Figure legends
Figure 1. Preparation and characterization of LNP particles formulated with IL-21 mRNA (LNP-IL-21). (A ) Schematic presentation of components used to generate LNP-IL-21 particles. (B ) The size distribution of three samples of LNP-IL-21measured using dynamic light scattering. (C ) Transmission electronic microscopy image of LNP-IL-21. (D ) Primary mouse hepatocytes cultured in 24-well plate were transfected with 0.3 μg of LNP-IL-21 or control LNP, or left untreated. At 2 days post transfection, IL-21 in culture medium was measured using ELISA. (E ) Primary mouse hepatocytes cultured in 96-well plate were transfected with 0.025-0.1 μg of LNP-IL-21, control LNP or left untreated. Cell viability was detected at day 1-4 post transfection using CCK-8 assays. (F ) Male BALB/c mice aged 6-8 weeks were injected with 10 μg of LNP-IL-21 or LNP via tail vein. At 3 days post injection, mice were sacrificed to collect major organs including heart, liver, spleen, lung and kidney. Intracellular IL-21 levels were determined using ELISA. LNP-IL-21 particles were stored at 4℃ for 0-6 weeks and used for further analysis. Measurement of LNP-IL-21 in particle size (G ) and mRNA concentration (H ). Same volumes of LNP-IL-21 particles stored for different weeks were transfected into primary mouse hepatocytes (I ) or injected into BALB/c mice (J , solid arrow) and IL-21 levels in culture medium or serum measured using ELISA. The number of mice (n ) in each group was indicated. Group means and SEM within groups were plotted and statistical significances calculated using unpaired two-tailed t -test. Dotted lines represented cut-off thresholds (F and J ). **p < 0.01; ***p < 0.001. DSPC, disaturated phosphatidyl choline. PDI, polymer dispersity index. w.p.i., weeks post injection of LNP.
Figure 2. LNP-IL-21 administration induced HBV clearance in BPS persistence mice. BPS persistence mice remaining positive serum HBV antigens for 4 weeks were injected (solid arrows) with two-dose LNP-IL-21injections, two weeks apart, or control LNP. Sera collected at indicated time points were analyzed for HBsAg (A ), HBsAb (B ), HBeAg (C ), HBV DNA (D , pooled serum), ALT (E ) and IL-21(F , pooled serum). Means and SEMs within group were presented with the group size (n ) indicated. Group positivity percentage data were presented for HBsAg, HBsAb and HBeAg (A-C , right panels). Dotted lines represented cut-off thresholds (A-C , left panels, and F ), quantification lower limit (D ) or pre-treatment baseline (E ). Statistical significances were calculated using log-rank (Mantel-Cox) test for group positivity percentages (A-C , right panels) or unpaired two-tailed t -test (E ). **p < 0.01; ***p < 0.001. w.p.i., weeks post injection of LNP. geq, genome equivalents.
Figure 3. LNP-IL-21 administration induced intrahepatic BPS replicons DNA clearance in BPS persistence mice. (A ) BPS persistence mice remaining positive serum HBV antigens for 4 weeks were injected with two-dose LNP-IL-21 injections (n = 6), two weeks apart, or control LNP (n = 6). Sera collected at indicated time points were analyzed for HBsAg. At 8 weeks post injection of LNP (w.p.i.), mice were sacrificed to collect liver tissues. (B ) HBV core-associated DNA, BPS replicons DNA in nuclear DNA and capsid proteins were extracted from liver tissues and subjected to Southern and Western blots respectively. Serum HBsAg status of respective mouse was indicated.
Figure 4. LNP-IL-21 administration induced HBV clearance in rcccDNA persistence mouse model. rcccDNA persistence mice remaining positive serum HBV antigens for 4 weeks were injected (solid arrows) with two-dose LNP-IL-21 injections, two weeks apart, or control LNP. Sera collected at indicated time points were analyzed for HBsAg (A ), HBsAb (B ), HBeAg (C ), HBV DNA (D , pooled serum), ALT (E ) and IL-21(F , pooled serum). Means and SEMs within group were presented with the group size (n ) indicated. Group positivity percentage data were presented for HBsAg, HBsAb and HBeAg (A-C , right panels). Dotted lines represented cut-off thresholds (A-C , left panels, and F ), quantification lower limit (D ) or pre-treatment baseline (E ). Statistical significances were calculated using log-rank (Mantel-Cox) test for group positivity percentages (A-C , right panels) or unpaired two-tailedt -test (E ). *p < 0.05; **p< 0.01; ***p < 0.001. w.p.i., weeks post injection of LNP. geq, genome equivalents.
Figure 5. LNP-IL-21 administration induced intrahepatic rcccDNA clearance in rcccDNA persistence mice. (A ) rcccDNA persistence mice remaining positive serum HBV antigens for 4 weeks were injected with two-dose LNP-IL-21 injections (n = 6), two weeks apart, or control LNP (n = 6). Sera collected at indicated time points were analyzed for HBsAg. At 8 weeks post injection of LNP (w.p.i.), mice were sacrificed to collect liver tissues. (B ) HBV core-associated DNA and capsid proteins extracted from liver tissues were measured using Southern and Western blots respectively. rcccDA and murine GAPDH (mGAPDH) were amplified from unclear DNA in liver tissues using PCR and subjected to 1% agarose electrophoresis for amplicon size check. Serum HBsAg status of respective mouse was indicated.
Figure 6. LNP-IL-21 treatment induced potent HBV-specific humoral and cellular immune responses in BPS persistence mice.(A ) BPS persistence mice remaining positive serum HBV for 4 weeks were injected with two-dose LNP-IL-21 injections, two weeks apart, or control LNP. Blood samples were collected at indicated time points. At 2 and 8 weeks post injection of LNP (w.p.i.), mice were sacrificed to collect liver or/and spleen tissues. (B ) H&E and anti-CD8 staining were performed on liver tissues. Scale bar: 100 μm. At 8 w.p.i., serum HBsAg (C ) and HBsAb (D ) were measured using ELISA, and HBsAg-specific B cells in PBMC from LNP-IL-21- or LNP-injected mice were measured using flow cytometry analysis (E ) with representative images of two mice in each group presented (F ). At 8 w.p.i., spleen cells from above two groups were stimulated with recombinant HBsAg or HBcAg proteins. After 3 days post stimulation, IFN-γ in culture medium was measured using ELISA. Group means and SEMs were presented with group sizes (n ) indicated. Statistical significances were calculated using unpaired two-tailed t -test. **, P < 0.01; ***, P< 0.001.
Figure 7. Transfer of PBMC co-stimulated with LNP-IL-21 and rHBsAg induced clearance of HBV serum markers and intrahepatic BPS repliocns DNA in BPS persistence mice.(A ) PBMC from untreated BPS persistence mice were cultured and treated with 1μg of LNP-IL-21 or LNP in presence of 15 μg/ml rHBsAg. After 72 hours, 1 × 106 PBMC were injected into each untreated BPS persistence mice via tail vein. Sera collected at indicated time points were analyzed for HBsAg (B ), HBsAb (C ), HBeAg (D ), ALT (E ) and IL-21 (F ). Means and SEMs within group were presented with the group size (n ) indicated. Dotted lines represented cut-off thresholds (B-D , and F ) or pre-treatment baseline (E ). (G ) At 4 weeks post injection of PBMC (w.p.i.), BPS replicons DNA and capsid proteins extracted from liver tissues were measured using Southern and Western blots respectively. Serum HBsAg status of respective mouse was indicated. Statistical significance was unpaired two-tailed t -test. *p < 0.05; **p< 0.01.