Immunization with Apical Membrane Antigen 1 Confers Sterile Infection-Blocking Immunity against ...

14 Pages·2013·1.61 MB·English

Immunization with Apical Membrane Antigen 1 Confers Sterile
Infection-Blocking Immunity against Plasmodium Sporozoite
Challenge in a Rodent Model
SophieSchussek,a,bAngelaTrieu,aSimonH.Apte,aJohnSidney,cAlessandroSette,cDeniseL.Doolana,b
QueenslandInstituteofMedicalResearch,InfectiousDiseasesProgramme,Herston,Queensland,Australiaa;UniversityofQueensland,SchoolofMedicine,Herston,
Queensland,Australiab;LaJollaInstituteofAllergyandImmunology,SanDiego,California,USAc
Apicalmembraneantigen1(AMA-1)isaleadingblood-stagemalariavaccinecandidate.Consistentwithakeyroleinerythro-
cyticinvasion,AMA-1-specificantibodieshavebeenimplicatedinAMA-1-inducedprotectiveimmunity.AMA-1isalsoex- D
pressedinsporozoitesandinmatureliverschizontswhereitmaybeatargetofprotectivecell-mediatedimmunity.Here,we o
w
demonstrateforthefirsttimethatimmunizationwithAMA-1caninducesterileinfection-blockingimmunityagainstPlasmo- n
diumsporozoitechallengein80%ofimmunizedmice.Significantlyhigherlevelsofgammainterferon(IFN-(cid:1))/interleukin-2 lo
a
(IL-2)/tumornecrosisfactor(TNF)multifunctionalTcellswerenotedinimmunizedmicethanincontrolmice.Wealsoreport d
thefirstidentificationofminimalCD8(cid:2)andCD4(cid:2)TcellepitopesonPlasmodiumyoeliiAMA-1.ThesedataestablishAMA-1as e
d
atargetofbothpreerythrocytic-anderythrocytic-stageprotectiveimmuneresponsesandvalidatevaccineapproachesdesigned f
r
toinducebothcellularandhumoralimmunity. o
m

h
t
t
p
The Plasmodium apical membrane antigen-1 (AMA-1) is an parasite-derived or heterogeneously expressed AMA-1 protein :
/
/
integralmembraneproteinwhichisessentialforthedevelop- can protect against blood-stage parasite challenge in rodent (P. ia
ment of apicomplexan parasites in their host environment (1). berghei, P. yoelii , orP. chabaudi) and nonhuman primate (P. i.a
The AMA-1 mRNA half-life peaks in blood-stage trophozoites, knowlesi)models(1,18–25).Additionally,AMA-1-inducedpro- s
m
andexpressionofAMA-1proteinismaximizedinthelateasexual tection has been associated with species-specific invasion and .
o
schizontstage(www.plasmoDB.org)(2).Althoughitisstructur- growth-inhibiting antibodies against AMA-1, providing protec- r
g
ally conserved across apicomplexa, some domains of AMA-1 tionuponpassivetransferinmice(21).Inhumansnaturallyex- /

showhighlevelsofaminoacidpolymorphism(3).Itisthought posedtomalaria,thereisahighprevalenceofAMA-1antibodies, o
n
thatthehostimmuneresponseprovidesthepredominantselec- withtiterscorrelatingtoage(26,27).Accordingly,effortstode- D
tive pressure for these interstrain variations, with the parasite velopanAMA-1-basedvaccinehavefocusedontheinductionof e
c
varyingkeytargetstoevadehostimmunity(1). persistent,high-titerantibodyresponsesandhavealmostexclu- e
Uponcontactwiththehostcell,the83-kDaAMA-1proteinis sivelyusedrecombinantproteinsformulatedinvariousadjuvants m
b
proteolyticallyprocessedintoits66-kDamatureform,whichis (1, 22, 24, 26, 28, 29). However, although strong antibody re- e
transported to the cell surface membrane (4). In blood-stage sponses and in vitro functionality have been reported in some r
2
merozoites,AMA-1isconcentratedattheapicalpoleandpoten- studies, AMA-1-based vaccines have failed to confer significant 5
tiallyparticipatesinthereorientationandattachmentofmerozo- protection in humans (30–33). There is evidence that AMA-1- , 2
itestoredbloodcells(RBC)(5,6).Recently,adirectinteraction specificCD4(cid:1)Tcellsmayplayaroleinblood-stageimmunity, 0
1
betweenAMA-1onthemerozoitesurfaceandtherhoptryneck sincetheefficacyofAMA-1immunizationdependsonthepres- 8
protein(RON)complexinsertedintotheRBCmembranepriorto ence of CD4(cid:1) T cells and adoptive transfer of AMA-1 specific b
y
invasionhasbeendescribed(7,8).ThisAMA-1–RONcomplexis CD4(cid:1)Tcelllinescouldprotectnudemiceagainstparasitizedred
g
conserved in apicomplexans, suggesting functional importance bloodcell(pRBC)challenge(34–37).Furthermore,blood-stage u
forhostcellinvasion(6,9).Uponcellentry,theAMA-1ectodo- vaccinetrialsofAMA-1asaprotein/adjuvantformulationhave es
mainisshed;thisprocessappearstobeessentialforinvasion,since reportedlyelicitedTcellresponsesproducinganumberofcyto- t
antibodiesthatinhibitsheddingalsoinhibitinvasion(1).There- kines,includinginterleukin-5(IL-5),IL-2,andgammainterferon
mainingcytoplasmicAMA-1tailplaysanessentialroleintrigger-
ing and maintaining intracellular replication of the parasite,
whichisdistinctfromitsroleininvasionofRBC,buttheexact
Received1May2013 Returnedformodification31May2013
functionofAMA-1remainsunknown(10–12). Accepted5July2013
The mechanisms of protection against malaria are also not Publishedaheadofprint8July2013
completelyunderstood,buttheyincludethegenerationofahu- Editor:J.H.Adams
moralresponsethatblocksparasiteentryintohostcellsandin- AddresscorrespondencetoDeniseL.Doolan,[email protected].
hibits intracellular parasite growth, as well as the induction of Supplementalmaterialforthisarticlemaybefoundathttp://dx.doi.org/10.1128
parasite-targetedcellularimmuneresponsesthatdirectlyandin- /IAI.00544-13.
directlypromotethekillingofintracellularparasitesandmediate Copyright©2013,AmericanSocietyforMicrobiology.AllRightsReserved.
protectionfromreinfection(13–17). doi:10.1128/IAI.00544-13
Studieshaveshownthatimmunizationwithcorrectlyfolded,
3586 iai.asm.org InfectionandImmunity p.3586–3599 October2013 Volume81 Number10
AMA-1ConfersSterileImmunityagainstSporozoites
(IFN-(cid:2)),aswellasmultifunctionalCD4(cid:1)cytokine-producingT onepassageoffrozenpRBCstock(derivedfromcryopreservedsporozo-
cellsandmemoryTcells(38–41). itesat14dayspostinfection)throughaBALB/cmouse.Allstudieswere
Expression of AMA-1 has been described in sporozoite and approvedbytheQIMRAnimalEthicsCommitteeandwereconductedin
bothearlyandlateliverstagesinadditiontoasexualbloodstages accordancewiththeAustralianCodeofPracticefortheCareandUseof
(4, 42, 43). A role for AMA-1 in sporozoite invasion has been AnimalsforScientificPurposes(2004).
Parasiteextract.P.yoeliiextractwasgeneratedfromP.yoelii17XNL
suggested(4),butitwasrecentlydemonstratedthatwhileAMA-1
grown in BALB/c mice to approximately 50% parasitemia. Blood was
mightmediatehostcellrecognitionaswellasparasiteorientation
collectedbycardiacpunctureanddilutedwith10volumesofFCAB(flow
andstabilizationofhepatocytebinding,itisnotessentialforin- cytometric assessment of blood) buffer (1(cid:3) phosphate-buffered saline
vasionanddifferentiationinsidehepatocytes(44).Thepresence
[PBS],0.5%fetalcalfserum[FCS],2mMEDTA)(51).Thebloodsample
ofAMA-1inthesporozoiteandliverstagessuggeststhatitmaybe
wascentrifugedat600relativecentrifugalforce(RCF)for10min,andthe
a potential target of preerythrocytic-stage immunity. However, cellpelletwasresuspendedin0.5%(wt/vol)saponin(Sigma-Aldrich,Cas-
althoughAMA-1hasbeenextensivelystudiedasacandidatean- tleHill,NSW,Australia)inFCABbufferandincubatedfor30minat
tigenforasexualerythrocyticmalariavaccines,informationonits 37°C.The sample was then twice drawn through a 30-gauge needle,
roleinpreerythrocyticimmunityisscarce.Therearenumerous washedwith20volumesofMilli-QH O,andcentrifugedasdescribed D
2
datasetsshowingthatAMA-1isrecognizedbyantibodiesfrom above.pRBCwerefixedinFCABfixationandlysisbuffer(1(cid:3)PBS,4% o
w
malaria-naiveindividualsimmunizedwithradiation-attenuated [wt/vol]paraformaldehyde,and0.0067%[wt/vol]saponin)for10min n
sporozoiteswhichdonotdevelopintomatureliverschizonts(45) at 37°C. The extract was resuspended in FCAB buffer and stored at lo
(cid:4)20°Cpriortouse.Allbuffersweresterilefiltered(0.2(cid:5)m)immedi- a
asdeterminedbyenzyme-linkedimmunosorbentassay(ELISA), d
atelybeforeuse.
indirect fluorescent-antibody tests (IFATs) (J. Sacci, personal Synthetic peptides. Putative PyAMA-1 CD4(cid:1) and CD8(cid:1) T cell ed
communication),andproteinmicroarraystudies(46).Thosedata
epitopeswerepredictedfromthecompletePyAMA-1proteinsequence fr
showthatAMA-1isaccessibletotheimmunesystemduringearly o
(www.plasmoDB.org)(52,53)usingcomputerizedmajorhistocompati-
m
liver-stagedevelopment.However,toourknowledge,thereareno bilitycomplex(MHC)bindingpredictionalgorithmsavailablefromthe

