Human papillomavirus vaccines

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1 DOI: /toag SAC REVIEW Keywords anogenital neoplasia, human papillomavirus, lower genital tract neoplasia, vaccines, viral antigens This article was commissioned and peer reviewed by the Scientific Advisory Committee (SAC). Human papillomavirus vaccines Alison Fiander Human papillomavirus-associated anogenital neoplasia poses a major health threat worldwide, with new cases of cervical carcinoma alone annually. The majority of deaths occur in developing countries but even in the developed world anogenital neoplasia is frequently challenging to manage and thus new treatment modalities are urgently required. The association between neoplasia and a viral infection provides the opportunity for immunological intervention, either by preventing initial infection (prophylactic vaccination) or by clearance of infected (low-grade disease) and/or neoplastic cells (highgrade and invasive disease). This article summarises the targets and effectors for immunological intervention, potential vaccines and the clinical trials carried out to date and considers the future for HPV vaccines. Author details Professor Alison N Fiander DM, FRCOG, MSc, Consultant Obstetrician and Gynaecologist, Department of Obstetrics and Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK. fianderan@lcf.ac.uk Introduction Human papillomavirus (HPV)-associated lower genital tract neoplasia (LGTN) affects an estimated 325 million people worldwide. Cervical cancer alone accounts for almost new cases annually, being the second most common cancer to affect women. The majority of deaths from cervical carcinoma occur in the developing world, where incidence figures often equate with mortality in the absence of healthcare facilities. Even in the developed world LGTN frequently presents management difficulties and new treatment modalities are urgently required. It has been suggested that in the absence of cervical screening the West would be experiencing an epidemic of cervical carcinoma, given the increasing prevalence of HPV infection. High-risk HPV The human papillomaviruses are small, highly conserved, nonenveloped DNA viruses, approximately 55 nm in diameter with a 72- sided icosahedral outer capsid coat comprising two structural proteins, L1 and L2. Within the capsid coat is a double-stranded, circular viral genome approximately 7.9 kilobases in length. The genome is divided into seven early (E) and late (L) regions or genes. High-risk HPV types are now recognised as playing a central aetiological role in cervical carcinogenesis (99.7% of cervical tumours contain HPV DNA) and other lower genital tract neoplasia. HPV16 and 18 are officially designated as human carcinogens, HPV16 being recognised as the most carcinogenic agent known to humans. Of over 130 known HPV types, around 20 infect the genital tract, ten or so being of intermediate or high risk for neoplasia, (HPV16, 18, 31, 33, 35, 45, 51, 52, 58 and 59).An understanding of HPV epidemiology, the natural history of infection and mechanisms of HPV carcinogenesis has led to the potential for immunological intervention. HPV-induced neoplastic transformation is associated with integration of part of the HPV genome into host cell DNA, leading to overexpression of the E6 and E7 oncoproteins, which interfere with cell cycle regulation by inhibiting the tumour suppressors p53 and retinoblastoma (Rb) protein (Figure 1). This leads to the accumulation of genetic damage, genetic instability and eventual emergence of a malignant phenotype. Viral antigens The association between neoplasia and a viral infection provides the opportunity for immunological intervention, either by preventing initial infection (prophylactic vaccination) or by clearance of infected (low-grade disease) and/or neoplastic cells (high grade and invasive disease). Viral antigens are clearly distinguishable as foreign antigens and, as such, are potentially 98

2 recognisable during immunological surveillance, albeit that HPV has evolved a number of strategies to evade immunological control. Neoplastic cells are marked unambiguously by the presence of tumour specific viral antigens, offering attractive targets for immunotherapy (therapeutic vaccination). Immunology and vaccines For prophylaxis, HPV-specific neutralising antibodies (NA), either of immunoglobulin A (IgA) or G (IgG) subclass, are required to prevent infection at the genital mucosal surface, directed against the L1 or L2 HPV capsid proteins. Once infection has occurred, cell-mediated immune responses involving virus specific CD8+ cytotoxic T lymphocytes and CD4+ helper T lymphocytes (Th), are required to clear infected cells, targeting the E6 and E7 oncoproteins in high-grade and possibly E1, E2 or E5 viral proteins in low-grade disease. Cytotoxic T lymphocytes (CTL) recognise short peptide fragments of early proteins