Tag Archives: Edward Jenner

Thomas Jefferson: Fighting Smallpox

Variolation was the world’s first practical measure to control smallpox. It was developed in China in the 11th century. The procedure involved inoculating uninfected individuals with material from the scabs of individuals who survived smallpox infection.

It was brought to England for the first time in 1721, by Lady Mary Wortley Montague—the wife of the British ambassador to Turkey—when she returned home after learning of the practice in Istanbul. It was brought to Colonial North America the same year by the prominent Puritan minister, Cotton Mather. New England was then experiencing a major smallpox epidemic.

Mather is perhaps best known for his role in the Salem witchcraft trials. He learned of variolation not from the British, but from his African slave, Onesimus, who had been inoculated as child in Africa. Onesimus was a “gift” to Mather in 1706, from his Boston congregation. Variolation was used in western Africa when Onesimus was a child.  The practice may have been brought there by caravans from Arabia. In any case, an enslaved African man played a key role in bringing variolation to North America.

In 1777, during the American War for Independence, General George Washington required the entire Continental Army to undergo variolation. Bearing in mind that more than two-thirds of the American casualties during the War resulted from disease, and that smallpox alone caused a total of about 100,00 deaths, some historians maintain that Washington’s policy of enforced variolation was his most important strategic decision of his entire military career.

Variolation nonetheless encompassed risks—a fatality rate of 1 to 2%—that would be unacceptable today. Not surprisingly then, the colonial and Revolutionary War periods were times when public fear and restrictive laws often prevented the use of variolation. Nonetheless, Thomas Jefferson was a lifelong advocate of smallpox-prevention measures. In 1766, Jefferson traveled to Philadelphia to undergo variolation, since the practice was banned in his native Virginia. As a lawyer in 1768, Jefferson defended a Norfolk doctor, whose house was burned down by a mob because he practiced variolation. In 1769, Jefferson placed a bill before the Virginia General Assembly to reduce the 1769 restrictions against variolation. In the 1770s and 1780s, he had his children and his enslaved servants (including Sally Hemings, his wife’s half-sister, and mother of several of his enslaved children) undergo the procedure.

In 1799, Boston physician and founder of Harvard Medical School, Benjamin Waterhouse, introduced Edward Jenner’s new cowpox-based smallpox vaccine to New England. Wanting to spread word of the vaccine to the rest of the new country, in 1781 Waterhouse sent a sample to his friend, Thomas Jefferson. At the President’s House in Washington, Jefferson selected an enslaved kitchen worker to be the first recipient of the vaccine. However, the vaccine did not take. So, Jefferson then had two of his slaves at Monticello undergo vaccination. When those vaccinations proved to be successful (as shown by exposure to actual smallpox), Jefferson serially transmitted the vaccine from the two original vaccines to almost fifty other slaves. By means of serial inoculations he then sent vaccine material to Washington (the city), and from there the vaccine traveled to Philadelphia and beyond. So, Thomas Jefferson, and his African slaves, played a seminal role in protecting many people in the new United States from smallpox.

Jefferson was an amateur, but serious scientist. He kept detailed notes of his observations, and corresponded with Jenner. Here is what he wrote to Waterhouse about the appearance of papules at the vaccination site:

As far as my observation went, the most premature cases presented a pellucid liquor the sixth day, which continued in that form the sixth, seventh, and eighth days, when it began to thicken, appear yellowish, and to be environed with inflammation. The most tardy cases offered matter on the eighth day, which continued thin and limpid the eighth, ninth, and tenth days. [http://www.smithsonianmag.com/smart-news/thomas-jefferson-conducted-early-smallpox-vaccine-trials-180954146/]

 

 

 

 

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Vaccine Research using Children

Children have been used in vaccine research since its very beginning, usually said to have been in 1796, when Edward Jenner inoculated 8-year-old James Phipps with cowpox, and then challenged young James with actual smallpox (1). However, earlier, in 1789, Jenner inoculated his own 10-month-old son, Edward Jr., with swinepox. Edward Jr. then came down with a pox disease, which he fortunately recovered from. His father then challenged him with smallpox.

Edward Jr. survived his exposure to smallpox. But, since Edward Sr. wanted to determine the duration of young Edward’s protection, he again challenged his son with smallpox in 1791, when the boy was two.  Edward Sr. inoculated his son yet again with smallpox when the boy was three. Fortunately, young Edward was resistant to each of the smallpox challenges his father subjected him to.

Jenner used several other young children in his experiments, including his second son, Robert, who was 11-months-old at the time. One of the children in Jenner’s experiments died from a fever; possibly caused by a microbial contaminant in an inoculum. [Microbes were not known in the late 18th century.]

We have no record of how Jenner (or his wife) felt about his use of his own children. However, there is reason to believe that Jenner felt some remorse over his use of James Phipps, who he referred to as “poor James.” Jenner looked after Phipps in later years, eventually building a cottage for him; even planting flowers in front of it himself.

By the 20th century, some of the most esteemed medical researchers were using children—in institutions for the mentally deficient—to test new drugs, vaccines, and even surgical procedures. These institutions were typically underfunded and understaffed. Several of them were cited for neglecting and abusing their residents. Moreover, their young patients were usually from poor families, or were orphans, or were abandoned. Thus, many of the children had no one to look out for their interests. In addition, research at these institutions was hidden from the public. [The goings-on at these institutions were, in general, hidden from the public, and most of the public likely preferred it that way.] Federal regulations that might have protected the children were not yet in existence, and federal approval was not even required to test vaccines and drugs.

In the early 1940s, Werner Henle, of the University of Pennsylvania, used children at Pennhurst—a Pennsylvania facility for the mentally deficient—in his research to develop an influenza vaccine. [Pennhurst was eventually  infamous for its inadequate staffing, and for neglecting and abusing its patients (2). It was closed in 1987, after two decades of federal legal actions.] Henle would inoculate his subjects with the vaccine, and then expose them to influenza, using an oxygen mask fitted to their faces.

Pennhurst, a state-funded Pennsylvania facility for the mentally deficient, was one of the most shameful examples of the neglect and mistreatment that was common at these institutions. It was the site of Werner Henle’s research in the 1940s to develop an influenza vaccine.
Pennhurst, a state-funded Pennsylvania facility for the mentally deficient, was one of the most shameful examples of the neglect and mistreatment that was common at these institutions. It was the site of Werner Henle’s research in the 1940s to develop an influenza vaccine.

Henle’s vaccine did not protect all of his subjects. Moreover, it frequently caused side effects. Additionally, Henle maintained (correctly?) that a proper test of a vaccine must include a control group (i.e., a group exposed to the virus, but not to the vaccine). Thus, he deliberately exposed unvaccinated children to influenza. Children who contracted influenza had fevers as high as 104o F, as well as typical flu-like aches and pains.

Despite Henle’s investigations at Pennhuerst, he was a highly renowned virologist, best known for his later research on Epstein Barr virus. See Aside 1.

      [Aside 1: While Henle was researching his influenza vaccine at Pennhurst, Jonas Salk concurrently worked on an influenza vaccine, using adult residents (ranging in age from 20 to 70 years) at the Ypsilanti State School in Michigan.]

Next, consider Hilary Koprowski, an early competitor of Jonas Salk and Albert Sabin in the race to develop a polio vaccine (3). By 1950, Koprowski was ready to test his live polio vaccine in people. [That was four years before Sabin would be ready to do the same with his live polio vaccine.] Koprowski had already found that his vaccine protected chimpanzees against polio virus. And, he also tested his vaccine on himself. Since neither he nor the chimpanzees suffered any ill effects, Koprowski proceeded to test his vaccine on 20 children at Letchworth Village for mentally disabled children, in Rockland County, NY.  [Like Pennhurst, Letchworth Village too was cited for inadequately caring for its residents.]  Seventeen of Koprowski’s inoculated children developed antibodies to the virus, and none developed complications.

Koprowski did not initiate his association with Letchworth. Actually, Letchworth administrators, fearing an outbreak of polio at the facility, approached Koprowski, requesting that he vaccinate the children. Koprowski gave each child “a tablespoon of infectious material” in half a glass of chocolate milk (4). Koprowski never deliberately infected the Letchworth children with virulent virus.

Koprowski reported the results of his Letchworth studies at a 1951 conference of major polio researchers, attended by both Salk and Sabin. When Koprowski announced that he actually had tested a live vaccine in children, many conferees were stunned, even horrified. Sabin shouted out: “Why did you do it? Why? Why (4)?” See Aside 2.

      [Aside 2: In the 1930s, Canadian scientist Maurice Brodie tested a killed polio vaccine in twelve children, who supposedly had been “volunteered by their parents (4).” For a short time Brodie was hailed as a hero. However, too little was known at the time for Brodie to ensure that his formaldehyde treatment had sufficiently inactivated the live polio virus. Consequently, Brodie’s vaccine actually caused polio in several of the children. After this incident, most polio researchers could not conceive of ever again testing a polio vaccine, much less a live one, in children.]

Neither Koprowski nor Letchworth Village administrators notified New York State officials about the tests. Approval from the state would seem to have been required, since Koprowski later admitted that he was certain he would have been turned down. And, it is not clear whether Koprowski or the school ever got consent from the parents to use their children. However, recall there were not yet any federal regulations that required them to do so.

Koprowski was untroubled by the uproar over his use of the Letchworth children, arguing that his experiments were necessary. Yet he later acknowledged: “if we did such a thing now we’d be put on jail…” But, he added, “If Jenner or Pasteur or Theiler (see Aside 2) or myself had to repeat and test our discoveries [today], there would be no smallpox vaccine, no rabies vaccine, no yellow fever vaccine, and no live oral polio vaccine.”  Moreover, he maintained that, secret or not, his use of the Letchworth children fit well within the boundaries of accepted scientific practice.

   [Aside 2: Nobel laureate Max Theiler developed a vaccine against yellow fever in 1937; the first successful live vaccine of any kind (5). Theiler formulated a test for the efficacy of his vaccine, which did not involve exposing humans to virulent virus. Sera from vaccinated human subjects were injected into mice, which were then challenged with the Yellow Fever virus.]

Koprowski referred to the Letchworth children as “volunteers (6).” This prompted the British journal The Lancet to write: “One of the reasons for the richness of the English language is that the meaning of some words is continually changing. Such a word is “volunteer.” We may yet read in a scientific journal that an experiment was carried out with twenty volunteer mice, and that twenty other mice volunteered as controls.” See Aside 3.

     [Aside 3: Koprowski was a relatively unknown scientist when he carried out his polio research at Letchworth. He later became a renowned virologist, having overseen the development of a rabies vaccine that is still used today, and having pioneered the use of therapeutic monoclonal antibodies. Yet, he is best remembered for developing the world’s first effective polio vaccine; several years before Salk and Sabin brought out their vaccines.

   Most readers of the blog are aware that the Salk and Sabin vaccines are credited with having made the world virtually polio-free. What then became of Koprowski’s vaccine? Although it was used on four continents, it was never licensed in the United States. A small field trial of Koprowski’s vaccine in 1956, in Belfast, showed that its attenuated virus could revert to a virulent form after inoculation into humans. Yet a 1958 test, in nearly a quarter million people in the Belgian Congo, showed that the vaccine was safe and effective. Regardless, the vaccine’s fate was sealed in 1960, when the U.S. Surgeon General rejected it on safety grounds, while approving the safer Sabin vaccine. Personalities and politics may well have played a role in that decision (3, 4).

  Interestingly, Sabin developed his vaccine from a partially attenuated polio virus stock that he received from Koprowski. It happened as follows. In the early 1950s, when Koprowski’s polio research was further along than Sabin’s, Sabin approached Koprowski with the suggestion that they might exchange virus samples. Koprowski generously sent Sabin his samples, but Sabin never reciprocated.

   Koprowski liked to say: “I introduce myself as the developer of the Sabin poliomyelitis vaccine (7).” He and Sabin had a sometimes heated adversarial relationship during the time when their vaccines were in competition. But they later became friends.]

Sabin was at last ready to test his polio vaccine in people during the winter of 1954-1955. Thirty adult prisoners, at a federal prison in Chillicothe, Ohio, were the subjects for that first test in humans. [The use of prisoners also raises ethical concerns.]

Recall Sabin’s public outcry in 1951 when Koprowski announced that he used institutionalized children to test his polio vaccine. In 1954, Sabin sought permission to do the very same himself; asserting to New York state officials: “Mentally defective children, who are under constant observation in an institution over long periods of time, offer the best opportunity for the careful and prolonged follow-up studies…”

Although Sabin had already tested his attenuated viruses in adult humans (prisoners), as well as in monkeys and chimpanzees, the National Foundation for Infantile Paralysis, which funded polio research in the pre-NIH days of the 1950s, blocked his proposal to use institutionalized children. Thus, Sabin again used adult prisoners at the federal prison in Ohio. With the concurrence of prison officials, virtually every inmate over 21 years-old “volunteered,” in exchange for $25 each, and a possible reduction in sentence. None of the prisoners in the study became ill, while all developed antibodies against polio virus.

Testing in children was still a necessary step before a polio vaccine could be administered to children on a widespread basis. But, Sabin’s vaccine could not be tested in children in the United States. Millions of American children had already received the killed Salk vaccine, and the National Foundation for Infantile Paralysis was not about to support another massive field trial of a vaccine, in children, in the United States (3).

Then, in 1959, after a succession of improbable events, 10 million children in the Soviet Union were vaccinated with Sabin’s vaccine (3). The Soviets were so pleased with the results of that massive trial that they next vaccinated all seventy-seven million Soviet citizens under 20 years-of-age with the Sabin vaccine. That figure vastly exceeded the number of individuals in the United States, who were vaccinated with the rival Salk vaccine during its field trials.

Next up, we have Nobel laureate John Enders who, in the 1950’s, oversaw the development of the first measles vaccine. Enders and co-workers carried out several trials of their attenuated measles vaccine; first in monkeys and then in themselves. Since the vaccine induced an increase in measles antibody titers, while causing no ill effects, they next tested it in severely handicapped children at the Walter E. Fernald State School near Waltham, Massachusetts.

