Monthly Archives: April 2014

Maurice Hilleman: Unsung Giant of Vaccinology

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

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

hillemanMaurice R. Hilleman: The greatest vaccinologist.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu

The 1918 influenza pandemic killed an estimated 50 million people worldwide, making it the deadliest epidemic in human history. And despite the passage of nearly a century, a number of unexplained mysteries remain concerning the 1918 pandemic virus. A mystery important to our story is that the 1918 virus suddenly and inexplicably disappeared from the world in the early 1920s. And, since influenza virus was not even identified until the 1930s, no samples of the 1918 influenza strain were isolated at the time of the pandemic. Therefore, the 1918 pandemic virus did not exist in the world until it was “resurrected” nearly 80 years later by Jeffery Taubenberger and his colleagues, who used new, state-of-the-art molecular techniques to accomplish that feat.

More recently, in 2011, two independent research groups, one led by Yoshihiro Kawaoka and the other by Ron Fouchier, modified an H5N1 bird flu (see Aside 5) from a form that does not spread between humans, to forms that very well might. The unmodified avian virus has thus far infected only about 600 humans, in almost all instances by close contact with a diseased bird. But, and importantly, the avian virus killed more than half of the infected humans; a fatality rate far greater than that of even the 1918 pandemic virus. Thus, the resurrection of the 1918 pandemic virus, and the creation of transmissible H5N1 avian influenza, may have brought into the world pathogens with the potential to unleash extraordinary devastation.

These stories are compelling scientifically, historically, and for the public policy issues that they raise. As usual, we begin with some background.

The initial outbreak of the 1918 influenza pandemic occurred in March of that year, at an Army training camp outside of Boston. Yet by the fall of 1918 it was being referred to as the “Spanish” flu, probably because Spain, as a non-combatant in World War I, then in its final year, did not censor news of the pandemic. The combatants, on the other hand, fearing that news of the pandemic might cause panic that might undermine their war efforts, repressed news of it.

By the end of the winter of 1918-1919, two billion people around the world contracted the pandemic influenza strain and, as noted above, estimates of the total number of fatalities range as high as 50 million. That amount is about twice as many as would die of AIDS worldwide during the entire first twenty years of the AIDS epidemic. Moreover, the 1918 influenza pandemic killed more people in a single year than the four-year bubonic plague that ravaged Europe from 1347 to 1351. In the United States alone there were an estimated twenty million cases (out of a population of 100 million at the time) and 850,000 dead, including 196,000 people killed during the single month of October1918.

spanish fluRed Cross workers remove victims of the 1918 influenza pandemic from a house in St. Louis.      St. Louis Post-Dispatch

[Aside 1: Influenza virus pandemics also occurred in 1957 (the “Asian” flu) and 1968 (the “Hong Kong” flu). However, those pandemics were much less devastating than the pandemic of 1918. The number of deaths in the United States from those latter flu pandemics is estimated to be 70,000 and 50,000, respectively.]

Bearing in mind the sheer devastation of the 1918 pandemic, consider Taubenberger’s following comments from 1997: “It is curious that the (1918) pandemic doesn’t seem to be part of the cultural memory, at least in the United States, although it was a huge event with a huge impact. Everyone hears about the Black Death in the 1300s, yet here was an infectious disease only 85 years ago that killed 40 million people and for some reason we don’t know about it.”

It also is rather curious that while the 1918 influenza pandemic killed an astonishingly large number of people, it did not cause any public panic. Apropos that, in my last posting, Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, I noted that the annual poliovirus outbreaks, in the pre-vaccine days of the 1940s and 1950s, did cause widespread public panic. Moreover, that was so despite the fact that poliomyelitis actually caused fewer fatalities than were caused by seasonal influenza, to which the public then and now seems rather indifferent.

Another of the mysteries associated with the 1918 pandemic is that the first cases in March 1918 were relatively benign. Then, in August, the mild infection suddenly changed into something astonishingly lethal. Initial outbreaks of the new lethal variant of the virus occurred almost simultaneously in three locations; France, Sierra Leone, and Boston, and then spread worldwide. The changed virus struck with a ferocity that stunned medical professionals.

