Antibody responses induced by Leish-Tec®, an A2-basedvaccine for visceral leishmaniasis, in a heterogeneous caninepopulation.

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j ourn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Antibody

responses

induced

by

Leish-Tec

®

,

an

A2-based

vaccine

for

visceral

leishmaniasis,

in

a

heterogeneous

canine

population

Miriam

C.

de

Souza

Testasicca

_,

_Mariana

_Silva

_dos

_Santos

_,

Leopoldo

Marques

Machado

_,

_Angela

_Vieira

_Serufo

_,

_Daniel

_Doro

_,

Daniel

Avelar

_,

_Ana

_Maria

_Leonardi

_Tibúrcio

_,

_Christiane

_de

_Freitas

_Abrantes

_,

George

Luiz

Lins

Machado-Coelho

_,

_Gabriel

_Grimaldi

_Jr.

_,

Ricardo

Tostes

Gazzinelli

_,

_Ana

_Paula

_Fernandes

c_{HertapeCalierSaudeAnimal,Juatuba,MG,Brazil} d_{LabtestDiagnostica,BeloHorizonte,MG,Brazil}

e_{DepartamentodeCiênciasMédicas,UniversidadeFederaldeOuroPreto,OuroPreto,MG,Brazil} f_{CentrodePesquisaGonc¸aloMoniz/Fiocruz,Salvador,BA,Brazil}

g_{DepartamentodeBioquímicaeImunologia,InstitutodeCiênciasBiológicas,UniversidadeFederaldeMinasGerais,BeloHorizonte,MG,} Brazil

h_{CentrodePesquisasRenéRachou/Fiocruz,BeloHorizonte,MG,Brazil} i_{UniversityofMassachusettsMedicalSchool,Worcester,MA,USA}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received6March2014

Receivedinrevisedform15April2014 Accepted22April2014 Keywords: Visceralleishmaniasis Vaccine Leish-Tec® Dogs A2antigen Antibodies

a

b

s

t

r

a

c

t

Zoonoticvisceralleishmaniasis(VL)isawidespreaddisease,anddogsarethemain reser-voirsforhumanparasitetransmission.Hence,developmentofaneffectivevaccinethat preventsdiseaseandreducesthetransmissionofVLisrequired.Aseuthanasiaof seropos-itivedogsisrecommendedinBrazilforVLepidemiologicalcontrol,toincludeanti-VL caninevaccinesasamasscontrolmeasureitisnecessarytocharacterizethehumoral responsesinducedbyvaccinationandiftheyinterferewiththereactivityofvaccinated dogsinserologicaldiagnostictests.Leish-Tec®isanamastigote-specificA2recombinant proteinvaccineagainstcaninevisceralleishmaniasis(CVL)thatiscommerciallyavailable inBrazil.Here,wetestedtheimmunogenicityofLeish-Tec®_{inaheterogeneousdog}

popula-tionbymeasuringA2-specificantibodyresponses.Healthydogs(n=140)ofvariousbreeds wereallocatedtotwogroups:onegroupreceivedLeish-Tec®(n=70),andtheothergroup receivedaplacebo(n=70).Anti-A2oranti-Leishmaniapromastigoteantigen(LPA)antibody levelsweremeasuredbyELISAinserumsamplescollectedbeforeandaftervaccination. Animmunochromatographictest(DPP)basedontherecombinantK28antigenwasalso usedforserodiagnosisofCVL.Vaccinatedanimals,exceptone,remainedseronegativefor anti-LPAtotalIgGandanti-K28antibodies.Conversely,seropositivityforanti-A2totalIgG antibodieswasfoundin98%ofanimalsaftervaccination.Thisvaluedecreasedto81.13%at 6monthsbeforerisingagain(98%),afterthevaccinationboost.Anti-A2IgG2andIgG1titers

∗ Correspondingauthorat:DepartmentofClinicalandToxicologicalAnalyses,FacultyofPharmacy,FederalUniversityofMinasGerais,Av.Antônio Carlos,6627,BeloHorizonte,MGCEP31270901,Brazil.Tel.:+553134096884;fax:+553134096985.

