Our October 12, 2017 post, Douglas Lowy, John Schiller, and the Vaccine Against Cervical Cancer, has reached a gratifying number of people. Since some readers might welcome a bit more background vis-à-vis the remarkable human papillomavirus (HPV) life cycle, or details concerning the use of virus-like particles (VLPs) in the experimental stages of the vaccine’s development, or how the vaccine might actually work, here are a few additional points.
The post noted that the replication cycle of HPV is regulated by the differentiation states of the cells making up the layers of an intact, stratified epithelium or mucosae. “Since the outer layer of the skin is comprised of dead cells, cutaneous HPV infection requires a break or puncture of the skin for the virus to access cells of the underlying germinal stratum of the epithelium. In the actively dividing basal cells, the viral genome replicates more frequently than the cellular genome, thus amplifying the viral genome copy number. However, because the viral genes that encode the capsid proteins are not expressed in these cells, progeny virus particles, which might induce an immune response, are not yet produced. As the basal cells differentiate and move up in the epithelium, the viral genomes replicate only once per cell cycle, on average, to maintain the viral genome copy number. Then, as the infected cells go through their final stages of differentiation in the outer layers of the epithelium, the virus life cycle switches to its productive phase. Capsid proteins are produced, and thousands of virus particles are generated from the each of the infected, terminally differentiated cells.” [How cellular differentiation regulates HPV gene expression and replication is detailed in the textbook, Virology: Molecular Biology and Pathogenesis.]
The post noted that by coupling its replication cycle to the differentiated state of the host cell within the stratified epithelium, HPV can produce progeny virus particles only in the terminally differentiated cells that comprise the outermost live cells of the epithelium. In this way, HPV productive infection does not activate an antiviral immune response. [The host’s immune response eventually does clear many HPV infections. Also, the incidence of HPV-associated lesions is higher in immunosuppressed patients.]
Here then is an additional key point. After the amplification stage, the viral genomes replicate in the basal cells, but only in conjunction with cellular DNA replication. In that way, the viral genome copy number is maintained in the basal cells. Moreover, and importantly, when the basal cells divide, one daughter cell remains behind as a basal cell, while the other daughter cell migrates up into the epithelium. Thus, one daughter cell will differentiate and thereby enable the virus to complete its replication cycle—at a level in the epithelium or mucosae beyond the reach of immune attack—while the other daughter cell remains behind in the basal layer, where it sustains the persistent infection.
Another consequence of this remarkable replication cycle is as follows. Since there are no blood or lymphatic vessels in the stratum of the epithelium or mucosae where the productive replication is occurring, the infection tends to remain localized, thereby giving rise to warts or tumors.
Since HPVs are difficult to study and propagate, one might ask how Lowy and Schiller were able to assess the antibody titers that were induced by inoculation with the HPV VLPs. The answer is that they used a pseudovirion-based immune assay. Pseudovirions are essentially VLPs that contain a plasmid that carries a reporter gene.
One last point. I believe it is generally the case that vaccines due not prevent virus infections per se. Rather, they enable the host to bring an infection under control more quickly, before symptoms might arise. Considering that cervical carcinomas may develop after years of virus persistence, despite a continuing immune response against the virus the whole time, how then might the vaccine protect against the cancer? Here is a thought. Bearing in mind that the human immune response naturally clears many HPV infections over time, perhaps the vaccine protects the host by enhancing immune surveillance to clear the infection before the emergence, or malignant progression of HPV-induced lesions. Or, perhaps the vaccine actually prevents infection.
The 2017 Lasker-DeBakey Prize for Clinical Research went to two virologists at the National Cancer Institute, Douglas Lowy, 75, and John Schiller, 64, for developing technologies that led to FDA-approved vaccines against human papillomavirus (HPV) strains that cause cervical carcinoma and other cancers. Lasker awards are considered the United States’ most prestigious biomedical research awards. They often precede a Nobel Prize in Physiology or Medicine. Thus, they are referred to as “America’s Nobels.” Eighty-seven Lasker awardees have gone on to win a Nobel.
Lowy and Schiller’s achievements were prompted by Harald zur Hausen’s 1983 discovery that two HPV subtypes, HPV-16 and HPV-18, together account for about 70% of all cervical cancers. Since more than 120 distinct HPV subtypes had been identified, the high degree of association of cervical carcinoma with only two of these subtypes provided compelling evidence for the viral etiology of cervical carcinoma. Later studies showed that HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are responsible for another 20% of cervical cancers. Thus, an HPV infection can be detected in virtually all cervical carcinomas. Harald zur Hausen was awarded a share of the 2008 Nobel Prize in Physiology or Medicine for his discovery. [His story is told in Harald zur Hausen, Papillomaviruses, and Cervical Cancer, posted June 19, 2015.]
Lowy and Schiller did not begin their work on papillomaviruses with the intent to produce a vaccine. Instead, like many papillomavirus researchers at the time, they were investigating how papillomavirus oncogene products affected cell growth and replication (i.e., how they cause cancer). Toward that end, they were making use of bovine papilloma virus (BPV) in their studies, rather than HPV. BPV was easier to work with than HPV, because BPV, but not HPV, could be studied in standard cell cultures (see Aside 1).
[Aside 1: The replication cycle of HPV depends upon the differentiation states of the cells making up the layers of an intact, stratified epithelium. Details are as follow. Since the outer layer of the skin is comprised of dead cells, cutaneous HPV infection requires a break or puncture of the skin for the virus to access cells of the underlying germinal stratum of the epithelium. In the actively dividing basal cells, the viral genome replicates more frequently than the cellular genome, thus amplifying the viral genome copy number. However, because the viral genes that encode the capsid proteins are not expressed in these cells, progeny virus particles, which might induce an immune response, are not yet produced. As the basal cells differentiate and move up in the epithelium, the viral genomes replicate only once per cell cycle, on average, to maintain the viral genome copy number. Then, as the infected cells go through their final stages of differentiation in the outer layers of the epithelium, the virus life cycle switches to its productive phase. Capsid proteins are produced, and thousands of virus particles are generated from the each of the infected, terminally differentiated cells. Thus, the HPV life cycle is regulated by the differentiated state of the host cell within the stratified epithelium. Because virus production is restricted to the outermost layers of the epithelium, the virus can evade the immune system, such that the infection can persist, and be passed on for years. However, in most instances, the host appears to eventually mount a successful immune response, which clears the infection.
The development of so-called organotypic raft cultures eventually made it possible to study HPV in cell culture. But one could produce only very limited amounts of the virus in that system.]
Working with BPV, Lowy and Schiller developed protocols they would later use when they turned their attention towards an HPV vaccine. One of these protocols was for an assay to measure the titer of neutralizing antibodies against BPV. Importantly, they also discovered that they could generate “virus-like particles” (VLPs), comprised only of the major BPV coat protein (L1). The BPV L1 proteins (which were generated by a baculovirus vector in insect cells) self-assembled into VLPs that were morphologically like actual BPV particles. What’s more, using their assay to measure the titer of neutralizing serum antibodies, they found that the VLPs induced neutralizing antibodies in rabbits that were effective against the actual virus. Importantly, since the VLPs did not contain viral genes, they could not cause cancer.
Again, using their assay for measuring the titer of neutralizing antibodies against BPV, Lowy and Schiller compared the immunogenicity of BVP VLPs, to that of individual BPV proteins. The VLPs indeed are more immunogenic than individual viral proteins, since they induced higher levels of neutralizing antibodies than were induced by individual L1 proteins (see Aside 2).
[Aside 2: The activation of antibody-producing B-cells is triggered by the cross-linking of their antigen-binding B-cell receptors, which is facilitated by the multimeric VLPs, but not by individual viral proteins.]
The innovations resulting from their work with BPV would enable Lowy and Schiller to overcome the formidable challenges they faced when working to develop the HPV vaccine. One obstacle was that HPV cannot replicate in standard cell cultures. Thus, it was difficult to study HPV, and importantly, it also was difficult to propagate it. Being able to propagate substantial amounts of the virus would be necessary to produce a vaccine.
Another obstacle to an HPV vaccine was the potentially unacceptable risk of inoculating people with a virus (either attenuated or killed) that contains known oncogenes. Lowy and Schiller overcame this impediment, and the one noted above, by implementing protocols they previously developed while researching BPV. Specifically, they generated HPV VLPs that were comprised only of the HPV L1 capsid protein, and which induced an immune response that produced protective antibodies. [They used the L1 protein of HPV-16; the most carcinogenic strain of HPV.] In addition, they developed cell lines, which contained high copy numbers of the plasmid that encoded the HPV L1 protein; a step which enabled them to scale-up production of the VLPs.
Together, these breakthroughs made a compelling case for the feasibility of an HPV vaccine. So, Lowy and Schiller prevailed upon several pharmaceutical companies to produce a vaccine in commercial amounts, and to see the vaccine through the clinical trials process. Most companies remained skeptical about the ultimate success of the vaccine. But two companies, Merck and GlaxoSmithKline (which later bought Merck), accepted the challenge. Thus, Merck developed Gardasil, while GlaxoSmithKline developed Cervarix. [The VLPs in Gardasil are produced in yeast, whereas the VLPs from Cervarix are produced in insect cells, via a recombinant baculovirus.]
Clinical trials showed that the Merck and the GlaxoSmithKline vaccines induce significant antibody titers against high-risk HPVs. The US FDA approved the respective HPV vaccines in 2006 and 2009.
The HPV vaccines have had a substantial effect on human health. Consider the following: Cervical cancer is the second most common cause of death from cancer among women worldwide. HPV infection is the cause of virtually all cases of cervical cancer. HPVs also cause 95% of anal cancers, 70% of oropharyngeal cancers (more common in men than in women), 65% of vaginal cancers, 50% of vulvar cancers, and 35% of penile cancers. Next, consider that, since Gardasil and Cervarix were introduced, HPV infection rates have dropped by 50 percent among teen-age girls in U.S., even though only a third of teens between 13 to 17 years-old have received the full course (3 shots) of the vaccine (see Aside 3).
[Aside 3: Current CDC recommendations are as follows: “All kids who are 11 or 12 years old should get two shots of HPV vaccine six to twelve months apart. Adolescents who receive their two shots less than five months apart will require a third dose of HPV vaccine…If your teen hasn’t gotten the vaccine yet, talk to their doctor or nurse about getting it for them as soon as possible. If your child is older than 14 years, three shots will need to be given over 6 months. Also, three doses are still recommended for people with certain immunocompromising conditions aged 9 through 26 years.”]
Although he HPV vaccines have significantly reduced the incidence of cervical cancer in the developed world, the rates of cervical cancer in the United States are needlessly high, in comparison to the rates in other industrialized nations. The HPV vaccines have a loweracceptance rate than other childhood vaccines in the United States, perhaps because many American parents, some of whom associate with the religious right, have reservations about vaccinating their children against a sexually transmitted disease. Other individuals, liberals as well as conservatives, may oppose vaccines in general because they distrust pharmaceutical companies, or because they resent government interference in their lives. In any case, the CDC found no evidence of any increase in sexual activity among teenage girls who received the vaccine. Nor did it report any major ill effects]. See Aside 4.
[Aside 4: Since HPVs alone account for about 5% of all human cancers worldwide, we might ask what percentage of human cancers have a viral etiology. Hepatitis C virus, a flavivirus, and hepatitis B virus, a hepadnavirus, cause hepatocellular carcinoma; Epstein-Barr virus (EBV), a herpesvirus, causes Burkitt’s lymphoma and nasopharyngeal carcinoma; human herpesvirus 8 (HHV-8), causes Kaposi’s sarcoma, the most frequent cancer seen in AIDS patients; the human T-lymphotropic retrovirus I (HTLV-I) induces adult T-cell leukemia; and Merkel cell polyomavirus (MCV) causes its eponymous cancer. Together, viruses may account for as many as 20% of all human cancers, and a similar percentage of all deaths due to cancer!
As shown by the HPV vaccine, and earlier by vaccines against hepatitis B, cancers that have a viral etiology might be prevented by vaccination. Apropos hepatitis B, in the late 1980s, Merck and GlaxoSmithKline developed the respective hepatitis B vaccines, Recombivax and Engerix. Like, the HPV vaccines, they are based on VLPs, and they have significantly reduced the incidence of HBV-associated hepatoma; once one of the most lethal cancers.
Bacterial and parasitic infections too may lead to cancer. For example, Heliobacter pylori infections may lead to stomach cancer, and Schistosoma, Opisthorchis, and Clonorchis have been linked to rectum and bladder cancers in areas of Northern Africa and Southeast Asia, where those pathogens are prevalent.]
Lowy and Schiller’s achievement stands out as a superb example of basic research translating into very considerable public health benefits. Moreover, it serves as a strong endorsement for government support of basic research. To these points, Schiller noted that companies would not likely have carried out the necessary basic research and development necessary to produce the HPV vaccine, considering the seemingly small likelihood of success, as suggested by earlier failed attempts to develop a vaccine.
At a September 6, 2017 press conference announcing the Lasker-DeBakey Clinical Medical Research Award, Lowry related that he first learned about vaccines in 1955, when he went with his mother, a physician, to a talk by Jonas Salk about his then new polio vaccine. “I learned far more about polio virus and the vaccine than was probably appropriate for a 12-year-old boy.” Many years afterwards, Lowy began his “extraordinarily effective” collaboration with Schiller, which has endured for more than 30 years.
Schiller said that a high point in his career was taking his daughter to get the vaccine he helped to create. “We first came up with the idea of the vaccine when she was born and it became available when she was 13 years old (1).”
A Revolutionary Vaccine, New York Times, September 6, 2017.
Variolation was the world’s first practical measure to control smallpox. It was developed in China in the 11th century. The procedure involved inoculating uninfected individuals with material from the scabs of individuals who survived smallpox infection.
It was brought to England for the first time in 1721, by Lady Mary Wortley Montague—the wife of the British ambassador to Turkey—when she returned home after learning of the practice in Istanbul. It was brought to Colonial North America the same year by the prominent Puritan minister, Cotton Mather. New England was then experiencing a major smallpox epidemic.
Mather is perhaps best known for his role in the Salem witchcraft trials. He learned of variolation not from the British, but from his African slave, Onesimus, who had been inoculated as child in Africa. Onesimus was a “gift” to Mather in 1706, from his Boston congregation. Variolation was used in western Africa when Onesimus was a child. The practice may have been brought there by caravans from Arabia. In any case, an enslaved African man played a key role in bringing variolation to North America.