Enders seemed somewhat more sensitive than either Henle or Koprowski to the ethics of using institutionalized children. Samuel L. Katz, the physician on Enders’ team, personally explained the trial to every Fernald parent, and no child was given the vaccine without written parental consent. [Federal guidelines requiring that step still did not exist.] Also, no child was deliberately infected with virulent measles virus.

Katz personally examined each of the inoculated Fernald children every day. None of these children produced measles virus, while all of them developed elevated levels of anti-measles antibodies. Also, the Fernald School had been experiencing severe measles outbreaks before the Enders team vaccinated any of its children. But, when the next measles outbreak struck the school, all of the vaccinated children were totally protected.

In 1963, the Enders vaccine became the first measles vaccine to be licensed in the United States. Several years later it was further attenuated by Maurice Hilleman (8) and colleagues at Merck. In 1971, it was incorporated into the Merck MMR (measles, mumps, and rubella) vaccine. See Aside 4.

    [Aside 4: Before Enders carried out his measles investigations he pioneered the growth of viruses in tissue culture. In 1949, Enders, and collaborators Thomas Weller and Frederick Robbins, showed that poliovirus could be cultivated in the laboratory. This development was crucial, allowing Salk and Sabin to grow a virtually unlimited amount of polio virus and, consequently, to develop their polio vaccines. In 1954, Enders, Weller, and Robbins were awarded the Nobel Prize for Physiology or Medicine for their polio virus work.]

It may surprise some readers that before the mid 1960s the so-called Nuremburg Code of 1947 comprised the only internationally recognized ethical guidelines for experimentation on human subjects. The Nuremburg Code was drawn up by an American military tribunal during the trial of 23 Nazi physicians and scientists for atrocities they committed while carrying out so-called “medical” experiments during World War II. [Sixteen of the 23 Nazis on trial at Nuremburg were convicted, and 7 of these were executed (see Note 1)].

The Nuremberg Code’s Directives for Human Experimentation contained strongly stated guidelines. Its tenets included the need to obtain informed consent (interpreted by some to prohibit research using children), the need to minimize the risks to human subjects, and the need to insure that any risks are offset by potential benefits to society.

But, despite the well-articulated principles of the Nuremberg Code, it had little effect on research conduct in the United States. Federal rules, with the authority to regulate research conduct, would be needed for that. So, how did our current federal oversight of research come to be?

A 1996 paper in the The New England Journal of Medicine, “Ethics and Clinical Research,” by physician Henry Beecher, brought to the fore the need for rules to protect human subjects in biomedical research (9). Beecher was roused to write the paper in part by the early 1960s experiments of Saul Krugman, an infectious disease expert at NYU. Krugman used mentally deficient children at the Willowbrook State School in Staten Island, New York, to show that hepatitis A and hepatitis B are distinct diseases (9). Also, before a hepatitis vaccine was available, Krugman inoculated the children with serum from convalescing individuals, to ask whether that serum might protect the children against hepatitis. Krugman exposed the children to live virus either by injection, or via milkshakes seeded with feces from children with hepatitis.

Krugman found that convalescent sera indeed conferred passive immunity to hepatitis. Next, he discovered that by infecting passively protected patients with live hepatitis virus he could produce active immunity. Krugman had, in fact, developed the world’s first vaccine against hepatitis B virus (HBV) (see Aside 4). [Although Krugman used mentally deficient institutionalized children in his experiments, his investigations were nonetheless funded in part by a federal agency; the Armed Forces Epidemiology Section of the U.S. Surgeon General’s Office.]

         [Aside 4: The first hepatitis B vaccine licensed for widespread use was developed at Merck, based on principles put forward by Nobel Laureate Baruch Blumberg, (10).]

Beecher was particularly troubled by two aspects of Krugman’s experiments. First, Krugman infected healthy children with live virulent virus. Beecher maintained that it is morally unacceptable to deliberately infect any individual with an infectious agent, irrespective of the potential benefits to society. [See reference 11 for an alternative view. “The ethical issue is the harm done by the infection, not the mere fact of infection itself.”]

Second, Beecher charged that the Willowbrook School’s administrators coerced parents into allowing their children to be used in Krugman’s research. The circumstances were as follows. Because of overcrowding at the school, Willowbrook administrators closed admission via the usual route. However, space was still available in a separate hepatitis research building, thereby enabling admission of additional children who might be used in the research.

Were the Willowbrook parents coerced into allowing their children to be used in the research there? Consider that the parents were poor and in desperate need of a means of providing care for their mentally impaired children. Making admission of the children contingent on allowing them to be used in the research might well be viewed as coercion. Yet even today, with federal guidelines now in place to protect human subjects, institutions such as the NIH Clinical Center admit patients who agree to participate in research programs. Is that coercion?

Beecher’s 1966 paper cited a total of 22 instances of medical research that Beecher claimed were unethical (9). Four examples involved research using children. Krugman’s work at Willowbrook was the only one of these four examples that involved vaccine research. Beecher’s other examples involved research using pregnant women, fetuses, and prisoners. But it was Beecher’s condemnation of Krugman’s hepatitis research at Willowbrook that is mainly credited with stirring debate over the ethics of using children in research.

Did Krugman deserve Beecher’s condemnation? Before Krugman began his investigations at Willowbrook, he plainly laid out his intentions in a 1958 paper in the New England Journal of Medicine (12). Importantly, Krugman listed a number of ethical considerations, which show that he did not undertake his Willowbrook investigations lightly. In fact, Krugman’s ethical considerations, together with his plans to minimize risks to the children, were not unlike the assurances one might now submit to an institutional review board (11).

Many (but not all) knowledgeable biomedical researchers claimed that Beecher misunderstood Krugman’s research and, thus, unjustly vilified him. Krugman was never officially censored for his Willowbrook investigations. Moreover, condemnation of Krugman did not prevent his election in 1972 to the presidency of the American Pediatric Society, or to his 1983 Lasker Public Service Award.

To Beecher’s credit, his 1966 paper was instrumental in raising awareness of the need to regulate research using human subjects. Beecher was especially concerned with the protection of children and, apropos that, the nature of informed consent.

In 1974, the National Research Act was signed into law, creating the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. The basic ethical principles identified by the Commission are summarized in its so-called Belmont Report, issued in 1978. Its tenets include minimizing harm to all patients, and the need to especially protect those with “diminished autonomy” or who are incapable of “self-determination.”  In addition, federal guidelines now require universities and other research institutions to have Institutional Review Boards to protect human subjects of biomedical research. [Reference 13 (available on line) contains a detailed history of the establishment of these policies.]  See Aside 6.

      [Aside 6: The infamous U.S. Public Health Service Tuskegee syphilis research program, conducted between 1932 and 1972, in which several hundred impoverished black men were improperly advised and never given appropriate treatment for their syphilis, also raised public awareness of the need to protect human subjects. More recently, research involving embryonic stem cells and fetuses has stoked an ongoing and heated public debate. Policies regarding this research are still not settled, with stem-cell research being legal in some states, and a crime in others. Other recent technological advances, such as DNA identification and shared databases, have been raising new concerns, such as the need to protect patient privacy. In response to these new developments, in June 2016, the US National Academies of Sciences, Engineering and Medicine released a report proposing new rules (indeed a complete overhaul of the 1978 Belmont Report) to deal with these circumstances. The Academy’s report has stirred debate in the biomedical community]

Note 1: The use of children in medical research makes many of us profoundly uneasy. We may be particularly troubled by accounts of the exploitation of institutionalized children, who comprised a uniquely defenseless part of society. Indeed, it was the very vulnerability of those children that made it possible for them to be exploited by researchers. Consequently, some readers may well be asking whether the activities of vaccine researchers Krugman, Koprowski, Sabin, Henle and others might have been comparable to that of the Nazis on trial at Nuremberg. So, I offer this cautionary interjection. While in no way condoning the vaccine researchers using institutionalized children, their work was carried out for the sole purpose of saving human lives. As Koprowski suggested above, if not for that work, we might not have vaccines against smallpox, rabies, yellow fever, and polio. Now, consider Josef Mengele, a Nazi medical officer at Auschwitz, and the most infamous of the Nazi physicians. [Mengele was discussed several times at Nuremberg, but was never actually tried. Allied forces were convinced at the time that he was dead, but he had escaped to South America.] At Auschwitz, Mengele conducted germ warfare “research” in which he would infect one twin with a disease such as typhus, and then transfuse that twin’s blood into the other twin. The first twin would be allowed to die, while the second twin would be killed so that the organs of the two children might then be compared. Mengele reputedly killed fourteen twin children in a single night via a chloroform injection to the heart. Moreover, he unnecessarily amputated limbs and he experimented on pregnant women before sending them to the Auschwitz gas chambers.

References:

  1. Edward Jenner and the Smallpox Vaccine, Posted on the blog September 16, 2014.
  2.  Pennhurst Asylum: The Shame of Pennsylvania, weirnj.com/stories/pennhurst-asylum/
  3.  Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.
  4.  Oshinsky D, Polio: An American Story, Oxford University Press, 2005.
  5. The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 2014.
  6.  Koprowski H, Jervis GA, and Norton TW. Immune response in human volunteers upon oral administration of a rodent-adapted strain of poliomyelitis virus. American Journal of Hygiene, 1952, 55:108-126.
  7.  Fox M, Hilary Koprowski, Who Developed First Live-Virus Polio Vaccine Dies at 96, N.Y. Times, April 20, 2013.
  8. Maurice Hilleman: Unsung Giant of Vaccinology, Posted on the blog April 14, 2014.
  9. Beecher HK. Ethics and clinical research. The New England Journal of Medicine, 1966, 274:1354–1360.
  10.  Baruch Blumberg: The Hepatitis B Virus and Vaccine, Posted on the blog June 2, 2016.
  11.  Robinson WM, The Hepatitis Experiments at the Willowbrook State School. science.jburrougs.org/mbahe/BioEthics/Articles/WillowbrookRobinson2008.pdf
  12. Ward R, Krugman S, Giles JP, Jacobs AM, Bodansky O. Infectious hepatitis: Studies of its natural history and prevention. The New England Journal of Medicine, 1958, 258:407-416.
  13.  Ethical Conduct of Clinical Research Involving Children. http://www.ncbi.nlm.nih.gov/books/NBK25549/

 

 

Louis Pasteur: One Step Away from Discovering Viruses

Louis Pasteur (1822-1895) is the subject of our first posting of the New Year. Pasteur was history’s greatest microbiologist and, perhaps, its most famous medical scientist. Pasteur was also an early figure in the history of virology for his 1885 discovery of a rabies vaccine; only the second antiviral vaccine and the first attenuated one (see Aside 1). However, the main point of this tale is that Pasteur let pass an especially propitious opportunity to discover that the rabies agent is one of a previously unrecognized class of microbes; a class that is fundamentally different from the already known bacteria. Its members are submicroscopic and grow only inside of a living cell. Pasteur was just one step away from discovering viruses.

Louis Pasteur
Louis Pasteur

[Aside 1: Attenuation is the conversion of a pathogenic microbe into something that is less able to cause disease, yet is still able to induce immunity. Edward Jenner’s 1798 smallpox vaccine, the world’s first vaccine, as well as the first antiviral vaccine, was not based on the principle of attenuation. Instead, it contained live, unmodified cowpox virus. Although hardly understood in Jenner’s day, his smallpox vaccine worked because cowpox, which is not virulent in humans, is immunologically cross-reactive with smallpox. Thus, the relatively benign cowpox virus induced immunity against the related, deadly smallpox virus (1).]

The distinctive nature of viruses would first begin to be revealed in 1887 by a scientist of much less renown than Pasteur; the Russian microbiologist Dmitry Ivanovsky. The virus concept would be further advanced in 1898 by the accomplished Dutch botanist Martinus Beijerinck (2). In any case, to better appreciate how anomalous it was that Pasteur did not discover viruses, we review the greatness of his earlier achievements. After that, we consider the opportune circumstance that he let go by.

Pasteur was a chemist by background. Thus, his first major scientific discovery, at 26 years of age, was as a chemist. It was his 1847 discover of molecular asymmetry; that certain organic molecules exist in two alternative molecular structures, each of which is the mirror image of the other. Additionally, pairs of these asymmetric molecules are chemically indistinguishable from each other, and balanced mixtures of them rotate the plane of polarized light.

Pasteur’s discovery of molecular asymmetry was one of the great discoveries in chemistry. Yet his research would take on a momentous new focus when he began to investigate the chemistry of fermentations. This new course was inspired by the fact that while asymmetric molecules are not generated in the laboratory, they are found in the living world. And, since asymmetric molecules are found among fermentation products, Pasteur hypothesized that fermentation is a biological process, which he proceeded to demonstrate in 1857, basically by showing that fermentation products did not arise in nutrient broth if any microbes that might have been present were either killed by heating or removed by filtration. What’s more, he showed that specific fermentations are caused by specific microorganisms. Additionally, he discovered that fermentation is usually an anaerobic process that actually is impaired by oxygen; a phenomenon known as the “Pasteur effect.” And, he put forward the notion of aerobic versus anaerobic microbes.

Pasteur put his experience studying fermentations to practical use when he came to the rescue of the French wine industry, which was on the verge of collapse because of the wine becoming putrefied. Pasteur showed that the problem was due to bacterial contamination, and then showed that the putrefaction could be prevented by heating the wine to 50 to 60 °C for several minutes; a procedure we now refer to as pasteurization. Wines are seldom pasteurized today because it would kill the organisms responsible for the wines maturing. But, as we know, pasteurization is applied to many contemporary food products, especially milk. Pasteur also aided the beer industry by developing methods for the control of beer fermentation.

Pasteur’s study of fermentations led to an experiment of historic significance for biology in general. In the 1860s, the ancient notion that life can arise spontaneously from nonliving materials, such as mud or water, was still widely believed. The emerging awareness of microbes in the 1860s did not change this belief. Instead, it led to the idea that fermentations and putrefactions result from the spontaneous generation of microbes. In 1862, Pasteur unequivocally dispelled this belief by a simple yet elegant experiment in which he made use of a flask that had a long bending neck that prevented contaminants from reaching the body of the flask. If the broth in the flask was sterilized by boiling, and if the neck remained intact, then the broth remained sterile. But, if the neck of the flask was broken off after the boiling, then the broth became opaque from bacterial contamination.

