An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection

Abstract

An animal model for human hepatitis B virus (HBV) tolerance is needed to investigate the mechanisms. This model will also facilitate therapeutic strategies for the existing 350 million patients with chronic hepatitis B. We established a mouse model by hydrodynamic injection of an engineered, replication-competent HBV DNA into the tail veins of C57BL/6 mice. In 40% of the injected mice, HBV surface antigenemia persisted for > 6 months. Viral replication intermediates, transcripts, and proteins were detected in the liver tissues of the injected mice for up to 1 year. The tolerance toward HBV surface antigen in this model was shown to be due to an insufficient cellular immunity against hepatitis B core antigen, as was documented in humans. This animal model will accelerate further genetic and mechanistic studies of human chronic hepatitis B infection.

Fig. 1.

HBV persistence in mice induced by hydrodynamic injection of HBV plasmids was determined by the genetic background of the recipients and the plasmid backbone. C57BL/6 or BALB/c mice were injected hydrodynamically with 10 μg of HBV plasmid. The HBsAg titer in the mouse serum was determined at the indicated time points by an EIA (Abbott Diagnostics), and the obtained S/N ratio was converted to nanograms per milliliter by applying a standard curve with known concentrations of HBsAg. (A) Titer of serum HBsAg in C57BL/6 or BALB/c mice after pAAV/HBV1.2 injection. The detection limitation is 17.76 ng/ml. (B) Positive rate of serum HBsAg in C57BL/6 (n = 12) or BALB/c (n = 9) mice receiving pAAV/HBV1.2 injection at different time points after injection. The data were analyzed by Kaplan–Meier analysis, and the difference was statistically significant (P < 0.00001). (C) Titer of serum HBsAg in C57BL/6 mice receiving pAAV/HBV1.2 or pGEM4Z/HBV1.2 hydrodynamic injection. (D) Positive rate of serum HBsAg in C57BL/6 mice receiving pAAV/HBV1.2 (n = 12) or pGEM4Z/HBV1.2 (n = 8) injection. The data were analyzed by Kaplan–Meier analysis, and the difference was significant (P < 0.00001). The cutoff value for determining HBsAg-positivity is 22 ng/ml. ∗, mice that experienced anti-HBs seroconversion for >3 weeks were not examined for the appearance of their serum HBsAg.

Fig. 2.

Long-term expression of HBV replication intermediates, transcripts, and proteins was observed in the livers of HBV carrier mice. Southern blotting (SB) and Northern blotting (NB) showed the input HBV DNA, relaxed-circle (RC) DNA, ssDNA, and 3.5-kb pregenomic and 2.4/2.1-kb surface mRNAs in the livers of C57BL/6 mice (A) and BALB/c mice (B) at day 3, 5, 7, 14, and 22 after hydrodynamic injection of pAAV/HBV1.2. (C) Southern and Northern blotting in the livers of HBsAg-positive or -negative C57BL/6 mice at day 70, 93, or 222 after hydrodynamic injection of pAAV/HBV1.2. (D and E) Immunohistochemical staining for HBsAg (cytoplasm) (D) and HBcAg (cytoplasm/nucleus) (E) in hepatocytes of HBsAg-positive mice at 381 dpi. (Original magnification: ×200.)

Fig. 3.

Tolerance toward HBsAg was noted in HBV carrier mice. (A) Serum anti-HBs in HBV carrier (n = 5) or naïve C57BL/6 (n = 4) mice before or after immunization with 5 μg of rHBsAg formulated in CFA (1.5 or 2 months after hydrodynamic injection of pAAV/HBV1.2 or PBS). (B) Serum anti-HBs in immune C57BL/6 (n = 4) or BALB/c (n = 6) mice before or after immunization with 5 μg of rHBsAg formulated in CFA once (1.5 or 2 months after hydrodynamic injection of pAAV/HBV1.2).

Fig. 4.

BALB/c mice produced more IFNγ than C57BL/6 mice after hydrodynamic injection of HBV plasmids. Shown are the numbers (A) and mean spot sizes (B) of HBcAg-specific IFNγ-producing cells in 1 × 10⁶ splenocytes from C57BL/6 or BALB/c mice receiving pAAV/HBV1.2 at day 3 or day 10 after hydrodynamic injection assayed by IFNγ ELISPOT in the presence of 0.3 μg/ml rHBcAg. (C) The copy number of IFNγ mRNA produced by splenocytes from C57BL/6 (n = 3) or BALB/c (n = 3) mice receiving pAAV/HBV1.2 or PBS at day 10 after hydrodynamic injection in the presence of 0.3 μg/ml rHBcAg in cultures. Copy number of mGAPDH mRNA was used for normalization. The experiments were repeated, and the results were consistent. The data were analyzed by t test, and the differences were statistically significant (∗, not detectable; ∗∗, P = 0.0262; ∗∗∗, P = 0.0084).

Fig. 5.

Preexisting HBcAg-specific immunity could prevent HBV persistence in these mice. C57BL/6 mice were immunized with 100 μg of pCDNA3.1(+)/HBc or PBS twice within a 2-week interval. Then, the HBcAg- or mock-immunized mice were injected hydrodynamically with pAAV/HBV1.2 at day 14 after boost. Shown are titers of serum HBeAg (A), HBsAg (B), or anti-HBs (C) in the HBcAg- or mock-immunized C57BL/6 mice after hydrodynamic injection. (D) Number of HBcAg-specific (black bar) or HBcAg_129–140-specific (I-A^b-restricted peptide) (gray bar) IFNγ or IL-2 spot-forming cells in 8 × 10⁵ splenocytes from HBcAg- or mock-immunized C57BL/6 mice receiving pAAV/HBV1.2 at day 10 after hydrodynamic injection assayed by IFNγ or IL-2 ELISPOT in the presence of 0.3 μg/ml rHBcAg. Experiments depicted in A–D were performed on five mice per group and were repeated. (E) Southern and Northern blotting by using the livers samples from HBcAg-immunized (I) or mock-immunized (M) C57BL/6 mice at days 1, 3, 5, 7, and 14 after hydrodynamic injection of pAAV/HBV1.2.

Fig. 6.

Adoptive transfer of HBcAg-specific immunity to HBV carrier mice could cure HBV persistence in these mice. Shown are titers of serum HBsAg (A) or anti-HBs (B) in the HBV carrier C57BL/6 mice adoptively transferred with splenocytes from HBcAg-immunized or vector-immunized C57BL/6 mice.