Synthetic Oligosaccharide-Based Vaccines Protect Mice from Clostridioides difficile Infections

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.

MALDI-TOF MS. Mass spectra were acquired with an Autoflex Speed MALDI-TOF system (Bruker Daltonics). Samples were spotted using the dried droplet technique with 2,5dihydroxyacetophenone (DHAP) as matrix on MTP 384 ground steel target plates (Bruker Daltonics). Samples were prepared by mixing 2 µL of desalted protein sample with 2 µL of DHAP matrix and 2 µL of 2 % (v/v) trifluoroacetic acid (TFA) prior to spotting. The mass spectrometer was operated in linear positive mode. Mass spectra were acquired over an m/z range from 30,000 to 210,000 and data was analyzed with the FlexAnalysis software provided with the instrument.
Pilot Immunization Studies with Glycoconjugate 14. Female, 6-8 weeks old C57BL/6 mice (Charles River) were immunized s.c. with an amount of 14 corresponding to 1 μg of glycan antigen per injection. The immunogen was diluted in sterile PBS to a final volume of 100 μL per injection. For immunizations with Alum adjuvant, 14 was pre-incubated with 1 μL per μg protein of Alum Alhydrogel (Brenntag) the day before, and the mixture was rotated for 24 h at 4 °C. For immunizations with Freund's adjuvant (Sigma-Aldrich), a solution of 14 was combined with an equal volume of Complete Freund's adjuvant (CFA) (priming immunization) or Incomplete Freund's adjuvant (ICFA) (boosting immunizations) to a homogeneous emulsion immediately before injection. For immunizations with AddaVax (similar to MF59) (InvivoGen) a solution of 14 was combined with an equal volume of AddaVax to a homogeneous emulsion immediately before injection (priming and boosting immunizations). The experiments were performed in strict accordance with the German regulations of the Society for Laboratory Animal Science and the European Health Law of the Federation of Laboratory Animal Science Associations and were approved by the Landesamt für Gesundheit und Soziales of Berlin, Germany (protocol number G0135/14). All efforts were made to minimize suffering.
Challenge Studies. 6-8 weeks old female C57BL/6 mice (Charles River, Sulzfeld) were used for immunization experiments. Each injection comprised a volume of 100 µL using sterile PBS as diluent. Sham-immunizations with PBS contained either 17.3 µL Alum or 50 µL AddaVax. One dose of CRM197 contained 17.3 µg protein with either 17.3 µL Alum or 50 µL AddaVax. One dose of 12 contained 7 µg protein, corresponding to 1 µg of 1, and 7 µL Alum. Each dose of 13 contained 16.7 µg protein, corresponding to 1 µg of 2, and 16.7 µL Alum. Each dose of 14 contained 14.3 µg protein, corresponding to 1 µg of 3, and 50 µL AddaVax. Each dose of 15 contained 15 µg protein, corresponding to 1 µg of 4, and 15 µL Alum. Each dose of 16 contained 17.3 µg protein, corresponding to 1 µg of 5, and either 17.3 µL Alum or 50 µL AddaVax. Each dose of formalin-inactivated TcdB contained 75 µg protein and 75 µL Alum. 13 days after the last immunization, mice were rendered susceptible to C. difficile infection with intraperitoneal (i.p.) injections of clindamycin (20 mg per kg body weight) for one day. 33 The next day, mice were challenged via oral gavage with 5x10 7 CFUs of the C. difficile strains M68 or VPI 10463. M68 is a clindamycin-resistant ribotype 017 strain isolated from a hospital outbreak in Dublin, Ireland, expressing toxin B (TcdB) but not toxin A (TcdA). 28,34 VPI 10463 is a highly virulent ribotype 087 strain expressing both toxins. 26,33 Intestinal colonization was quantified 5 days after the infection by determining the C. difficile CFUs in fecal suspensions that were plated at limited dilutions on selective T.C.C.F.A. agar plates and cultivated for 48 h at 37 °C under anaerobic conditions. The degree of colonization is displayed as CFUs per gram feces. Enterococcus spp. CFUs grown on blood agar plates served as control. Characteristic colonies were counted and identified at random by MALDI-TOF MS analysis. Histopathological analysis of colon samples was performed to determine the degree of colitis.
