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Prophylactic and Therapeutic Vaccines for Genital Papillomavirus Infection

      The development of potential therapeutic and prophylactic vaccines for human papillomavirus (HPV) infection is a very exciting area of HPV research. There are a number of features of HPV biology that makes the development of a vaccine particularly difficult, although there are several examples of vaccines that have had spectacular success in the prevention of other viral diseases. Our poor understanding of the immune response to HPV infection is the first problem. We do not understand the mechanism by which spontaneous clearing of warts is generated and therefore cannot particularly target this pathway in the development of a vaccine. Furthermore, there is no in vitro culture system nor an animal model for HPV. Another problem is that there is no ready source of live virus that might be exploited for a live attenuated viral vaccine, such as was used with poliovirus. Although most other viruses spend a portion of their life cycle in the systemic circulation where they are vulnerable to neutralizing antibodies, HPV remain exclusively in the epithelium and thus antibodies must transverse the basement membrane and reach the other layers of the skin or mucosa to be effective in preventing infection. Significant progress is being made in the development of potential vaccine candidates despite these and other confounding factors.

      Keywords

      Microbiology

      Human papillomaviruses (HPV) are small, nonenveloped icosahedral viruses with a diameter of about 55 nm containing 8–10 genes on circular double-stranded DNA. Three functional regions are contained in the HPV genome. Transcription enhancer and promoter elements are found in the long-control region. Open reading frames are found in the early region, the products of which control viral replication, transcription, and cellular transformation, and encode for E6 and E7 oncoproteins. Two capsid structural proteins (the L1 major and the L2 minor) are encoded in the late region. There is immunogenic, and thus vaccine potential, in each of these proteins. Humans can be infected by over 80 types of papillomavirus. High-risk HPV types include HPV 16, 18, 31, 35, 45, 51, 52, 56, 58 and 66 as they may cause squamous cell carcinoma and premalignant cervical lesions (
      • Lorinez A.
      • Jenson R.
      • Greenberg M.
      • Lancaster W.
      • Kurman R.
      Human papillomavirus infection of the cervix: relative risk associations of 15 common anogenital types.
      ;
      • Kiviat N.
      • Koutsky L.
      Specific human papillomavirus types as the causal agents of most cervical intraepithelial neoplasia: implications for current views and treatment.
      ;
      • Schiffman M.
      • Bauer H.
      • Hoover R.
      • et al.
      Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia.
      ;
      • Bosch F.
      • Manos M.
      • Munoz N.
      • et al.
      Prevalence of human papillomavirus in cervical cancer: a worldwide perspective.
      ). Over 90% of cervical carcinomas (
      • Gissmann L.
      • Wolnik L.
      • Ikenberg H.
      • Koldovsky U.
      • Schnurch H.
      • zur-Hausen H.
      Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers.
      ;
      • Brown D.
      • Bryan J.
      • Cramer H.
      • Fife K.
      Analysis of human papillomavirus types in exophytic condylomata acuminata by hybrid capture and Southern blot techniques.
      ;
      • Walboomers J.
      • De Roda Husman A.
      • Van Den Brule A.
      • Snijders P.
      • Meijer C.
      ) are associated with HPV types 16 and 18. At least 80% of anal carcinomas contain HPV DNA, type 16 being the most common (
      • Zaki S.
      • Judd R.
      • Coffield L.
      • Greer P.
      • Rolston F.
      • Evatt B.
      Human papillomavirus infection and anal carcinoma. Retrospective analysis by in situ hybridization and the polymerase chain reaction.
      ). Over 90% of condyloma acuminata are caused by low-risk HPV types 6 and 11.

