Subcutaneous inverse vaccination with PLGA particles loaded with a MOG peptide and IL-10 decreases the severity of experimental autoimmune encephalomyelitis

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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

Subcutaneous

inverse

vaccination

with

PLGA

particles

loaded

with

a

MOG

peptide

and

IL-10

decreases

the

severity

of

experimental

autoimmune

encephalomyelitis

Giuseppe

Cappellano

a,∗

_,

_Abiy

_Demeke

_Woldetsadik

a

_,

_Elisabetta

_Orilieri

a

_,

Yogesh

Shivakumar

a

_,

_Manuela

_Rizzi

c

_,

_Fabio

_Carniato

d

_,

_Casimiro

_Luca

_Gigliotti

a

_,

Elena

Boggio

a

_,

_Nausicaa

_Clemente

a

_,

_Cristoforo

_Comi

b

_,

_Chiara

_Dianzani

e

_,

Renzo

Boldorini

a

_,

_Annalisa

_Chiocchetti

a

_,

_Filippo

_Renò

c

_,

_Umberto

_Dianzani

a

a_IRCAD_and_Department_of_Health_Sciences,_“A._Avogadro”,_University_of_Eastern_Piedmont_“A._Avogadro”,_via_Solaroli_17,_Novara,_28100,_Italy

b_IRCAD_and_Department_of_{Translational}_Medicine,_Section_of_Neurology,_“A._Avogadro”,_University_of_Eastern_Piedmont_“A._Avogadro”,

viaSolaroli17,Novara,28100,Italy

c_Innovative_Research_Laboratory_for_Wound_Healing,_Department_of_Health_Sciences,_University_of_Eastern_Piedmont_“A._Avogadro”,

viaSolaroli17,28100Novara,Italy

d_Department_of_Science_and_Technology,_University_of_Eastern_Piedmont_“A._Avogadro”,_viale_Teresa_Michel_11,₁₅₁₂₁_Italy

e_Department_of_Drug_Science_and_Technology,_University_of_Turin,_via_Pietro_Giuria_9,₁₀₁₂₅_Torino,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24February2014

Receivedinrevisedform19June2014

Accepted8August2014

Availableonline20August2014

Keywords: PLGA Tolerogenicvaccine TREG Interleukin-10

a

b

s

t

r

a

c

t

“Inversevaccination”referstoantigen-specifictolerogenicimmunizationtreatmentsthatarecapable ofinhibitingautoimmuneresponses.Inexperimentalautoimmuneencephalomyelitis(EAE),ananimal modelofmultiplesclerosis(MS),initialtrialsusingpurifiedmyelinantigensrequiredrepeated injec-tionsbecauseoftherapidclearanceoftheantigens.ThisproblemhasbeenovercomebyDNA-based vaccinesencodingformyelinautoantigensaloneorincombinationwith“adjuvant”molecules,suchas interleukin(IL)-4orIL-10,thatsupportregulatoryimmuneresponses.PhaseIandIIclinicaltrialswith myelinbasicprotein(MBP)-basedDNAvaccinesshowedpositiveresultsinreducingmagneticresonance imaging(MRI)-measuredlesionsandinducingtolerancetomyelinantigensinsubsetsofMSpatients. However,DNAvaccinationhaspotentialrisksthatlimititsuseinhumans.Analternativeapproachcould betheuseofprotein-basedinversevaccinesloadedinpolymericbiodegradablelactic-glycolicacid(PLGA) nano/microparticles(NP)toobtainthesustainedreleaseofantigensandregulatoryadjuvants.Theaimof thisworkwastotesttheeffectivenessofPLGA-NPloadedwiththemyelinoligodendrocyteglycoprotein (MOG)35–55autoantigenandrecombinant(r)IL-10toinversevaccinatemicewithEAE.Invitro experi-mentsshowedthatuponencapsulationinPLGA-NP,bothMOG35-55andrIL-10werereleasedforseveral weeksintothesupernatant.PLGA-NPdidnotdisplaycytotoxicorproinflammatoryactivityandwere partiallyendocytosedbyphagocytes.Invivoexperimentsshowedthatsubcutaneousprophylacticand therapeuticinversevaccinationwithPLGA-NPloadedwithMOG35-55andrIL-10significantlyameliorated thecourseofEAEinducedwithMOG35-55inC57BL/6mice.Moreover,theydecreasedthehistopathologic lesionsinthecentralnervoustissueandthesecretionofIL-17andinterferon(IFN)-␥inducedbyMOG35-55 insplenicTcellsinvitro.ThesedatasuggestthatsubcutaneousPLGA-NP-basedinversevaccinationmay beaneffectivetooltotreatautoimmunediseases.

