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HBsAg-tagged tumour vaccine system eliminates solid tumours through virus-specific memory T cells

Nature Biomedical Engineering (2025)Cite this article

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Abstract

It is challenging for cancer vaccines to identify immunogenic antigens that are specifically and uniformly expressed on heterogeneous solid tumours and that can elicit production of T cells to lyse antigen-positive tumour cells and expand within the immunosuppressive tumour microenvironment. In contrast, microbial antigens are well-defined and robustly immunogenic and can activate specific memory T cells to eliminate microbes within the tumour microenvironment. Inspired by this, we developed a hepatitis B surface antigen (HBsAg)-tagged tumour vaccine system (H-TVAC). H-TVAC leverages HBsAg-specific memory T cells from a HBsAg mRNA vaccine to target and lyse HBsAg-tagged tumour cells using the vaccinia virus. This approach also elicits a tumour-specific immune response through epitope spreading by recruiting dendritic cells, thereby eliminating heterogeneous solid tumours. In various preclinical murine models, including the B16-OVA, B16F10, MC38, CT26, 4T1 and H22 hepatocellular carcinoma, as well as a B16F10 bilateral tumour model, H-TVAC demonstrates anti-tumour immune responses, improved survival rates and reduced metastasis and recurrence.

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Fig. 1: Construction and characterization of H-TVAC.
Fig. 2: H-TVAC exhibits robust immunotherapeutic effects and elicits tumour-specific immune memory in B16 tumour-bearing mice.
Fig. 3: H-TVAC demonstrates efficacy superior to that of tumour neoantigen vaccines, inducing epitope spreading to mutated neoantigens in B16 melanoma tumours.
Fig. 4: H-TVAC induces pronounced immunotherapeutic effects and elicits specific immune memory against MC38 tumour-mutated neoantigens.
Fig. 5: H-TVAC induces systemic immunotherapeutic effects and elicits tumour-specific immune memory against low-immunogenicity solid tumours, inhibiting recurrence and metastasis.
Fig. 6: H-TVAC induces anti-tumour effects against B16F10 bilateral tumours, B16F10 tumours where mRNA-H immunization was administered after tumour establishment and highly aggressive orthotopic H22 liver tumour models.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. Raw data for sequencing results are available via the National Institutes of Health at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1300966 (ref. 70). Source data are provided with this paper.

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Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (grant no. 82304428 to W.W.) and the Fundamental Research Funds for the Central Universities (grant no. 2632025ZD03 to W.W.), the Anti-tumor New Drug Rapid Transformation Public Service Platform of Jiangsu Province (grant no. BM2023002 to Y.Y.) and the Health Science and Technology Innovation Joint Project of Hainan Province, China (grant no. WSJK2024MS231 to Y.Y.). Additional financial support was provided through the Research Start-Up Funds from China Pharmaceutical University (grant no. 3150120054 to W.W.) as well as the Qingyun Project Research Fund (grant no. PB-QY012106 to W.W.) from Polaris Biology (Shanghai, China). We sincerely thank A. Lin for his guidance on the construction of the mRNA-LNP vaccine, and thank Y. Wang, J. Qiu and X. Wang from Polaris Biology for the comprehensive technical support for this project.

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Author notes

  1. These authors contributed equally: Wenguang Wang, Ya Chu, Lili Zhao, Miao Lv.

Authors and Affiliations

  1. Institute of Translational Medicine, China Pharmaceutical University, Nanjing, China

    Wenguang Wang, Ya Chu, Lili Zhao, Miao Lv, Luyao Wang, Fangfei Jin, Kaiyuan Hu, Xianjing Li, Weijun Zhao & Yong Yang

  2. School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, China

    Wenguang Wang, Ya Chu, Lili Zhao, Miao Lv, Luyao Wang, Fangfei Jin, Kaiyuan Hu, Xianjing Li, Weijun Zhao & Yong Yang

