Publications by authors named "Santseharay Ramirez"

38 Publications

Efficacy of Ion-Channel Inhibitors Amantadine, Memantine and Rimantadine for the Treatment of SARS-CoV-2 In Vitro.

Viruses 2021 10 15;13(10). Epub 2021 Oct 15.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Kettegård Alle 30, 2650 Hvidovre, Denmark.

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v13102082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8537953PMC
October 2021

Viral genome wide association study identifies novel hepatitis C virus polymorphisms associated with sofosbuvir treatment failure.

Nat Commun 2021 10 20;12(1):6105. Epub 2021 Oct 20.

Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 1SY, UK.

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease, worldwide. With the development of direct-acting antivirals, treatment of chronically infected patients has become highly effective, although a subset of patients responds less well to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies, that targets the HCV NS5B polymerase. We use pre-treatment whole-genome sequences of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We find three common polymorphisms in non-targeted HCV NS2 and NS3 proteins are associated with reduced treatment response. These polymorphisms are enriched in post-treatment HCV sequences of patients unresponsive to treatment. They are also associated with lower reductions in viral load in the first week of therapy. Using in vitro short-term dose-response assays, these polymorphisms do not cause any reduction in sofosbuvir potency, suggesting an indirect mechanism of action in decreasing sofosbuvir efficacy. The identification of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of systematic genome-wide analyses of viruses in uncovering clinically relevant polymorphisms that impact treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-25649-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8528821PMC
October 2021

Neutralisation titres against SARS-CoV-2 are sustained 6 months after onset of symptoms in individuals with mild COVID-19.

EBioMedicine 2021 Sep 19;71:103519. Epub 2021 Aug 19.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Background: Given the importance of neutralising antibodies in protection against SARS-CoV-2 infection, it is critical to assess neutralisation persistence long-term following recovery. This study investigated neutralisation titres against SARS-CoV-2 up to 6 months post-symptom onset in individuals with mild COVID-19.

Methods: Plasma neutralisation titres in convalescent COVID-19 individuals were determined at baseline and 6 months post-symptom onset using a cell culture infectious SARS-CoV-2 assay. Total SARS-CoV-2 spike-specific IgG and IgA binding was measured using a lectin capture ELISA and compared between timepoints and correlated to neutralising titres.

Findings: All 48 convalescent COVID-19 individuals were found to have detectable SARS-CoV-2 50% inhibitory dilution neutralisation titres (ID) at baseline and 6 months post-symptom onset with mean ID of 1/943 and 1/411, respectively. SARS-CoV-2 neutralisation titres peaked within 1-2 months post-symptom onset. However, 50% of individuals showed comparable ID at baseline and 6 months post-symptom onset. Both SARS-CoV-2 spike-specific IgG and IgA levels correlated well with neutralising titres. IgG binding was found to be sustained up to 6 months post-symptom onset, whereas IgA levels declined.

Interpretation: This study demonstrates durability of SARS-CoV-2 spike-specific IgG and neutralisation responses following recovery from mild COVID-19. Thus, all subjects included in this study might potentially have protective levels of neutralising antibodies 6 months post-symptom onset. This study also demonstrates a relationship between spike-specific IgA and neutralisation decline, with implications for long-term protection against SARS-CoV-2 infection.

Funding: Novo Nordisk Foundation, Independent Research Fund Denmark and Danish Agency for Science and Higher Education.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2021.103519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8375401PMC
September 2021

In vitro efficacy of artemisinin-based treatments against SARS-CoV-2.

Sci Rep 2021 07 16;11(1):14571. Epub 2021 Jul 16.

Max Planck Institute for Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration-response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC) in different cell types: 7-12 µg/mL), followed by artemether (53-98 µg/mL), A. annua extracts (83-260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-93361-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285423PMC
July 2021

SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor.

Vaccines (Basel) 2021 Jun 29;9(7). Epub 2021 Jun 29.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, 2650 Hvidovre, Denmark.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demonstrated the value of pursuing different vaccine strategies. Vaccines based on whole viruses, a widely used vaccine technology, depend on efficient virus production. This study aimed to establish SARS-CoV-2 production in the scalable packed-bed CelCradle 500-AP bioreactor. CelCradle 500-AP bottles with 0.5 L working volume and 5.5 g BioNOC™ II carriers were seeded with 1.5 × 10 Vero (WHO) cells, approved for vaccine production, in animal component-free medium and infected at a multiplicity of infection of 0.006 at a total cell number of 2.2-2.5 × 10 cells/bottle seven days post cell seeding. Among several tested conditions, two harvests per day and a virus production temperature of 33 °C resulted in the highest virus yield with a peak SARS-CoV-2 infectivity titer of 7.3 log 50% tissue culture infectious dose (TCID)/mL at 72 h post-infection. Six harvests had titers of ≥6.5 log TCID/mL, and a total of 10.5 log TCID were produced in ~5 L. While trypsin was reported to enhance virus spread in cell culture, addition of 0.5% recombinant trypsin after infection did not improve virus yields. Overall, we demonstrated successful animal component-free production of SARS-CoV-2 in well-characterized Vero (WHO) cells in a scalable packed-bed bioreactor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/vaccines9070706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8310283PMC
June 2021

Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 .

Antimicrob Agents Chemother 2021 08 17;65(9):e0268020. Epub 2021 Aug 17.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovregrid.5254.6, Hvidovre, Denmark.

