Immune responses induced by a BacMam virus expressing the E2 protein of classical swine fever

ImmunologyLetters125(2009)145–150

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ImmuneresponsesinducedbyaBacMamvirusexpressingtheE2proteinofclassicalswinefevervirusinmice

MiaoLi,Yu-FeiWang,YuWang,HuiGao,NaLi,YuanSun,Bing-BingLiang,Hua-JiQiu

DivisionofSwineInfectiousDiseases,NationalKeyLaboratoryofVeterinaryBiotechnology,HarbinVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciences,427MaduanStreet,150001Harbin,Heilongjiang,China

articleinfoabstract

Non-replicatingbaculovirus-mediatedgenetransferintomammaliancellshasbeendevelopedasavac-cinestrategyagainstanumberofdiseasesinseveralanimalmodels.Inthepresentstudy,theBacMamvector,abaculoviruspseudotypedwiththeglycoproteinfromvesicularstomatitisvirus,wasusedasarecombinantvectortoexpressclassicalswinefevervirus(CSFV)E2proteinunderthecontroloftheimmediateearly1(ie1)promoterfromshrimpwhitespotsyndromevirus.TheE2genewasef cientlyexpressedinbothinsectandmammaliancells.Intramuscularinjectionofmicewiththerecombinantbaculovirusresultedintheproductionofhigh-titersofCSFV-speci cneutralizingantibodies.Speci clymphoproliferativeresponsestoCSFVstimulationweredetectedinthesplenocytesoftheimmunizedmiceasdemonstratedbyCFSEstainingassayandWST-8assay.ThisstudydemonstratesthattheBacMamvirusvectorcanef cientlyexpresstheE2proteinandeffectivelyinduceimmuneresponsesagainstCSFV.Thisisa rststepinthedemonstrationthatthepseudotypedbaculovirus-deliveredCSFVE2genecanbeapotentialnon-replicatingvaccineagainstCSFVinfections.

©2009ElsevierB.V.Allrightsreserved.

Articlehistory:

Received23January2009

Receivedinrevisedform28June2009Accepted1July2009

Availableonline7July2009Keywords:

ClassicalswinefevervirusE2gene

BacMamvirus

RecombinantbaculovirusImmunogenicity

1.Introduction

Thebaculovirusexpressionsystemhasbeenextensivelyusedasvectorsforabundantexpressionofalargevarietyofforeignproteinsininsectcells.Recently,recombinantbaculovirusvectorscontainingmammaliancell-activepromoterelements(alsoknownasBacMamviruses)havebeenusedsuccessfullyforgenedeliv-eryintomammaliancelllineswithoutapparentviralreplication[1].RecentstudieshavedemonstratedthatBacMamvirusescanbeusednotonlyforef cientgenetransductionintomammaliancellsinvitro[2,3]butalsoforgenetransductioninvivo[4,5].ThemajoradvantageoftheBacMamvirusisthatitshowsef cientgenetransductionintoawidevarietyofcelllinesbydirectinfection.Numerouseffortshavebeenmadetoharnessthebaculovirusasavectorforgenetherapyandvaccinedevelopment.Theuseofbac-ulovirusasavectorforvaccinationwasinitiallydescribedbyAokietal.[6],whodemonstratedthatintramuscularinjectionofmicewitharecombinantbaculovirusexpressingthepseudorabiesvirusgBproteincouldelicitedameasurablehumoralresponse.Further-more,severalgroupshavedemonstratedthatdirectvaccinationwithrecombinantbaculovirusbyintramuscular,intraperitonealorintranasalinoculationcaninducetheproductionofhumoral

Correspondingauthor.Tel.:+8645185935041;fax:+8645185935041.E-mailaddress:huajiqiu@(H.-J.Qiu).0165-2478/$–seefrontmatter©2009ElsevierB.V.Allrightsreserved.doi:10.1016/j.imlet.2009.07.001

andcell-mediatedimmunityagainstvariousantigens[7–9].Morerecently,Wangetal.[10]reportedthatanimmuneresponsetotheGP5andMproteinsofporcinereproductiveandrespiratorysyn-dromeviruswaseliciteduponvaccinationwithabaculovirusvectorexpressingthevirusstructuralcomponent.

