This report describes virologic breakthrough after prolonged ETV therapy in two patients from phase II clinical trials. The results of genotypic and phenotypic analyses of samples from these patients suggest that clinically relevant resistance to ETV requires at least three different concomitant amino acid substitutions in HBV RT, in contrast to the single amino acid substitutions that lead to viral resistance to 3TC (1
) or ADV (2
Both patients had failed 3TC therapy prior to receiving ETV, and baseline viral isolates showed genotypic evidence of the signature 3TCr
substitutions rtL180M and rtM204V. Levine et al. previously reported that the intracellular accumulation of ETV-TP was such that potency against viruses with 3TCr
substitutions was retained (20
), a finding that was confirmed in clinical studies (4
). However, patient A had an unusually poor response to ETV, with a reduction in viral load of only about 2 log10
copies/ml after 1 year in comparison with similar phase II trial patients (4
). Further treatment of patient A with combined ETV (0.5 mg) and 3TC (100 mg) resulted in only modest fluctuations in viral load. From week 108 to week 133 (>2.5 years of therapy), virologic breakthrough was noted, with an increase in HBV DNA of 1.2 log10
copies/ml, along with a corresponding increase in ALT levels. The atypical clinical picture that emerged for patient A may have favored the development of viral resistance; the viral load was not reduced below 6 log10
copies/ml despite ETV treatment for more than 2.5 years. It is important to note that the currently indicated ETV dose of 1.0 mg for patients with 3TCr
HBV was established subsequent to the enrollment of patient A into clinical studies to further enhance the suppression of 3TCr
HBV. The ETV resistance genotype for patient A, however, first emerged during treatment with 0.5 mg of ETV (Table ).
Evaluation of samples from patient A resulted in the identification of a unique HBV RT substitution, rtM250V, that resides in conserved domain E of viral RT and appears to play a critical role in the expression of an ETV resistance phenotype. In a three-dimensional homology model of HBV and human immunodeficiency virus RTs, rtM250 lies in a position corresponding to the “primer grip” component that interacts with the 3′ terminus of the DNA template (8
). While the 3TCr
substitution rtM204V within the YMDD motif of RT likely affects the initial polymerase binding of deoxynucleoside triphosphate analog inhibitors, substitutions at residue 250 may affect other polymerization events, such as elongation. In vitro HBV cell culture and enzyme experiments with recombinant viruses from patient A revealed that the combination of 3TCr
substitutions rtL180M and rtM204V with substitution rtM250V is required for high levels of ETV resistance. It is intriguing to postulate that changes in more than one functional stage of the polymerase, involving multiple substitutions, operated to bring about ETV resistance in patient A.
Patient B was a liver transplant recipient who had failed previous HBV therapies that had been administered over the course of 8.5 years (3
). Multiple substitutions were noted in the RT domain of the baseline viral isolate, including those associated with failed 3TC or famciclovir therapy (1
; reviewed in reference 13
). Patient B initially responded to ETV treatment with a reduction in viral DNA levels of 2.35 log10
copies/ml by week 24, a decrease that was maintained through week 60. However, as with patient A, viral DNA levels were never sustained below 6 log10
copies/ml, and elevations in both viral DNA and ALT levels were noted at week 76 (1.5 years of therapy). Importantly, the basis for a suboptimal initial response to ETV in this patient, despite the use of the 1.0-mg dose, may lie in the combination of baseline substitution rtT184S and other 3TCr
substitutions. In keeping with this proposed model, cell culture results for patient B revealed an ~2-fold increase in the EC50
of the baseline virus relative to an identical virus carrying the wt threonine at position 184 (Table ). Substitution rtT184S has been found in some 3TCr
isolates in combination with changes at residues 180 and 204 (1
Genotypic analysis of patient B isolates showed simultaneous selection of substitutions rtT/S184G and rtS202I within the 3TCr
background beginning at week 68 of ETV therapy. This observation is consistent with the results of the cell culture phenotypic analysis (Table ), which showed that the quadruple mutant (rtL180M/rtT184G/rtS202I/rtM204V) exhibited the highest level of ETV resistance. We also observed that the recombinant virus containing 3TCr
and rtS202I substitutions in the absence of the rtT184G substitution was severely replication impaired for the production of extracellular HBV and for the yield of intracellular nucleocapsids isolated for polymerase studies (both <5% wt values) (data not shown). Like the 3TCr
substitutions rtL180M and rtM204V, rtT184G and rtS202I reside in domain B and domain C of polymerase, respectively. The rtT184 residue is conserved in alignments of 250 wt HBV genomes and among the human, woodchuck, and duck HBV RTs (9
). While serine 202 is conserved among HBV wt sequences, the corresponding residues in woodchuck HBV and duck HBV are alanine and threonine, respectively (9
). It is unclear what effects these differences would have in the context of 3TCr
changes, although ETV is a very potent inhibitor of both woodchuck and duck HBV infections. A model of 3TCr
HBV RT predicts that substitution rtL180M compensates structurally for substitution rtM204V, consistent with their coselection (1
). Perhaps the coselection of the changes at residues 184 and 202 in viruses from patient B represents similar compensatory changes in HBV polymerase.
