Tag Archives: SV40

Harald zur Hausen, Papillomaviruses, and Cervical Cancer

Harald zur Hausen (1936- ) was awarded a share of the 2008 Nobel Prize in Physiology or Medicine for discovering that papillomaviruses cause cervical cancer. He received the award jointly with Luc Montagnier and Françoise Barré-Sinoussi, who were given their portion for discovering HIV (1). Before getting on with zur Hausen’s story per se, we begin with bit of earlier history.

zur hausen Harald zur Hausen in 2008

Genital warts are benign epithelial tumors that have been known and associated with sexual promiscuity since the time of the ancient Greeks. In 1907 these lesions were unequivocally proven to be an infectious disease by Italian researcher, G. Ciuffo, who showed that they can be transmitted by filtered extracts of wart tissue; a finding which also implied that the etiologic agent is a virus. Ciuffo inoculated himself to advance his case.

Ciuffo’s finding is relevant to our story since members of the papillomavirus family of DNA viruses are the cause of warts. What’s more, and importantly, some papillomaviruses  also cause malignant cervical carcinomas.

In 1933 Richard Shope, at the Rockefeller Institute, became the first researcher to isolate a papillomavirus, the cottontail rabbit papillomavirus. Shope went on to show that this virus is the cause of skin papillomas in its rabbit host. This finding by Shope was the first to demonstrate that a DNA virus can be tumorigenic.

Years earlier, in 1911, Peyton Rous discovered that an RNA virus—the Rous sarcoma virus (the prototype retrovirus)—causes solid tumors in chickens. Peyton Rous was Richard Shope’s friend and colleague at the Rockefeller Institute. In 1934 Shope asked Rous to characterize the warts that the rabbit papillomavirus induces in jackrabbits. Rous found those warts to be benign tumors that could progress to malignant carcinomas.

Despite the earlier findings of Ciuffo, Shope, and others, the notion that genital warts in humans is a sexually transmitted malady was slow to gain acceptance. Oddly, perhaps, recognition of that truth was prompted by a 1954 report that American servicemen, who had been serving in Korea, were transmitting genital warts to their wives upon returning to the U.S (T. J. Barrett, et al., J. Am. Med. Assoc. 154:333, 1954). [Sexually transmitted diseases were a long-standing problem in the military. Servicemen were most often infected by sex workers who frequented the vicinity of military quarters.]

The key discoveries of this tale are Harald zur Hausen’s 1983 and 1984 findings that two human papillomavirus subtypes, HPV-16 and HPV-18, together account for about 70% of all cervical cancers. Considering that more than 120 distinct HPV subtypes have been identified, the high degree of association of cervical carcinoma with only two of these subtypes provided compelling evidence for the viral etiology of this malignancy. Later studies showed that HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are responsible for another 20% of cervical cancers. Indeed, an HPV infection is present in virtually all cervical carcinomas. See Aside 1.

[Aside 1: Cervical cancer was once the leading cause of cancer-related deaths in women in the United States. However, the number of cervical cancer deaths in the industrialized world decreased dramatically over the last 40 years, largely because of the Pap test, which can detect pre-cancer cervical lesions in their early stages. The CDC website reports 12,109 cervical cancer cases and 4,092 deaths from cervical cancer in the U.S. in 2011 (the most recent year for which data are available). Worldwide, cervical cancer was responsible for 275,000 deaths in 2008. About 88% of these deaths were in developing countries (J. Ferlay et al., Int. J. Cancer, 127:2893, 2010).]

Harald zur Hausen was a child in Germany during the Second World War, growing up in Gelsenkirchen-Buer, which was then a center for German coal production and oil refining and, consequently, a major target for allied bombing. [The city also contained a women’s sub-camp of the Buchenwald concentration camp. The Nazis used its prisoners for slave labor.] All members of zur Hausen’s family survived the war. However, zur Hausen’s primary education contained significant gaps because schools were closed during the allied bombing (2).

