Tag Archives: Nobel Prize

John Enders: “The Father of Modern Vaccines”

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References:

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

 

The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler

The first part of this posting tells how a U.S. Army medical board, headed by Walter Reed, confirmed that the transmission of yellow fever requires a mosquito vector. The second part tells the story of the yellow fever vaccine developed by Max Theiler.

Bearing in mind the enormous benefit to mankind of the polio vaccines developed by Jonas Salk and Albert Sabin (1), and that Maurice Hilleman developed nearly 40 vaccines, including those for measles, mumps, and rubella (2), it would appear remarkable that Theiler was the only one of these four individuals to be recognized by the Nobel committee. In fact, Theiler’s 1951 Nobel award was the only one ever given for a vaccine! In any case, while Theiler’s vaccine was a major step forward in the fight against yellow fever, it came after a perhaps more dramatic episode in the struggle against that malady. But first, we begin with some background.

Yellow fever was another of mankind’s great scourges. Indeed, it was once the most feared infectious disease in the United States. And, while we might want to say that science has “conquered” yellow fever, that statement would not be entirely accurate. Unlike polio and measles, which have nearly been eradicated by the vaccines against them, that is not so for yellow fever. The reason is as follows. Humans are the only host for polio and measles viruses. Consequently, those viruses might be completely eradicated if a sufficient percentage of humans were to comply with vaccination regimens. In contrast, the yellow fever virus infects monkeys that range over thousands of square miles in Africa and the Amazon jungle. Thus, even with massive vaccination of humans, it would be impossible to eliminate the yellow fever virus from the world.

According to the World Health Organization’s estimates, there are still about 200,000 cases of yellow fever per year, resulting in about 30,000 deaths, about 90% of which occur in Africa. The yellow fever virus itself is the prototype virus of the flavivirus family of single-stranded RNA viruses, which also includes dengue hemorrhagic fever virus, Japanese encephalitis virus, and West Nile encephalitis virus, among others.

yellow fever map

Yellow fever is somewhat unique among the viral hemorrhagic fevers in that the liver is the major target organ. Consequently, the severe form of yellow fever infection is characterized by hemorrhage of the liver and severe jaundice. But, as in infections caused by other virulent viruses, most cases of yellow fever are mild.

Interestingly, the name “yellow fever” does not have its origin in the yellowing of the skin and eyes that is characteristic of severe disease. Instead, it has its origin in the term “yellow jack,” which refers to the yellow flag that was flown in port to warn approaching ships of the presence of infectious disease.

Yellow fever originated in Africa. It is believed to have been brought to the New World by slave ships in the year 1596. As noted above (and discussed below), yellow fever transmission, from an infected individual or primate to an uninfected one, requires a specific vector, the Aedes aegypti mosquito. The sailing ships of the day inadvertently transported the disease across oceans via the mosquito larvae in their water casks.

Before getting to our stories proper, we note a pair of intriguing instances in which yellow fever profoundly affected New World history. In the first of these, yellow fever was a key factor that led Napoleon to sell the Louisiana Territory to the United States in 1803; an act that doubled the size of the United States. It happened as follows. After Napoleon seized power in France, he reinstated slavery in the French colony of Saint Domingue (now Haiti); doing so for the benefit of the French plantation owners there. In response, the rather remarkable Toussaint Breda (later called Toussaint L’Ouverture, and sometimes the “black Napoleon”) led a slave revolt against the plantation owners. In turn, in February 1802, Napoleon dispatched an expeditionary force of about 65,000 men to Haiti to put down the revolt. The rebellious slaves, many fewer in number than the French, cleverly retreated to the hills, believing that the upcoming yellow fever season would wreak havoc on the French force. And, they were correct. By November 1803, the French lost 50,000 of the original 65,000 men to yellow fever and malaria. Thus, in 1804, Napoleon had to allow Haiti to proclaim its independence, and then become the second republic in the Western Hemisphere. Moreover, there is evidence suggesting that Napoleon’s actual purpose in dispatching the expeditionary force was to secure control of France’s North American holdings. With his expeditionary force decimated by yellow fever and malaria, that was no longer possible and, consequently, Napoleon sold France’s North American holdings (the Louisiana Purchase) to the United States.

louisiana purchaseThe Louisiana Purchase, in green.

