State Key Lab of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Guangdong Laboratory of Computational Biomedicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
J Virol. 2020 Sep 29;94(20). doi: 10.1128/JVI.00667-20.
Since the first outbreak in 2013, the influenza A (H7N9) virus has continued emerging and has caused over five epidemic waves. Suspected antigenic changes of the H7N9 virus based on hemagglutination inhibition (HI) assay during the fifth outbreak have prompted the update of H7N9 candidate vaccine viruses (CVVs). In this study, we comprehensively compared the serological cross-reactivities induced by the hemagglutinins (HAs) of the earlier CVV A/Anhui/1/2013 (H7/AH13) and the updated A/Guangdong/17SF003/2016 (H7/GD16). We found that although H7/GD16 showed poor HI cross-reactivity to immune sera from mice and rhesus macaques vaccinated with either H7/AH13 or H7/GD16, the cross-reactive neutralizing antibodies between H7/AH13 and H7/GD16 were comparably high. Passive transfer of H7/AH13 immune sera also provided complete protection against the lethal challenge of H7N9/GD16 virus in mice. Analysis of amino acid mutations in the HAs between H7/AH13 and H7/GD16 revealed that L226Q substitution increases the HA binding avidity to sialic acid receptors on red blood cells, leading to decreased HI titers against viruses containing HA Q226 and thus resulting in a biased antigenic evaluation based on HI assay. These results suggest that amino acids located in the receptor-binding site could mislead the evaluation of antigenic variation by solely impacting the receptor-binding avidity to red blood cells without genuine contribution to antigenic drift. Our study highlighted that viral receptor-binding avidity and combination of multiple serological assays should be taken into consideration in evaluating and selecting a candidate vaccine virus of H7N9 and other subtypes of influenza viruses. The HI assay is a standard method for profiling the antigenic characterization of influenza viruses. Suspected antigenic changes based on HI divergency in H7N9 viruses during the 2016-2017 wave prompted the recommendation of new H7N9 candidate vaccine viruses (CVVs). In this study, we found that the L226Q substitution in HA of A/Guangdong/17SF003/2016 (H7/GD16) increased the viral receptor-binding avidity to red blood cells with no impact on the antigenicity of H7N9 virus. Although immune sera raised by an earlier vaccine strain (H7/AH13) showed poor HI titers against H7/GD16, the H7/AH13 immune sera had potent cross-neutralizing antibody titers against H7/GD16 and could provide complete passive protection against H7N9/GD16 virus challenge in mice. Our study highlights that receptor-binding avidity might lead to biased antigenic evaluation by using the HI assay. Other serological assays, such as the microneutralization (MN) assay, should be considered a complementary indicator for analysis of antigenic variation and selection of influenza CVVs.
自 2013 年首次爆发以来，甲型流感（H7N9）病毒不断出现，并已引发超过五波疫情。第五次疫情期间，血凝抑制（HI）检测显示 H7N9 病毒的疑似抗原性发生变化，促使更新了 H7N9 候选疫苗病毒（CVV）。在本研究中，我们全面比较了较早的 CVV A/Anhui/1/2013（H7/AH13）和更新的 A/Guangdong/17SF003/2016（H7/GD16）血凝素（HA）诱导的血清学交叉反应性。我们发现，尽管 H7/GD16 与用 H7/AH13 或 H7/GD16 免疫的小鼠和恒河猴血清的 HI 交叉反应性较差，但 H7/AH13 和 H7/GD16 之间的交叉中和抗体滴度相当高。H7/AH13 免疫血清的被动转移也能为小鼠提供针对 H7N9/GD16 病毒的致死性攻击的完全保护。分析 H7/AH13 和 H7/GD16 之间 HA 中的氨基酸突变发现，L226Q 取代增加了 HA 与红细胞唾液酸受体的结合亲和力，导致针对含有 HA Q226 的病毒的 HI 滴度降低，从而导致基于 HI 检测的抗原性评估出现偏差。这些结果表明，位于受体结合位点的氨基酸可能会误导对抗原变异的评估，而仅仅通过影响红细胞的受体结合亲和力，而不真正促进抗原漂移，就会产生偏差。我们的研究强调，在评估和选择 H7N9 和其他流感病毒亚型的候选疫苗病毒时，应考虑病毒受体结合亲和力和多种血清学检测方法的结合。HI 检测是分析流感病毒抗原特征的标准方法。在 2016-2017 年波次中，H7N9 病毒的 HI 发散表明存在疑似抗原性变化，促使推荐了新的 H7N9 候选疫苗病毒（CVV）。在本研究中，我们发现 A/Guangdong/17SF003/2016（H7/GD16）HA 中的 L226Q 取代增加了病毒与红细胞的受体结合亲和力，但对 H7N9 病毒的抗原性没有影响。尽管用早期疫苗株（H7/AH13）免疫产生的血清对 H7/GD16 的 HI 滴度较差，但 H7/AH13 免疫血清对 H7/GD16 具有强大的交叉中和抗体滴度，并能为小鼠提供针对 H7N9/GD16 病毒攻击的完全被动保护。我们的研究强调，受体结合亲和力可能会导致使用 HI 检测产生偏向性的抗原评估。其他血清学检测方法，如微量中和（MN）检测，应被视为分析抗原变异和选择流感 CVV 的补充指标。
