: Chronic hepatitis B (CHB) remains being a major public health threat, and currently existing CHB therapies have limited efficacy and side effects. We have recently developed a vaccine termed VVX001 based on a recombinant fusion protein consisting of the preS domain of the large surface protein of hepatitis B virus (HBV) fused to grass pollen allergen peptides. VVX001 has been shown to induce preS-specific antibodies in grass pollen allergic patients, and sera of immunized subjects inhibited HBV infection in vitro. : In this study we investigated if immunization with VVX001 can induce preS-specific antibodies in CHB using the adeno-associated virus (AAV)-HBV murine model of CHB. Six groups of C57BL/6 female mice (n = 6) were transduced with AAV-HBV or AAV-Empty, and after six weeks, they were immunized five times with 20 µg of aluminum hydroxide-adsorbed VVX001 or preS or vehicle (Alum alone). Serum samples were taken continuously. Two weeks after the last immunization, spleen and liver mononuclear cells were collected. Serum reactivity to preS and preS-derived peptides was assessed by ELISA. B-cell responses were measured by ELISPOT assay, and intrahepatic lymphocyte (ILH) counts were determined by FACS. HBV DNA, HBsAg, HBeAg, ALT, and AST were assessed using commercial kits. : Our results show that VVX001 induces preS-specific IgG antibodies that cross-react with different HBV genotypes A-H and are directed against the sodium taurocholate co-transporting polypeptide (NTCP) receptor binding site of preS both in mice with and without HBV. Actively immunized AAV-HBV-treated mice had a higher number of intrahepatic lymphocytes than vehicle-vaccinated and mock-transduced animals. : These findings encourage performing further trials to study the potential of VVX001 for therapeutic vaccination against CHB.