Ruth Mateu Ferrando , Luigi Lay Laura Polito , development Gold nanoparticle-based platforms forvaccine

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TECHNOLOGIES

DRUG DISCOVERY

TODAY

Gold nanoparticle-based platforms for vaccine development

Ruth Mateu Ferrando

¹

, Luigi Lay

^1,2,

* , Laura Polito

^3,

*

1DepartmentofChemistry,UniversityofMilan,ViaC.Golgi19,20133Milan,Italy

2CRCMaterialiPolimerici(LaMPo),UniversityofMilan,ViaC.Golgi19,20133Milan,Italy

3NationalResearchCouncil,CNR-SCITEC,ViaG.Fantoli16/15,20138Milan,Italy

Sincetheirdiscovery,therapeuticorprophylacticvac- cinesrepresentapromisingoptiontopreventorcure infections and other pathologies, such as cancer or autoimmunedisorders.Morerecently,amonganum- berofnanomaterials,goldnanoparticles(AuNPs)have emerged as novel tools for vaccine developments, thankstotheirinherentabilitytotuneandupregulate immuneresponse. Moreover,owing totheirfeatures, AuNPs can exert optimal actions both as delivery systemsandasadjuvants.Notwithstandingthepoten- tial huge impact in vaccinology, some challenges re- main before AuNPs in vaccine formulations can be translatedintotheclinic.Thecurrentreviewprovides anupdatedoverviewofthemostrecentandeffective applicationof goldnanoparticlesasefﬁcientmeansto developanewgenerationofvaccine.

Introduction

Vaccination is one of the most cost-effective strategies to preventdeathandmorbidityassociatedwithinfectiousdis- eases, given the induction of pathogen-specific humoral immuneresponses.Overthepastyears,vaccineshavebeen

one of the many crucial medical advancements that has contributed to an increase in life expectancy, conferred long-termprotectiveimmunitytothepopulationandsaved millionsoflivesandtheeventualeradicationofsomeviral andbacterialinfectiousdiseases[1,2].

Vaccinesprimetheimmunesystemtogenerateanadap- tiveresponse,characterizedbycellular(CD4⁺orCD8⁺T-cells) orantibodyresponses.Therefore,immunomodulationisin the forefront of the development of treatments for many pathologies.Theimmunesystemcanbestimulatedorsup- pressedtohelpinthefightagainstinfectiousdiseasesorother kind of pathologies (i.e. cancer or autoimmune diseases) [3,4].Vaccinescanconsistoflive-attenuatedorinactivated pathogens, purified proteins, or subunits based on small antigens (i.e. peptides or carbohydrates) that should elicit specificimmuneresponses.Withrespecttotheuseofwhole pathogen vaccines, subunit vaccines are usually safer to administer,buttheyaregenerallypoorlyimmunogenic.To overcomesuchissues, alternativeapproachescanbetaken, suchas(1)theuseofadjuvants(e.g.alum,squalene,toll-like receptors(TLRs)agonists,monophosphoryllipidA)thatcan beaddedtovaccineformulations inordertostimulatethe immune system [1]; (2) conjugation of the antigen to an immunogenic carrier (i.e. immunogenic proteins tetanus toxoid(TT),diphtheriatoxoid(DT)anditsgeneticallydetox- ifiedform(CRM₁₉₇))[2]or(3)usecarrierswhichcandisplay multiple copiesofantigens, evokingthemultivalenteffect andenhancingtheimmuneresponse[3,5].

Nanotechnologyrepresentsacutting-edgeapproach,able toaddresssomeofthemostrelevantvaccinologyconcerns.

Nanomaterials(NMs)withsizesbetween1and100nm(i.e., Editors-in-Chief

KelvinLam–SimplexPharmaAdvisors,Inc.,Boston,MA,USA HenkTimmerman–VrijeUniversiteit,TheNetherlands

*Correspondingauthors::L.Lay(luigi.lay@unimi.it),L.Polito(laura.polito@scitec.cnr.it)

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in the range of cellular components and microorganisms) haveemergedasinnovativetoolsinmanybiomedicalappli- cations, from diagnostics to drug delivery. Many NMs are characterizedby highbiocompatibility,morphological cus- tomization,reliablefunctionalization,andprolongedcircu- lation in blood [6,7]. NMs can be internalized by antigen presentingcells(APCs,suchasdendriticcells,DCs,ormacro- phages)viaendocytosis,stimulatingtheimmunesystemsby releasing cytokines that initiate the adaptive immune response (Fig. 1) [6,8,9]. Subsequently, DCs can migrate to thelymphnodeswheretheyinteractwithT-cellsandpresent non-self-peptides on major histocompatibility complexes (MHC) classI or II, producing cytokinesand driving antigen-specificCD8⁺orCD4⁺T-cellactivation[9].

In the interaction between immunesystem and nanomaterial, several crucial aspects need to be taken into consideration. To generate a biased immune response, particlesize,surfacechargeandshapemustbemodulated inarationalmannertofacilitatethecrossingofthephysi- ological barriers and the access to immune cells. Some excellentand recent reviewsreport critical surveysabout the modulation of the immune response by means of differentkindofnanomaterials[3,4,9,10].

Amongmanydifferentnanoparticleswhichincludelipids, self-assembledproteins,biopolymersandinorganicmaterials [5],properlyfunctionalizedgoldnanoparticles(AuNPs),represent one of the most promising nanomaterial in the

developmentofanewgenerationofvaccines[7,11].Inthe presentpaperwewillreviewthemostrecentresults(seeTable 1 fora complete summary) that evidence the potential of AuNPsasinnovativenano-vaccinesplatforms.

