Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles

Marc D Hein,Stefan Pöhlmann,Klaus Schughart,Sascha Y Kupke,Udo Reichl,Yvonne Genzel,Michael Winkler,Pavel Marichal-Gallardo,Prerna Arora

doi:10.1186/s12915-021-01020-5

Abstract

BackgroundInfections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential.ResultsIn 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.ConclusionIn summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.

Highlights

Infections with influenza A virus (IAV) cause high morbidity and mortality in humans
In summary, experiments demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent
DI244 production yields depend on multiplicity of DIP (MODIP) In order to produce DI244 without use of a helper virus, a purely clonal DI244 seed, generated by reverse genetics, and a genetically engineered suspension cell line expressing the viral PB2 protein, called MDCK-PB2(sus), was used

Summary

Introduction

Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential. Antivirals currently in use are small molecules like oseltamivir and zanamivir [4,5,6] Use of these drugs is compromised by the existence of circulating resistant IAV strains [7, 8].

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