Short communication

Diminished CD4+/CD25+ T cell and increased IFN-g levels occur in dogsvaccinated with Leishmune1 in an endemic area for visceralleishmaniasis

Valeria Marcal Felix de Lima a,*, Fabiana Augusta Ikeda a, Claudio N. Rossi a,Mary Marcondes Feitosa a, Rosemeri de Oliveira Vasconcelos b,Caris Maroni Nunes c, Hiro Goto d

a Departamento de Clınica, Cirugia e Reproducap Animal, Faculdade de Odontologia, Curso de Medicina Veterinaria,

Universidade Estadual Paulista ‘‘Julio de Mesquita Filho’’, Aracatuba Campus, SP, Brazilb Departamento de Patologia, Faculdade de Ciencias Agrarias e Veterinarias, Universidade Estadual Paulista ‘‘Julio de Mesquita Filho’’, Jaboticabal, SP, Brazilc Departamento de Apoio, Producao e Saude, Faculdade de Odontologia, Curso de Medicina Veterinaria,

Universidade Estadual Paulista ‘‘Julio de Mesquita Filho’’, Aracatuba Campus, SP, Brazild Departamento de Medicina Preventiva da Faculdade de Medicina, Universidade de Sao Paulo, SP, Brazil

Veterinary Immunology and Immunopathology 135 (2010) 296–302

A R T I C L E I N F O

Article history:

Received 31 March 2009

Received in revised form 5 November 2009

Accepted 30 December 2009

Keywords:

Visceral leishmaniasis

Vaccine

Th1 response

Cytokines

Zoonosis

A B S T R A C T

The Leishmune1 vaccine has been used in endemic areas to prevent canine visceral

leishmaniasis in Brazil, but cytokine production induced by vaccination has rarely been

investigated in dogs. This study aimed to evaluate the immune response of dogs

vaccinated with Leishmune FML vaccine (Fort Dodge) against total antigen of Leishmania

(Leishmania) chagasi (TAg) and FML. Twenty healthy dogs from Aracatuba, Sao Paulo,

Brazil, an endemic leishmaniasis area, received three consecutive subcutaneous injection

of Leishmune vaccine at 21-day intervals. PBMC were isolated before and 10 days after

completing vaccination and lymphoproliferative response and antibody production

against FML or total promastigote antigen were tested. Cytokines IFN-g, IL-4 and TNF-awere measured in culture supernatant and CD4+/CD25+ and CD8+/CD25+ T cell presence

was determined. Analysis of the data indicated that the vaccine conferred humoral

responses (100%) against both antigens and cellular immunity to FML (85%) and total

antigen (80%), the supernatant of cultured cells stimulated with TAg and FML showed an

increase in IFN-g (P< 0.05), and the vaccine reduced CD4+/CD25+ T cell presence

compared to that observed before vaccination. These responses may constitute part of the

immune mechanism induced by Leishmune.

� 2010 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

journal homepage: www.e lsev ier .com/ locate /vet imm

1. Introduction

Zoonotic visceral leishmaniasis is caused by Leishmania

(Leishmania) chagasi. It is endemic in America, Europe and

* Corresponding author at: Departamento de Clınica e Cirurgia e

Reproducao Animal, Faculdade de Odontologia, UNESP, Campus de

Aracatuba, Rua Clovis Pestana, 793, Bairro Jardim Dona Amelia, CEP

16050-680 Sao Paulo, Brazil. Tel.: +55 18 36361422.

E-mail address: vmflima@fmva.unesp.br (V.M.F. de Lima).

0165-2427/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2009.12.008

Asia with new dissemination areas being identifiedregularly. Transmission between vertebrate hosts occursby the bite of the phlebotomine hematophagous bite ofLutzomyia longipalpis. Dogs (Canis familiares) are consid-ered the most important urban reservoirs of L. (L.) chagasi

due to the high level of infection and the directtransmission to humans (Alvar et al., 2004).

Dogs have been shown to be highly susceptible toinfection and current treatments have shown limitedefficiency; thus, the development of a preventive vaccine is

V.M.F. de Lima et al. / Veterinary Immunology and Immunopathology 135 (2010) 296–302 297

fundamental. During the last few years, several vaccineshave been tested, including: live and inactivated vaccines,Leishmania purified fractions, recombinant antigens,recombinant bacteria expressing Leishmania antigen andplasmidial DNA (Gradoni, 2001). The vaccine to preventcanine visceral leishmaniasis that has been used recentlyin Brazilian endemic areas is composed of a purifiedfraction (FML), also known as Leishmune (Fort Dodge Ltda,Sao Paulo, Brazil).

