Immune responses induced by a BacMam virus expressing the E2

1、immune responses induced by a bacmam virus expressing the e2 immunologyletters125(2021)145150 contentslistsavailableatsciencedirect immunology letters journalhomepage:/locate / immuneresponsesinducedbyabacmamvirusexpressingthee2proteinofclassicalswinefevervirusinmice miaoli,yu-feiwang,yuwang,huigao,

2、nali,yuansun,bing-bingliang,hua-jiqiu divisionofswineinfectiousdiseases,nationalkeylaboratoryofveterinarybiotechnology,harbinveterinaryresearchinstitute,chineseacademyofagriculturalsciences,427maduanstreet,150001harbin,heilongjiang,china articleinfoabstract non-replicatingbaculovirus-mediatedgenetra

3、nsferintomammaliancellshasbeendevelopedasavac-cinestrategyagainstanumberofdiseasesinseveralanimalmodels.inthepresentstudy,thebacmamvector,abaculoviruspseudotypedwiththeglycoproteinfromvesicularstomatitisvirus,wasusedasarecombinantvectortoexpressclassicalswinefevervirus(csfv)e2proteinunderthecontrolo

4、ftheimmediateearly1(ie1)promoterfromshrimpwhitespotsyndromevirus.thee2genewasef cientlyexpressedinbothinsectandmammaliancells.intramuscularinjectionofmicewiththerecombinantbaculovirusresultedintheproductionofhigh-titersofcsfv-speci cneutralizingantibodies.speci clymphoproliferativeresponsestocsfvsti

5、mulationweredetectedinthesplenocytesoftheimmunizedmiceasdemonstratedbycfsestainingassayandwst-8assay.thisstudydemonstratesthatthebacmamvirusvectorcanef cientlyexpressthee2proteinandeffectivelyinduceimmuneresponsesagainstcsfv.thisisa rststepinthedemonstrationthatthepseudotypedbaculovirus-deliveredcsf

6、ve2genecanbeapotentialnon-replicatingvaccineagainstcsfvinfections. 2021elsevierb.v.allrightsreserved. articlehistory: received23january2021 receivedinrevisedform28june2021accepted1july2021 availableonline7july2021keywords: classicalswinefeverviruse2gene bacmamvirus recombinantbaculovirusimmunogenici

7、ty 1.introduction thebaculovirusexpressionsystemhasbeenextensivelyusedasvectorsforabundantexpressionofalargevarietyofforeignproteinsininsectcells.recently,recombinantbaculovirusvectorscontainingmammaliancell-activepromoterelements(alsoknownasbacmamviruses)havebeenusedsuccessfullyforgenedeliv-eryinto

8、mammaliancelllineswithoutapparentviralreplication1.recentstudieshavedemonstratedthatbacmamvirusescanbeusednotonlyforef cientgenetransductionintomammaliancellsinvitro2,3butalsoforgenetransductioninvivo4,5.themajoradvantageofthebacmamvirusisthatitshowsef cientgenetransductionintoawidevarietyofcellline

9、sbydirectinfection.numerouseffortshavebeenmadetoharnessthebaculovirusasavectorforgenetherapyandvaccinedevelopment.theuseofbac-ulovirusasavectorforvaccinationwasinitiallydescribedbyaokietal.6,whodemonstratedthatintramuscularinjectionofmicewitharecombinantbaculovirusexpressingthepseudorabiesvirusgbpro

10、teincouldelicitedameasurablehumoralresponse.further-more,severalgroupshavedemonstratedthatdirectvaccinationwithrecombinantbaculovirusbyintramuscular,intraperitonealorintranasalinoculationcaninducetheproductionofhumoral correspondingauthor.tel.:+8645185935041;fax:+8645185935041.e-mailaddress:huajiqiu

11、(h.-j.qiu).0165-2478/$seefrontmatter2021elsevierb.v.allrightsreserved.doi:10.1016/j.imlet.2021.07.001 andcell-mediatedimmunityagainstvariousantigens79.morerecently,wangetal.10reportedthatanimmuneresponsetothegp5andmproteinsofporcinereproductiveandrespiratorysyn-dromeviruswaseliciteduponvaccinationwi

12、thabaculovirusvectorexpressingthevirusstructuralcomponent. classicalswinefever(csf),whichiscausedbyclassicalswinefevervirus(csfv),isafatalswinediseaseandoneofdiseaseslistedbytheworldorganizationforanimalhealth(oie).thediseaseisendemicorepidemicinmanycountriesandcausesgreateconomiclosstotheswineindus

