HBV X protein-based therapeutic vaccine accelerates viral antigen clearance by mobilizing monocyte infiltration into the liver in HBV carrier mice - PubMed (original) (raw)
HBV X protein-based therapeutic vaccine accelerates viral antigen clearance by mobilizing monocyte infiltration into the liver in HBV carrier mice
Jau-Hau Horng et al. J Biomed Sci. 2020.
Abstract
Background: Hepatitis B virus (HBV) persistently infected about 250 million people worldwide, and a curative treatment remains an unmet medical need. Among many approaches to treat chronic hepatitis B (CHB), therapeutic vaccines have been developed for two decades, but none have yielded promising results in clinical trials. Therefore, dissection of HBV clearance mechanisms during therapeutic vaccination in appropriate models, which could give rise to new curative therapies, is urgently needed. Growing evidence indicates that prolonged and intensive exposure of antigen-specific T cells to viral antigens is a major cause of T cell exhaustion, and decreases anti-HBV immunity efficacy of therapeutic vaccination. HBV X protein (HBx) is expressed at low levels, and the understanding of its immunogenicity and potential in therapeutic CHB vaccines is limited.
Methods: HBV genome sequences from CHB patients were cloned into a pAAV plasmid backbone and transfected into immunocompetent mouse hepatocytes through hydrodynamic injection. Mice carrying > 500 IU/mL serum HBV surface antigen (HBs) for more than 4 weeks were considered HBV carriers mimicking human CHB and received 3 doses of weekly HBx vaccine by subcutaneous immunization. Serum HBV clearance was evaluated by monitoring serum HBs and HBV-DNA titers. Residual HBV in the liver was evaluated by western blotting for HBV core antigen. The splenic antigen-specific T cell response was quantified by a 15-mer overlapping peptide-stimulated interferon-γ enzyme-linked immunospot assay. Blood and hepatic immune cells were quantified by flow cytometric analysis.
Results: Our HBx-based vaccine induced systemic HBx-specific CD4+ and CD8+ T cell responses in HBV carrier mice and demonstrated significant HBs and HBV-DNA elimination. The protective effect persisted for at least 30 days without additional booster immunization. Different infiltrating myeloid cell subsets, each with distinctive roles during immune-mediated HBV clearance, were found in the liver of vaccinated mice. During vaccine therapy, inflammatory monocyte depletion resulted in sustained HBV clearance inhibition, whereas phagocytic monocyte-derived macrophage and Kupffer cell elimination resulted in only transient inhibition of vaccine-induced HBV clearance.
Conclusions: We report the potential role of HBx as a major immunogen in an HBV therapeutic vaccine and the significance of a liver-infiltrating monocyte subset during hepatic viral clearance.
Keywords: Chronic hepatitis B; Hepatic innate immunity; Immunotherapy; Myeloid cell.
Conflict of interest statement
JH Horng received a scholarship from TheVax Genetics Vaccine Company. WS Lin is a former employee of TheVax Genetics Vaccine Company. PJ Chen received research funding from TheVax Genetics Vaccine Company. The funding was limited to reimbursement for the necessary expenses for this research work. The other authors have no competing interests to report.
Figures
Fig. 1
TVGV-HBx exerts a protective function against hydrodynamic HBV exposure in persistence-prone CBA/CaJ mice. a Naïve CBA/CaJ mice (N = 9) received hydrodynamic injection of 10 μg of different genotypes of the pAAV/HBV1.2 plasmid on day 0. Blood samples were collected at the indicated time points, and the serum HBs titers are shown. b Naïve CBA/CaJ mice (N = 4) received TVGV-HBx or TVGV-E7 or remained untreated on days 0, 7 and 14. Splenocytes were collected on day 21 and subjected to an IFN-γ ELISpot assay. c-f Naïve CBA/CaJ mice (N = 4 ~ 5) received TVGV-HBx or CpG-ODN immunization every 7 days for 3 consecutive weeks. Mice received HDI transfection with 10 μg of pAAV/HBV1.2 plasmid one week after the last immunization. Mouse serum was sampled on days 7, 14, 21, and 28 after HDI transfection. Liver samples were collected at the endpoint of the experiment. c Serum HBs titers were determined. d Serum HBV-DNA was detected. e Liver HBc was detected by western blotting. f Liver HBV-DNA was detected. Statistics: Student’s _t_-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; COI, cut-off index; LoD, limit of detection; SFCs, spot-forming cells
Fig. 2
TVGV-HBx exerts therapeutic function in HBV carrier mice. HBV carrier mice (N = 5 ~ 8) received TVGV-HBx, TVGV-E7, CpG-ODN alone or PBS on days 0, 7 and 14. Mouse serum was sampled on days 0, 7, 14, 21, 32, 39 and 46, and liver samples were collected on day 70. a Serum HBs titers were determined. b Serum HBV-DNA was detected. c Serum anti-HBs titer on day 39. d Liver HBc was detected by western blotting. e Liver HBV-DNA was detected. The value of the undetectable Q-PCR sample was assigned as the detection limit for plotting. Statistics: Student’s _t_-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; LoD, limit of detection