Taken alone, Pasteur’s achievements that are enumerated above would have been sufficient to have ensured his lasting fame. Nevertheless, Pasteur’s greatest successes were yet to come. In 1867 he put forward the “germ theory of disease.” By this time, the existence of a variety of microorganisms, including bacteria, fungi, and protozoa, was already well established. Pasteur’s new proposal, that microorganisms might produce different kinds of diseases, was inspired by his earlier experimental findings that different microorganisms are associated with different kinds of fermentations, and by his 1865 finding that a microorganism was responsible for a disease in silkworms that was devastating the French silk industry.

After Pasteur proposed his germ theory of disease, Robert Koch (another giant in the history of medical microbiology) established that anthrax in cattle is caused by a specific bacterium, Bacillus anthracis. Koch had taken a sample from diseased cattle and then used his new method for isolating pure bacterial colonies on solid culture media to generate a pure culture of B. anthracis. Next, he inoculated healthy animals with a portion of the pure culture. The healthy animals then developed anthrax. Finally, he re-isolated B. anthracis from the inoculated animals. This sequence of isolation, infection, and re-isolation constitutes Koch’s famous postulates, which provide the experimental basis for establishing that a specific microorganism is responsible for a specific disease.

Even after Pasteur confirmed Koch’s anthrax findings in 1877, some members of the medical establishment still rejected the germ theory of disease, mainly because Pasteur was a chemist by background, rather than a physician. Nevertheless, Joseph Lister, an English surgeon, admired Pasteur’s work on fermentation and was impressed by Pasteur’s disproving of spontaneous generation. Based on Pasteur’s demonstration of the ubiquity of airborne microorganisms (another of his noteworthy achievements), Lister reasoned that infections of open wounds are due to microorganisms in the environment. The aseptic techniques that Lister then introduced were responsible for dramatically reducing infections during surgery.

The following is one of my favorite parts of this story. In 1879, Pasteur made his first important contribution to vaccinology, when he discovered, by accident, that he could attenuate the bacterium responsible for chicken cholera (now known to be a member of genus Pasteurella), and then use the attenuated microbe as a vaccine. It happened as follows. Pasteur instructed his assistant, Charles Chamberland, to experimentally inject chickens with the cholera bacterium so that he, Pasteur, might observe the course of the disease. Then, just before a summer holiday break, Pasteur directed Chamberland to inject the chickens with a fresh culture of the bacteria. Chamberland may have been preoccupied with thoughts of the upcoming holiday, because he forgot to inject the chickens before leaving. When he returned a month later, he carried out Pasteur’s instructions, except that he injected the chickens with the now aged bacteria. What happened next was most important. The chickens that were inoculated with the aged culture developed only a very mild form of the disease. After that, Pasteur had Chamberland inject those same chickens with freshly grown, presumably virulent bacteria. The chickens still did not develop disease.

It is not clear why Pasteur instructed Chamberland to inoculate the freshly grown culture into the chickens that earlier had received the aged culture. Perhaps it was an accident, or perhaps Pasteur saw an opportunity to carry out a possibly interesting experiment. (The chickens had survived a mild infection by the aged culture. Might they now be resistant to freshly grown virulent bacteria?) In any case, Pasteur repeated the entire sequence of inoculating the chickens with an aged culture and then challenging them with a fresh culture. The outcome was the same as before.

Pasteur correctly surmised that the aging process (actually, oxidation by exposure to air) had attenuated the bacteria. And, he learned by experimentation that the virulence of the cholera microbe could be reduced to any desired extent by controlling its exposure to air. Most importantly perhaps, he discovered that the attenuated bacteria could induce resistance to the virulent bacteria and, consequently, could be used as a vaccine. Pasteur’s chicken cholera vaccine was the first vaccine deliberately created in a laboratory. What’s more, it was the first attenuated vaccine. See Aside 2.

[Aside 2: During the years that Pasteur was carrying out his vaccine studies, nothing was known regarding the physiological basis of immunity, or the determinants of virulence, or of mutations, or the underlying mechanism of attenuation that changed a deadly microbe into a harmless one that still could induce immunity. Considering the intellectual milieu in which Pasteur carried out his investigations, it is all the more remarkable that he achieved so much. And while Pasteur’s interpretations for how his attenuated vaccines worked were far from accurate, they are still impressive for their plausibility. Initially, he thought that the attenuated organisms might simply compete with the virulent organisms for a limited availability of nutrients in the host. Later, he thought that the attenuated organisms might release a toxin that blocked growth of the virulent organisms. The notion, that the host might actually initiate its own defense, began to emerge in 1890 when Emil von Behring and Shibasaburo Kitasato discovered that a host factor neutralized the diphtheria toxin. Kitasato then put forward the theory of humoral immunity, proposing that a host serum factor could neutralize a foreign antigen. In 1891 Paul Ehrlich used the term “antibody” for the first time, in reference to those serum factors.]

This account of the cholera vaccine brings to mind Pasteur’s famous remark, “Chance only favors the prepared mind.” Yet in the context of our larger story, it is an ironic statement, considering that Pasteur later missed an auspicious opportunity to discover viruses. But, before getting to that, we briefly note Pasteur’s work on his anthrax vaccine.

In 1879 Pasteur began to develop an anthrax vaccine, which, like the cholera vaccine, would be based on his principle of attenuation. And, as in the case of the cholera vaccine, Pasteur attenuated the anthrax bacillus by exposing it to oxygen. [History records that Pasteur and his assistants developed a second approach to attenuate the anthrax bacillus, based on their discovery that when the bacilli are cultivated at 42 or 43 degrees centigrade, they do not develop the endospores that are necessary to cause a virulent infection.] In 1881 Pasteur carried out a dramatic public demonstration of the effectiveness of his air-oxidized anthrax vaccine in livestock, causing many doubters to accept the validity of his work. See Aside 3 and the end note.

[Aside 3: Currently, the only FDA-licensed anthrax vaccine for use in humans is BioThrax, produced by Emergent BioDefense Operations Lansing Inc. BioThrax is generated from an avirulent, nonencapsulated mutant of B. anthracis. It does not contain any living organisms. As suggested by the name of the manufacturer, BioThrax was produced mainly for the U.S. Department of Defense, for use in case B. Anthracis might be used as a biological weapon. Thus, BioThrax is not available to the public. People who are exposed to B. anthracis are now treated with antibiotics (e.g., ciprofloxacin and doxycycline).]

Pasteur turned his attention to rabies in1880, when the problem of rabid dogs in Paris was getting out of hand. Once again Pasteur sought to develop a vaccine, and once again he wanted to apply the principle of attenuation. But, early on, he found that he could not grow the rabies agent in pure culture. Thus, he was not able to isolate the rabies agent. Moreover, he would need to devise new procedures if he was to grow and attenuate it. His solution was to develop methods for cultivating the rabies agent in the spinal cords of live rabbits. His method for attenuation was then suggested by his assistant, Emile Roux, who had been studying survival of the rabies agent in pieces of rabbit spinal cord that he suspended inside a flask. Following Roux’s example, Pasteur attenuated the rabies agent by air-desiccating spinal cords taken from experimentally infected rabbits that earlier had died of rabies. Each successive day of desiccation resulted in greater attenuation of virulence, such that an extract from a spinal cord aged for 14 days could no longer transmit the disease. What’s more, those extracts could be used as inoculums that prevented rabies in dogs that later were challenged with the virulent microbe.

Pasteur, himself, took saliva samples from rabid dogs. In one such incident, he used a glass tube to suck up a few drops of deadly saliva from the mouth of a mad, squirming bulldog that was held down on a table by two assistants. The assistants wore heavy leather gloves.

Here is another of my favorite parts of this story. In 1885, nine-year-old Joseph Meister was bitten repeatedly by a rabid dog. Young Joseph’s desperate mother then brought her son to Pasteur, hoping that he might help Joseph. But, any attempt by Pasteur to treat the boy was sure to provoke controversy. Pasteur was not a medical doctor. Moreover, his rabies vaccine had never been successfully used in humans. Furthermore, attenuation and vaccination were still new and contentious concepts. For these reasons, Pasteur rejected many earlier requests for help from people in France, and from abroad as well. But, in Joseph’s case, Pasteur relented, convinced that the boy would die if he did not intercede.

Pasteur gave young Joseph a series of 13 injections, one each day, in which each successive injection contained a less-attenuated (stronger) virus. Pasteur dreaded inoculating Joseph with the last shot in the series; a one-day-old vaccine that was strong enough to kill a rabbit. Emile Roux wanted no part in this episode and, in fact, withdrew from the rabies study because of it. But, Joseph never developed rabies, and millions of people subsequently received Pasteur’s anti-rabies treatments. [Pasteur’s attenuated rabies vaccine may not have been entirely safe for humans. Modern rabies vaccines are generally killed virus vaccines, prepared by chemically inactivating tissue culture lysates.] See Asides 4 and 5.

[Aside 4: Post-infection rabies vaccination works and, indeed, is necessary because (for reasons that are still not entirely clear) the human immune response against a natural rabies infection is not able to prevent the virus from reaching the central nervous system, at which point the infection is invariably fatal. Importantly, the incubation period between the time of the bite and the appearance of disease can be more than several months, and is never less than two weeks. Consequently, there is a substantial window of opportunity for the vaccine to cause the virus to be inactivated at the site of the bite.]

[Aside 5: In 1888, Emile Roux, working at the Pasteur Institute (see below), would confirm the existence of the diphtheria toxin by showing that injecting animals with sterile filtrates of liquid cultures of Corynebacterium diphtheriae caused death with a pathology characteristic of actual diphtheria.]

Pasteur worked hard to isolate the rabies agent, but he wrongly presumed that he should be able to grow it in pure culture. Finally, in 1884, he conceded that he had not been able to isolate and cultivate the rabies agent in a laboratory media. So, might that failure alone have been sufficient to cause Pasteur to think of the rabies agent in new terms? Perhaps not, since, at the time, the inability to cultivate a microbial pathogen was assumed to be a laboratory failure, rather than a reason to hypothesize that that the agent was something other than a bacterium. [Even with the eventual awareness of the uniqueness of viruses, the inability of virologists to cultivate viruses outside of an animal would remain a mystery, as well as an obstacle, well into the early 1930s (3).]

Pasteur also got sidetracked while trying to isolate the rabies agent. In 1880 he injected a rabbit with the saliva of a child who died of rabies. He then examined the blood of the rabbit after it too succumbed to rabies. Using his microscope, Pasteur in fact saw a microbe in the rabbit’s blood, which he thought might be the rabies agent. However, he later found the same microbe in the saliva of normal children. Ironically, this microbe, which Pasteur at first thought might be the rabies agent, was actually Pneumococcus pneumoniae, a major bacterial pathogen that was correctly identified several years later by Albert Frankel. Thus, Pasteur missed the opportunity to identify a bacterial pathogen that is much more important in humans than rabies virus. Moreover, and importantly, Pasteur never did see the actual rabies agent under his microscope. Thus, he was aware that the rabies agent might be unusually small in comparison to the usual bacteria.

Here is another bit of irony. The item (apparatus?) that initially played the key role in distinguishing viruses from bacteria was invented in Pasteur’s laboratory. It was the unglazed terra cotta filter, conceived by Charles Chamberland, which he used to provide a good supply of sterile water for Pasteur’s lab. Chamberland allegedly developed these bacterium-proof filters while experimenting with a broken clay pipe that he bought from his tobacconist.

Bearing in mind that Pasteur was never able to grow the rabies agent in pure culture, and that he never saw the rabies agent under his microscope, might he have thought that it might be a submicroscopic infectious agent that is different from bacteria in some fundamental way? I have not come across any definitive answer to that question. But, I feel safe to say that it is unlikely that anyone other than Pasteur might have seriously considered that possibility. Regardless, Pasteur did not take the next logical step, which would have been to see if the rabies agent might pass through Chamberland’s filters. Had he done so, he could have isolated the rabies agent from the rabbit spinal cords, and he would have discovered “filterable viruses” (see below).

That crucial step was taken for the first time in 1887 by the Russian bacteriologist, Dmitry Ivanovsky, who used Chamberland filters in his investigations into the cause of tobacco mosaic disease. Ivanovsky could not propagate the tobacco mosaic agent (later known as the tobacco mosaic virus) in pure culture. However, because of his finding that the agent could actually pass through Chamberland’s filters, Ivanovsky is sometimes credited for discovering viruses. Yet Ivanovsky did not accept his own results. He still presumed that the disease was caused a bacterium, and he thought that the filters were defective or, instead, that the disease was due to a toxin produced by the bacterium.

In 1898, Martinus Beijerinck, unaware of Ivanovsky’s earlier work, also could not see or cultivate the tobacco mosaic agent. In addition, he too found that the agent passed through Chamberland filters. Beijerink expected, and perhaps even hoped that the filters would remove the agent from diseased plant extracts, so that he might prove it to be a bacterium. But despite his possible disappointment, Beijerinck went one major step further. He demonstrated that the filtered sap from a diseased plant did not lose its ability to cause disease after being diluted by repeated passage through new healthy plants. Consequently, the filterable agent was replicating in the plant tissue and, thus, could not be a toxin.

Little is recorded about Ivanovsky, aside from his four-page report on the tobacco mosaic disease (see Aside 6). In contrast, Beijerinck was a major scientist, who made numerous important contributions, including the discovery of nitrogen-fixing bacteria and bacterial sulfate reduction (4). Yet even Beijerinck found it difficult to conceive that the filterable, incredibly small, submicroscopic tobacco mosaic agent might be particulate in nature. Instead, he famously described it as a “contagious living fluid.” Nonetheless, Beijerinck, a botanist by background, is often considered to be the first virologist.

[Aside 6: Ivanovsky’s four-page paper would be unremarkable if it were not for the single sentence, “Yet I have found that the sap of leaves attacked by the mosaic disease retains its infectious qualities even after filtration through Chamberland filters.”]

