston. The others included the nonreplicating adenovirus vector vaccine from the University of Oxford, the adenovirus vector vaccine produced in Belgium, the replicating VSV vector vaccine originated in Canada, and the four versions of RNA vaccines produced in Germany. Details of the companies sponsoring these and all other candidates in the COVID-­19 vaccine landscape are provided in the appendix. The US Congress provided $10 billion to Operation Warp Speed; a

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further $3 billion came from the National Institutes of Health. One of the most important components of Operation Warp Speed was the directive to manufacture and stockpile vaccines at risk (financial risk, that is) in case they prove effective. This is exactly what happened in preparation for the great field trial of the Salk polio vaccine in 1954. The global collaborative endeavor to accelerate vaccine development came to fruition in December 2020, with the approval of RNA vaccines and soon after of a leading viral-vector vaccine. An advantage of these vaccines is the broad spectrum of immune responses they induce, preventing evasion by emerging variants.

CHAPTER SIX

Don’t Count on the Magic Bullet While working at the Institute of Experimental Therapy in Frankfurt am Main, Germany, at the turn of the 20th century, the immunologist Paul Ehrlich, who would receive the 1908 Nobel Prize in Physiology or Medicine for his work on the specificity of immunity, conceived the notion of an agent that would annihilate a specific pathogen with no collateral damage to the body. He named this hypothetical agent Zauberkugel—­the magic bullet. It would be wonderful to achieve the perfect vaccine: “Toll! Wir haben die Zauberkugel!” We may succeed, but it’s not very likely. More importantly, we don’t need to go that far. The measles vaccine results in immunity in 98 percent of those vaccinated, meaning almost everyone is completely protected, essentially for their lifetimes. But many highly successful vaccines do not achieve this. The Salk polio vaccine is one of them. The great field trial of 1954 showed that the Salk vaccine was 80 to 90 percent effective in preventing paralytic polio compared with the placebo control group. And when compared with a larger group of one million unvaccinated children, its apparent effectiveness was 60 to 80 percent across the three types of polio virus. Yet this was sufficient for the vaccine to put an end to polio as a major problem in the developed world. According to the WHO, most routine childhood vaccines are effective in 85 to 95 percent of recipients. At the lower end of effectiveness are the seasonal flu vaccines. Recent studies by the Centers for Disease Control and Prevention (CDC) in Atlanta,

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Georgia, show that flu vaccination reduces the risk of flu illness by between 40 and 60 percent in the general population during seasons when the flu vaccine is well-­matched to circulating flu viruses. This is immensely valuable in terms of serious illness. A 2014 study at centers of excellence across the United States showed that flu vaccination reduced children’s risk of admission to an intensive care unit (ICU) with a flu-­related illness by 74 percent during the years 2010 to 2012. A later study showed that between 2012 and 2015, flu vaccination among adults reduced the risk of being admitted to an ICU with flu by 82 percent. And in a highly susceptible group, elderly adults, flu vaccination has recently reduced the risk of flu-­associated hospitalizations by around 40 percent. Though imperfect, flu vaccinations are still highly worthwhile. In concert with these vaccination efforts, large-­scale immunization of young children with a live attenuated nasal vaccine called FluMist helps protect their family members, especially grandparents. Seasonal flu is difficult for vaccinologists. The flu virus avoids pre-­existing immunity by changing its coat every year. This change happens in two ways: drift and shift. Drift describes small changes in the viral coat that happen all the time. Shift is a sudden, major change, which only happens once in a while. Influenza is able to infect people, birds, pigs, horses, and other animals, making it more likely that cells will be infected by two different strains. When this happens, the genes of the two strains can combine to form a new virus. Two important flu proteins, whose job is to gain entry to human cells, protrude as spikes from the virus and exist in different forms. If these appear in a new combination, the human population has little pre-­existing immunity. This is very unlikely to happen with SARS-­CoV-­2. So far, researchers who are tracking the genetic changes in SARS-­CoV-­2 tell us that it seems relatively stable. It appears to be undergoing around two mutations per month during its

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spread around the globe. This is about one-­third to one-­half the rate of change seen in flu viruses. Coronaviruses also differ from flu viruses in another important way that works to reduce mutations. Coronaviruses proofread their genome each time they replicate, cutting out things that don’t seem right. In this way the viral genome resists change. As SARS-­CoV-­2 continues its spread around the globe, its genetic makeup is certainly changing, but the alterations so far have not led to any significant variations in virulence—only in transmissibility. Some mutations have been reported in the spike protein itself. Could these mutations compromise the effectiveness of vaccines? Studies with MERS-­CoV vaccines are reassuring here. When Sarah Gilbert and her colleagues at Oxford wanted to be sure that their ChAdOx1 MERS vaccine could cope with changes in spike protein composition, they used a panel of engineered viruses (called pseudotypes) expressing a wide range of inserted spike proteins. Antibodies from vaccinated volunteers could prevent these viruses from infecting target cells in tissue culture no matter which variant of the spike protein the viruses were using. Although increased virulence is unlikely to arise from changes in SARS-CoV-2, increased transmissability was observed in variants appearing in late 2020. These were the result of 17 changes, eight of which were in the spike protein. However, because vaccines deliver more than 1,000 amino acids to the immune system, this small proportion of changes is very unlikely to compromise vaccine efficacy. We can be sure that the immune response is effective against the virus in the majority of people who encounter it. This is why 80 percent of people with symptoms recover without hospitalization, and it is also why many have no symptoms at all. An in-­ depth study by Allessandro Sette and his colleagues at the La Jolla Institute of Immunology in Southern California made multiple, small synthetic peptides matching the sequence of SARS-­CoV-­2 proteins in order to study immunity in patients. They found that

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most patients recovering from SARS-­CoV-­2 infection had strong killer T-­cell responses and helper T-­cell responses against the virus, especially the spike protein. And these cellular responses correlate with high levels of antibodies, including antibodies in the mucosal surfaces lining the respiratory tract. As well as spike protein, responses to the core protein and the envelope protein were also detectable. But the most intriguing aspect of this study was that helper T-­cells able to recognize SARS-­CoV-­2 were present in 40 to 60 percent of people who had never been exposed to COVID-­19. This strongly suggests that immune responses to common cold coronaviruses can help fight SARS-­CoV-­2. In another US study, this one of more than 200 patients recovering from SARS-­CoV-­2 infection, antibodies specific for the receptor-­binding domain of the spike protein were seen in all patients one week after confirmed diagnosis. And the antibodies in these patients were capable of neutralizing the virus. The higher the antibody level, the greater the neutralizing power against SARS-­CoV-­2. Some researchers are concerned about how long this infection-­ induced immunity remains. Can we learn anything about the duration of antibody responses from previous epidemics? Following infection with SARS-­CoV-­1 (the virus causing the 2002–­2004 SARS outbreak), concentrations of antibodies remained high for approximately four to five months before declining slowly over the next two to three years. In a similar way, neutralizing antibodies following infection with MERS-­CoV persisted for up to three years in recovered patients. A further reason researchers were optimistic from the start is that in preclinical animal studies, a range of vaccines across classes were able to induce much higher levels of antibodies than those seen in recovering patients. When it comes to benign coronaviruses (cold viruses), the duration of natural immunity may be less than for serious infections. Reviewing previous studies, Derek Cummings of the Uni-

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versity of Florida and his colleagues concluded that while antibodies to SARS and MERS viruses persisted for up to three years, immunity to common cold viruses (of a single serotype) lasted little more than one year. But which of these patterns might apply to SARS-­CoV-­2? A study by Katie Doores and colleagues at Kings College London in 2020 indicated that in patients who experience only mild symptoms of COVID-­19, the persistence of antibodies is shorter than in more serious infections and resembles responses to common cold coronavirus infections. Persistence of antibodies is not the only factor in resistance to reinfection, however. The quality of antibodies and the levels of memory T-­cells are also important. One question asked by people who were worried about failure

in the search for a COVID-­19 vaccine relates to HIV. Why can we make a vaccine for COVID-­19 when we haven’t for HIV? In most infectious diseases, a good proportion of infected people make a successful immune response that defeats the pathogen naturally. This is very much the picture with SARS-­CoV-­2. But this doesn’t happen with HIV. In HIV-­positive patients, the immune system delivers a wide range of responses, but none eliminate the virus, and none stand out as being particularly effective. This makes it hard to know what kind of immunity to aim for with a vaccine. The fact that HIV lives in immune cells adds a further major complication. Some immune responses triggered by HIV actually help the infection. Certain interfering antibodies block neutralizing antibodies, while virus coated with so-­called enhancing antibodies can enter immune cells more easily. HIV hoodwinks the immune system by reducing MHC molecules on infected cells, which would otherwise flag them as infected and attract killer T-­cells. Another problem is the diverse nature of HIV viral subtypes: more than a dozen exist, with different geographic distributions. Constructing a vaccine to protect against all of them is an

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enormous challenge. Yet another obstacle is the subtle changes in the virus within individuals during the chronic infection, which keeps the virus one step ahead of the immune system. HIV can also hide in the DNA of the infected host as a so-­called provirus. When HIV enters a host T-­cell, HIV RNA is turned into HIV DNA. This HIV provirus can then become integrated with the host cell DNA and thereby evade immunity. Attempts to produce an HIV vaccine in the late 20th and early 21st centuries failed for these reasons. But in 2009, researchers devised a new approach. An initial live viral vector HIV vaccine first presented the immune system with elements of the virus. A second, booster dose confronted the immune system with a nonliving mix of purified HIV proteins mixed with the adjuvant alum. This combination of HIV antigens in two very different guises was the first vaccine to show a modest but significant protective effect of 31 percent. The search for more effective HIV vaccines continues. SARS-­CoV-­2 is much closer to other single-­strand RNA viruses such as mumps, for which highly effective vaccines already exist. But if vaccination against SARS-­CoV-­2 is problematic for whatever reason, then vaccination could become just one aspect of management in which newly discovered drug treatments will play a major role. Test and trace programs, surveillance technologies, and transmission prevention measures would also be used in concert to minimize the impact of the disease. But I don’t think this is the likely outcome. Does SARS-­CoV-­2 attempt to evade immunity? Not in the manifold ways that HIV employs. But there are some mechanisms SARS-­CoV-­2 uses that are not seen in its predecessor, SARS-­ CoV-­1. Both viruses rely on shape-­changes in the spike protein to enter human cells, and these changes are initiated by enzymes contributed by the target cell. The enzymes split the spike into two parts, unsheathing the membrane fusion elements that allow

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the virus to enter target cells in our lungs, liver, kidneys, and blood vessels. But the SARS-­CoV-­2 spike employs an extra target cell enzyme (furin), and this allows the new virus to keep its membranefusion elements hidden from the immune system until nearer the moment of entry. This hiding of receptor-­binding elements may reduce targeting by antibodies, but this kind of problem can be countered by vaccines consisting of just the binding domain itself or fragments of it. The RNA vaccines that encode just the receptor-­binding domain, for example, adopt this strategy. Once SARS-­CoV-­2 has gained a foothold, an additional devious immune evasion strategy may begin to exert its effects. Studies led by Kei Sato at the University of Tokyo suggest that one SARS-­CoV-­2 gene encodes a regulatory protein that inhibits the production of type I interferons. Interferons play a vital role in amplifying innate immune responses, switching up the activity of surveillance cells in the first line of immune defense. By down-­regulating interferon production, it seems that SARS-­CoV-­2 can dampen immune defenses at an early stage in a way not seen with SARS-­CoV-­1. Many other viruses are known to employ equivalent immune evasion strategies. Is SAR-­CoV-­2 capable of additional subterfuge in its battle with the human immune system? Researchers at the University of Pittsburgh and Cedars Sinai Medical Center in Los Angeles have raised one possibility. Their modeling studies of the SARS-­ CoV-­2 spike protein identified a potential sequence of amino acids forming a so-­called superantigen. Far from being really good antigens, superantigens confound the immune system by binding to the framework of T-­cell receptors, avoiding their key viral-­recognition component. By linking surveillance cells and T-­cells in this way, very large numbers of irrelevant T-­cells can become activated with no antiviral value. These may contribute to intense inflammation. The authors of the study suggested that a

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rare mutation of SARS-­CoV-­2, predicted to strengthen this putative super-antigen, might be responsible for pediatric inflammatory multisystem syndrome, the very rare COVID-­19 syndrome in children in which multiple organs become inflamed. But subsequent studies of the genetic sequences of virus samples from cases of this syndrome conducted by Judith Breuer and colleagues at University College London found no particular variant of SARS-­ CoV-­2 to be responsible. Instead, a range of viruses identical to that seen in the general population were detected in these children. There has been a great deal of research in drug management of COVID-­19. One early breakthrough was the antiviral drug remdesivir. In more than a thousand COVID-­19 patients studied by John H. Beigel and colleagues at the US National Institute of Allergy and Infectious Diseases, intravenous remdesivir shortened recovery time from around 15 days to 11 days. The report was published in the New England Journal of Medicine on May 22, 2020. The company that developed this drug, Gilead, is planning an inhalable version for administration through a nebulizer in an effort to benefit a wider range of patients. A second treatment breakthrough, at Oxford University, was announced in a press release on June 16, 2020. In a large study of potential drug treatments involving more than 11,000 patients, the drug dexamethasone was shown to reduce death by up to one-­ third in hospitalized patients with severe respiratory complications of COVID-­19. News reports characterized the drug as “old” and “cheap as chips.” Dexamethasone is a member of a miraculous family of drugs called corticosteroids, and it’s helpful to understand their discovery. In the 1930s, two endocrinologists, Philip Hench and Edward

Kendall, at the Mayo Clinic in Rochester, Minnesota, were working on secretions from the adrenal gland situated on top of the

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kidneys. One of the mysterious products that they discovered, “compound E,” seemed particularly interesting. They already knew that removal of such glands had marked effects in laboratory animals. Could compound E be a key player in adrenal gland function? World War II delayed their research, but by September 1948 Kendall and Hench had prepared a synthetic version of compound E in sufficient quantity to try it as a medicine. Armed with a good supply of the new substance, they decided to treat a middle-­aged woman suffering from severe rheumatoid arthritis. The symptoms of arthritis are reduced or disappear in pregnant or jaundiced women, suggesting that natural hormones control the disease. This knowledge was enough to persuade them to take the plunge. The patient was a serious case. She was suffering severe pain and joint swelling, and she could barely move. But as the series of injections started to take effect, her symptoms began to melt away. After a complete course of this natural anti-­ inflammatory agent, she seemed fully restored to health. She got up and walked away from the bed in which she’d been confined for years. The new therapy, soon to be called corticosteroid treatment, appeared to be a miraculous cure. Sadly, it soon became clear that compound E’s effects did not last. The magical new treatment could quickly and powerfully suppress inflammation and its devastating consequences in a way never seen before. But the underlying course of the crippling disease had not changed. And in the years that followed, the serious side effects of prolonged use of corticosteroids gradually emerged. Among them were weight gain, high blood pressure, thinning of the bones, increased risk of diabetes, susceptibility to infection, disturbed sleep, cataracts, and glaucoma. But Kendall and Hench’s discovery remained a massive advance. The horrors of uncontrolled inflammation could now be managed by the judicious use of corticosteroids, and this could be lifesaving. In 1950 Kendall, Hench, and their Polish collabora-

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tor Tadeus Reichstein received the Nobel Prize in Physiology or Medicine for their discovery. The reason dexamethasone is effective in seriously ill COVID-­19 patients is that life-­threatening symptoms are caused by intense inflammation. A host of intercellular messengers (small proteins) released by immune cells orchestrate and amplify the immune response and promote the attack on the invader. But in some circumstances things go too far, and the response begins to damage cells and organs, a course of escalating events that can prove fatal. The corticosteroid dexamethasone can calm this overreaction and save lives. When it came to doubts about the search for a coronavirus vac-

cine, one of the most frequent questions I was asked centered on the common cold: If we can’t immunize against the common cold, how could we hope to make a vaccine against SARS-­CoV-­2? But the common cold as we know it is very different from COVID-­19. Many different viruses cause colds and the list is quite long: rhinoviruses are most often to blame, and there are 99 different serotypes of these. Then there are coronaviruses, adenoviruses, influenza viruses, parainfluenza viruses, human respiratory syncytial viruses, human metapneumoviruses . . . In all there are more than 200 viral types associated with colds. Vaccination is impossible, and after all it’s just a minor illness. Who would go to the trouble and expense of deploying a common cold vaccine? There is an answer to this question. If you rely on the fitness and consistent high physical performance of your employees, and if your staff live together in very close proximity, and if you have ample financial resources, then you might want to deploy a cold vaccine against at least one of the viral culprits. And this is just what the US Army did in 1971 with a live virus vaccine against adenovirus. The use of this vaccine over several decades did much to establish the safety of attenuated adenoviruses in large

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populations, encouraging scientists over the last three decades to develop adenoviruses as vaccine vectors for other infectious diseases. The Oxford vaccine, ChAdOx1 nCoV-­19, one of the first selected for fast-­tracking by the US government, is based on a chimpanzee adenovirus, chosen to avoid pre-­existing antibodies to human adenoviruses. The Chinese Ad5-­vector vaccine exploits the same delivery principle using a human adenovirus. The first results obtained with this Chinese vaccine in human subjects were published in The Lancet on May 22, 2020. They were encouraging. A single dose of the Ad5-vector COVID-­19 vaccine induced antibody and T-­cell responses rapidly in most participants. T-­cell responses peaked at day 14 after vaccination, and antibodies peaked at day 28. Around three-­quarters of those vaccinated in the highest dose group developed antibodies that could neutralize the SARS-­CoV-­2 virus. As expected with a live virus vaccine of this type, killer T-­cell and helper T-­cell responses were observed in all dose groups. The public reaction to the results was mixed. “Underwhelming” was a term appearing in some news reports. But this is to misunderstand what is expected. As the authors took care to point out, the adenoviral vector in this study met with pre-­existing antibodies (due to previous encounters with natural cold viruses), and this reduced its effectiveness. COVID-­19 vaccines using chimpanzee adenovirus vectors should encounter this problem much less frequently. The vaccine, produced by Can Sino Biologics, is a promising pathfinder, produced at extraordinary speed. And since there weren’t great differences between the three dose levels, and only transient adverse events of the usual kind were encountered, the two lower doses of this vaccine were entered into phase II trials. There wasn’t long to wait: on July 20 the results of the phase II Can Sino trial were published in The Lancet. The trial had involved more than 600 volunteers and investigated the vaccine at two dose levels. Both neutralizing antibodies and T-­cell antiviral

