In a previous posting, it was related how, in 1965, David Tyrrell, at the Common Cold Unit in Salisbury, England, grew a virus from a schoolboy with a cold (1). That virus, designated B814, would be the first known human coronavirus. With the aid of Swedish surgeon Bertil Hoorn, B814 was grown in human trachea organ cultures that were inoculated with throat swabs taken from the sick boy. The main point of the previous posting was that the detection of B814 depended on the exceptional electron microscopy skills of June Almeida. In addition, Almeida noted that the morphology of B814 resembled that of the previously isolated infectious bronchitis virus (IBV) of chickens. She then concluded that B814 and IBV are members of a previously unclassified family of viruses. Then, based on Almeida’s interpretation of B814’s morphology, she, Tyrrell, and Tony Waterson (a colleague of Tyrrell who recommended that Almeida be recruited to Tyrrell’s team), dubbed the new family “coronaviruses.”
Even though B814 is recognized as the first known human coronavirus, listings of coronaviruses that infect humans, as compiled in medical virology books and in the journal literature, generally include only seven coronaviruses, none of which has been shown to be identical to B814. These seven coronaviruses include four human coronaviruses that cause common colds (HCoVs 229E, NL63, OC43, and HKUI), and three animal coronaviruses that recently emerged in, and are highly virulent in humans (SARS, MERS, and SARS-CoV2). Where then does B814 fit in this compilation of coronaviruses? In search of an answer, here is a brief history of the discoveries of the human coronaviruses.
In 1966, at about the same time that Almeida was carrying out her electron microscopy analysis of B814, Dorothy Hamre and John Procknow, at the University of Chicago, recovered 5 viral isolates from medical students with colds (2). One of these isolates, 229E, was examined by Almeida, who found its morphology to be identical to that of B814. Then, in 1967, Robert Chanock and coworkers at the NIH used the organ culture technique to isolate yet other strains, including OC43, whose morphology likewise resembled that of B814 (3).
229E and OC43 were virtually the only HCoVs being studied during the next thirty years, mainly because they were the easiest of the HCoV strains to work with. As for B814, it could only be grown in organ culture (1). In contrast, clinical samples of 229E could be grown directly in cell culture. And while OC43 was originally grown in organ culture, it was subsequently adapted to growth in suckling mouse brain, and then to growth in cell culture.
Both 229E and OC43 are clinically significant, as each caused multiple epidemics in the United States, at intervals of 2 to 3 years. Moreover, reinfection with each of these strains was common (4); a point that may be relevant to the current COVID-19 pandemic. In any case, in 1970 it was reported that B814 is not serologically identical to either OC43 or 229E (5). Little else seems to be known about B814.
The winter of 2002-2003 saw the emergence of the SARS coronavirus; a virus far more lethal in humans than either 229E or OC43. By the end of the SARS outbreak, a total of 8,096 cases were reported, of which 744 were fatal, for a fatality rate of about 12%. Prior to the SARS outbreak, coronavirus infections were regarded as merely mild nuisances. But the SARS outbreak led to an awareness that animal coronaviruses could represent a significant health threat to humans.
But yet other relatively mild HCoVs remained to be discovered. In 2004, researchers at the Erasmus Medical Center in the Netherlands isolated NL63 from a 7-month-old girl with coryza, conjunctivitis, fever, and bronchiolitis (6). The same year, another group of investigators in the Netherlands isolated a coronavirus, designated NL, from an 8-month-old boy with pneumonia (7). And, in 2004, researchers in New Haven, Connecticut, used a PCR-based approach to isolate a virus, designated the New Haven coronavirus (HCoV-NH), from young children with respiratory disease, (8).
NL63, NL, and NH are so-called group I coronaviruses. They are closely related to each other and may very well be the same species. In any case, these viruses are believed to be a significant cause of respiratory tract disease in infants and children. And, like SARS-CoV2, HCoV-NH has been associated with Kowasaki disease in children (9).
In 2005, researchers at the University of Hong Kong used RT-PCR to recover a novel human coronavirus from a sample taken earlier from a 71-year old man who presented in Hong Kong with a fever and a cold (10). HKU1 is a so-called group II coronavirus. As such, it is distinct from OC43, the only other known group II human coronavirus. [229E is a group I coronavirus. Sequence analysis shows SARS to be sufficiently different from any of the known human and animal coronaviruses for it to occupy a separate group.] It is not known whether B814, or any of the other uncharacterized HCoV strains from the 1960s (i.e., those strains that were grown only in organ culture) are similar or identical to the more recently isolated HCoVs.
MERS-CoV is the second deadly coronavirus to emerge in humans. It was first isolated from a patient in Saudi Arabia in 2012. It caused fewer than 2,500 confirmed cases worldwide. Yet it killed an astounding 35% of people with confirmed diagnoses. At present, we are in what is still the early phase of the SARS-CoV-2 (COVID-19) pandemic. SARS-CoV-2 is clearly far more transmissible than either SARS or MERS. Its fatality rate is not known but estimates are at about 1%. In any case, the above compendium hopefully includes all the coronavirus strains known to infect humans.
References:
Norkin, L.C. June Almeida and the Discovery of the First Human Coronavirus, Posted on the blog June 2, 2020.
Hamre, D., and J.J. Procknow, 1966. A new virus isolated from the human respiratory tract. Soc. Exp. Biol. Med. 121:190–193.
McIntosh, K., J.H. Dees, W.B. Becker, A.Z. Kapikian, and R.M. Chanock, 1967. Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease. Natl. Acad. Sci. USA. 57:933–940.
Callow, K.A., H.F. Parry, M. Sergeant, and D.A. Tyrrell, 1990. The time course of the immune response to experimental coronavirus infection of man. Infect. 105:435–446.
Bradburne, A.F., 1970. Antigenic relationships amongst coronaviruses. Gesamte Virusforsch. 31:352–364.
van der Hoek, L., K. Pyrc, M.F, Jebbink, et al., 2004, Identification of a new human coronavirus. Med. 10:368–373.
Fouchier, R.A., N.G. Hartwig, T.M. Bestebroer, et al., 2004. A previously undescribed coronavirus associated with respiratory disease in humans. Natl. Acad. Sci. USA. 101:6212–6216.
Esper, F., R.A. Martinello, R.P. Boucher, et al., 2005. Evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children. Infect. Dis. 191:492–498.
Esper, F., E.D. Shapiro, C. Weibel, D. Ferguson, M.L. Landry, and J.S. Kahn, 2005. Association between a novel human coronavirus and Kawasaki disease. Infect. Dis. 191:499–502.
Woo, P.C., S.K. Lau, C.M. Chu, et al., 2005. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. Virol. 79:884–895.
At this still early phase of the COVID-19 crisis, it is not clear who, if any, individual might emerge as a hero comparable in stature to say Carlo Urbani during the SARS outbreak, or Peter Piot during the Ebola and HIV outbreaks. Nonetheless, June Almeida has been noted in the media recently for her critical role in the discovery of the first human coronavirus. Almeida made other significant contributions to virology. Yet, for a major part of her career, her only formal scientific training was as a laboratory technician.
David Tyrrell recounts the discovery of the first human coronavirus in his book “Cold Wars: The Fight Against the Common Cold” (Oxford University Press, 2002). In 1966, Tyrrell and co-researchers at the Common Cold Unit in Salisbury, England, were taking nasal swabs of people experiencing common colds. They then inoculated human volunteers with the swabs. Next, samples that caused colds in the volunteers were inoculated into cell cultures, so that possible viral agents might be grown for further analysis. But Tyrrell ran up against a serious impediment. Some samples, which caused colds in the volunteers, did not appear to grow in cell cultures. Consequently, those samples could not be studied further. Nothing more could be ascertained regarding their taxonomy, mode of replication, or ability to induce and respond to antibodies. Nor might they be grown to produce a possible vaccine. Consequently, Tyrrell and coworkers sought a more reliable means for growing their isolates in the lab.
“We decided to look at things from the virus point of view…we were interested in viruses that normally grow in the cells of the nose. Perhaps some of them were quite unable to grow anywhere else.” But while “the cells of the human nose are by far the best environment in which to grow the cold virus, …from the researcher’s point of view, they have the disadvantage of being attached to the rest of the human body.”
Tyrrell learned of a possible solution to his dilemma from Sir Christopher Andrewes, a colleague who had been to Sweden to receive an honorary doctorate. While in Sweden, Andrewes met Bertil Hoorn, a surgeon at the University of Lund, who discovered that he could make organ cultures from human trachea tissue, that would support the growth of influenza virus. Hoorn established these cultures from small squares of airway-lining tissue that attached to specially treated surfaces of plastic petri dishes. The cultures were then maintained under liquid medium. They retained characteristics of the actual airway tissue in situ, such as beating cilia. If virus grew in these cultures, it would cause the cilia to cease beating. Moreover, progeny viruses could be recovered from the media.
Influenza viruses are a family of viruses that are distinct from the rhinoviruses already known to cause common colds. Nonetheless, Tyrrell recognized the potential of Hoorn’s discovery for his own work. Consequently, he invited Hoorn to come to Salisbury, to test whether his respiratory tract organ cultures might support the growth of cold viruses. Hoorn accepted Tyrrell’s invitation. He came by ferry from Sweden and arrived at the driveway of the CCU in his two-horsepower Citroën, crammed in the back with his glassware, fluids, and surgical instruments. At the CCU, Hoorn duplicated his influenza findings. Then, without exception, he was able to grow all of the cold virus samples that Tyrrell had at hand.
But, while Hoorn’s work was underway in Salisbury, Tyrrell’s team acquired new virus samples from a group of boarding-school boys with colds. Tyrrell was taken aback when one of these samples, dubbed B814, caused no apparent change in Hoorn’s organ cultures. Yet this was the case even though B814 appeared to replicate in the organ cultures, as shown by the fact that fluids from the inoculated cultures caused colds in human volunteers.
Tyrrell grew his first rhinovirus in cell culture in 1946. Yet B814 was, thus far, his only sample that did not cause cell damage (cytopathic effects) in Hoorn’s organ cultures. In the undifferentiated cell cultures that Tyrrell used earlier, rhinoviruses would cause the cells to darken, round up, and detach from the surface of the culture dish. In Hoorn’s organ cultures, rhinoviruses generally caused the cilia to stop beating. So, might B814 not be a rhinovirus?
There was another clue that B814 might not be a rhinovirus. Since rhinoviruses are not enclosed within lipid envelopes, they are not inactivated by fat solvents. But B814 was inactivated by fat solvents, suggesting that it has a lipid envelope and, consequently, is not a rhinovirus. [If B814 were in fact enveloped, that might explain why it caused no apparent damage in the Hoorn’s organ cultures. Enveloped viruses generally acquire their envelopes during their release from the cell. This occurs via a process called “budding,” which usually leaves behind an intact cell. In contrast, release of non-enveloped viruses is generally facilitated by cell damage.] But, without an image of B814, Tyrrell had no means to verify whether B814 is enveloped, and whether it is something other than a rhinovirus. Moreover, Tyrrell had no way of detecting other B814-like isolates, except by infecting volunteers.
Again, a colleague suggested a solution to a Tyrrell dilemna. Tony Waterson, a virologist at St Thomas’ Hospital in London (the same hospital that treated British Prime Minister Boris Johnson when he was suffering from COVID-19) had just hired an electron microscopist, June Almeida, who he claimed was extending the range of electron microscopy to new limits. In Tyrrell’s words: “It was generally believed at the time that you had to have concentrated and purified viruses to detect them with such an instrument. But she claimed that she would be able to find the virus particles in our organ cultures with her new, improved techniques. We were not too hopeful but felt it was worth a try.”
“Samples were prepared, each one infected with a well-known virus such as influenza or herpes, and included amongst them was the B814 virus. The cultures were put into bottles with coded labels and sent by train to London. June Almeida duly dabbed the surface of the tissues onto her microscope grids and examined each one. The results exceeded all our hopes. She recognized all our viruses, and her pictures revealed their structure beautifully. But, more importantly, she also saw virus particles in the B814 specimens!”
But what exactly was B814? As expected, it was not a rhinovirus. Rather, with its envelope and spikes, it looked more like influenza virus. Indeed, that was what most researchers believed it to be. But, based on the unique short spikey projections that Almeida detected on B814, she was quite certain that it represented a previously unknown family of viruses.
