HPV vaccines. Margaret Stanley* MB PhD Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK

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1 Best Practice & Research Clinical Obstetrics and Gynaecology Vol. 20, No. 2, pp , 2006 doi: /j.bpobgyn available online at 5 HPV vaccines Margaret Stanley* MB PhD Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK The ability to generate human papillomavirus virus (HPV)-like particles by the synthesis and selfassembly in vitro of the major virus capsid protein L1 has transformed our prospects for preventing cervical carcinoma in women. These particles provide vaccines that are immunogenic and safe, and data from proof-of-principle efficacy trials strongly suggest that they will protect against persistent HPV infection and cervical intraepithelial neoplasia. However, the duration of protection provided by these vaccines is not known, the antibody responses induced are HPVtype-specific and immunisation must occur pre-exposure to the virus. Second-generation vaccines could include an early antigen for protection post exposure and alternative delivery systems might be needed for the developing world. Therapeutic vaccines for low-grade intraepithelial disease are realistic but high-grade disease presents major hurdles for immunotherapies. Key words: cervical carcinoma; HPV; neutralising antibody; prophylactic vaccines: therapeutic vaccines; vaccines; virus-like particles. A wealth of epidemiological and molecular evidence has led to the conclusion that virtually all cases of cervical cancer, and its precursor intraepithelial lesions, are a result of infection with one or other of a subset of human papillomaviruses (HPVs). 1 The HPVs are a large family of viruses that infect skin and mucosae, where they induce epithelial proliferation aka warts. Between 30 and 40 HPV types infect the genital tract regularly or sporadically, and these types fall into two discrete groups: HPVs 6 and 11 and their relatives cause anogenital warts and are rarely detected in malignant anogenital disease. They are regarded as low-risk, non-oncogenic types. Genital warts, however, are the most common sexually transmitted viral infection; the incidence of these lesions is increasing and they represent a major cost to healthcare providers. HPV types 16, 18, 31, 33, 35, 45, 58 plus 8 10 other minor types (Table 1) are oncogenic viruses or high-risk HPVs (hrhpv) and are associated with a range of * Corresponding author. Tel.: C ; fax: C address: mas@mole.bio.cam.ac.uk /$ - see front matter Q 2005 Elsevier Ltd. All rights reserved.

2 280 M. Stanley Table 1. HPV types in anogenital malignancies. Lesion HPV types involved % Cases HPV positive Cervical carcinoma 16, 18, 31, 33, 35, 39, 45, 51, 52, 66, 58, 59, 66 O95% (26, 68, 73, 82 a ) Vulval carcinoma basaloid 16, 18 O50% warty 16, 18 O50% keratinising 16!10% Penile carcinoma basaloid 16, 18 O50% warty 16, 18 O50% keratinising 16!10% Vaginal carcinoma 16, 18 O50% Carcinoma of the anus 16, 18 O70% HPV16 is the dominant type in all anogenital malignancies, present in 60 70% of cervical carcinomas and at least 80% of HPV-positive vulval and penile carcinomas. a These types are relatively rare. anogenital cancers including cervix, vulva and anus in women and penis and anus in men. The most compelling associations are with cervical cancer and hrhpv DNA sequences are detected in almost 100% of cervical cancer biopsies and 90% of highgrade cervical intraepithelial neoplasia (CIN) precursor lesions. The associations are so strong that the hrhpvs are regarded as human carcinogens with a causal role in cancer of the uterine cervix in women. 2 Strategies for the control and treatment of genital HPV infections are therefore a matter of high priority. Traditionally, vaccines have represented a cost-effective means of preventing viral diseases and HPV should be no exception. Despite the complex biology of HPVs (they are absolutely host specific and can be produced only in trivial amounts in vitro) substantial progress has been made in vaccine development, particularly for prophylaxis, and there is now considerable and justified optimism that infection with the major genital HPVs can be prevented. THE INFECTIOUS CYCLE OF THE HPVS The HPV genome comprises 8 kb of double-stranded DNA and encodes a maximum of eight genes, six of which encode non-structural or early proteins E1, E2, E4, E5, E6 and E7 and two of which encode structural or late proteins L1 and L2 (Figure 1). The viral replication cycle is one in which viral infection is targeted to basal keratinocytes; high-level expression of viral proteins and viral assembly occur only in differentiating keratinocytes in the stratum spinosum and stratum granulosum of squamous epithelium. 3 This replication cycle is the key to understanding the pathogenesis and immunobiology of these viruses but knowledge of this process is limited in several key areas due mainly to the inability to infect cells in tissue culture with virus and achieve a complete infectious cycle in vitro. In consequence, much of the information on

