Systemic removal of host restriction factors enables rapid, scalable virus production for cell culture-based vaccine development.

The efficiency of cell culture-based vaccine production is fundamentally constrained by the antiviral defenses of host cells, creating a major bottleneck for rapid and large-scale vaccine manufacturing. Key antiviral proteins-such as RNase L, PKR, and JAK1 act as intrinsic brakes on viral replication, limiting efficient propagation of many clinically relevant viruses. To overcome this challenge, we generated HEK293T, Vero, and MDCK cell lines with targeted knockouts of multiple antiviral genes. Notably, these engineered cells maintained normal growth and viability while supporting markedly increased viral yields. Multi-gene deletions enhance the replication of both enveloped viruses, including influenza A virus, pseudotyped lentivirus, and porcine epidemic diarrhea virus (PEDV), and non-enveloped viruses such as coxsackievirus. The magnitude of enhancement scaled proportionally with the number of genes disrupted. By systematically removing host restriction factors, this platform provides a versatile and powerful strategy for accelerating viral propagation, offering a strong foundation for more efficient development and large-scale production of cell culture-based anti-viral vaccines.