免疫增强剂介导口蹄疫灭活抗原诱导长效体液免疫应答的机制研究

Foot-and-mouth disease is a highly contagious disease of cloven-hoofed animals, which caused by foot-and-mouth disease virus. Vaccination is the predominant strategy for the prevention and control of FMDV infection. However, the commercial FMDV inactivated vaccines have several drawbacks, such as slow speed of antibody generation, low levels of antibody titers, minimal duration of immunity and narrow protection across the many clades of viral strains. Many times of vaccination were needed in order to overcome the disadvantages of FMDV vaccines, in turn, caused huge economic cost. A new immunopotentiator CVC1302 was successfully screened which could significantly enhance the efficacy of porcine FMDV inactivated vaccine. When pigs immunized with just one dose of inactivated FMDV coupled with CVC1302, the immune persistence was as long as 6 months, which provide long-term of protective humoral immunity. In mice, CVC1302 significantly enhanced the titers of FMDV antibody as well as the numbers of long-lived plasma cells in bone narrow. This project would research the mechanism of inactivated FMDV immunopotentiator CVC1302 in mediating the establishment of long-term humoral immune responses in mice and pig models. Here are the details: the formation of long-lived plasma cells and germinal center; the activation of Tfh; the transcriptional level of key genes and the expression level of cytokine. Our aim is to reveal the mechanism of CVC1302 in mediating the establishment of long-term humoral immunity, and spark new insights into the development of novel immunopotentiator and novel vaccines.

口蹄疫是由口蹄疫病毒(FMDV)引起的偶蹄动物的一种高度接触性传染病。我国主要倚靠疫苗免疫防控该病，然而商品化FMDV灭活疫苗存在抗体产生缓慢、水平偏低、抗体持续期较短以及对变异毒株保护力差等缺点，在实际应用中，需进行多次免疫，不利于口蹄疫的疫情防控，造成了严重的经济损失。本实验室前期成功筛选到一种免疫增强剂CVC1302，配伍FMD灭活抗原后，1次免疫便为猪只提供长期有效的体液免疫力。动物试验结果显示，CVC1302可显著提高灭活抗原诱导产生的抗体滴度，且骨髓长寿浆细胞数目显著增多，但其作用机制尚不清楚。本项目拟在小鼠和猪两种模型中研究免疫增强剂CVC1302产生长效体液免疫应答的机制，分析其对长寿浆细胞及生发中心形成、Tfh细胞活化及关键基因转录水平及细胞因子表达水平的影响，阐明免疫增强剂介导FMD灭活抗原产生长效体液免疫应答的分子机制，为新型免疫增强剂及新型疫苗的开发提供理论依据。

免疫增强剂CVC1302由PRRs激动剂组成，配伍口蹄疫灭活疫苗（KV），1次免疫小鼠或猪只可提供长效体液免疫力，抗体滴度显著升高，骨髓长寿浆细胞（LLPC）数目显著增多。本项目研究CVC1302介导KV触发长效体液免疫应答的机制，研究内容包括生发中心应答、Tfh细胞活化及关键转录因子。. 首先，CVC1302于注射位点促进MIP-1α、MIP-1β和MCP-1的表达，募集抗原递呈细胞(APC)，单核细胞与巨噬细胞于免后1天数目达到峰值，树突状细胞于免后3天数目达到峰值； APCs的增多表明更多的抗原被捕获处理，向T、B细胞提供抗原信号。. 其次，CVC1302显著增多引流淋巴结处生发中心数目，且Tfh细胞百分比显著增加。iTAQ蛋白图谱发现，试验组小鼠淋巴结内与凋亡和自噬相关蛋白表达呈下调趋势，表明CVC1302使浆母细胞亲和力升高，凋亡比例降低，通过Tfh细胞正向选择，分化为高亲和力LLPC，与高亲和力抗体滴度增高相对应。. 最后，CVC1302显著提高骨髓LLPC数目，免后42天达到峰值，凋亡检测发现，CVC1302可显著抑制细胞凋亡(凋亡早期或凋亡晚期)，且相对定量PCR检测发现Mcl-1的转录水平显著提高，进一步解释流式检测结果。. 猪只试验发现，CVC1302显著提高KV诱导的抗体滴度，且IgG1与IgG2分型抗体水平也显著升高，且促进IL-4、IL-6和IFN-γ、IL-2分泌，进一步解释分型抗体水平升高。. 本项目系统阐释了CVC1302依赖于Tfh细胞对生发中心B细胞进行正向选择，分化为高亲和力LLPC，诱导机体触发长效体液免疫应答。. 本项目通过解析CVC1302的作用机制，为后续免疫增强剂的研制提供理论指导，研制更为有效的佐剂，提高疫苗免疫效力；一方面减少经济损失，降低养殖成本；一方面提升动物福利，减少免疫带来的应激等。