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

Abstract

Background Infections 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. Results 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. Conclusion In 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.

Cell culture-based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

“…We previously reported the batch production of pure DI244 particles in shake flasks for the use as an antiviral (Hein et al 2021a). To evaluate if process intensification strategies that are already used for non-modified MDCK suspension cells and IAV STV production (Wu et al 2021) would be applicable, a manually adjusted perfusion process in a STR was implemented.…”

“…The profile was based on the cell growth rate and metabolite uptake rates observed in a previous batch cultivation (Fig. S1) (Hein et al 2021a).…”

“…MDCK-PB2(sus) cells were used for production of propagation incompetent DI244 as described previously (Hein et al 2021a). Briefly, MDCK cells adapted to growth in chemically defined Xeno™ medium (Bissinger et al 2019) were genetically modified by retroviral transduction to express the viral PB2, encoded by IAV Seg1 vRNA.…”

“…The permeate flow rate was increased over the process time either manually or automatically to maintain a cell specific perfusion rate (CSPR) of approximately 200 pL/cell/day. For the manually adjusted perfusion rate, a perfusion profile was calculated based on the growth rate and metabolite consumption rates determined during the exponential phase of a previous batch cultivation (Hein et al 2021a). Here, the perfusion rate was increased stepwise every 12 hours.…”

“…This allowed propagation of IAV DIPs with a deletion in Seg1. We recently reported the production of a prototypic Seg1 DIP, called DI244, utilizing a suspension MDCK cell line expressing PB2 (MDCK-PB2(sus)) and the evaluation of the produced DI244 in animal experiments (Hein et al 2021a). Here, all mice infected with an otherwise lethal dose of IAV survived the infection when treated with DI244.…”