Neutralization of Clostridium difficile Toxin A with Single-domain Antibodies Targeting the Cell Receptor Binding Domain

Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario K1A 0R6, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2011; 286(11):8961-76. DOI: 10.1074/jbc.M110.198754
Source: PubMed


Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals
and nursing homes. The Gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) and toxin B (TcdB),
which are the major virulence factors responsible for C. difficile-associated disease and are targets for C. difficile-associated disease therapy. Here, recombinant single-domain antibody fragments (VHHs), which specifically target the cell receptor binding domains of TcdA or TcdB, were isolated from an immune llama phage
display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8), all shown to recognize conformational epitopes, were potent neutralizers of the cytopathic
effects of toxin A on fibroblast cells in an in vitro assay. The neutralizing potency was further enhanced when VHHs were administered in paired or triplet combinations at the same overall VHH concentration, suggesting recognition of nonoverlapping TcdA epitopes. Biacore epitope mapping experiments revealed that
some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased
neutralization. Further binding assays revealed TcdA-specific VHHs neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin. With favorable characteristics
such as high production yield, potent toxin neutralization, and intrinsic stability, these VHHs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal

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    • "The toxin neutralization capacity shown in mice exceeds the results provided from similar scFv-based constructs. In addition, several Nbs targeting bacterial toxins and snake venoms have been reported.42,77 A range of other human pathogens, such as Vibrio cholerae and Salmonella enterica, might also be suitable for Nb targeting. "
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    ABSTRACT: The discovery of naturally occurring, heavy-chain only antibodies in Camelidae, and their further development into small recombinant nanobodies, presents attractive alternatives in drug delivery and imaging. Easily expressed in microorganisms and amenable to engineering, nanobody derivatives are soluble, stable, versatile, and have unique refolding capacities, reduced aggregation tendencies, and high-target binding capabilities. This review outlines the current state of the art in nanobodies, focusing on their structural features and properties, production, technology, and the potential for modulating immune functions and for targeting tumors, toxins, and microbes.
    International Journal of Nanomedicine 01/2013; 8:4215-4227. DOI:10.2147/IJN.S39428 · 4.38 Impact Factor
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    • "The use of toxin neutralising antibody fragments in the gastrointestinal tract can potentially be used as treatment against other pathogens like Clostridium difficile, Vibrio cholera and E.coli O157:H. Recently a neutralising single domain antibody fragment (VHH) against Clostridium difficile toxin A was developed [31] illustrating this approach. We have previously shown that VHH can be produced at high levels using the described expression system and in addition have advantages over scFvs for expression in lactobacilli [25], so as more VHH are being developed their use expressed from engineered lactobacilli for therapies in the gastrointestinal tract will likely increase. "
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    ABSTRACT: Anthrax is caused by the bacterium Bacillus anthracis and is regarded as one of the most prominent bioterrorism threats. Anthrax toxicity is induced by the tripartite toxin complex, composed of the receptor-binding anthrax protective antigen and the two enzymatic subunits, lethal factor and edema factor. Recombinant lactobacilli have previously been used to deliver antibody fragments directed against surface epitopes of a variety of pathogens, including Streptococcus mutans, Porphyromonas gingivalis, and rotavirus. Here, we addressed whether or not anthrax toxins could be targeted and neutralised in the gastrointestinal tract by lactobacilli producing recombinant antibody fragments as a model system for toxin neutralisation in the gastrointestinal lumen. The neutralising anti-PA scFv, 1H, was expressed in L. paracasei as a secreted protein, a cell wall-anchored protein or both secreted and wall-anchored protein. Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis. Binding of secreted or attached scFv produced by lactobacilli to PA were verified by ELISA. Both construct were able to protect macrophages in an in vitro cytotoxicity assay. Finally, lactobacilli producing the cell wall attached scFv were able to neutralise the activity of anthrax edema toxin in the GI tract of mice, in vivo. We have developed lactobacilli expressing a neutralising scFv fragment against the PA antigen of the anthrax toxin, which can provide protection against anthrax toxins both in vitro and in vivo. Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens. The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.
    BMC Biotechnology 12/2011; 11(1):126. DOI:10.1186/1472-6750-11-126 · 2.03 Impact Factor
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    • "Recently, human scFvs derived from a phage display library were developed that neutralize the cytotoxicity of Shiga toxins produced by enterohemorragic E. coli bacteria [29]. The efficacy of single domain antibodies (VHH) to neutralize Clostridium difficile toxin A was demonstrated by the in vitro assay [28]. The ability to neutralize botulinum neurotoxin in mice was demonstrated using chimeric antibody generated by fusion of hybridoma-derived scFv with human Fc domain [26]. "
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    ABSTRACT: Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G₄S)₄ were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis.
    BMC Biotechnology 11/2011; 11(1):100. DOI:10.1186/1472-6750-11-100 · 2.03 Impact Factor
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