Our findings show that extensive analysis of HBV quasispecies dynamics aids understanding of the fine mechanisms of resistance to antiviral drugs. Experience with highly active antiretroviral therapy in HIV-infected patients shows that resistance to HIV RT inhibitors is acquired gradually, through the selection of preexisting resistant variants and gradual accumulation of new amino acid substitutions that confer stepwise increases in the level of drug resistance (reviewed in reference 4
). Schematically, partial resistance conferred by a substitution in a preexisting viral population impairs drug efficacy sufficiently to restore a level of replication compatible with the accumulation of further mutations. The latter may restore the in vivo replication capacity of the resistant virus, allowing viral replication to return to pretreatment levels. There are some exceptions to this rule, however, such as the M184V substitution in the HIV RT, which immediately confers full resistance to lamivudine together with a significant fitness hit (34
), and single mutations in the hydrophobic pocket of the HIV RT, which also confer strong resistance to nonnucleoside RT inhibitors (15
). HBV resistance to lamivudine appears to be more closely related to the latter mechanism, for the following reasons: (i) HBV lamivudine resistance is characterized by a rapid, one-step reincrease in viral replication to baseline levels (18
), as confirmed here; and (ii) full resistance can be conferred by a single substitution (V or I) at RT position 204, in the YMDD motif; this results in a more than 1,000-fold increase in resistance in vitro (1
), and it led to a return to baseline replication levels in our patients B and C.
Our results suggest that rtM204V and rtM204I are frequent preexisting polymorphisms in HBV-infected patients and that they appear randomly in viral populations which have a replication disadvantage relative to YMDD variants in the absence of lamivudine. Their selection during lamivudine therapy appears to depend principally on their presence at the outset of treatment, as only rtM204V variants were selected in our patients A and B, while only rtM204I variants were selected in patient C. In patient D, who harbored both variants prior to treatment, the two were selected together, and their dynamics fluctuated as additional mutations accumulated. A steady state was reached when YIDD variants overtook YVDD variants, even though both variants were still present at the end of follow-up. It is theoretically possible, however, that YMDD variants could arise after the initiation of therapy, given the relatively high levels of residual replication during lamivudine administration (Table ).
This study provides new information on the influence of amino acid substitutions located outside the C domain of HBV RT in vivo. Two substitutions, rtL180M and rtV173L, are frequently associated with the C domain during the onset of lamivudine resistance. Both partially restore the replication capacities of C-domain HBV mutants in vitro (1
). In our study, the in vivo replicative advantages conferred by these substitutions varied substantially from one patient to another and also from one time point to another in a given patient. Thus, the in vivo replication of HBV variants appears to be driven by multiple forces, including the following: (i) intrinsic replicative advantages potentially conferred to lamivudine-resistant variants by mutations accumulating outside domain C, such as rtL180M and rtV173L, and (ii) the fluctuating environment in which these variants replicate during lamivudine administration to a given patient, potentially favoring one variant population over another, regardless of their intrinsic replication capacity. Direct host protein-viral protein interactions and the host immune response probably play a key role in determining relative HBV variant replication capacities in vivo. As a result, the influence of specific resistance mutations on the development and establishment of HBV resistance to antiviral drugs in vivo cannot be inferred solely from in vitro replication experiments and cannot be fully predicted by structural analysis in silico.
Our results also have major implications for virological follow-up and diagnosis of resistance in the clinical setting. Currently, the most sensitive method for detecting early selection of resistant variants is to generate large numbers of clones at regular intervals during therapy. By analyzing approximately 30 clones per time point and carrying out a highly sensitive quantitative HBV DNA assay based on real-time PCR technology, we started to detect resistant variants between 2 and 4 months before virological breakthrough. However, this approach is too cumbersome to be used in clinical practice. Reverse hybridization with the line probe assay, which can detect variant populations representing approximately 10% of the HBV quasispecies, is usable in practice and was recently reported to reveal the emergence of resistance variants an average of 2 weeks before the HBV DNA load started to increase (26
). The time of virological breakthrough is too late for diagnosis of HBV resistance, because most of the quasispecies variants are already both resistant and highly fit. This is a particularly important issue, because lamivudine resistance is associated with gradual progression of liver disease (7