The adjuvanticity of Chiococca alba saponins increases with the length and hydrophilicity of their sugar chains

(1)

ContentslistsavailableatSciVerseScienceDirect

Vaccine

j o ur na l ho me p ag 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

The

adjuvanticity

of

Chiococca

alba

saponins

increases

with

the

length

and

hydrophilicity

of

their

sugar

chains

Dirlei

Nico

a

_,

_Ricardo

_Moreira

_Borges

b

_,

_Layza

_Mendes

_Brandão

a

_,

_Daniel

_Ferreira

_Feijó

a

_,

Daniele

Crespo

Gomes

a

_,

_Marcos

_Palatnik

c

_,

_Mauricio

_Martins

_Rodrigues

d

_,

Antonio

Jorge

Ribeiro

da

Silva

b

_,

_Clarisa

_Beatriz

_{Palatnik-de-Sousa}

a,∗

a_Departamento_de_{Microbiologia}_Geral,_Instituto_de_{Microbiologia}_“Paulo_de_Góes”,_Universidade_Federal_do_Rio_de_Janeiro_(UFRJ),_CP_68040,_21941-902_Rio_de_Janeiro,_Brazil

b_Núcleo_de_Pesquisas_de_Produtos_Naturais,_Universidade_Federal_do_Rio_de_Janeiro,_Rio_de_Janeiro,_Brazil

c_Hospital_{Universitário}_Clementino_Fraga_{Filho-Faculdade}_de_Medicina,_UFRJ,_CEP_21941-913_Rio_de_Janeiro,_Brazil

d_Centro_{Interdisciplinar}_de_Terapia_Gênica,_Universidade_Federal_de_São_Paulo_(UNIFESP),_04044-010_São_Paulo,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23June2011

Receivedinrevisedform3March2012

Accepted3March2012

Available online 15 March 2012

Keywords:

Saponins Adjuvants

QS21saponin

Chiococcaalba

Visceralleishmaniasis

FMLantigen

a

b

s

t

r

a

c

t

ThesaponinsofChiococcaalbaaretriterpenebidesmosidesthatcontainglycidicmoieties attached totheC-3andC-28carbonoftheiraglycone.Wedescribethattheiradjuvantpotentialincreasesin directrelationshiptothelengthandhydrophilicityoftheC-28attachedsugarchainwhichcontains: arabinose–rhamnoseintheCA2,arabinose–rhamnose–xyloseintheCA3X;arabinose–rhamnose–apiose intheCA3andarabinose–rhamnose–apiose–apioseintheCA4saponin.Thehydrophile/lipophilebalance calculatedforCA2was12.7,forCA3andCA3Xwas15.8andforCA419.9.Allsaponinswereformulated withtheFMLantigenformiceprophylaxisagainstvisceralleishmaniasis.Theimmuneresponsewas studiedusinganELISA-antibodyassayandmonitoringoftheintradermalresponse(IDR)to Leishma-niaantigens,thecytokineexpressioninsupernatantsandtheintracellularstainingofinvitrocultured splenocytes.Afterchallenge,significantincreasesofIgGandIgG2aantibodieswerenotedonlyintheCA4 vaccinatedmicethatshowedextendedIDR,higherIFN-␥productionbyCD8+andTNF-␣productionby CD4+Tcells,higherTNF-␣secretionandthehighestreductionoftheparasiteload(78%).Theincreasesin IDR,CD4–TNF-␣,CD8–IFN-␥andCD8–TNF-␣bytheCA4vaccinewerestrongcorrelatesofprotectionand weresignificantlycorrelatedtothedecreaseofparasiteload(p=−0.007).Protectiongeneratedbythe CA4vaccinewasmainlymediatedbyaCD4+TcellandaTNF-␣drivenresponsewithalower contribu-tionofCD8+Tcells,asconfirmedbyaninvivodepletionwithmonoclonalantibodiesandbyvaccination assaysinTNF-␣-receptorknock-outmice.OurresultsconfirmthatthesuperiorityoftheCA4saponinis relatedtothehigherhydrophilicityofitslongercarbohydratechain.C.albasaponinswerenon-toxicand onlythexylose-containingsaponinCA3Xwashemolytic(HD50=87␮g/ml).Theincreaseinsugarunits ofthesaponinsispositivelycorrelatedtotheincreaseofIDRandtothedecreaseofparasiteload.

1. Introduction

Leishmune®_,_the_first_licensed_vaccine _against_visceral leish-maniasishasa76–80%ofvaccineefficacy.Thisveterinaryvaccine protects98%ofvaccinateddogsandblocksthetransmissionofthe diseaseinendemicareas[1–3].IntheAmericasandthe Mediter-ranean,visceralleishmaniasisisanimmunosuppressivezoonotic diseasetransmittedfromdogstohumansthroughthebyteofasand flyvector[4].Thediseaseisfatalinhumansanddogsifuntreated

∗_{Corresponding}_author_at:_Instituto_de_{Microbiologia}_“Paulo_de_Góes”,_CCS,_UFRJ,

Avda.CarlosChagas373,CaixaPostal68040,CidadeUniversitária,IlhadoFundão,

21941-902RiodeJaneiro,Brazil.Tel.:+552125626742;fax:+552125608344/8028.

E-mailaddress:immgcpa@micro.ufrj.br(C.B.Palatnik-de-Sousa).

andtreatmentishighlytoxicandnotalwaysefficient.The epidemi-ologicalcontrolof thediseaseincludesthetreatmentofhuman cases, insectvector controlwithinsecticides and thecullingof seropositive/infecteddogs.Humanorcaninevaccinesareexpected tobeeffectivetoolsfortheprophylacticcontrolofepidemics[5]. Therecent caninevaccinations withtheLeishmune® _vaccine_in Brazilreducedtheincidenceofhumancases,humandeathsand dogprevalenceofvisceralleishmaniasisinendemicareas[6].In districtswherethevaccinationsoccurredthecanineandhuman incidencedecreasedorachievedastabilizedplateauwhilein non-vaccinateddistrictstheincidencesrose[6].

Leishmune®_is_the_FML-saponin_vaccine_[1,3,7,8]_composed_of theFML(Fucose Mannoseligand)antigen[9],a complex glyco-proteic fractionofLeishmania donovani,andaQuillajasaponaria saponinadjuvant(RiedeldeHaën-Sigma)[revisedin3].Themain

doi:10.1016/j.vaccine.2012.03.006

Open access under the Elsevier OA license.

(2)

3170 30 (2012) 3169–3179

activecomponentsoftheLeishmune®_adjuvant_are_the_well_known QS21saponinandthetwodeacylatedsaponinsthatonlydifferfrom theQS21duetotheabsenceofthehydrophobicmoiety[10].

Saponinsareastructurallydiverseclassofnaturalcompounds occurringinseveralplantspecies.Accordingtopreviousreports themostcommoncomponentsofthesaponincorearethe triter-penoidandsteroidaglycones towhichcarbohydrate chainsare attached[11].Theyexhibitfromonetothreestraightorbranched sugarchainswhichmostoftenincluded-glucose,l-rhamnose,d

-galactose,d-glucuronicacid,l-arabinose,d-xyloseord-fucose.The

sugarchaincancontainfromoneormoremonosaccharideresidues, andisusuallyattachedattheC-3ofthetriterpene[11].

The correlationbetween structure and function of saponins hasbeenthefocus ofintensiveresearch inorder todefinethe essentialmoieties for thedevelopmentof theadjuvant activity [10–15].Saponinswithsteroidbutnotwithtriterpeneaglycones areconsideredtobethemosthemolytic[12,16].Alternatively,the hemolyticor membranolyticactivityhasbeenattributedtothe oligosaccharidemoietyofsaponins[13,17–22].Andthesaponins withtwoglycidicchainsattachedtotheaglycone,called bidesmo-sidic[10,14],havebeenshowntobemoreimmunogenicthanthe monodesmosidicones[14].

IntheQS21saponin ofQ.saponariaMolina,theinductionof a lymphoproliferativeCD8+ T cellresponse is attributedtothe normonoterpene hydrophobic moiety while the induction of a TH1 response isrelated to theC-23-aldehyde of thetriterpene nucleus[15,23].ThedifferentspatialconformationoftheC-23 alde-hydegroupdefines thetype ofinduced immuneresponse [17]. Anenhanced humoralimmune responsewasobtainedusingan enrichedaxialaldehyde-containingsapogeninwhileanenhanced cellularimmuneresponse(increasedDTHandIFN-␥seralevels) thatdetermineda77%reductionofliverparasiticloadwasobtained usinganenrichedequatorialaldehyde-containingQuilA-sapogenin [17].

