We assessed here the antiviral activity of a novel treatment strategy for hepatitis B in chronically infected woodchucks by using L-FMAU/FTC combination therapy with or without the additional delivery of a IFN-γ gene via a recombinant adenovirus vector. The combination of L-FMAU and FTC significantly inhibited WHV replication in vivo. A rapid and significant decrease in viremia level by a mean 4 to 5 log10
was observed within 2 weeks and a maximal final drop of 9 log10
was observed for woodchuck w649. A >3-log10
reduction in virus titers within 2 weeks has been reported by Peek et al. in the sera of chronically infected WHV carriers after treatment with L-FMAU at a daily dose of 10 mg per kg of body weight (29
). Zhu et al. confirmed these results in a more recent study in WHV-infected woodchucks after 4 weeks of L-FMAU treatment (43
). In a long-term L-FMAU treatment study, the minimum WHV titers reached 106
copies/ml between weeks 20 and 30 (43
). Cullen et al. demonstrated that FTC at an intraperitoneal dose of 30 mg/kg induced a 56-fold (1.75-log10
) reduction in the WHV DNA level in serum over a 4-week treatment period (8
). Based on these reproducible historical controls for L-FMAU and FTC monotherapy, our observations suggest that the antiviral activity of FTC and L-FMAU was improved when the drugs were combined. This is consistent with the results obtained in vitro with the same compounds in the DHBV model (33
) and therefore deserves further evaluation.
The antiviral effect of L-FMAU/FTC bitherapy persisted over time and was characterized by a delayed viral rebound in four of eight animals with a long-term follow-up. Indeed, viremia remained undetectable by dot blot assay (<6.107 copies/ml) in serum samples 1 to >12 months after treatment cessation. Inhibition of viral replication was especially sustained in two animals (w649 and w652) which showed the lowest viremia levels at baseline. In these woodchucks, the intrahepatic levels of WHV DNA and serum viral load remained suppressed for several months. This prolonged antiviral effect was consistent with a dramatic decrease in cccDNA levels. The delay observed in rebound of viral replication suggests that the combination of L-FMAU and FTC may have induced, at least in some animals, a reduction of WHV DNA synthesis below a threshold level necessary to maintain steady-state levels of cccDNA. This latency could be a reflection of the time required to reestablish cccDNA pools through reinfection of new hepatocytes and/or reamplification of existing intracellular pools of cccDNA in infected cells.
Although there was no control group with single drug administration because of the limited access to this animal model, the antiviral activity of L-FMAU and FTC in combination was greater than that previously reported for other single antiviral drugs administered to WHV chronic carriers (6
). The sustained antiviral effect on WHV replication after drug withdrawal observed in the present study suggests that life-long therapy may not be required for long-term control of chronic HBV infection. This new concept should be confirmed by further experimental evidence in a larger number of animals.
Interesting findings also came from our analysis of the evolution of intrahepatic viral load. Southern blot analysis of intrahepatic DNA showed a significant loss in the level of cccDNA and DNA replicative forms throughout the bitherapy, with a much lower rate of decline for the cccDNA than the replicative DNA intermediates (18
). This effect may be due to the antiviral activity of L-FMAU, as previously suggested by Zhu et al. (43
). However, in the latter study, long-term treatment with L-FMAU alone allowed the emergence of WHV drug-resistant strains (41
). The results of our study suggests that the potency of short-term antiviral combination therapy with L-FMAU and FTC may significantly decrease the viral cccDNA pool in less than 4 weeks, thus delaying the rebound of viral replication after treatment cessation and the selection of drug-resistant mutants.
Two different mechanisms may account for the loss of cccDNA in the liver: (i) a reduction of cccDNA in individual infected cells (12
) and/or (ii) a replacement of infected cells by newly divided virus-free hepatocytes (13
). The results of the analysis of PCNA expression in the livers of infected animals are consistent with the hypothesis that a fraction of cccDNA may survive through mitosis, being distributed to daughter cells. Thus, the cccDNA copy number in infected cells should decline through dilution as infected cells divide. In that case, most hepatocytes that remain infected would have fewer copies of cccDNA. This in turn may explain, in some animals, the sustainability of viral suppression after drug withdrawal and the decrease of viral antigens.