      Immunology/Immunobiology Of Infection

      Stratified squamous epithelial cells are infected by HPV. El helicase and E2 transactivator are responsible for viral DNA replication and RNA transcription. The viral capsid proteins (L1 and L2) are produced in the upper layers of differentiating epithelium. Human papillomavirus-containing epithelial cells are shed from the surface of the skin. A relatively large area of skin, probably in the order of 2–3 mm2 (
      • Steele J.
      • Gallimore P.
      Humoral assays of human sera to disrupted and nondisrupted epitopes of human papillomavirus type 1.
      ), can be infected by the progeny of a single HPV-infected stem cell. As there is no cell lysis, there is no release of viral proteins and poor antigen presentation to the immune system. Antibody to both capsid proteins of the virus are produced, however, during the course of HPV infection (
      • Christensen N.
      • Kreider J.
      • Shah K.
      • Rando R.
      Detection of human serum antibodies that neutralize infectious human papillomavirus type 11 virions.
      ). After an average of 6 mo following infection this acquired immunity occurs and may be protective against reinfection, but is not likely to be therapeutic.
      The progression of HPV infection to clinical disease appears to be regulated by host immune responses to HPV. Both humoral and cell-mediated immune responses are elicited by HPV. A crucial role in modulating the effects of HPV, such as lesion persistence and spontaneous regression, is played by cellular immunity, particularly the T cell system. The fact that immunosuppressed individuals (transplant, lymphoma, HIV disease) have enhanced HPV proliferation and an increased frequency of HPV infection and associated disease (
      • Kast W.
      • Feltkamp M.
      • Ressing M.
      • Vierboom M.
      • Brandt R.
      • Melief C.
      Cellular immunity against human papillomavirus associated cervical cancer.
      ) illustrates the importance of cellular immunity. In addition, reduced numbers of Langerhans cells are found in CIN (
      • Sherman M.
      • Schiffman M.
      • Strickler H.
      • Hildesheim A.
      Prospects for a prophylactic HPV vaccine: rationale and future implications for cervical cancer screening.
      ), and dense infiltrates of T lymphocytes and macrophages are found in regressing warts.
      In benign lesions viral DNA remains extrachromosomal. The viral DNA is incorporated into the host chromosome in most cervical cancers. Two of the viral genes, E6 and E7, are consistently retained and expressed in cervical cancers. These same HPV types show transforming activity (
      • Schlegel R.
      • Phelps W.
      • Zhang Y-L.
      • Barbosa M.
      Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma.
      ) in cultured cells. Continued growth requires these transforming proteins that may also act as tumor rejection antigens.

      Clinical disease

      Most clinically apparent genital HPV infections are benign. Subclinical genital infections with HPV are extremely common. A spectrum of disease results from genital HPV infection ranging from asymptomatic infection to invasive cervical cancer. Based on their association with cervical cancer, HPV types have been divided into low and high risks. External genital warts are the most common clinical expression of infection with low-risk HPV (
      • Beutner K.
      • Wiley D.
      • Douglas J.
      • Tyring S.
      Genital warts and their treatment.
      ). The giant condyloma of Buschke-Lowenstein caused by HPV type 6 (and sometimes HPV 11) is a rare verrucous carcinoma of the external genital area. Weeks, months, or years may be required from the time of HPV infection to development of warts. Warts, once clinically apparent, may persist, spread, grow, spontaneously regress, and/or recur. A rare but devastating condition caused by perinatally acquired laryngeal low-risk HPV infection is termed juvenile laryngeal papillomatosis (JLP) and is usually due to infection with HPV 6 or 11.
      The clinical manifestations of high-risk HPV types include abnormal pap smears, low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), atypical squamous cells of uncertain significance (ASCUS), and atypical glandular cells of uncertain significance (AGUS) or, on biopsy, cervical intraepithelial neoplasia, carcinoma in situ, and invasive cervical cancer. Some anogenital cancers are strongly associated with infection with certain HPV types. Almost all invasive cervical cancers and cervical intraepithelial neoplasia (CIN) contain HPV DNA. Weeks to decades may pass between infection to the appearance of cervical dysplasia and cervical cancer. As the vast majority of women infected with high-risk HPV never develop cervical cancer, these HPV types are considered necessary but not sufficient for the development of cancer. Certain cofactors are necessary but are currently not well understood. Thus, it is believed that if the acquisition of HPV could be prevented, cervical cancer could be greatly reduced, if not eliminated.
      The clinical manifestations of the high-risk HPV types on the external genital area are papular lesions (Bowenoid papulosis) or erythematous scaly patches (Bowen's disease), which histologically have been classified as squamous cell carcinoma in situ or vulvar intraepithelial neoplasia, or penile intraepithelial neoplasia; or, in the vagina, as vaginal intraepithelial neoplasia. Rarely, invasive squamous cell cancer of the penis, vulva, or vagina results.
      The oncogenic potential of the high-risk HPV types is frequently found in the anus as well as the cervix, because both structures histologically have transformation zones where columnar epithelium changes to stratified squamous epithelium. Anal HPV infection has best been characterized in men, and its natural history is the subject of ongoing studies.

