All posts by lnorkin2013

Confronting Science Denialism in the Trump Era

Our last post discussed President Trump’s advocacy of a link between vaccines and autism, while also noting that Trump has taken that stance despite the fact that the “link” has been well-debunked by several large-scale scientific studies (1). Also, our post made a case for biomedical scientists to speak out against Trump’s anti-vaccine pronouncements.

So, 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 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 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, you will 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. 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 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).”

References

  1. President Trump’s Advocacy of the Debunked Link Between Vaccines and Autism, Posted on the blog January 26, 2017.
  2. Charles Sykesfeb, Why Nobody Cares the President Is Lying, NY Times, February 4, 2017.
  3. Melissa Leach, Accommodating dissent, Nature 450, p283, 22 November 2007, doi:10.1038/450483a.
  4. Diana Kwon, Will a March Help Science?, The Scientist, February 2, 2017.
  5. Wendy Palenfeb, When Canadian Scientists Were Muzzled by Their Government, NY Times, February 14, 2017.]

Addendum

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

President Trump’s Advocacy of the Debunked Link Between Vaccines and Autism

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).”

References:

  1. 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.
  2.  Andrew Wakefield and the Measles Vaccine Controversy, Posted on the blog February 9, 2015.
  3. Mnookin S, How Robert F. Kennedy, Jr. Distorted Vaccine Science, Scientific American, STAT on January 11, 2017, https://www.scientificamerican.com/.../how-robert-f-kennedy-jr-distorted-vaccine-scie…
  4. 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.”

To Resign over an Editorial Decision You Disagree With

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.

AIDS denialist, Peter Duesberg
AIDS denialist, Peter Duesberg

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-reviewed medical 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.

References:

1. Duesberg PH, et al., 2011. AIDS since 1984: no evidence for a new, viral epidemic – not even in Africa. Italian Journal of Anatatomy and Embryololgy 116:73–92. http://fupress.net/index.php/ijae/article/view/10336/9525

2. Duesberg P, 1987. Retroviruses as carcinogens and pathogens: expectations and reality. Cancer Res. 47:1199–220. PMID3028606.

3. Thabo Mbeki and the South African AIDS Epidemic, Posted on the blog July 3, 2014.

4. Duesberg PH, et al., 2009. WITHDRAWN: HIV-AIDS hypothesis out of touch with South African AIDS – A new perspective. Medical Hypotheses. doi:10.1016/j.mehy.2009.06.024. PMID19619953.

5. Who Discovered HIV, Posted on the blog, January 24, 2014.

6.  Corbyn Z. 2012. Paper denying HIV–AIDS link sparks resignation: Member of editorial board quits as editor defends publication. Nature doi:10.1038/nature.2012.9926.

7. Howard Temin: “In from the Cold, Posted on the blog December 14, 2013.

8. Stanley Pruisner and the Discovery of Prions: Infectious Agents Comprised Entirely of Protein, Posted on the blog December 15, 2016.

 

Stanley Pruisner and the Discovery of Prions: Infectious Agents Comprised Entirely of Protein

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.

Stanley B. Pruisner (left) and His Majesty the King, at the Nobel Prize Ceremony
Stanley B. Pruisner (left) and His Majesty the King, at the Nobel Prize Ceremony

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.”

References:

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.

6. Carlton Gajdusek, Kuru, and Cannibalism, Posted on the blog April 6, 2015.

7. Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013.

8. Stanley B. Prusiner – Biographical”. Nobelprize.org. Nobel Media AB 2014. Web. 24 Nov 2016. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/1997/prusiner-bio.html&gt;

9. Prusiner, SB. 1982. Novel proteinaceous infectious particles cause scrapie. Science 216:136–144.

10. Howard Temin: “In from the Cold, Posted on the blog December 14, 2013.

11. Chesebro B. 1998. BSE and Prions: Uncertainties About the Agent. Science 279:42-43.
DOI: 10.1126/science.279.5347.42

Elie Metchnikoff: The “Father of Innate Immunity”

The legend of Isaac Newton being struck on the head by a falling apple has long been enshrined in scientific lore. Likewise, there is the tale of Mendeleev suddenly grasping the relationship between the elements (i.e., discovering the Periodic Table) while struggling over how to organize them for a chemistry textbook he was writing. And, there is the myth of Kekule envisioning the benzene ring structure while dreaming of a snake grasping its own tail. Also, there are the fables of Ben Franklin and his Kite, Darwin and his finches, and Galileo dropping objects from the Leaning Tower of Pisa, among others.

