j our na l ho me page: www.elsev ier .com/ locate /vacc ine
evelopment of a recombinant toxin fragment vaccine for Clostridiumifficile infection
erzy Karczewskia,∗, Julie Zormana, Su Wanga, Matthew Miezeiewskid, Jinfu Xiea,eri Soringd, Ioan Petrescub, Irene Rogersb, David S. Thiriotc, James C. Cooka,ihaela Chamberline, Rachel F. Xoconostlea, Debbie D. Nahasa, Joseph G. Joycea,
ean-Luc Bodmera,1, Jon H. Heinrichsa, Susan Secorea
Merck Research Laboratories, Vaccine Basic Research, West Point, PA, United StatesMerck Research Laboratories, Laboratory Animal Resources, West Point, PA, United StatesMerck Research Laboratories, Vaccine Drug Product Development, West Point, PA, United StatesEurofins Laboratories, Lancaster, PA, United StatesAgile 1, Torrance, CA, United States
Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomem-branous colitis, a disease associated with significant morbidity and mortality. The disease is mostly ofnosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C.difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically todamage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associatedwith CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. Avaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe andimmunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In orderto eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing pro-cess of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could bea challenging and costly process.
Vaccines base on non-toxic fragments of genetically engineered versions of the toxins alleviate mostof these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdBand explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian
Please cite this article in press as: Karczewski J, et al. Development of a recombinant toxin fragment vaccine for Clostridium difficileinfection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.026
hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A)developed high titer IgG responses and potent neutralizing antibody titers. We also show here that therecombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacyequivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provideseveral advantages over toxoid TcdA/TcdB such as improvements in manufacturability.
1 Current address: Genocea Biosciences, Cambridge, MA, United States.
are an estimated 500,000 C. difficile infection (CDI) cases in theUnited States each year [4,5]. Infection with C. difficile, an anaerobic,gram-positive, spore-forming bacillus, usually occurs as a compli-cation of antibiotic therapy due to the disruption of normal colonicflora caused by the antibiotic treatment. The manifestations ofCDI range from asymptomatic colonization to symptomatic diseaseand include antibiotic-associated diarrhea and pseudomembra-nous colitis. Furthermore, disease may progress to toxic megacolon,sepsis, and death. Advanced age (≥65 years), antibiotic use,immunosuppression, health care exposure, duration of hospital-ization and serious underlying illness are consistently identified asrisk factors for CDI [1].
Please cite this article in press as: Karczewski J, et al. Development of a recombinant toxin fragment vaccine for Clostridium difficileinfection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.026
ARTICLE IN PRESSG ModelJVAC 15111 1–7
2 J. Karczewski et al. / Vaccine xxx (2014) xxx–xxx
The first line of treatment for C. difficile infections (CDI) isto discontinue ongoing antibiotic treatment and to administermetronidazole or vancomycin [1,2]. While this treatment is effec-tive in most cases, a significant percentage of patients undergorelapse. In addition, a newly identified, hypervirulent strain ofC. difficile called BI/NAP1/027 has been implicated in outbreaksassociated with increased morbidity and mortality since the early2000’s [3–8]. This epidemic strain is responsible for increased inci-dence of C. difficile-associated diarrhea related not only to antibioticexposure, but also associated with GI surgery, prolonged hospital-ization and immune-compromising conditions [9]. The emergenceof this and potentially other hypervirulent strains (such as 078)prompted the search for alternative methods to treat and controlinfections leading to CDI. In addition to the development of novelnarrow-spectrum antibiotics such as fidaxomicin [10], two otherapproaches are being pursued: therapeutic monoclonal antibodies[11] and prophylactic vaccination [12].
C. difficile produces two potent exotoxins: Toxin A (TcdA) andToxin B (TcdB). These toxins induce a broad range of pathologieslocally (inflammation, colonic epithelium damage) [13], and poten-tially could induce cardiotoxicity, as demonstrated in zebrafishembryos [14]. Both toxins are encoded on a 19 kb region of thegenome referred to as the pathogenicity locus or PaLOC [15] andfunction through glucosylation of Rho-family GTP-ases leading toa disruption of the gut mucosa [16].
TcdA and TcdB are large proteins (308 kDa and 270 kDa, respec-tively) and share a relatively high degree of amino acid sequencehomology (∼50%) and similar multi-domain structures. The toxinsare orthologous to the Large Clostridial Toxin family, which alsoincludes TcsL, TcsH, TcnA and TpeL [17,18]. The N-terminal, enzy-matic domain (ED) of each protein contains all enzymatic activitiesnecessary for glucosylation of several GTPases such as RhoA, Racand Cdc42 [19,20]. The central hydrophobic core constitutes a poreforming domain that mediates translocation of the ED to the hostcell cytoplasm, while the C-terminus of the protein consists ofa highly repetitive region referred to as the clostridial repetitiveoligopeptide (CROP) domain that binds cellular receptors.
Toxins enter cells by receptor-mediated endocytosis andundergo an auto-catalytic cleavage in the cytosol which releasesthe enzymatic domain. The ED subsequently catalyzes glucosyla-tion of small GTPases, leading to disaggregation of the cytoskeletonand cell death (for a review see [21]).
