Tag Archives: Renato Dulbecco

Harold Varmus: From English Literature Major to Nobel Prize-Winning Cancer Researcher

Harold Varmus and J. Michael Bishop changed cancer research in a fundamental way in the 1970s, when they discovered proto-oncogenes at the University of California at San Francisco (UCSF). Proto-oncogenes are cellular genes that normally play an important role in controlling cell division and differentiation. However, Varmus and Bishop found that proto-oncogenes can be altered by mutation, to become oncogenes that contribute to cancer. When Varmus and Bishop first began their collaboration in 1970, cancer research was, for the most part, focused on epidemiology (e.g., studies linking smoking to lung cancer) and empirical approaches to therapy (e.g., radiation and chemotherapy).

Harold Varmus, Cancer Researcher, Nobel Laureate, Director of the NIH
Harold Varmus, Cancer Researcher, Nobel Laureate, Director of the NIH

The discovery of proto-oncogenes is a pertinent topic for our Virology blog because it depended crucially on Varmus and Bishop’s earlier finding that retroviral oncogenes are mutated versions of cellular genes that retroviruses “captured” from their host cells. Varmus and Bishop hypothesized and then demonstrated that since retroviral oncogenes are versions of genes that actually are part of a normal cell’s genetic makeup, mutations in those genes, or their inappropriate expression, can lead to cancer. The v-src gene of Rous sarcoma virus was the first retroviral oncogene that Varmus and Bishop showed is derived from a cellular genome (1).

Varmus and Bishop continued searching for proto-oncogenes in the 1980s. Varmus also began investigating HIV (also a retrovirus and the cause of AIDS). In 1989 Varmus and Bishop were awarded the Nobel Prize in Physiology or Medicine for their discovery of proto-oncogenes.

In the early 1990s Varmus stepped out from his role as a research scientist to take up the cause of public funding for biomedical research. In 1993 President Bill Clinton acknowledged Varmus’ efforts in that regard, as well as his stature as a scientist, by appointing him to serve as Director of the National Institutes of Health (NIH). Thus, Varmus became the first Nobel laureate to head the NIH.

In 2000 Varmus left the NIH to accept the presidency of the Memorial Sloan-Kettering Cancer Center in New York. In 2010 Varmus returned to the NIH, this time appointed by President Barak Obama to serve as director of the National Cancer Institute (NCI). In 2015 Varmus was back again in New York where he is the Lewis Thomas University Professor of Medicine at Weill-Cornell Medical College.

Varmus was featured in two earlier blog postings. The first of these described how he mediated the dispute between Robert Gallo and Luc Montagnier over the right to name the AIDS virus (2). The second posting covered some of the political and social dilemmas Varmus faced during his days leading the NIH (3).

Here, we relate first how Varmus opted for a career in biomedical science and, second, how his collaboration with Bishop came about. This is an interesting tale because Varmus’ remarkable career as a science researcher, administrator, and spokesperson happened despite his initial intention to become a teacher of English literature. Indeed, his career in science did not begin until after he earned an M.A. degree in English from Harvard University, and then spent four years in medical school preparing for a career in clinical medicine.

We begin our story in 1950 as Varmus recounts how, as a ten-year-old, he witnessed his physician father receive a call that conveyed shocking news: “one of my mother’s favorite cousins, a robust man in the middle of his life, had just been diagnosed with leukemia. Of course, I did not know very much about leukemia, but I did know immediately from my parents’ expressions–and within a few weeks, from our cousin’s death—that his disease was a veritable tidal wave.” [All quotations are from Varmus’ book The Art and Politics of Science (4), in which he reflects back on his entire career.]

His cousin’s leukemia actually resulted from a mutation in one of the genes that Varmus would discover more than two decades later. And Varmus notes just how far the science in general had progressed during that 25-year interim: “…when my father heard about our cousin’s leukemia, biologists were not even sure that genes were made of DNA, had no idea how genetic information could be encoded in genes, and, of course, had no way of knowing that cancers are driven by mutations.”

Varmus was urged by his father to prepare for a career in medicine. Nonetheless, when Varmus enrolled as a freshman at Amherst College he strongly favored studying the humanities. Thus, he “toyed with the idea of majoring in philosophy (ultimately too abstract), physics (ultimately too hard), and English literature (ultimately selected).”

Throughout his undergraduate days, Varmus envisioned preparing for an academic career teaching literature. Still, he dutifully fulfilled premed requirements to keep open the possibility of obliging his father’s wishes that he become a medical doctor. Yet he never considered majoring in biology. “I couldn’t understand how some of my close friends (among them, some now distinguished scientists) could spend long afternoons and evenings incarcerated in a laboratory, when they could be reading books in a soft library chair or reciting poetry on Amherst’s green hills.”

Varmus began having doubts about his career choice when his Amherst College classmate Art Landy (later a well-known molecular biologist at Brown University) won an Amherst biology prize that allowed him to attend a 1961 international biochemistry meeting in Moscow. Importantly, Landy invited Varmus to accompany him to the Moscow meeting, where Varmus learned that Marshal Nirenberg had deciphered the genetic code. “Even though I did not understand its meaning or its importance at the time, I was not oblivious to the excitement around me…Scientists seemed likely to discover new, deep, and useful things about the world, and other scientists would be excited by these discoveries and eager to build on them. Would this be true of literary critics and teachers?”

Notwithstanding these misgivings, Varmus continued on his path to a career in English literature after graduating from Amherst College in 1961, earning an M.A. in English from Harvard in 1962 (his focus was on Anglo-Saxon poetry). But his uncertainties about his future only grew stronger. “Despite outward signs that I had chosen a life of studying and teaching literature, soon after starting my graduate work at Harvard I began to suffer some further internal doubts about abandoning medicine. The graduate curriculum in English literature was not especially onerous, but it felt like a prolongation of college. Most of my courses were heavily populated with Harvard and Radcliff undergraduates.” Varmus leaves the impression that he looked upon much of his course work at Harvard as a tiresome chore.

Varmus was also aware of the enthusiasm of former Amherst College classmates who were then studying at Harvard Medical School. “Occasionally, on Saturday mornings, I traveled across the Charles River to join some Amherst classmates at Harvard Medical School, while they sat in the Ether Dome at Massachusetts General Hospital, entranced by diagnostic dilemmas discussed at the weekly pathology conference. These stories struck me as far more interesting than those I was reading, and my medical school friends expressed general excitement about their work. They also seemed to have formed a community of scholars, with shared interests in the human body and its diseases and common expectations that they would soon be able to do something about those diseases…These Saturday excursions probably account for an influential dream I had one night about my continuing indecision. In that dream, my future literature students were relieved when I didn’t turn up to teach a class, but my future patients were disappointed when I didn’t appear. It seemed I wanted to be wanted.”

So, Varmus finally came to grips with his qualms about a career teaching English literature, hastily preparing an application to Harvard Medical School and biking across the frozen Charles River to deliver it just in time to meet the deadline. But it was to no avail, since the dean of admissions thought Varmus was “too inconstant and immature” for medical school.

Varmus next sent off an application to Columbia University’s College of Physicians and Surgeons (P&S). His interviewer at Columbia was an esteemed physician named David Seegal, who also happened to be rather literate. Seegal asked Varmus if he might translate the Anglo Saxon phrase Ich ne wat. “This was easy; it simply means ‘I don’t know.’” Seegal used his question as a lead-in to discuss why a physician might admit fallibility to a patient. In any case: “By the fall of 1962, I was happily enrolled at P&S, helped for the first, but not the last, time by someone’s exaggerated appreciation of my competence in two cultures.”

Now ensconced at P&S, Varmus thought he might become a psychiatrist; an ambition stoked by an interest in Freud and by his winning of an essay prize at P&S in psychiatry. But, he found his “first hour alone in a room with a psychotic patient to be more difficult and less interesting than an hour reading Freud.” So, Varmus’ interests in medical school turned from the “elusive mind” to the physical brain and then, more generally, to diseases that might be explained by known physiology and biochemistry.

When graduation from medical school was impending, Varmus had to consider his career options more deliberately than he had in the past. A key factor was the Vietnam War, which was in progress, and which he and many others of that era vehemently opposed. “I was determined not to serve in it. Medical graduates were subject to the draft; however, we did have the more palatable option of two years training at one of the agencies of the Public Health Service. For most of my classmates with academic ambitions similar to my own, the NIH was the favored choice. As the largest biomedical research campus in the world, it offered unequaled opportunities to learn virtually any form of biomedical research…”

Varmus confesses that he had a “woeful lack of laboratory credentials.” Nonetheless, he entered the competition for one of the coveted research slots at the NIH. But, because of his lack of research experience, he was not encouraged by most of the NIH laboratory chiefs who interviewed him. However, one of them, endocrinologist Jack Robbins, suggested to Varmus that he speak to Ira Pastan; a young endocrinologist who, at that time, was interested in the production of thyroid hormones.

As Varmus relates, “The recommendation proved to be wise and fateful. My schooling in literature turned out to be more important than my interest in endocrinology, Ira’s field, because Ira’s wife Linda, a poet, had often complained that Ira’s colleagues seldom talked about books…When matches were announced I was told I would become Ira’s first clinical associate, having been passed over by the more senior investigators. This outcome could not have been more fortunate.”

But, before Varmus could take up his position at the NIH, he received a “shocking phone call” from Pastan, to the effect that he (Pastan) was giving up his work on thyroid hormones because he and colleague Bob Perlman “had made a shocking discovery about gene regulation in bacteria.” Pastan and Perlman found that cyclic AMP is a major regulator of bacterial gene activity, and that it plays a similar role in animal cells—findings which led Pastan to pioneer the field of receptor biology in animal cells.

The discovery by Pastan and Perlman had important consequences for Varmus. First, it immediately forced him to give up his plan to train in endocrinology. Instead, Pastan assigned Varmus to find out whether cyclic AMP augments bacterial gene expression by increasing transcription of mRNA. Second, as explained below, Pastan’s new research direction led to Varmus’ introduction to and fascination with virology.

So, Varmus was now a budding molecular biologist. But, since he had no prior research experience, his first days in the Pastan lab were a near disaster, leading Pastan to half jokingly ask, “Now remind me why I took you into the lab.”

In any case, Varmus worked closely with Pastan to develop a molecular hybridization assay to measure transcription of E. coli lac mRNA. [Their specific the goal was determine whether the mechanism by which cyclic AMP reverses catabolite repression of the E. coli lac operon is by enhancing transcription of lac mRNA.] And, they used an E. coli phage, which had incorporated the lac operon into its genome, as their source of isolated lac operon DNA. Thus, Varmus was introduced to virology. [Aficionados, note, “These experiments with the lac operon proved to be analogous in several ways to experiments that revealed the first proto-oncogene a few years later.”]

The satisfaction that Varmus derived from his research in Pastan’s lab caused him to reconsider his aspirations for a career in clinical medicine, and instead to think about a future in biomedical research. He thought he might next try his hand at cancer research, motivated in part, by his mother’s breast cancer, first diagnosed in 1968, to which she succumbed two years later. But there were other factors at work as well. In particular, Varmus’ use of the E. coli phage in Pastan’s lab led to his fascination with virology. And his interest in virology was relevant to his new plans because the DNA and RNA tumor viruses held immense potential for cancer research. 1970s technology could not identify which one of the tens of thousands of cellular genes might have mutated to result in a cancer. However, that technology was potentially able to identify which of the handful of a tumor virus’ genes might underlie its ability to transform a normal cell into a tumor cell.

That line of thought led Varmus to apply for a research position in Renato Dulbecco’s lab at the Salk Institute. [Dulbecco would win a share of the 1975 Nobel Prize in Physiology or Medicine for his pioneering studies of the DNA tumor viruses (5).] However, reminiscent of Varmus’ unsuccessful application to Harvard Medical School, he was “rebuffed by not one but two letters from his (Dulbecco’s) secretary.”

