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

Josef Schulze-Horsel,Yvonne Genzel,Udo Reichl

doi:10.1186/1472-6750-8-45

Abstract

BackgroundIn 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.ResultsIn 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.ConclusionIt 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.

Highlights

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
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 MadinDarby canine kidney cells from bioreactor samples
Qualitative detection of different influenza A virus infections in Madin-Darby canine kidney (MDCK) cells According to the recommendations of WHO, influenza virus strains used for vaccine manufacturing are adapted each year depending on the expected prevalent virus strains

Summary

Introduction

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. Human influenza vaccines are still mainly produced in embryonated hen's eggs. Strong efforts are put into the development of cell culture-based vaccine production systems to overcome such limitations and drawbacks [1]. Several cell lines have been characterised for industrial influenza virus production, such as Vero, the human foetal retina cell line PER.C6 and Madin-Darby canine kidney (MDCK) cells [4,5,6,7]

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