Introduction

Chronic hepatitis B is a global health problem caused by the hepatitis B virus (HBV). Hepatitis B virus (HBV) is a hepatophilic bacterium that causes both acute and chronic diseases. It is estimated that 257 million people worldwide currently suffer from chronic HBV infection and are at high risk of developing liver diseases, including cirrhosis and hepatocellular carcinoma [1, 2]. However, currently used drugs cannot completely cure chronic HBV infection, and their long-term use may lead to the development of resistance and severe side effects [3]. Therefore, overcoming these limitations is crucial for developing novel therapeutic strategies and drugs for chronic HBV infections.

After successful invasion, HBV often actively escapes the surveillance of the host immune system by impairing the antiviral immune function of both the innate and adaptive immune systems. Chronic HBV infection causes abnormal functioning of innate immune cells, such as dendritic cells (DCs), macrophages, and natural killer cells [4], and interferes with the synthesis of type I interferons (IFNs) and the activation of related signaling pathways. The initiation of adaptive immune responses is affected by a functionally compromised innate immune system. In patients with chronic HBV infection, CD4+ and CD8+ T cells highly express immune checkpoint molecules, such as PD-1, LAG-3, and TIM-3, indicating functional exhaustion [5]. Moreover, B-cell function is disrupted, resulting in reduced production of neutralizing antibodies [6].

Therapeutic vaccine development is a hot research topic in chronic hepatitis treatment. Unlike prophylactic vaccines, therapeutic vaccines resolve the challenge of host immune tolerance to HBV and promote HBV clearance by HBV-specific T cells. Adjuvants, defined as components capable of enhancing the immunogenicity of antigens and shaping the strength of antigen-specific immune responses, are key components of therapeutic HBV vaccines [7,8,9]. However, the development of therapeutic HBV vaccines remains a significant challenge due to the lack of ideal adjuvants or delivery systems. The cGAS-STING signaling pathway is important for the innate immune system because it activates DCs and induces the production of type I IFN and IFN-stimulated genes via the TBK1/IRF3 axis [10]. In addition, STING activates signals such as NFκB, IFN regulatory factor 3 (IRF3), and STAT6, improving IFN antiviral functions [11, 12]. As adjuvants, STING agonists effectively strengthen humoral and CD8+ T-cell immune responses induced by influenza vaccines, producing long-term immune protection against H1N1, H3N2, H5N1, and H7N9 viruses in mice [13]. The SARS-CoV-2 vaccine with a STING agonist induces high-titer antibodies and strong T-cell immune responses [14]. In addition, DMXAA, a STING agonist, reportedly activates macrophage autophagy, resulting in HBV clearance through the epigenetic suppression of cccDNA [15]. Estefania Rodriguez-Garcia et al. also showed that an adenoviral vector expressing diguanylate cyclase (cGAS) could activate DCs and promote STING-dependent HBV clearance in HBV-carrier mice [16]. These studies suggest that STING agonists are potential adjuvants for therapeutic HBV vaccines.

However, STING agonists are small molecules that are rapidly distributed throughout the body and are incompatible with antigen-presenting cells (APCs), such as DCs in the lymph nodes, and may cause systemic toxicity [17]. As essential immune organs, the lymph nodes are the sites where the immune response to exogenous antigens is initiated. DCs present antigen peptides to T cells, leading to their differentiation into specialized effector T-cell subtypes [18,19,20]. With the aid of Tfh cells, B cells further differentiate into plasma cells that produce antibodies [21]. Antigen uptake by DCs in the lymph node is essential for vaccine-induced CD8+ T-cell-mediated cellular immune responses [19]. Therefore, targeted delivery of antigens and adjuvants to DCs in the lymph nodes and prolonged antigen retention are important for enhancing the effects of therapeutic vaccines. NPs with a suitable particle size of 10–100 nm can effectively deliver antigens to lymph nodes [22,23,24,25,26]. Thus, nanoparticle-loaded antigens administered subcutaneously, intradermally, or intramuscularly can be passively targeted to the lymph nodes via lymphatic capillaries. Furthermore, nanoparticle-loaded antigens are taken up by APCs more efficiently than free antigens and are effectively protected from environmental factors, such as pH, ions, proteases, and other factors in vivo [24, 25]. Additionally, compared with free antigens, nanoparticle-adsorbed or encapsulated antigens are released more slowly inside APCs, prolonging the antigen retention time of APCs and continuously stimulating APCs [27,28,29], which is beneficial for the activation of the adaptive immune response.

