The
Brazilian
Journal
of
INFECTIOUS
DISEASES
w w w . e l s e v i e r . c o m / l o c a t e / b j i d
Original
article
A
CssA,
CssB
and
LTB
chimeric
protein
induces
protection
against
Enterotoxigenic
Escherichia
coli
Samane
Bagheri
a,
Seyed
Latif
Mousavi
Gargari
a,∗,
Iraj
Rasooli
a,
Shahram
Nazarian
b,
Masoome
Alerasol
aaDepartmentofBiology,ShahedUniversity,Tehran,Iran bDepartmentofBiology,ImamHusseinUniversity,Tehran,Iran
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t
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o
Articlehistory: Received2June2013 Accepted16July2013
Availableonline3January2014
Keywords:
EnterotoxigenicEscherichiacoli CS6
Enterotoxin Chimericprotein
a
b
s
t
r
a
c
t
Objectives:EnterotoxigenicEscherichiacoli(ETEC),amajorcauseofdiarrheainchildrenunder 5,isanimportantagentfortraveler’sdiarrhea.Heat-labileenterotoxin(LT)andcolonization factors(CFs)aretwomainvirulencemechanismsinETEC.CS6isoneofthemostprevalent CFsconsistingoftwostructuralsubunitsviz.,CssA,CssB,necessaryforattachmenttothe intestinalcells.
Methods:Inthepresentresearch,achimerictrivalentproteincomposedofCssB,CssAand LTBwasconstructed.ThechimericgenewassynthesizedwithcodonbiasofE.colifor enhancedexpressionoftheprotein.Recombinantproteinswereexpressedandpurified. Micewereimmunizedwiththerecombinantprotein.Theantibodytiterandspecificityof theimmuneserawereanalyzedbyELISAandWesternblotting.Efficiencyoftheimmune seraagainstETECwasevaluated.
Results:Antibodyinductionwasfollowedbyimmunizationofmicewiththechimeric pro-tein.PretreatmentoftheETECcellswithimmunizedanimalantiseraremarkablydecreased theiradhesiontoCaco-2cells.
Discussion:Theresultsindicateefficacyoftherecombinantchimericproteinasaneffective immunogen,whichinducesstronghumoralresponseaswellasprotectionagainstETEC adherenceandtoxicity.
©2013 ElsevierEditoraLtda.Allrightsreserved.
Introduction
EnterotoxigenicEscherichiacoli(ETEC)isoneoftheimportant agentscausingdiarrheainchildrenunderfive yearsold in developingcountriesandisamajorcauseofdiarrheain trav-elerstothoseregions.1,2Thismicroorganismisresponsiblefor 400,000to800,000deathsperyear.3ETECstrainshaveseveral
∗ Correspondingauthor.
E-mailaddress:slmousavi@shahed.ac.ir(S.L.MousaviGargari).
virulencefactorslikeheat-labileenterotoxin(LT),heat-stable enterotoxin(ST)andcolonizationfactors(CFs).1CFsare sur-faceproteinsthatmediateadherencetointestinalmucosa,the firststeptobeginpathogenesis.4Followingadherence,either orbothLTorSTareexpressed,resultingindiarrhealdisease.5 Ofmorethan25 identifiedCFs,6 CS6isoneofthemost epidemiologically prevalent CF in many countries, often accountingforupto20–30%ofallclinicalisolates.1,4Thishas
1413-8670/$–seefrontmatter©2013 ElsevierEditoraLtda.Allrightsreserved. http://dx.doi.org/10.1016/j.bjid.2013.07.012
ledtointeresttotrytouseCS6aloneorincombinationwith otherantigensinanETECvaccine.1,7ThemorphologyofCS6 isnon-fimbrialandisexpressedeitheraloneorwithCS5or lesscommonlywithCS4onETECstrains.7,8
ThegenesassociatedwithCS6areexpressedasatypical bacterialoperon,consistingoffourgenesviz.,cssA,cssB,cssC, and cssD.7 CssAand CssBare structuralsubunits, whereas CssC and CssD are recognized tobe chaperone and usher respectively.9CssCproteinassistsinthefoldingoftheCssA and CssBstructural subunits, and CssD has been ascribed an usher function responsible for transport of CssA and CssBtothecellsurface.740kDaCssAand15.5kDaCssBare necessaryforattachmenttointestinalcells.4,7TheCssA inter-actedwiththehostcellfibronectinwhileCssBbindstocell surfacesulfatide.4,10 Studieshave indicatedthatantibodies againstbothstructuralsubunitsareessentialtoinhibitthe adhesion.1,4
LTtoxin consists of a pentameric ring of five identical binding(LTB)subunitsof11.5kDasurroundinganactive sub-unit(LTA)and bindstronglytothe intestinalreceptorGM1 onthesurfaceofeukaryoticcells.11,12 TheBsubunitmostly associateswithimmunityagainstLTandinducesboth sys-temicandmucosalantibodyresponses.13Sincemostclinical ETECisolatescan produceLTandhas adjuvantproperties, thereforeitisasuitablecandidateantigentoprovideanti-LT immunity.14,15
In order to provide protection, an ETEC vaccine should enclosethemostprevalentCFandaLTtoxoidorBsubunit ofLTtoxinuntilitblocksadherenceandtoxinactivity.5,16
Becauseliveattenuatedorkilledbacterialcellvaccinesare potentiallyassociatedwithtransientsideeffects,useof sub-unitvaccineshasbeen preferred.Thesubunitvaccines are considerablysafeimmunesystemstimulatorswiththeability totargetthesitewhereimmunityisrequired.17
Inthisstudyatrivalentrecombinantproteinwasdesigned againstETEC CS6strains. A construction containing struc-turalsubunitsofCS6 (CssB,and 100 aminoacidsfrom the C-terminalof the proteinCssA) and Bsubunit of LTtoxin wasdesigned.Theimmunogenicityofatrivalentrecombinant proteincodedbyasyntheticgeneisreported.
Materials
and
methods
Bacterialstrains,plasmidsandmedia
ETECstrainswereisolatedfromchildrenwithdiarrhea.The strainswerepreviouslycharacterizedforthepresenceofCFs andenteroxinsandusedforchallengepurpose.Plasmids pET-28aandpET-32awerefromNovagen(USA).E.coliBL21(DE3), DH5␣andTop10wereprocuredfromPasteurInstituteofIran. TheE.colistrainswere growninLuria–Bertani(LB)brothat 37◦C.
Insilicopredictionsandconstructionofchimericgene
Chimeric gene was designed based on the nucleotide sequence of ltb, cssA and cssB genes (Gen Bank accession numbers;M17874.1,GQ241328.1,UO4844.1,respectively).The sequenceswereusedtogenerateachimerictrivalentprotein
fusedtogether by(EAAAK)4hydrophobiclinkersinorderto findthebestepitopeexposingchimericantigen.Theinsilico geneanalysisandmultiparametergeneoptimizationofthe synthetic chimeric gene was performed using OPTIMIZER server.Itasserandphyresoftwarewereusedinorderto esti-matetertiarystructureprediction.Thechimericproteinswere analyzedforB-cellepitopesusingBcepredserver.Thegene encodingproteinwassynthesizedbyShineGeneMolecular Biotech,Inc.(Shanghai,China).
Expressionandpurificationofchimericprotein
Thesyntheticgene(cssA-cssB-ltB)subclonedintopET28awas transformedtoE.coliBL21(DE3)(Novagen).Thetransformants were grown inLuriaBertani (LB)brothsupplementedwith 30gofkanamycin/ml. Expressionofthe chimeric protein wasinducedatOD600of0.5byadditionof1mMisopropyl-
-d-galactopyranoside(IPTG)andanalyzedbysodiumdodecyl sulfatepolyacrylamidegelelectrophoresis(SDS-PAGE).Batch purification ofHis-taggedproteinsfromE. coliunder dena-turingconditions(Qiagen)wascarriedoutandvalidatedby SDS-PAGE.Thedenaturant(8Murea)wasremovedbystepwise dialysis.
Westernblotanalysis
TheproteinsseparatedbySDS-PAGEwereblottedonto nitro-cellulose membrane using transfer buffer (39mM glycine, 48mMTris–base,0.037%SDS,and20%methanol).The mem-branewasincubatedin10mlblockingbuffer(5%skimmilk in PBST [137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4 and
0.05%(v/v)Tween-20])at4◦Covernight.Themembranewas then incubatedina1:10,000dilutionofmiceanti-His-tag -conjugatedHRPandanti-CTX(Sigma)inthePBST,withmild shaking for 1h at 37◦C. Detection was carried out using HRPstainingsolutioncontaining3,3-diaminobenzidine(DAB) (Sigma)andH2O2.
Animalimmunization
Five-weekoldfemaleBALB/CmiceandNewZealandWhite female rabbitsprocuredfrom PasteurInstituteofIranwere randomlydividedintotestandcontrolgroups.Animalswere immunized four times. Each mouse received 20g recom-binant CS6 proteinalong withcomplete Freund’s adjuvant (SIGMA)subcutaneously.Boosterdosesof15and10g recom-binant proteins with incomplete Freund’s adjuvant were injectedafter20and35days,respectively.PBSservedas con-trol inparallel to the aforementioned procedure. The sera takenfromanimalbloodafterthesecondandthirdinjections werestoredat−70◦Cforfurtheranalyses.
DeterminationofserumIgGresponsestorecombinant proteins
IndirectELISAwasusedforantibodytitration.A96-wellELISA plates (Nunc)were coatedwith5gofCssA,CssBand LTB proteinsincoatingbuffer(64mMNa2CO3,136mMNaHCO3,
pH9.8)at4◦Covernight.Theplateswerewashedthreetimes withPBSTandthenon-specificsiteswereblockedwithskim
milksolution5%(w/v)inPBST.Theserumsamplesservingas primaryantibodieswereseriallydilutedto1:500inPBSTand addedtotheELISAplateswithincubationat37◦Cfor45min. TheplateswerewashedthriceinPBSTandGoatAnti-Mouse IgGAntibody(12,000inPBST)wasaddedtotheELISAplatesas thesecondaryantibody.Theplateswereincubatedfor30min at37◦Candwerethenwashed.Thewellsaddedwith100lof citratebuffercontaining0.06%(W/V)ofO-phenylenediamine dihydrochloride(OPD)(SIGMA)and0.06%(V/V)hydrogen per-oxidewere incubated atroom temperaturefor15min.The reactionwasstoppedwith100lof2MH2SO4andtheOD492
wasreadonamicroplatereader(Bio-Rad).
ETECbindinginhibitionassay
Caco-2cells(104/well)wereaddedandgrowntoconfluencein
96-wellplateswithDMEMsupplementedwith10%(v/v)FBS at37◦Cinacultureflask.ETECstrainwasgrowninLBbroth. Thebacterialsuspension(105/well)waspretreatedwith40l
ofimmunizedmiceantiserafor15minatroomtemperature. ThebacterialmixturewasaddedtoCaco-2cellsandincubated for2hatroomtemperature.Thesupernatantwasdecanted, andthecellswerewashedwithPBS.Afterdigestion,the sus-pendedbacteriawereseriallydilutedandincubatedinLBagar at37◦Cfor16h.Subsequently,thenumberofbacteriaadhered totheCaco-2cellswascounted.Theseraofnon-immunized micewereusedascontrol.
Anti-LTantibodyneutralization
TheproductionofLTtoxin neutralizingantibodieswas per-formedwith rabbitileal loop,cAMP immunoassayand Y-1 cellroundingassay.Therabbitileallooptestwascarriedout accordingtothemethodsdescribedbyDeandChatterjee.18 NewZealandWhiterabbits(1.5kg)werefastedfor24hbefore surgery. Therabbits were anesthetized bythe intramuscu-larinjectionoffentanyl (SIGMA)(0.3ml/kgofbody weight) andzolazepamchloridrate(0.4ml/kgofbodyweight).10-cm ligated ileal loops were constructed. ETEC at 1×108
con-centrationwereincubatedwithmouseserumraisedagainst the chimeric protein, for 30min. ETEC bacteria incubated withnon-immunemouseserumservedasnegativecontrol. Theabove bacterial mixtureand PBS were inoculated into each ligated loop using a 25-gauge needle. After 18h, the
Table1–ThepredictedB-cellepitopesfromfull-length chimericprotein.
Epitopes Aminoacidpositions Scores IVPENVSQVTVYPVYDEDYG 11 1 NYTSGDKEIPPGIYNDQVMV 76 0.992 LVNVNNPDQNMNYYIRKDSG 195 0.98 FYSNEAAAKEAAAKEAAAKE 263 0.975 DDSQSIIYQIVDDKGKKMLK 39 0.956 TFKSGATFQVEVPGSQHIDS 327 0.94 SVKENTSYTFSAIYTGGEYP 227 0.921 KSLDVNVNIEQNFIPDIDSA 126 0.894 RIIPVNYDSDPKLNSQLYTV 147 0.845 TELCSEYRNTQIYTINDKIL 292 0.825 ERMKDTLRITYLTETKIDKL 352 0.802 CssA
EAAAK linker EAAAK linker
CssB LTB
A
B
C
Fig.1–Designandmodeledtertiarystructureofchimeric
protein:(A)schematicconstructionofrecombinantchimeric
proteincontainingCssB,CssAandLTBboundtogetherby
appropriatelinkers,(B)aminoacidsequencesofchimeric
proteinand(C)modeledtertiarystructureofchimeric
proteinbyI-TASSERsoftware.Thismodelshowedaprotein
withthreemaindomainslinkedtogetherwithlinkers.
animals weresacrificed withintravenous3%pentobarbital, and zolazepam chloridrate (0.4ml/kg). For each ileal loop, thevolumeofsecretionwasmeasuredafterexcision. Experi-mentalinfectionoftherabbitswasperformedattheFaculty ofVeterinary Medicine,UniversityofTehran.Researchwas conductedincompliancewiththeAnimalWelfare Actand regulations related to experiments involving animals. The productionofantibodies thatneutralizedLTtoxinwasalso performedwithacAMPimmunoassaykit(Abnova)andCHO cells.CHOcellswereseededin24-wellsterilecultureplates atadensityof5×104cellsperwellandweregrownovernight
toapproximately80%confluence.0.1gofLTwasincubated with50lmiceserumatroomtemperaturefor1h,andthe mixturewasaddedtoeachwellforincubationat37◦Cin5% CO2for2hAfterwashes,thecellswerelysedwith0.1MHCl
and thesupernatantwasusedincAMPassayfollowingthe manufacturer’sprotocol.
Forcell roundingassay,Y-1mouseadrenalcells(Pasteur InstituteofIran)wereseededincellcultureplatesata con-centrationof5×104 cellsperwellandgrowntoconfluence
at37◦C/5% CO2 atmosphere. Y-1 cells were mixedwithLT
Fig.2–Expressionandpurificationofrecombinantchimericprotein:(A)SDS-PAGEanalysisoftherCS6expressionafter IPTGinduction.Lane1:proteinweightmarker.Lanes2,4:noninducedcells.Lane3:theinducedbacteria.(B)Purificationof rCS6proteinbyNi-NTAcolumn.Lane1:proteinweightmarker.Lane2:expressionofchimericprotein.Lane3:
flow-through.Lane4:columnwashedwithbufferD(pH=5.9).Lane5:columnwashedwithelutionbuffer(pH=4.5).Lane6: columnwashedwithmesBuffer.
incubatorat37◦Cfor10h.Themorphologicalchangeswere observedunderthecompoundmicroscope.
Statisticalanalysis
AllstatisticalanalyseswereconductedusingaSPSS12.0 sta-tistical program. Student’s t-test was used to analyze the dataforantibodyresponsesbetween immunizedand non-immunized groups; t-test was also used to evaluate the significanceofdifferences ininhibition ofETECbinding to Caco-2cells generatedbytestsera.A valueofp<0.05was consideredstatisticallysignificant.
Results
Codonoptimizationandtertiarystructureprediction
A chimeric trivalent protein fused together by (EAAAK)4 hydrophobiclinkersinordertofindthebestepitope expos-ing chimeric antigen (Fig. 1A and B). Codon optimization wasperformed toimprove the transcription efficiency and transcriptstability. Percentageofcodonhavingafrequency distributionof91–100inthe nativechimeric CS6genewas 66%,which was significantlyimproved to91% inthe opti-mized gene sequence. The overall GC content, which is a measure of transcriptional and translational efficiency, wasimprovedfrom38.96%to48.75%uponcodon optimiza-tion. There were five negative cis elements in the native CS6 gene sequence, which were removed after optimiza-tion.
Abinitiomodelingofthesyntheticsequencewasexploited toproduce3Dmodelsofthechimericprotein.Theresultof tertiarystructureofthechimericproteinconstructionusing I-TASSERshowedaproteinwiththreemaindomainslinked
together withlinker (Fig. 1C). B-cell epitopewas predicted and the epitopes having cutoff values >0.8 were selected (Table1).
Expressionandpurificationofrecombinantprotein
Thegenesweresubclonedinto pET28aandpET32avectors respectivelyandoverexpressedinE.coli(BL21DE3)(Fig.2A). The SDS-PAGE analysis showed the presence of a 47 KD recombinant chimericprotein.Thepurification of recombi-nantproteinswascarriedoutunderdenaturationcondition byNi-NTAaffinity(Fig.2B).Theexpressionofthe recombi-nant proteinwasconfirmedbythedetectionoftheprotein byWesternblotusinganti-His-tagand antiCTXantibodies (Fig.3).
Responsetoimmunizationwithrecombinantprotein
Animalswereimmunizedwiththerecombinantchimeric pro-tein. All immunizedanimalshad anti-CssA,anti-CssB, and anti-LTIgGantibodiesdetectedinserum(Fig.4).
InhibitionofETECbindingtoCaco-2cells
Assessment of Caco-2 cell monolayers revealed that ETEC cellspretreatedwithserumfromnon-immunizedmicewere distributed on the Caco-2 cells. However, pretreatment of the ETECcells with immunizedmiceantiseraconsiderably blockedtheirbindingtoCaco-2cells.Theimmuneseracould inhibit68±3.5%bacterialadhesionontothetargetcells. Com-parison ofpretreatment of the bacteria with non-immune antiserawiththatofimmunizedmiceserum,showeda con-siderablereductionintheiradhesiontoCaco-2cellsfollowed bypretreatmentoftheETECcellswithimmunizedmice anti-sera.
Fig.3–WesternblotanalysisrCS6:(A)usinganti
6X-His-tagantibodies.Lane1:proteinweightmarker.Lane 2:rCS6and(B)usinganti-CTXB.Lane1:proteinweight marker,Lane2:rCS6,Lane3:antibody.Negativecontrol (bacteriallysatewithoutIPTGinduction).
5 4 3 2 4.5 3.5 2.5 1.5 0.5 0 LTB Ser
um lgG antibody titers (log 10)
CssB CssA 1
Fig.4–Serumanti-LTB,-CssB,and-CssAantibodytitersof miceimmunizedwithchimericprotein.Titerscalculatedas log10oflastreciprocaldilutionabovecut-off.Errorbars representmean±SEM(n=3).
250 200 150 100 50 0 Immunised serum + LT
Cyclic AMP comcentr
ation (pmole/m1)
Non immunised serum + LT
Fig.5–Anti-LTantibodiesneutralizationassaywithcAMP immunoassay.Serumsamplesfromimmunizedorfrom thecontrolmiceweretestedtoneutralizeLTtoxin.Cyclic AMPconcentrationsoftreatedCHOcellsweremeasured usingacAMPkit.Meanvaluesareshown,anderrorbars representstandarddeviations.
Anti-LTtoxinantibodyneutralizationassay
For each ileal loop, the volume of secretion measured after excision was as follows: ETEC as a positive con-trol: 2.59±0.3g/cm, ETEC+ antisera: 0.51±0.01g/cm, PBS: 0.31±0.12g/cm. The fluid accumulation was not observed in rabbit ileal loops 18h post-infection with ETEC treated withtheimmunizedmiceserum.Incontrast,infectionwith standardETECwasaccompaniedbyanincreaseoffluid secre-tion. Data from the cAMP immunoassay kit showed that antibodies in serum and fecal samples of the immunized miceneutralizedLTandpreventedthetoxinfromstimulating intracellularcyclicAMPlevelsinCHOcells(Fig.5).No mor-phologicalchangeswereobservedinY1cellsincubatedwith LTpretreated withanti-chimericantibody, whereasnormal serumhadnoinhibitoryeffectsonrounding(Fig.6).
Discussion
AvaccineagainstETECcouldhaveasignificantimpacton mor-bidityandmortalitycausedbydiarrhealdiseasesinchildren livingindevelopingcountries.19 ETECvaccinedevelopment hasbeenbasedontwostrategiesofblockingadherenceand/or toxinactivity.5Atrivalentrecombinantproteinwasdesigned consistingofCssB,CssA,LTBsubunits.
TheproductionofchimerasconsistingofETECadhesion and enterotoxinBsubunitmay becapableofelicitingboth antitoxinandanti-adhesionimmuneresponses.16Chimeric methodreducestheneedforproducingthreeseparate anti-gens,whichwouldmakeitmorecost-effectiveandwouldease themanufacturingprocess.
InthepresentstudyLTBreportedtobeexpressedbyover half ofLTexpressingETECisolateswas used,thepresence
Fig.6–Inhibitoryeffectofanti-L2C3antibodyontheroundingofY-1cells.Y-1cellswereco-culturedwithLTpretreated withserumofimmunizedmice(A)ornormalserum(B).
ofantibodyagainstLTBcan blockthe bindingofthe toxin toepithelialcells.14,20LTBshowsadjuvanteffectthatelicited strong humoral and cellularimmune response againstthe vaccine components.15,21 On the other hand, although ST antigensareprevalentinETECisolates,theycannotbeused as vaccine components for their poor immunogenicity.11 CssB have been reported as a key factor for binding to hostcells7 andahundredaminoacidsfromtheC-terminal regionofCssAhavebeenreportedtointeractwiththehost cell fibronectin.4 We combined three fragments with link-ers (EAAAK)4. Chimeric proteins with a helical linker are thoughttohavea moreelongatedconformation compared to those with a flexible linker. (EAAAK)4 sequences were introduced between different domains for more flexibility andefficientseparation.Thesuccessfulexperienceofusing (EAAAK)4sequencesinchimericgenehaspavedthewayfor ittobringaboutrationallyacceptableresults.17The recombi-nantproteinwasconfirmedbywesternblotusinganti-His-tag -conjugatedHRP andanti-CTXB antiserumasLTB is struc-turally very similar to B subunit of cholera toxin (CTXB). ThepurifiedrCS6 wasused forimmunizationofmiceand rabbit.Inordertoachieveanoptimalinductionand power-fulresponse,weappliedboosterdosesatleastthreeweeks afterthefirstinjection.Thedosesweregraduallydecreased toincreaseaffinityand tofully activatetheB-cellimmune cascade. The ELISA test results indicated that the immu-nizedanimalsproducedhighertiterofanti-chimerantibody thaneachindividualgene(toeachindividualgenealone)and immunizationwithchimericrCs6stronglyprotectedanimals fromETECchallenges.Accordinglyasignificantdifferencein antibodytiterwas observedbetweenseraoftheimmunize animalsandcontrolgroups.Thefindingfurtherindicatesthat B-cellepitopesofthischimeric proteinmightbelocatedin threedistinctparts,selectedastheCssA,CssB,andLTB.
Theadhesioninhibitioncapacityoftheantibodiesraised againsttherecombinantchimerproteinassayedwithCaco-2 cellsrevealedthatpretreatmentofETECwithimmunizedmice antiseradecreasedtheiradhesionpropertiesandblockedtheir bindingtoCaco-2cells.Attachmenttoepithelialcellculture ofETECwaseffectivelyreducedinimmunizedmicecompared tothecontrol.Thisisconsistentwiththereportsindicating
thatCssA,CssBwerenecessaryforattachmenttointestinal cells.4,7
Theability ofthe antibodiesagainst rCS6to blockfluid accumulationwastestedinrabbitintestinalloops.Theresults indicatethatrCS6antibodyiscapableofbindingtothetoxin and neutralizingits effect.Vaccination witharecombinant chimerictrivalentimmunogencouldprotectthehostagainst ETEC.PretreatmentoftheETECcellswithimmunizedmice antiseraremarkablydecreasedtheiradhesionpropertiesand blockedtheirbindingtoCaco-2cells.
Sincemucosalimmunity,especiallytheproductionofIgA antibodies,isthoughttobeimportantforblockingthe attach-ment ofE.colitoepithelial cells,the latteraspect,suchas theapplicationofthechimericrecombinantproteininoral administration,shouldbeconsideredinfurtherimmunization studies.However,ithasbeendemonstratedwithCitrobacter rodentiumthatsecretoryIgAisnotnecessaryforpreventing bacterial colonization in mice using intimin as a vaccine antigen.22
Inconclusion,ourchimericproteinnotonlyinduced anti-toxinsneutralizingantibodies,butalsoinducedanti-adhesion antibodies forprotecting againstETEC. Thefindings are in supportofitsuse asanimmunogenproteintowardtaking advantage of its components in vaccine strategies against ETEC.
Conflicts
of
interest
Theauthorsdeclarenoconflictofinterests.
Acknowledgments
TheauthorswishtothankShahedUniversityforthesanction ofgrantstoconductthepresentstudy.
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s
1.WajimaT,SabuiS,FukumotoM,etal.Enterotoxigenic EscherichiacoliCS6geneproductsandtheirrolesinCS6
structuralproteinassemblyandcellularadherence.Microb Pathog.2011;51:243–9.
2. ByrdW,deLorimierA,ZhengZR,CasselsFJ. Microencapsulatedsubunitvaccineapproachto
enterotoxigenicEscherichiacoliandothermucosalpathogens. AdvDrugDelivRev.2005;57:1362–80.
3. YuJ,CasselsF,Scharton-KerstenT,etal.Transcutaneous immunizationusingcolonizationfactorandheat-labile enterotoxininducescorrelatesofprotectiveimmunityfor enterotoxigenicEscherichiacoli.InfectImmun.
2002;70:1056–68.
4. GhosalA,BhowmickR,BanerjeeR,etal.Characterizationand studiesofthecellularinteractionofnativecolonizationfactor CS6purifiedfromaclinicalisolateofenterotoxigenic Escherichiacoli.InfectImmun.2009;77:2125–35.
5. LapaJA,SincockSA,AnanthakrishnanM,etal.Randomized clinicaltrialassessingthesafetyandimmunogenicityoforal microencapsulatedenterotoxigenicEscherichiacolisurface antigen6withorwithoutheat-labileenterotoxinwith mutationR192G.ClinVaccImmunol.2008;15: 1222.
6. WalkerRI,SteeleD,AguadoT.AdHocE.Analysisofstrategies tosuccessfullyvaccinateinfantsindevelopingcountries againstenterotoxigenicE.coli(ETEC)disease.Vaccine. 2007;25:2545–66.
7. TobiasJ,LebensM,KallgardS,NicklassonM,Svennerholm AM.RoleofdifferentgenesintheCS6operonforsurface expressionofenterotoxigenicEscherichiacolicolonization factorCS6.Vaccine.2008;26:5373–80.
8. TobiasJ,SvennerholmAM,CarlinNIA,LebensM,HolmgrenJ. Constructionofanon-toxigenicEscherichiacolioralvaccine strainexpressinglargeamountsofCS6andinducingstrong intestinalandserumanti-CS6antibodyresponsesinmice. Vaccine.2011;29:8863–9.
9. WolfMK,HaanLAM,CasselsFJ,etal.TheCS6colonization factorofhumanenterotoxigenicEscherichiacolicontainstwo heterologousmajorsubunits.FEMSMicrobiolLett.
1997;148:35–42.
10.JanssonL,TobiasJ,JarefjällC,LebensM,SvennerholmAM, TenebergS.Sulfatiderecognitionbycolonizationfactor antigenCS6fromenterotoxigenicEscherichiacoli.PloSone. 2009;4:e4487.
11.NicklassonM.Studiesontheexpressionandregulationof enterotoxinsandcolonizationfactorsinEnterotoxigenic Escherichiacoli(ETEC):InstofBiomedicine.Dept.ofMedical MicrobiologyandImmunology;2008.
12.HardyS,HolmgrenJ,JohanssonS,SanchezJ,HirstTR. Coordinatedassemblyofmultisubunitproteins:
oligomerizationofbacterialenterotoxinsinvivoandinvitro. ProcNatlAcadSciUSA.1988;85:7109.
13.SvennerholmAM,SteeleD.Progressinentericvaccine development.BestPractResClinGastroenterol. 2004;18:421–45.
14.SalimianJ,SalmanianA,KhalesiR,MohseniM,MoazzeniS. AntibodyagainstrecombinantheatlabileenterotoxinB subunit(rLTB)couldblockLTbindingtogangliosideM1 receptor.IranJMicrobiol.2010;2:120.
15.MillarDG,HirstTR,SniderDP.Escherichiacoliheat-labile enterotoxinBsubunitisamorepotentmucosaladjuvant thanitscloselyrelatedhomologue,theBsubunitofcholera toxin.InfectImmun.2001;69:3476.
16.SvennerholmAM,TobiasJ.Vaccinesagainstenterotoxigenic Escherichiacoli.ExpertRevVacc.2008;7:795–804.
17.NazarianS,MousaviGargariSL,RasooliI,AmaniJ,BagheriS, AlerasolM.Aninsilicochimericmultisubunitvaccine targetingvirulencefactorsofenterotoxigenicEscherichiacoli (ETEC)withitsbacterialinbuiltadjuvant.JMicrobiolMethods. 2012;90:36–45.
18.DeSN,ChatterjeD.Anexperimentalstudyofthemechanism ofactionofVibriocholeraeontheintestinalmucous
membrane.JPatholBacteriol.1953;66:559–62.
19.ByrdW,MogSR,CasselsFJ.Pathogenicityandimmune responsemeasuredinmicefollowingintranasalchallenge withenterotoxigenicEscherichiacolistrainsH10407andB7A. InfectImmun.2003;71:13–21.
20.MudrakB,KuehnMJ.Heat-labileenterotoxinbeyondGM1 binding.Toxins.2010;2:1445–70.
21.daHoraVP,Conceic¸ãoFR,DellagostinOA,DoolanDL. Non-toxicderivativesofLTaspotentadjuvants.Vaccine. 2011;29:1538–44.
22.Ghaem-MaghamiM,SimmonsCP,etal.Intimin-specific immuneresponsespreventbacterialcolonizationbythe attaching-effacingpathogenCitrobacterrodentium.Infect Immun.2001;69:5597–605.