reportsdemonstratingthatAMA-1vaccinescanprotectagainst ImmuneEpitopeDataBase(www.IEDB.org)(54).Peptideswereselected h
t
PlasmodiumsporozoitechallengeintheabsenceofotherPlasmo- for MHC alleles expressed by BALB/c mice (MHC class I, H-2Kd and tp
diumantigens.Severalstudiesinmice,nonhumanprimates,and H-2Dd;MHCclassII,IAdandIEd).Aconsensusalgorithmapproachwas ://
humanshaveinvestigatedtheprotectivecapacityofmultiantigen chosentorankpredictedpeptidesaccordingtotheirscoresfromvarious ia
vaccinesagainstsporozoitechallenge,withnoconclusiveresults. predictionmethods(ANN,SMM,andComblib).ThetopscoringMHC i.a
IntheP.knowlesinonhumanprimatemodel,DNAprime/poxvi- classI(9-mer;n(cid:6)15)andMHCclassII(15-mer;n(cid:6)5)nonoverlapping sm
peptidespredictedtobindwithhighestaffinitywereselectedforstudy(see
rus boost immunization with a combination of four candidate .
o
Table S1 in the supplemental material). Peptides were synthesized by
vaccineantigens,i.e.,circumsporozoiteprotein(CSP),sporozoite r
MimotopesPtyLtd.(Clayton,VIC,Australia)atapurityof(cid:7)80%,resus- g
surface protein 2 (SSP2), AMA-1, and the 42-kDa fragment of pendedat50mg/mlin100%dimethylsulfoxide(DMSO),andstoredat o/
merozoite surface protein 1 (MSP142), led to self-limited, low- (cid:4)80°Cpriortouse.ApoolofallputativeCD8(cid:1)andCD4(cid:1)Tcellepitopes n
levelparasitemiain60%to80%ofrhesusmonkeys(47,48).In wasusedtoimmunizemice. D
humans, a virosome-based vaccine comprising peptides repre- Plasmid DNA and recombinant protein immunogens. P. yoelii ec
sentingtheP.falciparumCSPBcellrepeatandthesemiconserved 17XNLgenomicDNA(gDNA)wasextractedfrompRBCofBALB/cmice e
m
loopIofdomainIIIofAMA-1inducedverylimitedblood-stage 14daysafterinfectionwithP.yoelii17XNLsporozoites.PyAMA-1specific
b
protection (manifested primarily as a delay in development of primersweredesignedtoamplifythefull-lengthgenebasedontheanno- e
parasitemiain1of12volunteers)(49).However,inbothstudies, tated protein sequence (www.plasmoDB.org) using Amplify 3.1 (http: r
2
protectiveeffectscouldnotexplicitlybeassociatedwithdefined //engels.genetics.wisc.edu/amplify/;BillEngels,UniversityofWisconsin) 5
immuneresponsestoanyoneoralltargetantigens.Mostrecently, and OligoCalc (www.basic.northwestern.edu/biotools/oligocalc.html) , 2
atwo-componentadenovirus-vectoredvaccineconsistingofone softwaretools.OligonucleotideswerecommerciallysynthesizedbySigma- 0
Aldrich(CastleHill,NSW,Australia).Full-lengthPyAMA-1wascloned 1
adenovectorencodingtheP.falciparumCSPproteinandoneen- 8
codingAMA-1inducedmoderateTcellIFN-(cid:2)responsesagainst iDnHto5p(cid:8)V,Ra1n0d20pu(Vriificeadl,Iunsci.n,gSaannDeinegdoo,toCxAin),-farmeeplpifilaesdmiindEesxchtrearcictihoinackoilti by
bothantigensbutonlylowantibodyresponses,withnogrowth-
(EndoFreePlasmidGigakit;QiagenPtyLtd.,ChadstoneCentre,VIC, g
inhibitoryactivity.However,theprotectiveefficacyofthecombi- u
Australia).
e
nationoroftheAMA-1componentalonewasnotassessed(50). Forproteinexpression,acustompIVEX-HIS/HAvectorwascreated s
Here,wereportthefirststudytoinvestigatethetruepotential bymodifyingthepIVEX-HIS3.2vector(RocheAppliedScience,Castle t
ofAMA-1asapreerythrocytic-stagevaccinecandidate.Withthis Hill,NSW,Australia)toexpressaC-terminalhemagglutinin(HA)tag
goal,wehaveemployedtwoparallelstrategiesthattargetcellular (55).Thecell-freeproteinexpressionsystemRTS500HYE.coli(5Prime
immune responses against P. yoelii AMA-1 (PyAMA-1) using GmbH,Hamburg,Germany)wasusedtoproducerecombinantprotein,
bothaDNAprime/proteinboostregimenandaTcellepitope/ whichwasthenHispurifiedusingCobaltTalonresin(ClontechLabora-
adjuvantimmunizationapproach. tories,Inc.,MountainView,CA).Briefly,Talonresinwasequilibrated
with wash buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM
imidazole)andincubatedfor30minat4°CwiththeRTSproductsolution
MATERIALSANDMETHODS inwashbuffercontaining8Murea.Themixturewasthentransferredto
Miceandparasites.Specific-pathogen-freefemaleBALB/cmice(Animal anEcono-Pacchromatographycolumn(Bio-RadLaboratories,Glades-
ResourceCentre,Perth,Australia)wereimmunizedat6weeksofage.The ville,NSW,Australia),theendotoxinwasremovedwithwashbuffercon-
parasite strain used in all experiments was Plasmodium yoelii 17XNL. taining0.05%TritonX-114,andthepurifiedproteinwaselutedwith150
CryopreservedsporozoiteskindlyprovidedbyStephenHoffman(Sanaria mM imidazole. To exchange the elution buffer with PBS, the protein
Inc.,Rockville,MD,USA)wereusedforsporozoitechallenge.Parasitized eluatewasloadedontoa4-ml3KAmiconUltraFiltrationtube(Merck
redbloodcells(pRBC)usedforblood-stagechallengewereobtainedafter Millipore,Billerica,MA)andconcentratedinthreealternatingspinning
October2013 Volume81 Number10 iai.asm.org 3587
Schusseketal.
andwashingsteps.Theproteinconcentrationafterthefinalcentrifuga- fromday14today30aftereithersporozoiteorpRBCchallenge,byflow
tionstepwasmeasuredusingaNanoDropspectrometer(ThermoFisher cytometricassessmentofthebloodasdescribedpreviously(51).Briefly,3
Scientific) with an A /A ratio of 0.6 (A /A ratios of (cid:9)1.8 are (cid:5)lbloodwascollectedfromthetipofthetailanddilutedinto1mlFCAB
260 280 260 280
indicativeoflownucleicacidcontamination,andavalueof0.6ischarac- buffer.Then,30(cid:5)lofdilutedbloodwastransferredintoa96-wellV-bot-
teristicofpureprotein).Theendotoxinlevelofthepurifiedproteinwas tomplate(SarstedtAustraliaPtyLtd.,MawsonLakes,SA,Australia),and
assessedbyendpointchromogenicLimulusamoebocytelysate(LAL)as- cellswerefixedfor10minat37°Cwith4%(wt/vol)paraformaldehyde
say(QCL-1000,LonzaGroup,Basel,Switzerland)andrangedbetween0.5 and0.0067%(wt/vol)saponin(Sigma-AldrichCo.,St.Louis,MO,USA).
and0.9endotoxinunits(EU)/ml. TheparasitenucleicacidwasstainedwithDAPI(4=,6=-diamidino-2-phe-
Immunizationandchallenge.BALB/cmice(n(cid:6)5)wereimmunized nylindole)(1(cid:5)g/mlinFCABbuffer)for30minat4°C,andthepercentage
intwoindependentexperimentseitherwithtwointramusculardoses(in offluorescentcellswasmeasuredbyflowcytometryonahigh-throughput
thetibialisanteriormusclewiththedosesplitbetweentwolegs)of100(cid:5)g sampler(HTS)-equippedFACSCantoII(BDBiosciences,SanJose,CA).
plasmid DNA and one intraperitoneal dose of 10 (cid:5)g recombinant DatawereanalyzedusingFlowJosoftwareversion9.1(TreestarInc.,Ash-
protein formulated 50:50 in Alhydrogel (Brenntag Biosector, Den- land, OR, USA) and displayed as comparative area under the curve
mark) or with three subcutaneous doses of a pool of 20 synthetic (AUC):thepercentageofparasitizedRBCswasmeasuredatmultipletime
peptides(29.5(cid:5)g/peptide/dose,10%DMSO)formulatedwith12(cid:5)g pointsoverthecourseofinfection(dailyfromday3today14afterchal- D
AbISCO100(Isconova,Sweden)atthreeweeklyintervals. lengeandthentwiceaweekuntilday30afterchallenge),andthecurve o
w
Mice administered an empty pVR1020 prime followed by a 50:50 determinedbythepercentageofpRBCovertimewasanalyzedforimmu-
n
alum–1(cid:3)PBSboostwereusedasacontrolgroupforDNA/protein-vac- nizedandcontrolgroups.TheAUCofblood-stageparasitemiaovertime lo
cinatedmice,andmicevaccinatedwithadjuvantAbISCO100onlywere wascalculatedinordertotakeintoconsiderationthekineticsofdevelop- a
d
usedasacontrolgroupforpeptide/adjuvant-vaccinatedmice.Inaddi- mentofblood-stageparasitemiaandsubsequentrecoveryinimmunized
e
tion,eachexperimentincludedagroupofnaiveno-vaccinemiceforim- andcontrolgroups.Inordertocomparedifferentexperiments,theAUC d

