A gold glyco-nanoparticle carrying a Listeriolysin O peptide and formulated with Advax™ delta inulin adjuvant induces robust T-cell protection against listeria infection

ContentslistsavailableatScienceDirect

Vaccine

jo u rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

A

gold

glyco-nanoparticle

carrying

a

listeriolysin

O

peptide

and

formulated

with

Advax

TM

delta

inulin

adjuvant

induces

robust

T-cell

protection

against

listeria

infection

Estela

Rodriguez-Del

Rio

_,

_Marco

_Marradi

_,

_Ricardo

_{Calderon-Gonzalez}

_,

Elisabet

Frande-Cabanes

_,

_Soledad

_Penadés

_,

_Nikolai

_Petrovsky

_,

Carmen

Alvarez-Dominguez

a_{GrupodeGenómica,ProteómicayVacunas,InstitutodeInvestigaciónMarquésdeValdecilla(IDIVAL),Santander,Spain} b_{CICbiomaGUNE,P}◦_{deMiramón182,SanSebastian,Gipúzcoa,Spain}

c_CIBER-BBN,P◦_{deMiramón182,SanSebastian,Gipúzcoa,Spain}

d_{DepartmentofDiabetesandEndocrinology,FlindersUniversity,Adelaide5042,SA,Australia} e_{VaxinePtyLtd,FlindersMedicalCentre,Adelaide5042,SA,Australia}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5November2014

Receivedinrevisedform12January2015 Accepted25January2015

Availableonline7February2015

Keywords: Listeriosis Glyconanoparticles Dendriticcells Vaccines Advax

a

b

s

t

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a

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t

Inthesearchforaneffectivevaccineagainstthehumanpathogen,Listeriamonocytogenes(Listeria),gold

glyconanoparticles(GNP)loadedwithalisteriolysinOpeptideLLO91–99(GNP-LLO)wereusedtoimmunise

mice,initiallyusingadendriticcell(DC)vaccineapproach,butsubsequentlyusingastandardparenteral

immunisationapproach.Toenhancevaccineimmunogenicityanovelpolysaccharideadjuvantbased

ondeltainulin(AdvaxTM_{)wasalsoco-formulatedwiththeGNPvaccine.Confirmingpreviousresults,}

DCloadedinvitrowithGNP-LLOprovidedbetterprotectionagainstlisteriosisthanDCloadedinvitro

usingfreeLLOpeptide.TheimmunogenicityofGNP-LLOloadedDCvaccineswasfurtherincreasedby

additionofAdvaxTM_{adjuvant.However,asDCvaccinesareexpensiveandimpracticablefor}

prophylac-ticuse,wenextaskedwhetherthesameGNP-LLOantigencouldbeusedtodirectlytargetDCinvivo.

ImmunisationofmicewithGNP-LLOplusAdvaxTM_{adjuvantinducedLLO-specificT-cellimmunityand}

protectionagainstListeriachallenge.ProtectioncorrelatedwithanincreasedfrequencyofsplenicCD4+

andCD8+_{Tcells,NKcellsandCD8␣}+_{DC,andTh1cytokineproduction(IL-12,IFN-␥,TNF-␣,andMCP-1),}

post-challenge.EnhancedT-cellepitoperecruitmentpost-challengewasseeninthegroupsthatreceived

AdvaxTM_{adjuvant.ImmunisationwithGNP-LLO91–99plusAdvax}TM_{adjuvantprovidedequallyrobust}

Lis-teriaprotectionasthebestDCvaccinestrategybutwithoutthecomplexityandcost,makingthisahighly

promisingstrategyfordevelopmentofaprophylacticvaccineagainstlisteriosis.

©2015TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND

license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

CandidateprophylacticvaccinesagainstListeriamonocytogenes (Listeria)includeliveattenuatedpathogens[1–4],livevector-based

Abbreviations: APC,antigen-presentingcells;CFU,colonyformingunits;Ctsd, cathepsin-D;DC,dendriticcells;GNP,goldglyconanoparticles;i.p,intra-peritoneal; i.v,intra-venous;LLO,listeriolysinO;MHC,majorhistocompatibilitycomplex;MIIC, MHC-classIIantigenloadingcompartments;MØ,macrophages;LM,Listeria mono-cytogenes.

∗ Correspondingauthorat:GrupodeGenómica,ProteómicayVacunas.Instituto deInvestigaciónMarquésdeValdecilla(IDIVAL),Laboratorio124.EdificioIFIMAV, Avda.CardenalHerreraOria,s/n.39011Santander,Cantabria,Spain.

Tel.:+34942315515x74104;fax:+34942315517.

E-mailaddress:calvarez@humv.es(C.Alvarez-Dominguez).

1 _{Theseauthorscontributedequallyasseniorauthorsofthispaper.}

approaches[5–7]andsubunitvaccines[8,9].However,theuseof livevaccinesinat-riskimmunocompromisedindividualsincluding pregnantwomenposesmajorsafetyrisks,makinganon-living sub-unitvaccineamorepreferableapproach.Unfortunately,subunit vaccinesbythemselvesaregenerallyineffectiveduetooverallpoor immunogenicityandinability,inparticular,togeneratetheT-cell immunityrequiredforprotectionagainstintracellularorganisms suchasListeria.

