- Email: [email protected]
Cervical cancer, human papillomavirus and vaccines
Clinical Oncology (1993) 5:386-390 © 1993 The Royal College of Radiologists
Clinical Oncology
Review Article Cervical Cancer, Human Papillomavirus and Vaccines* S. A . K h a n 5th Year Medical Student, United Medical and Dental Schools of Guy's and St Thomas' Hospitals (UMDS), Guy's Hospital Campus, St Thomas' Street, London SE1 9RT, UK
Abstract. HPV-16 has been strongly implicated in the aetiology of cervical cancer a n d pre-malignant cervical intraepithelial neoplasia. Despite current technical difficulties it may soon be possible to treat these tumours with anti-HPV-16 vaccines. A prophylactic vaccine could, in theory, be developed to induce neutralizing antibodies to HPV-16 virions in genital secretions, and a therapeutic vaccine to elicit cytotoxic T-cell responses against established lesions. Keywords: Cervical cancer; H u m a n papillomavirus type-16; Vaccines
CERVICAL C A R C I N O M A
Cervical cancer kills almost 2000 women every year in England and Wales [1]. This number seems to be unaffected by policies of screening and treatment of precancerous changes in the cervix (cervical intraepithelial neoplasias (CIN)). Moreover, cervical cancer is the most c o m m o n female malignancy in developing countries, with approximately half a million new cases occurring world-wide each year [2]. The cancer carries an overall mortality rate of 60% [31. Epidemiological studies imply that cervical cancer is caused by a sexually transmitted infectious agent. The strongest candidate to emerge from recent evidence is the human papillomavirus (HPV), particularly H P V type 16 (Table 1) [4-8]. How H P V might induce cancer is unknown. It may act as a 'hit and run' carcinogen, as not all cervical carcinomas contain HPV, or, alternatively, it may simply be an indirect marker of sexual promiscuity - a known risk
*This paper is based on the manuscript which won second prize in the Royal College of Radiologists' Undergraduate essay in 1993. Correspondence and offprint requests to: S. A. Khan, 146 Bristol Road, Forest Gate, London E7 8QF, UK.
Table 1. Summary of the evidence for an association between HPV-16 and cervicalcancer The incidence of HPV-16 DNA in CIN lesions is proportional to their severity [4]. HPV-16 DNA can transform and immortalize human keratinocytes in vitro, however non-cancer associated HPVs cannot [5]. Polymerase chain reaction-detected HPV-16 DNA occurs in up to 90% of cervical cancer biopsies, but in only 10% of control subjects [61. HPV-16 E6 and E7 proteins inactivate endogenous tumour suppressor proteins p53 and pRB respectively [7]. Other animal PVs can cause cancers in their natural hosts [8].
factor for cancer of the cervix. More likely however, H P V may induce cervical cancer by inhibiting antioncogenes [7], the specific cellular defence mechanisms that act against cancer development. Public education and modification of life-styles could, in theory, reduce the risk of acquiring genital H P V infections and exposure to potential co-factors (for example smoking). However, as HPVs are linked to the majority of cervical cancers, there is also the possibility of preventing this tumour by immunizing against HPV-16. Indeed immunization can be used to prevent two other naturally occurring, virally-induced, animal carcinomas: feline leukaemia [9] and Marek's disease of chickens [10]. Recently there has also been widespread interest in trials for a vaccine against the Epstein-Barr virus which is implicated in Burkitt's lymphoma and human nasopharyngeal carcinoma [11]. It is envisaged that, in the UK, an HPV-16 vaccine might prevent the majority of cervical cancer deaths and avoid treatment of many of the estimated 250 000 cases of CIN per year [12]. Other HPVs, as well as causing benign warts (e.g. HPV-6 and -11) which may persist for several years before regressing, have been linked to some penile, anal, vulvar, lung, laryngeal and tongue carcinomas [13]. Thus, immunization against HPV-16 and other HPVs may have a significant impact upon the mortality and morbidity of several cancerous diseases, particularly in developing countries where the incidence of HPV-related cancer is high but the facilities for screening and early treatment are relatively poor.
Cervical Cancer, Human Papillomavirus and Vaccines
PAPILLOMAVIRUSES Virology Papillomavirus (PV) particles are small (55 nm diameter) and proteinaceous with an icosahedral structure of 72 capsomeres. The capsomeres are assembled from the major (L1) and minor (L2) capsid (or surface) proteins. Each virion contains a single copy of double stranded DNA. HPV type 16 DNA contains six early (El-E6) open reading frames (ORFs) of DNA which code for proteins involved in viral replication and transcription and two late ORFs (L1 and L2) coding for the capsid proteins [14]. PVs are highly species-specific and heterogenous, and, as HPV-16 has no known animal host, it might ultimately be possible to eradicate HPV-16 infection by universal immunization. Although HPV-12 is thought to be acquired primarily by sexual contact, vertical transmission of genital HPVs has been documented [15]. HPV-16 DNA has also been detected by polymerase chain reaction techniques in buccal mucosal cells, and HPV-16 antibodies have been detected in the sera of children with no history of sexual abuse [16]. Furthermore, HPV-16 DNA has been isolated from surgical instruments and even smoke from laser ablation of CIN lesions. Thus it seems HPV particles are very stable structures and infection might occur by routes other than sexual and vertical transmission. High grade CIN lesions and cervical carcinomas often have HPV-16 DNA integrated into the host genome. The E6 and E 7 0 R F s may be of particular significance as they can transform human and rodent cells in vitro [17]. These ORFs are also expressed in cervical cancers and have been shown to bind to host tumour suppressor proteins [7].