E-mailaddresses:anav@uai.com.br,apfernandes.ufmg@gmail.com(A.P.Fernandes).

http://dx.doi.org/10.1016/j.vetpar.2014.04.025

werealsoincreasedinvaccinatedanimalsrelativetocontrolanimals.Thesedataindicate thatLeish-Tec®_{isimmunogenicfordogsofdifferentgeneticbackgroundsandthathumoral}

responsesinducedbyvaccinationcanbedetectedbyA2-ELISA,butdonotinterferewith theLPA-ELISAandDPPdiagnostictestsforCVL.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Leishmaniasis is a vector-borne disease caused by

protozoan parasites of the genus Leishmania that

pri-marily affects the poorest regions of 98 countries

worldwide. Among the two million new human

infec-tions, 200,000–400,000 are cases of life-threatening

visceralleishmaniasis(VL),culminatinginapproximately

20,000–30,000deathseveryyear(Alvaretal.,2012).

Chil-drenandimmunocompromisedindividualsareespecially

affected, but adults with active disease are frequently

foundinfociofrecentparasiteintroduction(WorldHealth

Organization,2010).Concernsabouttreatmentfailurefor

VL are exacerbated by the emergence of antimicrobial

resistantparasitestrainsandbecauseofco-infectionwith

HIV(Chappuisetal.,2007).

AnthroponoticVLoccursinregionswhereLeishmania

(Leishmania)donovaniinfectionsareendemic(theIndian

sub-continent), whereas zoonotic VL occurs in areas of

transmissionofL.(L.)infantum(southernEurope,North

Africa,andWestandCentralAsia)orL.(L.)infantum

cha-gasi(LatinAmerica) (World Health Organization,2010).

Domesticdogs(Canisfamiliaris)areconsideredthemain

peridomesticand domestic reservoirsof parasites for L.

(L.) infantum and L. (L.) infantum chagasi (Alvar et al.,

2004;GrimaldiandTesh,1993).AscanineVLconstitutes

themajorsourceofparasitesfortransmissionofhuman

VL,prophylacticcontrolprogramsinBrazilhavefocused

oneuthanasiaofseropositivedogs(Lacerda,1994).These

euthanasiacampaignsareexpensiveandlabor-intensive,

and their relatively poor performance is aggravated in

practicebytheabsenceofa good markerforinfectious

statusin dogs(Courtenayet al.,2002).Hence,both the

efficacyandacceptabilityofthiscontrolstrategyis

increas-inglybeingchallenged(Chappuisetal.,2007;Romeroand

Boelaert,2010;Tesh,1995).TreatmentofVL-infecteddogs

isnotafeasiblecontrolstrategy,becausedrug-cureddogs

oftenrelapseandremainasourceofparasitesforthesand

flyvector(Alvaretal.,1994).

AssomeL. (L.) infantumchagasi-infected dogs

spon-taneouslyrecoverand maintainprotective immunity to

re-infections(Grimaldietal.,2012b),vaccinationseemsto

beafeasiblestrategytoprotectdogs(Chappuisetal.,2007),

andalsotoreducetheincidenceofhumancases.Inthis

con-text,vaccinationmaybeadoptedconcomitantlywithother

currentmeasuresofcontrolespeciallyifvaccinesdonot

induceseroconversionindiagnostictests.Inparticular,the

amastigote-specificA2antigen,administeredin

recombi-nantprotein,DNA,orviralvectorsvaccineformulationsor

intransfectedparasites,inducespartialprotectionagainst

L.(L.)donovani,L.(L.)amazonensis,andL.(L.)infantum

cha-gasiinfectionsinmice(Coelhoetal.,2003;Ghosh etal.,

2001a,b;Mizbanietal.,2009;Resendeetal.,2008;Zanin

etal.,2007).A2isalsooneofthefewcandidateantigens

thathavebeentestedinbeagledogsagainstexperimental

challengeandprovidedsignificantprotection(Fernandes

etal.,2008).Basedonthesefindings,avaccine

formula-tionconsistingofsaponinandtherecombinantproteinA2,

namedLeish-Tec®,waslicensedinBrazil.Recently,

Leish-Tec®hasbeenalsotestedindogslivinginendemicareasfor

CVL(Fernandesetal.,2014).Thatstudyhasdemonstrated

that92.9%ofdogsvaccinatedwithLeish-Tec® remained

clinically health. Moreover, as tested by xenodiagnosis,

vaccinated dogswerelessinfectious(5.4%) thancontrol

animals(36.6%)tothephlebotominevector.

