Synthetic PS-IIIGlycoconjugate Vaccines Based onSolid-Phase Assembled Well-Defined Oligosaccharides Confers Protectionagainst Clostridium difficile

Clostridium difficile (C. difficile), a Gram-positive bacterium responsible for life-threatening diarrhea, causes approximately 12 800 deaths annually in the United States, yet no licensed vaccine is currently available. PS-III, a cell-surface glycan composed of 6,6′-phosphodiester-linked di-N-acetylglucosamine (di-GlcNAc) repeats, has been identified as a promising vaccine antigen because of its potent immunogenicity. However, the lack of efficient synthetic method limited the availability of structurally defined PS-III antigens, hampering systematic immunological analysis. Herein, we report a solid-phase synthesis strategy employing Merrifield resin that enables efficient assembly of PS-III oligosaccharides up to a tridecasaccharide─the largest well-defined structure synthesized up to date. Three CRM197-conjugated vaccines bearing precisely defined glycotopes ranging from pentasaccharide to tridecasaccharide were constructed and evaluated in both in vitro and in vivo immunization studies. Glycan microarray profiling of sera from immunized mice revealed glycotope length-dependent IgG/IgM responses, with the nonasaccharide (1d-CRM197) and tridecasaccharide conjugate (1f-CRM197) eliciting the strongest binding. In a murine challenge model, most glycoconjugate vaccines conferred superior protection against C. difficile compared with inactivated whole cell bacteria vaccine. Notably, 1f-CRM197 induced a nearly complete bacterial killing in the opsonophagocytic assay. Collectively, we have established an efficient solid-phase synthesis for well-defined C. difficile PS-III glycans. The resulting CRM197-conjugate vaccines with short-to-long glycans showed potent activity in opsonophagocytic and murine models, paving the way for preclinical development of C. difficile glycoconjugate vaccines.