Immunology of PV Infections The competence of the patient's immune system has a major role in influencing the outcome of HPV-16 infections. Immunosuppression, whether hereditary, acquired or iatrogenic, encourages the progression of lesions to malignancy, and all branches of the human immune system seem to be involved in the immunological response to cervical cancer. The cellular (T-cell) rather than humoral (antibodies) branch of the immune system seems particularly important as there is an association between certain major histocompatibility (MHC) class II alleles (for example DQw3), which are intricately linked to T-cell responses, and to susceptibility to cervical cancer [18]. Nevertheless, neutralizing antibodies to HPV-16 in cervical secretions may prevent re-infections, and effective cell mediated immune responses probably explain why most untreated CIN lesions do not progress to malignancy. Non-specific immunological effector cells, including natural killer cells, are also important; their activity is depressed in patients with CIN [19]. This deficiency in local immunity, possibly due to co-factors such as smoking, may predispose to malignancy. Studies of naturally occurring PV-induced tumours
387
in animals, such as bovine PV type 4 which causes alimentary carcinomas in cattle, and cotton-tail rabbit PV infection which can cause skin cancers in rabbits, have provided important information. Unfortunately the study of HPV-16-specific immunity has been hampered by the lack of an animal model. There have also been difficulties in obtaining HPV antigens. HPVs cannot be propagated in vitro and CIN and malignant lesions contain few intact HPV-16 particles. HPV-16 virions have been produced in nude mice transplanted with a CIN-derived cell line containing episomal HPV-16 DNA, but the resulting yield is low [20]. Therefore studies of HPV16-specific immunity have relied almost exclusively upon synthetic antigens produced by recombinant DNA technology and protein chemistry. Encouragingly, however, HPV virions (type 31-b) have recently been synthesized in vitro using a raft cell culture system [21]. Antibodies to recombinant proteins and to synthetic peptides corresponding to HPV-16 early proteins have been detected in sera from patients with CIN, but these antibody titres are low [22]. Antibodies to HPV-16's surface proteins have also been studied: antibodies to an L2 fashion protein were detected in the sera of 47% of patients attending a sexually transmitted diseases clinic [16], and, amongst HPV16 DNA positive CIN patients, ELISA reactivity to a synthetic HPV-16 L1 peptide was found in 91% of patients' sera [23]. Unfortunately the proportion of patients in whom neutralizing antibodies to HPV-16 are actually induced has not yet been determined. The extent of HPV-16-specific cytotoxic T-lymphocyte (CTL) responses in human subjects is also unclear. However it is known that, as in many breast cancers, some cervical carcinomas fail to express MHC antigens, which are necessary for CTL immune responses to occur. The failure to demonstrate aggressive immune responses against HPV-16, however, does not necessarily mean that effective responses cannot be induced by immunization with anti-HPV-vaccines [241.
VACCINES AGAINST HPV-16-ASSOCIATED CANCERS The muliple stages of HPV-16 replication and carcinogenesis invite several strategies for the prevention and treatment of HPV-16-induced carcinomas. For example topical antiviral cytokines such as ol- and/3interferons have been used with variable success to treat genital HPV infections [25]. Other workers have inhibited the growth of cervical cancer cell lines in vivo using antisense HPV-16 E7 mRNA [26]. Here the possible development of vaccines against HPV-16 will be discussed (Fig. 1).
Prevention of HPV-16 Infections: Prophylactic Vaccines The ideal prophylactic vaccine for preventing primary HPV-16 infections would:
S. A. Khan
388
li.o HPU
E6
Ii
I E7
H
E
C
6
CERUICAL TIIMOUR
I
/
Fig. 1. Simplified diagram summarizing proposed vaccination
strategies against HPV-16-inducedcervical cancer. (a) A prophylactic vaccine might induce neutralizing antibodies against HPV capsid (surface) proteins L1 and L2, before enters cervical cells. (b) A therapeutic vaccine might induce CTL against HPV-16E6 and E7 proteins expressedin establishedlesions. 1. Induce a significant concentration of high affinity neutralizing antibodies in the genital tract; these antibodies are likely to be directed against the surface L1 and L2 proteins (Fig. la). 2. Confer long lasting immunity following a minimal number of administrations. 3. Be thermostable: such a vaccine would be invaluable in many developing countries. 4. Be cost effective. Although there is no conclusive evidence that HPV-16 capsid proteins induce neutralizing antibodies during HPV-16 infections, knowledge of other vial infections suggests that such antibodies could be induced and would be protective in cervical secretions. At present the prospects of producing significant quantities of native HPV-16 proteins, or attenuated forms of the virus, for use in vaccines, are distant. Other, non-tumorigenic, PVs are more easily available, but, as antibodies to PVs are type specific, they are unlikely to induce neutralizing antibodies against HPV-16. Currently, the most productive sources of HPV-16 immunogens are recombinant proteins and synthetic peptides. There have been conflicting reports about the efficacy of inducing neutralizing antibodies to bacterially derived L1 and L2 fusion proteins [27]. This suggests that, whilst PV capsid proteins do contain neutralizing epitopes, recombinant late proteins may be inefficient stimulators of antibody production. This is presumably because bacterially
derived recombinant proteins are poor imitations of native viral proteins. One probable reason for this is that they are not post-transcriptionally modified. Eukaryotically-expressed recombinant proteins may eventually prove to be a better option: baculovirusderived proteins are currently undergoing evaluation as immunogens against human immunodeficiency virus [28]. A recombinant sub unit vaccine based on the Epstein-Barr virus envelope glycoprotein gp340 is also currently undergoing human trials [11]. Antibodies to native proteins are commonly directed against 'conformation-dependent' epitopes (an epitope is the part of the protein that is recognized as being foreign and evokes an immune response). Hence synthetic peptides [29] made to mimic conformation-dependent epitopes of, for example HPV-16's surface proteins, may effectively induce neutralizing antibodies and thus be used in a prophylactic vaccine. Peptides containing T-helper cell epitopes would also have to be constructed for inclusion in such a vaccine to ensure a B-cell response as B cells are dependent on T-cell help for antibody production [30]. Encouragingly, T-helper cell epitopes have been mapped in human T-ceU lines [31]. More imaginative vaccine approaches, such as the use of human anti-idiotype antibodies which mimic HPV-16 neutralizing epitopes, might also be worth considering. The efficacy of the 'inanimate' vaccines described above may be improved by the incorporation of adjuvanIs such as aluminium hydroxide or immunostimulatory complexes (adjuvants formed by the conjugating proteins to Quil-A) [32]. Furthermore, although a common mucosal immune system exists, it may be worthwhile immunizing subjects locally via vaginal surfaces to elicit neutralizing antibodies in cervical secretions. Another attractive approach is the development of 'live' recombinant vaccines. Here DNA coding for protein epitopes is artificially introduced into another organism and is subsequently transcribed and expressed. A 16-amino acid HPV-16 L1 B-cell epitope has been expressed on a chimeric poliovirus and was immunogenic in rabbits [33]. Bacille CalmetteGu6rin (BCG) may be a particularly good immunogen carrier in such systems, as it can express large proteins which stimulate both humoral and cellmediated immunity [34]. It is also one of the few live attenuated vaccines that can be given at birth and confers long-lived immunity with a single dose. Virus-like particles (VLP) produced in eukaryotic systems might also' be good immunogens. HPV-16 VLPs have been produced via recombinant vaccinia virus [35]. Immunization with a recombinant rabiesvaccinia has been successfully used in foxes [36] and an HIV-vaccinia construct was found to induce immune responses in humans [37]. Despite recombinant D N A technology making the construction of a prophylactic HPV-16 vaccine a distinct possibility, there are potential problems: 1. The neutralizing epitopes of HPV-16 have not been determined; however their discovery is, hopefully, not far away. 2. Naturally occurring HPV-16 variants exist and these may evade neutralization by antibodies
I
Cervical Cancer, Human Papillomavirus and Vaccines
stimulated from the vaccine's original epitopes. This problem may be overcome if neutralizing epitopes common to all these variants are discovered. 3. It would take several years to determine any prophylactic vaccine's effectiveness for preventing CIN. Nevertheless, by immunizing prepubescent girls against HPV-16 and comparing with them the incidence of CIN amongst nonimmunized cohorts, the efficacy of such a vaccine could be assessed. The effect of anti-HPV-16 immunization on the incidence of cervical carcinoma would not be possible to assess as any latestage pre-malignant CIN lesions would ethnically have to be treated and not allowed to progress.
