WO2010108245A2 - Use of leishmania antigens in a diagnostic method, vaccine and therapy for leishmaniasis - Google Patents

Abstract

The present invention relates to the use of different recombinant antigens obtained from Leishmania chagasi or Leishmania infantum genes in assays for identifying, detecting and quantifying specific antibodies in biological material, including serum, plasma, saliva and urine from human beings, dogs and other Leishmania vertebrate hosts. These recombinant antigens, or genes or parts of genes that encode same, can be used for diagnosing leishmaniases, both the infection and/or disease. The present invention further relates to the use of these recombinant antigens, or genes or parts of genes that encode same, or formulations containing these antigens, for the treatment and/or immunisation of human beings, dogs and other vertebrate hosts, against leishmaniases.

Description

USE OF LEISHMANIA ANTIGENS IN DIAGNOSTIC METHOD, VACCINE AND THERAPY FOR LEISHMANIASIS

Field of the Invention

 The present invention relates to the use of identified antigens, or parts thereof, from Leishmania chagasi / Leishmania infantu genetic libraries for the purpose of identifying, detecting and quantifying specific antibodies in biological material, including serum, plasma, saliva. and urine from humans, dogs and other Leishmania vertebrate hosts. These recombinant antigens or their genes or part of the genes encoding them may be used for the diagnosis of leishmaniasis, whether infection and / or disease. The present invention further relates to the use of such recombinant antigens, or genes or part of the genes. which encode them or formulations containing such antigens for the treatment and / or vaccines of humans, dogs and other vertebrate hosts against leishmaniasis.

Background of the Invention

Leishmania is a genus of protozoa in the Trypanosomatidae family. There are about 20 species of the genus Leishmania capable of causing disease in humans. These protozoans, during their life cycle, infect an inherited hematophagous vector (of the genus Phlebotomus or of the genus Lutzomyia, both of the family Psychodidae, commonly called phlebotomus) and a vertebrate host. After the protozoan is acquired by the vector insect, it transforms into the host digestive tract. invertebrate, in an elongated form with apparent flagella that is called promastigote. Within the insect, promastigote forms adhere for a period of time to the villous cells of the digestive tract lining epithelium and, after developing biochemical and morphological changes (a process called metacyclogenesis), lose their adherence. During a new blood meal metacyclic promastigote forms may be transferred to the dermis of a vertebrate host. Thereafter, promastigote forms are rapidly phagocytized by cells of the mononuclear phagocytic system and, within vesicles called phagolysosomes, acquire an oval shape, with flagella almost completely restricted to the flagellar pocket, called amastigote form. Within phagolysosomes, amastigote forms can multiply, promote host cell disruption and reach the extracellular medium. Later, amastigote forms can be internalized by new phagocytic cells present in the skin, mucous membranes, and especially in organs with abundant mononuclear phagocytic system cells, such as the spleen, liver, bone marrow and lymph nodes. During intracellular parasitism and / or protozoan release by rupture or exocytosis of the host cells, the development of inflammatory and adaptive immune response as well as structural changes of host organs may occur.

Following the introduction of Leishmanla into a vertebrate host, for example, man or dog, pending control over parasite multiplication within phagocytes, the individual may present subclinical infection or exhibit varied clinical manifestations resulting from the involvement of the skin, mucosa or viscera. Individuals who develop visceral compromised disease, called visceral leishmaniasis, tend to present mainly anemia, hypoalbuminemia, hypergammaglobulinemia, weight loss, hepatomegaly, splenomegaly and, in the absence of specific treatment, most often die. In addition, in parallel with the presentation of clinical manifestations, individuals infected with Leishmania chagasi / infantum or Leishmania donovani and, more rarely, Leishmania archibaldi, which may be identical to Leishmania donovani [Jamjoom, MB, Ashford, RW, Bates, PA, Chance, ML, Emp, SJ, Watts, PC, and Noyes, HA (2004). Leishmania donovani is the only cause of visceral leishmaniasis in East Africa; previous descriptions of L. infantum and "L. archibaldi" from this region are a consequence of convergent evolution in the isoenzyme data. Parasitology 129, 399-409], Tropic Leishmania [Alborzi, A., Rasouli, M., and Shamsizadeh, A. (2006). Tropic-isolated Leishmania patient with visceral leishmaniasis in southern Iran. Am 3 Trop Med Hyg 74, 306-307] and Leishmania amazonensis [Barral, A., Pedral-Sampaio, D., Grimaldi Junior, G., Momen, H., McAhon-Pratt, D., Ribeiro de Jesus , A., Almeida, R., Badaro, R., Barral-Netto, M., Carvalho, EM, and et al. (1991). Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease. Am J Tcop Med Hyg 44, 536-546] who develop visceral leishmaniasis, produce a large amount of antibodies reactive to various Leishmania antigens.