While the rejection from Dulbecco was a disappointment, it would be another of the seemingly providential happenings in Varmus’ career. In the summer of 1969 he chanced to visit Harry Rubin, an eminent Rous sarcoma virus researcher at U Cal Berkeley. Rubin, who had earlier introduced Howard Temin to virology (another auspicious happening; see reference 6), told Varmus about a new group at UCSF that had begun to study retroviruses. Importantly, the goal of the UCSF group was to discover cancer-causing genes. Thus, Varmus stopped over at UCSF, where he met members of the group, including a smart young virologist named Mike Bishop. Varmus reports, “we recognized from the first moments that we were destined to work together.”

Varmus came to Bishop’s lab in 1970 as a postdoctoral fellow. However, their relationship quickly evolved to one of equals, and they made all of their major discoveries in the 1970s and 1980s as a team, and they rose together through the UCSF academic ranks. Bishop relates that their bond formed not just by a shared fascination with cancer viruses but “by our mutual love of words and language.” Varmus, for his part, notes that “after many years of ambivalence and indecision…I appeared to be headed in a clear direction, even if not towards medicine or literature.”

References:

1. Stehelin D, Varmus HE, Bishop JM, Vogt PK., 1976. DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260:170-173.

2. How the Human Immunodeficiency Deficiency Virus (HIV) Got Its Name, posted on the blog February 4, 2014.

3. The Politics of Science: Vignettes Featuring Nobel Laureate Harold Varmus during his Tenure as Director of the NIH, posted on the blog June 2, 2014.

4. Varmus, H. 2009. The Art and Politics of Science, (W. W. Norton & Company).

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

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

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Harald zur Hausen, Papillomaviruses, and Cervical Cancer

Harald zur Hausen (1936- ) was awarded a share of the 2008 Nobel Prize in Physiology or Medicine for discovering that papillomaviruses cause cervical cancer. He received the award jointly with Luc Montagnier and Françoise Barré-Sinoussi, who were given their portion for discovering HIV (1). Before getting on with zur Hausen’s story per se, we begin with bit of earlier history.

zur hausen Harald zur Hausen in 2008

Genital warts are benign epithelial tumors that have been known and associated with sexual promiscuity since the time of the ancient Greeks. In 1907 these lesions were unequivocally proven to be an infectious disease by Italian researcher, G. Ciuffo, who showed that they can be transmitted by filtered extracts of wart tissue; a finding which also implied that the etiologic agent is a virus. Ciuffo inoculated himself to advance his case.

Ciuffo’s finding is relevant to our story since members of the papillomavirus family of DNA viruses are the cause of warts. What’s more, and importantly, some papillomaviruses  also cause malignant cervical carcinomas.

In 1933 Richard Shope, at the Rockefeller Institute, became the first researcher to isolate a papillomavirus, the cottontail rabbit papillomavirus. Shope went on to show that this virus is the cause of skin papillomas in its rabbit host. This finding by Shope was the first to demonstrate that a DNA virus can be tumorigenic.

Years earlier, in 1911, Peyton Rous discovered that an RNA virus—the Rous sarcoma virus (the prototype retrovirus)—causes solid tumors in chickens. Peyton Rous was Richard Shope’s friend and colleague at the Rockefeller Institute. In 1934 Shope asked Rous to characterize the warts that the rabbit papillomavirus induces in jackrabbits. Rous found those warts to be benign tumors that could progress to malignant carcinomas.

Despite the earlier findings of Ciuffo, Shope, and others, the notion that genital warts in humans is a sexually transmitted malady was slow to gain acceptance. Oddly, perhaps, recognition of that truth was prompted by a 1954 report that American servicemen, who had been serving in Korea, were transmitting genital warts to their wives upon returning to the U.S (T. J. Barrett, et al., J. Am. Med. Assoc. 154:333, 1954). [Sexually transmitted diseases were a long-standing problem in the military. Servicemen were most often infected by sex workers who frequented the vicinity of military quarters.]

The key discoveries of this tale are Harald zur Hausen’s 1983 and 1984 findings that two human papillomavirus subtypes, HPV-16 and HPV-18, together account for about 70% of all cervical cancers. Considering that more than 120 distinct HPV subtypes have been identified, the high degree of association of cervical carcinoma with only two of these subtypes provided compelling evidence for the viral etiology of this malignancy. Later studies showed that HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are responsible for another 20% of cervical cancers. Indeed, an HPV infection is present in virtually all cervical carcinomas. See Aside 1.

[Aside 1: Cervical cancer was once the leading cause of cancer-related deaths in women in the United States. However, the number of cervical cancer deaths in the industrialized world decreased dramatically over the last 40 years, largely because of the Pap test, which can detect pre-cancer cervical lesions in their early stages. The CDC website reports 12,109 cervical cancer cases and 4,092 deaths from cervical cancer in the U.S. in 2011 (the most recent year for which data are available). Worldwide, cervical cancer was responsible for 275,000 deaths in 2008. About 88% of these deaths were in developing countries (J. Ferlay et al., Int. J. Cancer, 127:2893, 2010).]

Harald zur Hausen was a child in Germany during the Second World War, growing up in Gelsenkirchen-Buer, which was then a center for German coal production and oil refining and, consequently, a major target for allied bombing. [The city also contained a women’s sub-camp of the Buchenwald concentration camp. The Nazis used its prisoners for slave labor.] All members of zur Hausen’s family survived the war. However, zur Hausen’s primary education contained significant gaps because schools were closed during the allied bombing (2).

Despite the gaps in zur Hausen’s early education, he was keenly interested in biology and dreamed of becoming a scientist. Yet at the University of Bonn he opted to study medicine, rather than biology. After zur Hausen received his medical degree, he worked as a medical microbiologist at the University of Düsseldorf, where he enjoyed the opportunity that the University gave him to carry out research on virus-induced chromosomal aberrations.

Although zur Hausen was fascinated by his research, he was soon aware of the deficiencies in his scientific background. So, in 1966 he looked to enhance his proficiency as a scientist by securing a postdoctoral position in the laboratories of Gertrude and Werner Henle at the Children’s Hospital of Philadelphia.

The Henles were a German-born husband and wife research team, known for their work on flu vaccines. More apropos to our story, they are also known for demonstrating the link between the recently discovered Epstein-Barr virus (EBV; a herpesvirus) and infectious mononucleosis, as well as for showing that EBV is the etiologic agent of Burkitt’s lymphoma; a cancer found in parts of Africa. EBV was, in fact, the first virus associated with a cancer in humans. [Gertrude Henle’s mother was murdered by the Nazis in 1943.]

Although zur Hausen took part in the Henles’ experiments involving EBV, he did so grudgingly because he was intimidated by his inexperience in molecular biology. In his own words: “I urged Werner Henle to permit me to work with a different agent, namely adenovirus type 12, hoping that this relatively well established system would permit me to become acquainted with molecular methods. He reluctantly agreed. I started to work eagerly on the induction of specific chromosomal aberrations in adenovirus type 12-infected human cells…and, to please my mentor, I demonstrated electron microscopically the presence of EBV particles directly in… Burkitt’s lymphoma cells (2).”

In 1969 zur Hausen returned to Germany to take an appointment as an independent scientist at the University of Wurzburg. His research was now focused entirely on EBV. Specifically, he wanted to challenge the prevailing view that Burkitt’s lymphoma tumors are persistently infected with EBV (i.e., that the tumors continuously produce low levels of the virus).

I presume that zur Hausen was interested in this issue because it was reasonable to believe that EBV gene expression is necessary to maintain the neoplastic state of the Burkitt’s tumor cells. Persistent infection would be one means by which viral genes could be carried by the cells. But zur Hausen believed that EBV DNA might be maintained in Burkitt’s lymphoma cells, even if they did not produce EBV particles.

Werner Henle in Philadelphia (and also George Klein in Stockholm) sent zur Hausen a large number of Burkitt’s lymphoma cell lines and tumor biopsies to aid in his study. One of those cell lines, the Raji line of Burkitt’s lymphoma cells, did not produce EBV particles. Nevertheless, zur Hausen isolated sufficient EBV DNA from the Raji cells to prove that multiple copies of EBV DNA were maintained in them. This was the first time that tumor virus DNA was shown to be present in malignant human cells that were not producing virus. See Aside 2.

[Aside 2: In 1968 Renato Dulbecco and co-workers were the first to discover viral DNA integrated by covalent bonds into cellular DNA (J. Sambrook et al., Proc. Natl. Acad. Sci. U S A. 60:1288, 1968). They were studying cells transformed by the polyomavirus, SV40. Integration explained how SV40 genes could be stably maintained and expressed in transformed cells, in the absence of productive infection. Integration is now recognized as a key event in cell transformation by members of several virus families, including the polyomaviruses, papillomaviruses, and the oncogenic retroviruses.

The situation in the case of EBV, a herpesvirus, is different, as herpesviruses are able to enter into a latent state in host cells. In the latent state the viral genome is maintained as an episome, and only a subset of the viral genes (i.e., those concerned with latency) are expressed. The episomal viral genome is replicated by the cellular DNA replication machinery during the cell cycle S phase, and a viral gene product, EBNA-1, ensures that viral genomes are equally partitioned between the daughter cells. In 1978 George Klein and co-workers were the first to demonstrate episomal EBV DNA in a cell line derived from a Burkitt’s lymphoma biopsy (S. Koliais et al., J. Natl. Cancer. Inst. 60:991, 1978).]

In 1972, while zur Hausen’s attention was focused on EBV and Burkitt’s lymphoma, his research direction took a providential turn that would lead to his most important discoveries. It happened as follows.

Recent seroepidemiological evidence was suggesting a link between herpes simplex virus type 2 (HSV-2), a well known genital infection, and cervical cancer. Since HSV-2, like EBV, is a herpesvirus, and since zur Hausen had already demonstrated that EBV DNA is present in Burkitt’s lymphoma tumor cells, zur Hausen believed he was well positioned to search for HSV-2 DNA in cervical cancer biopsies. However, in this instance, all his repeated attempts failed.

Harald zur Hausen then came across anecdotal reports of genital warts converting to squamous cell carcinomas. Importantly, those genital warts were known to contain typical papillomavirus particles. Taking these two points into account, zur Hausen considered the possibility that papillomaviruses, rather than herpesviruses, might be the cause of cervical carcinomas. Indeed, his initial thought was that the genital wart papillomavirus might also be the etiologic agent for cervical carcinomas.

Thus, Harald zur Hausen began his foray into papillomavirus research. His first experiments found papillomavirus particles in benign plantar (cutaneous) warts. His next experiments demonstrated that there are multiple papillomavirus subtypes. [In brief, zur Hausen used in vitro-transcribed plantar papillomavirus RNA as a hybridization probe against the DNA from various plantar and genital warts. Only some of the plantar wart DNAs, and none of the genital wart DNAs, reacted with his planter wart RNA probe. Restriction endonuclease patterns of a variety of human papillomavirus isolates confirmed that the HPVs comprise a heterogeneous virus family.]

Harald zur Hausen’s next experiments sought to detect papillomavirus DNA in cervical carcinoma biopsies. However, his initial trials in this crucial undertaking were unsuccessful.  He was using DNA from HPV-6 (isolated from a genital wart) as a hybridization probe in those failed attempts. But zur Hausen and co-workers had at hand a number of additional HPV subtypes, from which they prepared other DNA probes. DNA from HPV-11 (from a laryngeal papilloma) indeed detected papillomavirus DNA in cervical carcinomas.