The Leishmune vaccine consists of a glucoproteicfraction containing a ligand for fucose and mannose(FML) and inhibits penetration of the promastigote andamastigote forms in mice macrophages in vitro (Palatniket al., 1989), it is present on the surface of the parasitethroughout the entire cycle (Palatnik-De-Sousa et al.,1993) and is a potent immunogen for mice, rabbits and aspecific antigen for human and canine serodiagnosis(Palatnik-De-Sousa et al., 1995; Borja-Cabrera et al., 1999).

The FML antigen as a vaccine proved to be safeprotective and highly immunogenic for dogs (Da Silvaet al., 2001). In a Brazilian area endemic for both humanand dog visceral leishmaniasis, a recent phase III trialshowed that 7 months after vaccination with FMLassociated with saponin, used as adjuvant, dogs presented97% seropositivity for FML and 100% presented positiveDHR (delayed hipersensitivity reaction) against L. donovani

lysate (Da Silva et al., 2001).In the study mentioned, 92% protection was observed

two years after vaccination and only 8% of the vaccinateddogs showed clinical signs of the disease. In contrast, 33%of nonvaccinated dogs developed the clinical disease.These data suggest that the vaccine induces long-termimmunity in the field (Da Silva et al., 2001).

Several other studies have been performed to deter-mine the protective effects of the vaccine (Da Silva et al.,2001; Borja-Cabrera et al., 2002; Nogueira et al., 2005),which suggest that this antigen induces protectiveimmunity, however the cytokines induced and character-ization of cell populations associated with resistance hasonly been studied to a limited extent. Given this fact, theimmune response triggered by vaccination in an endemicarea could be useful for understanding the patterns ofimmunity associated with resistance to Leishmania spp.infection.

2. Materials and methods

2.1. Study area

The study was conducted in Aracatuba, Sao Paulo State,Brazil, an endemic area for canine visceral leishmaniasis(CVL) and human visceral leishmaniasis.

2.2. Animals

Twenty (20) healthy male and female dogs owned byresidents of Aracatuba, SP, Brazil were included in thestudy. The owners signed a term permitting the collectionof samples and the local ethics in animal researchcommittee approved this study. Blood and bone marrowsamples were taken after anesthetizing the dogs by

intramuscular injection of ketamine (Fort Dodge, Campi-nas, SP, Brazil) (30 mg/kg) and xylazine (Calier do Brasil,Sao Paulo, Brazil) (2 mg/kg). From each dog, 1.0 ml of bloodwas collected by puncture of the dogs cephalic veins,clotted at room temperature for 4 h and subsequentlycentrifuged to extract the serum. The serum samples werestored at �20 8C prior to analysis. The dogs wereseronegative for Leishmania spp. specific antibodies, asdetermined by the indirect ELISA method (Lima et al.,2003), and were Leishmania spp. negative in cytologicaland PCR for bone marrow samples and normal blood. Thesedogs received three consecutive subcutaneous doses ofLeishmune1 (Fort Dodge, Campinas, SP, Brazil) at 21-dayintervals, in accordance with the manufacturer’s instruc-tions. Peripheral blood was collected before administrationof the first vaccine dose and 10 days after completingvaccination for cellular immunity analysis and to profilethe cytokines produced. New blood and bone marrowsamples were collected 6 and 12 months after vaccinationto assess possible infection by Leishmania spp. and todetermine the dogs immune responses. Infection wasverified by cytological analysis and PCR. In this protocol,each dog was its own control. The analyses of cellularimmunity and the profile of cytokines produced beforevaccination were used as control parameters.

Another 20 healthy male and female dogs owned byresidents of Aracatuba, SP, Brazil were included as aplacedo group and the animal handling procedures forsample collection for vaccinated and placebo group werethe same. The placebo dogs were followed throughout theexperimental period to evaluate the natural index ofinfection by PCR and ELISA, as described above.

No insecticide impregnated collars for canine VL wereused and the groups studied were mantained under thesame VL transmission rate.