The aim of the current study was to evaluate the efficacy of a vac-cine consisting of two separately expressed recombinant fragmentsof TcdB combined with full-length, formaldehyde-inactivated TcdA(Toxoid A). We demonstrate that hamsters immunized with thisvaccine developed high titer neutralizing antibodies and long-lasting (>3 months) protective immunity against lethal challengewith C. difficile. The inclusion of fragments encompassing the com-plete sequence of TcdB presents a novel and promising approachfor development of a safe and efficacious vaccine for prevention ofCDI as well as for potentially reducing costs of manufacturing.
2. Materials and methods
2.1. Cloning and expression of recombinant antigens
The genes encoding TcdB fragments, based on the sourcesequence of C. difficile strain VPI 10463 source sequence werecodon optimized for expression in E. coli, synthesized by Gene-script (Piscataway, NJ). The fragments were then cloned intoapET30a expression vector (EMD Millipore, Billerica, MA) withC-terminal His-tag, and transformed into competent BL21 (DE3)Escherichia coli cells (New England Biolabs, Ipswich, MA). Oneliter cultures were grown in Luria-Bertani (LB) broth containing
30 �g/mL kanamycin to an optical density (OD) at 600 nm of 0.6.Expression was induced by the addition of 1 mM isopropyl �-d-thiogalactopyranoside (IPTG). Incubation continued for 4 h at 37 ◦C.Cells were harvested by centrifugation and cell pellets were storedfrozen at −70 ◦C. TcdA fragment A-C1 was expressed using the Sac-charomyces cerevisiae expression system previously described byJansen et al. [22].
2.2. Extraction and purification of recombinant antigens
TcdB fragments were purified from E. coli cell pellets usingimmobilized metal affinity chromatography (IMAC) followed byion-exchange chromatography. Briefly, each E. coli pellet was sus-pended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 20 mMimidazole) supplemented with the protease inhibitor cocktailComplete® (Roche Diagnostics, Mannheim, Germany) and 2500units of nuclease (Benzonase®, EMD Chemicals, Gibbstown, NJ) andthe cell suspension was passed through a microfluidizer (Model110Y, Microfluidics Corp. Newton, MA). The lysate was clarifiedby centrifugation (20,000 × g, 30 min), filtered through a 0.22 �ExpressTM Plus filtration device (Millipore Corp., Billerica, MA)and loaded onto a 5 mL HiTrap IMAC FF resin (GE Healthcare Bio-Sciences Corp. Piscataway, NJ) equilibrated in 50 mM Tris pH 8.0,300 mM NaCl, 20 mM imidazole and eluted with a 20–250 mMlinear imidazole gradient in the same buffer. Fractions contain-ing TcdB fragments were pooled and diluted 5-fold with 20 mMTris, pH 7.4 and subsequently loaded onto a 6 mL Resource Qcolumn (GE Healthcare Bio-Sciences Corp. Piscataway, NJ). TcdBfragments were eluted with a NaCl gradient, dialyzed againstHEPES-buffered saline (10 mM Hepes, pH 7.4, 150 mM NaCl) andfiltered through a 0.22 � Supor® membrane filter (Pall Corporation,Port Washington, NY) to make the final product for immunizationstudies.
The TcdA-C1 (amino acids 1832–2710) was purified to homo-geneity from S. cerevisiae cell paste. Briefly, the cell paste wassuspended in 0.2 M MOPS, pH 7.0 and ammonium sulfate addedto 50% saturation at 4 ◦C with mixing, for 30 min. And the pre-cipitate was harvested by centrifugation (10,000 × g, 30 min, 4 ◦C),dissolved in 50 mM MOPS, pH = 7.0 containing 0.1% CHAPS anddiluted 50-fold with 50 mM MOPS pH 7.0. A-C1 fragment waspurified by sequential chromatography on the cation exchangecolumn containing POROS® 50HS resin (Applied Biosystems,Foster City, CA) followed by multimode chromatography onhydroxyapatite HA resin (CHT-Type II 5 mL, BioRad, Hercules,CA).
2.3. Biophysical characterization of recombinant fragment B-TC
The purified fragment B-TC was dialyzed into 10 mM phosphatepH 7.0 and the circular dichroism (CD) spectrum was acquiredfrom 300 to 180 nm at 20 ◦C using a Jasco J810 spectropolarime-ter. Protein concentration was determined by amino acid analysisand was used to convert the raw CD data to molar ellipticity.Secondary structure estimation was performed using the CDProanalytical software suite and the programs CDSSTR, SELCON3 andCONTINLL [23,24,25,26,27,28]. Analytical ultracentrifugation wasperformed using sedimentation equilibrium analysis on fragmentB-TC in 10 mM sodium phosphate, pH 7.0, 0.15 M NaCl at 20 ◦C. Theanalysis was carried out at rotor speeds of 6000 RPM, 8000 RPM, and10,000 RPM for 16 h in a Beckman XL-I analytical ultracentrifugeand the data was fitted using HeteroAnalysis, ver. 1.1.33 software(James Cole, Jeffrey Larry, Analytical Ultracentrifugation Facility,Biotechnology and Bioservices Center, University of Connecticut,Storrs, CT).
