Adjuvant-carrying synthetic vaccine particles
augment the immune response to encapsulated
antigen and exhibit strong local immune activation
without inducing systemic cytokine release
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Citation
Ilyinskii, Petr O., Christopher J. Roy, Conlin P. O’Neil, Erica A.
Browning, Lynnelle A. Pittet, David H. Altreuter, Frank Alexis, et
al. “Adjuvant-Carrying Synthetic Vaccine Particles Augment the
Immune Response to Encapsulated Antigen and Exhibit Strong
Local Immune Activation Without Inducing Systemic Cytokine
Release.” Vaccine 32, no. 24 (May 2014): 2882–2895.
As Published
http://dx.doi.org/10.1016/j.vaccine.2014.02.027
Publisher
Elsevier
Version
Final published version
Citable link
http://hdl.handle.net/1721.1/91494
Terms of Use
Creative Commons Attribution
ContentslistsavailableatScienceDirect
Vaccine
jou 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
Adjuvant-carrying
synthetic
vaccine
particles
augment
the
immune
response
to
encapsulated
antigen
and
exhibit
strong
local
immune
activation
without
inducing
systemic
cytokine
release
Petr
O.
Ilyinskii
a,∗,
Christopher
J.
Roy
a,
Conlin
P.
O’Neil
a,
Erica
A.
Browning
a,
Lynnelle
A.
Pittet
a,
David
H.
Altreuter
a,
Frank
Alexis
b,1,
Elena
Tonti
c,2,
Jinjun
Shi
b,
Pamela
A.
Basto
d,e,
Matteo
Iannacone
c,2,
Aleksandar
F.
Radovic-Moreno
d,e,
Robert
S.
Langer
d,e,
Omid
C.
Farokhzad
b,
Ulrich
H.
von
Andrian
c,
Lloyd
P.M.
Johnston
a,
Takashi
Kei
Kishimoto
aaSelectaBiosciences,Watertown,MA02472,USA
bLaboratoryofNanomedicineandBiomaterials,BrighamandWomen’sHospital,Boston,MA02115,USA cDepartmentofMicrobiologyandImmunobiology,HarvardMedicalSchool,Boston,MA02115,USA dDavidH.KochInstituteforIntegrativeCancerResearch,Cambridge,MA02139,USA
eHarvard-MITDivisionofHealthSciencesandTechnology,Cambridge,MA02139,USA
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Availableonline1March2014 Keywords:
Syntheticnanoparticlevaccine TLRagonist Adjuvant CpG R848 Resiquimod
a
b
s
t
r
a
c
t
Augmentationofimmunogenicitycanbeachievedbyparticulatedeliveryofanantigenandbyits co-administrationwithanadjuvant.However,manyadjuvantsinitiatestrongsystemicinflammatory reactionsinvivo,leadingtopotentialadverseeventsandsafetyconcerns.Wehavedevelopedasynthetic vaccineparticle(SVP)technologythatenablesco-encapsulationofantigenwithpotentadjuvants.We demonstratethatco-deliveryofanantigenwithaTLR7/8orTLR9agonistinsyntheticpolymer nanopar-ticlesresultsinastrongaugmentationofhumoralandcellularimmuneresponseswithminimalsystemic productionofinflammatorycytokines.Incontrast,antigenencapsulatedintonanoparticlesandadmixed withfreeTLR7/8agonistleadstolowerimmunogenicityandrapidinductionofhighlevelsof inflam-matorycytokinesintheserum(e.g.,TNF-␣andIL-6levelsare50-to200-foldhigheruponinjectionof freeresiquimod(R848)thanofnanoparticle-encapsulatedR848).Conversely,localimmunestimulation asevidencedbycellularinfiltrationofdraininglymphnodesandbyintranodalcytokineproductionwas morepronouncedandpersistedlongerwhenSVP-encapsulatedTLRagonistswereused.Thestronglocal immuneactivationachievedusingamodularself-assemblingnanoparticleplatformmarkedlyenhanced immunogenicityandwasequallyeffectivewhetherantigenandadjuvantwereco-encapsulatedina singlenanoparticleformulationorco-deliveredintwoseparatenanoparticles.Moreover,particle encap-sulationenabledtheutilizationofCpGoligonucleotideswiththenaturalphosphodiesterbackbone,which areotherwiserapidlyhydrolyzedbynucleasesinvivo.TheuseofSVPmayenableclinicaluseofpotent TLRagonistsasvaccineadjuvantsforindicationswherecellularimmunityorrobusthumoralresponses arerequired.
©2014TheAuthors.PublishedbyElsevierLtd.
Abbreviations:DC,dendriticcell;LN,lymphnode;ODN,oligonucleotide;PAMP,pathogen-associatedmolecularpattern;PAP,prostaticacidphosphatase; PO,phosphodiester;PS,phosphorothioate;PRR,patternrecognitionreceptor;SVP,syntheticvaccineparticle;Tfh,follicularhelperTcells.
∗ Correspondingauthor.Tel.:+16172318118;fax:+16179243454. E-mailaddress:PIlyinskii@selectabio.com(P.O.Ilyinskii).
1 Currentaddress:RhodesResearchCenter,ClemsonUniversity,Clemson,SC29634,USA.
2 Currentaddress:DivisionofImmunology,TransplantationandInfectiousDiseases,SanRaffaeleScientificInstitute,20132Milan,Italy.
http://dx.doi.org/10.1016/j.vaccine.2014.02.027
0264-410X/©2014TheAuthors.PublishedbyElsevierLtd.Open access under CC BY-NC-ND license.
1. Introduction
Vaccine adjuvants augment the immune response by pro-moting more effective antigen processing, presentation,and/or delivery[1].Aluminumsalts(alum)werefirstintroducedas vac-cineadjuvantsover 80years ago whenlittlewasknownabout thecellularor molecularmechanisms of theimmune response
[2],yetalumremainsthemostwidelyusedadjuvanttodaydue toitsdemonstrated safetyprofileand effectivenesswhen com-binedwithmanyclinicallyimportantantigens[3,4].However,alum isnotsufficientlypotenttoattainprotectiveresponsestopoorly immunogenicentities[5–9].Additionally,alumpreferentially pro-motesTh2typeresponses[2–4,10],whichmayexacerbateadverse inflammatoryreactionstosomerespiratorypathogens,suchasthe respiratorysyncytialvirus(RSV)[11],anddoesnotefficiently aug-mentcytotoxicTcellresponses,whicharenecessarytoprovide protectiveimmunityagainst manyviralantigensortherapeutic immunityagainstcancer-relatedantigens[12].Oneofthemain challengesofcurrentvaccinedevelopmentistoadvancethe clin-icalapplicationofnewlydevelopedandpotentadjuvantswithout compromisingsafety[12,13].
Noveladjuvantcandidateshaveemerged fromthediscovery ofpatternrecognitionreceptors(PRR)that recognize pathogen-associated molecular patterns (PAMP) and damage-associated molecularpatterns(DAMP) [14–17].ResearchonPRRshas pro-videdinsightintohowtheinnatesystemactivatesandmodulates the adaptive immune system [16]. Microbial PAMPs, such as lipopolysaccharides, single-stranded RNA, and bacterial DNA motifs,bindtoafamilyofPRRscalledToll-likereceptors(TLR)on innateimmune cells andstimulate antigenprocessingand pre-sentation[16–18].TLRs arewidelyexpressedondendritic cells (DC) and other professional APCs such as macrophages and B cells.WhilesomeTLRsareexpressedonthecellsurfaceandact as sensorsfor extracellular PAMPs (e.g.,lipopolysaccharides), a subsetofTLRmolecules(TLR3,7,8and9)areexpressedon endo-somalmembranesandbindnucleicacid-derivedmolecules,suchas single-strandedRNAofviraloriginforTLR7and8[19–24]and bac-terialunmethylatedDNAoligonucleotides(ODNs)containingCpG motifs(CpGODNs)forTLR9[14,25–28].TLRligandsofnaturaland syntheticoriginarepotentinducersofinnateimmuneresponses andhavebeenshowntoeffectivelystimulatethetransitionfrom aninnateimmuneresponsetoanadaptiveimmuneresponse.As such,TLRagonistshavebeenevaluatedaspotentialadjuvantsina varietyofapplications[4].
