Mechanisms of Ad5-Induced Immune Dysfunction

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Citation Alayo, Quazim A. 2016. Mechanisms of Ad5-Induced ImmuneDysfunction. Master's thesis, Harvard Medical School.

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33789913

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MechanismsofAd5-InducedImmuneDysfunction

QuazimA.AlayoMBBS

AThesisSubmittedtotheFacultyof

TheHarvardMedicalSchool

inPartialFulfillmentoftheRequirements

fortheDegreeofMasterofMedicalSciencesinImmunology

HarvardUniversity

Boston,Massachusetts.

May,2016

ii

ThesisAdvisor:Dr.DanH.BarouchMDPhD QuazimA.Alayo

MechanismsofAd5-InducedImmuneDysfunction

Abstract

ThefailureofanAdenovirus5(Ad5)-basedhumanimmunodeficiencyvirustype1(HIV-1)

vaccineintheSTEPtrialwarrantedadetailedevaluationoftheimmunologicalpropertiesof

Ad5.PreviousstudieshaverevealedthatimmunizationwithAd5inducesapartiallyexhaustedT

cellresponsebutthemechanismofAd5-inducedimmunedysfunctionisunknown.Using

classicalanimalmodels,ithasbeenshownthatalteringantigendose,andmodulatingthePD-

1/PD-L1signalingpathway,ormodulatingregulatoryTcells(Tregs)caninfluencethequalityof

memoryCD8Tcell.Therefore,weinterrogatedwhetherthesefactorsplaysimilarrolesinAd5-

induceddysfunction.Here,weshowthatreducingAd5vaccinedoseinduceshighlyfunctional

memoryCD8Tcellresponses,characterizedbylowerPD-1expression,highercytokineco-

expression,andanimprovedrecallexpansionfollowingaheterologousboost.Interestingly,we

showthatthedysfunctionalrecallofAd5-primedTcellsfollowinghigh-doseimmunizationmay

partlybemediatedbyaPD-1-dependentCD8Tcellintrinsicphenomenon,asblockadeofPD-L1

leadstoasubstantialimprovementinanamnesticTcellexpansion.Furthermore,weprovide

preliminarydatasuggestingthatTregmaynotplayacrucialroleinthedevelopmentofAd5-

induceddysfunction.Overall,ourdatacontributetotheunderstandingofthemechanismof

Ad5-inducedimmunedysfunction,andmayberelevantforimprovingvaccinationmodalities

forHIVandotherchronicviralinfection.

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TaleofContents

1. Chapter1:Background....................................................................................................1

1.1 Introduction.........................................................................................................................1

1.2 AdenovirusBiology..............................................................................................................3

1.3 AdenovirusasaVaccineVector............................................................................................5

1.4 HumanAdenovirus5VaccineVector....................................................................................6

1.5 Ad5-inducedImmuneDysfunction.......................................................................................8

1.6 FactorsInfluencingAd5-inducedImmuneDysfunction.........................................................9

1.6.1 VectorDose..........................................................................................................................10

1.6.2 ImmunoinhibitoryReceptors................................................................................................11

1.6.3 ConventionalandRegulatoryCD4Tcells.............................................................................13

1.6.4 AntigenPresentingCells,Co-stimulatoryandCo-inhibitoryMolecules...............................14

1.7 Summary...........................................................................................................................14

1.8 Figures...............................................................................................................................16

1.8.1 FigureA:PotentialfactorsinfluencingAd5-inducedimmunedysfunction..........................16

2. Chapter2:DataandMethods........................................................................................18

2.1 Introduction.......................................................................................................................18

2.1.1 Hypothesis............................................................................................................................18

2.1.2 Specificresearchquestions:.................................................................................................18

2.2 MaterialsandMethods......................................................................................................19

2.2.1 Miceandimmunizations......................................................................................................19

2.2.2 Isolationoflymphocytes.......................................................................................................19

2.2.3 Tetramerbindingassayandflowcytometry........................................................................20

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2.2.4 Intracellularcytokinestaining(ICS)assays...........................................................................21

2.2.5 Monoclonalantibodiestreatments......................................................................................21

2.2.6 InvitroTregsuppressionassay.............................................................................................22

2.2.7 Statisticalanalysis.................................................................................................................22

2.3 Results...............................................................................................................................23

2.3.1 Thekinetics,phenotypeandfunctionalityofvaccine-inducedCD8Tcellsfollowing

vaccinationwithdifferentdosesofAd5............................................................................................23

2.3.2 ImprovedanamnesticCD8TcellresponsesaftervaccinationwithlowdoseAd5vector...25

2.3.3 Excessiveup-regulationofPD-1onAd5-inducedmemoryCD8Tcellsimpairstheirrecall

response............................................................................................................................................27

2.3.4 SimilarTregsfrequencyandsuppressivefunctioninAd5-andAd26-immunizedmice.......29

2.4 Figures...............................................................................................................................31

2.4.1 Figure1:Kineticsofvaccine-elicitedCD8Tcellfollowingvaccinationwithdifferentdosesof

Ad5…………..........................................................................................................................................31

2.4.2 Figure2:Phenotypeandpolyfunctionalityofvaccine-inducedCD8Tcellfollowing

vaccinationwithdifferentdosesofAd5............................................................................................32

2.4.3 Figure3:LoweringtheprimingdoseofAd5vectorpartiallyimprovesthephenotypeand

therecallresponsesafterAd35boosting..........................................................................................33

2.4.4 Figure4:PrimingwithlowdoseAd5inaAd5-Ad35heterologousprime-boostregimen

producemorepolyfunctionalCD8Tcells..........................................................................................34

2.4.5 Figure5:Excessiveup-regulationofPD-1onAd5-inducedmemoryCD8Tcellsimpairstheir

recallresponse...................................................................................................................................35

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2.4.6 Figure6:PD-L1blockadepriortoprimingdoesnotaffectthekinetics,memoryconversion

oranamnesticresponseofAd5-inducedCD8Tcells.........................................................................36

2.4.7 Figure7:SimilarTregfrequenciesandsuppressivefunctionsinAd5-andAd26-immunized

mice………………………………………………………………………………………………………………………………………………37

3. Chapter3:Discussionandperspectives..........................................................................38

3.1 Limitations.........................................................................................................................44

3.2 Futuredirections................................................................................................................44

4. Bibliography..................................................................................................................47

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Figures Pages

1.8.1FigureA:PotentialfactorsinfluencingAd5-inducedimmunedysfunction 16

2.4.1 Figure1:Kineticsofvaccine-inducedCD8Tcellfollowing vaccinationwithdifferentdosesofAd5. 31

2.4.2 Figure2:Phenotypeandpolyfunctionalityofvaccine-induced CD8TcellfollowingvaccinationwithdifferingdosesofAd5. 32

2.4.3 Figure3:LoweringtheprimingdoseofAd5vectorpartiallyimprovesthephenotypeandtherecallresponsesafterAd35boosting. 33

2.4.4 Figure4:PrimingwithlowdoseAd5inaheterologousprime-boostregimen(Ad5/Ad35)producemorepolyfunctionalCD8Tcells. 34

2.4.5 Figure5:Excessiveup-regulationofPD-1onAd5-inducedmemoryCD8Tcellsimpairstheirrecallresponse. 35

2.4.6 Figure6:PD-1blockadepriortoprimingdoesnotaffectthekinetics,memoryconversionoranamnesticresponseofAd5-inducedCD8Tcells. 36

2.4.7 Figure7:SimilarTregfrequencyandsuppressivefunctioninAd5-andAd26immunizedmice. 37

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Tables Pages

TableA:SpectrumofexhaustedCD8Tstates 17

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Acknowledgement

Firstly,IwouldliketoexpressmyprofoundgratitudetoDrDanBarouchforacceptingmein

hislab,hisunwaveringsupport,andguidancethroughoutthedurationofmyresearch.

IwouldalsoliketoappreciateDrPabloPenaloza,forteachingmealltheprotocolsand

providingaday-to-dayguidanceasIcrawledupthelearningcurve.Pablo,thankyouforthose

insightfulcommentsandtheencouragingwords,especiallyduringthechallengingmoments.I

wishyouallthebestasyoumovetoNorthwesterntostartyournewlab.Also,aspecialthank

youtoDr.AlexanderBadamchi-Zadehforhelpingwiththeintramuscularinjections,reviewing

mythesis,andprovidingveryusefulcomments.Iwouldalsoliketoexpressmygratitudeto

ErynBlassforassistingwithsomeofmyexperiments,teachingmeagreatdealoflabetiquettes

andhelpingtoreviewmythesis.Mysincerethanksalsogoestoallotherpost-docs,graduate

studentsandothermembersoftheBarouchlabfortheirwarmthwelcomeandeagernessto

help.Inparticular,IamgratefultoJade(andhernewbornbaby),Ben,Lily,andEricafor

assistingwithmiceorderingandinjections.Jess,CrystalandLauren,thankyoufor

accommodatingmeinyourbay.

Myheartfeltgratitudetomyfamilymembersandfriendsfortheirsupportandprayers.I

wouldalsoliketothankthePRESSIDscholarshipboardforsponsoringme.Aboveall,myutmost

gratitudegoestotheAlmightyAllaahSWTforaidingmeandgrantingmesuccess.�

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ThisworkwasconductedwithsupportfromStudentsintheMasterofMedicalSciencesin

ImmunologyprogramofHarvardMedicalSchool.Thecontentissolelytheresponsibilityofthe

authoranddoesnotnecessarilyrepresenttheofficialviewsofHarvardUniversityandits

affiliatedacademichealthcarecenters.

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1. Chapter1:Background

1.1 Introduction

Vaccinesareoneofthemostimportantpublichealthtoolsinthefightagainstinfectious

diseases.Thegoalofvaccinesistogeneratelong-livedimmunologicalprotection,withmemory

immuneresponsethatcanpreventnaturalinfectionorsignificantlylessenthediseaseburden1.

Mostofthecurrentlylicensedvaccineshaveprimarilyconferredthisimmunologicalprotection

viatheinductionofhigh-affinityneutralizingantibodies.However,developinghighlyeffective

vaccinesagainstintracellularpathogenssuchashumanimmunodeficiencyvirus-1(HIV-1),

Mycobacteriumtuberculosis,Plasmodiumfalciparum,hepatitisCvirus(HCV)willalsorequire

thegenerationofpotentmemoryCD8Tcells2,3,4,5,6.Inordertoelicitapotentcellularimmune

response,thevaccineantigenmustbeprocessedendogenouslywithinantigenpresentingcells

andpresentedinthecontextofclassImajorhistocompatibilitycomplex(MHC-I)moleculesto

CD8Tcells.Untilrecently,achievingthisgoalhasbeenhamperedbyalackofvaccineplatforms

capableofsuch,whichhasledtothedevelopmentofnovelplatformsincludingDNAvaccines

andviralvectors.

DNAvaccinesaregeneticallyengineeredplasmidscontainingtheDNAsequenceofthe

antigenofinterestthatcanbedelivereddirectlyintotheappropriatetissue7.DNAvaccinesare

safeandcapableofelicitingprotectivecellularandhumoralimmuneresponsesinsmallanimal

models8,9.However,theirapplicationislimitedbytheirlowimmunogenicityinhumanand

otherprimates9.ThesuboptimalimmunogenicityofDNAvaccinesinprimatesmaypartlybe

2

duetobindingoftheDNAtoserumamyloidPcomponent(SAP),anegativeregulatorofthe

innateimmuneresponse10.ThelimitedefficacyDNAvaccinesledtothedevelopmentofmore

immunogenicviralvectorvaccines.

IncontrasttoDNAvaccines,viralvectorshavebeenshowntobehighlyimmunogenic,both

inpreclinicalandclinicalstudies11.Theyaretypicallyattenuatedvirusesthataregenetically

modifiedtocarryanantigentowhichanimmuneresponseisbeingsoughtintothebody.They

alsoelicitverypotentinnateimmuneresponsesthroughthebindingofspecificToll-like

receptors(TLRs)leadingtothegenerationofacytokinemilieuwhichmodulatetheadaptive

immuneresponse12.Theadaptiveimmuneresponseinducedbythesevectorsdiffer

quantitativelyandqualitativelybasedonthedistinctbiologicalpropertiesofthevector.Over

thelastcoupleofyears,awiderangeofvirusfamilieshavebeendevelopedandtestedas

vaccinevectorsforeitherhumanorveterinaryuse11,13,14,15.Amongthesewidearrayofviruses

whichincludecanarypoxvirus16,flavivirus17,lentivirus18,modifiedvacciniavirusAnkara19and

sendaivirus20,adenovirus21hasbeenthemostwidelystudiedreplication-defectivevaccine

vectors.Ofalltheadenovirusserotypesisolatedsofar,adenovirus5(Ad5)isthebest

characterized22.

However,followingthefailureofanAd5-basedHIVvaccineinclinicaltrials,adetailed

evaluationoftheimmunologicalpropertiesrevealedthatitelicitedadysfunctionalimmune

response23.Ithasbeensuggestedthatthisdysfunctionalresponse,coupledwiththeprevalent

pre-existingAd5-vectorimmunitymayhavecontributedtothefailureoftheAd5-based

vaccine.However,thepossiblebiologicalorimmunologicalparametersresponsibleforthe

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immunedysfunctionisnotfullyunderstood.Althoughthereareongoingpre-clinicaland

clinicalstudiesevaluatingtheimmunogenicityandprotectiveefficacyofotherserotypesof

adenoviruses12,understandingthebiologicalandimmunologicalpropertiesofAd5thatmay

havecontributedtothedysfunctionalTcellresponsewillhelpintheselectionofnovelvaccine

vectors,someofwhicharephylogeneticallyrelatedtoAd5.

1.2 AdenovirusBiology

Adenovirusesareafamilyofairbornevirusesthatinfectvirtuallyallmajorclassesof

vertebratescausingmildtoseverediseasesoftheairwaysandgastrointestinaltracts24.

Currently,morethan60distinctadenovirusserotypesofhumanoriginhavebeenidentified25.

Thesevirusesaresubdividedintosevendifferentserotypes(subgroupsAthroughG)basedon

theneutralizingantibodiesprofilesandthiscorrespondstothedivergenceofnucleotide

sequenceofthecapsidproteingenes25.