Antivirals targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PIs) against SARS-CoV-2 and their interactions with remdesivir, the only direct-acting antiviral approved for COVID-19 treatment. HCV PIs showed differential potency in short-term treatment assays based on the detection of SARS-CoV-2 spike protein in Vero E6 cells. Linear PIs boceprevir, telaprevir, and narlaprevir had 50% effective concentrations (EC) of ∼40 μM. Among the macrocyclic PIs, simeprevir had the highest (EC, 15 μM) and glecaprevir the lowest (EC, >178 μM) potency, with paritaprevir, grazoprevir, voxilaprevir, vaniprevir, danoprevir, and deldeprevir in between. Acyclic PIs asunaprevir and faldaprevir had ECs of 72 and 23 μM, respectively. ACH-806, inhibiting the HCV NS4A protease cofactor, had an EC of 46 μM. Similar and slightly increased PI potencies were found in human hepatoma Huh7.5 cells and human lung carcinoma A549-hACE2 cells, respectively. Selectivity indexes based on antiviral and cell viability assays were highest for linear PIs. In short-term treatments, combination of macrocyclic but not linear PIs with remdesivir showed synergism in Vero E6 and A549-hACE2 cells. Longer-term treatment of infected Vero E6 and A549-hACE2 cells with 1-fold EC PI revealed minor differences in the barrier to SARS-CoV-2 escape. Viral suppression was achieved with 3- to 8-fold EC boceprevir or 1-fold EC simeprevir or grazoprevir, but not boceprevir, in combination with 0.4- to 0.8-fold EC remdesivir; these concentrations did not lead to viral suppression in single treatments. This study could inform the development and application of protease inhibitors for optimized antiviral treatments of COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.02680-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8370243PMC
August 2021

Overcoming Culture Restriction for SARS-CoV-2 in Human Cells Facilitates the Screening of Compounds Inhibiting Viral Replication.

Antimicrob Agents Chemother 2021 06 17;65(7):e0009721. Epub 2021 Jun 17.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark.

Efforts to mitigate the coronavirus disease 2019 (COVID-19) pandemic include the screening of existing antiviral molecules that could be repurposed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Although SARS-CoV-2 replicates and propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (NUCs) often show decreased activity in these cells due to inefficient metabolization. SARS-CoV-2 exhibits low viability in human cells in culture. Here, serial passages of a SARS-CoV-2 isolate (original-SARS2) in the human hepatoma cell clone Huh7.5 led to the selection of a variant (adapted-SARS2) with significantly improved infectivity in human liver (Huh7 and Huh7.5) and lung cancer (unmodified Calu-1 and A549) cells. The adapted virus exhibited mutations in the spike protein, including a 9-amino-acid deletion and 3 amino acid changes (E484D, P812R, and Q954H). E484D also emerged in Vero E6-cultured viruses that became viable in A549 cells. Original and adapted viruses were susceptible to scavenger receptor class B type 1 (SR-B1) receptor blocking, and adapted-SARS2 exhibited significantly less dependence on ACE2. Both variants were similarly neutralized by COVID-19 convalescent-phase plasma, but adapted-SARS2 exhibited increased susceptibility to exogenous type I interferon. Remdesivir inhibited original- and adapted-SARS2 similarly, demonstrating the utility of the system for the screening of NUCs. Among the tested NUCs, only remdesivir, molnupiravir, and, to a limited extent, galidesivir showed antiviral effects across human cell lines, whereas sofosbuvir, ribavirin, and favipiravir had no apparent activity. Analogously to the emergence of spike mutations , the spike protein is under intense adaptive selection pressure in cell culture. Our results indicate that the emergence of spike mutations will most likely not affect the activity of remdesivir.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.00097-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406809PMC
June 2021

HCV genome-wide analysis for development of efficient culture systems and unravelling of antiviral resistance in genotype 4.

Gut 2021 Apr 8. Epub 2021 Apr 8.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Objective: HCV-genotype 4 infections are a major cause of liver diseases in the Middle East/Africa with certain subtypes associated with increased risk of direct-acting antiviral (DAA) treatment failures. We aimed at developing infectious genotype 4 cell culture systems to understand the evolutionary genetic landscapes of antiviral resistance, which can help preserve the future efficacy of DAA-based therapy.

Design: HCV recombinants were tested in liver-derived cells. Long-term coculture with DAAs served to induce antiviral-resistance phenotypes. Next-generation sequencing (NGS) of the entire HCV-coding sequence identified mutation networks. Resistance-associated substitutions (RAS) were studied using reverse-genetics.

Result: The in-vivo infectious ED43(4a) clone was adapted in Huh7.5 cells, using substitutions identified in ED43(Core-NS5A)/JFH1-chimeric viruses combined with selected NS5B-changes. NGS, and linkage analysis, permitted identification of multiple genetic branches emerging during culture adaptation, one of which had 31 substitutions leading to robust replication/propagation. Treatment of culture-adapted ED43 with nine clinically relevant protease-DAA, NS5A-DAA and NS5B-DAA led to complex dynamics of drug-target-specific RAS with coselection of genome-wide substitutions. Approved DAA combinations were efficient against the original virus, but not against variants with RAS in corresponding drug targets. However, retreatment with glecaprevir/pibrentasvir remained efficient against NS5A inhibitor and sofosbuvir resistant variants. Recombinants with specific RAS at NS3-156, NS5A-28, 30, 31 and 93 and NS5B-282 were viable, but NS3-A156M and NS5A-L30Δ (deletion) led to attenuated phenotypes.

