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Vaccine 28 (2010) 53–62

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

ecombinant Leishmania tarentolae expressing the A2 virulence gene as a novelandidate vaccine against visceral leishmaniasis

mir Mizbania,b, Tahereh Taheria, Farnaz Zahedifarda, Yasaman Taslimia, Hiva Azizi a,ayhan Azadmaneshc, Barbara Papadopouloud, Sima Rafati a,∗

Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, IranDepartment of Biotechnology, University College of Science, University of Tehran, Tehran, IranHepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, IranResearch Centre in Infectious Diseases, CHUL Research Centre and Department of Microbiology and Immunology, Faculty of Medicine,aval University, 2705 Laurier Blvd., Quebec (QC), Canada G1V 4G2

r t i c l e i n f o

rticle history:eceived 22 August 2009eceived in revised form1 September 2009ccepted 25 September 2009vailable online 8 October 2009

a b s t r a c t

Visceral leishmaniasis is the most severe form of leishmaniasis. To date, there is no effective vaccineagainst this disease. Many antigens have been examined so far as protein- or DNA-based vaccines, butnone of them conferred complete long-term protection. The use of live attenuated vaccines has recentlyemerged as a promising vaccination strategy. In this study, we stably expressed the Leishmania donovaniA2 antigen in Leishmania tarentolae, a non-pathogenic member of the genus Leishmania, and evaluated itsprotective efficacy as a live vaccine against L. infantum challenge. Our results show that a single intraperi-

eywords:isceral leishmaniasisecombinant Leishmania tarentolaeive vaccine2 gene

mmune response

toneal administration of the A2-recombinant L. tarentolae strain protects BALB/c mice against L. infantumchallenge and that protective immunity is associated with high levels of IFN-� production prior andafter challenge. This is accompanied by reduced levels of IL-5 production after challenge, leading to apotent Th1 immune response. In contrast, intravenous injection elicited a Th2 type response, character-ized by higher levels of IL-5 and high humoral immune response, resulting in a less efficient protection.All together, these results indicate the promise of A2-expressing L. tarentolae as a safe live vaccine against

visceral leishmaniasis.

. Introduction

Protozoan parasites of the genus Leishmania are causative agentsf a broad spectrum of diseases, collectively known as leishmania-is. The clinical symptoms of the disease range from asymptomaticelf-healing cutaneous lesions (CL, caused by Leishmania major, L.ropica, and L. mexicana species) to mucocutaneous leishmaniasiscaused by L. braziliensis) and severe visceral (VL) infections (causedy L. infantum, L. donovani, and L. chagasi) [1,2]. Currently, over 12illion people in 88 countries are infected with this parasite, 350illion are at the risk of infection worldwide and 1.5–2 million

ew cases are reported every year [3–7]. VL is the most severe sys-emic disease among the three main categories of leishmaniasis andffects 500,000 people every year [6]. Moreover, VL has emerged as

n opportunistic infection in HIV-1 infected patients in many partsf the world [8–10].

Currently, there is no effective vaccine against leishmaniasis andontrol of the disease is almost confined to chemotherapy. There are

∗ Corresponding author. Tel.: +98 21 66953311x2112; fax: +98 21 88742314.E-mail address: s rafati@yahoo.com (S. Rafati).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.09.114

© 2009 Elsevier Ltd. All rights reserved.

only a limited number of drugs available and each has its own dis-advantages. Conventional drugs require long-term administrationperiods and often induce serious side-effects due to their toxicity[11,12]. In addition increasing incidence of drug-resistant strainshas hampered the control of the disease by chemotherapy [13–17].Therefore, much attention has been given to developing effectivevaccines.

Although induction of lifelong protection against reinfection inrecovered people demonstrates that a protective vaccine can beachieved, an effective vaccine against human leishmaniasis hasyet to be discovered [18]. However, several vaccination strategieshave been tried against experimental leishmaniasis, with partic-ular emphasis on their efficacy against CL rather than VL [19,20].First-generation anti-leishmanial vaccines, composed of killed par-asites, were examined as an alternative but none has provided fullprotection thus far [18]. In addition, several antigens such as GP63[21,22], GP46 [23], p36/LACK [24,25], CPB and CPA [26], LD1 [27],

PSA-2 [28], TSA/LmSTI1 [29,30], PFR2 [31], HASPB1 [32], LeIF [33],LCR1 [34], and M2 [35] have been tested as recombinant proteins orDNA vaccines, but they have given only partial protection, and/orneed some clinically unacceptable adjuvants to induce proper Th1response in humans [36–38]. Also, it seems unlikely that a single

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ntigen can elicit a fully protective immunity against a complexarasite like Leishmania [36]. On the other hand, there is increas-

ng evidence that presence of a small number of live parasites isecessary to maintain durable immunity [39,40], and the only wayo meet this requirement is by using attenuated live vaccines. Liveaccines can simulate the natural course of infection more closely,resent a rich repertoire of antigens to the host immune system inhe way that the wild type parasite does and persist for a longerime, leading to a more potent and durable immunity. Using livevirulent organisms as a vaccine has proven to be more efficacioushan traditional subunit vaccines [41].

