Sequential mRNA vaccine-enabled quadruple therapy induces functional cure of HBV in rAAV8-1.3HBV carrier mice

Chronic hepatitis B (CHB) functional cure remains difficult to achieve with current treatments, primarily due to hepatitis B virus (HBV)-induced multilayered immune tolerance. We proposed a sequential strategy to gradually reduce viral antigen load, enhance antigen presentation, and promote cytotoxic T-cell memory formation to overcome this immune barrier: (1) viral suppression with antisense oligonucleotides (ASO) and entecavir (ETV); (2) dendritic cell (DC) priming with interferon α (IFN α ); and (3) induction of cytotoxic memory T cell responses with a codon-optimized messenger RNA (mRNA) vaccine (encoding full-length HBV large S protein) delivered via chitosan nanoemulsion (CNE). In recombinant adeno-associated virus serotype 8 with 1.3 copies of the hepatitis B virus genome (rAAV8-1.3HBV) carrier mice, this regimen induced durable functional cure in 50% of mice for 17 weeks. A key finding was that functional cure depended on achieving hepatitis B surface antigen (HBsAg) levels below 100 IU/mL before IFN α administration. This threshold was necessary to permit IFN α -mediated DC activation, which enabled vaccine-expanded and functionally restored HBV-specific CD8 + T cells, alongside dissolution of immunosuppressive networks. Non-responders (pre-IFN α HBsAg >100 IU/mL) displayed impaired DC–T cell interactions. These results provide a mechanistic and staged pharmacological strategy to overcome HBV immune tolerance and advance toward functional cure.