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.

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. CONCLUSION: 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.

Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes.

East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.

Antigen-presenting cells from calves persistently infected with bovine viral diarrhoea virus, a member of the Flaviviridae, are not compromised in their ability to present viral antigen.

The aim of this study was to assess whether the infection of antigen-presenting cells (APC) in vivo, evident in calves persistently infected (PI) with bovine viral diarrhoea virus (BVDV), compromised their ability to stimulate virus-specific T cell responses. Major histocompatibility complex (MHC) molecule-identical cattle were identified from the inbred family at the Institute for Animal Health. One was PI and immunotolerant to BVDV. Virus was not isolated from the remaining calves, which were classified as BVDV-immune or BVDV-naïve depending on the presence or absence of BVDV-specific antibodies in sera. Two-colour flow-cytometric analysis of PBMC from the PI calf showed that 40% of CD14(+) monocytes were infected in vivo. Monocytes from the PI calf (PI monocytes) were used as naturally infected ex vivo APC with CD4(+) or CD8(+) T cells isolated from the BVDV-naïve or BVDV-immune animals. PI monocytes stimulated proliferative responses with CD4(+) and CD8(+) T cells from BVDV-immune animals, but not from BVDV-naïve calves. This provided evidence for the presence of virus-specific CD4(+) and CD8(+) memory T cells after acute infection and indicated that ex vivo monocytes from PI, immunotolerant calves stimulated both MHC class I- and MHC class II-restricted T cell responses to BVDV. Additionally, naturally infected ex vivo monocytes cultured in vitro for 3 days stimulated effective T cell responses to the virus with which they were infected.