Chemical Synthesis Elucidates the Immunological Importance of a Pyruvate Modification in the Capsular Polysaccharide of Streptococcus pneumoniae Serotype 4

Abstract

Carbohydrate modifications are believed to strongly affect the immunogenicity of glycans. Capsular polysaccharides (CPS) from bacterial pathogens are frequently equipped with a pyruvate that can be placed across the 4,6-, 3,4-, or 2,3-positions. A trans-2,3-linked pyruvate is present on the CPS of the Gram-positive bacterium Streptococcus pneumoniae serotype 4 (ST4), a pathogen responsible for pneumococcal infections. To assess the immunological importance of this modification within the CPS repeating unit, the first total synthesis of the glycan was carried out. Glycan microarrays containing a series of synthetic antigens demonstrated how antibodies raised against natural ST4 CPS specifically recognize the pyruvate within the context of the tetrasaccharide repeating unit. The pyruvate modification is a key motif for designing minimal synthetic carbohydrate vaccines for ST4.

Glycan Arrays: From Basic Biochemical Research to Bioanalytical and Biomedical Applications

“…In the case of serotype 4, one galactose of the repeating unit is modified with a cyclic pyruvate ketal connecting the 2-and 3-hydroxy groups. Only a tetrasaccharide representing the complete repeating unit with this rare modification was bound by serum antibodies (39).…”

“…Coupling is usually performed at slightly alkaline conditions (a pH of approximately 8.5) and remaining functional groups are quenched with an alkaline ethanolamine solution, therefore, attention has to be maintained with regard to functional groups on glycans prone to alkaline hydrolysis. The reliable amine/NHS coupling is the routine immobilization chemistry in many laboratories (18,24,39,40). Recently, it has been shown that the commonly used NHS hydrogel slides retain polysaccharides that are not amine functionalized, allowing immobilization of activated small glycans and unmodified polysaccharides on the same surface (13,39).…”

“…The reliable amine/NHS coupling is the routine immobilization chemistry in many laboratories (18,24,39,40). Recently, it has been shown that the commonly used NHS hydrogel slides retain polysaccharides that are not amine functionalized, allowing immobilization of activated small glycans and unmodified polysaccharides on the same surface (13,39). In some cases, reactive groups orthogonal to amines are necessary for immobilization, for example, to achieve site-directed immobilization of glycans that contain amino sugars.…”

“…Glycan arrays were used to elucidate the differences between serum and mAb binding profiles and provided insights into how to improve the oligosaccharide antigens (46). Analysis of defined rabbit sera immunized with the respective CPS revealed candidates for immunization studies based on the CPSs of two important Streptococcus pneumoniae serotypes, serotype 1 and serotype 4, and pointed toward important antigenic determinants www.annualreviews.org • Applications of Glycan Arrays (39,42). For serotype 1, the rare monosaccharide 2-acetamido-4-amino-2,4,6-trideoxy-Dgalactose (AAT) was essential for antibody reactivity (42).…”

A Semi‐Synthetic Glycoconjugate Vaccine Candidate for Carbapenem‐Resistant Klebsiella pneumoniae

“…Long lasting, T-cell-dependent protection from bacterial infections can be provided by oligosaccharide antigens that are linked to a carrier protein, [12] as demonstrated by a semi-synthetic glycoconjugate vaccine against Haemophilus influenzae type b marketed in Cuba. [13] Semi-synthetic oligosaccharide vaccine candidates against Shigella , [14] meningococci, [15] and streptococci [16] are currently under preclinical evaluation.…”