Protective Efficacy Induced by Recombinant Clostridium difficileToxin Fragments
Rosanna Leuzzi,a Janice Spencer,b Anthony Buckley,b Cecilia Brettoni,a Manuele Martinelli,a Lorenza Tulli,a Sara Marchi,a
Enrico Luzzi,a June Irvine,b Denise Candlish,b Daniele Veggi,a Werner Pansegrau,a Luigi Fiaschi,a* Silvana Savino,a Erwin Swennen,a
Osman Cakici,a Ernesto Oviedo-Orta,a Monica Giraldi,a Barbara Baudner,a Nunzia D’Urzo,a Domenico Maione,a Marco Soriani,a
Rino Rappuoli,a Mariagrazia Pizza,a Gillian R. Douce,b Maria Scarsellia
Novartis Vaccines and Diagnostics SRL, Siena, Italya; Institute of Infection, Immunity and Inflammation, School of Medicine, Veterinary and Life Sciences, University ofGlasgow, Glasgow, Scotlandb
Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety ofclinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxinsreleased by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments toinduce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a ham-ster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding do-main fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutral-ized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Furthercharacterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acutephase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdBantibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxinproduction during C. difficile infection.
Clostridium difficile is a Gram-positive, sporulating, toxigenicbacterium which colonizes and infects both humans and ani-
mals (1, 2). Although asymptomatic carriage occurs in 4 to 20% ofthe healthy human population (3–8), susceptibility to C. difficile-associated infection (CDI) increases with age, hospitalization, im-munodeficiency, and antibiotic treatment. Clinical symptomsrange from mild to severe diarrhea with relapsing episodes. Com-plications include severe pseudomembranous colitis (PMC), toxicmegacolon, and sepsis (9, 10).
CDI accounts for 15 to 39% of cases of antibiotic-associateddiarrhea (11–13). The disease is particularly prevalent in healthcare facilities, where use of broad-spectrum antibiotic treatmentseverely disrupts the resident protective bowel flora, enabling C.difficile to colonize the gut and induce clinical symptoms throughthe production of two large exotoxins, TcdA and TcdB. TcdA andTcdB have molecular masses of 308 and 270 kDa, respectively (14,15). Like all members of the large clostridial toxin family, theycontain three distinct domains, namely, an N-terminal enzymaticdomain consisting of glucosyltransferase (GT) and cysteine pro-tease (CP) moieties, a central translocation (T) domain putativelygoverning their import into the host cell, and a C-terminal recep-tor binding domain (RBD) responsible for interaction with thecellular receptors (16).
In 2002, a hypervirulent C. difficile ribotype known as BI/NAP1/027 (or just ribotype 027) emerged in North America (17,18) and Europe (19, 20). Ribotype 027 strains displayed increasedseverity and mortality and were frequently associated with largeoutbreaks (17–20). This change in clinical profile has been linkedto alterations in resistance to fluoroquinolones (21), modifica-tions in toxin production (22), sporulation rates (23), and theability to infect a wider range of patients, including hospitalizedchildren and pregnant women (24, 25).
Management of severe CDI is currently based on the use of
vancomycin and metronidazole (26, 27). Recently, the novel an-tibiotic fixadomicin was reported to be more effective than van-comycin at preventing relapses (28).
Active or passive immunization can represent an alternativefor CDI prevention and treatment. Individuals with high levels ofserum IgG against TcdA and TcdB are protected from recurrentinfection (4, 29–31), and administration of monoclonal antibod-ies against TcdA and TcdB has been shown to be effective at pre-venting recurrences (32).
Compared to passive immunization, the development of aneffective prophylactic vaccine against C. difficile offers the oppor-tunity to provide long-lasting protection against disease. Prepara-tions of formaldehyde-inactivated toxoid from C. difficile culturesupernatants have been shown to be well tolerated and able toinduce seroconversion in clinical trials (33). However, long-last-ing protection, particularly against TcdB, remains challenging(34). To eliminate the intrinsic risk of contamination or incom-
Received 27 November 2012 Returned for modification 25 January 2013Accepted 17 May 2013
plete toxoid inactivation, recombinant polypeptides as potentialvaccine candidates have been considered in several studies. In par-ticular, TcdA and TcdB RBDs have been cloned and purified froma variety of hosts (35–39) and then evaluated for the ability to raiseprotective immunity. Fragments derived from the RBD of TcdAhave been shown to induce both systemic and mucosal neutraliz-ing antibodies in animal models (35–39).
Initial reports suggested that only anti-TcdA antibodies werenecessary to provide complete protection against C. difficile-asso-ciated disease (4, 30). However, the role of TcdB in pathogenesis ofCDI and the importance of anti-TcdB immunity in preventingdisease and recurrences were recently reevaluated (31, 40, 41).Moreover, the increased frequency of TcdA-negative, TcdB-posi-tive strains responsible for severe clinical symptoms (42–45) pro-vides strong evidence that TcdB is a key factor in C. difficile diseaseand clearly suggests that neither toxin can be ignored in the devel-opment of an effective vaccine. The existence of TcdA-negative,TcdB-positive strains able to cause disease in humans thereforeprovided the rationale for including TcdB fragments in a vaccineformulation.
