IMMUNOLOGICAL STUDIES OF T. CRUZI METACYCLIC STAGES

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Chagas' disease ensues after invasion and infection by metacyclic trypomastigotes (MCs) of Trypanosoma cruzi. Mice immunized with irradiation-attenuated MCs resist infections by virulent MCs. Fusions of B-cells from such immunized mice with myelomas generate hybridomas which after cloning, produce monoclonal antibodies (MAbs) that react with stage- and species-specific antigens (Ags), neutralize MCs, or may when passively transferred protect recipients. MAbs will be selected from this panel on the basis of their abilities to neutralize MCs and complex with epitopes on different MC Ags as shown by Western blot analyses (WBA). Antiidiotype antibodies (AidAbs) will be prepared against each MAb. MAb-immunoaffinity chromatography will be used to isolate MC Ags. The homogeneity and molecular weight of each Ag will be assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoroesis and WBA. The protein and polysaccharide composition of each Ag will be correlated with the effects of enzymatic and chemical treatments on serological reactivity. Parasitemias and mortalities will be monitored in mice immunized with each MC Ag or AidAb after challenge infections with MCs. Mice receiving passively transferred MAbs, similarly inoculated with MCs, will be evaluated to compare their resistance to infections with the actively immunized groups. Interactions between MAb-defined Ags and host cell receptors will be examined in cultured macrophages treated with each AidAb or normal gamma globulins. Macrophages will also be exposed to MCs treated with each MAb or the normal globulins. After intervals of incubation and washing, the numbers of adherent MCs and infected cells will be enumerated to determine whether AidAbs block receptors on macrophage surfaces or MAbs affect MC attachment or internalization. AidAb affinity chromatography isolated receptors will be defined biochemically and both the MC Ags and receptors will be localized by indirect fluorescent Ab microscopy. Antisera from Mc-immunized and -infected mice and antisera from humans with a cute and chronic Chagas' disease will be compared in serological tests with each MC Ag and AidAb to establish their reactivities and the temporal relationships of Ab synthesis. The research will define the vaccine potential of the MC Ags and AidAbs and may provide sensitive and specific reagents for the serodiagnosis of infections in humans.