Tag Archives: Harry Rubin

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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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.