Project Summary The goals of this R01 proposal include the molecular and immunological characterization of vaccine adjuvant activity associated with processes mediated by the protein Stimulator of Interferon Genes (STING). Vaccines comprised of inert antigen are often only weakly immunogenic and thus require co-administration of adjuvants to augment immunostimulation. Unfortunately, very few adjuvants are approved for clinical use and the mechanisms of adjuvant-associated enhancement are poorly understood. STING is a pattern recognition receptor that detects purine-containing cyclic dinucleotides (CDN) synthesized by either bacterial cyclases or the host enzyme cyclic GMP-AMP (cGAMP) synthase (cGAS) following its sensing of cytosolic DNA. Pharmacologic activation of STING greatly enhances vaccine efficacy as indicated by antibody and cell-mediated protection against diverse microbial pathogens. Furthermore, endogenously synthesized cGAMP is naturally released from cells in exosomes and virus particles. We exploited this phenomenon by constructing cell lines that express constitutively active cyclases that we then used to produce immunogenic virus-like particles (VLP). We now show that VLP derived from unrelated emerging flavi- and alphaviruses efficiently encapsulate bioactive CDN and stimulate STING-dependent activity in vivo. Since extracellular CDNs are highly susceptible to degradation and disperse quickly from the site of injection, we hypothesize that this simple approach represents a transformative technological improvement for harnessing STING-directed adjuvant effects in the draining lymph node. We also predict this will be a highly adaptable platform and propose to explore the extent to which it is applicable to potentially emerging virus types. Experimentally, this also represents a unique investigative model that will allow in vivo characterization of the innate and molecular correlates of adaptive immunity that are potentiated by targeted STING activity. We thus hypothesize that the enhancement of antigen-directed immune responses by STING-based adjuvants is functionally linked to key molecular and cellular processes that are discernable using our proposed vaccines and experimental systems. We plan to explore this using an integrated approach that uses molecular transcriptomic analysis, mouse models of immune profiling, and nonhuman primate models of viral growth and protective immunity devised by our group.