3 Figure 1. The HPV genome. HPV vaccines 281

4 282 M. Stanley the infectious cycle comes from natural infections in animals, particularly the rabbit, cow, and dog plus surrogate systems in rodents. PROPHYLACTIC HPV VACCINES Preclinical studies In general, prophylactic vaccines induce the generation of neutralising antibody to the pathogen and thus prevent disease on subsequent exposure. If this is to be the rationale for HPV immunoprophylaxis then evidence that serum antibody is protective in natural infections is required. Studies on antibody responses to HPV infections have traditionally been compromised by the inability to generate adequate amounts of virus for seroassays. However, studies exploiting natural papillomavirus infections in the dog, rabbit and cow, together with HPV1 and HPV11 infections in humans (all situations in which adequate amounts of virus could be obtained) showed clearly that there were serum responses to viral capsid proteins in individuals who were or had been infected (for review see Ref. 4 ). In the animal models, seropositive individuals were resistant to subsequent viral challenge. Neutralising antibody, directed to determinants on the L1 protein (the major coat or capsid protein) exposed on the outer surface of the intact virus particle (Figure 1), is generated in these individuals. These observations suggested that a vaccine generating such responses must contain L1 protein in the correctly folded, tertiary or native form. Technically, this was very difficult but a major experimental advance that showed that the L1 protein, when expressed by vectors such as recombinant baculovirus or yeast, self-assembles into virus-like particles (VLPs). 5,6 The L1 VLP is a conformationally correct, empty capsid (i.e. it contains no DNA) that appears morphologically identical to, and contains the major neutralising epitopes of, the native virion. L1 VLPs closely approximate the antigenic characteristics of wild-type virions and have been used extensively in seroepidemiological studies in humans. These large population-based studies have revealed that type-specific antibody responses are common during and after infection with genital HPVs. 7,8 These data suggested that the generation of neutralising antibody to L1 via L1 VLPs would be effective in prophylaxis and experimental studies that show immunogenicity and efficacy with L1 VLP vaccines in three animal models the dog, cow and rabbit support this notion. In the dog and rabbit, immunisation with L1 VLPs induced circulating neutralising antibody to the L1 capsid protein and the animals were completely resistant to challenge with large amounts of virus. 9,10 Importantly, in the rabbit model relatively long-term protection was induced by VLP immunisation. 11 Successful immunisation was species specific and the canine model showed that protection was mediated by serum antibody, because immunity could be passively transferred by serum from vaccinated animals. 9 The canine data were particularly relevant for genital HPVs because canine oral papillomavirus (COPV) is a mucosatrophic virus infecting the oral cavity whereas the rabbit papillomavirus is a cutaneous infection. Conformational epitopes on intact VLPs appear to be critical for the induction of neutralising IgG and for successful vaccination since denatured L1 protein fails to generate neutralising antibody and to protect against virus challenge.

5 HPV vaccines 283 Clinical trials L1 VLPs were clearly candidate immunogens for prophylactic vaccination in humans and are the current vaccines undergoing clinical trials. Dose-ranging Phase I studies in healthy subjects have shown that mg injected L1 VLPs given as three immunisations over 6 months are highly immunogenic and generate high titres of anti-l1 antibody In the studies reported to date, all VLP-immunised subjects, but no subjects in the placebo arms, have seroconverted and made anti-vlp antibody responses substantially greater (at least 1 3 logs) than that identified in natural infections. The dominant antibody responses are of the IgG1 subclass and have been shown to be neutralising by a variety of surrogate neutralisation assays. 12,16 Two L1 VLP vaccines are now in Phase III trials: (1) a bivalent HPV16/18 L1 VLP vaccine (formulated in a novel adjuvant comprising alum and 3-deacylated monophosphoryl A) developed by GlaxoSmithKline; and (2) a quadrivalent HPV16/ 18/6/11 L1 VLP vaccine (formulated in alum) from Merck Vaccines. Together, these randomised placebo-controlled multicentre trials will enrol more than women from a diversity of ethnic and socioeconomic backgrounds; the results are expected in The preliminary efficacy data from Phase II proof-of-principle trials for VLP vaccines are immensely encouraging. In a double-blind, placebo-controlled efficacy study, 2392 women aged between 16 and 23 years were randomised to receive vaccine (three doses 40 mg of yeast-derived HPV16 L1 VLP adjuvanted in alum at 0, 3 and 6 months) or placebo. 17 The primary endpoint of the trial was detection of persistent cervico-vaginal HPV16 infection, where this is defined as HPV6 DNA detected in two consecutive samples. Of the 1533 HPV16 naïve individuals followed for a median 17 months, HPV16 DNA was detected in none of the vaccinees (nz768) compared to 41 of the placebo group (nz765), nine of whom had HPVrelated CIN. 17 Another double-blind, placebo-controlled efficacy study randomised 1113 women to receive vaccine (three 20-mg doses of baculovirus-expressed HPV16 and HPV18 L1 VLPs adjuvanted in alum and MPA at 0, 1 and 6 months). The primary endpoint of the trial was detection of persistent HPV16 or HPV18 infection in cervicovaginal cells. In the according to protocol (ATP) group, 100% of vaccinees (nz366) were protected against persistent HPV16 or HPV18 infection, whereas in the placebo group (nz355) seven cases of HPV16 or HPV18 infection were detected in the 18 months after vaccination. Importantly, in the intention to treat (ITT) group in this study, 94% of vaccinees were protected despite an incomplete vaccination regime. 18 These data for protection against HPV16 or HPV 18 persistent infection have also been confirmed in a Phase II efficacy trial of the HPV6, 11, 16, 18 quadrivalent vaccine. 19 The combined incidence of persistent HPV6, HPV11, HPV16 or HPV18 infection was reduced in vaccinees (nz235) by 90% compared to placebo (nz233). Importantly, there were no cases of disease CIN or external genital warts (EGW) in the vaccinated group, but three cases of CIN and three cases of EGW in the placebos. The results from the various trials strongly indicate that VLP vaccination with HPV16 or HPV18 L1 VLPs in HPV16- or HPV18-DNA-negative women is safe and protective, preventing HPV16 or HPV18 infection (as measured by acquisition of HPV16 or HPV18 DNA) and the development of low-grade intraepithelial lesions.

