The site of administration influences both the type and the magnitude of the immune response induced by DNA vaccine electroporation

ContentslistsavailableatScienceDirect

Vaccine

jo u rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

The

site

of

administration

influences

both

the

type

and

the

magnitude

of

the

immune

response

induced

by

DNA

vaccine

electroporation

Gaëlle

Vandermeulen

_,

_Kevin

_Vanvarenberg

_,

_Ans

_De

_Beuckelaer

_,

_Stefaan

_De

_Koker

_,

Laure

Lambricht

_,

_Catherine

_Uyttenhove

_,

_Anca

_Reschner

_,

_Alain

_{Vanderplasschen}

_,

Johan

Grooten

_,

_Véronique

_Préat

a_{UniversitécatholiquedeLouvain,LouvainDrugResearchInstitute,AdvancedDrugDeliveryandBiomaterials,Brussels,Belgium} b_{DepartmentofBiomedicalMolecularBiology,GhentUniversity,Ghent,Belgium}

c_{UniversitécatholiquedeLouvain,LudwigInstituteforCancerResearch,BrusselsBranchanddeDuveInstitute,Brussels,Belgium}

d_{Immunology-Vaccinology,DepartmentofInfectiousandParasiticDiseases,FundamentalandAppliedResearchforAnimals&Health(FARAH),Facultyof}

VeterinaryMedicine,UniversityofLiège,Liège,Belgium

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14November2014

Receivedinrevisedform10March2015 Accepted4May2015

Availableonline14May2015 Keywords: DNAvaccine Electroporation Deliverysite Tumorvaccine HIVvaccine

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WeinvestigatedtheinfluenceofthesiteofadministrationofDNAvaccineontheinducedimmune response.DNAvaccineswereadministeredbyelectroporationatthreedifferentsites:tibialcranial mus-cle,abdominalskinandearpinna.AimingtodrawgeneralconclusionsaboutDNAvaccinedelivery,we successivelyusedseveralplasmidsencodingeitherluciferaseandovalbuminasmodelsorgp160andP1A asvaccinesagainstHIVandP815mastocytoma,respectively.Lowlevelsanddurationofluciferase trans-geneexpressionwereobservedafterelectroporationoftheabdominalskin,partlyexplainingitslower immunogenicperformanceascomparedtotheothersitesofadministration.AnalysesofOT-ICD8+and OT-IICD4+Tcellresponseshighlightedthedifferentialimpactofthedeliverysiteontheelicitedimmune response.Muscleelectroporationinducedthestrongesthumoralimmuneresponseandbothmuscleand earpinnasitesinducedcellularimmunityagainstgp160.Earpinnadeliverygeneratedthehighestlevelof CTLresponsesagainstP1Abutelectroporationofmuscleandearpinnawereequallyefficientindelaying P815growthandimprovingmicesurvival.Thepresentstudydemonstratedthatthesiteofadministration isakeyfactortobetestedinthedevelopmentofDNAvaccine.

©2015ElsevierLtd.Allrightsreserved.

1. Introduction

DNAvaccinesareanattractivestrategytoinduceimmune mem-ory. An obvious advantage over protein-based vaccines is that thesamemethodsofproduction,purification,qualitycontroland preservationcanbeusedforallvaccines[1].Inaddition,the pres-enceofimmunostimulatorymotifssuchasCpGthatarecapable toactivatetheinnateimmunesystemendowDNAvaccinewith intrinsic adjuvant propertieswhile transfectionof both antigen presentingcells(APCs)andsurroundingstructuralcellsallowsthe generationofhumoral,cellularandCTLimmuneresponses[2].The efficiencyofDNAvaccineshasalreadybeendocumentedinawide

∗ Corresponding authorat: Universitécatholique deLouvain, Louvain Drug ResearchInstitute, Avenue Mounier73,bteB1.73.12,1200 Brussels,Belgium. Tel.:+3227647309;fax:+3227647398.

