Epitope of dengue virus E protein detect human antibodies associated with mild disease: a potential peptide for vaccine development

brazjinfectdis2020;24(1):85–88

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Epitope

of

dengue

virus

E

protein

detect

human

antibodies

associated

with

mild

disease:

a

potential

peptide

for

vaccine

development

Gilma

Sánchez-Burgos

_,

_Carla

_{Herrera-Nájera}

_,

_Roberto

_{Cedillo-Rivera}

_,

Eric

Dumonteil

a_{InstitutoMexicanodelSeguroSocial,UnidadMédicadeAltaEspecialidad,HospitalRegional1“Lic.IgnacioGarcíaTéllez”,Mérida,} Mexico

b_{UniversidadAutónomadeYucatán,CentrodeInvestigacionesRegionales“Dr.HideyoNoguchi”,LaboratoriodeParasitología,Mérida,} Mexico

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Articlehistory:

Received27August2019 Accepted27October2019

Availableonline21November2019

Keywords: Denguevirus Epitopes Peptides Antibodies

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Theantigenicpotentialofsevenimmunogenicpeptidesofthedengueviruswasevaluated intheseraofpatientswithdengueconfirmedbyIgM/IgGserology.AntibodiesIgMandIgG againstdengueviruspeptideswereanalyzedbyELISAin31denguesero-positiveand20 sero-negativepatients.TheP5peptideshowedsignificantIgGimmunoreactivitymostlyin theseraofpatientswithdenguewithoutwarningsignsincomparisonwithpatientswith denguewithwarningsigns,correlatingwithmilddisease.Thisfindingsuggeststhatthe lowantibodyresponseagainstP5epitopecouldbeariskfactorforhighersusceptibility todenguevirusinfectionwithwarningsigns,andthatP5couldbeapotentialantigenfor vaccinedevelopment.

©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

IdentificationoftheB- and T-cellepitopesofdengue virus (DENV)withantigenicpotentialhasbeenimportantin under-standingthepathogenesisofdengueandtodevelopvaccines. Thus,it was observedthat T-cell responsestothe original infectingserotypemightlimitresponsesdirectedagainst

epi-∗ _{Correspondingauthor.}

E-mailaddress:gilmagburgos@gmail.com(G.Sánchez-Burgos).

1 _{Presentaddress:Universidad AutónomadeYucatán,Facultadde Medicina,UnidadInterinstitucional deInvestigaciónClínicay} Epidemiología,Mérida,Mexico.

2 _{Presentaddress:TulaneUniversity,SchoolofPublicHealthandTropicalMedicine,DepartmentofTropicalMedicine,NewOrleans,} USA.

topesthatareeithercross-reactiveorspecificforasecondary infecting serotype.1 _{In addition,the lowmagnitudeofHLA}

restricted responses of CD8+ _{by predicted epitopes}

corre-latedwithdiseasesusceptibility,whereashigher-magnitude responses were associated with more polyfunctional and

https://doi.org/10.1016/j.bjid.2019.10.009

1413-8670/©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Table1–DiagnosisandserologiccharacteristicsofDENVinfectioninthepopulationstudied.

ID Sex Ageyears TestPositive Diagnosis Dayspostonsetoffever OD490nm

1 F 26 IgM+IgG DWWS 3 0.0788 2 M 20 IgM+IgG DWWS 3 0.0900 3 M 20 IgM+IgG DWWS 6 0.0670 4 M 34 IgM+IgG DWWS 7 0.0926 5 M 18 IgM+IgG DWWS 6 0.0673 6 F 19 IgM+IgG DWWS 5 0.0680 7 M 26 IgM+IgG DWWS 5 0.0433 8 M 13 IgM+IgG DWWS 6 0.0948 9 F 16 IgM+IgG DWWS 6 0.0531 10 F 15 IgM+IgG DWWS 7 0.1071 11 M 21 IgM+IgG DWWS 4 0.0838

