项目名称：H7N9禽流感病毒全人源单克隆抗体中和表位鉴定及中和作用机制研究

A novel avian influenza A(H7N9) virus that infects humans, which was highly pathogenic with a high mortality, aroused attention from all over the world. Currently, there is no specific vaccines or drugs against H7N9 available. We had selected three monoclonal H7N9 neutralizing antibodies from two patients recovered from H7N9 infection in Jiangsu by using specific memory B cell screening and cloning. Enzyme-linked immunosorbent assay showed that all the three human neutralizing H7N9 antibodies could recognize HA protein of H7N9. Besides, the binding was not affected by the mutant of the known RBD epitope of HA, which indicated that the binding epitope was probably a new one. Investigation on the structure of the epitope and the key amino acids is essential for understanding the pathogenesis and antibody neutralization mechanism. Therefore, we plan to analyze the crystal structure of the compound of H7N9 neutralizing antibodies and HA protein by x-ray. In addition, site-directed mutation will be applied to explore the relationship between the function and the structure. These information are critical for the avian influenza A H7N9 vaccine or drugs.

H7N9禽流感病毒以其对人类的高致病性，高病死率，而受到全球关注。目前尚无针对H7N9禽流感病毒的疫苗或药物上市。我们前期通过HA特异性B细胞的单克隆筛选技术，从两例感染H7N9禽流感病毒的病人恢复期血液中筛选到了三株具有广谱中和活性的H7N9禽流感病毒全人源单克隆抗体。通过抗体与已知关键位点突变的HA蛋白结合试验发现，突变不影响抗体与HA蛋白的结合力，提示其可能是针对病毒抗原新的结合位点。认识具有中和作用抗体的抗原结合表位之结构特征，确定其起关键作用的氨基酸位点，可从结构上阐明H7N9禽流感病毒的致病机理和抗体中和作用机制。因此，本项目拟通过X-ray晶体衍射解析H7N9禽流感病毒全人源单克隆中和抗体与HA蛋白复合物晶体结构，同时结合定点突变的手段，通过体内外实验探讨抗体表位结构与功能之间的关系，为人用H7N9禽流感疫苗或新的靶向药物设计的研发提供科学依据。

H7N9禽流感病毒以其对人类的高致病性，高病死率，而受到全球关注。我们前期通过HA特异性B细胞的单克隆筛选技术，从感染H7N9禽流感病毒的病人恢复期血液中筛选到了三株具有广谱中和活性的H7N9禽流感病毒全人源单克隆抗体。本项目组通过对其中和效力最高的一株抗体6-137抗体与HA蛋白（H7N9/Shanghai/2013）的晶体，并通过X-ray晶体衍射对晶体结构进行了解析。解析晶体结果发现6-137抗体与H7的RBD区域结合，识别唾液酸结合位点，竞争结合CD23的受体识别位点，包括190-helix, the 130-loop, and the 220-loop，深入到pocket。结合部位广泛，单个位点的RBD区域突变不能降低6-137的结合能力和中和H7病毒的能力。通过定点突变对H7N9抗原位点6个（150，167，171，203，204，240）可使6-137抗体丧失与HA蛋白的结合力。本研究深入探讨6-137抗体表位结构与功能之间的关系，为人用H7N9禽流感疫苗或治疗性抗体的研发提供科学依据。此外，6-137抗体作为针对H7的广谱中和抗体可在H7N9的诊断试剂研制方面具有较好应用前景。