Elie Metchnikoff: The “Father of Innate Immunity”

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

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

Elie Metchnikoff
Elie Metchnikoff

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References:

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

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

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

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

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

 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Addendum:

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

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

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

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

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

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

References:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References

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

 

The Phage in the Letter

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

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

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

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

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

figure-6-11-virology

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

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

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

Hilary Koprowski: Genesis of a Virologist

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

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

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

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

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

koprowski

Hilary Koprowski in Warsaw (2007)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References:

   

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

 

 

 

 

 

John Enders: “The Father of Modern Vaccines”

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References:

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

 

“More support for clinical trials in children”

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.

Nature 535:465–466 (28 July 2016)

doi:10.1038/535465b

 

Vaccine Research using Children

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.

Pennhurst, a state-funded Pennsylvania facility for the mentally deficient, was one of the most shameful examples of the neglect and mistreatment that was common at these institutions. It was the site of Werner Henle’s research in the 1940s to develop an influenza vaccine.
Pennhurst, a state-funded Pennsylvania facility for the mentally deficient, was one of the most shameful examples of the neglect and mistreatment that was common at these institutions. It was the site of Werner Henle’s research in the 1940s to develop an influenza vaccine.

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.

References:

  1. Edward Jenner and the Smallpox Vaccine, Posted on the blog September 16, 2014.
  2.  Pennhurst Asylum: The Shame of Pennsylvania, weirnj.com/stories/pennhurst-asylum/
  3.  Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.
  4.  Oshinsky D, Polio: An American Story, Oxford University Press, 2005.
  5. The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 2014.
  6.  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.
  7.  Fox M, Hilary Koprowski, Who Developed First Live-Virus Polio Vaccine Dies at 96, N.Y. Times, April 20, 2013.
  8. Maurice Hilleman: Unsung Giant of Vaccinology, Posted on the blog April 14, 2014.
  9. Beecher HK. Ethics and clinical research. The New England Journal of Medicine, 1966, 274:1354–1360.
  10.  Baruch Blumberg: The Hepatitis B Virus and Vaccine, Posted on the blog June 2, 2016.
  11.  Robinson WM, The Hepatitis Experiments at the Willowbrook State School. science.jburrougs.org/mbahe/BioEthics/Articles/WillowbrookRobinson2008.pdf
  12. Ward R, Krugman S, Giles JP, Jacobs AM, Bodansky O. Infectious hepatitis: Studies of its natural history and prevention. The New England Journal of Medicine, 1958, 258:407-416.
  13.  Ethical Conduct of Clinical Research Involving Children. http://www.ncbi.nlm.nih.gov/books/NBK25549/

 

 

Baruch Blumberg: The Hepatitis B Virus and Vaccine

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.

Nobel Laureate Baruch Blumberg
Nobel Laureate Baruch Blumberg

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.

Notes:

[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).]

References:

  1. Krugman, S. 1976. Viral Hepatitis: Overview and Historical Perspectives. The Yale Journal of Biology and Medicine 49:199-203.
  1. Blumberg, B, Australia Antigen and the Biology of Hepatitis B, Nobel Lecture, December 13, 1976.
  1. Maurice Hilleman: Unsung Giant of Vaccinology, Posted on the blog April 24, 20143.
  1. Emma Brown (6 April 2011). “Nobelist Baruch Blumberg, who discovered hepatitis B, dies at 85”. The Washington Post.
  1. Carlton Gajdusek, Kuru, and Cannibalism, Posted on the blog April 6, 2015.
  1. Mikhail Balayan and the Bizarre Discovery of Hepatitis E Virus, Posted on the blog May 3, 2016.
  1. Segelken, H. Roger (6 April 2011). “Baruch Blumberg, Who Discovered and Tackled Hepatitis B, Dies at 85”. New York Times.
  1. Choo, Q. L., G. Kuo, A.J. Weiner, L.R. Overby, D.W. Bradley, and M. Houghton. 1989. Isolation of a cDNA clone derived from non-A, non-B viral hepatitis genome. Science 244:359-362.

 

Mikhail Balayan and the Bizarre Discovery of Hepatitis E Virus

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.

Hepatitis E Virus
Hepatitis E Virus

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.”

References:

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 Virology 1:1-9.