J. Karczewski et al. / Vaccine xxx (2014) xxx–xxx 3
2.4. Toxin inactivation and formulation
TcdA and TcdB (purified from C. difficile strain VPI10463, List Bio-logicals, Campbell, CA) and selected recombinant fragments (B-EDand B-TC) were reconstituted in 20 mM HEPES-buffered saline (pH7.4) at 0.3–0.4 mg/mL. Formaldehyde was added to a final concen-tration of 0.43% (v/v) and l-lysine was added to a final concentrationof 4.2 mg/mL. Inactivation proceeded for 18 days 4 ◦C and then theprotein solutions were dialyzed against 20 mM HEPES buffer (pH7.4), containing 100 mM sodium chloride and sterilized by passagethrough a 0.22 � filter.
Protein antigens were co-formulated with aluminum hydroxy-phosphate sulfate adjuvant (AHPS, Merck & Co, Inc.) andISCOMATRIXTM adjuvant (CSL Limited, Parkville, Australia) in salineto prepare each component of the vaccine.
2.5. Hamster immunogenicity and protection studies
Golden Syrian hamsters, (male, 90–120 g), were obtained fromCharles River Laboratories (Wilmington, MA), and were individ-ually maintained in filter-lid cages. Hamsters were immunizedintramuscularly in the left quadriceps on days 0, 21, 42 and 63with a 200 �L vaccine dose containing 10 �g of combined TcdBfragments and10 �g of TcdA toxoid. Control animals received adju-vant alone. Serum samples were collected via retro-orbital bleedingimmediately prior to each immunization and on day 77 and storedfrozen. For evaluation of vaccine efficacy against a lethal C. dif-ficile spore challenge, animals were oro-gavaged with one doseof 10 mg/kg clindamycin five days prior to spore administrationand 16 days following the final vaccine dose. C. difficile strainVPI 10463spores 2×ED90, ∼600 Cfu)were administered by oro-gavage and animals were monitored for symptoms of CDI or deathfor the duration of the study (21 days post-challenge).The Man-tel–Cox test was applied to assess statistical differences in survivalrates between groups. All hamster experiments were performedunder Institutional Animal Care and Use Committee guidelines asapproved by Merck and Co., Inc.
well plates were coated with either TcdA or TcdB at 50 ng/well or25 ng/well, respectively in PBS overnight at 4 ◦C. Wells were washedwith PBS containing 0.05% Tween 20 (PBS-T) and blocked in PBScontaining 1% bovine serum albumin (Sigma, St. Louis, MO). Serialdilutions (1:2) of hamster sera were incubated at room temperaturefor 1.5 h. Plates were then washed with PBS-T and incubated withgoat anti-hamster IgG conjugated to horseradish peroxidase (HRP)(Abcam, Cambridge, MA) for 1 h at room temperature. Followingan additional wash, 50 �L of 3,3′,5,5′-tetramethylbenzidine (TMB)substrate (Thermo Labsystems, Rockford, IL) was added and after15 min, the reaction was stopped with 50 �L 0.4 N sulfuric acid andthe absorbance measured at 450 nm (Powerwave X Select, Bio-TekInstruments, Winooski, VT). Antibodytiters were calculated usingfour parameter regression fitting of the titration curve using Prism5 for Windows software (GraphPad Software, Inc. La Jolla, CA).
2.6.2. Neutralization and cytotoxicity assayNative TcdA or TcdB was pre-incubated with test sera for 1.5 h
at 4 ◦C, applied to Vero cells grown in 384 well plates and incubatedin humidified CO2 incubators at 37 ◦C for 48 h. The cells were fixed,permeabilized, and stained with Alexa Fluor® 488 phalloidin and animage of the cell monolayer was acquired using the ImageXpress®
Velos Scanning Cytometer (Molecular Devices, LLC, Sunnyvale, CA).The total cell surface area in each well was plotted against the
Table 1Recombinant toxin fragments selected for in vivo evaluation, their molecularweigths and purification yields.
dilution of sera (for neutralization titers) or against the toxin con-centration (for cytotoxicity assessment) and titers were calculatedusing four parameter regression fitting using Prism 5 for Windowssoftware (GraphPad Software, Inc. La Jolla, CA).
3. Results
3.1. Design, preparation and formulation of recombinant Toxin Bfragments
We designed a vaccine composed of two recombinant fragmentscovering an entire sequence of TcdB (Table 1). The first fragment,B-ED comprised the N-terminal region (amino acids 1–543) andcontained the entire enzymatic domain of TcdB. To minimize thepossibility of cytotoxicity resulting from cellular uptake of thisfragment, four residues were mutated: W102A, D288A, E515Qand S518A, which have been shown to be critical for the cat-alytic activity of the enzyme [29–31]. The second component ofthe vaccine (B-TC) encompassed the translocation domain throughthe C-terminus (residues 545–2367); therefore, when combinedtogether, fragments B-ED and B-TC contained the entire amino acidsequence from full-length TcdB. The CROPS regions were identifiedas being immunodominant and shown to induce potent neutraliz-ing antibodies [32], we therefore prepared and evaluated severalshorter CROPS-derived fragments of TcdB (B-C0–B-C4) and one C-terminal fragment of TcdA (Table 1).
