IIET EROCLITICPEPTID ESENI IANCEIL-2PRODUCTIONANDRECOGNITION BREADTI IOFIIIV -SPECIFIC CDl;+TCI:L L S
by
©Krista Gladlll'y
A thesis subm itted to theSchoo l ofGraduateStudies in partialfulfi llmentoft he requirementsfor the degreeof
Mast erofSciencein Medicine
Immunolo g yandInf ectio usDiseases/Biorn cd iculSc ien ce s/Facultyof Medicine MemorialUniversity ofNewfoundland
September20 12
St.John's Newfo undland
Abs t r act
Hctcroclitic pcptidcs arc pep tide sequence variants with enhanced inununogcnicit yrelativeto reference or wildtypepcptidcs.Intherapeutic cancer vaccines.hctcrocl itic pcptidcsdramaticallyaugmentcell-mediatedimmunityagainst tumourantigensbyactivatingcytotoxic'I'lymphocytes(CTL)against tumour-associated sclf-pcptidcs. l lctcrocl itic peptidcs could likewise contribute to human immunodeficiency virus(l lIV)immunothera py bybroaden ingCTL reactivity and/orby selectively stimulatinginterlcuk in (IL)-2-product ion (correlat edwith disease contro land non progression) .To investigate this possibility. we screened peripher alblood monon uclear cells(PBMC)froml llv-infcc tcdsubj ects for intcrfero n-y(lI:N-y)and IL-2 productionbyenzyme-linkedinununosorbcntspot(ELISPOT)assaywithpeptide pools spanning themajor11IVantigens.Poolsstimulating IL-2prod uct ionwere dcconvolutcd with peptide matrices10identifythe individua l lSmcrsres ponsible.Nc fX3~9 1(1\2 - 7 ).
cf13 5~143(1\2 - X).GagIX~27(1\3 -2)and Gag77~X 5(1\2-Gag) were confirmed as
optimally defined 9merpcptides stimulatingIL-2productionbyPBI'vICfromtwo or more l llv-infcctcdindivid ualsinourcohort.Twenty-fourpotentiallyhctcrocl itic variantswere generatedwithconservativeandsemi-conservative aminoacidsubstitutionsat position s 3.5and7 ofthese 9mers.Anadd itional24 variantswere genera tedina simi larfashion with Gag147 ~155 (1l57-1). Gag433 (1\2-9). and non-I IIV pcptid cs, 1\2-Flu (GILG FVFT L)and irrelevantpeptid e.1\2-11'(LLDV PTI\I\V),I:ighty-sixlllv-inlcctcd andsixlllv-uninlcc tcdsubj ectswerethen tested forCDX''I'cell reactivityagain stone or more of thevari ant sets byELiSPOTassay. at oneormore timepoints.l lctcrocl itic
pcptidesenhanced11.-2productionby CDS+T cellsin50%.orcasesin whichan 11.-2 responsewasdetected and enhancedIFN-yproductionin25%.orcases in which anIF-y responsewasdetected . Variants that increased IF-yand/or 11.-2 production in compa rison to index pcptidcswercfurther testedforhetc rocliticpropert iesincellculture.
Proliferation. differentiation and breadth or reactivity were assessed by curboxyf l uo rcsccinsuccinimidylester(CFSI:)dilution. surfaceandintracellular llow cytomctryand cytotoxicity assays.I lctcrocliticpcptidesstimulated broader reactivit y or peptide-stimulated CDS'T cells.reduced the exhaustedphenotypeorrespondingCDS'T cells.and. insomecases. enhancedproliferationor respondingTce lls,Ilctcrocliric propertiesor particul arpcptidcsvaried between individualsand. in mosteases.within individualsatdifferenttimepoints.Although we do notknowthemechani smsbywhich thesepeptide act. itappearsthat thesepcptidesstimulate thesamesubsetsor CDS'T cellsasrefere ncepcpt idcsbutsomehowexert differentialsignalingeffects.Further insight into thesemechanismswouldprovideinform ation on hcterocliticpeptide design forIIIV CDS'Tcellcpitopcsandcontribute to abasicunderstandingor the relationship between T cell receptor:pept ide interactionsandT cellfunct ion.
Acknnwle dgc mc nts
Special thankstomy supervisor.Dr.Michael Grant. torgivingme theopportunity to work in hislabandforallhisguidance.support and patiencethroughoutthisproject.I thankthe membersofmy committee.Dr.RodneyRussell andDr. Sheila Drover.fo rthis critiquesandencouragement.Thankyou to all the contributing mem berson thisthis project. atasha l lollctt,JuliaPohli ng, and Kutrin Zipperlcn.Thankyoutomylabmates.
Katr in Zippcrlcn,Stephen Penne y.MaureenGallant.NatalieCampbell.StuciStapleton.
and KaylaHarris. for making the laband the other roomenjoya ble working environments.Thanksto my classmates.Lisal.cxhanc andAlex Dancygcr.loralways cele bratingthegood times and listeningduringthehardtimes (GoTripod Unit!).Very spec ial thanksto Ada m Woodla ndfo rlisteningto allofmy science mumble jumb leand forallhislove andsupport. Lastly.I thank and dedicatethisthesistomyfam ily,Mom.
Dad.Eric. Erinand Rebecca.fortheircontinued love andsupport ineverything Ido.
TubleutCun tc nts
Abstract ii
Acknowledgements iv
ListofTables... ...ix
ListorFigures x
ListorAbbreviations xii
ListorAppendices xvi
Chapter \ Introduction \
1.\ IIIV-ISummary \
\.1.\ Structure...
\.1.2 Tropism... ...5
1.\.3 Lifecycle 5
1.\4 Diagnosis orIIIV infection... ...X
1.\.5 l'athogcncsis.. . 9
1.1.6 DiagnosisorAIDS... .. 11
\.1.7 Antirctrovirul thcrapy 12
I.\.X Viralpersistence 13
1.2 CDX+TCells...
1.\ . \ The'Icellreceptor...
....14
.. 15
1.2.2 Peptideprocessingandloadingonto MI ICclass1 17
\.2.3 Peptiderecognition andact ivat ionorCDX'Tcells 20
1.2.4 'L ccllselect ion 22
1.2.4 CDX'T cellresponsein IIIVinfcction.... .. 26
\.3 Thera pe uticvaccines 30 1.3.1 The need fora therape utic IIIV vaccinc.i..; . 31
1.3.2 l'cpti de-bascdvaccincs u
1.3.3 Iletcrodi tic pcptidcs-thc iru sc in thcrapcutic canccr vaccincs andpotential uscintherapeuticIIIVvaccines 35 1.3.4 Centralmemory'I'cellsas atarget oft hera peuticIIIV
vaccines 37
1.4 Preliminary work 3X
1.5 Speci ficaims... ...42
1.5.\ Identifypcptidcsthatselectivelyaugment IL-2production byl llV-spccificCDX'Tcellsrelative to index pcptidcs...42 1.5.2 Investigate whetherIL-2-inducinghctc rocliticIIIVpcptidcs
augment proliferation and differentiationofCDX'Tcclls....43
\.5.3 MeasureTCk/pcpridcavidityofI1.-2 inducinghctcroclitic IIIVpcptidcsrclativctoindexpcp tidcs 44 1.5.4 Testthe abilityof11.-2 inducinghctcrocl iticIIIVpcpt idcs
togene ratebroadlyreactiveCTL... . 44
Chapter2Materialsand Mcthods... ... 46
2.1 Studyparticipants...
')') I'BMCIsolation...
. 46
...47 2.3 lipstcin-Bnrrvirus(EBV) uunsfonu at ionofBcclls., . 47
2.4 lll.Atyping... . 4X
2.5 ELlSI'OTassays...
2.6 llcterocliticpcptidcs...