Abbreviations: BCA,bicinchoninicacid;CHOs,Chinesehamsterovarycells;CNS,centralnervoussystem;CTL,cytotoxicTlymphocytes;DCM,dichloromethane;DLS,

dynamiclightscattering;EAE,experimentalautoimmuneencephalomyelitis;FDA,foodanddrugadministration;H&E,hematoxylin&eosin;IFN,interferon;IL,interleukin;

LPS,lipopolysaccharide;MCF-7,humanbreastadenocarcinomacellline;MBP,myelinbasicprotein;MFI-R,meanﬂuorescenceintensityratio;MRI,magneticresonance

imag-ing;MOG,myelinoligodendrocyteglycoprotein;MS,multiplesclerosis;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide;Ni-NTA,nickel-nitrilotriacetic

agarose;NIRF,near-infraredﬂuorescence;NK,naturalkiller;NP,nano/microparticles;OD,opticaldensity;PBMCs,peripheralbloodmononuclearcells;PBS,

phosphate-bufferedsaline;PDI,polydispersityindex;PLGA,polymericbiodegradablelactic–glycolicacid;PLP,proteolipidprotein;r,recombinant;PVA,poly/vinylalcohol;RR,relapsing

remittingform;RT,roomtemperature;s.c.,subcutaneousinjections;SEM,scanningelectronmicroscope;SE,standarderror;TH,Thelper;TEFF,effectorT;TGF,transforming

growthfactor;TNF,TumorNecrosisFactor;TREG,regulatoryT.

∗ Correspondingauthorat:InterdisciplinaryResearchCenterofAutoimmuneDiseases(IRCAD),andDepartmentofHealthSciences,UniversityofEasternPiedmont“A.

Avogadro”,viaSolaroli17,I-28100Novara,Italy.Tel.:+390321660658;fax:+39032162042.E-mailaddress:giuseppe.cappellano@med.unipmn.it(G.Cappellano).

http://dx.doi.org/10.1016/j.vaccine.2014.08.016

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5682 G.Cappellanoetal./Vaccine32(2014)5681–5689

1. Introduction

Autoimmunediseasesmaybeascribedtoadisturbedbalance betweentheactivatedeffector T(TEFF)cells that recognize self antigensandtheregulatoryT(TREG)cellsthatsuppressTEFF acti-vationand controlboththeimmuneandautoimmunereactions

[1–5].Severalimmunoregulatorymolecules,suchasIL-10,TGF-ß, andindoleamine2,3-dioxygenase(IDO),cansupportTREGfunction

[3,4,6,7].Inparticular,IL-10playsaroleinthedevelopmentand immunosuppressivefunctionofseveralTREGcelltypes[2]andisa crucialprotectivecytokineinseveralautoimmunediseases[3].

AdoptivetransferofseveraltypesofTREGcellsresultsin pro-tectiveactivityinanimalmodelsofautoimmunediseases,butthe translationofthesestudiestohumanshasbeenhamperedby sev-eralissues[8,9].Inparticular,thelowfrequencyofthesecells,their poorgrowthinculture,andtheirhighfunctionalplasticityinvivo havemadetheiruseasignificantchallenge.Antigen-specific strate-giesmaypotentiallycircumventtheselimitationsinvivo[10,11], buteffectiveapproaches torestoretheTEFF/TREGbalanceinvivo remainlargelyundefined[9].

Multiple sclerosis (MS) is characterized by an autoimmune responseagainsttheaxonsandmyelinsheathsofthecentral ner-voussystem(CNS)thatresultsinaxonallossanddemyelinization

[12].Severalmyelinproteins,suchasmyelinbasicprotein(MBP), proteolipidprotein(PLP),andmyelinoligodendrocyteglycoprotein (MOG),havebeenimplicatedastargetsofautoreactiveTcellsinMS anditsanimalmodel,experimentalautoimmune encephalomyeli-tis(EAE)[12].Accordingtothe“epitopespreading”model, few epitopesandantigensplayaroleintheinitiationorperpetuation ofthedisease,butthisreactivitytendstospreadduringthedisease course[13].

“Inverse vaccination” refers to antigen-specific tolerogenic immunization treatments that restore the TEFF/TREG balance andinhibit autoimmuneresponses. Inversevaccinationmaybe obtained by the prolonged treatment with high doses of the autoantigens.InEAE,systemicadministrationofmyelinantigens eitherpreventedortreatedEAE,butthedeliveryofrepeated mas-sivedoses of theseproteins were required for the therapeutic effect[14–16].ThisproblemhasbeenovercomebyDNA-based vaccinesencoding themyelinantigensalone orin combination withimmunomodulatorymolecules,suchasIL-4orIL-10,thatact as“adjuvants”forTREGfunction[17,18].TheEAEresultswereso promisingthattheypromptedafirstclinicaltrialinMSpatients, thatshowedsomepositiveresultsinreducingtheMRI-measured diseaseactivityandinducingantigen-specifictolerancetomyelin antigensinpatientswithrelapse-remitting(RR)MS[19].However, DNAvaccinationisstillcontroversialinMSsinceasecondphase 2trialshowednosignificantimprovementinmagneticresonance imaginglesionparametersandreductionofnewCNSlesionsonlyin thepatientsdisplayinghighconcentrationsofanti-MBPantibodies

[20].MoreoverwhileDNA-basedvaccinesallowforthesustained expressionofboththeantigensandtheadjuvantmolecules,itis notpossibletocontroltheirdoseorexpressionkinetics,andthis maybeaprobleminthecaseofadversereactions.