AdenovirusesaredoublestrandedDNAviruseswithanon-envelopedicosahedralshell,70-

100nmindiameter,enclosingabout~26-43kilobaseslonglineargenomicmaterial26.The

genomeismadeupofearlyandlategenes,classifiedbasedonthetimeofexpressionin

relationtoDNAreplication.Theearlygenes(E1a,E1b,E2a,E2b,E3,andE4)expressproteins

beforeDNAreplicationandtheyareinvolvedinactivatingandmodulatingtranscription,the

cell-cycle,cellsignaling,DNArepairandimmuneevasion27,28,29.Thelategenes(L1–L5)which

areexpressedfollowingDNAreplication,codemostlyforstructuralprotein30.E1,E2,andE4

geneproductsregulatetranscriptionoflategeneswhileE3geneproductssubvertthehost

4

immuneresponsebyaffectingtheintracellulartraffickingmachinery,preventingantigen

presentationandmodulatingcytokinepathways31.Geneticmanipulationoftheseearlygenesis

oftenemployedinordertoadaptadenovirusforuseasvectors

Structurally,theadenoviruscapsidisicosahedral,witheachicosahedroncontaining12

copiesofhexontrimersandapentamericpentonslocatedatthe12vertices32.Projectingfrom

thepentonbasesaretrimericfiberswhicharemadeupofadistalknobdomain,ashaft,andan

N-terminaltail32.Thedistalknobdomainofthefibersselectivelybindswithhighaffinityto

cellularreceptors,leadingtocellulartransduction.Entryreceptorsidentifiedsofarinclude

coxsackievirusBandadenovirusreceptor(CAR),CD46,sialicacids,desmoglein2,and

CD80/8633,34,35,36,37,38.ThemostcharacterizedincludethecoxsackievirusBandadenovirus

receptor(CAR),whichisexpressedmainlyonepithelialandendothelialcellsinhuman,non-

humanprimatesandmice34;andCD46,aregulatoryproteininthecomplementsystemthatis

expressedonmostcellsinhumans,yetrestrictedtothetestisandsperminmice39.

AdenovirusesbelongingtoserotypesA,C,D,E,andFusesCARforcellularattachmentwhile

CD46isusedbytheadenovirusesinserotypesBandD40.Inadditiontothedifferencein

receptorusage,adenovirusesalsodifferintheirintracellulartraffickingcharacteristicsand

associationwiththenuclearenvelope,withsomeexitingtheendosomalspacerapidlyfollowing

endocytosisandtranslocatingimmediatelytothenucleus(subgroupC)whileotherstrafficto

thelateendosomeandthereforeexhibitaslowerassociationwiththenucleus(subgroupsB

andD)41,42,43,44.Thedifferencesincellularreceptorandintracellulartraffickinginfluence

cellulartropism,particularlytheimmunecellsubsetsthesevirusesinteractwith,thepattern

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recognitionreceptors(PRRs)theyactivate,thespecifictissuesiteswherethetransgeneare

expressed,andthereforethecharacteristicsofinnateandadaptiveimmuneresponseactivated

bytheseviruses.Appreciationofhowthesepropertiesinfluencetheimmuneresponseis

particularlyimportantwhenadenovirusesareusedasvaccinevectors.

1.3 AdenovirusasaVaccineVector

Followingtheirdiscoveryoversixdecadesago45,adenoviruseshaveevolvedtobean

importantbiologicaltoolutilizedasmodelsystemprovidingsignificantinsightsintocomplex

cellularprocessessuchasDNAreplicationandtranscription,DNArepairresponses,oncogenesis

andalternateRNAsplicing46,47.Additionally,theirabilitytoreadilyinfecthumanandother

mammaliancellshavemadethemapopularchoiceforgenedelivery48.Theirapplicationin

genetherapywashoweverhinderedbytherobustimmuneresponsetheyinduceleadingtoa

shorteneddurationoftransgeneexpression49.Interestingly,thisstrongimmunogenicityhas

beenexploitedbyvaccinologistswiththedevelopmentofmanyadenoviralvaccinevectors.

Adenoviruseshavesomeuniquefeaturesthatmakethemparticularlysuitableforuseas

vaccinevectors12,22.First,theyhaveaverybroadcellulartropismwiththeabilitytoinfectand

expressitstransgeneinawidevarietyofrapidlydividingandnon-dividingcells.Secondly,they

arestableandcanbereadilygrownandpurifiedinlargequantities.Thirdly,theycause

relativelymilddiseaseinhumanandcomparedtolentiviralvectors,theydonotintegrateinto

thehostcellgenome,reducingtheriskofmalignanttransformation.Fourthly,theirgenomeis

wellcharacterizedandcanbeeasilymanipulatedtorenderthemreplication-defective.Thisis

typicallyachievedbydeletingtheearlygeneE1,whichisrequiredforadenovirusreplication.In

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addition,E3,anotherearlygeneimportantforhost-vectorimmuneinteraction,isoften

deleted.AlthoughnotessentialinE1-deletedadenovirus,deletionofE3orE3/E4regionsoffers

thebenefitofincreasingthetransgeneinsertioncapacitytoupto7.5kbptocarrymedium-sized

proteintransgenes50,51.Furthermore,vectorizedadenovirusesarethermostableandhavebeen

demonstratedinbothpreclinicalandclinicalstudiestobesafeforbothsystemicandmucosal

surfaceapplication22.Finally,andmostimportantly,replicationincompetentadenovirusesare

highlyimmunogenic,inducingrobustantibodyandcell-mediatedimmunitytotheinserted

transgeneswiththeabilitytoelicitbothsystemicandmucosalimmuneresponsefollowing

parenteraldelivery52,53,54.

1.4 HumanAdenovirus5VaccineVector

Ad5isthemoststudiedoftheadenovirusvectors.ItisaserotypeCvirusandtransduces

cellseitherthroughtheuseofthecoxsackie-adenovirusreceptor(CAR)34orinaCAR-

independentpathway55.Asastandalonevaccinevector,itisarguablythemostimmunogenic

humanadenovirusserotype.BallayA.etalwasthefirsttoreportthatrecombinant

replication–competentAd5carryinghepatitisBsurfaceelicitsantibodyresponseagainstthe

transgeneinrabbits51.Notlongafterwards,theabilityofE1-deletedreplication-incompetent

Ad5vectorsexpressingrabiesvirusglycopeptide(GP)toinduceprotectivetitersofneutralizing

antibodiesandrabies-virusspecificCD8andCD4Tcellswasdemonstrated56.Followingthis

importantfinding,replication-incompetentAd5hasbeentestedinseveralotherpreclinicaland

clinicalstudiesandshowntoelicitbothneutralizingantibodiesandcellularimmuneresponses

7

againstpathogenssuchasdenguevirus,ebolavirus,hepatitisCvirus,Epstein-Barrvirus,

Streptococcuspneumonia,Mycobacteriatuberculosis,Plasmodiumfalciparum,HIVamong

others57,58,59,60,61,62,63.

IntheHIVfield,Ad5expressingsimianimmunodeficiencyvirus(SIV)Gagpeptideconferred

aprotectiveimmuneresponsefollowingchallengeofrhesusmonkeyswithpathogenicSIV/HIV

chimeraSHIV89.6P10,butfailedtoprotectagainstamorestringent,neutralization-resistant

strainSIVmac23964,65.Subsequently,twolargePhaseIIbclinicaltrials(STEPinNorthAmerica

andPhambilitrialsinSouthAfrica)werecommenced66,67.Inthesestudies,theMerckAd5-Gag-

Pol-Nefvaccinewasadministeredthreetimestovolunteersthathadeithernoneormoderate-

to-hightitersofneutralizingantibodiesagainstAd5withtheaimofelicitingHIV-specificT-cell

responsescapableofprovidingcompleteorpartialprotectionfromHIV-1infectionora

decreaseinviralloadsetpointspost-infection.Bothstudieswerestoppedandunblinded

beforecompletionofenrollmentfollowinganinterimanalysisoftheSTEPtrialthatshoweda

lackofefficacy67.Evenmorealarmingwasasubsequentanalysisthatrevealedatrendtoward

higherHIV-1acquisitioninvaccinerecipientscomparedtoplacebocontrols,particularlyina

subgroupofuncircumcisedmenwithhighbaselineAd5seropositivity67.Althoughnot

completelyproven,aprobableexplanationforthisunexpectedfindingisthatthevaccineledto

anincreaseinthenumberofHIV-1targetcells,resultingfromtheactivationofAd5-specific

CD4Tcellsatmucosalsurfaces68,69.Furthermore,asecondAd5-basedPhaseIIbHIVvaccine

clinicaltrial(HVTN505)didnotshowprotectiveefficacyagainstHIV-1infectioninhuman70.Itis

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importanttonotehowever,thattheAd5usedinthisstudywasdifferentindesignfromthe

MerckAd5vector,andwasusedasaboostingvectorfollowingaDNAvaccineprime.

ThefailureoftheAd5vectorintheseclinicaltrialsledtoarenewedinterestintwoareasof

Advectorresearch.Firstisthedevelopmentandevaluationofrareradenovirusserotypesof

humanorigin50,71,aswellasserotypesderivedfromotherspeciessuchaschimpanzees72,73,74

andrhesusmonkeys75,andstructurallymodifiedAd5vectorexpressingheterologous

adenovirushexons76inordertocircumventtheproblemofpre-existingAd5vectorimmunity.

TheotherareaofintenseresearchistheanalysisofthebiologicpropertiesofAdvector-

inducedimmuneresponses23,77,78,79,80,81,82.Thesedetailedanalyseshaveshedmorelighton

theinherentpropertiesofAd5-inducedTcellresponses,highlightinghowtheseresponses

substantiallydifferfromthoseelicitedbyalternativeserotypeAdvectorsuchasadenovirus26

(Ad26)oradenovirus35(Ad35).

1.5 Ad5-inducedImmuneDysfunction

Themostsurprisingfindingfromthecomprehensiveevaluationofthephenotypeand

qualityofAdvector-inducedimmuneresponsewasthatAd5,comparedtootherserotype

adenoviruses,inducesapartiallyimpairedimmuneresponse.Thisobservationwasfirst

reportedbyYangetalwhentheyanalyzedthephenotypicprofileoftheimmuneresponses

elicitedbyanAd5vaccineinmice83.TheyobservedthatAd5-elicitedCD8Tcellsexhibita

protractedeffectorphenotypewithadelayedcontractionphase,aphenomenontheyargued

maybeduetotheprolongedantigenpresentationseenfollowingAd5immunization83.Inline

9

withthisobservation,othershaveshownthat,comparedtoalternativeserotypeAdvectors,

Ad5inducesapartiallydysfunctionalTcellresponseinmice.Ad5-inducedmemoryTcellswere

foundtoexpresslowlevelsofIL-7alphachainreceptorCD127,lymphoidhomingreceptor

CD62L,antiapoptoticmoleculeBcl-2,aswellashighlevelsofinhibitoryreceptorsPD-1andTim

323,81,82,84.AlthoughAd5immunizationinducesahighmagnitudeprimaryTcellresponse,

theseTcellsarelesspolyfunctionalwithreducedcapabilitytosecreteIFN-γ,TNF-α,andIL-2,

comparedtoalternativeserotypeadenovirus23,82.Furthermore,circulatingandtissueresident

antigen-specificmemoryCD8TcellsinducedbyAd5proliferatelessrobustlyfollowinga

secondaryexposure(boosting)toantigens81.Similarfindingshavealsobeenobservedinnon-

humanprimates79.ItisimportanttonotethatalthoughrecombinantE1-deletedAd5vector

doesnotreplicate,itinducesamemoryCD8Tcellpopulationthattosomeextent–exceptfor

itsfunctionality–isreminiscentofanexhaustedTcellresponseseeninmodelsofchronic

infectionorcancercharacterizedbypersistentantigenicstimulation85,andassuchsuggests

thatAd5-inducedCD8Tcellsmaybe“partiallyexhausted”(TableA).

1.6 FactorsInfluencingAd5-inducedImmuneDysfunction

InordertounderstandwhyAd5-inducedmemoryCD8Tcellsexhibitsuchdysfunctionalor

“partiallyexhausted”phenotypes,itisIttmportanttoconsiderthevirologicaland

immunologicalcuesthatinfluencethedevelopmentandmaintenanceofexhaustedCD8Tcells

intheclassicalimmuneexhaustionmodelofchronicinfection,andassesswhetherthese

factorsplaysimilarrolesinmediatingAd5-inducedimmunedysfunction,andmoreimportantly,

10

ifmodulationoftheseparameterswillleadtoanimprovementinthephenotypeandqualityof

Ad5-inducedmemoryCD8Tcells.Extrapolatingfromthisclassicalmodelandrelyingonour

currentunderstandingofthebiologyofAd5,possiblefactorsthatmayfavorthedevelopment

ofAd5-inducedimmunedysfunctioninclude1)vectordose,2)immuno-inhibitoryreceptors,3)

CD4Tcells-conventionalandregulatoryTcellsand4)antigenpresentingcellsandexpression

ofco-stimulatoryand/orco-inhibitorymolecules.(Figure.A).

1.6.1 VectorDose

Theamountanddurationofantigenexposureimpactantigen-specificCD8Tcellsboth

intermsofquantityandquality.Inanacuteinfectionmodel,antigendoseaffectsthenumber

ofnaïveCD8Tcellsrecruitedforactivationandtheirdifferentiationintoeffectorandmemory

CD8Tcells86.Whileinchronicinfections,suchasthechronicmodeloflymphocytic

choriomeningitisvirus(LCMV)infectioninmice,differentiationofprimedCD8Tcellsintoeither

anexhaustedormemoryphenotypeisdependentontheamountanddurationofantigen

exposure87,88.Likewise,inAdvectorvaccineresearch,thedoseofAdvectorimpactsthequality

oftransgene-specificCD8Tcelldifferentiationandfunctionality78.HighdoseAd5immunization

hasbeenshowntoinducetransgenespecificCD8TcellsbearingfeaturesofadysfunctionalT

cellpopulation:reducedfunctionalCD8TcellsasevidencedbylowerpercellexpressionofIFN-

γ,lowerfractionofcellscoproducingTNF-αorIL-2andIFN-γ;impairedmemoryconversion

withlowerexpressionofCD127andhighexpressionofKLRG-1;andanabsenceofacontraction

phase78,89.Conversely,reducingthedoseofAd5significantlyimprovessomeofthese

11

impairments78.However,theeffectofloweringtheprimingdoseofAd5onrecallexpansion

followingantigenre-exposurehasnotbeeninvestigatedtodate.Inaddition,itisalsonot

knownwhetherloweringthedosewillleadtoareducedexpressionofimmunoinhibitory

receptors,suggestingareducedimmuneexhaustion.