12 F Notknown IgM+IgG DWOWS 7 0.5737

13 F 50 IgM+IgG DWOWS 12 0.3612

14 M 13 IgM+IgG DWWS 12 0.3735

15 F 14 IgM+IgG DWOWS 6 0.3825

16 F 11 IgM+IgG DWWS 5 0.4125 17 M 30 IgM+IgG DWWS 7 0.2115 18 M 58 IgM+IgG DWWS 0 0.1840 19 F 35 IgG DWOWS 20 0.3040 20 F 57 IgG DWOWS 2 0.2597 21 F 44 IgG DWOWS 4 0.3125 22 F 68 IgG DWOWS 3 0.3535 23 F 46 IgG DWOWS 2 0.4725 24 M 16 IgG DWWS 7 0.2450 25 M 30 IgG DWOWS 4 0.7235 26 F 30 IgG DWWS 5 0.0415 27 M 31 IgG DWWS 7 0.0695 28 F 17 IgG DWWS 4 0.1535 29 F 37 IgG DWOWS 4 0.1001 30 F 30 IgG DWWS 4 0.0726 31 M 25 IgG DWOWS 6 0.0481

DWWS,denguewithwarningsigns;DWOWS,denguewithoutwarningsigns.Cutoffvalue0.3486.

protectiveCD8+ _{T cells.}2 _{A highthroughput study ofT-cell}

responsestoDENV-2peptidesrevealedasignificant associa-tionbetweenthemagnitudeofT-cellactivation,mostlybyNS3 proteinanddengueseverity.3Ontheotherhand,monoclonal antibodiesagainstepitopesofNS1cross-reactwithmolecules expressedbyplateletsorwithendothelialcells,whichcanbe associatedwithplasmaleakageinseveredengue.4–6

WereportedseveralB-andT-cellDENVepitopesidentified byanimmunoinformaticstrategy,sevenhighlyimmunogenic peptidesinducedmostlyantibodies(P5,P13,andP19)and/or CD4+_{Tcells(P15,P18,P20,andP22)inmice.}7 _Fourofthese

peptidesbelongtothe proteinNS5(P18,P19, P20,andP22), whereas P15, P13, and P5 are included in the amino-acid sequencesofNS4b,NS4a,andEproteins,respectively.Here, weanalyzedtheantigenicityofthesepeptidesbythedetection ofanti-DENVantibodiesintheseraofpatientswithdengue.

WeaimedtoidentifytheepitopesinDENVusefulfor evalu-ationofhumoralimmuneresponseagainstthefourserotypes relatedwithmildandsevereclinicalmanifestations,andtheir implicationsforvaccinedevelopment.

Serumsampleswereobtainedfrompatientswithaclinical and laboratory diagnosis of dengue at the Instituto Mexi-canodelSeguroSocialinYucatán,Mexico.Ethicalapproval was obtained from the Ethics Committee of the Institu-tion (Approval Code No. 2010-785-041). As surveillance is mandatory at the Instituto Mexicano del Seguro Social,

there isnorequirement forinformed consent,but all data were maintained in privacy. All sampleswere tested by a Dengue IgG/IgM Lafon® immunochromatographic cassette (Standard Diagnostics, Inc., Korea), with Dengue IgM Cap-tureELISA(E-DEN01M/E-DEN01M05)andDengueIgGCapture ELISA(E-DEN02GPanBio®,Brisbane,Australia).Sero-positive patients were furtherclassified into denguewithout warn-ing signs (DWOWS)or denguewith warningsigns (DWWS) groups according to the World Health Organization (WHO) classification.8 _{To compare the resultsemploying the 1997}

WHODengueCaseClassification,9_{thesero-positivepatients}

werealsogroupedintodenguefever(DF)ordengue hemor-rhagicfever(DHF).

Theimmunoreactivitiesofthereportedsyntheticpeptides P5,P13,P19, P15,P18, P20,andP22wereanalyzedbyELISA todetectIgMandIgGantibodiesintheseraofpatientswith dengueasreported.7_{Alltestswererunintriplicate.}

Statistical significancewas establishedat p<0.05 for all parameters. Median values of optical density (OD) 490nm between groups were compared by the non-parametric Mann–WhitneyUtestutilizingSPSSversion18statistical soft-ware.Estimationofriskorprotectionfactorwasdetermined byoddsratio(OR).

Thirty-one serum samples were confirmed with DENV infectionfrom14maleand17femalepatients,agebetween11 and68yearswithanaverageof28.61years.Thirteenpatients

brazj infect dis.2020;24(1):85–88

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Fig.1–IgGantibodiesdetectedwithP5inpatientswith denguewithoutwarningsigns(DWOWS)anddenguewith warningsigns(DWWS).LevelsofIgGantibodieswere detectedbyELISAwithP5peptideintheseraofpatients withDWOWSandintheseraofpatientswithDWWS. Mann–WhitneyUtestshowedastatisticallysignificant differencebetweenthemeanvaluesofOD490nmofthe groups:sero-negativeandDWOWS(p=0.004),DWOWSand DWWS(p=0.002).

wereonlyIgG-positiveand18wereIgG+IgM-positive(Table1). Therewere10patientsconfirmedwithmilddisease(DWOWS), and21hadseveredengue(DWWS).

