高效递呈外源抗原的乳酸菌表面展示系统的构建及其诱导黏膜免疫应答的机制

The efficacy of mucosal vaccines is usually suboptimal due to inefficient induction of mucosal immunity. To improve the efficiency of antigen presenting systems is the key to effectively activate mucosal immune responses. It has been demonstrated that targeting mucosal antigen-presenting cells (APCs) by targeting peptides can specifically enhance the ability of intestinal mucosal uptake of antigens. Besides, the internalins from certain pathogenic bacteria are able to improve the internalization of antigens in epithelial cells, resulting in effective mucosal immune responses. In this proposal, different combinations of APCs-targeting peptides and active internalins will be displayed on the lactobacillus cell surface by using various anchor proteins. Then a number of recombinant lactobacilli expressing two model antigens will be constructed and screened using cell adhesion assay, invasion assay and flow cytometry. Furthermore, the mucosal and systemic immune responses induced by the recombinant lactobacillus which can be effectively internalized by the mucosal epithelium cells and target mucosal APCs will be evaluated in animals. Finally, the effects of the recombinant lactobacillus on DCs to capture antigens and the regulation of the TLR-NF-κB signaling pathway will be examined to reveal the mechanism for inducing mucosal immune responses. This study is expected to develop a powerful antigen-delivery system that can induce efficient mucosal and systemic immune responses. This project will establish a universe platform for developing animal mucosal vaccines and elucidating the mechanism for mucosal immune responses.

黏膜免疫应答低下是制约黏膜疫苗发展的主要瓶颈，而提高抗原递呈效率是有效激活黏膜免疫应答的关键。研究证实，靶向抗原递呈细胞能特异性增强肠黏膜摄取抗原的能力，并且某些病原菌的内化蛋白能显著提高黏膜上皮细胞内化抗原的效率，进而增强黏膜免疫应答。本项目拟利用不同锚定蛋白将多种抗原递呈细胞靶向基序及内化蛋白两两组合展示于乳酸菌表面，以其为载体表达外源抗原，利用细胞黏附试验、入侵试验和流式细胞术筛选出高效内化于黏膜上皮细胞且靶向抗原递呈细胞的乳酸菌表面展示系统；进一步经动物试验验证上述抗原递呈系统诱导黏膜和全身免疫应答的水平；最后，分析经该系统递呈抗原对抗原递呈细胞跨黏膜上皮摄取抗原能力的影响以及对TLR-NF-κB信号通路的调控来揭示其诱导黏膜免疫应答的机制。本研究有望获得能高效激活黏膜和全身免疫应答的乳酸菌抗原递呈系统，为黏膜疫苗研发创建一个共性关键技术平台，同时为深入探讨黏膜免疫机制奠定基础。

黏膜免疫应答低下是制约黏膜疫苗发展的主要瓶颈，而提高抗原递呈效率是有效激活黏膜免疫应答的关键。研究证实，靶向抗原递呈细胞能特异性增强肠黏膜摄取抗原的能力，并且某些病原菌的内化蛋白能显著提高黏膜上皮细胞内化抗原的效率，进而增强黏膜免疫应答。本研究首先利用食品级诱导表达NICE系统，以其配套的载体pNZ8149分别融合表达猪瘟病毒E2蛋白与沙门氏菌侵袭蛋白RCK或耶尔森氏菌外膜蛋白OmpH，成功构建了两株重组乳酸乳球菌rL.lactis-E2-RCK和rL.lactis-E2-OmpH。经体外验证表达后，口服免疫家兔未能诱导CSFV特异性抗体和中和抗体以及免疫保护反应。随后，以乳酸乳球菌为载体构建了5株表达ASFV抗原蛋白的重组乳酸菌，将等量混合后的重组乳酸菌口服免疫猪只后未诱导ASFV特异性的p30和p72抗体，使用野生型ASFV进行接触感染后，免疫猪只全部产生病毒血症和排毒，重组乳酸乳球菌未能提供免疫保护。之后，本研究选择定植能力更强、定植时间更长的植物乳杆菌作为载体，以植物乳杆菌NC8作为宿主菌株，构建了表达CSFV E2蛋白的重组植物乳杆菌NC8-pSIP409-E2，Western blotting检测结果证实NC8-pSIP409-E2成功表达了与预期大小一致的E2蛋白；通过口服免疫小鼠后，抗体检测结果显示，与阴性对照组相比，重组植物乳杆菌NC8-pSIP409-E2免疫组小鼠血清中IgG抗体水平无明显变化，而粪便中sIgA抗体水平略有升高；进一步分离小鼠外周血淋巴细胞和脾脏淋巴细胞分别进行T淋巴细胞亚型分析和淋巴细胞增殖试验，结果显示与对照组相比，免疫组小鼠CD4+和CD8+ T细胞比例和淋巴细胞刺激指数显著升高，表明重组植物乳杆菌NC8-pSIP409-E2免疫小鼠后能够增强细胞免疫应答。同时，为了开发适用于研制黏膜疫苗的乳酸菌载体，优化抗原蛋白的表达和递呈，我们从大兴安岭地区的野猪体内分离乳酸菌，并对其进行了载体开发的初步研究，有望将其改造为口服疫苗载体诱导高水平的免疫应答。上述研究结果为进一步应用于猪瘟及非洲猪瘟等病原的黏膜疫苗的研制奠定了基础。