人肠道病毒交叉中和表位筛选及其嵌合病毒样颗粒疫苗的免疫保护性研究

Human enterovirus (EV) could cause diverse clinical diseases including aseptic meningitis, myocarditis and hand, foot and mouth disease, et al. More than 100 types of EVs have been identified to date, and different EV types circulate alternately or together, making it hard to control. It is therefore quite significant to develop a multivalent or broad-spectrum vaccine against various EV infections. EV epitope vaccines can induce virus-specific immunity and avoid harmful immune response. However, the broadened scope of immune protection and enhanced immunogenicity are required for the development of EV epitope vaccines. It was found by our research group and some others that cross-protective epitopes existed within EV genus; Hepatitis B virus core antigen (HBc), which can self-assemble into virus-like particle (VLP) and present exgenous epitopes on the VLP surface, could markedly improve immunogenicity of chimeric epitope peptides. On this basis, this study plans to further expand screening and idenification of intertypic cross-neutralizing epitopes, and determine their cross neutralization spectrum and neutralization effciency. Subsequently, we will optimize the combination of these broad-spectrum neutralizing epitopes and incorporate the fusion gene into HBc as a carrier to construct a multi-epitope chimeric VLP vaccine. Finally, the immunogenicity and multiple immune protections of the chimeric VLP vaccine against a variety of EVs in vivo and in vitro will be investigated. This project will lay foundation for the development of safe, effective and acceptable broad-spectrum vaccines against EVs.

人肠道病毒（EV）可引起无菌性脑膜炎、心肌炎、手足口病等复杂多样的临床疾病，迄今已有100多个型别；不同型交替或共同流行，使其难以控制。因此，开发多价或广谱性EV疫苗具有重要意义。EV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，但如何扩大免疫保护范围并增强免疫原性是其发展关键。包括本课题组在内的研究发现，EV存在型间交叉保护性抗原表位；可自组装成病毒样颗粒（VLP）并展示外源表位于表面的乙肝病毒核心抗原（HBc）可显著增强嵌合表位多肽的免疫原性。本项目拟在此基础上，进一步扩大筛选具有EV型间交叉中和性的抗原表位，确定其抗体交叉中和谱及中和效率；优化组合广谱性EV中和表位，以HBc为载体构建多表位嵌合VLP疫苗；免疫小鼠并观察该VLP疫苗的免疫原性及体内外多重免疫保护能力，为研制安全有效、价格低廉和易于接受的广谱性EV疫苗奠定基础。

人肠道病毒（EV）型别众多，引起的临床疾病复杂多样，筛选其交叉中和表位并构建表位嵌合的多价病毒样颗粒（VLP）疫苗具有重要意义。本项目① 利用了多种方法筛选EV各组主要型别EV的表位，鉴定到柯萨奇病毒A组10型（CA10）的2个中和表位vp1aa39-53和vp1aa162-176，前者为潜在的型间交叉中和表位，后者为可能的型内交叉中和表位，两者可作为候选表位用于CA10或EV广谱性表位疫苗的开发；鉴定到EV96的vp1aa281-304为型特异的非中和表位，可用于EV96感染诊断试剂的研制。② 在VLP疫苗的构建中，比较了以HBc为载体呈现多个EV表位的嵌合VLP疫苗与以分枝杆菌为载体的重组活疫苗的免疫效果，结果发现，前者具有很强的免疫原性，可赋予乳鼠对EV71、CA16的免疫保护，后者免疫原性较弱，不能赋予新生鼠的免疫保护作用；利用昆虫细胞—杆状病毒表达系统构建了多种重要EV的单价VLP，其中已完成免疫效果评价的CB5 VLPs疫苗的免疫原性和免疫保护效果优于CB5灭活病毒疫苗，可赋予新生小鼠完成的保护；成功构建了以EV71 VLP呈现多种EV表位的嵌合多价VLP疫苗，明确多种类型VLP在EV疫苗研制中的价值。③ 制备了手足口病主要型别EV的鼠适应株，构建了EV71、CA16、CA10的感染性克隆，建立了CA10、CA4的乳鼠感染模型，为研制EV疫苗的效果评价奠定了一定基础。