Protection against Clostridium difficile infection in a hamster model by oral vaccination using flagellin FliC-loaded pectin beads.

Clostridium difficile flagellin FliC is a highly immunogenic pathogen-associated molecular pattern playing a key role in C. difficile pathogenesis and gut colonization. Here, we designed an oral vaccine against C. difficile with FliC encapsulated into pectin beads for colonic release. Bead stability and FliC retention was confirmed in vitro using simulated intestinal media (SIM), while bead degradation and FliC release was observed upon incubation in simulated colonic media (SCM). The importance of FliC encapsulation into pectin beads for protection against C. difficile was assessed in a vaccination assay using a lethal hamster model of C. difficile infection. Three groups of hamsters orally received either FliC-loaded beads or unloaded beads in gastro-resistant capsule to limit gastric degradation or free FliC. Two other groups were immunized with free FliC, one intra-rectally and the other intra-peritoneally. Hamsters were then challenged with a lethal dose of C. difficile VPI 10463. Fifty percent of hamsters orally immunized with FliC-loaded beads survived whereas all hamsters orally immunized with free FliC died within 7 days post challenge. No significant protection was observed in the other groups. Only intra-peritoneally immunized hamsters presented anti-FliC IgG antibodies in sera after immunizations. These results suggest that an oral immunization with FliC-loaded beads probably induced a mucosal immune response, therefore providing a protective effect. This study confirms the importance of FliC encapsulation into pectin beads for a protective oral vaccine against C. difficile.

Clostridium difficile forms variable biofilms on abiotic surface.

Clostridium difficile can form biofilms. Thirty-seven strains were characterized for their ability to form a biofilm, adhesion on an inert surface and hydrophobicity. No correlation between the ability to form a biofilm and the strain virulence was highlighted. However, non-motile strains were not able to form a high biofilm.

Immunogenic properties of the surface layer precursor of Clostridium difficile and vaccination assays in animal models.

Clostridium difficile is an opportunistic pathogen causing gut inflammation generally associated with an intestinal dysbiosis due to antibiotics. Several virulence factors have been identified as playing a key role in gut colonization. The surface-layer proteins, comprised of two proteins, the high molecular weight SlpA (HMW-SLP) and the low molecular weight SlpA (LMW-SLP), are the most abundant proteins on the C. difficile surface. These two proteins are derived from the Cwp84-mediated cleavage of a single precursor protein SlpA. In this study, we assessed the immunogenic properties of a recombinant SlpA precursor derived from a toxigenic C. difficile strain (630) and its protective effect as a vaccine antigen co-administered with the cholera toxin as an adjuvant in both hamster and mouse models. First, we confirmed the immunogenicity of SlpA in humans. Sera from patients with C. difficile infection were analyzed by ELISA. Patients with CDI have a greater number of SlpA antibodies than healthy patients, confirming the immunogenicity of this protein during the pathogenic process. Then, rectal vaccination assays were performed in both conventional hamsters and mice. The animals' sera were sampled before and after vaccination, and were analyzed by ELISA. In addition, in the mouse model, feces were sampled after vaccination and IgA directed against SlpA were detected by ELISA. In both models, the intestinal colonization was evaluated by fecal bacterial count after challenge. Intra-rectal vaccination with SlpA and cholera toxin as an adjuvant induced a local and systemic humoral immune response in mice and hamsters potentially responsible for the weak decrease of C. difficile colonization in mice and the partial protection observed in a lethal-hamster model.