Dendriticcell(DC)vaccinesareproducedbyisolationofDCfrom asubject,invitroloadingoftheDCwiththerelevantvaccine anti-gen,andthenintravenousorsubcutaneousinjectionoftheliveDC backintothesubject.Basedontheirimprovedabilitytoinduce T-cellimmunity,DCvaccineshavebeenproposedassolutionsfor inductionofT-cellprotectionagainstintracellularinfections,such asHIV[10].OurgrouppreviouslyreportedaDCvaccineloadedwith a peptidefromtheListeriaprotein, glyceraldehyde3-phosphate http://dx.doi.org/10.1016/j.vaccine.2015.01.062

dehydrogenase(GAPDH), inducedgenerationof GAPDH-specific CD8+_andCD4+_{TcellsandconferredprotectionagainstListeria[11],}

consistentwiththeimportanceofT-cellresponsesagainstGAPDH inListeriaprotection[12–14].OtherListeriaantigensthatcontain CD4+_andCD8+_{T-cellepitopesandthatmightthereforeenhance}

theefficacyofaDC-basedListeriavaccineincludelisteriolysin(LLO), ActA,andp60[11,15,16].Todate,onlyT-cellimmunityagainstLLO hasbeenreportedinhumansrecoveringfromlisteriosis[17], sug-gestingapotentialroleofinprotection.Wethereforewishedto testwhetheraT-cellvaccinetargetingLLOcouldprotectagainst listeriosis.Inparticular,wesoughttotestwhethera nanoparticle-conjugatedLLOpeptidecouldincreaseDCvaccineefficacy[10,18] andalsowhetheranovelT-celladjuvantcouldenhanceLLOvaccine immunogenicity[19].Goldglyconanoparticles(GNPs)constitute a nanoscale metallic coreto which self-assembled monolayers ofcarbohydrateligands arecovalentlylinkedbymeansofthiol chemistry[20]. Duetotheirwater dispersibility, biocompatibil-ity,resistancetoenzymaticdegradation,easeofpreparation,and abilitytoincorporatedifferentligands,GNPsarehighlyversatile [21,22].Inthisstudy,therefore,wesoughttotestthepotentialofa glucose-labelledGNPpresentingtheLLO91–99peptide(GNP-LLO)to

protectagainstlisteriosisinamurinemodel.Inthefirstapproach, wesoughttoapplythepreviousapproachofloadingDCinvitro usingtheGNP-LLOcarrierpriortoinjectionoftheDCbackintothe animals.However,giventhelogisticaldifficultiesoftheDCvaccine approach,wealsowishedtotestwhether,ratherthanbeingused forDCvaccineloadinginvitro,theGNP-LLOantigencouldbeused forparenteralimmunisationalongthelinesofatraditionalvaccine. Given the poor T-cell responses induced by subunit vac-cines, we sought to further enhance GNP-LLO immunogenicity by use of AdvaxTM_{, a novel polysaccharide adjuvant derived}

frommicroparticlesofdelta ␤-d-[2-1]poly(fructo-furanosyl)␣-d-glucose(deltainulin) [25].In animalmodelsAdvaxTM_hasbeen

showntoenhancecellularplushumoralimmunityagainstabroad spectrumofantigensincludinginfluenza[26],Japanese encephali-tis[27],WestNilevirus[28],hepatitisB[29],andHIV[30]vaccines. AdvaxTM_{hasalsobeenshowneffectiveandsafeinhumantrialsof}

pandemicinfluenza[31]andhepatitisB[29],vaccines. 2. Methods

2.1.1. Peptidesandadjuvants

We used as immunogens, peptides representing known Lis-teriaTcellepitopes,namelyGAPDH1–22,LLO91–99andLLO189–201

[6,11,15].ImmunisationwithfreeLLO189–200andLLO190–201

pep-tideshaspreviouslybeenshowntoinduceCD4+_{Tcellresponses}

inmicebutdidnotconferListeriaprotection[11,32].Allpeptides weresynthesisedatCNB,CSIC,Madridfollowedbyhigh perfor-manceliquidchromatography(HPLC)andmassspectrometryusing aMALDI-TOF ReflexTM _{IV massspectrometer(Bruker Daltonics,}

Bremen,Germany).Peptidepuritywas>95%afterHPLC.AdvaxTM

adjuvant (Adv1) was supplied by Vaxine (Adelaide, Australia) [31,33–35], and was mixed with antigen by simple admixture immediatelybeforeimmunisationofmice.

2.1.2. Bacteria

L.monocytogenes10403Sstrain(LMWT_{)wasobtainedfromD.A.}

Portnoy(UniversityofCalifornia,Berkeley,CA,USA). 2.1.3. PreparationofgoldglyconanoparticlesbearingLLO91–99

peptides(GNP-LLO91–99)

LLO91–99peptidewithaC-terminalcysteamide(LLO91–99C(O)

NHCH2CH2SH,14mg,purity95%)waspurchasedfromGenScript

and5-(mercapto)pentyl-␤-d-glucopyranoside(GlcC5SH)was

pre-paredasreportedintheliterature[36].GNPpreparationisincluded inSupplementalinformation. Averagegolddiameter1.5±0.5nm (thesizehistograminFig.S1,panelAwasobtainedbycounting 600particlesintheTEMimage).

2.1.4. PreparationofDCvaccines

Bone-marrowDCweredifferentiatedwithGM-CSF,andCD11c+

cellsisolatedwithanti-mouseCD11c-coatedmagneticbeadsand MACS separation columns (Miltenyi Biotech Inc., Auburn, CA) [6,11] from 8 to 12 week-old female CD1, Balb/c or C57BL/6 mice.Bonemarrowmacrophages(DM)wereobtainedfromsame femursanddifferentiatedwithM-CSF,asdescribed[11].DCused wereMHC-II+_CD11c+_CD40+_CD11b+/−_CD86+/−_F4/80−_Gr-1− _andof

DMwereCD11b+_MHC-II+_F4/80+/−_CD11c−_Gr-1−_CD40−_CD86−_.

Cul-turedDCwereexvivoloadedwith50␮g/mlGAPDH1–22,LLO91–99

orLLO189–201peptidesorwith5␮g/mlofGNP-LLO91–99for24h.

