Immune responses induced by a BacMam virus expressing the E2(3)

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).