title: African horse sickness virus serotype 4 antigens, VP1-1, VP2-2, VP4, VP7 and NS3, induce cytotoxic T cell responses in vitro.
abstract: It was shown in a previous study that proliferating CD8+ T cells could be detected in immune horse peripheral blood mononuclear cells (PBMC) when stimulated with African horse sickness virus serotype 4 (AHSV4). In this study the cytotoxicity of CD8+ T cells were tested by using the fluorescent antigen-transfected target cells-cytotoxic T lymphocytes (FATT-CTL) assay, for both the virus and its individual proteins expressed in Escherichia coli. This CTL assay measures the killing of viral protein expressing cells. AHSV proteins were successfully expressed in E. coli using the pET102/D-TOPO expression vector and the effector cells were stimulated with these recombinant proteins or with live viable virulent AHSV4. The AHSV genes were amplified and cloned into the pIRES-hrGFP II (pGFPempty) vector and these plasmid vectors encoding antigen-green fluorescent protein (GFP) fusion proteins were used to nucleofect PBMC, the target cells. The elimination of antigen-GFP expressing cells by CTL was quantified by flowcytometry. VP1-1, VP2-2, VP4, VP7 and NS3, antigen-specific CD8+ T cells resulted in cell lysis suggesting that CTL may play a role in the immune response induced against the AHSV4 vaccine strain.
authors.affiliation: Agricultural Research Council-Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa.
authors.forename: F E
authors.initials: FE
authors.lastname: Faber
authors.affiliation: Agricultural Research Council-Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa.
authors.forename: M
authors.initials: M
authors.lastname: van Kleef
authors.affiliation: Agricultural Research Council-Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa.
authors.forename: S I
authors.initials: SI
authors.lastname: Tshilwane
authors.affiliation: Agricultural Research Council-Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa. Electronic address: PretoriusAL@arc.agric.za.
authors.forename: A
authors.initials: A
authors.lastname: Pretorius
mesh_terms: D000355:African Horse Sickness / Q000276:immunology / Q000517:prevention & control* / Q000821:virology; D000356:African Horse Sickness Virus / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000276:immunology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006736:Horses; D007114:Immunization; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D065288:Serogroup; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects* / Q000276:immunology / Q000821:virology
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: AHSV4; CD8+ T cells; FATT-CTL assay