A DNA-based vaccine for HIV-1 will have to deal with the diversity of this virus, as well as issues of potency and effectiveness. This proposal is part of a large program undertaken by this research group aimed to exploit the utility of DNA to ultimately develop a prophylactic vaccine for HIV-1. We have designed a prototypic DNA based vaccine for HIV-1 which includes attenuated gag/pol genes, attenuated accessory genes, a prototypic env/rev immunization cassette and is likely to require additional envelope subtypes in an effort to deal with viral diversity and to limit viral escape. Furthermore one question central question regarding this novel technology is its potency. Accordingly, this proposal will focus on the generation of improved DNA immunogens through engineering gene expression cassettes which co-deliver cytokine as well as other immunomodulatory gene sequences as part of the vaccine. We will directly test this concept in mice and in primates and in clinical studies. There are three projects and two cores which form this proposal. Project 1 will develop and test cytokines and co-stimulatory molecules as molecular adjuvants for HIV-1 immunization cassettes and in particular monitor the cellular effects of such vaccine manipulation. Project 2 will test the serological consequences as well as effects on chemokine production induced by the use of cytokines and co-stimulatory molecules as molecular adjuvants to enhance HIV-1 immunization cassettes. Project 3 will test a combined vaccine cocktail of gag/pol and env/rev vaccine cassettes first and then go on to test a subset of the molecular adjuvant cassettes in clinical studies. These studies will define the initial safety profile as well as test their ability to impact on their specific immune responsiveness in humans. These projects are supported by an experienced Central Immunology core as well as an experienced Molecular Biology core laboratory. The projects and cores and subcontracts are coordinated by the Central Administrative core. This program project brings together a seasoned, collaborative and highly interactive group of investigators who have moved DNA vaccines from concept to the lab bench and to the clinic. In this proposal we will develop the second generation of tools for optimizing this new technology in an attempt to maximize the effectiveness of these vaccines and to probe the relationship of directed immune responses to impact on viral replication in vivo. This program will directly test the ability of such second generation vaccines to impact on HIV replication in animal models and to modulate HIV immune responsiveness in humans by not just characterizing but by driving immune response direction in vivo.