The diagnosis of visceral leishmaniasis is often made by finding clinical manifestations, including those mentioned above, and finding Leishmania in material aspirated from internal organs, such as bone marrow and spleen. As the search for protozoan in material aspirated from internal organs is a little sensitive method and the collection of material is necessarily made through invasive manipulations, alternatively the clinical diagnosis is confirmed by serological tests. The most commonly used serological tests, indirect immunofluorescence (IFI), enzyme immunoassay (ELISA) and direct agglutination test (DAT), use Leishmania obtained from culture as a source of antigen. As different lots of the same Leishmania isolate, grown only at different times, may exhibit distinct antigenic composition, the reproducibility of these tests, especially in terms of sensitivity, is not always assured [Nolan, TJ and R. Herman. (1985) "Effects of long-term in vitro cultivation on Leishmania donovani promastigotes." J Protozool 32 (1): 70-5; Wilson, ME, K.K. Hardin, et al. (1989) "Expression of the major glycoprotein surface of Leishmania donovani chagasi in virulent and attenuated promastigotes." J Immunol 143 (2): 678-84]. Furthermore, in diagnostic tests based on the whole protozoan or parasite extract, false positive results occur quite frequently due to cross-reactivity with other parasites [Kar, K. (1995). Serodiagnosis of leishmaniasis. Crit Rev Microbiol 21, 123-152]. Currently available serological tests based on the use of only one recombinant antigen may have variable sensitivity, since not all sick and infected individuals produce antibodies against the same parasite antigen [Brandonisio, O., Fumarola, L . , Maggi, P., Cavaliere, R., Spinelli, R., and Pastore, G. (2002). Evaluation of a rapid immunochromatographic test for serodiagnosis of visceral leishmaniasis. Eur J Clin Microbiol Infect Dis 21, 461-464; Schallig, HD, Gentleman's Corner, M., and da Silva, ES (2002). Evaluation of the direct agglutination test and the dipstick test RK39 for the sero-diagnosis of visceral leishmaniasis. Mem Inst Aldo Cruz 97, 1015-1018; and Sundar, S., Singh, RK, Maurya, R., Kumar, B., Chhabra, A., Singh, V., and Rai, M. (2006). Serological diagnosis of Indian visceral leishmaniasis: direct agglutination test versus rK39 strip test. Txans R Soc Trop Med Hyg 100, 533-537]. Thus, it is important to expand the panel of recombinant antigens, through selection and production, for the development of more sensitive and specific serodiagnostic tests.

Visceral leishmaniasis is booming in Brazil and around the world. The methods used to control visceral leishmaniasis involve the treatment of human cases, the fight against insect vector and the control of reservoir infection, such as dogs. These methods are costly and laborious and therefore often cannot be applied continuously in such a way as to promote a permanent restriction on the incidence of the disease. A vaccine against leishmaniasis capable of preventing the development of disease, infection in vertebrate hosts, such as man or dog, or preventing transmission of the protozoan from the vertebrate host to the vector insect, should contribute to disease control.

