Some of the earliest postings have mysteriously disappeared from the blog. One of these, The Phage in the Letter, is one of my favorites. So, I am re-posting it. Hope you enjoy.
Here is a favorite story of mine that I first heard when I was a graduate student in the mid 1960’s. The major protagonists are Sidney Brenner, who was one of the giants of the “golden age of molecular biology,” and Norton Zinder, also one of the top researchers of the day. Brenner was the first molecular biologist to propose the idea of a messenger RNA, a concept validated by experiments he later did with Mathew Meselson and Francois Jacob. Zinder’s major contributions included the discovery that a bacteriophage can transfer bacterial genes from one bacterial cell to another, a phenomenon referred to as “transduction.” And, apropos this anecdote, Zinder also isolated the f2 bacteriophage, the first virus known to contain a genome composed of RNA, rather than DNA.
Bearing in mind how little was known in 1960, when Zinder isolated bacteriophage f2; the discovery of RNA phages had great potential for use in the study of fundamental molecular processes, such as protein synthesis, including its initiation and termination. Clearly, there were good reasons why molecular biologists of the day, including Brenner, wanted to obtain their own samples of f2 phage. So, as the legend goes, Brenner, among others, requested a sample of f2 from Zinder. And, Zinder wrote back to all, saying that the phage was not available.
Zinder may have thought that Brenner wanted the phage to study RNA replication, a topic that Zinder wanted to keep for himself. Now, here is the delightful part of the story. Knowing how carefree researchers can be in the laboratory, Brenner is said to have dipped Zinder’s letter in a culture of E. coli (the f2 host), thereby readily growing up a stock of f2 for himself.
Amusing as this story might be, the actual facts, at least according to a 1997 article by Brenner1, are as follows. First, after Zinder isolsted f2 phage from a New York sewer, he indeed declined to distribute the phage to the large number of researchers requesting it. Second, Brenner’s reason for wanting f2 was not to use it to work on RNA replication, but instead to use it to test bacteria for the presence of a sex factor. The bacterial sex factor is a gene that encodes a so-called pilus, which is present on male bacteria, enabling them to transfer genes to female bacteria. It also is the bacterial “organ” via which RNA phages enter bacterial cells, thus explaining Brenner’s stated interest in f2. [While it might be thought that f2 can only infect male bacteria, interestingly, male bacteria that are infected with f2 can transfer the virus to female bacteria via their pili. Thus, even bacteria have sexually transmitted infections.] Third, while Brenner may not have isolated f2 from Zinder’s letter, he indeed recommended a similar procedure to several other researchers. Brenner also confesses that he might have added to the original myth by hinting that the story actually might be true. In reality, Brenner isolated many RNA phages himself by taking sewerage from the Cambridge, Massachusetts, sewer treatment plant and plating it on bacteria expressing a sex factor.
The Micrograph shows an F-pilus emerging from an E. coli cell that is covered with icosahedral MS2 phage particles. At the end of the pilus, a filamentous fd phage has attached itself. The thicker thread emerging at the right is a bacterial flagellum. Figure 6.11, page 188, From Virology: Molecular Biology and Pathogenesis, by Leonard C. Norkin, ASM Press, 2010.
While Brenner’s work as a molecular biology pioneer may have justified a Nobel Prize, he received the award in 2002 for his later studies of the nematode Caenorhabditis elegans, in which his research group traced the fate of each cell from the zygote right through to the adult worm. Their work established C. Elegans as a model system that is now studied in hundreds of laboratories all over the world.
1Brenner, S. 1997. Bacteriophage Tales. Current Biology7:R736-737.
Several years before Jonas Salk and Albert Sabin developed their famous polio vaccines, Hilary Koprowski (1916-2013) in fact developed the world’s first effective, but much less well known polio vaccine (1, 2). Koprowski’s vaccine was used world-wide, but it was never licensed in the United States, ultimately losing out to Sabin’s vaccine.
Koprowski’s reputation was tarnished in 1950, when he tested his live polio vaccine on 20 children at Letchworth Village for mentally disabled children, in Rockland County, NY; an episode recounted in a recent posting Vaccine Research Using Children (1). Koprowski reported on the Letchworth Village trials at a 1951 conference of major polio researchers. Although his vaccine induced immunity in the children, and caused no ill effects, many scientists in the audience were horrified that he actually tested a live polio vaccine in human children. Afterwards, Sabin shouted at him: “Why did you do it? Why? Why?”
Although Koprowski’s polio vaccine was supplanted by the Salk and Sabin vaccines, his demonstration, that a live polio vaccine could be safe and effective, paved the way for Sabin to develop his live polio vaccine. Moreover, Sabin developed his vaccine from a sample of attenuated poliovirus that he received from Koprowski.
There is much more to tell about Koprowski. This posting relates some of the remarkable earlier events of his life, including his harrowing escape from Poland on the eve of the Second World War; a flight which inadvertently led to his career in virology. A subsequent posting will recount the now discredited, although sensational at the time, accusation that Koprowski’s polio vaccine gave rise to the HIV/AIDS epidemic.
Koprowski was born and grew up in Warsaw, where he earned a medical degree from Warsaw University in 1939. He also was an accomplished pianist, having studied piano from the age of 12 at the prestigious Warsaw Conservatory, where Chopin is said to have studied. Koprowski eventually earned a music degree from the Conservatory. He recalled, “…the first year I was the youngest and voted second best in the class (3).”
Hilary Koprowski in Warsaw (2007)
In 1938, while Koprowski was in medical school, he married classmate Irena Grasberg who, in later years, would wonder how they had found the time for their courtship. Each had to contend with a demanding medical school program, while Hilary’s piano studies at the Conservatory was a full time program in itself (3). Irena recalled a day before both of them had an anatomy exam, and Hilary had an important recital. Hilary practiced a recital piece, while simultaneously studying a chart on the music rack showing the bones of the hand; all the while as Irena read anatomy to him.
Koprowski eventually chose a career in medicine, rather than one in music. As he explained: “…the top of the music pyramid is much narrower than that of medicine, where there is more space for successful scientists (3).” Koprowski rated himself only fourth best in his class at the Warsaw Conservatory, and he needed to excel. Yet he may have underrated himself. His piano professor at the Conservatory was “greatly disappointed” when he chose to enter medicine (3). [After the 1944 Warsaw uprising, Koprowski’s piano professor was arrested and beaten to death by German soldiers (see below and 3).] In any case, Koprowski continued to play the piano, and he even did some composing in his later years.
Germany invaded Poland in September 1939, setting off the Second World War. As German bombs were falling on Warsaw, Koprowski answered the call for Polish men to go east, where Polish forces were organizing to resist the Germans. Irena, now pregnant, and Hilary’s mother went with him, while his father chose to remain behind. They made their way in a horse-drawn hay wagon, traveling at night to avoid German planes that were strafing the roads during the day. After a week or so on the road, they encountered refugees moving in the opposite direction. Those refugees told them that Russia had signed a pact with Germany and was now invading Poland from the east (Aside 1). So the three Koprowskis joined the flood of refugees moving to the east. When they arrived back in Warsaw, they found the city in ruins. Many of their friends and neighbors had been killed or were seriously wounded, and the city was occupied by German soldiers.
[Aside 1: The German–Soviet Non-aggression Pact was signed in Moscow in August 1939, as a guarantee of non-belligerence between Nazi Germany and the communist Soviet Union. Hitler broke the pact in June 1941 when Germany attacked Soviet positions in eastern Poland. Hitler had no intention of keeping to the pact. However, it temporarily enabled him to avoid having to fight a war on two fronts—against Britain and France in the west and the Soviet Union in the east.]
Once Germany had conquered Poland, German and Polish Jews began to be sent to concentration camps set up in Poland. The Koprowskis, who were Jewish (Salk and Sabin too were descendants of eastern European Jews), quickly made plans to leave Poland. Their first destination was to be Rome. Hilary’s father went there first to arrange living conditions for the family. To facilitate the escape of Hilary’s father from Poland, Hilary and Irena wrapped him in bandages, hoping that the authorities might gladly believe they were letting a very frail individual depart from the country.
Hilary, Irena, and Hilary’s mother then traveled by train from Warsaw to Rome. It was a harrowing trip. Irena was pregnant, and the Gestapo was roaming the trains. They feared that they might have been arrested at any time.
In Rome, the Koprowski family’s main concern was the safety of Irena and her unborn baby. Since Irena had an aunt in Paris, who would know of a good doctor there, the family thought that Paris would be a safe place for the baby to be born. Thus, Irena left for Paris, accompanied by Hilary’s father. She gave birth to Claude five days after arriving there.
Hilary did not go with Irena to France. If he had done so, he would have been impressed immediately into the Polish Army that was forming there to fight the Germans. Yet he knew that he would eventually have to leave Rome. Italy, under Mussolini’s leadership, was poised to enter the Second World War, as an Axis partner of Hitler’s Germany.
After Claude was born, Irena worked as a physician at a psychiatric hospital in Villejuif, just outside of Paris. She was the sole internist there for eight hundred patients. She kept Claude at the hospital, in a locked room, which she would slip to away every three hours to nurse him.
Back in Rome, Hilary continued to play the piano. In fact, he auditioned for, and was accepted by Rome’s L’Accademia di Santa Cecilia, which awarded him a second degree in music. Importantly, his skill at the keyboard enabled him to get visas for himself and his mother to enter Brazil, which the family hoped would be a safe haven. The best students from L’Accademia di Santa Cecilia were often in demand to play for events at the Brazilian embassy in Rome. Thus, on several occasions, Hilary played the piano at the embassy. Brazil’s consul general admired Hilary’s pianism and was pleased to arrange Brazilian entry visas for Hilary and his mother. See Aside 2.
[Aside 2: The day after Hilary arrived in Rome, he volunteered to serve as a medical examiner for a Polish draft board that was set up in the Polish embassy. The draft board’s activity at the embassy—recruiting Poles for the Polish Army—violated diplomatic protocol. In addition, Italy would soon be Germany’s Axis partner in the War. Moreover, Brazil, though neutral in the War, favored the Axis.]
Hilary and his mother had been making plans to leave Italy. Their destination was to be Spain, where they hoped they might unite with Irena, Claude, and Hilary’s father. From Spain, the family might then go to Portugal, where they could get a boat to Brazil. But, on the very day that Hilary and his mother were to leave Italy, Mussolini issued a proclamation banning any male of military age from leaving the country. So it happened that Hilary’s escape from Italy was blocked at the boat registration. However, his mother rose to the occasion, crying and pleading with the boat registration official that she was sick, that Hilary was her sole means of support, and that she could not go on without him. “The man looked at his watch and said he must go to lunch. He looked at us and said, ‘If the boat leaves before I return, that’s my bad luck (3).’” So, Hilary and his mother boarded the boat, which left before the official returned. [Hilary’s mother was a well-educated woman, and a dentist by profession.]