Classicalswinefever(CSF),whichiscausedbyclassicalswinefevervirus(CSFV),isafatalswinediseaseandoneofdiseaseslistedbytheWorldOrganizationforAnimalHealth(OIE).Thediseaseisendemicorepidemicinmanycountriesandcausesgreateconomiclosstotheswineindustryworldwide.TheCSFVgenomecontainsasingle,largeopenreadingframe(ORF)encodingapolyproteinthat,uponproteolyticprocessing,givesrisetofourstructuralpro-teinsandeightnon-structuralproteins[11].TheE2proteinisanenvelopeglycoprotein,whichisresponsibleforelicitingneutraliz-ingantibodiesininfectedanimalsandthusprotectingpigsagainstvirulentchallenge.

Atpresent,themostfrequentlyusedvaccineistheChineselapinizedattenuatedvaccine,i.e.C-strain,whichcaninducecom-pleteprotectionagainstCSF.However,amajordisadvantageofthisvaccineisthattheantibodiesinducedbythevaccinecannotbedis-tinguishedfromthoseinducedbywild-typeCSFVinfections.ItisimportanttodevelopnovelCSFVvaccinescapableofinducingrapidprotectioncombinedwiththeabilitytodifferentiateinfectedfromvaccinatedanimals.

OurpreviousstudiesdemonstratedthattheBacMamviruswithanEGFPreportergeneunderthecontroloftheshrimpwhitespotsyndromevirus(WSSV)immediateearly1(ie1)promoterexhib-

146M.Lietal./ImmunologyLetters125(2009)145–150

itedhightransductionef ciencyandhigh-levelexpressionofthereporterproteininmammaliancells[12].Inthispresentreport,weexploredthepotentialofthisvectorfordevelopingavaccineagainstCSFVbyconstructingtherecombinantbaculoviruscontain-ingthepolyhedrinpromoter-controlledvesicularstomatitisvirusglycoprotein(VSV-G)expressioncassetteandWSSVie1promoter-controlledE2expressioncassette.Wetestedthegeneexpressionofthisvectorinbothinsectandmammaliancells.Furthermore,weinvestigatedtheabilityofthepuri edrecombinantbaculovirustostimulatehumoralandcell-mediatedimmunityagainstCSFVinmicebyintramuscularinjection.OurresultsindicatethattheBacMamviruscanbeusedtodevelopanewgenerationofnon-replicativevectorvaccineagainstCSFVinfections.2.Materialsandmethods2.1.Virusandcells

TheCSFVShimenstrainusedinthisstudywasmaintainedintheHarbinVeterinaryResearchInstitute.RecombinantadenovirusrAdV-E2expressingtheCSFVE2protein[13]wasconstructedbyourlaboratory.Sf9cellswereculturedat27 CinGrace’smedia(Gibco,GrandIsland,USA)supplementedwith10%fetalbovineserum(FBS).HeLaandPK-15cellswereculturedinDulbecco’smodi edEagle’smedium(DMEM,Gibco,GrandIsland,USA)sup-plementedwith10%heat-inactivatedFBS,andincubatedat37 Cwith5%CO2.

2.2.Generationofrecombinantbaculoviruses

TheVSV-GgenewasPCRampli edfrompVSV-G(Clontech,PaloAlto,CA,USA)withtheprimerpair5 -CGGTCCGTATGAAGT-GCCTTTTGTAC-3 and5 -GGATCCGACTTTTATTTTACTTTCCAAGTCG-GTTC-3 .TheE2cDNAwasampli edfromtheCSFV(Shimenstrain)genomebyRT-PCRwiththeprimerpair5 -GCCGGATCCATGCGGC-TAGCCTGCAAGGAAGACTAC-3 and5 -ATTGCGGCCGCCTACACATC-CAGGTCAAACCAGTATTGATACTC-3 .Themodi edbaculovirustransfervectorwasconstructedbyinsertingtheVSV-Ggeneunderthecontrolofthepolyhedronpromoter(PPH)inthepFastbacHTBvector(Invitrogen,SanDiego,USA).