As Almeida was certain that she had identified a previously unknown type of virus, she, Tyrrell, and Waterson met to decide the new family’s name. In Tyrrell’s words: “So, what should we call them? ‘Influenza-like’ seemed a bit feeble, somewhat vague, and probably misleading. We looked more closely at the appearance of the new viruses and noticed that they had a kind of halo surrounding them. Recourse to a dictionary produced the Latin equivalent, corona, and so the name coronavirus was born.” Thus, Almeida revealed the first known human coronavirus, and was part of the trio that gave the virus family its name.
In 1965, Tyrell published a report on B814 in the British Medical Journal, referring to it as “a novel cold virus.” But, when Almeida submitted her paper on the structure of B814 to a peer-reviewed journal, it was rejected because the referees believed that her images of the new virus were just bad pictures of influenza virus. Almeida was finally able to publish her photographs of B814 in 1967, in the Journal of General Virology.
Before the discoveries of SARS and MERS, and now of COVID-19, virologists associated coronaviruses with clinically mild common colds. Consequently, coronaviruses were viewed as not particularly significant. In Tyrrell’s words: “Perhaps one of the common cold’s main problems is that it is not sufficiently virulent. ‘Familiarity breeds contempt’ is a proverb which could have been devised expressly to describe our attitude towards it. If it was more frequently life-threatening, like smallpox, polio, yellow fever, and AIDS, then perhaps research into its effects and a possible cure might have begun much earlier, been more widespread, and been maintained over a longer period.”
In any case, such sentiments help explain why Almeida moved on to investigating other viruses, rather than continuing with B814. In 1967, she produced the first electron micrographs of the rubella virus. And in 1970, she generated the first electron micrographs of hepatitis B virus (HBV), which she detected in the blood of a hemophiliac patient. That was an achievement of singular medical importance, since it was not yet clear whether the “Australia antigen” originally discovered in the blood of leukemia patients by Baruch Blumberg, and later in the blood of hepatitis B patients, might be an actual virus particle or a non-viral particle related to it, or, indeed the actual agent of hepatitis B. [See Note 1.] In fact, many scientists were skeptical of any relationship between the antigen and hepatitis B disease. But Almeida’s electron micrographs of a blood sample of a hemophiliac patient (Note 1), who tested positive for the Australia antigen, were instrumental in convincing the scientific community that the antigen is a component of the virus. Moreover, she convinced skeptics that HBV is the agent of hepatitis B. Furthermore, Almeida’s studies of individual differences in the immune response to HBV provided key insights into the variety of patient outcomes to the infection. And, Almeida provided the first high quality micrographs of HIV, the agent responsible for AIDS. These, and yet other important contributions, are testimony to Almeida’s outstanding scientific career. Yet, if not for the current COVID-19 crisis, she would not have come to the attention of the general public.
[Note 1: Leukemia and hemophiliac patients were more likely than the general population to have received multiple blood transfusions. Moreover, prior to 1972, 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. Thus, leukemia and hemophiliac patients were more likely than most to have been recipients of donated blood, some of which was contaminated with HBV. By 1972, all donated blood in the United States had to be screened for HBV. See: Baruch Blumberg: The Hepatitis B Virus and Vaccine, posted on the blog June 2, 2016.]
June Almeida (nee June Hart) was born in 1930, in Glasgow, Scotland. Although she excelled in science in secondary school, her family lacked the means to send her to university. Consequently, June trained to be a laboratory technician in the department of histopathology at the Glasgow Royal infirmary. She then had a similar position at St. Bartholomew’s Hospital in London.
In 1954 June married Venezuelan artist, Henry Almeida. Two years later, June and Henry immigrated to Canada. The move was a watershed event in June’s life, since it gave her the opportunity to take a technician position at the newly opened Ontario Cancer Institute (OCI), under immunologist and expert electron microscopist, Allan F. Howatson, who set June was on the path to becoming an expert and innovative electron microscopist in her own right.
The move to Canada was fortunate for June for yet another reason. Not having a university degree was less of an impediment to her in Canada than it would have been had she remained in the United Kingdom, where there was more emphasis on formal academic degrees. So, in Canada, with no university degree, June Almeida rose to the position of junior scientist at the OCI. Moreover, she was encouraged to pursue her own independent research. What’s more, her expertise at electron microscopy is credited with helping to establish the reputation of the OCI in the new fields of tumor viruses and structural virology. Indeed, Almeida was a key player in the OCI group that characterized a new tumor virus, recently discovered by Ludwig Gross, as the prototype virus of the polyomavirus family of small DNA tumor viruses.
Almeida moved back to London in 1964. It happened after she met Tony Waterson when he happened to be visiting Toronto. Waterson had just been appointed chair of microbiology at London’s St Thomas‘s Hospital Medical School, one of the most prestigious medical schools in the United Kingdom. In any case, Almeida’s micrographs so impressed Waterson, that he used the occasion of being in Toronto to recruit her to his research team in London. That is how Almeida had been with Waterson when he recommended her to Tyrrell, so that she might help him sort out B814.
Henry Almeida soon regretted the couple’s mutual decision to leave Canada. He wanted to return to Canada, but June was unwilling to move back. So, the couple divorced in 1967. June then raised their daughter Joyce, born in 1960, as a single parent, while continuing her demanding work with Waterson.
Photograph of June Almeida in the 1960s using a Philips EM300 electron microscope. Credit: Joyce Almeida, in L. Marks, 2020.
After spending three years at St Thomas’ Hospital, Almeida moved, with Waterson, to the Hammersmith Postgraduate Medical School, where she was appointed a research fellow in the Department of Virology. At Hammersmith, she carried out her important investigations of HBV. In 1970, she was promoted at Hammersmith to the position of Senior Lecturer. Then, in 1972, she was recruited to the Wellcome Research Laboratories in Beckenham, Kent, where she worked on vaccines and diagnostics for different viruses, including HBV. Moreover, her pioneering contributions to the development of immune-electron microcopy significantly advanced diagnostic virology in general. And, her techniques for resolving viral morphologies were especially important for classifying newly detected viruses. Almeida remained at the Wellcome Laboratories until her retirement in 1984.
Lara Marks (in What is Biotechnology?) states that Almeida authored or co-authored 103 peer-reviewed publications during her career—a notable record by any standards. Nonetheless, Almeida had always been modest about her accomplishments, claiming that she was just fortunate to be in the right place at the right time. Actually, it was her nearly unique proficiency at electron microscopy that drew others to her with their projects.
Also, bear in mind that for a major part of Almeida’s career, her only academic qualification was her training, while a teenager, to be a laboratory technician. In 1971 she was awarded a D.Sc. from The University of London, based on the submission of her highly regarded scientific publications.
In 1982 Almeida was remarried to Phillip Gardner, a clinical virologist who had a hand in developing immunofluorescence-electron microscopy—an important technique for basic research, as well as for the rapid diagnosis of many viral infections, especially of the respiratory tract. The couple met at a conference organized by the European Group for Rapid Virus Diagnosis, which Gardner had founded. Gardner had earlier suffered a serious heart attack that would force him to retire in 1984. June joined him in retirement the same year, and the couple moved to the seaside town of Bexhill. But even in retirement, June helped colleagues at St Thomas’ Hospital to generate some of the first high-quality micrographs of HIV. June Almeida died in Bexhill, England, on Dec. 1, 2007. She was 77.
References:
David Tyrrell and Michael Fielder, Cold Wars: The Fight Against the Common Cold, Oxford University Press, 2002.
Tyrrell D. A. J., and M. L. Bynoe. 1965. Cultivation of a novel type of common-cold virus in organ cultures. British Medical Journal1:1467-1470.
Almeida, J. D., and D. A. J. Tyrrell. 1967. The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture. Journal of General Virology1:175-178.
Dr. Arno G. Motulsky, recognized for having pioneered the field of “pharmacogenetics”—the genetic basis for differences in the way individuals respond to drugs—passed away on January 17, 2018 in Seattle, at the age of 94. Since Motulsky was not a virologist, his passing is noted here not for his scientific contributions but, rather, for his improbable achievements against very long odds and, especially, for the remarkable journey from which this story began—a journey which brings to mind a current issue before the public. It began as follows.
Dr. Arno Motulsky in his Seattle apartment in 2014. Credit Clare McLean/University of Washington
1938 saw a dramatic increase in violent anti-Jewish acts by the Nazi regime in Germany. In May 1939, 916 passengers, including 910 German Jews seeking to escape from the Nazis, were aboard the SS St. Louis; a liner of the Hamburg-America Line, which was bound for Cuba. Hundreds of thousands of other German Jews would not be able to get out.
Each of the passengers on the St. Louis had a visa from the Cuban government that was issued before the ship departed from Germany. However, unbeknownst to the passengers—but known to the shipping line—the President of Cuba, Federico Laredo, had invalidated the visas the day before the ship sailed. Thus, when the St. Louis arrived in Havana Harbor, the Cuban government would not allow the passengers to disembark. Instead, the Cuban authorities attempted to exploit the refugees’ situation in order to extort bribes from them. These bribes began at $500 a person and rose to $1,000,000! All the while the ship sat within yards of the shore.
Cuba had been practicing this sort of extortion before the incident involving the St. Louis. Manuel Benitez Gonzalez, the Director-General of the Cuban immigration office, routinely sold landing documents for $150 or more and, according to U.S. estimates, had amassed a personal fortune of $500,000 to $1,000,000. Eventually, Benitez’s corruption would lead to his resignation. However, corruption was not the only factor working against the refugees. Many Cubans saw them as competitors for scarce jobs. Moreover, Cuban sympathy for the refugees’ plight was also tempered by xenophobia and antisemitism. [News reports of the impending sailing of the St. Louis triggered anti-Semitic demonstrations in Cuba even before the ship left Germany.]
As the St. Louis sat at anchor in Havana Harbor, the American public raptly followed daily media accounts of the refugees’ plight. And despite urgent appeals from American sympathizers (non-Jewish as well as Jewish), the U.S. State Department and the White House decided not to intervene on behalf of the refugees. President Franklin Roosevelt did not answer telegrams from the refugees on the ship. The State Department did respond, wiring back to the passengers that they must “await their turns on the waiting list and qualify for and obtain immigration visas before they may be admissible into the United States.”
Roosevelt had the power to issue an executive order that would have allowed the St. Louis’ refugees to enter the United States. He had, in fact, permitted 15,000 German and Austrian Jews, who were in the United States on visitor’s permits, to stay, saying: “It would be a cruel and inhuman thing to compel them to leave here” for Germany.
Why then did Roosevelt and the State Department abandon the refugees on the St. Louis? American popular opinion, which was opposed to accepting more new arrivals, was a key factor. Indeed, about 80% of Americans were against easing immigration restrictions. A key reason was that the depression left many Americans out-of-work. Consequently, many were fearful of foreigners becoming competitors for scarce American jobs. Antisemitism, isolationism (in the years after World War I, many Americans were staunchly against the U.S. being drawn into European affairs), and xenophobia were no doubt factors as well.
The National Origins Act of 1924 permitted only a fraction of would-be emigres from Germany to enter the United States. The quotas that were set on countries were intended to favor “desirable” immigrants from Northern and Western Europe, while limiting less “racially desirable” immigrants, such as Southern Europeans, Eastern European Jews, and people born in Asia and Africa. Yet anti-German sentiment may have led American immigration officials to allow only one fourth of the German quota to be filled. Also note that three months before the St. Louis sailed to Cuba, the U.S. Congress let die a bill that would have admitted 20,000 Jewish children from Germany above the existing quota.
Critics contend that despite the political pressure on Roosevelt, he might have done more to raise public sympathy for the plight of the refugees. Moreover, once the United States was in the war, and the U.S. government became aware of Hitler’s intent to annihilate all Jews in Germany and in German controlled areas, the government tried to withhold that information from the public. And as the facts about the murder of the European Jews—concentration and extermination camps—leaked out, and despite pleas from Jewish leaders in Warsaw and elsewhere in Europe, Roosevelt would not act to impede the extermination program (e.g., by ordering the bombing of Auschwitz and the rail lines to it). Instead, Roosevelt maintained that winning the war was the only way to save the refugees, and winning the war was the absolute priority.