Inadditionbeingsafeforthetargetanimal(Schetters

and Gravendyck,2006), theA2 vaccinealsoneedstobe

immunogenic to heterogeneousdog populations.

More-over, given the importance of serodiagnostic tests in

veterinary routine practiceand epidemiological control,

CVLvaccinesshouldideallynotinduceseroconversionin

routineserologicaltests.Wethereforeperformedastudy

toevaluatethespecificity,levels,anddurationofantibody

responseselicitedbyLeish-Tec® vaccinationin out-bred

dogs.

2. Materialsandmethods

2.1. Ethicsstatement

Approval to perform this study was obtained from

the institutional review board, Comitê de Ética em

Experimentac¸ãoAnimal(CETEA)ofUniversidadeFederal

deMinasGerais(UFMG),undercertificate020/2009.UFMG

adheres to the standards outlined by relevant national

(CONCEA;BrazilianGovernmentCouncilforControlof

Ani-malExperimentation)andinternationalguidelinesforcare

anduseofanimalsinresearch.

2.2. Animals

For thisstudy,315dogswereinitiallyevaluated;the

dogswereofdifferentbreeds,livingin12openkennels,and

locatedineightdifferentregionsofthemetropolitanregion

ofthecityofBeloHorizonteinMinasGerais,Brazil,whichis

anendemicareaforVL.Kennelswereselectedafter

veteri-narianinspectionbasedontheirroutinesanitaryandcare

andwelfarepracticeswithanimals.Animalsweretreated

forhelminticinfectionsandhadanupdatedroutine

vacci-nationprogramforrabies,parvovirus,andothercommon

canineinfections.Thescreening processfortheanimals

was performed during three consecutive months (from

November2009toJanuary2010)andincludedlaboratory

testsforVLdiagnosisandanalysisofclinicalparameters,as

and serological positive results in at least one

evalua-tionwereexcluded.Insecticide-impregnatedcollarswere

adoptedduringthescreeningprocessandvaccination,but

removedthereafter.Theseprocedureswereadoptedinan

attempttominimizethecontactoftheselecteddogswith

Leishmaniaantigenspriortoorduringvaccination,which

could subsequently interfere with the analysis of

vac-cineseroconversion.Noothercontrolmeasures,besides

welfare maintenance of the animals and application of

standardsanitizationprocedures,wereadoptedduringthe

follow-upperiod.

2.3. Clinicalandlaboratorialevaluationofdogs

Biochemical and hematological analyses were

per-formedmonthlyforanimalscreeningorevery3months

aftervaccinationforclinicalevaluationoftheanimals,as

previouslydescribed.DNAwasextractedfromperipheral

blood samples collected at 6 and 14 months

post-vaccination employing theGenomic Prep MiniSpin Kit

(G&EHealthcare).PCRfordetectionofLeishmaniakDNA

inDNAextractedfromblood sampleswasperformedas

previouslyreported(Vitaleetal.,2004).Theoccurrenceof

vaccinationadverseeffectssuchasfever,pain,oredemaat

thesiteofvaccineadministrationwasactivelymonitored

byveterinariansfor72hafteradministrationofeach

vac-cineorplacebo dose. Afterthis period,allowners were

instructedtocontact veterinariansiftheanimals

mani-festedanysideeffects.

2.4. Vaccinationprotocol

Leish-Tec® consistsof100␮gofA2recombinant

pro-tein(rA2)and250␮gofsaponin(Riedel).Inall,140animals

withoutclinicalsignsofdiseaseandwithnegative

serolog-icaltestsforVLandanti-A2antibodiesinthreeconsecutive

monthlyevaluationswereselectedforthestudy.These140

dogswereallocatedintotwogroups:inthefirstgroup,70

dogswerevaccinatedwithLeish-Tec®subcutaneouslyon

days0,21,and42.Inthesecondgroup,70dogsreceived

a placebo containing saponin only, following the same

doseschedule.Eachkennelharboredhalf-placeboand

half-vaccinated dogs. Each animal received a microchip for

identification.Vaccineandplacebodoseswerecodedand

undistinguishableinappearancefromeachother.Doses

were administeredblindly by veterinarians,and kennel

ownerswerenotawareandcouldnotdistinguishbetween

vaccinated and placebo-treated animals. Animals were

maintainedinopenkennelsandfollowedforaperiodof

14months(fromFebruary2010toApril2011).Aboosting

vaccinedosewasadministered12months(February2011)

aftertheprimingvaccinationdoses.