CellswereanalysedforapoptosisbyFACSanalysisusing annexin-V-APCandH-1770(BD-Biosciences),forcellviabilitywithTrypan bluestaining,andcellsurfacemarkersbyFACS.

2.1.5. T-cellassays

T-cellanalysis,cytotoxicTlymphocytesresponsesanddelayed typehypersensitivity(DTH)analysisaredescribedinSupplemental information.

2.1.6. Preparationofpeptide-coupledGNPvaccines

GNPcarryingapproximately90%glucoseand10%LLO91–99(Fig.

S1,panelA)werepreparedbyreductioninsituofAu(III)saltwith sodiumborohydride,asdescribed[21,37].Theligands(LLO91–99

peptideandglucose)werederivatisedwiththiol-endinglinkersto attachthemtothegoldcore(Fig.S1,panelA).5-Mercaptopentyl ␤-d-glucopyranoside(GlcC5SH) witha shortaliphaticlinker, as

previouslyreported[36]wasusedasaligandpartnertocontrol thedensityofLLO91–99peptideontheGNPs.Glucoseisidealto

con-ferwaterdispersibilityandbiocompatibilitytotheGNPswithout interferingwiththeantigenicpropertiesofLLO91–99peptide[24].

LLOpeptidesandGNP-LLO91–99didnotadverselyaffectDC

viabil-ityorpromoteapoptosis(Fig.S1,panelBandDCcolumninTable S1).Theywerealsowelltoleratedat50␮g/mlconcentrationsin C57BL/6,CD-1orBalb/cmicestrains,(C57BL/6columninTableS1), whileat500␮g/mlconcentrationstheycauseddecreasedDC via-bilityandincreasedIL-1concentrationsuggestingpotentialtoxicity (TableS1)[38].

2.1.7. GNPimmunogenicityassessment

Weimmunisedmousefootpadswith0.1␮gofrecombinantLLO (LLOrec)andthenevaluatedT-cellproliferativerecallresponsesto

invitrostimulationwithLLO91–99,LLO189–201,GNP-LLO91–99,LMWT

lysate(LM-lysate)orLLOrecatdosesrangingfrom0to500␮g/ml.

T-cellproliferationwasdosedependentpeakingat50␮g/mlandthen decliningforallantigens(Fig.S1,panelC).GNP-LLO91–99induced

ahighlevelofT-cellproliferationequivalenttoLM-lysate(Fig.S1, panelC).LLO91–99andLLOrecinducedthenextbestT-cell

prolifera-tion,whileLLO189–201waslowest.TotestwhetherthehigherT-cell

recallresponseswithGNP-LLO91–99mightreflectimprovedMHC-I

antigenprocessingbylymphnodeAPC,wecomparedMHC-I pep-tidepresentationafterexvivoloadingofDCorDMwithLLO91–99

orGNP-LLO91–99.MHC-Imoleculeswereimmuno-precipitatedand

levelsofLLOpeptidesboundtoMHC-Iassessedbywestern-blot usingrabbitanti-LLOantibodies,aspreviouslyreportedforMHC-II molecules[5,11].GNP-LLO91–99resultedinhigherefficiency

DM(Fig.S1,panelD),withfarhigherpresentationbyDCthanDM (Fig.S1,panelD).

2.1.8. ExvivoactivationofDCvaccines

WeexaminedtheeffectofLLOpeptidecarriersonDCvaccine activation[6,10].ActivatedDCshowacharacteristicCD11c+

MHC-II+_CD40+_CD86+ _{phenotype[10,18].ToexamineDCactivationby}

LLOcarriers,atday5afterGM-CSFdifferentiation,DCwere puri-fiedasCD11c+ _{cells usingMACSmagneticbeads(Miltenyi)and}

loadedex vivo for 24h with 5␮g/ml of GNP-LLO91–99 in

pres-enceorabsenceofAdvaxTM_{adjuvant(50␮g/ml)orwith50␮g/ml}

of LLO91–99, LLO189–201,LM-lysate or left untreated (DC-CONT).

Next, thefrequency of CD11c+_MHC-II+ _{cells positive for CD40}+

orCD86+ _{DCwasassessed asmarkers of matureand activated}

DC (Fig.S2, panelA).DC-Listeria lysateinduced thehighest fre-quencyofMHC-IIhigh_{DCfollowedbyGNP-LLO}

91–99withorwithout

AdvaxTM_{,whichwerebothsignificantlyhigherthanLLO}

189–201(Fig.

S2,panelB).AsimilarpicturewasseenforCD11c+_MHC-II+_CD40+

orCD11c+_MHC-II+_CD86+_{DC.DifferencesbetweenGNPloadedand}

freepeptidewereevenmoremarkedwhenweanalysedthe fre-quencyofCD40+_CD86+_{doublepositiveDC(Fig.S2,panelC).}

2.1.9. Immunisation

DCvaccinesloadedinvitrousingeitherfreepeptideor GNP-conjugatedpeptidesareindicatedbyplacementof“DC”infrontof thespecificantigen.Ifthenameofthevaccinedoesnotcontain “GNP”thenthismeansDCloadingorimmunisationwasdonewith freepeptide.VaccinesusedwereeitherDCvaccines(DC-LLO91–99,

DC-LLO189–201,DC-GAPDH1–22,DC-GNP-LLO91–99, allatdoses of

1×106_{DCperimmunisation),orstandardvaccines(GNP-LLO} 91–99

at5␮gperimmunisation).Standardvaccineswereadministered withor without AdvaxTM _{adjuvant (250␮g per immunisation).}

Micewereimmunisedintra-peritoneally(i.p)(n=5),intravenously inthetailvein(i.v)(n=5)orleftnon-vaccinated(NV).Sevendays afterimmunisationmicewerechallengedi.pori.vwith5×103_CFU

ofListeriamicroorganisms/mouse[2].Serawerestoredat−80◦_C

tomeasurecytokinesbyFACSanalysis.Atterminationspleensand liverswerehomogenisedandListeriacolonyformingunits(CFUs) countedinbloodagarplates.