Todate,onlyonePRRligand,3-O-desacyl-4-monophosphoryl lipidA(MPL),aTLR4agonist,hasbeenincludedasanadjuvant ina FDA-orEMA-licensedvaccine.MPLadsorbedonto alumis utilized in the HPV vaccine Cervarix, licensed in the U.S. and Europe[29],andthehepatitisBvaccineFendrix,licensedinEurope
[30].Imiquimod,atopicallyadministeredTLR7agonist,hasbeen approvedfortreatmentofgenitalwarts,actinickeratosis,andbasal cellcarcinoma[31].OtherTLRagonists,suchaspoly(I:C)(TLR3), imidazoquinolinesotherthanimiquimod(TLR7,8,or7/8),andCpG ODNs(TLR9),havefailedthusfartoenterclinicalpracticeas par-enteraladjuvantsdespiteamultitudeofpromisingdataobtained inpreclinicalandclinicalstudies[32–36].Oneofthemain rea-sonsforthisfailureisthedelicatebalancebetweentheinduction ofaugmentedimmunogenicitybyTLRagonistsandsafetyconcerns, whichareoftenrelatedtothegenerationofsystemicinflammatory responses[19,37–39].
Severalgroupshaveutilizedmicro-andnanocarriers,suchas virus-likeparticles,liposomes,andPLGAparticles,toencapsulate adjuvants[40–42].Encapsulationofadjuvantsreducessystemic exposureof adjuvant and enhancesuptake by APCs. Nano-size virusesandparticlesdistributerapidlytothelocaldraininglymph nodewheretheyaretakenupbysubcapsularmacrophagesand
dendriticcells[41,43,44].Antigenscanalsobedeliveredin par-ticles to target efficient uptake by APCs [36,41,45,46]. Recent studiesshowthatco-encapsulationofbothantigenandadjuvant innanoparticulatecarriershavesynergisticeffectsinaugmenting immunitybytargetingbothantigenandadjuvanttotheendosomal compartmentofAPCs[36,42,46].
In this study, we evaluated the immune responses induced by synthetic vaccine particles (SVP) carrying covalently bound orentrappedTLRagonistco-deliveredwithencapsulatedantigen (eitherinthesameorinseparatenanoparticlepreparations).We hypothesizedthatsuchanapproachmayprovideatwo-pronged benefitbyenablingafocuseddeliveryofantigenandadjuvantand henceenhancingimmunogenicitywhilepreventingsystemic expo-sureof theTLRagonist,which canresultin excessivesystemic cytokine release. Indeed, encapsulation of TLR agonistchanged the dynamics of cytokine induction in vitro and in vivo. Sys-temiccytokineproductionobservedwithfreeresiquimod(R848) wassuppressedbyitsencapsulationwithinnanoparticles.Atthe sametime,SVP-encapsulatedTLRagonists,butnotfreeTLR ago-nists,promotedsustainedcytokineinductioninthelocaldraining lymph nodeas wellasa robust infiltration byAPCs and, later, by antigen-responsivecells. SVP-encapsulatedTLR7/8and TLR9 ligands augmented humoral and cellular immune responses to bothsolubleandnanoparticle-deliveredproteincomparedtothat observedwithfreeadjuvants.Furthermore,thisaugmentationdid notrequireco-encapsulationofantigenandTLRagonistinthesame SVP.Collectively,thesedataindicatethatSVPsmayenabletheuse ofpotentTLRagonistsasnoveladjuvantsbytargetingtheir activ-itytothedraininglymphnodeandminimizingsystemicexposure, therebyreducingadjuvant-relatedsideeffects.
2. Materialsandmethods
2.1. Mice
Six-toeight-week-oldfemaleC57BL/6micewerepurchased fromCharlesRiverLaboratories(Wilmington,MA,USA)orTaconic (Germantown,NY,USA).Allanimalprotocolswerereviewedand approvedbyIACUCinaccordancewithfederal,stateandcityof Cambridge(MA,USA)regulationsandguidelines.
2.2. Cellsandreagents
FreshmurinesplenocyteswerecultivatedinRPMIwith10%FBS andwereassayedin96-wellplatesat20,000–50,000cells/well.Cell linesJ774 (murinemacrophages),EL4 (H-2bmurinethymoma), and E.G7-OVA (EL4 cells transfected with full the length gene encoding chickenOVA)werepurchased fromtheATCC (Ameri-canTypeCultureCollection,Rockville,MD,USA)andgrownper manufacturer’srecommendations.R848waspurchasedfromEnzo LifeSciences(Farmingdale,NY,USA)orPrincetonGlobal Synthe-sis (Bristol, PA, USA). Phosphorothioate (PS) or phosphodiester (PO) forms of CpG-1826(5-TCCATGACGTTCCTGACGTT-3) were purchased eitherfromEnzoLifeSciencesor fromOligoFactory (Holliston,MA,USA).OVAwaspurchasedfromWorthington Bio-chemicalCorporation(Lakewood,NJ,USA).Recombinantprostatic acidphosphatase(PAP)wasexpressedinEscherichiacoliand puri-fiedbyVirogen(Watertown,MA,USA).Aluminumhydroxidegel (alum)waspurchasedfromSigma–Aldrich(St.Louis,MO,USA).
Thepoly(lactide)orpoly(lactide-co-glycolide)polymers(PLAor PLGA)werepurchasedfromLakeshoreBiomaterials(Birmingham, AL,USA)unlessotherwisespecified.Poly(lactide)-bl-poly(ethylene glycol)monomethyletherdiblockcopolymer(PLA-PEG-OMe)was prepared according to the literature [47,48]. Dichloromethane (CH2Cl2),acetonitrile,HPLCgradewater,triethylamine(TEA),and
trifluoroaceticacid(TFA)werepurchasedfromVWRInternational (Radnor,PA,USA).Dimethylsulfoxide(DMSO)waspurchasedfrom Sigma–Aldrich.FluorescaminewaspurchasedfromTokyo Chemi-calIndustryAmerica(Waltham,MA,USA).CellgroPBS1X(PBS)was purchasedfromMediatech,Inc.(Manassas,VA,USA).PLGA-R848 polymerwasprepared byPrinceton GlobalSynthesis. Polyvinyl alcoholwaspurchasedfromEMDMillipore(Billerica,MA,USA). 2.3. SVPpreparationandanalysis
All of the SVP were prepared using a double emulsion water/oil/watersystem[49].Briefly,thepolymerswereprepared at10%wt/volinCH2Cl2,andOVAwaspreparedat50mg/mLin PBS.InformulationswithoutOVA,wesubstitutedtheOVA aque-ousphasewithPBS.Emulsificationviasonicationwasperformed usingaBransonDigitalSonifiermodel250equippedwithamodel 102Cconverteranda1/8taperedmicrotipfromBranson Ultra-sonics(Danbury,CT,USA).Centrifugationwascarriedoutusinga BeckmanCoulterJ-30IcentrifugewithaJA-30.50rotor(Beckman Coulter,Brea,CA,USA).Theprimaryemulsionwascarriedoutina thickwalledglasspressuretubewithanaqueoustoorganicphase ratioof1:5.Followingabriefsonicationstep,EmprovePVA4–88 aqueoussolutionwasaddedtothepolymerorganicsolution(at avolumeratioof3:1PVAtoorganicphase),vortexmixed,and emulsifiedbysonication.Theresultantdoubleemulsionwasthen transferredintoabeakerunderstirringcontaining70mM phos-phatebufferpH8.0atavolumeratioof1partdoubleemulsion to7.5partsbuffer.Theorganicsolvent(CH2Cl2)wasallowedto evaporatefor2hunderstirring,andthenanoparticleswere recov-eredviacentrifugationat75,600rcfwithtwowashsteps.PBSwas usedforthewashsolutionsandthefinalresuspensionmedia.The washedSVPsuspensionwasstoredat−20◦C.
DeterminationofOVA loadingwasperformedusing the flu-orescamine test from Udenfriend et al. [50]. R848 and CpG loading were each determined by SVP hydrolysis followed by reversed-phaseHPLCanalysis.Briefly,nanoparticlesolutionswere centrifuged,andthepelletsweresubjectedtobasehydrolysisto release theadjuvant. R848 hydrolysis wascarried out at room temperature usingconcentrated ammoniumhydroxide. Results were quantified from the absorption of R848 at 254nm using mobilephasescomprisedofwater/acetonitrile/TFA.ForCpG analy-sis,NaOHwasusedatelevatedtemperature,withresultsquantified fromtheabsorptionofCpGat260nm.TheHPLCmobilephasesfor CpGanalysisusedacetonitrile/water/TEA.
