Trivalent live attenuated Influenza-SIV vaccines: efficacy and evolution of CTL escape in macaques

15 Pages·2013·4.24 MB·English

Trivalent Live Attenuated Influenza-Simian Immunodeficiency Virus
Vaccines: Efficacy and Evolution of Cytotoxic T Lymphocyte Escape in
Macaques
JeanetteC.Reece,aSheilajenAlcantara,aShayaranaGooneratne,aSinthujanJegaskanda,aThakshilaAmaresena,a
CarolineS.Fernandez,aKarenLaurie,bAeronHurt,bShelbyL.O’Connor,cMaxHarris,cJankaPetravic,dAlexeyMartyushev,d
AndrewGrimm,dMilesP.Davenport,dJohnStambas,a,eRobertDeRose,aStephenJ.Kenta
DepartmentofMicrobiologyandImmunology,UniversityofMelbourne,Parkville,Victoria,Australiaa;WHOCollaboratingCentreforReferenceandResearchonInfluenza,
NorthMelbourne,Victoria,Australiab;DepartmentofPathologyandLaboratoryMedicine,UniversityofWisconsin,Madison,Wisconsin,USAc;CentreforVascular
Research,UniversityofNewSouthWales,Kensington,NewSouthWales,Australiad;SchoolofMedicine,DeakinUniversity,WaurnPonds,Victoria,Australiae
Thereisanurgentneedforahumanimmunodeficiencyvirus(HIV)vaccinethatinducesrobustmucosalimmunity.CD8(cid:1)cyto-
D
toxicTlymphocytes(CTLs)applysubstantialantiviralpressure,butCTLstoindividualepitopesselectforimmuneescapevari- o
antsinbothHIVinhumansandSIVinmacaques.Inducingmultiplesimianimmunodeficiencyvirus(SIV)-specificCTLsmay w
n
assistincontrollingviremia.Wevaccinated10Mane-A1*08401(cid:1)femalepigtailmacaqueswithrecombinantinfluenzaviruses lo
expressingthreeMane-A1*08401-restrictedSIV-specificCTLepitopesandsubsequentlychallengedtheanimals,alongwithfive a
d
controls,intravaginallywithSIV .Seroconversiontotheinfluenzavirusvectorresultedandsmall,butdetectable,SIV-spe- e
mac251 d
cificCTLresponseswereinduced.TherewasaboostinCTLresponsesafterchallengebutnoprotectionfromhigh-levelviremia f
r
o
orCD4depletionwasobserved.AllthreeCTLepitopesunderwentacoordinatedpatternofimmuneescapeduringearlySIV
m
infection.CTLescapewasmorerapidinthevaccineesthaninthecontrolsatthemoredominantCTLepitopes.AlthoughCTL
h
escapecanincura“fitness”costtothevirus,aputativecompensatorymutation20aminoacidsupstreamfromanimmunodomi- tt
p
nantGagCTLepitopealsoevolvedsoonaftertheprimaryCTLescapemutation.WeconcludethatvaccinesbasedonlyonCTL :/
/
epitopeswilllikelybeunderminedbyrapidevolutionofbothCTLescapeandcompensatorymutations.Morepotentandpossi- jv
i.
blybroaderimmuneresponsesmayberequiredtoprotectpigtailmacaquesfromSIV. a
s
m
.
o
r
Thehumanimmunodeficiencyvirustype1(HIV-1)pandemic WerecentlydescribedaninfluenzavectorSIVvaccinemodel g/

o
continues to be a global health problem. Despite significant wherebySIVCTLepitopeswereinsertedintotheneuraminidase n
efforts,aneffectivevaccinehasnotyetbeendeveloped,anditis (NA)geneofmouse-adaptedattenuatedinfluenzaviruses.Infec- M
stillunclearwhatasuccessfulvaccinationapproachwillcomprise. tion of pigtail macaques via the respiratory route induced SIV- a
r
c
Attemptstoinducebroadlyreactiveneutralizingantibodieshave specificCTLresponses,butinpilotstudiestheinductionofonly h
thusfarbeenunsuccessful.Non-neutralizingantibodiesappearto one SIV-specific CTL response resulted in selection of immune 2
5
haveaffordedonlymodestprotectionintherecentThaiRV144 escapevariantsandwasnotprotectiveagainstSIVmac251challenge , 2
trial (1). Cytotoxic T lymphocyte (CTL) responses have been (22).WeexpandedourvectorstoincludethreeSIV-specificCTL 0
showntoassistinthecontrolofHIVinhumansandsimianim- 1
epitopes,allofwhichboostedCTLresponsesinsmallstudieson 3
munodeficiencyvirus(SIV)inmacaques(2–9).InductionofCTL SIV-infectedanimals.Inthepresentstudy,weanalyzedtheim- b
y
immunity by adenovirus vaccines was not protective in human munogenicity,efficacy,andevolutionofimmuneescapeSIVvari- g
trials (10), and a median of only one epitope-specific CTL re- u
antsaftervaccinationwithrecombinantinfluenzavirusesexpress- e
sponsewasinducedbytheregimen(11).Asieveanalysisofbreak- s
ingthreeSIV-specificCTLepitopesinpigtailmacaques. t
throughHIVsequenceintheSTEPadenovirusvaccinetrialdid,
however, show some impact of the CTL responses in terms of
MATERIALSANDMETHODS
selection for escape mutations (12). In the absence of complete
control of viremia, selection of immune escape variants can re- Animals. Fifteen naive female and two naive male colony-bred pigtail
duceviralreplicationcapacity(reduce“fitness”)andslowdown macaques(Macacanemestrina)werestudied,allexpressingthecommon
diseaseprogression.MutationsoutsidetheCTLepitopecan,how- majorhistocompatibilitycomplex(MHC)classIalleleMane-A1*08401
ever,partiallycompensateforthefitnesscostofCTLescapemu- (previouslynamedMane-A*10)asconfirmedusinghigh-throughputse-
tations (13–17). The degree to which compensatory mutations quencing(23).Animalswereanesthetizedintramuscularlywithketamine
negatefitnesscostimputedbyCTLescapemutationsinthevac-
cinationsettingisnotclear.
