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Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy

Published:April 06, 2017DOI:https://doi.org/10.1016/j.jhep.2017.03.027

      Highlights

      • Two HBV-infected patients with incomplete viral suppression despite dual entecavir (ETV) and tenofovir (TDF) therapy carried the rtS78T/sC69∗ HBV mutation.
      • The rtS78T mutation causes enhanced viral replication and reduced susceptibility to ETV and TDF in vitro.
      • The sC69∗ mutation causes truncation of HBs protein without defective HBV secretion or intracellular HBsAg retention.
      • This mutation selected during ETV + TDF therapy may predispose to treatment failure and HBV-related carcinogenesis.

      Background & Aims

      Patients chronically infected with the hepatitis B virus (HBV) and receiving long-term treatment with nucleoside or nucleotide analogues are at risk of selecting HBV strains with complex mutational patterns. We herein report two cases of HBV-infected patients with insufficient viral suppression, despite dual antiviral therapy with entecavir (ETV) and tenofovir (TDF). One patient died from aggressive hepatocellular carcinoma (HCC).

      Methods

      Serum samples from the two patients at different time points were analyzed using ultra-deep pyrosequencing analysis. HBV mutations were identified and transiently transfected into hepatoma cells in vitro using replication-competent HBV vectors, and functionally analyzed. We assessed replication efficacy, resistance to antivirals and potential impact on HBV secretion (viral particles, exosomes).

      Results

      Sequencing analyses revealed the selection of the rtS78T HBV polymerase mutation in both cases that simultaneously creates a premature stop codon at sC69 and thereby deletes almost the entire small HBV surface protein. One of the patients had an additional 261 bp deletion in the preS1/S2 region. Functional analyses of the mutations in vitro revealed that the rtS78T/sC69∗ mutation, but not the preS1/S2 deletion, significantly enhanced viral replication and conferred reduced susceptibility to ETV and TDF. The sC69∗ mutation caused truncation of HBs protein, leading to impaired detection by commercial HBsAg assay, without causing intracellular HBsAg retention or affecting HBV secretion.

      Conclusions

      The rtS78T/sC69∗ HBV mutation, associated with enhanced replication and insufficient response to antiviral treatment, may favor long-term persistence of these isolates. In addition to the increased production of HBV transcripts and the sustained secretion of viral particles in the absence of antigenic domains of S protein, this HBV mutation may predispose patients to carcinogenic effects.

      Lay summary

      Long-term treatment with antiviral drugs carries the risk of selecting mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhances replication, sustains exosome-mediated virion secretion and decreases susceptibility to antivirals, thereby representing a potentially high-risk mutation for HBV-infected individuals.

