人乳头瘤病毒样颗粒不同中和表位的特性与结构基础研究

Human papillomavirus (HPV) types 6, 11, 16 and 18 are the main causive agents cervical cancers or genital warts. Effective prophylactic vaccines against these four HPV types in Gardasil? or against two high risk types, Type 16 and 18, in Cervarix? have been developed based on recombinant HPV major capsid L1 virus-like particles (VLPs). The mechanism of the HPV vaccine is to elicit neutralizing and functional antibodies to protect the vaccines from HPV infection. Previous support from NSFC enabled the studies on the regulation on L1 expression in mammalian cells, the structure activity relationship of VLP based vaccines, and most importantly the determination of functional VLP-Fab complexes for recombinant HPV 11 and 16 VLPs. Different binding modes of between a neutralizing Ab and capsid L1 protein on the surface of VLPs were demonstrated with HPV as well as bovine papillomavirus. The exact binding sites or epitopes for various neutralizing Abs have not been delineated structurally. Here we proposed to perform some in-depth characterization on a panel of anti-HPV L1 mAbs for each type using a set of different assays, including pseudovirion neutralization assay, mAb foot-printing, competition pattern with human sera from natural HPV infected individuals, etc. The nature of the epitopes recognized by neutralizing mAbs will be studied in details. From these results, representative mAbs will be chosen from each category to prepare Ag:Ab (i.e., VLP:Fab or pentamer:Fab) immuno complexes for structural analysis using cryoEM 3D reconstruction or x-ray diffraction studies on the pentamer-Fab complexes. Pentamer, more amenable for crystallization, is the building block for full-sized VLP, with 72 pentamers per VLP. The neutralizing and immune dominant epitopes, recognized by these mAbs, will be mapped to details with near atom resolution with definitive identification of the amino acids being at the binding interface. With this information available, mutations will be performed on capsid protein L1 gene via the pseudovirion (L1+L2) construction for additional cancer-causing types (6,11,16, 18, 33 and 52) to elucidate the key neutralizing epitopes and mode of virus neutralization for different mAbs. Site-directed mutagenesis of the involved amino acids in the L1 capsid protein will be performed in VLP preparation and on the L1+L2 pseudovirions. Alteration on the binding or neutralizing activity of the mAb on the pseudovirions or binding to VLPs with designed mutations. More insightful understanding on the HPV neutralization mechanism and on the detailed structure of the clinically relevant epitopes will help to develop assays for analyzing critical quality attributes of the vaccine, to guide the process optimization of vaccine production, and to facilitate the design of improved and new generation of vaccine with higher efficacy or broader type coverage for caner-causing serotypes of HPV.

人乳头瘤病毒（HPV）6/11/16/18型是引起宫颈癌和尖锐湿疣的主要型别。在已上市宫颈癌疫苗中，其机制均为通过诱导机体产生中和抗体的方式来抵御病毒感染的。前期申请人等研究提出HPV6/11的中和抗体与病毒衣壳蛋白的不同结合模式，但通过解析HPV病毒样颗粒与中和抗体复合物的结构，探索表位与抗体的中和关系尚不清楚。本课题拟从不同HPV的单抗盘中，通过不同方法鉴定有代表性中和单抗，利用冷冻电镜和晶体衍射，解析高分辨率的抗体与HPV五聚体的复合物结构，精确定位HPV VLP与优势中和抗体的相互作用位点，并鉴定具体氨基酸及其结构特征，利用基于细胞的假病毒中和实验验证HPV（6/11/16/18/33/52）抗体结合位点，项目研究成果为中和机制与关键中和表位的结构基础提供科学依据，有利于现有疫苗生产的质量分析，保证生产稳健性，并为下一代广谱高效的HPV疫苗设计提供思路。

人乳头瘤病毒（HPV）6/11/16/18 型是引起尖锐湿疣或宫颈癌的主要型别。在已上市宫颈癌疫苗中，其机制均为通过诱导机体产生中和抗体的方式来抵御病毒感染的。前期申请人等研究提出HPV6/11 的中和抗体与病毒衣壳蛋白的不同结合模式，但通过解析HPV病毒样颗粒与中和抗体复合物的结构，探索表位与抗体的中和关系尚不清楚。本课题从HPV6/11/16/18/59的单抗盘中，基于假病毒细胞中和模型筛选出结合活性高、构象敏感、识别关键优势表位的中和单抗。利用冷冻电镜和晶体衍射，解析了高分辨率的HPV 衣壳蛋白结构，精确定位HPV VLP与优势中和抗体的相互作用位点，并鉴定具体氨基酸及其结构特征。利用基于细胞的假病毒中和实验验证HPV中和抗体结合位点。本研究利用冷冻电镜三维重构技术和X-射线晶体衍射获取了高分辨率的HPV59 L1衣壳蛋白近原子结构。同时，发现暴露于病毒表面的C末端臂-即“悬挂桥”结构参与了与宿主细胞的相互结合。该研究为HPV的衣壳蛋白结构和感染机制研究提供了重要基础，并为下一代HPV疫苗的分子设计提供思路。此外，我们为各个型别，尤其是HPV16建立了很大的单抗盘，包括一些人源单抗，从HPV16/18双价疫苗免疫接种者的记忆B细胞中分离出抗HPV16的中和单抗26D1。通过环替换和抗原关键位点的定点突变及Ag-Ab作用界面上关键残基的分析显示26D1识别表位与H16.V5中和抗体识别的构象表位存在部分重叠，并发现针对不同表位的中和性抗体，在发挥其病毒中和作用是有明显的协同作用。这些发现有助于提高我们对HPV自然感染或疫苗接种的免疫应答中HPV16 L1 VLP和人B细胞表位的抗原结构的理解及在未来的疫苗设计中可通过跨不同类型的特异性环替换诱导杂交VLP的交叉型保护，从而提供广谱保护性免疫。本课题通过HPV VLP 中和抗体复合物的解析将为中和单抗的深度认识与中和机制的探索提供科学依据。不同型别的单抗盘，包括一系列的功能性单抗也将成为抗原性质鉴定、疫苗放行/批签发实验的关键性工具，可以有力地保障生产稳健性及产品的稳定性分析与研究。