人乳头瘤病毒样颗粒表位的鉴定和中和机制研究

The correlation of HPV infection and the incidence of cervical cancers (accounting for ~10% of human cancers) has been unambiguously established, which led to the Nobel Prize for Physiology or Medicine to H. zur Hausen in 2008. Introduction of quadrivalent (Types 6, 11, 16 and 18) Gardasil? and bivalent (Types 16 and 18) .Cervarix? was shown to have caused significant reduction in HPV infection and/or pre-cancerous lesions. While these virus-like particle (VLP) based vaccines were shown to be safe and highly efficacious, the high resolution VLP structure and the key neutralizing epitopes have not been determined. The proposed work will expand VLP production of the cancer causing HPV types to nine types (16, 18, 31, 33, 35, 45, 52, 58, 59), to cover >95% of cervical cancers. A large panel of mouse monoclonal antibodies (mAb) will be produced for each type to map out the immunoreactivity and structural requirements of full length and truncated L1 based VLPs. In particular, the epitopes for the neutralizing mAbs based on the pseudovirion neutralization assays will be characterized in details as to the location of inter- or intra-capsomeric location, IgG vs. Fab neutralization efficiency, and ability to block the virus/pseudovirion attachment to the cells. Efforts will be given on near atomic resolution VLP structure on full size (T=7， icosahedral) assembly using cryoEM three dimensional reconstruction technique, coupled with x-ray crystal structure of capsomeres. For those VLP-Fab complexes which are amenable for cryoEM image acquisition and processing, VLP-Fab structures will be obtained to locate the epitopes and to ascertain the mechanism of virus neutralization. Preliminary data showed that there are at least three different ways of neutralizing Ab binding to VLPs. The knowledge gained from this work would benefit better understanding of the use larger space betmechanism of current vaccines, help to design future vaccines with broader coverage, and aid in understanding the virus neutralization mechanism and designing anti-viral drugs and therapeutic vaccines.

宫颈癌是高危型HPV感染所致，目前的宫颈癌疫苗是通过诱导产生中和抗体的方式来抵御病毒感染的。HPV的中和抗体与HPV 有着多种的结合方式及对应的中和机制，但基于HPV VLP-中和抗体免疫复合物的空间结构来探索抗原表位和中和机制的功能关系还未见报道。本项目拟首先通过筛选鉴定HPV抗体得到不同类型的中和抗体，利用冷冻电镜三维重建技术和X射线晶体衍射实验解析免疫复合物的结构，定位HPV VLP上的各类中和表位并确定其氨基酸组成和结构特征，然后构建基于表位结构的突变假病毒，利用细胞中和实验研究HPV VLP作为疫苗的中和机制，阐明HPV VLP表位结构与中和作用的定性和/或定量关系，从而应用于现有疫苗生产的质量控制，并为研制下一代宫颈癌疫苗提供参考和依据。

宫颈癌是高危型HPV感染所致，目前的宫颈癌疫苗可诱导机体产生保护性中和抗体以抵御病毒的感染，并预防宫颈癌等疾病的发生。已知HPV的中和抗体与其衣壳蛋白有多种的结合方式，但基于HPV VLP-中和抗体免疫复合物的空间结构来探索抗原表位和中和机制的功能关系还未见报道。本项目拟进行研究内容包括：（1）在HPV抗体库中筛选出不同类别的优势中和抗体；（2）制备抗原抗体免疫复合物，并进行结构解析；（3）通过分子模建等技术确定中和表位的空间结构和主要的氨基酸组成；（4）基于结构和模建结果，探索该中和抗体可能的中和机制。由于课题评审结果建议为短期研究（一年），并且经费资助改为小额资助（16.0万元），故评审专家建议研究目标更改为：“加强已获得中和抗体的生物学鉴定，明确1~ 3种HPV中和抗体的结合位点，以阐明可能的中和机制”。.结题时，通过本课题研究，利用X射线晶体衍射技术解析HPV58五聚体的晶体结构，并采用抗体同源模建获得结构模型，利用分子对接和相互作用界面分析等，鉴定了HPV58两种中和抗体的结合位点和作用方式，对指导疫苗设计和下一代的疫苗研究具有指导意义。另外，本研究还发现异源核糖核蛋白hnRNP H对于HPV L1蛋白形成和组装具有调控作用，并综述了HPV VLP的结构，组装和抗原性检测方法等进展。通过课题的实施，共发表SCI论文3篇，培养1名博士生和3名硕士生。