Recently, two chimeric recombinant vaccines against C. diffi-cile were proposed. The first combined the RBDs of both toxins ina single polypeptide chain (46), while the second was derived froma full-length TcdB protein in which the original RBD was replacedby the corresponding portion of TcdA (47). Both constructs wereprotective in vivo, priming further research on the development ofrecombinant polypeptides as potential vaccine candidates.
In this study, we explored the efficacy of a panel of rationallydesigned recombinant fragments derived from TcdA and TcdB asimmunogens. Our results demonstrate that coadministration ofthe glucosyltransferase domain of TcdB with a carboxyl-terminalfragment of the TcdA RBD protects hamsters against lethal chal-lenge with C. difficile and significantly reduces clinical signs ofinfection.
(This work was featured in poster presentations at the 4th In-ternational Clostridium difficile Symposium, Bled, Slovenia, 20 to22 September 2012.)
MATERIALS AND METHODSCloning of recombinant fragments and generation of mutants. All frag-ments reported in Fig. 1A, except TcdA-GT and TcdA-B1, were clonedinto the pET15b� vector (Novagen) by the polymerase incompleteprimer extension (PIPE) method (48). In brief, sequences coding for eachfragment were amplified by PCR from the C. difficile 630 genomic DNA,using the primers listed in Table S1 in the supplemental material. PCRsgenerated mixtures of incomplete extension products; by primer design,short overlapping sequences were introduced at the ends of these incom-plete extension mixtures, which allowed complementary strands to an-neal and produce hybrid vector-insert combinations. Escherichia coliHK100 cells (49) were then transformed with vector-insert hybrids. Singleampicillin-resistant colonies were selected and checked for the presence ofthe recombinant plasmid by PCR. Plasmids from positive clones wereisolated and subcloned into competent E. coli BL21(DE3) cells (Novagen).
TcdA-GT was expressed in Brevibacillus choshinensis, as describedelsewhere (50), while TcdA-B1 (previously referred as 14CDTA) was ex-pressed as described previously (35).
The PIPE method was employed to generate TcdA-GT (Y283A,D285A, and D287A), TcdA-CP (D589A, H655A, and C700A), TcdB-GT(D270A, R273A, Y284A, D286A, and D288A), and TcdB-CP (D587A,H653A, and C698A) mutants with abrogated enzymatic activity.
Expression and purification of recombinant fragments. A single col-ony of E. coli BL21(DE3) cells expressing each recombinant fragment was
inoculated into LB containing 100 �g/ml ampicillin and grown overnight(ON) at 37°C. The bacterial culture was diluted in fresh medium, andprotein expression was induced by addition of 1 mM IPTG (isopropyl-�-D-thiogalactopyranoside) to the culture during exponential growth phase.Cultures were then incubated for 4 h at 25°C. Expression of each recom-binant fragment was determined by SDS-PAGE using a NuPAGE gel sys-tem (Invitrogen).
All recombinant polypeptides were purified by immobilized-metalion affinity chromatography (IMAC), and buffer exchange was per-formed by use of a PD-10 desalting column (GE Healthcare) or by dialysis.Protein concentration was determined using the bicinchoninic acid(BCA) assay (Thermo Scientific). Protein purity was checked by SDS-PAGE using NuPAGE 4 to 12% Bis-Tris gels (Invitrogen) followed byCoomassie blue staining. Protein identity was confirmed by dot blottingusing sera raised against inactivated TcdA and -B.
FIG 1 (A) Design of recombinant TcdA and TcdB fragments of the enzymatic(ED) and binding (B) domains. Attempts to express fragments of the translo-cation domain (T) resulted in low yields and poor solubility, leading to theexclusion of such fragments from further analysis. (B) SDS-PAGE analysis ofpurified recombinant fragments. Apparent molecular masses are reported inkilodaltons.
Toxin purification. To provide native TcdA and TcdB toxins, C. dif-ficile strain VPI10463 was inoculated onto BHIS (brain heart infusionsupplemented with 5 mg/ml of yeast extract and 0.1% L-cysteine) plates.Colonies were recovered, inoculated into tryptone-yeast extract-mannitol(TYM) medium, and grown ON at 37°C under anaerobic conditions(Whitley MG 500 anaerobic system). Cultures were then diluted 1/100 infresh medium and incubated for 5 days at 37°C under anaerobic condi-tions. Samples were centrifuged, and the supernatants were filteredthrough a 0.22-�m-pore-size filter and concentrated (6�) by tangential-flow filtration.