2.3. Detection of L. chagasi and FML antibodies by indirect

ELISA

Screening of noninfected dogs (vaccinated and placebogroup) was performed by an ELISA immunoassay using a L.

(L.) chagasi total promastigote lysate as the antigen, asdescribed by Lima et al. (2003).

The test of FML-ELISA in immunized dogs wasperformed as described by Borja-Cabrera et al. (1999),with one modification, anti-IgG HRP-conjugate was usedinstead of protein A-HRP.

2.4. Cellular immunity assessment

The lymphoproliferation technique was used to assesscellular immunity of the vaccinated group. Fifteen milli-litre of peripheral blood from the vaccinated group wascollected from the dogs by puncture of the cephalic orjugular veins, using heparin as an anticoagulant agent.Peripheral blood mononuclear cells (PBMC) were isolatedin accordance with the recommendations of the manu-facturer Ficoll–Paque (Amersham Biosciences, Piscataway,NJ, USA). Lymphocyte proliferation was assessed beforevaccination and 10 days after completing vaccination. Thecells were placed into RPMI 1640 (Gibco Industries,

V.M.F. de Lima et al. / Veterinary Immunology and Immunopathology 135 (2010) 296–302298

Langley, OK, USA) media supplemented with 10% of calffetal serum, 1% L-glutamine, 5 mM of mercaptoethanol andantibiotics. 100 ml of the suspension containing5� 106 cells/ml was used for plating in 96-well-plates(Corning Inc., NY, USA) and the following controls wereincluded: concanavalin A (0.4 mg/well), total antigen oflysed promastigote form of L. (L.) chagasi MHOM/BR/00/MERO2 (105/well), was prepared as described by Lima et al.(2003) and FML (10 mg/well) was provided by Fort Dodge.The cultures were maintained at 37 8C with 5% CO2 for 5days. Cell proliferation was determined by thymidine H3

incorporation, 0.5 mCi/well was added to the cultures 24 hbefore the end of the culture period. The cultures werethen harvested onto glass fiber filter paper (FlowLaboratories, Rockville, ML, USA) using a cell harvester(Titertek, Huntsville, AL, USA). The incorporated radio-activity was assessed by liquid scintillation in a b-counter(Packard BioScience Company, Meriden, CT, USA). Thestimulation index was determined as the medium rate ofcpm of concanavalin A-stimulated cultures or antigensused and cpm of culture with media only (controls). Astimulation index higher than 2.5 indicated a positiveresponse (Cabral et al., 1992).

2.5. IFN-g, IL-4 and TNF-a quantification

Approximately 1� 105 of isolated PBMC were culti-vated in vitro with total antigen of the lysed promastigoteform of L. L. chagasi MHOM/BR/00/MERO2 (105/well),prepared as described by Lima et al. (2003), and FML(10 mg/well), provide by Fort Dodge, in a humidifiedenvironment at 37 8C with 5% CO2 for 24 h. Next, thesupernatant was individually collected by removing thecells in suspension and stored at �80 8C until themeasurement of cytokines by ELISA.

Cytokine quantification in culture supernatants derivedfrom PBMC was performed by capture ELISA using mousemonoclonal anti-canine IFN-g, IL-4 and TNF-a antibodies(mAb) and biotinylated polyclonal goat anti-canine IFN-g,IL-4 and TNF-a (R&D Systems, USA). 96-well-plates(Maxisorb, Nalgene Nunc International, Rochester, NY,USA) were coated with 1 mg/ml mAb for cytokine captureand 0.5 mg/ml detection antibodies, respectively. Recom-binant canine IFN-g, IL-4 and TNF-a (R&D Systems, USA)were used to generate standard curves. The test wasdeveloped with 3,30,5,50-tetramethylbenzidine (TMB; Pro-mega Corporation, Madison, WI, USA), in accordance withthe manufacturer’s instructions, and ODs were red whenusing a Spectra CountTM reader (Packard BioScienceCompany) using a 450 nm filter.