In this study, we prepared a co-delivering hepatitis B surface antigen (HBsAg) and cyclic diguanylate monophosphate (c-di-GMP) nanovaccine (named the PP-SG nanovaccine) using a copolymer (acryloyl acetone oxime-2-(N-ethyl-N-propyl amino) ethyl methacrylate (pAA-pEPEMA)). We demonstrated that this nanovaccine could target lymph nodes and increase antigen phagocytosis by DCs. In addition, the PP-SG nanovaccine safely and effectively cleared HBV in HBV-carrier mice by inducing an HBV-specific T-cell response and the production of anti-HBs. Therefore, these findings suggest that nanovaccines targeting lymph nodes and co-delivering HBsAg and STING agonists may be an efficient strategy for treating chronic HBV infection.

Materials and methods

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).

Results

Physicochemical properties of the HBV nanovaccine

Figure 1A shows a comprehensive schematic representation of the study design. STING agonists activate the cGAS-STING pathway and induce type I IFN production, resulting in DC activation and enhanced adaptive immune responses [10,11,12]. However, we found that chronic HBV infection affected the signaling pathway involved in the STING-mediated induction of host immune responses compared to that in healthy donors (HDs), accompanied by a significant decrease in the expression of p-IRF3 (Fig. S1). We prepared a nanovaccine that co-delivered HBsAg and the STING agonist c-di-GMP to restore the cGAS-STING pathway and activate DCs (Fig. 1B). In the initial phase of our investigation, we conducted a comprehensive analysis of the prepared nanovaccine, primarily focusing on particle size and zeta potential characteristics. The hydrated particle size of the nanovaccine formulation was meticulously assessed using the dynamic light scattering technique. The results of this analysis revealed that the nanovaccine exhibited a particle size range of 130–150 nm (Fig. 1C). The particle size underscores the colloidal nature of the nanovaccine formulations. The zeta potential of the nanovaccine, which is a crucial electrostatic aspect, was rigorously measured to assess the surface charge and overall stability of the formulation. Remarkably, the zeta potential was nearly neutral and approximately zero (Fig. 1D). The electrically neutral zeta potential suggested that the nanovaccine possesses minimal inherent surface charge, indicating a balanced electrostatic state. To visualize and characterize the structural attributes of the nanovaccine at a higher resolution, transmission electron microscopy (TEM) was used. TEM imaging confirmed the homogeneity of the nanovaccine formulation. The nanovaccine particles exhibited a consistent homogenous spherical morphology (Fig. 1E). This consistent spherical structure indicated a well-controlled and defined synthesis process, highlighting the precision with which the nanovaccine formulation was engineered. Furthermore, TEM analysis revealed that the actual particle size of the nanovaccine was 30–40 nm, albeit smaller than the hydrated dimensions because of the dry-state imaging nature of TEM, which corroborates the overall size distribution range obtained through particle size techniques. Collectively, these findings indicate a well-engineered, uniformly sized, and potentially effective nanovaccine formulation.

Fig. 1
figure 1

Schematic illustration of the study design and the characterization of the HBV nanovaccine. (A) A comprehensive schematic representation of the study design. (B) The preparation of HBV nanovaccines. (C) The particle size of the PP-SG vaccine. (D) The zeta potential of the PP-SG vaccines. (E) TEM image of the PP-SG vaccine. SG is a physical mixture of HBsAg and c-di-GMP; PP-S is a nanovaccine containing HBsAg; and PP-SG is a nanovaccine containing HBsAg and c-di-GMP. One representative of at least three independent experiments is shown. TEM, transmission electron microscopy; HBsAg, hepatitis B surface antigen, c-di-GMP, cyclic diguanylate monophosphate

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The PP-SG nanovaccine increases the antigen uptake capacity and function of APCs in the lymph node

The antigens must be taken up and processed by APCs to present the antigen peptides to T cells, inducing T-cell activation and immune responses in the lymph nodes [18, 20, 32]. To demonstrate whether the prepared nanovaccine could promote the phagocytosis of antigens by APCs in the lymph nodes, fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) was used as the antigen for the nanovaccine (PP-BSA). Six hours after subcutaneous administration, the levels of FITC-BSA in the DCs and macrophages in the lymph nodes were measured using flow cytometry. Compared with those of mice treated with FITC-BSA alone, the DCs and macrophages in the lymph nodes of mice treated with the PP-BSA nanovaccine showed increased uptake of FITC-BSA (Fig. 2A). In addition, when BMDCs (Fig. S2) were treated with the PP-BSA nanovaccine or BSA alone in vitro, the FITC-BSA levels in the PP-BSA-treated BMDCs were significantly greater than those in the BSA-treated BMDCs in a time-dependent manner (Fig. 2B). These results indicate that the nanovaccine increased the efficiency of antigen delivery.