13、tryworldwide.thecsfvgenomecontainsasingle,largeopenreadingframe(orf)encodingapolyproteinthat,uponproteolyticprocessing,givesrisetofourstructuralpro-teinsandeightnon-structuralproteins11.thee2proteinisanenvelopeglycoprotein,whichisresponsibleforelicitingneutraliz-ingantibodiesininfectedanimalsandthus

14、protectingpigsagainstvirulentchallenge. atpresent,themostfrequentlyusedvaccineisthechineselapinizedattenuatedvaccine,i.e.c-strain,whichcaninducecom-pleteprotectionagainstcsf.however,amajordisadvantageofthisvaccineisthattheantibodiesinducedbythevaccinecannotbedis-tinguishedfromthoseinducedbywild-type

15、csfvinfections.itisimportanttodevelopnovelcsfvvaccinescapableofinducingrapidprotectioncombinedwiththeabilitytodifferentiateinfectedfromvaccinatedanimals. ourpreviousstudiesdemonstratedthatthebacmamviruswithanegfpreportergeneunderthecontroloftheshrimpwhitespotsyndromevirus(wssv)immediateearly1(ie1)pr

16、omoterexhib- 146m.lietal./immunologyletters125(2021)145150 itedhightransductionef ciencyandhigh-levelexpressionofthereporterproteininmammaliancells12.inthispresentreport,weexploredthepotentialofthisvectorfordevelopingavaccineagainstcsfvbyconstructingtherecombinantbaculoviruscontain-ingthepolyhedrinp

17、romoter-controlledvesicularstomatitisvirusglycoprotein(vsv-g)expressioncassetteandwssvie1promoter-controllede2expressioncassette.wetestedthegeneexpressionofthisvectorinbothinsectandmammaliancells.furthermore,weinvestigatedtheabilityofthepuri edrecombinantbaculovirustostimulatehumoralandcell-mediated

18、immunityagainstcsfvinmicebyintramuscularinjection.ourresultsindicatethatthebacmamviruscanbeusedtodevelopanewgenerationofnon-replicativevectorvaccineagainstcsfvinfections.2.materialsandmethods2.1.virusandcells thecsfvshimenstrainusedinthisstudywasmaintainedintheharbinveterinaryresearchinstitute.recom

19、binantadenovirusradv-e2expressingthecsfve2protein13wasconstructedbyourlaboratory.sf9cellswereculturedat27 cingracesmedia(gibco,grandisland,usa)supplementedwith10%fetalbovineserum(fbs).helaandpk-15cellswereculturedindulbeccosmodi edeaglesmedium(dmem,gibco,grandisland,usa)sup-plementedwith10%heat-inac

20、tivatedfbs,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-pcrwi

21、ththeprimerpair5 -gccggatccatgcggc-tagcctgcaaggaagactac-3 and5 -attgcggccgcctacacatc-caggtcaaaccagtattgatactc-3 .themodi edbaculovirustransfervectorwasconstructedbyinsertingthevsv-ggeneunderthecontrolofthepolyhedronpromoter(pph)inthepfastbachtbvector(invitrogen,sandiego,usa). theegfpgenewasexcisedfr

22、ompegfp-n1(invitrogen,sandiego,ca,usa)andthewssvie1promoterwasdescribedpreviously12.togeneratetherecombinantbaculovirus,thewssvie1promoter-controllede2expressioncassettewasinsertedintothemodi edbaculovirustransfervectorandintegratedintothebaculovirusgenomewithindh10bactmthroughsite-speci ctransposit

23、ionaccordingtothemanufacturersprotocolprovidedwiththebac-to-bacsystem(invitrogen),resultinginbacmam/g-ie1-e2.recombinantbaculovirusbacmam/g-ie1-egfpwasgeneratedasdescribedabove.theviruswasfurtherampli edbypropagationinsf9cells.viruspuri cationwasperformedbyultracentrifugationasdescribedbefore7.theti

24、terofviruswasdeterminedbyastandardviralplaqueassayafterpassageinsf9cells14. 2.3.identi cationofrecombinantbaculovirus therecombinantbaculoviruswasidenti edbypcrusingthem13universalprimer(invitrogen),andtheexpressionwasfurthercon rmedbyimmuno uorescenceassay(ifa)andwesternblot.2.3.1.ifa sf9cellsgrown