2.1.10. FrequenciesofCD8+_-LLO

91–99specificTcells

ToconfirmthefrequencyofIFN-␥producingLLO91–99specific

CD8TcellsweusedrecombinantsolubledimericmouseH-2Kb_:Ig

(forC57BL/6mice)orH-2Kd_{:Ig(forBalb/cmice)fusionproteins}

fol-lowingthemanufacturer’sinstructions(DimerXI;BDBioscience) andaspreviouslydescribed[2,11].

2.1.11. Ethicsstatement

Thisstudywascarried outinaccordance withtheGuidefor theCareandUseofLaboratoryAnimalsoftheSpanishMinistry ofScience,ResearchandInnovation.TheCommitteeontheEthics ofAnimalExperimentsoftheUniversityofCantabriaapprovedthe protocol(PermitNumber:2012/06)thatfollowstheSpanish leg-islation(RD1201/2005).Allsurgerywasperformedundersodium pentobarbitalanaesthesia,andalleffortsweremadetominimise suffering.

2.1.12. Statisticalanalysis

For statistical analysis, Student’s t test was applied.ANOVA analysis was applied to cytokine measurements. P≤0.05 was

Fig.1.ExvivoloadingofDCvaccinesandTcellresponses.(A)T-cellproliferationin responsetostimulationinvitrowith50␮g/mlofLLO91–99inpopliteallymphnode

homogenates7daysafterfootpadinoculationwith1×105_{cellsofDCvaccinesor}

saline(NT)[14].Resultsshowthemean±SDofthestimulationindexcalculated asratioofproliferationinactivewells/proliferationofcontrolsamplesintriplicate (P<0.05).(B)Micewereimmunisedi.pwith5×103_{CFUofListeria/miceand7days}

laterDTHassaysperformed.Resultsareexpressedasthemeandifference±SDof footpadswellingat24and48hinactiveandcontrollimbs,representativeexample ofthreeindependentexperiments(P<0.05).

consideredsignificant.GraphPadsoftwarewasusedforgeneration ofgraphs.

3. Results

3.1. DCvaccinesloadedusingGNP-peptidecarriersinduceT-cell proliferativeresponsesagainstListeria

DendriticcellsstimulatebothCD4+_andCD8+_Tcellsin

listerio-sis[14].Toevaluatewhetherdendriticcell(DC)vaccinesloaded usingGNP-peptide carriersmight bemore efficientininducing T-cellresponsesthanDC loadedusingfree peptides,micewere immunisedwithDCthathadbeenloadedindifferentways.Seven dayspost-immunisation,popliteallymphnodeswerestimulated invitrowithLLO91–99peptideandantigen-specificT-cell

prolife-rativeresponsesmeasured.ImmunisationwithDCloadedinvitro withGNP-LLO91–99inducedhigherT-cellproliferationresponses

thanimmunisation withDC loadedusingfree LLO91–99 peptide,

indicatingtheimportanceforinductionofT-cellmemoryof attach-mentoftheLLO91–99peptidetotheGNPtoenableefficientloading

an equivalent T-cell proliferative response to a whole Listeria lysate,consistentwithefficientDCloadingandinductionofT-cell responsesbytheGNP-peptidecarrier(Fig.1A).DCloadedwitha CD4+_{restrictedepitope(LLO}

189–201)[11],failedtoinduceaT-cell

proliferativeresponse, suggestingtheDC vaccinespreferentially induceaCD8+_{T-cellresponse.}

3.2. DCvaccinesloadedusingGNP-peptidecarriersinduceDTH responsesinListeria-sensitisedmice

Next,we examinedthe delayedtype hypersensitivity (DTH) responsetotheDCvaccinesinpreviouslyListeria-sensitisedmice asaninvivomeasureoftheabilityofthedifferentDC vaccines toinduceT-cellactivation.TheaimwastoevaluatewhetherDC loadedinvitrowithGNP-LLO91–99 when injected intothe

foot-padofpreviouslysensitisedmicewouldtriggeratypicalTh1-type T-cellresponseasmeasuredbyDTH[7].WeinjectedListeria sensi-tisedmiceinthefootpadwithDCloadedinvitrowithLLO91–99or

LLO189–201freepeptidesorwithGNP-LLO91–99.Infact,DCloaded

withLLO189–201generatedgreaterDTHresponsesinthesensitised

micethanDCloadedwithLLO91–99orGNP-LLO91–99,suggesting

thatLLO189–201isnormallyadominantT-cellepitopeafterListeria

infection(Fig.1B).Interestingly,theDTHresponsetoDCloaded withGNP-LLO91–99inthepresenceofAdvaxTMadjuvantwas

sim-ilarinsizetotheDC-LLO189–201responsesuggestingtheLLO91–99

epitopecouldbeturnedintoadominantepitope,providingitwas presentedbyDCata sufficientlyhighlevel togetherwith suffi-cientco-stimulatorymoleculeexpressioninducedbytheAdvaxTM

adjuvant(Fig.1B).

3.3. ImmunisationwithGNP-loadedDCvaccinesprovides protectionofmiceagainstlisteriosis

We next assessed protection against listeriosis using the different DC vaccine vectors, DC-LLO91–99, DC-LLO189–201,

DC-GAPDH1–22,DC-Listerialysate,DC-GNP-LLO91–99,andemptyDCs

(DC-CONTROL)whencomparedtoimmunisationwithfree GNP-LLO91–99.DC-GAPDH1–22wasincludedasapositivecontrolsinceit

previouslyshowedgoodListeriaprotection[11].Micewere immu-nisedi.vwithasingledoseofvaccine.Althoughi.visanunusual immunisation route for standard vaccines, it is a typical route foradministration ofcellularDC vaccines [6].Seven days post-immunisationthemicewereadministeredaliveListeriachallenge. Fivedayspost-challenge,allmiceweresacrificedandliversand spleensrecoveredtocountListeriacolonyformingunits(CFU)and analysetheimmunecellpopulations.DC-GAPDH1–22and

DC-GNP-LLO91–99providedthegreatestListeriaprotection(∼97and99%CFU

reduction,respectively),followedbyDC-LLO91–99(∼94%)(Fig.2A).

