Prior to each annual flu season, health authorities recommend three or four virus strains for inclusion in the annual influenza vaccine: a type A:H1N1 virus, a type A:H3N2 virus, and one or two type B viruses. Antigenic differences between strains are found in the glycosylation patterns of the major influenza virus antigen, hemagglutinin (HA). Here we examine the glycosylation patterns of seven reference antigens containing HA used in influenza vaccine potency testing. These reagents are supplied by the Center for Biologics Evaluation and Research (CBER) or the National Institute for Biological Standards and Control (NIBSC) for use in vaccine testing. Those produced in hen egg, Madin-Darby canine kidney (MDCK), and insect (Sf9) expression systems were examined. They are closely related or identical to antigens used in commercial vaccines. The reference antigens studied were used in the 2014-2015 influenza season and included A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012. Released glycan and HA-specific glycopeptide glycosylation patterns were examined. We also examined the sensitivity of the single radial immunodiffusion (SRID) potency test to differences in HA antigen glycosylation. Based on deglycosylation studies applied using standard assay procedures, the SRID assay was not sensitive to any HA antigen glycosylation status from any cell system. Mapping of glycosites with their occupying glycan to functional regions, including antigenic sites, lectin interaction regions, and fusion domains, was performed and has implications for immune processing, immune responses, and antigenic shielding. Differences in glycosylation patterns, as dictated by the cell system used for expression, may impact these functions.

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