For passive transfer experiments, mice received 200 µL of pooled sera i.p. on days 0 and 1 with the challenge performed on day 0 two hours after the first serum transfer, using 5x10 7 CFUs of strain VPI 10463. mAbs were applied i.p. on days 0, 1 and 2 (100 µg per injection) and mice were challenged on day 0 two hours after the first antibody transfer, using 5x10 7 CFUs of VPI 10463. Animal experiments were performed in strict accordance with the German regulations of the Society for Laboratory Animal Science and the European Health Law of the Federation of Laboratory Animal Science Associations and were approved by the Regierung von Mittelfranken, Germany (AZ 54-2532.1-47/13).
Processing of Stool Samples for Microarray and ELISA. To fresh stool samples two volumes of PBS supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich) were added. After vigorous vortexing samples were incubated in ice for 1h and then centrifuged (10,000 x g for 20 min). The supernatant was recovered and used for further analyses.
Flow Cytometry. Formalin-inactivated C. difficile bacteria (strain M68 and strain VPI10463) were incubated with murine antisera diluted 1:20 in PBS for 1 h, washed three times with PBS, and stained with anti-mouse IgG FITC produced in goat (cat. no. F0257; BD Biosciences) diluted 1:100 in PBS for 1 h. After three washing steps with PBS, the bacterial cells were subjected to flow cytometry using a FACS Canto II instrument (BD Biosciences). About 2,000 events (strain M68) or 10,000 events (strain VPI 10463) were counted for each individual measurement.
Histopathology. Intestinal tissue obtained from the ascending, transverse and descending colon was fixed in 10% buffered formalin, embedded in paraffin, and cut into 2 µm thick sections. Colonic sections were deparaffinized, stained with H&E by the Department of Pathology of the FAU Erlangen-Nürnberg, and evaluated microscopically in a double-blinded manner. Briefly, the intestinal damage shown in the presented figures was evaluated in at least 5 colonic tissue sections per mouse using the following parameters: (a) inflammatory infiltrate, (b) submucosal edema, (c) epithelial damage and the number of intraepithelial lymphocytes (IELs) (d), lymph follicles (LF) (e) and crypt abscesses (CA) (f). A histologic grading of the severity of the tissue damage was performed for each parameter a-c (score between 0 and 3) and the number of IELs (score of 0: 0 IELs, score of 1: < 5 IELs, score of 2: < 10 IELs and score of 3: > 10 IELs) or lymph follicles (LF) and crypt abscesses (CA) (score of 0: 0 LF or CA, score of 1: 1 LF or CA, score of 2: 2 LF or CA and score of 3: 3 LF or CA), leading to a cumulative score between 0 (no signs of inflammation) and 18 (very severe inflammation and epithelial damage). For the initial challenge studies with strain M68, inflammation scores reflect the average of the three tissue sections with most prominent pathology obtained from at least five colon sections per mouse. For the repeat challenge studies, inflammation scores reflect the average of the three tissue sections with most prominent pathology obtained from at least 15 (M68) or 10 (VPI 10463) colon sections per mouse. Genome Res. 17, 377-386. 50. Matsuda, K., Tsuji, H., Asahara, T., Takahashi, T., Kubota, H., Nagata, S., Yamashiro, Y., and Nomoto, K. (2012) Sensitive quantification of Clostridium difficile cells by reverse transcriptionquantitative PCR targeting rRNA molecules. Appl. Environ. Microbiol. 78, 5111-5118. 51. Bandelj, P., Logar, K., Usenik, A. M., Vengust, M., and Ocepek, M. (2013) An improved qPCR protocol for rapid detection and quantification of Clostridium difficile in cattle feces. FEMS Microbiol. Lett. 341, 115-121. 52. Kubota, H., Sakai, T., Gawad, A., Makino, H., Akiyama, T., Ishikawa, E., and Oishi, K. (2014) Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools. PLoS One 9, e111684.  Histopathological analyses of colon tissue sections from mice infected with C. difficile VPI 10463. Subpanels 1-3: Signs of colitis in C. difficile VPI 10463 infected mice. Subpanels 4-5 and 10: Tissue sections of mice vaccinated with 15. Subpanels 6-9 show histopathological changes in sham-immunized, infected control mice, including: focal mucosal lesions (subpanel 6, red arrowhead), increased formation of apoptotic bodies in colon epithelial cells (subpanel 7, black arrowheads), granulocyte infiltration (subpanel 7, green arrowhead) and diffuse infiltration of the lamina propria by inflammatory immune cells (subpanels 8 and 9).