Figure 1.
Detection of mIFN-α in serum samples from the mice after HI.
Mice received HI with 10 µg of either HBV clones or pmIFN-α alone or combination of HBV clones with pmIFN-α. Mouse sera were collected at the indicated time points. The concentrations of mIFN-α protein were detected by mIFN-α ELISA kit. (A) Expression of mIFN-α in mice at 24 hpi. (B) Kinetics of mIFN-α expression in mice after HI. At least three serum specimens of the mice per group were analysed at the indicated time points.
Figure 2.
Kinetics of HBsAg, HBeAg and HBV DNA in sera of the mice after HI.
10 µg of pAAV-HBV-B alone or combined with 20 µg pmIFN-α were used for HI. (A) HBsAg and HBeAg levels in the sera of C57BL/6 mice after HI. The value of OD450 ≥ Cut-Off value means the sample was positive. (B) Real-time PCR detection of HBV DNA levels in the sera of the mice after HI, dotted line indicates detection limit. At least three mice per group were analysed.
Figure 3.
Application of mIFN-α expression vector reduced HBcAg expression in liver of the mice.
The mice injected with 10 µg of HBV clones alone or combined wiht 20 µg of pmIFN-α. (A) Immunohistochemical staining of the liver sections for HBcAg in hepatocytes of pAAV-HBV-A- (a), pAAV-HBV-A combined with pmIFN-α- (b), pAAV-HBV-B- (c) and pAAV-HBV-B together with pmIFN-α (d)-injected mice at 1, 10, 20, 30 and 50 dpi (Original magnification: 400X). The mice injected with 0.9% NaCl were used as negative control. Liver specimens of at least three mice per group (n≥3) were analyzed. Each sample was done in duplicate. (B) Frequencies of HBcAg positive cells in the mouse liver sections. The data were analyzed by t test, and the differences were statistically significant (* means p<0.05 and # means p<0.01).
Figure 4.
Induction of ISG15, OAS and PKR expression in the mouse liver after HI.
Mice received HI with 10 µg of HBV clones or pmIFN-α alone or combination of HBV clones and pmIFN-α. The mice injected with 0.9% NaCl or pAAV vector, were used as control. Total RNA was extracted form liver tissue of the mice at the indicated time points after HI and the levels of ISG (A), OAS (B) and PKR (C) mRNA were determined by quantitative real-time RT PCR. The β-actin mRNAs were quantified for normalization. Each sample was run in duplicate and at least three mouse liver specimens per group were analyzed (n≥3). Differences between the groups were analyzed by using the t test: * means p<0.05 and # means p<0.01.
Figure 5.
Expression levels of IL-6, IL-10 and TGF-β in the mouse liver after HI.
Mice received HI with 10 µg of HBV clones or pmIFN-α alone or combination of HBV clones and pmIFN-α. The mice injected with 0.9% NaCl or pAAV vector, were used as control. Total RNA was extracted form liver tissue of the mice at the indicated time points after HI and the levels of IL-6 (A), IL-10 (B) and TGF-β (C) mRNA were determined by quantitative real-time RT PCR. The β-actin mRNAs were quantified for normalization. Each sample was run in duplicate and at least three mouse liver specimens per group were analyzed (n≥3). Differences between the groups were analyzed by using the t test: * means p<0.05 and # means p<0.01.
Figure 6.
Detection of T-cell responses to HBsAg and HBcAg in mice received HI with HBV clones and pmIFN-α.
The specific T-cell responses to the full length HBsAg and HBcAg in mice were analyzed by ELISPOT assays after HI with HBV clones and pmIFN-α. The numbers of HBsAg and HBcAg specific IFNγ secreting cells in 2×105 splenocytes of the mice HI with pAAV-HBV-A and pmIFN-α (A) or HI with pAAV-HBV-B and pmIFN-α (B) at 10 d by IFNγ ELISPOT assay in the presence of 0.5 µg/ml full length HBsAg or HBcAg peptide. 5 µg/ml of ConA was used as positive control and 0.5 µg/ml of CMV was used as negative control (data not shown). The experiments were done in duplicate and spleen specimens of at least three mice per group were analyzed (n≥3). The data were analyzed by t test but no significant difference between the experimental groups was found (p>0.05).