Recombinant Viral-Vectored Vaccines Expressing Plasmodium chabaudi AS Apical Membrane ...

14 Pages·2012·0.78 MB·English

Recombinant Viral-Vectored Vaccines
Expressing Plasmodium chabaudi AS Apical
Membrane Antigen 1: Mechanisms of
Vaccine-Induced Blood-Stage Protection
This information is current as
of November 26, 2018. Sumi Biswas, Alexandra J. Spencer, Emily K. Forbes, Sarah
C. Gilbert, Anthony A. Holder, Adrian V. S. Hill and Simon
J. Draper
J Immunol published online 13 April 2012
http://www.jimmunol.org/content/early/2012/04/11/jimmun
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Copyright © 2012 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Published April 13, 2012, doi:10.4049/jimmunol.1101106
The JournalofImmunology
Recombinant Viral-Vectored Vaccines Expressing
Plasmodium chabaudi AS Apical Membrane Antigen 1:
Mechanisms of Vaccine-Induced Blood-Stage Protection
Sumi Biswas,* Alexandra J. Spencer,* Emily K. Forbes,* Sarah C. Gilbert,*
Anthony A. Holder,† Adrian V. S. Hill,* and Simon J. Draper*
ApicalmembraneAg1(AMA1)isoneoftheleadingcandidateAgsforinclusioninasubunitvaccineagainstblood-stagemalaria.
However,theefficacyofAb-inducingrecombinantAMA1proteinvaccinesinphaseIIa/bclinicaltrialsremainsdisappointing.In
thisarticle,wedescribethedevelopmentofrecombinanthumanadenovirusserotype5andmodifiedvacciniavirusAnkaravectors
encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost
regimeninBALB/cmice,arecapableofinducingstrongtransgene-specifichumoralandcellularimmuneresponses.Weshowthat
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this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in o
w
reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect n
wasinvestigatedbyinvivodepletionofCD4+Tcellsandadoptive-transferstudiesintonaiveandimmunodeficientmice.Depletion loa
d
ofCD4+Tcellsledtoalossofvaccine-inducedprotection.Adoptive-transferstudiesconfirmedthatefficacyismediatedbyboth ed
CD4+TcellsandAbsfunctioninginthecontextofanintactimmunesystem.Unlikepreviousstudies,theseresultsconfirmthat fro
Ag-specificCD4+Tcells,inducedbyaclinicallyrelevantvaccine-deliveryplatform,canmakeasignificantcontributiontovaccine m
h
blood-stageefficacyintheP.chabaudimodel.Giventhatcell-mediatedimmunitymayalsocontributetoparasitecontrolinhuman ttp
malaria,thesedatasupporttheclinicaldevelopmentofviral-vectoredvaccinesthatinducebothTcellandAbsagainstPlasmo- ://w
diumfalciparumblood-stage malariaAgs likeAMA1. TheJournalof Immunology,2012,188: 000–000. w
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M m
alaria remains a significant burden on global public inhibitory Abs in in vitro assays and in vivo malaria models. It
u
n
health,despitesomerecentandencouragingsuccesses is also frequently associated with naturally acquired immunity o
withregardtomalariacontrolincertainpartsofAfrica. and, thus, has classically been one of the leading candidate Ags l.o
rg
Thus,thedevelopmentofahighlyeffectivevaccine,whichcould forinclusioninasubunitvaccineagainsttheasexualblood-stage b/
helpinthecontrolandeventualeliminationofthisdisease,remains of the parasite (3). In an attempt to induce such protective Abs, y
g
animportantgoal(1).Vaccinesagainsttheasexualblood-stageof malaria vaccine developers have classically focused on recombi- u
e
s
malariainfectionhaveoftenaimedtoreproducenaturalimmunity nant protein-in-adjuvant vaccines. Typically, these require multi- t o
and have generally targeted Ags associated with the merozoite ple immunizations in animal models to induce Ab responses of n
N
surface or located in the apical organelles (2). Apical membrane a protective magnitude (4), and clinical trials of the efficacy of o
v
Ag 1 (AMA1) appears on the surface of merozoites after its re- such candidatevaccines remaindisappointing (2). em
lease from the micronemes, where it is a target of growth- An increasingly recognized alternative to protein-in-adjuvant b
e
formulations is the use of viral-vectored vaccines (5, 6) and, in r 2
6
particular, replication-defective adenoviruses of both human and , 2
*Jenner Institute,UniversityofOxford,OxfordOX37DQ,UnitedKingdom;and simianserotype,aswellaspoxviruses,suchasmodifiedvaccinia 01
†DivisionofParasitology,MedicalResearchCouncilNationalInstituteforMedical 8
virus Ankara (MVA). These vectors can express relatively large
Research,LondonNW71AA,UnitedKingdom
Ag constructs and, when deployed in an adenovirus-MVA heter-
ReceivedforpublicationApril18,2011.AcceptedforpublicationMarch16,2012.
ologousprime-boostregimen,wereshowninmiceandnonhuman
ThisworkwassupportedinpartbyEuropeanVirtualInstituteforMalariaResearch,a
EuropeanCommunityFP7-fundedprogram(Grantagreement242095).S.C.G.,A.V.S.H., primates to induce high levels of Ag-specific CD8+ and CD4+
andS.J.D.areJennerInvestigators.A.V.S.H.wassupportedbyaWellcomeTrust T cell responses, as well as high titers of Ab (7–12). The chim-
PrincipalResearchFellowship.S.J.D.isaMedicalResearchCouncilCareerDevel-
panzee adenovirus ChAd63 and MVA have both displayed an
opmentFellow(G1000527).S.B.wasfundedbyMalParTraining,aFrameworkPro-
gramme6-fundedMarieCurieActionunderContractMEST-CT-2005-020492.A.A.H. excellentsafetyprofileforuseinhumansasprophylacticvaccines
isfundedbytheU.K.MedicalResearchCouncil(U117532067). (13, 14), and the recombinant adenovirus-MVAvaccine-delivery
ThesequencespresentedinthisarticlehavebeensubmittedtoGenBank(http://www. platform has shown high-level efficacy against all three malaria
ncbi.nlm.nih.gov/nuccore/JF690775)underaccessionnumberJF690775.
life-cycle stages inpreclinical models (9,15–17).
AddresscorrespondenceandreprintrequeststoDr.SumiBiswas,JennerInstitute, Therehavebeennumerousstudiessuggestingthattheinduction
UniversityofOxford,OldRoadCampusResearchBuilding,RooseveltDrive,Oxford
OX37DQ,U.K.E-mailaddress:[email protected] of cellular immunity in conjunction with Ab responses may in-
creasetheefficacyofvaccinestargetingclassicalblood-stageAgs,
Theonlineversionofthisarticlecontainssupplementalmaterial.
leadingtorecentcallsforsuchanapproachtobetestedclinically
Abbreviationsusedinthisarticle:AdHu5,adenovirushumanserotype5;AMA1,
apicalmembraneAg1;AUC,areaunderthecurve;ICS,intracellularcytokine (18).StudiesusingbothsporozoiteandinfectedRBCinoculation
staining; i.d., intradermally; MSP1, merozoite surface protein 1; MVA, modified to immunize human volunteers have associated cellular immune
vacciniavirusAnkara;pRBC,parasitizedRBC;vp,viralparticle.
responses against blood-stage parasites with protective outcome
Copyright(cid:1)2012byTheAmericanAssociationofImmunologists,Inc.0022-1767/12/$16.00 (19,20).Similarly,threestudiesinthePlasmodiumyoeliimouse
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101106
2 VIRAL-VECTOREDVACCINESAGAINSTP.CHABAUDIAMA1
modelhaveassociatedCD8+Tcellresponsesagainstblood-stage vestigatethe mechanisms of vaccine-induced protectionfollowing
parasites or the merozoite surface protein 1 (MSP1) Ag with challenge with P. chabaudi blood-stage parasites. These vectors
protective immunity against pre-erythrocytic liver-stage infection induceAMA1-specificTcellandAbresponsesandprovideasys-
(7, 21, 22), and a more recent study even associated such CD8+ tem in which to dissect the protective contribution of both the
T cells with protective blood-stage immunity (23). Other studies cellular and humoral arms of the immune system. Depletion and
with Plasmodium yoelii have shown that the adoptive transfer of adoptive-transfer studies demonstrateanimportant contribution of
CD4+ T cell lines against Ags, such as MSP1 , into naive mice bothvaccine-inducedCD4+TcellsandAbstoprotectiveimmunity.
