Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants:
            <i>In Vitro</i>
            Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

Serre, Stéphanie B. N.; Jensen, Sanne B.; Ghanem, Lubna; Humes, Daryl; Ramírez, Santseharay; Li, Yiping; Krarup, Henrik; Bukh, Jens; Gottwein, Judith M.

doi:10.1128/aac.02929-15

Cited by 25 publications

(23 citation statements)

References 88 publications

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“…Our data thus highlight the potent antiviral efficacy of this drug that has been approved in combination with the NS5A inhibitor elbasvir for the treatment of genotype 1-infected patients. Previously published studies on the use of grazoprevir in vitro also support the notion that grazoprevir shows a uniquely high efficiency against different virus isolates (26,52,53).…”

Section: Con1supporting

confidence: 55%

“…Importantly, treatment with a grazoprevir-elbasvir combination regimen has achieved high SVR rates in genotype 1-infected patients (51). However, so far for genotype 1b, protease inhibitor efficacy and resistance could not be evaluated in the context of the complete viral life cycle, due to the lack of infectious viruses comprising a genotype 1b-specific NS3/4A protease region (19,26,52,53). The genotype 1b C5A cultures developed in this study and previous full-length infectious systems for genotype 1a (43,44) permitted us to evaluate the efficacy of NS3/4A-protease and NS5A inhibitors against genotype 1 in the context of the complete viral life cycle.…”

Section: Resultsmentioning

confidence: 99%

“…Briefly, Huh7.5 cells seeded on poly-D-lysine-coated 96-well plates (Nunc) were infected with HCV stock viruses for 24 h. Subsequently, infected cells were treated with different concentrations of specific drugs (purchased from Acme Bioscience, Palo Alto, CA) (45,52,53). Each drug concentration was tested in triplicate.…”

Section: Methodsmentioning

confidence: 99%

“…The genotype 1a viruses TNcc, H77Ccc, and HCV1cc were described previously (43,44). The TNcc virus stock was generated previously (53). For the H77Ccc and HCV1cc virus stocks, Huh7.5 cells were infected, and supernatants were collected and pooled when the viruses reached the peak of infection.…”

Section: Methodsmentioning

confidence: 99%

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Efficient Hepatitis C Virus Genotype 1b Core-NS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors

Pham

Ramírez

Carlsen

et al. 2017

Antimicrob Agents Chemother

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Hepatitis C virus (HCV) strains belong to seven genotypes with numerous subtypes that respond differently to antiviral therapies. Genotype 1, and primarily subtype 1b, is the most prevalent genotype worldwide. The development of recombinant HCV infectious cell culture systems for different variants, permitted by the high replication capacity of strain JFH1 (genotype 2a), has advanced efficacy and resistance testing of antivirals. However, efficient infectious JFH1-based cell cultures of subtype 1b are limited and comprise only the 5= untranslated region (5=UTR)-NS2, NS4A, or NS5A regions. Importantly, it has not been possible to develop efficient 1b infectious systems expressing the NS3/4A protease, an important target of directacting antivirals. We developed efficient infectious JFH1-based cultures with genotype 1b core-NS5A sequences of strains DH1, Con1, and J4 by using previously identified HCV cell culture adaptive substitutions A1226G, R1496L, and Q1773H. These viruses spread efficiently in Huh7.5 cells by acquiring additional adaptive substitutions, and final recombinants yielded peak supernatant infectivity titers of 4 to 5 log 10 focus-forming units (FFU)/ml. We subsequently succeeded in adapting a JFH1-based 5=UTR-NS5A DH1 recombinant to efficient growth in cell culture. We evaluated the efficacy of clinically relevant NS3/4A protease and NS5A inhibitors against the novel genotype 1b viruses, as well as against previously developed 1a viruses. The inhibitors were efficient against all tested genotype 1 viruses, with NS5A inhibitors showing half-maximal effective concentrations several orders of magnitude lower than NS3/4A protease inhibitors. In summary, the developed HCV genotype 1b culture systems represent valuable tools for assessing the efficacy of various classes of antivirals and for other virological studies requiring genotype 1b infectious viruses.

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Section: Con1supporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Efficient Hepatitis C Virus Genotype 1b Core-NS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors

Pham

Ramírez

Carlsen

et al. 2017

Antimicrob Agents Chemother

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“…Basic studies in infectious cell culture systems have shown great variation in their potency against different HCV variants and genotypes, and in their pattern of resistance [137,[219][220][221]; since they all target the protease active site substitutions conferring cross-resistance have been identified [12].…”

Section: Cyclophilin Inhibitors Against Hcvmentioning

confidence: 99%

The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control

Bukh

2016

Journal of Hepatology

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212

173

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The discovery of hepatitis C virus (HCV) in 1989 permitted basic research to unravel critical components of a complex life cycle for this important human pathogen. HCV is a highly divergent group of viruses classified in 7 major genotypes and a great number of subtypes, and circulating in infected individuals as a continuously evolving quasispecies destined to escape host immune responses and applied antivirals. Despite the inability to culture patient viruses directly in the laboratory, efforts to define the infectious genome of HCV resulted in development of experimental recombinant in vivo and in vitro systems, including replicons and infectious cultures in human hepatoma cell lines. And HCV has become a model virus defining new paradigms in virology, immunology and biology. For example, HCV research discovered that a virus could be completely dependent on microRNA for its replication since microRNA-122 is critical for the HCV life cycle. A number of other host molecules critical for HCV entry and replication have been identified. Thus, basic HCV research revealed important molecules for development of host targeting agents (HTA). The identification and characterization of HCV encoded proteins and their functional units contributed to the development of highly effective direct acting antivirals (DAA) against the NS3 protease, NS5A and the NS5B polymerase. In combination, these inhibitors have since 2014 permitted interferon-free therapy with cure rates above 90% among patients with chronic HCV infection; however, viral resistance represents a challenge. Worldwide control of HCV will most likely require the development of a prophylactic vaccine, and numerous candidates have been pursued. Research characterizing features critical for antibody-based virus neutralization and T cell based virus elimination from infected cells is essential for this effort. If the world community promotes an ambitious approach by applying current DAA broadly, continues to develop alternative viral- and host- targeted antivirals to combat resistant variants, and invests in the development of a vaccine, it would be possible to eradicate HCV. This would prevent about 500 thousand deaths annually. However, given the nature of HCV, the millions of new infections annually, a high chronicity rate, and with over 150 million individuals with chronic infection (which are frequently unidentified), this effort remains a major challenge for basic researchers, clinicians and communities.

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Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

Jensen¹,

Fahnøe²,

Pham³

et al. 2019

Hepatology

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Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance–associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1‐4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156‐RASs were not maintained. For genotypes 1 and 2, persistence of 156‐RASs depended on genome‐wide substitution networks, co‐selected under continued PI treatment and identified by next‐generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156‐RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre‐existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156‐RASs, we observed genome‐wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1‐6 revealed 156‐RASs as key determinants of high‐level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1‐3 156‐variants, which might pose a threat to clinically relevant combination treatments.

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Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

Cited by 25 publications

References 88 publications

Efficient Hepatitis C Virus Genotype 1b Core-NS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors

Efficient Hepatitis C Virus Genotype 1b Core-NS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors

The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

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