1.6.2 ImmunoinhibitoryReceptors

ImmunoinhibitoryreceptorssuchasPD-1(ProgrammedCellDeath1),Tim-3(T-cell

immunoglobulinandmucin-domaincontaining-3),and2B4(CD244)areinducedonTcells

immediatelyfollowingactivation,andatthisstage,theyserveassafeguardstocurtailexcessive

immuneresponse23,85.However,thesustainedup-regulationofimmunoinhibitoryreceptors

hasbeenrecognizedasaprominentfeatureofexhaustedCD8Tcells,wheretheyplaya

significantroleinnegativelyregulatingTcellfunction90.Themostwidelystudiedofthese

receptorsisPD-1,amemberoftheCD28superfamily,whichisinduciblyexpressedonTcells,B

cells,naturalkillerT(NKT)cells,andsomemyeloidcells91.Likeothermembersofthefamily,

PD-1transducessignalsonlyinthecontextofTcellreceptor(TCR)crosslinkingfollowingaTCR-

antigen-MHCengagement.PD-1hastwoligands:PD-L1andPD-L2.PD-L1,alsoknownasB7-H1,

hasabroadtissuedistributionandisexpressedonBcells,DCs,macrophages,BM-derivedmast

cells,andTcellsandperipheralepithelialandendothelialcellsuponstimulationby

proinflammatorycytokines,suchasinterferonsandtumornecrosisfactor92,93,94.PD-L2orB7-

DCismorerestrictivelyexpressedondendriticcellsandmacrophages93.Uponinteractionwith

itsligands,PD-1sendssignalsthatlimitproliferation,interferewithcytotoxicactivityandIFN-γ

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productionthroughtheup-regulationofthetranscriptionfactorBATFinCD8Tcells95,96.The

suppressivefunctionofPD-1isalsoachievedviathereductioninthedurationofDC:Tcell

contact,andthedirectdephosphorylationofproximalTCRsignalingthroughtherecruitmentof

SHP1andSHP2phosphatasesbytheimmunoreceptortyrosine-basedswitch(ITSM)motifon

theintracellularcytoplasmictailofthePD-1receptor97,98,99.Otherimmunoinhibitoryreceptors

thatareup-regulatedonexhaustedTcellsincludeCTLA4,Tim-3,LAG-2,CD160and2B4and

thesereceptorshavebeenshowntoacteitherindependentlyorinsynergywithoneanother

and/orwithPD-1toinfluencethedegreeofTcellexhaustion85.PartialrestorationofTcell

functioncanbeachievedbyblockingthesignalingpathwaysofthesereceptors(knownas

immunecheckpointblockade)100.

Asmentionedearlier,Ad5vectorelicitedgreaterpercentagesoftransgene-specificCD8

Tcellsco-expressingbothPD-1andTim-3orsingly-positiveforeitherPD-1orTim-323

(Penaloza-MacMaster,P.etal.unpublisheddata).Indeed,PD-1andTim-3expressiononAd5-

inducedTcellsisassociatedwithlessfunctionality(Penaloza-MacMaster,P.etal,unpublished

data)similartofindingsinthechronicmodelofLCMVinfectioninmice85orHIVinfectionin

humans101.Additionally,thehigherco-expressionofPD-1andTim-3correlateswithreduced

levelsofmemorymarkersonCD8Tcells,possiblyexplainingthepooranamnesticpotential

seenwithAd5immunization.Similarly,increasedPD-1expressionwasalsoobservedon

“helpless”memoryCD8TcellscomparedtomemoryCD8TcellsprimedinthepresenceofCD4

Tcells102.Interestingly,PD-1blockadeduringantigenre-exposureenhancedexpansionofthese

‘helpless’memoryCD8Tcells102,indicatingthatPD-1maylimitmemoryCD8Tcellexpansion.

13

Furthermore,PD-1blockadehasbeenshowntorestorepulmonaryCD8Teffectorfunctions

(degranulationandcytokineproduction)andenhancedviralclearanceinamodelofrespiratory

virusreinfection103.Itisnotknownwhetherasimilarimprovedrecallexpansionandrestoration

ofpolyfunctionalitywillbeseenfollowingcheckpointblockadeinthecontextofAd5

immunization.

1.6.3 ConventionalandRegulatoryCD4Tcells

ItiswellestablishedthatconventionalCD4TcellhelpisrequiredformemoryCD8Tcell

differentiationfollowingnaturalinfection104,105orvaccination106.IthasbeenreportedthatAd5

inducesadysfunctionalCD4TcellresponsecharacterizedbyhighPD-1expressionandIL-10

expression80,whileanalternativeserotypeadenovirus,Ad26,whichinducesamorefavorable

CD8Tcellresponsewithbetterphenotypicandqualitativeprofileefficientlyelicitsfunctional

CD4Tresponse79.TheimpairedCD4TcellhelpassociatedwithAd5maythereforebe

responsibleforAd5-inducedCD8Tcelldysfunction.Ontheotherhand,regulatoryCD4Tcells

(Tregs),whoseprimaryroleistocontrolanddampenimmuneresponse,havebeenshownto

helpinmaintainingCD8Tcellexhaustion,andtheablationofTregsinmicechronicallyinfected

withLCMVledtoasignificantrescueandexpansionofexhaustedLCMV-specificCD8Tcells107.

SinceAd5-inducedimmunedysfunctionexhibitssomeexhaustedTcellphenotype,itisnot

impossiblethatregulatoryTcellsmayplayaroleindrivingthisexhaustion.Thisishoweveryet

tobedetermined.

14

1.6.4 AntigenPresentingCells,Co-stimulatoryandCo-inhibitoryMolecules

Adenoviralvectorsdifferentiallyinteractwithantigenpresentingcells(APCs)suchas

dendriticcells(DCs)55duetotheirdistinctinvivobiologicalproperties108,109.Ithasbeenshown

thatAdvectorsformimmunecomplexeswithspecificneutralizingantibodieswhichaffect

transductionefficiencyandmaturationofdendriticcellsinaFcRandToll-likereceptor9(TLR9)

interaction68.ComparativeevaluationofthepotencyofimmunecomplexesformedbyAd5and

alternativeserotypeadenovirusvectorssuchasAd26andAd36showedthatimmune

complexesofAd5werestrongerinducersofDCmaturation(asmeasuredbytheup-regulation

ofco-stimulatorymoleculesandproductionofproinflammatorycytokines)comparedtoAd26

andAd35immunecomplexes68.ItishowevernotknownifthestrongerDCinductioncombined

withthemoreprotractedpersistenceofAd5influencestheimpairedimmuneresponseseenin

Ad5.Theroleofco-stimulatory(e.g.B7-1)andco-inhibitorymolecules(e.g.PD-L1)expressed

onantigenpresentingcellsfollowingAdvectortransductionandhowtheymaycontributeto

theimpairedimmuneresponseisnotfullyunderstood.

1.7 Summary

Vaccine-mediatedprotectionagainstintracellularinfectionssuchasHIV-1,malariaand

tuberculosiswillmostlikelyrequirethegenerationofpotenthumoralandcellularimmune

responses.UntilitsfailureintheHIV-1vaccinetrial(STEPtrial),Ad5wasoneofthemost

promisingvectorsusedforelicitingsuchresponses.Evidenceisaccumulatingthattheimmune

responseelicitedbyAd5-basedvaccinevectorsexhibitsadysfunctionalphenotype,andthat

15

thisdysfunctionmayhavecontributedtoitsfailure.Assessingtheimmunologicalandbiological

cuesthataffectasimilarlyimpairedTcellresponseinthechronicviralinfection(LCMV)model

mayhelpuselucidatethemechanismofAd5-inducedimmunedysfunction.

16

1.8 Figures

1.8.1 FigureA:PotentialfactorsinfluencingAd5-inducedimmunedysfunction

Aschematicrepresentationofthepossiblefactorsinfluencingthedevelopmentand

maintenanceofAd5-inducedimmunedysfunction

Ad5-inducedimmune

dysfunction

Virologicalfactors:VectordoseRouteofAdministrationCellulartropism

CD8Tcellintrinsic:ImmunoinhibitoryreceptorsEpigeneticmodifiers

CD8Tcellextrinsic:ImpairedCD4 helpRegulatoryTcellsAPCs+co-stimulatoryandco-inhibitorymoleculesCytokinemilieu

17

TableA:SpectrumofexhaustedCD8Tstates

FullyFunctionalMemoryCD8Tcells(e.g.AcuteLCMVinfection,Ad26-InducedmemoryCD8Tcells)

Ad5-inducedmemoryCD8Tcells

PartiallyexhaustedCD8Tcells(e.g.EarlychronicLCMVinfection)

SeverelyExhaustedCD8Tcell(e.g.LateChronicLCMVinfection)

Antigenload -- ?-/+ ++ ++++CD4help +++ ?++ ++ ---

IL-2 ++ - - -TNF-α +++ ++/- +/- -IFN-y +++ ++ ++ -

Cytotoxicity +++ ++ + -InhibitoryReceptors + ++ ++ +++

ProliferativePotential +++ + + -

Apoptosis - - +/- ++++

Classicalimmuneexhaustioninchronicinfectionsandcancersischaracterizedbyastep-wiseand

progressivelossofeffectorcapabilities,thesustainedupregulationofinhibitoryreceptors,and

the loss of self-renewal capabilities. Ad5-elicited CD8 T cells lie in a spectrum between fully

functionalmemoryCD8Tcellsgeneratedfollowingtheclearanceofanacuteinfection(e.g.Acute

LCMVinfectionoralternativeserotypeadenoviral(e.g.Ad26)immunization)andfullyexhausted

CD8TcellsfromchronicLCMVinfection.Theroleofvectorload/persistenceandCD4Tcellhelp

inmodulatingAd5-inducedimmuneexhaustionhasnotyetbeenfullydetermined.(Adaptedand

modifiedfromKahanSMetal.Virology.201385)

18

2. Chapter2:DataandMethods

2.1 Introduction

Replication-incompetenthumanadenovirusserotype5(Ad5)vectorelicitspotentand

protectiveCD8+Tcellresponsesinpreclinicalstudies.However,adetailedevaluationofthe

immunologicalpropertiesofAd5inmouseandnon-humanprimateshasrevealedthatAd5

elicitsapartiallyexhaustedTcellresponse,characterizedbylowfrequencyofpolyfunctionalT

cells,highexpressionofimmunoinhibitoryreceptors,dysfunctionalcytokinesecretion,andan

impairedmemoryrecall.Recently,ithasbeenshownthatfactorssuchasvectorprimingdose

orantigenload,expressionofimmunoinhibitoryreceptors,andregulatoryTcellsmaymodulate

thephenotype,qualityandanamnesticpotentialofmemoryTcells.Inthisstudy,we

investigatedthepossibleroleofvectordose,PD-1signalingpathway,andregulatoryTcellsin

modulatingAd5-inducedcellularimmuneresponse.

2.1.1 Hypothesis

Vectordose,PD-1signalingandregulatoryTcellsmodulateAd5-inducedcellular

immuneresponse.

2.1.2 Specificresearchquestions:

1. IsitpossibletoovercometheimpairedanamnesticimmuneresponseassociatedwithAd5

byalteringtheprimingdose?

2. WhatistheeffectofimmunoinhibitoryreceptorblockadeontherecallofAd5-induced

memoryCD8Tcells?

3. IstherearoleforregulatoryTcellsinmodulatingtheAd5-elicitedimmunedysfunction?

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2.2 MaterialsandMethods

2.2.1 Miceandimmunizations

Sixto8-week-oldfemaleC57BL/6mice(fromJacksonLaboratories)wereusedforall

immunizationexperiments.Replication-incompetent,E1/E3-deletedAd5,Ad26andAd35

vectorsexpressingSIVmac239Gagwerepreparedaspreviouslydescribed71,110,whilemodified

vacciniaAnkara(MVA)expressingSIVGag-Pol-EnvwaspreparedbytheNationalInstitutesof

Health(NIH).Micewereimmunizedintramuscularlyinbothhindlegmuscles(quadriceps)with

escalatingdoses(107,108,109or1010viralparticles(vp))ofAd5orAd26vectorspermouse.

Micewereboosted60dayspostprimeintramuscularlywithMVAatadoseof≤107plaque-

formingunits(PFU)orAd35-SIVmac239Gag(109vp).Intramuscular(i.m)injectionswere

administeredin100μlphosphate-bufferedsaline(PBS)solution(50μlperquadricep).Animals

werehousedattheBethIsraelDeaconessMedicalCentre(BIDMC)animalfacilityandall

experimentswereperformedaccordingtoapprovedIACUCprotocol.

2.2.2 Isolationoflymphocytes

Single-cellsuspensionsofbloodandtissuesweregeneratedaspreviouslydescribed71,

111.Briefly,mousebloodwascollectedinRPMI1640containing40U/mlheparin.PBMCswere

isolatedfromwholebloodusingFicoll-Hypaquedensitycentrifugationat1900rpmfor20min.

Liver,lymphnodeandspleenwerehomogenizedtosinglecellsuspensionbyforcingtissue

chunkthrougha100µmsterilecellstrainerusingthebarrelofa5-ccsyringe.Redbloodcells

werelysedusingammoniumchloride(ACK)LysingBuffer(ThermoFischer).Resultingcell

20

suspensionwaspelleted,re-suspendedinRPMIcontaining10%fetalcalfserumand

penicillin/streptomycin.Cellswerecountedthereafter.Homogenizedlivertissuewasappliedto

a44/67%Percollgradientandcentrifugedat2,000rpmfor10minat20oC.Theinterface

containingtheintrahepaticlymphocytepopulationwasharvestedandwashed,andthe

resultinglymphocytessuspensionwasre-suspendedinRPMIcontaining10%fetalcalfserum

andpenicillin/streptomycin,andcounted.

2.2.3 Tetramerbindingassayandflowcytometry

MHCclassItetramerstainingwasperformedonsinglecellsuspensionusinganH-2Db

tetramerfoldedaroundtheimmunodominantSIVGagAL11epitope(AAVKNWMTQTL)as

describedpreviously112.BiotinylatedclassImonomerswereobtainedfromtheNational

InstitutesofHealthTetramerCoreFacility(EmoryUniversity,GA)andtetramerisedin-house.

BackgroundstainingofCD8Tcellsfromnaïveanimalswas≤0.1%.Surfacestainingwas

performedwithanti-CD8a(53-6.7),-CD44(IM7),-CD127(A7R34),-CD62L(MEL-14),-KLRG1

(2F1)and-PD-1(RMP1-30).Transcriptionfactorstainingwasperformedbyfirstpermeabilizing

thecellswiththeFoxP3Fixation/PermeabilizationKit(eBioscience)andsubsequentlystaining

withanti-T-bet(4B10),–Eomes(Dan11mag)and–FoxP3(150D/E4).AllAntibodieswere

purchasedfromBDBiosciences,eBioscience,orBioLegend.Live/DeadFixableNear-IRstaining

kitwasobtainedfromLifeTechnologies.Afterstaining,cellswerewashedinPBScontaining2%

fetalbovineserum(FBS)andfixedusingcytofix/cytopermbuffer(BDBioscience).Sampleswere

acquiredonanLSRIIflowcytometer(BDBiosciences),anddatawereanalyzedusingFlowJo

21

version9.7.7(TreeStar).

