Sexual transmission via a mucosal surface is the most common route for the spread of HIV-1 infection yet little is known about vaccine induction of mucosal immunity to this or any other virus. Methods for evaluating the correlates of protective immunity also remain incompletely defined. It has been previously demonstrated that an immunization protocol using SIV subunits and an optimized adjuvant delivered by targeted iliac lymph node (TILN) injections protected against SIV rectal mucosal challenge while other routes and sites of immunization did not. Protection was correlated with a significant increase of IgA-secreting cells in the lymph nodes, in CD8-suppressor factor and B-chemokine production. Based on these data and awareness of homing patterns, localization of antigen-presenting cells (APCs) and activation state, we suggest that in humans, immunization in regional, draining lymph nodes will provoke more robust mucosal immune responses than systemic (deltoid) immunization and provide enhanced protection from natural HIV infection. As different vaccines may vary in their ability to stimulate both T and B cell mucosal immune responses, we aim to compare mucosal responses in human seronegative volunteers triggered by either TILN or deltoid immunization using two HIV-1 vaccines that are either vaccinia-based (TBC-IIlB) or canarypox-based (ALVAC vCP1452 with gpl40 boost). Mucosal immune responses will be assessed using rectal Iavage and endoscopic biopsies and will include comparisons of specific secretory IgA and IgG, specific CD8 and CD4 responses and B-chemokine production. Systemic immune responses will be tested in parallel. To provide a comparative frame of reference to assess the breadth and potency of vaccine elicited specific anti-HIV-l immune responses, the same mucosal and systemic immune responses will be characterized in HIV-seropositive individuals, including long term nonprogressors, highly exposed persistently seronegatives, chronically infected untreated and chronically infected individuals with suppressed plasma viral loads. This proposal addresses the need to characterize the role of site-specific immunization in eliciting HIV-specific mucosal responses. In addition, the experiments proposed allow a better understanding of HIV-l induced pathogenesis and virus specific responses in this highly vulnerable, sexually exposed, compartment. If the hypothesis is correct and TILN immunization elicits either more diverse or stronger mucosal immunity than peripheral immunization, these findings will impact the way in which HIV-specific vaccines are tested in the future, and may optimize the chances of developing efficacious vaccines, a task that has so far been elusive.