TheEGFPgenewasexcisedfrompEGFP-N1(Invitrogen,SanDiego,CA,USA)andtheWSSVie1promoterwasdescribedpreviously[12].Togeneratetherecombinantbaculovirus,theWSSVie1promoter-controlledE2expressioncassettewasinsertedintothemodi edbaculovirustransfervectorandintegratedintothebaculovirusgenomewithinDH10BACTMthroughsite-speci ctranspositionaccordingtothemanufacturer’sprotocolprovidedwiththeBac-to-Bacsystem(Invitrogen),resultinginBacMam/G-ie1-E2.RecombinantbaculovirusBacMam/G-ie1-EGFPwasgeneratedasdescribedabove.Theviruswasfurtherampli edbypropagationinSf9cells.Viruspuri cationwasperformedbyultracentrifugationasdescribedbefore[7].ThetiterofviruswasdeterminedbyastandardviralplaqueassayafterpassageinSf9cells[14].

2.3.Identi cationofrecombinantbaculovirus

Therecombinantbaculoviruswasidenti edbyPCRusingtheM13universalprimer(Invitrogen),andtheexpressionwasfurthercon rmedbyimmuno uorescenceassay(IFA)andWesternblot.2.3.1.IFA

Sf9cellsgrowninsix-wellplateswereinfectedwiththebac-ulovirus,BacMam/G-ie1-E2.Seventy-twohourspost-infection,thesupernatantwasharvestedandthecellswerewashedwith0.1M

phosphate-bufferedsaline(PBS,pH7.4).Thecellswerethenresus-pendedinPBSand xedonaglassslidewithcold100%acetonefor8min.The xedcellswerethenincubatedwithananti-E2mono-clonalantibody(mAb)(6E10)[15]for1hat37 C.FITC-conjugatedgoatanti-mouseIgG(Sigma,St.Louis,USA),atadilutionof1:128,wassubsequentlyincubatedwiththe xedcellsfor1hasthedetec-torantibody.The uorescencesignalwasdetectedwithaninverted uorescencemicroscope(Nikon,Japan).

2.3.2.Westernblot

Baculovirus-infectedSf9celllysatesweresubjectedto10%SDS–polyacrylamidegelelectrophoresis(PAGE)andWesternblotanalysisusinganti-E2mAbHQ06[16],andbaculovirus-expressedrecombinantE2protein[17]servedaspositivecontrol.Theprepara-tionandrunningofgels,transferofproteinsfromSDS–PAGEgelstonitrocellulose lters,andimmunoblottingwereperformedaccord-ingtostandardprotocols[13].2.4.Transductionofmammaliancells

HeLacellswereseededonto24-wellplatesandallowedtoincu-bateat37 Covernight.Priortotransduction,thespentmediumwasremovedandthecellswerewashedwith0.1MPBS(pH7.4).Thecellswerethenincubatedwiththerecombinantbaculovirusatamultiplicityofinfection(MOI)of100in0.5mlofPBS.Theplateswereplacedonarockingshakerfor8hat25 C[18].Aftertheincu-bationperiod,thevirussolutionswereaspiratedandthecellswerewashedwithPBSagain.Thewellswerereplenishedwith2mloffreshDMEMcontaining10%FBSandincubatedat37 C.Afterincu-bationat37 Cfor48h,thecellswere xedontheplatewithcold33%acetonefor30minatroomtemperatureforanalysisbyIFAasdescribedabove.CellsampleswerealsocollectedforanalysisbyWesternblotassayasdescribedabove.2.5.Immunizationofmice

Seven-week-oldfemaleBALB/cmice(purchasedfromtheLabo-ratoryAnimalCenterofHarbinVeterinaryResearchInstitute)wererandomlydividedintofourgroups(withsevenmiceineachgroup).Onegroupwasinjectedintramuscularly(i.m.)with108plaqueformingunits(PFU)ofBacMam/G-ie1-E2in500 lofPBS.Themiceimmunizedi.m.with108TCID50rAdV-E2[13]wereusedasposi-tivecontrols,whileBacMam/G-ie1-EGFPvaccinatedmiceservedasnegativecontrols.Thefourthgroupremainedunimmunized.Atfourweeksfollowingtheprimeimmunization,themicewereboostedwiththesamedoseoftheinocula.2.6.IndirectELISA

Serumsamplesfromtheimmunizedmicewerecollectedweeklybeforeandafterimmunization.Theseraweretestedforthepres-enceofCSFV-speci cantibodylevelsbyanindirectELISAasdescribedpreviously[19].

