BRPI1006646A2 - vaccine immunogenic composition and visceral leishmaniasis immunodiagnostic test kit - Google Patents

Abstract

IMMUNOGENIC COMPOSITION FOR VACCINE AND KIT FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIOSIS. The present invention describes an immunogenic composition for vaccine and immunodiagnostic test for Visceral Leishmaniasis. More specifically, the invention comprises a vaccine and a kit for the Leishmaniasis immunodiagnostic test, developed through the identification, production and selection of new antigens by means of proteomic analysis, bioinformatics, peptide synthesis and immunoassay. The high specificity of these antigens enables the realization of an effective vaccine against Leishmaniasis and a more efficient immunodiagnostic test for canine or human Visceral Leishmaniasis.

Description

IMMUNOGENIC COMPOSITION FOR VACCINE AND KIT FOR IMMEDIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS

The present invention describes an immunogenic composition for Visceral Leishmaniasis vaccine and immunodiagnostic test. More specifically, the invention comprises a vaccine and kit for Leishmaniasis immunodiagnosis testing, developed through the identification, production and selection of new antigens by proteomic analysis, bioinformatics, peptide synthesis and immunoassay. The high specificity of these antigens enables an effective Leishmaniasis vaccine and a more efficient immunodiagnostic test for canine or human Visceral Leishmaniasis.

Leishmaniasis are diseases caused by protozoa of the genus Leishmania (Ross R 1903. (1) Note on the bodies recently described by Leishman and Donovan and (2) Further notes on Leishman's bodies. Brit Med J 2: 1261-1401), order Kinetoplastida . About 30 species are grouped in the genus, and it is accepted that approximately 21 have the ability to infect humans (Herwaldt B 1999. Leishmaniasis. Lancet v.354, n.9185, p. 1191-1199; Ashford R 2000.The leishmaniasis as emerging and reemerging zoonoses (Int J Parasitol 30: 1269-1281). The disease occurs in 88 countries with approximately 12 million people infected and 350 million people at risk of infection (http://www.who.int/leishmaniasis/burden/en/).

Over the past 10 years, endemic regions have expanded and a considerable increase in the number of reported cases of the disease has occurred worldwide. As disease reporting is compulsory in only 32 of 88 countries affected by Leishmaniasis, a large number of cases are not accounted for. In fact, it is estimated that 2 million new cases (1.5 million for Cutaneous Leishmaniasis and 500,000 for Visceral Leishmaniasis) occur annually (http://www.who.int/leishmaniasis/burden/en/).

Depending on the Leishmania species and the host immune response, there are several clinical manifestations of the disease: Cutaneous, Mucocutaneous, Diffuse, and Visceral Leishmaniasis. Visceral Leishmaniasis (VL), the most severe form of the disease, is an anthroponosis in India and Central Africa and a zoonosis in the Mediterranean region and the Americas. The zoonotic form of Visceral Leishmaniasis (VL), caused by L. infantum (also called L chagasi), represents 20% of world human visceral leishmaniasis (100,000 cases annually) and its incidence is increasing in the urban and peri-urban areas of the tropics ( Dye C 1996. The Iogic of Visceral Leishmaniasis Contracts (Am J Trop Med Hyg 55 (2): 125-30).

The dog plays an important role within the epidemiology of zoonotic visceral yeishmaniasis, as it has been considered as the main domestic reservoir for human disease (Ashford D, David J, Freire M, David R, Sherlock I, Eulalius M, Sampaio, Badaro R 1998. Studies on contraction of visceral leishmaniasis: impact of dog contraction on canine and human visceral leishmaniasis in Jacobina, Bahia, Brazil Am J Trop Med Hyg v.59, n.1: 53-7; Alvar J, Canavate C , Molina R, Moreno J, Nieto J 2004. Canine leishmaniasis Adv Parasitol 57: 1-88). The importance of dogs as a reservoir is due to the frequent contact and proximity to humans, besides the fact that the animal may present asymptomatic infection, despite the high parasitemia on the skin and viscera (Madeira M, Schubach A, Schubach T, Leal C, Marzochi M 2004. Identification of Leishmania chagasi isolated from healthy symptomatic skin and asymptomatic dogs seropositive for leishmaniasis in the Municipality of Rio de Janeiro, Brazil (Braz J Infect Dis 8: 440-444).

