Construction of HCV-polytope vaccine candidates harbouring immune-enhancer sequences and primary evaluation of their immunogenicity in BALB/c mice.

An efficient vaccine against hepatitis-C virus (HCV) infection requires vigorous and focused CD8(+) T-cell responses against viral antigens. Due to immunosuppressive effect of HCV antigens, polytope vaccines comprising the minimal CD8(+)CTL epitopes are of peculiar concern. Herein, to provide information for construction of efficient HCV polytope vaccine candidates, one H-2D(d) (E2(405-414):E(2)) and two HLA-A*0201 (E1(363-372):E(1) and Core(35-44):C)-restricted CD8(+) T-cell epitopes of HCV were selected. By employing number of in silico analyses, the E(2)E(1)C linear format was predicted as optimum epitope consecution and after amplification by SOEing-PCR, the corresponding DNA sequence was cloned in pcDNA3.1+ vector. To further evaluate the role of immune-enhancer elements, a universal T-helper epitope (PADRE), endoplasmic reticulum signal sequence (ERss) and hepatitis-B surface-antigen (HBsAg) gene were fused separately or in combination to the E(2)E(1)C minigene. In vitro analyses of polytopes by different DNA/protein-based assays demonstrated proper transcription/expression of constructs in transfected cells. Measurement of the HBsAg-mediated particle secretion by ELISA indicated lack of secretion in the related polytopes. Results of delayed-type hypersensitivity (DTH) as a preliminary in vivo analysis, and confirmatory ELISPOT assays showed the proper processing and presentation of H-2D(d)-restricted-E(2) epitope and approved the enhancing effect of PADRE and ERss sequences but not HBsAg for the immune responses against E(2) in immunized BALB/c mice. Our results pointed to the value of in silico predictions and application of immune-enhancer elements as well as DTH analysis for design and primary in vivo evaluation of HCV polytopes, prior to costly transgenic studies on immunogenicity of HLA-A*0201 epitopes.