Tag Archives: Maurice Hilleman

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/

 

 

Advertisements

SV40-Contaminated Polio Vaccines and Human Cancer

In earlier postings, we recounted how in 1959 Bernice Eddy, at the U. S. National Institutes of Health (NIH), and then Maurice Hilleman, at Merck & Co, discovered a new virus, simian virus 40 (SV40), in early lots of the Salk and Sabin polio vaccines (1, 2). The virus inadvertently contaminated those vaccines because it was unknowingly present in the rhesus monkey kidney cell cultures in which the vaccines were grown. Hilleman gave SV40 its name. It was the 40th simian virus that the Merck lab found in its rhesus kidney cell cultures.

Next, in 1961, both Eddy and Hilleman discovered that inoculating SV40 into hamsters caused cancer in about half of the animals. But, by then, hundreds of millions of people worldwide had been inoculated with live SV40, via the contaminated polio vaccines!

[Aside 1: Since the Sabin vaccine contains live, attenuated poliovirus, whereas the Salk vaccine contained formaldehyde-inactivated poliovirus, it was initially thought that any issues stemming from SV40 contamination would be limited to the Sabin vaccine. However, about one in 10,000 SV40 particles survived the formaldehyde treatment used to inactivate poliovirus in the Salk vaccine. Thus, early lots of both the Salk vaccine and the Sabin vaccine were contaminated with live SV40. But, despite the initial belief that that only the Sabin vaccine posed a threat of SV40 contamination, recipients of the Salk vaccine, but not those receiving the Sabin vaccine, developed an antibody response against SV40. Apparently, any SV40 that may have been present in the orally administered Sabin vaccine was killed in the intestinal tracts of its recipients. But, while the antibody response seen in recipients of the injection-administered Salk vaccine would be consistent with an actual SV40 infection, that response might have been only against the SV40 in the vaccine itself (see the main text).]

In July 1961, the New York Times broke part of this story, reporting that Merck was withdrawing its vaccines because they were contaminated with a monkey virus. However, the Times article did not mention the cancer connection. In fact, the Times did not report that aspect of the story until more than a year later, causing some individuals to suspect that the government deliberately withheld that information from the public.

I do not know whether or not there was a deliberate intent by the government to withhold information. Joseph Smadel, Bernice Eddy’s immediate superior at the NIH and himself an eminent scientist,  dismissed Eddy’s finding of a tumor-inducing virus in early lots of the Salk vaccine, after he reviewed her data. Later, after Eddy reported her findings at an October 1960 cancer conference in New York, Smadel forbade her from speaking about the matter again in public without first clearing her remarks with him. However, Smadel was indeed concerned when similar findings were reported to him later by Hilleman. Was sexism a factor in the dismissal of Eddy’s results? Perhaps, but it also was alleged (in fact by Hilleman) that Eddy’s experiments were poorly controlled.

At any rate, Eddy had, in fact, presented her findings at an open scientific conference. What’s more, at around the same time, Hilleman too presented his findings in public, at a conference in Copenhagen. So, their discovery, while not purposely publicized, was not kept secret either.

Next, consider that the second report in the New York Times, which mentioned the cancer connection for the first time, was buried on page 27 of the newspaper. This fact points more to the inability of the press to appreciate and cover a complex scientific issue, than to an attempt by the government to suppress the story.

In any event, the government did not alert the public to the possible danger that might be lurking in the polio vaccines. Is it possible that the NIH did not appreciate that threat? This seemingly implausible explanation is consistent with the fact that the NIH did not begin to screen all new lots of the polio vaccines for SV40 until 1963.

Another perhaps more likely possibility is that the government simply believed that alerting the public might have irrevocably broken its confidence in the vaccines, ultimately causing far more disease than might have been caused by the vaccines themselves. Even so, an impending public health debacle, of unprecedented severity, could not yet have been ruled out.

We return to our main story after a bit of background on SV40.

SV40 is a member of the polyomavirus family of small, double-stranded DNA viruses. It is one of several polyomaviruses that can transform normal cells into tumor cells in cell culture, as well as induce tumors in laboratory animals. For that reason, and because SV40 is a relatively simple virus (its genome contains only about 5,200 bases pairs), it was intensively studied for what it might reveal about tumor genesis. Moreover, it also came to serve as an important model to investigate fundamental issues in eukaryotic molecular biology (3).

The polyomaviruses are widespread in their natural hosts, in which they give rise to lifelong, usually benign, persistent infections. The Asian rhesus macaque is the natural host for SV40. These facts explain why SV40 was not evident in the rhesus monkey kidney cell cultures that were used to propagate the early poliovirus vaccine lots. SV40 does not cause sufficient cytopathology in those rhesus macaque cell cultures to reveal its presence in them. However, culture fluids from those rhesus cell cultures caused extensive cytopathology when added to African green monkey kidney cell cultures. Indeed, that is how SV40 was discovered.

[Aside 2: The Human Polyomaviruses The JC polyomavirus (JCPyV) and the BK polyomavirus (BKPyV), each discovered in 1971, are the best known polyomaviruses that naturally infect humans. JCPyV and BKPyV are each ubiquitous in their human host, in which they typically give rise to lifelong, benign, persistent infections. Yet, JCPyV can give rise to a rare but fatal demyelinating disease, progressive multifocal encephalopathy (PML), in immunologically compromised individuals. And BKPyV can cause kidney disease, also in immunologically compromised persons.