munogenicityassaysandevaluationofprotectivecapacity. valuesforimmunizedmicewerenormalizedtotheAUCvaluesforno- fr
o
Twoweeksafterthelastimmunization,micewerechallengedwith vaccinecontrolsofthesameexperimentthatreceivedthesamechallenge
m
either103cryopreservedsporozoitesin1%mouseserumor103pRBCin dose.

PBSinavolumeof200(cid:5)lintravenouslyinthetailvein.Thesporozoite Tcellassays.Spleensanddraininglymphnodes(DLNs)ofimmu- h
t
t
dosewasbasedoncountsconductedatvialing;thepRBCdosewasdeter- nizedandunimmunizedmicewereharvestedatthetimeofchallenge(10 p
:
minedimmediatelypriortoinfection.Theprocessofcryopreservationis to14daysafterthethirdimmunization)andrestimulatedwithPyAMA-1 //
associatedwithalossofsporozoiteviability,andourchallengedoseof103 DNA or peptide pools using A20 cells (mouse B lymphocyte cell line, ia
cryopreservedsporozoitescorrespondstoachallengedoseofapproxi- ATCCTIB-208)asantigen-presentingcells(APCs).Briefly,A20cellswere i.a
mately140freshlydissectedsporozoites(56).Wehavepreviouslyestab- preincubatedwith5(cid:5)g/mlPyAMA-1peptidepoolortransfectedwith s
m
lishedthat100%ofmiceinoculatedwithasfewas50cryopreservedP. PyAMA-1plasmidDNA(5(cid:5)gDNA/5(cid:3)106cells)usingtheAMAXA
.
o
yoeliisporozoitesdevelopedblood-stageparasitemia(S.Schussek,sub- Nucleofectorsystem(kitV;LonzaGroup,Basel,Switzerland)according
r
mittedforpublication).Infectivitycontrols(nonimmunizednaivemice to the manufacturer’s instructions and then irradiated with approxi- g
/
challengedwithparasitesinparalleltothetestgroups)wereincludedinall mately16,666cGy(Cs-137irradiator).Splenocytesandlymphnodecells o
experiments,andalldevelopedblood-stageparasitemiafromday4after wereharvestedbypassingtheorgansthrougha70-(cid:5)mcellstrainer,fol- n

sporozoitechallenge(datanotpresented). lowedbyRBClysis.Then,5(cid:3)105splenocytesorlymphnodecells/well D
Assessmentofliver-stageparasiteburdenviaqRT-PCR.Liverswere werecoincubatedwith1.5(cid:3)105antigen-presentingA20cells/wellat37°C ec
harvestedat42hafterchallengeandhomogenizedin5mlRNeasylysis for6h(forintracellularcytokinestaining[ICS])or48h(forenzyme- e
m
buffer(RLT;QiagenPtyLtd.,ChadstoneCentre,VIC,Australia)with1% linked immunosorbent spot [ELISpot] assay and cytokine bead array
b
(cid:10)-2-mercaptoethanol (Sigma-Aldrich Co LLC., St. Louis, MO, USA). [CBA]).Theserestimulationtimesandconditionswerepreviouslyopti- e
RNAwasextractedfrom200(cid:5)lhomogenateusingtheQiagenRNeasy mizedfortheinductionofcytokineresponsesusingeachoftheabove- r
2
Minikit(QiagenPtyLtd.)accordingtothemanufacturer’sinstructions. mentioned assays. Mock-stimulated cells, coincubated with irradiated 5
cDNAwassynthesizedfrom2(cid:5)gRNAusingViloreversetranscriptase A20cellsnotpresentinganyantigen,wereincludedforeachgroupof ,
2
(LifeTechnologiesPtyLtd.,AppliedBiosystems,Mulgrave,VIC,Austra- immunizedmice. 0
lia)accordingtothemanufacturer’sinstructions.cDNAsynthesisreac- (i)IFN-(cid:1)ELISpotassay.TheIFN-(cid:2)ELISpotassaywasperformed 1
8
tionmixtureswereassembledin20(cid:5)loniceandthenincubatedfor10 essentiallyaspreviouslydescribed(58,59).Atotalof5(cid:3)105splenocytes/
b
minatroomtemperature,followedby2hat42°C.Reactionsweretermi- wellinDulbecco’smodifiedEagle’smedium/HighModifieddrypowder y

