NS5A Inhibitor Research Articles

High sustained virologic response in genotypes 3 and 6 with generic NS5A inhibitor and sofosbuvir regimens in chronic HCV in myanmar.

Exclusive HCV therapy clinical trials with genotype 6 patients in high prevalence areas have been sparse. We analysed the safety and efficacy of two generic, pangenotypic NS5A/NS5B combination oral DAA regimens, primarily in genotypes 3 and 6, in a real-world setting: (a) daclatasvir/sofosbuvir (DCV/SOF)±ribavirin (RBV) and (b) Velpatasvir/sofosbuvir (VEL/SOF±RBV). Between December 2015 and November 2017, data from 522 patients were analysed, 311 of whom were treated with DCV/SOF±RBV for 12/24weeks (genotype 3: n=193, genotype 6: n=89) and 211 were treated with VEL/SOF±RBV for 12/24weeks (genotype 3: n=83, genotype 6: n=77). Overall SVR rates were high for both DCV/SOF±RBV (96.1%, n=299/311) and VEL/SOF±RBV (95.3%, n=201/211), and there was a good adverse event profile. Treatment naïve status and inclusion of RBV (in advanced fibrosis/cirrhosis) were significant independent predictors of achieving SVR12, while type of DAA regimen was not predictive. In this large cohort of genotypes 3 (n=276) and 6 (n=166; n=127 unique subtype of 6c-l), high SVR rates of 94.9% (n=262/276) and 95.2% (n=158/166), respectively, were noted. In conclusion, generic and pangenotypic DCV/SOF and VEL/SOF±RBV regimens were highly effective and safe, in genotypes 3 and 6 chronic HCV in Myanmar. These efficacious pangenotypic regimens suggest that baseline genotype testing can be eliminated moving forward. While RBV may still be needed for those with advanced fibrosis/cirrhosis, in a global elimination strategy it would not be practical even if it does compromise SVR in a minority with difficult to treat characteristics.

In vitro resistance profile of hepatitis C virus NS5A inhibitor velpatasvir in genotypes 1 to 6.

Velpatasvir is a pan-genotypic hepatitis C virus (HCV) NS5A inhibitor, which is used with sofosbuvir for treatment of infection with HCV genotypes 1-6. In vitro resistance studies were performed to characterize NS5A changes that might confer reduced velpatasvir susceptibility in vivo. Resistance selection studies using HCV replicon cells for subtypes 1a, 1b, 2a, 2b, 3a, 4a, 5a and 6a identified NS5A resistance-associated substitutions (RASs) at nine positions, most often 28M/S/T, 31F/I/M/P/V and 93D/H/N/S. In subtype 1a, RASs were selected at positions 31 and/or 93, while in subtype 1b, replicons with two or more RASs at positions 31, 54 or 93 were selected. Y93H was selected in subtypes 1a, 1b, 2a, 3a and 4a. In subtype 5a or 6a, L31P or P32L/Q was selected, respectively. Velpatasvir susceptibility of 358 replicons from genotypes 1 to 6 containing one or more NS5ARASs was also evaluated. The majority (63%) of subtypes 1a and 1b single RAS-containing replicons retained susceptibility to velpatasvir (<2.5-fold change in EC50 ). High levels of resistance to velpatasvir were observed for six single mutants in subtype 1a, including M28G, A92K, Y93H/N/R/W and for one mutant, A92K, in subtype 1b. Most single mutants in subtypes 2a, 2b, 3a, 4a and 5a displayed low levels of reduced velpatasvir susceptibility. High-level resistance was observed for C92T and Y93H/N in subtype 2b, Y93H/S in 3a, and L31V and P32A/L/Q/R in 6a, and several double mutants in these subtypes. Overall, velpatasvir maintained activity against most common RASs that are known to confer resistance to first-generation NS5A inhibitors.

SVR12 rates higher than 99% after sofosbuvir/velpatasvir combination in HCV infected patients with F0-F1 fibrosis stage: A real world experience.