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responses were observed in the majority of vaccine recipients after a single dose. From early in its development, this vaccine had progressed in partnership with the Institute of Biology at China’s Academy of Military Medical Sciences, and on June 25, 2020, less than six months after the genetic structure of the SAR-­ CoV-­2 virus was first published, the Chinese military approved the vaccine (on the basis of phase II results) for use in military personnel as a “specially needed drug.” This constituted the historic first approval of a vaccine against COVID-­19. This chronicle of concerns about the chance of an effective

vaccine would be incomplete without mention of enhancing antibodies. These antibodies were observed in animal responses to some experimental vaccines against SARS and MERS. Re-­ exposure of the vaccinated animals to the virus occasionally exacerbated the infection. What was going on? Shouldn’t a successful battle with infection always protect against future reinfection? The problem was with the antibodies themselves. When immune cells bind the tail piece of antibodies, this triggers responses that cause inflammation. And when viral particles bound by these antibodies enter the cell, they encounter Toll-­ like receptors on internal membranes, which trigger still more inflammation. In the experimental vaccine trials described above, the existing antibodies in some of the previously vaccinated animals were ineffective against the virus but still triggered all of the inflammatory responses. Rather than providing protection from reinfection, the combination of weak antibodies and unchecked inflammation exacerbated (enhanced) the disease. The good news was that this problem was not observed in preclinical studies of the candidate COVID-­19 vaccines. Forewarned of the dangers of enhancing antibodies, vaccine designers strove to stimulate high levels of effective antibodies by using the spike protein, its receptor-­binding domain, and the regions that trigger

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membrane fusion. They’re also using adjuvants, delivery formats, and vaccine packaging more likely to steer the immune response away from a damaging pathway and into a protective pathway. The latter also tend to favor killer T-­cell responses against the virus. These endeavors proved successful when COVID-19 vaccines delivered efficacy rates of 70 to 95 percent, greatly exceeding the 50 percent protection required by regulators. Happily we have just gained a highly relevant benchmark for the success of immunization using a viral vector vaccine in another infection—­the first such vaccine to be licensed. For two years, in the remote northeastern corner of the Democratic Republic of the Congo, an epidemic of Ebola continued to rage. Ebola is a deadly pathogen. In this particular outbreak, 3,470 people were infected and 2,280 died. But the outbreak was declared over on June 29, 2020, largely thanks to a new vaccine developed by Merck. The vaccine achieved a protection level of more than 80 percent among the 300,000 people immunized. Those who were vaccinated but nevertheless showed signs of disease experienced a much milder illness. The Merck Ebola vaccine is a replicating viral vector vaccine, and there are 17 vaccines of this class in development to combat COVID-­19. To acceler ate the delivery of billions of doses of COVID-­19

vaccines, governments did not require licensure to roll out large-­ scale vaccination. Nevertheless, all vaccines must have first passed through the three phases of rigorous clinical trials to demonstrate their safety and effectiveness. For most vaccines, reaching this final goal depends on the successful outcome of phase III clinical trials in tens of thousands of people. Success also depends on continuing surges in infection rates in order to collect data on natural encounters with the virus experienced by vaccinated individuals. Several hundred such encounters are required in order to calculate the percentage of vaccinees protected by the vaccine.

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This is not difficult as surges continue when governments ease lockdowns—­encouraging a return to normal life—­to safeguard economic recovery. In addition to manufacturing large stocks of vaccines, Operation Warp Speed also financed phase III clinical trials. Multiple organizations and partnering activities facilitated these trials. Central to this effort is the Biomedical Advanced Research and Development Authority (BARDA), a US Department of Health and Human Services office responsible for the procurement and development of medical countermeasures against grave threats such as bioterrorism, pandemic influenza, and emerging diseases. BARDA is a young organization, formed in December 2006, and is housed close to the Capitol, not far from the historic National Mall in downtown Washington, DC. Because of the unprecedented acceleration of vaccine development, phase III trials of the leading RNA vaccine, produced by the Boston-­based company Moderna, commenced in July 2020. The study recruited 30,000 volunteers between 19 and 55 years of age. The effort also vaccinated additional groups of elderly volunteers, including those vulnerable to serious disease. Multiple trial sites in the United States were employed and also some international sites to maximize exposure to natural infection, again to prove the effectiveness of the vaccine. Other fast-­track vaccines, including the Oxford-­AstraZeneca vaccine, started phase III trials at the same time. Countries such as Brazil, facing a burgeoning epidemic, were included in tests of vaccine effectiveness. After confirmation of effectiveness in phase III trials, vaccines then receive an emergency use authorization by the FDA. Roll-­out to the general population follows in a phased process using stockpiled vaccine, beginning with groups most at risk of disease. As of this writing, there are 232 contenders in the COVID-­19

vaccine race according to the WHO database. There are 35 other

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live viral vector and 16 other RNA-­based vaccine approaches. Protein subunit, DNA, and inactivated whole-­virus vaccines were not included in the Operation Warp Speed plan at first pass, but they looked promising in preclinical and clinical studies and further developments would soon be announced. The success of the approved RNA and viral-vector vaccines attests to the success of Warp Speed and to the effectiveness of the wider international endeavor to expedite vaccine development. But ultimately it is not about crossing the finish line first. Because several vaccines exploit the same principles, with only subtle variations, we have seen multiple COVID-­19 vaccines. Availability, stability, convenience of use, number of required doses, and route of administration are all contributing factors for ultimate vaccine success.

CHAPTER SEVEN

Overcome the Hurdles One of the most challenging hurdles to the successful delivery of vaccines relates to the fears, doubts, and uncertainties surrounding the practice of vaccination. This isn’t new. In July 2003, opposition to polio vaccination in Northern Nigeria escalated suddenly. The leadership of both the prominent Muslim umbrella group Jama’atul Nasril Islam and Nigeria’s Supreme Council for Sharia questioned the polio vaccine’s safety and encouraged a boycott. The incumbent president of the council went further, publicly claiming that the vaccine contained antifertility drugs, viruses causing HIV/AIDS, and a simian virus likely to cause cancer. The implication was that polio vaccination was a Western plot against Islam. But by the end of 2004, the mood had changed. In a powerful public gesture, the Kano State governor, Ibrahim Shekarau, allowed President Obasanjo to publicly administer the vaccine drops to his one-­year-­old daughter. In 2006, the newly appointed Sultan of Sokoto, Muhammadu Sa’ad Abubakar III, the spiritual leader of Nigeria’s 70 million Muslims, became a great advocate of polio immunization. By the summer of 2019 Nigeria had seen no polio cases for three years and the nation now stands at the cusp of becoming polio-­free. The international service organization Rotary International continues to carry the WHO campaign forward. In the short film Plandemic, whose creators are against COVID-­19 vaccines, a disaffected researcher and a sympathetic interviewer together mourn the ruined career of the complainant,

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barely able to credit the catalog of wrongs the victim has suffered at the hands of “they,” the powerful “great force” manipulating the pandemic in order to make money from the sales of vaccines and drugs. The film’s argument hinges on an illusory idea of a powerful, cohesive force, encompassing multiple high offices in government, health care, philanthropy, and science, manipulating global events for personal gain at the cost of millions of lives. There is no “they.” There is no “great force.” In my book The End of Plagues: The Global Battle against Infectious Disease, I described the large-­scale antivaccination movement in England in the 19th century, largely motivated by the introduction of compulsory vaccination against smallpox. But the 20th century was also troubled by groundless fears about vaccines. In 1974 a research paper in the respected British Medical Journal described 36 children with epilepsy and linked their condition with vaccination against whooping cough (pertussis). A number of British experts then expressed concerns about adverse reactions to whooping cough vaccine, including convulsions and a shock-­like state. The vaccine quite often caused fever and distress in a minority of infants, from which they quickly recovered, but concerns about the risk of permanent brain damage increased, and over the next few years vaccination rates in Britain fell sharply, reaching a low of 31 percent in 1978. Inevitably, whooping cough rates went up, together with the serious complications associated with this dangerous infection. In the early decades of the 20th century, before the advent of a protective vaccine, whooping cough infected millions, and many thousands of children died. But as immunization helped control the disease, the discussion about vaccine safety loomed large for every parent. When, in 1979, the time came for my own small daughter to be immunized, I was as worried as any parent. It’s very hard to imagine your infant with a high fever, delirious and screaming at your desperate attempts to bring down her tempera-

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ture with tepid bathing and other anxious strategies. We went ahead with vaccination, and it did produce some fever and a great deal of parental anxiety, but our daughter recovered a day or two later. In the United States, allegations regarding the same DTP (diphtheria, tetanus, pertussis) vaccine would do much greater damage to vaccine confidence. A 1982 TV program titled DPT: Vaccine Roulette dramatically and graphically blamed the vaccine for catastrophic brain damage in infants. The program led to the formation of a new antivaccine group—­Dissatisfied Parents Together—­which would campaign successfully for representation on national vaccine safety committees and the introduction of vaccine safety monitoring and reporting systems. When numerous successful lawsuits, brought by personal injury lawyers against vaccine companies, began to threaten the existence of the vaccine manufacturing industry, the US government acted. In 1986 Congress passed the National Childhood Vaccine Injury Act, establishing a federal no-­fault system to compensate victims of injury caused by vaccines mandated by law. The majority of claims filed through this system, which gives claimants the benefit of any doubt, have been related to damage allegedly caused by DTP. In the 1990s, however, a series of large-­scale studies involving hundreds of thousands of children firmly established that there is no link between serious neurological illnesses in children and immunization with the DTP vaccine. The Institute of Medicine, an independent research institute devoted to investigating such matters, and the CDC, which completed a large-­scale study with the University of Washington in 1994, both confirmed this with detailed studies. Another study in 2001 comparing 340,000 children given DTP vaccine and 200,000 who remained unvaccinated showed no difference in the incidence of neurological disorders. The new antivaccination movement in the United States was based on groundless fears.

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A second major blow to the acceptance of safe vaccines occurred in 1998 when a paper by British surgeon Andrew Wakefield appeared in the UK medical journal The Lancet. Wakefield and his coresearchers studied 12 children, 9 of whom were autistic and suffering gastrointestinal problems and immunological deficiencies. The paper suggested that the onset of behavioral and gastrointestinal problems in these children was associated in time with the combination vaccine for measles, mumps, and rubella, commonly known as MMR. In the discussion, the authors speculated that the vaccine could have triggered the disorders. This study galvanized vaccine safety pressure groups and revived antivaccine sentiment in the United States and the United Kingdom. In England and Ireland, the uptake of MMR vaccination declined, and the incidence of measles and its complications increased. In 2004, the UK General Medical Council found Wakefield guilty of dishonesty and flouting ethical protocols in the conduct of the study, and The Lancet retracted the report. In the United States, which had become clear of measles in the year 2000, the reduced uptake of MMR vaccines due to these groundless fears about safety resulted in a measles outbreak in 2008. Eventually, 131 cases were reported, of which 11 percent were hospitalized. Measles causes an extremely unpleasant sickness in children, and 7 percent of those infected experience serious complications such as pneumonia, middle-­ear infections, and encephalitis (inflammation of the membranes surrounding the brain). The latter can precipitate fits and produce neurological disorders that persist for months. In Britain shortly before the introduction of vaccination in 1963, 1 in 5,000 children died from the infection. In 2001 and 2004, studies by the Institute of Medicine showed that MMR vaccine is not a cause of autism. Given continued public concern, other groups conducted similar vaccine safety studies. In 2014 Australian researchers at the University of Sydney

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analyzed all the studies on MMR vaccines. In this meta-­analysis, researchers processed data on more than 1.25 million children included in investigations conducted in several countries over 15 years. It found that there was no connection between autism or autistic spectrum disorder (ASD) and MMR vaccination. It also found that there was no connection between autism/ASD and thiomersal (a preservative used in some vaccines before 2001, though never in MMR). And there was no connection between autism/ASD and mercury (present in thiomersal). Still, in the year 2019, the United States saw 1,282 cases of measles across 31 states. The WHO has concluded that the rise in measles is a direct result of antivaccination movements. The risks posed by vaccines are extremely small compared with other hazards. The estimated chance of dying in a transportation accident in the United States is 1 in 6,000 each year; examples of real vaccine damage include the very rare cases of paralysis (1 in 2.7 million vaccinations) with the Sabin live virus polio vaccine. This live virus vaccine is no longer used in nations long free of polio. Allergies to egg proteins present in flu vaccines produced in chicken eggs can cause other problems. For several decades, vaccine manufacturers used gelatin to stabilize the MMR vaccine and prolong its shelf life, posing a problem for those with rare allergies to this animal protein. In 2009 a vaccine against swine flu (H1N1 influenza) used in several European countries caused rare cases of narcolepsy in recipients. Studies suggest that 1 in 52,000 recipients developed this sleep disorder. In 1998, a vaccine against the gut pathogen rotavirus, which causes diarrhea in infants, was introduced. This infection is fatal for many children in developing countries. A rare problem soon emerged in the form of intussusception, a bowel obstruction, in 1 in every 20,000 to 100,000 vaccine recipients. The early detection of this problem testified to the effectiveness of the Vaccine Adverse Event Reporting System. An oral rotavirus vaccine without this problem became available

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in 2006 and is now a part of routine childhood vaccinations in many nations. These genuine vaccine problems are almost never taken up as campaign issues by antivaccination groups. Unlike the 19th-­century antivaccination movement, formed in response to compulsory vaccination, the two great setbacks to disease prevention in the modern age—­the DTP controversy and MMR concerns—­began with bad science. But once the erroneous information is out there, it is very hard to put things right with consensus science. Perhaps the best chance we have is to point with certainty at the real cause of developmental disorders. In the case of autism, this is sadly impossible at the present time. But in the case convulsive seizures leading to arrested development, biomedical science can now explain the reason for the problem, which has no connection to the DTP vaccine. In 2006, Samuel Berkovic, working at the University of Melbourne, traced the source of the disorder to a specific genetic defect that affects sodium transport in the brain. Infants with this problem appear perfectly typical until the first seizure occurs in the early months of life. This means the illness appears suddenly, often, coincidentally, soon after vaccinations administered between one and two months. At last there is an explanation to salve the guilt and anguish of parents who believed their decision to vaccinate permanently harmed their children. In these cases, the damage is tragically both natural and inevitable. In 2011, Paul A. Offit, Chief of the Division of Infectious Diseases at the Children’s Hospital at the University of Pennsylvania School of Medicine published an important and illuminating account of childhood vaccine safety and the antivaccination movement: Deadly Choices: How the Anti-­ Vaccine Movement Threatens Us All. Antivaccination sentiment ran high in the 19th century and again in the last three decades of the 20th century, and this continued through the first two decades of the new millennium. But midcentury attitudes were very different. And nowhere more so

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than in the United States. When Franklin Roosevelt, inaugurated the National Foundation for Infantile Paralysis, later called the March of Dimes, a grassroots fervor to beat polio and discover a vaccine united the nation. The foundation would become the model for private charitable fundraising, quickly growing into the largest voluntary health organization in America. Its success in promoting awareness, mobilizing communities, sowing the seeds of hope, and collecting funds for the care of patients and the search for a cure would be unparalleled in the 20th century. One of its most successful strategies was to harness the power of show business. Its public relations operation developed promotional spots for radio and motion pictures, enlisting the help of movie stars and entertainers much loved by the American public. The first of these was Eddie Cantor, who came up with the “March of Dimes” slogan, adapted from the newsreel title “March of Time,” popular in all movie theaters at the time. On the morning of April 12, 1955, Jonas Salk’s vaccine, funded by the March of Dimes, was shown to be effective against polio. As the jubilant media announced the news, the nation celebrated as one. The New York Times headlined “Lasting Prevention of Polio Reported in Vaccine Trials,” while the San Antonio Light chose “Salk Vaccine Whips Polio.” “Polio Routed!” appeared in the New York Post. The public clamored for the vaccine and heralded Jonas Salk as a hero. I can draw many parallels to the fear of polio and the disruption to normal life in the middle of the 20th century and the COVID-­19 pandemic starting in 2020. In all cases, great hopes were vested in a vaccine. Although the WHO lists vaccine hesitancy as one of its concerns, I believe most people are likely to be convinced of the need for effective vaccines to end the emergency and achieve the resumption of normal life. Is it right to attribute all issues of vaccine refusal to superstition, conspiracy theories, and irrationality? Of course not. Much

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more subtle sociopolitical forces are at work, and these have been perceptively analyzed in a number of recent studies. Sociologist Jennifer Reich, in her book Calling the Shots: Why Parents Reject Vaccines, describes the practice of free-­riding: children who are unvaccinated through parental choice getting a “free ride” thanks to the immunity of those whose parents have complied with vaccination policy (accepting the small risk of adverse reactions). Reich’s research reveals that the majority of vaccine-­avoiding parents in the United States in the 21st century are well aware of this accusation. They maintain their sense of themselves as good people by arguing that since these other children are vaccinated, they are not at risk from their own unvaccinated children. And they believe that by devoting themselves to carefully nurturing their children, maintaining a healthy diet, good hygiene, and a loving, caring family environment, they as parents can best protect their own offspring from harm. Such parents may be hypervigilant. Despite an awareness of the fact that, if everyone behaved like them, dreaded childhood infections would return to levels seen before the introduction of immunization, their feelings of anxiety about adverse reactions to vaccines outweigh the cold calculation of infection risks in the modern world. Their mission as parents is to honor and protect the bodily integrity of their children, and this, of course, is a deeply rooted cultural imperative as well as a fundamental instinctive drive, especially for mothers. The fact that this puts at risk those undervaccinated children sharing the community—­the much larger group of youngsters from poorly resourced backgrounds who miss appointments because of disorganized parenting or underprivileged circumstances, does not weigh heavily against the deeply felt belief of vaccine avoiders that they are the only people properly qualified to protect their children. Mothers are central in the vaccination debate, and some experts believe that a shift in the cultural rhetoric is needed to ensure that moth-