  3. Liuzhou Municipal Liutie Central Hospital, Liuzhou, China

    Lihang Cen, Shuang Qin & Shuguang Zuo

  4. State Key Laboratory of Pharmaceutical Biotechnology, Medical School of Nanjing University and School of Life Sciences, Nanjing University, Nanjing, China

    Yiqiao Hu

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Contributions

W.W., Y.Y., Y.H. and S.Z. conceptualized and designed the research framework. Experiments were carried out and data analysis was performed by Y.C., L.Z., M.L., W.W., L.W., F.J., K.H., W.Z., L.C. and S.Q. The interpretation and discussion of the data were undertaken by W.W., Y.Y., W.Z., X.L. and S.Z. The drafting and preparation of the manuscript were managed by W.W., Y.C., L.Z., M.L., Y.Y., S.Z. and Y.H. All authors have meticulously read the final version of the paper and given their approval for its publication.

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Correspondence to Wenguang Wang, Weijun Zhao, Yiqiao Hu, Shuguang Zuo or Yong Yang.

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Extended data

Extended Data Fig. 1 Characterization of the mRNA-H vaccine.

a, Characteristics of mRNA-LNPs, including their size, zeta potential and polydispersity index. b, Quantification of HBsAg secretion by 293T cells 24 h after mRNA-H transfection using an ELISA assay (n = 3 biologically independent samples). c, HBsAb titres in the serum were measured with an ELISA kit 7 days after the last of two immunizations with mRNA-H. The reference HepB-CHO served as a commercially available benchmark for immunization with recombinant hepatitis B (n = 6 biologically independent samples). These experiments were repeated twice independently with similar results. d, Flow cytometry gating strategy for the HBsAg-specific T cells (IFN-γ+, gate: CD3+CD8+ T cells) under the stimulation of HBsAg peptides in the spleen 30 days after immunization with the HBsAg vaccine (0.25 mg per mouse). e, Treatment scheme for the analysis of HBsAg-specific memory T cells on days 76 and 390 in the spleen from mice immunized by mRNA-H (0.25 mg per mouse). f, Flow cytometry gating strategy for the HBsAg-specific memory T cells (IFN-γ+CD44+, gate: CD3+CD8+ T cells) under the stimulation of HBsAg peptides from the spleen of the treated mice in (e). g,h, Representative flow cytometry plots (g) and quantitative analysis (h) (day 76 n = 5 and day 390 n = 3; biologically, independent samples) of HBsAg-specific memory T cells. i, Representative flow cytometry plots of eGFP expression during the preparation of B16-HBsAg tumour cells. eGFP and neomycin are the positive fluorescence and resistance markers used for the generation and screening of B16F10-HBsAg cells, respectively. The generated B16F10-HbsAg cells were continuously screened in neomycin-containing medium for 2 weeks before the experiments. j, qPCR analysis of the HBsAg mRNA level in B16-HBsAg cells compared with control cells (B16F10 and B16F10-pc) (n = 3 biologically independent samples). These experiments were repeated twice independently with similar results. For b, c, h and j, data are presented as mean ± s.e.m., with statistical significance assessed using one-way ANOVA (b, c and j) or two-tailed Student’s t-test (h).

Source data

Extended Data Fig. 2 Characterization of the intra-tumoural VV-H spreading and HBsAg expression at 24, 48 and 72 h post intra-tumoural injection.