In 1983, two of Zur Hausen’s former students, Mathias Dürst and Michael Boshart, using HPV-11 DNA as a probe, isolated a new HPV subtype, designated HPV-16, from a cervical carcinoma biopsy. In the following year, they isolated HPV-18 from another cervical carcinoma sample. Harald zur Hausen’s group soon determined that HPV-16 is present in about 50% of cervical cancer biopsies, while HPV-18 is present in slightly more than 20%. [The famous HeLa line of cervical cancer cells contains HPV-18 DNA.]

Additional key discoveries took place during the next several years, including the finding that papillomavirus DNA is integrated into the cellular DNA of cervical carcinoma cells. This finding clarified how papillomavirus genes persist in the cancers, and also revealed that the cancers are clonal (see Aside 2, above). Moreover, while the integrated viral genomes often contain deletions, zur Hausen’s group found that two viral genes, E6 and E7, are present and transcribed in all cervical cancer cells. This finding implied that E6 and E7 play a role in initiating and maintaining the oncogenic state. [In 1990 Peter Howley and co-workers demonstrated that the interaction of the E6 gene product with the cellular tumor suppressor protein p53 results in the degradation of p53. In 1992 Ed Harlow and coworkers showed that the E7 gene product blocks the activity of the cellular tumor suppressor protein pRb. Reference 3 details the mechanisms of papillomavirus carcinogenesis.]

The above findings led to widespread acceptance that cervical carcinoma is caused by papillomaviruses. Yet acceptance was not immediate. The prevailing belief, that herpesviruses cause cervical carcinoma, was well-entrenched and slow to fade away. It was based on the observation that many women afflicted with cervical carcinoma also had a history of genital herpes. But, individuals infected with one sexually transmitted pathogen are often infected with others as well. Apropos that, genital warts were long thought to be associated with syphilis, and later with gonorrhea. In any case, in 1995 the World Health Organization officially accepted that HPV-16 and HPV-18 are oncogenic in humans.

Harald zur Hausen was awarded one half of the 2008 Nobel Prize for Medicine or Physiology for proving that cervical cancer is caused by human papillomaviruses. By the time of his award, his findings had led to key insights into the mechanism of HPV-mediated carcinogenesis and, importantly, to the development of a vaccine to prevent cervical cancer. See Aside 3.

[Aside 3: The first generation of Gardasil, made by Merck & Co., helped to prevent cervical cancer by immunizing against HPV types 16 and 18, which are responsible for an estimated 70% of cervical cancers. Moreover it also immunized against HPV types 6 and 11, which are responsible for an estimated 90% of genital warts cases. Apropos genital warts, there are 500,000 to one million new cases of genital warts (also known as condylomata acuminate) diagnosed each year in the United States alone.

The original vaccine was approved by the USFDA on June 8, 2006. An updated version of Gardasil, Gardasil 9, protects against the HPV strains covered by the first generation of the vaccine, as well as five additional HPV strains (HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58), which are responsible for another 20% of cervical cancers. Gardasil 9 was approved by the USFDA in December 2014.]

Harald zur Hausen reviewed the overall contribution of viruses to human cancer in his 2008 Nobel lecture (4). Some of his key points are as follows. HPVs were discussed above with respect to cervical carcinoma. HPVs also are associated with squamous cell carcinomas of the vagina, anus, vulva, and oropharynx. What’s more, 40% of the 26,300 cases of penile cancer reported worldwide in 2002 could be attributed to HPV infection.

Epstein-Barr virus too was discussed above. This member of the herpesvirus virus family causes nasopharyngeal carcinoma, as well as Burkitt’s lymphoma. Another herpesvirus, human herpesvirus 8, causes Kaposi’s sarcoma; the most frequent cancer affecting AIDS patients. Hepatitis B virus (HBV, a hepadnavirus), as well as hepatitis C virus (HCV, a flavivirus), causes hepatocellular carcinoma. The human T-lymphotropic virus 1 (HTLV-1), a retrovirus, induces adult T-cell leukemia. And the recently discovered Merkel cell polyomavirus (MCPyV) is responsible for Merkel cell carcinoma.

Harald zur Hausen estimated that viruses directly cause about 20% of all human cancers, and a similar percentage of all deaths due to cancer! And while 20% might seem to be a remarkably high figure for the extent of viral involvement in human cancer, zur Hausen suggests that it is actually a minimal estimate. That is so because it is difficult to determine that a particular virus is actually the cause of a cancer. Consequently, it is likely that other examples of viral involvement in human cancer will be discovered.

Harald zur Hausen gave two principal reasons for why it is difficult to establish that an infectious agent is the cause of a cancer in humans. First: “… no human cancer arises as the acute consequence of infection. The latency periods between primary infection and cancer development are frequently in the range of 15 to 40 years…” Second: “Most of the infections linked to human cancers are common in human populations; they are ubiquitous… Yet only a small proportion of infected individuals develops the respective cancer type.”

Viruses also contribute to the human cancer burden in an indirect way. For instance, HIV types 1 and 2 play an indirect role in cancer via their immunosuppressive effect, which is the reason for the extraordinarily high prevalence and aggressiveness of Kaposi’s sarcoma in AIDS patients.

Bacterial infections also contribute to the cancer burden. For example, Helicobacter pylori infections may lead to stomach cancer. What’s more, the parasites Schistosoma, Opisthorchis, and Clonorchis have been linked to rectum and bladder cancers in parts of Northern Africa and Southeast Asia, where they are prevalent.

Obviously, but important enough to state anyway, knowing that a particular cancer is caused by a particular infectious agent opens the possibility of developing a rational strategy to prevent that cancer. Gardasil is an exmple. A vaccine against HBV is also available, and one against HCV is under development.

References:

1. Who discovered HIV, Posted on the blog January 23, 2014.

2. MLA style: “Harald zur Hausen – Biographical”. Nobelprize.org. Nobel Media AB 2014. Web. 27 May 2015. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/2008/hausen-bio.html&gt;

3. Norkin, Leonard C. (2010) Virology: Molecular Biology and Pathogenesis. ASM Press, Washington, D.C. See Chapters 15 and 16.

4. Zur Hausen, Harold, The search for infectious causes of human cancers: where and why. Nobel Lecture, December 7, 2008.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Andre Lwoff
Andre Lwoff

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

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

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

Prophage
Prophage

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

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

Elie Wollman
Elie Wollman

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

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

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

Francois Jacob
Francois Jacob

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

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

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

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

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

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

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

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

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

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

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

Jacques Monod
Jacques Monod

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

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

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

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

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

References:

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

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

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

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

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

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

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

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

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

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

Andre Lwoff

Ernest Goodpasture and the Egg in the Flu Vaccine

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References:

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

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

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

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

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

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

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

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

Seymour Benzer: A Star of the “Golden Age of Molecular Biology”

An earlier blog posting told of how Max Delbruck, in 1950, summoned Renato Dulbecco to his office to propose that Dulbecco launch animal virus research at Caltech; where virology was still concerned solely with bacteriophages (1.) The background was as follows.

In the late 1940s, a wealthy Californian became ill with shingles (later known to be a delayed complication of chickenpox, caused by varicella-zoster virus, a herpesvirus). The man’s physician explained that nothing could be done for his shingles, and moreover, that virtually nothing was known about the viruses that infect humans. Auspiciously, the physician knew of the studies being done on bacteriophages at Caltech, and he also was aware that Caltech was the great center for such work. So, after explaining to his well-heeled patient that bacteriophages were only of theoretical interest regarding human disease, he suggested that the patient might help to develop a center at Caltech which might begin to study medically important viruses. The patient agreed, and since virology at Caltech was headed by Delbruck, the former physicist found himself with an endowment to study human viruses, with virtually no background for how to use it.

Delbruck tried to recruit Dulbecco to open up animal virus research at Caltech because Dulbecco, unlike others in the Caltech “Phage Group,” trained as a physician. However, Dulbecco was not the only one who Delbruck sought to enlist to take up the task that day. As Dulbecco tells us (2), “One day Seymour Benzer and I were called to his (Delbruck’s) office: Delbruck pointed out that animal virology appeared ready for major advances. Would either of us be interested in trying his hand at it? To me it sounded wonderful. I had been thinking perhaps with nostalgia, of my work with tissue cultures, years before, in Guiseppe Levi’s laboratory in Torino; so I immediately expressed my interest, before Benzer could say anything. Benzer, on the other hand, was not interested, so everything was settled without delay.”

Thus, it came to pass that Dulbecco was the one who launched the study of animal virology at Caltech and, moreover, the one who initiated quantitative animal virology in general. But, who was Benzer, and what became of him?

As an undergraduate at Brooklyn College in the late 1930s, Seymour Benzer considered majoring in biology. However, since the biology teaching of the day was largely concerned with taxonomy, which he had little interest in, he instead majored in physics. He continued his training in physics as a graduate student at Purdue University during the Second World War.

His doctoral research involved semi-conductors, as part of a project to develop better crystal rectifiers; a crucial component for radar. Benzer’s doctoral work as a physicist is notable since it is credited with contributing to the development of the first transistors.

Apropos the current story, while Benzer was studying physics at Purdue, he happened to read Erwin Schrodinger’s 1944 book: What is Life? A chapter in Schrodinger’s book, entitled “Delbruck’s Model,” especially intrigued Benzer; so much so that he considered switching back to biology.

[Aside 1: Schrodinger, the great Austrian physicist and Nobel laureate, was at the time an anti-Nazi émigré, living in Ireland. In that regard, see references 1 and 5.]

Why might What is Life? have made such a strong an impression on Benzer? It was largely because it was a time when the chemical nature of the genetic material, and its manner of replication and action, were not yet known. In fact, most biologists thought that proteins constitute the genetic material, while DNA was merely a structurally uninteresting, monotonous molecule, much like a starch.

[Aside 2: The classic 1952 blender experiment of Alfred Hershey and Martha Chase, together with the earlier (1944) transformation experiments of Avery, MacLeod, and McCarty, would eventually convince virtually everyone that DNA is the genetic material. Additionally, the 1953 discovery of the DNA structure by Watson and Crick, would immediately suggest a plausible mechanism by which DNA might be replicated.]

Given the state of knowledge in the mid 1940s, when genes were still thought to be comprised of protein, the models of the day to account for how genes might be replicated and expressed were neither convincing nor satisfying. Consequently, many scientists came to believe that it would be impossible to understand heredity and gene function in terms of the known laws of chemistry and physics.

In What is Life? Schrodinger sought to account for genetic attributes in terms of quantum mechanics. For instance, to explain how genes might preserve their structure, and store genetic information over the lifetime of an organism, while at 310 degrees above absolute zero, he suggested that genes might reside in an aperiodic crystal state, in which their atoms stay put in stable energy wells. The Delbruck model that Schrodinger cites, which so excited Benzer, “explains” gene mutations as different quantum mechanical energy levels of a gene (3). [The Delbruck model may actually have inspired Schrodinger to write What is Life?]

Bearing in mind that Schrodinger was a Nobel laureate, who discovered the immensely important wave equation (which expressed the movements of electrons in terms of wave mechanics rather than as particles), we can appreciate the impact that his following comment (in What is Life?) may have had on some physicists of the day: “From Delbruck’s general picture of the hereditary substance, it emerges that living matter, while not eluding the ‘laws of physics’ as established up to date, is likely to involve ‘other laws of physics’ hitherto unknown which, however, once they have been revealed will form just as integral part of this science as the former.”

The notion, that “other laws of physics” might be discovered by researching the genetic material, roused Benzer to enter, and indeed help to create the field now known as molecular biology (4, 5).

