EV71和CA16中和表位的交叉反应谱及其融合基因重组卡介苗的免疫保护性研究

Hand, foot and mouth disease (HFMD) is a common illness caused by many enteroviruses (EV), among which EV71 and CA16 are the main causative agents. No vaccine is currently available against this disease. Epitope vaccines of EV71 and CA16 can induce virus-specific immunity without autoimmunity. However, the enhanced immunogenicity and broadened protection scope are required for the epitope vaccines. Bacillus Calmette-Guerin (BCG) is an ideal multivalent vaccine vehicle and by self a potent adjuvant in experimental animals and man, and we found in the preliminary study that several conserved B-cell epitopes of EV71 and CA16 are potentially cross-reactive against a number of other EVs. In this project, we plan to validate the cross-immunity of these neutralization epitopes against other HFMD agents by a neutralization assay, and optimize the combination of intra- and inter-typic cross-protective neutralization epitopes from EV71 and CA16 and universal Th epitopes by using bioinformatics tools, and construct a recombinant BCG expressing a fusion peptide of these epitopes, and then investigate the immunological effects in mice and protective efficacy of the recombinant BCG against a variety of HFMD agents. This study will lay foundation and provide useful information for the development of a safe, effective and acceptable broad-spectrum vaccine against HFMD.

手足口病是以EV71和CA16为主的多种肠道病毒（EV）引起的常见传染病，目前缺乏临床可用疫苗。EV71和CA16表位疫苗在诱导病毒特异性免疫的同时可避免有害的免疫反应，但是如何进一步增强免疫原性和扩大免疫保护范围是其发展的关键。卡介苗是优良的多价疫苗投递工具兼高效佐剂；我们的前期工作发现，EV71和CA16的多个保守中和表位具有与多种其它EV交叉反应性潜能。本项目拟在此基础上，制备基于这些中和表位的抗体，以微量中和实验测定这些中和表位对手足口病其它病原的交叉免疫性；然后借助生物信息学工具优化EV71和CA16的型内和型间交叉反应性中和表位与通用Th细胞表位构成的融合多肽的连接组合，以卡介苗为载体构建分泌表达该融合多肽的重组卡介苗，免疫小鼠并观察该融合多肽的基因重组卡介苗诱导的免疫反应以及针对手足口病多种病原的免疫保护效率，为研制安全有效、价格低廉和易于接受的广谱性手足口病疫苗奠定基础。

手足口病（HFMD）是二十几种肠道病毒（EV）引起的儿童常见传染病。不断有EV型间交叉反应的报道；分枝杆菌的多种优点使其成为良好的重组疫苗投递工具。鉴于此，本项目主要目的是确定HFMD主要病原EV71和CA16中和表位的交叉反应谱，并构建基于其交叉中和表位和分枝杆菌作为活载体的重组疫苗。我们的研究发现，目前唯一可用的HFMD疫苗-EV71灭活疫苗对8种EV有弱的体外交叉保护作用，对CA9的中和滴度最高（1:16），保护能力与亲缘关系远近无明显关系；具有交叉反应潜能的EV71和CA16 的5个中和表位，仅EV71中VP2蛋白中一表位具有体内外对CA16的交叉保护能力；构建的呈递EV71交叉中和表位的病毒样颗粒疫苗和重组分支杆菌疫苗，前者具有很强的免疫原性，可赋予乳鼠对EV71的完全免疫保护以及对CA16的交叉免疫保护，后者可高效表达重组蛋白，但是免疫原性较弱，不能赋予新生鼠的免疫保护作用；通过传代驯化获得EV71、CA16、CA6和CA10的乳鼠适应性毒株，从而建立HFMD主要病原的乳鼠感染模型。这些研究结果和发现，为手足口病的疫苗研发、治疗评价和致病机制研究奠定了一定基础。