Identification of antigenic epitopes in the haemagglutinin protein of H7 avian influenza virus

The H7 subtype avian influenza virus (AIV) has been reported to infect not only poultry but also humans. The haemagglutinin (HA) protein is the major surface antigen of AIV and plays an important role in viral infection. In this study, five monoclonal antibodies (mAbs, 2F8, 3F6, 5C11, 5E2 and 5C12) against the HA protein of H7 virus were produced and characterized. Epitope mapping indicated that 103RESGSS107 was the minimal linear epitope recognized by the mAbs 2F8/3F6/5C11, and mAbs 5E2/5C12 recognized the epitope 103-145aa. The protein sequence alignment of HA indicated that the two epitopes were not found in other subtypes of AIV, and none of the five mAbs cross-reacted with other subtypes, suggesting these mAbs are specific to H7 virus. The epitope 103RESGSS107 was highly conserved among Eurasian lineage strains of H7 AIV, whereas three amino acid substitutions (E104R, E104K and E104G) in the epitope occurred in 98.44% of North-American lineage strains. Any of these single mutations prevented the mutated epitope from being recognized by mAbs 2F8/3F6/5C11; thus, these mAbs can distinguish between Eurasian and North-American lineages of H7 strains. Furthermore, the mAbs 2F8, 3F6 and 5C11 could be highly blocked with H7-positive serum in blocking assays, revealing that 103RESGSS107 may be a dominant epitope stimulating the production of antibodies during viral infection. These results may facilitate future investigations into the structure and function of HA protein, as well as surveillance and detection of H7 virus. RESEARCH HIGHLIGHTSFive mAbs against HA protein of H7 AIV were generated and characterized.Two novel epitopes 103RESGSS107 and 103-145aa were identified.The epitope 103RESGSS107 differs between Eurasian and North-American lineages.The mAbs 2F8, 3F6 and 5C11 could distinguish two lineages of H7 strains. Five mAbs against HA protein of H7 AIV were generated and characterized. Two novel epitopes 103RESGSS107 and 103-145aa were identified. The epitope 103RESGSS107 differs between Eurasian and North-American lineages. The mAbs 2F8, 3F6 and 5C11 could distinguish two lineages of H7 strains.H7亚型禽流感病毒（avian influenza virus, AIV）已被证实可感染家禽与人类。血凝素（haemagglutinin, HA）蛋白是AIV的主要表面抗原，在病毒感染过程中发挥关键作用。本研究制备并鉴定了5株针对H7病毒HA蛋白的单克隆抗体（monoclonal antibodies, mAbs，编号依次为2F8、3F6、5C11、5E2及5C12）。表位作图结果显示，103RESGSS107是mAbs 2F8、3F6、5C11识别的最小线性表位，而mAbs 5E2、5C12识别的表位为103-145位氨基酸。HA蛋白序列比对结果表明，上述两个表位均未在其他亚型AIV中被发现，且5株mAbs均未与其他亚型病毒发生交叉反应，提示这些mAbs对H7病毒具有高度特异性。103RESGSS107表位在欧亚分支H7 AIV毒株中高度保守，而北美分支H7 AIV毒株中有98.44%的该表位发生了3种氨基酸替换（E104R、E104K及E104G）。上述任意单一位点突变均可使突变后的表位无法被mAbs 2F8、3F6、5C11识别，因此这些mAbs可区分H7病毒的欧亚与北美分支。此外，阻断试验结果显示，mAbs 2F8、3F6及5C11可被H7阳性血清高效阻断，表明103RESGSS107可能是病毒感染过程中刺激抗体产生的优势表位。本研究结果可为后续HA蛋白的结构与功能研究，以及H7病毒的监测与检测工作提供重要支撑。研究亮点：制备并鉴定了5株针对H7 AIV HA蛋白的单克隆抗体；鉴定出两个全新表位：103RESGSS107与103-145位氨基酸；103RESGSS107表位在欧亚分支与北美分支H7毒株中存在序列差异；mAbs 2F8、3F6及5C11可区分H7病毒的两个分支。制备并鉴定了5株针对H7 AIV HA蛋白的单克隆抗体；鉴定出两个全新表位：103RESGSS107与103-145位氨基酸；103RESGSS107表位在欧亚分支与北美分支H7毒株中存在序列差异；mAbs 2F8、3F6及5C11可区分H7病毒的两个分支。