Papillomaviruses (PVs) infect the epithelia of animals and man where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is concerned with development of vaccines against HPV and other targets and elucidation of the PV life cycle. We have generated virus-like particles (VLPs) for HPV 16 and other PVs that consist of the L1 major capsid protein or L1 plus L2, the minor capsid protein. Parenteral injection of purifed VLPs induced high titers of neutralizing antibodies and protection from experimental challenge in animal models. Based upon these results, we have validated GMP grade VLPs and have completed phase 1 and phase 2 clinical trials of an HPV16 L1 VLP vaccine. Vaccinees, even those vaccinated in the absence of adjuvant, consistantly produced high titers of HPV16 psuedovirion neutralizing antibodies and reported only minor side effects. Specific IgG antibodies are found at the cervix. Although the titers at the cervix remain constant in women taking oral contraceptives, cervical titers vary considerably during the menstrual cycle, as does total cervical IgG. The clinical implications of this variation remain to be determined. In animal immunogenicity studies, we have determined that antibodies elicited by L1 VLPs of one HPV16 variant effectively neutralized infection by other HPV16 variants. Therefore, it appears that that only a single HPV16 serotype needs to be considered in designing a prophylactic vaccine. We are also developing alternative vaccine candidates, some for use in disease induced by HPV and others for use in diseases unlined to HPV infection. To increase the therapeutic potential of a VLP-based vaccine, we have incorporated non-structural HPV proteins into the VLPs as L2 fusion proteins. Vaccination with an HPV16 E7 or E7-E2 chimeric VLP generated a CD8 restricted T cell response that protected mice from tumor challenge using an E7 expressing tumor line and also induced regression of established tumors. Stategies for effecitive boosting of CTL responses to chimeric VLPs were explored. A method for generating clinical grade chimeric VLPs for a phase I trial has been developed, and the vaccine lots are undergoing preclinical evaluation. To break B-cell tolerance and induce antibody responses to a self-protein, we have displayed target self-polypeptides in an ordered array on a VLP surface. In mice, display of a mouse TNF peptide on the VLPs increased TNF IgG titers 1000-fold. Vaccination of mice with the conjugated VLPs, either protected them from experimentally induced rheumatoid arthritis or reduced the severity of disease. High density of self-antigen appears to be a critical determinant for abrogating B cell tolerance. Macaques vaccinated with VLPs displaying the N-terminus of macaque CCR5 produced high titers of antibodies that bound cell surface CCR5, blocked HIV infection of cultured cells, and increased the rate of viral clearance after challenge with a CCR5-tropic SHIV. These findings with self-proteins bound to VLPs suggest a general method for inducing auto-antibodies, with many basic and applied reasearch applications. To determine why HPV VLPs have proven to be such consistently potent immunogens in clinical trials, we have examined the interaction of VLPs with purified subsets of human peripheral blood-derived antigen presenting cells in vitro. Monocytes, macrophages and myeloid dendritic cells all rapidly bound and internalized VLPs. Interaction with VLPs, but not disorganized capsid subunits, resulted in rapid phenotypic maturation of myeloid dendritic cells and release of pro-inflammatory cytokines from monocytes and macrophages. Interestingly, the patterns of cytokine release by this viral inducer of innate immunity were quite distinct from those induced by LPS, a well-characterized bacterial inducer of innate immune responses. Human plamacytoid dendritic cells also bound and internalized VLPs, leading to release of INF-alpha. However, the VLPs did not induced their phenotypic maturation, and consequently the VLP reacted plasmacytoid dendritic cells did not induce T cells with an IL-10 expressing suppressor phenotype. Taken together, the results document the ability of papillomavirus VLPs to induce a variety of innate immune response and thereby explain the ability of VLPs to induce potent T and B cell responses, even in the absence of adjuvant. We have also examined the role of the minor capsid protein L2 in virion assembly, infection, and induction of neutralizing antibodies. We found that L2 associates with pentameric L1 capsomers but not intact L1 VLPs, indicating that the two capsid proteins associate at an intermediate stage of virion assembly. L2 was found to be required for genome encapsidation in vivo. In addition, mutation analysis indicated that L2 functions during the infectious process. Antibodies to specific peptides HPV6, 16, or 18 L2 were able to cross-neutralize heterlogous HPV types. In contrast L1/L2 VLPs did not induce cross-neutralizing antibodies. These results indicate that HPV virions contain cryptic or subdominant L2 neutralizing epitopes and that an L2 polypepdide vaccine may provide broad-spectrum protection from genital HPV infection. In addition, we have determined that papillomaviruses infect cells via a clathrin-dependent pathway, and that VLPs follow the same route as infectious virions. We have also developed a simple tissue culture procedure for the routine production and purification of PV pseudoviruses with titers of more than 1 billion transducing units per ml, which represents a million-fold increase over previous methods. The pseudovirus production strategy has been adapted to develop a new high throughput PV neutralization assay. The new neutralization assay is as easy to perform and at least as sensitive as the widely used VLP-based ELISA developed several by us several years ago. However the neutralization assay has the advantages of being more type-specific and directly measuring biologically relevant antibodies. This neutralization assay should have considerable utility in HPV natural history studies and vaccine trials. Other potential applications of the pseudovirus technology are being explored.
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