Attenuated strains using either long-term in vitro cultures [42]r selection for temperature sensitivity [43] or chemical mutage-esis [44], and parasite culture under drug pressure [45] are notasily applicable to human use, because there is always a riskf reversion of the organism to its virulent state. The advent ofene transfection technologies for Leishmania has made it possi-le to obtain attenuated organisms by targeting genes importantor virulence or intracellular survival of the parasite, such as dihy-rofolate reductase thymidylate synthase (DHFR-TS) [46], cysteineroteinase genes [47], HSP100 heat shock protein [48], SIR2 [49],PG2 [50], biopterin transporter BT1 [51], and the trypanothioneeductase [52]. Using intrinsically non-pathogenic species is also aromising approach, exemplified by BCG vaccine against Mycobac-erium tuberculosis infection.

Among different species of Leishmania, Leishmania tarentolae islizard parasite which is non-pathogenic to humans. The parasite

an differentiate into amastigotes, but is not able to persist longnough within mammalian macrophages [53,54]. Recent studiesave shown that it can be used as a live vaccine against L. dono-ani due to its capability to efficiently target antigen-presentingells, induce maturation of dendritic cells, and elicit a protectiveh1 immune response [53]. Interestingly, a recombinant L. taren-olae expressing HIV-1 Gag protein has been used as a candidateIV-1 vaccine and shown to induce strong cell-mediated immu-ity in mice and to decrease HIV-1 replication in an ex-vivo system55], suggesting that this species can be used as a promising live-ectored vaccine against intracellular pathogens.

We have recently shown that among some important virulenceactors, A2 is not present in L. tarentolae [56]. A2 was first identifiedn L. donovani as a gene family that is expressed only in the amastig-te stage [57]. A2 proteins are comprised mainly of a 10-amino acidepeated sequence and range in size from 45 to 110 kDa, depend-ng on the number of repeats [58]. A2-specific antibodies have beenetected in 90% of sera samples from VL patients, which confirmshat A2 is expressed in the human host [59]. In addition, a signif-cant protection against L. donovani and L. amazonensis infectionsssociated with both humoral and cellular immune responses haseen obtained when using A2 as a recombinant protein or a DNAaccine in BALB/c mice, and more recently in dogs [60–65].

In this study, we engineered a recombinant L. tarentolae express-ng the L. donovani-specific A2 protein and used it as a live vaccinegainst L. infantum infection in BALB/c mice. We provide evidencehat intravenous (i.v.) and to a larger extent intraperitoneal (i.p.)mmunization with the recombinant L. tarentolae-A2 strain elicitedavorable immune responses and significant levels of protectivemmunity against L. infantum infectious challenge.

. Materials and methods

.1. DNA constructs

The A2 gene (with 3′UTR sequences) was amplified fromKSNEOA2-1 vector [58] (kindly provided by Dr. Greg Mat-

ashewski, Microbiology and Immunology Department, McGill

28 (2010) 53–62

University, Montreal, Quebec, Canada) by hot-start PCR (Hot-Star kit, Qiagen) using the following primers: (forward,5′-TTGAAGCTTACCGAGCACAATGAAGATCC-3′); and (reverse,5′-AACAAGCTTAGCAGAGGAAGTCAGCAAGG-3′) including HindIIIrestriction sites indicated as underlined. For amplification, DNAwas denatured at 95 ◦C for 5 min, 30 cycles of 94 ◦C for 30 s, 61 ◦C for30 s and 72 ◦C for 90 s, and a cycle of 72 ◦C for 10 min. The amplifiedfragment was cloned into the pDrive cloning vector (Qiagen). Aftersequence confirmation, the A2 fragment was subcloned into theHindIII site of vector pNEO-GFP [66] upstream of the GFP geneto generate pNEO-GFP-A2, and the correct insert orientation wasconfirmed by restriction analysis.

2.2. Parasite growth and transfections

The L. tarentolae Tar II (ATCC 30267) parasites were grownat pH 7.2 and 26 ◦C in M199 medium (Sigma) supplementedwith 10% heat-inactivated fetal calf serum (FCS, Gibco), 40 mMHEPES, 0.1 mM adenosine, 5 �g/ml hemin and 50 �g/ml gentam-icin. For transfection, 4 × 107 log-phase parasites were washed andre-suspended in 300 �l of electroporation buffer (21 mM HEPES,137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose; pH 7.5)containing 15 �g of plasmid DNA, stored on ice for 10 min, and elec-troporated using Bio-Rad Gene Pulser Ecell at 450 V and 500 �F asdescribed previously [67]. Transfectants were selected with G418(Sigma).