natedbyheatingthesampleto85°Cfor5min.cDNAsampleswerestored (KDMEM)(SAFCGlobal,St.Louis,MO)(6mg/literfolicacid,36mg/ g
at(cid:4)20°C.P.yoelii18ScDNAwasquantifiedusinganewlydevelopedand literL-asparagine,116mg/literL-arginine,2g/litersodiumbicarbonate, ue
highlysensitivequantitativereversetranscription-PCR(qRT-PCR)assay 10mMHEPES,60mg/literpenicillin,100mg/literstreptomycin),10% s
(Schusseketal.,submitted)modifiedfromapreviouslydescribedproto- FCS,5nM(cid:10)-2-mercaptoethanol,and1.5mML-glutaminewereplatedat t
col(57)andusingacustom-madeTaqManprobeandprimers(Applied 100(cid:5)l/wellinto96-wellMultiScreenfilterplates(MilliporeMAIPS4510
Biosystems)andTaqManFastAdvancedMasterMix(AppliedBiosys- plates)precoatedwith100(cid:5)g/mlanti-mouseIFN-(cid:2)monoclonalanti-
tems, Life Technologies Australia). The mouse (cid:10) -microglobulin gene body(MAb)(BDBiosciences,SanJose,CA).Irradiatedpeptide-pulsedor
2
wasusedasthehousekeepinggene(AppliedBiosystemskit,at1/100)to antigen-transfectedA20APCsormitogencontrol(concanavalinA)was
normalize 18S cDNA threshold cycle (C ) values for analysis. Both P. addedat100(cid:5)l/well.Afterincubationat37°Cwith5%CO for36h,
T 2
yoelii18Sand(cid:10) -microglobulincDNAswerequantifiedusingastandard plateswerewashed6timeswith1(cid:3)PBSplus0.05%Tween20(PBST)and
2
curveofclonedcDNA,andtheratioofP.yoelii18ScDNAtototalmouse incubatedwith25(cid:5)g/mlbiotinylatedanti-mouseIFN-(cid:2)antibody(BD
cDNA((cid:10) -microglobulin)wascalculatedandnormalizedagainstvalues Biosciences,SanJose,CA)for3hat37°C.Plateswereagainwashedand
2
fortheno-vaccinecontrolgrouptogivethepercentageofreductionof incubatedwith1(cid:5)g/mlstreptavidin-horseradishperoxidase(HRP)(BD
liverparasiteburden(MicrosoftExcel2007,version12;MicrosoftCorpo- Biosciences,SanJose,CA)for1hatroomtemperature.Plateswerethen
ration,Redmond,WA,USA). washed3timeswithPBSTand3timeswithPBS,incubatedwith3-amino-
Assessmentofblood-stageparasitemiaviaflowcytometricassess- 9-ethylcarbazole(AEC)substratesolution(BDBiosciences,SanJose,CA)
mentofblood(FCABassay).Thekineticsandburdenofblood-stage for5min,floodedwithtapwatertostopthereaction,anddriedovernight.
infectionweremonitoreddailyfromday3today14and3timesaweek Spot-formingcells(SFCs)werecountedusingtheAIDELISpotreader
3588 iai.asm.org InfectionandImmunity
AMA-1ConfersSterileImmunityagainstSporozoites
(AIDELISpotreaderclassic;AID,SanDiego,CA).Datawereanalyzed package;GraphPad,SanDiego,CA)performedtocomparePyAMA-1
usingPrism4.02software(GraphPad,SanDiego,CA). immunizationgroupswithcontrolgroups.Themeanbackgroundcyto-
(ii)Cytokinebeadarray(CBA).Atotalof5(cid:3)105splenocytesand/or kineresponseinducedbymockstimulationwithAPCaloneineachgroup
lymph node cells/well were restimulated with 1.5 (cid:3) 105 A20 antigen- wassubtractedfromthemeanvaluesforrestimulatedcells.Tocompare
presentingcells(APCs)/wellinatotalvolumeof200(cid:5)lin96-wellround- separatetimepointsorcellpopulations,weanalyzedthedifference(cal-
bottomplates.Afterincubationfor48hat37°Cwith5%CO ,thecell culatedasaratio)betweentwomeasurementsofthesamesampleand
2
supernatantwascollectedforcomprehensiveanalysisofsecretedcyto- evaluatedthestatisticalsignificanceofdifferencesbetweenimmunized
kines(IL-1(cid:10),IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,IL-13,IFN-(cid:2),andtumor andcontrolgroupsusingthetwo-tailedMann-Whitneytest.Compari-
necrosisfactor[TNF])usingthemousecytometricbeadarrayflexkit(BD sons of multiple different stimulations (e.g., with individual peptides)
Biosciences,SanJose,CA).Briefly,amixofcaptureanddetectionbeads wereevaluatedbyone-wayanalysisofvariance(ANOVA)followedby
foreachcytokinewasincubatedwith5(cid:5)lsamplefor2hatroomtemper- two-tailedMann-Whitneytest.StatisticalanalysisofmultifunctionalT
ature.Thefluorescenceintensityofthedetectionbeadswasmeasuredon cell populations was conducted using Pestle program v1.6 and SPICE
aFACSArrayinstrument(BDBiosciences,SanJose,CA)andanalyzed softwarev5.1(60).Significancewasdefinedatthe5%level.
usingFlowJosoftware.
(iii)Intracellularcytokinestaining(ICS)andanalysisofmultifunc- RESULTS D
tionalTcellpopulations.Splenocytesand/orlymphnodecellswerere- Protectivecapacity.Theprimaryoutcomeoftheproposedstudy o
w
stimulatedasdescribedforCBAfor6handincubatedwith0.1%Golgi wastodeterminewhetherPyAMA-1-specificimmuneresponses n
Plug(BDBiosciences,SanJose,CA)fromthestartoftherestimulation couldreducetheliver-stageparasiteburdenorblood-stagepara- lo
periodat37°Cwith5%CO in96-wellV-shapedplates.Cellswerespunat a
2 sitemia following sporozoite challenge, in order to evaluate d
600RCFfor4min,washedwithFCABbuffer,andstainedforsurface
AMA-1 as a target of sterile disease-preventing immune re- e
moleculesCD4(mCD4-peridininchlorophyllprotein[PerCP]/Cy5;In- d
sponses.Toaddressthisquestion,BALB/cmicewereimmunized
vitrogen) and CD8 (mCD8-fluorescein isothiocyanate [FITC]; Bioleg- fr
with PyAMA-1 using DNA/protein or peptide/adjuvant ap- o
end)for30minat4°C.Cellswerethenwashedasbeforeandfixedwith1%
proaches and challenged with 103 infectious P. yoelii 17XNL m
paraformaldehydefor15minatroomtemperature.Cellswerewashed

