Panoramic Synthesis – Rome, 21.11.2008 – Forum

Australia/UK

Ian Frazer

Balzan Prize 2008 for Preventive Medicine, Including Vaccination

For his outstanding scientific achievement and lasting contribution to preventive medicine through his role in the development of a vaccine that promises to prevent virus-induced carcinoma of the cervix, which claims 250,000 lives every year.

Infection of the cervix with papillomavirus is necessary for initiation of cervical cancer, a discovery by Prof Harald zur Hausen in the early 1980s which was recently recognised by the award of the Nobel Prize for Medicine. Cervical cancer is a relatively rare and late consequence of infection with sexually transmitted virus, which most of us acquire and resolve without our ever knowing that we’ve had it. Cervical cancer nevertheless kills over 0.25million women worldwide each year, mostly in the developing world, and mostly while the woman is still rearing and educating her family. Screening using cytological tests to detect cancer or pre-cancer cells, or viral tests to detect persisting viral infection, can reduce the burden of cervical cancer, but is not widely available in the countries with the biggest problem with this disease.

I became interested in papillomavirus infection in the early 1980s, shortly after Prof zur Hausen and his colleagues made their seminal observations. At that time I was training as an immunologist with Prof Ian Mackay in Melbourne, Australia, having recently emigrated from Scotland. I observed that men whose immune system was damaged by infection with the virus we now call HIV/AIDS could not clear genital warts effectively. Warts are caused by a papillomavirus related to the virus responsible for cervical cancer. These viruses are rather unusual as they can infect only skin cells, and do not spread through the blood like most viruses. Also they do not kill the cells they infect, as other viruses commonly do, but actually make them live longer. I became interested in how the immune system, our defence against infection, handles this sort of infection, as the persistence of papillomavirus infection suggested that it seemed to evade our immune defences quite effectively. I didn’t set out to develop a vaccine to prevent papillomavirus infection, though vaccines were always somewhere on the agenda because they had been my interest since I started training as an immunologist. Rather, I wanted to understand what the immune mechanisms were, that might lead to clearance of papillomavirus infection. The research work on papillomavirus vaccine development that lead to the award of the Balzan prize was largely undertaken in collaboration with my late colleague, and then postdoctoral research fellow, Dr Jian Zhou during the early 1990s in Brisbane, and was part of a 25 year research program to understand the immunology of persisting papillomavirus infection, and hence to develop immune interventions to prevent cervical cancer, that is still ongoing!

Papillomaviruses (PV) were in the late 1890s among the first known viruses, or ‘‘filtrable agents’’ described. In the 1930s Peyton Rous showed that the Shope (cotton-tail rabbit) PV causes epithelial cancer in rabbits. However, human papillomaviruses were difficult viruses to work with experimentally, as they could not be grown in the laboratory, or in animals. This precluded the normal methods of viral classification according to their reactions with sera from infected patients. Human papillomaviruses were recognised to be responsible for skin and genital warts, but with the limited tools available there was little knowledge of their immunological properties. Since warts in humans “never” turned into cancer, there was little incentive to study these difficult viruses intensively. The association of papillomavirus infection with human epithelial cancer was first suggested by the work of Zur Hausen and colleagues in the late 1970s. Increasing availability of molecular techniques for cloning and eventually sequencing viral genes, which I had acquired along with my knowledge of viral immunology a the Walter and Eliza Hall institute in Melbourne, enabled work on papillomaviruses which had not previously been possible, and gave me the tools to work on the immunology of papillomavirus infection. Subsequently, improved methods for detecting papillomaviruses via DNA hybridization allowed extensive epidemiological studies by the International Agency for Cancer Research (IARC) and others, and these studies have eventually established that approximately 100% of cervical cancer, and 10-50% of other anogenital and head and neck cancers, can be attributed to ‘‘high risk’’ human PVs. But, in 1985, when I started studies on papillomavirus immunology, that was all in the future.