Fig. 2
figure 2

The PP-SG nanovaccine enhanced the antigen uptake capacity and maturation of BMDCs. (A) C57BL/6J mice were subcutaneously injected with PBS, FITC-BSA, or PP-BSA (FITC-BSA, 1.25 µg/mouse). Lymph nodes were excised 6 h after the injection, and flow cytometry was performed to analyze the FITC-BSA levels of macrophages (left) and DCs (right) from the lymph nodes. n = 3 for each group. (B) BMDCs were treated with PBS, BSA, or PP-BSA for 2, 4, or 8 h. Then, FITC-BSA levels were analyzed by flow cytometry. (C and D) BMDCs were treated with PBS, SG, PP-S, or PP-SG (HBsAg, 0.3 µg/ml; c-di-GMP, 1.5 µg/ml) combined with 5 µg/ml FITC-BSA for 36 h; after that, the expression of CD86 and MHC-II (C) and the phagocytosis of FITC-BSA (D) were measured by flow cytometry. SG is a physical mixture of HBsAg and c-di-GMP; PP-S is a nanovaccine containing HBsAg; and PP-SG is a nanovaccine containing HBsAg and c-di-GMP. The data are presented as the mean ± SEM (n ≧ 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. BMDCs, bone marrow-derived dendritic cells; HBsAg, hepatitis B surface antigen; c-di-GMP, cyclic diguanylate monophosphate

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Subsequently, BMDCs were stimulated with SG, PP-S, or PP-SG nanovaccines for 36 h in vitro. FITC-BSA was added to evaluate the effect of these vaccines on the phagocytic ability and maturation of BMDCs. Compared with the PBS-treated BDMCs, although the SG and PP-S vaccines increased the expression of MHC-II and CD86 on the BMDCs to some extent, the PP-SG nanovaccine significantly upregulated the expression of MHC-II and CD86 compared with the SG and PP-S nanovaccine-treated BDMCs (Fig. 2C). The level of FITC-BSA was greater in the PP-SG-treated BMDCs than in the BMDCs in the other treatment groups (Fig. 2D). These results suggest that the PP-SG nanovaccine promotes the phagocytic and antigen-presenting ability of DCs.

The PP-SG nanovaccine effectively clears HBV and prevents HBV reinjection

To evaluate the efficacy of the PP-SG nanovaccine against chronic HBV infection, an HBV-carrier mouse model was generated by administering a hydrodynamic injection of pAAV/HBV 1.2 and HBsAg, SG, PP-S, and PP-SG nanovaccines (Fig. 3A). Considering that prime-boost immunizations could elicit immune responses with greater quality, magnitude, and duration, here we also administered PP-SG nanovaccine in a prime-booster vaccination strategy. Consistent with previous reports [31, 33], HBsAg alone could not efficiently eliminate HBV in HBV-carrier mice (Fig. 3B–D). We observed that in the SG, PP-S, and PP-SG treatment groups, serum HBsAg levels were significantly reduced and were almost undetectable on day 7 after the last immunization (Fig. 3B), and no significant liver damage was observed (Fig. S3A and B). In addition, the serum HBV DNA (Fig. 3C), intrahepatic HBV DNA, total HBV RNA, total HBV 3.5 kb RNA (Fig. 3D), and HBcAg levels (Fig. 3E) were significantly reduced by the vaccines, especially in the PP-SG-immunized group. Notably, compared with the other vaccines, the PP-SG nanovaccine induced the highest level of protective anti-HBs IgG (Fig. 3F).