The results of experiments performed with HBV-transgenic mice supports the possibility that hepadnavirus replication intermediates may be cleared by noncytolytic processes induced by the expression of TH1 cytokines in the liver (5
). Zhou et al. assessed, in chronically infected woodchucks, the effect of lamivudine treatment combined with superinfection with a β-galactosidase-expressing adenovirus. These authors observed a short-term suppression of the WHV replication that coincided with the time of recovery from the adenovirus infection (42
). One possible explanation for this pattern of response is that the virus replication was temporarily suppressed by the activation of the host immune response to WHV. The transient reduction in cccDNA and replicative intermediates may be explained by cytotoxic-T-lymphocyte killing of the adenovirus-infected hepatocytes and/or by a bystander effect as described for HBV-transgenic mice infected with adenovirus (4
We failed to obtain evidence that Ad-IFN, directly expressing wIFN-γ rather than indirectly inducing its production, can deplete the cccDNA or decrease the number of infected cells in the liver. No significant antiviral effect of wIFN-γ delivery was observed after detailed analysis of serum and intrahepatic viral loads. For lack of suitable tools, the actual levels of woodchuck IFN-γ expression cannot currently be quantified; formally, this negative result might therefore be attributed to the failure of the Ad-IFN vector to deliver a biologically active wIFN-γ gene. However, several lines of evidence argue against this interpretation. First, the Ad-IFN vector induced, upon transduction into HEK 293 cells, secretion of an activity into the supernatants that protected the woodchuck WCH17 cell line from VSV infection, at similar if not higher dilutions than those previously reported (23
). Second, this activity was, as expected for IFN, species specific because the 293 supernatants, but not human IFN-γ, induced expression in the WCH17 cell line of a bona fide GBP mRNA. In at least five of the Ad-IFN-treated animals a band of similar mobility on Northern blots was induced (data not shown). More directly, animals that received L-FMAU/FTC+Ad-IFN showed clear signs of enhanced liver inflammation. All of these findings are indicative of the generation of biologically active wIFN-γ.
A second potential problem is ineffective delivery of the Ad-IFN vector into the woodchuck livers. However, consistent with previously reported data from studies using a homologous adenovirus (42
), specific RFP fluorescence could be detected in the majority of the hepatocytes 4 days postinoculation in the livers of the woodchucks that received the Ad-IFN vector (Table ). This finding therefore suggests that the Ad-IFN vector indeed reached the liver and was transcriptionally active in the hepatocytes.
Very recently, Summers et al. provided evidence that hepatocyte killing and regeneration play an important role in eliminating cccDNA during clearance of acute WHV infection (35
). Either the enhanced hepatocyte turnover associated with Ad-IFN transduction in our experiments was therefore insufficient in quantity and/or duration to cause a detectable antiviral effect, or chronically WHV-infected hepatocytes may qualitatively differ from acutely infected cells in their abilities to respond to IFN-γ even when viral replication is drastically reduced by chemotherapy. Notably, our negative results are consistent with a recent in vitro study by Lu et al., who showed that at a concentration of up to 1,000 U/ml, wIFN-γ was unable to inhibit WHV replication in primary hepatocytes from persistently WHV-infected woodchucks (23
In conclusion, we showed here that a combination of the two polymerase inhibitors, L-FMAU and FTC, may be able to induce a prolonged and sustained inhibition of viral replication in WHV-infected woodchucks. The delivery of wIFN-γ after the initial drop in viral load by the nucleoside bitherapy induced an inflammatory cell infiltration in the liver and an increase of cell turnover, which were, however, insufficient to enhance the antiviral effect of the polymerase inhibitor administration.