My initial observations in Melbourne were that warts were poorly cleared in patients with HIV with a damaged immune system, and also in patients with immune impairment from administration of the then increasingly available immune suppressants given for autoimmune disease and to protect kidney transplants. Papillomavirus infections resolve more slowly than most viral infections even in immunocompetent subjects, but the patients with HIV didn’t seem to clear these infections at all. Zur Hausen’s work suggested that persisting HPV infection of the cervix could cause cervical cancer, and along with a colleague in Melbourne, Dr Gabrielle Medley, I looked for persisting infection in the anogenital skin of the HIV positive patients. We found not only that they had persisting papillomavirus infection, but also that at least some of them had precancerous lesions of the anal canal. This was particularly interesting, as it suggested that HPV infection could cause cancer at more than one site. I was also intrigued that the association of immunosuppression with anal cancer that we had observed might be a first example in humans of immune surveillance against cancer, a hypothesis first made by Sir MacFarlane Burnett in the 1950s and without, at that time, good examples in either animals or humans. The idea that the immune system could defend against papillomavirus associated cancer led me to pursue development of an immunotherapeutic for cervical cancer, a goal I’m still pursuing in 2008! When I moved in 1985 from Melbourne to Brisbane, to take up a new position as director of the clinical immunology service at the Princess Alexandra Hospital, I decided that this would be the focus of the research work I undertook there. With Dr Robert Tindle, who joined the lab as a research fellow, I studied immune responses to the papillomavirus proteins associated with persistent infection and cancer. Two non-structural proteins, termed E6 and E7, which substantially alter epithelial cell growth, and seem necessary for cancer formation, continue to be expressed in HPV-associated cancers. Cell-mediated immune responses to these proteins, and also to another viral protein, E2, have subsequently been associated with regression of infection. The cellular mechanisms inducing wart regression remain uncertain to this day, although there may be clues in the ability of topically applied imiquimod, an activator of Toll-like receptor 7 (TLR7) and TLR8 and a promoter of local inflammation, to induce regression. Our initial animal studies demonstrated immunogenicity of the papillomavirus non-structural proteins. However to study immune responses to these proteins in infected humans, we needed humans cells expressing papillomavirus antigens as targets for an assay of cell-mediated immunity, and we also needed animal models of persistent epithelial infection without in?ammation, to study how the immune response determined the outcome of infection.

These needs led me to take sabbatical leave in 1989 to visit the laboratories of Prof Lionel Crawford and Prof Margaret Stanley, both acknowledged experts in HPV infection, in the department of Pathology at Cambridge University. I wished to learn how to clone viral genes from clinical samples and express these in epithelial tissues and transgenic animals. I found that E7, when over-expressed in cells, provoked early cell differentiation and death, which hindered the development of target cells for immunoassays, and animal models to study infection. An E7 transgenic mouse was subsequently made available to me during a sabbatical, in 1993, in the lab of Paul Lambert in Madison, and became a critical tool for our ongoing study, with Dr Lambert and many other colleagues, of therapeutic vaccines for papillomavirus infection. However, the Cambridge visit resulted in my meeting with Dr Jian Zhou, a Chinese doctor and scientist, at that time working with Lionel Crawford on expression of papillomavirus genes in mammalian cells using recombinant vaccinia virus. I could see that this work if successful would allow us to create targets for immune assays using human cells. Together, we came up with the idea that if papillomavirus genes when over expressed singly were lethal to cells, and natural HPV16 virus, the virus most responsible for cervical cancer, was not available as a laboratory reagent, we might be able to make targets for immune assays by construction of an artificial HPV16 papillomavirus and using it to infect cells to make targets for our assays. We thought this might be achieved by packaging the viral genome in the capsid proteins, and we eventually achieved this in 1993. However, in 1989, our visits to Cambridge were drawing to a close, and I persuaded Jian that we could work together if he and his wife, Xiao Yi Sun, came to Brisbane, which he did in late 1990. In early 1991, as part of our strategy for making synthetic HPV16, we demonstrated that expression of the two viral capsid proteins of HPV16 (L1 and L2) in monkey kidney cells, via a doubly recombinant vaccinia virus, resulted in assembly of virus-like particles (VLPs), visible with electron microscopy. This was a first convincing demonstration that HPV16 could actually form a capsid, as this virus had not been seen with electron microscopy in HPV16-associated clinical lesions. To achieve the production of the virus capsid, we expressed the major capsid protein of HPV16 (L1) from the second initiation codon in the L1 gene. We identified this initiation codon by comparing the gene sequences of the various PV L1 genes then sequenced. This primitive exercise in comparative genomics was undertaken with paper and pencil because our only lab computer was not up to the job. We and others including Rose in Rochester and Kirnbauer in the Lowy and Schiller lab at the NIH subsequently demonstrated that PV L1 genes of various HPV types, if expressed with more efficient eukaryotic expression systems, would self assemble into viral capsids (VLPs) without L2, albeit with somewhat reduced efficiency. L1 VLPs are deficient in the ability to package viral DNA, and for the production of infectious virions we needed L2, although better techniques for producing infectious pseudovirions have subsequently been developed by Roden and Schiller. However the virus like particles made with L1 alone resembled the natural virus physically, and more importantly, immunologically, and once we’d established this it seemed logical that the virus like particles might become the basis of a vaccine to prevent this unwanted infection that could cause cancer.

