Our overall goal is to understand how L2-specific antibodies effect broad protection against HPV infection. Here, we dissect why L2 and intra-membranous cleavage by gamma-secretase are required for infection, and how antibodies to L2 potently protect by neutralization at multiple steps during infection. In the initial funding perio we made the surprising observation that gamma-secretase is absolutely required for infection by HPV (Karanam et al., 2010), but only once HPV has begun to disassemble in the late endosome and gamma-secretase's proteolytic activity is required for escape from late endosomes of L2 in association with the viral genome. Recent findings suggest that L2 has a transmembrane-like domain between residues 45-67 that is essential for infection. We propose that the putative transmembrane region pierces the endosomal membrane, and gamma-secretase cleavage releases L2's carboxy terminus to bring the viral genome to the nucleus. Two key neutralizing epitopes in L2 reside either side of L2's putative transmembrane domain. Addition of L2 antibodies even several hours after the binding of virus to cells is neutralizing, suggesting blockade occurs late in infection. Therefore, we propose: Hypothesis 1: cleavage of L2 within its putative transmembrane region by gamma-secretase is critical for endosomal escape and is inhibited by L2-specific neutralizing antibodies.
Specific Aim 1 A: To determine the residues of L2 required for membrane insertion and whether they contribute to infectivity by facilitating endosomal escape.
Specific Aim 1 B: To determine the functional role and site of gamma secretase cleavage during HPV infection and the impact of L2-specific neutralizing antibodies and gamma secretase inhibitors on endosomal escape. Antibodies have generally been assumed to effect protection in a purely extracellular manner, as they are not generally found in the cytoplasm. Recently however, a TRIM21-dependent mechanism of antibody- dependent intracellular neutralization (ADIN) has been described. TRIM21 is a cytoplasmic high affinity Fc- receptor for the Fc of virally-associated antibody in the cytoplasm, triggering degradation of the antibody-bound virus. Our new findings suggest that ADIN may be important last line of defense in protection mediated by L2 antibodies. Hypothesis 2: the Fc-region of L2-specific neutralizing antibodies is important for mediating antibody- dependent intracellular neutralization (ADIN) and protection against papillomavirus challenge.
Specific Aim 2 A: To determine whether binding of the Fc of L2-specific antibodies to TRIM21 contributes to antibody-dependent intracellular neutralization and if this is restricted to particular epitopes.
Specific Aim 2 B: To determine the contribution of TRIM21 to protection by HPV vaccination.
Cervical and other anogenital cancers, and a subset of head and neck cancers are caused by 'high risk'human papillomaviruses (HPV) and account for 5% of all cancers worldwide (10% of all cancers in women worldwide). Immune suppressed patients exhibit more severe and progressive HPV disease, and consequently cervical cancer has been designated an HIV-associated malignancy. Low risk HPVs cause skin and genital warts, responsible for considerable morbidity and health care costs. Non-melanoma skin cancers in epidermodysplasia verruciformis and immune compromised patients are also linked to HPV. This proposal seeks to understand the role in HPV infection of the minor capsid protein L2, and examine how L2-specific antibodies can protect from the acquisition of HPV infections.
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