Vaccine 30 (2012) 4249– 4258Contents lists available at SciVerse ScienceDirectVaccinejou rn al h om epa ge: www.elsevier.com/locate/vaccineA novel fusion protein containing the receptor binding domains of C. difficiletoxin A and toxin B elicits protective immunity against lethal toxin and sporechallenge in preclinical efficacy modelsJing-Hui Tiana,3, Steven R. Fuhrmanna,1, Stefanie Kluepfel-Stahla,2, Robert J. Carmanb,Larry Ellingswortha, David C. Flyera,3,∗aIntercell, USA, 22 Firstfield Road, Gaithersburg, MD 20878, USAbTechLab Inc., Blacksburg, VA, USAa r t i c l e i n f oArticle history:Received 4 January 2012Received in revised form 12 March 2012Accepted 10 April 2012Available online 23 April 2012Keywords:Clostridium difficileClostridium difficile associated diseaseVaccineToxinFusion proteina b s t r a c tAntibodies targeting the Clostridium difficile toxin A and toxin B confer protective immunity to C. dif-ficile associated disease in animal models and provided protection against recurrent C. difficile diseasein human subjects. These antibodies are directed against the receptor binding domains (RBD) locatedin the carboxy-terminal portion of both toxins and inhibit binding of the toxins to their receptors. Wehave constructed a recombinant fusion protein containing portions of the RBD from both toxin A andtoxin B and expressed it in Escherichia coli. The fusion protein induced high levels of serum antibodiesto both toxins A and B capable of neutralizing toxin activity both in vitro and in vivo. In a hamster C.difficile infection model, immunization with the fusion protein reduced disease severity and conferredsignificant protection against a lethal dose of C. difficile spores. Our studies demonstrate the potential ofthe fusion protein as a vaccine that could provide protection from C. difficile disease in humans.© 2012 Elsevier Ltd. All rights reserved.1. IntroductionThe increased incidence of infectious diarrheal disease asso-ciated with Clostridium difficile and the marked morbidity andmortality resulting from such disease has made treatment of C.difficile associated disease (CDAD) a priority for the health caresystem [1,2]. Its virulence, spore forming ability and persistencecontribute to its identification as a leading cause of diarrhea inhospitals worldwide. As the major nosocomial pathogen respon-sible for gastrointestinal diseases ranging from mild diarrhea tofulminant pseudomembraneous colitis, CDAD has been primar-ily associated with antibiotic treatment, however it is also a riskassociated with immunosuppression, chemotherapy and gastroin-testinal procedures. Within the hospital setting, between 10 and25% of patients who undergo antibiotic treatment become infected[3,4]. Most illnesses resolve following discontinuation of antibioticAbbreviations: CDAD, Clostridium difficile associated disease; RBD, receptor bind-ing domains; C-TAB, carboxy-terminal toxin A and B; C-TAB.G5, C-TAB generation5; PRAS, pre-reduced anaerobically sterilized; MLD100, minimum 100% lethal dose;ED50, 50% effective dose.∗Corresponding author. Tel.: +1 240 671 6312; fax: +1 240 268 2100.E-mail addresses: dcflyer51@gmail.com, dflyer@novavax.com (D.C. Flyer).1Current address: Intrexon Corporation, Germantown, MD, USA.2Current address: Emergent Biosolutions, Gaithersburg, MD, USA.3Current address: Novavax, Inc. Rockville, MD, USA.treatment followed by administration of metronidazole or van-comycin, however, the incidence of clinical relapse and secondaryinfection 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 oftwo potent exotoxins, toxin A and toxin B. Both toxins are highmolecular weight (∼300 kDa) secreted proteins that possess mul-tiple functional domains [7]. The N-terminal domain of both toxinscontains ADP-glucosyltransferase activity that modifies Rho-likeGTPases. This modification induced by the C. difficile toxins causesa loss of actin polymerization and cytoskeletal changes resulting inthe disruption of the colonic epithelial tight junctions. This leadsto excessive fluid exudation into the colon and a resulting diar-rhea. The central domain of the toxins contains a hydrophobicdomain and is predicted to be involved in membrane transportof the ADP-glucosyltransferase domain from the endosome intothe cytoplasm. The carboxy-terminal domain of both toxins con-tains a series of repeating units of 21-, 30- or 50-amino acidsreferred to as “clostridial repetitive oligopeptides” [8,9]. Theserepetitive units comprise the toxins’ receptor binding domain(s)(RBD) responsible for the binding of toxin to cell surface oligosac-charide receptors on the target cells [10–12]. The repeat unitsare thought to exert their function by amplifying the toxin bind-ing affinity through an avidity effect [13]. Thirty-eight repetitiveoligopeptides have been identified in toxin A and 24 in toxin B[13].0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.vaccine.2012.04.045