In 1777, during the American War for Independence, General George Washington required the entire Continental Army to undergo variolation. Bearing in mind that more than two-thirds of the American casualties during the War resulted from disease, and that smallpox alone caused a total of about 100,00 deaths, some historians maintain that Washington’s policy of enforced variolation was his most important strategic decision of his entire military career.
Variolation nonetheless encompassed risks—a fatality rate of 1 to 2%—that would be unacceptable today. Not surprisingly then, the colonial and Revolutionary War periods were times when public fear and restrictive laws often prevented the use of variolation. Nonetheless, Thomas Jefferson was a lifelong advocate of smallpox-prevention measures. In 1766, Jefferson traveled to Philadelphia to undergo variolation, since the practice was banned in his native Virginia. As a lawyer in 1768, Jefferson defended a Norfolk doctor, whose house was burned down by a mob because he practiced variolation. In 1769, Jefferson placed a bill before the Virginia General Assembly to reduce the 1769 restrictions against variolation. In the 1770s and 1780s, he had his children and his enslaved servants (including Sally Hemings, his wife’s half-sister, and mother of several of his enslaved children) undergo the procedure.
In 1799, Boston physician and founder of Harvard Medical School, Benjamin Waterhouse, introduced Edward Jenner’s new cowpox-based smallpox vaccine to New England. Wanting to spread word of the vaccine to the rest of the new country, in 1781 Waterhouse sent a sample to his friend, Thomas Jefferson. At the President’s House in Washington, Jefferson selected an enslaved kitchen worker to be the first recipient of the vaccine. However, the vaccine did not take. So, Jefferson then had two of his slaves at Monticello undergo vaccination. When those vaccinations proved to be successful (as shown by exposure to actual smallpox), Jefferson serially transmitted the vaccine from the two original vaccines to almost fifty other slaves. By means of serial inoculations he then sent vaccine material to Washington (the city), and from there the vaccine traveled to Philadelphia and beyond. So, Thomas Jefferson, and his African slaves, played a seminal role in protecting many people in the new United States from smallpox.
Jefferson was an amateur, but serious scientist. He kept detailed notes of his observations, and corresponded with Jenner. Here is what he wrote to Waterhouse about the appearance of papules at the vaccination site:
As far as my observation went, the most premature cases presented a pellucid liquor the sixth day, which continued in that form the sixth, seventh, and eighth days, when it began to thicken, appear yellowish, and to be environed with inflammation. The most tardy cases offered matter on the eighth day, which continued thin and limpid the eighth, ninth, and tenth days. [http://www.smithsonianmag.com/smart-news/thomas-jefferson-conducted-early-smallpox-vaccine-trials-180954146/]
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.
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.”’
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.
George Klein, professor emeritus of tumor biology at the Karolinska Institute in Stockholm, where he worked with his wife Eva from the very beginning, passed away on December 10, 2016, at the age of 91. Klein was best known for discovering that Epstein-Barr virus (EBV)—the herpesvirus now known to cause infectious mononucleosis—causes two human cancers, Burkitt’s lymphoma and nasopharyngeal carcinoma. Moreover, Klein discovered that EBV triggers Burkitt’s lymphoma by facilitating a chromosomal translocation of the cellular c-myc oncogene, resulting in its constitutive expression. Klein also played pioneering roles in developing the concept of tumor-suppressor genes, and in opening the field of tumor immunology. Klein’s key discoveries are summarized below. But, first, Klein, like several other protagonists in these tales, was profoundly affected by events of the Second World War, and by the early days of the Cold War that followed.
George Klein’s Jewish family moved from Eastern Slovakia to Budapest in 1930. Nineteen-year-old George was working as an assistant secretary to the Jewish Council in Budapest when Nazi Germany began its occupation of Hungary in March 1944. Because George had been working for the Jewish Council, in April 1944 he chanced that to see the Vrba-Wetzler Report, known at the time as the “Auschwitz Report.” It was written by, and was secretly transmitted to the Jewish Council by Rudolf Vrba and Alfred Wetzler, two escapees from Auschwitz. It described firsthand the fate of Jews arriving at Auschwitz, and was meant to warn Hungary’s Jews, so that they might hide from, or rebel against their Nazi oppressors.
The Auschwitz report was not publicized in Hungary for reasons explained below. However, George’s supervisor at the Jewish Council gave him permission to tell his relatives and friends of what the report revealed. But they, like most Hungarian Jews, could not believe that such atrocities could actually be taking place. [During May, June, and July 1944, 437,000 Hungarian Jews were deported to Auschwitz; to be “resettled” according to the Nazis. But, in fact, most were murdered in the gas chambers.]
Klein was arrested and pressed into forced labor by the Nazis. Afterwards, since he knew the contents of the Auschwitz Report, he fled when he was about to be ordered to board one of the deportation trains to Auschwitz. Having escaped from almost certain death, he lived underground until January 1945, when the Russian Army liberated Budapest.
Forty-three years later, Klein was watching, Shoa, the monumental (nine-hour-long) French documentary film about the holocaust. Watching the movie, Klein chanced to see a man named Vrba (one of the six principal holocaust witnesses in the film) describe his experiences as a prisoner in Auschwitz. The events that Vrba recounted horrified Klein.
Later in the film, as Vrba described his escape from Auschwitz, Klein suddenly realized, “the report I had been given to read under a promise of secrecy in Budapest in May 1944—at the age of nineteen and at a time when deportations from the Hungarian countryside were at their peak—was identical to the Auschwitz Report of Vrba and Wetzler (1).”
Next in this remarkable tale, Klein decided to try to find Vrba, to “tell him of what enormous help his report had been to me. If I had not known what was awaiting me at the other end of the train trip, I would never have dared to risk an escape. It was not difficult to find Vrba, for it turned out that we were scientific colleagues. He is a professor of neuropharmacology in Vancouver, and I am now (in the Spring of 1987) sitting in a comfortable armchair in the faculty club at a Canadian university, talking with someone who, at first glance, seems quite ordinary. He impresses me as being relaxed and jovial. By now I have also read his book (Escape from Auschwitz, 1964), and I am aware that he has survived more death sentences than anyone else I have ever met (1).”
Vrba (1924–2006), was indeed a professor of pharmacology at the University of British Columbia; a position he held from 1976 until the early 1990s. Note that he and Wetzler were the first prisoners ever to escape from Auschwitz. Vrba’s real name was Walter Rosenberg. Rudolf Vrba was the nom de guerre he used after joining the resistance in his native Czechoslovakia. Afterwards, he made the change legal.
The horrors of the holocaust remained an obsession for Klein, although he was uncertain as to why that was so. “Was it to honor my murdered family, my murdered classmates? Or was it rather to steel myself against the darkest side of our human heritage?” In any case, Auschwitz and the holocaust were the main topics of conversation when Klein met with Vrba.
Vrba took Budapest’s Jewish Council to task for not widely broadcasting the warnings in the Auschwitz Report. He, and others, have alleged that Dr. Kastner, a well-known Zionist leader in Budapest, decided to keep the Report secret, in return for a promise from the Germans to allow sixteen-hundred people, as selected by Kastner, to safely emigrate from Hungary. Klein retorted that he knew Kastner from his work for the Jewish Council, and considered him to be a hero, because he had rescued many, while others tried to rescue only themselves or their own families. [In 1957, Kastner was murdered in Israel by a young man whose family was exterminated by the Nazis. Kastner remains a controversial figure to this day.]
Klein and Vrba next discussed whether dissemination of the Auschwitz Report might have caused Budapest’s Jews to revolt against the Nazi program of annihilation. Klein argued that of the dozen or so people that he warned, no one believed him. Vrba countered, “You were a mere boy. Why would anyone believe what you were saying? The Jews would certainly have believed their responsible leaders (1).” Nonetheless, Vrba conceded that even the prisoners at Auschwitz were in denial of what they could see with their own eyes: “…prisoners, who knew full well that no one ever returned from the gas chambers, repressed such knowledge as they themselves lined up for execution in front of the chamber doors.”
Klein asked Vrba how he is able to live and function in Vancouver, a pleasant and friendly place, where no one has the slightest concept of what he endured: “…you must go back constantly to those days. You are called in as a witness at trials of old Nazis or their followers, people who claim that the holocaust never happened. You try to describe something that cannot be described in any human language, you try to explain the incomprehensible, you want people to listen to something they do not want to hear (1).” Vrba, in fact, never did reveal his Auschwitz experience to his colleagues. Vrba explained: “What would have been the use? No one who has not experienced it can understand.” Their conversation went on for almost ten hours. Afterwards, they parted like old friends, despite any differences in their views.
In the Fall of that year, Klein was reunited with Vrba in Paris, together with another newfound friend, German scientist Benno Muller-Hill. In 1966, Muller-Hill was a graduate student in Walter Gilbert’s Harvard laboratory, when he purified the lac repressor; the first genetic control protein to be isolated. Muller-Hill then began a second career lecturing and writing about the role of Nazi doctors and scientists in the holocaust. Klein met Müller-Hill for the first time at a meeting at the Institute for Genetics in Cologne, and the two immediately developed a close friendship.
Muller-Hill was in Paris to visit colleagues at the Pasteur Institute, as well as to meet Vrba. Klein was visiting Paris after attending a scientific meeting in Lyon. Vrba was in Paris at the invitation from the French radio service to refute claims of the ultra-right French leader, Jean Marie Le Pen, that the Nazi gas chambers never existed, and that if the Nazis indeed had any intent to annihilate the Jews, it was merely one of many episodes of the war. [Marine Le Pen, currently a leader of France’s ultra-right National Front, and a candidate for the presidency of France, is Jean Marie’s daughter. She was recently taken to task for denying that French officials and police were complicit in the Nazi roundup of more than 13,000 French Jews in July 1942 (they were later deported to Auschwitz). Le Pen also calls for the deportation of all immigrants from France; a stance that mainly targets Muslims.]
Klein and his two companions ambled about Paris on a beautiful Fall afternoon. They strolled around the Luxembourg Gardens, then continued along the banks of the Seine, turned toward the Latin Quarter, and then stood before the façade of Notre Dame. Yet their minds were elsewhere. “Vrba suggested that we visit the holocaust memorial behind Notre Dame…That walk of only a few minutes took us from the noisy tourist crowd to the silence of the museum’s rooms, where you feel alone and isolated among the symbolic chains and barbed wire. A faint glow of sunlight came in through the narrow openings in the wall. We were surrounded by the voices of the victims…We were all completely speechless. Even Vrba’s macabre sense of humor and his sharp sarcasm had fallen silent for the moment (1).”
After they exited from the memorial, they sat down in a small bistro, where Klein asked his two companions whether German scientists and doctors were actual architects of the holocaust or, instead, merely passive followers. “Benno had concluded from his exhaustive documentation that, contrary to what many wanted so desperately to believe, the ‘euthanasia programs’…and the horrible human experiments… could not be ascribed to a small minority of madmen, opportunists, or charlatans. On the contrary, they had been carried out by quite ordinary and in some instances, eminent physicians and scientists… He (Verba) thought … that would not explain why so many apparently ordinary people took part in the murders without showing any signs of remorse, or how the annihilation program could have been carried out with such efficiency… The discussions between Benno and Vrba continued for several hours (1).”
The day became even more notable later, since Klein had arranged for the threesome to have dinner that evening with Francois Jacob. After a glass of sherry in Jacob’s Latin Quarter apartment, the foursome went to a small restaurant around the corner.
Francois Jacob, and fellow Pasteur Institute scientist Jacques Monod, were awarded Nobel Prizes for their work together on the regulation of lactose metabolism in E. coli (2). More apropos the current episode, Jacob and Monod each received France’s highest military honors for his service during the Second World War—Jacob for his heroism serving with the Free French forces, and Monod for his heroism in the Resistance (2). Yet Jacob’s harrowing escape from Nazi-occupied France at 19-years in age, and his wartime exploits as one of Charles De Gaul’s most highly decorated volunteers, were barely known to his three dinner companions.
At first, Klein was somewhat worried that his friends might not like each other. Jacob often found conversation to be difficult; partly because the thousands of pieces of shrapnel that he carried in his body from the war, made it hard for him to sit comfortably. [Jacob’s wartime wounds prematurely ended his surgical career, and led him to turn to a career in science (2).] But, the get-together didn’t go badly at all.
Conversation eventually turned to the issue of holocaust deniers, as well as to those who would put the past completely behind them. As they talked, the incongruity of the scene suddenly struck Klein. They were sitting in a “first-class Parisian restaurant, surrounded by elegant people, having a very nice dinner in the best French tradition.” “…why did the three of us, with Jacob listening, choose to spend that beautiful Saturday in Paris compulsively focusing our attention on the black birds? We were all citizens of free countries, living well in peaceful times. Were we haunted by feelings of guilt toward the dead? Were we afraid that the whole experience would recur if we let go? We knew that the wide and relentless river of history is rarely influenced by knowledge of the past. In no more than one or two generations, archives of extreme horror turn into scraps of faded paper, with no more influence than dried leaves. I suddenly felt that we were like a traveler with a fear of flying, forcing himself to stay awake and keep his seatbelt buckled during the entire flight, obsessed with the idea that the plane would surely crash if he were to fall asleep. But perhaps we had other motives. Perhaps we wanted to feel a solidarity with each other by selecting a more or less taboo subject for our conversation, one avoided by most others. Or did we try to perform a kind of autopsy, using our brains to understand what human minds are capable of at their worst? Have we appointed our brains to serve as the pathologist and the cadaver at the same time?”
The above recounts only a small sampling of Klein’s conversations with Vrba, Muller-Hill, and Jacob, during their day together in Paris. For more, see reference 1.
In January 1945, 19-year-old George Klein emerged from the Budapest cellar where had been hiding during the last weeks of the German occupation. He gazed on the dead soldiers, civilians, and horses that were frozen in the snow, and was struck by the thought that he had survived, despite the likelihood that he would have ended his 19 years in a Nazi gas chamber or a slave labor camp. However, with the city now in Russian hands, George faced a new threat to his freedom; the Russian patrols that were exporting young Hungarians to labor camps in Russia.
