3.3 The amino acid at position 158 in HA was major determinant for antigenic alteration of EA H1N1 SIVs.
Sequence alignment revealed that the HA1 proteins of GX/18 and GD/104 viruses differed by 25 amino acids (Figure 2A). And nine of them have been reported to be located on the antigenic sites Sa (blue), Sb (yellow) and Ca2 (red)(Jin et al., 2005, Tsibane et al., 2012, Koel et al., 2013), as shown in the stimulated 3D structure of GX/18 HA protein generated by using SWISS-MODEL (www. swissmodel.expasy.org) (Waterhouse et al., 2018)and PyMOL software (Figure 2B). To determine whether these nine amino acids contribute to the different antigenicity of GX/18 and GD/104 viruses, we generated four chimeric viruses in the GX/18 background. rGX/18-GDpool contained all the nine different amino acids of GD/104 virus, rGX/18-GDSa contained K156Q, G158E, and K166N mutations in Sa site, rGX/18-GDSb contained T187N, D188Y, S189R, and T193A mutations in Sb site, and rGX/18-GDCa2 contained S138A and N145K mutations in Ca2 site. The cross-reactivity of the four recombinant viruses with the mAb102-95 were evaluated. As shown in Figure 3, the reactive titers of rGX/18-GDSb and rGX/18-GDCa2 viruses with mAb102-95 were comparable to those of the parental rGX/18 virus and reached 1:2,560. However, the reactive titers of rGX/18-GDpool and rGX/18-GDSa were 8~32-fold lower than those of the parental rGX/18 virus and only reached 1:160 and 1:640, respectively. These results demonstrated that the nine different amino acids, especially the Sa antigenic site, played critical roles in viral antigenic drift.
Sa antigenic sites of the GX/18 and GD/104 viruses differed by three amino acids at positions 156, 158, and 166. To further refine the impact of specific mutation on antigenicity, the amino acid substitutions K156Q, G158E, and K166N were individually introduced into the GX/18 virus, and their cross-reactivity with mAb102-95 were assessed by HI tests. As shown in Figure 3, rGX/18-HA-K156Q and rGX/18-HA-K166N viruses had the similar reactive patterns with the parental rGX/18 virus, and the HI titers were comparable to the homologous ones. However, the reactive titer of rGX/18-HA-G158E with mAb102-95 was 8-fold lower than the homologous titer and only reached 1:640. Additionally, the E158G mutation substantially increased the cross-reactivity of rGD/104-HA-E158G with the mAb102-95 and the HI titer was up to 1:5,120. These results further indicated that a single amino acid substitution at position 158 in HA protein significantly affected viral antigenicity.