2.6. CD4+/CD25+ and CD8+/CD25+ T cell presence

The presence of CD4+/CD25+ and CD8+/CD25+ T cells wasmeasured before administration of the first vaccine dose and10 days after vaccination. 5� 105 isolated PBMC werecultivated with concanavalin A (0.4 mg/well), total antigen oflysed promastigote form of L. L. chagasi MHOM/BR/00/MERO2 (105/well), prepared as described by Lima et al.(2003), and FML (10 mg/well), provide by Fort Dodge. Thecultures were maintained at 37 8C with 5% CO2 for 24 h and

the nonadherent cells were collected for flow cytometryanalysis. The cells were suspended in 0.01 M phosphatebuffered saline solution (PBS) pH 7.2 with 1% calf serumalbumin, 0.1% azide and 20% calf fetal serum to block the Fcreceptors. The cells were double-stained with specificfluorochrome-conjugated antibodies: monoclonal fluores-cein isothiocyanate (FITC) conjugated mouse anti-canineCD4 antibody (Serotec, Kidlington, Oxford, UK), monoclonalfluorescein isothiocyanate (FITC) conjugated mouse anti-canine CD8 antibody (Serotec), and human anti-CD25conjugated to phycoerythrin (PE; Dako, USA) (Galkowskaet al., 1996) or control isotypes conjugated to FITC and PE(Serotec; Dako). The cells were analyzed using the FACSca-libur system (Becton Dickinson, Franklin Lakes, NJ, USA).

2.7. PCR protocols

DNA obtained from the bone marrow aspirates andperipheral blood was extracted after lysing by freezing andthawing 3 times and washing in 1� SSC buffer solution(NaCl 3 M, sodium citrate 0.3 M, pH 7.0). For digestion,300 ml of lysing solution was added (10% SDS in 0.2 Msodium acetate) together with 20 mg/ml proteinase K. Thesamples were incubated at 56 8C for 2 h and DNA wasextracted using the phenol/chloroform/isoamyl alcoholmethod (25:24:1) (Sambrook et al., 1989). After extraction,DNA was resuspended in 50 ml TE (10 mM Tris–HCl pH 8.0,1 mM EDTA pH 8.0) and incubated for 3 min at 60 8C. Thematerial was stored at �20 8C until analysis. The primers13A (30-GTG GGG GAG GGG CGT TCT-50) and 13B (30-ATTTTA CAC CAA CCC CCA GTT-50) were used (Rodgers et al.,1990) to amplify a 120-bp fragment of kDNA from the baseof the Leishmania spp. mini-circle. PCR was performed in60 ml of volume containing 30 pmol of each primer(Invitrogen Corporation, Carlsbad, CA, USA), 0.2 mM dNTPs(Invitrogen), 1.5 mM MgCl2 (Invitrogen), 5 U Taq DNAPolymerase (Invitrogen), 50 nM buffer solution, milliQwater and DNA. Amplification was performed in anEppendorf1 Mastercycler Thermocycler gradient withinitial heating to 95 8C for 5 min, followed by 33 cyclesat 95 8C for 1.5 min, 57 8C for 1.5 min and 72 8C for 2 min.Extension was carried out at 72 8C for 10 min and the finalproduct was stored at �20 8C until analysis. For eachreaction, a negative control (no DNA) and a positive controlwith DNA extracted from a L. chagasi promastigote culture(MHOM/BR00/MER02) were added. The amplified productwas analyzed by electrophoresis in 2% agarose gel followedby ethidium bromide staining and visualization under UVlight.

PCR to detect DNA Leishmania spp. was evaluated inhealthy dogs before (day 0) and one year after vaccination.

2.8. Statistical analysis

Statistical differences were analyzed using GraphPadPRISM 3 Software (San Diego, CA, USA). Considering thenonparametric nature of all data sets, the Wilcoxonmatched pairs test was used to access significantdifferences between pre- and postvaccination. In all cases,the differences were considered significant when theprobabilities of equality, p-values, were p< 0.05.

Fig. 1. Serum antibody titers (IgG) against total antigen of FML (a) and L.

chagasi (b) from dogs before and after vaccination. Data are presented as

absorbance at 490 nm and are expressed as the mean OD� SD.

Fig. 2. Lymphoproliferative response to total antigen from Leishmania (L.)

chagasi, FML antigen and concanavalin A 10 days after vaccination. The

data are represented as the stimulation index (SI).

V.M.F. de Lima et al. / Veterinary Immunology and Immunopathology 135 (2010) 296–302 299

3. Results and discussion

The increase in the number of cases of visceralleishmaniasis reported in Brazil, associated with the dogas the main reservoir in urban areas and the source ofinfection in humans via sandfly bites, added to the failureof chemotherapeutic procedures (Ikeda-Garcia et al., 2007)all indicate immunoprophylaxis as a promising alternativefor CVL prevention.

