H7N9禽流感HA蛋白免疫调控作用及其与病毒致病力关系研究

In last ten years, avian influenza viruses keep out-breaking in multiple countries and areas. Compared with seasonal influenza viruses, avian influenza patients show an unusually aggressive clinical course, with rapid deterioration and high fatality, which is more than 30% in H7N9 cases, and it causes tremendous society panic and seriously impacts livelihoods, the economy and international trade in affected countries. The pre-existing studies showed the high pathogenicity of avian influenza virus is strongly connected with its ability of miss-leading immune response and inducing a drastically strong immune reaction. However, so far, none of study has showed a pathogenetic factor in avian influenza viruses has a directly immune regulating activity. As showed in our preliminary data, we found H7N9 HA can bind on the surface of immune cells and regulate CD4+ T cells proliferation and differentiation, but further study is need for elucidating the regulation mechanism and its correlation with the immune regulating activity and pathogenesis of H7N9 virus. In present project, firstly, we plan to study the mechanism of H7N9 binding with immune cells, and attempt to locate the binding domain in HA protein and target protein of HA on the immune cells. Secondly, we will study on the mechanism of H7N9 HA regulating CD4 T cell proliferation and differentiation, and attempt to locate the target immune cell of HA and the correlated immune signal pathway. At last, we will investigate the effect of H7N9 HA in vivo by mouse model of influenza virus attacking, and attempt to study the in vivo immune regulating activity of H7N9 HA and its relationship with pathogenesis of influenza virus. The present study will dissect the pathogenesis mechanism of avian virus and may present new potential therapy target for avian influenza treatment.

近十几年以来，禽流感病毒在全球多个地区造成人群中的爆发流行。相对于季节性流感患者，禽流感患者表现出病危病死率高的特点（H7N9患者死亡率超过30%），因此易导致社会恐慌。已有研究表明，禽流感病毒的高致病性与其能诱导机体过强免疫反应密切相关，但是目前尚未发现相应的免疫调控致病因子。申请者初步实验结果显示H7N9 HA能与免疫细胞结合并调控CD4 T细胞的增殖和分化，但是其具体机理及其与病毒致病性和免疫调控能力的关系需要深入研究。本项目计划首先对H7N9 HA与免疫细胞结合机理进行研究以确定HA的结合功能域及相应的免疫细胞靶蛋白，并对H7N9 HA调控CD4 T细胞增殖/分化机理进行研究以确定其作用的靶细胞及相关免疫信号通路，最后将通过流感病毒小鼠攻击模型观察上述HA离体免疫调控作用的在体表现及其与病毒致病力的关系。本研究将为阐明禽流感病毒致病机理提供依据，并为将来研发对抗手段提供潜在靶标。

诸如H5N1, H7N9等禽流感病毒能通过跨种传播等方式威胁人民身体健康、公共卫生安全乃至社会经济正常运转。相对于H1N1和H3N2等季节性流感，禽流感病毒导致的呼吸道疾病表现出重症率和死亡率高的特点。有研究表明，重症禽流感病毒患者体内普遍存在诸如细胞因子风暴等免疫紊乱，提示禽流感病毒能够调控机体免疫反应。另有研究表明，禽流感流感病毒表面的HA蛋白与其致病性相关。为初步阐明禽流感病毒HA蛋白是否对免疫系统具有调控作用，我们研究了i) H7N9 HA蛋白梗部一10氨基酸大小免疫调控致病因子的致病机理；ii) H7N9 HA蛋白头部介导的与免疫细胞高亲和力结合的机理。研究结果表明，该10氨基酸免疫调控致病因子可以通过与整合素家族结合，刺激单核细胞释放多种炎症因子从而增强病毒的致病能力。而对H7N9HA蛋白与免疫细胞的亲和力的一系列相关研究表明H7N9禽流感病毒HA蛋白能高亲和力的和免疫细胞特别是B细胞结合并定位了H7N9 HA蛋白上介导结合的关键氨基酸。以H1N1病毒为骨架构建的含有H7N9 HA的H7N9-HA嵌合病毒能高效的感染B细胞并导致细胞凋亡和死亡。小鼠在体研究表明H7N9-HA嵌合体病毒能高效的感染小鼠肺部B细胞，降低外周B细胞水平并抑制骨髓B细胞发育。上述研究结果不仅发现了崭新的H7N9禽流感病毒致病机理并且也为对抗H7N9等禽流感病毒相关疾病提供了潜在的治疗靶标。此外，我们还利用本课题提供的研究平台对其他病毒包括呼吸道合胞病毒及沙粒病毒的免疫相关领域进行了初步的研究。