Pasteur was probably unaware of Ivanovsky’s findings, and he did not live long enough to know of Beijerinck’s. So, we do not know what he might have made of their results. Regardless, Pasteur remained one step away from making these discoveries himself.

In 1898, after the announcement of Beijerinck’s findings, Friederich Loeffler and Paul Frosch isolated the foot and mouth disease virus; the first virus isolated from animals. Next, in 1901, in Cuba, U.S. Army doctor Walter Reed isolated yellow fever virus (5); the first pathogenic virus of humans to be isolated. In 1903, Paul Remlinger, working at the Constantinople Imperial Bacteriology Institute, filtered and then isolated rabies virus. Despite these early achievements, it was not until 1938 that the development of the electron microscope made it possible to resolve that viruses are indeed particulate, rather than liquid in nature. See Aside 7.

[Aside 7: The term “virus” indeed appears in the scientific literature of Pasteur’s day. However, at that time “virus” referred to any microbe that might cause disease when inoculated into a susceptible human or animal. By the 1890s, the term “filterable virus” came into use, meaning an infectious agent which, like the tobacco mosaic virus, passed through filters that retained bacteria. But, bearing in mind that there was not even a consensus regarding the identity of the genetic material until the early 1950s, there would be no clear understanding of viruses until then. In fact, the classic, early 1950s blender experiment of Alfred Hershey and Martha Chase, which featured bacteriophage T4, played a key role in establishing DNA as the genetic material, while also elucidating the essentials of virus replication (2).]

In 1887 Louis Pasteur founded the Institute in Paris that bears his name. A minor irony is that the Pasteur Institute was founded as a rabies vaccine center. The Institute has since been the site of numerous major discoveries in infectious diseases. But we underscore here that it was the site where, in 1910, Constantin Levaditi and Karl Landsteiner demonstrated that poliomyelitis is caused by a filterable virus, and where Félix d’Herelle in 1917 discovered bacteriophages. And it was also the site where, in 1983, Luc Montagnier and Françoise Barré-Sinoussi were the first to isolate HIV (6).

In a fitting end to our story, when Joseph Meister grew up, he became the gatekeeper of the Pasteur Institute. Meister was still minding the gate at age sixty four when, in 1940, the Nazis invaded Paris. Legend has it that when Nazi soldiers arrived at the Institute and ordered Meister to open Pasteur’s crypt, rather than surrendering Pasteur’s resting place to the Nazis, Meister shot himself (7).

Pasteur Institute: Museum and Crypt
Pasteur Institute: Museum and Crypt

End note:

Science historian, Gerald L. Geison, wrote a controversial revisionist account of Pasteur’s achievements, that was based on Geison’s reading of Pasteur’s laboratory notes (8). Geison undermines Pasteur’s integrity and discredits some of his major accomplishments. For example, Geison asserts that Pasteur surreptitiously used the oxidation procedure of French veterinary surgeon, Henry Toussaint, when preparing his own widely acclaimed anthrax vaccine for its public demonstration.

Max Perutz, who shared the 1962 Nobel Prize for Chemistry with John Kendrew for their studies of the structures of hemoglobin and myoglobin, reviewed Geison’s book for The New York Review of Books (December 21, 1995). Perutz’s review, entitled The Pioneer Defended, contains a vigorous rebuttal of Geison’s claims. Geison responded to Perutz’s review in the April 4, 1996 issue of The New York Review of Books. Perutz’s counter-response immediately follows.

I make note of all this because Geison’s uncertain assertions are reported as unqualified fact in some accounts of Pasteur’s work. And, while Perutz’s representations are not entirely accurate, the review, the response, and the counter-response make a very interesting read.

References:

(1) Edward Jenner and the Smallpox Vaccine, Posted on the blog September 16, 2014.

(2) Norkin, L. C. Virology: Molecular Biology and Pathognesis, ASM Press, 2010. Chapters 1 and 2 review key developments towards the understanding of viruses.

(3) Ernest Goodpasture and the Egg in the Flu Vaccine, Posted on the blog November 26, 2014.

(4) Chun, K.-T., and D. H. Ferris,  Martinus Willem Beijerinck (1851-1931) Pioneer of general microbiology, ASM News 62, 539-543, 1996.

(5) The Struggle against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 2014.

(6) Who Discovered HIV?, Posted on the blog January 23, 2014.

(7) Dufour, H. D., and S. B. Carroll, (2013), History: Great myths die hard, Nature 502, 32–33. This note contains an update on the myth.

(8) Geisen, G. L., The Private Science of Louis Pasteur, Princeton University Press, 1996.

Ernest Goodpasture and the Egg in the Flu Vaccine

There is a cautionary note on the info sheet accompanying the influenza vaccine, which advises individuals who are allergic to eggs to speak with their doctors before receiving the vaccine. As most readers know, the reason for the warning is that the usual flu vaccine is grown in embryonated chicken eggs.

[Aside 1: The current trivalent influenza vaccine is prepared by inoculating separate batches of fertile chicken eggs; each with one of the three influenza strains (representing an H1N1, an H3N2, and a B strain) recommended by the WHO for the upcoming winter flu season. The monovalent viral yields are then combined to make the trivalent vaccine.]

But, why chicken eggs, and how did this state of affairs come to be? The backdrop to this tale is that until the third decade of the twentieth century, virologists were still searching for fruitful means to cultivate viruses outside of a live laboratory animal. This was so despite the fact that, as early as 1907, researchers had been developing procedures for maintaining viable tissues in culture. And, soon afterwards, virologists began to adapt tissue cultures as substrates for propagating viruses.

Yet as late as 1930, there were still only two antiviral vaccines—the smallpox vaccine developed by Edward Jenner in 1798 (1) and the rabies vaccine developed by Louis Pasteur in 1885. Bearing in mind that Jenner’s vaccine preceded the germ-theory of disease by a half century, and that Pasteur’s vaccine came 15 years before the actual discovery of viruses (as microbial agents that are distinct from bacteria), the development of these first two viral vaccines was fortunate indeed (2).

The principal factor holding up the development of new viral vaccines was that viruses, unlike bacteria, could not be propagated in pure culture. Instead, for reasons not yet understood, viruses could replicate only within a suitable host. And, notwithstanding early attempts to propagate viruses in tissue culture (reviewed below), developments had not yet reached a stage where that approach was fruitful enough to generate a vaccine. How then were Jenner and Pasteur able to produce their vaccines? See Aside 2 for the answers.

[Aside 2: Jenner, without any awareness of the existence of infectious microbes, obtained his initial inoculate by using a lance to pierce a cowpox postule on the wrist of a young milkmaid, Sarah Nelmes. Jenner then propagated the vaccine, while also transmitting immunity, by direct person-to-person transfer. (The rationale underlying Jenner’s vaccine, and his story, is told in detail in reference 1.)

Jenner’s live cowpox vaccine protected against smallpox because cowpox, which produces a relatively benign infection in humans, is immunologically cross-reactive with smallpox. Thus, inoculating humans with cowpox induces immunity that is active against cowpox and against smallpox as well. Jenner’s discovery of the smallpox vaccine, while not entirely fortuitous, was still providential, since immunity per se, as well as microbes, were unknown in Jenner’s day.

Following a successful worldwide vaccination program, smallpox was officially declared to be eradicated in 1977. The smallpox vaccine currently stockpiled in the United States contains live vaccinia; a virus that is immunologically related to cowpox and smallpox. Like cowpox, vaccinia causes a mild infection in humans.

The existing smallpox vaccine was grown in the skin of calves. It is now more than 40 years old and has not been used for years, but it is still believed to be effective.

Pasteur (probably the greatest and most famous microbiologist) was a pioneer of the germ theory of disease. Yet he developed his rabies vaccine more than a decade before the discovery of viruses. He did so by applying the same principle that he used earlier to produce a vaccine against cholera. That is, he “attenuated” the rabies agent. He began with virus that was contained in an extract from a rabid dog. Pasteur attenuated the virus for humans by successively passing extracts in the spinal cords of live rabbits, and then aging the last extracts in the series. Modern rabies vaccines are generally killed virus vaccines, prepared by chemically inactivating tissue culture lysates.]

In the years following the pioneering 19th century contributions of Pasteur, Koch, and Lister, and with the widespread acceptance of the germ theory of disease, microbiologists (that is, bacteriologists) appreciated the importance of working with “pure cultures” that could be grown in a sterilized medium. Yet this was proving to be impossible in the case of viruses. Moreover, as late as the 1930s, it was not understood why that should be so

At the very least, virologists would have liked to be able to cultivate viruses outside of a living animal host. The possibility of achieving that goal began to emerge when Ross G. Harrison, working at Johns Hopkins in 1907, became the first researcher to maintain bits of viable tissue outside of an animal. Harrison maintained frog neuroblasts in hanging drops of lymph medium. What’s more, under those conditions, the neuroblasts gave rise to outgrowths of nerve fibers.

In 1913, Edna Steinhardt became the first researcher to cultivate (or at least maintain) a virus (cowpox) in a tissue culture. Steinhardt did this by infecting hanging-drop cultures with corneal extracts from the eyes of cowpox-infected rabbits and guinea pigs. However, there was no methodology at the time for Steinhardt to determine whether the virus might have replicated in her tissue cultures.

In 1912, Alexis Carrel, working at the Rockefeller Institute, began a two-decade-long experiment that significantly increased interest in tissue culture. Carrel maintained tissue fragments from an embryonic chicken heart in a closed flask, which he regularly supplied with fresh nutrients. Later, he claimed that he maintained the viability of the culture for more than 20 years; well beyond the normal lifespan of a chicken. See Aside 3.

[Aside 3: Carrel’s experimental results could never be reproduced. In fact, in the 1960s, Leonard Hayflick and Paul Moorhead made the important discovery that differentiated cells can undergo only a limited number of divisions in culture before undergoing senescence and dying. It is not known how Carrel obtained his anomalous results. But, Carrel was an honored, if controversial scientist, having been awarded the 1912 Nobel Prize in Physiology or Medicine for pioneering vascular suturing techniques. In the 1930s Carrel developed an intriguing and close friendship with Charles Lindbergh, which began when Lindbergh sought out Carrel to see if Carrel might help Lindbergh’s sister, whose heart was damaged by rheumatic fever. Carrel could not help Lindbergh’s sister, but Lindbergh helped Carrel build the first perfusion pump, which laid the groundwork for open heart surgery and organ transplants. Carrel and Lindbergh also co-authored a book, The Culture of Organs. In the 1930s, Carrel, promoted enforced eugenics. During the Second World War, Carrel, who was French by birth, helped the Vichy French government put eugenics policies into practice. Moreover, he praised the eugenics policies of the Third Reich, leading to inconclusive investigations into whether he collaborated with the Nazis. Carrel died in November, 1944.]

In 1925 Frederic Parker and Robert Nye, at the Boston City Hospital, provided the first conclusive evidence for viral growth in a tissue culture. The virus was a strain of herpes simplex, which Parker and Nye received in the form of an extract from Ernest Goodpasture; soon to be the major character in our story. Parker and Nye established their first culture from the brain of a rabbit that was inoculated intracerebrally with an extract from an infected rabbit brain. The animal was sacrificed when in a convulsive state, and its brain was then removed aseptically. Small pieces of normal rabbit testes were added to pieces of brain in the cultures, to provide another potential host cell for the virus. Virus multiplication was demonstrated by inoculating diluents of subculture extracts into laboratory animals. A 1:50,000 diluent was able to transmit the infection.

At this point in our chronology, the pathologist Ernest Goodpasture, and the husband-wife team of Alice and Eugene Woodruff, enters our story. Goodpasture’s principal interest was then, as always, in pathology. He became interested in viruses while he was serving as a Navy doctor during World War I. But his focus was on the pathology of the 1918 influenza pandemic, which he studied in the first sailors stricken by the infection (3). He was later interested in herpetic encephalitis, and in how rabies virus made its way to the central nervous system, but always from the perspective of a pathologist.

Ernest Goodpasture. (I was unable to find a picture of Alice Woodruff.)
Ernest Goodpasture. (I was unable to find a picture of Alice Woodruff.)

In 1927, Eugene Woodruff was a newly graduated physician who joined Goodpasture in the Pathology Department at Vanderbilt University for training as a pathologist. Eugene’s wife, Alice, a Ph.D., came to the Vanderbilt Pathology Department a year later, as a research fellow in Goodpasture’s laboratory.

Goodpasture set Eugene Woodruff to work on fowlpox; a relative of smallpox, which, unlike cowpox, can not infect humans. Goodpasture was interested in the cellular pathology of fowlpox infection; specifically, in the nature of the inclusion bodies seen in fowlpox-infected cells. Using a micropipette, Woodruff was able to pick single inclusion bodies from infected chicken cells, and to then determine that inclusion bodies are intracellular crystalline arrays of the virus.

More apropos to our story, in the late 1920s, virologists still could not generate large amounts of virus that were free of bacteria and contaminating tissue elements. For that reason, Goodpasture believed that future important advancements in virology would require the development of methods to grow large amounts of virus in pure culture; an impossible goal. In any case, Goodpasture delegated Alice Woodruff to develop a method for growing fowlpox outside of a live chicken.

Goodpasture had already adapted Carrel’s tissue culture methods, which he used to maintain chick kidney tissue in culture. So, Alice’s first experiments were attempts to get fowlpox to propagate in cultures of chick kidney tissue. However, the virus stubbornly declined to grow in the tissue cultures. Goodpasture then suggested to Alice that she try to grow the virus in embryonated chicken eggs. But why did Goodpasture make that suggestion?

The answer isn’t clear. But, back in 1910, Peyton Rous and colleague James Murphy, at the Rockefeller Institute, fruitfully made use of fertile chick eggs to cultivate a virus, as described in Aside 4. However, Rous’ accomplishments, which eventually would be recognized as huge, were largely ignored for the next 50 or so years. (The reasons are discussed in reference 4.) Goodpasture may well have been unaware of Rous’ earlier work when he suggested to Alice that she try to cultivate fowlpox in chicken eggs. If so, then his suggestion to Alice may have been an original idea on his part, perhaps inspired by his thinking of the chick embryo as a sterile substrate that is enclosed in a naturally sterile container. On the other hand, he and Alice did note the earlier work of Rous and Murphy in the 1931 report of their own work. (In that paper, they state: “The production of experimental infection in the chorio-allantoic membrane has, however, been done only in the one instance where Rous and Murphy grew the virus of the Rous sarcoma.”). In any case, the chick embryo method for growing viruses had lain dormant for twenty years.