Despite the gaps in zur Hausen’s early education, he was keenly interested in biology and dreamed of becoming a scientist. Yet at the University of Bonn he opted to study medicine, rather than biology. After zur Hausen received his medical degree, he worked as a medical microbiologist at the University of Düsseldorf, where he enjoyed the opportunity that the University gave him to carry out research on virus-induced chromosomal aberrations.

Although zur Hausen was fascinated by his research, he was soon aware of the deficiencies in his scientific background. So, in 1966 he looked to enhance his proficiency as a scientist by securing a postdoctoral position in the laboratories of Gertrude and Werner Henle at the Children’s Hospital of Philadelphia.

The Henles were a German-born husband and wife research team, known for their work on flu vaccines. More apropos to our story, they are also known for demonstrating the link between the recently discovered Epstein-Barr virus (EBV; a herpesvirus) and infectious mononucleosis, as well as for showing that EBV is the etiologic agent of Burkitt’s lymphoma; a cancer found in parts of Africa. EBV was, in fact, the first virus associated with a cancer in humans. [Gertrude Henle’s mother was murdered by the Nazis in 1943.]

Although zur Hausen took part in the Henles’ experiments involving EBV, he did so grudgingly because he was intimidated by his inexperience in molecular biology. In his own words: “I urged Werner Henle to permit me to work with a different agent, namely adenovirus type 12, hoping that this relatively well established system would permit me to become acquainted with molecular methods. He reluctantly agreed. I started to work eagerly on the induction of specific chromosomal aberrations in adenovirus type 12-infected human cells…and, to please my mentor, I demonstrated electron microscopically the presence of EBV particles directly in… Burkitt’s lymphoma cells (2).”

In 1969 zur Hausen returned to Germany to take an appointment as an independent scientist at the University of Wurzburg. His research was now focused entirely on EBV. Specifically, he wanted to challenge the prevailing view that Burkitt’s lymphoma tumors are persistently infected with EBV (i.e., that the tumors continuously produce low levels of the virus).

I presume that zur Hausen was interested in this issue because it was reasonable to believe that EBV gene expression is necessary to maintain the neoplastic state of the Burkitt’s tumor cells. Persistent infection would be one means by which viral genes could be carried by the cells. But zur Hausen believed that EBV DNA might be maintained in Burkitt’s lymphoma cells, even if they did not produce EBV particles.

Werner Henle in Philadelphia (and also George Klein in Stockholm) sent zur Hausen a large number of Burkitt’s lymphoma cell lines and tumor biopsies to aid in his study. One of those cell lines, the Raji line of Burkitt’s lymphoma cells, did not produce EBV particles. Nevertheless, zur Hausen isolated sufficient EBV DNA from the Raji cells to prove that multiple copies of EBV DNA were maintained in them. This was the first time that tumor virus DNA was shown to be present in malignant human cells that were not producing virus. See Aside 2.

[Aside 2: In 1968 Renato Dulbecco and co-workers were the first to discover viral DNA integrated by covalent bonds into cellular DNA (J. Sambrook et al., Proc. Natl. Acad. Sci. U S A. 60:1288, 1968). They were studying cells transformed by the polyomavirus, SV40. Integration explained how SV40 genes could be stably maintained and expressed in transformed cells, in the absence of productive infection. Integration is now recognized as a key event in cell transformation by members of several virus families, including the polyomaviruses, papillomaviruses, and the oncogenic retroviruses.

The situation in the case of EBV, a herpesvirus, is different, as herpesviruses are able to enter into a latent state in host cells. In the latent state the viral genome is maintained as an episome, and only a subset of the viral genes (i.e., those concerned with latency) are expressed. The episomal viral genome is replicated by the cellular DNA replication machinery during the cell cycle S phase, and a viral gene product, EBNA-1, ensures that viral genomes are equally partitioned between the daughter cells. In 1978 George Klein and co-workers were the first to demonstrate episomal EBV DNA in a cell line derived from a Burkitt’s lymphoma biopsy (S. Koliais et al., J. Natl. Cancer. Inst. 60:991, 1978).]