BroaderCTLresponsesmayimpartmoresubstantialcontrol Received12October2012 Accepted15January2013
ofviremia(18,19),assuggestedbythe“heterozygousadvantage” Publishedaheadofprint23January2013
seeninsubjectswithawidercomplementofHLAalleles(20,21). AddresscorrespondencetoStephenJ.Kent,[email protected].
Alternatively,elicitingCTLresponsestoasmallnumberofcritical Copyright©2013,AmericanSocietyforMicrobiology.AllRightsReserved.
epitopespertinentforSIVcontrolmaybeamoreimportantfactor doi:10.1128/JVI.02645-12
forvaccinedesign(7).
4146 jvi.asm.org JournalofVirology p.4146–4160 April2013 Volume87 Number8
TrivalentLiveAttenuatedInfluenzaVirus-SIVVaccines
TABLE1Vaccinegroupsandregimens
SIV challenge
Influenzavirus-SIVvaccination(route,strain,dose[PFU])at: mac251
(dose[TCID ],
50
Testgroup n Animalno.a Wk0 Wk4 Wk9 Wk17 route)atwk25f
Vaccinees 8 25377,26359,B0527*, i.n.,H3N2-SIV,108 i.n.,H1N1-SIV,108 i.n.,H3N2-SIV,108 i.n.,H1N1-SIV,108 104,i.vag.
(n(cid:5)10) B0517,B0526,
B0440,B0547,
B0443*
2 45418†,C0933 i.vag.,H3N2-SIV,108 i.vag.,H1N1-SIV,108 i.n.,H3N2-SIV,108 i.n.,H1N1-SIV,108 104,i.vag.
Controls 3 19351,19530†,B0508, i.n.d,H3N2b,108 i.n.,H1N1c,108 i.n.,H3N2,108 i.n.,H1N1,108 104,i.vag.
(n(cid:5)7) 2 19341,C3754† i.vag.e,H3N2,108 i.vag.,H1N1,108 i.n.,H3N2,108 i.n.,H1N1,108 104,i.vag.
2 C3751‡,5873‡ 104,i.r.
a*,Macaqueseuthanizedatday10afterSIVchallenge;†,3macaquesnotinfectedaftertheSIV challengeatweek25wererechallengedwitha10-fold-higherdoseof
mac251
SIV (105TCID )atweek32;‡,malemacaquesinfectedwithSIV atthesametimeusedascontrolsforpyrosequencingstudies.
mac251 50 mac251
bX31(H3N2,A/HKx31).
cPR8(H1N1,A/PuertoRico/8/1939). D
dMacaquesimmunizedintranasally(i.n.). o
eMacaquesimmunizedintravaginally(i.vag.). w
fNotethatfemalemacaqueswerechallengedi.vag.,andmalemacaqueswerechallengedintrarectally(i.r.). n
lo
a
d
e
d
(10 mg/kg) prior to any procedures. All studies were approved by the vaccinationduringinfection(22).Briefly,swabswereanalyzedusinga
f
r
UniversityofMelbourneanimalethicscommittee. SuperScript III One-Step reverse transcription-PCR (RT-PCR) system o
m
Recombinantinfluenzavirus-SIVconstructs.Sixseparaterecombi- (Invitrogen)andprimersandaprobetargetingthematrixgeneusingthe

nantinfluenzaAvirusesusedinthetrialweregeneratedusinganeight- ABI7500real-timePCRsystem(AppliedBiosciences).Seroconversionto h
t
plasmid reverse genetics system as previously outlined and studied in influenzaAvirusinserialplasmasampleswasmeasuredbyahemagglu- tp
macaques(22,24–27).Briefly,thesixDNAconstructsencodedeightin- tinationinhibition(HI)assayaspreviouslydescribed(29).Briefly,25(cid:1)l ://
fluenzavirussegments,includingageneticallymanipulatedneuramini- ofinfluenzaviruswasincubatedatroomtemperaturewith2-folddilu- jvi.
dase (NA) segment containing one Mane-A*10-restricted CD8 T cell tions of sera treated with receptor-destroying enzyme (RDE) (Deka a
s
epitopesequence;eitherKP9(SIVGag164-172),KVA10(SIVTat114-123),or Seiken,Tokyo,Japan)for1h.Turkeyredbloodcells(25(cid:1)l[vol/vol]) m
KSA10(SIVTat87-96).TheCD8Tcellepitopeswereinsertedseparately wereadded,andhemagglutinationwasobserved30minlater.Titersare .o
intotheNAstalksoftwomouse-adaptedstrainsofinfluenzaAvirususing expressedasthereciprocalofthehighestdilutionofplasmawherehem- rg
recombinantPCRtechniques(25).Thesevirusessharethesameinternal agglutinationwasprevented. o/
gene segments but differ in their surface glycoprotein hemagglutinin SIVchallengeofpigtailmacaques.Allfemalemacaques(10recom- n
(HA)andNAgenes—X31(H3N2,A/HKx31)andPR8(H1N1,A/Puerto binant influenza virus-SIV-immunized animals and 5 influenza virus- M
Rico/8/1939).Thatis,theconstructsconsistedofX31-KP9,X31-KVA10, immunizedanimals)werechallengedi.vag.atweek25witha10450% ar
X31-KSA10,PR8-KP9,PR8-KVA10,andPR8-KSA10.Thecontrolinflu- c
enzavirusesconsistedofX31andPR8withouttheinsertedCD8Tcell Mtisisluleerc,uNltautrioeninafleIcntisvtietudtoesseo(TfCHIeDal5t0h))o.fISnIVadmdaict2i5o1n(,ktinwdolynparivoevimdeadlebmyNa-. h 2
epitopes. The stability of the inserted peptide epitopes within the ex- 5
caquesusedsolelyforpyrosequencingstudieswerechallengedintrarec- ,
pandedvirionswasconfirmedbysequencingpriortovaccination. tally(i.r.)witha104TCID doseofSIV (Table1).SIVinfectionwas 2
Infection of pigtail macaques with recombinant influenza virus- 50 mac251 0
assessedbymonitoringSIVplasmaRNAlevelsaspreviouslydescribed 1
SIV.Allthreeseparaterecombinantinfluenzavirus-SIVconstructs(ei- 3
therPR8orX31)wereadministeredto10vaccinatedanimals(Table1).A (22). Three animals that did not become infected after the initial SIV b
totalof108PFUofinfluenzaviruswasadministeredviatherespiratory cfohladl-lehniggheewrecroenrceecnhtraallteionngeodfiv.ivraugs.(710w5eTekCsIDlaterof(aStIVweek32)).Twwitohvaac1c0i-- y g
tract(intranasally[i.n.]andintratracheally)inphosphate-bufferedsaline 50 mac251 u
as previously described (22). Corresponding wild-type influenza virus nated animals (B0443 and B0527) were euthanized at day 10 to study e
(108PFU)wasadministeredto“control”animals.Allanimalsreceivedthe tissuesamplesafterSIVmac251challenge,sothetotalnumberofvaccinated st
animalsstudiedimmunologicallywas8.Animalsweremonitoredatserial
influenzavirusesviatherespiratorytractwiththeexceptionoffourma-
timepointsfortotalperipheralCD4Tcelldepletionaspreviouslyde-
caques(twovaccineesandtwocontrols)thatreceivedthefirsttwoinoc-
scribed(30).(cid:2)7highCD4(cid:3)Tcellsinthebloodwerealsoexaminedsinceit
ulationsintravaginally(i.vag.),followedbytwoinoculationsviatherespi-
haspreviouslybeendemonstratedthatareductionin(cid:2)7highCD4(cid:3)Tcells
ratorytract(Table1).Vaccinationsweregiveni.vag.basedontheresults
frompreviousmurinestudieswherei.vag.immunizationwithinfluenza inthebloodduringSIVinfectioninrhesusmacaquescorrelateswiththe
Avirusprovidedprotectivemucosalimmunityagainsthomosubtypicand lossofintestinallaminapropriaCD4(cid:3)Tcells,potentiallyprovidinga
heterosubtypicviruschallengeviatherespiratorytract(28).Asaresult,we noninvasivemethodtomeasurediseaseprogression(31).