Conclusion: Rapidly emerging complex evolutionary landscapes of mutations define the persistence of HCV-RASs conferring resistance levels leading to treatment failure in genotype 4. The high barrier to resistance of glecaprevir/pibrentasvir could prevent persistence and propagation of antiviral resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/gutjnl-2020-323585DOI Listing
April 2021

Lipid Droplets Accumulation during Hepatitis C Virus Infection in Cell-Culture Varies among Genotype 1-3 Strains and Does Not Correlate with Virus Replication.

Viruses 2021 02 28;13(3). Epub 2021 Feb 28.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, 2650 Hvidovre, Denmark.

Liver steatosis is a common complication of chronic hepatitis C virus (HCV) infection, which can result in accelerated liver fibrosis development, especially in patients infected with genotype 3a. The precise mechanisms of HCV-induced liver steatosis remain unclear, but it is often posited that increased intracellular lipid accumulation is the underlying cause of steatosis. To study experimentally how HCV infection in human liver derived cells by different genotypes and subtypes might affect lipid accumulation, we performed detailed cytofluorimetric and microscopy analyses of intracellular lipid droplets (LDs) in relation to the viral Core and to cell endoplasmic reticulum proteins. Following culture infection with HCV genotype 1a, 2a, 2b, 2c, and 3a strains, we found variable levels of intracellular LDs accumulation, associated to the infecting strain rather than to the specific genotype. Although two genotype 3a strains showed high levels of lipid accumulation, as previously observed, some strains of other genotypes displayed a similar phenotype. Moreover, the analyses of LDs size, number, and shape indicated that the apparent increase in lipid accumulation is due to an increase in the overall number rather than in the size of droplets. Finally, differences in total lipid content across genotypes did not correlate to differences in Core distribution nor Core levels. In conclusion, our study provides a quantitative in-depth analysis of the effect of HCV infection on LDs accumulation in cell-culture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v13030389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999684PMC
February 2021

Mutations Identified in the Hepatitis C Virus (HCV) Polymerase of Patients with Chronic HCV Treated with Ribavirin Cause Resistance and Affect Viral Replication Fidelity.

Antimicrob Agents Chemother 2020 11 17;64(12). Epub 2020 Nov 17.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Ribavirin has been used for 25 years to treat patients with chronic hepatitis C virus (HCV) infection; however, its antiviral mechanism of action remains unclear. Here, we studied virus evolution in a subset of samples from a randomized 24-week trial of ribavirin monotherapy versus placebo in chronic HCV patients, as well as the viral resistance mechanisms of the observed ribavirin-associated mutations in cell culture. Thus, we performed next-generation sequencing of the full-length coding sequences of HCV recovered from patients at weeks 0, 12, 20, 32 and 40 and analyzed novel single nucleotide polymorphisms (SNPs), diversity, and mutation-linkage. At week 20, increased genetic diversity was observed in 5 ribavirin-treated compared to 4 placebo-treated HCV patients due to new synonymous SNPs, particularly G-to-A and C-to-U ribavirin-associated transitions. Moreover, emergence of 14 nonsynonymous SNPs in HCV nonstructural 5B (NS5B) occurred in treated patients, but not in placebo controls. Most substitutions located close to the NS5B polymerase nucleotide entry site. Linkage analysis showed that putative resistance mutations were found in the majority of genomes in ribavirin-treated patients. Identified NS5B mutations from genotype 3a patients were further introduced into the genotype 3a cell-culture-adapted DBN strain for studies in Huh7.5 cells. Specific NS5B substitutions, including DBN-D148N+I363V, DBN-A150V+I363V, and DBN-T227S+S183P, conferred resistance to ribavirin in long-term cell culture treatment, possibly by reducing the HCV polymerase error rate. In conclusion, prolonged exposure of HCV to ribavirin in chronic hepatitis C patients induces NS5B resistance mutations leading to increased polymerase fidelity, which could be one mechanism for ribavirin resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01417-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674049PMC
November 2020

Insights into the unique characteristics of hepatitis C virus genotype 3 revealed by development of a robust sub-genomic DBN3a replicon.

J Gen Virol 2020 11;101(11):1182-1190

School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

Hepatitis C virus (HCV) is an important human pathogen causing 400 000 chronic liver disease-related deaths annually. Until recently, the majority of laboratory-based investigations into the biology of HCV have focused on the genotype 2 isolate, JFH-1, involving replicons and infectious cell culture systems. However, genotype 2 is one of eight major genotypes of HCV and there is great sequence variation among these genotypes (>30 % nucleotide divergence). In this regard, genotype 3 is the second most common genotype and accounts for 30 % of global HCV cases. Further, genotype 3 is associated with both high levels of inherent resistance to direct-acting antiviral (DAA) therapy, and a more rapid progression to chronic liver diseases. Neither of these two attributes are fully understood, thus robust genotype 3 culture systems to unravel viral replication are required. Here we describe the generation of robust genotype 3 sub-genomic replicons (SGRs) based on the adapted HCV NS3-NS5B replicase from the DBN3a cell culture infectious clone. Such infectious cell culture-adaptive mutations could potentially promote the development of robust SGRs for other HCV strains and genotypes. The novel genotype 3 SGRs have been used both transiently and to establish stable SGR-harbouring cell lines. We show that these resources can be used to investigate aspects of genotype 3 biology, including NS5A function and DAA resistance. They will be useful tools for these studies, circumventing the need to work under the biosafety level 3 (BSL3) containment required in many countries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jgv.0.001486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879556PMC
November 2020

Cell Culture Studies of the Efficacy and Barrier to Resistance of Sofosbuvir-Velpatasvir and Glecaprevir-Pibrentasvir against Hepatitis C Virus Genotypes 2a, 2b, and 2c.