Background and objectivesThe pangenotypic single tablet regimen of NS5B inhibitor sofobuvir (SOF) and NS5A inhibitor velpatasvir (VEL) is advised for 12 weeks in HCV-infected patients including those with compensated cirrhosis. Addition of ribavirin (RBV) may be considered in genotype 3 (GT3) with compensated and is recommended in decompensated cirrhosis. Real-life results with SOF/VEL are limited. To evaluate efficacy and safety in a large real-world-cohort including patients with different GTs and various fibrosis stages.DesignIn total, 1429 patients were treated with SOF/VEL 400/100 mg for 12 weeks in the Puglia registry between June 2017 and May 2018. 1319 (92.3%) reached week 12 post-treatment (SVR12) at the moment. Only 41 received RBV. Diagnosis of cirrhosis was based on transient elastography and/or APRI or FIB-4 scores. Sensitivity analysis in the population including all patients except non virological failure was conducted. Primary efficacy endpoint was the percentage of patients with SVR12.ResultsPatients’ mean age was 63.8 years, 42.3% had GT1. The majority were naïve and 735 (55.5%) F0/F2. Of the remaining 587, 282 had cirrhosis. SVR12 was 98.5%, 98.0% in GT1, 99.4% in GT2, 97.1% in GT3, 100% in GT4. Overall, SVR12 by sensitivity analysis was 99.4%; 99.7% among F0-F1. Among 218 PWID, SVR12 was 94.5%. Discontinuation rates were 3.7% among PWID and 0.7% among non-PWID (p = 0.004).ConclusionsSOF/VEL treatment of chronic HCV infection reaches very high cure rates in a variety of patients; including those with F0/F1 and PWID.

Discovery of velpatasvir (GS-5816): A potent pan-genotypic HCV NS5A inhibitor in the single-tablet regimens Vosevi® and Epclusa®.

Direct-acting antiviral inhibitors have revolutionized the treatment of hepatitis C virus (HCV) infected patients. Herein is described the discovery of velpatasvir (VEL, GS-5816), a potent pan-genotypic HCV NS5A inhibitor that is a component of the only approved pan-genotypic single-tablet regimens (STRs) for the cure of HCV infection. VEL combined with sofosbuvir (SOF) is Epclusa®, an STR with 98% cure-rates for genotype 1-6 HCV infected patients. Addition of the pan-genotypic HCV NS3/4A protease inhibitor voxilaprevir to SOF/VEL is the STR Vosevi®, which affords 97% cure-rates for genotype 1-6 HCV patients who have previously failed another treatment regimen.

SUN-519 Hypoparathyroidism after Harvoni Treatment for Hepatitis C

Background: Hepatitis C (Hep C) infection is one of the leading causes of end stage liver disease and hepatocellular carcinoma. Several advances have been made in the treatment of Hep C infection. Harvoni (ledipasvir and sofosbuvir) is one of the treatment options. There have been no case reports of hypocalcemia or hypoparathyroidism associated with Harvoni therapy. We report a case of hypocalcemia and permanent hypoparathyroidism that began after Harvoni therapy. Case Presentation: A 64-year old African American man presented with weight loss and was diagnosed to have Hep C. He was treated with 12 weeks course of Harvoni. He was first noted to have low calcium level at 8.4 (8.4 – 10.3 mg/dl), during the last week of Harvoni treatment. Several previous calcium levels were normal. Four months later, he presented to Emergency Department with numbness, palpitations, and muscle cramping. His calcium was then 6.6 mg/dl, albumin 4.3 (3.4 - 4.8 mg/dl), PTH 18 (15 - 65 pg/ml), 25-hydroxy vitamin D 11 (30 - 50 ng/ml), magnesium 2.2 (1.6 – 2.6 mg/dl), phosphorus 3.8 (2.3 - 4.7 mg/dl) and 24-hour urine calcium was less than 2. Oral calcitriol, ergocalciferol and calcium carbonate were initiated and his calcium normalized. His calcium remained normal on calcitriol and calcium carbonate, but twice he had to be treated in emergency department for symptomatic hypocalcemia after missing his medications. Recently, he began PTH (1-84) (NATPARA). He has been tolerating parathyroid hormone injections well. Discussion: Harvoni is a combination medication of Ledipasvir and Sofosbuvir. Ledipasvir is a NS5A inhibitor. NS5A is an RNA binding protein required for the activity of RNA polymerase of hepatitis C virus. Sofosbuvir is a viral RNA polymerase inhibitor. There have been no case reports of hypoparathyroidism occurring with Harvoni therapy in the literature. Hypoparathyroidism most commonly results after anterior neck surgery like thyroidectomy, parathyroidectomy, or anterior neck dissection. Other causes include neck irradiation, infections like HIV, infiltration of the glands by heavy metals such as iron or copper; genetic disorders of parathyroid gland development, parathyroid hormone synthesis and calcium sensing receptor mutations, and finally autoimmune etiologies. Our patient had none of the risk factors for developing hypoparathyroidism. Nevertheless, he developed permanent, symptomatic hypoparathyroidism towards the end of Harvoni treatment, making Harvoni as the most likely cause of his hypoparathyroidism. More long-term data on this drug is needed to assess the risk of hypoparathyroidism after Harvoni therapy. Conclusion: Hypoparathyroidism leading to hypocalcemia could be a potential complication of Harvoni treatment. Monitoring calcium and parathyroid hormone levels prior to initiation and periodically after completion of Harvoni treatment should be recommended for all patients.