Destroying Established Lesions: Therapeutic Vaccines A vaccine capable of inducing HPV-16-specific CTLs, which can destroy established HPV-16containing CIN lesions and cervical carcinomas, is an exciting possibility. A precedent for this approach exists: recombinant vaccinia containing T antigens of polyomavirus confers immunity to rats against syngeneic tumours induced with the full length viral genome [38]. E6 and E7 are the main HPV-16 proteins transcribed in cervical cancer cells [39] and it is thought that they probably represent tumour specific, CTL inducing antigens for most HPV-16 lesions. They would therefore be reasonable targets for a therapeutic vaccine (Fig. lb). Indeed, CTL elicited in mice by transfer of non-tumorigenic cell lines transferred with recombinant vaccinia E7, or infected with recombinant vaccinia E6 or E7, are protective in vivo for rats subsequently implanted with tumorigenic cell lines containing transfected HPV-16 DNA [40]. Unlike B cells, CTLs tend to recognize short (approximately 10 amino acids) peptide epitopes. HPV-16 E6/E7 synthetic peptides or proteins might therefore prove to be effective immunogens, particularly for the immunocompromised patient. Although protein immunogens are generally poor stimulators of CTLs, synthetic viral proteins have been used to induce CTL responses in experimental animals [41]. Inclusion of specific T-helper cell epitopes in vaccine constructs will probably by necessary for, or at least to potentiate, CTL immune responses, and, encouragingly, some murine T-helper epitopes on the HPV16 E7 protein have been identified. The use of mutant or subunit recombinant proteins as immunogens is also feasible, as the majority of CTL epitopes, being short peptides, would be unaltered. However T-cell epitopes bind to and form complexes with MHC antigens on antigen-presenting cells of the body's immune system, and T cells need to recognize both the foreign epitope as well as the MHC. Thus CTL immune responses are said to be MHC restricted. As there is tremendous MHC heterogeneity in the general population, any HPV-16 vaccine would therefore need to incorporate either several HPV-16 CTL epitopes, or a highly promiscuous ('public') epitope, to induce effective immunity amongst the general population. Encouragingly, a public murine T-helper cell epitope on HPV-16 E7
389
has been identified [42] and it is reasonable to suspect that similar CTL epitopes also exist on the HPV-16 E6 and E7 proteins. There are again potential problems preventing therapeutic vaccination strategies being applied to humans: 1. Some HPV-16-containing lesions may not be affected by CTL responses because they fail to express MHC class I antigens [43] which are necessary for tumour recognition by CTL, as described above. Prior administration of o-interferon, which 'upregulates' expression of MHC class I antigens in cells, might overcome this problem. 2. It is unknown if HPV-16 DNA is retained and expressed in cancer cells after metastasis. 3. Almost 20% of cervical carcinoma do not contain HPV-16, and therefore would not be susceptible to such a vaccine. A further necessary consideration, before vaccinating with HPV-16 recombinant or synthetic early proteins, is that E6 and E7 are potentially oncogenic and could therefore be dangerous for patient administration. However, the oncogenic regions of E6 and E7, which bind to endogenous tumour suppressors such as p53 and pRB and cause cell transformation, have been mapped [44] and thus it is feasible that truncated or mutant E6 and E7 proteins, lacking these oncogenic sites, might be constructed. How significantly this would compromise immunogenicity is unclear. Of critical importance in determining whether a CTL response develops is the method of immunization. CTLs are more efficiently induced when protein immunogens are formed intracellularly [29], therefore a vaccine designed to stimulate CTLs might best be delivered via 'live' recombinant viruses, providing the subject is immunocompetent. Systems such as vaccinia may be ideal for the expression of large protein molecules, whereas cocktails of HPVcontaining poliovirus chimeras could be suitable for presenting a variety of shorter T-cell epitopes to the mucosal immune system. One of the most pressing problems for the development of a therapeutic vaccine against HPV-16 is the construction of a safe delivery system. As well as vaccinia, strains of low pathogenicity, attenuated adenovirus and Salmonella typhimurium are now available, and are particularly attractive vectors as they can stimulate mucosal immunity [45]. The efficacy of therapeutic vaccines against HPV-16associated CIN and cervical carcinomas could be assessed by monitoring cervical smears and tumour regression respectively in immunized and nonimmunized cohort groups.
CONCLUSION There are obstacles to overcome before the prevention and treatment of cervical cancer with vaccines becomes a reality. Nevertheless recent progress in epitope mapping, genetic engineering and protein synthesis technology have made vaccine therapy an
390
exciting and tenable possibility for the near future. Indeed, two Phase I clinical trials have recently been launched (in the UK and in Australia) to determine the immunogenicity of therapeutic vaccination against HPV-16. Both trials involve live vaccinia/ HPV-16 recombinants expressing HPV-16 E7. Acknowledgements. I am very grateful to Dr J. Cason (Laboratory of Cancer Virology, St Thomas' Hospital, London) and Dr A. R. Timothy (Department of Radiotherapy and Oncology, St Thomas' Hospital, London) for their help and advice during the preparation of this paper. References 1. Macgregor JE, Teper S.Mortality for carcinoma of cervix uteri in Britain. Lancet 1978;ii:774-6. 2. Munoz N, Bosch FX. Epidemiology of cervical cancer. In: Munoz N, Bosch FX, Jensen OM, editors. Human papillomaviruses and cervical cancer. Lyon: IARC Scientific Publications No. 6 1989, 9-40. 3. Jordan M. Treatment of CIN by destruction. In: Jordan JA, Sharp F, Singer A. editors. Preclinical neoplasia of the cervix. London: Royal College of Obstetricians and Gynaecologists, 1982, 185-6. 4. Syrjanen K, Parkkinen S, Mantyjarvi R, et al. Human papillomavirus type as an important determinant of the natural history of human papillomavirus infections of the uterine cervix. Eur J Epidemiol 1985;1:180-7. 