Document Type : Original Article
Hepatitis and AIDS Department, NRGB Lab, Pasteur Institute of Iran, Tehran, Iran
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 to costly transgenic animal studies.
Materials and Methods: Four HLA-A2 and H-2d restricted CTL epitopes were selected and designed in three appropriate sequential tandems based on epitope and proteasomal cleavage predictions. The related nucleotide sequences were synthesized using SOEing PCR method and cloned into a pcDNA3.1 vector, either alone or fused to the small hepatitis B surface antigen (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 also assessed through the delayed-type hypersensitivity (DTH) assay in BALB/c mice.
Results: Due to in silico designs and optimizations, the polytope products of constructed plasmids were efficiently detected in vitro through common techniques and HBsAg-S-based particles were shown to be secreted into the culture media (up to 30%). Moreover, all plasmids were able to efficiently induce a positive DTH response while HBsAg-S fusion constructs indicated 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 transgenic mice.