Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse

Abstract

Objective: Polytope DNA vaccines, capable of focusing the cytotoxic T lymphocyte (CTL)response on critical epitopes, represent a promising approach in HCV immunotherapy. Nevertheless,due to controversial rules governing epitope processing and the low level expression/immunogenicity of recombinant polytope peptides, designing and primary expression/immunogenicity analysis of these vaccine types should be the first consideration prior tocostly transgenic animal studies.Materials and Methods: Four HLA-A2 and H-2d restricted CTL epitopes were selected anddesigned in three appropriate sequential tandems based on epitope and proteasomal cleavagepredictions. The related nucleotide sequences were synthesized using SOEing PCRmethod and cloned into a pcDNA3.1 vector, either alone or fused to the small hepatitis B surfaceantigen (HBsAg-S) gene. Following the preparation of polyclonal anti-sera, expression/secretion of polytopes was evaluated in Cos-7 cells by using immunofluorescence, Westernblot,dot blot, ELISA and RT-PCR techniques. The immunogenicity of the plasmids was alsoassessed through the delayed-type hypersensitivity (DTH) assay in BALB/c mice.Results: Due to in silico designs and optimizations, the polytope products of constructedplasmids were efficiently detected in vitro through common techniques and HBsAg-S-basedparticles were shown to be secreted into the culture media (up to 30%). Moreover, all plasmidswere able to efficiently induce a positive DTH response while HBsAg-S fusion constructsindicated a significant immunopotential effect towards the incorporated mouse epitopes.Conclusion: Designed polytope constructs of this study are efficiently expressed and processed.They have the required initial potency for further immunogenicity analysis in transgenicmice