Rabies virus nucleoprotein as a carrier for

RabiesvirusnucleoproteinasacarrierforforeignantigensMartinL.Koser*,JamesP.McGettigan*,GeneS.Tan†,MaryEllenSmith†,HilaryKoprowski†,BernhardDietzschold†,andMatthiasJ.Schnell*‡Departmentsof*BiochemistryandMolecularPharmacologyand†Microbiology,JeffersonMedicalCollege,ThomasJeffersonUniversity,Philadelphia,PA19107-5541ContributedbyHilaryKoprowski,May18,2004Rabiesvirus(RV)nucleoprotein(N)tightlyencapsidatesthegenomicandantigenomicRNAofRVtoformtheviralribonucle-oprotein(RNP)complex.Antigens,suchasN,presentedinahighlyorganizedstructurearesufficientandevendesirabletoactivateBcellstoproliferateandproduceantibodies.Inadditiontoactivat-ingBcellstoproliferate,ithasbeenshownthatRVNintheRNPcomplexinducespotentThelpercellresponsesresultinginlong-lastingandstronghumoralimmuneresponsesagainstRV.ThepossibilitytosystematicallyincorporateforeigngenesintothegenomeofRVandproducearecombinantvirusallowsustoexaminewhethertheimmunogenicityofforeignantigenscanbeenhancedbyincorporationintotheRVRNPstructure.TotestthishypothesisweconstructedarecombinantRVexpressingaRVN-GFPfusionprotein.ThechimericN-GFPfusionproteinwasefficientlyexpressedandincorporatedintoRVRNPandvirions.Moreover,therecombinantRNPinducesastronghumoralimmuneresponseagainstGFPinmice.Incontrast,miceinoculatedwithGFPaloneoracombinationofwild-typeRVRNPsandGFPdidnottriggeranyGFP-specifichumoralresponsesusingthesameimmu-nizationschedule.ThesedataindicatetheusefulnessofRV-basedvectorsaskilledvaccinesagainstotherinfectiousdiseases.nucleocapsid
fusionprotein
vaccineRabiesvirus(RV)isanonsegmentednegative-strandRNAviruswithintheRhabdoviridaefamily(1).TheRVgenomeis
12kbinsizeandencodesfivemonocistronicmRNAsencodingthenucleocapsidprotein(N),phosphoprotein(P),matrixprotein(M),thetransmembraneglycoprotein(G),andtheviralRNA-dependentRNApolymerase(L)(2).TheNefficientlyandspecificallyencapsidatestheviralRNAtoformtheribonucleoprotein(RNP)complex,whichprovidesthetem-plateforRNAtranscriptionandreplicationbytheviralpoly-merasecomplex,whichincludesthePandL(3,4).TheRVMbridgestheRNPwiththecytoplasmicdomainofRVG,whichisembeddedinthehostcell-derivedviralmembrane(5).TheextracellulardomainoftheRVGmediatesinfectionofthehostcell(6).Recombinantlive-viralvectorsexpressingforeignantigensefficientlyinducepotentcellularandhumoralimmuneresponsesagainsttheexpressedantigens.ThepossibilitythatRVcanbeusedasanexpressionvectorwasshownearlierwiththemodelgenechloramphenicolacetyltransferase,andtheresultsfromthesestudiesindicatedthatforeigngenescanbeexpressedstablyinRV(7).ThepossibilitythatRVcouldbeusedasanHIV-1vaccinewastested,anditwasshowninseveralstudiesthattheexpressionofHIV-1EnvorGagresultedinpotentimmuneresponsesdirectedagainstHIV-1(8–10).Inaddition,wedem-onstratedthatRVvaccinevectorstoleratelarge,multiplefor-eigngenes,upto6.5kb,andthatlive-viralRVvectorswithaverylowpathogenicpotentialcanbeconstructed(11).Moreover,ourresultshaveindicatedthatkilledRVvirionscontainingforeignglycoproteinssuchashepatitisCE2orHIV-1gp160inducestrongBcellresponsesinasmallanimalmodel(12,13).PartoftheimmuneprotectionfrompathogensrequiresatleastastrongBcell-mediatedhumoralresponse.Ithasbeenpreviouslyshownwithdifferentvirus-basedexpressionsystemsthatavarietyofviralparticlesorvirus-likeparticlescanserveascarriersofforeignBcellepitopeswithintheviralcapsidorenvelopeproteinoftheviralparticle.TheseepitopecarriersresultedinstrongBcellresponsesagainsttheexpressedforeignepitopesindicatingausefulnessofcarrierproteins(14–20).However,mostoftheseapproachesarerestrictedtoincorpora-tionofonlyshortpeptidesthatdefineepitopesduetotherestrictedcloningcapacitywithinthecarrierproteins.Wehy-pothesizedthatRVNasacarrierproteinforforeignepitopesmightcircumventthisproblem.Alargebodyofevidencesug-geststhatthefollowingfeatures,oracombinationofthem,areparticularlyimportanttoinducestronghumoralresponsesagainstcertainepitopesorantigens.(i)Epitopesorantigenspresentedinthecontextofatightly,highlyorganizedstructureactivateBcellstoproliferateandinduceIgMproduction(21,22).(ii)AstrongTcellantigenlinkedtoaBcellepitopeenhancestheBcell-specificimmuneresponse.Aproposthisstudy,theRVNisabletoprimeTcellsandinduceRVN-specificantibodies(23,24).(iii)Becauseantigen-presentingcellsplayanimportantroleinthedevelopmentofimmunity,thestronghumoralimmuneresponseagainstRVNsuggeststhatRVNmustbeefficientlypresentedbyantigen-presentingcells(24).Inaddition,thestabilityoftheantigencanbeimportant.RVRNPishighlyresistanttoproteolyticenzymes(25)andthereforeshouldalsosustainthepresentationofaforeignantigentotheimmunesystemoveralongtime.GFPwasusedinthepresentstudyasamodelantigeninoursystemtoshowthatRVNcanserveasacarrierofawholeforeignprotein.TheN-GFPfusionproteinwasefficientlyin-corporatedintoRVRNPsandvirions.Asshownbelow,humoralresponseswereonlydetectedwhenGFPwaspresentedbyRVRNPs,andnoBcellresponsesweredetectedbyrecombinantGFPalone,evenafterthreeimmunizationswithrecombinantGFP.MaterialsandMethodsPlasmidConstructionandGenerationofRecombinantViruses.TheplasmidspBNSP,pSPBN,andpSPBN-333havebeendescribedprevio