Key result
CRISPR-Cas9 genomic deletion of ISG15 in Vero cells resulted in a 70-fold and 87-fold increase in total viral particle production for influenza A virus and recombinant vesicular stomatitis virus, respectively.
Why the study?
Efforts to develop Vero cells as high-throughput vaccine manufacturing platforms have been limited by a lack of understanding of virus-host interactions during infection and cell-based virus production.
CRISPR-based deletion of the ISG15 gene in Vero cells significantly increases the production yield of influenza A and recombinant vesicular stomatitis viruses, offering a promising platform for high-throughput vaccine manufacturing.
Answers are grounded in this paper and cite their sources.
Identifies virus-host factors in Vero cells for IAV production; leaves open whether findings improve vaccine manufacturing platforms.
Despite their wide use in the vaccine manufacturing field for over 40 years, one of the main limitations to recent efforts to develop Vero cells as high‐throughput vaccine manufacturing platforms is the lack of understanding of virus‐host interactions during infection and cell‐based virus production in Vero cells. To overcome this limitation, this manuscript uses the recently generated reference genome for the Vero cell line to identify the factors at play during influenza A virus (IAV) and recombinant vesicular stomatitis virus (rVSV) infection and replication in Vero host cells. The best antiviral gene candidate for gene editing was selected using Differential Gene Expression analysis, Gene Set Enrichment Analysis and Network Topology‐based Analysis. After selection of the ISG15 gene for targeted CRISPR genomic deletion, the ISG15 genomic sequence was isolated for CRISPR guide RNAs design and the guide RNAs with the highest knockout efficiency score were selected. The CRISPR experiment was then validated by confirmation of genomic deletion via PCR and further assessed via quantification of ISG15 protein levels by western blot. The gene deletion effect was assessed thereafter via quantification of virus production yield in the edited Vero cell line. A 70‐fold and an 87‐fold increase of total viral particles productions in ISG15−/− Vero cells was achieved for, respectively, IAV and rVSV while the ratio of infectious viral particles/total viral particles also significantly increased from 0.0316 to 0.653 for IAV and from 0.0542 to 0.679 for rVSV‐GFP.
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Sène et al. (2022) studied Viral vaccine production. CRISPR-Cas9 genomic deletion of ISG15 vs. Wild-type Vero cells was evaluated on Total viral particles production (70-fold increase for IAV and 87-fold increase for rVSV). CRISPR-Cas9 genomic deletion of ISG15 in Vero cells resulted in a 70-fold and 87-fold increase in total viral particle production for influenza A virus and recombinant vesicular stomatitis virus, respectively.
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