Department of Pharmacology and Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Republic of Korea.
Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
J Hepatol. 2019 Jun;70(6):1093-1102. doi: 10.1016/j.jhep.2019.02.006. Epub 2019 Feb 20.
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 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.
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 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC value for wild-type HBV was 30 ± 0.5 µM, whereas the IC 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 <0.4 µM vs. IC = 0.4 µM, respectively).
Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC and 26.3-fold in IC. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high.
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) and 26.3-fold (IC) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.
富马酸替诺福韦二吡呋酯（TDF）是治疗慢性乙型肝炎病毒（HBV）感染最有效的核苷（酸）类似物之一。尚未报道由于对 TDF 的基因型耐药而导致表型耐药。本研究旨在描述赋予替诺福韦耐药性的 HBV 突变。
前瞻性纳入 2 例 TDF 治疗方案中病毒突破的患者。测序 HBV 逆转录酶基因。通过定点诱变构建了 11 个含有逆转录酶基因一系列突变的 HBV 克隆。通过 Southern blot 分析和实时 PCR 测定每个克隆的药物敏感性。通过超深度测序和克隆分析评估突变体的相对频率。
在 2 名患者的病毒分离物中发现了 5 种突变（rtS106C [C]、rtH126Y [Y]、rtD134E [E]、rtM204I/V 和 rtL269I [I]）。新型突变 C、Y 和 E 与耐药性相关。在药物敏感性测定中，野生型 HBV 的 IC 值为 3.8±0.6µM，而 CYE 和 CYEI 突变体的 IC 值分别为 14.1±1.8 和 58.1±0.9µM。野生型 HBV 的 IC 值为 30±0.5µM，而 CYE 和 CYEI 突变体的 IC 值分别为 185±0.5 和 790±0.2µM。替诺福韦耐药突变体和野生型 HBV 对衣壳组装调节剂 NVR 3-778 的敏感性相似（IC<0.4µM 与 IC=0.4µM，分别）。
我们的研究表明， quadruple (CYEI) 突变使 HBV 抑制所需的替诺福韦量在 IC 中增加了 15.3 倍，在 IC 中增加了 26.3 倍。这些结果表明，尽管遗传屏障很高，但仍可能出现对替诺福韦具有 10 倍以上耐药性的 HBV 突变体。
替诺福韦是治疗慢性乙型肝炎病毒感染最有效的核苷酸类似物，到目前为止，还没有发现对替诺福韦的耐药性超过 10 倍的乙型肝炎病毒突变，耐药性高达 8 年。在此，我们首次在 2 例替诺福韦治疗中发生病毒突破的患者中发现， quadruple (CYEI) 突变使替诺福韦的耐药性分别增加了 15.3 倍（IC）和 26.3 倍（IC）。