Fig. 3
figure 3

The PP-SG nanovaccine effectively eliminated HBV and protected against reinfection in HBV-carrier mice. (A) Schematic illustration of the HBV vaccination protocol for HBV carrier mice. HBV-carrier mice (pAAV-HBV1.2) were divided into five groups and immunized subcutaneously with PBS, SG, PP-S, or PP-SG (HBsAg, 0.3 µg/ml; c-di-GMP, 1.5 µg/ml) on days 0, 7, and 14. For the long-term immune memory assay, the immunized HBV-carrier mice were rechallenged with a hydrodynamic injection of 8 µg of the pAAV/HBV1.2 plasmid on day 59 after treatment initiation. (B-C) Serum was collected the day before each immunization, and the serum HBsAg levels were measured by CLIA (B). Serum HBV DNA levels were measured on day 21 after treatment (C). (D) HBV DNA, total HBV RNA, and total HBV 3.5 kb RNA in liver tissues were detected on day 21 by RT‒PCR, and the data are shown on a log10 scale. (E) HBcAg expression was analyzed on day 21 by immunohistochemical staining. (F) Serum anti-HBs IgG levels after different treatments were measured by ELISA. (G) Serum HBsAg was measured on days 2 and 4 after rechallenge using CLIA. (H) Serum anti-HBs IgG was analyzed on days 2 and 4 after rechallenge using ELISA. SG is a physical mixture of HBsAg and c-di-GMP; PP-S is a nanovaccine containing HBsAg; and PP-SG is a nanovaccine containing HBsAg and c-di-GMP. All data are presented as the mean ± SEM (n ≧ 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HBsAg, hepatitis B surface antigen; ELISA, enzyme-linked immunosorbent assay

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The major challenge of clinical CHB therapy is the high incidence of recurrence after therapy discontinuation. To determine whether vaccination can induce signs of the development of memory responses against HBV reinfection, the immunized HBV-carrier mice were rechallenged with a hydrodynamic injection of 8 µg of the pAAV/HBV1.2 plasmid on day 59 after treatment initiation, as previously reported [31]. We found that the number of mice with high HBsAg serum levels on days 2 and 4 post-rechallenge was lower in the PP-SG-immunized group than in the other treatment groups (Fig. 3G), which was accompanied by high levels of anti-HBs IgG (Fig. 3H). These results show that the PP-SG nanovaccine effectively induces long-lasting immunity against HBV in HBV-carrier mice.

The PP-SG nanovaccine augments the function of DCs in HBV-carrier mice

Chronic HBV infection impairs DC maturation and activation, resulting in immune escape and tolerance to HBV [34, 35]. DCs are divided into different subpopulations, with cDC1s and cDC2s presenting antigens mainly to CD8+ and CD4+ T cells, respectively [32, 36, 37]. Among the SG-, PP-S-, and PP-SG nanovaccine-treated HBV-carrier mice, we found that the PP-SG nanovaccine increased the percentage of hepatic DCs, exhibiting the strongest inhibitory effect on the expression of PD-L1 and a promotional effect on the expression of MHC-II, MHC-I, and CD86 in hepatic DCs (Fig. 4A and B). Although the PP-SG nanovaccine did not increase the percentage of hepatic cDC1s or cDC2s, it significantly reduced the expression of PD-L1 on hepatic cDC1s and cDC2s and increased the expression of MHC-II, MHC-I, and CD86 on cDC1s and cDC2s (Fig. 4C and D). We observed similar phenomena in DCs, cDC1s, and cDC2s in the spleen (Fig. S4A–C). These results suggest that the PP-SG nanovaccine can promote the maturation and activation of DCs in HBV-carrier mice, enhancing the generation of anti-HBV immune responses.

Fig. 4
figure 4

The PP-SG nanovaccine downregulated the expression of PD-L1 on DCs and activated DCs in HBV-carrier mice. HBV-carrier mice treated with PBS or vaccines were rechallenged with 8 µg pAAV/HBV 1.2 on day 59. The following analyses were performed on day 7 after the HBV rechallenge. (A) The strategy for gating hepatic mononuclear cells on DCs, cDC1s, and cDC2s. (B) The percentage of hepatic DCs and the expression of PD-L1, MHC-I, MHC-II, and CD86 on hepatic DCs. (C) The percentage of hepatic cDC1s and the expression of PD-L1, MHC-I, MHC-II, and CD86 on hepatic cDC1s. (D) The percentage of hepatic cDC2s and the expression of PD-L1, MHC-I, MHC-II, and CD86 on hepatic cDC2s. SG is a physical mixture of HBsAg and c-di-GMP; PP-S is a nanovaccine containing HBsAg; and PP-SG is a nanovaccine containing HBsAg and c-di-GMP. The data are presented as the mean ± SEM (n ≧ 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DC, dendritic cell; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; c-di-GMP, cyclic diguanylate monophosphate

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The PP-SG nanovaccine increases the proportion and reverses the exhaustion of HBV-specific CD8+T cells