5. Jewers RJ, Hildebrandt P, Ludlow JW, et al. Regions of human papillomavirus type-16 oncoprotein required for immortalization of human keratinocytes. J Virol 1992;66:132%35. 6. Labeit D, Back W, Weizsacker FV, et al. Increased detection of HPV-16 virus in invasive, but not early cervical cancers. J Med Virol 1992;36:131-5. 7. Tidy JA, Wrede D. Turnout suppressor genes: new pathways in gynaecological cancer. Int J Gynaecol Cancer 1992;2:1-8. 8. Sundberg JP. Animal papillomaviruses. In: Salzman NP, Howley PM editors. The papovaviridae. Volume II; The papillomaviruses. New York: Plenum. 1987, 40-103. 9. Mastro JM, Lewis MG, Mathes LE, et al. Feline leukaemia vaccine: Efficacy, contents and probable mechanism. Vet Immunol Immunopathol 1986;11:205-13. 10. Nazerian K. Marek's Disease: a herpes virus-induced malignant lymphoma of the chicken. In: Klein G, editor. Viral oncology. New York: Raven 1980, 665-82. 11. Morgan AJ. Epstein-Barr virus vaccines. Vaccine 1992;10:563-71. 12. Cuzick J, Terry G, Ho L, et al. Human papillomavirus type-16 DNA in cervical smears as a predictor of high-grade cervical cancer. Lancet 1992;339:959-60. 13. zur Hausen H, Schneider A. The role of papillomaviruses in human anogenital cancer. In: Salzman NP, Howley PM, editors. The papovaviridae. Volume II: The papillomaviruses. New York: Plenum, 1987, 245-63. 14. Pfister It, Fuchs P. Papillomaviruses: Particles, genome organisation and proteins. In: Syrjanen K, Gissman L, Koss LG, editors. Papillomaviruses and human disease. Berlin: Springer-Verlag, 1987, 6-15. 15. Sedlacek TV, Lindheim S, Eder C, et al. Mechanism for human papillomavirus transmission at birth. Am J Obstet Gynecol 1989;161:55-59. 16. Jenison SA, Xiu-ping Y, Valentine JM, et al. Evidence of prevalent genital-type human papillomavirus infections in adults and children. J Infect Dis 1990;162:60-9. 17. Munger K, Phelps WC, Bubb V, et al. The E6 and E7 genes of human papillomavirus type-16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol 1989;63:4417-21. 18. Klitz W. Viruses, cancer and the MHC. Nature 1992;356:17-8. 19. Tay SK, Jenkins D, Maddox PH, et al. Subpopulations of Langerhans cells in cervical neoplasia. Br J Obstet Gynaecol 1987 ;94:10-5. 20. Sterling J, Stanley M, Gatward G, et al. Production of human papillomavirus type-16 virions in a keratinocyte cell line. J
S . A . Khan Virol 1990;64:6305-7. 21. Meyers C, Frattini MG, Hudson JB, et al. Biosynthesis of human papillomavirus type 31b from a continuous cell line upon epithelial differentiation. Science 1992;257:971-3. 22. Cason J, Best JM. Antibody responses to human papillomavirus type-16 infections. Rev Med Virol 1991;1:201-9. 23. Cason J, Kambo PK, Best JM, et al Detection of antibodies to a linear epitope on the major coat protein (L1) of human papillomavirus type-16 in sera from patients with cervical intraepithelial neoplasia and children. Int J Cancer 1992;50:349-55. 24. DaviesDH, McIndoeGAJ, ChainBM. Current status review. Cancer of the cervix: Prospects for immunological control. Int J Exp Pathol 1991;72:239-51. 25. Einhorn N, Ling P, Stander H. Systemic interferon alpha treatment of human condylomata acuminata. Acta Obstet Gynecol Scand 1983;62:285-7. 26. Doeberitz MVK, Rittmuller C, zur Hausen H, et al. Inhibition of tumorigenicity of cervical cancer cells in nude mice by HPV E6 and E7 antisense mRNA. Int J Cancer 1992;51:831-4. 27. Jarrett WFH, Smith KT, O'Neil BW, et al. Studies on vaccination against papillomaviruses: Prophylactic and therapeutic vaccination with recombinant structural proteins. Virology 1991;184:33-4. 28. Barnes DM. AIDS vaccine trial OKed. Science 1987;237:973. 29. Geysen HM, Rodda SJ, Mason TJ. A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol Immunol 1986;23:709-16. 30. Klein J. T-lymphocyte responses In: Immunology. Oxford: Blackwell, 1990, 346-50. 31. Strang G, Hickling JK, McIndoe GAJ, et al. Human T-cell responses to human papillomavirus type-16 L1 and E6: Identification of T-cell determinants, HLA-DR restriction and virus type specificity. J Gen Virol 1990;71:423-31. 32. Jones PD, Tha-Hla R, Morein B, et al. Cellular immune responses in the murine lung to local immunization with influenza A virus glycoproteins in micelles and immunostimulatory complexes (ISCOMS). Scand J Immunol 1988;27:645-52. 33. Jenkins O, Cason J, Burke KL, et al. An antigenic chimera of poliovirus induces antibodies against human papillomavirus type-16. J Virol 1990;64:1201-6. 34. Aldovini A, Young RA. Humoral and cell-mediated immune responses to live recombinant B C G - H I V vaccines. Nature 1991 ;351:479-82. 35. Zhou J, Sun X-Y, Stenzel DJ, et al. Expression of vaccinia recombinant HPV-16 L1 and L 2 0 R F proteins in epithelial cells is sufficient for assembly of HPV virion-like particles. Virology 1991;185:251-7. 36. Brochier B, Kiery MP, Costy F, et al. Large scale eradication of rabies using a vaccinia-rabies vaccine. Nature 1991 ;354:520-2. 37. Zagury D, Leonard R, Fouchard M, et al. Immunization against AIDS in humans. Nature 1987;326:249-50. 38. Lathe R, Kieny MP, Gerlinger P, et al. Tumour prevention and rejection with recombinant vaccinia. Nature 1987; 326:878-80. 39. Smotkin D, Wettstein FO. Transcription of human papillomavirus type-16 early genes in cervical cancer and a cancerderived cell line and identification of the E7 protein. Proc Natl Acad Sci USA 1986;83:4680-4. 40. Chen L, Thomas EK, Hu SL, et al. Human papillomavirus type-16 nucleoprotein E7 is a tumor rejection antigen. Proc Natl Acad Sci USA 1991;88:110-4. 41. Takkahashi H, Takeshita T, Moein B, et al. Induction of CD8+ cytotoxic T-cells by immunization with purified HIV-1 envelope protein in ISCOMS. Nature 1990;344:873-5. 42. Tindle RW, Fernando GJ, Sterling JC, et al. A 'public' Thelper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-association human papillomavirus genotypes. Proc Natl Acad Sci USA 1991;88:55887-91. 43. Connor ME, Stern PL. Loss of MHC class I expression in cervical carcinomas. Int J Cancer 1990;46:1029-34. 44. Crook T, Tidy JA, Vousden KH. Degradation of p53 can be targeted by HPV E6 sequences distinct from those required for p53 binding and transactivation. Cell 1991; 67:547-56. 45. Frankling HT, Grossman RA, Bartelloni PJ, et al. Immunization with live type-7 and -4 adenovirus vaccines. J Infect Dis 1971;24:148-54.
