Patient samples

Peripheral blood samples from immune-tolerant CHB patients and healthy donors were collected from Qilu Hospital. This study was approved by the Institutional Review Board of Qilu Hospital of Shandong University (approval number: KYLL-2022(ZM)-1414) and was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all study participants. The inclusion criterion for all immune-tolerant CHB patients was an HBV DNA concentration > 2 × 104 IU/ml for hepatitis B e antigen (HBeAg)-positive patients who did not receive prior antiviral therapy. The exclusion criteria were liver disease of other etiologies (HCV, hepatitis D virus (HDV), alcoholic liver disease, nonalcoholic fatty liver disease, primary biliary cholangitis, autoimmune hepatitis, and hereditary metabolic liver disease), decompensated cirrhosis, HCC due to HBV or other etiologies, major systematic diseases, other malignancies, and so on. The clinical characteristics of the immune-tolerant CHB patients are shown in Supplementary Table 1.

Animals and reagents

Male C57BL/6J mice, 5–6 weeks old, were purchased from Beijing HFK Bioscience (Beijing, China). c-di-GMP was purchased from Invitrogen (Carlsbad, CA). HBsAg (HBsAg; H. polymorpha) was purchased from Dalian Hissen Biopharm. Co. Ltd. (Dalian, China).

Preparation and characterization of the HBV nanovaccines

The synthesis of pAA-pEPEMA and FITC-BSA has been described previously [30]. To formulate the HBV nanovaccines (containing HBsAg and c-di-GMP), the following procedures were performed: To 1 ml of 20 µg/ml HBsAg solution, 200 µl of 1 mg/ml c-di-GMP solution was added, followed by 2.8 ml of phosphate-buffered saline (PBS). The entire solution was mixed meticulously to obtain a final volume of 4 ml, which constituted the SG vaccine solution. The SG vaccine solution was determined to contain 5 µg of HBsAg and 50 µg of c-di-GMP per ml. The polymer pEPEMA-pAA was dissolved in sterile water at a concentration of 20 mg/ml, and the pH was maintained at 5.5. Additionally, 54 µl of 240 µg/ml HBsAg solution was dispersed in 1.5 ml of sterile water at a pH of 6.5. Optionally, 130 µl of 1 mg/ml c-di-GMP solution was added to the above dispersion. During continuous stirring, 150 µl of pEPEMA-pAA was added dropwise to the dispersion. After 10 min, the pH of the suspension was adjusted to 7.0 using a 0.1 M NaOH solution. The suspension was stirred for 2 h. The steps above were repeated, including pH adjustment and a further 6 h of stirring. The resulting formulations were denoted as PP-S (comprising pAA-pEPEMA and HBsAg) and PP-SG (containing pAA-pEPEMA, HBsAg, and c-di-GMP). These processes were performed meticulously to ensure accurate preparation of vaccine solutions with the specified components and concentrations. The nanovaccines were stored at 4 °C for further use. The particle size and zeta potential were measured using a Malvern Zetasizer Nano-ZS90 dynamic light scattering system (Malvern Instruments, Malvern, UK). The morphology of the nanovaccines was examined using TEM (JEM-100CX II, Jeol, Tokyo, Japan).

Preparation of BMDCs

Bone marrow cells were collected from the femurs and tibias of C57BL/6J mice under sterile conditions. After the red blood cells were lysed, the cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM glutamine, 0.1 mM nonessential amino acids, 5 ng/ml rmIL-4 (Peprotech, USA), and 10 ng/ml rmGM-CSF (Peprotech, USA) at 37 °C and 5% CO2 for 7 days. The medium was replaced on days 3 and 5, the BMDCs were harvested, and the proportion of CD11c+ cells was analyzed by flow cytometry on day 7.

HBV carrier mouse model

C57BL/6J mice aged 5–6 weeks were administered 8 µg of pAAV/HBV 1.2 plasmid (provided by Pei-Jer Chen from National Taiwan University College of Medicine, Taipei, Taiwan) through the tail vein by hydrodynamic injection. After 5–6 weeks, peripheral blood serum was collected to measure HBsAg levels. Mice with serum HBsAg concentrations above 500 ng/ml were considered HBV carriers, and intrahepatic DNA from these HBV-carrier mice confirmed the detection of HBV replicative intermediates, including HBV DNA and HBV RNA [31].

Mononuclear cell isolation

Single-cell suspensions from the liver, spleen and dLNs were isolated as described previously [31]. Briefly, PBS-perfused livers were passed through a 200-µm nylon cell strainer. The single-cell suspensions were centrifuged at 100 × rcf for 1 min to remove hepatocytes. The supernatants were centrifuged at 400 × rcf for 10 min to collect residual cells and then layered over 40% Percoll (GE Healthcare, Uppsala, Sweden). The hepatic mononuclear cells (MNCs) were harvested after centrifugation at 400 × rcf for 10 min, followed by RBC lysis and washing. The spleens and dLNs were passed through a 200-µm nylon cell strainer, and the precipitated cells were harvested, followed by RBC lysis and washing.

Immunization strategy and HBV challenge

HBV-carrier mice were vaccinated subcutaneously with PBS, SG, PP-S, or PP-SG nanovaccine containing 1 µg of HBsAg weekly for 3 weeks, and peripheral blood serum was collected the day before each immunization. For the HBV challenge assay, 8 µg of pAAV/HBV 1.2 was administered through the tail vein by hydrodynamic injection on day 59 after the last immunization, and peripheral blood serum was collected on days 61 and 63. All serum samples collected were stored at -80 °C for further use.