More recently, several additional human polyomaviruses have been discovered, by means of modern DNA amplification procedures. One of these viruses, the ubiquitous Merkel cell polyomavirus (MCPyV), is associated with a rare, aggressive human malignancy, Merkel cell carcinoma, and is the best candidate for an oncogenic human polyomavirus.]

We now resume our main story, with the following key points.

Despite the fact that the unintended exposure of millions of individuals to SV40 via the contaminated polio vaccines in the 1950s posed a potential public health crisis of immense proportions, it still is not clear whether SV40 is an agent of human disease. Moreover, it is not known whether SV40 is circulating in the human population. How can this be?

Early investigations into this matter in the 1960s were compromised by the fact that it was not apparent which individuals had actually received SV40-contaminated vaccines and which did not. That was so in part because the serological reagents and procedures of the day were not sensitive or accurate enough to generate unambiguous results. Additionally, population sample sizes were often too small to generate statistically significant results. [This was especially so in the case of the rare childhood tumors in which SV40 had been implicated.] And, since cancer is a disease that may take decades to emerge, it was possible that more time needed to pass before the virus might unequivocally reveal itself as a cause of human cancer. At any rate, since the substantial experimental data then available could neither establish nor absolve SV40 as a cause of cancer in humans, the NIH conceded that more research and better methods for detecting the virus would be needed to settle the issue.

The more recent development of extremely sensitive polymerase chain reaction (PCR)-based procedures, which can detect minute levels of specific DNA sequences, led to renewed interest in whether SV40 might be present in humans, and whether it might be an agent of human disease. Using PCR technology, several different research groups detected SV40 DNA in four types of human cancers; mesotheliomas, osteosarcomas, non-Hodgkin’s lymphomas, and childhood brain tumors. These findings were alarming because the four tumor types, in which SV40 was detected in humans, are the same tumors that SV40 induces experimentally in hamsters (i.e., mesothelioma, bone, lymphoma, and brain).

PCR procedures also detected SV40 DNA in individuals who never were inoculated with an SV40-contaminated vaccine. This too was disturbing because it raised the specter that SV40 might be circulating in the human population, spreading by horizontal transmission from one individual to another.

Yet the issue of SV40 in humans remains controversial because other studies, from other research groups, using similar PCR procedures, could not detect SV40 DNA in human tissues. In addition, newer, more sensitive and accurate serologic procedures could not demonstrate to everyone’s satisfaction that SV40 circulates in humans.

How might we explain how capable scientists, using powerful and proven techniques, can obtain such disparate experimental results? Ironically, the sensitivity of PCR itself may be a problem, since it increases the likelihood of false-positive results, which may occur from the slightest sample contamination. Thus, it is important to have suitable positive and negative control samples that might be processed side-by-side with test samples; a sometimes difficult criterion to fulfill. [Bearing the above in mind, consider the following example, in which a human mesothelioma sample was micro-dissected to separate normal tissue from the actual tumor. SV40 sequences were detected in the tumor, but not in the adjacent normal tissue, which served as an internal control.]

Another potential source of error stems from the widespread prevalence of human polyomaviruses (e.g., JCPyV, BKPyV, and MCPyV) in the human population, leaving open the possibility that these viruses, rather than SV40, are detected by the PCR-based procedures. But, with that possibility in mind, several researchers took the extra step of confirming the presence of SV40 sequences by direct sequencing of the PCR-amplified DNA. The ubiquitous human polyomaviruses are also a concern when carrying out serological procedures, since immune cross-reactivity between SV40 and these viruses remains a potential source of error.

Another problem is theoretical rather than technical. An underlying premise behind these studies is that the continued presence and expression of polyomaviral tumor genes are necessary for a tumor cell to express its tumor cell characteristics. Indeed, this was shown to be the case fifty years ago for cells transformed in culture by polyomaviruses. Thus, the absence of SV40 DNA, or SV40 tumor antigens, in a tumor is taken as evidence against SV40 as the cause of the tumor. However, there is some experimental evidence that the paradigm itself may not always be true. Cancers result from a complex multistage course of events and, in some instances, the virus may play a necessary role only at a particular point in the overall process. Thus, the absence of SV40 DNA, or SV40 antigens, in a tumor may not be definitive proof against viral involvement in the tumor process.

The above points help us to appreciate why there is no consensus regarding a role for SV40 in human cancer, and indeed whether SV40 might be circulating in humans. Yet we remain troubled by the fact that SV40 can transform a variety of cells in culture and can induce tumors in laboratory animals. And there are additional experimental findings that while contentious, cannot be easily ignored. For instance, the types of human cancers, in which SV40 DNA was detected by some researchers, are the same types of tumors that SV40 induces in laboratory animals. Also, infectious SV40 was isolated from a brain cancer of a 4-year-old child. [LT/pRb and LT/p53 complexes were identified in human brain tumors, consistent with current understanding of how SV40 induces neoplasia (3).]