Here we have the tale of Russian zoologist Elie Metchnikoff (1845-1916) who, in 1882, discovered leukocyte recruitment and phagocytosis as key elements in the body’s natural defenses. The mythical aspect of Metchnikoff’s discovery is that it allegedly happened while he was experimenting on starfish larvae. Metchnikoff was awarded a share the 2008 Nobel Prize in Physiology or Medicine for his discovery. German microbiologist Paul Ehrlich shared the 2008 award for his pioneering discoveries in humoral immunity.

Elie Metchnikoff
Elie Metchnikoff

We are fortunate to have Metchnikoff’s account of his 1882 epiphany, written in his own words shortly after he was awarded the Nobel Prize in 2008 (1).

“One day, as the whole family had gone to the circus to see some exceptional trained monkeys, while I had remained alone at my microscope and was following the life of motile cells in a transparent starfish larva, I was struck by a novel idea. I began to imagine that similar cells could serve the defense of an organism against dangerous intruders. Sensing that I was on to something highly interesting, I got so excited that I started pacing around, and even walked to the shore to gather my thoughts.

I hypothesized that if my presumption was correct, a thorn introduced into the body of a starfish larva, devoid of blood vessels and nervous system, would have to be rapidly encircled by the motile cells, similarly to what happens to a human finger with a splinter. No sooner said than done. In the shrubbery of our home, the same shrubbery where we had just a few days before assembled a ‘Christmas tree’ for the children on a mandarin bush, I picked up some rose thorns to introduce them right away under the skin of the superb starfish larva, as transparent as water. I was so excited I couldn’t fall asleep all night in trepidation of the result of my experiment, and the next morning, at a very early hour, I observed with immense joy that the experiment was a perfect success! This experiment formed the basis for the theory of phagocytosis, to whose elaboration I devoted the next 25 years of my life.

So, at a time when virtually nothing was known about the body’s natural defenses, Metchnikoff proposed that the mobile cells (later dubbed “phagocytes” or cell-eaters), which gathered around the thorns in the starfish larvae, were agents of healing. Moreover, he proposed that those cells are the first line of an organism’s defense against invading pathogens. Metchnikoff’s use of starfish larvae in his breakthrough experiment owed to his interest in marine invertebrates which, in turn, reflected his broad interest in natural history.

Metchnikoff’s passionate interest in science, natural history, and marine invertebrates developed early in his life. In 1870, when he was barely 25 years-old, he was appointed a professor of zoology and comparative anatomy at the University of Odessa; a position he resigned in 1882 because of limited research opportunities in Odessa, and because of political instability in the Ukraine after the assassination of Alexander II. Metchnikoff’s pioneering experiments that year were carried out at a private laboratory in Messina. [Later, during the Soviet Era, Odessa University was renamed Odessa I.I. Mechnikov National University, in Metchnikoff’s honor.]

In 1888 Louis Pasteur recruited Metchnikoff to the Pasteur Institute, where he would spend the remainder of his career. There, under the influence of Pasteur and Emile Roux (with whom he developed a close friendship), Metchnikoff turned his attention from simple organisms to experimental infectious disease and immunity.

By the late 1880s, Metchnikoff’s hypothesis that leukocyte recruitment and phagocytosis played a key role in host defense was garnering considerable attention. However, much of that attention was hostile, mainly because Paul Ehrlich, in Germany, was concurrently promoting the role of antisera in the body’s defenses.  The resulting feud between French scientists at the Pasteur Institute and Ehrlich’s colleagues in Germany was dubbed the “Immunity War.” [The “Immunity War” also may have reflected nationalistic feelings left over from the quite real Franco-Prussian war of July 1970 to May 1971.]

It was not until after Metchnikoff and Ehrlich shared their 1908 Nobel award that immunologists recognized that Metchnikoff’s phagocytes were a feature of “innate immunity,” while Ehrlich’s antibodies were a feature of “adaptive immunity.” Eventually both schools of thought would be integrated into our modern understanding of immunity. Metchnikoff would be recognized as the “Father of Innate Immunity,” while Ehrlich would be recognized as the pioneer of adaptive immunity (see the Aside). But, Metchnikoff’s early dispute with Ehrlich may be one reason why he avoided attending the 1908 Nobel Prize award ceremony. Metchnikoff presented a delayed Nobel lecture in Stockholm in 1909.

