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Papillomavirus: gy and clinical

plications for unotherapy

15th International Papillomavirus Workshop Gold Coast, Queensland,

Australia, 2-6 December 1996

First Daydream Island Tumour Immunology Meeting Daydream Island, Whitsunday Islands,

North Queensland, Australia,

6-9 December 1996

~)maviruses are a remarkable and fascinat- ~oup of associated with human pathogens !ases ranging from the humble wart to can- the intricacies of these associations were iussed recently at the annual, broad-ranging rnational Papillomavirus Workshop. Three ~s were prominent:

Human papilloma virus and the ' natural history of cervical cancer he relationship between infection with the high- !k genital human papilloma viruses (HPV16, ~V18 and their relatives) and cervical cancer Hnevitably dominated epidemiological stud- ~l~have often thought that epidemiologists, ~: descended in spirit from the medieval D logians who debated at length the number alng~Js who could fit on the head of a pin. ~arkably, at this meeting there was general

greement about HPV infection and the natural ;listory of cervical neoplasia. In most women,

infection with genital HPV (as detected by the presence of HPV DNA in cervical smears or scrapes) is a transient phenomenon; but in some women HPV DNA from high-risk viruses per- sists, and in these cases cervical intra-epithelial neoplasia (CIN) is usually present, and often of a high grade [Eduardo Franco, McGill University, Montreal, Canada; Mark Schiffman, National Institutes of Health (NIH), Bethesda, MD, USA]. Schiffman presented data to support the concept that the natural history of cervical neoplasia is, on the whole, a stepwise progression, with most cases of high-grade CIN arising from pre-exist- ing low-grade lesions. None the less, a sizeable minority of high-grade CIN arises in women with equivocal lesions. Crucially, almost no cases of CIN arise in HPV- women. This message was reinforced by data showing that detection using hybrid-capture of DNA from high-risk HPV in equivocal smears (smears that cannot be cat- egorized as neoplastic) was highly sensitive

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© 1997 Elsevier Science Ltd Plh S 1357-4310( 97 )01028-9 2 3 9

M e e t i n g s M O L E C U L A R M E D I C I N E T O D A Y , J U N E 1 9 9 7

and moderately specific for the identification of underlying CIN (Michelle Manos, Kaiser Permanente, CA, USA). Indeed, in my opinion, data presented at this meeting probably provide the strongest evidence so far that detection of HPV DNA and HPV typing are essential adjuncts to - and could eventually replace - cervical cytology.

HPV serology has become a 'respectable oc- cupation' with the advent of conformationally correct synthetic virus-like particles (VLPs). The utility of these reagents for seroepidemiology was demonstrated in many studies. Sero- positivity to HPV16 L1 capsid proteins is a good predictor for the subsequent development of cervical cancer (Keerti Shah, Johns Hopkins University, Baltimore, MD, USA) and for the development of CIN, although HPV DNA detec- tion is a more sensitive predictor than serology (Keng-Ling Chua, Karolinska Institute, Stockholm, Sweden). Sero-conversion occurs, on average, 8 months after the detection of HPV DNA (either low- or high-risk viruses) with anti- bodies persisting for 2-3 years (Joseph Carter, Fred Hutchinson Cancer Research Center, Seattle, WA, USA). Detection of antibody to HPV-capsid proteins is therefore a measure of past exposure to the virus but does not necess- arily correlate with current infection.

Vaccines and therapy The pace of development of both prophylactic and therapeutic vaccines was striking. The obvi- ous candidates for a prophylactic vaccine are VLPs, and the immunogenicity of these reagents in animal models was demonstrated by several groups. Bacterially expressed HPV L1 proteins were shown for the first time to assemble into VLPs (Wei Zhang, Cambridge University, Cambridge, UK); and recombinant Salmonella bacteria expressing the HPV16 L1 gene were shown to assemble HPV L1 into VLPs, showing promise as an oral vaccine (Denise Nardelli- Haefliger, University of Lausanne, Lausanne, Switzerland). There were promising results from a small Phase Ila clinical trial of a recombinant HPV6 L2-E7 fusion protein vaccine designed to treat genital warts. Vaccination, either alone or in conjunction with conventional therapy, was found to aid clearance of the virus and prevent the recurrence of genital warts (Stephen Thompson, Cantab Pharmaceuticals, Cambridge, UK).

