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ARTICLE IN PRESSVAC 7040 1–12

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Long-lasting protection against canine visceral leishmaniasis usingthe LiESAp-MDP vaccine in endemic areas of France:

Double-blind randomised efficacy field trial

Jean-Loup Lemesre a,∗, Philippe Holzmuller a, Rachel Bras Goncalves a,Gilles Bourdoiseau b, Christophe Hugnet c, Mireille Cavaleyra a, Gerard Papierok d

a Institut de Recherche pour le Developpement, UR 008 “Pathogenie des Trypanosomatidae”, Equipe 1,911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France

b Service de Parasitologie, Ecole Nationale Veterinaire de Lyon, 69280 Lyon, Francec Clinique Veterinaire, 26160, La Begude de Mazenc, France

d Bio Veto Test, 285, avenue de Rome, Z.A. Jean Monnet Sud, 83500 La Seyne Sur Mer, France

Received 4 April 2006; received in revised form 22 February 2007; accepted 28 February 2007

bstract

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for anffective vaccine against visceral leishmaniasis is pressing. Dogs constitute the major reservoir of Leishmania infantum/chagasi responsibleor human visceral leishmaniasis. We have recently demonstrated that the combination of naturally excreted/secreted antigens, easily purifiedrom culture supernatant of Leishmania infantum promastigotes (LiESAp) as vaccine antigen in formulation with muramyl dipeptide (MDP)s adjuvant, conferred 100% protection to dogs experimentally infected with L. infantum by inducing in vaccinees a significant, stable andong-lasting Th1-type cell response [Lemesre JL, Holzmuller P, Cavaleyra M, Bras Goncalves R, Hottin G, Papierok G. Protection againstxperimental visceral leishmaniasis infection in dogs immunised with purified excreted secreted antigens of L. infantum promastigotes.accine 2005; 23:2825–2840; Holzmuller P, Cavaleyra M, Moreaux J, Kovacic R, Vincendeau P, Papierok G, Lemesre JL. Lymphocytes ofogs immunised with purified excreted secreted antigens of L. infantum co-incubated with Leishmania-infected macrophages produce IFN-amma resulting in nitric oxide-mediated amastigote apoptosis. Vet. Immunol. Immunopathol. 2005, 106:247–257]. In this report, protectiongainst visceral leishmaniasis is investigated in naturally exposed dogs of endemic areas of the South of France vaccinated with LiESAp/MDPaccine. A double-blind randomised efficacy field trial was developed on a large-scale dog population composed of vaccinees (n = 205) andlacebo-treated animals (n = 209), which were prospectively studied for a 2-year period. 0f the initial 414 enrolled dogs, 340 (175 controls and65 vaccinees) were analysed for clinical, serological and parasitological studies at 24 months post-vaccination, after two sand fly seasons.trong seroconversion disclosed by an L. infantum indirect immunofluorescence antibody test (IFAT) associated with suspicious clinicalymptoms, considered an indication that the animals had an established progressive infection, was only observed in the placebo group. Theeropositive and/or symptomatic dogs were selected for further examination for possible Leishmania infection by culturing parasites from bone-arrow aspirate. The presence of leishmanial infection was also evaluated by means of the PCR analysis of bone marrow samples in all enrolled

ogs prior to vaccination and in all evaluated animals (175 controls and 165 vaccinees) at 24 months post-vaccination. After two transmissionycles completed, the Leishmania infection rate was 0.61% (1/165) in vaccinated dogs and 6.86% (12/175) in the placebo group. The efficacy

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Please cite this article in press as: Lemesre J-L, et al., Long-lasting protecvaccine in endemic areas of France: Double-blind randomised efficacy fi

f the vaccine was calculated to be 92% (P = 0.002). A clear difference between the dogs that received vaccine and those that received placeboas also established by the results of their immune status. Increased anti-LiESAp IgG2 reactivity and significant enhanced NO-mediated anti-

eishmanial activity of canine macrophages in response to higher IFN-� production by T cells were almost exclusively revealed in vaccinees.he LiESAp-MDP vaccine induced a significant, long-lasting and strong p2007 Published by Elsevier Ltd.

eywords: Canine visceral leishmaniasis; Excreted secreted antigens; LiESAp-MD

∗ Corresponding author. Tel.: +33 4 67 41 62 20; fax: +33 4 67 41 63 30.E-mail address: lemesre@mpl.ird.fr (J.-L. Lemesre).

264-410X/$ – see front matter © 2007 Published by Elsevier Ltd.oi:10.1016/j.vaccine.2007.02.083

rotective effect against canine visceral leishmaniasis in the field.

tion against canine visceral leishmaniasis using the LiESAp-MDPeld trial, Vaccine (2007), doi:10.1016/j.vaccine.2007.02.083

P vaccine; Protection; Immune responses

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. Introduction

Protozoa of the genus Leishmania are obligatory intra-ellular parasites of mammalian macrophages. They areransmitted via the bite of phlebotomine sandflies andause cutaneous, mucocutaneous, or visceral leishmaniasis.oonotic visceral leishmaniasis (VL) is one of the most

mportant emerging diseases. Wild canines and domesticogs are the major reservoirs of L. infantum in the Mediter-anean basin, extending to several Middle East and Asianountries, and of L. chagasi in South and Central America.he seroprevalence of canine VL (Can VL) in the Mediter-

anean region has been shown to range from 2% to 48% [1]nd higher infection rates are found in tropical America (upo 67% in highly endemic clusters) [2,3], and is therefore aerious problem of disease management.

Neglected by researchers and funding agencies, leishma-iasis control strategies have varied little for decades. Currenttrategies for control of zoonotic VL are based on vector con-rol and drug treatment or elimination of infected dogs [4].nterestingly, laboratory and field evaluations have shown thatmpregnated dog collars and topical application of insecti-ides protected domestic dogs from L infantum infections,ut might also reduce the risk of L infantum infection in chil-ren [5]. Conventional chemotherapies are often inadequate,oxic and expensive or are becoming less effective becausef the emergence of numerous resistances, a clear risk foruman health [6]. The impact of canine control by removalf seropositive infected dogs in reducing human VL preva-ence in endemic areas has been debated [2,7–11], but such

easures remain socially unacceptable, difficult to develop,xpensive and finally quite ineffective [9,11]. As most of thevailable methods for canine VL treatment and control aref limited effectiveness, the development of a dog vaccines highly desirable and would be the most practical and effi-ient control tool, reducing the dog-sandfly-dog peridomesticransmission cycle, probably important in maintaining trans-

ission to humans [12–14]. Moreover, vaccination againstL has received limited attention compared with cutaneous

eishmaniasis, although the need for an effective vaccine isressing for the control of this disease. In natural host modelsf visceral leishmaniasis, studies on the protective role of Tells and cytokines are limited and only a few reports in theiterature deal with a vaccine [15–23].

