Abstract
Clostridium difficile is a Gram-positive bacterium and is the most commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. Despite the emergence of epidemic C. difficile strains having led to an increase in the incidence of the disease, a vaccine against this pathogen is not currently available. C. difficile strains produce two main toxins (TcdA and TcdB) and express three highly complex cell-surface polysaccharides (PSI, PSII and PSIII). PSII is the more abundantly expressed by most C. difficile ribotypes offering the opportunity of the development of a carbohydrate-based vaccine. In this paper, we evaluate the efficacy, in naive mice model, of PSII glycoconjugates where recombinant toxins A and B fragments (TcdA_B2 and TcdB_GT respectively) have been used as carriers. Both glycoconjugates elicited IgG titers anti-PSII although only the TcdB_GT conjugate induced a response comparable to that obtained with CRM197. Moreover, TcdA_B2 and TcdB_GT conjugated to PSII retained the ability to elicit IgG with neutralizing activity against the respective toxins. These results are a crucial proof of concept for the development of glycoconjugate vaccines against C. difficile infection (CDI) that combine different C. difficile antigens to potentially prevent bacterial colonization of the gut and neutralize toxin activity.
Highlights
Clostridium difficile is a Gram-positive, spore-forming and toxin-producing anaerobic gastrointestinal pathogen that is the major cause of antibiotic-associated colitis
The two C. difficile recombinant toxin fragments, TcdA_B2 and TcdB_GT, derive from a fragment design of TcdA and TcdB assisted by computer modeling
PSII is composed of hexaglycosyl repeating units hold together by phosphodiester bonds [22], and the assigned structure has been confirmed by synthesis of the non-reducing end terminal phosphorylated repeating unit [31]
Summary
Clostridium difficile is a Gram-positive, spore-forming and toxin-producing anaerobic gastrointestinal pathogen that is the major cause of antibiotic-associated colitis. The virulence of C. difficile is conferred primarily by two large exotoxins, toxins A and B, and there is evidence that protection against severe CDI is mediated by systemic antibodies to TcdA and TcdB [6,7,8]. PSII, after conjugation to CRM197 (non-toxic mutant of diphtheria toxin) [28], a carrier protein widely used for the manufacturing of glycoconjugate vaccines [29], was formulated with the adjuvant MF59 and tested in Balb/C mice, inducing high levels of specific anti carbohydrate IgG, a class of antibodies which is generally relevant to induce protection against the sugar coated pathogens [23]. We have evaluated the immunological response of PSII-toxin based glycoconjugates in mouse, investigating the possible double role of the two TcdA_B2 and TcdB_GT fragments, as carrier protein for the PSII polysaccharide and antigens able to elicit antibodies with toxin neutralizing activity
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