Project Summary/Abstract Vaccines composed solely of antigens are often poorly immunogenic; increasing intensity and length of the induced immune response is required to achieve desirable vaccine efficacy. Adjuvants (adjuvare, to help) are biomolecules used for >100 years to enhance human immune responses to vaccine antigens. Vaccines containing adjuvants are spectacularly successful, with billions of doses administered to save millions of lives each year. Despite this success, there is only a limited set of FDA-approved adjuvants (e.g., aluminum salts, oil- in-water emulsions, CpG oligonucleotides, and an extract [aka QS-21] from the Chilean soapbark tree [Quillaja saponaria]) owing to the intrinsic toxicity of new adjuvant candidates, difficulty to source and produce them, and their poor ability to induce long-term immunogenicity. Ongoing efforts to tailor adjuvant bioactivity are limited because their structure-activity relationships and mechanisms of action are not fully understood. Herein, we propose to address this challenge by engineering adjuvants through iterative diversification of their molecular structures (synthetic biology) and deduction of their molecular-level immunogenic mechanisms (systems-level immunology), an approach that will enable the rapid discovery of improved adjuvants. Recently, we engineered yeast (Saccharomyces cerevisiae) to produce QS-21 from simple sugars by upregulating native yeast pathways and heterologously expressing 38 proteins from six other organisms. We also have extensive experience in profiling (in vitro and in vivo) immunogenicity (innate and adaptive) of small molecules (e.g., adjuvants) associated with both bacterial and viral antigens. Together, we are uniquely positioned to redesign the microbial biosynthetic pathway for QS-21 to access its scalable production as well as that of its natural (i.e., QS-7) and new-to-nature (i.e., a “core pharmacophore”) analogs (Specific Aim 1). QS-21, QS-7, and a “core pharmacophore” will be starting points for a rapid pipeline that iteratively studies their mechanisms of action through systems- and molecular-level immunology studies (Specific Aim 2) and diversifies their molecular structures using combinatorial synthetic biology approaches (Specific Aim 3). Ultimately, this interdisciplinary and innovative approach will establish a framework to discover the structure-activity relationships that govern adjuvant immunogenicity and apply this knowledge to design and deploy best-in-class adjuvants that transform the prevention and treatment of disease.