Concentrated supernatants were fractionated by precipitation withammonium sulfate at 45% saturation for TcdA enrichment and at 65%saturation for TcdB enrichment. After stirring for 3 h at 0°C, the precip-itate was concentrated by centrifugation at 9,000 � g for 30 min at 4°C.The pellet was resuspended in buffer A (50 mM Tris-HCl, pH 7.5, 50 mMNaCl) and then dialyzed at 4°C against two changes of buffer A. TcdA andTcdB were purified from the precipitated proteins by chromatographyusing two 5-ml HiTrap Q HP columns (GE Healthcare) connected inseries. A linear gradient from 50 to 1,000 mM NaCl in buffer A was appliedusing 30 column volumes (CV) at 2 ml/min. TcdA and TcdB were elutedin distinct peaks around 200 and 500 mM NaCl, respectively, and werepooled separately. The pool containing TcdB was dialyzed against buffer B(20 mM piperazine-HCl, pH 5.0, 50 mM NaCl), and TcdB was furtherpurified at pH 5.0 by chromatography on a 5-ml HiTrap Q HP columnequilibrated in buffer B. A segmented gradient from 300 to 600 mM NaClin buffer B was applied using 15 CV at 2 ml/min to elute nearly homoge-neous TcdB. The pool containing TcdA was dialyzed against buffer A andapplied to a second chromatography step on a 5-ml HiTrap Q HP columnequilibrated in buffer A. A segmented gradient from 50 to 250 mM NaClin buffer A was applied using 30 CV at 2 ml/min to elute nearly homoge-neous TcdA. Fractions were collected and analyzed by SDS-PAGE. Theabsence of cross contamination in TcdA and TcdB preparations was ver-ified by Western blotting with specific antibodies against each toxin. Pu-rified toxins were dialyzed against 50 mM Tris-HCl, 500 mM NaCl, and10% glycerol and then stored at �20°C until further use.
Toxin inactivation. After dialysis against phosphate-buffered saline(PBS), TcdA and -B were inactivated by treatment with formaldehydeunder the following conditions: for TcdA, 0.58 �M TcdA, 56 mM lysine,and 10 mM formaldehyde in PBS; and for TcdB, 0.93 �M TcdB, 10 mMlysine, and 3.9 mM formaldehyde in PBS. After 120 h on a rotary shaker at37°C, samples were dialyzed against 2 changes of a 500-fold volume of PBStwice for 24 h each. Samples were confirmed as being inactivated using thecell-based toxicity assay described below.
Mouse immunization. CD1 outbred mice (Charles River, Italy) re-ceived three intraperitoneal (i.p.) injections with 10 �g of recombinantproteins in PBS, on days 1, 21, and 35. Two groups of eight animals eachwere immunized with each antigen or combination of antigens. The firstgroup received the polypeptides adsorbed to aluminum hydroxide, whilethe second group received the antigens mixed with an oil-in-water emul-sion of 4.3% squalene oil, 0.5% Tween 80, and 0.5% Span 85 detergents(MF59 adjuvant) (51). Control mice were immunized with adjuvantalone, and serum samples were collected on day 49. Prior to each experi-ment, the appropriate amount of aluminum hydroxide for complete an-tigen adsorption was determined. Aluminum hydroxide was titratedagainst increasing quantities of protein in PBS. After ON incubation at4°C in a vertical rotator (PTR 25–360; Grant Instruments Ltd., Cam-bridge, United Kingdom), preparations were centrifuged and superna-tants were analyzed for the presence of unbound antigen. For all the poly-peptides, the minimum quantity of aluminum hydroxide required for fulladsorption was 2 mg/ml, and this dosage was used for all mouse immu-nizations. The final osmolarity of each formulation was optimized to0.300 � 0.06 osmol/kg of body weight by addition of 2 M NaCl and 10mM histidine, pH 6.5.
Enzyme-linked immunosorbent assay (ELISA). Microtiter plates(Greiner Bio-One) were coated ON at 4°C with 1-�g/ml purified TcdA or
TcdB. Wells were washed three times with PBS plus 0.1% Tween 20(PBS-T) and blocked with 2.7% polyvinylpyrrolidone (PVP; Serva) for 2 hat 37°C. After three washes with PBS-T, plates were incubated with mousesera diluted 1:1,000 for 2 h at 37°C, followed by incubation with alkalinephosphatase-conjugated anti-mouse antibodies diluted 1:2,000 in PBS-Tplus 1% bovine serum albumin (BSA) for 90 min at 37°C. Samples wereincubated with p-nitrophenyl phosphate (SigmaFast OPD; Sigma) atroom temperature for 30 min, and the reaction was stopped with 4 NNaOH. Optical density was analyzed using a plate reader at a dual wave-length of 405/620 to 650 nm. Antibody titers were quantified via interpo-lation against a reference standard curve.
Cytotoxicity and neutralization assays. IMR-90 human fibroblastswere obtained from the American Type Culture Collection (ATCC; Rock-ville, MD). Cells were grown to 80 to 90% confluence in 96-well plates inEagle’s minimum essential medium (EMEM) with 10% fetal calf serum.The minimal doses of TcdA and TcdB needed to cause 100% rounding in24 h (1 CTU100) were defined as 20 ng/ml for TcdA and 10 pg/ml for TcdB.
For the neutralization assay, 2-fold dilutions of mouse or hamster serafrom 1:16 to 1:32,000 were preincubated with 1 CTU100 of each toxin incell medium for 90 min at 37°C. Serum and toxin mixtures were thenadded to the cells and incubated for 16 to 18 h before analysis. Preimmunesera and/or sera from mice treated with adjuvant alone were used as neg-ative controls. The endpoint titer was defined as the reciprocal of thehighest dilution able to inhibit cell rounding. To check the absence ofcytotoxicity, recombinant TcdA-CP, TcdB-CP, TcdA-GT, and TcdB-GTwere serially diluted from 40 �g/ml to 20 ng/ml and added to the cells for24 h at 37°C; cells were observed after 16 to 18 h to evaluate morphologicalalterations. TcdA and TcdB (1 CTU100 each) were used as positive con-trols.