displayed on the surface of infected cells in conjunction with host encoded major histocompatibility complex (MHC) class 1 molecules. Helper T cells recognise longer peptide fragments complexed with MHC class 2 molecules and function to provide accessory signals, promoting CTL production or specific antibody production through cytokine induction. An effective CTL response requires a type-1 T helper (Th1) response characterised by production of interferon gamma and interleukin-2, whereas a type-2 T helper (Th2) response is characterised by interleukin-4 and interleukin-10 production and favours the production of antibodies. Generation of the correct type of T helper response may be critical for effective immunotherapy. Vaccines are composed of two components: an antigen specific component to generate immunity specifically against HPV and a nonspecific component that delivers the antigen to the immune system in an appropriate form. In some cases, the delivery system can act as an adjuvant, boosting the immune response to the specific antigen. Virus-like particles A major break through came in the discovery that capsid proteins L1 and L2 (or L1 alone) are able to self-assemble into virus-like particles (VLPs). The VLPs are structurally identical to native HPV virions but lack the viral DNA core. They are able to induce virus neutralising antibodies in both animals and humans. Potentially, there is the possibility of using chimeric VLPs that contain an early protein gene, to combine prophylactic and therapeutic approaches. SAC REVIEW Figure 1. A model of cervical carcinogenesis Subclinical Subclincial infection infection Low-grade Low CIN grade CIN High-grade CIN High grade CIN HPV integration Carcinogens long-term culture Further genetic mutation HPV infection HPV persistance Immortalisation Transformation Invasion E6.E7 p53.rb Hormones Oncogenes Failure of immune surveillance Metastasis Genetic instability Clonal selection KEY CIN = Cervical intraepithelial neoplasia HPV = human papillomavirus 99

3 SAC REVIEW Recombinant viruses Certain human viruses, but not HPV, elicit extremely potent antibody and cell-mediated immune responses.vaccinia virus was used in the eradication of smallpox and much is known about its clinical use. The establishment of recombinant vaccinia virus technology led to the use of this vector for the TA-HPV (Xenova, Slough, Berkshire, UK) vaccine, which was the first HPV vaccine to be tested in clinical trials. However, there are safety concerns over the use of a live virus vector, particularly in premalignant disease, and this has driven research into safer pox vectors and consideration of adenovirus vectors. Recombinant bacteria Bacteria also are efficient at stimulating T cell responses. HPV vaccine development in bacteria has largely focused on intracellular bacteria, such as listeria, salmonella and Bacillus Calmette- Guerin (BCG). BCG has a well-established safety profile for human vaccine use. Bacteria also have properties that facilitate mucosal delivery, allowing direct local application to mucosal lesions and oral or intranasal administration. Although studies have been undertaken in animal models, there has been no clinical testing of bacterially based HPV vaccines. Proteins and peptides CD8+ CTL recognise 9-11 amino acid peptides complexed to MHC class 1 molecules on a cell surface. Peptides themselves are likely to be poorly immunogenic, requiring adjuvant to promote sustained CD8+ T cell responses, and under some circumstances can have the unwanted effect of inducing tolerance. A disadvantage of the peptide approach is that people must express the appropriate human leucocyte antigen (HLA) molecule capable of binding immunising peptides. The need for a particular HLA type can be circumvented by using either longer peptides or full-length proteins to deliver all potential T cell peptide epitopes to the immune system. However, for optimal immunogenicity these need adjuvants. DNA vaccines The use of naked DNA vaccines has great appeal, based on simplicity, stability and cost. Vaccination with DNA intramuscularly can generate protective CD8+ T cell responses in murine models. DNA vaccines produce weaker immune responses than those enlisted by recombinant viral or bacterial vectors. However, a major advantage of DNA vaccination is that a certain amount of tailoring of vaccines can be done, e.g. adding cytokine genes or molecules involved in antigen processing and presentation. Full-length HPV genes cannot be used because of their oncogenicity. Dendritic cells Dendritic cells are potent antigen-presenting cells capable of direct activation of CD8+ T cells in vitro and in vivo. Dendritic cells could therefore act as a delivery system and cellular adjuvant to the above approaches, although this would require isolation and culture of dendritic cells from each patient. The labour intensive nature of such an approach