Evaluation of Resistance Associated Mutations and Host Prognostic Factors Determining Treatment Response in HCV Infected Pakistani Patients

Abstract

Hepatitis C, caused by the Hepatitis C virus (HCV), is a global health concern owing to its consequential effect on morbidity and mortality. The prevalence of this infection in Pakistan is highest, with approximately 10 million cases reported annually. The undefined nature of the current treatment makes it very difficult to pharmacologically characterize combination strategies, especially in the treatment of a significant proportion of patients that exhibit drug resistance and therapeutic failure. The evolution pace of HCV is quite high which may lead to failure of new direct-acting antiviral treatment. Previously, Peg IFN/RBV therapy was used to clear viremia but recent studies have reported that only few patients can respond to Peg IFN/RBV therapy which works by activating Natural Killer (NK) cells. Association of Killer Immunoglobulin Receptor present on NK cells and their ligand HLA C expressed on target cells play a significant role in viral clearance and genotype associated with KIR and HLA can affect the treatment response in Peg IFN therapy. The aim of the study is to undermine viral mutations in genotype 3a that are conferring resistance to Direct-Acting Antivirals (DAAs) and to identify prognostic host factors that can help determine if patient will be able to clear viremia or not. A total of 499 sequences were retrieved from 499 complete genomes of HCV genotype 3a available at Virus pathogen resource database. The consensus sequence obtained from 499 sequences was mutated with Resistance Associated Substitutions reported in literature. The 3D structures for wild type and mutant (containing RASs) NS5A and NS5B were generated, refined and simulated at 10 to determine the effect of RASs on catalytic domains of NS5A and NS5B. Afterwards, comparative docking was performed of wild type and mutant NS5A and NS5B with Daclatasvir and Sofosbuvir, respectively. It was revealed that RASs disrupted thand Sofosbuvir and may eventually lead to failure of treatment considering the significantly high mutation rate of HCV. The available treatment option Peg IFN/RBV therapy was found to be 60-70% effective but causes side effects in people who do not have activating KIRs that activate NK cells. So, to determine host prognostic factors such as activating and inhibitory KIR and their HLA ligands that can be used to determine treatment response before therapy, 82 samples from each Karachi and Gujrat were selected that were later categorized as responders or non-responders based on follow up data. The frequency of KIR and HLA genes in each population was determined and their association with treatment response was determined via chi-square and an algorithm as well as application was designed for clinician. Our analysis revealed that non-responders had more frequent inhibitory KIRs (i.e. KIR2DL1 and KIR2DL2) while more activating KIRs were present in responders that were able to clear viremia. Based on follow up data, 65% patients showed association of KIR and HLA genes with treatment response. The error rate was found to be 35% which is probably because of diet, lifestyle, and other factors. The pace at which the virus is mutating is making virus difficult to treat and can in turn lead to failure of therapy. Conversely, the application developed based on these results can help in determining treatment response in treatment naïve patients for existing Peg IFN/RBV therapy.e catalytic pockets of NS5A and NS5B which lead to lesser binding affinity of Daclatasvir