2.2.4 Intracellularcytokinestaining(ICS)assays

CytokineexpressionofGag-specificcellularimmuneresponsesinimmunizedmicewas

assessedbymultiparameterintracellularcytokinestaining(ICS)assaysaspreviously

described110,withsomemodifications.Briefly,lymphocytesisolatedfromtheblood(~5x105),

spleen(106)ortheliver(106)wereincubatedfor5hoursat37°Cwith0.2μg/mlofSIVGag

peptidepool(togetherwithbrefeldinA(GolgiPlug)andmonensin(GolgiStop).Afterincubation,

cellswerestainedwithanti-CD8(53-6.7),-CD44PacificBlue(IM7),-CD4(RM4-5)andLive/Dead

FixableNear-IRstainingkit.Afterwards,cellswerefixedandpermeabilizedwith

Cytofix/Cytopermpriortointracellularstainingwithanti-IFN-γ(XMG1.2),anti-TNF(MP6-XT22),

andanti-IL-2(JES6-5H4).AllICSreagentswerepurchasedfromBDBiosciences.

2.2.5 Monoclonalantibodiestreatments

PD-L1blockadewasachievedviainjectionof200microgram10F.9G2(BioXCell)atthe

timeofprimingorboostingimmunization.Theregimenconsistedoffivedosesevery3daysas

previouslydescribed100,withthefirstdosegivenadaypriortoimmmunization.Allantibodies

(ab)weredilutedinDPBSandadministeredintraperitoneally(i.p).RatIgG2b(BioXCell)was

usedascontrol.MemoryCD8Tcellrecallresponseswereassessedinbloodonday7,andin

bloodandtissuesonday15postboost.

22

2.2.6 InvitroTregsuppressionassay

InvitroTregsuppressionassaywasperformedaspreviouslydescribed113,withsome

modifications.Briefly,CD4Tcellswereenrichedbynegativemagneticselectionfromthe

spleenofadenoviralvector-immunizedFoxp3gfpmice(pooledfrom2spleenspergroup)using

anEasySep™MouseCD4TCellIsolationKit(StemcellTechnologies).AfterenrichmentofCD4T

cells,FoxP3+GFP+CD4TcellsweresortedusingaFACSAriaIIcellsorter(BDBioscience).Naive

CD8TcellswereisolatedfromthespleenofC57BL/6mice,labeledwithCellTraceViolet(CTV)

dye(5μM;LifeTechnologies)for10minatroomtemperatureinPBSandwashedwith

completemediabeforeculture.Forthesuppressionassay,2.5x104CTV-labelledCD8Tcells

wereco-culturedwithDynabeads®MouseT-ActivatorCD3/CD28beads(ThermoFischer

Scientific)ataratioof4to5(beads:CD8Tcells),andvaryingnumbers(0,1.5x103,3x103,6x103,

1.25x104,2.5x104)ofsortedFoxP3+GFP+CD4Tcellsperwellina96-wellflatbottomplate.After

72hofincubationat37oC,cellswereharvested,stainedwithanti-CD8a(53-6.7),-CD44(IM7),

and-CD4(RM4-5),-CD25(3C7)andLive/DeadFixableNear-IRstainingkit(RMP1-30).CD8T

cellswereanalyzedforCTVdilutionbyflowcytometry.

2.2.7 Statisticalanalysis

StatisticalanalysiswasperformedonGraphPadPrismversion6.0husingtwo-tailed

nonparametricMann-WhitneyUtest.

23

2.3 Results

2.3.1 Thekinetics,phenotypeandfunctionalityofvaccine-inducedCD8Tcellsfollowing

vaccinationwithdifferentdosesofAd5

Weinitiatedstudiesbyassessingthemagnitude,phenotypeandqualityoftransgene-

specificCD8TcellselicitedbydifferentdosesofAd5vector.Weimmunized6-8weekold

C57BL/6miceintramuscularly(i.m)withescalatingdosesofAd5(108,109and1010vp)

expressingtheGagantigenfromSIVstrainmac239.Gag-specificCD8Tcellresponsesin

peripheralbloodwereassessedlongitudinallyusinganMHCclassItetramerloadedwiththe

immunodominantepitopeAL11112.Micevaccinatedwith1010vpofAd26expressingthesame

antigenalsowereincludedascontrol(Fig.1A).Consistentwithpreviousreports78,81,alldoses

ofAd5elicitedsimilarpeakCD8Tcellresponsesatday15post-prime,butthelowdoseAd5

prime(108vp)wasassociatedwithmorepronouncedCD8Tcellcontractioncomparedtohigh

doseAd5prime(1010)(p<0.001)(Fig.1B-1C).

Next,weexaminedthephenotypeofDb-AL11+CD8Tcellsfollowingvaccinationwith

differentdosesofAd5.IthaspreviouslybeenshownthatAd5,comparedtoalternateserotype

AdvectorssuchasAd26andAd35,leadstoanup-regulationoftheimmunoinhibitoryreceptor,

PD-1onvaccine-elicitedCD8Tcells23,81.WethereforesoughttoassesswhetherPD-1up-

regulationonAd5-elicitedCD8Tcellsisdose-dependent.Wenoticedthatantigen-specificCD8

TcellsinmiceprimedwithlowdoseAd5expressedsignificantlylowerPD-1relativetohigh

doseAd5-primedmiceatthepeakoftheprimaryresponse(day15postimmunization(p.i))

24

(p<0.0001;Fig.2A)andatmemorytime-point(day60p.i)(p=0.0059;Fig.2B),suggestingthat

PD-1expressiononAd5-elicitedCD8Tcellsisdose-dependent.

Wefurtherassessedthecellsurfaceexpressionofkillercelllectin-likereceptorG1

(KLRG1)andIL-7receptoralpha-chain(CD127),twomarkersoftenusedtodelineateterminal

(KLRG1+CD127-)andmemoryprecursor(KLRG1-CD127+)effectorCD8Tcellsinmice114,115.We

foundthat,onday60postimmunization,theproportionofKLRG1+CD127–memoryprecursor

effectorcells(MPECs)inbloodwasreducedinthelowdoseAd5-immunizedgroupcompared

withthehighdosegroup(Ad5108vp,18%MPECs;Ad51010vp,10%MPECs;p<0.05),while

therewasacorrespondinghigherproportionofKLRG1+CD127–shortlivedeffectorcells(SLECs)

inmiceimmunizedwithhighdoseAd5relativetolowdoseAd5-immunizedmice(Ad5108vp,

45%MPECs;Ad51010vp,50%MPECs;p<0.05)(Fig.2C).Thissuggeststhatthereisa

preferentialdifferentiationofvaccine-inducedeffectorCD8Tcellstowardsthememory

precursorlineagefollowinglowdoseAd5immunizationcomparedwithhighdoseAd5

immunization.

PolyfunctionalTcellsareTcellswhichsecretemultiplecytokinesconcurrently.They

havebeenshowntoplayanessentialroleinthecontrolofHIVinfection,andcorrelatewith

protectioninsomevaccinemodels2,116,117,118.Therefore,weassessedtheabilityofvaccine-

elicitedmemoryCD8Tcellstosecretemultiplecytokinesfollowingex-vivoantigenre-

stimulationwithanoverlappingSIVGagpeptidepool.WeobservedthatlowdoseAd5induces

ahigherfrequencyofGag-specificCD8Tcellsco-expressingtwo(IFN-γandTNF-α)orthree

(IFN-γ,TNF-αandIL-2)cytokines,whilehighdoseAd5leadstotheinductionofmoreCD8T

25

cellsexpressingonlyIFN-γrelativetolowdoseAd5(P=0.0029(forAd5108vpversusAd51010

vp;IFN-γ,TNF-αandIL-2co-producers);P<0.0001(forAd5108vpversusAd51010vp;IFN-γand

TNF-αco-producers);P<0.0001(forAd5108vpversusAd51010vp;IFN-γonly)(Fig.2D).Forall

parametersexamined,thephenotypeandqualityofCD8TcellsinducedbylowdoseAd5

closelyresemblesthoseofAd26-inducedCD8Tcells(Fig.1B-1C;Fig.2A-2D).Altogether,these

findingsbuildonpreviousreportsbyshowingthatlowdoseAd5vectorisassimilarly

immunogenicashighdoseAd5andyetelicitsamorefunctionaltransgene-specificCD8Tcell

populationcharacterizedbylowerPD-1expression,enhancedmemoryconversionand

improvedfunctionality.

2.3.2 ImprovedanamnesticCD8TcellresponsesaftervaccinationwithlowdoseAd5vector

Ithaspreviouslybeenshownthat,comparedwithalternativeadenovirusserotype,Ad5-

inducedCD8Tcellsexhibitreducedanamnesticexpansionfollowingaheterologousvector

boost23,81orasecondaryantigenchallenge119.Wethereforesoughttoassesstherecall

expansionofthephenotypicallyfavorableCD8TcellinducedbylowdoseAd5comparedwith

CD8TcellsinducedbyhighdoseAd5.MicevaccinatedwithescalatingdosesofAd5orafixed

doseofAd26vectorsexpressingSIVGagwereboostedwithAd35vectorexpressingthesame

SIVGagtransgene(Fig.2A).Intriguingly,weobservedarobustrecallexpansionoflow-doseAd5

primedCD8TcellscomparedwithhighdoseAd5primebyday7post-boost(difference

betweenpreandpost-boostforlowdoseAd5was5.37-fold;forhighdoseAd5,1.92-fold;a2.8-

folddifference,p=0.0012)(Fig.3B-3D).Similarly,onday14and30postboost,therewasa

26

trendtowardsahighernumberofGag-specificCD8Tcellsinthegroupprimedwithlowdose

Ad5comparedwithhighdoseAd5-primedmice,althoughthiswasnotsignificant(datanot

shown)

Onday7postboost,weanalyzedthephenotypeofantigen-specificCD8Tcellsinblood.

WeobservedahigherexpressionofKi-67,amarkerofproliferativepotential,onGag-specific

CD8TcellsfromlowdoseAd5-primedmicecomparedwithhighdoseAd5-primed(Fig.2E)(p=

0.029),correlatingwiththeimprovedrecallexpansionseeninthelowdosegroup.Additionally,

granzymeBexpressiononGag-specificCD8TcellsinlowdoseAd5-vaccinatedmicewasgreater

thanthoseofhighdoseAd5-vaccinatedmicefollowingboost,suggestingimprovedcytotoxic

potential(Fig.2F)(p=0.029).Furthermore,antigen-specificCD8Tcellsinmiceprimedwithlow

doseAd5expressedsignificantlylowerPD-1relativetohighdoseAd5-primedmicefollowing

boost(Fig.3G)(p<0.0005).Nosignificantdifferenceswereobservedamongdosegroupsinthe

expressionofotherphenotypicmarkerssuchasCD127,KLRG-1orthelymphoidhoming

receptorCD62L(datanotshown).

Todeterminethefunctionalityofvaccine-elicitedCD8Tcellsfollowingheterologous

boost,weisolatedsplenocytesonday30postboostandanalyzedtheirabilitytoexpressIFN-γ,

TNF-αandIL-2concurrentlyafterre-stimulationwithGagpeptide.Weshowedthatthenumber

ofindividualvaccineelicitedCD8Tcellsco-expressingthreecytokineswassignificantlygreater

inlowdoseAd5-primedmicecomparedwithhighdoseAd5-primedmice(Fig.4A-4C).Inline

withthisfinding,wealsoobservedthatthefrequencyofGag-specificCD8Tcellsco-producing

IFN-γandTNF-αinmiceprimedwithlowdoseAd5was2.4%comparedwith0.8%inlowdose

27

Ad5-primedmice,a3folddifference(P=0.031),whilethefrequencyofGag-specificCD8Tcells

co-producingIFN-γandIL-2was4foldhigherinlowdoseAd5-primedmicethaninhighdose

Ad5primedmice(%ofCD8Tcellsco-producingIFN-γandIL-2:mean(lowdoseAd5)=0.3%,

mean(lowdoseAd5)=0.07%;P=0.0079).Representativeflowcytometryplotsofcytokineco-

expressiononGag-specificCD8Tcellsareshown(Fig.4D).Furthermore,wefoundthatthe

proportionofcellsco-expressingallthreecytokines(IFN-γ,TNF-αandIL-2)wassignificantly

higherinthelowdosegroupcomparedwiththehighdose(P=0.008)(Fig.4E).However,there

wasnodifferenceinthepercellexpressionofthesecytokines(datanotshown).Similar

findingswereseenfollowinganMVAboost(datanotshown).Takentogether,thesedata

suggestthatloweringthedoseofAd5atprimewillimproveanamnesticrecallofvaccine-

inducedmemoryCD8Tcells,theircytotoxicpotential,andfunctionality,withoutcompromising

themagnitudeofitsprimaryresponse.

2.3.3 Excessiveup-regulationofPD-1onAd5-inducedmemoryCD8Tcellsimpairstheir

recallresponse

HigherPD-1expressiononmemoryCD8Tcellshaspreviouslybeenshowntocorrelate

withimpairedmemoryrecallinsomemodelsofimmuneexhaustion102.Wethereforereasoned

thatthehigherPD-1expressiononhighdoseAd5-inducedmemoryCD8Tcellsmaybe

responsibleforthebluntingofrecallresponse.Datafrompre-clinicalandclinicalstudieshave

shownthatPD-1blockadecanbeachievedbyinvivoadministrationofeitheranti–PD-1oranti–

PD-1ligand(aPD-L1)100,120,121,122,123,124.ToassessthefunctionalsignificanceofPD-1expression

28

onAd5inducedmemoryCD8Tcells,weinhibitedthePD-L1:PD-1pathwaybyadministering

anti-PD-L1blockingantibody(aPD-L1)atthetimeofboostingwithaheterologousvector,Ad35

(Fig.5A).MicevaccinatedwithAd26wereusedascontrolsinceCD8Tcellselicitedbythis

vectorarenotassociatedwithhighPD-1expression23.Treatmentwithanti-PD-L1significantly

enhancedtherecallresponseofAd5-inducedmemoryCD8Tcellsfollowingboost,ascompared

withcontrols(3.8foldexpansioninPD-L1blockadegroup,comparedwith1.6foldexpansionin

isotypecontrolgroup,P=0.0317)(Fig.5B-5C).SimilartreatmentofAd26-inducedmemoryCD8

Tcellsdoesnotaltermemoryrecall.Thissuggeststhattheimprovementinrecallresponse

observedfollowinganti-PD-L1treatmentcanonlyoccurinthecontextofhighPD-1expression.