Mindful of the danger on the streets, George was yet eager to begin his medical studies. So, he cautiously dodged the Russian patrols as he made his way to Budapest’s medical school, only to find war-torn deserted buildings and dead soldiers there.
Undeterred by the situation in Budapest, George and a friend set out to Szeged, with the hope of attending the medical school there. The journey of 160 miles took the pair five days, by way of a variety of vehicles, including a Russian military truck. In any case, they were admitted to the Szeged university on the same day that they arrived. And while the school was a shadow of its former self, with all the professors having fled to the West, to George, it was a “previously forbidden paradise (3).”
George spent two years in Szeged, and then returned to Budapest when the University reopened there. Back in Budapest, George fell “desperately” in love with Eva Fisher, a fellow medical student. [George describes their whirlwind romance in reference 3.] George now faced a dilemma. Before he met Eva, he finalized plans to visit Stockholm (under the sponsorship of the Jewish Student Club there). But going to Stockholm would mean leaving Eva behind, under conditions in which travel back into Hungary could be risky. Nonetheless, George went to Stockholm, with Eva believing she would never see him again. Yet the trip would be a defining experience for George and, eventually, would be important for Eva too. In Stockholm, George would learn of, and be riveted by the research of renowned cell biologist Torbjörn Caspersson, at Stockholm’s Karolinska Institute.
Caspersson’s research so enthralled George that he diligently pressed Caspersson for a junior research assistantship in his laboratory. But once George had been accepted by Caspersson, he viewed his situation with a “mixture of ecstatic happiness and enormous anxiety.” “I knew virtually nothing…I was halfway through my medical studies…I was desperately in love with a girl whom I had only known during a summer vacation of eight days and who was on the other side of an increasingly forbidding political barrier (3).”
Despite these misgivings, George knew that his future lay in Sweden, rather than Hungary. He had been accepted into Caspersson’s laboratory, and Hungary was falling increasingly under totalitarian Soviet domination. But Eva was still in communist Hungary. So, George risked returning there with one goal; to marry Eva, and then to leave Hungary for good. “The reunion with Eva confirmed what we both already knew: we wanted to live and work together (3).”
But George and Eva didn’t have the necessary documents to get married, nor did Eva have a passport to leave Hungry. Moreover, communist bureaucrats made it increasingly difficult to obtain these documents. In some instances, up to six weeks might be needed. However, George and Eva were daring and resourceful. When told by a police officer that it would take at least three weeks to obtain a marriage license, George suddenly acted on impulse: “I had always heard others tell of such things but I myself had neither seen nor done it. I pulled a fairly modest bill out of my pocket and put it in the policeman’s hand. ‘Pardon me, how much time was it you said?’ ‘I’ll go get it at once,’ he answered (3).”
With similar persistence and ingenuity, George and Eva obtained all their necessary documents, and they were married that very day! One document, a certificate asserting that neither George nor Eva had a venereal disease, would normally require a three-week lab test. But they beseeched an older colleague, now a doctor at a children’s hospital, to write the certificate for them. Their colleague did so, on his Children’s Hospital stationary. George and Eva then went to the prefecture to be married, only to find a disagreeable marriage official, who was determined to leave work for the day. But, when the official leafed through their papers, and saw the venereal disease certificate written on Children’s Hospital stationary: “He laughed until tears ran down his cheeks. This was the funniest thing he had seen during his whole time in service.” He then gladly married the couple.
As the Iron Curtain descended about Hungary, George and Eva left for Sweden, where they would now continue their medical studies. What’s more, Eva joined George in Caspersson’s laboratory at the Karolinska Institute. The couple would work together at the Karolinska until George’s death at the age of 91. [Eva was born to Jewish parents in Budapest in 1925. In 1944 and 1945, she and several members of her family hid from the Nazis at the Histology Institute of the University of Budapest. Encouraged by Caspersson, Eva had an independent research career, while also collaborating with George. She is best known for discovering natural killer cells, and for generating the Burkitt’s lymphoma cell lines, which she and George studied together (see below).]
We conclude with a brief review of some of George Klein’s contributions to virology and to cancer research.
Tumor immunology: In 1960, George and Eva used methylcholanthrene to induce tumors in mice. Next, they surgically removed the tumors, killed them with irradiation, and inoculated them back into genetically compatible mice. Next, they challenged these mice with cells from a variety of different tumors, and showed that the immune systems of the inoculated mice rejected only those cancer cells that came from the original tumor. Thus, there are tumor-specific antigens that can be recognized by the immune system. See Aside 1.
[Aside 1: Importantly, the tumor resistance seen in these experiments did not arise spontaneously in the original tumor-bearing animals. Instead, it developed in the test mice, in response to sensitization with killed tumor cells. Thus, these experiments per se do not point towards an immune mechanism of tumor surveillance. Nonetheless, harnessing such a mechanism is currently a promising means of cancer therapy, and was a major theme in Klein’s thinking.]
The following year, Klein’s group showed that polyoma virus-induced tumors share a common antigen. Importantly, polyoma virus-induced tumors, and polyoma virus-transformed cells, were rejected irrespective of whether they released virus. Thus, antiviral immunity as such was neither necessary nor sufficient for tumor rejection. This was the first demonstration that tumors caused by a virus might share a common antigen. The Kleins, and others, later found similar “group-specific” transplantation antigens on other virus-induced tumors, including retrovirus-induced lymphomas.
Burkitt’s lymphoma: “Sometime in the mid-1960s, Eva suggested that we should use our experience on virus-induced murine lymphomas to examine a human lymphoma with a presumptive viral etiology. Could we detect group specific antibody responses that might be helpful in tracing a virus? Burkitt’s lymphoma (BL) was the obvious choice (3).” [Burkitt’s lymphoma, originally described by Dennis Burkitt in 1958, is a malignant B-cell lymphoma that is most prevalent in tropical Africa and New Guinea. It is the most common childhood cancer in equatorial Africa. Burkitt first proposed that the lymphoma might have a viral etiology, since its geographic distribution is like that of yellow fever, which is caused by a flavivirus. In 1964, Tony Epstein and Yvonne Barr, by means of electron microscopy, discovered a virus in cells which they cultured from BL tissue, thereby giving credence to Burkitt’s premise.]
Klein’s group identified a membrane antigen (MA) that was expressed in some BL-derived cell cultures. Werner and Gertrude Henle had previously discovered that the MA antigen is a structural protein from a newly discovered herpesvirus—the virus that Epstein and Barr first saw in 1964. Klein decided to call that virus the Epstein Barr virus (EBV). The MA antigen is now known to be one of the EBV envelope glycoproteins. Klein and collaborators later identified complement receptor type 2 (CR2), also known as the complement C3d receptor, as the cell surface attachment protein for the viral MA glycoprotein. CR2 receptors on B cells play a role in enabling the complement system to activate B cells.
By 1970, Klein’s group, in collaboration with Harald zur Hausen, found that the subset of BL-derived cell lines that express MA are, in fact, those that produce EBV. However, more than 90% of the BL cell lines, and all nasopharyngeal carcinomas, were found to contain multiple EBV genomes per cell, irrespective of whether they produced virus. Thus, only a subset of BL and nasopharyngeal carcinoma cells that harbor EBV genomes, actually produce the virus. During this time, the Henles discovered that EBV is the cause of infectious mononucleosis, and that EBV could immortalize normal B cells in culture.
Oncogene activation by chromosomal translocation: A sero-epidemiological study, begun in Uganda in 1971 by Geser and de-The, showed that children with a high EBV load are more likely to develop BL than are children with a low EBV load. Thus, the presence of EBV genomes in a B cell increases the likelihood of it turning into a BL. “But this is still not a satisfactory explanation; some essential element is obviously missing (3).”
What then is the missing event that gives rise to BL? A 1972 study by Manolov and Manolova, Bulgarian scientists working with the Kleins, found that a particular chromosomal marker, 14q+, was present in about 80% of BL tumors. After the Manolovs returned to Bulgaria, the Kleins, in collaboration with Lore Zech, used the chromosomal banding technique recently developed by Caspersson and Zech to examine the BL-cell chromosomes more precisely. They showed that the 14q+ marker was derived from chromosome 8, which broke at the same site (8q24) and underwent a reciprocal translocation with the short arm of either chromosome 2 or chromosome 22. All BLs carried one of the translocations.
Meanwhile, another research group found that carcinogen-induced mouse plasmacytomas are associated with an almost homologous chromosomal translocation. Thus, a common mechanism seemed to underlie two distinct types of tumors, in two distinct species. In each instance, a putative oncogene was translocated to an immunoglobulin locus, which might then have caused the oncogene to be constitutive expressed. A somewhat similar mechanism was reported earlier for the induction of bursal lymphomas in chickens by the avian leukosis virus (ALV) . In that instance, the cellular c-myc gene came under the control of the ALV provirus promotor. What’s more, Michael Cole’s group identified the transposed gene in BL, and in the mouse plasmacytomas, as c-myc. It is not yet clear how EBV infection promotes the chromosomal translocation.
Tumor suppressor genes: In the early 1970s, Klein, and collaborator Henry Harris, played a pioneering role in developing the concept of tumor suppressor genes. They found that when highly malignant mouse cells are fused with normal mouse cells, the hybrid cells are non-malignant when inoculated into genetically compatible mice. That is, tumorgenicity is suppressed by fusion with normal cells. However, tumorgenicity reappears after some apparently important chromosomes, contributed by the normal cell, are lost from the hybrid cells.
In the 1950s and 1960s, two French biologists at the Pasteur Institute, Francois Jacob and Jacques Monod, explained how genes are regulated in bacteria. Their studies of the “lac operon” of E. coli indeed opened up the field of gene regulation, and were a key development in the new science of molecular biology. Their experimental findings also implied the existence of an unstable intermediate between genes and protein synthesis, which eventually led to Jacob’s discovery, in collaboration with Sydney Brenner and Matt Meselson, of messenger RNA (1).
Jacob and Monod shared in the 1965 Nobel Prize for physiology or medicine for their breakthrough studies on gene regulation. Fellow Pasteur Institute scientist, Andre Lwoff, received a share of the award for his pioneering studies on the nature of lysogeny (i.e., how a bacteriophage’s genome can be incorporated into the genome of a host bacteria, and remain latent until being activated by an inducing factor).
In 2013, evolutionary biologist Sean B. Carroll published a book—Brave Genius: A Scientist, a Philosopher, and their Daring Adventures from the French Resistance to the Nobel Prize—that relates how wartime circumstances brought together Jacques Monod and his scientific colleagues Francois Jacob and Andre Lwoff (2). But while much of that story is already known (3), Carroll also tells us of the little known, but remarkable coming together of Monod and philosopher/writer Albert Camus, one of the intellectual giants of the 20th century. Coming from very different intellectual backgrounds, Monod and Camus forged a deep friendship, united in their opposition to tyranny and oppression. Carroll’s book was the inspiration for this post.
When Albert Camus learned that he had won the Nobel Prize for Literature in October 1957, he wrote to a few well-wishers, including an old friend in Paris:
My dear Monod,
I have put aside for a while the noise of these recent times in order to thank you from the bottom of my heart for your warm letter. The unexpected prize has left me with more doubt than certainty. At least I have friendship to help me face it. I, who feel solidarity with many men, feel friendship with only a few. You are one of these, my dear Monod, with a constancy and sincerity that I must tell you at least once. Our work, our busy lives separate us, but we are reunited again, in one same adventure. That does not prevent us to reunite, from time to time, at least for a drink of friendship! See you soon and fraternally yours.
Camus appears somewhat downcast in his note to Monod. At 43-years-in-age, he was the second youngest writer ever to receive the Nobel Prize for literature (Rudyard Kipling at 42 was the youngest), and he was worried that the ballyhoo surrounding the award might distract him from his writing. And, he was concerned that the prize might stir up additional contempt from critics of his writing, as well as from his leftist colleagues who opposed his condemnation of Soviet communism.
But, why did philosopher/writer Camus—an intimate of some of the greatest writers and artists of the mid-twentieth century, including Sartre and Picasso—write to scientist Monod, and acknowledge the special importance he placed on their friendship? Likewise, why did he assert: “I have known one true genius, Jacques Monod.” And, what is the same adventure that Camus refers to?
A brief background to our tale is as follows. In March 1939, Hitler took control of Czechoslovakia. Next, on September 1, Germany invaded Poland. On September 2nd, Poland’s allies, Britain and France, issued an ultimatum to Germany: withdraw or face war. On September 3rd, the ultimatum expired, Britain and France declared war on Germany, and the Second World War was underway; sort of. Although Germany went on to conquer Poland in a mere eight days, several months passed without further action. Then, in May 1940, Nazi Germany invaded and overran France in just six weeks. Marshall Pétain surrendered to the Germans, the French Forces were disbanded, the pro-Nazi Vichy government was put in place under former prime minister Pierre Laval and the 84-year-old Pétain, and the Nazi occupation of the defeated French nation began.
A few months before the Nazis invaded France, thirty-year old Jacques Monod was a doctoral student in zoology at the Sorbonne. [A polymath, he also founded a Bach choral group, and was an accomplished cellist, and seriously considered a career in music (4).] But as war with Germany loomed, Monod enlisted in the army—in the communication engineers—where he thought he might use his scientific talents if war were to break out. Consequently, Monod was serving on the front lines when the Germans invaded. France suffered the most colossal military disaster in its history, and Monod returned to his studies in Paris.
Life in France grew progressively harsher under the Nazis; beginning with subjugation, and followed by deportations, enslavement, and mass murder. Early on, Monod joined one of the first units of the French Resistance; a group of ethnologists and anthropologists at the Musée de l’Homme (Museum of Man).
One of Monod’s duties for the Musée de l’Homme group was to distribute its newspaper, at night. This seemingly simple task was extremely dangerous since capture could mean deportation to a concentration camp or execution. Monod, in fact, had several close escapes. On one occasion, the Gestapo raided his laboratory at the Sorbonne. Fortunately, since they were fearful of the viruses and radioactive isotopes in the lab, they didn’t search it as thoroughly as they might have. Otherwise, they might have found sensitive documents that Monod would hide inside the leg of a mounted giraffe outside his office. In any case, the Germans soon routed the short-lived Musée de l’Homme group
Monod’s wife, Odette, was the granddaughter of Zadoc Kahn, the former chief rabbi of France. Since the Vichy government soon began enacting Nazi policies, including anti-Jewish laws, and because of homegrown French anti-Semitism, Odette sought refuge for herself, and for her and Jacque’s twin sons (born in August 1939, four weeks before the war broke out), under assumed names, in a village outside of Paris. Meanwhile, Jacques had to register with the Vichy authorities as the spouse of a Jewish person.