Seymour Benzer (right), with Francis Crick in 1964
Seymour Benzer (right), with Francis Crick in 1964

[Aside 3: Surprisingly, Schrodinger himself seemed unaware of the earlier pioneering work of George Beadle, Boris Ephrussi, and Edward Tatum in the 1930s and early 1940s, which established the concept, “one gene, one enzyme;” later revised to “one gene, one polypeptide chain.” [Those ground-breaking biochemical genetic studies were carried out using the fungus Neurospora crassa.] Also, it is surprising that Schrodinger appears unaware that in 1940, Delbruck, together with Salvatore Luria and, eventually, Alfred Hershey, had already formed the “Phage Group,” which carried out its first experiments at the Cold Spring Harbor Laboratory on Long Island, NY, with the ultimate purpose of understanding the physical basis of heredity (4, 5).]

[Aside 4: James Watson refers in the following comment to an early time in his graduate student years at Indiana, while he was still deciding whose lab to join there: “Some weeks later in Luria’s flat, I first saw Max Delbruck, who had briefly stopped over in Bloomington for a day. His visit exited me, for the prominent role of his ideas in What is Life? made him a legendary figure in my mind. My decision to work with Luria had, in fact, been made so quickly because I knew that he and Delbruck had done phage experiments together and were close friends (6).]

Here now is one of my favorite parts of this story. Benzer, now leaning towards biology, was attending a meeting of the American Physical Society in Bloomington, Indiana, where he happened to accompany a friend to the home of the friend’s former classmate, who just happened to be the wife of Salvatore Luria. Benzer tells us, “I could not have been more impressed…and it was not long before he (Luria) had persuaded me to enroll in the phage course at Cold Spring Harbor. Thus I suddenly plunged into the biology business (6).”

[Aside 5: Incidentally, in 1936, Dulbecco was in Luria’s lab in Italy, while studying for his medical degree at the University of Torino. Having favorably impressed Luria, Dulbecco was later (after the Second World War and a brief stint in politics) invited to join Luria’s group at Indiana to study bacteriophages. Dulbecco and Watson shared a lab bench in Luria’s Indiana lab.]

Benzer next spent a postdoctoral year at the Oak Ridge Biology Division, and then had the choice of going to Salvador Luria’s laboratory at Indiana, or to Delbruck’s group at Caltech. Benzer relates, “…I asked Luria’s student James Watson for advice…Luria, he said, would be likely to ask me every day what I had done, whereas I might not see Delbruck for a week at a time. I chose to join Delbruck at Cal Tech (7).”

Benzer’s key contributions to the developing field of molecular biology took place mainly at Purdue, to which he returned after spending two years as a postdoctoral fellow in Delbruck’s Caltech lab. But first, here is a brief personal recollection. When I initially encountered genetics in high school in the 1950s, chromosomes were depicted as beads on a string, with the beads representing the genes. The beads (genes) were the units of function, determining whether you had blue or brown eyes, for example. An entire bead (each one representing a gene) was also the unit of mutation. Moreover, recombination occurred between the beads. Thus, each bead (gene) was the unit of function, mutation, and recombination.

By the late 1950s, it was reasonable to believe that a phage genome might well be one long thread of DNA. With that premise in mind, Benzer proposed that there might then be a uniform probability of recombination anywhere along the length of the phage genome. Note here the corollary notion that the unit of genetic function and the units of recombination, and perhaps mutation as well, are not necessarily the same physical entities.

Benzer carried out his experiments using T4 phage, specifically investigating the rII region of the T4 genome. Mutations in the T4 rII region cause infected cells to undergo premature (rapid) lysis, resulting in lower phage yields. The r (rapid lysis) mutants could be distinguished from wild-type T4 by their plaque morphology on E. coli strain B. Fortuitously, r mutants can not grow on E. coli strain K. Thus, T4 r mutants could be plaque-isolated on E. coli B and, if recombinants were to occur between r mutants, they might be detected on E. coli K.

When Benzer became aware of these facts, he realized that he had the ingredients at hand for a high resolution genetic system that might enable him to detect recombinants between mutations within the rII region; possibly even between mutations within the same gene. And, if one were to “run the genetic map into the ground” (as Delbruck put it), it might be possible to obtain recombination even between adjacent nucleotides.

So, Benzer infected E. coli K cells with pairs of independently isolated T4 rII mutants. And, as he hoped, he found that wild-type T4 recombinants indeed were generated, although at a very low frequency, which indicated that the rII mutations are very close together on the phage chromosome. But, and importantly, in addition to finding rare genetic recombinants between rII mutations, Benzer also found that certain pairs of rII mutants actually replicated together in E. coli K. That is, they complemented each other.

Next, Benzer found that the rII mutants could be placed in either of two groups, designated A and B. All A mutants complemented all B mutants, and visa versa. However, mutants within the same group could not complement each other. Moreover, for complementation to occur, the mutations also had to be on separate phage chromosomes; that is, they had to be in trans. Complementation did not occur if the mutations were on the same phage chromosome; that is, in cis. [In the trans orientation, one phage chromosome contains a wild-type rIIA region and a mutant rIIB region, while the other phage chromosome contains a mutant rIIA region and wild-type rIIB region. In the cis orientation, both mutations are on the same phage chromosome, and no wild-type RII regions are present.]

Thus, in addition to demonstrating that all of the rII mutations are very close together on the T4 chromosome, Benzer’s experimental results also showed that the rII mutations fall into two distinct complementation groups. The key question is the explanation for complementation between rIIA and rIIB mutants, but only when the mutations are expressed in trans. The answer is that the rIIA and rIIB regions of the phage chromosome are separate genetic units of function, each of which encodes a distinct polypeptide. Thus, if the rIIA and rIIB mutations are on separate phage chromosomes (i.e., in trans), then a wild-type A and a wild-type B polypeptide can be generated by the respective wild-type rII region of each chromosome, thereby enabling complementation.

Benzer dubbed the genetic units of function, as exemplified by the rIIA and rIIB regions, “cistrons,” since they are operationally defined by the cis-trans test (i.e., mutations in separate cistrons complement each other when expressed in trans, but not when expressed in cis). As expected, mutations in the same complementation group also cluster together on the phage chromosome, as shown by genetic mapping techniques.

To appreciate the immense significance of Benzer’s findings from his rII system, we need to remember that classical genetics made no distinction between genes as units that specified a particular phenotypic trait, versus units of mutation, or as units of recombination. Indeed, classical genetics envisioned a gene as a single indivisible unit that embodied all three of these properties. Benzer’s experiments thus provided the distinctions between genetic units of function (cistrons), versus units of recombination, and of mutation, making clear that a gene is a unit that encodes a polypeptide, whereas a single nucleotide is the minimal unit of mutation. And, recombination might occur even between single pairs of nucleotide bases.

Benzer’s cis-trans test was widely used to determine whether any two mutations are in the same or different functional genetic units. [Notice that the the cis-trans test reflects the earlier one gene-one protein (now one gene-one polypeptide chain) concept.] Today, the term “cistron” is rarely used. Instead, we simply say gene to imply the same meaning.

Benzer also examined a curious rII mutation, r1589, which contains a deletion that extends over portions of both the A and B cistrons, including the spacer region between them. This mutation leads to the production of a continuous polypeptide chain comprised of portions encoded by both the A and B cistrons. The study of r1589 led to important insights into how mRNA (yet to be discovered) is transcribed and then translated into protein.

By the 1960s, Benzer’s interest in genetic fine structure began to wane. Yet he was still publishing papers at a steady rate. The simultaneous appearance of several of his papers tempted Delbruck to append the following postscript to a letter from his wife to Benzer’s wife: “Dear Dotty, please tell Seymour to stop writing so many papers. If I gave them the attention his papers used to deserve, they would take all my time. If he must continue, tell him to do what Ernst Mayr asked his mother to do in her long daily letters, namely underline what is important (8).”

Benzer’s reaction was: “It is very difficult for me now to think of anything worthy of being underlined.” So, Benzer’s scientific focus shifted again; this time to developing a model system that might lead to insights into the genetic basis for behavior. He eventually settled on using Drosophila melanogaster, and founded the field of neurogenetics.

Seymour Benzer passed away in November, 2008. He received numerous awards for his research, including the National Medal of Science, but not the Nobel Prize, which many believed he deserved.

References

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

(2)  Dulbecco, Renato, The Plaque Technique and the development of Quantitative Animal Virology, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J. D. Watson eds., Cold Spring Harbor Laboratory of Quantitative Biology, 1966.

(3)  Stent, Gunther S., Introduction: Waiting for the Paradox, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J. D. Watson eds., Cold Spring Harbor Laboratory of Quantitative Biology, 1966.

(4) Norkin, Leonard C., Virology: Molecular Biology and Pathogenesis, ASM Press, 2010. Chapters 1 and 2 recount the beginnings, philosophy, and early contributions of the Phage Group.

(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013. This piece contains more background on Max Delbruck, Salvatore Luria, and the founding of the phage group, as well as some of my very favorite anecdotes.

(6) Watson, J. D., Growing up in the Phage Group, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J. D. Watson eds., Cold Spring Harbor Laboratory of Quantitative Biology, 1966.

(7) Benzer, Seymour,  Adventures in the rII region, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J. D. Watson eds., Cold Spring Harbor Laboratory of Quantitative Biology, 1966.

(8) Sidney Brenner: “Only Joking”, Posted on the blog January 7, 2014. This piece gives another glimpse into the personality of Max Delbruck.

The Politics of Science: Vignettes Featuring Nobel Laureate Harold Varmus during his Tenure as Director of the NIH

During his extraordinary career, Nobel laureate Harold Varmus practiced science and served science with distinction. The vignettes that follow are, for the most part, about Varmus’ service to science during his tenure (1993-1999) as director of the U.S. National Institutes of Health. But first, we begin with a brief account of Varmus’ most significant scientific accomplishment.

In 1976, Varmus and collaborator Michael Bishop reported that retrovirus oncogenes (cancer-causing genes) are versions of genes that actually are present in the genomes of normal cells (1). Indeed, retroviruses acquired their oncogenes by “capturing” them from the genomes of their host cells. Perhaps the most singularly important conclusion to be drawn from Varmus’ and Bishop’s finding is that since retroviral oncogenes are versions of genes that are actually part of a normal cell’s genetic makeup, mutations in those particular cellular genes, or the inappropriate expression of those genes, might lead to cancer.

Varmus’ and Bishop’s findings led to a mushrooming of discoveries in cell signaling, cell growth, and cell differentiation (see Aside 1). Moreover, their discoveries are increasingly relevant clinically, as recounted below in the main text.

[Aside 1: The v-src gene of Rous sarcoma virus was the first retroviral oncogene that Varmus and Bishop showed is a version of a cellular gene. Next, in 1978, Raymond Erikson and coworkers isolated the Src protein. Then, Erikson’s research group and that of Bishop and Varmus independently discovered that Src has protein kinase activity. Protein kinases add phosphate groups to a specific target protein, generally triggering its activity. (Actually, Src phosphorylates itself, thus regulating its own activity.)

At the time that Erikson isolated Src, all protein kinases were believed to add phosphate to serine and threonine residues on their target proteins. Then, in 1980, Tony Hunter and Bart Sefton discovered that Src adds phosphotes to tyrosine residues. Thus, Src was the first known protein tyrosine kinase.

Stanley Cohen then discovered that the epidermal growth factor (EGF) receptor too is a protein tyrosine kinase, underscoring the role of tyrosine kinases in the control of normal cell proliferation, while also affirming the notion that inappropriate phosphorylation of a cellular protein can lead to cancer.

These discoveries led to a burst of research activity in cell signaling, and to the discovery of additional tyrosine and serine/threonine protein kinases, many of which act in mitogenic signaling pathways. What’ more many of the cellular genes encoding these proteins were initially discovered as retroviral oncogenes. For details on these points, see Virology: Molecular Biology and Pathogenesis.]

