Virologists scoop

The discoverers of HIV and the cervical cancer virus share the Physiology Nobel.

THE discoverers of two viruses associated with serious human diseases have been chosen for this years Nobel Prize in Physiology or Medicine. One half of the prize money ($1.4 million) has gone to 72-year-old Harald zur Hausen of the German Cancer Research Centre, Heidelberg, for his discovery of human papilloma viruses [HPV] causing cervical cancer and the other half jointly to 61-year-old Francoise Barre-Sinoussi of the Pasteur Institute, Paris, and 76-year-old Luc Montagnier of the World Foundation for AIDS Research and Prevention, Paris, for their discovery of human immnunodeficiency virus [HIV].

The latter has, not unexpectedly, generated some controversy as it has ignored the contributions of the American researcher Robert Gallo, a name that is closely linked to HIV. Indeed, in a recent article on the Nobel Prize, the journal Scientific American included Gallos omission among the Top 10 Nobel Snubs. It was Gallos team at the National Cancer Institute of the U.S. National Institutes of Health (NIH) that proved that the acquired immune deficiency syndrome (AIDS) was indeed caused by HIV.

However, this finding, which was published in 1984 in Science, was clouded by controversy and charges of scientific misconduct when, a year later, the Pasteur Institute (where Montagnier was working at that time) sued Gallo for using one of its samples of HIV to draw his conclusion on the virus connection to AIDS. In 1992, a review panel of the National Academy of Sciences determined that the sample Gallo used was contaminated with material from the Pasteur Institute and accused him of intellectual recklessness of a high degree. That same year Gallo was also indicted for scientific misconduct by the Office of Research Integrity of the United States.

However, both charges were dismissed on appeal. In 1987, U.S. President Ronald Reagan and French Prime Minister Jacques Chirac defused the controversy and ended the legal battle by proclaiming that the two researchers were co-discoverers of the virus and agreeing to share the patent royalties between the two countries. Gallo and Montagnier accepted the compromise and later even co-authored two landmark accounts of HIV research in Scientific American in 1988 and later in Science in 2002, in which they credited each other with different aspects of the discoveries about HIV and its role in AIDS. The controversy, in fact, resurfaced in 2000, when the NIH carried out yet another investigation. But Gallo was cleared of any misconduct.

Montagnier and Barre-Sinoussi isolated the virus, identified as HIV-1, from a French patient with swollen lymph nodes. They also detected the activity of the enzyme reverse transcriptase that proved that the virus belonged to the family of retroviruses. (Retroviruses have an RNA, or ribonucleic acid, genome and use the reverse transcriptase to convert RNA into DNA, or deoxyribonucleic acid, which is then inserted into the genome of the hosts where they replicate.) From the virus morphological, biochemical and immunological properties, Montagnier and Barre-Sinoussi characterised HIV-1 as a lentivirus, a slow-acting retrovirus. This was the first human lentivirus to be discovered.

But Montagnier did not prove that HIV caused AIDS. That crucial link was provided by Gallo and co-workers a year later. His team also played an important role in developing the technology to grow HIV in culture and also made significant discoveries about HIV genes and how the virus entered cells. The Nobel Committee has, however, strongly defended its decision to award only the French researchers on the grounds that as per the wishes of the donor, Alfred Nobel, the award should be given only to a discovery.

The committee said: These two persons provided the virus. It is completely evident that the discovery was made in Paris. It is quite clear if you go to the scientific magazines. The virus discovery by the French scientists has, of course, led to epidemiological surveys, tracing of the origin of HIV-1, identification of novel steps in the retroviral replicative cycle, and evolution of various therapeutic as well as prophylactic strategies.

I am surprised [and] very sorry for Robert Gallo, Montagnier told Science NOW from Ivory Coast, where he was attending a meeting on HIV. It was important to prove that HIV was the cause of AIDS and Gallo had a very important role in that, he said. On the other hand, Gallo, who is now the co-director of the Institute of Human Virology at the University School of Medicine in Maryland, said that all the three recipients of the prize deserved it and that he was happy to see that the Nobel Assembly at long last had given an award to the HIV/AIDS field. He admitted that he was disappointed at being omitted. Yes, I am a little down about it, but not terribly, he told Science NOW. The only thing I worry about is that it may give people the notion that I might have done something wrong.

AIDS was discovered as a new serious medical syndrome in 1981, mainly in California and New York, which caused different life-threatening medical conditions. The clinical AIDS spectrum was defined as repeated opportunistic infections, specific malignancies and autoimmune phenomena occurring in hitherto healthy adults with no history of inherited disorders.

This immunodeficiency was seen to be associated with rapid elimination of CD4+ T cells (lymphocytes that express the protein CD4 and play an important role in maximising the immune response of the host) and antigen presenting cells (which enable T cells to express CD4+). Epidemiological studies had already established that AIDS was transmitted sexually, via placenta to foetuses and via transfusion by plasma and coagulated blood products. However, it was not initially obvious that AIDS was one disease and that the diversity of symptoms which involved almost all organ systems the immunodeficiency syndrome could have a single cause. By the end of 1982, several virus laboratories were trying to establish the cause of the AIDS.

