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Immunogenicity of Lyophilized MVA Vaccine for HIV-1 in Mice Model

By Kathy Flores,2014-06-02 02:21
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Immunogenicity of Lyophilized MVA Vaccine for HIV-1 in Mice Modelof,in,IN,MVA,for,HIV1,Mice,Model,mice,model

    Immunogenicity of Lyophilized MVA

    Vaccine for HIV-1 in Mice Model CHEM.RES.CHlNESEU.

    2007,23(3),329332

    ArticleID1005-9040(2007)-03329-04

    Availableonlineatwww.sciencedirect.com

    

    ScienceDirect

    H?.1inMiceModel

    ZHANGYizhe,JIANGChunlai,YUXianghui,LOUChaoping,ZHAODong

    hai,

    WUYongge,JINYinghua,LIUChengshanandKONGWei

    VaccineResearchCenter,CollegeofLifeScience,JilinUniversity,Changchun130021,P.R.China

    ReceivedFeb.8,2006

    HighlyattenuatedmodifiedvacciniaAnkara(MVA)issensitivetorepeatfreeze

    thawcycleandeasytoloseac-

    tivity.InordertomaketheactivityofMVAvaccineremainstableduringitsmanufacturing,storage,andadministra

    tion,thelyophilizationasagoodoptioncouldberesortedto;throughscreening,therightstabilizercompositionand

    itsproductionprocedurewereobtained.Thefinalmoisturecontentoffreezing

    driedrecombinantMVAHIVvaccine

    waslowerthan3%.ItCallbereconstitutedquicklyandshowsreSularphysicalappearanceandstablepotency./nv/vo

    functionalexperiment,miceweredividedrandomlyintotheliquidvaccinationgroup,thelyophilizedvaccination

group,andthecontrolgroup.HavingbeenDNAvaccinepriming,themicewereboostedtllado

    seof10pfuMVA

    HIVvaccine.whichproducedindistinguishableantibodytiterandcytotoxicT

    lymphocyte(CTL)levelcomparedtll

    thoseofliquidvaccinationgroup(P>0.05).TheseresultsdemonstratethatlyophilizedM

    VAvaccineCallinduce

    highimmunogenicityinmice.

    KeywordsRecombinantMVAVaccine;Lyophilize;Immunogenicity;Stability

    Introduction

    HighlyattenuatedmodifiedvacciniaAnkara

    (MVA)isgeneratedfromvacciniaAnkarastraincell viabilityassayv/ahundredsofserialpassagesinchick embryofibroblasts(CEF)cells.

    MVAlosesasubstantialpartof

    During

    coding

    theprocess,

    genomese

    quencesanddemonstratesasevererestrictionofrepli. cationonmammaliancells.

    whichturnsMVAvector

    extremelysafeforresearchandclinicaluse.Inaddi

    tion,thecomparativelysmallergenomemayenhance thecapacityofMVAintowhichlargerforeignse

    quencescanbecloned.LivevaccineswithMVAasthe vectorareabletoefficientlyinducehumoralandcel

    lularimmuneresponses[]againstantigensexpressedby therecombinantMVAs.Thus,MVAisoneofthemost widelyusedviralvectorsatpresent.Itservesasacan

    didatevaccinetoimmunizeagainstinfectiousdiseases

andcancer,whichhasbeenapopulartopicinmedi

    cine,biology,andveterinarysciences.

    However,MVAissensitivetorepeatfreezethaw

    cycleandeasytoloseactivity.Sofar,MVAbased

    vaccinehasbeenplacedinphosphatebufferedsaline (PBS)bufferandstoredataverylowtemperature(for example80oC1inlhefrozenstate.Owingtoitspoor thermostability,thesafecontinuumhandlingpractices, includingmaterials,equipmentandures,which maintainvaccinesbelow20oCfromthetimetheyare

    beingmanufacturedtothetimetheyareadministeredto patients,mustbeinsured.Furthermore,MVAshould avoidfreezethawcycling.Ifthe"coldchain"isbro. ken,asignificantlossofinfectivitymayoccuratany time.