[Aside 4: Rous and Murphy cut a small window into the shells of six-to-sixteen-day-old embryonated chicken eggs, and then placed a bit of a filtered, cell-free extract from a chicken sarcoma into each. By one week’s time there was a tumor mass growing in each of the inoculated embryos. These studies led to Rous’ 1911 report of a filterable, infectious agent, eventually named the Rous sarcoma virus, which causes sarcomas in chickens. The Rous sarcoma virus was the first virus known to cause solid tumors and, moreover, it was the prototype of a virus family that eventually would be known as the retroviruses (4).]

Alice Woodruff’s procedure for infecting the chicken eggs began with her making a small window in the egg shell, at the site of the air sac. (An egg cup served as the operating table, and the window was cut with a dentist’s drill.) She then inoculated the viral extract into the outermost layer of the chorio-allantoic membrane, which encloses the embryo and provides an air channel into its body. Alice then closed the window with a piece of glass, held in place with Vaseline.

Alice tried to maintain sterility at all stages of her procedure. Yet despite the elegance of her techniques, she had nothing to show for these efforts except dead embryos that were overgrown with mold or bacteria. She then turned to her husband, Eugene, who was working in a separate laboratory, down the hall from her lab.

Alice and Eugene, working together, developed procedures to sterilely remove fowlpox lesions from the heads of chicks. In brief, the chick heads were shaved and then bathed in alcohol. Then, the lesions were excised with sterile instruments. Next, the excised lesions were tested for bacterial or fungal contamination by incubating fragments in nutrient broth. If a lesion was sterile by that test, it was deemed fit to be inoculated into the eggs.

Eugene further contributed to the effort by applying a technique that he developed earlier; picking out individual inclusion bodies from fowlpox-infected cells. When he discovered that the inclusion bodies could be disrupted into individual virus particles by incubating them in trypsin, he was able to provide Alice with virtually pure virus that she could inoculate the eggs with.

As Greer Williams relates in Virus Hunters (5): “Then, one morning when she peeked into the window of an egg that had been incubating for about a week after she had infected it with the virus, she saw something different. This chick embryo was still alive…She removed the embryo from the shell and examined it. It had a swollen claw. ‘Could this be due to fowlpox infection?’…She went to Goodpasture and put the same question to him…”

In Alice’s own words, “I can’t forget the thrill of that moment when Dr. Goodpasture came into my lab, and we stood by the hood where the incubator was installed and I showed him this swollen claw from the inoculated embryo (5).”

The swollen claw indeed resulted from the fowlpox infection. This was shown by the fact that when bits of the swollen tissue were transferred to other embryos, they in turn induced more swollen tissue. Moreover, these swollen tissues contained fowlpox inclusion bodies. Additionally, when transferred to adult chickens, those bits of swollen tissue produced typical fowlpox lesions.

During the next year, Goodpasture, Alice Woodruff, and Gerritt Budding (a lab assistant, who dropped out of medical school to participate in the chick embryo work) reported that cowpox and herpes simplex viruses could also be grown in the embryonated chicken eggs.

Later studies by Goodpasture and Buddingh showed that each embryonated chicken egg could produce enough vaccinia to produce more than 1,000 doses of smallpox vaccine. They also showed, in a case-study involving 1,074 individuals, that the chick-grown smallpox vaccine works as well in humans as the vaccine produced by inoculating the skin of calves. Regardless, the chick vaccine never caught on to replace the long-established, but cruder calf-grown vaccine (see Aside 2).

Goodpasture placed Alice’s name ahead of his own on their report describing the propagation of fowlpox in chicken eggs. Alice says that Goodpasture was “over-generous” in that regard. Howevever, much of the day-to-day lab work resulted from her initiatives. Eugene’s name also came before Goodpasture’s on the report describing the inclusion body study.

Shortly after completing these studies, Alice left research to raise a family. Eugene’s name also disappeared from the virus literature. But in his case that was because his interests turned to tuberculosis.

In 1932, soon after the above breakthroughs in Godpasture’s laboratory,  Max Theiler and Eugen Haagen developed their yellow fever vaccine (6), which initially was generated in embryo tissue from mice and chickens. But, starting in 1937, production of the yellow fever vaccine was switched to the embryonated egg method, in part, to “cure” the live yellow fever vaccine of its neurotropic tendencies.

Recall our introductory comments regarding the warning that individuals allergic to eggs should get medical advice before receiving the standard flu vaccine. In 1941, Thomas Francis, at the University of Michigan, used embryonated chicken eggs to produce the first influenza vaccine (see Asides 5 and 6). Remarkably, even today, in the era of recombinant DNA and proteomics, this seemingly quaint procedure is still the preferred means for producing the standard trivalent flu vaccine (see Aside 1).

[Aside 5: Thomas Francis produced his 1941influenza vaccine in response to urging by U.S. Armed Forces Epidemiological Board. With the Second World War underway in Europe and Asia, and with the 1918 influenza pandemic in mind, there was fear that if an influenza epidemic were to emerge during the upcoming winter, it might impede the military training that might be necessary. An epidemic did not materialize that winter, but the vaccine was ready, and we were at war.]

[Aside 6: Thomas Francis was one of the great pioneers of medical virology. The same year that he developed his flu vaccine, Jonas Salk (recently graduated from NYU medical school) came to his laboratory for postgraduate studies. Francis taught Salk his methodology for vaccine development, which ultimately enabled Salk to develop his polio vaccine (7).]

Next, Hillary Koprowski developed a safer, less painful and more effective rabies vaccine that is grown in duck eggs, and that is still widely used. Why duck eggs? The reason is that duck eggs require four weeks to hatch, instead of the three weeks required by chicken eggs. So, duck eggs give the slow-growing rabies virus more time to replicate.

By any measure, the procedures for growing viruses in embryonated chicken eggs, developed by Ernest Goodpasture and Alice Woodruff, were a major step forward in vaccine development. Sir Macfarlane Burnet (a Nobel laureate for his work on immunological tolerance) commented 25 years later, “Nearly all the later practical advances in the control of viral diseases of man and animals sprang from this single discovery.”

Addendum 1: Several major advances in cell and tissue culture (the other means for growing viruses outside of an animal) happened after Woodruff and Goopasture reported the development of their embryonated egg method in 1931. For the sake of completeness, several of these are noted.

In 1933, George Gey, at Johns Hopkins, developed the roller tube technique, in which the tissue is placed in a bottle that is laid on its side and continuously rotated around its cylindrical axis. In that way, the media continually circulates around the tissue. Compared to the older process of growing tissues in suspension, the roller culture method allowed the prolonged maintenance of the tissues in an active state and, consequently, the growth of large amounts of virus. The roller tube technique also works very well for cell cultures that attach to the sides of the bottle. [Incidentally, Gey is probably best known for having established the HeLa line of human carcinoma cells from cancer patient, Henrietta Lacks. HeLa cells comprise the first known human immortal cell line and they have served as one of the most important tools for medical research. (See The Immortal Life of Henrietta Lacks, by Rebecca Skloot, 2010.)]

In 1948, John Enders, and colleagues Thomas Weller and Frederick Robbins, used Gey’s methods, to demonstrate for the first time that poliovirus could be grown in non-nervous tissue. This was significant because the potential hazard of injecting humans with nervous tissue was holding up the development of a polio vaccine.

Next, Renato Dulbecco and Marguerite Vogt, working at Caltech, developed procedures to grow large amounts poliovirus in cell culture, adding to the feasibility of an eventual polio vaccine (8). Additionally, Dulbecco and Vogt developed a plaque assay procedure to measure the titer of animal viruses grown in cell culture (7).

Addendum 2: The following excerpt tells of the chance encounter that led Howard Temin to become a virologist (4). Temin was the Nobel laureate who first proposed the retroviral strategy of replication, and who co-discovered reverse transcriptase.

“Howard Temin began working on Rous sarcoma virus in the 1950s, while a graduate student in Renato Dulbecco’s laboratory at Caltech (see reference 7 for more on Dulbecco). However, he worked under the direct supervision of Harry Rubin, an early star in the field, who was, at the time, a postdoctoral fellow in the Dulbecco lab. Nothing was known as yet about the replication of the RNA tumor viruses, as the retroviruses were then known. Moreover, little more was known about the molecular basis of cancer in the 1950s than was known in 1911, when Rous first isolated his virus; a state of affairs that would be much alleviated by future studies of the oncogenic retroviruses.

Rubin was a veterinarian by training, perhaps accounting for his somewhat unique appreciation of an oncogenic virus of chickens, well after even Rous himself had lost interest. And, Rubin was responsible for introducing other young investigators to the RNA tumor virus field, both at Caltech and later at UC Berkely.

Rubin’s mentorship of Temin began somewhat fortuitously, as follows. When they first met, Temin was actually doing his graduate research in another laboratory at Caltech, looking into the embryology of the innkeeper worm, Urechis caupo. But he was also serving as a laboratory assistant in the Caltech general biology course. In that capacity, he was dispatched to Dulbecco’s laboratory to obtain some fertilized chicken eggs for use in the general biology lab. Harry Rubin supplied the chicken eggs. But the chance visit from Temin gave Rubin the opportunity to tell Temin about the chicken sarcoma viruses that were being studied in the Dulbecco laboratory.

Rubin had just recently found that he could induce the neoplastic transformation of a normal chicken cell with a single Rous sarcoma virus particle. He then demonstrated that the transformed cell produced hundreds more transformed daughter cells in a week’s time. During their chance conversation, Rubin suggested to Temin that he (Temin) might make use of that observation to develop a quantitative tissue culture assay for Rous sarcoma virus. Sufficiently intrigued by Rubin’s proposition, Temin switched from embryology to virology and proceeded to develop a focus-forming cell culture assay for Rous sarcoma virus; an assay analogous in principle to a plaque assay. But instead of forming plaques of dead cells, the non-cytocidal Rous sarcoma virus induces the growth of visible foci of morphologically transformed neoplastic cells.”

[Addendum 3: Today, viruses are usually cultivated in readily available continuous cell lines. That said, when I first entered the field in 1970, as a postdoctoral studying the murine polyomavirus, my first task of the week was to prepare the baby-mouse-kidney and mouse-embryo primary cell cultures, which at that time served as the cellular host for that virus. This rather unpleasant chore was a reason I eventually turned to SV40, since I could grow that virus in continuous lines of monkey kidney cells.

References:

1. Edward Jenner and the Smallpox Vaccine, posted on the blog September 16, 2014.

2. Leonard C. Norkin, Virology: Molecular Biology and Pathogenesis, ASM Press, 2010. Chapter 1 tells how viruses were discovered and how their distinctive nature was brought to light.

3. Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu, posted on the blog April 15, 2014.

4. Howard Temin: “In from the Cold,” posted on the blog December 14, 2013.

5. Greer Williams, Virus Hunters, Alfred A. Knopf, 1960.

6. The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, posted on the blog May 12, 2014.

7. Renato Dulbecco and the Beginnings of Quantitative Animal Virology, posted on the blog December 3, 2013.

8. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, posted on the blog March 27, 2014.

Edward Jenner and the Smallpox Vaccine

The Greek historian, Thucydides, discovered twenty four centuries ago that smallpox survivors were resistant to subsequent smallpox episodes. Thucydides’ remarkable perception, more than two thousand years before awareness of infectious agents, may have influenced his fellow Athenians, since those who survived the infection comprised the few who were willing to care for those who fell ill. Thucydides’ insight was lost to Western medicine. However, the independent perception in China, that that smallpox survivors are entirely safe from a second attack, led to the development there, about 1,000 years ago, of an empirically based smallpox control strategy, in which uninfected individuals were protected by inhaling powder prepared from dried smallpox scabs. The scabs were from individuals who survived a mild smallpox infection. They were dried to further diminish the likelihood of the recipient undergoing a severe infection.

By 1700, the process had spread to Africa, India, Arabia, and the Ottoman Empire. The Arabians streamlined this approach by transferring the dried postular material on the point of a needle. Lady Mary Wortley Montague, the wife of the British ambassador to Turkey, had her children undergo the process in the early 18th century, and then brought the practice to Europe, where British physicians dubbed it “variolation.” [See Cotton Mather, Onesimus, George Washington, and Variolation, posted on the blog November 20, 2013, for an account of the introduction of variolation to the New World.]

As might be expected, variolation carried risks that would not be acceptable today. However, those risks were tolerable in 18th century Europe, when as many as one person in ten died of smallpox. We now have the smallpox vaccine, which was the first, and arguably the most successful vaccine ever put into practice. Remarkably, the smallpox vaccine was developed in 1798 by an English country doctor, Edward Jenner, a half-century before the germ theory of disease, and 100 years before the actual discovery of viruses!

At thirteen years of age, Jenner was apprenticed to an English surgeon; a mister Ludlow. While Jenner was in Ludlow’s service, he heard the doctor suggest to an ill milkmaid that she might be coming down with smallpox. The milkmaid replied that she could not get smallpox since she already had cowpox. The notion, that having had cowpox protects one against smallpox, may actually have been common among English country folk of the day, but it was just as commonly dismissed by physicians.

At 21 years of age, Jenner continued his training under the prominent British surgeon, John Hunter. When Jenner ran the milkmaid’s comment by Hunter, the great surgeon encouraged his young protégé to investigate the matter further.

Now, perhaps the most remarkable part of the story. History usually credits young James Phipps as the first person “vaccinated” by Jenner. And, while Phipps, in 1796, was the first individual Jenner inoculated with cowpox, and subsequently challenged with smallpox, he was, in fact, not the subject of Jenner’s first experiment. Instead, that person was Jenner’s first son, Edward, Jr., born in 1789. Jenner inoculated Edward Jr. with swinepox when the infant was only 10 months old!