2.7 CTLassays...
...49
. 52
. 53
2.X Prol i ferationassaysand flow cytornc try... . 5X
2.9 Statistica lAnalysis (}O
Chapter3Results (}I
3.1 A comparisonofIFN-yand/or11.-2inductionby referenceand
potentiallyhctcroc l iticpeptides... . (}I
3.2 EvolutionofIF -yand 11.-2 responses... . X4 .'..' Re ference andvariantpcp tidesstimulateoverlappingsubsetsof
ens'
'Icells X7304 A comparisonofCDX+T cell proliferationinducedbyreference
and hct cr ocliti cpcpt idc s.; . X()
3.5 Acompari sonofC DX+T celldiff erent iationandactivation induced byreference and hetcrocliticpcptidcs., ....97 3.() A comparison ofCDX+Tcell cytotoxicity induced by reference
and hctcroclitic pcptidcs.; ...101
3.7 EvolutionofCTLresponses JO()
3.X A comparisonofTC R/pept ide-IILAclassIinteraction avidityof
referenceand heteroclitic peptides... . 110
3.9 A comparisonof referenceandhcrcrocli ricpeptide-stimulated
C'l'L rcactivity. . 112
Chaptcr -l Discussion.... . II()
4.1 Identifi cationofIL-2-induci nghctcrocl iric pcpridcs., . II(}
4.2 Comparisonofrefere nc eandIL-2-induc inghct c ro cl iticpcpt id es driven proliferationandd ilkrenliation... . 120 4.3
' l \'IlIIIl)' , " " , 124
404 l lctcrocliticpcpiidcsstimulatc broadly rcactivcC'I'L. 12(}
4.5 l lctcrocliticIIIVpcptidc sand theirpotent ial in therapeuticIIIV vaccines.... ...127 4.() Study limitations andsuggestions forimprovement. 130
4.7 Conclusion 133
Rd'crenees... . 135
Appcndiccs..; . 147
Table2.6.1:A comparisonor referenceand correspondingvariant peptide
sequences 5-t
Table2.6.2:liLA- BindingPredictionslorReferen ce andVuriant Pcptidcs 55 Table3.1.1:Asummary or the numberor peoplewithpositiveIF-yand
11.-2responsesto oneor morepcptidesin eachpeptideset 63
Figure\.1.\ IIIV GeneStructure Figure1.1.2The structureor l llV Figure \.1.3 The LifeCycle or IIIV
Figure 1.2.\Thestructure orthe((:11TCR 1(,
Figure 1.2.21'0sitive andnegativeselectioninthethymus 23
Figure104.1TheGagandNcfmatriccs 40
Figure 3.1.\Comparisonor referenceandvariantstimulation or IFN-y
production I 65
Figure 3.1.2Comparisonorreferenceandvariantstimulationor IFN-y
productionII 67
Figure3.1.3ComparisonorreferenceandvariantstimulationorIFN-y
productionIII 69
Figure 3.104ComparisonofreferenceandvariantstimulationorlFN-y
production IV 72
Figure 3.1.5Comparisonofreferenceandvariantpeptidestimulationor11.-2
production I 75
Figure3.1.6Comparisonofreferenceandvariantpeptidestimulation01'11.-2
productionII 76
Figure 3.1.7Comparisonor referen ce andvariant pept idestimulationor 11.-2
product ion III 77
Figure 3.I.XRelationship between 11.-2and IFN-yresponses1 XO Figure3.1.9 Relationship between11.-2and IFN-yresponsesII X2 Figure3.2.\Evolution orpeptide-induced IFN-yand 11.-2responses X5 Figure3.3.\Responsesto combinationsofrefere nceandvariant pcpt idcs XX
Figure3.4.1ComparisonorCDX'Tcellprolifera tion inducedbyreference and
hctcrocl iticpcpt idcsl 91
Figure 3.4.2ComparisonorCDX'Tcellprol ife rat io ninducedbyreferenc eand
hctcroclit ic pcptidcsll 93
Figure3.4.3ComparisonorCDX'T cellprolif erati oninducedbyreferen ceand
hctcrocliti c pcptidcslll 95
Figure3.5.IComparisonorCD45RAand ),D-Iexpressiononrefere nceand hctcrocliticpepti de-stimulatedCFSE10\\andCFSEllIghCDX'T cells 99
Figure3.6. 1ComparisonorCDX'T cellcytotoxicity induced byreferenceand
hctcrocl iticpcptidcsI 102
Figure 3.6.2ComparisonorCDX'Tcel lcytotoxicityinducedbyreferenc eand
hctcrocliticpcptidcsll 104
Figure 3.6.3ComparisonorCDX'Tcellcytotox icityinducedbyrefere nceand
hctcrocl iticpcpt ideslll 107
Figure3.7.1Evolutionorpep tide-indu cedCT L responses lOX Figure 3.X.IComparisonofr eferenceandhctcroclitic peptide TCR/peptide-IILA
class lavid ity II I
Figure3.9.1Comparisonofreferenceand hctcrocl iticpcptidc-stimulatcd-C'l'L
reactivity 113
ListufAhhn'"iatiul\s
aminoacid ACD
AIDS AIED AIRE ALI' AI'C ART ATI' BLCL bp
CDR CFSE essDNA CTEC CT L
DC DMF DMSO DN DNA 1)1'
acid-c itra te-dex trose
Acquired Immun odefi ciency Syndrome acute infection earlydisease auto im muneregulator alkalinephosphatase antige npresentingcell nntirctrovira lthcra py ade nos ine triphosphate
EBV-transllll"lllcd B Iymphoblasto id cells base pair
complementarity-de terminingregion earboxylluorcsecin suee inimidylcster complemen tarysinglestrandedDNA cortical thym ic epithelialcells eytotoxie T lymphoeytc dendritic cells dimcth yll on n ami dc dimethylsulfoxide doublenegative dcoxyrib onuclcicucid double positive
DRiPs ds EBV ELISA ELISPOT
ER ERAAP Ell Fab FADD ITS gag
gp IIAART IICL
IIEV IIIIV-X III V
liLA IFN-y Ig IL ITA I\1
defectiveribosomalproducts do ublestra nded Epstein-BarrVirus
enzyme- linkedi mmunosorbentassay enzyme-linked imm unosorbentspot enve lope
endo plasmicreticulum
I:I{ aminopeptidaseassociatedwithantigen processing l llV-cxpo scdbutuninlcctcd indiv iduals antigen bindingfragment
l-as-As sociatedprotein with DeathDoma in Ictalcullseru m
group-specificantigen glyco protein highl y activeART hydrochloric acid high endo thelia lvenule humanherpesvirusX humanimmunode ficiencyvirus humanleu kocyte antigen intcrf cron -y
immunoglob ulin intcrlcu kin
immu noreceptort yrosine-basedact ivation mot if
lck Iymph ocytc-spcciticproteintyrosine kinase LMI' the low mo lecu larprotei n I,TNI' long-tcrmnon-p rogrcssor
LTR longterminal repeat
MI:CL multicatalytic endopeptid ase comple x subun it MI IC Major Histocomp at ibility Complex Mil' macro phageinflamm atoryprote in
mRNA messengerRNA
m<p macrophage
ncf negative-reg ulationfact or
NK natura lkiller
NNRTI non-nucleosideRTinhibitors NRTI nucleoti deRTinhibitor
I'BC pepti de-bindingcomplex
I'IH\l1C peripheral bloodmon onucle a rc ells I'BS phosphatebufferedsaline I'CR polymerasechain react ion pMllC pcptidc.Ml ICcomplex
pol polymerase
regu la torofviralexpression
RNA ribonucleicacid
RT rcvcrsctrunscriptasc
SlY simianimmunodeficiencyvirus
SIYmac SlYrhesusmacaque
singlestranded SSP SequenceSpecific Primer
TAP transporterassociatedwith antigen processing transacti vator
TCM central memory Tcclls
TCR reenreceptor
TEM effector memoryTcells TGF transfonu inggrowth (actor
TNF tumornecrosisfactor
variable
vir viralinfectivity
vpr viral proteinR
vpu viralprotcinU
WIIO Worldllealth Organization Zap-70 zeta-chain-associatedprotein kinase 70
ListIlfA(lIlcndicc s
Appendix A:Relationshipbetween11.-2andIF-yresponses I·n Appendix13:Compar ison orCDX'T cell cytotoxicit yinduced by reference and
hctcroclitic pcptidcsII 150
AppendixC:Comparison ofreference and hctcroclitic pcptidc-stimulntcd- C'll .
reactivity 151
Chapter I Introduction
1.1 IIIV-I summary
Thc World llealth Organiza tion(WI IO)estimates that 33 millio npeople worldwidearc infectedwithIIIVIII.Infect ion with IIIVleadsto a grad ual loss01' immune competenceallowi ngopportunistic infect ionsthat arcnotnorm allypathogenic to become lifethreaten ing andallow ingcertaincancers to occur. Mostuntreated individualsinfected withIIIV progressover timeto acquired imm unodeficiency syndrome (AIDS) 121.Transmission01'IIIV occurs throughtransfer01'infectedbod ily lluids.namelyblood.semen.vaginal fluid and breastmilk. withinwhichIIIV can be presentasfree virusor containedwithinaninfectedimmune cell.Thistransfercan occur through unsa fe sex, contaminated needles,contaminated breast milk. perinatal transmission or through infected blood products.although thelatterisnotascommon now due10improvementsin IIIV screen ing,Twotypes01'IIIV.IIIV-I and IIIV-2.have been identi fied.IIIV-I ismore prominentworldw ideandleadsto a more virulent infectionwhile IIIV-2 isconfined to WestAfricaand isnotassociated with
immun osuppression 131. This thesis will locus IIIV-1.
1.1.1 Str uctu r e
IllY isencodedfor by 9genes (Figure1.1.1) 141.Envelo pc«('111").group-specific antigen(ga g)andpolymerase(po!)genes arccommon to allretrovirusesandarcrequired tomake thestructural and enzymaticproteins.respectively. fo rtheform at ion ofnew virusparticles.Approx imatelyfou rt een"spikes"151.trimcrs ofthe ('/II'-cncodcd
glycoprotcins(gp)gpl20andgp-ll ,arcembeddedwithin the viralenvelope(Figure
1.1.2).gp l20 isimportant fordockingof virusparticlesonto hostcells161.whilegp41is required tor viralfusionandintcrnalization 17.XI.Beneaththe outerenvelope liesthe matrix.whichmaintainsthe integrityolthcvirus particleandseparatesthe viralenvelope from thecapsid.The viralcapsidismadeupof-2.000 copiesofgag-cncodcd p24and containstwosingle strandsof ll lV ribon ucleicacid(RNA)that arc tightlybound lothe nucleocapsid protein,p7.andthepo!-cncodcdenzymes.reversetrunscriptasc (RT)and intcgrasc.EachstrandofRNAhasa completecopyofthe virus'genes-('/II".gag.and
!IO!.and the remai ning6 regulatorygenes.transactivator (ta t),regulatorof viral expression(r(,I").ncgutivc-rcgulationfactor(1/(:/) .viral infectivity(vif ).viralproteinR tvpr)andviralproteinU(l'plI)(Figure 1.1.1).The regulatorygenes encode proteinsthat control infectivity, replicationand pathogenesisof IllY.'lhelongterm inalrepeat(LTR) ateitherend of eachstrandofRNAisinvolvedincontrolofIllY replication.Thepo]:
encodedenzymes.reverse transcriptasc.proteaseand intcgrasc arc responsiblefor copying the viral RNA gcnomcinto deoxyribo-nucle ic acid(DNA).processingimmature gagandgag/pol proteinsand integratingtheDNA copy oft he lilYgcnomc into the host DNA.rcspcctively.