    Thebasicdosageformofbiologicaltechnology

    medicineisfreezedrying.Lyophilization,atechnique commonlyemployedtoextendthestabilityoflabile compoundsatambienttemperatures,isconsideredasa practicalalternative.Thisreportcoversthecorrectly

    ophilizedMVAstabilizercompositionsandevaluatesthe immunogenicityoflyophilizedMVAHIVvaccinein

    mice.

    Experimental

    1PreparationofLiquidMVA-HIVVaccine

    MVAHIVexpressingHIV1B/CGag,Poland

    EnvwaskindlydonatedbyChangchunBCHTPharma

    ceuticalCompany.ItwasgrowninCEFcells

    .

    andcuhi

    ratedat37oC.Theinfectedcellsandcultureliquid werecollected.Thecellswerelysedandcentrifugedto removethecelldebris.Thesupernatantwascollected andprocessedunderultrasonicwavesandpurifiedvht sucrosedensitygradientcentrifugation,respectively, andtheviruswascollected.

    inaPBSsolutiontoobtain

    Theviruswasresuspended

    recombinantMVAvaccine

    SupportedbytheNationalNaturalScienceFoundationofChina(No.30371317).

    Towhomcorrespondenceshouldbeaddressed.E

    mail:weikong@jlu.edu.cn;ygwu@jlu.edu.cn

33OCHEM.RES.CHINESEUV01.23

    inliquidform.

    2VaccineStabilizers

    Thestabilizerscontaining3%trehalose.3%man. nito1.2%dextranand1.5%inositolin0.O1mol/L sodiumphosphatebufferatpH=7.2wereusedforthis study.

    3Lyophilization

    LyophilizationwascarriedoutonaShanghaiLYO- 0.5freeze.drier.Thevaccineandthestabilizerwere mixedinequalvolume.0.1mLofthemixturewasdis. pensedinasterile1.mLampoulevia1.Thevaccine vialswhichwerefrozenat40?for3handprevi.

    ouslysubjectedtothefirstlyophilizationat30?for

    10handthesecondlyophilizationat28?for5hata

    vacuumof6Pa.Batchesofthevaccinecontainingdif- ferentstabilizerformulationswerelyophilizedsimultane.

    ouslyunderidenticalconditionstocomparethequality intermsofresidualmoistureandtitrelossduringthe lyophilization.

    4MeasurementofResidualterContents

    Residualmoistureofthelyophilizedvaccinewas measuredviathermo.gravimetricmethoddescribedby Worralleta1.L6].

    Accordingtothismethod.theweight

    ofwaterlostfromthedriedvaccinewasexpressedasa percentage?

    5AnalysisofThermostabilityofLyophilizedVa- ccine

    Lyophilizedvaccinevialsforeachstabilizerwere exposedat4and37?.respectively.Sampleswere

    takenfromtherefrigeratorafter2,6,10,13,17and 21months.respectively.and37?after1,2,3and4

    weeks.Exposedsampleswerereconstitutedwith0.1 mLofdistilledwaterandtitratedinCEFcells.For eachtimepoint,twosamplesweretitratedandtheir averagelogarithmvalueoftiter(1g[V(pfu)/mL])was calculated.