We had been talking for some time with our colleagues at CSL, an Australian based vaccine company, about the possibility of making vaccines to treat papillomavirus infection, and when we saw that we could produce virus like particles which induced antibody responses in animals, we suggested to them that they might be interested in partnering with us on a project to make a vaccine to prevent papillomavirus infection. At this time, we had been presenting our findings at various international meetings, perhaps most significantly at the International papillomavirus workshop in Seattle in September 1991, and this presentation had aroused the interest of a number of vaccine companies including Merck and GSK. Over the next few years considerable further interest vaccines to prevent HPV infection was engendered by the strengthening evidence that two papillomavirus types (HPV16 and HPV18) were together responsible for the majority of cervical cancers. Studies by Campo and Jarrett had shown that cows were protected by infection with bovine papillomavirus against further viral challenge, and could also be protected against challenge by prior immunization with formalized virions prepared from cow warts. Protection was associated with antibody that could bind to bovine PV1 virions. Similar results were obtained for canine oral papillomavirus, which infected the dogs’ oral mucosa, by Bennett Jensen and colleagues. The canine oral PV model showed that virus like particles could induce protection against challenge with live virus, and that protection could be transferred to another animal by the immunoglobulin fraction of serum. These results suggested that vaccines to prevent human PV based on virus like particles would likely be successful if they induced neutralizing antibody. However, the human papillomavirus infections of greatest interest occurred at mucosal surfaces and there were no successful viral vaccines to prevent infection at mucosal surfaces, so there was considerable scepticism about the concept of producing a successful cervical cancer vaccine within the scientific community. Also, despite the extensive amino acid sequence homology (>80%) between the major capsid proteins of the different papillomaviruses, each papillomavirus genotype turned out to be a largely distinct serotype, at least as far as antibodies raised against the native virus structure are concerned. It therefore seemed likely that any papillomavirus vaccine would only protect against the virus types incorporated into the vaccines. In 1985, a critical step towards vaccine development occurred with the signing of an agreement between the University of Queensland, CSL, and Merck to enable Merck to develop under licence a vaccine for cervical cancer based on the virus like particle technology we had developed. This project was particularly championed at Merck by Dr Kathrin Jansen.

Two vaccines to help with cervical cancer prevention are now available, each based on the virus like particle technology that Jian Zhou and I developed back in 1991. Gardasil, produced by Merck, includes VLPs that are produced from recombinant yeast and correspond to two human papillomavirus types (HPV16 and HPV18) responsible for about 70% of cervical cancer and two types (HPV6 and HPV11) responsible for >90% of genital warts. Cervarix, produced by Glaxo Smith Kline (GSK), includes VLPs that are produced in insect cells via recombinant baculovirus and that correspond to HPV16 and HPV18. Several clinical trials of the two commercial vaccines have been undertaken in 18-to 26-year-old sexually active women. These trials have shown that the vaccines are 100% effective at preventing not only infection with the high-risk human PVs incorporated in the vaccines but also at preventing the resulting cervical precancer lesions and external anogenital lesions, including genital warts attributable to the vaccine incorporated human PV strains. The vaccines are protective against infection, but are not therapeutic for existing infections. They have proven safe in clinical trials, with only local pain and swelling, and very occasional allergic reactions, amongst over 29 million doses delivered worldwide over the last two years. For both vaccines, antibody responses are almost universal among immunized subjects previously naive to the relevant papillomavirus type. Peak antibody responses after three immunizations gradually fall over the first two years and then plateau at an amount well above the average observed in response to natural infection, suggesting that these vaccines will give long term protection against HPV infection and cervical cancer. The current vaccines can prevent the 70% of cervical disease caused by the two cancer causing HPV types in the vaccines, HPV16 and HPV18, though there is no reason why more types cannot be added to give 100% coverage .

The prerequisite for successful vaccination to prevent cervical cancer is delivery of vaccine before women are exposed to the viruses. Public health vaccination programs have therefore been introduced in a number of countries for routine immunisation of all 12 year old girls. My ongoing involvement in vaccine delivery programs has been aimed at demonstration that these vaccines can be deployed effectively in the developing world. The challenge is to deliver three doses of vaccine to 12 year old girls in countries where knowledge of cervical cancer is minimal, and there are no public health measures aimed at adolescents. With Australian and local colleagues, we have established vaccine programs in rural Nepal, and are about to start a program in Vanuatu. These countries, both largely dependent on subsistence farming and tourism, have very high incidence of cervical cancer, and have little prospect of establishing screening programs in the near future. I believe that my job with the cervical cancer prophylactic vaccines, which started with the technology, and then faced the challenge of finding and interesting commercial partners, will not be properly complete until we have managed to implement programs worldwide to protect women against cervical disease. The current vaccines can only be a part of that program, and screening will need to be implemented to protect the very many women, perhaps about 20 million, who already have an infection with a high risk HPV infection which if left untreated will go on to develop and die from cervical cancer.

My initial research goal was to develop therapeutic vaccines to treat patients with cervical cancer, and the precancer lesions that are detected by screening programs. This goal has not been forgotten. Using model systems that I have been given access to by many colleagues world wide, we have developed strategies for testing potential vaccines to treat persistent viral infections of skin, and particularly papillomavirus infection. We have learned much about the immunology of skin through this work, and have discovered that our simplistic approaches to immunotherapy were indeed too simple. Vaccines can be used to develop the right sort of immune response in animals and in humans. However, the immune effector cells seem able, or perhaps are persuaded , to ignore the skin cells expressing viral proteins. This occurs through a complex range of regulatory mechanisms, and my current research is focussed on laboratory and clinical trials of potential strategies for overcoming the regulatory mechanisms, and hence enabling development of vaccines which might be given safely to young women with persisting papillomavirus infection. It is my hope that these vaccines could be used in the developing world, and help to reduce the significant burden of cervical cancer that will continue to be experienced there for at least the next 50 years.