6 284 M. Stanley Vaccine endpoints A critical issue for HPV vaccines is how to ascertain vaccine efficacy. Traditionally, the measurable endpoint of vaccine efficacy is disease incidence. This is not feasible with cervical cancer for both practical and ethical reasons. Invasive cervical cancer is a disease with a long interval between HPV infection and clinical presentation. Furthermore, it can be prevented by detection and treatment. Invasive cervical cancer incidence cannot therefore be an endpoint and disease-relevant surrogate endpoints acceptable to national regulatory authorities have to be identified for HPV vaccines. There are two possible endpoints (neither of which are exclusive) virological and clinical. As HPV infection is necessary for the development of almost all cervical cancers 1, it could be argued that preventing HPV infection is an adequate measure of efficacy. Genital HPV infection can be incident (new detection of HPV DNA in cervicovaginal cells in a woman previously HPV DNA negative) or persistent (HPV DNA of the same type detected on two successive occasions 6 12 months apart in a woman previously HPV DNA negative). The available evidence indicates that persistent HPV infection is the most significant predictor of progression to CIN and by implication invasive cervical cancer, suggesting that prevention of persistent HPV infection should be a suitable surrogate endpoint. However, there is no real evidence that preventing persistent HPV infection impacts on cervical cancer development. There is, however, good evidence that hrhpv infection is the cause of CIN 23,24,a clinical disease requiring medical intervention and one that can be diagnosed histologically on biopsy. High-grade CIN (CIN2/3) is accepted as the immediate precursor of invasive cervical cancer and there is a powerful argument that a vaccine that prevented CIN2/3 would be effective in reducing the incidence of cervical cancer. In the USA, the FDA Vaccines and Related Biologicals Advisory Committee has concluded that for licensure in the USA of HPV vaccines the endpoint should be detection of CIN2/3 (or worse) by histology together with virological detection. It seems likely that this will be globally accepted. 25 DURATION OF PROTECTION AND IMMUNOLOGICAL CORRELATES Some important issues could impact on vaccination strategies and the deliverability of HPV vaccines. A key question is What is the duration of the protection induced by these vaccines will we need frequent booster immunisations? The available data from the Phase II trials indicate that antibody levels fall from the peak levels after immunisation to low but measurable levels that persist for at least 48 months after vaccination. This is encouraging because it mirrors the situation in the animal models where protection is long lasting despite low levels of circulating antibody. 11 However, the data from the trials covers a relatively short time span and, in reality, we do not know how long the protection induced by L1 VLPs will last. Relevant to this is the issue of whether exposure to virus after vaccination will act as a natural booster. There is no unequivocal evidence for or against this from the trials, but some encouragement from the fact that in natural infections 50% of women remain seropositive 10 years after the last detection of cervicovaginal HPV DNA (Carter et al, 2005, personal communication) and this does suggest some effect of natural boosting. The mechanisms by which protection is being effected by VLP vaccines are not fully understood. VLP vaccines elicit high titres of neutralising serum antibody and there is