      Graphical abstract

      Keywords

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      References

      Author names in bold designate shared co-first authorship

        • Stanaway J.D.
        • Flaxman A.D.
        • Naghavi M.
        • Fitzmaurice C.
        • Vos T.
        • Abubakar I.
        • et al.
        The global burden of viral hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study 2013.
        Lancet. 2016; 388: 1081-1088
        • European Association for the Study of the Liver
        EASL clinical practice guidelines: Management of chronic hepatitis B virus infection.
        J Hepatol. 2012; 57: 167-185
        • Zoulim F.
        • Locarnini S.
        Optimal management of chronic hepatitis B patients with treatment failure and antiviral drug resistance.
        Liver Int. 2013; 33: 116-124
        • Terrault N.A.
        • Bzowej N.H.
        • Chang K.M.
        • Hwang J.P.
        • Jonas M.M.
        • Murad M.H.
        • American Association for the Study of Liver D
        AASLD guidelines for treatment of chronic hepatitis B.
        Hepatology. 2016; 63: 261-283
        • Chang T.T.
        • Lai C.L.
        • Kew Yoon S.
        • Lee S.S.
        • Coelho H.S.
        • Carrilho F.J.
        • et al.
        Entecavir treatment for up to 5 years in patients with hepatitis B e antigen-positive chronic hepatitis B.
        Hepatology. 2010; 51: 422-430
        • Amini-Bavil-Olyaee S.
        • Herbers U.
        • Luedde T.
        • Trautwein C.
        • Tacke F.
        Impact of hepatitis B e antigen-suppressing mutations on the replication efficiency of entecavir-resistant hepatitis B virus strains.
        J Viral Hepat. 2011; 18: 804-814
        • Buti M.
        • Tsai N.
        • Petersen J.
        • Flisiak R.
        • Gurel S.
        • Krastev Z.
        • et al.
        Seven-year efficacy and safety of treatment with tenofovir disoproxil fumarate for chronic hepatitis B virus infection.
        Dig Dis Sci. 2015; 60: 1457-1464
        • Sheldon J.
        • Camino N.
        • Rodes B.
        • Bartholomeusz A.
        • Kuiper M.
        • Tacke F.
        • et al.
        Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir.
        Antivir Ther. 2005; 10: 727-734
        • Amini-Bavil-Olyaee S.
        • Herbers U.
        • Sheldon J.
        • Luedde T.
        • Trautwein C.
        • Tacke F.
        The rtA194T polymerase mutation impacts viral replication and susceptibility to tenofovir in hepatitis B e antigen-positive and hepatitis B e antigen-negative hepatitis B virus strains.
        Hepatology. 2009; 49: 1158-1165
        • Tong S.
        • Revill P.
        Overview of hepatitis B viral replication and genetic variability.
        J Hepatol. 2016; 64: S4-S16
        • Margeridon-Thermet S.
        • Shulman N.S.
        • Ahmed A.
        • Shahriar R.
        • Liu T.
        • Wang C.
        • et al.
        Ultra-deep pyrosequencing of hepatitis B virus quasispecies from nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI)-treated patients and NRTI-naive patients.
        J Infect Dis. 2009; 199: 1275-1285
        • Amini-Bavil-Olyaee S.
        • Herbers U.
        • Mohebbi S.R.
        • Sabahi F.
        • Zali M.R.
        • Luedde T.
        • et al.
        Prevalence, viral replication efficiency and antiviral drug susceptibility of rtQ215 polymerase mutations within the hepatitis B virus genome.
        J Hepatol. 2009; 51: 647-654
        • Shirvani-Dastgerdi E.
        • Pourkarim M.R.
        • Herbers U.
        • Amini-Bavil-Olyaee S.
        • Yagmur E.
        • Alavian S.M.
        • et al.
        Hepatitis delta virus facilitates the selection of hepatitis B virus mutants in vivo and functionally impacts on their replicative capacity in vitro.
        Clin Microbiol Infect. 2016; 22: e91-e96
        • Tacke F.
        • Gehrke C.
        • Luedde T.
        • Heim A.
        • Manns M.P.
        • Trautwein C.
        Basal core promoter and precore mutations in the hepatitis B virus genome enhance replication efficacy of Lamivudine-resistant mutants.
        J Virol. 2004; 78: 8524-8535
        • Yang Y.
        • Han Q.
        • Hou Z.
        • Zhang C.
        • Tian Z.
        • Zhang J.
        Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction.
        Cell Mol Immunol. 2016;
        • Amini-Bavil-Olyaee S.
        • Vucur M.
        • Luedde T.
        • Trautwein C.
        • Tacke F.
        Differential impact of immune escape mutations G145R and P120T on the replication of lamivudine-resistant hepatitis B virus e antigen-positive and -negative strains.
        J Virol. 2010; 84: 1026-1033
        • Cento V.
        • Van Hemert F.
        • Neumann-Fraune M.