I \
pI7 p24p7
MA CA NC
Group-spec lficanti ge n Encod esmatr ix (MA).ca psid(CA).nucle ocapsid(NC).andp2 proteins
En co d e sre ve rs et ranscript ase (RT).protease(P R)a ndin te~ rase(IN) enzymes
Cleave dintotwop rote ins:ep 12 0bin ds CD4andCC R5/CXCR4allowine vi ral atta ch me nt;gp41mediatesviru sfusion andinte rn alization
Facilit at esinfecti on ofmacro phagesand promotescel l-cyclearrest Allo w sexpo rt of uns p1ice da ndpartially splice d viralRNA Positive regu latorof viralRNAtran script ionan d pr om ot es CD4 lym pho cyte act iva tio n
Pr om ot esintracellulardegra dationofC D4andenhancesvi r ion release Overcom esinhibi t ionsbythe host facto rsAP OBEC3G andpro mot esmo re sta blerev e rse transcriptas e com plexes
Regulat or ofvira l expre ssion
ne] Negative-regulationfact or Pr om otesC0 4lvrnphccvt e activa t ion,blockscellsuicide,enhances infe ctivityandisassociat edwithdise aseprogressio n
Fi~lIrc1.1.1 \lIV Gcnc Structure. IIIV 'sRNA genomeenco des9genes. whic harc flankedby LTRs.Eachstra ndor RNAhasa com p letecopyor thevirus'genes-the:1 struc turalgenesCI/I'.gag. andpo] . and theremaining ()regulat o rygenes .tat,1'('\' .nc].vi],
\'111'and\'IJII.The regul ator yge nes enco de prot einstha t contro linfec tivity,replica tionand pathogen e s isorliIV.TheLTRsateithe rendoreachstrandorRNA arc invol ved in controlof I llVrepli ca tion.Ada ptedrrom l9.101.
Fi~u re1.1.2Thestr uct ure(IfIllY.The viralenvelopeiscomposedor a phospholipid bilayer.taken fromthe membrane or a humancel l when anewlyformed virusparticle buds.Embeddedthroughouttheviralenvelopearctrimcrsor{'III'-elH:mkdgp 120and gp-ll.whichmed iate viralauachmcntand fusiontohost cells.The matrixlaysbeneath the viralenvelope.IIma intainstheintegrity or the virusparticle andseparatesthe vira l envelopefromthe capsid.The viralcapsidismadeupor-2.000 copiesorgag-encoded p24 and containslWOsinglestrandsorIllYRNAthat arctightlybound tothe nucleocapsidproteinsand theflo/-eneodcd enzymes.RT andintcgrasc,Eachstrandor RNAhasa completecopy oft hevirus' genes. Ada ptedfrom1111.
1.1.2 Tropism
IIIV tropismisdependenton thehostcellreceptorsrequiredfixviral attachment.
TheCD4 rece ptor.expressedonCD41T cells.macrophagcs(m<jl).anddendriticcells (DC).actsasthe primary receptor1'01'gp120-mediatedbindingor IIIVto targetcells112.
131.In1996.it wasdiscoveredthatIIIVrelicson theusc orco-receptors. CCR5and CXCR4.tohelpmediatevirus fusionandpenetrationintothetargetcell 114-16 1.The importance orCCR5 asthepredomi nantco-receptor in IIIV-Iinfection 114.151was highlighted upon the observance that polyrnorphisms in CCR5.particularly a homozygous 32base pair(bp)deletioninthe codingregion(CCR5-L\32).reduced the risk ofl llVinfection regardless orthemode ortransmission I 17-191.lIowever.a few IIIV'CCR5-L\32individualswithaslower courseor disease existed.indicatingviral usc oranadditionalco-recep tor. CXCR4. These CXCR4-usingvariantvirusesusuallyhave altered tropismandpathogenic properties.Theyarc commonin late-stage subtypeB IIIV-1infection,in which there isoftenaco-receptorswitchfromCCR5 to CXCR4120.
211. IIIV canthusbeCCH5-ll'lIllie(H5),('XCH-I-lro lli e(.1-1)ordual-mixtropic:(H5-X-I) ,
1.1.3 Lifecycle
TheIIIVlife cycle(Figure1.1.3)beginswithvirusentry into the hostcell.Viral gp120bindsto intcgrinu41J7.thegut mucosalhomingreceptoronperiphera lTcells.
activating adhesion moleculesto establish thevirologicalsynapse1nI.gp120bindingor
Attachment/Bindin g
Cytoplasm Export
Vi ralRNA Nucleus ___ splicedrnRNi\
m V
Transcription~.~ ~~;~~I~~dr~~e~AmRNA
Preintegration Complex
-- y ~
Integration ln tcgrascr"
Fig u re 1.1.3 The LifeCycle of IIIV. The life cycleor IIIV beginswith fusion or the virus
parti cle to the hostcellsurfacc.jncdiutcd bythe intcract ionsofgp lZu.gp41.CD4andCCR5 and/orCXCR4.Afterfusion,the vira lcapsidand itscontentsarc released into thehostcell cytoplasm.Viral DNAisformedbyreve rsetranscript ion ofthe RNAgenome.Onceforme d.
theviral DNAistransportedinto thenucleu sand integ ratesintothe hostDNA.Upon
activationoft hecel l.TheDNAistranscribe dtomakenew vira l RNA.which willbeused as genomicRNA and tomakenewviral proteins.Thesemovetothe cellsurface andbuddi ng begins. Duringmaturation oft heviral particle.individu allll Vprote insarc releasedcleave d andreassemble to[ormthematr ix. capsid.nucleocapsid.RT and iutcgrasc,completing maturat ionoftheviruspa rticle.Adaptedfrom123-25j.
CD4 inducesa conformationalchangeingp 120 that allows interactionwithceR:> or CXCR4andallowsgp-ll-mcdiatcdfusionto thehostmembrane1261.Upon fusion.the viralcapsid isreleasedinto the cytoplasm1271andthesingle-stranded (ss)R Agenome isreleased.The viral RT reverse-transcribe sthessRNA intoacomplementary ss deoxyribonucleicacid(css DNA) copy.The RThasRase activity12Xlthatdegrade sthe viral RNAduringthisprocess andhasDNA-dependentD A polymer aseactivity1291 thatsynthesizes a.l'l'II.1'l'strand li·omthe llllli.l'l'II.1'l' cssDNAto forma double-stranded(ds) DNAcopyofthe IllVgenome.ThedsD NAmigratestothenucle usandisintegratedinto thehost genomebecom inganintegrated provirus 1301.
Activation ofCD4'T cellsinduces translocationoftranscr iptionfactorsNF"n andNFAT to thenucleus131.321.whichbindto promotersin theviralLTRof the integratedprovirusand initiate transcription of the viralgenome 1331.The lirstviral transcript s.whichencode severa l of the regulatoryproteins.arc multiply spliced.then exported and translatedin the cytoplasm.Singly spliced and unspliccdtranscripts arc translated intotheRT.intcgrase. viralprotease and the structuralprote ins cuv (polyp ro ici ngp I( 0).gag.and ncr. ThestructuralproteinsIorm the newvirus panicle aroundadditional unspliccd transcriptsthatconstitutethe IIIVgenome. Aftertranslation of the viralproteins.the virusis assembled.gp l 60 iscleaved into gp41andgpl20by protcascsintheCiolgicomplexand transportedto the plasmamembrane1341.Thegag andpolpolyprotcinsassociate withtheIIIVRNA at the plasma mem brane andbudd ing begins.Duringmaturat ion of theviral particle. thesepolyprotcinsarc cleavedand
reassemble toformthematrix. capsid.nucleocapsid.RT and intcgrasc,completing maturation ult hcvirus particle1351.
1.1... Dia gn osi s ofIIIVinfe cti on
Unless thereisahighindex orsuspicion. IllY infectionisnotnormallydiagnosed immediate ly.Thoseinfectedmayexperiencea flu-likeillness(fever,headache,tiredness.
andenlarged lymphnodes)in theacute phaseor infection.whichusuallydisappea rs within1-4weeksand iseasily mistakenfix another viralinfection1361.l llV-iutcctcd individualsarc highly infectiousduringthisstage dueto theabundanceorvirusin the peripheral bloodandgenitalfluids.Atscroconvcrsion.l llv-spcci ficantibodiesin the bloodcan bedetectedbythe enzyme-linkedirn m unosorbcntassay(ELISA)and Western blot1361.
TheELISA is generally usedto screen donated blood productsandfor general surveillanceor infection 1361.II' theserumcontainsantibod iesfor specificIllYantigens theywill bind to theIllYuutigcn-coatcdELISAplate.Because orthe variabilityorthis procedure,the lack ora comparableglobalstandardand the concernorfalsepositive responses.asingleELISA testcannothe usedtoconfirmIllYinfection,TheWestern blottest,likethe ELlS/\.isanantibodydetectiontl:stI 371.II'l ilV-spccificantibodiesarc presentin theserumtheywillattachtosome of theIllY proteinson the membraneand animageor bandswill he displayedon the photographicfilm.A positiveresult isone in
which at least one viral bandcorresponding to each gag.polandcnv proteinsispresen t.
lfI cwcrthan 3band s are detected.theresult s areinco ncl usiveandanother testisneeded . II'the personisrecentlyinfected (1-3month s) asufficientamountor antibodies
1'01'thesetestsmaynot yet be present.Inthis case.IIIVRAorDNAcanbedetected
using apolymera se chain reaction (PC R)-based methodtoconfirmthe presence or IIIV genet ic materialinthe blood1361.Thismethod. in quantitativeItm11.isnormall yusedto trackclinicalprogressionorIIIV infectionand tomonitortheeffect orantirctro virul therap ybutcanbe used10confirmanew infectionin highrisk individ uals.A highviral load is stronglycorrelatedto diseaseprogression and increa sedriskortransmission13XI.
IIIVinfecti onisconfirmedwhen asequenceortests.includinga repeator the initial ELISA testcombined with the Westernblottest andPCR testfor viralload.confirmIIIV infection1361.
1.1.5 Palho~cll csis
IIIVinfectscellsthat expressCD4andCCR5orCXC R4.part icularl y CD4'T cells.CD4+Tce lls are keyplayersor our immunesystem.Theyinteractwiththe innate immunesystemandare requiredlo ractiva tio nofBcellsandCT L. In the acutephase or IIIV infection. thereis an abundanceor virusinthe peripheralblood andgenitai lluids. a significantdecreaseinthe numbe rorcirculating CD4'T cellsanda markedincrease in antibody production and in the number or CDX+T cellsthatrecognizeand kill IIIV- infec ted cells.Followin g scroconvcrsion, whenHlV-spccifi c antibod iesare detectablein
the blood,theacute phase orviremia haspassedand thereisarebound in the numberor circulatingCD4'Tcel ls.Duringthisasymptomatic period.IllYrepli cation continues and thenumber orCD4'T cells gradually decl ines.When the numberorCJ)4'Tce lls full sbelow 500ccl ls/p lorbloodthesymptomaticphaseorinfectionhegins,markedby the appearance oroppo rtunisticinfecti onsand othersymptoms.Whenlliv-inl cctcd individuals becomeill with oneormore or the clinica lor indicatorillnesses,theyhave AIDS1391.
