Comparison of anti-hepatitis B virus activities of lamivudine and clevudine by a quantitative assay

Abstract

In this study, we used a quantitative assay to measure the concentration-dependent effects of antivirals on extracellular hepatitis B virus (HBV) DNA as well as on different cytoplasmic and nuclear forms of HBV DNA that participate in HBV replication. HBV recombinant baculovirus, which efficiently delivers the HBV genome to HepG2 cells, was used for this study because (i) antivirals can be administered prior to initiation of HBV infection or after HBV infection and (ii) sufficiently high HBV replication levels are achieved that HBV covalently closed circular (CCC) DNA can be easily detected and individual HBV DNA species can be quantitatively analyzed separately from total HBV DNA. The results showed that the levels of HBV replicative intermediate and extracellular DNA decreased in a concentration-dependent fashion following antiviral treatment. The 50% effective concentration (EC(50)) and EC(90) values and the Hill slopes differed for the different HBV DNA species analyzed. The data clearly indicated that (i) nuclear HBV DNAs are more resistant to antiviral therapy than cytoplasmic or extracellular HBV DNAs and (ii) nuclear HBV CCC DNA is more resistant than the nuclear relaxed circular form. This report presents the first in vitro comparison of the effects of two antivirals administered prior to initiation of HBV infection and the first thorough in vitro quantitative study of concentration-dependent antiviral effects on HBV CCC DNA.

FIG. 1.

Effect of posttreatment with lamivudine on HBV replication. HepG2 cells were seeded in 60-mm-diameter dishes, infected with HBV recombinant baculovirus at 100 PFU/cell, and treated with different concentrations of lamivudine starting at 4 days p.i. Cells were daily fed fresh medium with or without the drug. Drug treatment was continued for 7 days, at which time the cells were harvested and the levels of HBV RI and non-protein-bound nuclear HBV DNA were determined. Samples of conditioned medium exposed to the infected cells for 24 h prior to harvest were collected 7 days after the initiation of treatment and assessed by Southern blot analysis for extracellular HBV DNA. Three independent experiments were carried out. HBV DNA bands were visualized using a Phosphorimager. Digital images of the Southern blots for a representative experiment are shown for HBV extracellular DNA (A), HBV RI DNA (B), and non-protein-bound HBV DNA (C). The concentrations of lamivudine used are also indicated. HBV DNA standards were included in each gel subjected to Southern blot analysis, thereby making it possible to quantitate the amount of HBV DNA. HBV DNA bands were visualized and quantitated using a Phosphorimager and ImageQuant software (Molecular Dynamics). The data from the Southern blots were analyzed for the representative experiment shown and plotted (in picograms/culture/24 h against log lamivudine concentration) as HBV extracellular DNA (D), HBV RI DNA (E), and non-protein-bound HBV DNA (F). The average HBV DNA values for all three experiments were also determined and plotted (in picograms/culture/24 h against log lamivudine concentration) for HBV extracellular DNA (G), HBV RI DNA (H), and non-protein-bound HBV DNA (I). For each step in the replication of HBV DNA, it was possible to quantify from the Southern blots not only total HBV DNA but also individual species of HBV DNA. For HBV extracellular DNA, HBV DNA concentrations were calculated for RC-DS-SS (total) DNA (∗), RC-DS DNA (▴), and SS DNA (▾). For HBV RI, HBV DNA concentrations were calculated for RC-DS-SS (total) DNA (○), RC-DS DNA (▵), and SS DNA (□). For non-protein-bound HBV DNA, HBV DNA concentrations were calculated for RC DNA (♦) and CCC DNA (▪).

FIG. 2.

Effect of posttreatment with l-FMAU on HBV replication. HepG2 cells were seeded in 60-mm-diameter dishes, infected with HBV recombinant baculovirus at 100 PFU/cell, and treated with different concentrations of l-FMAU starting at 4 days p.i. Cells were daily fed fresh medium with or without the drug. Drug treatment was continued for 7 days, at which time the cells were harvested and the levels of HBV RI and non-protein-bound nuclear HBV DNA were determined. Samples of conditioned medium exposed to the infected cells for 24 h prior to harvest were collected 7 days after the initiation of treatment and assessed by Southern blot analysis for extracellular HBV DNA. Three independent experiments were carried out. HBV DNA bands were visualized using a Phosphorimager. Digital images of the Southern blots for a representative experiment are shown for HBV extracellular DNA (A), HBV RI DNA (B), and non-protein-bound HBV DNA (C). The concentrations of l-FMAU used are also indicated. HBV DNA standards were included in each gel subjected to Southern blot analysis, thereby making it possible to quantitate the amount of HBV DNA. HBV DNA bands were visualized and quantitated using a Phosphorimager and ImageQuant software (Molecular Dynamics). The data from the Southern blots were analyzed for the representative experiment shown and plotted (in picograms/culture/24 h against log l-FMAU concentration) as HBV extracellular DNA (D), HBV RI DNA (E), and non-protein-bound HBV DNA (F). The average HBV DNA values for all three experiments were also determined and plotted (in picograms/culture/24 h against log l-FMAU concentration) for HBV extracellular DNA (G), HBV RI DNA (H), and non-protein-bound HBV DNA (I). For each step in replication of HBV DNA, it was possible to quantify from the Southern blots not only total HBV DNA but also individual species of HBV DNA. For HBV extracellular DNA, HBV DNA concentrations were calculated for RC-DS-SS (total) DNA (∗), RC-DS DNA (▴), and SS DNA (▾). For HBV RI, HBV DNA concentrations were calculated for RC-DS-SS (total) DNA (○), RC-DS DNA (▵), and SS DNA (□). For non-protein-bound HBV DNA, HBV DNA concentrations were calculated for RC DNA (♦) and CCC DNA (▪).