Goldnanoparticlesinvaccinology

AuNPs have aroused huge interest in vaccinology, due to theirreliablesurfacefunctionalization,biocompatibility,size andshapecustomization,andopticalproperties.Moreover, owing to their inertness, AuNPs can be exploited both as deliveryagentsandasadjuvants, withtheabilitytoexten- sively enhance immune responses while assuring minimal toxicity[1].

AuNPsareusuallysynthesizedby areductivereaction of chloroauricacid(HAuCl₄),usingwell-establishedTurkevich orBrust-Schriffinsproceduresfollowedby surfacemodifica- tionandstabilization(Fig.2).Dependingonthedesiredsize andmorphology,theprecursorAu^IIIsaltisreducedtoAu⁰by means ofsodium borohydride, sodium citrate,or ascorbic acidwiththeeventualpresenceoftemplatingagent(i.e.cetyl trimethylammoniumbromide) [11,12]. Despitetheir inertness,AuNPsurfacecan befunctionalizedby formingstable bonds with sulfur-containing compoundsor by multilayer coating. This relatively straightforward functionalization confers AuNPs great versatility, extending their use into thebiomedical field [11–13]. Whendesigninga nano-gold platform,sizeandshape haveacrucial impactinterms of

I. Vaccination

III. Activation

IV. Maturation

II. Uptake

Nanomaterial

Innate immunityAdaptive immunity

Cellular Immunity

Humoral Immunity

Cytokines release

Antibodies production

Help Control

Immune tolerance

Drug Discovery Today: Technologies

Fig.1. Schematicrepresentationofinnateandadaptiveimmuneresponsesuponexposuretonanomaterial-basedvaccines.APCsprocesstheantigensand promoteTcellproliferationtowardscellular(cytokines),humoral(antibodies)ortolerogenicresponses.APC,antigenpresentingcell;CTL,cytotoxicT cells;B,Bcells;Th,Thelpercells;Treg,regulatoryTcells.

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antigenpresentation,cellular uptake, bloodclearance, bio- distribution and immunological response [14]. AuNPs, in fact,up-regulate theexpressionsofpro-inflammatorycyto- kines via different pathways depending ontheir geometry andmodeofadministration[3,6,9,10,15].Theirsizemostly affectstheuptakeandbiodistributionwhereastheirsurface functionalizationcanalterthecirculationtime[16].Allthese variablesare stronglyinterrelated andeventhough several authorshaveaddressedthisparamounttopic,nosystematic approachhasyetbeenfoundregardingthebestpreferredsize

and shape for every application [6,17,18]. Moreover, the functionalizationstepplaysapivotalroleconsidering that, whenexposedtobiologicalmedia,AuNPscanbecoatedby proteinsandotherbiomoleculesformingtheso-called‘bio- corona’[19,20].Theformationofsuchabio-coronacanhave majoreffectsonthebiodistributionandNPtargetingactivity, sincetheoriginalfunctionalizationcanbehidden.Another crucialaspectthatmustbeconsideredistheAuNPelectronic properties [7,12]. These noble metal NPs can absorb and convertelectromagneticradiationintoheatand,thus,they Table1. CompletesummaryofthemostrecentapplicationsofAuNPsinvaccinology, theirmorphology,andtheirﬁeldof applications.

AuNP(morphology) Application Surfacefunctionalization Experiment Ref

15nm(sphere) Adjuvant Flagellin_(1–161) Invivo(injected) [35]

20nm(sphere) Adjuvant SIINFEKLandpolyIC Invivo(injected) [13]

20and41nm(sphere)28

7.9nmand4112nm(rods)

Adjuvant PolyIC(40:400) Invivo(intranasal) [15]

70nm(nanostar) Adjuvant – Invivo(injected) [45]

2nm(sphere) Bacterialinfections

(S.pneumoniae)

Oligosaccharideepitopes S.pn14/OVA323–339

Invivo(injected) [25]

(S.pneumoniae)

Oligosaccharideepitopes S.pn14and19F

15.5nm(sphere) 51.2nm(nanourchins)

Bacterialinfections (GAS)

Tetraandhexarhamnosides ELISA [27]

15nm(sphere) Bacterialinfections (B.pseudomallei)

Hemagglutinin/FIgL/LPS Invivo(intranasal) [28]

15nm(sphere) Bacterialinfections (EHEC)

LomW/EscC Invivo(injected) [31]

(Listeria)

LLO91–99/GADPH1–22/Advax^TM Invivo(injected) [32–34]

2nm(sphere) Viralinfections

(HIV)

(oligo)mannosides/carboxyl-ending linker

2.3nm(sphere) Viralinfections (HIV)

Gagp17/dimanoside (Mana1–2Man)

Exvivo [41]

12nm(sphere) Viralinfections

(Inﬂuenza)

M2eprotein Invivo(intranasal) [43]

18nm(Sphere) Viralinfections

(Inﬂuenza)

H3N2/HA/FliC Invivo(intranasal) [44]

3nm(sphere) Cancer OVAprotein Invivo(injected) [47]

3–5nm(sphere) Cancer TFdisaccharide Invivo(injected) [49]

5–20nm(sphere) Cancer Tn(a-GalNAc) Invivo(injected) [51]

7and14nm(sphere) Cancer MUC-1glycopeptides Invitro(quartzcrystalmicrobalanceand

dot-blotImmunoassay)

[52]

15nm(sphere) Cancer MUC-1glycopeptides Invitro [53]

22–30nm(sphere) Cancer MUC-1glycopeptides/CD4T-cell

epitope

Invivo [54]