      Epidemiology/Expected Impact of an Effective Vaccine

      It has been estimated that 1% of sexually active persons in the U.S.A. have visible genital warts (
      • Koutsky L.A.
      • Galloway D.A.
      • Holmes K.K.
      Epidemiology of genital human papillomavirus infection.
      ). Because HPV 6 and 11 cause the majority (>90%) of genital warts, it is feasible to develop an effective bivalent vaccine for these low-risk infections. Genital warts consume healthcare resources and are emotionally troubling for the patients, although they are rarely associated with significant mortality or morbidity. Thus, immunization is an important goal. HPV infects about 50% of young sexually active women (
      • Bauer H.M.
      • Ting Y.
      • Greer C.E.
      • et al.
      Genital human papillomavirus infection in female University students as determined by a PCR-based method.
      ;
      • Koutsky L.
      • Holmes K.
      • Crirchlow C.
      A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection.
      ;
      • Ho G.
      Natural history of cervicovaginal papillomavirus infection in young women.
      ). The leading cause of cancer death in women under the age of 50 worldwide is cervical cancer (
      • Rowen D.
      • Lacey C.
      Toward a human papillomavirus vaccine.
      ). In countries where screening is unavailable or underused the impact of HPV infection is greatest. Vaccination against HPV is an attractive goal due to this strong association between sexually acquired HPV and cervical cancer. The majority of cervical carcinomas is associated with relatively few HPV types and animal models have consistently shown that both prophylactic and therapeutic vaccination is feasible. The worldwide public health implication of both preventive and therapeutic vaccines not only includes saving lives, but also decreasing the cost of screening and treating premalignant cervical disease. The annual cost of HPV-related disease has recently been estimated by the Institute of Medicine to be $10 billion (
      • Eng T.
      • Butler W.
      ).

      History/Background of Vaccine Research and other Strategies for Control

      The inability to culture HPV and host tropism of the virus has hampered vaccine development. Cottontail rabbit papillomavirus (CRPV) studies established over 60 y ago that antibodies elicited by the injection of intact virions protect against experimental challenge by the homologous viral type (
      • Shope R.
      Immunization of rabbits to infectious papillomatosis.
      ). Intact virions displaying immunodominant epitopes were found to be needed to induce protective antibodies (
      • Shope R.
      Immunization of rabbits to infectious papillomatosis.
      ;
      • Kidd J.
      The course of virus-induced rabbit papillomas as determined by virus, cells, and host.
      ;
      • Pilacinski W.
      • Glassman D.
      • Glassman K.
      • et al.
      Immunization against bovine papillomavirus infection.
      ;
      • Jin X.
      • Cowsert L.
      • Marchall D.
      • Reed D.
      • Pilacinski W.
      • Lim L.Y.
      • Jenson A.B.
      Bovine serological response to a recombinant BPV-1 major capsid protein vaccine.
      ;
      • Ghim S.
      • Christensen N.
      • Kreider J.
      • Jenson A.
      Comparison of neutralization of BPV-1 infection of C127 cells and bovine fetal skin xenografts.
      ). It was demonstrated that the L1 major capsid protein of HPV expressed in eukaryotic cells self-assembles into virus-like particles (VLP), which resemble authentic virions without the viral genome and its transforming genes. Neutralizing antibodies generated against confirmational epitopes found on the surface of native virions and VLP are sufficient to prevent infection both in vitro and in animal models.
      Immunotherapy is another strategy for HPV control. Cells involved in the host defense can be collected from patients with cervical cancer or a histocompatible donor and then grown and activated ex vivo by cytokines such as recombinant IL-2 and transferred back to the cancer patient as therapy in a process known as adoptive cellular transfer (
      • Hines J.
      • Ghim S.
      • Jenson A.
      Prospects for human papillomavirus vaccine development emerging HPV vaccines.
      ). This approach (
      • Boursnell M.
      • Rutherford E.
      • Hickling J.
      • Rollinson E.
      • Munro A.
      • Rolley N.
      Construction and characterization of a recombinant vaccinia virus expressing human papillomavirus proteins for immunotherapy of cervical cancer.
      ;
      • Krul M.
      • Tijhaar E.
      • Kleijne J.
      • Van Loon A.
      • Nievers M.
      • Schipper H.
      Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains.
      ) provided some protection against HPV 16 and 18 E6 and E7-positive tumors in mouse models.

