Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients

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. Methods Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis. Results Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC 50 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC 50 values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC 90 value for wild-type HBV was 30 ± 0.5 µM, whereas the IC 90 values for CYE and CYEI mutants were 185 ± 0.5 and 790 ± 0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3–778 (IC 50 vs. IC 50  = 0.4 µM, respectively). Conclusions Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC 50 and 26.3-fold in IC 90 . These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. Lay summary Tenofovir is the most potent nucleotide analogue for the treatment of chronic hepatitis B virus infection and there has been no hepatitis B virus mutation that confers >10-fold resistance to tenofovir up to 8 years. Herein, we identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC 50 ) and 26.3-fold (IC 90 ) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.