Abstract

Abstract A subset of human papillomaviruses (HPVs) has been implicated as the principal etiologic agents of cervical cancer. Cervical cancers consistently retain and express two of the viral genes, E6 and E7. Although infection with HPV seems to be necessary, other factors, such as cellular immune function, play an important role in determining whether cervical infection will regress, persist, or progress to cancer. The close relationship between viral infection and cancer makes HPV an attractive target for prophylactic and therapeutic vaccines. Candidate vaccines have been shown to have efficacy in animal models, and human clinical trials are planned or in progress. In addition to inducing benign papillomas of the skin and mucous membranes, some human papillomaviruses (HPVs) are clearly associated with the development of malignant epithelial tumors (1-3).1 These cancers include anogenital cancers, especially cancer of the cervix, which is the second most common cancer among women worldwide. A wealth of epidemiologic and molecular biologic data now points to an etiologic link between HPV infection and most cervical cancers. Recognition of the clinical importance of papillomaviruses has stimulated efforts to develop vaccines that may treat or prevent benign and malignant diseases associated with papillomavirus infection. This article considers the relationship between papillomavirus infection and cervical cancer and describes recent approaches to papillomavirus vaccines. Subset of Genital/Mucosal HPV Types Found in Cervical Cancer The HPV replicative cycle is limited to stratified squamous epithelia, with no viremic phase (4). Most of the virus gene expression and replication take place in suprabasal cells that are undergoing terminal differentiation, a strategy that seems designed to evade immune surveillance. More than 70 different HPV types have been identified on the basis of the sequence divergence between their DNA genomes (2). Their genomes, which are a closed, circular, double-stranded DNA approximately 8 kilobases in length, share a similar structural and genetic organization (5). Only a subset of the HPV types appears to regularly infect the genital epithelia. Some of these so-called genital/mucosal types, such as HPV6 and HPV11, are almost never found in cervical cancer and have thus been designated “ low-risk” viruses. Others, such as HPV16 and HPV18, are found regularly in cervical cancer and have therefore been designated “high-risk” HPV types (2,3,6). Many different HPV types have been found in cervical cancer. However, a recent analysis of almost one thousand cervical cancers from different regions of the world (6) showed that HPV16 was consistently the most common type, being present in about one half of the cancers from any region. HPV16, HPV18, HPV31, or HPV45 was detected in about 80% of the cancers in every region. HPV infection of the cervix precedes the onset of cancer by many years. Reliable epidemiologic evidence has shown that HPV infection is by far the most important risk factor (10-fold to 200-fold, compared with controls) for the development of cervical dysplasias, from which almost all cervical cancers arise (3,7-9). The peak in cervical cancer incidence is more than 20 years after the peak in incidence of high-risk genital HPV infection, suggesting that infection per se is insufficient to cause cancer. In most women, cervical infection even with a high-risk HPV is self-limited. Low-risk viruses do not seem to possess the intrinsic capacity to induce cervical cancer. Inactivation by High-Risk HPVs of Proteins Controlling Cell Proliferation Considerable progress has been made in identifying potentially important differences between high-risk and low-risk genital/mucosal HPVs (10). Normal human keratinocytes in culture have a finite life span, which is not increased when low-risk HPVs are introduced into them. However, high-risk types, in contrast to low-risk types, can reproducibly induce the immortalization of the keratinocytes. This ability to be passaged indefinitely is a characteristic of partial cellular transformation. Further analysis of high-risk HPV indicated that keratinocyte immortalization requires two of the viral genes, E6 and E7, which are the same two viral genes that are preferentially retained and expressed in cervical tumors (11,12). Biochemical analysis of the proteins encoded by E6 and E7 has shown that they form complexes with and inactivate specific cellular proteins that probably contribute to the limited life span of cultured keratinocytes and inhibit cell growth (10,13,14). These activities are much lower or are lacking in the E6 and E7 of low-risk viruses. The biological significance of such complexes with high-risk E6 and E7 has been shown most clearly for E6 and the p53 protein and for E7 and the pRB protein, which is encoded by the retinoblastoma tumor susceptibility gene RB. Since p53 and pRB normally control cell proliferation, abrogating these functions places the cells at much greater risk of malignant progression. As in the clinical situation, cultured keratinocytes that have been recently immortalized by high-risk HPV are often resistant to differentiation signals but are not fully transformed. Continued passage or the addition of an activated ras oncogene can, in some instances, render these cells tumorigenic for nude mice (15). Additional Changes Required for Tumorigenic Progression The long interval between HPV infection and the development of cervical cancer suggests that factors other than viral infection are required for progression to high-grade