Protein Priming Followed by a Replication-Competent VSV-GP Vector Boost Induces Sustained Immune Control in Therapeutic Hepatitis B Vaccination

<b>Background/Objectives</b>: Eliciting robust immune responses against the hepatitis B virus (HBV) through therapeutic vaccination holds promise for curing chronic hepatitis B. We previously developed the heterologous protein prime/viral vector boost clinical vaccine candidate, <i>TherVacB</i>. Here, we evaluated a replication-competent chimeric vesicular stomatitis virus vector (VSV-GP) as an alternative viral vector boost vaccine. <b>Methods</b>: A recombinant VSV-GP vector co-expressing HBV surface and core antigens (VSV-GP-HBs/c) was generated and characterized for antigen expression. Its immunogenicity, antiviral efficacy, and durability were assessed in HBV-naïve and HBV-carrier mice, using protein primed, viral vector-primed, and multi-viral vector boost regimens. <b>Results</b>: VSV-GP-HBs/c efficiently expressed both HBV antigens in vitro. A single immunization with VSV-GP-HBs/c induced only weak HBV-specific immune responses in vivo. Replacing protein priming with VSV-GP-HBs/c resulted in modest immune activation and limited antiviral effects in HBV-carrier mice. In contrast, substituting the modified vaccinia virus Ankara (MVA)-HBs/c boost in the <i>TherVacB</i> regimen with VSV-GP-HBs/c elicited robust HBV-specific antibody responses and strong CD4 and CD8 T-cell immunity, assessed by intracellular IFN-γ staining after peptide stimulation. This regimen achieved a substantial reduction in serum HBsAg levels, numbers of HBV-positive hepatocytes, and intrahepatic HBV-DNA, with antiviral efficacy comparable to that of the classical <i>TherVacB</i> regimen. Notably, a second viral vector boost did not enhance HBV-specific immunity or antiviral efficacy; instead, it promoted dominant vector-specific CD8 T-cell responses. Long-term analyses performed 10 weeks after the last vaccination further demonstrated that a single protein-prime/VSV-GP-HBs/c boost was sufficient to achieve sustained antiviral control. <b>Conclusions</b>: These findings identify VSV-GP-HBs/c as an effective boost vector for therapeutic hepatitis B vaccination and establish protein priming followed by a single viral vector boost as an optimal strategy for sustained antiviral immunity.