2.7.Serum-virusneutralizationtest(SNT)

Serumsampleswerecollectedat0,3and6weeksfollowingtheprimeimmunization,andheat-inactivatedfor45minat56 Cbeforetesting.TheSNTwascarriedoutin96-wellcultureplatesasdescribedelsewhere[20].Brie y,two-foldseriallydilutedseraweremixedwithanequalvolumeof100TCID50ofCSFVShimenstrainandincubatedat37 Cfor1h.Subsequently,eachoftheserum–virusmixtureswasaddedtoacon uentmonolayerofPK-15cellsin96-wellcultureplates.Theplateswereplacedinamoistchamberandincubatedina5%CO2incubatorat37 Cfor1h.After2–3days,thecultureplatewas xedfor20minwithcoldabsolute

M.Lietal./ImmunologyLetters125(2009)145–150

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Fig.1.SchematicrepresentationoftherecombinanttransfervectorspFB-G-ie1-E2andpFB-G-ie1-EGFP.ThedesiredVSV-GorE2expressioncassettewasinsertedwithinthepolyhedrinlocusthroughsite-speci ctranspositionemployingtheBac-to-Bacsystem.ie1,WSSVie1promoter;PPH,polyhedrinpromoter.

ethylalcohol.AfterextensivewashingwithPBS,theexpressionofCSFVwasdetectedbyIFAwithmAb6E10,followedbyincubationwithFITC-conjugatedgoatanti-mouseIgG(Sigma,St.Louis,USA).TheCSFVneutralizingantibody(NAb)titersweredeterminedandexpressedasthereciprocalofthehighestdilutionatwhichinfectionofthePK-15cellswasinhibitedin50%oftheculturewells.2.8.Lymphoproliferationassays

Threeweeksaftertheboosterimmunization,cellsuspensionswerepreparedfromthespleensoftheimmunizedmicebypress-ingthrougha nesieveintoRPMI1640medium(Gibco,GrandIsland,USA).Thecellswerethentreatedwithredbloodcelllysingbuffer(155mMNH4Cl,10mMNaHCO3,0.1mMEDTA;pH7.4).CSFVShimenstrainpropagatedinPK-15cellswasreleasedafterthreefreeze-thawcycles,andthentheviralsuspensionwascentrifugedat2000×gfor15min.Thesupernatantwascollectedandvirionswerepuri edbydifferentialcentrifugation.Thelymphoprolifera-tionassaybasedoncarboxy uoresceinsuccinimidylester(CFSE)stainingwasperformedusingpuri edCSFVvirionsasstimula-torsasdescribedpreviously[21].ThelymphoproliferationassaybasedonWST-8wasalsoperformedasdescribedpreviously[22,23]byusingtheCellCountingKit-8(DojindoMolecularTechnologies,Inc.,Japan),whichmeasurestheamountofwater-solubleformazanproducedbyWST-8.3.Results

3.1.Constructionandidenti cationofrecombinantbaculovirusBacMam/G-ie1-E2

Toexplorethepotentialofabaculovirus-basedvaccineagainstCSFV,weconstructedarecombinantbaculoviruscontaininganE2-expressioncassetteunderthecontrolofWSSVie1promoter,namedBacMam/G-ie1-E2(Fig.1).InfectionofinsectSf9cellswithBacMam/G-ie1-E2resultedinextensivecell–cellfusion.Thephe-notypewasduetothehigh-levelexpressionofVSV-Gproteinwithmembrane-fusionactivity(datanotshown).ExpressionoftheE2glycoproteinduetoinfectionwiththeBacMamviruswasdetectedbyIFAwithanti-E2mAb6E10[15](Fig.2A).Theexpressionwasfur-thercon rmedbyWesternblotanalysisofthetotalcellularextractwithanti-E2mAbHQ06[16].Aproteinofapproximately53kDawasdetectedinBacMam/G-ie1-E2infectedSf9celllysatesbutnotinnegativecontrolsamples(Fig.3A).