E-mailaddress:veronique.preat@uclouvain.be(V.Préat).

rangeofpreclinicalmodelsandseveralclinicaltrialsarecurrently ongoing[3].

ThedeliverymethodofDNAvaccinehasoftenbeendescribed asakeypointtoachievehighleveloftransfectioninvivo. Elec-troporationis anon-viraldeliverymethodbasedonmembrane destabilization and DNA electrophoresis, mediated by electric pulses.Efficientdeliveryofplasmidintomuscle,skin,tumorand othertissuesusinginvivoelectroporationhasthusbeen demon-strated[4–10].

SkinisanattractivesiteforDNAvaccineadministration,rich inAPCs[11].Moreover,intradermalinjectioncanbeeasily per-formed,simpleelectrodedevicessuchasplateelectrodesorless invasivemicroneedleelectrodescanbeapplied[12].Theskinofthe earpinnawasdescribedasaparticularlyeffectivesiteleadingto CTLresponseandprotectivetumorimmunity[13].Yet,alsomuscle hasbeenusedfrequentlyastargettissuefortheadministrationof DNAvaccines.Muscleiseasilyaccessiblebyinjectionthroughthe skinandrequireslowerdosesofDNAascomparedtotheother tis-suesasaconsequenceofitshightransfectionefficiency.Although http://dx.doi.org/10.1016/j.vaccine.2015.05.005

CD8+andOT-IICD4+Tcellswasstudied.

2. Materialsandmethods

2.1. Plasmids

pVAX2 wasobtained by replacing thecytomegalovirus pro-moter of pVAX1 (Invitrogen) by that of pCMV-␤ (Clontech). pVAX2-LUCencodesluciferase.Thecodonoptimized ovalbumin sequencewasorderedfromGeneArtandsubcloned intopVAX2 toconstructpVAX2-OVA.pcDNA3.1(−)-96ZM-gp160CD5(gp160) encodes the gp160 HIV type-1 (HIV-1) envelope glycoprotein. pMDLg/pRRE (gag) was purchased from Addgene (Fargo, ND). pVAX2-P1Aencodingthefull-lengthP1Aproteinwaspreviously constructed[17].PlasmidswerepreparedusingEndoFreePlasmid MegaorGigaKit(Qiagen,Venlo,Netherlands).Opticaldensityat 260nmwasusedtodetermineDNAconcentration.Plasmidquality wasassessed bythe260nm/280nmratioand agarosegel elec-trophoresis.PlasmidsdilutedinPBSwerestoredat−20◦_C. 2.2. Animals

Balb/c,C57BL/6 andDBA/2 femalemicewereobtainedfrom HarlanNederlandor Janvier France.Mice werebetween 6 and 8-week-old at the beginning of the experiments. They were anesthetizedbyintraperitonealinjectionofketamine(Anesketin, euroVet,Belgium)andxylazine(Sigma,Belgium).Before electropo-ration,micewereshavedusingarodentshaver(AesculapExacta shaver,AgnTho’s,Sweden).Allexperimentalprotocolsinmicewere approvedbytheEthicalCommitteeforAnimalCareandUseofthe MedicalSectoroftheUniversitécatholiquedeLouvain.

2.3. Plasmidinjectionandelectroporation

Injectionand electroporationproceduresofskin,muscleand earpinnawerepreviouslydescribed[6].Plasmiddosesand elec-troporationsettingsaresummarizedinTable1.Briefly,plasmids werefirstinjectedusinganinsulinsyringe.Forskinandear vac-cination,weinjectedeither15␮lintothedermisattwodifferent sitesor30␮lintotheexternalsideoftherightearpinna, respec-tively. 2mm spaced electrodes wereapplied todeliver a short highvoltagepulse(HV,700V/cm100␮s)immediatelyfollowed byalowvoltagepulse(LV,200V/cm400ms)usingaCliniporator system(Cliniporator,IGEA,Italy).Formuscleadministration,we injected30␮lintothelefttibialcranialmuscle,andplacedtheleg between4mmspacedelectrodestodelivereightsquare-wave elec-tricpulses(200V/cm20ms2Hz).ForthestudyoftheOT-IandOT-II proliferation,twositespermouseweretreated:bothtibialcranial muscles,leftand rightlowerabdominalquadrantsorbothears. Forallexperiments,conductivegelwasusedtoensureelectrical contactwiththeskin(Aquasonic100,Parker,USA).

