Background & Aims

Inhibitors of the hepatitis C virus (HCV) NS5A protein are a key component of effective treatment regimens, but the genetic heterogeneity of HCV has limited the efficacy of these agents and mutations lead to resistance. We directly compared the efficacy of all clinically relevant NS5A inhibitors against HCV genotype 1–7 prototype isolates and resistant escape variants, and investigated the effects of pre-existing resistance-associated substitutions (RAS) on HCV escape from treatment.

Methods

We measured the efficacy of different concentrations of daclatasvir, ledipasvir, ombitasvir, elbasvir, ruzasvir, velpatasvir, and pibrentasvir in cultured cells infected with HCV recombinants expressing genotype 1–7 NS5A proteins with or without RAS. We engineered HCV variants that included RAS identified in escape experiments, using recombinants with or without T/Y93H and daclatasvir, or that contained RAS previously reported from patients.

Results

NS5A inhibitors had varying levels of efficacy against original and resistant viruses. Only velpatasvir and pibrentasvir had uniform high activity against all HCV genotypes tested. RAS hotspots in NS5A were found at amino acids 28, 30, 31, and 93. Engineered escape variants had high levels of fitness. Pibrentasvir had the highest level of efficacy against variants; viruses with RAS at amino acids 28, 30, or 31 had no apparent resistance to pibrentasvir, and HCV with RAS at amino acid 93 had a low level of resistance to this drug. However, specific combinations of RAS and deletion of amino acid 32 led to significant resistance to pibrentasvir. For the remaining NS5A inhibitors tested, RAS at amino acids 28 and 93 led to high levels of resistance. Among these inhibitors, velpatasvir was more effective against variants with RAS at amino acid 30 and some variants with RAS at amino acid 31 than the other agents. Variants with the pre-existing RAS T/Y93H acquired additional NS5A changes during escape experiments, resulting in HCV variants with specific combinations of RAS, showing high fitness and high resistance.

Conclusions

We performed a comprehensive comparison of the efficacy of the 7 clinically relevant inhibitors of HCV NS5A and identified variants associated with resistance to each agent. These findings could improve treatment of patients with HCV infection.

中文翻译：

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NS5A 抑制剂对丙型肝炎病毒基因型 1-7 和逃逸变体的功效

背景与目标

丙型肝炎病毒 (HCV) NS5A 蛋白抑制剂是有效治疗方案的关键组成部分，但 HCV 的遗传异质性限制了这些药物的疗效，突变导致耐药性。我们直接比较了所有临床相关的 NS5A 抑制剂对 HCV 基因型 1-7 原型分离株和耐药逃逸变体的疗效，并研究了预先存在的耐药相关替代 (RAS) 对 HCV 逃避治疗的影响。

方法

我们测量了不同浓度的 daclatasvir、ledipasvir、ombitasvir、elbasvir、ruzasvir、velpatasvir 和 pibrentasvir 在感染了表达基因型 1-7 NS5A 蛋白（有或没有 RAS）的 HCV 重组体的培养细胞中的功效。我们设计了 HCV 变体，其中包括在逃逸实验中鉴定的 RAS，使用带有或不带有 T/Y93H 和 daclatasvir 的重组体，或者包含先前从患者报道的 RAS。

结果

NS5A 抑制剂对原始病毒和耐药病毒具有不同程度的功效。只有 velpatasvir 和 pibrentasvir 对所有测试的 HCV 基因型具有一致的高活性。NS5A 中的 RAS 热点位于氨基酸 28、30、31 和 93 处。工程逃逸变体具有高水平的适应度。Pibrentasvir 对变异体的疗效水平最高；在氨基酸 28、30 或 31 具有 RAS 的病毒对 pibrentasvir 没有明显的耐药性，而在氨基酸 93 具有 RAS 的 HCV 对这种药物具有低水平的耐药性。然而，RAS 和氨基酸 32 缺失的特定组合导致对 pibrentasvir 的显着耐药性。对于其余测试的 NS5A 抑制剂，氨基酸 28 和 93 的 RAS 导致高水平的耐药性。在这些抑制剂中，与其他药物相比，维帕他韦对氨基酸 30 处具有 RAS 的变异体和一些氨基酸 31 处具有 RAS 的变异体更有效。具有预先存在的 RAS T/Y93H 的变体在逃逸实验期间获得了额外的 NS5A 变化，导致 HCV 变体具有特定的 RAS 组合，显示出高适应性和高抗性。

结论

我们对 7 种临床相关的 HCV NS5A 抑制剂的疗效进行了全面比较，并确定了与每种药物耐药相关的变异。这些发现可以改善 HCV 感染患者的治疗。