2.5. Serologicalanalysisforthediagnosisofcanine

visceralleishmaniasis

The presence of anti-Leishmania promastigote

anti-bodies (LPA)wasinvestigatedmonthly,beforeandafter

vaccination,byindirectimmunofluorescence(IFI).Titers

higherthan1:40dilutionswereconsideredpositive.We

alsousedtheLeishmaniapromastigoteantigen(LPA)-based

ELISAkitsprovidedbythemanufacturer(Bio-Manguinhos,

OswaldoCruzFoundation,RiodeJaneiro,Brazil).ForELISA,

themanufacturerprovidesLPA-sensitizedELISAplatesand

recommendsaserumsampledilutionof1:100.Allassays

wereperformedusingnegativeandpositivecontrolsera.

InELISA,cut-offvaluesweredeterminedfromthe

aver-ageopticaldensityplustwostandarddeviationsobtained

fromnegativesera,followingthemanufacturer’s

recom-mendation.Anti-parasiteantibodieswerealsoinvestigated

throughtheDualPathPlatformtest(DPP)forCVL,

accord-ingtomanufacturer’sinstructions(Bio-Manguinhos).DPP

isarapidimmunochromatographictestforthedetection

ofCVLbasedontherecombinantantigensK39andK26

(Grimaldietal.,2012a;Marcondesetal.,2011),andithas

beenrecentlyapprovedforscreeningofCVLseropositive

dogsbytheBrazilianPublicHealthauthorities.Sera

sam-pleswererandomlyselectedfortestingwithDPPfromthe

placebo-treated(n=27)andLeish-Tec®vaccinatedgroups

(n=55,1monthaftervaccination;n=51,6monthsafter

vaccination;andn=45,14monthsaftervaccination).

2.6. ELISAforanti-A2totalIgGandspecificIgG1and

IgG2antibodies

Anti-A2-specific IgG, IgG1, and IgG2 subtype levels

were measured in pre-immune sera and at 1, 6, and

14 months after vaccination. ELISA tomeasure anti-A2

serological responses was performed using purified A2

recombinant protein, prepared as previously described

(Carvalho et al., 2002; Fernandes et al., 2008). Briefly,

96-well plates (Costar®, Corning, NY,USA) were

sensi-tizedwith rA2 (250ng/well) in coating buffer (0.015M

Na2CO3,0.03MNaHCO3,pH9.6)overnightat4◦C.Plates

were blocked with 2% non-fatty milk PBS at 25◦C for

1h,washedagainandthen100-␮Lserasamples,diluted

at1:100wereadded andincubatedfor 90minat 37◦C.

AfterthreewasheswithPBS/0.05%Tween20,

peroxidase-labeledantibodies specific to canineIgG, IgG1, or IgG2

isotypes(Sigma,MO,USA)dilutedat1:25,000,1:15,000,

or1:35,000,respectively,wereaddedfor1hat37◦C.The

plateswerewashedsixtimesandincubatedwithH2O2and

o-phenylenediamine(Sigma,MO,USA)forreaction

devel-opment.Reactionswerestoppedbytheadditionof50␮L

of2NH2SO4(Merck).Opticaldensitiesweredetermined

at492nmbyusingaVersaMaxELISAreader(Molecular

Devices,CA,USA).Allsampleswereanalyzedinablinded

manner.Cutoffvalueswerecalculatedforeachplatefrom

theaverageplus twostandard deviationsof absorbance

valuesobtainedfromthese5negativesera.

2.7. Immunoblotanalysis

Purified A2 recombinant protein (1␮g) was

sub-mitted to sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) and transferred onto PVDF

membranes (Resende et al., 2008). Membranes were

blocked with 5% non-fatty milk PBS and incubated

overnight at 4◦C prior to incubation with serum

samples from immunized or placebo dogs.

Peroxidase-conjugatedanti-canineIgG (Sigma, MO, USA)dilutedat

productswerevisualizedbytheadditionof

chloronaph-thol,diaminobenzidine,(Sigma,MO,USA)andH2O2.The

anti-A2monoclonalantibody(1:3000)wasusedasa

posi-tivecontrol.

