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Influenza viruInfluenza virus infections remain a major cause of morbidity and mortality, especially in the elderly. Today, human influenza vaccines are mainly produced in embryonated hen's eggs. Since several years there are increasing efforts to develop animal cell culture-based vaccine production systems to overcome the limitations and drawbacks of the existing production system. In cell culture-based vaccine production cell physiology during infection is of great importance for process characterization and optimization studies. The use of flow cytometry offers a powerful tool to monitor and correlate several physiological parameters in a single-cell approach.
Based on the results from process monitoring, the improvement of cell physiology to increase the overall virus yield is subject of recent research. Furthermore, the experimental data are the basis for mathematical modelling using population balances. By this modelling approach and a better understanding of the infection dynamics we aim towards a quantitative prediction of process behaviour and a systematic process optimization.
Here, we present flow cytometric data on the replication of human influenza A virus (H1N1) in adherent Madin Darby canine kidney (MDCK) cells in lab-scale bioreactors. Physiological parameters of interest are apoptosis, status of infection and viral nucleoprotein content per cell. The production of influenza virus particles was detected in culture supernatants by hemagglutination assay (HA) for total virus particles and by tissue culture infectious dose (TCID50) determination for infectious virus particle concentrations. For the quantification of cellular infection status and content of viral nucleoprotein in the host-cells we established a sensitive immunocytometric detection method using fluorochrome-labelled monoclonal antibodies against influenza nucleoprotein (NP). In contrast to titration methods such as HA and TCID50, the single cell-based immunocytometry is independent of dilution series and the number of infected cells. In particular this is of interest in case of vaccine production processes which are typically initiated at low multiplicities of infection. Apoptotic cell death occurring during virus propagation was detected via DNA strand breaks caused by endonuclease activity measured by flow cytometry and agarose gel electrophoresis.
Results confirm the close correlation of apoptosis with viral infection, as described in literature [1]. Moreover, the extend of apoptosis induction was found to be strongly depending on the culture conditions ds infections remain a major cause of morbidity and mortality, especially in the elderly. Today, human influenza vaccines are mainly produced in embryonated hen's eggs. Since several years there are increasing efforts to develop animal cell culture-based vaccine production systems to overcome the limitations and drawbacks of the existing production system. In cell culture-based vaccine production cell physiology during infection is of great importance for process characterization and optimization studies. The use of flow cytometry offers a powerful tool to monitor and correlate several physiological parameters in a single-cell approach.
Based on the results from process monitoring, the improvement of cell physiology to increase the overall virus yield is subject of recent research. Furthermore, the experimental data are the basis for mathematical modelling using population balances. By this modelling approach and a better understanding of the infection dynamics we aim towards a quantitative prediction of process behaviour and a systematic process optimization.
Here, we present flow cytometric data on the replication of human influenza A virus (H1N1) in adherent Madin Darby canine kidney (MDCK) cells in lab-scale bioreactors. Physiological parameters of interest are apoptosis, status of infection and viral nucleoprotein content per cell. The production of influenza virus particles was detected in culture supernatants by hemagglutination assay (HA) for total virus particles and by tissue culture infectious dose (TCID50) determination for infectious virus particle concentrations. For the quantification of cellular infection status and content of viral nucleoprotein in the host-cells we established a sensitive immunocytometric detection method using fluorochrome-labelled monoclonal antibodies against influenza nucleoprotein (NP). In contrast to titration methods such as HA and TCID50, the single cell-based immunocytometry is independent of dilution series and the number of infected cells. In particular this is of interest in case of vaccine production processes which are typically initiated at low multiplicities of infection. Apoptotic cell death occurring during virus propagation was detected via DNA strand breaks caused by endonuclease activity measured by flow cytometry and agarose gel electrophoresis.
Results confirm the close correlation of apoptosis with viral infection, as described in literature [1]. Moreover, the extend of apoptosis induction was found to be strongly depending on the culture conditions during the virus propagation phase. This could be shown for different multiplicities of infection and virus seeds.
[1] Morris, S.J., Nightingale, K., Smith, H., Sweet, C., Virology 2005, 335: 198-211.