2.3. RNA extraction and reverse-transcription PCR

For L. tarentolae, RNA samples were extracted from both pro-mastigote and amastigote-like forms of the parasite. L. tarentolaeamastigote-like forms were prepared by culturing the parasitesat pH 4.5 and 37◦C for 10 h. In the case of L. infantum, amastig-otes were obtained by infecting mouse peritoneal macrophageswith stationary-phase parasites. Briefly, peritoneal exudate cellsfrom naïve BALB/c mice were used as a source of residentmacrophages (MQ). The peritoneal cavity was washed with 5 mlcold RPMI 1640 (Sigma) media, and then the media containingperitoneal macrophages was recovered. Cells were cultured inRPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 50 nM2-mercaptoethanol, 10 mM HEPES and 40 �g/ml gentamicin ata density of 106 cells/well, and incubated at 37 ◦C in 5% CO2for 24 h. In order to infect MQ, the stationary-phase L. infantumpromastigotes were added at the ratio of 10:1 parasites ver-sus macrophages. The cultures were washed after 4 h to excludefree parasites and then incubated for 5 h. Infected attached MQswere collected using cold PBS. RNA was extracted using RNeasykit (Qiagen) and treated with RNase-free DNase for 30 min at37 ◦C to eliminate any remaining DNA. cDNA synthesis was per-formed using the Qiagen Omniscript RT Kit from 1 �g of RNA.To detect the A2 cDNA, two primers were designed to amplifya 154-bp region within the A2 3′-UTR starting at the proximityof A2 stop codon (AUF: 5′-GCAAGAGCAACAGCAGAGC-3′; AUR: 5′-CTTCCAACCTTGCGACTTTC-3′), and a hot-start PCR (Qiagen HotStarKit) was performed at 95 ◦C for 5 min, 40 cycles of 95 ◦C for 30 s,55 ◦C for 30 s and 72 ◦C for 45 s, and a cycle of 72 ◦C for 10 min.

2.4. Northern blot hybridization

Northern blot analysis was performed with the DIG System(Roche Applied Science) according to instructions of the manu-

facturer with minor modifications. For each sample, 3 �g of totalRNA was denatured, blotted onto Hybond + membrane (Roche), andUV cross-linked to the membrane with the intensity of 0.12 J/cm2.Using the “PCR DIG Probe Synthesis Kit” (Roche Applied Science), aDIG-labeled probe was prepared by PCR amplification of a 1.7 kb

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A. Mizbani et al. / V

equence containing A2 ORF and part of its 3′-UTR using ADFnd ADR primers. After incubating in prehybridization buffer (10%-lauroylsarcosine, 0.02% SDS (w/v), 10% Blocking reagent, 0.02%enhardt solution, 2.5% Herring sperm in SSPE 5×) at 65 ◦C for 5 h,

he probe was added and incubation continued for 16 h. After sev-ral washing steps using 2× SSPE, anti Dig AP conjugated antibody1:5000 in blocking buffer) were used and incubated for 30 mint room temperature. Hybridized probes were detected usingolor substrate solution according to manual of Roche detectionit.

.5. Fluorescence microscopy and flow cytometry analysis

Both promastigote and amastigote forms of L. tarentolae werexamined for GFP expression. Promastigotes were centrifuged in000 rpm for 15 min and after washing once with PBS, cells were re-uspended in Hayman solution and mounted on microscope slides.o obtain amastigotes, mouse peritoneal macrophages were cul-ured on glass coverslips for 24 h to attach to the surface, and thennfected with stationary-phase L. tarentolae as described above.overslips were examined for GFP-expressing intracellular para-ites 24 h post-infection. Green fluorescence was excited with a88 nm argon-ion laser and was captured using a combination of510–530 nm bandpass emission filter. For flow cytometric mea-

urement, both non-infected and infected macrophages with eitherNEO-GFP or pNEO-GFP-A2-transfected L. tarentolae were pre-ared as described above. After cell detachment on ice and washingith PBS, the infection rate was monitored using at least a total of

0,000 events counted by PAS flow cytometer (Partec, Germany)8 h post-infection. The results were analyzed using Flowmax soft-are (Partec, Germany).