The dog is an excellent model for studying VL. This species is the target of control measures and presents similarity of clinical and anatomopathological alterations with human VL. The canine experimental model can be used for drug testing, identification of marker molecules of infection and identification of antigens for diagnostic, prognostic and / or vaccine use in humans. In short, advancing knowledge of canine VL (CVI) can help prevent and treat human disease (Moreno J, Alvar J 2002. Canine leishmaniasis: epidemiological risk and the experimental model. Trends Parasito /, 18 (9): 399 -05).

In the areas of VL occurrence, control measures include canine and human diagnosis, reservoir elimination and patient treatment. Therefore, early diagnosis influences treatment success and transmission control. However, canine diagnosis faces a number of difficulties in view of the available methods.

Currently, there is a wide range of tests for diagnosis of the CVL. However, each has some negative point. Complement Fixation Reaction and Indirect Immunofluorescence Reaction, for example, may result in cross reactions with Chagas Disease (CD) and American Cutaneous Leishmaniasis (ATL). The PCR reaction has limited use due to its high cost and low adaptability to the field. The enzyme-linked immunosorbent assay (ELISA) with currently used antigens also demonstrates cross-reactions.

For the immunodiagnosis of human cases, the availability of tests is small. In the Brazilian market, practically the only option for use in humans, with good sensitivity and specificity, is the Indirect Immunofluorescence Reaction (RIFI), which is not yet ideal due to the possibility of cross-reactions due to the use of crude antigen. In addition, it is a test that requires accurate technical training, since interpretation of the result is subjective in cases of low anti-Leishmania antibody titers. The implementation of IFAT is more laborious than other serological methods, there is no possibility of automation and will always require the purchase of high cost equipment (immunofluorescence microscope).

Considering that most of the available methods have disadvantages and that the occurrence of cross reactions with other infections makes a correct diagnosis unfeasible, it is necessary to search for new antigens that allow the disease to be diagnosed with greater safety, sensitivity and specificity, both in dogs as well as humans. Among the different diagnostic methods, ELISA is the most widely used due to its low cost and ease of execution.

In order to improve the available immunodiagnostic tests, some methodologies have been developed, such as those described in the following patent documents: US 2010092938 - Novel and Practical Serological Assay for the Clinical Diagnosis of Leishmaniasis1 US 2007134671 - Oligonucleotides for detection of Ieishmaniasis and methods thereof , US 5965142 - Polypeptides and methods for detection of L. tropica infection, W09633414 - Compounds and methods for diagnosis of leishmaniasis, US 5942403 - Compounds and methods for detection of t. cruzi infection, PI9610679-4 - Leishmania antigens for use in the therapy and diagnosis of Leishmaniasis, PI9300775-2 - Serological diagnosis process for Canine and / or Human Visceral Ieishmaniasis, WO 2005/063803 - Polypeptides for the diagnosis and therapy of leishmaniasis, WO 1996/033414 - Compounds and methods for diagnosis of leishmaniasis, WO 1989/001045 - Leishmania-specific antigens, process for preparing them, antigenic profiles containing these antigens and their application for the diagnosis of visceral leishmaniasis.

These documents disclose compounds comprising polypeptides containing at least one immunogenic portion of at least one Leishmania antigen, or a variant thereof, for use in Leishmaniasis immunodiagnosis. The polypeptides described herein, however, are different from those used in the present invention and have been isolated by different approaches from that used in the present invention. The present invention describes the identification, production and selection of novel antigens by proteomic, bioinformatic analysis, peptide synthesis and immunoassay, allowing for a more specific and efficient immunodiagnostic test for canine or human visceral leishmaniasis.