33
can lead to the control of blood-stage parasitemia (24), although Thesedata,generatedwithavaccineplatformthatishighlysuited
notallvaccinatedorTcell-transfusedrecipientssurvive,despitea for human use (13, 14), support the further clinical translation of
significant reduction in parasite densities. We also reported pre- thisapproach.Giventheevidencethatcell-mediatedimmunitymay
viously that a human adenovirus serotype 5 (AdHu5)-MVAvac- also contribute to parasite control in human malaria, the develop-
cinationregimenagainsttheP.yoeliiMSP1 Agcaninducestrong mentofviral-vectoredvaccinestargetingAgs,suchasPlasmodium
42
Ag-specific cellular and humoral immune responses. In this case, falciparumAMA1,whichinducestrongcellularimmuneresponses
theCD8+TcellsagainstMSP1 providedpartialefficacyagainst inconjunctionwithAbs,maystandagreaterchanceofsuccessin
33
developing liver-stage parasites; however, depletion of either the protectinghumansagainstblood-stageparasitesincomparisonwith
CD8+orCD4+Tcellsubsetspriortoblood-stagechallengeof vaccinationstrategiesaimingtoinduceAbsalone.
immunized micehad noeffect onprotective outcome (7,9).
ThePlasmodiumchabaudichabaudistrainASparasitehaspro- Materials and Methods
videdanalternativeandhighlyinformativemodelforthestudyof Generationofviralvectorsexpressing AMA1 D
blood-stagemalariaimmunity,inwhichbothAbsandCD4+Tcells o
Theforwardprimer(59-ATGAAAGAAATATATTATATTGTAATT w
(acting ashelpercellsfor B cells, aswellasthrough Ab-indepen- n
TTGTGC-39)andreverseprimer(59-TTAATAGTATGGTTTTTCCAT lo
dentmechanisms)arereportedtobeimportant(25).Inthecaseof CAAAACTG-39)wereusedtoamplifytheAgfromgenomicP.chabaudi ad
infectionwith thisparasite,immunologically competentmicegen- chabaudi strain AS DNA by PCR, prior to ligation into a plasmid and ed
erateaprotectiveimmuneresponsethatinvolvesbothTh1andTh2- sequencing. The genomic DNAwas obtained from Dr. Jean Langhorne fro
type CD4+ T cells (26), with the importance of such cells dem- (National Institute of Medical Research). DNA sequencing of these am- m
plifiedregionswasthencompletedtoobtaintheaminoacidsequenceforP. h
onstrated in numerous depletion and adoptive-transfer studies in chabaudi chabaudi strain AS AMA1. The sequencing was done at the ttp
immunodeficient mice (27). During the acute stage of infection, a WellcomeTrustCentreforHumanGenetics,OxfordUniversity,andMWG ://w
Th1-typeAb-independentresponsehasbeenshowntocontrolinitial Biotech (Ebensburg, Germany). The sequence of P. chabaudi chabaudi w
peak parasitemia, with this effect mediated by the production of strainASAMA1differedfromthatofP.chabaudiadami(strainDS)by34 w
proinflammatory cytokines and effector molecules, such as IFN-g aa(SupplementalFig.1).Thevaccineconstructencodedtheectodomain .jim
ofP.chabaudichabaudistrainASAMA1(aa25–546)andincluded4aa m
andTNF-a(27).Onceparasitemiaisbroughtundercontrol,aTh2- substitutionstoremovepotentialsitesofN-linkedglycosylation([original u
typeCD4+Tcellresponseappearstopredominate.Thesecellswere aminoacidposition.newaminoacid]S-136.A,T-191.A,S-233. no
showntoproduceIL-4,IL-5,IL-6,andIL-10,aswellastoprovide A, S-251 . A). The native parasite N-terminal signal sequence, trans- l.o
membranedomain,andtheC-terminalcytoplasmicdomainwerenotin- rg
essentialhelpforAbproductionbyBcells(28,29).Bcell-depletion cluded. The construct was codon optimized for expression in mice and b/
studies showed that these cells per se are not required for early y
synthesizedbyGeneArt(Regensburg,Germany).Thetissueplasminogen g
control of parasitemia but are important for the resolution of the activator leader sequence replaced the native AMA1 signal sequence as ue
s
chronicphaseofinfectionandtheclearanceoftheparasite(30). previouslydescribed(12).Thisconstructwasusedtomakerecombinant t o
In agreement with these data, immunization studies with re- AdHu5 and MVA by methods that were described previously (9). The n
vectorcontrolvaccineswereAdHu5withthetransgenepromoterand N
combinant AMA1 in the P. chabaudi adami model also showed o
polyAtail,butnoantigenicinsert,andMVAexpressingGFP(37).AdHu5 v
animportantcontributionofCD4+Tcellsactingindependentlyof waschosenasamodeladenoviruswithoutintellectualpropertyrestrictions em
Absinvaccine-inducedprotection(31).Inthisstudy,asignificant thatissuitableforpreclinicalvaccinework.Theimmunogenicityand be
decreaseinacuteantiparasiteimmunitywasseenifCD4+Tcells efficacy of AdHu5 vaccines can be readily matched by clinically appli- r 2
twprraeinoresrfudtoseepdblelotweodidth-fsrtoaamgCeDimc4hm+alulTennizgceeedl.lSnloiimnremilaarlarliyos,erdaBthacgyeamlilnicsktn(onacuk/cnoouun)tsemmrviieccdee cA(in1ad1bbH,loe1uth2s5i,mna1oni5nad,nh1uva7amd)reiaaonnnuodpsvrtisihrmiuamstaeitCase;nhswAtaueddd6erien3ecosvev(in8irrtu)luysaseenssshdaoiirswneechhdouigmmthhpalayantrtiachmbleilmneiimucinnamolaugtnrneiinoamligscae(lna1nis3ctdiu,tsd1yai4oef)esf. 6, 2018
crypticepitope in AMA1 showed50% survival following P. cha-
Animalsandimmunizations
baudi adami challenge, and accelerated rates of de novo Ab pro-
duction against AMA1 in some mice (32). Comparable observa- Five-tosix-week-oldfemaleBALB/c(H-2d)mice(JohnRadcliffeHospital,
tions were madein a similar study following adoptive transfer of Oxford,U.K.orHarlan,U.K.)wereused,asstated,inallexperiments.RAG
transgenicCD4+TcellsthatrecognizedaH-2I-Edepitopewithin 1/2 knockout mice were a kind gift from Dr. Nicholas Jones (Nuffield
DepartmentofSurgery,UniversityofOxford).Thenudemicewereobtained
P. chabaudi MSP1 (33). A more recent study showed that low
33 fromHarlan.Allprocedureswereperformedaccordingtothetermsofthe
doses of killedblood-stage P. chabaudi chabaudi strain AS para- U.K. Animals (Scientific Procedures) Act Project License (PPL 30/2414)
sites,administeredinaluminumhydroxideadjuvantplustheTLR9 andPersonalLicense(PIL30/7792)andwereapprovedbytheUniversityof
agonist CpG oligodeoxynucleotide, can induce high-level cross- Oxford Animal Care and Ethical Review Committee. The vaccines were
dilutedinendotoxin-freeDulbecco’sPBSpriortoimmunization.Vaccines
strain, and even some cross-species, immunity that is dependent
were administered intradermally (i.d.) bilaterally into the ears in a total
on CD4+ T cells, IFN-g, and NO (34). However, none of these volumeof50mlforbothrecombinantadenovirusandMVA.Thedosesof
studiesusingrecombinantAMA1proteinformulatedinMontanide recombinantadenovirusvaccinesusedwere531010viralparticles(vp),
ISA720 adjuvant, transferred CD4+ T cell lines, or transgenic 131010vp,or13109vp;thedoseofrecombinantMVAvaccineusedwas
always13107PFU.Theintervalbetweentheprimeimmunizationwith
Tcells,orawhole-parasitebloodproductadministeredwithCpG,
AdHu5-P.chabaudichabaudistrainASAMA1andboostingwithMVA-P.
represent vaccine formulations that are easily translatable with
chabaudichabaudistrainASAMA1was8wkinallexperiments.
demonstrated adequate safetyprofilesforhumanuse (35,36).