Second, in 1882, France began its attempt to build a canal across the Isthmus of Panama. However, thousands of French workers succumbed to yellow fever, causing France to abandon the project. The United States was able to successfully take up the task in 1904; thanks to the deeds of the individuals in part I of our story, which now begins.

In May 1900, neither the cause of yellow fever, nor its mode of transmission was known. At that time, U.S. Army surgeon, Major Walter Reed, was appointed president of a U.S. Army medical board assigned to study infectious diseases in Cuba, with particular emphasis on yellow fever. Cuba was then thought to be a major source of yellow fever epidemics in the United States; a belief that was said to have been a factor in the American annexation of Cuba.

ReedMajor Walter Reed

When Reed’s board began its inquiry, a prevailing hypothesis was that yellow fever might be caused by the bacterium Bacillus icteroides. However the board was unable to find any evidence in support of that notion.

Another hypothesis, which was advanced by Cuban physician Dr. Carlos Juan Finlay, suggested that whatever the infectious yellow fever agent might be, transmission to humans requires a vector; specifically, the mosquito now known as Aedes aegypti. Reed was sympathetic to this idea because he noticed that people who ministered to yellow fever patients had no increased risk of contracting the disease, which indicated to Reed that people did not pass yellow fever directly from one to another.

Reed, as president of the medical board, is generally given major credit for unraveling the epidemiology of yellow fever. Yet there were other heroes in this story as well. Finlay, whose advice and experience were invaluable to Reed’s board, was one. He was the object of much ridicule for championing the mosquito hypothesis, at a time when there little evidence that might support it. In any case, Reed, in his journal articles and personal correspondences, gave full credit to Finlay for the mosquito hypothesis.

Acting Assistant Surgeon Major James Carroll was another hero. As a member of Reed’s board, Carroll volunteered to be bitten and, promptly, developed yellow fever. Major Jesse Lazear, also a board member, asked Private William Dean if he might be willing to be bitten. Dean consented, and he too contracted yellow fever. Fortunately, Dean and Carroll each recovered. Not so for Lazear. After allowing himself to be bitten, he died after several days of delirium.

Lazear’s contribution to gaining recognition of the mosquito hypothesis went significantly beyond his tragic martyrdom. When Reed examined Lazear’s notebook after his death, Reed found that it contained several key observations. First, Lazear had carefully documented that in order for a mosquito to be infected; it had to bite a yellow fever patient within the first three days of the patient’s illness. Second, twelve days then had to elapse before the virus could reach high enough levels in the insect’s salivary glands to be transmitted to a new victim.

The observations of the board, up to then, convinced Reed and the others that the mosquito hypothesis indeed was correct. Yet Reed knew that more extensive controlled experiments would be needed to convince the medical community. So, he directly supervised those experiments, which involved twenty-four more volunteers, each of whom may rightly be considered a hero.

Just as Reed benefited from Finlay’s insights, William C. Gorgas, Surgeon General of the U.S. Army, applied the findings of Reed’s board to develop vector control measures to combat urban yellow fever; first in Florida, then in Havana, Cuba, and next in Panama, where those measures enabled the United States to complete the canal in 1914. The last urban yellow fever outbreak in the United States occurred in New Orleans in 1905, and the last in the New World occurred in 1999 in Bolivia.

The vector control strategy works for urban yellow fever because the Aedes aegypti mosquitoes have a very short flight range and, consequently, the female mosquito does not stray far from the source of her blood meal before laying her eggs. Thus, it is only necessary to control the vector population in the immediate vicinity of human habitation. In practice, this is accomplished by draining potential mosquito breeding sites such as swamps and ditches, and destroying water-collecting objects such as discarded tires.

