Efficacy of NS5A Inhibitors Against Hepatitis C Virus Genotypes 1–7 and Escape Variants

Abstract

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. 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 orwithout RAS. We engineered HCV variants that included RAS identified in escape experiments, using recombinants with or without T/Y93H and daclatasvir, or thatcontained RAS previously reported from patients. NS5A inhibitors had varying levels of efficacy against original andresistant 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 hadnoapparent resistance to pibrentasvir, and HCV with RASat 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. Forthe remaining NS5A inhibitors tested, RAS atamino acids28 and 93 led to high levels of resistance. Among theseinhibitors, velpatasvir was more effective against variants with RAS at amino acid 30 and some variants withRAS at amino acid 31 than the other agents. Variants withthepre-existing RAS T/Y93H acquired additional NS5Achanges during escape experiments, resulting in HCV variantswith specific combinations of RAS, showing high fitness and high resistance. 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.