The study of the resistance of hepatitis viruses to direct-acting antivirals (DAA) is carried out in close collaboration with the ANRS through the National Observatory of HCV Resistance to DAAs backed by the national cohort HEPATHER (ANRS CO22), as well as the medical virology group of Coordinated Action 33 (AC33), both coordinated by Professors Jean-Michel Pawlotsky and Stéphane Chevaliez.
Several novel therapeutic options are now available for the treatment of chronic hepatitis C patients. In 2014-2015, France benefitted from compassionate access to sofosbuvir, simeprevir, and daclatasvir in a Temporary Use Authorization (TUA) cohort. Today, numerous molecules (sofosbuvir, simeprevir, daclatasvir, ledipasvir-sofosbuvir, paritaprevir/ritonavir, and ombitasvir, with or without dasabuvir, grazoprevir-elbasvir, and sofosbuvir-velpatasvir) have obtained marketing authorization. The efficacy of DAAs is high, with cure rates of greater than 90%, characterized by undetectable viral RNA 12 weeks after discontinuation of treatment. Most treatment failures are relapses, defined by the reappearance of detectable viral RNA in cured patients following cessation of treatment. Such relapses occur mostly during the first few weeks after the discontinuation of treatment. Analysis of the viral populations of patients experiencing treatment failure, by direct sequencing (“population sequencing”) or more sensitive next generation sequencing (UDPS) methods, show that most patients host viruses resistant to one or more of the administered DAAs. Resistance probably plays an important role in the non-response to treatment, but this needs to be evaluated.
The Primary Objectives of this national observatory are to measure the incidence of HCV resistance to DAAs and characterize resistance profiles in patients enrolled in the French cohort HEPATHER who experience treatment failure.
Secondary Objectives :
To date, nearly 6,000 patients have started treatment with at least one DAA for at least 12 weeks. Amongst them, 358 (6.5%) experienced treatment failure. Direct sequencing of the three regions targeted by antivirals show that most of the patients in treatment failure harbored resistant variants of one or more amino-acid substitutions conferring reduced sensitivity to one or more DAAs (preliminary results).
HCV Platform
Models of subgenomic replicons have been developed within the NRC.
The replicon system is based on a bicistronic chimeric construct engineered to have a domain consisting of the 5’ non-coding end of HCV, and an HCV internal ribosome entry site (IRES), the resistance gene to neomycin, and a luciferase-based reporter gene and a second domain consisting of the IRES of the EMCV (encephalomyocarditis virus), which directs the translation of the non-structural proteins of HCV. The construct ends with the 3’ non-coding end of the viral genome. The subgenomic replicons lack the sequences encoding the structural proteins p7 and NS2, which are not necessary for their replication. Candidate amino-acid substitutions are introduced by site-directed mutagenesis on one (or more) plasmid(s) and the phenotypic analysis is performed on transient replicons (replicons of subtype 1b or 2a) or “stabilized” replicons (replicons of genotype 3, 4 or 5) in the Huh-7.5 human hepatoma line. After site-directed mutagenesis and linearization of the plasmid(s), the product is translated in vitro via RNA polymerase T7. The RNA of the subgenomic replicon also generated is transfected into the Huh-7.5 human hepatoma line. After stabilization (for genotypes 3, 4, or 5), phenotypic tests are performed in the presence of various concentrations of the antivirals studied. Inhibition of viral replication is evaluated by measuring the activity of luciferase in the presence of various antivirals. The level of resistance of the mutants is measured by determining the effective concentration 50 (EC50) and 90 (EC90), which correspond to the antiviral concentrations which inhibit viral replication by 50% and 90%, respectively.
HBV Platform
We have developed and implemented a phenotyping platform for the characterization of HBV resistance to nucleos(t)ide analogs. It consists of a cellular model of HBV replication and infection, allowing characterization of the resistance conferred by the amino-acid substitutions observed in patients, as well as determination of the replicative capacity of resistant variants. The infectious model is based on transfection of a plasmid encoding the HBV genome (pCH-9/3091 of 3182pb) of genotype D, subtype A, under the control of a promoter, into Huh7 cells, a human hepatoma line (Nassal et al, 1992). The transfection is performed in a P3 laboratory; 120 hours later, the supernatant is recovered, filtered, and digested with DNAse to remove the residual plasmid. HBV DNA is then extracted and viral replication measured using a specific HBV qPCR.
Amino-acid substitutions observed in patients are inserted by site-directed mutagenesis and the plasmids are transfected using the same technique. Phenotypic tests are performed in the presence of various concentrations of nucleos(t)id analogs. Inhibition of viral replication is evaluated by qPCR measurement of the amount of HBV DAN produced in the cell-culture supernatant in the presence of various antivirals. The level of resistance of the mutants is measured by determining the effective concentration 50 (EC50) and 90 (EC90), which correspond to the antiviral concentrations which inhibit viral replication by 50% and 90%, respectively.