Barriers to Jewish immigrants were also in place across Latin America. [Note that the British refused to consider Palestine as a solution to the Jewish refugee problem.] Thus, Columbia, Chile, Paraguay, and Argentina rejected appeals from the Joint Distribution Committee (JDC, the major Jewish agency for relief overseas) for haven. And, when the JDC could not meet a deadline set by Cuba for the payment of ransoms, the St. Louis was forced to leave Havana Harbor. Remarkably, the German captain of the St. Louis, Gustav Schröder (who had been unaware of the Cuban government’s duplicity before the St. Louis sailed), appealed to the United States for haven. But his personal pleas were unheeded. What’s more, as the St. Louis sailed past Miami and the Florida coast, U.S. Coast Guard ships made certain that none of its passengers might escape to freedom. So, the St. Louis turned back to Europe, with sixteen-year-old Arno Motulsky among its passengers.
The German liner St. Louis being denied entrance to Havana, Cuba, in June 1939. Dr. Motulsky was aboard the ship with his mother and siblings when it was sent back to Europe. Credit Associated Press
But the St. Louis did not return to Germany. The JDC had arranged with Great Britain, France, Belgium, and the Netherlands for each of these countries to take one fourth of the refugees. Still, only the 288 refugees admitted to Great Britain would be safe, since the Nazis would overrun the other countries within months. Only a few would survive the Holocaust.
Motulsky was among the refugees who disembarked from the St. Louis in Belgium. He then spent a year in internment camps in Vichy France, where many other detainees died from starvation or typhoid. He reached the United States in June of 1941; ten days before his 18th birthday. His account of his flight to freedom, in his own words, is as follows:
“…By early 1939, my parents realized we would need to leave Germany. My dad had a brother in Chicago, so we hoped to go there, but in order to get out of Germany and into America, one needed a visa, which required a quota number that took a while to come up. So my father went to Cuba, where immigration permits were available, with the idea that we would join him later in Chicago. But over the next few months, as the situation deteriorated in Germany, my parents decided it would be best for my mother and brother and sister and me to join my father in Cuba and go to Chicago later.
We left Hamburg in May 1939, on a ship named the St. Louis. There were almost a thousand Jewish refugees on board. The voyage from Hamburg to Havana was normal. But before we could disembark in Havana, the Cubans declared our permits void and refused to let us land. The ship was German, but the captain, Gustav Schröder, was very sympathetic to us and sailed all over the Caribbean in search of a friendly port. Of course, we asked to land in America, but were denied. When we sailed close to Miami, US Coast Guard cutters and planes shooed us off.
So ultimately the ship had to return to Europe. I was 16, so still a little naive, but many of the passengers had been interned previously and knew what awaited us on our return. Some attempted suicide by jumping overboard. Telegrams were sent all over the world asking that we be allowed to disembark anyplace other than Germany. Miraculously, a few days before we would have arrived back in Germany, four other countries—England, France, Holland, and Belgium—each agreed to take one-fourth of the passengers. By luck of the draw, my mother and brother and sister and I were assigned to Belgium. So in June 1939, I started high school in Brussels.
Eleven months later, on May 1, 1940, my dad got his visa to move from Cuba to the US and went to Chicago. On the same day, our US visas came through as well. But by May 10, we hadn’t yet gotten out of Belgium, and the Germans invaded. Leaving was impossible. Since I was now 17, I was arrested by the Belgians as an enemy alien—ironically, as a German—and sent to an internment camp in France. As the Germans invaded France, we prisoners were moved further and further south, until we ultimately were interned in a camp in the Pyrenees, in Vichy-controlled France. The Vichy French, who collaborated with the Germans, sent the ethnic Germans back to Germany and kept the Jews interned. The internment camps had no food or sanitation. Many prisoners died, most from typhoid or starvation.
About ten months later, those of us with US visas were allowed to go to another camp, near Marseilles, where there was an American consulate. My visa had expired, but I pleaded for its renewal. It was ultimately granted, and in June 1941, ten days before my 18th birthday, I left France legally, crossed Spain into Portugal, and sailed from Lisbon to America. Ten days later and I wouldn’t have made it, because Franco did not allow males 18 or older to pass through Spain. A few months later, the Vichy French turned over all their internment camps to the Gestapo.
In August 1941, I arrived in America and joined my father in Chicago. Two years later, we learned that my mother and brother and sister had also survived. With the help of Belgian friends, they had crossed illegally into neutral Switzerland, where they were allowed to live unharmed until the end of the war. My family reunited in Chicago in early 1946.”
In Chicago, in 1942, 18-year-old Motulsky was examined on material he learned in the informal classes taught in the internment camps in France, and was then granted a high school certificate. Motulsky then enrolled in evening classes at Central YMCA College (which later became Roosevelt University) in Chicago, while working at a job during the day. He met his future wife, Gretel, in his English class at the college. He notes that Gretel was the better English student because she survived the war in England and America after leaving Germany in 1938. They were married in 1945.
Remarkably, in 1943, Motulsky was accepted by the medical school of the University of Illinois in Chicago. He also became a U.S. citizen, joined the Army, and was assigned to a specialized program at Yale for rapid training of young physicians. During his year at Yale, Motulsky completed the premedical courses that he still was lacking. More importantly, he also “took genetics with Donald Poulson and was hooked forever.”
In 1944 Motulsky served briefly as an orderly at an Army hospital near Boston, before returning to medical school at the University of Illinois, as a private first class. He was released from the Army in 1946, finished medical school in 1947, and began his residency in internal medicine at Chicago’s Michael Reese Hospital; supported by a fellowship in hematology. Under his fellowship mentor, biochemist and hematologist, Karl Singer, Motulsky began his foray into research, investigating hereditary hemoglobin disorders.
In 1951, the Korean War broke out and Motulsky was again in the Army; this time at a new hematology research unit at Walter Reed Hospital in Washington, DC. “At Walter Reed, we studied hemolytic anemias that might explain problems encountered by our soldiers… Singer had encouraged me to think about biochemical mechanisms, and here were genetic diseases, for which mechanisms could be studied to the direct benefit of patients.”
In 1953 Motulsky joined the faculty at to the University of Washington in Seattle, where he taught internal medicine and specialized courses in hematology. That same year, Watson and Crick determined the structure of DNA, rousing Motulsky to slip what he called “bootleg medical genetics” into his hematology lectures. In 1957 he founded the Division of Medical Genetics at UW; one of the first such divisions in the United States. [The Division of Medical Genetics at Johns Hopkins opened the same year.] But, before that, to broaden the scope of his knowledge, Motulsky spent eight months in the human genetics unit of Lionel Penrose at University College London. “Penrose’s department was the best in human genetics in Europe, including, in addition to Penrose himself, J.B.S. Haldane… People were very critical and helped me recognize excellent work.” Concurrently, Motulsky became interested in what is now called pharmacogenetics—”that is, differential reaction to drugs among people based on their genotypes.”
This blog post was meant to recount Motulsky’s remarkable personal journey during the early years of his life and career. Nonetheless, Motulsky’s considerable scientific contributions also need to be noted, if only briefly—in particular, his analyses of the genetic predisposition to a broad assortment of conditions, including heart disease, blood disorders, colorblindness, infections and immunity, hypertension, and alcoholism (for a concise account of his achievements, see his short autobiography referenced below). It is a good story. For now, note the comments of Francis Collins—a geneticist, and the director of the National Institutes of Health—which Collins made after Motulsky’s passing. “The relationship between heredity and the response to drug therapy — nobody was thinking about that until he started, 60 years ago. He anticipated it decades before science made it possible to get the answers that he dreamed of.” Moreover, “There were very few medical centers that considered genetics as being all that relevant to human medicine. It was more a study of fruit flies and mice, not humans.” Also note the comment of Claire King, Motulsky’s colleague at the University of Washington (and reporter of his autobiography), who discovered the role of certain genetic mutations in breast cancer. Because of Motulsky’s work in medical genetics, “the field is now integrated into every other field of medical practice and has become the soul of precision medicine.”
Motulsky ends his autobiography as follows: “Throughout my career, I have very much enjoyed the practice of medicine. Until I was 70 years old, I was an attending physician in general internal medicine. The human contact, to be able to help people, is enormously rewarding. And I always learned something. I was stimulated to ask new questions by seeing things that I hadn’t previously thought about. And with respect to medical genetics in particular, I know of no other field in science and medicine that is as fascinating. Medical genetics is closer to science than most specialties in medicine. It reveals exciting new biological phenomena. It has social implications and historical implications and ethical implications. I cannot imagine having done anything more exciting for the past 70 years.”
References
Dallek, R. Franklin Roosevelt and American Foreign Policy, 1932-1945. Oxford University Press. 1979.
Berenbaum, M. The World Must Know: The History of the Holocaust as told in the United States National Holocaust Memorial Museum. Little, Brown and Company, 1993.
Gradyjan, D. Arno Motulsky, a Founder of Medical Genetics, Dies at 94. Obituary, N.Y. Times, January 29, 2018
Motulsky, A. G. (as told to Mary-Claire King). 2016. The Great Adventure of an American Human Geneticist. Annual Review of Genomics and Human Genetics 17:1-15. https://doi.org/10.1146/annurev-genom-083115-022528
Addendum:
Several posts on the blog feature individuals whose lives were impacted in major ways by World War II and the Holocaust. In particular:
Günter Blobel, recipient of the 1999 Nobel Prize in Physiology or Medicine, died on February 18, 2018. Blobel is best known for his groundbreaking studies on an issue of fundamental importance to cell biology and virology—how newly synthesized proteins reach their correct location within or outside the cell.
Gunter Blobel (4)
In Blobel’s words: “Concurrently with or shortly after their synthesis on ribosomes, numerous specific proteins are unidirectionally translocated across or asymmetrically integrated into distinct cellular membranes. (1).” How this might be realized, now known as the “signal hypothesis,” was first put forward in 1971 by Blobel and colleague David Sabatini. The central feature of the hypothesis is “the occurrence of a unique sequence of codons, located immediately to the right of the initiation codon, which is present only in those mRNAs whose translation products are to be transported across (my note: or inserted into) a membrane (2).”
Blobel and postdoc Bernhard Dobberstein (one of the impressive cell biologists trained by Blobel) confirmed the hypothesis in a classic experiment based on an earlier observation by Cesar Milstein. Specifically, Milstein’s group found that proteins, produced by translation of immunoglobin light chain mRNA, in a cell-free translation system, contained about 20 amino acids at their N-terminal end that are not present on immunoglobin light chains that are secreted from cells. Blobel and Dobberstein confirmed that cell-free translation (on free ribosomes) of immunoglobin light chain mRNA indeed yields the larger form of the protein. However, when membranes were added to the system, the light chains that were synthesized were the same size as normally secreted light chains. What’s more, the ribosomes synthesizing the light chains were bound to the membranes, and the new light chains were resistant to digestion by added proteases, indicating that they had been secreted into microsomes (2).
The overall scheme is that those nascent proteins, which are destined for secretion, or insertion into membranes, contain a short signal sequence at their N-terminus, which causes their ribosome to attach to a membrane. The signal sequence is removed by a signal peptidase as the growing polypeptide passes through a channel in the membrane. Proteins destined to be transmembrane proteins also contain a hydrophobic stop-transfer sequence, which anchors them in the membrane. Note that important details of this fundamental cellular pathway were revealed by Blobel’s analysis of the strategy by which sindbis virus generates its envelope glycoproteins (3). [For a detailed review of this key step in the replication cycles of enveloped viruses, see Chapters 7 and 8 of L. Norkin, Virology: Molecular Biology and Pathogenesis, ASM Press, 2010.]
Gunter Blobel was born in May 1936, in the Silesian village of Waltersdorf, then in eastern Germany, and later a part of Poland. He is another of the scientists featured on the blog whose life was impacted by events of the Second World War. Others include Max Delbruck, Francois Jacob, Jacques Monod, Andre Lwoff, the Wollmans (Eugene, Elizabeth, and Elie), Renato Dulbecco, Harald zur Hausen, and George Klein.