Themechan ismbywhichIllY causes CD4'T cell depictionhascaused much debateover thelastfifteenyea rs.110ct al.1401showed thatin the presence or the proteaseinhibitorritonuvir, there wasa rapid redu ctionin theamount orfree virusinthe plasmaandasignificant increasein the number or circulating CD4'cells.Asdrug- resistant mutat ionsevo lved, the viral loadincreased and thenumberorcirculatingCD4' cells decreased140,411,suggesting thatIllY causesCD4'Tcell depletionbydirect cyto- pathic effect.Later.it was observed thatAfricannon-humanprimatesinfected with their spec ies-specificstrain orsimian immunode ficiencyviruses(SIYs)did notshow progressive CD4'Tcelldeclinesand manifestationsorimmunodeficiencydespite chronic viral replication1421.Schindler('{al. 1431reported thatCD4'Tcel l dep letion wasnot observedin thesehostsbecausenc rproteinsfromtheseSlYstrainsdown- regulate expressionor theT cellreceptor (TCR)-CD3comp lexleadi ngtodecreasedcell activationandapoptosiswhereasnc rproteinsfromIllY-I and the chimpanzee strainor SlYhadno effecton TCR-CD3surfaceexpression.This suggestedthatincreased immuneactivationandapoptosisleadstothedem ise orCJ)4'Tcclls.
10
Other mechanismsofIIIV pathoge nesishave alsobeensuggested.cm~'T cell- mediated killingof infectedCD4'T cellshaslongbeen consideredoneof these mechanisms144.4 51butcytotoxic killingofinfectedCD4'T cellsmayonly beeffective duringa certain"window period"ofinfection146. 471.Cellcycledysrcgulation,caused bychronicT cellactivat ion.accelerate dce ll turnover andanimbalance in cytokinc production.also leadstothedep letion of uuinlcc tcdCD4'andCDX'T cellsby causing perturba tionsin proteinmetabol ism14XI.CirculatingCD4' T ce llsmay alsobe sequestered insecondarylymphoidorgans1491.The relat ive contributionsof eachof thesemechan ism sonIIIVpathoge nesisandCD4'Tcell depletionremainsunclea r.
I.I.CJ l>ia:,:llIlsis llf AII>S
In Canada.if'anindivid ualhasreceivedapositiveresultfromIIIV testin g and has oneor more of the clinical illnesses.or indicator diseases. such aspulmonary tuberculosis.recurrentpneumo niaand invasivecervicalcancer. that person hasAIDS 139 1.SymptomsofopportunisticinfectionscommonwithAIDSincludecoma.extreme fatigue,fever. nausea .vomiti ng.severepersistent diarrhea.vision loss. weightloss and mentalsymptomssuchasconfusionand for getfuln ess1501.Kaposi's sarco ma. causedby the humanhcrpcsvirus-S (I ll IV-X).isthemost commonAlI)S- rda tedcancer.Itcauses redd ish-purplelesionson theskin that wereahallm ar kfeatur e of AIDS patientsinthe 19X<)"s.Otheropportunisticinfecti on sthat canoccur inAIDS patien tsinclud e. butarc not limited to candidiasis. toxoplasmosis. cytomegalovirus. herpessimplex. l'ncum ocvst is
[irovcci.tuberculosisandrecurrentpneumonia139.501.In untreatedIllYinfect ion.
progression to AIDS takesabout tenyears onaverage.However.sincethe introduction or antirctroviral therapy (ART). particularlyhighly activeART (IIAART ).progressionto AIDScanbedelayed1'01'severaldecadesor completely prevented.
1.1.7 Antiretruviraltherapy
Before ART wasavailable. peop le withAIDSwere unlikelytolive1'0 1'morethan ale wyears.Sincetheintroduction orARTin 19X71511and IIAARTin 19<)('1521 mortalityand morbidity in l IlVvinfcctcdpatientshasdramaticallydecreased 1531.ART therapieshavecome alongway sincetheywerefirstintroduced.Antirctroviral drugsarc morenumero us.diverse with respectto antirctroviru lclass.morepotent and lesstoxic than before,Current IIAARTisacombination therapyconsistingoracocktailorat least 3differentantirctrov iral drugsthatbelongto at least 2di ffere ntclassesorantirctroviral agents. Currently.sevenclassesor antirctroviralagentsexist.Theseinclude entry inhibitors.CCR5antago nists.nucleosideand nucleotid eRT inhibitors(NRT ls).non- nucleosideRT inhibitors(NNRTls).proteaseinhibitors.intcgrasc inhibitors1541 and maturation inhibitors1551.Entry inhibitorsinterfere with the binding.fusion andentryor thevirusintothehostcell.CCR5antagonists bindtoCCR5blocking gp120binding.
NRTlsaremodiliedDNAnuclcotidcsthatlack a3"hydroxylgroup.These arc incorporatedinto the viral DNA duringcDNA synthes is andresultin DNA cha in terminationasno addiiiona l nuclcotid cscanbe attached tothem.NNRT lsbind directlyto
12
theRTandinterfere with itsability tosynthesize the IIiVeDNAstrand from the viral RNA.Proteaseinhibitorsprevent cleavage orpolyprotcinsneeded to produce infectious viralparticleswhileintcgrascinhibitorsblockIIIV DNAintegrationinto its hostDNA.
Maturation inhibitorsarestillin development1551.They could potentiallyinhibitproper assem bly of thevirion and preventmaturation ofthe panicle.
Despitetheeff ective nessor IIAART.manyissues suchasthegenerationold rug- resistance mutations.non-adherenc e,unwanted shortand long-termsideeffec ts.cost,and highpillburden exist.Non-adherence,dueto poor access.drug abuse.pill burden.and unwantedsideeffec ts,allows viral replication to reboundleadingtothe generationor immuneescapeand drugresistancemutations15(J-5l\1.Howe ver.in manyregionswhere individualshave accessto state-of-the-art IIIVcare. the incidence and risk or antirctroviru lresistance and incomplet eviralsuppression isdeclining1591.Up untilthe recent IIIVPrevention TrialsNetwork (IIPTN)052randomizedtriall ()Oj.it wasthought thateven while l llV-infcctcdindividualshad IIAART-suppre ssedviral loads.theywere couldstillpasstheviruson to others.However,resultsoft he IIPTN052trial showed that earlyinitiationorARTis effi cacious in preventingIIIV transmission160. 611.
I.I.N Viral persistence
Despiteeffecti vetherapy.IIIVpersistsand continuestoinfect new hosts.
particu larly inareaswhere ART isunavailableor treatment sarenot initiated earlyin infcciton.IIIVpersistenceismainlydue tothenumberor mutationsthat can be genera ted
13
in minimal time periodsand toitsabilitytomaintainlatentinf ecti onasaprovirus.The viral RT lucksproofreadin gcapacityallowing ahigherror rate and multiplemutationsto occur 162/thusleadin gto the rapid developmentof viralquasispccicsincludingdrug- resistant andimmune escapevariantstrains.Insome cases.asingle mutationcanconfer resistancetosome drugs1631.whilea sequentialaccumulationof multiplemutationsis requiredto conferresistancelo ro the rs16-11. The mutations generatedin thepresenceof onedrugmayleadto cross-resistancein which the virusbecomesresistant todrugsto which it hasnever beenexposed 165.66/.This usuallycon fersresistance withinoneclass of inhibitor butis ofgreatconcernasitsignificantlylimitsthecombinationsofARTiluu can be used.However. inregionswherestate-of-the-artARTisavailable.theseproblems
1.2 CDIl+T cells
Hematopoietic thymocyteprecursorcells that migratefrom the bone marrowto the thymusmaturethere and become T cells.BothCD4'andCDX'T cellsmaturein the thymusand are distinguishedtrom eachotherbythe expressionof theirrespectiveco- receptors(CD4orCDX) and bytheirdifferen teffectorfunctions. CD4+T cellsare dividedintoseveralsubgroups(I'llL T1I2.T1117.T,cgs.ctc.) based onthe irdiffe rent cyioki ncproduct ionandeffec torfunctions.ActivatedCDX''I'ce lls(CTL) killvirus- infectedand tumorcells.Thefol lowingsection willaddressmany aspectsofCDX''I'cell
development and function includinghowthesecellsrecognize antigen. how theydevelop in thethymus. and most importantly theirroleinIIIVinfection,
1.2.1 Th~T cell receptor(TCR)
EachT cell has-30.000clonotypicTCRsonitssurface1671.Twodifferen tTCR hctcrodimcrs.theu:11TCRandy:i)TCRexist.Theu:11TCR.the 1•ocusofthis section. is expressed on mostT cells andishomologoustotheantigen bindingfragment(Fah)of immunoglobulin(Ig)16XI.They:i)TCRsrepresent aminorsubsetofTCRs.whichhave differentantigen-recogn ition propert iesfrom theu:flTCRs.EachTCRconsistsoftwo receptor chains.whicharedisulphidc-linkcd variable trans-membraneglycoprotcins anchored inthe membrane(Figure 1.2.1).The mem brane proximalregion ofeachchain istermedtheconstantregion(C).while the membranedistal regionof each chainisthe variable region (V).The amino-terminalvariable region isthesiteofantigen binding.
Crystalstructures oftheu:11TCR169-731haw revea led thatallbutthe Cu doma inofthe TCRaresimilarinstructuretothe domainsofIgs.Thelg-likc domainsof theTCRareconstructedfro mtwoIIsheetslinked bydisul fidebridgestoformaIIbarrel.