DC-LLO189–201 conferred nosignificantprotection(5%).Notably,

immunisationofmicewithfreeGNP-LLO91–99(asopposedtoDC

pre-loadedinvitrowithGNP-LLO91–99)conferredintermediate

pro-tection(∼60%CFUreduction).Controlnon-vaccinatedmice(NV) werenotprotectedandasexpectedhadenlargedspleensand gran-ulomatousliverspost-challenge(Fig.2A,righthandfigure). 3.4. ImmunisationwithGNP-loadedDCsincreasesthefrequency ofDCandCD8Tcellsinthespleenpost-challenge

Protectioninlisteriosisistypicallyreflectedbyincreasesinthe frequencyofsplenicmacrophages(MØ),naturalkillercells(NK), matureDC and activatedTcells post Listeriachallenge[14,39]. We therefore examined the cell populations in the spleens of immunisedmice5dayspost-challenge.Miceimmunisedwith DC-LLO91–99,DC-GAPDH1–22,DC-GNP-LLO91–99orfreeGNP-LLO91–99

particlesshowed anincreased frequencyof matureDC (70% of positivecells),CD8+_{Tcells(∼32%ofpositivecells),MØ(18–19%}

Table1

FrequenciesofLLO91–99-CD8+TcellsinducedbyDCvaccines.Splenocytesof

vacci-natedmiceincubatedwithrecombinantdimericH-2Kb_{:Igfusionproteinloaded}

withLLO91–99peptide.FrequenciesofCD8+-LLO91–99andIFN-␥producerswere

performedasdescribedinSection2.1.10.Resultsareexpressedasthemean±SD (P<0.05).

Vaccinationtype %Total dimer-CD8/LLO91–99 %Gated dimer-CD8/LLO91–99 DC-LM-lysate 0.05±0.01 0.48±0.01 DC-LLO91–99 0.06±0.01 1.22±0.02 DC-GNP-LLO91–99 0.17±0.02 3.42±0.02 GNP-LLO91–99 0.05±0.01 0.35±0.01 GNP-LLO91–99+Advax 0.20±0.01 3.21±0.02

ofpositivecells)and NK(10%ofpositive cells)intheirspleens,

post-challenge.OnlyDC-LLO189–201andDC-GAPDH1–22immunised

miceshowedanincreasedfrequencyofCD4+_{Tcellspost-challenge}

(Fig.2B).

3.5. ListeriaprotectionbyGNP-loadedDCisassociatedwithan enhancedTh1T-cellresponse

WenextevaluatedwhetherListeriaprotectionofimmunised mice was associated with enhanced Th1 cytokine production, as would be predicted for protection against an intracellular bacterium.ImmunisationwithDC-LLO91–99,DC-GAPDH1–22,

DC-GNP-LLO1–99 and, to a lesser extent, free GNP-LLO91–99 was

associatedwithhighlevelsofMCP-1, TNF-␣andIFN-␥ produc-tion,consistentwitha strongTh1response (Fig.2C).However, onlyDC-GAPDH1–22andDC-GNP-LLO1–99immunisationwas

asso-ciatedwithmeasurableIL-12production,post-challenge,(Fig.2C), which correlated withthesegroupshaving thegreatestListeria protection(Fig.2A)[6,11].Next,wecomparedtheabilityofthe differentvaccinestoinduceLLO-specificT-cellimmunity. DC-GNP-LLO91–99inducedahigherfrequencyofLLO-specificIFN-␥+CD8+

Tcells (3.42%), than inducedby DC-LLO91–99 (1.22%), (Table1).

The response induced by DC-GNP-LLO91–99 thereby compared

favourably to the 4.2% frequency of LLO-specific CD8+ _{T cells}

obtainedwiththemostprotectiveListeriavaccinewehadidentified todate,DC-GAPDH1–22,[11].

3.6. AdvaxTM_{adjuvantenhancesListeriaprotectionby}

GNP-LLO91–99

DC vaccines are expensive and impracticable. However, standard immunisation with free GNP-LLO91–99 vaccine only

provided intermediate levels of Listeria protection, (∼60% CFU reduction,Fig.2),thatwasinferiortotheprotectionobtainedwith theDCvaccines.Wewishedtotest,therefore,whether formula-tionofGNP-LLO91–99 withAdvaxTMadjuvant mightenhanceits

abilitytoprotectwhenadministeredthroughatraditionalvaccine approach.Miceimmunisedi.pwithasingledoseofGNP-LLO91–99

plusAdvaxTM_{didindeedexhibitenhancedprotection(84%)when}

compared to mice immunised with GNP-LLO91–99 alone (60%)

(Fig.3A).ThistherebyachievedcomparablelevelsofListeria pro-tection to those previously only achieved withthe DC vaccine approach.

Analysisofspleenpopulations5dayspost-challengeconfirmed thati.pimmunisationwithGNP-LLO91–99plusAdvaxTMadjuvant,

inducedasimilarfrequencyofmatureDC,CD8+_{TcellsandNKcells}

andahigherfrequencyofCD4+_{TcellswhencomparedtotheDC}

vaccine,DC-GNP-LLO91–99 (Fig.3B).Thissuggeststhatproviding

it wasformulated withAdvaxTM _{adjuvant, standard}

immunisa-tionwithGNP-LLO91–99wasasgoodorbetteratinducingT-cell

responsesandprotectionagainstListeriathanthemuchmore com-plexDCvaccineapproach.