Several approaches have been used to characterize anti-enic macromolecules excreted/secreted by microorganismshat are important in the establishment of immune andhysiologic interactions with the host and that are highly pro-ective in vaccine models [24–26]. Similarly, recent studiesndicated that the Leishmania promastigote culture filtrateroteins could elicit strong immunity in different hosts27–30] and protection in L. major-infected BALB/c mice

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Please cite this article in press as: Lemesre J-L, et al., Long-lasting protecvaccine in endemic areas of France: Double-blind randomised efficacy fi

31,32]. In a previous report, the protective potential ofhe excreted/secreted antigens of L. infantum promastigotesLiESAp) was demonstrated in an experimental model [30].

e showed that the combination of LiESAp in formulation

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ith muramyl dipeptide (MDP) induced a total protectiongainst canine experimental leishmaniasis [30]. Vaccine-nduced protection correlated with an early establishmentf a long-lasting predominantly Th1-type cellular immuneesponse specifically directed against LiESAp [29,30].

In this report, we show the efficacy of the LiESAp-MDPaccine in the field by means of a randomised controlled trial.e used a large-scale dog population of an endemic area of

he South of France, divided it into vaccine- and placebo-reated control groups, under conditions that equalised theirhance of exposure to natural infection. The efficacy of theaccine was monitored for 2 years (after two transmissionycles were completed) by clinical examination and serolog-cal and parasitological analyses. We used the anti-LiESApgG2 reactivity and the NO-mediated anti-leishmanial activ-ty of canine monocyte-derived macrophages (CM-DM) inesponse to higher IFN-� production by T cells as toolsor evaluation of humoral and cellular immune responses inogs.

. Materials and methods

.1. Animals and study design

Dogs living outdoor in endemic areas of the South ofrance with a high prevalence of leishmaniasis over a distancef 500 km between Beziers (Herault) and Menton (Alpesaritimes) along the Mediterranean coast were screened

n clinical and serological criteria by investigators of 18eterinary clinics. The majority of the dogs screened wereunting dogs, with some guard dogs and dogs from breedingacilities. Animals with suspected clinical manifestations ofeishmaniasis were excluded. The initial serological screen-ng disclosed by an L. infantum indirect immunofluorescencentibody test (IFAT, Fluoleish®, Bio Veto Test, La Seyneur Mer, France) and/or the Speed Leish® test (Bio Vetoest, La Seyne sur Mer, France) excluded seropositive dogs,ven those showing a low level of anti-Leishmania antibodiesweak positive reaction at 1:100 dilution). Healthy seroneg-tive dogs were admitted to the study even though they wereolymerase chain reaction (PCR) positive at the bone marrowxamination [33].

Consent was obtained from the dogs’ owners, who werenformed of the risk of the procedures and the require-

ent for a 2-year follow-up. All the animals included inhis investigation had to live outdoor without any treatmentor leishmaniasis and for ectoparasites (permethrin, fipronin,tc.), or use of sandfly repellent delthamethrine collar neck-ace (ScaliborR, Intervet, Holland). Every dogs included inhis study were treated following the guidelines for animalxperimentation of the National Veterinary School of Lyon

tion against canine visceral leishmaniasis using the LiESAp-MDPeld trial, Vaccine (2007), doi:10.1016/j.vaccine.2007.02.083

ENVL), and experiments were done in accordance with 145

he institutional guidelines in order to keep animal suffer- 146

ng as minimal as possible. Protocols were submitted to and 147

pproved by the ENVL ethics committee.

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.2. Vaccine, vaccination and follow-up

The LiESAp vaccine preparations (vaccine batches015A, 0023, 0114) used in this study were produced underood manufacturing conditions using a WHO reference L.nfantum strain (MHOM/MA/67/ITMAP-263) and following

previously described methodology [29,30]. Briefly, pro-astigotes were grown in CDM/LP culture medium [34,35].hen parasite concentration reached 2–3 × 107 promastig-

tes per millilitre in a 6-day period, culture was centrifuged2000 × g, 20 min, 4 ◦C) to remove parasites. The supernatantas collected, filtered (0.2 �m-pore-size filter, Millipore,illerica, MA, USA) to eliminate removal promastigotes,oncentrated approximately 100-fold and dialysed by ultra-ltration with a 3-kDa-cutoff filter unit (Pall). Proteinoncentration was determined according to the Bradfordethod (Bio-Rad, Laboratories). Placebo was obtained from

nparasitised culture medium and processed as described forhe parasitised culture supernatant.

Vaccine doses were composed of 100 �g lyophilizediESAp antigen and 200 �g MDP reconstituted in 1 mlaCl 0.9% sterile saline solution. Corresponding lyophilizedlacebo control was treated with 1 ml sterile saline. Thenrolled dogs were immunized with two subcutaneous injec-ions of 100 �g LiESAp supplemented with 200 �g MDP orith similar injections of normal saline placebo, 3–4 weeks

part (basic vaccination). Twelve months after basic vacci-ation, a third dose was injected (complete vaccination).

Ninety-three injected dogs were particularly followed upvery 2 weeks for up to 4 months for safety evaluation.eneral tolerance was investigated by means of clinical

xamination and general health evaluation. The site of theaccine injection was checked and the type and size of reac-ions, such as erythema, induration or ulcer, were recordedfter each injection.

Dogs were followed up for a 2-year period correspondingo two sand fly activity periods. Clinical signs were recordednd blood samples were taken prior to vaccination, at 6 and 12onths post-basic vaccination and at 6 and 12 months post-

omplete vaccination. Bone marrow samples were collectedn EDTA-tubes in all enrolled dogs prior to vaccination andn all evaluated animals from both placebo and vaccinatedroups at 24 months post-basic vaccination.