      Vaccine strategies

      Prophylactic vaccines

      Protective anti-HPV antibodies to prevent infection are induced by HPV subunit vaccines. Antigenic targets in most prophylactic vaccine studies consist of L1 and L2. Fusion proteins, vaccinia virus recombinants, plasmids, and VLP are used in preparation of these vaccines. Molecular techniques (
      • Rose R.
      • Bonnez W.
      • Reichman R.
      • Garcea R.
      Expression of human papillomavirus type 11, L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles.
      ) are used to produce VLP that are spherical 50 nm structures resembling hollow viral capsids. These VLP lack oncogenic DNA, but possess structurally intact viral capsid proteins and may be used in enzyme-linked immunosorbent and hemagglutination assays to detect humoral responses to HPV (
      • Sherman M.
      • Schiffman M.
      • Strickler H.
      • Hildesheim A.
      Prospects for a prophylactic HPV vaccine: rationale and future implications for cervical cancer screening.
      ).

      Therapeutic vaccines

      Therapeutic vaccination may be used to eliminate residual cancer, cause regression of existing CIN or warts, or prevent progression of infection or low-grade disease to higher-risk lesions. Potential targets in the development of therapeutic vaccines include HPV E6 and E7 epitope peptides selectively maintained and expressed during malignant progression. Human leukocyte antigen (HLA)-specific, human cytotoxic T lymphocytes (CLT) induced against E6 and E7 peptides have been shown to cause lysis of HLA-specific HPV-positive cervical carcinoma cell lines (
      • Feltkamp M.
      • Smits H.
      • Vierboom M.
      Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16- transformed cells.
      ;
      • Alexander M.
      • Salgaller M.
      • Celis E.
      • Sette A.
      • Barnes W.A.
      • Rosenberg S.A.
      • Steller M.A.
      Generation of tumor-specific cytolytic T lymphocytes from peripheral blood of cervical cancer patients by in vitro stimulation with synthetic human papillomavirus type 16, E7 epitopes.
      ) in in vitro studies.

      Animal models

      Because HPV is species-specific, it does not cause disease in animals, so extrapolating data to humans must be done with caution. Encouraging results have been obtained, however, using species-specific PV. Experimental infection in vivo can be prevented with immunization with proteins produced in bacteria or immunization with vaccinia vectors that express L1 and/or L2, although low levels of neutralizing antibodies are induced (
      • Pilacinski W.
      • Glassman D.
      • Glassman K.
      • et al.
      Immunization against bovine papillomavirus infection.
      ;
      • Jarrett W.
      • Smith K.
      • O'Neil B.
      • et al.
      Studies on vaccination against papillomaviruses: prophylactic and therapeutic vaccination with recombinant structural proteins.
      ;
      • Lin Y-L.
      • Borenstein L.
      • Selvakumar R.
      • Ahmed R.
      • Wettstein F.
      Effective vaccination against papilloma development by immunization with L1 or L2 structural protein of cottontail rabbit papillomavirus.
      ). On the other hand, VLP vaccines are strongly immunogenic. Experimental infection with native virus specific to beagle dogs (COPV) (
      • Bell J.
      • Sundberg J.
      • Ghim S.
      • Newsome J.
      • Jenson A.
      • Schlegel R.
      A formalin inactivated vaccine protects against mucosal papillomavirus infection: a canine model.
      ;
      • Suzich J.
      • Ghim S.
      • Palmer-Hill F.
      Systemic immunizations with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.
      ), cottontail rabbits (CRPV) (
      • Donnelly J.
      • Martinez D.
      • Jansen K.
      • Ellin R.
      • Montgomery D.
      • Liu M.
      Protection against papillomavirus with a polynucleotide vaccine.
      ;
      • Lin Y.
      • Borenstein L.
      • Ahmed R.
      • Wettstein F.
      Cottontail rabbit papillomavirus L1 protein-based vaccines: protection is achieved only with a full-length, nondenatured product.
      ;
      • Breitburd F.
      • Kirnbauer R.
      • Hubbert N.
      • et al.
      Immunization with virus-like particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection.
      ;
      • Christensen N.
      • Reed C.
      • Cladel N.
      • Han R.
      • Kreider J.
      Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus.
      ), and calves (BPV) (
      • Kirnbauer R.
      • Chandachud L.
      • O'Neil B.
      • et al.
      Virus-like particles of bovine papillomavirus type 4 in prophylactic and therapeutic immunization.
      ) can be prevented by prior immunization with homologous VLP. Both species and type specific protection is produced by neutralizing antisera. Confirmational epitopes present on the surface of VLP also help determine protection.
      Systemic vaccination in animal models has produced protection of mucosal surfaces against the natural transmission of PV. HPV-11 L1 VLP-specific IgG present in the cervicovaginal secretions of monkeys parenterally immunized with HPV-11 L1 VLP is sufficient to neutralize HPV-11 in the athymic mouse xenograft system (
      • Lowe R.
      • Brown D.
      • Bryan J.
      • 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.
      ).
      Therapeutic vaccines may target early antigens, including E1, E6, and E7 proteins. Regression of tumors derived from HPV 16-transformed oncogenic cell lines (
      • Meneguzzi G.
      • Cerni C.
      • Kieny M.
      Immunization against human papillomavirus type 16 tumor cells with recombinant vaccinia viruses expressing E6 and E7.
      ) have been observed in rats vaccinated with vaccinia virus expressing HPV 16 E7. Prevention and regression of CRPV tumors were both achieved using an E1 vaccine derived from recombinant Listeria monocytogenes (
      • Jensen E.
      • Selvakumar R.
      • Shen H.
      Recombinant Listeria monocytogenes vaccination eliminates papillomavirus induced tumors and prevents papilloma formation from viral DNA.
      ). A mechanism to eliminate cells undergoing productive viral infection is provided by T cell responses induced by HPV-16 L1 VLP (
      • Dupuy C.
      • Buzoni-Gatel D.
      • Touze A.
      • Cann P.
      • Bout D.
      • Coursaget P.
      Cell mediated immunity induced in mice by HPV 16, L1 virus-like particles.
      ;
      • Luxton J.
      • Rose R.
      • Coletart T.
      • Wilson P.
      • Shepherd P.
      Serological and T -helper cell responses to human papillomavirus type 16, L1 in women with cervical dysplasia or cervical carcinoma and in healthy controls.
      ).
      Studies with animal models have not evaluated authentic routes of PV infection or the longevity of immune responses, or established that tumors can be eliminated with vaccination, despite these encouraging results.