Clinical Oncology
Review Article Cervical Cancer, Human Papillomavirus and Vaccines* S. A . K h a n 5th Year Medical Student, United Medical and Dental Schools of Guy's and St Thomas' Hospitals (UMDS), Guy's Hospital Campus, St Thomas' Street, London SE1 9RT, UK
Abstract. HPV-16 has been strongly implicated in the aetiology of cervical cancer a n d pre-malignant cervical intraepithelial neoplasia. Despite current technical difficulties it may soon be possible to treat these tumours with anti-HPV-16 vaccines. A prophylactic vaccine could, in theory, be developed to induce neutralizing antibodies to HPV-16 virions in genital secretions, and a therapeutic vaccine to elicit cytotoxic T-cell responses against established lesions. Keywords: Cervical cancer; H u m a n papillomavirus type-16; Vaccines
CERVICAL C A R C I N O M A
Cervical cancer kills almost 2000 women every year in England and Wales [1]. This number seems to be unaffected by policies of screening and treatment of precancerous changes in the cervix (cervical intraepithelial neoplasias (CIN)). Moreover, cervical cancer is the most c o m m o n female malignancy in developing countries, with approximately half a million new cases occurring world-wide each year [2]. The cancer carries an overall mortality rate of 60% [31. Epidemiological studies imply that cervical cancer is caused by a sexually transmitted infectious agent. The strongest candidate to emerge from recent evidence is the human papillomavirus (HPV), particularly H P V type 16 (Table 1) [4-8]. How H P V might induce cancer is unknown. It may act as a 'hit and run' carcinogen, as not all cervical carcinomas contain HPV, or, alternatively, it may simply be an indirect marker of sexual promiscuity - a known risk
*This paper is based on the manuscript which won second prize in the Royal College of Radiologists' Undergraduate essay in 1993. Correspondence and offprint requests to: S. A. Khan, 146 Bristol Road, Forest Gate, London E7 8QF, UK.
Table 1. Summary of the evidence for an association between HPV-16 and cervicalcancer The incidence of HPV-16 DNA in CIN lesions is proportional to their severity [4]. HPV-16 DNA can transform and immortalize human keratinocytes in vitro, however non-cancer associated HPVs cannot [5]. Polymerase chain reaction-detected HPV-16 DNA occurs in up to 90% of cervical cancer biopsies, but in only 10% of control subjects [61. HPV-16 E6 and E7 proteins inactivate endogenous tumour suppressor proteins p53 and pRB respectively [7]. Other animal PVs can cause cancers in their natural hosts [8].
factor for cancer of the cervix. More likely however, H P V may induce cervical cancer by inhibiting antioncogenes [7], the specific cellular defence mechanisms that act against cancer development. Public education and modification of life-styles could, in theory, reduce the risk of acquiring genital H P V infections and exposure to potential co-factors (for example smoking). However, as HPVs are linked to the majority of cervical cancers, there is also the possibility of preventing this tumour by immunizing against HPV-16. Indeed immunization can be used to prevent two other naturally occurring, virally-induced, animal carcinomas: feline leukaemia [9] and Marek's disease of chickens [10]. Recently there has also been widespread interest in trials for a vaccine against the Epstein-Barr virus which is implicated in Burkitt's lymphoma and human nasopharyngeal carcinoma [11]. It is envisaged that, in the UK, an HPV-16 vaccine might prevent the majority of cervical cancer deaths and avoid treatment of many of the estimated 250 000 cases of CIN per year [12]. Other HPVs, as well as causing benign warts (e.g. HPV-6 and -11) which may persist for several years before regressing, have been linked to some penile, anal, vulvar, lung, laryngeal and tongue carcinomas [13]. Thus, immunization against HPV-16 and other HPVs may have a significant impact upon the mortality and morbidity of several cancerous diseases, particularly in developing countries where the incidence of HPV-related cancer is high but the facilities for screening and early treatment are relatively poor.
Cervical Cancer, Human Papillomavirus and Vaccines
PAPILLOMAVIRUSES Virology Papillomavirus (PV) particles are small (55 nm diameter) and proteinaceous with an icosahedral structure of 72 capsomeres. The capsomeres are assembled from the major (L1) and minor (L2) capsid (or surface) proteins. Each virion contains a single copy of double stranded DNA. HPV type 16 DNA contains six early (El-E6) open reading frames (ORFs) of DNA which code for proteins involved in viral replication and transcription and two late ORFs (L1 and L2) coding for the capsid proteins [14]. PVs are highly species-specific and heterogenous, and, as HPV-16 has no known animal host, it might ultimately be possible to eradicate HPV-16 infection by universal immunization. Although HPV-12 is thought to be acquired primarily by sexual contact, vertical transmission of genital HPVs has been documented [15]. HPV-16 DNA has also been detected by polymerase chain reaction techniques in buccal mucosal cells, and HPV-16 antibodies have been detected in the sera of children with no history of sexual abuse [16]. Furthermore, HPV-16 DNA has been isolated from surgical instruments and even smoke from laser ablation of CIN lesions. Thus it seems HPV particles are very stable structures and infection might occur by routes other than sexual and vertical transmission. High grade CIN lesions and cervical carcinomas often have HPV-16 DNA integrated into the host genome. The E6 and E 7 0 R F s may be of particular significance as they can transform human and rodent cells in vitro [17]. These ORFs are also expressed in cervical cancers and have been shown to bind to host tumour suppressor proteins [7].
