Since 2/1/00, substantial progress has been made on the first two Specific Aims of the original proposal, focused on development, optimization and utilization of a manual cytokine flow cytometry (CFC) assay for the detection of antigen-induced IFNy responses by CD4+ and CD8+ human T cells specific for the human immunodeficiency virus, type 1 (HIV-1). In studies that are currently ongoing, we anticipate that additional progress will be made in the use of this "Gag-IFN'l, CFC assay" to determine whether various HIV-1 vaccines may induce specific CD4+ and/or CD8+ T cell immunity against HIV-1 in vivo. This work has generated 7 papers published in peer-reviewed journals and 5 abstracts presented at international meetings. Our objective now is to assess laboratory correlates of immune protection by automating new flow cytometric assays of antigen-specific immune responses, to identify candidate laboratory correlates of immune protection by comparing the results of these assays with results from other assays of immune phenotype and function in long-term nonprogressors (LTNP), untreated subjects with progressive HIV-1 disease, and recipients of candidate HIV-1 vaccines, and to examine HIV-1-specific immune responses in HIV-l-infected individuals who appear to exhibit significant immune protection from HIV-1 disease. Specific Aim 1 To develop, optimize, and standardize the methods, hardware, and software for automated stimulus-response flow cytometric assays of immune function. The manual Gag-IFN_, CFC assay will be configured into an automated assay platform, amenable for use with additional stimulants and for the measurement of additional intracellular and cell surface markers. Specific Aim 2 To cross-sectionally compare Gag-IFN_ CFC results to those obtained from other assays of immune phenotype and function in a) long-term nonprogressors, b) untreated subjects with progressive HIV-1 disease, and c) recipients of candidate HIV-1 vaccine(s). The automated Gag-IFNy CFC assay will be compared to CFC assays which measure additional intracellular and cell surface markers; and to more traditional measures of CD4+ and CD8+ T cell function (e.g., proliferation and cytolysis). Such comparisons will be made by cross-sectional analysis of two distinct groups of HIV-l-infected patients (long-term nonprogressors and untreated subjects with progressive HIV-1 disease) and two distinct groups of HIV- vaccinees ("responders" and "nonresponders" as tested by lymphoproliferation, 5_Cr release, and ELISPOT). Specific Aim 3 To prospectively characterize antigen-specific immunity in HIV-l-infected individuals who appear to exhibit significant immune protection from HIV-1 disease. Using a cohort of patients with partial virologic control, a prospective analysis will be carried out to define the temporal relationship between loss of immune function, as measured in CFC assay(s) developed in the first two Aims, and disease progression.