TheQ.saponaria saponins,whichlack thehydrophobic moi-etyof QS21, arecapable of inducing increasesin DTH, CD4+ T lymphocytesin spleen, IFN-␥ in vitro,body weight gain and a pronouncedreductionofparasiteburdenintheliver,suggesting thattheimmunoprotectivepotentialofthesaponinreliesmoreon itscarbohydratechainsthanonitshydrophobicattachedmoiety [10].SimilartoQS21,theCP05saponinofCalliandrapulcherrimais composedofatriterpenenucleuswithtwocarbohydratefractions attachedtoC-3andC-28,respectively,andonehydrophobic moi-etyacylatedtoasugarattachedtoC-28[24].Thechemicalremoval ofthehydrophobicmonoterpenemoietyofCP05didnotinterfere withtheprotectionbuttheremovalofoneortwoofthe carbo-hydratechains,however,abolishedprotectionanddeterminedan increaseoftheparasiteloadindicatingthat,aspostulatedforother saponins[25–27],and inthecase oftheCP05saponin also,the inductionofprotectionisdirectlyrelatedtothepresenceofthe carbohydratemoieties[14].

Consideringtherelevanceofthecarbohydratemoietiestothe adjuvantpotentialofsaponins,andtheevidencethatthe immuno-protectivepotentialincreasesindirectrelationtothenumberof sugarunitsonthecarbohydratechains[19,22]thiswork investi-gated,twosaponinsofChiococcaalba(CA3andCA4)[28]which differonlyinonesugarunit.Thesetwosaponinswerecomparedin themurinevaccinationagainstvisceralleishmaniasiswiththeFML antigen.TheQS21-containingsaponinadjuvantoftheLeishmune® vaccine(saponinR)wasusedasapositivecontrol.

TheCA3andCA4saponinsofC.albaaretwotypicalGlucuronide Oleanane-typeTriterpeneCarboxylic Acid3,28-O-Bisdesmosides (GOTCAB).Their structures were recentlyelucidated [28]. Both shareatriterpenenucleustowhichaglucuronicacidisattached atC-3andanapiose–rhamnoseandarabinosechainisattached atC-28 (Fig. 1).TheCA4 shows thesametriterpene and sugar

chainswithoneadditionalapioseunit1→3linkedtothe rham-noseunitoftheC-28carbohydratechain(Fig.1).TheQS21saponin ontheotherhandismorecomplexbutalso,similartotheC.alba saponins,showsatriterpenenucleustowhichisattachedone glu-curonicacidatC-3,substitutedwithgalactoseandxyloseanda 5-sugar-unitchain(rhamnose–xylose–apioseandfucoseand ara-binose)atC-28thatalsoincludesahydrophobicmoietycomposed of2normonoterpenes[29].

Inthisinvestigationwepursuedtheanalysisoftheadjuvant potentialsofCA3andCA4saponinsofC.albaaimingtoidentifyifthe additionofonesugarunithasanyimpactontheimmunoprotective potentialofthesaponin.

2. Materialsandmethods

2.1. Ethicalstatements

AllmousestudiesfollowedtheguidelinessetbytheNational InstitutesofHealth,USAandtheInstitutionalAnimalCareandUse Committeeapprovedtheanimalprotocols(Biophysics Institute-UFRJ,Brazil,protocolIMPPG-007).

2.2. IsolationofsaponinsofC.alba(L.)Hitch

Samples of C. alba were collected in Nova Friburgo, Rio de Janeiro, Brazil. The botanical identification was made by Dr. Sebastião Neto, and a voucher specimen (RB395399) hasbeen depositedintheHerbarium oftheRiodeJaneiroBotanical Gar-den.Air-driedandpowderedrootsofC.alba(400g)wereextracted withethanol.Theextractwasevaporatedandtheresidueobtained (12g)wassuspendedinwaterandsuccessivelypartitionedwith methylenechlorideandbutanol.Thebutanolfractionswere com-bined,evaporatedandtheresidue(4g)wassuspendedinmethanol and subjected to controlled precipitation with diethyl ether. The precipitate (2g) wasfractionated bycolumn chromatogra-phy(octadecylsilane,60cm×20cm) usingH2Owithincreasing

proportions of methanol (0–100%) to obtain 10 fractions. TLC tests carried out withLiebermann–Bouchard and sulfuric orci-nolreagentstogetherwiththeobservationofanabundantfoam formation,allowedtheidentificationofthesaponinenriched frac-tions. Furtherpurification wascarried out withreversed-phase (octadecylsilane)preparativeHPLCusingmethanol:0.02%aqueous trifluoroaceticacid(60:40;v/v)toobtain48mgofCA3(Chiococca saponinII)and78mgofCA4(ChiococcasaponinI)[28].Wealso collectedandidentifiedtwoothersaponinsofC.albatobeusedas controls:theCA2(18mg)andtheCA3X(10mg)(Fig.1).Allsaponins (CA4,CA3,CA3XandCA2)shareatriterpenenucleustowhicha glucuronicacidisattachedatC-3andarhamnoseandarabinose containingchainisattachedatC-28(Fig.1).TheCA3XandCA3have athirdsugarattached1→4totherhamnoseunit.Thisthirdsugaris xyloseinCA3XandapioseinCA3.TheCA4saponinhas,inaddition tothe1→4linkedapiosepresentinCA3,afourthapioseunit,1→3 linkedtotherhamnoseunitoftheC-28carbohydratechain(Fig.1). Thehydrophile–lipophilebalance(HLB)valueofthesaponinswas calculatedtheoreticallybytheDaviesandRiedelmethod[30] con-sideringtheirchemicalstructureaspreviouslydescribedbyBorges etal.[28]andrepresentedinFig.1.Thevaluewascalculatedby integratingthenumberofeachfunctionalgroupcomposingthe saponinmoleculewiththegroupunitdefinedbytheDaviesmethod (HLB=7+

hydrophilicgroups−

lipophilicgroups)[30].

2.3. Hemolyticassay

(3)

Saponins of

Chiococca alba

_{(L.) Hitch}

O HO

HO OH HOOC

O

OH HO O Me

O HO

HO HO

O

O OH

HO CA3 HLB = 15.8

glucuronicacid

apiose rhamnose

arabinose

O HO

HO OH HOOC

O

OH HO HO

Me

O

O OH

HO CA2 HLB = 12.7

glucuronic acid

rhamnose arabinose

3β-hydroxyolean-12,15-dien-28-oicacid

O HO

HO OH HOOC

O

OH O O Me

O HO

HO HO

O

O OH

HO

O HO

HO HO CA4 HLB = 19.9

glucuronic acid

apiose

O HO

HO OH HOOC

O

OH HO O Me

O

O OH

HO

O HO

HO OH CA3X HLB = 15.8

glucuronic acid

xylose

Fig.1.ChemicalstructureoftheCA4,CA3,CA3XandCA2saponinsisolatedfromtherootsofChiococcaalba(L.)Hitch.

CA3,CA3XandCA2saponinsinsalinesolution.Mixtureswere incu-batedfor30minat37◦_C_and_centrifuged_at₇₀_×_g_for₁₀_min._Free hemoglobininthesupernatantswasmeasuredbyabsorbanceat 415nm[21].Salineanddistilledwaterwereincludedasminimal andmaximalhemolyticcontrols.Thehemolyticpercentdeveloped bythesalinecontrol wassubtractedfromallgroups. The adju-vantconcentrationinducing50%ofthemaximumhemolysiswas consideredastheHD50(graphicalinterpolation).Eachexperiment

includedtriplicatesateachconcentration.Aseriesof3independent experimentswasperformedfortheanalysisofeachHD50.Human

redbloodcellsforthehemolyticassaywereobtainedfromhealthy adultbloodbankdonors(HospitalUniversitárioClementinoFraga Filho,UniversidadeFederaldoRiodeJaneiro,RJ,Brazil).Thered bloodcellsuspensionwaspreparedbyfinallydilutingthepelletto 0.5%insalinesolution.

2.4. Invivosaponintoxicity

Toxicity(assessedbylethality,localpain,localswelling,andloss ofhair)wastestedinthevaccinatedmicethatreceived100␮gof eitherRiedeldeHaënoreachoneoftheC.albasaponins formu-latedwiththeFMLantigen,asthreeweeklydoses.Themicewere monitoredforsevendaysaftereachvaccinedose.