In Spain, Hilary and his mother stayed at a hotel in Barcelona. Despite the wartime conditions, they were able to communicate, if only sporadically, with Irena and Hilary’s father, who were still in France. Then, after Germany invaded France in 1940, Irena, Claude, and Hilary’s father reunited with Hilary and his mother in Barcelona. [The escape of Irena, Claude, and Hilary’s father from France was far more harrowing than the escape of Hilary and his mother from Italy (See 3 for details).]
The family now needed to get to Portugal, where they could then get a boat to Brazil. Irena had already obtained Portuguese visas for herself and for Claude. But Hilary and his mother only had visas for Brazil. Hilary’s applications for visas at the Portuguese embassy were repeatedly denied, until a fellow Pole at Hilary’s Barcelona hotel advised him of the obligatory bribe that must accompany visa applications. The advice was right-on, and the family (minus Hilary’s father, who chose to go to England) sailed for Brazil without further incident.
In Brazil, Irena found work in Rio de Janeiro as a nurse. But she soon managed to secure a position as a pathologist at the largest hospital in the city. Hilary, on the other hand, could not find a job in medicine and, so, he turned to teaching piano. After six months of teaching unenthusiastic piano students, Hilary by chance recognized a man on the street in Rio who happened to be a former schoolmate from Warsaw. The man also happened to be working at the Rockefeller Foundation’s outpost in Rio. He told Hilary that the Foundation was looking for people, and he also told Hilary who he should contact there. Hilary interviewed at the Foundation the next day, and was told to report for work the day after that.
The Foundation assigned Hilary to research how well, and for how long the attenuated yellow fever vaccine—developed by Nobel laureate Max Theiler in 1935 (4) —might protect against yellow fever. The disease was endemic in Brazil, and it was actually the Rockefeller Foundation’s first priority.
Hilary’s supervisor at the Foundation was Edwin Lennette; a staff member of the International Health Division of the Rockefeller Foundation, assigned to its Brazilian outpost, specifically because of his interest in yellow fever. In 1944, Lennette would be reassigned to the Rockefeller Foundation laboratory in Berkeley, California, where he would establish the first diagnostic virology laboratory in the United States. Indeed, Lennette is known as one of the founders of diagnostic virology. But, in Brazil, he introduced Hilary Koprowski to virology.
Hilary’s apprenticeship under Lennette was going very well. It would result in nine papers—published between 1944 and 1946— that Hilary would co-author with Lennette. Moreover, Lennette was interested in other viruses, in addition to yellow fever. Thus, their co-authored papers included studies of Venezuelan equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, and West Nile virus, as well as yellow fever.
Most importantly, Koprowski’s work under Lennette introduced him to Max Theiler’s methods and approach to viral attenuation. In brief, Theiler found that propagating yellow fever virus in an unnatural host—chick embryos—caused the virus to adapt to that host, thereby reducing its capacity to cause disease in humans. Koprowski would later acknowledge that Theiler provided him with a “most encouraging model” for attenuating poliovirus. [Koprowski attenuated poliovirus by propagating it first in mice and then in rats. Recall that Sabin developed his live polio vaccine from attenuated poliovirus that he received from Koprowski (1).] See Asides 3 and 4.
[Aside 3: The rabies vaccine, which Louis Pasteur developed in 1885, is often referred to as the first attenuated virus vaccine. Nevertheless, while Pasteur did passage his vaccine virus in rabbit spinal cords, the virus may have been killed when the spinal cords were later dried for up to fourteen days. Also, in Pasteur’s day, nothing was known about immunity or mutation, and viruses had not yet been identified as microbes distinct from bacteria. The yellow fever vaccine developed by Max Theiler at the Rockefeller Institute (now University) in New York may have been the first deliberately attenuated viral vaccine.]
[Aside 4: Koprowski and Lennette were among the first researchers to observe that infection by one virus (yellow fever, in this instance) might inhibit the growth of another unrelated virus (West Nile virus, in this instance). That is, they had inadvertently detected what later would be known as interferon. Yet while they looked for an anti-viral substance in their tissue culture media, and while their results suggest that it actually was there, they stated in their summary that nonspecific anti-viral factors were not present (5). Koprowski and Lennette collaborated again in the 1970s; this time to investigate subacute sclerosing panencephalitis, a rare late complication of measles infection that results in neurodegeneration.]
Hilary continued to give piano recitals in Brazil, regretting only that he did not have time to practice the piano as much as he would have liked. Nonetheless, his piano playing expanded his circle of friends to include musicians, artists and writers, in addition to his fellow scientists. Moreover, Irena was satisfied with her medical practice, and with the many friends and rich social life that she and Hilary had in Brazil.
Earlier, in 1940, while Hilary was still in Rome, and expecting that the family would soon have to leave Europe, he believed that the United States would likely be the best destination for them. Thus, he applied to the United States for visas. He had nearly forgotten those applications when, in 1944, their numbers came up.
The Koprowski family now faced somewhat of a dilemma. It was happily settled in Brazil, and had no prospects in the United States. On the other hand, the Rockefeller Foundation’s yellow fever project was drawing to a close, and the Foundation was planning to leave Rio. Importantly, coming to America was now a “dream come true (3)”. So, in December 1944, the Koprowskis boarded an aging steamer in Brazil, and sailed under wartime blackout conditions, through German submarine-infested waters, for New York City.
During Hilary’s his first days in America, he used the Rockefeller Institute library in Manhattan to work on manuscripts reporting his research in Brazil. During one of his visits to the Rockefeller, he happened to meet Peter Olitzky (Aside 5), an early polio researcher there, who arranged for Hilary to meet Harold Cox, the director of the virology department at Lederle Laboratories, in Pearl River, New York. Hilary interviewed with Cox, who offered him a research position at Lederle, which Hilary accepted. Meanwhile, Irena was appointed an assistant pathologist at Cornell Medical College in Manhattan.
[Aside 5: In 1936, Olitzky and Sabin collaborated on a study at the Rockefeller Institute, which, although carefully done, wrongly concluded that poliovirus could attack nerve cells only; a result that did not bode well for the development of an attenuated polio vaccine.]
At Lederle, Hilary began the experiments that led to the world’s first successful polio vaccine. In 1950 he tested the live vaccine in eighteen mentally disabled children at Letchworth Village (1). None of these children had antibodies against poliovirus before he vaccinated them, but each of them was producing poliovirus antibodies after receiving the vaccine. Importantly, none of the children suffered ill effects. What’s more, Koprowski did not initiate the test. Rather, a Letchworth Village physician, fearing an outbreak of polio at the facility, came to Koprowski’s office at Lederle, requesting that Koprowski vaccinate the Letchworth children (1).
Vaccine Research Using Children, Posted on the blog July 7, 2016.
Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.
Roger Vaughan, Listen to the Music: The Life of Hilary Koprowski. Springer-Verlag, 2000.
The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 4014.
Lennette EH, Koprowski H., 1946. Interference between viruses in tissue culture, Journal of Experimental Medicine, 83:195–219.
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.
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).
Vaccine Research Using Children, Posted on the blog July 7, 2016.
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.
Our last posting, Vaccine Research using Children (July 7, 2016), addressed the history and ethics of testing vaccines in children. For a rather different take on the issue of children in biomedical research, see the appended Nature editorial, More support for clinical trials in children (Nature 535:465-466, 2016), which considers the use of children in cancer research. It raises issues that are similar to those on the earlier blog post (e.g., the problem of informed consent). More importantly, it raises dissimilar ones, which arise from the unique dilemma of cancer in children.
More support for clinical trials in children
US lawmakers should give drug firms the confidence to test pediatric cancer therapies.
27 July 2016
A cancer diagnosis is a shock, but adults with the disease can take some comfort in the numerous treatments available to them — both through clinical trials and as drugs that are already on the market. Children cannot. Because they make up only 1% of US patients with cancer, children are a low priority for pharmaceutical companies that want to launch an effective drug quickly. The hassle of a pediatric clinical trial may not seem worth it until after the drug has proved to be safe and effective in adults. This process can take decades, leaving children with therapies that are sometimes almost obsolete.
To access therapies early, parents of these children can turn to compassionate-use programs, in which companies give experimental drugs to people who are in desperate need. In the United States, firms that agree to provide medicines in this way will ask the Food and Drug Authority for emergency permission, which is almost always granted.
This system, although helpful for some, is rife with complications. Patients and their families report difficulties in applying for such programs, and say that they rarely receive responses. Companies that withhold a drug — because it is in short supply or not right for a patient — can find themselves on the receiving end of critical social-media campaigns highlighting individual patients. And firms worry that if a person dies or is harmed while taking a drug, it could hurt the drug’s chances of being approved. No one knows how many requests parents make and how often companies approve them, but anecdotally, firms often deny drugs on the grounds that they have not been tested in children.
Proper clinical trials for childhood cancer drugs are scarce. Designing a clinical trial is never simple, but adding children to the picture complicates the process immensely. Children are not just ‘small adults’ — they metabolize drugs in very different ways. It is difficult to predict from adult or animal studies whether a chemotherapy drug will be more or less toxic in a child, and at what dose. The process of obtaining informed consent for children participating in a trial can also be more complicated. And companies fear that the death of a child — even if unrelated to the treatment — could bring bad publicity for a new drug.
“Legal loopholes often prevent children with cancer from accessing new drugs.”
Recent years have seen attempts to make more drugs available to treat children. In the United States, a 2003 law known as the Pediatric Research Equity Act (PREA) requires that companies develop a plan for how they will test experimental drugs in children, although many trials are exempted. A second law, called the Best Pharmaceuticals for Children Act, motivates companies to perform pediatric clinical trials by granting an extra six months of market exclusivity for the adult drug.
Overall, these laws have been successful, leading to hundreds of drug labels being updated with information for use in children. But legal loopholes often prevent children with cancer from accessing new drugs. For instance, therapies for conditions that do not affect children — such as Alzheimer’s disease — are exempt from the PREA. And exemptions intended for such diseases have been broadly applied to cancer. For example, therapies that are being trialed in adults with breast cancer are exempted because children do not get that cancer, even if the drug could treat a childhood cancer in a different organ.
