Collaborative Research: Cell-free glycoprotein synthesis technology for point-of-care vaccine biomanufacturing

The bacterial cell surface is decorated with polysaccharide structures including capsular polysaccharides and O-polysaccharides that can be formulated as vaccine subunits and used to protect humans against a diverse array of life-threatening bacterial infections. Conjugate vaccines, a special type of subunit vaccine whereby polysaccharide antigens are linked to a protein carrier, are among the safest and most effective methods for inducing humoral and/or cellular immunity against pathogenic bacteria. Unfortunately, current technology for making conjugate vaccines is technically complex and relies on living cells, which necessitates highly centralized manufacturing, skilled operators, specialized equipment, refrigeration, and cold-chain distribution. To address these limitations, this proposal seeks to create a scalable, cell-free biosynthesis technology for generating potent glycoconjugate vaccines against specific pathogens, with the potential for portable, on-demand production and development in resource-poor settings. This will involve the seamless integration of experimental and computational approaches to develop and optimize an all-in-one-pot cell-free glycoprotein synthesis (CFGpS) system that contains all of the necessary biosynthetic machinery for coordinated transcription, translation, and glycosylation of FDA-approved vaccine carrier proteins. A complementary technology, referred to as shotgun scanning glycomutagenesis, will be developed to comprehensively evaluate glycosylation efficiency as a function of conjugation site and/or loading density of antigens, parameters which will then be correlated with vaccine immunogenicity. Finally, the safety, scalability, economic viability, and portability of the CFGpS platform will be optimized to generate highly productive lysates that can be stored in freeze-dried formats and subsequently reconstituted simply by adding water.