GLYCOSYLATION SITE OCCUPANCY OF THE HEMAGGLUTININ GLOBULAR HEAD IS IMPLICATED IN THE IMMUNE RESPONSE TO INFLUENZA A VIRUS

Abstract

Abstract Objective N-linked glycosylation of the hemagglutinin (HA) affects host immune responses to influenza A virus (IAV). This study determines the significance of N-linked glycosylation site occupancy of the HA globular head in the generation of effective antibody responses to IAV in mice. Methods We grew A(H3N2)2013 IAV in the presence of an inhibitor of N-linked glycosylation, NGI-1, which reduces glycosylation in mammalian cells by targeting the oligosaccharyltransferase. Mass spectrometry-based analysis of this virus revealed reduction of glycosylation in six out of twelve HA N-linked glycosylation sites, compared to virus grown without the inhibitor. The globular head of HA was affected by NGI-1 treatment. The ability of NGI-1-treated HA to generate immune responses was assessed by immunization of naïve mice with two 10 μg HA doses followed by measurement of serum antibody titers by hemagglutination inhibition and ELISA tests. Results Reduced occupancy of N-linked glycosylation sites IAV HA globular head correlated with a significant reduction in cross-strain antibody responses. Serum antibody titers directed toward recombinant HAs from A(H3N2) IAVs of 2003–2013 were from 2 to 4 times lower in mice immunized with HA from NGI-1-treated virus than in the mice immunized with HA from the fully glycosylated virus. Similarly, titers of the antibodies that are correlated with protection against influenza disease, measured by hemagglutination inhibition assays, were significantly lower in mice immunized with NGI-treated virus than those immunized with fully-glycosylated virus. Conclusion Glycosylation site occupancy of the HA globular head affects the immune response to IAV.