Immunology of PV Infections The competence of the patient's immune system has a major role in influencing the outcome of HPV-16 infections. Immunosuppression, whether hereditary, acquired or iatrogenic, encourages the progression of lesions to malignancy, and all branches of the human immune system seem to be involved in the immunological response to cervical cancer. The cellular (T-cell) rather than humoral (antibodies) branch of the immune system seems particularly important as there is an association between certain major histocompatibility (MHC) class II alleles (for example DQw3), which are intricately linked to T-cell responses, and to susceptibility to cervical cancer [18]. Nevertheless, neutralizing antibodies to HPV-16 in cervical secretions may prevent re-infections, and effective cell mediated immune responses probably explain why most untreated CIN lesions do not progress to malignancy. Non-specific immunological effector cells, including natural killer cells, are also important; their activity is depressed in patients with CIN [19]. This deficiency in local immunity, possibly due to co-factors such as smoking, may predispose to malignancy. Studies of naturally occurring PV-induced tumours
387
in animals, such as bovine PV type 4 which causes alimentary carcinomas in cattle, and cotton-tail rabbit PV infection which can cause skin cancers in rabbits, have provided important information. Unfortunately the study of HPV-16-specific immunity has been hampered by the lack of an animal model. There have also been difficulties in obtaining HPV antigens. HPVs cannot be propagated in vitro and CIN and malignant lesions contain few intact HPV-16 particles. HPV-16 virions have been produced in nude mice transplanted with a CIN-derived cell line containing episomal HPV-16 DNA, but the resulting yield is low [20]. Therefore studies of HPV16-specific immunity have relied almost exclusively upon synthetic antigens produced by recombinant DNA technology and protein chemistry. Encouragingly, however, HPV virions (type 31-b) have recently been synthesized in vitro using a raft cell culture system [21]. Antibodies to recombinant proteins and to synthetic peptides corresponding to HPV-16 early proteins have been detected in sera from patients with CIN, but these antibody titres are low [22]. Antibodies to HPV-16's surface proteins have also been studied: antibodies to an L2 fashion protein were detected in the sera of 47% of patients attending a sexually transmitted diseases clinic [16], and, amongst HPV16 DNA positive CIN patients, ELISA reactivity to a synthetic HPV-16 L1 peptide was found in 91% of patients' sera [23]. Unfortunately the proportion of patients in whom neutralizing antibodies to HPV-16 are actually induced has not yet been determined. The extent of HPV-16-specific cytotoxic T-lymphocyte (CTL) responses in human subjects is also unclear. However it is known that, as in many breast cancers, some cervical carcinomas fail to express MHC antigens, which are necessary for CTL immune responses to occur. The failure to demonstrate aggressive immune responses against HPV-16, however, does not necessarily mean that effective responses cannot be induced by immunization with anti-HPV-vaccines [241.
VACCINES AGAINST HPV-16-ASSOCIATED CANCERS The muliple stages of HPV-16 replication and carcinogenesis invite several strategies for the prevention and treatment of HPV-16-induced carcinomas. For example topical antiviral cytokines such as ol- and/3interferons have been used with variable success to treat genital HPV infections [25]. Other workers have inhibited the growth of cervical cancer cell lines in vivo using antisense HPV-16 E7 mRNA [26]. Here the possible development of vaccines against HPV-16 will be discussed (Fig. 1).
Prevention of HPV-16 Infections: Prophylactic Vaccines The ideal prophylactic vaccine for preventing primary HPV-16 infections would:
S. A. Khan
388
li.o HPU
E6
Ii
I E7
H
E
C
6
CERUICAL TIIMOUR
I
/
Fig. 1. Simplified diagram summarizing proposed vaccination
strategies against HPV-16-inducedcervical cancer. (a) A prophylactic vaccine might induce neutralizing antibodies against HPV capsid (surface) proteins L1 and L2, before enters cervical cells. (b) A therapeutic vaccine might induce CTL against HPV-16E6 and E7 proteins expressedin establishedlesions. 1. Induce a significant concentration of high affinity neutralizing antibodies in the genital tract; these antibodies are likely to be directed against the surface L1 and L2 proteins (Fig. la). 2. Confer long lasting immunity following a minimal number of administrations. 3. Be thermostable: such a vaccine would be invaluable in many developing countries. 4. Be cost effective. Although there is no conclusive evidence that HPV-16 capsid proteins induce neutralizing antibodies during HPV-16 infections, knowledge of other vial infections suggests that such antibodies could be induced and would be protective in cervical secretions. At present the prospects of producing significant quantities of native HPV-16 proteins, or attenuated forms of the virus, for use in vaccines, are distant. Other, non-tumorigenic, PVs are more easily available, but, as antibodies to PVs are type specific, they are unlikely to induce neutralizing antibodies against HPV-16. Currently, the most productive sources of HPV-16 immunogens are recombinant proteins and synthetic peptides. There have been conflicting reports about the efficacy of inducing neutralizing antibodies to bacterially derived L1 and L2 fusion proteins [27]. This suggests that, whilst PV capsid proteins do contain neutralizing epitopes, recombinant late proteins may be inefficient stimulators of antibody production. This is presumably because bacterially
derived recombinant proteins are poor imitations of native viral proteins. One probable reason for this is that they are not post-transcriptionally modified. Eukaryotically-expressed recombinant proteins may eventually prove to be a better option: baculovirusderived proteins are currently undergoing evaluation as immunogens against human immunodeficiency virus [28]. A recombinant sub unit vaccine based on the Epstein-Barr virus envelope glycoprotein gp340 is also currently undergoing human trials [11]. Antibodies to native proteins are commonly directed against 'conformation-dependent' epitopes (an epitope is the part of the protein that is recognized as being foreign and evokes an immune response). Hence synthetic peptides [29] made to mimic conformation-dependent epitopes of, for example HPV-16's surface proteins, may effectively induce neutralizing antibodies and thus be used in a prophylactic vaccine. Peptides containing T-helper cell epitopes would also have to be constructed for inclusion in such a vaccine to ensure a B-cell response as B cells are dependent on T-cell help for antibody production [30]. Encouragingly, T-helper cell epitopes have been mapped in human T-ceU lines [31]. More imaginative vaccine approaches, such as the use of human anti-idiotype antibodies which mimic HPV-16 neutralizing epitopes, might also be worth considering. The efficacy of the 'inanimate' vaccines described above may be improved by the incorporation of adjuvanIs such as aluminium hydroxide or immunostimulatory complexes (adjuvants formed by the conjugating proteins to Quil-A) [32]. Furthermore, although a common mucosal immune system exists, it may be worthwhile immunizing subjects locally via vaginal surfaces to elicit neutralizing antibodies in cervical secretions. Another attractive approach is the development of 'live' recombinant vaccines. Here DNA coding for protein epitopes is artificially introduced into another organism and is subsequently transcribed and expressed. A 16-amino acid HPV-16 L1 B-cell epitope has been expressed on a chimeric poliovirus and was immunogenic in rabbits [33]. Bacille CalmetteGu6rin (BCG) may be a particularly good immunogen carrier in such systems, as it can express large proteins which stimulate both humoral and cellmediated immunity [34]. It is also one of the few live attenuated vaccines that can be given at birth and confers long-lived immunity with a single dose. Virus-like particles (VLP) produced in eukaryotic systems might also' be good immunogens. HPV-16 VLPs have been produced via recombinant vaccinia virus [35]. Immunization with a recombinant rabiesvaccinia has been successfully used in foxes [36] and an HIV-vaccinia construct was found to induce immune responses in humans [37]. Despite recombinant D N A technology making the construction of a prophylactic HPV-16 vaccine a distinct possibility, there are potential problems: 1. The neutralizing epitopes of HPV-16 have not been determined; however their discovery is, hopefully, not far away. 2. Naturally occurring HPV-16 variants exist and these may evade neutralization by antibodies
I
Cervical Cancer, Human Papillomavirus and Vaccines
stimulated from the vaccine's original epitopes. This problem may be overcome if neutralizing epitopes common to all these variants are discovered. 3. It would take several years to determine any prophylactic vaccine's effectiveness for preventing CIN. Nevertheless, by immunizing prepubescent girls against HPV-16 and comparing with them the incidence of CIN amongst nonimmunized cohorts, the efficacy of such a vaccine could be assessed. The effect of anti-HPV-16 immunization on the incidence of cervical carcinoma would not be possible to assess as any latestage pre-malignant CIN lesions would ethnically have to be treated and not allowed to progress.