Yet, notwithstanding the force of the above arguments, impressive evidence has been presented against a role for SV40 in human cancer. And, if SV40 indeed were responsible for human cancer on a large scale, then it is rather certain that there would be little if any uncertainty in that regard.

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

2. Maurice Hilleman: Unsung Giant of Vaccinology, posted on the blog, April 24, 2014

3. Virology: Molecular Biology and Pathogenesis, Leonard C. Norkin, ASM Press, 2010.

The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler

The first part of this posting tells how a U.S. Army medical board, headed by Walter Reed, confirmed that the transmission of yellow fever requires a mosquito vector. The second part tells the story of the yellow fever vaccine developed by Max Theiler.

Bearing in mind the enormous benefit to mankind of the polio vaccines developed by Jonas Salk and Albert Sabin (1), and that Maurice Hilleman developed nearly 40 vaccines, including those for measles, mumps, and rubella (2), it would appear remarkable that Theiler was the only one of these four individuals to be recognized by the Nobel committee. In fact, Theiler’s 1951 Nobel award was the only one ever given for a vaccine! In any case, while Theiler’s vaccine was a major step forward in the fight against yellow fever, it came after a perhaps more dramatic episode in the struggle against that malady. But first, we begin with some background.

Yellow fever was another of mankind’s great scourges. Indeed, it was once the most feared infectious disease in the United States. And, while we might want to say that science has “conquered” yellow fever, that statement would not be entirely accurate. Unlike polio and measles, which have nearly been eradicated by the vaccines against them, that is not so for yellow fever. The reason is as follows. Humans are the only host for polio and measles viruses. Consequently, those viruses might be completely eradicated if a sufficient percentage of humans were to comply with vaccination regimens. In contrast, the yellow fever virus infects monkeys that range over thousands of square miles in Africa and the Amazon jungle. Thus, even with massive vaccination of humans, it would be impossible to eliminate the yellow fever virus from the world.

According to the World Health Organization’s estimates, there are still about 200,000 cases of yellow fever per year, resulting in about 30,000 deaths, about 90% of which occur in Africa. The yellow fever virus itself is the prototype virus of the flavivirus family of single-stranded RNA viruses, which also includes dengue hemorrhagic fever virus, Japanese encephalitis virus, and West Nile encephalitis virus, among others.

yellow fever map

Yellow fever is somewhat unique among the viral hemorrhagic fevers in that the liver is the major target organ. Consequently, the severe form of yellow fever infection is characterized by hemorrhage of the liver and severe jaundice. But, as in infections caused by other virulent viruses, most cases of yellow fever are mild.

Interestingly, the name “yellow fever” does not have its origin in the yellowing of the skin and eyes that is characteristic of severe disease. Instead, it has its origin in the term “yellow jack,” which refers to the yellow flag that was flown in port to warn approaching ships of the presence of infectious disease.

Yellow fever originated in Africa. It is believed to have been brought to the New World by slave ships in the year 1596. As noted above (and discussed below), yellow fever transmission, from an infected individual or primate to an uninfected one, requires a specific vector, the Aedes aegypti mosquito. The sailing ships of the day inadvertently transported the disease across oceans via the mosquito larvae in their water casks.

Before getting to our stories proper, we note a pair of intriguing instances in which yellow fever profoundly affected New World history. In the first of these, yellow fever was a key factor that led Napoleon to sell the Louisiana Territory to the United States in 1803; an act that doubled the size of the United States. It happened as follows. After Napoleon seized power in France, he reinstated slavery in the French colony of Saint Domingue (now Haiti); doing so for the benefit of the French plantation owners there. In response, the rather remarkable Toussaint Breda (later called Toussaint L’Ouverture, and sometimes the “black Napoleon”) led a slave revolt against the plantation owners. In turn, in February 1802, Napoleon dispatched an expeditionary force of about 65,000 men to Haiti to put down the revolt. The rebellious slaves, many fewer in number than the French, cleverly retreated to the hills, believing that the upcoming yellow fever season would wreak havoc on the French force. And, they were correct. By November 1803, the French lost 50,000 of the original 65,000 men to yellow fever and malaria. Thus, in 1804, Napoleon had to allow Haiti to proclaim its independence, and then become the second republic in the Western Hemisphere. Moreover, there is evidence suggesting that Napoleon’s actual purpose in dispatching the expeditionary force was to secure control of France’s North American holdings. With his expeditionary force decimated by yellow fever and malaria, that was no longer possible and, consequently, Napoleon sold France’s North American holdings (the Louisiana Purchase) to the United States.

louisiana purchaseThe Louisiana Purchase, in green.

Second, in 1882, France began its attempt to build a canal across the Isthmus of Panama. However, thousands of French workers succumbed to yellow fever, causing France to abandon the project. The United States was able to successfully take up the task in 1904; thanks to the deeds of the individuals in part I of our story, which now begins.

In May 1900, neither the cause of yellow fever, nor its mode of transmission was known. At that time, U.S. Army surgeon, Major Walter Reed, was appointed president of a U.S. Army medical board assigned to study infectious diseases in Cuba, with particular emphasis on yellow fever. Cuba was then thought to be a major source of yellow fever epidemics in the United States; a belief that was said to have been a factor in the American annexation of Cuba.