Inthisstudy,wecharacterizetheimmunogenicityandprotective Total IgGELISA
efficacy of a prime-boost vaccination regimen with viral-vectored TheAbsinducedbyvaccinationweremeasuredbyELISA,aspreviously
vaccines targetingP.chabaudichabaudistrainASAMA1andin- described(9).Briefly,recombinantP.chabaudichabaudistrainASprotein
TheJournal ofImmunology 3
wasadsorbedto96-wellNunc-ImmunoMaxisorpplatesataconcentration 5 min before the supernatant was transferred to another tube. This was
of5mg/mlinPBS.P.chabaudichabaudistrainASproteinwasakindgift centrifugedat12,000rpmfor30mintoremoveanyremaininginsoluble
from Dr. Jean Langhorne. The P. chabaudi chabaudi strain AS parasite material. The protein concentration of the lysate was measured using
lysatepreparationisdescribedbelowandwascoatedataconcentrationof aNanoDrop.
2mg/mlinPBS.Afterblocking,theserumwasincubatedfor2h,andthe
boundAbs were detected usingalkaline phosphatase-conjugated goat InvivoCD4+Tcelldepletion
anti-mouse IgG (whole molecule) (Sigma-Aldrich, Dorset, U.K.) diluted CD4+Tcellsweredepletedusinganti-CD4GK1.5(ratIgG2a)mAb,as
1:5000.SerumAbendpointtitersweretakenasthex-axisinterceptofthe
previouslydescribed(7).The mAbwas purified usingproteinG affinity
dilution curve at an absorbance value 3 SD greater than the OD for
405 chromatographyfromhybridomaculturesupernatants.Micewereinjected
naivemouseserum(typicalcut-offOD405forpositivesera=0.15).Serum i.p.with200mgmAbdilutedinPBSondays22,21,and0(withrespect
fromnaivemicewaspooledandusedascontrolsforallELISAs.
to challenge on day 0), and the control mice were given normal rat
Multiparameter flowcytometry polyclonalIgG(Sigma-Aldrich)purifiedinthesameway.Thedegreeof
in vivo CD4+ T cell depletion was assessed by flow cytometry in the
CytokinesecretionbyPBMCsandsplenocyteswasassayedbyintracellular PBMCsofrepresentativedepletedandundepletedcontrolmice,anditwas
cytokinestaining(ICS).PBMCsandsplenocyteswereprepared,andthe showntobe.98%.Adifferentanti-CD4mAbclone(RM4.4)wasusedto
ICSassaywasperformedaspreviouslydescribed(7).Briefly,100mlcells stainCD4,toconfirmthattheCD4+Tcellsweredepleted(ratherthanthe
were added to a 96-well U-bottom plate. Fifty microliters of 1 mg/ml stainingmAbbindingbeingblockedbyGK1.5)(SupplementalFig.2).
brefeldin A (GolgiPlug; BD Biosciences) and 50 ml peptide diluted
incompletemediumwereaddedtothetestwells.Individualorpooled AdoptivetransferofCD4+Tcells
peptides (Peptide Protein Research, Hampshire, U.K.) are described in
Splenocyteswerepreparedfromimmunizedmice(asdescribedinthetext),
TablesIandIIandwereaddedatafinalconcentrationof1mg/ml.Inthe and CD4+ T cells were isolated using the CD4+ T Cell Isolation Kit
controlunstimulatedwells,GolgiPlugandmediumwereadded.Cellswere (MACS; Miltenyi Biotec) by depletion of non-CD4+ T cells (negative
incubatedat37˚C5%CO for5h.Afterincubation,thecellswereleftat D
4˚Covernight.Thenextd2ay,cellsweresurfacestainedfor30minat4˚C saenldectthioen)c.eBllsriewfleyr,espcloeunnotceydtesuswinegreareCsuAsSpYendceoduninte4r0(SmclhaM¨rfAeCSSysbtuefmfesr,, ow
cBtwshciSifieiteAnchnBcpaPetleeusrar)emCd-fPleoiala-rubCbte2iiyllo0ei5nzd.me5iad–ninncditoiniav-ntmijd1u4ou0g˚ua0CalstleemyadnCladsDa1snt34ehtsie(-scnmCeldoyoinntbuoteysrfieaLtxciT/CteCr3laDly4ut8it)looaadnprilely(furcomtlsreotedansaieonicnleh5udP3tAiB-fob6oSn..r7Cw(3)Be0ialtDlhnmsd0wiB.n1Peio%raae--t Racanegeldaulistn4liso0ntfgmCbelDnioM,8tGiaAne-C(rcLmSoynab-j2nuuy)fg,f)ae.CtrCeD/1de4l3Al5sRbw1(0ceB7ore2cc2keth0tlale)si,nlwDccaXoesnn5tta,radiiCfdnuDeignd1eg.1dTbbaeit(onM3tmi0na0i-ccc-3ro1on)l,jgiutaegfnroadsrt/e1T1de03rm-m11Ai10n9b7, nloaded fro
wasaddedtothis,mixed,andincubatedfor10minat4˚C.Afterthein- m
4˚Cwithallophycocyanin-conjugatedanti-mouseIFN-g(cloneXMG1.2), cubation,30mlMACSbufferand20mlAnti-BiotinMicrobeads/13107 h
aiFnnITt2iC-0m0-coomunsljeuPgIBLaSt-e2cdo(acnnltotaini-neminJoEguSs1e6%-T5NHfoF4r-)ma.aA(lcifntleo.rnSesat1mai4np5il-ne2sgC,w1ce1erl)el,saawnndearlPyeEzre-ecdsouunssjpuiengngadteaednd cweiltlhsbwuefrfeerabdydeadddainndgi1n0c3ubathteedlafboerl1in5gmvionluamt4e˚aCn.dCceelnlstrwifeurgeedthaetn3w00as3hegd ttp://w
LSRIIFlowCytometer(BDBiosciences)andFlowJov8.8(TreeStar).The fpoernd1e0dminin5.0T0hemlsubpuefrfnear/t1an3tw1a0s8dtoistcaalrcdeeldls,.andthecellpelletwasresus- ww
Bcoomolbeiannatgiaotnes.plPaetfsotlremvw1.a6saunsdedSwPIitChEinvd5iv.0idsuoafltwgaatrees(t3o8c)rewaeterereusspeodnstoe MagneticseparationofthecellswasperformedusingaMACSseparator .jim
andLScolumn(MACS;MiltenyiBiotec).Columnswereattachedtothe
analyzetheTcell-responseprofiles.Backgroundresponsesinunstimulated m
magnetsintheMACSseparatorandpreparedbyrinsingwith3mlMACS u
controlcellsweresubtractedfromthepeptide-stimulatedresponse. n
buffer.Thecellsuspensionwasaddedtothecolumninunitsof10mland o
Parasitesandblood-stagechallenge allowedtopassthrough.Theeffluentwascollected,whichcontainedthe l.o
unlabeledcells(i.e.,theCD4+Tcell-enrichedfraction).Thecolumnswere rg
P.chabaudichabaudistrainASparasiteswerekindlyprovidedbyDr.Jean thenwashedfourtimeswith3mlMACSbuffer,andtheflow-throughwas b/
y
Langhorne.Fivedayspriortochallenge,afrozenaliquotofP.chabaudi collectedintothesametubeastheeffluent.Thecellswerethenspundown g
chabaudi strain AS-infected blood was thawed and injected i.p. into andresuspendedinPBS,andthecellcountwasdetermined.Aftercounting ue
oafdbolnooordBwAaLsBc/ocumntoeudse(.3O9)n,tahneddtahyeopfecrcheanllteanggeeo,fthpeanrausmitbemeriaofwRaBsCmse/ma-l trheseucseplelns,dtehdeyinwaesrueitcaebnlterivfuogluemdeatof30P0B3Sfgorfionrje5ctmioinn.,Aantdottahleopfe9lle3tw10as7 st on