[Aside: Innate immunity is so named because it is present at birth and remains unchanged throughout life. It is the body’s first response to an invasive pathogen. Innate immunity is fast because it recognizes molecular patterns that are characteristic of broad classes of microorganisms; doing so via receptors that are encoded in the germ line. In contrast, the adaptive immune system is highly specific, recognizing determinants that are unique to each invader; doing so via receptors that are not encoded in the germ line. The adaptive immune system also has a memory. The price for the adaptive system’s specificity is that activation can take 1 week or longer. Innate immunity is the more primitive of these systems. It is present in primordial invertebrates, including insects, worms and mollusks. In contrast, adaptive immunity is seen only in vertebrates.]

How true to fact is the starfish-based tale of Metchnikoff’s discovery? A recent review by Siamon Gordon (Oxford professor of cellular pathology) suggests that Metchnikoff’s own personal account may not be entirely accurate (2).  For instance, a review of the early scientific literature shows that at the time of Metchnikoff’s discovery, phagocytosis had already been described by others.  Intriguingly, a description of phagocytosis appeared in the 1862 novel Fathers and Sons by Turgenev; an author admired by Metchnikoff. In Turgenev’s novel, “the description is given by a nihilist doctor, Yevgeny Bazarov, who, like Metchnikoff, used the microscope to make his own observations (2).”

Nonetheless, Gordon asserts that Metchnikoff indeed carried out the starfish experiments which led to the discovery. Moreover: “What distinguishes his (Metchnikoff’s) discovery from other early descriptions is that he followed up the initial observation with a program of striking experiments, which convinced him that this was a far-reaching process of general biological significance (2).” [Another review by Gordon summarized Metchnikoff’s many considerable contributions (3), some of which are noted below (see Note).]

The “myth” of Metchnikoff’s discovery, like all such myths, often convey a misimpression of the nature of scientific discovery, since they do not sufficiently acknowledge the intense efforts, sustained over considerable periods of time, which are generally necessary to produce major breakthroughs. But, these myths are fun and they do enhance the lay-public’s awareness of science.

Metchnikoff became somewhat of a public celebrity in his later years when he advocated eating yogurt to promote good health and long life (4). Apropos our larger story, Metchnikoff’s promotion of yogurt consumption was inspired by his interest in phagocytes. It was based on his beliefs that 1) the infirmities of old-age happen when phagocytes are transformed from defenders against infection into destroyers of healthy tissue by autotoxins (i.e., toxins that harm the organisms in which they are produced) derived from “putrefactive bacteria” residing in the colon, 2) that these degenerative changes could be prevented by inhibiting the colon’s putrefactive bacteria, and 3) that the host-friendly lactate-producing bacteria in yogurt would inhibit the putrefactive bacteria in the colon. [Metchnikoff regarded the colon as a “vestigial cesspool,” which does little more than provide a reservoir for putrefactive bacteria.]

Metchnikoff’s yogurt-eating regimen attracted numerous adherents for a time, but it eventually fell out of favor (indeed it even was satirized), since the premises on which it was based were never verified. Nonetheless, the medical community has recently been using Lactobacillus acidophilus to effectively treat several conditions, including pediatric antibiotic-associated diarrhea, acute infectious diarrhea, and persistent diarrhea in children. So, might Metchnikoff also be viewed as the “father” (or grandfather perhaps) of the current probiotics craze?

References:

1. Metchnikoff E: My stay in Messina (Memories of the  past, 1908); in Souvenirs, Editions en Languese Etrangeres. Moscow, 1959 (translated from the French by Claudine Neyen). (w.karger.com/doi/10.1159/000443331)

2. Gordon S. 2016. Elie Metchnikoff, the Man and the Myth. Journal of Innate Immunity, 8:223-227.

3. Gordon S. 2008. Elie Metchnikoff: Father of natural immunity. European Journal of Immunology, 38:3257-3264.

4. Mackowiak P. 2013. Recycling Metchnikoff: Probiotics, the Intestinal Microbiome and the Quest for Long Life. Frontiers in Public Health. 1-3.