25、insix-wellplateswereinfectedwiththebac-ulovirus,bacmam/g-ie1-e2.seventy-twohourspost-infection,thesupernatantwasharvestedandthecellswerewashedwith0.1m phosphate-bufferedsaline(pbs,ph7.4).thecellswerethenresus-pendedinpbsand xedonaglassslidewithcold100%acetonefor8min.the xedcellswerethenincubatedwith

26、ananti-e2mono-clonalantibody(mab)(6e10)15for1hat37 c.fitc-conjugatedgoatanti-mouseigg(sigma,st.louis,usa),atadilutionof1:128,wassubsequentlyincubatedwiththe xedcellsfor1hasthedetec-torantibody.the uorescencesignalwasdetectedwithaninverted uorescencemicroscope(nikon,japan). 2.3.2.westernblot baculovi

27、rus-infectedsf9celllysatesweresubjectedto10%sdspolyacrylamidegelelectrophoresis(page)andwesternblotanalysisusinganti-e2mabhq0616,andbaculovirus-expressedrecombinante2protein17servedaspositivecontrol.theprepara-tionandrunningofgels,transferofproteinsfromsdspagegelstonitrocellulose lters,andimmunoblot

28、tingwereperformedaccord-ingtostandardprotocols13.2.4.transductionofmammaliancells helacellswereseededonto24-wellplatesandallowedtoincu-bateat37 covernight.priortotransduction,thespentmediumwasremovedandthecellswerewashedwith0.1mpbs(ph7.4).thecellswerethenincubatedwiththerecombinantbaculovirusatamult

29、iplicityofinfection(moi)of100in0.5mlofpbs.theplateswereplacedonarockingshakerfor8hat25 c18.aftertheincu-bationperiod,thevirussolutionswereaspiratedandthecellswerewashedwithpbsagain.thewellswerereplenishedwith2mloffreshdmemcontaining10%fbsandincubatedat37 c.afterincu-bationat37 cfor48h,thecellswere x

30、edontheplatewithcold33%acetonefor30minatroomtemperatureforanalysisbyifaasdescribedabove.cellsampleswerealsocollectedforanalysisbywesternblotassayasdescribedabove.2.5.immunizationofmice seven-week-oldfemalebalb/cmice(purchasedfromthelabo-ratoryanimalcenterofharbinveterinaryresearchinstitute)wererando

31、mlydividedintofourgroups(withsevenmiceineachgroup).onegroupwasinjectedintramuscularly(i.m.)with108plaqueformingunits(pfu)ofbacmam/g-ie1-e2in500 lofpbs.themiceimmunizedi.m.with108tcid50radv-e213wereusedasposi-tivecontrols,whilebacmam/g-ie1-egfpvaccinatedmiceservedasnegativecontrols.thefourthgrouprema

32、inedunimmunized.atfourweeksfollowingtheprimeimmunization,themicewereboostedwiththesamedoseoftheinocula.2.6.indirectelisa serumsamplesfromtheimmunizedmicewerecollectedweeklybeforeandafterimmunization.theseraweretestedforthepres-enceofcsfv-speci cantibodylevelsbyanindirectelisaasdescribedpreviously19.

33、 2.7.serum-virusneutralizationtest(snt) serumsampleswerecollectedat0,3and6weeksfollowingtheprimeimmunization,andheat-inactivatedfor45minat56 cbeforetesting.thesntwascarriedoutin96-wellcultureplatesasdescribedelsewhere20.brie y,two-foldseriallydilutedseraweremixedwithanequalvolumeof100tcid50ofcsfvshi

34、menstrainandincubatedat37 cfor1h.subsequently,eachoftheserumvirusmixtureswasaddedtoacon uentmonolayerofpk-15cellsin96-wellcultureplates.theplateswereplacedinamoistchamberandincubatedina5%co2incubatorat37 cfor1h.after23days,thecultureplatewas xedfor20minwithcoldabsolute m.lietal./immunologyletters125

35、(2021)145150 147 fig.1.schematicrepresentationoftherecombinanttransfervectorspfb-g-ie1-e2andpfb-g-ie1-egfp.thedesiredvsv-gore2expressioncassettewasinsertedwithinthepolyhedrinlocusthroughsite-speci ctranspositionemployingthebac-to-bacsystem.ie1,wssvie1promoter;pph,polyhedrinpromoter. ethylalcohol.aft