        • Mirabelli C.
        • Di Maio V.C.
        • Salpini R.
        • et al.
        Anti-HBV treatment induces novel reverse transcriptase mutations with reflective effect on HBV S antigen.
        J Infect. 2013; 67: 303-312
        • Kim H.
        • Lee S.A.
        • Kim D.W.
        • Lee S.H.
        • Kim B.J.
        Naturally occurring mutations in large surface genes related to occult infection of hepatitis B virus genotype C.
        PLoS One. 2013; 8e54486
        • van Breugel P.C.
        • Robert E.I.
        • Mueller H.
        • Decorsiere A.
        • Zoulim F.
        • Hantz O.
        • et al.
        Hepatitis B virus X protein stimulates gene expression selectively from extrachromosomal DNA templates.
        Hepatology. 2012; 56: 2116-2124
        • Chen J.
        • Liu Y.
        • Zhao J.
        • Xu Z.
        • Chen R.
        • Si L.
        • et al.
        Characterization of novel hepatitis B virus PreS/S-gene mutations in a patient with occult hepatitis B virus infection.
        PLoS One. 2016; 11e0155654
        • Xiang K.H.
        • Michailidis E.
        • Ding H.
        • Peng Y.Q.
        • Su M.Z.
        • Li Y.
        • et al.
        Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA.
        J Hepatol. 2017; 66: 288-296
        • Betz-Stablein B.D.
        • Topfer A.
        • Littlejohn M.
        • Yuen L.
        • Colledge D.
        • Sozzi V.
        • et al.
        Single-molecule sequencing reveals complex genome variation of hepatitis B virus during 15 years of chronic infection following liver transplantation.
        J Virol. 2016; 90: 7171-7183
        • Warner N.
        • Locarnini S.
        The antiviral drug selected hepatitis B virus rtA181T/sW172∗ mutant has a dominant negative secretion defect and alters the typical profile of viral rebound.
        Hepatology. 2008; 48: 88-98
        • Shirvani-Dastgerdi E.
        • Schwartz R.E.
        • Ploss A.
        Hepatocarcinogenesis associated with hepatitis B, delta and C viruses.
        Curr Opin Virol. 2016; 20: 1-10
        • Huang S.F.
        • Chen Y.T.
        • Lee W.C.
        • Chang I.C.
        • Chiu Y.T.
        • Chang Y.
        • et al.
        Identification of transforming hepatitis B virus S gene nonsense mutations derived from freely replicative viruses in hepatocellular carcinoma.
        PLoS One. 2014; 9e89753
        • Tenney D.J.
        • Levine S.M.
        • Rose R.E.
        • Walsh A.W.
        • Weinheimer S.P.
        • Discotto L.
        • et al.
        Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to Lamivudine.
        Antimicrob Agents Chemother. 2004; 48: 3498-3507
        • Ciftci S.
        • Keskin F.
        • Cakiris A.
        • Akyuz F.
        • Pinarbasi B.
        • Abaci N.
        • et al.
        Analysis of potential antiviral resistance mutation profiles within the HBV reverse transcriptase in untreated chronic hepatitis B patients using an ultra-deep pyrosequencing method.
        Diagn Microbiol Infect Dis. 2014; 79: 25-30
        • Colonno R.J.
        • Rose R.
        • Baldick C.J.
        • Levine S.
        • Pokornowski K.
        • Yu C.F.
        • et al.
        Entecavir resistance is rare in nucleoside naive patients with hepatitis B.
        Hepatology. 2006; 44: 1656-1665
        • Tenney D.J.
        • Rose R.E.
        • Baldick C.J.
        • Levine S.M.
        • Pokornowski K.A.
        • Walsh A.W.
        • et al.
        Two-year assessment of entecavir resistance in Lamivudine-refractory hepatitis B virus patients reveals different clinical outcomes depending on the resistance substitutions present.
        Antimicrob Agents Chemother. 2007; 51: 902-911
        • van Hemert F.J.
        • Berkhout B.
        • Zaaijer H.L.
        Differential binding of tenofovir and adefovir to reverse transcriptase of hepatitis B virus.
        PLoS One. 2014; 9e106324
        • Warner N.
        • Locarnini S.
        • Kuiper M.
        • Bartholomeusz A.
        • Ayres A.
        • Yuen L.
        • et al.
        The L80I substitution in the reverse transcriptase domain of the hepatitis B virus polymerase is associated with lamivudine resistance and enhanced viral replication in vitro.
        Antimicrob Agents Chemother. 2007; 51: 2285-2292
        • Quarleri J.
        Core promoter: a critical region where the hepatitis B virus makes decisions.
        World J Gastroenterol. 2014; 20: 425-435
        • Glebe D.
        • Urban S.
        Viral and cellular determinants involved in hepadnaviral entry.
        World J Gastroenterol. 2007; 13: 22-38
        • Seeger C.
        • Mason W.S.
        Molecular biology of hepatitis B virus infection.
        Virology. 2015; 479–480: 672-686