Vaccine xxx (2017) xxx–xxx

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Clostridium difficile chimeric toxin receptor binding domain vaccineinduced protection against different strains in active and passivechallenge models

http://dx.doi.org/10.1016/j.vaccine.2017.06.0620264-410X/� 2017 The Authors. Published by Elsevier Ltd.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Abbreviations: RBD, receptor binding domain; CDI, C. difficile infection; Q-toxin,quadravalent toxin vaccine; T-toxin, trivalent toxin vaccine; MLD, maximal lethaldose; HAC, human artificial chromosome.⇑ Corresponding author at: 20 Firstfield Road, Gaithersburg, MD 20878, USA.

E-mail addresses: jhtian@Novavax.com (J.-H. Tian), gglenn@novavax.com(G. Glenn), dflyer@novavax.com (D. Flyer), zhou@Novavax.com (B. Zhou), yliu@Novavax.com (Y. Liu), esullivan@sabbiotherapeutics.com (E. Sullivan), hwu@sabbiotherapeutics.com (H. Wu), jcummings@Novavax.com (J.F. Cummings),lellingsworth@Novavax.com (L. Elllingsworth), gsmith@novavax.com (G. Smith).

Please cite this article in press as: Tian J-H et al. Clostridium difficile chimeric toxin receptor binding domain vaccine induced protection against dstrains in active and passive challenge models. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.062

Jing-Hui Tian a, Gregory Glenn a, David Flyer a, Bin Zhou a, Ye Liu a, Eddie Sullivan b, Hua Wub,James F. Cummings a, Larry Elllingsworth a,⇑, Gale Smith a

aNovavax, Inc, 20 Firstfield Road, Gaithersburg, MD, USAb SAB Biotherapeutics, 2301 E. 60th Street, Sioux Falls, SD, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 April 2017Received in revised form 19 June 2017Accepted 20 June 2017Available online xxxx

Keywords:Clostridium difficileChimeric fusion protein vaccineTherapeutic immunoglobulin

Clostridium difficile is the number one cause of nosocomial antibiotic-associated diarrhea in developedcountries. Historically, pathogenesis was attributed two homologous glucosylating toxins, toxin-A(TcdA) and toxin-B (TcdB). Over the past decade, however, highly virulent epidemic strains of C. difficile(B1/NAP1/027) have emerged and are linked to an increase in morbidity and mortality. Increased viru-lence is attributed to multiple factors including: increased production of A- and B-toxins; productionof binary toxin (CDT); and the emergence of more toxic TcdB variants (TcdB(027)). TcdB(027) is more cyto-toxicity to cells; causes greater tissue damage and toxicity in animals; and is antigenically distinct fromhistorical TcdB (TcdB(003)). Broadly protective vaccines and therapeutic antibody strategies, therefore,may target TcdA, TcdB variants and CDT. To facilitate the generation of multivalent toxin-based C. difficilevaccines and therapeutic antibodies, we have generated fusion proteins constructed from the receptorbinding domains (RBD) of TcdA, TcdB(003), TcdB(027) and CDT. Herein, we describe the development of atrivalent toxin (T-toxin) vaccine (CDTb/TcdB(003)/TcdA) and quadravalent toxin (Q-toxin) vaccine(CDTb/TcB(003)/TcdA/TcdB(027)) fusion proteins that retain the protective toxin neutralizing epitopes.Active immunization of mice or hamsters with T-toxin or Q-toxin fusion protein vaccines elicited the gen-eration of toxin neutralizing antibodies to each of the toxins. Hamsters immunized with the Q-toxin vac-cine were broadly protected against spore challenge with historical C. difficile 630 (toxinotype 0/ribotype003) and epidemic NAP1 (toxinotype III/ribotype 027) strains. Fully human polyclonal antitoxin IgG wasproduced by immunization of transgenic bovine with these fusion proteins. In passive transfer studies,mice were protected against lethal toxin challenge. Hamsters treated with human antitoxin IgG werecompletely protected when challenged with historical or epidemic strains of C. difficile. The use of chi-meric fusion proteins is an attractive approach to producing multivalent antitoxin vaccines and therapeu-tic polyclonal antibodies for prevention and treatment of C. difficile infections (CDI).� 2017 The Authors. Published by Elsevier Ltd. This is anopenaccess article under the CCBY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Clostridium difficile is a spore-forming, Gram-negative anaerobethat is the leading cause of infectious diarrheal disease in hospitalsworldwide [1,2]. The clinical presentation of C. difficile infection(CDI) ranges from asymptomatic carriers to mild diarrhea to fulmi-nant pseudomembraneous colitis. In the United States, CDI isresponsible for 500,000 infections [3] and healthcare costs exceed-ing $3 billion [4]. Risk factors include antibiotic treatment of hos-pitalized patients receiving suppressive immunotherapy andchronic care elderly patients. In the US hospital setting, 10–25%of patients receiving antibiotic treatment develop CDI, relapse

ifferent

2 J.-H. Tian et al. / Vaccine xxx (2017) xxx–xxx

following antibiotic treatment is 20% (61,400 recurrences), and thedeath rate is >9% (27,000 deaths) [3].