An alternative method to obtain the sustained release of antigensandadjuvantsmaybetoloadtheminsolid biodegrad-ableparticles.Polymericbiodegradablelactic–glycolicacid(PLGA) particlesareattractivecarriersduetotheirbiodegradability, bio-compatibilityandapprovalbytheFoodandDrugadministration (FDA)[21].Theymaintaineffectiveconcentrationsoftheloaded proteinsfor prolongedperiods of times by trappingthem in a hydratedpolymernetworkthat enablestheirslowrelease [21]. PLGAallowsfortheﬁne-tuningofthedegradationrateand sub-sequentantigenreleasefromseveraldaystomorethanoneyear by modulating the polymer lactide–glycolide ratio, the molec-ularweight, and thecrystal proﬁle [22]. The controlledand/or

sustainedreleaseofantigensandimmunemodulatorsfrom poly-mericmaterialshasbeenused previouslytoinducethe proper immuneeffectorresponse[23].TheuseofPLGA-NPcanenhance toleranceinductioninsomedeliverysettings,suchasnasal vacci-nation[24].Moreover,arecentstudyshowedthattheintravenous infusionofeitherpolystyreneorPLGAparticleschemically cou-pledwithanencephalitogenicpeptidecanpreventandcureEAE

[25].Thatstrategystemmedfrompreviousobservationsshowing thatautoantigenicpeptidescoupledtoapoptoticleukocytescan induceantigen-speciﬁctoleranceinmodelsofautoimmune dis-eases,allergy,andtransplantationbyinducingthedepletionand anergyoftheantigen-speciﬁclymphocytes.However,akey limi-tationofthisapproachwasthatitrequiredsystemic(intravenous) injectionofthevaccineandwasnoteffectiveuponlocal(suchas subcutaneous)injection[25].Thisimpliesthattheintrinsicrisksof lifethreateningadversereactions(suchassystemicanaphylaxis) mightdiscourageuseofthesevaccinesinhumans.

Theaimofthepresentstudywastodevelopanovelapproachfor inversevaccinationbasedonthesustainedreleaseofautoantigens andadjuvantsfromPLGA-NPdeliveredsubcutaneously.

2. Materialsandmethods

2.1. PLGAproductionandcharacterization

PLGANPwerepreparedbyamodifieddoublesolvent evapora-tionmethod[26].NanoparticlescontainingMOG35-55(MOG-PLGA), IL-10(IL10-PLGA),6-coumarin(C6-PLGA)orIR783(IR783-PLGA) fluorescentdyeswereproducedbyaddingMOG35-55(200␮g), IL-10(200_␮g),6-coumarin(C6)(6mg)orIR783(6mg)tothePLGA solutionduringthepreparation.ThereleaseofMOG35-55andIL-10 wereevaluatedbydialysisinPBSat37◦C,andtheproteinsreleased werequantifiedbytheBCAassay(forMOG35-55)andbyELISA(for rIL-10).DetailsaregiveninSupplementaryinformation.

2.2. Invitrobiologicalanalyses

Cell toxicity was assessed in humanbreast adenocarcinoma cellline(MCF-7)usingtheMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)assay.

Proinﬂammatoryactivitywasassessed onhumanperipheral bloodmononuclearcells(PBMCs)thatwereculturedwithPLGA particles and/or bacterial lipopolysaccharide (LPS; 1␮g/ml) for 48h.Then,TumorNecrosisFactor(TNF)-␣secretioninthe super-natantwasmeasuredbyELISA.

ThecellularuptakeofPLGA-NPwasinvestigatedinMCF-7cells incubatedwithC6-PLGA-NPat37◦Cfor4h.Then,thecellswere analyzedbyconfocallasermicroscopyandﬂowcytometry.

LymphocyteactivationwasassessedinEAEmousespleencells culturedinthepresenceandabsenceof10␮g/mlMOG35-55for5 d.Then,thesupernatantswerecollected,andthelevelsofIFN-␥, IL-10,andIL-17wereevaluatedbyELISA.

Histologic analysisof the EAElesions wasperformed inthe spinal cords ﬁxed in 10% formalin. Sectionswere stained with hematoxylin&eosin(H&E)andwithLuxolfastbluedyeformyelin staining. Detailsof thesemethods are given in Supplementary information.