Note: “His (Metchnikoff’s) notable observations include proof that organisms were taken up by an active process, involving living, and not just scavenged dead organisms; acidification of vacuoles, digestion and destruction of degradable particles including many infectious microbes including bacteria, spirochaetes and yeasts; uptake of host cells, e.g. erythrocytes, often nucleated for ready identification, from diverse species, as well as spermatocytes; and carmine dye-particles, used as an intravital marker of phagocytosis. Metchnikoff emphasized observations in living systems, combining microscopy and staining with neutral red and other histological labels to evaluate the acidity of vacuoles, viability and fate of ingested organisms. The bacteria examined included Cholera vibrio, Bacillus pyocyaneum, Bacillus anthracis and its spores, Mycobacterium (human, avian and bovine), plague bacilli, Streptococci and Gonococci, and some of these were studied in combination. He demonstrated killing by leukocytic enzymes (‘cytase’). Metchnikoff made important contributions to understanding the entire process of inflammatory recruitment, described at length in his lectures on comparative inflammation. He observed diapedesis through vessel walls, aggregation of leukocytes at sites of inflammation and their tendency to fuse, and he dissected the role of endothelial, epithelial and mesenchymal cells, as well as of lymphatic drainage and nervous elements in the classic hallmarks of inflammation (oedema, rubor, calor, dolor, loss of function) and repair. By using simple organisms, he discovered the central role of phagocytosis in diverse biologic models. This work led naturally to studies on the clearance and fate of organisms after experimental administration via a variety of routes, e.g. intravenous, intraperitoneal, subcutaneous and even the anterior chamber of the eye (3).”

 

A Most “Elegant” Experiment: Sydney Brenner, Francois Jacob, Mathew Meselson, and the Discovery of Messenger RNA

What was the most “elegant” experiment ever? Many molecular biologists, who were active during the so-called “golden age” of the 1950s and 1960s, might opt for the 1958 experiment of Mathew Meselson and Franklin Stahl, which demonstrated the semiconservative replication of DNA (1). My choice is the 1960 experiment by Sidney Brenner, Francois Jacob, and Matt Meselson, which established the existence of messenger RNA (mRNA) (2). The story behind the discovery is an appropriate topic for the blog since bacteriophage T2 had a key role to play. It is told here, largely through the words of one of its contributors, Pasteur Institute scientist and Nobel laureate, Francois Jacob (3).

Imagine for the moment that we are back in the late 1950s, at a time when the precise role of RNA was not yet known. However, pertinent evidence was accumulating, which implied that RNA had a role in protein synthesis. For example, cellular RNA levels correlated with the levels of protein synthesis.

But what might the role of RNA be? The example of eukaryotic cells seemed to indicate that DNA could not directly serve as the template for protein synthesis. The DNA in those cells is contained within the membrane-bounded nucleus, whereas protein synthesis occurs in the cytoplasm. Might RNA then serve as an intermediate information carrier?

Jacob, and others, knew that protein synthesis took place in the cytoplasm, on tiny granules called ribosomes. Moreover, “for each gene there were corresponding ribosomes specifically charged with producing the corresponding protein (3).” This remark might seem to suggest an accurate view of protein synthesis. Nonetheless, the understanding of ribosomes at the time was fundamentally wrong. Each gene was thought to be transcribed to a unique RNA that became an integral component of a ribosome. Moreover, that integral RNA was thought to confer on the ribosome the specificity to support the synthesis of only the one protein that corresponded to that particular RNA—a scenario under which an entire ribosome needed to be produced de novo to support the translation of a gene.

With that view of ribosomes in mind, Jacob was troubled by the results from an earlier experiment, carried out in 1957 by Arthur Pardee, Jacob himself, and Jacques Monod—the famous (and also quite “elegant”) PaJaMa experiment (4). In this experiment, the Lac gene of an Hfr (male) strain of E. coli is transferred to a Lac-minus, F-minus (female) strain. [This experiment is famous because it was carried out under experimental conditions which enabled the three researchers to demonstrate the existence of a previously unknown regulatory molecule; the “repressor.”] What troubled Jacob was that the Lac gene of the donor E. coli strain was expressed “immediately upon entry of the gene…”—a result not in accord with the thinking of the day about the nature of ribosomes, and the way in which they translated genes into proteins.

It seemed impossible to Jacob that ribosomes, which are complex structures composed of proteins and RNA, could be produced quickly enough to enable the virtually immediate translation of the transferred Lac gene, as had been seen in the PaJaMa experiment. What’s more, the prevailing view of ribosomes also did not fit “with the existence of units of activity recently baptized ‘operons,’ that contained several genes. Nor with a regulation functioning directly on the DNA through the intermediary of a switch, now called an ‘operator.’”