After Reed’s board was disbanded, he made yet another key contribution to the wiping out of yellow fever. The focus of the board had been on the means of yellow fever transmission; not with the infectious agent itself. In 1901, at the suggestion of William Welch, an eminent Johns Hopkins pathologist, Reed and James Carroll (who nearly died of yellow fever after being experimentally infected while in Cuba), asked whether yellow fever might be caused by a filterable virus. Indeed, they found that they could infect volunteers by inoculating them with filtered serum taken from yellow fever patients. What’s more, theirs was the very first demonstration of a human illness being caused by a filterable agent. That is, yellow fever was the first human illness shown to be caused by a virus. [Pasteur developed an attenuated rabies vaccine in 1885, more than a decade before the discovery of viruses. Remarkably, this most brilliant of experimentalists did not recognize that he was dealing with a previously unknown, fundamentally distinct type of infectious agent; the topic of a future posting.]

[Aside: Walter Reed spent the early years of his Army career at different posts in the American west. The Mount Vernon Barracks in Alabama, which served as a prison for captured Apache Native Americans, including Geronimo, was a particularly interesting stop for Reed. Captain Walter Reed, serving as post surgeon in the 1880s, looked after Geronimo and his followers.]

Part II of this posting concerns the development of Max Theiler’s yellow fever vaccine. But first, here is a bit more background.

Vector control measures ended yellow fever epidemics in most, but not all urban centers worldwide. Outbreaks have not occurred in the United States for more than a century. However, jungle yellow fever still persists in areas of Sub-Saharan Africa and, to a lesser extent, in tropical South America. Individuals who are infected in the jungle by wild mosquitoes can then carry the virus to densely populated urban areas, where Aedes aegypti mosquitoes can transmit the virus from one individual to another. [Vector-mediated, human-to-human transmission happens because the level of yellow fever virus in the blood of an infected person becomes high enough for the infected person to transmit the virus to a biting mosquito. In this regard, the yellow fever virus is an exception to the generalization that humans are a “dead end” host for arthropod-borne (arbo) viruses.]

Fortunately, people who live in high risk areas for yellow fever can be protected by vaccination. Indeed, the World Health Organization’s strategy for preventing yellow fever epidemics in high risk areas is, first, to mass immunize the population, and then to routinely immunize infants. [Vaccinated American or European visitors to West Africa or the Amazon need not be concerned about yellow fever. However, the risk to an unvaccinated person of acquiring yellow fever during a two-week stay at the height of the transmission season (July through October), is estimated to be 5%. Individuals wanting to enter or return from countries where yellow fever is endemic may need to show a valid certificate of vaccination. ]

Part II of our story, concerning Max Theiler and the development of the yellow fever vaccine now begins.

Even as late as the 1920s, some reputable bacteriologists remained unconvinced by the earlier findings of Reed and Carroll that yellow fever is caused by a filterable agent. Instead, they persisted in the belief that the illness is caused by a bacterium. The notion of a bacterial etiology for yellow fever was finally put to rest after A. H. Mahaffy in 1927 discovered that the yellow fever agent could be propagated and cause illness in Asian rhesus monkeys. With an experimental animal now at hand, yellow fever workers were able to prove conclusively that the disease is caused by a virus. [Mahaffy drew the virus he used in his experiments from a 28-year-old African man named Asibi, who was mildly sick with yellow fever. That isolate, referred to as the Asibi strain, will play an important role later in this anecdote.]

Regardless of the significance of the discovery that the yellow fever virus could be propagated in rhesus monkeys, Max Theiler had to contend with the fact that these monkeys were quite expensive; especially for a not yet established young investigator. [They cost the then princely sum of about $7.00 apiece.] As for mice, while they could be bred for pennies apiece, other researchers were not able infect them via the usual practice of inoculating them under the skin or in the abdomen. However, Theiler took a cue from Pasteur’s inability to propagate the rabies virus in laboratory rabbits until he put the virus directly into their brains. Thus, in 1929 Theiler attempted to do the same with yellow fever virus in mice.

