Construction of pIRES2 vector encoding truncated NS3 of HCV and IL-18 for DNA vaccine studies

Abstract

Introduction: Long term complications of hepatitis C virus (HCV) infection include fibrosis, cirrhosis, and hepatocellular carcinoma and although the disease is treatable with the newly introduced direct acting antivirals, but factors such as drug resistance, viral genotype and adverse effects can limit the effectiveness of therapy. Therefore, development of effective, safe and affordable prophylactic and therapeutic vaccines is a global goal. This study was undertaken for construction of a vector containing the coding region for a truncated form of nonstructural protein 3 (NS3) of HCV and IL-18 cytokine gene fused to murine Fcy2a as molecular adjuvant for assessment as a DNA vaccine candidate. Methods: A truncated form of NS3 was amplified and cloned in pIRES2 containing IL-18 hybrid gene. Expression of the truncated NS3 and bioactivity of IL-18 fusion protein were assessed in transfected HEK293 T cells. Physicochemical properties determination, secondary structure, 3D modeling and T-cell epitope predictions were made using various online tools. Results: In silico analysis of truncated NS3 predicted a molecular weight of 32.35 kDa containing epitopic regions with the highest scores for binding to MHC complex. 3D model of IL-18 showed that fusion with murine Fc had not impaired the structure. HEK293 T cells were successfully transfected with pIRES-IL18-NS3 construct and expression of truncated NS3 with an apparent molecular weight of approximately 32 kDa was confirmed by Western blotting. Conclusion: Despite the advantages, widespread use of DNA vaccination has been hampered by low immunogenicity. Truncated NS3 was expressed albeit in low amount with bioactive IL-18 fusion proteinas a molecular adjuvant Immunogenicity assessment of this novel combination as a DNA vaccine candidate is underway.