The “perplexity prevailing in the Pasteur group” led to a new line of thought— “either direct synthesis of the protein on DNA itself, with no intermediary; or production of an unstable intermediary, probably an RNA with rapid renewal. But the former hypotheses seemed highly improbable and the latter without a chemical basis, without any trace of a molecule that could substantiate it.”

In 1959 Jacob attended a colloquium on microbial genetics in Copenhagen, where he intended to discuss this conundrum. “A small group attended, including notably Jim Watson, Francis Crick, Seymour Benzer, Sydney Brenner, Jacques (Monod), and even the physicist Niels Bohr. Courteous as ever, Jim Watson spent most of the sessions ostentatiously reading a newspaper. So, when it came time for him to speak, everyone took from his pocket a newspaper and began to read it”

When Jacob’s turn to speak came, he raised the possibility of a need for an unstable intermediary, which he called X. “No one reacted. No one batted an eyelash. No one asked a question. Jim continued to read his newspaper.”

“A new opportunity to discuss protein synthesis arose around Easter 1960 in Cambridge (England), in Sydney’s small apartment in King’s College, where he was a Fellow.” Although the meeting that morning was casual, several heavy hitters were present, including Francis Crick, Leslie Orgel, and Ole Maaloe, in addition to Jacob and Brenner.

Crick and Brenner discussed the results of a recent experiment carried out by Pardee and Monica Riley (Pardee’s student at the time). “They had succeeded in charging the DNA of male bacteria with radioactive phosphorus; in making them transfer to females the gene of galactosidase; in letting it synthesize the enzyme for some minutes; and then in destroying the gene through the disintegration of the radioactive phosphorus. The result was clear: once the gene was destroyed, all synthesis stopped. No gene, no enzyme. Which excluded any possibility of a stable intermediary.” [Recall the thinking that stable ribosomes contained an integral RNA that conferred its specificity.]

“At this precise point, Francis and Sydney leaped to their feet. Began to gesticulate. To argue at top speed in great agitation. A red-faced Francis. A Sydney with bristling eyebrows. The two talked at once, all but shouting. Each trying to anticipate the other. To explain to the other what had suddenly come to mind. All this at a clip that left my English far behind. For some minutes, it was impossible to follow them, just as it would have been impossible for them to follow a discussion in French between Jacques (Monod) and me. What had set off Francis and Sydney was, once again, a connection between the lactose system and phage. After infecting the colon bacillus, certain highly virulent phages blocked the synthesis of new ribosomes. As had been shown by two American Researchers, Elliot Volkin and Lazarus Astrachan, the only RNA then synthesized had two remarkable properties: on the one hand, unlike ribosomal RNA, it had the same base composition as DNA; on the other hand, it renewed itself very quickly. Exactly the properties required for what we called X, the unstable intermediary we had postulated for galactosidase. Why, in Paris, when we were looking for a support material for X, had we not thought of this phage RNA? Why had I not thought of it? Ignorance? Stupidity? Oversight? Misreading of the literature? Failure of judgment? A little of all these, no doubt. A mixture that, as in a detective novel, had made us fail to spot the murderer, the molecule responsible. In the last analysis, however, what mattered was that X, the unstable intermediary, was materializing…it had to be shown that all this was not a dream; that this RNA of the phage was indeed the unstable intermediary functioning in the synthesis of proteins: the issue that we and Sydney immediately decided to take up. …” See Aside 1.

[Aside 1: Volkin and Astrachan, at the Oak Ridge National Laboratory in Tennessee, showed that there actually are two kinds of RNA seen during phage infection—a stable type found in ribosomes (now known as ribosomal RNA, which does not have the same base composition as the DNA ), and an unstable, rapidly turning over type, that has the same base composition as the viral DNA, but not the bacterial DNA (5). Transfer RNA remained to be discovered.]

That afternoon, Jacob and Brenner found out that they each had been invited to spend a month (June) at the California Institute of Technology. Brenner’s invitation came from Matt Meselson, and Jacob’s from Max Delbruck. “A unique opportunity to work together to demonstrate the nature and role of X.” Importantly, Meselson recently developed a technique that would make the discovery possible.