In 1972, while zur Hausen’s attention was focused on EBV and Burkitt’s lymphoma, his research direction took a providential turn that would lead to his most important discoveries. It happened as follows.

Recent seroepidemiological evidence was suggesting a link between herpes simplex virus type 2 (HSV-2), a well known genital infection, and cervical cancer. Since HSV-2, like EBV, is a herpesvirus, and since zur Hausen had already demonstrated that EBV DNA is present in Burkitt’s lymphoma tumor cells, zur Hausen believed he was well positioned to search for HSV-2 DNA in cervical cancer biopsies. However, in this instance, all his repeated attempts failed.

Harald zur Hausen then came across anecdotal reports of genital warts converting to squamous cell carcinomas. Importantly, those genital warts were known to contain typical papillomavirus particles. Taking these two points into account, zur Hausen considered the possibility that papillomaviruses, rather than herpesviruses, might be the cause of cervical carcinomas. Indeed, his initial thought was that the genital wart papillomavirus might also be the etiologic agent for cervical carcinomas.

Thus, Harald zur Hausen began his foray into papillomavirus research. His first experiments found papillomavirus particles in benign plantar (cutaneous) warts. His next experiments demonstrated that there are multiple papillomavirus subtypes. [In brief, zur Hausen used in vitro-transcribed plantar papillomavirus RNA as a hybridization probe against the DNA from various plantar and genital warts. Only some of the plantar wart DNAs, and none of the genital wart DNAs, reacted with his planter wart RNA probe. Restriction endonuclease patterns of a variety of human papillomavirus isolates confirmed that the HPVs comprise a heterogeneous virus family.]

Harald zur Hausen’s next experiments sought to detect papillomavirus DNA in cervical carcinoma biopsies. However, his initial trials in this crucial undertaking were unsuccessful.  He was using DNA from HPV-6 (isolated from a genital wart) as a hybridization probe in those failed attempts. But zur Hausen and co-workers had at hand a number of additional HPV subtypes, from which they prepared other DNA probes. DNA from HPV-11 (from a laryngeal papilloma) indeed detected papillomavirus DNA in cervical carcinomas.

In 1983, two of Zur Hausen’s former students, Mathias Dürst and Michael Boshart, using HPV-11 DNA as a probe, isolated a new HPV subtype, designated HPV-16, from a cervical carcinoma biopsy. In the following year, they isolated HPV-18 from another cervical carcinoma sample. Harald zur Hausen’s group soon determined that HPV-16 is present in about 50% of cervical cancer biopsies, while HPV-18 is present in slightly more than 20%. [The famous HeLa line of cervical cancer cells contains HPV-18 DNA.]

Additional key discoveries took place during the next several years, including the finding that papillomavirus DNA is integrated into the cellular DNA of cervical carcinoma cells. This finding clarified how papillomavirus genes persist in the cancers, and also revealed that the cancers are clonal (see Aside 2, above). Moreover, while the integrated viral genomes often contain deletions, zur Hausen’s group found that two viral genes, E6 and E7, are present and transcribed in all cervical cancer cells. This finding implied that E6 and E7 play a role in initiating and maintaining the oncogenic state. [In 1990 Peter Howley and co-workers demonstrated that the interaction of the E6 gene product with the cellular tumor suppressor protein p53 results in the degradation of p53. In 1992 Ed Harlow and coworkers showed that the E7 gene product blocks the activity of the cellular tumor suppressor protein pRb. Reference 3 details the mechanisms of papillomavirus carcinogenesis.]