may limit vaccination to small cohorts. History of HPV vaccines The production of HPV vaccines does not come without challenges. Some of these reflect characteristics of the virus itself and the process of carcinogenesis; others are the challenges of stimulating an effective immune response to a mucosal infection. Papillomavirus infection is highly speciesspecific and, since HPV does not cause disease in animals, it is difficult to test vaccines for human use in animal models. However, animal models investigating animal papillomavirus infection or transgenic mouse models incorporating HPV antigens have demonstrated effective prophylaxis and therapeutic intervention, thereby establishing proof of concept. Animal models can never completely mimic the interaction between HPV and human host cells, necessitating the move into early phase studies of HPV vaccines in human subjects. The real thing : clinical studies of HPV vaccines Clinical studies of HPV vaccines are summarised in Table 1. Prophylactic vaccines The first proof of principal study of an HPV16 L1 VLP (Merck Research Laboratories) was published in In this randomised, doubleblind study, 2392 young women received three doses of placebo or vaccine. At the end of the follow-up period (median 17.4 months), the vaccine showed 100% efficacy (P < 0.001) for persistent HPV16 infection. 1 All nine cases of 100

4 HPV16-related cervical intraepithelial neoplasia occurred among placebo recipients. A further placebo-controlled, randomised phase IIb trial was presented at the EUROGIN Conference 2003.This trial used an HPV16 and HPV18 VLP vaccine (GSK Biologicals). Young women (n = 1100) received three doses of placebo or vaccine at zero, one and six months.a 99.8% seroconversion rate was seen, with only one woman failing to seroconvert. High titres of neutralising antibody were seen, times higher than those seen in natural infections. At one year of follow up the VLP was 100% effective in preventing persistent HPV infection. More work is required to identify the longevity of protection, the number of doses required and the development of multivalent vaccines to cover the majority or all high risk HPV types in both developed and developing countries. A quadrivalent vaccine covering HPV16, HPV18, SAC REVIEW Table 1. Clinical trials of anti-hpv vaccination for anogenital neoplasia Author Vaccine Participants Response Koutsky HPV16 L1 VLP (Merck) % protection against persistent HPV16 infection (17 months of follow up) EUROGIN 2003 HPV16/18 VLP (GlaxoSmithKline) % protection against persistent HPV Conference, Paris infection (1 year of follow up) Borysiewicz et al TA-HPV recombinant vaccinia 8 recurrent/advanced cervical cancer HPV specific CTL in 1/3 HPV16 and 18 E6/E7 1 patient disease free > 7 years Ressing et al HPV16 E7 peptides 15 HLA A2 Recurrent/residual cervical cancer 2/15 stable disease > 1 year Adams et al Dendritic cells + tumour lysate 8 recurrent/advanced cervical cancer 2/8 HPV16 specific CTL 1/8 T-helper response 1 stable disease 6/12 Frazer et al HPV16 E7 fusion protein + adjuvant 5 advanced cervical cancer 2/3 proliferative T-cell responses Stellar et al E7 lipopeptide vaccine 12 advanced cervical cancer HLA A2 E7 peptide specific CTL responses Muderspach et al E7 peptide with adjuvant 10 high-grade CIN/VIN 3/18 complete response E7 peptide with adjuvant and HPV16 + 6/18 partial response T helper epitope + lipid tail HLA A2 10/16 E7 specific responses 8 high-grade CIN/VIN HPV16 + HLA A2 Fiander et al TA-HPV 12 CIN3 3/12 HPV18 specific CTL response Davidson et al TA-HPV 18 VIN3 1/18 complete response 8/18 partial response 4 new ELISPOT responses Baldwin et al TA-HPV 12 VIN 1/12 complete clinical response 4/12 partial response 6 ELISPOT responses Tristram et al TA-HPV 11 high-grade AGIN No clinical responses Klencke et al Encapsulated plasmid DNA (ZYC101) 12 high-grade AIN 3/12 partial response 10/12 direct ELISPOT response Palefsky et al HspE7 (BCG heat-shock protein 80 evaluable high-grade AIN 75% partial response at 6/ HPV16 E7) 95% response at 15/12 (51% partial response, 44% complete response) Frazer et al Cervax16 TM (HPV16 E6E7 protein 31 CIN1-3 No clinical responses with matrix adjuvant) LLETZ at 7 weeks 12/20 T-helper response Kitchener et al Prime-boost strategy: 29 high-grade AGIN 1 complete response TA-CIN 5 partial responses TA-HPV 15/27 symptomatic improvement AGIN = anogenital intraepithelial neoplasia, AIN = anal intraepithelial neoplasia, BCG = Bacillus Calmette-Guerin, CIN = cervical intraepithelial neoplasia, CTL = cytotoxic T lymphocytes, HLA = human leucocyte antigen, LLETZ = large loop excision of the transformation zone,vin = vulval intraepithelial neoplasia,vlp = virus-like particles 101