NoneofthesevenP5,P13,P19P15,P18,P20,andP22 pep-tidesdetectedIgMantibodiesinseropositivesamples(data notshown).VerylowOD490nmlevelswereobservedbyELISA IgGutilizing P13, P19, P15, P18, P20, and P22. OnlyP5 pep-tidedetectedvariablelevelsofIgGantibodiesinserafrom11 seropositive-patientswithDWOWS(ODaverage, 0.354)and 20 patients withDWWS (OD average,0.130). A statistically significant difference of median OD 490nm values of IgG wasobservedbetweenthesegroups(Mann–WhitneyUtest, p=0.002)(Fig.1).MedianODvaluesamong16patientsunder 25yearsofageand15patientsover25yearswasnot signifi-cantlydifferent(Mann–WhitneyUtest,p=0.252).

The strong association between mild dengue and immunoreactivity of IgG antibodies against P5 was con-firmed by means of OR=0.09 (range, 0.01–0.608). Similar results were obtained when seropositive patients were grouped according to the 1997 WHO Guidelines (data not shown).

Keydifferences intheimmunodominance ofDENV pro-teinsformodulationoftheadaptativeimmuneresponsehave been reported. In a previous study, we reported synthetic peptidesfromDENVepitopesthatinduceddifferentimmune responsesinimmunizedmice.7

TheP18,P19,P20,andP22epitopesoftheNS5protein acti-vatedT CD4+ _{and Bcells inmice,but theywere unableto}

showsimilaractivationinhumans,ascanbeinferredfromour negativeIgGandIgMimmunoreactivityresultsintheseraof patientswithdengue.Incontrast,Rivinoetal.demonstrated thatCD8+ _{cellspreferentially target NS3and NS5epitopes,}

whereas CD4+ _{and Blymphocytes recognize E, C,and NS1}

epitopes.10_{TheimmunogenicpeptideP5}7_{islocatedindomain}

IIIofproteinE,whichhasbeenimplicatedasareceptor bind-ingdomain,andseveralneutralizingmonoclonalantibodies havetheirepitopesinthisdomain.11,12 _{Inmice,P5induced}

moderate antibody levels and more T CD8+ _{cell response,}

suggestinga lessercapacityforB-cellinduction.7 _However,

differentlevelsofIgGantibodiesweredetectedintheserum samplesofseropositivepatientswithdengueemployingthe P5peptideinELISAtests.AstheP5peptideis89%conserve amongthefour DENVserotypes,it wouldbeexpectedthat theP5peptidewouldbeabletoreactsimilarlywithantibodies frompatientsinfectedwithdifferentDENV.However,theP5 peptidehasasingleamino-acidvariationinposition4(R/K) inDENV-2andDENV-4,whichcouldbethereasonfor non-reactivityinsomepatients.

All patients but three with DWWS showed high lev-els of antibodies detected more than three days after the onset of fever, suggesting that P5 could detect antibodies during the early convalescent phase of secondary dengue infections. Furthermore, patients with the mild form of the disease had higher levels ofIgG antibodies up to four and even seven days post-onset of fever, compared to patientswithDWWS, accordingtobothWHOclassification systems.8,9

In addition, a strong association between high levels of anti-P5 antibodies and mild disease presentation was observed(OR=0.09),suggestingthat,insecondaryinfections, lowantibodyresponseagainstP5peptidecould playarole inhighersusceptibilityfordevelopingwarningsigns. There-fore,thiscouldberelevantforthedevelopmentofapotential vaccine. Inthissense, wedemonstratedtheantigenicityof the P5peptide, in addition toits immunogenicity inmice, increase its potentialasavaccine candidate againstDENV infection.

In conclusion, a newantigenic epitopefrom the E pro-tein of DENV previously identified in silico was able to detect IgG antibodies in the sera of humans with mild DENV infection, showing its potential use for studies on denguepathogenesis,diseasediagnosisandvaccine develop-ment.

Funding

ThepresentstudywassupportedbyConsejoNacionalde Cien-ciayTecnología(grantnumberSALUD-2007-01-68909).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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