Immune responses induced by a BacMam virus expressing the E2(5)

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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