      Clinical studies

      Immunology and efficacy studies

      Currently numerous clinical studies are underway throughout the world and a few have obtained data as described below.
      In a phase I study with 65 healthy volunteers a vaccine specific for HPV type 11 was found to be both safe and immunogenic (
      • Reichman R.
      • Bonnez W.
      • O'Brien D.
      • et al.
      A phase I study of a recombinant virus-like particle vaccine against human papillomavirus type 11 in healthy adult volunteers.
      ). The major capsid protein, L1, was placed in a recombinant virus, expressed in insect cells, and VLP were produced. Volunteers seronegative for HPV-11 were given vaccine composed of 3, 9, 30, or 100 µg of VLP or placebo at 0, 4, and 16 wk. Neutralizing antibody titers against HPV 11 of 1:1000 or greater were achieved in seven of 10 subjects who received the 3 µg dose, nine of 10 who received the 9 µg dose, all 12 who received the 30 µg dose, and all 10 who received the 100 µg dose. The vaccine was well tolerated at all doses.
      No significant toxicities, an antivaccine antibody response in all eight participants, and HPV-specific antibody response in three of eight participants resulted from a recombinant vaccine virus expressing E6 and E7 epitope peptides of HPV 16 and 18 in a phase I/II study in advanced cervical carcinoma (
      • Borysiewiez L.
      • Fiander A.
      • Nimako M.
      • Winkinson G.
      • Westmoreland D.
      • Evans A.
      A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer.
      ).
      Good humoral responses were produced in open-label studies with an L2 E7 fusion protein vaccine, incorporating an alum adjuvant combined with conventional therapy for the treatment of anogenital warts in men, although lymphoproliferative responses were variable (
      • Lacey C.
      • Monteiro E.
      • Thompson H.
      A phase IIa therapeutic vaccine for genital warts.
      ;
      • Rowen D.
      • Thompson H.
      • O'Neill B.
      A phase IIa open study to evaluate the safety, immunogenicity and clinical response to TA-GW given in combination with cryotherapy to men with genital warts.
      ).