30nm(sphere) Cancer SIINFEKLOVApeptide Invivo(injected) [55]

24nm(Sphere) Cancer pCMV-MART1 Invivo(injected) [56]

17nm(sphere) Cancer PD-L1siRNA/STAT3siRNA Invivo(injected) [57]

45x15nm(rods) Cancer IDOsiRNA Invivo(injected) [58]

30nm(sphere)50 nm(star)60 nm(cage)30–40 nm(prism)

ParasiteInfections (P.falciparum)

CHrPfs25 Invivo(injected) [63]

60nm(sphere) Non-infectiousdiseases (Diabetes)

AhR/proinsulin/ITE Invivo(injected) [65]

2–4nm(sphere) Non-infectiousdiseases (Diabetes)

Proinsulin(PIC19-A3) Invivo Exvivo

[66]

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can be exploited for targeting and therapeutic purposes.

Notably,theapplicationofAuNPsinnanomedicinecovers severalfieldsfromdrugdeliverytodiagnosticsandtherapeu- tics, making these nanomaterials extremely versatile and promising.

Inthepresent paper,themost recentexamplesofAuNP exploitation against bacterial infections, viral infections, cancer,parasiteinfections andnon-infectiousdiseases,will bereviewed.

Bacterialinfections

Diseases caused by bacterial infections can varyfrommild symptomstolethalsepsis.Suchinfectionscanhaveastrong impactonpublichealth,worsenedbyalonglistofantibiotic- resistant bacteria for which new and efficient preventive therapiesareurgentlyneeded[2,21].Bacteriacellenvelopes arecharacterizedbyadensearrayofglycans:Gram-negative bacteriaarecoatedbyapeptidoglycanwallsurroundedbya lipopolysaccharide (LPS) membrane, while Gram-positive lack the outer lipid membrane and present only a thick polysaccharidelayer.Thesepolysaccharidesprotectthebac- teriaandarethemainwayofinteractionofthepathogens withthehostimmunesystem,representingthemajorviru- lencefactorandthus,theoptimaltargetforvaccinedesign.

Nevertheless, it iswell knownthat themajor drawbackof polysaccharide-basedvaccinesisthelimitedimmunogenicity (especially for infants, elderly and immunocompromised patients)duetotheinductionofTcell-independentimmune responses. This issue can be overcome by conjugating a

fragmentofthecapsularpolysaccharidetoanimmunogenic carrierprotein(i.e.TT,DT,CRM₁₉₇)abletostimulateBcell maturationtomemorycells.Suchsuccessfulstrategypaved the road for thedevelopment of promising new carbohydrate-basedvaccines,comprisedofnaturalpolysaccharidesor well-definedsyntheticoligosaccharides[2,22].Nevertheless, somedrawbacksrelatedtoimmuneinterferencephenomena, referred to as carrier epitope suppression, can lead to a reductionoftheanti-carbohydrateimmuneresponseagainst glycoconjugateformulations.Therefore,acontinuouseffort isdevotedtothedevelopmentofnewproteincarriersortheir replacementwith shorter peptidesor innovative nano-car- riers[2,22,23].Inthiscontesttoenhancethepoorimmuno- genicity of carbohydrate antigens, AuNPs offer many advantages and represent optimal and versatile platforms [5,24]. The controlled surface functionalization can afford a well-mimic of the natural glyco-cluster, leveraging the multivalenteffecttoinduceanenhancedimmuneresponse.

AuNPshavebeenusedasvaccinecandidatesagainstStrepto- coccus pneumonia by coating 2nm AuNPs with different ratios ofthe branched tetrasaccharide unit b-^D-Galp-(1–4)- b-^D-Glcp-(1–6)-[b-^D-Galp-(1–4)-]b-^D-GlcpNAc-(1–)ofS.pneumonia capsular polysaccharide serotype 14 and a T-helper peptide,ovalbumin323–339peptide(OVA323–339)[25].The simultaneous coating of the saccharide antigen and a T- helper peptide was essential to trigger anti-saccharide antibodies and avoid theepitope suppression issue. More- over, the same nanoparticle surface can display different type of antigens and immunogenic peptides or adjuvants I. Synthesis

II. Functionalization

III. Applications

Vaccine Adjuvants

Bacterial Vaccines

Viral Vaccines

Anti-parasite Vaccines

Autoimmune Diseases (a) Covalent conjugation

(b) Electrostatic multilayerd functionalization

Multi-shaped AuNPs HAuCl₄

Fig.2. I.Synthesis:AuNPscanbesynthesizedfromAu^IIIsalts,ﬁnelytuningsize,andshape.II.Functionalization:a.ReliableAu-Schemistryisusedto functionalizedgoldsurfacewithligands,proteinsandoligoorpolysaccharides.b.Surfacefunctionalizationcanbeaccomplishedbyamultilayeredstrategy.

III.Applications:avastnumberofdifferentﬁeldscanhavegreatadvantageusingAuNPs:theywillbereviewedintheirmostrecentprogressinthispaper.

