To better understand pathogenesis and protection in toxoplasmosis, proteins which elicit cytolytic T lymphocytes (CTL) will be further characterized and tested for ability to protect. Toxoplasma peptide which stimulates protective CTL when bound to MHC Class I Ld (called TgLdP) will be identified. This will be accomplished by affinity chromatography, acid disruption of Ld-peptide complex, HPLC isolation, NMR and sequencing. T2-Ld cells express Ld on their surface and efficiently bind Ld restricted peptides extracellularly. MHC bound peptides unique to infected cells will be tested for biological activity measured as ability to render T2-Ld cells targets for anti-Toxoplasma CTL. The protein from which this peptide originates (TgLd) will be identified, either by comparison with known T. gondii protein sequences or by screening our T. gondii DNA library with antibody to multiple antigenic peptides and sequencing the gene identified. As an alternative approach for identifying TgLdP, T. gondii peptides with the Ld motif will be tested for ability to render T2-Ld cells targets for T. gondii specific CTL lines and a protective CTL clone. When a peptide is found to be a CTL target, it will be studied for ability to elicit protective CTL when administered as a lipopeptide. Immunoelectronmicroscopy with antibodies to TgLd will establish its location within the parasite and also its movement from the parasitophorous vacuole into the host cell cytoplasm and through a MHC Class I presentation pathway. Effect of CTL on intracellular T. gondii will be determined. Mechanism of CTL effect will be studied by immunizing perforin deficient mice and determining if they can generate T. gondii specific CTL. To determine if human CTL recognize T. gondii immunodominant proteins and identify their MHC restriction, human CTL responses to TgLd or other Toxoplasma proteins will be tested by generating target cells with defined MHC Class I haplotypes which express selected T. gondii proteins. To identify whether there are resistant human MHC haplotypes, similar to murine Ld, susceptible H-2b mice expressing human MHC Class I transgenes will be tested for their resistance to parasitemia and cyst formation. Proteins which elicit protective CTL will be studied for ability to protect using our models of peroral and congenital infection. Effect of administration with adjuvant or expression by an avian pox construct on duration of CTL activity and protection will be compared. These proteins will be tested first for superantigen or mitogenic activity as this would impact on their use in protective preparations. The proposed studies will define protein(s) that confer protection against T. gondii by eliciting CTL.
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