TheSVPconcentrationwasdeterminedgravimetrically.Briefly, aliquotsofSVPwerecentrifugedat108,800rcftopelletoutthe nanoparticles.Thesupernatantwasdiscarded,andthepelletwas dried(72h,37◦C,atmosphericpressure).SVPconcentrationswere calculatedbycomparingtheweightofthemicrotubeateachofthe followingsteps:empty,withthenanoparticlesolution,withthe supernatantdiscarded,andthenaftertheincubatordryingstep. 2.4. Vaccination
Groupsof3–10micewereinjecteds.c.inthehindlimbwithPBS vehiclecontainingSVP-formulatedorfreeantigensandadjuvants eitherinbothlimbs(30lvolumeperasingleinjectionsite,60l total)orinasinglelimb(60ltotalvolume).ThestandardSVP injectiondosewas100gperanimal(unlessspecifiedotherwise). A single time-point injection was used in cytokine production andTcellinductionexperiments,andprime-boostregimens(2–3 immunizationswith14or28-dayintervals;detailedinfigure leg-ends)wereusedin experimentsassessing antibodygeneration. Intranasalinoculationinbothnares(60ltotalvolume)wasdone atasingletime-pointunderlightanesthesia.
2.5. DeterminationofantibodytitersbyELISA
96-Well Costar plates (CorningInc., Corning,NY, USA)were coatedwith100lperwellofOVAprotein(5g/mL)orprostatic acidphosphatase(PAP)protein(1g/mL;Virogen)andincubated overnight at 4◦C. Plates were washed three times with 0.05% Tween-20inPBS,300ldiluent(1%caseininPBS;ThermoFisher, Waltham,MA,USA)wasaddedtoeachwelltoblocknon-specific binding,andplateswereincubatedforatleast2hatroom tem-perature(RT).Plateswerewashedasdescribedabove,andserum sampleswereseriallydiluted3-folddowntheplateandincubated for2hatRT.Twocolumnsofstandardswereincludedoneachplate (anti-OVA monoclonal antibody, Abcam, Cambridge, MA, USA) startingat0.25g/mLanddiluted3-folddowntheplate.Naive mouseserumwasusedasanegativecontrol.Plateswerewashed anddetectionantibody(eitherbiotinylatedgoatanti-mouseIg(BD Biosciences,SanJose,CA,USA)orhorseradishperoxidase (HRP)-conjugatedgoatanti-mouseIgG(Abcam))wasaddedtoeachwell. Forantibodyisotyping,goatanti-mouseIgG1(SouthernBiotech, Birmingham,AL,USA)andanti-mouseIgG2c(BethylLaboratories, Montgomery,TX,USA)(bothHRP-conjugated)wereused.Plates wereincubatedinthedarkfor1hatRTandwashed(threetimes, withat leasta 30-s soak betweeneach wash). For plates with biotinylatedantibodies,plateswereincubatedfor30mininthe darkatRTwithstreptavidin–HRP(BDBiosciences)and washed (threetimes,withatleasta30-ssoakbetweeneachwash).TMB substrate(BDBiosciences,SanJose,CA,USA)wasadded,andplates wereincubated for10 or 15minin thedark. Thereaction was stoppedbyaddingstopsolution(2NH2SO4)toeachwell,andthe ODwasmeasuredat450nmwithsubtractionofthe570nmreading usingaVersamaxplatereader(MolecularDevices,Sunnyvale,CA, USA).DataanalysiswasperformedusingSoftMaxProv5.4 (Molecu-larDevices).Afour-parameterlogisticcurve-fitgraphwasprepared withtheserumdilutiononthex-axis(logscale)andtheODvalue onthey-axis(linearscale),andthehalfmaximumvalue(EC50)for eachsamplewasdeterminedtocalculateantibodytiter.
2.6. Lymphnodepreparationforlymphocytecultivation
Fourdaysposts.c.injectionwithSVPorfreeantigen(aloneor withTLRagonist),miceweresacrificed,drainingpopliteallymph nodes aseptically removed and digested for 30min at 37◦C in 400U/mLcollagenasetype4(Worthington,Lakewood,NJ,USA). Singlecellsuspensionswerepreparedbyforcingdigestedlymph nodesthrougha70-mnylonfiltermembrane,thenwashedin PBScontaining2%FBSandcountedusingaCountess®cellcounter (LifeTechnologies,Carlsbad,CA,USA).Lymphnodederived lym-phocyteswerethen seeded at 5×106cells/mLin 96-wellplate (round-bottom)andculturedforanadditional4daysinRPMI-1640 supplementedwith10%(v/v)heatinactivatedFBS,10U/mL recom-binanthumanIL-2,50M2-ME,andantibiotics(penicillin-Gand streptomycinsulphate,bothat100IU/mL).
2.7. Invitrocytotoxicity
OVA specific cytolytic activity in vitro was determined via lactatedehydrogenase(LDH)releaseCytoTox96Assay(Promega, Madison, WI, USA) according to manufacturer’s recommenda-tions. Briefly, effector lymphocytes were cultured in limiting dilution either alone or with appropriate target cells, EL4 or E.G7-OVA at 37◦C for 18h. CTL activitywas assessed by mea-suring relative LDH with maximum and spontaneous release values measured against LDH within supernatants of effector target combinations. Specific lysis was calculated as follows:
R848 (nM) 1 10 100 1000 10000 TN F-αα (pg/ ml ) 0 1000 2000 3000 4000 5000 6000 SVP-R848 R848, free R848 (nM) 0.1 1 10 100 1000 10000 TN F-αα (pg /m l) 0 20 40 60 80 100 120 140 SVP-R848 R848, free PS-CpG 1826 (nM) 0.1 1 10 100 1000 TN F -αα (pg /m l) 0 200 400 600 800 1000 1200 SVP-PS-CpG 1826 PS-CpG 1826, free
A
B
C
D
CpG 1826 (nM) 1 10 100 1000 10000 TN F-αα(pg/ m l) 100 1000 10000 SVP-PS-CpG 1826 PS-CpG 1826, free SVP-PO-CpG 1826 PO-CpG 1826, freeFig.1.Adjuvantencapsulationinnanoparticles(SVP)resultsinsuppressedTNF␣inductioninmurinecellsinvitro.SVPcontainingTLR7/8agonistR848(A,B)orTLR9agonists PO-CpG1826(C)orPS-CpG1826(D)wereaddedtoJ774cells(A,C)orfreshmousesplenocytecultures(B,D)inparallelwithfreeadjuvantsintriplicates.Theamountof TNF-␣inculturesupernatantswasmeasured6h(splenocytes)or16h(J774)afterincubation.Representativeresultsoutoftwoseparateexperimentswithgroupsofthree miceareshown.
percent specific lysis (%)=100×[(experimental−T cell sponta-neous)/(targetmax−targetspontaneous)].
2.8. Invivocytotoxicity
OVA-specific cytolytic activity in vivo was determined as described[51]at6daysafterasingleimmunization.Briefly, spleno-cytesfromsyngeneicnaïvemicewerelabeledwitheither0.5M, or5MCFSE,resultinginCFSElowandCFSEhighcellpopulations, correspondingly.CFSEhigh cells wereincubatedwith1g/mL of SIINFEKLpeptideat37◦Cfor1h,whileCFSElow cellswere incu-batedinmediumalone.Bothpopulationsweremixedina1:1ratio andinjectedintoimmunizedorcontrolanimals(i.v.,2.0×107cells total).After18-hincubation,spleenswereharvested,processed and analyzed by flow cytometry. Specificcytotoxicity was cal-culatedbasedonacontrolratioofrecovery(RR)innaïvemice: (percentageofCFSElowcells)/(percentageofCFSEhighcells).Percent specificlysis(%)=100×[1−(RRofcellsfromnaivemice/RRofcells fromimmunizedmice)or100×[1−(RRnaive/RRimm)].
2.9. Determinationofcytokineconcentrationinseraandculture supernatants
Free or SVP-encapsulatedTLR agonists were seriallydiluted in tissue culture medium and added to J774 cells or fresh murine splenocytes. Culture supernatants were collected after 6–48handassayedforTNF-␣andIL-6byELISA(BDBiosciences, CA, USA). Local cytokine secretion was determined in culture
Fig.2.NanoparticleencapsulationofbothantigenandTLR7/8agonist(R848), whetherco-encapsulatedorinseparateparticles,resultsina higherhumoral immuneresponsethanutilizationoffreeTLR7/8agonistand/orfreeantigen.Groups of5–10micewereimmunized(3times,4-weekintervals,s.c.,hindlimb)with variouscombinationsofSVP-OVAorfreeOVAand/orSVP-R848orfreeR848,as indicated.ThesamedoseofOVA(5.6g)wasusedinallexperimentalgroupsand thesamedoseofR848(6.8g)wasusedinallgroupsinwhichR848was admin-istered.AntibodygenerationwasmeasuredbyELISAattheindicatedtime-points andispresentedaslog10ofEC50withstandarddeviations.
