Synthetic Oligosaccharide-Based Vaccines Protect Mice from Clostridioides difficile Infections.

Felix Broecker,Paulina Kaplonek,Peter H Seeberger,Erik Wegner,Armin Ensser,Maria Bräutigam,Christopher E Martin,Bruna M S Seco,Christoph Daniel,Jochen Mattner,Frederick Pfister

doi:10.1021/acschembio.9b00642

Abstract

Infections with Clostridioides difficile (formerly Clostridium difficile) have increased in incidence, morbidity, and mortality over the past decade. Preventing infections is becoming increasingly important, as frontline antibiotics become less effective and frequently induce recurrence by disrupting intestinal microbiota. The clinically most advanced vaccine approaches prevent symptoms once C. difficile infection is established by inducing immunity to secreted clostridial cytotoxins. However, they do not inhibit bacterial colonization and thereby favor asymptomatic carriage. Synthetic oligosaccharides resembling the C. difficile surface glycans PS-I, PS-II, and PS-III are immunogenic and serve as basis for colonization-preventing vaccines. Here, we demonstrate that glycoconjugate vaccine candidates based on synthetic oligosaccharides protected mice from infections with two different C. difficile strains. Four synthetic antigens, ranging in size from disaccharides to hexasaccharides, were conjugated to CRM197, which is a carrier protein used in commercial vaccines. The vaccine candidates induced glycan-specific antibodies in mice and substantially limited C. difficile colonization and colitis after experimental infection. The glycoconjugates ameliorated intestinal pathology more substantially than a toxin-targeting vaccine. Colonization of the gut by C. difficile was selectively inhibited while intestinal microbiota remained preserved. Passive transfer experiments with anti-PS-I serum revealed that protection is mediated by specific antiglycan antibodies; however, cell-mediated immunity likely also contributed to protection in vivo. Thus, glycoconjugate vaccines against C. difficile are a complementary approach to toxin-targeting strategies and are advancing through preclinical work.

Highlights

Clostridioides dif ficile is the leading cause of antibiotic-induced bacterial colitis and diarrhea worldwide.[1,2] Risk factors for C. diff icile infections (CDIs) include broad-spectrum antibiotic use, hospitalization, and advanced age.[3]
ACS Chemical Biology previously reported that synthetic PS-I, PS-II, and PS-III oligosaccharides (Figure 1A) are immunogenic in mice when linked to the CRM197 carrier protein, which is a nontoxic mutant of diphtheria toxin that allows for efficient covalent attachment of synthetic oligosaccharides and is used in licensed glycoconjugate vaccines.[16−23] In 2011, we reported the synthesis of the hexasaccharide repeating unit of PS-II 3 that, when linked to CRM197, was immunogenic in mice and was used to generate PS-II specific monoclonal antibodies.[16]
Cross-linking 1, 2, and 4 to CRM197 was achieved with di-p-nitrophenyl adipate (DNAP),[20,25] whereas, for 3 and 5, di-N-succinimidyl adipate (DSAP) was used, as coupling with DNAP was inefficient (Figure 1 B)

Summary

■ INTRODUCTION

Clostridioides dif ficile is the leading cause of antibiotic-induced bacterial colitis and diarrhea worldwide.[1,2] Risk factors for C. diff icile infections (CDIs) include broad-spectrum antibiotic use, hospitalization, and advanced age.[3]. 17/20 mice vaccinated with C. dif f icile-specific glycoconjugates developed detectable levels of IgG to the respective oligosaccharides. No glycan-specific serum antibody binding to inactivated bacteria was detected, but it appeared that CRM197 reactive IgG bound nonspecifically to C. dif ficile to some degree (Supplemental Figures 4A− 4C in the Supporting Information). III disaccharide 4) led to a 96.8% reduction of C. dif f icile colonization This confirmed our previous studies in which a CRM197 glycoconjugate with PS-III tetrasaccharide 10 reduced colonization with C. diff icile by 96% and 83.3%, compared to nonimmunized and CRM197 immunized mice, respectively.[21] both 4 and 10 can evoke protective IgG in mice, with 4 being easier to synthesize,[19] which is an advantage toward costefficient vaccines.

Conclusions

■ EXPERIMENTAL METHODS

■ ACKNOWLEDGMENTS

■ REFERENCES