Fourloops of[Istrands at the outeredgeoftheIIbarrel(o n nthehypcrvariab lcregionsof theVregion oreachchain.Theseloopsforma complementarysurfaceto aparticular antigenandassucharecalledcomplemen tarity-deter miningregions(CDRs).TheC«
domaindinerslromthe othersin thathal l' orit isconnected totheC]]domain lim llinga IIsheet while theotherhalI'iscomposedorlooselypackedstrands joinedto anu-hclix by
15
achain ~chain
Var iabl eRegio n(V)
Const antR egio n(C)
Tran sm embraneR egion
• Carbohyd rat e -ss-Disulph ideBond
Fi!-: lI r~ 1.2.1The st r u ct u r eof the u:ll TCI{. TheTCI{is composed of two transme mb ra negpchai ns,u andII.Thememb ran eproxim alregion ofthe extrace llula r portionor eac hcha in istermed theconsta nt reg ion (C). whilethemembranedistalregi on oft heextrace llularportionofeachchai n isthe varia b le region(V).Theami no- tc n ui nnl varia ble region isthe siteorantigenbindin g .Bothcha insare anchore dinthememb rane viaa shortstalkseg m ent.This seg me ntcontainsa cyst eine residu e tha t Ionusadisulfid e bond ,linkingthetwochains.The hyd roph obictra nsm e mbr an esegmentoreac hchain conta ins positi velycharg edresidu es.Adaptedrrom1741.
16
adisulfide bond.Gene rcarran gcmcms,junction diversity andrando m pa iringoru and1\ cha insresultin highvariabilityorTCRs. part icul arlyin thehighl yvariable CDR]region that interactsdirectlywith the antigenicpeptide,CDR IandCDR2arc lessvariable as theyarc requ ired1'01'interactio nwithsc lf-MlLCmolecules and thus.musthaveconserved regionstomaintainreco gnition16XI.
1.2.2 PeptideprocessingandInadin~nntnMileclassI
CDX+'I'cellsrecognizepeptid e antigensthroughthe TCRonlyinthe contextor selfmajor histocom patibil itycomplex(MlK') classI molecu les(human leukocyte antigens(lILA) in humans).MI ICclassIisexpressedon nearlyall nucleated cellsas wellasplatelets,Duringthe inflammatoryresponse.cytok incs suchasIFN-yand tumor necrosisfactor (TNF)-uincreasethe expressionorMI IC classI moleculeson thecell surface,The maj orityorthepcptidcspresentedbyMI IC ClassIarc derived from newly synthesizedproteinsinthecytosolalthoughasmaller fraction isderivedfrom uuuur c proteins175.761.These proteinsarcdegradedthroughthe proteo lyticactivityor the protcasomc,acylindr icalstructure formed bytheadenosine triphosphate (ATI')- dependentassociatio northe proteolyticcoreparti cleandtheregulatory particlesthat makeup the baseandthe lid1771.
The protcasomcexistsin twoforms:I)The constitutiveprotcasomc. whichis present inallsomatic cellsand2)theimmunoprotcasomc,which ispresentonlyin antigen-presentingcells(AI'Cs)andinflame d tissues.WhcnAI'Carcstimul.ucd with
17
IF -yo the lowmolecularprotein (I.Ml')2.Uvl1'7and multicatalyticendopepti dase complexsubunit(MECL)- lor the im muno pro tcasomcaresynthesized.Theserep lace constitutivesub unitsin thenewly synthesized prot ca sorn c17XI. TheA'lP<dc pc nd c n t activity or theimmuno protcaso mc isregulated byI'A2X. whichbindstotheterminal ringsor the protc a som ctoformaprot ea se- a ct ivat o r com p le x thatopens thepro teol yt ic core part icle. strongly regulat ing the degrad ationand release or endogenously- synthesized pcptidcs1791. Theoligopc p tidcs rel easedrangefrom4-20amino acid residu esin length and terminatemostlywith basic and hydrophobi cresidues,SuchC- terminiarefavou redIll!'uptakebythetransporterassociatedwithantigenprocessing (TAP)and are requiredIll!'binding10MI ICclass lmolcculcs17XI,
Oncereleasedlromtheprotcasom c.themajorityor thepcpt idcsarefurther degradedIIII'usein proteinsynthes is orene rgyprodu ction.Theremaind er bind to chaperones.whic hprotectthemIromdcgrad arionuntiltranslocationintothelumen of'the endoplas micretic u lum(ER).Transportinto the ERisachievedinanATI'-depe l1lknl mannerbythe heterodim cric TAPcompl ex, an integralER mem brane protein"hose expressionis signifi cantly enha ncedinthepres e nceorIFN-yIXOI.The TAPcom p lex preferentiallytransportspcptidcsX-16residuesinlength, withpcptidesorthe appropriate lengthIll!'bindingto !'vII ICclassI(X-I I residues)mosteffic ientlytransported.Once insidethe ER.peptideprecursorsthatarcnot attheappropriate MI IC classI-bind ing lengtharetrimmedat theNstcnninusbytheER aminopeptidaseassociatedwithantigen processing(ERAAI')to generate pcptidc sthatcanbepresented byMIIC class I lXII, MI ICclassImoleculesaresynthesizedandloaded withpeptid einthe ER.This
IX
processismed iatedbyseveral molecular chaperonessuch as culncx in and culrciiculin.
whichprevent degradationof properly fo lded proteinsand allowstable assembl yof the MIICclassI:peptidecomplex (pMIIC). ER-resident prote ins.including ERpS7.the TAP-associatedglycoprotein(tupasin).and the TAP hctcrodimcr interact with thenewly formedcomplex.'la pasin.whichhas anER retentionsignal in itscytoplasm icdomain IX2j.mediatesthe critica l interaction of theMIICclassI hctcrodimcr with TAP. allowing peptidebindingIX]I.Allertransportintothe ER viaTAP.pcptidcs are loadedinthe(11(12 domain of MIICclassI molecules.Low affi nity pcptidcsdonotcause the release ofthe MIIClromthe peptide-bind ing complex (I'BC).whilehigh affi nitypc piidcsmay cause conformationalchanges that completethefold ingof the MIIC classI molecule resultin g in itsrelease from the I'BC andsubsequent display at thecellsurface IX4j.
Each individualcarriesa setof(, classicalMIIC ClassI alleles:oneA. one Band oneC alle le from eachparent. These allelesare highlypolymorphi c.particularlyin the aminoacidsformingthe peptide-bindinggroove.Suchpolymorphisms are likelythe resultofevoluti onarypressureto maxim izepeptidebindingdiversityin the presence of multiple emergingpathogens.lach MIICclassI moleculehasa preferredpeptide- binding moti f:however.with the lackof structural datathese are not all known.All.
however. are functionally complex:they interactat differentsites with peptides.j 12-m.the (1:11TCR.the CDXcorccc ptorand naturalkiller cell(NK)inhibitorymolecule sIXSI.
I')
1.2.3 Peptiderecuguitiun and activatiun ufCDS+T cells
MI ICclassl molccul cspresentpcptid csin the peptide bindin g groove.formedby twoflanki ngu-hcliccsanda110 01'ofantiparallcljf-strundsIX61.Peptide anchor residues interactwiththeMI ICclassI binding cle ft inspecificitypock etsthroughaseries or hydrogenbondsand ion icinteract ionsleavingtheupwardpoin tingside chains availuhlc till'interaction with theTCRIX7. XXI.Norma lly.MI ICclassIpresent sauto logous pcptidcsderived bydegradat ion or defective riboso malproducts(DRiPs)and mature proteinsthat have reached the endortheirnatur allife.Since Tcell precursorsthat recognizesclf-pcptidcs arc eliminatedinthe thymu s.the presentatio nor these autologous pcptidcsbyMIICclassIistypicallyignoredby T cells.In con trast.Tcellsbearing receptorsthatrecognizeforeignpcptidcsboundtosclf-MlK'classImatureandarc highly specifi cforfore ign antigensin thecontextofsclf-Ml fCIX91.
TheTCRaloneallowstil!'pcptidc/MlIC (pM I IC)recogn itionbut isnotsufficient for intrace llularsignalling.Other molecules.CD3y. CD3b.andCD3l:(CD3complcx) and theTCR~chain.whichcontain conservedimmunorcccp tor tyro sine-basedactivation motifs(lTAMs).arc requ iredtill' theexpressionorthe TCRon thesurface orthe celland till' thesignalling cascade thatfollo ws antigen recogniti on190-921.The interaction or the TCRcomplex(TCRandCD3complex) with pMllCis stabilized bythecm:co-rece ptor onCDX'T cells1931.pMl lCrecognition bythe TCRenhanceslymph ocyte-spec ifi c protei n tyro sinekinase(Lck)recruitm entto theTCRcomplex 1941.l.ckphosphorylatcs theITA MsortheCD3 chainsand theTCR~chain. whichallowsthe~-cha in-assoc i a t ed
20
proteinkinase 70 (Zap-70)to bind.ZAP-70 isthenphosphorylatedbyl.ckinitiati ngan intrace llularsignallingcascadeinvolvingmany other proteinsthatultimatelyresult sin cytokincproduction,proliferation,diffcrcntiar ionand/oractivation-inducedcell death IlJ5.lJ61.Thisprocessis also dependent oncostim ula tory recepto rssuchas CD2X IlJ71 andotherauxiliary adhesionmolecul essuch asintcgrin sIlJXI.which allow form ation or the immunological synapse.CD2X isexpressed on T eelIsand isligatedbyB7onAPCs.
