Tag Archives: Jonas Salk

Julius Youngner: Slighted Polio Vaccine Pioneer

This is a tale of the hurt that a junior investigator might feel when a senior investigator takes the lion’s share of the credit for the junior investigator’s crucial breakthroughs. Jonas Salk, who conceived and oversaw the development of the first widely used polio vaccine, is the senior investigator in this anecdote. Julius Youngner, the last surviving member of the original vaccine research team that Salk assembled in the early 1950s at the University of Pittsburgh, is the slighted assistant. Youngner later had his own distinguished career. He passed away in April of this year. Here is their story.

After earning his Ph.D. in microbiology, Youngner was drafted into the World War II U.S. Army, which assigned him to the Manhattan Project, to test the toxicity of uranium salts. Youngner first learned the purpose of the Manhattan Project when the first atomic bomb was dropped on Japan.

After the war, Youngner worked as a commissioned officer for the U.S. Public Health Service. This was a significant stop in his career, since it was there that he first became interested in viruses and cell culture. But, since there was no opportunity for him to pursue that interest in Bethesda, he began to look elsewhere. Thus, it happened in 1949 that Salk recruited Youngner to join his vaccine research team in Pittsburgh, after a mutual acquaintance told Salk that Youngner was eager to work on viruses and cell culture.

Jonas Salk and Julius Youngner at the University of Pittsburgh, early 1950s

Salk hoped that Youngner might find a way to generate enough cells from monkey kidney tissue to support mass-production of the vaccine. Youngner, on his own, then developed the use of the proteolytic enzyme, trypsin, to disperse tissue fragments into individual cells, thereby generating many more cells from a given amount of tissue. Indeed, Youngner could generate enough cells to support manufacture of the vaccine. This was his first key contribution to the vaccine project. “Trypsinization” remains a mainstay of modern cell culture.

Youngner’s next major contribution to the vaccine enterprise was his development of a rapid analytical test that had two crucial applications. First, recalling that the Salk vaccine contains an inactivated virus, Youngner’s so-called “color test” made it possible to quickly screen batches of the vaccine for any live virus that might have survived the inactivation process.  Second, Youngner’s test made it possible to quickly test the vaccine’s ability to induce anti-poliovirus antibodies (1). [Youngner based his color test on an earlier observation by John Enders, Tom Weller, and Fred Robbins, that metabolic activity (as indicated by a drop in pH) was less in cultures inoculated with live virus than in control cultures (2, 3). In Youngner’s test, a color change of phenol red, resulting from a shift in pH, served as an indicator of virus activity, or of antibody activity.]

Some sources credit Youngner with having devised the process for inactivating the virus. But, that is correct in a very limited sense only. Salk selected incubation in formalin as the means to disable the virus. In truth, Salk learned of that approach a decade earlier while doing postgraduate studies under Thomas Francis at the University of Michigan. Francis was then using formaldehyde to produce his killed influenza vaccine (2).

What’s more, Salk’s choice of formalin to generate his polio vaccine was bold. Earlier, in the 1930s, Canadian scientist Maurice Brodie tested a formalin-killed polio vaccine in twelve children, with disastrous results. Several of the children developed paralytic poliomyelitis (4).

Clearly, too little exposure to formalin could leave enough live virus to cause paralytic poliomyelitis or death. On the other hand, too much exposure could so badly damage the virus’ proteins that they might no longer induce an immune response against the live virus. Brodie did not have analytical procedures to ensure that he had inactivated his vaccine to safe levels. In contrast, it was clear to Salk that getting the correct balance would be vital to his vaccine project, and Youngner’s color test was the means for doing so. Youngner used his test to determine that six days of incubation in a 1:4,000 formalin solution would result in one live virus particle in 100 million doses of the vaccine (5).

Since Youngner’s inactivation curve was based on only a few data points, and since it was likely that the slope of the curve might flatten out after a time, Salk added a margin of safety of six extra days. Thus produced, the vaccine induced antibody production in monkeys, while showing no signs of causing paralysis or other problems.

By 1954, 800,000 children had been successfully immunized against polio in the first clinical trial of the vaccine. In April 1955, the outcome of the trial would be announced to a very grateful public.

By 1957, Salk’s vaccine team at Pittsburgh was no longer needed, and was dispersing. Salk was making plans to leave Pittsburgh for California, where he would found the prestigious Salk Institute. Youngner, now 34 years-old, remained at Pittsburgh, where he would begin his own distinguished career.

Although Youngner was now independent of Salk, he remained bitter over his former boss’s failure to acknowledge the underlings who had labored so diligently behind the scenes to bring the vaccine to fruition. “The first rule we learned was to call him ‘Dr Salk,’ never Jonas. He would speak to us through a wall of notes and memos…Here was a guy who could always find an hour to brief some reporter at the local Chinese restaurant, but could never find the time to sit down with his own people (6).”

Youngner was particularly appalled by events involving the paper he wrote describing his color test. “After I had what I considered to be a good draft…I gave my copy to Jonas for his comments. It should be noted this was 1954, the pre-Xerox, pre-word-processing era. I had made a working transcript of the paper for my own use and it was this copy that I handed to him. Also, it should be noted that the title page had the authors listed as ‘J.S. Youngner and E.N. Ward (6).’” Elsie Ward, who served as Youngner’s technician, was a zoologist who specialized in growing viruses.

Salk intended to read Youngner’s manuscript while away on a trip.  When Salk returned a week later, he claimed that he had lost the manuscript, but that he had jotted down some notes from which he was able to produce a draft of his own. Youngner was rather incredulous that a person as meticulous and disciplined as Salk could lose such an important manuscript. Youngner’s skepticism was further roused by the fact that Salk’s version contained all the data in Youngner’s original manuscript. Salk explained that incongruity, alleging that he found Youngner’s tables, but not the text.

In any case, Youngner was especially upset by a specific change Salk made to the title page of the manuscript: “The authors were now ‘Jonas E. Salk, J.S. Youngner, and Elsie N. Ward.’ When I (Youngner) questioned the change, Jonas said that since he had to reconstruct the whole paper it was only fair that his name go first…It was obvious to me then, and is more so now, that he considered the advance in this paper a major one and he wanted his name associated with it, even though at the time he had done nothing in the lab (no kidding!) or of an advisory nature to initiate or carry out the work (6).”

Youngner could grudgingly accept that project leaders often used their senior position to appear as co-authors, or even principal authors, on papers emanating from their labs, even if their contributions were minimal. What troubled Youngner in this instance was not that Salk pulled rank, but rather his seeming duplicity.

In yet another instance—the 1955 public announcement of the successful outcome of the clinical trial—Youngner again sensed “a pattern of deception on Salk’s part to take undue credit for the discoveries of others (6).” Salk advocated for the announcement to happen at the University of Pittsburgh. However, the National Foundation for Infantile Paralysis (better known as the “March of Dimes”), which funded the vaccine project, chose the University of Michigan in Ann Arbor as the site for the announcement. That was where Michigan professor Thomas Francis supervised the evaluation of the field trial. [Note that the NIH was not able to fund research back then the way it can today. Thus, the polio vaccine project was supported nearly entirely by private donations to the National Foundation.]

Thomas Francis spoke first. Then, when Salk spoke, he acknowledged the more prominent players in the vaccine project, including Thomas Francis, Harry Weaver (director of research at the National Foundation), Tom Rivers (chairman of the advisory committees on research and vaccines for the  National Foundation), and Basil O’Connor (law partner of Franklin Roosevelt, recruited by Roosevelt in 1928 to raise funds for polio patients at Roosevelt’s Warm Springs Foundation, and a co-founder with Roosevelt of the National Foundation in 1938; (2)). Salk then acknowledged various deans and trustees at the University of Pittsburgh. Yet, he made no mention whatsoever of his dedicated coworkers in his laboratory. They had been expecting at least some recognition from their boss.

Some of Salk’s defenders argued that Salk had acted in the best scientific tradition by prefacing his printed remarks with the phrase, “From the Staff of the Virus Laboratory by Jonas E. Salk, M.D.” But, this was small consolation to Youngner and others of Salk’s coworkers, who expected to be individually acknowledged for their exhausting work on behalf of the life-saving vaccine. Indeed, they felt betrayed.

At any rate, the 1955 announcement of the success of the polio vaccine field trials was joyously received by the public. And while Youngner remained embittered over Salk’s slighting of his coworkers, he nonetheless understood that from the point of view of the National Foundation, “it was much easier to continue raising money when you have a hero, and they had an enormous public relations department that took up Jonas’ name as the hero, which he deserved…But in the meantime, Jonas was, how shall I say, not very generous to his colleagues and he made sure that nobody else was ever mentioned (6).”

The following excerpt is from Polio: An American Story (6). “In September 1963, Salk returned to Pittsburgh to attend the unveiling of his portrait in the auditorium of the University’s medical complex, a stone’s throw from the hospital where he had done his historic polio research. Before the ceremony, Salk told Dean George Bernier that he wished to speak privately with his former assistant, Julius Youngner, now a distinguished professor at the school of medicine. The two men hadn’t talked or crossed paths since Salk’s move to California in 1961. Salk saw the meeting as a courtesy to the only remaining member of his laboratory staff; Youngner had a different agenda. Speaking softly, he recalled, he slowly released the ‘hurt’ he had bottled up for more than thirty years. ‘Do you still have the speech you gave in Ann Arbor in1955? Have you ever reread it?’ Youngner began. ‘We were in the audience, your closest colleagues and devoted associates, who worked hard and faithfully for the same goal that you desired…Do you remember who you mentioned and who you left out? Do you realize how devastated we were at that moment and ever afterward when you persisted in making your coworkers invisible? Do you know what I’m saying,’ I asked. He answered that he did…Jonas was clearly shaken by these memories and offered little response.’…The two men engaged in some uncomfortable small talk before Dean Bernier returned to escort them to the ceremony. Speaking later to a reporter, Youngner admitted, ‘I got a lot of things off my chest. I’m beyond the point where I pull my punches with him. I think it was the first time he ever heard it so graphically.’ Asked if he had any regrets about working for Salk, Youngner replied: ‘Absolutely not. You can’t imagine what a thrill that gave me. My only regret is that he disappointed me.”’

