Engineering a replication-tunable HSV-1 strain for a safe and effective live-attenuated vaccine

Live-attenuated vaccines represent a promising strategy for preventing herpes simplex virus type 1 (HSV-1) infection, but achieving an optimal balance between attenuation and immunogenicity remains a significant challenge. Here, we engineered a replication-tunable HSV-1 strain, M4, by fusing the essential replication factor UL54 to a destabilizing domain, enabling ligand-dependent control of viral replication. Additionally, we deleted four viral genes (UL23, LAT, UL13 and US12) to reduce neurovirulence and enhance immune activation. Without the stabilizing ligand, M4 exhibited severely impaired replication, while ligand addition restored replication, facilitating efficient production of virions. In vivo studies confirmed that M4 is highly attenuated, eliciting robust HSV-1-specific humoral and cellular immune responses that conferred full protection against lethal viral challenge. Transcriptomic and immune profiling revealed that M4 preferentially activated adaptive immunity while minimizing inflammatory responses associated with neurovirulence. This study provides a novel strategy for developing safe and effective live-attenuated herpesvirus vaccines.