Fig.3.NanoparticleencapsulationofbothantigenandTLR7/8agonist(adjuvant)resultsinhigherlocalandsystemiccellularimmuneresponsesthanutilizationoffree antigenoradjuvant.A–totaldraininglymphnode(LN)cellcount,B–SIINFEKL-positiveCD8+Tcellsindraininglymphnode(absolutenumbers–graybars,leftY-axis;
percentageoftotalCD8+Tcells–hatchedbars,rightY-axis),C–flowcytometricanalysisofSIINFEKL-positiveCD8+Tcellsexvivo,D–flowcytometricanalysisof
SIINFEKL-positiveCD8+Tcellsafterinvitroexpansion,E–invitrocytotoxicactivityofLN-derivedcellsagainstOVA-expressingtargetcells,andF–invivocytotoxicityagainst
SIINFEKL-pulsedsyngeneicsplenocytes.Groupsof3–6micewereinjected(s.c.,hindlimb)eitherwithSVP-encapsulatedorfreeOVAand/orR848incombinationsindicated. At4daysafterinjection,totalcellsinthepoplitealLNswereisolatedandcounted(A).LNcellswerethenstainedforsurfacemarkerseitherimmediately(B,C)orafter4 daysofinvitroexpansiononIL-2withoutfeeder/stimulatorcells(D).Thepercentageofantigen-specificCTLstainedwithSIINFEKL-MHCclassIpentamerandanti-CD8 antibodywasassessedbyflowcytometry(indicatedinboxesinCandD;samplesfromtworepresentativeanimalsshown).Invitrocytotoxicactivitywasassessedafter invitroexpansionofLNcells,asdescribed.Thedifferencebetweenspecific(E.G7-OVAtransfectedtargetscells)andnon-specific(EL-4parentalcells)cytotoxicityisshown (E).TheamountoffreeorSVP-encapsulatedOVAandR848ing/injectiondoseisindicatedinparentheses.Theeffector-to-targetratio(E:Tratio)isdepictedinthex-axis. InvivoCTLactivity(F)wasassessedinmice(n=2–4/group)injecteds.c.withSVP-encapsulatedorfreeOVAeitheraloneorwithfreeorSVP-encapsulatedR848,asindicated. ThetotaldoseofOVAwasthesameacrossalltreatmentgroups,andthetotaldoseofR848wasthesameforallgroupsreceivingR848.Specificinvivocytotoxicitywas determinedasdescribedinSection2at6dayspost-immunization.Allexperimentspresentedinthisfigurewererun2–3times.
Time after injection (hrs) 0 4 24 48 72 IF N-γγ, pg /m l 0 5000 10000 15000 20000 25000 30000
Time after injection (hrs)
0 4 24 48 72 RA NT ES, p g /m l 0 200 400 600 800 1000 1200 1400 1600 1800 SVP-OVA-R848 SVP-OVA SVP-OVA + R848, free
Time after injection (hrs)
0 4 24 48 72 IL -12 (p40 ), p g/ml 0 20 40 60 80 100 120 140
Time after injection (hrs)
0 4 24 48 72 IL-1ββ , pg/m l 0 20 40 60 80 100 120
A
B
C
D
Fig.4. LocalcytokinesareinducedbySVP-encapsulated,butnotfree,TLR7/8agonistR848.Micewereinjecteds.c.inbothhindlimbswitheitherSVP-OVA-R848,SVP-OVA, orSVP-OVAadmixedwithfreeR848.Atthetimesindicated,poplitealLNsweretakenandincubatedinvitroovernight.Culturesupernatantswerecollectedandanalyzed forthepresenceofspecificcytokinesbyELISA.A–IFN-␥,B–RANTES,C–IL-12(p40),D–IL-1.AverageoffourLNspertime-pointisshown.
Table1
LocallymphadenopathyinductionbyadministrationofSVP-encapsulatedbutnot freeTLR7/8agonist.
Day SVP-R848 SVP(noadjuvant) SVP(noadjuvant)+R848
0 1.00 1.00 1.00
1 1.50 1.36 1.08
4 3.19 1.53 1.03
7 8.06 1.53 0.84
Micewereinjectedsubcutaneously(hindlimb)withSVP-R848,SVP(noadjuvant) orSVP(noadjuvant)admixedwithfreeR848.PoplitealLNsweretakenatvarious timesasindicated.Totallymphnodecells(normalizedtoday0values)inallgroups (averageof3micepertime-point)areshown.
supernatantsafterbriefinvitroincubationofdraininglymphnodes (LNs) fromimmunized animals. Briefly, poplitealLN were har-vestedatdifferentintervalsafterinjectionofSVP,freeTLRagonist, or PBS and were incubatedindividually (completeLN, without processing)for16hin 300lofsupplemented RPMI-1640with FBSasdescribedabove.Culturesupernatantswerethenassayed
Table2
LocallymphadenopathyafteradministrationofSVP-encapsulatedTLR7/8agonist andantigeninnaïvevs.pre-immunizedmice.
Day SVP-R848,totalcellsinLN
Naïvemice Immunemice
0 1.00 1.00
3 4.33 10.14
8 8.04 9.86
14 5.36 6.93
21 4.14 6.60
Naive orpreviously immunizedmice were injectedwith SVP-R848 with co-encapsulatedantigen(hindlimb,s.c.).PoplitealLNsweretakenatvarioustimes asindicated.Totallymphnodecellcounts(normalizedtoday0values)inallgroups (averageof3micepertime-point)areshown.
formurinecytokinesbyELISAusingspecifickits(BDBiosciences)or bymultiplexELISAbiomarkerassays(AushonBioSystems,Billerica, MA,USA).CytokinelevelsdeterminedintheculturesfromLNsof PBS-immunizedanimalswereusedastheinitialtime-point(0h). Table3
LocallymphadenopathyandimmunecellpopulationexpansionafteradministrationofSVP-encapsulatedTLR7/8agonist. Day Totalcells CD11c+B220−
(mDC) CD11c+B220+ (pDC) B220+CD11c− (Bcells) F4/80+GR1− (Mø) F4/80−GR1+ (Gran) CD3+(Tcells) CD3−CD49b+ (NK) CD3+CD49b+ (NKT) 0 1.00 1.00 1.00 1.00 1.000 1.00 1.00 1.00 1.00 1 1.50 3.15 1.36 0.98 1.97 3.52 1.52 1.03 0.62 4 3.19 6.50 8.14 3.56 1.16 14.20 2.54 9.64 6.72 7 8.06 7.00 7.77 11.09 2.51 18.43 4.39 8.57 8.25
Micewereinjecteds.c.(hindlimb)withSVP-R848,SVP(noadjuvant),orSVP(noadjuvant)admixedwithfreeR848.PoplitealLNsweretakenattimesindicated.Totallymph nodecellswerecounted,stainedforvarioussurfacemarkers,asindicated,andanalyzedbyflowcytometry.Cellnumberswerenormalizedtoday0values.Datashownin tablearefromSVP-R848injectedmice.NomarkedvariationinLNcellpopulationswasseeninmiceinjectedwith“noadjuvant”-SVPor“noadjuvant”-SVPadmixedwith freeR848(notshown).Mø–macrophages,mDCandpDC–myeloidandplasmacytoidDC,Gran–granulocytes,NKT–NKTcells.
Similarly,systemiccytokinelevelsinpooledorindividualserum samplesdrawnfromvaccinatedanimalsviaterminalbleedsat dif-ferenttime intervalsafterinoculationweremeasuredbyELISA. CytokinelevelsfromtheseraofPBS-immunizedanimalswere con-sideredastheinitialtime-point(0h).Allexperimentsoncytokine measurementinvivowereruntwoorthreetimesyieldingsimilar resultsforeachexperimentalgroup.
