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A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs

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A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs
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  Vaccine (2008)  26 , 623—638 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/vaccine A killed  Leishmania  vaccine with sand fly salivaextract and saponin adjuvant displaysimmunogenicity in dogs Rodolfo Cordeiro Giunchetti a , b , ∗ , Rodrigo Corrˆea-Oliveira b ,Olindo Assis Martins-Filho c , Andr´ea Teixeira-Carvalho a , c ,Bruno Mendes Roatt a , Rodrigo Dian de Oliveira Aguiar-Soares a ,Wendel Coura-Vital a , Raquel Tropia de Abreu a , Luiz Cosme Cotta Malaquias d ,Nelder Figueiredo Gontijo e , Cl´audia Brodskyn f  , Camila Indiani de Oliveira f  ,Dirceu Joaquim Costa f  , Marta de Lana g , h , Alexandre Barbosa Reis a , b , h , ∗ a Laborat´orio de Imunopatologia, N ´ucleo de Pesquisas em Ciˆencias Biol´ogicas, Instituto de Ciˆencias Exatas e Biol´ogicas,Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil b Laborat´orio de Imunologia Celular e Molecular, Instituto Ren´e Rachou, Fundac¸˜ao Oswaldo Cruz, Belo Horizonte,Minas Gerais, Brazil c Laborat´orio de Biomarcadores de Diagn´ostico e Monitorac¸˜ao, Instituto Ren´e Rachou, Fundac¸˜ao Oswaldo Cruz,Belo Horizonte, Minas Gerais, Brazil d N ´ucleo de Pesquisa em Imunologia, Universidade Vale do Rio Doce, Governador Valadares, Minas Gerais, Brazil e Laborat´orio de Fisiologia de Insetos Hemat´ofagos, Departamento de Parasitologia, Instituto de Ciˆencias Biol´ogicas,Universidade Federal de Minas Gerais, Belo Horizonte, Brazil f  Laborat´orio de Imunoparasitologia, Centro de Pesquisas Gonc¸alo Moniz Fundac¸˜ao Oswaldo Cruz, Salvador, Bahia, Brazil g Laborat´orio de Doenc¸a de Chagas, N´ucleo de Pesquisas em Ciˆencias Biol´ogicas, Instituto de Ciˆencias Exatas e Biol´ogicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil h Departamento de An´alises Cl´ınicas, Escola de Farm´acia, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil Received 6 October 2007; received in revised form 14 November 2007; accepted 21 November 2007Available online 17 December 2007 KEYWORDS Canine visceralleishmaniasis;Cell and humoralimmune response;Flow cytometry Summary  A vaccine against canine visceral leishmaniasis (CVL), comprising  Leishmaniabraziliensis  promastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was eval-uated in dog model, in order to analyse the immunogenicity of the candidate vaccine. Thevaccine candidate elicited strong antigenicity in dogs in respect of specific SGE and  Leishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogsexhibitedmolecularweightsof35and45kDa,andwererelatedtotheresistancepatternagainst Leishmania  infection. Immunophenotypic analysis revealed increased circulating CD21 + B-cells ∗ Corresponding authors at: Laborat´orio de Imunopatologia, N´ucleo de Pesquisas em Ciˆencias Biol´ogicas, Instituto de Ciˆencias Exatas e Biol´ogicas, Universidade Federal de Ouro Preto, Campus Universit´ario, Morro do Cruzeiro, 35.400-000, Ouro Preto, Minas Gerais, Brazil.Tel.: +55 31 3559 1694; fax: +55 31 3559 1680. E-mailaddresses: giunchetti@nupeb.ufop.br (R.C. Giunchetti), alexreis@nupeb.ufop.br (A.B. Reis). 0264-410X/$ — see front matter © 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2007.11.057  624 R.C. Giunchetti et al. and CD5 + T-cells, reflected by higher counts of CD4 + and CD8 + T-cells. The observed interac-tion between potential antigen-presenting cells (evaluated as CD14 + monocytes) and lymphocyteactivation status indicated a relationship between innate and adaptive immune responses. Thehigher frequency in  L. chagasi  antigen-specific CD8 + T-lymphocytes, and their positive associationwith intense cell proliferation, in addition to the progressively higher production of serum nitricoxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies onimmunological response after challenge with  L. chagasi  may provide important information thatwill lead to a better understanding on vaccine trial and efficacy.