36、erextensivewashingwithpbs,theexpressionofcsfvwasdetectedbyifawithmab6e10,followedbyincubationwithfitc-conjugatedgoatanti-mouseigg(sigma,st.louis,usa).thecsfvneutralizingantibody(nab)titersweredeterminedandexpressedasthereciprocalofthehighestdilutionatwhichinfectionofthepk-15cellswasinhibitedin50%oft

37、heculturewells.2.8.lymphoproliferationassays threeweeksaftertheboosterimmunization,cellsuspensionswerepreparedfromthespleensoftheimmunizedmicebypress-ingthrougha nesieveintorpmi1640medium(gibco,grandisland,usa).thecellswerethentreatedwithredbloodcelllysingbuffer(155mmnh4cl,10mmnahco3,0.1mmedta;ph7.4

38、).csfvshimenstrainpropagatedinpk-15cellswasreleasedafterthreefreeze-thawcycles,andthentheviralsuspensionwascentrifugedat2000gfor15min.thesupernatantwascollectedandvirionswerepuri edbydifferentialcentrifugation.thelymphoprolifera-tionassaybasedoncarboxy uoresceinsuccinimidylester(cfse)stainingwasperf

39、ormedusingpuri edcsfvvirionsasstimula-torsasdescribedpreviously21.thelymphoproliferationassaybasedonwst-8wasalsoperformedasdescribedpreviously22,23byusingthecellcountingkit-8(dojindomoleculartechnologies,inc.,japan),whichmeasurestheamountofwater-solubleformazanproducedbywst-8.3.results 3.1.construct

40、ionandidenti cationofrecombinantbaculovirusbacmam/g-ie1-e2 toexplorethepotentialofabaculovirus-basedvaccineagainstcsfv,weconstructedarecombinantbaculoviruscontainingane2-expressioncassetteunderthecontrolofwssvie1promoter,namedbacmam/g-ie1-e2(fig.1).infectionofinsectsf9cellswithbacmam/g-ie1-e2resulte

41、dinextensivecellcellfusion.thephe-notypewasduetothehigh-levelexpressionofvsv-gproteinwithmembrane-fusionactivity(datanotshown).expressionofthee2glycoproteinduetoinfectionwiththebacmamviruswasdetectedbyifawithanti-e2mab6e1015(fig.2a).theexpressionwasfur-thercon rmedbywesternblotanalysisofthetotalcell

42、ularextractwithanti-e2mabhq0616.aproteinofapproximately53kdawasdetectedinbacmam/g-ie1-e2infectedsf9celllysatesbutnotinnegativecontrolsamples(fig.3a). 3.2.expressionofrecombinantbaculovirusbacmam/g-ie1-e2inmammaliancells totestwhethertheobtainedbaculoviruscanexpressthee2geneinnon-insectcelllines,wetr

43、ansducedthemammalianhelacellswithbacmam/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

44、(b)byifausingmab6e10.thecellswere xedfor72hor48hafterinfectionortransductionandanalyzedbyifausingtheprimaryantibody6e10andsecondaryfitc-conjugatedgoatanti-mouseigg.uninfectedsf9andhelacellsservedasnegativecontrols. wasfurthercon rmedbywesternblotanalysisofthetotalcellularextractusinganti-e2mabhq0616

45、(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.anindirectelisawasusedtomonito

46、rthee2-speci cantibodylevels.theresultsshowedthatmiceimmunizedwithbacmam/g-ie1-e2orradv-e2developedsigni cantantibodyresponsesasdetectedbyelisainwhichpuri edbaculovirus-expressede2protein17wascoatedasantigen.meanwhilethemiceimmunizedwithbacmam/g-ie1-egfpdidnotseroconvertwithjustanon-speci cantibodyr

47、esponsebelowthreshold,whilethenon-immunizedmiceshowedonlyabackgroundantibodylevel.afurtherincreaseinantibodylevelswasobservedoneweekaftertheboosterimmu-nizationwithbacmam/g-ie1-e2.theserumantibodylevelsinthebacmam/g-ie1-e2-immunizedgroupsweresigni cantlyhigherthanthoseintheradv-e2-immunizedgroupat5a