ReedMajor Walter Reed

When Reed’s board began its inquiry, a prevailing hypothesis was that yellow fever might be caused by the bacterium Bacillus icteroides. However the board was unable to find any evidence in support of that notion.

Another hypothesis, which was advanced by Cuban physician Dr. Carlos Juan Finlay, suggested that whatever the infectious yellow fever agent might be, transmission to humans requires a vector; specifically, the mosquito now known as Aedes aegypti. Reed was sympathetic to this idea because he noticed that people who ministered to yellow fever patients had no increased risk of contracting the disease, which indicated to Reed that people did not pass yellow fever directly from one to another.

Reed, as president of the medical board, is generally given major credit for unraveling the epidemiology of yellow fever. Yet there were other heroes in this story as well. Finlay, whose advice and experience were invaluable to Reed’s board, was one. He was the object of much ridicule for championing the mosquito hypothesis, at a time when there little evidence that might support it. In any case, Reed, in his journal articles and personal correspondences, gave full credit to Finlay for the mosquito hypothesis.

Acting Assistant Surgeon Major James Carroll was another hero. As a member of Reed’s board, Carroll volunteered to be bitten and, promptly, developed yellow fever. Major Jesse Lazear, also a board member, asked Private William Dean if he might be willing to be bitten. Dean consented, and he too contracted yellow fever. Fortunately, Dean and Carroll each recovered. Not so for Lazear. After allowing himself to be bitten, he died after several days of delirium.

Lazear’s contribution to gaining recognition of the mosquito hypothesis went significantly beyond his tragic martyrdom. When Reed examined Lazear’s notebook after his death, Reed found that it contained several key observations. First, Lazear had carefully documented that in order for a mosquito to be infected; it had to bite a yellow fever patient within the first three days of the patient’s illness. Second, twelve days then had to elapse before the virus could reach high enough levels in the insect’s salivary glands to be transmitted to a new victim.

The observations of the board, up to then, convinced Reed and the others that the mosquito hypothesis indeed was correct. Yet Reed knew that more extensive controlled experiments would be needed to convince the medical community. So, he directly supervised those experiments, which involved twenty-four more volunteers, each of whom may rightly be considered a hero.

Just as Reed benefited from Finlay’s insights, William C. Gorgas, Surgeon General of the U.S. Army, applied the findings of Reed’s board to develop vector control measures to combat urban yellow fever; first in Florida, then in Havana, Cuba, and next in Panama, where those measures enabled the United States to complete the canal in 1914. The last urban yellow fever outbreak in the United States occurred in New Orleans in 1905, and the last in the New World occurred in 1999 in Bolivia.

The vector control strategy works for urban yellow fever because the Aedes aegypti mosquitoes have a very short flight range and, consequently, the female mosquito does not stray far from the source of her blood meal before laying her eggs. Thus, it is only necessary to control the vector population in the immediate vicinity of human habitation. In practice, this is accomplished by draining potential mosquito breeding sites such as swamps and ditches, and destroying water-collecting objects such as discarded tires.

After Reed’s board was disbanded, he made yet another key contribution to the wiping out of yellow fever. The focus of the board had been on the means of yellow fever transmission; not with the infectious agent itself. In 1901, at the suggestion of William Welch, an eminent Johns Hopkins pathologist, Reed and James Carroll (who nearly died of yellow fever after being experimentally infected while in Cuba), asked whether yellow fever might be caused by a filterable virus. Indeed, they found that they could infect volunteers by inoculating them with filtered serum taken from yellow fever patients. What’s more, theirs was the very first demonstration of a human illness being caused by a filterable agent. That is, yellow fever was the first human illness shown to be caused by a virus. [Pasteur developed an attenuated rabies vaccine in 1885, more than a decade before the discovery of viruses. Remarkably, this most brilliant of experimentalists did not recognize that he was dealing with a previously unknown, fundamentally distinct type of infectious agent; the topic of a future posting.]

[Aside: Walter Reed spent the early years of his Army career at different posts in the American west. The Mount Vernon Barracks in Alabama, which served as a prison for captured Apache Native Americans, including Geronimo, was a particularly interesting stop for Reed. Captain Walter Reed, serving as post surgeon in the 1880s, looked after Geronimo and his followers.]

Part II of this posting concerns the development of Max Theiler’s yellow fever vaccine. But first, here is a bit more background.

Vector control measures ended yellow fever epidemics in most, but not all urban centers worldwide. Outbreaks have not occurred in the United States for more than a century. However, jungle yellow fever still persists in areas of Sub-Saharan Africa and, to a lesser extent, in tropical South America. Individuals who are infected in the jungle by wild mosquitoes can then carry the virus to densely populated urban areas, where Aedes aegypti mosquitoes can transmit the virus from one individual to another. [Vector-mediated, human-to-human transmission happens because the level of yellow fever virus in the blood of an infected person becomes high enough for the infected person to transmit the virus to a biting mosquito. In this regard, the yellow fever virus is an exception to the generalization that humans are a “dead end” host for arthropod-borne (arbo) viruses.]

