Molecular Diversity of EpsteinBarr Virus IgG and IgA Antibody Responses in Nasopharyngeal Carcinoma.docx

Molecular Diversity of EpsteinBarr Virus IgG and IgA Antibody Responses in Nasopharyngeal Carcinoma.docx

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MolecularDiversityofEpsteinBarrVirusIgGandIgAAntibodyResponsesinNasopharyngealCarcinoma

Chapter2

MolecularDiversityofEpstein-BarrVirusIgGandIgAAntibodyResponsesinNasopharyngealCarcinoma:

AComparisonofIndonesian,Chinese,

andEuropeanSubjects

JajahFachiroh1,TabithaSchouten3,BambangHariwiyanto1,DewiKParamita1,AhmadHarijadi1,SofiaMHaryana1,MunHNg2,andJaapMMiddeldorp3

1GadjahMadaUniversity,Yogyakarta,Indonesia,

2DepartmentofMicrobiology,QueenMary'sHospital,Hongkong,China

3DepartmentofPathology,VrijeUniversiteitMedicalCenter,Amsterdam,TheNetherlands

TheJournalofInfectiousDiseases2004;190:

5362

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Chapter2

Abstract

Epstein-Barrvirus（EBV）-specificimmunoblotanalysiswasusedtorevealthemoleculardiversityofimmunoglobulin（Ig）GandIgAantibodyresponsesagainstEpstein-Barrnuclearantigen（EBNA）,earlyantigen（EA）,andviralcapsidantigen（VCA）inserumsamplesfrompatientswithnasopharyngealcarcinoma（NPC）andcontrolsubjects,byuseofimmunofluorescenceassay（IFA）.Controldonors（np150）showedIgGresponsestofewEBVproteinsVCA-p18,VCA-p40,EBNA1,andZebra-andsporadicallyweakIgAreactivitytoEBNA1andVCA-p18.PatientswithNPCstage1（np6）hadsimilarresponsepatterns.PatientswithNPCstage24（np132）showedsignificantlymorediverseIgGandIgAresponsestoEAandVCAproteins-VCA-p18/-p40,EBNA1,Z-encodedbroadlyreactiveactivator,andEAd-p47/54,-DNAse,thymidinekinase,and-p138.NocorrelationwasfoundbetweenIFAtitersandthenumberofEBVproteinsrecognizedbyIgGorIgA.OurresultsrevealdissimilaritybetweenEBVpolypeptidesrecognizedbyIgGandIgAantibodies,whichsuggestsindependentBcelltriggeringevents.

INTRODUCTION

Epstein-Barrvirus（EBV）,ag-herpesvirus,iswellestablishedinthehumanpopulationandisefficientlytransmittedbymucosalsecretions.EBVinfectionusuallyoccurssilentlyearlyinlife,butitmaybesymptomaticwheninfectionisdelayeduntiladolescence[13].EBVisalsoahumancarcinogenthathasbeenimplicatedinthedevelopmentofmalignanciesoflymphoidandepithelialorigin,includingBurkittlymphoma,Hodgkindisease（HD）,immunodeficiency-relatedBcelllymphoma,extranodalT/NKcelllymphomas,gastriccarcinoma,andnasopharyngealcarcinoma（NPC）[2,46].MostEBV-associatedmalignanciesshowadistinctgene-expressionpattern[7]andareaccompaniedbyaberrantantibodyresponsestovariousEBVproteinsandantigencomplexes[8].

