Antigenic evolution of human influenza H3N2 neuraminidase is constrained by charge balancing

As one of the main influenza antigens, neuraminidase (NA) in H3N2 virus has evolved extensively for more than 50 years due to continuous immune pressure. While NA has emerged as an effective vaccine target recently, biophysical constraints on the antigenic evolution of NA remain largely elusive. Here, we apply deep mutational scanning to characterize the local fitness landscape in an antigenic region of NA in six different human H3N2 strains that were isolated around 10 years apart. The local fitness landscape correlates well among strains and the pairwise epistasis is highly conserved. Our analysis further demonstrates that local net charge governs the pairwise epistasis in this antigenic region. In addition, we show that residue coevolution in this antigenic region can be predicted by charge states and pairwise epistasis. Overall, this study demonstrates the importance of quantifying epistasis and the underlying biophysical constraint for building a predictive model of influenza evolution.