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

PeerJ

article

Oraphan Mayuramart¹  Witthaya Poomipak²  Somruthai Rattanaburi¹  Kritsada Khongnomnan³  Songtham Anuntakarun¹  Suthat Saengchoowong³  Tanit Chavalit³  Naphat Chantaravisoot³  Sunchai Payungporn¹ 

[1] Research Unit of Systems Microbiology, Faculty of Medicine, Chulalongkorn University;Research Affairs, Faculty of Medicine, Chulalongkorn University;Department of Biochemistry, Faculty of Medicine, Chulalongkorn University;Center of Excellence in Systems Biology, Faculty of Medicine, Chulalongkorn University

关键词: CRISPR-Cas9;    IRF7;    MDCK;    Influenza;    Interferon;    Vaccine production;   

DOI  :  10.7717/peerj.13989

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

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.

【 授权许可】

CC BY