.6. Western blot analysis

Promastigote, amastigote, and amastigote-like forms of the par-sites were prepared as described above. Cells were then harvestedy centrifugation at 3000 rpm for 15 min and washed in PBS. Theellets were immediately lysed in 2× SDS-PAGE sample buffer4.5 mM Tris–HCl, pH 6.8, 10%, v/v glycerol, 2%, w/v SDS, 5%, v/v-mercaptoethanol, 0.05%, w/v bromophenol blue) on ice and thenoiled for 5 min. Samples were then loaded on a 15% SDS-PAGE.he gels were transferred onto a nitrocellulose membrane usingBio-Rad wet blotting system and incubated with blocking solu-

ion (PBS with 0.15% Tween 20 and 2.5% BSA) for 1 h. Washingas performed 3 times with 0.15% Tween 20 in PBS, and blotsere incubated overnight with anti-A2 monoclonal antibody C9

kindly provided by Dr. Greg Matlashewski) as the first antibodyt a 1:100 dilution. The membranes were washed three timesnd incubated for 90 min with peroxidase-conjugated goat anti-ouse IgG (1:1000, Sigma) as secondary antibodies. Unbound

econdary antibodies were removed by washing as describedbove. Diaminobenzidine tetrahydrochloride (DAB, Sigma) weresed as the substrate to detect the desired bands of the pro-eins.

.7. Immunization and challenge infection

Six groups of 8–10-week-old female BALB/c mice (n = 8) werebtained from the breeding stock maintained at the Pasteur Insti-ute of Iran. Two different routes of immunization, including i.p. and.v. were chosen. In the case of i.p. immunization, total number of

× 106 stationary-phase promastigotes were used. In the case of i.v.dministration, 2 × 108 stationary-phase L. tarentolae promastig-tes were injected. Groups 1 (i.v. route) and 2 (i.p. route) weremmunized with L. tarentolae expressing the GFP only and groups

(i.v. route) and 4 (i.p. route) with recombinant L. tarentolae-A2.

28 (2010) 53–62 55

The fifth group received PBS only and the sixth group correspondsto naïve animals. Six weeks later, animals were challenged with107 virulent stationary-phase L. infantum promastigotes throughthe lateral tail vein. The parasite burden in the spleens and liverswas quantitatively determined 4 weeks after challenge by real timePCR.

2.8. Determination of antibody responses

Sera were taken from all groups before and 4 weeks after chal-lenge. Total IgG, IgG1 and IgG2a were measured using SolubleLeishmania Antigen (SLA) as coating antigen and performed byELISA. Briefly, 96-well plates (Greiner) were coated with 10 �g/mlSLA in PBS and incubated at 4 ◦C overnight. Then plates wereblocked with 100 �l of 1% BSA in PBS for 2 h at 37 ◦C. 100 �l ofserum samples (1:500 dilutions) was then added and incubated for2 h at 37 ◦C. After three washes, horseradish peroxidase-conjugatedgoat anti-mouse IgG (1:5000, Sigma) or biotinylated rabbit anti-mouse IgG1 (1:10,000) or IgG2a (1:1000, Zymed) were added andincubated for 2 h at 37 ◦C, and then plates were incubated for1 h at 37 ◦C with streptavidine-conjugated horseradish peroxidase(BRL, Gaithersburg, MD). After four washes, plates were devel-oped with O-phenylenediamine (OPD) as substrate. Reactions werestopped with 4N sulfuric acid and the absorbance was measuredat 492 nm.

2.9. Cytokine assay

Four mice from each group were sacrificed before and also 4weeks after challenge and spleens were homogenized. After lysisusing ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3 and 0.1 mMNa2EDTA), splenocytes were washed and re-suspended in com-plete RPMI medium (RPMI-1640 supplemented with 5% FCS, 1%l-glutamine, 1% HEPES, 0.1% 2ME, 0.1% gentamicin). Cells werethen seeded at a density of 2 × 105 cells/well in the presence of SLA(10 �g/ml) or medium alone. Concanavalin A (Con A; 5 �g/ml) wasalso used in all experiments as the positive control. Plates wereincubated for 5 days at 37 ◦C in 5% CO2 humidified atmosphere.The IFN-� and IL-5 production in supernatants of splenocyte cul-tures was measured by sandwich ELISA kits (R&D, Minneapolis, MN,USA), according to the manufacturer’s instructions. All experimentswere run in duplicates.

2.10. Real time PCR

Real time PCR was used to quantify parasite burden inspleens and livers 4 weeks after challenge. Four mice from eachgroup were sacrificed and genomic DNA was extracted from10 mg of spleen and 30 mg of liver tissues using DNeasy Blood& Tissue kit (Qiagen). Two set of primers were designed byusing Primer Express software version 1.3.1 (Applied Biosys-tems), targeting a region of kinetoplastid minicircle DNA of L.infantum (forward: 5′-GCGCATACTGCAGTGAATTGAA-3′; reverse:5′-CTACCCGGAGGACCAGAAAAGT-3′). Absolute copy number of thetarget sequence was measured using Applied Biosystem 7500 realtime PCR system. L. infantum genomic DNA was used in 10-folddilutions corresponding to 2 × 105 parasites and used in real timePCR to draw the standard curve. For quantification of parasites intissues, 600 ng of DNA was subjected to the reaction containing

tiFast SYBR Green Master Mix in total volume of 25 �l. All reactionswere performed in duplicate. Conditions for PCR amplification wereas follows: 95 ◦C for 5 min; 40 cycles consisting of 95 ◦C for 15 s, 60◦

C for 20 s, and 72 ◦C for 40 s. Specific amplification of the targetregion was confirmed by gel electrophoresis of the PCR products.

