Discussion
HA is the most important antigenic protein of H9N2 virus, which stimulates host chicken to product the HA-specific neutralizing antibodies. HA mutations in antigenic sites promoted the virus to escape from antibody-based immune responses in host. Several studies reported HA mutations that affect the antigenic variation of H9N2 virus. In these studies, HA mutations from different H9N2 antigenic variants are mainly located at or near the receptor binding sites in HA, most of which were selected with HA-specific mAbs in vitro (Peacock et al., 2016; Jin et al., 2019; Kaverin et al., 2004; Okamatsu,Sakoda,Kishida,Isoda, & Kida, 2008; Ping et al., 2008; Wan et al., 2014; Zhu et al., 2015). Few studies on the HA mutations selected with pAbs driving from inactivated vaccine in vivo and the contribution of single HA mutation in H9N2 virus to antigenic variation or immune escape were reported. We previously identified 15 antigenic variants in HA gene passaged with or without selection pressure of H9N2 inactivated vaccine (Su et al., 2020; Jin et al., 2018). Here, the contribution of the 15 HA mutations to antigenic variation and immune escape were studied with a ≥4-fold change in HI titers of standard antiserum as significant antigenic change, which result in escape from antibodies (Klingen,Reimering,Guzmán, & McHardy, 2018; Wang et al., 2017).
The amino acids located at or around receptor binding sites of HA are the major determinants of the antigenicity of influenza virus, which were confirmed in A/H3N2 virus, avian-origin A/H5N1, swine-origin A/H3N2, and horse-origin A/H3N8(Lewis et al., 2011; Hensley et al., 2009; Lewis et al., 2016). The mutations S145N, Q164L, A168T and M224K are near the receptor binding site. The mutations A198V and Q234L are at the receptor binding site of HA (Figure 1A). In particular, the positions 145, 164, 168, and 234 on H9 were mapped as the antigenic sites by mAbs, where the mutations S145N, Q164K, A168T, and L234Q occurred (Ping et al., 2008; Kaverin et al., 2004; Wan et al., 2014; Zhu et al., 2015). All the above mutations, including S145N, Q164L, A168T, M224K, A198V, and Q234L, had the potential to alter the antigenicity of H9N2 virus. Of these potential mutations, only A198V HA mutation caused ≥4-fold decreased readouts of HI titers or cross-MN titers. The receptor binding site in HA of H9N2 virus includes the residues at positions 109, 161, 163, 191, 198, 202 and 203, of which all are conservative except the residues at the position 198. About 90% of H9N2 wild viruses in China possess V or T at position 198 in HA (R. Zhu et al., 2018). Additionally, the A198V mutation occurred in all of the passaged viruses occurring antigenic variation in SPF chicken embryos or SPF chickens with or without homologous vaccine antibodies (Su et al., 2020; Jin et al., 2018), suggesting that the HA mutation A198V played a key role in the process of adaptive evolution of F/98 strain.
HI or MN titers were determined by viral antigenically distinct or viral receptor binding avidity. Li et al. (Li et al., 2013c) found single N145K mutation in H3 increased viral receptor binding, and did not prevent anti-145N sera binding. Single K145N mutation in H3 reduced reactivity to anti-145K sera, which consistent with the report of Doud et al. (Doud,Hensley, & Bloom, 2017) that only some specific amino acid residue at antigenic sites can alter the viral antigenicity. In our study, A198V substitution promoted viral escape from pAbs-neutralizing reaction by enhancing the receptor binding activity significantly while A or V at the position 198 in HA did not prevent virus-antibody binding. In virus elution assay, we found that the mutation A198V initially inhibited the virus elution from the red blood cell surface, and subsequently the elution of the virus rF/HAA198V was similar to that of the F/98 strain. As further data shown, the mutation A198V with high receptor avidity in HA also led to an increase in NA activity significantly, whereas the enhanced NA activity did not enough maintain the functional balance between HA and NA. The strong receptor binding avidity prevented the release of the virus rF/HAA198V from cells, but did not affect viral entry into host cells and infectivity, which was unexpected. These results suggested that the strong receptor binding avidity is not conducive to maintain the functional balance between HA receptor binding affinity and NA activity or the viral release from cell surface. Naturally, to restore the viral fitness, the compensatory mutations that increase avidity or modulate NA function are required (Das et al., 2011). That is why HA receptor binding avidity drove AIV antigenic drift (Hensley et al., 2009b), which might be a potential biological significance of the mutation A198V in the evolution of H9N2 virus.
Hensley et al. reported that a positive correlation was shown between receptor binding avidity and escape from antiserum (Hensley et al., 2009b). Sealy et al. characterized H9N2 viruses isolated in Pakistan during 2014-2016 and found association of increased receptor binding avidity with escape from antibody-based immunity (Sealy et al., 2018). So we postulated that A198V mutation should contribute viral immune escape in vivo . Expectedly, we found that the antibody induced by the F/98 vaccine could provide 100% protection against the challenge by the F/98 virus, and 83.3% protection against the challenge by the rF/HAA198V virus, and the antibody induced by the rF/HAA198V vaccine could provide 83.3% protection against the challenge by either the F/98 virus or the rF/HAA198V virus. Thus, although single A198V mutation slightly affected cross-protection in vivo , it broke down the full protective efficiency of the inactivated vaccine of paternal F/98 virus, suggesting that single A198V mutation with strong receptor binding avidity facilitated the virus rF/HAA198V to escape from the antibodies induced by the vaccine of the paternal virus F/98 or its own. The slight contribution of A198V to viral immune escape might be related to the phenomenon that homologous vaccine antibodies could not provide acceptable protection for vaccinated chicken flocks in recent years (Li et al., 2019b).
In summary, our finding revealed that the mechanism of A198V mutation promoting virus escape from pAbs in vitro , and the contribution to escape from the antibody-based immunity. A198V substitution promoted H9N2 virus escape from pAbs-neutralizing reaction by enhancing the receptor binding activity and did not prevent Abs binding physically, which led to the decreased readouts of cross-MN titers significantly whereas slightly affected cross-protection in vivo . These data remind us that the HA receptor binding avidity, antibody-virus binding and cross-protection should be taken into account synthetically when we monitor the new emerging of the antigenic variation.
Funding: This research was funded by the National Natural Science Foundation of China (32172802, 31672516, 31172300, 30670079), supported by the Grant No. BE2016343 from Jiangsu province, the Jiangsu University and College Natural Science Foundation (12KJA230002), the Doctoral Program of Higher Education of China (20133250110002) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Conflicts of Interest: All the authors have declared that no conflicts of interest exist regarding to the publication of the data in this manuscript.