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.cattle ͉ East Coast fever ͉ immunoscreening ͉ protozoan parasite ͉ vaccination A single inoculation with a potentially lethal dose of Theileria parva sporozoites and simultaneous treatment with a longacting oxytetracycline induces solid immunity to homologous and, in certain instances, heterologous parasite challenge (1, 2). This methodology has been adopted as a live vaccine for the control of East Coast fever (ECF) (3). The long-lasting immunity to ECF contrasts with the partial immunity to malaria that develops after only several years of exposure to T. parva-related Plasmodium spp. (4). Manufacture and delivery of the live ECF vaccine is difficult to sustain, but it has enabled elucidation of the dominant protective immune response against the disease. Kinetic and adoptive cell transfer studies (5, 6) have demonstrated that protection of cattle is mediated by MHC class I-restricted CD8 ϩ cytotoxic T lymphocytes (CTL) that destroy schizontinfected lymphocytes, the pathogenic life-cycle stage of T. parva. In addition, there is a strong correlation between the specificity of the CTL response and cross-immunity profiles of distinct parasite strains (2). The identification of schizont antigens targeted by CTL from T. parva-immune cattle has been elusive but should pave the way for the development of a subunit vaccine against ECF and provide a long-term solution to a socioeconomically important constraint to livestock agriculture in Africa (7). We adopted two approaches to antigen identification, both dependent on screening of transiently transfected antigenpresenting cells with fully characterized CTL (8, 9) from live vaccine-immunized cattle of diverse bovine leukocyte antigen (BoLA) MHC class I genotypes. First, in a targeted gene approach, we immunoscreened genes that were predicted by using preliminary sequence data from one of the four T. parva chromosomes (10) to contain a secretion signal. The approach was ...

Various pathogens have been shown to infect antigen-presenting cells and affect their capacity to interact with and stimulate T-cell responses. We have used an antigenically identical pair of noncytopathic (ncp) and cytopathic (cp) bovine viral diarrhoea virus (BVDV) isolates to determine how the two biotypes affect monocyte and dendritic cell (DC) function. We have shown that monocytes and DCs are both susceptible to infection with ncp BVDV and cp BVDV in vitro. In addition, monocytes infected with ncp BVDV were compromised in their ability to stimulate allogeneic and memory CD4+ T cell responses, but DCs were not affected. This was not due to down-regulation of a number of recognized co-stimulatory molecules including CD80, CD86 and CD40. Striking differences in the response of the two cell types to infection with cytopathic virus were seen. Dendritic cells were not susceptible to the cytopathic effect caused by cp BVDV, whereas monocytes were killed. Analysis of interferon (IFN)-a/b production showed similar levels in monocytes and DCs exposed to cp BVDV, but none was detected in cells exposed to ncp BVDV. We conclude that the prevention of cell death in DCs is not associated with enhanced production of IFN-a/b, as proposed for influenza virus, but is by a distinct mechanism.

BackgroundImmunity 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.ResultsOf 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-γ 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.ConclusionThe 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.

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