      Vaccine development

      A number of vaccines are in various stages of development (Table I). There are a number of observations that would seem to indicate that effective HPV vaccination is feasible although modern pivotal efficacy trials with HPV have not been completed. These observations are as follows:
      • 1
        more persistent and more frequent clinical expression of HPV infection (which is usually more difficult to treat) results from immunosuppression;
      • 2
        vaccine studies with animal papillomavirus systems have produced favorable results;
      • 3
        many infected humans never express their clinical lesions;
      • 4
        external genital warts, nongenital warts, and HPV-related cervical lesions often regress spontaneously;
      • 5
        infiltrates of regressing warts often contain CD4 lymphocytes and macrophages;
      • 6
        autologous vaccines have been reported to be successful in humans.
      Table ICandidate HPV vaccines
      Peptide vaccines
       HLA-A0201 HPV-16 lipopeptides
      • Steller M.A.
      • Schiller J.T.
      Human papillomavirus immunology and vaccine prospects.
       HPV 16 E7 PADRE
      • Feltkamp M.
      • Smits H.
      • Vierboom M.
      Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16- transformed cells.
       HPV 16 E7 “ISCAR” conjugate
      • Tindle R.W.
      • Croft S.
      • Herd K.
      • Malcolm K.
      • Geczy A.F.
      • Stewart T.
      • Fernando G.J.
      A vaccine conjugate of ISCAR immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants.
      Protein-based vaccines
       HPV 16 E6-E7
      • Mallarios J.
      • Quinn C.
      • Arnold F.
      • Macfarlan R.
      Cell-mediated immune responses to recombinant HPV-16, E6E7hh protein generated by immunization with creating lscomatrixTM adjuvant.
       HPV 16 E7 + BCG H5/65
      • Zhu X.
      • Tommasino M.
      • Vousden K.
      • et al.
      Both immunization with protein and recombinant vaccinia virus can stimulate CTL specific for the E7 protein of human papillomavirus 16 in H-2 (D) mice.
       HPV 16 E7
      • Hariharan K.
      • Braslawsky G.
      • Barnett R.S.
      • Berquist L.G.
      • Huynh T.
      • Hanna N.
      • Black A.
      Tumor regression in mice following vaccination with human papillomavirus E7 recombinant protein in ProvaxTM.
       HPV 16 E2
      • Heineman L.
      • Amer M.
      • Van Nest G.
      • Hibma M.
      An immune response to human papillomavirus type 16 early protein E2 following immunization with adjuvant MF 59 in a mouse model.
      ;
      • Hibma M.H.
      • Amer M.
      • Heinemann L.
      • Van Nest G.
      Immune responses to the human papillomavirus type 16 early protein E2 following several methods of immunization.
       HPV 16 E4 with HbeAg
      • El Mehdaoui S.
      • Touze A.
      • Coursaget P.
      Production of chimeric human papillomavirus 16, E4 – hepatitis B core recombinant particles.
      Virus-like particles
       HPV 11 L1
      • Reichman R.
      • Balsley J.
      • Carlin D.
      • et al.
      Evaluation of the safety and immunogenicity of a recombinant HPV-11, L1 virus like particle vaccine in healthy adult volunteers.
       HPV 6 L1
      • Zhang L.F.
      • Zhou J.
      • Shao C.
      • et al.
      A phase II trial of HPV 6 B virus like particles as immunotherapy for genital warts.
       HPV 16 L1
      • Da Silva D.M.
      • Nieland J.D.
      • Greenstone H.L.
      • Schiller J.T.
      • Kast W.M.
      Chimeric papillomavirus virus-like particles induce antigen-specific therapeutic immunity against tumours expressing the HPV-16 E7 protein.
      Viral vector vaccines
       Vaccinia HPV 16 L1
      • Cooney E.L.A.C.
      • Collier P.D.
      • Greenberg R.W.
      • et al.
      Safety of and immunological response to a recombinant vaccinia virus vaccine expressing HIV envelope glycoprotein.
      ;
      • Gao L.
      • Chain B.
      • Sinclair C.
      • et al.
      Immune response to human papillomavirus type-16, E6 gene in a live vaccina.
       Vaccinia HPV 16-E7
      • Lin K.Y.
      • Guamieri F.G.
      • Staveley-O'Carroll K.F.
      • Levitsky H.I.
      • August J.T.
      • Pardoll D.M.
      • Wu T.C.
      Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen.
       Vaccinia HPV 16 + 18 E6 + E7
      • Borysiewiez L.
      • Fiander A.