2.3. Invivoanalyses

FemaleC57BL/6micewerepurchasedfromHarlan(Horst,The Netherlands)andwerehousedunderspeciﬁcpathogen-free con-ditionsattheanimalfacilityattheUniversityofEasternPiedmont “A.Avogadro”(Novara).Theexperimentswereperformedin accor-dancewiththeguidelinesoftheInstitutionalEthicalCommitteefor AnimalExperimentation.

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Fig.1. PhysicalandchemicalanalysisofPLGA-NP.(A)SEMmicrographsofblank-PLGAand(B)theparticlesizedistributionofblank-PLGA,IL10-PLGAandMOG-PLGA

assessedbyDLSanalysis;(C)Invitrocumulativerelease[%]ofMOG35-55andhumanIL-10fromPLGA-NPinPBS(pH7.4)at37◦C.Thedataareshownasthemean±SEfrom

2independentexperiments.

ThebiodistributionofPLGA-NPwasassessedinC57BL/6mice afterasubcutaneous(s.c.)injectionofIR783-PLGAorPBS.After7 d,themicewereeuthanized,andtheirorganswereanalyzedwith anIVIS®imagingsystem(PerkinElmer).

Prophylactic and therapeutic vaccination was performed in femaleC57BL/6micewithEAEinducedaspreviously described

[27]. Mice(4–8 weeksold) werevaccinated bys.c. injectionof PLGA-NPloadedwitheither2␮gMOG35-55ormouseIL-10.The NPweredissolvedin PBS,andtheinjectionsweregiveninone ﬂank either (i) on days-30 and -15 (prophylactic vaccination) fromEAE induction (day 0)or (ii)on days8 and 22 after EAE induction (therapeutic vaccination). Each experiment involved 5–10mice/group. Details are given in Supplementary informa-tion.

2.4. Statisticalanalysis

The statistical analyses were performed using the Mann–WhitneyU-testcalculatedwithGraphPadInstatsoftware (GraphPadSoftware,SanDiego,CA).

3. Results

3.1. PhysicalandchemicalcharacterizationofPLGA-NP

We produced PLGA 65:35 lyophilized nanoparticles that were unloaded (blank-PLGA) or loaded with either

MOG35-55 (MOG-PLGA) or rIL-10 (IL10-PLGA). PLGA-NP were dispersedindistilledwaterandanalyzedforsizedistributionand zetapotential;theparticles werealsoimagedusing ascanning electronmicroscope (SEM).Similarresultswere obtainedusing blank-PLGAparticlesandMOG-PLGAorIL10-PLGAparticles.The SEMshowedasphericalshapewithasmoothsurfacelackingany evidentirregularities(Fig.1A).Thesizedistributionwasevaluated bydynamiclightscattering(DLS)andranged100–1000nmwith apeakapproximately200nmandapolydispersityindex(PDI)of approximately0.2forallformulations(Fig.1B).Thisbehaviorcan berelated to thenegative chargedensity (␨-potential=−19mV forPLGA;−21.1forIL-10and-−24.9mVforMOG35-55)exposed onthesurfaceoftheparticlesasaconsequenceofthepresenceof carboxylatesmoietiesinthestructure.

The loadingefﬁciencyof MOG35-55 and IL-10wasevaluated bycalculating theamountof proteinrecoveredafter dissolving MOG-PLGAandIL10-PLGAinNaOH(1M)andneutralizingbyHCl (1N).Theresultsshowedthattheloadingefﬁciencywas approxi-mately23%forrIL-10and90%forMOG35-55.Thisdifferencemaybe ascribedtothedifferentsizesofthesemolecules(19kDaforrIL-10 and2.5kDaforMOG35-55).

The invitro release of humanIL-10or MOG35-55 from their respectivePLGA preparationswasassessed byincubating these particles inPBS at 37◦C and samplingthesupernatant weekly. TheinvitroreleaseproﬁlesshowedthatPLGAprovidedsustained releaseofeachproteinforseveralweekswithsimilarkineticsfor eachprotein(Fig.1C).

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3.2. BiologicalcharacterizationofthePLGA-NP

The cell toxicity of the PLGA preparations was assessed by performinganMTTassayonMCF-7cellsincubatedwithtitrated amounts(0.6,6, 60, and600␮g/ml)of blank-PLGA.Theresults showednosigniﬁcanttoxicityatanydose(datanotshown).

Thepro-inﬂammatory activityof blank-PLGAand IL10-PLGA wasevaluatedbyassessingtheabilityoftheparticlestoinducethe secretionofTNF-_␣inhumanPBMCs.HumanPBMCswereincubated withtitratedamounts(0,6,60,600␮g/ml)ofeachPLGA prepa-rationinthepresenceandabsenceofLPS(1␮g/ml),and TNF-␣ secretionintotheculturesupernatantwasassessedafter48hby ELISA.Atconcentrations≥60␮g/ml,theblank-PLGAinduced TNF-␣secretionandincreasedtheresponsetoLPS.Bycontrast,none oftheIL10-PLGAdosesinducedTNF-␣secretion,andeachdose inhibitedthatinducedbyLPS(Fig.2).

