Supporting data for "Egg-adaptive mutations and antigenicity shaped by natural variants on hemagglutinin: improving the design of seasonal influenza (H3N2) vaccine"

Seasonal influenza vaccines have been developed for more than half a century and considered to be the most cost-effective approach to prevent influenza or associated severe outcomes. Nevertheless, the vaccine effectiveness ranges from only 40% to 60% when vaccine virus matches the circulating viruses well and varies between seasons. The lowest vaccine effectiveness can often be seen in protection against human H3N2 virus. This can be partially due to egg-adaptive mutations in egg-based vaccines, which remain the prevalent type of vaccine formulation compared to cell-based and recombinant hemagglutinin, the major antigen of virus envelope. Egg-adaptive mutations facilitate the binding to α2,3-linked sialylated glycan receptors in eggs, via the receptor-bindings site of hemagglutinin that overlaps with several antigenic sites under higher selection pressure, for higher viral yield. Thus, the occurrence of egg-adaptive mutations can potentially alter the immunogenicity and result in the reduction of vaccine effectiveness. Identifying factors that influence the preference of egg-adaptive mutation can help optimize the effectiveness of egg-based influenza vaccine.During the past evolution of human H3N2 viruses, antigenic drifts were found to be accompanied by changes of hemagglutinin structure and receptor binding mode influenced by several natural mutations adjacent to the receptor-binding site. These changes could also subsequently influence the fitness of adaptive mutations. As little is known about the antigenic effects and rules of selecting egg-adaptative mutations in egg-based human H3N2 vaccines, this study aims to investigate the mechanism behind egg-adaptation based on the natural evolution of hemagglutinin in recent human H3N2 virus.In this study, I found that recently emerged egg-adaptive mutations contributed to an altered immunogenicity and antigenicity of human H3N2 vaccine in a mouse model. Using deep mutational scanning and mutagenesis experiments, the preference of egg-adaptive mutations was found to be strain or clade-dependent and constrained by natural variants on hemagglutinin of human H3N2 virus, making it predictable when selecting vaccine virus. However, the preference could change with the evolution of hemagglutinin in circulating influenza viruses. Using X-ray crystallography and glycan array, two coevolving natural mutations on hemagglutinin were further identified to cause major changes of receptor binding mode in more recent human H3N2 virus. The coevolution was mediated by the epistatic interaction between them, which coordinates to maintain the functional receptor binding, restrict the preference of egg-adaptive mutations, and possibly contribute to major antigenic changes. By neutralization assay on plasma from human, potential antigenic mismatch was indicated by the overall low capacity in neutralizing recent human H3N2 viruses. Such mismatch also involved the coevolving natural variants identified above.Altogether, this study reveals that preference of egg-adaptative mutations is constrained by natural variants on hemagglutinin and changes as these natural variants evolve. At least two natural variants coevolve to cause major changes of receptor binding mode, determine the preference of egg-adaptive mutations, and possibly immune escape. These highlight the important of intensive surveillance on natural mutations for selecting better vaccine strains.

季节性流感疫苗的研发已有半个多世纪历史，被认为是预防流感及其相关严重病症最具成本效益的手段。然而，当疫苗病毒与流行病毒匹配度较高时，疫苗有效性仅为40%至60%，且不同流感季的有效性存在差异；其中针对人类H3N2病毒的保护效力往往最低。这一现象部分源于鸡胚疫苗中的蛋适应性突变（egg-adaptive mutations）：相较于细胞基疫苗与重组血凝素（hemagglutinin，病毒包膜的主要抗原），鸡胚疫苗仍是当前主流的疫苗剂型。蛋适应性突变通过血凝素的受体结合位点（该位点与多个抗原位点存在重叠，且处于较高选择压力下），促进病毒结合鸡胚内的α2,3-连接唾液酸化糖蛋白受体，以提升病毒滴度。因此，蛋适应性突变的出现可能改变疫苗的免疫原性，进而导致疫苗有效性下降。明确影响蛋适应性突变偏好性的因素，有助于优化鸡胚流感疫苗的保护效果。在人类H3N2病毒的既往进化过程中，抗原漂移现象往往伴随血凝素结构与受体结合模式的改变，而这类改变受受体结合位点附近的若干自然突变调控；上述变化后续还会影响适应性突变的病毒适配性。由于目前对鸡胚来源人类H3N2疫苗中蛋适应性突变的抗原效应及选择规则尚不清楚，本研究旨在基于近期人类H3N2病毒血凝素的自然进化，探究蛋适应性背后的分子机制。本研究发现，新近出现的蛋适应性突变可改变小鼠模型中人类H3N2疫苗的免疫原性与抗原性。借助深度突变扫描（deep mutational scanning）与诱变实验，本研究发现蛋适应性突变的偏好性具有毒株或进化枝（clade）依赖性，且受人类H3N2病毒血凝素上的自然变异体约束，这使得疫苗毒株的筛选具备可预测性。但该偏好性可随流行流感病毒血凝素的进化发生改变。借助X射线晶体学与糖芯片（glycan array）实验，本研究进一步鉴定出血凝素上的两个协同进化自然突变，其可在近期人类H3N2病毒中引发受体结合模式的显著改变。该协同进化由二者之间的上位性互作（epistatic interaction）介导，二者协同维持功能性受体结合能力，约束蛋适应性突变的偏好性，并可能促成显著的抗原性改变。通过对人类血浆开展中和实验，研究发现其中和近期人类H3N2病毒的整体能力较弱，提示存在潜在的抗原错配；该错配同样与上述鉴定出的协同进化自然变异体相关。综上，本研究揭示：蛋适应性突变的偏好性受血凝素上的自然变异体约束，并随这些变异体的进化发生改变；至少有两个自然变异体通过协同进化引发受体结合模式的显著改变，进而决定蛋适应性突变的偏好性，且可能介导免疫逃逸。上述发现凸显了加强自然突变监测以筛选更优疫苗毒株的重要性。