      • Nimako M.
      • Winkinson G.
      • Westmoreland D.
      • Evans A.
      A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer.
      Bacterial vectors
      Salmonella typhimurium HPV 16 E6 and E7
      • Krul M.
      • Tijhaar E.
      • Kleijne J.
      • Van Loon A.
      • Nievers M.
      • Schipper H.
      Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains.
      ;
      • London L.P.
      • Chatfield S.
      • Tindle R.W.
      • Herd K.
      • Gao X.M.
      • Frazer I.
      • Dougan G.
      Immunization of mice using Salmonella typhimurium expressing human papillomavirus type-16 E7 epitopes inserted into hepatitis-B virus core antigen.
      Salmonella typhimurium HPV16 L1
      • Nardelli-Haflinger D.
      • Roden R.B.S.
      • Benyacoub J.
      • et al.
      Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium, elicit mucosal and systemic neutralizing antibodies in mice.
      Streptococcus gordovii HPV16 E7
      • Jensen E.
      • Selvakumar R.
      • Shen H.
      Recombinant Listeria monocytogenes vaccination eliminates papillomavirus induced tumors and prevents papilloma formation from viral DNA.
      The possibility that a genital HPV vaccine might not be effective, on the other hand, is suggested by the following factors that make HPV vaccines particularly challenging:
      • There is no clear definition of protective immunity, nor is the necessary response to particular antigens well understood. Antibodies appear to protect against infection in animal systems, and cell-mediated immune responses are required for wart regression or protection against the development of warts.
      • With HPV the route and frequency with which the immunized subject is exposed naturally may be more chronic and repetitive than with other viral infections for which we have effective vaccines. The level of immunity required to protect against frequent genital exposure is unknown. Viral infections for which we currently have vaccines are not characterized by frequent (potentially daily) exposure for years.
      Although both prophylactic and therapeutic vaccines are feasible (Table II), prophylactic vaccines are the most appealing, based on experience with animal PV and in humans, with a number of viral vaccines in clinical trials.
      Table IIPotential clinical trial end-points
      High-risk genital HPV
       Prophylactic
        Acquisition of infection
        Shortening duration of infection
        Decrease in frequency of cytologic abnormalities
        Decrease in duration of cytologic abnormalities
       Therapeutic
        Resolution of cytologic abnormalities
        Regression of dysplasia (monotherapy or combination therapies)
        Regression of cancer (monotherapy or combination therapies)
      Low-risk genital HPV
       Prophylactic
        Acquisition of infection
        Development of warts
       Therapeutic
        Monotherapy or combination therapies
        Prevention of recurrence after treatment
      A vaccine to control genital HPV probably would need to involve immunization of both men and women. Our knowledge of detection of high-risk HPV in women, the attack rate of HPV in women, and the natural history of HPV in women, however, is much greater than our information about infectivity, attack rate, and natural history of HPV in men.
      Development of vaccination strategy and rational design of clinical trials is difficult without this information. The lack of a readily available standardized and validated serologic test to determine who is “immune” and who is susceptible is another obstacle to HPV vaccine development and implementation. VLP ELISA and pseudovirion infectivity assays serve as surrogate markers. In addition, the detection of viral DNA and cervical lesions will characterize patients at entry and serve as markers to determine vaccine efficacy.
      There are major psychosocial challenges in addition to the scientific challenges. We do not know if the public is ready to vaccinate their children for sexually transmitted diseases. In addition, small but vocal groups question the value of current childhood vaccinations. There is very little awareness of HPV among the general public, and the risk of acquiring HPV and the reality that cervical cancer is a sexually transmitted disease are unfamiliar concepts outside the medical community.