sured by counting a Giemsa-stained thin-blood smear. This was used to CD4+Tcellswasinjectedi.v.ina200-mlvolume/mouse2dbeforepRBC N
o
calculate the number of parasitized RBCs (pRBCs)/ml of blood. Blood challenge. v
from the donor mousewas diluted accordingly in PBS 1 0.2% glucose em
supplementedwith10%naivemouseserum(Sigma-Aldrich)togive13 Statisticalanalysis b
e
107 pRBCs/ml, unless otherwisestated.Micewere injectedwith 100 ml Datawere analyzed for statistical significance usingGraphPad Prismv5 r 2
this solution (1 3 106 pRBCs) i.v. via the tail vein. From day 4 post- 6
challenge, the parasitemia was monitored daily by Giemsa-stained thin- (GraphPadSoftware,SanDiego,CA).Thenormalityofthedatasetwas , 2
determined using the Kolmogorov–Smirnov one-sample test. For non- 0
bloodsmear,andthehealthstatuswasmonitoredtwicedailyduringacute 1
parametricdata,aMann–WhitneyUtestwasusedtocomparetwogroups. 8
parasitemia.P.chabaudichabaudistrainASnonlethalinfectioninnaive
Parametricdatawerecomparedeitherusingthettest(forcomparingtwo
wild-typeBALB/cmicereachesamaximumparasitemiaof50–60%andis
groups) or one-way ANOVA (for comparing more than two groups). A
thencontrolled.P.chabaudiadamistrainDSandstrainDKparasiteswere
posthocDunnettcorrectionwasusedtocomparetestgroupstoonecontrol
kindlyprovidedbyDr.AlisonCreasey(InstituteofImmunologyandIn-
group,whereasaBonferronicorrectionwasusedwhenallofthegroups
fectionResearch,EdinburghUniversity,U.K.).Challengeswereperformed
werecomparedwitheachother.ELISAtiterswerelogarithmically(log10)
asdescribedforP.chabaudichabaudistrainAS.
transformedtonormalizethedataandenableamorestringentparametric
Blood-stageP.chabaudichabaudi strainASparasitelysate analysis.The areaunder the curve (AUC) was calculatedto analyzethe
challengedata,andthedifferentgroupswerecomparedbyANOVA.Ap
MicewereinjectedwithP.chabaudichabaudistrainAS-infectedpRBCs, value#0.05wasconsideredsignificant.
andthe parasitemiawas monitoreduntilit reached ∼20–30%.Themice
werethensacrificedandbledbycardiacpunctureinto103heparin(final Results
heparin concentration 30 U/ml blood), and the blood was pooled and
storedonice.Thebloodwascentrifugedat2500rpmfor5min,andthe P.chabaudichabaudiASAMA1sequence andvaccinedesign
supernatant was removed carefully using a pipette. The pellet was then
ThecompletecodingsequenceofP.chabaudichabaudistrainAS
resuspendedin5mlPBSandmixedbyinvertingthetubefiveorsixtimes.
AMA1 was not available, so primers were designed against con-
APlasmodipurfilter(Euro-Diagnostika)waspreparedwithPBS,andthe
samplewaspassedthroughthefilterusinga20-mlsyringe.Theoriginal servedregionsbycomparingsequences fromrelated strains (40),
tubewaswashedwithPBSandfilteredtwice.Thecellswerewashedafter and the sequence was determined (GenBank accession number
filtrationwith10mlPBSandresuspendedin8mlPBS+0.06%saponin, JF690775, http://www.ncbi.nlm.nih.gov/nuccore/JF690775) (Sup-
mixed,andincubatedatroomtemperaturefor10min.Aftertheincuba-
plemental Fig. 1). Avaccine construct (termed P. chabaudi cha-
tion,thesamplewascentrifugedat2500rpmfor5min,andthepelletwas
washedtwicein5mlPBSandresuspendedin1mlPBS.Thiswasthen baudistrainASAMA1)wassubsequentlydesignedthatcontained
freeze-thawedthreetimestolysethecellsandcentrifugedat2500rpmfor the ectodomain of P. chabaudi chabaudi strain AS AMA1, with
4 VIRAL-VECTOREDVACCINESAGAINSTP.CHABAUDIAMA1
fourpotentialsitesofN-linked glycosylationremoved(described TableI. P.chabaudichabaudistrainASAMA1peptidestestedbyICS
inMaterialsandMethods).Theconstructwascodonoptimizedfor
expressioninmice,synthesized,andusedtogeneraterecombinant AminoAcid CD4or
AdHu5andMVAvectors,asdescribedinMaterialsandMethods. Pool Peptide# AminoAcidSequence Position CD8
Pool1 P7 LINPWEKFMEKYDIE 51–65 CD4
AbresponsesinducedbyAdHu5-MVAP.chabaudichabaudi
P8 EKFMEKYDIEKVHGS 56–70
strainASAMA1 prime-boostvaccination P9 KYDIEKVHGSGIRVD 61–75
BALB/cmicewereimmunizedi.d.with531010vpofAdHu5-P. P10 KVHGSGIRVDLGEDA 66–80
P11 GIRVDLGEDARVENQ 71–85
chabaudi chabaudi strain AS AMA1 and boosted 8 wk later i.d. P12 LGEDARVENQDYRIP 76–90
with 1 3 107 PFU of MVA-P. chabaudi chabaudi strain AS P13 RVENQDYRIPSGKCP 81–95
AMA1.This8-wkintervalwasshowntobeimportantforbothAb P14 DYRIPSGKCPVMGKG 86–100
and T cell responses when using these two vectors in a prime- P15 SGKCPVMGKGITIQN 91–105
P16 VMGKGITIQNSKVSF 96–110
boost vaccination regimen (9, 41). Serum was obtained 2 wk
Pool2 P25 ANLKLMYKDHKEILA 141–155 CD8
postprime (day 14), preboost (day 55), and 2 wk postboost (day P26 MYKDHKEILALNDMS 146–160
70).ThetotalIgGresponseagainsttheectodomainofP.chabaudi Pool3 P29 LCAKHASFYVPGTNV 161–175 CD8
chabaudistrainASAMA1wasmeasuredbyELISA.Inagreement P30 ASFYVPGTNVNTAYR 166–180
Pool4 P74 SFPCDIYKKKIAEEI 386–400 CD8
with other murine data for the AdHu5 vector, there were detect-
P75 IYKKKIAEEIKVMNV 391–405
ableAMA1-specificIgGresponses2wkaftertheAdHu5priming Pool5 P79 GNGTIQFPRIFISDD 411–425 CD4
immunization, and these increased over time to become signifi- P80 QFPRIFISDDKESLK 416–430
cantly higher at day 55. MVA administration on day 56 boosted Pool6 P85 SSCNFFVCNCVEKRQ 441–455 CD4 Do
theAb levelsignificantly,asmeasured onday70(Fig. 1). P86 FVCNCVEKRQFISEN 446–460 wn
lo
TcellresponsesinducedbyAdHu5-MVAP.chabaudichabaudi sequTehnecetaobfleeashchowpsepthtiedes.ixThpeepftairderigphotolcsoltuemstendinbdyicIaCtSes,awshwetehlelratshethpeeapmtidineopaocoilds ade