.3. Assessment of disease development and leishmanialnfection

Dogs were monitored for subsequent development of theisease by IFAT measurements for the detection of totalgG anti-Leishmania antibodies according to the manufac-urer’s instructions (Fluoleish®, BVT, La Seyne sur Mer,rance, batch 99452) and routine screening of the animals

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Please cite this article in press as: Lemesre J-L, et al., Long-lasting protecvaccine in endemic areas of France: Double-blind randomised efficacy fi

or the appearance of classical clinical signs such as weightoss, cachexia, apathy, anorexia, uveitis, alopecia, exfoliativeermatitis, ulcerative skin lesions, mucosa paleness, ony-hogryphosis and presence of popliteal lymphoadenopathies

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nd renal failure. Any dog with positive serology and/oruspicious clinical signs for visceral leishmaniasis waselected for further examination for possible leishmanialnfection. Parasite establishment was assessed by collectingone marrow aspirates of seropositive and/or symptomaticogs followed by culturing parasites in NNN (Novy-icolle-McNeal) biphasic medium. Bone marrow samples

approximately 500 �l) were cultured in NNN containingml of RPMI-20% inactivated foetal calf serum (FCS) forweek. Subcultures were seeded weekly with 0.5 ml of cul-

ure sample in NNN medium containing 3 ml of RPMI-20%VF (four subcultures). The presence of parasites was deter-ined by direct examination for 20 min of the microtiter cells

n an inverted microscope at 400 × magnification. When par-sites were observed, the sample from which the aspirate wasaken was considered as parasite-positive.

The possible presence of Leishmania DNA was alsossayed in bone marrow samples of all the enrolled dogsrior to immunization and all the evaluated animals at 24onths post-vaccination by PCR analysis using primers that

mplify the conserved region of the DNA minicircles, asreviously described [30,36]. Briefly, bone marrow samplesere collected in EDTA tubes for DNA isolation. The DNA

luted from a chromatographic procedure (5% Chelex,io-Rad Laboratories) was precipitated with sodium acetatend ethanol and suspended in 20 �l of TE (10 mM Tris–HClH 8, 1 mM EDTA pH 8). Leishmania infantum-specificligonucleotides primers Lm5 (TGGTGTAAAATAG-CCAGGTGG) and Lm3G (CCTACCCGCGGGACCA-AAAAG) were used to amplify a 700-base-pair (bp)

ragment of the conserved region of the minicircle moleculesf the Leishmania mitochondrial DNA (kDNA), using 45ycles of 92/58/70 ◦C. The reaction was conducted using5 pmol of each nucleotide, 200 �M of a dNTPs mixture,.5 mM MgCl2, 10 mM Tris–HCl pH 8.3 and 2 U of Taqolymerase (APLIGENE). This assay detects kDNA from ateast one organism. The 700-bp amplification products werenalysed by electrophoresis on 4% agarose gels followedy ethidium bromide staining and visualization under UVight. All reactions were performed with a negative controlhere no DNA was added to the mixture and a positive

ontrol containing genomic DNA isolated from five culturedromastigotes.

.4. Detection of specific IgG2 to LiESAp bynzyme-linked immunosorbent assay (ELISA)

Immune and control dogs’ sera were tested by a standardLISA procedure for antibody levels to LiESAp. Briefly, sera

rom immune or control dogs were added in triplicate at 1/50ilution in PBS containing 0.05% Tween-20 to 96-well platesreviously coated with LiESAp (1 �g per well, lot 0011).

tion against canine visceral leishmaniasis using the LiESAp-MDPeld trial, Vaccine (2007), doi:10.1016/j.vaccine.2007.02.083

fter 1-h incubation at 37 ◦C, plates were washed exten- 251

ively with PBS-0.05% Tween-20 and incubated for 30 min 252

t 37 ◦C with secondary antibody (horseradish peroxidase- 253

onjugated sheep anti-dog IgG2, 1/5000). After three washes 254

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n PBS-0.05% Tween-20, plates were developed with OPDubstrate (with H2O2 in citrate buffer) and absorbance wasead at 492 nm. Sera from healthy dogs were assayed and theeans of absorbance values plus three standard deviationsere determined. The cut-off of LiESAp-ELISA assay was

alculated as Abs 492 nm: 0.128. Positive and negative con-rol sera were used in each assay run. Results are expresseds mean values of triplicates.

.5. Assessment of anti-leishmanial activity of canineonocyte-derived macrophages

Macrophage killing ability expressed as the percent-ge of parasite index inhibition in vitro was evaluated asescribed before [29,30]. Briefly, canine monocyte-derivedacrophages (CM-DM) were prepared by differential adher-

nce of PBMC. CM-DM were obtained by maintaining thedherent cells for 5 additional days at 37 ◦C in 5% CO2.acrophage viability was determined by the trypan blue dye

xclusion test greater than 96% and non-specific esterasetaining (Sigma, St. Louis, MO, USA) showed that morehan 95% of the cells were macrophages. Non-adherent cellsi.e. lymphocytes) were washed with RPMI medium andultured in 25 cm2 ventilated tissue culture flasks at 37 ◦Cn 5% CO2 until use. Lymphocyte viability was also deter-

ined by the trypan blue dye exclusion test and was greaterhan 98%. CM-DM were infected with stationary-phase pro-

astigotes of L. infantum at a parasite:macrophage ratio of:1 for 2 h and 30 min at 37 ◦C with 5% CO2 in LabTek®

6-well glass chamber slides. Non-internalised parasitesere removed by gentle washing. Infected macrophagesere then cultured for 72 h in the presence or absence of

utologous lymphocytes at a 2:1 lymphocyte:macrophageatio. In addition, cell activation experiments were con-ucted both with and without 1 mM nitro-l-arginine, aompetitive inhibitor of the type II NO synthase (NOS-I), or 1 mM nitro-l-arginine plus 2 mM l-arginine toeverse NOS-II inhibition. The changes in the percentages ofnfected cells and the number of amastigotes per macrophagefter treatment were indicative of the anti-leishmanialctivity. Both parameters were estimated by microscopicxamination of Giemsa-stained preparations of duplicatexperiments and were used to calculate the percentage ofarasitic index (PI) inhibition, PI = 100 − [(mean numberf amastigotes per macrophage × percentage of infectedacrophages in treated wells)/(mean number of amastig-

tes per macrophage × percentage of infected macrophagesn untreated wells)] × 100.

.6. Measurement of nitrite oxide production andytokines

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Please cite this article in press as: Lemesre J-L, et al., Long-lasting protecvaccine in endemic areas of France: Double-blind randomised efficacy fi

After 72 h co-culture of infected macrophages with autol-gous lymphocytes, cell supernatants were assayed for NO2

−y the Griess reaction according to the manufacturer’snstructions (Nitrite colorimetric assay from Alexis Biochem-

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cal), which is usually employed to evaluate NO production.ata are expressed as nmol NO2

−/105 cells/72 h. The Griesseagent was modified according to Pinelli et al. [37].