© 2007 Elsevier Ltd. All rights reserved. Introduction The New World sand fly,  Lutzomyia longipalpis , is an impor-tant vector of   Leishmania chagasi , the etiological agent of visceral leishmaniasis (VL) [1]. The act of probing and feed-ing by an infected female sand fly introduces into the hostboth secretions from the salivary gland of the fly and pro-mastigotes of   L. chagasi  [2]. It is known that the saliva of  L.longipalpis possessesapyrase,anticoagulant,vasodilatoryand immunomodulatory activities that could facilitate eva-sion of the inflammatory and immune responses of the host[3—7]. Furthermore, initial studies with  Leishmania major  [8] demonstrated that sand fly saliva exacerbates host infec-tion, and similar findings have been reported for a numberof different species of   Leishmania  [2,9—11].Mice that had been exposed to homogenates of the sali-vary glands of   L. longipalpis  or to bites from uninfectedsand flies were protected from infection when challengedwith either needle-inoculated  L. major   plus sand fly saliva[12] or with bites from sand flies infected with  L. major  [13].Morrisetal.[14]reportedthatanimalsvaccinatedwith maxadilan, a potent vasodilator and immunomodulator from L. longipalpis , were protected against  L. major   infection.Additionally, mice that had been vaccinated with a 15kDasalivary protein (PpSP15) from sand flies were immune toinfection by  L. major   when challenged with promastigotesand saliva [15]. These data support the hypothesis thatinduction of an immune response to salivary proteins of thesand fly may facilitate a protective immune response against Leishmania  infection.Although an effective vaccine against human and caninevisceral leishmaniasis (CVL) is not yet available, much efforthas been expended in this area in recent years and severalcandidate vaccine antigens have been studied extensivelyin dogs. A recent strategy for the development of a vaccineagainst leishmaniasis has been based on the use of purifiedfractions from parasite extracts (FLM antigen) or from para-site cultures (excreted/secreted antigens), frequently usingsaponin as adjuvant, and some encouraging results havebeen reported [16—25].However, in the search for a potential vaccine, target-ing a single protein might not be an adequate approach,and the selection of multiple proteins as candidates maybe required [7,26]. In the development of a vaccine against leishmaniasis, a strategy based on a combination of sandfly salivary gland extract (SGE) and  Leishmania  antigensmight be appropriate. Moreover, although the current strat-egy for vaccination against leishmaniasis is based on theuse of recombinant antigens, whole parasite vaccines arestill attractive in terms of cost, safety, and stability of their biochemical composition and antigenicity. Trials utiliz-ing such vaccines have already been undertaken [27], andseveral studies involving the dog model have revealed thatcrude antigen vaccines elicit strong cell reactivity against Leishmania  antigens [28—31]. Unfortunately, the detailedimmune status of the experimental animals following SGEvaccination was not evaluated within the framework of these studies.Dogs represent the most important domestic reservoirsof   L. chagasi  [32], and a vaccine against CVL would be animportant tool in the control of human VL by decreasingdramatically the infection pressure of   L. chagasi / L. infan-tum  [33—37]. A better understanding of the canine immuneresponse to sand fly salivary proteins could be of significantassistance in defining alternative vaccination strategies bywhich to control CVL as well as human VL. The present studyconstitutes the first detailed analysis of immunogenicity inexperimental dogs that had received a promising CVL vac-cine composed of killed  L. braziliensis  together with saponinadjuvant and SGE. Material and methods Details of the study were presented to and approved by theEthical Committee for the Use of Experimental Animals of the Universidade Federal de Minas Gerais, Belo Horizonte-MG, Brazil. Sand flies and salivary gland extracts Closed colonies of   L. longipalpis  were maintained at 25 ◦ Cand 60—80% relative humidity according to a published pro-tocol[38].SGEwaspreparedusingthemethodofCavalcanteet al. [6] in which the acini of salivary glands of 4-day-old,mated, but non-blood