nature14053 Broad CTL response is required to clea

LETTERdoi:10.1038/nature14053BroadCTLresponseisrequiredtoclearlatentHIV-1duetodominanceofescapemutationsKaiDeng1,MihaelaPertea1,2,AnthonyRongvaux3,LeyaoWang4,ChristineM.Durand1,GabrielGhiaur5,JunLai1,HollyL.McHugh1,HaipingHao6,HaoZhang7,JosephB.Margolick7,CaganGurer8,AndrewJ.Murphy8,DavidM.Valenzuela8,GeorgeD.Yancopoulos8,StevenG.Deeks9,TillStrowig3,PritiKumar10,JanetD.Siliciano1,StevenL.Salzberg2,11,RichardA.Flavell3,12,LiangShan3&RobertF.Siliciano1,13Despiteantiretroviraltherapy(ART),humanimmunodeficiencyvirus(HIV)-1persistsinastablelatentreservoir1,2,primarilyinrestingmemoryCD41Tcells3,4.ThisreservoirpresentsamajorbarriertothecureofHIV-1infection.Topurgethereservoir,pharmacolog-icalreactivationoflatentHIV-1hasbeenproposed5andtestedbothinvitroandinvivo6–8.Akeyremainingquestioniswhethervirus-specificimmunemechanisms,includingcytotoxicTlymphocytes(CTLs),canclearinfectedcellsinART-treatedpatientsafterlatencyisreversed.HereweshowthatthereisastrikingallornonepatternforCTLescapemutationsinHIV-1Gagepitopes.UnlessARTisstartedearly,thevastmajority(.98%)oflatentvirusescarryCTLescapemutationsthatrenderinfectedcellsinsensitivetoCTLsdirectedatcommonepitopes.Tosolvethisproblem,weidentifiedCTLsthatcouldrecognizeepitopesfromlatentHIV-1thatwereunmutatedineverychronicallyinfectedpatienttested.Uponstimulation,theseCTLseliminatedtargetcellsinfectedwithautologousvirusderivedfromthelatentreservoir,bothinvitroandinpatient-derivedhuman-izedmice.ThepredominanceofCTL-resistantvirusesinthelatentreservoirposesamajorchallengetoviraleradication.Ourresultsdemonstratethatchronicallyinfectedpatientsretainabroad-spectrumviral-specificCTLresponseandthatappropriateboostingofthisresponsemayberequiredfortheeliminationofthelatentreservoir.HIV-1establisheslatentinfectioninrestingCD41Tcells3,4.RecenteffortstoeradicateHIV-1infectionhavefocusedonreversinglatencywithoutglobalT-cellactivation5.However,inducingHIV-1geneexpres-sioninlatentlyinfectedcellsisnotsufficienttocausethedeathofthesecellsiftheyremaininarestingstate9.BoostingHIV-1-specificimmuneresponses,includingCTLresponses,mayberequiredforclearanceofthelatentreservoir9.CTLshaveasignificantroleinsuppressingHIV-1replicationinacuteinfection10–14.Becauseofthisstrongselectivepres-sure,HIV-1quicklyacquiresmutationstoevadeCTLrecognition12,13,15–18.CTLescapehasbeenstudiedprimarilythroughtheanalysisofplasmavirus12,13,16,18–20,andCTL-basedvaccineshavebeendesignedbasedonconservedepitopes21,22.AsystematicinvestigationofCTLescapeinthelatentreservoirwillbeofgreatimportancetotheongoingCTL-basedviruseradicationefforts,becauselatentHIV-1probablyrepresentsthemajorsourceofviralreboundaftertreatmentinterruption.EarlierstudieshavesuggestedthepresenceofCTLescapemutationsinpro-viralDNA15,17,butitremainsuncleartowhatextentthelatentreservoirinrestingCD41TcellsisaffectedbyCTLescape,whethermutationsdetectedinproviralDNAarerepresentativeoftheverysmallfractionofprovirusesthatarereplicationcompetent,and,mostimportantly,whethertheCTLresponsecanrecognizeandclearinfectedcellsafterlatencyisreversed.ToinvestigateCTLescapevariantsinthelatentreservoir,wedeepsequencedtheproviralHIV-1DNAinrestingCD41Tcellsfrom25patients(ExtendedDataTable1).Amongthem,10initiatedARTduringtheacutephase(AP;within3monthsofinfection)whiletheother15initiatedARTduringthechronicphase(CP)ofinfection.Thesequenc-ingwasfocusedonGagbecauseitisanimportanttargetoftheCTLre-sponse23andishighlyconserved,whichfacilitatesthedetectionofescapevariants.OurdatashowthatpreviouslydocumentedCTLescapevar-iantscompletelydominatetheviralreservoirsofnearlyallCP-treatedpatients(ExtendedDataFig.1andSupplementaryTable1).ThistrendisespeciallyobviousforseveralwellcharacterizedCTLepitopes:thehumanleukocyteantigen(HLA)-A2-restrictedepitopeSLYNTVATL(SL9),theHLA-A3-restrictedepitopeRLRPGGKKK(RK9)andtheHLA-B57/58-restrictedepitopeTSTLQEQIGW(TW10)(Fig.1aandExtendedDataFig.1,highlightedincolouredboxes.).Intheseepitopes,closeto100%ofthesequencesharbouredescapemutations.Comparisonofmuta-tionfrequenciesbetweenHLAallele-relevantand-irrelevantepitopesinCP-treatedpatientssuggeststhattheCTLescapemutationsidentifiedarespecifictoeachpatient’sHLAtype(Fig.1b).Bycontrast,exceptforSL9frompatientAP01andRK9frompatientAP08,fewifanyCTLescapemutationswerearchivedinAP-treatedpatients(Fig.1candEx-tendedDataFig.1).ThestrikingdifferencebetweenAP-andCP-treatedpatients(Fig.1c)indicatesthat,unlesstreatmentisinitiatedwithinthefirstseveralmonthsofinfection,thelatentreservoirbecomesalmostcompletelydominatedbyvariantsresistanttodominantCTLresponses.Toconfirmthatvariantsdetectedathighfrequencyinthelatentres-ervoirrepresentfunctionalCTLescapemutants,cellsfromsevenCP-treatedsubjectsweretestedforreactivitytosyntheticpeptidesrepresentingwild-typeandmutantversionsoftherelevantepitopes.Asexpected,therewereonlyminimalresponsestopreviouslydocumentedCTLescapemutantsbypatientCD81Tcells,andnodenovoresponsewasdetected(Fig.1dandExtendedDataFig.2).Incontrast,alltestedsubjectsretainedastrongresponsetopeptidesrepresentingthewild-typeep