CD8+Tcells(OT-I) + +++ −

CD4+Tcells(OT-II) − ++ +

gp160HIV

25gofgp160and27.6gofgag(4.7pmolofeachplasmid)

TotalIgGtiters − + ++

IgG1 + ++ IgG2a + + ␥-IFN − ++ + IL-2 + ++ ++ IL-4 − + − IL-10 − − − P815tumor 50gofpVAX2-P1A CTLresponse − ++ +

Efficacyagainstchallenge − + +

2.4. Luciferaseexpression

Balb/cmicewereinjectedwith50␮gofpVAX2-LUCand elec-troporated.Tofollowtheluciferaseexpression,micewereinjected i.p.withluciferin(150mg/kg)in100␮lofPBS.Opticalimageswere acquiredat severaltime points usingan IVIS Spectrum system (XenogenCorporation,USA).

2.5. OT-IandOT-IIproliferation

Tcellswereisolatedfromspleenand lymphnodesof trans-genicOT-IandOT-IImiceusingCD8+andCD4+TcellisolationkitII mouse(MiltenyiBiotec,TheNetherlands).SubsequentlytheTcells werelabeledwithCFSE(carboxyfluoresceindiacetatesuccinimidyl ester)byincubating50×106_{cell/mlwith5␮MCFSE for10min} at37◦C.Thereactionwasblockedbyaddingice-coldPBS(Lonza, Belgium)+10%serum.2×106_{OT-IorOT-IIcellswereinjectedinto} thetailveinofC57BL/6mice.Theyweretreated2dayslaterby pVAX2-OVAinjectionandelectroporation.Foreachsiteof admin-istration,twoinjectionswereperformedforatotaldoseof5␮gof plasmid.Miceweresacrificed4dayslatertocollectthedraining lymphnodesforsinglecellsuspensionpreparation.Flow cytomet-ricmeasurementwasperformedona triple-laser(B-V-R)LSR-II withFACSDivasoftware(BectonDickinson,Belgium).Analysiswas donewithFlowJoSoftware(Treestar,USA).Cellswerestainedwith aqualivedead(Invitrogen,Belgium),␣-CD16/CD32,CD19APC-Cy7, CD8PerCP,CD3V450(allBDBiosciences),dextramerSIINFEKL H-2kbPE(Immudex,Denmark).

2.6. gp160HIVmodel

BALB/cmicewereimmunizedbyco-injectionof25␮gofgp160 plasmid and 27.6_␮g of gag plasmid, followed by electropora-tion [18]. They receivedone priming and two boosts, 2 and 4 weeks later. Twoweeks after delivery of the last boost, blood

Fig.1.Effectofthedeliverysiteontransgeneexpression.Aplasmidcodingfortheluciferasereportergenewasdeliveredbyelectroporationofthemuscle,theearpinnaorthe skinatday0(n=6pergroup).Luciferaseexpressionwasfollowedinliveanimalsatdays1,13,30and85byinjectingluciferinandimagingthemicewithabioluminescence camera.Statisticalanalysis:one-wayANOVAwithTukeypost-test.*pvalue<0.05,**pvalue<0.01,***pvalue<0.001.

