人源抗H7、H10 流感病毒HA单克隆抗体及其中和表位研究

Group 2 influenza A viruses, avian origin H7N9 and H10N8, pose a heavy threat on public health. To date, there is still no effective vaccine available and, viral strains resistant to Nueraminidase inhibitors have emerged gradually. Human monoclonal antibodies (mAbs) against hemagglutinin (HA) of influenza virus has the huge potential for prophylaxis and treatment of group 2 virus infection in future. The neutralization ability of anti-HA mAbs depends on the conformational location and mutability of the epitopes on HA antigen. Anti-HA stem mAbs are very strain specific and quickly lose efficacy against drifted strains. In contrast, anti- HA stem mAbs are potential for broadly neutralization both group 1 and/or group 2 viruses. However, the ability and efficacy of human mAbs binding to and neutralizing group 2 HA were still unclear. There is increasing interest in antibodies that can effectively used in treatment of patients infected with group 2 virus. We recruit survivors of persons infected with H7N9 and H10N8 virus as study subjects to screen and isolate human anti-HA mAbs from their memory B cells using single cell culture. After identification of the binding capacity to group 2 HAs, we also test their neutralization effect on group 2 virus, including H3N2, H7N9 and H10N8 virus. In addition, we employ site-directed mutagenesis to predict epitopes of broadly neutralizing mAbs and potential escape mutants on HA. Thus provide scientific evidences for development of universal vaccine and antiviral strategies against group 2 influenza virus.

禽源H7N9和H10N8病毒在group 2流感病毒中最具公共卫生威胁，人源抗血凝素（HA）单克隆抗体（mAbs）具备预防和治疗group 2病毒感染的潜力。mAbs的中和能力因其表位的构象位置和突变能力不同而异。抗HA头部mAbs具高度株特异性，而HA茎部mAbs则可对group 1和/或group 2病毒具有广谱中和活性。然而，人源抗HA mAbs对group 2病毒的广谱中和活性尚不明确。本研究以自然感染H7N9、H10N8病毒的存活病例为研究对象，采用单细胞培养法筛选、鉴定和纯化人源抗HA mAbs，分析其与group 2 HA的结合能力和对group 2病毒（H3N2、H7N9、H10N8）的广谱中和活性，采用点突变技术预测mAbs的中和表位及潜在逃逸突变。从而为设计group 2 HA通用抗原、抗病毒治疗方案提供理论依据。

H7N9和H10N8流感疫情在国内均造成了较高病死率。虽然神经氨酸酶抑制剂磷酸奥司他韦（达菲）是甲型流感首选治疗药物，但耐药株业已出现和扩散，故研制备选抗体药物或被动免疫疗法是应对流感大流行的当务之急。本研究分选培养H7N9患者记忆性B细胞并克隆抗体基因，表达和纯化抗体筛选人源H7单抗并鉴定结合和中和活性。制备鼠源H7/H10单抗，建立双抗体夹心ELISA法（DAS-ELSIA）以快速检测H7/H10亚型禽流感病毒抗原。制备靶向H7N9病毒的人源化N9嵌合抗体，鉴定其抑制病毒释放和扩散的功能特性，以期为设计和开发具有临床诊断和治疗应用价值的HA和NA单克隆抗体提供科学依据。.通过B细胞抗体基因克隆和测序，从1#样本中获得24种VH胚系基因和14种Vκ胚系基因；从3#样本中获得17种VH胚系基因和12种Vκ胚系基因。表达纯化筛选获得5株人源单克隆抗体，抗体56B11-2/4具有神经氨酸酶抑制活性，其IC50约为52.46μg/ml；抗体10F10-13/13为广谱结合型抗体，对N9蛋白和多种亚型HA蛋白均具强结合活性。利用杂交瘤技术制备了12株H7N9和8株H10N8病毒鼠源HA单克隆抗体。利用H7单抗2B6和5E9以及H10单抗5C1和2E9，建立了高灵敏度和强特异度的H7/ H10亚型病毒抗原DAS-ELISA快检方法，对A/Guangdong/17SF003/2016(H7N9)和A/Jiangxi/346/2013(H10N8)的最低检测限均为0.5HAU/50μl，重组H7和H10蛋白的最低检测限均为10ng/ml，且均呈剂量反应关系。筛选得到3株特异性抗H7N9病毒人源N9嵌合抗体1E2，3E3及5D1，它们与AH1-N9蛋白的EC50值分别为0.048 µg/ml，0.028 µg/ml和0.675 µg/ml；1E2和3E3对AH1-N9蛋白具有极强亲和力且两者识别的抗原表位相似但与5D1识别的抗原表位不同；3株N9嵌合抗体均能抑制N9酶活及耐药N9酶活，其中3E3 的NA酶活抑制能力最强；1E2和3E3，尤其3E3可显著抑制病毒释放并阻止病毒扩散感染。.本研究制备的人源单克隆抗体10F10-13/13、1E2和3E3具有较强的流感病毒抗体药物开发应用潜力；建立的DAS-ELISA方法具有H7/H10亚型病毒快速检测及疫苗HA含量测定的应用前景。