Abstract
The tetrasaccharide (2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-α-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranose) from the major exosporium protein (BclA) of Bacillus anthracis has been proposed as a target for development of diagnostics and immune therapy or prophylaxis. While the immunodominant character of the anthrose residue has been previously elucidated, the role of the stereochemical configuration of the downstream rhamnose is unknown. Because the linkage of this residue to the GlcNAc bridging the glycan and the protein is lost during isolation of the tetrasaccharide, its α- and β-glycoforms have been synthesized. Herein, we prepared neoglycoconjugates from a series of fragments of the tetrasaccharide, including the complete α- and β-tetrasaccharide glycoforms, a 2-demethoxylated version of the α-tetrasaccharide, and the α- and β-trirhamnosides and CRM(197). By immunization of mice, we showed that the anti α- and β-tetrasaccharide serum equally recognized both glycoforms. In contrast the sera produced following immunization with the α- and β-trirhamnoside fragments exhibited higher recognition for their own antigens than for their anomeric counterparts. The anti α- and β-tetrasaccharide sera recognized Sterne spores in a comparable fashion. ΔBclA spores not expressing the major exosporium protein were also recognized by the same sera, while mutants that produced the carbohydrate antigen with deletion of either rhamnose or anthrose were not. The tetrasaccharide could, therefore, be expressed in proteins other than BlcA. This work proves that α- and β-tetrasaccharide are equally potent immunogens.