Mechanisms of Papillomavirus Neutralization

描述（由申请人提供）：我们的总体目标是了解L2特异性抗体如何对HPV感染产生广泛的保护作用。在这里，我们剖析了为什么L2和γ-分泌酶的膜内切割是感染所必需的，以及L2抗体如何在感染过程中的多个步骤中通过中和而有效地保护。在最初的资助期间，我们进行了令人惊讶的观察，即γ-分泌酶是HPV感染所绝对需要的（Karanam等人，2010），但只有当HPV开始在晚期内体中分解时，γ-分泌酶的蛋白水解活性才是从与病毒基因组相关的L2的晚期内体逃逸所需的。最近的研究结果表明，L2在残基45-67之间具有对感染至关重要的跨膜样结构域。我们认为，假定的跨膜区刺穿内体膜，γ-分泌酶裂解释放L2的羧基末端，将病毒基因组带到细胞核。L2中的两个关键中和表位位于L2的推定跨膜结构域的任一侧。即使在病毒与细胞结合后几小时加入L2抗体也是中和的，这表明阻断发生在感染后期。因此，我们建议：假设1：γ-分泌酶在其推定的跨膜区域内切割L2对于内体逃逸是关键的，并且被L2特异性中和抗体抑制。具体目标1A：确定膜插入所需的L2残基，以及它们是否通过促进内体逃逸而导致感染性。具体目标1B：确定HPV感染期间γ分泌酶裂解的功能作用和位点，以及L2特异性中和抗体和γ分泌酶抑制剂对内体逃逸的影响。 抗体通常被认为以纯粹的细胞外方式实现保护，因为它们通常不在细胞质中发现。然而，最近已经描述了抗体依赖性细胞内中和（ADIN）的TRIM 21依赖性机制。TRIM 21是细胞质中病毒相关抗体Fc的细胞质高亲和力Fc受体，触发抗体结合病毒的降解。我们的新发现表明ADIN可能是L2抗体介导的保护作用的重要最后一道防线。假设二：L2特异性中和抗体的Fc区对于介导抗体依赖性细胞内中和（ADIN）和保护免受乳头瘤病毒攻击是重要的。具体目标2A：确定L2特异性抗体的Fc与TRIM 21的结合是否有助于抗体依赖性细胞内中和，以及这是否限于特定表位。具体目标2B：确定TRIM 21对HPV疫苗接种保护作用的贡献。

DESCRIPTION (provided by applicant): 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 1A: To determine the residues of L2 required for membrane insertion and whether they contribute to infectivity by facilitating endosomal escape. Specific Aim 1B: 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 2A: 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 2B: To determine the contribution of TRIM21 to protection by HPV vaccination.