3.2.ExpressionofrecombinantbaculovirusBacMam/G-ie1-E2inmammaliancells

TotestwhethertheobtainedbaculoviruscanexpresstheE2geneinnon-insectcelllines,wetransducedthemammalianHeLacellswithBacMam/G-ie1-E2.TheIFAresultshowedthatBacMam/G-ie1-E2ef cientlytransducedthetestedcelllinesasindicatedbythepositive uorescentcells(Fig.2B).The

expression

Fig.2.DetectionoftheE2proteinexpressioninBacMam/G-ie1-E2-infectedSf9cells(A)orBacMam/G-ie1-E2-transducedHeLacells(B)byIFAusingmAb6E10.Thecellswere xedfor72hor48hafterinfectionortransductionandanalyzedbyIFAusingtheprimaryantibody6E10andsecondaryFITC-conjugatedgoatanti-mouseIgG.UninfectedSf9andHeLacellsservedasnegativecontrols.

wasfurthercon rmedbyWesternblotanalysisofthetotalcellularextractusinganti-E2mAbHQ06[16](Fig.3B).

3.3.CFSV-speci cantibodieselicitedbyBacMam/G-ie1-E2inmice

TodeterminewhethertheBacMamvirusexpressingE2pro-teincaninduceCSFV-speci chumoralimmuneresponsesinvivo,BALB/cmicewereimmunizedi.m.with108PFUofBacMam/G-ie1-E2,BacMam/G-ie1-EGFP,or108TCID50/mouseofrAdV-E2.AnindirectELISAwasusedtomonitortheE2-speci cantibodylevels.TheresultsshowedthatmiceimmunizedwithBacMam/G-ie1-E2orrAdV-E2developedsigni cantantibodyresponsesasdetectedbyELISAinwhichpuri edbaculovirus-expressedE2protein[17]wascoatedasantigen.MeanwhilethemiceimmunizedwithBacMam/G-ie1-EGFPdidnotseroconvertwithjustanon-speci cantibodyresponsebelowthreshold,whilethenon-immunizedmiceshowedonlyabackgroundantibodylevel.Afurtherincreaseinantibodylevelswasobservedoneweekaftertheboosterimmu-nizationwithBacMam/G-ie1-E2.TheserumantibodylevelsintheBacMam/G-ie1-E2-immunizedgroupsweresigni cantlyhigherthanthoseintherAdV-E2-immunizedgroupat5and6weekspost-immunization(P<0.05)(Fig.4).

TodetectCSFV-speci cNAbtiters,SNTwasperformedat0,3and6weeksfollowingprimeimmunization.TheresultsshowedthatmiceimmunizedwithBacMam/G-ie1-E2orrAdV-E2developeddetectableNAbtitersat3weeksfollowingprimeimmunization.AfurtherincreaseinNAbantibodytiterswasobservedat6weeksfollowingprimeimmunization;whereasthemiceimmunizedwithBacMam/G-ie1-EGFPdidnotdevelopadetectableantibodyresponseagainstCSFVevenat6weeksfollowingprimeimmuniza-tion.TheCFSV-speci cneutralizinglevelintheBacMam/G-ie1-E2groupwassigni cantlyhigherthanthatintherAdV-E2-immunizedgroup6weeksfollowingprimeimmunization(P<0.05)(Fig.5).

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Fig.3.WesternblotanalysisoftheE2expressioninBacMam/G-ie1-E2-infectedSf9cells(A)orBacMam/G-ie1-E2-transducedHeLacells(B)usinganti-E2mAbHQ06[22].Lanes1and6:prestainedproteinmolecularweightmarker(JingmeiBiotech,Beijing,China);Lanes2and10:baculovirus-expressedrecombinantE2protein[17]aspositivecontrol;Lane3:BacMam/G-ie1-E2infectedSf9cells;Lane4:BacMam/G-ie1-EGFPinfectedSf9cells;Lane5:normalSf9cellsasnegativecontrol.(B)Lane7:normalHeLacellsservedasnegativecontrol;Lane8:totalcellularextractsfromHeLacellstransducedwithBacMam/G-ie1-EGFP;Lane9:totalcellularextractsfromHeLacellstransducedwithBacMam/G-ie1-E2.

