Neuraminidase antigenic drift of Influenza A virus H3N2 clade 3c.2a viruses alters virus replication, enzymatic activity and inhibitory antibody binding

In the 2014-2015 influenza season a novel neuraminidase (NA) genotype emerged in the Johns Hopkins Center of Excellence for Influenza Research and Surveillance (JH CEIRS) surveillance network as well as globally. This novel genotype encoded a glycosylation site at position 245-247 in the NA protein from clade 3c.2a H3N2 viruses. In the years following the 2014-2015 season, this novel NA glycosylation genotype quickly dominated the human H3N2 population of viruses. To assess the effect this novel glycosylation has on virus fitness and antibody binding, recombinant viruses with (NA Gly+) or without (NA Gly-) the novel NA glycosylation were created. Viruses with the 245 NA Gly+ genotype grew to a significantly lower infectious virus titer on primary, differentiated human nasal epithelial cells (hNEC) compared to viruses with the 245 NA Gly-genotype, but growth was similar on immortalized cells. The 245 NA Gly+ blocked human and rabbit monoclonal antibodies that target the enzymatic site from binding to their epitope. Additionally, viruses with the 245 NA Gly+ genotype had significantly lower enzymatic activity compared to viruses with the 245 NA Gly-genotype. Human monoclonal antibodies that target residues near the 245 NA glycosylation were less effective at inhibiting NA enzymatic activity and virus replication of viruses encoding an NA Gly+ protein compared to ones encoding NA Gly-protein. Additionally, a recombinant H6N2 virus with the 245 NA Gly+ protein was more resistant to enzymatic inhibition from convalescent serum from H3N2-infected humans compared to viruses with the 245 NA Gly-genotype. Finally, the 245 NA Gly+ protected from NA antibody mediated virus neutralization. These results suggest that while the 245 NA Gly+ decreases virus replication in hNECs and decreases enzymatic activity, the glycosylation blocks the binding of monoclonal and human serum NA specific antibodies that would otherwise inhibit enzymatic activity and virus replication. Author Summary Influenza virus infects millions of people worldwide and leads to thousands of deaths and millions in economic loss each year. During the 2014/2015 season circulating human H3N2 viruses acquired a novel mutation in the neuraminidase (NA) protein. This mutation has since fixed in human H3N2 viruses. This mutation at position 245 through 247 in the amino acid sequence of NA encoded an N-linked glycosylation. Here, we studied how this N-linked glycosylation impacts virus fitness and protein function. We found that this N-linked glycosylation on the NA protein decreased viral replication fitness on human nasal epithelial cells (hNEC) but not immortalized Madin-Darby Canine Kidney (MDCK) cells. We also determined this glycosylation decreases NA enzymatic activity, enzyme kinetics and affinity for substrate. Furthermore, we show that this N-linked glycosylation at position 245 blocks some NA specific inhibitory antibodies from binding to the protein, inhibiting enzymatic activity, and inhibiting viral replication. Finally, we showed that viruses with the novel 245 N-linked glycosylation are more resistant to convalescent human serum antibody mediated enzymatic inhibition. While this 245 N-linked Glycan decreases viral replication and enzymatic activity, the 245 N-linked glycosylation protects the virus from certain NA specific inhibitory antibodies. Our study provides new insight into the function of this dominant H3N2 NA mutation and how it impacts antigenicity and fitness of circulating H3N2 viruses.