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ers are empowered to make informed decisions for their children while not receiving all the blame when things go wrong. Such are the reasons, Reich and others argue, for vaccine resistance in the modern world: an individual sense of entitlement to public resources without a shared responsibility to the community. This is the position adopted by parents claiming the right to make their own choices because of their high levels of care and commitment to their own families. Experts believe that the parental debate about whether to conform to mandated state vaccination schedules would benefit from a shift away from individual rights, liberties, and self-­ determination toward societal needs and benefits whose fulfillment returns advantages for all. Among the recommendations for improvement that a modern sociological analysis distills is the need to alter perceptions of vaccines as only for individual benefit. Marketing vaccines to the public should also promote their great societal advantages, an aspect well understood for a vaccine like rubella, a disease that can be damaging to fetuses in early pregnancy. Every child receives this vaccine, not for their own benefit but to protect pregnant women from infection. Rebalancing perceptions of individual liberty versus collective responsibility and the good of the community is essential in the drive to reduce elective vaccine avoidance. Greater transparency in vaccine policy is another area for improvement. Many parents are concerned that they do not know how doctors decide on vaccine schedules (the frequency and number of vaccine doses) and fear such doses may overload their children, given that routine childhood vaccines now protect against 18 different diseases. But this is not the case. A small number of antigens from each pathogen can now be used to generate immunity, totaling less than a single traditional smallpox vaccination, for example. In the United States, the CDC generates vaccine schedules. Within the CDC, the Advisory Committee on

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Immunization Practices (ACIP) recommends schedules for states to adopt and enforce through school attendance regulations. But those who generate the schedules have a responsibility to maintain the trust of parents and practice transparency in potential conflicts of interest. Concerns arise when prominent regulators take jobs in profit-­making companies that manufacture vaccines. That only very modest revenues are generated by vaccines is less important than public perceptions. Historian Elena Conis, in her book Vaccine Nation: America’s Changing Relationship with Immunization, describes the social factors that dictate vaccine use and perceptions. For example, she explains how infections, like chicken pox (varicella), once perceived as mild and part of the passage of childhood become designated as a “threat” when new vaccines are introduced, creating an imperative to vaccinate that didn’t exist before. Now, in the United Kingdom, children are not routinely vaccinated against varicella, and the vaccine is only offered to individuals likely to come into contact with people vulnerable to severe effects from chicken pox, such as those receiving chemotherapy. The 21st-­century debate about vaccine hesitancy and vaccine avoidance often seems in danger of overshadowing the fact that the great majority of parents strongly support routine vaccination. A 2014 study in the United States found that 80 percent of parents believe that preschool children should stay up to date with their vaccination schedule and 74 percent said they would consider removing their child from a care facility in which 25 percent or more users were unvaccinated. This did not mean that awareness of the risk of adverse reactions was absent. A quarter of respondents expressed concerns. But the signs are that trust in vaccines is on the increase. It is to be hoped that the experience of the COVID-­19 pandemic will lead to a further strengthening in the approval of vaccines as an effective strategy against childhood infections and a pivotal weapon against emergent epidemic

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diseases. When Howard Bauchner, editor-­in-­chief of the Journal of the American Medical Association, interviewed vaccinologist Paul Offit on June 3, 2020, he was asked to name his greatest concern about the response to COVID-­19. His answer was not about vaccine hesitancy. Instead he said his greatest fear was that standards of vigilance in vaccine safety would be relaxed in the haste to deliver COVID-­19 vaccines. Such a relaxation would betray the trust that the great majority of people place in vaccination. In addition to the suspicions, doubts, and uncertainties sur-

rounding the introduction of new vaccines, a host of purely practical difficulties must be overcome in order to provide vaccine doses on a global scale. China, as manufacturer to the world of low-­cost consumer goods, may be better placed than some nations in terms of facilities that can readily be repurposed to manufacture vials, syringes, and the like, essential for vaccine production. In the United States the logistics of reaching the target quantity of 300 million doses of COVID-­19 vaccine in 2021 began to be addressed at the outset of the operation to fast-­track vaccines and produce vaccine stocks “at risk.” Companies in the United States such as Corning Pharmaceutical Technologies have been the traditional suppliers of scientific glassware since 1879. In a memorable phrase, the vice president and general manager of the company, Brendan Mosher, reportedly assured authorities that “glass won’t be the critical bottleneck” in the delivery of COVID-­19 vaccines. Other commentators, however, took the view that it could take two years to produce the required syringes and needles to vaccinate the US population and that a long-standing world shortage of the sand used to make medical-­grade glass vials could impact targets. Becton Dickinson, the world’s largest manufacturer of syringes, is also a US company, founded in the same year as Corning. By July 2020, the US government had placed an initial order of 190 million syringes and also assisted the company in the expansion

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of their manufacturing capacity. As a multinational operation, BD has facilities in Canada, Mexico, Argentina, Brazil, Chile, and Colombia, as well as in Europe. But since 1995, the company has also acquired large operations in Asia. Becton Dickinson has a substantial base in Suzhou, Jiangsu Province, in East China and a large-­capacity plant in the Indian city of Bawal, close to New Delhi. Here, over a billion medical devices with disposable needles and syringes are produced in a highly automated process meeting global standards. The nature of manufacturing operations in the 21st century means that the provision of materials to deliver COVID-­19 vaccines is already a global affair. US organizers are aiming to acquire 700–­800 million syringes for the entire COVID-­19 campaign, although reaching this figure is by no means guaranteed. Once vaccine doses have been manufactured, another hurdle on the road to vaccinating large populations is the delivery of vaccines to those in most need. This entails challenges under a number of headings. Policymakers generally agree that vulnerable groups should be vaccinated first during a global emergency, but identifying and prioritizing subgroups for the earliest interventions is sensitive and fraught with difficulties. The elderly are most at risk in the current global emergency, and targeting subgroups living in supported environments makes sense, given concerns about institutional transmission. Frontline workers in health and social care should also be given priority. During the first eight months of the COVID-­19 pandemic, it became clear that certain ethnic groups had a significantly elevated risk of serious illness and death. Prioritizing these groups for vaccination, although politically sensitive, can serve to ease strains on health-­care facilities as well as benefiting those in most need. Related to this is the question of prioritizing individuals with existing health conditions such as diabetes and cardiovascular disease. Those who must work in close proximity to others to fulfill public needs may also justify early targeting.

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Public scrutiny of decision making is essential during the roll-­ out of COVID-­19 vaccines. To this end, senior figures at the US National Institutes of Health began working with agencies such as the National Academy of Medicine in order to improve transparency. The CDC Advisory Committee on Immunization Practices is also responsible for producing guidance codes during operations of this kind. Those with commercial experience of vaccine delivery caution that immunizing a sufficient proportion of the population to reduce the transmission of a pandemic infection would take six months to a year; and this is an optimistic timescale. Throughout the complex organization and implementation of vaccine delivery, media campaigns to keep the public informed and promote the uptake of vaccination are a major resource for success. From a global perspective, can the delivery of a vaccine to end a pandemic benefit from experience with other new vaccines in the 21st century? The WHO estimates that just over 80 percent of the world’s children now have access to immunization, while an increasing number also have access to new vaccines. But the scaling up of immunization programs and the introduction of vaccination at times of emergency are bound to place strains on distribution systems. One problem that WHO experts have identified is the unnecessary layering of distribution networks. In the traditional model, a central store supplies regional stores, which then feed provincial stores and district stores, which in turn supply local health centers. But these conventions were established in the previous century, when lines of communication were relatively weak. In the digital age, when low-­cost technology has transformed communication networks, such structures are redundant. Stores for each administrative level are no longer needed when the person from the health center can call the central store directly. For nearly the last decade, the work of modernizing the global distribution of vaccines has been under-

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way, but a global emergency inevitably means that distribution becomes a bottleneck. A global perspective on vaccine roll-­out must include the problem of vaccine uptake around the world. In 2014 the WHO formed the Strategic Advisory Group of Experts (SAGE) to investigate the problem, and the group has continued to update findings. The WHO approach was intensely pragmatic. Among their conclusions were the importance of recognizing the complexity of the issue. The most effective interventions countering vaccine hesitancy were those combining several strategies. These included directly targeting unvaccinated or undervaccinated communities; increasing local knowledge of how vaccines work and what they achieve; improving convenience and easy access to vaccines; targeting minority religious-­belief groups and the health-­care workers serving them; mandating vaccines or imposing sanctions for nonvaccination; the use of reminder and follow-­up measures; mass media communications; and recruitment of influential leaders to promote vaccination. The latter are known as social mobilizers, and their use was pivotal in the global campaign to eliminate polio in the second decade of the 20th century. In some areas of Pakistan, localized resistance stemmed from the persistent belief that the polio program was a plot to sterilize Muslims, or that the contents of the vaccine were not halal (that is, not produced in accordance with Islamic laws that govern products consumed by Muslims). One role of social mobilizers was to explain and dispel such myths about the vaccine. Mobilizers used passages from the Qur’an and the Hadith to remind people of the importance of the health of children in Islam. Pronouncements by highly respected Islamic clerics also helped to demonstrate that polio vaccine is not anti-­Islamic. These effective persuasion measures gave religious leaders the power to convince families to vaccinate their children. Another key challenge to success in the roll-­out of vaccines in a global emergency is the issue of equity in vaccine distribu-

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tion. During discussions in August 2020, some commentators highlighted the problem of how competition between nations could undermine the provision of COVID-­19 vaccines around the world. An adversarial approach and nationalistic inclinations are an ever-­present problem in global emergencies. Some governments act quickly to ban exports of personal protective equipment and ventilators, and buy up drug supplies; the same policies may impact fair vaccine distribution. Concerns began to be raised after Russia was accused of cyber espionage, targeting information on the UK Oxford vaccine. This matter seemed strange from the outset because technical and clinical details of the Oxford vaccine had been promptly published at every stage of its discovery and clinical testing. The same was true for Chinese vaccines in the pipeline. Not long afterward, the Russians announced the licensing of their own vaccine. But the issues of geopolitics in vaccine distribution have less to do with stealing technical know-­how and more to do with putting national interest ahead of altruism. In the 2009 swine flu epidemic, despite pledges to ensure equitable distribution of swine flu vaccine, wealthy countries, including the United States and Australia, banned exports until their own populations had been fully inoculated. The CDC estimates that up to 575,000 people, largely in the developing world, may have died as a result. It is in the interest of all nations that vaccines be distributed in the most efficient and cost-­effective way. The multinational status of pharmaceutical companies can aid this process, especially when noncommercial, nongovernmental initiatives are introduced to foster equitable programs. In January 2017, the Wellcome Trust together with the Bill and Melinda Gates Foundation inaugurated a new organization: the Coalition for Epidemic Preparedness Innovations (CEPI), a global partnership between public, private, philanthropic, and civic organizations with centers in Oslo, London, and Washington, DC. Its mission is

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to accelerate the development of vaccines against emerging infectious diseases and enable access to these vaccines for people of all nations during outbreaks. CEPI works closely with another long-­ established group—­the Global Alliance for Vaccines and Immunisation (Gavi). Created with funding from the Gates Foundation in the year 2000, Gavi is a public-­private partnership, bringing together United Nations agencies, governments, vaccine companies, charities, and civil society. Its goal is the sustained improvement of childhood immunization in poor countries and accelerated access to new vaccines. In line with these global objectives, pharma giant AstraZeneca has agreed a multimillion-­dollar collaboration with CEPI and Gavi to support the manufacture and distribution of its COVID-­19 vaccine to low-­and middle-­income nations on a not-­for-­profit basis. AstraZeneca is just one of several global pharma companies working with Gavi in this way to deliver vaccines for the developing world. In the task of ending global pandemics through vaccination, the WHO remains the principle international resource. Against pandemic infectious disease, nobody wins the race until everyone wins. Organizations such as COVAX—­which brings together Gavi, CEPI, and the WHO—­recognize this. COVAX aims to accelerate the development and manufacture of COVID-­19 vaccines and to guarantee fair and equitable access for every country in the world. The timetable for delivering success, however, looks somewhat different from this idealistic picture. Most public health experts agree that NGOs (nongovernmental organizations) will never have the financial purchasing power of the industrialized countries, and altruism is not likely come into play until the pressing needs of the wealthier nations have been fulfilled.

CHAPTER EIGHT

Embrace Many Solutions I’ve spent much of my career making predictions and then conducting experiments to prove them wrong. In one chapter in my working life, I tested seven quite different predictions relating to the same theory. I asked the questions and received the answers, one by one. It felt like a solemn conversation without words. And when it was over, my theory remained true. Yet the hope of applications for my theory—­a revolutionary drug for chronic infections, including HIV, hepatitis B, and cancer—­came to an end in the closing years of the 20th century. Only in malignant melanoma were there anecdotal stories of success with the new drug I had discovered, and my colleagues failed to see a significant commercial opportunity. My experimental predictions were right, but my dreams of new drugs were never realized. As you can tell from this book, I still enjoy making predictions, and I remain an optimist. But when it comes to COVID-­19 vaccines, we did not have to predict or embrace one winner; multiple different vaccines succeeded and at the same time provided a fast track to manufacturing and distribution. When I first drafted this book, six classes of COVID-­19 vaccines had shown themselves to be effective in provoking antibody and cellular immune responses. Nonreplicating viral vector vaccines work by infecting tissue cells and exposing the immune system to all the danger molecules present in a live virus. Replicating viral vector vaccines do the same but also grow in the body and infect new cells. Nonliving subunit

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protein vaccines powerfully stimulate the immune response to key vaccine antigens through the inclusion of adjuvants, which amplify the body’s response. Nonliving whole-­virus vaccines do the same but contain all the components of the pathogen. DNA vaccines deliver key antigens to the immune system in the form of proteins encoded in the engineered DNA, and the body’s own cells manufacture these proteins. Packaging and special delivery ploys ensure the vaccines reach the right place to stimulate immune defenses. RNA vaccines work in the same way but with fewer intermediate steps in protein production. Some versions of this class of vaccine also reproduce and amplify themselves in the cells of the body. Success with nucleic acid vaccines, especially RNA vaccines, will break new ground, as there are no existing human vaccines of this kind. Duration of protection may further differentiate those vaccines that induce an initial strong protective response. Single-­dose and oral and nasal vaccines may have an advantage for ease of use around the globe. Self-­amplifying vaccines would provide a radical new resource for the future. I hope this book has given you an understanding of the basics of how all these vaccines work, whether to protect against COVID-­19 or future pandemics. On July 7, 2020, as the vaccines selected for fast-­tracking by Operation Warp Speed moved swiftly toward phase III trials, a surprise addition to this elite club was unexpectedly announced. Had there been some crucial further thinking? A late-­stage brainstorming session? In fact it was obvious, really. An entity of considerable importance was entirely missing from the high-­powered race. Just to remind ourselves, Warp Speed had chosen from three vaccine classes: nonreplicating viral vector vaccines, replicating viral vector vaccines, and RNA-­based vaccines. But one major class had been left out—­the protein subunit vaccines. In these vaccines, proteins are wrapped up in intriguing (at least for an immune system) parcels, and the proteins mixed with adjuvants

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or linked to danger signals. On July 7, 2020, the program elevated the Novavax protein candidate to Warp Speed status. Novavax agreed to supply the US government with 100 million doses—­ enough vaccine to immunize 50 million people—­assuming the product proved safe and effective. Delivery of the full amount was planned for February 2021. It is a highly promising vaccine, and an interesting one, too. Novavax, a Maryland-­based company not far from the National Institute of Allergy and Infectious Diseases in Bethesda, uses the baculovirus in insect cells to produce a stable, full-­length version of the spike protein. In mice and baboons, this adjuvanted protein vaccine (whose spike is not susceptible to break up by enzymes) produces strong neutralizing antibodies and good T-­cell–­mediated immune responses of the right kind to combat SARS-­CoV-­2. The reason it can do this has everything to do with the adjuvant, Matrix-­M. Matrix-­M is not science fiction but it is a classic. In the 1980s, vaccinologists began developing small particles made of lipids normally present in the membranes of healthy cells. These are the same class of structure as the lipid nanoparticles used in RNA vaccines. These cage-­like constructions, designed to enclose protein antigens, are just the right size to be taken up by immune surveillance cells. But there’s more to it than that. In the case of Matrix-­M, the potent adjuvant has been incorporated as an integral part of the structure (or matrix) of these packages. The recipe is simple: one part lipid A, one part lipid B, one part protein antigen, and one part adjuvant molecule. But what is this mysterious adjuvant? It’s a plant product which shares an important characteristic with the lipids in the matrix—­it is amphipathic. This means it has a hydrophilic (water-­ loving) end and a hydrophobic (water-­averse) end. This ensures that it integrates well with the rest of the matrix. The matrix is therefore not a passive parcel but a package bristling with potent

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immunostimulatory tools. We have already discussed the adjuvant molecule itself. It is saponin, the product of the evergreen soapbark tree of South America. It’s also present in Coca-­Cola, imparting the much-­loved foamy effervescence to the world’s favorite soft drink. Of course, the amount of saponin present in soft drinks is very small indeed. But injected into the body in much larger amounts, saponin can prove a toxic molecule, disruptive to cell membranes. There are ways around this problem, however. One strategy is to purify fractions of the product in order to isolate molecules with low toxicity. Another is to incorporate saponin in lipid matrix particles, which allows it to become an effective adjuvant at very low (nontoxic) concentrations. Novavax has adopted this adjuvant in their SARS-­CoV-­2 protein subunit vaccine. But how does saponin work? It’s a pro-­inflammatory molecule that tends to bind to cholesterol, producing micropores in cell membranes, and these effects may contribute to its adjuvant activity. Not everything about its powerful adjuvant effect is understood, but it does have an additional crucial feature. It contains an aldehyde which reacts covalently with cell-­surface molecules to form Schiff bases—­an event which, as we have seen, provides a potent costimulatory stimulus to T-­cells responding to antigens presented by surveillance cells. For me, although oral Schiff base–­ forming drugs to strengthen immune responses never secured a place as medicines, this use of a Schiff base–­forming adjuvant constitutes a fulfilling end to my own exploratory journey of discovery. At this point, it might be useful to again remember polio. In

the United States in 1952, polio infected 57,628 people, killed 3,145, and paralyzed 21,269. In the United States in 2020, numbers infected by SARS-­CoV-­2 were measured in millions, with death rates of around 3 percent. The figures speak for themselves.