Fig.2. GNP-LLO91–99vaccineprotectsagainstlisteriosis.DCvaccinesweretestedforabilitytoprotectagainstlisteriosis.(A)C57BL/6micewerevaccinatedoncei.vwith

variousDCvaccinevectors(1×106_{cells)(DC-CONT,DC-LMlysate,DC-GAPDH}

1–22,DC-LLO91–99,DC-LLO189–201,DC-GNP-LLO91–99),solubleGNP-LLO91–99(5␮g/ml)ornot

vaccinated(NV)(n=5mice/group)and7dayslaterchallengedi.vwith5×103_{CFUofListeria.ListeriacountsinspleenhomogenatesexpressedasCFU(mean±SD)obtained}

fromtriplicatesamplesofthreeindependentexperiments(P<0.01).Imagescorrespondingtospleensandliversofnon-vaccinated(NV)versusGNP-LLO91–99vaccinated

mice(GNP-LLO91–99).(B)Percentagesofdifferentspleencellpopulationsareshown:MØs,Bcells,NK,CD4Tcells,CD8Tcells,DCi,DCmquantifiedinspleenhomogenates

byFACS.Resultsexpressedasthemean±SDofthepercentageofpositivecells(P<0.05).(C)Levelsofpro-inflammatorycytokines(MCP-1,TNF-␣,IFN-␥,IL-6,IL-10,IL-12) wereanalysedinmouseserabyCBA.Resultsexpressedascytokineconcentration(pg/ml)ofmean±SD,P<0.05.

Fig.3. EffectofAdvaxTM_{adjuvantonGNP-LLO}

91–99vaccineprotection.(A)C57BL/6micewerevaccinatedi.pwithDC-LLO91–99orDC-GNP-LLO91–99(1×106cells),orsoluble

GNP-LLO91–99(5␮g/ml)withorwithoutAdvaxadjuvant(1␮g/ml)ornotvaccinated(NV)(n=5mice/vaccinevector).Sevendayspost-immunisationtheywerechallenged

i.pwith5×103_{ofListeria(LM}WT_{).Listeriacountsinspleenhomogenates5dayspost-challenge(CFUmean±SD)ofthreeindependentexperiments(P<0.01).(B)Spleen}

homogenates5dayspost-challengewereanalysedbyFACS(mean±SDofpercentagesofpositivecells,P<0.05).(C)SplenicDCfrommicevaccinatedasinAwereanalysed toquantitateCD11c+_MHC-II+_CD8␣+_{DC(percentagesofMHC-II}+_CD8␣+_{positivecells(mean±SD)ofthreedifferentexperiments,P<0.05).(D)Serumcytokinelevels5days}

3.7. Advax-adjuvantedGNP-LLO91–99increasessplenicIL-12+

CD8˛+_{DCpost-challenge}

CD8␣+_{DCsplayacriticalroleinpresentationofListeria}

anti-gens toprotective CD4+ _{and CD8}+ _{Tcells and, in particular, in}

IL-12productionthatcorrelateswithprotection[39,40].We there-foreanalysedsplenicDC content,post-challenge. Thefrequency ofsplenicCD8␣+_{DCwasalmostdoubledfrom10%to18%when}

GNP-LLO91–99 wasformulated withAdvaxTM adjuvant (Fig.3C).

Thiscontrastedwithalowfrequency(2–3%)ofCD8␣+_DCinthe

spleenofnon-vaccinatedmicepost-challenge.Notably, immun-isationwithfreeGNP-LLO91–99plusAdvaxTMadjuvantgenerated

a higher frequency of splenic CD8␣+ _{DC than theDC vaccines}

(DC-GNP-LLO91–99 orDC-LLO91–99)(10%)(Fig.3C).Hence,when

formulatedwithAdvaxTM_{adjuvant,standardimmunisationwith}

GNP-LLO91–99 wasasgoodorbetterthanthemorecomplexDC

vaccineapproachatinducingsplenicCD8␣+_{DCthatplayacritical}

roleinlisteriaprotection

3.8. FormulationofGNP-LLO91–99vaccinewithAdvaxTM

adjuvantenhancesTh1cytokineresponses

SinceformulationofvaccineswithAdvaxTM_increasedthe

fre-quencyofbothCD8+_{TcellsandCD8␣}+_{DCinthespleenpost-Listeria}

challenge,wenextexaminedserumcytokinelevelspost-challenge insplenocytesofmiceimmunisedwithorwithoutAdvaxTM

adju-vant.MiceimmunisedwithGNP-LLO91–99plusAdvaxTMexhibited

significantly greaterMCP-1, TNF-␣and IFN-␥ production when comparedtomiceimmunisedwithGNP-LLO91–99alone,

consis-tent with AdvaxTM _{driving an enhanced Th1-response against}

Listeria. Cytokinelevels in mice immunised withGNP-LLO91–99

plus AdvaxTM _{were equivalentto those seen withthe DC}

vac-cine,DC-GNP-LLO91–99(Fig.3D,upperpanel).Immunisationwith

GNP-LLO91–99plusAdvaxTMwasalsoassociatedwithsignificantly

greatersplenocyteIL-12and IL-10production andreduced IL-6 productionwhencomparedtoimmunisationwithGNP-LLO91–99

alone(Fig.3D,lowerpanel),consistentwiththeAdvaxTM_adjuvant

enhancingtheTh1responseagainstListeria.