strainASAMA1 prime-boostvaccination c(uosnitnagintehdepepuibtloipsheepdreCdDic4ti+onTseccetliloenpoitfotpheesS(3Y2F)PoErITpHredWicetbedsiCteD;h8t+tpT://cwewllwe.psyitfoppeeis- d fro
A previous study mapped both immunodominant and cryptic thi.de). m
epitopes within P. chabaudi adami AMA1 following immuniza- h
taigoaninosftBPA. cLhBa/bcamudiciecwhaitbhaurAdigs(t3ra2i)n.TAhSeATMceAll1rebsypoAndseHsui5n-dMucVeAd vested 2 wk after AdHu5-MVA P. chabaudi chabaudi strain AS ttp://w
AMA1immunization(day70),wererestimulatedwiththepeptide w
immunization were measured in this study by ICS following pools for 5 h, surface stained for CD4 and CD8, and then intra- w
restimulation of splenocytes with peptides corresponding to the cellularlystainedforIFN-g,TNF-a,andIL-2.Pools1and5were .jim
previously reported CD4+ T cell epitopes, as well as predicted identified as containing peptides that were recognized by CD4+ m
CD8+ T cell epitopes. Six peptide pools containing 15 mer pep- un
T cells, leading to the production of IFN-g (Fig. 2A). Similarly, o
tides,overlappingby10aa,weretested.Thepeptidesweredivided TNF-a+and IL-2+CD4+Tcellresponses werealsomeasuredin l.o
into pools according to whether they were reported to contain rg
CD4+ T cell epitopes (32) or predicted CD8+ T cell epitopes, as cweelrlserneostiCmDul8a+teIdFwNi-tgh+PToolce1llanredspPoonosle5s(ddeatteacnteodtsthoowanny).oTfhtehree by/
well as according to the position of the peptide within the Ag g
peptidepoolstested(Fig.2B).Theindividualepitopeswithinthe u
e
(TableI).Pool1contained10peptides(P7–P16)thatincludedthe s
known immunodominant CD4+ T cell epitope. Pool 2 (P25 and rmesepnot.ndTihnigspsehpotwideedptohoaltstwheereCtDh4en+ iIdFeNn-tigfi+edTinceallrerpesepaotnesxepseriin- t on
P26), Pool 3 (P29 and P30), and Pool 4 (P74 and P75) each N
BALB/c mice against the ectodomain of P. chabaudi chabaudi o
containedtwopeptidesandwerepredictedtobeH-2dCD8+Tcell strain AS AMA1 were against epitopes present within two pep- vem
epitopesfromknownH-2classI-bindingmotifs.Pool5(P79and tides: P16 (within Pool 1) and P79 (within Pool 5) (Fig. 2C–E). b
e
P80)andPool6(P84andP85)alsocontainedtwopeptideseach, These cells produced all three cytokines assayed, but the per- r 2
whichwere publishedbyGoodetal.(32)tobeCD4+Tcellim- 6
munodominant epitopes. Splenocytes from BALB/c mice, har- cteenndtaegdetsoobfeIFslNig-hgt–ly(Fhiigg.h2eCrt)haanndthTeNpFe-rac–esnetacgreetsinogfcceellllss(sFeicgr.et2iDng) , 201
8
IL-2 (Fig.2E).
Two CD4+ T cell epitopes had now been identified, and these
were in agreement with those reported to be induced following
immunization with recombinant AMA1 protein in adjuvant (32).
In this study using a viral-vectored vaccination regimen, it re-
mained possible that T cell responses to other epitopes were in-
duced in addition to those identified after protein immunization.
To confirm whether any other T cell responses were induced
againsttheAMA1Ag,15merpeptidesoverlappingby10aawere
synthesized that covered the entire ectodomain of P. chabaudi
chabaudi strain AS AMA1 contained within the vaccine insert
(SupplementalTableI).Splenocytes,isolatedasbefore,weresub-
FIGURE 1. Ab responses induced by AdHu5-MVA P. chabaudi cha- sequently restimulated with seven overlapping peptide pools (10
baudi strain AS AMA1 prime-boost vaccination. BALB/c mice (n = 5)
peptidesineachpool)ornopeptide(Unstim)andassayedbyICS.
wereprimedwith531010vpofAdHu5-P.chabaudichabaudistrainAS TheCD4+TcellepitopesP16andP79,identifiedintheprevious
AMA1 and boosted 8 wk later with 1 3 107 PFU of MVA-P. chabaudi
set of experiments (Fig. 2C), were included as positive controls.
chabaudistrainASAMA1.TheAMA1-specifictotalIgGresponseagainst
TherewerenofurtherCD4+IFN-g+Tcellresponsesidentifiedin
recombinant P. chabaudi chabaudi strain AS AMA1 ectodomain was
measuredbyELISAintheserumatdays14(postprime),55(preboost),and any of the peptide pools (Fig. 3A); however, a very weak CD8+
70(postboost).Eachpointindicatestheresponsefromanindividualmouse, IFN-g+TcellresponsewasmeasuredinthreeoffivemiceinPool
andthelinelinksthemean.**p#0.01,***p#0.001,pairedttest. 11(Fig. 3B).
TheJournal ofImmunology 5
FIGURE 2. Identification of
splenic T cell responses to AMA1
followingAdHu5-MVAP.chabaudi
chabaudi strain AS AMA1 immu-
nization.Micewereimmunizedwith
thesameregimenasinFig.1.Two
weeks postboost (day 70), spleno-
cyteswereisolatedandrestimulated
with pools of overlapping peptides
(P1–P6,TableI)ornopeptide(Un-
stim).Cellsweresurfacestainedfor
CD4andCD8,intracellularlystained
forIFN-g,andassessedbyflowcy- D
tometry to determine the percent- ow
age of responding cells per total nlo
CD4+(A)orCD8+(B)Tcellsubset. a
d
The bars represent the mean re- ed
sponse 6SEM(n=5).Inarepeat fro
experiment,splenocyteswererestim- m
ulatedwithindividualpeptidesfrom h
PanodolP180(P)7ortonPo1p6e)potridPeo(oUln5st(iPm7)9. ttp://w
w
CellsweresurfacestainedforCD4,
w
TinNtrFac-eallu(Dla)r,lyasntdainILed-2fo(rEI)F,Na-ngd(aCs)-, .jim
m
sessed by flow cytometry to de- u
n
termine the percentage of respond- o
ing cells/total CD4+ T cell subset. l.o
Tsphoensbear6s SreEpMres(nen=t 5th).e mean re- byrg/
g
u
e
s
t o
n
N
o
v
e
m
b
e
r 2
6
, 2
0
The functionality of the P16- and P79-specific splenic CD4+ with an increase in the overall magnitude of the Ag-specific re- 1
8
TcellswasalsoanalyzedaftertheAdHu5-P.chabaudichabaudi sponse.
