SUMMARY Adjuvants able to induce long lasting type-1 cellular immunity, including both Th1 CD4+ T cells and cytotoxic CD8+ T cells, are highly sought out for the development of vaccines against intracellular pathogens that evade antibodies. We have identified a powerful new class of adjuvant that elicits protective type-1 biased responses. These adjuvants are synthetic RNA oligonucleotides derived from defective viral genomes (DDOs) and trigger strong immune responses by engaging cellular RIG-I-like receptors (RLRs). DDOs contain a unique immunostimulatory motif that we identified as essential for their ability to trigger RLR signaling. In mice, DDOs induce localized expression of type I IFNs and other cytokines and promote accumulation of DCs in the draining lymph node. In addition, DDOs promote a type I IFN-dependent IgG2b/c-biased antibody response able to protect mice from lethal virus challenge and induce IFNγ-producing antigen-specific CD4+ and CD8+ T cells. Moreover, DDOs synergize with the squalene-based adjuvant AddaVax, providing strong type-1 immunity bias to vaccines adjuvanted with AddaVax+DDOs. These data support our central hypothesis that DDOs represent a new class of adjuvants that stimulate the RLR/type I IFN signaling axis to drive optimal long-lived type-1 humoral and cellular immunity. Experiments in this proposal use the combined expertise and unique set of tools of the Lopez and Scott laboratories to assess the quality, longevity, and protective capacity of DDO-induced T cell responses during vaccination, and to characterize specific molecular and cellular mechanisms responsible for directing the type-1 immune response after DDO administration. Specifically, in Aim 1 we will use our ability to track DDOs in vivo, together with a series of reporter and transgenic mice, to identify the early interactions of DDOs with cells of the immune system, in particular DCs, and to identify potential key targets for the development of type -1 biased responses. In Aim 2, we will assess the development and quality of T helper 1 cells, cytotoxic T cells, T follicular helper cells, and tissue resident T cells in response to a vaccine adjuvanted with DDOs alone or in combination with AddaVax, and assess the role of type I IFNs in establishing these responses. In Aim 3, we will test the ability of DDOs to induce CD4+-mediated protection against the intracellular protozoan parasite Leishmania, since protection against this parasite is dependent upon CD4+ Th1 cells and is independent of antibodies, and we will directly assess the ability of DDOs to induce protective T cell responses using a model influenza vaccine in ferrets.