The overall goal of this project is the identification and analysis of the human T-lymphocyte responses to discrete Onchocerca volvulus antigens which are associated with the immune-mediated ocular and dermal disease characteristic of infection with this parasite. The identification of discrete T-cell antigens in onchocerciasis has been hampered by a paucity of available parasite material and the difficulty in separating crude extracts into distinct antigens. The recent demonstration of the feasibility of expressing immunologically significant T-cell epitopes from isolated cDNA clones of O. volvulus, combined with the ability to expand and maintain T-cell clones in continuous culture will enable us for the first time to precisely map the T-cell determinants of onchocercal disease. Specific experimental design is: 1) Differential identification utilizing T-cell immunoblotting analysis and T-lymphocyte clones (TLC), of native T- cell antigens uniquely associated with ocular and skin disease. The TLC will be produced utilizing crude O. volvulus antigen and PBMC collected from well-defined patient subgroups. Antisera raised to the selected antigens will be used to screen cDNA libraries. 2) Examination of the response of the TLC made in 1 above, to recombinant antigens selected as a result of screening either the T-cell immunoblots or patient IgG responses (see Project 2). Blastogenesis, lymphokine production (IL-2, IL-4, g-IFN), cell surface markers and MHC restriction of the TLC will be examined. 3) Definition utilizing proteolytic fragments and overlapping peptides of individual T-cell epitopes associated with O. volvulus induced disease. TLC for epitope mapping will be produced by stimulation of patient PBMC's with those recombinant antigens most associated with each of ocular and skin disease as defined by lymphokine production as well as by field studies in Project 1 and guinea pig studies in Project 4. 4) Examination of the response of the TLC produced in 3 above to antigens derived from the different life-cycle stages of O. volvulus. Both forest and savannah region disease associated recombinant antigens will be developed and patient PBMC from the two separate regions will be crossed over in order to clarify issues basic to the biology and immunology of onchocerciasis. Definition and characterization of defined T-cell antigens of O. volvulus will impact on many crucial aspects of disease control programs including diagnosis, modulation of the immune sequelae of infection and ultimately on vaccine development.
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