Convergent evolution of the N156K mutation in A(H1N1)pdm09 hemagglutinin contributes to antigenic drift and cluster transition.

The continuous antigenic evolution of influenza A(H1N1)pdm09 poses a persistent challenge to vaccine efficacy. While recent viral lineages have diverged significantly from ancestral strains, the precise molecular determinants driving the latest antigenic cluster transitions remain incompletely characterized. Here, using the broadly neutralizing monoclonal antibody 32D6 as a molecular probe, we identified a critical escape mechanism of antibody evasion in recent A(H1N1)pdm09 isolates. Through pseudovirus neutralization assays and LASSO regression analysis, we identified residue 156 in the Sa antigenic site as the key determinant of escape. Furthermore, molecular dynamics (MD) simulations and MM-GBSA calculations indicated that the N156 K mutation reduces the binding free energy at the antibody-antigen interface due to steric hindrance and electrostatic repulsion. Notably, while the N156 K mutations caused a significant reduction in pseudovirus infectivity within the earlier GD19 genetic backbone, it was fully accommodated in recent lineages. Phylogenetic analysis revealed the rapid, convergent fixation of N156 K across global isolates. These findings provide insights into the mechanism of the recent GD19-to-VI19 antigenic transition and highlight the hypothesized role of compensatory mutations in sustaining viral fitness during antigenic evolution.