Wealsodidnotobserveanydifferentialup-regulationofPD-L1expressiononTcellsorDCs

followingAd5vaccinationcomparedtoAd26immunization(datanotshown),suggestingthat

theeffectisaCD8Tcellintrinsicphenomenon.Therewerenodifferencesinexpressionprofiles

ofphenotypicmarkerssuchasCD62L,CD127,orKLRG-1ontheexpandedGag-specificmemory

CD8Tbetweenbothtreatmentgroups(datanotshown).Onday14postboost,weisolated

splenocytesandassessedthefunctionalityofGagspecificCD8Tcellsasdescribedearlier.Inthe

Ad5-immunizedgroup,wefoundahigherfrequencyofIFN-γ-producingGag-specificCD8Tcells

(P=0.04)andTNF-αproducingGag-specificCD8Tcells(P=0.03)followinganti-PD-L1-treatment

comparedtountreated(Fig.5Dand5E).TherewasnosignificantdifferenceinfrequencyofIL-

2-producingGag-specificCD8Tcellsbetweenbothgroups(Fig.5F).However,therewasatrend

towardsamorepolyfunctionalantigen-specificCD8Tcellsinthetreatmentgroupcompared

withthoseuntreated(Fig.5G).

29

TodeterminetheeffectofblockingPD-1signalingpathwayontheprimaryimmune

responsesofAd5vectorandassessifthisearlyblockadewillleadtoasimilarimprovementin

memoryrecallasseenwhenthePD-1pathwaywasblockedatalatertime-point,we

administeredanti-PD-L1treatmentatthetimeofAd5prime(Fig.6A).Micewerefollowed

longitudinallytoassesstetramerkineticsinblood,andboostedwithAd35onday60post

immunizationtoassessmemoryrecall.WeobservedthatPD-1blockadedoesnotalterthe

kineticsofprimaryresponseinbothAd5-orAd26-primedmice.Inaddition,therewasno

differenceinexpressionlevelofsurfacemarkerslikeCD62L,CD127andKLRG-1(datanot

shown),andcytokineco-expressionongag-specificCD8Tcellswassimilarinbothtreatedand

controlgroup(Fig.6C).Inaddition,PD-L1blockadeatprimedoesnotleadtoasignificant

enhancementofmemoryrecallfollowingaheterologousAd35boost(Fig.6Eand6F).Taken

together,thesedatasuggestthattheestablishmentofan“immunedysfunction”state

characterizedbyup-regulationofPD-1isrequiredforPD-1blockadetohaveasignificanteffect

onrecallresponse

2.3.4 SimilarTregsfrequencyandsuppressivefunctioninAd5-andAd26-immunizedmice

RegulatoryTcellshavebeenshowntomodulateCD8Tcellactivation,proliferation,

differentiationandmemoryrecall125.IncreasedfrequencyandsuppressivefunctionofFoxP3+

CD4TcellshavebeenreportedinLCMVclone13infection,amousemodelofchronicinfection,

inwhichPD-1isup-regulated107,126.AsapreludetodeterminingwhetherTregsmayplayarole

intheimpairedmemoryrecallassociatedwithAd5-elicitedCD8Tcells,wesoughttodetermine

30

whetherTregsfollowingAd5immunizationaredifferentiallyactivatedcomparedtoalternative

serotypeAdvectorimmunizedmice.WevaccinatedFoxP3gfpmicewithAd5expressingSIVGag

orAd26expressingSIVGagandassessedthefrequencyofFoxP3+CD4Tcellsatthepeakof

infection(day15p.i)(Fig.7A).TregsisolatedfromnaïvemiceorLCMVclone13infectedmice

servedascontrols.Asexpected,thefrequencyofTregswassignificantlyhigherinLCMVclone

13infectedmicecomparedtonaïveorAd-immunizedmice.However,thenumberand

frequencyofFoxP3+CD4TcellsinthespleenandbloodofAd5immunized,Ad26immunized

andnaïvemicewassimilar.RepresentativeflowcytometryplotsareshowninFig.7A.Toassess

whetherthereisadifferenceinthesuppressivecapacityofTregsinducedbythesevectors,we

isolatedsplenocytesfromAd5orAd26vaccinatedmice,enrichedforCD4Tcellsfollowedby

FACSsortingforGFP+Tregs.Weco-cultureisolatedTregswithCellTraceViolet(CTV)-labeled

naiveCD8Tcells(targetcells)isolatedfromnaïvemice,inthepresenceofantiCD3/antiCD28

beads,andassessedCD8Tcellproliferation72hoursfollowingincubation.Interestingly,we

observedthatatpeakprimaryresponse(day15postimmunization),TregsfromAd5

immunizedandAd26immunizedmiceweresimilarlysuppressiveofCD8proliferation,with

bothbeinglesssuppressivecomparedwithnaïve,andTregsfromClone13infectedmicebeing

themostsuppressiveamongstallgroups(Fig.7B-7C).Althoughmorerigorousinvestigations

arerequired,theseresultssuggestthatregulatoryTcellsmaynotplayanimportantroleinthe

impairedimmunerecallseenwithAd5,furthersupportingthehypothesisthattheimpaired

immuneresponseseenfollowingAd5immunizationisprobablyaCD8Tcellintrinsic

phenomenon.

31

2.4 Figures

2.4.1 Figure1:Kineticsofvaccine-elicitedCD8Tcellfollowingvaccinationwithdifferent

dosesofAd5

(A)Experimentaloutline.(B)RepresentativeflowcytometryplotsshowingthepercentagesofGag-specificCD8TcellsinbloodfollowingimmunizationwithAd5-SIVGag(108,109,and1010)vporAd26-SIVGag(1010vp)(C)NumberofGag-specificCD8Tcellsinblood.Dataarerepresentativeofatleastthreeindependentexperiments,withn=4-5micepergroupperexperiment.Errorbarsrepresentstandarderrorsofthemeans(SEM).;***,P<0.001;ns;notsignificant.

32

2.4.2 Figure2:Phenotypeandpolyfunctionalityofvaccine-inducedCD8Tcell

followingvaccinationwithdifferentdosesofAd5.

(AandB)MFIofPD-1onGag-specificCD8Tcellsinbloodat(A)peaktimepoint(Day15p.i)andat(B)memorytimepoint(Day60p.i)followingimmunizationwithindicateddosesofAd5andAd26.(C)Proportion(%)ofGag-specificCD8TcellsthatexpressvariouscombinationsofCD127/KLRG1atday60followingAd5orAd26vaccination.(D)Proportion(%)oftotalGag-specificCD8Tcellsinbloodco-expressing3(IFN-γ,TNF-αandIL-2)cytokines,2(IFN-γandTNF-α)cytokinesor1(IFN-γ)cytokineatday60 followingAd5orAd26 vaccination.Data are representativeof at least three independentexperiments,withn=4-5micepergroupperexperiment.Errorbarsrepresentstandarderrorsofthemeans(SEM).*,P<0.05;**,P<0.01;***,P<0.001;****,P<0.001

Ad5 (108vp)

Ad5 (109vp)

Ad5 (1010vp)

Ad26 (1010vp)

0

100

200

300

400

500

PD-1

(MFI

)

****

*******

Day 15 Post-primeA

Ad5 (108vp)

Ad5 (109vp)

Ad5 (1010vp)

Ad26 (1010vp)

0

25

50

75

100

125

PD-1

(MFI

)

**

***

Day 60 Post-primeB

Ad5 108vp

45

186

31

Ad5 109vp

47

15

33

5

Ad5 1010vp

50

104

36

Ad26 1010vp

35

28

13

24

CD127- KLRG1+ (SLEC)CD127+ KLRG1- (MPEC)CD127+ KLRG1+CD127- KLRG1-

C

| |

+

| +

+

+ +

+

0

5

1020

40

60

80

100

% o

f G

ag-s

peci

fic C

D8+

T c

ells

pr

oduc

ing

indi

cate

d cy

toki

nes

IFN-γTNF-αIL-2

Ad5 (108vp)Ad5 (109vp)Ad5 (1010vp)Ad26 (1010vp)

**

****

****

**

**

ns

****

**

***

D

33

2.4.3 Figure3:LoweringtheprimingdoseofAd5vectorpartiallyimprovesthe

phenotypeandtherecallresponsesafterAd35boosting.

(A)Experimentaloutline.(B)RepresentativeFACSplotsshowingthepercentagesofGag-specificCD8TcellsinbloodpreandpostAd35boost.(C)NumberofGag-specificCD8Tcellsinblood.(D)Summaryshowingthefold-increaseofGag-specificCD8TcellsinbloodafterAd35boost.(E)MFIofKi-67onGag-specificCD8TcellsinbloodonDay7postAd35Boost.(F)MFIofGranzymeBonGag-specificCD8TcellsinbloodonDay7postAd35Boost.(G)MFIofPD-1onGag-specificCD8TcellsinbloodonDay7postboost.Dataarerepresentativeoftwoindependentexperiments,withn=4-5micepergroupperexperiment.Errorbarsrepresentstandarderrorsofthemeans(SEM).*,P<0.05;**,P<0.01;ns,notsignificant;vp,viralparticle.

34

2.4.4 Figure4:PrimingwithlowdoseAd5inaAd5-Ad35heterologousprime-boost

regimenproducemorepolyfunctionalCD8Tcells.

(A-C)NumberofsplenicGag-specificCD8Tcellsexpressing(A)IFN-γ,(B)TNF-α,and(C)IL-2onday30 followingAd35boost (D) RepresentativeFACSplots showing thepercentagesofGag-specificCD8Tcells inspleenco-expressing IFN-γandTNF-α,or IFN-γand IL-2 followingAd35boost.(E)Proportion(%)oftotalGag-specificCD8Tcellsinspleenco-expressing3(IFN-γ,TNF-αandIL-2)cytokines,2(IFN-γandTNF-α)cytokinesor1(IFN-γ)cytokineatday30followingAd35boost.Dataarerepresentativeoftwoindependentexperiments,withn=4-5micepergroupperexperiment.Errorbarsrepresentstandarderrorsofthemeans(SEM).*,P<0.05;**,P<0.01;ns,notsignificant;vp,viralparticle.

| |

+

| +

+

+ +

+

1

2

3

4

20

40

60

80

100

% o

f G

ag-s

peci

fic C

D8+

T c

ells

pr

oduc

ing

indi

cate

d cy

toki

nes

IFN-γ

TNF-αIL-2

**

Ad5 1 x 108 vp

Ad5 1 x 109 vp

ns

Ad5 1 x 1010 vp

Ad26 1 x 1010 vp

*

6.32 0.124

0.11193.4

5.33 0.0674

0.059994.5

7.86 0.219

0.060891.9

16.7 0.487

0.068382.7

4.41 2.04

0.14493.4

4.58 0.824

0.19194.4

6.45 1.63

0.11391.8

13.4 3.79

0.32782.5

TNF-! IL-2

IFN-γ

Ad5108vp

Ad5109vp

Ad51010

vp

Ad261010

vp

D

E

35

2.4.5 Figure5:Excessiveup-regulationofPD-1onAd5-inducedmemoryCD8Tcells

impairstheirrecallresponse.

(A)Experimentaloutline.(B)NumberofGag-specificCD8TcellsinbloodfollowingAd35boost+/-aPD-L1orIgGtreatment.(C)Summaryshowingthefold-increaseofGag-specificCD8TcellsinbloodafterAd35boost+/-aPD-L1orIgGtreatment.(D-F)PercentageofsplenicGag-specificCD8Tcellsexpressing(D)IFN-γ,(E)TNF-α,and(F)IL-2onday14followingAd35boost+/-aPD-L1orIgGtreatment.(G)Proportion(%)oftotalGag-specificCD8Tcellsinspleenco-expressing3(IFN-γ,TNF-αandIL-2)cytokines,2(IFN-γandTNF-α)cytokinesor1(IFN-γ)cytokineatday30following Ad35 boost in Ad5-primed mice. Data are representative of two independentexperiments,withn=4-5micepergroupperexperiment.Errorbarsrepresentstandarderrorsofthemeans(SEM).*,P<0.05;**,P<0.01;ns,notsignificant;vp,viralparticle.

36

2.4.6 Figure6:PD-L1blockadepriortoprimingdoesnotaffectthekinetics,memory

conversionoranamnesticresponseofAd5-inducedCD8Tcells

(A)Experimentaloutline.(B)NumberofGag-specificCD8TcellsinbloodfollowingAd5orAd26immunization +/- aPD-L1 or IgG treatment. (C) Representative FACS plots showing thepercentagesofGag-specificCD8Tcells inspleen(D60post immunization)co-expressingIFN-γand TNF-α, or IFN-γ and IL-2 following Ad5 or Ad26 immunization +/- aPD-L1 or control IgGtreatment.(D)NumberofGag-specificCD8TcellsinbloodfollowingAd35boost(E)Summaryshowing the fold-increase of Gag-specific CD8 T cells in blood after Ad35 boost. Data arerepresentative of two independent experiments,with n=4-5mice per groupper experiment.Errorbarsrepresentstandarderrorsofthemeans(SEM).ns,notsignificant;vp,viralparticle.

TNF-! IL-2

IFN-γ

37

2.4.7 Figure7:SimilarTregfrequenciesandsuppressivefunctionsinAd5-andAd26-

immunizedmice.

(A)Experimentaloutline.(B)RepresentativeFACSplotsshowingthepercentagesoffoxP3+CD4Tcellsinspleenonday15followingvaccinationwithAd51010vpandAd261010vp.ControlarenaïvemiceandLCMVclone13infectedmice.(C)SuppressionofCD8TcellproliferationbyTregsisolatedatday15followingvaccinationwithAd51010vpandAd261010vp.Numbersrepresentpercentageofproliferatingcells.(D)SummaryshowingpercentageproliferationofCD8Tcellsatday15followingvaccinationwithAd51010vpandAd261010vpDataarerepresentativeoftwoindependentexperiments,withn=4-5micepergroup.Suppressionassaywasdoneintriplicates.Errorbarsrepresentstandarderrorsofthemeans(SEM).;**,P<0.01;ns,notsignificant;vp,viralparticle.

38

3. Chapter3:Discussionandperspectives

TheinductionofhighmagnitudeCD8Tcellresponseshasbeenshowntocorrelatewith

vaccine-mediatedprotectionagainstebola,malariaandSIV3,4,127.Althoughasingle

administrationofanAdvectorcanelicitdetectableCD8Tcellresponses,heterologousprime-

boostregimensaretypicalusedtogeneratehighermagnituderesponsesinconventional

vaccinationregimes.Asastandalonevaccine,Ad5elicitshighmagnitudeCD8Tcellresponse

thatconfersomeprotectioninpreclinicalstudies78.However,followingaheterologousboost,

theseprimaryCD8Tcellsexhibitanimpairedproliferativecapacity,whichsubsequently

impactsprotectiveefficacy23.FurtherevaluationhasrevealedthattheprimaryCD8Tcells

inducedbyAd5vaccinationdisplayapartiallyexhaustedphenotypicprofilecharacterizedby

highexpressionofimmunoinhibitoryreceptors,poorpolyfunctionality,andanimpaired

memoryconversion,comparablewithchronicLCMVinfectioninmice23,83.Inthisstudy,we

evaluatedthemechanismunderlyingAd5-inducedimpairedimmuneresponsesbyassessing

theeffectofreducingvectordose,blockingPD-1signalingpathway,andevaluatingtreg–

mediatedsuppressionfollowingAd5immunization.