7 HPV vaccines 285 evidence that serum antibody titres correlate with the level of protection. 17 It seems unlikely that local mucosal antibody mediates the protection because, although cervicovaginal lavage fluid harvested from immunised primates neutralises HPV particles 26, only 50% of women immunised with HPV11 L1 VLPs develop local mucosal anti-hpv antibody. 14 The assumption, therefore, is that serum IgG transudates across the cervical epithelium in a sufficiently high concentration to bind to virus particles, preventing infection, although other scenarios such as intracellular neutralisation are feasible. The strength of serum anti-l1 antibody levels and the persistence of the antibody response might be critical immune correlates for measuring the level of protection, and data from vaccine failures will be critical in this context. TYPE SPECIFICITY Another important issue is type specificity. The neutralising antibodies generated by the L1 VLP vaccines appear to be type specific. Thus, it is expected that immunisation with HPV16 L1 VLPs will protect against HPV16 infection but not apparently against any of the 34 other genital HPVs. Similarly, HPV18 L1 VLPs protect against HPV18 infection but no other HPV, except possibly for HPV45. The cumulative prevalence of HPV types in cervical cancer is shown in Figure 2 and it is clear that increasing the number of types in the vaccine, e.g. HPV16, 18, 31, 45 and 59, would prevent more than 80% of cancers, although to prevent more than 90% of cancers at least a further six types would need to be added. The current generation of VLP vaccines contains only HPV16 and HPV18 and, assuming that HPV16 accounts for 50 60% and HPV18 for 10 12% of cervical cancer cases, even in the best-case scenario with 100% vaccine coverage of the target population only 60 70% of cervical cancers would be prevented. If the current HPV prophylactic vaccines are introduced for mass immunisation in countries with effective cervical cancer screening programmes, such as the UK, these programmes will also have to continue, unless significant cross-protection is induced by VLP immunisation. The general view, at the present, is that unless significant Squamous cell carcinoma Adeno - and adenosquamous carcinoma Figure 2. Cumulative prevalence of HPV types in women with cervical cancer. Published with permission from the Journal of Family Planning and Reproductive Healthcare.

8 286 M. Stanley cross-protection can be demonstrated by the current vaccines, broad protection from genital HPV disease will require polyvalent vaccines incorporating several serotypes. If cross-protection does occur, then the mechanism is unclear. The available evidence is that the neutralising antibodies generated by L1 VLPs are type specific. The only exceptions are HPV6 and HPV11 27, HPV31 and HPV33, and HPV18 and HPV45 28, which appear to share a neutralising epitope. There is a common neutralising linear epitope but the levels of antibody generated against this determinant are considered to be too low to be effective in protection. 29 The role of cell-mediated immunity in the protection generated by VLP vaccines is also not clear. Experimentally, VLPs alone in the absence of adjuvant induce a strong cell-mediated response. 30 In the Phase I trials, T cell responses to VLPs have been measured by lymphoproliferation and cytokine assays 31,32 and, interestingly, in one study some cross-reactivity to HPV16 VLPs was observed. 13 This suggests that T helper epitopes are conserved across serologically distinct genotypes, but whether this translates into cross-protection remains to be demonstrated. It is assumed (and animal models support this) that these vaccines will be effective only if delivered before exposure to virus. Genital HPV infection is usually sexually transmitted and immunisation must therefore precede the sexual debut, implying that the target population for vaccination will be 9- to 10-year-old prepubertal girls. This is achievable in the UK context but might be difficult in other cultural and social settings, particularly in developing countries. Nonetheless, there might be possible benefits of high levels of serum antibody in individuals already exposed to infection and the effects of vaccinating women with L1 VLP vaccines after exposure should be evaluated in longterm studies. HPV VACCINES IN DEVELOPING COUNTRIES The results from the VLP vaccine trials are immensely encouraging and exciting but the great burden of cancer of the cervix is in women in the underdeveloped world, where L1 VLP vaccines are far from ideal. Vaccines for women in the developing world must be cheap and easily delivered. The VLP vaccines currently in trial are likely to be expensive, require medical or paramedical personnel for delivery and a cold chain. Antigen delivery to the mucosal surface rather than parenteral immunisation via intranasal or oral routes might overcome the delivery problems. Both nasal and parenteral immunisation with HPV16 VLPs elicited HPV16-neutralising antibodies in genital secretions in mice 33 but only mucosal immunisation induced neutralising titres of antibodies throughout the oestrous cycle. 34 Oral immunisation with VLPs is an attractive option but in initial studies only poor antibody responses were generated. 35 However, when co-administered with a heatlabile Escherichia coli enterotoxin or CpG DNA as adjuvant, HPV16 and HPV18 VLPs resulted in high titres of anti-vlp antibody. 36 A potentially important development is the demonstration that complete protection can be achieved in rabbits by a single intranasal immunisation with an L1 vaccine delivered via an attenuated recombinant vesicular stomatitis virus (VSV). 37 Viruses such as VSV are exceptionally effective vectors for recombinant proteins because the antigen load that can be delivered in a single immunisation is considerable. However, these viruses are important human and animal pathogens in many parts of the world and the risks attendant on their use, even when attenuated, in widespread immunisation of humans in these geographic locations would require extensive evaluation.

