Field randomized trial to evaluate the efficacy of the Leish - Tec vaccine against canine visceral leishmaniasis in anendemic area of Brazil.

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ContentslistsavailableatScienceDirect

Vaccine

jo u rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

Field

randomized

trial

to

evaluate

the

efﬁcacy

of

the

Leish-Tec

®

vaccine

against

canine

visceral

leishmaniasis

in

an

endemic

area

of

Brazil

Shara

Regina-Silva

a

_,

_Ana

_Maria

_Leonardi

_Tibúrcio

_Feres

b

_,

_João

_Carlos

_Franc¸

_a-Silva

c

_,

Edelberto

Santos

Dias

a

_,

_Érika

_Monteiro

_Michalsky

a

_,

_Hélida

_Monteiro

_de

_Andrade

c

_,

Eduardo

Antonio

Ferraz

Coelho

d

_,

_Gustavo

_Meirelles

_Ribeiro

e

_,

_Ana

_Paula

_Fernandes

f

_,

George

Luiz

Lins

Machado-Coelho

e,∗

a_Centro_de_Pesquisas_René_Rachou,_Fiocruz,_Belo_Horizonte,_Minas_Gerais,_Brazil b_Inova_{Biotecnologia}_Saúde_Animal_Ltda,_Juatuba,_Minas_Gerais,_Brazil

c_Departamento_de_{Parasitologia,}_Universidade_Federal_de_Minas_Gerais,_Belo_Horizonte,_Minas_Gerais,_Brazil d_Setor_de_Patologia_Clínica,_Colégio_Técnico,_Universidade_Federal_de_Minas_Gerais,_Belo_Horizonte,_Minas_Gerais,_Brazil e_Escola_de_Medicina,_Universidade_Federal_de_Ouro_Preto,_Ouro_Preto,_Minas_Gerais,_Brazil

f_Faculdade_de_Farmácia,_Universidade_Federal_de_Minas_Gerais,_Belo_Horizonte,_Minas_Gerais,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22December2015 Receivedinrevisedform1March2016 Accepted8March2016

Availableonline18March2016 Keywords:

Randomizedﬁeldtrial Caninevisceralleishmaniasis Vaccine

A2antigen Leish-Tec® Efﬁcacy

a

b

s

t

r

a

c

t

Background:Acaninevaccineremainsapromisingapproachforeffectivecontrolofvisceralleishmaniasis

(VL),givenitscomplexepidemiologyinareaswherezoonoticVLisprevalent.Leish-Tec®_is_a

recombi-nantvaccine,basedontheLeishmaniaA2antigen,againstcanineVL(CVL).Itis,since2014,thesingle

commercialvaccinelicensedinBrazil.Here,Leish-Tec®efﬁcacywasestimatedthrougharandomized

ﬁeldtrial(RFT),inahighlyVLendemicarea.

Methods:TheRFTwasconductedfrom2008to2010inanendemicareaofsoutheasternBrazil,presenting

aCVLseroprevalenceof41.9%.Eighthundredforty-sevenseronegativedogswererandomlyselectedto

receiveLeish-Tec®_(n₌₄₂₉₎_or_placebo_(n₌_418)._Animals_were_followed_up_by_clinical,_serological,_and

parasitologicalexamsfor18months.TheCVLincidenceinbothgroupswascomparedthroughproportion

analysis.

Results:AsigniﬁcantreductioninthenumberofcasesofCVLwasobservedinthevaccinegroup,as

comparedwiththeplacebogroup,whetherefﬁcacywasestimatedaccordingtoparasitologicalresults

(71.4%;95%CI:34.9–87.3%;p=0.001;riskratio=0.287),byaddingresultsofxenodiagnosisand

parasito-logicalexams(58.1%;95%CI:26.0–76.3%;p=0.002;riskratio=0.419).Amongtheanimalsthatconverted

toapositiveanti-A2serology,efﬁcacyreached80.8%(95%CI:37.6–94.1%,p=0.001;riskratio=0.192).

Xenodiagnosishasdetectedareductionof46.6%(p=0.05)intransmissiontosandﬂiesfromvaccinated

animalspresentinganti-A2positiveserology.

Conclusion:TheLeish-Tec®vaccineprovedsigniﬁcantlyeffectiveforprophylaxisofCVL,afternatural

challengeassuredbytransmissionofLeishmaniaparasites,inahighlyendemicarea.Noteworthy,this

reporthasunveiledthecomplexityofperformingaRFTforanti-CVLvaccinesinBrazil,whichmaybe

helpfulfordesigningoffuturestudies.

∗ Correspondingauthorat:UniversidadeFederaldeOuroPreto,Campus Univer-sitárioMorrodoCruzeiro,EscoladeMedicina,OuroPreto,MinasGerais,Zipcode: 35400-000,Brazil.Tel.:+553135591004;fax:+553135591001.