Destroying Established Lesions: Therapeutic Vaccines A vaccine capable of inducing HPV-16-specific CTLs, which can destroy established HPV-16containing CIN lesions and cervical carcinomas, is an exciting possibility. A precedent for this approach exists: recombinant vaccinia containing T antigens of polyomavirus confers immunity to rats against syngeneic tumours induced with the full length viral genome [38]. E6 and E7 are the main HPV-16 proteins transcribed in cervical cancer cells [39] and it is thought that they probably represent tumour specific, CTL inducing antigens for most HPV-16 lesions. They would therefore be reasonable targets for a therapeutic vaccine (Fig. lb). Indeed, CTL elicited in mice by transfer of non-tumorigenic cell lines transferred with recombinant vaccinia E7, or infected with recombinant vaccinia E6 or E7, are protective in vivo for rats subsequently implanted with tumorigenic cell lines containing transfected HPV-16 DNA [40]. Unlike B cells, CTLs tend to recognize short (approximately 10 amino acids) peptide epitopes. HPV-16 E6/E7 synthetic peptides or proteins might therefore prove to be effective immunogens, particularly for the immunocompromised patient. Although protein immunogens are generally poor stimulators of CTLs, synthetic viral proteins have been used to induce CTL responses in experimental animals [41]. Inclusion of specific T-helper cell epitopes in vaccine constructs will probably by necessary for, or at least to potentiate, CTL immune responses, and, encouragingly, some murine T-helper epitopes on the HPV16 E7 protein have been identified. The use of mutant or subunit recombinant proteins as immunogens is also feasible, as the majority of CTL epitopes, being short peptides, would be unaltered. However T-cell epitopes bind to and form complexes with MHC antigens on antigen-presenting cells of the body's immune system, and T cells need to recognize both the foreign epitope as well as the MHC. Thus CTL immune responses are said to be MHC restricted. As there is tremendous MHC heterogeneity in the general population, any HPV-16 vaccine would therefore need to incorporate either several HPV-16 CTL epitopes, or a highly promiscuous ('public') epitope, to induce effective immunity amongst the general population. Encouragingly, a public murine T-helper cell epitope on HPV-16 E7
389
has been identified [42] and it is reasonable to suspect that similar CTL epitopes also exist on the HPV-16 E6 and E7 proteins. There are again potential problems preventing therapeutic vaccination strategies being applied to humans: 1. Some HPV-16-containing lesions may not be affected by CTL responses because they fail to express MHC class I antigens [43] which are necessary for tumour recognition by CTL, as described above. Prior administration of o-interferon, which 'upregulates' expression of MHC class I antigens in cells, might overcome this problem. 2. It is unknown if HPV-16 DNA is retained and expressed in cancer cells after metastasis. 3. Almost 20% of cervical carcinoma do not contain HPV-16, and therefore would not be susceptible to such a vaccine. A further necessary consideration, before vaccinating with HPV-16 recombinant or synthetic early proteins, is that E6 and E7 are potentially oncogenic and could therefore be dangerous for patient administration. However, the oncogenic regions of E6 and E7, which bind to endogenous tumour suppressors such as p53 and pRB and cause cell transformation, have been mapped [44] and thus it is feasible that truncated or mutant E6 and E7 proteins, lacking these oncogenic sites, might be constructed. How significantly this would compromise immunogenicity is unclear. Of critical importance in determining whether a CTL response develops is the method of immunization. CTLs are more efficiently induced when protein immunogens are formed intracellularly [29], therefore a vaccine designed to stimulate CTLs might best be delivered via 'live' recombinant viruses, providing the subject is immunocompetent. Systems such as vaccinia may be ideal for the expression of large protein molecules, whereas cocktails of HPVcontaining poliovirus chimeras could be suitable for presenting a variety of shorter T-cell epitopes to the mucosal immune system. One of the most pressing problems for the development of a therapeutic vaccine against HPV-16 is the construction of a safe delivery system. As well as vaccinia, strains of low pathogenicity, attenuated adenovirus and Salmonella typhimurium are now available, and are particularly attractive vectors as they can stimulate mucosal immunity [45]. The efficacy of therapeutic vaccines against HPV-16associated CIN and cervical carcinomas could be assessed by monitoring cervical smears and tumour regression respectively in immunized and nonimmunized cohort groups.
CONCLUSION There are obstacles to overcome before the prevention and treatment of cervical cancer with vaccines becomes a reality. Nevertheless recent progress in epitope mapping, genetic engineering and protein synthesis technology have made vaccine therapy an
390
exciting and tenable possibility for the near future. Indeed, two Phase I clinical trials have recently been launched (in the UK and in Australia) to determine the immunogenicity of therapeutic vaccination against HPV-16. Both trials involve live vaccinia/ HPV-16 recombinants expressing HPV-16 E7. Acknowledgements. I am very grateful to Dr J. Cason (Laboratory of Cancer Virology, St Thomas' Hospital, London) and Dr A. R. Timothy (Department of Radiotherapy and Oncology, St Thomas' Hospital, London) for their help and advice during the preparation of this paper. References 1. Macgregor JE, Teper S.Mortality for carcinoma of cervix uteri in Britain. Lancet 1978;ii:774-6. 2. Munoz N, Bosch FX. Epidemiology of cervical cancer. In: Munoz N, Bosch FX, Jensen OM, editors. Human papillomaviruses and cervical cancer. Lyon: IARC Scientific Publications No. 6 1989, 9-40. 3. Jordan M. Treatment of CIN by destruction. In: Jordan JA, Sharp F, Singer A. editors. Preclinical neoplasia of the cervix. London: Royal College of Obstetricians and Gynaecologists, 1982, 185-6. 4. Syrjanen K, Parkkinen S, Mantyjarvi R, et al. Human papillomavirus type as an important determinant of the natural history of human papillomavirus infections of the uterine cervix. Eur J Epidemiol 1985;1:180-7. 