againandstainedforintracellularcytokinesIL-2(mIL-2–phycoerythrin sporozoitesor103parasitizedredbloodcells(pRBC).Theparasite h
t
[PE];Biolegend),IFN-(cid:2)(mIFN-(cid:2)–allophycocyanin[APC];MiltenyiBiotec), burdensinliverandbloodwereassessedbyquantitativereal-time tp
:
andTNF(mTNF-PE/Cy7;Bioscience)overnightat4°Cinthedark.Samples PCR(qRT-PCR)orflowcytometricassessmentofblood(FCAB), //
wereacquiredonanHTS-equippedFACSCantoIIusingFACS-DiVasoft- respectively,andcomparedtothoseincontrolmice. ia
ware(BDBioscience). (i) Liver-stage parasite burden following sporozoite chal- i.a
ThepercentageofIFN-(cid:2)-,IL-2-,orTNF-positiveCD4(cid:1)andCD8(cid:1) lenge.ImmunizationwitheitherDNA/proteinorpooledpeptide sm
splenocytepopulationswascalculatedbysubtractingtheunstimulated epitopesinadjuvantsignificantlyreducedparasiteburdeninthe .
o
backgroundcontrolvaluefromthePyAMA-1-restimulatedsampleusing
liverfollowingsporozoitechallenge,(Fig.1A)asdeterminedby r
OvertonsubtractionintheFlowJoPopulationComparisontool(FlowJo g
scoelfltswpaoresivtievresifoonr9o.n1e;,TarneyesttwaroI,nocr.)a.lTlothdreifefeorfenthtieatmeethaesupreedrcecnyttoagkeinseosf, q(mRTea-nP;CsRta.nTdhaerdlivdeerv-isattaigoenp[aSrDas]it(cid:6)eb2u.9rd)einnwmaicsereimdumceudnbizyed95w.1i%th on/
Booleangates(FlowJosoftwareversion9.1;TreestarInc.)werecreatedfor PyAMA-1 DNA/protein compared to no-vaccine controls (Fig. D
IFN-(cid:2)-,IL-2-,orTNF-producingCD4(cid:1)andCD8(cid:1)Tcellpopulations. 1A)(P(cid:6)0.0195)andby51.6%(mean;SD(cid:6)27.5)inmiceim- ec
Thebackgroundfrequenciesofcytokine-producingpopulationsinun- munizedwithPyAMA-1peptide/adjuvantcomparedtotheadju- e
stimulatedcontrolsweresubtractedusingthePestleprogramv1.6,and vant-onlycontrols(Fig.1A)(P(cid:6)0.0119). m
b
thresholding,visualization,andstatisticalanalysisofdatawereperformed (ii)Blood-stageparasitemiafollowingsporozoiteorpRBC e
using SPICE software v5.1 (http://exon.niaid.nih.gov/spice/ ) 6(0). The challenge. In parallel groups, mice challenged with infectious r
2
median fluorescence intensity (MFI) of the three cytokines in single-, sporozoites (preerythrocytic stage) or pRBC (blood stage) were 5
double-, or triple-positive T cell populations relates to the amount of monitored for development of blood-stage parasitemia via the , 2
cytokinesecretedfromeachofthesepopulations.Bymultiplyingthefre- FCABassay(51).ToinvestigatetheeffectofAMA-1immuniza- 0
quencyofamultifunctionalTcellpopulationbytheMFIoftheindividual 1
tionontheentirecourseofinfection,wecalculatedtheAUCof 8
cytokinesforthispopulation,theintegratedMFI(iMFI)wascalculatedas
blood-stageparasitemiaovertimeandnormalizedAUCvaluesof b
anindicatorofTcellfunctionality(61). y
immunizedmicetoAUCvaluesofcontrolmicechallengedatthe
Indirectfluorescent-antibodytest(IFAT)byflowcytometry.The g
sametime. u
antiparasiteantibodytiterwasdeterminedbyflowcytometryasdescribed
e
previously(62)(seeFig.S1inthesupplementalmaterial).Briefly,seraof Sterileprotectionagainstsporozoitechallengeasindicatedby s
immunized and unimmunized mice were collected 2 weeks after each thecompleteabsenceofblood-stageparasitemiawasobservedin t
immunizationand21daysafterchallenge,and30(cid:5)lofserumdilutedin 80% of mice immunized with PyAMA-1 DNA/protein (P (cid:6)
FCABbuffer(1/100prechallengeand1/300postchallenge)wasincubated 0.0159).Intheremaining20%,blood-stageparasitemiawasre-
with6(cid:5)lofparasiteextractfor20minatroomtemperature.Antibodies duced by 65%, indicating partial protection against sporozoite
bindingtotheP.yoeliiparasiteextractwerestainedwithsecondarygoat challenge. However, there was no significant protection against
anti-mouseIgG-DyLight405(Biolegend)oramixofanti-mouseIgG1- thedevelopmentofblood-stageparasitemiainmiceimmunized
APC(cloneA85-1),anti-IgG2a–FITC(cloneR19-15),anti-IgM–PE/Cy7
withthePyAMA-1peptidepool(Fig.1B).
(cloneR6-60.2),andanti-IgE–PE(cloneRME-1)(allfromBDBiosci-
Neither PyAMA-1 plasmid DNA/protein nor peptide/adju-
ences)for15minat4°C.Followinganotherwash,thesampleswerere-
vant immunization conferred protection against blood-stage
suspendedin35(cid:5)lofFCABbufferandanalyzedonanHTS-equipped
challenge(Fig.1C).
FACSCantoII.PostacquisitiondataanalysiswasperformedwithFlowJo
OurdatademonstraterobustPyAMA-1-inducedprotectionat
software(seeFig.S1inthesupplementalmaterial).Allcalculationswere
performedusingMicrosoftExcel. the preerythrocytic stage following sporozoite challenge, as evi-
Statisticalanalysis.Geometricmeanresponseswerecalculatedand denced by a 98% reduction in liver-stage parasite burden and
thenonparametric,two-tailedMann-Whitneytest(Prism4.02software sterileimmunityin80%ofmice(absenceofparasitemia)follow-
October2013 Volume81 Number10 iai.asm.org 3589
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FIG1ProtectivecapacityofPyAMA-1plasmidDNAprime/proteinboostimmunizationandpeptide/adjuvantimmunization.Mice(n(cid:6)5/group,represen- w
n
tativeoftwoindependentexperiments)wereimmunized3timesat3weeklyintervalswithplasmidDNAencodingPyAMA-1andboostedwithPyAMA-1protein lo
orwithPyAMA-1poolsofpredictedPyAMA-1TcellepitopesformulatedinAbISCOadjuvant.Micewerechallengedat14daysafterthelastimmunizationwith
a
1,000P.yoeliisporozoitesor1,000pRBC.ProtectionwasassessedbyquantitativeRT-PCR(qRT-PCR)andFCABassay.(A)qRT-PCRofPy18SrRNAintotal d
liverRNA42haftersporozoitechallenge,normalizedagainsttheparasiteliverburdenininfectivitycontrolmiceimmunizedwithemptyvector.(B)Parasitemia e
d
inthebloodmeasuredasareaunderthecurve(AUC)aftersporozoitechallenge,normalizedagainsttheAUCforinfectedemptyvectorcontrol-immunizedmice.
(C)Parasitemiaintheblood(AUC)afterblood-stagechallenge,normalizedagainsttheAUCforinfectivitycontrolmiceimmunizedwithemptyvector. fr
Statisticalanalysiswasconductedbyatwo-tailedMann-Whitneytest;significancewasdefinedasaPvalueof(cid:9)0.05.n.s.,notsignificant. om

h
t
t
p
ingDNA/proteinimmunization.ThesedataestablishAMA-1asa CD4(cid:1)(7.1-fold[P(cid:9)0.0001])andCD8(cid:1)(6.3-fold[P(cid:6)0.0002]) :/
/
keytargetofpreerythrocytic-stageimmunity. TcellswasalsoincreasedinPyAMA-1DNA/proteinimmunized ia
i.
Immunologicalresponse.Toobtaininformationontheun- micecomparedtono-vaccinecontrols,asmeasuredbyICS.No- a
derlying immune responses mediating AMA-1-induced protec- tably,Th1cytokineswerepreferentiallysecretedbyCD8(cid:1)Tcells sm
tion against sporozoite challenge, we analyzed cellular immune compared to CD4(cid:1) T cells for all three cytokines (IFN-(cid:2), P (cid:6) .
o
responsesagainstthewholeantigenordefinedCD4(cid:1)orCD8(cid:1)T 0.0079;IL-2,P(cid:6)0.0079;TNF,P(cid:6)0.0119)(Fig.2B;seeFig.S2in r
g
cellepitopesandmeasuredantibodyresponsesagainstwholepar- thesupplementalmaterial).PyAMA-1peptideimmunizationin- /
asite. ducedasignificant5-foldincreaseinIFN-(cid:2)spot-formingcellsas on
(i)Cellularresponses.Protectionagainstpreerythrocytic- measuredbyELISpotassay(P(cid:6)0.0079)(Fig.2A),significantly D
stage malaria (directed at the parasite-infected hepatocyte) is higherfrequenciesofIFN-(cid:2)expressingCD8(cid:1)Tcells(6.1[P(cid:9) e
thought to be mediated mainly by CD8(cid:1) T cells and Th1-type 0.0001)asmeasuredbyICS,andasignificantincreaseofIFN-(cid:2) ce
CD4(cid:1) T cells producing IFN-(cid:2), IL-2, and TNF (13). Thus, we secretionasdetectedbyCBAinspleens(2.5-fold[P(cid:6)0.0119)but m
measuredIFN-(cid:2),IL-2,andTNFproductionbysplenocytesfrom notlymphnodesofimmunizedmicecomparedtovaluesforcon- be
AMA-1-immunizedmiceviaELISpotassay,intracellularcytokine trolsgivenadjuvantonly(Fig.2BandC).IL-2-expressingCD4(cid:1) r
2
staining (ICS), and cytokine bead array (CBA). Recent studies (3.5-fold[P(cid:6)0.0005])andCD8(cid:1)(3.7-fold[P(cid:6)0.002])Tcells 5
,
suggestthatcellsexpressingmorethanonecytokinehavebetter werealsoincreasedinimmunizedcomparedtono-vaccinecon- 2
effectorfunctionthansingle-cytokine-expressingcellsandmight trolmiceintheICSassay,andincreasedsecretionofIL-2from 0
1
correlatewithprotectionagainstinfectionsrequiringTcells(38, spleens(3.9-fold[P(cid:6)0.0120])wasdetectedbyCBA.However, 8