FIG. 3.

Effect of pretreatment with lamivudine on HBV replication. HepG2 cells were seeded in 60-mm-diameter dishes and infected with HBV recombinant baculovirus at 100 PFU/cell at 24 h after plating. Treatment with lamivudine was initiated 16 h prior to HBV baculovirus infection and continued during and after infection for 7 days. Cells were daily fed fresh medium with or without the drug. At 7 days p.i., the cells were harvested and the levels of HBV RI and non-protein-bound nuclear HBV DNA were determined. Samples of conditioned medium exposed to the infected cells for 24 h prior to harvest were collected 7 days p.i. and assessed by Southern blot analysis for extracellular HBV DNA. Three independent experiments were carried out. HBV DNA bands were visualized using a Phosphorimager. Digital images of the Southern blots for a representative experiment are shown for HBV extracellular DNA (A), HBV RI DNA (B), and non-protein-bound HBV DNA (C). The concentrations of lamivudine used are also indicated. HBV DNA standards were included in each gel subjected to Southern blot analysis, thereby making it possible to quantitate the amount of HBV DNA. HBV DNA bands were visualized and quantitated using a Phosphorimager and ImageQuant software (Molecular Dynamics). The data from the Southern blots were analyzed for the representative experiment shown and plotted (in picograms/culture/24 h against log lamivudine concentration) as HBV extracellular DNA (D), HBV RI DNA (E), and non-protein-bound HBV DNA (F). The average HBV DNA values for all three experiments were also determined and plotted (in picograms/culture/24 h against log lamivudine concentration) for HBV extracellular DNA (G), HBV RI DNA (H), and non-protein-bound HBV DNA (I). For each step in replication of HBV DNA, it was possible to quantify from the Southern blots not only total HBV DNA but also individual species of HBV DNA. For HBV extracellular DNA, HBV DNA concentrations were calculated for RC-DS-SS (total) DNA (∗), RC-DS DNA (▴), and SS DNA (▾). For HBV RI, HBV DNA concentrations were calculated for RC-DS-SS (total) DNA (○), RC-DS DNA, (▵) and SS DNA (□). For non-protein-bound HBV DNA, HBV DNA concentrations were calculated for RC DNA (♦) and CCC DNA (▪).

FIG. 4.

Effect of pretreatment with l-FMAU on HBV replication. HepG2 cells were seeded in 60-mm-diameter dishes and infected with HBV recombinant baculovirus at 100 PFU/cell at 24 h after plating. Treatment with l-FMAU was initiated 16 h prior to HBV baculovirus infection and continued during and after infection for 7 days. Cells were daily fed fresh medium with or without the drug. At 7 days p.i., the cells were harvested and the levels of HBV RI and non-protein-bound nuclear HBV DNA were determined. Samples of conditioned medium exposed to the infected cells for 24 h prior to harvest were collected 7 days p.i. and assessed by Southern blot analysis for extracellular HBV DNA. Three independent experiments were carried out. HBV DNA bands were visualized using a Phosphorimager. Digital images of the Southern blots for a representative experiment are shown for HBV extracellular DNA (A), HBV RI DNA (B), and non-protein-bound HBV DNA (C). The concentrations of l-FMAU used are also indicated. HBV DNA standards were included in each gel subjected to Southern blot analysis, thereby making it possible to quantitate the amount of HBV DNA. HBV DNA bands were visualized and quantitated using a Phosphorimager and ImageQuant software (Molecular Dynamics). The data from the Southern blots were analyzed for the representative experiment shown and plotted (in picograms/culture/24 h against log l-FMAU concentration) as HBV extracellular DNA (D), HBV RI DNA (E), and non-protein-bound HBV DNA (F). The average HBV DNA values for all three experiments were also determined and plotted (in picograms/culture/24 h against log l-FMAU concentration) for HBV extracellular DNA (G), HBV RI DNA (H), and non-protein-bound HBV DNA (I). For each step in replication of HBV DNA, it was possible to quantify from the Southern blots not only total HBV DNA but also individual species of HBV DNA. For HBV extracellular DNA, HBV DNA concentrations were calculated for RC-DS-SS (total) DNA (∗), RC-DS DNA (▴), and SS DNA(▾). For HBV RI, HBV DNA concentrations were calculated for RC-DS-SS (total) DNA (○), RC-DS DNA (▵), and SS DNA (□). For non-protein-bound HBV DNA, HBV DNA concentrations were calculated for RC DNA (♦) and CCC DNA (▪).