In Blobel’s words (4): “1945 was also a turning point in my life. Until then my childhood was a perfect 19th century idyll. In the cold and snow-rich Silesian winters there were hour-long rides on Sundays in horse-drawn sleighs to my maternal grandparent’s farm to have lunch and to spend the afternoon. The house was a magnificent 18th century manor house in the nearby Altgabel with a great hall that was decorated with hunting trophies. In the summer, of course, horse-drawn landauers were used as means of transportation. The way to school was a long one. We went there on foot and as a pack, usually consisting of one or two of my seven brothers and sisters and of children from neighboring houses.
At the end of January 1945, we had to flee from the advancing Russian Red Army. My father, a veterinarian stayed behind for a few more days and left only hours before the Red Army moved in. My fourteen-year-old brother, Reiner, drove my mother, my youngest brother, an older brother, the two younger sisters and me in a small automobile to relatives west of Dresden in Saxony. On the way there we drove through Dresden. We entered the city from the eastern hills. Its many spires and the magnificent cupola of the Frauenkirche (die Steinerne Glocke, the Stone Bell) were a magnificent sight even for the untrained eye of a child. Driving through Dresden, I still remember the many palaces, happily decorated with cherubs and other symbols of the baroque era. The city made an indelible impression on me. Only a few days, later, on February 13, 1945, we saw from a distance of about 30 kilometers a fire-lit, red night sky reflecting the raging firestorm that destroyed this great jewel of a city in one of the most catastrophic bombing attacks of World War II. It was a very sad and unforgettable day for me.
The months before and after the end of World War II were chaotic and miserable. None of my relatives had enough space to accommodate our large family leaving us divided among several relatives in different villages. There was no communication and little food. On September 9, 1945, we learned of the death of my beautiful oldest sister Ruth who, at age 19, was killed in an air raid on a train she was travelling in on April 10, 1945. She was buried in a mass grave near the site of the attack in Schwandorf, Bavaria. Ruth was born when my mother was just 20. The two had a sisterly relationship. My mother grieved over Ruth’s death until the end of her own life.
Fortunately, things took a turn for the better, when my father was able to continue his veterinarian practice in the charming medieval Saxon town of Freiberg. Most members of our family were reunited there by 1947. We lived in a nice villa surrounded by a large garden on the edge of town. My way to school was along the old medieval city wall. For only 40,000 inhabitants, Freiberg had a rich cultural life with a 175-year-old theater. Most impressive were the musical performances in the magnificent gothic cathedral, the Dom, with the splendid great Silbermann organ. Each week Bach cantatas were performed. The great choral works of Bach, Mozart and Haydn were regularly performed and at the highest artistic level at the major religious holidays. I even participated in singing in the cantus firmus of Bach’s Matthäus Passion. So, it was almost like a 19th century idyll again, this time in a small medieval town instead of a country village.
However, there was now the ever more oppressive regime of East Germany to deal with on a daily basis. When I graduated from high school in 1954 I was not allowed to continue my education at a university because I was considered a member of the “capitalist” classes. [My note:, Gunter was labeled “a member of the capitalist classes,” and barred from universities, for refusing to join a Communist youth group.] Fortunately, at that time, i.e., before the Berlin Wall, it was possible to escape and to travel freely to West Germany. So, on August 28, Goethe’s birthday, I left Freiberg for Frankfurt on the Main in West Germany. The train left in the morning and in the afternoon, it passed Weimar, where Goethe spent most of his life, and then Eisenach, where Bach was born and in the evening it arrived in Frankfurt, Goethe’s birthplace.”
Several paragraphs later:
“In 1994, I founded Friends of Dresden, Inc., a charitable organization, with the goal to raise funds in the U.S. to help rebuild the Frauenkirche in Dresden. The rebuilding of many of the historic monuments of Dresden is one of the most exciting consequences of German reunification and the liberation from communism. It is a childhood dream come true.
It was one of the great pleasures of my life to donate the entire sum of the Nobel Prize [my note: $960,000], in memory of my sister Ruth Blobel, to the restoration of Dresden, to the rebuilding of the Frauenkirche and the building of a new synagogue. This donation also serves to express my gratitude to my fellow Saxons. They received us with open arms when we had to flee Silesia. I spent a wonderful period of my life there and they gave me a thorough and valuable education. A few thousand dollars will also be donated for the restoration of an old baroque church in Fubine/Piemonte/ltaly, the home town of my wife’s father, Sebastanio Maioglio. We have spent many happy summers there in the parental home of my wife.” [My notes: the Frauenkirche was destroyed by the Allied bombing, and the Nazis destroyed the synagogue was in 1938. Globel also took up the rebuilding of the Paulinerkirche, the university church of the University of Leipzig, which had been blown up by the communist regime of East Germany in 1968.]
Blobel earned a degree in medicine from the University of Tubingen in 1960. However, after his internship in Germany, he decided that as a doctor he was merely treating symptoms, and that to get at causes he must turn to research. Blobel earned a doctorate in oncology from the University of Wisconsin in 1967. Next, he was a postdoctoral fellow at the Rockefeller Institute, under Nobel laureate, George Palade, and eventually became a full professor at the Institute in 1976, where he remained active until his recent death.
References:
Blobel, G. (1980). Intracellular protein topogenesis, Proc Natl Acad Sci USA, 77:1496-1500.
Blobel, G., and B. Dobberstein. (1975). Transfer of proteins across membranes. 1. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol, 67:835-851.
Bonatti, S., R. Cancedda, and G. Blobel (1979). Membrane biogenesis. In vitro cleavage, core glycosylation, and integration into microsomal membranes of sindbis virus glycoproteins. J Cell Biol, 80:219-230. DOI: 10.1083/jcb.80.1.219
Seasonal influenza outbreaks cause between 250,000 to 500,000 deaths word-wide each year (according to 2008 WHO estimates). What’s more, unpredictable pandemics, of which there were four in the 20th and the 21st centuries (1918, 1957, 1968, and 2009) pose a still greater threat. The worst of these pandemics, the 1918 Spanish flu outbreak, claimed an estimated 50 to 100 million lives globally (according to 2014 WHO estimates).
The human population was “only” 1.9 billion individuals during the 1918 pandemic, whereas there now are about 8 billion people inhabiting our planet. Thus, a future pandemic might be far more catastrophic than the 1918 episode. Moreover, since pandemic strains are derived in part from zoonotic influenza viruses, the constant rise in livestock numbers, intensive farming, and the numbers of animals being transported around the world, combine to facilitate the genetic mixing and evolution of influenza viruses, and the chance of an animal influenza virus becoming able to jump to humans and causing a pandemic.
Unlike vaccines against other viruses (e.g., measles), the seasonal flu vaccine needs to be updated each year to keep up with the antigenic changes that continually occur in influenza viruses. This creates several problems, the first of which is that individuals need to be re-vaccinated each year. And, since it takes months to produce a vaccine, when the updated vaccine is at last ready, it may not be a particularly good match against the new season’s strains. The current vaccine is only about 30 percent effective, which accounts at least in part for the unusually severe flu season we are currently experiencing. And while the efficacy of the current vaccine may be atypically low, even in good years the match is less than optimal.
Pandemics present a much greater challenge to vaccine makers, since pandemics may be vastly more severe than seasonal outbreaks, and since an entirely new vaccine is needed against pandemic viruses. That latter is so because, as noted, pandemic strains are derived in part from zoonotic influenza viruses, which by-and-large are antigenically distinct from strains already circulating in the human population, that humans already express immunity against. Consider the example of the 2009 pandemic. Because an entirely new vaccine was needed to meet the threat of the pandemic virus, the vaccine was not available until after the first wave of infection had already occurred. Fortunately, the 2009 pandemic virus was relatively mild.
A “universal” flu vaccine, that could provide lifelong protection against all seasonal strains of influenza, as well as provide protection against a pandemic virus, would be a most crucial and significant breakthrough. A major international workshop, entitled “Pathway to a Universal Influenza Vaccine,” was convened June 28 and 29, 2017, by the U.S. National Institute of Allergy and Infectious Diseases, to identify gaps in our knowledge that need to be addressed to develop such a vaccine (1).
Participants noted shortcomings in our understanding of the epidemiology, transmission, natural history, and pathogenesis of influenza. Among the issues specifically mentioned: “influenza surveillance is lacking in certain regions of the developing world and globally in certain high-risk groups… gaps in knowledge include the relationship between symptoms, viral shedding and transmission, as well as the level of protection needed to interrupt transmission.”
The host factors that influence influenza disease severity were also acknowledged to be poorly understood. To that point, participants addressed the need to better understand how pre-existing immunity—which might result from multiple natural influenza infections, as well as from repeated vaccinations—might affect how that person’s immune response will respond to future infections and, importantly, how past exposures might affect the efficacy of a vaccine. As stated in the meeting report: “Recent data provide strong epidemiologic evidence that infection with the influenza strain circulating during one’s childhood elicits a lifelong immunologic imprint that impacts responses to novel strains and can help protect against unfamiliar HA subtypes from the same phylogenetic group as the original infecting virus…The potential consequences of imprinting infants with vaccines versus natural exposure need to be carefully assessed.” [The HA protein is the so-called hemagglutinin, which serves the virus as its attachment protein.]
Participants also noted gaps in our understanding of the underlying B and T cell immune mechanisms that are induced by both natural infections and vaccinations. Further study of these responses was recommended so that we might be better able to stimulate them.
As might be expected, it is singularly important to identify the antigens that might be the most promising targets of a universal vaccine. To that point: “…most areas of the HA head are subject to antigenic change, and therefore unlikely to yield a broadly protective immune response. Neutralizing antibody responses to conserved regions such as the HA stalk, and non-neutralizing antibodies such as those directed at the neuraminidase (NA), and matrix 2 ectodomain, merit further study…The importance of each site may differ for pandemic versus seasonal influenza.” [The HA head region binds to receptors on the cell surface. After the bound virus is taken into the cell by receptor-mediated endocytosis, the low pH within endosomes triggers a conformational rearrangement of the HA stalk region, which mediates fusion of the viral envelope with the endosomal membrane, thereby releasing the viral cores into the cytosol. Since mutations within the stalk region might disrupt its membrane fusion function, such mutations are generally selected against. Researchers have recently had success developing antibodies that target the neuraminidase protein at the viral surface.]
Participants acknowledged that animal models play an important role in influenza research, especially when studying pandemic viruses. Nonetheless, “Animal models have limitations including the inability to mimic the human experience regarding genetic background, lifetime exposure to natural influenza infection or vaccine, viral susceptibility…and determinants of immune response and protection.” Thus, the participants noted that “a human a challenge model will be a crucial tool for vaccine development, as it can help answer fundamental questions about influenza immunity and serve as a mechanism for rapidly testing the efficacy of new products…Expansion of this resource should be a top priority…”
Day two of the workshop consisted of a rapporteur session on key conclusions, chaired by David Baltimore and Anthony S. Fauci (1)
Participants agreed that a robust collaboration between government agencies, academia, and industry would be needed to translate the fruits of basic research into a universal influenza vaccine. To that point, a January 24, 2018 editorial in Nature (doi: 10.1038/d41586-018-01070-w) asserted: “…advocates rightly argue that the research and development of a universal flu vaccine — ultimately the only effective defense against future pandemics — merits a program equivalent in scale to the Manhattan Project.” Yet the US government last year invested just $75 million on universal flu vaccine research and development.
Our October 12, 2017 post, Douglas Lowy, John Schiller, and the Vaccine Against Cervical Cancer, has reached a gratifying number of people. Since some readers might welcome a bit more background vis-à-vis the remarkable human papillomavirus (HPV) life cycle, or details concerning the use of virus-like particles (VLPs) in the experimental stages of the vaccine’s development, or how the vaccine might actually work, here are a few additional points.
The post noted that the replication cycle of HPV is regulated by the differentiation states of the cells making up the layers of an intact, stratified epithelium or mucosae. “Since the outer layer of the skin is comprised of dead cells, cutaneous HPV infection requires a break or puncture of the skin for the virus to access cells of the underlying germinal stratum of the epithelium. In the actively dividing basal cells, the viral genome replicates more frequently than the cellular genome, thus amplifying the viral genome copy number. However, because the viral genes that encode the capsid proteins are not expressed in these cells, progeny virus particles, which might induce an immune response, are not yet produced. As the basal cells differentiate and move up in the epithelium, the viral genomes replicate only once per cell cycle, on average, to maintain the viral genome copy number. Then, as the infected cells go through their final stages of differentiation in the outer layers of the epithelium, the virus life cycle switches to its productive phase. Capsid proteins are produced, and thousands of virus particles are generated from the each of the infected, terminally differentiated cells.” [How cellular differentiation regulates HPV gene expression and replication is detailed in the textbook, Virology: Molecular Biology and Pathogenesis.]
