Elsevier

Journal of Hepatology

Volume 70, Issue 6, June 2019, Pages 1093-1102
Journal of Hepatology

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

https://doi.org/10.1016/j.jhep.2019.02.006Get rights and content

Highlights

  • Among oral antivirals for HBV infection, only tenofovir has revealed no genotypically resistant HBV.

  • However, there are patients with incomplete viral response during tenofovir-containing treatment.

  • We consistently identified 7 common mutations including rtS106C (C), rtH126Y (Y), rtD134E (E), and rtL269I (I).

  • The mutations C, Y, and E were novel mutations associated with drug resistance.

  • 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.

  • All tenofovir-resistant mutants with/without entecavir resistance were susceptible to a novel capsid assembly modulator.

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.

Introduction

Worldwide, an estimated 2 billion people are infected with hepatitis B virus (HBV), and 686,000 people die from complications due to HBV each year.1 Despite the development of nucleos(t)ide analogue (NA) drugs, antiviral therapy of HBV infection remains a major clinical issue. Due to both the viral persistence and heterogeneity of the HBV genome, the emergence of drug-resistant mutants is inevitable. The development of drug resistance is associated with poor prognosis. Problems arising from drug resistance include hepatitis flares, reversion of histologic improvement, and sometimes severe exacerbation of illness, hepatic decompensation, or death.2

Both entecavir and tenofovir (which is an active moiety of tenofovir disoproxil fumarate [TDF] or tenofovir alafenamide [TAF]) are approved as first-line therapeutic options for chronic hepatitis B (CHB) in current international guidelines due to their high potency and low resistance of the virus.[3], [4] Although entecavir has a high genetic barrier to resistance, resistance to entecavir has been reported with a significant incidence rate, especially in patients with genotypic resistance to lamivudine and a prior history of lamivudine treatment.[5], [6]

Meanwhile, HBV that displays clinical resistance to tenofovir has not been reported. Although TDF showed inferior efficacy in adefovir-experienced patients,7 there was no detectable genotypic resistance to TDF after 8 years of therapy in patients with CHB.8 Although the rtA194T mutation was reported to decrease tenofovir sensitivity by increasing the IC50 value in in vitro analysis, it does not confer tenofovir resistance in vivo nor is it associated with partial tenofovir drug resistance.[9], [10], [11] A recent article reported that rtS78T/sC69* was related to tenofovir resistance, but the IC50 values were increased by only 1.6-fold compared to wild-type.12 Moreover, a recent randomized control study reported that viral response to TDF monotherapy was comparable to that to TDF and entecavir combination therapy in patients with multidrug resistance (MDR).[13], [14] However, daily clinical practice shows that there are patients with a partial response to TDF varying from 0.8% to 24% and some patients developed viral breakthrough despite good adherence to TDF.[15], [16] Therefore, we suspected the existence of genotypic resistance to tenofovir.

In this study, we aimed to identify the presence of tenofovir-resistant HBV by collecting blood samples from patients who showed viral breakthrough during TDF-based treatment and to characterize the responsible mutations in vitro.

Section snippets

Patients

This study included 2 patients who developed viral breakthrough during TDF-containing treatment from 2 university-affiliated hospitals in Korea. Detailed flow of patient enrollment is provided in Supplementary methods and Fig. S1. Viral breakthrough is defined as an increase in the HBV DNA level of more than 1 log10 IU/ml compared to the nadir during therapy.17 All participants provided written informed consent before enrollment and blood of each patient was sampled at the time of viral

Mutation profiles of the HBV polymerase RT isolated from TDF-resistant patients

HBV DNA was isolated from the serum of the first patient (Patient #1) after she developed viral breakthrough during treatment with TDF-containing regimens. We constructed the HBV 1.2-mers where the original RT gene was replaced with the patient-derived ones and obtained 8 clones (Clones 1-1, 1-2, 1-3, 1-4, 1-6, 1-7, 1-8, and 1-13) from Patient #1. Each clone was sequenced to analyze the quasispecies of the entire RT gene.

Among the 8 clones isolated from Patient #1 after TDF treatment, 4 clones

Discussion

In this study, we identified a quadruple tenofovir-resistant mutation in HBV isolated from the sera of 2 patients with clinical resistance to tenofovir treatment and confirmed in vitro tenofovir resistance. To the best of our knowledge, this is the first report of HBV mutants with both clinical and in vitro resistance to TDF treatment. A novel CYE triple mutation reduced tenofovir susceptibility and a quadruple CYEI mutation conferred complete resistance to tenofovir. Ultra-deep sequencing

Financial support

Park ES was supported by the National Research Foundation of Korea (NRF) grant funded by Korea Government (MSIP) (No. NRF-2016R1A2B4007531) and by extramural grants from the Korea National Institute of Health (No. 2016-ER5101-00). Lee JH was supported by the Seoul National University Hospital Research Fund (No. 03-2016-0380). Ju YS was supported by Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), which was funded by the Ministry of Health &

Conflict of interest

These authors disclose the following: Dr. Lee JH reports receiving lecture fee from GreenCross Cell, Daewoong Pharmaceuticals, and Gilead Korea; Dr. Yu SJ reports lecture fee from Bayer HealthCare Pharmaceuticals; Dr. Kim YJ, research grants from Bristol-Myers Squibb, Roche, JW Creagene, Bukwang Pharmaceuticals, Handok Pharmaceuticals, Hanmi Pharmaceuticals, Yuhan Pharmaceuticals, Samjin Pharmaceuticals, and Pharmaking, and lecture fees from Bayer HealthCare Pharmaceuticals, Gilead Science, MSD

Authors’ contributions

Study concept and design: Lee JH and Kim KH. Provision of study patients and acquisition of clinical data: Lee JH, Lee YB, Chae HB, Yu SJ. Provision of study materials: Jeong N, Choi BS, and Park YK. In vitro experiments: Park ES, Lee AR, Kim DH, Ahn SH, Sim H, Park S, Kang HS, Won J, Ha YN, Shin GC, and Kim KH. Biostatistical analysis and interpretation: Lee JH, Kim KH, Park ES, Lee AR, Kim DH, Yu KS, An Y, and Ju YS. Drafting of the manuscript: Park ES, Yoo JJ, Lee JH, and Kim KH. Critical

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