How to Make a Vaccine
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Jenner Institute,
Immune Cell Tasks
“angiotensin-converting enzyme 2” or ACE2,
the Cutter Incident, took place in the manufacturing phase.
Frederick Sanger, one of only four people to win the Nobel Prize twice.
This opened the door to the development of both DNA and RNA vaccines. Two years later, Stephen Johnston at the
In line with the new picture of immunity, the fragments of germs (pathogens) stimulating the production of antibodies soon became known as antigens.
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.
Tonegawa became, in 1987, the first Japanese scientist to receive the Nobel Prize for Physiology or Medicine (though perhaps Kitasato ought to have been honored in this way 80 years before).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 sees; Miller removing the thymus and pondering the resulting loss of immunity; Tonegawa conceiving of shuffled genes; and Janeway intuiting the importance of danger.
The advent of genetic engineering allowed scientists to create 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.
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.
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.
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.
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, for an early COVID-19 front-runner 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.
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 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
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 8 <Você alcançou o limite de recortes para este item>
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 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.
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.
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 in this way until their own integrity becomes exhausted or they’re picked off by the immune system.
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.
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.
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.
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 antibodies 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.
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 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.”
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 regulating the immune response. Regulatory T-cells closed down the response when all the cowpox virus was cleared.
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.
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—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.
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.
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 experiment 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.