IL-21-based therapies induce clearance of hepatitis B virus persistence in mouse models - PubMed (original) (raw)
. 2019 May 31;9(13):3798-3811.
doi: 10.7150/thno.35331. eCollection 2019.
Affiliations
- PMID: 31281514
- PMCID: PMC6587353
- DOI: 10.7150/thno.35331
IL-21-based therapies induce clearance of hepatitis B virus persistence in mouse models
Zhongliang Shen et al. Theranostics. 2019.
Abstract
Chronic hepatitis B virus (HBV) infection causes hepatitis, liver cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA (cccDNA) is the sole viral transcription template and not affected by current treatment options, constituting a key determinant of HBV persistence. Novel therapeutics with demonstrable effectiveness against cccDNA are required. Methods: Previously, we established an HBV persistence mouse model using replicon plasmid derived from a clinical isolate (termed BPS) and identified IL-21 as a potent clearance-inducer. We also described another persistence mouse model based on cccDNA mimics produced in vivo termed recombinant cccDNA (rcccDNA). In this work, effectiveness of IL-21-based gene and cellular therapies was tested using these models. Results: In both models of HBV persistence, single injections with adeno-associated virus (AAV) expressing murine IL-21 highly efficiently induced clearance of both HBV markers from serum, and more importantly, BPS DNA and rcccDNA from mouse liver. Mechanistically, IL-21-induced clearance was associated with activation and liver infiltration of CD8+ T cells, and CD8 antibody injections negatively affected AAV-IL-21 effectiveness. More notably, adoptive transfer of CD8+ T cells from AAV-IL-21-cured mice engendered clearance in acceptor HBV persistence mice. Furthermore, cured mice were protected against re-challenge with long-lived memory. Most significantly, infusion of splenocytes from treatment-naïve mice stimulated ex vivo with IL-21 protein and HBV antigen could also induce clearance in treatment-naïve mice. Conclusion: These data demonstrate IL-21-based gene and cellular therapies as valid candidates for treating chronic HBV infections, with potential in removing cccDNA-harboring hepatocytes via activated CD8+ T cells accompanied by long-term protective memory.
Keywords: IL-21; cellular immunity; clearance; hepatitis B virus; mouse model.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interest exists.
Figures
Figure 1
AAV-IL-21 treatment induced HBV clearance in BPS persistence mice. BPS HDI mice remaining positive for serum HBV antigens for 4 weeks were injected (solid arrows) with 2 × 1011 geq of AAV-IL-21 or AAV-Ctrl, or left untreated. Sera collected at indicated time points were analyzed for HBsAg (A), HBsAb (B), HBeAg (C), HBV DNA (pooled sera, D), ALT (E) and IL-21 (F). Means and SEMs within group are presented with the group size (n) indicated. Group positivity percentage data are presented for HBsAg, HBsAb and HBeAg (A-C, right panels). Dotted lines represent cut-off thresholds (A-C, left panels), quantification lower limit (D) or pre-treatment baseline (E). Data are compared to that of the untreated group and statistical significance calculated using log-rank (Mantel-Cox) test (A-C, right panels) and unpaired two-tailed _t_-test (E and F). *p < 0.05; **p < 0.01; ***p < 0.001. w.p.i., weeks post injection of AAV. geq, genome equivalents.
Figure 2
AAV-IL-21 treatment cleared intrahepatic BPS DNA from BPS persistence mice. BPS HDI mice remaining positive for serum HBV antigens for 4 weeks were injected with 2 × 1011 geq of AAV-IL-21 (n = 3), AAV-Ctrl (n = 2), or left untreated (n = 2). Serum HBsAg was tested every two weeks. At 10 weeks post injection (w.p.i.) of AAV, mice were sacrificed and liver samples taken (A). Two AAV-IL-21 treated mice had displayed HBsAg negativity for over 4 weeks by the time of sacrifice. BPS plasmid in nuclear DNA extracted from liver tissue samples were measured using quantitative realtime PCR (B) and Southern blot (C) with serum HBsAg status indicated. Data from each mouse and group means are presented (B). Naïve, mice without BPS HDI (n = 2).
Figure 3
AAV-IL-21 treatment induced HBV clearance in rcccDNA persistence mice. Cre Tg C57BL/6 mice positive for serum HBV antigen for 4 weeks after Ad/prcccDNA injection were injected (solid arrows) with 2 × 1011 geq of AAV-IL-21, AAV-Ctrl, or left untreated. Sera collected at indicated time points were analyzed for HBsAg (A), HBsAb (B), HBeAg (C), HBV DNA (pooled sera, D), ALT (E) and IL-21 (F). Means and SEMs within group are presented with the group size (n) indicated. Group positivity percentage data are presented for HBsAg, HBsAb and HBeAg (A-C, right panels). Dotted lines represent cut-off thresholds (A-C, left panels), quantification lower limit (D) or pre-treatment baseline (E). Data are compared to that of the untreated group and statistical significance calculated using log-rank (Mantel-Cox) test (A-C, right panels) and unpaired two-tailed _t_-test (E and F). *p < 0.05; **p < 0.01; ***p < 0.001. w.p.i., weeks post injection of AAV. geq, genome equivalents.
