PCR-BasedIn VitroSynthesis of Hepatitis C Virus NS3 Protease for Rapid Phenotypic Resistance Testing of Protease Inhibitors

Abstract

bProtease inhibitors (PIs) targeting the hepatitis C virus (HCV) NS3 protease, such as telaprevir, have significantly improved the sustained virologic response (SVR) rates of HCV genotype 1 antiviral therapy. Given the expanding antiviral therapy regimen, fast HCV PI resistance assays are urgently needed. In this view, we have developed a novel phenotypic resistance test for HCV PIs based on in vitro synthesis of patient-derived HCV NS3 protease and subsequent enzymatic testing in a fluorescent readout. The enzymatically active HCV NS3 proteases were synthesized from PCR-derived templates by an Escherichia coli S30 extract system. Tests of the protease genes with known mutations for telaprevir resistance showed that the phenotypic resistance test was fast, with a total turnaround time of <10 h, and was fully in agreement with the previous resistance results. The initial tests with 38 treatment-naive serum samples showed that the method was significantly less laborious and faster than currently available phenotypic resistance assays of HCV NS3 PIs. Chronic infection with the hepatitis C virus (HCV) affects an estimated 160 million individuals worldwide (1) and leads to severe liver diseases, such as fibrosis, cirrhosis, and hepatocellular carcinoma (2). Since the early 2000s, pegylated interferon (PEG-IFN) and ribavirin (RBV) have been used in the standard of care (SOC) for treatment of chronic hepatitis C and result in a sustained virologic response (SVR) for 80% of patients infected with HCV genotype 2 or 3. However, in patients infected with genotype 1, SVR rates with the SOC reach only 42 to 46% (3, 4). In 2011, two protease inhibitors (PIs), boceprevir and telaprevir, were approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency as the first two direct-acting antivirals (DAAs) for the treatment of patients infected with chronic HCV genotype 1. Clinical studies showed that these PIs improved the SVR rates to HCV genotype 1 in treatment-naive and previously treated patients when compared to the standard dual-treatment regimen (5-8). Thus, the current SOC recommended for HCV genotype 1 infection is a triple therapy combining PEG-IFN, ribavirin, and a protease inhibitor, either boceprevir or telaprevir (9). Because of the narrow spectrums of activity and the side effects of the two approved PIs, second-generation PIs with broad genotypic coverage and a high genetic barrier for resistance are being developed actively (10). Given the expected expanding antiviral therapy regimen using the PIs, fast HCV PI resistance assays are urgently needed.The current methods used for testing the PI resistance of HCV are normally genotypic assays based on determining the individual mutation pattern of a patient's virus population. Genotypic methods, such as population-sequencing methods (11, 12), clonal sequencing (13), the TaqMan mismatch amplification mutation assay (TaqMAMA) (14), and ultradeep pyrosequencing (15, 16), have been developed. However, due to the high replication rate of HCV and i...

Virologic Tools for HCV Drug Resistance Testing

“…Enzyme assays are laborious and time-consuming. A new, more accessible assay has been developed, based on a coupled in vitro transcription/translation system, with a total turnaround time of less than 10 h [ 46 ].…”