3.9. FormulationofGNP-LLO91–99withAdvaxTMadjuvant

enhancesIFN-producingListeria-specificCD4+_andCD8+_Tcells

Next, we evaluated the effect of AdvaxTM _{on the ability of}

standardimmunisationswithGNP-LLO91–99toinduceLLO-specific

CD8+ _andCD4+_{TcellsusingIFN-␥asaTh1subsetmarker.Five}

dayspostListeriachallenge,miceimmunisedwithGNP-LLO91–99

alonehadadetectablefrequency(0.59%)ofLLO91–99specific

IFN-␥+CD8+_{Tcells,butasexpectedhadnoresponsesagainsttheCD4}+

Tcellepitopenotcontainedinthevaccine(Fig.3E,upperpanel). ImmunisationwithGNP-LLO91–99plusAdvaxTMnotonlyresulted

inasignificantincreaseinthefrequencyofLLO91–99specific

IFN-␥+CD8+ _{Tcells(0.89%) butalsoresultedintheappearanceofa}

significantnumberofIFN-␥+CD4+_{TcellsrecognisingtheCD4}

epi-tope,LLO189–201,notpresentinthevaccine(Fig.3E,lowerpanel).

ThustheAdvaxadjuvantenhancedtheabilityoftheListeria vac-cinetoprimeforaprocessofT-cellepitopespreadinginresponse toListeriainfection.

Toconfirm theabove findings,theability of AdvaxTM _when

addedtoeitherDCvaccinesorfreeGNP-LLO91–99vaccinetoinduce

LLO91–99-specific CD8+ cells was tested using H2-Kb-LLO91–99

dimerstolabelandidentifytheantigen-specificTcells (H2-Kd

-LLO91–99dimerswereusedwithsimilarresults,datanotshown).

AdditionofAdvaxTM_toGNP-LLO

91–99resultedina4-foldincrease

in the frequency of LLO91–99-specific CD8+ cells versus

GNP-LLO91–99vaccinealone,andwassuperiortoallothervaccine

for-mulationsincludingtheDCvaccine,DC-GNP-LLO91–99(Table1,left

handcolumn).TheeffectsofAdvaxTM_{wereevenmorepronounced}

whenH2-Kb_-LLO

91–99positivecellsweregatedforIFN-␥,with

∼10-foldhigherfrequencyofIFN-␥expressingH2-Kb_-LLO

91–99positive

cellsinmicevaccinatedwithGNP-LLO91–99plusAdvaxversusmice

vaccinatedwithfreeGNP-LLO91–99alone(Table1,righthand

col-umn).ThisconfirmedthatprovidingthevaccineincludedAdvaxTM

adjuvant,standardimmunisationwithGNP-LLO91–99wassuperior

toallothervaccineapproaches, includinguseofthebest GNP-loadedDCvaccine,atinducingListeria-specificIFN-␥+_CD8+_Tcells,

thecriticalcellsinvolvedinprotection. 4. Discussion

The objective of this study wasto identify an optimal vac-cineformulationtoprotectagainstlisterosis.Listeriaprotectionis largelydependentonCD4+_andCD8+_{Tcellsratherthan}

antibody-mediated immunity[14,17].Livevector vaccinesare inherently unsuitableforListeriavaccinesgiventheirriskofcausingserious infectioninimmune-compromisedindividuals.Asanalternative, cellularDC-basedvaccineswhereDCareloadedwithListeria anti-gensexvivo[6,11,19]andinjectedbackintothehosthavebeen proposedforinductionof protectivecellularimmunity.We ini-tiallydesignedaDCvaccinebasedonloadingofDCwithLLO91–99

peptideusingconjugatedGNP(GNP-LLO91–99)andshowedthat

thisprovidedahighefficiencyofDCloading,invitro[22,23]. DC-GNP-LLO91–99 inducedCD8+Tcellimmunityinimmunisedmice

thattranslatedintoprotectionagainstListeriachallenge.However DCvaccinesareimpracticalandexpensivetomake,necessitating thatwefindanalternativestrategyfordeliveryofourGNP-based vaccine. To overcometheseproblems, we next tested whether standardimmunisationwithGNP-LLO91–99ratherthanusingitto

loadDCinvitro,couldtargetandloadDCinvivo.However,only par-tialprotectionwasobtainedbystandardimmunisationwithfree GNP-LLO91–99vaccine,inferiortothatobtainedwiththeDCvaccine.

To overcome this problem, we next formulated the free GNP-LLO91–99antigenwithAdvaxTM,apolysaccharideadjuvantshown

effectiveandsafeinabroadrangeofanimalmodelsandhuman trials[25–31,33–35].Inadditiontoastronghumansafetyrecord, AdvaxTM_{wasselectedbasedonitsabilitytoinducerobustCD4}+

andCD8+_{Tcellresponses[30,35].Asaproofofprinciplewefirst}

showedthatAdvaxTM_{enhancedtheimmunogenicityoftheDC}

vac-cine,GNP-LLO91–99.ThisfindingsuggestsAdvaxadjuvantmayoffer

benefitsforDCvaccinesforotherindicationssuchascancer,where thecostandcomplexityofDCvaccinesislessanissue.However, givenourdesiretomoveawayfromtheDCvaccineapproachfor Listeria,themostimportantfindingwasthatAdvaxTM_{significantly}

enhanced the immunogenicityof GNP-LLO91–99 when

adminis-teredasastandardvaccine.Notably,thistranslatedintoimproved Listeriaprotectiontoalevelwehadpreviouslyonlyachievedusing DCvaccines.Theenhancedprotectionobtainedwiththe Advax-adjuvantedGNP-LLO91–99vaccinewasassociatedwithanincreased

frequencyofsplenicCD4+ _andCD8+ _{Tcells,NKcellsandCD8␣}+

DC.ThelatterhavebeenreportedtoplayacriticalroleinListeria protectionviaabilitytoproduceIL-12andactivateIFN-␥ produc-ingCD4+ _{and CD8}+ _{Tcells [39,40]. Notably,immunisation with}