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Hope on the horizon in 1953 existed in the shape of a single vaccine (inactivated whole virus) and two live attenuated vaccines in the pipeline. Three vaccines, of which two would ultimately be licensed. For COVID-­19 there are at least 232 vaccines in the development pipeline. The vaccines are all variations on a common theme—­activating an immune response. The simple term vaccine can have a hundred different meanings depending on the context, tone, color, and inflection vaccinologists have chosen to employ. This brings us back to shi, the single syllable repeated many times in subtly different—­or not so different—­ways, and to the poet whose lions turned to stone. Yuen Ren Chao, the author of the poem that begins this book, was born in the port city of Tianjin, where the ancient Chinese empire met the sea, but he grew up to become a professor of Oriental languages and literature at the University of California at Berkeley. His mission was to explain and convey meaning across barriers, whatever the challenges. One of his most admired publications on languages appeared in the Bulletin of the Institute of History and Language of the Central Academy in 1934. In it, he argues that there are few correct or incorrect answers to any problem—­there are multiple solutions. What is important is embracing many solutions to achieve your goal.

EPILOGUE

In November 2020, as global COVID-19 cases topped 55 million and deaths soared above one million, pharmaceutical giant Pfizer announced the results of an interim analysis of its phase III vaccine trial. This mRNA vaccine was produced by BioNTech, a small company founded in 2008 in Mainz, Germany, by Uğur Şahin and Özlem Türeci, first generation Turkish immigrants to that country. RNA vaccination depends on an astonishing feat of minimalism in which the vaccine is reduced to just a fragment of the viral genetic code. In this case, the Pfizer vaccine delivers only the surface spike protein of SARS-CoV-2. A full analysis of the results confirmed that in 43,538 trial participants of diverse ethnicity and age, half of whom received a placebo, the Pfizer vaccine proved safe and was 95 percent effective in preventing COVID19 infection. Researchers documented 170 cases of symptomatic infection, some serious. These spectacular results exceeded expectations. Results from the Boston-based US company Moderna followed quickly. Their mRNA vaccine proved safe and was 94.5 percent effective in preventing infection in a study of more than 30,000 volunteers of diverse ethnicity and age. Researchers documented 95 cases of symptomatic COVID-19 infections in the study population, some serious. Although the rapid discovery and development of these specific mRNA vaccines against a novel pathogen was entirely unprecedented, this speed and success was made possible by three decades of pioneering research in the field of nucleic acid vaccination. On November 23, AstraZeneca announced success in the phase III trial of the Oxford adenoviral-vector vaccine, publish-

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ing a full report in The Lancet two weeks later. In a pooled, statistically approved, interim analysis of 11,636 participants, the vaccine was 70.4 percent effective in preventing infection. This figure was the mean of two subgroups—those receiving two standard vaccine doses several weeks apart (62.1 percent protection) and those receiving a half dose followed by a full dose (90.0 percent protection). The high protection afforded by a lower priming dose was discovered by accident—serendipity is practically traditional in the field of vaccinology—and, once confirmed, will allow for optimal dosing in the vaccination program. The study further demonstrated protection against asymptomatic infection in those receiving the lower initial dose. Reports from Russia indicated that its adenoviral vector vaccine had also proved more than 90 percent effective. On December 9, Sinopharm’s inactivated whole-virus vaccine proved 86 percent effective in phase III trials and received approval in the United Arab Emirates, one of 10 nations conducting trials of this leading Chinese vaccine. Success in three vaccine classes—mRNA, adenoviral vector, and inactivated whole-virus vaccines—gave confidence that other vaccines would also prove effective, including subunit vaccines, the largest category in the COVID-19 vaccine landscape. From the moment Wuhan virologists shared the sequence of the novel coronavirus with specialists around the globe in January 2020, the world of vaccinology was ready to respond. Previous epidemics had prepared them. The lessons of SARS and MERS had been learned. Diverse vaccine technology platforms stood ready and waiting. The virus, though novel, was likely to prove tractable. The new field of nucleic acid vaccination was poised to come of age. But something more was needed. Perhaps the pivotal breakthrough of 2020 was not the science but the unparalleled orchestration of the international collaborative endeavor, largely funded

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by national governments. The combination of an unprecedented pursuit of overlapping trials without compromising safety, the parallel manufacture of vaccines, the fusion of phases, the rolling assessment of clinical data encouraged by regulators, and the rapid and free access to information for all has proved to be the true discovery of the modern pandemic age.

ACKNOWLE DGMENTS

Writing this book would have been impossible without the spirit of openness that has prevailed across the international scientific community ever since Chinese scientists first shared the genetic sequence of the new coronavirus, SARS-­CoV-­2, on global platforms early in January 2020. For me, the Chinese word that best describes this new ethos is 开放政策, rendered phonetically as Kāifàng zhèngcè, which simply means “open door.” It is, I believe, unprecedented. Never before have I experienced such openness, nor benefited from such a privileged level of access to scientific studies immediately relating to the current global emergency but also increasing our understanding and capabilities in preparation for threats of future pandemic disease. Throughout the global emergency, traditional publishing models, too slow to respond to a fast-­moving pandemic, have been overlaid by a new system for rapidly delivering scientific information. Expert investigators have embraced open publishing platforms and preprint servers to share their findings as rapidly and as freely as possible in a manner not seen before. By the summer of 2020, around 850 COVID-­19–­related preprints were being published each week. And in response to this openness, a new species of democratic vigilance has been added to more traditional critical appraisals in order to police and preserve the integrity of rapidly published information, quickly calling out flawed data that occasionally slips through. I am extremely grateful to all those authors, publishers, editors, journalists, journal-­producers, and policy makers around the globe who together continue to deliver scientific data and

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assure its quality so widely and so freely. The gateway to all these resources has, of course, been my desktop computer, programmed to access the full range of scholarly publications in all their technical complexity and depth. But there are also windows to a much more human level of enlightenment. I have moved effortlessly, for example, between a formal multi-­agency analysis of strategies to counter vaccine hesitancy in diverse cultures of the world to the cozy informality of a relaxed exchange between Dr. Howard Bauchner, Editor-­in-­Chief of the Journal of the American Medical Association, and authorities such as Dr. Anthony Fauci, Director of the National Institute of Allergy and Infectious Diseases, and Dr. Paul Offit, Chief of the Division of Infectious Disease at Children’s Hospital of Philadelphia, reflectively sharing insights from the comfort of their living rooms. For these experiences, I am truly thankful. It is a pleasure to thank Professor Sarah Gilbert of Oxford University for providing valuable information and for kindly making corrections to the manuscript at an early stage. Professor Judith Breuer of University College London has generously helped me on some key points, and Professor Patricia Woo, also of University College, has been a constant source of helpful information and discussion. I am indebted to my former colleagues at the National Institutes of Allergy and Infectious Diseases, Cambridge University, Wellcome Research Laboratories, Burroughs Wellcome, Glaxo Wellcome, and GlaxoSmithKline for countless discussions on the subject of immunology and the developing science of vaccinology in various phases of my career, all of which have equipped me to write this book. Collaborations with GSK Biologicals in Rixensart, Belgium, have proved an important source of insight for explaining the vaccine technologies in this account. In particular, I am grateful to Dr. Nathalie Garçon. For enlightening me on the ups and downs of clinical drug development, I am grateful

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to Dr. Catherine Heitman of Burroughs Wellcome. I thank Tony Phillips of Glaxo Wellcome and Lee Roberts and his colleagues at PowderJect in Madison, Wisconsin, for our unforgettable joint adventure in pioneering gene-­gun DNA vaccine development in the 1990s. For so many invaluable discussions on the complexities of immunology over the years, I am indebted to Dr. John Tite, Dr. Sara Brett, and Professor Foo Yew “Eddy” Liew. This book is dedicated to my students, one of whom, Gao Xiaoming, was jointly supervised with Eddy Liew, John Tite, and Sara Brett. For excellent academic supervision for all three students I thank Professor Adrian L. W. F. Eddleston of Kings College London and Professor Francis E. G. Cox of the London School of Hygiene and Tropical Medicine. For long-­term technical support at work I remain grateful to Barbara Pearce. For intensive technical support at home, I am very grateful to a masked and shoeless Khalifa Bellili for his efforts, including a new computer hard drive at the height of my research and writing activities. Heartfelt thanks to my agent Peter Tallack of the Science Factory for encouraging me to write this book, for balancing and extending it in scope and vision, and for surefootedly guiding it to a home with the right publisher. I am hugely indebted to Joseph Calamia, senior editor at the University of Chicago Press, for his valuable improvements to the text, his close attention to detail, and his tireless insistence on clarity and broad accessibility. I am grateful for his patience with the formidable complexities of immunology and the ultimately satisfying exercise of together making things understandable for general readers. While searching for ways to do this, I came across this story from Ed Yong in The Atlantic, (published August 5, 2020), which he credited to immunologist Jessica Metcalf of Princeton University. An immunologist and a cardiologist are kidnapped. The kidnappers threaten to shoot one of them but promise to spare whoever has made the greater contribution to humanity. The cardiologist says,

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“Well, I’ve identified drugs that have saved the lives of millions of people.” Impressed, the kidnappers turn to the immunologist. “What have you done?” they ask. The immunologist says, “The thing is, the immune system is very complicated . . .” And the cardiologist says, “Just shoot me now.” I am happy to report that, faced with these same complexities, Joe Calamia remained cheerfully stoic. Warm thanks also to Jenni Fry for her excellent copyediting, smoothing, clarifying, and correcting throughout, making the book still more accessible. It is a great pleasure to thank my lifelong friends for critical readings of parts of the manuscript. In particular I am indebted to Stuart Bevan, Andy Dray, Karen Jackson, Maureen Lewis, and Sue Cox for their constructive comments. I also appreciate the readings undertaken by Rodney and Pauline Hillman and the advice they kindly provided. Heartfelt thanks to my daughters Chloe Rhodes and Leonie Rhodes for their wisdom and support. Lastly I thank my wife, Heather Rhodes, who at one point wasn’t sure if she had another book in her but happily found that she did. I am ever grateful to her for support, unfailing encouragement, and sound advice.

APPENDIX

C O V I D - ­1 9 VA C C I N E S A N D VA C C I N E C A N D I D A T E S

COVID‑19 vaccine developer / manufacturer

Vaccine class

Type of candidate vaccine

Coronavirus antigen delivered

Route of administration and dosage

University of Oxford / AstraZeneca (UK)

nonreplicating viral vector

chimpanzee adenovirus (ChAdOx1‑S)

SARS‑CoV‑2 spike protein

intramuscular; 1+ dosing occasions

CanSino Biological Inc. / Beijing Institute of Biotechnology (China)

nonreplicating viral vector

adenovirus type 5 vector

SARS‑CoV‑2 spike protein

intramuscular; 1+ dosing occasions

Gamaleya Research Institute (Russia)

nonreplicating viral vector

adenovirus type 26 plus adenovirus type 5

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Janssen Pharmaceutical Companies / Johnson & Johnson (Belgium)

nonreplicating viral vector

adenovirus type 26

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Sinovac (China)

inactivated

SARS-­CoV‑2 whole virus

Multiple antigens

intramuscular; 2 dosing occasions

Wuhan Institute of Biological Products / Sinopharm (China)

inactivated

SARS-­CoV‑2 whole virus

Multiple antigens

intramuscular; 2 dosing occasions

Beijing Institute of Biological Products / Sinopharm (China)

inactivated

SARS-­CoV‑2 whole virus

Multiple antigens

intramuscular; 2 dosing occasions

Moderna / NIAID (USA)

RNA

lipid nanoparticle encapsulated mRNA (mRNA‑1273)

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

BioNTech / Fosun Pharma / Pfizer (Germany)

RNA

three-­lipid nanoparticle encapsulated mRNA s

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

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COVID‑19 vaccine developer / manufacturer

Vaccine class

Type of candidate vaccine

Coronavirus antigen delivered

Route of administration and dosage

Novavax (USA)

protein subunit

full-­length recombinant SARS-­CoV‑2 glycoprotein nanoparticle vaccine adjuvanted with Matrix-­M

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Anhui Zhifei Longcom Biopharmaceutical / Institute of Microbiology, Chinese Academy of Sciences (China)

protein subunit

adjuvanted recombinant protein (receptor-­ binding domain dimer)

SARS‑CoV‑2 spike protein; receptor-­ binding domain

intramuscular; 2 or 3 dosing occasions

CureVac (Germany)

RNA

mRNA

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Institute of Medical Biology, Chinese Academy of Medical Sciences (China)

inactivated

SARS-­CoV‑2 whole virus

Multiple antigens

intramuscular; 2 dosing occasions

SARS-­CoV‑2 whole virus

Multiple antigens

intramuscular; 2 dosing occasions

Research Institute for Biological Safety Problems (Republic of Kazakhstan) Inovio Pharmaceuticals / International Vaccine Institute (USA)

DNA

DNA plasmid vaccine with electroporation

SARS‑CoV‑2 spike protein

intradermal; 2 dosing occasions

Osaka University / AnGes / Takara Bio ( Japan)

DNA

DNA plasmid vaccine plus adjuvant

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Cadila Healthcare Limited (India)

DNA

DNA plasmid vaccine

SARS‑CoV‑2 spike protein

intradermal; 3 dosing occasions

Genexine Consortium (South Korea)

DNA

DNA plasmid vaccine (GX‑19)

SARS‑CoV‑2 spike protein

Intramuscular 2 dosing occasions

Bharat Biotech (India)

inactivated

SARS-­CoV‑2 whole virus

multiple antigens

intramuscular; 2 dosing occasions

A P P E N DIX / 139

COVID‑19 vaccine developer / manufacturer

Vaccine class

Type of candidate vaccine

Coronavirus antigen delivered

Route of administration and dosage

Kentucky Bioprocessing, Inc. (USA)

protein subunit

recombinant protein (receptor-­ binding domain)

SARS‑CoV‑2 spike; receptor-­ binding domain

intramuscular; 2 dosing occasions

Sanofi Pasteur / GSK

protein subunit

full-­length recombinant SARS-­CoV‑2 glycoprotein adjuvanted with AS03

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Arcturus / Duke‑NUS (Singapore)

RNA

mRNA

SARS‑CoV‑2 spike protein

intramuscular; 1+ dosing occasions

ReiThera / LEUKOCARE / Univercells (Italy, Germany, Belgium)

nonreplicating viral vector

chimpanzee adenovirus

SARS‑CoV‑2 spike protein

intramuscular; 1+ dosing occasions

Clover Biopharmaceuticals Inc. / GSK / Dynavax (China)

protein subunit

native-­like trimeric subunit spike protein vaccine

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Vaxine Pty Ltd / Medytox (Australia)

protein subunit

recombinant spike protein with Advax adjuvant

SARS‑CoV‑2 spike protein

intramuscular; 1+ dosing occasions

University of Queensland / CSL / Seqirus (Australia)

protein subunit

molecular clamp–­ stabilized spike protein adjuvanted with squalene oil‑in‑water adjuvant (MF59)

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Medigen Vaccine Biologics Corporation / NIAID / Dynavax (USA)

protein subunit

modified, stabilized SARS-­CoV‑2 glycoprotein adjuvanted with CpG (CpG1018)

modified SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

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COVID‑19 vaccine developer / manufacturer

Vaccine class

Type of candidate vaccine

Coronavirus antigen delivered

Route of administration and dosage

Instituto Finlay de Vacunas (Cuba)

protein subunit

adjuvanted recombinant protein (receptor-­ binding domain)

SARS‑CoV‑2 spike protein; receptor-­ binding domain

intramuscular; 2 dosing occasions

FBRI SRC VB VECTOR, Rospotrebnadzor, Koltsovo (Russia)

protein subunit

adjuvanted peptide

not specified

intramuscular; 2 dosing occasions

West China Hospital, Sichuan University (China)

protein subunit

recombinant protein (receptor-­ binding domain)

SARS‑CoV‑2 spike protein; receptor-­ binding domain

intramuscular; 2 dosing occasions

Institute Pasteur / Themis / University of Pittsburgh CVR / Merck Sharp & Dohme (France)

replicating viral vector

measles viral vector

SARS‑CoV‑2 spike protein

intramuscular; 1+ dosing occasions

Beijing Wantai Biological Pharmacy / Xiamen University (China)

replicating viral vector

influenza viral vector (receptor-­ binding domain)

SARS‑CoV‑2 spike protein; receptor-­ binding domain

intranasal; 1 dosing occasion

Imperial College London (UK)

RNA

lipid nano‑particle encapsulated self‑replicating mRNA

SARS‑CoV‑2 spike protein

intramuscular; 1+ dosing occasions

People’s Liberation Army (PLA) Academy of Military Sciences / Walvax Biotech (China)

RNA

mRNA

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Medicago, Inc. (Canada)

virus‑like particle

AS03 adjuvanted plant‑derived virus-­like particle

SARS‑CoV‑2 spike protein

intramuscular; 2 dosing occasions

Source: Adapted from “World Health Organization Draft Landscape of COVID-­19 Candidate Vaccines,” September 9, 2020.

