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Virus Genes

, Volume 40, Issue 1, pp 44–52 | Cite as

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

  • Arash Arashkia
  • Farzin RoohvandEmail author
  • Arash Memarnejadian
  • Mohammad Reza Aghasadeghi
  • Sima Rafati
Article

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-2Dd (E2405–414:E2) and two HLA-A*0201 (E1363–372:E1 and Core35-44:C)-restricted CD8+ T-cell epitopes of HCV were selected. By employing number of in silico analyses, the E2E1C 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 E2E1C 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-2Dd-restricted-E2 epitope and approved the enhancing effect of PADRE and ERss sequences but not HBsAg for the immune responses against E2 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.

Keywords

Hepatitis C virus Polytope vaccine PADRE Endoplasmic reticulum signal sequence DTH assay 

Notes

Acknowledgements

A.A. received a fellowship from graduate school of Institute Pasteur to pursue this study in the Ph.D. programme. Portions of this study were presented as oral presentations in symposium of Research on Infectious Diseases—A Global Challenge (26–27 June 2008, Paris) and the first international vaccine congress (9–11 December 2007, Amsterdam). This work was financially supported by Pasteur Institute of Iran.

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Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Arash Arashkia
    • 1
  • Farzin Roohvand
    • 1
    Email author
  • Arash Memarnejadian
    • 1
  • Mohammad Reza Aghasadeghi
    • 1
  • Sima Rafati
    • 2
  1. 1.Hepatitis & AIDS Department-NRGB LabPasteur Institute of IranTehranIran
  2. 2.Molecular Immunology and Vaccine Research LabPasteur Institute of IranTehranIran

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