Spore preparation. C. difficile strains 630 and B1 were grown in BHIbroth under anaerobic conditions for 7 to 10 days. Cultures were thenpelleted by centrifugation and vortexed every 10 min for 1 h before cen-trifugation for 10 min. The pellet was then treated with 1% Sarkosyl inPBS for 1 h at room temperature and again pelleted by centrifugation,followed by incubation ON at 37°C with lysozyme (10 mg/ml) in 125 mMTris-HCl buffer (pH 8.0). The sample was treated in a sonicating waterbath (3 pulses of 3 min each; Branson model 1510 instrument) beforecentrifugation through a 50% sucrose gradient for 20 min. The pelletwas incubated in 2 ml of PBS containing 200 mM EDTA, 300 ng/mlproteinase K, and 1% Sarkosyl for 30 min at 37°C before centrifugationthrough a 50% sucrose gradient for 20 min. The final pellet was thenwashed twice in sterile distilled water before finally being resuspendedin 1 ml of sterile water. Spore preparations were stored at �80°C priorto use. Serial 10-fold dilutions of the spore preparations were inocu-lated onto BHI agar plates to determine the number of spores capableof germinating.
Hamster immunization and C. difficile challenge. Experiments onfemale Golden Syrian hamsters (Harlan Olac, United Kingdom) wereperformed in strict accordance with the requirements of the Animals (Sci-entific Procedures) Act 1986. Prior approval for these procedures wasgranted by the University of Glasgow Ethical Review Panel and by the UKHome Office (project license 60/4218).
Three weeks prior to the start of vaccination, telemetry chips (Vi-talview Emitter) were inserted by laparotomy into the body cavities of allanimals. These chips allowed continuous monitoring of the body temper-ature and activity of each animal. Hamsters were immunized i.p. at days 0,14, 28, and 42 with 50 or 20 �g of each antigen in PBS mixed at a 1:1volume ratio with the MF59 adjuvant, diluted to a final volume of 200�l/dose with PBS. Control hamsters were immunized with adjuvant aloneor PBS. Three weeks after the last immunization, animals received 30mg/kg of clindamycin phosphate orogastrically and were challenged thefollowing day with 1,000 spores of C. difficile strain B1 or 630 administeredby oral gavage (52). Animals were monitored for symptoms of infection,including onset and duration of loose stools (wet tail); hamsters showinga drop in temperature of more than two degrees (35°C) or losing more
than 10% of their body mass were culled (52). Animals surviving for morethan 2 weeks postchallenge were culled to provide an endpoint to theexperiment. Sera were collected before culling.
To exclude the interference of infection with the antitoxin immuneresponse, the neutralization activity of serum collected before challengefrom a hamster vaccinated with the combination of TcdA-B2, TcdA-GT,TcdB-B3, and TcdB-GT (TcdA-B2�TcdA-GT�TcdB-B3�TcdB-GT)was also analyzed.
Measurement of toxins in gut samples. Production of C. difficile tox-ins was detected in vitro by using filtered cecum contents taken postmor-tem as described previously (53). Briefly, monolayers of Vero cells(kidney epithelial cells) or HT29 cells were washed with preheated sterilePBS before addition of serially diluted filtered gut content in supple-mented EMEM and then were incubated for 18 h at 37°C with 5% CO2.Cells were washed with PBS, fixed in 1% formalin for 10 min, and thenwashed again. Fixed adherent cells were stained with Giemsa stain for 30min, washed before addition of 0.1% SDS, and left to stand for 1 h. Theoptical density at 600 nm was taken using an ELx808 Ultra microplatereader (Bio-Tek Instruments) and compared with those of noninfectedhamster cecum and colon gut contents as a negative control. If the toxindilution was able to cause cell toxicity (cell rounding), loss of cell adher-ence was observed, resulting in reduced staining and a reduced opticaldensity of the wells. Results were expressed as log reciprocal titers (53).Toxin levels were analyzed using the nonparametric two-tailed Mann-Whitney test.
Dot blot analysis. Twofold dilutions of native TcdA and TcdB, rang-ing from 100 down to 3.12 ng, were spotted on a nitrocellulose membrane.After blocking in 10% milk in PBS with 0.05% Tween 20, membraneswere incubated with hamster gut washes (see above) diluted 1:100 andthen incubated with horseradish peroxidase (HRP)-conjugated second-ary anti-hamster antibodies. Spots were visualized with a Super SignalWest Pico chemiluminescent substrate kit (Pierce).
Histology. Cecum samples were prepared for simple histology as pre-viously described (52). Cecum pathology was scored in a blinded fashion,grading neutrophil accumulation (0, no neutrophil accumulation; 1, localacute neutrophil accumulation; 2, extensive submucosal neutrophil accu-mulation; and 3, transmural neutrophilic infiltrate), hemorrhagic conges-tion (0, normal tissue; 1, engorged mucosal capillaries; 2, submucosalcongestion with unclotted blood; and 3, transmural congestion with un-clotted blood), hyperplasia (0, no epithelial hyperplasia; 1, 2-fold increasein thickness; 2, 3-fold increase in thickness; and 3, 4-fold or greater in-crease in thickness), and percentage of epithelial barrier involvement(0, no damage; 1, less than 10% of mucosal barrier involved; 2, less than50% of mucosal barrier involved; and 3, more than 50% of mucosal bar-rier involved). Results are expressed as the mean pathology score perstrain for each criterion and are reported in Fig. S5 in the supplementalmaterial.