Since the effectiveness of VL control measures has been inconsistent, canine vaccination may be an effective way to reduce transmission (Gradoni L. 2001. An update on antileishmanial vaccine candidates and prospects for a canine Leishmania vaccine. Vet Parasitol 100 (1-2): 87-103). Different candidate immunogens for vaccine development include dead parasites, purified fractions, Leishmania recombinant antigens, and antigen-coding DNA (Melby PC. 2002. Vaccination against cutaneous leishmaniasis: current status. Am J Clin Dermatol 3 (8): 557-70). In the canine model, different VL vaccine strategies have been used. Vaccination with dead promastigotes or indefinite mixtures of antigens induce cellular immune response and partial protection against infection (Gradoni, 2001). Purified L. donovani fucose mannose (FML) ligand (Borja-Cabrera GP1 Correia Bridges NN1 da Silva VO, Paraguay de Souza E, Santos WR, Gomes EM, et al. Long Iasting protection against canine kala-azar using the FML (Saponin vaccine in an endemic area of Brazil (São Gonçalo do Amarante). Vaccine) and several different recombinant antigens obtained from purified proteins also confer at least partial protection against L infantum infection in dogs (Saldarriaga OA, Travi BL, Park W, Perez LE, Melby PC 2006. Immunogenicity of a multicomponent DNA vaccine against visceral Iemanmaniasis in dogs Vaccine 24: 1928-1940.).

The development of vaccines against this parasite is the main goal of the World Health Organization. A large number of studies have shown that different vaccine formulations offer significant protection against Leishmania in a wide variety of animal models (Kedzierski L1 Zhu Y, Handman E Leishmania vaccines: progress and problems Parasitology 2006, 133, S87-112 Drummelsmith J, Brochu V, Girard I, Messier N1 Ouellette M. Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance Molecular Cell Proteomics 2003, 2: 146-55, by Oliveira Cl, IP Birth, Barrai A, Soto M, Barral-Netto M. Challenges and perspectives on vaccination against leishmaniasis, Parasitol Int. 2009, 19.) . Currently, only four vaccines are licensed for use: one used for humans in Uzbekistan, one used for immunotherapy in Brazil, and two vaccines for prophylactic dogs in Brazil (Palatnik-de-Sousa CB. Vaccines for leishmaniasis in the fore coming 25 Vaccine 2008 Mar 25,26 (14): 1709-24] Fernandes AP, Costa MMS, Rabbit EA, Michalick MS, by Freitas E1 Melo MN, Luiz Tafuri W, Resende Dde M, Hermont V, Abrantes Cde F, Gazzinelli RT Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle dogs vaccinated with recombinant A2 protein (Vaccine. 2008, 26, 5888-5895.).

However, the effectiveness of vaccines remains partial, and therefore the development of highly effective vaccines is necessary. The development of Leishmania vaccines is a major challenge due to insufficient knowledge of the pathogenicity and complexity of the immune response required for protection. In addition, a small number of Leishmania antigens have been tested as a vaccine, suggesting that there are still many potential antigens to be evaluated. (Matlashewski, G. Leishmania infection and virulence. Microbiol Immunol. 2001, 190, 37-42 El Fakhry Y, Ouellette M1 Papadopoulou Β. A proteomic approach to identify developmentally regulated proteins in Leishmania infantum. Proteomics. 2002, 2, 1007-1117 ; Dea-Ayuela MA, Rama-Iniguez S, Bolas-Fernández F. Proteomic analysis of antigens from Leishmania infantum promastigotes Proteomics 2006, 6: 4187-4194; Chappuis F, Sundar S1 Hailu A, Ghalib H, Rijai S, Peeling RW, Alvar J, Boelaert M. Visceral leishmaniasis: What are the needs for diagnosis, treatment and control? Nat. Rev. Microbiol 2007, 5, 873-882; de Oliveira Cl, IP Birth, Barrai A, Soto M, Barral -Netto M. Challenges and perspectives on vaccination against leishmaniasis ParasitoL Int. 2009, 19; Herrera-Najera C, Pina-Aguilar R, Xacur-Garcia F, Ramirez-Sierra MJ, Dumonteil E. Mining the Leishmania genome for novel antigens and vaccine candidates, Proteomics, 2009, 9: 1293-301.).

