Because CCC DNA is stably maintained in infected hepatocytes, clearance of HBV infection is dependent on the relatively slow turnover of infected cells, and antiviral therapy of chronic hepadnavirus infections with nucleoside analogs requires long-term administration to eradicate viral infection (30
). However, prolonged antiviral treatment with lamivudine may be associated with the selection of resistant strains in a significant proportion of patients (11
). Adefovir is a promising nucleotide analog that exhibits activity against wild-type HBV as well as lamivudine-resistant HBV mutants (15
). Since the kinetics of viral clearance in adefovir-treated patients were reported to be faster than with lamivudine, we investigated the effect of adefovir on CCC DNA formation and amplification in the duck model of HBV infection.
In primary hepatocyte cultures infected with DHBV in vitro, adefovir induced a concentration-dependent inhibition of viral DNA synthesis. These data are consistent with the observation of Heijtink et al. (18
) and Kruining et al. (24
), who also showed a potent inhibitory effect of adefovir on both DHBV and HBV replication in vitro. However, despite strong inhibition of the viral DNA replicative intermediates and viral CCC DNA synthesis, CCC DNA was not cleared from cultured hepatocytes. Our results demonstrated a greater suppression of DHBV replication by adefovir than by lamivudine, in agreement with previous data obtained with avian cells transfected with the DHBV genome (42
). On the other hand, lamivudine was shown to be as effective as or more effective than adefovir in human hepatoma cell lines transfected with the HBV genome (14
). These differences in activity may reflect differences between the species of hepadnavirus and of the cell type. In our in vivo study, adefovir was used intraperitoneally because it has a poor oral bioavailability. The intraperitoneal route makes it possible to obtain in blood circulation the same concentration of adefovir as that resulting from oral administration of the prodrug, adefovir dipivoxil (8
). This in vivo study also showed that adefovir inhibits DHBV replication but is not able to eradicate viral CCC DNA from the liver, in keeping with other observations with the duck model with adefovir (18
) or other compounds (31
). Our data are also consistent with those of others, who showed, using woodchuck hepatocyte cultures treated long term with different nucleoside analogs, including lamivudine (34
) and adefovir (9
), that viral CCC DNA is a very stable molecule whose half-life may match that of the infected hepatocytes.
Besides the observation that adefovir administration inhibits DHBV viral DNA synthesis in hepatocytes in vitro and in vivo, our study revealed new information regarding its antiviral activity. In primary hepatocyte cultures, administration of adefovir 1 day prior to inoculation with virus delayed and inhibited DHBV genome replication and amplification of viral CCC DNA more potently than lamivudine. However, as observed with lamivudine, adefovir was unable to completely prevent the initial formation of CCC DNA. The in vivo administration of adefovir 1 day prior to viral inoculation and maintained administration of the drug for 3 weeks postinoculation confirmed our in vitro data. The results showed a more significant reduction of DHBV replication by adefovir than by lamivudine, but CCC DNA that was formed despite antiviral therapy was the source of renewed viral production after the cessation of therapy. These data suggest that in our experimental conditions, adefovir could inhibit and delay but not prevent the infection of naive hepatocytes and spread of virus in the liver. Potentially, pretreatment for more than 1 day prior to inoculation may allow time for higher concentrations of the drug and the active metabolite, adefovir diphosphate, to accumulate inside cells and confer a more potent effect. Adefovir, which inhibits both HBV and DHBV reverse transcriptase (42
), was unable to prevent the conversion of RC-DNA to transcriptionally active CCC DNA, suggesting that the polymerase activity of the viral reverse transcriptase may not be the only critical determinant involved in the repair reactions. Köck and Schlicht strongly suggested that cellular enzymes may be sufficient for conversion of virion RC-DNA into CCC-DNA (23
). Nonetheless, their analysis was limited, like ours, to possible enzymatic targets which are blocked by nucleoside analogs. Since the viral polymerase mediates, among other functions, the transport of the hepadnavirus genome into the nucleus (22
), it is possible that both cellular and viral enzymes may be involved in viral CCC DNA formation but that adefovir has no influence on the repair reaction.
Several studies have showed that viral CCC DNA clearance may involve lysis of infected cells or may be hastened by increased cell turnover (13
). In our primary hepatocyte culture experiments, adefovir did not exhibit significant cellular toxicity. The inhibitory effect on viral CCC DNA amplification may therefore be explained by the potent suppression of the synthesis of viral replication intermediates. Newbold et al. demonstrated the existence of two subpopulations of CCC DNA, depending on the interaction of this viral DNA form with nucleoproteins (36
). They also hypothesized that these two different populations of CCC DNA may have different half-lives (5
). In our in vitro and in vivo experiments, we can speculate that the unstable form of CCC DNA may explain the observation of a decline in total viral CCC DNA over time in primary embryonic duck hepatocyte culture, while the more stable form may explain the lack of complete clearance of CCC DNA both in tissue culture and in vivo.
The faster kinetics of viral clearance observed in chronic hepatitis B patients receiving adefovir therapy (44
) than with lamivudine treatment (38
) may therefore be better explained by a more potent inhibition of hepadnavirus genome replication and its subsequent consequences on CCC DNA amplification rather than a direct effect on the initial formation of viral CCC DNA.
In our study, although a potent reduction of viremia was observed during adefovir therapy, the persistence of DHBV DNA in the liver indicated that the duration of adefovir treatment was not sufficient to clear DHBV-infected hepatocytes. Moreover, these results substantiate the necessity for maximal inhibition of viral replication to increase the initial phase of viral clearance to prevent further cycles of infection of naive hepatocytes. Since adefovir is unable to block the initial formation of CCC DNA, new hepatocytes will continue to be infected as long as residual circulating virions are present in the bloodstream. Once the clearance of free virus from plasma is achieved, the duration of adefovir therapy required to achieve viral elimination would depend only on the persistence of CCC DNA and the longevity of hepatocytes (32
). It is therefore important to further evaluate combination treatments to obtain a synergistic effect on the first phase of viral clearance, as this was elegantly suggested by in vitro experiments with lamivudine, penciclovir, and adefovir (6
), but also with lamivudine and famciclovir in patients (27
Persistence of virus during therapy may result from the long half-life of viral CCC DNA in the liver (49
) as well as the presence of inaccessible extrahepatocytic reservoirs, such as bile duct epithelial cells, a compartment of DHBV replication previously shown to be resistant to penciclovir and lamivudine therapy but susceptible to adefovir (37
). These data and our results suggest that viral clearance during nucleoside analog therapy could be enhanced by protecting uninfected cells from residual circulating virions by using additional agents, such as neutralizing antibodies (33
), cytokines (16
), or other pathways. In fact, adefovir was also reported to exhibit immunomodulatory effects, including stimulation of natural killer cell activity and interferon alpha production (4
). Other approaches, such as combining antiviral therapy with DNA-based vaccination to induce a specific antiviral TH1 immune response (41
), should be further evaluated as strategies to enhance viral clearance.