Vaccine 27 (2009) 4601–4608

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Vaccination with EtpA glycoprotein or flagellin protects against colonizationwith enterotoxigenic Escherichia coli in a murine model

Koushik Royc, David Hamiltond, Marguerite M. Ostmannb, James M. Fleckensteina,c,e,∗

a Medicine, University of Tennessee Health Science Center, Memphis, TN, United Statesb Research Services, University of Tennessee Health Science Center, Memphis, TN, United Statesc Veterans Affairs Medical Center; Departments of Medicine, University of Tennessee Health Science Center, Memphis, TN, United Statesd Comparative Medicine, University of Tennessee Health Science Center, Memphis, TN, United Statese Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN, United States

a r t i c l e i n f o

Article history:Received 28 January 2009Received in revised form 7 May 2009Accepted 27 May 2009Available online 11 June 2009

Keywords:

a b s t r a c t

Enterotoxigenic Escherichia coli (ETEC) remain a leading cause diarrheal illness, prompting a search forvaccine targets that led to the recent discovery of EtpA, a secreted adhesin of ETEC that acts by bridgingflagella and host cells. In a murine model, immunization with recombinant EtpA glycoprotein inhibitedcolonization by two EtpA-producing human ETEC strains, H10407 and E24377A. In addition, vaccinationwith recombinant flagellin (serotype H11) generated antibodies that specifically recognized the tips offlagella from E24377A expressing a heterologous flagellar serotype (H28) and afforded significant protec-tion against colonization. EtpA and/or flagellin could be valuable subunit antigens in the formulation of

Adhesins, BacterialEscherichia coliEscherichia coli VaccinesDF

a broadly protective ETEC vaccine.Published by Elsevier Ltd.

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. Introduction

Infectious diarrhea remains a formidable problem in developingountries, accounting for more than one fifth of all deaths in chil-ren under the age of five [1]. The enterotoxigenic Escherichia coliETEC), along with rotavirus and Shigella accounts for the major-ty of these deaths [2]. ETEC remains the major cause of traveller’siarrhea [3] and causes sporadic outbreaks in industrialized coun-ries including the United States [4]. In addition, ETEC along withther enteric pathogens, likely contributes significantly to child-ood morbidity including delayed development in areas wherehese diseases are endemic [5].

Enterotoxigenic E. coli have in common the ability to produceeat-labile and/or heat stable enterotoxins that elicit watery diar-hea when delivered to the small intestine. In the classic paradigmor ETEC pathogenesis, this process is mediated by a variety of

ntigenically distinct fimbrial appendages known as colonizationactors (CFs) [6].

Epidemiologic data demonstrating a decrease in symptomaticTEC infections with age suggest that an ETEC vaccine could be

∗ Corresponding author at: Veterans Affairs Medical Center, Memphis, 1030 Jef-erson Avenue, Memphis, TN 38119, United States. Tel.: +1 901 523 8990x6447;ax: +1 901 577 7273.

E-mail address: jflecke1@tennessee.edu (J.M. Fleckenstein).

264-410X/$ – see front matter. Published by Elsevier Ltd.oi:10.1016/j.vaccine.2009.05.076

developed to mimic effective immunity that develops followinginfection with these organisms [7,8]. CFs, remain a central criticalfocus for vaccine development [9], and recent investigations intomore highly conserved CF tip adhesin molecules show significantpromise as protective antigens [10]. The substantial antigenic het-erogeneity among E. coli that produce these toxins [11–13] predictthat the ideal ETEC vaccine would potentially need to incorporatemultiple antigenic targets to achieve protection against a wide vari-ety of strains. In addition, epidemiology studies have suggested thatantigens other than CFs might induce protective immunity againstETEC [14].

In a recent search for novel virulence proteins that also mightbe investigated as vaccine targets, we identified EtpA [15], a large170 kD glycoprotein adhesin that is secreted by H10407. EtpA isencoded on the etpBAC locus comprised of genes for a two-partnersecretion (TPS) system that includes: EtpB an outer membranetransport protein, EtpA the secreted exoprotein, and EtpC a possi-ble glycosyltransferase involved in glycosylation of EtpA. Previousstudies have demonstrated that EtpA is required for optimal colo-nization of the murine intestine, and similarly that mice vaccinatedwith a truncated 110 kD, non-glycosylated version of EtpA are

protected against subsequent intestinal colonization with ETECH10407 [16].

Interestingly, we have recently demonstrated that EtpAenhances the ability of ETEC to colonize the small intestine in aunique fashion: by bridging the ends of bacterial flagella and host

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eceptors of the intestinal mucosa [17]. The peritrichous flagellaf E. coli are long filaments of approximately 20,000 monomersf the protein flagellin (FliC). Each FliC molecule is composed ofighly conserved amino and carboxy terminal regions, and a centralariable central portion. The conserved regions of these moleculesnteract and are normally buried in the flagellar shaft upon poly-

erization, while the central portion of the molecule is oriented tohe exterior of the filament and is the basis for “H” serotyping of E.oli. New flagellin molecules are secreted into the nascent flagellarhaft and are incorporated at the distal tip of the growing filaments they are captured by a rotating pentameric FliD cap complex.hrough loss or displacement of this FliD, cap conserved regionsf flagellin are exposed at the flagellar tips and available to bindtpA.

Our earlier studies have demonstrated that these interactionsf EtpA with conserved regions of flagellin are critical for ETEC todhere to intestinal epithelial cells and for colonization of the smallntestine. Similarly, we demonstrated that flagellin expression isequired for optimal colonization of mice and that vaccination ofice with flagellin (serotype H48) afforded significant protection

gainst colonization by H10407 (expressing heterologous flagellarerotype H11) [17].

Collectively, these earlier studies suggested that either EtpAr its cognate ligand at the ends of flagella, conserved regions ofhe flagellin molecule, could be useful in ETEC vaccines. However,

any questions remain regarding the utility of EtpA or flagellins protective antigens. First, glycosylation of bacterial proteinsan potentially influence their antigenicity or host recognition18–21]. Because we have demonstrated that production of EtpAs a recombinant glycoprotein can facilitate its purification andnhance stability of the protein [15,22], we sought to determine

hether the full-length glycoprotein would also serve as suitable

mmunogen. Also, we wished to examine whether immunizationith flagellin would afford protection against colonization by ETEC

trains in addition to the H10407 prototype. Finally, we questionedhether vaccination with both antigens simultaneously would be

able 1acterial strains and plasmids.

trains

train Description

nterotoxigenic E. coli strainsB7A ETEC strainb. origin: ietnam; serotype O148:H28H10407 ETEC straina. origin: angladesh; serotype O78:HE24377A ETEC strainb. origin: Egypt; serotype O139:H28;

EtpA+Jf876 �lacZYA::KmR derivative of H10407

ecombinant E. coli strainsTop10 F- mcrA �(mrr-hsdRMS-mcrBC) �80lacZ�M15

�(araleu) 7697 galU galK rpsL (StrR) endA1 nupjf1696 Top10(pJL017)(pJL030); AmpR, CmR

lasmidslasmid DescriptionpTrcHisB Expression plasmid for polyhistidine fusions, AmpJL017 etpBA cloned into pBAD/Myc-His A, with EtpA in

and 6His coding regions.pJL030 etpC gene cloned into pACYC184, CmRpKR001b Full-length FliC (H48) (AA residues M1-G498) fr

pTrcHis BpKR008 Full length H11 flagellin amplified from ETEC H1pKR011 Residues N174-L385 from FliC (H11) amplified frompMMO001 Full-length fliC (H28) gene from E24377A cloned

pTrcHisBpMMO002 Region encoding serotype-specific H28 sequence

and HindIII sites of pTrcHisB

enomic DNA sequence:a Wellcome Trust Sanger Institute, Cambridge, UK.b jcvi: J. Craig Venter Institute.

(2009) 4601–4608

more or less effective in preventing colonization than administra-tion of either single antigen.

The advent of high-throughput microbial sequencing now per-mits rapid assessment of potential molecular targets in a varietyof strains within a given pathotype [23]. Currently, DNA genomicsequence data are emerging from three ETEC strains H10407,E24377A, and B7A (Table 1) [24]. These data revealed that the ETECE24377A genome encodes a nearly identical etpBAC locus [24], andrecent studies have indicated that EtpA is produced by E24377A[25].

Here we demonstrate that immunization with full length EtpAglycoprotein affords significant protection against subsequent col-onization by ETEC H10407 or E24377A. Furthermore, vaccinationwith recombinant flagellin provided robust protection against colo-nization by E24377A expressing a heterologous serotype of flagellinsuggesting that this strategy could be applied to strains other thanthe H10407 ETEC prototype.

2. Materials and methods

2.1. Bacterial strains

Enterotoxigenic E. coli strain E24377A was kindly supplied byDr. Stephen Savarino, and was derived from cGMP stock held atthe Walter Reed Army Institute of Research (WRAIR) Pilot Biopro-duction Facility. This stock, used to generate DNA for sequencingof the E24377A genome [24] is fully virulent in human clinicalchallenges studies [26]. ETEC H10407 was also obtained from GMPstocks maintained at WRAIR and is fully virulent in human clinicalchallenge studies.

2.2. Cloning and expression of recombinant antigens

Recombinant, polyhistidine-tagged EtpA glycoprotein was pre-pared as previously described [25]. Briefly, strain j1696 was usedto express recombinant EtpA for these studies. This strain con-

Reference(s)

; CS6; LT+/ST+; EtpA− [38,39]11;CFA/1; LT+/ST+; EtpA+ [40,41]CFA/II (CS1/CS3); LT+/ST+; [36]

[30]

�lacX74 recA1 araD139G

Invitrogen

[17]

InvitrogenpR Invitrogen-frame with C-terminal myc [17]

[17]om MG1655 as 6His fusion in [17]

0407 [17]H10407 [17]

into BglII and HindIII sites of This study

(E24377A) cloned into BglII This study

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colonies from plates were examined for presence of the eltABoperon encoding the ETEC heat labile toxin by PCR using theprimers jf030204.1 (5′-CCCCAGTCTATTACAGAA-3′) and jf030204.2(5′-CTAGTTTTCCATACTGAT-3′) [28].

Fig. 1. EtpA glycoprotein is immunogenic following nasal immunization of mice.Open symbols represent pre-immunization baseline values (n = 10) obtained bykinetic ELISA for sera (each at 1:1024 dilution, open circles) stool (undiluted

K. Roy et al. / Vacci

ists of E. coli Top10 transformed with both the pJL017 plasmidwhich contains etpB gene for the TpsB transporter protein and thetpA gene cloned in frame at its 3’ end with myc and polyhistidinencoding regions on pBAD/mycHisA) as well as another plasmid,JL030 (which contains the etpC gene required for optimal secretionnd glycosylation of EtpA). jf1696 (maintained at −80 ◦C in frozenlycerol stocks) was grown overnight in Luria broth (2 ml) contain-ng Ampicllin (100 �g/ml) and chloramphenicol (15 �g/ml), dilutednto fresh media and grown in side-arm flasks to OD600 of ≈0.5.ollowing induction of recombinant myc-His(6) tagged EtpA with.0002% arabinose, bacterial culture supernatants were concen-rated using 30,000 MWCO filters. Purified, recombinant full-lengthtpA glycoprotein was then recovered directly from concentratedulture supernatants by metal affinity chromatography using cobaltTalon) resin [22]. Recombinant myc-polyhistidine-tagged EtpAlycoprotein was eluted from the resin with imidazole (250 mM)nd then dialyzed against phosphate buffered saline (PBS), pH 7.4

Glycosylation of the resulting EtpA-myc-His(6) recombinantrotein was confirmed using digoxigenin glycan detection assays previously described for the native protein [15]. Briefly, recom-inant EtpA was first separated by SDS-PAGE, and transferred toitrocellulose. Hydroxyl groups of sugars on immobilized glycocon-

ugate proteins were then oxidized with 10 mM sodium metaperi-date, and incubated with DIG-3-0-succinyl-�-aminocaproic acidydrazide, and probed with anti-digoxigenin antibody labeled withlkaline phosphatase. Alkaline phosphatase activity was detectedsing 4-Nitro blue tetrazolium chloride and 5-Bromo-4-chloro-3-

ndolyl-phosphate (NBT/BCIP) (Dig Glyan Detection Kit, Roche).To clone the full-length H28 flagellin sequence, primers

f030106.1 (5′-AATAATAGATCTATGGCACAAGTCATTAATACC-3′) andf030106.2 (5′-AATAATAAGCTTTACCCTGCAGCAGAGACAGAAC-3′)ere used to amplify a 1750 fragment from E24377A genomicNA. This was digested with BglII and HindIII (underlined inrimer sequences) and cloned in-frame with the region encod-

ng an amino terminal polyhistidine tag on pTrcHisB yieldingMMO001. Multiple E. coli flagellin peptide sequences corre-ponding to different serotypes and the predicted E24377Aequence encoding serotype H28 flagellin were aligned to iden-ify the serotype-specific region of the E24377A H28 flagellin.his sequence corresponding to amino acids 178-491 of theagellin H28 peptide was amplified using primers jf082508.25′-AATAATAGATCTGGCGGGGCTGTGGCTAAT-3′), and jf082508.15′-AATAATAAGCTTTTTATAGATCGGCGGTGGTTGC-3′) which wasloned into BglII and HindIII sites on pTrcHisB to create pMMO002.lasmids were then introduced into E. coli Top10, (which does notroduce native flagellin, and was used as the host strain for proteinxpression) [27]. Recombinant flagellins were then produceds polyhistidine-tagged proteins as previously described [17].riefly, Top10 carrying respective recombinant flagellin expressionlasmids was grown to mid-log phase and induced with 0.1 mM

sopropyl ß-d-thiogalactoside (IPTG) for 4 h. Pellets were lysed inacterial protein extraction reagent (B-PER, Thermo Scientific), andecombinant proteins were recovered from clarified supernatantsy metal affinity chromatography using Talon beads. Purifiedroteins were eluted in imidazole and dialyzed against PBS prioro use in immunizations.

.3. Mouse immunizations

Groups of 10-20 ICR [28] mice were immunized intranasallyith either IVX908 (Protollin®) [29] (7.5 �g), IVX908

7.5 �g) + recombinant full-length EtpA glycoprotein (rEtpAgp)30 �g), or IVX908 (7.5 �g) + recombinant, polyhistidine-taggedH48) flagellin (rFliC) on days 0, 14, 28. Sera were collected on day

prior to immunization (pre-immune) and 7–14 days followinghe last vaccination (post-immune).

2009) 4601–4608 4603

Additional experiments were then conducted in which groupsof 10 mice each were immunized with either IVX908 alone as con-trols, IVX908 + rEtpAgp, IVX908 + rFliC, or a combination of bothrEtpAgp + rFliC with IVX908 adjuvant to test the effect of combiningboth EtpA and flagellin in a single vaccination. Each of these mice(40 total) received only two vaccinations on days 0, 14.

2.4. Mouse colonization studies

Prior to challenge with bacteria, mice received streptomycin(5 g/liter) in the drinking water for 48 h to eradicate normal flora.Food was withheld 12 h prior to challenge and replaced with ster-ile water without antibiotics. Cimetidine was then administeredby intraperitoneal injection (50 mg/kg) 2 h prior to inoculationwith ETEC. Mice were subsequently challenged by gavage witheither ≈1 × 104 cfu of E24377A (on day 7 following the last immu-nization) or 1 × 104 cfu of H10407 (on day 14 following the lastimmunization) as previously described [16]. The second group of40 mice that received two vaccinations of either antigen or thecombination of rEtpAgp and rFliC were challenged on day 14 fol-lowing their second immunization with approximately 1 × 105 cfuof jf876, a derivative of H10407 bearing a KmR marker in lacZYA[30].

The day prior to challenge, fecal pellets were collected afterplacing mice in separate enclosures 5–6 fresh fecal pellets werecollected, and immediately resuspended in 1.5 ml extraction buffercontaining Tris (10 mM), NaCl (100 mM), Tween-20 (0.05%), Sodiumazide (5 mM) pH 7.4.

Approximately 24 h following challenge, mice were sacrificed,and blood was obtained immediately by cardiac puncture. Seg-ments of ileum were harvested and solubilized in saponin torelease attached bacteria as previously described [28], then platedonto Luria agar plates (in the case of H10407 and E24377Awild type strains), or Luria agar containing kanamycin (50 �g/ml)in the case of jf876. In each experiment, selected bacterial

fecal antibody extracts, open triangles). Closed symbols (n = 20) in each grouprepresent post-immune values. Pre and post-immune values were compared byMann–Whitney U-test. Dashed horizontal lines represent geometric means. Insetshows detection of EtpA glycoprotein (>) from concentrated supernatants of wildtype (WT) H10407 (left) and the recombinant, purified myc-polyhistidine-taggedprotein (rEtpAgp) using dig-glycan reagents.

4604 K. Roy et al. / Vaccine 27 (2009) 4601–4608

Fig. 2. immunization with full-length recombinant flagellin stimulates production of antibodies which recognize highly conserved epitopes of multiple flagellins. (a) Mice(n = 10) immunized with recombinant flagellin (serotype H11) develop serum antibodies against conserved, but not serotype-specific regions of H28 and H48 flagellins.Shown are kinetic ELISA data obtained at a dilution of 1:200 of all sera (pre-immune values were subtracted except for H48[1-173] data which compares pre and post-immunevalues). p-values obtained using two-tailed Mann–Whitney U-test (***p < 0.0001). Dashed horizontal lines represent geometric mean values. (b) Fecal antibodies from samemice. (c) Alignment of the amino acid sequences of E. coli flagellin molecules from different serotypes used in these studies demonstrating highly conserved amino andcarboxy regions of each molecule. (Amino acids are colored based on RasMol amino color scheme tables http://www.rasmol.org/software/RasMol 2.7.4.2 Manual.html –a (lineH

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minocolours) Represented in the alignment are flagellins belonging serotypes H4810407, respectively.

.5. Assessment of immune responses following vaccination

After immunization with recombinant proteins, sera from miceere tested for reactivity to EtpA, and the full-length, serotype

pecific and conserved regions of flagellins (FliCH48, FliCH11,nd FliCH28). Briefly, the respective recombinant polyhistidine-agged proteins were diluted to a final concentration of 4 �g/mln 0.1 M NaHCO3 buffer, pH 8.6 and used to coat wells ofLISA plates overnight at 4 ◦C. Plates were then washed withBS (Tris buffered saline) containing 0.05% Tween-20, andlocked for 1 h at 37 ◦C with 1% BSA in TBS-T (Blocker, Thermocientific). Dilutions of immune and preimmune mouse seraere prepared in 1% BSA in TBS-T. After incubation for 1 h

t 37 ◦C, plates were washed with TBS-T, and secondary goatnti-mouse (IgA,IgM,IgG) antibody was added at a final concen-ration of 1:10,000. After incubation for 1 h at 37 ◦C, plates were

ashed and developed with TMB Peroxidase Substrate [3,3’,5,5’-

etramethylbenzidine]. Kinetic absorbance measurements [31]ere determined at a wavelength of 620 nm, and acquired at 30 s

ntervals using a Molecular Devices Spectramax 340PC microplateeader. All data were recorded and analyzed using SoftMax Pro

1), H28 (line 2) and H11 (line 3) from MG1655 (K12), and ETEC strains E24377a, and

software v5.0.1 and reported as the Vmax expressed as milli-units/min.

2.6. In situ immunogold labeling

Sera from mice immunized with H11 flagelllin (FliC) was pooledand affinity purified using 10 �g of recombinant full-length FliC(H11, amino acids 1–488) immobilized on nitrocellulose as the tar-get antigen. After elution in 100 mM glycine, pH 2.5 and subsequentneutralization with 1 M Tris, pH 8.0 antibodies [32] were concen-trated using centrifugal spin filters accompanied by buffer exchangewith PBS, pH 7.4. Rabbit antibodies raised against rEtpA wereprepared by cross-absorption against both recombinant FliD andrecombinant FliC (H48) immobilized on nitrocellulose. Unboundantibody was then affinity-purified against 10 �g of rEtpA as notedabove [15].

2.7. Amino acid alignments

Alignment of full-length flagellin peptide sequences from E. coliserotypes H56, H10, H9, H4, H2, H7, H11 (from H10407), H48 with

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Fig. 3. Conserved regions of flagellin and EtpA localize to the tips of E24377A flagella.(a) in situ immunogold labeling experiment demonstrating that mice immunizedwith recombinant full-length H11 flagellin develop serum antibodies against con-

bodies that significantly inhibited adherence of both H10407, andE24377A relative to IVX908-vaccinated controls (Fig. 4). Likewise,sera from mice vaccinated with H11 flagellin significantly inhibitedadherence by either of these strains.

Fig. 4. Antisera from mice immunized with EtpA and flagellin inhibit adherence ofEtpA-producing ETEC strains in vitro. Shown at left (circles) are Caco-2 cell adher-

K. Roy et al. / Vacci

28 from E24377A was performed using protein alignment algo-ithm in CLC Main Workbench (v 4.0.1) (CLC Bio).

. Results

.1. Immune response to EtpA glycoprotein and recombinantagellin

Prior studies have indicated that mice immunized with a 110 kDaon-glycosylated recombinant fragment of EtpA are protectedgainst subsequent intestinal colonization with ETEC strain H1040716]. However, as noted above, glycosylation of bacterial proteinsan affect their immunogenicity [18]. Furthermore, the stability oftpA like other bacterial glycoproteins appears to be influencedavorably by glycosylation [15,33,34]. Since our original discovery ofhe etpBAC operon in ETEC we have recently optimized the produc-ion of full-length EtpA glycoprotein from recombinant E. coli so thatt can be purified in either tagged or native forms [22,25]. As each ofhese issues could critically affect the utility of EtpA as an effectiverotective immunogen, we elected to examine the immunogenicityf the full-length EtpA glycoprotein in mice as previously described16].

Following intranasal immunization with rEtpA glycoprotein andhe adjuvant IVX908 (Protollin®), mice mounted significant anti-ody responses in both serum (p < 0.0001) and stool (p = 0.0002)elative to IVX908 (adjuvant only) controls (Fig. 1). These studiesndicate that when administered under the conditions employedere, EtpA retains its immunogenicity in its fully glycosylated

orm.We have also recently demonstrated that mice immunized with

ecombinant full length flagellin (serotype H48) mount signifi-ant antibody responses to conserved regions of flagellin and thatice vaccinated in this fashion were significantly protected from

ubsequent colonization with ETEC expressing another serotypef flagellin (H11) [25]. We surmised therefore that serotypeeterminant regions of flagellin were irrelevant in providing pro-ection against ETEC colonization and that immunization withny full-length flagellin should theoretically afford protectiongainst bacteria expressing heterologous flagellin molecules (other

serotypes). As an additional test of this hypothesis we vac-inated mice with full-length recombinant (H11) flagellin. Miceaccinated in this fashion mounted robust serum (Fig. 2a) and fecalFig. 2b) antibody responses to both the full-length and serotypepecific regions of the H11 molecule. These antibodies recognizedhe full-length H28 and H48 antigens, but not the correspond-ng serotype-specific regions of these molecules. Conversely, thesentibodies did recognize the highly conserved amino-terminalamino acids 1–173) of flagellin, a region that is virtually identical in. coli flagellin molecules as illustrated by alignment of amino acidequences of H48, H28, and H11 flagellins from MG1655, E24377A,nd H10407, respectively (Fig. 2c). Collectively, these data lenddditional support to the idea that vaccination with full-length flag-llin will engender immune responses directed at highly conservedegions of flagellin molecules.

.2. Antisera from vaccinated mice recognize EtpA and flagellin athe tips of ETEC flagella and inhibit bacterial adherence

We have recently demonstrated that EtpA must interact withonserved regions of flagellin molecules available exclusively at theips of ETEC H10407 flagella in order to promote bacterial adherence

n vitro and for efficient intestinal colonization in vivo [17]. In thesetudies, we also found that mice immunized with H11 flagellineveloped antibodies that identified conserved regions of flagellint the tips of the flagella from the EtpA-producing ETEC strain24377A (O139:H28) as shown by in situ immunogold labeling

served regions of H28 flagellin available at the tips of enterotoxigenic Escherichia colistrain E24377A (serotype O139:H28) (labeled here with anti-mouse gold (10 nm)conjugate). (b,c) Co-localization of conserved regions of flagellin (10 nm particles,closed arrows) and EtpA (5 nm particles, open arrows).

(Fig. 3a). Furthermore, these studies demonstrated co-localizationof EtpA with conserved regions of flagellin at the tips of H28 flag-ella from E24377A (Fig. 3b–c), providing additional evidence thatthe interaction of these proteins is not limited to a single prototypestrain of ETEC.

These same mouse antisera were tested for their ability to inhibitETEC adherence to Caco-2 intestinal cell lines in vitro. In theseassays, mice vaccinated with EtpA glycoprotein developed anti-

ence data for ETEC strain H10407 (078:H11) in the presence of antisera from micevaccinated with adjuvant only (IVX908, control); with EtpA; or with recombinantflagellin (H11). Shown at right (squares) are adherence data for ETEC strain E24377Ausing the same sera. (*p 0.028 by Mann–Whitney U-test). Horizontal bars for eachgroup represent geometric mean numbers of cell-associated bacteria obtained fromquadruplicate wells.

4606 K. Roy et al. / Vaccine 27 (2009) 4601–4608

Fig. 5. Immunization with recombinant glycosylated EtpA or flagellin affords het-erologous protection against colonization by ETEC. (a) Immunization of mice withrecombinant, full length EtpA glycoprotein prevents colonization by ETEC H10407.(b) Immunization of mice with either recombinant, full-length EtpA glycopro-tein or flagellin (FliCH48) prevents colonization against challenge with E24377A(O139:H28). Dashed horizontal lines represent geometric mean values. Statisticalcvt(

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Table 2protection against colonization following vaccination with rEtpAgp and/or flagellin.

Immunization group Colonizing bacteria*(cfu/ml intestinal lysate)

Fold reduction p-value†

IVX908 control 4530 ± 1563 – –IVX908 + rEtpAgp 1486 ± 259 3.1 0.001IVX908 + rFliCH48 1457 ± 184 3.1 0.0002IVX908 + rFliCH48 /rEtpAgp 821 ± 133 5.5 <0.0001

us to investigate whether EtpA could be useful in a vaccine for ETEC.While we have shown that immunization with either a recom-

binant fragment of EtpA [16] or full-length flagellin (H48) couldprotect mice from colonization by the prototypical ETEC strainH10407 [25], it was not clear that these results could be extrap-

omparisons were made using two-tailed Mann–Whitney analysis. In 7/10 miceaccinated with flagellin, no ETEC bacteria could be recovered from the small intes-ine. These are represented at the theoretical lower limit of detection on the graph1 cfu/ml).

.3. EtpA glycoprotein and flagellin protect mice against ETEColonization

Similar to earlier studies with a 110 kDa fragment of EtpA [16],e found that immunization of mice with the full-length recom-inant EtpA glycoprotein (rEtpAgp)(≈170 kDa), afforded significantrotection against intestinal colonization by two different EtpA-xpressing ETEC strains, H10407 (p < 0.0001) (Fig. 5a), and E24377Ap < 0.0008) (Fig. 5b) when compared to control mice immunizedith only the adjuvant, IVX908 (Protollin). Likewise, we found

hat vaccination of mice with recombinant full-length H11 anti-en provided robust protection against subsequent challenge with24377A, which expresses a different serotype of flagellin (H28)p < 0.0001) (Fig. 5b).

We also questioned whether rEtpAgp and flagellin could besed together to provide additional protection. Therefore, we con-ucted experiments in which groups of 10 mice were vaccinatedith rEtpAgp, recombinant H48 flagellin (rFliCH48), or both antigens

*Mice in these studies were challenged with at 1 × 105 cfu of jf876, a derivative ofETEC H10407 with a kanamycin resistance marker in lacZYA (lacZYA::KmR).

† By Mann–Whitney non parametric testing.

simultaneously. Mice vaccinated with either rEtpAgp or rFliCH48mounted robust serum antibody responses to their respective anti-gens, while those vaccinated simultaneously with both antigenshad slightly lower antibody responses to either protein. Intestinalchallenge, with a 10-fold higher inoculum of bacteria (≈1 × 105 cfu)than used in prior studies, demonstrated approximately a 3-foldreduction in colonization of mice vaccinated with either antigenrelative to controls, while vaccination with both antigens was sig-nificantly (p = 0.009) more effective (nearly 6 fold reduction incolonization) than either antigen alone (Table 2). These data pro-vide additional evidence that the EtpA glycoprotein; its bacterialligand, highly conserved regions of flagellin molecules; or perhapsa combination of these molecules, could be incorporated in thedevelopment of ETEC vaccines Fig. 6.

4. Discussion

EtpA, initially discovered by selection for potential secreted vir-ulence proteins using transposon mutagenesis, is a member of alarge family of exoproteins secreted by two-partner secretion. Thisfamily includes filamentous hemagglutinin (FHA), a component ofpresent acellular pertussis vaccines [35]. Like FHA and a number ofother proteins in this family, EtpA appears to promote epithelial celladherence and colonization of mucosal surfaces [16,25]. The struc-tural and functional relationship of EtpA to molecules like FHA led

Fig. 6. serum immune responses to EtpA (circles) and flagellin (triangles) in micevaccinated with either antigen alone or in combination. Values represent total (IgG,IgM, IgA) responses obtained by kinetic ELISA at a 1:1024 dilution of all sera. p-valuesobtained by 2-tailed Mann–Whitney testing.

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lated to other EtpA-producing strains. Furthermore, productionf intact recombinant EtpA is greatly facilitated by its expressions a glycoprotein, leading us to examine both the immunogenic-ty and protective efficacy of this molecule. The recent completionf the E. coli E24377A genome sequence [24], an ETEC isolate forhich there is clear documented virulence in human clinical trials

36], and which secretes EtpA [25], also led us to examine whethere could extend these observations to other ETEC strains whichroduce the same glycoprotein.

Here we demonstrate that the EtpA full-length glycoproteinetains immunogenicity in this model, and that immunizationeduces intestinal colonization by both H10407 and E24377A, sim-lar to our prior experience in using the non-glycosylated 110 kDaEtpA fragment as an immunogen. Additionally, in experimentseported here, vaccination with flagellin H11 offered robust pro-ection against E24377A (0139:H28). Finally, studies included hereemonstrate that vaccination with EtpA and flagellin together couldnhance the efficacy of a vaccine targeting this unique mechanismf colonization.

Certainly, it is not clear that reductions in intestinal colonizationn our mouse model can be used to predict the utility of EtpA or simi-ar antigens in ameliorating or preventing human disease. However,olonization is thought to be an important step in facilitating deliv-ry of the known enterotoxins LT and ST to their respective targetsnd the ultimate development of diarrhea.

Preliminary studies demonstrated that EtpA is secreted by aariety of ETEC strains from geographically disparate sources [15].hese strains belong to multiple serotypes and express a variety ofolonization factors. Clearly, however additional studies are neededo define more carefully the molecular epidemiology of EtpA, asell as the extent to which glycosylation is conserved among EtpA-roducing strains. Given the known plasticity of E. coli genomes

ncluding ETEC [12,37], it is exceedingly unlikely that any single tar-et antigen would offer sufficient protection to prevent infection byhe panoply of ETEC strains that cause disease in humans.

Further studies will also be needed to assess the immunogenic-ty of EtpA. While this protein is recognized following experimentalnfection of mice with ETEC, and is immunogenic in mice afteraccination [16], additional studies are needed to assess the recog-ition of these proteins during the course of natural infection inumans.

The animal model of colonization used here to investigatehe utility of these antigens cannot completely mimic disease inumans. One important limitation is that while mice become col-nized with human ETEC strains [28], these animals do not getiarrhea. Nevertheless, the important role that colonization plays

n pathogenesis would suggest that these studies offer importantarly preclinical validation of the potential implementation of thesentigens as vaccine targets.

cknowledgements

The authors wish to thank Katherine Troughton of the Neu-osciences Imaging Center at UTHSC for her assistance withransmission electron microscopy. We thank Dr. James Dale andis laboratory for providing IVX908 (originally obtained from IDiomedical) for use as an adjuvant in these studies, and Dr. Harryourtney for his thorough review of the manuscript.

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