Figure 4
AAV-IL-21 treatment cleared intrahepatic rcccDNA from rcccDNA persistence mice. Cre Tg C57BL/6 mice positive for serum HBV antigen for 4 weeks after Ad/prcccDNA injection were injected with 2 × 1011 geq of AAV-IL-21 (n = 3), AAV-Ctrl (n = 3), or left untreated (n = 3). Serum HBsAg were tested as indicated. At 10 weeks post injection (w.p.i.) of AAV, mice were sacrificed and liver samples taken (A). All AAV-IL-21 treated mice had been HBsAg-negative for over 4 weeks at sacrifice. RcccDNA in liver nuclear DNA was measured using quantitative realtime PCR and normalized against GAPDH genomic DNA in the same sample (B). Statistical significances are calculated using unpaired two-tailed _t_-test. ***p < 0.001. ns, not significant. Reaction mixtures from (B) were subjected to agarose electrophoresis for amplicon size check (C). Serum HBsAg status of respective mouse was indicated (B and C). naïve, Cre Tg C57BL/6 mice without Ad/prcccDNA injection (n = 2).
Figure 5
Adoptive transfer of CD3+ and CD8+ T cells from AAV-IL-21 cured BPS persistence mice induced HBV clearance in treatment-naïve BPS persistence mice. BPS persistence mice were transferred with CD3+, CD4+, or CD8+ T cells from AAV-IL-21-cured BPS persistence mice (serum HBsAg negative for over 4 weeks), or left untreated. Sera collected at indicated time points were analyzed for HBsAg (A), HBsAb (B), HBeAg (C), and ALT (D). Means and SEMs within group are presented with the group size (n) indicated. Group positivity percentage data are presented for HBsAg, HBsAb and HBeAg (A-C, right panels). Dotted lines represent cut-off thresholds (A-C, left panels) or pre-transfer baseline (D). Data are compared to that of the untreated mice and statistical significance calculated using log-rank (Mantel-Cox) test (A-C, right panels) and unpaired two-tailed _t_-test (D). *p < 0.05; **p < 0.01; ***p < 0.001. w.p.i., weeks post injection of AAV.
Figure 6
Adoptive transfer of CD3+ and CD8+ T cells from AAV-IL-21 cured BPS persistence mice induced clearance of intrahepatic BPS DNA. BPS persistence mice were transferred with CD3+ (n = 3), CD4+ (n = 3), or CD8+ (n = 3) T cells from AAV-IL-21-cured BPS persistence mice (serum HBsAg negative for over 4 weeks), or left untreated (n = 3). Sera collected at indicated time points were analyzed for HBsAg. At 11 weeks post injection (w.p.i.) of T cells, mice were sacrificed and liver samples taken (A). All CD3+ and CD8+ T cell recipient mice had displayed HBsAg negativity for over 4 weeks by the time of sacrifice. BPS plasmid in liver nuclear DNA extracted was measured using quantitative realtime PCR (B) and Southern blot (C). Serum HBsAg status of mice are indicated (B-C). Data from each mouse and group means are presented (B). Statistical significances are calculated using unpaired two-tailed _t_-test. **p < 0.01; ***p < 0.001. ns, not significant.
Figure 7
Transfer of splenocytes stimulated with IL-21 and HBsAg induced HBV clearance in treatment-naïve BPS persistence mice. Splenocytes from untreated BPS persistence mice were cultured in the presence of 100 ng/ml recombinant mouse IL-21 (rIL-21) with or without 15 μg/ml recombinant HBsAg, or without stimulation. After 60 hours, 1 × 108 splenocytes were injected into each BPS persistence mice via tail vein. Sera collected at indicated time points were analyzed for HBsAg (A), HBsAb (B), HBeAg (C) and ALT (D). Means and SEMs within group are presented with the group size (n) indicated. Group positivity percentage data are presented for HBsAg, HBsAb and HBeAg (A-C, right panels). Dotted lines represent cut-off thresholds (A-C, left panels) or pre-transfer baseline (D). Data are compared to mice transferred with unstimulated splenocytes and statistical significance calculated using log-rank (Mantel-Cox) test (A-C, right panels) and unpaired two-tailed _t_-test (D). ***p < 0.001. w.p.i., weeks post splenocytes injection.
Figure 8
Transfer of splenocytes stimulated with IL-21 and HBsAg induced clearance of intrahepatic BPS DNA. Splenocytes from untreated BPS persistence mice were cultured in the presence of 100 ng/ml recombinant mouse IL-21 (rIL-21) with or without 15 μg/ml recombinant HBsAg, or without stimulation. After 60 hours, 1 × 108 splenocytes were injected into each untreated BPS persistence mice via tail vein. Sera collected at indicated time points were analyzed for HBsAg. At 10 weeks post injection (w.p.i.) of splenocytes, mice were sacrificed and liver samples taken (A). BPS plasmid in liver nuclear DNA extracted was measured using quantitative realtime PCR (B) and Southern blot (C). Serum HBsAg status of mice are indicated (B and C). Data from each mouse and group means are presented (B). Statistical significances are calculated using unpaired two-tailed _t_-test. **p < 0.01; *p < 0.05.
References
- World Health Organization: Hepatitis B, Fact Sheet. Revised 18 July 2018. http://www.who.int/en/news-room/fact-sheets/detail/hepatitis-b.
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