E-mailaddress:[email protected](G.L.L.Machado-Coelho).

1. Introduction

Visceral leishmaniasis (VL) is a protozoan parasitic disease that, if untreated, leads to high mortality rates. VL, similarly to other leishmaniasis forms, is a neglected disease, resulting in 20,000–40,000deaths[1].In zoonotic VLtransmissionareas, domestic dogsare reservoirs ofL. (L.)infantum (Mediterranean basin)andL.(L.)infantumchagasi(SouthAmerica)parasitesfor humaninfection[2–4].

http://dx.doi.org/10.1016/j.vaccine.2016.03.019

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Dogsarealsovery susceptibletoinfectionand maydevelop severesymptoms,endingtodeath[3,5].Theavailable chemother-apeutic treatment is limited [6] and once clinically cured, asymptomaticanimalsmaystilltransmittheinfection.Therefore, treatmentisnotrecommendedasamasscontrolmeasureinBrazil [4].ProphylacticcontrolprogramsinBrazilhavefocusedon eutha-nizingseropositivedogs[7].However,controlcampaignsarevery expensiveandtheirrelativelypoorsuccessrateisaggravatedbythe lackofhighlysensitive,speciﬁcbiomarkersfortheinfectious sta-tusindogs[7–9].Consequently,VLhasexpandedtoareaswhereits occurrencehadnotbeenpreviouslyreported[7,9–10].Therefore, vaccinationemergesasapotentialstrategytoprotectdogsfrom beinginfectioustothesandﬂyvector,therebyreducing transmis-sionrates.

Currently, Leish-Tec®, a vaccine formulation containing the recombinantproteinA2ofL.(L.)donovaniandsaponin,istheunique commercialCVLvaccinelicensedinBrazil.Severalpre-clinicaltrials inmicehaveprovidedevidenceoftheprotectiveresponsesinduced byvaccinationwiththeA2antigen,againstVL[11–16].InRhesus monkeys,animpressiveprotectiveeffectwasinducedby prime-boostingvaccinationprotocolsusingrecombinantA2proteinand adenovirusexpressingA2[17].Leish-Tec® wasshowntoinduce protectiveimmunityinbeagledogsagainstahighdoseintravenous infectionwithL.(L.)infantumchagasi[18].Moreover,Leish-Tec® doesnotinduceseroconversioninvaccinatedanimals,an impor-tantrequirementforCVLvaccineswheneuthanasiaofseropositive dogsisrecommended[19].Vaccinationalsoreducedsigniﬁcantly theinfectiousnessofdogstosandﬂies,asdemonstratedby xeno-diagnosis[20].

Here,we reporttheresultsofa randomizedﬁeld trial(RFT) carriedoutinanendemicareaforVL,toevaluatetheefﬁcacyof Leish-Tec®.

2. Methods 2.1. Ethicsstatement

TheResearch Ethics Committeeof the Federal University of OuroPreto(CEP/UFOP,Brazil)approvedthis RFT(documentNo. 58/2008).ThisRFTwasalsoregisteredintheBrazilianMinistryof Agriculture(MAPA,Brazil),undertheofﬁcialdocument 028/2013-CPV/DFIP/MAPA.

2.2. Vaccine

Thedosesof theLeish-Tec® vaccine wereproduced by Her-tapeSaúdeAnimal(Juatuba,MinasGerais,Brazil),andstandardized tocontain100_␮g/mLofrecombinantA2proteinand500_␮g/mL of saponin, as adjuvant. The vaccine doses had the same for-mulation of the commercially available vaccine. Dogs received three 1.0mL doses administered through subcutaneous injec-tion at 21-day intervals [18,19]. Placebo and vaccine doses were coded at Hertape Saúde Animal; had the same appear-ance and were given to the animals by ﬁeld veterinarians, blindly.

2.3. Samplesizeestimation

Asamplesizeof568dogsineachgroupwasestimated, accord-ingtoFleiss[21],usingthefollowingassumptions:(a)a1/1ratio betweenvaccinatedandplacebogroups;(b)50%vaccine effective-ness;(c)10%annualCVLincidenceinPorteirinha[22,23];(d)power (0.90);(e)signiﬁcantdifference5%;and(f)20%estimatedlosses. Sensitivityof96%wasconsideredwhenapplyingdiagnosistestsin parallel[24]. Seronegative n=252 Seronegative n=253 Vaccine group n=429 Placebo group n=418 Losses=4 Losses=13 3 doses n=425 3 doses n=405

End of immunologic window (252)

Seropositive=124 (excluded) Seropositive=147

(excluded)

Losses=28 Losses=26

Losses=566

Serum Samples Time zero serologic

Seronegative n=847 Seropositive n=98 Seronegative n=1,511 Seropositive n=1,088 Canine population N=2,615

Eluate of dried blood sample on filter paper Losses=16

Losses = 49

Negative (n=188) Parasitologic positive (n=5)

Losses=49 Negative (n=168) Parasitologic positive (n=17) Xenodiagnosis positive (n=12) Xenodiagnosis positive (n=8)

Both exames positives (n=7) Both exames positives (n=2)

Fig.1.Flowchartshowingscreening,samplingandlossesofdogsincludedinthe randomizedﬁeldvaccinetrial,inPorteirinha,MinasGerais,Brazil,2008.

