Many diseases that were once widespread are now controlled or have been eradicated through the use of vaccines. However, there is an increasing need for new adjuvants to potentiate candidate vaccines against other important pathogens such as Malaria, HIV and TB. Such new adjuvants may help replace live attenuated vaccines with subunit or killed antigens, which are safer, easier to combine with other antigens, and have shorter development and implementation cycles. New adjuvant strategies exploit the activation of innate immune signals which results in a vigorous adaptive immune response to vaccine antigens. One novel adjuvant is Venezuelan equine encephalitis (VEE) replicon particles (VRP). These particles perform a single replication round with no further propagation and have proven safety as vaccine vectors in humans. Using non- mucosal routes of inoculation, VRP demonstrated strong mucosal and systemic adjuvant activity for important vaccine antigens such as H5N1 and Polio vaccines, Malaria soluble proteins and Norovirus VLPs. This adjuvant effect was observed in four different mouse strains as well as in non-human primates. In macaques the adjuvant amplified the response to an off-the-shelf vaccine for seasonal influenza by over twenty-fold and significantly increased protection from challenge. The robust induction of mucosal immunity by parenteral immunization is a novel and valuable feature of this adjuvant, and is superior to that achieved by cholera toxin, a model mucosal adjuvant. To date, development of strong mucosal immunity has required either antigen delivery into the mucosal site or use of live attenuated vaccines. Despite these promising data, the regulatory approval of vaccines containing VRP-adjuvant will require about a better understanding of its mechanism of action. Such mechanistic studies of the VRP-adjuvant effect will also benefit the field of viral immunity by improving our understanding of the role of dendritic cells (DCs) and innate immune signaling pathways in response to alphaviruses. In addition, these studies will benefit future vaccine design by identification of pathways that can be targeted in order to achieve stronger immunity after vaccination. Our published and preliminary data indicate that VRP infect specific subsets of dendritic cells (DCs), inducing the activation of an innate immune response. During the next four years we will focus our research to investigate how VRP infection of DCs and the activation of the innate immune response are linked to the adjuvant effect of VRP. We expect to shed light on how VRP-triggered innate immune responses by DCs help to establish an adaptive immune response against viruses and soluble antigens. An understanding of the VRP mechanism of action might also address safety concerns and improve the design of preclinical and clinical studies. Last but not least, VRP may be a valuable tool for augmentation of DC-activation during DC-transfer based therapies in vaccines against cancer and chronic infectious diseases. PUBLIC HEALTH RELEVANCE: Our proposal is aimed at understanding the mechanism of action of a novel potentiator of the immune system, a necessary step for his use approval. This potentiator could result in safer, more effective, and more easily-developed vaccines. We expect that our results will increase the knowledge of how our immune system responds to viruses and vaccines.