To investigate the immune response triggered by FMLvaccine in dogs, 20 vaccinated and 20 controls, wereexamined prior to vaccination to detect visceral leishma-niasis in the study group, cytology and PCR showed noLeishmania spp. and were seronegative for Leishmania spp.specific antibody (data not shown). Prior to immunization,all dogs included in the experiments were asymptomaticand healthy, one year after immunization they wereanalyzed for symptoms and the presence of parasite DNA.Clinical signs of disease, such as alopecia, skin ulcers,cachexia, onychogryphosis and Leishmania spp. specificantibody and DNA were observed in 45% of the controlgroup, while no parasite DNA was detected in the vaccinatedgroup; these data suggest that vaccination induced long-term immunity. These results are similar to those observedby Da Silva et al. (2001) and Nogueira et al. (2005).

To investigate the mechanisms associated with post-vaccination protection, cellular and humoral immunity tototal promastigote antigen from Leishmania spp. and FMLwere assessed. The mean titers of anti-FML IgG antibodiesbefore vaccination (n = 20) were OD 0.173� 0.05, with thehighest levels observed 10 days after vaccination(0.912� 0.11; P< 0.05), 6 months (0.450� 0.12; P< 0.05)and one year (0.342� 0.11; P< 0.05), as shown in Fig. 1a.These results are similar to several studies that show thatvaccination with Leishmune vaccine induces a stronghumoral response against the FML antigen (Borja-Cabreraet al., 2002; Da Silva et al., 2001). Similarly, the mean titers ofIgG anti total Leishmania promastigote antigen beforevaccination was OD 0.121� 0.06, with the highest levelsobtained 10 days after vaccination (0.806� 0.14; P< 0.05),after 6 months (0.254� 0.11; P< 0.05) and after one year(0.215� 0.11; P< 0.05), as shown in Fig. 1b.

The present study showed that vaccination withLeishmune induced antibody that reacted with totalpromastigote antigen from Leishmania spp., and that100% of the dogs produced these antibodies. Total antigenis frequently used in RIFI and ELISA assays to detect thedisease, so in endemic areas, the suspected infection invaccinated dogs should be monitored by improvedserological methods using recombinant antigens or bymolecular methods, such as PCR.

The high antibody levels observed could be related toprotection. Recently Bourdoiseau et al. (2009) reportedthat antibodies induced by vaccination interfered not onlywith the intracellular parasite survival and multiplication,but also with the binding and/or internalization ofpromastigotes by macrophage, so further studies arerequired to clarify the role of these antibodies inprotection.

Although humoral immunity was stimulated by vacci-nation, several studies involving mice (Murray et al., 2006),

humans (Kurkjian et al., 2006) and dogs (Pinelli et al.,1995) reported that cellular immunity is responsible forprotective responsiveness to visceral leishmaniasis.

Fig. 2 shows the lymphoproliferative responses of PBMCto antigen of lysed promastigote form of L. (L.) chagasi andFML and concanavalin A on day 10 after completingvaccination; i.e., 73 days after initiating immunization.Before vaccination, only 1 dog presented a positivelymphoproliferative response to total antigen (data notshown). All the dogs studied presented a positive lympho-proliferative response to concanavalin A and this responsewas similar before and after vaccination. After vaccination,85% of the dogs presented a positive lymphoproliferativeresponse to FML antigen and 80% presented this response to

Fig. 3. IFN-g, IL-4 and TNF-a quantification by ELISA in culture

supernatant from PBMC of dogs before and 10 days after vaccination,

*P< 0.05 between means before and after vaccination groups.

V.M.F. de Lima et al. / Veterinary Immunology and Immunopathology 135 (2010) 296–302300

total antigen. The stimulation index showed heterogeneity,probably due to the use of mixed breed dogs, in addition toage and sex.

