Measuring Site-specific Glycosylation Similarity between Influenza a Virus Variants with Statistical Certainty

Abstract

Antigenic drift in influenza A virus results in poor vaccine effectiveness. Accumulating N-linked glycosylation sites on the envelope protein hemagglutinin shield antigenic regions from adaptive immune responses. Quantitatively understanding how glycosylation similarity correlates with changes in protein sequence variation or viral expression platforms is necessary for improving vaccine design. These results presented demonstrate that a mutant strain of influenza has measurably distinct glycosylation compared to its wild-type counterpart.

Graphical Abstract

Highlights

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  Changes in N-linked glycosylation in influenza A virus affect antigenicity of the virus.

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  Glycosylation similarity can be quantified, even in heterogeneously glycosylated proteins.

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  Glycosylation is measurably and site-specifically distinct in influenza from related strains.

Abstract

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between WT IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.