title: A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology.
abstract: BACKGROUND
  Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8+ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL.
  
  
  RESULTS
  Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-gamma ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8+ T cell responses were observed during the protective immune response against sporozoite challenge.
  
  
  CONCLUSIONS
  The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.
authors.affiliation: International Livestock Research Institute, P, O, Box 30709, Nairobi 00100, Kenya. s.graham@vla.defra.gsi.gov.uk
authors.forename: Simon P
authors.initials: SP
authors.lastname: Graham
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Honda
authors.forename: Roger
authors.initials: R
authors.lastname: Pellé
authors.forename: Duncan M
authors.initials: DM
authors.lastname: Mwangi
authors.forename: E Jane
authors.initials: EJ
authors.lastname: Glew
authors.forename: Etienne P
authors.initials: EP
authors.lastname: de Villiers
authors.forename: Trushar
authors.initials: T
authors.lastname: Shah
authors.forename: Richard
authors.initials: R
authors.lastname: Bishop
authors.forename: Pierre
authors.initials: P
authors.lastname: van der Bruggen
authors.forename: Vishvanath
authors.initials: V
authors.lastname: Nene
authors.forename: Evans L N
authors.initials: EL
authors.lastname: Taracha