[Aside 2: An earlier posting on the blog, Renato Dulbecco and the Beginnings of Quantitative Animal Virology, noted that Renato Dulbecco shared the 1975 Nobel Prize for Physiology or Medicine, in recognition of his opening up the study of transformation by the DNA tumor viruses (i.e., the polyomaviruses, papillomaviruses, and adenoviruses). How then did analysis of transformation by the oncogenic retroviruses (i.e., the RNA tumor viruses) complement analysis of the DNA tumor viruses?

As suggested above; studies of the oncogenic retroviruses led to the identification of cellular signaling pathways that positively govern cell replication (i.e., that trigger cell growth). In contrast, studies of the DNA tumor viruses led to insights into cellular processes that negatively regulate cellular replication; in particular, processes affected by the key cellular tumor suppressor protein, p53, which activates apoptosis in cells that attempt to divide without having appropriately passed cell cycle checkpoints. The DNA tumor viruses affect transformation by inactivating tumor suppressor proteins. See Virology: Molecular Biology and Pathogenesis for details.]

[Aside 3: Varmus tells us that early in his career, in the late 1960s, he looked for places and people that might offer research training in the tumor viruses. “However, when I wrote to the already famous virologist Renato Dulbecco, at the Salk Institute in La Jolla, just North of San Diego, for a postdoctoral position, I was rebuffed by not one but two letters from his secretary (2).” See Aside 2.]

In the days before Varmus and Bishop published their findings, many cancer researchers actually were reluctant to believe that cancer has an underlying genetic basis. This was partly because it was not yet possible to clone and sequence genes, and there were no other apparent methods by which to identify putative cancer-related genes. [The means by which Varmus and Bishop made their breakthrough discovery are recounted in the Appendix, below.] In recognition of their discovery, Varmus and Bishop were awarded the 1989 Nobel Prize for Physiology or Medicine.

Varmus looked back on all aspects of his career in his 2009 autobiography, The Art and Politics of Science (2). [Unless otherwise noted, all of the quotations that follow are from Varmus’ 2009 memoir.] Here, are his remarks on the clinical significance of his and Bishop’s Nobel Prize-winning findings:

“In recent years, after our prize was awarded, mutant proto-oncogenes and the proteins they encode have become critical tools for the classification of cancers and promising targets for drugs and antibodies—treatments that have, in some cases, proven to be effective for a significant and growing number of cancers, including leukemias and lymphomas, lung, gastrointestinal, and kidney cancers: and cancers of the breast.”

In 1993, Varmus was named by President Bill Clinton to serve as Director of the U.S. National Institutes of Health; a position he held through 1999. As such, he was the only Nobel laureate to ever serve as the NIH director and he was also the first NIH director to also run an active laboratory. What’s more, during his tenure as director, he managed to nearly double the NIH’s research budget.

nih clinical center NIH Clinical Center

One of Varmus’ major responsibilities as the NIH director, and also one of his most contentious ones, was to apportion research dollars among the individual NIH institutes and programs. Why was there contention? As might be expected, the directors of the individual institutes actively advocate for their shares of the NIH budget. But, a further source of contention was Congress, in which the most ardent NIH supporters were generally motivated by their interest in a particular disease or program. What’s more, public advocacy groups likewise championed their own favored disease. Consequently, as Varmus explains:

“Apart from the difficulties of predicting where and how discoveries will arise, the priority-setting process can be ugly—for instance, when advocates refuse to recognize, or to care, that funds for their disease must come from funds being spent elsewhere, including funds used for a disease important to another group of advocates.”

Here is one such instance that Varmus notes:

“One of my first exposures to this problem occurred soon after I arrived at the NIH, when I received a call from my own former congresswoman, Nancy Pelosi, asking me to add $50 million to the budget for AIDS research. As the representative from one of the districts most heavily affected by the epidemic, her wishes were understandable. Since she was a member of the House Appropriations Committee for the NIH, she was in a position to try to increase funds for AIDS research when the subcommittee was debating the size of the NIH budget, without taking the money from some other research program. But, in the period of spending caps, she had presumably been unsuccessful in negotiations with her fellow committee members and was now trying to fulfill a promise to her constituents by asking me to shift funds from some other budget categories into the OAR (Office of AIDS Research) account. I declined as politely as I could.”

Varmus notes that it can difficult to refuse such requests (demands?) when they come from powerful people; especially so when the come from the President. For instance, President Bill Clinton “requested” that $10 million more be spent on spinal cord research; this coming after he spent an afternoon with recently paralyzed actor, Christopher Reeve.

“But the President’s wishes are always obeyed. When the next accounting was made of disease-specific spending at the neurology institute (formerly known as the National Institute of Neurological Diseases and Stroke, or NINDS), the funds for spinal cord research were accordingly higher, and funds for other purposes were lower.”

Here is an additional example, this time involving the vice-president:

“But Vice-President Al Gore posed a potentially serious dilemma for the NIH late in 1997 when he proposed that the National Cancer Institute (NCI) should receive a much larger share than the other institutes in the record-breaking $1 billion budget increase that the president was going to request for the NIH for fiscal year 1999. Possibly as a result of promises made to cancer research advocates, possibly because of personal concerns about cancer (his sister died of lung cancer at an early age), possibly because cancer research was popular politically, Gore asked that the cancer institute’s budget grow at twice the rate accorded the others.”

Varmus continues:

“I was very unhappy about this. The differential rates of growth were not in accord with clearly defined medical needs or with carefully considered scientific opportunities. No major changes in disease rates or outcomes and no sudden developments in cancer research made the needs for the NCI any greater than those for brain disorders, metabolic diseases, or infections. By any measure, the NCI was already the largest institute by a considerable margin, and Gore’s plan would further accentuate the differences. And, of course, there would be strong negative reaction from the supporters of the other institutes when the plan was announced. But, he was the vice-president, and conceivably the next president, so the idea of arguing with him on this issue was not appealing.”

However, Varmus had an ally in Donna Shalala, the Secretary of Health and Human Services (HHS), who supported his position. And, with her help, Varmus was able to take the issue directly to Gore:

“… we were able to reach a rapprochement when I pointed out that many institutes did cancer research, not simply the NCI, and he was very pleased to learn this. That gave us an opening for a compromise: we would ensure a relatively large increase for cancer research, but it would be spread among all the institutes that could be said to do cancer research.”

[Aside 4: Varmus was featured in an earlier posting on the blog that recounted how, in 1986; he resolved the dispute between Luc Montagnier and Robert Gallo over the right to name the AIDS virus (3). It’s been said that Varmus developed diplomatic skills while resolving the naming dispute that served him well as Director of the NIH. The following comment from Varmus shows his subtle diplomacy when interacting with the directors of other government agencies that he competed against:

“Often the best way to support the NIH and science in general was to make a magnanimous gesture toward the other agencies, emphasizing their importance in an increasingly interdisciplinary world of science and hoping the gesture would be reciprocated. This strategy was appreciated by my colleagues in other disciplines, helped to dispel jealousies about our fiscal success, and is remembered as a hallmark of my time at the NIH.”]

Irrespective of any political considerations, the setting of research priorities is an inherently difficult process. The following quotation points up the often conflicting scientific and public health considerations that Varmus took under consideration when determining research priorities. And, bearing in mind his recounting of Nancy Pelosi’s request for additional funding for AIDS, these remarks also demonstrate that he was hardly insensitive to the AIDS issue:

“For much of my time at the NIH, I was castigated by advocates for research on heart disease because the NIH was spending about as much on AIDS research as on studies of heart disease, even though there were about twenty times more deaths from heart disease than from AIDS in the United States each year. The arguments tended to ignore other important facts: that AIDS was a new and expanding disease, that it is infectious, that it is devastating large parts of the world, or that age-adjusted death rates from heart disease have fallen by two-thirds in the past 50 years.”

Elsewhere Varmus notes: “Of course, very different impressions can be produced by the use of different criteria—the number of people living with a condition, the number who die from it each year, the age adjusted death rate, the number of healthy individuals at risk, the number diagnosed each year, the annual medical expenditures, the annual cost to society, or the degree of pain and suffering. These are legitimate aspects of the nation’s burden of disease, but they are crude tools for deciding how to spend research dollars appropriately.”

Another difficulty that Varmus had to contend with was that laypeople, both in Congress and in public advocacy groups, often did not appreciate that science usually works best when scientists are free to investigate the particular issues that most intrigue them. And, when biomedical scientists follow their own inclinations, they often focus on basic or fundamental questions that may seem to have no apparent clinical relevance. Yet, and importantly, the knowledge gained from untargeted basic research may have a more positive affect on the understanding and treatment of a particular disease than all of the clinical research specifically targeted at that disease. [Indeed, the Nobel Prize-winning research of Varmus and Bishop is a good example of that very point.]

Speaking to that notion, Varmus said the following in a June 2009 interview with Catherine Clabby in American Scientist:

“Look at what pride people take now in advances made in diabetes and cancer research and infectious disease research. Almost all of it is based on recombinant DNA technology, genomics and protein chemistry. These are methods that grew out of basic science that was funded for years and years in a non-categorical way.”

Still and all, while basic research often may lead to significant clinical advances, Varmus acknowledges that the NIH still must have programs that are targeted at public health concerns:

“One of the potential strengths of the NIH is its ability to encourage scientists throughout the country to pay greater attention to underserved and deserving problems, even when the opportunities may not be obvious. Simply by encouraging attention to such problems—autism, rare neurological diseases, imaging methods, emerging infections, or bioengineering, to mention a few areas promoted during my tenure—new ideas may emerge to create those opportunities.”

But, Varmus adds:

“In this regard, the NIH must walk a narrow line: to respond responsibly to public health needs and yet to provide the freedom for investigators to exercise their imaginations as freely as possible.”

[Aside 5: An earlier posting on the blog, Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, described how the National Foundation for Infantile Paralysis financed the crusade against polio in the pre-NIH days of the 1950s. But, the Foundation’s efforts went beyond merely raising money for research. It also attempted to provide direction to the research, which often placed it at odds with its grantees. That was so because the principal goal of Harry Weaver, the Foundation’s director of research, was to bring a vaccine to the public. In contrast, most of the Foundation’s grantees were more interested in investigating basic virological issues, such as poliovirus transmission, replication, and dissemination.]

Research involving human embryonic stem cells was a particularly contentious issue that Varmus dealt with as NIH director. Stem cell research “attracted controversy mainly because the cells are obtained from human embryos, linking stem cell research to historical battles over abortion and over the legal and moral status of the human embryo and fetus.”

Yet Varmus took up the cause for stem cell research because “embryonic stem cells were likely to have the potential to develop into many specific tissue types…if so they could be used to repair damaged tissues or to treat chronic degenerative diseases of the brain or spinal cord, endocrine organs (such as pancreatic islets), muscles, joints, or other tissues.”

In 1993 Varmus assembled the Human Embryo Research Panel, tasked to advise him on what types of stem cell research might be suitable for federal funding. Not surprisingly, an immediate hullabaloo followed the panel’s recommendation that in vitro fertilization might be used to create embryos, from which stem cells could then be derived. Varmus remarked on the reaction to the panel’s recommendation as follows:

“Although well received by scientists who were watching its work, the panel’s report ignited a storm of government opposition; even within the liberal Clinton administration…the White House was in shock from the Democratic Party’s loss of control of both congressional chambers in the midterm elections held a month earlier. Democrats across the nation, especially those at the highest ranks of the Clinton administration, were concerned about a shift in the electorate toward the conservative policies of Newt Gingrich and his Republican revolutionaries, and already anxious about the presidential election of 1996…I remember getting a call from Leon Panetta, then the White House chief of staff, telling me that I was supposed to repudiate some of the panel’s recommendations, in particular any that might permit the use of federal funds to create embryos for research purposes. I refused to reject the recommendations of my panel summarily. I was not fired, as the tone of Panetta’s call had threatened.”