Thisligationprovides an essenti alco-stimul atory signal thatpromotesTCR -indueed tyrosine phosphor ylation or the TCRSchainand ZA P-70. whichincreasesTce ll proli feratio nby augmentingIL-2product ionand preventstheinduct ion or ancrgy and celldeath,
Onceactiva ted throughthis mechanism.CDX+Tcellsbecome effectoren .CT L mediatecytotoxickilling olv irus-in fcc tcdand tumorcellsthroughtwodominant co ntact- depend en tmechanisms:gra nule cxocytosi sand therecept or-medi ated Fas-Associatcd prot e inwith DcathDomain (FADD) pathway(revie wedin IlJlJJ).Activation orC()X'T cellsresultsinthe uprcgulat iouorcytokincreceptorsand inducestheexpressionor granulecomponents such aspcrf orinandgranzym cs(seri nepretenses).Performis a C}t-dcpcndentpore- form ingproteinthatrnultimcrizcsin membranes andisresponsible
1'0 1'theentry orgranzy m csintota rgetcells.however.the mech anismbywhichthisoccurs
isunclear1100I.GranzymcsA and Bare the mostabundantgrunzytuc»and areeach capableor protcolyti callyactivating ce ll death pathw ays.which ultimatelycause fragmentationor thetargetcell'sDNA andsubseq uentcell deathIIO\. 1021.Theperform pathwayisthe predomin antpathwa yusedforC'I'Ls u cd ia tcdkillingor targetcellslikely
21
dueto itsefficiency.Cytotoxicgranulesarcreleasedwithin minutesortargetcell recognition andcan he reoriented after eachencount erallowing C'lL mcdia tcd killingor multiple target cells 11031.The FADD-mcdiatcdpathwaywhichincludesmembers olt hc T Freceptorpathway.TFRI. TRAILRandmostcommon ly Fas1104-1061.is also involvedinC'll.vmcd iatcdkilling or target cells.However.unlike inthe granule cxocytotispath way.verylillieligand(c.g, Fasl.)is stored andsothere isagreater lag time(1-2h)between recognitionand killingofeach target cc111991.Activationorthis pathwayrelicson the release of'certain ligands(c.g.Fasl.)whichbind tothe irreceptors.
resultingin therecru itmentorFADDandcaspasc X.Thisleadstothe activationora cascade or protcascs,particularly caspascs.which then cleave varioussubstrates responsible1'01'thechanges associated withapoptosis suchasnuclear membrane breakdownandDNAcleavage11071.
1.2A Tcl'Ilsclccliu n
Tccllsarc derivedfrom hematopo ietic stem cellsin thebonemarrowthatmigrate throughthe bloodtomatu re inthe thymus.alymph ocpithcl ial organinthe upperanterior thoraxIIOXI.Matu ration requiresthestepwise completionofseveral processesincluding rearrangementor TCRgenes,tornuuionora completeTCR.and positive and negative selection thatensuressclf-MlICrestrictionandself-tolerance (Figure 1.2.3)11091.
Tolerance isthestate ori mmunological nonrcsponsivcncssin the presence oraparticular antigen.It is achieved throughthe deletionofautorcuctivcTcellsduringmaturation
Capsule
o ~
Mllt~(ostillluatory Moleculev
Fi!-:ure 1.2.2Positive and ne!-:ativese lectio n in thethymu s. CD4/CDX doublepositive
(D P) thymocytcsundergoTCR ge nerearrangementsintheco rtex.DPce llsexpressing appro pria teTCR sreceive a "resc uesignal"throughthesei r-p MI IC:TCR inte ract ionwith cortica l thymic epithelialce lls(CTE C) .whichinducesDP maturat iontotheCD4'X-or CD4-X' single positive (SP)stage. whilece llsexpressingTCRsthatlackspecificityIIII' scll-M lK'undergo apo ptosis.Positivel y sel ect edcellsenter the medulla.Inthemedull a.
high-avidityTCk/sclf-pc ptidc.Ml lt. ligationswithmedullarythymic epi thelia lcel ls (MT EC)orDClead stonegati vesel ectionorse lf-reactive thymoc ytcs.Adaptedfrom 11091·
23
(cent ra ltolera nc e) and thesup p ressio norau to rcac tiv cTcells thathave escaped eliminationi n thc pcrip hcry(pcr iphcra l lolerancc) .
Onenteringthethymus. immature lymph ocytescommiucd tothethymocyte lineageinteractwiththethymicstromain the cortexand bcgindiff erentiation alongthe T cel l lineagepat hway. At the end or thisphase (-1 week).thethym oc yt cs express distinctive marke rs or the Tcell lineage suchas CD2but donotexpressmarkers or matureTcell s(CD].CD4orCDX).The se ce llsarc calleddouble-negativ e(DN) thymo cytcsbecausetheylack CD4 andCDX.TheDN phaseisfurthersubd ivided into tou rstages(DNI.DN2.DN]and DN4)basedonthe presenceand absenceorcertain su rfac emolecul esand thesta teor TCR chainge ne rearrangem ent 11101.II'gene rcarrungc mcutor the[Ichainlocus isunsuccessfulin the D]stage.thecells die by apoptosi s.Suc cessfu lge nercarrungcmc ntresult sin the ex p ressio nor a com p le te pre- TCR . acombinationortheIIchainandasurrogateprc-Tf.R u chain.atthe cellsurfa ce 11101.The ce llsthen ente rtheD 4stag ein whichprolife ration occurs .The assemb lyor theprc-TtRwiththeCD]moleculesarrestsfurtherI~chainrearrangementandleads10 the expressionofbothCD4 andCDXonthecel lsurf ace.When thesedouble positive (DI') thy mo c ytesceasetoproliferate.theuchai n locusrearra nge sanda com p le teu:11 TCR isproduced.
Thefate ofeachDI' cell dependsonthe abilityoritsncwTf.Rto interact with a seir-pMI IC complex,a processknownaspositivese lec tio n,Thisprocessoccursin the cortexofthethymu sand ismediatedbycorticalthymicepithe lialce lls(CT EC ).Recepto r editing,inwhichsequential roundsor rearrangementat theTeRu locusoccurs.at this
stage increases thelikelihoodor success fulsel f-Mi ll' restriction 11111.1)1'cells expressingappropriate TCR srecei ve a"rescuesignal" throughthescll:'pMl ll':Tl'R interaction.which inducesDP maturati onto thecoasorCD4-X'single positi ve (SP) stage11121.while cells expressingTCksthatlack speci fici ty1'0 1'sclf-Ml IC (-95'1.,) undergo apoptosis 11131.Chcmok incs that directthe Tcellforfurther maturationwithin thethymicmedulla arcalso producedduringthistime11141.Negativese lection,in which cellsexpressinghigh aviditysell-reactive TCRarc deleted,occursthroughoutthymic development11151.but ismostlyconccmratcdin the medulla 11111.In themedulla.this processisdepend ent on thehigh avidity'l'Ck.sclf-pcpridc.scl f-M ll'C interactionand costimulatorysignals [rumAPe.. particularl ythe interactio n orCD2X(onthymocytcs) and 137 (on DCs)1110.1171.Othercostimulatorysignalsthrough CD40 providedby medull arythymi c epithelialcellsand DCsIIIXI.and through CDSandCD43on Tcells 11191 alsohave aroleinclonal deletion,Negativeselectionoccurs to alesserextentin the thymic cortex.wherethereisalack or costimulatoryAPCs.l lcrc,clonaldeletio nor scmimaturcTcellswithstrong TCRbindin ginthe absenceor costimul.uion isFas- dcpcndcntllZtl],
In order1(11'successfulclonal deleti on or potenti all ysel f-reac tiveT cellsto occur.
theremust bea complete rcprcscnu nion orsel f-antige nwithin the thymus.The
expressionor man y of thesetissu e-specif i cantigens in thethymicmedulla iscontrolled by genessuchasthe auto immun eregulator (AIRE)gene 11211. AIR!::defi ciency inmice andinhumansresults inautoimmunity.highlightin gits importance inclonal deleti on and centra l tolerance11221.II' acellis to lerant totissue -spe cificantigensandsurvivesboth
25
positiveand negativeselection.it leavesthethymusandcirculatesin the peripheryasan immunocompetentsinglepositive(SI') matu re'I'cell. Thismeansthatselectionofthe'I' cellrepertoireisbased on therecognition ofoneorafewsclf-MlK'moleculesmodilied bythe bindingof a vastarrayofsclf-pcptidcs.Thus.therecognit ion ofsclf-pcptidcs below a certain threshold ofavidityisultimatelyresponsiblefo rpositiveselection01''1' cells.The diverse repertoire01''1'cellsthatis selected recognizesfo reign pcptidcsbound tothesamesclf-MlK'molecule astheselectingsel f-peptide in the perip hery.Thismeans that foreignpcptidesthat arc recogn ized inthe peripheryarc actuallyall hctcroclitic variant sofsclf-pcptidcs,l lctcroclitic variantsarcslightalterations ofnatu rally-occurring pcptidcsthatenhanceTCI{ recognition 11231.Wewillapply thisconcept togenerate potentiallyhctc rocl itic variantsof IIIVpcptidcs forpossibleusc inthera peuticvaccines (discussedfurther below).
1.2.5 ens'Teell responsein IIIV infection
Severalfindingssuggest animportantrolefo r CDX''I'cellsincontrollingIIiV infection.Studiesby both Koup£:1al.11241 and Borrow£'1al.11251 indicatedCTL controlofIIiV replicationduringacuteinfectionbymonit oringthe level ofl llVvspccific CT L activity in relationto plasmaviremiain l llV-infcctcdstudyparticipants.Inboth studies.participantswhoshowedanincreaseinCTL activityduringacuteinfection showedaconcomitantdecline inviremia.whereas those who hadlow or undetectable CT L activityhadprolonged symptomsand persistentlyhigherlevelsof viremia.CDX''I'
26
cell-mediatedcontro lorl llVvinfcct ionwas alsosuggested[romill"i l'tlCDS·Tce ll depletionstudiesin the SIV rhesusmacaque(SIVmac) model or AIDS.These stud ies showed a rapid and mark cdincreaseinviremia in acute 1126.1271 and chronic Ilnl iulectionwhen CDS·'I'cells\\"CITeliminated.l lowc vcr,upon the reappearanceor SIV- spec ifi cCDS·'I'cells.theincrease in viremia wassuppressed11271.Therapidgeneration or sequencemutat ionswithin inun unodo minantCTLcpiiopcs112S- 1321provides add itio nalevide nceorCTLpressureon IIIVreplic at ion asdoestheassocia tio nor particu lar l ll.A classIalleles(Bnand BS7)withlongterm nonpro grcssion (rev iewed in IIJ 3. IJ 4i).
