Immunogenicity and cellular protein associations of the papillomavirus minor capsid protein

by Embers, Monica E.

Human papillomavirus (HPV) infection is associated with a variety of diseases involving cutaneous or mucosal epithelia. Genital infection with certain high-risk papillomavirus types is a primary cause of cervical cancer. The papillomaviruses are comprised of an 8 kb double-stranded circular genome encapsidated by two structural proteins. The major capsid protein, L1 is the predominant element in the physical assemblage of the viral capsid, while a minor structural element, L2, is far less abundant, but necessary for infectivity. Antibody responses to the major capsid protein have been well characterized. Humoral immunity to the L1 protein immunogen, assembled into virus-like particles (VLP), is vigorous and generally type-specific. Although VLP-based vaccines elicit protective virus-neutralizing antibodies, they are encumbered by the type-specific immunity generated, when tremendous inter- and intra-type diversity is exhibited by the over 100 different HPVs identified to date. Thus, the L2 protein has been assessed for its ability to generate neutralizing antibody responses that may display cross-reactivity. Aside from a probable role in genome encapsidation, L2 may also function in early events of host cell infection and in the disruption of nuclear architecture prior to capsid assembly. An examination of the cellular proteins with which L2 associates could provide clues as to the specific function of this protein in viral infection and virion morphogenesis. iv The primary objectives of this thesis project were: 1) to derive monoclonal antibodies specific for L2 proteins from several different virus types; 2) To assess antibody responses against a specified region of L2 with regard to antigen reactivity, specificity, the ability to neutralize virus, and protect animals against experimental papillomavirus infection; and 3) in a limited fashion, to examine L2-associated proteins. L2 proteins from rabbit and human papillomaviruses were cloned, expressed, purified and used as immunogens in a variety of vaccine regimens. L2 was found to be a poor immunogen, for which the purity and quantity of L2 antigen affected the yield of antigen-specific hybridomas significantly. Three hybridomas specific for rabbit oral papillomavirus (ROPV) L2 and one targeting HPV-16 L2 were generated. Additionally, two hybridomas that produced antibodies specific for L2-associated proteins were isolated. These reagents may provide useful tools for studies on the structural and functional properties of L2. The region consisting of amino acids 108-120 from HPV-16 L2 has been reported to elicit cross-neutralizing antibodies, by experimental “pseudovirion” neutralization. An assessment of the immunogenic properties of this peptide and its HPV type 6 and 11 homologue, extended HPV L2 peptides incorporating additional conserved residues, and peptides from the corresponding regions of ROPV and cottontail rabbit papillomavirus (CRPV), was undertaken. Antibody responses to peptide immunogens exhibited heterologous peptide cross-reactivity, but this did not translate into protein reactivity in all cases. HPV-16 L2 peptide antiserum exhibited moderate levels of crossneutralization, as determined by an in vitro assay of authentic HPV-11 neutralization utilizing quantitative RT-PCR, where the extended peptides elicited generally better v responses. The immunization of rabbits with peptides encompassing the relatively divergent complementary region of L2 from ROPV and CRPV resulted in virtually complete protection from high-dose viral infection with the cognate virus. This resulted predominantly, if not completely from the induction of L2-specific virus-neutralizing antibodies. Thus, although further studies are warranted, this region of L2 possesses the ability to elicit neutralizing antibodies that could potentially protect from an array of human viral types. Heat shock proteins (Hsps) assist in the proper folding and localization of a vast range of polypeptides. Their roles in viral processes, including virion assembly and nuclear targeting of viral proteins have been widely reported. A notable “contaminant” of L2 preparations was theorized to be Hsp70. Indeed, this protein was shown to bind L2 specifically and was detected in capsid protein preparations, including virus-like particles and virion extracts. Additionally, Hsp70 was detected in VLP prepared from cell lysates, but not in those derived from long-term culture supernatants, suggesting provisional association, perhaps in the process of capsid assembly. vi