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.11. Statistical analysis

All data comparisons were examined for significance by one-ay ANOVA and Student’s t-test using ‘SigmaPlot’ software.ifferences were considered statistically significant when P < 0.05.

. Results

.1. Generation of recombinant L. tarentolae expressing the A2ene

Stably expressing A2 L. tarentolae cells were generated byntroducing the pNEO-GFP-A2 vector into L. tarentolae by elec-

ig. 1. Expression of A2 in the recombinant L. tarentolae strains as well as in L. infantum. L.ike parasites were obtained by culturing the promastigotes at 37 ◦C and pH 4.5 for 10 h anPCR reaction on cDNA samples with primers targeting a 154-bp region within A2 3′-UTromastigote and amastigote-like forms of transfected parasites. (B) Northern dot blot aarasites, with a DIG-labeled probe consisted of a 1.7-kb region of A2. A2 is expressed in bmastigote stage. (C) Western blot analysis for A2 protein. Protein samples were preparnd amastigotes were used as negative and positive controls, respectively. Results show thife stages, in contrast to L. infantum, which expresses A2 only in amastigotes. (D) Express

ouse intraperitoneal macrophages (right) 24 h post-infection.

28 (2010) 53–62

troporation, as indicated in Materials and methods. To confirmthe expression of A2 by L. tarentolae at the mRNA level, RNAwas extracted from promastigote and amastigote cultures andused for cDNA synthesis. A PCR reaction using primers to amplifya short sequence within the 3′-UTR of A2 mRNA resulted ina PCR product of the expected size, which confirms the A2mRNA expression in the recombinant strain (data not shown).To determine whether A2 expression is stage-specific or not,RNA was extracted from promastigote and amastigote-like forms

L. tarentolae A2-recombinant parasites (both recombinant par-asites transfected with pNEO-GFP-A2 and pKSNEO-A2-1) andreverse transcribed. PCR reaction was performed using thecDNA with exactly the same conditions mentioned above. Incontrast to L. infantum where A2 is specifically expressed in

tarentolae was transfected with either pKSNEO-A2-1 or pNEO-GFP-A2. Amastigote-d RNA was extracted from these cells as well as from promastigotes. (A) Results ofR. L. infantum amastigote was used as positive control. The A2 mRNA is present innalysis of total RNA extracted from L. tarentolae promastigote and amastigote-likeoth forms of L. tarentolae, in contrast to L. infantum, which expresses A2 only in the

ed and subjected to Western blot analysis as described. L. infantum promastigotesat A2 is expressed in transfected L. tarentolae in both promastigote and amastigote

ion of GFP by recombinant L. tarentolae-A2 promastigotes (left) and amastigotes in

A. Mizbani et al. / Vaccine 28 (2010) 53–62 57

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ig. 2. Percentage of mouse peritoneal macrophages infected with transfected L. tar8 h post-infection. Uninfected macrophages were used as a negative control (A). In. tarentolae pNEO-GFP. The experiment was repeated three times and similar resul

mastigotes, in L. tarentolae pNEO-GFP-A2 and pKSNEO-A2 trans-ectants A2 is expressed similarly in both developmental stagesFig. 1A).

Consistently, Northern blot analysis using an A2-specific probehows that A2 is expressed at approximately equal level in bothorms of the A2-transfected L. tarentolae, in contrast to L. infan-um which expresses A2 only in amastigotes, as demonstrated inig. 1B. Both promastigote and amastigote-like forms of L. tarentolaes well as L. infantum were also examined for A2 protein expression.otal protein was obtained and subjected to Western blot analysiss described in Section 2. Results presented in Fig. 1C show that2 protein is expressed in both promastigote and amastigote-like

orms of transfected L. tarentolae. Overall, the results of RT-PCR,orthern blot hybridization and Western blot analysis are consis-

ent with the exogenously provided A2 gene in L. tarentolae beingonstitutively expressed. Interestingly, the 3′UTR of the A2 mRNAs not able to promote amastigote-specific regulation, as it does in. infantum. Although further investigation is required to draw anyefinitive conclusions, we hypothesize that the mechanism or theactors responsible for stage-specific regulation of the A2 gene are

issing or not functional in L. tarentolae.Expression of GFP in transfected parasites in both promastigote

nd amastigote stages was confirmed by fluorescence microscopyFig. 1D). Mouse intraperitoneal macrophages were infected

ith GFP-expressing L. tarentolae and the infectivity rates of

he recombinant L. tarentolae-A2 parasites were compared to L.arentolae-GFP by measuring fluorescence levels by FACS. As shownn Fig. 2, recombinant L. tarentolae-GFP-A2 (48.60%) were close to-fold more infective than L. tarentolae-GFP (27.44%).

e with either pNEO-GFP (B) or pNEO-GFP-A2 (C), as determined by flow cytometryity rates are almost doubled in the case of A2-expressing L. tarentolae compared toe obtained.

