B Cells and Antibody Play Critical Roles in the Immediate De(2)

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VOL.77,2003BCELLSANDANTIBODYPROTECTAGAINSTWNVINFECTION2579

inNewYorkstatein2000(14)andobtainedfromLauraKramer(Albany,N.Y.).TheinitialisolatewasharvestedafterinoculationofamosquitohomogenateintoVerocells(passage0).Allcellcultureandinvivostudiesusedastock(2 108PFU/ml)ofthisvirusthatwaspropagated(passage1)onceinC6/36cells.VirusesweredilutedinHanks’balancedsaltsolutionand1%heat-inactivatedfetalbovineserumforinjectionintomice.

Mouseexperiments.AllmiceusedintheseexperimentswerederivedfromtheinbredC57BL/6Jstrain(H-2b).Thewild-typeC57BL/6Jandcongenic MTmice(strainB6-Igh6-6tm1Cgn)werepurchasedfromJacksonLaboratories(BarHar-bor,Maine).ThecongenicRAG1mice(strainB6-RAG1tm1Mom)wereagiftfromE.Unanue,WashingtonUniversitySchoolofMedicine).Themiceusedforallstudieswerebetween8and12weeksofageandwereinoculatedsubcutaneouslywithWNVbyfootpadinjectionafteranesthetizationwithxylazineandketamine.MouseexperimentswereapprovedbyandperformedaccordingtotheguidelinesoftheWashingtonUniversitySchoolofMedicineAnimalSafetyCommittee.DifferencesinsurvivaltimesandoutcomewereanalyzedbyKaplan-Meieranal-ysisandthelogranktest.

Adoptivetransferandpassiveseraadministrationexperiments.Wild-typemicethatsurvivedprimaryWNVinfectionweremaintainedfor28days.Atthispoint,spleenswereharvestedandcellswerereleasedafterdissection.Erythro-cyteswereremovedafterFicollgradientcentrifugation,andtheremainingcellswerequantitatedandphenotyped.Basedonimmunostainingwithdirectlycon-jugatedMAbs,thesplenocytepopulationwasfoundtoconsistofBcells(45%),CD4 Tcells(30%),andCD8 Tcells(20%).Insomeexperiments,Bcellswereisolatedtogreaterthan95%purityafternegativeselectionwithantibody-coatedmagneticbeads(MiltenyiBiotechInc.,Auburn,Calif.).Splenocytes(10 106)orpuri edBcells(4 106)wereresuspendedinendotoxin-freephosphate-bufferedsaline(PBS)andinjectedintotheperitoneumofRAG1-de cientmice1daypriortoinfectionwithWNV.

Serumsampleswereisolatedfromnaïve,infected(day4postinfection),orimmune(day28postinfection)mice,heat-inactivatedfor30minat56°C,andstoredat 80°C.Analiquotwasreservedforbindingandneutralizationtiters(seebelow).Forpassivetransferexperiments,micewereadministered0.5mlofserumintraperitoneally1daypriortoandafterinoculationwith102PFUofWNV.

Quantitationofviralburdeninmice.Foranalysisofvirusintissuesofinfectedmice,organswererecoveredaftercardiacperfusionwithPBSanddissection,cooledonice,weighed,homogenizedusingaBead-Beaterapparatus,andti-tratedforvirusbyperformanceofaplaqueassayonBHK-21cellsasdescribedpreviously(13).Serumsampleswereobtainedfromwholebloodbyphlebotomyoftheaxillaryveinimmediatelypriortosacri ce.ViralRNAwasharvestedfromthawedaliquots(10 l)ofserumbyusingaQia-AmpviralRNArecoverykit(Qiagen,PaloAlto,Calif.).ForanalysisofviralRNAintissuesofinfectedmice,afterdissection,tissuesamplesweresnap-frozeninliquidnitrogen,thawedinaguanidiniumisothiocyanatedenaturingsolution,passedoverasilicabindingresin(RNEasykit;Qiagen),andelutedinRNase-freewater.ViralRNAwasquantitatedbyreal-time uorogenicreversetranscriptasePCR(RT-PCR)usinganABI7000sequencedetectionsystem(AppliedBiosystems,FosterCity,Calif.)accordingtoapreviouslypublishedprotocol(27).Foreachtissuesample,aparallelreal-timeRT-PCRwasperformedtoquantify18SrRNA(AppliedBio-systems)tocontrolfortheamountoftissueintheoriginalsample.StandardWNVandrRNAcurveswererunoneachplate.Sampleswereruninduplicate,andthedataobtainedfromseveraldifferentmiceforaparticularexperimentalconditionwereaveraged.ThelevelsofWNVRNAintissueswerenormalizedforrRNAcontentsothatthedatawereultimatelyexpressedasthegeneticequivalentsofWNVRNAperunitof18SrRNA.

