Hepatitis B virus e antigen loss during adefovir dipivoxil therapy is associated with enhanced virus-specific CD4+ T-cell reactivity

Abstract

Weak T-cell reactivity to hepatitis B virus (HBV) is thought to be the dominant cause for chronic HBV infection. Treatment with adefovir dipivoxil (ADV) increases the rate of HBV e antigen (HBeAg) loss; however, the immune mechanisms associated with this treatment response are not understood. Serial analysis of HBV-specific CD4+ T-cell reactivity was performed during 48 weeks of therapy with ADV and correlated with treatment outcome for 19 HBeAg-positive patients receiving ADV (n = 13) or the placebo (n = 6). We tested T-cell reactivity to HBV at seven protocol time points by proliferation, cytokine production, and enzyme-linked immunospot assays. A panel of serum cytokines was quantitated by cytokine bead array. ADV-treated patients showed increased CD4+ T-cell responses to HBV and lower serum levels of cytokines compared to those of placebo-treated patients. Enhanced CD4+ T-cell reactivity to HBV, which peaked at treatment week 16, was confined to a subgroup of ADV-treated patients who achieved greater viral suppression (5.3 +/- 0.3 log(10) copies/ml [mean +/- standard error of the mean {SEM}] serum HBV DNA reduction from baseline) and HBeAg loss, but not to ADV-treated patients with moderate (3.4 +/- 0.2 log(10) copies/ml [mean +/- SEM]) viremia reduction who remained HBeAg positive or to patients receiving the placebo. In conclusion, T-cell reactivity to HBV increases in a proportion of ADV-treated patients and is associated with greater suppression of HBV replication and HBeAg loss.

FIG. 1.

Comparison of CD4⁺ T-cell reactivity to HBcAg in patients receiving ADV or the placebo. *, there was a significant increase in the frequencies of IFN-γ-producing CD4⁺ T cells at TW16 compared to baseline values for ADV-treated patients (P = 0.03). **, at TW40 and TW48, patients receiving ADV had significantly higher frequencies of IFN-γ-producing CD4⁺ T cells than did patients receiving placebos (P = 0.03). The bars represent means ± SEMs.

FIG. 2.

Relationship between serum HBV DNA levels and the frequency of virus-specific CD4⁺ T cells during the study period for the three groups of patients presented in Table 2. *, patients in group 1 had significantly lower HBV DNA levels at TW48 than did patients in groups 2 (P = 0.011) and 3 (P = 0.003). **, patients in group 2 had significantly lower HBV DNA levels at TW48 than did patients in group 3 (P = 0.028). The bars represent the means ± SEMs for the frequency of HBV-specific CD4⁺ T cells. Error bars indicate standard deviations.

FIG. 3.

HBcAg-specific T-cell proliferation according to virological response. Patients in group 1 had a higher number of positive tests results for T-cell proliferation to HBcAg than did patients in group 3 (P = 0.03).

FIG.4.

Time course analysis of serum ALT levels, virological parameters, and the frequency of IFN-γ-producing CD4⁺ T cells during the study period. (a and b) For two representative patients from group 1, an increased frequency of IFN-γ-producing CD4⁺ T cells preceded or coincided with HBeAg loss, without ALT flare. (c) A representative patient from group, who received ADV treatment, but had moderate reduction in viremia, remained HBeAg⁺ and showed no increase in the frequency of virus-specific, IFN-γ-producing T cells. IFN_γ, IFN-γ; ND, not determined.

FIG.4.

Time course analysis of serum ALT levels, virological parameters, and the frequency of IFN-γ-producing CD4⁺ T cells during the study period. (a and b) For two representative patients from group 1, an increased frequency of IFN-γ-producing CD4⁺ T cells preceded or coincided with HBeAg loss, without ALT flare. (c) A representative patient from group, who received ADV treatment, but had moderate reduction in viremia, remained HBeAg⁺ and showed no increase in the frequency of virus-specific, IFN-γ-producing T cells. IFN_γ, IFN-γ; ND, not determined.

FIG. 5.

Comparison of serum cytokine profiles using cluster analysis of normalized values. The levels of all cytokines measured, or measured separately for IL-12p70, IFN-γ, and IL-2, were significantly higher in the placebo (PLB)-treated group than in the ADV-treated group. At TW16, the difference was even more apparent between PLB- and ADV-treated groups, while there was no difference between the two ADV-treated subgroups with or without HBeAg loss (group 1 [G1] or group 2 [G2], respectively). Error bars indicate standard deviations.