TheilerlMax Theiler

Theiler’s attempts to infect the mice by intracranial injection were a success. All of the inoculated mice died within several days. Surprisingly, the dead mice did not display the liver or renal pathology characteristic of yellow fever. Instead, the mice appeared to have succumbed to inflammation of their brains. Thus, the yellow fever virus appeared to be neurotropic in mice. Also, Theiler himself contracted yellow fever from one of his inoculated mice. He was fortunate to survive.

A fortuitous result of Theiler’s perilous bout with yellow fever was that he had become immune to the virus, as revealed by the presence of antiviral antibodies in his blood. Importantly, Theiler’s acquired immunity to the virus validated the possibility of developing an attenuated yellow fever vaccine. And, in a sense, Theiler was inadvertently the first recipient of the nascent vaccine he soon would be developing.

Theiler also determined that the virus could be passed from one mouse to another. And, while the virus continued to cause encephalitis in mice, it caused yellow fever when inoculated back into monkeys; quite a unique and striking set of findings. But, and crucially significant, while continued passage of the virus in mice led to its increased virulence in those animals, the virus was concurrently losing its virulence in monkeys. [In 1930, Theiler moved from the Harvard University School of Tropical Medicine to the Rockefeller Foundation’s Division of Biological and Medical Research. The Rockefeller Foundation shared facilities with the Rockefeller Institute (now University); although it was otherwise administratively separate from it.]

Since the mouse-passed virus was becoming attenuated in monkeys, Theiler’s belief in the possibility of generating an attenuated yellow fever vaccine was bearing out. However, because the mouse-passed virus remained neurovirulent in mice, Theiler was reluctant to inoculate that virus into humans. In an attempt to solve this problem, Theiler turned from passing the virus in the brains of live mice and, instead, began passing the virus in mouse tissue cultures.

Theiler carried out seventeen different sets of trials to further attenuate the virus. In the 17th of these, Theiler used the wild Asibi strain, isolated earlier by Mahaffy. Initially, this virus was extremely virulent in monkeys, in which it caused severe liver damage. But, after passing the virus from culture to culture several hundred times, over a period of three years, a flask labeled 17D yielded the virus that was to become the famous 17D yellow fever vaccine.

Theiler never gave a satisfactory accounting for the “D” in the “17D” designation, and for what, if anything became of A, B, and C. Regardless, the genesis of 17D was as follows. Theiler initially took an Asibi sample that had been multiplying in mouse embryo tissue and continued passing it in three separate types of minced chicken embryo cultures. One of these sets contained whole minced chicken embryos, and was designated 17D (WC). A second set contained chick embryo brain only, and was designated 17D (CEB). In the third set, the brains and spinal cords were removed from the otherwise whole chick embryo tissue cultures. This set, alone among all the sets, generated an attenuated virus that did not induce encephalitis when injected directly into monkey brains. Indeed, Theiler removed the central nervous systems from the chicken tissue in this set of cultures, in the express hope of generating just such an attenuated virus. And, by hook or by crook, the virus emerging from that particular set of passages became the vaccine that is now known simply as 17D.

Field tests of Theiler’s yellow fever vaccine were underway in 1937 in Brazil, and were successfully completed by 1940. In 1951 Theiler was awarded the Nobel Prize in Physiology or Medicine for developing the vaccine.

Next, we return to a point noted above, and discussed in two earlier postings. Neither Jonas Salk nor Albert Sabin were awarded Nobel prizes for developing their polio vaccines (1). And, Maurice Hilleman was never awarded a Nobel Prize, despite having developed nearly 40 vaccines, including those for measles, mumps, and rubella (2). Indeed, Max Theiler’s Nobel Prize for the yellow fever vaccine was the only Nobel Prize ever awarded for a vaccine! Why was that so?