TcdB fragments were expressed in E. coli and purified from sol-uble extracts in a two-step process including IMAC followed byanion exchange chromatography. A similar strategy was initiallyattempted to produce recombinant fragments of TcdA, includingthe larger fragment A-TC and the shorter CROPS-derived fragmentA-C1; however, expression yields as well as solution stability didnot allow for production of sufficient quantities of proteins nec-essary to support in vivo studies. Expression of the A-C1 fragmentin S. cerevisiae followed by sequential chromatography on cationexchange and hydroxyapatite chromatography allowed the desiredproduct to be purified to homogeneity (>95%). The overall purifica-tion yields from E. coli extracts ranged from 39 to 1836 mg/L of E. coliculture, as shown in Table 1. The purified TcdA and TcdB fragmentsmigrated on SDS-PAGE at a molecular weight corresponding to themonomeric form of each protein, as calculated from the amino acid
of a recombinant toxin fragment vaccine for Clostridium difficile.026
sequence (Fig. 1A). The purity of all antigens was greater than 90%with very little evidence of degradation, as confirmed by Westernblot analysis (data not shown).
4 J. Karczewski et al. / Vaccine xxx (2014) xxx–xxx
Fig. 1. (A) Analysis of recombinant toxin fragments by SDS/PAGE. 5 �g of each pro-tein was separated on NuPage 4–12% gel and stained with Coomassie Blue. (B)Cytotoxic activity of native and recombinant toxin fragments. The potency of frag-mTt
fia�nspt�t7
aQ5
Table 2Combinations of TcdA TcdB antigens selected for in vivo evaluation in clindamycin-induced enterocolitis model.
ents or unmodified combinations was measured in a cell-based cytotoxicity assay.he values for all recombinant fragments are plotted as the highest concentrationsested (no cytotoxicity was observed at these concentrations).
Secondary structure estimation and solution-based size of puri-ed fragment B-TC (207 kDa) was determined by circular dichroismnd analytical ultracentrifugation, respectively. CD indicated 22%-helix, 29% �-sheet, 21% turns, and 29% unordered compo-ents suggesting that the recombinant antigen adopted a complexecondary structure not dominated by any single structural com-onent. In comparison, previously published results have reportedhat full length native TcdB is composed of 36% �-helix, 21%-sheet, 18% turns and 25% unordered components [33]. Sedimen-
Please cite this article in press as: Karczewski J, et al. Development
ation equilibrium analysis of B-TC yielded a molecular weight of26,745 Da, consistent with the formation of trimers in solution.
Recombinant fragments and combinations thereof were evalu-ted in a cellular-based cytotoxicity assay. Studies by Genisyuerek
317
Group 9 AAHS/IMX /IMX
et al. [33] demonstrated that a number of chimeric TcdB variants,even when lacking the domain critical for translocation could retaintheir full cytotoxic activity. For all recombinant antigens, includingthe enzymatic domain B-ED as well as combinations of B-ED withC-terminal fragments (B-TC, B-C0 and B-C1), we detected no cyto-toxic activity even at micromolar concentrations (Fig. 1B) whereasTcdA and TcdB exerted potent cytotoxic effects with an EC50 of1.8 pM and 0.02 pM, respectively.
Table 2 presents the various combinations of recombinant frag-ments selected for direct comparison with a benchmark vaccineconsisting of full-length TcdA and TcdB purified from the C. difficilebacterium and inactivated by treatment with formaldehyde (group1). A two-segment vaccine containing recombinant fragments B-ED and B-TC (groups 2 and 3) was tested as formaldehyde treated(group 2) and untreated combination. In addition, several addi-tional combinations including B-TC, B-C0 and B-C1 (groups 3–5),B-TC fragment alone (group 6), A-C1, B-ED, and B-TC (group 7), andA-C1 and native TcdB (group 8) were tested.
3.2. Immunogenicity of the recombinant fragments
Hamsters were immunized four times with the antigen groupslisted in Table 2 and the post-dose four sera was evaluated by ELISA.All immunized animals developed IgG antibodies specific for TcdAas measured by ELISA (Fig. 2A) as well as antibodies to TcdB (Fig. 2B).There were no statistically significant differences between titers ingroups 1–8 as measured by Mantel–Cox test. No C. difficile-specificIgG response was detected in the adjuvant control group 9 (Fig. 2Aand B). We also evaluated fragments B-C2, B-C3 and B-C4 represent-ing shorter fragments of CROPS, however these fragments failed toinduce potent IgG responses (data not shown) and therefore thesefragments were excluded from subsequent evaluation.
3.3. Serum neutralizationactivity
Neutralizing antibody titers were evaluated inpost-dose4immune sera. All groups immunized with recombinant anti-gen combinations (Groups 2–8) developed neutralizing antibodiesagainst both TcdA and TcdB (Fig. 2C and D, respectively). We did notobserve statistically significant differences in groups 1–8 in neu-tralization titers as measured against TcdA (Mantel–Cox test). Inthe case of TcdB, a statistically significant increase in neutralizingantibody titers was observed in groups 2 and 3 as compared to tox-oid group 1. The remaining groups (4–8), did not reach statisticalsignificance (p > 0.05) relative to group 1. Control animals receivingadjuvant alone (group 9) did not produce measurable antibodiesthat neutralized the cytotoxic effects of either TcdA or TcdB (Fig. 2C
of a recombinant toxin fragment vaccine for Clostridium difficile.026
and D). Similarly, preimmune sera obtained from all of the animalswas incapable of neutralizing toxicity at the lowest dilution tested(data not shown).
