Currently there are no HIV prophylactic vaccines available that are capable of either generating durable protection from infection or reducing viral load post-infection in humans. Many factors may be involved, including insufficient levels of dendritic cell (DC) 'danger' signals during presentation of antigen by the vaccine, and insufficient DC intracellular signaling induced by these 'danger' (adjuvant) molecules. There is also evidence that combinations of adjuvant signals, such as combinations of TLR agonists, can markedly increase the strength of the antigen-specific immune response. Certain TLR agonist combinations, such as mixtures of TLR2, TLR3, and TLR9 agonists, have been shown to synergistically increase both correlates of immunity and protection from viral challenge in mouse models. Other models have shown protection from tumor challenge with mixtures of TLR7 agonists and CD40 stimulation. This project will test a novel class of vaccine adjuvants composed of chimeric proteins that artificially induce constitutive TLR signaling. Importantly, these chimeric proteins can be encoded within DNA and viral vector vaccines, one of the limitations of TLR agonists. Just as significantly, these chimeric proteins will provide 'always on' costimulatory signals and cannot be downregulated at the level of the TLR receptor. These proteins are anticipated to be constitutively active in the transfected DC, leading to DC-mediated immune activation. Preliminary data will be presented that these chimeric proteins are potent immune activators in dendritic cells and macrophages. This activation leads to a Th1 cytokine response and can adjuvant antigen-specific T cell responses to HIV-1 Gag antigen. These chimeric proteins have never before been tested as HIV adjuvants, and present a novel and innovative class of HIV vaccine adjuvant. In addition, this proposal will explore using these chimeric proteins to replicate the synergy observed with combinations of TLR agonists, or combinations of TLR agonists and CD40 costimulation. Published data is presented supporting the hypothesis that combinations of TLR intracellular signaling are synergistic. It is proposed that combinations of our novel chimeric proteins can be used to generate gene-based TLR-like synergistic combinations. It is also proposed that these combinations can markedly enhance DNA and viral vector based HIV vaccines in vivo in a mouse vaccinia-gag challenge model. Overall, the project aims to improve the strength and efficacy of DNA and viral vector HIV vaccines through the use of these chimeric proteins. Combinations of these chimeric proteins, encoded within DNA and viral vectors, have the potential to replicate the adjuvant effect of TLR agonist combinations and generate a more effective anti-HIV immune response, an immune response with the potential to protect vaccinated individuals from HIV challenge or reduce viral setpoint after HIV infection.