hypothesizedthati.vag.inoculationmightresultinsufficientinfectionto CD8(cid:1)CTLresponsesbytetrameranalysis.SIV-specificCD8Tcell
initiateanimmuneresponseinmacaques. responsesweremeasuredusingfreshwholeheparinizedbloodbytetramer
Thetimingofinfluenzavirus-SIVvaccinationwasasfollows:week0, stainingofCD3(cid:3)CD8(cid:3)lymphocytesusingfluorescentMane-A01*084tetra-
X31influenzavirus-SIV;week4,PR8influenzavirus-SIV;week9,X31 mericproteinfoldedaroundeithertheKVA10(SIVTat114-123),theKSA10
influenzavirus-SIV;andweek17,PR8influenzavirus-SIV(Table1).Cor- (SIVTat ),ortheKP9(SIVGag )peptideepitopeaspreviously
87-96 164-172
respondingwild-typeinfluenzavirus(108PFU)wasadministeredtofive described (22, 32). Expression of memory markers was assessed using a
“control”animalsasfollows:week0,PR8influenzavirus;week4,X31 monoclonal antibody cocktail of anti-CD3-AF700 (clone SP34-2), anti-
influenzavirus;week9,X31influenzavirus;andweek17,PR8influenza CD4-PE Cy7 (clone L200), anti-CD8-APC-H7 (clone SK1), anti-CD28
virus.Infectionwithinfluenzaviruswasmonitoredbyquantitatinginflu- PerCPCy5.5(cloneL293),andanti-CD95fluoresceinisothiocyanate(clone
enzavirusRNAlevelsinserialsswabsoftheupperrespiratorytractpost- DX2).Expressionofthemucosalhomingmarker(cid:4)4(cid:2)7integrinontetramer-
April2013 Volume87 Number8 jvi.asm.org 4147
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positivecellswasmeasuredbycounterstainingwithratanti-humanintegrin H3N2(X31)andH1N1(PR8)strains(fourvaccinationsintotal).
(cid:2)7APC(BDPharmingen,catalogno.551082,cloneFIB504)(31). Five macaques in the control group were inoculated with wild-
Real-timePCRforCTLescapeatKP9.Toquantifythevirallevelsof type influenza virus strains at 108 PFU (n (cid:5) 5; Table 1). The
wild-type(WT)orescapemutant(EM)quasispeciesattheKP9epitope, vaccinegroupwasgivenamixtureofthreerecombinantinfluenza
weusedastrain-specificreal-timePCRassaydevelopedinthelaboratory viruses (108 PFU of each) containing either KP9, KVA10 and
(33,34).Thisassayusesaforwardprimerspecificforeitherthenucleotide
KSA10insertedintotheneuraminidasestalkofH1N1(PR8)and
mutationencodingthedominantK165RKP9escapemutantorwild-type H3N2(X31)(n(cid:5)10;Table1)aspreviouslydescribed(22,40).