2.5. ImmunizationandparasitechallengebyLeishmaniachagasi

Eight-week-oldfemaleBalb/cmice,received3dosesof150␮g oftheFMLantigen[9]and100␮gofeithertheCA3,CA4saponins ofC.albaoroftheSigma-RiedeldeHaën16109saponin[reviewed in3]ontheback, throughthescroute,at weeklyintervals.At thebeginning of week 4, micewere challenged with3×107_L.

chagasiamastigotesobtainedfrominfectedhamsterspleens.The strain usedfor challengein this study (IOC-L 3324) was origi-nallyisolated fromthespleen ofan infecteddog ofAndradina,

SãoPaulo,BrazilandtaxonomicallycharacterizedasLeishmaniaL. chagasibytheCLIOC-WDCM731(InstitutoOswaldoCruz Leishma-niacollection,RiodeJaneiro,Brazil).Fifteendaysafterinfection, micewereeuthanizedwithetherandtheparasiteloadwas evalu-atedinGiemsa-stainedliversmearsandexpressedinLDUvalues (LeishmanDonovanunitsofStauber=numberofamastigotesper 600livercellnuclei/mgofliverweight)asdescribed[reviewedin 3].Theincreasein totalbody weightand liver/corporalrelative weightwerealsorecordedasclinicalsignsofVL.Control exper-imentsinBalb/cfemalemicealsoincludedgroups treatedwith saponinsCA2andCA3X.

2.6. Detectionofantibodies

Sevendaysafterimmunizationand15daysafterinfectionwith L.chagasi,antibodies ofserawere measuredbyanELISA assay againstFMLantigenaspreviouslydescribed[31],using2␮g anti-genperwellandProtein-Aperoxidase(KPL,Kirkegaard&Perry Laboratories, Inc.)or goatanti-mouseIgG1, IgG2a, IgG2b,IgG3, IgMandIgAhorseradishperoxidaseconjugatedantibodies (South-ern,BiotechnologyAssociates,Birmingham,AL,USA)ina1:1000 dilutioninblockingbuffer. ThereactionwasdevelopedwithO -phenyldiamine (Sigma), interrupted with1N sulfuric acid, and monitoredat492nm.Eachindividualserumwasanalyzedin trip-licateindouble-blindtests.Positiveandnegativecontrolserawere includedineachtest.Resultswereexpressedasthemeanofthe absorbancevalues(492nm)ofthe1/100dilutedseraofeach ani-mal.

2.7. AssessmentofthespecificTcellimmunity

(4)

3172 30 (2012) 3169–3179

wereinjectedintradermally,intherighthindfootpad,with107

freeze–thawedstationaryphaseLeishmaniadonovani promastig-otes(LD-1SSudanstrain)(200␮gofprotein)in0.1mlsterilesaline solution.ThefootpadthicknessesweremeasuredwithaMitutoyo apparatus,bothbeforeand0,24and48hafterinjection. Inject-ingeachanimalwith0.1mlsalineinthelefthindfootpadserved ascontrol.Ateachmeasurement,thevaluesofthesalinecontrol weresubtractedfromthereactionduetotheLeishmaniaantigen. PreviousexperimentscarriedoutinBalb/cmiceandCBhamsters demonstratedthat24hafterinoculationsalinetreatedfootpads returnedtobaselevels[32].

WealsocomparedtheIDRinducedinimmunizedandin chal-lenged mice by theinjection of either the promastigote lysate (200␮g of protein),or the FML antigen (100␮g), or theNH36 recombinantprotein(100␮g),in0.1mlofsalinesolution.

Furtheranalysesofcellularimmuneresponseswascarriedout using106 _splenocytes_after₅ _days_of _in_vitro_culturing _at₃₇◦_C and5%CO2 inRPMImediumand/or5␮gofrecombinantNH36,

themain antigenic component of theFML antigen [31]. Secre-tionof IFN-␥ and TNF-␣ was evaluatedin the supernatants of invitroculturedsplenocytesbyanELISAassay,usingtheBiotin Ratanti-mouseIFN-␥(cloneXMG1.2),thepurifiedRatanti-mouse IFN-␥ (clone R4-6A2) and the Mouse TNF ELISA Set IIkit (BD BiosciencePharmingen)accordingtothemanufacturer’s instruc-tions. Flowcytometryanalysis (FACSanalysis) in a FACScalibur apparatuswasperformedaftersplenocyteimmunostainingwith anti-CD4 (clone GK1.5) or anti-CD8-FITC (clone 53-6.7) mono-clonalantibodies(R&Dsystems,Inc.).Theintracellularproduction ofIFN-␥,TNF-␣and IL-10cytokinesbyCD4+ and CD8+ Tcells wasdeterminedusing10mg/mlbrefeldin(Sigma)for4hat37◦_C and5%CO2followedbywashingwithFACSbuffer(2%fetalcalf

serum,0.1%sodiumazideinPBS).Cellswerelabeledfor20min at 4◦_C _in _the _dark _with_rat _anti-mouse _CD4FITC _and _CD8FITC (R&Dsystems)inFACSbuffer(1/100).Afterthattheywerefixed with4%paraformaldehyde,washedandtreatedwithFACSbuffer with0.5%saponin (Sigma)for20minatroomtemperatureand thenfurtherstainedwithIFN-␥-APC,TNFPEandIL-10PE mono-clonalantibodies(BD-Pharmingen),1/100dilutedinFACSbuffer with 0.5% saponin for 20min, and finally washed and resus-pended in FACS buffer. A total of 30,000 cells were analyzed byflowcytometryinaBectonDickinsonFACScaliburapparatus, and furtheranalyzed using WinMDI(Windows Multiple Docu-mentInterfaceFlowCytometryApplication)Version2.8software [31].

InvivodepletionofCD4+orCD8+Tcellswasperformedby treat-ingCA4saponinandFMLvaccinatedmicewithGK1.5or53.6.7rat IgGMAbondays2,4and6beforechallengeandonday7after chal-lenge.ControlmicereceivedtheCA4-FMLvaccineand0.05mLof ratserumthroughtheintraperitonealroute,equivalentto0.25mg ofIgG,ornudemiceasciticfluidscontaining0.25mgofanti-CD4+ and/oranti-CD8+antibodies.AsdeterminedbyFACSanalyses,the efficacyof depletionofCD4+ or CD8+ spleen cells before chal-lengewasof99.94%or96%inanti-CD4+oranti-CD8+treatedmice, respectively.Theefficacyofdepletiontreatmentwasmonitoredby theincreaseinliverparasiteloadandliverrelativeweight,15days afterinfection.

RandomlyselectedfemaleTNFKOmice(n=15)andtheir wild-type(WT)littermates(n=15),generatedonaC57BL/6background, wereusedintheseexperiments.Groupsoffivemicewere vacci-natedwithCA3orCA4saponinincombinationwithFML-antigen orwithsalineandwereinjectedviathetailveinwith3×107

ham-sterspleen-derivedL.chagasiamastigotes(IOC-L3324).TheIDR wasdeterminedafterimmunizationand15daysafterinfection, visceralinfectionwasmonitoredmicroscopicallyusing Giemsa-stainedliverimprints,andliverparasiteburdensweremeasured inliversbycountinginablindedfashiontheamastigotesper600

cellnucleiandmultiplyingthisnumberbytheliverweightin mil-ligrams(LDUunits).

2.8. Statisticalanalysis

Differences between means were compared by the Kruskall–Wallis(KW)andMann–Whitney(MW)non-parametrical tests(Analyze-it).Fortheanalysisofdependentdataofthesame individualsbeforeandafterinfectiontheWilcoxonSigned-Rank two-tailedtestwasused,whichisthenon-parametricalternative of thet-test forcorrelated samplesof theVassarStats program (http://faculty.vassar.edu/lowry/wilcoxon.html) [33]. Correlation coefficientanalysiswasdeterminedusingaPearsonbivariate,two tailedtestofsignificance(SPSSforwindows).

3. Results

3.1. Assessmentoftheanti-FMLhumoralresponse

Aftercomplete immunization significant differences in anti-FMLantibodieswerefoundamongtreatmentsforIgM,IgG,IgG1, IgG2a,IgG2bandIgG3(p<0.01forallantibodytypes)butnotfor IgAantibodies(p=0.7331).TheCA3,CA4andRsaponinsraisedthe IgM,IgG1andIgG3antibodylevelsabovetherespectivesaline con-trols(Fig.2).TheCA3vaccineinduced54%and76%oftheIgMand theIgG1absorbencyvaluesinducedbythesaponinRpositive con-trol,respectively.TheCA4vaccine,ontheotherhand,induced62% and82%ofthetotalIgMandIgG1responsegeneratedbysaponin R,respectively.WeconcludethatafterimmunizationbothC.alba saponinsinducedapredominantIgM,IgG3andIgG1anti-FML anti-bodyresponse.