Although Varmus wasn’t fired for his independence, the Clinton White House quickly issued an executive order forbidding the use of federal funds to create human embryos for research. Varmus attributed the political pushback to the undue influence (“on the conduct of science in a diverse society”) of a few conservative religious groups. Varmus went on to say:

“Few arguments can seem as insulting to medical scientists as the claim that we are ethically irresponsible when we toil to extract stem cells from donated early human embryos, which would otherwise be destroyed, and use them for beneficial, potentially lifesaving purposes.”

Varmus lamented the fact that President George W. Bush limited federal funding for stem cell research during his administration. Nevertheless, stem cell research was being done even during the Bush presidency, although it was supported by the private sector and by several states (California, New York, Massachusetts, Wisconsin and others). Yet because potential stem cell investigators would need to obtain funding from less well-endowed non-federal sources to do this research, it is likely that many were discouraged from entering the field.

Varmus also fought a difficult and frustrating battle to secure federal funding for needle-exchange programs. By way of background, intravenous drug abusers were accounting for one-quarter of all new HIV infections in the United States. And, while other industrialized nations had needle-exchange programs that were successful at reducing the number of new HIV infections, many powerful individuals in the United States, including General Barry McCaffrey, head of the Clinton White House Office on Drug Control, regarded efforts to make drug use safer to be the equivalent of condoning drug use.

In 1998, HHS Secretary Donna Shalala, using evidence compiled by Varmus, advised President Bill Clinton that needle exchange programs were proven to be effective at preventing HIV transmission and, moreover, they did not increase drug use. Nevertheless, the President did not lift the ban on federal support for needle exchange programs.

By coincidence, the day that Clinton announced his decision not to lift the ban, Varmus and his wife were having dinner with Rahm Emanuel, who, at that time, was a domestic policy advisor in the Clinton White House. Interestingly, the liberal Emanuel was not sympathetic to lifting the ban. Instead, he believed that doing so would open the Democratic Party to charges that it was soft on drugs. At any rate, not lifting the ban did not enable the Democrats to regain either house of Congress. Varmus adds:

“The only satisfaction we received was the later admission by Bill Clinton, speaking at an international AIDS conference in Spain, less than two years after he left the White House, that his failure to lift the ban on funding needle exchange was wrong and one of the worst decisions he made during his presidency.”

Another of the good fights that Varmus fought on behalf of science was to establish new approaches to publishing scientific papers. His purpose was to enhance access to the scientific literature by taking advantage of new opportunities being offered by the internet and by new computational tools. His efforts resulted in two important new ways in which scientific research is published, stored, and retrieved; specifically, public digital libraries and “open access” publishing.

Varmus credits Stanford biologist Pat Brown with pushing him, in 1998, to think about improving access to the scientific literature by making the most of the internet. Brown had earlier worked with Varmus and Bishop on retroviral integration in the 1980s.

Varmus was still the NIH director when he helped to launch PubMed Central; the NIH’s full-text public digital library for the biomedical sciences, and the first of its kind. [In contrast to PubMed Central, PubMed is the NIH’s on-line archive of titles, authors, and abstracts. Access to full text articles was possible via PubMed, but only if one had a personal subscription to the particular journal, or had access via their institution.] But, many scientists and journal publishers were initially opposed to PubMed Central. Consequently, in 2000, after Varmus had left the NIH, he and Brown, together with Mike Eisen, a computational biologist at Berkeley (who had worked as a post-doc with Brown), took more vigorous steps to promote it:

“Pat, Mike, and I wrote a short declaration of purpose—we called it a pledge, publishers called it a boycott—in which we said that one year hence, the signatories would no longer submit articles, provide reviewing or editing services, or purchase individual subscriptions to journals that had not agreed to deposit their articles with PubMed Central.”

Thirty thousand scientists worldwide signed the pledge, but most didn’t. One reason for the lack of wider support was that leading scientists typically strove to have their papers published in the most prestigious journals, and most of those journals had not bought in to the open access idea. Journal publishers were opposed because their revenues from subscriptions would be undermined by the open access model.

Since most journal publishers were not willing to participate in PubMed Central, Varmus, Brown, and Eisen decided to found open access journals themselves. They began by creating two outstanding journals, PLoS (for Public Library of Science) Biology and PLoS Medicine. Their business model was to use author’s fees to cover publication costs, usually paid from research grants. [Incidentally, no favorably reviewed paper would be turned away for inability to pay the fee. All of the PLoS journals can be seen by anyone, anytime, at http://www.plos.org.]

The public access situation began to change dramatically in 2007 when a coalition of leading scientists, open access publishers (including PLoS), and concerned members of Congress advocated for a policy that would require all scientific papers reporting NIH-funded research to be deposited in PubMed Central. This cause came to fruition when President George W. Bush signed the 2008 appropriations bill, which included a clause making the NIH public access policy the law of the land.

So, wrapping things up, considering that the NIH director’s job can grind one down with its “incessant and inevitable conflicts,” why did Varmus put up with it? His answer is he enjoyed it:

“Above all, there was the pride, excitement, and (at times) historical significance of being the leader of the largest funding agency for medical research in the world. The position represents medical science and the good things it does for the country, if not the world. I felt this when working within the administration, when speaking to members of Congress, when talking to reporters, and when addressing the public at commencement exercises, and elsewhere.”

Varmus left the NIH to become President of the Memorial Sloan-Kettering Cancer Center in New York City; a position he held from 2000 until 2010. He then returned to the NIH, where he serves as director of the National Cancer Institute.

In the Epilogue to his memoir (2), Varmus refers to the work of scientists, and its potential benefit to society, as follows:

“Scientists may work and compete as individuals, but the competitive efforts are ultimately directed to the construction of a common edifice, knowledge of the natural world. There are few other fields in which such fierce independence serves the public good in such a transparently shared fashion”

But he adds: “…our knowledge does not improve the societies in which we live unless other kinds of actions, both political and pragmatic, are taken.”

References:

1. Stehelin D, Varmus HE, Bishop JM, Vogt PK., 1976. DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260:170-173.

2. Varmus, H. 2009. The Art and Politics of Science, (W. W. Norton & Company)

3. How the Human Immunodeficiency Deficiency Virus (HIV) Got Its Name, on the blog

Appendix:

Varmus and Bishop turned to nucleic acid hybridization to test their hypothesis that v-src might be a version of a cellular gene. They could not use the complete Rous sarcoma virus genome as a probe for the putative cellular src gene, because the complete virus genome might have detected endogenous retrovirus sequences within the cellular genome, rather than a cellular src gene per se. So, they needed to generate a more specific probe.

In the days before recombinant DNA procedures, Varmus and Bishop cleverly generated their specific src probe by making use of a transformation-defective mutant of Rous sarcoma virus, isolated earlier by Peter Vogt. The important feature of this mutant virus was that its src gene was deleted.

Varmus and Bishop generated their src-specific probe by first using reverse transcriptase to make a radioactively labeled, single-stranded DNA copy of the entire standard Rous sarcoma virus genome, which contains the src gene. This cDNA was then fragmented and annealed to an excess of RNA genomes of the src deletion mutant. The only DNA fragments that did not anneal were those containing only src sequences. These single-stranded DNA fragments could be separated from the annealed product and used as the src nucleic acid hybridization probe.

Using their cDNA probe, Varmus and Bishop were able to demonstrate the presence of src not only in the genomes of normal chicken cells, but also in the genomes of many other vertebrates as well, including humans (reference 1). These experimental findings led to the remarkable conclusions that the cellular src gene was present early in vertebrate evolution and that it has remained conserved to this day.

More experiments of this kind demonstrated that other highly oncogenic retroviruses contain other oncogenes of their own, which likewise have their counterparts in normal cell genomes. Indeed, each of the known retroviral oncogenes corresponds to a gene present in a normal cellular genome, and each of these retroviral oncogenes appears to be derived from a cellular genome.

But, why did Varmus and Bishop suspect that v-src might have originated as a cellular gene? In part it was because Steve Martin had earlier isolated a mutant of Rous sarcoma virus that was temperature-sensitive for transformation, but not for replication. Why would a virus carry a gene that it did need for it to replicate?

 

 

 

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

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

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

iron lung

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

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

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

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

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

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

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

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

sabinAlbert Sabin, who developed the live polio vaccine.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

salk instSalk Institute for Biological Studies

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

 

 

 

 

 

 

Howard Temin: “In from the Cold”

Howard Temin persevered during years of neglect and ridicule until his iconoclastic provirus hypothesis was at last accepted.  This story also features David Baltimore, and personal recollections of Temin and Baltimore.

Concurrently, but independent of each other, Howard Temin and David Baltimore discovered reverse transcriptase in 1970; one of the most dramatic and important findings in the history of molecular biology. The impact of this discovery was so huge that Temin and Baltimore had to wait a mere five years for their contribution to be recognized by the Nobel Committee. Yet the path to the Nobel Prize was far from easy for Temin. Rather, it is a tale of unwavering conviction, persistence, and courage. We begin though with a bit of earlier history.

In 1911, Peyton Rous reported the existence of a filterable, infectious agent that causes sarcomas in chickens. This agent, eventually named the Rous sarcoma virus, was the first virus known to cause solid tumors (1). Moreover; it was the prototype of a virus family that initially was called the RNA tumor viruses (and also the leukoviruses,1).  But, after Temin’s unexpected and radical provirus hypothesis was finally accepted, the family was named the retroviruses.

Here is how Rous described his findings in his original 1911 report: “A transmissible sarcoma of the chicken has been under observation in this laboratory for the past fourteen months, and it has assumed of late a special interest because of its extreme malignancy and a tendency to widespread metastasis. In a careful study of the growth, tests have been made to determine whether it can be transmitted by a filtrate free of the tumor cells . . . . Small quantities of a cell-free filtrate have sufficed to transmit the growth to susceptible fowl.”

Current students might be surprised that Rous’ singularly novel finding of a tumorigenic virus generated only scant interest back in the day. But, bear in mind that nothing whatsoever was known yet regarding the genetic and molecular basis of cancer or, in fact, of the nature of viruses. What’s more, medical researchers in the early 1900s did not recognize the relevance of a transmissible cancer in chickens to malignancies in humans (1).

Indeed, Rous’s unique observations were not appreciated until the 1950s, when it was shown that other viruses (DNA tumor viruses, as well as other RNA tumor viruses) could cause sarcomas, mammary tumors, and leukemias in mice. Only then did the study of tumor viruses become a respectable and even a mainstream pursuit.

Eventually, in 1966, Rous was recognized by the Nobel Committee for his 1911 discovery (2)! This 55-year hiatus is the longest in the history of the Nobel Prize. Bearing in mind that Nobel Prizes are not awarded posthumously, Rous was fortunate to have longevity on his side. He was 87-years-old when awarded the Prize.

This brief anecdote concerning Peyton Rous is relevant to our main account of Howard Temin for multiple reasons. First, the independent discovery of reverse transcriptase by Temin and Baltimore, and the resultant acceptance of Temin’s provirus hypothesis, were the crucial keys to elucidating the unique replication strategy of the retroviruses, of which Rous’ virus was the prototype. Second, reminiscent of the indifferent response of the scientific community to Rous’ discovery, Temin too had to wait years for his contribution to be acknowledged by his peers. However, unique to Temin’s circumstance, during his nearly ten-year wait, he endured being ridiculed by some, while being ignored by most, yet all the time holding fast to his convictions.

Interestingly, during Temin’s 1975 Nobel lecture, he commented as follows on the indifferent response of the scientific community to Rous’ findings at the time they were first reported, and indeed for the next 40 or more years afterwards as well: “Although Rous and his associates carried out many experiments with Rous sarcoma virus, as the virus is now called, and had many prophetic insights into its behavior, they and other biologists of that time did not have the scientific concepts or the technical tools to exploit his discovery.”

