新城疫病毒样颗粒诱导树突状细胞成熟与迁移的黏膜免疫机制研究

Virus-like particles (VLPs) are self-assembled, highly structured, hollow particles constituted with one or more structural proteins of a virus, which have similar immunogenicity like intact virus. VLPs are replication as well as infection incompetent, lacking regulatory proteins as well as infectious nucleic acids. Virus-like particles (VLPs) are increasingly being considered as ideal vaccine candidates or vaccine vectors because they offer significant advantages over many currently used vaccines or developing vaccine technologies. The inductive phase of mucosal immunity leads to trafficking of primed lymphocytes and antigen-presenting cells through regional lymph nodes, providing opportunity for interactions between the peripheral and mucosal compartments of the immune system. It has application potentials for the special "homing" functions and "homing molecules" of lymphocytes in the prevention and control of respiratory and other mucosal immune-related diseases. In this study, NDV virus-like particles (NDV VLPs) and chimeric NDV VLPs expressing influenza virus HA protein (NDV-HA cVLPs) will be constructed by baculovirus expression system. Firstly, it is to evaluate the self-adjuvant effect of NDV VLPs and its application potential as a platform to display foreign proteins. Secondly, it is to explore the mechanisms of mucosal immunity of maturation and migration of DCs induced by NDV VLPs in vitro and in vivo so as to establish a theoretical basis of NDV VLPs in prevention and control of infectious diseases, cancer, allergic diseases.

病毒样颗粒（VLPs）是由病毒的一种或多种结构蛋白装配而成的结构化的空心蛋白颗粒，与完整病毒免疫原性相似，无复制和感染能力，具有理想疫苗及其载体的诸多优势。黏膜免疫在诱导阶段启动初始淋巴细胞与抗原递呈细胞向局部淋巴结迁移，从而介导黏膜和外周免疫之间的相互作用。这种淋巴细胞特殊的"归巢"功能及"归巢分子"在呼吸系统相关黏膜免疫的疾病预防中具有应用潜能。本项目以新城疫病毒VLPs（NDV VLPs）与树突状细胞（DCs）为研究靶点，利用杆状病毒表达系统构建NDV VLPs与嵌合表达流感病毒HA蛋白的NDV-HA cVLPs，通过免疫攻毒实验评估NDV VLPs在黏膜免疫应答中的自体佐剂效应及其作为外源蛋白展示平台的应用潜能，然后通过体外刺激黏膜DCs和体内黏膜免疫，探索NDV VLPs诱导DCs成熟与迁移的黏膜免疫机制，为NDV VLPs在传染病、肿瘤、过敏性疾病防治中的应用奠定理论基础。

本研究为揭示新城疫病毒样颗粒（NDV VLPs）诱导树突状细胞（DC）成熟与迁移的黏膜免疫机制，首先利用昆虫-杆状病毒表达系统组装并纯化了NDV VLPs，该病毒粒子具有典型的囊膜结构，其表面展现高密度病毒糖蛋白，随后在动物模型中证实NDV VLPs具有良好的免疫原性，能引起较高水平的特异性体液、细胞和局部黏膜免疫应答以及提供完全的免疫保护效力。采用体外重组GM-CSF和IL-4联合诱导分化的DCs具有典型树枝状突起，其表面标志分子CD11c和MHC-II均高表达。体外试验结果显示，DCs有效摄取VLPs并促进初始型T细胞分化；VLPs诱导DC上调成熟标志分子表达以及增加炎性细胞因子分泌；TLR4和NF-κB抑制剂处理会阻断VLPs诱导DC成熟；VLPs上调DC胞内NF-κB、IκB以及IKK分子的磷酸化表达，表明NDV VLPs诱导DC成熟通过TLR4-IKK-IκB-NF-κB信号通路实现；NDV VLPs诱导DC迁移依赖CCR7-CCL19/CCL21轴的作用，且CCL21作用更显著。体内试验结果显示，负载VLPs的DCs在体内迁移同样依赖CCR7-CCL19/CCL21轴，促使引流淋巴结和脾脏间形成趋化因子浓度差；VLPs早期招募外周DCs经由引流淋巴结向脾脏迁移，随后激活初始型T细胞。以NDV VLPs为外源蛋白载体平台，构建的多种嵌合VLPs具有较好的免疫原性和免疫保护效果，可作为多联、多价疫苗候选株为新型疫苗研制提供策略。综上，本研究揭示NDV VLPs诱导体内、外DC成熟与迁移的细胞与分子机制，充实并填补了VLPs激活天然免疫应答研究领域的空白，为深入解析VLPs激活天然免疫应答机制提供科学依据，同时也为NDV VLPs在传染病防治中的应用奠定理论基础和技术平台。