Long-ter m non-progrcssors (LTN l's)exhibit naturalimmune controltoIIIV whichallows stable norm al CD4·'I'cellcountsandcontinuo us low10undetectable viral loads.Becauseor the lack or ananima l model or protection.studiesor LTI'sandIIIV- exposedbut uninfcctcd individuals(EU)have beenused todefine correlatesor protection.Whilevigorous CDS·'I'cellresponseshave been implicated in the mainten anceorlow viralload in1.'1'I'sIUS.U61 andseronegativityinEUIIJ7-IJ91.
nosingleCDS''I'cellfunction hasproven10correlatewith protection or control. LTNl's do.however, possessagreater proport ion orpolylun ctionull ilVvspccificCDS·'I'cells relativetol llv-infcctcd progrcssors11401.HlV-spcc ificCDS·'I'cellsfromLTNl'scan releaseseveralcytokincssimultaneously(IFN-y.11.-2.TNF-u.mq,inflammatoryprote in (1'v111')- 1I1.and pcrforin)1140.1411. have greater prolifcr.uivccapacit y1142.1431and have theability10dranuu icallyuprcgulate grunzymc Bandpcrfo rinprodu ct ionnltcr long-termculture 144.1441.
Otherspecificproperties orCDX+Tcells havebeenproposedas cor relates or protectiveimmu nityinLT PsandIIIV controllers.Chenet(/1.11451 round that111.1\- B27-restrictedCDX'T cellsincontrollers were better able to inhibitviralreplication throughtargetingor the immunodominantGagcpiiopc.Thiswasalsoassociatedwith distinctTCRclonotypcs,characterized by superiorcontrolorIIIV-Ireplication in vitro.
greater cross-reactivityto cpitopevariantsandenhanced load ingand delivery ofpcrforin.
Similarly. Turnbull('( al.11461have shown thatcpitopc-spccificresponseswithenhanced cross-recognitionorsequence variantswere moststronglycorrclurcd to delayed progressionto disease,Theyalsoreportedthatthe rateor diseaseprogressionis determined bythequality orresponsestocertaincriticalepitopesand thatthisis influenced by propertiesor the dominant TCRs used 1'01' cpitopc recognition.
Importantly.11.-2production bypolyfunctionalHlV-spccificCDX'T cellshasalsoken correlatedwith enhanced viralsuppression11411.Almeidact(/1.11471reported that polyfunctionali tyand potent lllv-suppressivcactivityorlIlv-spccificCDX'T cellsarc a resultorincreased antigen sensitivity. while Ilararict al.114XI argue thatCDX'Tcel ls withtheseattributesarc eq uippedwithlow-avidityTCRsand thushave low antigen sensitivity.Such debatesmustbe resolved in ordertoproperly defineCDX'T cell correlatesorprotection.It isimportanttonote aswellthatCDX'TCl:lI responsesmay not hethe onlyprotectivemeasureinIIIVprotect ion.Emu('(al.11491 compared111.1\
polymorphismsandl ll v-spccific CDX'TcellresponsesamongLTN l's. non-cont rollers (high level viremia),and"ARTsuppressed'individ uals(undetectable IIIVRNI\while onI\RT).Inthis study.while thema jority orLTNl'sdidhavehigherIIIV-spec ili c CDX'
'I'cellresponsesthantheothergroups.many1.'1'Pslacked protectivelll.AclassI allelesand evidence 01''1'cell-mediatedcontrol.It is.however.importantto note that only gag.envand pol peptide poolswere usedto detectCDX''I'cell responses,Thusit remainspossible thatthese individualshave detectableCDX''I'cell respon sestoother IIIV proteins.
DefininghowCDX''I'cellsinhibi t IIIV is oneor the mostcriticalquestion sthat need to beaddress edinorde rto developefficaciousprotect iveandtherapeuticvaccines.
Inadd itio n.wemustalsodetermin ewhich CDX''I'cellsarcresponsiblefor viralcontrol.
Severalsubsetsor CDX''I'cellsincludingnaive.effec torand memor y.exist.Upon activation.naive'I'cellsbecomeeff ector'I'ce lls(CTL)ordiffc rc nti. ucintoa diverse arrayor memory'I'cells,The processbywhichanaive cellentersintoaneffectorlineage ormemorylineage isnot entirelyclear.Changet al.11501show that a dividing'I'cell initially respondingto a microbeasymmetr icallydividesintotwodaughtercells eac h with adifferentcellratespecifi cation:atermina leffectorlineageandamemorylineage , Sarkarctal.11511sugges t thatcellsthatreceive prolonged antigenicstimulatio n during thelaterstagesor infect ion are more like lyto become terminallydifferentiated effector ce lls.while thosewho do not endure prolongedantigenstimulation andhave the ability tomake11.-2becomememory'I'cells, Perhapsbothor thesemechan ismsarc involved or maybe thememoryphenotypedependsentirelyontheantigenichistoryor each'I'cell.
CTLsproduceeffec tormolecules. suchaspcrforin andgranzymeB.andarc responsibletor thetargeteddest ruct ion or virus-infectedor tumorcells.Theyarc ter minallydifferentiatedandshort-lived. altho ugh5-10'%orthese cells survivelong-term
29
andcanbe reactiva ted1'01'protectionupon reinfection with the same pathogen1152.15]1.
Memory Tcellsdiff erfrombothnaive andeff ect orTcellsandcan hefurther subd ivided intoeffec to rmemoryT cells(TI~!)orcentral memoryTcells(Tn!).These memor yT cellsubsets arcde finedbyloc ation.funct ion and phenotype.Tl' ~larc long-lived cells that.uponeach exposure totheirspecifi c antigen.generateanacceleratedimmune response.TheyexpressCD62LandCCR7. which allows themto crosshighendo thelia l vcnulcs(1IEVs)andenter thelymphnodesfromthebloodstream11541.Tl'~1produce 11.- 2whichallowsthemto sustai n their own surv ival butarc poorprodu ccrs ofcffcc tor cytokines,suchasIFN-y.unt ilthey arc reactivated11551.TI~1arcshorter-lived. reside in non-lymph oidtissuesandexpresslowlevelsorcytot oxiceffec tormo lecule ssuchas granzymcBandperforin1155.1561.1\ study byFreelctul.11571that investigated the ability ofcellsfrom variousdifferentiation stagestoinhibitlllVreplication showed that earlymemorystagesthroughterminaleffec torswere able to effectivelyinhibit viral repl icat ion[0 similardegrees.Howe ver. given thestrong correlatio nbetween the sustainedpresen ceorI llV-spccificcus'Tn!and lon g-termnonprogression115li.1591.
it appearsthat
cos'
Tn!arc vitaltoIIIVcontrolandas suchshould be atarget or both therapeutic and prophylact ic vaccines.I.J Thcrupcutic vaccincs
Twogenera lclassesor vacci nesexist: preventati vevaccinesand therapeutic vaccines.I'reventati ve vaccinesare admin isteredtouninfcctedindividualstoprovide
30
protectionagainstfutureinfec tion.These vaccinesoftenconsistoflive attenuatedor inactivatedmicroorganisms. orpartsof thosemicroorganisms. thatstimulate thebody's immunesystem andprimeimmune cellstorespond quicklyagainst subsequentin fec tio n.
Preventativevaccinesarceffectivebecauseof immunologicalmemory.whichallmvsthe adaptive immunesystemto easilyrecognizeand destroypathogensit hasalreadyseen, Preventative vaccinesarc available fix a number viralinfectio ns11601and arc responsible for theglobaleradicationof smallpox 11611 and ncar eradicationof poliomyeliti s11621.
Therapeutic vaccine s.unlike preventativevaccines. modulate the ongoing immune responsein unresolved orchronicconditions.Theytypical lyinducecell- mediatedimmunityrather thanantibody- and complement-media tedimmunity(although some vaccine initiativesaim for neutralizing responses)byenhancingexistingor generating new immuneresponsestowardschronicpathogensor tumor antigens.
Therapeuticvaccinationcanbe achieved through bothpassivetransferand active immunization toselectivelyreduce immunopathology.Theirpotential usc inIIIV infect ion and mechanismsbywhichalleffectivetherapeuticIIIV vaccinemaybe designed arc discussedin the followin g subsections.
1.3.1 The needfor a therapeutic IIIVva ecine
ForII;\ ;\ RT to beeffect ive,strict adhe renceisrequired.Initially.II;\ ;\RT regimens signifi cantly impacteddaily life asspec ialdietaryandstrict scheduling
31
require mentsweredemandedinordertopreventthe development ordrug-resistance mutations156.65. 661. owadays . manydrugscanhecomb ined into asinglepill. which reducespill burden andscheduling requ irements.l lowcvcr, notallcombi nationsordrugs arcavailablein this single dose.therefore ,ifapersondevelops several drug-resistance mutat ionstheymayhavetoresorttoharsherIIAART regimens.IIAART is alsovery toxic and evenshort-termuscisassociatedwiththel llv-associatcd lipodystroph y syndrome11631.andcardiovascularandbone disease116~I.Therapeuticvaccination wouldreducetheneedtor toxicandexpensiveARTandwouldofferseve raladva ntages overcurrentARTsuch asfewershort-and long-termsideeffec ts,longerlastingeffec ts followingasingleadministration.and a redu ctioninthenumberorART-resistance mutations.Previous andcurrent clinicaltrialssuggestthat thesideeffectsassociated with therapeuticvaccination wouldbesimilarto those or approved vaccines,suchas inflammat ionat the siteor injection and mildflu-likesymptoms11651. The ultimategoal or a therapeuticvaccine wouldbe to generateananti-I IIVimmune response that would complete ly clear the infection.howe ver.mostresearchers thinkthisisnotpossiblein IIIV infect ion duetothe virus'abilitytoremainlaten t for decades11651.Aneffec t i ve therapeuticIIIV vaccinecould.however,preventor delaytheneed II II'ART by sustainingimmunological fitnessand thelevelandstability orCl)~'T cells.thus allowingviralcontrolwithout the added toxicity orART.