2.4. Studydesignandpopulation

The RFT was conducted from 2008 to 2010, in urban and ruralareasofthemunicipalityofPorteirinha,MinasGerais,Brazil. PorteirinhaisaVLendemicareaandnocontrolmeasures, includ-ingeuthanasiaofseropositivedogs,insecticidespraying,oruseof impregnatedcollars,hadbeenappliedintheeightyearspriorto thisstudy[22,23].

Fig.1showsaﬂowchartofthescreeningandselectionofthe caninepopulation.Initially,acanineserologicalcensus(N=2615) wasperformedintheurbanneighborhoodsandruraloutskirtsof Porteirinha.ThenumberofVLseropositivedogspercityblockwas determined,withanoverallCVLprevalenceof41.9%, correspond-ingto31.6%withinthecitylimitsand80.1%in theruralareas. ReactivedogsinatleastoneserologicalexamcrudeELISA(cELISA) orindirectﬂuorescenceantibodytest(IFAT)wereexcludedfrom thevaccinationtrial,correspondingto1088animals.Atotalof1511 seronegativedogswerepre-selectedtobeincludedinthetrial.

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Fourmonthslater,beforevaccination(timezero), serological testing(cELISA,IFATandKalazardetectTM_(KD))_was_repeated_in these1511dogs,toﬁnallydetermineeligibilityoftheseanimals.Of those,98animalswereexcludedduetopositiveserology.Other566 seronegativedogswerealsoexcluded,duetotheowners’refusal toparticipate,animal’sdeath,escapeorclosedhouses.Theﬁnal sampleof 847dogs,negativebyserologicalanalysis,were ran-domlyallocatedintotwogroups:vaccinated(n=429)andplacebo (n=418).Forrandomizationasimplemethodwasused, consist-ingofblocksorsetsoffourcodes(twolettersforvaccineandtwo forplacebo).Thus,fouranimals,twoallocatedintheplaceboand twointhevaccinatedgroup,composedeachblock.Veterinarians activelymonitoredtheoccurrenceofanyadverseeffectssuchas fever,pain,oredemaatthesiteofvaccineadministration,overa periodof72haftervaccination.

2.5. Inclusionandexclusioncriteria

Onlyhealthydogswereeligibleforthestudy,asclinically deter-minedbyveterinarians,whetherapurebredormongrel,regardless ofgender,atleastfourmonthsoldandseronegativeforCVL. Clin-icalexaminationincludedevaluationforclinicalsignsofCVL[25]. Exclusioncriteriawerethepresenceofotherpathologiesor posi-tiveserologyforCVL,duringtheimmunologicalwindow.Animals wereconsideredasseropositiveifbothcELISAandIFATresulted positive.CVLnegativeanimals,inclinicalandserological evalua-tions,wereincludedintheRFT,onlyaftertheirownershadagreed totakepartinthestudy,followinganinterviewtoexplainthe pur-poseofthestudyandaftertheyhadsignedaStatementofInformed Consent.

Eligible dogs were dewormed and received two doses of Multi-Dog®(HertapeSaúdeAnimal)apolyvalentvaccine, admin-istered42and21days,beforeLeish-Tec®doses.Eachanimalwas microchipped(AnimalTAG®,KorthRFIDLtd.,SãoCarlos,SãoPaulo, Brazil),identiﬁedbyafour-digitcode,andthenregisteredinthe projectdatabase.

Alldogsweremaintainedintheiroriginalhouseholdsand nat-urally exposed to Leishmania spp. infection(natural challenge). Veterinariansconductedmonthlyclinicalexaminationsinalldogs. BloodsampleswerecollectedeverythreemonthsforCVL serolog-icaltesting.Aperiodof252dayswasconsideredasimmunological window[24,25],i.e.,aperiodinwhichanimalsmaymanifest clini-calandlaboratoryresultspositiveforCVLduetopreviousinfection. ThiswindowperiodwasalsobasedonthereportbyCourtenayetal. [8],whichdescribedacohortofnaivedogsexposedtonatural infec-tioninanendemicarea.Inthiscohort,dogsbecameinfectiousto sandﬂiesinaverage333days(105daysafterseroconversion).

