Non‐Targeting shRNA‐Encoded Plasmid DNA Enhances Protective Immunity Through RIDD‐RIG‐I Signaling Pathway in the Zika Virus Animal Model

DNA vaccines offer advantages such as low production cost, rapid manufacturing, and enhanced stability for transport and storage, making them suitable for addressing tropical and emerging infectious diseases. However, their limited immunogenicity in humans often necessitates multiple booster doses or high antigen quantities, posing a significant barrier to broader application. To overcome this limitation, we developed a DNA vaccine encoding the Zika virus (ZIKV) structural proteins (prME) that incorporates a non-targeting short hairpin RNA (shRNA) as an intrinsic molecular adjuvant. This shRNA is not designed to silence host genes; rather, it enhances innate immune "RIG-I" signaling by selectively activating the IRE1α-dependent regulated IRE1-dependent decay pathway. Mechanistically, shRNA overexpression is inferred to increase the load on the RNA-induced silencing complex, potentially triggering IRE1α oligomerization and subsequent upregulation of TXNIP. These events generate cytoplasmic RNA fragments that activate RIG-I and downstream antiviral responses. In murine models, the vaccine elicited strong ZIKV-specific humoral and cellular immune responses. In AGB6 mice, it significantly increased neutralizing antibody titers, reduced viremia, and improved survival. This approach enhances the immunogenicity of DNA vaccines without targeting host genes, providing a scalable and adaptable platform for infectious disease prevention.