ThecellularuptakeofthePLGAnanoparticleswasassessedby incubatingMCF-7cellsfor4hwitheithercoumarin6(C6),a fluores-centlipophilicdyethatemitsintheFL-1channel,orC6-PLGA.The cellularuptakeofthefluorescentsubstancewasthenassessedby bothconfocalmicroscopyandflowcytometry.Theresultsshowed that C6-PLGA waspartially taken up byphagocytes in a dose-dependentmanner,andnosubstantialuptakewasdetectedwith C6alone(Fig.3AandB).

The in vivo biodistribution of the PLGA preparations was assessedbyinjectingmicesubcutaneouslywithPLGA-NPloaded withtheIR783infrareddye(IR783-PLGA)orPBS(control).After 7 d, mice were euthanized, and their organs were analyzed by ﬂuorescence imaging. As shown in Fig. 3C, strong signals were detected in the brain, heart, kidneys and, especially, the

Fig.2.TNF-␣productionwasassessedbyELISAinPBMCsculturedwith

blank-PLGAandIL-10(human)inthepresenceorabsenceoflipopolysaccharide(LPS)at

1␮g/ml.Statisticalanalysiswasperformedwiththenon-parametricANOVAtest;

thedataarethemean±SEfrom5experiments.*Signiﬁcantlydifferent(p<0.05)

fromblank-PLGA;signiﬁcantlydifferent(p<0.001)fromblank-PLGAplusLPS.

lungs.Mildsignalsweredetectedintheliver,spleenandlymph nodes.

3.3. InversevaccinationofmicewithEAE

WeevaluatedtheinvivoeffectoftreatmentswithMOG35-55 -based PLGA inverse vaccines in EAE induced in C57BL/6 mice.

Fig.3.CellularuptakeandbiodistributionofPLGA-NP.(A)and(B)MCF-7cellswereincubatedwith120␮g/mlofC6-PLGA-NPfor4h,andﬂuorescenceuptakewasassessed

byconfocallaserscanningmicroscopy(A)andﬂowcytometry(B);representativeresultsfromthreeindependentexperimentsareshown.(C)Micewereinjectedwith

IR783-PLGA,andtheheart(H),liver(L),spleen(S),lung(L),kidney(K),brain(B)andlymphnodes(Ly)werecollectedafter7dandanalyzedbynear-infraredﬂuorescence

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Fig.4.ProphylacticinversevaccinationofEAEmicewithdifferentPLGApreparations.Individual(upperpanels)andcumulative(lowerpanel)resultsfromtwoindependent

experiments.DailyclinicalscoresofEAEinmicetreatedwithblank-PLGA,MOG-PLGA,IL10-PLGAorMOG-PLGAplusIL10-PLGAondays-30and-15priortoEAEinduction.

Experiment1:5–8mice/group;experiment2:8–10mice/group.Resultsareshownasmean±SE.TheMann–WhitneyU-testwasusedtocomparetheclinicalscores:

IL10-PLGA+MOG-PLGAvsPLGA(*),IL10-PLGA(§),MOG-PLGA(#);*,§,#p<0.05.

Mice(n=5–10/group)werevaccinatedvias.c.injectionofeither blank-PLGA,MOG-PLGA,IL10-PLGAorMOG-PLGAplusIL10-PLGA in one flank ondays-31 and -15 prior to EAE induction (pro-phylacticinversevaccination).Theresultsfromtwoindependent experimentsshowedthat,atseveraltimepoints,treatmentwith MOG-PLGAplus IL10-PLGAsignificantly inhibited EAE develop-ment comparedto theothertreatments (Fig.4).Moreover,the average daily disease score calculated on the cumulative data fromboth experiments wassignificantly lower in micetreated with MOG-PLGA plus IL10-PLGA (mean±SE, 1.3±0.17) than in those treated with blank-PLGA (1.8±0.23, p<0.05) or IL10-PLGA(1.8±0.22, p<0.05). Asmall effectwasdisplayed alsoby MOG-PLGAalone (1.5±0.19)which inhibited EAE comparedto blank-PLGAandIL-10-PLGAaloneatsometimepoints,butno sig-nificantdifferencesweredetectedatthelevelofaveragedisease scores(Fig.4).Bycontrast,IL10-PLGAalonehadnoeffect.No vac-cinationdelayedthediseaseonset.