Also exempted are drugs for ‘orphan’ diseases that affect fewer than 200,000 people in the United States. The number of orphan designations has skyrocketed in recent years — the improved ability to define the molecular basis of an individual’s cancer means that diagnoses have become increasingly subdivided, and the majority of approved cancer drugs now carry this orphan designation.
Legislation is now attempting to close those loopholes. The Research to Accelerate Cures and Equity (RACE) for Children Act, introduced to the US Congress on 14 July, would require companies to apply the PREA to any therapy with a molecular target that is relevant to both an adult and a childhood disease. It would also end the exemption for orphan diseases. Last July, the European Medicines Agency passed similar rules to make it more difficult for companies to avoid testing drugs in children. This applies when the disease has a common mechanism in adults and children, unless the drug is likely to be unsafe in children.
With Congress now out of session and focused on the upcoming US election, the RACE for Children Act is unlikely to advance before next year. But when lawmakers pick it up, they should also address problems with compassionate-use programs — and ensure a transparent and useful process for people to gain access to unapproved drugs. They should also encourage companies to make more drugs available through market incentives, and provide increased protection should something go wrong.
Children have been used in vaccine research since its very beginning, usually said to have been in 1796, when Edward Jenner inoculated 8-year-old James Phipps with cowpox, and then challenged young James with actual smallpox (1). However, earlier, in 1789, Jenner inoculated his own 10-month-old son, Edward Jr., with swinepox. Edward Jr. then came down with a pox disease, which he fortunately recovered from. His father then challenged him with smallpox.
Edward Jr. survived his exposure to smallpox. But, since Edward Sr. wanted to determine the duration of young Edward’s protection, he again challenged his son with smallpox in 1791, when the boy was two. Edward Sr. inoculated his son yet again with smallpox when the boy was three. Fortunately, young Edward was resistant to each of the smallpox challenges his father subjected him to.
Jenner used several other young children in his experiments, including his second son, Robert, who was 11-months-old at the time. One of the children in Jenner’s experiments died from a fever; possibly caused by a microbial contaminant in an inoculum. [Microbes were not known in the late 18th century.]
We have no record of how Jenner (or his wife) felt about his use of his own children. However, there is reason to believe that Jenner felt some remorse over his use of James Phipps, who he referred to as “poor James.” Jenner looked after Phipps in later years, eventually building a cottage for him; even planting flowers in front of it himself.
By the 20th century, some of the most esteemed medical researchers were using children—in institutions for the mentally deficient—to test new drugs, vaccines, and even surgical procedures. These institutions were typically underfunded and understaffed. Several of them were cited for neglecting and abusing their residents. Moreover, their young patients were usually from poor families, or were orphans, or were abandoned. Thus, many of the children had no one to look out for their interests. In addition, research at these institutions was hidden from the public. [The goings-on at these institutions were, in general, hidden from the public, and most of the public likely preferred it that way.] Federal regulations that might have protected the children were not yet in existence, and federal approval was not even required to test vaccines and drugs.
In the early 1940s, Werner Henle, of the University of Pennsylvania, used children at Pennhurst—a Pennsylvania facility for the mentally deficient—in his research to develop an influenza vaccine. [Pennhurst was eventually infamous for its inadequate staffing, and for neglecting and abusing its patients (2). It was closed in 1987, after two decades of federal legal actions.] Henle would inoculate his subjects with the vaccine, and then expose them to influenza, using an oxygen mask fitted to their faces.
Henle’s vaccine did not protect all of his subjects. Moreover, it frequently caused side effects. Additionally, Henle maintained (correctly?) that a proper test of a vaccine must include a control group (i.e., a group exposed to the virus, but not to the vaccine). Thus, he deliberately exposed unvaccinated children to influenza. Children who contracted influenza had fevers as high as 104o F, as well as typical flu-like aches and pains.
Despite Henle’s investigations at Pennhuerst, he was a highly renowned virologist, best known for his later research on Epstein Barr virus. See Aside 1.
[Aside 1: While Henle was researching his influenza vaccine at Pennhurst, Jonas Salk concurrently worked on an influenza vaccine, using adult residents (ranging in age from 20 to 70 years) at the Ypsilanti State School in Michigan.]
Next, consider Hilary Koprowski, an early competitor of Jonas Salk and Albert Sabin in the race to develop a polio vaccine (3). By 1950, Koprowski was ready to test his live polio vaccine in people. [That was four years before Sabin would be ready to do the same with his live polio vaccine.] Koprowski had already found that his vaccine protected chimpanzees against polio virus. And, he also tested his vaccine on himself. Since neither he nor the chimpanzees suffered any ill effects, Koprowski proceeded to test his vaccine on 20 children at Letchworth Village for mentally disabled children, in Rockland County, NY. [Like Pennhurst, Letchworth Village too was cited for inadequately caring for its residents.] Seventeen of Koprowski’s inoculated children developed antibodies to the virus, and none developed complications.
Koprowski did not initiate his association with Letchworth. Actually, Letchworth administrators, fearing an outbreak of polio at the facility, approached Koprowski, requesting that he vaccinate the children. Koprowski gave each child “a tablespoon of infectious material” in half a glass of chocolate milk (4). Koprowski never deliberately infected the Letchworth children with virulent virus.
Koprowski reported the results of his Letchworth studies at a 1951 conference of major polio researchers, attended by both Salk and Sabin. When Koprowski announced that he actually had tested a live vaccine in children, many conferees were stunned, even horrified. Sabin shouted out: “Why did you do it? Why? Why (4)?” See Aside 2.
[Aside 2: In the 1930s, Canadian scientist Maurice Brodie tested a killed polio vaccine in twelve children, who supposedly had been “volunteered by their parents (4).” For a short time Brodie was hailed as a hero. However, too little was known at the time for Brodie to ensure that his formaldehyde treatment had sufficiently inactivated the live polio virus. Consequently, Brodie’s vaccine actually caused polio in several of the children. After this incident, most polio researchers could not conceive of ever again testing a polio vaccine, much less a live one, in children.]
Neither Koprowski nor Letchworth Village administrators notified New York State officials about the tests. Approval from the state would seem to have been required, since Koprowski later admitted that he was certain he would have been turned down. And, it is not clear whether Koprowski or the school ever got consent from the parents to use their children. However, recall there were not yet any federal regulations that required them to do so.
Koprowski was untroubled by the uproar over his use of the Letchworth children, arguing that his experiments were necessary. Yet he later acknowledged: “if we did such a thing now we’d be put on jail…” But, he added, “If Jenner or Pasteur or Theiler (see Aside 2) or myself had to repeat and test our discoveries [today], there would be no smallpox vaccine, no rabies vaccine, no yellow fever vaccine, and no live oral polio vaccine.” Moreover, he maintained that, secret or not, his use of the Letchworth children fit well within the boundaries of accepted scientific practice.
[Aside 2: Nobel laureate Max Theiler developed a vaccine against yellow fever in 1937; the first successful live vaccine of any kind (5). Theiler formulated a test for the efficacy of his vaccine, which did not involve exposing humans to virulent virus. Sera from vaccinated human subjects were injected into mice, which were then challenged with the Yellow Fever virus.]
Koprowski referred to the Letchworth children as “volunteers (6).” This prompted the British journal The Lancet to write: “One of the reasons for the richness of the English language is that the meaning of some words is continually changing. Such a word is “volunteer.” We may yet read in a scientific journal that an experiment was carried out with twenty volunteer mice, and that twenty other mice volunteered as controls.” See Aside 3.
[Aside 3: Koprowski was a relatively unknown scientist when he carried out his polio research at Letchworth. He later became a renowned virologist, having overseen the development of a rabies vaccine that is still used today, and having pioneered the use of therapeutic monoclonal antibodies. Yet, he is best remembered for developing the world’s first effective polio vaccine; several years before Salk and Sabin brought out their vaccines.
Most readers of the blog are aware that the Salk and Sabin vaccines are credited with having made the world virtually polio-free. What then became of Koprowski’s vaccine? Although it was used on four continents, it was never licensed in the United States. A small field trial of Koprowski’s vaccine in 1956, in Belfast, showed that its attenuated virus could revert to a virulent form after inoculation into humans. Yet a 1958 test, in nearly a quarter million people in the Belgian Congo, showed that the vaccine was safe and effective. Regardless, the vaccine’s fate was sealed in 1960, when the U.S. Surgeon General rejected it on safety grounds, while approving the safer Sabin vaccine. Personalities and politics may well have played a role in that decision (3, 4).
Interestingly, Sabin developed his vaccine from a partially attenuated polio virus stock that he received from Koprowski. It happened as follows. In the early 1950s, when Koprowski’s polio research was further along than Sabin’s, Sabin approached Koprowski with the suggestion that they might exchange virus samples. Koprowski generously sent Sabin his samples, but Sabin never reciprocated.
Koprowski liked to say: “I introduce myself as the developer of the Sabin poliomyelitis vaccine (7).” He and Sabin had a sometimes heated adversarial relationship during the time when their vaccines were in competition. But they later became friends.]
Sabin was at last ready to test his polio vaccine in people during the winter of 1954-1955. Thirty adult prisoners, at a federal prison in Chillicothe, Ohio, were the subjects for that first test in humans. [The use of prisoners also raises ethical concerns.]
Recall Sabin’s public outcry in 1951 when Koprowski announced that he used institutionalized children to test his polio vaccine. In 1954, Sabin sought permission to do the very same himself; asserting to New York state officials: “Mentally defective children, who are under constant observation in an institution over long periods of time, offer the best opportunity for the careful and prolonged follow-up studies…”
Although Sabin had already tested his attenuated viruses in adult humans (prisoners), as well as in monkeys and chimpanzees, the National Foundation for Infantile Paralysis, which funded polio research in the pre-NIH days of the 1950s, blocked his proposal to use institutionalized children. Thus, Sabin again used adult prisoners at the federal prison in Ohio. With the concurrence of prison officials, virtually every inmate over 21 years-old “volunteered,” in exchange for $25 each, and a possible reduction in sentence. None of the prisoners in the study became ill, while all developed antibodies against polio virus.
Testing in children was still a necessary step before a polio vaccine could be administered to children on a widespread basis. But, Sabin’s vaccine could not be tested in children in the United States. Millions of American children had already received the killed Salk vaccine, and the National Foundation for Infantile Paralysis was not about to support another massive field trial of a vaccine, in children, in the United States (3).