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simultaneously. In a recent example, 2nm AuNPs were functionalizedwithfourcomponentstodevelopacandidate againstdifferentserotypesofS.pneumoniae,amajorpathogen causing pneumonia with over 2 million deaths annually (Fig.3)[21,26].AuNPswereengineeredwith:(1)twosynthet- icoligosaccharideepitopesofS.pneumoniaeserotype14(the tetrasaccharideunit)and19F (trisaccharide(b-^D-ManpNAc- (1!4)-a-^D-Glcp-(1!2)-a-^L-Rhap-(1!]);(2)aT-helperovalbu- min323–339peptide;(3)D-glucose.Afterimmunizationin mice,glyconanoparticlesinducedimmuneresponseprimar- ilydirected towardsS. pneumoniae14 comparable withthe commercially available PCV13 vaccine [26]. This result emphasizestheexcitingadvantagesarisingfromanaccurate designandsynergisticactionofAuNPsurfacefunctionaliza- tion.Recently,our grouppreliminaryevaluatedtheroleof AuNPgeometrydesigningoligorhamnan-functionalizedNPs toaddressinfectionsbyaGram-positiveGroupAStreptococ- cus(GAS),abacteriumresponsibleforalargerangeofinfec- tions in developing countries. Spherical and urchinated AuNPs functionalized with the synthetic tetra and hexa- rhamnosides were able to inhibit the binding of specific polyclonalserummuchbetterthantheunconjugatedoligo- saccharides,regardlessthedifferentmorphologies[27].

Burkholderia pseudomallei is a Gram-negative bacterium, common to tropical and subtropical climates, that causes melioidosis in humansand othermammals. Theinfection

fromB.pseudomalleipresentsavarietyofclinicalmanifesta- tions,rangingfromsofttissueabscessestofulminantsepsis.

Currently, melioidosis treatments are limited because the bacterium isinherently resistantto antibioticsandnovac- cinesareavailableorinadvancedclinicaltrials.Recently,a reversevaccinology approachhas beensuccessfullyapplied toidentifynewimmunogenicB.pseudomalleiproteins:hem- agglutininandFIgL.Such proteinshavebeen incorporated into a nanoglycoconjugate vaccine formulation based on 15nmAuNPalready coatedwithhighlyantigeniccapsular lipopolysaccharides(LPS)ofthebacteria [28].Thesamere- searchgrouppreviouslydemonstratedthattheincorporation ofLPSontoAuNPsshowedsignificantprotectioninmurine and nonhuman primate models of inhalational glanders, enhancingtheimmuneresponseandincreasingtheprotec- tionagainstthecloselyrelatedpathogenB.mallei[29,30].The latest and promising results based on 15nm glyco-AuNPs withthenewimmunogenicproteinsevidencedthatimmu- nizedanimalsgeneratedbothantigen-andLPS-specificIgG titers in mice. Additionally, immunization demonstrated between90%and100%survivalfollowingalethalchallenge withB.pseudomallei[28].

Areversevaccinologystrategyhasbeenexploitedalsoto identifytwonewimmunogenic proteins,LomWandEscC, thathavebeen covalentlylinked onAuNPsurfacetochal- lengeenterohemorrhagicEscherichiacoli(EHEC)O157:H7,a

Fig.3. AuNPsfunctionalizedwith4componentcomprisedof(1)synthetictetrasaccharideepitopeofS.pneumoniaeserotype14(b-^D-Galp-(1–4)-b-^D- Glcp-(1–6)-[b-^D-Galp-(1–4)-]b-^D-GlcpNAc-(1–));(2)synthetictrisaccharideepitopeofS.pneumoniaeserotype19F(b-^D-ManpNAc-(1!4)-a-^D-Glcp- (1!2)-a-^L-Rhap-(1!]);(3)aT-helperovalbumin323–339peptidetoevokeanti-saccharideantibodies;(4)D-glucoseasinnercomponenttoensurewater solubility.

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humanpathogenthatcausesdiarrheaandhemorrhagiccoli- tisworldwide[31].Sinceantibiotictreatmentiscontra-indi- cated,vaccinationrepresentsthebestalternativetoprevent such infections. Proteins were conjugated through a thio- linker to 15nm AuNPs and injected subcutaneously into mice; the new systems elicited strong humoral responses withhightiterofIgGandsIgAtowardseachantigen.

AuNPsrepresentapromisingalternativetodevelopanew typeofvaccineagainstlisteriosis,inducedbyListeriamono- cytogenes (Listeria), a bacterium that causes opportunistic infections in immuno-compromised patients and cerebral listeriosisinfetuses andnewborns. AuNPswere functional- izedwithtwopeptidesfromListeria’svirulencefactors,lis- teriolysin O (LLO) 91–99 and glyceraldehyde-3-phosphate- dehydrogenase(GAPDH)1–22peptideantigens.Inbothcases peptide-functionalizedAuNPs wereextensively studiedand provedtheirefficacyinvitroandinvivo,inducingCD8⁺T-cell immunity, response enhanced by the co-formulation with Advax^TM,acarbohydrate-basedadjuvantderivedfrommicro- particles of deltab-^D-[2–1]poly(fructo-furanosyl)a-^D-glucose (deltainulin)[32,33].Moreover,GADPH-AuNPsdemonstrat- ed theirability to pass theplacenta barrier and to protect neonatesinfectedthroughvertical transmissionofthebac- teria,inducinghighlevelofIL-12andadecrementofIL-6in newbornsfromvaccinatedmothers[34].

Recently,AuNPswerefavorablyusedalsoagainstPseudo- monasaeruginosa.AuNPsconjugatedtoN-terminaldomains ofP.aeruginosaflagellin(flagellin(1–161))elicitedhightiters ofanti-flagellinIgGantibodiesabletorecognizeandneutral- izethegram-negativeopportunisticbacteriuminmicemod- els[35].

