RNAi-mediated HBV antigen shutdown enhances the antiviral immune effects of PEGIFNα via T and B cell crosstalk remodeling

Backgrounds/Aims: PEGylated interferon-α (PEGIFNα) shows promise in treating chronic hepatitis B (CHB), yet patient response remains suboptimal. While suppressing hepatitis B virus (HBV) antigens by RNA interference (RNAi) could enhance PEGIFNα efficacy in CHB patients, the underlying immunological mechanisms remain obscure. Methods: Using our newly established extracellular humanized IFNAR (IFNAR-hEC) mouse model of chronic HBV infection, we evaluated the efficacy of a GalNac-conjugated siRNA (GalNac-siHBV) alone or in combination with PEGIFNα. Phenotypic and functional characteristics of immune cells were assessed by flow cytometry, ELISpot, and single-cell RNA sequencing (scRNA-seq). Results: High circulating HBsAg reduced antiviral effects and immune responsiveness of PEGIFNα. Combined PEGIFNα and RNAi therapy synergistically and durably suppressed HBsAg (~4log10 IU/mL, vs PBS) and achieved HBsAg seroconversion in ~30% of mice, outperforming either monotherapy. Mechanistically, PEGIFNα enhanced global T and B cell function, whereas combined therapy further amplified HBV-specific T and B cell responses. scRNA-seq analysis indicated that combined therapy attenuated inhibitory B cell-B cell interactions, strengthened MHC-I-mediated T cell crosstalk, and enhanced MHC-II signaling networks across B cells and hepatocytes/Cd8+ T cells, collectively improving the lymphocyte functionality and facilitating HBsAg seroconversion. Reinforcing MHC-I/II signaling with agonistic antibodies (α4-1BB, αCD40) or IL-2-Fc further potentiated antiviral immune responses of combinational regimen. Conclusions: Combined RNAi and PEGIFNα therapy exerts synergistic antiviral effects by relieving HBV antigen-induced immune tolerance and orchestrating a functional T cell-B cell crosstalk network centered on MHC-I/II signaling. Enhancing antigen presentation pathways represents a promising adjunctive strategy to improve functional cure rate of CHB.