Temporal changes in sialoglycan binding profile of human H3N2 influenza viruses

The human H3N2 influenza viruses was introduced into the human population in 1968 as a novel pandemic virus that emerged through genetic reassortment between human H2N2 and avian influenza virus of H3 subtype. A switch in receptor binding specificity is a critical step for influenza interspecies transmission, as avian influenza viruses typically recognize alpha 2, 3-linked sialiosides while human influenza viruses recognize alpha 2,6-linked sialosides. Further, the receptor binding domain (RBD) that mediates binding to sialosides is located at the head of hemagglutinin (HA) protein and is surrounded by antigenic sites targeted by antibodies. As such, the RBD of H3N2 viruses may continue to evolve under the pressure of host humoral response as well as the need to optimize binding to sialosides that are present in human airway epithelial cells. The development of glycan array platform has enabled us to efficiently assess influenza binding profiles to various glycans. Analyzing the temporal changes of the receptor binding profile of human H3N2 influenza virus will help us to understand viral evolutionary path under the complex selection pressures posed by the host, and if there is any potential evolutionary constrain for these viruses over time. Here, twenty-three vaccine or vaccine-like strains and 6 animal isolates were examined binding properties using a synthetic glycan microarray. Our results showed dynamic changes in receptor recognition profile and a gradual decline in the number of glycans that can be recognized by human H3N2 influenza viruses from 1975 to 2018. Importantly, four linear glycans (6’SLN, 6’SLN+sulfate, 6’SLN2-L, and 6’SLN3-L) were commonly recognized by H3N2 human influenza viruses from 1975 to 2018, with the recent H3N2 isolates showing an increased binding affinity for the longer linear glycans 6’SLN2-L and 6’SLN3-L. We also compared the glycan binding pattern of 6 pairs of cell-grown and egg-grown human H3N2 isolates. Our results confirmed that the egg-grown viruses showed distinct receptor binding patterns than their cell-grown counterparts due to egg adaptive mutations at residues xxx and xxx that may change HA antigenicity as recently reported.Glycan microarray may be used as a risk assessment tool for profiling of influenza virus receptor-binding specificity of zoonotic influenza viruses. With the aim for constructing a “Smart Glycan Array”, we also determining the receptor binding profiles of swine and avian influenza viruses of H3N2 subtype. Three swine H3N2 viruses isolated in 2003, 2011, 2018 similarly recognized the four linear glycans as the human H3N2 viruses. Notably, the swine viruses also showed binding for alpha 2,6-linked multi-branched N-glycans that was only recognized by early human H3N2 strains. In contrast, three avian influenza viruses isolated from 2009, 2014, and 2018 showed comparable binding to alpha 2,3-linked sialosides. In conclusion, these findings suggest a gradual decline in glycan species used by human H3N2 influenza viruses over time. Further investigation on the biological function and the distribution of the four N-linked α2–6-sialosides favored by the human seasonal H3N2 viruses will help us to better define the tropism of human H3N2 viruses at human airway.

1968年，人类H3N2流感病毒作为由人类H2N2流感病毒与H3亚型禽流感病毒通过基因重排产生的新型流感大流行病毒，首次进入人类群体。流感病毒跨物种传播的关键步骤之一是受体结合特异性的转变，因为禽流感病毒通常识别α2,3连接的唾液酸苷，而人类流感病毒则识别α2,6连接的唾液酸苷。此外，介导与唾液酸苷结合的受体结合域（RBD）位于血凝素（HA）蛋白的头部，并围绕着抗体靶向的抗原位点。因此，在宿主体液反应的压力以及优化与存在于人类气道上皮细胞中的唾液酸苷结合的需求下，H3N2病毒的RBD可能会持续进化。糖蛋白阵列平台的发展使我们能够高效地评估流感病毒对各种糖蛋白的结合特性。分析人类H3N2流感病毒受体结合谱的时序变化，有助于我们理解病毒在宿主复杂选择压力下的进化路径，以及这些病毒在时间上的潜在进化限制。在此，我们通过合成糖蛋白微阵列对二十三个疫苗或类似疫苗株和六个动物分离株的绑定特性进行了研究。我们的结果显示，从1975年到2018年，人类H3N2流感病毒的受体识别谱发生了动态变化，能够被人类H3N2流感病毒识别的糖蛋白数量逐渐减少。值得注意的是，从1975年到2018年，H3N2人类流感病毒普遍识别了四种线性糖蛋白（6’SLN、6’SLN+硫酸盐、6’SLN2-L和6’SLN3-L），最近的H3N2分离株显示出对较长的线性糖蛋白6’SLN2-L和6’SLN3-L的增强结合亲和力。我们还比较了6对细胞培养和鸡蛋培养的人类H3N2分离株的糖蛋白结合模式。我们的结果证实，由于在残基xxx和xxx处的蛋适应突变可能改变HA抗原性，鸡蛋培养的病毒与细胞培养的对应病毒显示出不同的受体结合模式。糖蛋白微阵列可能被用作一种风险评估工具，用于分析人畜共患病流感病毒的受体结合特异性。为了构建“智能糖蛋白阵列”，我们还确定了猪和禽H3N2亚型流感病毒的受体结合谱。2003年、2011年和2018年分离的三个猪H3N2病毒与人类H3N2病毒相似地识别了四种线性糖蛋白。值得注意的是，猪病毒还显示出对仅被早期人类H3N2株识别的α2,6连接的多分支N糖蛋白的结合。相比之下，2009年、2014年和2018年分离的三个禽流感病毒显示出与α2,3连接的唾液酸苷的相似结合。总之，这些发现表明，人类H3N2流感病毒所使用的糖蛋白种类随着时间的推移而逐渐减少。进一步研究人类季节性H3N2病毒所偏爱的四种N连接α2–6唾液酸苷的生物功能和分布，将有助于我们更好地定义人类H3N2病毒在人类气道中的嗜性。