sampleswerecollectedand anELISAassaywasperformed. 96-wellplates werecoated at 4◦C overnightwith 5␮g/ml of HIV gp160protein(Fitzgerald,USA)dilutedinPBS.Afterwashingwith PBS/Tween200.05%,plateswereblockedwith5%drymilkinPBS for2hat37◦C. Plateswerethenwashedand incubatedfor2h at37◦Cwithserial2-folddilutionsofserasamplesdilutedin1% dry milkin PBS.Afterwashing, peroxidase-labeled LO-MGCOC-2 (IMEX, UCL,Brussels, Belgium)and TMB substrate revelation (Calbiochem,USA) wereusedtodeterminetotal immunoglobu-lintiters,definedasthedilutionfactorgivinganopticaldensityat 450nmequaltothelimitofquantification(LOQ,meanblankvalue plus10SDs).Isotypesofanti-gp160antibodiesweredetermined usingappropriatesecondary antibodies(LO-MG1-13, LO-MG2A-9).Miceweresacrificed18daysafterthesecondboostandtheir spleenswerecollectedaseptically.Redbloodcellswereremoved withACKlysisbuffer(Lonza,USA)andsplenocyteswashedwith PBS, countedand adjusted toa concentrationof 106_cells/well. Theywereculturedin96-welltissuecultureplates(Becton Dick-inson,Belgium)in RPMI1640medium supplementedwith10% FBS,1%penicillin/streptomycin, 1%sodiumpyruvate,5_×10−5M 2-mercapto-ethanoland10%MEM(Gibco,Belgium)aspreviously described[6].Cellswererestimulatedby theadditionof100␮l ofsupernatantfromgp160plasmid-transfected293Tcells,100␮l concanavalinA(5␮g/ml)asapositivecontrolor100␮lofculture mediumasanegativecontrol.After48hofincubation(37◦C,5% CO2),supernatantswerecollectedandassayedforIFN-␥,IL-2, IL-4andIL-10(DuoSetELISAdevelopmentkits,R&DSystemsEurope Ltd,UK).

2.7. P815tumormodel

DBA/2micewereimmunizedbyinjectionof50␮gof pVAX-P1Aandelectroporation.Twoboostsweresimilarlyapplied2and 4weeksafterthepriming.Asapositivecontrol,micewere immu-nized by two intra-peritoneal injections of 106 _{L1210.P1A.B7.1} livingcells[19].Afterimmunization,micewereeitherusedto ana-lyzetheCTLresponseor challenged[17].Briefly,theperipheral bloodlymphocyteswereisolated andrestimulatedinvitrowith L1210.P1A.B.7.1cells.Lyticactivitywasmeasuredinachromium releaseassay[20,21].Specificlyticunit(LU) wasdefined asthe numberofcellsthatlyse50%of104_{targetcellsin4h.Asachallenge,} 106 _{P1A-expressing P815B cells were injected subcutaneously} intotheflankandthetumorsize(i.e.length×width×height,in mm3_{)wasmeasuredwithanelectronicdigitalcaliper.Micewere}

sacrificed when the volume of the tumor became larger than 1500mm3_{orwhentheywereinpoorcondition.}

2.8. Statisticalanalyses

t-Test, one-way ANOVA with Tukey post-test and two-way ANOVAwithBonferronipost-testwereused.Statisticalanalyses wereperformedusingthesoftwareGraphPadPrism5forWindows.

3. Results

3.1. Thedeliverysiteinfluencedthemagnitudeandtheduration ofluciferaseexpressionafterelectroporation

Luciferasereportergenewasusedinordertostudytheimpact ofthedeliverysiteonthelevelofDNAvaccineexpression(Fig.1). After1day,geneexpressionwasobservedinallmice.Thosetreated bymuscleelectroporationshowedthehighestlevelofexpression whichwas28-foldand93-foldcomparedtothoseobtainedafter electroporationofearpinnaandskin,respectively.After13days, theexpressionlevelsdroppedtobelowthelimitofquantification formicetreatedintotheabdominalskin.After1month, expres-sionwasstilldetectableinthemicetreatedintothemuscleorthe earpinna.Finally,85daysaftertreatment,lowlevelsofexpression weremeasuredwhatevertheDNAadministrationsite.

