A novel fusion protein containing the receptor binding domains of C. difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models
Highlights
► C. difficile ToxinA:toxin B fusion protein. ► Induction of toxin neutralizing antibody. ► Immunoprotection against an in vivo C. difficile spore challenge.
Introduction
The increased incidence of infectious diarrheal disease associated with Clostridium difficile and the marked morbidity and mortality resulting from such disease has made treatment of C. difficile associated disease (CDAD) a priority for the health care system [1], [2]. Its virulence, spore forming ability and persistence contribute to its identification as a leading cause of diarrhea in hospitals worldwide. As the major nosocomial pathogen responsible for gastrointestinal diseases ranging from mild diarrhea to fulminant pseudomembraneous colitis, CDAD has been primarily associated with antibiotic treatment, however it is also a risk associated with immunosuppression, chemotherapy and gastrointestinal procedures. Within the hospital setting, between 10 and 25% of patients who undergo antibiotic treatment become infected [3], [4]. Most illnesses resolve following discontinuation of antibiotic treatment followed by administration of metronidazole or vancomycin, however, the incidence of clinical relapse and secondary infection with multi-resistant organisms can be as high as 25% within this group [2], [5].
The pathogenicity of C. difficile is mediated by the release of two potent exotoxins, toxin A and toxin B. Both toxins are high molecular weight (∼300 kDa) secreted proteins that possess multiple functional domains [7]. The N-terminal domain of both toxins contains ADP-glucosyltransferase activity that modifies Rho-like GTPases. This modification induced by the C. difficile toxins causes a loss of actin polymerization and cytoskeletal changes resulting in the disruption of the colonic epithelial tight junctions. This leads to excessive fluid exudation into the colon and a resulting diarrhea. The central domain of the toxins contains a hydrophobic domain and is predicted to be involved in membrane transport of the ADP-glucosyltransferase domain from the endosome into the cytoplasm. The carboxy-terminal domain of both toxins contains a series of repeating units of 21-, 30- or 50-amino acids referred to as “clostridial repetitive oligopeptides” [8], [9]. These repetitive units comprise the toxins’ receptor binding domain(s) (RBD) responsible for the binding of toxin to cell surface oligosaccharide receptors on the target cells [10], [11], [12]. The repeat units are thought to exert their function by amplifying the toxin binding affinity through an avidity effect [13]. Thirty-eight repetitive oligopeptides have been identified in toxin A and 24 in toxin B [13].
Animal studies have demonstrated that protection against CDAD and disease relapse correlates with the presence of anti-toxin serum antibody. Immunization of mice and hamsters with inactivated toxin (toxoid) and various toxin fragments induced protective immunity which is associated with high levels of toxin-neutralizing antibody [14], [15], [16], [17], [18], [19]. These results have been supported by the use of anti-toxin antibody in passive transfer studies which further showed that that titers of serum antibodies to toxin A and toxin B correlate with levels of protection [14], [20], [21]. Antibodies to both toxin A and toxin B were required to provide optimal protection against morbidity and mortality, as well as CDAD relapse and C. difficile reinfection [22], [23], [24].
Human studies indicated a protective role for anti-toxin antibodies in CDAD outcome. In one study, a significant correlation between serum anti toxin A antibody concentrations and protection from CDAD was observed [3]. In patients colonized with C. difficile and treated with antibiotics, those with low levels of anti-toxin serum IgG were 48 times more likely to develop CDAD. In a second study, protection from C. difficile relapse correlated with the early development of anti-toxin A antibody [25]. Additional studies have shown that the administration of pooled human IgG containing anti-toxin antibodies led to clinical improvement in patients with severe or protracted CDAD [26], [27], [28]. In a recent phase 2 clinical trial, the passive transfer of monoclonal antibodies to toxins A and B provided protection against recurrent C. difficile diarrhea in human subjects [29].
Observations obtained in a number of studies have indicated that the critical antigenic determinants recognized by anti-toxin antibodies are localized to the repetitive oligopeptides contained within the carboxy-terminal RBD of both toxin A and toxin B. Immunization with toxin A fragments containing only the repetitive oligopeptide sequences induced toxin neutralizing antibody and was protective in hamsters receiving a lethal C. difficile spore challenge [19], [20], [21], [29], [30]. Human monoclonal antibodies against toxins A and B shown to confer protection against CDAD in hamsters [23] and providing protection against recurrent C. difficile diarrhea in human subjects [31] have also been mapped to this region. These observations indicate that blocking the binding of the toxins to their receptors is critical for the prevention of CDAD.
In this study we have constructed a recombinant fusion protein containing portions of the carboxy-terminal RBD from both toxin A and toxin B. The carboxy-terminal toxin A and B fusion protein (C-TAB) was evaluated in multiple animal models for its immunogenicity, and its ability to induce toxin neutralizing antibody and in vivo anti-toxin protective immunity. The data presented in this report demonstrate that the C-TAB fusion protein represents a novel and highly efficacious vaccine candidate for the protection against CDAD.
Section snippets
Animals
Female C57BL/6 mice 6–7 weeks of age were purchased from Charles River Labs, Wilmington, MA and were maintained under specific-pathogen-free conditions. Female Cynomolgus monkeys 4–6 years of age (body weight 2.0–4.0 kg) were provided by Avanza Laboratories, Gaithersburg, MD. Golden Syrian adult female hamsters weighing ∼100 g (6–7 weeks of age) were purchased from Harlan Laboratories, Bristol, TN and were housed individually in micro-isolator cages (Micro-Vent Environmental System; Allentown
Construction and purification of the C-TAB fusion protein
The gene encoding the C-TAB.G5 fusion protein was constructed as described in Section 2. The fusion protein (Fig. 1) contains the carboxy-terminal portion of the toxin A RBD fused to the carboxy-terminal portion of the toxin B RBD separated by a 4 amino acid linker sequence (arg–ser–met–his). The fusion protein was constructed to contain 19 of the 39 repetitive oligopeptides found in the full length toxin A RBD and 23 of the 24 repetitive oligopeptides found in the full length toxin B RBD.
Discussion
The pathogenesis of CDAD is mediated by two C. difficile secreted toxins, toxin A, both a cytotoxin and endotoxin, and toxin B, a more potent cytotoxin. The action of these toxins on the intestinal epithelium results in the stimulation of intestinal fluid secretion and the release of proinflammatory mediators which results in mucosal injury, inflammation of the colon and diarrhea. The role of serum anti-toxin antibodies in mediating systemic and mucosal protection against CDAD in both animal
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Development and verification of an enzyme-linked immunosorbent assay for the quantification of toxoid A and toxoid B from Clostridioides difficile
2021, Journal of Immunological MethodsCitation Excerpt :A novel fusion protein containing the RBD of both C. difficile toxin A and B has shown to induce anti-toxin A and anti-toxin B antibodies in mice, hamsters and monkeys, which were capable of neutralising both toxins in vitro and in vivo. This fusion protein has also demonstrated protective efficacy in the hamster C. difficile associated-disease (CDAD) model by reducing the severity and delaying the time to onset of CDAD and significantly reducing mortality following a spore challenge (Tian et al., 2012). Therefore, toxoids are important for inducing protective immunity.
- 1
Current address: Intrexon Corporation, Germantown, MD, USA.
- 2
Current address: Emergent Biosolutions, Gaithersburg, MD, USA.
- 3
Current address: Novavax, Inc. Rockville, MD, USA.