Accordingly,271dogswereexcludeddue topositive results inserologicaltestsduringtheimmunologicalwindow.These ani-malswerenotsubmittedtoacompletefollowup,sincesomewere euthanizedbypublichealthauthoritiesandotherwerelostdue tonaturalintercurrences.Theoutcomeamongtheremainingdogs variedaccordingtothediagnosticmethod:137werepositivein parasitologicaltests,and14inthexenodiagnosis.Ofthose,only 17animalspresentedthetypicalsymptomsofCVL.Alltheother hadonlypositiveserology(n=120).Theremainingseronegative animalswerefollowedupfor557days,includingtheperiodof vaccination(time0)andtheimmunologicalwindow(252days). 2.6. Outcomevariablesandcasedeﬁnition

After the immunological window, dogs presenting positive serology(cELISAandIFAT)wereconsideredaspotentialVLcases. Theywereeuthanizedandsubmittedtoparasitological examina-tion.Bonemarrowsmearsandimprintsoftissuefragments(skin, mesentericlymphnodes,andspleenbiopsy)onglassslideswere

stainedwithGiemsaandexaminedmicroscopically.PotentialVL caseswereconsideredpositiveVLcasesonlyafterthedetection ofparasitesinatleastonedog’stissue,inanyofthe parasitolo-gicaltests,includingbonemarrowcultures.Directparasitological examsandhistopathologywereperformedaccordingtostandard procedures[23,26].

2.7. BonemarrowcultureandidentiﬁcationofLeishmania species

Bonemarrowculturewasperformedaspreviouslydescribed [19].GenomicDNAextractedfromparasitesisolatedfrombone marrowaspirateswassubmittedtokDNAPCR-RFLPanalysisfor identificationofLeishmaniaspecies,aspreviouslydescribed,with modifications[27].TypingofL.(L.)amazonensisisolateswere con-firmedbyAva-Idigestion.

2.8. Serologicaltests

cElisa(BiomanguinhosInstitute,RiodeJaneiro,Brazil)wasused forserologicalscreeningandIFAT(BiomanguinhosInstitute,Riode Janeiro,Brazil),asaconfirmatorytest.KD(InbiosNDI)wasalsoused asaconfirmatorytest.Alltestswerecarriedoutinaccordancewith manufacturers’instructions.Nineserologicalevaluationswere per-formed,for longitudinal follow upof allanimals. Measuringof serologicalanti-A2responseswereperformedbyELISAusing puri-fiedA2recombinantprotein,aspreviouslydescribed[19,28]. 2.9. Xenodiagnosis

Xenodiagnosiswasperformedaftertheimmunologicalwindow period,startingatday497.Thenumberofselecteddogs(n=154, 77 ineachgroup) wasbasedonthefollowing assumptions:(a) statisticalsignificance(0.05);(b)power(0.90);(c)theproofof bidi-rectional(ortwo-tailed)hypothesis;(d)anestimatedproportion of50%ofvectorinfectionbyLeishmaniainnon-vaccinatedinfected dogs[29];(e)anexpecteddifferenceof50%betweengroups;(f) 10%sampleloss.Alldogsallocatedinrandomizedblockslodging atleasttwoanimalswithserologicalpositiveresults,wereselected toensurethatxenodiagnosiswasperformedinablindedmanner. Thephlebotomine sandflieswereF1off springsfroma Lut-zomyialongipalpis colony of insects provided by CpqRR/Fiocruz (Brazil). They were transported to the Porteirinha (640km), in appropriatedcontainersundercontrolledhumidityand tempera-ture.Xenodiagnosiswasperformedaspreviouslydescribed[30]. The presence of Leishmania spp. DNA wasinvestigated by PCR amplificationofa120bpsegmentfromtheconservedminicircle ofkDNAfortheLeishmaniagenus[31].

2.10. Statisticalanalysis

An external clinical monitor (ECM) reviewed data files and lockedthemafterthevaccinationprocedures.Initially,eachdog wasidentifiedasaconfirmedornegativecaseforLeishmaniaspp. infection,accordingtoparasitologicaldiagnosis.Datamanagement, descriptive analysesofbaseline characteristics(frequenciesand means±SD),foreachtrialgroupandbivariateanalysesof differ-encesbetweenproportions(Fisherexacttest)andmeans(t-tests) wereperformedwithSPSS20software(SPSSInc.,Chicago,IL,USA). Vaccineefficacywasestimatedaccordingtoeitherparasitological definitionofcasesorbyaddingxenodiagnosisandparasitological results.Analyseswerealsostratifiedaccordingtoanti-A2 serologi-calconversiontoestimateefficacy.RRistheratioofCVLincidence inthevaccinegroupascomparedtotheplacebogroupandused tocalculatedefficacyas100×(1−relativerisk(RR)).Onlythose subjectswho completedallvaccinationdoses wereincludedin

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theefﬁcacyanalyses.Differencesbetweengroupswereconsidered signiﬁcantwhenp≤0.05.