The post noted that by coupling its replication cycle to the differentiated state of the host cell within the stratified epithelium, HPV can produce progeny virus particles only in the terminally differentiated cells that comprise the outermost live cells of the epithelium. In this way, HPV productive infection does not activate an antiviral immune response. [The host’s immune response eventually does clear many HPV infections. Also, the incidence of HPV-associated lesions is higher in immunosuppressed patients.]
Here then is an additional key point. After the amplification stage, the viral genomes replicate in the basal cells, but only in conjunction with cellular DNA replication. In that way, the viral genome copy number is maintained in the basal cells. Moreover, and importantly, when the basal cells divide, one daughter cell remains behind as a basal cell, while the other daughter cell migrates up into the epithelium. Thus, one daughter cell will differentiate and thereby enable the virus to complete its replication cycle—at a level in the epithelium or mucosae beyond the reach of immune attack—while the other daughter cell remains behind in the basal layer, where it sustains the persistent infection.
Another consequence of this remarkable replication cycle is as follows. Since there are no blood or lymphatic vessels in the stratum of the epithelium or mucosae where the productive replication is occurring, the infection tends to remain localized, thereby giving rise to warts or tumors.
Since HPVs are difficult to study and propagate, one might ask how Lowy and Schiller were able to assess the antibody titers that were induced by inoculation with the HPV VLPs. The answer is that they used a pseudovirion-based immune assay. Pseudovirions are essentially VLPs that contain a plasmid that carries a reporter gene.
One last point. I believe it is generally the case that vaccines due not prevent virus infections per se. Rather, they enable the host to bring an infection under control more quickly, before symptoms might arise. Considering that cervical carcinomas may develop after years of virus persistence, despite a continuing immune response against the virus the whole time, how then might the vaccine protect against the cancer? Here is a thought. Bearing in mind that the human immune response naturally clears many HPV infections over time, perhaps the vaccine protects the host by enhancing immune surveillance to clear the infection before the emergence, or malignant progression of HPV-induced lesions. Or, perhaps the vaccine actually prevents infection.
The 2017 Lasker-DeBakey Prize for Clinical Research went to two virologists at the National Cancer Institute, Douglas Lowy, 75, and John Schiller, 64, for developing technologies that led to FDA-approved vaccines against human papillomavirus (HPV) strains that cause cervical carcinoma and other cancers. Lasker awards are considered the United States’ most prestigious biomedical research awards. They often precede a Nobel Prize in Physiology or Medicine. Thus, they are referred to as “America’s Nobels.” Eighty-seven Lasker awardees have gone on to win a Nobel.
Douglas Lowy and John Schiller
Lowy and Schiller’s achievements were prompted by Harald zur Hausen’s 1983 discovery that two HPV subtypes, HPV-16 and HPV-18, together account for about 70% of all cervical cancers. Since more than 120 distinct HPV subtypes had been identified, the high degree of association of cervical carcinoma with only two of these subtypes provided compelling evidence for the viral etiology of cervical carcinoma. Later studies showed that HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are responsible for another 20% of cervical cancers. Thus, an HPV infection can be detected in virtually all cervical carcinomas. Harald zur Hausen was awarded a share of the 2008 Nobel Prize in Physiology or Medicine for his discovery. [His story is told in Harald zur Hausen, Papillomaviruses, and Cervical Cancer, posted June 19, 2015.]
Lowy and Schiller did not begin their work on papillomaviruses with the intent to produce a vaccine. Instead, like many papillomavirus researchers at the time, they were investigating how papillomavirus oncogene products affected cell growth and replication (i.e., how they cause cancer). Toward that end, they were making use of bovine papilloma virus (BPV) in their studies, rather than HPV. BPV was easier to work with than HPV, because BPV, but not HPV, could be studied in standard cell cultures (see Aside 1).
[Aside 1: The replication cycle of HPV depends upon the differentiation states of the cells making up the layers of an intact, stratified epithelium. Details are as follow. Since the outer layer of the skin is comprised of dead cells, cutaneous HPV infection requires a break or puncture of the skin for the virus to access cells of the underlying germinal stratum of the epithelium. In the actively dividing basal cells, the viral genome replicates more frequently than the cellular genome, thus amplifying the viral genome copy number. However, because the viral genes that encode the capsid proteins are not expressed in these cells, progeny virus particles, which might induce an immune response, are not yet produced. As the basal cells differentiate and move up in the epithelium, the viral genomes replicate only once per cell cycle, on average, to maintain the viral genome copy number. Then, as the infected cells go through their final stages of differentiation in the outer layers of the epithelium, the virus life cycle switches to its productive phase. Capsid proteins are produced, and thousands of virus particles are generated from the each of the infected, terminally differentiated cells. Thus, the HPV life cycle is regulated by the differentiated state of the host cell within the stratified epithelium. Because virus production is restricted to the outermost layers of the epithelium, the virus can evade the immune system, such that the infection can persist, and be passed on for years. However, in most instances, the host appears to eventually mount a successful immune response, which clears the infection.
The development of so-called organotypic raft cultures eventually made it possible to study HPV in cell culture. But one could produce only very limited amounts of the virus in that system.]
Working with BPV, Lowy and Schiller developed protocols they would later use when they turned their attention towards an HPV vaccine. One of these protocols was for an assay to measure the titer of neutralizing antibodies against BPV. Importantly, they also discovered that they could generate “virus-like particles” (VLPs), comprised only of the major BPV coat protein (L1). The BPV L1 proteins (which were generated by a baculovirus vector in insect cells) self-assembled into VLPs that were morphologically like actual BPV particles. What’s more, using their assay to measure the titer of neutralizing serum antibodies, they found that the VLPs induced neutralizing antibodies in rabbits that were effective against the actual virus. Importantly, since the VLPs did not contain viral genes, they could not cause cancer.
Again, using their assay for measuring the titer of neutralizing antibodies against BPV, Lowy and Schiller compared the immunogenicity of BVP VLPs, to that of individual BPV proteins. The VLPs indeed are more immunogenic than individual viral proteins, since they induced higher levels of neutralizing antibodies than were induced by individual L1 proteins (see Aside 2).
[Aside 2: The activation of antibody-producing B-cells is triggered by the cross-linking of their antigen-binding B-cell receptors, which is facilitated by the multimeric VLPs, but not by individual viral proteins.]
The innovations resulting from their work with BPV would enable Lowy and Schiller to overcome the formidable challenges they faced when working to develop the HPV vaccine. One obstacle was that HPV cannot replicate in standard cell cultures. Thus, it was difficult to study HPV, and importantly, it also was difficult to propagate it. Being able to propagate substantial amounts of the virus would be necessary to produce a vaccine.
Another obstacle to an HPV vaccine was the potentially unacceptable risk of inoculating people with a virus (either attenuated or killed) that contains known oncogenes. Lowy and Schiller overcame this impediment, and the one noted above, by implementing protocols they previously developed while researching BPV. Specifically, they generated HPV VLPs that were comprised only of the HPV L1 capsid protein, and which induced an immune response that produced protective antibodies. [They used the L1 protein of HPV-16; the most carcinogenic strain of HPV.] In addition, they developed cell lines, which contained high copy numbers of the plasmid that encoded the HPV L1 protein; a step which enabled them to scale-up production of the VLPs.
Together, these breakthroughs made a compelling case for the feasibility of an HPV vaccine. So, Lowy and Schiller prevailed upon several pharmaceutical companies to produce a vaccine in commercial amounts, and to see the vaccine through the clinical trials process. Most companies remained skeptical about the ultimate success of the vaccine. But two companies, Merck and GlaxoSmithKline (which later bought Merck), accepted the challenge. Thus, Merck developed Gardasil, while GlaxoSmithKline developed Cervarix. [The VLPs in Gardasil are produced in yeast, whereas the VLPs from Cervarix are produced in insect cells, via a recombinant baculovirus.]
Clinical trials showed that the Merck and the GlaxoSmithKline vaccines induce significant antibody titers against high-risk HPVs. The US FDA approved the respective HPV vaccines in 2006 and 2009.
The HPV vaccines have had a substantial effect on human health. Consider the following: Cervical cancer is the second most common cause of death from cancer among women worldwide. HPV infection is the cause of virtually all cases of cervical cancer. HPVs also cause 95% of anal cancers, 70% of oropharyngeal cancers (more common in men than in women), 65% of vaginal cancers, 50% of vulvar cancers, and 35% of penile cancers. Next, consider that, since Gardasil and Cervarix were introduced, HPV infection rates have dropped by 50 percent among teen-age girls in U.S., even though only a third of teens between 13 to 17 years-old have received the full course (3 shots) of the vaccine (see Aside 3).
[Aside 3: Current CDC recommendations are as follows: “All kids who are 11 or 12 years old should get two shots of HPV vaccine six to twelve months apart. Adolescents who receive their two shots less than five months apart will require a third dose of HPV vaccine…If your teen hasn’t gotten the vaccine yet, talk to their doctor or nurse about getting it for them as soon as possible. If your child is older than 14 years, three shots will need to be given over 6 months. Also, three doses are still recommended for people with certain immunocompromising conditions aged 9 through 26 years.”]
Although he HPV vaccines have significantly reduced the incidence of cervical cancer in the developed world, the rates of cervical cancer in the United States are needlessly high, in comparison to the rates in other industrialized nations. The HPV vaccines have a loweracceptance rate than other childhood vaccines in the United States, perhaps because many American parents, some of whom associate with the religious right, have reservations about vaccinating their children against a sexually transmitted disease. Other individuals, liberals as well as conservatives, may oppose vaccines in general because they distrust pharmaceutical companies, or because they resent government interference in their lives. In any case, the CDC found no evidence of any increase in sexual activity among teenage girls who received the vaccine. Nor did it report any major ill effects]. See Aside 4.
[Aside 4: Since HPVs alone account for about 5% of all human cancers worldwide, we might ask what percentage of human cancers have a viral etiology. Hepatitis C virus, a flavivirus, and hepatitis B virus, a hepadnavirus, cause hepatocellular carcinoma; Epstein-Barr virus (EBV), a herpesvirus, causes Burkitt’s lymphoma and nasopharyngeal carcinoma; human herpesvirus 8 (HHV-8), causes Kaposi’s sarcoma, the most frequent cancer seen in AIDS patients; the human T-lymphotropic retrovirus I (HTLV-I) induces adult T-cell leukemia; and Merkel cell polyomavirus (MCV) causes its eponymous cancer. Together, viruses may account for as many as 20% of all human cancers, and a similar percentage of all deaths due to cancer!
As shown by the HPV vaccine, and earlier by vaccines against hepatitis B, cancers that have a viral etiology might be prevented by vaccination. Apropos hepatitis B, in the late 1980s, Merck and GlaxoSmithKline developed the respective hepatitis B vaccines, Recombivax and Engerix. Like, the HPV vaccines, they are based on VLPs, and they have significantly reduced the incidence of HBV-associated hepatoma; once one of the most lethal cancers.
Bacterial and parasitic infections too may lead to cancer. For example, Heliobacter pylori infections may lead to stomach cancer, and Schistosoma, Opisthorchis, and Clonorchis have been linked to rectum and bladder cancers in areas of Northern Africa and Southeast Asia, where those pathogens are prevalent.]
Lowy and Schiller’s achievement stands out as a superb example of basic research translating into very considerable public health benefits. Moreover, it serves as a strong endorsement for government support of basic research. To these points, Schiller noted that companies would not likely have carried out the necessary basic research and development necessary to produce the HPV vaccine, considering the seemingly small likelihood of success, as suggested by earlier failed attempts to develop a vaccine.
At a September 6, 2017 press conference announcing the Lasker-DeBakey Clinical Medical Research Award, Lowry related that he first learned about vaccines in 1955, when he went with his mother, a physician, to a talk by Jonas Salk about his then new polio vaccine. “I learned far more about polio virus and the vaccine than was probably appropriate for a 12-year-old boy.” Many years afterwards, Lowy began his “extraordinarily effective” collaboration with Schiller, which has endured for more than 30 years.