First,wecorroboratedearlierfindingsthatthereductionofvectordosesignificantly

improvesthephenotypicandqualitativeprofilesoftransgene-specificCD8Tcellswithout

affectingthemagnitudeoftheprimaryimmuneresponse.Wethenextendedthisobservation

byshowingthatvectordosereductionandPD-1signalingblockaderesultinanimprovementin

Ad5-inducedimmunedysfunctionasevidencedbytheimprovedmemoryrecallfollowing

boost.Finally,weshowedthat,comparedtoalternativeserotypeadenovirus,Ad5

39

immunizationdoesnotdifferentiallyaffectthemagnitudeorsuppressivefunctionofTregs.

Takentogether,thesedataprovidesomemechanisticunderstandingofAd5-inducedimmune

dysfunction.

Inbroadterms,thefactorsinfluencingimmuneresponseelicitedbyAdvectorsmaybe

categorizedintovirologicalandimmunologicalfactors(FigureA).Possiblevirologicalfactors

includetheserotypeoftheAdvector,itscellularreceptorspecificity,intracellulartrafficking

routeemployedbythevector,thevectordoseorrouteofadministration.Intermsof

immunologicalfactorsinfluencingAd5-inducedimmunedysfunction,thesemayeitherbeCD8T

cellintrinsicorCD8Tcellextrinsic.Likelyintrinsicfactorsareimmunoinhibitoryreceptors

expressedonCD8Tcells,transcriptionfactors(e.g.T-betandEomes)orepigeneticfactors,

suchasDNAexpressionregulatoryenzymes(e.g.histonedeacetylases(HDAC)),similartowhat

hasbeenobservedinchronicLCMVinfection128,129.ExtrinsicCD8Tcellfactorsincludethe

innateimmunesystem,suchasinnatecytokineandchemokineprofilesorthesubsetof

dendriticcellstransduced.Otherextrinsicfactorsincludetheinductionofco-stimulatoryorco-

inhibitorymoleculese.g.PD-L1ontransducedantigenpresentingcells,ortheeffectofother

cellsoftheadaptiveimmunesystemsuchasconventionalCD4TcellsorregulatoryTcells.All

thesefactorsareknowntoplayimportantrolesinthedevelopmentandmaintenanceofTcell

exhaustioninclassicalmodelsofimmuneexhaustion.

Itisknownthatthedoseanddurationofantigenicstimulationprofoundlyaffectthe

phenotype,differentiation,functionality,andproliferativecapacityofeffectorTcells

produced130,131,132,133.ThetypicalprimaryCD8Tcellresponsefollowinganexposuretoa

40

rapidly-clearedantigenasseeninacuteinfectionorvaccinationcanbedividedintothree

phases:(1)anexpansionphase,characterizedbyactivationandrapidexpansionofantigen-

specificnaïvecellsintoeffectorTcells,thatmediateantigenclearance;followedby(2)a

contractionphase,inwhichapproximately90-95%oftheeffectorCD8T-cellpopulation

undergoapoptoticcelldeath,leavingasmallsubsetofcellthatgothroughthe(3)memory

differentiationandmaintenancephase,inwhichstable,polyfunctionalmemoryCD8Tcellsare

maintainedviahomeostaticproliferation134.Incontrast,persistentantigenstimulationasseen

inchronicinfectioninmiceusuallyleadstoamuchdelayedCD8Tcellcontractionphasedueto

antigenpersistence135.IthasbeenshownthatAd5immunizationleadstoaprotractedCD8T

cellexpansionphase,withlittleornoimmunecontraction23,81,83,84,136.Inourstudy,we

showedthatreducingthedoseofAd5leadstoamorepronouncedCD8Tcellcontraction,

similartowhatisobservedfollowingimmunizationwithalternativeserotypeAdvectors23,81,

MVA136,orfollowinganacuteLCMVinfection137.Ithasalsobeenshownthatthedurationand

persistenceofantigenicstimulationisdependentontheAdvectortypeandvectordosage138.

Although,ourstudydidnotdirectlyassessthepersistenceofantigenstimulationfollowing

administrationofdifferentdosesofAd5vector,wehypothesizethattheimprovedcontraction

weobservedislikelyduetoareductioninamountanddurationofantigenexpressionfollowing

lowdoseAd5immunization.

UsingtheLCMVmiceinfectionmodel,Joshietal.showedthateffectorCD8Tcellscanbe

differentiatedintoSLEC(CD127-KLRG1+)andMPEC(CD127+KLRG1-)populationsbasedon

expressionofbothCD127andKLRG1onCD8+Tcells114.SLECs,whichareshortlived,

41

predominateinsituationsofantigenpersistencebutundergosignificantapoptosisfollowing

antigenclearance,whileMPECsdevelopedintolong-lived,self-renewingmemoryCD8Tcells.In

addition,comparedtoSLECs,MPECsaremore“functionallymature”withanincreasedcapacity

toproliferateandproduceIL-2inadditiontoIFNγandTNF-α,i.e.greaterpolyfunctionality.We

observedthatalowdoseAd5immunizationleadstoahigherproportionofCD8Tcells

differentiatingintotherelativelymorestableMPECswithimprovedpolyfunctionality(IFN-

γ+TNF-α+IL-2+).Thisimprovedmemorydifferentiationprobablyresultedintheenhancedrecall

expansionweobservedfollowingheterologousboostingwithAd35(Fig,2C-D)orMVA(data

notshown).WealsoshowedthatthereisalowerpercellPD-1expressionfollowinglowdose

Ad5immunizationcomparedtohighdoseAd5.Sincethedurationofantigenicstimulationis

alsoknowntoaffecttheexpressionofthesemarkers139,140,141,142,wespeculatethattheoverall

improvementseenwithlowerdoseofAd5isaresultofreducedantigenpersistence.

TherouteofadministrationofAd5vectorimmunizationmayalsoinfluencetheimmune

responseelicited,suchthatintravenousadministrationleadstoamoredysfunctionalimmune

responsecomparedtoothermodesofadministration.Intravenousinjectionofadenovirus5

leadstoarapidaccumulationoftheviruswithintheliverduetoitshepatictropism143.High

viralloadintheliverleadstoTcellexhaustionanddepletionofantigen-specificcytotoxicT

lymphocytes144,145.Therefore,anotherplausibleexplanationforAd5-immunedysfunctionis

thathighdoseAd5immunizationresultsinasignificantlyhighernumberofviralparticles

migratingtotheliver,wheretheycancontributetothedevelopmentofdysfunctionalTcells.

Thispossibilityneedstobefurtherexplored.

42

ExhaustedTcellsseenduringchronicinfectionssuchasHIV,malaria,HCV,andHBVin

humans,orchronicLCMVinfectioninmiceareknowntoup-regulateimmunoinhibitory

receptorssuchasPD-1.ThePD-1/PD-L1signalingpathwayisinvolvedinnormalimmune

responses,andseveralregulatoryprocessesrangingfromtolerancetoTcellexhaustion.The

significantreductioninPD-1expressionfollowinglowdoseAd5immunization,coupledwiththe

improvedrecallresponsesuggeststhatthehigherPD-1expressionmaybemediatingthe

impairedrecallresponseassociatedwithhigh-doseAd5.Indeed,Fuseetel.showedthat

“helpless“CD8Tcells(withoutCD4Tcellshelp)highlyexpressPD-1andexhibitimproverecall

responsefollowingPD-1blockade102.Similarly,weobservedthatblockingPD-1signalingviaan

invivoPD-L1blockadeledtoanimprovedmemoryrecallfollowingsecondaryantigen

encounter.Interestingly,thisobservationwasseenonlywhentheblockadewasdoneatthe

timeofboosting,withnoeffectseenwithPD-L1blockadeatthetimeofprimingwithAd5.The

failureofPD-L1blockadeatprimecouldbeduetotheneedforan“exhausted”state

(upregulationofPD-1)tobeestablishedbeforetheeffectofblockingPD-L1:PD-1interaction

canbeseen.PD-L1isexpressedonTcells,Bcells,DCs,andisup-regulatedfollowingIFN-γ

treatment.However,therewasnodifferentialup-regulationofPD-L1ontransducedDCinthe

draininglymphnodefollowingAd5immunizationcomparedtoAd26immunization(Penaloza-

MacMaster,P,etal,unpublisheddata).OurdatathereforesuggestthattheintrinsicPD-1

signalingmaybepartlymediatingAd-5inducedimmunedysfunction.

Tregsplayamajorroleinmaintainingperipheraltolerance,inadditiontocontrolling

immuneresponsetoinfections.TheirroleinmaintainingTcellsinanexhaustedstatewas

43

demonstratedbythestrikingrescueofexhaustedTcellsfollowingTregsablation.Inacancer

vaccinemodel,vaccinationwithalphavirusvectorexpressinghumanpapillomavirus(HPV)type

16peptidesdidnotresultinchangesinthenumberand/oractivityofTregs,andthedepletion

ofTregsdidnotimprovetheefficacyofthisvaccineagainsttumours146.However,alphavirus

vectorelicitedimmuneresponsesarenotknowntoexhibitimmuneexhaustion.WithCD8T

cellsinducedbyAd5exhibitingapartialexhaustedphenotypeandthesignificantroleTregs

playinmaintainingexhaustedTcells,itispossiblethatregulatoryTcellsmaybecontributingto

theimpairedTcellsresponseseenfollowingAd5immunization.Ourstudydidnotfindany

differenceinthemagnitudeandsuppressivefunctionofTregsfollowingAd5orAd26

immunization.WechoseAd26ascontrolbecauseitdoesnotelicitadysfunctionalimmune

responselikeAd523.AlthoughfurtherstudiesareneededtocompletelyexcludearoleforTregs,

ourdataprovideahintthattheregulatoryTcellmaynotplayasignificantroleinmodulating

Ad5-inducedimmunedysfunction.

Insummary,ourdatasuggestthatantigenicstimulationandtheimmunoinhibitory

receptorPD-1playsasignificantroleinmodulatingAd5-inducedimmunedysfunction.The

absenceofaneffectofAd5immunizationonTregsandtheexpressionofPD-L1onantigen

presentingcellsalsosuggestthattheimmunedysfunctionseenwithAd5isprimarilyaCD8

intrinsicphenomenon.Althoughpre-existingvectorimmunitymayimpedetheuseofAd5in

futureclinicalvaccinetrials,particularlyinregionsofhighseroprevalence,themechanistic

implicationofthedatashownherewillbeusefulinselectingandtestingothersimilarviral

vectorsforfuturevaccinedevelopment.

44

3.1 Limitations

Theimmunogenicitydatapresentedinthisstudywereacquiredbyassessingimmune

responseslongitudinallyinblood,withveryfewevaluationoftheresponsesintissues.Itis

possiblethattheimmuneresponseinbloodmayslightlydifferfromthoseoftissues-both

lymphoidandnonlymphoidtissues.Thedecisiontofocusonbloodwasduetotheneedto

evaluatememoryrecall.Theassessmentoftissueimmuneresponsepriorofboostwillrequire

sacrificingtheanimalwhichwillprecludeaninternallycontrolledevaluationofthemagnitude

ofimmuneexpansion.Inaddition,theexperimentsreportedherewereallcarriedoutinmice.

Whethersimilarfindingswillbeseeninnon-humanprimatesandhumansisyettobe

determined.

3.2 Futuredirections

TheimprovedimmuneresponseseenfollowingimmunizationwithlowerdoseAd5maybe

duetoanacceleratedantigenclearance.Futureexperimentsmayutilizeinvivoimagingstudies,

transgenetranscriptPCRandmeasurementofinvivoproliferationofadoptivelytransfer

antigen-specificTcellstocomparethedurationoftransgeneexpressionfollowinglowdoseand

highdoseAd5immunization,andcomparingAd5tootherserotypeadenovirussuchasAd26.In

ourstudy,themagnitudeoftransgene-specificmemoryCD8Tcellswassignificantlyhigherin

highdoseAd5-primedmicecomparedtolowdoseAd5-primedmicepriortoheterologous

boostbecauseoftheprofoundimmunecontractionseenwithlowdoseAd5immunization.

Futureadoptivetransferexperimentswillnormalizeforthenumberofantigenspecificmemory

cellstransferredintocongenicallydistinctmicebeforeheterologousboost.Futureexperiments

45

willalsohelptodetermineifAd5-induceddysfunctionistrulyaCD8Tcellintrinsic

phenomenonorwhetheritisduetodifferencesintheimmuneenvironment.Inaddition,an

assessmentoftheamountofvectorreachingtheliverfollowingintramuscularimmunization

withhighdoseAd5,andthedegreeofhepatotoxicity,ifany,willhelptodetermineifliver

sequestrationofAd5vectorcontributestotheimpairedresponse.

WeshowherethatPD-L1blockadeleadstoanimprovementinimmunerecallfollowing

heterologousboosteventhoughPD-L1expressionisnotdifferentiallyup-regulatedonantigen

presentingcellsfollowingAd5comparedtoAd26immunization.Itisthereforeimportantto

determineiftreatmentwithPD-1blockingantibodywillleadtoasimilarimprovementin

immuneexpansion.Inaddition,futurestudiesshouldalsoassessifacombinationofdose

reductionandPD-1signalingblockadewillleadtoasynergisticimprovementinimmunerecall.

TheeffectoflowdoseAd5immunizationontheexpressionofotherimmmunoinhibitory

moleculessuchasTim-3andCTLA-4,andwhetherblockadeofthesereceptorswillsimilarly

leadtoimmunerescueshouldalsobedetermined.

OurpreliminaryfindingsalsosuggestthatTregsmaynotplayacriticalroleinmodulating

Ad5-inducedimmunedysfunction.Futureexperimentshoulddeterminetheeffectof

immunizationonTregmagnitudeandfunctionatlater(memory)timepoints.Itisalso

importanttodeterminemoreconclusivelytheroleofTregsbyassessingtheeffectofTreg

depletionusingtheFoxP3DTRmousemodel.Finally,andmostimportantly,futureresearch

shouldassesstheprotectiveefficacyoftheimprovedimmuneresponseobservedfollowinglow

doseAd5immunizationandPD-1signalingblockadeastheultimategoalofvaccinesisto

46

protectagainstpathogensorsignificantlylessendiseaseburden.

47

4. Bibliography

1. Kaech,S.M.,Wherry,E.J.&Ahmed,R.EffectorandmemoryT-celldifferentiation:implicationsforvaccinedevelopment.NatRevImmunol2,251-262(2002).