Proteomic studies with Leishmania have shown the difference in protein expression between amastigotes and promastigotes and the possibility of using "proteomic serology" as an appropriate approach for antigenicity mapping in leishmaniasis. Recently, a study using 2-DE of L. infantum promastigote extract, followed by Western Blot with immunized rabbit serum and EM1 analysis allowed the identification of several relevant antigenic proteins (Dea-Ayuela M, Rama-Iniguez S, Bolás). -Fernández F 2006. Proteomic analysis of antigens from Leishmania infantum promastigotes (Proteomics 6: 4187-4194). In a similar approach, several L. donovani antigens were identified using sera and parasites isolated from Indian patients, thus concluding that the proteome-serology approach yields a comprehensive and highly resolved representation of the L. donovani antigenicity and the specificity of the antimicrobial immune response. leishmania in VL patients (Forgber M, Basu R, Roychoudhury K, Theinert S, Roy S, Sundar S, Walden P 2006. Mapping antigenicity of parasites in Leishmania donovani infection by proteome serology. PLoS ONE 1: e40). All of these studies used axenic culture amastigotes. The present invention demonstrates the use of promastigote protein extract for identification of antigens that will participate in the immunogenic Leishmaniasis vaccine composition.

Several patent documents demonstrating the use of Leishmania polypeptides in immunogenic vaccine composition have been found. Examples are: WO 2009143006 - Recombinant polyprotein vaccines for the treatment and diagnosis of leishmaniasis, WO 2007121184 - Compounds and methods for the diagnosis and treatment of leishmaniasis, WO 2006110915 - Vaccine formulations for leishmania, US 2006073170 - Vaccine complex for preventing or treating leishmaniasis, CA 2503932 - Polypeptides of leishmania major and polynucleotides encoding same and vaccinal, therapeutical and diagnostic applications thereof, WO 02098359 - Leishmania antigens for use in therapy and diagnosis of leishmaniasis. None of them, however, use the same proteins and / or peptides demonstrated in the present invention, besides not showing their isolation by proteomic analysis. The present invention describes the identification, production and selection of new antigens by proteomic, bioinformatic analysis and immunoassay, allowing the production of a more specific and efficient vaccine for canine or human visceral leishmaniasis.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - Protein extracts of L. chagasi promastigote forms were fractionated by 2-DE using 18 cm strips, pH 4-7 and 12% SDS-PAGE. The gels were stained with comassie (A) or transferred to nitrocellulose membranes (B, C and D) and incubated with naturally infected or uninfected dog sera (E) and stained with total anti-IgG (B and E). Peroxidase-conjugated anti-IgG2 (D) IgGI (C). Spots recognized only by infected animals and identified by MS (Mass Spectrometry) are highlighted. Figure 2 - To assess antibody reactivity to membrane synthesized peptides, the relative intensity (IR) between infected and uninfected sera for the same peptide was calculated. Peptides with IR> 2.0 were considered reactive. The proteins and peptide sequences are shown in Table 2.

Figure 3 - To assess antibody reactivity to membrane synthesized peptides, the relative intensity (IR) between infected and uninfected sera for the same peptide was calculated. We considered reactive peptides with IR> 2.0. The proteins and peptide sequences are shown in Table 3.

DETAILED DESCRIPTION OF TECHNOLOGY

Protein Obtaining

Leishmania chagasi promastigotes (MCAN / BR / 2000 / BH400) were cultured at 23 ° C in Schneider medium (Gibco BRL) supplemented with 10% fetal bovine serum (Sigma), penicillin 200U (Sigma), streptomycin IOM (Sigma) ). These forms were used in the exponential growth phase for protein extraction.

To obtain the proteins the parasites were washed 3 times in Schneider medium (Gibco BRL) with centrifugation at 200g for 5 minutes at 4 ° C. The cell pellet was homogenized in an Iise buffer (8M urea, 2 M thiourea, 4% CHAPS, 65 mM dithiothreitol - DTT, 40 mM Tris-base and GE Healhtcere protease inhibitor cocktail) at a ratio of 500 μί. Iise buffer for 109 parasites. Samples were incubated for 1 hour at room temperature and centrifuged at 20,000g for 15 minutes at room temperature. The supernatant (protein extract) was kept at -70 ° C. The protein was quantified by 2D-Quant kit (GE Healthcare, USA) according to the manufacturer's instructions.