      Potential vaccines

      Peptide vaccines

      Peptide vaccines have obvious therapeutic appeal, particularly if capable of binding to HLA and generating cytotoxic T lymphocytes. Peptide vaccines are in early trials as candidate therapeutic vaccines for cervical cancer.

      Proteins

      Production of fusion proteins of all HPV gene products is now possible, but which of these or which combinations should be studied in clinical trials is not known. An HPV6 L2-E7 fusion protein has been shown to be immunogenic, and perhaps therapeutic, in the treatment of genital warts in humans.

      Virus-like particles

      Chimeric VLP can be formed by combining L1 or L1 plus L2 with early proteins. Clinical trials are being conducted with an HPV 11 VLP (
      • Reichman R.
      • Bonnez W.
      • O'Brien D.
      • et al.
      A phase I study of a recombinant virus-like particle vaccine against human papillomavirus type 11 in healthy adult volunteers.
      ,
      • Reichman R.
      • Balsley J.
      • Carlin D.
      • et al.
      Evaluation of the safety and immunogenicity of a recombinant HPV-11, L1 virus like particle vaccine in healthy adult volunteers.
      ), an HPV6 L1 VLP (
      • Zhang L.F.
      • Zhou J.
      • Shao C.
      • et al.
      A phase II trial of HPV 6 B virus like particles as immunotherapy for genital warts.
      ), and an HPV 16 L1 VLP (
      • Da Silva D.M.
      • Nieland J.D.
      • Greenstone H.L.
      • Schiller J.T.
      • Kast W.M.
      Chimeric papillomavirus virus-like particles induce antigen-specific therapeutic immunity against tumours expressing the HPV-16 E7 protein.
      ).

      Viral and bacterial vectors

      Viral and bacterial vector vaccines are potentially equivalent to live attenuated HPV vaccines. Humoral and CTL responses could potentially be generated with such vaccines, which could be polyvalent. Early clinical trials in human cervical cancer patients are evaluating viral vector vaccines such as a vaccinia vaccine expressing modified forms of E6 and E7 from HPV 16 and 18 (
      • Borysiewiez L.
      • Fiander A.
      • Nimako M.
      • Winkinson G.
      • Westmoreland D.
      • Evans A.
      A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer.
      ), which appear to be immunogenic.

      Other approaches

      It is theoretically possible to elicit virus-specific immune responses utilizing direct introduction into the host of viral DNA coding for viral antigens. This approach has only been investigated in the rabbit system thus far.

      Low-risk types

      The goal of vaccination with low-risk types is primary prevention of HPV acquisition. If this goal cannot be achieved, it is hoped that vaccination can at least prevent the development of external genital warts. There is a lack of precise knowledge about infectivity, susceptibility, attack rate, and time from infection to the development of external genital warts. Although the presence or absence of such lesions would represent a clear clinical end-point, designing clinical trials in terms of sample size and duration of follow-up is challenging without this information.
      Therapeutic end-points could be achieved with a low-risk HPV vaccine, either by accelerating the resolution of genital warts, enhancing the efficacy of current therapies, or preventing wart recurrence after current therapy has produced a wart-free state. The precise frequency and factors that favor or prevent “spontaneous” resolution of genital warts are poorly understood. Complete resolution of genital warts over a few months appears to be in the range of 0%-20% based on the placebo arm of controlled clinical trials (
      • Beutner K.
      • Richwald G.
      • Wiley D.
      • Reitano M.
      • the AMA Expert Panel on External Genital Warts
      External genital warts: report of the American Medical Association Consensus Conference.
      ).
      Most current therapies have about a 50% complete response rate. Increasing complete responses from 50% to 80% by supplementing current treatment with a vaccine would require a trial using hundreds of patients.
      If one hopes to decrease recurrences after conventional therapy, similar challenges exist. There have been no studies prospectively powered and designed to establish the frequency of or time to recurrence following treatment of genital warts with any modality, although published recurrence rates with most modalities appear to be high. In order to design vaccine trials adequately, this information will be required.

      High-risk HPV types

      Prevention of cervical cancer is the obvious aim of vaccination against high-risk HPV types, but it is not a practical end-point for human vaccine trials. Because the biopsy may alter the natural history, histologic end-points are not feasible. Several potential surrogate end-points are of potential use: the acquisition of infection, the virologic persistence, and a decrease in the proportion of subjects who experience cytologic abnormalities.
      In theory the best way to prevent cervical cancer is to prevent the acquisition of high-risk HPV infection, but this may also be the greatest challenge. The lack of a serologic test to accurately identify those who are either susceptible or immune is one of the many problems with this end-point. While virgins would be susceptible, limiting enrolment to virgins has some logistic limitations.
      The persistence of HPV, not just the acquisition of a short-lived infection, is a critical step in the pathway to cancer, which may help explain why only a minority of those infected develop cervical cancer. Cervical cancer may thereby potentially be prevented using a vaccine to decrease the proportion of the population with persistent HPV. Inherent limitations in terms of interpretation and reproducibility exists with the use of a screening cytologic test as a clinical end-point.

      Conclusion

      We have come a long way on the path to a vaccine considering that HPV cannot be grown in vitro and that until very recently there have been no serologic tests for HPV. Although large-scale clinical trials are now underway, it is difficult to predict how soon we will have effective prophylactic and/or therapeutic HPV vaccines.

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