Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions.

Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). The first-wave of approved NS3 protease inhibitors (PIs) were Telaprevir and Boceprevir, which are currently discontinued. Simeprevir is a second-wave PI incorporated into the Brazilian hepatitis C treatment protocol. Drug resistance plays a key role in patients' treatment regimen. Here, we developed a simple phenotypic assay to evaluate the impact of resistance mutations in HCV NS3 protease to PIs, using a protein expression vector containing wild type NS3 protease domain and NS4A co-factor. We analyzed the impact of five resistance mutations (T54A, V36M, V158I, V170I and T54S+V170I) against Telaprevir, Boceprevir and Simeprevir. Protein purifications were performed with low cost methodology, and enzymatic inhibition assays were measured by FRET. We obtained recombinant proteases with detectable activity, and IC50 and fold change values for the evaluated PIs were determined. The variant T54A showed the highest reduction of susceptibility for the PIs, while the other four variants exhibited lower levels of reduced susceptibility. Interestingly, V170I showed 3.2-fold change for Simeprevir, a new evidence about this variant. These results emphasize the importance of enzymatic assays in phenotypic tests to determine which therapeutic regimen should be implemented.

Hepatitis C Virus-Infected Responders and Relapsers to Treatment Show Similar Genetic Profiles of IL28B and IL10 Single Nucleotide Polymorphisms.

Genotype 1 of hepatitis C virus (HCV) is the most prevalent worldwide. Pegylated-interferon and ribavirin therapy is still used in the developing world but has less efficiency in this genotype. Single nucleotide polymorphisms (SNPs) rs12979860 and rs8099917 (IL28B) and rs1800896, rs1800871, and rs1800872 (IL10) are related to treatment outcome, but previous studies clustered nonresponse and relapse patients. The aim of this study is to analyze the frequency of those SNPs in HCV genotype 1 for response, nonresponse, or relapse. Patients were classified according to treatment outcome. Genomic DNA was extracted by blood samples and SNPs were defined by PCR and sequencing. Data analysis was performed with R project. The frequency of rs12979860 CC was similar among responders (0.48) and relapsers (0.46) and lower among nonresponders (0.18). The same trend was observed for rs8099917 TT. rs12979860 CC showed a protective effect for relapsers compared to nonresponders (OR = 0.25) as it occurs with responders (OR = 0.17). Haplotypes 12979860/C rs8099917/T were associated with protection against the nonresponder phenotype compared to responders (OR = 0.27) or relapsers (OR = 0.37). Frequency of rs12979860 and rs8099917 is different between relapsers and nonresponders, but similar between relapsers and responders.