The above findings led to widespread acceptance that cervical carcinoma is caused by papillomaviruses. Yet acceptance was not immediate. The prevailing belief, that herpesviruses cause cervical carcinoma, was well-entrenched and slow to fade away. It was based on the observation that many women afflicted with cervical carcinoma also had a history of genital herpes. But, individuals infected with one sexually transmitted pathogen are often infected with others as well. Apropos that, genital warts were long thought to be associated with syphilis, and later with gonorrhea. In any case, in 1995 the World Health Organization officially accepted that HPV-16 and HPV-18 are oncogenic in humans.

Harald zur Hausen was awarded one half of the 2008 Nobel Prize for Medicine or Physiology for proving that cervical cancer is caused by human papillomaviruses. By the time of his award, his findings had led to key insights into the mechanism of HPV-mediated carcinogenesis and, importantly, to the development of a vaccine to prevent cervical cancer. See Aside 3.

[Aside 3: The first generation of Gardasil, made by Merck & Co., helped to prevent cervical cancer by immunizing against HPV types 16 and 18, which are responsible for an estimated 70% of cervical cancers. Moreover it also immunized against HPV types 6 and 11, which are responsible for an estimated 90% of genital warts cases. Apropos genital warts, there are 500,000 to one million new cases of genital warts (also known as condylomata acuminate) diagnosed each year in the United States alone.

The original vaccine was approved by the USFDA on June 8, 2006. An updated version of Gardasil, Gardasil 9, protects against the HPV strains covered by the first generation of the vaccine, as well as five additional HPV strains (HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58), which are responsible for another 20% of cervical cancers. Gardasil 9 was approved by the USFDA in December 2014.]

Harald zur Hausen reviewed the overall contribution of viruses to human cancer in his 2008 Nobel lecture (4). Some of his key points are as follows. HPVs were discussed above with respect to cervical carcinoma. HPVs also are associated with squamous cell carcinomas of the vagina, anus, vulva, and oropharynx. What’s more, 40% of the 26,300 cases of penile cancer reported worldwide in 2002 could be attributed to HPV infection.

Epstein-Barr virus too was discussed above. This member of the herpesvirus virus family causes nasopharyngeal carcinoma, as well as Burkitt’s lymphoma. Another herpesvirus, human herpesvirus 8, causes Kaposi’s sarcoma; the most frequent cancer affecting AIDS patients. Hepatitis B virus (HBV, a hepadnavirus), as well as hepatitis C virus (HCV, a flavivirus), causes hepatocellular carcinoma. The human T-lymphotropic virus 1 (HTLV-1), a retrovirus, induces adult T-cell leukemia. And the recently discovered Merkel cell polyomavirus (MCPyV) is responsible for Merkel cell carcinoma.

Harald zur Hausen estimated that viruses directly cause about 20% of all human cancers, and a similar percentage of all deaths due to cancer! And while 20% might seem to be a remarkably high figure for the extent of viral involvement in human cancer, zur Hausen suggests that it is actually a minimal estimate. That is so because it is difficult to determine that a particular virus is actually the cause of a cancer. Consequently, it is likely that other examples of viral involvement in human cancer will be discovered.

Harald zur Hausen gave two principal reasons for why it is difficult to establish that an infectious agent is the cause of a cancer in humans. First: “… no human cancer arises as the acute consequence of infection. The latency periods between primary infection and cancer development are frequently in the range of 15 to 40 years…” Second: “Most of the infections linked to human cancers are common in human populations; they are ubiquitous… Yet only a small proportion of infected individuals develops the respective cancer type.”

Viruses also contribute to the human cancer burden in an indirect way. For instance, HIV types 1 and 2 play an indirect role in cancer via their immunosuppressive effect, which is the reason for the extraordinarily high prevalence and aggressiveness of Kaposi’s sarcoma in AIDS patients.

Bacterial infections also contribute to the cancer burden. For example, Helicobacter pylori infections may lead to stomach cancer. What’s more, the parasites Schistosoma, Opisthorchis, and Clonorchis have been linked to rectum and bladder cancers in parts of Northern Africa and Southeast Asia, where they are prevalent.