2.8. Statisticalanalysis

Samplesizeestimationwasperformedassuminga

cri-terion of 5% for significance and a power of 95%. The

estimationsconsideredaprobabilityofinfectionof10%(or

8.7%ofseroconversiontoA2)among thenon-protected

dogs,basedontheincidenceofCVLintheendemicarea

ofBeloHorizonte,aseroconversionof70%among

vacci-nateddogs,andalossrateof50%duringthestudy.Based

onthese parameters, 25 dogs in each group would be

requiredforstatisticalsignificance.Forstatisticalpurposes,

absorbancevaluesweretransformedintologarithmic

val-ues{log10[(100)(x)+1]}. Differencesamongabsorbance

values wereproportional tothemeanvalues (Snedecor

andCochran,1989;Stoltetal.,2003).ThelevelsofIgG1,

IgG2, and total IgG antibodies weresubmitted to

split-plotANOVAanalysis,consideringthegroups(vaccinated

orplacebo)asthebetween-subjectfactor(wholeplots)and

thetimeofobservation(months)asthewithin-subject

fac-tor(subplot).Theinteractionfactorswerecomparedwith

theresidualforsubplotswithinwholeplots (Stoltetal.,

2003).Two-tailedP<0.05wasconsideredsignificant.

3. Results

Attheendofthescreeningperiod,14%oftheanimals

initiallyevaluatedweresuspectedofhavingVL,basedon

thepresenceofsuggestivesymptoms;17%were

seroposi-tivefortotalIgGantibodiesagainstpromastigoteparasite

antigens;22%presentedotherintercurrencesthatresulted

intheirexclusion;and2%died.Fromtheremaining45%,

140dogswereselectedforuseinthistrial.

Vaccinatedanimalsweremonitoredforadverseeffects,

suchas fever, pain, abscesses, ulceration or edema. No

significantreactionsthat couldbe associated with

vac-cineadministrationweredetectedintheanimals(datanot

shown),indicatingthatthedoseswerewelltoleratedby

theanimals.

Anti-A2totalIgGantibodylevelsweremeasuredtotest

vaccineimmunogenicity. Dataarepresentedin Fig.1as

apercentage of seropositiveanimals.Ahighproportion

(98%)ofLeish-Tec®vaccinatedanimalsdisplayedpositive

anti-A2antibodytiters1monthaftervaccination.This

per-centagewasreduced6monthsaftervaccination(81.13%).

Nevertheless,thepercentageofanimalspositivefor

anti-A2antibodiesreturnedtotheinitialvalues(98%),2months

aftertheboostingvaccinedose(14monthsafterthe

prim-ingvaccination). The reduction in anti-A2 IgG antibody

levelsinvaccinatedanimalsisalsoshowninFig.2.Itshould

benotedthatalthoughthelevelsofanti-A2antibodieswere

decreasedat6months,81.13%oftheanimalsstillpresented

antibodylevels above the establishedcut-off values. In

contrast,mostplacebo-treatedanimals(76.71%)remained

negativeforanti-A2antibodies.Thehighestrateofpositive

animalsforanti-A2antibodiesamongtheplacebo-treated

Fig.1.Percentageofplacebo-treatedandLeish-Tec®_{-vaccinateddogs} presentinganti-Leishmaniapromastigoteantigens(LPA)oranti-A2 anti-bodies,asdetectedbyELISA.Positiveseraweredefinedbasedonoptical density(OD)readingsabovecut-offvalues.Cut-offvalueswere deter-minedforeachELISAplatefromtheaverageopticaldensityplustwo standarddeviations obtainedfrom5negativesera.Eachsamplewas testedinduplicate.

dogs(23.81%)wasdetectedonthesixmonths

measure-ment.

ELISAwasperformedforanimalsfromboth

placebo-treated and Leish-Tec® vaccinated groups to detectthe

presenceofanti-Leishmaniapromastigote(anti-LPA)

anti-bodies, with the goal of testing whether vaccination

induced cross-reactive anti-promastigote antibodies. In

contrasttotheanti-A2totalantibodies,animalsfromthe

vaccinatedorplacebogroupsremainednegativefortotal

IgG antibodies against LPA throughout the experiment

(Fig.1), except for two animals (2.85%) in placebo and

one(1.42%)inthevaccinatedgroup.Similarresultswere

observedusingtheDPPtestinaselectedpanelofserum

samplesfromplacebo-treatedandLeish-Tec®-vaccinated

animals(Table1),i.e.,animalsthatwerepositiveinELISA

werealsopositiveinDPP.