dysplasia and tumor development. Both virus-specific factors and immune reactivity appear to be important. While the viral DNA in benign lesions remains extrachromosomal, it is integrated into the host genome in most cervical cancers. During progression, viral expression is usually limited to E6 and E7. Other changes associated with progression likely involve E6- and E7-induced chromosome instability that results from deregulation of cellular growth-control genes and telomerase activity (16). Persistence of viral infection is associated with progression (17). The likelihood of progression to invasive cancer appears to depend in part on the relative oncogenic potential of the HPV type (18). Some evidence also suggests that genotypic variants within a high-risk type may affect the potential for progression to high-grade dysplasia (19). It remains to be determined whether these apparent differences in likelihood of progression mainly represent biological differences between the viruses or differences in host response. Cellular immunity to the viral infection seems to represent a critical determinant of whether dysplastic lesions will develop, regress, persist, or progress. HPV16-infected patients with more severe cervical cytology are less likely to show positive cellular immune responses to E6 and E7 antigens than are patients with less severe cytology or with a history of previous HPV16 infection (20,21). There is some evidence that loss of major histocompatibility complex (MHC) class I expression may allow some lesions to evade immune surveillance and progress more rapidly (22). Progression is more common in long-term renal transplant patients on immunosuppressive therapy or in women who are human immunodeficiency virus (HIV) positive, indicating the important role of cellular immune function in host defense (3). Vaccination Against HPV Infection The recognition that HPV infection plays the central etiologic role in cervical cancer has fostered efforts to develop vaccines against HPV. Both prophylactic and therapeutic forms of vaccines are under development (23-28). They seek, respectively, to prevent infection or to induce regression of established infection via immune recognition of specific HPV-encoded proteins or peptides. Such vaccines can be delivered either directly as protein, as DNA that encodes and expresses the requisite viral protein(s), or by heterologous viral vectors (29). General Considerations A major barrier to developing a practical vaccine is that most critical HPV-immune determinants are likely to be type specific because the proteins of different HPV types are quite divergent at the amino acid level. This limitation implies that protection induced by protein from a given HPV type is likely to be type specific. It will therefore be necessary either to have a specific vaccine for each HPV type or to incorporate viral protein from an appropriate spectrum of HPV types in a polyvalent vaccine. Further difficulties are that HPV does not cause disease in animals and is not infectious for them. This means that animal studies must be carried out with animal papillomaviruses, with grafted human material in immunologically suppressed hosts, or with model systems that incorporate specific HPV genes or proteins. It is not always clear whether experimental results obtained with these animal models will apply directly to clinical HPV infection in humans. Prophylactic Vaccines Encouraging results have come from animal studies of vaccines to prevent papillomavirus infection. Consistent protection (90%-100%) has been obtained by immunizing animals with virus-like particles (VLPs) composed of the major structural viral protein L1 (30-32). The papillomavirus capsid is primarily composed of 360 molecules of L1 protein, and L1, when expressed in the absence of other papillomavirus genes, can self-assemble into VLPs that are morphologically and immunologically similar to infectious papillomavirus (33,34). Since the VLPs are produced by genetically engineered cells that do not contain the nonstructural viral genes, such as E6 or E7, the VLPs do not contain the papillomavirus DNA genome, are not infectious, and cannot cause neoplastic changes in cells. Immunization with VLPs (30) or with the L1 gene (35) has produced substantial protection in rabbits against experimental challenge with the Shope cottontail rabbit papillomavirus (CRPV), which induces cutaneous papillomas that can progress to malignant squamous cell carcinomas. VLP immunization can also prevent experimental oral mucosal infection in dogs by canine oral papillomavirus (31) and in cows by bovine papillomavirus type 4 (BPV4) (32). In these studies, the protection, which can be passively transferred by antibodies from immune animals to nonimmune animals, is mediated by antibodies directed against conformational epitopes that are present on the VLP as well as on infectious papillomaviruses. Since the conformational epitopes are type specific (36-38), protection is type specific (30). The efficacy of these protocols seems to be limited to prevention; BPV4 VLP immunization did not induce regression of established BPV4 papillomas (32). These promising animal studies are leading to the testing of a candidate prophylactic HPV vaccine in humans. In addition to deciding which HPV types to include in such a vaccine, it will be desirable to optimize the adjuvant, dosage, and route of administration. Efforts to improve the mucosal immunity induced by VLPs may increase their efficacy against mucosal genital infection. However, there are reasons to believe that preventing genital mucosal