With Odette and the children concealed, Monod joined the most militant unit in the Resistance; the Communist-led Franc-Tireurs (Free Shooters) group. Monod was not then a Communist Party member. Nonetheless, he joined the Franc-Tireurs since they actually were fighting the Germans—assassinating German officers in the streets and carrying out sabotage. One of his missions for the Franc-Tireurs took him to Geneva—through the Alps to avoid arrest—to request money for arms from the United States Office of Strategic Services; the precursor of the present Central Intelligence Agency.
By this time, Monod had gone completely underground. He wore a disguise during the day, slept in safe houses at night, and stayed away from his laboratory at the Sorbonne. But then, Andre Lwoff, the head of microbial physiology at the Pasteur Institute, offered Monod a refuge and a place to work in his laboratory at the Pasteur Institute. Monod then led a double-life. By day, as Monod, he worked on his experiments at the Pasteur Institute. At night, he carried out his duties for the Franc-Tireurs, as “Marchal” (from a character in a novel by Stendhal), and as commander “Malivert.” [Lwoff too had been active in the Resistance, gathering intelligence for the Allies, while also hiding downed American airmen in his apartment.]
Monod was resolutely committed to the Resistance, while also maintaining a productive research program. At the Pasteur Institute, he and his student, Alice Audureau, made key discoveries that would lead to the later breakthroughs he would make with Jacob. [For instance, Monod and Audureau discovered mutations in E. coli genes that caused the induction of lactose metabolism; a finding that would have important implications concerning gene action and regulation.] Moreover, he was devoted to Odette and their twin sons, and managed to make frequent clandestine visits to see them.
Monod took on increasing responsibilities in the Franc-Tireurs, as more members of the group were discovered and executed by the Germans. In fact, by the time of the allied invasion of Normandy in June 1944, Monod, had become chief of staff of the operations bureau for the National Resistance Organization; a position from which his three predecessors had disappeared (4). As such, Monod prepared battle plans for the allied surge to Paris. He also arranged parachute drops of weapons, railroad bombings, and mail interceptions.
Interestingly, Monod also recruited to the Resistance renowned French chemist, John Frédéric Joliot-Curie (Aside 1), who devised a unique recipe for Molotov cocktails, which were the Resistance’s principal weapon against German tanks. In addition, Monod organized the general strike that facilitated the liberation of Paris. Then, after the liberation of Paris, he became an officer in the Free French Forces, and a member of General de Lattre de Tassigny’s general staff.
[Aside 1: John Frederick Joliet was working as an assistant to Marie Curie, when he married Marie’s daughter, Irene. Afterwards, both John Frederick and Irene changed their surnames to Joliot-Curie. In 1935, the couple was awarded the Nobel Prize in Chemistry for their seminal research on radioactivity. John Frederick then worked at the Collège de France on controlled chain reactions. His work on that was cited by Albert Einstein in his famous 1939 letter to President Franklin Roosevelt, warning Roosevelt of the possibility of a nuclear weapon: “In the course of the last four months it has been made probable through the work of Joliot in France as well as Fermi and Szilard in America—that it may be possible to set up a nuclear chain reaction in a large mass of uranium, by which vast amounts of power and large quantities of new radium-like elements would be generated. Now it appears almost certain that this could be achieved in the immediate future…This new phenomenon would also lead to the construction of bombs…” The Nazi invasion ended Joliet-Curie’s nuclear research. Nevertheless, he managed to smuggle his research notes out of France to England.]
Francois Jacob, a Jewish, nineteen-year old 2nd-year medical student, was planning on a career in surgery when the German occupation of France began in the Spring of 1940. Resolved to carry on the fight against Hitler, Jacob left medical school and boarded one of the last boats for England. In London, he was one of the first of the French to join Charles de Gaulle’s Free French Forces. He wanted to enroll in a combat unit, but, despite his incomplete medical training, he was commissioned as a medical doctor, and then served as a medical officer in North Africa. His surgical career was prematurely cut short in August 1944, when he was severely wounded at Normandy; by a bomb dropped from a German Stuka dive bomber. At the time, he was tending to a dying officer.
Unable to practice surgery after the war because of his wartime wounds, Jacob eventually turned to a career in science. He was accepted at the Pasteur Institute, where he beseeched Lwoff (Monod’s host at the Pasteur Institute) to serve as his mentor. Lwoff rebuffed Jacob several times, but finally agreed to take the young doctor under his wing. Then, in the cramped quarters of Lwoff’s laboratory at the Pasteur, Jacob and Lwoff’s student, Elie Wollman, began a fruitful collaboration that produced key insights into bacterial conjugation and the regulation of lysogeny (Aside 2). After that, Jacob and Monod forged their extraordinary collaboration that would lead to their Nobel Prizes. Note that Jacob’s earlier work with Wollman, on lysogenic induction, would provide the underpinning for his later work on gene regulation with Monod (3).
[Aside 2: Elie Wollman, born in 1917, was Jewish. In 1940, he escaped from the Nazis in Paris and then worked underground in the Resistance as a physician. His parents, Eugene and Elizabeth Wollman, were Pasteur Institute scientists who were seized by the Nazis in 1943 and sent to Auschwitz. They were never heard from again (3).]
In December of 1939, our other main protagonist, twenty-six-year-old Albert Camus, was an unknown, aspiring writer, working as a reporter and editor for a newly founded left-wing newspaper, Alger Republican, in his native Algeria; which was then under French control. Camus was completely opposed to the war, which he saw as “another unnecessary, avoidable, disastrous, absurd chapter of history that would consume the lives of those who did not make it or wish for it.” His antiwar editorials in the Alger Republican outraged French government officials who were calling for unity against Germany. The government finally shut down the newspaper, leaving Camus unemployed. So, Camus returned to France, where the prospects for employment were now better because wartime mobilizations had left many businesses shorthanded. See Aside 3.
[Aside 3: Camus started writing The Stranger while in Algeria, basing it on people and places he knew there. His purpose in The Stranger was to express how one might react to his philosophical notion of the “absurd”—the disconnect between our desire for a rational existence, and the actual world, which appears confused and irrational—in the form of a novel. Meursault, the narrator, and principle character in The Stranger, shows no grief over his mother’s death, no remorse over having committed an unintended murder, and no belief or interest in god. Even while Meursault was awaiting the guillotine, he was reconciled to “the tender indifference of the world.” Meursault’s honesty in describing his feelings makes him a ‘stranger’ in the setting of the novel, and seals his fate.]
Camus was not called up for military service when he returned to France, because he had contracted tuberculosis in Algeria, when he was 17 (Aside 4). Nonetheless, he twice attempted to enlist—the second time when the French Army was on the verge of surrender to the Nazis—to express his solidarity with those who were being drafted. In any case, the military rejected him each time because of his tuberculosis. So, he managed to get a job in Paris as a layout designer for the newspaper Paris-Soir.
[Aside 4: In the pre-antibiotic era, tuberculosis was often fatal, and the 17-year-old Camus indeed had a close brush with death. That experience had a profound effect on the “precocious philosopher,” who made notes on the question of “how, in the light of the certainty of death, one should live life.”]
Parisians began fleeing from their city when the German invasion began in May of 1940. Then, in June, as the Germans were on the verge of entering Paris, the stream of refugees became a flood, with about 70 percent of the city’s metropolitan population of nearly five million eventually taking flight from the city. All Parisian newspapers stopped publishing. However, Paris-Soir hoped to resume its operations in the south, with a reduced staff. Thus, Camus joined the stream of refugees, driving an automobile (almost all the paper’s regular drivers had been drafted), with a Paris-Soir executive as his passenger. After Camus and his passenger were well on their way, Camus suddenly realized that in the rush to vacate from Paris, he may have left his manuscript for The Stranger behind in his room. “He jumped out of the car and threw open the trunk, and was relieved to find in his valise the complete text of The Stranger.” See Aside 5.
[Aside 5: In 1885, Joseph Meister, at nine-years-of-age, was the first recipient of Louis Pasteur’s rabies vaccine and, as an adult, was caretaker of the Pasteur Institute; a position that he still held at the start of the Nazi occupation in 1940. In despair over the fall of France, and wrongly believing that German bombs killed his family after he sent them away, he went to his apartment, closed the windows, and turned on the gas in his stove (5, 6).]
Camus went with Paris-Soir to Clermont-Ferrand. There, the paper began to publish again, using printing facilities made available by Pierre Laval, the former premier, and now architect of the Petain Vichy government. But with the paper now under Laval’s control, it began publishing anti-Semitic articles, and other articles in support of the Vichy government. Camus did not write any of these items. In any case, he was let go by Paris-Soir after the draftees of the 1940s were discharged and could return to work. Camus then went back to Algeria, where he completed The Stranger.
In 1942, with The Stranger about to be published in France, Camus suffered a nearly fatal relapse of his tuberculosis. He wanted to return to France for treatment in the Massif Central mountain range, but several months would pass before Algerian authorities gave him permission to do so. Then, upon returning to Paris, he would have a purpose that would totally engage him.
One night, under an assumed name (because of the need for secrecy in the Resistance), Camus stole into the clandestine headquarters of Combat (the journalistic arm and voice of the French Resistance), to implore the staff to take him on since he “had already done a little journalism” and would be happy to help in any way. Like Monod, Camus then led a double-life, carrying out his duties at Combat, as “Bauchard.” At first, he helped to select and edit articles, and prepare the paper’s layout. Then, in 1943 he became the paper’s editor, and wrote stirring editorials, exhorting Frenchmen to act against the German occupiers. By the time The Stranger was published in 1942, his recognition as Camus led to his acceptance into the literary and artistic circle that included Sartre, Simone de Beauvier, and Picasso.
Camus was suffering from recurrent bouts of tuberculosis all the while that he was carrying out his work at Combat. Nonetheless, as Camus, he also published his essay, The Myth of Sisyphus, which, like The Stranger, contemplates the experience of the Absurd (see Aside 3, above). And he also wrote The Plague, which depicts a city’s response to an outbreak of bubonic plague; perhaps a metaphor for the Nazi occupation. Remarkably, no one at Combat had an inkling that the man who at first had been editing and arranging pages for them as Bauchard was in fact the now renowned Camus. See Aside 6.
[Aside 6: Among laypeople, Jacques Monod is perhaps best known for his “popular” book, Chance and Necessity, published in 1970, and a bestseller in its day. Monod’s Chance and Necessity, and Camus’ The Myth of Sisyphus, are each relevant here because they point up how Camus influenced Monod’s view of the meaning of life. While Camus took a philosophical approach to that issue, Monod’s assessment was also informed by his knowledge of life’s fundamental molecular mechanisms. With the 1953 discovery by Watson and Crick of the molecular structure of DNA, it was apparent how accidental, random, unpredictable mutations in the sequence of bases in DNA were the source of all biological diversity. Thus, Monod knew that all living forms, including humans, are the products of chance genetic mutations and circumstances: “Man at last knows that he is alone in the unfeeling immensity of the universe, out of which he emerged only by chance. Neither his destiny nor his duty have been written down. The kingdom above or the darkness below: it is for him to choose.” [Monod’s title, Chance and Necessity, is from Democritus’ dictum “Everything in the universe is the fruit of chance of chance and necessity.”]
That we live in a world that is indifferent to our hopes and suffering was the reason for Monod to inquire into the meaning of life, which, for Camus, was “the most urgent of questions.” Camus was often branded an existentialist, but unlike many contemporary existentialist thinkers, Camus vehemently rejected nihilism. In The Myth of Sisyphus, he wrote that Sisyphus gave his life meaning by choosing to believe that he remained the master of his own fate, even though he was condemned to rolling his rock uphill each day, only to have it roll back down.
On the opening page of Chance and Necessity, Monod includes a lengthy quotation from the closing paragraphs of The Myth of Sisyphus. “The struggle itself towards the heights is enough to fill a man’s heart…One must imagine Sisyphus happy.” Camus is advocating that we oppose the certainty of death in an uncaring Universe by living life to the fullest. For Monod, life is like Sisyphus, pushing its rock uphill. The end might be bleak, but “the struggle towards the heights is enough to fill a man’s heart.”]
By 1944, the liberation of Paris was imminent, Combat went from a monthly publication to a daily one, and the paper chanced to circulate in the open. Camus was still writing his editorials anonymously. And when his identity was finally revealed, his inspiring, eloquent words resulted in his widespread public acclaim.
Monod and Camus were very likely aware of each other at this point in our saga, but they had not yet met. Their meeting would happen after the liberation of France, and it would be in response to a new totalitarian threat; from the Soviet Union. It transpired as follows.
In 1948, Monod was working full-time on his research at the Pasteur Institute, when events in the Soviet Union moved him to write a stirring editorial that appeared on the front page of Combat. [Camus had left Combat the previous year, after it became a commercial paper.] Monod’s piece was in response to a pseudoscientific doctrine advanced by Stalin’s head of Soviet agriculture, Trofim Lysenko, which asserted that organisms could swiftly change their genetic endowment in response to a new environment. [Lysenko’s doctrine is reminiscent of discredited Lamarckian doctrine, also known as heritability of acquired characteristics—i.e., the premise that if an organism changes to adapt to an environment, it can pass on those changes to its offspring.] Lysenko based his doctrine on his purported discovery of a means to enable winter wheat to be sown in the spring.
Stalin embraced Lysenkoism—during an acute grain shortage in Russia—since it was in accord with his ideology to create the New Soviet Man. Stalin also banned all dissent against Lysenko’s doctrine. Consequently, traditional Russian geneticists were exiled or murdered, Mendelian genetics was no longer practiced in the Soviet Union, and Soviet agriculture suffered severely.
Monod was roused to write his editorial after French Communist newspapers began to widely disseminate Lysenko’s doctrine in France. One Party newspaper proclaimed Lysenko’s discovery “A Great Scientific Event,” and further asserted that the notion of evolution by natural selection was a racist form of thinking, in harmony with Nazi doctrine (7). Another Party newspaper condemned Mendelian genetics for being “bourgeois, metaphysical and reactionary,” while claiming that it must be false because it is reactionary; having been invented by an Austrian monk. In Contrast, Lysenkoism is true because it is progressive and proletarian.