Obviously, but important enough to state anyway, knowing that a particular cancer is caused by a particular infectious agent opens the possibility of developing a rational strategy to prevent that cancer. Gardasil is an exmple. A vaccine against HBV is also available, and one against HCV is under development.

References:

1. Who discovered HIV, Posted on the blog January 23, 2014.

2. MLA style: “Harald zur Hausen – Biographical”. Nobelprize.org. Nobel Media AB 2014. Web. 27 May 2015. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/2008/hausen-bio.html&gt;

3. Norkin, Leonard C. (2010) Virology: Molecular Biology and Pathogenesis. ASM Press, Washington, D.C. See Chapters 15 and 16.

4. Zur Hausen, Harold, The search for infectious causes of human cancers: where and why. Nobel Lecture, December 7, 2008.

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SV40-Contaminated Polio Vaccines and Human Cancer

In earlier postings, we recounted how in 1959 Bernice Eddy, at the U. S. National Institutes of Health (NIH), and then Maurice Hilleman, at Merck & Co, discovered a new virus, simian virus 40 (SV40), in early lots of the Salk and Sabin polio vaccines (1, 2). The virus inadvertently contaminated those vaccines because it was unknowingly present in the rhesus monkey kidney cell cultures in which the vaccines were grown. Hilleman gave SV40 its name. It was the 40th simian virus that the Merck lab found in its rhesus kidney cell cultures.

Next, in 1961, both Eddy and Hilleman discovered that inoculating SV40 into hamsters caused cancer in about half of the animals. But, by then, hundreds of millions of people worldwide had been inoculated with live SV40, via the contaminated polio vaccines!

[Aside 1: Since the Sabin vaccine contains live, attenuated poliovirus, whereas the Salk vaccine contained formaldehyde-inactivated poliovirus, it was initially thought that any issues stemming from SV40 contamination would be limited to the Sabin vaccine. However, about one in 10,000 SV40 particles survived the formaldehyde treatment used to inactivate poliovirus in the Salk vaccine. Thus, early lots of both the Salk vaccine and the Sabin vaccine were contaminated with live SV40. But, despite the initial belief that that only the Sabin vaccine posed a threat of SV40 contamination, recipients of the Salk vaccine, but not those receiving the Sabin vaccine, developed an antibody response against SV40. Apparently, any SV40 that may have been present in the orally administered Sabin vaccine was killed in the intestinal tracts of its recipients. But, while the antibody response seen in recipients of the injection-administered Salk vaccine would be consistent with an actual SV40 infection, that response might have been only against the SV40 in the vaccine itself (see the main text).]

In July 1961, the New York Times broke part of this story, reporting that Merck was withdrawing its vaccines because they were contaminated with a monkey virus. However, the Times article did not mention the cancer connection. In fact, the Times did not report that aspect of the story until more than a year later, causing some individuals to suspect that the government deliberately withheld that information from the public.

I do not know whether or not there was a deliberate intent by the government to withhold information. Joseph Smadel, Bernice Eddy’s immediate superior at the NIH and himself an eminent scientist,  dismissed Eddy’s finding of a tumor-inducing virus in early lots of the Salk vaccine, after he reviewed her data. Later, after Eddy reported her findings at an October 1960 cancer conference in New York, Smadel forbade her from speaking about the matter again in public without first clearing her remarks with him. However, Smadel was indeed concerned when similar findings were reported to him later by Hilleman. Was sexism a factor in the dismissal of Eddy’s results? Perhaps, but it also was alleged (in fact by Hilleman) that Eddy’s experiments were poorly controlled.

At any rate, Eddy had, in fact, presented her findings at an open scientific conference. What’s more, at around the same time, Hilleman too presented his findings in public, at a conference in Copenhagen. So, their discovery, while not purposely publicized, was not kept secret either.

Next, consider that the second report in the New York Times, which mentioned the cancer connection for the first time, was buried on page 27 of the newspaper. This fact points more to the inability of the press to appreciate and cover a complex scientific issue, than to an attempt by the government to suppress the story.

