title: Trypsin promotes efficient influenza vaccine production in MDCK cells by interfering with the antiviral host response.
abstract: Trypsin is commonly used in Madin-Darby canine kidney (MDCK) cell culture-based influenza vaccine production to facilitate virus infection by proteolytic activation of viral haemagglutinin, which enables multi-cycle replication. In this study, we were able to demonstrate that trypsin also interferes with pathogen defence mechanisms of host cells. In particular, a trypsin concentration of 5 BAEE U/mL (4.5 μg/mL porcine trypsin) used in vaccine manufacturing strongly inhibited interferon (IFN) signalling by proteolytic degradation of secreted IFN. Consequently, absence of trypsin during infection resulted in a considerably stronger induction of IFN signalling and apoptosis, which significantly reduced virus yields. Under this condition, multi-cycle virus replication in MDCK cells was not prevented but clearly delayed. Therefore, incomplete infection can be ruled out as the reason for the lower virus titres. However, suppression of IFN signalling by overexpression of viral IFN antagonists (influenza virus PR8-NS1, rabies virus phosphoprotein) partially rescued virus titres in the absence of trypsin. In addition, virus yields could be almost restored by using the influenza strain A/WSN/33 in combination with fetal calf serum (FCS). For this strain, FCS enabled trypsin-independent fast propagation of virus infection, probably outrunning cellular defence mechanisms and apoptosis induction in the absence of trypsin. Overall, addition of trypsin provided optimal conditions for high yield vaccine production in MDCK cells by two means. On the one hand, proteolytic degradation of IFN keeps cellular defence at a low level. On the other hand, enhanced virus spreading enables viruses to replicate before the cellular response becomes fully activated.
authors.affiliation: Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106, Magdeburg, Germany.
authors.forename: Claudius
authors.initials: C
authors.lastname: Seitz
authors.forename: Britta
authors.initials: B
authors.lastname: Isken
authors.forename: Björn
authors.initials: B
authors.lastname: Heynisch
authors.forename: Maria
authors.initials: M
authors.lastname: Rettkowski
authors.forename: Timo
authors.initials: T
authors.lastname: Frensing
authors.forename: Udo
authors.initials: U
authors.lastname: Reichl
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D018929:Cell Culture Techniques; D002460:Cell Line; D004285:Dogs; D007252:Influenza Vaccines / Q000302:isolation & purification*; D007372:Interferons / Q000378:metabolism; D009975:Orthomyxoviridae / Q000254:growth & development* / Q000302:isolation & purification*; D013678:Technology, Pharmaceutical / Q000379:methods; D014357:Trypsin / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D007252:Influenza Vaccines; D007372:Interferons; D014357:Trypsin