FURTHER READING

Chapter ONE: Understand the Virus

General Reading

Andiman, Warren A. Animal Viruses and Humans, a Narrow Divide: How Lethal Zoonotic Viruses Spill Over and Threaten Us. Philadelphia: Paul Dry Books, 2018. Crawford, Dorothy H. Deadly Companions: How Microbes Shaped Our History (Oxford Landmark Science). Updated ed. Oxford: Oxford University Press, 2018. de Kruif, Paul. Microbe Hunters. Boston: Houghton Mifflin Harcourt, 2002. First published 1926 by Harcourt Brace Jovanovich. Enjuanes, Luis, ed. Coronavirus Replication and Reverse Genetics. New York: Springer, 2004. Honigsbaum, Mark. The Pandemic Century: A History of Global Contagion from the Spanish Flu to Covid-­19. London: Virgin, 2020. Horton, Richard. COVID-­19 Catastrophe: What’s Gone Wrong and How to Stop It Happening Again. Cambridge: Polity Press, 2020. Li, Fang. “Structure, Function, and Evolution of Coronavirus Spike Proteins.” Annual Reviews of Virology 3 (2016): 237–­61. MacKenzie, Debora. COVID-­19: The Pandemic That Never Should Have Happened, and How to Stop the Next One. London: Bridge Street Press, 2020. McIntosh, Kenneth. “Coronaviruses: A Comparative Review.” In Current Topics in Microbiology and Immunology, edited by W. Arber, R. Haas, W. Henle, P. H. Hofschneider, N. K. Jerne, P. Koldovský, H. Koprowski, O. Maaløe and R. Rott, 87–­119. New York: Springer, 1974. Mosely, Michael. Covid-­19: What You Need to Know about the Coronavirus and the Race for the Vaccine. London: Short Books, 2020. Oldstone, Michael B. A. Viruses, Plagues, and History: Past, Present and Future. 2nd ed. New York: Oxford University Press, 2020.

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Quammen, David. Spillover: Animal Infections and the Next Human Pandemic. New York: Norton, 2013. Singhal, Tanu. “A Review of Coronavirus Disease-­2019 (COVID-­19).” Indian Journal of Pediatrics 87 (2020): 281–­86. Tyrrell, David, and Michael Fielder. Cold Wars. Oxford: Oxford University Press, 2002. Waltner-­Toews, David. Coronavirus. Rev. ed. Vancouver: Greystone Books, 2020. Zheng, Jun. “SARS-­CoV-­2: An Emerging Coronavirus That Causes a Global Threat.” International Journal of Biological Sciences 16 (2020): 1678–­85. Specific Reading

Almeida, June D., D. M. Berry, C. H. Cunningham, D. Hamre, M. S. Hofstad, L. Mallucci, K. McIntosh, and D. A. J. Tyrrell. “Virology: Coronaviruses.” Nature 220 (1968): 650. Almeida, June D., and David A. Tyrrell. “The Morphology of Three Previously Uncharacterized Human Respiratory Viruses That Grow in Organ Culture.” Journal of General Virology 1 (1967): 175–­78. Andersen, Kristian G., Andrew Rambaut, W. Ian Lipkin, Edward C. Holmes, and Robert F. Garry. “The Proximal Origin of SARS-­ CoV-­2.” Nature Medicine 26 (2020), 450–­55. Bao, Linlin, Wei Deng, Baoying Huang, et al. “The Pathogenicity of SARS-­CoV-­2 in hACE2 Transgenic Mice.” Nature 583 (2020): 831–­33. Beaudette, Frederick R, and Charles B. Hudson. “Cultivation of the Virus of Infectious Bronchitis.” Journal of the American Veterinary Medical Association 90 (1937): 51–­58. Corbett, Kizzmekia, S. Darin K. Edwards, Sarah R. Leist, et al. “SARS-­CoV-­2 mRNA Vaccine Design Enabled by Prototype Pathogen Preparedness.” Nature 586 (2020): 567–­71. https://doi. org/10.1038/s41586-­020-­2622-­0. Estola, T. “Coronaviruses, a New Group of Animal RNA Viruses.” Avian Diseases 14, (1970): 330–­336. Grant, Oliver C., David Montgomery, Keigo Ito, and Robert J. Woods. “3D Models of Glycosylated SARS-­CoV-­2 Spike Protein Suggest Challenges and Opportunities for Vaccine Development.” Preprint, submitted May 1, 2020. https://doi. org/10.1101/2020.04.07.030445.

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Heald-­Sargent, Taylor, and Tom Gallagher. “Ready, Set, Fuse! The Coronavirus Spike Protein and Acquisition of Fusion Competence.” Viruses 4, (2012): 557–­80. Hoffmann, Markus, Hannah Kleine-­Weber, Simon Schroeder, Marcel A. Müller, Christian Drosten, and Stefan Pöhlmann. “SARS-­CoV-­2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.” Cell 181 (2020): 271–­80. Hu, Ben, Lei-­Ping Zeng, Xing-­Lou Yang, et al. “Discovery of a Rich Gene Pool of Bat SARS Related Coronaviruses Provides New Insights into the Origin of SARS Coronavirus.” PLoS Pathogens 13 (2017): e1006698. https://doi.org/10.1371/journal.ppat.1006698. Mason, Robert J. “Pathogenesis of COVID-­19 from a Cell Biology Perspective.” European Respiratory Journal 55 (2020): 2000607. https://doi.org/10.1183/13993003.00607-­2020. Merad, Miriam, and Jerome C. Martin. “Pathological Inflammation in Patients with COVID-­19: A Key Role for Monocytes and Macrophages.” Nature Reviews 20 (2020): 355–­62. Robson, Fran, Khadija Shahed Khan, Thi Khanh Le, Clement Paris, Sinem Demirbag, Peter Barfuss, Palma Rocchi, and Wai-­Lung Ng. “Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting.” Molecular Cell 79 (2020): 1–­18. Schalk, Arthur F., and M. C. Hawn. “An Apparently New Respiratory Disease of Baby Chicks.” Journal of the American Veterinary Medical Association 78 (1931): 413–­23. Shang, Jian, Yushun Wan, Chuming Luo, Gang Ye, Qibin Geng, Ashley Auerbach, and Fang Li. “Cell Entry Mechanisms of SARS-­ CoV-­2.” Proceedings of the National Academy of Sciences of the United States of America 117 (2020): 11727–­11734. Tyrrell, David A. J., J. D. Almeida, C. H. Cunningham, et al. “Coronaviridae.” Intervirology 5 (1975): 76–­82. Wang, Qihui, Yanfang Zhang, Lili Wu, et al. “Structural and Functional Basis of SARS-­CoV-­2 Entry by Using Human ACE2.” Cell 181 (2020): 894–­904. Wang, Xuemei, Li Tao Li, Shubing Zhang, Lianzi Wang, Xian Wu, and Jiaqing Liu. “The genetic sequence, origin, and diagnosis of SARS-­ CoV-­2.” European Journal of Clinical Microbiology & Infectious Diseases 39 (2020): 1629–­35. Weiss, Susan R., and Sonia Navas-­Martin. “Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome

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Coronavirus.” Microbiology and Molecular Biology Reviews 69 (2005): 635–­64. Wrapp, Daniel, Nianshuang Wang, Kizzmekia S. Corbett, Jory A. Goldsmith, Ching-­Lin Hsieh, Olubukola Abiona, Barney S. Graham, and Jason S. McLellan. “Cryo-­EM Structure of the 2019-­ nCoV Spike in the Prefusion Conformation.” Science 367 (2020): 1260–­63. Zirui Tay, Matthew, Chek Meng Poh, Laurent Rénia, Paul A. MacAry, and Lisa F. P. Ng. “The Trinity of COVID-­19: Immunity, Inflammation and Intervention.” Nature Reviews Immunology 20 (2020): 363–­74. Chapter TWO: Explore the Immune System

General Reading

Cohen, Irun R. Tending Adam’s Garden. New York: Elsevier, 2004. Davis, Daniel M. The Beautiful Cure: The Revolution in Immunology and What It Means for Your Health. London: Bodley Head, 2018. Davis, Daniel M. The Compatibility Gene. London: Allen Lane, 2013. Delves, Peter J., Seamus Martin, Dennis R. Burton, and Ivan Roitt. Roitt’s Essential Immunology. 13th ed. London: Wiley Blackwell, 2017. Murphy, Kenneth M., and Casey Fox. Janeway’s Immunobiology. 9th ed. Oxford: Garland Science, Taylor & Francis Group, 2016 . Paul, William E. Fundamental Immunology. 7th ed. Philadelphia: Lippincott, Williams & Wilkins, 2012 . Richtel, Matt. An Elegant Defense: The Extraordinary New Science of the Immune System. New York: Harper Collins, 2019. Silverstein, Arthur M. A History of Immunology. 2nd ed. New York: Academic Press, Elsevier, 2009. Sompayrac, Lauren. How the Immune System Works. 6th ed. London: Wiley Blackwell, 2019. Specific Reading

Behring, Emil A. “Serum Therapy in Therapeutics and Medical Science.” In Nobel Lectures, Physiology or Medicine 1901–­1921. Amsterdam: Elsevier Publishing Company, 1967.

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Behring, Emil, and Kitasato Shibasaburō. “Ueber das Zustandekommen der Diphtherie-­Immunität und der Tetanus-­Immunität bei Thieren.” Deutsche Medizinische Wochenschrift 16 (1890): 1113–­14. Billingham, Rupert E., Leslie Brent, and Peter B. Medawar. “Actively Acquired Tolerance of Foreign Cells.” Nature 172 (1953): 603–­6. Burnet, Frank M. The Clonal Selection Theory of Acquired Immunity. Cambridge: Cambridge University Press, 1959. Burnet, Frank M. “Immunological Recognition of Self.” In Nobel Lectures, Physiology or Medicine 1942–­1962, Amsterdam: Elsevier Publishing Company, 1964. Edelman, Gerald M., B. A. Cunningham, W. E. Gall, P. D. Gottlieb, U. Rutishauser, and M. J. Waxdal. “The Covalent Structure of an Entire Gamma-­G Immunoglobulin Molecule.” Proceedings of the National Academy of Sciences of the United States of America 63 (1969): 78–­85. Gao, Xiaoming, and John Rhodes. “An Essential Role for Constitutive Schiff Base Forming Ligands in Antigen Presentation to Murine T Cell Clones.” Journal of Immunology 144 (1990): 2883–­90. Gibson, T., and P. B. Medawar. “The Fate of Skin Homografts in Man.” Journal of Anatomy 77 (1963): 299–­310. Janeway, Charles A., Jr. “Approaching the Asymptote? Evolution and Revolution in Immunology.” Cold Spring Harbor Symposium on Quantitative Biology 54 (1989): 1–­13. Janeway, Charles A., Jr., Christopher C. Goodnow, and Ruslan Medzhitov. “Immunological Tolerance: Danger—­Pathogen on the Premises!” Current Biology 6 (1996): 519–­22. Miller, Jacques F. A. P. “The Golden Anniversary of the Thymus.” Nature Reviews in Immunology 11 (2011): 489–­95. Miller, Jacques F. A. P. “Immunological Function of the Thymus.” Lancet 2 (1961): 748–­49. Miller, Jacques F. A. P. “Immunological Significance of the Thymus of the Adult Mouse.” Nature 195 (1962): 1318–­19. Mitchell, Graham. F., and Jacques F. A. P. Miller. “Cell to Cell Interaction in the Immune Response. II. The Source of Hemolysin-­ Forming Cells in Irradiated Mice Given Bone Marrow and Thymus or Thoracic Duct Lymphocytes.” Journal of Experimental Medicine 128 (1968): 821–­37. Mitchison, Nicholas A. “T-­cell–­B-­cell Cooperation.” Nature Reviews Immunology 4 (2004): 308–­12.

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Mosmann, Timothy R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman. “Two Types of Murine Helper T Cell Clone. I Definition According to Profiles of Lymphokine Activities and Secreted Proteins.” Journal of Immunology 136 (1986): 2348–­57. Porter, Rodney R. “Chemical Structure of γ-­Globulin and Antibodies.” British Medical Bulletin 19 (1963): 197–­201. Porter, Rodney R. “Lecture for the Nobel Prize for Physiology or Medicine 1972. Structural Studies of Immunoglobulins.” Scandinavian Journal of Immunology 34 (1972): 381–­89. Rhodes, John. “Evidence for an Intercellular Covalent Reaction Essential in Antigen Specific T Cell Activation.” Journal of Immunology 143 (1989): 1482–­89. Rosenthal, Alan S., and Ethan M. Shevach. “Function of Macrophages in Antigen Recognition by Guinea Pig Lymphocytes I. Requirement for Histocompatible Macrophages and Lymphocytes.” Journal of Experimental Medicine 138 (1973): 1194–­1212. Sharma, Preeti, Pradeep Kumar, and Rachna Sharma. “The Major Histocompatibility Complex: A Review.” Asian Journal of Pharmaceutical and Clinical Research 10 (2017): 33–­36. Shevach, Ethan M., and Alan S. Rosenthal. “Function of Macrophages in Antigen Recognition by Guinea Pig Lymphocytes II. Role of the Macrophage in the Regulation of Genetic Control of the Immune Response.” Journal of Experimental Medicine 138 (1973): 1213–­29. Tonegawa, S. “Somatic Generation of Antibody Diversity.” Nature 302 (1983): 575–­81. Zheng, Biao, Sara Brett, John P. Tite, M. Robert Lifely, Thomas A. Brodie, and John Rhodes. “Galactose Oxidation in the Design of Immunogenic Vaccines.” Science 256 (1992): 1560–­63. Zinkernagel, Rolf M., and Peter C. Doherty. “Immunological Surveillance against Altered Self Components by Sensitised T Lymphocytes in Lymphocytes Choriomeningitis.” Nature 251 (1974): 547–­ 48. Zinkernagel, Rolf M., and Peter C. Doherty. “Restriction of in Vitro T Cell-­Mediated Cytotoxicity in Lymphocytic Choriomeningitis within a Syngeneic or Semiallogeneic System.” Nature 248 (1974): 701–­2.

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Chapter THREE: Discover a Vaccine

General Reading

Allen, Arthur. Vaccine: The Controversial Story of Medicine’s Greatest Lifesaver. New York: Norton, 2007. Baron, John. Life of Edward Jenner MD with Illustrations from His Doctrines and Selections from His Correspondence. 2 vols. London: H. Colburn, 1838. Bazin, Hervé. The Eradication of Smallpox. Translated by Andrew and Glenise Morgan. London: Academic Press, 2000. Foege, William H. House on Fire: The Fight to Eradicate Smallpox. Berkeley: University of California Press, 2011. Glynn, Ian, and Jennifer Glynn. The Life and Death of Smallpox. London: Profile Books, 2004. Henderson, Donald A. Smallpox: The Death of a Disease. Amherst, NY: Prometheus Books, 2009. Oshinsky, David M. Polio: An American Story. Oxford: Oxford University Press, 2005. Rhodes, John. The End of Plagues: The Global Battle against Infectious Disease. New York: Palgrave Macmillan, 2013. Williams, Gareth. Angel of Death: The Story of Smallpox. London: Palgrave Macmillan, 2010. Williams, Gareth. Paralyzed with Fear: The Story of Polio. London: Palgrave Macmillan, 2013. Specific Reading

Bahl, Kapil, Joe J. Senn, Olga Yuzhakov, et al. “Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses.” Molecular Therapy 25 (2017): 1316–­27. Baxby, Derek. Vaccination: Jenner’s Legacy. Berkeley: Jenner Educational Trust, 1994. Baxby, Derek, and William Hanna. “Review of Studies in Smallpox and Vaccination.” Reviews in Medical Virology 12 (2002): 202–­9. Beall, Otho T., and Richard H. Shryock. Cotton Mather: First Significant Figure in American Medicine. Baltimore: Johns Hopkins University Press, 1954.

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Berlanda Scorza, Francesco, and Norbert Pardi. “New Kids on the Block: RNA-­Based Influenza Virus Vaccines.” Vaccines 6 (2018): 20. https://doi.org/10.3390/vaccines6020020. Cai, Ying, Stephen Rodriguez, and Henry Hebel. “DNA Vaccine Manufacture: Scale and Quality.” Expert Review of Vaccines 8 (2009): 1277–­91. Clarke, David K., R. Michael Hendry, Vidisha Singh, et al. “Live Virus Vaccines Based on a Vesicular Stomatitis Virus (VSV) Backbone: Standardized Template with Key Considerations for a Risk/Benefit Assessment.” Vaccine 34 (2016): 6597–­6609. Coffman, Robert L., Alan Sher, and Robert A. Seder. “Vaccine Adjuvants: Putting Innate Immunity to Work.” Immunity 33 (2010): 492–­503. Donnelly, John J., Jeffrey B. Ulmer, and Margaret A. Liu. “DNA Vaccines.” Life Sciences 60 (1996): 163–­72. Folegatti, Pedro M, Mustapha Bittaye, Amy Flaxman, et al. “Safety and Immunogenicity of a Candidate Middle East Respiratory Syndrome Coronavirus Viral-­Vectored Vaccine: A Dose-­Escalation, Open-­Label, Non-­Randomised, Uncontrolled, Phase 1 trial.” Lancet Infectious Diseases 20 (2020): 816–­26. Fosbroke, Thomas Dudley. Berkeley Manuscripts. London: John Nichols, 1821. Franco-­Paredes, Carlos, Lorena Lammoglia, and José Ignacio Santos-­ Preciado. “The Spanish Royal Philanthropic Expeditions to Bring Smallpox Vaccination to the New World and Asia in the 19th Century.” Clinical Infectious Diseases 41 (2005): 1285–­89. Gilbert, Sarah C., and George M. Warimwe. “Rapid Development of Vaccines against Emerging Pathogens: The Replication-­Deficient Simian Adenovirus Platform Technology.” Vaccine 35 (2017): 4461–­64. Glenny, Alexander T., C. G. Pope, H. Waddington, and U. Wallace. “Immunological Notes: XVII–­XXIV.” Journal of Pathological Bacteriology 29 (1926): 31–­40. Glenny, Alexander T., and H. J. Südmersen. “Notes on the Production of Immunity to Diphtheria Toxin.” Journal of Hygiene 20 (1921): 176–­220. Henderson, D. A. “Smallpox Eradication—­the Final Battle.” Journal of Clinical Pathology 28 (1975): 843–­49. Jenner, Edward. An Inquiry into the Causes and Effect of the Variolae

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Vaccinae—­A Disease Discovered in Some of the Western Counties of England, Particularly Gloucestershire, and Known by the Name of the Cow Pox. London: Sampson Low, 1798. Karikó, Katalin, Michael Buckstein, Houping Ni, and Drew Weissman. “Suppression of RNA Recognition by Toll-­like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA.” Immunity 23 (2005): 165–­75. Kovesdi, Imre, and Susan J. Hedley. “Adenoviral Producer Cells.” Viruses 2 (2010): 1681–­1703. Leitner, Wolfgang W., Han Ying, and Nicholas P. Restifo. “DNA and RNA-­Based Vaccines: Principles, Progress and Prospects.” Vaccine 18 (1999): 765–­77. Luca, Simona, and Traian Mihaescu. “History of BCG Vaccine.” Maedica (Buchar) 8 (2013): 53–­58. Pardi, Norbert, Michael J. Hogan, Frederick W. Porter, and Drew Weissman. “mRNA Vaccines –­a New Era.” Nature Reviews: Drug Discovery 17 (2018): 261–­79. Robert-­Guroff, Marjorie. “Replicating and Non-­Replicating Viral Vectors for Vaccine Development.” Current Opinion in Biotechnology 18 (2007): 546–­56. Sabin, Albert B., and L. R. Boulger. “History of Sabin Attenuated Poliovirus Oral Live Vaccine Strains.” Journal of Biological Standardization 1 (1973): 115–­18. Salk, Jonas E., U. Krech, and J. S. Younger. “Formaldehyde Treatment and Safety Testing of Experimental Poliomyelitis Vaccines.” American Journal of Public Health 44 (1954): 563–­70. Sloane, Hans, and T. Bart. “An Account of Inoculation by Sir Hans Sloane, Bart. Given to Mr. Ranby, to Be Published, Anno 1736.” Philosophical Transactions of the Royal Society 49 (1755): 516–­20. Tang, De Chu, Michael DeVit, and Stephen A. Johnston. “Genetic Immunization Is a Simple Method for Eliciting an Immune Response.” Nature 356 (1992): 152–­54. Wang, Ruobing, Denise L. Doolan, Thong P. Le, et al. “Induction of Antigen-­Specific Cytotoxic T Lymphocytes in Humans by a Malaria DNA Vaccine.” Science 282 (1998): 476–­80. Wolff, Jon A., R. W. Malone, P. Williams, W. Chong, G. A. Acsadi, A. Jani, and P. L. Felgner. “Direct Gene Transfer into Mouse Muscle in Vivo.” Science 247 (1990): 1465–­68.

