IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production

PeerJ. 2022 Sep 21:10:e13989. doi: 10.7717/peerj.13989. eCollection 2022.

Abstract

The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-based production is limited by host interferons (IFN). Interferon regulating factor 7 (IRF7) is a transcription factor for type-I IFN that plays an important role in regulating the anti-viral mechanism and eliminating viruses. We developed IRF7 knock-out MDCK cells (IRF7-/ - MDCK) using CRISPR/Cas9 technology. The RNA expression levels of IRF7 in the IRF7-/ - MDCK cells were reduced by 94.76% and 95.22% under the uninfected and infected conditions, respectively. Furthermore, the IRF7 protein level was also significantly lower in IRF7-/ - MDCK cells for both uninfected (54.85% reduction) and viral infected conditions (32.27% reduction) compared to WT MDCK. The differential expression analysis of IFN-related genes demonstrated that the IRF7-/ - MDCK cell had a lower interferon response than wildtype MDCK under the influenza-infected condition. Gene ontology revealed down-regulation of the defense response against virus and IFN-gamma production in IRF7-/ - MDCK. The evaluation of influenza viral titers by RT-qPCR and hemagglutination assay (HA) revealed IRF7-/ - MDCK cells had higher viral titers in cell supernatant, including A/pH1N1 (4 to 5-fold) and B/Yamagata (2-fold). Therefore, the IRF7-/ - MDCK cells could be applied to cell-based influenza vaccine production with higher capacity and efficiency.

Keywords: CRISPR-Cas9; IRF7; Influenza; Interferon; MDCK; Vaccine production.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics
  • Dogs
  • Factor VII / genetics
  • Humans
  • Influenza Vaccines* / genetics
  • Influenza, Human* / genetics
  • Interferon Type I* / genetics
  • Madin Darby Canine Kidney Cells
  • Orthomyxoviridae*
  • Technology
  • Virus Replication / genetics

Substances

  • Influenza Vaccines
  • Factor VII
  • Interferon Type I

Grants and funding

Funding was provided by the Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University and the National Research Council of Thailand (NRCT) [N41A640077]. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.