Fig. 3
TVGV-HBx induces an HBx-specific T cell response and posttreatment immune memory in HBV carrier mice. a-b HBV carrier mice (N = 4) received TVGV-HBx on days 0, 7 and 14. Splenocytes were isolated at day 21 and subjected to ex vivo CD4+ or CD8+ T cell depletion or left untreated. Splenocyte preparations were subjected to 15-mer overlapping HBx peptide-stimulated IFN-γ ELISpot assays. a Normalized spot counts of untreated splenocytes. b Pairwise comparative results for untreated splenocytes versus CD4+ cell- or CD8+ cell-depleted splenocytes. c-d HBV carrier mice (N = 3 ~ 4) received TVGV-HBx or CpG-ODN alone on days 0 and 7. Intrahepatic lymphocytes were isolated on day 10. Isolated cells were analyzed by flow cytometry to evaluate T cell subpopulations. c Frequency of liver CD8+ T cells among total CD45+ intrahepatic lymphocytes. d Frequency of liver CD11a+CD8+ and CD11a−CD8+ T cells among CD45+ intrahepatic lymphocytes. e-f HBV carrier mice (N = 5) were immunized with TVGV-HBx or CpG-ODN on days 0, 7 and 14 and rechallenged with HDI of 10 μg of pAAV/HBV1.2 plasmid on day 53. Blood samples were collected at the time points indicated on the plots, and liver samples were collected on day 84. e Serum HBs titer were determined. f Liver HBc was detected by western blotting. Statistics: Student’s _t_-test. **, p < 0.01; ***, p < 0.001; NS, not significant; SFCs, spot-forming cells
Fig. 4
Comparison of the immunogenicity of HBV core antigen and that of HBV X protein. a-c HBV carrier mice (N = 3) received vaccination with 3.125 μg of rHBc or rHBx supplemented with 0.625 μg of CpG-ODN on days 0, 7 and 14. Mouse serum was sampled on days 0, 7, 14, 21 and 35. (a) Serum HBs titers were determined. b Serum anti-HBs antibody titer on day 35. c Serum anti-HBc antibody titer on day 35. Lower values represent higher antibody production because the anti-HBc assay is a competition assay. d HBV carrier mice (N = 3 ~ 4) received vaccination with 6.25 μg of rHBc or rHBx supplemented with 1.25 μg of CpG-ODN on days 0, 7, 14 and 22. Mice were boosted once with the same dose 7 days before splenocyte isolation. Splenocytes were separately stimulated with HBx-, HBs-, or HBc-derived 15-mer overlapping peptide pool and subjected to an IFN-γ ELISpot assay. The result was normalized to the spot counts per million cells. LoD, limit of detection; SFCs, spot-forming cells
Fig. 5
CpG-ODN-induced monocyte mobilization regulates TVGV-HBx-mediated HBV clearance. a-b HBV carrier mice (N = 3) received TVGV-HBx on day 0. Gemcitabine (40 mg/kg) was given by intraperitoneal injection on days 0 and 1. CLs (200 μL) were given by intravenous injection on day 0. PBMCs were isolated on day 2. PBMCs were stained with fluorochrome-conjugated anti-CD11b, anti-CD115, anti-Ly6C and anti-Ly6G antibodies and then subjected to FACS analysis. a Frequency of CD11b+CD115+Ly6G− monocytes among total PBMCs. b Representative FACS plots of Ly6C+ monocyte subsets (gated by CD11b + CD115 + Ly6G-) before and after vaccination and drug administration. The cell frequencies of each subpopulation are shown on the plot. c-d HBV carrier mice (N = 4 ~ 5) were administered TVGV-HBx vaccination (black arrow with the dosage indicated, 100 μg of antigen plus 20 μg of CpG-ODN as 1.0x) and a monocyte-depleting drug (red arrow) at the indicated time points. Blood samples were collected at the indicated time points. c Serum HBs titer after TVGV-HBx vaccination and CL treatment. d Serum HBs titer after TVGV-HBx vaccination and gemcitabine treatment. e Splenocyte IFN-γ ELISpot results (mice N = 5) after TVGV-HBx vaccination and gemcitabine treatment. The vaccination and drug administration protocol were the same as in (d), and the splenocytes were harvested on day 14. Cells were stimulated with HBx-derived 15-mer overlapping peptides. Statistics: Student’s _t_-test. *, p < 0.05; **, p < 0.01; NS, not significant; LoD, limit of detection; SFCs, spot-forming cells; GEM, gemcitabine
Fig. 6
Liver monocyte subpopulation changes after TVGV-HBx vaccination and monocyte-depleting drug treatment. a-b HBV carrier mice (N = 4) received TVGV-HBx on day 0. Gemcitabine (40 mg/kg) was given by intraperitoneal injection on days 0 and 1. CLs (200 μL) were given by intravenous injection on day 0. Total intrahepatic leukocytes were collected on day 2 and subjected to flow cytometric analysis. The isolated cell count of each cell subpopulation was calculated by multiplying the actual isolated CD45+ leukocyte count per liver by the subpopulation frequency among isolated cells. a Total isolated cell count of all F4/80lowCD11b+Ly6G− monocytes. b Total isolated cell counts of the indicated monocyte subsets and F4/80highCD11b+ KCs. c Schematic Venn diagram of liver myeloid cell subpopulations depleted by CL or gemcitabine treatment. Statistics: Student’s _t_-test. *, p < 0.05; **, p < 0.01; NS, not significant
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