3.2. Immunization with live recombinant L. tarentolae A2protects mice against L. infantum challenge

We first examined whether immunization with the A2-recombinant L. tarentolae was protective against L. infantuminfectious challenge. Different groups of mice were immunizedeither with recombinant L. tarentolae parasites expressing GFP onlyor with L. tarentolae-GFP-A2 via the i.p. or i.v. routes and challengedwith virulent stationary-phase L. infantum 6 weeks later. The degreeof protection against infection was determined by quantifying para-site burden in the spleen and the liver by real time PCR 4 weeks afterchallenge. As shown in Fig. 3, immunization with A2-recombinant L.tarentolae via the i.p. route drastically (p < 0.05) reduced the infec-tion levels in both the liver (Fig. 3A) and the spleen (Fig. 3B) incontrast to the naïve control mice (group 5) (p = 0.008 and 0.01,respectively). Indeed, almost no parasites were detected in thespleens of mice vaccinated with the A2-recombinant L. tarentolae(Fig. 3B). Interestingly, this decrease in parasite burden was morepronounced in the case of A2-recombinant L. tarentolae-immunizedmice than in mice vaccinated with L. tarentolae expressing theGFP only. However, in contrast to liver, no significant differenceswere observed between the spleen parasite burdens in these twogroups when the vaccine regimens were administered by the i.v.route (p = 0.13). Results of immunization with A2-recombinant

L. tarentolae indicate that there is a highly significant differencebetween the levels of protection elicited depending on the routeof immunization, i.p. vs. i.v. In this case, the i.p. immunizationseems to result in higher levels of protection as compared to thei.v. immunization. These data emphasize the importance of the

58 A. Mizbani et al. / Vaccine

Fig. 3. Protection levels of immunized BALB/c mice challenged with L. infantum.Groups of BALB/c mice (n = 8) were immunized with GFP recombinant or A2-recombinant L. tarentolae via i.p. or i.v. routes. Groups 1 (i.v. route) and 2 (i.p. route)were immunized with L. tarentolae-GFP and groups 3 (i.v. route) and 4 (i.p. route)with recombinant L. tarentolae expressing A2. Group 5 received PBS and group 6corresponds to naïve animals. Six weeks after immunization, mice were challengedi 6

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.v. with 5 × 10 L. infantum promastigotes. Four mice were killed from each groupweeks after challenge and parasite burden in the liver (A) and the spleen (B)as quantified by real time PCR. The data are represented as the mean ± S.E. and

xperiment was performed two times.

mmunization route in stimulating the right branch of the immuneesponse.

.3. Protective immunity is associated with a potent Th1 response

The above experiments demonstrate that immunization withecombinant L. tarentolae expressing A2 confers a significantrotection against L. infantum infection in mice. We thereforeharacterized the type of immune response contributing to protec-ive immunity. We first focused on the cellular immune responseecause it is well established that high levels of protection againsteishmania infections require the presence of a strong Th1 cell-ediated immunity [68–70]. We therefore determined the level

f IFN-� and IL-5 production in cultures of splenocytes isolatedrom immunized and control mice in response to L. infantumLA as a recall antigen. Levels of IFN-� and IL-5 were comparedetween different groups after the infectious challenge as shown

n Fig. 4. Splenocytes from mice immunized via the i.p. route with

he recombinant L. tarentolae A2 produced significantly higherevels (∼1.7-fold) of IFN-� (p = 0.001) after challenge in compar-son to mice immunized with L. tarentolae expressing GFP onlyFig. 4A), while similar levels of IL-5 were detected in both groupsFig. 4B). Furthermore, significantly higher levels of IL-5 were gen-

28 (2010) 53–62

erally observed after i.v. administration as compared to the i.p.immunization (p = 0.02), which along with IFN-� data confirm thati.p. injection leads to a stronger Th1 response. Immunization withboth GFP recombinant and A2-recombinant L. tarentolae by the i.p.route resulted in a higher IFN-� production levels in comparisonto the i.v. immunization, further stressing the substantial role ofthe route of immunization in the type of the immune response(Fig. 4A and B). Moreover, the post-challenge IFN-� production wasincreased significantly (p = 0.002) in comparison to the IFN-� lev-els before challenge in the group immunized with A2-expressing L.tarentolae via the i.p. route (Fig. 4A).

