Monophosphoryl lipid A (MLA) is a detoxified form of TLR4 agonist LPS that is approved for use as a vaccine adjuvant in Europe and is nearing approval for use in the US. Extensive testing shows that MLA, in its clinical grade from, MLA adjuvant, is as safe as the current adjuvant standard, alum, but is far more effective at generating protective antibody responses. In spite of this extensive testing for safety and efficacy, very little is known about the mechanism by which MLA efficiently triggers TLR4 signaling while showing so little of the endotoxic properties of its parent compound, LPS. TLR4 is unique among TLRs in that it signals by activating both MyD88 and TRIF signaling pathways. This proposal will test the central hypothesis that MLA's adjuvant effects are associated with preferential stimulation of the TLR4/Trif pathway. Because MLA is manufactured from endotoxin, synthetic forms are preferred by vaccine manufactures. However, the synthetic forms that are feasible to manufacture on a mass scale have variations of structure relative to MLA that cause them to have a wide range of distinct adjuvant functions. These functional variations are interesting, but also make it more difficult to predict which will ultimately be best suited for widespread use in prophylactic vaccination. This proposal is designed to elucidate the molecular basis for MLA's TRIF- biased adjuvant effects on T and B cell so that its success can be replicated and improved in next generation synthetic forms. The specific aims of this proposal are: I. To identify the molecular mechanism(s) of MLA's Trif-biased signaling in monocytes. II. To identify TLR4 signaling pathways required for low toxicity adjuvant effects on T cells. III. To identify pro-inflammatory requirements for TLR4-mediated adjuvant effects on B cells. PUBLIC HEALTH RELEVENCE: Non-infectious vaccines are typically the safest way to protect against infectious disease and bioterrorism, but vaccines are relatively inefficient unless adjuvants that boost the human immune response are added. This proposal will decipher the means by which new adjuvants that are just beginning to be used in human vaccines influence the responses of T cells, an important component of the immune system. Our work will allow next- generation adjuvants to be improved for use in new vaccines against a broad array of infectious threats.