Production of IAV DIPs for Antiviral Treatment

Motivation

Influenza A virus (IAV) causes annual epidemics and occasional pandemics and is therefore considered as a major human pathogen. Resistances against current antivirals have been found for various IAV strains, therefore there is clearly a need for novel treatment modalities. Promising candidates for antiviral treatment are defective interfering particles (DIPs). Conventional IAV DIPs are naturally occurring virus mutants with large internal deletions in one of their eight viral RNA (vRNA) segments. As DIPs miss the genetic information necessary for replication, they can only replicate in a co-infection with infectious standard virus (STV), compensating for the missing gene function. Apart from conventional DIPs that carry deletions, we recently discovered a novel type of IAV interfering particle, which contains various nucleotide substitutions in its segment 7 vRNA (but no internal deletions). This DIP, called OP7, also strongly inhibits the propagation of STV.

Aim of the Project

So far, only an egg-based method was described for production of DIPs. In this project, we develop suspension cell culture-based processes for manufacturing of conventional DIPs and OP7. As DIP replication typically relies on a co-infection, a seed virus containing both DIP and infectious STV is required, that demands UV inactivation of STV in DIP harvests before administration to organisms. To overcome this limitation, we also are use genetically engineered cell lines that can complement the defect of DIPs, allowing propagation of pure DIP populations in the absence of STV. For analysis of the production process, we use a diversity of virus quantification assays, PCR-based methods to monitor DIP propagation, mass spectrometry to detect mutated DIP proteins and biological interference assay to determine antiviral properties in vitro. Moreover, downstream processing approaches are applied to purify and concentrate the DIP harvests from bioreactors. Finally, the produced DIP material will be tested in the mouse models.

References

Hein, M. D.; Chawla, A.; Cattaneo, M.; Kupke, S. Y.; Genzel, Y.; Reichl, U.: Cell culture–based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system. Applied Microbiology and Biotechnology 105, pp. 7251 - 7264 (2021)
Hein, M. D.; Arora, P.; Marichal-Gallardo, P.; Winkler, M.; Genzel, Y.; Pöhlmann, S.; Schughart, K.; Kupke, S. Y.; Reichl, U.: Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles. BMC Biology 19 (1), 91 (2021)
Hein, M. D.; Kollmus, H.; Marichal-Gallardo, P.; Püttker, S.; Benndorf, D.; Genzel, Y.; Schughart, K.; Kupke, S. Y.; Reichl, U.: OP7, a novel influenza A virus defective interfering particle: production, purification, and animal experiments demonstrating antiviral potential. Applied Microbiology and Biotechnology 105, pp. 129 - 146 (2021)
Kupke, S. Y.; Riedel, D.; Frensing, T.; Zmora, P.; Reichl, U.: A Novel Type of Influenza A Virus-Derived Defective Interfering Particle with Nucleotide Substitutions in Its Genome. Journal of Virology 93 (4), 01786-18 (2019)
Bdeir, N.; Arora, P.; Gärtner, S.; Hoffmann, M.; Reichl, U.; Pöhlmann, S.; Winkler, M.: A system for production of defective interfering particles in the absence of infectious influenza A virus. PLoS One 14 (3), e0212757 (2019)
Marichal-Gallardo, P.; Pieler, M.; Wolff, M. W.; Reichl, U.: Steric exclusion chromatography for purification of cell culture-derived influenza A virus using regenerated cellulose membranes and polyethylene glycol. Journal of Chromatography A 1483 (3), pp. 110 - 119 (2017)
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