    6MeasurementofTitersofMVAViruses

    TodeterminetheMVAtiter.aseriesofdilutedso. 1utionsofvirusstockwaspreparedandusedtoinfect CEFcells.At24hafterinfection.thecellswerefixed andimmtmostainedwithapolyclonalvacciniavirusan. tiserumandasecondaryantibody.Thenumberof stainedfociwascountedandmultipliedbythedilution factortoexplessthetiterininfectiousunits/mL. 7MicemadImmunizationSchedule

    FemaleBalb/cmiceweredividedintotheliquid vaccinationgroup,thelyophilizedvaccinationgroup, andthecontrolgroup.Themiceofliquidvaccination groupandlyophilizedvaccinationgroupwereimmu

    nizedintramuscularlywith100gDNAvaccine (D-GPE)at2-weekintervals.Onthe6thweek,liquid vaccinationgroupmicewereboostedintraperitoneally withliquid10'pfuMVA-HIVinPBS,

    lyophilizedva-

    ccinationgroupmicewereboostedintraperitoneallywith 10'pfuMVA.HIVafterlyophilization.Controlgroup micewereinoculatedwithPBS.

    8AntibodyDetection

    Molt11IBcell(stablyexpressestheHIV-protein cellline)supernatantwascollectedandthencentri. fugedat26000r/min.TheprecipitationwasreSUS- pendedandusedtodetectthemousebloodserumby Westerblotanalysis.

    9CTLAssay

    LymphocytesshouldbeculturedinRPMI.1640 (containing3%ofBovinecalfsera)(Phenolrede)

    mediumat1×10cells/mL.Fourdilutionswerepre- paredatratiosofeacheffectorcell:targetcell=100:1: 33:1:10:1and3.3:1.Theplatewasincubatedin 5%CO,at37?for45min,towhich10LofLysis

    bufferwasadded.Afterincubationatloomtemperature for1h.theplatewascentrifugedat250gfor4min. 50Lofaliquotoftheaboveobtainedsupernatantwas transferredtoafresh96.wellplatefollowedbyadding 50Lofreconstitutedsubstratemixtoeachwel1.The

    platewascoveredwithfoilandincubatedfor30minat roomtemperatureand50Lofstopsolutionwasadded toeachwel1.Atlaststep,theabsorbancewasmeas. uredontIleplatereaderatawavelengthof490nm. Results

    1PhysicalCharacters

    Havingbeenstoredfor2monthsat37?,the-

    pearanceofthelyophilizedMVA.HIVvaccinestillisin goodcondition.whiteincolorandinpowderform (Fig.1).Furthermore,itCallbereconstitutedrapidly withdistilledwater.Theresidualmoistureofthevac. cineafterfreeze.dryingisbelow3%.

    Fig.1AppearanceoflyophilizedMVA-HIVvaccine storedatthe21stdayat37?

    2ThermostabilityofFreeze-driedMVA??HIVVac?? cine

    ThetiterofMVAHIVvaccineafterlyophilization

No.3ZHANGYi.zheeta1.331

    wasdecreasedby0.12lg[V(pfu)/mL](Table1).As forlyophilizedvaccine,Table2shows0.581g[V (pfu)/mL]titerand1.011g[V(pfu)/mL]titerre

    ducedwhenheatedat37?for14and21days.re

    spectively.Thelosswasverysmal1.Asforliquidvac

    cine,Table2shows0.901g[V(pfu)/mL]titerand 3.231g[V(pfu)/mL]titerreducedwhenheatedat 37?f0r7and21days.respectively.Wealsoas

    sessedlong?-termstabilityoflyophilizedMVA?-HIV storedat4__8?(Table3),adropofapproximately

    0.4lg[V(pfu)/mL]intiterwasobtainedhavingbeen

    storedfor13months,butthetiteroftheliquidvaccine droppedbyapparently1.31g[V(pfu)/mL]having beenstoredfor2months.Thesedatashowlyophilized vaccinevirusesarestableafteralengthyexposureto highertemperature(37?)comparedwiththecurrent

    liquidvaccinepreparations.

    Table1StabilityofrecombinantMVAvaccine beforeandafterlyophilized'

    ?Resultsaretheaveragesof10vialsofeachpreparation.Alltitres

    expressedaslg[p~)/mL].

    Table2Acceleratedthemostabilityofrecombinant MVAvaccineat37?'

