Combination therapy of directly acting antivirals (DAA's) for the treatment of chronic HCV infections has proven to be a highly effective strategy to cure chronic infections with this virus. Here we studied, using HCV as an example, how to best design in vitro studies that explore the combined antiviral efficiency of combinations of three or more DAA's. To that end we used a HCV NS3 protease inhibitor, a NS5A targeting compound and two non-nucleoside NS5B polymerase inhibitors (each one targeting a different drug binding site). We demonstrate, employing HCV subgenomic replicon containing Huh 9–13 hepatoma cells, that quadruple therapy with these 4 different DAA's each at 1x their EC₇₅, results in a highly efficient inhibition of viral replication. This is further reflected in the rapid clearance of the HCV replicon from the host cell. By contrast, neither equipotent combinations that consist of either molecules alone at 4x EC₇₅ nor triple combinations at 1.33x the EC₇₅ resulted in clearance. In contrast to the quadruple combo, drug-resistant variants emerged under mono-treatment and in most triple combo's. These data thus demonstrate that quadruple combinations at total suboptimal concentrations [i.e. concentrations at which neither mono- nor triple therapy is sufficiently potent] result rapidly in a pronounced antiviral efficacy. Altogether, this work provides an example as to how to design studies to explore the antiviral efficacy of combinations of more than two compounds.

事实证明，直接作用抗病毒药（DAA's）的联合疗法可用于治疗慢性HCV感染，是治疗该病毒慢性感染的高效策略。在这里，我们以HCV为例研究了如何最佳设计体外研究，以探索三个或更多DAA组合的联合抗病毒效率。为此，我们使用了HCV NS3蛋白酶抑制剂，靶向NS5A的化合物和两种非核苷NS5B聚合酶抑制剂（每种靶向不同的药物结合位点）。我们证明，使用含有Huh 9-13肝癌细胞的HCV亚基因组复制子，可以对这4种不同的DAA进行四重疗法，每种疗法的EC ₇₅均为其1倍。导致高效抑制病毒复制。HCV复制子从宿主细胞中的快速清除进一步反映了这一点。相比之下，在4x EC _75下既不由单独一个分子组成的等价组合，也不在1.33x EC _75下由三分子组合组成的等价组合导致通关。与四联组合相反，在单次治疗和大多数三联组合中出现了耐药性变异。因此，这些数据表明，在总的次优浓度（即单药或三药均无效的浓度）下，四联组合可迅速产生显着的抗病毒功效。总之，这项工作为如何设计研究以探索两种以上化合物组合的抗病毒功效提供了一个实例。