5'-triphosphate siRNA targeting HBx elicits a potent anti-HBV immune response in pAAV-HBV transfected mice

RNA with 5'-triphosphate (3p-RNA) is recognized by RNA sensor RIG-I (retinoic acid-inducible gene I protein). Previously, we reported that small interfering RNA targeting HBx (3p-siHBx) could confer potent anti-hepatitis B virus (HBV) efficacy via HBx silencing and RIG-I activation. However, the characteristics of innate and adaptive immunity especially exhaustion profiles in the liver microenvironment in response to 3p-siHBx therapy have not been fully elucidated. Here, we observed that 3p-siHBx more significantly inhibited HBV replication in vivo. 3p-siHBx enhanced natural killer (NK) cell activation with KLRG1 and CD69 upregulation and interferon (IFN)-γ secretion. 3p-siHBx significantly reversed the exhaustion phenotype of CD8⁺ T cells, and augmented CD8⁺ T cell activation and function. Importantly, 3p-siHBx disrupted the differentiation of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg), accompanied by the reduction of the immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β. 3p-siHBx also enhanced dendritic cell maturation. Further investigation showed that RIG-I was involved in 3p-siHBx-induced IFN-α, IFN-β, and IFN-λ production. Moreover, RIG-I activation in HBV⁺ hepatocytes would improve the recruitment of CD8⁺ T cells and NK cells. These results reveal that 3p-siHBx therapy can improve the immune microenvironment in HBV-carrier liver and inhibit HBV replication, indicating the potential utility of RIG-I ligands as molecular adjuvants for viral vaccines or candidate drugs.