Immune responses induced by a BacMam virus expressing the E2(4)

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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.