Epilogue:

Jonas Salk is deservedly celebrated for developing the killed polio vaccine. That vaccine, together with Albert Sabin’s live attenuated vaccine, which followed soon afterwards, has nearly eradicated polio worldwide. Importantly, Sabin and other polio researchers believed that only a live vaccine could induce a level of immunity sufficient to protect against a challenge with live virulent virus. Nonetheless, Salk persevered in his conviction that a killed vaccine could protect against polio, and he was right.

Salk founded the prestigious Salk Institute in 1963. Yet he never himself made another notable contribution to science.

Youngner may be best known for his work on the Salk vaccine. Yet he had a distinguished career of his own at the University of Pittsburgh after Salk left. Youngner is especially noted for his contributions to interferon research. These include his finding that non-viral agents could trigger interferon induction in animals. And, in collaboration with colleague Samuel Salvin, he identified a second type of interferon, now known as gamma-interferon. Youngner also helped to explain the antiviral-effect of interferon, and he was the first researcher to demonstrate that some viruses express countermeasures against interferon.

Youngner also made important findings in the area of persistent virus infections. Importantly, he demonstrated that defective viral variants, including temperature-sensitive mutants, can play a role in the establishment and maintenance of viral persistence; doing so by impairing (modulating) the replication of the wild-type parental viruses. Based on that principle, Youngner sought to develop dominant-negative mutants of influenza virus as a novel means of anti-influenza therapy. In addition, Youngner and colleague Patricia Dowling developed a novel live attenuated vaccine against equine influenza virus, based on a cold-adapted influenza virus, which can replicate only at the temperatures found in the respiratory tract. That live vaccine was the first to prevent a serious respiratory disease of horses.

Julius Youngner, 2010

References:

  1. Salk, J.E., Youngner, J.S, Ward, E.N. (1954). Use of Color Change of Phenol Red as the Indicator in Titrating Poliomyelitis Virus or Its Antibody in a Tissue–Culture System,” American Journal of Epidemiology. 60: 214–230.
  2. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.
  3. John Enders: “The Father of Modern Vaccines,” Posted on the blog August 4, 2016.
  4. Vaccine Research using Children, Posted on the blog, July 7, 2016.
  5. Williams, G., Paralysed with Fear: The Story of Polio, Palgrave Macmillan, 2013.
  6. Oshinsky, D.M., Polio: An American Story, Oxford University Press, 2005.
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Hilary Koprowski’s Oral Polio Vaccine: The Bizarre Claim that it was the Source of HIV in Humans

Jonas Salk and Albert Sabin are justly celebrated for developing their respective polio vaccines which, together, have nearly eradicated polio worldwide. However, it was Hilary Koprowski (1916-2013) who actually developed the world’s first safe and effective polio vaccine, doing so several years before Salk and Sabin brought out their more famous vaccines (1). In fact, Koprowski’s oral polio vaccine was used throughout the world between 1957 and 1960. But, it was never licensed in the United States, where the U.S. Surgeon General rejected it in favor of Sabin’s more highly attenuated oral vaccine. [By the way, Sabin developed his vaccine from a sample of attenuated poliovirus that he received from Koprowski.] In any case, Koprowski was the first to demonstrate the practicality of an oral polio vaccine.

An earlier posting told how Koprowski’s reputation was sullied when, in 1950, he tested his live polio vaccine in 20 patients at Letchworth Village; a facility for mentally disabled children in Rockland County, NY (2). Another posting told of Koprowski’s harrowing escape from Poland on the eve of World War II, and of his serendipitous introduction to virology in Brazil, where he sought refuge from the Nazis (3). Here we relate another episode in Koprowski’s tumultuous life; the 1990s assertion that his oral polio vaccine was responsible for the onset of the HIV/AIDS epidemic, when it was administered, between 1957 and 1960, to nearly a quarter million people in the former Belgian Congo. But first, some background.

On June 5, 1981, the Morbidity and Mortality Weekly Report (a publication of the U.S. Centers for Disease Control) told of five sexually active gay men who were suffering from a lung disease caused by the protozoan Pneumocystis carinii. Importantly, those men also presented with “profoundly depressed numbers of thymus-dependent lymphocytes.” That CDC report was singularly notable since it brought to light the onset of a strange and deadly new disease, which soon would be named the acquired immunodeficiency disease or AIDS. Within two years, a “new” virus, which was later termed the human immunodeficiency virus (HIV), was isolated and shown to be the cause of AIDS (4).

The general public, as well as the biomedical community, wanted to know the origin of HIV, and how and where it entered the human population. Research would show that HIV likely crossed into humans from particular subspecies of chimpanzees, unknowingly and on multiple occasions during the 20th century. However, two 1990s publications—a 1992 Rolling Stone article by writer Tom Curtis (5) and The River, A Journey to the Source of HIV and AIDS, a 1999 book by British journalist Edward Hooper (6)—proposed a rather different hypothesis; that Koprowski’s oral polio vaccine gave rise to the HIV/AIDS epidemic.

At the heart of the accusation was, first, the claim that some of Koprowski’s vaccine lots were propagated in primary monkey or chimpanzee tissue that harbored the related simian immunodeficiency virus (SIV). Second, they alleged that SIV was transmitted to the Congolese via the contaminated vaccine and, third, that SIV evolved into HIV in humans.

In the Rolling Stone article, Curtis rightly noted that Koprowski indeed grew his vaccine in monkey cells, and Curtis stated so again in a 1992 letter to Science (7). Curtis also asserted that 87% of the 39 confirmed cases of HIV-positive blood samples that were collected in Africa before 1981 came from towns within 100 miles of sites where the Koprowski’s vaccine was administered (5, 7).

Koprowski responded to Curtis’ charges in his own letter to Science (8). First, he addressed the claim that the vaccine harbored SIV: “After the original batch of the type II polio vaccine was produced in cotton rat brain, all other batches were produced in kidney tissue obtained from rhesus monkeys (Macaca mulatta) captured either in India or the Philippines… Curtis’ speculation that we could have used in our production kidney tissue from other species of monkeys that might have harbored a simian immunodeficiency virus (SIV) or an HIV virus has no basis in fact.”

Next, Koprowski addressed the claim that the outbreak of HIV correlated geographically to the regions where the vaccine was administered: “Curtis has theorized that the ‘African epidemic was spawned by a contaminated polio vaccine administered from 1957 to 1960 to at least 325,000 people in Rwanda, Burundi and the former Belgian Congo.’ He has stated that the area of vaccination of children in Ruzizi Valley in 1958 corresponds to ‘roughly to another map . . . the one identifying the regions of highest HIV [human immunodeficiency virus] infection in equatorial Africa.’ This is completely wrong. Ruzizi Valley, where 215,504 subjects were vaccinated in 1958, is located in the northwestern part of the Republic of Burundi, not in the Kivu district of Zaire, an area where Curtis placed ‘the lion’s share of their [Koprowski and his associates] samples (8).’” See Aside 1.

[Aside 1: Koprowski justified taking his dispute with Curtis to Science as follows: “As a scientist, I did not intend to debate Tom Curtis when he presented his hypothesis about the origin of AIDS in Rolling Stone. The publication of his letter in Science (29 May, p. 1260), however, transferred the debate from the lay press to a highly respected scientific journal. I would now like to state my views, based on facts, in order to counter and thereby repudiate Curtis’ hypothesis about the origin of AIDS (8).]

Curtis received considerable pushback from the biomedical community. Yet his Rolling Stone article seems to have been an earnest and sober attempt to put forward a credible premise for how HIV might have crossed into humans. Before Curtis wrote the piece, he first interviewed several top retrovirologists and polio researchers, including Robert Gallo, William Haseltine, Joseph Melnick, Albert Sabin, and Jonas Salk, as well as Koprowski; asking each probing questions concerning the plausibility of his premise. ‘“You can’t hang Koprowski with that,’ Albert Sabin growls at me… Sabin insists that the AIDS virus won’t survive swallowing…Dr. Robert Gallo and other retrovirus researchers acknowledged to me; no one knows for sure… Salk… flatly refused to discuss the subject (5).”

Curtis defended his Rolling Stone article in his 1992 letter to Science, writing: “…I think any fair-minded reader will recognize that I took great pains not to demonize medical science in general or any individual research scientist.”  To that point, Curtis acknowledged in the Rolling Stone: “Like Salk and Sabin, Koprowski had the best intentions: He wanted to eradicate a debilitating and deadly scourge.” Nonetheless, in Science, Curtis added: “As for the assertion that there is not a ‘picogram of evidence” supporting the theory, that is flat-out wrong. There is a strong, if circumstantial case.”

Turning now to The River, bear in mind that it was published seven years after Curtis published his Rolling Stone article. During that interim, significant evidence had accumulated, and had been reported in scientific journals, repudiating the charge that Koprowski’s vaccine was responsible for the HIV outbreak. What’s more, the CDC had issued an official statement that the “weight of scientific evidence does not support the idea.”