Evidence was already mounting towards the retroviral origin for the acquired immunodeficiency seen in patients: clusters of patients affected, the transmission via filtered blood products and loss of CD4+ T helper lymphocytes.

A working group headed by Montagnier and Barre-Sinoussi at the Pasteur Institute collaborated with clinicians to establish this hypothesis. Usually, a generalised disease and swelling of the lymph nodes precede AIDS. In 1983, the two scientists isolated cells from the swollen lymph nodes of patients, an early sign of acquired immune defect. The idea being that in the early stage of infection, lymphocytes should still have CD4+ cells, which the virus targeted. This was the strategy that led to the Nobel Prize-winning discovery of HIV.

A hypothesis that was gaining ground at that time was that the cause of AIDS was a close relative of human T-lymphotropic virus type 1 (HTLV-1), the first human retrovirus to be isolated by Gallo. However, the virus isolated by Montagnier and Barre-Sinoussi did not react significantly with the reagents specific to HTLV. This suggested the presence of a new, previously unidentified virus. Cultured cells from patients were found to generate an extracellular release of viral particles that could infect lymphocytes from healthy individuals and newborns. The virus isolate was, therefore, initially called lymphadenopathy associated virus (LAV) in 1983.

Barre-Sinoussi and Montagnier also studied a pair of siblings with haemophilia B (treated with Factor VIII). One was healthy but the other had symptoms of AIDS. A virus similar to LAV was isolated in both cases with typical lentivirus morphology, distinct from HTLV types. They called this the immunodeficiency associated virus (IDAV-1 and IDAV-2 respectively). They also isolated the virus from diseased patients and demonstrated that the virus could cause severe deterioration of cells (cytopathy). However, unlike the other known cancer-causing retroviruses, the novel retrovirus did not induce uncontrolled cell growth. Instead, the virus required cell activation for replication and mediated fusion of white blood cells.

Antibodies to the new virus also began to be discovered soon in individuals at risk for AIDS as well as those with AIDS and AIDS-related conditions. This lent strong support to the hypothesis of Barre-Sinoussi and Montagnier that LAV/IDAV was crucial to the disease. In subsequent studies, the two isolated the same type of virus from sexually infected individuals, haemophiliacs and blood-transfused patients. Evidence was also seen in mother-to-child transmission. In 1984, Gallos group at the NIH also detected a novel HTLV-like virus from many patients with AIDS or pre-AIDS symptoms. The virus shared some properties with HTLV-1 and HTLV-2 and was denoted as HTLV-3. But it was found to have greater similarities with LAV-1. Jay Levys group in San Francisco also identified a virus of the lentivirus group, which was structurally related to LAV-1 and HTLV-3 from AIDS patients and patients with lymphadenopathy, and called it AIDS-associated retrovirus (ARV). The American and French teams later agreed that LAV-1/IDAV-1 & 2/HTLV-3 and ARV were all the same type of virus. In 1985, an international taxonomy consortium chosen to name the virus called it HIV-1.

The discovery also made HIVs molecular cloning possible. This led to the understanding of important details of HIVs replication cycle and how the virus interacts with the host. This, in turn, led to the development of diagnostic tools for the identification of patients and the screening of blood products, which has contained the spread of the pandemic. This has also resulted in the development of several classes of antiretroviral therapy.

Despite these advances, serious problems remain to be tackled through research. Most notably, progress in developing an HIV-1 vaccine is restricted by an incomplete understanding of immunity to HIV-1 infection and the induction of appropriate immune responses. Lastly, there is no therapeutic cure visible in the horizon owing to the inherent difficulty in eliminating either the pro-virus in infected cells or the cells themselves, as AIDS patients carry a latent pool of cells with the potential of virus replication after long dormant periods. Montagnier said in a recent interview: Even after 20 years, we are still fighting this virus, very strongly, and the AIDS epidemic is still spreading in Africa. So the fight is not finished. But, nevertheless, 25 years after HIV-1 was discovered, remarkable insight has been gained into the nature of the infection and the disease.

Harald zur Hausen, the winner of the other half of the Nobel award, went against the prevalent dogma and postulated in the early 1970s that an oncogenic human papilloma virus caused cervical cancer, the second-most common cancer among women. HPV was a virus hitherto known to infect the skin and other mucous membranes. In general, carcinomas of the anogenital tract, in particular cervical cancer, had been for long believed to be caused by a sexually transmitted agent.

On the basis of sero-epidemiological data, the prevailing medical view was that the tumour was caused by herpes simplex virus type 2 (HSV-2). This was also supported by the induction of cervical cancer in animals. However, zur Hausen consistently failed to find the HSV-2 DNA in cervical cancer cells when applying a certain molecular technique in situ, which he had successfully used in the identification of the Epstein-Barr virus from different carcinoma types.