2.10. Flowcytometry
Atdifferenttime-pointsafterinjectionwithSVP,freeTLR ago-nistorPBS,miceweresacrificed,drainingpopliteallymphnodes harvestedanddigestedfor30minat37◦Cin400U/mLcollagenase type4(Worthington,Lakewood,NJ,USA).Singlecellsuspensions werepreparedbyforcingdigestedlymphnodesthrougha70-m nylonfiltermembrane,thenwashedinPBScontaining2%FBSand countedusingaCountess®cellcounter(LifeTechnologies, Carls-bad,CA,USA).Cellswerestainedpairwisewithantibodiesagainst thefollowingmousesurfacecellmolecules:B220andCD11c,CD3 andCD49b,F4/80andGr1(BDBiosciences,CA,USA).Thegating logicwasasfollows:plasmacytoiddendriticcells(CD11c+,B220+),
myeloiddendriticcells(CD11c+,B220−),Bcells(CD11c−,B220+),
granulocytes(GR-1+,F4/80−),macrophages(GR-1−,F4/80+),NKT
cells(CD49b+,CD3+),NKcells(CD49b+,CD3−),andTcells(CD49b−,
CD3+).Similarly,SIINFEKL-loadedpentamers(Proimmune,Oxford,
UK),wereusedalongwithanti-mouseCD8andCD19(togateout non-specificpentamerbinding).Cellsampleswerethenwashed andimmediatelyanalyzedbyflowcytometry.Datawereanalyzed withFlowJosoftware(TreeStarInc.,Ashland,OR,USA).
3. Results
3.1. NanoparticleencapsulationofTLRagonistschangesthe patternofinflammatorycytokineinductioninvitro
TLR7/8(R848) and TLR9(CpG ODN 1826; mouse-specific B-typeCpGODN)agonistswereencapsulatedinsyntheticpolymer nanoparticlesand tested fortheirabilitytoinducecytokinesin vitro.R848waschemicallyconjugatedtoPLGAandusedforSVP for-mulationasPLGA-R848,andCpGODNwaspassivelyentrappedinto SVPasdescribedinSection2.Naturaloligonucleotidesequences containaphosphodiester(PO)backbone,whichissusceptibleto rapidhydrolyticcleavagebynucleasesinvivo.Nuclease-resistant CpGsequenceswithaphosphorothioate(PS)backbonehavebeen showntohavesuperioractivitytoPO-CpGinvivo.BothPSandPO formsoftheimmunostimulatoryCpGODN1826sequence(PS-CpG 1826andPO-CpG1826)wereevaluated.SVP-encapsulatedR848 inducedlowerlevelsofTNF-␣fromsplenocytesandespeciallyfrom murinemacrophagesthanidenticalamountsoffreeR848(Fig.1A andB).SimilarprofileswereseenwhenPS-CpG1826andPO-CpG 1826sequencesweretestedinfreeorSVP-encapsulatedform.Not surprisingly,PO-CpG1826wasalesspotentinducerofTNF-␣ pro-ductionthanPS-CpG1826,withitsSVP-encapsulatedformbeing nearlyinactive,eveninthemoresensitiveJ774cells(Fig.1Cand D).IL-6productioninvitrofollowedthesamepatternasTNF-␣ (datanotshown).However,astaticinvitrosystemdoesnot cap-turepotentialdifferencesinbiodistributionandpharmacokinetics offreeadjuvant versusnanoparticle-encapsulatedadjuvant that areexpectedinvivo.
3.2. NanoparticleencapsulationofTLR7/8agonistincreasesits adjuvantactivitywhileantigenencapsulationincreases immunogenicityinvivo
Theadjuvantactivityofnanoparticle-encapsulatedR848 (SVP-R848) was assessed in vivo in immunogenicity studies with
0 4 24 48 144 IF N -γγ , pg /m l 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 SVP-R848 (I) R848, free (I) SVP-R848 (C) R848, free (C)
Time after injection (hrs)
0 4 24 48 144
IL
-12
(p
40
), pg
/ml
0 200 400 600 800 1000 1200 1400 1600 1800Time after injection (hrs)
0 4 24 48 144
IL
-1
ββ
, pg
/m
l
0 20 40 60 80 100 120 140 160 180 200A
B
C
Fig.5.Focusedlocalcytokineinductionafterasingle-siteinjection with SVP-encapsulated,butnotfree,TLR7/8agonistR848.Micewereinjecteds.c.inasingle hindlimbwitheitherSVP-R848orfreeR848.Atthetimesindicated,poplitealLNs fromtheinjectionside(ipsilateral,I)andfromtheoppositeside(contralateral,C) werecollectedandincubatedinvitroovernight.Culturesupernatantswere ana-lyzedforthepresenceofspecificcytokinesbyELISA.A–IFN-␥,B–IL-12(p40),C– IL-1.Averageoftwomicepertime-pointisshown.
a model antigen, OVA (Fig. 2). The potency of free and SVP-encapsulatedR848 toinduce antibodiestoOVA was compared inastandardprime-boostimmunizationregimen.Bothfreeand nanoparticle-encapsulatedforms of OVA weretested (OVA and SVP-OVA,respectively).Additionally,R848andOVA wereeither co-encapsulated in the same particle (SVP-OVA-R848) or were admixed as separateparticles (SVP-R848 and SVP-OVA). When admixedwithsolubleOVA,SVP-R848resultedinnearlya10-fold
Time after injection (hrs) 0 0.5 1 1.5 3 6 TN Fαα ,,pg /m l 0 200 400 600 800
Time after injection (hrs)
0 0.5 1 1.5 3 6 24 IL -12 (p 40 ), pg /m l 0 1000 2000 3000 4000 5000
Time after injection (hrs)
0 0.5 1 1.5 3 6 IL -6 , pg /m l 0 2000 4000 6000 8000 10000 12000 14000 16000 SVP-OVA-R848 SVP-OVA SVP-OVA + R848
Time after injection (hrs)
0 0.5 1 1.5 3 6 24 MC P -1 ( p g /m l) 0 5000 10000 15000 20000 25000 30000 35000
Time after injection (hrs)
0 3 6 24 48 R AN TE S (p g /m l) 0 200 400 600 800 1000
Time after injection (hrs)
0 3 6 24 IP -1 0 ( pg /m l) 0 2000 4000 6000 8000 10000 12000 14000
A
B
C
D
E
F
Fig.6.Strongsystemiccytokineinductionafterinjectionoffree,butnotSVP-encapsulatedTLR7/8agonistR848.Micewereinjecteds.c.witheitherSVP-OVA-R848,SVP-OVA orSVP-OVAadmixedwithfreeR848andbledattimesindicated.ThesameamountoffreeorSVP-encapsulatedR848wasusedforeachgroup(6.8g).Serumsampleswere collectedatthetimesindicatedandanalyzedforindividualcytokinesbyELISA.Averagecytokineconcentrationisshown(threesamplespergrouppereachtime-point).A– TNF-␣,B–IL-6,C–RANTES,D–IP-10,E–IL-12(p40),F–MCP-1.
increaseinimmunogenicitycomparedtofreeR848aftertwoor threeinjections(Fig.2).SVP-R848exceededthepotencyofalum, anadjuvantinnumerouscommerciallyapprovedvaccines,byan evenhighermargin(antibodytiterEC50valuesforanimals immu-nizedwithOVAinalumwerebelowthecut-offlevelfortheassay). Notably,thepresentationofOVAbySVPalsoresultedinamarked increaseofantibodyresponse(byatleast2–3ordersof magni-tude)comparedtofree OVA withor withoutalum.Additionof freeR848toSVP-OVAfurtherincreasedimmunogenicity,especially afteroneortwoinjections,butitseffectwasnotpronouncedafter thethirdvaccination.FreeR848wasalsoinferiortoencapsulated R848whetheritwasco-encapsulatedwithOVA(SVP-OVA-R848) orpresentinaseparateparticle(SVP-OVA+SVP-R848).Onaverage, co-encapsulationofOVAandR848ledtoa0.5-logincreasein anti-bodytitercomparedtoutilizationoffreeR848,whileadmixingof SVP-OVAwithSVP-R848wasmorepotentinantibodygeneration
thanadditionofafreeR848toSVP-OVAbyanorderofmagnitude (Fig.2).WhileadditionoffreeR848toSVP-OVAledtoaclearTh1 shiftinantibodyresponseaftertwoinjections(IgG1:IgG2cratios of0.28vs.3.13atday40forSVP-OVA+R848andSVP-OVA, corre-spondingly),thedifferencewasevenmorepronouncedifR848was SVP-encapsulated(IgG1:IgG2cratiosof0.08forSVP-OVA-R848and 0.11forSVP-OVA+SVP-R848).