3.2. Tcellresponsewasdependentonthedeliverysite

Toinvestigatetheimpactofthedeliverysiteontheinductionof Tcellresponsesinvivo,CFSE-labeledOT-ICD8+orOT-IICD4+Tcells weretransferredintomicethatwerethentreatedbypVAX2-OVA electroporation.ThedraininglymphnodeswerecollectedandOT-I andOT-IIproliferationassessedbyflowcytometry.After electro-porationofthemuscleswith5␮gofplasmid,OT-Icellsremained undividedwhileaslightproliferationwasobservedforOT-IIcells (Fig.2AandB,respectively).Afterelectroporationoftheabdominal skin,OT-IcellsdividedwhileOT-IIcellsfailedtoproliferate.Finally, thestrongerproliferationwasobservedforbothOT-IandOT-IIcells afterelectroporationoftheearpinnasbutthedivisionofOT-Icells appearedtobehigherthanOT-IIcellswith7.5%and47.5%of undi-videdcells,respectively.Together,theseresultsprovideevidence thatthedeliverysitehasastronginfluenceonCD8+andCD4+T cellresponsesinvivo.

Fig.2.FlowcytometryhistogramsofOT-I(panelA)andOT-II(panelB)invivo proliferation.5␮goftheovalbuminplasmidwasdeliveredbyelectroporationinto themuscle,theabdominalskinortheearpinnaatday0(n=3pergroup).Tcells proliferationwasassessed4dayslater.

3.3. Muscleelectroporationelicitedthestrongesthumoral responseagainstgp160

Strongimmuneresponsesagainstgp160wereobtainedinmice whena plasmidencoding thegp160HIV-1envelopewas com-binedwithaplasmidcodingforthegagvirionstructuralprotein [18].Here, afterone primingand twoboosts deliveredevery2 weeks, mice that were vaccinated intramuscularly showed the highestanti-gp160IgG levels.Levels ofIgGagainstgp160were alsosignificantlyincreasedwhentheDNAvaccinewasdelivered intradermallyintotheearpinnabutremainedverylowand sim-ilartotheuntreatedcontrolmicewhendeliveredintothedermis oftheabdomen(Fig.3A).Theanti-gp160IgGtiterswere1.6times and16timeshigherafterimmunizationintothemuscleas com-paredtotheearpinnaandabdominalskin,respectively(Fig.3B). Toinvestigateifthedeliverysitecouldalsoinfluencedthebiasof theimmuneresponse,wemeasuredtheIgG1andIgG2aspecific titers.Bothisotypesweredetectedafterimmunizationby electro-porationintothemuscleandtheearpinna,indicatingthatboth Th1andTh2armsoftheimmunesystemwerestimulated. How-ever,whereas thesetwoadministrationsitesresultedinsimilar IgG2atiters,IgG1titersweresignificantlyincreasedafter intramus-culardelivery(Fig.1C).TheresultingIgG1/IgG2aratioswere1.1and 5.1forearpinnaandmuscledeliverysitesrespectively,suggesting

Fig.3.Influenceofthedeliverysiteonthehumoralimmuneresponsedirected togp160measured2weeksaftertheendoftheimmunization.Primingandtwo boostsweredeliveredintothemuscle(n=6),theabdominalskin(n=5)ortheear pinna(n=6)bygp160andgagplasmidsinjectionandelectroporation.Naivemice remaineduntreated(n=5).PanelA:totalanti-gp160IgGasafunctionofsera dilu-tionmeasuredbyELISA.PanelB:totalanti-gp160IgGtiters.PanelC:anti-gp160 titersfortheIgG1andIgG2aisotypesformiceimmunizedbytheearpinnaorthe musclesite.Graphsrepresentthemeanvalues(±SEM).Statisticalanalysis:one-way ANOVAwithTukeypost-testascomparedtonaive(panelB)andt-test(panelC)*p value<0.05,**pvalue<0.01,***pvalue<0.001.

theoccurrenceofastrongerhumoralresponseagainstgp160after muscleelectroporation.

