title: Design and production of a multiepitope construct derived from hepatitis E virus capsid protein.
abstract: The aim of this study was to design a high density multiepitope protein, which can be a promising multiepitope vaccine candidate against Hepatitis E virus (HEV). Initially, conserved and antigenic helper T-lymphocyte (HTL) epitopes in the HEV capsid protein were predicted by in silico analysis. Subsequently, a multiepitope comprising four HTL epitopes with high-affinity binding to the HLA molecules was designed, and repeated four times as high density multiepitope construct. This construct was synthesized and cloned into pET-30a (+) vector. Then, it was transformed and expressed in Escherichia coli BL21 cells. The high density multiepitope protein was purified by Ni-NTA agarose and concentrated using Amicon filters. Finally, the immunological properties of this high density multiepitope protein were evaluated in vitro. The results showed that the high density multiepitope construct was successfully expressed and purified. SDS-PAGE and Western blot analyses showed the presence of a high density multiepitope protein band of approximately 33 kDa. Approximately 1 mg of the purified protein was obtained from each liter of the culture media. Moreover, the purified multiepitope protein was capable of induction of proliferation responses, IFN-γ ELISPOT responses and IFN-γ and IL-12 cytokines production in a significant level in peripheral blood mononuclear cells (PBMCs) isolated from HEV-recovered individuals compared to the control group. In conclusion, the newly produced multiepitope protein can induce significant T helper type 1 responses in vitro, and can be considered as a novel strategy for the development of HEV vaccines in the future.
authors.affiliation: Department of Microbiology and Parasitology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran; Persian Gulf Biomedical Research Center, Bushehr University of Medical Sciences, Bushehr, Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Taherkhani
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Farshadpour
authors.forename: Manoochehr
authors.initials: M
authors.lastname: Makvandi
mesh_terms: D000328:Adult; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology*; D016022:Case-Control Studies; D016207:Cytokines / Q000096:biosynthesis; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D005260:Female; D016751:Hepatitis E / Q000276:immunology / Q000378:metabolism; D016752:Hepatitis E virus / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology; D055815:Young Adult
chemical_list: D036022:Capsid Proteins; D016207:Cytokines; D000939:Epitopes; D018984:Epitopes, T-Lymphocyte; C400971:ORF2 protein, Hepatitis E virus; D014764:Viral Proteins
keywords: IFN-γ ELISPOT; ORF2; cell proliferation assay; cellular immune responses; cytokines; hepatitis E virus