Viralinfections

Theactionsagainstviralinfectionshavebeenatthecenterof scientificresearchforcenturiesandthedevelopmentofeffi- cient vaccines ableto induce specific immunity and long- lastingmemoryremainsthemaingoalinthisfight.Almost fourdecadesafteritsdiscoveryin1983,humanimmunode- ficiency virus(HIV) continues to bea major global public health concern.Thehugepotentialto inducemucosalim- munitymakesAuNPsidealplatformsforintranasalapplica- tions andfortopical treatment,designing vaccinesagainst sexuallytransmitteddiseasessuchasHIV.Nowadaysthereis onlyoneHIVvaccineinhumanclinicaltrials(RV144),and theprogress has been hampered given thelack ofrealistic animalmodelstovalidatethenanovaccines[36].Antiretro- viral(ART)therapieshaveproventofightinfection,prolong thelife-expectancyofinfectedindividualsandlowertherisk ofinfection.However,theydonotprovideacureandindi- vidualstakingthislifelongmedicationmustovercomeissues associated with side effects, drug resistance, strict dosing regimensandhighcosts,whichmakesthisstrategyunfeasi- ble for developing countries. Therefore, an effective HIV

vaccineisurgentlyneededtopreventinfectionandprogres- sion of theacquired immune deficiencysyndrome (AIDS).

Amongthemanychallengestoachievethisgoal,oneofthe greatestisHIVdiversity,reflectedbythepresenceofsubtypes, circulatingrecombinantforms,andcontinuousviralevolu- tiontoevadethehostimmunesystem[37].Hence,effective vaccines need to (1) elicit broadly neutralizing antibodies (bNAbs)capable ofneutralizingthemajorityofcirculating HIV strains and(2) generate strong CD4⁺ andCD8⁺ T-cell responsesabletokillvirallyinfectedcellswithoutrepresent- ingahazard.Mostvaccinedevelopershavefocusedonenve- lopeglycoproteins(Env)ontheviralmembrane,whichare the sole target for bNAbs against HIV. However, despite decadesofefforts,thereisnoavaccineabletoinducesuffi- cient bNAbs and generate protective immunity [37,38].

AuNPs aim toovercome current limitations by inducing a synergistichumoralandcellularimmuneresponse.Thesim- ilarstructuresofthe‘antennas’constitutedbyhigh-mannose N-glycanspresentontheHIV-1envelopeglycoproteingp120 were proposed by simultaneously incorporating tetra- and penta-mannosidesonAuNPsurface.Theseglycansnotonly constituteanepitopefortheHIVbroadlyneutralizinganti- body2G12,butalsotargetDC-SIGNreceptorsinvolvedinthe firststepsofHIVinfection.Mannose-AuNPs,invitro,inhib- itedHIVgp120bindingtoDC-SIGNandelicitingIgG2G12 [39]. (Oligo)mannosideAuNPs havebeen also leveragedto control the conformationof the V3 loop peptide, derived from the third variable region of HIV gp120 protein, the major immunogenic domain of HIV-1 [40]. Displaying on thesame nanoparticles both (oligo)mannosides and acar- boxyl-ending linker, the negative charged nanoplatforms wereabletostabilizeeitherthea-helix orb-strandV3 con- formation.RabbitsimmunizedwithsuchAuNPssuccessfully triggeredantibodiesabletorecognizearecombinantgp120.

Toenhancethepoorimmunogenicityofthehighmannose oligosaccharides, AuNPs carrying simultaneously di-man- noseandGagp17,aHIV-peptideantigen,weresynthesized.

Ex vivoexperiments with monocyte-derived dendritic cells (MDDCs) and lymphocytes from HIV-1 infected patients showed that such AuNPs increased HIV-specific CD4⁺ and CD8⁺proliferationandinducedhighlyfunctionalcytokine secretion compared with free peptides. Altogether, results suggest that these formulations not only inducea T_H1 re- sponsebutalsopromoteacombinedT_H1/T_H2profilewhich could activate B cells enhancing the humoral arm of the immune system. Thus, co-delivery of HIV-1 antigens by AuNPscouldinducenotonlyHIV-specificcellularresponse but also an antibody response, like that exerted by the broadlyneutralizing2G12antibody[41].

Greateffortsarededicated totheidentificationofauni- versal vaccine against seasonal influenza, which puts greateconomicpressureonpublichealthcaresystemsworld- wide. Unfortunately, a new influenza vaccine needs to be

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developedeveryyearaccordingtothenewcirculatingstrains.

Therefore, a universal influenza vaccine focused on type- specificaminoacidsequencesandconformationalepitopes wouldbehighlydesirable[42].Inapreliminarystudy,12nm AuNPswerefunctionalizedwiththehighlyconservedextra- cellularregionofthematrix2protein(M2e)ofinfluenzaand intranasallyinjectedinmice,inducingM2e-specificIgGse- rumantibodies.Theco-administrationofAuNPsandCpG,as adjuvant, strongly stimulated M2e-specific IgG antibodies, inducing cross-protection against diverse influenza viral strains [43]. A more recent approach is instead based on 18nm AuNPs conjugated with recombinant trimetric A/

Aichi/2/68 (H3N2), hemagglutinin (HA)and TLR5 agonist flagellin(FliC)[44].Combiningtheintranasalinvivoadmin- istrationandinvitroresults,theconjugatedAuNPsresulted able to effectively induce a strong immune response by activatingDCsandTcells.

Polyelectrolyte self-assembled AuNPs proved to be very efficientasinnovativenanoplatforms.Zhangeta.prepared multilayered,self-assembledAuNPsby alternate deposition of polyinosinic-polycytidylic acid (polyIC) and OVA257–264

octapeptideSIINFEKL[13].Theformerisananionicnucleic acidTLR3 agonistwithstrongadjuvantpropertieswhereas thelatteractsasapeptideantigenactingascationicanchor.