Influenza’s genetic variability is a well known characteristic of the virus. [Indeed, it is the reason why the flu vaccine needs to be re-formulated each year.] Regardless, it is not clear how the 1918 pandemic virus suddenly became so deadly. Many of the fatalities resulting from our yearly seasonal influenza epidemics are due to pneumonia caused by opportunistic bacterial pathogens. And, while bacterial pneumonia also killed many during the 1918 pandemic, the 1918 virus itself was quickly lethal in many individuals. Some patients had massively hemorrhaged lungs, and were effectively drowning in their own blood; a scenario more reminiscent of the pathology of Ebola virus than of the fevers and aches typically associated with seasonal influenza infections.

Indeed, the 1918 pandemic virus was utterly unique in how quickly it could kill; literally overnight. There are anecdotes of people leaving for work in the morning feeling fine, and then succumbing on their way. One story tells of four women in a bridge group playing together until 11:00 in the evening. By morning, three of them had died.

Another puzzling feature of the 1918 virus was that it tended to kill the hale and hearty; individuals between the ages of 25 and 34, in the primes of their lives. In contrast, seasonal influenza epidemics cause the most fatalities in the elderly, the very young, the chronically ill, and people with weakened immunity.

The lower mortality rates among the elderly during the1918 pandemic is possibly explained by their prior exposure to an influenza strain serologically related to the 1918 pandemic virus, thus providing them with a measure of protective immunity against the pandemic virus. [On this point, and others related to the biology of influenza virus, see chapter 12 of Virology: Molecular Biology and Pathogenesis.]

The higher mortality rate among individuals between the ages of 25 and 34 is sometimes attributed to the fact that the pandemic occurred during the last year of World War I; a time when many individuals in this most susceptible group were living in crowded army camps, which predisposed them to the opportunistic bacterial infections responsible for many of the influenza fatalities in the pre-antibiotic era. Yet the virus itself was extraordinarily lethal, as noted above. Moreover, the “crowded army camp theory” can not explain why the same pattern of disease was seen in the populations of countries that did not participate in the war. So, these mysteries remain.

Recalling that the 1918 pandemic virus was absent from the world after the early 1920s, we now tell the story of Jeffery Taubenberger. In March of 1997, Taubenberger and his colleagues at the Armed Forces Institute of Pathology (AFIP) in Washington, D.C. startled virologists when they reported the sequence of the hemagglutinin (HA) gene of the 1918 pandemic virus. So, how was Taubenberger able to sequence the HA gene of a virus that was nonexistent for nearly 80 years?

[Aside 2: Influenza HA proteins are located in the viral envelope. They bind to the receptor on the target cell, and then promote fusion of the viral envelope with the plasma membrane of the target cell.]

[Aside 3: Jeffery Taubenberger is currently at the National Institute of Allergy and Infectious Diseases. The Armed Forces Institute of Pathology closed its doors in September 2011. It was founded in 1862 as a museum for specimens taken from American Civil War casualties. Over the years, the Institute’s specimen collection became legendary, and it became known for its role in diagnosing difficult civilian, as well as military cases. Moreover, its staff has included some of America’s greatest pathologists.]

Taubenberger was hired by the AFIP to create a state-of-the-art molecular pathology laboratory. Towards that end, his unit, which included molecular biologist Ann Reid, developed new procedures to recover nucleic acids from tissue samples that were fixed in formaldehyde and embedded in paraffin. Although pathologists routinely examine fixed tissues, molecular analysis of those specimens had not been possible, since the fixation can destroy nucleic acids.

Taubenberger’s initial involvement with influenza was not based on an interest in influenza per se. Instead, his intention was merely to showcase his Institute’s new procedures, and also its vast collection of specimens that had been assembled over the past century. With those purposes in mind, Taubenberger and Ann Reid put in a request for fixed tissue samples from soldiers who had succumbed during the 1918 flu pandemic.

Expecting a long wait, Taubenberger and Reid were themselves surprised when the Institute’s automated recovery system successfully retrieved their samples from the 3 million others in the AFIP collection, within a few seconds of receiving their request. The samples contained flecks of tissue from soldiers killed by the flu pandemic 80 years earlier. They were taken by doctors who, of course, had no knowledge at the time of what might be causing the soldiers’ illness.