Then, in 1959, after a succession of improbable events, 10 million children in the Soviet Union were vaccinated with Sabin’s vaccine (3). The Soviets were so pleased with the results of that massive trial that they next vaccinated all seventy-seven million Soviet citizens under 20 years-of-age with the Sabin vaccine. That figure vastly exceeded the number of individuals in the United States, who were vaccinated with the rival Salk vaccine during its field trials.
Next up, we have Nobel laureate John Enders who, in the 1950’s, oversaw the development of the first measles vaccine. Enders and co-workers carried out several trials of their attenuated measles vaccine; first in monkeys and then in themselves. Since the vaccine induced an increase in measles antibody titers, while causing no ill effects, they next tested it in severely handicapped children at the Walter E. Fernald State School near Waltham, Massachusetts.
Enders seemed somewhat more sensitive than either Henle or Koprowski to the ethics of using institutionalized children. Samuel L. Katz, the physician on Enders’ team, personally explained the trial to every Fernald parent, and no child was given the vaccine without written parental consent. [Federal guidelines requiring that step still did not exist.] Also, no child was deliberately infected with virulent measles virus.
Katz personally examined each of the inoculated Fernald children every day. None of these children produced measles virus, while all of them developed elevated levels of anti-measles antibodies. Also, the Fernald School had been experiencing severe measles outbreaks before the Enders team vaccinated any of its children. But, when the next measles outbreak struck the school, all of the vaccinated children were totally protected.
In 1963, the Enders vaccine became the first measles vaccine to be licensed in the United States. Several years later it was further attenuated by Maurice Hilleman (8) and colleagues at Merck. In 1971, it was incorporated into the Merck MMR (measles, mumps, and rubella) vaccine. See Aside 4.
[Aside 4: Before Enders carried out his measles investigations he pioneered the growth of viruses in tissue culture. In 1949, Enders, and collaborators Thomas Weller and Frederick Robbins, showed that poliovirus could be cultivated in the laboratory. This development was crucial, allowing Salk and Sabin to grow a virtually unlimited amount of polio virus and, consequently, to develop their polio vaccines. In 1954, Enders, Weller, and Robbins were awarded the Nobel Prize for Physiology or Medicine for their polio virus work.]
It may surprise some readers that before the mid 1960s the so-called Nuremburg Code of 1947 comprised the only internationally recognized ethical guidelines for experimentation on human subjects. The Nuremburg Code was drawn up by an American military tribunal during the trial of 23 Nazi physicians and scientists for atrocities they committed while carrying out so-called “medical” experiments during World War II. [Sixteen of the 23 Nazis on trial at Nuremburg were convicted, and 7 of these were executed (see Note 1)].
The Nuremberg Code’s Directives for Human Experimentation contained strongly stated guidelines. Its tenets included the need to obtain informed consent (interpreted by some to prohibit research using children), the need to minimize the risks to human subjects, and the need to insure that any risks are offset by potential benefits to society.
But, despite the well-articulated principles of the Nuremberg Code, it had little effect on research conduct in the United States. Federal rules, with the authority to regulate research conduct, would be needed for that. So, how did our current federal oversight of research come to be?
A 1996 paper in the The New England Journal of Medicine, “Ethics and Clinical Research,” by physician Henry Beecher, brought to the fore the need for rules to protect human subjects in biomedical research (9). Beecher was roused to write the paper in part by the early 1960s experiments of Saul Krugman, an infectious disease expert at NYU. Krugman used mentally deficient children at the Willowbrook State School in Staten Island, New York, to show that hepatitis A and hepatitis B are distinct diseases (9). Also, before a hepatitis vaccine was available, Krugman inoculated the children with serum from convalescing individuals, to ask whether that serum might protect the children against hepatitis. Krugman exposed the children to live virus either by injection, or via milkshakes seeded with feces from children with hepatitis.
Krugman found that convalescent sera indeed conferred passive immunity to hepatitis. Next, he discovered that by infecting passively protected patients with live hepatitis virus he could produce active immunity. Krugman had, in fact, developed the world’s first vaccine against hepatitis B virus (HBV) (see Aside 4). [Although Krugman used mentally deficient institutionalized children in his experiments, his investigations were nonetheless funded in part by a federal agency; the Armed Forces Epidemiology Section of the U.S. Surgeon General’s Office.]
[Aside 4: The first hepatitis B vaccine licensed for widespread use was developed at Merck, based on principles put forward by Nobel Laureate Baruch Blumberg, (10).]
Beecher was particularly troubled by two aspects of Krugman’s experiments. First, Krugman infected healthy children with live virulent virus. Beecher maintained that it is morally unacceptable to deliberately infect any individual with an infectious agent, irrespective of the potential benefits to society. [See reference 11 for an alternative view. “The ethical issue is the harm done by the infection, not the mere fact of infection itself.”]
Second, Beecher charged that the Willowbrook School’s administrators coerced parents into allowing their children to be used in Krugman’s research. The circumstances were as follows. Because of overcrowding at the school, Willowbrook administrators closed admission via the usual route. However, space was still available in a separate hepatitis research building, thereby enabling admission of additional children who might be used in the research.
Were the Willowbrook parents coerced into allowing their children to be used in the research there? Consider that the parents were poor and in desperate need of a means of providing care for their mentally impaired children. Making admission of the children contingent on allowing them to be used in the research might well be viewed as coercion. Yet even today, with federal guidelines now in place to protect human subjects, institutions such as the NIH Clinical Center admit patients who agree to participate in research programs. Is that coercion?
Beecher’s 1966 paper cited a total of 22 instances of medical research that Beecher claimed were unethical (9). Four examples involved research using children. Krugman’s work at Willowbrook was the only one of these four examples that involved vaccine research. Beecher’s other examples involved research using pregnant women, fetuses, and prisoners. But it was Beecher’s condemnation of Krugman’s hepatitis research at Willowbrook that is mainly credited with stirring debate over the ethics of using children in research.
Did Krugman deserve Beecher’s condemnation? Before Krugman began his investigations at Willowbrook, he plainly laid out his intentions in a 1958 paper in the New England Journal of Medicine (12). Importantly, Krugman listed a number of ethical considerations, which show that he did not undertake his Willowbrook investigations lightly. In fact, Krugman’s ethical considerations, together with his plans to minimize risks to the children, were not unlike the assurances one might now submit to an institutional review board (11).
Many (but not all) knowledgeable biomedical researchers claimed that Beecher misunderstood Krugman’s research and, thus, unjustly vilified him. Krugman was never officially censored for his Willowbrook investigations. Moreover, condemnation of Krugman did not prevent his election in 1972 to the presidency of the American Pediatric Society, or to his 1983 Lasker Public Service Award.
To Beecher’s credit, his 1966 paper was instrumental in raising awareness of the need to regulate research using human subjects. Beecher was especially concerned with the protection of children and, apropos that, the nature of informed consent.
In 1974, the National Research Act was signed into law, creating the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. The basic ethical principles identified by the Commission are summarized in its so-called Belmont Report, issued in 1978. Its tenets include minimizing harm to all patients, and the need to especially protect those with “diminished autonomy” or who are incapable of “self-determination.” In addition, federal guidelines now require universities and other research institutions to have Institutional Review Boards to protect human subjects of biomedical research. [Reference 13 (available on line) contains a detailed history of the establishment of these policies.] See Aside 6.
[Aside 6: The infamous U.S. Public Health Service Tuskegee syphilis research program, conducted between 1932 and 1972, in which several hundred impoverished black men were improperly advised and never given appropriate treatment for their syphilis, also raised public awareness of the need to protect human subjects. More recently, research involving embryonic stem cells and fetuses has stoked an ongoing and heated public debate. Policies regarding this research are still not settled, with stem-cell research being legal in some states, and a crime in others. Other recent technological advances, such as DNA identification and shared databases, have been raising new concerns, such as the need to protect patient privacy. In response to these new developments, in June 2016, the US National Academies of Sciences,Engineering and Medicine released a report proposing new rules (indeed a complete overhaul of the 1978 Belmont Report) to deal with these circumstances. The Academy’s report has stirred debate in the biomedical community]
Note 1: The use of children in medical research makes many of us profoundly uneasy. We may be particularly troubled by accounts of the exploitation of institutionalized children, who comprised a uniquely defenseless part of society. Indeed, it was the very vulnerability of those children that made it possible for them to be exploited by researchers. Consequently, some readers may well be asking whether the activities of vaccine researchers Krugman, Koprowski, Sabin, Henle and others might have been comparable to that of the Nazis on trial at Nuremberg. So, I offer this cautionary interjection. While in no way condoning the vaccine researchers using institutionalized children, their work was carried out for the sole purpose of saving human lives. As Koprowski suggested above, if not for that work, we might not have vaccines against smallpox, rabies, yellow fever, and polio. Now, consider Josef Mengele, a Nazi medical officer at Auschwitz, and the most infamous of the Nazi physicians. [Mengele was discussed several times at Nuremberg, but was never actually tried. Allied forces were convinced at the time that he was dead, but he had escaped to South America.] At Auschwitz, Mengele conducted germ warfare “research” in which he would infect one twin with a disease such as typhus, and then transfuse that twin’s blood into the other twin. The first twin would be allowed to die, while the second twin would be killed so that the organs of the two children might then be compared. Mengele reputedly killed fourteen twin children in a single night via a chloroform injection to the heart. Moreover, he unnecessarily amputated limbs and he experimented on pregnant women before sending them to the Auschwitz gas chambers.
Edward Jenner and the Smallpox Vaccine, Posted on the blog September 16, 2014.
Pennhurst Asylum: The Shame of Pennsylvania, weirnj.com/stories/pennhurst-asylum/
Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.
Oshinsky D, Polio: An American Story, Oxford University Press, 2005.
The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 2014.
Koprowski H, Jervis GA, and Norton TW. Immune response in human volunteers upon oral administration of a rodent-adapted strain of poliomyelitis virus. American Journal of Hygiene, 1952, 55:108-126.
Fox M, Hilary Koprowski, Who Developed First Live-Virus Polio Vaccine Dies at 96, N.Y. Times, April 20, 2013.
Hepatitis B virus (HBV), one of mankind’s most important pathogens, infects about 2 billion people worldwide, and more than 500 million individuals are life-long carriers of the virus; with most in Asia. HBV causes acute and chronic cirrhosis, as well as hepatocellular carcinoma. In point of fact, HBV is the 10th leading cause of death in the world! The serendipitous discovery of HBV, and the development of the first HBV vaccine, happened as follows. [See Note 1 for a brief review of the remarkable HBV replication strategy].
