An important goal of this project continues to be the generation and testing of maximally efficient expression vectors for specific antigens. Our hypothesis is that the DNA vaccine dose is suboptimal for many human applications; therefore, increased efficiency is necessary for practical human DNA vaccines. We have generated a set of optimized expression vectors for HIV and SIV. HIV vectors are developed for eventual human clinical trials. These vectors are studied in macaques for immunogenicity and ability to protect against challenge with Simian/Human Immunodeficiency Virus hybrid viruses (SHIV). Several of our vectors were used in clinical trials sponsored by our previous CRADA collaborator (Wyeth). In parallel, SIV expression vectors are developed and studied in the most faithful model system for human AIDS, i.e., challenge of Rhesus macaques by SIV, a virus closely related to HIV, which causes very similar pathology to human AIDS. Our results have shown that optimized DNA expression vectors in the absence of any other form of vaccine boosting are able to protect rhesus macaques from high viremia after challenge with a highly pathogenic SIVmac251 challenge. In addition, we have developed powerful new DNA and protein co-immunization protocols that increase the magnitude, rapidity and longevity of immune responses. To further improve vaccine efficiency we study the intrinsic properties of the different candidate antigens. We take advantage of the ability to manipulate the form of expressed antigen by recombinant DNA technology. We have shown that modulating the form, stability and cellular fate of the DNA-produced antigens has profound effects on their immunogenicity and the type of response generated. We perform comparative studies to develop optimal forms of several antigens. Results in rhesus macaques verified that the form of expressed antigen affects the type and magnitude of immune response. We study several different antigen forms to achieve optimal immune response and to address the variability of HIV strains circulating worldwide. We compare the immune response generated by either mixes of native antigens, mosaics, centralized and consensus candidates, and also antigens containing only conserved elements of HIV proteins. Such comparisons may lead to further optimization of a protective immune response. We have recently shown that vaccination with conserved elements vaccine constructs have the ability to alter the hierarchy of immune response and to direct it towards conserved elements, which are found in all HIV clades. On the basis of these data, we have proposed a clinical trial to test the ability of Conserved Element vectors to provide broader immune response in humans. The methodology and vectors used for DNA vaccination of macaques have shown that we produce a strong, broad and long-lasting immunity, which is able to contain virus replication and prevent disease development. More recently, we showed that DNA in combination with an adjuvanted protein is able to delay or prevent infection after repeated low dose virus challenge. DNA vaccination is emerging as the strongest and most effective vaccination procedure for the development of cellular immunity in humans, based on clinical trials using the same methods and vectors we co-developed for macaques. These results strongly suggest that DNA vaccination will have many practical clinical applications. We have used our understanding of gene regulation to develop non-pathogenic strains of SIV, which are maintained in macaques for more than 10 years, yet they do not cause any disease. These animals develop a strong protective immune response and are able to resist high viremia and disease development even after challenge with wild-type SIV. We showed that these animals develop neutralizing antibodies against difficult-to-neutralize SIVmac239, and that CD8 cells contribute to the protective effect. We have also shown that these animals develop high levels of cytotoxic CD4 cells, which contribute to viral control. This macaque model is important for the further understanding of the pathogenic mechanisms leading to AIDS, the virus interactions in different tissues and the components of the immune system contributing to protection from disease development. In addition to prophylactic vaccination against AIDS, the same methodologies were used in therapeutic vaccination protocols. A strong boost of cellular immune responses and subsequent control of viremia was observed in macaque studies, suggesting that therapeutic vaccination may contribute to long-term virus control. These results have also implications for the development of methods to apply DNA vaccine methodology to therapeutic cancer vaccines. We wish to combine therapeutic vaccination with additional methods to boost immune response and specific cell killing by cytotoxic cells. We therefore will use hetIL-15 to further boost cytotoxic cells responses based on our completion of studies demonstrating the safety of hetIL-15 in infected macaques. We have shown that inclusion of hetIL-15 in therapeutic vaccination results in high levels of cytotoxic cells.