2. Betts,M.R.,Casazza,J.P.&Koup,R.A.MonitoringHIV-specificCD8+Tcellresponses

byintracellularcytokineproduction.ImmunolLett79,117-125(2001).3. Sullivan,N.J.etal.CD8+cellularimmunitymediatesrAd5vaccineprotectionagainst

Ebolavirusinfectionofnonhumanprimates.NatMed17,1128-1131(2011).4. Reyes-Sandoval,A.etal.Prime-boostimmunizationwithadenoviralandmodified

vacciniavirusAnkaravectorsenhancesthedurabilityandpolyfunctionalityofprotectivemalariaCD8+T-cellresponses.InfectImmun78,145-153(2010).

5. Behar,S.M.,Woodworth,J.S.&Wu,Y.Nextgeneration:tuberculosisvaccinesthat

elicitprotectiveCD8+Tcells.ExpertRevVaccines6,441-456(2007).6. Shoukry,N.H.etal.MemoryCD8+Tcellsarerequiredforprotectionfrompersistent

hepatitisCvirusinfection.JExpMed197,1645-1655(2003).7. Tang,D.C.,DeVit,M.&Johnston,S.A.Geneticimmunizationisasimplemethodfor

elicitinganimmuneresponse.Nature356,152-154(1992).8. Ulmer,J.B.etal.HeterologousprotectionagainstinfluenzabyinjectionofDNA

encodingaviralprotein.Science259,1745-1749(1993).9. Schalk,J.A.etal.PreclinicalandclinicalsafetystudiesonDNAvaccines.HumVaccin

2,45-53(2006).10. Wang,Y.etal.SerumamyloidPcomponentfacilitatesDNAclearanceandinhibits

plasmidtransfection:implicationsforhumanDNAvaccine.GeneTher19,70-77(2012).

11. Draper,S.J.&Heeney,J.L.Virusesasvaccinevectorsforinfectiousdiseasesand

cancer.NatRevMicrobiol8,62-73(2010).12. Barouch,D.H.&Picker,L.J.NovelvaccinevectorsforHIV-1.NatRevMicrobiol12,

765-771(2014).13. Skinner,M.A.,Laidlaw,S.M.,Eldaghayes,I.,Kaiser,P.&Cottingham,M.G.Fowlpox

virusasarecombinantvaccinevectorforuseinmammalsandpoultry.ExpertRevVaccines4,63-76(2005).

48

14. Xu,Y.etal.Prime-boostbacillusCalmette-Guerinvaccinationwithlentivirus-

vectoredandDNA-basedvaccinesexpressingantigensAg85BandRv3425improvesprotectiveefficacyagainstMycobacteriumtuberculosisinmice.Immunology143,277-286(2014).

15. Teigler,J.E.etal.ThecanarypoxvirusvectorALVACinducesdistinctcytokine

responsescomparedtothevacciniavirus-basedvectorsMVAandNYVACinrhesusmonkeys.JVirol88,1809-1814(2014).

16. Canarypoxvirusasavaccinevector.Lancet339,1448-1449(1992).17. Bonaldo,M.C.,Caufour,P.S.,Freire,M.S.&Galler,R.Theyellowfever17Dvaccine

virusasavectorfortheexpressionofforeignproteins:developmentofnewliveflavivirusvaccines.MemInstOswaldoCruz95Suppl1,215-223(2000).

18. Hashimoto,D.etal.Intratrachealadministrationofthird-generationlentivirus

vectorencodingMPT51fromMycobacteriumtuberculosisinducesspecificCD8+T-cellresponsesinthelung.Vaccine26,5095-5100(2008).

19. Howles,S.etal.VaccinationwithamodifiedvacciniavirusAnkara(MVA)-vectored

HIV-1immunogeninducesmodestvector-specificTcellresponsesinhumansubjects.Vaccine28,7306-7312(2010).

20. Moriya,C.etal.IntranasalSendaiviralvectorvaccinationismoreimmunogenicthan

intramuscularunderpre-existinganti-vectorantibodies.Vaccine29,8557-8563(2011).

21. Lasaro,M.O.&Ertl,H.C.Newinsightsonadenovirusasvaccinevectors.MolTher17,

1333-1339(2009).22. Tatsis,N.&Ertl,H.C.Adenovirusesasvaccinevectors.MolTher10,616-629(2004).23. Penaloza-MacMaster,P.etal.Alternativeserotypeadenovirusvaccinevectorselicit

memoryTcellswithenhancedanamnesticcapacitycomparedtoAd5vectors.JVirol87,1373-1384(2013).

24. Knipe,D.M.&Howley,P.M.Fieldsvirology,6thedn.WoltersKluwer/Lippincott

Williams&WilkinsHealth:Philadelphia,PA,2013.25. Robinson,C.M.etal.Molecularevolutionofhumanadenoviruses.SciRep3,1812

(2013).

49

26. Russell,W.C.Updateonadenovirusanditsvectors.JGenVirol81,2573-2604(2000).

27. Nevins,J.R.Controlofcellularandviraltranscriptionduringadenovirusinfection.

CRCcriticalreviewsinbiochemistry19,307-322(1986).28. Weitzman,M.D.FunctionsoftheadenovirusE4proteinsandtheirimpactonviral

vectors.Frontiersinbioscience:ajournalandvirtuallibrary10,1106-1117(2005).29. Burgert,H.G.etal.Subversionofhostdefensemechanismsbyadenoviruses.Current

topicsinmicrobiologyandimmunology269,273-318(2002).30. Larsson,S.,Svensson,C.&Akusjärvi,G.Controlofadenovirusmajorlategene

expressionatmultiplelevels.JournalofMolecularBiology225,287-298(1992).31. Windheim,M.,Hilgendorf,A.&Burgert,H.G.ImmuneevasionbyadenovirusE3

proteins:exploitationofintracellulartraffickingpathways.Currenttopicsinmicrobiologyandimmunology273,29-85(2004).

32. Rux,J.J.&Burnett,R.M.Adenovirusstructure.HumGeneTher15,1167-1176

(2004).33. Arnberg,N.,Edlund,K.,Kidd,A.H.&Wadell,G.Adenovirustype37usessialicacidas

acellularreceptor.JVirol74,42-48(2000).34. Philipson,L.&Pettersson,R.F.TheCoxsackie-AdenovirusReceptor—ANew

ReceptorintheImmunoglobulinFamilyInvolvedinCellAdhesion.In:Doerfler,W.&Böhm,P.(eds).Adenoviruses:ModelandVectorsinVirus-HostInteractions:ImmuneSystem,Oncogenesis,GeneTherapy.SpringerBerlinHeidelberg:Berlin,Heidelberg,2004,pp87-111.

35. Short,J.J.,Vasu,C.,Holterman,M.J.,Curiel,D.T.&Pereboev,A.Membersof

adenovirusspeciesButilizeCD80andCD86ascellularattachmentreceptors.VirusRes122,144-153(2006).

36. Fleischli,C.etal.SpeciesBadenovirusserotypes3,7,11and35sharesimilar

bindingsitesonthemembranecofactorproteinCD46receptor.JGenVirol88,2925-2934(2007).

37. Wang,H.etal.Desmoglein2isareceptorforadenovirusserotypes3,7,11and14.

NatMed17,96-104(2011).

50

38. Li,H.etal.Adenovirusserotype26utilizesCD46asaprimarycellularreceptorandonlytransientlyactivatesTlymphocytesfollowingvaccinationofrhesusmonkeys.JVirol86,10862-10865(2012).

39. Gaggar,A.,Shayakhmetov,D.M.&Lieber,A.CD46isacellularreceptorforgroupB

adenoviruses.NatMed9,1408-1412(2003).40. Zhang,Y.&Bergelson,J.M.Adenovirusreceptors.JVirol79,12125-12131(2005).41. Gastaldelli,M.etal.Infectiousadenovirustype2transportthroughearlybutnotlate

endosomes.Traffic(Copenhagen,Denmark)9,2265-2278(2008).42. Chardonnet,Y.&Dales,S.EarlyeventsintheinteractionofadenoviruseswithHeLa

cells.II.Comparativeobservationsonthepenetrationoftypes1,5,7,and12.Virology40,478-485(1970).

43. Miyazawa,N.etal.FiberswapbetweenadenovirussubgroupsBandCalters

intracellulartraffickingofadenovirusgenetransfervectors.JVirol73,6056-6065(1999).

44. Miyazawa,N.,Crystal,R.G.&Leopold,P.L.Adenovirusserotype7retentioninalate

endosomalcompartmentpriortocytosolescapeismodulatedbyfiberprotein.JVirol75,1387-1400(2001).

45. Rowe,W.P.,Huebner,R.J.,Gilmore,L.K.,Parrott,R.H.&Ward,T.G.Isolationofa

cytopathogenicagentfromhumanadenoidsundergoingspontaneousdegenerationintissueculture.ProceedingsoftheSocietyforExperimentalBiologyandMedicine.SocietyforExperimentalBiologyandMedicine(NewYork,N.Y.)84,570-573(1953).

46. Berget,S.M.,Moore,C.&Sharp,P.A.Splicedsegmentsatthe5'terminusof

adenovirus2latemRNA.ProcNatlAcadSciUSA74,3171-3175(1977).47. Weitzman,M.D.,Carson,C.T.,Schwartz,R.A.&Lilley,C.E.Interactionsofviruseswith

thecellularDNArepairmachinery.DNArepair3,1165-1173(2004).48. Crystal,R.G.Adenovirus:thefirsteffectiveinvivogenedeliveryvector.HumGene

Ther25,3-11(2014).49. Jooss,K.&Chirmule,N.Immunitytoadenovirusandadeno-associatedviralvectors:

implicationsforgenetherapy.GeneTher10,955-963(2003).50. Maxfield,L.F.etal.AttenuationofReplication-CompetentAdenovirusSerotype26

VaccinesbyVectorization.ClinVaccineImmunol22,1166-1175(2015).

51

51. Eloit,M.,Gilardi-Hebenstreit,P.,Toma,B.&Perricaudet,M.Constructionofadefectiveadenovirusvectorexpressingthepseudorabiesvirusglycoproteingp50anditsuseasalivevaccine.JGenVirol71(Pt10),2425-2431(1990).

52. Haut,L.H.etal.Robustgenitalgag-specificCD8+T-cellresponsesinmiceupon

intramuscularimmunizationwithsimianadenoviralvectorsexpressingHIV-1-gag.EurJImmunol40,3426-3438(2010).

53. Bangari,D.S.&Mittal,S.K.Developmentofnonhumanadenovirusesasvaccine

vectors.Vaccine24,849-862(2006).54. Sharpe,S.etal.Singleoralimmunizationwithreplicationdeficientrecombinant

adenoviruselicitslong-livedtransgene-specificcellularandhumoralimmuneresponses.Virology293,210-216(2002).

55. Lore,K.etal.Myeloidandplasmacytoiddendriticcellsaresusceptibleto

recombinantadenovirusvectorsandstimulatepolyfunctionalmemoryTcellresponses.JImmunol179,1721-1729(2007).

56. Xiang,Z.Q.,Yang,Y.,Wilson,J.M.&Ertl,H.C.Areplication-defectivehuman

adenovirusrecombinantservesasahighlyefficaciousvaccinecarrier.Virology219,220-227(1996).

57. Jaiswal,S.,Khanna,N.&Swaminathan,S.Replication-defectiveadenoviralvaccine

vectorfortheinductionofimmuneresponsestodenguevirustype2.JVirol77,12907-12913(2003).

58. Gao,W.etal.EffectsofaSARS-associatedcoronavirusvaccineinmonkeys.Lancet

362,1895-1896(2003).59. Sullivan,N.J.,Sanchez,A.,Rollin,P.E.,Yang,Z.Y.&Nabel,G.J.Developmentofa

preventivevaccineforEbolavirusinfectioninprimates.Nature408,605-609(2000).

60. Seong,Y.R.etal.ImmunogenicityoftheE1E2proteinsofhepatitisCvirusexpressed

byrecombinantadenoviruses.Vaccine19,2955-2964(2001).61. Duraiswamy,J.etal.InductionoftherapeuticT-cellresponsestosubdominant

tumor-associatedviraloncogeneafterimmunizationwithreplication-incompetentpolyepitopeadenovirusvaccine.CancerRes64,1483-1489(2004).

62. Pinto,A.R.etal.InductionofCD8+TcellstoanHIV-1antigenthroughaprimeboost

regimenwithheterologousE1-deletedadenoviralvaccinecarriers.JImmunol171,6774-6779(2003).

52

63. Lubeck,M.D.etal.Long-termprotectionofchimpanzeesagainsthigh-doseHIV-1

challengeinducedbyimmunization.NatMed3,651-658(1997).64. Wilson,N.A.etal.Vaccine-inducedcellularimmuneresponsesreduceplasmaviral

concentrationsafterrepeatedlow-dosechallengewithpathogenicsimianimmunodeficiencyvirusSIVmac239.JVirol80,5875-5885(2006).

65. Shiver,J.W.etal.Replication-incompetentadenoviralvaccinevectorelicitseffective

anti-immunodeficiency-virusimmunity.Nature415,331-335(2002).66. Gray,G.E.etal.SafetyandefficacyoftheHVTN503/Phambilistudyofaclade-B-

basedHIV-1vaccineinSouthAfrica:adouble-blind,randomised,placebo-controlledtest-of-conceptphase2bstudy.LancetInfectDis11,507-515(2011).

67. Buchbinder,S.P.etal.Efficacyassessmentofacell-mediatedimmunityHIV-1

vaccine(theStepStudy):adouble-blind,randomised,placebo-controlled,test-of-concepttrial.Lancet372,1881-1893(2008).

68. Perreau,M.,Pantaleo,G.&Kremer,E.J.Activationofadendriticcell-Tcellaxisby

Ad5immunecomplexescreatesanimprovedenvironmentforreplicationofHIVinTcells.JExpMed205,2717-2725(2008).

69. Benlahrech,A.etal.Adenovirusvectorvaccinationinducesexpansionofmemory

CD4TcellswithamucosalhomingphenotypethatarereadilysusceptibletoHIV-1.ProcNatlAcadSciUSA106,19940-19945(2009).

70. Hammer,S.M.etal.EfficacytrialofaDNA/rAd5HIV-1preventivevaccine.NEnglJ

Med369,2083-2092(2013).71. Abbink,P.etal.Comparativeseroprevalenceandimmunogenicityofsixrare

serotyperecombinantadenovirusvaccinevectorsfromsubgroupsBandD.JVirol81,4654-4663(2007).

72. Tatsis,N.etal.Chimpanzee-originadenovirusvectorsasvaccinecarriers.GeneTher

13,421-429(2006).73. Farina,S.F.etal.Replication-defectivevectorbasedonachimpanzeeadenovirus.J

Virol75,11603-11613(2001).74. Dudareva,M.etal.Prevalenceofserumneutralizingantibodiesagainstchimpanzee

adenovirus63andhumanadenovirus5inKenyanchildren,inthecontextofvaccinevectorefficacy.Vaccine27,3501-3504(2009).

