Tuning intracellular immunity by Nodamura virus B2 protein enhances self-amplifying RNA activity

Abstract Self-amplifying RNA (saRNA) offers a powerful approach for vaccine development, as its replicative nature within cells provides prolonged antigen expression together with self-adjuvanticity. However, its potency is often limited by innate immune responses, particularly those triggered by intracellular double-stranded RNA (dsRNA), highlighting the need for strategies that can boost saRNA efficacy without compromising its immunostimulatory properties. Here we show that the cis -expression of the Nodamura virus (NoV) B2 protein markedly enhances saRNA-driven transgene expression by alleviating key cell-intrinsic antiviral effectors. In stem cells, NoV B2 limits the accumulation of Dicer-generated viral small RNAs, consistent with suppression of antiviral RNA interference, while in somatic cells, it prevents protein kinase R (PKR)-driven translational shutdown. NoV B2 sequesters dsRNA at the cell periphery, shielding it from antiviral effectors without suppressing type I interferon induction or signalling, thereby preserving saRNA’s immunostimulatory properties, while enhancing its transgene expression potential. These findings reveal a strategy to overcome intracellular antiviral restriction of saRNA while preserving adjuvanticity, with implications for the design of more effective saRNA-based vaccines and therapeutics.