手足口病四价表位嵌合病毒样颗粒疫苗的构建及其免疫原性和免疫保护性研究

Hand, foot and mouth disease (HFMD) is caused by multitudinous types of enterovirus (EV), among which EV71, CA16, CA6 and CA10 are main causative agents in recent years. There is no significant cross protection between them, and EV71 monovalent vaccine is difficult to control HFMD’s transmission. Therefore, developments of multivalent vaccine against the four EVs are of great importance. According to studies that EV71 chimeric virus-like particles (VLPs) presenting CA16 neutralizing epitopes could provide anti-EV71 and anti-CA16 protection, and preliminary bioinformatics analysis, we assume that some peptides on the surface of EV71-VLPs could be replaced by a number of epitopes of other EV types, which preserved immune protection against important pathogens(EV71) and conferred protection against other EVs. Therefore, in this project, based on the identification of CA16, CA6 and CA10 linear neutralizing epitopes, we firstly plan to confirm epitopes and its combination by bioinformatics method, then construct recombinant tetravalent epitope chimeric VLPs expression vectors, produce VLPs in yeast cell, identify morphology of VLPs and surface display of each chimeric epitopes, finally analyze the immunogenicity in mice and evaluate the immunoprotection against the four EVs in vitro and in vivo. This study will lay foundation for the development of a safe, acceptable and broader-immunoprotection vaccine against HFMD.

手足口病由众多型别肠道病毒（EV）引起，EV71、CA16、CA6和CA10是造成其近年流行的主要病原，它们之间无明确的交叉保护，EV71单价疫苗难以控制其流行。因此，研发针对该四种EV的多价疫苗具有重要意义。根据嵌入CA16表位的EV71病毒样颗粒（VLPs）疫苗可产生针对EV71和CA16的双重保护，以及前期生物信息学分析，我们推测：EV71-VLPs表面部分肽段可被多个其他型别EV表位替换，其保留针对重要病原EV71的免疫保护性的同时可赋予对其他EV的免疫保护。因此，本项目拟在已鉴定的CA16、CA6和CA10线性中和表位的基础上，首先借助生物信息学方法确定拟嵌合的三种EV表位及其组合，然后构建四价表位嵌合VLPs重组表达载体，酵母表达并鉴定其形态和表位展示情况，最后免疫小鼠检测其免疫原性以及针对该四种EV的体内外免疫保护性，为研制安全价廉且免疫保护更广泛的手足口病疫苗奠定基础。

手足口病由众多型别肠道病毒(EV)引起，EV71、CA16、CA6和CA10是造成其近年流行的主要病原，它们之间无明显的交叉保护，研制多价疫苗以及构建EV动物感染模型具有重要意义。(1)本项目在已鉴定的CA6、CA10和CA16中和表位的基础上，以EV71 VLP为骨架，应用昆虫细胞杆状病毒表达系统，成功构建了EV71三价（EV71-VLP Chi3 ）和四价（EV71-VLP Chi4 ）表位嵌合VLPs，Western blot确认主要抗原蛋白VP1正确表达，透射电镜确定VLPs形态与灭活病毒形态一致，EV71-VLP Chi3 疫苗针对 CA16 和 CA10 以及 EV71-VLP Chi4 疫苗针对CA16、CA6或CA10的免疫原性和免疫保护性与灭活 EV71 疫苗相比，得到了明显提升，且两者针对EV71的免疫原性和免疫保护性无显著差异，这说明通过表位嵌合构建的 EV71-VLP Chi3 和 EV71-VLP Chi4 疫苗在保留了原有免疫原和免疫保护力的同时，扩大了其保护范围；(2)利用昆虫细胞杆状病毒表达系统构建了CB5单价VLP，其免疫原性和免疫保护效果优于CB5灭活病毒疫苗，可赋予新生小鼠100%保护，单价CB5 VLPs疫苗的构建成功为混合多价疫苗的研制奠定了基础；(3)构建了CA10的感染性克隆，并建立了标准化 CA10 感染 ICR 乳鼠模型，经CA10感染乳鼠的四肢肌、小肠和肺组织出现明显病变特征，为疫苗和抗病毒药物的评价，以及模型菌株的保存和共享创造了条件。