That evening, at a party given by Crick and his wife, Jacob and Brenner discussed the experiment that they were envisioning. But: “It was difficult to isolate ourselves at such a brilliant, lively gathering, with all the people crowding around us, talking, shouting, laughing, singing, dancing. Nevertheless, squeezed up next to a little table as though on a desert island, we went on, in the rhythm of our own excitement, discussing our new model and the preparations for experiments at Caltech.”

In their new concept of protein synthesis: “The ribosomes had lost all specificity. They had become simple machines for assembling amino acids to form proteins of any kind, like tape recorders that can play any kind of music depending on the magnetic tape inserted in them. In protein synthesis, it was X, the unstable RNA copied on a gene, that had to play the role of the magnetic tape, associating with the ribosomes to dictate to them a particular sequence of amino acids corresponding to a particular protein.” Thus, the experiment would be to “show that the unstable RNA, synthesized after infection of a colon bacillus by the virulent phage, associated with pre-existing ribosomes, synthesized before infection, to produce the proteins of the phage.”

A key problem would be to distinguish ribosomes made before infection from any ribosomes that might be made after infection. Their solution would be provided by Matt Meselson’s new technique in which “he marked macromolecules by cultivating bacteria in heavy isotopes before putting them back in a normal environment. Using ultracentrifugation, he could then separate the marked molecules along gradients of density…”

Thus, the plan was to grow cells for several generations in medium containing the heavy isotopes 15N and 13C as the sole nitrogen and carbon sources, respectively. In this way, essentially all ribosomes present in the cells would be “heavy”. Next, the cells would be washed and placed in medium containing the normal isotopes, 14N and 12C. Then, the cells would immediately be infected with the phages. Any new ribosomes made after the infection got underway would be “light”.

Here is a key point. Recall that Volkin and Astrachan showed that the only RNA that is made after infection is the unstable RNA, which has the same base composition as the phage DNA. [That is so because the phage shuts down host transcription and translation.] Consequently, this phage RNA can be specifically labeled by adding 32P to the infected cultures (5). Brenner, Jacob, and Meselson hoped to find this rapidly turning-over phage-specific RNA in the density gradients, in association with the old heavy ribosomes that were made before infection. “If we were right, if our hypothesis was correct, the radioactivity of the RNA had to be associated, in the gradients, with the band of “heavy” ribosomes.”

However: “We were not succeeding.” The problem that was frustrating their efforts was that the ribosomes were unstable in the density gradients. “In vain did we try to check through the experiment, to modify it, to change a detail here and there. It was now three weeks since Sydney Brenner and I had arrived at the California Institute of Technology. We had come for the sole purpose of carrying out this experiment with Matt Meselson. An experiment that we had no doubt was going to change the world. But the gods were still against us. Nothing worked.”

“Our fine confidence at the start had evaporated. Disheartened, Meselson had departed-to get married! Sydney and I talked about going back to Europe. In a burst of compassion, a biologist by the name of Hildegaard had taken us under her wing and, to give us a change of scene, driven us to a nearby beach. There we were, collapsed on the sand, stranded in the sunlight like beached whales. My head felt empty. Frowning, knitting his heavy eyebrows, with a nasty look, Sydney gazed at the horizon without saying a word. Never yet had I seen Sydney Brenner in such a state. Never seen him silent…And our time was running out. For, come what may, Sydney and I had decided to leave at month’s end.”

“Hildegaard tried to tell us stories to lighten the atmosphere. But we were not listening. Suddenly, Sydney gives a shout. He leaps up, yelling, “The magnesium! It’s the magnesium!” Immediately we get back in Hildegaard’s car and race to the lab to run the experiment one last time. We then add a lot of magnesium… Sydney had been right. It was indeed the magnesium that gave the ribosomes their cohesion. But the usual quantities were insufficient in the density gradients used to separate heavy and light compounds. This time we added plenty of magnesium. The result was spectacular. Eyes glued to the Geiger counter, our throats tight, we tracked each successive figure as it came to take its place in exactly the order we had been expecting. And as the last sample was counted, a double shout of joy shook the basement at Caltech…This was merely one experiment, performed in extremis… But we now knew that we had won. That our conception explained the transfers of information in the synthesis of proteins…Scarcely was the experiment over than we gave a seminar at Caltech to demonstrate the existence of X and its role as magnetic tape. No one believed us. The next day we left, each to his own home. The bet had paid off. In the nick of time.”