9 HPV vaccines 287 If VLP assembly is not a prerequisite for the induction of neutralising antibodies then cheaper and perhaps more stable vaccines could be developed. The L1 protein of COPV when expressed in E. coli as a glutathione S transferase (GST) fusion protein formed capsomeres/pentamers but did not assemble into VLPs. Despite this, the capsomeres reacted with conformation-specific anti-copv antibodies and animals immunised with capsomeres were protected from high-dose viral challenge. 38 Both HPV11 L1 39 and HPV33 L1 40 capsomeres display neutralising epitopes, raising the prospect of a low cost second-generation vaccine. An important issue is whether the L1 vaccines will both be and remain efficacious in immunocompromised subjects, particularly those who are HIV infected. In developing countries there is limited access to effective antiretroviral therapy, raising questions of safety, immunogenicity and the induction of effective immune memory in such individuals. Clinical trials in such populations are needed to address this. FREQUENTLY ASKED QUESTIONS Four questions are often posed about polyvalent HPV vaccines: If HPV31, HPV45 and HPV59 and/or others are included as well as HPV16 and HPV18, will we get immunological equivalence to achieve 80 90% prevention of cancer? The answer to this is probably yes, because in the double-blind, placebocontrolled study for evaluation of the efficacy of the HPV6, 11, 16, 18 quadrivalent vaccine, comparable antibody titres were induced to each of the vaccine components. 19 Will we need different cocktails of HPV types for different populations? There is no answer to this at the present. The HPV-type distribution in cervical cancers is generally consistent worldwide but several reports using highly sensitive HPV detection and typing systems have found geographical variability. 1 If we control types that are currently the most common, will other rarer types take their place? This is another unanswerable question at the present and should be addressed by long-term studies in vaccinated populations. Should we immunise boys as well as girls? Genital HPV infections are predominantly sexually transmitted and vaccinating males will be necessary if herd immunity is to be achieved. However, all HPV vaccine trials to date have been in women and there is an almost total absence of information about the natural history of HPV16/HPV18 infection in men, a gap that will need to be filled if HPV vaccines are to be delivered to prepubertal males. One should not underestimate the difficulties of ascertaining vaccine efficacy in males because the problems of adequately sampling extensive areas of anogenital skin for accurate assessments of HPV DNA presence or absence and/or clinical HPV-related 16/18 disease are formidable. A disease endpoint (incidence of external genital warts) for HPV6/11 is, however, realistic. THERAPEUTIC VACCINES Although the development of prophylactic vaccination against HPV is exciting, realistically these interventions are at least a decade away and several decades must elapse before any effects will be evident. In the interim there are large numbers of infected individuals at risk for benign and malignant HPV-related disease: the need to

10 288 M. Stanley develop effective immunotherapies remains a priority. The induction of strong cellmediated responses as opposed to antibody is certainly central to any therapeutic vaccine strategy and may be critical for long-term immunity in prophylaxis. It is important to define therapeutic in this context; there are there possible scenarios: (1) A vaccine designed to be effective post exposure (2) A vaccine that could be effective against low-grade disease (EGW and CIN 1) (3) A vaccine for high-grade intraepithelial disease and cancer. The antigenic targets in 1 and 2 might be identical but only the oncoproteins E6 and E7 are possible targets for 3 because these are the only viral proteins that will be expressed in all high-grade lesions or cancer. 41 Post-exposure vaccines Post-exposure vaccines are worth more than a passing consideration. The communities with the highest incidence of cervical cancer are predominantly in the developing world and, in many societies, immunising young girls before the sexual debut for social and/or cultural reasons might not be easy; immunising women would pose fewer problems. HPV testing, if adopted as the primary screening modality in developed societies in particular, might identify incident infection as opposed to clinical disease, a situation that would raise anxiety in many women; a post exposure vaccine could have a place in the management of such cases. Evidence from animal models of infection gives insight into the early proteins against which effective T cell responses are generated and which, therefore, are candidate antigens for therapeutic vaccines. In the dog and rabbit models of natural infection, immunisation with vaccines encoding E1 or E2 genes modified to increase antigen expression protects against challenge with live virus 42 44, implying that these are good vaccine targets for post-exposure vaccines or therapeutic vaccines for low-grade disease. Therapeutic vaccines for high-grade intraepithelial disease and invasive cancer Low-grade and high-grade intraepithelial disease should be considered separately when discussing therapeutic vaccines. In low-grade disease, permissive viral replication is the norm and all early viral proteins will be expressed, the lesions are genetically stable and in an immunocompetent individual an effective therapeutic vaccine should result in lesion clearance and no recurrence. In cervical cancers and most CIN2/3, viral gene expression is deregulated and the E6 and E7 genes are constitutively expressed. The continued expression of these oncogenes is essential for progression to, and maintenance of, the malignant phenotype. In effect, therefore, there are only two possible antigenic targets, E6 and E7, because these are the only viral proteins that will be expressed in all high-grade CIN. The approach of deliberate immunisation with HPV16 E6 and/or E7 and the generation of antigen-specific cytotoxic T lymphocyte (CTL) as an immunotherapy for HPV-associated cancer has been tested with a wide array of potential vaccine delivery systems in transplantable rodent tumour models (for a review, see Ref. 45 ). HPV-expressing cancers in mice are relatively easy to cure but human HPV-induced cancers have been, to date, largely refractory to the approaches successful in rodents (for a review, see Ref. 46 ).