Schiller said that a high point in his career was taking his daughter to get the vaccine he helped to create. “We first came up with the idea of the vaccine when she was born and it became available when she was 13 years old (1).”
References:
A Revolutionary Vaccine, New York Times, September 6, 2017.
Variolation was the world’s first practical measure to control smallpox. It was developed in China in the 11th century. The procedure involved inoculating uninfected individuals with material from the scabs of individuals who survived smallpox infection.
It was brought to England for the first time in 1721, by Lady Mary Wortley Montague—the wife of the British ambassador to Turkey—when she returned home after learning of the practice in Istanbul. It was brought to Colonial North America the same year by the prominent Puritan minister, Cotton Mather. New England was then experiencing a major smallpox epidemic.
Mather is perhaps best known for his role in the Salem witchcraft trials. He learned of variolation not from the British, but from his African slave, Onesimus, who had been inoculated as child in Africa. Onesimus was a “gift” to Mather in 1706, from his Boston congregation. Variolation was used in western Africa when Onesimus was a child. The practice may have been brought there by caravans from Arabia. In any case, an enslaved African man played a key role in bringing variolation to North America.
In 1777, during the American War for Independence, General George Washington required the entire Continental Army to undergo variolation. Bearing in mind that more than two-thirds of the American casualties during the War resulted from disease, and that smallpox alone caused a total of about 100,00 deaths, some historians maintain that Washington’s policy of enforced variolation was his most important strategic decision of his entire military career.
Variolation nonetheless encompassed risks—a fatality rate of 1 to 2%—that would be unacceptable today. Not surprisingly then, the colonial and Revolutionary War periods were times when public fear and restrictive laws often prevented the use of variolation. Nonetheless, Thomas Jefferson was a lifelong advocate of smallpox-prevention measures. In 1766, Jefferson traveled to Philadelphia to undergo variolation, since the practice was banned in his native Virginia. As a lawyer in 1768, Jefferson defended a Norfolk doctor, whose house was burned down by a mob because he practiced variolation. In 1769, Jefferson placed a bill before the Virginia General Assembly to reduce the 1769 restrictions against variolation. In the 1770s and 1780s, he had his children and his enslaved servants (including Sally Hemings, his wife’s half-sister, and mother of several of his enslaved children) undergo the procedure.
In 1799, Boston physician and founder of Harvard Medical School, Benjamin Waterhouse, introduced Edward Jenner’s new cowpox-based smallpox vaccine to New England. Wanting to spread word of the vaccine to the rest of the new country, in 1781 Waterhouse sent a sample to his friend, Thomas Jefferson. At the President’s House in Washington, Jefferson selected an enslaved kitchen worker to be the first recipient of the vaccine. However, the vaccine did not take. So, Jefferson then had two of his slaves at Monticello undergo vaccination. When those vaccinations proved to be successful (as shown by exposure to actual smallpox), Jefferson serially transmitted the vaccine from the two original vaccines to almost fifty other slaves. By means of serial inoculations he then sent vaccine material to Washington (the city), and from there the vaccine traveled to Philadelphia and beyond. So, Thomas Jefferson, and his African slaves, played a seminal role in protecting many people in the new United States from smallpox.
Jefferson was an amateur, but serious scientist. He kept detailed notes of his observations, and corresponded with Jenner. Here is what he wrote to Waterhouse about the appearance of papules at the vaccination site:
As far as my observation went, the most premature cases presented a pellucid liquor the sixth day, which continued in that form the sixth, seventh, and eighth days, when it began to thicken, appear yellowish, and to be environed with inflammation. The most tardy cases offered matter on the eighth day, which continued thin and limpid the eighth, ninth, and tenth days. [http://www.smithsonianmag.com/smart-news/thomas-jefferson-conducted-early-smallpox-vaccine-trials-180954146/]
This is a tale of the hurt that a junior investigator might feel when a senior investigator takes the lion’s share of the credit for the junior investigator’s crucial breakthroughs. Jonas Salk, who conceived and oversaw the development of the first widely used polio vaccine, is the senior investigator in this anecdote. Julius Youngner, the last surviving member of the original vaccine research team that Salk assembled in the early 1950s at the University of Pittsburgh, is the slighted assistant. Youngner later had his own distinguished career. He passed away in April of this year. Here is their story.
After earning his Ph.D. in microbiology, Youngner was drafted into the World War II U.S. Army, which assigned him to the Manhattan Project, to test the toxicity of uranium salts. Youngner first learned the purpose of the Manhattan Project when the first atomic bomb was dropped on Japan.
After the war, Youngner worked as a commissioned officer for the U.S. Public Health Service. This was a significant stop in his career, since it was there that he first became interested in viruses and cell culture. But, since there was no opportunity for him to pursue that interest in Bethesda, he began to look elsewhere. Thus, it happened in 1949 that Salk recruited Youngner to join his vaccine research team in Pittsburgh, after a mutual acquaintance told Salk that Youngner was eager to work on viruses and cell culture.
Jonas Salk and Julius Youngner at the University of Pittsburgh, early 1950s
Salk hoped that Youngner might find a way to generate enough cells from monkey kidney tissue to support mass-production of the vaccine. Youngner, on his own, then developed the use of the proteolytic enzyme, trypsin, to disperse tissue fragments into individual cells, thereby generating many more cells from a given amount of tissue. Indeed, Youngner could generate enough cells to support manufacture of the vaccine. This was his first key contribution to the vaccine project. “Trypsinization” remains a mainstay of modern cell culture.
Youngner’s next major contribution to the vaccine enterprise was his development of a rapid analytical test that had two crucial applications. First, recalling that the Salk vaccine contains an inactivated virus, Youngner’s so-called “color test” made it possible to quickly screen batches of the vaccine for any live virus that might have survived the inactivation process. Second, Youngner’s test made it possible to quickly test the vaccine’s ability to induce anti-poliovirus antibodies (1). [Youngner based his color test on an earlier observation by John Enders, Tom Weller, and Fred Robbins, that metabolic activity (as indicated by a drop in pH) was less in cultures inoculated with live virus than in control cultures (2, 3). In Youngner’s test, a color change of phenol red, resulting from a shift in pH, served as an indicator of virus activity, or of antibody activity.]
Some sources credit Youngner with having devised the process for inactivating the virus. But, that is correct in a very limited sense only. Salk selected incubation in formalin as the means to disable the virus. In truth, Salk learned of that approach a decade earlier while doing postgraduate studies under Thomas Francis at the University of Michigan. Francis was then using formaldehyde to produce his killed influenza vaccine (2).
What’s more, Salk’s choice of formalin to generate his polio vaccine was bold. Earlier, in the 1930s, Canadian scientist Maurice Brodie tested a formalin-killed polio vaccine in twelve children, with disastrous results. Several of the children developed paralytic poliomyelitis (4).
Clearly, too little exposure to formalin could leave enough live virus to cause paralytic poliomyelitis or death. On the other hand, too much exposure could so badly damage the virus’ proteins that they might no longer induce an immune response against the live virus. Brodie did not have analytical procedures to ensure that he had inactivated his vaccine to safe levels. In contrast, it was clear to Salk that getting the correct balance would be vital to his vaccine project, and Youngner’s color test was the means for doing so. Youngner used his test to determine that six days of incubation in a 1:4,000 formalin solution would result in one live virus particle in 100 million doses of the vaccine (5).
Since Youngner’s inactivation curve was based on only a few data points, and since it was likely that the slope of the curve might flatten out after a time, Salk added a margin of safety of six extra days. Thus produced, the vaccine induced antibody production in monkeys, while showing no signs of causing paralysis or other problems.
By 1954, 800,000 children had been successfully immunized against polio in the first clinical trial of the vaccine. In April 1955, the outcome of the trial would be announced to a very grateful public.
By 1957, Salk’s vaccine team at Pittsburgh was no longer needed, and was dispersing. Salk was making plans to leave Pittsburgh for California, where he would found the prestigious Salk Institute. Youngner, now 34 years-old, remained at Pittsburgh, where he would begin his own distinguished career.
Although Youngner was now independent of Salk, he remained bitter over his former boss’s failure to acknowledge the underlings who had labored so diligently behind the scenes to bring the vaccine to fruition. “The first rule we learned was to call him ‘Dr Salk,’ never Jonas. He would speak to us through a wall of notes and memos…Here was a guy who could always find an hour to brief some reporter at the local Chinese restaurant, but could never find the time to sit down with his own people (6).”
Youngner was particularly appalled by events involving the paper he wrote describing his color test. “After I had what I considered to be a good draft…I gave my copy to Jonas for his comments. It should be noted this was 1954, the pre-Xerox, pre-word-processing era. I had made a working transcript of the paper for my own use and it was this copy that I handed to him. Also, it should be noted that the title page had the authors listed as ‘J.S. Youngner and E.N. Ward (6).’” Elsie Ward, who served as Youngner’s technician, was a zoologist who specialized in growing viruses.
Salk intended to read Youngner’s manuscript while away on a trip. When Salk returned a week later, he claimed that he had lost the manuscript, but that he had jotted down some notes from which he was able to produce a draft of his own. Youngner was rather incredulous that a person as meticulous and disciplined as Salk could lose such an important manuscript. Youngner’s skepticism was further roused by the fact that Salk’s version contained all the data in Youngner’s original manuscript. Salk explained that incongruity, alleging that he found Youngner’s tables, but not the text.
In any case, Youngner was especially upset by a specific change Salk made to the title page of the manuscript: “The authors were now ‘Jonas E. Salk, J.S. Youngner, and Elsie N. Ward.’ When I (Youngner) questioned the change, Jonas said that since he had to reconstruct the whole paper it was only fair that his name go first…It was obvious to me then, and is more so now, that he considered the advance in this paper a major one and he wanted his name associated with it, even though at the time he had done nothing in the lab (no kidding!) or of an advisory nature to initiate or carry out the work (6).”
Youngner could grudgingly accept that project leaders often used their senior position to appear as co-authors, or even principal authors, on papers emanating from their labs, even if their contributions were minimal. What troubled Youngner in this instance was not that Salk pulled rank, but rather his seeming duplicity.
In yet another instance—the 1955 public announcement of the successful outcome of the clinical trial—Youngner again sensed “a pattern of deception on Salk’s part to take undue credit for the discoveries of others (6).” Salk advocated for the announcement to happen at the University of Pittsburgh. However, the National Foundation for Infantile Paralysis (better known as the “March of Dimes”), which funded the vaccine project, chose the University of Michigan in Ann Arbor as the site for the announcement. That was where Michigan professor Thomas Francis supervised the evaluation of the field trial. [Note that the NIH was not able to fund research back then the way it can today. Thus, the polio vaccine project was supported nearly entirely by private donations to the National Foundation.]
Thomas Francis spoke first. Then, when Salk spoke, he acknowledged the more prominent players in the vaccine project, including Thomas Francis, Harry Weaver (director of research at the National Foundation), Tom Rivers (chairman of the advisory committees on research and vaccines for the National Foundation), and Basil O’Connor (law partner of Franklin Roosevelt, recruited by Roosevelt in 1928 to raise funds for polio patients at Roosevelt’s Warm Springs Foundation, and a co-founder with Roosevelt of the National Foundation in 1938; (2)). Salk then acknowledged various deans and trustees at the University of Pittsburgh. Yet, he made no mention whatsoever of his dedicated coworkers in his laboratory. They had been expecting at least some recognition from their boss.
Some of Salk’s defenders argued that Salk had acted in the best scientific tradition by prefacing his printed remarks with the phrase, “From the Staff of the Virus Laboratory by Jonas E. Salk, M.D.” But, this was small consolation to Youngner and others of Salk’s coworkers, who expected to be individually acknowledged for their exhausting work on behalf of the life-saving vaccine. Indeed, they felt betrayed.
At any rate, the 1955 announcement of the success of the polio vaccine field trials was joyously received by the public. And while Youngner remained embittered over Salk’s slighting of his coworkers, he nonetheless understood that from the point of view of the National Foundation, “it was much easier to continue raising money when you have a hero, and they had an enormous public relations department that took up Jonas’ name as the hero, which he deserved…But in the meantime, Jonas was, how shall I say, not very generous to his colleagues and he made sure that nobody else was ever mentioned (6).”
