Variation of Site-Specific Glycosylation Profiles for Influenza Glycoproteins from Different Vaccines and Recombinant Sources

Description:
Influenza is a viral disease responsible for the death of ~500,000 people annually worldwide. As a result, vaccination against influenza is one of the most common immunizations. The antigens of interest within these vaccines include the viral glycoproteins hemagglutinin and neuraminidase. The study of glycosylation on viral glycoproteins is important in order to understand immune evasion and cell binding. However, the accurate measurement of site-specific glycan distributions on glycoproteins and determination of their variability presents a significant analytical challenge. In this study, variation of glycosylation is assessed between different preparation and digestion conditions in order to determine typical levels of glycan variation, which has the potential to confound conclusions drawn from experimental data. Glycoproteins were obtained from three sources; recombinant hemagglutinin and neuraminidase produced from HEK293 cell line, single-strain egg-based vaccines, and a commercial quadrivalent egg-based vaccine. Site-specific glycosylation profiles of intact glycopeptides were produced using multi-enzymatic methods and mass spectrometric evaluation on an orbitrap Fusion Lumos Tribrid nLC-MS/MS system. Variation in glycan abundances and distribution was most pronounced between different strains of virus (match factor = 383), whereas replicates resulted in low variation (match factor = 957). Glycan variation was also measured based on differences between vendors, lots, batches, protease digestion, and intra-protein site. The most abundant glycans on recombinant influenza glycoproteins were monofucosylated complex type such as Hex5HexNAc4dHex1 and Hex6HexNAc5dHex1NeuAc1, whereas the egg-based vaccines were primarily high-mannose of Hex6HexNAc2 and Hex7HexNAc2. Overall, this work develops methods for both accurate and reproducible measurements of these distributions to potentially reduce uncertainty in vaccine development.

Speaker: Zachary Goecker - National Institute of Standards and Technology