Impact of novel NS5A resistance-associated substitutions of hepatitis C virus detected in treatment-experienced patients

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) in the NS5A region impair the efficacy of NS5A inhibitors. In this study, we evaluated the characteristics of the novel RASs observed in treatment-failure patients, A92K and a deletion at P32 (P32del), and the susceptibility of viruses with these RASs to various anti-HCV reagents by using JFH-1 based recombinant HCV with NS5A from a genotype 1b Con1 strain (JFH1/5ACon1). We introduced A92K or P32del solely or in combination with Q24K, L28M, R30Q or L31F into the NS5A of JFH1/5ACon1. Viruses harboring R30Q/A92K showed high extracellular core antigens and infectivity titers, whereas the other viruses with RASs showed low replication levels and infectivity titers. All the viruses with A92K or P32del were markedly resistant to ledipasvir, velpatasvir and elbasvir. Interestingly, viruses with R30Q/A92K were more susceptible to grazoprevir than viruses without RAS. All the viruses had a similar susceptibility to ribavirin and sofosbuvir. In conclusion, combination RASs R30Q/A92K enhanced virus production whereas other RASs impaired virus replication. Both A92K and P32del conferred severe resistance even to second generation NS5A inhibitors. However, these viruses were susceptible to grazoprevir, ribavirin and sofosbuvir. Thus, combination regimens with these reagents may eradicate viruses harboring A92K or P32del.

SAT-437-An mTor-based immunosuppression reduces the antiviral efficacy of NS3/4A and NS5A inhibitors for HCV genotype 1b treatment in vitro

SAT-437-An mTor-based immunosuppression reduces the antiviral efficacy of NS3/4A and NS5A inhibitors for HCV genotype 1b treatment in vitro

Subtype-Specific Prevalence of Hepatitis C Virus NS5A Resistance Associated Substitutions in Mainland China

Resistance associated substitutions (RASs) can reduce the efficacy of direct-acting antiviral agents (DAAs) targeting hepatitis C virus (HCV) and lead to treatment failure. Clinical data of HCV NS5A RASs prevalence are limited in China and need to be investigated. A total of 878 unique patient samples with different genotypes (GT) (1b: n = 489, 2a: n = 203, 3a: n = 60, 3b: n = 78, 6a: n = 48) were collected from around mainland China by KingMed Laboratory and analyzed for NS5A RASs distribution by Sanger sequencing. Phylogeographic analyses based on NS5A domain 1 sequences indicated circulation of both locally and nationally epidemic strains. Relatively high frequency of Y93H (14.1%) was only detected in GT1b but not in other subtypes. High frequency of L31M was found in both GT2a (95.6%) and GT3b (98.7%) sequences. Due to the overlapping incidence of A30K, 96% of GT3b isolates had NS5A RASs combination A30K + L31M, which confers high levels of resistance to most NS5A inhibitors. No RASs were detected in GT6a strains. Meanwhile, baseline NS5A RASs fingerprints were also evaluated in 185 DAA treatment-naive GT1b patients with next generation sequencing method. Patients presenting with Y93H had statistically higher entropy of HCV NS5A sequences. Taken together, subtype-specific distribution patterns of NS5A RASs were observed. GT1b patients with higher HCV complexity tend to have a greater chance of Y93H presence, while GT3b patients are naturally resistant to current NS5A inhibitors and their treatment may pose a challenge to real-world DAA application.