Apropos our Virology blog, this experiment also showed that viruses subvert the cellular protein synthesis machinery for their own ends.

Addendum:

Nobel laureate Sidney Brenner was the main subject of two earlier posts—The Phage in the Letter, reposted September 8, 2016 and Sidney Brenner: Only Joking, January 5, 2014 (6, 7). Each of these posts highlighted Brenner’s mischievous sense of humor. Jacob offers more insight into Brenner’s personality in his account of the episode on the beach with Hildegaard: “There we were, collapsed on the sand, stranded in the sunlight like beached whales. My head felt empty. Frowning, knitting his heavy eyebrows, with a nasty look, Sydney gazed at the horizon without saying a word. Never yet had I seen Sydney Brenner in such a state. Never seen him silent. On the contrary, he was an indefatigable talker at every opportunity. A tireless storyteller, able to discourse for days and nights on end. Interminable monologues on every conceivable subject. Science, politics, philosophy, literature, anything that cropped up. With stories he made up as he went along. Generously laced with jokes. With nasty cracks, too, at the expense of just about everyone. An excellent actor, he could render a speech in Hungarian, a lecture in Japanese. Mimic Stalin or Franco. Even himself. He went without a break from one register to another. A sort of fireworks whose effects he gauged from the expressions of the people around him.”

In the September 8th reposting I wrote: “While Brenner’s work as a molecular biology pioneer may have justified a Nobel Prize, he received the award in 2002 for his later studies of the nematode Caenorhabditis elegans, in which his research group traced the fate of each cell from the zygote right through to the adult worm. Their work established C. Elegans as a model system that is now studied in hundreds of laboratories all over the world (6).”

Jacob collaborated with Jacques Monod to elucidate the genetic switch that regulates beta-galactosidase synthesis in E. coli. Their collaboration established the concepts of regulator genes and operons, for which they shared in the 1965 Nobel Prize for physiology or medicine.

François Jacob (left), with Jacques Monod and André Lwoff. This Pasteur Institute threesome shared the 1965 Nobel Prize for Physiology or Medicine
François Jacob (left), with Jacques Monod and André Lwoff. This Pasteur Institute threesome shared the 1965 Nobel Prize for Physiology or Medicine “for their discoveries concerning genetic control of enzyme and virus synthesis.” Lwoff’s share of the award was for his pioneering studies of lysogeny (8).

In 1940, Jacob, who was Jewish, left medical school in occupied France to join Free French Forces in London. He then served as a medical officer in North Africa, where he was wounded, and was wounded again, this time severely, at Normandy in August 1944. Monod too was active in the French Resistance, during the Nazi occupation of Paris. He eventually become chief of staff of the French Forces of the Interior. In that capacity, he helped to prepare for the Allied landings in Normandy. Monod and Jacob each received France’s highest honors for their wartime service. For more on Jacob, see Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob, posted January 28, 2015 (8).

Matt Meselson (still at Harvard at 86 years in age) is best known for showing that DNA replication is semi-conservative and for his part in the discovery of messenger RNA. Jacob tells us that at the time of their collaboration at Cal Tech: “He (Meselson) was haunted by the Cold War, by the need to establish better relations with the Soviet Union. In his soft voice, he could discourse for hours on strategy, tactics, nuclear arms, the Rand Corporation, first strikes, reprisals, annihilation.” Meselson later helped to persuade President Richard Nixon to renounce biological and chemical weapons, and to support an international treaty (the 1972  Biological Weapons Convention) banning the use of biological agents.

References:

  1. Meselson M and FW Stahl, 1958. The Replication of DNA in Escherichia coli, Proceeding of the National Academy of Sciences USA. 44:671–82.
  2. Brenner S, F Jacob, and M Meselson, 1961. An Unstable Intermediate Carrying Information from Genes to Ribosomes for Protein Synthesis, Nature 190:576-80.
  3. Francois Jacob, The Statue Within: An Autobiography, English language translation copyright 1988 Basic Books Inc.
  4. Pardee, AB, F Jacob, and J Monod, 1959. The genetic control and cytoplasmic expression of ‘inducibility’ in the synthesis of β-galactosidase by coli, Journal of Molecular Biology 1:165–178.
  5. Volkin E and L Astrachan. 1956. Phosphorus Incorporation in Escherichia coli Ribonucleic Acid after Infection with Bacteriophage T2. Virology 2:149-161.
  6. The Phage in the Letter. Reposted on the blog September 8, 2016.
  7. Sidney Brenner: Only Joking. Posted on the blog January 5, 2014 (find under Archives, January 2014).
  8.  Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob. Posted on the blog January 28, 2015.

