主动递送黏膜系统的乳酸菌活载体构建及其诱导黏膜免疫机制的研究

Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive alternatives to attenuated pathogens, for mucosal delivery strategies. According to the characteristics of mucosal immunity and gastrointestinal infections, exploring new disease prevention strategy against mucosal disease is very necessary. Lactic acid bacteria play an important role in the prevention gastrointestinal diseases and infections as major candidate carrier strain. Rencently, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture, Research has yielded an abundance of vaccine candidates against mucosal infections, but only few mucosal vaccines have been registered for use. .We show here that engineered lactic acid bacteria LAB outer membrane anchored vector (LAB-Vectors) are an easily yielded vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. By expression of foreign antigens derived from bacterial pathogens, such as Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A (InlA)；Staphylococcus aureus fibronectin-binding protein A (FnBPA); and Salmonella typhimurium InvA (InvA), which can efficiently internalize and deliver vectored- heterologous proteins in epithelial cells, and together expression IBDV-VP2 recombinant vaccines are being developed that aim to stimulate mucosal immunity. Screening of effective combination of vaccine delivery systems, we get an active and effective presentation heterologous proteins vaccine delivery systems. These delivery systems have improved the performance and versatility of inducing mucosal immunity to recombinant antigens in animal mucsal system. our work signals the possibility of LAB-Vectors as a tunable technology platform for a new generation of prophylactic and therapeutic mucosal vaccines.

开发“高效和安全”的黏膜疫苗递送系统是解决当前黏膜疫苗“有效”激活黏膜免疫的关键科学问题。本研究利用抗感染益生乳酸菌高通量免疫评价技术，筛选抗感染、抗耐受及免疫调节土著益生乳酸菌作为活载体，同时表达和锚钉展示两种异源蛋白：抗原蛋白VP2和内化蛋白InlA或FnBP或InvA（来源于病原菌的粘附侵袭蛋白），两种蛋白的表达和锚钉经过8套诱导表达和5套锚钉控制系统的组合和优选，以达到“适时和适量”分别表达并锚钉的目的，经体外筛选表达异源抗原蛋白和锚钉内化粘附蛋白并能高效内化黏膜上皮“适时和适量”的优化表达和锚钉展示组合，最后经动物实验筛选“主动递送系统”“有效”激活黏膜免疫且不产生黏膜损伤和炎症反应的活载体递送系统。该主动递送黏膜系统利用了内化蛋白的粘附和内化作用，有望达到“高效激活”黏膜免疫和共同黏膜免疫效应的目的，对深入探讨黏膜免疫机制、开发黏膜疫苗，探索控制动物和人类黏膜疫病具有重大意义。

随着现代免疫技术和生物工程技术的发展，主流常规活疫苗和灭活疫苗已经远远不适应现代养殖业的发展，开发“高效和安全”的黏膜疫苗是当前畜禽业发展中迫切需要解决的关键科学问题。乳酸菌作为递送系统载体系统是递送抗原和保证生物安全的完美结合，许多乳酸菌活载体系统并不能“高效”激活黏膜免疫系统，存在着内化抗原较弱及不能“理想地”激活黏膜和系统免疫的重大缺点，“主动内化”即“主动递送”是实现乳酸菌活载体“有效激活”黏膜免疫的必要策略。.本研究开发了以食品级乳酸菌为宿主载体，以食品安全级选择标记的表达载体系统，以鸡传染性法氏囊病毒保护性抗原VP2作为递送的外源抗原，以无毒力的内化蛋白加强内化效率，经过多种“主动内化蛋白”载体的构建、表达与锚钉的测试和内化效率的筛选，最终构建成功了以OmpH和RCK为内化蛋白的重组乳酸菌主动递送活载体系统。.研究表明，重组乳球菌共表达M细胞靶向配体OmpH和IBDV-VP2蛋白，动物实验表明，在没有任何佐剂的情况下，重组L. lactis VP2-OmpH菌株能高效激活免疫应答并起到保护作用，在注射和口服免疫的鸡中，能产生100%和80%的对鸡超强毒vvIBDV的攻毒免疫保护。此外，我们构建的表达沙门氏菌内化蛋白RCK和IBDV抗原VP2的重组乳球菌，注射免疫组和口服免疫组的攻毒成活率分别为100%和80%，可产生了高水平中和抗体，结果表明，r-L.lactis-OptiVP2-RCK诱导特异性中和抗体介导免疫反应，抵御超强毒IBDV（vvIBDV）的攻击。本研究获得了可以快速递送抗原的、快速产生中和抗体的重组乳酸菌主动递送活载体系统，为新型食品安全级乳酸菌活载体的构建奠定了重要的基础。.本研究构建的“主动”免疫递送乳酸菌活载体系统，申报发明专利3项，获得发明专利2项，发表SCI论文2篇，中文核心3篇。相关产品《重组乳酸菌（r-L.Lactis-VP2-RCK）表达鸡传染性法氏囊病毒VP2和沙门氏菌外膜RCK基因融合蛋白基因工程亚单位灭活疫苗》已完成在黑龙江省的中间试验，正在申报环境释放。从实践和理论上探索实现真正意义上的食品安全级和生物安全级的高效疫苗递送系统奠定了基础。