Please cite this article in press as: Karczewski J, et al. Development of a recombinant toxin fragment vaccine for Clostridium difficileinfection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.026
ARTICLE IN PRESSG ModelJVAC 15111 1–7
J. Karczewski et al. / Vaccine xxx (2014) xxx–xxx 5
Fig. 2. Systemic antibody response in the individual sera samples collected at day 77. (A and B) ELISA titers were measured against TcdA (Panel A) and TcdB (Panel B). (C andD) Neutralizing titers for TcdA (Panel C) and TcdB (Panel D) were measured. The average titers (n = 9) ± SEM are shown.
3.4. In vivo efficacy of fragment based vaccine in hamsterchallenge model
The clindamycin-induced lethal enterocolitis model in GoldenSyrian hamsters is commonly utilized for studying the mecha-nism of C. difficile infections as well as protection mediated byvaccines [34] and antibodies [32] as well as small molecule antibi-otics [35]. To evaluate the efficacy of our recombinant vaccines,hamsters immunized with vaccine combinations (group 1–8) orwith adjuvant control (group 9) were treated with clindamycin, todisrupt the gut microbiota and render the treated animals suscep-tible to subsequent challenge with C. difficile spores (Fig. 3A). Fivedays following treatment, all animals were challenged with a lethaldose of C. difficile spores VPI10463and monitored for developmentof disease symptoms. We observed that all animals in the adju-vant control group developed diarrhea (wet tail) and died within42–80 h after challenge (group 9, Fig. 3B). The extent of protec-tion in the vaccinated groups varied significantly. We observednear complete protection in the toxoid-vaccinated animals (group1, Fig. 3B). In this group, eight out of nine hamsters survived for atleast 3 weeks post challenge. We observed similar complete pro-tection in group 2, which received the recombinant two-segmentTcdB vaccine combined with toxoid A. Despite being protected fromdeath, significant weight loss was evident in both groups 1 and 2.All animals in these groups regained weight and resumed a nor-mal diet within 2–3 weeks post challenge (Fig. 3D). No diseaserelapse was observed up to two months post-challenge. In addition,statistically significant protection, compared to adjuvant controlwas observed in groups 3–6, with 80–90% survival at 1 week postchallenge and the overall survival between 40% and 70% recordedat the end of the study. Mantel–Cox analysis revealed no statis-tically significant differences in the survival between groups 3–6(P = 0.36–05). Partial protective efficacy was observed in groups 7
and 8 which were immunized with the recombinant fragment ofTcdA (A-C1) combined with either the two-segment recombinantTcdB (group 7) or with native TcdB (group 8) as shown in Fig. 3C.Survival in both groups was improve das compared to the adju-vant control (P < 0.007), however these two vaccine combinationsperformed substantially poorer than the full-length toxoid combi-nation (group 1) with overall 20% and 40% survival (in groups 8 and7, respectively) at the completion of the study.
4. Discussion
Prevention of nosocomial C. difficile infections represents amajor unmet medical need. Development of an efficacious vac-cine is a major focus in multiple leading academic and industrialresearch centers. An investigational vaccine comprised of formalin-inactivated full length toxoids TcdA and TcdB prepared from C.difficile bacterial culture is currently undergoing clinical trials [36].In order to produce a vaccine from the native organism, the cyto-toxicity of purified toxins must be reduced by several orders ofmagnitude, typically via chemical inactivation, such as formalde-hyde treatment. A phase 1 study demonstrated the relative safetyof such a toxoid vaccine in a small group of healthy volunteers[37]. Recombinant expression technology represents a convenientplatform for the high yield production of protein subunit vaccineswhile offering the added advantage of enabling genetic alterationsvia site-directed mutagenesis or truncations to obtain antigens ofdesired properties. In this study we report the development ofa vaccine candidate shown to be officious in hamsters and com-prising detoxified fragments of TcdB produced in a recombinantexpression system and combined with full length TcdA. The cyto-toxicity of native TcdB was eliminated by expressing, purifying andformulating two individual segments of the protein then combiningthe formulations for immunization. We found that immunization of
6 J. Karczewski et al. / Vaccine xxx (2014) xxx–xxx
Fig. 3. Evaluation of C. difficile vaccines in Golden Syrian hamster model of CDI.(A) The outline of in vivo challenge model. (B) The groups of hamsters (n = 9) thatwere immunized with recombinant TcdB fragments combined with Toxoid A werechallenged with C. difficile spores and the survival monitored. (C) Hamsters wereimmunized with recombinant fragment of TcdA (A-C1) combined with Toxoid B,challenged with C. difficile spores and the survival measured. (D) Surviving hamstersin group 1 and 2 were monitored for weight changes for the duration of the studya
hncsvkowdI(TT
The efficacy of recombinant C. difficile vaccines has beenreported by others. A vaccine based on genetically fusing the C-terminal domains of TcdA and TcdB with a four amino acid linkerand formulated with aluminum hydroxide was recently reportedby Tian et al. [38]. This chimeric antigen was efficacious in a ham-ster model using C. difficile strain 630 as the challenge. We believethat the current results, in which we challenged hamsters immu-nized with our vaccine constructs with the higher toxin-producingstrain VPI 10463 [39], represents a more rigorous demonstration ofefficacy.