Two-Dimensional Electrophoresis (2-DE)

The protocol described above was used (Paba J, Santana JM, Teixeira AR, Sources W, Sousa MV, Ricart CA. 2004. Proteomic analysis of the human pathogen Trypanosoma cruzi. Proteomics 4 (4): 1052-9.). was not transferred, but stained by Coomassie colloidal- CBB G-250 (Neuhoff V, Arold N, Taube D, Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear nanogram sensitivity using Coomassie Brilliant Blue G- 250 and R-250. Electrophoresis 1988 Jun; 9 (6): 255-62.).

WB stained gels and membranes (Figure 1) were digitized and the image analyzed by computer program to be normalized and to identify the position of the proteins in the gel revealed in WB1 using the isoelectric point (pl) and mass markers as reference. molecular (MM).

Membrane images were analyzed to select spots that are recognized by sera from infected animals, discarding those that are recognized by sera from uninfected animals (nonspecific reactions). The spots selected as immunogenic and without nonspecific reactions in the acute and chronic phases of canine infection were removed from the gels for identification by mass spectrometry (MS = MS).

Protein Digestion

The selected spots were removed from the gel manually. Each spot was placed in a 1.5mL tube and washed 3 times in 10ΟμΙ of 25mM ammonium bicarbonate and 50% v / v acetonitrile. After drying, the gel fragments were rehydrated with 10μl trypsin (Promega) at 20 ng / μΙ in 25mM ammonium bicarbonate for 30 minutes at 4 ° C. Excess solution was removed and the gel pieces were washed with 10 μl of the same solution without the addition of trypsin and left at 37 ° C for 16h. The extracted peptides were washed twice for 15 minutes with 30μΙ of 50% acetonitrile / 5% formic acid. Then the trypsin-digested material was concentrated in SpeedVac (Savant) to a volume of approximately 10μl and desalted by Zip-Tip® (C18 resin; P10, Millipore Corporation, Bedford1 MA). The peptides were eluted from the column in 50% acetonitrile / 5% formic acid and frozen at -20 ° C until time of use. Mass Spectrometry - MALDI-T0F-T0F

The samples were mixed to the matrix [10mg / mL R-cyano-4-hydroxycinnamic acid (Aldrich, Milwaukee, Wl) in 50% acetonitrile / 0.1% trifluoroacetic acid] and applied to the plate for analysis on 4700 proteomics analyzer ( Applied Biosystems, Foster City, CA). Both MS and MS / MS were purchased with the 200-Hz neodymium-doped nyttrium aluminum garnet (Nd: YAG) laser. The spectra were edited using the FIexAnaIysis and Biotools programs.

The spots were identified by searching the database using the MASCOT program (http: // www.matrixscience.com) and using the National Center for Biotechnology (NCBI) database. The search parameters were: molecular mass deviation tolerance between 100-200ppm, carbamidomethylation, maximum of a uncleaved cleavage site and methionine oxidation. This assay allowed the identification of 45 candidate proteins for use in diagnosing CVL, including in the acute phase of infection. Table 1 shows only the 10 of these 45 proteins that were used in the immunogenic composition for vaccine and visceral leishmaniasis immunodiagnostic test kit. Table 1 - Proteins of L. chagasi promastigote forms recognized by canine sera in WB with 2-DE gel and identified by mass spectrometry (MS / MS), selected for vaccine and visceral leishmaniasis immunodiagnosis test kit.