A Party member’s position on Lysenko indeed had become a gauge of his commitment to Stalin’s Soviet cause. But for Monod, the Soviet embrace of Lysenko was “senseless, monstrous, unbelievable.” As expected, Monod’s article was strongly condemned by the powerful French Communist Party, which enjoyed broad support from both intellectuals and workers; many of whom saw the Soviet Union as a model for a French socialist state. In any case, the Party’s strong backlash inspired Monod to “make his life’s goal a crusade against anti-scientific, religious metaphysics, whether it be from Church or State.” Importantly, a separate consequence of the Lysenko affair was that it influenced François Jacob to focus his research in the field of genetics. See Aside 7.
[Aside 7: Ironically, the observation that Jacob and Monod initially set out to explain looked remarkably like Lysenkoism. When E. coli are fed a solution of glucose and lactose, they grow rapidly until glucose—their preferred carbon source—is depleted. Only then, they turn to metabolizing lactose. But, in contrast to Lysenko’s doctrine, Jacob and Monod showed that when E. coli “adapts” to lactose, it does so without changing its genes. Instead, the genes encoding the enzymes that metabolize lactose lie dormant until lactose induces them, under conditions in which glucose is not available. That is, Jacob and Monod determined that lactose regulates lactose metabolism in the cell by acting as an inducer of genes that already exist in the cell; as opposed to lactose causing the cell to undergo a Lamarckian acquisition of a genetic characteristic. In so doing, Jacob and Monod created the now well-established paradigm of inducers, regulators, regulator genes, and operators.]
While Monod was crusading against Lysenkoism, Camus was having his own feud, in public, with Sartre, who had chastised him for his anti-Soviet stance. Camus had once been a Communist, in Algeria, mainly because he was troubled by the way in which the European French treated the native Algerians. However, he was never very sympathetic to the Marxist cause. Monod too had once been a member of the Communist Party; but only because it enabled him to have a voice in the running of the Resistance. In any case, Camus seized upon Monod’s condemnation of Lysenkoism in his feud with Sartre.
Our two main protagonists finally met when Camus co-founded the anti-Stalin, anti-totalitarian Groupes de Liaison Internationale. Monod attended one of the group’s meetings. There, he, and Camus, discovering that they shared much in common, forged their friendship. Carroll writes: “Camus, who so treasured the sense of solidarity that existed among the Resistance, had in Monod a new comrade who shared both the deep bond of that wartime experience and an unqualified opposition to a new common enemy.”
As noted, Monod’s views on the meaning of life owed much to Camus. Likewise, Camus learned from Monod. Camus not only used Monod’s case against Lysenko in his dispute with Sartre, but he also “borrowed” from Monod in The Rebel; in which Camus argued that revolution inevitably leads to tyranny. In any event, after Camus and Monod had separately fought the Nazis, they were now united against another oppressor—the totalitarian state run by Stalin. [Camus’ anti-Soviet stance cost him the friendships of many French intellectuals on the left. He and Sartre never spoke to each other again.]
Monod was also troubled by the situation of scientists working under Eastern European Soviet regimes. In 1959, he organized the escape into Austria of Hungarian biochemist Agnes Ullman (who participated in the failed Hungarian uprising of 1956), and her husband, also a scientist. Earlier, in 1958, Agnes Ullman managed to visit Monod at the Pasteur Institute, and confided to him that she and her husband wanted to defect from Hungary. Monod maintained contact with the Ullmans in Hungary, using coded messages, written in invisible ink, which turned blue when exposed to iodine. The Ullmans finally crossed into Austria, hidden underneath a bathtub, in a compartment of a pull-along camping trailer. See Aside 8.
[Aside 8. Agnes Ullmann, became Monod’s long-time close collaborator at the Pasteur Institute. Now retired, she was carrying out research at the Pasteur Institute as recently as 2012; 53 years after her rescue from Hungary. At the Institute, she collaborated with Monod on characterizing the lac operon promoter, on complementation between β-galactosidase subunits, and on the role of cAMP in overcoming the repressive effect of glucose (catabolite repression) on lactose metabolism in E. coli.]
There are numerous other instances in which Monod stepped forward to fight injustice and defend human rights. In 1952, he wrote a letter in Science that might have been “ripped from today’s headlines.” It protested the U.S. government’s rejection of visa requests for himself and other Europeans who had once been Communists. Monod also condemned the treatment of Jews in the Soviet Union, while continuing to speak out against Soviet totalitarianism in general. And, in 1965, shortly after Monod, Lwoff, and Jacob received word of their Nobel Prizes, they publicly appealed to the French government to allow the use of contraceptives, and the legalization of abortion. See Aside 9.
[Aside 9: Jacob too was devoted to the defense of human rights. He chaired a committee of the French Academy of Sciences that supported persecuted scientists living under totalitarian regimes, and he worked for the release of those who had been imprisoned for their political views. Moreover, he forcefully advocated for the public support of the biological and medical sciences. What’s more, Jacob also had a distinguished writing career that produced a series of acclaimed books, including The Logic of Life: A History of Heredity; Of Flies, Mice and Men;The Possible and the Actual, and his memoir, The Statue Within. In Joshua Lederberg’s review of the latter for The Scientist, he stated: “As a work of literature, it evokes unmistakable overtones of Rousseau, Proust, and Sartre.” In Jon Beckwith’s view, all of Jacob’s books are “written in a fluid and elegant style” Others refer to the “clarity and grace” of Jacob’s writing. See reference 8 for more on Jacob.]
In 1966, Martin Luther King Jr. and Harry Belafonte visited France to raise funds for the Southern Christian Leadership Conference (SCLC). Remarkably, Monod was chosen to introduce King to a crowd of 5,000 people at Paris’ Palais des Sports. Belafonte was introduced by French singer and actor Yves Montand (9).
The intellectual lives of Monod and Camus played out in entirely different areas. Yet the parallels were striking. Each, in his way, searched for meaning in life. Moreover, each put his life on the line to oppose ignorance, injustice, and totalitarianism. And, it is clear from their correspondences that they were dear to each other. Here is the note from Monod that elicited Camus’ response at the top of this post.
My dear Camus,
My emotion and my joy are profound. There were many times when I felt like thanking you for your friendship, for what you are, for what you managed to express with such purity and strength, and that I had likewise experienced. I wish that this dazzling honor would also appear to you, in some small part, as a token of friendship and of personal, intimate recognition. I would not dare coming to see you right now, but I embrace you fraternally.
This piece ends with a few personal thoughts. Jacques Monod was a Nobel Prize-winning scientist, a hero of the French Resistance, a rescuer of persecuted scientists from behind the Iron Curtain, and a leading voice against tyranny and oppression. And, he was also blessed with dashing good looks. I remember well the women among my fellow graduate students in the 1960s finding him to be very attractive. But, on a more serious note: Today, when political and religious blocs dismiss evidence-based science in favor of alternative ‘facts’ in order to advance their ideologies, and when they are tacitly aided by a press that all too often gives equal validity to all points of view, and while scientists seem to be groping for an effective response, one can hope that scientists with the courage, eloquence, and eminence of Jacques Monod and Francois Jacob might emerge to take up the cause of science and reason. Meanwhile, it is especially important for young scientists, and the public, to be aware of the examples set by these men. See Aside 10.
[Aside 10: The following is from a March 8, 2017 editorial in Nature. “Last week, state legislators in Iowa introduced a bill that would require teachers in state public schools to include ‘opposing points of view or beliefs’ in lessons on topics including global warming, evolution and the origins of life… Since last month, Indiana, Idaho, Alabama, Texas, Oklahoma and Florida have all introduced and discussed similar tweaks to the way in which they want to educate their children… Although these proposed changes are typically presented by their supporters as giving teachers the chance to discuss genuine scientific controversies, in truth they are (very) thinly veiled attempts to pursue political and religious agendas that have no place in school science lessons — for whatever age. They seek to import the alternative facts and misleading rhetoric of the new federal government and to impose it on children who deserve much better from those elected to serve them.”]
As Trump does when rejecting the findings of climate scientists, he similarly misrepresents and ignores the vast amount of scientific evidence that confirms the safety and effectiveness of vaccines. And this is happening while he asserts almost daily that any facts, which call his positions to account, are “fake.” Moreover, his millions of followers, who feast on his “alternative facts,” can pass them on to others with a click. See Aside 1.
[Aside 1: Trump surrogate, Scottie Nell Hughes, “explained” that everybody now had their own way of interpreting whether a fact was true or not. “There’s no such thing, unfortunately, anymore as facts,” she declared. Thus, “a large part of the population” will pick and choose whatever “alternative facts” confirm their views (2).]
Many biomedical scientists now feel an urgent need to speak out against vaccine non-compliance. Yet others argue that scientists hurt the cause when they take political sides. Nonetheless, science is founded on honesty and rigor. And, if scientists do not speak out when their findings are distorted or ignored by politicians who put forward policies that harm the public, who else will? So, our concern here is to consider how we might effectively engage not only anti-vaxxers, but science denialists in general. It is important that we consider this, since we have not been especially effective in the past at curtailing science denialism (e.g., re evolution and human-caused global warming).
A key prerequisite for effective communication is that each party listen to, and acknowledge the others point of view. This may be difficult to accomplish with science denialists under any circumstance. But it is most difficult in public discussions, where a group of committed denialists is unlikely to allow the free and open discussion that is essential. Even if you should happen to get your points out, hard-core denialists in the audience will probably not consider them (see Asides 2 and 3). So, in front of a group, address your remarks to the skeptical and undecided members of your audience, rather than to the stanch denialists.
Your chance of influencing undecided or skeptical individuals is much greater in a one-on-one discussion. But whether before a group, or in a one-on-one discussion, your major asset and advantage is that the scientific consensus supports your position. Focus on the evidence.
[Aside 2: Hard-core denialists provide but one example of a more general phenomenon that is well known to social scientists; people zealously resist challenges to their most strongly held beliefs. Moreover, studies show that threatening those beliefs has the effect of people clinging to those beliefs even more fervently; the so-called “worldview backfire effect.” Thus, the stronger your evidence-based arguments against the vaccine-autism link might be, the stronger your disputants might cling to their anti-vaxxer position. The reason is the same as that which makes religious and political zealots immovable. See Aside 3.]
[Aside 3: Moses Maimonides (1138-1204), who many consider to be the greatest Jewish philosopher, confronted dogmatists in the 12th century, when writing his Guide to the Perplexed; his attempt to reconcile the Old Testament bible with what he considered to be the irrefutable scientific worldview put forth by Aristotle and other eminent Greek philosophers. In brief, Maimonides argued that the bible should not be taken literally but, instead, should be read metaphorically. Then, it could be entirely consistent with the truths arrived at through science and reason. Yet, Maimonides realized that most people did read the bible literally, and that to challenge their traditional point of view would be equivalent to challenging their faith itself. Thus, he realized that his arguments would be listened to by only a small group of the most open-minded readers.]
University of Sussex social anthropologist, Melissa Leach, suggests that scientists need to be more empathetic to the personal and cultural beliefs that cause people to reject scientific evidence (3). To that point, scientists need to listen to and understand the reasons why denialists seek alternatives to science, before they might be heard in turn. And scientists must be careful not to imply to science deniers that they are ignorant or irrational (see Aside 4). “Dismissing public and political concerns about health interventions as unscientific, irrational or misled fails to do justice to the different perspectives in play… It is why we see backlashes to even the best-intentioned initiatives (3).” In addition, scientists should not fall into the trap of advocating for an abstract principle. If you are perceived as an advocate for a policy, you may lose trust as an unbiased knowledge broker. So, stick to the evidence. Patiently and clearly connect the dots.
[Aside 4: It may surprise some that science denialists do not sort cleanly along income or education demographics. For instance, the movement to forgo vaccinations has become popular in some more liberal and affluent communities; the organic grocery demographic. Also, consider the example of conservative columnist George Will; an obviously well-educated and sophisticated individual, who nonetheless steadfastly maintains that since climate change happened naturally in the past, we cannot know that human-caused carbon pollution will cause harmful climate changes in the future. Others have noted that Will’s logic is equivalent to saying that since nonsmokers died of lung cancer in the past, we cannot know that cigarette smoking is a cause of lung cancer now. George Will also is not moved by the fact that there is a consensus among climate scientists—based on the accumulation of massive evidence—that human-caused carbon emissions are changing the climate. Climate scientists are now as certain of that conclusion as biomedical scientists are that cigarette smoking causes lung cancer.]
Better communication with science denialists is not easy for reasons noted above. Moreover, many science denialists have learned to rebut the consensus view by cherry-picking “scientific” evidence that might cast doubt on the consensus view; irrespective of whether their selected evidence came from poorly conducted experiments. Moreover, denialists may throw their “alternative facts” at you so fast that, in refuting them, you exhaust your energy and patience well before you get to make your own argument (see Aside 5). And there still will be vociferous politicians, who will continue to misrepresent and ignore science, to advance their own agendas.
[Aside 5: To that point, in 2013 Italian programmer Alberto Brandolini put forward Brandolini’s law (also known as the “Bullshit Asymmetry Principle”). It states: “The amount of energy needed to refute bullshit is an order of magnitude bigger than to produce it.”]
In early February 2017, scientists across the United States began to plan a March for Science, to take place in Washington on April 22; Earth Day. Are organized marches an effective way to promote a pro-science agenda? Some scientists say that the march might be counterproductive. For instance, Geologist Robert Young, of Western Carolina University, argued that the march “could deepen the divide between conservatives and liberals, reinforce the idea that scientists are a political interest group…There’s a section of the American electorate—whether we like to acknowledge it or not—that has become skeptical of science. . . I don’t think that scientists standing in Washington, giving speeches and holding signs, is going to convince those people that they need to pay attention to our concerns… Somehow, as a community, those of us who care about science need to find a way to communicate with those folks…It has to be direct communication or ways that we have not imagined yet (4).”