Quantitationofantibodies.Thetiterofneutralizingantibodieswasdeter-minedbyastandardplaquereductionneutralizationassay(23).Experimentswereperformedinduplicate,andplaqueswerescoredvisually.Theresultswereplotted,andtheplaquereductionneutralizationtiterfor50%inhibition(PRNT50)wasdetermined.Todeterminethespeci cimmunoglobulinG(IgG)andIgMtiters,aWNVantigenenzyme-linkedimmunosorbentassay(ELISA)wasused(45).Brie y,WNV-infectedoruninfected(control)BHK21celllysateswereadsorbedtoMaxi-Sorpmicrotiterplates(NalgeNuncInternational,Roch-ester,N.Y.).Nonspeci cbindingsiteswereblockedafterincubationwithblock-ingbuffer(PBS,0.05%Tween20,3%bovineserumalbumin,3%horseserum)for1hat37°C.Plateswerethenincubatedwithserialdilutionsofheat-inacti-vatedserumfrominfectedmicefor1hat4°C.Afterextensivewashing,plateswereincubatedseriallywithbiotin-conjugatedgoatanti-mouseIgMorIgG(SigmaChemical)andhorseradishperoxidase-conjugatedstreptavidin(SigmaChemical)at4°Canddevelopedafteradditionoftetramethylbenzidinesubstrate(SigmaChemical).Opticaldensities(ODs)at450nmweredeterminedwithanautomaticELISAplatereader(MolecularDevices).TheODvalueforthe

controlantigenwassubtractedfromtheviralantigenwellstoobtaintheadjustedODvalueforeachsample.

Insomeexperiments,serumsamplesweredepletedofIgMbychemicalorimmunologicmeans.ChemicaldepletionofIgMwasperformedbytreatingserawith0.05M -mercaptoethanolinsalinefor1hat37°C(35,42).Immunologicdepletionwasperformedasfollows:serumsampleswereincubatedtwicewithanequalvolumeofanti-IgM-speci cagarosefor1hat4°C.Aftercentrifugation,thesupernatant(1/4dilutionoftheoriginalsample)wastitratedforthePRNT.Isotypedepletionwascon rmedbyELISA.

Immunohistochemistry.Immediatelyaftereuthanasia,organswereperfusedwith4%paraformaldehydeinPBS.Freshlyremovedbrainswereimmersedin4%paraformaldehydeinPBSovernight.Six-micrometer-thicktissuesectionswerecutwithamicrotomeafterparaf nembeddingandweredriedovernight.Deparaf nizationandantigenrecoverytreatmentswereperformed(31).Slideswereincubatedwithratanti-WNVorcontrolserum,biotinylatedgoatanti-ratIgG,streptavidin-conjugatedhorseradishperoxidase,anddiaminobenzidine.Slideswerethencounterstainedwithhematoxylinandreviewedbymicroscopy.

RESULTS

Tode nehowtheimmunesystemprotectsagainstdissem-inatedWNVinfection,amousemodelofinfectionwasdevel-opedinaninbredstrain(C57BL/6J)thathadanarrayofavailablegeneticde cienciesinindividualcellsandmediatorsintheimmunesystem.We rstcharacterizedinfectioninwild-typeC57BL/6Jmicewithalowpassage(P 1)viralisolatethatwasobtainedfromtheNewYorkstateepidemicin2000(14).TheuseofthisstrainwasimportantbecauseWNVstrainsisolatedfromNorthAmericaaremorevirulentthanthoseisolatedfromotherregionsoftheworld(4).Aperipheralrouteofinoculationviathefootpadwasutilizedtomorecloselymimicnaturalinfectionandtofacilitateananalysisofviralreplicationandspread.InfectionwiththeNewYorkstrainofWNVparalleledhumandisease.Sevento10daysaftersubcutaneousinoculation,micedevelopedevidenceofCNSinfection,withasubsetprogressingtoparalysisanddeath;immunohistochemistrydocumentedWNVinfectioninCNSneurons(Fig.1A).Duringthecourseofinfection,ap-proximately60%ofthewild-typemicedevelopedsigni cantlevelsofinfectiousvirusintheirbrains.Incontrast,asubset(approximately20to40%)ofwild-typemiceeitherneverdis-seminatedWNVintothebrainorhadviralloadsthatwerebelowthelevelofdetectionofourplaqueassay.Animalsthatsuccumbedtoinfectionshowedsimilarclinicalsigns24to48hpriortodeath,includingfurruf ing,weightloss,hunchbackposture,andlimbparalysis.Inadult8-to12-week-oldmice,acharacteristicdose-responsecurvewasnotobserved(Fig.2A).Althoughlowerdoses( 1PFU)wereassociatedwithbetteroutcome,therewasnosigni cantdifferenceinmortalityratesathigherdoses(35to45%mortalityat102to106PFU).Evenatthehighestinoculatingdose(107PFU),lessthan50%ofthewild-typeanimalssuccumbedtoinfection,althoughallanimalsdevelopedclinicalsignsandviraltissueburdenconsistentwithinfection(datanotshown).Thisdose-insensitivesurvivalcurveissimilartothatobservedafterinfectionofmicewithMurrayValleyencephalitisvirus(30),acloselyrelated avivirus.

ToassesstheroleoftheimmunesysteminlimitingWNVinfection,preliminaryexperimentswereperformedwithcon-genicRAG1micethatlackedbothBandTcells.RAG1micewereextremelyvulnerabletoinfection.Evenatthelowestdose(102PFU)tested,100%ofthemicerapidlysuccumbedtoinfection(P 0.0001)(Fig.2B).VirologicanalysisrevealedextremelyhightitersofWNV( 108PFU/goftissue)inthe

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