抗流感病毒N9 抗体结构、遗传基础及功能研究

The neuraminidase of influenza virus is the targeting glycoprotein of the only anti-viral drugs available in clinical practice. This viral antigen could also induce humoral response in host. Via inhibiting viral releasing from infected cells, the clinical severity could be alleviated by anti-NA antibodies while the infection might deteriorate due to the antibody-dependent enhancement (ADE) in influenza. Although the importance of anti-heamagglutinin(HA) immunity has been extensively investigated, far less is known about NA epitopes of anti-NA as compared to anti-HA, as well as their structure and genetic basis and function. Previously, we discovered a high titter of anti-N9 in the patients infected with avian influenza A (H7N9) even early on day 7 after illness onset. Some of the anti-N9 monoclonal antibodies could cross-react with H7 protein. The antibodies use some specific genes, such as VH1-69, VH3-23, VH4-34, VH4-4, VK3-20 and VK1-5. Here, we will define the epitopes of the cross-reactive anti-N9 antibody, their structure and gnetic basis and functions to develop a potential anti-viral strategy.

流感病毒神经氨酸酶(NA)是目前临床主要抗病毒药物的靶向蛋白。该蛋白也是重要抗原，能诱导机体的抗体应答。抗NA抗体可抑制病毒释放减轻临床严重程度，但也能通过抗体依赖的感染增强效应（ADE）加重病情。较病毒血凝素（HA）抗体,对NA抗体的表位、结构及功能认识还不足。我们发现，在发病7天，禽流感病毒H7N9患者体内便能检测到高滴度的抗N9抗体，部分抗N9单抗也能结合H7抗原，且抗体编码基因存在一定偏好性。本研究将进一步明确抗N9抗体识别的抗原表位、 结构特点及功能，从而推动新型抗病毒策略的发展。

流感病毒神经氨酸酶(NA)是目前临床主要抗病毒药物的靶向蛋白。该蛋白也是重要抗原，能诱导机体的抗体应答。抗NA抗体可抑制病毒释放减轻临床严重程度，但也能通过抗体感染增强效应（ADE）加重病情。较病毒血凝素（HA）抗体,对NA抗体的结构、遗传及功能认识还不足。本课题研究了1)季节性流感病毒灭活疫苗接种者、H7N9患者血清抗N9抗体；2)季节性流感病毒灭活疫苗接种者、H7N9患者外周血B细胞的抗H7、N9抗体可变区基因特点及抗体功能; 3)人源化鼠源抗N9单抗；4）H3N2感染人群抗N2血清抗体（与N9同为Group2 NA）的应答及变异。发现H7N9感染人群有血清抗H7、N9功能性抗体；流感灭活苗免疫人群几乎无血清抗N9交叉抗体，但2009H1N1疫苗接种个体有高滴度的N9应答，即能检测到血清抗N9结合性、NA酶活抑制性抗体（NI），也分离到识别H7、N2、N9抗原的两株人源单抗39BD10和39C；儿童H3N2感染者血清N2抗体应答强于成人，阳性应答率及NI抗体滴度明显高于成人；该N2抗体对既往人群中流行的季节性N2及禽流感N2交叉反应，识别N2抗原的329糖基化及E344表位，对2020年后流行的N2(E344K)不反应。获得的鼠源抗体人源化的嵌合抗N9单抗1E2和3E3，这两株抗体识别抗原表位重叠, 能抑制NA酶活及耐药N9神经氨酶活，体外细胞实验中能抑制病毒释放，阻止病毒进一步感染细胞。两株嵌合抗体能特异性识别H7N9 N9蛋白，KD值达pM。获得的人源单抗39BD10和39C，39C单抗识别H7、N2、N9抗原，具有ADE及ADCC效应，缺乏NI及中和活性，初步认识了这些抗体的特性及遗传基础。