Antimicrob Agents Chemother 2020 02 21;64(3). Epub 2020 Feb 21.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Copenhagen, Denmark

The introduction of highly efficient therapies with direct-acting antivirals (DAA) for patients with chronic hepatitis C virus (HCV) infection offers exceptional opportunities to globally control this deadly disease. For achieving this ambitious goal, it is essential to prevent antiviral resistance against the most optimal first-line and retreatment DAA choices. We performed independent comparisons of the efficacy and barrier to resistance of pangenotypic DAA regimens for HCV genotype 2 infections, using previously and newly developed efficient cell culture-adapted strains of subtypes 2a, 2b, and 2c. With the applied experimental cell culture conditions, combination treatment with the sofosbuvir-velpatasvir or glecaprevir-pibrentasvir DAA regimen was efficient in eradicating HCV infections; in contrast, single-drug treatments frequently led to viral escape. Sequence analysis of drug targets from recovered viruses revealed known resistance-associated substitutions (RAS) emerging in the NS3 protease or NS5A after treatment failure. These RAS were genetically stable after viral passage, and viruses with these RAS exhibited significant phenotypic resistance. After sofosbuvir treatment failure, only a genotype 2a virus harbored NS5B RAS S282T and thus had decreased susceptibility to nucleotide analogs (nucs). However, in most cases, viral escape from sofosbuvir led to other NS5B substitutions but drug susceptibility was maintained, and in one case, no changes in NS5B were detected. For a genotype 2b virus, after treatment failure with sofosbuvir-velpatasvir, the efficacy of retreatment with glecaprevir-pibrentasvir was maintained due to the high barrier to resistance and low cross-resistance of pibrentasvir. Our findings suggest the slight superiority of glecaprevir-pibrentasvir against genotype 2b in culture, which could have potential therapeutic interest meriting more definitive investigations in the clinic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01888-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038289PMC
February 2020

Ribavirin inhibition of cell-culture infectious hepatitis C genotype 1-3 viruses is strain-dependent.

Virology 2020 01 28;540:132-140. Epub 2019 Sep 28.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark. Electronic address:

Ribavirin remains relevant for successful treatment of chronic hepatitis C virus (HCV) infections in low-income settings, as well as for therapy of difficult-to-treat HCV patients. We studied the effect of ribavirin against cell-culture adapted HCV of genotypes 1, 2 and 3, representing ~80% of global infections. TNcc(1a) was the most sensitive to ribavirin, while J6/JFH1(2a) was the most resistant. ECs ranged from 21 μM (95%CI: 20-22 μM) to 189 μM (95%CI: 173-207 μM). Substitutions at position 415 of NS5B resulted in little or no change to ribavirin sensitivity (0.7-0.9 fold) but conferred moderate drug resistance during extended treatment of genotype 1 (1.8-fold). NS5A and NS5B sequences could alter ribavirin sensitivity 2-4-fold, although their contribution was not simply additive. Finally, we detected limited accumulation of mutations associated with ribavirin treatment. Our findings show that the antiviral effect of ribavirin on HCV is strain-dependent and is influenced by the specific sequence of multiple HCV nonstructural proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2019.09.014DOI Listing
January 2020

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

Hepatology 2019 09 5;70(3):771-787. Epub 2019 Jun 5.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.30647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6772116PMC
September 2019

HCV genotype 1-6 NS3 residue 80 substitutions impact protease inhibitor activity and promote viral escape.

J Hepatol 2019 03 3;70(3):388-397. Epub 2018 Nov 3.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address:

Background & Aims: Protease inhibitors (PIs) are of central importance in the treatment of patients with chronic hepatitis C virus (HCV) infection. HCV NS3 protease (NS3P) position 80 displays polymorphisms associated with resistance to the PI simeprevir for HCV genotype 1a. We investigated the effects of position-80-substitutions on fitness and PI-resistance for HCV genotypes 1-6, and analyzed evolutionary mechanisms underlying viral escape mediated by pre-existing Q80K.

Methods: The fitness of infectious NS3P recombinants of HCV genotypes 1-6, with engineered position-80-substitutions, was studied by comparison of viral spread kinetics in Huh-7.5 cells in culture. Median effective concentration (EC50) and fold resistance for PIs simeprevir, asunaprevir, paritaprevir, grazoprevir, glecaprevir and voxilaprevir were determined in short-term treatment assays. Viral escape was studied by long-term treatment of genotype 1a recombinants with simeprevir, grazoprevir, glecaprevir and voxilaprevir and of genotype 3a recombinants with glecaprevir and voxilaprevir, next generation sequencing, NS3P substitution linkage and haplotype analysis.

Results: Among tested PIs, only glecaprevir and voxilaprevir showed pan-genotypic activity against the original genotype 1-6 culture viruses. Variants with position-80-substitutions were all viable, but fitness depended on the specific substitution and the HCV isolate. Q80K conferred resistance to simeprevir across genotypes but had only minor effects on the activity of the remaining PIs. For genotype 1a, pre-existing Q80K mediated accelerated escape from simeprevir, grazoprevir and to a lesser extent glecaprevir, but not voxilaprevir. For genotype 3a, Q80K mediated accelerated escape from glecaprevir and voxilaprevir. Escape was mediated by rapid and genotype-, PI- and PI-concentration-dependent co-selection of clinically relevant resistance associated substitutions.

Conclusions: Position-80-substitutions had relatively low fitness cost and the potential to promote HCV escape from clinically relevant PIs in vitro, despite having a minor impact on results in classical short-term resistance assays.