Analysis of hepatitis C virus genotype 3 resistance to direct acting antivirals

Treatment of hepatitis C virus (HCV) with direct acting antivirals (DAAs), results in a sustained virological response (SVR) that varies according to the viral genotype. Genotype 3 is the second most prevalent in Brazil and presented the lowest response among all genotypes, this was associated with the occurrence of resistance substitutions in the viral genome. The aim of this study was to analyze genotype 3 viruses circulating in Brazilian patients who do not respond to treatment with Daclatasvir to investigate the presence of novel substitutions within NS5A, and to investigate the role of these substitutions in resistance to different NS5A inhibitors in vitro. A total of 60 patients have been analyzed, including 4 relapsers. Samples collected before (all patients) and after treatment (relapsing patients) were analysed by RNA extraction, cDNA synthesis, amplification by Nested-PCR and direct Sanger sequencing. Many previously undefined substitutions were indeed observed. These included S98G which, although detected in 15 % of pre-treatment samples, substitution appeared in 75 % of post-treatment samples from the non-responding patients and only in 10.7 % of responding individuals. For post-treatment samples in relapsing patients, this substitution had a prevalence of 50 %. Due to the high prevalence, this substitution showed potential to be associated with therapy failure. The phenotypes of this substitution and others identified in the study are being evaluated in the context of a genotype 3a subgenomic replicon and infectious virus to determine their effect on DAA resistance and/or potential fitness cost.

No need to discontinue hepatitis C virus therapy at the time of liver transplantation.

ObjectivesDirect antiviral agents (DAA) has dramatically improved the therapy outcome of hepatitis C-virus (HCV) infection, both on the waiting-list and post liver transplantation (LT). DAA are generally well-tolerated in patients with mild to moderate liver and kidney failure, but some DAAs are contraindicated in patients with severe dysfunction of these organs. Today there are few studies of peri-LT DAA use and treatment is commonly discontinued at the time of LT. We report here our experience of DAA therapy given continuously in the perioperative LT period in a real-life setting in Sweden.MaterialIn total 10 patients with HCV-cirrhosis, with or without hepatocellular carcinoma, and a median age of 60.5 years (range, 52–65) were treated with DAAs on the waiting list for LT, and continued in the early postoperative period without any interruption, on the basis of not having reached a full treatment course at the time of LT. Sofosbuvir and a NS5A inhibitor with or without ribavirin, or sofosbuvir and ribavirin only, were given. The distribution of genotypes was genotype 1 and 3, in 4 and 6 patients, respectively. Six of the 10 patients had previously been treated with IFN-based therapy.ResultsThere were no adverse events leading to premature DAA discontinuation. All recipients achieved a sustained viral response 12 weeks after end-of-treatment (SVR12). At the time of LT the median MELD-score was 16.5 (range 7–21), CTP-score 9.0 (range 5–10), creatinine 82.5 μmol/L (range 56–135, reference 60–105), bilirubin 33 μmol/L (range 16–79, reference 5–25) and PK-INR 1.5 (range 1.1–1.8). The median duration of DAA therapy was 60 days (range 18–132) pre-LT, 54 days post-LT (range 8–111 days) and in total 15.5 weeks (range 12–30 weeks).ConclusionInterferon-free DAA therapy of HCV-infection given in the immediate pre- and post-operative LT period is safe, well-tolerated and yields high SVR rates.

A novel, potent, and orally bioavailable thiazole HCV NS5A inhibitor for the treatment of hepatitis C virus

A medicinal chemistry program based on the small-molecule HCV NS5A inhibitor daclatasvir has led to the discovery of dimeric phenylthiazole compound 8, a novel and potent HCV NS5A inhibitor. The subsequent SAR studies and optimization revealed that the cycloalkyl amide derivatives 27a-29a exhibited superior potency against GT1b with GT1b EC50 values at picomolar concentration. Interestingly, high diastereospecificity for HCV inhibition was observed in this class with the (1R,2S,1′R,2′S) diastereomer displaying the highest GT1b inhibitory activity. The best inhibitor 27a was found to be 3-fold more potent (GT1b EC50 = 0.003 nM) than daclatasvir (GT1b EC50 = 0.009 nM) against GT1b, and no detectable in vitro cytotoxicity was observed (CC50 > 50 μM). Pharmacokinetic studies demonstrated that compound 27a had an excellent pharmacokinetic profiles with a superior oral exposure and desired bioavailability after oral administration in both rats and dogs, and therefore it was selected as a developmental candidate for the treatment of HCV infection.

Comparison of Naturally Occurring Resistance-Associated Substitutions Between 2008 and 2016 in Chinese Patients with Chronic Hepatitis C Virus Infection.