Fortunately, people who live in high risk areas for yellow fever can be protected by vaccination. Indeed, the World Health Organization’s strategy for preventing yellow fever epidemics in high risk areas is, first, to mass immunize the population, and then to routinely immunize infants. [Vaccinated American or European visitors to West Africa or the Amazon need not be concerned about yellow fever. However, the risk to an unvaccinated person of acquiring yellow fever during a two-week stay at the height of the transmission season (July through October), is estimated to be 5%. Individuals wanting to enter or return from countries where yellow fever is endemic may need to show a valid certificate of vaccination. ]

Part II of our story, concerning Max Theiler and the development of the yellow fever vaccine now begins.

Even as late as the 1920s, some reputable bacteriologists remained unconvinced by the earlier findings of Reed and Carroll that yellow fever is caused by a filterable agent. Instead, they persisted in the belief that the illness is caused by a bacterium. The notion of a bacterial etiology for yellow fever was finally put to rest after A. H. Mahaffy in 1927 discovered that the yellow fever agent could be propagated and cause illness in Asian rhesus monkeys. With an experimental animal now at hand, yellow fever workers were able to prove conclusively that the disease is caused by a virus. [Mahaffy drew the virus he used in his experiments from a 28-year-old African man named Asibi, who was mildly sick with yellow fever. That isolate, referred to as the Asibi strain, will play an important role later in this anecdote.]

Regardless of the significance of the discovery that the yellow fever virus could be propagated in rhesus monkeys, Max Theiler had to contend with the fact that these monkeys were quite expensive; especially for a not yet established young investigator. [They cost the then princely sum of about $7.00 apiece.] As for mice, while they could be bred for pennies apiece, other researchers were not able infect them via the usual practice of inoculating them under the skin or in the abdomen. However, Theiler took a cue from Pasteur’s inability to propagate the rabies virus in laboratory rabbits until he put the virus directly into their brains. Thus, in 1929 Theiler attempted to do the same with yellow fever virus in mice.

TheilerlMax Theiler

Theiler’s attempts to infect the mice by intracranial injection were a success. All of the inoculated mice died within several days. Surprisingly, the dead mice did not display the liver or renal pathology characteristic of yellow fever. Instead, the mice appeared to have succumbed to inflammation of their brains. Thus, the yellow fever virus appeared to be neurotropic in mice. Also, Theiler himself contracted yellow fever from one of his inoculated mice. He was fortunate to survive.

A fortuitous result of Theiler’s perilous bout with yellow fever was that he had become immune to the virus, as revealed by the presence of antiviral antibodies in his blood. Importantly, Theiler’s acquired immunity to the virus validated the possibility of developing an attenuated yellow fever vaccine. And, in a sense, Theiler was inadvertently the first recipient of the nascent vaccine he soon would be developing.

Theiler also determined that the virus could be passed from one mouse to another. And, while the virus continued to cause encephalitis in mice, it caused yellow fever when inoculated back into monkeys; quite a unique and striking set of findings. But, and crucially significant, while continued passage of the virus in mice led to its increased virulence in those animals, the virus was concurrently losing its virulence in monkeys. [In 1930, Theiler moved from the Harvard University School of Tropical Medicine to the Rockefeller Foundation’s Division of Biological and Medical Research. The Rockefeller Foundation shared facilities with the Rockefeller Institute (now University); although it was otherwise administratively separate from it.]

Since the mouse-passed virus was becoming attenuated in monkeys, Theiler’s belief in the possibility of generating an attenuated yellow fever vaccine was bearing out. However, because the mouse-passed virus remained neurovirulent in mice, Theiler was reluctant to inoculate that virus into humans. In an attempt to solve this problem, Theiler turned from passing the virus in the brains of live mice and, instead, began passing the virus in mouse tissue cultures.

Theiler carried out seventeen different sets of trials to further attenuate the virus. In the 17th of these, Theiler used the wild Asibi strain, isolated earlier by Mahaffy. Initially, this virus was extremely virulent in monkeys, in which it caused severe liver damage. But, after passing the virus from culture to culture several hundred times, over a period of three years, a flask labeled 17D yielded the virus that was to become the famous 17D yellow fever vaccine.

Theiler never gave a satisfactory accounting for the “D” in the “17D” designation, and for what, if anything became of A, B, and C. Regardless, the genesis of 17D was as follows. Theiler initially took an Asibi sample that had been multiplying in mouse embryo tissue and continued passing it in three separate types of minced chicken embryo cultures. One of these sets contained whole minced chicken embryos, and was designated 17D (WC). A second set contained chick embryo brain only, and was designated 17D (CEB). In the third set, the brains and spinal cords were removed from the otherwise whole chick embryo tissue cultures. This set, alone among all the sets, generated an attenuated virus that did not induce encephalitis when injected directly into monkey brains. Indeed, Theiler removed the central nervous systems from the chicken tissue in this set of cultures, in the express hope of generating just such an attenuated virus. And, by hook or by crook, the virus emerging from that particular set of passages became the vaccine that is now known simply as 17D.

Field tests of Theiler’s yellow fever vaccine were underway in 1937 in Brazil, and were successfully completed by 1940. In 1951 Theiler was awarded the Nobel Prize in Physiology or Medicine for developing the vaccine.