IFN-� and IL-4 amounts were determined in culture super-atants from 48-h and 72-h co-cultures, respectively, bytwo-site sandwich Enzyme-Like Immunosorbent Assay

ELISA) using specific anti-dog IFN-� and anti-dog IL-4 andiotin–avidin system antibodies (R & D Systems, Minneapo-is, MN, USA), as previously described [29,30]. Absorbancealues were read at 490 nm in an automatic microplate readerWallac Victor2TM 1420 Multilabel counter, Perkin Elmerife Sciences). Standard curves for IFN-� and IL-4 were cal-ulated using recombinant canine proteins (R & D Systems).

.7. Statistical analysis

To test the statistical significance of the differencesetween groups, we used the 95% confidence interval of theeans. Means of continuous variables were compared usingstandard t-test. The X2- and Fisher’s exact tests were used inomparing proportions. All measurements and cellular assaysere done at least twice. A P value of ≤0.05 was considered

ignificant.

. Results

.1. Enrolment results

Seven hundred and seventy one dogs living outdoor inndemic areas of the South of France were screened on clin-cal and serological criteria between January and February.ix dogs were excluded for suspected clinical manifestationsf leishmaniasis. Most exclusion (46.3%) resulted from pos-tive or weak positive Leishmania serology. Four hundrednd fourteen healthy dogs (53.5% females and 46.5% males)ithout a previous history of visceral leishmaniasis, nor anti-eishmania antibodies in the blood were considered eligibleor the assay and included in this study. The majority ofhe dogs screened were adults (93.5%) and 6.5% were pup-ies (<6 months). Bone marrow PCR examinations of allnrolled dogs (414) revealed that 13 animals (3.14%) wereeishmanial DNA-positive as determined with the presencef a weak 700-bp amplification product. Healthy seroneg-tive dogs with bone marrow-PCR positive were includedn the study. The 414 enrolled dogs were double-blind ran-omly assigned and injected either by LiESAp-MDP vaccinen = 205) or placebo (n = 209).

The sand fly activity period is estimated to occur betweenay and October in endemic areas of the South of France.asic vaccination completion was achieved prior to the first

and fly season (April–May) and a third dose was injected

tion against canine visceral leishmaniasis using the LiESAp-MDPeld trial, Vaccine (2007), doi:10.1016/j.vaccine.2007.02.083

2 month post-basic vaccination (April). Follow-up assess- 353

ents were performed after the first season (6 months and 12 354

onth post-basic vaccination) and before (12 months) and 355

fter (18 and 24 months) the second sand fly activity period. 356

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f the initial enrolled 414 dogs, 340 were analysed for clinicalxamination, and serological and parasitological investiga-ions at 24 month post- basic vaccination. Sixty six dogs (29lacebos and 37 vaccinees, 15.9%) were eliminated from thetudy because of death with no relation to leishmaniasis or theaccine studied (hunting accident for the majority) or theirisappearance (lost, change in residence, etc.). Eight dogs5 controls and 3 vaccinees) did not undergo bone marrowxamination at 24 months, due to owner refusals.

.2. Safety evaluation

The LiESAp-MDP vaccine was safe and well-tolerated inaccinated dogs. Only a mild local reaction was observed in

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any vaccinated dogs, particularly after the second injection.o local and/or general adverse reactions were observed after

he complete vaccination. The overall tolerance of the vaccinen dogs appeared satisfactory.

cvlw

able 1ist of naturally exposed controls and vaccinated dogs that showed positive cliniost-vaccination

og numbers Vaccinated (V)placebo (P)

Clinical manifestationssuggestive of leishmaniasis

Level of IgGby IFAT (≥

B 027 P + 1:12800M 007 P + 1:3200J 014 P − 1:3200

MP 004 P + 1:3200JP 030 P − 1:1600R 015 P + 1:800JP 118 P + 1:400B 120 P − 1:400GC 030 P − 1:200C 062 P − −MP 024 P − −J 047 P − −JP 046 P − 1:400MP 027 P − 1:100MP 042 P − 1:100R 001 P − 1:100B 113 P − 1:100

GC 072 V − 1:400M 002 V − 1:200M 009 V − 1:200MP 010 V − 1:200JP 100 V − 1:200C 048 V − 1:200GC 030 V − 1:100MP 022 V +a −J 008 V +b −J 052 V + −F 012 V − −

erum samples were examined by immunofluorescence antibody test (IFAT) accordrance; batch 99452). The IFAT result was regarded as positive when a 1:100 dilutiulturing parasites in NNN biphasic medium in dogs that developed positive serologPCR) analysis in all evaluated dogs at the end of the experiment.FAT, indirect immunofluorescent antibody test; NNN, Novy-Nicolle-McNeal biph* Not determined.

** Owner refused bone marrow sampling.a Bladder tumour.b Babesiosis.

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PRESSxxx (2007) xxx–xxx 5

.3. Prevention of disease development and protectiono infection

The efficacy of the LiESAp/MDP vaccine was evaluatedfter completion of two transmission cycles by clinical exam-nation and serological and parasitological analyses.

Prior to immunization, all animals included in the experi-ent were asymptomatic and seronegative by IFAT. Three

undred and forty eight dogs (180 controls and 168 vac-inated) were analysed for symptoms and for the presencef specific anti-Leishmania antibodies to assess the rate oferoconversion at the end of the experiment. Suspiciousisease symptoms and/or specific anti-parasite antibodiesnly appeared between the 12th and 18th month after vac-

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ination. Table 1 gives the list of dogs from control and 386

accinated groups that showed positive clinical and/or sero- 387

ogical examination at the end of the experiment, and that 388

ere investigated further by means of culture and PCR, as 389

cal, and/or serological, and/or parasitological examinations at 24 months

antibodies1/100)

Parasites in bonemarrow NNN

PCR Conclusive leishmaniasisdiagnosis

+ + YES+ − YES+ + YES+ + YES+ − YES+ + YES+ + YES+ − YES+ − YESND* + YESND* + YESND* + YESND** ND** ND**

− − NO− − NO− − NO− − NO

− − NO− − NO− − NO− − NO− − NO− − NO− − NO− − NO− − NO− − NOND* + YES

ing to the manufacturer’s instructions (Fluoleish®, BVT, La Seyne sur Mer,on of the serum gave fluorescence. Leishmanial infection was evaluated byy and/or suspicious clinical and by means of the polymerase chain reaction

asic medium for Leishmania culturing; PCR, polymerase chain reaction.

