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Background
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.
Results
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. Specific binding of monoclonal antibodies was shown for influenza A virus strains of different subtypes (H1N1, H1N5, H3N8) and host specificity (human, equine, swine). At high multiplicity of infection in a bioreactor, the onset of influenza virus replication was detected at about 2 to 4 h post infection by immunocytometry. In contrast, a significant increase in titre by hemagglutination assay was detected at the earliest 4 to 6 h post infection. In addition to the quantification of the infected cell fraction, influenza A virus nucleoprotein and matrix protein copy numbers in cells could be estimated from the measured fluorescence intensities. The fluorescence intensity threshold for infected cell quantification was equivalent to 76 000 nucleoprotein and matrix protein 1 copies per cell, which corresponds to 19 virions at the most.
Conclusions
It is shown that immunocytometry is a sensitive and robust method for the monitoring of viral infection and the quantification of virus protein content 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.
© 2008 Schulze-Horsel et al; licensee BioMed Central Ltd.
[accessed June 6, 2008]
