基于新型微环DNA技术平台CRIM的H9N2禽流感广谱性原位靶向纳米疫苗激活特异性细胞免疫机制研究

H9N2禽流感病毒（AIV）具有高度变异性，严重危害我国家禽生产。本项目利用延迟裂解型沙门菌及新型微环DNA平台（CRIM）构建表达H9N2 AIV保守性抗原（HA2, 3M2e）的菌株，口服免疫后抗原在体内表达并自组装为病毒样颗粒（CRIM-VLPs）或铁蛋白纳米颗粒(CRIM-DLnano)，进而利用靶向鸡树突细胞（chDCs）表面DEC205分子的单链抗体增强chDCs对纳米颗粒的捕获及抗原递呈作用，攻毒保护实验评价对不同亚系H9N2 AIV的保护效果。通过细胞分选、转录组测序等手段揭示靶向纳米疫苗经“晚期溶酶体-MyD88途径”上调炎性因子及天然免疫受体表达以促进chDCs分化成熟，阐明chDCs活化后增强特异性CD8+T细胞免疫的分子机制。本项目为创制H9N2 AIV广谱性口服纳米疫苗奠定基础，同时为其他重要畜禽疫病新型口服疫苗研究开辟新思路。

H9N2 subtype Avian influenza virus (AIV) still post great threat to poultry production due to its high frequency of variation. In this study we are planning to take use of both regulated delayed lysis Salmonella and novel Cre Recombinase-mediated In vivo Minicircle DNA (CRIM) platform to construct a novel broad-spectrum vaccine, which could synthesize the protective conserved antigen of H9N2 AIV (HA2, 3M2e) in vivo after oral immunization and then self assembled to virus like particles (VLPs) or nanoparticle when fused with ferritin protein, yielding CRIM-VLPs and CRIM-DLnano, respectively. Furthermore, we will take use of the single chain variable fragment targeting to DEC205 located on the surface of chicken dendritic cells (chDCs) to target nanoparticles to chDCs, resulting in enhanced particle capture by chDCs and antigen presentation. Then chicken challenge study will be performed to evaluate the protective effects against different species of H9N2 subtype AIV, in addition, the involvement of "late lysosome-MyD88 route" to upregulate levels of inflammation cytokines, innate immune receptors and to enhance CD8+ T cellular immune response during the activation of chDCs will be determined by cell separation, RNA-seq and other approaches. The results of this study will lay a foundation for the development of broad-spectrum oral nano vaccine for H9N2 AIV, in addition, it also open up a novel field for the application of oral vaccines in the control of both poultry and mammal diseases.