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samples of 55 dogs (22 vaccinees and 33 placebos). The 473

results obtained are summarised in Fig. 1. Of the 33 dogs 474

that received placebo, 84.8% of them remained negative dur- 475

ing the entire follow-up period. Moreover, the data shown in 476

Fig. 1. Changes in the IgG2 anti-LiESAp antibody absorbency values withtime in serum samples of naturally exposed vaccinated dogs and placebo-

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ell as the data corresponding to the leishmanial DNA-ositive animals detected in all evaluated dogs from bothroups at 24 months post-basic vaccination. As can be seen inable 1, anti-Leishmania antibodies disclosed by IFAT wereevealed in 14 of 180 placebo-injected dogs (incidence ratef 7.8%) and in 7 of 168 vaccinated dogs (incidence rate of.2%). The titres of sera equal or above the cut-off in dogshat received placebo (mean 1:594) were not significantlyifferent (P = 0.2) than those in vaccinees (mean 1:200). Inter-stingly, a strong seroconvertion was only observed in thelacebo group of dogs. As shown in Table 1, the number ofogs that yielded titres of sera above the 1:400 dilution wereignificantly higher (P < 0.05) in the placebo group (6) thann vaccinees (0). Five placebo-injected dogs and three vacci-ated dogs presented suspicious clinical symptoms. Two outf 3 vaccinated dogs were affected by other diseases (bladderumour and babesiosis, respectively). All placebo dogs withisease symptoms gave positive titres of anti-Leishmaniantibodies, whereas none of the vaccinated dogs with clinicalanifestations were antibody-positive (Table 1). The number

f symptomatic dogs displaying specific antibodies against. infantum was significantly higher (P < 0.05) in the placeboroup (5/180) than in vaccinated group (0/168) (Table 1).

The data corresponding to the number of parasites and/oreishmanial DNA-positive animals detected in vaccinatednd unvaccinated dogs after two transmission cycles com-leted are summarized in Table 1. Among the 348 dogsvaluated by serology at 24 months post-vaccination, 340175 controls and 165 vaccinees) underwent bone marrowxamination. One healthy seropositive dog (placebo) and

healthy seronegative animals were not analysed due towner’s refusals for bone marrow sampling.

Parasite detection by NNN culture from bone marrowspirates was found to be very sensitive in confirming theresence of leishmanial infection in dogs that developed pos-tive serology and/or suspicious clinical symptoms. As seenn Table 1, parasites were isolated in nine of 175 placebo-njected dogs (5.14%), whereas none of the vaccinees yieldedositive cultures. The difference may be considered to beighly significant (P = 0.0036).

Additionally, leishmanial infection was also evaluated byeans of the PCR analysis for Leishmania DNA detection

n the bone-marrow samples of the 414 healthy seronegativenrolled dogs and of the 340 evaluated dogs at 24 monthsost-vaccination. Among the 13 healthy seronegative dogsith bone marrow-PCR positive included in the study (8accinees and 5 controls), six (2 vaccinees and 4 controls)ere lost at follow-up and 7 (6 vaccinees and 1 control) gaveegative PCR results at 24 months. The number of positiveCR for leishmanial DNA significantly decreased during thetudy in vaccinated dogs, suggesting that the LiESAp/MDPaccine should be able to reduce the rate of infection which

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Please cite this article in press as: Lemesre J-L, et al., Long-lasting protecvaccine in endemic areas of France: Double-blind randomised efficacy fi

ventually resulted in decrease rate of parasite transmission.As shown in Table 1, eight dogs that received placebo and

ne vaccinated dog became bone marrow PCR positive at 24onths post-vaccination. Four out of the 320 seronegative

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ealthy evaluated dogs were found as bone marrow PCR posi-ive (one vaccinated and 3 controls). Combined culture and/orCR results increased the difference between the two groupshile the Leishmania infection rate was 0.61% (1/165) inaccinated dogs and 6.86% (12/175) in animals that receivedlacebo (Table 1). This difference between vaccinated andontrol dogs was considered as highly significant (P = 0.002).ased on leishmanial DNA and/or parasite detection in bonearrow aspirates, the LiESAp-MDP vaccine efficacy against

nfection in dogs was evaluated at 92%.

.4. Anti-LiESAp responses in dogs

The ELISA technique was also used to investigate caninentibody IgG2 anti-LiESAp responses in vaccinated and con-rol dogs throughout the duration of the study. Anti-LiESApgG2 reactivity was checked in all animals before vaccinedministration and 6 months after the basic vaccination. Prioro immunisation, the LiESAp-ELISA assay for IgG2 antibod-es was negative in sera samples of all dogs included in thexperiment. Interestingly, 98.2% of vaccinees and 2.3% ofogs that received placebo became positive 6 months afterhe basic immunisation. Moreover, the level of anti-LiESApgG2 antibodies was significantly higher (P < 0.005) in theaccinated group (0.308 ± 0.019) than in the control group0.091 ± 0.028). The levels of anti-LiESAp IgG2 reactiv-ty were also monitored before and 6, 12 (just before theooster), 18 and 24 months (6 and 12 months after the booster,espectively) after placebo or vaccine administration in sera

tion against canine visceral leishmaniasis using the LiESAp-MDPeld trial, Vaccine (2007), doi:10.1016/j.vaccine.2007.02.083

njected controls. Fifty-five dogs (33 placebo and 22 vaccinees) were alsoollowed up before and every 6 months after the vaccine or placebo adminis-ration. The cut-off value of the LiESAp-ELISA assay is 0.128 (absorbancet 492 nm). Positive and negative control sera were included in each assayun. Results are expressed as the mean values of triplicates.

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Table 2Anti-leishmanial activity by canine macrophages of placebo (n = 19) and vaccinated (n = 15) dogs after in vitro infection with L. infantum promastigotes and72-h co-culture with autologous lymphocytes

Placebo dogs Vaccinated dogs

Dog Leishmanicidal activity(percentage inhibition ofparasite index: cut-off = 30%)

Dog Leishmanicidal activity(percentage inhibition ofparasite index: cut-off = 30%)

7–9 months post-vaccination HC 046 11.8% HC 047 24.1%HC 049 12.0% HC 048 71.6%LB 028 8.9% HC 051 78.8%LB 029 0.3% LB 059 41.6%LB 030 2.4% LB 064 49.1%LB 076 9.5% LB 077 65.4%Mean ± S.D. 7.5 ± 4.9% Mean ± S.D. 51.1 ± 20.6%

