Eun-Sook Park1, Ah Ram Lee1, Doo Hyun Kim1, Jeong-Hoon Lee2, Jeong-Ju Yoo3, Sung Hyun Ahn1, Heewoo Sim1, Soree Park1, Hong Seok Kang1, Juhee Won1, Yea Na Ha1, Gu-Choul Shin1, So Young Kwon4, Yong Kwang Park5, Byeong-Sun Choi5, Yun Bin Lee6, Nakcheol Jeong7, Yohan An8, Young Seok Ju9, Su Jong Yu10, Hee Bok Chae11, Kyung-Sang Yu12, Yoon Jun Kim10, Jung-Hwan Yoon10, Fabien Zoulim13, Kyun-Hwan Kim14. 1. Department of Pharmacology and Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Republic of Korea. 2. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Electronic address: pindra@empal.com. 3. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Gastroenterology and Hepatology, Soonchunhyang University Bucheon Hospital, Gyeonggido, Republic of Korea. 4. Department of Internal Medicine, School of Medicine, Konkuk University, Seoul, Republic of Korea. 5. Division of AIDS, Center for Immunology and Pathology, Korea National Institute of Health, Korea Center for Disease Control and Prevention, Osong, Chungbuk, Republic of Korea. 6. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Gyeonggido, Republic of Korea. 7. Department of Chemistry, Korea University, Seoul, Republic of Korea. 8. Biomedical Science and Engineering Interdisciplinary Program, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea. 9. Biomedical Science and Engineering Interdisciplinary Program, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea; Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea. 10. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. 11. Department of Internal Medicine, Chungbuk National University College of Medicine, Cheongju, Republic of Korea. 12. Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine, Seoul, Republic of Korea. 13. INSERM Unité 1052, Cancer Research Center of Lyon, Hospices Civils de Lyon, Lyon University, Lyon, France. 14. Department of Pharmacology and Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Republic of Korea; KU Open Innovation Center, Konkuk University, Seoul, Republic of Korea; Research Institute of Medical Sciences, Konkuk University, Seoul, Republic of Korea. Electronic address: khkim10@kku.ac.kr.
Abstract
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 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. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3-778 (IC50 <0.4 µM vs. IC50 = 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 IC50 and 26.3-fold in IC90. 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 (IC50) and 26.3-fold (IC90) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.
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 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. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3-778 (IC50 <0.4 µM vs. IC50 = 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 IC50 and 26.3-fold in IC90. 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 (IC50) and 26.3-fold (IC90) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.
Authors: Peter A Revill; Thomas Tu; Hans J Netter; Lilly K W Yuen; Stephen A Locarnini; Margaret Littlejohn Journal: Nat Rev Gastroenterol Hepatol Date: 2020-05-28 Impact factor: 46.802
Authors: Jolynne Mokaya; Anna L McNaughton; Phillip A Bester; Dominique Goedhals; Eleanor Barnes; Brian D Marsden; Philippa C Matthews Journal: Wellcome Open Res Date: 2020-06-29
Authors: Son V Chu; Son T Vu; Hang M Nguyen; Ngan T Le; Phuong T Truong; Van T T Vu; Thuy T B Phung; Anh T V Nguyen Journal: J Clin Microbiol Date: 2021-07-28 Impact factor: 5.948