A second chimeric-based vaccine in which the C-terminal CROPSdomain of TcdB variant carrying three detoxifying amino acidmutations was replaced by the corresponding CROPS segment ofTcdA was recently described by Wang et al. [40]. Animals immu-nized with this construct displayed protective immunity up to 80 hpost challenge. These results are in good agreement with the sur-vival outcome we report in the current study.
Interestingly, our current study demonstrated that formalde-hyde treatment of a B-ED and B-TC fragment vaccine resulted in asignificant improvement of efficacy relative to the untreated com-bination (Fig. 3, group 2 vs. group 3). The mechanism for thisenhanced efficacy is not entirely understood. Binding antibodytiters as measured by ELISA or neutralizing antibody titers as mea-sured by the cytotoxicity assay were not increased in group 2,suggesting that a mechanism not directly related to the neutraliza-tion of the cytotoxic effect may exist. Such effects may be relatedto improvements in structural stability of the antigenic site bytreatment with formaldehyde. The biophysical characterizationsreported herein demonstrate that B-TC is a structurally complexmolecule containing multiple �-helical and �-sheet componentsas determined by CD analysis. Analytical centrifugation using equi-librium sedimentation analysis shows that the B-TC fragment formstrimeric complexes in solution. The formation of similar multimericstructures has been reported previously for native toxins usingdifferential light scattering (DLS) analysis [33,41]. This multimer-ization is believed to be a physiologically relevant phenomenon andcould play a role during pore formation following endocytosis ofTcdB (reviewed in [42]). Formaldehyde-crosslinked TcdB was alsoshown to exhibit an improved thermal stability as compared withuntreated TcdB [33]. It is likely that the B-TC fragment undergoessimilar modifications following formaldehyde treatment whichmay lead to improved stability and enhanced vaccine efficacy.
The mechanism of protection in the hamster lethal challengemodel may be complex in that factors other than anti-toxin anti-bodies may play a role. Our current study demonstrates that levelsof anti-toxin IgG and neutralizing antibody elicited by recombinantfragment vaccine are comparable to those elicited by full lengthinactivated toxoid, yet these titers do not correlate well with thefinal extent of protection measured three weeks post-challenge. Itappears plausible that neutralizing antibody titers measured in ourcytotoxicity assay reflect a reduction in TcdB binding to host cells,most likely mediated by antibodies directed against the CROPSregion. However, other regions of the toxin proteins may still playa role in progression of the disease and it is possible that a broaderrepertoire of antibodies to those regions is elicited by the toxoidvaccine, A recent report by Marozsan et al. [43] demonstrated thatmonoclonal antibodies against the enzymatic domain of TcdB com-bined with an anti TcdA antibody supported long-lasting protectionin a hamster model of CDI. In contrast, a different set of mono-clonal antibodies, directed against the C-terminal domains of TcdAand TcdB, only partially protected hamsters from lethal challengewith C. difficile. This study supported the role of the enzymaticdomain in development of protective immunity. It would be ofinterest to evaluate a combination of antibodies directed against
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
nd continued for an additional month.
amsters with a formaldehyde treated two-segment TcdB compo-ent combined with a full-length, formaldehyde inactivated TcdAomponent provided long-lasting (>3 months) immunity with100%urvival against lethal challenge with C. difficile. Moreover thisaccine was as efficacious as a full-length toxoid vaccine. To ournowledge this is the first report to demonstrate equivalent efficacyf a recombinant TcdB and toxoid antigen, where both moleculesere evaluated within the same experiment, at the same antigenose and formulation and using the same C. difficile challenge strain.
n contrast, we observed only partial efficacy in all other groupsgroups 3–8), immunized with one of several shorter fragments of
Please cite this article in press as: Karczewski J, et al. Development of a recombinant toxin fragment vaccine for Clostridium difficileinfection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.026
cdB and/or one fragment of TcdA in combination with full-lengthcdB toxoid.
two distinct regions of TcdB to better understand the contributionof multiple epitopes to protection [43] Additional animal efficacy
Please cite this article in press as: Karczewski J, et al. Development of a recombinant toxin fragment vaccine for Clostridium difficileinfection. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.026
ARTICLE IN PRESSG ModelJVAC 15111 1–7
J. Karczewski et al. / Vaccine xxx (2014) xxx–xxx 7
studies will be required to define which regions of TcdA and TcdBmediate protection from challenge and thus are critical for inclu-sion in a final vaccine composition. Similarly, additional work isrequired to understand the effect of chemical and genetic inactiva-tion on the structure, stability and antigenicity of both native andrecombinant toxins and fragments thereof. Finally, adjuvant andformulation considerations will be important to produce vaccinecandidates that offer long-lived protective efficacy in humans.
We have attempted to conduct a similar study with TcdA, how-ever our attempts to produce set of analogous fragments were notsuccessful. The preliminary results with A-C1 encourage furtherresearch to establish an alternative expression platform allowingproduction of sufficient quantities of antigens to support in vivostudies.
