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

Abstract

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 recently emerged as an effective vaccine target, biophysical constraints on the antigenic evolution of NA remain largely elusive. Here, we apply combinatorial mutagenesis and next-generation sequencing 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 is correlated with the pairwise epistasis between charge states. Overall, this study demonstrates the importance of quantifying epistasis and the underlying biophysical constraint for building a model of influenza evolution.

Prevalence and mechanisms of evolutionary contingency in human influenza H3N2 neuraminidase

“…These observations substantiate the involvement of epistasis in NA antigenic drift, which corroborates with our recent study on a seven-residue antigenic region that contains residues 344 and 369 (Wang et al, 2021). Specifically, our recent study shows that charge balancing gives rise to epistasis in the antigenic evolution of NA (Wang et al, 2021). Such result is highly parallel to the specific epistasis between K385N and N387K in the present study, which is governed by non-additive stability effect due to electrostatic interaction.…”

“…Moreover, mutations at residue 344, which locates at antigenic region II, can lead to antibody escape (Air et al, 1985; Colman et al, 1983). These observations substantiate the involvement of epistasis in NA antigenic drift, which corroborates with our recent study on a seven-residue antigenic region that contains residues 344 and 369 (Wang et al, 2021). Specifically, our recent study shows that charge balancing gives rise to epistasis in the antigenic evolution of NA (Wang et al, 2021).…”

“…The NA head domains of SD93 and Mos99 differed by 18 mutations (Figure S1C). Since multiple mutations may be required simultaneously to restore the fitness of N387K in SD93, we have decided to use a high-throughput approach that coupled combinatorial mutagenesis and next-generation sequencing (Wang et al, 2021;Wu et al, 2018Wu et al, , 2020 to identify permissive mutations for N387K. Briefly, we constructed a mutant library for SD93 that contained all possible combinations of 10 mutations (n = 2 10 = 1024 variants), namely E248G, R249K, I265T, Y336H, R338L, N339D, K344E, S346G, E369K, and G381E (Figure 2C), in the genetic background of N387K.…”

“…The natural function of NA is to cleave the sialic acids from cellular receptors to facilitate virus release and from newly synthesized HA to prevent virion aggregation (McAuley et al, 2019). After entering the human population in 1968, H3N2 has accumulated around 75 amino acid mutations in NA, which account for 16% of the entire protein (Wang et al, 2021). Such an extensive evolution of NA can at least be partly attributed to epistasis, where the fitness of a mutation depends on genetic background.…”

“…Such an extensive evolution of NA can at least be partly attributed to epistasis, where the fitness of a mutation depends on genetic background. For example, our recent study indicates that the emergence of mutations in an antigenic region of human H3N2 NA is contingent on the evolution of other parts of the sequence (Wang et al, 2021). Similarly, the emergence of oseltamivir resistance in seasonal H1N1 virus is contingent on permissive mutations (Abed et al, 2011; Bloom et al, 2010; Duan et al, 2014).…”