fed, female sand flies were dissectedin 0.8% unbuffered saline, broken by sonication for 10s andcentrifuged at 10,000 ×  g  for 2min. The supernatant wascollected and stored  − 70 ◦ C until required for use. Study animals and vaccination Twenty-five mongrel male and female dogs that had beenborn and reared in the kennels of the Instituto de CiˆenciasExatas e Biol´ogicas, Universidade Federal de Ouro Preto,Ouro Preto, Minas Gerais, Brazil, were vaccinated atthe age of 7 months against rabies (Tecpar, Curitiba-PR,Brazil), canine distemper, type 2 adenovirus, coronavirus,parainfluenza, parvovirus and leptospira (Vanguard ® HTLP5/CV-L; Pfizer Animal Health, New York, NY, USA), and  A killed  Leishmania  vaccine with sand fly saliva extract and saponin adjuvant 625treated with an anthelmintic. The absence of specificanti- Leishmania  antibodies was confirmed by indirect flu-orescence immunoassay. Ouro Preto city is considereda non-endemic area for visceral leishmaniasis in Brazil.Besides negative serology by IFAT, others additional effec-tive approaches were performed aim to rule out  Leishmania infection such as sprayed the kennels of the UFOP withpyrethroid insecticide and protected all their extensionthroughout an appropriated and security stainless steelgauze as recommended by Brazilian Ministry of Healthy.Animals were treated within four experimental groups:(i) the control group C ( n =10) received 1mL of sterile 0.9%saline; (ii) Sap group ( n =5) received 1mg of saponin asproposed by Borja-Cabrera et al. [39] and Santos et al.[25] in 1mL sterile 0.9% saline; (iii) the Sal group ( n =5)received SGE prepared from five acini of salivary glands of  L. longipalpis  in 1mL sterile 0.9% saline; (iv) the LBSal group( n =5) received 600  g of   Leishmania braziliensis  promastig-ote protein (Lb; prepared as described previously [30]) plusSGE (as above) in 1mL sterile 0.9% saline; and (v) the LBSap-Sal group ( n =5) received 600  g of Lb plus 1mg of saponintogether with SGE in 1mL sterile 0.9% saline. The type of saponin used is a  Quilaja saponaria  Molina extract [40,41]now commercialized by Sigma Chemical Co., St. Louis, MO,USA.Inallcases,animalsreceivedthreesubcutaneousinjec-tions in the right flank at intervals of 4 weeks. Local and/or general reactions to immunization A veterinarian checked all animals throughout the courseof the study and also provided full medical support asrequired. Dogs were monitored particularly closely for 2weeksfollowingeachinjection.Ageneraltolerancetovacci-nation was established from an overall evaluation (includingrectal temperature measurements) of the health of theanimal. Local tolerance was determined by direct visualexamination, and when lesions were observed they weremeasured at 24h intervals over a period of 72h after eachinjection. Collection of blood samples Samples (5mL) of peripheral blood were collected from thejugular vein of each dog and transferred to tubes containingan amount of EDTA sufficient to produce a final concen-tration of 1mg/mL. Blood samples were stored at roomtemperature for up to 12h prior to processing. A Coultercounter (Miami, FL, USA) model MD18 was employed in orderto determine the absolute count of lymphocytes in eachsample. Humoral immune response Immunogenicity was evaluated by the determination of anti-bodies raised against SGE and a soluble lysate of   L. chagasi antigen (MHOM/BR/1972/BH46) (SLcA) according to conven-tional enzyme-linked immunosorbent assays (ELISA) [42,43]. The wells of MaxiSorp TM (Nalge Nunc Intl., Rochester, NY,USA) 96-well microplates were coated with SGE (at a con-centration of one pair of salivary glands/well) or SLcA(2  g/well), serum samples were added at dilutions of 1:100(SGE-ELISA) or 1:80 (SLcA-ELISA), the wells were washedand peroxidase-conjugated goat anti-dog IgG1 or sheep anti-dog IgG and IgG2 (Bethyl Laboratories Inc., Montgomery,TX, USA) added at dilutions of 1:1000 (IgG1), and 1:16000(IgG and IgG2). The wells were then re-washed, substrateandchromogen( o -phenylenediamine;Sigma—AldrichCo.,StLouis, MO, USA) added, and absorbance recorded at 405nm(SGE-ELISA) or 492nm (SLcA-ELISA) on a Multiskan ® MCC340 (Labsystems, Helsinki, Finland) automatic microplatereader. Western blot of SGE Western blot analysis of   L. longipalpis  SGE was performedaccording to a published method [12]. Briefly, a sample of SGE containing  ca . 