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Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles.
Hein, Marc D Arora, Prerna Marichal-Gallardo, Pavel Winkler, Michael Genzel, Yvonne Pöhlmann, Stefan Schughart, Klaus Kupke, Sascha Y Reichl, Udo
Hein, Marc D
Arora, Prerna
Marichal-Gallardo, Pavel
Winkler, Michael
Genzel, Yvonne
Pöhlmann, Stefan
Schughart, Klaus
Kupke, Sascha Y
Reichl, Udo
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2021-05-03
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Abstract
In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived.
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BMC Biol. 2021 May 3;19(1):91. doi: 10.1186/s12915-021-01020-5.
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en
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1741-7007
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Attribution 4.0 International