The molecular basis of antigenic variation among A(H9N2) avian influenza viruses

Avian influenza A(H9N2) viruses are an increasing threat to global poultry production and, through zoonotic infection, to human health where they are considered viruses with pandemic potential. Vaccination of poultry is a key element of disease control in endemic countries, but vaccine effectiveness is persistently challenged by the emergence of antigenic variants. Here we employed a combination of techniques to investigate the genetic basis of H9N2 antigenic variability and evaluate the role of different molecular mechanisms of immune escape. We systematically tested the influence of published H9N2 monoclonal antibody escape mutants on chicken antisera binding, determining that many have no significant effect. Substitutions introducing additional glycosylation sites were a notable exception, though these are relatively rare among circulating viruses. To identify substitutions responsible for antigenic variation in circulating viruses, we performed an integrated meta-analysis of all published H9 haemagglutinin sequences and antigenic data. We validated this statistical analysis experimentally and allocated several new residues to H9N2 antigenic sites, providing molecular markers that will help explain vaccine breakdown in the field and inform vaccine selection decisions. We find evidence for the importance of alternative mechanisms of immune escape, beyond simple modulation of epitope structure, with substitutions increasing glycosylation or receptor-binding avidity, exhibiting the largest impacts on chicken antisera binding. Of these, meta-analysis indicates avidity regulation to be more relevant to the evolution of circulating viruses, suggesting that a specific focus on avidity regulation is required to fully understand the molecular basis of immune escape by influenza, and potentially other viruses.

甲型禽流感A(H9N2)亚型病毒（Avian influenza A(H9N2)）对全球家禽养殖业的威胁日益加剧，且可通过人畜共患感染危及人类健康，被视作具备大流行潜力的病毒。家禽免疫接种是疫区国家防控该疫病的核心举措，但抗原变异株的持续出现始终挑战着疫苗的防护效力。本研究采用多技术联用的研究策略，探究H9N2抗原变异的遗传基础，并评估不同免疫逃逸分子机制的作用。我们系统测试了已发表的H9N2单克隆抗体（monoclonal antibody）逃逸突变株对鸡抗血清结合活性的影响，发现多数突变株未产生显著作用。值得注意的例外是引入额外糖基化位点的氨基酸替换，尽管这类替换在流行毒株中相对罕见。为筛选出驱动流行毒株抗原变异的关键替换位点，我们整合所有已发表的H9血凝素（haemagglutinin，HA）序列与抗原数据，开展了集成化荟萃分析。随后通过实验验证了该统计分析结果，并为H9N2抗原位点分配了多个全新的氨基酸残基，所得分子标记可用于解释田间疫苗失效现象，同时为疫苗筛选决策提供参考。本研究发现，除直接调控表位结构的免疫逃逸机制外，其他途径同样发挥关键作用：其中，增加糖基化修饰或提升受体结合亲和力的氨基酸替换，对鸡抗血清结合活性的影响最为显著。在上述两类机制中，荟萃分析结果显示受体结合亲和力调控与流行毒株的进化关联更为紧密。这提示，若要全面解析流感病毒（乃至其他潜在病毒）免疫逃逸的分子基础，需将研究重点聚焦于亲和力调控机制。