Comprehensive evaluation of hepatitis B virus reverse transcriptase substitutions associated with entecavir resistance.

UNLABELLED: Virologic resistance emerging during entecavir (ETV) therapy for hepatitis B virus (HBV) requires three substitutions in the viral reverse transcriptase (RT), signifying a high barrier to resistance. Two of these substitutions are associated with lamivudine resistance (LVDr) in the tyrosine-methionine-aspartate-aspartate (YMDD) motif (rtM204V and rtL180M), whereas the other occurs at one or more positions specifically associated with ETV resistance (ETVr): rtT184, rtS202, or rtM250. Although a variety of substitutions at these primary ETVr positions arise during ETV therapy, only a subset give rise to clinical virologic breakthrough. To determine the phenotypic impact of observed clinical and potential new ETVr substitutions, a comprehensive panel of clones containing every possible amino acid at the three primary ETVr positions in LVDr HBV was constructed and analyzed in vitro. A range of replication capacities was observed for the panel, but none of the mutations rescued replication of the LVDr mutant to the wild-type level. More clones with residue rtS202 substitutions were severely impaired than those at rtT184 or rtM250. A wide variety of ETV susceptibilities was observed, ranging from approximately eight-fold (no increase over the LVDr parent) to greater than 400-fold over the wild-type. A correlation was identified between clinically observed substitutions and those displaying higher in vitro replication and resistance, especially those from virologic breakthrough patients. CONCLUSION: The high number of tolerated and resistant ETVr substitutions is consistent with models predicting that the mechanism for ETVr is through enhancement of LVDr changes in the RT deoxyribonucleotide triphosphate (dNTP)-binding pocket.

Entecavir resistance is rare in nucleoside naïve patients with hepatitis B.

Comprehensive monitoring of genotypic and phenotypic antiviral resistance was performed on 673 entecavir (ETV)-treated nucleoside naïve hepatitis B virus (HBV) patients. ETV reduced HBV DNA levels to undetectable by PCR (<300 copies/mL, <57 IU/mL) in 91% of hepatitis B e antigen (HBeAg)-positive and -negative patients by Week 96. Thirteen percent (n = 88) of the comparator lamivudine (LVD)-treated patients experienced a virologic rebound (> or =1 log increase from nadir by PCR) in the first year, with 74% of these having LVD resistance (LVDr) substitutions evident. In contrast, only 3% (n = 22) of ETV-treated patients exhibited virologic rebound by Week 96. Three ETV rebounds were attributable to LVDr virus present at baseline, with one having a S202G ETV resistance (ETVr) substitution emerge at Week 48. None of the other rebounding patients had emerging genotypic resistance or loss of ETV susceptibility. Genotyping all additional ETV patients with PCR-detectable HBV DNA at Weeks 48, 96, or end of dosing identified seven additional patients with LVDr substitutions, including one with simultaneous emergence of LVDr/ETVr. Generally, ETV patients with LVDr were detectable at baseline (8/10) and most subsequently achieved undetectable HBV DNA levels on ETV therapy (7/10). No other emerging substitutions identified decreased ETV susceptibility. In conclusion, ETVr emergence in ETV-treated nucleoside naïve patients over a 2-year period is rare, occurring in two patients with LVDr variants. These findings suggest that the rapid, sustained suppression of HBV replication, combined with a requirement for multiple substitutions, creates a high genetic barrier to ETVr in nucleoside naïve patients.