Alfred Nobel, in his will, specified that the award for Physiology or Medicine shall be for a discovery per se; not for applied research, irrespective of its benefits to humanity. With that criterion in mind, the Nobel committee may have viewed the contributions of Salk and Sabin as derivative, requiring no additional discovery. [Hilleman’s basic discoveries regarding interferon should have been sufficient to earn him the award (2). The slight to him may have been because the Nobel committee was reluctant to give the award to an “industrial” scientist. Hilleman spent the major part of his career at Merck & Co.]

So, what was there about Theiler’s yellow fever vaccine that might be considered a discovery? Hadn’t Pasteur similarly developed an attenuated Rabies vaccine in 1885?

Perhaps the “discovery” was Theiler’s finding that passage of the Asibi strain of yellow fever virus in chick embryo cultures, which were devoid of nervous system tissue, generated attenuated yellow fever virus that was no longer neurovirulent in mice and monkeys. But, consider the following.

Theiler indeed believed that removing the brains and spinal cords from the chick embryo cultures in which 17D had been serially passed was the reason why the virus lost its neurovirulence. Nevertheless, as a serious scientist he needed to confirm this for himself. So, he repeated the long series of viral passages under the same conditions as before. But, this time, there was no loss of neurovirulence. Thus, a cause and effect relationship, between the absence of the brains and spinal cords from the tissue cultures and the emergence of non-neurovirulent virus, was not confirmed.

So, perhaps the Nobel committee merely paid lip service to the directives in Alfred Nobel’s will. In any case, Theiler’s 17D yellow fever vaccine has had a virtually unblemished safety record, and is regarded as one of the safest and most effective live-attenuated viral vaccines ever developed.

Theiler’s unshared 1951 Nobel award paid him $32,000. At the time, he resided in Hastings-on-Hudson; a village in Westchester County, NY, from which he commuted to the Rockefeller labs. Theiler’s next door neighbor in Hastings-on-Hudson was Alvin Dark, the star shortstop of the New York Giants. Nobel laureate Max Theiler was known to fellow commuters from Hastings-on-Hudson as the man who lives next door to Alvin Dark.

Virus Hunters, by Greer Williams (Alfred A, Knoff, 1960) was my major source for the material on Max Theiler.

1. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science. On the blog.

2. Maurice Hilleman: Unsung Giant of Vaccinology. On the blog.

 

 

 

Maurice Hilleman: Unsung Giant of Vaccinology

In January 2005, more than 100 of the world’s most renowned biomedical researchers got together to pay tribute to the 85-year-old Maurice Hilleman. When it was Hilleman’s turn to address the gathering, he alluded to them as his “peers in the world of science.” Referring to Hilleman’s gracious comment, science journalist Alan Dove wrote: “By any objective measure, a gathering of Maurice Hilleman’s scientific peers would not fill a telephone booth.” (1)

Hilleman truly was a giant in the history of virology. But, if you have only a vague idea of who Hilleman was or of his achievements, you are not alone. Anthony Fauci, director of the U.S. National Institutes of Allergy and Infectious Diseases, who was present at the gathering, noted: “Very few people, even in the scientific community, are even remotely aware of the scope of what Maurice has contributed….I recently asked my post-docs whether they knew who had developed the measles, mumps, rubella, hepatitis B and chickenpox vaccines. They had no idea,” Fauci said. “When I told them that it was Maurice Hilleman, they said, ‘Oh, you mean that grumpy guy who comes to all of the AIDS meetings?’”

hillemanMaurice R. Hilleman: The greatest vaccinologist.