RESULTSDesign and expression of recombinant fragments. The presenceof epitopes inducing neutralizing antibodies was explored alongthe TcdA and TcdB sequences by expressing the recombinantfragments summarized in Fig. 1A.
Nucleic acid sequences of TcdA and TcdB from C. difficilestrain 630 were used as templates for subcloning. As suggested bythe tripartite organization of both toxins, the glucosyltransferase(GT) and cysteine protease (CP) regions were subcloned. More-over, the RBDs were subdivided into several fragments.
Fragment design was assisted by computer modeling. TheTcdA and TcdB binding domains both consist of a succession ofshort and long sequence repeats (54). The X-ray structure of theTcdA C-terminal fragment (residues 2208 to 2710) revealed thatthe repetitive sequence folds into a repetitive solenoid-like struc-ture (54), and this provided the rationale to model the entire cell
binding domains of TcdA (TcdA-B) and TcdB (TcdB-B) by usingthe available atomic coordinates as a template. To preserve theoriginal fold, the TcdA-B and TcdB-B portions were then de-signed by avoiding interruptions of the structural repeats pre-dicted by computer modeling (see Fig. S1 in the supplementalmaterial). TcdA-B was subdivided into three different fragmentsthat were progressively shortened at the N terminus (called TcdA-B1, TcdA-B2, and TcdA-B3). One additional fragment, calledTcdA-B4, corresponded to a region near the N terminus of TcdA-B3. TcdB-B was subdivided into fragments TcdB-B1, TcdB-B2,and TcdB-B3, which overlapped at the carboxyl terminus. Recom-binant GT and CP portions of the N-terminal domains were mu-tated in their catalytic active sites to remove the enzymatic activity.Abrogation of toxicity was assessed by an in vitro assay on humanIMR-90 cells (see Fig. S2). Electrophoretic profiles of the proteinsused for immunization were evaluated by Coomassie staining ofan SDS-PAGE gel (Fig. 1B).
Immunogenicity. Systemic antibody responses to toxin frag-ments were analyzed by measuring antigen-specific IgG titers inthe sera of immunized mice (see Fig. S3 in the supplemental ma-terial). RBD fragments, with the exception of TcdA-B4, werehighly immunogenic. Lower titers were detected against the enzy-matic portions, although a significant IgG response was inducedby TcdB-GT. IgG titers were comparable and were independent ofthe adjuvant formulation. Sera against TcdA fragments showedvery low IgG titers against TcdB and vice versa, indicating that theimmune response against each toxin fragment was poorly cross-reactive.
In vitro toxin neutralization. To verify the functionality of theantibodies, the capacity to neutralize the cytotoxicity of TcdA andTcdB was investigated in vitro (Table 1). All antibodies against theRBD fragments neutralized TcdA, except those elicited by TcdA-B4. Remarkably, antibodies directed to TcdA-GT had neutralizingactivity despite the low overall IgG titer (as measured by ELISAagainst TcdA). Compared with that of TcdA, TcdB cytotoxicitywas inhibited by higher serum concentrations, with neutralizingtiters elicited only by the longer TcdB-B3 fragments and TcdB-GT. Overall, no correlation was observed between ELISA titersand the toxin neutralization capability of single fragments; more-over, none of the fragments were able to neutralize both toxins.Mice were therefore immunized with combinations of fragmentsin an attempt to achieve concurrent neutralization of TcdA andTcdB. Such an approach resulted in the production of sera able toneutralize both toxins in vitro. An example of such cross-neutral-ization is reported in Fig. 2.
In general, neutralizing titers indicated that the componentsperformed with comparable efficiencies when used individually orin combination (Table 1).
TcdA-B1 and TcdB-GT fragments were also combined in asingle chimeric polypeptide (TcdA-B1/TcdB-GT). This proteininduced antibodies with efficient neutralizing activity againstTcdA but not against TcdB (Table 1), suggesting that the neutral-izing epitopes of TcdB-GT were compromised in the chimericprotein. Overall, this screening indicated that while a limited por-tion of the TcdA RBD was sufficient to neutralize TcdA, the entireTcdB domain was necessary to induce functional antibodiesagainst the cognate toxin. It also highlighted the ability ofTcdB-GT to elicit neutralizing antibodies against TcdB. The frag-ment combinations TcdA-B1�TcdB-B3, TcdA-B1�TcdB-GT,and TcdA-B2�TcdB-GT emerged as the most promising and
were subsequently tested for efficacy in the hamster model ofdisease.
Protective efficacy of toxin fragments. The protective effica-cies of fragments in a hamster model of infection are summarizedin Table 2. Hamsters were vaccinated i.p. with four doses of anti-gens. For these studies, we decided to include MF59 as an adjuvantbecause of its well-established ability to induce effective long-termprotection in the elderly, which is the estimated target populationfor a C. difficile vaccine (55, 56).
Three weeks after the last vaccination, animals were treatedwith clindamycin to disrupt their intestinal commensal flora andwere challenged with C. difficile spores given by oral gavage. Vac-cination efficacy was tested using strains 630 and B1, which showsignificant differences in severity of disease within this model (52).Survival and clinical signs were evaluated for 14 days postchal-lenge to provide an indication of vaccine efficacy. Control groupsincluded naive animals and animals treated with adjuvant only,which were not protected following challenge with both strains ofC. difficile.