Lay Summary: Among all clinically relevant hepatitis C virus protease inhibitors, voxilaprevir and glecaprevir showed the highest and most uniform activity against cell culture infectious hepatitis C virus with genotype 1-6 proteases. Naturally occurring amino acid changes at protease position 80 had low fitness cost and influenced sensitivity to simeprevir, but not to other protease inhibitors in short-term treatment assays. Nevertheless, the pre-existing change Q80K had the potential to promote viral escape from protease inhibitors during long-term treatment by rapid co-selection of additional resistance changes, detected by next generation sequencing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jhep.2018.10.031DOI Listing
March 2019

Current status and future development of infectious cell-culture models for the major genotypes of hepatitis C virus: Essential tools in testing of antivirals and emerging vaccine strategies.

Antiviral Res 2018 10 27;158:264-287. Epub 2018 Jul 27.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address:

In this review, we summarize the relevant scientific advances that led to the development of infectious cell culture systems for hepatitis C virus (HCV) with the corresponding challenges and successes. We also provide an overview of how these systems have contributed to the study of antiviral compounds and their relevance for the development of a much-needed vaccine against this major human pathogen. An efficient infectious system to study HCV in vitro, using human hepatoma derived cells, has only been available since 2005, and was limited to a single isolate, named JFH1, until 2012. Successive developments have been slow and cumbersome, as each available system has been the result of a systematic effort for discovering adaptive mutations conferring culture replication and propagation to patient consensus clones that are inherently non-viable in vitro. High genetic heterogeneity is a paramount characteristic of this virus, and as such, it should preferably be reflected in basic, translational, and clinical studies. The limited number of efficient viral culture systems, in the context of the vast genetic diversity of HCV, continues to represent a major hindrance for the study of this virus, posing a significant barrier towards studies of antivirals (particularly of resistance) and for advancing vaccine development. Intensive research efforts, driven by isolate-specific culture adaptation, have only led to efficient full-length infectious culture systems for a few strains of HCV genotypes 1, 2, 3, and 6. Hence research aimed at identifying novel strategies that will permit universal culture of HCV will be needed to further our understanding of this unique virus causing 400 thousand deaths annually.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.antiviral.2018.07.014DOI Listing
October 2018

Recombinant hepatitis C virus genotype 5a infectious cell culture systems expressing minimal JFH1 NS5B sequences permit polymerase inhibitor studies.

Virology 2018 09 20;522:177-192. Epub 2018 Jul 20.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address:

The six major epidemiologically important hepatitis C virus (HCV) genotypes differ in global distribution and antiviral responses. Full-length infectious cell-culture adapted clones, the gold standard for HCV studies in vitro, are missing for genotypes 4 and 5. To address this challenge for genotype 5, we constructed a consensus full-length clone of strain SA13 (SA13fl), which was found non-viable in Huh7.5 cells. Step-wise adaptation of SA13fl-based recombinants, beginning with a virus encoding the NS5B-thumb domain and 3´UTR of JFH1 (SA13/JF372-X), resulted in a high-titer SA13 virus with only 41 JFH1-encoded NS5B-thumb residues (SA13/JF470-510cc); this required sixteen cell-culture adaptive substitutions within the SA13fl polyprotein and two 3´UTR-changes. SA13/JF372-X and SA13/JF470-510cc were equally sensitive to nucleoside polymerase inhibitors, including sofosbuvir, but showed differential sensitivity to inhibitors targeting the NS5B palm or thumb. SA13/JF470-510cc represents a model to elucidate the influence of HCV RNA elements on viral replication and map determinants of sensitivity to polymerase inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2018.05.020DOI Listing
September 2018

Ribavirin-induced mutagenesis across the complete open reading frame of hepatitis C virus genotypes 1a and 3a.

J Gen Virol 2018 08 21;99(8):1066-1077. Epub 2018 Jun 21.

1​Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Ribavirin (RBV) has been used for the last 20 years to treat patients with chronic hepatitis C virus (HCV) infection. This pluripotent drug is believed to induce mutagenesis in HCV RNA. However, for cell-cultured HCV (HCVcc) this phenomenon has only been investigated in genotype 2a recombinants. Here we studied the mutations that developed in HCVcc of genotypes 1a and 3a treated with RBV or ribavirin triphosphate (RBV-TP) compared to non-treated controls. Analysis was performed on the amplified full-length open reading frame (ORF) of recovered viruses following next-generation sequencing and clonal analyses. Compared to non-treated controls, the spread of TNcc(1a) and DBN3acc(3a) HCVcc was delayed by RBV and RBV-TP at concentrations of 40 µM or higher. The delay in HCVcc spread was associated with increased new single-nucleotide polymorphisms (SNP). Significantly higher numbers of new SNP were observed in TNcc(1a) viruses treated with RBV or RBV-TP compared to matched non-treated controls. RBV or RBV-TP treatment led to significantly increased proportions of new G-to-A and C-to-U SNP compared to non-treated TNcc(1a). Clonal analyses confirmed a significantly increased mutation rate in RBV-treated TNcc(1a). Synonymous pairwise distances increased in both viruses across the complete ORF under RBV and RBV-TP treatment compared to controls. Consensus-shifts in single samples of RBV- or RBV-TP-treated TNcc(1a) viruses occurred in proteins E1, p7, NS3 and NS4B. No non-synonymous consensus changes were observed in DBN3acc(3a). This study supports a biased G-to-A and C-to-U mutagenic effect of RBV and RBV-TP throughout the entire ORF of HCV genotypes 1a and 3a.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jgv.0.001095DOI Listing
August 2018

HCV Genotype 6a Escape From and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models.