In the early 1940’s, during World War II, British doctor, F. O. MacCallum, was the first to suggest that an infectious agent might cause hepatitis. MacCallum was assigned to produce a yellow fever vaccine for British soldiers. That was how he happened to notice that soldiers tended to come down with hepatitis a few months after receiving the yellow fever vaccine.
It was fortunate that MacCallum also knew of hepatitis cases in children who received inoculations of serum from patients convalescing from measles and mumps (a means of protection against those viruses before vaccines were available), and of hepatitis cases in blood transfusion recipients, and of cases following treatments with unsterilized reused syringes.
To explain these coincidences, MacCallum hypothesized that hepatitis might be transmitted by a factor in human blood. And, since hepatitis could be transmitted by inoculation with serum that had been filtered, MacCallum proposed that the hepatitis factor might be a virus. [In 1947 MacCallum reported that hepatitis could be spread by food and water that had been contaminated with fecal material, as well as by blood. He coined the term hepatitis A for the form of the disease spread by food and water, and hepatitis B for the form transmitted via blood.] See Aside 1.
[Aside 1: The following episode, described in MacCallum’s own words (1), occurred in England during World War II: “One day in 1942, I received a message to go to Whitehall to see one of the senior medical advisers and when I arrived I was asked ‘What is this yellow fever vaccine and how dangerous is it?’ After explaining its constitution and the possibility of a mild reaction four to five days after inoculation, I was told that the Cabinet was at that moment debating whether or not Mr. Churchill should be allowed to go to Moscow, which he wished to do in a few days’ time. The yellow fever vaccine was theoretically essential before he could fly through the Middle East, but I explained that no antibody would be produced before seven to ten days so that there would be little point in giving the vaccine. It was finally decided that the vaccine would not be used, and the administrators would take care of the situation. Several months later I received an irate call from the Director of Medical Services of the RAF, who had been inoculated with the same batch of vaccine which would have been used for Mr. Churchill, and was informed that the D. G. had spent a very mouldy Christmas with hepatitis about 66 days after his inoculation…I will leave it to you to speculate on what might possibly have been the effect on the liver of our most famous statesman and our ultimate fate if he had received the icterogenic vaccine.”]
With the advent of cell culture in the 1950s, researchers hoped that a hepatitis agent might soon be cultivated in vitro. Nonetheless, HBV was not discovered until 1966. What’s more, the discovery did not involve growing the virus in cell culture. And, reminiscent of the case of MacCallum above, the discovery was made by a researcher, Baruch S. Blumberg, who was not even working on hepatitis. Rather, Blumberg was interested in why individuals varied in their susceptibilities to various illnesses.
Blumberg sought to answer that question by identifying possibly relevant genetic differences between population groups, which, in the pre-molecular biology era, might be revealed by differences in their blood proteins. Thus, in the early 1950s, Blumberg, then working at the NIH, began collecting a panel of blood samples from diverse populations throughout the world.
Blumberg looked for serum protein variations (i.e., serum protein polymorphisms) by asking if sera from multiply-transfused individuals (defined by Blumberg as persons who received 25 units of blood or more) might contain antibodies that reacted with proteins in the serum samples of his panel. His rational, in his own words, was as follows: “We decided to test the hypothesis that patients who received large numbers of transfusions might develop antibodies against one or more of the polymorphic serum proteins (either known or unknown) which they themselves had not inherited, but which the blood donors had (2).” In other words, patients who received multiple transfusions were more likely than others to have antibodies against polymorphic serum proteins in donor blood, and those antibodies might also react with polymorphic serum proteins in the samples from his panel. See Aside 2.
[Aside 2: Blumberg used the Ouchterlony double-diffusion agar gel technique in these experiments. Serum samples to be tested against each other were placed in opposite wells of a gel. The proteins they contained could then diffuse through the gel. Antigen-antibody complexes that formed between the two samples appeared as white lines in the gel.]
Hemophilia and leukemia patients were well-represented in Blumberg’s collection of serum samples from multiply-transfused individuals. And, a serendipitous aspect of Blumberg’s experimental approach was that he used these samples to probe for serum protein polymorphisms in samples from geographically diverse populations. Thus it happened that Blumberg detected a cross-reaction between a New York hemophilia patient’s serum and a serum sample from an Australian aborigine. But what could these two individuals have had in common that might have triggered the cross-reaction?
His curiosity thus aroused, Blumberg and collaborator, Harvey Alter, of the NIH Blood Bank, tested the hemophilia patient’s serum against thousands of other serum samples. Blumberg and Alter may have been surprised to find that whatever the antigen in the Aborigine’s serum was that reacted with the hemophilia patient’s serum, reactivity against that antigen was common (one in ten) in leukemia patients, but rare (one in 1,000) in normal individuals. In any case, because the antigen was first identified in an Australian aborigine, it was termed the Australia antigen.
Bear in mind that Blumberg’s original purpose was to explain why individuals differed in their susceptibilities to various illnesses. Thus, Blumberg at first believed that he detected an inherited blood-protein that predisposes people to leukemia. However, additional experiments showed that the antigen was more common in older individuals than in younger ones; a finding more consistent with the possibility that the antigen might be associated with an infectious agent.
Blumberg’s first clue that the Australia antigen might be associated with hepatitis came to light when he tested serum samples from a 12-year old boy with Down syndrome. The first time that the boy was tested for the Australia antigen, he was negative. However, several months later, when retested, the boy was positive. Moreover, sometime during that interim, the boy also developed hepatitis.
Blumberg, and other researchers, carried out additional experiments, which confirmed that the Australia antigen indeed associated with hepatitis. In addition, the antigen was more frequently detected in hepatitis sufferers than in individuals with other liver diseases. Thus, the Australia antigen was a marker of hepatitis in particular and not of liver pathology in general. See Aside 3.
[Aside 3: Blumberg had a personal reason motivating him to identify the cause of hepatitis. His technician (later Dr. Barbara Werner) became ill with hepatitis, which she almost certainly acquired in the laboratory. Fortunately, she underwent a complete recovery.]
In 1970, British pathologist David Dane and colleagues at Middlesex Hospital in London, and K. E. Anderson and colleagues in New York, provided corroborating evidence that hepatitis is an infectious disease. Using electron microscopy, they observed 42-nm “virus-like particles” in the sera of patients who were positive for the Australia antigen. In addition, they saw these same particles in liver cells of patients with hepatitis.
What then is the Australia antigen? Actually, it is the surface protein of the 42-nm HBV particles; now known as the hepatitis B surface antigen (HBsAg). Since HBV particles per se were described for the first time by David Dane, they are sometimes referred to as Dane particles.
Now we can explain Blumberg’s early finding, that individuals who received multiple transfusions (e.g., leukemia and hemophilia patients) were more likely than the general population to have antibodies against the Australia antigen. Those individuals were more likely than the general population to have received donated blood and, thus, were more likely to have been recipients of blood contaminated with HBV. At that time, a large percentage of the blood supply came from paid donors, at least some of whom were syringe-sharing, intravenous drug abusers and, consequently, more likely than most to be HBV carriers. In 1972 it became law in the United States that all donated blood be screened for HBV. See Note 2.
But it was important to protect all people from HBV; not just transfusion recipients. In 1968, Blumberg, now at the Fox Chase Cancer Center in Philadelphia, and collaborator Irving Millman, hypothesized that HBsAg might provoke an immune response that would protect people against HBV and, consequently, that a vaccine could be made using HBsAg purified from the blood of HBV carriers. In Blumberg’s own words: “Irving Millman and I applied separation techniques for isolating and purifying the surface antigen and proposed using this material as a vaccine. To our knowledge, this was a unique approach to the production of a vaccine; that is, obtaining the immunizing antigen directly from the blood of human carriers of the virus (1).” The Fox Chase Cancer Center filed a patent for the process in 1969.
Blumberg was willing to share his method and the patent with any pharmaceutical company willing to develop an HBV vaccine for widespread use. Nonetheless, the scientific establishment was somewhat slow to accept his experimental findings and his proposal for making the vaccine. Then, in 1971, Merck accepted a license from Fox Chase to develop the vaccine. In 1982, after more years of research and testing, Maurice Hillman (3) and colleagues at Merck turned out the first commercial HBV vaccine (“Heptavax”). Producing an HBV vaccine, without having to cultivate the virus in vitro, was considered one of the major medical achievements of the day. See Notes 3 and 4.
The consequences of Blumberg’s vaccine were immediate and striking. For instance, in China the rate of chronic HBV infection among children fell from 15% to around 1% in less than a decade. And, in the United States, and in many other countries, post-transfusion hepatitis B was nearly eradicated.
Moreover, Blumberg’s HBV vaccine was, in a real sense, the world’s first anti-cancer vaccine since it prevented HBV-induced hepatocellular carcinoma, which accounts for 80% of all liver cancer; the 9th leading cause of death. Jonathan Chernoff (the scientific director of the Fox Chase Cancer Center, where Blumberg spent most of his professional life) stated: “I think it’s fair to say that Barry (Blumberg) prevented more cancer deaths than any person who’s ever lived (4).”
In 1976 Blumberg was awarded the Nobel Prize in Physiology or Medicine for “discoveries concerning new mechanisms for the origin and dissemination of infectious diseases.” He shared the award with Carlton Gajdusek, who won his portion for discoveries regarding the epidemiology of kuru (5). See Note 5.
Blumberg claimed that saving lives was the whole point of his career. “This is what drew me to medicine. There is, in Jewish thought, this idea that if you save a single life, you save the whole world, and that affected me (7).” See Aside 4.
[Aside 4: Blumberg received his elementary school education at an orthodox yeshiva in Brooklyn, and he attended weekly Talmud discussion classes until his death. Interestingly, Blumberg graduated from Far Rockaway High School in Queens, N.Y.; also the alma mater of fellow Nobel laureates, physicists Burton Richter and Richard Feynman.]
As we’ve seen, Blumberg’s landmark discovery of HBV sprang from a basic study of human genetic polymorphisms. In Blumberg’s own words, “… it is clear that I could not have planned the investigation at its beginning to find the cause of hepatitis B. This experience does not encourage an approach to basic research which is based exclusively on specific-goal-directed programs for the solution of biological problems (1).”
Saul Krugman (Note 4) had this to say about Blumberg’s discovery: “It is well known that Blumberg’s study that led to the discovery of Australia antigen was not designed to discover the causative agent of type B hepatitis. If he had included this objective in his grant application, the study section would have considered him either naïve or out of his mind. Yet the chance inclusion of one serum specimen from an Australian aborigine in a panel of 24 sera that was used in his study of polymorphisms in serum proteins…led to detection of an antigen that subsequently proved to be the hepatitis B surface antigen (1).” See Note 6.