5 SAC REVIEW HPV31 and HPV45 would cover 80% of cervical cancer, although other high-risk types are clustered in certain regions, e.g. HPV39 and HPV59 in Central and South America. There is some evidence that L2 VLPs may have greater cross neutralising potential than L1 VLPs. Experts predict that a safe and effective prophylactic HPV vaccine will be available within five years. Therapeutic vaccines Progress in therapeutic vaccine development has been slower for a variety of reasons, including: the debate over the optimal recipient group; when to incorporate immunotherapeutic interventions into conventional management; the use of agents with an uncertain risk profile and the problem of early clinical studies in immunocompromised patients with advanced stage cervical carcinoma. The first clinical trial used TA-HPV (Xenova), a recombinant vaccinia virus encoding HPV16 and 18, E6 and E7 open reading frames. Eight women with recurrent or advanced cervical cancer received one dose by dermal scarification. 2 Three women developed an HPVspecific antibody response and one of three evaluable women developed HPV-specific CTL responses nine weeks following vaccination.this woman has shown disease remission and is disease free seven years later. The majority of women with late-stage disease appeared to be immunocompromised 3 and therefore later studies with TA-HPV were in women with earlier-stage cancer (EORTC trial) 4 or preinvasive disease. While initial trials concentrated on finding evidence of immunological responses to vaccination, later trials are attempting to assess clinical responses.ta-hpv has been the subject of three further immunotherapy trials in high grade LGTN. One complete response was seen in each of two studies conducted among women with vulval intraepithelial neoplasia, as well as a number of partial responses (reduction in lesion size by greater than 50% in 8 of 18 and 4 of 12 women). 5,6 An E7 peptide vaccine with adjuvant in highgrade anogenital neoplasia was followed by complete responses in three of 18 patients and partial responses in a further six recipients, 7 although spontaneous regression might be expected in up to 30% of patients with highgrade anogenital neoplasia. Encouraging clinical responses were presented at the 20th International HPV Conference in 2002, using a BCG heat-shock protein (BCGhsp) and an HPV16 E7 fusion protein (Stressgen) in 80 patients with high-grade anal intraepithelial neoplasia. Six months after vaccination there was a 75% partial response rate, although no complete responses. Follow-up to fifteen months showed an overall response rate of 95% (51% partial and 44% complete). 8 This study suggests that clinical responses following immunotherapy may take some time: the elucidation of the duration of response requiring additional follow-up. Three partial responses were observed in another study of 12 patients with high-grade anal intraepithelial neoplasia, which used four doses of an encapsulated plasmid DNA vaccine (ZYC101, Zycos Inc.). 9 The need for an adequate follow-up period was again noted. More recently it has been suggested that heterologous prime-boost vaccination strategies may enhance immune responses. 10 Initial clinical results of a prime-boost trial have been reported in which 29 patients with high-grade anogenital neoplasia received three doses of a recombinant fusion protein HPV16 L2E6E7 (TA-CIN, Xenova), followed by a single dose of TA-HPV. 11 Three months after completion of the vaccination schedule, one complete and five partial responses were observed. Interestingly, 15 of 27 women who were symptomatic showed improvement in symptomatology. It can be seen from the above studies that clinical responses are being observed as a result of therapeutic vaccination, albeit at suboptimal frequency. The challenge now is to optimise vaccine design (dose, vector, adjuvant), vaccination strategy and route of administration, in order to produce reliable clinical responses. For a mucosal pathogen such as HPV, mucosal vaccination or vaccination at the site of disease in LGTN may be of greater benefit. 12 The future of HPV vaccines Although the prospect of a prophylactic vaccine is imminent there are still major issues to be addressed, including that of public education related to the acceptance of a sexually transmitted disease vaccine and the understanding of the relationship between HPV, cervical cancer and other LGTN. The issues of whether or not to vaccinate men as well as women and at what age also need addressing. Prophylactic vaccines are likely to produce type-specific protection, which may not be absolute, and therefore the possible negative effect on subsequent uptake of screening requires consideration. 102