CD8+ T cells, especially CD11ahi CD8αlo T cells, which are antigen-specific CD8+ T cells, play a crucial role in HBV clearance [31, 32]. Therefore, we evaluated the effects of the PP-SG nanovaccine on HBV-specific CD8+ T cells in HBV-carrier mice. We found that the proportion of HBV-specific CD8+ T cells in the liver significantly increased in mice vaccinated with the SG, PP-S, or PP-SG nanovaccine compared to that in mice treated with PBS (Fig. S4 and 5 A–B). Although the proportion of HBV-specific CD8 + T cells in the PP-SG nanovaccine group was not greater than that in the SG and PP-S vaccine groups, we observed that the PP-SG nanovaccine had the strongest downregulatory effect on the immune checkpoint molecules PD-1, LAG-3, and TIM-3 (Fig. 5C). The proportions of HBV-specific CD8+ T cells with single (PD-1+, TIM-3+, and LAG-3+), double (PD-1+ TIM-3+, PD-1+ LAG-3+, and TIM-3+ LAG-3+), or triple (PD-1+ TIM-3+ LAG-3+) immune checkpoint molecule expression were also analyzed, which showed that the PP-SG nanovaccine-treated mice had the lowest proportion of exhausted HBV-specific CD8+ T cells (Fig. 5D). These results show that the PP-SG nanovaccine can disrupt chronic HBV infection-induced immune tolerance, increase the proportion of HBV-specific CD8 + T cells, and reverse the exhaustion of these cells.

Fig. 5
figure 5

The PP-SG nanovaccine increased the proportion and reversed the exhaustion of HBV-specific CD8+T cells in HBV-carrier mice. HBV-carrier mice were subcutaneously injected with PBS, SG, PP-S, or PP-SG (HBsAg, 0.3 µg/ml; c-di-GMP, 1.5 µg/ml) on days 0, 7, and 14. The following analyses were performed on day 7 after the HBV rechallenge. (A–B) The proportion of HBV-specific CD8+ T cells in the liver was measured by flow cytometry. (C) The expression of immune checkpoint molecules, such as PD-1, LAG-3, and TIM-3, on HBV-specific CD8+ T cells in the liver was analyzed by flow cytometry. (D) Pie charts showing the proportions of cells expressing 0–3 of the immune checkpoint molecules tested. SG is a physical mixture of HBsAg and c-di-GMP; PP-S is a nanovaccine containing HBsAg; and PP-SG is a nanovaccine containing HBsAg and c-di-GMP. The data are presented as the mean ± SEM (n ≧ 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen

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The PP-SG nanovaccine promotes the proliferation and activation of CD8+T cells in HBV-carrier mice

CD8+ T-cell-mediated cellular immune responses are critical for developing therapeutic HBV vaccines. Ki-67 and ICOS levels reflect the proliferation and activation of CD8+ T cells, respectively. We found that SG and the PP-S nanovaccine increased the levels of Ki-67 and ICOS in hepatic CD8+ T cells from HBV-carrier mice, and this effect was more significant in PP-SG nanovaccine-treated mice (Fig. 6A and B), accompanied by the upregulation of IFN-γ, TNF-α, perforin, and IL-2 (Fig. 6C). The proportions of hepatic multifunctional CD8+ T cells expressing single (IFN-γ+, TNF-α+, IL-2+, and perforin+), double (IFN-γ+ IL-2+, IFN-γ+ TNF-α+, IFN-γ+ perforin+, IL-2+ TNF-α+, IL-2+ perforin+, TNF-α+ perforin+), triple (IFN-γ+ IL-2+ TNF-α+, IFN-γ+ IL-2+ perforin+, IFN-γ+ TNF-α+ perforin+), or quadruple (IFN-γ+ TNF-α+ IL-2+ perforin+) cytokines were significantly increased in the HBV-carrier mice treated with the PP-SG nanovaccine (Fig. 6D). Similar phenomena were observed for CD8+ T cells in the lymph nodes (Fig. 6E and F). These results indicate that the PP-SG nanovaccine promotes proliferation and augments the function of CD8+ T cells.

