Most cellular immune responses, including those to HIV-1-infected cells that may be dominated by cytolytic CD8+ T cells. require the activity of CD4+ T cells. In immunocompromised individuals, such as those with HIV-1, boosting CD4+ T cell responses may be important to induce or maintain protective immunity. In order to stimulate antigen-specific CD4+ T cells, complexes of major histocompatibility complex (MHC) class II molecules bound to antigen-derived peptides must be presented on the surface of antigen presenting cells. These complexes are generated in intracellular antigen processing compartments within the endocytic pathway. Foreign proteins access the processing compartments primarily by non-specific internalization in most antigen presenting cells, but exceptions suggest that specifically targeting antigens to these compartments via receptors greatly increases the efficiency with which antigens are processed and presented. Direct targeting and the consequent improved efficiency of antigen processing and CD4+ T cell responses is thus a consideration in development of HIV-1-specific vaccines. Because different endosonial compartments may contribute to antigen processing through production of distinct sets of peptides, the applicants propose to target a model immunogenic HIV-1 antigen, nef, to distinct endosomal compartments, and monitor, by both in vitro and in vivo assays, the relative ability of differentially targeted nef to be presented by MHC class II molecules. To do this, the investigators plan to use a chimeric protein approach appending nef to well-characterized compartmental targeting signals. Chimeric proteins will be expressed in transfected cells or in mice by DNA immunization, and both biochemical and cellular aspects of antigen presentation will be assessed. There are two specific aims, namely: (1) to target the HIV-1 nef gene product to the cytosol, the cell surface, or to early and late endosomes in a model antigen-presenting cell (a cultured mouse B cell line), and to determine the effect of differential targeting on the array of peptides presented by surface MHC class II molecules; and (2) to determine the consequences for nef-specific cellular and humoral immune responses in mice inoculated with DNA encoding chimeric nef proteins targeted to distinct subcellular compartments.
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