After challenge, on the other hand, significant differences between groups were found in anti-FML IgG (p<0.0001), IgG1 (p<0.0001), IgG2a (p<0.0001), IgG2b (p=0.0094) and IgG3 (p=0.0003)butnotforIgA(p=0.5164)orIgM(p=0.0783) antibod-ies.Asdisclosedbeforechallenge,theIgG1andtheIgMantibodies werestronglyenhancedbyallthesaponins(Fig.2).Inthecaseof IgM,asignificantenhancementwasalsonotedafterinfectionin thesalinecontrols.FollowingtheRsaponinpositivecontrol,the CA4saponinraisedmoreIgGandIgG2aantibodiestotheFML anti-genthantheCA3saponin(Fig.2).Indeed,theaverageabsorbanceof

Fig.2. Antibodyanalysis.Barsrepresentthemean±SEoftheabsorbancevalues

ofanti-FMLantibodiesfrom1/100dilutedserumoftwoindependentexperiments

usingn=6–8micepertreatmentineachexperiment,afterimmunizationandafter

challengewith3×107_amastigotes._Significant_differences_to_the_saline_controls

(*);tothesaponinRvaccinepositivecontrol();andtotheFML-CA3vaccine

treatment(䊉)asdisclosedbytheKruskall–Wallis(KW)andMann–Whitney(MW)

(5)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

after immunization after challenge

IDR to Leishmania lysate

skin test (mm)

24h 48h 24h 48h

* *

**

*

**

sal

CA3

CA4

R

A B

Fig.3.Intradermalresponsetotheleishmanialantigen,24h(left)and48h(right)aftercompleteimmunization(A)andafterchallenge(B)with3×107_amastigotes_of_L.

chagasi.Resultsof2independentexperimentsusingn=6–8micepertreatmentineachexperiment,areshownasindividualIDRmeasures.Horizontalfulllinesrepresentthe

meanvalues.*p<0.05indicatessignificantdifferencesfromthesalinetreatedcontrolsand**fromalltreatmentsasdisclosedbytheKruskall–Wallis(KW)andMann–Whitney

(MW)non-parametricaltests.

CA4increasedfrom0.564beforeto1.189afterinfection(p=0.0079) whiletheaverageforCA3vaccinatedmicedidnotsignificantly changed(from0.718to0.689;p=0.114).Furthermore,theCA4sap vaccineIgG2aresponseafterinfectionwasnotstatisticallydifferent fromthesaponinRvaccine.Allsaponinsraisedequivalentlevels ofIgG1abovethesalinecontroland onlytheRsaponin signifi-cantlyenhancedtheIgGbandIgG3antibodiesabovesalinecontrols (Fig.2).TheIgAantibodies,ontheotherhand,wereenhancedinall groupsafterchallenge(Fig.2).

ThepredominanceoftheCA4saponin,althoughonlymodest afterimmunization,wasmoreevidentafterinfection.Indeed, com-paredtotherespectiveantibodytitersbeforeinfection,significant increasesweredetectedintheCA4saponinvaccinatedmiceafter challengefor IgA (p=0.0032), IgM (p=0.0124), IgG (p=0.0414), IgG2a(p=0.0061)andIgG2b(p=0.0349)antibodieswhiletheCA3 saponinvaccineonlyshowedanincreaseoftheIgA(p=0.0016) andIgMantibodies(p=0.0045).Theseresultsconfirmthehigher potencyofthe4sugarchainCA4saponin(Fig.1)intheinduction ofanti-FMLspecificantibodiesthatwasfurtherenhancedafterthe infectivechallenge.

3.2. Assessmentoftheintradermalresponsetoleishmanial antigen(IDR)

Thecellularimmuneresponsewasinitiallyevaluatedbythe intradermalreactionagainstLeishmanialysate(IDR)(Fig.3).IDR wasmeasuredintherighthindfootpadsandsubtractedfromthe valuesofthelefthindfootpadinjectedonlywithsaline.At24h afterimmunization,theIDRresponsewassignificantlyhigherfor theRsaponincomparedtoalltheothergroupsandalsohigherfor theCA3(mean=0.06mm)andCA4(mean=0.08mm)thanforthe salinecontrol(mean=0.02mm)(Fig.3A).At48honlytheRand CA4sustainedthisresponseindicatingthesuperiorityofCA4over theCA3saponinofC.alba.Afterchallenge,onlytheRsaponin vac-cinesustainedtheenhancedIDR(Fig.3B).Therewasnosignificant variation,beforeandafterinfection,inthemagnitudeoftheIDR responseinducedbytheCA3(p=0.8103at24handp=0.6818at 48h)orbytheCA4vaccines(p=0.3898at24handp=0.2801at 48h)(Fig.3AandB).

In an additional experiment we compared the abilities of the Leishmania promastigote lysate, the FML antigen that was usedfor vaccinationandtheNH36recombinant protein,which is the main antigenic component of FML for the induction of IDR (Fig. 4).Althoughthesame trend described inFig. 3Awas

observed, the predominance of the CA4 IDR against the Leish-mania lysate was in this experiment even more pronounced (mean=0.416mmand 0.430at24h,beforeandafterchallenge, respectively)(Fig.4AandC).TheCA3vaccine,ontheotherhand, showedmeans=0.202and0.217at24h,beforeandafterchallenge, respectively(Fig.4AandC).Inthisexperiment,thepredominance oftheCA4 saponinvaccinewassustainedeven afterchallenge. IDR reactionsafterinjectionwitheitherFML orNH36 antigens werehigherinmicevaccinatedwithCA4thanwithCA3saponin. While allreactionsto promastigote lysateweresustained after challenge,theIDRtoFMLorNH36antigensshowedtobereduced (Fig.4CandD).

3.3. AssessmentofthespecificTcellimmunitybyflowcytometry analysisandintracellularproductionofcytokines

Followingtheanalysis of thecellularimmune response, the increaseofthepercentsofspleenLeishmania-specificTcellsafter challengewasevaluatedbyfluorescentcytometryanalysis(Fig.5). WeobservedthatonlytheCA4vaccineincreasedboththeCD4+ andtheCD8+Leishmania-specificTcellproportionsoverthesaline controlswhiletheCA3vaccineincreasedonlytheCD8+specificT cellproportions(Fig.5).TherewasnodifferencebetweentheCA3 andCA4vaccinestothegoldstandardR.Finally,thesplenocytes werealsolabeledthroughtheICSmethodandtheresultsareshown asdoublepositivecells(Fig.6).WeobservedthattheCA4vaccine inducedenhancementsoftheTNF-␣-producingCD4+Tcellsandof theIFN-␥-producingCD8+-TcellswhiletheCA3vaccineinduced theincreaseoftheIFN-␥-producingCD4+-Tcellproportions.No significantvariationsamongtreatmentswereobservedinthe pro-portionsregardingtheTNF-␣ortheIL-10productionbytheCD8+ Tcells.

3.4. ProtectiveefficacytoinfectionbyL.chagasi

(6)

3174 30 (2012) 3169–3179

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

sal

CA3

CA4

R

after immunization

24h 48h

skin test (mm) skin test (mm)

lysate FML NH36 lysate FML NH36

*

A

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

B

*

lysate FML NH36 lysate FML NH36

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

*

C

D

Fig.4.Comparisonofantigensfortheintradermalresponse,at24h(A)and48h(B)aftercompleteimmunizationand24h(C)and48h(D)afterchallengewith3×107

amastigotesofL.chagasi.Resultsofoneexperimentusingn=4–5micepertreatmentfortheLeishmaniapromastigotelysate,n=2–3fortheFMLantigenandn=2forthe

NH36recombinantprotein.ResultsareshownasindividualIDRmeasures.Horizontalfullblacklinesrepresentthemeanvalues.*p<0.05indicatessignificantdifferences

amongtreatments,asdisclosedbytheKruskall–Wallis(KW)andMann–Whitney(MW)non-parametricaltests.

(p<0.0125)henceconfirmingthesuperiorityoftheCA4saponinin protectionagainstvisceralleishmaniasis(Fig.7).Thegaininbody weightalongtheexperimentinducedbyRsaponinwassuperiorto thatofthesalinecontrols(p=0.0407)butnotsignificantly differ-entfromtheincreasesintheCA3andCA4saponinvaccinatedmice (notshown).

