Identification of theileria parva vaccine candidate antigens recognised by cytotoxic t lymphocytes from Zebu cattle

Abstract

East Coast fever (ECF), caused by Theileria parva, a tick-borne intracellular apicomplexan parasite, is a highly fatal lymphoproliferative disease of cattle. Immunity against T. parva has previously been shown to be mediated through lysis of schizont infected cells by Major histocompatibililty complex (MHC) class I restricted CD8+ cytotoxic T lymphocytes (CTL). A strategy has recently been developed to identify CTL target schizont antigens and has provided a solid basis for the development of a subunit vaccine against ECF. To date CTL target antigens have been identified using CTL derived from Bos taurus and Boran (B. indicus) cattle immunised with the Muguga stock of T. parva. It has been hypothesised that additional antigens are required to formulate a sub-unit vaccine that would protect the out bred cattle population at risk from a highly heterogeneous T. parva population. This study aimed to extend and expand the process of vaccine candidate antigen identification by employing CTL obtained from genetically diverse East African zebu cattle immunised with the cocktail of T .parva stocks that constitute the FAO 1 live vaccine that protects cattle across the ECF endemic areas. T. parva specific CD8+ polyclonal CTL lines were generated by repeated in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from ITM immunised zebu cattle with T. parva infected lymphoblasts. CTL were confirmed to express a CD3+ CD8+ phenotype and specifically lyse autologous T. parva infected cells in 5 IChromium release cytotoxicity assays. CTL lines were tested for recognition of immortalised skin fibroblasts (iSF) infected with recombinant Modified Vaccinia Ankara strain (MV A) viruses expressing the previously identified schizont antigens. Only CTL from one calf (BY 120), showed specific recognition of these antigens; CTL responded specifically to iSF infected with MVA expressing antigen Tp2. Synthetic peptides were employed to identify a novel CTL epitope and analysis of the consensus sequences of positive peptides suggested that the minimal length antigenic peptide was the 10mer Tp2138-147 (KTSIPNPCKW). CTL were next used to immunoscreen iSF transiently transfected with 96 cDNA encoding secreted/membrane bound proteins and 644 cDNA pools derived from a T parva (Muguga) schizont cDNA expression library. CTL from calf BY126 specifically responded to two cDNA pools. All the other CTL failed to recognize transfected iSF. This study has demonstrated that CTL isolated from Zebu cattle immunized with a cocktail of T parva stocks recognize novel schizont antigens and continued immunoscreening is required to identify these antigens, which will constitute valuable additions to the vaccine candidates currently being evaluated.