甲型流感病毒血凝素头部区跨亚型广谱中和抗体的作用机制研究

Influenza virus causes a long-term threat to global health. In recent year, broadly neutralizing antibody against hemagglutinin (HA bnAbs) have emerged as a promising approach for combating influenza. Most previously described HA bnAbs are specific to the HA stem region. In contrast, only a small number of HA bnAbs targeting the HA head region have been identified. Notably, it has been reported that HA head bnAbs may exhibit more potent anti-influenza activity than HA stem bnAbs. Thus, it is necessary to make clear the action mechanism of HA head bnAbs. Encouragingly, we have identified several cross-lineage HA bnAbs against influenza B viruses and cross-subtype HA bnAbs against influenza A viruses, suggesting that there should exist some highly conserved epitopes on the HA head. In this study, we will generate more cross-subtype HA head bnAbs against seasonal H1, pandemic-H1, and H5 subtype influenza viruses, and further demonstrate their potency and breadth of anti-influenza activity in vitro and in vivo. Also, anti-viral mechanism analysis and epitope structure determination of several representative antibodies will be revealed. This study will provide some promising antibody candidates for the development of prophylactics or therapeutics against influenza virus infection and may inform the design of a truly universal influenza vaccine.

流感病毒长期危害人类健康。近年来发现的血凝素广谱中和单抗(HA bnAbs)为研制抗流感药物和通用型流感疫苗指明新方向。已报道的HA bnAbs多识别血凝素茎部区，罕有识别血凝素头部区。研究显示识别血凝素头部区的HA bnAbs比识别血凝素茎部区的HA bnAbs有更强的抗病毒效果，因此，研究识别血凝素头部区HA bnAbs抗病毒作用机制具有重要意义。近期我们先后发现识别血凝素头部区的乙型流感跨亚系HA bnAbs和甲型流感跨亚型HA bnAbs，提示血凝素头部区存在高保守性中和表位，为识别血凝素头部区HA bnAbs的筛选与研究提供了科学依据。本项目拟以识别H1、pan-H1和H5亚型流感血凝素头部区的跨亚型广谱中和单抗为对象，系统研究其体内外抗病毒活性，阐明其抗病毒作用机制，并解析其识别表位的结构特征与关键氨基酸组成，为研制新型抗流感药物和通用型流感疫苗提供科学依据。

研发能够提供持久、长效、广谱保护的通用型流感疫苗和广谱高效的新型抗流感病毒药物已成为流感病毒学领域的重要研究方向。近年来，流感病毒血凝素广谱中和表位的发现，为通用流感疫苗研究带来新希望，除了靶向血凝素蛋白颈部区的广谱中和单抗，新的研究发现血凝素蛋白头部区也存在未知的保守功能性表位，值得深入探索。为此，本研究首先基于前期筛选到的甲型流感HA头部区广谱中和抗体C12H5，深入完善流感广谱中和抗体作用机制研究平台，应用X-射线晶体衍射和冷冻电镜对C12H5和HA的免疫复合物进行结构解析，完成对血凝素头部区广谱中和表位的结构特征及关键氨基酸组成的图谱绘制，并初步探索了基于头部区广谱中和表位设计表位疫苗免疫原的可行性；为深入认识流感血凝素头部区广谱中和抗体筛选策略与表位特征，本项目应用序贯免疫策略成功筛选到识别乙型流感血凝素头部区的广谱中和抗体7G6-IgM，并在小鼠和雪貂模型中评估了7G6-IgM的体内外广谱抗病毒活性，结果提示乙型流感血凝素蛋白头部区同样存在诱导跨亚型广谱中和抗体的保守表位，且靶向HA头部区的IgM亚型抗体可通过更高的亲和力和空间位阻作用发挥更强的血凝抑制活性和抗病毒功能；此外，本项目应用去糖基化酶处理血凝素蛋白，免疫小鼠并结合抗体组学、Single B cell技术，筛选到若干具有保护活性的甲型流感广谱功能性抗体，体外实验中可抑制H3N2和H7N9亚型HA蛋白HA0的剪切，体内试验中可提供对H3N2和H7N9跨年份跨亚型不同毒株的广谱保护效果，结果提示可通过糖基化修饰与优化免疫筛选策略改变血凝素蛋白的免疫应答图谱，实现血凝素蛋白表面糖链依赖的隐匿性保守功能表位的免疫聚焦。综上，本项目通过探索靶向流感病毒血凝素蛋白头部区广谱保护性抗体的筛选与机制研究，为后续基于结构生物学的通用流感疫苗的研发提供了科学依据。