Background & Aims
Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance.
Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase (RT) was sequenced. Eleven HBV clones harboring a series of mutations in the RT gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. Relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis.
Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from two patients. The mutations C, Y, and E were novel mutations associated with drug resistance. In assays for drug susceptibility, the IC50 value for wild-type HBV was 3.8±0.6 μM, whereas the IC50 values for CYE and CYEI mutants were 14.1±1.8 and 58.1±0.9 μM, respectively. The IC90 value for wild-type HBV was 30±0.5 μM, whereas the IC90 values for CYE and CYEI mutants were 185±0.5 and 790±0.2 μM, respectively. All tenofovir-resistant mutants had similar susceptibility to a capsid assembly modulator, NVR 3-778 (IC50 < 0.4 μM), compared with wild-type (IC50 = 0.4 μM).
Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC50 and 26.3-fold in IC90. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high.
Keywords : capsid assembly modulator; entecavir; nucleotide analogue; CYEI; rtS106C; rtH126Y; rtD134E; rtL269I