TheeffectofthevaccinationontheimmuneresponseandCNS lesionswasevaluatedbycomparingtheanti-MOGspleen lympho-cyteresponseandthehistopathologiclesionsinmicetreatedwith MOG-PLGAplusIL10-PLGAandthosetreatedwithblank-PLGA.The anti-MOGresponsewasassessedbyincubating spleen lympho-cytescollectedatday21(thediseasepeak)afterEAEinductionwith MOG35-55andassessingcellproliferation(by3H-thymidine incor-poration)andthesecretionofIFN-␥,IL-17,andIL-10intheculture supernatants(byELISA)aftera 5d culture.Theresultsshowed that vaccination with MOG-PLGA plus IL10-PLGA signiﬁcantly inhibitedthesecretionofIL-17andIFN-␥comparedtovaccination withblank-PLGA,whereas nosigniﬁcanteffect oncell prolifer-ationwasdetected(Fig.5A).ThesecretionofIL-10wasalways

undetectable(datanotshown);thisobservationisinaccordance withdatareportedbyotherauthors[28].

Thehistopathologicanalysiswasperformedonthespinalcords ofmicecollectedatday21afterEAEinduction.Lightmicroscopy analysis revealed that the mice treated with blank-PLGA dis-played a higher inflammatory cell infiltrate than those treated withIL10-PLGAplusMOG-PLGA,butthisdifferencewasnot sta-tisticallysignificant(Fig.5B).However,detectionofCD3+ _T_cells byimmunofluorescenceshowedthattheywerestrikinglymore abundantintheformerthaninthelattergroup.Moreover,myelin stainingshowedasignificantlydramaticlossofmyelininthemice treatedwithblank-PLGA,whereasmicetreatedwithMOG-PLGA plusIL10-PLGAshowedmildmyelindamageonlyinthesubpial myelin(Fig.5B).

Finally, we assessed theeffect oftreatment withthe differ-entPLGApreparationsdeliveredafterEAEinduction(therapeutic inversevaccination).Mice(n=8–10/group)wereinjecteds.c.with either blank-PLGA, MOG-PLGA, IL10-PLGA or MOG-PLGA plus IL10-PLGA on days 8 and 22 after EAE induction. The results from two independentexperiments showedthat, at mosttime points, treatmentwith MOG-PLGAplus IL10-PLGAsigniﬁcantly inhibitedEAE developmentcompared totreatmentwith blank-PLGAorIL-10-PLGA(Fig.6).Moreover,theaveragedailydisease scorecalculated onthecumulativedatafrombothexperiments wassigniﬁcantlylowerinthemicetreatedwithMOG-PLGAplus IL10-PLGA(mean±SE,1.2±0.15)comparedtothosetreatedwith blank-PLGA(1.8±0.24,p<0.05)orIL10-PLGA(1.8±0.23,p<0.05) orMOG-PLGA(1.6±0.20,p<0.05)(Fig.6).Bycontrast,MOG-PLGA andIL-10-PLGAhadnosubstantialeffect.Novaccinationdelayed thediseaseonset.

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Fig.5.TcellresponseandhistopathologiclesionsinEAEmicevaccinatedwithblank-PLGAorMOG-PLGAplusIL10-PLGA.(A)Spleenlymphocyteswerecollected21dafter

EAEinductionandculturedwithMOG35-55;cellproliferationandthesecretionofIFN-␥andIL-17intheculturesupernatantswereassessedaftera5dcultureperiod.(B)

Histopathologicanalysisofthespinalcordscollectedfrommice21dafterEAEinduction;dataareshownasrepresentativestainingsandquantitativeanalysisperformedin

multipleexperiments(bargraphs).H&Estaining:(a)blank-PLGA,(b)MOG-PLGAplusIL10-PLGA(arrowsmarktheinflammatoryinfiltrate).Immunofluorescencestainingof

CD3+_T_cells:_(c)_blank-PLGA,_(d)_MOG-10_plus_IL-10_PLGA;_KB_(myelin)_staining:_(e)_blank-PLGA,_(f)_MOG-PLGA_plus_IL10-PLGA;_arrows_indicate_myelin_vacuoles._{Magniﬁcation}

20×.Thedataarethemean±SEoftheresultsfrom6to8mice.StatisticalanalysiswasperformedwiththeMann–WhitneyU-test(*p<0.05).

4. Discussion

Thisworkshowsthats.c.inversevaccinationwithamixture ofPLGAloadedwithMOG35-55andIL-10iseffectiveatdecreasing diseaseseverityinmouseEAE.

Polymericmaterialshavebeeninvestigatedfortheirabilityto inducetheproperimmuneeffectorresponses,andinsomecases, topromotetolerance.Becausetheinductionandmaintenanceof immune toleranceis inﬂuenced bythe persistenceof the anti-genand can besupported bytolerogenic cytokines,theuse of biodegradablemicro-nanoparticlesisidealforinversevaccination becausetheycansupportasustainedreleaseofseveralentrapped molecules[23].