sequence.BrieflycDNAwasmadebyreversetranscribingRNAextracted
All animals received the influenza viruses via the respiratory
from EDTA-anticoagulated plasma and amplified by quantitative RT-
PCRusingbothWTandEMprimersspecificforKP9onanEppendorf tractwiththeexceptionoffourmacaques(twovaccineesandtwo
Realplex cycler. Analysis was performed using the Eppendorf Realplex controls)thatreceivedthefirsttwoinoculationsi.vag.(Table1)
software,wherebaselinesweresettwocyclesearlierthanrealreported sincewehypothesizedthati.vag.inoculationmightresultinsuf-
fluorescence. ficientinfectiontoinitiateanimmuneresponsebasedonprevious
PyrosequencingforescapeatKP9,KVA10,andKSA10.Toexamine murine studies (28). To evaluate humoral recognition of influ-
the kinetics of CD8(cid:3) T cell selection, we isolated and pyrosequenced enzavirususingthedifferentroutesofdeliveryoftheinfluenza
plasmavirusfrom15femaleanimalsinthevaccinetrialandtwoaddi-
virus-SIVconstruct,wemeasuredserumhemagglutinationinhi-
tionalmaleunvaccinatedcontrolanimalsat10serialtimepointsfromday
bition(HI)titerstobothhomologousparentviruses(H1N1and
10toweek20afterSIVinfectionaspreviouslydescribed(13).Briefly,viral D
H3N2) (Fig. 1). HI titers to PR8 (H1N1) and X31 (H3N2) for o
RNAfromEDTAanticoagulatedplasmawasextractedusingtheQIAamp w
macaquesgiventwoi.vag.immunizationsfollowedbytworespi-
MinElutevirusspinkitorQIAampUltrasensRNAkit(Qiagen,Valencia, n
CA).ViralRNAwasreversetranscribedandamplifiedusingtheSuper- ratoryimmunizationsinthecontrolgroupandthevaccineegroup lo
a
ScriptIIIOne-StepRT-PCRsystemwithPlatinumTaqDNAPolymerase comparedtothoseforcontrolsandvaccineesgivenallimmuni- d
HighFidelity(Invitrogen,Carlsbad,CA)andMID-taggedprimers(cata- zationsviatherespiratorytractareshowninFig.1.Influenzavirus e
d
log no. 454; Sigma Aldrich) spanning Gag164-172 (KP9; KKFGAEVVP) infectionoccurredinallmacaquesaftertworespiratoryinocula- fr
andTat (KVA10;KKETVEKAVA)andTat (KSA10;KKAKAN tions(Fig.1BandD).ThemeanHItitersweresignificantlyhigher o
114-123 87-96 m
TSSA).TheRT-PCRconditionswereasfollows:50°Cfor15min;94°Cfor inbothcontrolsandvaccineesgiventwodosesofinfluenzavirus
2min;40cyclesof94°Cfor15s,58°Cfor30s,and68°Cfor50s;andthen h
via the respiratory tract compared to intravaginally (controls: t
t
68°Cfor5min.DNAfromPCRproductswaspurifiedbycuttingout PR8,P(cid:5)0.02;X31,P(cid:5)0.03;vaccinees:PR8,P(cid:5)0.05;X31,P(cid:5) p:
appropriatebandsfroma1%agarosegelandusingaQiagengelextraction //
kAiut.stAramlipanlicGonensowmeereRpeoseoalrecdhaFtaecqiluitiymuoslianrgraatRiooschane/d45s4eqsuyestnecmed.Abmyitnhoe o0.u0s2ly[Mimamnnu-nWizheidtnveiya];tFhieg.v1a)g.inAaslatrreascutltr,etcheeivfeodurthaeniirmtahlisrdpraevnid- jvi.a
acidvariationwithinthethreeepitopes(GagKP9,TatKVA10,andTat fourthinoculationsviatherespiratorytract.Thesemacaquessub- sm
KSA10)wasexaminedusingacustomdataanalysispipelineinGalaxy,the sequentlyseroconvertedtoinfluenzavirus,withHItiterssimilar .o
opensourcesystemforprocessingnext-generationsequencedataasde- tomacaquesthathadreceivedallvirusesviatherespiratorytract rg
scribed previously (18, 35–37). Sequence reads were translated and (Fig. 1). Influenza virus RNA could be recovered from nose or /
o
alignedintosixreadingframesandcomparedtoareferenceSIVsequence larynxswabs2daysafterthefirstandsecondinoculationsinmost n

using BLAT (BLAST-Like Alignment Tool) (18, 38). Reads with low- animals immunized by the respiratory route but was recovered M
qualitybaseswithinepitopeswerediscarded. a
lessofteninanimalsinoculatedi.vag.(Fig.1E). r
Calculationofescaperates.WecalculatedtheescaperateEbetween c
TcellimmunogenicityaftervaccinationandSIVchallenge. h
twotimepointstiandti(cid:3)1(seetheboldlinesegmentsinFig.7A)assum- Toassesstheimmunogenicityoftherecombinantinfluenza-SIV 2
ing exponential growth/decay of viral strains during this time interval 5
(39): vaccinations,weanalyzedserialfreshbloodsamplesforthepres- , 2
(cid:1) (cid:2) enceofKVA10-,KSA10-,andKP9-specificCD8Tcellsusingspe- 0
E(cid:1) 1 ln fEM(ti(cid:2)1) fWT(ti) , cifictetramers.Modest,butsignificantKVA10andKSA10CD8T 13
ti(cid:2)1(cid:3)ti fEM(ti) fWT(ti(cid:2)1) cellresponsesinvaccineescomparedtocontrolswereobserved2 b
y
wheref andf arethefractionsofEMandWTinthetotalviralload. weeksafterthefirstimmunizationand1weekafterthefinalvac- g
TheEtMimeatwWhTichWTdropsto50%(t )wasestimatedusinglinear cination at week 18 (Fig. 2A ; *,P (cid:7) 0.05). CTL responses to u
50% e
interpolationoffractions.Forpolymorphicescape,wedefinedtheescape KVA10andKSA10inallvaccineeswereabovethethresholdof s
t
mutantascomprisingallsequencesdifferentfromtheWTattheconsid- detection(mean(cid:3)3(cid:8)thestandarddeviationoftheindividual
eredepitope(fEM(cid:5)1(cid:6)fWT).Inthiscase,t50%liesattheintersectionof tetramerresponsesofallanimalspriortovaccination)onatleast
fWTandfEMlines(seeFig.7A). three separate occasions postvaccination (Fig. 2B). In contrast,
onlyoneCTLresponsetoKVA10atonetimepointinthecontrol
RESULTS groupwasabovethisthresholdofdetection.
Influenzavirus-SIVinoculationofmacaques.Inducingbroader Incontrast,thefrequencyofKP9-specificCD8Tcellresponses
CTLresponsesisanimportantgoalofCTL-basedHIVvaccines. invaccineespostvaccinationabovethethresholdofdetectionwas
WedevelopedsixseparateinfluenzavirusesexpressingthreeSIV lower(only7of10vaccineeshadresponsesabovethethreshold).