ELISA

HBsAg levels were measured using a hepatitis B surface antigen test kit (Autobio, China). Anti-HBs IgG levels were detected using an HBV surface antibody diagnostic kit (Wantai Biopharm, China). The levels of alanine aminotransferase (ALT) were detected by an alanine aminotransferase (ALT/GPT) test kit (Nanjing Jiancheng Bioengineering Institute, China).

HBV DNA and RNA detection

Serum HBV DNA was detected using the Hepatitis B Virus Nucleic Acid Quantification Kit (Da An Gene, China). Genomic DNA was extracted from liver tissues using a genomic DNA extraction kit (Tiangen, China). RNA from the liver tissues was extracted using TRIzol (CW Biotech, China), and cDNA was synthesized using a HiFiScript cDNA synthesis kit (CW Biotech, China). HBV DNA and RNA were analyzed by quantitative real-time PCR (LightCycler 480 II, Roche, USA) using SYBR Green mix (CW Biotech, China). The primers used are listed below: β-ACTIN-real-F: 5′-CATTGCTGACAGGATGCAGAAGG-3′; β-ACTIN-real-R: 5′-TGCTGGAAGGTGGACAGTGAGG-3′; HBV-total-real-F: 5′-TCACCAGCACCATGCAAC-3′; HBV-total-real-R: 5′-AAGCCACCCAAGGCACAG-3′; HBV-3.5 kb-RNA-real-F: 5′-AAGCCACCCAAGGCACAG-3′; HBV-3.5 kb-RNA-real-R: 5′-GAGGCGAGGGAGTTCTTCT-3′; HBV-DNA-real-F: 5′-CACATCAGGATTCCTAGGACC-3′; and HBV-DNA-real-R: 5′-GGTGAGTGATTGGAGGTTG-3′.

Hematoxylin-eosin staining and immunohistochemical analyses

Mouse livers were isolated, fixed in 4% formalin, and embedded in paraffin. Liver tissue was cut into 5-µm sections. After dewaxing and hydration, the sections were stained with hematoxylin & eosin (H&E) for histopathological evaluation. For immunohistochemical analysis, tissue sections were dewaxed, hydrated, and placed in citrate buffer for antigen repair. After blocking with goat serum (Boster, China), the sections were stained with an HBcAg antibody (Gene Tech Co., Ltd., China) overnight at 4 ℃ and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (ZSGB-bio Co., Ltd., China). After 15 min, DAB (ZSGB-bio Co. Ltd., China) substrate was added dropwise to the sections, which were subsequently stained with hematoxylin. After dehydration and permeabilization, the sections were mounted with a neutral resin. Images were captured using Image-Pro Plus software.

Flow cytometry

For surface molecules, mononuclear cells were blocked with rat serum at 4 °C for 30 min and then stained with the appropriate antibodies for 30 min at 4 °C. Unbound antibodies were removed by washing with PBS. For intracellular cytokines, mononuclear cells were seeded into 96-well plates at 2 × 106 cells per well with RPMI 1640 supplemented with 30 ng/ml PMA (Beyotime, China), 1 µg/ml ionomycin (Beyotime, China), 100 U/ml IL-2 (Changchun Institute of Biological Products Co., Ltd., China), and 5 µg/ml BFA (Biolegend, USA) at 37 °C and 5% CO2 for 4 h. Then, the cells were collected and blocked with rat serum at 4 °C for 30 min. After staining for surface markers, the cells were fixed in 4% paraformaldehyde solution at 4 °C for 30 min in the dark and then permeabilized with saponin solution. After blocking, the corresponding antibodies were added to label the intracellular molecules. All data were obtained using a FACSCalibur flow cytometer (BD Biosciences, USA) or a FACSCelesta flow cytometer (BD Biosciences, USA) and analyzed using FlowJo 10. The following fluorescently labeled antibodies were used: eFluor 450 anti-mouse Ki-67 (Cat. #48-5698-82), Brilliant Violet 605 anti-mouse PD-L1 (Cat. #12-5982-83), FITC anti-mouse CD4 (Cat. #11-0041-85), FITC anti-mouse CD11b (Cat. #12-0112-83), FITC anti-mouse perforin (Cat. #11-9392-82), PE anti-mouse ICOS (Cat. #12-9949-81), PE/Cyanine7 anti-mouse TNF-α (Cat. #25-7321-82), PerCP/Cyanine5.5 anti-mouse CD3e (Cat. #45-0031-82), PerCP/Cyanine5.5 anti-mouse CD40 (Cat. #48-5698-82), APC anti-mouse PD-1 (Cat. #65-0866-14), APC anti-mouse CD11c (Cat. #17-0114-82) from eBioscience (California, USA). Brilliant Violet 421 anti-mouse IFN-γ (Cat:# 505830), Brilliant Violet 650 anti-mouse CD86 (Cat:# 105036), Brilliant Violet 785 anti-mouse TIM-3 (Cat:# 119725), Brilliant Violet 785 anti-mouse I-A/I-E (MHC-II) (Cat:# 107645), FITC anti- mouse CD3e (Cat:# 100204), PE anti-mouse H-2Kb/H-2Db (MHC-I) (Cat:# 114608), PE/DazzleTM594 anti-mouse CD8α (Cat:# 100762), PE/DazzleTM594 anti-mouse IL-2 (Cat:# 503840), PerCP/Cyanine5.5 anti-mouse CD11a (Cat:# 101124) from Biolegend (San Diego, USA).

Analysis of the dataset

Gene Ontology enrichment analysis was performed using previously published data (accession number: GSE182159) to analyze gene expression and GSVA enrichment associated with STING-mediated induction of host immune responses.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (v6.0; GraphPad Software, La Jolla, CA, USA). The data were analyzed using an unpaired Student’s t-test or one-way analysis of variance. All the data are presented as the means ± SEMs, and P < 0.05 was considered to indicate statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).