Howard M. Temin – via Nobelprize.org. Nobel Media AB 2013

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

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

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

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

Temin’s assay is truly quantitative, since the number of foci of transformed cells that appear in a Rous sarcoma virus-infected cell culture is proportional to the concentration of virus particles in the inoculum. Indeed, Temin’s assay was the first quantitative assay for viral transformation in general, and the first quantitative assay for a retrovirus. Its development was all the more impressive at the time because cell culture was still in its infancy. Importantly, Temin’s assay opened up the study of retroviruses in cell culture, enabling others to eventually make the connection between viral carcinogenesis and the genetic basis of cancer. [Most importantly, the oncogenes of the RNA tumor viruses have their counterparts in the normal cell genome.] But Temin’s more far reaching contribution was still to come. Its genesis was as follows.

As noted above, Rous sarcoma virus is not cytocidal. Consequently, it is able to stably transform chicken cells, while at the same time replicating in them. From Temin’s point of view in the late 1950s, these facts were consistent with the attractive but still untested premise that virus-mediated transformation might result from the continuous presence and expression of virus genes in the transformed cells (3). But how then might one account for the fact that Rous sarcoma virus could likewise stably transform rat cells, which do not support replication of the avian Rous sarcoma virus? To do so, one must either abandon the paradigm that continued viral gene expression maintains the transformed cell phenotype, or one must somehow explain how a viral RNA genome might persist in a line of cells that do not support replication of the virus.

At this early time in the history of retrovirology, it was discovered that bacteriophage λ (lambda) could stably integrate its DNA genome into the chromosome of its host bacterium; a state in which the temperate bacteriophage genome might stably persist for many cell generations. So, did the example of the temperate bacteriophages influence the radical hypothesis that Temin was about to put forward? That question is considered below. In any case, Temin proposed that the Rous sarcoma virus genome likewise persists in eukaryotic host cells in a similar integrated state.

Temin of course realized that such an option would not be possible unless the Rous sarcoma virus single-stranded RNA genome might somehow be transcribed into a double-stranded DNA form. Accordingly, that is precisely what Temin hypothesized. In 1964, Temin’s premise became known as the provirus hypothesis, which explicitly proposes that Rous sarcoma virus generates a DNA copy of its RNA genome, which is then integrated into the cellular DNA as a provirus and stably maintained in that state in clones of transformed cells.

Temin’s provirus hypothesis was iconoclastic in the extreme, largely because it was proposed at the time that the “central dogma of molecular biology” was taking hold. The central dogma, as expounded by Francis Crick in 1958, maintained that information in biological systems always “flows” from DNA to RNA and then to protein. Indeed, the central dogma had taken such a strong hold on thinking at the time that Temin’s provirus hypothesis was regarded as the scientific equivalent of heresy. Consequently, Temin had to fight a long, lonely battle against the criticism and ridicule that his hypothesis generated. Even Harry Rubin, who recruited Temin to the RNA tumor virus field and who served as his first mentor, was candid in his disdain for the provirus hypothesis.

It may now seem odd that the central dogma should have exercised such a strong influence, since RNA viruses were already known and appeared to be an exception to the dogma as then stated. In this regard, Temin noted the following in his 1975 Nobel lecture. “Studies with the newly discovered RNA bacteriophage [see The Phage in the Letter] and with animal RNA viruses, especially using the antibiotic actinomycin D, indicated that RNA viruses transferred their information from RNA to RNA and from RNA to protein and that DNA was not directly involved in the replication of these RNA viruses.”

Temin was a serious researcher, and he indeed carried out experiments to test his radical hypothesis during the ten years that he was being ignored or else scoffed at. Moreover, his experimental results were entirely consistent with his proposal. However, the experimental results generated by the technology of the day could not provide the compelling proof that was demanded to verify a hypothesis that seemingly challenged such strongly entrenched beliefs as enunciated by the central dogma.

Temin’s early experimental findings included the following. First, he reported that actinomycin D, which inhibits transcription from a DNA template, impaired replication of Rous sarcoma virus in cultured chicken cells. Yet while this experimental finding is consistent with the provirus hypothesis, the result also might be explained if viral replication were dependent on the expression of particular cellular genes. Second, Temin showed that inhibitors of DNA synthesis impeded Rous sarcoma virus replication. Yet one could argue that cellular DNA synthesis rather than viral DNA synthesis is necessary for virus replication, perhaps as part and parcel of establishing an intracellular milieu conducive to viral replication. Third, Temin carried out nucleic acid hybridization experiments to demonstrate the presence of Rous sarcoma virus DNA in virus-transformed rat cells. Results of those hybridization experiments also supported the provirus hypothesis. Nevertheless, the hybridization technology of the 1960s could not generate data that was compelling enough to convince Temin’s critics.

In Temin’s Nobel lecture, he said the following about how his experimental findings were received by his colleagues: “Based on the results of these experiments, I proposed the DNA provirus hypothesis at a meeting in the Spring of 1964—the RNA of infecting Rous sarcoma virus acts as a template for the synthesis of viral DNA, the provirus, which acts as a template for the synthesis of progeny Rous sarcoma virus RNA. . . . At this meeting and for the next 6 years this hypothesis was essentially ignored.”

A crucial sticking point preventing acceptance of the provirus hypothesis was that no enzyme was known that could copy RNA into DNA. Thus, the breakthrough that led to widespread acceptance of Temin’s hypothesis came in 1970, when Temin (at the University of Wisconsin) and David Baltimore (at MIT) concurrently, but independently, showed that the previously unknown enzyme, reverse transcriptase, is present in RNA tumor virus particles.Temin found the enzyme in Rous sarcoma virus, while Baltimore found it in the related murine leukemia virus.

In recognition of their independent discoveries, Temin and Baltimore were awarded Nobel Prizes in Physiology or Medicine in 1975. Renato Dulbecco shared in that award for his work on the DNA tumor viruses [see Renato Dulbecco and the Beginnings of Quantitative Animal Virology]. One of Dulbecco’s findings, relevant in the current context, is that neoplastic transformation by the polyomaviruses is associated with the stable integration of their DNA genomes into the host cell genome.

Temin and Baltimore had somewhat different motivations for carrying out their breakthrough experiments. Temin’s entire research career was dedicated to studying the RNA tumor viruses and his primary goal became to validate his provirus hypothesis.  His impetus to seek a reverse transcriptase activity in the retrovirus particle came about as follows. In 1969, in a study that never was reported in its entirety, Temin and his postdoc, Satoshi Mizutani, did experiments demonstrating that the alleged Rous sarcoma virus DNA synthesis could occur in the absence of de novo protein synthesis. This experimental finding implied (to believers at least) that the DNA polymerase activity that catalyzed the synthesis of the viral DNA was present before infection. With no precedent for such a cellular activity, Temin and Mizutani sought the putative polymerase in the virus particle.

In contrast, Baltimore had been studying polymerases generated by other RNA viruses, specifically, ones that transcribe RNA from RNA. Earlier, with his wife, Alice Huang, and other colleagues, he sought to explain why the purified single-stranded RNA genome of vesicular stomatitis virus (VSV) is not infectious, while the purified single-stranded RNA genome of poliovirus is. The contrasting infectivities of the VSV and poliovirus RNA genomes led Baltimore, Huang, and their coworkers to hypothesize that VSV has an obligate requirement for a particle-associated polymerase activity. Earlier, they found that during infection, the poliovirus genome functions as an mRNA after it is released from the virus particle into the cytoplasm. Consequently, the purified poliovirus genome likewise may function as an mRNA in transfection experiments. In contrast to the poliovirus case, they found that the VSV RNA genome is complementary to the mRNAs that encode that virus’s proteins. Accordingly, to account for how VSV might initiate transcription upon infection, they looked for, and found, an RNA-dependent transcriptase activity within VSV particles. From this background and point of view, Baltimore sought a reverse transcriptase activity in retrovirus particles.

David_Baltimore_NIH
David Baltimore in the 1970’s. Image via the National Library of Medicine (image in public domain).

During his Nobel lecture, Baltimore reflected on how his earlier experiences might have led him to his discovery of the retrovirus reverse transcriptase, as follows. “If we look back to virology books of 15 years ago, we find no appreciation yet for the variety of viral genetic systems used by RNA viruses. Since then, the various systems have come into focus, the last to be recognized being that of the retroviruses (“RNA tumor viruses”). As each new genetic system was discovered, it was often the identification of an RNA or a DNA polymerase that could be responsible for the synthesis of virus-specific nucleic acids that gave the most convincing evidence for the existence of the new system. . . .” [This line of thought underlies the Baltimore classification system. See my personal recollection of David Baltimore, below.]

Baltimore spoke after Temin at the Nobel ceremony. The following item from Baltimore’s Nobel lecture is cited here for his comments about his co-award recipient: “In his Nobel lecture, Howard Temin has outlined how he was led to postulate a DNA intermediate in the growth of RNA tumor viruses. Although his logic was persuasive and seems in retrospect to have been flawless, in 1970 there were few advocates and many skeptics. Luckily, I had no experience in the field and so no axe to grind—I also had enormous respect for Howard dating back to my high school days when he had been the guru of the Summer School I attended at the Jackson Laboratory in Maine. So I decided to hedge my bets—I would look for either an RNA or a DNA polymerase in virions of RNA tumor viruses. . . .”

Temin died in 1994 from lung cancer. He was only 59 years of age and never was a smoker. His lung cancer was an adenocarcinoma, a type not linked to smoking. In fact, Temin was a zealous crusader against smoking. Even at the Nobel Prize ceremony in his honor, he reprimanded smokers (including Swedish princesses) in the audience.

After Temin’s death, Baltimore wrote the following: “Ten years in the scientific wilderness is a long time; few have had to bear the silence of their colleagues for so long. I can remember meetings in the 1960s when Howard would present his latest data supporting the provirus notion only to be greeted by either skeptical questions or quiet, polite disbelief. Howard’s conviction that there had to be a provirus never seemed to waver over the whole decade. He knew he was right—and he was—but what fortitude it took to keep looking for the experiment that would show it! My first reaction when I realized that I had seen the reverse transcriptase was to call Howard, because I so much wanted him to know that he was vindicated in his commitment to the idea of a provirus. But he had already found out for himself.”

Howard Temin and David Baltimore published their independently discovered, singularly important finding in back-to-back papers in the British journal Nature (4, 5).

In 1972, Hill and Hillova provided further proof that RNA tumor virus genomes persist in transformed cells in the form of a DNA copy. They did so by demonstrating that purified DNA from a Rous sarcoma virus-transformed cell could produce infectious virus when transfected into a normal cell.

Indisputable evidence of reverse transcription led to the RNA tumor viruses becoming known as the retroviruses. What’s more, it led to the realization that the central dogma, as initially stated by Crick, is not always valid. Exceptions include other viruses such as the hepatitis B viruses, which actually have double-stranded DNA genomes, yet make use of reverse transcription to replicate those genomes. And, there are the cellular retroelements and telomeres, which also bring reverse transcription into play. Importantly, knowledge of the reverse transcription step in the retrovirus life cycle also led to a more complete understanding of HIV infection and the pathogenesis of AIDS. What’s more, the reverse transcriptase enzyme is a critical tool of modern molecular biology.