    Lyophilized8.O17.67(0.34)7.43(0.58)7.00(1.O1)6.77(1.24) Liquid7.596.69(0.90)5.21(2.38)4.36(3.23)ND ?Resultsaretheaveragesof10vialsofeachpreparation.ND,not detected.AUtitlesexpressedaslg[pfu)/mL]. Table3Long-termstabilityofrecombinant ,,Avaccineat4—名?'

    ?Resultsaretheaveragesof10vialsofeachpreparation.ND.not done.AUfitresexpresseda8lg[(p~)/mL].

    3HumoralImmuneResponses

    Thebloodserumofmicewhichwereimmunized byrecombinantMVAvaccines,weredilutedaccording to1:50dilution.AntiP,JantibodyofHIVwasusedto

    detectthehumoralimmuneresponseinthebloodserum withWesternblottechnique.Fig.2demonstratesthat themiceinthetwovaccinesgroupsproducethesame levelofantibody.

    4CTLAssay

    espleenceHsfromBalb/cmicewereprepared

to4dillutionsforeachexperimentalgroup(Lseffector

    P24——

    Fig.2Westernblotanalysisoftheantibodylevelof lyophilizedandliquidMVAvaccineilnmu- nizedmice

    1.MicePuAbpositivecontrol;2.1yophilizedMVA vaccine;3.1iquidMVAvaccine;4.PBSnegativecon- tro1.

    cel1.TheP815P7Gwasthetargetcel1.ThenCTL

    assaysforthevaccinewascarriedoutbycytoTox96 LDHassay.Fig.3indicatesthattheC11Linducedby lyophilizedMVA?-HIVissimilartothatinducedbyli?- quidMVAHIVvaccineinmice(P>0.05,students t-test).

    E:T

    Fig.3CTLofspleen-derivedlymphocytesfrommice inmluniz~!withlyophilizedandliquidMVA vaccine

    a.LyophilizedMVA-HIVvaccine;b.1iquidMVA-HIV vaccine;c.PBScontro1.

    Discussion

    ThecompoundHIVvaccine.whichconsistsofa plasmidDNAencodingHIVfragment(gag,pol,env namedDNAGPEandarecombinantMVAvaccine

    encodingthesameantigen(namedMVAHIV)hasbeen

    approvedbySFDAofChinatoproceedwithclinicaltri

    a1.Itsimmunizationprocedureincludesprimingimmu

    nizationwithDNAGPEfollowedbyaboostingimmuni

    zationwithMVAHIVagainstHIVchallenge.ACom

binationofDNAGPEwithMVAHIVcangenerate

    strongerspecificimmunityagainstHIVthanthesingle entityapplication.Thusitcallprovideabetterprotec

    tionagainstAIDS.Butthestricttemperaturedepend

    enceoftheliquidMVAformulationlimitsitsefficient deliverytoruralandremotemountainareas.Alyophi

    一摹一?IsAl009d?

332CHEM.RES.CHINESEUV01.23

    lizedMVAformulationwithgoodthermostabilitycan fectivelyeliminatethisconcern.Althoughfreezedry

    itusually

    ef-micewereprimedwith100gofDNAvaccine

    lS

    methodinpreservinglivebiologicalsamples, leadstoproteindenaturationanddropincell viability[7]-Thus,selectingtheoptimalstabilizerin thefreezefryingprocessofthevaccineisacriticalstep invaccineproduction.Thisisdirectlyrelatedtothe

    titerandstabilityofthevaccine.

    Carbohydrate,polyalcoholandaminoacidsen

    hancet}Iehydrophobieinteractionbetweenproteinby influencingt}Ieconformationofwatermolecule.thus preventingtlleheatdenaturationandenhancingthesta

    bilityofproteinsinsolution.Whenwaterisremoved

    duringdrying,thestabilizersformahydrogenbond t}It}Ieproteinaswaterdoes.therebypreservingthe nativestructureduringtheprocessofthermodynamic (DGPE)andafterprimingfor6weeks,theywerela

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