Similarly, nanoparticle-encapsulated OVA and R848 induced stronglocalandsystemiccellularimmuneresponses(Fig.3). Injec-tionofnanoparticle-encapsulatedR848ledtoasignificantinflux ofcellsintodraininglymphnodes(LN)evenafterasingle inocula-tion(Fig.3A).Moreover,utilizationofencapsulatedR848(whether withfreeorco-encapsulatedOVA)elevatedboththepercentage andabsolutequantitiesoflocallyinducedantigen-specificCD8+T cells,asassessedbyreactivitywithMHCclassIpentamers con-tainingthedominantovalbuminepitope,SIINFEKL(Fig.3BandC).
Time after inoculation (hrs) 0 0.5 1 1.5 3 6 IL -12 (p 4 0 ), pg /m l 0 1000 2000 3000 4000
Time after inoculation (hrs)
0 0.5 1 1.5 3 6 TN Fαα , p g /ml 0 50 100 150 200 250
Time after inoculation (hrs)
0 1 1.5 3 6 IL -6 , pg /m l 0 50 100 150 200 250 300 SVP-OVA-R848 SVP-OVA SVP-OVA + R848
Time after inoculation (hrs)
0 0.5 1 1.5 3 6 MC P -1 , pg /m l 0 2000 4000 6000 8000 10000 12000 14000
A
B
C
D
Fig.7. Systemiccytokineinductionafterintranasalinoculationoffree,butnotSVP-encapsulatedTLR7/8agonistR848.Micewereinoculatedintranasallywitheither SVP-OVA-R848,SVP-OVA,orSVP-OVAadmixedwithfreeR848andbledattimesindicated.ThesameamountoffreeorSVP-encapsulatedR848wasusedforeachgroup(3.8g). SerumsampleswerecollectedatthetimesindicatedandanalyzedforindividualcytokinesbyELISA.Averagecytokineconcentrationisshown(threesamplespergroupper eachtime-point).A–TNF-␣,B–IL-6,C–IL-12(p40),D–MCP-1.
Cellsinducedbyco-encapsulatedR848andOVAexhibitedahigher proliferativepotentialthanwheneitherfreeR848orfreeOVAwas utilized,asevidencedbyinvitroexpansionofOVA-specificCD8+ Tcells(Fig.3D)andtheircytotoxicactivity(Fig.3E).Theinvivo cytotoxicactivitywasassessedat6daysafterasingleinjectionof nanoparticle-encapsulatedorfreeOVAinthepresenceorabsence offreeornanoparticle-encapsulatedR848.SIINFEKL-pulsed syn-geneictargetcellswereeliminatedefficientlyinvivoonlyifboth OVAandR848weredeliveredinencapsulatedform(Fig.3F).The levelofinvivocytotoxicactivitywasmaintainedforseveraldays afterasingleinjection(datanotshown).Theadmixof nanoparticle-encapsulatedOVAwithfreeR848ortheadmixoffreeOVAwith nanoparticle-encapsulatedR848 induced poorin vivo cytotoxic activity(Fig.3F).
3.3. Rapidinfiltrationofdraininglymphnodesbyinnateand adaptiveimmunecellsuponinjectionof
nanoparticle-encapsulatedR848
R848-bearingnanoparticlesinducedaprofoundincreasein cel-lularitywithinthedraininglymphnodesat4daysafterasingle inoculation (Fig. 3A). Further analysis of cellularity within the draininglymphnodesafters.c.injectionshowedthatLNinfiltration startsasearlyas1dayafterinoculation,reachesapeakat7–8days, andismaintainedforatleast3weeks(Tables1and2).Theincrease inlymphnodecellularitywasevenmorerapidandpronouncedin micethatwerepreviouslyimmunizedwithSVP(10-foldincrease inthepoplitealLNcellcountat1dayafterinoculation,Table2). Nosignificantcellinfiltrationofthedraininglymphnodewasseen ifSVPlackingR848wereusedeitheraloneoradmixedwithfree R848(Table1).
AdetailedanalysisofintranodalcellpopulationsafterSVP-R848 injectionshowedarapidincreaseinthenumberofinnateimmune cells,suchasgranulocytesandmyeloidDC,inthedrainingLN,with theirnumbersincreasing3-foldwithin24hafterasingle injec-tion(Table3).Therewasalsoanearlyelevationinmacrophage cellnumbersinthedraininglymphnode,whileincreasesinother APCsubtypes(plasmacytoid DCandBcells)wereobservedata slightlylatertime-point.Interestingly,amongthepopulations ana-lyzed,onlyeffectorcellsoftheadaptiveimmuneresponse(TandB cells)showedacontinuedexpansionfromday4today7(Table3).
3.4. NanoparticleencapsulationofTLR7/8agonistinducesstrong localcytokineproduction
Stronglocalimmuneactivationbynanoparticle-encapsulated R848wasfurthermanifestedbycytokineproductioninthe drain-ingLNmilieu(Figs.4and5).At4haftersubcutaneousinjection, highlevelsofIFN-␥,RANTES,IL-12(p40)andIL-1weresecreted byLNsfromanimalsinjectedwithSVP-OVA-R848,whilethe pro-ductionofthesecytokinesbyLNsfrommiceinjectedwithfreeR848 wasclosetothebackgroundlevel(Fig.4).Inparticular,theamount ofIFN-␥secretedbyLNsfrommiceinjectedwithSVP-OVA-R848at 4hwasmorethan100-foldhigherthanthatfromtheLNsofmice injectedwithSVP-OVAandanequalamountoffreeR848(Fig.4A), whileRANTESwaselevatedmorethan27-fold(Fig.4B). Produc-tionofallofthesecytokinesintheLNwasmaintainedforatleast 72hafterinjectionofSVP-OVA-R848,withlevelsofIL-12(p40)and IL-1remainingnearlystable(Fig.4CandD),andlevelsofIFN-␥ andRANTES,whiledecreasing,remaining4-to20-foldhigherthan thebackground.Incontrast,inoculationoffreeR848ledtoonlya modestincreaseoflocalcytokineproductionat4h,whichreturned
tobackgroundlevelsby24hafteradministration.LevelsofIP-10 andMCP-1inLNsfromSVP-OVA-R848-injectedanimalswerealso elevatedinasimilarfashion(datanotshown).
Thestrikingdifferenceinlocalcytokineproductionafter admin-istrationofnanoparticle-encapsulatedversusfreeR848(Fig.4)was alsoevidentbycomparingcytokineproductionintheipsilateral draininglymph nodeversus thecontralaterallymph nodeafter injectioninasinglehindlimb(Fig.5AandB).Thesustained expres-sionofIFN-␥,IL-12(p40),andIL-1wasseenintheipsilateralLN at4–48hafterinjectionofSVP-R848,butnotinthecontralateral lymphnode.Incontrast,freeR848inducedamodestelevationof IL-12(p40)andIFN-␥inboththeipsilateralandcontralaterallymph nodes(Fig.5B).ThelevelofIFN-␥observedintheipsilaterallymph nodefollowinginjectionoffreeR848was50-foldlowerthanthat inducedbySVP-R848(Fig.5A).NoinductionofIL-1byfreeR848 wasseen(Fig.5C).
3.5. NanoparticleencapsulationofTLR7/8agonistR848 attenuatestheproductionofsystemicinflammatorycytokines
WhilenanoparticleencapsulationofR848enhanced immuno-genicityandlocalinductionofimmunecytokines,theproduction ofsystemicinflammatory cytokinesbySVP-R848wasmarkedly suppressedcomparedtothatobservedwithfreeR848aftereither subcutaneous or intranasal inoculation (Figs. 6 and 7, respec-tively).In particular,4hafter subcutaneousinoculation, serum concentrations ofearly inflammatory cytokinesTNF-␣and IL-6 were50–200timeshigheriffreeR848wasused(Fig.6AandB). Serumcytokinelevelsweresimilarinanimalsinoculatedwith SVP-OVAwithorwithoutencapsulatedR848.Similardifferenceswere observedwithsystemicproductionofRANTES(Fig.6C). SVP-OVA-R848inducedmodestlevelsofIP-10,IL-12(p40),andMCP-1,which wereapproximately5–10timeslower thanthat observedafter injectionofSVP-OVAadmixedwithfreeR848(Fig.6D–F).
Patterns of systemic cytokine expression profiles after intranasal deliveryof either free or encapsulatedR848 (Fig.7) weresimilartothose seenafters.c. delivery.SerumTNF-␣and MCP-1wereonlyweaklyinducedbySVP-R848,withlevels10-to 100-foldlowerthanthoseinducedbyfreeR848(Fig.7AandD), whilelevelsofIL-6andIL-12(p40)inductionwere5timeslower (Fig.7BandC).