3. Results

3.1. Demographicandgeographicdistributionofdogs

Table1showsthenumberofdogsallocatedinthevaccineand placebogroupsbycitylocation.Therewerenosigniﬁcant differ-encesinratiosbetweenthevaccineandplacebo groups,orthe distributionin ruraland urbanareas,indicating that successful randomizationofvaccinedoseswasachieved.

After the immunological window, 559 animals remained includedintheRFT.Table2describestheirserologicaland para-sitologicalresultsinalltests,duringthechallengeperiodofthe RFT.Themajorityoftheanimalswasasymptomaticandremained seronegative(n=356, 63.7%),throughoutthestudy.In contrast, only5.6%(n=31)ofanimalspresentedsuggestiveclinicalsignsof CVL.Parasitologicalexamswereperformedin65seropositive ani-mals.Ofthose,34dogsresultedparasitologicalnegativeandwere notincludedintheestimationofefficacy,duetotheuncertainty intheirinfectiousstatus.Confirmedcases,i.e.,animalspresenting positiveparasitologicalresultsinatleastoneparasitologicaltest, correspondingto5.6%(n=31)ofthefinalsample,wereincluded forefficacyestimation.Thus,thefinalsampleforefficacy estima-tioncorrespondedto356negativeanimalsand31cases.Ahigh degreeofdiscordancewasobservedinserologicalanalysis,since only6.6%(n=37/559)oftheanimalswereconcomitantlypositive intwoserologicaltests(cELISA+IFAT),while13.5%(44/325)were positiveonlyinKD,19.3%(108/559)incELISAand20.9%(117/559) inIFAT.

Table1

Randomizationofdogsreceivingvaccineorplacebodoses,inruralandurbanareas inPorteirinha,MinasGerais,Brazil.

Group Ruralarea N(%) Urbanarea N(%) Total N(%) pvalue Vaccine 50(50.5) 380(50.8) 430(50.8) Placebo 49(49.5) 368(49.2) 417(49.2) 0.955 Total 99 748 847 Table2

Parasitologicalandserologicalresultsindogs,aftertheimmunologicalwindow. Finalclassiﬁcation Diagnostictest No.ofpositive/No.of

evaluateddogs(%) Conﬁrmedcasesa _Parallel

Parasitological Exams 31/65(47.7%)b Non-conﬁrmed casesc Xenodiagnosis 20/71(28.1%) Serology(ELISA andIFAT) 37/559(6.6%) ELISA 108/559(19.3%) IFAT 117/559(20.9%) Rapidtest-Kalazar DetectTM 44/325(13.5%) Negatived _356/559_(63.7%) Total 559

a_Parasites_were_detected_in_at_least_one_of_the_parallel_{parasitological}_exams,

includingbonemarrowculture,directparasitologicalexam(imprintorsmearin microscopicslides)ofskin,lymphnodes,spleenandbonemarrowor histopatho-logicalanalysis.

b _Nine_animals_were_also_positive_in_{xenodiagnosis.}

c _{Non-conﬁrmed}_cases_were_not_considered_for_efﬁcacy_estimation.

d _Asymptomatic_animals,_presenting_negative_results_in_all_serological_diagnostic

tests,appliedlongitudinallythroughoutthestudyperiod.

Fig.2. Anti-A2speciﬁcserologicalresponsesinanimalsvaccinatedwith Leish-Tec®.Levelsofanti-A2IgG,IgG1,andIgG2antibodiesinpre-immunesera(time 0)andaftervaccination(time73)weredetectedbyELISA.Eachbarrepresentsthe average±standarddeviationfortheopticaldensityineachgroup.Oneasterisk indi-catesstatisticallysigniﬁcantdifferencesbetweenplaceboandvaccinatedanimals atthesametimepoint,andtwoasterisks,thedifferencesbetweenantibodylevels atdifferenttimepointsforthesamegroupofanimals.

Asampleof71animalswasalsotestedinxenodiagnosis, result-ingin40.8%(29/71)positivedogs.Ofthose,ninewerealsopositive inparasitologicaltests.Asexpectedforasamplecomposed pre-dominantlyofasymptomaticanimals,alargedegreeofdiscordant resultswasobservedamongtheCVLdiagnostictoolsused,mainly xenodiagnosisorserologicaltests.GiventheuncertainintheCVL diagnosisamonganimalsclassifiedasnon-confirmedcases,they werenotincludedinthesampleusedtoestimateefficacy.