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Nonetheless, Hooper’s assertions in The River were more immoderate than those made earlier by Curtis. Hooper’s argument began with the fact that before the mass trial of the Koprowski vaccine in the Congo, the vaccine was tested first in a colony of chimpanzees living near Stanleyville (now Kisangani) —the headquarters of the vaccine campaign. [The animals’ caretakers were vaccinated concurrently. In fact, the successful immunization of those workers provided the justification for the ensuing first ever mass trial of an oral polio vaccine in humans.]

Hooper then noted that the Stanleyville chimpanzee colony was maintained by Philadelphia’s Wistar Institute (where Koprowski developed the vaccine). Hooper next alleged that Wistar scientists took kidneys from those chimpanzees back to Philadelphia, where they used them to produce the cell cultures in which they grew more of the vaccine. Hooper’s argument continues with the assertion that the chimpanzees carried SIV, which thus contaminated the vaccine, and that the SIV evolved into HIV after being introduced into humans via the vaccine.

In response to Hooper’s claims, the Wistar Institute engaged three independent laboratories to test 40-year-old leftover vaccine lots for the presence of HIV and SIV, and also for chimpanzee mitochondrial DNA. The combined results of those studies, which were reported at a 2000 meeting of the Royal Society of London, failed to support the claims put forward by Hooper, nor did they support the earlier clams advanced by Curtis. The vaccine lots did not contain either HIV or SIV, nor was there any evidence that any of the lots were grown in chimpanzee cells. See Aside 2.

[Aside 2: Stanley Plotkin (1932, currently an adviser at the vaccine firm Sanofi Pasteur) was a Wistar scientist who, in the 1950s, collaborated with Koprowski on the polio vaccine project. In a 2001 paper, Plotkin disputed Hooper’s charge that Wistar scientists were oblivious to the threat of extraneous agents in their primary cell cultures (9). Plotkin added: “This is the strangest paper I have ever given, belonging perhaps more to the world of literary exegesis than to the world of science. However, it is time that the true history be told… to correct the misrepresentations that have been widely disseminated by The River (Hooper 1999) and subsequently by articles written about the book…The river has been praised for its precise detail and wealth of footnotes, but one can be precise without being accurate (9).”]

Hooper was not to be dissuaded by the reproach of the science community. Instead, he fought back. He dismissed the fact that tests of 40-year-old leftover vaccine lots did not find any evidence of SIV, HIV, or chimpanzee DNA, claiming that the particular vaccine lots that were produced in chimpanzee cells were no longer in existence and, thus, were not tested.

Even if Hooper were correct on that particular point, his allegations against the Koprowski vaccine were discredited by several other lines of evidence. For instance, the SIV strain in the Stanleyville chimpanzees was phylogenetically distinct from all strains of HIV (10). Thus, even if the SIV carried by those chimpanzees had somehow contaminated the Koprowski vaccine, it could not have been the progenitor of HIV in humans. To that point, other studies showed that the chimpanzee virus that is the precursor of HIV actually originated in west-central Africa; not in the Congo.

Moreover, a comparison of HIV samples taken over time leads to the estimate that the crossover of SIV into humans occurred sometime during the1920s and 1930s, and perhaps even before that; at any rate, decades before Koprowski’s African vaccine program. [That analysis assumes that the rate of change of HIV has been constant over time.]

Earlier, in 1993, Koprowski filed a defamation suit against Curtis and Rolling Stone. Just before Koprowski was scheduled to give a deposition, his lawyers reached a settlement, in which Koprowski was awarded $1 in damages. However, in addition to that symbolic award, the magazine agreed to publish a “retraction” of sorts, which (in December 1993) stated in part: “The editors of Rolling Stone wish to clarify that they never intended to suggest in the article that there is any scientific proof, nor do they know of any scientific proof, that Dr. Koprowski, an illustrious scientist, was in fact responsible for introducing AIDS to the human population or that he is the father of AIDS…”

Hooper, on the other hand, has stood by his assertion that the Koprowski oral polio vaccine (OPV) program in the Congo was responsible for the emergence of HIV. He maintains a current web site—AIDS Origins: Edward Hooper’s Site on the Origins of AIDS—which, in a December 2015 update, stated: “Though members of the “bushmeat school” would have you believe otherwise, the arguments for the OPV/AIDS hypothesis grow consistently stronger as more information becomes available.” [The bushmeat or hunter theory holds that the HIV precursor was transmitted to humans when a human hunter was bitten or cut while hunting or butchering a monkey or ape for food. It is considered the simplest and most plausible explanation for the cross-species transmission of HIV to humans.] Elsewhere on the site, Hooper states: “In the years since 1992, I and many others (including the great evolutionary biologist, Bill Hamilton) have examined further evidence from many different sources, and found that OPV is in fact a far more compelling theory of origin than bushmeat.”

Hooper has gone so far as to suggest that the biomedical community is engaged in an organized cover-up of the OPV-HIV connection: “Because of the enormous implications of the hypothesis that AIDS may be an unintended iatrogenic (physician-caused) disease, it is almost inevitable that this theory will engender heated opposition from many of those in the scientific establishment, and those with vested interests (11).” See Aside 3.

[Aside 3: Conspiracy theories about the origin of AIDS—particularly that HIV was man-made and deliberately introduced into humans—first appeared in the late 1980s and abounded in the 1990s. They gained especial traction in the African American Community. Some may recall Reverend Jeremiah Wright, President Barak Obama’s former pastor, whose comments on several subjects raised a storm in the media (causing Obama to ultimately disassociate himself from Wright). One of those comments was that “the U.S. government invented AIDS to destroy people of color.”]

Although Hooper’s claims have been discredited by rigorous scientific testing, The River was well-received in the popular press. Consequently, and sadly, the book’s anti-vaccine sentiments gained credibility in the public; stirring a distrust of vaccines that set back global efforts to eradicate polio, while also discouraging many Americans from having their children vaccinated against polio and other diseases as well. To that point, Koprowski concluded his 1992 letter to Science as follows: “Tremendous efforts were made by scientists to save children from paralytic polio. The current anxiety among parents of children who have been or are going to be vaccinated against polio followed dissemination by the lay press of unproved theories of the origin of AIDS. This was unnecessary and harmful, particularly since the vaccine was tested thoroughly before any vaccination was done; the vaccine was and continues to be safe (8).”

Yet the story does not end on so simple a moral lesson. As asserted by noted retrovirologist Robin Weiss: “Yet one lesson to be learned from considering OPV as a source of HIV is how plausibly it might have happened and how cautious we need to be over introducing medical treatments derived from animal tissues, such as live, attenuated vaccines… (12).”

To Weiss’ point, recall that early lots of both the Salk and Sabin polio vaccines were unknowingly contaminated with simian virus 40 (SV40) (13). What’s more, the contaminated vaccines were administered to hundreds of millions of people world-wide, before SV40 was even discovered! In fact, SV40 was discovered as a contaminant of those vaccines. The early polio vaccine lots were contaminated with SV40 because that virus was unknowingly present in the rhesus monkey kidney cell cultures in which the vaccines were grown. Afterwards, it was discovered that SV40 causes tumors in newborn hamsters. We owe it to good fortune that SV40 was not a serious threat to humans.

Curtis was well aware of the SV40 story when he wrote the Rolling Stone article. “There is evidence that all three pioneers (Koprowski, Salk, and Sabin) used vaccines inadvertently contaminated with viruses from a species dangerously close to our own. If the Congo vaccine turns out not to be the way AIDS got started in people, it will be because medicine was lucky, not because it was infallible (5).”

References

  1. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.
  2. Vaccine Research Using Children, Posted on the blog July 7, 2016.
  3. Hilary Koprowski: Genesis of a Virologist, Posted on the blog August 26, 2016.
  4. Who discovered HIV? Posted on the blog January 23, 2014.
  5. T Curtis, The origin of AIDS, Rolling Stone, no. 626 (19 March 1992)
  6. E Hooper, The River, A Journey to the Source of HIV and AIDS, Little Brown & Co, 1999.
  7. T Curtis, 1992. Possible origins of AIDS. Science 256: 1260-1261.
  8. H Koprowski, 1992. AIDS and the polio vaccine. Science 257:1026-1027.
  9. SA Plotkin, 2001. Untruths and consequences: the false hypothesis linking CHAT type1 polio vaccination to the origin of human immunodeficiency virus. Philosophical Transaction of the Royal Society of London. Series B, Biological Sciences 356:815-823.
  10. Worobey M, Santiago ML, Keele BF, et al., 2004. Origin of AIDS: contaminated polio vaccine theory refuted. Nature 6985:820.
  11. E Hooper, 2001. Experimental oral polio vaccines and acquired immune deficiency syndrome. Philosophical Transaction of the Royal Society of London. Series B, Biological Sciences 356:803-814.
  12. RA Weiss, 2001. Natural and iatrogenic factors in human immunodeficiency virus transmission. Philosophical Transaction of the Royal Society of London. Series B, Biological Sciences 356:947-953.
  13. SV40-Contaminated Polio Vaccines and Human Cancer, Posted on the blog July 24, 2014.

 

Hilary Koprowski: Genesis of a Virologist

Several years before Jonas Salk and Albert Sabin developed their famous polio vaccines, Hilary Koprowski (1916-2013) in fact developed the world’s first effective, but much less well known polio vaccine (1, 2). Koprowski’s vaccine was used world-wide, but it was never licensed in the United States, ultimately losing out to Sabin’s vaccine.

