In full response to RFA-AI-20-004, we propose to study the mechanisms of cooperativity between NKT cell and TLR adjuvants. We have previously shown that we could use a combination of NKT cell and TLR7 adjuvants to produce high affinity protective anti-bacterial glycan vaccines. Our goal is now to understand the mechanisms of this cooperative effects between the two adjuvants to optimize it and produce a vaccine formulation that promotes protection after a single administration. In addition, we contend that mechanistic studies will also allow the limitation of the potential side effects of adjuvants. To this end, our proposal is built on two separate and complementary specific aims: Aim 1: Evaluate endosomal TLR ligands for their ability to enhance NKT cell responses and their B cell helper activities. We have produced nanomolar affinity anti-glycan antibodies by combining NKT cell and TLR7 agonists. This response was dependent on NKT and CD4 T cell help. We hypothesize that each endosomal TLR ligand may have a different enhancing effect in a combinatorial usage. We will investigate NKT, T follicular helper, dendritic, and B cell responses by single cell technologies for RNA and protein expression in animal models of vaccination for each TLR/NKT cell pair that we will study. B cell receptor sequencing and pathogen challenges will be used to measure the efficacy of each combination. Aim 2: Evaluate the importance of the co-delivery of the two adjuvants to particular cell types and endosomal/lysosomal compartments. We hypothesize that the physical aspects of adjuvanticity with respect to antigen uptake and delivery to processing compartments are critical. By co-delivering antigen and adjuvants to particular cells and within chosen compartments, using synthetic chemistry to control their pairing and separation, mechanisms of adjuvant activity will be examined. The biological consequences of this molecular based design of adjuvant combinations will be evaluated with respect to dosage, toxicity, and efficacy. We expect to develop robust formulations that induce protection after a single vaccine administration. Our entire proposal is focused exclusively on vaccines aimed at developing protective B cell immunity, and our model system is a conjugate vaccine against Streptococcus pneumoniae.