HPV infection may not require the induction of mucosal immunoglobulin (Ig) A. In addition to the animal studies that show protection against experimental oral infection, transudation of IgG into vaginal secretions can be induced by systemic immunization with purified protein (39). Furthermore, it is believed that initiation of genital HPV infection occurs only when there is sufficient trauma to allow the virions to come in contact with the proliferating epithelial cells, which are located in the basal layer of the epithelium. This hypothesis is supported by the tentative identification of α 6 integrin, which is not expressed in suprabasal cells, as a candidate receptor for papillomaviruses (40). It is reasonable to expect that trauma which was sufficient to abrade the epithelium would usually be associated with exudation of systemic IgG into the abraded area, where it might neutralize the HPV virions. Therapeutic Vaccines Therapeutic vaccines might be used in various settings, including the treatment of invasive cancers, as adjunct therapy to prevent recurrence or metastasis, against dysplasias, or in benign disease. A major theoretical obstacle to developing such vaccines is that the immunologic determinants for viral persistence or regression remain poorly defined (41,42), although it is clear that patients with impaired cellular immunity are at increased risk of persistent HPV infection and carcinogenic progression. Most efforts have been directed toward using the E6 and E7 proteins, or peptides derived from them, largely because these are the viral proteins that are retained and expressed in cervical tumors (43). However, vaccines directed against benign lesions would not need to be limited to these two proteins. For example, E1 and E2, which are required for the viral DNA to be maintained as an extrachromosomal element that is unintegrated in the host DNA, represent potentially interesting targets for benign lesions. Since these papillomavirus proteins are not expressed on the cell surface, there is little potential for antibody-dependent cytotoxicity to mediate regression. Instead, potentially effective cytotoxic responses will probably require a vaccine that induces the presentation of small virally encoded peptides to antigen-presenting cells. In cells that possess class I molecules, the normal process of partial intracellular degradation of cytoplasmic or nuclear viral proteins can, following the binding of small viral peptides to the class I molecules, lead to the induction of antigen-specific reactivity of CD8-positive cytotoxic T lymphocytes (CTLs). Introduction of the relevant viral gene or protein into target cells is normally required to develop virus-specific CTLs. Papillomavirus genes can be introduced into cells as naked DNA, which is a relatively inefficient process, or as part of a viral vector such as vaccinia virus, which is usually more efficient. Rodents immunized with HPV16 E6 or HPV16 E7, delivered either in vaccinia virus vectors containing one of the two viral genes or in killed tumor cells that expressed one of the genes and the gene for immune co-stimulatory protein B7, were protected against subsequent challenge with tumor cells expressing the corresponding viral protein (44-46). A recombinant vaccinia human safety trial of an HPV16 and HPV18 E6 and E7 virus has been completed (47), and others are under way or planned (28). A CTL response can also be induced by the viral protein itself if it is taken up by cells in a manner that leads to its partial degradation and presentation with class I antigen. Although soluble protein by itself does not usually generate such a response, injection of rabbits with bacterially derived CRPV E1 or E2 protein was shown to increase the rejection rate of CRPV-induced papillomas (48). The precise immunologic mechanism underlying this effect remains to be established. However, class I-dependent CD8 CTLs can be reproducibly induced if viral protein is presented in particulate form (49), as part of a VLP, in combination with some adjuvants, or encapsidated into liposomes. Another alternative is to immunize with small viral peptides, which, following their binding to empty class I molecules on cell surfaces, can induce cytotoxic CD8 T cells (50). A difficulty associated with this approach is that the immunogenicity of a given peptide is genetically determined by the class I alleles present in a given individual, which means that only some individuals will respond to even an “ immunogenic” peptide. Also, small peptides are often quite unstable in vivo(51). In some tumor models, immunologic rejection is mediated by antigen-specific CD4 T cells rather than by CD8 T cells (52,53). While the class I CD8 pathway is characteristic of cytoplasmic and nuclear proteins, CD4 T cells can be activated by the processing of endogenously expressed membrane-associated proteins for processing and presentation by MHC class II molecules to CD4 cells. To induce E7-specific CD4 cells, a study (54) used genetic engineering to target the ordinarily nuclear HPV16 E7 protein to the lysosomal compartment by adding a lysosomal membrane targeting signal to E7. Such a lysosomally targeted E7 protein, which was introduced into mice via a vaccinia virus vector carrying the engineered E7 gene, was highly effective in protecting mice against tumors derived from a mouse epithelial cell line transformed by HPV16 E6 and E7 genes and a ras oncogene (55). In this tumor model, protection by the lysosomal E7 protein was found to depend on both CD4 and CD8 cells. 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