Aims: The presence of pre-existing hepatitis C virus (HCV) resistance-associated substitutions (RASs) could attenuate viral susceptibility to direct-acting antiviral agents. The aim of this study was to better understand the differences among HCV RASs over time. We compared the prevalence and characteristics of naturally occurring HCV RASs in the NS3, NS5A, and NS5B genes between 2008 and 2016 in Chinese patients chronically infected with HCV genotypes (GT) 1b, 2a, 3a, 3b, and 6a. Methods: HCV RNA was extracted after serum samples were collected from 242 patients at treatment baseline, including 120 samples in 2008 and 122 samples in 2016. Reverse transcription and nested PCR were performed, and the PCR products of the NS3, NS5A, and NS5B regions were sequenced using the Sanger sequencing method. Finally, RASs were identified from the different viral strains. Results: In GT1b, the overall frequency of NS5A RASs in 2016 was significantly higher than that in 2008 (42.0% vs. 18.4%; p = 0.002). Among NS5A RASs, the most frequently detected RAS was Y93H (5.3% in 2008 vs. 15.9% in 2016; p = 0.035), which confers medium- to high-level resistance to the NS5A inhibitors: daclatasvir (DCV), ledipasvir (LDV), ombitasvir (OMV), and elbasvir. The frequency of NS5A L28 (low-level resistance to DCV/LDV/OMV) in 2016 was also higher than that in 2008 (11.6% vs. 1.3%; p = 0.027). In addition, the highest frequency of clinically relevant NS3 RASs was S122G/A/T (69.7% in 2008 and 72.5% in 2016) in HCV GT1b isolates, which had medium-level resistance to simeprevir and asunaprevir, followed by Y56F (7.9% in 2008 and 14.5% in 2016), which confers resistance to paritaprevir. Although NS5B C316N had the highest substitution rate in GT1b (80.2% in 2008 and 91.3% in 2016), it was associated with low-level resistance to sofosbuvir and dasabuvir. However, HCV RASs were rarely detectable at baseline in other genotypes or subtypes except GT1b in this study. Conclusion: The frequency of NS5A RASs in 2016 was significantly higher than that in 2008, especially at the L28 and Y93 substitution positions, which may be due to their better fitness compared with wild-type viruses.

A pan-genotypic Hepatitis C Virus NS5A amplification method for reliable genotyping and resistance testing

BackgroundChronic infection with the Hepatitis C Virus (HCV) is associated with the risk of progressive liver disease. Although, HCV treatment options and viral cure rates have tremendously increased over the last decade, all currently licensed combination therapies contain inhibitors of the replication complex NS5A. Resistance-associated substitutions (RAS) in NS5A can limit the efficacy of therapy; however, resistance testing is routinely not recommended for all patients. Notably, pan-genotypic combinations have been approved, however the correct identification of the HCV genotype is still required for treatment decisions and is a good predictor for treatment success. ObjectiveThe aim of this study was the establishment of a pan-genotypic NS5A amplification method for reliable genotyping and simultaneous resistance testing in a fast and cheap routine diagnostic setup. Study designPan-genotypic degenerated nested PCR primer were designed and tested in 262 HCV-patients. The collection included samples from genotypes 1–7 and the median viral load was 1.07 × 106 IU/ml (range 248-21 × 106 IU/ml). ResultsAmplification of the expected 747bp fragment was successful in 257 of 262 (98.1%) samples including samples <1000 IU/ml. The direct comparison of the genotype information obtained with core sequencing to those obtained by NS5A prediction showed high concordance (97.3%) and discrepancies occurred only for relatively rare subtypes. Resistance analysis using Geno2Pheno[HCV] showed NS5A-RAS in 23 of 257 (8.9%) of samples. ConclusionsWe successfully developed a routine diagnostic method for pan-genotypic amplification of NS5A. This amplicon can be used for simultaneous genotyping and resistance testing for enhancing and improving routine HCV diagnostic.

Combinations of two drugs among NS3/4A inhibitors, NS5B inhibitors and non-selective antiviral agents are effective for hepatitis C virus with NS5A-P32 deletion in humanized-liver mice.

