Published Ahead of Print 18 February 2014. 2014, 82(5):1823. DOI: 10.1128/IAI.01394-13. Infect. Immun. 

D. Clements and Weiping ZhangXiaosai Ruan, Donald C. Robertson, James P. Nataro, John AntibodiesPeptide in Inducing Neutralizing Anti-STaFused to Double Mutant Heat-Labile Toxin

coliToxoids of Enterotoxigenic Escherichia Characterization of Heat-Stable (STa)

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Characterization of Heat-Stable (STa) Toxoids of EnterotoxigenicEscherichia coli Fused to Double Mutant Heat-Labile Toxin Peptide inInducing Neutralizing Anti-STa Antibodies

Xiaosai Ruan,a* Donald C. Robertson,b James P. Nataro,c John D. Clements,d Weiping Zhang,a* The STa Toxoid Vaccine ConsortiumGroup

The Center for Infectious Disease Research and Vaccinology, South Dakota State University, Brookings, South Dakota, USAa; Diagnostic Medicine/Pathobiology, KansasState University College of Veterinary Medicine, Manhattan, Kansas, USAb; University of Virginia School of Medicine, Charlottesville, Virginia, USAc; Tulane University Schoolof Medicine, New Orleans, Louisiana, USAd

A long-standing challenge in developing vaccines against enterotoxigenic Escherichia coli (ETEC), the most common bacteriacausing diarrhea in children of developing countries and travelers to these countries, is to protect against heat-stable toxin typeIb (STa or hSTa). STa and heat-labile toxin (LT) are virulence determinants in ETEC diarrhea. LT antigens are often used in vac-cine development, but STa has not been included because of its poor immunogenicity and potent toxicity. Toxic STa is not safefor vaccines, but only STa possessing toxicity is believed to be able to induce neutralizing antibodies. However, recent studiesdemonstrated that nontoxic STa derivatives (toxoids), after being fused to an LT protein, induced neutralizing antibodies andsuggested that different STa toxoids fused to an LT protein might exhibit different STa antigenic propensity. In this study, weselected 14 STa toxoids from a mini-STa toxoid library based on toxicity reduction and reactivity to anti-native STa antibodies,and genetically fused each toxoid to a monomeric double mutant LT (dmLT) peptide for 14 STa-toxoid-dmLT toxoid fusions.These toxoid fusions were used to immunize mice and were characterized for induction of anti-STa antibody response. The re-sults showed that different STa toxoids (in fusions) varied greatly in anti-STa antigenicity. Among them, STaN12S, STaN12T, andSTaA14H were the top toxoids in inducing anti-STa antibodies. In vitro neutralization assays indicated that antibodies induced bythe 3�STaN12S-dmLT fusion antigen exhibited the greatest neutralizing activity against STa toxin. These results suggested3�STaN12S-dmLT is a preferred fusion antigen to induce an anti-STa antibody response and provided long-awaited informationfor effective ETEC vaccine development.

Diarrhea remains a leading cause of death in children youngerthan 5 years who live in developing countries (1). Enterotoxi-

genic Escherichia coli (ETEC) strains (i.e., E. coli strains producingenterotoxins) are the most common bacteria causing diarrhea,particularly in children younger than 1 year from developingcountries (2). ETEC diarrhea is responsible for an estimated an-nual death rate of 300,000 to 500,000, with the vast majority beingchildren younger than 5 years (3, 4). ETEC strains are also theleading cause of diarrhea in children and adults who travel fromdeveloped countries to countries or regions where ETEC is en-demic and in military personnel deployed in these areas and is alsoa threat to immunocompromised patients (3, 5–7). The virulencedeterminants of ETEC in diarrhea are bacterial adhesins and en-terotoxins. Adhesins mediate initial ETEC bacteria attachment tohost epithelial cells and subsequent colonization at host small in-testines. Attachment and colonization bring ETEC bacteria inclose proximity, which allows ETEC to deliver enterotoxins tohost epithelial cells. Enterotoxins, mainly heat-labile toxin (LT)and heat-stable toxin type Ib (human-type STa or hSTa, whichdiffers from type Ia STa or pSTa associated with ETEC diarrhea inanimals and also from heat-stable toxin type II or STb), disruptfluid homeostasis in host small intestinal epithelial cells to causeelectrolyte-rich fluid hypersecretion through activation of intra-cellular adenylate cyclase (by LT) or guanylate cyclase (by STa),which directly leads to diarrhea (8). Fluid hypersecretion disarraysthe mucin layer over host small intestinal epithelial cells and altersmicrovilli tight junction, which in return enhances ETEC bacterialcolonization at host small intestines (9–11). An ideal ETEC vac-

cine should induce antiadhesin immunity to block ETEC attach-ment and to prevent bacterial colonization at host small intestinesand also antitoxin immunity to neutralize both LT and STa toxins(12–14). However, there are currently no vaccines available toeffectively protect against ETEC diarrhea.

Key technical challenges would have to be overcome to developan effective ETEC vaccine. These include the immunological het-erogeneity among ETEC strains or virulence determinants, thepotent toxicity of LT and STa toxins, and the poor immunogenic-ity of the STa molecule. Progress has been made in developingantiadhesin vaccines by targeting multiple CFA adhesins whichare expressed by the most prevalent and the most virulent ETECstrains (15, 16) and also in inducing anti-LT immunity protectingagainst LT by using the nontoxic LTB subunit, a homologous chol-era toxin (CT) B subunit, or LT toxoids (17, 18). However, the

Received 30 October 2013 Returned for modification 30 November 2013Accepted 10 February 2014

Published ahead of print 18 February 2014

Editor: S. M. Payne

Address correspondence to Weiping Zhang, wpzhang@vet.k-state.edu.

* Present address: Xiaosai Ruan and Weiping Zhang, Kansas State UniversityCollege of Veterinary Medicine, Diagnostic Medicine/Pathobiology Department,Manhattan, Kansas, USA.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01394-13

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development of anti-STa immunity or vaccines against STa hasbeen much hampered (12, 14, 19). Indeed, due to its potent tox-icity and poor immunogenicity, this small STa (19 amino acidlong for human-type STa; 18 amino acid long for porcine-typeSTa) has been considered unsafe and unsuitable as a vaccine com-ponent (20). Nontoxic STa peptides would be safe as vaccine an-tigens, but they were found unable to induce neutralizing anti-STaantibodies (20). Thus, it has been suggested that STa toxicity andantigenicity are associated and that only toxic STa antigens areable to induce neutralizing antibodies (20).

Data from recent studies, however, indicated that nontoxicSTa antigens can induce neutralizing antibodies against STa toxin.Full-length STa, of human-type or porcine-type, were shown to beless toxic or nontoxic after a single amino acid was substituted,and they became immunogenic and elicited neutralizing anti-STaantibodies after being genetically fused to a nontoxic monomericLT (1A:1B; not 1A:5B holotoxin structured protein) peptide (21,22). It was also suggested that STa mutated at different amino acidresidues or at the same residue but with different replacementamino acids differed not only in toxicity reduction and antigenicstructure but also likely in stimulation the of anti-STa antibodyresponse when fused to an LT toxoid peptide (23). More recently,a study indicated that additional copies of an STa toxoid carried byan LT-STa toxoid fusion additively enhanced induction of anti-STa antibody response (24).

To further characterize STa toxoids in the stimulation of neu-tralizing anti-STa antibody response and to examine the potentialapplication of STa toxoids in antitoxin vaccine development, wefirst constructed a mini-STa toxoid library and selected a panel ofSTa toxoids based on their toxicity, anti-STa antigenic propensity,and biochemical properties. Each STa toxoid was then geneticallyfused to a monomeric double mutant LT (dmLT; LTR192G/L211A)peptide to create different STa-toxoid-dmLT toxoid fusions. TheseSTa-toxoid-dmLT toxoid fusions were used in mouse immuniza-tion and were examined for the induction of neutralizing anti-STaantibody responses. To further enhance the stimulation of anti-

STa antibody response, we genetically fused three copies of eachSTa toxoid to one monomeric dmLT peptide in this study.

MATERIALS AND METHODSBacterial strains and plasmids. The E. coli strains and plasmids used inthe present study are listed in Table 1. Toxoid fusion recombinant strains8751, 8752, and 8753, which were constructed previously (22), were usedas templates to construct STa-toxoid-dmLT toxoid fusions. Vector pUC19(Promega, Madison, WI) was used to clone STa toxoids, and vectorpET28� (Novagen, Madison, WI) was used to clone and express toxoidfusion genes. E. coli BL21 (GE Healthcare, Piscataway, NJ) was used as thehost strain. Recombinant E. coli strains were cultured in Luria broth sup-plemented with ampicillin (100 �g/ml) or kanamycin (30 �g/ml).

Cloning and mutation of STa gene estA. The human-type STa gene(estA) encoding hSTa toxin was isolated from E. coli H10407 genomic DNAand was cloned into vector pUC19 as described previously (23). Briefly,the estA gene was PCR amplified with the primers hSTapUCHindIII-F(5=-GCGCAAAGCTTCTGATTTTGAT-3=; the HindIII restriction site isunderlined) and hSTapUCBamHI-R (5=-AGCCACGGCGGATCCAAATATAAAGGG-3=; the BamHI restriction site is underlined). PCR productswere purified using gel electrophoresis, digested with HindIII and BamHIrestriction enzymes (New England BioLabs, Ipswich, MA), and ligatedinto vector pUC19. After the verification of STa protein expression andenterotoxicity, the cloned native STa gene had residue 3, 4, 8, 9, 12, 13, 14,16, or 17 mutated (but not residues 6, 7, 10, 11, 15, and 18, which areinvolved in disulfide bond formation). Replacement amino acid residueswere selected, respectively, based on biochemistry properties, such asmolecule weight, accessible surface area, and hydrophobicity or hydro-philicity.

Splice overlapping extensions (SOE) PCRs with specifically designedPCR primers were used to mutate the estA gene as described previously(23). Mutated genes were verified initially by DNA sequencing. After se-quencing verification, these STa mutated genes were examined for proteinexpression by measuring reactivity to anti-STa antibodies using STa com-petitive ELISA and for toxicity by examining stimulation of cyclic GMPlevels in T-84 cells using an EIA cGMP enzyme-linked immunosorbentassay (ELISA; Assay Design, Ann Arbor, MI) as described previously (21).

STa-toxoid-dmLT toxoid fusion construction. A group of STa toxoidsthat had their toxicity reduced or eliminated but retained reactivity to

TABLE 1 E. coli strains and plasmids used in this study

Strain Relevant properties Plasmid Source or reference

BL21 F� ompT hsdS (rB� mB

�), gal dcm GE Healthcare8751 Fusion 2b construct, BL21/pfusion-2b LT192-L-STa13/pET28� 228752 Fusion 3b construct, BL21/pfusion-3b STa13-gly-pro-LT192/pET28� 228753 Fusion 4b construct, BL21/pfusion-4b LT192A1-gly-pro-STa13-LTA2-B/pET28� 229064 STa recombinant strain, BL21 p8835, STa in pUC19 This study9355 3�STaS3F-dmLT strain, BL21 p3�STaS3F-dmLT in pET28� This study9330 3�STaE8Q-dmLT strain, BL21 p3�STaE8Q-dmLT in pET28� This study9357 3�STaL9A-dmLT strain, BL21 p3�STaL9A-dmLT in pET28� This study9359 3�STaN12I-dmLT strain, BL21 p3�STaN12I-dmLT in pET28� This study9331 3�STaN12S-dmLT strain, BL21 p3�STaN12S-dmLT in pET28� This study9361 3�STaN12T-dmLT strain, BL21 p3�STaN12T-dmLT in pET28� This study9332 3�STaP13A-dmLT strain, BL21 p3�STaP13A-dmLT in pET28� This study9333 3�STaP13F-dmLT strain, BL21 p3�STaP13F-dmLT in pET28� This study9363 3�STaA14H-dmLT strain, BL21 p3�STaA14H-dmLT in pET28� This study9334 3�STaA14Q-dmLT strain, BL21 p3�STaA14Q-dmLT in pET28� This study9335 3�STaA14T-dmLT strain, BL21 p3�STaA14T-dmLT in pET28� This study9336 3�STaA14V-dmLT strain, BL21 p3�STaA14V-dmLT in pET28� This study9337 3�STaT16K-dmLT strain, BL21 p3�STaT16K-dmLT in pET28� This study9374 3�STaT16 M-dmLT strain, BL21 p3�STaT16 M-dmLT in pET28� This study9353 3�STa-dmLT strain, BL21 p3�STa-dmLT in pET28� This study8955 Negative control, BL21/pET28� pET28� 23

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polyclonal antibodies derived against native STa were selected to be ge-netically fused to a monomeric dmLT peptide. In contrast to the 1A:5Bholotoxin-structured dmLT (LTR192G/L211A) (25), this monomeric dmLTmolecule is a single peptide that consisted of only one copy of the Asubunit and one copy of the B subunit. It was derived by modifying thenative LT eltAB genes with disruption of the cistron gene structure (be-tween the eltA gene and eltB gene) and the removal of nucleotides codingthe B subunit signal leading peptide.

Three copies of each STa toxoid were genetically fused to a monomericdmLT at its N terminus, at its C terminus, and between the A1 segmentand the A2 segment, respectively, using an SOE PCR described previously(24). However, instead of using a monomeric tmLT (LTS63K/R192G/L211A)peptide (24), we used a monomeric dmLT (LTR192G/L211A) peptide as thecarrier protein. Briefly, three nucleotide segments, which were generatedfrom three separate SOE PCRs, were fused together to form a single openreading frame coding an STa-toxoid-dmLT toxoid fusion (Fig. 1A). Thefirst nucleotide segment was produced by overlapping two regular PCRproducts: one was amplified with the primers T7-F (5=-TAATACGACTCACTATAGGG-=3) and STa-toxoid-R, and the other was amplified with theprimers STa-toxoid-F and LT73-R (5=-AGACTGTCCTGCTAAGTGAGC

ACT-=3). Since plasmid p8752 (STa13-gly-pro-LT192/pET28�) was usedas the DNA template, the first segment consisted of nucleotides coding thefirst copy of a STa toxoid (at the N terminus of the fusion) and the first 75amino acids of LT (N terminus). The second nucleotide segment wasgenerated by connecting of another set of PCR products: one was ampli-fied with the primers LT73-F (5=-AGTGCTCACTTAGCAGGACAGTCT-=3) and STa-toxoid-R, and the other was amplified with the primersSTa-toxoid-F and LT211-R (5=-TGATTGATATTTCCTGGCATATATTGT-=3). Since plasmid p8753 (LT192A1-gly-pro-STa13-LTA2-B/pET28�) wasused as the DNA template in these two PCRs, the second segment con-tained nucleotides encoding the second copy of an STa toxoid (betweenA1 and A2 of the LTA peptide) and the LTA peptide encoding amino acids68 to 216 (but with residues 192 and 211 mutated). The third nucleotidesegment included nucleotides of part of the LTA peptide which encodeamino acids 204 to 240, the entire LTB peptide, and the third copy of a STatoxoid (at the C terminus of the fusion, with a stop codon). This segmentwas produced by joining two PCR products: one was amplified with theprimers LT211-F (5=-CAGAATCTGAGCACAATATATGCCAG-=3) andSTa-toxoid-R, and the other was amplified with the primers STa-toxoid-Fand T7-R (5=-TGCTAGTTATTGGTCAGGGGT-=3), with plasmid p8751(LT192-L-STa13/pET28�) as the template (Fig. 1A). Primers STa-toxoid-Fand STa-toxoid-R were specifically designed to mutate the estA gene forindividual STa toxoid genes (Table 2).

Each STa-toxoid-dmLT fusion gene was further amplified with the PCRprimers T7-F and T7-R and purified by using gel electrophoresis. PurifiedPCR products were digested with NheI and EagI restriction enzymes (Bio-Lab) and cloned into expression vector pET28� (Novagen). Toxoid fu-sions were expressed as 6�His-tagged proteins in E. coli BL21 by follow-ing standard protocols (26). In addition, the native STa gene was fused tothe modified dmLT gene for fusion STa-dmLT.

Expression and detection of toxoid fusion proteins. The expressionof each toxoid fusion protein in E. coli BL21 was examined in a standardsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)as described previously (24). Briefly, each toxoid fusion recombinantstrain was cultured at 37°C in 500 ml of Luria-Bertani medium supple-mented with kanamycin (30 �g/ml). Overnight-grown bacteria were in-duced with isopropyl-1-thio-�-D-galactoside (IPTG; 100 �M) for 4 h af-ter the culture optical density (OD) reached 0.5. Bacterial culture wascentrifuged at 5,000 � g for 20 min, and pellets were suspended with 5 mlof B-PER (bacterial protein extraction reagent in phosphate buffer; Pierce,Rockford, IL) for total protein extraction (with a majority of proteins asinclusion bodies; in denaturing buffer). Recombinant 6�His-tagged tox-oid fusion proteins were further extracted from total insoluble proteins toa purity of �90% with Ni-nitrilotriacetic acid agarose according to themanufacturer’s protocol (Qiagen, Valencia, CA). Extracted 6�His-tagged proteins were refolded using a Novagen protein refolding kit ac-cording to the manufacturer’s protocol (Novagen).

A 100-ng portion of refolded 6�His-tagged protein from each toxoidfusion strain was analyzed using 10 to 12% SDS-PAGE gels and an immu-noblot assay. Rabbit anti-CT (1:3,300; Sigma) and protein A-purifiedanti-STa antisera (1:5,000; Robertson Laboratory) were used as primaryantibodies, respectively. IRDye-labeled goat anti-rabbit IgG (1:5,000; LI-COR, Lincoln, NE) were used as the secondary antibody to detect eachfusion protein with a LI-COR Odyssey premium infrared gel imagingsystem.

In addition, the expression of each toxoid fusion protein was exam-ined in an enzyme-linked immunosorbent assay (ELISA). One hundrednanograms of each toxoid fusion protein was coated to each well of anImmulon 2HB plate (Thermo Scientific, Rochester, NY). Rabbit anti-CT(1:3,000) and anti-STa (1:3,000) sera were used as primary antibodies, andhorseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:3,000;Sigma) was used as the secondary antibody. OD values were measuredwith a plate reader at a 405-nm wavelength, after 25 min of incubationwith 100 �l of 3,3=,5,5=-tetramethylbenzidine (TMB; Microwell Peroxi-dase Substrate System [2-C]; KPL, Gaithersburg, MD).

FIG 1 Construction and detection of STa-toxoid-dmLT toxoid fusions. (A)Illustration of an STa-toxoid-dmLT toxoid fusion gene. Three copies of eachSTa-toxoid gene were genetically fused to the 5= end, between A1 and A2 of LTA,and the 3= end of a monomeric dmLT peptide gene (LTR192G/L211A; as a singleopen reading frame) using splicing overlap extension PCRs. Primers: 1, T7-F;2, STa-toxoid-F; 3, STa-toxoid-R; 4, LT73-R; 5, LT73-F; 6, LT211-R; 7, LT211-F; 8,T7-R. Primers 2 and 3 were used to mutate the STa gene for different STatoxoids (Table 2). Primers 1 and 3 and primers 2 and 4 were, respectively, usedin two PCRs (which generated two nucleotide fragments to be fused) to pro-duce the first segment of the fusion gene, which included the first copy of a STatoxoid and the first 75 amino acids of the LT A subunit (at the N terminus).Primers 5 and 3 and primers 2 and 6 were used in two other PCRs (for twoother fragments to be fused) to generate the second segment of the fusion gene,which consisted of the second copy of a STa toxoid and amino acids 68 to 216of the LT A subunit. Primers 7 and 3 and primers 2 and 8 were used in anothertwo PCRs (for another two fragments to be fused) to create the third segmentof the fusion gene, which contains the third copy of a STa toxoid, amino acids204 to 240 of the LT A subunit and the LT B subunit (1 to 100 amino acids).These three segments were connected in an SOE PCR for a single open readingframe coding a STa-toxoid-dmLT toxoid fusion protein. (B) Western blot todetect each STa-toxoid-dmLT fusion protein with anti-CT antibodies. A fusionprotein (100 ng) separated in 10 to 12% PAGE gel was detected using rabbitanti-CT antiserum (1:3,300; Sigma) and IRDye-labeled goat anti-rabbit IgG(1:5,000; LI-COR). (C) Western blot to detect toxoid fusion proteins withanti-STa antibodies. Fusion proteins (100 ng each) were detected with proteinA-purified rabbit anti-STa antiserum (1:5,000) and IRDye-labeled goat anti-rabbit IgG (1:5,000; LI-COR). The total protein extracted from host strain8955 was used as the negative control (�). Lane M is the protein marker (inkilodaltons; Precision Plus Protein Pre-stained standards; Bio-Rad).

STaN12S-dmLT Is Optimal for Anti-STa Antibodies

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Toxicity detection of toxoid fusion proteins. The in vitro enterotoxicactivity of each toxoid fusion was determined by measuring stimulation ofintracellular cyclic AMP (cAMP) and cGMP in T-84 cells with EIA cAMPand cGMP ELISA kits (Assay Designs). Toxoid fusions would stimulate anincrease of intracellular cAMP and cGMP level if they retain LT and STatoxicity. As described previously (21, 22, 24), 100 ng of each toxoid fusionprotein (in 150 �l of phosphate-buffered saline [PBS]), 10 ng of choleratoxin (CT [Sigma]; in 150 �l of PBS, as an LT positive control, which ishighly homologous to LT structurally and functionally), or 2 ng of puri-fied STa toxin (from the Clements laboratory, in 150 �l of PBS, as an STapositive control) was added to each microplate well (in duplicate) that wasseeded with 105 T-84 cells. After incubation in a CO2 incubator for 1 h (forcGMP) or 3 h (for cAMP), T-84 cells were gently washed with 1 ml of PBS(2�) and then lysed. Cell lysates were collected and measured for intra-cellular cAMP or cGMP levels (pmol/ml) using EIA ELISAs according tothe manufacturer’s protocol and with Assay Blaster software (Enz LifeSciences, Farmingdale, NY).

Mouse immunization with each toxoid fusion. Five 6- to 8-week-oldfemale adult BALB/c mice (Charles River Laboratories International, Inc.,Wilmington, MA) as a group were immunized intraperitoneally with eachrefolded toxoid fusion protein. Each mouse was primarily injected with 200�g of refolded fusion protein (in 200 �l) and 200 �l of Freund completeadjuvant (Sigma). Five mice in the control group were each intraperitoneallyinjected with 200�l of Freund complete adjuvant and 200�l of protein buffer(0.02 M Tris-HCl). Two booster injections at the same dose as the primaryinjection but with Freund incomplete adjuvant followed at biweekly intervals.Serum and fecal suspension samples (1 g of feces was suspended in 5 ml offecal reconstitution buffer [10 mM Tris, 100 mM NaCl, 0.05% Tween 20, 5mM sodium azide, pH 7.4; 1:6 dilution]) (22, 27) were collected from eachmouse prior to immunization and 10 to 12 days after each immunization andwere then stored at �80°C until use. On day 37, mice were anesthetized withCO2 and exsanguinated. Animal studies complied with the Animal WelfareAct according to National Research Council guidelines (28) and were ap-proved and supervised by state veterinarians and university Institutional An-imal Care and Use Committees.

Anti-LT and anti-STa antibody titration. Anti-LT and anti-STa IgGantibodies in serum and IgA antibodies in fecal suspension samples ofeach mouse were examined in ELISAs as described previously (21, 22, 24).Briefly, 100 ng of CT (an LT homologue which has been commonly usedas a coating antigen to titrate anti-LT antibodies) in 100 �l of antigencoating buffer (15 mM Na2CO3, 35 mM NaHCO3; pH 9.6) was used tocoat each well of an Immulon 2HB plate to titrate anti-LT IgG and IgAantibodies, whereas 10 ng of STa-ovalbumin conjugates in 100 �l of STaELISA buffer (29) was coated at each well of a Costar plate (Corning, Inc.,Corning, NY) to titrate anti-STa IgG and IgA antibodies. After incubationat 37°C for 1 h and then at 4°C overnight, the plates were washed withPBST (PBS plus 0.05% Tween 20) and blocked with 5% (for anti-STa) or10% (for anti-LT) nonfat milk-PBST (150 �l per well) at 37°C for 1 h.After washes with PBST (3� for anti-STa and 5� for anti-LT), wells wereincubated with 2-fold serial dilutions of serum samples (100 �l; at aninitial dilution of 1:200 in 5% milk-PBST for anti-LT or of 1:25 dilution in1% milk-PBST for anti-STa) or fecal suspension samples (1:20 dilutionfor anti-LT or at 1:15 to 1:20 for anti-STa in 5% milk-PBST) for 1 h at37°C. All samples were examined in triplicate. Wells were washed (fivetimes with PBST) and incubated with HRP-conjugated goat anti-mouseIgG (a 1:5,000 dilution for anti-LT or a 1:3,000 dilution for anti-STa) orIgA (1:1,000 dilution; Sigma) at 100 �l per well for 1 h at 37°C. The plateswere washed again (three times with PBST, followed by two times withPBS) and incubated with a TMB microwell peroxidase substrate system(2-C; KPL) at 100 �l per well for 20 min at room temperature. The ODwas measured with a plate reader at a 405-nm wavelength. Antibody titerswere calculated as the highest dilution that produced OD readings of �0.3above the background readings and were displayed in a log10 scale aspreviously described (21, 22, 24).

Anti-LT and anti-STa antibody neutralization assays. Serum and fe-cal suspension samples pooled from mice in each group were examinedfor in vitro antibody neutralization activities against STa and CT toxins byusing EIA cAMP and cGMP kits (Assay Design) and T-84 cells. CT andSTa toxins stimulate increases in cAMP and cGMP, respectively, in T-84cells. Neutralizing antibodies would neutralize toxins and prevent CT orSTa from stimulating an increase of intracellular cAMP or cGMP level incells; therefore, neutralizing activity against CT and STa from antibodiesin serum and fecal suspension samples of the immunized mice can bemeasured. As described previously (21, 24), serum (30 �l of serum pooledfrom each group, 6 �l per mouse; a 1:33.3 dilution in a final volume of 1ml) or fecal (30 �l, 1:6 diluted fecal suspension, a 1:200 final dilution)samples were incubated with 2 ng of STa toxin or 10 ng of CT (diluted in150 �l of Dulbecco modified Eagle medium [DMEM]/F-12 medium) for1 h at room temperature. Incubated solution was brought up to 300 �lwith DMEM/F-12 medium and transferred to T-84 cells (105 cells in 700�l of culture medium), which was pretreated with 1 mM IBMX (3-isobu-tyl-1-methylxanthine; Sigma). After incubation of 1 h (STa in cGMP) to 3

TABLE 2 STa-toxoid forward and reverse (STa-toxoid-F, STa-toxoid-R)PCR primers designed to mutate the STa gene (estA) for STa toxoidswhich were fused to a modified LT toxoid gene for STa-toxoid-dmLTtoxoid fusionsa

STatoxoid Primer Nucleotide sequence (5=-3=)3F hSTaS3F-F ATG AAT AGT TTC AAT TAC TGC TGT

hSTaS3F-R ACA GCA GTA ATT GAA ACT ATT CAT

8Q hSTaE8Q-F AAT TAC TGC TGT CAA TTG TGT TGThSTaE8Q-R ACA ACA CAA TTG ACA GCA GTA ATT

9A hSTaL9A-F TAC TGC TGT GAA GCA TGT TGT AAThSTaL9A-R TGG ATT ACA ACA TGC TTC ACA GCA

12I hSTaN12I-F GAA TTG TGT TGT ATC CCT GCT TGThSTaN12I-R ACA AGC AGG GAT ACA ACA CAA TTC

12S hSTaN12S-F GAA TTG TGT TGT AGC CCT GCT TGThSTaN12S-R ACA AGC AGG GCT ACA ACA CAA TTC

12T hSTaN12T-F GAA TTG TGT TGT ACC CCT GCT TGThSTaN12T-R ACA AGC AGG GGT ACA ACA CAA TTC

13A hSTaP13A-F TTG TGT TGT AAT GCA GCT TGT ACChSTaP13A-R GGT ACA AGC TGC ATT ACA ACA CAA

13F hSTaP13F-F TTG TGT TGT AAT TTT GCT TGT ACChSTaP13F-R GGT ACA AGC AAA ATT ACA ACA CAA

14H1 hSTaA14H-F TGT TGT AAT CCT CAT TGT ACC GGGhSTaA14H-R CCC GGT ACA ATG AGG ATT ACA ACA

14Q hSTaA14Q-F TGT TGT AAT CCT CAG TGT ACC GGGhSTaA14Q-R CCC GGT ACA CTG AGG ATT ACA ACA

14T hSTaA14T-F TGT TGT AAT CCT ACA TGT ACC GGGhSTaA14T-R CCC GGT ACA TGT AGG ATT ACA ACA

14V hSTaA14V-F TGT TGT AAT CCT GTA TGT ACC GGGhSTaA14V-R CCC GGT ACA TAC AGG ATT ACA ACA

16K hSTaT16K-F AAT CCT GCT TGT AAG GGG TGC TAThSTaT16K-R ATA GCA CCC CTT ACA AGC AGG ATT

16M hSTaT16 M-F AAT CCT GCT TGT ATG GGG TGC TAThSTaT16 M-R ATA GCA CCC CAT ACA AGC AGG ATT

a Nucleotides that are underlined indicate mutations.

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h (CT in cAMP) at 37°C in a CO2 incubator, the cells were washed andlysed. Cell lysates were collected and measured for intracellular cAMP orcGMP levels (pmol/ml) using cAMP and cGMP ELISA kits, respectively.CT or STa, alone or mixed with a serum or fecal suspension of the controlgroup to show enterotoxicity in the stimulation of cAMP or cGMP, andcell culture medium (without toxin or antibodies) to show a baseline ofELISA were used as controls.

Statistical analysis. Data were analyzed by using the SAS for Win-dows, version 8 (SAS Institute, Cary, NC). The results are presented asmeans � the standard deviations. A Student t test was used to compare thedifferent treatment groups. Calculated P values of 0.05 were regarded assignificant when treatments were compared using two-tailed distributionand two-sample equal or unequal variance.

RESULTSFourteen STa toxoids were identified for toxoid fusion con-struction. By substituting amino acid 3 (S), 4 (N), 8 (E), 9 (L), 12(N), 13 (P), 14 (A), 16 (T), or 17 (G) of STa with different residues,we generated 47 STa toxoid candidates for a mini-STa toxoid li-brary. These STa toxoid candidates were examined for toxicity(stimulation of the cGMP level) by using a cGMP EIA kit withT-84 cells and were assessed for antigenic structure (reactivity toanti-STa antibodies) by using an STa competitive ELISA with an-tiserum against native STa. The data showed that these STa deriv-atives differed from each other in toxicity reduction and also ac-tivity, reacting to anti-STa antibodies. Some mutants, such asSTaN4Y and STaT16S, were as toxic as the native STa in the stimu-lation of intracellular cGMP in T-84 cells. In contrast, other mu-tants, for example, STaA14Q, STaA14L, STaN12K, and STaE8Q, exhib-ited no or little activity in stimulating cGMP levels in T-84 cells.The data from the STa competitive ELISA showed that some mu-tants, such as STaT16R, STaT16M, STaT16Q, and STaA14T, retained asimilar level of activity reacting to anti-STa antiserum as the nativeSTa. However, others, including STaE8G, STaL9K, STaL9Q, andSTaN12K, showed no or low reactivity to anti-STa antiserum.

Fourteen STa mutants—STaS3F, STaE8Q, STaL9A, STaN12I,STaN12S, STaN12T, STaP13A, STaP13F, STaA14H, STaA14Q, STaA14T,STaA14V, STaT16K, and STaT16M—that showed no or little entero-toxicity but a level of reactivity similar to anti-STa antiserum asthe native STa were selected for toxoid fusion construction. Threecopies of each of these 14 STa toxoids were fused to a monomericdmLT peptide. DNA sequencing verified that each toxoid fusioncarried three copies of a STa toxoid gene and one copy of a mo-nomeric dmLT gene and formed a single open reading frame en-coding a single peptide (Fig. 1A). In addition, a fusion with threecopies of the native STa and one copy of the monomeric dmLTwas also constructed and was verified from DNA sequencing.

Each toxoid fusion protein was expressed and showed no en-terotoxicity. Fourteen STa-toxoid-dmLT toxoid fusions and thenative STa fusion protein 3�STa-dmLT were examined in West-ern blot with anti-CT and anti-STa antiserum. The detectable pro-teins from all fusion strains had a molecular mass about 48 kDa,the expected size of all fusion proteins (Fig. 1B and C). In addition,the ELISA data indicated that all fusion proteins were recognizedby the anti-LT and anti-STa antiserum. This finding confirmedthat toxoid fusion proteins were expressed by each recombinantfusion strain (Table 3).

In vitro toxicity assays showed that enterotoxicity was not de-tected with any of the fusion proteins (including the native STafusion). T-84 cells incubated with 100 ng of each fusion proteinshowed no increase in intracellular cAMP levels (an average of

0.96 pmol/ml) or cGMP levels (an average of 8.31 pmol/ml).These levels are similar to those observed in cells incubated withthe negative controls: cell culture medium (0.98 pmol/ml ofcAMP, 7.8 pmol/ml of cGMP) and protein buffer (1.06 pmol/mlof cAMP, 6.8 pmol/ml of cGMP). In contrast, T-84 cells incubatedwith 2 ng of STa showed a significant increase in cGMP (72 � 2.83pmol/ml; P 0.01) and showed a significant increase in cAMPwhen incubated with 10 ng of CT (�200 pmol/ml; P 0.01)(Table 4).

All toxoid fusions elicited anti-LT and anti-STa antibodies,but anti-STa antibodies varied among and within immunizedgroups. Among immunized mice, anti-LT and anti-STa IgG an-

TABLE 3 ELISA OD values obtained with anti-CT and anti-STa antiserato verify the expression of each STa-toxoid-dmLT fusion protein

Fusion proteina Mean OD � SD

Anti-CTb

STa 1.05 � 0.033F 1.4 � 0.028Q 1.32 � 0.09A 1.5 � 0.0312I 1.5 � 0.0712S 1.4 � 0.0312T 1.36 � 0.0313A 1.41 � 0.0113F 1.49 � 0.0714H 1.34 � 0.0314Q 1.28 � 0.0214T 1.32 � 0.0414V 1.45 � 0.0716K 1.36 � 0.0616M 1.29 � 0.02(–) 0.11 � 0.05

Anti-STac

STa 1.95 � 0.073F 1.94 � 0.028Q 1.69 � 0.09A 1.97 � 0.0312I 1.84 � 0.0512S 1.95 � 0.0412T 1.71 � 0.013A 1.86 � 0.0513F 1.71 � 0.0614H 1.93 � 0.0714Q 1.89 � 0.0314T 1.91 � 0.0514V 1.89 � 0.0216K 2.1 � 0.0116M 2.04 � 0.01(–) 0.34 � 0.11

a Fusion proteins: STa, fusion 3�STa-dmLT; 3F, 3�STaS3F-dmLT; 8Q, 3�STaE8Q-dmLT; 9A, 3�STaL9A-dmLT; 12I, 3�STaN12I-dmLT; 12S, 3�STaN12S-dmLT; 12T,3�STaN12T-dmLT; 13A, 3�STaP13A-dmLT; 13F, 3�STaP13F-dmLT; 14H, 3�STaA14H-dmLT; 14Q, 3�STaA14Q-dmLT; 14T, 3�STaA14T-dmLT; 14V, 3�STaA14V-dmLT; 16K,3�STaT16K-dmLT; 16M, 3�STaT16 M-dmLT. Each purified fusion protein (100 ng perwell) was used to coat ELISA 2HB plate (in triplicate). PBS was used as the negativecontrol (–).b Rabbit anti-CT (1:3,000; Sigma) and HRP-conjugated goat-anti-rabbit IgG (1:3,000;Sigma) were used as the primary and secondary antibodies to detect the LT peptide ofeach fusion.c Protein A-purified rabbit anti-STa serum (1:3,000; Robertson laboratory) and HRP-conjugated goat anti-rabbit IgG (1:3,000) were used to detect the STa toxoid of eachfusion protein.

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tibodies were detected in serum, and IgA antibodies were detectedin fecal suspension samples. Anti-LT IgG antibodies in serumsamples were detected at an average titer of 3.17 � 0.10 (in log10)among all immunized groups, but not in the control mice (Table5). Anti-LT IgG antibody titers in mice immunized with the tox-oid fusion were not significantly different than titers in mice im-munized with 3�STa-dmLT fusion (P � 0.05) (Table 5). In con-

trast, anti-STa IgG antibody titers in the serum samples variedgreatly among groups immunized with different fusion antigens,ranging from 0.73 � 1.03 to 3.43 � 0.29 (Fig. 2). Comparedto the unimmunized control group, all immunization groups ex-cept those immunized with 3�STa-dmLT, 3�STaS3F-dmLT,3�STaP13F-dmLT, 3�STaA14T-dmLT, and 3�STaA14V-dmLTwere significantly different in detection of anti-STa IgG antibodiesin serum samples. However, compared to the anti-STa IgG titer inmice immunized with 3�STa-dmLT, only titers in serum samplesof the mice immunized with 3�STaN12S-dmLT, 3�STaA14H-dmLT,3�STaN12T-dmLT, and 3�STaN12I-dmLT were significantly differ-ent (P 0.05) (Fig. 2). Among them, mice immunized with3�STaN12S-dmLT, 3�STaA14H-dmLT, and 3�STaN12T-dmLT hadthe greatest anti-STa serum IgG titers detected (Fig. 2). The differ-ences among these three groups, however, were not significant (P 0.78 for 3�STaN12S-dmLT versus 3�STaN12T-dmLT; P 0.24 for3�STaN12S-dmLT versus 3�STaA14H-dmLT; P 0.44 for3�STaN12T-dmLT versus 3�STaA14H-dmLT).

For every mouse in these three groups and in the groups im-munized with 3�STaL9A-dmLT and 3�STaN12I-dmLT, an anti-STa IgG antibody response was detected. In contrast, mice in theremaining groups showed a wide range of intragroup variationsupon the induction of an anti-STa antibody response (Fig. 2). Forexample, while for one mouse anti-STa IgG was detected at a titer

TABLE 4 Detection of in vitro enterotoxic activity of each fusionprotein, measured by the stimulation of intracellular cAMP and cGMP(pmol/ml) in T-84 cells with EIA cAMP and cGMP ELISA kits (AssayDesigns)

Fusion proteina

Mean pmol of cAMP orcGMP/ml � SD

cAMPb

STa 5.4 � 0.563F 5.2 � 0.078Q 10 � 0.79A 9.2 � 0.8512I 10.5 � 1.5612S 10.4 � 0.8412T 6.4 � 0.5713A 6.8 � 0.2813F 8.6 � 1.4114H 11 � 0.2814Q 8.4 � 3.3914T 6.8 � 1.1314V 6.2 � 0.8516K 9.3 � 1.5616M 10.4 � 0.57CT(�) �200Medium (–) 7.8 � 0.99Buffer (–) 6.8 � 1.41

cGMPc

STa 1.05 � 0.013F 1.26 � 0.348Q 1.10 � 0.149A 1.0 � 0.5612I 1.05 � 0.0712S 0.84 � 0.0512T 1.2 � 0.1413A 1.05 � 0.0713F 0.62 � 0.5914H 0.88 � 0.0414Q 0.96 � 0.1314T 1.05 � 0.1414V 0.81 � 0.3016K 0.88 � 016M 0.7 � 0.42STa(�)d 72 � 2.83Medium (–) 0.98 � 0.25Buffer (–) 1.06 � 0.20

a Fusion proteins are labeled as in Table 3. Each fusion protein (100 ng) was used tostimulate cAMP or cGMP in T-84 cells for toxicity detection (in duplicate). Theintracellular cAMP and cGMP levels serve as indicators of LT and STa enterotoxicity,respectively.b Intracellular cAMP levels in T-84 cells were used to measure LT enterotoxicity. A totalof 10 ng of CT (cholera toxin) was used as the positive (toxic) control; cell culturemedium and protein buffer were used as the negative (nontoxic) controls.c Intracellular cGMP levels in T-84 cells were used to measure STa enterotoxicity. Atotal of 10 ng of STa was used as the positive (toxic) control; cell culture medium andprotein buffer were used as the negative (nontoxic) control.d Purified STa toxin was used as the positive control.

TABLE 5 Mouse serum anti-LT IgG antibody titers

Mouse immunizationgroup (fusionantigen)a

Mean IgG titer(log10) � SDb Pc

STa 3.15 � 0.053F 3.13 � 0.09 0.588Q 3.16 � 0.14 0.849A 3.17 � 0.14 0.7412I 3.15 � 0.10 0.9912S 3.17 � 0.11 0.7112T 3.27 � 0.09 0.1013A 3.21 � 0.08 0.1513F 3.15 � 0.06 0.9214H 3.18 � 0.11 0.6914Q 3.22 � 0.07 0.1414T 3.15 � 0.11 0.9614V 3.16 � 0.22 0.1416K 3.24 � 0.06 0.0816M 3.15 � 0.12 0.99Control 0 � 0 0.001a Groups of mice (n 5 animals per group) immunized with different fusion antigens:STa, mice immunized with fusion 3�STa-dmLT; 3F, 3�STaS3F-dmLT; 8Q, 3�STaE8Q-dmLT; 9A, 3�STaL9A-dmLT; 12I, 3�STaN12I-dmLT; 12S, 3�STaN12S-dmLT; 12T,3�STaN12T-dmLT; 13A, 3�STaP13A-dmLT; 13F, 3�STaP13F-dmLT; 14H, 3�STaA14H-dmLT; 14Q, 3�STaA14Q-dmLT; 14T, 3�STaA14T-dmLT; 14V, 3�STaA14V-dmLT; 16K,3�STaT16K-dmLT; 16M, 3�STaT16M-dmLT; and control, mice injected with proteinbuffer and Freund adjuvant. Anti-LT IgG in serum sample of each immunized orcontrol mouse was titrated by ELISA using CT (100 ng per well of H2B plates) as thecoating agent and HRP-conjugated goat-anti-mouse IgG (1:5,000) as the secondaryantibodies.b Means and standard deviations of the anti-LT IgG titers in each immunization groupand the control group are shown. Antibody titers were calculated from the highestdilution of each serum sample that produced an ELISA OD of 0.3 above thebackground and are presented in log10 scale.c P values were calculated by using a Student t test comparing mouse antibody titers ineach group to titers in the STa group, mice immunized with fusion 3�STa-dmLT,which was used as a reference for toxoid fusions.

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as great as 3.8 log10, for the remaining four mice from the samegroup no anti-STa IgG was detected in their serum samples.

Anti-STa IgA antibody titers detected in fecal suspension sam-ples varied among immunized groups as well. Greater anti-STaIgA antibody titers were detected in mice in groups immunizedwith 3�STaT16M-dmLT (1.51 � 0.62), 3�STaL9A-dmLT (1.14 �0.82), and 3�STaN12S-dmLT (1.11 � 0.66). Compared to anti-STa IgA detected in the fecal samples of mice immunized with3�STa-dmLT (0.88 � 0.74), however, the titers of these threegroups were not significantly different (P 0.19, 0.62, and 0.62).Indeed, compared to 3�STa-dmLT, none of the toxoid fusions,except 3�STaS3F-dmLT, which had no anti-STa IgA detected,showed a significant difference in the stimulation of anti-STaIgA antibodies (P � 0.05). However, compared to the IgA titerof the control group, anti-STa IgA titers in mice immunizedwith 3�STaT16M-dmLT (P 0.006), 3�STaT16M-dmLT (P 0.02),3�STaA14Q-dmLT (P 0.01), 3�STaA14H-dmLT (P 0.048),3�STaN12T-dmLT (P 0.03), 3�STaN12S-dmLT (P 0.02),and 3�STaL9A-dmLT (P 0.02) were significantly different.Only the group immunized with the 3�STaN12S-dmLT or the3�STaT16M-dmLT toxoid fusion displayed an anti-STa IgA antibody

response that was detected in all five mice (Fig. 3). No anti-LT oranti-STa IgG or IgA antibodies were detected in serum or fecal sus-pension samples of the control mice.

Elicited anti-STa antibodies exhibit in vitro neutralizing ac-tivities against STa toxin. In vitro antibody neutralization assaysindicated that serum samples pooled from each immunized groupshowed neutralizing activity against STa toxin but with variationsamong groups. Serum samples pooled from the groups immu-nized with 3�STa12S-dmLT, 3�STa14H-dmLT, 3�STa16K-dmLT,and 3�STa-dmLT showed stronger neutralizing activities againstSTa toxins, since serum samples from these groups prevented STatoxin from stimulating cGMP levels in T-84 cells (Fig. 4). Theintracellular cGMP levels in T-84 cells incubated with 2 ng STatoxin and the pooled serum samples of mice immunized withthese four fusions were 1.65 � 0.21, 4.65 � 0.21, 5 � 0.14, and7.7 � 0.42 pmol/ml, respectively. These cGMP levels differed sig-nificantly from the cGMP in cells incubated with STa and serum ofcontrol mice (62 � 0 pmol/ml; P 0.01) or STa alone (72 � 2.83pmol/ml; P 0.01). When the STa toxin dose was increased to 5or 10 ng, only serum sample pooled from the 3�STaN12S-dmLT-immunized group maintained strong neutralizing activity, show-ing cGMP levels of 0.28 � 0.03 and 1.16 � 0.18 pmol/ml, respec-tively, in the incubated T-84 cells.

Pooled fecal samples (in a dilution of 1:200; versus 1:33.3 inserum) were also analyzed but showed mild or moderate neutral-izing activity against STa toxin (Fig. 5). Lower cGMP levels weredetected in T-84 cells incubated with STa and the fecal samples ofmice immunized with 3�STaN12I-dmLT (13.5 � 0.71 pmol/ml),3�STaP13A-dmLT (14 � 0 pmol/ml), 3�STaN12T-dmLT (16.3 �1.1 pmol/ml), 3�STaE8Q-dmLT (16.5 � 0.71 pmol/ml), or3�STaN12S-dmLT (18.8 � 0.35 pmol/ml). The mean cGMP level

FIG 2 Mouse serum anti-STa IgG antibody titers. Anti-STa IgG antibodies inserum samples of the immunization and control groups (five mice per group)were titrated in ELISA using STa-ovalbumin conjugates (10 ng per well ofCostar plates; coating agent) and HRP-conjugated goat anti-mouse IgG (1:3,000; the secondary antibodies). The anti-STa antibody titer was calculatedfrom the highest dilution of a serum sample that produced an ELISA OD of�0.3 (above the background). Each dot represented a mouse IgG titer, and thebar indicated the mean titer of the group. Mouse group labels (on the x axis)indicated the immunization groups (immunized with each different fusionantigen) and the control group (�). The mean antibody titer (mean � thestandard deviation) and P value (compared to titers of mice in the groupimmunized with fusion 3�STa-dmLT) were calculated for the groups immu-nized with different fusions—STa, 3�STa-dmLT (0.76 � 1.69); 3F,3�STaS3F-dmLT (0.73 � 1.03, P 0.97); 8Q, 3�STaE8Q-dmLT (2.22 � 1.52,P 0.19); 9A, 3�STaL9A-dmLT (2.73 � 0.91, P 0.051); 12I, 3�STaN12I-dmLT (2.63 � 0.57, P 0.047); 12S, 3�STaN12S-dmLT (3.43 � 0.29, P 0.008); 12T, 3�STaN12T-dmLT (2.89 � 0.52, P 0.027); 13A, 3�STaP13A-dmLT (2.21 � 1.46, P 0.17); 13F, 3�STaP13F-dmLT (0.96 � 0.89, P 0.82);14H, 3�STaA14H-dmLT (3.14 � 0.43, P 0.016); 14Q, 3�STaA14Q-dmLT(2.1 � 1.46, P 0.26); 14T, 3�STaA14T-dmLT (1.44 � 1.49, P 0.52); 14V,3�STaA14V-dmLT (1.18 � 1.67, P 0.70); 16K, 3�STaT16K-dmLT (2.74 �1.54, P 0.089); 16M, 3�STaT16M-dmLT (2.12 � 1.35, P 0.35)—and forthe control group (0 � 0, P 0.37).

FIG 3 Mouse anti-STa IgA antibody titers, detected in fecal suspension sam-ples of each immunized or control mouse. Anti-STa IgA in fecal suspensionsamples of the immunization groups and the control group (five mice pergroup) were titrated in ELISA with STa-ovalbumin conjugates (10 ng per well,Costar plates) as the coating agent and HRP-conjugated goat anti-mouse IgA(1:1,000) as secondary antibodies. The antibody titer was calculated from thehighest dilution of a fecal suspension sample that produced an ELISA OD of�0.3 above the background, and results are presented in a log10 scale. Each dotrepresented a mouse IgA antibody titer, and the bar was the mean titer of thegroup. Groups (labeled on the x axis) were mouse groups immunized witheach fusion antigen and the control group (“�” column), of which fecal sus-pension samples were titrated for anti-STa IgA antibodies.

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in T-84 cells incubated with STa toxin and the fecal samples of thecontrol mice was 62 � 0 pmol/ml, and the mean cGMP level incells incubated with cell culture medium was 0.98 � 0.25 pmol/ml(Fig. 5).

Neutralization activity against CT toxin was detected in theserum and fecal samples of the immunized groups. The averagecAMP level in T-84 cells incubated with 10 ng of CT and the serumsample pooled from each immunized group was 17 pmol/ml,whereas the average cAMP level in cells incubated with the toxinand the serum of the control group was 37 pmol/ml. When fecalsuspensions were used in neutralization assays, an average ofcAMP level in T-84 cells incubated with CT and samples of eachimmunized group was detected at 10.3 pmol/ml, but the cAMPlevel in T-84 cells incubated with toxin and the fecal suspension ofthe control group was 21 pmol/ml.

DISCUSSION

Recent studies indicated that nontoxic STa, after being geneticallyfused to a monomeric LT toxoid peptide, can elicit moderatelyneutralizing anti-STa antibodies (21, 22, 24). That indicates a pre-viously suggested STa dilemma, of which STa has to possess en-terotoxicity (which is not safe as a vaccine component) to be ableto induce neutralizing antibody response (20), can be solved.These findings led to the current effort toward further character-ization of STa toxoids for the induction of antibody response andthe search for optimal STa toxoid(s) that induce neutralizing an-tibodies against STa toxin. Shown differences in toxicity reduction

and more importantly antigenic structure alteration, individualSTa toxoids were thought to be different in induction of neutral-izing antibody response (23). A thorough examination of all po-tential STa toxoids would help to identify toxoids that have max-imum reduction in enterotoxicity but minimum alteration in STaantigenic structure. The antigenic propensity and perhaps the tox-icity of these optimal STa toxoids, however, could be altered afterbeing genetically fused to or chemically conjugated to a carrierprotein, which is required to facilitate anti-STa antibody response.Therefore, toxoid fusions or chemical conjugates have to be ex-amined essentially to identify STa-related antigens that induceneutralizing anti-STa antibodies. However, construction of tox-oid fusions and the expression of fusion proteins, or the purifica-tion of STa toxoids and then conjugation to a carrier, is labor-intensive and also technically challenging (some STa toxoidscannot be purified based on current protocols [J. D. Clements,unpublished data]), especially if a large number of STa toxoids areto be examined.

In the present study, we preselected a panel of STa toxoids froma mini-STa toxoid library to construct STa-toxoid-dmLT toxoidfusions and then used these fusions for mouse immunization tocharacterize STa toxoids for the induction of neutralizing anti-body response and to identify optimal STa toxoid(s) or more pre-cisely toxoid fusion antigen(s) for future ETEC antitoxin vaccinedevelopment. Here, a monomeric dmLT peptide was served as aprotein carrier to enhance STa toxoids for anti-STa immunoge-nicity and also an antigen used to induce an anti-LT antibodyresponse. This monomeric dmLT peptide, however, probablydoes not possess adjuvant activity as the holotoxin structured

FIG 4 Mouse serum in vitro antibody neutralizing activity against STa toxin.Intracellular cGMP levels in T-84 cells measured with an EIA cGMP ELISA kit(Assay Design) were used to show anti-STa antibody neutralization activity. Ifantibodies in serum (or fecal suspension) are neutralizing against STa toxin,these antibodies will prevent the toxin from stimulating intracellular cGMP inT-84 cells, which will result in a low cGMP level (lower than that of the controlgroup [the “�” column]). The serum sample (30 �l; in a final dilution of1:33.3) pooled from each immunization group or the control group (�) wasincubated with STa toxin (2 ng, in 150 �l of cell culture medium) for 1 h atroom temperature, and the serum-toxin mixture was added to T-84 cells (1 mlof final volume with cell culture medium). The intracellular cGMP level in theT-84 cells was measured after 1 h of incubation, with the mean cGMP level andstandard deviation (from two replicates) of each group indicated as columnsand lines. Labels on the x axis indicate the mouse groups immunized withdifferent fusion antigens and the control group (“�”), from which serumsamples were used for the antibody neutralization assay. STa toxin (withoutserum sample) was used as the control to show STa toxicity in stimulation ofcGMP, and cell culture medium (without STa toxin and serum) was used asthe control to show baseline cGMP level in T-84 cells.

FIG 5 Mouse fecal suspension in vitro antibody neutralization activity againstSTa toxin, with intracellular cGMP levels in T-84 cells used to measure neu-tralizing activity of antibodies in fecal suspension samples against STa toxin. Afecal suspension sample (30 �l; in a final dilution of 1:200) pooled from eachimmunized group or the control group (5 mice per group) was incubated withSTa toxin (2 ng; in 150 �l cell culture medium), and the fecal-toxin mixturewas added to T-84 cells. The intracellular cGMP level in T-84 cells was mea-sured by using an EIA cGMP ELISA kit (Assay Design), with columns and linesindicating the mean cGMP levels and standard deviations. A low cGMP level(lower than that of the control group) indicated antibody neutralizing activityagainst STa toxin. Group labels (on the x axis) indicate mice immunized withdifferent fusion antigens and the control mice, for which fecal suspensionsamples were examined for STa neutralizing activity. STa toxin only (toxinwithout fecal sample) to show enterotoxicity in the stimulation of cGMP andcell culture medium (no STa toxin and anti-STa antibodies) to show the base-line cGMP level in T-84 cells were included as controls.

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dmLT does. When a similar monomeric peptide, tmLT, was used,the toxoid fusion 3�STaA14Q-tmLT alone (without Freund or CTadjuvant) was unable to stimulate anti-STa or anti-LT antibodies(24). It will be interesting to examine currently constructed toxoidfusions for stimulation of anti-STa and anti-LT antibodies whenthe holotoxin structured dmLT (instead of Freund adjuvant) isused as the adjuvant.

STa enterotoxicity was thought to be tightly associated withantigenic propensity, especially of the toxicity domain (from res-idues 6 to 18) (30). Changes in the amino acid residues of a smallmolecule, such as STa, which consists of only 19 amino acids, 6 ofwhich form three disulfide bonds, were considered to have signif-icant effects upon alteration of its antigenic structure and entero-toxicity. It has been noted that STaG17S retains an antigenic struc-ture very similar to that of native STa based on its reactivity toantiserum against native STa and that it also possessed a level oftoxicity similar to that of STa (23). After screening 47 mutated STamolecules, we noted that some STa toxoids exhibited an associa-tion between the antigenic structure and enterotoxicity, but suchan association did not occur with other toxoids. It was found thatSTaT16K, STaT16M, STaA14V, and STaN12S had levels of reactivity toanti-STa antiserum similar to that of native STa, but they showedno STa toxic activity in vitro. That indicates that STa toxoids canmaintain an original antigenic structure or epitope topology af-ter their toxicity is eliminated. More interestingly, the nontoxicSTaT16K, STaT16M, and STaN12S molecules seemed to retain theirantigenic topology after being fused to the monomeric dmLT pep-tide, as indicated by the ELISA data (Table 3) and the fact thatthese fusions induced stronger anti-STa antibody responses inimmunized mice (Fig. 2).

It was unexpected that the fusion with native STa, 3�STa-dmLT, was not the top antigen in the induction of an anti-STaantibody response. Indeed, the sera of mice immunized with fu-sion 3�STa-dmLT had the lowest overall anti-STa IgG antibodytiters detected; four of five immunized mice did not develop ananti-STa immune response. That suggests that native STa anti-genic topology was substantially altered after being geneticallyfused to the monomeric dmLT peptide. However, the one mousethat developed anti-STa antibody response had the greatest anti-STa IgG titer detected in the serum sample. It is unknown to usthat how the genetic fusion process affected the STa or STa toxoidantigenic propensity and why such a wide range variation oc-curred in the induction of anti-STa antibody responses amongthese immunized mice. Regardless, native STa has to be pre-ex-cluded from vaccine development, since unintentional proteolyticcleavage of native STa from toxoid fusions by cellular proteases ordissociation of toxic STa from conjugates raises safety concerns.

Our data showed that variation in the induction of an anti-STaantibody response also existed among groups immunized withdifferent toxoid fusions and among individual mice within thegroup immunized the same toxoid fusion. Of five mice in an im-munized group, only one (3�STa-dmLT), two (3�STaS3F-dmLT,3�STaA14v-dmLT), three (3�STaP13F-dmLT, 3�STaA14Q-dmLT,3�STaA14T-dmLT), or four mice (3�STaE8Q-dmLT, 3�STaP13A-dmLT, 3�STaT16K-dmLT, 3�STaT16M-dmLT) had anti-STa IgGdetected in the serum samples. Even in the group of which all micedeveloped anti-STa IgG antibody response, the antibody titers ofindividual mice varied greatly, except the group immunized withthe 3�STaN12S-dmLT. In contrast to the variation in anti-STaantibody response, the anti-LT IgG antibody response was de-

tected in all immunized mice and at a very similar titer among andwithin the immunized groups (Table 5). The causes of such “hit-or-miss” in the induction of a host anti-STa antibody response bythese toxoid fusions are unclear to us. However, the poorly immu-nogenic nature of STa likely attributes to the inconsistency in in-ducing anti-STa antibody response. Increasing a sampling sizelikely will be helpful to further characterize the anti-STa antibodyinduction from these toxoid fusions. However, the present studywas repeated in a separate laboratory, which brought up an overallsampling size of 10 mice per group, and the anti-STa antibodyresponse pattern was not much changed (data not shown).

This study showed that toxoid fusions 3�STaN12S-dmLT,3�STaA14H-dmLT, and 3�STaT16K-dmLT were the top candi-dates in eliciting anti-STa IgG antibodies in mice. Serum samplesof mice immunized with these fusion antigens exhibited strongerneutralizing activity against the STa toxin. Among them,3�STaN12S-dmLT was the most effective at inducing a neutraliz-ing anti-STa antibody response. Only the serum sample pooledfrom mice immunized with the toxoid fusion 3�STaN12S-dmLTwas able to neutralize higher doses of STa toxin, since it prevented5 ng and even 10 ng of STa from stimulating any increase in cGMPin T-84 cells. Even when 20 ng of STa (a 10-fold increase) wasincubated with this serum sample, T-84 cells had only 10% cGMPlevel of those incubated with the toxin and the serum sample of thecontrol mice (data not shown). In addition, in fecal samples frommice immunized with 3�STaN12S-dmLT, more anti-STa IgA an-tibodies were detected that also displayed better neutralizationactivity against STa toxin. Future studies of in vivo antibody neu-tralization assays, such as the infant suckling mouse assay, or evena piglet challenge study, will further define neutralization activityagainst STa toxin from antibodies induced by this toxoid fusion.

Induction of the strongest anti-STa antibody response by this3�STaN12S-dmLT was confirmed from the study carried out inthe separate laboratory (data not shown). The data from thatmouse immunization study, using the same toxoid fusion anti-gens and the same immunization protocol, showed that the serumsamples of the mice immunized with 3�STaN12S-dmLT showedthe greatest anti-STa IgG antibody titers and also the strongestneutralizing activity against STa toxin detected (data not shown).Although continued construction of toxoid fusions with differentSTa toxoids and additional screening of fusions for anti-STa anti-body responses likely will reveal new findings, the toxoid fusion3�STaN12S-dmLT currently should be primarily considered fornext-step animal challenge or human volunteer studies in ETECantitoxin vaccine development.

The effective prevention of ETEC diarrhea requires antitoxinimmunity to neutralize the enterotoxicity of both toxins and alsoanti-adhesin immunity to block bacterial adherence and to pre-vent ETEC colonization at host small intestines (14). Anti-STaand anti-LT antibody responses induced by this 3�STaN12S-dmLT toxoid fusion or other fusions or conjugates alone may notbe sufficient for effective protection against ETEC diarrhea. Fu-ture studies should include the 3�STaN12S-dmLT fusion antigenamong promising ETEC vaccine candidates that induce anti-ad-hesin immunity against multiple ETEC adhesins, or coadminis-tration with a product that induces broadly protective anti-adhe-sin immunity may be desirable for developing effective vaccinesagainst ETEC diarrhea.

STaN12S-dmLT Is Optimal for Anti-STa Antibodies

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ACKNOWLEDGMENTS

Financial support for this study was provided by National Institutes ofHealth AI083897 (W. Zhang), EVI/PATH STa Toxoid Vaccine Consor-tium project, and the South Dakota Agricultural Experiment Station. Thisstudy was also supported by all members of the STa Toxoid Vaccine Con-sortium Group: J. P. Nataro (University of Virginia School of Medicine),E. M. Barry (University of Maryland School of Medicine), J. D. Clements(University of Tulane School of Medicine), W. Zhang (South Dakota StateUniversity), D. H. Francis (South Dakota State University), H. Sommer-felt (University of Bergen), P. Puntervoll (University of Bergen), A. LouisBourgeois (PATH), and R. Walker (PATH).

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