3.6. NanoparticleencapsulationofTLR9agonistpermitseffective utilizationofimmunostimulatoryphosphodiesterCpGODNs invivo
We next assessed the adjuvant activity of a nanoparticle-encapsulatedTLR9agonist,CpG-1826,inimmunogenicitystudies withamodelantigen,OVA.ItisknownthataPOformofCpGis subjecttorapiddegradationbynucleases[36,46]andthereforethe backbone-modifiedPSformisusuallyemployedinvivo.We rea-sonedthatnanoparticleencapsulationmayprotectthePOform fromprematuredegradationandenable useof PO-CpGinvivo. Co-administrationofnanoparticle-encapsulatedOVAandPO-CpG 1826inducedantibodytiters comparabletothat obtainedwith nanoparticle-encapsulatedOVAadmixedwiththesamedoseof free PS-CpG1826 (Fig.8A).Animalsimmunizedwiththesame dosesoffreeOVAadmixedwithfreePS-CpG1826exhibited 20-to40-foldlowerantibodytiters(Fig.8A).Increasingthedoseof free OVA and free PS-CpG 1826did not increase theantibody titerscomparedtoSVP-encapsulatedOVA andPO-CpG(Fig.8B). When another antigen, prostatic acid phosphatase (PAP), was evaluated,PS-CpG1826wasinferiorbynearlytwoordersof magni-tudeinantibodyinductioncomparedtonanoparticle-encapsulated PAPandPO-CpG1826(Fig.8C).Nanoparticleentrapmentof PS-CpG1826 didnot leadto higherimmunogenicitycompared to
SVP-OVA + S VP-PO-CpG SVP-OVA + P S-CpG OVA +PS-Cp G A n ti-O V A Ig G E C50 ( Log 10 ) 3 4 5 6 7 Day 26 Day 44
SVP-OVA + SVP-PO-CpG OVA (5x) + PS-CpG (5x)
An ti-O V A I g G E C50 (Log1 0) 3 4 5 6 7 Day 26 Day 40 SVP-PAP-PO-CpG PAP (50x) + PS-CpG (2x)
An
ti-P
AP
Ig
G
E
C
50
(L
og1
0)
3 4 5 6 7Day 19
Day 31
A
B
C
Fig.8. HigherhumoralimmunogenicityofSVP-encapsulatedantigencoupledwith freeTLR9agonistPS-CpGorwithSVP-encapsulatedTLR9agonistPO-CpG.Mice (5/group)wereimmunized(s.c.,hindlimb)withtheindicatedSVPor combina-tionsoffreeantigenandPS-CpG1826.Antibodytitersweredeterminedattimes indicated.Micewereimmunizedeithertwo(A,C)orthree(B)timeswith2-week intervalsbetweenimmunizations.OVA(A,B)andprostaticacidphosphatase(PAP) (C)proteinswereevaluatedastestantigens,eitherinfreeformorencapsulatedin SVP.InPanelB,thetotaldoseofbothfreeOVAandfreePS-CpG1826wasfivetimes greaterthanthatadministeredintheSVP-treatedgroups.InPanelC,theamount offreePAPusedwas50timeshigherthanthatofSVP-encapsulatedPAP,andthe amountoffreePS-1826wastwotimeshigherthanusedinSVP-encapsulatedform (C).NoadjuvanteffectoffreePO-1826wasdetected(datanotshown).
entrapped PO-CpG1826,while utilizationof freePO-CpG 1826 resultedinnoaugmentationofimmunogenicity(datanotshown). When nanoparticle-encapsulated OVA and PO-CpG 1826 were compared tofree OVAand free PS-CpG1826 intheirability to inducespecificCTLsinvivo,thecombinationoftheformer was moreeffectiveevenif10timesmorefreeOVAand5timesmore freePS-CpG1826wereused(Fig.9).
Fig.9.NanoparticleencapsulationofbothantigenandTLR9agonistPO-CpG(adjuvant)resultsinahigherlocalcellularimmuneresponsethanutilizationoffreeantigenand adjuvant.Groupsof2–3micewereinjected(s.c.,hindlimb,onceortwicewith2-wkinterval)withacombinationofSVP-encapsulatedOVAandSVP-encapsulatedPO-CpG 1826,freeOVAadmixedwithfreePS-CpG1826,orPBSasindicated.TheamountoffreeOVA(50g)wastentimeshigherandtheamountoffreePS-CpG1826(20g) wasfivetimeshigherthanthatpresentwithinSVP(5and4g,respectively).Tissuesweretakenat5daysafterthelastimmunizationandrestimulatedinvitrofor7days withmitomycin-treatedE.G7-OVAcells.LNcellswerestainedwithsurfacemarkerantibodiesandanalyzedbyflowcytometry.Toprow–poplitealLNcultures(initiated5 daysafterasingleimmunization),bottomrow–splenocytecultures(initiated5daysafterprime-boostimmunization).Representativeflowcytometrydotplotsfromtwo separateexperimentsareshown.ThenumbersindicatethepercentageofCD8+TcellsthatarespecificfortheOVASIINFEKLpeptide(percentshareofCD8+CD19−cells).
Nosignificantinductionofinflammatorycytokines(TNF-␣, IL-6)inserumwasseenwhenfreeorencapsulatedPOandPSformsof CpG-1826weretested,whilefreePS-CpG1826inducedthe produc-tionofIL-12(p40)tothesamelevelsasnanoparticle-encapsulated PO-CpG1826(Table4).NanoparticleentrapmentofPS-CpG1826 ledtoelevatedandsustainedlocalproductionofIFN-␥,IL-12(p40), andIL-1,whichexceededthatoffreePS-CpG1826(usedin10-fold excess,Fig.10),closelyparallelingresultsseenwhenfreeand SVP-encapsulatedR848werecompared(Fig.7).Nocytokineinduction fromcontralateralLNwasobservedafterSVP-PS-CpGinoculation (Fig.10).
4. Discussion
TLR7/8 and TLR9 agonists have shown great promise as immunomodulatingtherapeuticagents[52–61;reviewed in36]
andasadjuvantsforDNA-[62]andprotein-basedvaccines[63–67]. BothR848andCpGODNswereseenasattractivecandidatesfor sys-temicuseinavarietyofsettings[seesummariesin12,31,36,40,68]
duetoTLR7/8andTLR9distributioninimmunecellsandresulting
Table4
Systemic cytokine induction after subcutaneous administration of SVP-encapsulatedorfreeTLR9agonist.
Cytokinepeakconcentration(pg/mL)
Miceinjectedwith TNF-␣ IL-6 IL-12(p40)
SVP-OVA 1.4 67.2 43.9
SVP-OVA+SVP-PO-CpG1826 11.2 105.2 569.4
SVP-OVA+PO-CpG1826 2.2 79.6 44.0
SVP-OVA+PS-CpG1826 12.8 58.7 448.0
Micewereinjectedsubcutaneously(hindlimb)witheitherSVP-OVAaloneor com-binedwithSVP-PO-CpG1826,freePO-CpG1826,orfreePS-CpG1826andbledat 0.5,1,1.5,and3hafterinjection.Peakaveragecytokineconcentrationinserumis shown(threesamplespergroupforeachtime-point).
ability of these compounds to specifically activate APCs (i.e., dendritic cell, monocyte/macrophage, and B cell populations). However,broaduseofTLR7/8and9agonistsasparenteraldrugs andadjuvantshasbeenlimitedduetosystemictoxicity[13,69], which is directlyrelated tosystemic cytokine release [70]. We hypothesizedthatencapsulationofaTLRagonistintoa nanopar-ticlecarriermayattenuatesystemiccytokineinductionandthus enableitsuseasaparenterallyadministeredadjuvant. Nanopar-ticle deliveryof TLR7/8 or TLR9 agonists would have multiple benefits,including(1)minimizingsystemicexposureoftheTLR agonist,(2)deliveringofadjuvanttolymphnodesviadirectflow ofnanoparticlesthroughdraininglymphatics[43,44],(3) promot-inguptakeintoendosomalvesiclesofAPC,whereTLR7,8,and9 areexpressed, and (4)providing a sustainedrelease oftheTLR agonistfromananocarrierratherthanabolusdelivery.Moreover, nanoparticleencapsulationofbothantigenandadjuvantmayhave asynergisticbenefitbyenablingco-deliveryofbothantigenand adjuvanttoAPCsasdemonstratedearlierformicroparticledelivery vehicles[40,46].