Young’s remarks provoked a notable backlash on Twitter, with most scientists coming out in favor of the march. Also, consider the outcome of a 2012 march in Ottawa, by Canadian scientists opposed to the anti-science policies of Canada’s conservative Harper government (Aside 5). The Canadian march did not diminish the credibility of the participants, nor did it lead to polarization of the public. Instead, by bringing the Harper government’s anti-science policies to the public’s attention, the march may have helped to elect the more pro-science government of Justin Trudeau in 2015. So, one might hope that an American march might have a positive effect here, even if only to stem the tide of misinformation being fed to the American public.
[Aside 5: Canadian scientists protested the Harper government’s restrictions against free communication between scientists and the media; particularly communications that opposed the government’s pro-industry environmental policies. Scientists who did not comply with the Harper government’s restrictions might have their research programs terminated. In the U.S., in December 2016, then President-elect Trump asked the Department of Energy for the names of career employees and contractors who attended U.N. climate talks over the past five years. He also requested emails of those meetings. The DOE responded with a statement saying that Trump’s request had “unsettled” many in its workforce, that the DOE would “be forthcoming with all [publicly] available information,” but that it would withhold “any individual names.”]
There is no middle ground between objective science and unsubstantiated “alternative facts.” As stated most eloquently by Wendy Palenfeb: “Evidence and objective reality are the foundation of successful policy and governance. Openness is as vital to science as it is to democracy. We cannot allow hard-won knowledge to be ignored or distorted (5).”
Charles Sykesfeb, Why Nobody Cares the President Is Lying, NY Times, February 4, 2017.
Melissa Leach, Accommodating dissent, Nature450, p283, 22 November 2007, doi:10.1038/450483a.
Diana Kwon, Will a March Help Science?, The Scientist, February 2, 2017.
Wendy Palenfeb, When Canadian Scientists Were Muzzled by Their Government, NY Times, February 14, 2017.]
Prompted by President Trump’s comments asserting a link between vaccines and autism, on February 7, 2017, more than 350 medical and professional organizations sent the President a letter stating that vaccines are a safe and most effective means for protecting the health of children and adults and saving lives. The text of the letter, and its signatories, can be accessed from: The week in science: 10–16 February 2017. Nature 542, (16 February 2017) doi:10.1038/542276a.
The following is from an April 11, 2017 editorial in Nature,Naturesupports the March for Science: “Finally, to the critics, yes it is true that the march blurs the lines between science and politics. But that line is already much fuzzier than some try to argue. It is possible to care about science and scientific thinking while ignoring the political context in which it operates. But it is difficult to do that and demand change at the same time.”
On March 28, 2014, more than a year before Donald Trump announced his candidacy for the Presidency of the United States, he tweeted: “Healthy young child goes to doctor, gets pumped with massive shot of many vaccines, doesn’t feel good and changes – AUTISM. Many such cases!”
Although Trump’s anti-vaccine sentiment has not been a secret, he nonetheless took the medical community by surprise when, on January 10, 2017, just days before he was sworn in as the 45th President of the United States, he met with anti-vaccine activist, Robert Kennedy Jr., at Trump Tower in Manhattan, where, per Kennedy, Trump asked him to head a new government commission on vaccine safety (1).
Kennedy claimed that representatives of Trump’s transition team approached him before the meeting to ask whether he would be interested in participating in a vaccine inquiry. Moreover, he stated that Trump’s chief strategist, Stephen K. Bannon; Trump’s counselor, Kellyanne Conway; and then Vice President-elect Mike Pence also attended the meeting. A few hours later, a spokesperson for Trump confirmed that Trump was “exploring the possibility of forming a committee on autism,” but added that no final decisions had been made (1).
The “possibility” that Trump might form a committee on vaccines and autism (irrespective of who heads it) raises fears in the medical community that, by doing so, Trump would give a sense of legitimacy to the discredited anti-vaccine point of view, which, in turn, would give many parents misinformation regarding the crucial need to get their children vaccinated. Vaccines are safe and effective. What’s more, they have prevented more human (especially childhood) suffering and death than any other measure in history! If Kennedy’s panel (or any other action by Trump, which reflected his “alternative” view of vaccines) led to even a small decrease in vaccination rates, the result would be the otherwise preventable deaths of children, including infants too young to be vaccinated (2), as well as the elderly.
The idea that vaccines might cause autism first gained widespread attention in 1998 after the British medical journal, The Lancet, published a study involving only 12 children, by former British surgeon, Andrew Wakefield, which claimed to find a link between the measles vaccine and autism. However, an investigation by the British Medical Council later found that data in The Lancet paper was fraudulent. Moreover, Wakefield’s study received financial support from lawyers representing parents of autistic children; a conflict of interest that Wakefield did not disclose. The British Medical Journal took the extraordinary step of publishing a report in which it concluded that Wakefield’s study was not simply bad science, but a deliberate and elaborate fraud. The Lancet paper was retracted and Wakefield was stripped of his medical license. A subsequent large scale study by the U.S. Institute of Medicine, involving more than a half million children, found no evidence whatsoever of any connection between vaccines and autism (2).
Some individuals, including Kennedy, believe that thimerosal (a mercury compound once added to some vaccines as a preservative) is the link between vaccines and autism. However, thimerosal was added only to killed vaccines (e.g., the vaccines against diphtheria, whooping cough, and tetanus), whereas the MMR vaccine—the original source of the vaccine controversy—is a live vaccine. What’s more, all vaccinations in the United States have been thimerosal-free since 2001, while new cases of childhood autism have not abated since then. Furthermore, extensive studies by the US Centers for Disease Control (CDC), and by the US Institute of Medicine, could not find any connection between thimerosal and autism (2). At first, Kennedy completely ignored these studies, but later asserted that these government agencies were participating in a major cover-up (3).
Considering: 1) the overwhelming scientific evidence against the anti-vaccine point of view, 2) the extensive expert advice available to Trump from physicians and biomedical scientists both within and outside the government and, 3) the unceasing federal oversight of vaccine safety (by the both the CDC and the FDA), why would Trump reopen this issue at all, especially via a panel headed by a layperson, when doing so under any conditions will undermine public health? Is it to distract the public’s attention from more politically troubling issues, or is it merely a play to his base, or does Trump actually believe what he says?
Ben Carson, a physician and former presidential aspirant, and now Trump’s pick to head the Department of Housing and Urban Development, framed the vaccine issue as a matter of government infringement on the peoples’ liberties; a point of view that resonates with the political right (see Aside 1.), as does Trump’s bizarre view, as tweeted in 2012, that: “The concept of global warming was created by and for the Chinese in order to make U.S. manufacturing noncompetitive.”
[Aside 1: Carson, a physician by background, ignores the crucial concept of herd immunity. People who cannot get vaccinated (e.g., young infants, pregnant women, children suffering from leukemia or other immune deficiencies) are yet protected from measles by herd immunity; that is, the immunity in the entire population that results when a high enough percentage of individuals has been vaccinated. When that level of compliance is attained, there are not enough susceptible individuals in the population to sustain the chain of transmission. Thus, vulnerable individuals, who cannot be vaccinated, pay the price for vaccine noncompliance by those who opt out.]
What might Trump’s position on vaccines portend for those biomedical scientists and physicians who would publicly oppose his anti-vaccine sentiments? For a hint, this past December Trump’s transition team asked the DOE for a list of its employees who worked on climate change, or who had attended climate change meetings, thereby raising the specter of repercussions against those who do not adhere to Trump’s stance on the climate change issue. Would the prospect of such repercussions undermine the willingness of physicians and scientists to speak out against Trump’s stance on vaccines?
This past week, Tom Price, Trump’s pick to head the US Department of Health and Human Services (HHS), rejected the claim that vaccines are linked to autism. He did so during his confirmation hearing before the Senate Finance Committee, thus offering some hope that the Trump White House might not pursue its debunked stance on vaccines. Nonetheless, bearing in mind Trump’s unpredictability, and his alternative view of reality regarding other issues, scientific and otherwise, scientists must remain vigilant, and be willing to speak out against policy decisions based on ideological political agendas or “alternative” views of reality, rather than sound scientific evidence.
“Scientists, medics and commentators who have fought vaccine disinformation in the past must take a deep breath and return to the fray. There is no need to wait for this commission to be announced officially. There is no need to wait until it issues its findings. There is no cause to be surprised if it shows little regard for science — or even if it targets scientists who speak out in favor of vaccination… Lives are at stake (4).”
Shear MD, Haberman M, and Belluck P, Anti-Vaccine Activist Says Trump Wants Him to Lead Panel on Immunization Safety. NY Times January 11, 2017.
Andrew Wakefield and the Measles Vaccine Controversy, Posted on the blog February 9, 2015.
4. Trump’s vaccine-commission idea is biased and dangerous. Nature 541:259, 2017. doi:10.1038/541259a
Addendum: The following is from the January 11, 2017 NY Times report (1).
Both Mr. Trump and Mr. Kennedy have described themselves as “pro-vaccine.” But they have repeatedly expressed concerns about what they claim is a link between vaccines and the development of autism. At a Republican presidential debate in September 2015, Mr. Trump described knowing people personally who had seen a cause and effect.
“Autism has become an epidemic,” Mr. Trump said in the debate. “Twenty-five years ago, 35 years ago, you look at the statistics, not even close. It has gotten totally out of control.”
“I am totally in favor of vaccines,” he added. “But I want smaller doses over a longer period of time. Same exact amount, but you take this little beautiful baby, and you pump — I mean, it looks just like it’s meant for a horse, not for a child, and we’ve had so many instances, people that work for me.”
Mr. Trump has also repeatedly used Twitter to spread his concerns about the safety of vaccines. In particular, he has often raised doubts about giving children vaccines in a single large dose rather than several smaller ones… Mr. Kennedy said Mr. Trump “believes in those anecdotal stories” about the dangers of vaccines. He said the president-elect “says if you have enough anecdotal stories saying the exact same thing, that you can’t dismiss the validity.”
What would you do if you were serving on the editorial board of a scientific journal which had just published a manuscript that you knew was seriously flawed. Moreover, you knew that publication of the manuscript might seriously undermine global public health? That was the circumstance of cell biologist Klaudia Brix, Professor of Cell Biology, Jacobs University Bremen, Germany, when, in 2011, the Italian Journal of Anatomy and Embryology (IJAE)—the official publication of the Italian Society of Anatomy and Histology—published a paper by infamous AIDS denialist, Peter Duesberg, which reiterated his already discredited argument that HIV (the human immunodeficiency virus) does not cause AIDS (1). Brix resigned in protest from the IJAE editorial board. But why is that noteworthy? Remarkably, she was, for a time, the only member of the journal’s 13-person editorial board to do so, despite other members having similar misgivings over the decision to publish the paper. Afterwards, Heather Young, an anatomy and neuroscience researcher at the University of Melbourne, likewise resigned from the IJAE editorial board. Here is the background to this state of affairs.
Peter Duesberg is not the only AIDS denialist. However, he has been the most infamous of the AIDS denialists. HIV is a retrovirus, and Duesberg is the only AIDS denialist who also happens to be an expert retrovirologist. In fact, Duesburg was at one time a highly esteemed retrovirologist. In 1985 he was elected to the U.S. National Academy of Sciences; mainly for his 1970 discovery, with Peter Vogt, of the first known retroviral oncogene—the Rous sarcoma virus v-src.
Duesberg first put forward his denialist view in a 1987 paper in Cancer Research (2), which asserted that AIDS results from drug abuse, parasitic infections, malnutrition, and antiretroviral drugs. In Duesberg’s assessment, HIV is just another opportunistic infection. He has maintained that view since then, despite overwhelming evidence to the contrary. Consequently, he is looked upon as a pariah by the scientific community.
Even though Duesberg’s denialist views have been rejected by AIDS experts, Duesberg’s standing as a retrovirologist enabled him to yet influence some public health officials. In 2000, Duesberg was serving on a panel advising Thabo Mbeki (President of South Africa after Nelson Mandela) on how to manage the South African AIDS outbreak. Although Mbeki was an able and intelligent leader, he accepted Duesberg’s denialist view that HIV was not the cause of the South African AIDS epidemic. Thus, Mbeki allowed the South African outbreak to get completely out of control (3). Two independent studies later concluded that over 300,000 South African AIDS deaths would not have occurred if the Mbeki government’s public health policy had not followed the denialist view. Many thousands of South African AIDS victims, including infants, would have been spared infection if the government had publicized that AIDS is an infectious disease, and if it had made antiretroviral drugs available, particularly to pregnant women (1). See Asides 1 and 2.
[Aside 1: The reasons why Mbeki assented to Duesberg’s denialist view are not clear. One possibility is that Mbeki held strong anti-colonialist and anti-West sentiments—born of having come of age during South Africa’s apartheid era—which led him to see his country’s AIDS crisis as a means by which the West sought to exploit his nation. To that point, he may have doubted the efficacy of expensive antiretroviral drugs, which were available only from large Western pharmaceutical companies. Moreover, the cost of treating the 5 million or more HIV-infected South Africans with those drugs would have exceeded the annual health department budget of his poverty-stricken nation by a factor of ten. Mbeki did accept that AIDS is the consequence of a breakdown of the immune system. But he was inclined to believe (or at least claimed) that poverty, bad nourishment, and ill health, rather than a virus, led that breakdown; a stance that enabled him to justify treating poverty in general, rather than AIDS in particular. Duesberg defended Mbeki in his publications, denying that hundreds of thousands of lives were lost in South Africa because of the unavailability of anti-retroviral drugs. But in 2002, after Mbeki suffered political fallout from the consequences of having acceded to Duesberg’s views, he tried to distance himself from the AIDS denialists, and asked that they stop associating his name with theirs.]
[Aside 2: The 2000 International AIDS Conference was taking place in Durban (a city in the South African province of KwaZulu-Natal) at the same time that Mbeki’s AIDS panel was convening in Johannesburg. Consequently, the denialist views expressed by Mbeki’s panel were also being heard in Durban. This prompted the so-called “Durban Declaration,” signed by over 5,000 scientists and physicians, and published in Nature, which proclaimed that the evidence that HIV causes AIDS is “clear-cut, exhaustive and unambiguous”.]
Well before Duesberg submitted his paper to IJAE, the arguments put forward in the paper had already been appraised and rebuffed by the scientific community. Indeed, the paper had previously been rejected by several other journals. The first submission was to the Journal of Acquired Immune Deficiency Syndromes (JAIDS), a peer-reviewedmedical journal covering all aspects of HIV/AIDS. The JAIDS editors found that Duesberg’s contentions in the paper were based on a selective reading of the scientific literature, in which he dismissed all the vast evidence that HIV is the etiologic agent of AIDS. Not surprisingly, JAIDS rejected the paper, with one peer reviewer even warning that Duesberg and co-authors could face criminal charges if the paper were published.