In 1999, Blumberg’s scientific career took a rather curious turn when he accepted an appointment by NASA administrator, Dan Goldin, to head the NASA Astrobiology Institute. There, Blumberg helped to establish NASA’s search for extraterrestrial life. Blumberg also served on the board of the SETI Institute in Mountain View, Calif.
Blumberg passed away on April 5, 2011, at 85 years of age.
[Note 1: HBV is the prototype virus for the hepadnavirus family, which displays the most remarkable, and perhaps bizarre, viral replication strategy known. In brief, in the cell nucleus, the cellular RNA polymerase II enzyme transcribes the hepadnavirus circular, double-stranded DNA genome, thereby generating several distinct species of viral RNA transcripts, all of which are exported to the cytoplasm. The largest of these viral transcripts is the pregenomic RNA; a transcript of the entire circular viral DNA genome, as well as an additional terminal redundant sequence. Remarkably, the pregenomic RNA is then packaged in nascent virus capsids, within which it is reverse transcribed by a virus-encoded reverse transcriptase activity, thereby becoming an encapsulated progeny hepadnavirus double-stranded DNA genome. Thus, reverse transcription is a crucial step in the replication cycle of the hepadnaviruses, as it is in the case of the retroviruses. But, while the retroviruses replicate their RNA genomes via a DNA intermediate, the hepadnaviruses replicate their DNA genomes via an RNA intermediate.]
[Note 2: The highly sensitive radioimmunoassay (RIA) technique, developed by Rosalyn Yallow and Solomon Berson, is the basis for the test that screens the blood supply for the Australia antigen. The story behind this assay is worthy of note here because it is yet another example of serendipity in the progress of science. In brief, Yallow and Berson sought to develop an assay to measure insulin levels in diabetics. Towards that end, they happened to find that radioactively-labeled insulin disappeared more slowly from the blood samples of people previously given an injection of insulin than from the blood samples of untreated patients. That observation led them to conclude that the treated patients had earlier generated an insulin-binding antibody. And, from that premise they hit upon the RIA procedure. Using their insulin test as an example, they would add increasing amounts of an unlabelled insulin sample to a known amount of antibody bound to radioactively labeled insulin. They would then measure the amount label displaced from the antibody, from which they could calculate the amount of unlabelled insulin in the test sample. Their procedure has since been applied to hundreds of other substances. RIA is simpler to carry out and also about 1,000-fold more sensitive than the double-diffusion agar gel procedure that Blumberg used to identify the Australia antigen. Yallow and Berson refused to patent their RIA procedure, despite its huge commercial value. Yallow received a share of the 1977 Nobel Prize in Physiology or Medicine for her role in its development. Berson, died in 1972 and did not share in the award.]
[Note 3: Making Heptavax directly from the blood of human HBV carriers was somewhat hindered because it required a continuing and uncertain supply of suitable donor blood. Moreover, there was concern that even after purifying the HBsAg, and treating it with formalin to inactivate any infectivity, the vaccine might yet contain other live dangerous viruses. Concern increased in the early 1980s with the emergence of HIV/AIDS, since much of the HBV-infected serum came from donors who later developed AIDS. Thus, in 1990 Heptavax was replaced in the United States by a safer genetically engineered (i.e., DNA recombinant) HBV vaccine, which contained no virus whatsoever. That vaccine was the first to be made using recombinant DNA technology. Moreover, it was yet another instance in which Hilleman played a key role in the development of a vaccine (3).]
[Note 4: In 1971, Saul Krugman, working at NYU, was actually the first researcher to make a “vaccine” against HBV. Krugman’s accomplishment began as a straightforward inquiry into whether heat (boiling) might kill HAV (see Note 5). Finding that it did, Krugman repeated his experiments; this time to determine whether boiling might likewise kill HBV in the serum of HBV carriers. As Krugman expected, boiling indeed destroyed HBV infectivity. But, to his surprise, while the heated serum was no longer infectious, it did induce incomplete, but statistically significant protection against challenge with live HBV. Krugman considers his “vaccine” discovery, like Blumberg’s discovery of HBV, to have resulted from “pure serendipity” (1).
Krugman could not answer whether HBsAg per se in his crude vaccine induced immunity. However, Hilleman, in 1975, using purified HBsAg, as per Blumberg’s concept, showed that HBsAg indeed induced immunity against an intravenous challenge with HBV.
Krugman also carried out key studies on the epidemiology of hepatitis, demonstrating that “infectious” (type A) hepatitis is transmitted by the fecal-oral route, while the more serious “serum” (type B) hepatitis is transmitted by blood and sexual contact.
Krugman reputation was somewhat tarnished because he used institutionalized disabled children as test subjects in the experiments that led to his vaccine. While that practice astonishes us today, it was not unheard-of in the day. In any event, it did not prevent Krugman’s election in 1972 as president of the American Pediatric Society, or his 1983 Lasker Public Service Award.]
[Note 5: Gajdusek’s reputation was later sullied when he was convicted of child molestation (5).]
[Note 6: In 1973 and 1974, research groups led by Stephen Feinstone and Maurice Hilleman (3) discovered hepatitis A virus (HAV), a picornavirus.
After the discoveries of HAV and HBV, it became clear that blood samples cleared of HAV and HBV could still transmit hepatitis. In 1983 Mikhail Balayan identified a virus, now known a hepatitis E virus (the prototype of a new family of RNA viruses), as the cause of a non-A, non-B infectious hepatitis (6).
In 1989, a mysterious non-A, non-B hepatitis agent, now known as hepatitis C virus (a flavivirus), was identified by a team of molecular biologists using the cutting-edge molecular biology techniques of the day (8).]
Krugman, S. 1976. Viral Hepatitis: Overview and Historical Perspectives. The Yale Journal of Biology and Medicine 49:199-203.
Blumberg, B, Australia Antigen and the Biology of Hepatitis B, Nobel Lecture, December 13, 1976.
There have been several instances in which medical researchers, for the sake of mankind, allowed themselves to be infected with a potentially deadly pathogen. A well known example involved the discovery that the Aedes aegypti mosquito is the vector for yellow fever (1). Here we consider a less known and slightly bizarre example in which Mikhail S. Balayan, of the Russian Academy of Medical Sciences in Moscow, discovered the hepatitis E virus.
But first, hepatitis refers to an inflammatory disease involving the liver. Four unrelated viruses, hepatitis A, hepatitis B, hepatitis C, and hepatitis E viruses cause epidemic viral hepatitis (see Aside 1). Hepatitis E was initially identified in 1980 as a non-A, non-B infectious hepatitis. The differences between hepatitis A, B, and E virus infections are as follows. Hepatitis A and hepatitis E are similar, insofar as the etiologic agent of each usually gives rise to an acute (i.e., self-limiting) infection and illness. In contrast, hepatitis B and hepatitis C viruses usually give rise to persistent infections that may lead to chronic hepatitis, cirrhosis, and liver cancer. The mortality rate for hepatitis E is generally “only” about 1% to 2%. Yet, hepatitis E is unusual among hepatitis viruses for its severity in pregnant woman, in whom the fatality rate may reach 20%.
[Aside 1: For aficionados, hepatitis A is a picornavirus, hepatitis B is a hepadnavirus (a DNA retrovirus), and hepatitis C is a flavivirus. Hepatitis E-like viruses were originally classified as calciviruses. However, sequencing of their RNA genomes revealed that they are more similar to rubella virus, a togavirus, than to the calciviruses. Yet they are different enough from togaviruses to merit their own family. The prototype is the hepatitis E virus, discovered by Balayan. Like hepatitis A virus, it is spread by the fecal-oral route. Hepatitis E virus is found worldwide, but it is most problematic in developing countries.]
Here then is Balayan’s tale. In 1983 Balayan was investigating an outbreak of non-A, non-B hepatitis in Tashkent; now the capital city of Uzbekistan. Balayan wanted to bring patient samples back to Moscow to study. However, he had no means for refrigerating the samples. Moreover, he may not have had permission from his supervisors to return with the samples. So, he solved his dilemma by a rather extreme form of self sacrifice—he drank a pooled filtrate of patient stool samples. He is said to have made his private inoculum more palatable by first mixing it with yogurt.
Belayan’s efforts were not for naught since, after returning to Moscow, he indeed came down with hepatitis, as he presumably desired. In fact, he became seriously ill. He then began to collect his own stool samples, in which he detected, by electron microscopy, 32 nm virus particles that produced a hepatitis-like illness when inoculated into monkeys. Balayan then observed a virus in the stool of these monkeys that appeared to be identical to the virus in the original patient samples, which he transported in, and recovered from himself.
Belayan’s virus looked like hepatitis A virus in electron micrographs. But, he could show that it was not hepatitis A virus. He already had antibodies against the hepatitis A virus, and these did not react with the new virus.
Balayan mentions himself in his original report (2), as follows: “Hepatitis E virus (HEV) was first identified in the excreta of an experimentally infected human volunteer and further confirmed by similar findings in clinical specimens from patients with acute jaundice disease different from hepatitis A and B.”
1. The Struggle Against Yellow fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 2014.
2. Balayan, M.S., 1983. Hepatitis E virus infection in Europe: Regional situation regarding laboratory diagnosis and epidemiology. Clinical and Diagnostic Virology1:1-9.
In 1911 Peyton Rous, at the Rockefeller Institute, discovered the Rous sarcoma virus; the first virus known to cause solid tumors (1). Although Rous’ eponymous virus also would be known as the prototype retrovirus, his discovery generated only scant interest at the time, and would not be recognized by the Nobel Committed until 65 years later! [Nobel prizes are not awarded posthumously. Fortunately, Rous had longevity on his side. He died 4 years after receiving the prize, at age 87.]
In 1976 Harold Varmus and J. Michael Bishop, then at the University of California San Francisco, discovered that the Rous sarcoma virus oncogene, v-src, as well as the oncogenes of several other tumorgenic retroviruses, actually were derived from cellular genes that normally play an important role in controlling cell division and differentiation (2). Moreover, Varmus and Bishop showed that these cellular “proto-oncogenes” can be altered by mutation, to become “oncogenes” that contribute to cancer. [Varmus and Bishop received the 1989 Nobel Prize in Physiology or Medicine for their discovery of proto-oncogenes.]
But what is the actual activity of the protein coded for by the normal cellular c-src, and by v-src as well? The story of that discovery is rather delightful and begins as follows.
