Immune responses induced by a BacMam virus expressing the E2(2)

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