Characterization and analytical issues on an influenza A virus production process

Different approaches for a mammalian cell culture based production process for influenza vaccines are currently in development. Each approach needs optimization based on analytical data collected from many small scale experiments. In the presentation results for different cultivation methods (rollerbottles, stirred tank or wave bioreactor microcarrier system) for a production process with adherent MDCK cells are discussed. Analytical data on cell numbers, metabolism, host cell protein level, cell cycle and virus titer are shown

High yields of Influenza A virus in MDCK cells are promoted by an insufficient IFN-induced antiviral state

Because of their high susceptibility to infection with various influenza virus strains, Madin Darby canine kidney (MDCK) cells have been widely used as a substrate for influenza virus isolation and vaccine production. However, MDCK cells are also interferon competent and the type I interferon (IFN) response is commonly thought to be a factor strongly inhibiting virus replication. Therefore, the inhibition of influenza virus replication by IFN signalling was analysed for an adherent MDCK cell line used in vaccine manufacturing. Depending on the respective virus strain different levels of IFN induction and a corresponding up-regulation of the IFN induced myxovirus resistance protein 1 (Mx1) were observed. Suppression of IFN induction by transient expression of the viral NS1 protein enhanced replication of an influenza virus lacking NS1, but not wild type strains. In agreement with this, stimulation of cells with MDCK cell-derived IFN prior to infection resulted only in a decrease of replication rate, but not in a change of final yields for wild type influenza viruses. This lack of IFN-induced antiviral activity correlated with missing anti-influenza activity of MDCK Mx proteins. No inhibitory effect on viral polymerase activity was found for canine Mx1 and Mx2 in minireplicon assays. In conclusion, in MDCK cells IFN expression is not a limiting factor for influenza virus replication and this might be caused partially by a lack of anti-influenza activity of canine Mx proteins. Copyright © 2010 by the Society for General Microbiology. [accessed September 21st, 2010

Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells

Influenza viruses are respiratory pathogens and can cause severe disease. The best protection against influenza is provided by annual vaccination. These vaccines are produced in embryonated chicken eggs or using continuous animal cell lines. The latter processes are more flexible and scalable to meet the growing global demand. However, virus production in cell cultures is more expensive. Hence, further research is needed to make these processes more cost-effective and robust. We studied influenza virus replication dynamics to identify factors that limit the virus yield in adherent Madin-Darby canine kidney (MDCK) cells. The cell cycle stage of MDCK cells had no impact during early infection. Yet, our results showed that the influenza virus RNA synthesis levels out already 4 h post infection at a time when viral genome segments are exported from the nucleus. Nevertheless, virus release occurred at a constant rate in the following 16 h. Thereafter, the production of infectious viruses dramatically decreased, but cells continued to produce particles contributing to the hemagglutination (HA) titer. The majority of these particles from the late phase of infection were deformed or broken virus particles as well as large membranous structures decorated with viral surface proteins. These changes in particle characteristics and morphology need to be considered for the optimization of influenza virus production and vaccine purification steps. Moreover, our data suggest that in order to achieve higher cell-specific yields, a prolonged phase of viral RNA synthesis and/or a more efficient release of influenza virus particles is required. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-016-7542-4) contains supplementary material, which is available to authorized users