Consider this. Hilleman developed nine of the 14 vaccines routinely recommended in current vaccine schedules. These are the vaccines for the measles, mumps, rubella, hepatitis A, hepatitis B, and chickenpox viruses, and for meningococcal , pneumococcal, and Haemophilus influenzae bacteria. Moreover, he was the first to forecast the arrival of the 1957 Asian flu and, in response, led the development of a flu vaccine that may have saved hundreds of thousands or more lives worldwide (2). And, independently of Robert Huebner and Wallace Rowe, he discovered cold-producing adenoviruses, and developed an adenovirus vaccine. Overall, Hilleman invented nearly 40 vaccines. And, he was a discoverer of simian virus 40 (SV40). If the above accomplishments were not enough to ensure his fame, he also was the first researcher to purify interferon, and the first to demonstrate that its expression is induced by double-stranded RNA.

[Aside: I first became aware of Maurice Hilleman 44 years ago. It was in the context of his 1959 discovery of SV40, which I came across only because I was beginning my post-doctoral studies of the related murine polyomavirus. Bernice Eddy, at the U. S. National Institutes of Health (NIH), was probably the first to discover SV40, which she detected in early lots of the Salk polio vaccine (3). Hillman, then at Merck & Co, independently discovered the same virus in rhesus monkey kidney cell cultures, in which the polio vaccine was being produced. Hilleman gave SV40 its name. It was the 40th simian virus the Merck lab found in the monkey kidney cells. In 1961, both Eddy and Hilleman found that inoculating SV40 into hamsters causes tumors in the animals. Merck withdrew its polio vaccine from the market. But, by then, live SV40 had been unknowingly injected into hundreds of millions of people worldwide! More on this in a future posting.]

We begin our account of Hilleman’s achievements with his development of the mumps vaccine. In the days before the vaccine, mumps struck about 200,000 children in the United States, annually. Yet except in rare circumstances, the infection was mild, and was generally regarded as a childhood rite of passage. There is a sweetness to the story of the mumps vaccine that I hope you might enjoy.

The tale began at about 1:00 AM, on March 21, 1963, when 5-year-old Jeryl Lynn Hilleman ambled into her father’s bedroom complaining of a sore throat. Jeryl Lynn’s father felt his daughter’s swollen glands, and knew in a flash that it was mumps. And, while I suspect that many lay parents back in the day would also have recognized Jeryl Lynn’s symptoms, few would have done what her father did after first comforting his daughter. Although it was already past midnight, Maurice hopped into his car and drove the 20 minutes to his lab at Merck & Co. to pick up some cotton swabs and beef broth. Returning home, he then awakened Jeryl Lynn, gently swabbed her throat, and immersed the swabs in the nutrient broth. Next, he drove back to his lab and put the inoculated broth in a freezer.

Hilleman made the early A.M. dashes to his lab and back because he had to leave in the morning for a conference in South America, and his daughter’s infection might have cleared by the time he returned home from there. So, upon his return from South America, Hilleman, thawed the frozen sample from his daughter’s throat and inoculated it into chick embryos. Serial passage of the mumps virus in the chick embryos eventually generated attenuated mumps virus that in 1967 would serve as a live mumps vaccine.

The virus in the vaccine was dubbed the Jeryl Lynn strain, in honor of its source. Years later, an adult Jeryl Lynn Hilleman noted that her father had a need to be “of use to people, of use to humanity.” She added: “All I did was get sick at the right time, with the right virus, with the right father.”

We’ll have a bit more to say about the mumps vaccine shortly. But first, a few words about measles and rubella.

If mumps was not a major killer, measles certainly was. Before Hilleman and his colleagues introduced their measles vaccine (Rubeovax) in 1962, there were 7 to 8 million measles fatalities worldwide each year, and virtually all of the victims were children. Hilleman developed his attenuated measles vaccine from a measles strain isolated earlier by John Enders. Hilleman attenuated the Enders isolate by putting it through 80 serial passages in different cell types.