a, Flow cytometry gating strategy of tumour cells infected with VV-H at 24, 48 and 72 h after intra-tumoural injection of VV-H (2 × 107 pfu per mouse). The VV-infected tumour cells were stained with anti-mouse CD45 antibody (PECy7) and anti-VV antibody (FITC), and identified as the CD45−VV+ population. b, Representative flow cytometry plots of the VV-H infected tumour cells. c, Ex vivo immunofluorescence images of B16F10 tumours infected with VV-H and intra-tumoural HBsAg expression at 24, 48 and 72 h after intra-tumoural injection of VV-H (2 × 107 pfu per mouse). Tumour sections were stained with an anti-VV antibody (yellow), an anti-HBsAg antibody (green) and DAPI (blue) to visualize VV-H, HBsAg and tumour nuclei, respectively. Scale bar, 50 μm. d, Quantitative analysis of tumour cells infected by VV-H and intra-tumoural HBsAg expression from the treated mice shown in (c) (n = 3 biologically independent samples). The proportion of infected cells and HBsAg expression levels was quantified by calculating the fluorescence area of VV-H or HBsAg relative to the DAPI-stained area. For d, data are presented as mean ± s.e.m.

Source data

Extended Data Fig. 3 Representative images of B16-OVA tumour-bearing mice and flow cytometric analysis of T-cell populations.

a, Images of B16-OVA tumour-bearing mice at the therapeutic endpoint. b, Flow cytometry gating strategy for intra-tumoural CD3+CD8+ T cells. c,d, Representative flow cytometry plots of intra-tumoural CD3+CD8+ T (c) and CD3+CD4+ T cells (d).

Extended Data Fig. 4 Analysis of T-cell and NK cell populations following H-TVAC administration with or without targeted depletion.

a, Treatment scheme for B16-OVA tumour-bearing mice with or without depletion of CD4+ T cells, CD8+ T cells and NK cells with H-TVAC as indicated. Depletion was achieved through the intraperitoneal administration of blocking antibodies targeting CD4+ T cells (10 mg kg−1), CD8+ T cells (10 mg kg−1) and NK cells (10 mg kg−1) on days 11, 13 and 15, respectively. b, Representative flow cytometry gating strategy for intra-tumoural CD8+ T cells (gate: CD45+CD3+), CD4+ T cells and NK cells (gate: CD45+). c,d, Representative flow cytometry plots of intra-tumoural CD8+ T cells, CD4+ T cells (c) and NK cells (d). e–g, Quantification of CD8+ T cells (e), CD4+ T cells (f) and NK cells (g) in the blood 3 days after various treatments (n = 3 biologically independent samples). For e–g, data are presented as mean ± s.e.m., with statistical significance assessed using one-way ANOVA (e–g) followed by Tukey’s multiple comparison test.

Source data

Extended Data Fig. 5 H-TVAC triggers a robust immune response in B16F10 tumour-bearing mice.

a, Treatment scheme for B16F10 tumour-bearing mice. b, Averaged tumour growth curves for B16F10 tumour-bearing mice (n = 8 biologically independent animals). c,d, Quantitation of intra-tumoural CD8+ T cells (c) and CD4+ T cells (d) at the therapeutic endpoint by flow cytometry (mRNA-H+VV-C and H-TVAC n = 7 and others n = 6; biologically independent samples). e, Representative flow cytometry plots of intra-tumoural CD3+CD8+ T and CD3+CD4+ T cells (gate: CD45+ cells). For b–d, data are presented as mean ± s.e.m., with statistical significance assessed using two-way ANOVA (b) and one-way ANOVA (c and d) followed by Tukey’s multiple comparison test.

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Extended Data Fig. 6 Characterization of intra-tumoural HBsAg-specific memory T cells.

a, Treatment regimen for the analysis of intra-tumoural HBsAg specific memory T cells (mRNA-H, 0.25 mg kg−1; VV-H, 2 × 107 pfu per mouse). b, Representative flow cytometry plots of intra-tumoural HBsAg specific memory T cells. c, Flow cytometry gating strategy for the intra-tumoural HBsAg specific memory T cells (IFN-γ+CD44+, gate: CD3+CD8+ T cells) under the stimulation of HBsAg peptides. d, Flow cytometry gating strategy for exhausted (PD-1+TIM-3+, PD-1+LAG-3+ and TIM-3+LAG-3+) and functional phenotype (IFN-γ+TNF+) analysis of intra-tumoural HBsAg-specific memory T cells (gate: IFN-γ+CD44+; the gate for saline group, CD44+). e–h, Representative flow cytometry plots of the exhausted and functional phenotype analysis of intra-tumoural HBsAg-specific memory T cells. i, Quantification of IFN-γ+ TNF+ T cells in (h). For i, data are presented as mean ± s.e.m. (n = 6 biologically independent animals), with statistical significance assessed using a two-tailed Student’s t-test.