Interestingly, substitution rtI169T, located in conserved domain B of polymerase, was selected in both patients, although it was found not to be responsible for the majority of resistance. Its role was tested by using recombinant viruses from patient A, and it was found to modestly increase the level of ETV resistance—more than two- to threefold—in two clones containing 3TCr
and rtM250V substitutions while not affecting resistance in a third. Perhaps substitution rtI169T confers an ancillary or adaptive change and is analogous to substitution rtV173L found after the development of other 3TCr
Discontinuation of 3TC therapy often results in the reappearance of the wt virus (5
). This was not the case for patients A and B, despite treatment with ETV alone for at least 1 year (Tables and ). The virologic advantage of reduced ETV susceptibility may account for this finding, although shifts from 3TCr
substitutions to wt residues during ETV therapy have been observed for other patients (unpublished observations). Nevertheless, retention of the 3TCr
substitutions along with additional substitutions in viral RT was essential for the development of ETV resistance in these patients.
Substitutions in HBV polymerase are often accompanied by changes in HBsAg encoded in an overlapping reading frame, including those in response to antiviral therapy (27
). The ETV-related RT substitutions rtI169T, rtT184G, and rtS202I seen in patients A and B result in HBsAg changes sF161L, sL176V, and sV194F, respectively. The results reported here support the concept that RT changes and not HBsAg substitutions conferred the decreased susceptibility to ETV seen in these patients. First, resistance was evident at the level of in vitro endogenous HBV polymerase activity, as determined by using cores that lack HBsAg. Second, resistance to ETV was seen in cultures when both extracellular (Tables and ) and intracellular (data not shown) nucleocapsids were assayed. Since the HepG2 transfection system is incapable of multiple infectious cycles involving virus release and reinfection, HBsAg arguably does not influence the levels of intracellular replicated HBV DNA within nucleocapsids. However, the experiments described here did not address the interaction of the resulting HBsAg substitutions and the patient's immune system in the control or selection of ETV-resistant viruses.
It is unclear which enzymatic functions of HBV polymerase are affected by the ETV resistance substitutions identified in the patients reported here. While phenotypic resistance to ETV in cell cultures was also evident in the in vitro enzyme assays, the magnitude of resistance appeared to differ. This result was also observed for viruses and enzymes with 3TCr
substitutions and phenotypes (20
). ETV-TP is a potent inhibitor of all three polymerase functions—priming and first (minus)- and second (plus)-strand DNA synthesis (23
)—and all of these functional activities would be operational in the cell culture system. In contrast, the in vitro endogenous polymerase assay primarily evaluates functional steps postpriming. Nonetheless, it will be interesting to determine whether the various substitutions influence different polymerase activities.
In contrast to findings for 3TC, the overall results of evaluations to date suggest that the emergence of ETV-resistant variants likely will be infrequent in both patients who have not had treatment and patients who have been treated with 3TC (4
; unpublished results). Of the two ETV-resistant variants identified, both were discovered in patients who had undergone treatment and contained key signature substitutions (rtL180M and rtM204V) associated with 3TCr
prior to ETV treatment. Furthermore, the results described here indicate that resistance to ETV requires the presence of 3TCr
substitutions, since the changes that emerged during ETV therapy had only modest effects on ETV susceptibility when tested alone.
Given its greater intrinsic antiviral potency and range of inhibitory activities, along with a predicted lower incidence of viral resistance over the course of long-term therapy, ETV offers the potential for an effective, safe, and durable treatment for HBV infections.