In any event, the government did not alert the public to the possible danger that might be lurking in the polio vaccines. Is it possible that the NIH did not appreciate that threat? This seemingly implausible explanation is consistent with the fact that the NIH did not begin to screen all new lots of the polio vaccines for SV40 until 1963.

Another perhaps more likely possibility is that the government simply believed that alerting the public might have irrevocably broken its confidence in the vaccines, ultimately causing far more disease than might have been caused by the vaccines themselves. Even so, an impending public health debacle, of unprecedented severity, could not yet have been ruled out.

We return to our main story after a bit of background on SV40.

SV40 is a member of the polyomavirus family of small, double-stranded DNA viruses. It is one of several polyomaviruses that can transform normal cells into tumor cells in cell culture, as well as induce tumors in laboratory animals. For that reason, and because SV40 is a relatively simple virus (its genome contains only about 5,200 bases pairs), it was intensively studied for what it might reveal about tumor genesis. Moreover, it also came to serve as an important model to investigate fundamental issues in eukaryotic molecular biology (3).

The polyomaviruses are widespread in their natural hosts, in which they give rise to lifelong, usually benign, persistent infections. The Asian rhesus macaque is the natural host for SV40. These facts explain why SV40 was not evident in the rhesus monkey kidney cell cultures that were used to propagate the early poliovirus vaccine lots. SV40 does not cause sufficient cytopathology in those rhesus macaque cell cultures to reveal its presence in them. However, culture fluids from those rhesus cell cultures caused extensive cytopathology when added to African green monkey kidney cell cultures. Indeed, that is how SV40 was discovered.

[Aside 2: The Human Polyomaviruses The JC polyomavirus (JCPyV) and the BK polyomavirus (BKPyV), each discovered in 1971, are the best known polyomaviruses that naturally infect humans. JCPyV and BKPyV are each ubiquitous in their human host, in which they typically give rise to lifelong, benign, persistent infections. Yet, JCPyV can give rise to a rare but fatal demyelinating disease, progressive multifocal encephalopathy (PML), in immunologically compromised individuals. And BKPyV can cause kidney disease, also in immunologically compromised persons.

More recently, several additional human polyomaviruses have been discovered, by means of modern DNA amplification procedures. One of these viruses, the ubiquitous Merkel cell polyomavirus (MCPyV), is associated with a rare, aggressive human malignancy, Merkel cell carcinoma, and is the best candidate for an oncogenic human polyomavirus.]

We now resume our main story, with the following key points.

Despite the fact that the unintended exposure of millions of individuals to SV40 via the contaminated polio vaccines in the 1950s posed a potential public health crisis of immense proportions, it still is not clear whether SV40 is an agent of human disease. Moreover, it is not known whether SV40 is circulating in the human population. How can this be?

Early investigations into this matter in the 1960s were compromised by the fact that it was not apparent which individuals had actually received SV40-contaminated vaccines and which did not. That was so in part because the serological reagents and procedures of the day were not sensitive or accurate enough to generate unambiguous results. Additionally, population sample sizes were often too small to generate statistically significant results. [This was especially so in the case of the rare childhood tumors in which SV40 had been implicated.] And, since cancer is a disease that may take decades to emerge, it was possible that more time needed to pass before the virus might unequivocally reveal itself as a cause of human cancer. At any rate, since the substantial experimental data then available could neither establish nor absolve SV40 as a cause of cancer in humans, the NIH conceded that more research and better methods for detecting the virus would be needed to settle the issue.

The more recent development of extremely sensitive polymerase chain reaction (PCR)-based procedures, which can detect minute levels of specific DNA sequences, led to renewed interest in whether SV40 might be present in humans, and whether it might be an agent of human disease. Using PCR technology, several different research groups detected SV40 DNA in four types of human cancers; mesotheliomas, osteosarcomas, non-Hodgkin’s lymphomas, and childhood brain tumors. These findings were alarming because the four tumor types, in which SV40 was detected in humans, are the same tumors that SV40 induces experimentally in hamsters (i.e., mesothelioma, bone, lymphoma, and brain).