Next, we return to a point noted above, and discussed in two earlier postings. Neither Jonas Salk nor Albert Sabin were awarded Nobel prizes for developing their polio vaccines (1). And, Maurice Hilleman was never awarded a Nobel Prize, despite having developed nearly 40 vaccines, including those for measles, mumps, and rubella (2). Indeed, Max Theiler’s Nobel Prize for the yellow fever vaccine was the only Nobel Prize ever awarded for a vaccine! Why was that so?

Alfred Nobel, in his will, specified that the award for Physiology or Medicine shall be for a discovery per se; not for applied research, irrespective of its benefits to humanity. With that criterion in mind, the Nobel committee may have viewed the contributions of Salk and Sabin as derivative, requiring no additional discovery. [Hilleman’s basic discoveries regarding interferon should have been sufficient to earn him the award (2). The slight to him may have been because the Nobel committee was reluctant to give the award to an “industrial” scientist. Hilleman spent the major part of his career at Merck & Co.]

So, what was there about Theiler’s yellow fever vaccine that might be considered a discovery? Hadn’t Pasteur similarly developed an attenuated Rabies vaccine in 1885?

Perhaps the “discovery” was Theiler’s finding that passage of the Asibi strain of yellow fever virus in chick embryo cultures, which were devoid of nervous system tissue, generated attenuated yellow fever virus that was no longer neurovirulent in mice and monkeys. But, consider the following.

Theiler indeed believed that removing the brains and spinal cords from the chick embryo cultures in which 17D had been serially passed was the reason why the virus lost its neurovirulence. Nevertheless, as a serious scientist he needed to confirm this for himself. So, he repeated the long series of viral passages under the same conditions as before. But, this time, there was no loss of neurovirulence. Thus, a cause and effect relationship, between the absence of the brains and spinal cords from the tissue cultures and the emergence of non-neurovirulent virus, was not confirmed.

So, perhaps the Nobel committee merely paid lip service to the directives in Alfred Nobel’s will. In any case, Theiler’s 17D yellow fever vaccine has had a virtually unblemished safety record, and is regarded as one of the safest and most effective live-attenuated viral vaccines ever developed.

Theiler’s unshared 1951 Nobel award paid him $32,000. At the time, he resided in Hastings-on-Hudson; a village in Westchester County, NY, from which he commuted to the Rockefeller labs. Theiler’s next door neighbor in Hastings-on-Hudson was Alvin Dark, the star shortstop of the New York Giants. Nobel laureate Max Theiler was known to fellow commuters from Hastings-on-Hudson as the man who lives next door to Alvin Dark.

Virus Hunters, by Greer Williams (Alfred A, Knoff, 1960) was my major source for the material on Max Theiler.

1. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science. On the blog.

2. Maurice Hilleman: Unsung Giant of Vaccinology. On the blog.

 

 

 

Maurice Hilleman: Unsung Giant of Vaccinology

In January 2005, more than 100 of the world’s most renowned biomedical researchers got together to pay tribute to the 85-year-old Maurice Hilleman. When it was Hilleman’s turn to address the gathering, he alluded to them as his “peers in the world of science.” Referring to Hilleman’s gracious comment, science journalist Alan Dove wrote: “By any objective measure, a gathering of Maurice Hilleman’s scientific peers would not fill a telephone booth.” (1)

Hilleman truly was a giant in the history of virology. But, if you have only a vague idea of who Hilleman was or of his achievements, you are not alone. Anthony Fauci, director of the U.S. National Institutes of Allergy and Infectious Diseases, who was present at the gathering, noted: “Very few people, even in the scientific community, are even remotely aware of the scope of what Maurice has contributed….I recently asked my post-docs whether they knew who had developed the measles, mumps, rubella, hepatitis B and chickenpox vaccines. They had no idea,” Fauci said. “When I told them that it was Maurice Hilleman, they said, ‘Oh, you mean that grumpy guy who comes to all of the AIDS meetings?’”

hillemanMaurice R. Hilleman: The greatest vaccinologist.

Consider this. Hilleman developed nine of the 14 vaccines routinely recommended in current vaccine schedules. These are the vaccines for the measles, mumps, rubella, hepatitis A, hepatitis B, and chickenpox viruses, and for meningococcal , pneumococcal, and Haemophilus influenzae bacteria. Moreover, he was the first to forecast the arrival of the 1957 Asian flu and, in response, led the development of a flu vaccine that may have saved hundreds of thousands or more lives worldwide (2). And, independently of Robert Huebner and Wallace Rowe, he discovered cold-producing adenoviruses, and developed an adenovirus vaccine. Overall, Hilleman invented nearly 40 vaccines. And, he was a discoverer of simian virus 40 (SV40). If the above accomplishments were not enough to ensure his fame, he also was the first researcher to purify interferon, and the first to demonstrate that its expression is induced by double-stranded RNA.

[Aside: I first became aware of Maurice Hilleman 44 years ago. It was in the context of his 1959 discovery of SV40, which I came across only because I was beginning my post-doctoral studies of the related murine polyomavirus. Bernice Eddy, at the U. S. National Institutes of Health (NIH), was probably the first to discover SV40, which she detected in early lots of the Salk polio vaccine (3). Hillman, then at Merck & Co, independently discovered the same virus in rhesus monkey kidney cell cultures, in which the polio vaccine was being produced. Hilleman gave SV40 its name. It was the 40th simian virus the Merck lab found in the monkey kidney cells. In 1961, both Eddy and Hilleman found that inoculating SV40 into hamsters causes tumors in the animals. Merck withdrew its polio vaccine from the market. But, by then, live SV40 had been unknowingly injected into hundreds of millions of people worldwide! More on this in a future posting.]