Jenner could not have known about microbes, and he left no records revealing his purpose in inoculating Edward Jr. with swinepox. It may be relevant that cowpox was relatively rare at the time, while a similar pox disease was more common in pigs. Regardless, Jenner’s baby son became sick on the eighth day with a pox disease, from which he fortunately recovered. Then, his father proceeded to challenge him with genuine smallpox!

Fortunately, Edward Jr. also resisted his father’s experimental attempt to transmit smallpox to him. His father tried again in 1791, when the boy was two, and again when he was three. Edward Jr. resisted each of Edward Senior’s smallpox challenges, most likely because the swinepox virus immunized him against smallpox. We can only guess how Mrs. Jenner regarded these happenings.

Jenner also used several other young children in his experiments, including his 11-month-old second son, Robert. One of these children died from a fever, possibly from a contaminant (streptococcus?) in the vaccine. In those days one could hardly know what might be in a vaccine.

In Jenner’s famous and classic experiment involving James Phipps, he used a lance to pierce a cowpox postule on the wrist of a young milkmaid, Sarah Nelmes. He then scratched James twice on the arm with the lance. Six weeks afterwards, Jenner challenged James with smallpox from a postule on the body of a smallpox patient. The smallpox challenge caused only a slight inflammation on James’ arm, indicating what now would be recognized as an immune reaction. During the next 25 years or so, Jenner challenged James twenty more times with smallpox, with never any sign of the disease.

JENNER Edward Jenner administering the first smallpox vaccination in 1796.  Painting by Ernst Board.

Not much else is known about James Phipps, who was only 8 years old when he was first inoculated by Jenner. Additionally, nothing is known about James’ parents and whether they may have consented to Jenner’s use of James. However, Jenner referred to his young subject as “poor James,” and looked after him in later years, suggesting he may have felt some remorse. Moreover, he eventually built a cottage for James and even planted flowers in front of it himself. Little is known of Sarah Nelmes.

Thankfully, the sorts of experiments Jenner carried out cannot be done today. Yet because of his efforts, the once dreaded smallpox virus now exists only in the laboratory.

More than a century would have to pass before it could be appreciated that the protection against smallpox that was generated by inoculation with cowpox and swinepox depended on the facts that these two viruses are immunologically cross-reactive with smallpox virus and that they produce a relatively benign infection in humans. [When contemporary vaccinologists develop vaccines to protect against viral diseases, they are essentially tapping into biological mechanisms that have been perfected through eons of natural selection. Indeed, the principal fact exploited by vaccinologists is that natural infection, by many different viruses, results in lifelong immunity against the same virus.]

Some final points:

It is possible that Jenner was not the first to use cowpox to vaccinate against smallpox. However, he was the first to eliminate the cow from the procedure. That is, he transmitted immunity from person-to-person, without the need for an infected cow. Nevertheless, he hung in his house the hide of the cow, which had initially given Sarah Nelmes cowpox.

Although Jenner demonstrated that his vaccine could be passed indefinitely from person-to-person, neither he, nor anyone else at the time, had the insight that this indefinite passage meant that the active agent in the vaccine must be able to replicate.

References:

Greer Williams: Virus Hunters, Alfred A. Knopf, 1960.

Cotton Mather, Onesimus, George Washington, and Variolation, posted on the blog November 20, 2013.

Related Postings:

Smallpox in the New World: Vignettes featuring Hernan Cortes, Francisco Pizarro, and Lord Jeffry Amherst, posted on the blog February 24, 2014.

Notable Individuals Who Survived Smallpox and One Who Didn’t: Featuring Abraham Lincoln, Elizabeth I, Josef Stalin, and Pocahontas, posted on the blog March 10, 2014.

Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science

Paralytic poliomyelitis was one of the world’s most feared diseases during the first half of the 20th century. However, the dread of poliovirus ended abruptly with the advent of the poliovirus vaccines in the 1950s. This posting tells the stories of the key players in the race to develop a polio vaccine. In particular, it features the rivalry between Jonas Salk and Albert Sabin, the two main contenders in the pursuit. While their vaccines together have led to the near disappearance of poliovirus worldwide, neither was recognized by the Nobel committee for his achievement. We begin with some background.

Poliovirus has long been especially interesting to me, both as a virologist and personally as well. The reason is that I was a child and young teenager during the annual polio epidemics of the 1940s and early 1950s, and can vividly remember the panic that set in early every summer of the pre-vaccine days, when the first neighbor or schoolmate was stricken. You were kept home from school and couldn’t even play outside. A visit to a hospital in those times was associated with the pitiful sight of young polio victims in the iron lungs that filled the wards, and even the hallways of hospitals back then.

iron lung

Not even the emergence of AIDS in the early 1980s evoked fear comparable to that brought on by poliomyelitis. Yet despite the dread of poliomyelitis, the disease actually struck many fewer victims than was commonly perceived by the public. The number of poliomyelitis cases in the United States was typically 20,000 to 30,000 per year in the 1940s and 1950s, while influenza still typically kills 40,000 to 50,000 Americans annually. Yet most individuals, then and now seem indifferent to influenza. What’s more, even the 1918 “Spanish Flu” epidemic, which was arguably the most devastating epidemic in human history, did not cause any panic, despite the fact that during the single month of October 1918, it killed 196,000 people in the United States alone! Estimates of the total number killed worldwide by the 1918 Spanish Flu range between 20 million and 50 million.

So, how can we explain the terror brought on by poliomyelitis? It wasn’t simply because poliovirus struck suddenly, without any warning. So did the “Spanish Flu.” Rather, paralytic poliomyelitis mainly struck children, adolescents and young adults. In contrast, influenza mainly threatens the elderly. And, in truth, most parents are far more emotionally invested in their children’s well-being than in that of their parents or themselves. Furthermore, the sight of a child in an iron lung or leg braces (affected legs became atrophied and deformed) was truly heart rending.

The fear evoked by poliomyelitis was permanently ended in the United States and in much of the developed world as well, by the emergence of Salk’s killed polio vaccine in 1955. Sabin’s live attenuated vaccine followed soon after. [Live vaccines generally contain attenuated (weakened) variants of the virulent virus, which can replicate and induce immunity, but which cannot cause disease.] The response of the public to Salk’s vaccine was so great that he was hailed as a “miracle worker.” Nevertheless, and despite the fact that the vaccines created by Salk and Sabin have nearly ridden the world of poliovirus, neither man would ever be recognized by the Nobel committee.

salk Salk’s public acclaim was resented by his colleagues.

Most virologists of the day strongly favored live vaccines over killed ones, based on the belief that only a live vaccine could induce a level of immunity sufficient to protect against a challenge with live virulent virus. Indeed, the effectiveness of live vaccines had been established much earlier by Jenner’s smallpox vaccine (1798) and Pasteur’s rabies vaccine (1885). Jenner’s smallpox vaccine actually contained live cowpox virus, which was similar enough immunologically to variola to protect against smallpox, while not being able to cause smallpox itself. Pasteur’s rabies vaccine contained live rabies virus that was attenuated by passages through rabbit spinal cords. [Adapting the virus to grow in rabbits attenuated its virulence in humans, while not impairing its ability to induce immunity.] So, bearing in mind the well-established precedence of attenuated vaccines, why did Salk seek to develop a killed vaccine?

In 1941, Thomas Francis, one of the great pioneers of medical virology, working at the University of Michigan, developed a killed influenza vaccine. Providentially, in the same year, Jonas Salk (recently graduated from NYU medical school) came to Francis’ laboratory for postgraduate studies. In Francis’ lab, Salk learned his mentor’s methods for producing his killed influenza vaccine and assisted in its field trials.

Salk’s experience in Francis’ laboratory led him to believe in the potential of a killed poliovirus vaccine. And, Salk learned practical procedures from Francis that would be valuable in his pursuit of that objective. These included the use of formaldehyde to kill the virus, the use of adjuvants to enhance the immunogenicity of the killed vaccine, and protocols for conducting field tests.

In contrast to Salk, Sabin firmly believed that a live attenuated vaccine would be vastly superior to a killed one. And, although Salk won the race to produce an actual vaccine, Sabin had been doing polio research well before the younger Salk emerged on the scene. Indeed, Sabin made several important contributions to the field; some of which are discussed below. For now, we mention that in 1936, Sabin and colleague Peter Olitsky demonstrated that poliovirus could be grown in cultured human embryonic nervous tissue. While this might appear to be a key step towards the development of a vaccine, Sabin and Olitsky feared that nervous tissue might cause encephalitis (inflammation of the brain and spinal cord) when injected into humans.

sabinAlbert Sabin, who developed the live polio vaccine.

John Enders, at the Children’s Hospital of Boston, is the next key player in our story. Enders believed that poliovirus should be able to grow in non-nervous tissue, particularly tissue from the alimentary canal, as suggested to him by the amount of the virus that was present in the feces of many patients. So, in 1948, Enders, and colleagues Thomas Weller and Frederick Robbins, succeeded in growing poliovirus in cultured non-nervous tissue, including skin, muscle, and kidney. As a result of Ender’s work, sufficient amounts of poliovirus could now be grown, free from the hazards of nervous tissue, thereby enabling the mass production of a vaccine.

[Aside: Enders, Weller, and Robbins maintained their tissue samples in culture using the roller culture procedure, in which a horizontally positioned bottle is laid on its side and continuously rotated around its cylindrical axis. In comparison to the older process of growing tissues in suspension, the roller culture method enabled the prolonged maintenance of the tissues in an active state and, consequently, the growth of large amounts of virus. For readers who read Renato Dulbecco and the Beginnings of Quantitative Animal Virology (on the blog), note that Dulbecco developed procedures for growing pure cell types as flat adherent monolayer cultures, thereby making possible quantitative plaque assays of animal viruses.]

In 1954, Enders, Weller, and Robbins shared the Nobel Prize in Physiology or Medicine for their contribution described above. What’s more, the Nobel award to Enders, Weller, and Robbins was the only Nobel award ever given in recognition of polio research! Ironically, Ender’s true interests actually lay elsewhere; with measles. He would later develop a measles vaccine. [Enders has been referred to as the “Father of modern vaccinology.”]

The next key player of note in our story is not a person but, rather, a foundation; the “National Foundation for Infantile Paralysis,” which led and financed the crusade against polio in the pre-NIH days of the 1950s. The National Foundation was actually an outgrowth of the Georgia Warm Springs Foundation, a charitable organization founded by Franklin D. Roosevelt, himself crippled by polio. However, after Roosevelt became president of the United States, he was too polarizing a figure (particularly after his “court-packing” scheme in 1937) to head up a philanthropic organization. Consequently, in 1938, Roosevelt announced the formation of the nonpartisan National Foundation for Infantile Paralysis.

roosevelt Photos of Franklin Roosevelt in a wheel chair are rare and were not shown to the public, which was generally unaware that he was paralyzed from the waist down.

[Aside: The National Foundation was initially funded by the contributions of wealthy celebrities who attended Roosevelt’s yearly birthday bashes. At one of these fundraisers, the vaudevillian, Eddie Cantor, jokingly urged the pubic to send dimes directly to the president. And, bearing in mind the fear evoked by polio, the public, perhaps not recognizing the joke, did exactly that, flooding the White House with nearly three million dimes. And so, the slogan “March of Dimes,” for the Foundation’s grass-roots fund-raising campaign, came to be. And, it was not coincidental that a dime (the Roosevelt dime) was issued in 1946 to memorialize the late president.

In 1950, a March of Dimes chapter in Phoenix, Arizona held a “Mother’s March on Polio,” in which volunteers went door-to-door raising money for polio research. People were urged to leave their porch lights on to show that the volunteers would be welcome. The Phoenix initiative soon spread to other locals, and the Mother’s March became a nationwide annual event.]

The role of the National Foundation went beyond merely raising money for research. It also attempted to provide direction to the research, which often placed it at odds with its grantees. This was the case because Harry Weaver, the director of research at the National Foundation, was focused on bringing a vaccine to the public. In contrast, most of the Foundation’s grantees were largely motivated by their desire to understand basic virological issues, such as poliovirus transmission, replication, and dissemination. What’s more, they believed that there was still too much to be known about poliovirus and poliomyelitis before a vaccine might be a realistic possibility.

[Aside: Apropos the sentiment of some poliovirus researchers that there was too much yet to be known before a polio vaccine might be possible, Jenner’s 1798 smallpox vaccine was developed a half century before the germ theory of disease was proposed, and 100 years before the actual discovery of viruses. It was based on the empirical observation that milkmaids seemed to be “resistant” to smallpox; apparently because they had been exposed earlier to cowpox. The initial smallpox vaccine simply contained matter from fresh cowpox lesions on  the hands and arms of a milkmaid. It was then serially passed from one individual to another; a practice eventually ended because of the transmission of other diseases. And, Pasteur’s 1885 rabies vaccine too was developed before viruses were recognized as discrete microbial entities.]

Sabin’s objection to the Foundation’s priority of having a vaccine available as quickly as possible was somewhat more personal. Since a killed vaccine should be more straightforward and, therefore, quicker to develop than an attenuated one (see below), Sabin believed that Weaver’s sense of the urgent need for a vaccine would lead him to favor supporting Salk’s killed vaccine over his attenuated one. Moreover, Sabin felt that he was being shunted aside. And, Since Sabin remained firm in his belief in the superiority of a live vaccine; he also felt that Weaver’s main concern of having a vaccine available as quickly as possible, would compromise the efficacy of the vaccine that would be implemented in the end.

[Aside: Back in the Enders laboratory, Thomas Weller and Frederick Robbins wanted to enter the polio vaccine race. But, Enders viewed the project as boring and routine; a view pertinent to the question of why Salk and Sabin were never recognized by the Nobel Committee. Furthermore, Enders didn’t believe that a killed vaccine could ever provide adequate protection against polio, or that a live vaccine would be possible without years more of research.]

Sabin’s worry that a killed vaccine would be faster to develop than an attenuated one was borne out when, in1953, Salk was preparing to carry out a field-test of his killed vaccine. Yet Sabin and other poliovirus researchers remained inclined to move slowly, placing them in opposition to Harry Weaver’s sense of urgency. Moreover, Sabin and the other polio investigators were also piqued at the National Foundation for promoting Salk’s vaccine to the public and, also, for promoting Salk himself as a miracle worker. The Foundation’s reason for publicizing Salk was to stir up public enthusiasm for its fund raising campaigns. And Salk indeed was becoming the symbol of the miracles of medical research to an admiring public.