strain AS AMA1 prime (day 14) and the MVA-P. chabaudi cha-
ProtectiveefficacyofAdHu5-MVAP.chabaudichabaudistrain
baudi strain AS AMA1 boost (day 70) (Fig. 4). Triple-positive
ASAMA1 vaccinationagainstblood-stagechallenge
cells were defined as cells that simultaneously produce the three
cytokinesassayed(IFN-g,TNF-a,andIL-2)(42),double-positive TheprotectiveefficacyoftheAdHu5-MVAP.chabaudichabaudi
cellssimultaneously produceanytwoof thethree cytokines, and strain AS AMA1 prime-boost vaccination regimen was assessed
single-positive cells produce only one cytokine.For the P16 epi- by blood-stage challenge with P. chabaudi chabaudi strain AS
tope,thenumberoftriple-positivecellsproducingIFN-g,TNF-a, parasites. Immunized micewere challenged 2 wk postboost (day
and IL-2 did not change significantly after the boost (Fig. 4A). 70) i.v. with 105 P. chabaudi chabaudi strain AS pRBCs. Naive
However, there was a significant increase (∼2–5-fold) in the unimmunized control BALB/c micewere challenged atthesame
number of double-positive cells producing IFN-g and IL-2 or time. Parasitemia was monitored from day 4 postchallenge by
IFN-g and TNF-a (p = 0.02 and p = 0.004, respectively, Mann– Giemsa-stainedthin-bloodsmears,andtheresultsareexpressedas
WhitneyUtest).CellsproducingonlyTNF-awerenotdetectable, thepercentageofinfectedRBCs(Fig.5).GiventhatP.chabaudi
and therewas no significant increase in the number of cells pro- chabaudistrainASinfection innaiveBALB/cmiceisnonlethal,
ducing only IFN-g or IL-2 after the MVA boost. For the P79 protective efficacy was assessed by comparing parasitemia be-
epitope, the functionality profile of the CD4+ T cells was very tween the groups. Significantly lower mean parasitemia was ob-
similar to that of P16 (Fig. 4B). Overall, these data showed an served in the AdHu5-MVA P. chabaudi chabaudi strain AS
increasedfrequencyofCD4+TcellsproducingIFN-gandIL-2or AMA1-vaccinated mice in comparison with the naive controls
IFN-gandTNF-aaftertheboosterimmunization,inconjunction fromthetimeofpatency(day4)untilday7.Atthetimeofpeak
6 VIRAL-VECTOREDVACCINESAGAINSTP.CHABAUDIAMA1
5).Meanpeakparasitemiawasagainsignificantlyreducedinthe
vaccinated mice in comparison with the naive controls at days 6
and 7 postchallenge (Fig. 6A). However, CD4+ T cell depletion
resulted in the loss of vaccine efficacy in immunized mice, as
reflected by increased peak parasitemias on days 6–7 that were
comparable to those of undepleted naive controls (Fig. 6B), thus
confirming an important contributory role for vaccine-induced
CD4+ T cells to protection in this model. Interestingly, the vac-
cinateddepletedmicealsofailedtocleartheparasitesasearlyas
didthenondepletedmice,andasecondwaveofparasitemiawas
observedthatpeakedonday13.Analmostidenticalsecondwave
wasalsoobservedinthenaivedepletedanimals;overalltheyfared
theworst,demonstratingachronicpatentparasitemiaat30dpost-
infection(Fig.6C).Also,inagreementwithapreviousreport(31),
thenaiveCD4+ T cell-depleted micesufferedthe highestpeak
parasitemias ondays6–7(Fig. 6D).
RoleofP.chabaudi chabaudistrainASAMA1-specificand
whole-parasiteAbsinclearance
FIGURE3. TcellepitopemappingwithintheentireectodomainofP. D
chabaudichabaudistrainASAMA1.Micewereimmunizedwiththesame Unlike the control of peak parasitemia, parasite clearance in the ow
rreegstiimmeunlaatesdinforFi5g.h1w. iTthwosewveenekosveprolastpbpoionsgtp(edpatyid7e0p),oosplslecnoonctyatiensinwge1r0e tPh.echfaabstaeurdiramteodoeflpisaarattsriitbeutceldeatoraBncceelolsbsaenrdveAdbsin(2t7h,e45d)e.pTlehtuesd, nloa
d
peptideseach(Pool7–13,SupplementalTableI)ornopeptide(Unstim). vaccinatedmiceincomparisonwithnaivedepletedcontrols(Fig. ed
TIFhNe-cge.lTlshweefirgeutrheesnhsouwrfsatcheesptaeirnceedntfaogreCoDf4CDor4C+DIF8Na-ngd+icnetlrlasc(eAll)uolarrClyDf8o+r 6spCe)cicfioculAdbbseindutheetovatchceinpatreedsemnciceeotfhavtacarceinaeb-liendtuocceodnAtroMlAan1d- from
IrFepNr-egse+nctetlhlse(mB)e.dRiaens.ponsesfromindividualmiceareshown,andthelines cIAglMeGaArAp1baprtariostetietresinsaiagnnadtihnwesthlaroetlecero-pmsatarbagisneiatoenftlyiPns.faectechtawiobenar.euTdmiheecarhseaufboreardeu,dbsiyesrEturLmaiInStoAAtaS1l http://w
w
parasitemia (day 7), the vaccinated mice had significantly lower mopostchallenge(Fig.7A).Inagreementwith thepreviouslyre- w
levelsofinfection(geomean6SD=24.662.6%versus43.46 ported protein-vaccine study (31), there was no difference in .jim
5.6%inthenaivecontrols,p=0.002).Thesedataconfirmedthat P. chabaudi chabaudi strain AS AMA1-specific Ab titers in the m
u
thevaccinecouldleadtosignificantcontrolofpeakparasitemiain vaccinated depleted versus nondepleted mice. A de novo P. cha- n
o
these mice, although the rate of parasite clearance following this baudichabaudistrainASAMA1-specificAbresponsewasdetected l.o
was comparable between thevaccinees and controls. It was con- in the non-CD4+ T cell-depleted naive control mice, but the titers brg/
firmedinarepeatexperimentthatthiscontrolofpeakparasitemia were significantly lower than those seen in the vaccinated mice. y
g
occurs followingAg-specificimmunization. The same resultwas There was no detectable P. chabaudi chabaudi strain AS AMA1- u
e
obbinsaenrvt/ecdonfotrlololwAidnHguc5haalnlednMgeVoAfvmaiccceinimesm(SuunpizpeldemweinthtanloFnirge.c3oAm)-. sCpDec4i+ficTAcebllrehseplponisseniencetshsearnyaifvoer-ddeepnleotveod Pm.icche,absauugdgiescthinagbatuhdait st on
N
InvivodepletionofCD4+Tcellspriortochallengeresultsin strainASAMA1AbproductionfromBcellsduringinfection. ov
Interestingly, the response to the whole P. chabaudi chabaudi e
a lossofvaccine-inducedprotection m
strainASparasitelysatewassimilarinthenondepletedvaccinated b
e
PreviousworkanalyzingprotectiveimmunemechanismsintheP. and naive groups, whereas there was a significantly lower Ab r 2
cbhyabaactuivdeiaidmammiumniozadteilondemwiotnhstrreacteodmabirnoalentfoArMAbAs1eiothrerpainsdsiuvceelyd rdeespploentesdegtorotuhpeswcohmolpeapreadrawsiittehptohsetctwhaollnenognedeipnlettheedCgDro4u+psT(Fciegll. 6, 20
1
transferredfromtheseraofimmunizedrabbits(40,43),aswellas 7B). Given that the naive CD4+ T cell-depleted mice were still 8
for CD4+ T cells acting independently of, or in concert with, parasitemic1mopostchallenge(Fig.7C),itisinterestingtonote
AMA1-specific Abs (31, 32). Thus, the contribution of CD4+ that these mice possessed no P. chabaudi chabaudi strain AS
Tcellsinvaccine-inducedprotectioninthisP.chabaudichabaudi AMA1-specific Abs but had similar parasite lysate-specific Ab
strain AS model was initially assessed by in vivo CD4+ T cell levels compared with thevaccinated depleted mice (who cleared
depletion prior to blood-stage challenge. The anti-mouse CD4 the infection faster). However, both thevaccinated and the naive
GK1.5(ratIgG2a)mAbwasreportedtodepleteCD4+Tcellsfor nondepleted mice cleared their parasites at the same time (Fig.
up to 10 d, after which they are replenished to ∼50% of the 6A),althoughthenaivemicepossessedsignificantlylowerlevels
originallevelby4wk(44).Twogroupsofmicewereimmunized ofP.chabaudichabaudistrainASAMA1-specificAbs(Fig.7A).
as beforewith the AdHu5-MVA P. chabaudi chabaudi strain AS Takentogether,thesedatasuggestthatbothP.chabaudichabaudi
AMA1vaccineregimen.CD4+Tcellsweredepletedinonegroup strain AS AMA1-specific Abs and Abs to other parasite Ags are
ofthevaccinatedmice,aswellasinonegroupofnaivecontrols, able tocontributeto parasiteclearance.
prior to i.v. challenge with 106 P. chabaudi chabaudi strain AS
AdoptivetransferofCD4+Tcellsfromvaccinated miceto
pRBCs. The undepleted vaccinated and naive mice were admin-
naive andimmunocompromisedmice
istered thesame amount ofnormal rat polyclonalIgG.