11 HPV vaccines 289 Several therapeutic vaccines aiming at the elimination of such established HPV infections and HPV-induced malignancies are being developed and tested in human beings (reviewed in Refs ). However, the formidable problems facing vaccines for CIN2/3 and invasive cancer relate to the neoplastic phenotype. These lesions are genetically unstable with the potential to rapidly evolve immune escape mechanisms. Tolerance to viral antigens, modulation of the cytokine milieu and down regulation of MHC class I alleles on the neoplastic keratinocytes are associated with progressive CIN and vulval intraepithelial neoplasia (VIN) 51,52, and invasive cancers. These are tough barriers for immunotherapy and dendritic cells loaded with E7 or E6/E7 peptide or protein appear to be the most potent inducers of antigen specific CD8C and CD4C CTL in cervical cancer patients It is important to remember that only a proportion of CIN2/3 progress to invasive carcinoma, although the size of the progressive fraction is not known. There is also evidence that CIN2/3 regress, presumably by immune mechanisms (but there is no unequivocal evidence for this), indicating that immune escape mechanisms are not inevitable in CIN. In view of this, it is distinctly possible that there will be a spectrum of responses to therapeutic vaccination, ranging from complete through partial to no clearance of the clinical disease. If this scenario is correct then it is critical to identify those immune parameters associated with clinical efficacy of therapeutic vaccines. There is good evidence for an important role for the IFNg-associated HPV16-specific T cell response in the control of progressive cervical disease. 56,57 In view of these results, the data from three Phase II vaccination trials of high-grade VIN, using a recombinant vaccina virus encoding modified HPV16/18 E6 and E7 genes has been re-evaluated to focus on the relationship between clinical efficacy and the vaccine-induced HPV16- specific IFNg-associated T cell response. The design of these trials permitted the combination of all patient data, giving an enlarged group of vaccinated patients, and the results of the analysis show that those patients with a vaccine-induced HPV16-specific Th1 response display a significantly higher clinical response rate than the group of nonreacting patients (van der Burg, personal communication 2005). This is encouraging, but the fact remains that therapeutic vaccines for high-grade and malignant anogenital disease have been disappointing so far and this is an area in which basic research is essential if progress is to be made and these vaccines are to have clinical utility. SUMMARY Immunoprophylaxis with HPV L1 VLP vaccines should, in theory, make cervical cancer a preventable disease, and the next 2 3 years should see the licensing of the first generation of these vaccines. All the evidence suggests that to achieve the level of efficacy that will be cost effective, the vaccines will have to be delivered before the sexual debut and, in reality, this means that prepubertal girls will form the vaccinated population. The take-up of such vaccines will depend on social attitudes, public health policies and economics. Although the development of prophylactic vaccination against HPV is exciting, realistically these interventions are at least a decade away and several decades must elapse before any effects will be evident. In the interim there are large numbers of infected individuals at risk for benign and malignant HPV-related disease: the need to develop effective immunotherapies remains a priority. The induction of strong cellmediated responses as opposed to antibody is certainly central to any therapeutic

12 290 M. Stanley vaccine strategy and may be critical for long-term immunity in prophylaxis. Polynucleotide and recombinant viral vaccines encoding non-structural viral proteins show therapeutic and prophylactic efficacy in animal models and are candidate immunotherapies for established low grade benign genital infections. Vaccines designed to elicit CTLs specific for the HPV oncoproteins E6 and E7 show immunogenicity and efficacy in transplantable tumour models in rodents. In clinical trials these vaccines are immunogenic and safe but show limited efficacy and further scientific developments are needed.() Research agenda can L1 HPV16/18 vaccines induce protection against other hrhpv types? are there long-term differences in the prevalence of HPV types in vaccinated and non-vaccinated populations? what is the natural history of hrhpv infection in men? Will HPV L1 vaccines protect men against HPV-associated anogenital disease? can vaccines inducing strong cell-mediated immunity to HPV early proteins have therapeutic efficacy in benign and malignant HPV associated anogenital disease? REFERENCES 1. Bosch FX & de Sanjose S. Human papillomavirus and cervical cancer burden and assessment of causality. Journal of the National Cancer Institute Monographs 2003; 3 13 [chapter 1]. 2. IARC monographs on the evaluation of carcinogen risks to humans. human papillomaviruses, vol. 64. Lyon, France: World Health Organization International Agency for Research on Cancer 1995 (Meeting of IARC Working Group on 6 13 June 1995); Stanley MA. Pathobiology of human papillomaviruses. In Grand RA (ed.) Viruses, Cell Transformation and Cancer. Amsterdam: Elsevier, 2001, pp Stanley MA. Genital papillomaviruses prospects for vaccination. Current Opinion in Infectious Diseases 1997; 10: *. Zhou J, Sun XY, Stenzel DJ et al. Expression of vaccinia recombinant HPV 16 L1 and L2 ORF proteins in epithelial cells is sufficient for assembly of HPV virion like particles. Virology 1991; 185: *. Kirnbauer R, Booy F, Cheng N et al. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proceedings of the National Academy of Sciences of the United States of America 1992; 89: Nonnenmacher B, Hubbert NL, Kirnbauer R et al. Serologic response to human papillomavirus type 16 (HPV 16) virus like particles in HPV 16 DNA positive invasive cervical cancer and cervical intraepithelial neoplasia grade III patients and controls from Colombia and Spain. The Journal of Infectious Diseases 1995; 172: Carter JJ, Koutsky LA, Hughes JP et al. Comparison of human papillomavirus types 16, 18, and 6 capsid antibody responses following incident infection. The Journal of Infectious Diseases 2000; 181: *. Suzich JA, Ghim SJ, Palmer Hill FJ et al. Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas. Proceedings of the National Academy of Sciences of the United States of America 1995; 92: Breitburd F, Kirnbauer R, Hubbert NL et al. Immunization with virus-like particles from cotton tail rabbit papillomavirus (CRPV) can protect against experimentally CRPV infection. Journal of Virology 1995; 69:

13 HPV vaccines Christensen ND, Reed CA, Cladel NM et al. Immunization with viruslike particles induces long term protection of rabbits against challenge with cottontail rabbit papillomavirus. Journal of Virology 1996; 70: Harro CD, Pang YY, Roden RB et al. Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine. Journal of the National Cancer Institute 2001; 93: Evans TG, Bonnez W, Rose RC et al. A Phase 1 study of a recombinant viruslike particle vaccine against human papillomavirus type 11 in healthy adult volunteers. The Journal of Infectious Diseases 2001; 183: Fife KH, Wheeler CM, Koutsky LA et al. Dose-ranging studies of the safety and immunogenicity of human papillomavirus Type 11 and Type 16 virus-like particle candidate vaccines in young healthy women. Vaccine 2004; 22: Ault KA, Giuliano AR, Edwards RP et al. A phase I study to evaluate a human papillomavirus (HPV) type 18 L1 VLP vaccine. Vaccine 2004; 22: White WI, Wilson SD, Bonnez W et al. In vitro infection and type-restricted antibody-mediated neutralization of authentic human papillomavirus type 16. Journal of Virology 1998; 72: Koutsky LA, Ault KA, Wheeler CM et al. A controlled trial of a human papillomavirus type 16 vaccine. The New England Journal of Medicine 2002; 347: Harper DM, Franco EL, Wheeler C et al. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet 2004; 364: Villa LL, Costa RL, Petta CA et al. Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial. The Lancet Oncology 2005; 6: Ho GY, Bierman R, Beardsley L et al. Natural history of cervicovaginal papillomavirus infection in young women. The New England Journal of Medicine 1998; 338: Liaw KL, Glass AG, Manos MM et al. Detection of human papillomavirus DNA in cytologically normal women and subsequent cervical squamous intraepithelial lesions. Journal of the National Cancer Institute 1999; 91: Schlecht NF, Kulaga S, Robitaille J et al. Persistent human papillomavirus infection as a predictor of cervical intraepithelial neoplasia. Journal of the American Medical Informatics 2001; 286: Koutsky LA, Holmes KK, Critchlow CW et al. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection. The New England Journal of Medicine 1992; 327: Herrero R, Hildesheim A, Bratti C et al. Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica. Journal of the National Cancer Institute 2000; 92: Pagliusi SR & Teresa Aguado M. Efficacy and other milestones for human papillomavirus vaccine introduction. Vaccine 2004; 23: Lowe RS, Brown DR, Bryan JT et al. Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast. The Journal of Infectious Diseases 1997; 176: Christensen ND, Reed CA, Cladel NM et al. Monoclonal antibodies to HPV-6 L1 virus-like particles identify conformational and linear neutralizing epitopes on HPV-11 in addition to type-specific epitopes on HPV-6. Virology 1996; 224: Giroglou T, Sapp M, Lane C et al. Immunological analyses of human papillomavirus capsids. Vaccine 2001; 19: Combita AL, Touze A, Bousarghin L et al. Identification of two cross-neutralizing linear epitopes within the L1 major capsid protein of human papillomaviruses. Journal of Virology 2002; 76: Ohlschlager P, Osen W, Dell K et al. Human papillomavirus type 16 L1 capsomeres induce L1 specific cytotoxic CTL lymphocytes and tumour regression in C57BL/6 mice. Journal of Virology 2003; 77: Emeny RT, Wheeler CM, Jansen K et al. Priming of human papillomavirus type 11-specific humoral and cellular immune responses in college-aged women with a virus-like particle vaccine. Journal of Virology 2002; 76:

14 292 M. Stanley 32. Pinto LA, Edwards J, Castle PE et al. Cellular immune responses to human papillomavirus (HPV)-16 L1 in healthy volunteers immunized with recombinant HPV-16 L1 virus-like particles. The Journal of Infectious Diseases 2003; 188: Balmelli C, Roden R, Potts A et al. Nasal immunization of mice with human papillomavirus type 16 viruslike particles elicits neutralizing antibodies in mucosal secretions. Journal of Virology 1998; 72: Nardelli Haefliger D, Roden R, Balmelli C et al. Mucosal but not parenteral immunization with purified human papillomavirus type 16 virus-like particles induces neutralizing titers of antibodies throughout the estrous cycle of mice. Journal of Virology 1999; 73: Rose RC, Lane C, Wilson S et al. Oral vaccination of mice with human papillomavirus virus-like particles induces systemic virus-neutralizing antibodies. Vaccine 1999; 17: Gerber S, Lane C, Brown DM et al. Human papillomavirus virus-like particles are efficient oral immunogens when coadministered with Escherichia coli heat-labile enterotoxin mutant R192G or CpG DNA. Journal of Virology 2001; 75: Reuter JD, Vivas Gonzalez BE, Gomez D et al. Intranasal vaccination with a recombinant vesicular stomatitis virus expressing cottontail rabbit papillomavirus L1 protein provides complete protection against papillomavirus-induced disease. Journal of Virology 2002; 76: Yuan H, Estes PA, Chen Y et al. Immunization with a pentameric L1 fusion protein protects against papillomavirus infection. Journal of Virology 2001; 75: Rose RC, White WI, Li M et al. Human papillomavirus type 11 recombinant L1 capsomeres induce virusneutralizing antibodies. Journal of Virology 1998; 72: Fligge C, Giroglou T, Streeck RE et al. Induction of type-specific neutralizing antibodies by capsomeres of human papillomavirus type 33. Virology 2001; 283: zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nature Reviews Cancer 2002; 2: Moore RA, Santos EB, Nicholls PK et al. Intraepithelial DNA immunisation with a plasmid encoding a codon optimised COPV E1 gene sequence, but not the wild-type gene sequence completely protects against mucosal challenge with infectious COPV in beagles. Virology 2002; 304: Moore RA, Walcott S, White KL et al. Therapeutic immunisation with COPV early genes by epithelial DNA delivery. Virology 2003; 314: Brandsma JL, Shlyankevich M, Zhang L et al. Vaccination of rabbits with an adenovirus vector expressing the papillomavirus E2 protein leads to clearance of papillomas and infection. Journal of Virology 2004; 78: Stanley MA. Progress in prophylactic and therapeutic vaccines for human papillomavirus infection. Expert Review Vaccines 2003; 2: Adams M, Borysiewicz L, Fiander A et al. Clinical studies of human papilloma vaccines in pre-invasive and invasive cancer. Vaccine 2001; 19: Frazer IH. Prevention of cervical cancer through papillomavirus vaccination. Nature Reviews in Immunology 2004; 4: Baldwin PJ, van der Burg SH, Boswell CM et al. Vaccinia-expressed human papillomavirus 16 and 18 e6 and e7 as a therapeutic vaccination for vulval and vaginal intraepithelial neoplasia. Clinical Cancer Research 2003; 9: Davidson EJ, Boswell CM, Sehr P et al. Immunological and clinical responses in women with vulval intraepithelial neoplasia vaccinated with a vaccinia virus encoding human papillomavirus 16/18 oncoproteins. Cancer Research 2003; 63: Smyth LJ, Van Poelgeest MI, Davidson EJ et al. Immunological responses in women with human papillomavirus type 16 (HPV-16)-associated anogenital intraepithelial neoplasia induced by heterologous prime-boost HPV-16 oncogene vaccination. Clinical Cancer Research 2004; 10: Glew SS, Connor ME, Snijders PJ et al. HLA expression in pre invasive cervical neoplasia in relation to human papilloma virus infection. European Journal of Cancer 1993; 29A: Abdel Hady ES, Martin Hirsch P, Duggan Keen M et al. Immunological and viral factors associated with the response of vulval intraepithelial neoplasia to photodynamic therapy. Cancer Research 2001; 61: Santin AD, Bellone S, Palmieri M et al. Induction of tumor-specific cytotoxicity in tumor infiltrating lymphocytes by HPV16 and HPV18 E7-pulsed autologous dendritic cells in patients with cancer of the uterine cervix. Gynecologic Oncology 2003; 89:

15 HPV vaccines Adams M, Navabi H, Jasani B et al. Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)] (Ampligen R). Vaccine 2003; 21: Nonn M, Schinz M, Zumbach K et al. Dendritic cell-based tumor vaccine for cervical cancer I: in vitro stimulation with recombinant protein-pulsed dendritic cells induces specific T cells to HPV16 E7 or HPV18 E7. Journal of Cancer Research and Clinical Oncology 2003; 129: Welters MJ, de Jong A, van den Eeden SJ et al. Frequent display of human papillomavirus type 16 E6- specific memory t-helper cells in the healthy population as witness of previous viral encounter. Cancer Research 2003; 63: de Jong A, van Poelgeest MI, van der Hulst JM et al. Human papillomavirus type 16-positive cervical cancer is associated with impaired CD4CT-cell immunity against early antigens E2 and E6. Cancer Research 2004; 64: