Evaluation of a synthetic peptide from the Taenia saginata 18 kDa surface/secreted oncospheral adhesion protein for serological diagnosis of bovine cysticercosis

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ContentslistsavailableatScienceDirect

Acta

Tropica

j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / a c t a t r o p i c a

Evaluation

of

a

synthetic

peptide

from

the

Taenia

saginata

18 kDa

surface/secreted

oncospheral

adhesion

protein

for

serological

diagnosis

of

bovine

cysticercosis

Rafaella

Paola

Meneguete

Guimarães-Peixoto

a,∗

_,

_Paulo

_Sérgio

_Arruda

_Pinto

a

_,

Marcus

Rebouc¸

as

Santos

b

_,

_Marcelo

_Depólo

_Polêto

c

_,

_Letícia

_Ferreira

_Silva

a

_,

Abelardo

Silva-Júnior

b

a_Universidade_Federal_de_Viç_osa,_Departamento_de_{Veterinária,}_Laboratório_de_Inspeç_ão_de_Produtos_de_Origem_Animal,_Campus_{Universitário,}_Viç_osa, MinasGerais,Brazil

b_Universidade_Federal_de_Vic¸_osa,_Departamento_de_{Veterinária,}_Laboratório_de_Virologia_Animal,_Campus_{Universitário,}_Vic¸_osa,_Minas_Gerais,_Brazil c_Universidade_Federal_do_Rio_Grande_do_Sul,_Centro_de_{Biotecnologia,}_Porto_Alegre,_Rio_Grande_do_Sul,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received23March2016

Receivedinrevisedform7October2016 Accepted14October2016

Availableonline17October2016 Keywords: Taeniasaginata Bovinecysticercosis ELISA Oncosphericprotein Syntheticpeptides

a

b

s

t

r

a

c

t

BovinecysticercosisisazoonoticinfectionwidelyspreadthroughoutBrazil,creatingaburdenonhygiene maintenanceandtheeconomy.Diagnosisofcysticercosisusuallyreliesonpostmorteminspectionof car-cassesinslaughterhouses.Thisdetectionmethodprovidesonlylowsensitivity.Recentadvancements haveimprovedtheperformanceofserologictests,suchasELISA,providinggreatersensitivityand speci-ficity.Theobjectiveofthecurrentstudywastoidentifyandevaluateasyntheticpeptidederivedfrom theTaeniasaginata18kDaoncosphericsurfaceproteinforthediagnosisofbovinecysticercosisinELISA. TestperformanceoftheidentifiedpeptidewascomparedtoanELISAbasedonaheterologouscrude Taeniacrassicepsantigen(Tcra),widelyusedforthesero-diagnosisofbovinecysticercosis.Basedonthe primarysequenceofaninsilicostructuralmodelofthe18kDaprotein,anepitoperegiondesignated EP1wasselected(46-WDTKDMAGYGVKKIEV-61).Thepeptidederivedfromthisregionyielded91.6% (CI=80–96%)sensitivityand90%(CI=82–95%)specificitywhenusedinanELISA,whereasthecrude antigenyielded70%(CI=56–8%)sensitivityand82%(CI=73–89%)specificity.Thus,weconcludethatEP1 hashigherdiagnosticpotentialfordetectingbovinecysticercosisthanthecrudeantigenTcra.

1. Introduction

Bovinecysticercosisisaparasiticinfection.It’scausedbythe larvalstageofthehumanintestinalparasiteTaeniasaginata.Itis ratherafoodhygienicandeconomicproblemthanahealth prob-lemwithsevereimpact,andiswidelyspreadthroughoutBrazil, causingconcerninmeatpackingdepotsaswellasamongrural live-stockproducers.InBrazil,thereportedaverageprevalenceis1.05%, whichvariesamongFederalStates(Dutraetal.,2012).

Usually, bovine cysticercosis is diagnosed during a routine postmortemexaminationofcarcassesatofﬁcialmeatinspection in slaughterhouses. The examination consists of standard cuts throughthemuscletissue forthedetectionofcysticerci(Brasil, 1971).However,someinfectedcarcassesaremissedduringthis

∗ Correspondingauthor.

E-mailaddress:rafaella.peixoto@ufv.br(R.P.M.Guimarães-Peixoto).

procedure,asdemonstratedbyaprobabilisticmodeldevelopedby

Kyvsgaardetal.(1990),whichshowedthatover85%ofinfected animalsmaybemissedduringroutinemeatinspection.Thelow sensitivity of routine post-mortem examination is reported by several authors (Dorny et al., 2000; Eichenbergeret al., 2013; Guimarães-Peixotoetal.,2015),andwiththatcomesnew emerg-ingalternativesforthediagnosisofcysticercosis,asanexample,

Eichenbergeretal.(2011)thatsuggestedtheuseofanauxiliary modelbyadditionalcutsinthemuscle;Wanzalaetal.(2003), rec-ommendincreasingtheareaandnumberoflocationsinspected; andcurrently,ithasbeenemployedtheuseofauxiliary serolog-icaltestssuchasELISAidentifyingthediseaseintheanimalwith greaterprecision(Dornyetal.,2000;Pintoetal.,2006;Monteiro etal.,2004;Allepuzetal.,2012;Guimarães-Peixotoetal.,2015).It isnoteworthythat,inaretrospectivestudyofdatafromtheFederal InspectionServiceofBrazil,Souzaetal.(2007)foundthat94%of carcassesparasitizedbycysticerciismonocysticercosis(onlyone cysticercusinroutineinspectionsites).

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TheTaeniacrassicepsantigenhasbeenusedinELISAassaysfor thediagnosisofcysticercosissinceitfeaturesproteinshomologous tothoseofTaeniasaginata.AnotheradvantageofusingtheTaenia crassicepsantigenistheeaseofhandlingofcysticerciinthe labo-ratoryandthatstandardizedreproducibleresultscanbeobtained usinglow-costmaterialsforimmunologicalassays(Larraldeetal., 1990).However, theaccuracyof thetest maybecompromised bythelackofspeciesspeciﬁcity.Manyresearchersemphasizethe needtoimplementserologicELISAtestsasauxiliaryroutine inspec-tionmethods. Thiswould require furtherevaluation of various laboratoryprotocolsinordertoincreasethetests’accuracyand efﬁciency(Monteiroetal.,2006;OgunremiandBenjamin,2010; Eichenbergeretal.,2011;Allepuzetal.,2012).

TheindirectELISAtest isnotyetreliable inthedetectionof bovinecysticercosisinnaturallyinfectedanimals(Monteiroetal., 2006;Allepuzetal.,2012)orinchroniccasesofthedisease,due tolowamountofcirculatingantibodiesinbovine.Therefore,new technologiesarebeingimplementedandutilized,including molec-ulartechniques,suchassyntheticpeptidesandpredictionoftheir productioninheterologousexpressionsystems,withthegoalof improvingtheperformanceoftheimmunologicaltest(Ferreretal., 2003,2007;Parkhouseetal.,2008).

Initslarvalform,T.saginataexpressesproteinsthatmediatethe adhesionoftheparasitetohostcells.Theseproteinspresentone ortwoﬁbronectintype-IIIdomains,whichconferadhesionability (Gonzalezetal.,2007).Amongthebindingproteins,the18kDa pro-teinischaracterizedasanimportantadhesin(Bonayetal.,2002) andpotential vaccineimmunogenprotecting fromcysticercosis (Lightowlers etal.,1996).The 18kDaoncosphere protein (HP6-TSAG)isasecretion/excretionantigen(Ferreretal.,2007).Theuse ofTaeniasaginataproteinHP6hasbeenreportedforthediagnosis ofbothanimalandhumancysticercosis(Ferreretal.,2003,2007; Abuseiretal.,2007).

Antigen-speciﬁcantibodiesmaybecapturedfromserum sam-plesviathesubstitutionofshort,syntheticpeptidesintheplaceof theproteinantigenepitopes(AndresenandBier,2009).Innovative computationalapproachesresultsinthepredictionofacceptable tohigh-precisionofmappingofantigenicepitopes,facilitatingthe selection ofpeptides used for immunodiagnostics (Zimic et al., 2011;Wangetal.,2014).Withinthiscontext,theaimofthepresent studywastocharacterizeandevaluatethediagnosticpotentialofa syntheticpeptidederivedfromtheT.saginata18kDaoncopsheric antigeninanELISAforthedetectionofbovinecysticercosis.

2. Materialsandmethods

2.1. Computationalcharacterization

Preliminary,assemblyofstructuralmodelswasperformedfor theTaeniasaginata18kDaproteinusingasequencewasobtained fromGenBank(AccesscodeADO86979.1).Thestructuralmodels weregeneratedbyI-TASSER(Yangetal.,2015)andPhyre2servers (Kelleyetal.,2015)andthetwobestresultsfromeachserverwere comparedin thiswork.Additionally, transmembraneregions of theproteinswerepredictedbyusingtheTMHMMserver(Krogh etal.,2001).PossibleN-andO-glycosylationsitesfromthe pri-maryaminoacidsequencewerepredictedbyusingtheNetNGIyc 1.0(Guptaetal.,2004)andNetOGIyc4.0(Steentoftetal.,2013) servers.

2.2. Selectionandpreparationofantigenicpeptide

Anepitopemapwasassembledbasedonthethree-dimensional modelpredictedfor18kDaproteinanditsprimarystructure.Four methods were utilized: Bepipred® (Larsen et al., 2006), which

usesacombinationofParker’shydrophobicityscales(Parkeretal., 1986), Levitt’s secondary structure (Levitt, 1978), and the hid-denMarcovmodel;ABCpred®,whichutilizesanartiﬁcialneural network(SahaandRaghava,2004);AAPpred®,whichutilizesan aminoacidpairantigenicityscale(Chenetal.,2007);andELIPRO®, whichpredictsdiscontinuousepitopes(conformational)by analyz-ingregionsaccessibletosolventsandﬂexibilityina3Dstructure (Ponomarenko et al., 2008).Ultimately, theresulting data con-vergedinepitopeEP1(46-WDTKDMAGYGVKKIEV-61),whichwas subsequentlysynthesized(Genscript®,USA)with>95%purity.

2.3. Serumsamples

Thesamplesofbovineserumusedinourstudyconstitutedfive distinctgroups, as follows:Group1 (G1, n=60) wascomposed of experimentally infected bovine blood samples, which were previously collectedat differentpost-infectionperiods froman establishmentwithoutahistoryofcysticercosis.Ninemalebovines, livestockofmixedDutch-Zebubreeds,wereinoculatedwitha par-asiticloadof120.000eggsofTaeniasaginata.Thetapewormsample waspreviouslyobtainedbyahumandonor,andtapewormspecies wasconfirmedbydirectmicroscopy.Cysticercosiswasconfirmed inallanimalsbelongingtoG1atmeat-inspectionina slaughter-house.Group2(G2,n=60)consistedofsamplesfromnaturally infectedanimalswhosecysticercosisdiagnosishadbeenmade dur-ingroutinepost-mortemsurveillanceconductedinslaughterhouses supervisedbytheofficialinspectionalservice.Group3(G3,n=60) constitutedofsamplesfromlivestockslaughteredincommercial slaughterhousesthat testednegativefor cysticercosisandother diseasesduring routinepost-mortem inspection.Lastly, Group4 (G4,n=28)consistedofsamplesfromlivestockthattestednegative forcysticercosis,butpresentedwithotherdiseases(tuberculosis (n=8),hydatidosis(n=8),andfasciolosis(n=12)),tocheckpossible cross-reactionwithcysticercosis.

2.4. IndirectELISA

2.4.1. Syntheticpeptide-basedELISA

TheoptimaldilutionsforthereagentsusedintheELISAassays were determined via block titration. The immunoassay plates (Thermo.Nunc,Maxisorp)wereinitialtreatedwithPolypep®2% (SigmaP5163)for10hat25◦C,followedbythreewasheswithPBS, pH7.4.Thepeptidesweredilutedwith0.5M(1␮g/mL) carbonate-bicarbonatebuffer,pH9.4,andincubatedintheplatesovernightat 4◦C.Afterthreewashes(with0.15Msalinesolution,pH7.4, con-taining0.05%Tween20),theplateswereblocked(with1%Molico® denaturedpowderedmilkinPBS)andincubatedfor1hat37◦C.The livestockserumsampleswerediluted1:25intheblockingsolution with0,05%Tween20,for1hat37◦C.Followingthreetimes wash-ing,anrabbitanti-bovineIgGantibodyconjugatedtoperoxidase (A5295,SigmaChemicalCo.,StLouis,MO,USA)wasaddedtothe platesata1:1.250dilution,followedbyincubationandwashing stepsasdescribedabove.Thespectrophotometricdetectionwas initiatedviaincubationwithasolutionof0.1%o-phenylenediamine dihydrochloride(OPD)and0.003%H2O2in0.2Mcitrate-phosphate

buffer, pH5.0, for30min37◦C. Thereactionwasstopped with H2SO4 (4N), and the plates were read in a spectrophotometer

(BiotekInstruments)ata492nmwavelength.Allreagentswere addedtotheplatesatavolumeof100_␮L/well,excepttheblocking solution,at200␮L/well.

2.4.2. Taeniacrassicepsantigen-basedELISA

Taeniacrassiceps larvalantigenswereobtainedvia intraperi-tonealinoculationofBALB/cfemalemice,followingthe method-ologyaccordingtoVazetal.(1997).

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After collection, the cysticerci were immediately frozen (−20◦_C)._Posteriorly,_the_cysticerci_were_lyophilized_at₂₅◦_C_and

afterthat,homogenizerandmixedwitha0.15Msalinesolution, resultinginaﬁnalconcentrationof6.5–10%.Thedilutedcysticerci werethenhomogenizedoniceusingatissuehomogenizer (Pot-ter)andthencentrifugedat17.400gfor30minat4◦C.Aprotease inhibitor(PMSF, SigmaChemicalCo, St.Louis,MO, USA,P7626, 0.25M–10␮L/mL)wasaddedtothesupernatantandtheantigen wasstored(₋₂₀◦C)untilused.

Thepolystyreneplatesweresensitizedwithdilutedantigens (40␮g/mL)ina0.5Mbufferedsolutionofcarbonate-bicarbonate, pH9.6.The ELISAplatewascoatedfor1hat 37◦C. Afterthree washesinsalinesolutioncontaining0.05%Tween20,thereactive siteswereblocked(using5%denaturedmilkinPBS,pH7.4)for1h at37◦C.Afteradditionalthreewashes,thesampleswerediluted in1%denaturedmilkinPBS,pH7.4.Theplateswerethen incu-batedfor30minat37◦C. Afterwashing,peroxidase-conjugated anti-IgGantibody(A5295,SigmaChemicalCo.,StLouis,MO,USA) (1:5000)wasadded toeach well,andtheincubationand wash procedures wererepeated.The chemiluminescent reactionwas initiatedusingasolutionof0.1%o-Phenylenediamine dihydrochlo-ride(OPD)P8287(SigmaChemical Co.;StLouis,MO, USA) and 0.003%H2O2in0.2Mcitrate-phosphatebuffer,pH5.0.Aftera 5-min-incubation,thereactionwasstoppedusing4N H2SO4,and theplateswerereadinaspectrophotometeratawavelengthof 492nm.Allreagentsappliedtotheplateswereappliedatavolume of100␮L/well,withtheexceptionofthe200␮Lblockingsolution. 2.5. Determinationoftestperformance

Thereactivityofthepeptideswasevaluatedbasedontheresults obtainedfromtheELISAtestsEachELISAreactionwasperformed intriplicateandthemeanopticaldensities(OD)werecalculated. TheODvalueswerenormalizedtoastandardreferenceplate,and thecorrectionfactor wascalculatedaccordingtoPassos(1993). Thecut-off pointwasselectedbasedonthemeanODobtained fromanalysesof ﬁve negativeserum-control samples collected fromlivestockraisedinisolation,keptundercontrolledconditions andslaughteredunderrigorousinspection,includingtwostandard deviations.

To determinetheperformance of the ELISA test, wereused serum-controlgroupsdistributedasfollows:analysisofantibody kinetics (G1) to evaluate the diagnosis performance; to calcu-late thesensitivity (G2), specificity (G3-G4) considering allthe negativesamplesforcysticercosisafterinspectionandpotential cross-reactions.Thetests’sensitivity,specificity,positiveand neg-ativepredictivevaluesandaccuracywerecalculatedbasedonthe recommendationsofAltman,Bland(1994)andAkobeng(2006).To testtheperformanceratesitwastakenintoaccountaconfidence intervalof(CI)95%.

2.6. Ethicscommitteeapproval

Thenormsofconductfortheuseofanimalsinresearchfromthe EthicsCommitteeforAnimalExperimentationoftheFederal Uni-versityofVic¸osawerefollowedaccordingtotheProcessreference 20/2011CEUA/UFV.

3. Results

Structuralmodelsof 18kDa proteinweregenerated using I-TASSERandPhyre2serversthroughcomparativemodeling,fold recognitionandabinitioalgorithms.Amongtheresultingmodels, theoneswithhigherscoresfromeachserverwereselectedformore in-depthcomparisons.Interestingly,thestructureswiththe high-estscoreswereobtainedforanproteinmodelusingaﬁbronectin

Fig.1. Three-dimensionalmodelofthe18kDaprotein.(a)Overlappingof pre-dictedmodelsbyI-TASSER(inblue)andPhyre2(ingreen).Intranslucentsilver, theN-terminalstructuremodeledbyI-TASSERandnotconsideredinthisstudy. (b)LocalizationofthepredictedepitopeEP1(inred).Inblue,theputativesitesof N-glycosylation(ASN74andASN92)andO-glycosylation(THR116).(For interpre-tationofthereferencestocolourinthisﬁgurelegend,thereaderisreferredtothe webversionofthisarticle.)

type-IIIdomainofhumanneuralcelladhesionmolecule(PDBID 2DOC),leadingtoC-scoreof−1.99inI-TASSERandConﬁdenceof 95.4%inPhyre2.Bothmodelsconvergedtoaverysimilarfold,with arootmeansquare deviation(RMSD)valueof1.167Å(Fig.1a). Duetothesimilaritybetweenbothmodels,allstructuralfeatures of18kDaproteinwerediscussedinthisworkusingthe3Dstructure obtainedbyPhyre2.

Since18kDaproteinisknowntobeamembraneprotein(Bonay etal.,2002),furtheranalyseswerecarriedoutusingtheTMHMM serverinordertopredicttransmembranehelicalregions.Despite thehighlyhydrophobicregion5-FGLILLVAVVVLA-17,theserver didnotpredictsequenceasatransmembranehelixwithhigh prob-abilityofoccurrence(datanotshown).However,region1–17was predictedtocontainasignalpeptidewithameanscoreof94.4%, conﬁrmingthehypothesisofanN-terminaltransmembraneregion in18kDaprotein.Therefore,thepredictedN-terminalstructureof 18kDawasnottakenintoaccountinthisstudy,duetothehigh probabilitythatthisregionformsatransmembranestructure.

In addition, since 18kDa protein is located in the outer membrane and it contains a signal peptide, it is possible that glycosilationpathwayscouldbeinvolved in maturation. There-fore, glycosilation sites predictions were performed using the NetNGlyc1.0andNetOGlyc4.0servers.Bothserverspredicted N-glycosylationsitesatASN74andASN92,andanO-glycosylation siteatTHR116(Fig.1b),withscoresof0.7303,0.5054and0.7021, respectively.

The resultsof thebioinformatics programs usedfor epitope prediction of18kDa proteinmostly convergedin theregionof aminoacidof46-WDTKDMAGYGVKKIEV-61.Thepeptidederived from this region was subsequently named EP1, being used for

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Fig.2. ReactivityofbovineserumsamplesintheELISAtestandcut-off(dottedline).Assaysconductedusing(a)EP1peptideasanantigen(Cutoff0,881);(b)Taeniacrassiceps heterologousantigen(Cutoff0,359).

theposteriorsanalyses.EP1inourmodelwasexposedtoa sol-ventinabeta-hairpinwithoutanypredictedsitesofglycosylation (Fig.1b).Theantibodydiscriminationbythepeptideandits diag-nosticpotentialwasmeasuredbyitsperformanceinELISA.The EP1 antigen was detected in most of the positive serum con-trols(n=112/120)(animalsexperimentallyornaturallyinfected) (Fig. 2), while the majority of negative animal serum samples (n=80/88)didnot reactwiththeEP1 peptide.Therefore, these resultsindicatethatEP1enableddistinctionbetweenpositiveand negativesamplesintheELISAassay.Theperformancerateswere calculated and wereobtainedthe following results:91.6% sen-sitivity(CI=80–96%), 90%speciﬁcity (CI=82–95%), 85% positive predictivevalue(PPV)(CI=75–93%),93%negativepredictivevalue (NPV)(CI=86–97%)and91%accuracy.

4. Discussion

Themajorrecentimprovementsinbiomolecularmodeling tech-niqueshavegreatlyimprovedtheaccuracyoftheoreticalstructural models(Figueiredoet al.,2014).Based onthis background,we generatedstructural modelsofTaenia saginata18kDa oncopsh-ericprotein,usingcomputationalapproaches. Basedonthefold recognitionstepsandabinitiocalculationsusedforthecreation of this model, thefolding convergence and low RMSDindicate thatourmodelislikelyandaccuratestructuralrepresentationof 18kDaprotein.Itisimportanttonotethattheﬁnalmodelswere verysimilartotheonedescribedbyKyngdonetal.(2006)forthe TaeniasoliumTSOL18protein,whichhas61%identitywiththe Tae-niasaginata18kDaproteinandanalogousfunction.Furthermore,

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gly-cosylationsiteinTSOL18,aswasalsopredictedinourmodel,thus furtherincreasingtheconﬁdenceinourmodel.

Withtheaimof improvingdiagnostictestsagainstdifferent pathogens,variousstudieshaveattemptedtomapBlymphocyte epitopesinamannersimilartothatpresentedinthisstudy(Meloen etal.,2003).Themaintechniqueusedtoidentifyconformational epitopesreliesonathreedimensionalstructuralrepresentationof theproteinofinterest,aswellasinferencesregardingregionsor aminoacidresiduesthatmayinteractwithantibodies.Despitethis attractivemethod,themajorityofthestudiesdescribelinearand notconformationalepitopes(Vitaetal.,2010).Themain explana-tionforthisdiscrepancyisthatvariousproteinspresentachallenge inplausibleresolutionoftertiarystructure,becauseofeitherthe highcostandtimerequiredforcrystallography,orincomputational molecularmodeling.Forourresearch,weemployedfourmethods ofepitopeprediction,includingoneconformationalepitope predic-tionmethodandthethreelinearpredictionmethods.Itisimportant toemphasizethatonlylinearepitopeswerepredicted,whichwere basedonconvergentregionspredictedbythesoftwareprograms andregionsnotdescribedinotherstudies.

The immunodominant oncosphere antigens are considered immunetargetsandmayalsohavepreventiveanddiagnostic appli-cations (Ferrer et al., 2003).The 18kDa oncospheric protein is termedHP6(Ferreretal.,2003,2007;Gonzalezet al.,2011)or TSA-18(Lightowlersetal.,1996;Jabbaretal.,2010).

Thefollowingperformanceindicatorswereobtainedfromthe ELISA test using the heterologous antigen (T. crassiceps): 70% sensitivity (CI=56–80%), 82% speciﬁcity (CI=73–89%), 72% PPV (CI=60–84%),80%NPV (CI=70–87%),and78%accuracy.Theuse ofEP1inanELISAyieldedslightlyhighervaluesthanthosefrom theheterologousantigen,indicatinganimprovedperformanceof thediagnostictest.WhileEP1wasabletodetect91.6%ofsamples fromthenaturallyinfectedanimals,theTaeniacrassicepscrude het-erologousantigenonlydetected70%ofthesamples.Itisknown thatthereisahomologybetweenthe18kDaproteinofTaenia sagi-nataanddifferentspeciessuchas:Taeniamulticeps–Tm18(85.5%), Taeniasolium–TSOL18(61.5%),Taeniaovis–To18(79.7%),Taenia asiaticaTASI18(95.5%) eTaeniahydatigena.Ainfecc¸ãoem bovi-nossóocorrecomaTaeniamulticeps(Avciogluetal.,2011)eTaenia hydatigena(DadaandBelino,1978),however,itisimportanttonote thattheserumcontrolsamples(G1,G2,G3,G4)areobtainedfrom animalsthathaveundergonepost-morteminspectionanddiscard apossibleco-infectionwiththeseparasites.

Ferreretal.(2003)synthesizedsixpeptidesderivedfromTaenia saginataproteins,usingtheantigenicindexofJamesonandWolf (JamesonandWolf,1988).Thetwopeptides(HP6-2andHP6-3) derivedfrom18kDaproteinwereabletodifferentiateserumsfrom cysticercosis-positiveand-negativeanimalsinasmallersample size.Itisimportanttostate,incontrasttoEP1,whichislocatedin anexposedbeta-harpin,HP6-2ismostlyburiedinourmodel,while HP6-3ismainlylocatedintheproteinC-terminalregionand, there-fore,exposedtointeractions(datanotshown).Thisdifferencein locationmightrevealdifferencesintheperformanceofdiagnostic testsusingthedistinctpeptides;agreaterexposureofthe pep-tideintheproteinstructureincreasestheprobabilityofinteraction withantibodies.Immunodominantpeptideshaveadirecteffecton disease,asevidencedbytheirhighafﬁnityforthehostantibodies releasedduringinfection.Thus,amorethoroughunderstandingof thebiologyoftheimmunodominantpeptideswillgreatlyaidthe attemptstoimprovetheperformanceofimmunodiagnostictests (Noyaetal.,2003).

Thus,incomparisontousingtheheterogenicantigen,theuse oftheEP1peptidemayenableimproveddetectionofcysticerciin naturallyinfectedanimalsand/orinanimalswith monocysticer-cosis.Animalsnaturallyinfectedhavelowamountsofcirculating antigens,whichnormallyhinderaccuratediagnoses.Accordingto

Allepuzetal.(2012),thecostofimplementingofamoresensitive toolforthediagnosisofbovinecysticercosisshouldbecompared withthe associated beneﬁtsfor humanhealth, given thegreat potentialoflimitingthetransmissioncycleofbovine cysticerco-sis.Theuseofanimproveddiagnostictoolforbovinecysticercosis wouldalsobeneﬁtthelivestock-farmingsectorbyreducing eco-nomicloss.

TheELISAtesthasbeenusedinresearchworldwide(Pintoetal., 2000;Ferreretal.,2003,2007;Monteiroetal.,2006;Allepuzetal., 2012; Guimarães-Peixoto et al., 2015).It shouldbe noted that theELISAshouldbeabletodetectanimalsatdifferentstagesof infection(viableornon-viablecysticerci),andsimilarly,showing satisfactoryperformance(Pintoetal.,2006;Monteiroetal.,2006; Minozzoetal.,2004;Guimarães-Peixotoetal.,2015).According

Smithetal.(1991)lowlevelsinlowcyst-burdenproduced anti-bodieshindertheselectionofacutoffpointandinterpretationof ﬁndingsinserologicaltestssuchasELISA,especiallyin monocys-ticercosis(Thomaz-Soccoletal.,2010).

IntheperformanceofanELISAtest,itisimportanttoconsider theamplitudevalueofthedifferencebetweenthepositiveand neg-ativecontrolsera(Guimarães-Peixotoetal.,2015;Silvaetal.,2015). Thus,theaimofthisresearchwastosetupalaboratoryscenario usingtheopticaldensityvaluesadjustedtoastandardplateto com-paretheperformanceofpeptideEP1withtheantigenTcra,thusthe backgroundarereduced.

5. Conclusion

In thepresent study,acomputationalapproach wasusedto select a peptide withgreat potential for use in serologic diag-nosis of bovinecysticercosis. Theselected EP1 peptideenabled discriminationbetweensamplesfromanimalspositiveornegative forcysticercosis.TheEP1peptidealsoexhibitedimproved perfor-mancecomparedtotheheterologousantigenofTaeniacrassiceps.

Theincreasinguseofcomputationaltoolstoobtainstructural dataofbiomoleculeshassigniﬁcantlyimprovedour understand-ingofantigen-antibodyinteractions.Inthisstudy,thecreationof aconformationalmodelof18kDaproteinprovidedatheoretical frameworkforasuitableandrationalmethodologicaldesignthat enabledtheidentiﬁcationofanewepitopewithpotentialusein thediagnosisofbovinecysticercosis.

Acknowledgements

Theauthorswould liketothankFapemig (ResearchSupport Foundation of Minas Gerais), CAPES (the Coordination for the ImprovementofHigherEducationPersonnel),andCNPq(National CounselofTechnologicalandScientiﬁcDevelopment)forthegiven supportandVivianeAssaoforthegraphicalabstract.

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