The relevance of the in vitro neutralization data to protectionwas confirmed in vivo, as hamsters immunized with the individualTcdA-B1 or TcdB-B3 fragment succumbed to infection after thechallenge, indicating that immunity to both toxins was necessaryfor protection. However, when hamsters were immunized withboth TcdA-B1 and TcdB-B3, protection appeared to correlatewith disease severity for the different strains: full protection wasobserved after challenge with 630, a strain known to cause lesssevere pathology, while the same combination ensured an 83%survival rate against strain B1. In contrast, vaccination with TcdA-B1�TcdB-GT conferred 100% protection against both strains,even when the vaccine dosage was reduced to 20 �g/dose. Com-binations of three and four toxin fragments, including both bind-ing and glucosyltransferase domains, did not increase the level ofprotection induced by TcdA-B1�TcdB-GT (Table 2). Besides
preventing fatal infection, vaccination significantly reduced theseverity of disease. All control animals infected with strain 630 orB1 showed the typical acute response with occurrence of diarrheafollowed by death, while surviving animals developed mild diar-rhea or no signs of disease followed by full recovery. Remarkably,after challenge with strain 630, total inhibition of diarrhea wasobserved in animals vaccinated with TcdA-B1�TcdB-GT.
Vaccinated and surviving animals showed high levels of toxinneutralizing antibodies in the sera 14 days after infection. Con-versely, sera from hamsters which did not survive the challengeshowed very low or no neutralization titers, suggesting that toxinneutralization correlated closely with protection (Table 2). Thepresence of toxin-specific IgGs in the intestinal lumens of animalsvaccinated with TcdA-B1�TcdB-GT was also investigated. Al-though the response to TcdA was higher than that to TcdB in theacute phase of infection, rising amounts of anti-TcdB antibodieswere detectable at the endpoint (Fig. 3).
Postvaccination analysis. To further evaluate the effects ofvaccination with TcdA-B1�TcdB-GT, toxin levels produced dur-ing infection and gut histology were examined both in the acutephase of infection (48 h postchallenge) and at recovery (14 dayspostchallenge). Equivalent levels of TcdA and TcdB were detect-able in both control and vaccinated hamsters at 48 h postchallenge(Fig. 4). However, while severe gut inflammation accompanied byepithelial necrosis and polymorphonuclear leukocyte (PMN) in-flux was observed in unvaccinated animals (green and black ar-rows in Fig. 5B), the tissue from vaccinated hamsters showed lessepithelial damage and a limited amount of PMN infiltrate (greenand black arrows in Fig. 5C). Hyperplasia, associated with theappearance of mucin-producing cells, and crypt-to-tip length in-creases were observed (red arrows in Fig. 5B and C), particularly inthe lower colon. Protected animals showed significantly lower lev-els of toxin within the intestinal lumen 14 days after infection,despite the presence of large numbers of C. difficile colonies asso-
TABLE 1 Neutralization titers of sera from mice immunized with toxin A and B fragmentsa
ciated with the intestinal tissue (data not shown). The gut epitheliaappeared to revert to normality, with an absence of polymorphinflux (Fig. 5D). Interestingly, while no hyperplasia of the cecumwas evident, it persisted in the terminal colon (red arrows inFig. 5D).
Overall, postvaccination analyses indicated that the presenceof antibodies neutralizing both toxins strongly limited gut epithe-lial damage and mediated recovery from disease.
DISCUSSION
There is well-established evidence that protection against severeCDI is mediated by systemic antibodies to TcdA and TcdB (10,34). In this study, we evaluated the ability of recombinant toxinfragments to induce robust immunity against lethal challengewith C. difficile. The use of recombinant proteins is an attractivestrategy for vaccine design, as antigen fragments can be engi-neered to meet all the quality standards required during large-scale production, excluding issues of incomplete inactivation anddestruction of conformational epitopes associated with the use offormaldehyde-detoxified toxoid.
Initial screening in vitro revealed that immunization of micewith TcdB fragments induced relatively low neutralization titers.
The limited activity of the anti-TcdB antibodies could be ex-plained in part by the difference in cytotoxicity of TcdB, which hasbeen reported to be 1,000 times more potent in vitro than TcdA(14). A second striking difference between the toxins was the lo-calization of the protective epitopes. While the RBD domain ofTcdA was clearly predominant in inducing neutralizing antibod-ies to this toxin, both TcdB-B3 and TcdB-GT induced antibodiesin mice that were comparably efficient at neutralizing TcdB. Thisobservation was subsequently confirmed in hamster experiments.All animals immunized with the TcdA-B1�TcdB-GT and TcdA-B1�TcdB-B3 combinations survived challenge with C. difficilestrain 630. Unlike the control animals, hamsters immunized withTcdA-B1�TcdB-B3 were protected from death, although mostsuffered a single episode of self-limiting diarrhea. In contrast, an-imals immunized with TcdA-B1�TcdB-GT and challenged withstrain 630 did not develop diarrhea, suggesting that this combina-tion provided enhanced protection. The hypothesis was con-firmed by the observation that only vaccination with TcdA-B1�TcdB-GT fully protected all animals after challenge with theB1 strain, with the vaccine remaining fully protective even whenthe formulation was reduced to 20 �g/dose. Remarkably, the ad-
FIG 2 In vitro neutralization assay on IMR-90 human fibroblasts. Images show the morphology of control cells (A) and cells incubated with 1 CTU100 of TcdA(C) or TcdB (D). (E and F) Cell rounding was inhibited by serum against TcdA-B1�TcdB-GT. (E) TcdA (1 CTU100) plus 1/8,000-diluted serum; (F) TcdB(1 CTU100) plus 1/256-diluted serum. The negative control was the corresponding preimmune serum in the presence of 1 CTU100 of TcdA (B) or TcdB (notshown). The complete panel of images showing the titration of neutralizing activities is reported in Fig. S4 in the supplemental material.
