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Zusammenfassung:
The envelope of influenza A is spiked with two glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA as the most abundant protein on the virus surface, triggers the strongest immunogenic response. Each HA monomer contains 3 to 9 N-linked glycans, depending on the virus strain. The functional role of these glycans is still not completely understood. However, previous glycosylation studies have shown that structural modifications of these glycans 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 cultivation conditions in vaccine production. Further modifications in the structure can occur during inactivation- and downstream processing steps. Hence, monitoring and controlling the glycosylation pattern during the virus production process can be crucial to obtain maximum production yields and to ensure the immunogenicity of the antigens.
In this study we present a method, allowing the comparison of influenza A/PR/8/34 (H1N1) virus HA N-glycosylation pattern produced in different cell lines (MDCK, VERO) and egg derived virus. The virus is concentrated and purified by “g-force-step-gradient-centrifugation” directly from cell culture supernatants. Afterwards viral proteins are separated by SDS-PAGE followed by enzymatical cleavage of HA N-glycans from the protein in gel with PNGase F. An aliquot of the N-glycan pool is labeled with 8-Aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) by reductive amination for monitoring by capillary gel electrophoresis with laser induced fluorescence (CGE-LIF). N-glycans are than analyzed on two levels: first generating fingerprints and second by sequential exoglycosidases digestion. Additional information on the N-glycan structure are obtained by CGE experiments where N-glycans with known structure were spiked to the samples. The developed method provides a promising tool for monitoring the HA N-glycosylation pattern during the major steps of up- and downstream process during the influenza virus vaccine production.