TheincreasesinIDRaftervaccinationandinfectionwerestrong correlatesof protectionand weresignificantlycorrelatedtothe decreaseofparasite load(p=−0.007) andtothegain in corpo-ralweight(p=0.0001).TheincreasesinCD4–TNF-␣(p<−0.001), CD8–IFN-␥(p<−0.002)andCD8–TNF-␣(p<−0.015)expressingT cellproportionwerealsocorrelated tothedecrease ofparasite load.

0 25 50 75

percent of splenocytes

T cells

sal

CA3 CA4 R

CD4+ T cells CD8+ T cells *

*

Fig.5.AnalysisoftheNH36-specificcellularimmuneresponseafterchallenge.

Resultsareshownastheindividualpercentofsplenocytes,stainedwithanti-CD4

(cloneGK1.5)oranti-CD8-FITCmonoclonalantibodies,after5daysinvitroculture

with5␮gofrecombinantNH36protein.Horizontalbarsrepresentthemeansofone

experiment(n=6–7micepertreatment).*p<0.05indicatesdifferencesfromthe

salinetreatedcontrols,asdisclosedbytheKruskallWallis(KW)andMannWhitney

(MW)non-parametricaltests.

3.5. InvivodepletionassayofCD4+andCD8+Tcells

TherelativecontributionoftheNH36-specific-CD4+andCD8+T cellproducingcellswasevaluatedinaninvivodepletionassaywith monoclonalantibodies(Fig.8).Incorrelationtowhatwasdetected forthespecificincreaseoftheCD4+Tcells(Fig.5),theTNF-␣–CD4+ producingTcells(Fig.6),onlythetreatmentwithanti-CD4+ mon-oclonalantibodyinduceda66%increaseinthetotalLDUcountsof micevaccinatedwithCA4saponin,indicatingamaincontribution ofCD4+Tcells(Fig.8;p>0.05)tothevaccineinducedprotection. Ontheotherhand,theprotectiveeffectoftheCA3-vaccineis medi-atedby bothCD4+ and theCD8+ Tcellcontributions sincethe anti-CD4+antibodytreatmentinduceda 43%andtheanti-CD8+ antibodyinduceda16%increaseofthetotalLDUcountsofCA3 vaccinatedmice,respectively(Fig.8).Thisisinagreementwiththe increaseofthepercentofCD8+NH36-specificTcellsbytheCA3 vaccine(Fig.5)andoftheIFN-␥-CD4+producingTcells(Fig.6). TheincreasesinIDR,CD4–TNF-␣,CD8–IFN-␥andCD8–TNF-␣by theCA4vaccinewerestrongcorrelatesofprotectionandwere sig-nificantlycorrelatedtothedecreaseofparasiteload(p=−0.007).

3.6. TNF-˛contributiontoprotection

(7)

IFN- -CD4+

IFN- -CD8+

0.0

0.5 1.0 1.5 2.0

*

TNF- -CD4+

0.0 0.5 1.0 1.5 2.0

_*

IL-10-CD4+

0.0 0.5 1.0 1.5 2.0

cytokine producing cells (%) cytokine producing cells (%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

sal

CA3

CA4

R

*

TNF- -CD8+

0.0 0.5 1.0 1.5 2.0 2.5 3.0

IL-10-CD8+

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Fig.6. DevelopmentofNH36-specificcellularimmuneresponseasdisclosedbyintracellularstaininganalysisofsplenocytesinvitroculturewithNH36afterL.chagasi

infection.Anti-CD4-FITCandanti-CD8-FITCantibodieswereusedforlabelingthecellsurfacesandanti-IFN-␥-APC,anti-TNF-␣-PEandanti-IL-10-PEforintracellularstaining.

Dotsrepresenttheindividualresultsof:IFN-␥–CD4+,TNF-␣–CD4+,IL-10–CD4+,IFN-␥–CD8+,TNF-␣–CD8+andIL-10–CD8+producingTcells.Horizontalfulllinebars

representmeansoftwoindependentexperiments(n=6–7micepertreatmentforeachexperiment).Asinglemeasurewasobtainedforeachantibodyandeachmouse.

*p<0.05indicatesdifferencesfromthesalinetreatedcontrolsasdisclosedbytheKruskall–Wallis(KW)andMann–Whitney(MW)non-parametricaltests.

protectioninvisceralleishmaniasis,onlytheCA4-saponinvaccine (mean=596LDUunits)inducedasignificantreductionof50%ofthe parasiteload(Fig.9).TheTNF-␣-receptordeficientmicelostthe abilitytoclearamastigotesfromtheliverandshowedamean con-trolvalue(2185LDU)56%greaterthanthecontrolwildtypegroup (1200LDU).ProtectionduetotheCA4saponinwasnotobservedin theTNF-␣-receptordeficientmice.

sal CA3 CA4 R 0

250 500 750 1000

sal

CA3

CA4

R

Liver LDU values

*

Fig.7.Protectiveefficacyofsaponin-vaccinatedmiceagainstL.chagasiinfection.

TheindividualL.chagasiliverparasiteloadofvaccinatedandcontrolgroupsis

expressedinLDUvalues(numberofamastigotesper600livercellnuclei/mgofliver

weight).ResultsrepresenttheindividualliverLDUvaluesofmicefromtwo

inde-pendentexperiments(n=6–7micepertreatmentforeachexperiment).Horizontal

fulllinesrepresentthemeanvaluesanddashedhorizontallinesrepresenttheSE

values.*p<0.05indicatessignificantdifferencesasdisclosedbytheKruskall–Wallis

(KW)andMann–Whitney(MW)non-parametricaltests.

3.7. Correlationbetweentheincreaseofthesugarchainand cytokinesecretion

(8)

3176 30 (2012) 3169–3179

0 500 1000 1500 2000

sal CD4

anti-CD8 rat

serum

anti-CD4+CD8

CA3-FML vaccine

rat serum

anti-CD4

anti-CD8

anti-CD4+CD8

CA4 FML-vaccine

*

Liver LDU units

Fig.8.Developmentofcell-mediatedimmuneresponseasdisclosedbyinvivodepletionwithanti-CD4+andanti-CD8+monoclonalantibodies.Leishmaniachagasi

parasite-loadinmicevaccinatedwithCA3-FMlandCA4-FMLvaccinesandtreatedwithratserum,anti-CD4+oranti-CD8+orthecombinationofanti-CD4+andanti-CD8+MAbs.

Micethatreceivethevaccineswerealsotreatedwithratserum(ratIgG)ascontrolsforantibodytreatment.ResultsrepresenttheindividualliverLDUvaluesofmicefrom

oneexperiment(n=5micepertreatment).Horizontalfulllinesrepresentthemeanvalues.*p<0.05indicatessignificantdifferencesbetweentreatmentsasdisclosedbythe

Kruskall–Wallis(KW)andMann–Whitney(MW)non-parametricaltests.

3.8. Correlationbetweentheincreaseofthesugarchain,IDRand thedecreaseofparasiteload

Furthermore,theimpactoftheincreaseoftheC-28attached sugarchain ofC.alba wascompared intheBalb/c micemodel, usingtheCA2andtheCA3Xsaponins(Fig.1)ascontrols.TheIDR responsewasenhancedonlybytheCA4andtheRsaponinabove thesalinecontrols(Fig.11).Incorrelationtothat,onlytheCA4and theRsaponinreducedtheparasiteloadwhencomparedtosaline control(Fig.11),confirmingthesuperiorityofCA4.Thereduction determinedbyCA4wasstrongerthanthatofCA2andCA3X,and, asdescribed inFig.7,notdifferentfromtheprotectioninduced byCA3.MaximalparasiteloadreductionwasachievedbytheR saponincontrolgroup(Fig.11).Therewasapositivecorrelation betweentheincreaseinIDRmeasuresandinthenumberofsugar unitsattachedtothetriterpene-C-28(p<0.0001).Supportingour hypothesisofthesuperiorityoftheCA4saponin,ontheotherhand, theLDUvalues decreased withtheincrease of thesugar chain (p=−0.014).

0 1000 2000 3000 4000 5000

liverLDU values

* 0.0 0.1 0.2 0.3 0.4 0.5

skin test (mm)

*

wild type TNF-α-receptor-KO wild type TNF-α_-receptor-KO

sal CA3 CA4 R

Fig.9. TNF-␣isrequiredforefficientparasiteclearanceintheliver.C57BL/6mice

wildtypeandTNF-␣-receptorknock-out(TNF-␣-KO)werevaccinatedwithCA3

orCA4saponinandFMLortreatedwithsalineonlyandfurtherchallengedwith

L.chagasiamastigotes.Theintradermalreactiontopromastigotelysate,at24h

afterantigeninjection,isexpressedinmillimeters.TheindividualL.chagasiliver

parasiteloadvaluesare expressedinLDU values(numberofamastigotesper

600livercellnuclei/mgofliverweight)(n=5micepertreatment).Horizontalfull

linesrepresentthemeanvalues.*p<0.05indicatessignificantdifferencesas

dis-closedbytheKruskall–Wallis(KW)andMann–Whitney(MW)non-parametrical

tests.