3.4. Thesiteofelectroporationinfluencedcytokinessecretionby gp160restimulatedsplenocytes

Tofurtherstudytheroleoftheadministrationsiteonthe gen-erationofimmuneresponse,secretionofvariouscytokineswas measuredafterrestimulationofsplenocytescollectedfromanimals immunizedbygp160andgagplasmidsinjectionand electropora-tion(Fig.4).IFN-_␥wassignificantlyinducedwhentheearpinna orthemusclesitewereusedbutremainedlowwhenthe abdom-inalskinsitewasusedforimmunization.Asignificantincreaseof

Fig.4. IFN-␥,IL-2,IL-4andIL-10concentrationsdeterminedaftergp160-restimulationofsplenocytesfor48h.RestimulationwithconcanavalinAandculturemediawere usedascontrols.Graphsrepresentthemeanvalues(±SEM)afterimmunizationintothemuscle(n=6),theabdominalskin(n=5)ortheearpinna(n=6).Statisticalanalysis: one-wayANOVAwithTukeypost-test.*pvalue<0.05,**pvalue<0.01,***pvalue<0.001ascomparedtonaive(restimulatedwithgp160).

IL-2wasobservedforalltheDNAvaccineadministrationsitesbut higherlevelswereobtainedafterimmunizationthroughearpinna ormuscle.SecretionofIL-4washigherfor miceimmunizedby electroporationofearpinna.NosignificantincreaseofIL-10was observedwhateverthedeliverysite.

3.5. EarpinnawastheoptimalsitetogenerateaCTLresponse ADNAvaccineencodingthemouseP1Atumorantigenwas pre-viouslyreportedtodelaytheoutgrowthofP815mastocytoma,a preclinicalmodelforhumantumorsexpressingMAGEtumor anti-gens[17].TheCTLresponsegeneratedbythevaccinewasevaluated byachromiumreleaseassayafterinvitrorestimulationofimmune spleen cells. NoCTLreactivity wasdetectablewhen micewere immunizedbytheabdominalskinsite.Incontrastherewith,aclear anti-P1Aresponsewasobservedwhenthevaccinewasdelivered intothemuscleandwasathisstrongestwhentheDNAvaccinewas administeredthroughearpinna(Fig.5A).

3.6. Muscleandearpinnaelectroporationwereequallyefficient indelayingP815tumorgrowthandimprovingmousesurvival

VaccinatedmicewerechallengedwithP1A-expressingP815B cells.MiceimmunizedwithL1210.P1A.B7.1asapositivecontrol wereefficientlyprotectedagainstthetumorcells.Electroporation oftheP1Avaccineintotheabdominalskinfailedtoinduce rejec-tionofP815Bcellsoreventodelaytumorgrowth(Fig.5BandC). Interestingly,electroporationofthesamevaccineintothe mus-cleandtheearpinnasignificantlyimpairedP815tumorgrowth andpromoteda longtermsurvivalofapproximately40%ofthe

treatedmice,equaltomicevaccinatedwiththereferencevaccine, L1210.P1A.B7.1.

4. Discussion

Thisstudyrevealsastronginfluenceofthedeliverysiteonthe typeandmagnitudeoftheimmuneresponseselicitedbyDNA vac-cineelectroporationassummarizedinTable1.

The skinis quiteoften selected for theimmunization stud-iesduetoitseasyaccessibility. However,electroporationofthe abdominalskinresultedin verylow immuneresponsesagainst both gp160HIV andP815tumorvaccines. Thiscouldbepartly explained by thelow level of protein expression we observed afterintradermalelectroporation.Moreover,electroporationofthe skincompletely failedtoinduceCD4+ T cells proliferationin a mousemodelusingTCRtransgenicOT-IITcells.Althoughsome CD8+ T cellreactivity was observed in the same experimental setupaswellassecretionofIL-2afterimmunizationwithgp160 DNAvaccine,thecellularimmuneresponseelicitedwasnot suffi-cienttogenerateaprotectiveimmuneresponseagainsttheP815 tumor.