5. Jewers RJ, Hildebrandt P, Ludlow JW, et al. Regions of human papillomavirus type-16 oncoprotein required for immortalization of human keratinocytes. J Virol 1992;66:132%35. 6. Labeit D, Back W, Weizsacker FV, et al. Increased detection of HPV-16 virus in invasive, but not early cervical cancers. J Med Virol 1992;36:131-5. 7. Tidy JA, Wrede D. Turnout suppressor genes: new pathways in gynaecological cancer. Int J Gynaecol Cancer 1992;2:1-8. 8. Sundberg JP. Animal papillomaviruses. In: Salzman NP, Howley PM editors. The papovaviridae. Volume II; The papillomaviruses. New York: Plenum. 1987, 40-103. 9. Mastro JM, Lewis MG, Mathes LE, et al. Feline leukaemia vaccine: Efficacy, contents and probable mechanism. Vet Immunol Immunopathol 1986;11:205-13. 10. Nazerian K. Marek's Disease: a herpes virus-induced malignant lymphoma of the chicken. In: Klein G, editor. Viral oncology. New York: Raven 1980, 665-82. 11. Morgan AJ. Epstein-Barr virus vaccines. Vaccine 1992;10:563-71. 12. Cuzick J, Terry G, Ho L, et al. Human papillomavirus type-16 DNA in cervical smears as a predictor of high-grade cervical cancer. Lancet 1992;339:959-60. 13. zur Hausen H, Schneider A. The role of papillomaviruses in human anogenital cancer. In: Salzman NP, Howley PM, editors. The papovaviridae. Volume II: The papillomaviruses. New York: Plenum, 1987, 245-63. 14. Pfister It, Fuchs P. Papillomaviruses: Particles, genome organisation and proteins. In: Syrjanen K, Gissman L, Koss LG, editors. Papillomaviruses and human disease. Berlin: Springer-Verlag, 1987, 6-15. 15. Sedlacek TV, Lindheim S, Eder C, et al. Mechanism for human papillomavirus transmission at birth. Am J Obstet Gynecol 1989;161:55-59. 16. Jenison SA, Xiu-ping Y, Valentine JM, et al. Evidence of prevalent genital-type human papillomavirus infections in adults and children. J Infect Dis 1990;162:60-9. 17. Munger K, Phelps WC, Bubb V, et al. The E6 and E7 genes of human papillomavirus type-16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol 1989;63:4417-21. 18. Klitz W. Viruses, cancer and the MHC. Nature 1992;356:17-8. 19. Tay SK, Jenkins D, Maddox PH, et al. Subpopulations of Langerhans cells in cervical neoplasia. Br J Obstet Gynaecol 1987 ;94:10-5. 20. Sterling J, Stanley M, Gatward G, et al. Production of human papillomavirus type-16 virions in a keratinocyte cell line. J
S . A . Khan Virol 1990;64:6305-7. 21. Meyers C, Frattini MG, Hudson JB, et al. Biosynthesis of human papillomavirus type 31b from a continuous cell line upon epithelial differentiation. Science 1992;257:971-3. 22. Cason J, Best JM. Antibody responses to human papillomavirus type-16 infections. Rev Med Virol 1991;1:201-9. 23. Cason J, Kambo PK, Best JM, et al Detection of antibodies to a linear epitope on the major coat protein (L1) of human papillomavirus type-16 in sera from patients with cervical intraepithelial neoplasia and children. Int J Cancer 1992;50:349-55. 24. DaviesDH, McIndoeGAJ, ChainBM. Current status review. Cancer of the cervix: Prospects for immunological control. Int J Exp Pathol 1991;72:239-51. 25. Einhorn N, Ling P, Stander H. Systemic interferon alpha treatment of human condylomata acuminata. Acta Obstet Gynecol Scand 1983;62:285-7. 26. Doeberitz MVK, Rittmuller C, zur Hausen H, et al. Inhibition of tumorigenicity of cervical cancer cells in nude mice by HPV E6 and E7 antisense mRNA. Int J Cancer 1992;51:831-4. 27. Jarrett WFH, Smith KT, O'Neil BW, et al. Studies on vaccination against papillomaviruses: Prophylactic and therapeutic vaccination with recombinant structural proteins. Virology 1991;184:33-4. 28. Barnes DM. AIDS vaccine trial OKed. Science 1987;237:973. 29. Geysen HM, Rodda SJ, Mason TJ. A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol Immunol 1986;23:709-16. 30. Klein J. T-lymphocyte responses In: Immunology. Oxford: Blackwell, 1990, 346-50. 31. Strang G, Hickling JK, McIndoe GAJ, et al. Human T-cell responses to human papillomavirus type-16 L1 and E6: Identification of T-cell determinants, HLA-DR restriction and virus type specificity. J Gen Virol 1990;71:423-31. 32. Jones PD, Tha-Hla R, Morein B, et al. Cellular immune responses in the murine lung to local immunization with influenza A virus glycoproteins in micelles and immunostimulatory complexes (ISCOMS). Scand J Immunol 1988;27:645-52. 33. Jenkins O, Cason J, Burke KL, et al. An antigenic chimera of poliovirus induces antibodies against human papillomavirus type-16. J Virol 1990;64:1201-6. 34. Aldovini A, Young RA. Humoral and cell-mediated immune responses to live recombinant B C G - H I V vaccines. Nature 1991 ;351:479-82. 35. Zhou J, Sun X-Y, Stenzel DJ, et al. Expression of vaccinia recombinant HPV-16 L1 and L 2 0 R F proteins in epithelial cells is sufficient for assembly of HPV virion-like particles. Virology 1991;185:251-7. 36. Brochier B, Kiery MP, Costy F, et al. Large scale eradication of rabies using a vaccinia-rabies vaccine. Nature 1991 ;354:520-2. 37. Zagury D, Leonard R, Fouchard M, et al. Immunization against AIDS in humans. Nature 1987;326:249-50. 38. Lathe R, Kieny MP, Gerlinger P, et al. Tumour prevention and rejection with recombinant vaccinia. Nature 1987; 326:878-80. 39. Smotkin D, Wettstein FO. Transcription of human papillomavirus type-16 early genes in cervical cancer and a cancerderived cell line and identification of the E7 protein. Proc Natl Acad Sci USA 1986;83:4680-4. 40. Chen L, Thomas EK, Hu SL, et al. Human papillomavirus type-16 nucleoprotein E7 is a tumor rejection antigen. Proc Natl Acad Sci USA 1991;88:110-4. 41. Takkahashi H, Takeshita T, Moein B, et al. Induction of CD8+ cytotoxic T-cells by immunization with purified HIV-1 envelope protein in ISCOMS. Nature 1990;344:873-5. 42. Tindle RW, Fernando GJ, Sterling JC, et al. A 'public' Thelper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-association human papillomavirus genotypes. Proc Natl Acad Sci USA 1991;88:55887-91. 43. Connor ME, Stern PL. Loss of MHC class I expression in cervical carcinomas. Int J Cancer 1990;46:1029-34. 44. Crook T, Tidy JA, Vousden KH. Degradation of p53 can be targeted by HPV E6 sequences distinct from those required for p53 binding and transactivation. Cell 1991; 67:547-56. 45. Frankling HT, Grossman RA, Bartelloni PJ, et al. Immunization with live type-7 and -4 adenovirus vaccines. J Infect Dis 1971;24:148-54.