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Extended Data Fig. 7 Characterization of all intra-tumoural T cells at the therapeutic endpoint.

a, Treatment regimen for the analysis of intra-tumoural T cells at the therapeutic endpoint (mRNA-H 0.25 mg kg−1 and VV-H 2 × 107 pfu per mouse). b, Flow cytometry gating strategy for intra-tumoural exhausted T cells (PD-1+TIM-3+, PD-1+LAG-3+ and TIM-3+LAG-3+). HBsAg specific memory T cells were determined as IFN-γ+CD44+ cell populations under the stimulation of HBsAg peptides (gate: CD3+CD8+). c–e. Quantification of intra-tumoural PD-1+LAG-3+(c), PD-1+TIM-3+(d) and TIM-3+LAG-3+ T cells (e) (n = 7 biologically independent samples). f–h. Representative flow cytometry plots of the exhausted T cells, including PD-1+TIM-3+(f), PD-1+LAG-3+(g) and TIM-3+LAG-3+ T cells (h). i,j, Immunohistochemical staining (i) and quantification (j) of intra-tumoural granzyme B secretion (n = 3 biologically independent samples). k,l, Immunohistochemical staining (k) and quantification (l) of intra-tumoural perforin secretion (n = 3 biologically independent samples). For c–e, j and l, data are presented as mean ± s.e.m., with statistical significance assessed using two-tailed Student’s t-test (c–e), and one-way ANOVA (j and l) followed by Tukey’s multiple comparison test.

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Extended Data Fig. 8 Safety evaluation of the H-TVAC system.

a–o, Serum biochemical indexes (a–i) and haematological indexes (j–o) of CT26 tumour-bearing mice 14 days after treatment: AST (a), ALT (b), GGT (c), CKI (d), TGL (e), CHOL (f), BUN (g), TP (h), TBI (i), HCT (j), RBC (k), HGB (l), MCH (m), MCV (n) and MCHC (o) (saline and mRNA-H + saline n = 7 and others n = 8; biologically independent samples). p, images of HE staining in major organs (heart, liver, spleen, lung and kidney) 14 days post-treatment. For a–o, data are presented as mean ± s.e.m., with statistical significance assessed using two-way ANOVA (a–o) followed by Tukey’s multiple comparison test.

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Extended Data Fig. 9 An overview of the H-TVAC platform for universal cancer vaccine and its advantages over traditional tumour neoantigen vaccines.

H-TVAC leverages microbial-specific memory T cells, induced by microbial vaccines, to lyse tumour cells tagged with microbial antigens. This approach facilitates the efficient elimination of tagged tumour cells via virus-specific memory T cells. Furthermore, tumour cell lysis, combined with DC recruitment mediated by the VV, enhances tumour-specific immune responses through epitope spreading, thereby promoting the eradication of heterogeneous solid tumours. Compared with traditional tumour neoantigen vaccines, H-TVAC therapy provides significant advantages. First, H-TVAC utilizes well-defined, conserved and highly immunogenic microbial antigens, effectively eliciting microbial-specific memory T cells. In contrast, tumour neoantigens are heterogeneous, highly mutated and require a complex screening process. Despite these efforts, their immunogenicity remains low. Second, unlike tumour-specific T cells that often become exhausted within the TME, microbe-specific memory T cells can resist this exhaustion and stimulate robust tumour-specific immune responses through epitope spreading.

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Wang, W., Chu, Y., Zhao, L. et al. HBsAg-tagged tumour vaccine system eliminates solid tumours through virus-specific memory T cells. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01555-w

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