The following excerpt is from Polio: An American Story (6). “In September 1963, Salk returned to Pittsburgh to attend the unveiling of his portrait in the auditorium of the University’s medical complex, a stone’s throw from the hospital where he had done his historic polio research. Before the ceremony, Salk told Dean George Bernier that he wished to speak privately with his former assistant, Julius Youngner, now a distinguished professor at the school of medicine. The two men hadn’t talked or crossed paths since Salk’s move to California in 1961. Salk saw the meeting as a courtesy to the only remaining member of his laboratory staff; Youngner had a different agenda. Speaking softly, he recalled, he slowly released the ‘hurt’ he had bottled up for more than thirty years. ‘Do you still have the speech you gave in Ann Arbor in1955? Have you ever reread it?’ Youngner began. ‘We were in the audience, your closest colleagues and devoted associates, who worked hard and faithfully for the same goal that you desired…Do you remember who you mentioned and who you left out? Do you realize how devastated we were at that moment and ever afterward when you persisted in making your coworkers invisible? Do you know what I’m saying,’ I asked. He answered that he did…Jonas was clearly shaken by these memories and offered little response.’…The two men engaged in some uncomfortable small talk before Dean Bernier returned to escort them to the ceremony. Speaking later to a reporter, Youngner admitted, ‘I got a lot of things off my chest. I’m beyond the point where I pull my punches with him. I think it was the first time he ever heard it so graphically.’ Asked if he had any regrets about working for Salk, Youngner replied: ‘Absolutely not. You can’t imagine what a thrill that gave me. My only regret is that he disappointed me.”’
Epilogue:
Jonas Salk is deservedly celebrated for developing the killed polio vaccine. That vaccine, together with Albert Sabin’s live attenuated vaccine, which followed soon afterwards, has nearly eradicated polio worldwide. Importantly, Sabin and other polio researchers believed that only a live vaccine could induce a level of immunity sufficient to protect against a challenge with live virulent virus. Nonetheless, Salk persevered in his conviction that a killed vaccine could protect against polio, and he was right.
Salk founded the prestigious Salk Institute in 1963. Yet he never himself made another notable contribution to science.
Youngner may be best known for his work on the Salk vaccine. Yet he had a distinguished career of his own at the University of Pittsburgh after Salk left. Youngner is especially noted for his contributions to interferon research. These include his finding that non-viral agents could trigger interferon induction in animals. And, in collaboration with colleague Samuel Salvin, he identified a second type of interferon, now known as gamma-interferon. Youngner also helped to explain the antiviral-effect of interferon, and he was the first researcher to demonstrate that some viruses express countermeasures against interferon.
Youngner also made important findings in the area of persistent virus infections. Importantly, he demonstrated that defective viral variants, including temperature-sensitive mutants, can play a role in the establishment and maintenance of viral persistence; doing so by impairing (modulating) the replication of the wild-type parental viruses. Based on that principle, Youngner sought to develop dominant-negative mutants of influenza virus as a novel means of anti-influenza therapy. In addition, Youngner and colleague Patricia Dowling developed a novel live attenuated vaccine against equine influenza virus, based on a cold-adapted influenza virus, which can replicate only at the temperatures found in the respiratory tract. That live vaccine was the first to prevent a serious respiratory disease of horses.
George Klein, professor emeritus of tumor biology at the Karolinska Institute in Stockholm, where he worked with his wife Eva from the very beginning, passed away on December 10, 2016, at the age of 91. Klein was best known for discovering that Epstein-Barr virus (EBV)—the herpesvirus now known to cause infectious mononucleosis—causes two human cancers, Burkitt’s lymphoma and nasopharyngeal carcinoma. Moreover, Klein discovered that EBV triggers Burkitt’s lymphoma by facilitating a chromosomal translocation of the cellular c-myc oncogene, resulting in its constitutive expression. Klein also played pioneering roles in developing the concept of tumor-suppressor genes, and in opening the field of tumor immunology. Klein’s key discoveries are summarized below. But, first, Klein, like several other protagonists in these tales, was profoundly affected by events of the Second World War, and by the early days of the Cold War that followed.
From an announcement for a 2015 symposium at the Karolinska Institute, honoring George and Eva on the occasion of their 90th birthdays
George Klein’s Jewish family moved from Eastern Slovakia to Budapest in 1930. Nineteen-year-old George was working as an assistant secretary to the Jewish Council in Budapest when Nazi Germany began its occupation of Hungary in March 1944. Because George had been working for the Jewish Council, in April 1944 he chanced that to see the Vrba-Wetzler Report, known at the time as the “Auschwitz Report.” It was written by, and was secretly transmitted to the Jewish Council by Rudolf Vrba and Alfred Wetzler, two escapees from Auschwitz. It described firsthand the fate of Jews arriving at Auschwitz, and was meant to warn Hungary’s Jews, so that they might hide from, or rebel against their Nazi oppressors.
The Auschwitz report was not publicized in Hungary for reasons explained below. However, George’s supervisor at the Jewish Council gave him permission to tell his relatives and friends of what the report revealed. But they, like most Hungarian Jews, could not believe that such atrocities could actually be taking place. [During May, June, and July 1944, 437,000 Hungarian Jews were deported to Auschwitz; to be “resettled” according to the Nazis. But, in fact, most were murdered in the gas chambers.]
Klein was arrested and pressed into forced labor by the Nazis. Afterwards, since he knew the contents of the Auschwitz Report, he fled when he was about to be ordered to board one of the deportation trains to Auschwitz. Having escaped from almost certain death, he lived underground until January 1945, when the Russian Army liberated Budapest.
Forty-three years later, Klein was watching, Shoa, the monumental (nine-hour-long) French documentary film about the holocaust. Watching the movie, Klein chanced to see a man named Vrba (one of the six principal holocaust witnesses in the film) describe his experiences as a prisoner in Auschwitz. The events that Vrba recounted horrified Klein.
Later in the film, as Vrba described his escape from Auschwitz, Klein suddenly realized, “the report I had been given to read under a promise of secrecy in Budapest in May 1944—at the age of nineteen and at a time when deportations from the Hungarian countryside were at their peak—was identical to the Auschwitz Report of Vrba and Wetzler (1).”
Next in this remarkable tale, Klein decided to try to find Vrba, to “tell him of what enormous help his report had been to me. If I had not known what was awaiting me at the other end of the train trip, I would never have dared to risk an escape. It was not difficult to find Vrba, for it turned out that we were scientific colleagues. He is a professor of neuropharmacology in Vancouver, and I am now (in the Spring of 1987) sitting in a comfortable armchair in the faculty club at a Canadian university, talking with someone who, at first glance, seems quite ordinary. He impresses me as being relaxed and jovial. By now I have also read his book (Escape from Auschwitz, 1964), and I am aware that he has survived more death sentences than anyone else I have ever met (1).”
Vrba (1924–2006), was indeed a professor of pharmacology at the University of British Columbia; a position he held from 1976 until the early 1990s. Note that he and Wetzler were the first prisoners ever to escape from Auschwitz. Vrba’s real name was Walter Rosenberg. Rudolf Vrba was the nom de guerre he used after joining the resistance in his native Czechoslovakia. Afterwards, he made the change legal.
The horrors of the holocaust remained an obsession for Klein, although he was uncertain as to why that was so. “Was it to honor my murdered family, my murdered classmates? Or was it rather to steel myself against the darkest side of our human heritage?” In any case, Auschwitz and the holocaust were the main topics of conversation when Klein met with Vrba.
Vrba took Budapest’s Jewish Council to task for not widely broadcasting the warnings in the Auschwitz Report. He, and others, have alleged that Dr. Kastner, a well-known Zionist leader in Budapest, decided to keep the Report secret, in return for a promise from the Germans to allow sixteen-hundred people, as selected by Kastner, to safely emigrate from Hungary. Klein retorted that he knew Kastner from his work for the Jewish Council, and considered him to be a hero, because he had rescued many, while others tried to rescue only themselves or their own families. [In 1957, Kastner was murdered in Israel by a young man whose family was exterminated by the Nazis. Kastner remains a controversial figure to this day.]
Klein and Vrba next discussed whether dissemination of the Auschwitz Report might have caused Budapest’s Jews to revolt against the Nazi program of annihilation. Klein argued that of the dozen or so people that he warned, no one believed him. Vrba countered, “You were a mere boy. Why would anyone believe what you were saying? The Jews would certainly have believed their responsible leaders (1).” Nonetheless, Vrba conceded that even the prisoners at Auschwitz were in denial of what they could see with their own eyes: “…prisoners, who knew full well that no one ever returned from the gas chambers, repressed such knowledge as they themselves lined up for execution in front of the chamber doors.”
Klein asked Vrba how he is able to live and function in Vancouver, a pleasant and friendly place, where no one has the slightest concept of what he endured: “…you must go back constantly to those days. You are called in as a witness at trials of old Nazis or their followers, people who claim that the holocaust never happened. You try to describe something that cannot be described in any human language, you try to explain the incomprehensible, you want people to listen to something they do not want to hear (1).” Vrba, in fact, never did reveal his Auschwitz experience to his colleagues. Vrba explained: “What would have been the use? No one who has not experienced it can understand.” Their conversation went on for almost ten hours. Afterwards, they parted like old friends, despite any differences in their views.
In the Fall of that year, Klein was reunited with Vrba in Paris, together with another newfound friend, German scientist Benno Muller-Hill. In 1966, Muller-Hill was a graduate student in Walter Gilbert’s Harvard laboratory, when he purified the lac repressor; the first genetic control protein to be isolated. Muller-Hill then began a second career lecturing and writing about the role of Nazi doctors and scientists in the holocaust. Klein met Müller-Hill for the first time at a meeting at the Institute for Genetics in Cologne, and the two immediately developed a close friendship.
Muller-Hill was in Paris to visit colleagues at the Pasteur Institute, as well as to meet Vrba. Klein was visiting Paris after attending a scientific meeting in Lyon. Vrba was in Paris at the invitation from the French radio service to refute claims of the ultra-right French leader, Jean Marie Le Pen, that the Nazi gas chambers never existed, and that if the Nazis indeed had any intent to annihilate the Jews, it was merely one of many episodes of the war. [Marine Le Pen, currently a leader of France’s ultra-right National Front, and a candidate for the presidency of France, is Jean Marie’s daughter. She was recently taken to task for denying that French officials and police were complicit in the Nazi roundup of more than 13,000 French Jews in July 1942 (they were later deported to Auschwitz). Le Pen also calls for the deportation of all immigrants from France; a stance that mainly targets Muslims.]
Klein and his two companions ambled about Paris on a beautiful Fall afternoon. They strolled around the Luxembourg Gardens, then continued along the banks of the Seine, turned toward the Latin Quarter, and then stood before the façade of Notre Dame. Yet their minds were elsewhere. “Vrba suggested that we visit the holocaust memorial behind Notre Dame…That walk of only a few minutes took us from the noisy tourist crowd to the silence of the museum’s rooms, where you feel alone and isolated among the symbolic chains and barbed wire. A faint glow of sunlight came in through the narrow openings in the wall. We were surrounded by the voices of the victims…We were all completely speechless. Even Vrba’s macabre sense of humor and his sharp sarcasm had fallen silent for the moment (1).”
After they exited from the memorial, they sat down in a small bistro, where Klein asked his two companions whether German scientists and doctors were actual architects of the holocaust or, instead, merely passive followers. “Benno had concluded from his exhaustive documentation that, contrary to what many wanted so desperately to believe, the ‘euthanasia programs’…and the horrible human experiments… could not be ascribed to a small minority of madmen, opportunists, or charlatans. On the contrary, they had been carried out by quite ordinary and in some instances, eminent physicians and scientists… He (Verba) thought … that would not explain why so many apparently ordinary people took part in the murders without showing any signs of remorse, or how the annihilation program could have been carried out with such efficiency… The discussions between Benno and Vrba continued for several hours (1).”
The day became even more notable later, since Klein had arranged for the threesome to have dinner that evening with Francois Jacob. After a glass of sherry in Jacob’s Latin Quarter apartment, the foursome went to a small restaurant around the corner.
