Structural validation and immunogenic characterization of Streptococcus pneumoniae serotype 22A capsular polysaccharide relative to serotype 22F

Streptococcus pneumoniae serotype 22A is a significant contributor to invasive pneumococcal disease (IPD) in selected regions and is closely related to the vaccine-included serotype 22F. The repeating unit (RU) structure of serotype 22A capsular polysaccharide (CPS) was reported in a patent. In this study, we performed a comprehensive structural validation combining chemical derivatization, high-resolution MS, and NMR spectroscopy to confirm the RU structure, O-acetylation pattern, as well as its immunological characterization relative to 22F. The RU of 22A CPS was confirmed as: [→3)-β-D-Galf-(1 → 2)-α-L-Rhap-(1 → 4)-β-D-GlcAp-(1 → 4)-(α-D-Galp-(1 → 3))-β-L-Rhap-(2-OAc)(1 → 4)-β-D-Glcp-(1→]_n. It is highly similar to that of 22F, with the key difference being a side chain α-d-galactopyranose in 22A versus α-d-glucopyranose in 22F. O-acetylation was also identified as an important structural feature. Immunogenicity was assessed using 22A and 22F CPS-CRM197 conjugates in rabbits. Both vaccines elicited robust antibody responses. Glycan microarray analysis revealed cross-reactive binding between 22A and 22F, while also demonstrating that O-acetyl group is a key contributing factor for antibody recognition. These findings provide the detailed structural characterization of 22A CPS, establish its distinction from 22F, and highlight the roles of side chain sugar and O-acetylation in immunogenicity, offering guidance for pneumococcal vaccine design.