在TDF-ETV联合治疗期间选择高度复制和部分多药耐药的rtS78T HBV

StephenW

Research Article
Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy
.
1
    Department of Medicine III, RWTH-University Hospital Aachen, Aachen, Germany

2
    Department of Molecular Biology, Princeton University, Princeton, NJ, USA

3
    Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain

4
    Liver Unit, Vall d’Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain

5
    Medical Care Centre, Dr Stein and Colleagues, Mönchengladbach, Germany

6
    Roche Diagnostics SL, Sant Cugat del Vallès, Spain

Received 7 September 2016, Revised 1 March 2017, Accepted 16 March 2017, Available online 7 April 2017.
crossmark-logo
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.

StephenW

研究论文
在TDF-ETV联合治疗期间选择高度复制和部分多药耐药的rtS78T HBV聚合酶突变
。
1
    德国亚琛亚琛亚特兰大医院第三医学系

2
    普林斯顿大学分子生物系，普林斯顿，新泽西州，美国

3
    Centro deInvestigaciónBiomédicaen Red de EnfermedadesHepáticasy Digestivas（CIBERehd），Instituto de Salud Carlos III，Madrid，Spain

4
    肝脏股，Vall d'Hebron研究所（VHIR），巴塞罗那自治大学（UAB），西班牙巴塞罗那

五
    医疗中心，Stein博士和同事，德国门兴格拉德巴赫

6
    Roche Diagnostics SL，Sant Cugat delVallès，西班牙

于2017年9月7日收到，2017年3月1日修订，2017年3月16日接受，2017年4月7日在线。
CROSSMARK车标
https://doi.org/10.1016/j.jhep.2017.03.027

强调

•

    两个HBV感染患者，尽管双重恩替卡韦（ETV）和替诺福韦（TDF）治疗仍然进行了rtS78T / sC69 * HBV突变的不完全病毒抑制。
•

    rtS78T突变引起病毒复制增加，体外对ETV和TDF的易感性降低。
•

    sC69 *突变引起HBs蛋白的截短，而不会有HBV分泌不良或细胞内HBsAg保留。
•

    在ETV + TDF治疗期间选择的这种突变可能易于治疗失败和HBV相关的致癌作用。

背景与目标

长期感染乙型肝炎病毒（HBV）并接受核苷或核苷酸类似物长期治疗的患者有选择具有复杂突变型HBV病毒株的风险。我们在这里报告了HBV病毒抑制不足的两例病例，尽管使用恩替卡韦（ETV）和替诺福韦（TDF）进行双重抗病毒治疗。一名患者死于侵袭性肝细胞癌（HCC）。
方法

使用超深度焦磷酸测序分析分析来自不同时间点的两名患者的血清样品。 HBV突变被鉴定，并使用复制型HBV载体体外瞬时转染肝癌细胞，并进行功能分析。我们评估复制功效，抗病毒药物的抗性和对HBV分泌的潜在影响（病毒颗粒，外来体）。
结果

在两种情况下，测序分析显示rtS78T HBV聚合酶突变的选择同时在sC69上产生过早终止密码子，从而几乎消除了整个小的HBV表面蛋白。其中一名患者在preS1 / S2区域内还有261 bp的缺失。体外突变的功能分析显示，rtS78T / sC69 *突变，但不是preS1 / S2缺失，显着增强了病毒复制，并赋予对ETV和TDF的易感性降低。 sC69 *突变导​​致HBs蛋白截短，导致商业HBsAg检测检测受损，不引起细胞内HBsAg滞留或影响HBV分泌。
结论

与增强复制相关的rtS78T / sC69 * HBV突变和抗病毒治疗反应不足可能有利于长期持续存在这些分离株。除了在不存在S蛋白的抗原结构域的情况下，HBV转录物的产生量增加以及病毒颗粒的持续分泌之外，HBV突变可能会使患者发生致癌作用。
总结

用抗病毒药物进行长期治疗具有选择乙型肝炎病毒（HBV）突变的风险。我们在这里报告了两例对双重替诺福韦和恩替卡韦治疗反应不足的患者。分子分析确定了一个不同的突变，rtS78T / sC69 *，其消除HBsAg检测，增强复制，维持外来体介导的病毒粒子分泌并降低对抗病毒药物的易感性，从而代表HBV感染个体潜在的高风险突变。