CTL epitopes that have previously been shown to induce SIV- ThemagnitudeofKP9-specificTcellsresponsespostvaccination
specificCD8(cid:3)TcellsresponsesinSIV-infectedpigtailmacaques werealsolower;however,theseresponseswereofsimilarmagni-
when administered via the respiratory tract (22) and evaluated tudetothosemeasuredinaprevioustrial,wheretwomacaques
these vaccines in naive macaques. To assess the infectivity and werealsoinoculatedviatherespiratorytractwiththeinfluenza
immunogenicity of recombinant influenza viruses expressing virus-KP9vaccine(22).
threeSIVTatKVA10,TatKSA10,andGagKP9epitopes,weim- To address whether SIV-specific immune responses were
munized10Mane-A*10(cid:3)pigtailmacaquesoveraperiodof119 primedbyvaccination,weanalyzedtetramer-specificCD8Tcell
days. Influenza inoculation used two alternating doses of both responses following SIV challenge. All female macaques
mac251
4148 jvi.asm.org JournalofVirology
TrivalentLiveAttenuatedInfluenzaVirus-SIVVaccines
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FIG1Vaccinationofpigtailmacaqueswithrecombinantinfluenzavirus-SIV.Acomparisonbetweenroutesofdeliveryofinfluenzavirus(controls)and
recombinantinfluenzavirus-SIVvaccine(vaccinees)givenintravaginallyorviatherespiratorytract.Hemagglutinationinhibition(HI)titersinserialplasma
samplestoPR8(H1N1,shownassolidlines)andX31(H3N2,shownasdashedlines)areillustrated.Linesrepresentindividualanimals;HAtiterstoPR8andX31
forthesameanimalareshownwiththesamesymbol,andindividualanimalsareidentifiedbyspecificsymbolsasshowninthelegend.HItitersofcontrols
administeredinfluenzavirusviathevaginaltractandrespiratorytract(A)andtherespiratorytractonly(B)areshown.HItitersofvaccineesadministered
recombinantinfluenzavirus-SIVvaccineviathevaginaltractandrespiratorytract(C)andtherespiratorytractonlyarealsoshown(D).(E)Recoveryofinfluenza
virusRNAfromnose,larynx,andvaginalswabsfromvaccineesadministeredinfluenzavirus-SIVviatherespiratorytract(black),controlsadministered
influenzavirusviatherespiratorytract(gray),andvaccineesandcontrolsadministeredviathevaginaltract(white).RNAwasrecoveredbyPCRatday0andday
2afterthefirstandsecondinfluenzavirusandinfluenzavirus-SIVvaccinations.
werechallengedintravaginally(i.vag.)andthetwomalemacaques lenge using tetramers showed that KVA10 and KP9 CD8 T cell
withchallengedintrarectally(i.r.)witha104TCID ofSIV responsesinvaccineeswerebothearlierandsignificantlyhigherat
50 mac251
8weeksafterthefinalvaccination(or105TCID ofSIV if 3weekspostchallengecomparedtothecontrols(Fig.2A,KVA10,
50 mac251
theydidnotbecomeinfectedaftertheinitialchallenge).Ananal- P(cid:5)0.011;KP9,P(cid:5)0.045[Mann-Whitneytest]),despitemini-
ysisoftheSIV-specificCD8TcellresponsesafterSIV chal- mal KP9 CD8(cid:3) T cell responses after vaccination (Fig. 2B). It
mac251
April2013 Volume87 Number8 jvi.asm.org 4149
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4150 jvi.asm.org JournalofVirology
TrivalentLiveAttenuatedInfluenzaVirus-SIVVaccines
shouldalsobenotedthatperipheralKSA10-specificCD8(cid:3)Tcell P(cid:5)0.05;KP9,P(cid:5)0.002]andvaccinees[KVA10,P(cid:5)0.006;
responses were first detected at 5 weeks postchallenge, which is KSA10, P (cid:5) 0.001; KP9, P (cid:5) 0.01] calculated using the Wil-
considerablylaterthanfortheKP9-andKVA10-specificCD8(cid:3)T coxon test, Fig. 3C). The decline in (cid:2)7high tetramer-positive
cellresponsesthatwerefirstobservedat2weekspostchallenge. cells in the periphery was observed in both controls and vac-
Inductionof(cid:2)7integrin-expressingSIV-specificCD8Tcells cineesandmaybeexplainedbytheirmigrationtoSIV-infected
afterinfluenzavirus-SIVvaccinationandSIVchallenge.Studies mucosal tissues after SIV infection or death of this particular
in vitro have demonstrated that cells expressing high levels of subsetofcells.
(cid:4)4(cid:2)7((cid:4)4(cid:3)(cid:2)7high)canbindtoHIVandSIV withhighaffinity, OutcomeofSIVchallenge.Eightweeksafterthefinalvaccina-
smm
suggestingthatthesecellscouldbeearlytargetsforviralinfection tion, all 15 vaccinated female macaques were challenged i.vag.
invivo(41).Theroleof(cid:4)4(cid:2)7hasalsobeendemonstratedinthe witha104TCID ofSIV Twoadditionalmalemacaques,
50 mac251.
mucosalhomingofCD8TcellsinmacaquemodelsofSIVand addedtothestudytobeusedascontrolsforthepyrosequencing
otherviralinfections(42,43),andithasbeenfoundthatgp120 studies,werechallengedi.r.witha104TCID doseofSIV
50 mac251
binds to and signals via an activated form of (cid:4)4(cid:2)7 on CD4(cid:3) T (Table1).Fourteenoftheseventeenanimalsbecameinfectedafter
lymphocytes (41). In addition, intravenous administration of a this initial challenge with SIV ; however, three macaques
mac251
recombinantrhesusmonoclonalantibodyagainstthe(cid:4)4(cid:2)7into (one vaccinee and two controls) did not become infected. As a
rhesusmacaquesjustpriortoandduringacuteSIVinfectionre- consequence,thesethreemacaqueswererechallengedwitha10-
D
sultedinasignificantdecreaseintheplasmaandgastrointestinal fold-higherdoseofSIV (105TCID )atweek32,afterwhich o
mac251 50 w
tissue viral load compared to control SIV-infected rhesus ma- theybecameinfected.Therewasnosignificantdifferenceinmean
n
caques (44). To assess the potential for the influenza virus-SIV viralloads(Fig.4A)orsurvival(Fig.4B)betweeninfluenzavirus- lo
a
inoculationstoinducemucosalhomingCD8Tcells,weexamined SIV-vaccinatedanimalsandinfluenzavirus-vaccinatedanimals. d
SIVKVA10-,KSA10-,andKP9-specificCD8Tcellsforhighlevels Themean((cid:9)SE)peakviralloads2weeksafterchallengebetween e
d
of(cid:2)7expression((cid:2)7high).Althoughthefrequencyofvaccine-in- vaccinatedandcontrolgroupswerealmostidentical:7.70((cid:9)0.54) f
r
ducedtetramer-specificCD8Tcellswaslow,theproportionof versus7.73((cid:9)0.71)log copies/ml,respectively.Althoughthere o
10 m
high (cid:2)7(cid:3) KVA10-, KSA10-, and KP9-specific CD8 T cells was wasasubtledifferenceintheviralloadsbetweencontrolsandvaccin-
h
greaterafterthefourthinfluenzavirus-SIVboost(week18)com- ees during chronic infection, with the viral loads in the vaccinees t
t
p
paredto1weekafterthefirstinfluenzavirus-SIVboost(datanot beingslightlylowerthanthecontrols(Fig.4A),thedifferencewasnot :
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shown). This suggests that the mucosal vaccination may have in- foundtobestatisticallysignificantwhenwecomparedtheaverage jv
ducedSIV-specificCD8Tcellswithmucosalhomingcapabilities. chronicviralloadsfromweek5andweek20betweencontrolsand i.a
Sincemacaqueswereimmunizedwiththeinfluenzavirus-SIV vaccineesusingtheMann-Whitneytest(P(cid:5)0.15). sm
vaccine via the respiratory tract, we examined mucosal homing LossofCD4(cid:1)Tcellsand(cid:2)7highCD4(cid:1)Tcellspostchallenge. .