[Aside: In a previous posting, we noted that Enders, together with colleagues Thomas Weller and Frederick Robbins, shared a Nobel Prize in Physiology or Medicine for growing poliovirus in non-nervous tissue (3). Apropos the current story, bear in mind that Salk and Sabin developed polio vaccines that have nearly rid the world of this once dread virus. Nevertheless, the Nobel award to Enders, Weller, and Robbins was the only Nobel award ever given in recognition of polio research!]

Rubeovax was somewhat tainted by its side effects; mainly fever and rash. While these reactions were successfully dealt with by combining Rubeovax with a dose of gamma globulin, in 1968 Hilleman’s group developed a new, more attenuated measles strain by passage of the Rubeovax virus 40 more times through animal tissues. Hilleman dubbed the new measles strain “Moraten,” for “More Attenuated Enders.” The new measles vaccine, Attenuvax, was administered without any need for gamma globulin.

Our chronicle continues with the rubella vaccine. Rubella poses its greatest danger to fetuses of non-immune pregnant woman, particularly during the first trimester of pregnancy. In up to 85% of these women, infection will result in a miscarriage or a baby born with severe congenital abnormalities. An outbreak of rubella began in Europe in the spring of 1963, and quickly spread worldwide. In the United States, the 1963 rubella outbreak resulted in the deaths of 11,000 fetuses, and an additional 20,000 others born with birth defects (e.g., deafness, heart disease, cataracts).

Hilleman had been working on a rubella vaccine at the time of the 1963 outbreak. But, he was persuaded to drop his own vaccine and, instead, refine a vaccine (based on a Division of Biologics Standards’ rubella strain) that was at the time too toxic to inoculate into people. By 1969 Hilleman was able to attenuate the DBS strain sufficiently for the vaccine to be approved by the FDA.

Next, and importantly, Hilleman combined the mumps, measles, and rubella vaccines into the single trivalent MMR vaccine, making vaccination and, hence, compliance vastly easier. Thus, MMR was a development that should have been well received by many small children and their mothers, as well as by public health officials.

In 1978 Hilleman found that another rubella vaccine was better than the one in the trivalent vaccine. Its designer, Stanley Plotkin (then at the Wistar Institute), was said to be speechless when asked by Hilleman if his (Plotkin’s) vaccine could be used in the MMR. Merck officials may also have been speechless, considering their loss in revenues. But for Hilleman, it was simply the correct thing to do.

Like Jonas Salk and Albert Sabin before him (3), Maurice Hilleman was never awarded a Nobel Prize. There is no obvious reason for the slight in any of these three instances. In Salk’s case, it may have been because Alfred Nobel, in his will, specified that the award for Physiology or Medicine shall be for a discovery per se; not for applied research, irrespective of its benefits to humanity. But, Max Theiler received the Nobel Prize for producing a yellow fever vaccine. What’s more, the Nobel committee seemed to equivocate regarding the discovery that might have been involved in that instance. Regardless, the Nobel award to Theiler was the only Nobel Prize ever awarded for a vaccine! [A more complete accounting of the development of Theiler’s yellow fever vaccine can be found in The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, now on the blog.]

Sabin had done basic research that perhaps merited a Nobel Prize (3). But, the Nobel committee may have felt uneasy about giving the award to Sabin, without also recognizing Salk. Or, perhaps the continual back-and-forth carping between supporters of Salk and Sabin may have reduced enthusiasm in Stockholm for both of them.