3.4.Cell-mediatedimmunityelicitedbyBacMam/G-ie1-E2inmice

Tocharacterizethecell-mediatedimmuneresponses,themiceweresacri cedandthesplenocyteswereisolatedat7weeksfollowingprimeimmunizationwithBacMam/G-ie1-E2,BacMam/G-ie1-EGFPorrAdV-E2.AfterstainingwithCFSE,thecellswerestimulatedwithCSFV(10 g/ml,200 leachwell).Afterincubatingfor5days,the uorescenceintensitiesofthecellsweredetectedby owcytometry.TheresultsshowedthatthecellsobtainedfromBacMam/paredwiththecontrolgroup,percentagesofproliferatedCD4+TandCD8+T-cellsweresigni cantlyhigherforthegroupsimmunizedwithBacMam/G-ie1-E2(P<0.01)andrAdV-E2(P<0.01).Onaver-age,percentagesofproliferatedCD4+T-cellforBacMam/G-ie1-E2,rAdV-E2orBacMam/G-ie1-EGFPgroupswere4.07%,4.01%and0.36%,respectively,andpercentagesofproliferatedCD8+T-cellforthesethreegroupswere4.06%,3.98%and0.35%,respectively(Fig.6).

Atthesametime,thelymphoproliferativeresponseswerealsomeasuredusingWST-8.SplenocyteswerestimulatedwithCSFVfor80h,andWST-8solutionwasadded.TheOD450nmofeachsamplewasmeasuredandthestimulationindex(SI)wascalculatedusingthefollowingformula:SI=(meanOD450nm

of

Fig.5.DetectionofCSFV-speci cserumneutralizingantibodylevelsinducedinthevaccinatedmice.ThreegroupsofsevenmicewereeachintramuscularlyinjectedtwicewithBacMam/G-ie1-E2,BacMam/G-ie1-EGFPorrAdV-E2,respectively.Pooledserumsamplesfromeachgroupweretestedat0,3and6weeksfollowingprimeimmunizationbyserum-virusneutralizationtesttodetermineantibodytiterstotheCSFVShimenstrain.*P<0.05.

CSFV-stimulatedcells)/(meanOD450nmofunstimulatedcells).TheSIoftheBacMam/G-ie1-E2group(P<0.01)andrAdV-E2group(P<0.01)wassigni cantlyhigherthanthatofthecontrolgroup,whiletherewerenosigni cantdifferencesbetweenthetwoformergroups(Fig.7).4.Discussion

TheBacMamviruses,carryingtargetgenesunderthecontrolofanappropriatemammaliancell-activepromoter,haveprovidedanalternativeandattractivechoiceforgenedeliveryduetotheirsafetyandimprovedef cacy[24].TheBacMamvirusesprovideuswithapromisingplatformtostudygenefunctionsandtodevelopnon-replicatingvectorvaccinesandgenetherapystrategies[25–27].Baculovirusvectorsaresaferthanotherconventionalviralvectors(suchasadenovirus),becausetheydonotreplicateinmammaliancellsandhavenomarkedcellulartoxicity.Furthermore,asagenetransfervector,thebaculovirusretainssomeadditionaladvantages:largeinsertioncapacityofforeignDNAsequences(>38kb),poten-tiallyhighviraltiters,simplemanipulation,andeaseofpreparation.

Fig.4.DetectionofserumantibodylevelsinducedbytherecombinantbaculovirusinmicebyindirectELISA.ThreegroupsofsevenmicewereintramuscularlyinjectedtwicewithBacMam/G-ie1-E2,BacMam/G-ie1-EGFP,orrAdV-E2ata4-weekinterval.SerumsamplesfromeachgroupwerecollectedweeklyandtestedbyanindirectELISA[19]inwhicheachwellofa96-wellmicroplatewascoatedwith200ngofthepuri edbaculovirus-producedE2protein[17]ina96-wellmicroplate.AdashedlineindicatesthepositivecutoffvalueofELISA(OD450nm=0.35,whichisthemeanvaluesofnegativeseraplusthree-foldstandarderrors).*P<

0.05.

