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

Abstract

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.

A Review of Protein- and Peptide-Based Chemical Conjugates: Past, Present, and Future

“…Taking the idea of targeted conjugation further, some researchers have used non-native amino acids (nnAA) and click chemistry to generate a conjugate vaccine in which the pathogenic polysaccharide is directly conjugated to a conserved protein antigen from the same pathogen species [ 140 ]. Specifically, in developing a vaccine for the group A carbohydrate (GAC) and polyrhamnose core thereof (GAC PR ) from Streptococcus pyrogens [ 141 , 142 , 143 , 144 ], investigators sought to use the autologous protein antigen streptolysin O (SLO) on the basis of it being a key virulence factor [ 142 ].…”

“…Specifically, in developing a vaccine for the group A carbohydrate (GAC) and polyrhamnose core thereof (GAC PR ) from Streptococcus pyrogens [ 141 , 142 , 143 , 144 ], investigators sought to use the autologous protein antigen streptolysin O (SLO) on the basis of it being a key virulence factor [ 142 ]. These studies used a C -terminal truncation of SLO previously known to elicit a neutralizing immune response in rodents [ 142 ] that was genetically modified to convert specific solvent-exposed lysine and/or arginine residues to the non-native amino acid residue p-azidomethyl phenylalanine (pAMF) and expressed by cell-free protein synthesis [ 140 ]. These modified carrier proteins were then reacted with a dibenzocyclooctyne (DBCO)-derivatized form of GAC PR to yield glycoconjugate vaccines [ 140 , 145 ].…”

“…These studies used a C -terminal truncation of SLO previously known to elicit a neutralizing immune response in rodents [ 142 ] that was genetically modified to convert specific solvent-exposed lysine and/or arginine residues to the non-native amino acid residue p-azidomethyl phenylalanine (pAMF) and expressed by cell-free protein synthesis [ 140 ]. These modified carrier proteins were then reacted with a dibenzocyclooctyne (DBCO)-derivatized form of GAC PR to yield glycoconjugate vaccines [ 140 , 145 ]. These conjugate vaccines produced by nnAA click chemistry were compared to conventional GAC PR -SLO conjugates produced by reductive amination, and it was found that only the former retained immunogenicity of the SLO carrier and, in turn, conferred antibody-mediated protection in vivo [ 140 ].…”

“…These modified carrier proteins were then reacted with a dibenzocyclooctyne (DBCO)-derivatized form of GAC PR to yield glycoconjugate vaccines [ 140 , 145 ]. These conjugate vaccines produced by nnAA click chemistry were compared to conventional GAC PR -SLO conjugates produced by reductive amination, and it was found that only the former retained immunogenicity of the SLO carrier and, in turn, conferred antibody-mediated protection in vivo [ 140 ].…”

Carriers and Antigens: New Developments in Glycoconjugate Vaccines

“…Furthermore, Kapoor et al also target the GAC oligosaccharides from GAS. However, they found an application in the use of the secreted toxin antigen streptolysin O (SLO) as the protein carrier [ 84 ]. Additionally, Romero-Saavedra et al described the use of two enterococcal proteins (secreted antigen A and the peptidyl-prolyl cis-trans isomerase) as a carrier for the Enterococcus faecalis polysaccharide di-heteroglycan [ 85 ].…”