Fig. 6
figure 6

The PP-SG nanovaccine induced the proliferation and activation of CD8+T cells. HBV-carrier mice were subcutaneously injected with PBS, SG, PP-S, or PP-SG (HBsAg, 0.3 µg/ml; c-di-GMP, 1.5 µg/ml) on days 0, 7, and 14. The following analyses were performed on day 7 after the HBV rechallenge. (A) The proliferation of CD8+ T cells in the liver was analyzed by flow cytometry and is presented as histograms showing the percentage and MFI of Ki-67. (B) The activation of CD8+ T cells in the liver was analyzed by flow cytometry and is presented as histograms showing the percentage and MFI of ICOS. (C and E) Mononuclear liver and lymph node cells were stimulated with PMA/ionomycin in the presence of BFA (5 µg/ml) and IL-2 (100 U/ml) for 4 h. The expression of IFN-γ, TNF-α, perforin, and IL-2 on CD8+ T cells in the liver (C) and lymph nodes (E) was analyzed by flow cytometry. (D and F) The pie charts show the proportion of cells expressing between 0 and 4 of the cytolysis-associated molecules tested (n ≧ 5). SG is a physical mixture of HBsAg and c-di-GMP; PP-S is a nanovaccine containing HBsAg; and PP-SG is a nanovaccine containing HBsAg and c-di-GMP. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HBsAg, hepatitis B surface antigen; c-di-GMP, cyclic diguanylate monophosphate

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The PP-SG nanovaccine regulates the function of CD4+T cells

During HBV infection, CD4+ T cells participate in the anti-HBV immune response by regulating the activity and function of other immune cells, including CD8+ T and B cells [38, 39]. However, similar to CD8+ T cells, CD4+ T cells overexpress immune checkpoints in patients with chronic HBV infection, resulting in impaired CD4+ T-cell function. Therefore, we examined the influence of the PP-SG nanovaccine on CD49d+ CD11ahi CD4+ T cells and HBV-specific CD4+ T cells [33, 40] in HBV-carrier mice. The results showed that the PP-SG nanovaccine effectively reduced the expression of PD-1, LAG-3, and TIM-3 on HBV-specific CD4+ T cells in the liver (Fig. S5 and 7 A), with the lowest single (PD-1+, TIM-3+, and LAG-3+); dual (PD-1+ TIM-3+, PD-1+ LAG-3+, and TIM-3+ LAG-3+); and triple (PD-1+ TIM-3+ LAG-3+) immune checkpoint molecule expression on HBV-specific CD4+ T cells (Fig. 7B). Furthermore, the PP-SG nanovaccine significantly upregulated Ki-67 and ICOS expression in splenic CD4+ T cells (Fig. 7C and D). Moreover, the PP-SG nanovaccine enhanced the production of IFN-γ, TNF-α, and IL-2 by hepatic CD4+ T cells (Fig. 7E) and increased the proportion of multifunctional CD4+ T cells expressing single (IFN-γ+, TNF-α+, and IL-2+), dual (IFN-γ+ IL-2+, IFN-γ+ TNF-α+, and IL-2+ TNF-α+), or triple cytokines (IFN-γ+ IL-2+ TNF-α+) (Fig. 7F). These results indicated that the PP-SG nanovaccine promoted the activation and multifunctionality of CD4+ T cells.

Fig. 7
figure 7

The PP-SG nanovaccine reversed chronic HBV infection-induced exhaustion of CD4+T cells. HBV-carrier mice were subcutaneously injected with PBS, SG, PP-S, or PP-SG (HBsAg, 0.3 µg/ml; c-di-GMP, 1.5 µg/ml) on days 0, 7, and 14. The following analyses were performed on day 7 after the HBV rechallenge. (A) The expression of immune checkpoint molecules, such as PD-1, LAG-3, and TIM-3, on HBV-specific CD4+ T cells in the liver was analyzed by flow cytometry. (B) The pie charts show the proportions of cells expressing 0–3 of the immune checkpoint molecules tested. The expression of Ki-67 (C) and ICOS (D) on CD4+ T cells in the spleen was analyzed via flow cytometry. (E) Mononuclear cells of the liver were stimulated with PMA/ionomycin in the presence of BFA (5 µg/ml) and IL-2 (100 U/ml) for 4 h. The expression of IFN-γ, TNF-α, and IL-2 on CD4+ T cells was analyzed by flow cytometry. (F) The pie charts show the proportion of cells expressing 0–3 of the tested cytolysis-associated molecules. SG is a physical mixture of HBsAg and c-di-GMP; PP-S is a nanovaccine containing HBsAg; and PP-SG is a nanovaccine containing HBsAg and c-di-GMP. All the data are presented as the means ± SEMs (n ≧ 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HBsAg, hepatitis B surface antigen; c-di-GMP, cyclic diguanylate monophosphate; HBV, hepatitis B virus