<table> table see original document page 12 </column> </row> <table>

Epitope Prediction

The amino acid sequences of the 45 identified proteins were processed using two distinct approaches:

1) Localization of linear B-cell epitopes by prediction by the "BCPreds" software (available at http-.//ailab.cs.iastate.edu/bcpreds/) and "ABCPred" (available at http: // www. imtech.res.ΐη / Γθρήβνθ / θόορΓβάή, from sequence similarity and alignment using mathematical models (Yang X, Yu X 2009. An introduction to epitope prediction methods and software. Rev Med Virol 19: 77-96). In silico mapping, the epitopes generated by both software were compared to select those resulting from overlapping predictions of both software simultaneously, and it was established that the size of these peptides should be between 9 and 15 amino acids.

2) Localization of linear B-cell epitopes by BEPIPRED prediction by a combination of mathematical models (Improved method for predicting linear B-cell epitopes. Jens Erik Pontoppidan Larsen, Ole Lund and Morten Nielsen. Immunome Research 2: 2, 2006). After in silico mapping, peptides with a score greater than 2.0 were selected for synthesis.

Thus, 180 peptides resulting from the overlapping of the prediction programs "BCPreds" and "ABCPred" (data not shown) and coincidentally 180 different peptides resulting from analysis by the BEPIPRED program (data not shown) were selected.

The sequences identified in the mapping indicate which peptides of each protein are most likely to interact with antibodies. It should be noted that different peptides were selected from the same protein. These were synthesized in duplicate by the Spot Synthesis method described below and tested with a pool of sera from infected and uninfected dogs by L. chagasi.

Cellulose Membrane Peptide Synthesis: Spot Synthesis Method Amino acids are deposited in minimal volume (0.6 μΙ) with the aid of an automatic micropipettor, allowing to obtain approximately 50 nanomoles of peptide per point. Multiple synthesis is performed in synthesizer (Abimed Spot Synthesis-ASP222) and the amino acid distribution plan as well as the determination of the protocols of the various peptides are defined in a Multipeps computer program (Molina F, Laune D, Gougat C, Pau B, Granier C 1996. Improved performances of Spot multiple peptide synthesis (PeptRes 9: 151-155). Free hydroxyl groups on the cellulose membrane are used as anchor points for peptide synthesis. These groups are conjugated through stable binding with 8 to 10 polyethylene glycol (PEG) 1 units in order to move the peptide away from the support and provide greater stability in peptide binding to the membrane.

Peptide synthesis begins at the C-terminus of the last amino acid of the given sequence. With the deprotection of the protective 9-fluorenylmethyloxycarbonyl-bonded group (Fmoc) by the addition of piperidine (20% in Dimethylformamide - DMF), the amine functions are recovered and can be visualized by bromophenol blue staining.

The amino acids are then activated by diisopropylcarbodiimide / 1-hydroxybenzotriazole (DIC / HOBT) (150 μΙ for each amino acid) and deposited for restarting another cycle, with activators providing a binding yield ranging from 74 to 87% per cycle. Amino acid replacement always begins with arginine, as it is the most labile amino acid, and each amino acid is deposited twice per cycle. Binding reactions are monitored by changing the coloration of the spots from blue to yellowish green.

Free or unreacted NH2 functions are acetylated (10% acetic anhydride in DMF) to prevent formation of irregular peptides or other undesirable bonds. The Fmoc protecting group of the next amino acid is again cleaved in basic medium by piperidine to verify binding by bromophenol staining. The membrane is washed with methanol and dried in fresh air. Then the membrane is reallocated on the synthesizer for another cycle.

By the Spot synthesis method, the size of the constructed peptide is limited to approximately 15 amino acids (Laune L1 Molina F, Ferrières G, Villard S, Bès C1 Rieunier F1 Chardes T, Granier C 2002. Application of the Spot method for the Identification of peptides and amino acids from the antibacterial paratope that contribute to antigen binding (J Immunol Method 267, 53-70), as doubts remain about the binding quality of very elongated or large peptides. At the end of synthesis, initially protected amino acid side groups are deprotected by the addition of trifluoracetic acid (TFA) associated with dichloromethane and triethylsilane. Finally, the peptides are covalently attached to the membrane.

