H5N6禽流感神经氨酸酶人源抗体筛选与抗病毒机制研究

The highly pathogenic avian influenza virus (HPAIV) H5N6 has a case fatality rate greater than 65%, which is a serious hazard to human health. Neuraminidase (NA) is a type of important functional glycoproteins found on the surface of influenza viruses, the discovery of broadly protective antibodies against the NA protein in recent years is creating a promising direction for the development of high-efficacy anti-influenza drugs and vaccines. So far, there are no reports on the study about the antibodies against the HPAIV-H5N6-NA, it is currently unclear how to induce this kind of antibodies, how strong the antiviral activities of these antibodies in vitro and in vivo can be and which mechanisms the antibodies exert to inhibit viruses. Therefore, it is of great importance to conduct related studies on the HPAIV-H5N6-NA antibodies. In our previous studies, we have collected abundant clinical specimens such as PBMCs and sera from the patients infected with HPAIV-H5N6, established an efficient technical platform for the development of human antibodies. And we have obtained two HPAIV-H5N6-NA specific human antibodies by using this technical platform. This proposal aims to continue to raise more human antibodies with effective antiviral activity against NA of HPAIV-H5N6, characterize the antiviral activities of these antibodies in vitro and in vivo, and illuminate the antiviral mechanisms of them. The expected results of this study will facilitate the development of novel antiviral drugs and vaccines against HPAIV-H5N6.

H5N6亚型高致病性禽流感病毒（HPAIV-H5N6）人感染致死率超过65%，严重危害人类健康。神经氨酸酶（NA）是流感病毒的重要功能性膜蛋白，近年来发现的识别NA蛋白的广谱保护性抗体为研制抗流感高效药物和疫苗指明新方向。至今尚无HPAIV-H5N6-NA特异性抗体报道，该类抗体的诱导规律和体内外抗病毒活性如何、通过何种作用机制发挥抗病毒作用等科学问题，目前均不明确，因此，HPAIV-H5N6-NA特异性抗体的相关研究具重要的科学意义。前期研究中申请人团队已积累了丰富的H5N6感染患者的PBMCs和血清等标本、建立了人源抗体制备技术平台并已筛选到两株HPAIV-H5N6-NA人源抗体，本研究拟在此基础上，进一步筛选高抗病毒活性的HPAIV-H5N6-NA人源单抗，系统研究其体内外抗病毒活性，阐明其抑制病毒的具体作用机制，为HPAIV-H5N6新型抗病毒药物和高效疫苗研究提供科学依据。

流感病毒严重危害人类健康，季节性流感病毒每年在全球造成数百万重症病例，其中数十万人死亡，H5N6亚型高致病性禽流感病毒人感染致死率超过65%，近年来发现的识别NA蛋白的广谱保护性抗体为研制抗流感高效药物和疫苗指明新方向。高致病性禽流感特异性抗体的相关研究具重要的科学意义。成功建立起基于抗原特异性单B细胞分选技术和总B细胞培养的两项全人源抗体制备平台，基于该平台，对多份H5N6、H1N1流感病毒感染患者和数位健康志愿者的外周血单核细胞（PBMCs）试验标本开展抗原特异性全人源抗体的制备工作。成功制备出八株特异性针对H5N6-NA的全人源抗体，其中，抗体TH003-7-4B和TH003-18-14D具良好的抗病毒活性；应用酶联免疫吸附试验（ELISA），证实抗体TH003-7-4B和TH003-18-14D对H5N6毒株的NA蛋白和超离纯化病毒均具良好的反应性，而对其他亚型的流感病毒的NA蛋白和超离纯化病毒未显示出特异反应性，说明该两株抗体为H5N6 NA蛋白特异性抗体。在基于补体的细胞毒性试验中（CDC），抗体TH003-7-4B和TH003-18-14D可通过CDC效应清除被A/Yunnan/DQ001/2015病毒感染的细胞；在动物体内保护实验中，抗体TH003-18-14D可部分保护被毒株A/Yunnan/DQ001/2015或A/Shenzhen/Th002/2016致死剂量感染的小鼠，并在一定程度上减轻了病毒感染导致的小鼠体重的下降；而抗体TH003-7-4B则未显示出体内抗病毒保护效果。. 此外，本项目研发了一批流感病毒广谱鼠源单克隆抗体，并鉴定了其体内外抗病毒活性及机制。本研究的相关研究结果，为流感病毒新型广谱药物和疫苗的研发，提供了重要科学依据。