C. difficile pathogenicity is mediated by two high molecularweight extotoxins, toxin-A (TcdA) and toxin-B (TcdB), which aresecreted as single large polypeptides with similar functionaldomains [5]. The sequence homology between the toxins is 63%[6], however, TcdA and TcdB neutralizing antibodies are notcross-neutralizing [7]. The N-terminal domain of both toxins isan ADP-glucosyltransferase (GT) that mediates glycosylation ofRho GTPase resulting in disruption of colonic epithelial tight junc-tion integrity with excessive fluid loss [8,9]. Adjacent to the GTdomain is the autocatalytic cysteine protease (CP) domain, whichfunctions to proteolytically cleave and activate GT within the acidicenvironment of endosomes [10,11]. The central hydrophobicdomain (PT) is responsible for pore formation within endosomalmembranes and aids in transport of activated GT into the cytosolof intoxicated cells [11]. The C-terminal domain consists of a seriesof repeating units that make up the receptor binding domains(RBD). The RBD binds toxins to cell-surface receptors on colonicepithelial cells which stimulate endocytosis, endosome formation,and uptake of holotoxin (Fig. 1A) [12,13].

The amino acid sequences of TcdA are highly conservedbetween the historical strain 630 (toxinotype 0/ribotype 03) andthe more virulent epidemic strain B1/NAP1/027 (toxinotype III/ribotype 027), while TcdB has greater sequence differences (overall92% identity). TcdB variant from NAP1 (TcdB(027)) is 1000-foldmore cytotoxic and 4-fold more lethal in mice than TcdB fromstrain 630 (TcdB(003)) [14,15]. The greatest sequence differencesoccur within the RBD (overall 88% identity). Epitope mapping stud-ies have identified 11 unique antigenic epitopes within the RBDbetween the two strains and antibodies are not cross-neutralizing [15].

Binary toxin (CDT) is a third virulence factor that is produced bythe hypervirulent NAP1 strain. Although its role in pathogenesis isnot fully understood, CDT is believed to contribute to increasedmorbidity and mortality [16–18]. CDT consists of two toxin com-ponents (Fig. 1A). CDTa is the enzymatically active componentwith ADP-ribosyltransferase activity which modifies G-actin andblocks polymerization of F-actin resulting in disruption of micro-tubule organization [16]. CDTb is the receptor binding componentthat is activated by serine protease to release two smaller peptides.The larger peptide forms a heptamer that binds the cell surfacereceptor and mediates internalization [16,19].

Vaccination is a viable strategy for preventing CDI. Non-toxinbased strategies include bacterial surface proteins such as flagellarcomponents (FliC and FliD), adhesins (Cwp66) and surface polysac-charides (PSI, PSII and PSI) that are broadly expressed on the sur-face of different C. difficile ribotypes [20–22]. Strategies targetingthe C. difficile toxins for prevention of CDI must take into consider-ation the role of multiple virulence factors and the emergence oftoxin variants that are antigenically distinct from historical strains.To this end, we generated novel chimeric fusion proteins compris-ing the RBD of TcdA, TcdB(003), TcdB(027), and CDT. The immuno-genicity and in vivo protection by multivalent vaccines andtransgenically produced human antitoxin immunoglobulins wereevaluated in mice toxin challenge and hamster CDI models.

2. Methods

2.1. Design of constructs

Chimeric fusion proteins were constructed to encode RBD ofTcdA, TcdB(003), TcdB(027), and CDTb. The RBD amino acid sequencefor TcdA was derived from C. difficile strain VPI 10463 (ATCC43255), NCBI P16154 (toxinotype 0/ribotype 003); TcdB(003) from

Please cite this article in press as: Tian J-H et al. Clostridium difficile chimeric tostrains in active and passive challenge models. Vaccine (2017), http://dx.doi.o

strain VPI 10463 (ATCC 43255), NCBI P18177 (toxinotype 0/ribo-type 003); TcdB(027) from strain CD196, NCBI WP_009888442.1(toxinotype III/ribotype 027); and CDTb from strain CD196, Gen-Bank ABS57477.1 (toxinotype III/ribotype 027).

The coding sequences for TcdA RBD (truncated with 19 of 38repeats), TcdB(003) and TcdB(027) RBDs (24 repeats each), and CDTbwere codon optimized for expression in insect cells (GenScript).

The nucleotide sequences encoding the CDTb gene fragment(amino acids 1–835), TcdA RBD (1314 base pairs [bp], 6816–8130 bp), and TcdB(003) RBD (1608 bp, 5493–7098 bp) were PCRamplified from the synthesized gene. PCR-amplified gene frag-ments were digested with restriction enzyme: CDTb with BamHI/NheI; TcdB(003) RBD with NheI/XbaI; and TcdA RBD with XbaI/Hin-dIII. After digestion, the three genes were ligated into the BamH1and HindIII sites of pFastBac1 (Invitrogen). The plasmid encodingthe three RBDs was used to construct a recombinant Autographacalifornica Multiple Nuclear Polyhedrosis Virus (AcMNPV) bac-ulovirus using the Bac-to-Bac baculovirus expression system(Invitrogen) in Spodoptera frugiperda (Sf9) insect cells to expressthe trivalent fusion protein, hereafter referred to as T-toxin.