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Chapter FOUR: Develop Vaccines

General Reading

Baschieri, Selene, ed. Innovation in Vaccinology: From Design, through to Delivery and Testing. New York: Springer, 2012. Bloom, Barry R., and Paul-­Henri Lambert. The Vaccine Book. Philadelphia: Academic Press, Elsevier, 2003. Plotkin, Stanley A., ed. History of Vaccine Development. New York: Springer, 2011. Singh, Manmohan, and Indresh K. Srivastava, eds. Development of Vaccines: From Discovery to Clinical Testing. Hoboken, NJ: Wiley, 2011. Thomas, Sunil, ed. Vaccine Design: Methods and Protocols: Volume 1: Vaccines for Human Diseases. New York: Springer, 2016. von Gabain, Alexander, and Christoph Klade, eds. Development of Novel Vaccines Skills, Knowledge and Translational Technologies. New York: Springer, 2012. Wen, Emily P. S., Narahari S. Pujar, and Ronald Ellis. Vaccine Development and Manufacturing. Hoboken, NJ: Wiley, 2015. Specific Reading.

Chen, Huaqing, Simon Hall, Brian Heffernan, Neil T. Thompson, Mark V. Rogers, and John Rhodes. “Convergence of Schiff Base Costimulatory Signaling and T-­Cell Receptor Signaling at the Level of Mitogen Activated Protein Kinase ERK2.” Journal of Immunology 159 (1997): 2274–­81. Detoc, Maelle, A. Gagneux-­Brunon, F. Lucht, and E. Botelho-­Nevers. “Barriers and Motivations to Volunteers’ Participation in Preventive Vaccine Trials: A Systematic Review.” Expert Review of Vaccines 16 (2017): 467–­77. Ferran, Maureen C., and Gary R. Skuse. Recombinant Virus Vaccines: Methods and Protocols. New York: Springer, 2017. Foged, Camilla, Thomas Rades, Yvonne Perrie, and Sarah Hook, eds. Subunit Vaccine Delivery. New York: Springer, 2015. Giese, Matthias. Introduction to Molecular Vaccinology. New York: Springer, 2016. Greenwood, Brian. “The Contribution of Vaccination to Global Health: Past, Present and Future.” Philosophical Transactions of the

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Royal Society Series B. 369 (2014): 20130433. Hall, Simon R., and John Rhodes. “Schiff Base Mediated Costimulation Primes the TCR-­Dependent Calcium Signalling Pathway in CD4 T-­cells.” Immunology 104 (2001): 50–­57. Kahn, Rebecca, Annette Rid, Peter G. Smith, Nir Eyal, and Marc Lipsitch. “Choices in Vaccine Trial Design in Epidemics of Emerging Infections.” PLoS Med 15 (2018): e1002632. https://doi. org/10.1371/journal.pmed.1002632 2018. Kozlowski, Pamela A., ed. Mucosal Vaccines: Modern Concepts, Strategies, and Challenges. New York: Springer, 2012. Lukashevich, Igor S., and Haval Shirwan, eds. Novel Technologies for Vaccine Development. New York: Springer, 2015 . Mehrotra, Devan V. “Vaccine Clinical Trials: A Statistical Primer.” Journal of Biopharmaceutical Statistics 16 (2006): 403–­14. Offit, Paul A. The Cutter Incident: How America’s First Polio Vaccine Led to the Growing Vaccine Crisis. New Haven, CT: Yale University Press, 2005. Rhodes, John. “Covalent Chemical Events in Immune Induction: Fundamental and Therapeutic Aspects.” Immunology Today 17 (1996): 436–­41. Rhodes, John. “Discovery of Immunopotentiatory Drugs: Current and Future Strategies.” Clinical and Experimental Immunology 130 (2002): 363–­69. Rhodes, John, Huaqing Chen, Simon Hall, Julian E. Beesley, David C. Jenkins, Peter Collins, and Biao Zheng. “Therapeutic Potentiation of the Immune System by Costimulatory Schiff Base-­Forming Drugs.” Nature 377 (1995): 71–­75. Rinaldi, Monica, Daniela Fioretti, and Sandra Lurescia, eds. DNA Vaccines: Methods and Protocols. New York: Springer, 2014. Saul, Allan. “Models of Phase 1 Vaccine Trials: Optimization of Trial Design to Minimize Risks of Multiple Serious Adverse Events.” Vaccine 23 (2005): 3068–­75. Singh, Kavita, and S. Mehta. “The Clinical Development Process for a Novel Preventive Vaccine: An Overview.” Journal of Postgraduate Medicine 62 (2016): 4–­11. Voordouw, B., R. F. Breiman, and J. Clemens. “Guidelines on Clinical Evaluation of Vaccines: Regulatory Expectations.” WHO Technical Report series no. 924 (2004).

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Chapter FIVE: Evaluate the Contenders

General Reading

Bar-­Zeev, Naor, and Tom Inglesby. “COVID-­19 Vaccines: Early Success and Remaining Challenges.” Lancet 396, no. 10255 (2020): 868–­69. https://doi.org/10.1016/S0140-­6736 (20)31867-­5. Chen, Wen-­Hsiang, Ulrich Strych, Peter J Hotez, and Maria Elena Bottazzi. “The SARS-­CoV-­2 Vaccine Pipeline: an Overview.” Current Tropical Medicine Reports 7 (2020): 61–­64. Chtarbanova, Stanislava, and Jean-­Luc Imler. “Microbial Sensing by Toll Receptors: A Historical Perspective.” Arteriosclerosis, Thrombosis, and Vascular Biology 31 (2011): 1734–­38. Frantz, Phanramphoei N., Samaporn Teeravechyan, and Frédéric Tangy. “Measles-­Derived Vaccines to Prevent Emerging Viral Diseases.” Microbes and Infection 20 (2018): 493–­500. Fuller, Deborah H., and Peter Berglund. “Amplifying RNA Vaccine Development.” New England Journal of Medicine 382 (2020): 2469–­ 71. Funk, Colin D., Craig Laferrière, and Ali Ardakani. “A Snapshot of the Global Race for Vaccines Targeting SARS-­CoV-­2 and the COVID-­19 Pandemic.” Frontiers in Pharmacology 11 (2020). https://doi.org/10.3389/fphar.2020.00937. Gao, Qiang, Linlin Bao, Haiyan Mao, et al. “Development of an Inactivated Vaccine Candidate for SARS-­CoV-­2.” Science 369, no. 6499 (2020): 77–­81. https://doi.org/10.1126/science.abc1932. Kim, Young Chan, Barbara Dema, and Arturo Reyes-­Sandoval. “COVID-­19 Vaccines: Breaking Record Times to First-­in-­Human Trials.” npj Vaccines 5, no. 34 (2020). https://doi.org/10.1038/ s41541-­020-­0188-­3. Liu, Margaret A., and Jeffrey B. Ulmer. “Human Clinical Trials of Plasmid DNA Vaccines.” Advances in Genetics 55 (2020): 25–­40. Lurie, Nicole, Melanie Saville, Richard Hatchett, and Jane Halton. “Developing Covid-­19 Vaccines at Pandemic Speed.” New England Journal of Medicine 382 (2020): 1969–­73. Robert-­Guroff, Marjorie. “Replicating and Non-­Replicating Viral Vectors for Vaccine Development.” Current Opinion in Biotechnology 18 (2007): 546–­56. Slaoui, Moncef, and Matthew Hepburn. “Developing Safe and Effec-

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tive COVID Vaccines —­Operation Warp Speed’s Strategy and Approach.” New England Journal of Medicine 383 (2020): 1701–­3. https://doi.org/10.1056/NEJMp2027405. Specific Reading

Alharbi, Naif K., Eriko Padron-­Regalado, Craig P. Thompson, et al. “ChAdOx1 and MVA Based Vaccine Candidates against MERS-­CoV Elicit Neutralising Antibodies and Cellular Immune Responses in Mice.” Vaccine 35 (2017): 3780–­88. Berger, Imre, Daniel J Fitzgerald, and Timothy J Richmond. “Baculovirus Expression System for Heterologous Multiprotein Complexes.” Nature Biotechnology 22 (2004): 1583–­87. Coleman, J. Robert, Dimitris Papamichai, Steven Skiena, Bruce Futcher, Eckard Wimmer, and Steffen Mueller. “Virus Attenuation by Genome-­Scale Changes in Codon Pair Bias.” Science 320 (2008): 1784–­87. Cotter, Catherine A., Patricia L. Earl, Linda S. Wyatt, and Bernard Moss. Preparation of Cell Cultures and Vaccinia Virus Stocks. Current Protocols in Microbiology 39 (2015): 14A.3.1–­14A.3.18. https:// doi.org/10.1002/9780471729259.mc14a03s39. Di Pasquale, Alberta, Scott Preiss, Fernanda Tavares Da Silva, and Nathalie Garçon. “Vaccine Adjuvants: From 1920 to 2015 and Beyond.” Vaccines 3 (2015): 320–­43. Feng, Jicai, Faces in the Crowd: 36 Extraordinary Tales of Tianjin. Translated by Olivia Milburn. London: Sinoist Books, 2019. Folegatti, Pedro M., Katie J. Ewer, Parvinder K. Aley, et al. “Safety and Immunogenicity of the ChAdOx1 nCoV-­19 Vaccine against SARS-­CoV-­2: A Preliminary Report of a Phase 1/2, Single-­Blind, Randomised Controlled Trial.” Lancet 396, no. 10249 (2020): 467–­ 78. http://doi.org/10.1016/S0140-­6736(20)31604-­4. Garçon, Nathalie, and Alberta Di Pasquale. “From Discovery to Licensure, the Adjuvant System Story.” Human Vaccines and Immunotherapeutics 13 (2017): 19–­33. Jackson, Lisa A., Evan J. Anderson, Nadine G. Rouphael, et al. “An mRNA Vaccine against SARS-­CoV-­2 —­Preliminary Report.” New England Journal of Medicine 383 (2020): 1920–­31. http://doi. org/10.1056/NEJMoa2022483.

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Logunov, D. Y., I. V. Dolzhikova, O. V. Zubkova, et al. “Safety and Immunogenicity of an rAd26 and rAd5 Vector-­Based Heterologous Prime-­Boost COVID-­19 Vaccine in Two Formulations: Two Open-­Label, Non-­Randomised Phase 2/2 Studies from Russia.” Lancet 396 (2020): 887–­97. http://doi.org/10.1016/S0140-­ 6736(20)31866-­3. McKay, Paul F., Kai Hu, Anna K. Blakney, et al. “Self-­Amplifying RNA SARS-­CoV-­2 Lipid Nanoparticle Vaccine Candidate Induces High Neutralizing Antibody Titers in Mice.” Nature Communications 11 no. 3523 (2020). https://www.nature.com/articles/s41467-­020-­ 17409-­9. Mercado, Noe B., Roland Zahn, Frank Wegmann, et al. “Single-­Shot Ad26 Vaccine Protects against SARS-­CoV-­2 in Rhesus Macaques.” Nature 586 (2020): 583–­88. https://doi.org/10.1038/s41586-­020-­ 2607-­z. Nusslein-­Volhard, Christiane W. E. “Mutations Affecting Segment Number and Polarity in Drosophila.” Nature 287 (1980): 795–­801. Rollier, Christine S., Alexandra J. Spencer, Karen Colbjorn Sogaard, Jared Honeycutt, Julie Furze, Migena Bregu, Sarah C. Gilbert, David Wyllie, and Adrian V. S. Hill. “Modification of Adenovirus Vaccine Vector-­Induced Immune Responses by Expression of a Signalling Molecule.” Scientific Reports 10, no. 5716 (2020). https:// doi.org/10.1038/s41598-­020-­61730-­8. RTS,S Clinical Trials Partnership. “Efficacy and Safety of RTS,S/AS01 Malaria Vaccine with or without a Booster Dose in Infants and Children in Africa: Final Results of a Phase 3, Individually Randomised, Controlled Trial.” Lancet 386 (2015): 31–­45. Schiller, John T., Xavier Castellsagué, and Suzanne M. Garland. “A Review of Clinical Trials of Human Papillomavirus Prophylactic Vaccines.” Vaccine 30 (2012): F123–­F138. Shukarev, Georgi, Benoit Callendret, Kerstin Luhn, et. al. “A Two-­ Dose Heterologous Prime-­Boost Vaccine Regimen Eliciting Sustained Immune Responses to Ebola Zaire Could Support a Preventive Strategy for Future Outbreaks.” Human Vaccines and Immunotherapeutics 13 (2017): 266–­70. Smith, Trevor R. F., Ami Patel, and Kate E. Broderick. “Immunogenicity of a DNA Vaccine Candidate for COVID-­19.” Nature Communications 11, no. 2601 (2020).

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Sun, Hong-­Xiang, Yong Xie, and Yi-­Pin Ye. “Advances in Saponin-­ Based Adjuvants.” Vaccine 27 (2009): 1787–­96. van Doremalen, Neeltje, Teresa Lambe, Alexandra Spencer, et al. “ChAdOx1 nCoV-­19 Vaccination Prevents SARS-­CoV-­2 1 Pneumonia in Rhesus Macaques.” Nature 586 (2020): 578–­82. https://doi. org/10.1038/s41586-­020-­2608-­y. Vogel, Annette B., Laura Lambert, Ekaterina Kinnear, et al. “Self-­ Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses.” Molecular Therapy 26 (2018): 446–­455. Xia, Shengli, Kai Duan, Yuntao Zhang, et al. “Effect of an Inactivated Vaccine against SARS-­CoV-­2 on Safety and Immunogenicity Outcomes: Interim Analysis of Two Randomized Clinical Trials.” Journal of the American Medical Association 324, no.10 (2020): 951–­60. https://doi.org/10.1001/jama.2020.15543. Zhang, Yan-­Jun, Gang Zeng, Hong-­Xing Pan, et al. “Immunogenicity and Safety of a SARS-­CoV-­2 Inactivated Vaccine in Healthy Adults Aged 18–­59 Years: Report of the Randomized, Double-­Blind, and Placebo-­Controlled Phase 2 Clinical Trial.” medRxiv preprint, 2020. https://doi.org/10.1101/2020.07.31.20161216. Zhu, Daming, and Wenbin Tuo. “QS-­21: A Potent Vaccine Adjuvant.” Natural Products Chemistry and Research 3, no. 4 (2016). https:// doi.org/10.4172/2329-­6836.1000e113. Zhu, Feng-­Cai, Xu-­Hua Guan, Yu-­Hua Li, et al. “Immunogenicity and Safety of a Recombinant Adenovirus Type-­5-­Vectored COVID-­19 Vaccine in Healthy Adults Aged 18 Years or Older: A Randomised, Double-­Blind, Placebo Controlled, Phase 2 Trial.” Lancet 396, no. 10249 (2020): 479–­88. https://doi.org/10.1016/S0140-­ 6736(20)31605-­6. Zhu, Feng-­Cai, Li-­Hua Hou, Jing-­Xin Li, et al. “Safety and Immunogenicity of a Novel Recombinant Adenovirus Type-­5 Vector-­Based Ebola Vaccine in Healthy Adults in China: Preliminary Report of a Randomised, Double-­Blind, Placebo-­Controlled, Phase 1 Trial.” Lancet 385 (2015): 2272–­79.

