一株针对H7N9亚型禽流感病毒血凝素蛋白茎部的单克隆抗体的表征

Characterization of a monoclonal antibody against the hemagglutinin stem of H7N9 subtype avian influenza virus

摘要:

流感病毒血凝素蛋白(hemagglutinin, HA) 的茎部区相对保守，是广谱疫苗、抗体与病毒检测制剂的重要靶点。前期研究获得了一株与H7N9亚型禽流感病毒HA蛋白茎部多肽(aa 428–452) 反应的单克隆抗体(5D3-1B5)。为系统评价其生物学特性，本研究测定了5D3-1B5的抗体效价(IgG、血凝抑制与病毒中和滴度)，鉴定了抗体识别的抗原表位与编码基因的序列及结构，并评价了抗体与不同亚型流感病毒的交叉反应性。结果显示，5D3-1B5不具有针对H7N9病毒的血凝抑制与病毒中和活性，但是与HA蛋白的反应性较强; 基于多肽的酶联免疫吸附试验与噬菌体表面展示随机多肽文库筛选的方法，鉴定了5D3-1B5识别的表位是位于HA茎部C螺旋区的⁴³¹W-⁴³³Y-⁴³⁷L基序，且含有该表位突变的重组H7N9病毒失去了与抗体的反应性; 测定了抗体可变区的基因序列并定位了轻链与重链可变区的互补决定区; 抗体可变区的结构模拟与分子对接则验证了抗体与HA蛋白茎部C螺旋区的结合; 值得注意的是，5D3-1B5与group 1和2不同亚型的流感病毒具有广谱反应性。因此，5D3-1B5具有作为流感病毒广谱检测与抗体治疗制剂的应用潜力，研究结果也为认识H7N9亚型禽流感病毒HA蛋白的抗原表位特征提供了新的信息。

关键词:禽流感病毒;血凝素蛋白茎部;单克隆抗体;抗原表位;交叉反应性

Abstract:

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428–452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (⁴³¹W-⁴³³Y-⁴³⁷L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.

Key words:avian influenza virus;hemagglutinin stem;monoclonal antibody;antigenic epitope;cross-reactivity