H7N9诱导记忆B细胞产生交叉反应性H3杆部抗体的研究

Antibodies that target highly conserved epitopes on the stalk region of influenza HA display broad reactivity with group 1 and/or group 2 viruses. However, complete HA configuration is required to induce the stalk-reactive antibodies, and there is minimal chance of activating the rare memory B cells that recognize conserved and subdominant epitopes in the HA stalk in the presence of dominant epitopes in the global head. It is reported that memory B cells recognizing conserved epitopes are induced and produce cross-reactive antibodies because the shifted HA of pandemic H1N1viruses is missing many of the dominant and variable epitopes recognized by the majority of preexisting influenza-specific memory B cells. Our previous study showed that some of adults exposed to influenza viruses or immunized with vaccines possess stalk-reactive memory B cells which secreted low levels of cross-reactive antibodies. It is unclear that H7N9 viruses induce immuno-protecting IgG by memory B cells recognizing conserved epitopes in HA stalk. In this study, peripheral blood will be collected from health controls and donors immunized with influenza vaccines to analyze the HA stalk-reactive IgG and H3-specific memory B cells. B cells will be isolated from those subjects who possess stalk-reactive IgG and memory B cells recognizing H3 HA. Six days after B cells are stimulated with inactivated H7N9 virus, HA stalk-reactive antibodies and BCR CDR3 repertoire will be detected. The immune-protection of stalk-reactive antibodies will be assessed through attacking mice with H7N9 viruses. Our results will put forward a novel strategy for influenza prevention.

针对流感病毒HA杆部表位的抗体具有广泛交叉反应性，能够与多种不同亚型病毒交叉结合。但是杆部抗体的诱导需要完整的HA空间构型，且通常情况下处于劣势。研究证实H1N1抗原性变异导致头部特异的记忆B细胞缺乏抗原刺激，处于劣势的杆部记忆B细胞被激活，产生交叉反应性抗体。我们在前期研究中证实部分健康人和流感疫苗接种者体内存在H3杆部记忆B细胞，可被诱导产生低水平的杆部抗体。但H7N9是否能诱导H3记忆B细胞产生具有免疫保护作用的杆部抗体尚未见报道。本研究拟采集遵义地区健康人及流感疫苗接种者外周血，用高通量mPlex-Flu法和ELISpot法筛选H3杆部抗体和H3记忆B细胞阳性个体,分离外周血B细胞，经灭活H7N9病毒刺激后，分析H7N9诱导的H3杆部抗体水平及其交叉反应，分析B细胞BCRCDR3组库变化，并在小鼠体内观察H7N9诱导的H3杆部抗体及其免疫保护作用，为流感的预防提供新的思路和策略。

血凝素（HA）是流感病毒的重要抗原，由高度变异的头部和相对保守的杆部组成。由HA杆部抗原刺激产生的抗体能与多种不同亚型流感病毒结合，具有交叉保护性。然而，针对HA的抗体应答以头部为优势，HA杆部处于竞争劣势。2013年爆发了禽流感病毒H7N9流行，死亡率高达40%。H7N9与H3N2属于同一进化组，其HA杆部高度相似。本研究探讨H7N9病毒对H3杆部记忆B细胞二次应答的影响，为后续通用流感疫苗的研究奠定基础。首先构建了嵌合血凝素cH5/3及H5头部重组杆状病毒，用纯化的cH5/3蛋白建立了高敏感性的抗H3杆部抗体检测体系。经ELISpot法分析发现健康志愿者外周血中存在H3杆部记忆B 细胞。高敏感性Luminex、ELISA及HI法证实志愿者血清中存在抗H1、H3抗体以及抗H1、H3杆部抗体。灭活H7N9病毒在体外可刺激健康人外周血记忆B细胞产生H3杆部抗体，诱导产生的杆部抗体可与H1N1、H5N1、H7N9及H9N2发生交叉结合反应。C57BL/6 小鼠接种灭活H3N2病毒60天后，接种灭活 H7N9 病毒 A/shanghai/1/2013可诱导小鼠产生抗H3杆部抗体，诱导产生的杆部抗体对H4N6病毒感染具有免疫保护作用。健康人B细胞经H7N9刺激6天后，采用单细胞培养技术获得分泌抗H3杆部单克隆抗体的B细胞，经ELISA证实该细胞产生的抗体滴度高达106，抗H3杆部单克隆抗体不与H5头部反应，抗体特异性好。为了探讨再次接触流感病毒后健康人体内HA杆部记忆B细胞的扩增情况，本研究采用高通量测序技术分析疫苗接种前后外周血记忆B细胞BCR CDR3组库的变化。健康志愿者接种流感疫苗后H1、H3抗体滴度增高，记忆B细胞BCRCDR3组库发生偏移，共享克隆数量减少，HA杆部相关V基因频率增加，HA杆部记忆B细胞克隆发生扩增。本课题经体内及体外实验证实，H7N9可诱导H3杆部记忆B细胞二次应答，产生具有广泛交叉反应的杆部抗体，为新型通用流感疫苗的研究提供了新的策略。已于本领域权威期刊Vaccine等发表论文7篇。