This result differs from data obtained by Borja-Cabreraet al. (2002) and Da Silva et al. (2001), who investigatedcellular immunity by delayed hypersensitivity reaction(DHR) in vaccinated dogs using total antigen from L.

donovani lysate and obtained 100% positive DRH aftervaccination. DRH can differ in relation to the lymphopro-liferative response during the immune response toLeishmania spp. antigens (Fernandez-Bellon et al., 2005).In endemic areas, DRH to total antigen must be attributedto vaccination and not to infection, when infection ismonitored by molecular methods. Da Silva et al. (2001)performed PCR to detect Leishmania spp. DNA; however,only 5 dogs were studied, while Borja-Cabrera et al. (2002)did not use molecular methods.

To evaluate the immunological profile associated withlymphoproliferative immune responses, the cytokines IFN-g, IL-4 and TNF-a were measured in supernatants of thePBMC cultures prior to immunization (day 0) andpostimmunization (day 73). The levels of IFN-g showeda significant difference (P< 0.05) after vaccination in thesupernatants stimulated with total antigen and FML fromthe vaccinated group. IL-4 and TNF-a levels were similarfor these periods, as shown in Fig. 3.

IL-4 and TNF-a results confirms previous reportsshowing that IL-4 is not increased by Leishmune (Araujoet al., 2009); however, IFN-g levels increased significantlyafter vaccination in culture supernatant stimulated withtotal antigen from Leishmania spp. and FML. These resultsare similar to those obtained by Araujo et al. (2009). IFN-gcan stimulate macrophages and increase cytotoxic T cellactivity, induce nitric oxide production, increase MHCmolecules and costimulatory signals, and polarize theimmune response to Th1. In murine models and curedhuman patients, IFN-g is associated with resolution ofinfection (Garg et al., 2006; White et al., 1992).

The production of IFN-g induced by the vaccinationcould be related to the protection observed in endemicareas by our group and other researchers (Da Silva et al.,2001; Borja-Cabrera et al., 2002; Nogueira et al., 2005). TheIFN-g detected in culture supernatant confirmed that theimmune profile of vaccinated dogs showed a patternrelated to Th1 activation (Rutten et al., 2008).

In a recent vaccine study, the reduction in theaccumulation of parasite-specific T of T reg cells (CD4+/CD25+) in secondary lymphoid tissue compared withcontrol values appeared to be an indicator of partialvaccine success against murine leishmaniasis (Carrionet al., 2008); Fig. 4 shows that CD4+/CD25+ percentages inthe PBMC decreased significantly after immunization(P< 0.05) (Fig. 3a), while the percentages of CD8+/CD25+ were similar before and after vaccination(Fig. 3b). The decrease in the CD4+/CD25+ T cell populationafter the vaccination, could be related to postvaccinationprotection, because this population has a regulatoryactivity by producing IL-10 and TGF-beta (Belkaid, 2003).

The role of T regulatory cells in VL has been reportedand discussed, though it continues to be controversial. Treg cells do not appear to produce IL-10 in spleen cells from

infected humans (Nylen et al., 2007), although in L.

donovoni infected mice, a significant increase in thefrequency of IL-10 CD25+ T cells was observed comparedto naıve mice (Stager et al., 2006). CD4+CD25+ T cells fromL. infantum infected mice were capable of secreting IL-10,though nonspecifically and during the early phase ofinfection, implicating a minor role for CD4+CD25+T IL-10producing T regs during visceral L. infantum infection(Rodrigues et al., 2009). No previous studies have beenperformed on infected or vaccinated dogs.

In conclusion, the vaccinated dogs showed cellular andhumoral response to total antigen from Leishmania spp.and FML, while the immune response promoted dimin-ished CD4+/CD25+ T cell presence and increased IFN-g;

Fig. 4. Box-plot representation of the frequency of CD4+/CD25+ T cells (a)

and CD8/CD25 T cells (b) from PBMC before and after vaccination. PBMC

was cultivated with Con-A, FML and total antigen from Leishmania (L.)

chagasi, after 24 h the nonadherent cells were dyed with monoclonal

antibodies and analyzed by flow cytometry before (1) and 10 days after

vaccination (2). The horizontal and vertical lines in each box represent the

median and range (95% confident interval) (*P< 0.05).

V.M.F. de Lima et al. / Veterinary Immunology and Immunopathology 135 (2010) 296–302 301

these patterns may constitute part of the immunemechanism induced by Leishmune. Further studies arerequired to elucidate other immunological featurestriggered by vaccination with Leishmune and determinewhich are crucial to protection in an endemic area.

Acknowledgement

The authors are grateful to the FAPESP for financiallysupporting this project.

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