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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

a

,

Kevin

Vanvarenberg

a

,

Ans

De

Beuckelaer

b

,

Stefaan

De

Koker

b

,

Laure

Lambricht

a

,

Catherine

Uyttenhove

c

,

Anca

Reschner

d

,

Alain

Vanderplasschen

d

,

Johan

Grooten

b

,

Véronique

Préat

a,∗

aUniversitécatholiquedeLouvain,LouvainDrugResearchInstitute,AdvancedDrugDeliveryandBiomaterials,Brussels,Belgium
bDepartmentofBiomedicalMolecularBiology,GhentUniversity,Ghent,Belgium

cUniversitécatholiquedeLouvain,LudwigInstituteforCancerResearch,BrusselsBranchanddeDuveInstitute,Brussels,Belgium

dImmunology-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

a

b

s

t

r

a

c

t

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:[email protected](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
dx.doi.org/10.1016/j.vaccine.2015.05.005

(2)

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)byincubating50×106cell/mlwith5␮MCFSE for10min
at37◦C.Thereactionwasblockedbyaddingice-coldPBS(Lonza,
Belgium)+10%serum.2×106OT-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

(3)

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 106cells/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%of104targetcellsin4h.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
1500mm3orwhentheywereinpoorcondition.

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.

(4)

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

(5)

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

(6)

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
tumorvolumesafterchallengewith106P1A-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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