Glycosylation of Influenza A Virus Hemagglutinin

The genome of a wide range of viruses, including the influenza A virus, is enclosed in a lipid envelope which are generally acquired in the final step of virus assembly. The influenza A envelope is spiked with two glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA is the most abundant protein on the virus surface, triggering the strongest immunogenic response. It plays an important role during virus attachment, endocytosis and membrane fusion. Mature HA forms homotrimers whereas the monomers are present as polypeptide precursor (HA0). Conformational changes, affecting membrane fusion, dissociate HA0 into the subunits: HA1 and HA2. Both subunits are glycosylated and each monomer contains 3 to 9 N-linked glycans, depending on the virus strain. The structure of the individual glycans depends mainly on virus subtype and on the host cell. Biological function of HA-glycans is still not fully understood. However, it has been demonstrated that they play an important role in the intracellular transport of HA and the viral replication regulation. Structural modifications can influence virus attachment to the host cell, and therefore change viral replication dynamics and its immunogenicity. The glycosylation pattern of viral proteins is affected by the glycosylation machinery of the host cell and their cultivation conditions. Further modifications in the structure can occur during inactivation- and downstream processing steps. Hence, monitoring and controlling the glycosylation pattern during the production process can be crucial for obtaining maximum production yields and for securing immunogenicity of the antigens. In the present study we characterize the N-glycan structure of influenza A/PR/8/34 (H1N1) and influenza A/NM/1/93 (H3N8) virus HA produced in Madin Darby canine kidney (MDCK) cells via the following method. In a first step viral proteins are separated by SDS-PAGE. Next, HA N-glycans are enzymatically cleaved from the protein in gel with PNGase F. Than a part of the glycan pool is labelled with 8-Aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) by reductive amination. These fluorescently conjugated glycans are further characterized by capillary gel electrophoresis (CGE). This analysis generates fingerprints of HA N-glycan mixtures with a detection limit in a low fmolar range. The unconjugated N-glycans are analyzed by mass spectrometry. Further structural analysis of the HA N-glycans is obtained by sequential sequencing implementing a reagent array analysis method (RAAM). This involves the digestion of the oligosaccharides with exoglycosidases monitored by CGE and mass spectrometry. Glycan characterization indicated a low sialation level and high level of terminal ï¢-galactose of HA N-glycans of influenza H1N1. These results go in line with experimental results obtained by lectin affinity chromatography. Together, the methods demonstrated here represent a promising tool to monitor HA-glycosylation during the major steps of up- and downstream process for the influenza virus vaccine production