The emergence of a deletion mutant at hepatitis C virus (HCV) NS5A-P32 (P32del) has recently been reported in a subset of chronic hepatitis C patients who experience virologic failure after direct-acting antiviral drug (DAA) treatment. This mutation confers extremely high resistance to NS5A inhibitors. No effective treatment has been established for cases with this mutation. We used a JFH1-based recombinant virus with NS5A from a genotype 1b strain to introduce a P32del mutation. We inoculated human hepatocyte chimeric mice with sera from a patient with ledipasvir/sofosbuvir therapy failure carrying a genotype 1b HCV with NS5A L31M and P32del or from a DAA-naïve patient carrying wild-type virus. JFH1-based chimeric viruses with P32del showed sufficient levels of replication for in vitro assay despite the suppression of viral growth and infectious virus production. Variants with P32del exhibited severe resistance to all tested NS5A inhibitors, including daclatasvir, ledipasvir, elbasvir and velpatasvir, but were as susceptible to NS3/4A inhibitors, NS5B inhibitors, interferon alfa-2b, and ribavirin as wild-type viruses in the in vitro assay. The P32del mutant virus caused persistent infection in all inoculated chimeric mice with high viral titer and frequency. The virus was resistant to the ledipasvir/GS-558093 (a nucleotide analog inhibitor of NS5B polymerase) regimen but susceptible to either simeprevir plus GS-558093 or peg-interferon alfa-2b, compared to the wild-type virus. Therapies combining at least two drugs among NS3/4A inhibitors, NS5B inhibitors and non-selective antiviral agents may be effective for HCV-infected patients with NS5A-P32del.

Discovery of novel pan-genotypic HCV NS5A inhibitors containing a novel tetracyclic core

A series of novel tetracyclic core-containing HCV NS5A inhibitors has been discovered. Incorporation of tetrahydropyran-substituted amino acid moiety improved their potency and yielded HCV NS5A inhibitors with a minimum potency shift from the GT1a strain compared to other genotypes and mutants. Compounds 53 and 54 showed the best potency profile and had reasonable half-times in rat PK studies. However, further optimization of their oral bioavailability is still needed in order to advance them for further development. [BMCL ABSTRACT] ©2000 Elsevier Science Ltd. All rights reserved.

Population Pharmacokinetic Analysis of Daclatasvir, Asunaprevir, and Beclabuvir Combination in HCV-Infected Subjects.

A fixed-dose combination of daclatasvir (pangenotypic NS5A inhibitor), asunaprevir (NS3/4A protease inhibitor), and beclabuvir (nonnucleoside NS5B inhibitor) was approved for hepatitis C virus treatment in Japan. The objectives of the analyses were to develop the daclatasvir, asunaprevir, and beclabuvir population pharmacokinetic models for the combination regimen. First, an original population pharmacokinetic model was developed using the data in non-Japanese hepatitis C virus-infected subjects. The model was subsequently updated after a phase 3 study in Japanese hepatitis C virus-infected subjects was available. A total of 11,382, 11,300, and 10,728 pharmacokinetic records from 1,228 subjects were included for daclatasvir, asunaprevir, and beclabuvir in the updated model, respectively. Daclatasvir and beclabuvir pharmacokinetics (PK) were described by a 1-compartment model with linear elimination and asunaprevir PK was described by 2-compartment model with linear elimination. Cirrhosis, baseline, and time-varying ALT were significant covariates on asunaprevir apparent oral clearance. Asian subjects had greater asunaprevir and beclabuvir exposures than white subjects. The effects of all covariates on daclatasvir PK were modest and not considered clinically significant. With the exception of race on asunaprevir and beclabuvir PK, no other parameters for daclatasvir, asunaprevir and beclabuvir population PK models were meaningfully impacted during the refinement with Japanese subjects.

Frequent Antiviral Treatment Failures in Patients Infected With Hepatitis C Virus Genotype 4, Subtype 4r.

Hepatitis C virus (HCV) genotype 4 is highly heterogeneous. HCV subtype 4r has been suggested to be less responsive to direct-acting antiviral (DAA) drug treatment than other genotype 4 subtypes. Among 537 DAA-treated patients who experienced a virological failure (VF) in France between 2015 and 2018, 121 (22.5%) were infected with genotype 4 and 27 of them (22.3%) with subtype 4r; subtype 4r was thus over-represented as compared to its prevalence in the French general population. Population sequencing of the nonstructural protein (NS) 3, NS5A, and NS5B genes was performed in all subtype 4r patients at treatment failure and in 6 at baseline, whereas full-length HCV genome sequencing was performed in two baseline and three treatment failure samples by means of an original shotgun metagenomics method based on deep sequencing. At treatment failure, all subtype 4r patients harbored two to three dominant NS5A resistance-associated substitutions (RASs), including at least L28A/C/I/M/V and L30R. Among 13 patients exposed to sofosbuvir and an NS5A inhibitor (daclatasvir, ledipasvir, or velpatasvir), 5 (38.5%) also harbored NS5B S282C/T RASs at treatment failure. An additional patient harbored S282C/T RASs at treatment failure by deep sequencing. Prevalence of S282C/T RASs at treatment failure was significantly higher in patients infected with genotype 4r than with other genotypes, including other subtypes of genotype 4. Conclusion: The lower rates of sustained virological response in patients infected with subtype 4r are related to the frequent preexistence at treatment baseline and subsequent selection by DAA treatment of both NS5A and NS5B S282 RASs. Our study suggests that these patients should be identified and receive a triple DAA combination regimen as first-line treatment.