dition of other components to the TcdA-B1�TcdB-GT combina-tion had a detrimental effect on animal survival. This wasparticularly evident for formulations with lower antigen concen-trations, where the addition of TcdA-GT and TcdB-B3 was unableto eliminate clinical signs or enhance the neutralization titers ofimmune sera. TcdA-B1�TcdB-GT was therefore the minimalcombination necessary and sufficient to ensure the survival of allvaccinated hamsters and induce neutralizing antibody titers ableto prevent the onset of disease. This evidence opens new perspec-tives on the use of recombinant antigens to vaccinate against CDI.It is widely accepted that C. difficile vaccines should be based onthe generation of antibodies interfering with the initial bindingand internalization of TcdA and TcdB within the host cells. Forthis reason, an immune response toward protective epitopes ofboth toxins is of fundamental importance.
Historically, the first evidence that antitoxin immunity canprotect hamsters against lethal challenge was obtained by immu-nizing animals with formalin-inactivated toxoid from culture fil-trates (57–59). Significantly, Torres and colleagues compared theeffectiveness of toxoid preparations at different antigen doses andimmunization routes (60). They showed that a combination ofmucosal and systemic immunization induced protection againstboth the diarrheal symptoms of infection and lethality in ham-sters. The combined use of mucosal and parenteral administrationof both toxoids was further investigated by Giannasca et al., whoused more-purified toxoid preparations and replaced the i.p.route with the clinically acceptable intramuscular injection route(61). Full protection was achieved only in animals vaccinated with
FIG 3 IgG antibodies against toxin A (A) and toxin B (B) in cecum samplesfrom hamsters vaccinated with TcdA-B1�TcdB-GT. Twofold dilutions ofTcdA and TcdB (100 ng to 3 ng) were serially spotted onto a nitrocellulosemembrane. Dot blots were then performed with filtered cecum samples takenfrom vaccinated animals in the acute phase of infection (48 h postchallenge)(hamsters 1 and 2) and at the experimental endpoint (14 days postchallenge)(hamsters 3 to 8). Control animals were treated with adjuvant only and in-fected under the same experimental conditions (hamsters 9 and 10).
TABLE 2 Protection against C. difficile disease in hamsters vaccinated with recombinant TcdA and TcdB fragments with MF59 adjuvanta
Challenge strain and vaccine antigen(s)
% Survival (no. ofsurvivors/total no.of animals) Duration of diarrhea
Anti-TcdA neutralizationtiter
Anti-TcdBneutralization titer
Strain 630TcdA-B1�TcdB-B3 100 (6/6) 5 h 55 min � 3 h 25 min 4,667 � 1,764 558 � 121TcdA-B1�TcdB-GT 100 (6/6) None 8,000 � 0 512 � 0Tcd-B1/TcdB-GT chimera 60 (3/5) 16 h 57 min � 7 h 39 min 1,667 � 333 (32 � 0) 512 � 0 (16 � 0)Adjuvant alone 0 (0/7) 4 h 5 min � 0 h 44 minc
Naive animals 0 (0/7) 3 h 37 min � 0 h 33 minc
Strain B1TcdA-B1 0 (0/6) NDc ND NDTcdB-B3 0 (0/6) NDc 0 426 � 85TcdA-B1�TcdB-B3 83 (5/6) 24 h 46 min � 7 h 43 min 2,000 � 0 (32 � 0) 512 � 0 (16 � 0)TcdA-B1�TcdB-GT 100 (6/6) 7 h 53 min � 2 h 34 min 1,200 � 5,060 341 � 85TcdA-B2�TcdB-GT 100 (6/6) 12 h 19 min � 3 h 4 min 8,000 � 0 256 � 0TcdA-B1�TcdB-B3�TcdB-GT 83 (5/6) 8 h 8 min � 4 h 59 min 1,000 � 0 (32 � 0) 2,000 � 0 (32 � 0)TcdA-B2�TcdA-GT�TcdB-B3�TcdB-GT 83 (5/6) 6 h 1 min � 1 h 28 min 6,667 � 1,333 (512 � 0) 512 � 0 (0)Adjuvant alone 0 (0/10) 3 h 2 min � 0 h 22 minc
Naive animals 0 (0/10) 2 h 34 min � 0 h 17 minc
Strain B1 with reduced antigen dose (20�g/dose)
TcdA-B1�TcdB-GT 100 (8/8) 7 h 25 min � 2 h 6 min 10,000 � 1,852 112 � 10TcdA-B2�TcdB-GT 37 (3/8) 9 h 39 min � 4 h 44 min 2,133 � 5,333 (8,000 � 0) 128 � 0 (0)TcdA-B2�TcdA-GT�TcdB-B3�TcdB-GTb 86 (6/7) 15 h 15 min � 2 h 3 min 8,000 � 0 (512 � 0) 256 � 0 (0)
a Neutralization titers were determined with pooled sera from protected animals at the experimental endpoint. Values represent geometric means for 3 to 5 independentexperiments � SE. Titers in parentheses refer to single or pooled sera from unprotected animals. ND, not determined.b Sample sera from vaccinated animals were collected before the challenge and showed neutralization titers comparable to those measured at the experimental endpoint (data notshown).c Diarrhea was observed until the body temperature dropped below 35°C.