 The immune system of vertebrate animals, which is made up of organs, tissues, cells and molecules, distributed throughout the body, is capable of developing adaptive (or specific) immune responses that are efficient in controlling the multiplication of invading microorganisms and thus preventing the establishment of diseases promoted by such microorganisms [Pulendran, B. (2004). Modulating vaccine responses with dendritic cells and Toll-like receptors. Immunol Rev 199, 227-250].

 Adaptive immune responses can be grouped into humoral immune responses (which may be T independent or T-dependent) and cellular immune responses.

In T-dependent humoral immune responses, cytokine-producing CD4 T-lymphocytes such as IL-4, IL-5, IL-10 and IL-13 stimulate B lymphocytes to differentiate and produce immunoglobulins, which function as effector molecules [Mosmann, TR , and Sad, S. (1996). The expanding universe of T-cell subsets: Thl, Th2 and more. Immunol Today 17, 138-146; and Coffman, RL (2006). Origins of the T (H) 1-T (H) 2 mode ^: a personal perspective. Nat Immunol 7, 539-541] - In cellular immune responses, also called Thl-type immune responses, CD4 T lymphocytes, cytokine producers such as interferon gamma (IFN-γ), interleukin (IL-2) and tumor necrosis factor (TNF) stimulate macrophage microbicidal activity. and / or the cytolytic activity of CD8 T lymphocytes (cytotoxic lymphocytes), resulting in the destruction of microorganisms present in intracellular compartments [Mosmann, TR, and Sad, S. (1996). The expanding universe of T-cell subsets: Thl, Th2 and more. Immol Today 17, 138-146; and, Sher, A., and Coffman, R, L. (1992). Regulation of immunity to parasites by T cells and T cell-derived cytokines. Ann Rev Imnol 10, 385-409].

In vertebrate hosts, such as man and dog, besides the mouse, the latter, used as an experimental model, control over the multiplication of Leishmania (infection) and, consequently, over the establishment of disease, is associated with development of Th1 type specific cell immune response. In this type of immune response, activated CD4 (Leishmania antigen) specific T cells mainly produce interferon gamma and IL-2 cytokines, and the first one favors activation of phagocytic cells capable of destroying Leishmania within phagolysosomes by producing of nitric oxide and oxygen free radicals. Most humans suffer from visceral leishmaniasis when they are specifically treated with drugs such as pentavalent antimonials (pharmaceutical specialties: Glucantime or Pentostan), or amphotericin B, among others, develop clinical cure and independently remaining in an endemic area, therefore, subject to re-infection, do not relapse or have a new episode of disease. Acquired resistance after chemotherapy is associated with the appearance of a Thl-specific cellular immune response.

 On the other hand, dogs that develop visceral leishmaniasis and undergo specific chemotherapy treatment, most often with the same drugs used in human treatment, although they develop reduced parasitic load and remission of clinical manifestations, often relapse. after stopping drug administration, even when in the absence of reinfection [Slappendel, RJ and E. Teske. (1997) "The effect of intravenous or subcutaneous administration of meglumine antimonate (Glucantime) in dogs with leishmaniasis. A randomized clinical trial." Vet Q 19 (1): 10-3]. During the period of clinical remission, dogs tend to have a specific cellular immune response that disappears with disease relapse. These facts suggest that additional therapeutic methods, such as immunotherapy, are probably needed to induce a lasting resistance state against visceral leishmaniasis in the dog.

Visceral leishmaniasis occurs in 65 countries and is a serious public health problem, especially for India, Bangladesh, Nepal, Sudan, and Brazil. In several countries over the last decades, it has been difficult to maintain control of viscerotropic Leishmanía infection and there has been an increase in the number of cases. infection and disease in both humans and dogs.

 Both a vaccine and an effective immunotherapy against Canine Visceral Leishmaniasis (LVC), which establish a resistance state against infection, preventing the development of the disease and / or promoting sterilization of the infection and / or preventing the transmission of leishmania from dog to phlebotomus, would be very useful in controlling the endemic. Reagents capable of both prevention and treatment would be ideal. These would be highly desirable for several reasons, such as: (a) a canine vaccination and / or immunotherapy campaign would be far less costly and time consuming than the current dog elimination program, and even than a possible human vaccination program. (b) its acceptance would be wide in the communities, as it would avoid the sacrifice of dogs; (c) dogs with resistance induced by vaccination and / or immunotherapy would remain in the area, decreasing the intake of new susceptible dogs (which occurs promptly after elimination of dogs).

