Clinical Emergence of Entecavir-resistant Hepatitis B Virus Requires Additional Substitutions in Virus Already Resistant to Lamivudine

Entecavir (ETV) exhibits potent antiviral activity in patients chronically infected with wild-type or lamivudine (3TC)-resistant (3TC(r)) hepatitis B virus (HBV). Among the patients treated in phase II ETV clinical trials, two patients for whom previous therapies had failed exhibited virologic breakthrough while on ETV. Isolates from these patients (arbitrarily designated patients A and B) were analyzed genotypically for emergent substitutions in HBV reverse transcriptase (RT) and phenotypically for reduced susceptibility in cultures and in HBV polymerase assays. After 54 weeks of 3TC therapy, patient A (AI463901-A) received 0.5 mg of ETV for 52 weeks followed by a combination of ETV and 100 mg of 3TC for 89 weeks. Viral rebound occurred at 133 weeks after ETV was started. The 3TC(r) RT substitutions rtV173L, rtL180M, and rtM204V were present at study entry, and the additional substitutions rtI169T and rtM250V emerged during ETV-3TC combination treatment. Reduced ETV susceptibility in vitro required the rtM250V substitution in addition to the 3TC(r) substitutions. For liver transplant patient B (AI463015-B), previous famciclovir, ganciclovir, foscarnet, and 3TC therapies had failed, and RT changes rtS78S/T, rtV173L, rtL180M, rtT184S, and rtM204V were present at study entry. Viral rebound occurred after 76 weeks of therapy with ETV at 1.0 mg, with the emergence of rtT184G, rtI169T, and rtS202I substitutions within the preexisting 3TC(r) background. Reduced susceptibility in vitro was highest when both the rtT184G and the rtS202I changes were combined with the 3TC(r) substitutions. In summary, infrequent ETV resistance can emerge during prolonged therapy, with selection of additional RT substitutions within a 3TC(r) HBV background, leading to reduced ETV susceptibility and treatment failure.