In 1978, Raymond Erikson and coworker Marc Collette, then at the University of Colorado Medical Center, were the first researchers to isolate the Src protein. They accomplished this by first preparing lysates from avian and mammalian cells, which had been transformed in culture into tumor cells by Rous sarcoma virus. Next, they precipitated those lysates with antisera from rabbits that bore Rous sarcoma virus-induced tumors. The premise of their strategy was that antibodies from the tumor-bearing rabbits would recognize and precipitate proteins that were specific to cells transformed by the virus .
With the Src protein now in hand, Ericson and Collette next sought its function. They initially asked whether Src might have protein kinase activity (i.e., an activity that adds a phosphate group to a protein.). This was a reasonable possibility because protein phosphorylation was already known to play a role in regulating various cellular processes, including cell growth and differentiation.
Ericson and Collette tested their premise by incubating their Src immunoprecipitates with [γ-32P] ATP (i.e. 32P-labelled adenosine triphosphate). In agreement with their proposal, they found that the antibody molecules in the Src immunoprecipitates had been phosphorylated. [Note that Src’s protein kinase activity was simultaneously and independently discovered by Varmus and Bishop.]
Ericson and Collette also carried out control experiments that were particularly revealing. When the same rabbit antisera was used to immunoprecipitate extracts from normal cells, or extracts from cells infected with a transformation-defective mutant of Rous sarcoma virus, no signs of protein kinase activity were seen in those immunoprecipitates. What’s more, the protein kinase activity was found to be temperature sensitive in immunoprecipitates from cells infected with a mutant Rous sarcoma virus that was temperature-sensitive for transformation.
These control experiments confirmed that the protein kinase activity in the immunoprecipitates was coded for by the virus. What’s more, they confirmed that the kinase activity of the retroviral Src protein plays an essential role in transformation. Furthermore, when taken with the earlier findings of Varmus and Bishop, they implied that the kinase activity of the cellular Src protein plays a key role in the control of normal cell proliferation.
While Erickson and coworkers were carrying out the above experiments in Denver, Walter Eckhart and TonyHunter, at the Salk Institute, were looking into the basis for the transforming activity of the mouse polyomavirus middle T (MT) protein. [Unlike Rous sarcoma virus, which is a retrovirus, the mouse polyomavirus is a member of the Polyomavirus family of small DNA tumor viruses. SV40 is the prototype Polyomavirus.]
Since Erickson’s group was finding that Src expresses protein kinase activity, Eckhart and Hunter asked whether the polyomavirus MT protein might likewise be a protein kinase. Thus, as Erickson and Collette had done in the case of Src, Eckhart and Hunter examined immunoprecipitates of MT to see if they too might express a protein kinase activity, and found that indeed they did.
Interestingly, it was not known at the time of these experiments that MT actually does not express any intrinsic enzymatic activity of its own. Instead, MT interacts with the cellular Src protein to activate its protein kinase activity. See Aside 1.
[Aside 1: For aficionados, MT is a membrane-associated protein that interacts with several cellular proteins. Importantly, the phosphorylation events carried out by MT-activated Src cause a variety of signal adaptor molecules [e.g., Shc, Grb2, and Sos] and other signal mediators [e.g., PI3K and PLCγ] to bind to the complex, thereby triggering a variety of mitogenic signaling pathways. These facts were not yet known when Eckhart and Hunter were doing their experiments.]
At the time of these experiments, serine and threonine were the only amino acids known to be phosphorylated by protein kinases. In fact, Erikson and Collette, as well as Varmus and Bishop, believed that threonine was the amino acid phosphorylated by the Src kinase (see below). Consequently, Hunter asked whether the polyomavirus MT protein likewise would phosphorylate threonine. [Recall that MT actually does not express any intrinsic enzymatic activity of its own.]
Hunter’s experimental procedure was relatively straightforward and reminiscent of Erikson’s and Collette’s. It involved incubating immunoprecipitates of MT with [γ-32P]ATP, hydrolyzing the immunoglobulin, and then separating the amino acids in the hydrolysate by electrophoresis. But, to Hunter’s surprise, the position of the labeled amino acid in his electropherogram did not correspond to that of either threonine or serine.
Hunter was well aware that tyrosine is the only other amino acid with a free hydroxyl group that might be a target for the MT kinase activity. And, while there was no precedent for a tyrosine-specific protein kinase, Hunter proceeded to ask whether the polyomavirus MT protein indeed might phosphorylate tyrosine.
Hunter began by synthesizing a phosphotyrosine molecule that could be used as a standard marker against which to compare the labeled amino acid in a repeat of his earlier experiment. And, to his pleasure, Hunter found that the amino acid that was phosphorylated by the MT kinase activity ran precisely with the phosphotyrosine standard marker in his new electropherograms.
But why had other researchers not detected tyrosine phosphorylation earlier? It was partly because phosphotyrosine accounts for only about 0.03% of phosphorylated amino acids in normal cells. The remaining 99.97% are phosphoserine and phosphothreonine. But, again, that is not the entire explanation. The rest is truly precious.
In Hunter’s own words, he was “too lazy to make up fresh buffer” before doing his experiments. Had the buffer been fresh, its pH would have been the usual 1.9; a pH that, unbeknownst to all at the time, does not separate phosphotyrosine from phosphothreonine during the electrophoresis procedure. The pH of the old buffer that Hunter used in his experiment had inadvertently dropped to 1.7; a pH at which phosphotyrosine is resolved from phosphothreonine. That fact enabled Hunter to discriminate phosphotyrosine from phosphothreonine for the first time. Thus, Hunter attributes his hugely important discovery to his laziness.
The finding that tyrosine is the amino acid phosophorylated by the polyomavirus MT protein kinase activity led Hunter and his Salk Institute-colleague Bart Sefton to ask whether Src too might phosphorylate tyrosine, rather than serine or threonine (4). Indeed, they found that the retroviral Src protein, as well the normal cellular Src protein, function as tyrosine-specific protein kinases. [Recall that it became clear only later that MT actually has no intrinsic enzyme activity of its own and that it acts through Src.] Moreover, the levels of phosphotyrosine were 10-fold higher in cells infected with wild-type Rous sarcoma virus than in control cells, consistent with the premise that Src’s protein tyrosine kinase activity accounts for the altered growth potential of those cells.
Subsequently, Stanley Cohen, at Vanderbilt University, discovered that the epidermal growth factor (EGF) receptor contains an intrinsic protein-tyrosine kinase activity, further underscoring the importance of protein-tyrosine kinases in the normal control of cell proliferation. [Cohen shared the 1986 Nobel Prize in Physiology or Medicine with Rita Levi-Montalcini for their discoveries of growth factors, including EGF.] Subsequent studies identified additional receptor protein-tyrosine kinases, such as the fetal growth factor (FGF) receptor, and non-receptor protein-tyrosine kinases, such as Abl, each of which activates a mitogenic intracellular signaling pathway.
Tony Hunter and coworkers went on to demonstrate that protein-tyrosine kinases play key roles in additional crucial cellular processes, including cellular adhesion, vesicle trafficking, cell communication, the control of gene expression, protein degradation, and immune responses. Moreover, discoveries regarding the role of protein-tyrosine kinases in cell transformation and cancer gave rise to a promising new rational approach to cancer therapy; i.e., the targeting of protein-tyrosine kinases. For example, the drug Gleevec, which inhibits activation of the Abl and platelet-derived growth factor (PDGF) tyrosine kinases, was approved by the U.S. Food and Drug Administration for the treatment of chronic myelogenous leukemia and several types of gastrointestinal tumors.
Much has happened since our lasting posting on the Brazilian Zika outbreak (1). In particular, the major topic of our last posting was the uncertainty regarding whether Zika virus causes congenital birth defects. Recent findings may be settling the issue.
One reason for the earlier uncertainty was that although Zika virus has spread to more than a dozen countries since its discovery in Uganda more than 50 years ago, Brazil remained the world’s only country in which the virus was associated with microcephaly. However, in February 2016, Brazil’s neighbor, Colombia, now the world’s second-most Zika-affected country, reported its first cases of birth defects linked to Zika.
More direct and compelling evidence for Zika as an agent of microcephaly was reported early this March in the New England Journal of Medicine (2). Ultrasound examination of Zika-infected pregnant woman revealed that 29 percent of them carried fetuses suffering “grave outcomes, including fetal death, placental insufficiency, fetal growth restriction, and CNS injury.” Zika infection of the mothers was confirmed by reverse-transcriptase–polymerase-chain-reaction assays of blood and urine specimens. “To date, 8 of the 42 women in whom fetal ultrasonography was performed have delivered their babies, and the ultrasonographic findings have been confirmed.”
Although the above study examined only 88 women, all at one clinic in Rio de Janeiro, an article in the New York Times (March 5, 2016) quotes Anthony Fauci, the director of the National Institute of Allergy and Infectious Diseases as saying, “Now there’s almost no doubt that Zika is the cause.”
Another notable report described a case of a pregnant woman who, while living in Brazil, came down with a Zika-like feverish illness at the end of the first trimester of her pregnancy (3). The mother opted to abort her 29-week-old fetus after it showed signs (by ultrasonography) of microcephaly—subsequently confirmed by autopsy of the fetus. Importantly, a flavivirus was visualized in the fetal brain by electron microscopy, and the entire Zika genome (unambiguously identified by reverse-transcriptase–polymerase-chain-reaction assay) was recovered from it.
Next, we consider a new finding that Zika can be present in breast milk. Whereas Zika is an arthropod-borne virus that is transmitted primarily by its mosquito vector, our first posting on the Brazilian Zika outbreak noted at least one instance in which Zika was transmitted via a blood transfusion (4). In addition, there were reports of Zika being sexually transmitted (5). Now there is a report of Zika virus in the breast milk of a mother in New Caledonia (6).
The woman was feverish in July 2015 when she arrived at the hospital to give birth. Nevertheless, she breast fed her apparently healthy baby immediately after delivering it. Samples of the mother’s serum and breast milk then tested positive for Zika virus by reverse-transcriptase–polymerase-chain-reaction assay, while a test of a serum sample from the 3-day–old baby was ambiguous. The mother’s fever, now accompanied by a characteristic Zika rash, persisted for the next several days. Nonetheless, she and her baby were each healthy when they left the hospital.
This report would appear to raise considerable concern that a Zika-infected mother might transmit the virus to her baby via her breast milk. All the same, the US Centers for Disease Control and Prevention (CDC) maintains that the benefits of breastfeeding outweigh the theoretical risks of Zika virus infection via breast milk, and recommends that infected women should breastfeed.
