To study prevention of AIDS, we have generated efficient DNA expression vectors, which are evaluated as vaccines against SIV and HIV. This work is based on our previous recognition that RNA elements (called INS) present within the gag/pol and env coding regions of HIV are responsible for nuclear retention and instability of the transcripts in the absence of Rev, and that these elements can be eliminated by changing the RNA composition without affecting the amino acid sequence. These RNA-optimized gag and env expression vectors mediate the development of protective immune responses in vaccinated macaques when used as DNA only as well as DNA prime vaccine modality. We have now shown that that a combination of DNA vaccine vectors producing native and modified antigens are able to induce immune responses able to protect from high viremia in the rhesus macaque/SIVmac251 model. The introduced modifications of the proteins led to more efficient secretion of the SIV antigens resulting in increased cellular and humoral immune responses in the vaccinated rhesus macaque. Our goal has been to further improve DNA mediated immune responses using different approaches. In collaboration with other investigators, we have studied immunogenicity of SIV genes either as DNAs or as part of viral vectors. We have shown that these DNAs provide an excellent prime in DNA prime-recombinant Herpes boost studies inducing protective immune responses. We investigated the contribution of cytokines co-administered as molecular adjuvant to further improve the efficacy of DNA vaccination. We found that the use IL-12 and IL-15 plasmid DNAs as adjuvants resulted in greatly improved immunogenicity in mice and macaques. Another important new development is that we have shown that DNA delivery via in vivo electroporation elicits greatly improved cellular immune responses and these responses are long-lasting. Thus, these results indicate that improved gene delivery and expression dramatically improves immunogenicity and effectiveness of DNA vaccination. We have investigated the effect of DNA vaccination during antiretroviral treatment (ART). We have found that vaccination of SIV-infected rhesus macaques induces potent immune responses that are able to control viremia after ART interruption. Thus, DNA vaccination has great potential to be used as an additional therapeutic modality. We have found that DNA vaccination can be applied repeatedly leading to further control of viremia. To design better vaccine strategies, it is important to dissect correlates of protective immunity. Among the 'controllers' are animals from our vaccine studies as well as animals infected with live-attenuated SIV strains serve as model to study the underlying mechanisms. Such animals allow us to dissect the cellular and viral determinants that contribute to disease development and they also provide a unique resources to study mechanisms leading to protective immunity. These studies will provide critical information about the establishment and maintenance of host immune responses during chronic retroviral infections with distinct pathogenic outcomes and will aid in our design to improve DNA vaccination studies.