Particulate hepatitis B core antigen (HBcoreAg) is a potent immunogen used as a vaccine carrier platform. HBcoreAg produced in E. coli encapsidates random bacterial RNA (bRNA). Using the heterologous protein-prime, viral-vector-boost therapeutic hepatitis B vaccine TherVacB, we compared the properties of different HBcoreAg forms. We explored how the content of HBcoreAg modulates antigen stability, immunogenicity, and antiviral efficacy. bRNA was removed from HBcoreAg by capsid disassembly, followed by reassembly in the absence or presence of specific nucleic acid-based adjuvants poly I:C or CpG. The morphology and structure of empty, bRNA-containing and adjuvant-loaded HBcoreAg were monitored by electron microscopy and nuclear magnetic resonance spectroscopy. Empty, bRNA-containing or adjuvant-loaded HBcoreAg were applied together with HBsAg and with or without nucleic acid-based external adjuvants within the TherVacB regimen in both wild-type and HBV-carrier mice. While HBcoreAg retained its structure upon bRNA removal, its stability and immunogenicity decreased significantly. Loading HBcoreAg with nucleic acid-based adjuvants re-established stability of the capsid-like antigen. Immunization with poly I:C- or CpG-loaded HBcoreAg induced high antibody titers against co-administered HBsAg. When applied within the TherVacB regimen, they activated vigorous HBcoreAg- and HBsAg-specific T-cell responses in wild-type and HBV-carrier mice, requiring a significantly lower dose of adjuvant compared to externally added adjuvant. Finally, immunization with adjuvant-loaded HBcoreAg mixed with HBsAg led to long-term control of persistent HBV replication in the HBV-carrier mice. Adjuvant-loaded HBcoreAg retained capsid integrity and stability, was as immunogenic in vivo as externally adjuvanted HBcoreAg, requiring lower adjuvant levels, and supported immunity against co-administered, non-adjuvanted HBsAg. Thus, adjuvant-loaded HBcoreAg represents a promising novel platform for vaccine development. Hepatitis B core antigen (HBcoreAg) recapitulates the capsid of the HBV that hosts the viral genome. Produced recombinantly, it is not infectious but emerges as a potent immunogen in vaccine development. In this preclinical study, we show that loading HBcoreAg with defined nucleic-acid-based adjuvants on the one hand stabilizes the HBcoreAg with standardized capsid content and, on the other hand, efficiently promotes the immunity of HBcoreAg and a co-administered antigen, allowing for reduced adjuvant doses. Therefore, adjuvant-loaded HBcoreAg not only serves as an encouraging option for therapeutic hepatitis B vaccines, but could also act as an efficient adjuvant delivery system for other types of vaccine.

Background/Objectives: 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, TherVacB. Here, we evaluated a replication-competent chimeric vesicular stomatitis virus vector (VSV-GP) as an alternative viral vector boost vaccine. Methods: 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. Results: 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 TherVacB 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 TherVacB 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. Conclusions: 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.