Interestingly, the discovery of reverse transcriptase led Crick to state that he never excluded the possibility that RNA might serve as a template to make DNA, but instead only the possibility that a protein might serve as the template to make a nucleic acid. Moreover, Crick’s use of the word “dogma” itself caused him additional embarrassment. In his autobiography, What Mad Pursuit, he wrote the following: “I called this idea the central dogma, for two reasons, I suspect. I had already used the obvious word hypothesis in the sequence hypothesis, and in addition I wanted to suggest that this new assumption was more central and more powerful. … As it turned out, the use of the word dogma caused almost more trouble than it was worth. Many years later Jacques Monod pointed out to me that I did not appear to understand the correct use of the word dogma, which is a belief that cannot be doubted. I did apprehend this in a vague sort of way but since I thought that all religious beliefs were without foundation, I used the word the way I myself thought about it, not as most of the world does, and simply applied it to a grand hypothesis that, however plausible, had little direct experimental support.”

We conclude this part of the posting by returning to an issue raised above: did the discovery of bacteriophage lysogeny prompt Temin to put forward the provirus hypothesis? Here are Baltimore’s comments on this question: “Although the pregnant analogy to known lysogenic bacteriophage might have guided Howard, people who were at Caltech at that time assure me that Howard was unlikely to have arrived at the notion of a DNA intermediate through this route. Apparently, the influence of Max Delbrück (6)—who was totally committed to the study of lytic phages and did not really believe in the importance of phage lysogeny—was so great that there was little discussion of lysogeny at Caltech then. Furthermore, Howard has minimized the importance of lysogeny as a precursor to his concepts. Therefore, he must have arrived at the concept of a DNA intermediate simply from the persuasive power of such a concept to explain the properties of the transformed state. He was particularly influenced by the morphological difference between cells transformed by particular Rous sarcoma virus variants, which he felt had to mean that the viral genome continued forever to affect the transformed cell.” Temin, in his Nobel lecture, indeed cites the mutant studies to which Baltimore refers as a key factor in the genesis of the provirus hypothesis.

Personal Recollections of Howard Temin and David Baltimore

I briefly met Howard Temin in 1971, when he visited my postdoctoral advisor, Tom Benjamin. Several years later, when I was a still young assistant professor at UMass, I had the chance to spend more time with Howard when he came to present a seminar in my department. After the seminar, I had the privilege of taking Howard to lunch where, much to my delight, he asked me to tell him about my own research. What struck me during this incident was how attentive and responsive Howard was to all I was telling him. He instantly understood the implications of each experiment I described and his mind always seemed to be one experiment ahead of me. When we were finished, I mentioned this impression to him. His response, delivered in a most unassuming way, was “Lenny, I’ve seen a lot of science. There are only so many experiments one can do, and I’ve seen them all.”

Shortly afterwards, while investigating simian virus (SV40) entry into cells, I became interested in the intracellular signals that SV40 appeared to transmit from the cell surface. At the time, cell signaling was already a very big field with a vast literature. However, very little was known or published about virus-induced signaling. I soon found myself snowed under by the enormity of the new-to-me cell signaling literature. Then, I suddenly realized that I was seeing one or more of the same four basic experimental approaches in each of the papers I was reading.  Recalling my lunch with Howard; there are only so many experiments one can do, and I’d become familiar with all that I would need.

David Baltimore (born in 1938) is currently Professor of Biology at Caltech, where he earlier served as President. Before that, he served as a professor at MIT and as President of Rockefeller University. I never had the pleasure of meeting David Baltimore. Nevertheless, he influenced my career in a major way as follows. I earned my Ph.D. in 1969 in the area of bacterial genetics. Then, I spent an all too brief two years as a post-doc studying transformation by the mouse polyomavirus. Next, it seemed I was all too suddenly an assistant professor at UMass, where I was expected to teach an animal virology course to advanced undergraduates and graduate students.

Here then was my dilemma. I was still an expert bacterial geneticist. But, while I was knowledgeable regarding transformation by the tumor viruses, I was far from being an expert animal virologist. Moreover, I never actually had a virology course. My predicament was compounded further by the fact that the virology textbooks of the day were for the most part descriptive. Thus, I was at a loss as to how I might deliver three 50-minute virology lectures per week to advanced undergraduates and graduate students.

With my back seemingly against the wall, I rather fortuitously came across a 1971 review article by David Baltimore, in which he put forward his Baltimore classification system (7).  In brief, the Baltimore classification system is based on the notion that the nature of a viral genome (e.g., double-stranded DNA, plus-strand RNA, minus-strand RNA, etc.) largely determines its expression strategy. Moreover, there were only six classes, or basic strategies in the original Baltimore scheme, and each of the formal virus families fit into one of the Baltimore classes. Although the Baltimore system is not a formal means for classifying viruses, I immediately recognized its didactic potential. Indeed, I had my answer regarding how to teach virology.

At first, I followed Baltimore’s review article, initially basing my lectures on the references therein, which I updated with more recent articles over the next several years. But, the field was progressing too rapidly to go on in this way, so I had to turn to textbooks. However, none of these books was organized around the Baltimore classification system, which remained the key concept around which I structured my lectures. Indeed, to this day I remain convinced that the best way to teach virology is to discus viruses in the context of virus families, with the Baltimore system as the organizing principle.

Over the years I fantasized about writing a book that might present the field in this way. Virology: Molecular Biology and Pathogenesis was the fulfillment of that vision.

References and notes:

1. An oncogenic virus, later known to be closely related to the Rous sarcoma virus, actually was discovered 3 years earlier by Vilhelm Ellereman and Olaf Bang. They found that leukemia in birds could be transmitted by a filterable agent from leukemic cells or by serum from leukemic birds. However, leukemia was not then recognized as cancer, so the significance of this discovery, like Rous’, went unrecognized.

2. The 1966 Nobel Prize in Physiology or Medicine was divided equally between Peyton Rous “for his discovery of tumour-inducing viruses” and Charles Brenton Huggins “for his discoveries concerning hormonal treatment of prostatic cancer”.

3. Shortly after the discovery of reverse transcriptase, Steve Martin, at the time a graduate student in Harry Rubin’s laboratory (at UC Berkeley where Rubin was then a professor) reported the isolation of a mutant of Rous sarcoma virus that was temperature-sensitive for transformation. Importantly, cells transformed by the mutant virus at its permissive temperature reacquired the normal cell phenotype when incubated at the non-permissive temperature. Thus, expression of a viral oncogene (src in this instance) is required to both initiate and maintain the transformed cell phenotype.

4. Baltimore, D. 1970. RNA-dependent DNA polymerase in virions of RNA tumor viruses. Nature 226:1209-1211.

5. Temin, H. M., S.Mizutani. 1970. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226:1211-1213.

6. See the earlier posting, Max Delbruck, Lise Meitner, Niels Bohr,  and the Nazis.

7. Baltimore, D. 1971. Expression of animal virus genomes. Bacteriol Rev. 35:235-41.

Renato Dulbecco and the Beginnings of Quantitative Animal Virology

In an earlier posting [Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis], we mentioned that when James Watson was a graduate student in Salvatore Luria’s lab at the University of Indiana in the late 1940’s, he shared a lab bench with another future Nobel laureate, Renato Dulbecco. Dulbecco happened to be in Luria’s lab because earlier, in 1936, when Dulbecco was studying for a medical degree at the University of Torino in Italy, he favorably impressed Luria, who was then a professor at Torino. Later, in 1947, after Dulbecco had spent a short stint in politics in Italy, Luria invited Dulbecco to join his Indiana group to study bacteriophages. Hence, Dulbecco came to share a lab bench with Watson. In the summer of 1949, Dulbecco moved on to the California Institute of Technology, to join Max Delbruck’s phage group to further his inquiry into bacteriophages. But, providence was to intervene, as follows.

In the late 1940s, a wealthy Californian became ill with shingles; a late complication of chickenpox, caused by varicella-zoster virus, a herpesvirus. The man’s physician explained that nothing could be done for his shingles, and moreover, that virtually nothing was known about the viruses that infect humans. Auspiciously, the physician knew of the studies being done on bacteriophages at Caltech, and he also was aware that Caltech was the great center for such work. So, after explaining to his well-heeled patient that bacteriophages were only of theoretical interest regarding human disease, he suggested that the patient might help to develop a center at Caltech that might begin to study medically important viruses. The patient agreed, and since virology at Caltech was headed by Delbruck, the former physicist found himself with an endowment to study human viruses, with virtually no background for how to use it. So, Delbruck summoned to his office Dulbecco, who had trained to be a physician, and proposed that Dulbecco give animal viruses a try. Dulbecco was delighted by the idea and, together with Marguerite Vogt (also in Delbruck’s group) he developed procedures to grow poliovirus in cell culture. Additionally, Dulbecco and Vogt developed a plaque assay procedure to measure the titer of animal viruses grown in cell culture. Importantly, the plaque assay also made it possible to plaque-purify attenuated poliovirus variants; crucial to the development of the Sabin live-attenuated polio vaccine. And, apropos the major point of this vignette, this is how quantitative animal virology came to be.

Plaques produced by Western equine encephalitis virus on chick embryo fibroblasts (left) and by poliovirus on HeLa cells, a line of cells derived from a human cervical carcinoma (right). Photo by R. Dulbecco; Figure 1.6, page 18, From Virology: Molecular Biology and Pathogenesis, by Leonard C. Norkin, ASM Press, 2010.
Plaques produced by Western equine encephalitis virus on chick embryo fibroblasts (left) and by poliovirus on HeLa cells, a line of cells derived from a human cervical carcinoma (right). Photo by R. Dulbecco; Figure 1.6, page 18, From Virology: Molecular Biology and Pathogenesis, by Leonard C. Norkin, ASM Press, 2010.

Dulbecco, remarking on the importance of the plaque assay for animal viruses, noted that subsequent biochemical and molecular studies would have been much less meaningful without reference to the multiplication cycle revealed by the plaque assay. Interestingly, although this was apparent to the phage workers, it was not equally obvious to most animal virologists of the day.

Before moving on, we might note that Marguerite Vogt came to Caltech in 1950 to work with Delbruck, who introduced her to Dulbecco, thus initiating the long and productive collaboration of Dulbecco and Vogt. Following their ground-breaking poliovirus studies, they went on to study the tumorigenic mouse polyomavavirus, demonstrating that infection of normal cells in culture with mouse polyomavirus resulted in neoplastic transformation of the cells into tumor cells. What’s more, this transformation was associated with the integration of the viral genome into that of the host cell. Moreover, a subset of the viral genes continued to be expressed in the transformed cells. Importantly, these studies gave credence to the notion that cancer has an underlying genetic basis. Indeed, the subsequent identification by others of those viral genes that are expressed in virally transformed cells led to singularly important insights into the molecular basis of cancer. [In 1973, Vogt established her own research program, looking into the immortalization of cancer cells, and the roll of telomeres in the origin of cancer.]

Renato Dulbecco.  Image via the National Library of Medicine (image in public domain).
Renato Dulbecco. Image via the National Library of Medicine (image in public domain).

For his studies revealing the link between genetic mutations and cancer, Dulbecco shared the 1975 Nobel Prize for physiology or medicine with his former student, Howard Temin, and David Baltimore. The latter two individuals simultaneously and independently discovered the enzyme, reverse trancriptase, which enables retroviral genomes to be reverse-transcribed and then incorporated into cellular genomes (another of my favorite stories and the subject of a future posting). Dulbecco took no part in these studies, but he did teach Temin and Baltimore approaches that led to their discoveries. Vogt was never recognized by the Nobel Committee for her contributions, which many regard as an oversight.

Bearing in mind events recounted in the earlier posting, Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, note that Dulbecco served as a medical officer in the Italian Army during the Second World War, eventually being ordered to the Russian front. During a stopover in Warsaw, he happened to see Jewish slave laborers wearing yellow stars, and was horrified to learn that they would be killed when their work was completed. He later referred to that episode as his “turning point.” In Russia he was wounded and sent back to Italy to recuperate. After his recovery, and the collapse of Italian fascism, he joined the resistance against the German occupation, attending to wounded partisans.