In summary, our wok shows that a fragment-based recombi-nant vaccine comprising two segments of TcdB, combined with fulllength toxoid A was highly immunogenic in a hamster lethal chal-lenge model of CDI. The vaccine stimulated development of robustneutralizing antibody titers and demonstrated complete protectionwhich was equivalent to a full length TcdA/TcdB toxoid vaccine.This approach presents an attractive and novel pathway for furtherQ6development of a low cost, efficacious and potentially safer vaccineto prevent CDI infections.
Acknowledgements
We thank Mark Knower, Patricia Rebbeck and Noelle Dahl-Parisifor their help in developing andperforming the hamster model ofCDI.
References
[1] Lo Vecchio A, Zacur GM. Clostridium difficile infection: an update on epi-demiology, risk factors, and therapeutic options. Curr Opin Gastroenterol2012;28(January (1)):1–9.
[2] Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, et al. Clinicalpractice guidelines for Clostridium difficile infection in adults: 2010 update bythe ociety for healthcare epidemiology of America (SHEA) and the infectiousdiseases society of America (IDSA). Infect Control Hosp Epidemiol 2010;31(May(5)):431–55.
[3] Loo VG, Bourgault AM, Poirier L, Lamothe F, Michaud S, Turgeon N, et al. Hostand pathogen factors for Clostridium difficile infection and colonization. N EnglJ Med 2006;365(November (18)):1693–703.
[4] McDonald LC, Killgore GE, Thompson A, Owens Jr RC, Kazakova SV, Sambol SP,et al. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med2005;353(December (23)):2433–41.
[5] Muto CA. Why are antibiotic-resistant nosocomial infections spiraling out ofcontrol? Infect Control Hosp Epidemiol 2005;26(January (1)):10–2.
[6] Pepin J, Valiquette L, Alary ME, Villemure P, Pelletier A, Forget K, et al. Clostrid-ium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: achanging pattern of disease severity. CMAJ 2004;171(August (5)):466–72.
[7] Zar FA, Bakkanagari SR, Moorthi KM, Davis MB. A comparison of vancomycinand metronidazole for the treatment of Clostridium difficile-associated diarrhea,stratified by disease severity. Clin Infect Dis 2007;45(August (3)):302–7.
[8] Pituch H, Bakker D, Kuijper E, Obuch-Woszczatynski P, Wultanska D, NurzynskaG, et al. First isolation of Clostridium difficile PCR-ribotype 027/toxinotype III inPoland. Pol J Microbiol 2008;57(3):267–8.
[10] Venugopal AA, Johnson S. Fidaxomicin: a novel macrocyclic antibiotic approvedfor treatment of Clostridium difficile infection. Clin Infect Dis 2011;54(February(4)):568–74.
[11] Lowy I, Molrine DC, Leav BA, Blair BM, Baxter R, Gerding DN, et al. Treatmentwith monoclonal antibodies against Clostridium difficile toxins. N Engl J Med2010;362(3):197–205.
[12] Kotloff KL, Wasserman SS, Losonsky GA, Thomas Jr W, Nichols R, Edelman R,et al. Safety and immunogenicity of increasing doses of a Clostridium difficiletoxoid vaccine administered to healthy adults. Infect Immun 2001;69(February(2)):988–95.
[13] Lyerly DM, Krivan HC, Wilkins TD. Clostridium difficile: its disease and toxins.Clin Microbiol Rev 1988;1(January (1)):1–18.
[14] Hamm EE, Voth DE, Ballard JD. Identification of Clostridium difficile toxin B car-diotoxicity using a zebrafish embryo model of intoxication. Proc Natl Acad SciU S A 2006;103(September (38)):14176–81.
[15] Cohen SH, Tang YJ, Silva Jr J. Analysis of the pathogenicity locus in Clostridiumdifficile strains. J Infect Dis 2000;181(February (2)):659–63.
[16] Riegler M, Sedivy R, Sogukoglu T, Castagliuolo I, Pothoulakis C, Cosentini E,et al. Epidermal growth factor attenuates Clostridium difficile toxin A- and B-induced damage of human colonic mucosa. Am J Physiol 1997;273(November(5 Pt 1)):G1014–22.
[17] Amimoto K, Noro T, Oishi E, Shimizu M. A novel toxin homologous to largeclostridial cytotoxins found in culture supernatant of Clostridium perfringenstype C. Microbiology (Reading, England) 2007;153(April (Pt 4)):1198–206.
[18] von Eichel-Streiber C, Boquet P, Sauerborn M, Thelestam M. Large clostridialcytotoxins – a family of glycosyltransferases modifying small GTP-binding pro-teins. Trends Microbiol 1996;4(October (10)):375–82.
[19] Spyres LM, Qa’Dan M, Meader A, Tomasek JJ, Howard EW, Ballard JD. Cytoso-lic delivery and characterization of the TcdB glucosylating domain by using aheterologous protein fusion. Infect Immun 2001;69(January (1)):599–601.
[20] Jank T, Giesemann T, Aktories K. Rho-glucosylating Clostridium difficile toxinsA and B: new insights into structure and function. Glycobiology 2007;17(April(4)):15R–22R.
