表面展示NDV F/HN蛋白的细菌样颗粒通过chTLR2亚类分子介导黏膜免疫机制研究

As an emerging technology for surface display on lactic acid bacteria without genetic modification, Bacterium-like particles (BLPs) have be used as an adjuvant and vaccine display system for mucosal delivery. Antigens are displayed on the particle surface as a fusion protein containing a peptidoglycan binding domain, called the protein anchor, that attaches non-covalently and with high avidity to the peptidoglycan wall, resulting in antigen display particles. As a novel development for mucosal vaccine, BLPs have shown a safe and efficient unique advantage in mammals by activating the host TLR2 signaling pathway. The TLRs of poultry are different from that of mammals. It is not sure whether BLPs have the same effect and mechanism in poultry as that in mammals. In this study, we will take Newcastle disease virus (NDV) as a model for poultry pathogens and construct the BLPs which display both the F and HN glycoproteins of NDV (NDV-BLPs). The main purpose of this study is to explore the action and mechanism of chTLR2 subtype molecules when NDV-BLPs induce maturation of dendritic cells and activate adaptive responses through direct interaction with lymphocytes. The systematic study on local mucosal and systemic immune responses of poultry induced by NDV-BLPs through activation of chTLR2/MyD88 signaling pathway will fill the blank of mucosal immunological mechanism involved BLP-based vaccines and display a broad space for development and application prospect of BLPs as mucosal vaccine delivery platform for poultry. This results will also provide a new scientific basis for development of mucosal vaccine or immune modulators for Newcastle disease and other infectious diseases in poultry.

细菌样颗粒（BLPs）是一种新型非遗传修饰型乳酸菌表面展示技术，外源蛋白可通过锚钩蛋白锚定于经热酸处理而得的乳酸菌肽聚糖骨架表面，形成空心抗原展示颗粒。通过激活宿主TLR2信号通路，BLPs作为黏膜疫苗在哺乳动物上显示出了安全、高效的独特优势。而家禽chTLR与哺乳动物不同，BLPs在家禽上是否具有同样的效应和机制尚不得而知。本研究拟以新城疫病毒（NDV）为家禽模式病原，构建同时展示囊膜糖蛋白F和HN的细菌样颗粒（NDV-BLPs），并重点探索家禽chTLR2亚类分子在NDV-BLPs诱导DCs细胞成熟以及直接激活淋巴细胞中的作用与机制，系统研究其通过激活chTLR2/MyD88信号通路增强家禽局部黏膜及系统免疫应答效应和方式，评价BLPs作为家禽黏膜疫苗构建平台的潜力。本研究将填补BLPs诱导家禽黏膜免疫应答机制的空白，为新城疫等家禽传染病新型黏膜疫苗和免疫调节剂的研发提供新的理论依据。

细菌样颗粒（Bacterium-like particles，BLPs）是一种新型抗原展示与黏膜递送平台，其有效性在哺乳动物上已得到证实，而家禽BLP黏膜疫苗的研发及相关免疫机制的研究相对滞后。本研究以家禽新城疫病毒（NDV）为模式病原，采用具有翻译后修饰功能的杆状病毒-昆虫细胞真核表达系统，分别构建了表达基因Ⅶ型NDV的F和HN蛋白融合锚钩基序PA的重组杆状病毒rBAC-F-PA和rBAC-HN-PA，将重组杆状病毒表达上清与制备的裸露乳酸乳球菌BLPs共孵育，分别构建了表面展示F、HN和同时展示F/HN的细菌样颗粒BLPs-F、BLPs-HN和BLPs-F/HN。BLPs主要成分为肽聚糖是TLR2的激动剂，我们证明BLPs主要通过由chTLR2t1和chTLR1t1亚型组成的二聚体激活鸡固有免疫系统，诱导树突状细胞的成熟和巨噬细胞上调IFN-γ、IL-1β和IL-6和iNOS的表达，且通过FITC标记后检测发现，表面展示抗原蛋白的NDV-BLPs同样能被骨髓源树突状细胞和巨噬细胞系HD11这两种抗原递呈细胞高效地摄入，激活chTLR2t1/chTLR1t1/MyD88信号通路，其他chTLR可能也参与了该过程。将BLPs-F、BLPs-HN、BLPs-F/HN及BLPs-F+H（BLPs-F和BLPs-HN混合）滴鼻免疫雏鸡后，可检测出高水平特异性ELISA和中和抗体，及Th1/Th2混合型细胞免疫，更重要的是，诱导产生的局部SIgA抗体可全面封闭病毒入侵途径。以NDV强毒株NA-1进行致死性攻击，结果显示，与单抗原BLPs-F、BLPs-HN免疫组相比，无论双抗原的BLPs-F+HN混合免疫组还是共展示的BLPs-F/HN组均能提供更高保护水平，其中BLPs-F+HN混合免疫组的保护率可达90%，为基因Ⅶ型新城疫新型亚单位黏膜疫苗的研发奠定了坚实的基础。本项目首次将BLPs应用于家禽传染病黏膜免疫，填补了BLPs诱导家禽黏膜免疫应答机制的空白，也为其他诸如禽流感等家禽呼吸系统传染病的免疫防控提供重要借鉴。