Hilary Koprowski’s Oral Polio Vaccine: The Bizarre Claim that it was the Source of HIV in Humans

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

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

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

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

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

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

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

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

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

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

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

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

index

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References

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

 

The Phage in the Letter

Some of the earliest postings have mysteriously disappeared from the blog. One of these, The Phage in the Letter, is one of my favorites. So, I am re-posting it. Hope you enjoy.

Here is a favorite story of mine that I first heard when I was a graduate student in the mid 1960’s. The major protagonists are Sidney Brenner, who was one of the giants of the “golden age of molecular biology,” and Norton Zinder, also one of the top researchers of the day. Brenner was the first molecular biologist to propose the idea of a messenger RNA, a concept validated by experiments he later did with Mathew Meselson and Francois Jacob. Zinder’s major contributions included the discovery that a bacteriophage can transfer bacterial genes from one bacterial cell to another, a phenomenon referred to as “transduction.” And, apropos this anecdote, Zinder also isolated the f2 bacteriophage, the first virus known to contain a genome composed of RNA, rather than DNA.

Bearing in mind how little was known in 1960, when Zinder isolated bacteriophage f2; the discovery of RNA phages had great potential for use in the study of fundamental molecular processes, such as protein synthesis, including its initiation and termination. Clearly, there were good reasons why molecular biologists of the day, including Brenner, wanted to obtain their own samples of f2 phage. So, as the legend goes, Brenner, among others, requested a sample of f2 from Zinder. And, Zinder wrote back to all, saying that the phage was not available.

Zinder may have thought that Brenner wanted the phage to study RNA replication, a topic that Zinder wanted to keep for himself. Now, here is the delightful part of the story. Knowing how carefree researchers can be in the laboratory, Brenner is said to have dipped Zinder’s letter in a culture of E. coli (the f2 host), thereby readily growing up a stock of f2 for himself.

Amusing as this story might be, the actual facts, at least according to a 1997 article by Brenner1, are as follows. First, after Zinder isolsted f2 phage from a New York sewer, he indeed declined to distribute the phage to the large number of researchers requesting it. Second, Brenner’s reason for wanting f2 was not to use it to work on RNA replication, but instead to use it to test bacteria for the presence of a sex factor. The bacterial sex factor is a gene that encodes a so-called pilus, which is present on male bacteria, enabling them to transfer genes to female bacteria. It also is the bacterial “organ” via which RNA phages enter bacterial cells, thus explaining Brenner’s stated interest in f2. [While it might be thought that f2 can only infect male bacteria, interestingly, male bacteria that are infected with f2 can transfer the virus to female bacteria via their pili. Thus, even bacteria have sexually transmitted infections.] Third, while Brenner may not have isolated f2 from Zinder’s letter, he indeed recommended a similar procedure to several other researchers. Brenner also confesses that he might have added to the original myth by hinting that the story actually might be true. In reality, Brenner isolated many RNA phages himself by taking sewerage from the Cambridge, Massachusetts, sewer treatment plant and plating it on bacteria expressing a sex factor.

figure-6-11-virology

The Micrograph shows an F-pilus emerging from an E. coli cell that is covered with icosahedral MS2 phage particles.  At the end of the pilus, a filamentous fd phage has attached itself. The thicker thread emerging at the right is a bacterial flagellum. Figure 6.11, page 188, From Virology: Molecular Biology and Pathogenesis, by Leonard C. Norkin, ASM Press, 2010.

While Brenner’s work as a molecular biology pioneer may have justified a Nobel Prize, he received the award in 2002 for his later studies of the nematode Caenorhabditis elegans, in which his research group traced the fate of each cell from the zygote right through to the adult worm. Their work established C. Elegans as a model system that is now studied in hundreds of laboratories all over the world.

1Brenner, S. 1997. Bacteriophage Tales. Current Biology 7:R736-737.

Hilary Koprowski: Genesis of a Virologist

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

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

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

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

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

koprowski

Hilary Koprowski in Warsaw (2007)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References:

   

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

 

 

 

 

 

John Enders: “The Father of Modern Vaccines”

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References:

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