[21] Voth DE, Ballard JD. Clostridium difficile toxins: mechanism of action and rolein disease. Clin Microbiol Rev 2005;18(April (2)):247–63.
[22] Jansen KU, Rosolowsky M, Schultz LD, Markus HZ, Cook JC, Donnelly JJ,et al. Vaccination with yeast-expressed cottontail rabbit papillomavirus (CRPV)virus-like particles protects rabbits from CRPV-induced papilloma formation.Vaccine 1995;13(November (16)):1509–14.
[23] Johnson WC. Analyzing protein circular dichroism spectra for accurate sec-ondary structures. Proteins 1999;35(May (3)):307–12.
[24] Provencher SW, Glockner J. Estimation of globular protein secondary structurefrom circular dichroism. Biochemistry 1981;20(January (1)):33–7.
[25] Sreerama N, Venyaminov SY, Woody RW. Estimation of the number ofalpha-helical and beta-strand segments in proteins using circular dichroismspectroscopy. Protein Sci 1999;8(February (2)):370–80.
[26] Sreerama N, Woody RW. A self-consistent method for the analysis of proteinsecondary structure from circular dichroism. Anal Biochem 1993;209(February(1)):32–44.
[27] Sreerama N, Woody RW. Poly(pro)II helices in globular proteins: identificationand circular dichroic analysis. Biochemistry 1994;33(August (33)):10022–5.
[28] Sreerama N, Woody RW. Estimation of protein secondary structure fromcircular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTRmethods with an expanded reference set. Anal Biochem 2000;287(December(2)):252–60.
[29] Busch C, Hofmann F, Gerhard R, Aktories K. Involvement of a conservedtryptophan residue in the UDP-glucose binding of large clostridial cytotoxinglycosyltransferases. J Biol Chem 2000;275(May (18)):13228–34.
[30] Jank T, Giesemann T, Aktories K. Clostridium difficile glucosyltransferase toxinB-essential amino acids for substrate binding. J Biol Chem 2007;282(November(48)):35222–31.
[31] Reinert DJ, Jank T, Aktories K, Schulz GE. Structural basis for the function ofClostridium difficile toxin B. J Mol Biol 2005;351(September (5)):973–81.
[32] Babcock GJ, Broering TJ, Hernandez HJ, Mandell RB, Donahue K, BoatrightN, et al. Human monoclonal antibodies directed against toxins A and Bprevent Clostridium difficile-induced mortality in hamsters. Infect Immun2006;74(November (11)):6339–47.
[33] Salnikova MS, Joshi SB, Rytting JH, Warny M, Middaugh CR. Physical charac-terization of Clostridium difficile toxins and toxoids: effect of the formalde-hyde crosslinking on thermal stability. J Pharm Sci 2008;97(September(9)):3735–52.
[34] Torres JF, Lyerly DM, Hill JE, Monath TP. Evaluation of formalin-inactivatedClostridium difficile vaccines administered by parenteral and mucosal routes ofimmunization in hamsters. Infect Immun 1995;63(December (12)):4619–27.
[35] Ochsner UA, Bell SJ, O’Leary AL, Hoang T, Stone KC, Young CL, et al. Inhibitoryeffect of REP3123 on toxin and spore formation in Clostridium difficile, andin vivo efficacy in a hamster gastrointestinal infection model. J AntimicrobChemother 2009;63(May (5)):964–71.
[36] Foglia G, Shah S, Luxemburger C, Pietrobon PJ. Clostridium difficile: developmentof a novel candidate vaccine. Vaccine 2012;30(June (29)):4307–9.
[37] Greenberg RN, Marbury TC, Foglia G, Warny M. Phase I dose finding studiesof an adjuvanted Clostridium difficile toxoid vaccine. Vaccine 2012;30(March(13)):2245–9.
[38] Tian JH, Fuhrmann SR, Kluepfel-Stahl S, Carman RJ, Ellingsworth L, Flyer DC.A 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 models. Vaccine 2012;30(June (28)):4249–58.
[39] Merrigan M, Venugopal A, Mallozzi M, Roxas B, Viswanathan VK, JohnsonS, et al. Human hypervirulent Clostridium difficile strains exhibit increasedsporulation as well as robust toxin production. J Bacteriol 2010;192(October(19)):4904–11.
[40] Wang H, Sun X, Zhang Y, Li S, Chen K, Shi L, et al. A chimeric toxin vaccine pro-tects against primary and recurrent Clostridium difficile infection. Infect Immun2012;80(August (8)):2678–88.
[41] Palace GP, Lazari P, Norton K. Analysis of the physicochemical interac-tions between Clostridium difficile toxins and cholestyramine using liquidchromatography with post-column derivatization. Biochim Biophys Acta2001;1546(March (1)):171–84.
[42] Pruitt RN, Lacy DB. Toward a structural understanding of Clostridium difficiletoxins A and B. Front Cell Infect Microbiol 2012;2(March):28.
[43] Marozsan AJ, Ma D, Nagashima KA, Kennedy BJ, Kang YK, Arrigale RR, et al. Pro-tection against Clostridium difficile infection with broadly neutralizing antitoxinmonoclonal antibodies. J Infect Dis 2012;206(September (5)):706–13.
