Immune effectors required for hepatitis B virus clearance

Abstract

To better define the mechanism(s) likely responsible for viral clearance during hepatitis B virus (HBV) infection, viral clearance was studied in a panel of immunodeficient mouse strains that were hydrodynamically transfected with a plasmid containing a replication-competent copy of the HBV genome. Neither B cells nor perforin were required to clear the viral DNA transcriptional template from the liver. In contrast, the template persisted for at least 60 days at high levels in NOD/Scid mice and at lower levels in the absence of CD4(+) and CD8(+) T cells, NK cells, Fas, IFN-gamma (IFN-gamma), IFN-alpha/beta receptor (IFN-alpha/betaR1), and TNF receptor 1 (TNFR1), indicating that each of these effectors was required to eliminate the transcriptional template from the liver. Interestingly, viral replication was ultimately terminated in all lineages except the NOD/Scid mice, suggesting the existence of redundant pathways that inhibit HBV replication. Finally, induction of a CD8(+) T cell response in these animals depended on the presence of CD4(+) T cells. These results are consistent with a model in which CD4(+) T cells serve as master regulators of the adaptive immune response to HBV; CD8(+) T cells are the key cellular effectors mediating HBV clearance from the liver, apparently by a Fas-dependent, perforin-independent process in which NK cells, IFN-gamma, TNFR1, and IFN-alpha/betaR play supporting roles. These results provide insight into the complexity of the systems involved in HBV clearance, and they suggest unique directions for analysis of the mechanism(s) responsible for HBV persistence.

Fig. 1.

Viral gene expression in wild-type and knockout mice. Serum HBsAg levels (open bars) were quantified by enzyme-linked immunosorbant assay (ELISA). Data represent averages (n ≥ 3). The average number of HBcAg⁺ hepatocytes in the liver (filled bars) was quantified by counting at least 100 grids per liver (×20 magnification) for at least three animals for each strain. All strains examined had comparable levels of serum HBsAg and HBcAg⁺ hepatocytes on day 4.

Fig. 2.

Control of HBV replication and clearance of the transcriptional template in wild-type and knockout mice. (A) Clearance of the transcriptional template was monitored by real-time PCR assay using probes that amplify a sequence on the backbone of the input plasmid. (B) Replication of the viral genome was monitored by real-time PCR assays using two primer sets specific for HBV and the input plasmid. Ratios of HBV:input plasmid greater than 2 indicated the presence of replicative DNA intermediates. (C) HBcAg-positive hepatocytes were quantified by counting at least 100 grids per liver (×20 magnification) for at least three animals for each strain. *, data were not collected at this time point.

Fig. 3.

Frequency and localization of HBcAg in wild-type and knockout mice. Representative sections stained for the presence of HBcAg are presented. Images were collected at ×40 magnification. (A) C57BL/6, day 4. All strains exhibited similar frequency and distribution of HBcAg on day 4. (B) CD8 knockout, day 20. CD4-, TNFR1-, and Fas-deficient animals exhibited similar frequency and distribution of HBcAg at this time point. (C) Fas knockout, day 60. CD4-, TNFR1-, and Fas-deficient animals exhibited similar frequency and distribution of HBcAg at this time point. (D) NOD/Scid, day 60. C57BL/6 mice were representative of the frequency and distribution of HBcAg staining observed in all strains examined on day 4. By day 20, C57BL/6 mice and mice deficient in B cells, perforin, or IFN-α/βR had no detectable HBcAg. In contrast, NOD/Scid animals and animals deficient in CD8, CD4, NK cells, IFN-γ, TNFR1, or Fas exhibited HBcAg in both the nucleus and cytoplasm of hepatocytes on day 20. IFN-γ- and NK cell-deficient animals cleared HBcAg by day 60 whereas animals deficient in CD8, CD4, Fas, or TNFR1 exhibited nuclear HBcAg at comparable frequencies between days 30 and 60. NOD/Scid animals exhibited hepatocytes with both nuclear and cytoplasmic HBcAg at stable levels between days 30 and 60.

Fig. 4.

The HBV HBcAg93-specific intrahepatic CD8⁺ T cell response. Intrahepatic lymphocytes were isolated from mice on day 14 after transfection and then stained ex vivo with FITC-anti-CD8 and the HBV HBcAg93-dimer followed by a PE-anti-mouse Ig secondary antibody. The percentage of intrahepatic CD8⁺ T cells specific for HBcAg93 was measured and averaged (n ≥ 3). Values plotted were expressed as a percentage of all CD8⁺ T cells normalized against C57BL/6.