The membrane constructed containing the various peptides can be analyzed by colorimetric immunoassays. The ability of synthetic peptides to bind to antibodies is assessed by immunoassays and they can be reproduced several times using the same membrane after regeneration (Frank R 1992. Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support (Tetrahedron 48: 9217-9232).

Immunoassay

Serological assays were used for sera from dogs with L. chagasi infection in the acute and chronic phases, sera from animals free of this parasitosis and also serums from dogs with Trypanosoma cruzi.

Sera from dogs in the acute phase of L. chagasi infection came from experimentally infected animals and were collected up to 60 days after inoculation. Sera from animals with LV1 T. cruzi (chronic phase) and from healthy dogs were obtained from a library.

Membrane-bound peptide immunoassay

Membranes containing the synthetic peptides were incubated for approximately 16 to 18 hours with blocking solution [PBS pH 7.4, 0.2% Tween-20, 4% sucrose and 5% BSA] at room temperature. After blocking, the membranes were washed and incubated with the test sera for 120 min at room temperature. After further washes, the conjugate (total anti-IgG, anti-IgG1, anti-IgG2 or canine alkaline phosphatase conjugated antibodies) was added and kept under stirring for 120 minutes. After three washes with PBS containing 0.2% Tween-20 and three subsequent PBS washes with stirring at room temperature, the alkaline phosphatase substrate (5-bromo-4-chloro-3-indolylphosphate p-toluidine) and the chromogen (nitroblue tetrazolium chloride) BCIP / NBT, Promega. The reaction, interrupted after three membrane washes with distilled water, can be visualized by the presence of a bluish precipitate on the most reactive peptides (Frank, 1992).

Color intensity was calculated using Image Master Platinum® (GE) software, comparing the recognition profile of a membrane incubated with a pool of uninfected dog sera against another membrane containing the same peptides, incubated with a pool of sera. infected dogs. Thus, it was possible to compare the color intensity, thus obtaining the ratio between infected and uninfected sera. Only spots with a ratio of 2 or greater are considered valid. These spots were produced again in Spot synthesis for evaluation with individual sera, both positive and negative.

At the end of the immunological assays, the membranes were documented and then subjected to a regeneration treatment for reuse. The membranes were treated with DMF, reagent A (8M urea, 1% SDS) and reagent B (acetic acid / ethanol / water 60:30:10) to remove precipitated molecular complexes on the peptides.

EXAMPLE 1: SELECTION OF SPECIFICALLY RECOGNIZED PEPTIDES THROUGH COMPARISON OF POSITIVE SERIES WITH LV NEGATIVES

After immunoassay of the membranes containing the synthesized peptides, an analysis of the relative intensity of the peptides was performed, calculated by the ratio between the color intensity of the reaction with positive and negative sera using the computer program (Platinum 7.0). Thus, it was possible to select specifically recognized peptides by comparing positive and negative LV sera (Tables 2 and 3).

The 24 peptides described in Table 2 showed no nonspecific reaction (with uninfected sera) and no cross-reaction with serum from dogs infected with T.cruzi. These peptides correspond to 16 different proteins. The relative intensity of the selected peptides is shown in Figure 2. Likewise, 29 other peptides (which recognized serum from dogs infected with L. chagasi but not sera from control animals or infected with T. cruzi) were selected and described. in Table 3. These peptides correspond to 9 different proteins. The relative intensity of the selected peptides is shown in Figure 3.

These results were summarized in Table 4.

Table 2: Proteins mapped to B epitopes and peptides synthesized using overlapping prediction programs "BCPreds" and "ABCPred". Only selected peptides after immunoassay are shown.

<table> table see original document page 17 </column> </row> <table> <table> table see original document page 18 </column> </row> <table>

Table 3: Proteins mapped to B epitopes and peptides synthesized to score greater than 2.0 using the BEPIPRED prediction program. Only selected peptides after immunoassay are shown.

<table> table see original document page 18 </column> </row> <table> <table> table see original document page 19 </column> </row> <table> Table 4 - Numerical survey of investigated proteins and peptides until selection

<table> table see original document page 20 </column> </row> <table>

In synthesis, 53 synthetic peptides with potential for sensitive and specific diagnosis of Visceral Ieishmaniasis were selected. Among these 53 peptides, 35 belong to 9 different hypothetical proteins, which means that they are quite promising for further diagnostic tests, since these hypothetical proteins had not yet been explored for this potential.