53

75. Abbink,P.etal.Constructionandevaluationofnovelrhesusmonkeyadenovirusvaccinevectors.JVirol89,1512-1522(2015).

76. Roberts,D.M.etal.Hexon-chimaericadenovirusserotype5vectorscircumventpre-

existinganti-vectorimmunity.Nature441,239-243(2006).77. Schirmbeck,R.,Reimann,J.,Kochanek,S.&Kreppel,F.Theimmunogenicityof

adenovirusvectorslimitsthemultispecificityofCD8T-cellresponsestovector-encodedtransgenicantigens.MolTher16,1609-1616(2008).

78. Quinn,K.M.etal.Comparativeanalysisofthemagnitude,quality,phenotype,and

protectivecapacityofsimianimmunodeficiencyvirusgag-specificCD8+Tcellsfollowinghuman-,simian-,andchimpanzee-derivedrecombinantadenoviralvectorimmunization.JImmunol190,2720-2735(2013).

79. Liu,J.etal.Magnitudeandphenotypeofcellularimmuneresponseselicitedby

recombinantadenovirusvectorsandheterologousprime-boostregimensinrhesusmonkeys.JVirol82,4844-4852(2008).

80. Larocca,R.etal.Adenovirus5vaccinationelicitsIL-27,whichimpairsCD4andCD8

Tcellfunctionality(VAC12P.1113).TheJournalofImmunology194,213.214(2015).81. Tan,W.G.etal.Comparativeanalysisofsimianimmunodeficiencyvirusgag-specific

effectorandmemoryCD8+Tcellsinducedbydifferentadenovirusvectors.JVirol87,1359-1372(2013).

82. Teigler,J.E.etal.Hexonhypervariableregion-modifiedadenovirustype5(Ad5)

vectorsdisplayreducedhepatotoxicitybutinduceTlymphocytephenotypessimilartoAd5vectors.ClinVaccineImmunol21,1137-1144(2014).

83. Yang,T.C.etal.TheCD8+Tcellpopulationelicitedbyrecombinantadenovirus

displaysanovelpartiallyexhaustedphenotypeassociatedwithprolongedantigenpresentationthatnonethelessprovideslong-termimmunity.JImmunol176,200-210(2006).

84. Tatsis,N.etal.AdenoviralvectorspersistinvivoandmaintainactivatedCD8+T

cells:implicationsfortheiruseasvaccines.Blood110,1916-1923(2007).85. Kahan,S.M.,Wherry,E.J.&Zajac,A.J.Tcellexhaustionduringpersistentviral

infections.Virology479-480,180-193(2015).86. Kaech,S.M.&Ahmed,R.MemoryCD8+Tcelldifferentiation:initialantigen

encountertriggersadevelopmentalprograminnaivecells.NatImmunol2,415-422(2001).

54

87. Mueller,S.N.&Ahmed,R.HighantigenlevelsarethecauseofTcellexhaustion

duringchronicviralinfection.ProceedingsoftheNationalAcademyofSciences106,8623-8628(2009).

88. Richter,K.,Brocker,T.&Oxenius,A.AntigenamountdictatesCD8+T-cellexhaustion

duringchronicviralinfectionirrespectiveofthetypeofantigenpresentingcell.EuropeanJournalofImmunology42,2290-2304(2012).

89. Holst,P.J.,Ørskov,C.,Thomsen,A.R.&Christensen,J.P.QualityoftheTransgene-

SpecificCD8+TCellResponseInducedbyAdenoviralVectorImmunizationIsCriticallyInfluencedbyVirusDoseandRouteofVaccination.TheJournalofImmunology184,4431-4439(2010).

90. Odorizzi,P.M.&Wherry,E.J.Inhibitoryreceptorsonlymphocytes:insightsfrom

infections.JImmunol188,2957-2965(2012).91. Ishida,Y.,Agata,Y.,Shibahara,K.&Honjo,T.InducedexpressionofPD-1,anovel

memberoftheimmunoglobulingenesuperfamily,uponprogrammedcelldeath.Emboj11,3887-3895(1992).

92. Freeman,G.J.etal.EngagementofthePD-1immunoinhibitoryreceptorbyanovel

B7familymemberleadstonegativeregulationoflymphocyteactivation.JExpMed192,1027-1034(2000).

93. Latchman,Y.etal.PD-L2isasecondligandforPD-1andinhibitsTcellactivation.

NatImmunol2,261-268(2001).94. Yao,S.&Chen,L.PD-1asanimmunemodulatoryreceptor.CancerJ20,262-264

(2014).95. Wei,F.etal.StrengthofPD-1signalingdifferentiallyaffectsT-celleffectorfunctions.

ProcNatlAcadSciUSA110,E2480-2489(2013).96. Quigley,M.etal.TranscriptionalanalysisofHIV-specificCD8+Tcellsshowsthat

PD-1inhibitsTcellfunctionbyupregulatingBATF.NatMed16,1147-1151(2010).97. Chemnitz,J.M.,Parry,R.V.,Nichols,K.E.,June,C.H.&Riley,J.L.SHP-1andSHP-2

associatewithimmunoreceptortyrosine-basedswitchmotifofprogrammeddeath1uponprimaryhumanTcellstimulation,butonlyreceptorligationpreventsTcellactivation.JImmunol173,945-954(2004).

98. Riley,J.L.PD-1signalinginprimaryTcells.ImmunolRev229,114-125(2009).

55

99. Sheppard,K.A.etal.PD-1inhibitsT-cellreceptorinducedphosphorylationoftheZAP70/CD3zetasignalosomeanddownstreamsignalingtoPKCtheta.FEBSLett574,37-41(2004).

100. Barber,D.L.etal.RestoringfunctioninexhaustedCD8Tcellsduringchronicviral

infection.Nature439,682-687(2006).101. Cai,G.etal.PD-1ligands,negativeregulatorsforactivationofnaive,memory,and

recentlyactivatedhumanCD4+Tcells.CellImmunol230,89-98(2004).102. Fuse,S.etal.RecallresponsesbyhelplessmemoryCD8+Tcellsarerestrictedbythe

up-regulationofPD-1.JImmunol182,4244-4254(2009).103. Erickson,J.J.,Rogers,M.C.,Hastings,A.K.,Tollefson,S.J.&Williams,J.V.Programmed

death-1impairssecondaryeffectorlungCD8(+)Tcellsduringrespiratoryvirusreinfection.JImmunol193,5108-5117(2014).

104. Shedlock,D.J.&Shen,H.RequirementforCD4Tcellhelpingeneratingfunctional

CD8Tcellmemory.Science300,337-339(2003).105. Janssen,E.M.etal.CD4+Tcellsarerequiredforsecondaryexpansionandmemory

inCD8+Tlymphocytes.Nature421,852-856(2003).106. Provine,N.M.etal.LongitudinalrequirementforCD4+Tcellhelpforadenovirus

vector-elicitedCD8+Tcellresponses.JImmunol192,5214-5225(2014).107. Penaloza-MacMaster,P.etal.InterplaybetweenregulatoryTcellsandPD-1in

modulatingTcellexhaustionandviralcontrolduringchronicLCMVinfection.JExpMed211,1905-1918(2014).

108. Lemiale,F.etal.Noveladenovirusvaccinevectorsbasedontheenteric-tropic

serotype41.Vaccine25,2074-2084(2007).109. Perreau,M.etal.Contrastingeffectsofhuman,canine,andhybridadenovirus

vectorsonthephenotypicalandfunctionalmaturationofhumandendriticcells:implicationsforclinicalefficacy.JVirol81,3272-3284(2007).

110. Liu,J.etal.ModulationofDNAvaccine-elicitedCD8+T-lymphocyteepitope

immunodominancehierarchies.JVirol80,11991-11997(2006).111. Wherry,E.J.etal.LineagerelationshipandprotectiveimmunityofmemoryCD8T

cellsubsets.NatImmunol4,225-234(2003).

56

112. Barouch,D.H.etal.ImmunogenicityofRecombinantAdenovirusSerotype35VaccineinthePresenceofPre-ExistingAnti-Ad5Immunity.TheJournalofImmunology172,6290-6297(2004).

113. Brincks,E.L.etal.Antigen-specificmemoryregulatoryCD4+Foxp3+Tcellscontrol

memoryresponsestoinfluenzavirusinfection.JImmunol190,3438-3446(2013).114. Joshi,N.S.etal.Inflammationdirectsmemoryprecursorandshort-livedeffector

CD8(+)TcellfatesviathegradedexpressionofT-bettranscriptionfactor.Immunity27,281-295(2007).

115. Kaech,S.M.etal.Selectiveexpressionoftheinterleukin7receptoridentifieseffector

CD8Tcellsthatgiverisetolong-livedmemorycells.NatImmunol4,1191-1198(2003).

116. Beveridge,N.E.etal.ImmunisationwithBCGandrecombinantMVA85Ainduces

long-lasting,polyfunctionalMycobacteriumtuberculosis-specificCD4+memoryTlymphocytepopulations.EurJImmunol37,3089-3100(2007).

117. Darrah,P.A.etal.MultifunctionalTH1cellsdefineacorrelateofvaccine-mediated

protectionagainstLeishmaniamajor.NatMed13,843-850(2007).118. Seder,R.A.,Darrah,P.A.&Roederer,M.T-cellqualityinmemoryandprotection:

implicationsforvaccinedesign.NatRevImmunol8,247-258(2008).119. Penaloza-MacMaster,P.etal.Augmentedreplicativecapacityoftheboostingantigen

improvestheprotectiveefficacyofheterologousprime-boostvaccineregimens.JVirol88,6243-6254(2014).

120. Brown,K.E.,Freeman,G.J.,Wherry,E.J.&Sharpe,A.H.RoleofPD-1inregulating

acuteinfections.CurrOpinImmunol22,397-401(2010).121. West,E.E.etal.PD-L1blockadesynergizeswithIL-2therapyinreinvigorating

exhaustedTcells.JClinInvest123,2604-2615(2013).122. Brahmer,J.R.etal.Safetyandactivityofanti-PD-L1antibodyinpatientswith

advancedcancer.NEnglJMed366,2455-2465(2012).123. Topalian,S.L.etal.Safety,activity,andimmunecorrelatesofanti-PD-1antibodyin

cancer.NEnglJMed366,2443-2454(2012).124. Velu,V.etal.EnhancingSIV-specificimmunityinvivobyPD-1blockade.Nature

458,206-210(2009).

57

125. Josefowicz,S.Z.,Lu,L.F.&Rudensky,A.Y.RegulatoryTcells:mechanismsofdifferentiationandfunction.AnnuRevImmunol30,531-564(2012).

126. Park,H.J.etal.PD-1upregulatedonregulatoryTcellsduringchronicvirusinfection

enhancesthesuppressionofCD8+TcellimmuneresponseviatheinteractionwithPD-L1expressedonCD8+Tcells.JImmunol194,5801-5811(2015).

127. Barouch,D.H.etal.Protectiveefficacyofadenovirus/proteinvaccinesagainstSIV

challengesinrhesusmonkeys.Science349,320-324(2015).128. Wherry,E.J.&Kurachi,M.MolecularandcellularinsightsintoTcellexhaustion.Nat

RevImmunol15,486-499(2015).129. Zhang,F.etal.EpigeneticmanipulationrestoresfunctionsofdefectiveCD8(+)T

cellsfromchronicviralinfection.MolTher22,1698-1706(2014).130. Bachmann,M.F.etal.Long-livedmemoryCD8+Tcellsareprogrammedby

prolongedantigenexposureandlowlevelsofcellularactivation.EurJImmunol36,842-854(2006).

131. Badovinac,V.P.,Porter,B.B.&Harty,J.T.CD8+Tcellcontractioniscontrolledby

earlyinflammation.NatImmunol5,809-817(2004).132. Haring,J.S.,Badovinac,V.P.&Harty,J.T.InflamingtheCD8+Tcellresponse.

Immunity25,19-29(2006).133. Badovinac,V.P.&Harty,J.T.Programming,demarcating,andmanipulatingCD8+T-

cellmemory.ImmunolRev211,67-80(2006).134. Kalia,V.,Sarkar,S.&Ahmed,R.CD8T-cellmemorydifferentiationduringacuteand

chronicviralinfections,vol.684,2010.135. Wherry,E.J.,Blattman,J.N.,Murali-Krishna,K.,vanderMost,R.&Ahmed,R.Viral

persistencealtersCD8T-cellimmunodominanceandtissuedistributionandresultsindistinctstagesoffunctionalimpairment.JVirol77,4911-4927(2003).

136. Pillai,V.K.etal.Differentpatternsofexpansion,contractionandmemory

differentiationofHIV-1Gag-specificCD8Tcellselicitedbyadenovirustype5andmodifiedvacciniaAnkaravaccines.Vaccine29,5399-5406(2011).

137. Murali-Krishna,K.etal.Countingantigen-specificCD8Tcells:areevaluationof

bystanderactivationduringviralinfection.Immunity8,177-187(1998).

58

138. Quinn,K.M.etal.AntigenexpressiondeterminesadenoviralvaccinepotencyindependentofIFNandSTINGsignaling.JClinInvest125,1129-1146(2015).

139. Ibegbu,C.C.etal.Expressionofkillercelllectin-likereceptorG1onantigen-specific

humanCD8+Tlymphocytesduringactive,latent,andresolvedinfectionanditsrelationwithCD57.JImmunol174,6088-6094(2005).

140. Velu,V.etal.Elevatedexpressionlevelsofinhibitoryreceptorprogrammeddeath1

onsimianimmunodeficiencyvirus-specificCD8Tcellsduringchronicinfectionbutnotaftervaccination.JVirol81,5819-5828(2007).

141. Vollbrecht,T.etal.ImpactofchangesinantigenlevelonCD38/PD-1co-expression

onHIV-specificCD8Tcellsinchronic,untreatedHIV-1infection.JMedVirol82,358-370(2010).

142. Joshi,N.S.&Kaech,S.M.EffectorCD8Tcelldevelopment:abalancingactbetween

memorycellpotentialandterminaldifferentiation.JImmunol180,1309-1315(2008).

143. Alemany,R.&Curiel,D.T.CAR-bindingablationdoesnotchangebiodistributionand

toxicityofadenoviralvectors.GeneTher8,1347-1353(2001).144. Krebs,P.,Scandella,E.,Odermatt,B.&Ludewig,B.Rapidfunctionalexhaustionand

deletionofCTLfollowingimmunizationwithrecombinantadenovirus.JImmunol174,4559-4566(2005).

145. Crispe,I.N.,Dao,T.,Klugewitz,K.,Mehal,W.Z.&Metz,D.P.TheliverasasiteofT-cell

apoptosis:graveyard,orkillingfield?ImmunolRev174,47-62(2000).146. Walczak,M.etal.RoleofregulatoryT-cellsinimmunizationstrategiesinvolvinga

recombinantalphavirusvectorsystem.AntivirTher16,207-218(2011).