o
characteristicsofSIV-specificCD8TcellsafterSIVchallenge(Fig. AdecreaseintotalperipheralCD4Tcellspostchallengewasob- rg
3). A progressive decline in the proportion of peripheral (cid:2)7high served in both vaccinee and control groups, with no significant /
o
SIVKVA10-,KSA10-,andKP9-specificCD8Tcellswasobserved difference between the groups (Fig. 4C). It has previously been n
out to week 15 after SIV challenge. Note that the percentage of reportedthatmemoryCD4(cid:3)Tcellsinperipheralbloodexpress- M
(cid:2)7high tetramer-positive cells could only be measured once te- inghighlevelsofthe(cid:4)4(cid:2)7receptorarepreferentiallyinfectedand ar
c
tramer-specific CTLs appeared in the periphery, explaining the depletedduringacuteSIVinfection,andthisdepletioncorrelated h

absencesintimepointvaluesinthefigures.Forinstance,periph- withdepletionofgutCD4Tcellsinrhesusmacaques(31).We 2
5
eral KSA10-specific cells were not measured in the majority of thereforemeasured(cid:2)7highCD4Tcellsinserialbloodsamples,and ,
animalsuntilweek5,whereasKP9-specificandKVA10-specific flowcytometrydotplotsofCD95(cid:3)(cid:2)7highCD4(cid:3)Tcellsofarep- 20
1
CTLresponsesweremeasuredatweek2.Flowcytometrydotplots resentativevaccinatedanimalareshowninFig.5A.Adecreasein 3
fromarepresentativevaccinatedmacaqueshowingthedeclinein theproportionofCD95(cid:3)(cid:2)7highCD4Tcellsinmostanimalswas b
CD95(cid:3)(cid:2)7highCD8(cid:3)SIVtetramer-positivecellsaftertheemergence observed at week 2 to 3 during acute infection (31, 45) when y
g
ofSIVtetramerpositivecellsintheperipherycomparedtoatweek15 examining individual macaques before and after challenge with u
e
areshowninFig.3A.Thisdeclineisalsoevidentwhenexaminingthe SIV (Fig.5B).Surprisingly,however,thisdeclinewasonly s
proportionsof(cid:2)7highCD95(cid:3)CD8(cid:3)SIVtetramer-positivecellsin tranmsiaecn25t1,andbyweek10afterSIVchallenge,theproportionsof t
individualanimalsatserialtimepointsafterSIV challenge(Fig. peripheralCD95(cid:3)(cid:2)7highCD4Tcellshadreturnedtoprechallenge
mac251
3B).Thedecreaseintheproportionof(cid:2)7highCD95(cid:3)CD8(cid:3)SIV levels.ItisalsointerestingthattheproportionsofCD95(cid:3)(cid:2)7high
tetramer-positive cells shortly after challenge compared to CD4Tcellsinbloodinallmacaques,withtheexceptionofone,
duringchronicinfectionwasstatisticallysignificant(mean(cid:9) increased at day 10 after SIV challenge prior to this decrease
thestandarderror[SE]ofcontrols[KVA10,P(cid:5)0.01;KSA10, (Fig.5B).Therewasnosignificantdifferenceintheproportionof
FIG2DetectionofSIVGag(KP9)andTat(KVA10andKSA10)specificCD8Tcellsinperipheralblood.Pigtailmacaquesweregivenfourvaccinationsatweeks
0,4,9,and17withrecombinantinfluenzavirus-SIV(vaccinees)orinfluenzavirus(controls)andchallengedintravaginallyatweek25withpathogenicSIV .
mac251
(A)Themeans((cid:9)SE)oftheproportionofCD8TcellsstainedforKVA10/ManeA1*08401,KSA10/ManeA1*08401,andKP9/ManeA1*08401tetramersatweeks
2and18postvaccinationandweeks3and5postSIV challengeweremeasuredbyusingflowcytometry.ProportionsofSIV-tetramerpositiveCD8(cid:3)Tcells
mac251
invaccineessignificantlyhigherthancontrolsasdeterminedbytheMann-Whitneytest(P(cid:7)0.05)arehighlightedwithanasterisk(*).(BandC)Proportionsof
CD8 T cells stained for KVA10/ManeA1*08401, KSA10/ManeA1*08401, and KP9/ManeA1*08401 tetramers for individual macaques in vaccinees (open
symbols,solidlines)andcontrols(solidsymbols,dashedlines)aftervaccination(B)andafterSIV challenge(C).Thehorizontallineindicatesthethreshold
mac251
ofdetection(mean(cid:3)3(cid:8)thestandarddeviationoftheindividualtetramerresponsesofallanimalspriortovaccination).Symbolsforindividualanimalsare
showninFig.1.