61,63).Furthermore,Tcellsexpressingmorethanonecytokine TNFwasonlymarginallyincreasedinICSaswellasCBAassaysin b
y
simultaneously might have a higher capacity to develop into bothTcellpopulationsandbothtissues,respectively.Asnotedfor
g
memorycells(61).Thus,wealsoanalyzedourICSdataintermsof DNA/proteinimmunization,thereweresignificantlymoreIFN- u
simultaneousproductionofanytwoorallthreeTh1cytokines, (cid:2)-, IL-2-, or TNF-expressing CD8(cid:1) T cells than CD4(cid:1) T cells es
IFN-(cid:2),IL-2,andTNF.Additionally,weinvestigatedtheextended (IFN-(cid:2),P(cid:6)0.0079;IL-2,P(cid:6)0.0119;TNF,P(cid:6)0.0117)(Fig.3C; t
cytokineprofileinducedbyeachimmunizationregimebyanalyz- see Fig. S2 in the supplemental material) in spleen samples of
ingthesupernatantsofsplenocytesandlymphnodecellsafterin AMA-1peptide/adjuvant-immunizedmice.
vitro restimulation with PyAMA-1 for a panel of 10 cytokines Overall these data show that both PyAMA-1 immunization
(IFN-(cid:2),IL-2,IL-4,IL-5,IL-6,IL-10,IL-12p70,IL-13,IL-1(cid:10),and strategiesinducedrobustTh1cytokineresponses,althoughthere
TNF)usingCBA. wassomeinconsistencyinthefinedetailsoftheresponseasnoted
(a) Th1 type cytokines IFN-(cid:1), IL-2, and TNF. PyAMA-1 byus(50)andothers(64)previously.Theantigen-specificIFN-(cid:2)
DNA/proteinimmunizationincreasedthenumberofIFN-(cid:2)-ex- response as detected by ELISpot assay and CBA was lower in
pressingCD4(cid:1)andCD8(cid:1)splenocytescomparedtothoseforno- PyAMA-1DNA/protein-immunizedmicethaninpeptide/adju-
vaccinecontrolsasmeasuredbyELISpotassay(2-fold[notsignif- vant-immunizedmice,butsimilarlevelsofIFN-(cid:2)weremeasured
icant]), ICS (CD4(cid:1), 2-fold [P (cid:6) 0.0201]; CD8(cid:1), 7.8-fold [P (cid:9) insplenocytesofbothimmunizedgroupsbyICS.Bothimmuni-
0.0001]), and CBA (1.6-fold [P (cid:6) 0.0159]) after restimulation zationstrategiesalsoinducedantigen-specificTNFresponses,al-
withPyAMA-1invitro(Fig.2).ThepercentageofIL-2-positive thoughtheTNFresponsewashigherinDNA/protein-immunized
CD8(cid:1)Tcells(4.6-fold[P(cid:9)0.0001])aswellasTNF-expressing micethaninpeptide/adjuvant-immunizedmicewhenmeasured
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FIG2IFN-(cid:2),IL-2,andTNFresponsesatthetimeofchallenge.(A)IFN-(cid:2)ELISpotassayofsplenocytesrestimulatedinvitrowiththePyAMA-1peptidepoolfor h
36h.(B)IntracellularcytokinestainingforIFN-(cid:2),IL-2,andTNFforCD4(cid:1)(whitebars)andCD8(cid:1)(blackbars)cellpopulationsafterDNA/proteinimmuni- t
t
zation (I) or peptide/adjuvant immunization (II) and restimulation for 6 h. Data were analyzed using FACS-DiVa software and the FlowJo population p
comparisontool.(C)CBAforIFN-(cid:2),IL-2,andTNFsecretionofsplenocytesafterrestimulationwithPyAMA-1for48h.ELISpotdata(SFC/millionsplenocytes) ://
arepresentedasindividualdatapointsfor5mice(representativeoftwoindependentexperiments;n(cid:6)10).ICS(percentcytokineexpression)andCBA(pg/ml ia
secretedcytokineaftercorrectionforbackgroundinemptyvectorcontrolgroups)dataarerepresentedasthemeanvalue(n(cid:6)10mice,twoindependent i.a
experiments).Statisticalsignificancecomparedtono-vaccineoradjuvant-onlycontrolswasdeterminedbyatwo-tailedMann-Whitneytest(***,P(cid:9)0.0005;**, s
P(cid:9)0.005;*,P(cid:9)0.05). m
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by ICS (CD8(cid:1) T cells, P (cid:6) 0.0071; CD4(cid:1) T cells, P (cid:6) 0.0007). nized mice than in adjuvant-only controls (triple positive, 10% on
Furthermore,therobustantigen-specificIL-2responsesinduced [P(cid:6)0.018];double-positiveIFN-(cid:2)/IL-2andIL-2/TNF,14%and D
byeitherPyAMA-1DNA/proteinorPyAMA-1peptide/adjuvant 17.5%,respectively[P(cid:6)0.018])(Fig.3C).However,andincon- e
c
immunizationdetectedbyICSwerenotmeasurableintheCBA. trast to the case for PyAMA-1 DNA/protein immunization, e
ConsistentwitharoleforCD8(cid:1)Tcellsinpreerythrocytic-stage PyAMA-1 peptide/adjuvant immunization was very poor in in- m
immunity, PyAMA-1-specific CD8(cid:1) responses were more pro- ducingamultifunctionalCD8(cid:1)Tcellpopulation(Fig.3D);only be
nouncedthanCD4(cid:1)responsesinbothimmunizedgroups(Fig.2; theIL-2/TNFdouble-positiveCD8(cid:1)Tcellpopulationwassignif- r
2
seeFig.S2inthesupplementalmaterial). icantly greater in PyAMA-1 peptide/adjuvant-immunized mice 5
(b) Multifunctional T cells. The relative distribution of the thaninadjuvant-onlycontrols(33.5%[P(cid:6)0.018]). , 2
different CD4(cid:1) and CD8(cid:1) T cell populations expressing every TheamountofcytokineexpressedinmultifunctionalTcells 0
1
possible combination of IFN-(cid:2), IL-2, and TNF following wasgenerallyhigherthanthatinsingle-cytokine-producingcells 8