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Fig. 8
figure 8

STING agonist-based lymph node-targeted nanovaccine against chronic HBV infection. Chronic HBV infection affects the signaling pathway involved in STING-mediated induction of host immune responses in DCs, especially the p-IRF3 pathway. Based on this, we prepared a lymph node-targeted pAA-pEPEMA nanovaccine that co-delivered HBsAg and c-di-GMP to restore the cGAS-STING pathway and activate DCs, resulting in HBV elimination, disrupting chronic HBV infection-induced immune tolerance and increasing multifunctional antigen-specific CD8+ T and CD4+ T-cell responses. Moreover, the nanovaccine-immunized mice achieved partial anti-HBs IgG seroconversion and a long-lasting immune response against HBV reinjection, indicating that the nanovaccine is a promising vaccine candidate for clinical chronic hepatitis B therapy

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Discussion

The difficulty in curing chronic HBV infection is associated with HBV escape from immune surveillance in patients with chronic HBV infection. Studies have shown that chronic HBV infection inhibits the activity of APCs, such as DCs and macrophages [5, 34, 41]. In addition, chronic HBV infection induces the expression of immune checkpoint molecules, such as PD-1 and LAG-3, on T cells, blocking costimulatory signals and enhancing coinhibitory signals between DCs and T cells [4, 42,43,44]. All these factors interfere with antiviral immune responses, leading to HBV persistence. In turn, the long-term presence of HBV induces T-cell immune tolerance, resulting in functional exhaustion. In addition, HBV can increase the number of immunosuppressive cells or molecules, such as Tregs and IL-10, creating an immunosuppressive microenvironment [9, 45, 46]. Therefore, reversing the immunosuppressive microenvironment and activating immune cell function is key to curing chronic HBV infection [7, 9].

The activation of DCs in lymph nodes is important for activating CD8+ T-cell-mediated cellular immune responses, and the targeted delivery of antigens and adjuvants to DCs in lymph nodes enhances vaccine-induced immune responses. The cGAS-STING pathway is important for DC activation and is a promising target for activating cellular immunity [10, 47, 48]. In this study, we found that chronic HBV infection impaired STING-mediated induction of the host immune response signaling pathway compared with that in healthy donors; therefore, we chose the STING agonist c-di-GMP as an adjuvant to prepare a new therapeutic HBV vaccine in combination with HBsAg to restore the cGAS-STING pathway and activate DCs. Generally, vaccines prepared by physical mixing of HBsAg and c-di-GMP cannot target the lymph nodes. Moreover, as a small molecule, c-di-GMP is rapidly distributed throughout the body, with the risk of a systemic inflammatory response at high doses and difficulty remaining in the lymph nodes for a sufficient time [17]. Although intralymph node injection can overcome this obstacle to a certain extent, this method still has limitations, such as its cumbersome operation and small-dose administration. Based on these results, we used nanotechnology to overcome these limitations.

NPs can enter lymph nodes through lymphatic capillaries owing to their small particle size [22,23,24]. Nanodelivery systems protect antigens from environmental influences and safely deliver antigens and adjuvants to APCs [25]. Moreover, given that they are similar in size and composition to cells, nanoparticles can be rapidly taken up by cells through endocytosis [49]. Nanoparticles carrying HBsAg have a greater ability to induce antibody production than free HBsAg [50, 51]. In addition, the slow biodegradation rate of nanovaccines prolongs the residence time of antigens in APCs, augmenting the processing of antigens by APCs and the initiation of subsequent adaptive immune responses [27, 28]. Therefore, we prepared HBsAg with a c-di-GMP nanovaccine, named PP-SG using pAA-pEPEMA. To evaluate the efficacy of the PP-SG nanovaccine for HBV clearance and the underlying mechanisms, a physical mixture of HBsAg and c-di-GMP (SG) and a nanovaccine containing HBsAg (PP-S) were used as controls.

Using FITC-BSA, we first confirmed that the pAA-pEPEMA nanovaccine could target the lymph nodes, facilitate antigen delivery, and prolong antigen retention time in the lymph nodes in vivo. The “prime-boost” immunization strategy is conducive to activating immune responses; therefore, we vaccinated HBV-carrier mice three times in alternate weeks. Previous studies have shown that inflammatory stimulation alone, such as poly I: C or CpG administration [31, 52], cannot drive antigen-specific CD8+ T-cell responses or pathogen clearance, so we excluded the effects of c-di-GMP alone on HBV clearance during treatment. Our results showed that the SG, PP-S, and PP-SG vaccines effectively reduced serum HBsAg levels. However, both SG and PP-S had weaker effects on hepatic HBV RNA and DNA levels than did the PP-SG nanovaccine, while the PP-SG nanovaccine induced high levels of anti-HBs IgG. The PP-SG nanovaccine induced long-term immune memory against HBV reinjection in HBV-carrier mice. Therefore, the pAA-pEPEMA nanovaccine exhibited advantages for HBV clearance in HBV-carrier mice, and we focused on changes in the immune system upon PP-SG nanovaccine treatment.