Koprowski’s reputation was tarnished in 1950, when he tested his live polio vaccine on 20 children at Letchworth Village for mentally disabled children, in Rockland County, NY; an episode recounted in a recent posting Vaccine Research Using Children (1). Koprowski reported on the Letchworth Village trials at a 1951 conference of major polio researchers. Although his vaccine induced immunity in the children, and caused no ill effects, many scientists in the audience were horrified that he actually tested a live polio vaccine in human children. Afterwards, Sabin shouted at him: “Why did you do it? Why? Why?”

Although Koprowski’s polio vaccine was supplanted by the Salk and Sabin vaccines, his demonstration, that a live polio vaccine could be safe and effective, paved the way for Sabin to develop his live polio vaccine. Moreover, Sabin developed his vaccine from a sample of attenuated poliovirus that he received from Koprowski.

There is much more to tell about Koprowski. This posting relates some of the remarkable earlier events of his life, including his harrowing escape from Poland on the eve of the Second World War; a flight which inadvertently led to his career in virology. A subsequent posting will recount the now discredited, although sensational at the time, accusation that Koprowski’s polio vaccine gave rise to the HIV/AIDS epidemic.

Koprowski was born and grew up in Warsaw, where he earned a medical degree from Warsaw University in 1939. He also was an accomplished pianist, having studied piano from the age of 12 at the prestigious Warsaw Conservatory, where Chopin is said to have studied. Koprowski eventually earned a music degree from the Conservatory. He recalled, “…the first year I was the youngest and voted second best in the class (3).”

koprowski

Hilary Koprowski in Warsaw (2007)

In 1938, while Koprowski was in medical school, he married classmate Irena Grasberg who, in later years, would wonder how they had found the time for their courtship. Each had to contend with a demanding medical school program, while Hilary’s piano studies at the Conservatory was a full time program in itself (3). Irena recalled a day before both of them had an anatomy exam, and Hilary had an important recital. Hilary practiced a recital piece, while simultaneously studying a chart on the music rack showing the bones of the hand; all the while as Irena read anatomy to him.

Koprowski eventually chose a career in medicine, rather than one in music. As he explained: “…the top of the music pyramid is much narrower than that of medicine, where there is more space for successful scientists (3).” Koprowski rated himself only fourth best in his class at the Warsaw Conservatory, and he needed to excel. Yet he may have underrated himself. His piano professor at the Conservatory was “greatly disappointed” when he chose to enter medicine (3). [After the 1944 Warsaw uprising, Koprowski’s piano professor was arrested and beaten to death by German soldiers (see below and 3).] In any case, Koprowski continued to play the piano, and he even did some composing in his later years.

Germany invaded Poland in September 1939, setting off the Second World War. As German bombs were falling on Warsaw, Koprowski answered the call for Polish men to go east, where Polish forces were organizing to resist the Germans. Irena, now pregnant, and Hilary’s mother went with him, while his father chose to remain behind. They made their way in a horse-drawn hay wagon, traveling at night to avoid German planes that were strafing the roads during the day. After a week or so on the road, they encountered refugees moving in the opposite direction. Those refugees told them that Russia had signed a pact with Germany and was now invading Poland from the east (Aside 1). So the three Koprowskis joined the flood of refugees moving to the east. When they arrived back in Warsaw, they found the city in ruins. Many of their friends and neighbors had been killed or were seriously wounded, and the city was occupied by German soldiers.

[Aside 1: The German–Soviet Non-aggression Pact was signed in Moscow in August 1939, as a guarantee of non-belligerence between Nazi Germany and the communist Soviet Union. Hitler broke the pact in June 1941 when Germany attacked Soviet positions in eastern Poland. Hitler had no intention of keeping to the pact. However, it temporarily enabled him to avoid having to fight a war on two fronts—against Britain and France in the west and the Soviet Union in the east.]

Once Germany had conquered Poland, German and Polish Jews began to be sent to concentration camps set up in Poland. The Koprowskis, who were Jewish (Salk and Sabin too were descendants of eastern European Jews), quickly made plans to leave Poland. Their first destination was to be Rome. Hilary’s father went there first to arrange living conditions for the family. To facilitate the escape of Hilary’s father from Poland, Hilary and Irena wrapped him in bandages, hoping that the authorities might gladly believe they were letting a very frail individual depart from the country.

Hilary, Irena, and Hilary’s mother then traveled by train from Warsaw to Rome. It was a harrowing trip. Irena was pregnant, and the Gestapo was roaming the trains. They feared that they might have been arrested at any time.

In Rome, the Koprowski family’s main concern was the safety of Irena and her unborn baby. Since Irena had an aunt in Paris, who would know of a good doctor there, the family thought that Paris would be a safe place for the baby to be born. Thus, Irena left for Paris, accompanied by Hilary’s father. She gave birth to Claude five days after arriving there.

Hilary did not go with Irena to France. If he had done so, he would have been impressed immediately into the Polish Army that was forming there to fight the Germans. Yet he knew that he would eventually have to leave Rome. Italy, under Mussolini’s leadership, was poised to enter the Second World War, as an Axis partner of Hitler’s Germany.

After Claude was born, Irena worked as a physician at a psychiatric hospital in Villejuif, just outside of Paris. She was the sole internist there for eight hundred patients. She kept Claude at the hospital, in a locked room, which she would slip to away every three hours to nurse him.

Back in Rome, Hilary continued to play the piano. In fact, he auditioned for, and was accepted by Rome’s L’Accademia di Santa Cecilia, which awarded him a second degree in music. Importantly, his skill at the keyboard enabled him to get visas for himself and his mother to enter Brazil, which the family hoped would be a safe haven. The best students from L’Accademia di Santa Cecilia were often in demand to play for events at the Brazilian embassy in Rome. Thus, on several occasions, Hilary played the piano at the embassy. Brazil’s consul general admired Hilary’s pianism and was pleased to arrange Brazilian entry visas for Hilary and his mother. See Aside 2.

[Aside 2: The day after Hilary arrived in Rome, he volunteered to serve as a medical examiner for a Polish draft board that was set up in the Polish embassy. The draft board’s activity at the embassy—recruiting Poles for the Polish Army—violated diplomatic protocol. In addition, Italy would soon be Germany’s Axis partner in the War. Moreover, Brazil, though neutral in the War, favored the Axis.]

Hilary and his mother had been making plans to leave Italy. Their destination was to be Spain, where they hoped they might unite with Irena, Claude, and Hilary’s father.  From Spain, the family might then go to Portugal, where they could get a boat to Brazil. But, on the very day that Hilary and his mother were to leave Italy, Mussolini issued a proclamation banning any male of military age from leaving the country. So it happened that Hilary’s escape from Italy was blocked at the boat registration. However, his mother rose to the occasion, crying and pleading with the boat registration official that she was sick, that Hilary was her sole means of support, and that she could not go on without him. “The man looked at his watch and said he must go to lunch. He looked at us and said, ‘If the boat leaves before I return, that’s my bad luck (3).’” So, Hilary and his mother boarded the boat, which left before the official returned. [Hilary’s mother was a well-educated woman, and a dentist by profession.]

In Spain, Hilary and his mother stayed at a hotel in Barcelona. Despite the wartime conditions, they were able to communicate, if only sporadically, with Irena and Hilary’s father, who were still in France. Then, after Germany invaded France in 1940, Irena, Claude, and Hilary’s father reunited with Hilary and his mother in Barcelona. [The escape of Irena, Claude, and Hilary’s father from France was far more harrowing than the escape of Hilary and his mother from Italy (See 3 for details).]

The family now needed to get to Portugal, where they could then get a boat to Brazil. Irena had already obtained Portuguese visas for herself and for Claude. But Hilary and his mother only had visas for Brazil. Hilary’s applications for visas at the Portuguese embassy were repeatedly denied, until a fellow Pole at Hilary’s Barcelona hotel advised him of the obligatory bribe that must accompany visa applications. The advice was right-on, and the family (minus Hilary’s father, who chose to go to England) sailed for Brazil without further incident.

In Brazil, Irena found work in Rio de Janeiro as a nurse. But she soon managed to secure a position as a pathologist at the largest hospital in the city. Hilary, on the other hand, could not find a job in medicine and, so, he turned to teaching piano. After six months of teaching unenthusiastic piano students, Hilary by chance recognized a man on the street in Rio who happened to be a former schoolmate from Warsaw. The man also happened to be working at the Rockefeller Foundation’s outpost in Rio. He told Hilary that the Foundation was looking for people, and he also told Hilary who he should contact there. Hilary interviewed at the Foundation the next day, and was told to report for work the day after that.

The Foundation assigned Hilary to research how well, and for how long the attenuated yellow fever vaccine—developed by Nobel laureate Max Theiler in 1935 (4) —might protect against yellow fever. The disease was endemic in Brazil, and it was actually the Rockefeller Foundation’s first priority.

Hilary’s supervisor at the Foundation was Edwin Lennette; a staff member of the International Health Division of the Rockefeller Foundation, assigned to its Brazilian outpost, specifically because of his interest in yellow fever. In 1944, Lennette would be reassigned to the Rockefeller Foundation laboratory in Berkeley, California, where he would establish the first diagnostic virology laboratory in the United States. Indeed, Lennette is known as one of the founders of diagnostic virology. But, in Brazil, he introduced Hilary Koprowski to virology.

Hilary’s apprenticeship under Lennette was going very well. It would result in nine papers—published between 1944 and 1946— that Hilary would co-author with Lennette. Moreover, Lennette was interested in other viruses, in addition to yellow fever. Thus, their co-authored papers included studies of Venezuelan equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, and West Nile virus, as well as yellow fever.