After JAIDS rejected the paper, Duesberg submitted a revised version to Medical Hypotheses (4). Like the original paper sent to JAIDS (as well as the version accepted by IJAE), the paper submitted to Medical Hypotheses contained data cherry-picked to cast doubt on HIV as the cause of AIDS. Nonetheless, Medical Hypotheses accepted the paper. However, the paper never went to press. But first, what was the explanation for the seemingly bizarre decision to accept the paper?
The answer laid in the fact that Medical Hypotheses was the only journal of its parent publisher, Elsevier, that did not use peer review; instead relying on its editorial board to select papers for publication. In any case, before the accepted paper went to press, prominent AIDS researchers, including Nobel laureate Francoise Barre-Sinoussi (co-discoverer that HIV is the cause of AIDS, 5), complained to Elsevier that the paper would have a negative impact on global healthcare, and requested that the paper be withdrawn.
Elsevier responded to these protests by asking the editors of another of its journals, The Lancet, to oversee a peer review of the paper. The Lancet editor sent the paper to five external reviewers, each of whom found that it contained numerous errors and misinterpretations, and that it could threaten global public health if it were published. Elsevier then permanently withdrew the paper. Elsevier also instituted a peer-review policy at Medical Hypotheses (and fired the journal’s editor, who resisted the change).
The Medical Hypotheses incident resulted in more notoriety for Duesberg when the University of California, Berkley, where Duesberg is still a professor of molecular and cell biology, bought charges of misconduct against him for making false scientific claims in the paper, and for a conflict-of-interest issue. Apropos the latter, Duesberg did not reveal that co-author David Rasnick had earlier worked for Matthias Rath, a German doctor and vitamin entrepreneur, who sold vitamin pills as a therapy for AIDS. Duesberg was later cleared of both charges. But the next iteration of paper, to IJAE, did not respond to these allegations.
Duesberg regarded Elsevier’s actions as another example of “censorship” imposed by the “AIDS establishment.” Undeterred however, he submitted a revision of the paper to IJAE, which that journal then accepted, prompting Klaudia Brix and Heather Young to resign from that journal’s editorial board. The IJAE paper contained the same cherry-picked data and discredited assertions that were rejected earlier by JAIDS and Elsevier. Moreover, publication of the paper still posed a threat to global public health. What then was behind the IJAE decision to publish?
Here is what happened. The paper was “peer-reviewed” by IJAE, but by only two reviewers; one of whom was Paolo Romagnoli, the IAJE editor-in-chief, who is neither a virologist or an epidemiologist but, instead, a cell anatomist. Consequently, the paper underwent only one external review, and there is no information regarding whether the lone external reviewer was an AIDS expert. One board member (who did not resign) later commented: “Only one [external] reviewer in my mind is not enough for manuscripts of a sensitive nature… (6)” [But this comment too is a bit troubling. Bearing in mind that the paper contained numerous errors and misinterpretations, would those have been okay if the paper were not of a “sensitive nature”?]
One also might ask why a journal that specialized in anatomy and embryology would consider a paper about the cause of AIDS. To that point; Klaudia Beix gave, as a reason for her resignation from the IJAE board, her belief that a journal should function within its scientific “scope” (6). So how did Romagnoli, the IJAE editor-in-chief, justify his decision to consider the paper? He did so by asserting that it dealt with “issues related to the biology of pregnancy and prenatal development and with the tissues of the immune system (6).” But despite Romagnoli’s contention, the only mention of embryology in the paper was a short comment in the abstract: “We like to draw the attention of scientists, who work in basic and clinical medical fields, including embryologists, to the need of rethinking the risk-and-benefit balance of antiretroviral drugs for pregnant women, and newborn babies (1).”
As for Romagnoli’s reliance on only two reviewers, he justified that stance on the fact that the reviewers had concurring opinions. Moreover, he claimed that his criteria for selecting reviewers—apparently irrespective of their expertise—was to choose individuals (himself included) who he believed would not reject a paper merely because it challenged prevailing opinion.
But is there any possibility that Duesberg might be right? The answer is virtually none whatsoever. An earlier post noted: “…the evidence that HIV causes AIDS is, without exaggeration, overwhelming. Consider just the following. Data from matched groups of homosexual men and hemophiliacs show that only those who are infected with HIV ever develop AIDS. Moreover, in every known instance where an AIDS patient was examined for HIV infection, there was evidence for the presence of the virus. These data have been available for years, and Duesberg should have been aware of them. What is more, there has been the enormous success of antiretroviral therapy in changing AIDS from a nearly invariably fatal disease, into a manageable one, for many HIV-infected individuals (3).”
Even so, Duesberg is not regarded as a pariah by AIDS experts merely because his views concerning the connection between HIV and AIDS challenge accepted wisdom. Instead, as asserted by Harvard University AIDS epidemiologist, Max Essex, Duesberg has sustained a “dangerous track of distraction that has persuaded some people to avoid treatment or prevention of HIV infection (6)”.
A scientist mounting a long-time challenge to the “establishment,” and being ridiculed for his views, before eventually being vindicated, makes for a very good story. However, such instances are very rare. Exceptions include Howard Temin (7) who hypothesized reverse transcription, and Stanley Pruisner (8) who hypothesized prions—infectious agents that contain no nucleic acid genome. Both researchers had to endure widespread ridicule for several years. But, and importantly, irrefutable evidence eventually accumulated to support their hypotheses. And, finally, both were awarded Nobel Prizes. But Duesberg has not been vindicated and, almost certainly, he never will be.
Stanley Prusiner (1942) received the 1997 Nobel Prize in Physiology or Medicine for discovering the agents responsible for the transmissible spongiform encephalopathies—diseases so named because the brains of afflicted subjects contain numerous holes or vacuoles, which give them a spongy (“spongiform”) appearance under the microscope. These diseases include scrapie in sheep, bovine spongiform encephalopathy (“mad cow disease”) in cattle, and Creutzfeldt-Jacob disease (CJD) and kuru in humans. Each of these diseases is invariably fatal.
Pruisner’s discovery was iconoclastic in the extreme because the etiologic agents of these diseases, which can multiply and kill, are comprised entirely of protein! Since they can replicate, despite carrying no genetic information, they defy the very foundation of biology. Pruisner dubbed them “prions”—an acronym he derived from “proteinaceous infectious particle.” The spongiform encephalopathies are now more commonly called “prion diseases.”
How might an infectious agent, which contains no genetic information, replicate? Pruisner provided the answer. Prion proteins (PrPSc) (Sc for scrapie, the prototype prion disease) are misfolded forms of corresponding normal cellular proteins (PrPC), which are generally present in all vertebrates, and which are particularly plentiful in the brain. The PrPSc isoforms act as templates that cause the normally configured proteins to refold into the PrPSc configuration. Once underway, this conversion process might escalate exponentially. In that way PrPSc isoforms “replicate,” and their accumulation in the brain leads to the characteristic prion disease pathology. See Aside 1.
[Aside 1: Prion diseases were once referred to as “slow virus diseases,” where “slow” referred to the course of the disease, rather than the agent. All the prion diseases have a clinically unapparent incubation period that may last for as long as 50 years. But once symptoms emerge, the duration of the clinical stage is only a matter of months, and invariably ends in death. The length of the incubation period appears to be inversely correlated with the level of PrPC. The actual cause of cell death in prion diseases is not known.]
The medical relevance of Pruisner’s discovery of prions, and of their mode of replication, may be much more significant than merely their association with the relatively rare infectious prion diseases. That is so because similar aggregates of misfolded proteins have since been observed in the much more widespread Alzheimer’s and Parkinson’s diseases, as well as Lou Gehrig’s (ALS) and Huntington’s diseases. Misfolded PrP-like proteins associated with Alzheimer’s disease include amyloid-β and tau. In Parkinson’s disease, these aggregates are comprised of α-synuclein. [The entire family of the PrP-like misfolded proteins are referred to as amyloids.] Thus, Pruisner’s discovery may have significant implications for the diagnosis and treatment of much more prevalent neurodegenerative diseases and dementias. See Aside 2.
[Aside 2: Potential therapies include the targeting of toxic species of PrP with monoclonal antibodies or with other ligands that bind to amyloid aggregates. Apropos that, in November 2016, the drug solanezumab, which targets amyloid, failed in a clinical trial to determine whether it might help people with mild dementia. Critics of the “amyloid hypothesis” (who still remain; see below) cited the failed trial as evidence against the amyloid premise. For a review of evidence in support of the amyloid hypothesis, and for analyses of the meaning of the failed solanezumab trial, see references 1 and 2. For a current review of the field, see reference 3.]
If Alzheimer’s and Parkinson’s diseases, as well as other neurodegenerative disorders such as ALS, indeed resemble the transmissible prion diseases, as disorders of protein conformation, then they also suggest new disease paradigms. Some of these diseases are infectious, while others are sporadic, or genetic. However, and importantly, some of these diseases may be transmitted by several of these various ways that determine the frequency and distribution of a disease in a population. For instance, Pruisner’s group demonstrated that CJD can be an infectious as well as a familial disease (see below). In the latter instance, it results from a particular mutation in the cellular PrP gene. Indeed, more than 20 mutations in PrP are now known which underlie inherited prion diseases. See Asides 3 and 4.
[Aside 3: It is not surprising that Pruisner’s proposal of an entirely new type of infectious agent—one comprised entirely of protein—met with considerable skepticism. Reflecting on the rather vicious ridicule that some of the naysayers subjected him to, Pruisner wondered “how the course of scientific investigation might have proceeded had transmission studies not been performed until after the molecular genetic lesion had been identified (4).”]
[Aside 4: Might Alzheimer’s disease be transmissible? There is no epidemiological evidence to suggest that possibility. However, bearing in mind that infectious prion diseases, such as CJD, can be transmitted during medical or surgical procedures (e.g., corneal transplant), it is reasonable to suggest that Alzheimer’s too might be transmitted by a physician’s or surgeon’s treatment. Other known routes of iatrogenic CJD transmission include injection of pituitary hormones obtained from cadavers, and intracerebral exposure to contaminated neurosurgical instruments. Apropos the latter possibility, amyloid-β adheres stubbornly to metal surfaces, and prions are highly resistant to sterilizations that would inactivate a true virus. Yet, because of the already high prevalence of Alzheimer’s disease in the population, and a possible decade-long non-clinical incubation period, the risk of iatrogenic transmission in that instance is not yet known. New (and expensive) methods have been developed for removing amyloids from surgical instruments, but they are not widely used because of the uncertainty of the danger of iatrogenic transmission.]
Here now is the story of Pruisner’s discovery of prions, with a nod toward how he persevered in the face of the widespread disbelief and scorn that his discovery engendered.
Pruisner first became interested in neurological diseases as a third-year medical student at Penn, during his rotation on the neurology service (5). However, that exposure to neurological diseases did not immediately affect his career goals. Instead, in his fourth year, he satisfied his desire to do research by investigating oxidative metabolism of brown fat cells. Nonetheless, his research on fat cells excited him enough to envision a career as a physician-scientist. “I was astonished that people actually got paid to solve puzzles every day—what a fantastic way to make a living (5).”
Pruisner continued his medical training as an intern at the University of California, San Francisco (UCSF). Providentially perhaps, he found his internship to be demanding enough to dissuade him of any thoughts of a career practicing clinical medicine. So, Pruisner spent the next three years at the NIH researching enzyme regulation in bacteria; an experience that he found gratifying enough to decide that a career in medical research would be his goal.
Next, Pruisner had to choose a research area. He was still interested in neurological diseases. “With its billions of neurons, its ability to affect all aspects of human activity and its endless mysteries, the brain seemed a perfect subject for research… (5).” To gain the background he would need for his new calling, Pruisner decided to carry out an “abbreviated residency” in neurology at UCSF.
The next step in Pruisner’s path would be choosing a solvable research problem. Here now is an example of one of those fortuitous happenings that can make a scientific career and, with a bit of luck, lead to a singularly important scientific breakthrough. “It was during my residency at UCSF that I encountered a patient with a rare progressively debilitating illness called Creutzfeldt-Jacob disease (or CJD), and the mysteries surrounding this illness launched my scientific studies for the next four decades (5).”
Pruisner was intrigued as well as perplexed by his CJD patient. She had suddenly developed severe intellectual and memory deficits, and myoclonus (jerky movements in her muscles). But more puzzling: “She exhibited no signs of an infectious disease…she did not have a fever, and she had no increase in white blood cells in either her blood or cerebrospinal fluid (5).” Yet CJD is a transmissible disease, as shown earlier by Carleton Gajdusek’s finding that the illness could be passed to a chimpanzee by injecting it with a brain extract from a dead human CJD patient. See Aside 5.
[Aside 5: Gajdusek also experimentally transmitted kuru, via homogenates of human patient biopsies, to chimpanzees. Moreover, his epidemiological studies showed that kuru was transmitted among the Fore people of New Guinea via ritual cannibalism. For more on this, and for an account of how Bill Hadlow first suggested that scrapie and kuru might have a similar underlying basis, see reference 6.]
Gajdusek also found that CJD symptoms did not emerge in injected chimpanzees until months after the inoculation; a finding that agreed with the prevailing view in the scientific community that the scrapie-like diseases are caused by “slow viruses”—a term originally coined by Bjorn Sigurdsson in 1954 while he was working on scrapie in Icelandic sheep. Gajdusek referred to the scrapie agents as “unconventional viruses,” although he had no knowledge of how they might differ from “conventional viruses.”
Pruisner became fascinated by the prospect of once-and-for-all defining the nature of the agents responsible for the transmissible spongiform encephalopathies. However, more experienced, and cautious colleagues at UCSF saw the scrapie project as fraught with too many pitfalls, and tried to steer Pruisner away from it. But he would not be deterred.
One of Pruisner’s UCSF colleagues alerted him to papers by radiation biologist Tikvah Alper, who incidentally trained with Lisa Meitner (7). Alper noted other bizarre properties of the scrapie agents. In brief, she found them to be extremely resistant to UV light and X-rays, which should have inactivated any “conventional” virus by damaging its DNA or RNA genome. And: “Since a single X-ray photon should be sufficient to kill a single scrapie agent, Alper was able to calculate the minimal size of the agent. She estimated that it was less than one-hundredth the size of a typical virus (5).”