a combination of rectal immunization with E. coli heat-labiletoxin adjuvant and intramuscular injection of alum-adjuvantedtoxoids. The absence of antitoxin antibodies in feces suggestedthat circulating antibodies were responsible for protection of vac-cinated hamsters. Subsequent studies investigated the efficacy ofseveral recombinant toxin fragments by systemic vaccination.Vaccine candidates included the entire RBD from TcdA (62),smaller TcdA internal fragments (35), and, more recently, chime-ric proteins that incorporate regions from both toxins into a singlepolypeptide chain (46, 47). Such fusion proteins have been testedwith alumimun hydroxide at doses ranging from 10 �g (47) up to100 �g (46). Full protection from death has been observed only at
higher antigen doses after challenge with the less virulent strain630.
Overall, the TcdA-B1�TcdB-GT combination reported in thepresent study appears to be a promising vaccine candidate, as fourdoses of 20 �g were able to protect hamsters against severe infec-tion with strain B1. Whether a vaccine containing such a combi-nation could potentially be optimized by reducing the total num-ber of injections is an aspect that requires further investigation.
Our results clearly indicate that the protective epitopes of TcdBare not exclusively localized in the RBD and emphasize the impor-tance of carrying out an initial screening to identify the mostpromising vaccine candidates. These findings align with previousstudies showing that TcdB neutralizing epitopes are locatedwithin the N-terminal GT domain (47). The importance of theTcdB-GT domain was also revealed by epitope mapping of hu-manized monoclonal antibodies for passive immunotherapy (32).
The absence of a clear correlation between immunogenicityand protective efficacy suggests a biased immune response towardimmune-dominant nonneutralizing epitopes. From a vaccineperspective, the dissection of the toxin polypeptides into recom-binant fragments provides the double advantage of reducing an-tigen size and enhancing the population of neutralizing antibod-ies.
Our analysis in vivo revealed that antibody-mediated toxinneutralization is effective at the level of the epithelial barrier. In-deed, toxin levels in the gut lumens of vaccinated animals recov-ering from diarrhea 48 h after challenge and those found in con-trol animals were equivalent. Therefore, the reduction in diarrhealepisodes observed in protected animals could be associated withearly toxin production, which causes limited damage to the vas-culature. The resulting lesion allows permeation of the immuneserum containing neutralizing antibodies. The protective effect ofsuch infiltration was appreciable from inspection of gut tissues ofvaccinated animals, as initial damage and neutrophil infiltrationwere limited and were followed by tissue repair.
It remains to be determined whether the higher protectioninduced in hamsters by combinations containing TcdB-GT is dueto the ability of specific antibodies to limit cell binding and uptakeof TcdB or whether the direct neutralization of the enzymaticdomain is able to protect against the effects of still-uncharacter-
FIG 4 Toxin A and B levels in hamsters vaccinated with the TcdA-B1�TcdB-GT combination. Values indicate the fold dilutions required to eliminate cellrounding. Filtered cecum samples were taken from vaccinated animals in the acute phase of infection (�; 48 h postchallenge) and at the experimental endpoint(Œ; 14 days postchallenge). Toxin levels in the cecum samples of animals at day 14 were significantly lower (��, P � 0.0014 for toxin A and P � 0.0015 for toxinB) than those measured in control animals (acute disease) or vaccinated animals at 48 h. Control animals (�) were treated with adjuvant only and infected underthe same experimental conditions. Toxin levels were evaluated at the acute end stage of the infection.
FIG 5 Histopathology of hamster gut tissues following immunization withTcdA-B1�TcdB-GT and challenge with C. difficile B1. Samples were takenfrom control hamsters (A), unvaccinated hamsters during the acute phase ofinfection (B), and vaccinated hamsters at 48 h (C) and 14 days (D) postchal-lenge. Arrows indicate an influx of PMNs (black), hyperplasia (red), and epi-thelial disruption (green). Histopathological scores are reported in Fig. S5 inthe supplemental material.
ized extracellular enzymatic activity of the toxin. Whichever is thecase, TcdA-B1�TcdB-GT appears to generate significant protec-tion, providing new hope for reducing the impact of CDI.
ACKNOWLEDGMENTS
This work was partially supported by MIUR (Italian Ministry of Univer-sity and Research) (grant PON01_00117) and Regione Toscana (grantPOR CREO FESR 2007-20013). A.B. was supported by The WellcomeTrust (grant 086418).
We are grateful to John Telford for a helpful discussion, to GiorgioCorsi for artwork, and to Antonietta Maiorino for manuscript review.
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