EXAMPLE 2: EFFECTIVENESS OF THE IMMUNODIAGNOSTIC TEST

In order to test the efficiency of the immunodiagnostic test, a new synthesis of the 24 selected peptides (Table 2) was performed on cellulose membranes. Each of these peptides was tested with different individual canine sera, which had varying anti-Leishmania antibody titers. With the new membranes, the test was also performed once again with T. cruzi infected canine sera for cross reaction investigation (Table 5). Table 5: Individual canine sera, with different antibody titers, tested in immunoassays involving cellulose membranes containing the 24 selected peptides.

<table> table see original document page 21 </column> </row> <table>

It was observed that the peptide recognition profile was variable among different sera. However, there were some peptides that were recognized by most of the sera tested. All 24 peptides tested were recognized by at least one individual serum (Table 5). Therefore, it can be verified that these peptides have a great immunogenic potential.

EXAMPLE 3: EVALUATION OF MOST PROMISING PROTEINS FOR VACCINE USE

To evaluate, among the identified proteins, which would be the most promising for vaccine use, all were mapped using the NetCTL program (www.cbs.dtu.dk/services/NetCTL). This program is a combined algorithm for predicting HLA class I binding, proteasome cleavage, and transporter associated with antigen processing (TAP) transport efficiency simultaneously on the amino acid sequences of a given protein (Peters, B., Bulik S. , Tamper, R., Endert, PMV and Holzhutter, HG Identifying MHC class I epitopes by predicting the TAP transport efficiency of epitope precursors (J. Immunol. 2003, 171, 1741-1749.). The ability of each protein to bind to 10 different HLA supertypes (A1, A2, A3, A24, B7, B8, B27, B44, B58 and B62) by bioinformatics was also evaluated. Thus, proteins that have a higher percentage of HLA I epitopes and / or greater ability to bind to different HLA supertypes were selected as antigens in vaccine compositions (Table 6).

Table 6 - Proteins that, according to NetCTL mapping, present higher percentage of epitopes for HLA I.

<table> table see original document page 22 </column> </row> <table>

Claims (10)

1. IMMUNOGENIC COMPOUNDS FOR VISCERAL LEISHMANIASIS, characterized in that they are selected from a group comprising at least one of the proteins or peptides represented by Seq ID Nos. 1 to 63. Immunogenic compounds for visceral leishmaniasis according to claim 1, characterized in that they generate cellular and / or humoral immune response. 3. VISCERAL LEISHMANIASIS IMMUNODIAGNOSTIC TEST KIT, characterized in that it comprises at least one of the proteins or peptides represented by Seq ID NOs 1 to 63, attached to a support. Visceral leishmaniasis immunoassay test kit according to claim 2, characterized in that the support is preferably an ELISA microtiter plate or a membrane. Visceral Leishmaniasis Immunodiagnosis Test Kit according to Claims 2 and 3, characterized in that it uses immunodiagnostic techniques selected from the group comprising ELISA, Western blot, Dot blot and Immunochromatography. 6. Leishmaniasis vaccine, characterized in that it comprises at least one of the proteins represented by Seq ID NO 56 to 63, or part thereof, in combination with at least one adjuvant. Leishmaniasis vaccine according to Claim 6, characterized in that the adjuvant is selected from a group comprising CpG-containing oligonucleotides, TLR agonists, saponins, AGPs or a combination thereof. Leishmaniasis vaccine according to claims 6 and 7, characterized in that it is administered in an amount sufficient to induce protective immune response in mammals. Leishmaniasis vaccine according to any one of claims 6 to 8, characterized in that it is administered by the intramuscular, subcutaneous, oral or parenteral routes. Leishmaniasis vaccine according to any one of claims 6 to 9, characterized in that it is for immunization, prophylaxis or treatment of mammals at risk of contracting or suffering from Leishmaniasis.