Advax-adjuvantedGNP-LLO91–99enhancedproductionoftheTh1

cytokines,IL-12,IFN-␥,TNF-␣,andMCP-1,whilereducingtheTh2 cytokine,IL-6.Interestingly,immunisationwithAdvax-adjuvanted

werestimulatedfor5hwithLLO91–99orLLO189–201peptidesthenintracellularcytokinestainingperformed.HistogramsshowthefrequencyofLLO189–201-specificCD4+Tcells

andfrequencyofthesemakingIFN-␥producers(leftplots)andthefrequencyofLLO91–99-specificCD8+TcellsandfrequencyofthesemakingIFN-␥(rightplots).Experiments

Fig.4.HypotheticalmodeltoexplaintheeffectofAdvaxTM_{adjuvantonGNP-LLO}

91–99protectionagainstlisteriosis.Diagramdepictingpredictedeventswhenmicearevaccinated

withsolubleGNP-LLO91–99carriersinthepresenceorabsenceofAdvaxTMadjuvant(step1)andthenchallengedwithliveListeria(step2).Explanationofthemodelis

describedintextoftheSection4.

GNP-LLO91–99 wasalsoassociated withincreasedproduction of

theanti-inflammatorycytokine,IL-10,butunlikeotherstudiesthis increasedIL-10wasnotassociatedwithenhancedListeria suscep-tibility[32].IL-10maynormallyactasaninversemarkerofIFN-␥ productionasIL-10isdownregulatedbyIL-12andIFN-␥andvice versa.BecauseIL-12andIFN-␥aredominantindeterminingListeria protectionandwerebothmarkedlyincreasedbyAdvax-adjuvanted GNP-LLO91–99,theIL-10 evenalthough increased wouldnot be

expected to block IFN-␥-mediated protection in this situation, butmayactuallyhavebeenbeneficialbyreducinginflammatory damageinresponsetoListeriainfection.Immunisationwith Advax-adjuvanted GNP-LLO91–99 resultedin an increasedfrequency of

LLO91–99specificIFN-␥+CD8+Tcells,post-challenge.More

surpris-ingwastheincreasedfrequencyofLLO189–201specificIFN-␥+CD4+

Tcells, asthisCD4+_{epitope wasnotpresent inthevaccinethe}

micereceivedsotheseCD4+_{Tcellsmusthavebeengeneratedin}

responsetotheListeriaitself.Thissuggeststhatmiceimmunised withAdvaxTM_{adjuvanthaveagreatercapacityforT-cellepitope}

spreadingafterListeriachallenge,therebyexplainingthebroader T-cellrecognitionofListeriaepitopesinthemicethatreceivedthe Advax-adjuvantedGNP-LLO91–99vaccine.

OurmodelforhowvaccinationwithAdvax-adjuvanted

GNP-LLO91–99 provides enhanced protection against listeriosis is

depictedinFig.4.Inbrief,injectionofsolubleGNP-LLO91–99

carri-ers(step1)targetstheconjugatedpeptidetoconventionalsplenic DCthatarestimulatedtoamaturephenotypeandcanthenprime LLO91–99-specificCD8+Tcells(step3).UponexposuretoListeria

(step2),theseCD8+ _{Tcellsbecomeactivatedandproduce}

IFN-␥thatinturnenhancesDCIL-12productionandconsequentNK cellactivation.ATh1feedbackloopisgenerated,whichassiststhe inductionofinflammatoryandactivatedDCandMØthat phago-cytoseandkill thebacteria(step4).AdvaxTM _{adjuvant recruits}

andinteractswithDCresultinginanincreaseinmatureDCand, inparticular,CD8␣+_{DC,whichafterloadingwithGNP-conjugated}

LLO91–99,migratetothesplenicwhitezoneswheretheybecome

activatedand induce expansionof LLO-specific CD4+ _{and CD8}+

T cells (step 5). Upon exposure to Listeria, these LLO-specific

CD8+ _{Tcells becomeactivatedandinducehighlevelsof IFN-␥,}

IL-12, MCP-1, and TNF-␣.These Th1cytokinesenhance Listeria phagocytosisanddestruction,whileincreasedIL-10mayhelp pre-ventexcessinflammation(step6).CD8+_{Tcellsgeneratedbythe}

Advax-adjuvantedvaccineenhanceepitopespreadingbyinducing DCtoexpressadditionalListeriaepitopes,therebyallowing recruit-mentofadditionalCD4+ _{TcellsableinresponsetotheseMHC-II}

epitopes.Thispromotesfurtherexpansionandactivationof LLO-specificTcellsincludingIFN-␥producingLLO189–201-specificCD4+

Tcells(step7)thatsynergisewithIFN-␥-producingCD8+_Tcellsin

Listeriacontrol.

Overall, these results support further development of GNP-peptide carriers together with AdvaxTM _{adjuvant as a vaccine}

platformwithpotentialapplicabilitytoprophylacticimmunisation againstListeria,plusotherbroaderuses.

Acknowledgements

This study was supported in part by grants founded by MINECO, SAF2009-08695 and SAF2012-34203 and by IDIVAL grants:API2012/03/SAF2009-08695andAIP2014/SAF2012-34203 (toC. A-D)and grantCTQ 2011-27268(to S.P).Developmentof AdvaxadjuvantwassupportedbycontractHHSN272200800039C from the National Institute of Allergy and Infectious Diseases, NationalInstitutesofHealth(toN.P).Thispaper’s contentsare solely the responsibility of the authorsand do not necessarily representtheofficialviewsofthefunders.Weacknowledgethe laboratory technical assistance of L. Vazquez-Rioja and IDIVAL institutionalassistanceofC.Santacruz-LlatawithFACSanalysis.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.vaccine.2015. 01.062.

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