PyAMA-1immunizationwassignificantlydifferentfromtherel- followingPyAMA-1DNA/proteinimmunization,asevidencedby b
ativedistributionofthosepopulationsinno-vaccinecontrolmice theiMFI(Fig.4A).Triple-positiveCD4(cid:1)andCD8(cid:1)Tcellpopu- y
g
for DNA/protein (CD4(cid:1), P (cid:6) 0.0176; CD8(cid:1), P (cid:6) 0.0047) and lationsaccountedfor29.5%and37.8%,respectively,ofthetotal u
peptide/adjuvant(CD4(cid:1),P(cid:6)0.0059;CD8(cid:1),P(cid:6)0.0068)immu- IFN-(cid:2)expression(Fig.4A),andwithinthetotalpopulationsof es
nization(Fig.3A). IL-2-andTNF-expressingCD8(cid:1)Tcells,61%and39.5%,respec- t
ThenumberofIFN-(cid:2)/IL-2/TNFtriple-positiveTcellswassig- tively,weretriplepositive(Fig.4A).Similarly,IL-2andTNFex-
nificantlyincreasedforCD4(cid:1)andCD8(cid:1)Tcellpopulationsex- pressionindouble-positiveCD4(cid:1)(45%oftotalIL-2and56.2%of
tractedfromspleensofPyAMA-1DNA/protein-immunizedmice total TNF) and double-positive CD8(cid:1) (36.4% of total IL-2 and
compared to no-vaccine controls (CD4(cid:1), 20% [P (cid:6) 0.018]; 59.1% of total TNF) T cells was increased compared to that in
CD8(cid:1),25%[P(cid:6)0.05])(Fig.3AandB).Additionally,theper- single-cytokine-expressing T cell populations after PyAMA-1
centageofIFN-(cid:2)/IL-2double-positiveCD4(cid:1)andIL-2/TNFdou- DNA/proteinimmunization(Fig.4A).However,andconsistent
ble-positiveCD4(cid:1)andCD8(cid:1)TcellswasincreasedinPyAMA-1 with an increase in the frequency of IFN-(cid:2)-expressing CD4(cid:1) T
DNA/protein-immunizedmicecomparedtono-vaccinecontrols cellscomparedtothatinno-vaccinecontrols(17.5%;P(cid:6)0.018)
(CD4(cid:1) IFN-(cid:2)/IL-2, 20% [P (cid:6) 0.018]; CD4(cid:1) and CD8(cid:1) IL-2/ (Fig. 3B), IFN-(cid:2) single-positive cells comprised approximately
TNF,18%and33%,respectively[P(cid:6)0.018])(Fig.3). 48.4% and 33.6% of the CD4(cid:1) and CD8(cid:1) T cell populations,
ConsistentwiththeprofilemeasuredfollowingDNA/protein respectively(Fig.4A).
immunization, multifunctional CD4(cid:1) T cell populations were Incontrast,followingimmunizationwithPyAMA-1peptide/
also significantly higher in PyAMA-1 peptide/adjuvant-immu- adjuvant,thehighestcontributiontoIFN-(cid:2)andTNFexpression
October2013 Volume81 Number10 iai.asm.org 3591
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FIG3MultifunctionalTcellresponsesinducedbyPyAMA-1immunization.(A)Thedistributionoftriple-cytokine-positive(red),double-cytokine-positive
(orange),single-cytokine-positive(blue),andcytokine-negative(gray)TcellpopulationsforPyAMA-1-immunizedandcontrolmiceispresentedaspiecharts.
Statisticalevaluationofpiechartswasdoneusingapermutationtest,comparingeachdistributionagainsttherespectivecontrolgroup;significancewasdefined
asaPvalueof(cid:9)0.05.(BtoE)AcomparisonofsplenicPyAMA-1-specificproductionofIFN-(cid:2),TNF,andIL-2inimmunized(black)versuscontrol(gray)mice
inCD4(cid:1)Tcells(B)andCD8(cid:1)Tcells(C)ofDNA/protein-immunizedorCD4(cid:1)Tcells(D)andCD8(cid:1)Tcells(E)ofpeptide/adjuvant-immunizedmice,assessed
atthetimeofchallengeasdescribedinthelegendtoFig.1.Dataarepresentedasrelativefrequencyforeachpopulationaftercorrectionforbackgroundcytokine
productioninunstimulatedcells,conductedusingPestleprogramv1.6andSPICEsoftwarev5.1.Thestatisticalsignificanceofresultsforimmunizedversus
controlgroupswasdeterminedusingtheWilcoxonranktest(*,P(cid:9)0.05).
3592 iai.asm.org InfectionandImmunity
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FIG4 IFN-(cid:2),IL-2,andTNFproductioninducedbyPyAMA-1immunization.ThecontributionsofdifferentCD4(cid:1)andCD8(cid:1)Tcellpopulationstototal rg
/
cytokineproductionareillustratedinpiechartsbasedontheintegratedmedianfluorescenceintensity(iMFI),calculatedbymultiplyingthefrequencyofcells
o
producingagivencytokinebythemagnitudeofthecytokineresponse(measuredasmedianfluorescenceintensity).(A)iMFIofsplenocytesafterPyAMA-1 n
DNA/proteinimmunization;(B)iMFIofsplenocytesafterpeptide/adjuvantimmunization. D
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was made by single-positive CD4(cid:1) T cells (IFN-(cid:2), 92.2%; IL-2, P(cid:6)0.0195andDLNP(cid:6)0.0159)butnotDNA/protein(Fig.5). m
88.7%; TNF, 83.5%) and CD8(cid:1) T cells (IFN-(cid:2), 61.3%; IL-2, IL-4secretionwassignificantlyhigher(23.2-fold;P(cid:9)0.0001)in be
87.2%;TNF,68.4%)ratherthanmultifunctionalcells(Fig.4B). thespleensthaninthelymphnodesofPyAMA-1peptide/adju- r
2
Verylowlevelsofcytokineswereexpressedintriple-positiveand vant-immunizedmice(Fig.5B)andsignificantlyhigherinspleno- 5
double-positive CD4(cid:1) T cells (Fig. 4B). Consistent with the cytesofPyAMA-1peptide/adjuvant-thanDNA/protein-immu- , 2
above-notedincreaseinthefrequencyofIL-2/TNFdouble-posi- nizedmice(93-fold;P(cid:6)0.0001)(Fig.5A).Significantlevelsof 0
1
tive CD8(cid:1) T cells compared to that in adjuvant-only controls, IL-12andIL-13werealsodetectedinlymphnodes(IL-12,P(cid:6) 8
87.2%ofthetotalIL-2and31.6%ofthetotalTNFwereproduced 0.0079;IL-13,P(cid:6)0.0286)butnotspleensofpeptide/adjuvant- b
bydouble-positiveCD8(cid:1)Tcells(Fig.4B). immunizedmice(Fig.5B).Allothermeasuredcytokineresponses y
g
Overall,PyAMA-1DNA/proteinimmunization,butnotpep- weremarginal. u
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tide/adjuvant immunization, induced robust multifunctional (ii)Identificationofimmunodominantepitopes.Therobust s
CD4(cid:1)andCD8(cid:1)Tcellresponseswithapreferencetowardhigh IFN-(cid:2) responses induced in our studies as reported above pro- t
triple-positive and IL-2/TNF double-positive CD8(cid:1) T cell re- vided the opportunity to identify, for the first time, minimal
sponses. CD4(cid:1)andCD8(cid:1)TcellepitopesonPyAMA-1.Understandingthe
(c)Othercytokineresponses.UsingCBA,wemeasuredthe IFN-(cid:2) response to the different immunization strategies could
quantitiesofIL-12p70,IL-1(cid:10),IL-6,IL-13,IL-4,IL-5,andIL-10 also allow us to identify differences in the immunodominant
secretedinthesupernatantofsplenocytesfromPyAMA-1-immu- epitopesrecognized.Indeed,weidentifiedrobustIFN-(cid:2)responsesto
nizedmicerestimulatedinvitrowithfull-lengthPyAMA-1ora fivepeptidesofthepoolof20putativeTcellepitopes(15CD8(cid:1)
PyAMA-1peptidepoolfor48h.Toprovideadditionalinforma- and5CD4(cid:1)peptidespredictedtobindwithhighaffinitytoMHC
tiononthePyAMA-1-specificcellularresponses,wealsoprofiled classIorIImolecules)inmiceimmunizedwithPyAMA-1pep-
cytokine secretion by cells harvested from the draining lymph tide/adjuvant (Fig. 6 and Table 1). Four of these five immuno-
nodes. dominant PyAMA-1 epitopes are MHC class I specific (H-2Kd
SignificantlevelsofIL-4andIL-5weredetectedinbothspleno- peptideI-1,H-2DdpeptideI-10,H-2KdpeptideI-3,andH-2Dd
cytesandlymphnodesofmiceimmunizedwithpeptide/adjuvant peptideI-6),andoneisMHCclassIIspecific(H-2IAdpeptide
(forIL-4,spleenP(cid:6)0.0347andDLNP(cid:6)0.0317;forIL-5,spleen II-4),indicatingaCD8(cid:1)Tcellbiasforthepeptideimmunization.
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FIG 5Cytokine bead array analysis of cytokine responses induced by PyAMA-1 immunization. (A) Splenocytes from mice immunized with PyAMA-1
n
DNA/proteinrestimulatedinvitrowithPyAMA-1DNA-transfectedA20cells.(B)I,splenocytesfrommiceimmunizedwithPyAMA-1peptidepool/AbISCO100 lo
adjuvantrestimulatedinvitrowithPyAMA-1peptidepool.II,lymphnodecellsfrommiceimmunizedwithPyAMA-1peptidepool/AbISCO100adjuvant
a
restimulatedinvitrowithPyAMA-1peptidepool.Dataarepresentedinpg/mlaftercorrectionforbackgroundcytokineproductioninemptyvectoror d
adjuvant-onlycontrols.Statisticalsignificancecomparedtocontrolswasdeterminedbyatwo-tailedMann-Whitneytest(**,P(cid:9)0.005;*,P(cid:9)0.05). e
d

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Adominancehierarchywasnoted,withtwooftheepitopes(H- peptidesarealsotargetsofPyAMA-1-specificCD4(cid:1)orCD8(cid:1)T m

2Kd peptide I-1 and H-2Dd peptide I-10) recognized by similar cellresponses. h
t
numbers of IFN-(cid:2) secreting cells as the complete peptide pool Sincepolymorphisminparasiteepitopestargetedbyhostim- tp
:
(Fig.6). muneresponsesisproblematicforvaccinedevelopment(65),we /
/
The IFN-(cid:2) response to individual peptides following DNA/ nextanalyzedthepolymorphismoftheidentifiedimmunodomi- ia
i.
proteinimmunizationwasmuchlessrobustthanthatfollowing nantepitopes.Surprisingly,giventhatAMA-1ishighlypolymor- a
s
peptide/adjuvant immunization. Nevertheless, we were able to phic(1),allfiveimmunodominantPyAMA-1epitopesidentified m
identifyoneH-2Kdbindingpeptide(I-2)andthreeH-2Ddbind- inourstudieswerehighlyconserved((cid:7)66%)acrossrodentspe- .
o
ingpeptides(I-7,I-13,andI-15)thatwererecognizedbya2-fold- cies(Table1andFig.7AandB),andtwoofthefiveepitopes(I-3 r
g
highernumberofcellscomparedtocontrols(Table1;seeFig.S3 andII-4)showedhighlevelsofconservationacrossallanimaland /
o
in the supplemental material); these responses were not signifi- humanspecies(59to88%)(Fig.7AandBandTable1). n
cantly different from the background and did not overlap the Twoofthefivenewlyidentifiedepitopes(I-6andI-10)overlap D
epitopes identified following peptide immunization (Table 1). peptide sequences shown to be recognized by malaria-exposed e
c
Additionalstudiesarenecessarytoestablishwhetherornotthese adultsinKenya(66)(PL186in25%ofperipheralbloodmononu- e
m
clear cell [PBMC] donors and PL192 in 18% of PBMC donors,
b
respectively), and a third (I-3) overlaps with cryptic epitopes e
showntobetargetedbyThelpercellsassociatedwithprotective r
2
antibodiesinP.chabaudi(35).Oneoftheputativeepitopesiden- 5
,
tifiedfollowingDNA/proteinimmunization(I-2)wasalsoiden- 2
tifiedinmalaria-exposedadultsinKenya(PL190in10to12%of 0
1
PBMC donors) (66), suggesting that this sequence does in fact 8

