基于口蹄疫抗原表位的T细胞免疫佐剂研究

Foot-and-mouth disease (FMD) causes serious harm to the development of global animal husbandry, resulting in huge economic losses. At present, we adopt a mandatory immunization-based comprehensive prevention and control measures to prevent and control FMD in China, which has urgent need to develop new type vaccines and immune adjuvants with more secure and high performance to prevent and control FMD outbreak effectively.. Regarding helper T cell epitopes (Th epitope) of foot and mouth disease virus proteins as the research objects in the study, we synthesized candidate Th epitope peptides using chemical synthesis combined with bioinformatics analysis methods. Th cell epitopes of FMDV were identified through the establishment of identification platform of SLA-II class (swine MHC II) restricted epitopes in vitro and activity determination method of Th epitope peptide, and then Th cell epitope maps of FMDV were draw. Moreover, new type T-cell immunoadjuvant were prepared by using combinatorial design theory and methods based on identified Th epitope, and swine were immunized with vaccine contained T-cell immunoadjuvant. The humoral immune response and cellular immune response level of immunized swine were determined and immune effects of the new T-cell immune adjuvants were valuated. The study is expected to enhance the humoral immune responses and make up the shortcoming of lack of cellular immune responses of immune animals, and lays the research foundation for the development of new type FMD vaccine and immune adjuvant that stimulated humoral immune response and cellular immune responses simultaneous, which has significant social and economic significance and application prospects.

口蹄疫严重危害世界各国畜牧业发展，造成巨大的经济损失。目前我国采取以强制免疫注射为主的综合性防控措施来预防和控制口蹄疫，急需研发更加安全、高效的疫苗及免疫佐剂，有效防控口蹄疫疫情发生。本项目以口蹄疫各病毒蛋白中辅助性T细胞表位(Th表位)为研究对象，结合生物信息学分析方法，采用化学合成技术合成口蹄疫病毒候选Th表位多肽。通过建立猪SLA-Ⅱ类限制性表位体外鉴定平台及Th表位肽活性测定方法鉴定FMDV Th细胞表位，绘制FMDV Th表位图谱。同时，将鉴定的Th表位应用组合型设计理论和方法制备成新型T细胞免疫佐剂，配制疫苗免疫猪群，测定其体液免疫应答和细胞免疫应答水平，评价新型T细胞免疫佐剂的免疫效果。该研究有望增强免疫动物的体液免疫应答并弥补细胞免疫应答不足的缺陷，为研制能同时激发特异性体液免疫和细胞免疫应答的口蹄疫新型疫苗及免疫佐剂奠定研究基础，具有重大的社会经济意义和应用前景。

口蹄疫严重危害世界各国畜牧业发展，造成巨大的经济损失。目前我国采取以强制免疫注射为主的综合性防控措施来预防和控制口蹄疫，急需研发更加安全、高效的疫苗及免疫佐剂，有效防控口蹄疫疫情发生。本项目以口蹄疫病毒蛋白中辅助性T细胞表位为研究对象，旨在研发基于口蹄疫抗原表位的T细胞免疫佐剂。项目实施顺利，总体进展良好，完成了项目原定的研究计划及任务，在国内外学术期刊上正式发表研究论文5篇，其中英文论文2篇、中文核心期刊论文3篇；获授权专利2项，国家二类新兽药1项；培养博士后1名、研究生2名。 . 根据项目研究内容及目标任务要求，完成了以下研究工作：（1）综合生物信息学分析、猪SLA-Ⅱ类复合体限制性多肽表位筛选及文献汇总结果，绘制了口蹄疫（FMDV）各基因Th表位图谱。（2）基于Th表位图谱，应用组合型设计理论和方法设计4种含FMDV非结构蛋白不同表位、拷贝数及排列组合的的组合型Th表位肽。（3）应用基因工程手段构建4种组合型Th表位肽的重组表达质粒，使用原核表达系统重组表达并优化了重组蛋白的表达量、可溶性及稳定性，纯化制备了2种水溶性候选T细胞免疫佐剂用于免疫研究及效果评价。（4）使用制备的T细胞免疫佐剂配制疫苗免疫猪群，经口蹄抗体检测试纸和ELISA检测血清抗体水平及效价，结果表明两种佐剂均能辅助增强主抗原产生更高水平的中和抗体，辅助B细胞有效增强免疫猪群的特异性体液免疫应答水平。（5）建立了以A型口蹄疫VP1蛋白为基础的A型口蹄疫病毒抗体ELISA检测方法，为后续评价2种水溶性T细胞佐剂在A型口蹄疫疫苗中的免疫效果奠定基础。（6）T细胞佐剂疫苗免疫猪群后细胞免疫应答评价结果表明，两种佐剂均能促进免疫猪群的T淋巴细胞增殖，且极显著高于未添加佐剂组。. 该研究获得两种有效的基于口蹄疫抗原表位的T 细胞免疫佐剂，增强了免疫动物的体液免疫应答并弥补了细胞免疫应答不足的缺陷，为研制能同时激发特异性体液免疫和细胞免疫应答的口蹄疫疫苗免疫佐剂奠定了研究基础，具有重大的社会经济意义和应用前景。