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IF=2.2181.IntroductionMerozoitesurfaceprotein-1ofmalariaparasites,presentonthesurfaceofmerozoiteandplayingakeyroleintheinvasionprocess,isoneoftheleadingvaccinetargetsforbothP.falciparumandP.vivaxmalaria.MSP-1occursasa200kDaprecursorandundergoesstep-wiseproteolyticprocessingresultinginaglycosyl-phosphatidylinositol-anchored42kDaproteinfragment(MSP-142)onthesurfaceoffreemerozoite.AlargenumberofstudieshaveshowntheprotectivepotentialofMSP-1fragments(42and19kDa)ofP.falci-parumandP.yoelii.DNAvaccineshaveemergedasapromisingapproachforavarietyofinfectiousagentsincludingmalariaparasites.DNAbasedvaccinesareparticularlyimportantfordevelopingmultistage/multiantigenvaccinesforcomplexparasiteslikemalariatherebyinducingspecificandprotectiveimmunityagainstdifferentlifecyclestages(pre-erythrocytic,erythrocyticandsexualstages)ofmalaria.TheadvantagesofDNAvaccinesaretheeaseoftheirproductionandtheirabilitytoinduceresponseswithoutanyexogenousadjuvant,whichisanabsoluterequirementforproteinbasedvaccineformulations.DNAvaccineshavebeenshowntoinducestronghumoralandcellularresponsestomalarialantigensinimmunized.However,clinicaltrialsofthesecandidatevaccineswhenusedaloneorinrepeatedhomologousboostingregimenshavebeendisappointing,withshortlivedlowlevelsofinducedspecificT-cellsresponses.Sequentialimmunizationwithdifferentplasmids/vectorsknownasheterologousprime-boostingappearstobeabetterapproachandhasbeenshowntoinduceenhancedandpersistentlevelsofantibodyandcellmediatedimmuneresponsesagainstmalaria.Inheterologousprime-boostingstrategytheprimingwasdonewithDNAfollowedbytheboosterimmunizationwitharecombinantprotein,arecombinantviralvaccine,orexposuretotheliveorganism.InthepresentstudywehaveevaluatedtheimmunogenicityofaDNAvaccineencodingMSP-142ofP.vivaxusingprime-boostingstrategyinBALB/cmice.WehavealsocomparedtheimmunogenicityofPvMSP-142recombinantproteininBALB/cmice.2.MaterialsandmethodsDNAvaccineconstructsencodingP.vivaxMSP-142InvitrotransfectionofmammaliancellswithP.vivaxMSP-142–pcDNA3.1plasmidMonoclonalantibodiesagainstP.vivaxMSP-142LocalizationofP.vivaxMSP-142byindirectimmunofluorescenceSDS–PAGEandimmunoblottingPurificationofrecombinantP.vivaxMSP-142expressedinE.coliPurificationofplasmidforimmunizationImmunizationstudiesinmiceEnzymelinkedimmunosorbentassayDeterminationofIgGsubclassesCellproliferationassayDetectionofcytokinesIFN-γ,IL-2andIL-12byELISA3.Results

3.1.ConstructionofaP.vivaxmerozoitesurfaceprotein-142-pcDNA3.1plasmidDNAvaccineanditsinvitroexpressioninCOS1cells3.2.AntibodyresponsesinimmunemiceagainstP.vivaxmerozoitesurfaceprotein-142-pcDNA3.1DNAvaccineconstructandrecombinantPvMSP-142protein3.3.MeasurementofP.vivaxmerozoitesurfaceprotein-142specificIgGisotypesinimmunemicesera3.4.InvitroTcellresponsesagainstP.vivaxmerozoitesurfaceprotein-1423.5.InvitrocytokinesproductionagainstP.vivaxmerozoitesurfaceprotein-1424.DiscussionsInthepresentstudy,wehavepreparedaplasmidDNAconstructencodingP.vivaxPvMSP-142antigen.TheexpressionofPvMSP-142proteinwascheckedbyinvitrotransfectionofCOS-1mammaliancellswithPvMSP-142-pcDNA3.1plasmidDNAconstruct.ImmunogenicityofPvMSP-142DNAvaccineconstructwasinvestigatedbyprimingtheBALB/cmiceeitherwithPvMSP-142plasmidDNAorwithrecombinantPvMSP-142proteinandboostingwithrecombinantPvMSP-142protein.InthepresentstudywehaveconductedDNAvaccinationforP.vivaxMSP-142andcomparedtheimmunogenicityofDNAvaccineconstructwithcorrespondingproteinvaccinationinBALB/cmice.ImmunizationofmicewithrecombinantPvMSP-142resultedintheinductionofhightitresofanti-rPvMSP-142antibodiesthanthatofDNAimmunization.However,theantibodytitresobservedinPvMSP-142plasmidDNAimmunizedmiceweresignificantlyhigherthanthemiceinthecontrolgroup(vectorimmunized).DNAvaccineplasmidsencodingmerozoitesurfaceprotein-1frommurinemalariahasbeenshowntoprovideprotectionagainstmalariainfection.Theimportanceofheterologousprime-booststrategyinmalariainfectionhasbeendemonstratedbyanumberofstudies.Inthepresentstudy,primingwithP.vivaxMSP-142plasmidDNAandboostingwithrecombinantPvMSP-142proteinexhibitedasignificantincreaseintheantibodyresponsesinDNA/ProteingroupcomparedtoDNA/DNAgroupasitwasevidentoncomparingthe1ODtitreaswellasendpointtitrevaluesofdifferentimmunizedgroups.TheseresultssuggestthattheprimingwithplasmidDNAandboostingwithrecombinantproteinisessentialfortheenhancementofimmuneresponses.However,theantibodytitresinProtein/ProteingroupweresignificantlyhigherthanDNA/Proteingroup.ThismaybeattributedtotheuseofFCA/IFAforproteinimmunizationwhichresultedinhighlevelsofantibodyinProtein/Proteingroup.Previousstudieshaveshownthatcellmediatedimmuneresponses,inadditiontoantibodyresponses,arerequiredforprotectionagainstmalaria.PresentstudyonmeasuringTcellresponsesinmiceafterDNAimmunizationandproteinboostingexhibitedthatTcellproliferationwassignificantlyenhancedinDNA/ProteinandProtein/Proteingroups.Elevatedlevelsofcytokines(IL-2,IL-12,andIFN-γ)andhightitresofantigenspecificIgGisotypes(IgG1>IgG2b>IgG2a)indicatedthatbothTh1andTh2subsetsofThelpercellsmaybeproducedduringimmunizationofmiceinthepresentstudy.Thistypeofresponses,i.e.,theactivationofbothTh1andTh2cells,isconsideredidealforablood-stagevaccine.OurfindingsonDNAprimeandproteinboostinghavealsoindicatedthatamongtheDNAimmunizedgroups,primingwithDNAandboostingwithproteinclearlyelevatedthelevelsofallthecytokinesinDNA/ProteingroupsuggestingthatDNAcanprimethemicebutitselfisnotsufficientenoughtoenhancetheimmuneresponses.5.ConclusionsInthepresentstudy,wehavecloned42kDafragmentofP.vivaxmerozoitesurfaceprotein-1(PvMSP-142)ineukaryoticexpressionvectorpcDNA3.1.WecheckedtheinvitroexpressionofPvMSP-142-pcDNA3.1inCOS-1cellline.Indirectfluorescentassayandwesternblottingconfirmed