Immune responses to glycoconjugate vaccines rely on a balance between polysaccharide length and glycosylation density.

Glycoconjugate vaccines are effective against bacterial infections. Key characteristics for optimal immunogenicity are the carbohydrate-to-protein ratio and consequent glycosylation density. Directing the conjugation to specific sites is beneficial when the protein acts as carrier and antigen, however a caveat of this approach is the limited number of conjugated glycans attached which might reduce the anti-glycan immune response. We investigated if polysaccharide length is crucial for optimizing immunogenicity in single-site selective conjugates. We tested conjugates from meningococcal serogroup A and C, and pneumococcal serotype 8 and 14 polysaccharides of varying lengths. Meningococcal protein fHbp was used as carrier. Site-selective conjugates were obtained by thiol-maleimide addition at a C-terminal cysteine, whereas random conjugates were prepared via classic lysine coupling. Results showed that for meningococcal type C and pneumococcal serotypes 8 and 14, oligosaccharides over 15-20 repeating units elicited robust anti-glycan responses, even when attached at a single site. Differently, the less immunogenic meningococcal type A required longer glycan chains for a strong response. Additionally, increasing glycosylation by random conjugation did not enhance polysaccharide immunogenicity but reduced the protein immunogenicity. These findings emphasize that an optimal balance between glycan length and density is essential for effective glycoconjugate vaccine design.