Immunogenicity when utilizing adenovirus serotype 4 and 5 vaccines expressing circumsporozoite protein in naïve and Adenovirus (Ad5) immune mice

Amalfitano, Amalfitano Andrea, Schuldt, Aldhamen, Godbehere-Roosa Sarah, Seregin, Kousa

Description

Induction of potent long lasting effector T cell responses against liver stage malaria antigens strongly correlates with protection from malaria. While Adenovirus serotype 5 (Ad5) based malaria vaccine platforms have the ability to induce potent effector T cell responses against transgenes, high rates of pre-existing Ad5 immunity in malaria endemic regions has prompted study of alternative Ad serotype based malaria vaccines as replacements for Ad5 based malaria vaccines. The research described in this article examines the utility of alternative serotype adenovirus serotype 4 (Ad4) expressing a sporozoite surface protein (circumsporozoite protein (CSP)) (Ad4-CSP) to induce immune responses against CSP. The immunogenicity of Ad4-CSP was also tested in homologous and heterologous prime boost vaccinations in both Ad5 naïve and Ad5 immune backgrounds as compared to use of Ad5-CSP. Results In Ad5 naïve animals, use of Ad4-CSP priming vaccinations followed by boosting with Ad5-CSP (Ad4-CSP/Ad5-CSP) maximally increased the numbers of CSP specific cytokine secreting cytotoxic T cells relative to repeated use of Ad5-CSP. The Ad4-CSP/Ad5-CSP regimen also induced equivalent levels of CSP specific cell killing as did homologous prime-boost vaccinations with Ad5-CSP, despite stimulating lower numbers of CSP specific cytotoxic T cells. Priming with Ad4-CSP followed by a homologous boost resulted in significantly less CSP specific humoral responses than any other vaccination regimen tested in Ad naïve animals. In Ad5 immune animals, addition of Ad4-CSP in homologous or heterologous prime boost resulted in inductions of higher CSP specific responses than animals repeatedly vaccinated with Ad5-CSP alone. However, the observed responses were well below those observed in similarly treated Ad naïve mice. Conclusions While the Ad4-CSP/Ad5-CSP and Ad5-CSP/Ad5-CSP vaccination regimens resulted in equivalent CSP specific killing in Ad naïve animals, Ad4-CSP/Ad5-CSP achieved this result with a lower percentage of CSP specific CD8 + T cells and a higher number of IFNγ secreting cells, suggesting that the Ad4-CSP/Ad5-CSP vaccination regimen elicits more efficient cytotoxic T cells. In Ad5 immune animals use of Ad4-CSP improved CSP specific immune responses as compared to repeated use of Ad5-CSP, but could not achieve the levels of immunogenicity observed when the same vaccine regimens were used in Ad naïve animals. These data indicate the existence of some level of immunological cross-reactivity between these two adenovirus subgroups. Based on these results, it is suggested that future studies .

Sujets

• Adenoviridae
• Malaria
• Vaccine
• Heterologous
• Homology (biology)
• Prime
• Boost

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 2
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Schuldt
etal.MalariaJournal
2012,
11
:209
http://www.malariajournal.com/content/11/1/209

RESEARCH

OpenAccess

Immunogenicitywhenutilizingadenovirus
serotype4and5vaccinesexpressing
circumsporozoiteproteininnaïveand
Adenovirus(Ad5)immunemice
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*
ahGodbehere-Roosa
3
,SergeySSeregin
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Abstract
Background:
InductionofpotentlonglastingeffectorTcellresponsesagainstliverstagemalariaantigensstrongly
correlateswithprotectionfrommalaria.WhileAdenovirusserotype5(Ad5)basedmalariavaccineplatformshave
theabilitytoinducepotenteffectorTcellresponsesagainsttransgenes,highratesofpre-existingAd5immunityin
malariaendemicregionshaspromptedstudyofalternativeAdserotypebasedmalariavaccinesasreplacementsfor
Ad5basedmalariavaccines.Theresearchdescribedinthisarticleexaminestheutilityofalternativeserotype
adenovirusserotype4(Ad4)expressingasporozoitesurfaceprotein(circumsporozoiteprotein(CSP))(Ad4-CSP)to
induceimmuneresponsesagainstCSP.TheimmunogenicityofAd4-CSPwasalsotestedinhomologousand
heterologousprimeboostvaccinationsinbothAd5naïveandAd5immunebackgroundsascomparedtouseof
Ad5-CSP.
Results:
InAd5naïveanimals,useofAd4-CSPprimingvaccinationsfollowedbyboostingwithAd5-CSP(Ad4-CSP/
Ad5-CSP)maximallyincreasedthenumbersofCSPspecificcytokinesecretingcytotoxicTcellsrelativetorepeated
useofAd5-CSP.TheAd4-CSP/Ad5-CSPregimenalsoinducedequivalentlevelsofCSPspecificcellkillingasdid
homologousprime-boostvaccinationswithAd5-CSP,despitestimulatinglowernumbersofCSPspecificcytotoxic
Tcells.PrimingwithAd4-CSPfollowedbyahomologousboostresultedinsignificantlylessCSPspecifichumoral
responsesthananyothervaccinationregimentestedinAdnaïveanimals.InAd5immuneanimals,additionof
Ad4-CSPinhomologousorheterologousprimeboostresultedininductionsofhigherCSPspecificresponsesthan
animalsrepeatedlyvaccinatedwithAd5-CSPalone.However,theobservedresponseswerewellbelowthose
observedinsimilarlytreatedAdnaïvemice.
(Continuedonnextpage)