Serumsamplesfromtheanimalswerealsoexamined

byELISAtoassessanti-A2IgG2-andIgG1-specificantibody

responses.AsshowninFig.2,levelsofanti-A2IgG2were

significantly higher in Leish-Tec®-vaccinated animals

thanincontrolplaceboanimals.Anti-A2IgG2levelswere

decreasedinvaccinateddogsat6months,butincreased

Table1

DetectionofseropositivedogsusingtherapidDPPrK28fusionprotein chromatographicimmunoassayfortestingselectedserafromvaccinated andcontroldogs.

Numberofpositivesamples/totalsamplestested Monthsaftertheprime-vaccination

Groups 1 6 14a

Vaccinated 0/55 0/51 0/45

Placebo 0/27 2/27 2/27

Fig.2.Anti-A2specificserologicalresponsesinanimalsvaccinatedwithLeish-Tec®.Levelsofanti-A2IgG,IgG1,andIgG2antibodiesinpre-immunesera andatdifferenttimepointsaftervaccinationweredetectedbyELISA.Cut-offvaluesweredeterminedforeachELISAplatefromtheaverageopticaldensity plustwostandarddeviationsobtainedfrom5negativesera,foreachIgGisotypes.Eachbarrepresentstheaverage±standarddeviationfortheoptical densityineachgroup.Asteriskindicatesthatdifferencesbetweenplaceboandvaccinatedanimalsarestatisticallysignificant,astestedusingsplitplot varianceanalysis.

again to higher levels2 months after the last booster

vaccination. Leish-Tec®-vaccinated dogs displayed

increasedlevelsofIgG1antibodiesat1monthafterthe

priming vaccination dose. However, IgG1 levels were

decreased at 6 months and remained lower after the

boostervaccinedose.Anti-A2IgG2levelswerehigherthan

anti-A2 IgG1 levels, leading to IgG2/IgG1 ratios higher

than1,invaccinateddogs(datanotshown).Lowlevelsof

anti-A2IgG2andIgG1antibodieswereobservedinplacebo

animals, which remainedunchanged during the

exper-iment. To confirmthe specificity of antibodyresponses

from Leish-Tec® immunized animals, an immunoblot

analysis with recombinant A2 protein was performed.

The sera of vaccinated animals recognizeda protein of

52kDa,whichwasalsodetectedbyananti-A2monoclonal

antibody(Fig.3).

Only two animals in thecontrol placebo group

pre-sentedseroconversionofanti-LPAELISAassociatedwith

symptoms characteristic of VL, including

lymphadeno-pathy,periocularalopecia,onychogryphosis, andweight

loss.Theseanimalsalsopresentedalteredleukocytecounts

andalbumin/globulinratio(datanotshown).One

vacci-natedanimal presented positive resultsin theanti-LPA

ELISAandLeishmaniakDNA-specificPCR,butshowedno

signsofdiseaseattheendoftheexperiment.

4. Discussion

Here,wetestedwhetherLeish-Tec®isimmunogenicfor

a heterogeneousdogpopulation. Itisworthmentioning

thatthisstudydidnotaimtotestvaccineefficacy,sincea

differentexperimentaldesign(placebocontrolled,

double-blindedfieldtrial),includingalargersampleofanimalsand

complete randomizationwouldberequiredtoapproach

efficacywithstatisticalsignificance.

SaponiniscommonlyusedasadjuvantincanineVL

vac-cinesforinductionoftypeIimmuneresponses(Marcondes

etal.,2011,2013;Vandepapeliereetal.,2008),although

it maycause adverse reactions(Fernandes et al.,2014;

Marcondesetal.,2011;Vandepapeliereetal.,2008).Inthis

study,Leish-Tec®doseswerewelltoleratedbytheanimals,

aspreviouslyreported(Fernandesetal.,2008,2014).