6–8 months post-booster PMP 013 0.0% PMP 015 72.5%PMP 019 0.0% PMP 022 67.0%PMP 024 0.0% PMP 010 50.2%PMP 025 11.0% LM 009 62.0%PMP 004 0.0% PGC 072 73.0%RR 015 0.0% SJP 100 46.9%MJ 014 0.1% GJ 043 58.0%PGC 030 2.0% DM 002 69.0%SJP 030 23.0% DM 003 48.0%SJP 046 15.0%HB 027 5.2% Mean ± S.D. 60.7 ± 10.4%LB 120 0.0%DM 007 0.0%Mean ± S.D. 4.3 ± 7.4%

Total 5.3 ± 6.8% Total 58.5 ± 14.9%

Macrophage-killing ability is expressed as the percentage of parasite index inhibition evaluated 7–9 months after the basic vaccination in 12 dogs (six placeboa and ninf tandard

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nd six vaccinees) and 6–8 months after the booster in 22 dogs (13 placebosor dogs with Leishmania status appear in bold. Values represent means ± s

ig. 1 clearly showed that the levels of anti-LiESAp IgG2ntibodies significantly increased in all 22 vaccinated dogs 6onths after the basic vaccination (P < 0.001) and 6 months

fter the booster (P < 0.001). By contrast, 1 year after theasic vaccination (just before the booster), dogs from theaccinated group showed a baseline low level of LiESApeactivity, which significantly increased 6 months after theooster. In vaccinees, IgG2 levels were found significantlyigher 1 year after the booster than 1 year after the basicaccine administration (Fig. 1).

.5. Killing capacities by canine-infected macrophagesxposed to autologous lymphocytes

Macrophage killing ability expressed as the percentagef parasite index inhibition after 72 h co-culture with lym-hocytes was evaluated ex vivo 7–9 months after the basicaccination and 6–8 months after the booster in 34 dogs (19lacebos and 15 vaccinees) (Table 2). Before co-cultures,M-DM from placebo dogs were infected in a manner simi-

ar to those from the vaccinated animals (data not shown). As

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Please cite this article in press as: Lemesre J-L, et al., Long-lasting protecvaccine in endemic areas of France: Double-blind randomised efficacy fi

hown in Table 2, 72 h of exposure to autologous lymphocytesf L. infantum-infected macrophage derived from placebo-njected dogs did not induce any significant leishmanicidalffect, both after basic vaccination and after the booster

mNro

e vaccinees). The cut-off value for the assay is ≥30%. The results obtaineddeviation of duplicate experiments.

parasite index inhibition of 7.5 ± 4.9% and 4.3 ± 7.4%,espectively). In contrast, Leishmania killing capacities byanine-infected macrophages exposed to autologous lympho-ytes derived from vaccinated dogs increased significantlyfter vaccine administration (51.1 ± 20.6%, P < 0.01) andere higher after the booster (60.7 ± 10.4%, P < 0.01). Our

esults showed a significant increase in the macrophageeishmania-killing capacity in LiESAp-vaccinated dogs.nterestingly, a strong anti-leishmanial activity even persistedhroughout a long post-immunisation period after both basicaccination (7–9 months) and booster (6–8 months).

.6. Nitric oxide release, IFN-γ and IL-4 productions inupernatants of co-cultured cells

The accumulation of NO2− in culture fluids of co-cultured

ells correlated with the intracellular killing of L. infan-um amastigotes (Fig. 2A). We showed that NO levels inM-DM from vaccinated dogs were significantly higher

P < 0.01) than those observed in cells from dogs that receivedlacebo. As those of pre-immune dogs (n = 34), infected

tion against canine visceral leishmaniasis using the LiESAp-MDPeld trial, Vaccine (2007), doi:10.1016/j.vaccine.2007.02.083

acrophages of placebo-injected dogs (n = 19) produced low 519

O levels (4.61 ± 0.75 and 4.58 ± 1.75 nmol/105 cells/72 h, 520

espectively) when they were co-cultured with their autol- 521

gous lymphocytes. In contrast, NO release by infected 522

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Fig. 2. Nitrite (A) and IFN� (B) contents in supernatants of co-culturedcells from naturally exposed placebo (n = 19) and vaccinated (n = 15) dogs.Leishmania-infected macrophages were incubated in medium alone or withautologous lymphocytes. Nitrite concentration as an indicator of NO pro-duction by canine macrophages was determined in supernatant of 72 hco-cultured cells using the Griess reaction before immunisation, 7–9 monthsafter the basic vaccination in 12 dogs (six placebo and six vaccinees) and 6–8mpte

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Conths after the booster in 22 dogs (13 placebos and nine vaccinees). IFN-�roduction by canine peripheral lymphocytes was determined by ELISA inhe same samples. Values represent means ± standard deviation of triplicatexperiments.

o-cultured macrophages from vaccinated dogs (n = 15)eached 17.5 ± 3.1 nmol/105 cells/72 h (Fig. 2A). Both NOroduction and leishmanicidal effect were significantlyeduced in the presence of 1 mM nitro-l-arginine, a NO syn-hase competitive inhibitor, and were almost totally restoredy the addition of 2 mM l-arginine (data not shown).

The IFN-� and IL-4 contents in supernatants of co-ultured cells from placebo-injected or vaccinated dogsere also evaluated. Supernatants of co-cultured cells

rom pre-immune dogs expressed IFN-� levels in theange of 0.07 ng/ml. No significant increase in IFN-�mount was observed in supernatants of infected co-ultured macrophages from dogs in the placebo group0.14 ± 0.09 ng/ml) (Fig. 2B). In contrast, supernatants ofanine infected macrophages upon activation with autolo-

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Please cite this article in press as: Lemesre J-L, et al., Long-lasting protecvaccine in endemic areas of France: Double-blind randomised efficacy fi

ous T cells from vaccinated dogs exhibited a significantlyigher IFN-� activity (1.68 ± 0.43, P < 0.01) than those fromlacebo animals (Fig. 2B). IL-4 production measured inupernatants of co-cultured cells from placebo-injected dogs

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as low and did not differ significantly from that determinedn supernatants of cells derived from vaccinated animals (dataot shown).