Their interest now aroused, Taubenberger and Reid began to screen paraffin-embedded, formaldehyde-fixed patient specimens for influenza sequences, using then new, extremely sensitive molecular techniques (reverse- transcription polymerase chain reaction [RT-PCR] amplification of HA gene fragments). They hoped to increase their chance of success by focusing on specimens that showed severe lung disease. The rationale was that these samples would have come from victims who died quickly, before the virus might have cleared. [Influenza generally clears the lungs within days of the infection.] Regardless, they looked in vain for a year, until they came to a sample from Private Roscoe Vaughn, who died in September 1918 at Fort Jackson, SC., during the peak of the pandemic. In Private Vaughn’s fixed cells they found small segments of influenza-like RNA. Then, to be certain that these RNA segments were indeed from the 1918 pandemic virus, they resumed their search for positive samples until they found one from a soldier who died at Camp Upton, NY, also in September 1918. After thus confirming that their samples contained RNA segments from the actual 1918 pandemic virus, they were able to generate the complete sequence of it’s HA gene. Interestingly, the HA gene of the 1918 pandemic virus was unlike that of any other influenza HA gene that had been sequenced to date.

Having thus succeeded at reconstructing the HA gene of the 1918 virus, the next step would be to reconstruct its entire genome. However, from the very small amounts of tissue in the formaldehyde-fixed autopsy samples, Taubenberger doubted ever being able to do so. What follows is my favorite part of the story.

Dr. Johan Hultin, a 73-year-old retired pathologist, unexpectedly provided a solution to the AFIP group’s dilemma. Years earlier, in 1951, when Hultin was a graduate student at the University of Iowa, he attempted to grow live influenza virus from Alaskan Inuit victims of the 1918 pandemic, whose bodies remained buried in the Alaskan permafrost over the subsequent years. It was Hultin’s hope that the virus might have been preserved in those frozen victims. However, all his attempts to grow the virus were unsuccessful.

Hultin’s failure caused him to abandon his graduate studies and, instead, become a pathologist. Then, in 1997, after he was already retired, he happened to read the report from Taubenberger’s group describing how they reconstructed the HA gene of the 1918 pandemic virus. The report rekindled Hultin’s memories of his own earlier attempts in 1951 to grow the virus. Now, excited by his thought that the frozen bodies of the Alaskan victims might contain influenza genome fragments, from which it might be possible to reconstruct the entire genome, he wrote to Taubenberger, offering to return immediately to Alaska to obtain fresh specimens. Taubenberger agreed and, thus, Hultin eagerly returned to Alaska in 1997. There, he deliberately took tissue samples from a particularly obese woman, hoping that the combination of her fat and the permafrost might have preserved the influenza genomes. Hultin’s reasoning may indeed have saved the day, since Taubenberger’s group was able to generate the entire genome of the 1918 virus from these samples and, subsequently, to grow up the virus itself.

After Taubenberger and his co-workers successfully brought the 1918 pandemic virus “back to life,” they then tested its virulence in mice. Not surprisingly, the pandemic virus was extraordinarily lethal in the mouse model. However, the explanation for the exceptional virulence of the virus was not revealed by its genetic sequence per se. But, once the technology was available to recover gene sequences of the 1918 virus, it became technologically feasible to identify which genes of the 1918 virus accounted for its extreme virulence. Some readers may need to read the following brief aside to fully appreciate this part of the story.

[Aside 4: Most viruses contain all of their genes on a single chromosome. In contrast, the influenza genome is comprised of eight distinct single-stranded RNA segments. Five of these segments encode a single protein, while three of these segments encode two different proteins. The segmented nature of influenza genomes has important consequences in nature, as follows.

If a cell were simultaneously infected with two different influenza strains, then the genomic segments of the two strains might randomly re-assort to produce brand new strains. Indeed, this is precisely how pandemic strains are believed to arise in nature. In those instances, a human influenza genome re-assorts with the genome of a zoonotic virus, usually an avian one. In fact, the 1918 pandemic virus is at least partly avian in origin.]

Bearing in mind that influenza viruses contain segmented genomes (Aside 4), and that re-assortment of genomic segments between different strains occurs in nature, several research groups, each working independently, sought to determine which of the genomic segments of the 1918 pandemic virus might be responsible for its extraordinary virulence. In brief, it was possible to experimentally substitute each of the genomic segments of a benign influenza strain with the corresponding genomic segment of the 1918 pandemic virus. [The individual influenza gene segments were reverse transcribed and then inserted into individual plasmids. Recombinant viruses were then generated by microinjecting different combinations of these plasmids into cells in culture.] These viruses were then screened for their virulence in mice.

The results of these experiments showed that several different genes from the 1918 virus contributed to its virulence. These included the viral genes that encode two of the envelope proteins; the HA protein described above and the neuraminidase (NA), which promotes virus release from cells. The viral polymerase also contributed to its virulence.

