Abstract
In the 2014–2015 influenza season a novel neuraminidase (NA) genotype was detected in global human influenza A surveillance. This novel genotype encoded an N-linked 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 N-linked glycan has on virus fitness and antibody binding, recombinant viruses with (NA Gly+) or without (NA Gly-) the 245 NA glycan 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 glycan 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 245 NA glycan blocks the binding of monoclonal and human serum NA specific antibodies that would otherwise inhibit enzymatic activity and virus replication.
Highlights
Each year seasonal influenza accounts for 3 to 5 million incidences of severe disease and up to 650,000 deaths [1]
This mutation at position 245 through 247 in the amino acid sequence of NA encoded an N-linked glycosylation. We studied how this N-linked glycan impacts virus fitness and protein function. We found that this Nlinked glycan on the NA protein decreased viral replication fitness on human nasal epithelial cells but not immortalized Madin-Darby Canine Kidney (MDCK) cells
We determined this glycan decreases NA enzymatic activity, enzyme kinetics and affinity for substrate. We show that this N-linked glycan at position 245 blocks some NA specific inhibitory antibodies from binding to the protein, inhibiting enzymatic activity, and inhibiting viral replication
Summary
Each year seasonal influenza accounts for 3 to 5 million incidences of severe disease and up to 650,000 deaths [1]. The anti-HA protein antibodies inhibit virus entry into cells and provide an immune pressure which leads to the emergence of virus strains with mutations in HA antigenic sites [2, 3]. This antigenic drift leads to escape from vaccine- and infection-induced immunity and results in the need to change influenza vaccine strains on a fairly frequent basis. There is renewed interest in generating influenza vaccines that provide broader and stronger protection against several virus strains [4,5,6] and the other major influenza surface glycoprotein, the neuraminidase (NA) protein, has emerged as a potential candidate for such a universal influenza vaccine [6]. The neuraminidase activity removes sialic acid from host cell membrane bound proteins and viral HA and NA proteins at late times post infection, allowing viral particles to efficiently bud and spread to other respiratory epithelial cells [7, 11]
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