68 research outputs found
Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production
<p>Abstract</p> <p>Background</p> <p>In cell culture-based influenza vaccine production the monitoring of virus titres and cell physiology during infection is of great importance for process characterisation and optimisation. While conventional virus quantification methods give only virus titres in the culture broth, data obtained by fluorescence labelling of intracellular virus proteins provide additional information on infection dynamics. Flow cytometry represents a valuable tool to investigate the influences of cultivation conditions and process variations on virus replication and virus yields.</p> <p>Results</p> <p>In this study, fluorescein-labelled monoclonal antibodies against influenza A virus matrix protein 1 and nucleoprotein were used for monitoring the infection status of adherent Madin-Darby canine kidney cells from bioreactor samples. Monoclonal antibody binding was shown for influenza A virus strains of different subtypes (H1N1, H1N2, H3N8) and host specificity (human, equine, swine). At high multiplicity of infection in a bioreactor, the onset of viral protein accumulation in adherent cells on microcarriers was detected at about 2 to 4 h post infection by flow cytometry. In contrast, a significant increase in titre by hemagglutination assay was detected at the earliest 4 to 6 h post infection.</p> <p>Conclusion</p> <p>It is shown that flow cytometry is a sensitive and robust method for the monitoring of viral infection in fixed cells from bioreactor samples. Therefore, it is a valuable addition to other detection methods of influenza virus infection such as immunotitration and RNA hybridisation. Thousands of individual cells are measured per sample. Thus, the presented method is believed to be quite independent of the concentration of infected cells (multiplicity of infection and total cell concentration) in bioreactors. This allows to perform detailed studies on factors relevant for optimization of virus yields in cell cultures. The method could also be used for process characterisation and investigations concerning reproducibility in vaccine manufacturing.</p
Distributed Modeling and Parameter Estimation of Influenza Virus Replication During Vaccine Production
Quantification system for the viral dynamics of a highly pathogenic simian/human immunodeficiency virus based on an in vitro experiment and a mathematical model
<p>Abstract</p> <p>Background</p> <p>Developing a quantitative understanding of viral kinetics is useful for determining the pathogenesis and transmissibility of the virus, predicting the course of disease, and evaluating the effects of antiviral therapy. The availability of data in clinical, animal, and cell culture studies, however, has been quite limited. Many studies of virus infection kinetics have been based solely on measures of total or infectious virus count. Here, we introduce a new mathematical model which tracks both infectious and total viral load, as well as the fraction of infected and uninfected cells within a cell culture, and apply it to analyze time-course data of an SHIV infection <it>in vitro</it>.</p> <p>Results</p> <p>We infected HSC-F cells with SHIV-KS661 and measured the concentration of Nef<it>-</it>negative (target) and Nef<it>-</it>positive (infected) HSC-F cells, the total viral load, and the infectious viral load daily for nine days. The experiments were repeated at four different MOIs, and the model was fitted to the full dataset simultaneously. Our analysis allowed us to extract an infected cell half-life of 14.1 h, a half-life of SHIV-KS661 infectiousness of 17.9 h, a virus burst size of 22.1 thousand RNA copies or 0.19 TCID<sub>50</sub>, and a basic reproductive number of 62.8. Furthermore, we calculated that SHIV-KS661 virus-infected cells produce at least 1 infectious virion for every 350 virions produced.</p> <p>Conclusions</p> <p>Our method, combining <it>in vitro </it>experiments and a mathematical model, provides detailed quantitative insights into the kinetics of the SHIV infection which could be used to significantly improve the understanding of SHIV and HIV-1 pathogenesis. The method could also be applied to other viral infections and used to improve the <it>in vitro </it>determination of the effect and efficacy of antiviral compounds.</p
Zellphysiologische Charakterisierung von Zellkulturen in der Influenza-Impfstoffproduktion
Untersuchungen zur Replikation von Influenzaviren während der Impfstoffproduktion mittels Durchflusszytometrie und polaritätsspezifischer Realtime-PCR
Single-cell approach in influenza vaccine production : apoptosis and virus protein production
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
Influenza vaccine production : flow cytometric monitoring of apoptosis and virus protein production in mammalian cell culture
Today, human influenza vaccines are still mainly produced in embryonated henâs eggs. However, strong efforts are put into the development of cell culture-based vaccine production systems to overcome limitations and drawbacks of this production system. In cell culture-based vaccine production cellular physiology during infection is of great importance for process characterization and subsequent optimization approaches. The use of flow cytometry offers a powerful tool to monitor and correlate several physiological parameters on a single-cell level. Here, we present flow cytometric data on the replication of human influenza A virus in adherent Madin-Darby canine kidney cells in lab-scale stirred-tank bioreactors. Physiological parameters of interest are apoptosis, status of infection and viral nucleoprotein content per cell. For the quantification of cellular infection status and content of viral nucleoprotein in the host-cells we established a sensitive immunocytometric detection method. In contrast to virus titration methods, this single cell-based immunocytometry is independent of dilution series and the number of infected cells. This is of interest especially in case of vaccine production processes, which are typically initiated at low multiplicities of infection. Apoptotic cell death occurring during virus propagation was measured via DNA strand breaks and pancaspase activity. Results obtained from monitoring vaccine production processes confirm a close correlation of apoptosis and influenza virus infection. Separate analysis of adherent and detached cell populations showed strong differences regarding status of infection and degree of apoptosis. The extend of apoptosis induction was found to be strongly dependent on culture conditions during the virus propagation phase. Based on these results, options to influence cell physiology to increase overall virus yields are being investigated. Together with mathematical models using population balances a better understanding of infection dynamics and host-cell interaction should be obtained to support a quantitative prediction of process behaviour and systematic process optimization [1]. [1] Sidorenko, Y., Schulze-Horsel, J., Voigt, A., Reichl, U., Kienle, A., 2007, Stochastic population balance modeling of influenza virus replication in vaccine production processes, submitte
Monitoring of Cell Physiology in Influenza Vaccine Production by Flow Cytometry
The cell cycle distributions of adherent Madin-Darby canine kidney cells grown for vaccine production were investigated. During cell growth phase cell cycle distributions for all cultivations were similar whereas during infection and mock-infection significant differences in S-phase contents were observed
Single-cell approach in influenza vaccine production : apoptosis and virus protein production
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