An early hypothesis to explain the virulence of the 1918 pandemic virus was based on the contention that the virus acquired and expressed the HA gene, and perhaps the NA gene as well, of an avian influenza strain. Consequently, there might have been little if any immunity in the human population against the pandemic virus. However, Taubenberger’s group found that laboratory-generated recombinant viruses, which contained both the HA and the NA proteins of the 1918 pandemic virus, induced higher levels of inflammation in the mouse model than were induced by more benign influenza viruses. That is, the laboratory-generated recombinant viruses were actually more immunogenic than benign influenza strains. While this finding might not have been predicted, it actually is consistent with the extreme lung pathology seen in humans during the pandemic. At any rate, more research still needs to be done to better understand the virulence of the 1918 virus.

Taubenberger’s group also found some important differences between the viruses in samples from individuals infected early in the 1918 pandemic, when the virus was relatively benign, and the viruses in individuals infected after the virus became vastly more virulent. In the earlier cases, the HA protein was more like that found in avian influenza strains, while later cases had an HA protein somewhat more like that found in human influenza strains. Presumably, the avian HA gene underwent changes that adapted the virus to disseminate and spread more easily in its human host.

[Aside 5: There are 16 known serologically distinct types of the influenza HA protein in nature; only three of which, H1, H2, and H3 are found in human influenza strains. There are nine known serologically distinct types of the NA protein, of which N1, N2, and N3 are most commonly found in human strains. The 1918 pandemic virus was an H1N1 strain. Pandemic viruses generally arise when a current seasonal human strain acquires a new HA gene from an avian influenza. Other genes also may be acquired from the avian virus in addition to the HA gene. Thus, the 1957 Asian flu was H2N2, and the 1968 Hong Kong flu was H3N2. See the following aside.]

[Aside 6: In April 2009, a novel H1N1 virus (see the above aside), which originated in swine, was found in humans in the United States, Mexico, Canada, and elsewhere. Although this virus turned out to be relatively benign, its emergence caused widespread panic, due in part to the non-stop updates of new cases in the media, which created the false impression that a killer pandemic was sweeping through the country.

In May, 2009, Vice President Joe Biden told a national TV audience that he would tell members of his own family not to go anywhere where they might be in a confined space, such as an airplane, subway or classroom. But, in fairness to Biden and the media, it was net yet clear that the virus was relatively mild.

Initially, the virus was referred to as the swine flu. But, Biden’s boss, President Barack Obama, in deference to the U.S. pork industry (people were afraid they might catch the virus by eating pork), began to deliberately call this virus “the H1N1 virus.” The new designation stuck. And while it does characterize the 2009 swine flu, it likewise characterizes the vastly more lethal 1918 pandemic virus, as well as a current seasonal influenza strain. Thus, the 2009 virus was hardly the H1N1 virus.

The world was of course fortunate that the 2009 H1N1 swine flu outbreak turned out to be relatively mild. Many millions of people might have been killed. Will the public remember the episode and, consequently, be complacent in the face of a future outbreak, doubting the credibility of government warnings?]

An earlier influenza outbreak, which indeed startled virologists, took place in 1997, when the first cross-species transmission of an avian H5N1 influenza to a human was documented. The patient, a child succumbed, and there were additional lethal human infections that followed. Indeed, the H5N1 virus killed about half of the individuals it infected; a fatality rate far greater than that of even the 1918 pandemic virus. Fortunately, during the past 17 years, the virus has not adapted to spread readily from person to person. Instead, the vast majority of the 600 humans, who were estimated to have been infected, acquired the virus by close contact with diseased birds.

Next, in September 2011, Yoshihiro Kawaoka at the University of Wisconsin and Ron Fouchier of Erasmus Medical Center in Rotterdam, shocked virologists when they announced that they and their colleagues had created variants of the H5N1 virus that could be transmitted between ferrets; often considered a good model for transmission in humans. What’s more, Fouchier’s group deliberately modified the virus so that it might be transmitted through the air; a very significant modification, since transmission of avian influenza viruses between their avian hosts is via the fecal-oral route, whereas mammalian influenza viruses are transmitted via the respiratory route.