40  g of protein (equivalent to 40 pairsof salivary glands from  L. longipalpis ) was submitted to SDS-PAGE on NuPAGE TM Novex bis—Tris gels (4—12%, 1.0mm, 2D;Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellu-lose. The membrane was cut into strips, blocked overnightwith Tris HCl buffer (pH 8.0) containing 150mM NaCl plus5% non-fat milk, and incubated with dog serum (1:50 dilu-tion) in blocking buffer. Following incubation in anti-dog IgGalkaline phosphatase conjugate (1:4000 dilution; Promega,Madison, WI, USA), bands were visualized by the additionof alkaline phosphatase substrate (Promega, Madison, WI,USA). Immunophenotyping Unlabelled canine monoclonal antibodies (mAbs) anti-CD5(rat-IgG2a: clone YKIX322.3), anti-CD4 (rat-IgG2a: cloneYKIX302.9), anti-CD8 (rat-IgG1: clone YCATE55.9) were usedin an indirect immunofluorescence procedure in whichpooled normal rat serum (diluted 1:6000) was employedas isotypic control, and fluorescein isothiocyanate (FITC)-labelled IgG sheep anti-rat polyclonal antibody was used asthe secondary antibody. Non-specific binding of the second-step reagent was blocked with pooled normal sheep serumin phosphate buffered saline (PBS) containing 10% foetalbovine serum (Gibco, Grand Island, NY, USA).FITC-labelled mouse anti-human-CD21 (mouse-IgG1:clone IOBla), phycoerythrin (PE)-Cy5-conjugated mouseanti-human-CD14 (mouse-IgG2a: clone T¨UK4), RPE-conjugated mouse anti-mouse MHC-I (mouse-IgG2b:clone 2G5) and RPE-conjugated hamster anti-mouse CD80(Armenian hamster-IgG2, clone 16-10A1) mAbs were usedin a direct immunofluorescence procedure. In order todetermine the optimal dilutions for each assay, mAbs weretitred in PBS containing 1% bovine serum albumin and 0.1%sodium azide. Unlabelled mAbs, anti-CD14 and anti-MHC-ImAbs were purchased from Serotec (Oxford, UK), anti-CD21was from Immunotech Co. (Marselle, France) and anti-CD80was from BD Bioscience Pharmingen (Franklin Lakes, NJ,USA).Microplate assays for immunophenotyping canine wholeblood leukocytes (WBL) in fresh blood samples and in periph-eral blood mononuclear cells (PBMC) obtained after  in vitro stimulation, were carried out as described by Giunchetti etal. [30] and Reis et al. [44].  626 R.C. Giunchetti et al. Flow cytometry Flow cytometric measurements were performed on a FAC-Scan instrument (Becton Dickinson, Moutain View, CA, USA)interfaced to an Apple G3 workstation running Cell-Questsoftware (Becton Dickinson) for both data acquisition andanalysis. A total of 15,000 events were acquired for eachpreparation. Canine WBL were selected on the basis of theircharacteristic forward (FSC) and side (SSC) light-scatterdistributions. Following FSC and SSC gain adjustments,the lymphocytes were selected by gating on the FSC  ver-sus  SSC graph. Fluorescence was evaluated from FITC, PEand PE-Cy5 spectra, respectively, on FL1, FL2 and FL3 insingle-histogram representations. Monocytes were analysedby fluorescence intensity detection on single histogramsobtained directly from un-gated leukocytes. A marker wasset as an internal control for non-specific binding in orderto encompass >98% of unlabelled cells, and this marker wasthen used in the analysis of data for a given animal. Theresults were expressed as the percentage of positive cellswithin the selected gate for cell surface markers presentingbimodal distributions (CD5, CD4, CD8 and CD21). Semi-quantitative analyses were carried out for the cell surfacemarkers MHC-I and CD80, which exhibited unimodal distri-butions, in order to evaluate differential expression, andthe results were expressed as mean fluorescence channel(MFC) on a log scale. Data were also expressed as abso-lute counts in order to allow the normalization of valuesobtained from groups presenting different overall leukocytecounts. The absolute counts for lymphocytes and monocyteswere calculated as (global leukocyte counts × percentageof lymphocytes or monocytes in hematoscopy)/100. Theabsolute counts for lymphocyte subsets and monocyteswere determined, respectively, from (absolute lymphocytecounts × percentage of fluorescent positive cells