48、nd6weekspost-immunization(p0.05)(fig.4). todetectcsfv-speci cnabtiters,sntwasperformedat0,3and6weeksfollowingprimeimmunization.theresultsshowedthatmiceimmunizedwithbacmam/g-ie1-e2orradv-e2developeddetectablenabtitersat3weeksfollowingprimeimmunization.afurtherincreaseinnabantibodytiterswasobservedat6

49、weeksfollowingprimeimmunization;whereasthemiceimmunizedwithbacmam/g-ie1-egfpdidnotdevelopadetectableantibodyresponseagainstcsfvevenat6weeksfollowingprimeimmuniza-tion.thecfsv-speci cneutralizinglevelinthebacmam/g-ie1-e2groupwassigni cantlyhigherthanthatintheradv-e2-immunizedgroup6weeksfollowingprime

50、immunization(p0.05)(fig.5). 148m.lietal./immunologyletters125(2021) 145150 fig.3.westernblotanalysisofthee2expressioninbacmam/g-ie1-e2-infectedsf9cells(a)orbacmam/g-ie1-e2-transducedhelacells(b)usinganti-e2mabhq0622.lanes1and6:prestainedproteinmolecularweightmarker(jingmeibiotech,beijing,china);lane

51、s2and10:baculovirus-expressedrecombinante2protein17aspositivecontrol;lane3:bacmam/g-ie1-e2infectedsf9cells;lane4:bacmam/g-ie1-egfpinfectedsf9cells;lane5:normalsf9cellsasnegativecontrol.(b)lane7:normalhelacellsservedasnegativecontrol;lane8:totalcellularextractsfromhelacellstransducedwithbacmam/g-ie1-

52、egfp;lane9:totalcellularextractsfromhelacellstransducedwithbacmam/g-ie1-e2. 3.4.cell-mediatedimmunityelicitedbybacmam/g-ie1-e2inmice tocharacterizethecell-mediatedimmuneresponses,themiceweresacri cedandthesplenocyteswereisolatedat7weeksfollowingprimeimmunizationwithbacmam/g-ie1-e2,bacmam/g-ie1-egfpo

53、rradv-e2.afterstainingwithcfse,thecellswerestimulatedwithcsfv(10 g/ml,200 leachwell).afterincubatingfor5days,the uorescenceintensitiesofthecellsweredetectedby owcytometry.theresultsshowedthatthecellsobtainedfrombacmam/paredwiththecontrolgroup,percentagesofproliferatedcd4+tandcd8+t-cellsweresigni can

54、tlyhigherforthegroupsimmunizedwithbacmam/g-ie1-e2(p0.01)andradv-e2(p0.01).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%,respectivel

55、y(fig.6). atthesametime,thelymphoproliferativeresponseswerealsomeasuredusingwst-8.splenocyteswerestimulatedwithcsfvfor80h,andwst-8solutionwasadded.theod450nmofeachsamplewasmeasuredandthestimulationindex(si)wascalculatedusingthefollowingformula:si=(meanod450nm of fig.5.detectionofcsfv-speci cserumneu

56、tralizingantibodylevelsinducedinthevaccinatedmice.threegroupsofsevenmicewereeachintramuscularlyinjectedtwicewithbacmam/g-ie1-e2,bacmam/g-ie1-egfporradv-e2,respectively.pooledserumsamplesfromeachgroupweretestedat0,3and6weeksfollowingprimeimmunizationbyserum-virusneutralizationtesttodetermineantibodyt

57、iterstothecsfvshimenstrain.*p0.05. csfv-stimulatedcells)/(meanod450nmofunstimulatedcells).thesiofthebacmam/g-ie1-e2group(p0.01)andradv-e2group(p0.01)wassigni cantlyhigherthanthatofthecontrolgroup,whiletherewerenosigni cantdifferencesbetweenthetwoformergroups(fig.7).4.discussion thebacmamviruses,carr

58、yingtargetgenesunderthecontrolofanappropriatemammaliancell-activepromoter,haveprovidedanalternativeandattractivechoiceforgenedeliveryduetotheirsafetyandimprovedef cacy24.thebacmamvirusesprovideuswithapromisingplatformtostudygenefunctionsandtodevelopnon-replicatingvectorvaccinesandgenetherapystrategies2527.baculovirusvectorsaresaferthanotherconventionalviralvectors(suchasadenovirus),becausetheydonotreplicateinmammaliancellsandhavenomarkedcellulartoxicity.furthermore,asagenetransfervector,thebaculovirusretainssomeaddi