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Mechanisms of Papillomavirus Neutralization

乳头瘤病毒中和机制

基本信息

批准号：
7817027
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/7817027
关键词：
Address Animals Antibodies Binding Binding Sites Biochemical Genetics Capsid Capsid Proteins Cell physiology Cell surface Cells Conflict (Psychology)Cryoelectron Microscopy Data 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 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)：持续感染致癌的人乳头瘤病毒(HPV)类型之一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。虽然已知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，但我们发现，L2免疫动物通过中和抗体提供了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L2在感染过程中的作用机制和L2依赖的中和作用，合理地开发一种单一、廉价的泛致癌HPV预防性疫苗。我们发现L2是适当的细胞内运输所必需的，并与细胞蛋白Synaxin-18和VAV-2结合。L2中和抗体允许病毒与细胞单层结合，但它们的表位与相互作用的细胞蛋白的结合部位重叠，这表明：L2抗体依赖的中和作用是通过阻止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，我们提出了特定的目标1：表征L2特异性中和单抗在感染过程中对病毒粒子的运输和与细胞蛋白相互作用的影响，以及特定目标2：在HPV L2中产生防止与细胞蛋白相互作用的优良突变体，并确定它们对HPV假型病毒粒子与感染性比的影响。文献中关于L2功能的相互矛盾的报告可能反映了用于研究乳头瘤病毒分子病毒学的过多实验模型的差异。器官型木筏模型是最接近复制整个HPV生命周期的“黄金标准”。因此，我们提出了具体的目标3：利用病毒整个生命周期的器官型RAFT模型，在病毒基因组的背景下验证L2单抗的中和活性和突变型HPV16L2的表型。最后，我们提出了具体目标4：用冷冻电子显微镜和图像重建技术确定HPV16与中和性和非中和性L2特异性单抗结合的三维结构。这些研究将确定L2和细胞蛋白之间的保守相互作用，这些蛋白对感染至关重要，并成为保护性抗体的靶标。