UndifferentiatedNPCis100%associatedwithEBVandformsanunusualtumorwithintriguingepidemiologicalandbiologicalcharacteristics[610].ThehighestincidenceisfoundinpersonsofChineseethnicitylivinginsouthernChina,HongKong,Taiwan,andSingapore.IntermediateincidenceoccursincertainAfricanandMediterraneanpopulations,inInuitsfromGreenlandandAlaska,andinMalaysfromSingaporeandMalaysia.AlowincidenceisfoundinAmericanandEuropeanwhites,Hispanics,andJapanese.InIndonesia,NPChasanoverallintermediateincidence（3.9cases/100,000population）similartothatinMalaysia.However,intheYogyakartaarea,NPCconstitutes21.8%oftumorsinmenand7.9%oftumorsinwomen,whichranksNPCasthemostfrequenttumorinmenandthefourthmostfrequenttumorinwomen[11].TheresultsofseroepidemiologicalstudieshaveindicatedacloserelationshipbetweenEBVinfectionandNPC,asrevealedbyelevatedIgGand,especially,IgAresponsestoEBVviralcapsidantigen（VCA）,earlyantigen（EA）,andEpstein-Barrnuclearantigen（EBNA）complexes[8,12].ElevatedtotalandEBV-specificserumIgAlevelsareindicativeofNPCstage[8,13,14]andcanprecedetumordevelopmentby15years,

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MolecularDiversityofEpstein-BarrVirusIgGandIgAAntibodyResponsesinNPC

whichsuggeststhatareactivationofEBVinfectionplaysaroleintumordevelopment[14,15].Inaddition,adeclineinanti-EBVantibodyresponsesafterradiotherapymayhaveprognosticvalue[16].Atpresent,theindirectimmunofluorescenceassay（IFA）isstillusedasthereferencestandardfortheserodiagnosisofEBVinNPC[2,8,12,14].Thismethod,however,isdifficulttostandardizeandisnotsuitableforlarge-scaletestingindevelopingcountries;itisgraduallybeingreplacedbymore-definedEIAs[17-20].Ofimportance,IFAdoesnotprovideinsightintothemolecularbasisofanti-EBVresponses,becauseEBV-infectedcellseachcontainamultitudeofdifferentEBVproteinsthatcanserveasthetargetforantibodyinteraction[21-23].

RecentmolecularserologicaltestingapproachesinthediagnosisofNPChavefocusedontheuseofdefinedrecombinantEBVproteins.Tedeschietal.[24]showedthatantibodiesagainsttheZ-encodedbroadlyreactiveactivator（Zebra）proteinareregularlyfoundamongpatientswithNPC.Othershaveproposedtheanti-ZebraIgGantibodytitertobeaprognosticmarkerforNPC[16].However,anti-ZebraIgGantibodiesaredetectablein74%ofhealthyEBVcarriers,accordingtotheresultsofasensitiveimmunoblotassay[22]-thishasbeenconfirmedbyexchangingblindserumsamples（I.JoabandJ.M.M.,unpublisheddata）.EBVDNAseneutralizingantibodieshavebeenfoundin83%-94%ofpatientswithNPC[25],andtheyappeartobeagoodmarkerforNPCscreeningandprognosis.However,thereisnocorrelationbetweenthelevelofanti-DNAseantibodiesandantibodytitertoVCAorEA-D,accordingtotheresultsofIFAserologicaltesting[26].Stolzenbergetal.[27]detectedIgAantibodyagainstrecombinantDNAseinpatientswithNPCbutrarelyinpatientswithotherEBV-relatedmalignancies.Shimakageetal.[28]suggestedtheuseoftheEBNA1-IgAserumlevelasaprognosticmarkerformonitoringpatientswithNPCafterradiationtherapy,andFoongetal.[29]suggestedtheuseofserumandsalivaryIgAlevelsagainstanEBNA1Gly-AlarepeatpeptideasasuitableNPCmarker.Connollyetal.[20]suggestedtheuseofthymidinekinase（TK）astheantigeninIgA-ELISAforthediagnosisofandscreeningforNPC.Dardarietal.[30]indicatedthatthecombinationofIgG-ZebraandIgA-EA[（p54）+（p138）]shouldbeused.Mostrecently,Chanetal.[31]proposedthecombineduseofEBNA1-IgAandZebra-IgGasthebestpredictorforNPC.ItmaybeobviousfromtheabovethatthereisnoconsensusontheuseofdefinedEBVproteinsinserologicaltestingforthediagnosisandprognosisofNPC.

