Abundant Drug-Resistant NS3 Mutants Detected by Deep Sequencing in Hepatitis C Virus-Infected Patients Undergoing NS3 Protease Inhibitor Monotherapy

Abstract

The high genetic variation of hepatitis C virus (HCV) results in rapid selection of drug resistance mutations (DRMs) during monotherapy with direct-acting antivirals (DAAs). It has been proposed that each possible single mutant preexists in infected individuals; however, the levels of preexisting DRMs are too low to be directly quantified in most patients using current techniques. In this study, we evaluated the presence of DRMs in HCV-infected patients treated with the HCV protease inhibitors GS-9256 or GS-9451 as monotherapy using deep sequencing in 137 longitudinal samples from 45 patients. Software was developed to analyze deep-sequencing results with an assay cutoff of 0.25%. No NS3 DRMs that confer resistance to GS-9256 and GS-9451 (R155K, A156T, and D168V/E) were observed in 33 baseline samples at >0.25%. In contrast, these and other substitutions at NS3 positions 155, 156, and 168 were detected in 19/27 patients at day 2 (24 h) and 21/21 at day 4 (84 h) of monotherapy but not in placebo-treated patients. Based on the DRM growth kinetics during drug treatment, pretreated NS3 mutations at amino acids 155, 156, and 168 were estimated on average at 0.025% and 0.015% per genotype 1a and 1b HCV-infected patients, respectively. Relative fitness of the DRM viruses was shown to be significantly lower than the wild type. Deep-sequencing analyses of NS3 protease inhibitor-treated HCV-infected patients suggest a limit of HCV viral load suppression of 3.6 to 3.8 log 10 with NS3 protease inhibitor monotherapy that does not suppress the identified preexisting NS3 DRMs and thus a need for a combination therapy.

Hepatitis C virus genetic variability and evolution

“…This finding strongly evidences the need for a combined therapy to durably treat HCV infection [105] . In addition, NGS technology has already been implemented in order to study the transmission event of HCV among injection drug users.…”

“…Consequently, as they may be present at different low-level frequencies, this may lead to varying degrees of viral response and therefore the mutant genome will become enriched upon treatment with HCV inhibitors. Hence, determining the natural levels of low frequency resistant variants before starting a treatment might be relevant to better predict viral response to HCV inhibitors [105] .…”

The emergence of NS5B resistance associated substitution S282T after sofosbuvir‐based treatment

“…This suggests that S282T has a poor fitness in the absence of drug pressure, and S282T is therefore unlikely to be detected at measurable frequency in untreated patients. In comparison, baseline NS3, NS5A, and NS5B non‐nucleoside inhibitor (NNI) RASs has been detected in 10%‐90% of DAA‐naïve patients depending on genotype and subtype27, 28, 29, 30, 31; for example, NS5A L31M has been detected in >50% of GT2a patients 32. Due to the high error rate of HCV polymerase, substitutions at all sites in the HCV genome can exist within the viral quasispecies33, 34; however, the lack of S282T compared with other RASs suggests that not all positions in the HCV genome has the same allowance for genetic variability.…”

Deep Sequencing Reveals Mutagenic Effects of Ribavirin during Monotherapy of Hepatitis C Virus Genotype 1-Infected Patients

“…Also, in patients undergoing therapy with IFN-␣ plus ribavirin, an increased mutation rate and a mutational spectrum with increased G-to-A and C-to-U transitions were detected during treatment based on E1/E2 and NS5A sequencing studies (28). A greater nucleotide sequence variation with an increase in C-to-U transitions within NS3 and NS5B was also observed for patients treated with a NS3 inhibitor and a NS5B inhibitor in combination with ribavirincontaining regimens (29). In contrast, other results showed only transient (30) or no (31) increases in substitution rates in NS5B and NS3/4A, respectively.…”