3.4. Immunization via the i.v. route induces higher level ofantibody production against L. infantum SLA

The level of total IgG, IgG1 and IgG2a antibodies against SLAfrom both L. infantum and L. tarentolae was determined by ELISAbefore and 4 weeks after challenge in all groups. As shown in Fig. 5,although IgG2a levels are similar in all groups immunized with L.tarentolae prior or after challenge with L. infantum, immunizationthrough the i.v. route leads to considerably higher levels (2–4-fold)of IgG1 antibodies in comparison to the i.p. route. Interestingly, incontrast to groups 1 and 2 (immunized with L. tarentolae-GFP, i.v.and i.p. respectively), the level of IgG1 did not increase in groups3 and 4 (immunized with L. tarentolae-GFP-A2, i.v. and i.p. respec-tively) both before and after challenge. In other words, only i.p.immunization with L. tarentolae has kept the IgG1 production atlow levels after challenge (although more pronounced in group 4in comparison to group 3). This is consistent with cytokine produc-tion which shows that IFN-� levels increase also in the case of i.p.immunization with recombinant L. tarentolae A2.

4. Discussion

Despite several efforts towards a vaccine development againstvisceral leishmaniasis (VL), no effective vaccine currently exists forhuman use. It is well established that infection with Leishmaniaresults in a natural protective immunity against subsequent chal-lenges. Based on this fact, leishmanization has been used for manyyears to generate protection, however, at the expense of unpleasantclinical episodes in many cases. Therefore, the induction of protec-tive immunity using either inactivated or attenuated live vaccineswould be a significant step in the control of the disease [71], andpreclinical data suggest that this approach holds the most promisefor an anti-leishmanial vaccine [19]. Furthermore, different experi-mental studies both in human and mice support this strategy. Nylénet al. [72] showed that live parasites were more effective in inducingthe production of IFN-� from human PBMCs than killed parasites.It is also demonstrated that differences in early cytokine responsein mice inoculated with live and killed parasites are primarily dueto loss of infectivity [73]. Infection with live parasites (virulent andavirulent) leads to substantial activation and IFN-� production fromboth CD4+ and CD8+ T cells whereas inoculation of killed parasitesonly leads to activation of CD4+ T cells [73]. Another report also indi-cates that live parasites are important for maintaining Leishmaniaspecific effector cells that mediate immediate protection follow-ing secondary challenge in contrast to killed parasites, which aredefective in their ability to maintain effector as well as memorycells [74].

In this study, we generated a recombinant L. tarentolae strain

expressing the L. donovani A2 gene shown to play a role in viscer-alization [75] and evaluated its potential as a live vaccine againstVL. Interestingly, the A2-expressing recombinant parasites showedhigher macrophage infectivity in comparison to GFP-expressing L.tarentolae used as a control. Immunization (i.v. and i.p.) of BALB/c

A. Mizbani et al. / Vaccine 28 (2010) 53–62 59

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ig. 4. Cytokine levels detected in immunized mice after stimulation with L. infan2-recombinant L. tarentolae via the i.v. or the i.p. route and challenged with L. infa

ust before and also 4 weeks after challenge (A). IL-5 production levels were measureere done in duplicate.

ice with recombinant L. tarentolae A2 elicited a strong protectivemmunity against virulent L. infantum challenge, manifested by aramatic decrease in parasite burdens in the liver and the spleenf immunized mice. IFN-� production upon antigen stimulation

ig. 5. Analysis of humoral immune response in vaccinated mice. Groups of mice (n = 8)ix weeks later, mice were challenged with virulent L. infantum promastigotes. Levels ofeeks after challenge. Plates were coated with 1 �g/well of either L. infantum or L. tarentooled sera are represented as the mean ± S.E. Similar results were observed when ELISAuplicate.

oluble Leishmania Antigen (SLA). Mice were immunized with GFP recombinant or6 weeks later. IFN-� levels were measured in supernatants of splenocytes isolatedr challenge (panel B). The data are represented as the mean ± S.E. All measurements

indicates that protection is associated with a Th1 cell-mediatedimmunity accompanied by reduced levels of the Th2 type IL-5 pro-duction. Interestingly, although IFN-� production is also inducedin groups of mice immunized with wild type L. tarentolae, cytokine

were injected via the i.v. or i.p. routes with GFP or A2-recombinant L. tarentolae.total IgG, IgG1, and IgG2a were measured by ELISA just before challenge and also 4olae SLA and sera samples were diluted 1:500. Optical density values at 490 nm for

