RESUMO
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