Still,verylittledetailisknownabouttheoverallmoleculardiversity（complexity）ofanti-EBVIgGandIgAantibodyresponsesinpatientswithNPC[23].Moreover,acomparisonofantibodyprofilesamongpatientswithNPCwhoareofdifferentgeneticbackgroundhasnotbeendescribed.ThestudydescribedhereprovidesinsightintothemolecularbasisofEBV-specificIgGandIgAantibodyresponsesinpatientswithNPCofdefinedtumorstagefromJavanese（Indonesia）,Chinese（HongKong）,andwhite（Europe）origin,comparedwiththoseinregionalnon-NPCcontrolsubjectsandhealthyEBVcarriers.OurparallelanalysisofIgGandIgAresponsesrevealeddifferencesinEBVantigenrecognitionprofiles,whichsuggestsindependentBcelltriggering.

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Chapter2

SUBJECTS,MATERIAL,ANDMETHODS

Serumsamplesandantibodies.SerumsamplesfromIndonesianJavanese（nonChinese）subjectsconsistedofsamplesfromapanelof135patientswithhistologicallyconfirmedNPC,5patientswithnon-NPCheadandneckcancer（allofwhichwerecollectedattheDepartmentofEar,Nose,andThroat[ENT],Dr.SardjitoGeneralHospital,Yogyakarta）,and70healthydonorsobtainedfromthelocalRedCrossbloodbank.TheNPCserumsampleswereobtainedonthefirstvisitofpatientstoENTduring2001-2003.FromallpatientswithNPC,nasopharyngealand/orlymph-nodebiopsysampleswereobtainedandconfirmedhistologicallyforthepresenceofundifferentiatedcarcinomacellsandthepresenceofEBV,byEBER1,2insituhybridization,byuseoftheDakoPNAkit（Dako）andbyimmunohistochemistry（Labvision）withEBNA1-andlatentmembraneprotein（LMP）1-specificmonoclonalantibodiesOT1X[32]andOT21C[23,33],respectively.NPCstagingwasdonebyENTexaminationandcomputedtomographyscanandwasclassifiedaccordingtothe1997UnionInternationalCancerControl（UICC）classification.

SerumsamplesfrompersonsofChineseethnicitylivinginHongKongwereprovidedasablindpanelandincludedsamplesfrom40healthydonors,35patientswithheadandneck-relatednon-NPCtumors,and40patientswithhistologicallyconfirmedNPC（obtainedbyM.H.N.）.TheEBVserologicalprofileoftheChinesepanelwasanalyzedwithoutknowledgeoftheclinicaldiagnosis.VCAandEAIgGandIgAantibodytitersweredeterminedbystandardIFAtechniques,accordingtothemethodofHenleandHenle[8],andthisinformationwasrevealedonlyafterbreakingthecode.

Serumsamplesfrom7whitepatientswithNPCwereobtainedfromhospitalsinGermany,theUnitedKingdom,andTheNetherlands.Oneseries（np5）offollow-upsamplesfromawhiteDutchpatientwithNPCwasobtainedfromtheVrijeUniversiteitmedicalcenter,Amsterdam,TheNetherlands.Allserumsampleswerestoredat20°Cuntiluse.

Monoclonalandpolyclonalmonospecificantiserumsampleswere

producedbytheimmunizationofanimalswithsyntheticpeptidesorpurifiedrecombinantproteins,asdescribedelsewhere.AntibodiestodefinedEBVproteinsconsistedofOT13B（anti-EA-p138;BALF2）[34],rabbitanti-EBNA1（BKRF1）[35],rabbitanti-DNAse（BGLF5）[27],OT14E（anti-EA-p47;BMRF1）[36],BZ-1（anti-Zebra;BZLF1）[37],OT41A（anti-VCA-p40;BdRF1）[38],andOT15E（anti-VCA-p18;BFRF3）[39].

Cellcultureandantigenpreparation.ThesuperinducibleP3HR1-derivedcelllineHH514.c16was