*Correspondence:[e-mail]
1
GeneticsProgram,MichiganStateUniversity,2240EBiomedicaland
PhysicalSciencesBuilding,EastLansing,MI48824,USA
2DepartmentofPediatrics,MichiganStateUniversity,EastFeeHall,East
Lansing,MI48824,USA
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Schuldtetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.

Schuldt
etal.MalariaJournal
2012,
11
:209
http://www.malariajournal.com/content/11/1/209

Page2of14

(Continuedfrompreviouspage)
Conclusions:
WhiletheAd4-CSP/Ad5-CSPandAd5-CSP/Ad5-CSPvaccinationregimensresultedinequivalentCSP
speci
+
fickillinginAdnaïveanimals,Ad4-CSP/Ad5-CSPachievedthisresultwithalowerpercentageofCSPspecific
CD8TcellsandahighernumberofIFN
γ
secretingcells,suggestingthattheAd4-CSP/Ad5-CSPvaccination
regimenelicitsmoreefficientcytotoxicTcells.InAd5immuneanimalsuseofAd4-CSPimprovedCSPspecific
immuneresponsesascomparedtorepeateduseofAd5-CSP,butcouldnotachievethelevelsofimmunogenicity
observedwhenthesamevaccineregimenswereusedinAdnaïveanimals.Thesedataindicatetheexistenceof
somelevelofimmunologicalcross-reactivitybetweenthesetwoadenovirussubgroups.Basedontheseresults,itis
suggestedthatfuturestudiesshouldundertakesimilarlystringentanalysesofalternativeAdserotypestoestablish
theireffectivenessasreplacementsforAd5.
Keywords:
Serotype5,Serotype4,Adenovirus,Malaria,Circumsporozoiteprotein,Vaccine,Heterologous,
Homologous,Prime,Boost

Background
utilizedrAd5sgeneticallyengineeredtoexpressCSPin
Despiteuseofprophylacticmedicationsandvectorcon-humanandmousemodelsofmalaria[6,19,20].However,
trol,malariacontinuestobeoneoftheworld
’
smostpre-existingAd5immunityiscommoninregions
deadlyhealthconcernsclaimingthelivesofalmostwheremalariaisendemic,andthepresenceofneutral-
1millionpeopleannually.Theprotozoanparasite,izingantibodiesagainstAd5hasbeenshowntohinder
Plasmodiumfalciparum
,accountsforabout90%ofAd5basedvaccineefficacy[21-23].Ithasbeen
thesedeaths[1].Numerous
P.falciparum
targetedvac-hypothesizedthattheuseofalternativeserotypebased
cinestudiesarecurrentlyunderwayineffortstoelimin-rAdsmayinduceimprovedimmunogenicresponsesto
atethisdangerouskiller.The
P.falciparum
derivedantigensirrespectiveofpre-existingAd5immunity,for
circumsporozoiteprotein(CSP)isthemoststudiedandexampleinHIVvaccinedevelopment[24,25].There
commonlyusedantigenforthepurposeofdevelopingaareatleast52differenthumanAdenovirusserotypes.
vaccineagainstmalaria[2-6].CSPisabundantontheAdenovirusserotypesaredividedintosubgroupsA-F
surfaceofthesporozoite,andisalsopresentinthebasedprimarilyonanti-seraneutralization.SinceAd5is
plasmamembraneandcytosolofplasmodiuminfectedamemberofsubgroupC,itishypothesizedthatalterna-
hepatocytes.CSPisa58kDproteincomposedofativeserotypesfromothersubgroupswouldnotbeneu-
C-terminuscontainingthethrombospondin-liketypeItralizedbyAd5antibodyandtherefore,couldstillbe
repeatregion(TSRinvolvedinliversinusoidattach-utilizedtoinfectcellsandstimulateimmunitytoan
ment),acentralregionof[NANP]repeats,andaexpressedtransgene.Useofalternativeserotypebased
N-terminalsitethatwhenincontactwiththeliversi-Advectorscanserveotherimportantpurposesaside
nusoidiscleavedexposingtheTSR[7-9].fromstimulatingimmuneresponsesinAd5immune
OftheseveralmalariavaccinevectorsthattargetCSP,patients.Heterologousprimeboostregimenswherethe
themostsuccessfultodateisavaccineformulationthatprimeandboostvaccinationsarederivedfromtwodif-
consistsofanovelfusionproteinbetweenthehepatitisferentAdserotypesbasedvaccinescanprovidegreater
Bsurfaceprotein(HBsAg)andCSP,andadditionaladju-inductionsofimmunitythanhomologousprimeboost-
vants.Thisformulation,referredtoasRTS,S/AS01B,isingwithasingleAdserotypebasedvaccine[26-28].
currentlyinaphase3clinicaltrial[10].ThisvaccinehasInthiscontext,Ad4basedvectorsmaybepromising
beenabletoconferprotectionto56%ofvaccinatedindi-foruseinmalariaspecificapplications.Theefficacyand
viduals[3,10-15].Althoughpromising,theresultsalsosafetyofAd4vaccineplatformshasbeenestablished.
suggestthatmorepotentimmuneresponsesmaybeForinstance,astheprincipalserotypecausingAcute
requiredtoachievehigherlevelsofprotection.ForthisRespiratoryDisease(ARD)inmilitaryrecruits,anorally
reasonothervectorsandimmunogenicstrategiesin-administered,liveAd4viruswasutilizedfordecadesin
corporatingCSParebeingpursuedineffortstodevelopvaccinationsofrecruitsagainstARD[29-32].Morere-
ahighlyefficacious,malariaspecificvaccine.cently,Ad4basedvaccineshavebeensuccessfully
Recombinantadenovirusserotype5(rAd5)basedutilizedinHIVvaccinestrategiesindogandchimpanzee
vaccinesareimportantinthisregardastheyhavebeenmodels[24,25].Thisresearcharticleanalysestheability
confirmedtoelicitpotentadaptiveresponsesagainstofanAd4-basedmalariaspecificvaccineexpressingCSP
expressedtransgenes[16-18].Multiplestudieshavetostimulatepotentimmuneresponseswhenusedin