Most importantly, Koprowski’s work under Lennette introduced him to Max Theiler’s methods and approach to viral attenuation. In brief, Theiler found that propagating yellow fever virus in an unnatural host—chick embryos—caused the virus to adapt to that host, thereby reducing its capacity to cause disease in humans.  Koprowski would later acknowledge that Theiler provided him with a “most encouraging model” for attenuating poliovirus. [Koprowski attenuated poliovirus by propagating it first in mice and then in rats. Recall that Sabin developed his live polio vaccine from attenuated poliovirus that he received from Koprowski (1).] See Asides 3 and 4.

[Aside 3: The rabies vaccine, which Louis Pasteur developed in 1885, is often referred to as the first attenuated virus vaccine. Nevertheless, while Pasteur did passage his vaccine virus in rabbit spinal cords, the virus may have been killed when the spinal cords were later dried for up to fourteen days. Also, in Pasteur’s day, nothing was known about immunity or mutation, and viruses had not yet been identified as microbes distinct from bacteria. The yellow fever vaccine developed by Max Theiler at the Rockefeller Institute (now University) in New York may have been the first deliberately attenuated viral vaccine.]

[Aside 4: Koprowski and Lennette were among the first researchers to observe that infection by one virus (yellow fever, in this instance) might inhibit the growth of another unrelated virus (West Nile virus, in this instance). That is, they had inadvertently detected what later would be known as interferon. Yet while they looked for an anti-viral substance in their tissue culture media, and while their results suggest that it actually was there, they stated in their summary that nonspecific anti-viral factors were not present (5). Koprowski and Lennette collaborated again in the 1970s; this time to investigate subacute sclerosing panencephalitis, a rare late complication of measles infection that results in neurodegeneration.]

Hilary continued to give piano recitals in Brazil, regretting only that he did not have time to practice the piano as much as he would have liked. Nonetheless, his piano playing expanded his circle of friends to include musicians, artists and writers, in addition to his fellow scientists. Moreover, Irena was satisfied with her medical practice, and with the many friends and rich social life that she and Hilary had in Brazil.

Earlier, in 1940, while Hilary was still in Rome, and expecting that the family would soon have to leave Europe, he believed that the United States would likely be the best destination for them. Thus, he applied to the United States for visas. He had nearly forgotten those applications when, in 1944, their numbers came up.

The Koprowski family now faced somewhat of a dilemma. It was happily settled in Brazil, and had no prospects in the United States. On the other hand, the Rockefeller Foundation’s yellow fever project was drawing to a close, and the Foundation was planning to leave Rio. Importantly, coming to America was now a “dream come true (3)”.  So, in December 1944, the Koprowskis boarded an aging steamer in Brazil, and sailed under wartime blackout conditions, through German submarine-infested waters, for New York City.

During Hilary’s his first days in America, he used the Rockefeller Institute library in Manhattan to work on manuscripts reporting his research in Brazil. During one of his visits to the Rockefeller, he happened to meet Peter Olitzky (Aside 5), an early polio researcher there, who arranged for Hilary to meet Harold Cox, the director of the virology department at Lederle Laboratories, in Pearl River, New York.  Hilary interviewed with Cox, who offered him a research position at Lederle, which Hilary accepted. Meanwhile, Irena was appointed an assistant pathologist at Cornell Medical College in Manhattan.

[Aside 5: In 1936, Olitzky and Sabin collaborated on a study at the Rockefeller Institute, which, although carefully done, wrongly concluded that poliovirus could attack nerve cells only; a result that did not bode well for the development of an attenuated polio vaccine.]

At Lederle, Hilary began the experiments that led to the world’s first successful polio vaccine. In 1950 he tested the live vaccine in eighteen mentally disabled children at Letchworth Village (1). None of these children had antibodies against poliovirus before he vaccinated them, but each of them was producing poliovirus antibodies after receiving the vaccine. Importantly, none of the children suffered ill effects. What’s more, Koprowski did not initiate the test. Rather, a Letchworth Village physician, fearing an outbreak of polio at the facility, came to Koprowski’s office at Lederle, requesting that Koprowski vaccinate the Letchworth children (1).

References:

   

  1. Vaccine Research Using Children, Posted on the blog July 7, 2016.
  2. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.
  3. Roger Vaughan, Listen to the Music: The Life of Hilary Koprowski. Springer-Verlag, 2000.
  4. The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 4014.
  5. Lennette EH, Koprowski H., 1946. Interference between viruses in tissue culture, Journal of Experimental Medicine, 83:195–219.

 

 

 

 

 

John Enders: “The Father of Modern Vaccines”

John Enders (1897- 1985) was one of the subjects of a recent posting, Vaccine Research Using Children (1). In the 1950s, Enders used severely handicapped children at the Walter E. Fernald State School in Massachusetts to test his measles vaccine—a vaccine that may have saved well over 100 million lives. Irrespective of the ethical issues raised by the incident at the Fernald School, Nobel laureate John Enders was one of the most highly renowned of virologists, and there is much more to his story, some of which is told here.

John F. Enders, November 17, 1961
John F. Enders, November 17, 1961

Enders grew up in West Hartford, Connecticut. His father, who was CEO of the Hartford National Bank, left the Enders family a fortune of $19 million when he passed away. Thus, John Enders became financially independent, which may help to account for his rather atypical path to a career in biomedical research.

Enders was under no pressure to decide on a vocation, and had no particular objective in mind when he enrolled at Yale University in 1915. In 1917 (during the First World War) he interrupted his Yale studies to enlist in the Naval Reserve. He became a Navy pilot and then a flight instructor. After three years of naval service, Enders returned to Yale to complete his undergraduate studies.

After Enders graduated from Yale he tried his hand at selling real estate in Hartford. However, selling real estate troubled him, in part because he believed that people ought to know whether or not they wanted to buy a house, rather than needing to be sold (2, 3). Thus, Enders considered other callings, finally deciding to prepare for a career teaching English literature.

What might have motivated that particular choice? Here is one possibility. During the years when Enders was growing up in West Hartford, his father handled the financial affairs of several celebrated New England writers, including Mark Twain. [The young Enders always admired Twain’s immaculate white suits whenever he visited the Enders home (3).] So, perhaps Enders’ early exposure to eminent writers among his father’s clients planted the seed for his interest in literature. In any case, Enders enrolled at Harvard to pursue graduate studies in preparation for his new calling.

Enders received his M.A. degree in English Literature from Harvard in 1922. Moreover, he was making substantial progress towards his Ph.D., when his career took yet another rather dramatic turn; one reminiscent of that taken later by Harold Varmus, who likewise did graduate studies in English literature at Harvard, with the intent of becoming an English teacher (4).

The changes in the career plans of both Enders and Varmus—from teaching English literature to biomedical research—were prompted by the friends each had who were at Harvard Medical School. Varmus’ friends were his former classmates from Amherst College. Enders first met his friends from among his fellow boarders at his Brookline rooming house.

Dr. Hugh Ward, an instructor in Harvard’s Department of Bacteriology and Immunology, was one of the friends Enders met at his rooming house. Enders wrote, “We soon became friends, and thus I fell into the habit of going to the laboratory with him in the evening and watching him work (5).” Enders was singularly impressed by Ward’s enthusiasm for his research (5).

During one of the trips that Ward and Enders made to the laboratory, Ward introduced Enders to Hans Zinsser, Head of Harvard’s Department of Bacteriology and Immunology. Zinsser was an eminent microbiologist, best known for isolating the typhus bacterium and for developing a vaccine against it.

Enders soon became fascinated by the research in Zinsser’s lab. So, at 30-years-of-age, and on the verge of completing his Ph.D. in English Literature, Enders changed career plans once again; this time to begin studies toward a doctorate in bacteriology and immunology, under Zinsser’s mentorship.

Zinsser, a distinguished microbiologist, was also a sufficiently accomplished poet to have some of his verses published in The Atlantic Monthly. That aspect of Zinsser likely impressed the literate Enders, who described his mentor as: “A man of superlative energy. Literature, politics, history, and science-all he discussed with spontaneity and without self-consciousness. Everything was illuminated by an apt allusion drawn from the most diverse sources, or by a witty tale. Voltaire seemed just around the corner, and Laurence Sterne upon the stair. . . . Under such influences, the laboratory became much more than a place just to work and teach; it became a way of life (3).”

Enders was awarded his Ph.D. in Bacteriology and Immunology in 1930. Afterwards, he remained at Harvard, as a member of the teaching staff, until 1946, when he established his own laboratory at the Children’s Medical Center in Boston.

Why might Enders have been satisfied staying so long at Harvard, for the most part as Zinsser’s underling? Perhaps that too might be explained by his financial independence. In any case, in 1939, while Enders was still at Harvard, he initiated the singularly significant course of research for which he is best remembered.

In 1939, in collaboration with Dr. Alto Feller and Thomas Weller (then a senior medical student), Enders began to develop procedures to propagate vaccinia virus in cell culture. After achieving that goal, the Enders team applied their cell culture procedures to propagate other viruses, including influenza and mumps viruses.

Enders and his coworkers were not the first researchers to grow viruses in cell culture. However, they were the first to do so consistently and routinely. Thus, the Enders lab launched the “modern” era of virus research in vitro. Virology could now advance much more quickly than before, since most virologists would no longer need to grow, or study their viruses only in live animals.

A recurrent theme on the blog is that key scientific discoveries may well be serendipitous. The case in point here was the unforeseen 1949 discovery by Enders and his young collaborators, Tom Weller and Frederick Robbins, that poliovirus could be grown in cultured cells. That crucial discovery made it possible for Jonas Salk and Albert Sabin to generate a virtually unlimited amount of poliovirus and, thus, to create their polio vaccines. Importantly, the discovery happened at a time when polio researchers believed that poliovirus could grow only in nerve cells. Their dilemma was that nerve cells could not be cultured in the laboratory.