representatrueTcellepitope. b
y
(iii) Humoral responses. Antibodies against sporozoite and
g
merozoiteproteinsareknowntoinhibitparasiteentryintohost u
e
cells, and antibodies against parasite proteins expressed on the s
surfaceofinfectedhepatocytesorerythrocytescanmediateinhi- t
bition of parasite development (13, 15, 67, 68). To determine
whetherantibodiescouldpotentiallycontributetotheobserved
reductionofblood-stageparasitemiaaftersporozoitechallengein
PyAMA-1DNA/protein-immunizedmice,wemeasuredthelevel
ofantibodiesbindingtowholeblood-stageparasitesintheseraof
FIG6 ImmunodominantPyAMA-1epitopesidentifiedafterpeptide/adju- immunized and unimmunized mice before and 23 days after
vantimmunization.IFN-(cid:2)productionbysplenocytesharvestedfrommice
sporozoite challenge (see Fig. S1 in the supplemental material).
immunizedwithpeptide/adjuvantat10daysafterthethirdimmunizationand
restimulatedwithindividualpeptidesorPyAMA-1peptidepoolinvitrois The level of parasite-specific IgG antibody was significantly in-
shown.Dataarepresentedasmeanspot-formingcells(SFCs)forindividual creased in mice immunized with PyAMA-1 DNA/protein (P (cid:6)
mice(n(cid:6)5),witherrorbarsrepresentingthetotalrangeofvalues.Thedotted 0.0079)andpeptide/adjuvant(P(cid:6)0.0079)comparedtocontrols
linerepresentsthemeanbackgroundreactivityofunstimulatedcells.Statisti-
(Fig.8).
calsignificanceforthemeannumberofSFCsinrestimulatedcellscomparedto
Next, we analyzed IgG subtypes, measuring the levels of
adjuvant-onlycontrolswasdeterminedbyone-wayANOVAandatwo-tailed
Mann-Whitneytest(**,P(cid:9)0.01;*,P(cid:9)0.05). mIgG2a(correlatingtohumanIgG1)andmIgG1(correlatingto
3594 iai.asm.org InfectionandImmunity
AMA-1ConfersSterileImmunityagainstSporozoites
TABLE1PyAMA-1immunodominantpeptidesandtheirconservationacrossrodentandprimatemalaria-causingspecies
SFCs/millionsplenocytesa Conservation(%)b
Peptide MHCclass Sequence Location(residues) DNA/protein Peptide Rodent Primate Cross-species
I-1 I YYIFILCSI 5–13 (cid:4) (cid:1)(cid:1)(cid:1) 81.5 64.8 54.0
I-3 I QYIAENNDV 455–463 (cid:1) (cid:1)(cid:1) 77.8 72.2 58.7
I-6 I EGPNQVISE 23–31 (cid:4) (cid:1)(cid:1) 70.4 NA 36.8
I-10 I FTPEKIENY 221–229 (cid:4) (cid:1)(cid:1)(cid:1) 74.1 59.3 49.2
II-4 II NDKNFIATTALSSTE 366–380 (cid:4) (cid:1)(cid:1) 87.5 76.0 71.1
I-2 I SLCAKHTSL 160–168 (cid:1) (cid:4) 77.8 61.1 50.8
I-7 I DKPVRSGGL 118–126 (cid:1) (cid:1) 66.7 74.1 52.2
I-13 I AFPETDVHI 127–135 (cid:1) (cid:4) 66.7 55.6 52.4
I-15 I GKGYNWGNY 339–347 (cid:1) (cid:4) 77.8 72.2 75.7
AMA-1 (cid:1)(cid:1) (cid:1)(cid:1)(cid:1)(cid:1) 48.3 54.9 51.6
aIFN-(cid:2)ELISpotresponsetoAMA-1-expressingcellsorAMA-1peptidepool.(cid:1)(cid:1)(cid:1),(cid:7)100;(cid:1)(cid:1),(cid:7)50;(cid:1),(cid:7)25. D
bConservationwasdeterminedbyCLUSTALWalignmentofannotatedproteinsequences(www.plasmoDB.org)(52)fordefinedPyAMA-1peptidesequences(classIorclassII)or o
w
full-lengthAMA-1protein.Theanalysiswassplitintoconservationacrossallrodentspecies(rodent),conservationacrossallhumanandnonhumanprimatespecies(primate),and
n
conservationacrossallPlasmodiumspecies(cross-species).NA,notapplicable. lo
a
d
e
humanIgG2)inimmunizedmicebeforeandafterchallenge(see zationstrategiesagainstsporozoitechallengeandestablishedthat d

Fig. S4 in the supplemental material). The ratio of mIgG2a to AMA-1isatargetofsterileinfection-blockingimmuneresponses fr
o
mIgG1levelspostchallengewassignificantly(P(cid:6)0.0278)higherin directedatthepreerythrocyticparasitestage.Asimmunodomi- m
DNA/protein-immunizedmicethaninno-vaccinecontrols(2.28(cid:11) nantepitopesforAMA-1havenotbeenpreviouslyidentified,we
h
0.4comparedto0.46(cid:11)0.05),consistentwithabiastowardacyto- utilized a whole-antigen immunization strategy incorporating tt
p
philic antibody response. Neither IgM nor IgE was elevated by immunization with PyAMA-1 plasmid DNA and recombinant :
/
/
PyAMA-1immunizationcomparedtocontroltreatment. protein in order to maximize immune responses to all possible ia
AMA-1epitopes;DNA/proteinstrategieshavepreviouslyproved i.
a
DISCUSSION successful in inducing protection in animal models of malaria s
m
Anumberofstudiesinanimalmodels(summarizedinreference (69).ThePyAMA-1peptide/adjuvantstrategy,ontheotherhand,
.
1)confirmthatAMA-1isatargetofantibody-mediatedprotective wasdesignedtofocusimmuneresponsesonputativeCD4(cid:1)and o
r
immunityagainstblood-stagePlasmodiumparasites.AMA-1has CD8(cid:1)TcellepitopespredictedtobindBALB/cMHCclassIand g
/

thusbeenconsideredahigh-prioritytargetantigenforablood- classIImoleculeswithhighaffinity.Weelectedtoformulatethe o
n
stagemalariavaccine.However,AMA-1isalsoexpressedinPlas- poolofpredictedpeptideepitopeswiththeadjuvantAbISCO100,a
D
modiumsporozoitesandliverstages(4,42,43)andmaytherefore commercially available ISCOM-matrix formulation consisting of e
also be a target of protective preerythrocytic-stage immune re- cholesterol,phospholipid,andacombinedstructureoftwosaponin c
e
sponses.AroleforAMA-1intheinductionofprotectiveimmu- fractionsthathaspreviouslydescribedtogenerateabalancedim- m
nityagainstpreerythrocyticPlasmodiumstageshasnotbeendem- muneresponsewithbothTh1andTh2characteristics(70). b
e
onstrated yet. Here, we performed the first evaluation of the In independent experiments, sterile protection assessed as r

immunogenicity and protective capacity of PyAMA-1 adminis- completeabsenceofblood-stageparasitemiawasinducedin80% 2
5
teredviaDNAprime/proteinboostorpeptide/adjuvantimmuni- ofmiceimmunizedwithPyAMA-1DNA/protein(Fig.1B).The ,

2
0
1
8

b
y

g
u
e
s
t
FIG7ConservationofimmunodominantAMA-1epitopesacrossPlasmodiumspecies.ImmunodominantepitopesweredefinedbyDNA/protein(A)or
peptide/adjuvant (B) immunization. PY01580, P. yoelii; PBANKSA_0900, P. berghei; PCHAS_093100, P. chabaudi; PF11_0344, P. falciparum 3D7;
PFIT_PF_0344,P.falciparumIT;PKH_093110,P.knowlesi;PVX_092275,P.vivax(www.plasmoDB.org)(52).Gray,conservedacrosstworodentPlasmodium
species;darkturquoise,conservedacrosstwohumanspecies;lightturquoise,conservedacrossfouranimalandhumanspecies.
October2013 Volume81 Number10 iai.asm.org 3595