Interestingly, the skin of the ear pinna showed particular features that drastically changed the outcome of DNA vaccine electroporation.First,ahumoralresponsewasnoticeableafter elec-troporationofthegp160HIVDNAvaccine,withthepresenceof bothIgG1andIgG2aisotypes.Inthesamemodel,IFN-␥,IL-2and IL-4weresecretedafterexvivostimulationofsplenocyteswiththe gp160envelopeprotein.Immunizationthroughtheearpinnasite alsoresultedinhighCTLresponseandallowedasignificanttumor growthdelayand alongtermsurvival ofvaccinated miceafter

Fig.5. Influenceofthedeliverysiteontheimmuneresponseagainstthetumor antigenP1A.TheP1ADNAvaccinewasdeliveredbyelectroporationofthemuscle (n=10),theabdominalskin(n=10)ortheearpinna(n=9).Miceimmunizedbyi.p. injectionsofL1210.P1A.B7.1cells(n=10)andnaivemice(n=10)wereusedas con-trols.PanelA:P1A-specificCTLresponsesafterimmunization.PanelB:evolutionof tumorvolumesafterchallengewith106_{P1A-expressingP815Bcells.Graph}

repre-sentsthemeantumorsize(±SEM).PanelC:survivalcurvesafterP815challenge. Statisticalanalysis:one-wayANOVAwithTukeypost-test(panelA)andtwo-way ANOVAwithBonferronipost-test(panelB).*pvalue<0.05,**pvalue<0.01,***p value<0.001ascomparedtonaive.

Inconclusion,thepresentworkunderlinestheneedfora con-scious, careful and rational choice of the delivery site for the electroporationofDNAvaccines.Fourdifferentmodelshavebeen used in this study which highlightedgeneral trends aboutthe influenceofthedeliverysiteontheimmuneoutcomeandcould thereforeguidethechoiceoftheelectroporationsitefor preclin-icalevaluationofDNAvaccinecandidates.Theskinoftheearpinna couldbeselectedasthesiteofchoicewhenapronouncedcellular immuneresponseisrequiredsuchasinthecaseofcancervaccines orvaccinesagainstintracellularpathogens.Theintramuscularsite shouldbepreferredwhenahumoralresponseisneededafter elec-troporationoftheDNAvaccineorwhenboththehumoralandthe cellulararmsoftheimmunedefensemustbeactivated.In addi-tiontothesiteofdelivery,severalparametersshouldbetakeninto accountforthedevelopmentofDNAvaccinessuchastheplasmid itself(backbone,genepromoter,sequenceoptimization,secretion signal,etc.)ortheelectrodedesignandabetterunderstandingof thesefactorswillbecriticalforthefutureofDNAvaccinestrategies.

Acknowledgments

We aregratefultoBernardUcakar,Nathalie Lecouturierand DominiqueDonckersfortheirtechnicalhelp.Franc¸oisHippeauis acknowledgedforhiscontributiontothisworkandCédricSzpirer (DelphiGenetics)forhelpfuldiscussions.Theauthorsalsothank PascalBigeyandDanielScherman(UniversityParisV)forthegift ofpVAX2 and pVAX2-LUCplasmids and GeorgeDickson(Royal HollowayUniversityofLondon)forthegp160plasmid.Thiswork wassupportedbygrantsfromtheBelgianFondsNationaldela RechercheScientifique(F.R.S.-FNRS)andfromtheWalloonregion throughDNAVAC,acertifiedBioWinproject(DG06).Gaëlle Vander-meulenisapostdoctoralresearcheroftheFondsdelaRecherche Scientifique-FNRS.

Conflictofintereststatement:Theauthorsdeclarethattheyhave noconflictofinterest.

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