Department of Bioengineering, McGill University, Montreal, Québec, Canada.
Biotechnol Bioeng. 2022 Oct;119(10):2794-2805. doi: 10.1002/bit.28190. Epub 2022 Jul 30.
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.
尽管 Vero 细胞在疫苗制造领域已经使用了 40 多年，但最近将 Vero 细胞开发为高通量疫苗生产平台的努力主要受到以下限制：缺乏对感染过程中病毒-宿主相互作用以及基于细胞的 Vero 细胞中病毒生产的理解。为了克服这一限制，本文利用最近生成的 Vero 细胞系参考基因组，鉴定甲型流感病毒（IAV）和重组水疱性口炎病毒（rVSV）在 Vero 宿主细胞中感染和复制时起作用的因素。使用差异基因表达分析、基因集富集分析和基于网络拓扑的分析，选择最佳抗病毒基因候选物进行基因编辑。选择靶向 CRISPR 基因组缺失的 ISG15 基因后，分离 ISG15 基因组序列用于 CRISPR 向导 RNA 设计，并选择敲除效率得分最高的向导 RNA。然后通过 PCR 确认基因组缺失验证 CRISPR 实验，并通过 Western blot 进一步评估 ISG15 蛋白水平的定量来评估。此后，通过编辑的 Vero 细胞系中病毒产量的定量评估基因缺失效果。在 ISG15 Vero 细胞中，IAV 和 rVSV 的总病毒颗粒产量分别增加了 70 倍和 87 倍，而 IAV 的感染性病毒颗粒/总病毒颗粒的比例也从 0.0316 显著增加到 0.653，rVSV-GFP 的比例从 0.0542 增加到 0.679。
