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.