Abstract
BackgroundOpportunistically nosocomial infections in hospitalized patients are often related to Clostridium difficile infections (CDI) due to disruption of the intestinal micro-flora by antibiotic therapies during hospitalization. Clostridial exotoxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) in the host intestine, disrupt the intestinal barrier leading to acute inflammation and diarrhea. The C-terminal receptor binding domain of TcdA (A-rRBD) has been shown to elicit antibody responses that neutralize TcdA toxicity in Vero cell cytotoxicity assays, but not effectively protect hamsters against a lethal dose challenge of C. difficile spores. To develop an effective recombinant subunit vaccine against CDI, A-rRBD was lipidated (rlipoA-RBD) as a rational design to contain an intrinsic adjuvant, a toll-like receptor 2 agonist and expressed in Escherichia coli.ResultsThe purified rlipoA-RBD was characterized immunologically and found to have the following properties: (a) mice, hamsters and rabbits vaccinated with 3 μg of rlipoA-RBD produced strong antibody responses that neutralized TcdA toxicity in Vero cell cytotoxicity assays; furthermore, the neutralization titer was comparable to those obtained from antisera immunized either with 10 μg of TcdA toxoid or 30 μg of A-rRBD; (b) rlipoA-RBD elicited immune responses and protected mice from TcdA challenge, but offered insignificant protection (10 to 20 %) against C. difficile spores challenge in hamster models; (c) only rlipoA-RBD formulated with B-rRBD consistently confers protection (90 to 100 %) in the hamster challenge model; and (d) rlipoA-RBD was found to be 10-fold more potent than A-rRBD as an adjuvant to enhancing immune responses against a poor antigen such as ovalbumin.ConclusionThese results indicate that rlipoA-RBD formulated with B-rRBD could be an excellent vaccine candidate for preclinical studies and future clinical trials.
Highlights
Nosocomial infections in hospitalized patients are often related to Clostridium difficile infections (CDI) due to disruption of the intestinal micro-flora by antibiotic therapies during hospitalization
In our previous study [30], a recombinant A-rRBD based on the consensus sequence of Clostridium difficile toxin A (TcdA) identified from different C. difficile strains obtained from the NCBI protein database and three truncated fragments of receptor binding domain (RBD) corresponding to the N-terminal, middle, and C-terminal parts (F1, F2 and F3, respectively) were designed and expressed in E. coli
The results demonstrate that 3 × 0.3 μg of rlipoA-RBD elicits neutralization titer >128 and provides full protective immune responses in mice against C. difficile TcdA challenge, and strongly suggests rlipoA-RBD is a good candidate for CDI vaccine developments
Summary
Nosocomial infections in hospitalized patients are often related to Clostridium difficile infections (CDI) due to disruption of the intestinal micro-flora by antibiotic therapies during hospitalization. Immunization with the receptor binding domain (RBD) of C. difficile toxins as an antigen formulated with different adjuvants has been shown to elicit toxin-neutralizing antibody responses and protect mice from toxin or bacteria challenges [20,21,22,23,24,25,26]. Recombinant TcdB RBD (B-rRBD) was purified, characterized biologically and immunologically, and found to have the following properties: (a) capable of binding to the cell surface of both Vero and Caco-2 cells and entering into the cytosol; (b) showing no hemagglutinin activity (HA); (c) functioning as a toll-like receptor agonist activating dendritic cell maturation; (d) in the absence of adjuvant, eliciting anti-TcdB neutralizing antibody responses that could weakly cross-neutralize
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