Clonal evolution of multi-drug resistant hepatitis B virus during entecavir rescue therapy

Abstract

Background: Analyzing the mutation pattern of multi-drug resistance is important in the treatment of chronic hepatitis B (CHB). In this study, the evolutionary pattern of multi-drug resistant (MDR) mutations was investigated in patients who received entecavir (ETV) rescue therapy.Methods: Eight CHB patients with lamivudine (LAM)- and adefovir (ADV)-resistant mutations showing partial virological response to ETV and subsequent ETV plus ADV therapy were enrolled. Clonal evolution of mutation pattern was investigated. Direct sequencing, multiplex restriction fragment mass polymorphism (RFMP), and clonal analysis were compared for the utility of affecting on clinical decision of antiviral therapy. The binding affinity of tenofovir (TDF) with MDR hepatitis B virus (HBV) was investigated by molecular modeling.Results: Among 160 clones at baseline, wild type HBV was present in 62 (38.8%), LAM-resistant mutations in 49 (30.6%), and ADV-resistant mutations in 55 (34.4%) clones. LAM-resistant mutations increased to 67.5% at the end of ETV therapy, and increased more to 69.4% at 12th month of ETV plus ADV therapy. ETV-resistant mutations reached to 46.3% at the end of ETV therapy, but slightly decreased to 38.8% at 12th month of ETV plus ADV therapy. ADV-resistant mutations decreased to 3.1% at the end of ETV therapy, but slightly increased to 9.4% at 12th month of ETV plus ADV therapy. When the frequency of mutations in 8 sites of HBV reverse transcriptase (rt) polymerase (rt 180, rt204, rt181, rt236, rt169, rt184, rt202, and rt250) was investigated in 32 samples, direct sequencing detected 36 (14.1%), multiplex RFMP 71 (27.7%), and clonal analysis 90 (35.2%) mutations. All 36 mutations detected by direct sequencing revealed identical results in the respective analyses by clonal analysis and multiplex RFMP. Among 28 mutations which revealed discordant results between clonal

analysis and multiplex RFMP, clonal analysis detected 24 (85.7%) mutations which RFMP did not. However, all mutations exceeding 40% of total clones by clonal analysis were also detected by multiplex RFMP. Although the docking of TDF with rtM204V/I+rtA181T/V mutants or rtM204V/I+rtN236T mutants were both available, the latter were disturbed by conformational change of rtN236T.Conclusions: The clonal evolution of mutation pattern in MDR HBV revealed the selection of LAM-resistant ( -resistant) HBV during ETV rescue therapy, and the main reason for suboptimal response might be due to LAM-resistance ( -resistance) which predominated over ADV resistance. Multiplex RFMP showed high sensitivity for detecting LAM-resistant ( -resistant) mutations taking less time and labor than clonal analysis. The potent drug TDF showed strong binding affinity with MDR HBV regardless of their co-location.