Yet by virtually any measure, Hilleman’s achievements vastly exceeded those of Salk, Sabin, Theiler, and just about everyone else. His basic interferon work alone should have earned him the Prize. Hilleman’s group demonstrated that certain nucleic acids stimulate interferon production in many types of cells, and detailed interferon’s ability to impede or kill many viruses, and correctly predicted its efficacy in the treatment of viral infections (e.g., hepatitis B and C), cancers (e.g., certain leukemias and lymphomas), and chronic diseases (e.g., multiple sclerosis). What’s more, Hilleman developed procedures to mass-produce and purify interferon. And, regarding his unmatched achievements as a vaccinologist, he did more than merely emulate Pasteur’s procedures for developing attenuated viral vaccines. His hepatitis B vaccine was the first subunit vaccine produced in the United States. It was comprised of the hepatitis B surface antigen (HBsAg), which Hilleman purified from the blood of individuals who tended to be infected with hepatitis B virus (e.g., IV drug abusers). Subsequently, to avoid the potential danger of using human blood products in the vaccine, Hilleman developed recombinant yeast cells that produced the HBsAg. And, Hilleman’s meningococcal vaccine was the first vaccine to be based on polysaccharides, rather than on a whole pathogen or its protein subunits.

So, why then was Hilleman bypassed by the Nobel committee? John E. Calfree, in The American, wrote: “As the 80-plus-year-old Hilleman approached death, Offit and other academic scientists lobbied the Nobel committee to award Hilleman the Nobel Prize for Medicine, based partly on his vaccine work and partly on his contributions to the basic science of interferons. The committee made clear that it was not going to award the prize to an industry scientist.” (4) [Paul Offit, referred to here, is the co-developer of the rotavirus vaccine, Rotateq, and a biographer of Hilleman.]

Calfree also notes that Hilleman’s tendency towards self effacement, and his absence from the academic and public spotlight, may also have worked against him. And, unlike Salk, whose name was closely linked to his polio vaccine (3), Hilleman’s name was never associated with any of his nearly forty vaccines. [Yet in the case of Jonas Salk, his public acclaim is generally believed to have hurt him in the eyes of his colleagues and of the Nobel committee.]

Considering the enormity of Hilleman’s contributions, his anonymity was really quite remarkable. As Calfree relates: “In one of the most striking of the dozens of anecdotes told by Offit, Hilleman’s death was announced to a meeting of prominent public health officials, epidemiologists, and clinicians gathered to celebrate the 50th anniversary of the Salk polio vaccine. Not one of them recognized Hilleman’s name!”

With Hilleman’s public anonymity in mind, we conclude our account with the following anecdote. In 1998, a Dr. Andrew Wakefield became a celebrity and hero in the eyes of the public. How this happened, and its consequences are troubling for several reasons, one of which is that it brought undeserved suffering to the self-effacing and benevolent Maurice Hilleman. The Wakefield incident merits, and will have a full-length blog posting of its own. But for now, in 1998 Wakefield authored a report in the prestigious British journal The Lancet, in which he claimed that the MMR vaccine might cause autism in children. The story had a bizarre series of twists and turns, with Wakefield and co-authors eventually issuing a retraction. The immediate cause of the retraction was the disclosure that Wakefield, on behalf of parents of autistic children, had accepted funding to investigate a link between the MMR vaccine and autism. The purpose of the investigation was to determine whether a legal case against the vaccine manufacturer might have merit. In addition to the obvious conflict of interest, Wakefield’s paper had serious technical flaws as well. At any rate, a number of independent studies subsequently demonstrated that there is no causal link between the MMR vaccine and autism. And, in 2010 Wakefield was barred by the British Medical Society from the practice of medicine. But the harm had been done. Hilleman had become the recipient of hate mail and death threats. And, more important to Hilleman I expect, many worried parents, even today, prevent their children from receiving the MMR vaccine (5). Ironically, the very success of the MMR vaccine enabled people to forget just how devastating measles and rubella could be.  Maurice Hilleman succumbed to cancer on April 11, 2005.

1. Nature Medicine 11, S2 (2005)
2. Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu  On the blog.
3. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science  On the blog
4. Calfree, J.E., Medicine’s Miracle Man , The American, January 23, 2009
5. Reference 4 contains a somewhat similar tale, in which a 1992 article in Rolling Stone attributed the emergence of HIV to Hillary Koprowski’s polio vaccine. It created a sensation but, as might be expected, there was no evidence to support its premise.