within lym-phogate)/100 and (global leukocyte counts × percentageof fluorescent positive cells within un-gated monocytes)/100. In vitro  assays PBMC were isolated from 20mL samples of heparinizedblood that had been layered onto 10mL of Ficoll—Hypaquedensity gradient (Histopaque ® 1.077; Sigma Chemical Co.)and centrifuged at 450 ×  g  for 40min at room temper-ature. The separated PBMC were resuspended in GibcoRPMI 1640 medium, homogenized, washed twice with RPMI1640, centrifuged at 450 ×  g  for 10min at room tempera-ture, homogenized and finally resuspended in RPMI 1640 at10 7 cells/mL. In vitro  assays were carried out in cell culture mediumcomprising RPMI 1640 supplemented with streptomycin(100mg/mL), penicillin (100U/mL),  L -glutamine (2mM),  -mercaptoethanol (5 × l0 − 5 M) and 10% heat-inactivatedfoetal calf serum. Lymphoproliferation assays were per-formed in 96-well flat-bottomed tissue culture plates(Corning, New York, NY, USA), each well containing150  L of supplemented RPMI medium. Aliquots (25  L)of PBMC (2.5 × 10 5 cells/well) were added to triplicatewells together with 25  L of   L. braziliensis  soluble anti-gen (SLbA; 25  g/mL) or 25  L of SLcA (25  g/mL), obtainedaccording to Reis et al. [42,43], for the antigenic stimu-lus assays. In the mitogenic stimulus assays, 25  L aliquotsof PBMC (2.5 × 10 5 cells/well) were added to triplicatewells together with 25  L of phytohaemagglutinin (PHA;2.5  g/mL; Sigma—Aldrich Chemie Gmbh, Taufkirchen, Ger-many). Assay mixtures were incubated under a humidified5% CO 2  atmosphere at 37 ◦ C for 3 (mitogenic-stimulatedcultures) or 5 days (antigenic-stimulated cultures).  3 H-thymidine (1  Ci; Sigma Chemical Co.) was added to eachwell 6h before the end of the incubation period. Thecells were subsequently harvested onto glass fibre fil-ters and the incorporation of radioactivity determinedby liquid scintillation counting. Control assays were pre-pared exactly as above, employing 25  L aliquots of PBMC(2.5 × 10 5 cells/well) but with 25  L of RPMI 1640 mediumreplacing the stimulant, and were incubated for an appro-priate time. Proliferation responses were expressed in termsof mean counts/min in triplicate wells, whilst the stimula-tion index was calculated as (mean proliferation responseof cultures stimulated by SLbA or SLcA/mean proliferationresponse of unstimulated cultures).In order to investigate the immunophenotypic fea-tures, PBMC were cultured in 48-well flat-bottomed tissueculture plates (Costar, Cambridge, MA, USA), each wellcontaining 650  L of supplemented RPMI medium. Aliquots(50  L) of PBMC (5.0 × 10 5 cells/well) were added to trip-licate wells together with 100  L of SLbA (25  g/mL)or 100  L of SLcA (25  g/mL). Control assays were pre-pared as above but employing 50  L aliquots of PBMC(5.0 × 10 5 cells/well) and 100  L of RPMI 1640 mediumreplacing the stimulant. Incubations were carried out undera humidified 5% CO 2  atmosphere at 37 ◦ C for 5 days, afterwhich the PBMC were removed for immunophenotypingand the supernatants were collected for further assay asdescribed below. Nitric oxide (NO) levels in serum As an indirect measurement of NO production, nitrite levelswere determined in serum samples using the Griess reaction[45,46]. All reagents employed in the assay were purchasedfrom Sigma—Aldrich Co. Nitrate reductase (1U/mL; 10  L),6mM NADPH (10  L) and 200mM FAD (10  L) were addedto 100  L of serum diluted 1:2 in distilled water. Afteran overnight incubation at 37 ◦ C, the sample was depro-teinized by the addition of 1/20 volume of zinc sulphate(300g/L), and the mixture centrifuged at 10,000 ×  g  for15min. An aliquot (100  L) of Griess reagent (0.1% naph-thylethylendiamine dihydrochloride, 1% sulphanylamide and5% phosphoric acid) was added to the supernatant and, fol-lowing 10min incubation in the dark at room temperature,the absorbance was measured at 540nm in an automaticmicroplate reader. Each sample was assayed in duplicate.The concentration of nitrite (NO 2 − ) was determined byinterpolation from a standard curve constructed by plottingthe absorbance values of standard sodium nitrate solutionsagainst their corresponding concentrations. The correla-tion plot was linear in the range 0—100  mol/L. Data