Thesenanoparticleswereefficientlyinternalizedbyprimary dendriticcells,resultinginactivationandpresentationofthe antigens,promoting high levelsofantigen-specific CD8⁺T cells response,compared tomice treatedbyco-administra- tionofantigen(SIINFEKL)andadjuvant(polyIC).Inanother example, a low-molecular-weight synthetic RNA adjuvant, polyinosinic-polycytidylic acid(uPIC(40:400)), wasusedto electrostatically coat spherical and rod-shapedAuNPs [15].

The needle-free intranasal administration ofuPIC(40:400)- goldnanorodstogetherwithinfluenzavirushemagglutinin showedanenhancedadjuvanticityofuPIC(40:400),leading tothesuppressionofinfluenzaviralinfectioninmicewhile maintaining lowinflammatory cytokine production.Inter- estingly, a shape-dependent adjuvant effect of AuNPs has beenreportedinanothercontributioninwhichtheaddition of gold nanostars to virus-like particle nanoformulations powerfullyactivatedmacrophagesandinducedspecificim- muneresponseagainstfoot-and-mouthdisease(FMD)[45].

Cancer

In 1893, W. Coley postulated for the first time that the immune system was able to recognize and set a response againsttumors,pavingthewaytowardsthedevelopmentof cancer vaccines as a potential alternative to conventional cancertherapies[46].Asopposedtoprophylacticvaccinesto preventinfectiousdiseases,therapeuticcancervaccineswork toactivateormodulatethebody’simmuneresponseagainst malignancies.Cancercellsexpressaseriesoftumor-associat- edantigens(TAAs),towhichcytotoxicTcellresponsesare

difficultto induce.Thus,theeffectivenessofcurrentvacci- nationapproachesislimitedbytheweakimmune-stimulating capacityof tumorantigens. AuNPs constitutes asmart template forthemultivalentpresentationofTAAantigens, co-deliveryofantigensandadjuvantsandhasproventoactas anefficientadjuvant,enhancingimmunogenicity[47].Tu- mor-associated carbohydrate antigens (TACA’s) are glycan chainsspecifically andaberrantly expressed onthe surface ofmalignantcellsandthus,areconsideredpromisingtargets forcancerimmunotherapy[48].

Mucins are a family ofaberrant proteinshighly overex- pressed on many tumor cells. They primarily exist as two domains:onelargeextracellularsubunitandasmaller,trans- membranecytosolicdomain.Theextracellulardomaincon- sistsoftandem repeat(TR)motifsofabout20aminoacids thatcontainalargedegreeofserineandthreonineO-glyco- sylated.Themostprevalent tumorassociated carbohydrate antigenspresentonmucinsaretheTn(GalNAca-O-Ser/Thr), the Thomsen Friedenreich (TF) (Galb1–3GalNAca-O-Ser/

Thr), and the sialyl-Tn (Neu5Aca2–6-GalNAca-O-Ser/Thr) TACAs.A strongantibodyresponse tothese antigensleads to a better prognosis and less aggressive tumors. Among mucins,MUC-4,issparseorabsentonnormalpancreastissue butaberrantly expressedinpancreatic cancermakingit an importanttarget forsolidtumors.Spherical3–5nmAuNPs werecoatedwithtumor-associatedglycopeptidesconsisting inthecellsurfacemucinMUC-4,functionalizedwiththeTF disaccharide antigen at different sites. The subcutaneous immunizationofmice, comprisedoffunctionalizedAuNPs andapeptideadjuvantderivedfromproteinC3d,showeda small butencouraging andstatisticallysignificantimmune responsewithproductionofbothIgMandIgGisotypes[49].

Veryrecently,theresultsobtainedusingthisnanoconstruct wereexploitedtoproduceamonoclonalantibodyspecificfor theN-terminalglycosylatedpeptidedomainthatrepresentsa novelimmunogentarget,interesting todevelop diagnostic antibodies orimmunotherapies[50].A simpleapproachto thesynthesis ofanticancervaccineswithout theneedof a typicalproteincomponentisbasedonapolymericversionof thetumor-associatedTnmonosaccharideantigen.Byexploit- ing the reversible addition-fragmentation chain transfer (RAFT) polymerization, the polymers were conjugated to AuNPs, producing peptide-free nanoscale glycoconjugate.

AuNPs,invivo,inducedasignificantproductionofIgGanti- bodies,selectivetotheTnantigenglycanandcross-protective immunity toward mucin glycoproteins displaying Tn antigens[51].

ThetetheredmucinglycoproteinMUC-1isover-expressed andwithadistinctlyalteredglycanpatternonepithelialtumor cells,thereforeitrepresentsatargetstructureinthedevelop- ment of effective carbohydrate-based cancer vaccines.

Nevertheless,thenaturalglycopeptideantigenhaslowimmu- nogenicityandaT-cellindependentresponse,drawbacksthat

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couldbeovercomebyusingmultivalentcarriers.Novelrobust nanoplatformsabletosuccessfully presentMUC1-glycopep- tideantigensmultivalentlytotheircorrespondingantibodies havebeenstudied[52].Threedifferent glycosylatedMUC-1 partialstructureswithafull 20amino acids,containing tandem repeats domain and a spacer (to guarantee flexibility and distance to the nanoparticle core) were synthesized using solid-phasepeptidesynthesis.Such glycopeptidesstructures werethenusedtocoatthegoldsurfaceof7and14nmAuNPs, developinginnovativeandreliableMUC-1functionalizedgold colloidsabletodetectselectivelyprimaryanti-MUC1antibody as provedby quartz crystalmicrobalanceandby adot-blot immunoassay.Inanotherapproach,MUC-1wasexploitedto coat15nmAuNPsandusedtotreat monolayersofperitoneum- derivedmacrophages[53].TheMUC-1-AuNPspowerfullyacti- vatedmacrophages,releasingcytokinesasTNF-,IL-6, IL-10, andIL-12.Moreover,apredominantM1polarizationofmacro- phagesresultedafterMUC-1-AuNPsexposure,representingan importantachievementasM1macrophagesplayacrucialrole in antigen presentation and tumor vaccine generation. In anotherapproach,PEGylatedAuNPs(inarangefrom22to 30nm)werefunctionalizedwithaglycopeptidesequencede- rived from MUC-1 covalently linked toCD4 T-cell peptide epitope (P30from TetanusToxoid)[54]. Such nanosystems wereevaluatedandtheirabilitytoinduceselectiveantibodies waspreliminarytestedinvivoonasmallnumberofanimals:

after immunization, mice showed significant Th1and Th2 mediatedimmuneresponsesdirectedtotheglycopeptidean- tigen.

Peptideantigenscanalsobeusedtoinduceanimmunologic response: the coupling to AuNPs has a double advantage, preventingpeptidedegradationandallowingatargeteddeliv- erytoAPCs.AuNPswereassessedforthedeliveryofanexoge- nous ovalbumin (OVA) peptide antigen to evaluate the therapeuticeffectinaB16-OVAmicetumormodel.Thesub- cutaneousadministrationAuNP-OVAinducedstrongantigen- specificresponses in miceand cytokinerelease inbone marrow- derivedDCcultures.ThesefindingssupporttheuseofAuNPsas effectivepeptidevaccinecarrierswiththepotentialofusing lowerandsaferadjuvantdosesduringvaccination[55].

Inrecentyears,AuNPshavedemonstratedpromisingpoten- tialasvehiclesfortargeteddeliveryofnucleicacidsgiventheir ability to protect genetic material from degradation by nucleases. A recent study presented AuNPs covalently functionalizedwithashikimoylgroupasDCtargetingligand andatransfectionenhancingguanidinylligand.AuNPswere electrostatically coupledwith amelanoma antigen (pCMV- MART1)encodedDNA. Thenanovaccinedemonstratedthe abilityto deliverDNAtoDCsinvitroandinvivowith high efficiency,inducinglong-lastingprotectiveimmunityaswell astherapeuticeffectsagainstmurinemelanoma[56].Unlike DNAvaccines,RNAvaccinesarelesslikelytocausesideeffects duetotheirrapiddegradationandclearance.Shortinterfering

RNA(siRNA)hasshown promisingpotentialasamolecular approachtodown-regulatespecificgeneexpressionincancer cells. A mechanism of tumor immune evasion consists in immunecheckpointinteractionsbetweenligands expressed ontumorcells(e.g.PD-L1)andreceptorsonthesurfaceofT- cells(e.g.PD-1)toinhibitcytotoxicT-cellsproliferation.Gulla et al. designed positively charged AuNPs bearing a tumor targetingpeptide(CGKRK)complexed electrostaticallywith PD-L1siRNAandSTAT3siRNA,withtheaimtodownregulate immune checkpoint proteins and prevent tumor evasion.

Intraperitoneal administration in melanoma-bearing mice revealedselectiveaccumulationofnanoconjugatesintumor tissues,tumorgrowthinhibitionandenhancedoverallsurviv- abilitywhencomparedtountreatedanimals[57].Indoleamine 2,3-oxygenase(IDO)isanimmuneregulatorymoleculethat inhibitsanti-tumorimmunityby inducingTcell apoptosis.

Zhangetal.explored theimmunostimulatoryeffectofgold nanorods (4515nm) functionalized simultaneously with gene silencingIDO siRNA and mannose targeting DCs for betterinternalizationofRNA.Thenanosystemsnotonlysuc- cessfullyachievedDC-targeteddeliveryofsiRNAbutinduced efficientDC-specificgenesilencinginvitroandinvivo.Thein vivoadministrationtolungcarcinomabearingmicepromoted DCmaturationandhigherantigen-specificTcellproliferation, attenuatingtumorgrowth[58].

Many reports have proven the ability of functionalized AuNPs to enhance antigen presentation while stimulating immuneresponsesforeffectivevaccinationagainsttumors.

Nonetheless,additionalstudiesarerequiredtobetterunder- stand the complex interaction between tumors, immune systemandnanovaccinesinvivotoeventuallytranslatethese promisingresultsintoclinicaltrials.Asinnateimmunityisa keyfactorintumorgrowth,thepotentialoffutureimmuno- therapycanbelinkedtothedevelopmentofnanomaterials forthespecificmodulationofinnateimmunitycells.

Parasitesinfections

AuNPshave evokedstronginterest intheparasitologyand entomologyfields,andtheirbioactivityhasbeenexamined on many insect species of economic relevance [59–61].

Amongmanydangerousparasites,Plasmodiumfalciparumis themaincauseofmalaria,whichremainsahealthconcernin developingcountries, responsiblefor up 283 million cases and755,000deathsworldwide.Besidesthedevelopmentof new drugsto preventand treatthedisease, addressing the parasite’ssexualstagesbymeansofmalaria’s transmission- blockingvaccines,representsapioneeringtarget.Pfs25isa surfaceprotein identified as targetantigenin P.falciparum andexpressed in zygotes and ookinetes. Therefore, anim- mune response against Pfs25 could affect the detrimental development of parasites by controlling vector-mediated transmission. Pfs25 is characterized by a complex tertiary structure and four EGF-like repeat motifs, therefore it is

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difficulttoobtainahomogeneousproductinnativeconfor- mation.Recently, the expression ofcodon-harmonized recombinant Pfs25(CHrPfs25) inanappropriate monomeric conformationhasbeenreportedandelicitedpotentmalaria transmission-blockingantibodiesinmice[62].Theseencour- agingresultsweremagnifiedwhenCHrPfs25wascovalently immobilized onto different size and shape AuNP surface (spherical, star, cages, and prism) or simpler admixed.

