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

Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production-2

Negative controls: uninfected cells (I), mock-infected stained cells, harvested 2 h p.i. (II); infected cells: equine influenza A virus-infected, harvested 18 h p.i. (III), equine influenza A virus-infected, harvested 6 h p.i. (IV).<p><b>Copyright information:</b></p><p>Taken from "Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production"</p><p>http://www.biomedcentral.com/1472-6750/8/45</p><p>BMC Biotechnology 2008;8():45-45.</p><p>Published online 30 Apr 2008</p><p>PMCID:PMC2408585.</p><p></p

Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production-5

Sample not analyzed by flow cytometry due to cell lysis); : total virus particle concentration measured by hemagglutination assay (∘ ± standard error of the method); : infectious virus concentration determined via virus infectivity assay (TCID, ∙ ± standard error of the method).<p><b>Copyright information:</b></p><p>Taken from "Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production"</p><p>http://www.biomedcentral.com/1472-6750/8/45</p><p>BMC Biotechnology 2008;8():45-45.</p><p>Published online 30 Apr 2008</p><p>PMCID:PMC2408585.</p><p></p

Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production-7

I): uninfected cells; (II): cells infected with equine influenza A virus H3N8 (18 h p.i.); (III): cells infected with porcine influenza A virus H1N5 (18 h p.i.); (IV): cells infected with human influenza A virus H1N1 (18 h p.i.).<p><b>Copyright information:</b></p><p>Taken from "Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production"</p><p>http://www.biomedcentral.com/1472-6750/8/45</p><p>BMC Biotechnology 2008;8():45-45.</p><p>Published online 30 Apr 2008</p><p>PMCID:PMC2408585.</p><p></p

Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production-8

Negative controls: uninfected cells (I), mock-infected stained cells, harvested 2 h p.i. (II); infected cells: equine influenza A virus-infected, harvested 18 h p.i. (III), equine influenza A virus-infected, harvested 6 h p.i. (IV).<p><b>Copyright information:</b></p><p>Taken from "Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production"</p><p>http://www.biomedcentral.com/1472-6750/8/45</p><p>BMC Biotechnology 2008;8():45-45.</p><p>Published online 30 Apr 2008</p><p>PMCID:PMC2408585.</p><p></p

Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production-4

with equine influenza virus in a lab-scale bioreactor (1 l wv, moi = 3.0); duplicate measurements (symbols) and profile of the corresponding mean (lines).<p><b>Copyright information:</b></p><p>Taken from "Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production"</p><p>http://www.biomedcentral.com/1472-6750/8/45</p><p>BMC Biotechnology 2008;8():45-45.</p><p>Published online 30 Apr 2008</p><p>PMCID:PMC2408585.</p><p></p