title: Construction of HCV-polytope vaccine candidates harbouring immune-enhancer sequences and primary evaluation of their immunogenicity in BALB/c mice.
abstract: 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.
authors.affiliation: Hepatitis & AIDS Department-NRGB Lab, Pasteur Institute of Iran, Pasteur Ave, Tehran 1316943551, Iran.
authors.forename: Arash
authors.initials: A
authors.lastname: Arashkia
authors.forename: Farzin
authors.initials: F
authors.lastname: Roohvand
authors.forename: Arash
authors.initials: A
authors.lastname: Memarnejadian
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Aghasadeghi
authors.forename: Sima
authors.initials: S
authors.lastname: Rafati
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry* / Q000276:immunology*; D019556:COS Cells; D002522:Chlorocebus aethiops; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005260:Female; D015234:HLA-A Antigens / Q000276:immunology; D016174:Hepacivirus / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D013601:T-Lymphocytes / Q000276:immunology; D014765:Viral Vaccines / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D018984:Epitopes, T-Lymphocyte; D015234:HLA-A Antigens; D014765:Viral Vaccines