(622a) A Low-Cost, Thermostable, Cell-Free Protein Synthesis Platform for on-Demand Production of Vaccines

Bacterial infections are a significant health threat to the global population, particularly for the 2.1 billion people without access to clean water. Glycoconjugate vaccines prevent infections by training the immune system to respond to species-specific glycans found on the bacterial cell surface, such as capsular or O-antigen polysaccharides. A glycoconjugate vaccine is comprised of a glycan antigen conjugated to an immunogenic carrier protein. There are muliple approved conjugate vaccines, however the cost and complexity of chemical conjugation and the reliance on the cold chain limit distribution to many resource-limited settings. An alternative approach relies on bioconjugation and can be interfaced with cell-free protein synthesis to produce glycoconjugate vaccines in a simple batch reaction that can be distributed without reliance on cold-chain distribution. Specifically, cell-free protein production systems can be freeze-dried and used for decentralized small-scale manufacturing at the point-of-care, enabling access and distribution of products. However, freeze-dried cell-free conjugate vaccine synthesis reactions have not yet been evaluated for stability above ambient temperature, and vaccine cost is still limiting for low resource settings. In this work, we highlight strategies to decrease the cost and increase the stability of the freeze-dried cell-free protein synthesis reactions when stored at temperatures up to 50°C. First, we identify lyoprotectants, or sugar additives that can increase the stability of freeze-dried reactions. Notably, we identify lyoprotectants that that can also act as high-yielding energy substrates, replacing one of the most expensive cell-free protein synthesis reagents: phosphorylated energy substrates. We then optimize the reaction formulation to further drive down the cost while still maintaining protein synthesis activity necessary for producing vaccine doses after storage at temperatures up to 50°C. Lastly, we apply these improvements to synthesize conjugate vaccines for preventing bacterial infections common in resource-limited settings. Overall, this work will enable the synthesis of low-cost, thermostable conjugate vaccines that are both pertinent and accessible.