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 HPV types, most frequently HPV 16. 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 recently 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. 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. 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. Mice and macaques vaccinated with VLPs displaying the N-terminus of macaque CCR5 produced high titers of antibodies that bound cell surface CCR5 and blocked HIV infection of cultured cells. These findings suggest a general method for inducing auto-antibodies, with many basic and applied reasearch applications. To begin to determine how VLP vaccination is able to induce potent B and T cell responses in the absence of adjuvant and without local inflammation, we have examined the interaction of VLPs with immature mouse bone marrow-derived dendritic cells (BMDCs). BMDCs rapidly bound and internalized VLPs. Interaction with VLPs, but not disorganized capsid subunits, resulted in rapid phenotypic maturation of BMDCs, but delayed release of pro-inflammatory cytokines, relative to a well characterized inducer, bacterial lipopolysacharide. VLP activated BMDCs induced a Th1 dominated primary T cell response in vitro. The results provide evidence that pattern recognition of virion surfaces by dendritic cells can play a central role in anti-virion immunity. 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.