Research ArticleIdentification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients
Graphical abstract
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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These 4 authors are co-first authors.