Anidealnanoparticleforinversevaccinationshoulddisplaylow celltoxicity,lowproinﬂammatoryactivity,sustainedreleaseofthe vaccineactivecomponents,andsloweliminationbyphagocytesto allowtheirpersistenceinvivotopromoteregulatoryratherthan

effectorimmunefunctions.TheMTTassayonMCF-7cellsshowed thatourparticleswerenottoxic,whichisinaccordancewithdata reportedbyothergroups[29].

Regarding the proinflammatory activity of PLGA, the data reportedintheliteraturearecontroversial.Nicoleteetal.showed thatPLGAnano/microparticlespersecanactivatetheinflammatory response;inparticular,microparticles(usedat1mg/ml)withasize rangingfrom5to7␮minducedtheproductionofhigheramounts ofTNF-␣inmurinemacrophagesthanthoseinducedby nanopar-ticles [30,31]. In our experimentalsetting, blank-PLGA induced TNF-␣productionatdoses≥60␮g/ml,butthisproinflammatory effectwasnotdetectedwhenusingIL10-PLGA;instead,these parti-clesinhibitedtheTNF-_␣secretioninducedbyLPS.However,itmust benotedthattheinvivoexperimentsshowedthattheEAEonset wasearlierintheprophylacticvaccinationthaninthetherapeutic vaccinationwhichsuggeststhatpretreatmentwiththePLGA-NP maypreconditiontheinductionofEAEbystimulatingtheinnate

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Fig.6. TherapeuticinversevaccinationofEAEmicewithdifferentPLGA-NPpreparations.Individual(upperpanels)andcumulative(lowerpanel)resultsfromtwoindependent

experiments.DailyclinicalscoresofEAEinmicetreatedwithblank-PLGA,MOG-PLGA,IL10-PLGAorMOG-PLGAplusIL10-PLGAonday8and22afterEAEinduction.

Experiment1:5–8mice/group;experiment2:8–10mice/group.Resultsareshownasmean±SE.TheMann–WhitneyU-testwasusedtocomparetheclinicalscores:

IL10-PLGA+MOG-PLGAvsPLGA(*),vsMOG-PLGA(#),IL10-PLGA(§),#,*,§,p<0.05.

immunity.ThisisinlinewithworksshowingthatPLGAparticles maysupportimmunizationinclassicalvaccinationsettings[32].

ThereleaseofMOG35-55andIL-10fromthePLGA-NPwas sus-tained,andbothmoleculeswerereleasedforseveralweeksinvitro. Theparticleswerepartiallyendocytosed,aneffectthatisbeneﬁcial forantigenpresentation,butthisdidnotsubstantiallyaffecttheir invivobiodistribution.

TheinvivoexperimentsinEAEshowedthatvaccinationwitha mixtureofMOG-PLGAandIL10-PLGAdisplayedasigniﬁcant pro-tectiveeffectanddecreasedtheseverityofthedisease.Thiseffect wasdetectednotonlywhenvaccinationwasperformedina pro-phylacticsetting,i.e.,beforeEAEinduction,butalsowhenitwas performedinatherapeuticsetting,i.e.,afterthediseaseonset.Thus, itmaybeapotentialtreatmentforpatientswithMS.

TheprotectiveeffectofIL10-PLGAandMOG-PLGAwas accom-paniedbydecreasedhistopathologiclesionsinthespinalcordand decreasedlymphocytesproductionofIFN-␥andIL-17-inresponse toMOG35-55invitro.Theseresultssuggestthatthisinverse vacci-nationinhibitstheautoantigen-speciﬁcTH1andTH17responses thatarecrucialinthepathogenesisofMSandEAE.Bycontrast, MOG-PLGAhadaminimaleffectthatwasdetectableonlyinthe prophylactictreatment.Moreover,IL10-PLGAhad noprotective effectinanyprotocol,whichisinaccordancewithpreviousdata fromCannellaetal.showingthattreatmentwithIL-10doesnot protectfromEAE[33].Theseresultsareinlinewiththoserecently reportedbyPeine etal.showingeffectivenessofEAE treatment bycodeliveryofMOGanddexamethasoneinacetalateddextran microparticles[34].Therefore,deliveryoftheautoantigentogether withimmunosuppressiveagents,suchasIL-10ordexamethasone, iscrucialtosuppresstheautoimmuneresponse.Dataobtainedwith tolerogenicDNAvaccinesindicatethattheseimmunosuppressive

agentsmayactas“inverseadjuvants”supportingtheinductionof tolerance.Lackofthese“inverseadjuvants”mayexplainthe fail-ureofclinicaltrialsusingautoantigensor“alteredpeptideligands” alonewhichwereterminatedbecauseoflackofsubstantialclinical effectsanddevelopmentofhypersensitivityreactions[35].

