Mechanisms of Papillomavirus Neutralization

乳头瘤病毒中和机制

基本信息

批准号：
7655517
负责人：
Richard Bruce Roden
金额：
$ 28.04万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-19 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7655517
关键词：
Address Animals Antibodies Binding Binding Sites Biochemical Genetics Capsid Capsid Proteins Cell physiology Cell surface Cells Conflict (Psychology)Cryoelectron Microscopy Data Developed Countries Developing Countries Development Dissection Epitopes Escherichia coli Experimental Models Gardasil Genotype Goals Gold Human Papillomavirus Human papilloma virus infection Human papillomavirus 16 Immunity In Vitro Infection L1 viral capsid protein Life Cycle Stages Literature Location Malignant neoplasm of cervix uteri Mediating Minor Modeling Molecular Virology Monoclonal Antibodies Oncogenic Oryctolagus cuniculus Pan Genus Papillomavirus Papillomavirus Infections Phenotype Prevention Preventive Process Proteins Reporting Research Personnel Screening procedure Serotyping Vaccination Vaccines Viral Viral Genome Virion Virus Virus Diseases Virus-like particle base image reconstruction monolayer mutant neutralizing antibody neutralizing monoclonal antibodies particle prevent programs syntaxin 18 three dimensional structure trafficking transmission process

项目摘要

DESCRIPTION (provided by applicant): Persistent infection by one of >15 oncogenic Human papillomavirus (HPV) types is a necessary cause of cervical cancer. Papillomavirus has only two capsid proteins; L1 and L2. Although L1 is known to form the capsid and mediates initial binding to cells, the functions of L2 remain to be determined. Whereas vaccination with L1 virus-like particles induces type-restricted immunity, we show that vaccination of animals with L2 provides broad immunity from viral challenge via neutralizing antibodies. Therefore, our long-term goal is the rational development of a single, inexpensive pan-oncogenic HPV preventive vaccine through dissection of mechanisms relating to L2 function in the infectious process and L2-dependent neutralization. We find that L2 is required for appropriate intracellular trafficking and binds to the cellular proteins syntaxin-18 and VAV-2. L2 neutralizing antibodies permit virus binding to cell monolayers, but their epitopes overlap the binding sites of interacting cellular proteins, suggesting: L2 antibody-dependent neutralization occurs by preventing interaction between L2 and key cellular proteins that mediate papillomavirus trafficking. To address this hypothesis we propose Specific aim 1: Characterize the effect of L2-specific neutralizing monoclonal antibodies upon the trafficking of virions and interaction with cellular proteins during infection, and Specific aim 2: Generate fine mutants within HPV L2 that prevent interaction with cellular proteins and determine their influence upon the particle to infectivity ratio of HPV pseudotype virions. Conflicting reports of L2 function in the literature likely reflect differences in the plethora of experimental models applied to study the molecular virology of papillomavirus. The organotypic raft model is the 'gold standard' that most closely replicates the entire HPV life cycle. Therefore we propose Specific aim 3: Validate the neutralizing activity of the L2 monoclonal antibodies and the phenotype of mutant HPV16 L2 in the context of the viral genome using the organotypic raft model of the complete viral life cycle. Finally we propose Specific Aim 4: Determine the three dimensional structure of HPV16 bound to neutralizing and non-neutralizing L2-specific monoclonal antibodies by cryo- electron microscopy and image reconstruction. These studies will identify conserved interactions between L2 and cellular proteins that are critical to infection and targeted by protective antibodies.

描述（由申请人提供）：> 15 种致癌人乳头瘤病毒 (HPV) 类型之一的持续感染是宫颈癌的必要原因。乳头瘤病毒只有两种衣壳蛋白； L1 和 L2。尽管已知 L1 形成衣壳并介导与细胞的初始结合，但 L2 的功能仍有待确定。虽然接种 L1 病毒样颗粒会诱导类型限制性免疫，但我们表明，给动物接种 L2 病毒疫苗可通过中和抗体提供针对病毒攻击的广泛免疫力。因此，我们的长期目标是通过剖析感染过程中L2功能和L2依赖性中和作用的相关机制，合理开发单一、廉价的全致癌HPV预防疫苗。我们发现 L2 是适当的细胞内运输所必需的，并与细胞蛋白 Syntaxin-18 和 VAV-2 结合。 L2 中和抗体允许病毒与单层细胞结合，但其表位与相互作用的细胞蛋白的结合位点重叠，这表明：L2 抗体依赖性中和是通过阻止 L2 与介导乳头瘤病毒运输的关键细胞蛋白之间的相互作用而发生的。为了解决这一假设，我们提出具体目标 1：表征感染过程中 L2 特异性中和单克隆抗体对病毒粒子运输以及与细胞蛋白相互作用的影响，以及具体目标 2：在 HPV L2 内产生精细突变体，防止与细胞蛋白相互作用并确定它们对 HPV 假型病毒颗粒的颗粒与感染性比率的影响。文献中关于 L2 功能的相互矛盾的报道可能反映了用于研究乳头瘤病毒分子病毒学的大量实验模型的差异。器官筏模型是最接近地复制整个 HPV 生命周期的“黄金标准”。因此，我们提出具体目标 3：使用完整病毒生命周期的器官筏模型在病毒基因组背景下验证 L2 单克隆抗体的中和活性和突变型 HPV16 L2 的表型。最后，我们提出具体目标 4：通过冷冻电子显微镜和图像重建确定与中和性和非中和性 L2 特异性单克隆抗体结合的 HPV16 的三维结构。这些研究将确定 L2 和细胞蛋白之间保守的相互作用，这些相互作用对感染至关重要，并且是保护性抗体的目标。