Antibodies were selected as markers of vaccine

immunogenicity or exposure to infection because they

areeasilymeasuredandusefulintheveterinaryroutine

practice. Moreover, CVL vaccines in Brazil are licensed

only for individual prophylaxis and it is recommended

bymanufacturesandpublichealth authoritiesthatonly

seronegativeanimalsshouldreceivethesevaccines.A2is

actuallyoneofthefewamastigote-specificantigenstested

Fig. 3.Immunoblot analysis of anti-A2 IgG antibodies in sera of placeboandLeish-Tec®_{vaccinatedanimals.TherecombinantA2protein} (1␮g/well)wastransferredfromSDS-PAGEtonitrocellulosemembranes andincubatedwith1:200seradilutionsofselectedserasamples.Lane1: proteinmolecularweightmarker;Lane2:anti-A2monoclonalantibody; Lane3:seraofplaceboanimal;Lane4:serafromLeish-Tec®vaccinated animal

forboth,diagnosisorvaccinedevelopment(Carvalhoetal., 2002;Fernandesetal.,2008).However,thereisno

com-merciallyavailabletestforCVLdiagnosisusingthisantigen.

ConsideringthatA2isacomponentofLeish-Tec®,anassay

todetectanti-A2antibodiescouldbeeasilyappliedto

eval-uateimmunizationwiththisvaccineinthefield.Thisassay

mayprovideevidencethatagivenanimalisresponsiveor

nottovaccination,andcouldthusbeindicativeof

Leish-Tec®immunogenicity.Inagreement,inthisstudy,98%of

thevaccinated dogs had anti-A2antibody levelshigher

thanthecut-offfornegativeresults,indicatingthat

Leish-Tec®isimmunogeniceveninahighlyheterogeneousdog

population.

TheA2-basedELISAalsoallowedthefollowupanti-A2

humoralresponses,afterprimingandboost vaccination.

Sincethelevelsanti-A2antibodiesdecreased overtime,

asexpectedforaproteinbased-vaccine,anannual

boost-ing vaccination may be required for recall of immune

responses.Inagreement,anti-A2antibodylevelsincreased

afteradministrationofasingleLeish-Tec®dose,asdetected

2monthsaftertheboostingvaccinedose(14monthsafter

theprimingvaccination).

Anotherimportant findingwasthe low incidenceof

cross-reactiveanti-LPAantibodies,aftervaccination,which

hasimplicationsnotonlyforindividualdiagnosisofCVLin

veterinarianroutinepractice,butalsoforepidemiological

surveys of CVL seropositive dogs. The current

serolog-ical tests (ELISA, IFI, and DPP) for VL adopted by the

Brazilianpublichealthauthoritiesarebasedon

promastig-ote antigens (Grimaldi et al., 2012a; Marcondes et al.,

2013).Theinductionofcross-reactingantibodiestoLPAor

K28,theantigensusedintheofficialserodiagnostictests,

maybeproblematicfordogowners,veterinarians,and

pub-lichealthauthorities whenmanaginganimalsthathave

receivedvaccinesthatmayinduceseroconversionforthese

surveydiagnostictests.

Duetoitspossibleimpactinthecurrentcontrol

meas-ures,thisaspecthasbeenrecentlyaddressedforthetwo

commerciallyavailableCVLvaccinesinBrazil,Leishmune®

(Marcondesetal.,2011,2013)andLeish-Tec®(Fernandes

et al., 2014). Since A2 is an amastigote antigen,

Leish-Tec® isnotexpectedtoinducecross-reactiveantibodies

againstthepromastigoteantigensusedindiagnostictests.

However, in one study it has been argued that

Leish-Tec® induced seroconversionin30.9% ofthevaccinated

animals,astestedbyLPA-ELISA(Fernandesetal.,2014),

contrasting with our findings. These discrepant results

may bewell attributed to differences in the

methodo-logicalproceduresandcriteriaadoptedforscreeningand

inclusionofdogsineachstudy.Althoughinbothstudies

only LPA-ELISA seronegative dogs were included, it is

wellknownthatthecurrentCVLdiagnosistestspresent

importantlimitationsregardingsensitivityandspecificity.

Consequently,a single point serologicalevaluation may

notbesufficientlyrigorousforscreeningdogsthat

after-wardswillbetestedforcross-reactivityaftervaccination.

Therefore, in the present study, only dogs negative in

serological tests for three consecutive months were

included,tominimizetheinterferenceofaprevious

infec-tionor contact withparasites, in serological responses,

after vaccination. Continuous exposure to parasite

antigens of animals living in endemic areas during

vaccination may also result in cross-reactive humoral

responses.Inthiscontext,tounequivocallydemonstrate

that vaccination does not induce seroconvertion, in

addition to the rigorous screening procedures, in this

study, insecticide-impregnated collarswere maintained

inanimalsduringthescreeningprocessandvaccination.