Enders, Weller, and Robbins were not working on polio, nor did they have any immediate intention of working on polio when they made their finding. In fact, when the thirty-year-old Robbins (see Aside 1) came to work with Enders, he proclaimed that he wanted to work on any virus, except polio (6).

[Aside 1: Weller was one year older than Robbins. Both had been Army bacteriologists during the Second World War, and they were classmates and roommates at Harvard Medical School when they came to Enders for research experience. Robbins’ father-in-law, John Northrop, shared the 1946 Nobel Prize in chemistry with James Sumner and Wendell Stanley (7). In 1954, Robbins joined his father-in-law as a Nobel laureate (see below).]

The Enders team was trying to grow varicella (the chicken pox virus) when, on a whim; they made their critical discovery. It happened as follows. While attempting to propagate varicella virus in a mixed culture of human embryonic skin and muscle cells, they happened to have some extra flasks of the cell cultures at hand. And, since they also had a sample of poliovirus nearby in their lab storage cabinet; they just happened to inoculate the extra cell cultures with polio virus.

The poliovirus-infected cultures were incubated for twenty days, with three changes of media. Then, Enders, Weller, and Robbins asked whether highly diluted extracts of the cultures might induce paralysis in their test mice. When those highly diluted extracts indeed caused paralysis in the mice, they knew that poliovirus had grown in the cultures. See Aside 2.

[Aside 2: Whereas Enders, Weller, and Robbins did not have pressing plans to test whether poliovirus might grow in non-neuronal cells, they probably were aware of already available evidence that poliovirus might not be strictly neurotropic. For instance, large amounts of poliovirus had been found in the gastrointestinal tract.]

Despite the exceptional significance of their discovery, Robbins said, “It was all very simple (6).” Weller referred to the discovery as a “fortuitous circumstance (6).” Enders said, “I guess we were foolish (6)”—rather modest words from a scholar of language and literature. See Aside 3.

[Aside 3: Current researchers and students might note that Enders’ entire research budget amounted to a grand total of two hundred dollars per year! The lab did not have a technician, and Weller and Robbins spent much of their time preparing cells, media, and reagents, as well as washing, plugging, and sterilizing their glassware.]

In 1954, Enders, Weller, and Robbins were awarded the Nobel Prize for Physiology or Medicine for their polio discovery. Interestingly, they were the only polio researchers to receive the Nobel award. The more famous Salk and Sabin never received that honor (8).

If Enders were so inclined, might he have produced a polio vaccine before Salk and Sabin? Weller and Robbins wanted to pursue the vaccine project, and Enders agreed that they had the means to do so. In fact, Weller actually had generated attenuated poliovirus strains by long-term propagation of the virus in culture; a first step in the development of a vaccine (3). Yet for reasons that are not clear, Enders counseled his enthusiastic young colleagues to resist the temptation (6). See Aside 4.

[Aside 4: Enders may have spared Weller and Robbins the sort of anguish that Salk experienced when some of his killed vaccine lots, which contained incompletely inactivated poliovirus, caused paralytic poliomyelitis in some 260 children (8).]

The Enders poliovirus group began to disperse, beginning in 1952 when Robbins became a professor of pediatrics at Western Reserve. Weller left in 1954 to become chairman of the Department of Tropical Public Health at Harvard.

Regardless of whether Enders might have regretted not pursuing the polio vaccine, he soon would play a hands-on role in the development of the measles vaccine. The first critical step in that project occurred in1954, at the time when the Salk polio vaccine was undergoing field trials. It was then that Enders and a new young coworker, pediatric resident Thomas Peebles (Aside 5), succeeded in cultivating measles virus in cell culture for the first time.

[Aside 5: Enders was known for nurturing bright young investigators. His latest protégé, Tom Peebles, spent four years in the Navy, as a pilot, before enrolling at Harvard Medical School. Peebles graduated from medical school in 1951, and then did an internship at Mass General, before coming to the Enders lab to do research on infectious diseases in children. When Enders suggested to Peebles that he might try working on measles, Peebles eagerly accepted.]

Here is a piece of the measles vaccine story that happened before Peebles’ success growing the virus in cell culture. At the very start of the vaccine project, Enders and Peebles were stymied in their attempts to get hold of a sample of measles virus to work with. Their quest for the virus began with Peebles searching the Enders laboratory freezers for a sample. Finding none there, Peebles next inquired at Boston area health centers; still without success. After several more months of fruitless searching, Peebles received an unexpected phone call from the school physician at the Fay School (a private boarding school for Boys in a Boston suburb), telling him about a measles outbreak at the school. Peebles immediately rushed to the school, where he took throat swabs, as well as blood and stool samples from several of the school’s young patients. He then rushed back to the Enders laboratory, where he immediately inoculated human infant kidney cells with his samples. [Enders obtained the cells from a pediatric neurosurgeon colleague, who treated hydrocephalus in infants by excising a kidney, and shunting cerebrospinal fluid directly to the urethra.]

Peebles monitored the inoculated kidney cell cultures for the next several weeks, hoping for a sign of a virus replicating in them. Seeing no such indication of a virus in the cultures, Peebles made a second trip to the Fay School, which, like the first trip, was unproductive.

On a third trip to the school, Peebles obtained a sample from an 11-year-old boy, David Edmonston. The sample from young Edmonston indeed seemed to affect the kidney cell cultures. Still, Peebles needed to carry out several additional experiments before he could convince a skeptical Enders and Weller—first, that a virus had replicated in the cultures and, second, that it was measles. Peebles convinced the two doubters by demonstrating that serum from each of twelve convalescing measles patients prevented the virus from causing cytopathic effects in the inoculated cell cultures. That is, the convalescent serum neutralized the virus. The measles virus growing in those cultures was named for its source. It is the now famous Edmonston strain.

Enders, in collaboration with Drs.Milan Milovanovic and Anna Mitus, next showed that the Edmonston strain could be propagated in chick embryos (3). Then, working with Dr. Samuel Katz (1), Enders showed that the egg-adapted virus could be propagated in chicken cell cultures.

By 1958, Enders, Katz, and Dr. Donald Medearis showed that the Edmonston measles virus could be attenuated by propagating it in chicken cells. Moreover, the attenuated virus produced immunity in monkeys, while not causing disease (3). Thus, the attenuated Edmonston strain became the first measles vaccine. [Tests of the vaccine in humans led to the episode at the Fernald School (1).]

The Enders measles vaccine was attenuated further by Maurice Hilleman at Merck (9). In 1971 it was incorporated into the Merck MMR combination vaccine against measles, mumps, and rubella (9, 10).

The MMR vaccine has had a remarkable safety record, and it was widely accepted until 1997; the time when the now discredited claim that the vaccine is linked to autism first emerged (10). However, even prior to the MMR/autism controversy, vaccine non-compliance was already a problem. But, in that earlier time, parents were declining to have their children vaccinated, not because of safety issues, but rather because they questioned the severity of measles. Ironically, that was why David Edmonston refused to have his own son receive the vaccine.

Despite receiving the Nobel Prize for his polio work, Enders maintained that developing the measles vaccine was more personally satisfying to him and more socially significant (3).

References:

  1. Vaccine Research Using Children, Posted on the blog July 7, 2016.
  2. John F. Enders-Biographical, The Nobel Prize in Physiology or Medicine 1954. From Nobel Lectures, Physiology or Medicine 1942-1962, Elsevier Publishing Company, Amsterdam, 1964.
  3. Weller TH, Robbins FC, John Franklin Enders 1897-1995, A Biographical Memoir www.nasonline.org/publications/…/endersjohn.pdf [An excellent review of Enders’ life and career.]
  4. Harold Varmus: From English Literature Major to Nobel Prize-Winning Cancer Researcher, Posted on the blog January 5, 2016.
  5. John F. Enders, “Personal recollections of Dr. Hugh Ward,” Australian Journal of Experimental Biology 41:(1963):381-84. [This is the source of the quotation in the text. I found it in reference 3.]
  6. Greer Williams, Virus Hunters, Alfred A. Knopf, 1960.
  7. Wendell Stanley: First to Crystallize a Virus, Posted on the blog April 23, 2015.
  8. .Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.
  9.  Maurice Hilleman: Unsung Giant of Vaccinology, Posted on the blog April 24, 2014.
  10.  Andrew Wakefield and the Measles Vaccine Controversy, Posted on the blog February 9, 2015.

 

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/

 

 

Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob

I am intrigued by the genealogies of our leading scientists, since their mentors too were often preeminent scientists. Earlier postings noted the example of Jonas Salk, who did postgraduate studies under Thomas Francis; one of the great pioneers of medical virology, perhaps best known for developing the first influenza vaccine (1, 2). James Watson, who did his doctoral studies in Salvatore Luria’s laboratory, and Renato Dulbecco, who trained under both Luria and Max Delbruck (3), are other examples. In fact, Watson and Dulbecco shared a lab bench in Luria’s lab. Howard Temin did his doctoral (and postdoctoral studies too) in Dulbecco’s lab (4). And Delbruck, who hugely influenced the new science of molecular biology, did his doctoral studies under Max Born, the 1954 Nobel Laureate in physics. Moreover, Delbruck later served as an assistant to Lisa Meitner (5).

Important research paths were undertaken, and major contributions were made, which resulted from less formal interactions between budding young scientists and top scientists of the day. Howard Temin’s chance encounter with Harry Rubin, while on a mission to Dulbecco’s lab, is a case in point (4).

Our last posting told how Louis Pasteur came within a whisker of adding the discovery of viruses to his list of extraordinary achievements (6). Robert Koch played a part in that story for developing his famous postulates, which provided the standard for demonstrating that a particular microbe causes a particular disease.

