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 ...

The production of cell wall-degrading enzymes (wall depolymerases) by plant pathogenic fungi is under catabolite (glu-cose) repression. In Saccharomyces cerevisiae , the SNF1 gene is required for expression of catabolite-repressed genes when glucose is limiting. An ortholog of SNF1 , ccSNF1 , was isolated from the maize pathogen Cochliobolus car-bonum , and ccsnf1 mutants of HC toxin-producing (Tox2) and HC toxin-nonproducing (Tox2) strains were created by targeted gene replacement. Growth in vitro of the ccsnf1 mutants was reduced by 50 to 95% on complex carbon sources such as xylan, pectin, or purified maize cell walls. Growth on simple sugars was affected, depending on the sugar. Whereas growth on glucose, fructose, or sucrose was normal, growth on galactose, galacturonic acid, maltose, or xylose was somewhat reduced, and growth on arabinose was strongly reduced. Production of HC toxin was normal in the Tox2 ccsnf1 mutant, as were conidiation, conidial morphology, conidial germination, and in vitro appressorium formation. Activities of secreted-1,3-glucanase, pectinase, and xylanase in culture filtrates of the Tox2 ccsnf1 mutant were reduced by 53, 24, and 65%, respectively. mRNA expression was downregulated under conditions that induced the following genes encoding secreted wall-degrading enzymes: XYL1 , XYL2 , XYL3 , XYL4 , XYP1 , ARF1 , MLG1 , EXG1 , PGN1 , and PGX1. The Tox2 ccsnf1 mutant was much less virulent on susceptible maize, forming fewer spreading lesions; however, the morphology of the lesions was unchanged. The Tox2 ccsnf1 mutant also formed fewer non-spreading lesions, which also retained their normal morphology. The results indicate that ccSNF1 is required for biochemical processes important in pathogenesis by C. carbonum and suggest that penetration is the single most important step at which ccSNF1 is required. The specific biochemical processes controlled by ccSNF1 probably include, but are not necessarily restricted to, the ability to degrade polymers of the plant cell wall and to take up and metabolize the sugars produced.

The production of cell wall-degrading enzymes (wall depolymerases) by plant pathogenic fungi is under catabolite (glucose) repression. In Saccharomyces cerevisiae , the SNF1 gene is required for expression of catabolite-repressed genes when glucose is limiting. An ortholog of SNF1 , ccSNF1 , was isolated from the maize pathogen Cochliobolus carbonum , and ccsnf1 mutants of HC toxin-producing (Tox2 ؉ ) and HC toxin-nonproducing (Tox2 ؊ ) strains were created by targeted gene replacement. Growth in vitro of the ccsnf1 mutants was reduced by 50 to 95% on complex carbon sources such as xylan, pectin, or purified maize cell walls. Growth on simple sugars was affected, depending on the sugar. Whereas growth on glucose, fructose, or sucrose was normal, growth on galactose, galacturonic acid, maltose, or xylose was somewhat reduced, and growth on arabinose was strongly reduced. Production of HC toxin was normal in the Tox2 ؉ ccsnf1 mutant, as were conidiation, conidial morphology, conidial germination, and in vitro appressorium formation. Activities of secreted ␤ -1,3-glucanase, pectinase, and xylanase in culture filtrates of the Tox2 ؉ ccsnf1 mutant were reduced by 53, 24, and 65%, respectively. mRNA expression was downregulated under conditions that induced the following genes encoding secreted wall-degrading enzymes: XYL1 , XYL2 , XYL3 , XYL4 , XYP1 , ARF1 , MLG1 , EXG1 , PGN1 , and PGX1 . The Tox2 ؉ ccsnf1 mutant was much less virulent on susceptible maize, forming fewer spreading lesions; however, the morphology of the lesions was unchanged. The Tox2 ؊ ccsnf1 mutant also formed fewer nonspreading lesions, which also retained their normal morphology. The results indicate that ccSNF1 is required for biochemical processes important in pathogenesis by C. carbonum and suggest that penetration is the single most important step at which ccSNF1 is required. The specific biochemical processes controlled by ccSNF1 probably include, but are not necessarily restricted to, the ability to degrade polymers of the plant cell wall and to take up and metabolize the sugars produced. INTRODUCTIONA major barrier to the penetration and spread of potential pathogenic organisms is the plant cell wall, and all of the major groups of cellular plant pathogens are known to make extracellular enzymes that can degrade cell wall polymers. Although the involvement of wall-degrading enzymes and their genes in penetration, pathogen ramification, plant defense induction, and symptom expression has been studied extensively, conclusive evidence for or against a role for any particular enzyme activity in any aspect of pathogenesis has been difficult to obtain (Walton, 1994).The major obstacle to addressing the function of walldegrading enzymes has been redundancy: all of the pathogens that have been studied in detail have multiple genes for any particular enzyme activity. Thus, most fungal strains mutated in wall-degrading enzyme genes-by either conventional (e.g., Cooper, 1987) or molecular (e.g., Scott-Craig et al., 1990) methods-retain at least som...

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