April2013 Volume87 Number8 jvi.asm.org 4151
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FIG3Expressionof(cid:2)7integrinonSIV/Mane-A1*08401tetramer-positiveCD8Tcells.TheexpressionofperipheralCD95(cid:3)(cid:2)7highcellsonCD3(cid:3)CD8(cid:3)SIV
tetramer-positivecellsafterchallengewithSIV wasdeterminedusingflowcytometry.(A)Flowcytometrydotplotsfromarepresentativevaccineemacaque
showingtheproportionofCD95(cid:3)(cid:2)7highcellsmwach2e5n1CD3(cid:3)CD8(cid:3)tetramer-positivecellsfirstappearintheperiphery:atweek2postchallengeforKVA10-and
KP9-positiveCD8(cid:3)Tcellsandatweek5forKSA10-positiveCD8(cid:3)Tcellsandduringchronicinfectionat15weeksafterSIVchallenge.(B)Individualvaccinees
(opensymbols,solidlines)andcontrols(solidssymbols,dashedlines)macaquesshowingtheproportionofCD95(cid:3)CD3(cid:3)CD8(cid:3)SIVtetramer-positivecellswith
(cid:2)7highexpressionatserialtimepointsafterSIV challenge.SymbolsforindividualanimalsareasdefinedinFig.1.(C)Means(cid:9)theSEoftheproportions
ofCD95(cid:3)(cid:2)7highcellsonCD3(cid:3)CD8(cid:3)SIVtetrammaec2r5-1positivecellsonvaccinees((cid:1))andcontrols((cid:1))atweek2afterSIVchallengeforKVA10andKP9,atweek
5afterSIVchallengeforKSA10,andatweek15afterSIVchallenge.
CD95(cid:3) (cid:2)7high CD4 T cells in blood between the vaccinees and aredistributionofcellsbetweentheperipheryandthemucosa.
controls(Fig.5C). Thatis,CD95(cid:3)(cid:2)7highCD4Tcellsmigratefromthemucosatothe
Anincrease,followedbyatransientdeclineandthenareturn peripheryduringacuteinfection,leadingtoanincreaseinCD95(cid:3)
tobaseline,ofperipheralCD95(cid:3)(cid:2)7highCD4Tcellsmayrepresent (cid:2)7highCD4Tcellsintheperiphery.Shortlyafter,CD95(cid:3)(cid:2)7high
4152 jvi.asm.org JournalofVirology
TrivalentLiveAttenuatedInfluenzaVirus-SIVVaccines
rhesusmacaques(31),thephysiologicalsignificanceof(cid:4)4(cid:2)7dur-
ingSIVinfectioninpigtailmacaquesmaybeminimal.
EvolutionofimmuneescapeofSIV-specificCD8Tcellsin-
duced by influenza virus-SIV vaccines. A possible explanation
forthelackofcontrolofSIVviremiadespitetheprimingofmul-
tipleCTLresponseswastherapidevolutionofimmuneescape.
We initially quantified CTL escape at the KP9 Gag epitope in
plasma viral RNA at serial time points from day 10 to week 20
usingastrain-specificPCRassay(33,34)andfoundthatvaccina-
tionwithrecombinantinfluenzavirus-SIVgenerallyresultedin
anearliergenerationofimmuneescapevariantsattheGagKP9
CD8Tcellepitopecomparedtothecontrolanimalsinoculated
with influenza virus alone. The findings for KP9 escape in two
representativevaccineesandcontrolsarepresentedinFig.6A.
AlthoughtheGagKP9CTLepitopeusuallyescapesinamono-
morphicway(K165Ratposition2),thetwopreviouslyobserved
D
Tatepitopes(KSA10,KVA10)escapeinpolymorphicways(46) o
w
acrossanimals,soastrain-specificreal-timePCRassayisnotsuit-
n
ablefortheseepitopes.Asaresult,toassessescapeattheKSA10 lo
a
andKVA10epitopes,weperformedpyrosequencingacrossthese d
epitopes,aswellastheKP9epitope. e
d
AnalysisofimmuneescapefromplasmaRNAattheGagKP9 f
r
epitope using pyrosequencing showed that immune escape not o
m
onlyoccurredearlierinvaccinees,oftenoccurring14daysafter
h
inoculation,butalsooccurredveryrapidly(i.e.,atahighrate). t
t
p
Thesametworepresentativevaccineesandcontrolsshowinges- :
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capeatKP9bypyrosequencingareillustratedinFig.6B.Pyrose- jv
quencing was able to confirm results obtained using the KP9- i.a
specificPCRassay,asillustratedinFig.6A(33,34).Inaddition, sm
pyrosequencing revealed early and very rapid escape at the Tat .
o
KVA10andKSA10epitopes(Fig.6CandD).Asmentionedabove, rg
thisescapewaspolymorphicinnature.Thedivergentnatureof /
o
escapeatthetwoTatepitopesincontrolandvaccineeanimalsis n

representedinFig.6CandD. M
a
Sinceweobtainedanaverageof500readsforeachsequencing r
c
timepointusingpyrosequencing,wecouldconfidentlydetectthe h

emergenceofarangeofviralvariantswithescapemutationswith 2
5
frequenciesaslowas1%ofcirculatingvirus,whichisconsistent ,
2
withtherecentlypublishedpyrosequencingerrorrate(18). 0
1
The rate of CTL escape provides an estimate of the pressure 3

appliedbytheCTLresponse(39,47).Wecalculatedbothmaximal b
y
escape rate assuming exponential growth/decay of WT and EM
g
strainsduringthistimeinterval(shownasboldlinesinFig.7A) u
e
(39)andthetimefortheEMtoreach50%ofthetotalvariants s
SFIIVG.E4aOchumtcoamcaequoefSwIaVsmcahc2a5ll1enchgaeldleinngteraovfagminaaclalqyuweisthva1c0c4inTatCeIdDwithSIiVnfluenzaa-t (indicated as “t50%” in Fig. 7A). For polymorphic escape, we t
50 mac251
week25(8weeksafterthefinalimmunization).Threemacaques(onevaccinee definedtheescapemutantascomprisingallsequencesdifferent
andtwocontrols)thatdidnotbecomeinfectedwererechallengedintravaginally fromWTattheconsideredepitope.Theanalysisofthetimetaken
witha10-fold-higherdoseofSIV (105TCID )atweek32,afterwhichthey
mac251 50 for the virus to escape from 100 to 50% wild-type (WT) at the
cameinfected.Bloodsamplesfromvaccinees((cid:1))andcontrols((cid:2))wereanalyzed
forSIVRNAbyRT-PCR.(A)Mean((cid:9)theSE)ofviralloads.(B)Kaplan-Meier threeCTLepitopesrevealedthatKP9andKVA10escapeinthe
survivalgraphforSIV-infectedpigtailmacaques.(C)CD4Tcelllossforeach vaccineesemergedsignificantlyearlierthaninthecontrolgroup.