The Pasteur article also noted that in 1898 Friedrich Loeffler and Paul Frosch isolated the foot and mouth disease virus; the first virus isolated from animals. However, the piece did not point up that Loeffler had trained under Robert Koch. Also, it did not underscore the special significance of what Loeffler and Frosch achieved. In brief, by the 1890s Dmitry Ivanovsky and Martinus Beijerinck had independently discovered that the agent responsible for tobacco mosaic disease passes through bacterium-proof filters. Nevertheless, neither Ivanovsy nor Beijerinck appreciated the implication of their observation. Ivanovsky believed his filters might be defective, while Beijerinck thought the disease was caused by a “living liquid.” In contrast, Loeffler and Frosch, in addition to isolating the first virus that is pathogenic in animals, also carefully considered all possible explanations for their experimental findings, and then were the first to conclude the existence of a kind of microbe too small to be retained by bacterium-proof filters, and too small to be seen under a microscope, and that will not grow on laboratory culture media. They also correctly predicted that smallpox, cowpox, cattle plague, and measles are similarly caused by a “filterable virus.”

Loeffler made another major discovery, fourteen years earlier, in 1884, when he used his mentor’s postulates to identify the bacterium that causes diphtheria, Corynebacterium diphtheriae. Importantly, Loeffler also discovered that when he injected C. diphtheriae into animals, the microbe did not need to spread to the tissues it damaged. This observation led Loeffler to propose the bacteria were secreting a poison or toxin that spread to the remote sites and caused disease there.

Loeffler’s idea of a toxin was a new concept that subsequently was confirmed by Emile Roux, who had been Louis Pasteur’s assistant (6). Using bacterium-proof filters developed by Charles Chamberland in Pasteur’s lab, Roux showed that injecting animals with sterile filtrates of C. diphtheriae cultures caused death with a pathology characteristic of actual diphtheria. Roux was also a co-founder of the Pasteur Institute, where he was responsible for the production of diphtheria anti-toxin; the first effective diphtheria therapy. See Aside 1.

[Aside 1: Earlier, Roux suggested the approach Pasteur used to generate attenuated rabies virus for the Pasteur rabies vaccine (aging spinal cords from rabbits that succumbed to experimental rabies infections of their spinal cords). Roux later withdrew from the rabies project because of a disagreement with Pasteur over whether the rabies vaccine might be safe for use in humans (6).]

So, Loeffler and Roux trained under Koch and Pasteur, respectively. But why might toxin production by C. diphtheriae interest virologists. Well, in 1951, Victor Freeman at the University of Washington showed that the lethal toxins produced by C. diphtheriae (and by Clostridium botulinum as well) are the products of lysogenic bacteriophage carried by the bacteria. This was shown by the finding that avirulent strains of these bacteria became virulent when infected with phages that could be induced from virulent strains. So, are diphtheria and botulism due to bacteria or to viruses? Our chain of genealogies continues with a selective history of lysogeny.

Almost from the beginning of phage research (bacteriophage were discovered independently by Frederick Twort in Great Britain in 1915 and by Félix d’Hérelle in France in 1917), some seemingly normal bacterial cultures were observed to generate phage. Initially, this phenomenon was thought to be a sign of a smoldering, steady state kind of persistent phage infection. Then, during the 1920s and 1930s, the French bacteriologists, Eugene Wollman and his wife Elizabeth, working together on Bacillus megatherium at the Pasteur Institute, provided evidence that instead of a steady state infection, the phage actually enter into a latent form in their host cells; a form in which they might be harmlessly passed from one cell generation to the next. [Considering the state of knowledge back then, note the insightfulness of Eugene Wollman’s 1928 comment, “the two notions of heredity and infection which seemed so completely distinct and in some ways incompatible, . . . almost merge under certain conditions.”] See Aside 2.

[Aside 2: Since some bacterial strains would, on occasion, spontaneously undergo lysis and release bacteriophage, the cryptic bacteriophage they carried were called “lysogenic.” Thus, it is a bit odd that “lysogeny” eventually came to refer to the temperate relationship between these phages and their host cells.]

In the late 1930s, the Wollmans developed a close friendship with Andre Lwoff, their new colleague at the Pasteur Institute. The Wollmans introduced Lwoff to their ideas about lysogeny, but, as Lwoff confesses, he was not then impressed by bacteriophage (7).

The Nazi occupation of Paris during the Second World War began in 1940. From then on, the Jewish Wollmans were prevented from publishing their research findings. Nevertheless, they continued their research at the Pasteur Institute until 1943, when they were seized by the Nazis and sent to Auschwitz. They never were heard from again. Their friend, Lwoff, grieved their loss and became active in the French resistance, gathering intelligence for the Allies, while also hiding downed American airmen in his apartment.

After the war, Lwoff received several honors from the French government for his efforts against the Nazis. He also returned to his research at the Pasteur Institute, studying the genetics of Moraxella; a bacterial pathogen of the human respiratory tract. Because of his work as a microbial geneticist, he was invited to the 1946 Cold Spring Harbor Symposium, where he met Max Delbruck. And as happened to others, meeting Delbruck resulted in Lwoff being seduced by bacteriophage.

Andre Lwoff
Andre Lwoff

Back in Paris, Lwoff’s passionate interest in phages was heightened further by discussions with Jacques Monod, a friend of Max Delbruck, and Lwoff’s neighbor in the attic of the Pasteur Institute. Although Monod was Lwoff’s junior colleague (in fact, it was Lwoff who first stirred Monod’s interest in microbiology), Lwoff’s conversations with the future Nobel Laureate resulted in Lwoff becoming intensely fascinated by lysogeny, which he began to study in 1949 (7).

Because of Lwoff’s earlier friendship with the Wollmans, he chose to study a lysogenic strain of B. megatarium. And, making use of techniques he learned from Renato Dulbecco during a brief stint at Cal Tech, he was able to follow a single lysogenic bacterium, which enabled him to observe that a bacterium could go through multiple rounds of replication without liberating virus. What’s more, he discovered that the phages are released in a burst when the cell lyses, thereby dispelling the still current notion that phages are liberated continuously by lysogenic bacteria. Furthermore, Lwoff showed that lysogenic bacteria usually do not contain phage particles, since none are detected when the cells are experimentally lysed with lysozyme; confirming the earlier (1937) findings of the Wollmans.

Lwoff went on to show that temperate phage genomes are maintained in a previously unknown integrated state in their host cell, and he gave the integrated phage genomes a name, “prophage.” He also discovered, unexpectedly, that irradiating lysogenic bacteria with ultraviolet light could induce the temperate phages to emerge from their latent state, and then replicate in, and lyse their host cells. And, he discovered that the phages lyse their host bacterial cells by producing enzymes that destroy bacterial cell walls.

Prophage
Prophage

Lwoff’s elucidation of the fundamental nature of lysogeny in bacteria would later provide a paradigm for the DNA tumor viruses, the herpesviruses, the oncogenic retroviruses, and HIV. He was awarded a share of the 1965 Nobel Prize for physiology or medicine for his lysogeny research. He shared the award with his fellow Pasteur Institute scientists, François Jacob and Jacques Monod, who received their awards for their pioneering studies of gene regulation in E. Coli.

A rather intriguing aspect of this story is that Lwoff was joined in his research on lysogeny at the Pasteur Institute by Elie Wollman; the son of Eugene and Elizabeth. Elie, born in 1917, escaped from the Nazis in Paris in 1940 and worked in the French resistance as a physician. In 1946, after the war, he came to the Pasteur Institute, where he took its microbiology course and then became Lwoff’s research assistant. Then, in 1947, Elie too happened to meet Max Delbruck (in Paris in this instance) and was invited to join the Cal Tech phage group, where he spent the next two years. See Aside 3.

Elie Wollman
Elie Wollman

[Aside 3: By the early 1940s, the then young Cal Tech “phage group,” headed by Max Delbruck, was on its way to becoming the World’s great center for phage research (5). However, the American group had little interest in lysogeny, since Delbrück neither believed in it, nor saw its importance. Instead, Delbruck was totally committed to the study of lytic phages. Then, during the late 1940s, Delbruck began to lose interest in molecular biology and looked for new research directions. When he thought of turning his attention to brain function, he asked his group to put together a series of seminars based on papers written by prominent neuroscientists of the day. Elie Wollman was the only member of the Cal Tech group who declined to participate in that endeavor, since he was totally committed to bacteriophage. Moreover, Elie was the one who finally convinced Delbruck that “such a thing as lysogeny does exist (7).”

Elie himself tells us that when he looked into a bibliographical index at Cal Tech, he came across an index card referring to his parent’s 1937 paper, which reported their finding that lysogenic cells contain a non-infectious form of the phage (8). “Delbruck’s comment on the card was “Nonsense.”]

After Eli’s two-year stint with Delbruck in Pasadena, he returned to the Pasteur Institute. Meanwhile, Francois Jacob had come to the Institute in the hope of beginning a research career in genetics under the tutelage of either Lwoff or Monod. Before that, in 1940, Jacob, who also was Jewish, left medical school in occupied France to join Free French Forces in London. He then served as a medical officer in North Africa, where he was wounded, and was later severely wounded at Normandy in August 1944, ending his dream of becoming a surgeon.