In 1974, zur Hausen made the bold jump and postulated HPV to be the causative agent for cervical cancer. His argument was that tumour cells, if transformed by a virus, would harbour the viral genetic information integrated into the host genome. This causality criterion that the viral genome should be persistently present and expressing in the cells added one more factor to the classical Kochs postulates on the criteria for infection. He suggested that the HPV-DNA could exist in a non-productive state in the tumours that is, not engaged in viral replication and production of viral particles and should be detectable though specific searches.

Zur Hausen said in a recent interview: I reported for the first time in public in 1974 at a meeting in Florida, where there was a meeting specifically scheduled for HSV-2 in cervical cancer, our negative results in trying to find HSV-2 in cervical cancer cells. And at that time I stated that it would be very worthwhile to look rather into genital papilloma viruses, because the reasoning for me was that I had surveyed the literature and found a number of anecdotal reports on malignant conversion of genital warts. My statements were not well received, and I felt a lonely voice at the meeting.

It is interesting to know that zur Hausen was not interested in the papilloma virus right from the beginning. He was mainly interested in infectious agents in human cancer. So papilloma virus came as the most likely candidate in my viewpoint, says the Nobel laureate. He pursued this idea for over 10 years by searching tumour cells for such viral DNA. However, the search was made difficult by the fact that only parts of the DNA were integrated into the host genome.

Though he found typical viral particles in biopsies of all types of genital tumours and warts, he discovered that certain types were negative for the virus itself. So he made another bold hypothesis of genetic diversity among HPV types. His team subsequently used DNA purified from virus particles of different warts and tumours to develop probes for distinct molecular markers in HPV isolates from many patients. This led to the identification of multiple HPV strains 1-3 and the conclusion that only certain HPV types caused cancer while others led to warts and benign tumours.

Recounting the discovery, zur Hausen says: So there was not, as initially assumed, only one papilloma virus type but a multitude of different, different types. But we concentrated on trying to isolate the viruses from genital warts because initially we suspected that the same virus may be responsible for cervical cancer development. In 1979-80, we could finally identify HPV6. Subsequently, we could detect another one, HPV11, which was also in genital warts and laryngeal papillomatosis. In fact, in the subsequent period we were disappointed not to find HPV6 and 11 in cervical cancer, with one rare exception. But the probes helped us to identify HPV16 and 18 subsequently. That was in a way for us a breakthrough because at that time interest was awakening very substantially and quite a large number of groups were collaborating and starting to work on papilloma viruses, and ask us for the probes, so we dispersed them very freely throughout the world.

These discoveries by zur Hausen led to a paradigm shift in the field. The premise developed by him has led to a characterisation of the natural history of HPV infection, an understanding of the HPV-induced cancer-causing mechanisms and the development of vaccines against HPV. Of course, there were incidents of some who had used zur Hausens probes going on to claim patents on some of the virus types. Improved molecular techniques and accumulated epidemiological data have since confirmed his original discoveries. Indeed, in some studies as much as 99.7 per cent of the cervical cancers investigated have been found to be HPV-positive.

Today, we know more than 100 HPV genotypes of which 40 infect the genital tract, and about 15 of these put women at high risk for cancer. In a vast majority of cases, the immune system clears the HPV infection before it causes harm. Fifty to 80 per cent of a sexually active population becomes HPV-infected during a lifetime. The actual life-long risk for cervical cancer in those infected is not known and depends on the type of HPV infection plus the genetic and immunological background. Studies of 3,000 women from 25 countries found HPV16 and 18 in over 70 per cent of malignant cases, with only minor geographical differences. The next five most prevalent types account for another 20 per cent, whereas the remaining 10 per cent are caused by other HPV-types.

About 500,000 new cases of cervical cancer are diagnosed every year with a 10-fold higher incidence in developing countries, resulting in 250,000 deaths. It affects about one per 120 women and kills about nine per 100,000 per year. Zur Hausens discovery more than 30 years ago that a restricted number of HPV types cause cervical cancer has led to a hope for vaccines today. A quadrivalent vaccine available today includes immunogens for four types of HPV (6, 11, 16 and 18) and has been shown to be effective in protecting against certain forms of the infection. A divalent vaccine has also been developed based on proteins from HPV16 and 18. The two HPV vaccines together provide protection against infection types that cause approximately 70 per cent of invasive cervical cancers worldwide.

However, whether these vaccines are effective in preventing not only cervical lesions but also cervical cancer and death must await epidemiological data in the coming decades, notes the background note from the Nobel Foundation. In addition, the duration of the vaccines protection is unclear. Since the vaccines aim to protect against strains that cause 70 per cent of cervical cancers, will other strains emerge as a major cause of the tumours? These are open questions, but zur Hausens work has shown that there is a much stronger link between infectious diseases and cancer than previously thought.