Salmonella, Mycobacterium and Histoplasma are facultative intracellular pathogens that live inside phagosomes of host macrophages. They all cause AIDS-defining illnesses, and the investigator's long-term goal is to understand the development of immunity against such pathogens. CD4+ T cells are also required for immune mice to resist virulent Salmonella, providing a model of protective host functions which can successfully combat a macrophage-tropic infection. However, the specific bacterial antigens (Ags) recognized by Salmonella-immune hosts are largely unknown. Two proteins expressed in the surface-exposed """"""""compartment"""""""" of Salmonella are recognized by CD4+ T cells from immune mice. One is a flagellar protein also recognized by T cells from humans immunized with Salmonella. The other is an unidentified protein expressed by most Enterobacteriaceae, including E. coli, Yersinia, Shigella, and Enterobacter. Both proteins are regulated in a fashion suggesting part of the Salmonella intracellular survival strategy is to down-regulate expression of bacterial surface Ags recognized by CD4+ T cells.
In AIM I, the diversity of Salmonella Ags recognized by CD4+ T cells from immune mice will be determined using SDS-PAGE fractionated bacteria as Ag, and bacterial expression of these Ags will be characterized with respect to compartmentalization and regulation using T cell clones. The studies will provide insight into the nature of Ags recognized by CD4+ T cells, the environmental signals affecting bacterial processing Salmonella for T cell responses.
In AIM 2, genes encoding Ags recognized by T cell clones will be identified by expression cloning or sequencing analysis of biochemically purified Ags. This work may reveal gene products useful as markers of cellular immunity to Salmonella in humans.
In AIM 3, murine infection with Salmonella strains expressing a model Ag in various compartments of the bacterial cell will be used to directly test if compartmentalization of bacterial Ag alters its significance for surveillance by T cells. Primary and secondary CD4+ T cell responses generated by these stains will be quantified using ELISPOT and flow cytometry, and the effectiveness of an Ag-specific immune response against these strains will be tested in vivo. These studies will provide insight into the nature of Ags recognized by CD4+ T cells responding to pathogens similarly adapted for life in phagosomes.
In AIM 4, the functional importance of Ags identified in AIMS 1 & 2 will be determined by testing purified Ags for their ability to stimulate protective immunity against challenge by virulent Salmonella. The protective Ags identified will be excellent candidates for components of subunit vaccines and markers of cellular immunity in humans.
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