3.9. Assessmentofthehydrophile–lipophilebalance(HLB)value, hemolyticindexandinvivotoxicityofsaponins

Thehydrophile/lipophilebalancecalculationperformed accord-ingtotheDaviesandRiedelmethoddisclosedanHLB=12.7forCA2, HLB=15.8forbothCA3andCA3XandanHLB=19.9forCA4saponin confirmingitshigherhydrophilicity.

The analysis of the hemolytic capacity of C. alba saponins (Table1)disclosedthatsaponinsCA2,CA3andCA4shareahigh HD50(175␮g/ml)whichmeansthattheyarepoorlyhemolyticand

thatthehemolyticcapacity,differentlyfromwhathappenswith theHLB,doesnotincreaseinpositivecorrelationwiththenumber ofsugarunitslinkedtothesapogenin.ThesaponinCA3X,with3 sugarunitsattachedtoC-3oftriterpene,butnotapiose,induced, ontheotherhand,astronghemolysis(HD50=87␮g/ml)(Table1).

The control saponin R, was as expected the most hemolytic (HD50=35␮g/ml).Furthermore,thesafetyanalysisdetected

nei-therlethalitynorlocalpainorswelling(Table1)foranyoftheC. albavaccines.Onlylossofhairatthelocalofinjectionwasdetected inthe5micetreatedwiththeQS21containingsaponinR.

4. Discussion

The increase in hemolyticactivities of C. alba saponins was not correlated to theincrease in the sizeof the C-28 attached carbohydratechain.Incontrast,theCA3andCA3Xsaponinsthat bothhavethreesugarunitsinthatchainstronglydifferedintheir hemolyticcapabilities.Saponin CA3Xwhichhasaxylose termi-nalunitinducedstronghemolysiswhilesaponinCA3thatshows anapiose unit insteadwasmuch less hemolytic. In correlation withourfindings,theQS21adjuvantiscomposedoftwoisomers thatincludeeitherapiose(QS21-Api)orxylose(QS21-Xyl)asthe terminalsugarresiduewithinthelineartetrasaccharidesegment, inaratioof65:35,respectively[34].ThesaponinQS21-Xylwas marginallymoretoxicthanQS21-ApiortheQS21mixture. Over-allmiceweight losswasgreatest intheSQS21-Xyl groups and althoughonemouseofbothgroupsdiedoverthecourseof immu-nizations,themiceintheQS21-Xylgroupshowedtheworstclinical status.Ontheotherhand,theQS21-Xyltreatedmiceinduceda higherIgMandIgGresponse[34].

(9)

IL-10

0 1000 2000 3000 4000 5000

saline FML+sapCA2 FML+sapCA3 FML+sapCA3x FML+sap4 FML+sapR

pg/ml

IFN

saline FML+sa

0 1000 2000

pg/ml

TNF-0

1000

2000

3000

pg/ml

*

Fig.10. DevelopmentofNH36-specificcellularimmuneresponse.ELISAofcytokinesinsupernatantsofmicesplenocytes.IFN-␥,TNF-␣andIL-10wereassayedinthe

supernatantsofsplenocytesofcontrolandvaccinatedmiceafter5daysofinvitroculturewiththeNH36antigen.Datacorrespondtoindividualresultsofoneexperiment

with4–5miceofeachvaccinegroupandsalinecontrolobtainedaftersacrifice.Horizontalfulllinesrepresentthemeanvalues.*p<0.05indicatessignificantdifferencesto

thesalinecontrolasdisclosedbytheKruskall–Wallis(KW)andMann–Whitney(MW)non-parametricaltests.

Fig.11.Parasiteloadreductionincreaseswithsugarchainofsaponins.Balb/cmice

werevaccinatedwiththeCA2,CA3,CA3X,CA4orRsaponinandFMLortreated

withsalineonlyandfurtherchallengedwithL.chagasiamastigotes.The

intrader-malreactiontopromastigotelysate,at24hafterantigeninjection,isexpressed

inmillimeters.TheindividualL.chagasiliverparasiteloadvaluesareexpressed

inLDUvalues(numberofamastigotesper600livercellnuclei/mgofliverweight)

(n=5micepertreatment).Horizontalfulllinesrepresentthemeanvalues.*p<0.05

indicatessignificantdifferencesasdisclosed bytheKruskall–Wallis (KW)and

Mann–Whitney(MW)non-parametricaltests.

Table1

Toxicityinvivoandhemolyticeffectofsaponins.

Saponin Localpain Swelling Lossofhair Lethality HD50

R(QS21) 0/5 0/5 5/5 0/5 35␮g/ml

CA4 0/5 0/5 0/5 0/5 175␮g/ml

CA3 0/5 0/5 0/5 0/5 175␮g/ml

CA3X 0/5 0/5 0/5 0/5 87␮g/ml

CA2 0/5 0/5 0/5 0/5 175␮g/ml

Note:Resultsareexpressedasnumberofmicepergroupthatshowedany reac-tivitywithin7daysaftereachoneofthethreesubcutaneousinjectionsofeach vaccinecontaining100␮gofsaponininthebackofeachanimal.HD50istheadjuvant

concentrationinducing50%ofthemaximumhemolysis.

terminalsugarunitisoptimizedwhencomparedtothe configu-rationofthesamegroupsinxylose.Thiswouldexplainalsothe reasonfortheincreasedadjuvantpotentialofCA4whichhasan additionalapioseunit.

TheCA4saponinofC.albain formulationwithFMLinduced ahigherresponseafterchallenge,significantincreasesinIgGand IgG2aanti-FMLantibodieswhichwereabsentintheCA3-saponin. These resultsconfirmthe relevanceof theaddition ofa fourth unitofapiose1→3linkedtotherhamnoseresidueoftheC-28 attachedsugar chain in theinductionof theanti-FML humoral response.Asexpectedforapositiveadjuvantcontrol,theglobal humoralresponseinducedbythesaponinQS21containingsaponin Rvaccinewasthehighest.Theintensityofthehumoralresponse generatedbysaponinshasbeenshowntoberelatedtothe pres-enceofcarbohydratemoietiesattachedtothetriterpenenucleus [14,17,25]andthisresponseincreasesindirectproportiontotheir length[22].Thisexplainswhy,eveninasaponinwithonly4sugar unitschain(CA4),thedifferenceofjustonesugarresidue[28]may accountforasignificantincreaseinIgGandIgG2aspecific antibod-ieswhicharethesubclassesofantibodiespreviouslyshowntobe correlatedtoprotection.

Inourinvestigation,allsaponinsincreasedtheIgG1 antibod-ies.Thishumoralresponseisinducedbywholesaponins[23]but seemstobecorrelated tothecarbohydrate deprivedsapogenin nuclei[14,17].AglobalincreaseofIgMandIgG3antibodiesbyall adjuvantswasdescribedwhichisexpectedtooccurinresponseto carbohydrateenrichedantigens[35]andsaponins[14,17].

(10)

3178 30 (2012) 3169–3179

thecapabilityofsaponinstoinduceantibodyresponsesincreases withtheirhydrophilicity.Amongbidesmosidic(twosugarchains) soyasaponins,soyasaponinA1 withthree sugarsattachedto C-3induced strongertotal-IgG andIgG1antibody responsesthan soyasaponin A2 withonly two sugar attached to C-3 [25]. An identicalconclusionwasobtainedbyBernardoetal.[19]working withthePSAGLEsaponinofAlbiziasaman.Formonodesmosidic (one sugar chain) soyasaponins, the ranking in terms of anti-bodyresponsewassoyasaponinI(-glcA-gal-rha)>soyasaponinII (-glcA-ara-rha)>soyasaponinIII(-glcA-gal)[25].Thismeansthat atrisaccharide(soyasaponinIandII)chainismorepotentthana disaccharideone(soyasaponinI),andthataresidueofgalactose inthetrisaccharidechainofsoyasaponinIthat exposesoneOH groupturnsthesaponinmorepotentthanaresidueofarabinose whichlacksthisOHgroup(soyasaponinII)[25].Therefore,among saponinsofthesamesugarchainlength,themorehydrophilicthe sugarcomponentsare,themorepotentthehumoralresponseis. TheC-28attachedchainoftheC.albaCA3saponiniscomposed of arabinose–rhamnose–apiose. The addition of one additional apiose sugar unit in the CA4 saponin is then expected to add hydrophilicitytothesaponin[25]increasingitsadjuvant poten-tial.OurresultswithsaponinsofC.albatherefore,stronglysupport thepreviousconclusionsofOdaetal.[25]statingthatthe adju-vantactivitytendedtoincreasewiththesugarsidechainlength andtheHLBvalue.Indeed,thisinvestigationreportedHLBvalues of15.8and19.9forCA3andCA4saponins,respectively.TheHLB ofQS21saponin,whichisa highlypotentstandardadjuvant, is 36.3[25].ThesaponinsofC.albadisplaylowerHLBvaluesthan QS21andbidesmosidicsoyasaponinsbuthigherHLBvaluesthan monodesmosidicsoyasaponins[25].