Gastroenterology 2018 06 15;154(8):2194-2208.e12. Epub 2018 Feb 15.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Background & Aims: Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance.

Methods: The consensus sequences of strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants. We studied the efficacy of NS5A and NS5B inhibitors in concentration-response assays. We examined the effects of long-term culture of Huh7.5 cells incubated with velpatasvir and sofosbuvir singly or combined following infection with passaged full-length HK2 or HK6a recombinant viruses. Resistance-associated substitutions (RAS) were identified by Sanger and next-generation sequencing, and their effects on viral fitness and in drug susceptibility were determined in reverse-genetic experiments.

Results: Adapted full-length HCV genotype 6a recombinants HK2cc and HK6acc had fast propagation kinetics and high infectivity titers. Compared with an HCV genotype 1a recombinant, HCV genotype 6a recombinants of strains HK2 and HK6a were equally sensitive to daclatasvir, elbasvir, velpatasvir, pibrentasvir, and sofosbuvir, but less sensitive to ledipasvir, ombitasvir, and dasabuvir. Long-term exposure of HCV genotype 6a-infected Huh7.5 cells with a combination of velpatasvir and sofosbuvir resulted in clearance of the virus, but the virus escaped the effects of single inhibitors via emergence of the RAS L31V in NS5A (conferring resistance to velpatasvir) and S282T in NS5B (conferring resistance to sofosbuvir). Engineered recombinant genotype 6a viruses with single RAS mediated resistance to velpatasvir or sofosbuvir. HCV genotype 6a viruses with RAS NS5A-L31V or NS5B-S282T were however, able to propagate and escape in Huh7.5 cells exposed to the combination of velpatasvir and sofosbuvir. Further, HCV genotype 6a with NS5A-L31V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor.

Conclusions: Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle for this important genotype. The combination of velpatasvir and sofosbuvir is required to block propagation of original HCV genotype 6a, which quickly becomes resistant to single inhibitors via the rapid emergence and persistence of RAS. These features of HCV genotype 6a could compromise treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2018.02.017DOI Listing
June 2018

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

Gastroenterology 2018 04 22;154(5):1435-1448. Epub 2017 Dec 22.

Copenhagen Hepatitis C Program, Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address:

Background & Aims: 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.

Methods: 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 or without RAS. We engineered HCV variants that included RAS identified in escape experiments, using recombinants with or without T/Y93H and daclatasvir, or that contained RAS previously reported from patients.

Results: NS5A inhibitors had varying levels of efficacy against original and resistant 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 had no apparent resistance to pibrentasvir, and HCV with RAS at 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. For the remaining NS5A inhibitors tested, RAS at amino acids 28 and 93 led to high levels of resistance. Among these inhibitors, velpatasvir was more effective against variants with RAS at amino acid 30 and some variants with RAS at amino acid 31 than the other agents. Variants with the pre-existing RAS T/Y93H acquired additional NS5A changes during escape experiments, resulting in HCV variants with specific combinations of RAS, showing high fitness and high resistance.

Conclusions: 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2017.12.015DOI Listing
April 2018

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

Antimicrob Agents Chemother 2017 06 24;61(6). Epub 2017 May 24.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

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 direct-acting 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 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.00037-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444172PMC
June 2017

Robust HCV Genotype 3a Infectious Cell Culture System Permits Identification of Escape Variants With Resistance to Sofosbuvir.

Gastroenterology 2016 11 22;151(5):973-985.e2. Epub 2016 Jul 22.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Background & Aims: Direct-acting antivirals (DAAs) effectively eradicate chronic hepatitis C virus (HCV) infection, although HCV genotype 3a is less responsive to these drugs. We aimed to develop genotype 3a infectious cultures and study the effects of inhibitors of NS5A and NS5B and resistance to sofosbuvir-the only nucleotide analog approved for treatment of chronic HCV infection.

Methods: The developed HCV genotype 3a full-length genome (DBN3a), with a strain-DBN coding sequence, modified NS5B consensus sequence, pS52 untranslated regions, and coding mutations from a culture-efficient JFH1-based core-NS5A (DBN) recombinant, was transfected into Huh7.5 cells. The efficacy of selected DAAs was determined in dose-response assays, in which the number of HCV-infected cells was measured after incubation with different concentrations of the specific DAA. Long-term culture of infected Huh7.5 cells with increasing concentrations of sofosbuvir was used to promote selection of HCV-resistant variants.

Results: We engineered a DBN3a variant with 17 substitutions (DBN3a) that had replication and propagation kinetics in Huh7.5 cells comparable with prototype J6/JFH1. The adaptive mutations also produced culture-efficient DBN-based recombinants with NS5B from HCV genotype 3a strains S52 and DH11. Compared with genotype 1a, genotype 3a was less sensitive to daclatasvir, ledipasvir, and elbasvir, but equally sensitive to ombitasvir, velpatasvir, beclabuvir, dasabuvir, MK-3682, and sofosbuvir. Exposure of Huh7.5 cells infected with DBN3a to sofosbuvir led to identification of an escape variant with substitutions in NS5B, including the resistance-associated substitution S282T. This variant showed increased infectivity of Huh7.5 cells, compared with DBN3a, and was genetically stable in cell cultures without sofosbuvir. Sofosbuvir, MK-3682, dasabuvir, or combinations of sofosbuvir and ledipasvir or sofosbuvir and velpatasvir had decreased efficacy against infection with the DBN3a sofosbuvir escape variant.

