Generation and Characterisation of HCV Genotype 1 Viruses Most Recent Common Ancestor

Abstract

Background: Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope glycoproteins, E1 and E2, interact with host cell receptors and provide a target for neutralising antibodies. Past vaccine studies using unmodified E2 proteins have failed to convincingly generate broadly neutralising antibody responses. Objectives: This study sought to generate and evaluate an immune-focused, vaccine candidate for HCV. Methodology: A synthetic construct based on most recent common ancestral sequence (MRCA) of HCV genotype 1 viruses was generated using sequences available from the Los Alamos HCV database [720 sequences (360 subtype 1a and 360 subtype 1b sequences)], after exclusion of epidemiologically-related sequences. Soluble E2 (sE2) proteins were generated by stably transfected S2 cells and purified using Strep- tag purification and size exclusion chromatography. The MRCA construct was subsequently interrogated using a linear (AP33) and conformational (1:7) monoclonal antibodies directed at E2. A full length E1E2 construct was used for production of HCV pseudoparticles (HCVpp). The infectivity of the HCVpp was measured in the presence of monoclonal antibodies; AP33 1:7 and AR3A. Results: Monomeric proteins of the MRCA generated using a Drosophila expression system were conformationally intact when examined by the monoclonal antibody 1:7 that targets the conformational epitope on E2 responsible for interaction with the CD81 receptors. The full length MRCA E1E2 construct showed functionality in the HCV pseudo-particle (HCVpp) system. The MRCA HCVpp construct was susceptible to neutralisation by AP33, 1:7 and AR3A, in dose- dependant manner. Conclusion: This study demonstrates the generation of a functional construct that could be used as a vaccine candidate in a potential vaccine approach to minimise the problem of genetic diversity between the vaccine construct and contemporary viruses.