plates were coated with freeze/thaw (F/T) antigen. All samples were measured in

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evels are increased in the group immunized with the recombi-ant L. tarentolae A2 and especially when the vaccine regimen isdministered via the i.p. route. This highlights the effect of A2ntigen in eliciting a potent Th-1 type response. This is consis-ent with epitope mapping studies on A2, which have shown thateveral A2 epitopes located in the repetitive sequences are recog-ized by CD8+ T cells and also an epitope in its N-terminal part isecognized by IFN-�-producing Th1-associated CD4+ T cells [64].ur data are also in agreement with previously published reportssing A2 in a vaccine regimen as part of a recombinant protein61,63] or a DNA vaccine [65] or a live recombinant viral vec-or [64] demonstrating the potential of A2 antigen to induce bothumoral and cellular immune responses and protective immunity

n mice and more recently in vaccinated dogs [60]. Furthermore,ild type L. tarentolae has been previously shown to be protective

gainst L. donovani infection [53]. Recently, it has been reportedhat qualitative differences in early immune response with live par-sites contribute to their ability to induce protective immunity. Theongevity of effectors cells and differences in their ability to inducend maintain different subsets of memory cells are among theost important factors [73]. In fact, we propose that these factors

ave possibly played a role in potentiating the protective immu-ity elicited by A2 in the presence of the recombinant L. tarentolaeector.

Characterization of the antibody response also corroborated theytokine assay findings. It has been shown previously that Th2ellular response is associated with increased level of IgG1 anti-ody subclass [76], whereas Th1 response, characterized by high

evels of IFN-� production, promotes the generation of IgG2a sub-lass [77]. Therefore, it can be concluded that maintaining thegG1 level at a constant level after challenge and before chal-enge, as observed in the group immunized with A2-recombinant. tarentolae through the i.p. route, is probably due to the induc-ion of a more pronounced Th1 response. In addition, it haseen well established that the increased level of IgG1 antibod-

es titers is indicative of the L. infantum load [78]. The antibodyesponse shown here indicates that the least IgG1 level is associ-ted with the least parasite burden, both observed in the groupmmunized with the recombinant L. tarentolae A2 via the i.p.oute.

Studies on the effect of immunization route on the type oflicited immune response (Th1 or Th2) have shown that immu-ization via the i.p. rather than the i.v., s.c. and i.m. routes results

n higher levels of IFN-� and IgG2a antibody, and in lower levelsf Th2-associated cytokines (IL-4 and IL-10) and IgG1 antibodyubclass [79]. Here, we show that intravenous immunization,s opposed to intraperitoneal immunization, elicited a Th2-likeesponse, characterized by significantly lower level of IFN-�, higherevels of IL-5, and high humoral immune response, resulting iness efficient protection. Recently, it has been reported that spleenells of BALB/c mice immunized with L. donovani promastig-te membrane antigens (LAg) through different routes (e.g. s.c.,.m., and i.v. in comparison to i.p.) have an increased capacityo produce pre-challenge TGF-� during an in vitro antigen-recallesponse [80]. Interestingly, i.p. immunization induced produc-ion of pre-challenge IFN-�, IL-12 and IL-4 with a Th1 bias.xposure to antigen-stimulated splenocyte supernatants of i.p.ut not s.c. immunized mice activated macrophages for in vitroarasite killing. Therefore, the route of injection could induce dif-erent immunological responses in animal model such as BALB/c

ice.

In conclusion, the results presented here demonstrate that

2-expressing recombinant L. tarentolae shows a promise as anffective live vaccine against L. infantum infection. L. tarentolaeepresents a completely safe live vector, as previously reported53]. There is no sign of any pathology with L. tarentolae even

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28 (2010) 53–62

in immunocompromised SCID mice. Although non-pathogenic, L.tarentolae can efficiently target antigen-presenting cells and differ-entiate inside macrophages [53]. It is clearly incapable of promotingdisease, however, our PCR analysis data indicated that this parasitecan be found in the spleen and the liver of mice infected with theA2-recombinant L. tarentolae, albeit at low levels, 4 weeks afterimmunization (data not shown). Therefore, it appears that L. tar-entolae is able to persist at very low levels in mice. In addition, A2per se, has proven to be a promising vaccine candidate, capableof inducing potent Th1 immune response against L. infantum infec-tious challenge. A2 has also been associated with the visceralizationprocess [75]. It is therefore possible that L. tarentolae expressing A2may be better armed to survive within the host than the wild typeL. tarentolae strain. We are currently pursuing this hypothesis inour laboratory.

Acknowledgments

We are thankful to Prof. Greg Matlashewski (Departmentof Microbiology and Immunology, McGill University, Montreal,Canada) for providing us with vector pKSNEOA2-1. We would alsolike to thank Shima Safayeian for genomic DNA preparation.

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