preS1–HBsAg Fusion mRNA Vaccine Enhances Immune Responses and Suppresses Virus in a Chronic Hepatitis B Mouse Model
Background: Combining antigens to revive weakened immunity in chronic infection
A messenger RNA (mRNA) vaccine fusing preS1 with hepatitis B surface antigen (HBsAg) lowered viral markers and induced both T-cell and antibody responses in a chronic hepatitis B mouse model. This preclinical study was published in Nature Communications. It did not establish therapeutic efficacy in humans or elimination of viral reservoirs.
Preventive vaccines based on HBsAg protect against new infections but have limited ability to treat chronic infection. Immune tolerance resulting from persistent antigen exposure and impaired T-cell function constrain responses to therapeutic vaccines. The researchers sought an antigen combination that would strengthen immune responses while retaining HBsAg’s role in inducing antibodies.
Key findings: Distinct antigen roles and seroconversion after additional stimulation
The researchers compared four mRNA constructs: S-HBsAg alone, and preS1, HBxAg, or HBcAg each fused to S-HBsAg. The preS1 fusion did not include the preS2 region. Each mRNA was formulated in lipid nanoparticles (LNPs) for administration.
The experiments used male mice in which a hepatitis B virus (HBV) genome was delivered using an adeno-associated virus (AAV) vector. The delivered HBV genome corresponded to genotype D and serotype ayw. Vaccination began four weeks after vector injection, with three doses given at weekly intervals. The HBsAg-only group received 10 μg of mRNA per dose; the other constructs were administered at corresponding equimolar amounts. The main comparisons of viral markers included five mice per group.
Among the candidates tested, the preS1–HBsAg group showed superior results in reducing serum and intrahepatic HBV DNA and intrahepatic viral antigens. Intrahepatic HBV RNA was also reduced at week 13 after vector injection. In an experiment involving additional stimulation with recombinant HBsAg and incomplete Freund’s adjuvant after vaccination, anti-HBsAg antibody seroconversion was observed in 4 of 5 mice (80%). This proportion reflects the additional-stimulation condition and does not represent a cure rate in patients.
preS1 alone also lowered several viral markers, but anti-HBsAg antibodies were not detected. Compared with preS1 alone, the fusion vaccine enhanced preS1-specific CD8⁺ T-cell responses while also inducing an antibody response. This supports the interpretation that preS1 drives antiviral T-cell responses, while HBsAg contributes to antibody induction and immune enhancement.
In mechanistic experiments using a model antigen, linking the antigen to HBsAg increased antigen-presenting cell (APC) activation and antigen presentation. Macrophage experiments supported direct mRNA uptake followed by endogenous antigen presentation via major histocompatibility complex class I (MHC-I), following intracellular protein synthesis, as the main pathway. Cross-presentation following reuptake of secreted antigen made a small contribution. This adjuvant-like effect was pronounced under conditions of mRNA delivery and intracellular expression; it does not mean that circulating HBsAg itself has the same function in people with chronic infection.
Significance and outlook: Combination effects and remaining questions in natural-infection models
In the interferon-α (IFN-α) combination group, researchers observed fewer intrahepatic cells positive for HBV core antigen (HBcAg) and improvements in some memory T-cell phenotypes. However, differences in serum HBsAg and HBV DNA compared with the vaccine-only group were not statistically significant. In the safety assessment, body weight remained stable at the tested doses and during the observation period, and no persistent rise in liver enzymes or treatment-related liver tissue damage was identified. These data do not establish long-term safety in humans.
The AAV model evaluates immune control of hepatocytes expressing HBV antigens, but does not adequately reproduce viral entry during natural infection or repeated reinfection. Changes in or elimination of the covalently closed circular DNA (cccDNA) reservoir, and the durability of the effect under reinfection pressure, were not directly tested. Applying results from male mice with a single HBV genotype to patients will require further validation across diverse genotypes and in models that better reflect natural infection.
The study design and results are presented in the full paper.
Therapeutic vaccination for chronic hepatitis B (CHB) remains challenging, as persistent immune tolerance to hepatitis B surface antigen (HBsAg) impedes anti-HBsAg seroconversion. Here we show that screening antigen combinations incorporating HBsAg identifies preS1-HBsAg as the optimal pairing. The resulting dual-antigen mRNA vaccine elicits robust hepatitis B virus (HBV)-specific immune responses in chronic HBV mouse models, leading to near-complete viral genome clearance, marked reduction of multiple HBV antigens, and serological conversion. Mechanistic analyses reveal that preS1 serves as the primary driver of HBV-specific T cell responses, while HBsAg contributes to both anti-HBsAg seroconversion and intrinsic adjuvant activity. Specifically, when delivered in mRNA form, HBsAg promotes antigen-presenting cell (APC) activation, enhances antigen presentation, and amplifies preS1-specific CD8⁺ T cell responses. Furthermore, combining the preS1-HBsAg vaccine with interferon-α (IFN-α) enhances antiviral efficacy and immune memory while maintaining a favorable safety profile. These findings establish preS1-HBsAg mRNA vaccination as a promising and translatable therapeutic strategy for functional cure of CHB.
This study provides a basis for evaluating T-cell and antibody responses together when selecting antigens for therapeutic vaccines. preS1 contributed to virus-specific T-cell responses, while HBsAg expressed from mRNA contributed to antibody induction and an adjuvant-like effect. In follow-up development, this combination could be tested across diverse HBV genotypes and in models that better reflect natural infection and reinfection, comparing antigen reduction, antibody responses, liver injury, and durability of the effect. IFN-α combinations should also be assessed by distinguishing serum viral markers from intrahepatic and immune measures. Changes in memory T-cell phenotypes alone cannot establish long-term prevention of relapse. These findings inform further preclinical study design; they do not support determining doses for individual patients or stopping existing treatment.