The goal of our proposal is to understand how alphavirus replicon particles (nVRPs) function as adjuvants. nVRPs stimulate a protective systemic and mucosal immune response when co-delivered with a target antigen even when delivered at a non-mucosal site. nVRPs appear to activate the innate immune response, but it is not known how this occurs. Activation of the innate immune response is a crucial event required for protective immunity. By determining how nVRPs activate the innate immune response, our studies may lead to improved rational design and targeting of new adjuvants (such as nVRPs) that can improve both existing and new vaccines to diseases such as tuberculosis, HIV, dengue virus and hepatitis C virus. In our proposal, we will determine which nVRP components activate the innate immune response. We will test the role of translation and replication of nVRP RNA in activating the innate immune response in murine L929 and bone marrow-derived dendritic cells (BMDCs). If we are able to define a particular component(s) of the nVRP that induces the innate immune response, we will test the ability of this component to function as an adjuvant when co-delivered with a target antigen in mice. In our proposal, we will also investigate the innate immune pathways important for responding to nVRP infection. We will investigate the roles of particular innate immune proteins through siRNA knockdown in L929 cells and BMDCs and through using BMDCs from knockout mice. These cells will be infected with nVRPs and measured for activation of the innate immune response through the detection of activated transcription factors and by measuring pro-inflammatory cytokines and type 1 interferons in supernatants from infected cells. We will also use in vitro assays that mimic activation of CD8 T cells by dendritic cells in the lymph node. After BMDCs are infected with nVRPs they stimulate activation of CDS T cells in vitro and we will investigate which innate immune pathways in BMDCs mediate this activity. Finally, we will test the nVRP adjuvant activity in vivo using mice knocked out for particular innate immune proteins. By determing the nVRP components that activate the innate immune response and which innate immune receptors are important for the nVRP adjuvant effect;our studies may lead to adjuvant formulations that contain a cocktail of molecules that specifically activates one or more pathways of the innate immune response. PUBLIC HEALTH RELAVENCE: We will determine which adjuvant components and innate immune proteins are important for stimulation of a protective immune response. Our findings may lead to improved design of adjuvants that can increase the effectiveness of both existing and new vaccines.