Theseprocedureswereadoptedattemptingtominimize

thecontactoftheselecteddogswithLeishmaniaantigens

prior or during vaccination, which could subsequently

interfere with the analysis of vaccine seroconversion.

Ideally,maintenanceofanimalsinthin-screened,instead

ofopenedkennelsshouldhavebeenadopted,aimingto

improve the control measures. However, it seems that

the lack of this additional preventive strategy did not

interferesignificantlyinseroconvertionrates,sinceonly

low cross-LPAreactiveantibodylevelsweredetectedin

Leish-Tec®326vaccinatedanimalswhentestedeitherby

ELISAorDPP.

Theanti-A2IgG2antibodyresponsewassimilartothat

observedinourpreviousstudy,inbeagledogs,asantibody

titerswerehighandinducedshortlyafterthepriming

vac-cinationscheme,andanincreasedproportionofanimals

remainedseropositiveinsubsequentmeasurements.

How-ever,levelsofanti-A2IgG1werealsohigher,contrasting

withpreviousresults(Fernandesetal.,2008).This

discrep-ancymaybeattributedtothedifferencesinthestudied

populations. Nevertheless, the anti-A2 IgG2/IgG1 ratios

werehigherthan1atall-timepointsaftervaccination(data

notshown),suggestingthepredominanceofTh1immune

responsesinLeish-Tec®vaccinateddogs.Furthermore,as

previouslyreported,thelevelsofanti-A2IgG1andIgG2a

antibodiesandtheIgG2a/IgG1ratiomaybeindicativeof

protective immunityelicited bythis vaccine(Fernandes

et al., 2008).In other studies,levels of IgG2antibodies

raisedafterimmunizationwithdifferentvaccine

formula-tionswerealsocorrelatedwithincreasedIFN-production

and Th1 cellularimmunity (Carrillo and Moreno, 2009;

Morenoetal.,2012;Vitoriano-Souzaetal.,2012).

More-over, in vaccinated animals, IgG2antibodies have been

associatedwithopsonizationandcomplementfixationthat

maypromoteclearanceof extracellularamastigotesand

inhibitionoftheirinfectivity,thuscontributingtothe

resis-tanceprofileinducedbyvaccination(Bourdoiseauetal.,

2009).

However, infected dogs frequently present a mixed

Th1/Th2cytokineprofile.AlthoughIgG1levelshavebeen

linked to symptomatic CVL, the immune responses in

an infectedanimal exposed toa varietyof antigensare

complex and may havehampered theestablishment of

correlations between a single marker and either

resis-tanceorsusceptibilitytoinfection(CarrilloandMoreno,

2009; Moreno et al., 2012; Reis et al., 2010).

Addi-tionally, insufficient data from vaccination studies are

available to establish such correlations. The different

nature ofantigens and adjuvantsused in anti-CVL

vac-cines, modifyingtheinducedimmune responsesfurther

hinderscomparisons(Day,2007;Fernandesetal.,2008;

Marcondes et al., 2013; Roatt et al., 2012; Vitoriano-Souza et al., 2012). Therefore, the contribution of

protective responsesstill requires furtherinvestigation.

Hence,anti-A2IgG2orIgG1antibodiesmaynotbedirectly

associatedwithprotectiveresponses,butshouldratherbe

takenasmarkersofimmunogenicityforvaccinationwith

Leish-Tec®.

Inconclusion,thefindingsreportedhereindicatethat

vaccinationwithLeish-Tec®(i)isimmunogenicandsafe

foraheterogeneousdogpopulation,(ii)doesnotinduce

antibodiesthataredetectablebyroutineCVL

immunodi-agnostictests,(iii) requires administrationofan annual

vaccineboost,and(iv)canbemonitoredbyA2-basedELISA.

Conflictofintereststatement

Theauthorsdeclarenoconflictofinterest.

Acknowledgements

ThisworkwassupportedbytheNationalInstituteof

Science and Technologyfor Vaccines/Conselho Nacional

deDesenvolvimento eTecnológico(CNPq),Fundac¸ãode

AmparoàPesquisadoEstadodeMinasGerais(FAPEMIG;

GrantAPQ-00304-10,ProgramadePesquisasparaoSUS)

andHertapeCalierSaúdeAnimal.Thefundershadnorole

instudydesign,datacollectionand analysis,decisionto

publish,orpreparationofthemanuscript.

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