Conclusions: We developed a system for highly efficient culture of HCV genotype 3a. Genotype 1a has a high genetic barrier to resistance for sofosbuvir, whereas resistance to this DAA can be induced in genotype 3a. We therefore isolated HCV genotype 3a variants with reduced sensitivity to sofosbuvir, with increased fitness and with cross-resistance to other NS5B inhibitors. These findings indicate that sofosbuvir escape variants could compromise the effectiveness of nucleotide analogs against HCV. GenBank accession numbers: KX280712-KX280716.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2016.07.013DOI Listing
November 2016

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.

Antimicrob Agents Chemother 2016 06 23;60(6):3563-78. Epub 2016 May 23.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.02929-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879388PMC
June 2016

Quasispecies dynamics in hepatitis C liver transplant recipients receiving grafts from hepatitis C virus infected donors.

J Gen Virol 2015 Dec;96(12):3493-3498

Liver Unit, Lab. Malalties Hepàtiques, Vall d'Hebron Institut de Recerca, Hospital Vall d'Hebron, CIBERehd, Universitat Autònoma de Barcelona, Barcelona, Spain.

The allocation of liver grafts from hepatitis C virus (HCV)-positive donors in HCV-infected liver transplant (LT) recipients leads to infection with two different viral populations. In a previous study, we examined quasispecies dynamics during reinfection by clonal sequencing, which did not allow an accurate characterization of coexistence and competition events. To overcome this limitation, here we used deep-sequencing analysis of a fragment of the HCV NS5B gene in six HCV-infected LT recipients who received HCV-infected grafts. Successive expansions and contractions of quasispecies complexity were observed, evolving in all cases towards a more homogeneous population. The population that became dominant was the one displaying the highest mutant spectrum complexity. In four patients, coexistence of minority mutants, derived from the donor or the recipient, were detected. In conclusion, our study shows that, during reinfection with a different HCV strain in LT recipients, the viral population with the highest diversity always becomes dominant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jgv.0.000289DOI Listing
December 2015

Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance.

Antimicrob Agents Chemother 2015 Dec 21;59(12):7426-36. Epub 2015 Sep 21.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Various protease inhibitors (PIs) currently are becoming available for treatment of hepatitis C virus (HCV). For genotype 1, substitutions at NS3 protease positions 155, 156, and 168 are the main determinants of PI resistance. For other genotypes, similar substitutions were selected during PI treatment but were not characterized systematically. To elucidate the impact of key PI resistance substitutions on genotypes 2 to 6, we engineered the substitutions R155A/E/G/H/K/Q/T, A156G/S/T/V, and D/Q168A/E/G/H/N/V into HCV recombinants expressing genotype 2 to 6 proteases. We evaluated viral fitness and sensitivity to nine PIs (telaprevir, boceprevir, simeprevir, asunaprevir, vaniprevir, faldaprevir, paritaprevir, deldeprevir, and grazoprevir) in Huh7.5 cells. We found that most variants showed decreased fitness compared to that of the original viruses. Overall, R155K, A156G/S, and D/Q168A/E/H/N/V variants showed the highest fitness; however, genotype 4 position 168 variants showed strong fitness impairment. Most variants tested were resistant to several PIs. Resistance levels varied significantly depending on the specific substitution, genotype, and PI. For telaprevir and boceprevir, specific 155 and 156, but not 168, variants proved resistant. For the remaining PIs, most genotype 2, 4, 5, and 6, but not genotype 3, variants showed various resistance levels. Overall, grazoprevir (MK-5172) had the highest efficacy against original viruses and variants. This is the first comprehensive study revealing the impact of described key PI resistance substitutions on fitness and PI resistance of HCV genotypes 2 to 6. In conclusion, the studied substitutions induced resistance to a panel of clinically relevant PIs, including the newer PIs paritaprevir, deldeprevir, and grazoprevir. We discovered complex patterns of resistance, with the impact of substitutions varying from increased sensitivity to high resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01953-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649233PMC
December 2015

Nationwide experience of treatment with protease inhibitors in chronic hepatitis C patients in Denmark: identification of viral resistance mutations.

PLoS One 2014 1;9(12):e113034. Epub 2014 Dec 1.

Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark; Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Background And Aims: The first standard of care in treatment of chronic HCV genotype 1 infection involving directly acting antivirals was protease inhibitors telaprevir or boceprevir combined with pegylated-interferon and ribavirin (triple therapy). Phase III studies include highly selected patients. Thus, treatment response and development of viral resistance during triple therapy in a routine clinical setting needs to be determined. The aims of this study were to investigate treatment outcome and identify sequence variations after triple therapy in patients with chronic HCV genotype 1 infection in a routine clinical setting.

Methods: 80 patients, who initiated and completed triple therapy in Denmark between May 2011 and November 2012, were included. Demographic data and treatment response were obtained from the Danish Database for Hepatitis B and C. Direct sequencing and clonal analysis of the RT-PCR amplified NS3 protease were performed in patients without cure following triple therapy.

Results: 38 (47%) of the patients achieved cure, 15 (19%) discontinued treatment due to adverse events and remained infected, and 27 (34%) experienced relapse or treatment failure of whom 15 of 21 analyzed patients had well-described protease inhibitor resistance variants detected. Most frequently detected protease variants were V36M and/or R155K, and V36M, in patients with genotype 1a and 1b infection, respectively.

Conclusions: The cure rate after triple therapy in a routine clinical setting was 47%, which is substantially lower than in clinical trials. Resistance variants towards protease inhibitors were seen in 71% of patients failing therapy indicating that resistance could have an important role in treatment response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113034PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249835PMC
September 2015

Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77.

J Virol 2015 Jan 29;89(1):811-23. Epub 2014 Oct 29.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Unlabelled: The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10(4.0) focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively.