vaccinegroup.Themeanvalues((cid:9)theSE)ofthepercentageofCD4Tcellsat Thatis,50%WTvirusattheKP9andKVA10epitopeoccurred
baselinearegiven.Therewerenosignificantdifferencesbetweenvaccineesand significantly earlier in vaccinees compared to controls (Mann-
controls. Whitneytest;KP9,50(cid:9)11daysversus38(cid:9)12days,P(cid:5)0.02;
KVA10,31(cid:9)6daysversus21(cid:9)1days,P(cid:5)0.037;Fig.7BandC)
but did not occur significantly faster for KSA10 (90 (cid:9) 14 days
CD4Tcellsareeitherkilledormigratebacktotheperipheryand versus 68 (cid:9) 5 days, P (cid:5) 0.73) (Fig. 7D). In addition to escape
return to baseline levels as a result of an equilibration forming occurringearlier,therateofescapewasshowntobealsosignifi-
betweenredistributionandlossofcellsbetweenthemucosaand cantlyquickeronlyforKVA10(Mann-Whitneytest;P(cid:5)0.0041)
theperiphery.Althoughtheseresultsarenotconsistentwiththe (Fig.7C).TherapidemergenceofKP9andKVA10escapemu-
loss of (cid:2)7high CD4 T cells observed after challenge with SIV in tantsmayexplainthefailuretoobservesignificantlylowerviremia
April2013 Volume87 Number8 jvi.asm.org 4153
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FIG5Expressionof(cid:2)7highintegrinonperipheralCD4Tcells.(A)FlowcytometrydotplotdemonstratingtransientlossofCD4(cid:3)CD95(cid:3)(cid:2)7highTcellsinblood rc
3(fiwlleeedkssyamftberolcsh)ailnlebnlgoeodwibtehfoSrIVeamnacd25a1ftienraSIrVepmraecs2e5n1tcahtaivlleevnagcecfionratineddimviadcuaaqlume.a(cBaq)uPerso.pSoyrmtiboonlssofofr(cid:2)i7nhdigihviCdDua4l(cid:3)anTimcealllssoafrevaacscdinefieense(doipneFnigsy.m1.b(Col)s)Maenadncso(n(cid:9)trtohles h 25
SE)oftheproportionsof(cid:2)7highCD4Tcellsinbloodinvaccineesandcontrols. ,
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levelsinvaccinatedanimalscomparedtocontrols.Theemergence efficacy,wehypothesizedthatrapiddevelopmentofcompensa- b
y
ofbothKSA10-specificCTLandKSA10escapemutantviruswas tory mutations may have evolved. We therefore examined the
g
delayed in both vaccinated and control animals, and occurred pyrosequencingdataoutsidetheimmunodominantepitopesfor u
e
significantly after the establishment of a “set-point” viral load. evidenceofmutationslinkedtotheGagK165Rmutation. s
t
Thissuggeststhatthisepitopemaynotbecrucialtoviralcontrol Wefoundthatamutation20aminoacidsupstreamoftheKP9
intheseanimals.Thefailureofvaccinationtosignificantlyaccel- epitope(V145A)thatcommonlyfollowedtheemergenceofthe
eratethedevelopmentofKSA10responsesmaysuggestthatthe K165Rmutationandwasdetectedin9of15animals.Thismuta-
delayed response occurs as a result of late presentation of this tion always emerged at a later time point relative to the KP9
epitope,whichisnotacceleratedbyvaccination. (K165R)escapemutation.TheV145Amutationcouldpotentially
Compensatorymutations.Compensatorymutationsoutside beeitheranewlyidentifiedescapemutationoracompensatory
anepitopecanpartiallyrestorethereplicativecapacityofviruses. mutationthatoccurstoovercomethesubstantialfitnesscostof
CertainpatternsoflinkedmutationsassociatedwithCD8(cid:3)Tcell theK165Rmutation.ThelikelihoodthattheV145Amutationis
epitopeescapeinhighlyconservedregionsmayalsoleadtovari- compensatoryisstronglysupportedby(i)significantlevelsofthe
ablelevelsofviralfitness(13).TheK165RGagCTLescapemuta- V145Amutationabovebackgroundsequencingerrors((cid:10)0.9%)
tionhasbeenshowntoincurasignificantfitnesscostsincethe only developed as the percentage of the K165R mutation ap-
mutationrapidlyrevertstoWTfollowinginvivopassageinMHC- proached100%andnotbeforeand(ii)thefactthatnoCD4or
mismatchedanimals(39,48).Thismightsuggestthat,intheab- CD8 T cell responses were detected across the V145A site from
sence of compensatory mutations, the more rapid CTL escape peripheral blood mononuclear cell samples obtained from ani-
observedinthevaccineeswouldincurafitnesscostandleadto malsdespiterepeatedeffortstodetectthemusingpeptide-stimu-
reducedviremia.Sincewedidnotinfactobserveanyvirological latedintracellularcytokinestainingassays(datanotshown).
4154 jvi.asm.org JournalofVirology
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FIG6EvolutionofKP9,KVA10andKSA10CTLescape.(A)ExamplesofK165RCTLescapemutationkineticsbyusingastrain-specificreal-timePCRassayinserial
plasmaRNAsamplesfromcontrolmacaques(leftpanel)andvaccinees(rightpanel).(BtoD)ExamplesofCTLescapebypyrosequencingincontrols(leftpanels)and
vaccinees(rightpanels)forthethreeCTLepitopesKP9(B),KVA10(C),andKSA10(D).TheCTLaminoacidsequenceisshowninthefirstcolumn,withthepercentage
ofthesequenceinthesubsequentcolumnsandthetimepointafterSIVchallengeatthetopofthecolumn.Themutationidentifiedisshownateachtimepoint,withthe
totalreadsshowninthebottomrow.Commonvariantsateachtimepointareshaded.Rarervariantsaccountfortheremainingsequences.
April2013 Volume87 Number8 jvi.asm.org 4155