Immunogenicitywasevaluatedbycomparinganti-A2humoral responses,measuredinserasamplescollectedimmediatelybefore (time0)andaftertheprimingvaccinationprotocol(time73),from bothvaccinatedandplaceboanimals.AsshowninFig.2,after vac-cination,anti-A2totalIgG,IgG2,andIgG1antibodylevelsincreased signiﬁcantlyinvaccinateddogs,ascomparedtotheantibodylevels detectedattimezeroortothelevelspresentedbyplaceboanimals attime73.

Table3showstheproportionofCVLcaseswithinthevaccine andplacebogroups.Asignificantreductioninthenumberofcases ofCVLwasobservedinthevaccinegroup,ascomparedtoplacebo, reachinganoverall efficacy,basedontheparasitological defini-tionofcasesof71.4%(95%CI:34.9–87.3%;p=0.001;RR=0.287).By addingresultsofxenodiagnosistoparasitologicalexams,efficacy remainedhighandsignificant(58.1%;95%CI:26.0–76.3%;p=0.002; RR=0.419).Amongtheanimalsthatconvertedtoapositive anti-A2 serology, efficacy was80.8% (95% CI:37.6–94.1%, p=0.001; RR=0.192).

Table4describesthedistributionofLeishmaniaspecies, iden-tified from 41 bone marrow cultures, between vaccinated and placebogroups.Asexpected,L.(L.)infantumchagasiwasthemost prevalentspecies(85.4%;n=35),whereasL.(L.)amazonensiswas identifiedin14.6%(n=6)ofthecultures.Nosignificantdifferences wereobservedinthedistributionoftheseparasitespecieswithin eachgroup.

ResultsfromxenodiagnosesarepresentedinTable5.Leishmania spp.kDNAwasdetectedin29 outofthe71sandflies’ samples, correspondingtoanoverallprevalence40.8%.Theprevalenceof positivesandflypoolsthatfedinanimalsoftheplaceboand vac-cinatedgroupswas44.2%(19outof43pools)and35.7%(10out28 pools),respectively(p=0.48).However,asignificant(p=0.05)of reductionininfectivitytosandflieswasobservedshouldthe ani-malsbestratifiedaccordingtoanti-A2serologicalresponses.While similar proportionsof negative (n=4)and positive (n=4)sand

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Table3

Leish-Tec®_efﬁcacy,_as_determined_by_{parasitological}_exams,_{xenodiagnosis}_and_anti-A2_serology,_in_Porteirinha,_Minas_Gerais,_Brazil.

Group CVLcriteriaa _CVL_criteriab _CVL_criteriac

Positive Negative Total Positive Negative Total Positive Negative Total Vaccine n(%) 7 (3.6) 188 (96.4) 195 (100.0) 15 (7.4) 188 (92.6) 203 (100.0) 3 (2.4) 122 (97.6) 125 (100.0) Placebo n(%) 24 (12.5) 168 (87.5) 192 (100.0) 36 (17.7) 168 (82.3) 204 (100.0) 24 (12.5) 168 (87.5) 192 (100.0) Total n(%) 31 (8.0) 356 (92.0) 387 (100.0) 51 (12.5) 356 (87.5) 407 (100.0) 27 (8.5) 290 (91.5) 317 (100.0)

a_According_to_{parasitological}_exams:_imprinting,_culture,_or_{histopathology}_of_dog_tissues_(skin,_lymph_nodes,_spleen_and_bone_marrow),_p₌_0.001;_risk_ratio₌_0.287;

efﬁcacy=71.4%(95%CI:34.9–87.3%).

b_According_to_{parasitological}_exams_plus_{xenodiagnosis,}_p₌_0.002;_risk_ratio₌_0.419;_efﬁcacy₌_58.1%_(95%_CI:_{26.0–76.3%).} c _According_to_anti-A2_serology,_p₌_0.001;_risk_ratio₌_0.192;_efﬁcacy₌_80.8%_(95%_CI:_{37.6–94.1%).}

Table4

DistributionofLeishmaniaspeciesisolatedfrombonemarrowofinfecteddogsas determinedbykDNAPCR-RFLP,inPorteirinha,MinasGerais,Brazil.

Group L.(L.)infantum chagasi n(%) L.(L.) amazonensis n(%) Placebo 20(87.0) 3(13.0) Vaccine 15(83.3) 3(16.7) Total 35(85.4) 6(14.6) Asymptomaticanimals 24(68.6) 6(100) pvalue=0.74. Table5

FrequenciesofLeishmaniaspp.DNA,asdetectedbyPCR,inLutzomyialongipalpis phlebotominesandﬂies’poolsfeeding(xenodiagnosis)onplaceboandvaccinated dogs,inPorteirinha,MinasGerais,Brazil,2010.