TcdB(027) RBD (1608 bp, 5493–7098) digested with Spel/HinIIIwas fused to the C-terminus of the trivalent fusion gene to formthe plasmid and baculovirus construct encoding the RBD of all fourtoxins, which was similarly expressed in Sf9 cells to produce thequadravalent fusion protein, hereafter referred to as Q-toxin.

2.2. Purification of T-toxin and Q-toxin chimeric fusion proteins fromcell lystates

Baculovirus-infected Sf9 cells were lysed with 0.2% Tergiltol NP-9 in 25 mM Tris buffer (pH 8.0), 250 mM NaCl and 2 mg/mL leu-peptin. Fusion proteins were purified with Fractogel EMD TMAE,phenyl HP and 30Q column chromatography. Purified T-toxinand Q-toxin were formulated in 25 mM Tris and 250 mM NaCl(pH 8.0) at 4.0 mg/mL.

2.3. SDS-PAGE and western blot

Purified T-toxin and Q-toxin were electrophoresed on 4–12%NuPAGE� gels (Invitrogen) and stained with SimplyBlueTM or usedfor Western blots. Blots were treated with chicken anti-TcdA(1:4000; US Biologicals), chicken anti-TcdB (1:10,000; US Biologi-cals), or rabbit anti-CDTb (1:5000; Covance) serum as primaryantibodies and species-specific secondary antibodies, alkalinephosphatase-conjugated goat anti-chicken or rabbit IgG (1:5000;SeraCare).

2.4. Spore preparation

C. difficile strain 630 (BAA-1382, toxinotype 0/ribotype 003) andstrain B1/NAP1/027 (BAA-1805, toxinotype III/ribotype 027) wereobtained from American Type Culture Collection (ATCC). Eachstrain was grown on trypticase soy + 5% sheep blood agar platesfor five days under anaerobic conditions at 37 �C. Spores were har-vested and heat-shocked at 60 �C for 15 min. Inoculum was pre-pared by diluting stock spore suspensions in DMEM to 1–2 � 108

spores/mL (NAP1) or 1 � 103 spores/mL (strain 630). Titers wereverified by dilution plating on C. difficile spores overnight underanaerobic conditions at 37 �C. Spore concentrations were3.5 � 107 CFU/mL (NAP1) and 3.0 � 102 CFU/mL (strain 630).

2.5. Toxins

TcdA and TcdB(003) were purchased from List Biological Labora-tories and TcdB(027) from Native Antigen Company. Binary toxin(CDTa + CDTb) was produced by Novavax.

xin receptor binding domain vaccine induced protection against differentrg/10.1016/j.vaccine.2017.06.062

GT CP PT RBD (38 repeats)

017284819674451

N- -C

GT CP PT RBD (24 repeats)

663215617083451

-CN-

TcdAMW 308 kDa

TcdB(003)MW 270 kDa

876

-CN-

1

CDTbMW 98.8 kDa

GT CP PT RBD (24 repeats)