Advances in antiviral therapy were also re- ported with promising clearance rates for genital warts treated with Imiquimod T M (Dick Miller, 3M Pharmaceuticals, Minneapolis, MN, USA) and impressive results both in vitro and in vivo in experimental systems with antisense oligonu- cleotides to HPV6 E1 and HPV11 E1 (David

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Szymkowski, Roche Research Centre, Welwyn, UK).

V i rus -kera t inocy te interact ions Papillomaviruses infect basal keratinocytes, but these cells are heterogeneous; the identification of a subset of basal keratinocytes with enhanced clonogenicity that overexpress the %1~ 4 integrin (Pritinder Kaur, Institute of Medical and Veterinary Sciences, Adelaide, Australia) com- plemented intriguingly the evidence that the a~ integrin subunit is the cellular receptor for HPV (Bruce MacMillan, University of Queensland, Brisbane, Australia). The absolute dependence on keratinocyte differentiation for virus repli- cation and assembly is an obstacle with which papillomavirologists wrestle. However, the ability to generate virus from cells transfected with viral genomes and grown in organotypic culture (Craig Meyers, Pennsylvania State University Medical Center, Hershey, PA, USA), and the use of these systems for genetic analysis (Laimonis Laimins, NorthWestern University Medical School, Chicago, IL, USA), mark a significant advance. The interactions of the HPV onco- proteins E6 and E7 with the tumour suppressor proteins p53 and RB are well known, but the application of the yeast two-hybrid screen has revealed new cellular partners for these mol- ecules: the human homologue (hTID56) of the Drosophila tumour suppressor protein TID56 interacts with the C-terminal domain of HPV16 E7 (Boris Schilling, Harvard Medical School, Boston, MA, USA) and a new HPV16 E6-binding protein was shown to have homology to MCM proteins, which are key regulators of eukaryotic DNA replication (Christian Kuhne, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy).

However, HPV does not make E6 and E7 pro- teins in order to cause cancer; elegant experi- ments in mice made transgenic for HPV16 E6 and/or E7 under the crystallin promoter (causing lens-specific expression) have shown that ex- pression of these proteins in differentiating ep- ithelia alters the regulation of the opposing, but balanced, processes of cell proliferation and cell death (Ann Griep, University of Wisconsin, Madison, Wl, USA). Expression of HPV E7 alone in the lens of the transgenic mouse results in fail- ure of these cells to withdraw from the cell cycle and differentiate; subsequently, apoptosis is in- duced by p53-dependent and -independent pathways. This HPV E7-induced apoptosis can be rescued by co-expression of HPV E6 or fi- broblast growth factor 2.

This workshop marked a change in emphasis in papillomavirus research; there is a move to- wards investigating the virology and biology of

these viruses to provide insights into cell cycle control, cell differentiation and induction of ma- lignant disease. Importantly, there was welcome evidence of progress in prevention and therapy, with new developments in antivirals and im- munotherapies.

Tumour immunology After the five-day workshop, the papillomavirolo- gists finished their deliberations and moved on to the second meeting. This took place in a tropi- cal paradise, but the science competed well with the hedonistic combination of coral reefs, sea and sun. The mood of the meeting was, how- ever, sombre and realistic, with a frank analysis of the problems faced in tumour immunotherapy. A proof of principle of immunotherapy against tumours was provided in a mouse model in which dendritic cells were loaded with peptides (Kees Melief, University Hospital, Leiden, The Netherlands). However, the real proof of these approaches must come from human trials, and Martin Kast (Loyola University, Chicago, IL, USA) and Melief described a Phase I trial in those patients with advanced HPV16 DNA ÷ cer- vical cancers who also have the human leuko- cyte antigen HLA-A2. The patients were immun- ized with peptides from HPV16 E7 protein shown previously to bind to HLA-A2. Although HPV16 E7-specific cytotoxic T lymphocytes (CTLs) were not detectable in 6 of the 13 patients im- munized, the procedure was safe and the pa- tients were clinically stable. Peter Stern (Paterson Institute for Cancer Research, Manchester, UK) discussed HLA downregulation in turnouts; this was a timely reminder that the development of a cancer is a process of selec- tion, with the emergence of a genetically unsta- ble and heterogeneous subpopulation of tumour cells. These will have run the gauntlet of host cytotoxicity, whether mediated by natural killer (NK) cells or CTLs, and the tumour escape- mutants are ready and able to respond to any new selection pressure placed upon them by therapy. For this reason, the lessons learnt in conventional cancer chemotherapy should be heeded by cancer immunotherapists.