不同型别五聚体杂合的人乳头瘤病毒样颗粒的组装机制和型交叉免疫原性的研究

Human papillomavirus (HPV) is the main causative agent associated with the diseases of cervical cancer, genital watts. There are more than 170 types of HPV isolates circulating in human being, of which at least 15 are cancer-causing (known as high-risk type) and 2 are watts-inducing (low-risk type). The major neutralization antibodies by HPV are highly type-specific and lower titer while natural viral infection. The newly emerging HPV 9-valent vaccine Gardasil® 9 comprises of 9 types of yeast-derived HPV VLPs with total 270μg dosage, which seems unrealistic to include more 8 types HPV VLPs to expand prevention coverage. How to elicit high-titer cross-type neutralization with vaccination by limited numbers of potency immunogens is the core scientific issue on HPV biology and immunology. In our previous study, two types of assembly-deficient HPV L1 mutants, C175A and C428A, can hybrid assemble into novel HPV VLPs, and more than 2 types of HPV L1 can involve in hybrid assembly by the paring mutations, the resultant VLPs are capable to induce hetero-type neutralization antibodies in mice. In the present grant application, we endeavor to decipher the assembly mechanism and structural basis for hybrid-featured neutralization epitopes by structural biology, and to evaluate the immunogenicity profile of various hybrid HPV VLPs. With the above insights, we hope to elucidate the structural evolution of HPV L1 protein and the structural basis for the cross-type neutralization-induced epitopes. These outcomes from the study are expected to guide pan-HPV vaccine design and development, and provide a typical paradigm for the new discipline – structural vaccinology, in which vaccine design is based on the knowledge of structural biology.

人乳头瘤病毒（HPV）感染导致宫颈癌、尖锐湿疣等疾病，型别超过170种，主要致病型别至少17种，刚上市的HPV九价疫苗采用野生型HPV病毒样颗粒（VLP），剂量达270μg，难以进一步增加型别扩大保护范围。野生型HPV VLP诱导的中和抗体高度型特异性，无法实现交叉保护。如何设计能够诱导出更多型别中和抗体的免疫原，实现一种免疫原对多种型别HPV的感染保护是下一代HPV疫苗研究的核心科学问题。我们前期发现两种型别HPV L1突变体C175A和C428A五聚体可互补形成新型杂合VLP，可诱导5种不同型别中和抗体。本项目深入研究杂合VLP的结构特征、组装机制及其诱导交叉中和抗体的规律，以期阐明HPV L1衣壳分子杂合的组装机制和结构基础，揭示L1蛋白结构的分子进化和型特异性表位特征，利用杂合VLP方式再现隐匿HPV衣壳中的广谱中和表位，从而，为下一代HPV广谱疫苗研究提供有效的免疫原和理论依据。

HPV 型别众多，流行病学研究表明能引起恶性肿瘤的型别至少有 15 种。目前上市的HPV九价苗对宫颈癌的保护率约90%，难以通过进一步增加价数来扩大疫苗的保护范围，下一代的疫苗研究主要是设计具有诱导型交叉中和抗体的免疫原，在控制疫苗潜在副反应的同时增加对其他10%宫颈癌的保护。前期研究发现两种型别HPV L1突变体C175A和C428A五聚体可以互补形成新型杂合VLP颗粒，本研究拟通过HPV16和HPV52的两型杂合组装探究杂合颗粒组装的化学剂量比，并利用低温电镜重构解析杂合颗粒的五聚体排布，探索三种及以上型别五聚体的杂合组装，分析杂合VLP与各型别HPV中和抗体的反应性及其免疫血清中抗体的中和活性。为下一代HPV广谱疫苗设计提供有效的免疫原和理论依据。.本课题按照预定的目标已完成各项指标：确定了HPV16和HPV52杂合颗粒组装的最优化学剂量比为等摩尔组装，杂合颗粒组装的二硫键数目是野生型的一半；利用低温电镜三维重构技术解析了杂合颗粒HPV16C175A-HPV52C428A与型特异抗体免疫复合物的冷冻电镜结构，显示杂合颗粒中不同五聚体呈现随机分布，和生化实验的等摩尔组装现象一致；不同型别HPV五聚体的组装证明杂合组装与HPV的型别无关，是一种无型别特异的随机组装，理论上可以将72种五聚体同时组装到单一杂合颗粒上，实验中进行了2~9种型别五聚体的杂合颗粒的组装；分析了两型杂合VLP的抗原表位谱的改变，通过单克隆抗体筛选证实杂合颗粒上存在表位能诱导新的型别交叉中和抗体，2~9种型别的杂合体颗粒免疫实验显示，除了诱导同型高滴度的中和抗体以外，还产生了其他型别的中和抗体；本研究利用杂合颗粒组装制备了与上市疫苗Gardasil9剂量一样的9型杂合颗粒，并验证这种颗粒的理化性质和稳定性与野生型相当，免疫实验显示9型杂合颗粒和Gardasil9诱导的同型中和抗体滴度相当，且产生一定的交叉中和抗体，从而为多型别HPV疫苗的研究提供一种新的免疫原理性设计方式，并为高变和多变病毒疫苗的疫苗分子设计提供了参考。.通过本课题实施，共发表SCI文章11篇。申请专利11项，获得国内授权专利4项，获得国际授权专利7项。培养硕士研究生4名，博士研究生2名。