Itisnoteworthythatourvaccinationwaseffectivebythes.c. route.Thismarksa keydifferencefromtheinversevaccination performedbyGettsetal.withMOG35-55covalentlylinkedtothe surfaceofeitherpolystyrenebeadsorPLGAparticles[25].These authorsobtainedastrikingprotectiveeffectusingboth prophylac-ticandtherapeutictreatments,butthiseffectstrictlydependedon theintravenousdeliveryofthevaccine,whichmayrestrictitsuse inhumansbecauseofthepotentialriskofanaphylacticreactions. Theeffectofthisintravenousvaccineseemedtobemediatedby theinductionofTcellanergy,asindicatedbyastronginhibition oflymphocyteproliferation;thiswaspossiblydrivenby tolero-genicmacrophagestakinguptheantigenafterscavengerreceptors recognizedthenanoparticlescovalentlylinkedtotheselfpeptide. By contrast,ourvaccineuses particlesthat slowlyrelease both theautoantigenandIL-10.ItdoesnotinhibitTcellproliferation, butinsteadinhibitsthesecretionofIFN-␥andIL-17.Therefore,it seemstoinduceimmunedeviationinsteadofanergyandopens thewaytofurtherstudiesaimingtoamelioratethemixtureof autoantigensandimmunosuppressiveadjuvants.Ourdatadonot supportthepossibilitythattheprotectiveeffectwasmediatedby CD4+_CD25+_FoxP3+_T

REGcellssincetheirnumbersweresimilarin thespleensof micetreatedwithblank-PLGA andthose treated withIL-10-PLGAplusMOG-PLGA(datanotshown).Thisisinline withotherreports[34],butitdoesnotruleouttheinvolvement ofotherpopulationsofregulatorycellsorCD4+_CD25+_FoxP3+_T

REG cellscompartimentalizedinothertissues[36,37].

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The in vivo biodistribution of the PLGA-NP was intriguing becausesubstantialamountsweredetectableinseveralorgansone weekaftertheinjection.Thisbiodistributionisinaccordancewith worksshowingthatparticles<50nmrapidlydiffusefromthe injec-tionsite,whereasthose>1–5␮mtendtostayattheinjectionsite

[38].Becausethesizesofourparticlesrangedfrom100nmto1␮m, itisconceivablethatthesmallestofthemwerereadilyableto dif-fuse.Itisnoteworthythatamongtheotherorgans,thestrongest signalwasdetectedinthelungs.Thelungshavebeenshownto playakeyrole inlicensingTcells toenterCNS[39].Thesignal inthebrain isalso intriguingbecauseit suggeststhat the vac-cinemightexertanimmunoregulatoryeffectdirectlyatthesite oftheautoimmuneaggression.Thiseffectwasnotsimply ascrib-abletoanaspeciﬁcimmunosuppressiveactivityofIL-10because noeffectwasdetectedusingIL10-PLGAalone.Becausechanging theparticlesizewouldprofoundlyaffectthebiodistribution, fur-therworkisneededtoassesstheroleofthePLGAparticlesizein theimmunosuppressiveeffect.

Akeyproblemfortranslatingthisapproachtohumansisthat, inMS,thetargetautoantigensarenotfullyknownandmayinvolve molecules differentfrom MBP, MOG and PLP knownto play a roleinEAEandMS.Moreover,theautoreactivityisheterogeneous indifferentpatientsandchanges duringthediseasecoursedue toepitope spreading[12].However,severalworks showedthat tolerogenicvaccination,too,mayinduceanepitopespreadingof thetolerance and maygradually involve also autoantigens not includedinthevaccine.Moreover,ameliorationof thevaccines mightbeobtainedusingmixturesofautoantigens[40,41].

Inconclusion,thisworkshowedthatPLGA-basedinverse vac-cinationdelivered subcutaneously may be effective in treating autoimmunediseases. Current treatments for autoimmune dis-eases(i.e.,MS)arebasedonimmunosuppressive therapies that affecttheglobalimmuneresponseanddisplaysigniﬁcanttoxicity, especiallywhenchronictherapyisneeded.Evennovel biotechno-logicaldrugsdonotsolvethisproblembutinsteadaddthefurther problemofadramaticincreaseintheeconomiccostsofthe ther-apy.Effectivetherapieswithlowcostandlowtoxicityareneeded bypatients and by thepublichealth systems.Inverse vaccina-tionwould meetthesecriteriabecauseitiscapableofinducing anautoantigen-speciﬁcimmunosuppressionwiththepotentialof beinglowcostandlonglasting.

Conﬂictofinterest

Theauthorsstatethatthereisnoconﬂictofinterest.

Acknowledgments

ThisworkwassupportedbyCompagniadiSanPaolo(Torino), FondazioneCariplo(Milano),FondazioneAmicidiJean(Torino), AssociazioneItalianaRicercasulCancro(Milano),FondazioneCassa diRisparmiodiCuneo.

AppendixA. Supplementarydata

Supplementarymaterialrelated tothis article canbefound, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2014.08.016.

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