Non-Native Amino Acid Click Chemistry-Based Technology for Site-Specific Polysaccharide Conjugation to a Bacterial Protein Serving as Both Carrier and Vaccine Antigen

[Image: see text] Surface-expressed bacterial polysaccharides are important vaccine antigens but must be conjugated to a carrier protein for efficient antigen presentation and development of strong memory B cell and antibody responses, especially in young children. The commonly used protein carriers include tetanus toxoid (TT), diphtheria toxoid (DT), and its derivative CRM197, but carrier-induced epitopic suppression and bystander interference may limit the expanded use of the same carriers in the pediatric immunization schedule. Recent efforts to develop a vaccine against the major human pathogen group A Streptococcus (GAS) have sought to combine two promising vaccine antigens—the universally conserved group A cell wall carbohydrate (GAC) with the secreted toxin antigen streptolysin O (SLO) as a protein carrier; however, standard reductive amination procedures appeared to destroy function epitopes of the protein, markedly diminishing functional antibody responses. Here, we couple a cell-free protein synthesis (CFPS) platform, allowing the incorporation of non-natural amino acids into a C-terminally truncated SLO toxoid for the precise conjugation to the polyrhamnose backbone of GAC. The combined immunogen generated functional antibodies against both conserved GAS virulence factors and provided protection against systemic GAS challenges. CFPS may represent a scalable method for generating pathogen-specific carrier proteins for multivalent subunit vaccine development.