Study of resistance-associated substitutions in chronic HCV patients under direct-acting antivirals therapeutic regimens

Abstract

Hepatitis C virus (HCV) is a deadly virus infecting 4% of the global population through chronic hepatitis C infection of the liver. Pakistan has the second‐largest HCV burden in the world. Hepatitis C treatment has been transformed by the development of direct‐acting antivirals (DAAs). HCV replication cycle is inhibited by these drugs mainly interfering with the activity of nonstructural proteins of HCV. Three DAA classes (inhibitors of the NS3/NS4A protease, inhibitors of the NS5A complex, and inhibitors of the NS5B polymerase) have been developed and approved. However, the rapid advancement of HCV treatment presents a great challenge to clinicians in optimizing therapy in terms of efficacy and safety profile, drug combinations and durations to individual patients considering comorbidities, degree of fibrosis, adherence, and antiviral resistance. The first part of the thesis deals with the clinical effectiveness in terms of sustained virological response (SVR), predictors of SVR, and safety of available second-generation generic DAAs in Pakistani chronic HCV patients (n = 993) of genotype 3 (GT3) in 2015 to 2019. Combinations of two drugs from three classes achieved high (>90%) SVR rates and were well tolerated against treatment naïve chronic HCV patients as compared to cirrhotic and previously treated patients. Patients’ education status, treatment arm, viral load, and alanine aminotransferase (ALT) had a statistically significant association with SVR at 12 weeks. The chance of achieving a cure was the same whether a person receive sofosbuvir (SOF)

Abstract xvii + daclatasvir (DCV) or SOF/velpatasvir (VEL) in chronic HCV patients of GT3 (n = 1500) performed in 2019-2020. Adding ribavirin (RBV) and extending the duration from 12 weeks to 24 weeks enhanced the SVR rates in cirrhotic patients. SOF/VEL were more effective and tolerable with fewer adverse events in chronic and cirrhotic patients than SOF+ DCV. However, the significant predictors of non SVR were old age and cirrhosis. Skin rashes and oral ulcers were the major side effects reported in patients receiving SOF-based combinations. The second part of the thesis deals with the emergence and persistence of clinically relevant resistance-associated substitutions (RASs) in HCV NS5A and NS5B genes at baseline and after treatment for understanding the role of RASs in DAAs-based treatment failures (n = 76). Sequence and mutational analyses of NS5 genes in treatment failing patients revealed that the prevalence of RAS in NS5A increased compared to their natural frequencies. However, there were no RASs in NS5B gene. NS5A RASs present at the baseline persisted through the treatment period and were enriched with emerging RASs during the treatment, i.e., paired RASs (A30K + A62S + Y93H) and (A62T + Y93H) frequency 0 - 28.6%, 21.4 - 35.7% pre- and post- DCV treatment, respectively. Similarly, the paired RAS (A62S + Y93H) frequency increased from 16.6 to 50% from pre-to post-VEL treatment, respectively. Most of the paired RASs emerged during the treatment period, indicating their fitness under drug pressure and leading to treatment failure. The most prevalent NS5A RAS was Y93H found in all treatment-failing patients followed by A62S/T and A30K. The presence of RASs may be one of the causes of treatment failures in 26.3% (20/76) of patients. Amino acid substitutions were present at baseline in most of the patients against NS5A and NS5B inhibitors with a minor impact on treatment outcome. Patients with baseline Y93H and/or A30K relapse more frequently than patients harboring A62S/T. In silico functional analysis of mutant HCV-NS5A proteins associated with resistance to DAAs therapy predicted the effectiveness of different NS5A inhibitors for the treatment of difficult-to-treat patients. The docking analyses revealed that substitution Y93H is significant in maintaining a preferred docking pose at the interface of a dimer. Whereas, enhanced interactions were observed with substitutions A21T, V46G, and W47R. Moreover, the drugs Velpatasvir and Pibrentasvir exhibited highest GOLD scores and preferred binding mode with the target mutant proteins compared to Daclatasvir, Elbasvir, and Ledipasvir. Overall based on the findings of the current study, it is inferred that the generic DAAs achieved high SVR12 rates. Old age and cirrhosis were significant predictors of treatment failure. Treatment-failing patients harbored NS5A RASs, the most frequent were A30K (5/20), A62S/T (20/20), and Y93H (20/20). Direct-resistance testing is recommended for optimizing re-treatment strategies in treatment-failing patients.