Theextra-apioseofCA4saponindeterminestheincreaseofthe C-28sugarchainlengthand,inthisway,ofthesaponin hydrophilic-ity.In order to confirmthat HLB isa crucial factorinfluencing theadjuvanticityoftheCA4saponinweusedascontrols,theCA2 andCA3XsaponinsofC.alba,thathaveshortersugarchainssince theyaresynthesizedinearlierstepsofthebiosyntheticpathway. Indeed,thetriterpenenucleusissynthesizedfirstandthesugar unitsaddedinsequencetoitsC-28byspecificglycosyltransferases [36].OurresultsconfirmedthecorrelationbetweenincreasedHLB andincreasedadjuvantcapabilities.

Asexpectedforprotectiongeneratedagainstvisceral leishmani-asis[31,37–44]protectioninducedbyCA4saponindeterminedthe decreaseofliverLDUandtheincreasesofIDRandofTNF-␣–CD4+ producingcellsand TNF-␣secretion.Theprotectioninducedby theCA4-vaccinewasmediateda CD4+ Tcelland TNF-␣-driven response.Thiscouldindicatetheexistenceofanearlyeffector cell-responsegenerated bythevaccine[45].TNF-␣isconsideredto bethemostubiquitouscytokineanditisproducedbymost acti-vatedCD4+Tcells[reviewedin45]generatedunderconditions thatfavorTH1-celldifferentiation.Ithasprovedtobeimportant inprotectionagainstvisceralleishmaniasis[37–44,46,47].A sus-tainedoranoverallincreasedglobalorLeishmania-specificCD4+ Tcellexpansionisexpectedinprotectionagainstvisceral leish-maniasis[31,48].Inourinvestigation,theCA4saponin,withthe longestsugarchainwastheonlyonecapableofenhancingtheCD4+ Leishmania-specificTcellpopulation.

Also,supportingourresults,astudyoftherelationshipbetween hemolyticandadjuvantactivityandstructureofprotopanaxadiol andprotopanaxatriol-typesaponinsfromtherootsofP.notoginseng showedthatthenumber,lengthandpositionofsugarsidechains, andthetypeandthelinkageoftheglycosylgroupinthestructure ofthesesaponinsdidnotonlyaffecttheadjuvantpotentials,but alsohadsignificanteffectsonthenatureoftheimmuneresponses [20,21].

Weconcludethattheadditionofone sugarunitintheC-28 attached glycosidic moiety provides a significant increase of

adjuvanticity and protective capability of the C alba saponins. Ourresultsencouragethenewsynthesisorremodelingofnatural saponinsbyadditionalglycosylation,aimingtherationale devel-opmentofeffectiveadjuvants.Thishasproventobefeasiblesince themostfrequentisomerofQS21saponinwhichhasalsoapioseas theterminalsugarunitofitsC-28chain,wasobtainedbychemical synthesiswhilemaintainingtheadjuvantcapabilityofthenatural product[34].

Acknowledgments

We thank James Huntington for providing the use of the Analyze-itprogramandDavidStrakerfortheEnglishlanguage edit-ingofthismanuscript.WearealsoverygratefultoprofessorsPedro PauloXavierElsasandMariaIgnezCapellaGasparforprovidingthe transgenicmiceusedinthisinvestigation.

Conflictofintereststatement:Theauthorsdeclarethatthereis noconflictofinterestregardingthepresentworkandthesponsors had norole in studydesign, data collectionand analysis, deci-siontopublish,orpreparationofthemanuscript.Fundingsupport: Thisworkwassupportedby theBrazilian theNationalCouncil ofScientificandTechnologicalDevelopment(CNPQ,Fellowships andUniversalGrant500992/2008-8)andbytheResearch Foun-dation of the State of Rio de Janeiro (FAPERJ, Fellowships and GrantsE-26/110305/2007,E-26/110132/2007,E-26/100416/2007, E-22/102733/2008)andaPIBICCNPQ-UFRJfellowshipfor under-graduatestudents.

References

[1]daSilvaVO,Borja-CabreraGP,CorreiaPontesNN,ParaguaideSouzaE,LuzKG,

PalatnikM,etal.AphaseIIItrialofefficacyoftheFML-vaccineagainstcanine

kala-azarinanendemicareaofBrazil(SãoGonc¸alodoAmarante,RN).Vaccine

2001;19:1068–81.

[2]Borja-CabreraGP,SantosFN,BauerFS,ParraLE,MenzI,MorgadoAA,etal.

ImmunogenicityassayoftheLeishmune®_vaccine_against_canine_visceral

leish-maniasisinBrazil.Vaccine2008;26:4991–7.

[3] Palatnik-de-SousaCB,BarbosaAF,OliveiraSM,NicoD,BernardoRR,SantosWR,

etal.TheFML-vaccineagainstcaninevisceralleishmaniasis:fromthesecond

generationtothesyntheticvaccine.ExpRevVaccines2008;7:833–51.

[4] TeshR.Controlofzoonoticvisceralleishmaniasis.Isittimetochange

strate-gies?AmJTropMedHyg1995;52:287–92.

[5]Dye C. The logic ofvisceral leishmaniasis control. Am J Trop Med Hyg

1996;55:125–30.

[6] Palatnik-de-SousaCB,Silva-AntunesI,MorgadoAA,MenzI,PalatnikM,Lavor

C. Decreaseofthe incidenceofhumanand caninevisceralleishmaniasis

afterdogvaccinationwithLeishmune®_in_Brazilian_endemic_areas._Vaccine

2009;27:3505–12.

[7] Borja-CabreraGP,CruzMendesA,ParaguaideSouzaW,OkadaLYH,

Trivel-latoFAA,KawasakiJKA,etal.EffectiveImmunotherapyagainstcaninevisceral

leishmaniasiswiththeFML-vaccine.Vaccine2004;22:2234–43.

[8]SantosFN,Borja-CabreraGP,MiyashiroLM,GrechiJ,ReisAB,MoreiraMAB,

etal.Immunotherapyagainstexperimentalcaninevisceralleishmaniasiswith

thesaponinenriched-Leishmune®_vaccine._Vaccine_{2007;25:6176–90.}

[9]Palatnik CB, BorojevicR, PreviatoJO,Mendonc¸a-Previato L.Inhibition of

Leishmaniadonovaniinternalizationintomurinemacrophagesbychemically

definedparasiteglycoconjugateligands.InfectImmun1989;57:754–63.

[10]Oliveira-Freitas E,Casas CP, Borja-Cabrera GP,Santos FN, NicoD, Souza

LOP,etal.AcylatedanddeacylatedsaponinsofQuillajasaponariamixture

as adjuvants forthe FML-vaccine against visceralleishmaniasis. Vaccine

2006;24:3909–20.

[11]PodolakI,GalantyA,SobolewskaD.Saponinsascytotoxicagents:areview.

PhytochemRev2010;9:425–74.

[12]TakechiM,TanakaY.Haemolytictimecoursedifferencesbetweensteroidand

triterpenoidsaponins.PlantaMed1995;61:76–7.

[13] Osbourn A. Saponins and plant defense—a soap story. Trends Plant Sci

1996;1:4–9.

[14]NicoD,SantosFN,Borja-CabreraGP,PalatnikM,PalatnikdeSousaCB.

Assess-mentofthemonoterpene,glycidicandtriterpene-moieties’contributionsto

theadjuvantfunctionoftheCP05-saponinofCalliandrapulcherrimaBenth.

Vaccine2007;25:649–58.

[15]SoltysikS,WuJY,RecchiaJ,WheelerDA,NewmanMJ,CoughlinRT,etal.

Struc-ture/functionstudiesofQS-21adjuvant:assessmentoftriterpenealdehyde

andglucuronicacidrolesinadjuvantfunction.Vaccine1995;13:1403–10.