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Chapter SIX: Don ’t Count on the Magic Bullet

General Reading

Chakraborty, S., B. M. Veeregowda, R. Deb, and B. M. Chandra Naik. “An Overview of the Immune Evasion Strategies Adopted by Different Viruses with Special Reference to Classical Swine Fever Virus.” Intech (2013). http://dx.doi.org/10.5772/55435. Crowcroft, Natasha S., and Nicola P. Klein. “A Framework for Research on Vaccine Effectiveness.” Vaccine 36 (2018): 7286–­93. Helmy, Yosra A., Mohamed Fawzy, Ahmed Elaswad, Ahmed Sobieh, Scott P. Kenney, and Awad A. Shehata. “The COVID-­19 Pandemic: A Comprehensive Review of Taxonomy, Genetics, Epidemiology, Diagnosis, Treatment, and Control.” Journal of Clinical Medicine 9, no. 4 (2020): 1225. https://doi.org/10.3390/jcm9041225. Valent, Peter, Bernd Groner, Udo Schumacher, et al. “Paul Ehrlich (1854–­1915) and His Contributions to the Foundation and Birth of Translational Medicine.” Journal of Innate Immunity 8 (2016): 111–­20. Specific Reading

Arvin, Ann M., Katja Fink, Michael A. Schmid, Andrea Cathcart, Roberto Spreafico, Colin Havenar-­Daughton, Antonio Lanzavecchia, Davide Corti, and Herbert W. Virgin. “A Perspective on Potential Antibody-­Dependent Enhancement of SARS-­CoV-­2.” Nature 584 (2020): 353–­63. Beigel, John H., Kay M. Tomashek, Lori E. Dodd, et al. “Remdesivir for the Treatment of Covid-­19 —­Preliminary Report.” New England Journal of Medicine 383 (2020): 1813–­26. https://doi. org/10.1056/NEJMoa2007764. Cao, Zhiliang, Lifeng Lui, Lanying Du, Chao Zhang, Shibo Jiang, Taisheng Li, and Yuxian He. “Potent and Persistent Antibody Responses against the Receptor-­Binding Domain of SARS-­CoV Spike Protein in Recovered Patients.” Virology Journal 7 (2010): 229. www.virologyj.com/content/7/1/299. Denison, Mark R., Rachel L. Graham, Eric F. Donaldson, Lance D. Eckerle, and Ralph S. Baric. “Coronaviruses: An RNA Proofreading Machine Regulates Replication Fidelity and Diversity.” RNA Biology 8 (2011): 270–­79.

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Ferdinands, Jill M., Lauren E. W. Olsho, Anna A. Agan, et al. “Effectiveness of Influenza Vaccine Against Life-­Threatening RT-­PCR-­ Confirmed Influenza Illness in US Children, 2010–­2012.” Journal of Infectious Diseases, 210 (2014): 674–­83. Grandjean, Louis, Anja Saso, Arturo Torres, et al. “Humoral Response Dynamics Following Infection with SARS-­CoV-­2.” medRxiv preprint, posted July 2020. https://doi.org/10.1101/2020.07.16.20155663. Grifoni, Alba., Daniela Weiskopf, Sydney I. Ramirez, et al. “Targets of T Cell Responses to SARS-­CoV-­2 Coronavirus in Humans with COVID-­19 Disease and Unexposed Individuals.” Cell 181, no. 7 (2020): 1489–­1501.e15. https://doi.org/10.1016/j.cell.2020.05.015. Henao-­Restrepo, Ana Maria, Anton Camacho, Ira M Longini, et al. “Efficacy and Effectiveness of an rVSV-­Vectored Vaccine in Preventing Ebola Virus Disease: Final Results from the Guinea Ring Vaccination, Open-­Label, Cluster-­Randomised Trial (Ebola Ça Suffit!).” Lancet 389 (2017): 505–­18. Hongying Chenga, Mary, She Zhanga, Rebecca A. Porrittb, Moshe Arditi, and Ivet Bahar. “An Insertion Unique to SARS-­CoV-­2 Exhibits Superantigenic Character Strengthened by Recent Mutations.” bioRxiv preprint, posted May 2020. https://doi. org/10.1101/2020.05.21.109272. Horby, Peter, Wei Shen Lim, Jonathan Emberson, et al. “Effect of Dexamethasone in Hospitalized Patients with COVID-­19: Preliminary Report.” medRxiv preprint, posted June 2020. https://doi.org /10.1101/2020.06.22.20137273. Huang, Angkana T., Bernardo Garcia-­Carreras, Matt D. T. Hitchings, et al. “A Systematic Review of Antibody Mediated Immunity to Coronaviruses: Antibody Kinetics, Correlates of Protection, and Association of Antibody Responses with Severity of Disease.” bioRxiv preprint, posted May 2020. https://doi. org/10.1101/2020.05.21.109272. Johnson, Welkin E., and Ronald C. Desrosiers. “Viral Persistence: HIV’s Strategies of Immune System Evasion.” Annual Review of Medicine 53 (2002): 499–­518. Karasavvas, Nicos, Erik Billings, Mangala Rao, et al. “The Thai Phase III HIV Type 1 Vaccine Trial (RV144) Regimen Induces Antibodies That Target Conserved Regions within the V2 Loop of gp120.” AIDS Research and Human Retroviruses 28 (2012): 1444–­57.

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Khalaf Alharbi, Naif, Eriko Padron-­Regalado, Craig Thompson, et al. “ChAdOx1 and MVA Based Vaccine Candidates against MERS-­CoV Elicit Neutralising Antibodies and Cellular Immune Responses in Mice.” Vaccine 35 (2017): 3780–­88. Konno, Yoriyuki, Izumi Kimura, Keiya Uriu, et al. “SARS-­ CoV-­2 ORF3b is a Potent Interferon Antagonist whose Activity is Increased by a Naturally Occurring Elongation Variant.” Cell Reports 32, no. 12 (2020). https://doi.org/10.1016/j.celrep.2020.108185. Lewnard, Joseph A., and Sarah Cobey. “Immune History and Influenza Vaccine Effectiveness.” Vaccines 6, no. 28 (2018). https://doi. org/10.3390/vaccines6020028. Li, Taisheng, Jing Xie, Yuxian He, et al. “Long-­Term Persistence of Robust Antibody and Cytotoxic T Cell Responses in Recovered Patients Infected with SARS Coronavirus.” PLoS ONE 1, no.1 (2006): e24. https://doi.org/10.1371/journal.pone.0000024. Luo, Fan, Fan-­Lu Liao, Hui Wang, Hong-­Bin Tang, Zhan-­Qiu Yang, and Wei Hou. “Evaluation of Antibody-­Dependent Enhancement of SARS-­CoV Infection in Rhesus Macaques Immunized with an Inactivated SARS-­CoV Vaccine.” Virologica Sinica 33 (2018): 201–­4. MacLean, Oscar A., Richard J. Orton, Joshua B. Singer, and David L. Robertson. “No Evidence for Distinct Types in the Evolution of SARS-­CoV-­2.” Virus Evolution 6, no. 1 (2020). https://doi. org/10.1093/ve/veaa034. Monto, Arnold S. “Francis Field Trial of Inactivated Poliomyelitis Vaccine: Background and Lessons for Today.” Epidemiological Reviews 21 (1999): 7–­23. Pachetti, Maria, Bruna Marini, Francesca Benedetti, et al. “Emerging SARS‑CoV‑2 Mutation Hot Spots Include a Novel RNA‑Dependent‑RNA Polymerase Variant.” Journal of Translational Medicine 18, no. 179 (2020). https://doi.org/10.1186/s12967-­020-­02344-­6. Pang, Juanita, Florencia A. T. Boshier, Nele Alders, Garth Dixon, and Judith Breuer. “No Evidence of Viral Polymorphisms Associated with Paediatric Inflammatory Multisystem Syndrome Temporally Associated With SARS-­CoV-­2 (PIMS-­TS).” medRxiv preprint, posted July 2020. https://doi.org/10.1101/2020.07.07.20148213. Payne, Daniel C., Ibrahim Iblan, Brian Rha, et al. “Persistence of Antibodies against Middle East Respiratory Syndrome Coronavirus.”

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General Reading

Conis, Elena. Vaccine Nation: America’s Changing Relationship with Immunisation. Chicago: Chicago University Press, 2016.

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Durbach, Nadja. Bodily Matters: The Anti-­Vaccination Movement in England 1853–­1907. Durham: Duke University Press, 2005. Offit, Paul. How the Anti-­Vaccine Movement Threatens Us All. Philadelphia: Basic Books, 2015. Reich, Jennifer A. Calling the Shots: Why Parents Reject Vaccines. New York: New York University Press, 2018. Willrich, Michael. Pox: An American History. New York: Penguin Books, 2011. Specific Reading

Barlow, William E., Robert L. Davis, and John W. Glasser. “The Risk of Seizures after Receipt of Whole-­Cell Pertussis or Measles, Mumps, and Rubella Vaccine.” New England Journal of Medicine 345 (2001): 656–­61. Becton Dickinson and Co. Celebrating the First One Hundred: 1897–­ 1997. Franklin Lakes, NJ: Becton Dickinson and Co., 1997. Berkovic, Samuel F., Louise Harkin, Jacinta M. McMahon, et al. “De-­ Novo Mutations of the Sodium Channel Gene SCN1A in Alleged Vaccine Encephalopathy: A Retrospective Study.” Lancet Neurology 5 (2006): 488–­92. Dawood, Fatimah S., A. Danielle Iuliano, Carrie Reed, et al. “Estimated Global Mortality Associated with the First 12 Months of 2009 Pandemic Influenza A H1N1virus Circulation: a Modelling Study.” Lancet Infect Diseases 12 (2012): 687–­95. de Oliveira, Lúcia Helena, and Claudio José Struchiner. “Vaccine-­ Associated Paralytic Poliomyelitis: A Retrospective Cohort Study of Acute Flaccid Paralyses in Brazil.” International Journal of Epidemiology 29 (2000): 757–­63. Gale, James L., Purushottam B. Thapa, and Steven G. F. Wassilak. “Risk of Serious Acute Neurological Illness After Immunization With Diphtheria-­Tetanus-­Pertussis Vaccine: A Population-­Based Case-­Control Study.” Journal of the American Medical Association 271 (1994): 37–­41. Gavi: The Vaccine Alliance. “COVAX: Ensuring Global Equitable Access to COVID-­19 Vaccines.” YouTube video. July 24, 2020. https://www.youtube.com/watch?v=5opR6x6NMpQ&feature=emb_title.

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Gouglas, Dimitrios, Mario Christodoulou, Stanley A. Plotkin, and Richard Hatchett. “CEPI: Driving Progress Toward Epidemic Preparedness and Response.” Epidemiological Reviews 41, no. 1 (2019): 28–­33. https://doi.org/10.1093/epirev/mxz012. Graham, Margaret B. W., and Alec T. Shuldiner. Corning and the Craft of Innovation. Oxford: Oxford University Press, 2001. Institute of Medicine. Report: Immunization Safety Review: Measles-­ Mumps-­Rubella Vaccine and Autism. Washington, DC: National Academy Press, 2001. Institute of Medicine. Report: Immunization Safety Review: Vaccines and Autism. Washington, DC: National Academy Press, 2004. Jefferson, Tom, Melanie Rudin, and Carlo Di Pietrantonj. “Systematic Review of the Effects of Pertussis Vaccines in Children.” Vaccine 21 (2003): 2003–­2014. Lane, Sarah, Noni E. MacDonald, Melanie Marti, and Laure Dumolard. “Vaccine Hesitancy around the Globe: Analysis of Three Years of WHO/UNICEF Joint Reporting Form Data-­2015–­2017.” Vaccine 36 (2018): 3861–­67. Miller, Elizabeth, Nick Andrews, Lesley Stellitano, Julia Stowe, Anne Marie Winstone, John Shneerson, and Christopher Verity. “Risk of Narcolepsy in Children and Young People Receiving AS03 Adjuvanted Pandemic A/H1N1 2009 Influenza Vaccine: Retrospective Analysis.” British Medical Journal 346 (2013): f794. Neil, Stuart J. D., and Edward M. Campbell. “Fake Science: XMRV, COVID-­19, and the Toxic Legacy of Dr. Judy Mikovits.” Aids Research and Human Retroviruses 36, no. 7 (2020): 545–­49. https:// doi.org/10.1089/aid.2020.0095. Pana, Daniel, Shirley Szec, Jatinder S. Minhas, et al. “The Impact of Ethnicity on Clinical Outcomes in COVID-­19: A Systematic Review.” EClinicalMedicine (published by The Lancet) 23 (2020): 100404. https://doi.org/10.1016/j.eclinm.2020.100404. Taylor, Luke E., Amy L. Swerdfeger, and Guy D Eslick. “Vaccines Are Not Associated with Autism: An Evidence-­Based Meta-­Analysis of Case-­Control and Cohort Studies.” Vaccine 32 (2014): 3623–­29. World Health Organization: “SAGE Working Group dealing with Vaccine Hesitancy -­Systematic Review of Strategies,” 2014. Yen, Catherine, Kelly Healy, Jacqueline E. Tate, Umesh D. Parashar, Julie Bines, Kathleen Neuzil, Mathuram Santosham, and A. Dun-

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can Steele. “Rotavirus Vaccination and Intussusception –­Science, Surveillance, and Safety: A Review of Evidence and Recommendations for Future Research Priorities in Low and Middle Income Countries.” Human Vaccines and Immunotherapeutics 12 (2016): 2580–­89. Chapter EIGHT: Embrace Many Solutions

Specific Reading

Chao, Yuen Ren. “The Non-­Uniqueness of Phonemic Solutions of Phonetic Systems.” Bulletin of the Institute of History and Philology 4 (1934): 363–­98. Keech, Cheryl, Gary Albert, Iksung Cho, et al. “Phase 1–­2 Trial of a SARS-­CoV-­2 Recombinant Spike Protein Nanoparticle Vaccine.” New England Journal of Medicine, 2020. https://doi.org/10.1056/ NEJMoa2026920. Lövgren Bengtsson, Karin, Bror Morein, and Albert D. M. E. Osterhaus. “ISCOM Technology-­Based Matrix M™ Adjuvant: Success in Future Vaccines Relies on Formulation.” Expert Review of Vaccines 10 (2011): 401–­3. https://doi.org/10.1586/erv.11.25. Morein, Bror, B. Sundquist, S. Höglund, K. Dalsgaard, and A. Osterhaus. “ISCOM, a Novel Structure for Antigenic Presentation of Membrane Proteins from Enveloped Viruses.” Nature 308 (1984): 457–­60. Soltysik, Sean, Jia-­Yan Wu, Joanne Recchia, Deborah A. Wheeler, Mark J. Newman, Richard T. Coughlin, and Charlotte R. Kensil. “Structure/Function Studies of QS-­21 Adjuvant: Assessment of Triterpene Aldehyde and Glucuronic Acid Roles in Adjuvant Function.” Vaccine 13 (1995): 1403–­10.

INDEX

Abubakar, Muhammadu Sa’ad, 108 Ad5 vector COVID-­19 vaccine, 57, 103 adenoviral vectors, 50, 79–­81, 83, 103 adenovirus serotype 5 (Ad5), 79, 82 adenovirus serotype 26 (Ad26), 82 adenoviruses, 48–­49, 78–­79, 102–­3 adjuvants, 33, 43, 45, 57, 61, 63, 69–­74, 84, 88–­89, 105, 125–­27 aldehydes, 61, 127 allergies, 112 Almeida, June, 8 alphaviruses, 90 alum (aluminum salts), 43, 69, 72 Andersen Kristian, 11 angiotensin-­converting enzyme 2 (ACE2), 9, 68, 91, 95 animal studies, 5, 55, 68, 96, 104 anthrax, 42–­43 antibiotic, 47­–­48 antibodies, 16–­20, 23, 27–­30, 41–­43, 47, 50, 55, 57, 64–­65, 69, 77, 96; enhancing, 104–­5; genetic coding, 29–­30; horse, 18, 72; molecular structure, 19–­ 21; persistence and quality of, 97 antigens, 16, 19, 21, 27, 42, 48, 54, 61–­62, 78, 82, 125; transplantation, 24–­25 antisera, 78 antitoxin, 15 antivaccination movement, 109–­13 arthritis, rheumatoid, 101 AS03, 74 AstraZeneca, 106, 123, 129, 135 autism, 111–­13 Bacillus Calmette-­Guérin, 46 bacteria, 6, 43, 47–­48, 63

bacterial toxins, chemical inactivation of, 72 baculovirus, 70–­71, 126 bats, 5–­6, 11, 50, 78 Bauchner, Howard, 118 B-­cells, and memory B-­cells, 21–­23, 29, 41 Beaudette, Fred, 6 Becton Dickinson, 118–­19 Behring, Emil von, 14–­16, 60 Beigel, John H., 100 Beijerinck, Martinus, 6 Berkovic, Samuel, 113 β-­propiolactone, 68 Beth Israel Deaconess Medical Center, 82 biological oils, 73 Biologics License Application, 58 Biomedical Advanced Research and Development Authority (BARDA), 106 BioNTech, 129 bioterrorism, 106 bird viruses, xi bone marrow, 28–­29 boosting effect, 80 Boylston, Zabdiel, 37 Breinl, Friedrich, 16 Breuer, Judith, 100 British Cancer Research Campaign (Cancer Research UK), 54 camels, 50 Camp Funston, x–­xi Can Sino Biologics, 103 cancer, 62–­63, 84, 124