663215617083451

-CN-

TcdB(027)MW 270 kDa

N- 835 aa

CDTb

24 repeats, 536 aa

19 repeats, 438 aa

TcdB(003) RBD

TcdARBD

T-toxin fusion protein MW 205 kDa

N- 835 aa

CDTb

24 repeats, 536 aa

19 repeats, 438 aa

TcdB(003) RBD

TcdARBD

Q-toxin fusion protein MW 268 kDa

24 repeats, 536 aa -C

-C

TcdB(027)RBD

CDTa MW 53 kDa

N- -C

1 420

ADP- ribosyltransferase RBD

835 aa

42 aa

42 aa

Clostridium difficile Toxin A, Toxin B and Binary Toxin

Construc�on of Trivalent and Quadravalent Toxin Fusion Proteins

43 42

A

B

kDa

250

150

10075

50

37

25

2015

1 2 3 4 5

T-toxin→ Q-toxin→

C D E F

Fig. 1. Functional domains of toxin A (TcdA), toxin B (TcdB), and binary toxin (CDT) used to construct the chimeric trivalent and quadravalent toxin fusion proteins. (A) TcdAand TcdB share common functional domains including the enzymatic glucosyltransferase (GT) domain, autocatalytic cysteine protease (CP) domain, pore-formingtranslocation (PT) domain, and receptor binding domain (RBD). The binary toxin (CDT) consists of the enzymatic ADP-ribosyltransferase component (CDTa) and receptorbinding component (CDTb). CDTb contains a 42 amino acid (aa) signal sequence with two serine-type proteolytic cleavage sites (arrow) which, when cleaved, generates a20 kDa and 75 kDa fragment. (B) The chimeric trivalent toxin fusion protein (T-toxin) was generated by joining the full-length coding sequence for CDTb with the RBD ofTcdB(003) containing 24 repeats and the truncated RBD of TcdA with 19 repeats. The expressed T-toxin fusion protein consists of 1813 aa. The chimeric qudravalent toxinfusion protein (Q-toxin) was generated by fusing the full-length coding sequence for CDTb to the RBD of TcdB(003) containing 24 repeats, the RBD of TcdA truncated at 19repeats, and the RBD of TcdB(027) containing 24 repeats. The expressed Q-toxin fusion protein consists of 2359 aa. (C) SDS-PAGE of purified T-toxin (lanes 2 and 3) migrateswith a molecular weight of 205 kDa and Q-toxin (lanes 4 and 5) migrates with a molecular weight of 268 kDa. T-toxin and Q-toxin purity was >90% as determined by SDS-PAGE scanning densitometry. Western blot analysis of T-toxin (lanes 2 and 3) and Q-toxin (lanes 4 and 5) fusion proteins probed with rabbit anti-CDTb (D), chicken anti-TcdB(E), and chicken anti-TcdA (F) specific antibodies. Molecular weight marker (lane 1).

J.-H. Tian et al. / Vaccine xxx (2017) xxx–xxx 3

Please cite this article in press as: Tian J-H et al. Clostridium difficile chimeric toxin receptor binding domain vaccine induced protection against differentstrains in active and passive challenge models. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.06.062

4 J.-H. Tian et al. / Vaccine xxx (2017) xxx–xxx

2.6. Murine immunogenicity and toxin challenge model

All animal studies were carried out in compliance with the NIHguide for the care and use of laboratory animals (NIH PublicationNo. 8023, revised 1978). Mouse studies were conducted in accor-dance with Noble Life Sciences’ IACUC approved protocols. FemaleC57BL/6 mice (6–8 weeks old) were immunized IM on Days 0 and14 with T-toxin (30 or 100 mg) or Q-toxin (100 mg) formulated with50 mg aluminum hydroxide (alum), or PBS (control). Serum wascollected 18 days after the second dose. Mice were challengedintraperitoneally (IP) 3 weeks after the second immunization witha 100% minimal lethal dose (MLD100%) of TcdA, TcdB(003), or CDTa/CDTb.

2.7. Hamster immunogenicity and spore challenge model

This study was conducted by Aragen BioSciences with an IACUCapproved protocol. Golden Syrian hamsters (HsdHan:Aura; HarlanLaboratories), males aged 5–7 weeks and 70–100 g, received 3immunizations at 3-week intervals with 30 mg Q-toxin and120 mg alum, or PBS (control), administered IM in alternatingthighs. Two weeks after the third immunization serum was col-lected and animals were treated with 10 mg/kg clindamycin IP.One day later, animals were challenged by gavage with strain630 or NAP1 spores.

2.8. Mouse and hamster ELISA methods

Mouse and hamster sera were evaluated for antibodies to thetoxins by ELISA. Briefly, 96-well MaxiSorp microtiter plates(Thermo Scientific) were coated with each toxin (2 mg/mL). Fivefoldserial dilutions of sera were added to plates. Bound antibodieswere detected with horseradish peroxidase-conjugated goat anti-mouse IgG or rabbit anti-hamster IgG (Southern Biotech). 3,30,5,50-tetramethylbenzidine (TMB) substrate (Sigma) was added andthe reaction stopped with TMB Stop Buffer (Scytek Laboratories).Plates were read at 450 nm with a SpectraMax Plus plate reader(Molecular Devices); results were analyzed using SoftMax Pro soft-ware. Titers were reported as the reciprocal dilution that resultedin a reading of 50% the maximum OD450nm. Titer values recordedas below the lower limit of detection (LLOD) were assigned a titer50 for calculating GMT.

2.9. In vitro toxin neutralization assay

Twofold serial dilutions of mouse or hamster sera were pre-pared in 96-well, flat-bottom tissue culture plates (Thermo Scien-tific). An equal volume (50 mL) of assay medium (1� DMEM with5% heat-inactivated FBS, 1� NEAA, 0.3% dextrose, 1� penicillin/streptomycin/glutamine, 0.006% Phenol Red) containing 2� mini-mum cytotoxic dose of TcdA, TcdB, or CDT was added to dilutedserum and incubated for 1 h at 37 �C. Vero cells (CCL-81, ATCC)suspended (7.5 � 104 cells/mL) in 50 mL medium and 150 mL min-eral oil (Sigma) were added and plates were incubated for 6–7 daysat 37 �C. After incubation, plates were observed for well color.Media and toxin-treated control wells were red/reddish-pink; cellcontrol wells were yellow/yellow-orange. For each sample dilu-tion, the last well that was yellow/yellow-orange was recordedas the endpoint neutralizing-antibody titer. Titer values recordedas <LLOD were assigned a value of 5 for calculating GMT.

2.10. Generation of hyperimmune human antitoxin IgG intranschromosomal bovine

Transchromasomal (Tc) bovine are triple knockouts in theendogenous bovine immunoglobulin genes (IGHM�/� IGHML1�/�

Please cite this article in press as: Tian J-H et al. Clostridium difficile chimeric tostrains in active and passive challenge models. Vaccine (2017), http://dx.doi.o

IGL�/�) and carry a human artificial chromosome (HAC) vector, isK-cHACD [23–26]. The HAC consists of two human chromosome frag-ments: the A14 fragment with the entire human immunoglobulinheavy chain locus except that the IGHM constant region remainsbovine and the key regulatory sequences were bovinized; andhuman chromosome 2 fragment with the entire humanimmunoglobulin j light chain locus [23–26]. Tc bovine producefully-human polyclonal antibodies (SAB BioTherapeutics; SAB).

Tc bovine was immunized via IM injection at 3-week intervalswith T-toxin (2 mg � 3 doses) followed by Q-toxin (5 mg � 3doses) formulated with SAB proprietary adjuvant (SAB-adj-1).Hyperimmune plasmas were collected after 3 vaccinations withT-toxin and after 3 additional vaccinations with Q-toxin byplasmapheresis (Baxter Healthcare, Autopheresis C Model 200).Fully-human IgG (hIgG) was purified from plasma [26] collectedafter 3 vaccinations with T-toxin (referred to as anti-T-toxin hIgG)and after 3 additional vaccinations with Q-toxin (referred to asanti-T + Q-toxin hIgG). hIgG was formulated in 10 mM glutamicacid, 262 mM D-sorbitol, 0.05 mg/mL Tween80, pH 5.5, at30.89 mg/mL for anti-T-toxin hIgG and 51.71 mg/mL for anti-T+ Q-toxin hIgG.

2.11. Murine passive protection

Female C57BL/6 mice (7–8 weeks old) were administered 2 mganti-T-toxin hIgG or PBS IP. Mice were challenged IP 24 h later witha MLD100% of TcdA, TcdB(003), or CDTa plus CDTb and observed for7 days.

2.12. Hamster passive protection

This study was conducted by Ricerca Biosciences’ under anIACUC approved protocol. Male Golden Syrian hamsters (Crl:LVG(SYR); Charles River Laboratories), aged 45 days and 113–117 g,received anti-T-toxin or anti-T + Q-toxin hIgG (10 or 60 mg), orPBS (control), IP (2 mL) daily (Days -4 to -1). Clindamycin (Sigma)was administered SC (50 mg/kg) on Day -1. Hamsters wereinfected on Day 0 by gavage with strain 630 or NAP1 and observedfor 8 days.

2.13. Statistical analysis

Survival data were analyzed by Mantel-Cox log-rank test usingGraphPad Prism software.

3. Results

3.1. Constructs, expression, and purification of T-toxin and Q-toxin

Constructs encoding the C-terminal RBD of TcdA, TcdB, andCDTb were generated to produce T-toxin and Q-toxin. T-toxinwas constructed with full-length CDTb fused with TcdB(003) RBDand truncated TcdA RBD. Q-toxin was constructed by addingTcdB(027) RBD from NAP1 to the C-terminus of the T-toxin(Fig. 1B). T-toxin or Q-toxin was expressed by infection of Sf9 cellswith recombinant baculovirus. Recovery of purified T-toxin and Q-toxin was 267 and 154 mg/L, respectively. T-toxin and Q-toxinmigrate in SDS-PAGE gels with molecular weights of 205 kDa and268 kDa, respectively, and purity of >90% (Fig. 1C). Western blotanalysis with toxin-specific antibodies confirmed expression ofCDTb, TcdB, and TcdA in each fusion protein (Fig. 1D–F).

xin receptor binding domain vaccine induced protection against differentrg/10.1016/j.vaccine.2017.06.062

J.-H. Tian et al. / Vaccine xxx (2017) xxx–xxx 5

3.2. Immunogenicity of T-toxin and Q-toxin fusion proteins in mice

To evaluate immunogenicity of T-toxin, mice received twoimmunizations given 2 weeks apart. Serum IgG titers followingimmunization were high for TcdA, TcdB, and CDT, however,toxin-neutralizing antibody (TNA) titers were modest (Table 1).Three weeks after the second immunization, mice were challengedwith TcdA, TcdB(003), or CDT. All T-toxin vaccinated animals werecompletely protected from intoxication with TcdA and CDT and83% survived TcdB(003) challenge. In contrast, there were no sur-vivors in the control groups receiving TcdA or TcdB(003) and only25% survived CDT challenge (Table 1).

The immunogenicity of Q-toxin was compared to T-toxin.Immunization with Q-toxin and T-toxin resulted in generation ofcomparable IgG and TNA titers to each of the three toxins(Fig. 2A and B, Table 1). Mice were challenged 3 weeks after thesecond immunization with TcdB(003). The group vaccinated withQ-toxin had 80% survival (p = 0.0043), while 65% (p = 0.018) ofthe T-toxin group survived challenge. In contrast, only 20% sur-vived toxin challenge in the control group (Fig. 2C, Table 1). Takentogether, these studies demonstrate that vaccination with thefused RBD of each toxin elicits generation of high IgG titers andneutralizing antibodies that protect (65–100%) againstintoxication.

3.3. Protective efficacy of Q-toxin vaccine in hamster CDI model

Q-toxin was designed to include the RBD of TcdB variantsderived from strain 630 (TcdB(003)) and NAP1 (TcdB(027)) and,therefore, should be broadly protective. Hamsters immunizedthrice at 3-week intervals with Q-toxin produced high IgG titersand TNA to all three toxins (Fig. 3A and B). After clindamycin treat-ment, animals infected with C. difficile strain 630 had 90% survival,while animals infected with NAP1 had 75% survival. All animals inthe placebo group died within 48–72 h following infection witheither strain (Fig. 3C and D).

3.4. Generation of hyperimmune human antitoxin IgG in Tc bovine

Human polyclonal antibodies were produced by immunizing Tcbovine with T-toxin and Q-toxin. One Tc bovine was immunized at3-week intervals thrice with T-toxin to generate anti-T-toxin hIgGfollowed by three additional immunizations with Q-toxin togenerate anti-T + Q-toxin hIgG. Titers following 3 immunizationswith T-toxin were determined for TcdA (5.2 � 106), TcdB(003)

(4.1 � 106), and CDTb (4.5 � 106). These IgG titers did notchange following immunization with Q-toxin (TcdA = 6.0 � 106,TcdB(003) = 9.4 � 106, TcdB(027) = 4.6 � 106, and CDTb = 3.9 � 106).Toxin-neutralizing titers after immunization with T-toxin werehighest for TcdA (TNA = 12,000), 7.5-fold lower for TcdB(003) and

Table 1Evaluation of immunogenicity and protection against lethal C. difficile toxin challenge in m

Vaccinea Dose Serum IgG Titer (GMT ± SEM)b

TcdA TcdB CDTb

Placebo 0 <LLOD <LLOD <LLODT-toxin 30 mg 197,931 ± 56,367 118,432 ± 15,464 245,150 ±T-toxin 100 mg 173,993 ± 33,980 34,682 ± 10,885 171,470 ±Q-toxin 100 mg 147,280 ± 15,508 97,370 ± 16,375 249,367 ±

a C56BL/6 mice (n = 6–10/group) were vaccinated IM on Days 0 and 14 with T-toxin ob Sera were collected 18 days after the second vaccination.c Three weeks after the second dose mice were challenged intraperitoneally with a

(0.75 mg).d Not determined; animals in these treatment groups were only challenged with TcdB

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CDT (TNA = 1600), and 19-fold lower for TcdB(027) (TNA = 640).Toxin-neutralizing titers for all toxins were increased after 3additional vaccinations with Q-toxin: TcdA increased 4-fold(TNA = 51,000), TcdB(003) increased 32-fold (TNA = 51,200),TcdB(027) increased 40-fold (TNA = 25,600), and CDT increased2-fold (TNA = 3200) (Table S1).

3.5. Protective efficacy of anti-T-toxin hIgG in mouse intoxicationmodel

The protective efficacy of anti-T-toxin hIgG was assessed inmice receiving lethal toxin challenge. Mice received a single injec-tion of anti-T-toxin hIgG or PBS and were challenged with TcdA,TcdB(003), or CDT one day following treatment. All mice in the con-trol groups died within 24–48 h. In contrast, 90–100% of mice trea-ted with anti-T-toxin hIgG survived the 7-day post-challengeperiod (Fig. 4).

3.6. Passive protection by anti-T-toxin hIgG and anti-T + Q-toxin hIgGin hamster CDI model

In an initial study, hamsters were treated with anti-T-toxinhIgG (10 or 60 mg) or PBS administered daily for 4 days and werechallenged with NAP1 one day after final treatment with hIgG andclindamycin. All animals in the control group died within 6 daysfollowing challenge and only 25% of animals in the anti-T-toxinhIgG group survived the 7-day observation period regardless ofdose (Fig. 5A). The poor survival was likely because the T-toxinused to generate anti-T-toxin hIgG was constructed using onlyTcdB(003), while animals were challenged using the hypervirulentNAP1 strain with TcdB(027). To test this hypothesis, the protectiveefficacy of anti-T + Q-toxin hIgG, which was prepared by immuniz-ing Tc bovine with both T-toxin and Q-toxin, was evaluated. In thisstudy, hamsters were treated daily for 4 days with anti-T + Q-toxinhIgG (10 or 60 mg) or PBS and were challenged with C. difficilestrain 630 or NAP1 one day after treatment. All animals in the con-trol group died within 24–48 h following challenge, while 40%treated with 10 mg anti-T + Q-toxin hIgG and 90–100% treatedwith 60 mg anti-T + Q-toxin hIgG survived the 8-day observationperiod (Fig. 5B-C). These results are consistent with the differencesin antigenic profiles of TcdB(003) and TcdB(027) and their lack ofcross-neutralizing epitopes.

4. Discussion

CDI is a toxin-based gastrointestinal disease with significantmortality and morbidity. Hos defense against severe or fatal dis-ease is mediated through antitoxin antibodies [27]. Here we eval-uated the feasibility of producing a single multivalent antitoxinvaccine that retains toxin-neutralizing epitopes from the RBDs of

ice vaccinated with T-toxin and Q-toxin.

TNA (GMT)b Toxin challenge survivors at7 days post challengec

TcdA TcdB CDT TcdA TcdB CDT

<LLOD <LLOD <LLOD 0 0 25%137,244 18 8.4 8.3 100% 83% 100%17,569 320 80 80 NDd 65% NDd

39,370 640 160 80 NDd 80% NDd

r Q-toxin adjuvanted with 50 mg alum, or PBS (control group).

MLD100% of TcdA (1 mg), TcdB(003) (2 mg), or a mixture of CDTa (0.25 mg) and CDTb

.

xin receptor binding domain vaccine induced protection against differentrg/10.1016/j.vaccine.2017.06.062

Fig. 2. Immunogenicity of T-toxin and Q-toxin fusion proteins in mice. Groups of female C57BL/6 mice (N = 10/group) were immunized IM on Days 0 and 14 with T-toxin(100 mg) or Q-toxin (100 mg) adjuvanted with alum (50 mg), or PBS (control group). Serum was collected 18 days after the second vaccination. Serum IgG titers to TcdA,TcdB(003), and CDTb were determined by ELISA (A). Vero cells were used to determine toxin-neutralizing antibody (TNA) titers using pooled serum (B). Mice received a lethaldose (MLD100% = 2.0 mg) of TcdB(003) administered IP 21 days after the second immunization (C). *Significance was determined by Mantel-Cox log-rank test comparing the T-toxin or Q-toxin groups to the PBS control group.

6 J.-H. Tian et al. / Vaccine xxx (2017) xxx–xxx

TcdA, variants of TcdB, and CDT. We reasoned that such a vaccineshould provide broad protection against diverse strains causinghuman disease. TcdA and TcdB are exotoxins that are structurallysimilar; however, neutralizing antibodies to each toxin are notcross-protective [6,7]. The role of TcdB as a major contributor topathogenesis has been confirmed by the occurrence of A�B+ C. dif-ficile isolates that produce severe gastrointestinal disease [28].TcdB produced by NAP1 and strain 630 are antigenically differentand have as many as 11 distinct epitopes in the RBD. In order tobroaden the protective efficacy of an antitoxin vaccine, the Q-toxin was constructed with TcdB RBD derived from both TcdB(003)

and TcdB(027) (Fig. 1B). Hamsters vaccinated with Q-toxin devel-oped antitoxin antibodies and were protected against infection

Please cite this article in press as: Tian J-H et al. Clostridium difficile chimeric tostrains in active and passive challenge models. Vaccine (2017), http://dx.doi.o

with strain 630 (90% survival), and NAP1 (75% survival)(Fig. 3C and D).

The role of CDT as a third major C. difficile toxin remains to befully defined. Mechanistically, CTD functions to disrupt thecytoskeletal structure of enterocytes and is highly toxic in themouse toxin challenge model (Table 1). Further, hamsters vacci-nated with attenuated forms of TcdA + TcdB + CDT are partially tofully protected (67–100% survival) against lethal challenge withNAP1 (TcdA+ TcdB+ CDT+) or 8864 (TcdA� TcdB+ CDT+) strains,while animals vaccinated with attenuated bivalent TcdA + TcdBdid not survive challenge with CDT+ strains [29]. Further evidencefor its role as a significant virulence factor in CDI is the presenceof CDT in epidemic NAP1 strains [17,18]. Strategies aimed at

xin receptor binding domain vaccine induced protection against differentrg/10.1016/j.vaccine.2017.06.062

C. difficile strain 630 C. difficile B1/NAP1/027 C D

A B

Fig. 3. Immunogenicity of Q-toxin fusion protein in hamsters. Male hamsters (N = 8/group) were immunized IM 3 times at 21-day intervals with 30 mg Q-toxin adjuvantedwith 120 mg alum, or PBS (control group). Two weeks after the third dose, blood was collected from the orbital plexus. Serum IgG titers to TcdA, TcdB(003), and CDTb weredetermined by ELISA (A). Toxin-neutralizing antibody titers for each toxin were determined in the Vero cell assay (B). Two weeks after the third immunization, all animalswere treated with clindamycin (10 mg/kg) IP one day prior to spore challenge and were challenged by gavage with 200 cfu C. difficile strain 630 (C) or with 500 cfu C. difficilestrain B1/NAP1/027 (D). Animals were observed for 8 days post challenge.

