Three-year assessment of entecavir resistance in chronic hepatitis B patients and the mechanism of viral breakthrough by the resistance substitutions

Abstract

Background/Aim: Persistence of lamivudine-resistance (LVDr)mutations in chronic hepatitis B patients who switched to ente-cavir (ETV) is worrisome because LVDr was shown to enhancethe risk of development of the ETV-resistance (ETVr). To assessfrequency of ETVr and the specific substitutions associated with viral breakthrough (BT) during 3-years ETV therapy and exam-ine the mechanism of the BT on the basis of molecular dockingsimulation study using the hepatitis B virus (HBV) polymerasemodel. Methods: One-hundred patients with chronic hepatitis B(45 from Japan, 25 USA, 30 Hong Kong) including 41 LVD-refractory patients and 59 naïve patients were enrolled. Drugresistant substitutions (T184SCGA/ILFM, 184ILFM, S202G,S202C) were assessed by recently developed INNO-LiPA ETVTDF (v3) add-on DR v2 assays. To examine the mechanism ofpossible involvement of the mutations in the drug resistance,three-dimensional (3D) homology for the HBV-RT domain wasmodeled, based on HIV-RT-DNA structure, demonstrating tenta-tive action of ETV against HBV-RT. Results: In 41 LVD-refractorypatients, the ETVr substitutions were present in 2 (cumulativeprevalence: 4.9%) cases at baseline and emerged in 6(14.6%), 10 (24.4%) and 13 (44.8%) cases at weeks 48, 96,and 144, respectively. There was no evidence of ETVr substitu-tions in naïve patients during the 144 weeks period. Eleven(26.8%) of 41 LVD-refractory patients treated with ETV devel-oped BT within 60 to 144 weeks of the therapy. The ETVr sub-stitutions (T184SCGA/ILFM or S202G) were associated withBT (adjusted Odds Ratio [OR] 141.12, 95%CI 6.94-2870.20;OR 201.25, 95%CI 11.22-3608.65, respectively) as revealedby multivariate logistic regression model analysis of the cohortsadjusted by age, gender, “2 log reduction of HBV DNA at 1year” and presence of LVDr (L180M and/or M204V). Molecu-lar docking simulation showed that the triple amino acid sub-stitutions (L180M, M204V, S202G or T184L) within HBV-RTinhibited ETV binding by altering the affinity of the tentativeattachment site (demonstrated by increase in the minimal dis-tance between ETV and the binding site, from 1.28 to 2.23angstrom, and decrease in binding potential from -97 to -86Kcal/mol). Conclusions: INNO-LiPA is useful for early detectionof ETVr. The molecular docking simulation suggested that theETVr could account for the resistance phenotype to ETV in vivo.High and long-term efficacy of ETV was demonstrated in naïvepatients, whereas ADV add-on LVD should be considered as afirst-, or second-line treatment option for LVD-refractory patientsdue to high risk of BT during ETV treatment.