R848isahighlypotentTLR7/8agonistthatrapidlydistributes throughout the body and exhibits a short half-life [12]. While imiquimod, ananalogof R848which is 100-foldless potent,is licensedasatopicaldrugforgenitalwarts,actinickeratosis,and basalcellcarcinoma[31],clinicaldevelopmentofR848asa topi-caldrugandasanorally-delivereddrugwasdiscontinueddueto itsnarrowtherapeuticwindowrelatedtoitsshortinvivohalf-life andsystemicside-effects.Ourresultsdemonstratethat encapsulat-ingR848maygreatlyincreaseitstherapeuticwindow.FreeR848 administereds.c.inducedserumTNF-␣andIL-6levelsthatwere 50-to200-foldhigherthanthatobservedwithSVP-encapsulated R848.ThesystemicproductionofTNF-␣,IL-6,andRANTESwas sup-pressedinSVP-R848-injectedanimalstobackgroundlevels,while systemicinductionofIP-10andMCP-1wasalsogreatly attenu-ated.Thereductioninsystemiccytokineproductionislikelydueto deliveryofnanoparticlestothelocaldraininglymph,directuptake
Time after injection (hrs) 0 4 24 48 144 IL -1 ββ,, pg /ml 0 50 100 150 200 250
Time after injection (hrs)
0 4 24 48 144 IL -1 2( p 40 ), p g /m l 0 1000 2000 3000 4000 5000
Time after injection (hrs)
0 4 24 48 144 IF N -γγ (p g /m L ) 0 20000 40000 60000 80000 SVP-PS-CpG (I) PS-CpG, free (I) SVP-PS-CpG (C) PS-CpG, free (C)
A
B
C
Fig.10.FocusedlocalcytokineinductionbySVP-encapsulated,butnotfreeTLR9 agonistCpG.Micewereinjecteds.c.inasinglehindlimbwitheitherSVP-PS-CpG 1826orfreePS-CpG1826.Atthetimesindicated,poplitealLNsfromtheinjection side(ipsilateral,I)andfromtheoppositeside(contralateral,C)weretakenand incubatedinvitroovernight.Culturesupernatantswerecollectedandanalyzedfor cytokinepresencebyELISA.A–IFN-␥,B–IL-12(p40),C–IL-1.Averageoftwomice pergrouppertime-pointisshown.
byAPCs,andsustainedreleaseofR848overtime.Consistentwith thishypothesis,weobservedastrongandsustainedlocalimmune activationfollowingsubcutaneousadministrationofSVP-R848,as evidencedbycellularinfiltrationofthedrainingLNbyAPCfollowed byeffectorcells,leadingtoprolongedlocalproductionofIFN-␥, IL-12(p40)andIL-1.In contrast,onlylow levelsofLN cellular infiltrationandlocalcytokineproductionwereseenupon adminis-trationoffreeTLR7/8agonist.Notably,SVPencapsulationofR848 ledtoastronginductionofcellularimmuneresponses(bothlocal andsystemic)evenafterasingleimmunization,whilefreeR848 wasnearlyinactive.
OurresultsconfirmandadvancetherecentfindingsofTacken etal.whoreportedthatnanoparticleencapsulationofTLR3and 7/8agonistsattenuatedtheserumcytokinestormandenhanced immunogenicity[71].Inthiscase,R848waspassivelyentrapped withinthenanoparticleandrequiredantibody-mediatedDC tar-getingfor delivery. The reduction incytokine storm(measured atasingletime-point)wasnotaspronouncedasobservedinthe presentstudywiththeSVP-basedplatform,eventhoughweused 20-foldmoreR848[71].ThisdifferencemaybeduetoouruseofSVP thatcontainedR848covalentlylinkedtothePLGApolymerwithan acid-labilebond,adesignintendedtoconstrainR848releasetothe acidicenvironmentwithintheendosome.
SVPencapsulationofaTLR9agonist,CpG-1826,alsoprovided significantbenefit.CpG-1826belongstotype BCpG,capableof activatingBcellsandinducingtheproductionofproinflammatory cytokines[14,72,73].CpG-1826encapsulationwithinSVPprovided forhigherlocalcytokineproductionand,whenco-deliveredwith encapsulatedantigen,resultedinhigherimmuneresponsesthan antigenadmixedwithfreeCpG-1826.UnmodifiedCpGcontainsa nuclease-labilephophodiesterbackbone(PO-CpG)whichisknown to berapidly degraded in vivo, thus parenterally administered freeCpGmustbemodifiedtocontainanucleaseresistant phos-phorothioatebackbone(PS-CpG)tobeactiveinvivo.Importantly, SVPencapsulationenabledutilizationofthenon-phosphorothioate formof CpG(i.e.,PO-CpG) withthesameefficiency asPS-CpG. TheuseofPO-CpGinSVPsmayfurtherreducethepotentialfor systemicimmuneactivation,asanyPO-CpGthatleaksoutofthe nanoparticleswillberapidlydegraded.
Nanoparticleencapsulationofbothantigenandadjuvantmay have a synergisticbenefitby enabling co-deliveryofboth anti-genand adjuvant toAPC.TheSVPtechnologyallows foreither covalentornon-covalententrapmentofaTLRagonistaswellas covalentandnon-covalentpresentationofantigenonthesurface orwithinthenanoparticle.TheSVPsaredesignedtoreleasetheir payloadinthelowpHenvironmentoftheendolysosomal compart-mentofAPC,whichcontainsTLR7,8,and9aswellasMHCclassII molecules.Thesustainedandconcomitantreleaseofantigenand adjuvantfromSVPscouldalsocontributetomorepotentimmune responsesandbettermemorycellgeneration.Ourdatashowthat adjuvantandantigencanbedeliveredinseparatenanoparticles. Theabilitytoutilizeindependentlyformulatedantigen-and TLR-agonist-carryingnanoparticlesmaybeadvantageousformodular andflexiblevaccinedesign.Forexample,atwoparticleapproach canprovideflexibilityindosingtooptimizetheratioof adjuvant-to-antigenforaparticularapplication.
Whilevaccineshavebeenaneffectiveandcost-efficienthealth careinterventionfortheprophylaxisofmanyinfectiouspathogens, new vaccine technology and more potent adjuvants may be requiredtodevelopeffectivetherapeuticvaccinesforchronic infec-tions, intracellularpathogens, and non-infectiousdiseases, such as cancer. Theimmune systemis keyed torespondto particu-lateantigens,suchasvirusesandbacteria.Syntheticnanoparticles canmimickeyelementsofviralparticlesbyco-deliveringantigen with strong TLR agonists and effectively induce robust cellu-lar andhumoralimmunity.Finally, onecanenvision that other immunomodulatoryagentscouldbeincorporatedintoSVPsto fur-therfine-tunetheimmuneresponsebytargetingspecificsubsets ofimmunecells,suchasCD8Tcells,Th1cells,Th2cells,Tfh,Th17 cells,Tregulatorycells,Bcells,andNKTcells.
Collectively,thedatareportedheresuggestanapproachto uti-lizeTLRagonistsasparenterallyadministeredvaccineadjuvants inaclinicalsettingwhileminimizingtheriskofsystemicadverse reactions.Co-encapsulationofantigenhastheaddedbenefitof co-deliveryofadjuvantandantigendirectlytoAPCs.TheSVPapproach iscurrentlybeingevaluatedinpre-clinicalstudiessuchascancer andchronicinfections,wheretraditionaladjuvantsareinadequate,
andinaPhase1clinicalstudyforsmokingcessation,wherehigh concentrationsofantibodiesagainstnicotinearethoughttobe nec-essaryfortherapeuticefficacy.
Acknowledgement
WethankAditiChalishazar,IngridSolteroandAlyssaRaguefor theirexperttechnicalhelp.
Conflictof interest:Petr Ilyinskii,ChristopherRoy, ConlinO’Neil, EricaBrowning,LynnellePittet,DavidAltreuter,LloydJohnston, and Takashi Kei Kishimoto are employees and shareholders of SelectaBiosciences.RobertLanger,OmidFarokhzadandUlrichH. vonAndrianarefoundersandshareholdersofSelectaBiosciences. Frank Alexis,Elena Tonti, JinjunShi, PamelaA. Basto, Aleksan-darF.Radovic-MorenoandMatteoIannaconereportnoconflict ofinterest.
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