Alper’s findings clearly suggested the provocative idea that the scrapie agent does not contain a nucleic acid genome. Yet Pruisner, like everyone else, held to the belief that some sort of “novel” virus was responsible for her unusual results. “What else could the “scrapie agent” be? There was nothing else (5).”
Nonetheless, in the back of Pruisner’s mind, he did not completely dismiss “…the most startling interpretation: All the data might be pointing to an infectious particle devoid of nucleic acid and thus with no apparent way to replicate (5).” Pruisner indeed was intrigued by this radical possibility. And, notwithstanding the advice of more experienced colleagues, Pruisner, a former chemistry major, believed that the scrapie problem would be easy. “It’s just a problem in protein chemistry (5).” And, if it were true that the scrapie agents contain no genetic information, “then it would be worth an enormous effort to decipher the structure (5).”
Pruisner’s first step would be to isolate the scrapie agent from brain homogenates; a feat not accomplished by Alper, nor by anyone else. To monitor his progress towards purification, Pruisner planned to assay his fractions by means of a biological assay, making use of the 1961 finding by British scientist, Richard Chandler, that scrapie disease could be transmitted from one mouse to another via injection of brain homogenates. Pruisner would employ the so-called “endpoint dilution” procedure, in which the titer of a sample is the last dilution (e.g., 1/2, 1/4, 1/8, etc.) able to induce scrapie infection.
But, as predicted by others, complications soon materialized: “…so little was known about the physical nature of the mysterious scrapie agent that hundreds of fractions would have to undergo titration measurements (5).” Additionally, Pruisner’s assay would require 60 mice to measure the titer of each sample. What’s more, since the scrapie incubation period could be a year or longer, some titrations might very well require that long as well. Consequently, Pruisner might have to maintain thousands of mice during this entire time. And, in the end, the assay might not be sensitive enough to show small increases in purity.
The above issues alone may explain why no one before Pruisner tried to systematically investigate the scrapie agent’s molecular nature. But, there is more. Even if Pruisner’s assays were sufficiently sensitive, the critical experiments would need to be repeated before they might be published. And, his findings would still need to be confirmed by others before they might be accepted. Furthermore, before Pruisner could make headway on this hugely expensive project, he would need to acquire a grant to support it. The NIH—the usual source for large biomedical research grants—appeared unlikely to provide that support, since its Virology Study Section held the view that a slow virus causes scrapie and that the issue should be approached as a virological problem, rather than by Pruisner’s chemical approach. Nonetheless, Pruisner was undeterred, “The hubris of youth was all that propelled me forward (5).”
The NIH indeed rejected Pruisner’s application for support of his scrapie project. However, Pruisner did obtain modest funding at UCSF from the Howard Hughes Medical Institute, which enabled him to set out on his project. Meanwhile, William Hallow and Carl Eklund, at the Rocky Mountain Laboratory in Hamilton, Montana, had been studying scrapie pathogenesis in sheep and goats, and had made some failed “hit-or-miss” attempts to define the molecular nature of the agent. When they met Pruisner, his more systematic approach impressed them, and they then taught him “an immense amount” about scrapie. Importantly, they helped him to characterize the scrapie agent’s sedimentation behavior—a key step towards purifying it.
Pruisner then began to produce his first experimental results, which were curious in the least: “I had anticipated that the purified scrapie agent would turn out to be a small virus and was puzzled when the data kept telling me that our preparations contained protein but not nucleic acid (8).”
But while Pruisner’s findings raised the possibility that he might be on to something new and exciting, not all was going well for him. He lost his funding at UCSF from the Howard Hughes Medical Institute. Worse yet, UCSF told him that he would not be promoted to tenure. But, the tenure decision was reversed, and because he had by now developed a starting point for his studies, and because his early results suggested that the project might yield intriguing new findings, he was awarded modest support from the NIH, as well as more substantial funding from the R. J. Reynolds Company (really).
Pruisner’s rate of progress was significantly enhanced when he found that he could shorten the length of time needed for his assays by moving from mice to hamsters, in which 70 days were required, rather than the 360 days needed in mice. And, he also, redesigned his measurement method. By 1982, in addition to the results of his biochemical analysis (which implied that the scrapie agent was comprised entirely of protein), he also found that scrapie infectivity could be reduced by treatments that alter proteins, but not by treatments that alter nucleic acids. Believing that he now had sufficient data to support his premise that the scrapie agent is comprised only of protein, he published his findings in a paper in Science (9).
Pruisner introduced the term “prion” for the first time in the 1982 Science paper (9). But in doing so, he set off a “firestorm (8).” Most virologists were skeptical of his findings, and some competitors, who had been working on scrapie and CJD, were incensed by his claims. “At times the press became involved since the media provided the naysayers with a means to vent their frustration at not being able to find the cherished nucleic acid that they were so sure must exist. Since the press was usually unable to understand the scientific arguments and they are usually keen to write about any controversy, the personal attacks of the naysayers at times became very vicious (8).” Nonetheless, Pruisner was confident that he was right: “Despite the strong convictions of many, no nucleic acid was found; in fact, it is probably fair to state that Detlev Riesner (Aside 6) and I looked more vigorously for the nucleic acid than anyone else (8).”
[Aside 6: Detlev Riesner had been studying viroids when he met Pruisner. These agents, which mimic viruses, are small, naked, single-stranded, circular RNAs, that infect plants. They were discovered by Theodore O. Diener, a Swiss plant pathologist. Since the small size of viroids is consistent with Alper’s X-ray data, which showed that the scrapie agent too is extremely small, Pruisner sought out Riesner to inquire whether viroids might be the causative agents of CJD. The meeting between the two researchers led to a long-time collaboration, and both contributed to the discovery that the scrapie agents do not contain nucleic acids.]
By the next year Pruisner had isolated the scrapie prion protein, and the following year Leroy Hood (who helped found the human genome program) determined a portion of its amino acid sequence. Meanwhile, skeptics kept searching for a nucleic acid-containing scrapie agent. And while they never succeeded in their efforts to overturn the wealth of evidence Pruisner was accumulating in support of the prion hypothesis, the mystery of how prions might replicate still remained to be solved. Toward that end, Pruisner collaborated with Charles Weissmann to clone the cellular gene encoding the prion protein.
“Once cDNA probes for PrP became available, the PrP gene was found to be constitutively expressed in adult uninfected brain. This finding eliminated the possibility that PrPSc stimulated the production of more of itself by initiating transcription of the PrP gene… (8).” Moreover, with the isolated PrP proteins in hand, it was clear that PrPSc was not the translational product of an alternatively spliced mRNA. [“The entire open reading frame of all known mammalian and avian PrP genes resides within a single exon (8).”] Furthermore, PrPSc was not the result of a posttranslational modification of PrPC.
Pruisner and coworkers next considered the possibility that PrPC and PrPSc differed only in their conformations. However, bear in mind that the molecular biology dogma of the day held that the amino acid sequence of a protein specifies only one biologically active conformation of the protein. Consequently, the idea that PrPC and PrPSc differed only in their conformations “was no less heretical than that of an infectious protein (8).” Nonetheless, the results of structural studies indeed bore out the conformation premise. See Aside 7.
[Aside 7: For aficionados: “Fourier transform infrared (FTIR) and circular dichroism (CD) studies showed that PrPC contains about 40% α-helix and little β-sheet, while PrPSc is composed of about 30% α-helix and 45% β-sheet. Nevertheless, these two proteins have the same amino acid sequence!” The PrPSc structure enables amyloids to form their characteristic tightly interacting, many stranded and repetitive intermolecular β-sheets. Readers interested in these, and additional structural studies might begin with Pruisner’s Nobel lecture (4).]
Pruisner and coworkers then carried out a series of telling experiments that began to unlock the mystery of prion replication. The first step was to generate mice that lacked both copies of the mouse PrPC gene. Importantly, this treatment rendered these mice completely resistant to mouse PrPSc. However, when hamster PrPC genes were incorporated into the genomes of these mice, and were expressed in them, these transgenic mice then were susceptible to hamster PrPSc. However, the mice remained resistant to mouse PrPSc. Thus, in mice, the hamster PrPC transgene product was required to promote the replication of hamster scrapie prions, whereas the mouse PrPC protein was required to promote the replication of the mouse scrapie prions.
These results show that the scrapie PrP isoform and the normal cellular PrP protein each play crucial roles in the transmission and pathogenesis of prion disease. Importantly, these results are completely consistent with the “misfolding hypothesis,” in which the scrapie isoform catalyzes the conversion of the normal cellular PrP protein into the scrapie conformation.
The finding that expression of the hamster PrPC promotes (and indeed is required for) replication of the hamster PrPSc, but does not promote replication of the mouse PrPSc, is an example of the “species barrier” to prion infection—in which the passage of prions between species is generally restricted. Differences in the amino acid sequence homology between the PrPSc of one species and the PrPC of another species, which might impair their interaction, readily explain the species barrier. See Aside 8.
[Aside 8: Interestingly, despite prions not having genomes, prion “mutation” can occur, in the sense that prions encoded by the same PrP gene may assume different conformations, thereby giving rise to a kind of prion “polymorphism,” which may enable prions to cross species barriers by a process of conformational selection.]
In 1990, in another series of key experiments, Pruisner’s research group discovered a mutation of the human PrP gene (a leucine substitution at codon 102), which appeared to be linked to Gerstman-Straussler-Scheinker syndrome (a very rare, exclusively inheritable, progressive spongiform encephalopathy in humans). Next, they generated a recombinant mouse PrP gene that encoded the leucine substitution at codon 102. Importantly, transgenic mice, which expressed the recombinant PrP gene, developed a scrapie-like disease with many of the pathological features of Gerstman-Straussler-Scheinker syndrome. What’s more, inoculates, which contained brain extracts from those mice, transmitted the disease to inoculated mice. Thus, Pruisner’s group demonstrated that a prion protein, containing a single amino acid substitution, can be the cause of a human familial prion disease.
Only a portion of Pruisner’s contributions up until he received his 1997 Nobel award were noted in the above narrative. For a more complete review of his work until then, see his 1997 Nobel lecture (4). Pruisner is still investigating neurodegenerative and dementing diseases at the UCSF School of Medicine, where he also serves as the director of its Institute for Neurodegenerative Diseases.
As already noted, Pruisner’s career is also remarkable for his having persevered in authenticating his iconoclastic protein-only prion hypothesis, despite the continuing and widespread skepticism and ridicule from colleagues in the scientific community. In that regard, his story is reminiscent of Howard Temin’s after announcing his discovery of reverse transcription by the RNA tumor viruses; which eventually would be re-designated “retroviruses” (10).
Pruisner himself was unprepared for the level of resistance to his discovery of prions: “…this created a rather harrowing and arduous journey for more than a decade…Many argued that I was spewing heresy and I had to be wrong (5).” What’s more, the ferocity of some of the personal attacks against Pruisner, particularly those in the media, were affecting his family.
But what might have explained the extent of the enmity on the part of some naysayers? Could it have simply been that the prion hypothesis conflicted with molecular biology dogma of the day? Or could it have been, as Pruiner suggested, that some critics perhaps were reacting to their frustration at not being able to find the nucleic acid, which they were sure had to be there?
Resigned to the criticism, Pruisner stated: “When there is a really new idea in science, most of the time it’s wrong, so for scientists to be skeptical is perfectly reasonable.” And, stoically, Pruisner’s answer to his critics was to keep producing data. “The incredulity of my colleagues only strengthened my conviction that scientists have a responsibility to convince their skeptics of the validity and importance of discoveries that run counter to prevailing opinions, and they can do so only by performing experiments that challenge their own hypotheses. Sometimes the road of testing and retesting is long and arduous—such was the case for me (5).” Moreover, and to Pruisner’s credit as a scientist, he also attributed his tenacity to his fascination with prions.
By the late 1980s, enough scientific data (particularly the knockout mouse studies) had emerged to begin garnering a measure of acceptance for the protein-only prion hypothesis. Then, in 1996, with the appearance in Britain of the first human cases of mad cow disease (incidentally identified as such using techniques originally developed by Pruisner), prions were suddenly a hot topic in the media. And, the very next year Pruisner was awarded the Nobel Prize.
Did the spotlight on mad cow disease and prions influence the Nobel committee’s decision? Pruisner conceded that “It didn’t hurt.” And, he graciously admits to the part that luck may have played in his exceptional career. “Extremely intelligent men and women can toil for years in the vineyards of science and never be fortunate enough to make a great discovery. And then there are a few people who are recipients of mammoth doses of good luck. The infectious pathogen that we now call a prion might well have turned out to be an atypical virus—not nearly as interesting as an infectious protein…or another group instead of mine might have discovered prions; that sort of preemption happens all the time in science (5)”
Yet some of Pruisner’s critics remained skeptical of the prion hypothesis even after he was awarded the Nobel Prize. For example, consider the following excerpts from a 1998 Science paper by Bruce Chesebro (11): “Although the notion that “protein only” can account for the infectious agent has received considerable publicity as a result of the Nobel prize award to S. Prusiner for the discovery of prions, the fact remains that there are no definitive data on the nature of prions… There are arguments both for and against the hypothesis that abnormal PrP itself is the transmissible agent, but on either side of this controversy no argument is as yet completely convincing … Clearly, we are in the very early stages of exploration of this subject. It would be tragic if the recent Nobel Prize award were to lead to complacency regarding the obstacles still remaining. It is not mere detail, but rather the central core of the problem, that remains to be solved.”
1. Abbott A. 2016. The red-hot debate about transmissible Alzheimer’s. Nature 531: 294–297 doi:10.1038/531294a
2. Abbott A and Dolgin E. 2016. Failed Alzheimer’s trial does not kill leading theory of disease. Nature doi:10.1038/nature.2016.21045
3. Collinge J. 2016. Mammalian prions and their wider relevance in neurodegenerative diseases. Nature 539:217–226.
4. Pruisner SB, Prions, Nobel Lecture, December 8, 1997.
5. Pruisner S. Madness and Memory: The Discovery of Prions-A New Biological Principle of Disease, Yale University Press, 2014.