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Fig.6.LymphoproliferativeresponsesintheimmunizedmicemeasuredbyCFSEassay.SplenocytesobtainedfromthemiceimmunizedwithBacMam/G-ie1-E2,BacMam/G-ie1-EGFPorrAdV-E2werestainedwithCFSEandincubatedwithCSFVfor5days.**P<0.01.

However,invivogenedeliverybybaculovirusishamperedbyitsinactivationbyserumcomplement[28],thuslimitingitsapplicationsingenetherapyandvaccinedevelopment.Recently,abaculoviruspseudotypedwithVSV-Gemergedasapromisinggene-deliveryvectorduetoitsef ciencyintransducingvariousmammaliancellsandresistancetoinactivationbyanimalsera[29,30].TheVSV-Gproteinwasshowntoenhancetheescapeofbaculovirusvectorsfromintracellularendosomes,increasingthetransductionef ciencyofthevirus[31].Furthermore,theVSV-G-pseudotypedbaculoviruscouldtransduceareportergeneintothecerebralcortexandtestisofmicebydirectinvivoinoculation[32].Inthisstudy,thepseudotypedbaculoviruswasgeneratedcarryingtheVSV-Ggeneunderthecontrolofthepolyhedronpromoter.

Asrecombinantbaculovirusesarebecomingapromisingtoolforgenetransferinvitroandinvivo,anef cientpromoterthatmediatesgeneexpressioninbothinsectcellsandmammaliancellswillfacilitatethedevelopmentofabaculovirus

vector-delivered

Fig.7.ThesplenocyteproliferationresponsesintheimmunizedmicemeasuredbyWST-8assay.SplenocyteswereseparatedfromtheimmunizedmiceandculturedwithCSFVfor80h.WST-8wasusedtomeasuretherelativeamountsofviablecells.TheOD450nmvaluesofsamplesweredeterminedandtheSIwascalculated.**P<0.01.

mammaliancellgeneexpression.Ourpreviousstudydemonstratedthattheie1promoterofWSSVisabaculovirus-independentshut-tlepromoterbetweeninsectcellsandmammaliancells[12].Inthisstudy,theVSV-G-modi edbaculoviruscarryingtheE2geneunderthecontroloftheie1promoterwasconstructed.TheresultsshowedthattheCSFVE2proteincanbeef cientlyexpressedanddeliveredinbothinsectandmammaliancells.Inperformingthetransduction,weinoculatedthecellswiththevirusinPBSfor8hat25 C.Thismethodcontrastedwithotherpublishedprotocolsinwhichtransductionoccurredforcertainhoursat37 Cusingthegrowthmedium(e.g.DMEM)[2,3,12,14].WefoundthatHeLacellscouldbereadilytransducedandef cientlyexpressthereporterpro-tein(datanotshown),consistentwiththepreviousreports[7,33].Theprotocolalsoavoidspotentialphysicalvirusinactivationdur-ingultracentrifugationandpuri cation,thusmakingiteasiertoconvertthisbaculovirus/mammaliancellsystemintoatoolforeukaryoticproteinproductiononalargerscale.

Toexplorethepotentialofthebaculovirus-basedvaccineagainstCSFV,theimmunogenicityofBacMam/G-ie1-E2wastheninvesti-gatedthroughintramuscularimmunizationofBALB/cmicewiththepuri edvirions.TheELISAresultsshowedthattheantibodylevelsinducedbythemiceimmunizedwiththeBacMam/G-ie1-E2viruswerehigherthanthoseoftherecombinantadenovirus-immunizedmice,whiletheEGFP-containingconstructinducedlow-levelnon-speci cantibodies.MoststudiesofCSFV-infectedanimalshaveconcentratedonthehumoralimmuneresponsetothevirusbydetectingandanalyzingtheactivityofserum-virusneutralizingantibodies[34,35]duetotheirobservedessentialroleinprotec-tiveimmunity.Tocon rmtheELISAresultswithafunctionalassay,SNTwascarriedouttodetecttheCSFV-speci chumoralimmuneresponseselicitedbyBacMam/G-ie1-E2inmice.Themiceinocu-latedwithBacMam/G-ie1-E2developedanti-CSFVNAbtitersthatwerehigherthanthoseoftherecombinantadenovirus-immunizedmice,whereastheNAbtiterselicitedbyBacMam/G-ie1-EGFPinmicewereconsistentlyundetectable.Thesigni cantantibodyresponsefromtherecombinantBacMamvirus-vaccinatedmiceindicatesthatthebaculovirusvectorcanef cientlyexpresshighimmunogeniclevelsoftheE2-proteininvivo.