PCR procedures also detected SV40 DNA in individuals who never were inoculated with an SV40-contaminated vaccine. This too was disturbing because it raised the specter that SV40 might be circulating in the human population, spreading by horizontal transmission from one individual to another.

Yet the issue of SV40 in humans remains controversial because other studies, from other research groups, using similar PCR procedures, could not detect SV40 DNA in human tissues. In addition, newer, more sensitive and accurate serologic procedures could not demonstrate to everyone’s satisfaction that SV40 circulates in humans.

How might we explain how capable scientists, using powerful and proven techniques, can obtain such disparate experimental results? Ironically, the sensitivity of PCR itself may be a problem, since it increases the likelihood of false-positive results, which may occur from the slightest sample contamination. Thus, it is important to have suitable positive and negative control samples that might be processed side-by-side with test samples; a sometimes difficult criterion to fulfill. [Bearing the above in mind, consider the following example, in which a human mesothelioma sample was micro-dissected to separate normal tissue from the actual tumor. SV40 sequences were detected in the tumor, but not in the adjacent normal tissue, which served as an internal control.]

Another potential source of error stems from the widespread prevalence of human polyomaviruses (e.g., JCPyV, BKPyV, and MCPyV) in the human population, leaving open the possibility that these viruses, rather than SV40, are detected by the PCR-based procedures. But, with that possibility in mind, several researchers took the extra step of confirming the presence of SV40 sequences by direct sequencing of the PCR-amplified DNA. The ubiquitous human polyomaviruses are also a concern when carrying out serological procedures, since immune cross-reactivity between SV40 and these viruses remains a potential source of error.

Another problem is theoretical rather than technical. An underlying premise behind these studies is that the continued presence and expression of polyomaviral tumor genes are necessary for a tumor cell to express its tumor cell characteristics. Indeed, this was shown to be the case fifty years ago for cells transformed in culture by polyomaviruses. Thus, the absence of SV40 DNA, or SV40 tumor antigens, in a tumor is taken as evidence against SV40 as the cause of the tumor. However, there is some experimental evidence that the paradigm itself may not always be true. Cancers result from a complex multistage course of events and, in some instances, the virus may play a necessary role only at a particular point in the overall process. Thus, the absence of SV40 DNA, or SV40 antigens, in a tumor may not be definitive proof against viral involvement in the tumor process.

The above points help us to appreciate why there is no consensus regarding a role for SV40 in human cancer, and indeed whether SV40 might be circulating in humans. Yet we remain troubled by the fact that SV40 can transform a variety of cells in culture and can induce tumors in laboratory animals. And there are additional experimental findings that while contentious, cannot be easily ignored. For instance, the types of human cancers, in which SV40 DNA was detected by some researchers, are the same types of tumors that SV40 induces in laboratory animals. Also, infectious SV40 was isolated from a brain cancer of a 4-year-old child. [LT/pRb and LT/p53 complexes were identified in human brain tumors, consistent with current understanding of how SV40 induces neoplasia (3).]

Yet, notwithstanding the force of the above arguments, impressive evidence has been presented against a role for SV40 in human cancer. And, if SV40 indeed were responsible for human cancer on a large scale, then it is rather certain that there would be little if any uncertainty in that regard.

1. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical   Science, posted on the blog, March 27, 2014

2. Maurice Hilleman: Unsung Giant of Vaccinology, posted on the blog, April 24, 2014

3. Virology: Molecular Biology and Pathogenesis, Leonard C. Norkin, ASM Press, 2010.

Maurice Hilleman: Unsung Giant of Vaccinology

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

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

hillemanMaurice R. Hilleman: The greatest vaccinologist.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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