Francois Jacob, and fellow Pasteur Institute scientist Jacques Monod, were awarded Nobel Prizes for their work together on the regulation of lactose metabolism in E. coli (2). More apropos the current episode, Jacob and Monod each received France’s highest military honors for his service during the Second World War—Jacob for his heroism serving with the Free French forces, and Monod for his heroism in the Resistance (2). Yet Jacob’s harrowing escape from Nazi-occupied France at 19-years in age, and his wartime exploits as one of Charles De Gaul’s most highly decorated volunteers, were barely known to his three dinner companions.
At first, Klein was somewhat worried that his friends might not like each other. Jacob often found conversation to be difficult; partly because the thousands of pieces of shrapnel that he carried in his body from the war, made it hard for him to sit comfortably. [Jacob’s wartime wounds prematurely ended his surgical career, and led him to turn to a career in science (2).] But, the get-together didn’t go badly at all.
Conversation eventually turned to the issue of holocaust deniers, as well as to those who would put the past completely behind them. As they talked, the incongruity of the scene suddenly struck Klein. They were sitting in a “first-class Parisian restaurant, surrounded by elegant people, having a very nice dinner in the best French tradition.” “…why did the three of us, with Jacob listening, choose to spend that beautiful Saturday in Paris compulsively focusing our attention on the black birds? We were all citizens of free countries, living well in peaceful times. Were we haunted by feelings of guilt toward the dead? Were we afraid that the whole experience would recur if we let go? We knew that the wide and relentless river of history is rarely influenced by knowledge of the past. In no more than one or two generations, archives of extreme horror turn into scraps of faded paper, with no more influence than dried leaves. I suddenly felt that we were like a traveler with a fear of flying, forcing himself to stay awake and keep his seatbelt buckled during the entire flight, obsessed with the idea that the plane would surely crash if he were to fall asleep. But perhaps we had other motives. Perhaps we wanted to feel a solidarity with each other by selecting a more or less taboo subject for our conversation, one avoided by most others. Or did we try to perform a kind of autopsy, using our brains to understand what human minds are capable of at their worst? Have we appointed our brains to serve as the pathologist and the cadaver at the same time?”
The above recounts only a small sampling of Klein’s conversations with Vrba, Muller-Hill, and Jacob, during their day together in Paris. For more, see reference 1.
In January 1945, 19-year-old George Klein emerged from the Budapest cellar where had been hiding during the last weeks of the German occupation. He gazed on the dead soldiers, civilians, and horses that were frozen in the snow, and was struck by the thought that he had survived, despite the likelihood that he would have ended his 19 years in a Nazi gas chamber or a slave labor camp. However, with the city now in Russian hands, George faced a new threat to his freedom; the Russian patrols that were exporting young Hungarians to labor camps in Russia.
Mindful of the danger on the streets, George was yet eager to begin his medical studies. So, he cautiously dodged the Russian patrols as he made his way to Budapest’s medical school, only to find war-torn deserted buildings and dead soldiers there.
Undeterred by the situation in Budapest, George and a friend set out to Szeged, with the hope of attending the medical school there. The journey of 160 miles took the pair five days, by way of a variety of vehicles, including a Russian military truck. In any case, they were admitted to the Szeged university on the same day that they arrived. And while the school was a shadow of its former self, with all the professors having fled to the West, to George, it was a “previously forbidden paradise (3).”
George spent two years in Szeged, and then returned to Budapest when the University reopened there. Back in Budapest, George fell “desperately” in love with Eva Fisher, a fellow medical student. [George describes their whirlwind romance in reference 3.] George now faced a dilemma. Before he met Eva, he finalized plans to visit Stockholm (under the sponsorship of the Jewish Student Club there). But going to Stockholm would mean leaving Eva behind, under conditions in which travel back into Hungary could be risky. Nonetheless, George went to Stockholm, with Eva believing she would never see him again. Yet the trip would be a defining experience for George and, eventually, would be important for Eva too. In Stockholm, George would learn of, and be riveted by the research of renowned cell biologist Torbjörn Caspersson, at Stockholm’s Karolinska Institute.
Caspersson’s research so enthralled George that he diligently pressed Caspersson for a junior research assistantship in his laboratory. But once George had been accepted by Caspersson, he viewed his situation with a “mixture of ecstatic happiness and enormous anxiety.” “I knew virtually nothing…I was halfway through my medical studies…I was desperately in love with a girl whom I had only known during a summer vacation of eight days and who was on the other side of an increasingly forbidding political barrier (3).”
Despite these misgivings, George knew that his future lay in Sweden, rather than Hungary. He had been accepted into Caspersson’s laboratory, and Hungary was falling increasingly under totalitarian Soviet domination. But Eva was still in communist Hungary. So, George risked returning there with one goal; to marry Eva, and then to leave Hungary for good. “The reunion with Eva confirmed what we both already knew: we wanted to live and work together (3).”
But George and Eva didn’t have the necessary documents to get married, nor did Eva have a passport to leave Hungry. Moreover, communist bureaucrats made it increasingly difficult to obtain these documents. In some instances, up to six weeks might be needed. However, George and Eva were daring and resourceful. When told by a police officer that it would take at least three weeks to obtain a marriage license, George suddenly acted on impulse: “I had always heard others tell of such things but I myself had neither seen nor done it. I pulled a fairly modest bill out of my pocket and put it in the policeman’s hand. ‘Pardon me, how much time was it you said?’ ‘I’ll go get it at once,’ he answered (3).”
With similar persistence and ingenuity, George and Eva obtained all their necessary documents, and they were married that very day! One document, a certificate asserting that neither George nor Eva had a venereal disease, would normally require a three-week lab test. But they beseeched an older colleague, now a doctor at a children’s hospital, to write the certificate for them. Their colleague did so, on his Children’s Hospital stationary. George and Eva then went to the prefecture to be married, only to find a disagreeable marriage official, who was determined to leave work for the day. But, when the official leafed through their papers, and saw the venereal disease certificate written on Children’s Hospital stationary: “He laughed until tears ran down his cheeks. This was the funniest thing he had seen during his whole time in service.” He then gladly married the couple.
As the Iron Curtain descended about Hungary, George and Eva left for Sweden, where they would now continue their medical studies. What’s more, Eva joined George in Caspersson’s laboratory at the Karolinska Institute. The couple would work together at the Karolinska until George’s death at the age of 91. [Eva was born to Jewish parents in Budapest in 1925. In 1944 and 1945, she and several members of her family hid from the Nazis at the Histology Institute of the University of Budapest. Encouraged by Caspersson, Eva had an independent research career, while also collaborating with George. She is best known for discovering natural killer cells, and for generating the Burkitt’s lymphoma cell lines, which she and George studied together (see below).]
George and Eva, at the Karolinska Institute, 1979
We conclude with a brief review of some of George Klein’s contributions to virology and to cancer research.
Tumor immunology: In 1960, George and Eva used methylcholanthrene to induce tumors in mice. Next, they surgically removed the tumors, killed them with irradiation, and inoculated them back into genetically compatible mice. Next, they challenged these mice with cells from a variety of different tumors, and showed that the immune systems of the inoculated mice rejected only those cancer cells that came from the original tumor. Thus, there are tumor-specific antigens that can be recognized by the immune system. See Aside 1.
[Aside 1: Importantly, the tumor resistance seen in these experiments did not arise spontaneously in the original tumor-bearing animals. Instead, it developed in the test mice, in response to sensitization with killed tumor cells. Thus, these experiments per se do not point towards an immune mechanism of tumor surveillance. Nonetheless, harnessing such a mechanism is currently a promising means of cancer therapy, and was a major theme in Klein’s thinking.]
The following year, Klein’s group showed that polyoma virus-induced tumors share a common antigen. Importantly, polyoma virus-induced tumors, and polyoma virus-transformed cells, were rejected irrespective of whether they released virus. Thus, antiviral immunity as such was neither necessary nor sufficient for tumor rejection. This was the first demonstration that tumors caused by a virus might share a common antigen. The Kleins, and others, later found similar “group-specific” transplantation antigens on other virus-induced tumors, including retrovirus-induced lymphomas.
Burkitt’s lymphoma: “Sometime in the mid-1960s, Eva suggested that we should use our experience on virus-induced murine lymphomas to examine a human lymphoma with a presumptive viral etiology. Could we detect group specific antibody responses that might be helpful in tracing a virus? Burkitt’s lymphoma (BL) was the obvious choice (3).” [Burkitt’s lymphoma, originally described by Dennis Burkitt in 1958, is a malignant B-cell lymphoma that is most prevalent in tropical Africa and New Guinea. It is the most common childhood cancer in equatorial Africa. Burkitt first proposed that the lymphoma might have a viral etiology, since its geographic distribution is like that of yellow fever, which is caused by a flavivirus. In 1964, Tony Epstein and Yvonne Barr, by means of electron microscopy, discovered a virus in cells which they cultured from BL tissue, thereby giving credence to Burkitt’s premise.]
Klein’s group identified a membrane antigen (MA) that was expressed in some BL-derived cell cultures. Werner and Gertrude Henle had previously discovered that the MA antigen is a structural protein from a newly discovered herpesvirus—the virus that Epstein and Barr first saw in 1964. Klein decided to call that virus the Epstein Barr virus (EBV). The MA antigen is now known to be one of the EBV envelope glycoproteins. Klein and collaborators later identified complement receptor type 2 (CR2), also known as the complement C3d receptor, as the cell surface attachment protein for the viral MA glycoprotein. CR2 receptors on B cells play a role in enabling the complement system to activate B cells.
By 1970, Klein’s group, in collaboration with Harald zur Hausen, found that the subset of BL-derived cell lines that express MA are, in fact, those that produce EBV. However, more than 90% of the BL cell lines, and all nasopharyngeal carcinomas, were found to contain multiple EBV genomes per cell, irrespective of whether they produced virus. Thus, only a subset of BL and nasopharyngeal carcinoma cells that harbor EBV genomes, actually produce the virus. During this time, the Henles discovered that EBV is the cause of infectious mononucleosis, and that EBV could immortalize normal B cells in culture.
Oncogene activation by chromosomal translocation: A sero-epidemiological study, begun in Uganda in 1971 by Geser and de-The, showed that children with a high EBV load are more likely to develop BL than are children with a low EBV load. Thus, the presence of EBV genomes in a B cell increases the likelihood of it turning into a BL. “But this is still not a satisfactory explanation; some essential element is obviously missing (3).”
What then is the missing event that gives rise to BL? A 1972 study by Manolov and Manolova, Bulgarian scientists working with the Kleins, found that a particular chromosomal marker, 14q+, was present in about 80% of BL tumors. After the Manolovs returned to Bulgaria, the Kleins, in collaboration with Lore Zech, used the chromosomal banding technique recently developed by Caspersson and Zech to examine the BL-cell chromosomes more precisely. They showed that the 14q+ marker was derived from chromosome 8, which broke at the same site (8q24) and underwent a reciprocal translocation with the short arm of either chromosome 2 or chromosome 22. All BLs carried one of the translocations.
Meanwhile, another research group found that carcinogen-induced mouse plasmacytomas are associated with an almost homologous chromosomal translocation. Thus, a common mechanism seemed to underlie two distinct types of tumors, in two distinct species. In each instance, a putative oncogene was translocated to an immunoglobulin locus, which might then have caused the oncogene to be constitutive expressed. A somewhat similar mechanism was reported earlier for the induction of bursal lymphomas in chickens by the avian leukosis virus (ALV) . In that instance, the cellular c-myc gene came under the control of the ALV provirus promotor. What’s more, Michael Cole’s group identified the transposed gene in BL, and in the mouse plasmacytomas, as c-myc. It is not yet clear how EBV infection promotes the chromosomal translocation.
Tumor suppressor genes: In the early 1970s, Klein, and collaborator Henry Harris, played a pioneering role in developing the concept of tumor suppressor genes. They found that when highly malignant mouse cells are fused with normal mouse cells, the hybrid cells are non-malignant when inoculated into genetically compatible mice. That is, tumorgenicity is suppressed by fusion with normal cells. However, tumorgenicity reappears after some apparently important chromosomes, contributed by the normal cell, are lost from the hybrid cells.
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