. Discussion

Control of leishmaniasis remains a source of grave con-ern worldwide. Visceral leishmaniasis is a severe diseasend both symptomatic and asymptomatic dogs might be con-idered a source of sandfly parasites [38,39]. The presence ofdog-sandfly-dog peridomestic transmission cycle is proba-ly important in maintaining transmission to humans [4,40].anines infected with VL are not only a serious veterinaryroblem throughout parts of southern Europe, the Middleast, and Central and South America, but also a major publicealth concern because there are no satisfactory therapeutictrategies, as dogs respond poorly to anti-leishmanial treat-ent and place humans at greater risk of infection [6].As most of the available methods for leishmaniasis treat-

ent and control are of limited effectiveness, there is nown urgent need for new, low-cost drugs and/or new ther-peutic interventions such as a vaccine, which is highlyesirable [29]. Even if development of an acceptable vaccines not an easy task, leishmaniasis remains one of the promis-ng parasitic diseases for vaccine development [14,41,42].n zoonotic visceral leishmaniasis (ZVL) foci, where dogsre the unique domestic reservoir, a significant reduction ineishmania transmission would be expected if we could com-ine effective preventative measures as vaccine, impregnatedog collars and topical application of insecticides. Futureontrol for ZVL should be probably an integrated vaccinelus collar strategy.

Although considerable progress has been made over theast decade in understanding the immune mechanisms under-ying protective responses, identifying potential candidatentigens, and implementing these principles with differentuccess rates in animal models [43,44], very few candidateaccines have progressed beyond the experimental stage.nfortunately, vaccination strategies have been confined to

xperimental animal models which may not entirely repli-ate the disease in dogs or humans. Dogs must be considereds the best animal model for VL in which relevant immuno-ogical studies and vaccine could be performed. The diseaseattern in dogs and humans is similar, with a long periodf asymptomatic infection following by wasting, anaemia,nlarged lymph nodes, and fever. As in humans, the infec-ion remains asymptomatic in some dogs [44]. One of theew differences is the presence of skin lesions in the dogs,nly detected in severely immunosuppressed humans [14].he recent advances in canine genomics and the develop-ent of specific antibodies and cell-surface markers provide

tion against canine visceral leishmaniasis using the LiESAp-MDPeld trial, Vaccine (2007), doi:10.1016/j.vaccine.2007.02.083

reater opportunities for researchers to take advantage of this 591

odel. Dog populations are an important reservoir of viscer- 592

lizing Leishmania in many endemic areas, and vaccination 593

f these animals would presumably constitute a major step 594

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ARTICLEJ.-L. Lemesre et al. /

owards control of the infection [12]. Recent attempts withither L. major or L. braziliensis promastigote preparations16,17], the FML antigen of L. donovani [22,23,45], or theiESAp vaccine of L. infantum [29,30] have shown promisingesults in dogs, a natural reservoir for Leishmania parasites.n clinical trials in humans, whole killed vaccines with BCGs an adjuvant failed to confer protection against cutaneouseishmaniasis [46,47] or visceral leishmaniasis [48].

Our laboratory has successfully developed a completelyefined medium that readily supports the continuous in vitroultivation of infective promastigotes of most Leishmaniapecies, without compromising parasite growth rates [34,35].ccess to this serum-free culture system paved the way to

he purification of antigens naturally excreted/secreted byarasites from filtered culture supernatants. Serum-free tech-ology is of great interest for research that moves closer toherapeutic applications since it can provide low-cost, well-efined soluble parasite molecules with native conformationhat could be used as a vaccine candidate against visceraleishmaniasis [30]. Indeed, the conformation of antigens haseen shown to play a major role in the induction of T cell-ediated immunity and to have important implications for

he design of vaccines against leishmaniasis. Correct post-ranslational modifications and protein folding of antigens

ay be important not only for the induction of neutralizingntibodies but also for the development of protective CD4+

cell responses [49].We recently found that LiESAp-MDP vaccine could elicit

otent activation of the immune system in canines [29]. Wehowed that vaccination with two subcutaneous injectionst a 3-week interval of 100 �g LiESAp in formulation withDP fully protected beagle dogs experimentally infectedith 108 virulent L. infantum promastigotes inoculated eithermonths or 8 months after vaccine administration [30].oreover, the use of LiESAp-MDP vaccine to treat dogsith visceral leishmaniasis resulted in a long-lasting clini-

al improvement [50]. Here, we evaluate the efficacy of aouble dose of the combination of LiESAp as vaccine anti-en and MDP as an adjuvant against visceral leishmaniasisn naturally exposed dogs in various endemic areas of theouth of France in a double-blind randomised efficacy field

rial, where a large-scale dog population was prospectivelytudied for a 2-year period, after two seasons of sand flyctivity. Two subcutaneous injections of 100 �g LiESAp sup-lemented with 200 �g MDP 3–4 weeks apart, complyingith a basic vaccination regimen and a booster injection 1ear after the basic were safe and well tolerated and protectedogs against natural infection in field trial conditions, withn efficacy rate of 92%. The results at 2 years of follow-uphowed that highly increased levels of total anti-leishmanialgG antibodies were exclusively detected in control dogs.his study revealed a cumulative serological incidence of

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Please cite this article in press as: Lemesre J-L, et al., Long-lasting protecvaccine in endemic areas of France: Double-blind randomised efficacy fi

.8% in placebo dogs found positive for IgG antibodies react-ng in IFAT. This value does not differ from the usual expectedate in endemic areas of the South of France, where an averagerevalence on order of 4–8% of the total canine population

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as been reported, with values exceeding 30% in some loca-ions [15]. Interestingly, placebo-injected dogs developed aignificantly higher proportion of strong seroconversion asso-iated with the appearance of suspicious clinical symptoms,hich are strong markers of infectiousness. Indeed, in dogs,igh levels of specific IgG antibodies have been related toathophysiological disorders and the active phase of the dis-ase [51,52]. Of interest also was the demonstration thatiESAp-MDP vaccine was successful in controlling para-ite burden in bone-marrow aspirates from vaccinated dogsnd significantly decreased the rate of L. infantum infec-ion from 6.86% to 0.61% in dogs. The inclusion of healthyeronegative dogs to the study even though they were PCRositive at the bone marrow examination was motivated byhe recent demonstration that a high proportion of dogs

ay show such type of “subpatent infection” during sev-ral years, without developing positive serology and culture,r even showing conversion to PCR-negative bone marrow33]. Moreover, the diagnosis of canine visceral leishmania-is by veterinarians is traditionally performed through simplend non-invasive procedures (quantitative serological testsnd clinical examination) whereas more aggressive diagnos-ic methods, oriented towards the detection of the parasitesere rarely used. Based on leishmanial DNA and/or parasiteetection in bone marrow aspirates, the cumulative frequencyf Leishmania infection was found more than 11-fold highern control animals than in vaccinees. Given recent stud-es that clearly indicate that asymptomatic dogs may act aseservoirs for parasite transmission to phlebotomine sandflies38,39,53], an effective vaccine against canine leishmaniasisas to achieve high protection from leishmanial infection.ndeed, a vaccine that only prevents severe disease may notecessarily be useful in the control of zoonotic Leishmaniaransmission. It should be noted that a comparison by meansf the PCR analysis for Leishmania DNA detection in bone-arrow samples at the beginning and at the end of the study

howed that the use of LiESAp-MDP vaccine significantlyeduced the rate of leishmanial infection in included vacci-ated dogs with bone marrow-PCR positive. Altogether, ouresults point out the remarkable potential of this formulationot only to reduce the rate of infection but also to reducehe rate of transmission, which eventually should result in aower rate of incidence in humans as well.