In fairness to the polio researchers who dissented with the National Foundation’s single minded emphasis on bringing a vaccine to the public, there were valid reasons for believing that the Foundation might be moving too quickly. So, consider the following excerpts from a letter that Sabin wrote to his rival, Salk: “…this is the first time they (the Foundation) have made a public statement based on work which the investigator has not yet completed or had the opportunity to present…in a scientific journal…Please don’t let them push you to do anything prematurely or to make liters of stuff for Harry Weaver’s field tests until things have been carefully sorted out, assayed, etc., so that you know what the score is before anything is done on a public scale.”

While Sabin’s advice to Salk seems eminently sensible, Sabin had never before shown any inclination to look out for Salk’s interests. So, might Sabin be sending a non-too-subtle warning to Salk that he could either play by the traditions of the scientific community, or face the consequences of playing to the interests of the Foundation? For his part, Salk was well aware of what was happening and he was indeed embarrassed by the adulation of the press; correctly sensing that it was compromising his standing with his colleagues.

[Aside: The media, in the person of the legendary broadcaster, Edward R. Murrow, provided Salk with a notable and very satisfying moment in the public spotlight. During an April, 1955 interview on the CBS television show See it Now, Murrow asked Salk: “Who owns the patent on this vaccine?” To which, Salk replied: “Well, the people, I would say. There is no patent. Could you patent the sun?”

While Salk’s answer to Murrow endeared him even more to the public, some colleagues questioned whether it might have been disingenuous. Both the University of Pittsburgh, where Salk carried out his work, and the National Foundation, which financed it, indeed had been looking into the possibility of patenting Salk’s vaccine. But, when patent attorneys sought to determine if there was a basis for a patent, Salk readily acknowledged that his vaccine was, for the most part, based on tried and true procedures developed by others.

In point of fact, Salk’s critics held him in low esteem largely because there was little about his vaccine that was innovative. Indeed, Sabin once quipped: “You could go into the kitchen and do what he (Salk) did.” But in fairness to Salk, he never claimed that his vaccine was unique. Instead, in the face of much skepticism, his point had always been that a killed vaccine could protect against polio. He persevered and he was right.

Note that Sabin too gave his vaccine to the world gratis.]

By 1954, field tests of Salk’s vaccine went ahead on a massive scale, involving nearly 1.5 million schoolchildren nationwide. The tests were overseen by Thomas Rivers, an eminent virologist who, at the time, was Director of the Rockefeller Institute. Like most virologists, Rivers favored a live vaccine as the ultimate solution to polio. Nevertheless, he believed that the world couldn’t wait ten or more years for an ideal vaccine, when a satisfactory one might be available at present.

With 57,879 cases of poliomyelitis in the United States in 1952, the peak year of the epidemic, Harry Weaver’s sense of the urgent need for a vaccine was widely shared by the public. Unsurprisingly then, the public eagerly supported the 1954 field test of Salk’s vaccine, as indicated by the fact that 95% of the children in the test received all three required vaccinations. [Killed vaccines require multiple doses. That is so because the first dose only primes the immune system. The second or third dose then induces the primed immune system to produce protective antibodies against the virus. Inoculation with a live vaccine resembles a natural infection and, consequently, a single dose is sufficient to induce immunity.]

The field test of Salk’s vaccine was unprecedented in its size. What’s more, it was supported entirely by the National Foundation, which strenuously opposed outside interference from the federal government. In actuality, the Foundation considered federal funding for polio research to be a “Communistic, un-American…scheme.”

[Aside: President Dwight Eisenhower, a Republican and a fiscal conservative, also believed that the federal government had no proper a role in health care. Consequently, Eisenhower took little interest in his Department of Health, Education, and Welfare (HEW). What’s more, Eisenhower’s Secretary of HEW, Oveta Culp Hobby, was even more conservative in that regard than Eisenhower himself. In 1955, after the field trials showed the Salk vaccine to be a success, and with the public clamoring for it, there were insufficient amounts of the vaccine available to meet the public’s demands. Thus, even some Republicans were stunned to learn that the Eisenhower administration had taken no actions whatsoever to watch over production of the vaccine or its distribution, believing that this was in the province of the drug companies. When pressed on this, Mrs. Hobby responded: “I think no one could have foreseen the public demand.”

Not surprisingly, American drug companies lobbied intensely to keep vaccine production under their own control. A different scenario played out in Canada, where the government viewed polio as a national crisis, and took control of its vaccination program, with overwhelming public support.]

All did not go well for Salk and his vaccine after the successful 1954 field tests. In April 1955, more than 200,000 children were inoculated with a stock of improperly inactivated vaccine made by Cutter Laboratories; one of the five companies that produced the vaccine in 1955. [The others were Eli Lilly, Parke-Davis, Wyeth, and Pitman-Moore.] The Cutter vaccine caused 40,000 cases of abortive poliomyelitis (a form of the disease that does not involve the central nervous system), and 56 cases of paralytic poliomyelitis; 5 of which were fatal. What’s more, some of the children inoculated with the Cutter vaccine transmitted the vaccine virus to others, resulting in 113 more cases of paralytic poliomyelitis and 5 fatalities.

A congressional investigation blamed the “Cutter incident” on the NIH Laboratory of Biologics Control, for insufficiently scrutinizing the vaccine producers. In point of fact, the NIH did little testing on its own. Instead, it mainly relied on reports from the National Foundation, whose agenda was to proceed with the vaccinations. Yet the NIH did have an early, in-house warning of potential problems with the Cutter vaccine, which it failed to act on. Bernice Eddy, a staff microbiologist at the NIH, reported to her superiors that the Cutter vaccine caused paralysis when inoculated into monkeys. However, no action was taken in response to Eddy’s warning. [In 1959, Eddy discovered simian virus 40 (SV40) in monkey kidney tissue that was used for vaccine production. By that time, live SV40 had unknowingly been injected into hundreds of millions of people worldwide; perhaps the subject of a future blog posting.]

Salk was exonerated of any fault in the Cutter incident. Moreover, after that episode, not a single case of polio in the United States would be attributed to Salk’s vaccine. Nevertheless, while Salk’s killed vaccine was perfectly safe when properly prepared, the Cutter incident led to the perception that it was unsafe. Consequently, Salk’s killed vaccine was eventually replaced by Sabin’s live attenuated one. Ironically, as we will see, the perception that Salk’s vaccine was dangerous led to its replacement by a more dangerous one.

Sabin’s work on his live polio vaccine began in 1951 and, like Salk; he was supported by the National Foundation. Sabin’s task was more difficult than Salk’s because it is more straightforward to kill poliovirus, than it is to attenuate it. [The attenuated virus must be able to replicate in the digestive tract and induce immunity, yet be unable to damage the nervous system.] But Sabin persisted, sustained by his conviction that a live vaccine would invoke stronger, longer-lasting immunity than a killed vaccine. Sabin attenuated his vaccine by successive passages through monkey tissue, until the live virus could no longer cause paralysis when inoculated directly into chimpanzee spinal cords.

[Aside: At this early date, live-vaccine-proponents could not have known that only a live vaccine could activate T-cell mediated immunity, which is generally necessary to clear a virus infection. Instead, their preference for live vaccines was based on the simpler, but correct notion that inoculation with a live vaccine would more closely approximate a natural infection. Also, since the vaccine virus is alive, vaccinated individuals might transmit it to unvaccinated ones, thereby inducing immunity in the latter as well. On the other hand, the attenuated vaccine poses a deadly threat to individuals with impaired immune systems, such as AIDS patients and individuals on immunosuppressive regimens following organ transplants.]

In 1954, a successful small-scale test of Sabin’s vaccine was carried out, which involved thirty adult human prisoners at a federal detention facility. The promising outcome of this test warranted a larger field-trial of Sabin’s vaccine. But, several obstacles stood in the way. First, the National Foundation was not inclined to support another massive field trial, now that Salk’s vaccine was already in use. Second, the Foundation was still reeling from the Cutter incident, and had no inclination to be caught up in another such debacle. Third, it would be virtually impossible to conduct the trials in the United States, since millions of American children had already been inoculated with Salk’s vaccine. The ensuing course of events was rather remarkable.

By 1956, poliomyelitis had become a serious public health crisis in the former Soviet Union. Consequently, a delegation of Russian scientists came to the United States to meet with Salk and consult with him on how to produce his vaccine. However, the Russians were disposed to meet with other polio researchers as well. Thus, Sabin seized this opportunity to invite the Russians to visit his laboratory at the University of Cincinnati, where he was able to tout his live vaccine to them. Sabin’s pitch was apparently effective, as he secured an invitation from the Russians to visit the Soviet Union, where he spent a month, further hyping his vaccine.

[Aside: While Sabin was in Russia, the Russians requested from him a sample of his live vaccine. So, when Sabin returned to the United States, he sought permission from the State Department to send the Russians the samples they requested. The State Department approved the request; but it did so over objections from the Defense Department, which was concerned that the vaccine virus might have “biological warfare applicability.”]

With the incidence of poliomyelitis on the rise in the Soviet Union, the Soviet Health Ministry needed to quickly decide which vaccine to adopt; Salk’s or Sabin’s. The Russians were already producing the Salk vaccine, but were unable to consistently maintain its efficacy from one batch to another. So, the Soviets invited Salk to visit Russia, so that he might help them to solve the problems they were having producing his vaccine.

Salk then made a decision that he would long regret. Because of pressure from his wife to spend more time with his family, Salk turned down the Russian invitation. The upshot was that the Russians turned instead to Sabin. In 1959 they vaccinated 10 million children with vaccine strains sent to them by Sabin. Soviet results with the Sabin vaccine were so promising that the Soviet Health Ministry decided to then use it to vaccinate everyone under 20 years of age. A total of seventy-seven million Soviet citizens were vaccinated with Sabin’s vaccine, vastly exceeding the number vaccinated during field trials of the Salk vaccine in the United States.

The U.S. Public Health Service did not endorse the Sabin vaccine for use in the United States until 1961. By then, the Salk vaccine had virtually eliminated polio from the country. Nevertheless, Sabin’s vaccine supplanted Salk’s in the United States and in much of the rest of the world as well.

Yet all did not go well with Sabin’s vaccine either. As noted above, after the Cutter incident, there were no cases of poliomyelitis in the United States that could be attributed to Salk’s vaccine. In contrast, Sabin’s vaccine caused about a dozen polio cases per year, a frequency of about one case per million vaccinated individuals. At least some of these cases resulted from the ability of the attenuated virus to revert to a more virulent form. What’s more, reverting viruses posed a threat to non-vaccinated individuals in the population. For instance, in 2000/2001, there were 21 confirmed cases of poliomyelitis in the Dominican Republic and Haiti, which were traced to a single dose of the Sabin vaccine that was administered during the preceding year. [As noted in an above Aside, since the Sabin vaccine is alive, vaccinated individuals might transmit the vaccine virus to unvaccinated individuals.]

In actual fact, the few cases of poliomyelitis that now occur in the West are vaccine-related, resulting from the rare reversions of Sabin’s vaccine. Ironically, the Sabin vaccine, which played a crucial role in the near eradication of polio from the world, had become an obstacle to the complete eradication of the virus. In 2000, the U.S. Centers for Disease Control (CDC) recommended the complete return to the Salk vaccine in the United States. However, the Sabin vaccine would continue to be used in much of the developing world.

[Aside: Several polio hotspots remain in the world. Three major ones are Pakistan, Afghanistan, and Nigeria. Recent outbreaks have also occurred in Syria and Somalia. In each of these instances, social and political climates make it difficult to carry out eradication campaigns.

As recently as March 2014, militants attacked a polio vaccination team in northwest Pakistan, detonating a roadside bomb and then opening fire on their convoy, killing 12 of their security team, and wounding dozens more. Some Pakistani religious leaders denounced the vaccination campaign in Pakistan as a cover for spying or as a plot to sterilize Muslim children.

In the developed world there is a very different problem. Ironically, the great success with which the polio vaccines eradicated the virus in the West has created conditions there in which poliomyelitis might make a most unwelcome return. That has come about because too many parents in the developed world now view polio as ancient history, and have become complacent about having their children vaccinated. What’s more, some parents are heeding unsubstantiated warnings that the risks of vaccines are greater than the risks of the viruses. Consequently, the frequency of vaccinated individuals in the West is declining to the point where the West may be susceptible to outbreaks sparked by imported cases. These issues will be discussed at length in a subsequent posting.]

We turn now to an issue raised at the outset of this posting; neither Salk nor Sabin was recognized by the Nobel Committee for his contribution. That is so, despite the fact that their individual efforts, taken together, have virtually eliminated polio from the world.

Max Theiler, at the Rockefeller Institute, is relevant regarding the Nobel issue, and for several other reasons as well. First, Theiler took an early interest in Sabin’s career during Sabin’s years at the Rockefeller (1935 to 1939). Second, during those years Theiler was working on a live attenuated vaccine for yellow fever. Like most virologists of the day, Theiler believed that only a live vaccine could provoke significant long-lasting immunity. And, Theiler’s thinking on this matter likely influenced Sabin’s later approach to a polio vaccine. Thirdly, and important in the current context, in 1951 Theiler was awarded the Nobel Prize in Physiology or Medicine for his yellow fever vaccine. Fourth, Theiler’s Nobel Prize was the only one ever awarded for the development of a virus vaccine!

Why was Theiler’s Nobel award the only one ever given for the development of a virus vaccine? In addition, recall that John Enders, Thomas Weller, and Frederick Robbins shared the 1954 Nobel Prize for Physiology or Medicine, for demonstrating that poliovirus could be propagated in non-nervous tissue. Moreover, the Nobel Prize shared by Enders, Weller, and Robbins was the only one ever given in recognition of polio research! Why weren’t Salk and Sabin recognized as well? Didn’t they also contribute substantially “to the benefit of mankind;” a standard for the award, as specified by Alfred Nobel?

Apropos these questions, it may be relevant that Alfred Nobel also specified that the prize for physiology or medicine should recognize a “discovery” per se. With that criterion in mind, the Nobel committee may have viewed the contributions of Salk and Sabin as derivative, requiring no additional discovery. In contrast, the discovery of Enders, Weller, and Robbins, refuted the previously held belief that poliovirus could be grown only in nervous tissue; a breakthrough that paved the way to the vaccines.

But then, what was there about Theiler’s yellow fever vaccine that might be considered a discovery? Hadn’t Pasteur developed an attenuated Rabies vaccine in 1885? And, what of Jenner’s earlier 1798 smallpox vaccine, comprised of live cowpox virus?

To the above points, Sven Gard, at the Karolinska Institute, and a member of the Nobel committee for Physiology or Medicine, wrote the following in his evaluation of Theiler’s prior 1948 Nobel nomination: “Theiler can not be said to have been pioneering. He has not enriched the field of virus research with any new and epoch-making methods or presented principally new solutions to the problems, but he has shown an exceptional capacity to grasp the essentials of the observations, his own and others, and with safe intuition follow the path that led to the goal.”

Despite the seeming inconsistency between Gard’s comments and Nobel’s instruction that the prize be awarded for a discovery, Gard nonetheless concluded that Theiler’s contributions indeed merited the Nobel award. [Incidentally, Theiler’s 1948 Nobel nomination was a detailed six-page-long document, written and submitted on his behalf by Albert Sabin!]

To the same point, Hilding Bergstrand, also at the Karolinska Institutet, and chairman of the Nobel Committee for Physiology and Medicine, said the following during his otherwise laudatory speech honoring Theiler at the 1951 Nobel Prize ceremony: “The significance of Max Theiler’s discovery must be considered to be very great from the practical point of view, as effective protection against yellow fever is one condition for the development of the tropical regions—an important problem in an overpopulated world. Dr. Theiler’s discovery does not imply anything fundamentally new, for the idea of inoculation against a disease by the use of a variant of the etiological agent which, though harmless, produces immunity, is more than 150 years old.”

Even Theiler himself agreed that he had not done anything fundamentally new. But then, what might Bergstrand have had in mind when referring to Theiler’s discovery? Perhaps it was Theiler’s finding that passage of the Asibi strain of yellow fever virus in chick embryos, which were devoid of nervous systems, generated viable, non-neurotropic attenuated yellow fever virus. If so, then did that discovery fulfill the condition for the Nobel award, as specified by Alfred Nobel? And, if that is the case, then might this discovery have been what makes Theiler’s contribution more worthy than those of Salk and Sabin in the eyes of the Nobel committee? [A more detailed account of Max Theiler’s yellow fever vaccine, particularly with regard to the “discovery” noted here, can be found in The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiller, now on the blog.]

The seemingly trivial distinction between the worthiness of Theiler’s contribution from that of Salk and Sabin, suggests that we may need to look elsewhere for answers to why Salk and Sabin were bypassed by the Nobel committee. One reason suggested in the case of Salk is that in the elitist world of big-time science, he had never spent time at a prestigious Research institution like the Rockefeller. Yet he did carry out postgraduate studies in association with the eminent Thomas Francis. So perhaps he was passed over by the Nobel committee because it did not see anything innovative about his vaccine. Or, perhaps it was because he allowed himself to be promoted as a celebrity by the March of Dimes, thereby causing resentment among his colleagues.

But, how then might we explain the case of Sabin? Sabin had not been used by the National Foundation to promote its fund-raising. And, he had done research at the Rockefeller Institute. Moreover, Sabin made seminal contributions to the poliovirus field before and after beginning his vaccine work. As noted above, Sabin and Peter Olitsky demonstrated that poliovirus could be grown in cultured human embryonic nervous tissue. Moreover, Sabin provided experimental evidence that the poliovirus port of entry is the digestive tract, rather than the respiratory tract, as was previously thought. And, Sabin established that the incidence of poliomyelitis tended to be highest in urban populations which had the highest standards of sanitation.

[Aside: Sabin’s finding, that the poliovirus route of entry is via the alimentary tract, validated the premise that poliomyelitis might be prevented by a live oral vaccine. In contrast, Salk’s killed vaccine needed to be injected. An advantage of a vaccine being administered by the oral route, particularly in developing countries, is that trained medical personnel are not required for its administration. On the other hand, the killed vaccine is safer. The few cases of poliomyelitis that now occur in the West are vaccine-related, resulting from rare reversions to virulence of the attenuated virus.]

[Aside: Why was the incidence of poliomyelitis highest in urban populations that had the highest standards of hygiene? Polio infection tends to be milder in the very young, perhaps because they are partially protected by maternal antibodies. But, in areas with high standards of hygiene, infection tends to occur later in life, when maternal antibodies have waned, and the infection can then be more severe.

Before this was appreciated, poliomyelitis was thought to originate in the slums and tenements of cities, and then spread to the cleaner middle-class neighborhoods. Thus, during polio outbreaks in New York City, there were instances when slums and tenements were quarantined, and city dwellers fled to the suburbs, all to no avail.]

Were Sabin’s discoveries noted above, taken together with his vaccine, worthy of a Nobel Prize? In any case, Sabin indeed had been nominated for the Nobel award by numerous colleagues, including Enders. So, why was Sabin never awarded the Nobel Prize? Perhaps the Nobel committee could not recognize Sabin without also recognizing Salk, which it may have been reluctant to do for reasons noted above. Or, as has been suggested, the continual back-and-forth carping between supporters of Salk and Sabin may ultimately have diminished enthusiasm in Stockholm for both of them.

Salk (in 1956) and Sabin (in 1965) each received the prestigious Lasker Award for Clinical Research (often seen as a prelude to the Nobel) and, earlier, in 1951, Sabin was elected to the U.S. National Academy of Sciences. In contrast, Salk was the only prominent polio researcher not elected to the Academy. And regarding the Nobel Prize, Salk once joked that he didn’t need it, since most people thought he had already won it.

In 1963 Salk founded the prestigious Salk Institute for Biological Studies in La Jolla, California. Francis Crick (1), Renato Dulbecco (2), and Leo Szilard (3), each of whom is featured elsewhere on the blog, were among the eminent scientists recruited by Salk to the La Jolla campus. Bearing in mind Salk’s alienation from other medical researchers of the day, we might enjoy his remark “I couldn’t possibly have become a member of this institute if I hadn’t founded it myself.” Jonas Salk died of congestive heart failure in 1995 at the age of 80. He remains one of the most venerated medical scientists ever.

salk instSalk Institute for Biological Studies

[Aside: Salk married Dora Lindsay in 1939, right after he graduated from NYU medical school. But, the marriage eventually fell apart, and the couple divorced in 1968.

In 1970, Salk married the artist Francois Gilot, who had been the mistress of Pablo Picasso for nearly ten years and with whom she had two children. Salk and Gilot met in 1969, at the home of a mutual friend in Los Angeles. They remained married until Salk’s death in 1995.

The following is from an April 27, 2012 article in Vogue by Dodie Kazanjian, entitled Life after Picasso: Francois Gilot.

“On a trip to Los Angeles in 1969, a friend introduced her to Jonas Salk. She had no interest in meeting him—she thought scientists were boring. But soon afterward, he came to New York and invited her to have tea at Rumplemayer’s. ‘He didn’t have tea; he ordered pistachio and tangerine ice cream,’ she recalls. ‘I thought, Well, a scientist who orders pistachio and tangerine ice cream at five o’clock in the afternoon is not like everybody else!’ He pursued her to Paris and a few months later asked her to marry him. She balked. “I said, ‘I just don’t need to be married,’ and he said, ‘In my position, I cannot not be married.’ He gave me two pieces of paper and told me to write down the reasons why I didn’t want to get married.” She complied. Her list included: ‘I can’t live more than six months with one person’; ‘I have my own children’; ‘I have my career as a painter and have to go here and there’; ‘I’m not always in the mood to talk. Et cetera, et cetera, et cetera.’

Salk looked at the list and said he found it ‘quite congenial.’ They were married in 1970 and were together until he died in 1995. ‘It worked very well,’ she says, because after all we got along very well.’”]

Albert Sabin became president of the prestigious Weizmann Institute of Science in Israel, but stepped down in November 1972 for health reasons. He passed away in 1993 at the age of 86. Unlike in the case of Salk, and despite the fact that he never was awarded the Nobel Prize, Sabin’s standing among his colleagues always remained high.

Before concluding, we note two other important contenders in the quest for a polio vaccine. The first of these was Isabel Morgan, the daughter of the great geneticist, Thomas Hunt Morgan. Isabel Morgan nearly produced a killed polio vaccine before Salk succeeded in doing so. Working at Johns Hopkins, she generated formalin-inactivated poliovirus preparations that indeed protected monkeys against intracerebral injections of live poliovirus. However, Morgan gave up her research in 1949 to marry and raise a family. At that time, Salk had barely begun his work. But, if Morgan had remained in the race, Salk may yet have beaten her to the finish line, since she was reluctant to test her vaccine on human subjects.

Hilary Koprowski was the other noteworthy contender in the race to a polio vaccine. Koprowski was a Polish Jew who immigrated to Brazil in 1939, after Germany invaded Poland. He later came to the United States, where, in 1945, he was hired by Lederle Laboratories to work on a project to develop a live polio vaccine. Koprowski’s foray into polio had a few interesting happenings. Moreover, he went on to have a renowned career as a virologist. Thus, we discuss him in a bit more detail.

[Aside: Salk and Sabin also were Jewish. And Sabin too was born in Poland. In 1921 he immigrated with his family to the United States, at least partly to escape persecution of Jews in his birth-land.]

Koprowski began his work at Lederle before John Enders developed methods for growing poliovirus in monkey kidney cell cultures. Consequently, Koprowski attenuated his live vaccine by passaging it in mouse brains in vivo. In 1950, several years before Sabin’s vaccine was ready for testing, Koprowski found that his vaccine indeed protected chimpanzees from challenge with virulent poliovirus. Koprowski then tested his live vaccine in humans; first on himself, and then on 19 children at a New York State home for “feeble minded” children.

Koprowski was still an unknown figure in the scientific community when he made the first public presentation his test findings. This happened at a 1951 National Foundation roundtable that was attended by the major polio researchers of the day, including Salk and Sabin. The conferees were aghast upon hearing that Koprowski had actually tested his live vaccine, grown in animal nerve tissue, on children. Koprowski’s response was simply that someone had to take that step. Also, it didn’t help Koprowski’s standing with his academic colleagues that he was employed by Lederle. In those pre-biotech days, he was looked down on as a “commercial scientist.”

Human testing was of course a necessary step in the development of this or any human vaccine. What’s more, using cognitively disabled children as test subjects was a common practice back then. So, the actual concern of Koprowski’s colleagues was that he inoculated human subjects with a vaccine that was grown in animal brains. Koprowski also may have been treading on shaky legal ground, since it is not clear whether he ever obtained consent from the children’s parents.

[Aside: The only guidelines for such tests back then were the so-called Nuremburg Code of 1947, which was formulated in response to Nazi “medical” experiments. Informed consent was one of the Nuremburg guidelines, which, in the case of children, meant consent from a parent or guardian. Note that federal approval was not required to test vaccines or drugs in those days.]

Irrespective of whatever uproar Koprowski caused by testing his vaccine on helpless institutionalized children, he indeed had a live polio vaccine in 1949; several years before Salk and Sabin brought out their vaccines. However, Koprowski’s vaccine began its demise soon afterwards. A small field trial in Belfast showed that the attenuated virus could revert to a virulent form after inoculation into humans. But, bearing in mind that there was not yet any alternative to his vaccine, Koprowski firmly believed that the greater risks of natural poliovirus infections justified its use.

The fate of Koprowski’s vaccine was sealed in 1960, when the U.S. Surgeon General approved the Sabin vaccine for trial manufacture in the United States, while rejecting Koprowski’s vaccine on safety grounds. Tests showed that Sabin’s vaccine was the less neurovirulent of the two vaccines in monkeys. Sabin had carefully tested plaque-isolated clones of his attenuated viral populations for neurovirulence in monkeys, and he then assembled his vaccine from the least neurovirulent of these clones. Moreover, by this time millions of children in the Soviet Union had had been successfully immunized with the Sabin vaccine.

Koprowski left Lederle Laboratories in 1957 after clashing with its management. After that, he became Director of the Wister Institute in Philadelphia. He then transformed the then moribund Wistar into a first class research organization.

The relationship between Koprowski and Sabin was quite adversarial at the time their vaccines were in competition, but they later became friends. In 1976, Koprowski was elected to the U.S. National Academy of Sciences, an honor shared with Sabin, bit never afforded to Salk.

Here is one last bit on Koprowski. Recall that early lots of the Salk and the Sabin vaccines unknowingly contained live SV40, which had been injected into hundreds millions of people worldwide. While the unknown presence of a live tumor virus in a vaccine must be one of a vaccinologist’s worst nightmares, this finding did not attract the attention of the public. In contrast, a 1992 article in Rolling Stone, which attributed the emergence of HIV to Koprowski’s polio vaccine, created a sensation. The premise of the article was that Koprowski’s vaccine was produced in chimpanzee cells that were contaminated with simian immunodeficiency virus (SIV), which then mutated into HIV when inoculated into humans. As might be expected, there was no evidence to support that premise. Indeed, PCR analysis could not detect SIV or HIV in the supposedly contaminated vaccine lots, and records from Koprowski’s laboratory showed that his vaccine was never grown in chimpanzee cells. So, faced with the possibility of a lawsuit, Rolling Stone issued a retraction.

Readers, who enjoyed the above account of the rivalry between Jonas Salk and Albert Sabin, may also enjoy the account of the rivalry between Robert Gallo and Luc Montagnier in Who Discovered HIV? More on the same topic can be found in How the Human Immunodeficiency Virus (HIV) Got its Name. For a very different kind of rivalry, that between Howard Temin and David Baltimore, see Howard Temin: In From the Cold.

1. Howard Temin: “In from the Cold” On the blog.

2. Renato Dulbecco and the Beginnings of Quantitative Animal Virology On the blog.

3. Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis On the blog.