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

PEGylated interferon-alpha (PEGIFNalpha) demonstrates promising therapeutic outcomes against chronic hepatitis B (CHB), whereas patient response to PEGIFNalpha therapy remains unsatisfied. Shutdown of hepatitis B virus (HBV) antigens by RNA interference (RNAi) could enhance PEGIFNalpha efficacy in CHB patients, whereas the underlying immunological mechanisms remain obscure. We performed studies by utilizing our newly established extracellular humanized IFNAR (IFNAR-hEC) mice. An in-house constructed small interfering RNAs (GalNac-siHBV) was administrated to mice either alone or in combination with PEGIFNalpha. The phenotypic and functional characteristics of peripheral and organ-specific immune cells were assessed by flow cytometry, ELISpot, RNA sequencing (RNA-seq), and single-cell RNA-seq (scRNA-seq) analysis. Our results demonstrated that combined treatment with PEGIFNalpha and RNAi exerted a synergistic and prolonged inhibition of HBsAg (~4log10 IU/mL, vs PBS) and induced a higher incidence of HBsAg seroconversion (~30%), comparing with either monotreatment. Mechanistically, combined therapy improved the functionality of global T and B cells, triggered increased anti-HBs producing B cells, and enhanced IFNgamma-producing T cells. scRNA-seq analysis revealed that the combined therapy reduced inhibitory B cell-B cell interaction, enhanced MHC-I signaling mediated T cell-T cell communication, and improved T cell-B cell crosstalk, thus improving the functionality of T and B cells. Enhanced MHC-II signaling networks across B cells and hepatocytes/Cd8+ T cells further promoted HBsAg seroconversion in the combined treatment groups. These results together provided scientific rationale and lessons for the combination of the two towards better therapeutic efficacy.