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Clostridium difficile is responsible for thousands of deaths each year and a vaccine would be welcomed, especially one that would disrupt bacterial maintenance, colonization and persistence in carriers and convalescent patients. Structural explorations at the University of Guelph (ON, Canada) discovered that C. difficile may express three phosphorylated polysaccharides, named PSI, PSII and PSIII; this review captures our recent efforts to create vaccines based on these glycans, especially PSII, the common antigen that has precipitated immediate attention. The authors describe the design and immunogenicity of vaccines composed of raw polysaccharides and conjugates thereof. So far, it has been observed that anti-PSII antibodies can be raised in farm animals, mice and hamster models; humans and horses carry anti-PSII IgA and IgG antibodies from natural exposure to C. difficile, respectively; phosphate is an indispensable immunogenic epitope and vaccine-induced PSII antibodies recognize PSII on C. difficile outer surface.

Campylobacter jejuni infection is now the main cause of diarrhea-related illnesses in humans. An efficacious vaccine for the traveler and developing world market would be welcomed. We are engaged in the discovery and characterization of serotype-specific C. jejuni capsule polysaccharides (CPSs) to study their role in virulence and as protective vaccine antigens. Our prototype conjugate vaccine with serotype HS23 CPS (strain 81–176) has been shown to fully protect non-human primates against diarrhea inflicted by C. jejuni HS23, but ultimately, a useful CPS-based vaccine will have to be multivalent. To this end, we describe here the creation of a CPS-conjugate vaccine against C. jejuni serotype HS15. Structural analysis revealed that a repeating block consisting of l -α-arabinofuranose (Ara) and 6-deoxy- l -α- gulo -heptopyranose (6d- gulo -Hep) comprised the CPS of serotype HS15 type strain ATCC 43442 [→3)-α- l -Ara f -(1→3)-6d- l -α- gulo -Hep p (1→] n . Strategically, the non-reducing end of the CPS was activated and used in the attachment of CPS to CRM 197 to yield a conjugate vaccine. A serological assessment of the CPS HS15 –CRM 197 conjugate with an anti-HS15 polyclonal antibody confirmed the conservation of antigenic epitopes, and subsequent inoculation of mice with CPS HS15 –CRM 197 revealed that this conjugate was indeed capable of raising anti-CPS HS15 antibodies.

Clostridium difficile is responsible for severe diarrhea in humans that may cause death. Spores are the infectious form of C. difficile, which germinate into toxin-producing vegetative cells in response to bile acids. Recently, we discovered that C. difficile cells possess three complex polysaccharides (PSs), named PSI, PSII, and PSIII, in which PSI was only associated with a hypervirulent ribotype 027 strain, PSII was hypothesized to be a common antigen, and PSIII was a water-insoluble polymer. Here, we show that (i) C. difficile spores contain, at least in part, a d -glucan, (ii) PSI is not a ribotype 027-unique antigen, (iii) common antigen PSII may in part be present as a low molecular weight lipoteichoic acid, (iv) selective hydrolysis of PSII yields single PSII repeat units, (v) the glycosyl diester–phosphate linkage affords high flexibility to PSII, and (vi) that PSII is immunogenic in sows. Also, with the intent of creating a dual anti-diarrheal vaccine against C. difficile and enterotoxin Escherichia coli (ETEC) infections in humans, we describe the conjugation of PSII to the ETEC-associated LTB enterotoxin.

Clostridium difficile is the most common cause of antimicrobial-associated diarrhea in humans and may cause death. Previously, we discovered that C. difficile expresses three polysaccharides, named PSI, PSII, and PSIII. It has now been established that PSII is a conserved antigen abundantly present on the cell-surface and biofilm of C. difficile. In contrast, the expression of PSI and PSIII appears to be stochastic processes. In this work, the total chemical synthesis of the PSI pentasaccharide repeating unit carrying a linker at the reducing end, α-l-Rhap-(1→3)-β-d-Glcp-(1→4)-[α-l-Rhap-(1→3)]-α-d-Glcp-(1→2)-α-d-Glcp-(1→O(CH2)5NH2, was achieved by a linear synthesis strategy from four monosaccharide building blocks. The synthesized PSI pentasaccharide was conjugated to a subunit of C. difficile exotoxin B yielding a potential dual C. difficile vaccine. More significantly, sera from healthy horses were shown to contain natural anti-PSI IgG antibodies that detected both the synthetic non-phosphorylated PSI repeat and the native PSI polysaccharide, with a slightly higher recognition of the native PSI polysaccharide.

A method for the controlled conjugation of polysaccharides (PSs) to carrier proteins based on the stoichiometric oxidation of PSs with 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) is described. In this scheme, a PS can be stoichiometrically activated, by the formation of minimal amounts of carboxylic acids with TEMPO, and subsequently coupled to a carrier protein through amide bond formation. Our exploratory studies included a number of readily available glucans, which were oxidized with several combinations of TEMPO-sodium hypochlorite preparations followed by conjugation to BSA. Following these investigations, more structurally complex bacterial capsular PSs were stoichiometrically oxidized with TEMPO-sodium hypochlorite and conjugated to BSA. Hypochlorite was deemed to be the reaction component responsible for the extent of oxidation. Collectively, the data showed this approach to be effective in designing PS-conjugates with no disruption to the structural integrity and immunochemical properties of the PS, and thus has the potential to become a reliable method for glycoconjugate vaccine synthesis.