Targeting a host-cell entry factor barricades antiviral-resistant HCV variants from on-therapy breakthrough in human-liver mice

Vercauteren, Koen; Brown, Richard J. P.; Mesalam, Ahmed Atef; Doerrbecker, Juliane; Bhuju, Sabin; Geffers, Robert; Eede, Naomi Van Den; McClure, C. Patrick; Troise, Fulvia; Verhoye, Lieven; Baumert, Thomas F.; Farhoudi, Ali; Cortese, Riccardo; Ball, Jonathan K.; Leroux‐Roels, Geert; Pietschmann, Thomas; Nicosia, Alfredo; Meuleman, Philip

doi:10.1136/gutjnl-2014-309045

Cited by 21 publications

(24 citation statements)

References 39 publications

(42 reference statements)

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“…Human liver-chimeric mice used in this study were produced as previously reported (Meuleman et al, 2005) and represent some of the monotherapy arm animals from Vercauteren et al (2015a). Mice were infected with a serum-derived virus (titer 2 × 10 5 IU/ml) from a chronically HCV infected patient (genotype 1b, P05) (Fafi-Kremer et al, 2010).…”

Section: Methodsmentioning

confidence: 99%

“…Ciluprevir is a peptidomimetic macrocyclic compound that binds non-covalently to the active site of the HCV NS3 serine protease. Viremia was tracked as previously described (Vercauteren et al, 2015a). …”

Section: Methodsmentioning

confidence: 99%

“…RNA extraction, cDNA synthesis and amplicon generation were performed according to Vercauteren et al (2015a). Briefly, viral RNA was extracted from EDTA-plasma using a Viral RNA MiniElute kit (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

“…NGS data were analyzed as previously described (Irving et al, 2014; Vercauteren et al, 2015a) with minor modifications. The nucleotide consensus of the P05 NS3 protease population (540bp) was determined and used as a reference sequence in all subsequent assemblies.…”

Section: Methodsmentioning

confidence: 99%

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Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy

et al. 2017

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Due to the highly restricted species-tropism of Hepatitis C virus (HCV) a limited number of animal models exist for pre-clinical evaluation of vaccines and antiviral compounds. The human-liver chimeric mouse model allows heterologous challenge with clinically relevant strains derived from patients. However, to date, the transmission and longitudinal evolution of founder viral populations in this model have not been characterized in-depth using state-of-the-art sequencing technologies. Focusing on NS3 protease encoding region of the viral genome, mutant spectra in a donor inoculum and individual recipient mice were determined via Illumina sequencing and compared, to determine the effects of transmission on founder viral population complexity. In all transmissions, a genetic bottleneck was observed, although diverse viral populations were transmitted in each case. A low frequency cloud of mutations (<1%) was detectable in the donor inoculum and recipient mice, with single nucleotide variants (SNVs) > 1% restricted to a subset of nucleotides. The population of SNVs >1% was reduced upon transmission while the low frequency SNV cloud remained stable. Fixation of multiple identical synonymous substitutions was apparent in independent transmissions, and no evidence for reversion of T-cell epitopes was observed. In addition, susceptibility of founder populations to antiviral therapy was assessed. Animals were treated with protease inhibitor (PI) monotherapy to track resistance associated substitution (RAS) emergence. Longitudinal analyses revealed a decline in population diversity under therapy, with no detectable RAS >1% prior to therapy commencement. Despite inoculation from a common source and identical therapeutic regimens, unique RAS emergence profiles were identified in different hosts prior to and during therapeutic failure, with complex mutational signatures at protease residues 155, 156 and 168 detected. Together these analyses track viral population complexity at high-resolution in the human-liver chimeric mouse model post-transmission and under therapeutic intervention, revealing novel insights into the evolutionary processes which shape viral protease population composition at various critical stages of the viral life-cycle.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy

et al. 2017

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“…The binding models of HCV NS3/4A protease were predicted using the AutoDock program (Vercauteren et al, 2015). Two crystal structures of NS3/4A (PDB code: 4A92 and 2GVF) were used with an ensemble-docking protocol (Hu et al, 2013).…”

Section: Methodsmentioning

confidence: 99%

Identification of novel anti-hepatitis C virus agents by a quantitative high throughput screen in a cell-based infection assay

et al. 2015

Antiviral Research

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Hepatitis C virus (HCV) poses a major health threat to the world. The recent development of direct-acting antivirals (DAAs) against HCV has markedly improved the response rate of HCV and reduced the side effects in comparison to the interferon-based therapy. Despite this therapeutic advance, there is still a need to develop new inhibitors that target different stages of the HCV life cycle because of various limitations of the current regimens. In this study, we performed a quantitative high-throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of ~ 350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial ~ 3,000 molecules that showed inhibitory activity for further structural and functional analyses. We were able to assign the majority of these compounds to specific stage(s) in the HCV life cycle. Three of them are direct inhibitors of NS3/4A protease. Most of the compounds appear to act on novel targets in HCV life cycle. Four compounds with novel structure and excellent drug-like properties, three targeting HCV entry and one targeting HCV assembly/secretion, were advanced for further development as lead hits. These compounds represent diverse chemotypes that are potential lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. In addition, they represent novel molecular probes to explore the complex interactions between HCV and the cells.

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Hepatitis C subtyping assay failure in UK patients born in sub‐Saharan Africa: Implications for global treatment and elimination

Adeboyejo

King

Tsoleridis

et al. 2022

Journal of Medical Virology

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Background and Aims: The newly developed direct-acting antivirals have revolutionized the treatment of chronic hepatitis C virus (HCV), with cure rates as high as 98% in some cohorts. Although genome sequencing has demonstrated that some subtypes of HCV naturally harbor drug resistance associated substitutions (RAS), these are often overlooked as "rarities." Furthermore, commercial subtyping assays and associated epidemiological findings are skewed towards Western cohorts and whole-genome sequencing can be problematic to deploy without significant infrastructure and training support. We thus aimed to develop a simple, robust and accurate HCV subtyping pipeline, to optimize and streamline molecular detection and sequence-based typing of diverse RAS-containing subtypes.Methods: HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction (PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools.Results: Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis.

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Targeting a host-cell entry factor barricades antiviral-resistant HCV variants from on-therapy breakthrough in human-liver mice

Abstract: (2016) Targeting a host-cell entry factor barricades antiviral-resistant HCV variants from on-therapy breakthrough in human-liver mice. Gut, 65 (12).

Cited by 21 publications

References 39 publications

Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy

Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy

Identification of novel anti-hepatitis C virus agents by a quantitative high throughput screen in a cell-based infection assay

Hepatitis C subtyping assay failure in UK patients born in sub‐Saharan Africa: Implications for global treatment and elimination

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