Schuldt
etal.MalariaJournal
2012,
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:209
http://www.malariajournal.com/content/11/1/209

homologousorheterologusprimeboostregimenswith
anAd5vaccinealsoexpressingCSP,bothinthecontext
ofAd5naïveandAd5immuneanimals.
Methods
Vectorconstruction
Theopenreadingframe(ORF)ofthe
P.falciparum
CSP
gene,composedofacodonoptimizedconsensusofsev-
eral
P.falciparum
CSPsequences,wasincorporatedinto
anE1,E3deletedadenovirusserotype5vectorunderthe
controlofacytomegalovirus(CMV)enhancer/promoter
elementaspreviouslydescribed[33].ThesameCSPcon-
sensussequencewasincorporatedintoanE1,E3deleted
adenovirusserotype4vectorundertheexpressional
controlofthesameCMVenhancer/promoterelement.
Ad4vectorconstructionwasperformedaspreviously
describedwithanAd4recombinationbasedproduction
system[34].
Animalprocedures
AllanimalprocedureswereapprovedbytheMichigan
StateUniversityInstitutionalAnimalCareandUse
Committee(IACUC).6
–
8weeksoldmaleBALB/cJmice
wereinjectedintramuscularly(IM)intothetibialisan-
terioroftherighthindlimb.Totalinjectedvolumewas
20
μ
l.Splenocytesandplasmawerecollected.Allproce-
dureswithrAdswereperformedunderBSL-2,andall
vectortreatedanimalsweremaintainedinABSL-2con-
ditions.Careformicewasprovidedinaccordancewith
PHSandAAALACstandards.
AELISELISA-basedantibodyassayswerecompletedasprevi-
ouslydescribed[16].High-bindingflatbottom96-well
plateswerecoatedwith0.2
μ
gofpurifiedCSPperwell
inavolumeof100
μ
Landincubatedovernightat4°C.
PlateswerewashedwithPBS-Tween(0.05%)thentrea-
tedwithblockingbuffer(3%bovineserumalbumin)for
1houratroomtemperature.Plasmafromadnaïveani-
malswasdiluted(1:100,1:200,1:400)inblockingbuffer.
PlasmafromAdimmuneanimalswasanalysedwith-
outdilution.Sampleswereincubatedfor1houratroom
temperature.WellswerewashedwithPBS-Tween
(0.05%)andHRPantibody(Bio-Rad)wasaddedat
1:4,000dilutioninPBS-Tween.Tetramethylbenzidine
(TMB)(Sigma-Aldich)wasaddedtoeachwellandthe
reactionwasstoppedwith2Nsulfuricacid.Platesare