Group A2classiﬁcation kDNAPCR Total

Negative Positive Placebo 24(55.8) 19(44.2) 43 Vaccinated Positive 11(73.3) 4(26.7) 15 Negative 4(50.0) 4(50.0) 8 Noinformation 3(60.0) 2(40.0) 5 Total 18(64.3) 10(35.7) 28 Total 42(59.2) 29(40.8) 71

pvalue=0.48(notstratiﬁed−placebo×vaccinated).

p value=0.05 (stratiﬁed according to positive serology for A2−placebo×vaccinated).

ﬂy’poolswereobserved,afterfeedinginanti-A2negativedogs,

onlyfouroutof15pools(26.7%)feedingonvaccinatedanimals

werepositive.Therefore,anti-A2seropositivevaccinatedanimals

(73.3%)werelessinfectioustosandﬂies,correspondingto46.6%

reductionintransmissiontosandﬂies.

4. Discussion

This RFT was carried out in a highly endemic area of VL

[22,32–33].Mostprobablyduetothelackofapplicationcontrol measuresforalongperiod(from2004to2008),thecanine sero-prevalenceinPorteirinhawashigherthanthatusedtoestimate samplesize,reaching41.9%.Nonetheless,thehighCVLprevalence inPorteirinhaallowedasignificantchallengefordemonstrating Leish-Tec®efficacy.Moreover,Leish-Tec®efficacywastestedina nativeheterogeneouspopulation,previouslyexposedtoinfection, anextremeconditionandchallengingscenario,thatbadlyneeds controlinterventions.

Differentstudydesignshavebeenusedtotestefﬁcacyof anti-CVLvaccines,includingtestingonnativeanimalsorthroughthe introduction of naïve animals, in endemic areas [34–36]. Both conditionsmay signiﬁcantlyshape the responsetovaccination.

Applicationofanimmunologicalwindowisrequired,when test-ing efﬁcacyin nativeanimals, allowingexclusion of previously exposedanimalsorthoseinfectedduringscreeningand vaccina-tion.Inagreement,thisstudyincludedanimmunologicalwindow. ThecriteriaappliedinBrazilforVLepidemiologiccontrol (serolog-icaltestsforscreeningofandeuthanasiaofseropositiveanimals) wasadopted,inordertotestLeish-Tec®intheconditionsitmight beused,inendemicareas[23].

Byassuminganimprovedsensitivityofparallelparasitological tests,inthisRFT,estimationofefficacywasbasedonresultsof para-sitologicaltests,mostlyduetothepredominanceofasymptomatic animalsinthefinalsample,andtothelowsensitivityand speci-ficityofserologicaltests.Accordingtothesecriteria,anddespitethe hightransmissionpressure,theresultsofthisRFTshowedthat vac-cinationwithLeish-Tec®resultedinsignificantefficacy(71.4%,CI 34.9–87.3%).Moreover,consideringonlyvaccinatedanimals, pro-tectionlevelsreached96.4%,accordingtoparasitologicalcriteria.

ItislargelyknowthatCVLdiagnosisinasymptomaticanimals posesonethemainchallengesintheveterinaryroutine,giventhe limitationsofserologicalandothernon-invasivediagnostictests andthelackofpathognomonicCVLsigns[25].Inendemicareas,the lowpositivepredictivevalueofserologicaldiagnostictestsimpairs diagnosisofdogsastruepositive,duetocross-reactionswithother pathogens[37].Inagreement,awidediscordanceamongresults ofallserologicaltestswasobservedforanimalsduringthe chal-lengeperiod,contributingfortheuncertaintyregardingtheuseof serologicalresultsforefﬁcacyestimation.Incontrast,therewasa highconcordancebetweentheserologicalandtheparasitological analysesperformed.Amongthe220seropositivedogssubmitted toeuthanasia,214werepositiveinparasitologicalanalysis(data notshown).Ontheotherhand,thenegativeanimalswere clini-callyhealthandpresentednegativeserologicalresults,throughout thelongitudinalserologicalfollowup.

Leish-Tec® doesnot induce anti-promastigotecross-reactive antibodies, but raises specific cellular and humoral immune responsesindogs[18–19,38].Hence,anti-A2antibodyresponses wereusedhereasbiomarkersofimmunogenicityofLeish-Tec®and asanauxiliarytoolforinterpretingvaccineefficacy.Inagreement, vaccineefficacyamongdogsthatrespondedtovaccinationwith increasedanti-A2antibodylevelswashigher(80.8%),ascompared totheoverallefficacy(71.4%). Itis noteworthythat,in another study,seropositivity for anti-A2total IgG antibodieswasfound in98%ofvaccinatedanimalsshortly,aftervaccination[19].This valuedecreased to81.13% six monthslater, beforerising again (98%)afterthevaccinationboost,asexpectedforaproteinvaccine. Antibodyresponsesinducedbyvaccinationmaybealsoaffected severalfactors,includingconcomitantinfections,andpour nutri-tion status of animals. In addition, in natural populations, not all individuals respondequallyto antigenicstimulation, due to MHCrestriction.For instance,trialsthatevaluatedthefirst vac-cineagainsthumanAmericancutaneousleishmaniasis(Leishvacin)

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showedthatregardlessofthepopulation,10–30%ofindividualsdo notconvertthetestofMontenegrotopositive,45daysafter vacci-nation[39,40].Nonetheless,theadjuvantusedisanotheraspectto beconsideredinordertoimprovecellularandimmuneresponses inducedbyvaccination.