Drug resistant variants of hepatitis C virus genotype 1b in Russia: analysis of aminoacid substitutions in NS5a and core proteins

Aim. To determine the prevalence of amino acid substitutions in hepatitis C virus NS5a and core proteins which areassociated with resistance to direct-acting antivirals and interferon in genotype 1b (HCV-1b) strains circulating in Russia. Materials and methods. Nucleotide sequences of NS5a (n=93) and core (n=30) of HCV-1b were obtained using direct sequencing of respective amplified genome fragments. The search for resistance associated substitutions was performed for amino acid positions 28, 29, 30, 31, 32, 58, 62, 92, 93 of NS5a, and 70 and 91 amino acid positions of core proteins, respectively. Results. The total proportion of HCV-1b strains carrying resistance associated substitutions in NS5a was 22,6% (21/93). The total detection rate of L31M and Y93H substitutions that are associated with resistance to the majority of NS5a inhibitors was 10,8%. Less clinically significant substitutions L28M, R30Q, P58S/T, A92T were detected too. The proportion of infections caused by HCV-1b strains that are potentially resistant both to interferon and NS5a inhibitors was 10% (10/30). Conclusion. Testing of HCV-1b infected patients for background resistance profile could be a useful tool to prevent the choosing of initially ineffective treatment regimen.

Prevalence and Factors Related to Natural Resistance-Associated Substitutions to Direct-Acting Antivirals in Patients with Genotype 1 Hepatitis C Virus Infection.

This study aimed to assess the prevalence of natural resistance-associated substitutions (RASs) to NS3, NS5A and NS5B inhibitors in 86 genotype 1 Hepatitis C Virus (HCV)-infected patients from Buenos Aires, Argentina, and to determine their effect on therapy outcome. Additionally, virological, clinical and host genetic factors were explored as predictors of the presence of baseline RASs. NS3 RASs (39.2%) were more prevalent than NS5A RASs (25%) and NS5B RASs (8.9%). In the three regions, the frequencies of RASs were significantly higher in HCV-1b than in HCV-1a. The prevalence of Y93H, L159F and Q80K were 1.3%, 6.3% and 2.5%, respectively. IFNL3 CC genotype was identified as an independent predictor of the presence of baseline RASs in NS5A and NS3 genes (p = 0.0005 and p = 0.01, respectively). Sustained virologic response was achieved by 93.3% of the patients after receiving direct-acting antivirals (DAAs), although 48.7% of them showed baseline RASs related to the DAA-regimen. Notably, the prevalence of clinically relevant RASs in the three genes was lower than that observed around the world. The baseline presence of RASs in both subtypes did not appear to affect therapy outcome. These results support the need to evaluate resistance patterns in each particular country since RASs´ prevalence significantly vary worldwide.

THE CURRENT CHALLENGE OF HCV-RNA RESISTANCE AGAINST DIRECT-ACTING ANTIVIRAL DRUGS.

The HCV-RNA (Hepatitis-C RNA) diagnosed patients are recommended interferon free Direct-Acting Antiviral (DAAs) therapy. Although the treated patients are reported viral resistance to DAAs, the genotype 3a infection is challenge to NS5A inhibitors leading to cirrhosis. Therefore, the patients with chronic Hepatitis-C treated with DAAs, nucleotide analogue inhibitors or NS5B protein have high risk of resistance. Breakthrough or relapse is not common with these drugs when used as monotherapy.NS3-4A protease inhibitors and non-nucleoside inhibitors have low susceptibility to resistance. Sofosbuvir-resistant variants have been discovered that disappear after treatment withdrawal. Patients who experience relapse after an INF-free treatment have large fraction of drug resistant viruses.