A Biologically Optimized PEG-IFN-α2 Variant Exhibits Potent Anti-HBV and Immunomodulatory Efficacy

Human interferon-alpha (IFN-α) includes 13 subtypes, but only IFN-α2 (a/b) is clinically approved for chronic hepatitis B (CHB) therapy, limited by adverse effects and suboptimal response rates. Previously, we identified IFN-α14 as a superior anti-HBV subtype compared to IFN-α2, leading to the engineering of the IFN-α2-EIFK variant, which mimics IFN-α14's enhanced antiviral activity through targeted mutations at receptor-binding sites. However, the in vivo anti-HBV effects of IFN-α2-EIFK remained unexplored due to its short plasma half-life and lack of immunocompetent murine systems suitable for evaluating human IFN-α activity. Using an immunocompetent IFNAR-hEC mouse model and HBV-infected human-liver chimeric mice etc, we demonstrated that N-terminally PEGylated IFN-α2-EIFK (PEG-IFN-α2-EIFK) suppressed HBV replication and antigen expression approximately 10-fold more effectively than PEG-IFN-α2 at equivalent mass doses, while concurrently activating IFN-γ-like signaling pathways in both hepatocytes and immune cells. Subsequent single-cell analysis of intrahepatic immune cells from IFNAR-hEC mice showed that PEG-IFN-α2-EIFK was associated with enhanced antigen-presenting capacity of macrophages, dendritic cells (DCs), and B cells. In addition, PEG-IFN-α2-EIFK was found to preferentially promote M1-like macrophage polarization, and modulate PD-L1/PD-1 interactions between macrophages and CD8+ T cells, accompanied by attenuation of an exhaustion-associated CD8+ T cell state. In conclusion, this study demonstrates that PEG-IFN-α2-EIFK exhibits greater anti-HBV activity and immunomodulatory potency than PEG-IFN-α2 under equivalent mass dosing, supporting its potential as an enhanced therapeutic candidate for CHB.