While muchresearchhasbeendirected at therapeutic vacc ination. thereisno therapeutic vacc ine approved for humanuscIIMll.Severalchallenges lacethe develo pmentoraneff ec t iv etherape uticIIIV vaccine.Thevirus'ability toproliferate
32
quickly and remain quiescent forman y years desp iteCTL pressu remeans athe rapeutic vaccine could help contro l the infection butwillnever clear it. Also,due to thevariation inIIIVstra insandlimited resistanceto closelyrelatedstrains.auniversa l vaccine is unlikely,ThedisappointingMERCKSteptrialIJ671.althougha preventativevacc ine trial. lenuswithtwoimportantquestions that needto beansweredfill"effectivevaccine design- J)WhichCTLfunct ionscorrela te withprotection'!2)Whatmagnitude or responseinassayscorrela tes to aneffec t iveCTLresponse invivo'!Answerstothese questions willallowusto surpasscurrentchallen gesand designbetterfuturevaccines,
1.3.2 Pcptidc-h ascd vaccines
Severalapproac hestotherapeutic vaccination forIIIVinfectionhave been and/or arc currentlybeingexplored,Theseincludetheusc orwhole inact ivated IIIVIIMq.
recombinantD1\andviral vectors1169-J7JI.dendritic cells1172-174 1.proteinsubunits and pcptidcs1175.1761.Peptide-based vaccines.specifica lly cpitopc-bascdpeptide vaccines.arc or particularinterestbecausetheyallow theability toselect theminimal immunogenic region orspec ificprotein antige nsthat canbetargetedby aspecific cell type (primarily CT L)toinduceeffec tiveand preciseimmuneresponses.l'cptidcsarc produced easilyandsafe ly,theref ore,canbe produced onalargescale.andquite economically.II' injected.pcptidcs cannotreverttoa virulent formorintegrateinto host DNI\.concernsassociated with liveand attenuated vacci nesand DNI\ vaccines.
respective ly. Pept ide-basedvaccinescanalsobedesignedto contain multiple cpitopcsor
oneormore patho gens.which.given theII l.Avrcstrictionor thecpito pcs.isanecessityiI' abroadlyapplicable vaccine isdesired.Broad vaccinat ion strategiesshouldalsoincl ude theincorporat ion orpcptidcsthat elicitimmune respon sesacrossavarietyorIllY clades.
whichmaybe achieved bybasin gpeptidedesign onconserved regionsinvirus strains.
Onesuchstrategyistheuseor mosaic vaccinestrateg ies whichuse a computational approac h todesignpolyvalent T cell-based vacc ines11771.This strategy maximizes coverageorcross-clade IllYsequences.
Altho ugh there are many adva ntagestopeptide-ba sed vaccines.thereare also manychallenges.Suchvaccines musttakethepresence orBandTcell cpitopc s and lH.Avrcstriction intoconside ration. Incancerimmunothera py. approachessuchasthe Tubingcnapproac h 117XI.which combinesgcno rni cs. pcpt idomi cs,bioinform at ics and T- cellimmunology.allowthe idcm ific.uion .selectionandvalida tion or largenumbersor MIIC/lILA class l-associatcd pcptidcsfromtumor-associated antigens1'01'usein vaccines. Peptid estability. delive ry and lowimmunogcnicit y alsoposedifficultiesin generatingeff ectivepeptide-basedvaccinesbut thesemaybe overcom ethroughpeptide modifi cat ion.Peptid e stabilitycan be improvedthroughthe incorp orati on orII-amino- acidsintothe cpitopc,which increasesMIIC-bind ingaffi nityandallowsprotease- resistance 1179.IXOI.and throughmodi ficationsor theaminoandcarboxy termin i.which alsoallowprotease- res istanceand improve the enzymati c stahilityor thepeptideIIXII. Pep tidedel iverystrategieshavebeen significantly improved throughtheuse or recom binant cytokincadj uvantsIIX2.IX] I.oil-emulsion-type adjuvantsIIX41andrecent developmentsin Iiposomc .uni crop articl c and nanop articlcdelivery systems(reviewedin
34
IIXSI).Impro vement sin deliveryresult inenhanced immunogcnicit y.humun ogcn icity canalsohe improved through slightcpitopc modifi cat ions.in which oneorseveralamino acidsarc substituted with others.l lctcroclit icpcpti dcs.which arcthe IllCUS of this rcscarchprojcCl. rcprcsc nta na pproac h toc pitopc mod iticat ion.
1.3.3 IIctcmclitic pcptidcs - theirIISCin therapeutic canccr vaccines and potential usein therapeutic IIIV vaccines
The termhctc rocliti cisdefin ed as "deviatingfromordi nary formsor rules··I IX(ll.
l lctcrocl itic pcptidcsarcslightaltera tio nsofnaturall y-occurr ing orindexpcptidcsthat enhanceTCRrecognitio n 1123I.Theydonotjustdeviatefromtheindexpept ideinter ms ofsequencebut deviatefrom theindexpeptid ein termsoftheirfunctional ity.l lctcroclit ic pcptidcs stimulatemorepotent immuneresponsesagainsttheirrespective indexpeptide than the index pepti deitsclfllX71.Todate,thisphenomen onhasbeenemployedmost effectivelyin therapeuticcancer vacc inesto overco mesuboptimalMI IC/TeRaffi nities and T cell toleranceto tumour-associated antige ns IIXX-1911.Tumour-associated antigensarcgenerallyse lf'antigensthatbecome overcx prcsscdintransfonuc dcel lsIIX9.
1901.Theseantige nsseldomelicitan immuneresponse asmost T ..:..:1Iswithhighor even moderateavid ityforscllpcptidcs arceliminatedduringTcellselectio ninthethym us.In animalmodelsandinvitro human systems,hctcroclit icpcptidcshavebeenshownto break Tc..:11tolera nceand dramaticall y augmentcell-mediatedimmunityagainsttumour - associ.ucd antige ns IIXX-190I.Slanskyctill.11921 attribute thisenhancedantigen-
35
specifi c antitumorimmunity to increasedstability or the pMIIC:TCR complex. which resultsin increased avidityand residence timeor this complex.However. theability or hctcrocliticpcpiid cstohypcrstirnulatcT cellsandgenerate high avidityinteractionsisnot sufficientonitsown to eliminate tumors.The Tcells thatbecomeprimed byhigh avidity hctcrocliiicpcptidcsmustbe able toundergofurther activationandexpansiondrivenby the loweravidity.poorly immunogenic indexpcptidcs,allowing theselective destruction oftransformedcellsthatovcrcxprcsstheindex sell-peptide 11901.Resultslrorncurrent clinical trials1193.1941highlight thepromiseorthisapproach.Basedonthese results\W propose that the same principlemaybe applied to IIIVinfect io nto selective lyaugmen t l llVvspccificCDXtT ccllresponses.
In astudy by Settectal.IIXXI.potentiallyhctcroclitic variantsweregenerated fromseveral IILI\ -1\2and1\3-rcstridc d rumor-associatedpcpiidcs (9mcrsand lOmcrs) andfrom two IILI\- 1\2-rcstrictcd viralpcptidcs, I\BV1'01.455 and IIIV1'01.476 (both 9mcrs).Peptide analoguesthat demonstratedhctcroc liticactivi ty.regardlessor index peptideorigin. each had aminoacid substitutionsthat were orconservativeor scmiconscrvativcnatureatpositions3.5.or 7but never at positionsI. 4. 6 or X.The hcterocliticpcptidcsidentified,includingtwo fill'theIIIV1'01.476indexpeptide.
produced up to a107-fiJld increase inT cellsensitivitytopeptid estimulationandwere assoc iated with highermagnituderesponsesIIXXI. X-raycrystnllography-infcrrcd3- dimensionalstructure or IILI\ -1\2and 1\3pMIIC:TCR complexesshowthatsidechains or aminoacidsat positions 3.5 and7in the IILI\- 1\2and1\3 bindingpcptid csinteract directlywith the CDR3regionsoft he TCR u and1\cha ins1195.1961thus suggcsting that
36
hctcroclicityisaresultorenhancedTCRreco gn ition. ThestudybySellectu].IIXXIalso includedatab le orsimilarity scoresfill'eachaminoacidin relation tothe others.These relationshipswere quantifie dbyaveragingtherankcoefficientscorefill'to lerability or pointmutatio nswithinaprotein (Dayho ffPAM250).hydrophobicity (anaverageor Kytc/Dooliu lcand l-auchcrc/I'liskusca les). andaminoacidsidechain volume(measured by1120 displacement)foreachaminoacidpair.We proposethathcicrocliticvariantsor IIIVindexpcpti dcscan begenerated inthesame mannerasin the studybySenectal.
IIXXIandincorp oratedintotherapeu tic IIlV vaccinedesign.
1.3... Cen t r a l mcmorvT cells as atar~et(IftherapeuticIIIV vaccines
Thesustainedpresenceor IIIV-speeifie CDX'Tn!cellsinLTNP isstrongly correlatedwithcontro lorIIlV replicatio nandslower disease progression 115X.1591. The
Tn! lineage is characterized by the CD3'CDX'CIH 5RATD2X'CCR7'CD I27'
phenotype11971.Tl' ~lproduceIL-2.undergoself-renewal.haveabroadTCRreperto ire andserve asprogen itorsor neweff ec torand mem oryT cells119XI.Most importantly.
Tl'~1appeartobe extremelyrarein progressiveIIlVin fectio n1144.1971.Renewal orTn! appears tobe dependenton low-le vel antigen persistence andcontinuedprolil crutio n.
llowc vcr, in casesorchronicinfection.when highviral loadspersist.renewalisless frequent leadingto adecline inTn! populat ions1151.1971.Therequirementsfor commitment tothelong-livedTl'~llineageisnotful lyunderstoodbut islikelydesignated earlyduringT cellsresponsesinvol ving Al'C vpriming 119XI.Furtherinsightintothis
37