Importance: Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02877-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301150PMC
January 2015

Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors.

Gastroenterology 2014 Mar 18;146(3):812-821.e4. Epub 2013 Nov 18.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Background & Aims: Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5' untranslated region-NS5A (5-5A) from genotypes 1-6 and 2a(JFH1) NS5B-3' untranslated region, and tested the effects of NS3 protease and NS5A inhibitors on these recombinants.

Methods: The HCV 5-5A recombinants with previously identified mutations in the NS3-helicase (F1464L), NS4A (A1672S), and NS5B (D2979G) were adapted and improved, by incorporating additional recovered mutations that increased their propagation in Huh7.5 cells. Concentration-response profiles were determined for each DAA agent in replicate infected Huh7.5 cells.

Results: Developed efficient 1a(H77), 1a(TN), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A recombinants did not require mutations after viral passage in the NS3 protease or NS5A domain-I regions targeted by the drugs. They were inhibited in a concentration-dependent manner by the NS3 protease inhibitors telaprevir, boceprevir, asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK-5172 and by the NS5A inhibitor daclatasvir. The 1a(TN) 5-5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5-5A recombinants as surrogates for full-length viruses in DAA testing. Compared with the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all protease inhibitors, and the 4a(ED43) recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared with other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency observed, but with genotype-dependent activity.

Conclusions: The mutations F1464L, A1672S, and D2979G permitted the development of efficient HCV recombinants comprising genotype-specific 5' untranslated region-NS5A (5-5A), which include the natural NS3 protease and NS5A domain-I drug targets. The robust replication of adapted 5-5A recombinants allowed for direct comparison of NS3 protease and NS5A inhibitors against HCV strains of genotypes 1-6.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2013.11.009DOI Listing
March 2014

Hypervariable region 1 deletion and required adaptive envelope mutations confer decreased dependency on scavenger receptor class B type I and low-density lipoprotein receptor for hepatitis C virus.

J Virol 2014 Feb 20;88(3):1725-39. Epub 2013 Nov 20.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Hypervariable region 1 (HVR1) of envelope protein 2 (E2) of hepatitis C virus (HCV) serves important yet undefined roles in the viral life cycle. We previously showed that the viability of HVR1-deleted JFH1-based recombinants with Core-NS2 of H77 (H77(ΔHVR1), genotype 1a) and S52 (S52(ΔHVR1), genotype 3a) in Huh7.5 cells was rescued by E2 substitutions N476D/S733F and an E1 substitution, A369V, respectively; HVR1-deleted J6 (J6(ΔHVR1), genotype 2a) was fully viable. In single-cycle production assays, where HCV RNA was transfected into entry-deficient Huh7-derived S29 cells with low CD81 expression, we found no effect of HVR1 deletion on replication or particle release for H77 and S52. HCV pseudoparticle assays in Huh7.5 cells showed that HVR1 deletion decreased entry by 20- to 100-fold for H77, J6, and S52; N476D/S733F restored entry for H77(ΔHVR1), while A369V further impaired S52(ΔHVR1) entry. We investigated receptor usage by antibody blocking and receptor silencing in Huh7.5 cells, followed by inoculation of parental and HVR1-deleted HCV recombinants. Compared to parental viruses, scavenger receptor class B type I (SR-BI) dependency was decreased for H77(ΔHVR1/N476D/S733F), H77(N476D/S733F), S52(ΔHVR1/A369V), and S52(A369V), but not for J6(ΔHVR1). Low-density lipoprotein receptor (LDLr) dependency was decreased for HVR1-deleted viruses, but not for H77(N476D/S733F) and S52(A369V). Soluble LDLr neutralization revealed strong inhibition of parental HCV but limited effect against HVR1-deleted viruses. Apolipoprotein E (ApoE)-specific HCV neutralization was similar for H77, J6, and S52 viruses with and without HVR1. In conclusion, HVR1 and HVR1-related adaptive envelope mutations appeared to be involved in LDLr and SR-BI dependency, respectively. Also, LDLr served ApoE-independent but HVR1-dependent functions in HCV entry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02017-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911595PMC
February 2014

Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors.

Hepatology 2014 Feb 13;59(2):395-407. Epub 2013 Dec 13.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Unlabelled: Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only exist for genotypes 1a and 2a. We developed efficient culture systems for the epidemiologically important genotype 2b. Full-length molecular clones of patient strains DH8 and DH10 were adapted to efficient growth in Huh7.5 cells by using F1468L/A1676S/D3001G (LSG) mutations. The previously developed J8cc prototype 2b recombinant was further adapted. DH8 and J8 achieved infectivity titers >4.5 log10 Focus-Forming Units/mL. A defined set of DH8 mutations had cross-isolate adapting potential. A chimeric genome with the DH10 polyprotein coding sequence inserted into a vector with J8 untranslated regions was viable. Importantly, we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct-acting antivirals. Nonstructural protein (NS)5B inhibitors sofosbuvir, mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX-222 and filibuvir only inhibited 1a. Genotype 2b strains were least sensitive to seven lead protease inhibitors, including MK-5172 with high overall potency. NS5A inhibitor daclatasvir was exceptionally potent, but efficacy was affected by the HCV strain.

Conclusion: Highly efficient HCV full-length 2b culture systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors sofosbuvir, mericitabine, and BI207127, show cross-activity against full-length 1a, 2a, and 2b viruses, but important sensitivity differences exist at the isolate level. Infectious cultures for different HCV strains will advance studies on viral biology and pathogenesis and promote individualized patient treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.26660DOI Listing
February 2014
-->