Vaccine vectors based on Venezuelan equine encephalitis virus (VEE) offer a number of advantages for immunization against human immunodeficiency virus (HIV). These include a high margin of safety, high antigen expression levels, up to l mg per 107 cells in culture or 20% of total cell protein, and induction of balanced humoral and cellular immune responses that show protection against disease in a simian immunodeficiency virus (SIV)/macaque challenge model. In addition, the VEE replicon particles (VRP) naturally target to and express in lymphoid tissues, an optimal site for induction of an immune response, and they demonstrate sustained efficacy of priming and booster immunization over multiple simultaneous or sequential inoculations of the same individual. These latter two characteristics clearly distinguish VRP from other viral vector systems. In this project, we propose to determine l) the genetic and immunological basis for the lymph node targeting phenotype of VRP and 2) the ability of VRP to immunize in the face of pre-existing anti-VEE immunity. These properties will be correlated with the level and character of the immune response induced to vectored HIV genes. Not only will the results of these experiments contribute significantly to the design of a VRP-based HIV vaccine candidate, but they also will provide fundamental information regarding 1) the role of lymph node and/or dendritic cell (DC) targeting in induction of an immune response, 2) the initial stages of viral infection in an immune host, and 3) the interactive effects of expression level, duration of expression, and cell targeting on immunogenicity of HIV proteins expressed from viral vectors.