In this experiment, a similar degree of protective efficacy was Tofurtherassessthecontributionofvaccine-inducedCD4+Tcells
observed for the AdHu5-MVA P. chabaudi chabaudi strain AS and Abs to the initial control of blood-stage parasite densities,
AMA1 vaccination regimenin undepleted mice, eventhoughthe a number of adoptive-transfer studies was undertaken. BALB/c
challenge dose was increased to 106 pRBCs (Fig. 6A) compared mice were immunized exactly as before, and the spleen and se-
withachallengewith105pRBCsinthepreviousexperiment(Fig. rumwereharvestedfromeachmouse2wkaftertheMVAboost.
TheJournal ofImmunology 7
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://w
w
w
.jim
m
u
n
o
FIGURE4. MultifunctionalityofP16-andP79-specificCD4+Tcells.MicewereimmunizedwiththesameregimenasdescribedinFig.1.Two l.o
w(deaeyk7s0p)o,sstpplreinmoecywteitshwAedreHrue5s-tPim.cuhlaatbeaduwdiitchhtahbeaPu1d6isatnrdaiPn7A9SpeApMtidAe1o(rdnaoyp1e4p)tiadned(2Unwsktimpo)sftobroo5sht.wCiethllsMwVeAre-Ps.ucrfhaacbeasutdaiincehdafboaruCdiDs4tr,aiinntrAacSelAluMlaArl1y byrg/
stainedforIFN-g,TNF-a,andIL-2,andassessedbyflowcytometrytodeterminethepercentageofrespondingcells/totalCD4+Tcellsubset.Total g
u
responsesofeachcytokinetoP16(A)andP79(B)areshownateachtimepoint.Themultifunctionalityor“quality”ofCD4+Tcellswasanalyzedusing es
SPICEsoftware. t o
n
N
o
MicewereeitherimmunizedwithAdHu5-MVAvectorsencoding trols did not express P. chabaudi chabaudi strain AS AMA1, ve
m
P. chabaudi chabaudi strain AS AMA1 or with an identical reg- thusenablinganassessmentoftheprotectiveroleofP.chabaudi b
imen using vectorcontrols expressing norAg. Thesevectorcon- chabaudistrainASAMA1-specificCD4+ Tcellsandconfirming er 2
that any protective effect observed was not due to viral vector 6
immunization per se. Splenic CD4+ T cells from the vaccinated , 20
1
mice were isolated by negative selection (as described in Mate- 8
rialsandMethods),andtheseorpooledseraweretransferredinto
naive BALB/c mice (Fig. 8A–C). All mice were subsequently
challenged with 106P. chabaudichabaudistrainAS pRBCs.
In agreement with the previous result (Fig. 6A), therewas a
significantly lower parasitemia (AUC analysis, p = 0.008) in the
AdHu5-MVA P. chabaudi chabaudi strain AS AMA1-vaccinated
control group in comparison with naive controls (Fig. 8A). This
effectwasmirrored inthenaivewild-typemicethatreceivedthe
FIGURE 5. Efficacy of AdHu5-MVA P. chabaudi chabaudi strain AS CD4+ T cell transfer from vaccinated mice (p = 0.008, AUC
AMA1immunizationagainstblood-stagechallenge.BALB/cmice(n=6) versusnaivecontrols).Moreover,althoughthemicethatreceived
werevaccinatedwith531010vpofAdHu5-P.chabaudichabaudistrain the CD4+ T cells from the Ad_M control-immunized controls
ASAMA1andboosted8wklaterwith13107PFUofMVA-P.chabaudi showed transiently lower parasitemias than the naive controls on
chabaudi strain AS AMA1. Two weeks after the MVA boost (day 70), days 4 and 5, therewassubsequently no significant difference in
thesemiceandsixnaiveunvaccinatedcontrolswerechallengedi.v.with
the AUC analysis (p = 0.69), unlike that observed in the mice
105 nonlethal P. chabaudi chabaudi strain AS pRBCs. Parasitemia was receiving CD4+ T cells from P. chabaudi chabaudi strain AS
monitoreddailyfromday4byGiemsa-stainedthin-bloodsmears,andthe
AMA1-immunized mice (Fig. 8A, 8B). These data confirm that
resultsareexpressedasthepercentageofinfectedRBCs.Thedifferencein
vaccine-induced P. chabaudi chabaudi strain AS AMA1-specific
parasitemiabetweenthetwogroupswastakenasthemeasureofvaccine
efficacy. The mean percentage parasitemia 6 SEM are shown. ***p # CD4+Tcellsareanimportantcontributingfactortothecontrolof
0.001,Mann–WhitneyUtest. initial peak parasitemiainthis model.
8 VIRAL-VECTOREDVACCINESAGAINSTP.CHABAUDIAMA1
FIGURE6. EffectofinvivoCD4+Tcelldepletion
onvaccineefficacy.TwogroupsofBALB/cmice(n=
6/group) were vaccinated with 5 3 1010 vp of
AdHu5-P. chabaudi chabaudi strain AS AMA1 and
boosted 8 wk later with 1 3 107 PFU of MVA-P. D
chabaudichabaudistrainASAMA1.Thesemiceand o
w
two groups of naive controls were challenged i.v. n
14dpostboostwith106pRBCs.Onegroupeach of loa
vaccinated and naive mice were depleted of CD4+ de
d
aTsicteemllsi,aawsadsemscornibiteodreidndMaialtyerbiyalGsiaenmdsaM-settahiondeds.tPhianr-- from
blood smears, and the results are expressed as the h
percentage of infected RBCs (mean 6 SEM). The ttp
difference in parasitemia between the groups was ://w
takenasthemeasureofvaccineefficacy.(A)Vacci- w
w
ndeapteldetendo(nNdaeipvleetNedD)(mAdic_eM.(BN)DVa)ccainndatendaidveeplneotend- .jim
(Ad_M D) and nondepleted (Ad_MND)mice.(C) m
u
Vaccinated depleted (Ad_M D) and naive depleted no
(Naive D) mice. (D) Naive depleted (Naive D) and l.o
nondepleted (Naive ND) mice. *p # 0.05, **p # rg
0.01,***p#0.001,Mann–WhitneyUtest. b/
y
g
u
e
s
t o
n
N
o
v
e
m
b
e
r 2
6
, 2
0
1
8
Interestingly, serum transfers into normal naive BALB/c mice knockout mice(BALB/c RAG1/22/2),which lack Tand B cells.