Next, we consider some recent history. On February 1, 2016 the WHO declared that Brazil’s Zika outbreak is as an international public-health emergency. But, uncharacteristically, the WHO put forth this pronouncement despite the fact that the scientific community was still not sure of the threat that Zika poses to humans. In point of fact, this was the first instance in which the WHO proclaimed its highest level of alarm for an agent of uncertain danger. [The CDC likewise elevated its Zika virus surveillance program to its highest priority level.]
Why did the WHO make its frightening declaration when the threat posed by Zika was still not clear? Obviously, a failure to take immediate action might allow the Zika outbreak to get well out of hand, with possibly devastating consequences.
In contrast to the hurried response by the WHO to the Zika outbreak, that agency responded more leisurely to the 2013/2014 West African Ebola outbreak, which did get out of control, and which persists even to this day. So, perhaps the more urgent response of the WHO to the Zika outbreak reflects a lesson learned from the Ebola affair.
But, why did the WHO wait longer before responding to the West African Ebola outbreak? One reason is because it was strongly criticized for “overreacting” to the risk posed by the 2009 H1N1 influenza epidemic—which turned out to be far less threatening than originally feared.
While the WHO may have learned a lesson from its somewhat unhurried response to the Ebola outbreak, its more urgent February 1, 2016 Zika declaration did not go far enough for some observers, since it stopped short of advising pregnant women not to travel to Zika-affected regions. For that reason, the WHO has been accused of taking political considerations into account, to the detriment of good public health policy. Any travel ban—even one aimed only at pregnant women—would be embarrassing and costly to Brazil, which has been moving ahead with its plans to host the Olympic Games this summer. Still, hundreds of thousands of people from around the world, including female spectators and participants, some of whom may be pregnant, are expected to attend.
Lastly, we note that the Zika outbreak has been stirring up a fierce religious debate in Latin America; a debate that is actually challenging the very authority of the Catholic Church in the hemisphere. But first, an earlier posting on the blog recounted how in 2002 Colin Powell, at the time Secretary of State in the George W. Bush administration, advocated that sexually active young people should use condoms to protect themselves against HIV/AIDS (7). Powell’s advocacy of condom usage was contrary to the Bush administration’s strongly held abstinence-only approach for preventing sexual transmission of HIV. Moreover, then as now, Powell’s stance was contrary to the official position of the Catholic Church on artificial contraceptives. Nevertheless, Powell asserted, “I certainly respect the position of the Holy Father and the Catholic Church. In my own judgment, condoms are a way to prevent infection. Therefore, I not only support their use, I encourage their use among people who are sexually active and need to protect themselves.”
Now, presumably in response to reports that Zika virus might be transmitted sexually, Pope Francis declared on February 18, 2016—during a mid-air news conference on his flight from Mexico back to Rome—that contraceptives could be used to block the spread of Zika virus. That same day, the WHO advised the sexual partners of pregnant women to use condoms, or to abstain from sex, if they live in a Zika-affected area, or if they are returning from one of those areas. Also, several Latin American governments asked their female citizens to delay getting pregnant.
Those suggestions, whether from Latin American governments, or from the WHO, offended many Latin American women, in part because of the strict anti-abortion laws, and laws that restrict access to contraceptives in some of those countries. Moreover, the situation is compounded in some regions of the hemisphere by rampant sexual violence against women. In any event, the Pope’s pronouncement intensified an angry debate over contraception, and abortion as well, that was already underway in Latin America.
Pope Francis did not condone abortion, which he referred to as an “absolute evil.” But, he did make a point of justifying his statement condoning contraception by citing as a precedent a 1960 judgment by Pope Paul VI, which permitted nuns in the Belgian Congo, who were in danger of being raped, to use contraceptives.
Pope Francis’ remarks, such as “avoiding pregnancy is not an absolute evil,” has encouraged Latin American opponents of the church’s longstanding ban on the use of artificial contraceptives to campaign harder against those policies. In any case, the Pope’s pronouncement, and the heated response it is provoking, shows that the Zika outbreak is now impacting religious institutions. And, as noted by Ana Ayala, the director of the Global Health Law Program at Georgetown University, “The pope’s positioning on this subject can significantly shift how governments see access to contraception.” See Aside 1.
[Aside 1: Ayala’s comment can be found in a February 18, 2016 article in the New York Times, entitled “Francis Says Contraception Can Be Used to Slow Zika”, by Simon Romero and Jim Yardley. This piece offers an extensive account of the response in Latin America, and elsewhere, to the Pope’s comments. “While international researchers are still trying to prove definitely a link between Zika and microcephaly, the pope’s comments on contraception seemed to catch up to the reality in parts of the hemisphere where many Catholics pay little heed to the church’s teachings on birth control.”]
Zika Virus, Part 2: The Link to Birth Defects, Is It Real?, Posted on the blog February 23, 2016.
Brasil, P., Pereira, J.P., Gabaglia, C.J., et al., Zika Virus Infection in Pregnant Women in Rio de Janeiro — Preliminary Report, N. Engl. J. Med., March 4, 2016DOI: 10.1056/NEJMoa1602412
Mlakar, J., Korva, M., Tul, M., et al., Zika Virus Associated with Microcephaly, N. Engl. J. Med. 2016; 374:951-958 March 10, 2016 DOI:10.1056/NEJMoa1600651
Zika Virus: Background, Politics, and Prospects, Posted on the blog February 4, 2016.
Foy, B.D., K. C. Kobylinski, J.L. Foy, et al., 2011. Probable Non–Vector-borne Transmission of Zika Virus, Colorado, USA, Emerg Infect Dis.17: 880–882.6.
Gravitational waves were detected for the first time this past February by the Laser Interferometer Gravitational-Wave Observatory (LIGO), which consists of two widely separated installations within the United States — one in Livingston, Louisiana and one in Hanford, Washington. Before LIGO, there was no technology able to detect these vanishingly weak waves. Consequently, LIGO’s accomplishment has generated considerable excitement in the physics and astronomy communities. First, it confirmed the existence of gravity waves; a key prediction of Einstein’s 1915 theory of general relativity. Second, and remarkably, LIGO detected gravitational waves that were emitted during the final fraction of a second of the merger of two black holes, which were over a billion light-years away (one light-year is about 5.88 trillion miles)! What’s more, LIGO’s findings in fact proved the existence of black holes. Prior to LIGO’s achievement, the existence of black holes was widely accepted, but based only on indirect evidence.
Physicists and astronomers are also excited by the potential of gravitational-wave detectors to shed light on other basic concerns, such as determining whether gravitational waves travel at the speed of light—an important issue since it would answer whether gravity is transmitted by particles having no mass. These detectors may also enable astronomers to measure the rate at which the universe is expanding, and perhaps observe the effect of dark energy on space.
Most exciting perhaps, gravitational wave detectors may enable astronomers to see almost all the way back to the big bang. Until now, astronomers could only see as far back as 380,000 years after the big bang, when the universe became transparent to light and other electromagnetic radiation. However, gravitational waves would have traveled unhindered through the newborn universe. By scrutinizing gravitational waves from the infant universe, cosmologists hope to learn more about its beginning and, perhaps, even uncover evidence for the existence of other universes. Moreover, gravitational wave detectors might even lead to a “theory of everything (1).”
Scientists from other disciplines, as well as lay people, might very well marvel at the sheer ingenuity and persistence of the physicists and engineers who designed LIGO; a result of decades of instrument research and development. But first, here is a very brief account of gravity waves.
Einstein’s theory of general relativity predicts that matter emits gravity waves. These waves disturb the fabric of space, in fact causing the distances between objects to ebb and flow in an oscillatory manner. However, these oscillations are far too small to have been detected prior to LIGO.
Here is Lawrence M. Krauss’ account in the New York Times of the LIGO technical achievement (2). “To see these waves, the experimenters built two mammoth detectors, one in Washington State, the other in Louisiana, each consisting of two tunnels about 2.5 miles in length at right angles to each other. By shooting a laser beam down the length of each tunnel and timing how long it took for each to be reflected off a mirror at the far end, the experimenters could precisely measure the tunnels’ length. If a gravitational wave from a distant galaxy traverses the detectors at both locations roughly simultaneously, then at each location, the length of one arm would get smaller, while the length of the other arm would get longer, alternating back and forth …To detect the signal they observed they had to be able to measure a periodic difference in the length between the two tunnels by a distance of less than one ten-thousandth the size of a single proton. It is equivalent to measuring the distance between the earth and the nearest star with an accuracy of the width of a human hair….If the fact that this is possible doesn’t astonish, then read these statements again. This difference is so small that even the minuscule motion in the position of each mirror at the end of each tunnel because of quantum mechanical vibrations of the atoms in the mirror could have overwhelmed the signal. But scientists were able to resort to the most modern techniques in quantum optics to overcome this.” See Asides 1 and 2.
[Aside 1: Lawrence M. Krauss is a theoretical physicist and director of the Origins Project at Arizona State University. He is the author of “A Universe from Nothing: Why There is Something Rather than Nothing.”]
[Aside 2: Interestingly, the LIGO detectors had just been turned on for their first observing run when they discovered a clear signal emanating from the colliding black holes. Also, recall that these black holes were over a billion light-years away.]
Krauss later says, “Too often people ask, what’s the use of science like this, if it doesn’t produce faster cars or better toasters. But people rarely ask the same question about a Picasso painting or a Mozart symphony. Such pinnacles of human creativity change our perspective of our place in the universe. Science, like art, music and literature, has the capacity to amaze and excite, dazzle and bewilder. I would argue that it is that aspect of science — its cultural contribution, its humanity — that is perhaps its most important feature (2).”
Also, consider the following from an editorial in the New York Times. “The curiosity of our species knows no bounds; more remarkably, neither does our capacity for satisfying it. And that is truly wonderful in itself, even if it doesn’t lead to a better toaster (3).” See Aside 3.
[Aside 3: The development of LIGO was made possible by support from the National Science Foundation. “By coincidence, at about the same time that the LIGO discovery was announced, the U.S. House of Representatives passed a bill requiring that National Science Foundation grants be justified ‘in the national interest.’ It is doubtful that LIGO would have survived such political meddling (3).”]
“The Theory of Everything,” Posted on the blog September 15, 2015.
Lawrence M. Krauss, Finding Beauty in the Darkness, Opinion in Sunday Review, New York Times, February 14, 2016.
The Editorial Board, New York Times, February 17, 2016