Kawaoka’s group randomly mutated the HA gene of the H5N1 virus, until they found mutations that caused it to attach to human receptors, instead of to bird receptors. Then, they replaced the HA gene from the 2009 H1N1 “swine flu” strain (Aside 6) with the mutated H5 HA gene, thereby creating a virus that contained the mutated avian HA gene, and the remaining genes from the 2009 H1N1 virus. In contrast, Fouchier’s group examined the possibility that the H5N1 virus might acquire the ability to transmit via the respiratory route by mutation alone; without re-assortment. They began by giving the H5N1 virus three mutations previously identified in the HA genes of the 1918, 1957, and 1968 pandemic viruses.

Fouchier’s virus indeed was lethal in ferrets. In contrast, Kawaoka’s virus did not kill the animals, and was no more pathogenic in ferrets than the 2009 H1N1 swine virus. But, recombinant viruses that that arise in nature might have unpredictable and very different pathogenicities. And, bear in mind that both research groups in fact demonstrated that H5 avian viruses might acquire the ability to infect mammals.

Now, consider that the resurrected 1918 pandemic virus is essentially identical to the virus that claimed up to 50 million lives during the 1918 pandemic. Moreover, consider that up to now H5N1 viruses have not been able to readily transmit between humans. But, if either of the H5N1 viruses developed in Wisconsin and Rotterdam is indeed transmissible between humans, while retaining a measure of its virulence, it might be even more life-threatening than even the 1918 pandemic H1N1 virus.

In view of the above, one may well ask what reasons could possibly justify creating such potentially dangerous viruses. A common rationalization is that these experiments provide insights into the genetic changes that might happen in nature to generate deadly pandemic viruses. A potential benefit of that knowledge might then be to enable surveillance against the emergence of such viruses, thus providing a window of opportunity to develop strategies to cope with the threat and minimize its consequences.

But regardless of the possibly enormous benefits that might result from the types of experiments described above, one could easily imagine important arguments against doing these experiments. Clearly, resurrecting the 1918 pandemic virus brought an extremely deadly pathogen back to life. And, the experiments in Rotterdam and Wisconsin may likewise have given rise to very lethal viruses. Moreover, the accidental release of these viruses, even from the most secure facility, is not all far-fetched. In this regard, in 2003 and 2004 the SARS virus “escaped” from three different Asian laboratories. Furthermore, while these experiments might be done safely in a very few laboratories in the United States and Europe, there is no global mechanism to insure that they would be done safely elsewhere. What’s more, there is concern that terrorist groups might gain possession of these viruses, or perhaps even replicate the work that gave rise to them.

So what is the bottom line? The issue is not simply whether the research is dangerous. It clearly is. And, the issue is not simply whether the research holds the promise of real and important benefits. While some potential benefits may have been overstated, they yet may one day be considerable. Thus, the real question is whether the potential benefits of the research outweigh its here-and-now risks. Experts have taken opposite positions on this question, and a heated debate goes on.

Yet a new issue arose with regard to the H5N1 experiments; specifically, whether or not the work ought to be reported in scientific journals. This issue arose over concern that the transmissible H5N1 variants might fall into the hands of individuals or groups with evil intentions or, perhaps, even be made by them. Consequently, in December 2011, the U.S. National Science Advisory Board for Biosecurity (NSABB) made the unprecedented recommendation to censor the papers that reported the work of the Rotterdam and Wisconsin groups. The papers were, at the time, under review at Nature and Science. The NSABB worried that publication of “the methodological and other details could enable replication of the experiments by those who would seek to do harm.” Thus, the NSABB recommended that the general conclusions of the papers, but not their methodologies, might be published. Later, in February 2012, a World Health Organization committee recommended that the studies be published in full.

As might be expected, there is no consensus in the scientific community over this censorship issue. On the one hand, constraints on communication are inherently incompatible with free scientific inquiry and would hinder progress in a field that significantly impacts human health. Moreover, would scientists devote years to investigating dangerous viruses, only to have their work censored in the end? On the other hand, should not the scientific community bear at least some responsibility for keeping the fruits of its research from being misused by those who would do harm? Few scientists would prefer to have individuals who are not practicing scientists, and who don’t always understand the science, making these judgments in their place. [I find it interesting that these other individuals are often referred to as bioethics, biosecurity, or bioterror “experts,” and wonder what makes them so.]

References

Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ, Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. 2012. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336:1534-1541.

Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson A, Katsura H, Watanabe S, Li C, Kawakami E, Yamada S, Kiso M, Suzuki Y, Maher EA, Neumann G, Kawaoka Y. 2012. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486:420-428.

Taubenberger JK, Reid AH, Krafft, AE, Bijwaard, KR, and Fanning TG. 1997. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science 275:1793-1796.