We begin our account of Hilleman’s achievements with his development of the mumps vaccine. In the days before the vaccine, mumps struck about 200,000 children in the United States, annually. Yet except in rare circumstances, the infection was mild, and was generally regarded as a childhood rite of passage. There is a sweetness to the story of the mumps vaccine that I hope you might enjoy.

The tale began at about 1:00 AM, on March 21, 1963, when 5-year-old Jeryl Lynn Hilleman ambled into her father’s bedroom complaining of a sore throat. Jeryl Lynn’s father felt his daughter’s swollen glands, and knew in a flash that it was mumps. And, while I suspect that many lay parents back in the day would also have recognized Jeryl Lynn’s symptoms, few would have done what her father did after first comforting his daughter. Although it was already past midnight, Maurice hopped into his car and drove the 20 minutes to his lab at Merck & Co. to pick up some cotton swabs and beef broth. Returning home, he then awakened Jeryl Lynn, gently swabbed her throat, and immersed the swabs in the nutrient broth. Next, he drove back to his lab and put the inoculated broth in a freezer.

Hilleman made the early A.M. dashes to his lab and back because he had to leave in the morning for a conference in South America, and his daughter’s infection might have cleared by the time he returned home from there. So, upon his return from South America, Hilleman, thawed the frozen sample from his daughter’s throat and inoculated it into chick embryos. Serial passage of the mumps virus in the chick embryos eventually generated attenuated mumps virus that in 1967 would serve as a live mumps vaccine.

The virus in the vaccine was dubbed the Jeryl Lynn strain, in honor of its source. Years later, an adult Jeryl Lynn Hilleman noted that her father had a need to be “of use to people, of use to humanity.” She added: “All I did was get sick at the right time, with the right virus, with the right father.”

We’ll have a bit more to say about the mumps vaccine shortly. But first, a few words about measles and rubella.

If mumps was not a major killer, measles certainly was. Before Hilleman and his colleagues introduced their measles vaccine (Rubeovax) in 1962, there were 7 to 8 million measles fatalities worldwide each year, and virtually all of the victims were children. Hilleman developed his attenuated measles vaccine from a measles strain isolated earlier by John Enders. Hilleman attenuated the Enders isolate by putting it through 80 serial passages in different cell types.

[Aside: In a previous posting, we noted that Enders, together with colleagues Thomas Weller and Frederick Robbins, shared a Nobel Prize in Physiology or Medicine for growing poliovirus in non-nervous tissue (3). Apropos the current story, bear in mind that Salk and Sabin developed polio vaccines that have nearly rid the world of this once dread virus. Nevertheless, the Nobel award to Enders, Weller, and Robbins was the only Nobel award ever given in recognition of polio research!]

Rubeovax was somewhat tainted by its side effects; mainly fever and rash. While these reactions were successfully dealt with by combining Rubeovax with a dose of gamma globulin, in 1968 Hilleman’s group developed a new, more attenuated measles strain by passage of the Rubeovax virus 40 more times through animal tissues. Hilleman dubbed the new measles strain “Moraten,” for “More Attenuated Enders.” The new measles vaccine, Attenuvax, was administered without any need for gamma globulin.

Our chronicle continues with the rubella vaccine. Rubella poses its greatest danger to fetuses of non-immune pregnant woman, particularly during the first trimester of pregnancy. In up to 85% of these women, infection will result in a miscarriage or a baby born with severe congenital abnormalities. An outbreak of rubella began in Europe in the spring of 1963, and quickly spread worldwide. In the United States, the 1963 rubella outbreak resulted in the deaths of 11,000 fetuses, and an additional 20,000 others born with birth defects (e.g., deafness, heart disease, cataracts).

Hilleman had been working on a rubella vaccine at the time of the 1963 outbreak. But, he was persuaded to drop his own vaccine and, instead, refine a vaccine (based on a Division of Biologics Standards’ rubella strain) that was at the time too toxic to inoculate into people. By 1969 Hilleman was able to attenuate the DBS strain sufficiently for the vaccine to be approved by the FDA.

Next, and importantly, Hilleman combined the mumps, measles, and rubella vaccines into the single trivalent MMR vaccine, making vaccination and, hence, compliance vastly easier. Thus, MMR was a development that should have been well received by many small children and their mothers, as well as by public health officials.

In 1978 Hilleman found that another rubella vaccine was better than the one in the trivalent vaccine. Its designer, Stanley Plotkin (then at the Wistar Institute), was said to be speechless when asked by Hilleman if his (Plotkin’s) vaccine could be used in the MMR. Merck officials may also have been speechless, considering their loss in revenues. But for Hilleman, it was simply the correct thing to do.