6 The sustainability of vaccine programmes in developing countries is questionable with current VLP vaccines. Second-generation vaccines are required with lower production and distribution costs and multivalent activity. Ideally, such a vaccine would combine prophylactic and therapeutic aspects within its design, thereby treating the generation of today while protecting the generation of tomorrow. Advances in the field of therapeutic vaccination are continuing. Improvements in immunological endpoint assays have enabled detection of immunological responses to vaccination with much greater reliability. In a study of TA-HPV immunotherapy in high-grade anogenital neoplasia, T-cell responses, as measured by enzymelinked immunospot assays, were detectable in the vast majority of recipients post vaccination (S Man, personal communication, 2003). Costs surrounding the design and production of effective therapeutic vaccines are extremely high, begging the question as to who can, or should, fund research in this area. For the foreseeable future, the need for therapeutic vaccines will remain, as the management of anogenital neoplasia is often problematic and prophylactic vaccination will not impact on the millions worldwide already affected by HPV- associated disease. References 1. Koutsky LA, Ault KA,Wheeler CM, Brown DR, Barr E, Alvarez FB, et al. A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 2002;347: Borysiewicz LK, Fiander A, Nimako M, Man S, Wilkinson GW,Westmoreland D, et al. A recombinant vaccinia virus encoding human papillomavirus type 16 and type 18, Ee6 and Ee7 proteins as immunotherapy for cervical cancer. Lancet 1996;347: Fiander AN, Adams M, Evans AS, Bennett AJ, Borysiewicz LK. Immunocompetent for immunotherapy? A study of the immunocompetence of cervical cancer patients. Int J Gynecol Cancer 1995;5: Kaufmann AM, Stern PL, Rankin EM, Sommer H, Nuessler V, Schneider A, et al. Safety and immunogenicity of TA-HPV, a recombinant vaccinia virus expressing modified human papillomavirus (HPV)-16 and HPV-18 E6 and E7 genes, in women with progressive cervical cancer. Clin Cancer Res 2002;8: Davidson EJ, Boswell CM, Sehr P, Pawlita M, Tomlinson AE, McVey RJ, et al. Immunological and clinical responses in women with vulval intraepithelial neoplasia vaccinated with a vaccinia virus encoding human papillomavirus 16/18 oncoproteins. Cancer Res 2003;63: Baldwin PJ, van der Burg SH, Boswell CM, Offringa R, Hickling JK, Dobson J, et al. Vaccinia-expressed human papillomavirus 16 and 18 E6 and E7 as a therapeutic vaccination for vulval and vaginal intraepithelial neoplasia. Clin Cancer Res 2003;9: Muderspach L,Wilczynski S, Roman L, et al. A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are HPV16 positive. Clin Cancer Res 2000;6: Palefsky J, Goldstone S, Neefe J. HSPE7 treatment of high grade anal dysplasia: final results of an open-label trial and interim long term follow-up. 20th International Papillomavirus Conference, October , Paris. 9. Klencke B, Matijevic M, Urban RG, Lathey JL, Hedley ML, Berry M, et al. Encapsulated plasmid DNA treatment for human papillomavirus 16- associated anal dysplasia: a phase I study of ZYC101. Clin Cancer Res 2002;8: van der Burg SH, Kwappenberg KM, O Neill T, Brandt RM, Melief CJ, Hickling JK, et al. Pre-clinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens. Vaccine 2001;19: Kitchener H,Tristram A, Davidson E,Tomlinson A, Sterling J, Dobson J, Man S & Fiander A. A multicentre study of a prime-boost vaccination strategy in women with anogenital intraepithelial neoplasia. Paper presented at EUROGIN April , Paris. 12. Gallichan WS, Rosenthal KL. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J Exp Med 1996;184: Ressing ME, van Driel WJ, Brandt RM, et al. Detection of T helper responses, but not of human papillomavirus-specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma. J Immunother 2000;23: Adams M, Borysiewicz L, Fiander A, Man S, Jasani B, Navabi H, et al. Clinical studies of human papilloma vaccines in pre-invasive and invasive cancer. Vaccine 2001;19: Frazer I,Tindle R, Fernando G, Malcolm K, Herd K, McFadyn S, et al. Safety and immunogenicity of HPV16E7/algammulin. In:Tindle RW, editor. Vaccines for Human Papillomavirus Infection and Anogenital Disease. Austin,TX: RG Landes Bioscience; p Steller MA, Gurski KJ, Murakami M, Daniel RW, Shah KV, Celis E, et al. Cell-mediated immunological responses in cervical and vaginal cancer patients immunized with a lipidated epitope of human papillomavirus type 16 E7. Clin Cancer Res 1998;4: Fiander A, Man S, Nimako M, Evans A S, Adams M, Hickling J, et al. Clinical HPV vaccination programme utilising recombinant vaccinia virus encoding E6 and E7 proteins of HPV16 and 18. Paper presented at the 17th International Papillomavirus Conference 9 15 January 1999, Charleston, USA. 18. Tristram A, Man S, Smith K, Fiander A. Clinical effects of a vaccine (TA-HPV) in anogenital intraepithelial neoplasia. Paper presented at the 20th International HPV conference, October , Paris. 19. Frazer IH, Quinn M, Nicklin J,Tan J, Perrin L, Ng P, et al. A randomised placebo controlled trial of immunotherapy for CIN. Paper presented at 20th International Papillomavirus Conference October , Paris. 103