Fig. 4. Passive protection of mice treated with Tc bovine anti-T-toxin hIgG and challenge with C. difficile toxins. Female C57BL/6 mice (N = 10/group) were treated IP with asingle dose of purified hyperimmune anti-T-toxin hIgG (2 mg) or PBS (control group). All animals received a MLD100% of TcdA (0.2 mg), TcdB(003) (0.5 mg), or a mixture of CDTa(0.75 mg) + CDTb (2.0 mg) administered IP. Animals were observed daily for 7 days post-toxin challenge for mortality and morbidity.

J.-H. Tian et al. / Vaccine xxx (2017) xxx–xxx 7

developing broadly protective antitoxin vaccines should includeTcdA, variants of TcdB, as well as CDT as targets.

Fully-human polyclonal antitoxin produced by hyperimmuniz-ing Tc bovine with T-toxin and Q-toxin induced high titer, neutral-izing activity against all toxins (Table S1). Passive transfer ofanti-T-toxin hIgG completely protected (90–100% survival) micereceiving lethal toxin challenge (Fig. 4). However, hamsters treatedwith anti-T-toxin hIgG were poorly protected (25% survival) whenchallenged with hypervirulent NAP1. The poor protection withanti-T-toxin hIgG was likely due to limited cross-reactivity of

Please cite this article in press as: Tian J-H et al. Clostridium difficile chimeric tostrains in active and passive challenge models. Vaccine (2017), http://dx.doi.o

neutralizing antibodies between TcdB(003) and TcdB(027). Tobroaden protection, passive transfer of anti-T + Q-toxin hIgG fromTc bovine additionally immunized with Q-toxin, which alsoincludes TcdB(027), broadly protected hamsters infected withstrains 630 or NAP1 (Fig. 5B and C).

Our findings are supportive of previous reports of functionaland antigenic differences between TcdB produced by historicaland epidemic strains of C. difficile. Sequence variation in theRBD may account for increased cytotoxicity, pathogenicity andantigenicity of TcdB(027). Further, recent C. difficile molecular

xin receptor binding domain vaccine induced protection against differentrg/10.1016/j.vaccine.2017.06.062

0

20

40

60

80

100

Days Post ChallengePe

rcen

t sur

viva

l

PBS

Anti-T-toxin hIgG (60 mg)

Anti-T-toxin hIgG (10 mg)

*p=0.03

C. difficile B1/NAP1/027 A

0

25

50

75

100PBS

Anti-T+Q-toxin hIgG(60mg/kg)

Anti-T+Q-toxin hIgG(10 mg/kg)

Day

Per

cent

sur

viva

l

*p<0.001

C. difficile strain 630 B

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 80

25

50

75

100

DayP

erce

nt s

urvi

val

*p<0.001

C. difficile B1/NAP1/027 C

Fig. 5. Passive protection of hamsters treated with Tc bovine anti-T-toxin hIgG or anti-T + Q toxin hIgG and challenged with C. difficile strain 630 or hypervirulent strain B1/NAP1/027. Male hamsters (N = 8/group) were treated IP with PBS (control groups) or 10 or 60 mg anti-T-toxin hIgG (A) or anti-T + Q-toxin hIgG (B and C) administered dailyfor four days (Days -4, -3, -2 and -1). Clindamycin (50 mg/kg) was administered SC one day prior to oral spore challenge (Day -1). Hamsters treated with anti-T-toxin hIgGreceived C. difficile B1/NAP1/027 (5 � 107 cfu). Hamsters treated with anti-T + Q-toxin hIgG received 150 cfu C. difficile strain 630 or 5 � 107cfu C. difficile B1/NAP1/027 bygavage. *p-values were determined by Mantel-Cox log-rank comparing active groups to PBS control.

8 J.-H. Tian et al. / Vaccine xxx (2017) xxx–xxx

epidemiology studies in the UK have identified a total of 17different allelic forms of the tcdB gene obtained from a collectionof 1290 clinical isolates [30]. These observations also suggestvaccines and therapeutic antibody strategies targeting TcdB as anantigen will need to consider targeting multiple antigenic variantsof TcdB.

Financial support

This work was fully funded and supported by Novavax, Inc andSAB Biotherapeutics, Inc. Novavax provided salary support for JHT,GG, DF, BZ, LF, JFC, YL, LE and GS. SAB Biotheapuetics providedsalary support for ES, HW and JJ. Study design, data analysis andeditorial contributions to this publication were provided by theauthors.

Conflicts of interest

Authors from Novavax, Inc. and SAB Biotherapeutics, Inc arecurrent or past employees of for profit organizations and authorsfrom these organizations own stock or hold stock options in theirrespective companies. The authors have submitted and pendingpatent applications related to the work. These interests do not alterthe author’s adherence to policies on sharing data and materials.

Acknowledgments

The authors thank Maggie Lewis for editorial assistance in thepreparation of this manuscript, Mimi Gueber-Xabier for assistancewith animal studies and Jin-An Jiao for purification of Tc bovineproduced human antitoxin immunoglobulins.

Please cite this article in press as: Tian J-H et al. Clostridium difficile chimeric tostrains in active and passive challenge models. Vaccine (2017), http://dx.doi.o

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.vaccine.2017.06.062.

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