Francois Jacob
Francois Jacob

Initially, Jacob was spurned by both Lwoff and Monod, but was finally taken on by Lwoff, who suggested that he, Jacob, start work on “the induction of the prophage.” Jacob confesses he had no idea what that meant, but he accepted the project. Thus it came to pass that Francois Jacob and Elie Wollman established a particularly close and friendly collaboration, in which they turned their attention to the lambda prophage of E. coli. Their initial goal was to clarify the events of bacterial conjugation so that they might then understand the phenomenon whereby a temperate phage carried by a lysogenic bacterium is activated to undergo vegetative replication when that bacterium conjugates with, and transfers its integrated phage genome to a non-lysogenic bacterium.

To accomplish their goal, Wollman and Jacob began with experiments to locate the lambda genome on the chromosome of the lysogenic cell, and to follow its transfer during conjugation into a non-lysogenic recipient cell. A key feature of their experimental approach was conceived by Wollman (8). It was simply to interrupt conjugation between a lysogenic donor (Hfr) cell and a non-lysogenic recipient (F-minus) cell, at various times, by using a kitchen blender to break the mating cells apart. Using the blender to interrupt conjugation, and also using bacterial strains in which the recipient bacteria contained a set of mutations, and plating the mating mixture on selective media, Wollman and Jacob were able to measure the length of time required for each of the corresponding wild-type genes to be transferred from the Hfr donor cells to the F-minus recipient cells. Indeed, the time intervals between the appearances of each wild type gene in the recipient cells directly correlated with the distances between the genes, as independently determined by recombination frequencies. Thus, the interrupted mating approach gave Wollman and Jacob a new means to construct a genetic map of the bacterium, while also enabling them to locate the integrated phage genome on that map. Their experimental approach also allowed Wollman and Jacob to establish that, during conjugation, the donor cell’s genome is transferred linearly to the recipient cell. [The designation “Hfr” was coined by William Hayes because Hfr strains yielded a high frequency of recombinants when crossed with female strains.]

Importantly, Wollman and Jacob’s study of the activation of a lambda prophage when it enters a non-lysogenic F-minus recipient (a phenomenon they called “zygotic induction”), showed that the temperate state of the lambda prophage is maintained by some regulatory factor present in the cytoplasm of a lysogenic bacterium, but which is absent from a non-lysogenic one. It led to the discovery of a “genetic switch” that regulates the activation of the lysogenic bacteriophage, and of a phage-encoded repressor that controls the switch. These findings are among the first examples of gene regulation, and are credited with generating concepts such as the repressor/operator, which were firmed up by Jacob and Monod in their Nobel Prize-winning studies of the E. coli lac operon. See Aside 4.

[Aside 4: At the time of Wollman and Jacob’s interrupted mating experiments, kitchen blenders had not yet made their way to European stores. Eli was aware of these appliances only because of his earlier stint at Cal Tech. He bought a blender for his wife before returning to France, and then “borrowed” it for these experiments.]

Wollman and Jacob went on to demonstrate that the fertility or F factor, which confers maleness on the donor bacteria, can exist either in an integrated or an autonomous state. Indeed, this was the first description of such a genetic element, for which they coined the term “episome;” a term now largely replaced by “plasmid.”

Wollman and Jacob also determined that the E. coli chromosome is actually a closed circle. The background was as follows. Only one F factor is integrated into the chromosome of each Hfr strain, and that integration occurs at random. And, since the integrated F factor is the origin of the gene transfer process from the Hfr cell to the F-minus cell, interrupted mating experiments with different Hfr strains gave rise to maps with different times of entry for each gene. However, when these time-of-entry maps were taken together, their overlapping regions gave rise to a consistent circular map. The discovery of the circular E. coli chromosome was most intriguing, because all previously known genetic maps were linear. See Aside 5.

[Aside 5: The bacterial strain used by Wollman and Jacob in their study of zygotic induction was, in fact, the original laboratory strain of E. coli (i.e. E. coli K12) that was isolated in1922 from a patient with an intestinal disorder. In 1951, Esther Lederberg discovered that K12 is lysogenic. The discovery happened when she accidentally isolated non-lysogenic or “cured” derivatives of E. coli K12 that could be infected by samples of culture fluid from the parental K12 strain, which sporadically produced low levels of phage. Esther gave the lysogenic phage its name, lambda.

Esther was the wife of Joshua Lederberg, who received a Nobel Prize in 1958 for discovering sexual conjugation in bacteria, and the genetic recombination that might then ensue. Prior to Lederberg’s discoveries, genetic exchange and recombination were not believed to occur in bacteria. Lederberg’s Nobel award was shared with George Beadle and Edward Tatum (the latter was Lederberg’s postdoctoral mentor) for their work in genetics.

Joshua Lederberg, working with Norton Zinder (9), also discovered transduction, whereby a bacterial gene can be transferred from one bacterium to another by means of a bacteriophage vector. And, working together with Esther, Joshua discovered specialized transduction, whereby lambda phage transduces only those bacterial gene sequences in the vicinity of its integration site on its host chromosome. Esther and Joshua also worked together to develop the technique of replica plating, which enabled the selection of bacterial mutants from among hundreds of bacterial colonies on a plate and, more importantly perhaps, to provide direct proof of the spontaneous origin of mutants that have a selective advantage.]

In 1954 Elie Wollman was appointed a laboratory head in his own right at the Pasteur Institute. He retired from research in 1966 to become vice-director of the Institute, which he then rescued from a severe financial crisis. He continued to serve in that role for the next 20 years, while garnering numerous prestigious awards for his research and service.

Francois Jacob earned his doctorate in 1954 for his lysogeny studies. Then, realizing that he and Jacques Monod, his senior neighbor in the Pasteur Institute attic, were actually studying the same phenomenon, gene repression, he entered into a hugely productive collaboration with Monod that led to the elucidation of the genetic switch that regulates beta-galactosidase synthesis in E. coli (9). Their collaboration established the concepts of regulator genes, operons, and messenger RNA, for which they shared in the 1965 Nobel Prize for physiology or medicine, as noted above. See Asides 6 and 7.

Jacques Monod
Jacques Monod

[Aside 6: One of Jacob and Monod’s first experiments was the famous 1957 PaJaMa experiment, carried out in collaboration with Arthur Pardee, who was then on sabbatical at the Pasteur Institute. In brief (for aficionados), a Lac-positive, Hfr strain was grown in an inducer-free media, and then mated, still in an inducer-free media, with a Lac-minus, F-minus strain. (Note that the deletion in the Lac-minus, F-minus strain included the LacI gene, which encodes the yet to be discovered lac repressor.) As expected, in the absence of inducer, no beta-galactosidase is detected initially. But, after the donor DNA sequence, which bears the normal Lac genes (including LacI), is transferred to the Lac-minus recipient, it initially finds no repressor in the recipient cell and begins to synthesize beta-galactosidase. Then, as the donor cell’s lac repressor gene begins to be expressed in the recipient cell, in the inducer-free media, expression of the donor cell’s beta-galactosidase gene ceases. The PaJaMa experiment thus showed that the genetic regulation of enzymatic induction depends on a previously unknown regulatory molecule, the repressor.

Notice the similarity between the rationale for the PaJaMa experiment and that of the earlier Wollman and Jacob experiment on zygotic induction. In each instance, a process regulated by a repressor is suddenly in the repressor-free environment of a recipient cell.]

[Aside 7: In June of 1960, Francois Jacob, Matt Meslson, and Sidney Brenner came together in Max Delbruck’s Cal Tech lab to carry out an experiment that confirmed the existence of messenger RNA. The key to the experiment was their ability to distinguish ribosomes present in the cell before infection from ribosomes that might have been made after infection. They cleverly did that by incorporating heavy isotopes into ribosomes before infection, so that they might be separated in a density gradient from ribosome made after infection. Then, they showed that RNA produced by T2 phage in E. Coli associates with ribosomes that were synthesized by the cell entirely before infection. Furthermore, the new phage-specific RNA directs the synthesis of phage-specific proteins on those “old” ribosomes. I vote for this experiment as the most elegant in the entire history of molecular biology (11).]

Incidentally, during the Nazi occupation of Paris, Monod too was active in the French Resistance, eventually becoming chief of staff of the French Forces of the Interior. In that capacity, he helped to prepare for the Allied landings in Normandy. Monod and Jacob each received France’s highest honors for their wartime service.See Aside 7.

[Aside 7: I am singularly intrigued by the experiences of Andre Lwoff, Elie Wollman, Francois Jacob, and Jacques Monod during the Second World War. References 3 and 5 recount the wartime experiences of Renato Dulbecco and of Max Delbruck, and of other great scientists of the time. Other posts on the blog give accounts of virologists courageously placing themselves in harm’s way under different circumstances. Examples include pieces featuring Ciro de Quadros, Carlo Urbani, Peter Piot, and Walter Reed.]

References:

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

(2) Ernest Goodpasture and the Egg in the Flu Vaccine, Posted on the blog November 25, 2014.

(3) Renato Dulbecco and the Beginnings of Quantitative Animal Virology, Posted on the blog, December 4, 2013.

(4) Howard Temin: “In from the Cold,” Posted on the blog December 16, 2013
(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013.

(6) Louis Pasteur: One Step Away from Discovering Viruses, Posted on the blog January 7, 2015.

(7) Lwoff, Andre, The Prophage and I, pp. 88-99, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J.D. Watson eds., Cold Spring Harbor Laboratory Press, 1966.

(8) Wollman, Elie L, Bacterial Conjugation, pp. 216-225, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J.D. Watson eds., Cold Spring Harbor Laboratory Press, 1966.

(9) “The Phage in the Letter,” Posted on the blog November 4, 2013.

(9) Francois Jacob, Nobel Lecture, December 11, 1965.

(11) Norkin, Leonard C., Virology: Molecular Biology and Pathogenesis, ASM Press, 2010.

Andre Lwoff

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.

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.