Theanti-A2IgG2/IgG1 ratioswere higherthan1, suggesting thepredominanceofTh1immuneresponsesinLeish-Tec® vacci-nateddogs,assimilarlyobservedinthephaseIItrialinbeagledogs [18],andinanheterogeneousdogpopulationkeptinkennels[19]. Saponinwastheadjuvantselectedtoformulateallthethree com-merciallyavailableCVLvaccines,sinceitinducesTypeIimmune responseswhenaddedtovaccineformulations[18,41–42].As pre-viouslyreported,thelevelsofanti-A2IgG1andIgG2antibodies andtheIgG2/IgG1ratiowerecorrelatedwithspeciﬁchighIFN-␥ andlowIL-10levels,inLeish-Tec®vaccinatedbeagledogs[18]orin otherstudieswithdifferentvaccineformulations[41,43].IgG2 anti-bodieshavebeenassociatedwithopsonizationandcomplement activationinvaccinatedanimals[44].

L.(L.)amazonensis wasidentified in14.6%of thecultures, in agreementwithotherreportsinBrazil[45–48].Still,thepresence oftwodistinctLeishmaniainfectingspeciesseemednottohave influencedtheresultsofLeish-Tec®vaccineefficacy.L.(L.) ama-zonensisalsocontains and expressA2 genesequences [28] and micevaccinationwiththeA2antigenresultedinsignificant pro-tection,providingpre-clinicalevidencethattheprotectiveeffect ofLeish-Tec® maybeextended toL.(L.)amazonensis infections indogs[11,15].Therefore,we believethatthedifferenceinthe percentage ofinfectingspecies observed inour assayis inline withtherateofprevalenceofthesespeciesinthearea,andnot byachangeinvaccineprotection.Althoughthereareno popula-tionalbasis’studiestoestimatetheprevalenceofL.amazonensis indogsandhumans,itisnoteworthythatBarraletal.[49]found that,among144humanVLpatientsinBahia,35%wereinfectedby L.amazonensis.

Xenodiagnosisisanimportantstrategyforaccessingthe poten-tialfor transmission of a given animal, though limited by low sensitivity and specificity in field conditions [20,29–30,50–53]. Nonetheless,by adding xenodiagnosesand parasitological find-ings,Leish-Tec®efficacyremainedsignificantlyhigh(58.1%;95% CI:26.0–76.3%;p=0.002).Moreover,vaccinationalsoinduceda sig-nificantreduction(46.6%)inLeishmaniaspp.transmissiontosand fliesthatfedinanti-A2seropositivevaccinateddogs.Inagreement, inanotherstudy,only5.4%ofanimalsvaccinatedwithLeish-Tec® wereinfectioustosandflies,inanendemicarea,ascomparedtoa positiverateof36.6%amongcontroldogs[20].Thereby,Leish-Tec® seemstoinduceanappreciablereductioninLeishmania transmis-siontosandflies.

The poor nutritional and immunological status of animals, besidesexposuretootherinfectionsmayhaveaffectedsigniﬁcantly efﬁcacy.Losses exceeding50% and much higher than expected (20%),alsoconstitutedanimportantdrawback.Lossesweremainly duetothepopulationrefusaltodeliverthedogsfortesting. How-ever,therewerenodifferencesinlossesbetweenthevaccinated andplacebogroups.

Finally,thisreporthasunveiledthecomplexityofperforminga RFTforanti-CVLvaccines,inBrazil,giventheepidemiological, diag-nostic,logistic,andotherissues,whichmaybehelpfulfordesigning futurestudies.

5. Conclusion

TheLeish-Tec®vaccinewassigniﬁcantlyeffectivefor prophy-laxisCVL,afternaturalchallengeassuredbyahightransmission exposuretoparasites,notwithstandingtheRFToperatingand logis-ticallimitations.

Acknowledgements

WewouldliketothankHertapeSaúdeAnimalSAforﬁnancial support,thelocalgovernmentofPorteirinha,Ms.VivianWalterdos Reisforyourtechnicalhelpduringthisstudy,andthePorteirinha ZoonosisCenterteamfortheircollaborationincollectingdataand samples.Wealsowanttoexpressourthankstotheownerswho allowedtheirdogstoparticipateinthistrial.

Conﬂictofinterest:None. References

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