Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-gamma

Abstract

Aim: To evaluate the in vitro anti-HBV activity of recombinant human IFN-gamma, alone and in combination with lamivudine.

Methods: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization.

Results: IFN-gamma at 0.1 to 5 microg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5 microg/L, IFN-gamma also suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-gamma showed no additive effect, sequential treatment first with lamivudine and then IFN-gamma was found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2 micromol/L lamivudine for two days, followed by 1 microg/L IFN-gamma for another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2 micromol/L lamivudine for two days, followed by 5 microg/L IFN-gamma for six days showed a 72% reduction in HBV cccDNA pool.

Conclusion: This in vitro study warrants further evaluation of a combination of IFN-gamma and lamivudine, especially in IFN-alpha non-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.

Figures

Figure 1

Construction of the recombinant Baculo-HBV virus (Bac-HBV). A: The recombinant transfer vector, pBacHep1.6 (10.5 kb) together with the 1.6 × genome length HBV cloned in an orientation opposite to the polyhedrin promotor (pPol) in vector pBacPAK9. The construct contains HBV-ORFs: PreS/S (1x), X (2x), P (1x), PreC/C (2x) and Enh I (2x), Enh II (2x) elements; B: HBsAg expression in culture medium upon transduction with 100 moi of Bac-HBV; C: Dot blot analysis of total HBV DNA in HepG2 cells transduced with 100 moi of Bac-HBV. Different amounts of HBV DNA were used as quantitation standards, as indicated; D: Densitometric analysis and quantitative representation of the results shown in C.

Figure 2

A time course HBsAg profile (ELISA) of transduced HepG2 cells, treated with variable doses of lamivudine and IFN-γ. A: Effect of lamivudine used at concentrations of 0.02, 0.2, and 2.0 μmol/L; B: Effect of IFN-γ used at concentrations of 0.1, 1.0 and 5.0 μg/L.

Figure 3

Representative Southern blot (A) and quantitative (densitometry) analysis (B) of effect of IFN-γ (5.0 μg/L) treatment on HBV DNA replication in transduced HepG2 cells. Synthesis of viral replicative intermediates: relaxed coil (RC), double stranded linear (DSL), covalently closed circular (CCC), and single stranded (SS) DNA forms in treated (•) and untreated (□) cells.

Figure 4

Effects of sequential treatment with “low/high-dose” of lamivudine and IFN-γ on HBV gene expression and total DNA load in transduced HepG2 cells. A: HBsAg profile; B: total HBV DNA upon sequential treatment of transduced cells. 1: Lamivudine (0.02 or 2.0 μmol/L) 6 d; 2: IFN-γ (1.0 μg/L) 6 d; 3: Lamivudine (0.02 or 2.0 μmol/L) 2 d + IFN-γ (1 μg/L) 4 d; 4: Untreated 2 d + IFN-γ (1.0 μg/L) 4 d; 5: IFN-γ (0.1 or 5 μg/L) 2 d + lamivudine (0.2 μmol/L) 4 d; 6: Untreated 2 d + lamivudine (0.2 μmol/L) 4 d; 7: Untreated control.

Figure 5

Effects of sequential treatment with “high-dose” of lamivudine and IFN-γ (A) on HBeAg expression in transduced HepG2 cells (B). 1: Lamivudine (2.0 μmol/L) 6 d; 2: IFN-γ (5.0 μg/L) 6 d; 3: Lamivudine (0.2 μmol/L) 2 d + IFN-γ (5 μg/L) 4 d; 4: Untreated 2 d + IFN-γ (5 μg/L) 4 d; 5: Lamivudine (0.2 μmol/L) 2 d and untreated 4 d; 6: Untreated control.

Figure 6

Effect of sequential treatment with “high-dose” of Lamivudine and IFN-γon HBV cccDNA pool in transduced HepG2 cells. The figure shows densitometric analysis of the 2 kb cccDNA form. 1: Untreated control; 2: Lamivudine (0.2 μmol/L) 8 d; 3: Lamivudine (0.2 μmol/L) 2 d + IFN-γ (5.0 μg/L) 6 d.