wereexpressed as means of NO production at T0 (immediatelyprior to the application of the first dose of vaccine), T1 (15days after the application of the first dose of vaccine), T2  A killed  Leishmania  vaccine with sand fly saliva extract and saponin adjuvant 627(15 days after the application of the second dose of vac-cine) and T3 (15 days after the application of the third doseof vaccine). Statistical analysis Statistical analyses were performed with the aid of Prism 4.0 software package (Prism Software, Irvine, CA,USA). Normality of the data was established using theKolmogorov—Smirnoff test. One-way analysis of variance(ANOVA) and Tukey post tests were used to investigate dif-ferences between groups with respect to humoral immuneresponses and immunophenotypic profiles. Student’s  t -testswere employed to evaluate differences in mean valuesdetermined in  in vitro  assays of humoral immune responseanti-SGE or stimulated cultures and control cultures pre-pared at T0 and T3. Associations between phenotypicfeatures in circulating leukocytes or between phenotypicfeatures and cell proliferation were investigated usingPearson’s rank correlation. In all cases, differences wereconsidered significant when  P   values were <0.05. Results Local induration was the major characteristic of adverse reaction during immunization withLBSapSal Vaccination was not associated with pain, fever, hyper-thermia, lymphadenopathy or any other general adversereactions. Furthermore, no local adverse reactions wereobserved in vaccinated animals, with the exception of mildinduration reactions in some dogs vaccinated with Sap andLBSapSal (Table 1). In spite of the presence of nodulesin some dogs that received saponin as adjuvant (Sap andLBSapSal) these did not result in the formation of ulceratedlesions. Serum from dogs vaccinated with LBSapSalexhibited elevated anti-SGE immunoglobulinisotype levels, reflecting an intense immunogenicreaction, and showed reactivity against threedifferent SGE proteins in Western blot analysis The specific anti-SGE humoral responses of the four groupsof experimental animals were determined at T0 (immedi-ately prior to the application of the first dose of vaccine)and at T3 (15 days after the application of the thirddose of vaccine). Significant ( P  <0.05) increases in theserum levels of anti-SGE total IgG, IgG1 and IgG2 wereobserved at T3 in dogs of the LBSapSal group com-pared with those of the C, Sal and LBSal groups (Fig. 1,left panels). Additional analyses revealed positive associ-ations between total IgG and IgG1 ( P  =0.0244;  r  =0.6994),between IgG and IgG2 ( P  <0.0001;  r  =0.9418) and betweenIgG1 and IgG2 ( P  =0.0174;  r  =0.7260) in the LBSapSalgroup at T0 and T3 (Fig. 1, middle panels). Westernblot analysis (Fig. 1, right panel) indicated that anti-bodies produced in dogs that had been vaccinated withpreparations containing SGE reacted with three differentproteins present in sand fly saliva. Thus, serum samplesderived from animals of the LBSapSal group showed sig-nificant reactivities against an SGE protein of molecularweight 35kDa, serum samples from the Sal, LBSal andLBSapSal groups were active against a 45kDa SGE protein,whilst serum samples from the Sal and LBSapSal groupswere active against a 71kDa SGE protein (Fig. 1, rightpanel). LBSapSal elicited an intense production of anti- L.chagasi  immunoglobulin isotypes The serum levels of anti- L. chagasi  immunoglobulins at T0,T1 (15 days after the application of the first dose of vac-cine), T2 (15 days after the application of the second doseof vaccine) and T3 (15 days after the application of the third Table 1  Local alterations in the inoculum region measured 72h after inoculation with saponin (Sap) and a candidate vaccinecomposed of   Leishmania braziliensis  promastigote protein plus saponin plus sand fly salivary gland extract (LBSapSal)Group Animal code Nodule size (cm)T1 a T2 b T3 c Sap  n =5#03 — — —#05 2.5 × 1.5 4.0 × 4.5 —#14 — — —#20 — 2.0 × 3.0 2.0 × 2.0#27 — — —LBSapSal  n =5#18 — — —#23 — — 1.0 × 1.0cm#28 — — —#31 2.3 × 1.8cm — —#38 — — — a T1, 72h after the first dose. b T2, 72h after the second dose. c T3, 72h after the third dose.
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