CHrPfs25deliveredwithvariousgoldnanoparticleselicited stronganti-Pfs25antibodytitersinmiceandtheantibodies fromimmunizedmice serashowedpromisingtransmission blocking efficacy in mosquito (An. gambiae) by means of membranefeedingassays[63].

Non-infectiousdiseases

With vaccines keeping at bay many infectious diseases, chronicautoimmunediseaseshavebecomeahealthconcern of modern times. Autoimmunity arises from an abnormal immuneresponsetoautologousproteinswhichleadstothe activationofBandTcellsagainstnormaltissues.Currently, thetreatmentofautoimmunedisordersreliesontheuseof immunosuppressive drugs which compromises the entire immunesystem,increasingsideeffectsandsystemictoxici- ties. Asopposedto traditional treatments,immunotherapy approaches aimto reestablisha normalT cell response by inducing regulatoryT cells(Tregs).Nanoparticlescarryinga disease-relevantautoantigenincombinationwithimmuno- modulatorymoleculescanpromoteantigenspecificTregsand thereforesuppressautoimmunity.Unfortunately,onlyafew disease-specificantigensareknownsuchasmyelininmulti- ple sclerosis (MS), insulin in Type 1 diabetes (T1D), and collageninrheumatoidarthritis(RA)[9].Thearylhydrocar- bonreceptor(AhR)isaligand-activatedtranscriptionfactor inducingtolerogenicDCsthatpromotethedifferentiationof Tregs.Moreover,AhRactivationlimitstheabilityofDCsto promotethedifferentiationofpathogenicTcellsasalready provedforMSapplication[64].Thisapproachwaspowerful forT1D:AuNPswereloadedwithanendogenousAhRligand, 2-(1⁰H-indole-3⁰-carbonyl)-thiazole-4-carboxylicacidmethyl ester(ITE)andproinsulin,abcellantigentargetedbyauto- immune T cells. Administration of the NPs to non-obese diabetic (NOD) mice suppressed the development of the pathologyandinducedaSOCS2-dependenttolerogenicDC phenotypecharacterizedbytheinhibitionofnuclearfactor kBsignaling, a decreased ability to activeTeff cells and an increaseddifferentiationofFoxp3⁺T_regs cells.However,the abilityofthisapproachtoameliorateT1Dsymptomswasnot statisticallysignificant[65].Inasimilarmanner,proinsulin autoantigen (PIC19-A3) was covalently attached to ultra- smallAuNPs,previouslycoatedwithcarbohydrates(glucose or mannose and glutathione). Intradermal administration intoexvivohumanskinusing aminimallyinvasivemicro- needleprovedtheirabilitytodiffusetoAPC-richepidermal

layer.In vitrostudiesshowedthatAuNP-peptidecomplexes impairedmaturationofDCsintoreactiveTcells,promoting the generation of Tregs: clinical studies are underway to determine if this system can indeed act as a platform for antigenspecifictoleranceinduction[66].

Conclusions

Developmentofprophylacticandtherapeuticvaccinesusing nanomaterials constitutes an emerging and promising re- searchfield.RecentliteraturedemonstratesthatAuNPs,with theiruniqueproperties,representidealplatformstowardsa neweraofvaccinology.Theirhighsurfaceareaandstraight- forwardfunctionalizationallowsimultaneousandmultiva- lent antigen presentation and make them excellent candidates for innovative nano-constructs in the field of vaccine development. AuNPs are readily endocytosed by APCs, affecting increased immunological responses while reducing antigen dosage. Therefore, we expect that some of these nano-formulations will be translated into clinical practice,butthiscanonlybeachievedifmajorchallengesare addressed.First,ahighlevelofconsistencyinthelarge-scale production, characterization, and reproducibility of engi- neeredAuNPsmust beensured.Abettercomprehensionof thenanoparticle interaction with theimmune system and theirinvivobiodistributionispivotaltoacceleratetheratio- naldesignofmoreencouragingnanoformulations.Tocor- rectly face this issue, it is fundamental to consider the potentialformationofbio-coronaaroundAuNPsurfaces.A careful evaluation of such bio-corona formation and the developmentofprotocolsorstrategytoavoidsuchphenom- enon must beconsideredinthefuture nanovaccinedevel- opments.Anothercriticalpointisthelackofrepresentative animal models which justifies the differences from very promising results fromanimal teststo poorlyencouraging outcomesinpreliminaryhumantrials.Finally,thefateofthe nanoparticles and their biodegradability must be carefully evaluated,makingnanotoxicologyagrowingresearchfieldin whichweneedtofocusourattention.

Conﬂictofinterest Nonedeclared.

Acknowledgements

WegratefullyacknowledgefinancialsupportbytheEuropean Union’s Horizon 2020 research and innovation program undertheMarieSkodowska-Curie grant‘NanoCarb’,agree- mentno.814236

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Ruth Mateu Ferrando , Luigi Lay Laura Polito *, * development Gold nanoparticle-based platforms forvaccine

Gold nanoparticle-based platforms for vaccine development

Ruth Mateu Ferrando

, Luigi Lay

* , Laura Polito

*

Ruth Mateu Ferrando , Luigi Lay Laura Polito , development Gold nanoparticle-based platforms forvaccine