[16]SantosWR,BernardoRR,Pec¸anhaLT,PalatnikM,ParenteJP,PalatnikdeSousa

(11)

mediterraneasaponinonthehumoralresponsetotheFMLantigenofLeishmania

donovani.Vaccine1997;15:1024–9.

[17]Palatnik deSousa CB,SantosWR,Casas CP,ParaguaideSouza E,Tinoco

LW,daSilvaBP,etal.Protectivevaccinationagainstmurinevisceral

leish-maniasis usingaldehyde-containingQuillajasaponariasapogenins.Vaccine

2004;22:2470–9.

[18]RönnbergB,FekaduM,BehboudiS,KenneL,MoreinB.Effectsofcarbohydrate

modificationofQuillajasaponariaMolinaQH-Bfractiononadjuvantactivity,

cholesterol-bindingcapacityandtoxicity.Vaccine1997;15:1820–6.

[19] BernardoRR,SantosWR,ParenteJP,PalatnikdeSousaCB.Vaccinationagainst

experimentalKala-azarwithsaponinsofAlbiziasaman(PsAsuandPsAgle)and

theFMLantigenofLeishmaniadonovani.MEMIOC1997;92(Suppl.1):285.

[20]SunHX,QinF,YeYP.Relationshipbetweenhemolyticandadjuvant

activ-ityandstructureofprotopanaxadiol-typesaponinsfromtherootsofPanax

notoginseng.Vaccine2005;23:5533–42.

[21] SunHX,YangZG,YeYP.Structureandbiologicalactivityof

protopanaxatri-oltypesaponinsfromtherootsofPanaxnotoginseng.IntImmunopharmacol

2006;6:14–25.

[22]SunHX,XieY,YeYP.Advancesinsaponin-basedadjuvants.Vaccine2009;27:

1787–96.

[23] MarcianiDJ.Vaccineadjuvants:roleandmechanismsofactioninvaccine

immunogenicity.DrugDiscovToday2003;8:934–43.

[24]SilvaBP,SoaresJBRC,ParaguaideSouzaE,PalatnikM,PalatnikdeSousaCB.

Pulcherrimasaponin,fromtheleavesofCalliandrapulcherrima,asadjuvantfor

visceralleishmaniasis.Vaccine2005;23:1061–71.

[25]OdaK,MatsudaH,MurakamibT,KatayamaS,OhgitaniT,YoshikawaM.

Rela-tionshipbetweenadjuvantactivityandamphipathicstructureofsoyasaponins.

Vaccine2003;21:2145–51.

[26]BomfordR, StapletonM,WinsorS,BeesleyJE,JessupEA,PriceKR,etal.

AdjuvanticityandISCOMformationbystructurallydiversesaponins.Vaccine

1992;10:572–7.

[27] KensilCR,SoltysikS,PatelU,MarcianiD.Structure/functionrelationshipon

adjuvantsfromQuillajasaponariaMolina.In:BrownF,ChanockRM,

Gins-bergHS,LernerRA,editors.Vaccines,vol.92.NewYork:ColdSpringHarbor

LaboratoryPress;1992.p.35–40.

[28] BorgesRM,TinocoLW,SouzaFilhoJD,BarbiNS,SilvaAJR.Twonewoleanane

saponinsfromChiococcaalba(L.)Hitch.JBrazChemSoc2009;20:1738–41.

[29]JacobsenNE,FairbrotherWJ,KensilCR,LimA,WheelerDA,PowellMF.

Struc-ture ofthesaponin adjuvant QS-21andits base-catalyzedisomerization

productby1_H_and_natural_abundance13_C_NMR_{spectroscopy.}_Carbohydr_Res

1996;280:1–14.

[30]Davies JT,RidealEK.Interfacialphenomena.NewYork/London:Academic

Press;1961.

[31] Nico D, Claser C, Travassos LR, Palatnik M, Soares IS, Rodrigues MM,

etal.VaccinationwiththeLeishmune®_’s_nucleoside_hydrolase_maps_the

C-terminaldomainasthetargetoftheprotectiveimmuneresponse.PLoSNTD

2010;4:e866,1–13.

[32] PalatnikdeSousaCB,BunnMorenoMM,ParaguaideSouzaE,BorojevicR.

Vac-cinationwiththeFMLantigen(FucoseMannoseLigand)ofLeishmaniadonovani

protectshamstersfromexperimentalvisceralleishmaniasis.JBrazSocAdvSci

CiênciaeCultura1994;46:290–6.

[33]TheWilcoxonSigned-RankTest.Conceptsandapplicationsofinferential

statis-tics.Chapter12a,p.1.http://faculty.vassar.edu/lowry/webtext.html.

[34]RagupathiG,DamaniP,DengK,AdamsMM,HangJ,GeorgeC,etal.

Preclin-icalevaluationofthesyntheticadjuvantSQS-21anditsconstituentisomeric

saponins.Vaccine2010;28:4260–7.

[35]SnapperCM,RosasF,MoormanMA,JinL,ShanebeckK,KlinmanDM,etal.

IFN-gammaisapotentinducerofIgsecretionbysort-purifiedmurineBcells

activatedthroughthemIg,butnottheCD40,signalingpathway.IntImmunol

1996;8:877–85.

[36] HostettmannK,MarstonA.Saponins.In:Chemistryandpharmacologyof

nat-uralproducts:saponins.CambridgeUniversityPress;1995.

[37] MurrayHW,HariprashadJ.Interleukin12iseffectivetreatmentforan

estab-lishedsystemicintracellularinfection:experimentalvisceralleishmaniasis.J

ExpMed1995;181:387–91.

[38]TaylorAP,MurrayHW.Intracellularantimicrobialactivityintheabsenceof

interferon-gamma:effectofinterleukin-12inexperimental visceral

leish-maniasisininterferon-gammagene-disruptedmice.JExpMed1997;185:

1231–9.

[39]Selvapandiyan A, Dey R, Nylen S, Duncan R, Sacks D, Nakhasi HL.

Intracellularreplication-deficientLeishmaniadonovaniinduces long-lasting

protectiveimmunityagainstvisceralleishmaniasis. J Immunol2009;183:

1813–20.

[40]MannaPP,ChakrabartiG,BandyopadhyayaS.Innateimmunedefensein

vis-ceralleishmaniasis:cytokinemediatesprotectiverolebyallogeneiceffector

cells.Vaccine2010;28:803–10.

[41]LiewFY,LiY,MillotS.Tumornecrosisfactor-␣synergizeswithIFN-␥in

mediat-ingkillingofLeishmaniamajorthroughtheinductionofnitricoxide.JImmunol

1990;145:4306–10.

[42]AlvesCF,deAmorimIF,MouraEP,RibeiroRR,AlvesCF,MichalickMS,etal.

ExpressionofIFN␥,TNF␣,IL-10andTGF␤inlymphnodesassociateswith

par-asiteloadandclinicalformofdiseaseindogsnaturallyinfectedwithLeishmania

(Leishmania)chagasi.VetImmunolImmunopathol2009;128:349–58.

[43]Sanchez-RobertE,AltetL,AlberolaJ,Rodriguez-CortésA,OjedaA,

López-FuertesL,etal.Longitudinalanalysisofcytokinegeneexpressionandparasite

loadin PBMC in Leishmania infantum experimentally infected dogs. Vet

ImmunolImmunopathol2008;125:168–75.

[44]DeLeonardisF,GovoniM,LoMonacoA,TrottaF.Visceralleishmaniasisand

antiTNF␣therapy:casereportandreviewofliterature.ClinExpRheumatol

2009;27:502–6.

[45] SederRA,DarrahPA,RoedererM.Tcellqualityinmemoryandprotection

implicationsforvaccinedesign.NatRev2008;8:247–58.

[46]MurrayHY,JungbluthA,RitterE,MontelibanoC,MarinoMW.Visceral

leish-maniasisinmicedevoidoftumornecrosisfactorandresponsetotreatment.

InfectImmun2000;68:6289–93.

[47] StanleyAC,RiveraFL,HaqueA,SheelM,ZhouY,AmanteFH,etal.Criticalroles

forLIGHTanditsreceptorsingeneratingTcell-mediatedimmunityduring

Leishmaniadonovaniinfection.PLoSPathog2011;7:e1002279.

[48] GuargaJL,MorenoJ,LucientesJ,GraciaMJ,Peribá ˜nezMA,AlvarJ,etal.Canine

leishmaniasistransmission:higherinfectivityamongstnaturallyinfecteddogs

tosandfliesisassociatedwithlowerproportionsofThelpercells.ResVetSci