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，我们剖析了为什么感染需要L2和伽马分泌酶的膜内切割，以及L2抗体如何在感染过程中通过多个步骤的中和来有效保护。在最初的资助期，我们做出了令人惊讶的观察，即感染HPV绝对需要伽马分泌酶(Karanam等人，2010年)，但只有当HPV在晚期内体中开始分解，并且伽玛分泌酶的蛋白分解活性才需要与病毒基因组相关联的L2晚期内体逃逸。最近的发现表明，L2在残基45-67之间有一个跨膜型结构域，这是感染所必需的。我们认为，假定的跨膜区穿透内体膜，伽玛分泌酶裂解释放L2‘S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‘S的跨膜区两侧。甚至在病毒与细胞结合的几个小时后，L2抗体的添加也是中和的，这表明阻断发生在感染的后期。因此，我们提出：假设1：L2在其假定的跨膜区内被伽马分泌酶切割是内体逃逸的关键，并被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促进感染性。目的1B：探讨人乳头瘤病毒(HPV)感染过程中伽玛分泌酶裂解的功能作用和部位，以及L2特异性中和抗体和伽马分泌酶抑制剂对内体逃逸的影响。抗体通常被认为是以一种纯粹的细胞外方式发挥保护作用，因为它们通常不存在于细胞质中。然而，最近，一种依赖于TRIM21的抗体依赖的细胞内中和(Adin)机制已被描述。TRIM21是一种细胞质内高亲和力的Fc受体，可在细胞质中结合病毒相关抗体的Fc，从而引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2：L2特异性中和抗体的Fc区对于介导抗体依赖的细胞内中和(Adin)和抵抗乳头瘤病毒攻击是重要的。具体目标2A：确定L2特异性抗体Fc与TRIM21的结合是否有助于抗体依赖的细胞内中和，以及这是否仅限于特定的表位。具体目标2B：确定TRIM21对接种HPV疫苗的保护作用。

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.