Ithasbeenshownthatcell-mediatedimmunityalsoplaysanimportantroleinantiviralmechanismswherebothCD8+andCD4+T-cellsareessentialforthecontrolofCSFVinfections[36].Thereisastronglinkbetweenvirus-speci cCD8+T-cellfunctionandtheef ciencyofregulatoryCD4+helperT-cells[37].Therefore,weevaluatedthecell-mediatedimmuneresponses,speci callythelymphoproliferativeresponsestoCSFV,intheimmunizedmicebytwomethods(WST-8andCFSEassays)inthisstudy.WST-8isamod-i edMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide]forassessinglymphoproliferativeresponses.Theprinci-plesfortheWST-8andMTTassaysaresimilar,yettheWST-8assayisfaster,simpler,andmorereproducibleandsensitivethantheMTTassay.Signi cantlymphoproliferativeresponsesoftheimmu-nizedmiceweredetectedbythetwoassays.TheCFSEassayshowedthat,comparedwiththecontrolgroup,theCD8+andCD4+T-cellsproliferationelicitedbyimmunizationwithBacMam/G-ie1-E2andrAdV-E2immunizedmiceclearlyproliferatedwhenculturedwithCSFV(P<0.01),whichisconsistentwiththeresultsoftheWST-8assay.

Wechoseamousemodeltoinitiallytestourvaccinereportedherebecauseofthecommerciallyavailabletoolstoevaluatetheimmuneresponses.Additionally,mousemodelshavebeenwidelyusedtoevaluatecandidatevaccinesbeforeclinicaltrialsinnaturalhosts.ArecombinantbaculovirusdisplayingtheE2proteinofCSFVwasalsorecentlyevaluatedinamousemodelbyXuandLiu[38].However,weutilizedseveralapproacheswhichdistinguishourworkfromtheirstudy:(1)Weproducedandtestedabaculovirus-deliveredDNAvaccine,whereastheotherreportusedabaculovirus

150M.Lietal./ImmunologyLetters125(2009)145–150

producedsubunitvaccine.Ourstudyisthe rsttoevaluatetheCSFVE2immunogenexpressedfromtheBacMamvirusundertheie1promoter(ashuttlepromoterbetweenmammalianandinsectcells[12]).Incontrast,XuandLiuutilizedaconventionalrecombi-nantbaculoviruswiththetypicalinsectp10promotertoproducetheE2protein.(2)Weassessedcell-mediatedimmunityinducedbytheBacMamvirususingCFSEandWST-8assays,inadditiontoevaluatingtheantibodyresponses,whiletheotherreportonlyevaluatedtheneutralizingantibodiesinducedbytherecombinantbaculovirus.

Insummary,thepresentstudydemonstratedthattherecombi-nantpseudotypedbaculovirusmediatedef cientgenedeliveryandexpressionofCSFVE2proteininbothinsectcellsandmammaliancells.Importantly,weshowedthatdirectvaccinationwiththeBacMamvirusinducedCSFV-speci chumoralandcell-mediatedimmuneresponses.OurstudyprovidessupportingevidencefortheuseoftheBacMamvirusasavectorforgeneticimmunization.Consideringthesafetyandcost-effectivenessofproducinghigh-titerrecombinantbaculovirus,ourvaccinehasahighpotentialfordevelopmentasapracticalandeffectivevaccineagainstCSF.Tofur-thervalidateour ndings,wewillperformvaccinationstudiesandevaluateprotectionfromchallengeinpigsinthefuture.Acknowledgement

ThisworkwassupportedbyagrantfromTheNational863Pro-gramofChina(2006AA10A204).References

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