fromAMA1-orcontrolvaccinatedmiceshowedaresultsimilarto PreviousstudiesshowedthatnudemiceinfectedwithP.chabaudi
thatforCD4+Tcelltransfer(Fig.8C).Themicereceivingserum chabaudistrainASdevelopachronicparasitemiaandexhibithigh
frommicevaccinatedwithP.chabaudichabaudistrainASAMA1 mortality (33, 46), with a similarly poor outcome in RAG22/2
had significantly lower initial parasite densities in comparison knockout mice, many of which succumb within the first 15 d of
with naive controls (AUC, p = 0.01). The serum transfer from infection (31, 33). In comparison with previous reports for the
vectorcontrol-immunizedmiceledtonodifferenceinparasitemia lethalchallengeofnudemicethatwerenotreconstituted(31,33),
comparedwiththenaivemice(AUCnotcalculatedbecauseoneof thetransferofCD4+TcellsfromP.chabaudichabaudistrainAS
themicediedonday9postchallenge).Takentogether,thesedata AMA1-immunized micewas sufficient to control the initial peak
suggestthatbothP.chabaudichabaudistrainASAMA1-specific parasitemias, albeit at a higher level than that seen when naive
CD4+ T cells and Abs can contribute to the control of initial normal mice were recipients (Fig. 8D). A similar result was ob-
parasitemia inimmunocompetent recipients. tained in the RAG1/2 knockout mice, and both immunocompro-
CD4+Tcell-transferstudieswerealsoperformedusingathymic misedstrainsshowedcomparableparasitedensitiesuptoday11,
nude mice (BALB/c nu/nu), which lack T cells, and RAG1/2 followedbyasecondsmallerwaveofparasitemiapeakingonday
TheJournal ofImmunology 9
munization, neither the P16 nor the P79 sequences were highly
conserved between these four P. chabaudi strains. P16 was con-
servedinallbutP.chabaudiadamiDS,whereasthesequenceof
P79 was different from the vaccine P. chabaudi chabaudi AS
strain in all of the other three strains compared (Table II). The
heterologous peptides P16B and P79B, encoding the alternative
sequences,wereusedtorestimulatesplenocytesfromimmunized
mice, as described before; as expected, there was no detectable
response to these heterologous peptides (Fig. 9). Given that the
P16epitopeispartiallyconserved,wesoughttoascertainwhether
theresponsetothisepitopealone(intheabsenceofstrain-specific
Abs) could lead to initial control of parasite densities following
heterologousparasitechallenge.However,therewasnoprotection
observed in this experiment against the heterologous strain P.
chabaudi adamiDK(SupplementalFig.3B).
Discussion
The AMA1 Ag remains a leading candidate Ag for inclusion in
a subunit vaccine against blood-stage P. falciparum malaria.
D
However, despite extensive efforts, candidate protein-in-adjuvant o
w
vaccines tested for efficacy in phase IIa/b trials have shown n
lo
minimalorstrain-specificefficacy(47,49).Inthisstudy,wechar- a
d
acterized in detail the immunogenicity and protective efficacy of ed
FIGURE7. P.chabaudichabaudistrainASAMA1-andparasite-spe- a prime-boost vaccination regimen using an alternative human- fro
cificAbresponsespostchallenge.TotalIgGserumAblevelsinthemice m
compatible vaccine-delivery platform encoding the P. chabaudi
shownin Fig. 6 were analyzed by ELISA 1 mopostchallenge (day 33). h
IndividualresponsesplusthemedianareshownforP.chabaudichabaudi chabaudi strain AS AMA1 Ag. The P. chabaudi rodent malaria ttp
strainASAMA1ectodomain(A)andwholeP.chabaudichabaudistrain model remains a key example of how cellular immunity, acting ://w
AS parasite lysate (B). Significant differences in Ab levels between the independently of Abs, can significantly contribute to immune w
w
ddiifafnerpeenrtcegnrotaugpesoafrepairnadsiicteamtedia.o(Cn)dTayhe33c.o*rrpes#po0n.d0i5n,g**inpd#ivid0u.0a1l,a*n*d*pm#e- cthoensterodlatoafcaosncfiernmdinthgatevairrlayl--pvheacsteorebdlovoadc-csitnaegseaprearcaaspitaebmleiao,fainnd- .jim
m
0.001,ANOVA. ducingCD4+Tcellresponsesthatcanfulfillthatrole.Todevelop u
n
viralvectorsencodingP.chabaudichabaudistrainASAMA1,the o
13,anobservationpreviouslyattributedtotransferofsuboptimal complete sequence of the Ag was obtained from sequencing a l.org
CD4+ T cell numbers (46). However, only two of five RAG1/2 PCRfragmentamplifiedfromparasitegenomicDNA.Theentire b/
knockoutmiceultimatelysurvivedincomparisonwithfouroffive ectodomain was subsequently included in the viral vaccine y g
nudemiceand,inagreementwithsimilarstudiesusingbothnaive transgene insert, given that this was previously shown in this ue
s
RwAildG-tykpneocokrotruatnsmgiecneicthCaDt4s+uTrvicveelldssdpidecinfioctfcolreMarStPh1e33in(3fe3c)t,iothne, mAdoHdeul5-tPo. icnhdaubcaeudpirochteacbtaivuediimstrmaiunneASresApMonAs1esv(a3c1ci,n4e0p).rimThede t on N
whereasthesurvivingnudemiceclearedtheparasitesbyday30. a detectable Ab response that increased over time and that was ov
GiventhatnudemicepossessBcells,unlikeRAGknockoutmice, significantlyboostedaftertheadministrationofMVA-P.chabaudi em
the trend for better survival and parasite clearance is consistent chabaudi strain AS AMA1. These data are in accordance with be
with reports detailing the importance of B cells with regard to previous findings in the P. yoelii model using viral vectors r 2
6
these two outcomes. expressing MSP142 or the circumsporozoite protein, in which an , 2
0
8-wkintervalbetweenprimeandboostwasshowntoinducehigh- 1
TcellresponsetoheterologouspeptidesfromotherP.chabaudi 8
titerAbs,aswellasstrongTcellresponses(9,37).Thekineticsof
strains
the Ab response are similar to those seen following vaccination
Sequencepolymorphismhasremainedasignificantobstacletothe with AdHu5 expressing other Ags, such as Ebola virus glyco-
development of Ab-inducing AMA1 protein subunit vaccines (3, protein(50)orOVA(51),inwhichtheAbresponseincreasedby
47).AbresponsesinducedagainstAMA1areallelespecific,and asimilarorderofmagnitudeovertimeandthenreachedaplateau
vaccinationwithonealleledoesnotconfercross-strainprotection: by6 wkpostvaccination.
aneffectclearlyshowninassaysofP.falciparuminvitrogrowth Analysis of the T cell responses in the spleen of immunized
inhibition(48),aswellashomologousversusheterologousstrain BALB/c mice, using overlapping peptides spanning the entire
challenge studies in mice following P. chabaudi AMA1 protein lengthoftheP.chabaudichabaudistrainASAMA1Ag,revealed
immunization (41). Having shown a protective contribution of two CD4+ T cell epitopes. These peptides (P16 and P79) corre-
vaccine-induced CD4+ T cells, it was of interest to ascertain spondedtothosethatwerepreviouslydescribedbyAmanteetal.
whether the induction of cellular immunity by the AdHu5-MVA (32) after immunization with recombinant AMA1 protein-in-
vaccinescould helptoimprovecross-strain protection. adjuvant.Theinvestigatorsreferredtotheseasimmunodominant
Initially, the amino acid sequences of the two CD4+ T cell epitopes, in contrast to cryptic epitopes, which only elicit de-
epitopes, P16 and P79, were compared across various parasite tectable responses in vitro following immunization of mice with
strains: P. chabaudichabaudi AS(vaccine strain), P.chabaudi the relevant peptide(s). Thus, the data generated in this study,
adami 556/DK, P. chabaudi adami CB, and P. chabaudi adami usingviral-vectoredvaccines,areinagreementwiththeirprevious
DS. As previously reported for the immunodominant epitopes observations, with the exception of a very weak CD8+ T cell re-
mapped in P. chabaudi adami DS following AMA1 protein im- sponse, which was also detected in some mice, to one of the