DCs are crucial antigen-presenting cells. In patients with chronic HBV infection, the function of DCs is impaired, which is an important factor for T-cell dysfunction. DCs are divided into plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) and further divided into cDC1s and cDC2s with different functions [32, 36, 37]. cDC1s possess a cross-presentation function, presenting exogenous antigens via MHC-I to induce CD8+ T-cell activation, which is essential for activating antiviral immune responses. We found that the PP-SG nanovaccine significantly increased the levels of MHC I/II and the costimulatory molecule CD86 on cDC1s, indicating its superior ability to activate CD8+ T cells against pathogens.

Cellular immune responses mediated by antigen-specific CD8+ T cells are essential for HBV clearance. However, it has been shown that antigen-specific CD8+ T cells in patients with chronic HBV infection are often exhausted, characterized by the upregulation of immune checkpoint molecules, such as PD-1, LAG-3, and TIM-3 [4, 5, 43, 44]. High levels of immune checkpoint molecules on CD8+ T cells are closely associated with poor HBV clearance. Therefore, reversing HBV-specific CD8+ T-cell exhaustion is an important strategy for disrupting the immune-tolerant microenvironment in patients with chronic HBV infection. We found that the PP-SG nanovaccine increased the proportion of HBV-specific CD8+ T cells in HBV-carrier mice, accompanied by increased production of IL-2, IFN-γ, TNF-α, and perforin. Notably, the PP-SG nanovaccine significantly downregulated the expression of PD-1, LAG-3, and TIM-3 in CD8+ T cells, as well as PD-L1 expression on DCs, especially cDC1s. These findings suggest that the PP-SG nanovaccine can attenuate coinhibitory signals and reverse antigen-specific CD8+ T-cell exhaustion induced by chronic HBV infection. Additionally, STING-mediated Type I IFN induction in DCs is essential to trigger T cell-mediated immune responses [53,54,55], and we believe that the changes in inflammatory cytokines such as type I IFN, IL-2, IFN-γ, and TNF-α are involved in PP-SG nanovaccine-induced anti-HBV effects.

cDC2s deliver antigens to CD4+ T cells mainly via MHC-II and play a complementary role in inducing and maintaining both neutralizing antibodies and CD8+ T-cell responses to facilitate HBV clearance [37, 44, 56]. CD4+ T cells can provide activating factors to assist in the differentiation of naïve CD8+ T cells into effector CD8+ T cells [57]. Additionally, CD4+ T cells can help activate B cells, promoting the production of neutralizing antibodies [38]. Upon activation, CD4+ T cells differentiate into functionally distinct subsets, such as Th1, Th2, and Th17 cells. Th1 cells are involved in the regulation of the immune response by secreting cytokines, such as IFN-γ, TNF-α, and IL-2 [58]. The impaired function of CD4+ T cells in patients with chronic HBV infection disrupts antiviral immune responses [44, 59, 60]. We found that the PP-SG nanovaccine enhanced antigen presentation and maturation of cDC2s and facilitated anti-HBs IgG seroconversion against HBV, which is the ideal endpoint of HBV treatment [61]. Subsequently, the PP-SG nanovaccine promoted the proliferation and activation of CD4+ T cells, with downregulated expression of immune checkpoint molecules, such as PD-1, LAG-3, and TIM-3. Notably, the PP-SG nanovaccine upregulated the expression of cytokines, such as IL-2, IFN-γ, and TNF-α, on CD4+ T cells and promoted Th1-type cellular immune responses, which are beneficial to the CD8+ T-cell immune response.

In conclusion, the pAA-pEPEMA nanovaccine can target lymph nodes, enhance the uptake of antigens by DCs, and promote the maturation and activation of DCs in HBV-carrier mice. The numbers of multifunctional antigen-specific CD8+ T and CD4+ T cells were increased, and the levels of immune checkpoint molecules were decreased. Thus, the PP-SG nanovaccine induced significant HBV-specific cellular and humoral immune responses for HBV clearance and long-term immune memory to prevent HBV reinjection (Fig. 8). Thus, this study identified a potential therapeutic nanovaccine candidate for treating chronic HBV infection. Considering that CHB induces immune tolerance by impairing the host immune system, the PP-SG nanovaccine might induce stronger HBV-specific cellular and humoral immune responses in WT mice than in HBV-carrier mice and could also be a promising candidate for prophylactic HBV vaccines.