Like Jonas Salk and Albert Sabin before him (3), Maurice Hilleman was never awarded a Nobel Prize. There is no obvious reason for the slight in any of these three instances. In Salk’s case, it may have been because Alfred Nobel, in his will, specified that the award for Physiology or Medicine shall be for a discovery per se; not for applied research, irrespective of its benefits to humanity. But, Max Theiler received the Nobel Prize for producing a yellow fever vaccine. What’s more, the Nobel committee seemed to equivocate regarding the discovery that might have been involved in that instance. Regardless, the Nobel award to Theiler was the only Nobel Prize ever awarded for a vaccine! [A more complete accounting of the development of Theiler’s yellow fever vaccine can be found in The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, now on the blog.]

Sabin had done basic research that perhaps merited a Nobel Prize (3). But, the Nobel committee may have felt uneasy about giving the award to Sabin, without also recognizing Salk. Or, perhaps the continual back-and-forth carping between supporters of Salk and Sabin may have reduced enthusiasm in Stockholm for both of them.

Yet by virtually any measure, Hilleman’s achievements vastly exceeded those of Salk, Sabin, Theiler, and just about everyone else. His basic interferon work alone should have earned him the Prize. Hilleman’s group demonstrated that certain nucleic acids stimulate interferon production in many types of cells, and detailed interferon’s ability to impede or kill many viruses, and correctly predicted its efficacy in the treatment of viral infections (e.g., hepatitis B and C), cancers (e.g., certain leukemias and lymphomas), and chronic diseases (e.g., multiple sclerosis). What’s more, Hilleman developed procedures to mass-produce and purify interferon. And, regarding his unmatched achievements as a vaccinologist, he did more than merely emulate Pasteur’s procedures for developing attenuated viral vaccines. His hepatitis B vaccine was the first subunit vaccine produced in the United States. It was comprised of the hepatitis B surface antigen (HBsAg), which Hilleman purified from the blood of individuals who tended to be infected with hepatitis B virus (e.g., IV drug abusers). Subsequently, to avoid the potential danger of using human blood products in the vaccine, Hilleman developed recombinant yeast cells that produced the HBsAg. And, Hilleman’s meningococcal vaccine was the first vaccine to be based on polysaccharides, rather than on a whole pathogen or its protein subunits.

So, why then was Hilleman bypassed by the Nobel committee? John E. Calfree, in The American, wrote: “As the 80-plus-year-old Hilleman approached death, Offit and other academic scientists lobbied the Nobel committee to award Hilleman the Nobel Prize for Medicine, based partly on his vaccine work and partly on his contributions to the basic science of interferons. The committee made clear that it was not going to award the prize to an industry scientist.” (4) [Paul Offit, referred to here, is the co-developer of the rotavirus vaccine, Rotateq, and a biographer of Hilleman.]

Calfree also notes that Hilleman’s tendency towards self effacement, and his absence from the academic and public spotlight, may also have worked against him. And, unlike Salk, whose name was closely linked to his polio vaccine (3), Hilleman’s name was never associated with any of his nearly forty vaccines. [Yet in the case of Jonas Salk, his public acclaim is generally believed to have hurt him in the eyes of his colleagues and of the Nobel committee.]

Considering the enormity of Hilleman’s contributions, his anonymity was really quite remarkable. As Calfree relates: “In one of the most striking of the dozens of anecdotes told by Offit, Hilleman’s death was announced to a meeting of prominent public health officials, epidemiologists, and clinicians gathered to celebrate the 50th anniversary of the Salk polio vaccine. Not one of them recognized Hilleman’s name!”

With Hilleman’s public anonymity in mind, we conclude our account with the following anecdote. In 1998, a Dr. Andrew Wakefield became a celebrity and hero in the eyes of the public. How this happened, and its consequences are troubling for several reasons, one of which is that it brought undeserved suffering to the self-effacing and benevolent Maurice Hilleman. The Wakefield incident merits, and will have a full-length blog posting of its own. But for now, in 1998 Wakefield authored a report in the prestigious British journal The Lancet, in which he claimed that the MMR vaccine might cause autism in children. The story had a bizarre series of twists and turns, with Wakefield and co-authors eventually issuing a retraction. The immediate cause of the retraction was the disclosure that Wakefield, on behalf of parents of autistic children, had accepted funding to investigate a link between the MMR vaccine and autism. The purpose of the investigation was to determine whether a legal case against the vaccine manufacturer might have merit. In addition to the obvious conflict of interest, Wakefield’s paper had serious technical flaws as well. At any rate, a number of independent studies subsequently demonstrated that there is no causal link between the MMR vaccine and autism. And, in 2010 Wakefield was barred by the British Medical Society from the practice of medicine. But the harm had been done. Hilleman had become the recipient of hate mail and death threats. And, more important to Hilleman I expect, many worried parents, even today, prevent their children from receiving the MMR vaccine (5). Ironically, the very success of the MMR vaccine enabled people to forget just how devastating measles and rubella could be.  Maurice Hilleman succumbed to cancer on April 11, 2005.

1. Nature Medicine 11, S2 (2005)
2. Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu  On the blog.
3. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science  On the blog
4. Calfree, J.E., Medicine’s Miracle Man , The American, January 23, 2009
5. Reference 4 contains a somewhat similar tale, in which a 1992 article in Rolling Stone attributed the emergence of HIV to Hillary Koprowski’s polio vaccine. It created a sensation but, as might be expected, there was no evidence to support its premise.