FcRn介导的抗细粒棘球蚴靶向黏膜疫苗的构建及其增强体液免疫应答的机制研究

Echinococcosis patients are mainly infected because of the Echinococcus granulosus eggs or larvae invading the intestinal mucosa. An effective vaccine should prevent the early infection of the mucosa or the further spread of pathogenic microorganisms. However, the intercellular tight junctions of epithelial monolayers lining the mucosal surfaces render them impervious to macromolecule diffusion. The neonatal Fc receptor, FcRn, can recognize the immunoglobulin IgG, and mediate the transport of IgG across polarized epithelial cells lining mucosal surfaces. Our previous study found that, the FcRn-targeted mucosal vaccines can significantly improve the humoral immune response. In this study, FcRn-mediated anti-Echinococcus granulosus targeted mucosal vaccines will be built using baculovirus surface display technology by mimicking the FcRn-IgG transport pathway. How FcRn-targeted vaccines induce Tfh proliferation thereby providing help to B cells immunization will be explained by using the methods of flow cytometry,laser confocal microscopy and real-time PCR. The mechanisms of FcRn-targeted vaccines enhancing T-cell-dependent antibody reactions are futher investigated by blocking the signaling pathway between Tfh cells and B cells. The results will provide a new theoretical foundation for FcRn-mediated anti-Echinococcus granulosus targeted mucosal vaccines enhancing the humoral immune response.

包虫病患者主要由于细粒棘球蚴虫卵或幼虫侵入肠黏膜而发生感染，有效的疫苗可预防黏膜早期感染或阻止病原微生物进一步扩散。然而，由于黏膜上皮间的紧密连接，导致抗原无法穿过上皮细胞而发挥有效的黏膜免疫。FcRn是识别免疫球蛋白IgG 的受体，并可介导IgG胞转穿过黏膜上皮细胞。我们前期研究发现，FcRn靶向疫苗可显著提高体液免疫应答。本项目拟通过模拟FcRn-IgG胞转通路，利用杆状病毒表面展示技术构建FcRn介导的抗细粒棘球蚴靶向黏膜疫苗；采用流式细胞术、激光共聚焦术、实时定量PCR等方法观察FcRn靶向疫苗诱导Tfh细胞增殖及促进其辅助B细胞免疫应答的作用；通过阻断Tfh细胞与B细胞间的信号通路，观察FcRn靶向疫苗对T细胞依赖性抗体反应的影响，明确Tfh细胞在FcRn靶向疫苗增强T细胞依赖性抗体反应中的作用地位，为FcRn介导的抗细粒棘球蚴靶向黏膜疫苗增强体液应答的免疫机制提供新的理论基础。

本项目通过模拟FcRn-IgG胞转通路，利用杆状病毒表面展示技术成功构建了FcRn介导的抗细粒棘球蚴靶向黏膜疫苗载体pBacSC-Eg95-wFc(mFc)、pBacSC-Eg.ferritin-wFc(mFc)及pBacSC-Eg95-Eg.ferritin-wFc(mFc)，并成功获得了表面展示Eg95-wFc(mFc)、Eg.ferritin-wFc(mFc)及Eg95-Eg.ferritin-wFc(mFc)融合蛋白的重组病毒粒子Bac-Eg95-wFc(mFc)、Bac-Eg.ferritin-wFc(mFc)及Bac-Eg95-Eg.ferritin-wFc(mFc)。通过体外Transwell试验表明，重组病毒粒子Bac-Eg95-wFc在FcRn的介导下可以跨越极化的MDCK-FcRn细胞，而Bac-Eg95-mFc病毒粒子不能胞转。为了进一步验证重组病毒体内胞转情况，首先我们检测了FcRn受体在不同日龄不同脏器的ICR小鼠表达情况，结果表明，FcRn受体在ICR小鼠的心脏、肝脏、脾脏、肺脏、肾脏、十二指肠、气管、肌肉和鼻腔中都有不同程度的表达，尤其在断奶前后（15-25日龄）FcRn表达差异较大。通过鼻腔免疫小鼠发现Bac-Eg95-wFc组小鼠产生的IgG抗体水平显著高于Bac-Eg95-mFc、Bac以及PBS组，表明Bac-Eg95-wFc在FcRn的介导下能有效的跨越小鼠鼻粘膜上皮进入血液中，提高血液中的抗体水平。为了进一步探讨Bac-Eg95-wFc增强体液免疫应答的机制，通过FCM、Real-time RT-PCR及ELISA试验初步证实，Bac-Eg95-wFc可明显促进Tfh细胞的分化（CD4+CXCR5+）、GC B细胞的分化（B220+GL-7+）以及提高Tfh细胞转录因子Bcl-6的表达和细胞因子IL-21的分泌水平。这些结果为下一步深入研究FcRn介导的抗细粒棘球蚴靶向黏膜疫苗的保护效力及机制奠定工作基础。