An interesting point in this study is the demonstration oflong-term establishment of an antigen-specific protective

mmune response in vaccinated dogs. Clear differences inoth humoral and cellular immune responses between theogs that received vaccine and those that received placeboere also established by the results of their immune sta-

us. These differences were indicative of different regulatoryechanisms in protected and non-protected animals. Anal-

sis of antibody isotype responses provides a convenient

tion against canine visceral leishmaniasis using the LiESAp-MDPeld trial, Vaccine (2007), doi:10.1016/j.vaccine.2007.02.083

urrogate marker of Th1 and Th2 CD4+ T cell differen- 703

iation [20,54]. We therefore analysed the specific isotype 704

esponse against LiESAp vaccine throughout the duration 705

f the study in vaccinated and control dogs. We reasoned 706

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hat determination of the antibody isotype profile in vac-inated dogs should provide an indication of the LiESApaccine impact on Th subset development. It is noteworthyhat a very significant increase in IgG2 antibodies to LiESApas observed in practically all the vaccinated animals after

he basic vaccination and after the booster, whereas only.3% of the dogs that received placebo exhibited positiventi-LiESAp IgG2 reactivity. These sera showed a constantesponse mainly against the 54-kDa antigen, as previouslyescribed with sera of LiESAp-MDP-vaccinated animals inn experimental trial [30]. This result is strong evidence thathe IgG2 increased response against an excreted/secreted4-kDa antigen was highly correlated with LiESAp vac-ine protection. Interestingly, this response was totally absentn naturally infected dogs, suggesting that dogs unable toontrol L. infantum infection did not generate this differen-ial isotype humoral immune response. These results appearo support those of several authors indicating that in dogshe differential IgG1/IgG2 response is a better indicator ofhe outcome of infection than total IgG [54,55]. It maye that IgG2-increased responses are associated with thexpansion of the Th1-type T cells producing IFN-� andL-2 cytokines, promoting resistance/protection to infection.his provides evidence that monitoring the LiESAp-specific

gG2 increased response in dogs might be an indirect andasy way to distinguish sera samples of vaccinated ornfected dogs in large-scale studies in the field. This fur-her argues for the LiESAp potential in regards to vaccineevelopment.

To further characterise the protective immune responseenerated in vaccinated dogs, we used the recently describedx vivo infection model of canine macrophages [29,30].illing of Leishmania parasites by activated macrophages

s critical in resolving an infection with an intracellularathogen. Canine monocyte-derived macrophages (CM-DM)ere first infected with L. infantum promastigotes and

o-cultured with autologous lymphocytes. After 72 h of incu-ation, we evaluate the CM-DM leishmanicidal capacityn vaccinated and control dogs. Enhanced production ofFN-� and NO, correlated with high leishmanicidal activity,ere exclusively evidenced in vaccinated dogs and persisted

hroughout a long post-immunisation period after both basicaccination and booster. These results indicate that incu-ation of L. infantum-infected CM-DM with autologousymphocyte of vaccinated animals led to the proliferation ofFN-�-producing LiESAp-specific T cells rather than Th2-L-4-induced expansion, as determined by the production ofhigh level of IFN-� and a low level of IL-4 in supernatantsf co-cultured cells. An increase in IFN-� resulted in activa-ion of canine macrophages that produced sufficient amountsf NO to display intracellular Leishmania killing. Using annhibitor of NO production, we show that NO contributes

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Please cite this article in press as: Lemesre J-L, et al., Long-lasting protecvaccine in endemic areas of France: Double-blind randomised efficacy fi

o the anti-leishmanial activity, which is mediated by the l-rginine-NO metabolic pathway. More recently, we showedhat the leishmanicidal effect observed in canine activated

acrophages was the consequence of intracellular amastig-

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tes undergoing cell death and exhibiting a classical featuref apoptosis: oligonucleosomal DNA fragmentation [29,56].ogether, our results demonstrate that the LiESAp-MDP vac-ine is able to elicit a protective cytokine phenotype in theain reservoir of zoonotic VL. A significant, stable and long-

asting Th1-type cell response was induced in vaccinatedogs. Furthermore, our results are in agreement with the viewhat the inability to generate a Th1-cell response rather thanhe presence of a Th2 response may be responsible for suscep-ibility [20,57,58]. Monitoring these parameters in vaccineesan be an indirect, easy and useful way of following therotective cellular immune response achieved in dogs immu-ised with the LiESAp-vaccine in large-scale studies in theeld.

Overall, our results support the view that low rate of anti-eishmanial antibodies in blood, absence of external clinical

anifestation, absence of parasites and leishmanial DNA in aone-marrow sample, anti-LiESAp IgG2 increased responsend enhancement of NO-mediated anti-leishmanial activityf canine macrophages in response to higher IFN-� produc-ion by specific LiESAp T cells were highly correlated withellular immune reactions related to a protective immuneesponse and were indicative of the non-infectious conditionf the vaccinated dog.

We conclude that the LiESAp/MDP vaccine induced aignificant, long-lasting and strong protective effect againstanine visceral leishmaniasis both in experimental infectednd in naturally exposed dogs.

cknowledgements

This investigation received financial support fromNVAR (Agence Francaise de l’Inovation) Provence-Alpes-ote d’Azur (PACA). We are very grateful to the Nationaleterinary School of Lyon (ENVL). The technical assistancey R Kovacic is gratefully acknowledged. We thank dog own-rs for their collaboration and the veterinarians that activelyarticipated in the efficacy field trial: Drs Bassine Y., Baroche., Berardi L., Berthie M., Bertrand A., Bruchon-Hugnet C.,e D., Chiocca S., Deveze M., Duffoset-Gauthier I., Duval., Escoffier K., Guardiola J., Guirard L., Hubert B., Huguet

., Jean E., Laborde C., Lacombre B., Laumonier M., Mathieu., Molho M., Negrel A., Petrau-Gay C., Peyre De Fabrigues., Pfister G., Puech M. P., Rabuel R., Riviere L., Saunier F.,egard F., Simon J.P. and Ville-Fiacre C.

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