164 / I N D E X

Cancer Agency Genome Sciences Centre (British Columbia), 3 Cantor, Eddie, 114 Caroline, princess of Wales, 36–­37 Cedars Sinai Medical Center (Los Angeles), 99 cell lines, 58, 65, 68, 71 Centers for Disease Control and Prevention (CDC), 93, 110, 116, 120, 122 ChAdOx1, and ChAdOx1 nCoV-­19, 50, 79–­81, 95, 103 charitable organizations, in vaccine development, 60 Charles IV of Spain, 42 chicken bronchitis virus, 6 chicken pox, 117 childhood vaccines. See immunization, of children chimpanzees, 50, 79, 103. See also primates, used in laboratories China National Health Commission, 10 cholera, 42–­43, 45 clinical data collection, 10 clinical fever, 26 clinical trials (testing): xiii, 56, 58, 69–­ 70, 74, 76–­77, 79–­80, 85–­86, 89; phase IV trials, 59. See also vaccine development Clynav, 86 Coalition for Epidemic Preparedness Innovations (CEPI), 122–­23 codons, and codon deoptimization, 76 Cold War, 44 common cold, 7–­8, 48, 92, 96–­97, 102 compound E, 101 Conis, Elena, 117 contact monitoring, 10–­11 Corning Pharmaceutical Technologies, 118 coronaviruses, 3, 6–­10, 50, 54, 96, 10; in bats, 5–­6; differing from flu viruses, 95. See also SARS-­CoV corticosteroids, 100–­102

costimulatory signals, 32, 127 COVAX, 123 COVID-­19: compared to HIV, 97–­98; pandemic, ix–­x, 66, 114, 117, 119; symptoms, 9, 95; treatment, 98–­100 cowpox, 38–­42, 81 coxsackievirus, guinea pig, 5 Crick, Francis, 7, 16 Cummings, Derek, 97 Cutter Incident, 58–­59 cytokines, 23, 41 danger signals, 32, 34, 41, 64, 70–­76, 84, 87–­88, 126 deforestation, 1 dexamethasone, 100, 102 Dinarello, Charles, 25 diphtheria, 15–­16, 18, 43, 72 Dissatisfied Parents Together, 110 DNA, 8, 16, 47–­48, 52; mutations and recombinations, 30; synthetic, 65 Doherty, Peter, 24–­25, 27, 60 Doores, Katie, 97 dosage: optimal, 57, 91, 129; in trials, 69–­ 70, 77, 80–­82, 89, 91, 103 doses: number needed for efficacy, 43, 45, 84, 116; quantities for distribution, 64, 90, 105, 107, 118, 126 DPT (diphtheria, tetanus, pertussis) vaccine, 110, 113 drift, changes in the viral coat, 94 drug management, of COVID-­19, 98, 100 Ebola, 49, 51, 75, 78, 81–­83, 105 Edelman, Gerald, 19–­20 effectiveness (efficacy), of vaccines, 55–­ 57, 59, 67, 83, 93, 105–­6 Ehrlich, Paul, 93 electroporation, 85 empirical methodology, 46 emulsion, 73–­74 Enders, John, xiii

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epidemic disease, naming of, xi epidemic diseases, emergent. See infectious diseases, emergent epidemics, global, 3 epidemiological data collection, 10 Escherichia coli (E. coli), 63 Esperanto, 68 European Medicines Agency (EMA), 56, 58, 63, 86 Fauci, Anthony, 25–­26 fermentation systems, bacterial, 65 fermentation vessels (tanks), 47, 58–­59, 63, 65 Fewster, John, 39 flu pandemic. See influenza pandemic of 1918 flu vaccines, xii–­xiii, 57 FluMist, 94 formaldehyde, 45, 68 Fosbroke, Thomas Dudley, 38 fractionation techniques, 64 Francis, Thomas, 45 Freund, Jules, 72 Fulin (emperor), xiv Gamaleya Research Institute of Epidemiology and Microbiology, 83 Gao Qiang, 68 Gardner, James, 39 Gates Foundation, 60, 122–­23 gene gun, 51–­52 gene therapy, 49 genetic (genome) sequencing, of viruses, 3, 5, 7, 10–­11, 77 genetic code, 30 genetic engineering, 32, 47–­48, 63, 70–­ 71, 75, 84 genetic material, 5–­7, 10, 30, 47–­49, 63, 66, 84, 86 Gilbert, Sarah, 50, 79, 95 Gilead, 100 Glaxo Wellcome (GSK), 52, 86

Glenny, Alexander, 43, 72 Global Alliance for Vaccines and Immunisation (Gavi), 123 Global Initiative for Sharing All Influenza Data, 10 gold, as a carrier for plasmid DNA, 51 Gowans, James, 21 Guangdong, 5 Guangzhou, 1–­2 guinea pigs, used in laboratories, 3–­4, 15, 72 Hamre, Dorothy, 7 Haurowitz, Felix, 16 Hawn, M. C., 6 hemoglobin, 62 Hench, Philip, 100–­102 hepatitis B, 48, 63, 73–­74, 124 Hill, Adrian, 50 Hilleman, Maurice, xiii, 46, 48, 87 HIV, 8, 63, 81–­82, 87, 92, 124; lack of a vaccine, 97–­98 Hoffman, Stephen L., 52 Hudson, Charles, 6 Human Genome Project, 65 human papilloma virus (HPV), 73, 84, 87 Huanan Seafood Wholesale Market, 10 immune cell tasks, 22 immune response, 21, 23, 27–­28, 31–­33, 41–­43, 45, 49–­51, 55, 57; antibody and cell-­mediated, 47, 76; T-­cell–­ mediated, 50, 52, 57, 126 immune system: and HIV, 97–­98; human, ix, xiii, 13, 23, 33–­34, 41–­43, 47, 49–­51, 62–­64, 70, 79; in mice, 65 immunity, 16; against COVID-­19, 95; cellular or cell-­mediated, 21, 23, 27; clonal theory of, 17; evasion by viruses, 98–­99; infection-­induced, 96; natural, 95–­97 immunization (vaccination), x, xiv, 105;

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anniversary, ix, xiii; deliberate, 35, 37; of children, 69, 93–­94, 110, 113, 115, 120, 123; schedule, 57, 116–­17; trials, 35–­36, 45. See also vaccine development immunology, ix, xiii, 14, 53, 71; instructive theories, 16 immunopotentiatory drug, 62 Imperial College London, 90 infection, 3, 14–­15, 17, 41, 55; natural, 89, 95, 106; rates, 57, 105 infectious diseases, emergent, 91, 106, 117–­18, 123 inflammation, xii, 21, 55, 72, 100–­102, 104 influenza, 68, 73–­74, 79, 87, 92, 102, 106; seasonal, 92–­94 influenza pandemic of 1918, xi inoculation, xiv, 35–­36 Institut Pasteur, 83 Institute of Biology, Academy of Military Medical Sciences (China), 104 Institute of Experimental Therapy, 93 Institute of Medicine, 110–­11 intellectual property, in vaccine development, 59, 79 interferon, 23, 99 intussusception, 112 Investigational New Drug application, 56 isolation, 10 Ivanovsky, Dimitri, 6 Janeway, Charles, 33, 61, 72, 87 Jenner, Edward, 38–­42, 80–­81 Jerne, Neils, 16 Jesty, Benjamin, 40 Johnston, Stephen, 51 K’ang-­hsi, xiv Karolinska Institute, 85 Kendall, Edward, 100–­102

Kerr, Randy, 35 Kirkpatrick, Charles, 25 Kitasato Shibasaburō, 14–­16, 31, 60 Koch, Robert, 14–­15 Kunkel, Henry, 19 Landsteiner, Karl, 18 Lassa, 82 leukemia, 28 lipopolysaccharides, 73 Liu, Margaret, 86–­87 live markets, 1, 5, 10–­12 lockdowns, 11, 106 London Medical Society, 39 lymphocytes, 21, 23, 27–­28, 41 macaques. See primates, used in laboratories Macfarlane Burnet, Frank, 16–­17, 60 magic bullet (Zauberkugel), 93 Maitland, Charles, 36 malaria, x, 52, 57, 64, 74, 79 Malenkov, Georgi, 44 malignant melanoma. See cancer Marburg, 82 martial arts, 13 Mather, Cotton, 37 Matrix-­M, 126, 136 Mayr, Anton, 81 McDermott, Katherine, 72 measles, xiii, 46, 75, 83, 93, 111–­12 Medawar, Peter, 21, 28 membrane fusion element, 9 meningitis, 24, 80 Merck, 51, 87, 105 mercury, 112 MERS-­CoV, 81, 95–­96 metapneumoviruses, 102 MHC (major histocompatibility complex) molecules, 25–­27, 31–­33, 41, 47, 75 mice (and rats), used in laboratories, 51–­

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52, 65, 69, 71, 79, 126 Middle East Respiratory Syndrome (MERS), 49–­50, 79, 81 Miller, Jacques ( Jacques Meunier), 27–­ 29, 31, 61 Mitchell, Graham, 28 MMR (measles, mumps, rubella) vaccine, 111–­13 Moderna, 106, 129 modified Vaccinia Ankara (MVA), 81–­82 monophosphoryl lipid A (MPL), 73–­ 74, 84 Montague, Mary Wortley, 37 Mosher, Brendan, 118 Mosquirix, 74 mRNA-­1273, 89 mumps, 46, 75, 98 myeloma proteins, 19 nanoparticle system, 89–­90 narcolepsy, 112 National Academy of Medicine, 120 National Childhood Vaccine Injury Act, 110 National Foundation for Infantile Paralysis (March of Dimes), 44–­45, 114 National Institute of Allergy and Infectious Diseases, xiv, 86, 100, 126 National Microbiology Laboratory (Canada), 51 Negri, Adelchi, 7 Nelmes, Sarah, 39 Nossal, Gus, 28 Novavax, 126–­27 nucleic acid, 7, 42, 51–­52, 63–­65, 84–­ 87, 125 Nüsslein-­Volhard, Christiane, 87–­88 Offit, Paul A., 113, 118 Onesimus, 37 Operation Warp Speed, 91, 106–­7, 125–­ 26

Oxford vaccine, 129; and Oxford COVID Vaccine Trial Group, 79–­81, 91, 95, 100, 103, 106, 122, 135 pandemic disease, international efforts in combating, x pandemic, declaration of, 12 pangolins, 12 papaya, and papain, 18–­20 parainfluenza viruses, 102 Pasteur, Louis, 42–­43 patents, for vaccine discoveries, 60–­61 pathogens, 41–­43, 49, 54, 62, 67 Pauling, Linus, 16 Pearce, Barbara, 32 pediatric inflammatory multisystem syndrome, 100 peptides, 75, 96 pharmaceutical companies, 57–­58, 122. See also by name PharmaJet technology, 85 Phipps, James, 39, 41–­42 pig viruses, xi plague, 45 Plandemic (film), 108–­109 plasmids, 47–­48, 51, 63–­65 pneumonia, cluster of cases in Wuhan, 10, 66 polio, x, xiii, 38, 44–­47, 58–­59, 112, 114, 121, 127. See also Sabin polio vaccine; Salk polio vaccine polio immunization, in Nigeria, 108 polysorbate 80, 74 Porter, Rodney Robert, 18–­20, 60 potency, 52, 87 PowderJect, 52 Press, Elizabeth, 20 primates, used in laboratories, 55, 69, 79, 126 prime-­boost platform, 82 Procknow, John, 8 programmed cell death, 31

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proof of concept, 60 pseudotypes, 95 Pfizer, 129 QS21, 74 rabies, 43 Ramon, Gaston, 72 recombinant DNA technology. See genetic engineering Reich, Jennifer, 115–­16 Reichstein, Tadeus, 102 remdesivir, 100 replicase, 90 research: fundamental versus applied, xiv; government-­sponsored, 60 respiratory syncytial virus, 81, 102 restriction enzymes, 47 rhinoviruses, 7, 102 RNA, 8–­10, 65, 71, 75–­76; messenger, 86; polymerase, 90; self-­amplifying, 90–­91 Roosevelt, Franklin D., 44, 114 Rosenthal, Alan, 25–­26 Rotary International, 108 rotavirus, 112 Royal Experiment, 36–­37 rubella, 46, 76, 111, 116 Rutter, William, 48 Sabin, Albert, xiii, 45–­47 Sabin polio vaccine, 76, 112 safety: of vaccines, 55–­57, 67, 77, 82–­83, 86, 89, 105, 109–­11, 118; standards, in the manufacture of vaccines, 59 Şahin, Uğur Salk, Jonas, xiii, 35, 45–­46, 67, 114 Salk polio vaccine, 35, 58, 67–­68, 92, 93, 114 salmon pancreas disease, 86 salmonella, 73 Sanger, Frederick, 18 saponin, 74, 127

SARS-­CoV (SARS coronavirus), 3, 5–­6, 10 SARS-­CoV-­1, 10, 71, 77, 96, 98 SARS-­CoV-­2, 10–­12, 57, 66, 75, 79–­80, 82, 96, 98, 126; compared to SARS-­ CoV-­1, 98–­99; genetic sequencing, 77; mutations (genetic changes), 94–­ 95; numbers infected and death rate, 2–­3, 66, 127; spike protein, 68–­71, 74 Sato, Kei, 99 Schalk, Arthur, 6 Schiff bases, 32, 61–­62, 127 science, pure versus practical, 53–­54 Scripps Research Institute, 11 seasonal flu. See influenza, seasonal serotypes, 79, 82 Sette, Allessandro, 95 severe acute respiratory syndrome (SARS), 2–­3, 5, 71 Shattock, Robin, 90 Shekarau, Ibrahim, 108 Shevach, Ethan, 26 Shi, Zheng-­Li, 5 shift, changes in the viral coat, 94 shigella, 43 sickle cell anemia, 62 side effects, 57, 59 Sinopharm, 129 skin grafting, 21, 24, 28 Sloane, Hans, 36 smallpox, x, xiii–­xiv, 7, 36–­42, 81, 109, 116 soapbark tree, 74, 127 social mobilizers, 121 Spanish flu, xi–­xii squalene, 74 Stanley, Wendell, 7 Strategic Advisory Group of Experts (SAGE), 121 superantigen, 99–­100 surveillance cells, 22–­23, 26, 31–­33, 41, 47, 61, 73, 84 swine flu (H1N1 influenza), 112, 122

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T-­cells, 21–­29, 31–­33, 41–­42, 47, 55, 60–­ 62, 103; helper, 23, 89, 96; killer, 23–­ 24, 41, 47, 50, 52, 57, 75, 80–­81, 88, 96; memory, 42, 47, 97; regulatory, 23, 42 test and trace programs, for SARS-­ CoV-­2, 10, 98 testing, in animals. See animal studies tetanus, 15, 18, 43 thiomersal, 112 thymus, 27–­29, 31 Tianjin, 76–­79, 128 tissue-­culture cells, 44–­46, 49, 55 tobacco mosaic virus, 7 Toll-­like receptors, 88, 104 Tonegawa, Susumu, 29–­30, 61 toxins, 15 traditional medicines, 1, 12 transmission (transmissibility), 12, 95, 98, 119–­20 transmission, from animals to humans, xi, 2, 12. See also zoonosis; zoonotic epidemic tuberculosis, 46, 87; mycobacteria, 72 tucaresol, 63 Türeci, Özlem, 129 typhoid, 43, 45 Tyrrell, David, 7–­8, 48 UK General Medical Council, 111 Ulmer, Jeffery, 52 undervaccination, 115 University of Pittsburgh, 99 University of Sydney, 111 US Army, adopting adenoviral vaccines, 48, 102 US Department of Health and Human Services, 82, 106 US Food and Drug Administration (FDA), 56, 58, 63 US National Institutes of Health, 91, 120 vaccination: history of, ix–­x, 35–­42; rates, 109

vaccines: acceptability, avoidance (refusal), or hesitancy, xiii, 114, 117–­18, 121; agricultural and veterinary, 86; bacterial, 46; for COVID-­19, 63, 124, 128, 135–­38; delivery to the immune system (technology, formats, packaging), 55–­56, 103, 105, 135–­38; delivery devices and techniques, 39, 51, 85, 118–­20; delivery of doses to populations, xiii, 105, 108, 119–­20, 124; developing-­world, 57, 60, 85; distribution, xiii, 120–­22, 124; emergency use authorization by the FDA, 106; first approval, 104; live, 59; licensure, 105; manufacturing, xiii, 42, 58, 64, 67–­68, 86, 91, 106, 118–­19, 124; platforms, 53, 55, 65, 129; policy, and transparency, 116–­17; self-­amplifying, 90–­91, 125; stockpiled, 91, 106; stocks, produced at risk, 91–­92, 118; synthetic (subunit), 33–­34; transferred via people, 42 Vaccine Adverse Event Reporting System, 59, 112 vaccine classes, 42, 44, 67, 84, 91, 124, 135–­38; adenoviral, 48, 51, 91, 129; DNA, 85, 107, 125, 136; inactivated whole-­virus, 67–­70, 107, 125, 128, 129, 135–­36; killed, 44–­46; mRNA, 85–91, 99, 107, 125, 129, 135–38; live attenuated, 44, 48, 75–­76, 128; live viral vector, 107; live virus, 42, 46; nonliving, 43; nonliving protein, 48, 125; nonreplicating viral vector, 76–­ 83, 91, 124–­25, 135, 137; nucleic acid (genetic), 51–­52, 63–­65, 84, 86, 125, 129; protein subunit, 70–­75, 107, 125–­ 27, 129, 136–­38; recombinant protein (genetically engineered), 48; replicating viral vector, 83–­84, 105, 124–­25, 138; virus-­like particle, 84, 138 vaccine development, 53–­54; stage one, exploratory, 54–­55; stage two,

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preclinical, 55–­56; stage three, phase I trials, 56–­57; stage four, phase II trials, 57, 60, 103; stage five, phase III trials, 57, 105–­6, 125, 129; stage six, regulatory review and approval, 58–­59 vaccine race: for COVID-­19, 70, 79, 82–­ 85, 107; for polio, 44 Vaccine Safety Datalink, 59 vaccine schedule. See immunization (vaccine) schedule vaccinia, 81–­82 vaccinology, xiii, 129 Valenzuela, Pablo, 48 varicella. See chicken pox variolation, 36–­38, 40 velvetbean moth, 71 venture capital, in vaccine development, 60 Vero, 68 vesicular stomatitis virus (VSV), 50–­51, 83, 91 viral contagious, author’s experience with, 3–­5 viral variations, 68 viral vectors, 48–­49, 58, 63, 78–­79, 81 viruses: discovery of, 6–­8; natural (wild-­ type), 11, 48 vitamin E, 73–­74

Wakefield, Andrew, 111 Wang Shi’er, 67 Watson, James, 7, 16 Weissman, Drew, 87–­88 Wellcome Foundation, Research Laboratories, and Trust, xiv, 8, 31, 43, 54, 62, 122 whooping cough, 43, 45, 109 wild-­type viruses. See viruses, natural (wild-­type) Wolff, Jon A., 51, 65, 86 Wolff, Sheldon, 25 World Health Organization (WHO), 2, 10, 12, 58, 63, 66, 93, 107–­8, 112, 114, 120–­23 Wu Xueqin, 12–­13 Wuhan, 10–­12, 66, 77; lockdown, 11, 66 Wuhan Institute of Virology, 5 Yuen Ren Chao, 1, 128 Zika, 81–­82 Zinkernagel, Rolf, 24–­25, 27, 60 zoonosis, 2 zoonotic epidemic, 2–­3 zoonotic viruses, 3, 5, 49, 78