 For the reasons mentioned above, there is still a lack of infection control tools such as vaccines, immunotherapeutic formulations and methods for disease diagnosis and detection of infection, especially in blood donors and individuals (man and dog) from endemic areas. Accordingly, the present invention aims to eliminate said shortage.

Summary of the Invention A first object of the present invention is to provide recombinant antigens identified from Leishmania chagasi / Leishmania infantum gene libraries. More specifically, such recombinant antigens are encoded by the genes, or parts of the genes, given below: Lcil, Lci2, Lci3, Lci4, Lci5, Lci6, Lcil, LciS, Lci9, cilO, Lcill, Lcil2, and Lcil3. As illustrated in the invention such recombinant antigens may be used to identify, detect and quantify specific antibodies in biological material, including serum, plasma, saliva and urine, obtained from humans, dogs and other vertebrate hosts of Leishmania. It is thus intended to use these recombinant antigens or their genes or part of the genes encoding them for the diagnosis of leishmaniasis, whether infection or disease.

 It is a further object of the invention to use such recombinant antigens, or genes or part of the genes encoding them or formulations containing such antigens, for the treatment and / or vaccines for humans, dogs and other vertebrate hosts against leishmaniasis.

Brief Description of the Drawings

 Figure 1 is a schematic representation of the coding regions of cDNA clones containing segments with repetitive motifs.

Figure 2 is a schematic representation of the coding regions contained within fragments of genomic clones containing segments with repetitive motifs. Figure 3 shows a comparison between the sequence of the 39 amino acid repeating domains found corresponding to the inserts of clones Lci2A and Lci2B and the consensus sequence of the repeating domains included in the patented rK39 sequence. Figure 3A shows the patented consensus sequence for the rK39 repetitive domain. Figure 3B lists all repetitive domains present in clones Lcl2A and Lci2B with amino acids that diverge from the consensus sequence of rK39 are underlined. The consensus sequence derived from the recombinant Lci2B antigen domains (rLci2B) is shown in Figure 3C for illustrative purposes.

 Figure 4 shows a dipstick assay performed on a longitudinally folding flexible plastic strip (P) in its central region, where small rectangles of nitrocellulose paper (a-h) were bonded with antigens or solutions containing control proteins.

 Figure 5 shows a representative analysis of some of the selected recombinant proteins used in immunodiagnostic ELISA assays.

Figure 6 shows the result of two ELISA sets performed with the rLcilA, rLci2B, rLci4A, rLci6A, rLci7A, rLcilQA-II, rLcil2A-II, and rLcil3A antigens and rLci8A-I, rLci9A-I, and rLci9A-I antigens. rLcillA-I and sera from dogs naturally infected by Leishmania chagasi / Leishmania infantum (parasitologically confirmed cases) or from healthy dogs from a non-endemic area (negative control). Each symbol corresponds to the result, in optical density, obtained from an individual serum. The horizontal line (side by side of the graph) corresponds to the mean plus three standard deviations of the results obtained with the control group sera.

 Figure 7 shows the ELISA results performed with the rLcilA, rLci2B, rLci6A, rLci7A, rLcilOA-II and rLcil2A-II antigens and sera from human patients with visceral leishmaniasis (LV) and from healthy subjects (negative control). Each symbol corresponds to the result, in optical density, obtained from an individual serum. The horizontal line (side by side of the graph) corresponds to the mean plus three standard deviations of the results obtained with the control group sera.

 Figure 8 shows the result of a lateral flow test using an equal mixture of rLcilA and rLci2B antigens.

 Figure 9 shows the evaluation of the immune response of saline, rLci2B or rLci2B injected mice in combination with saponin. Figure 9 further shows the lymphoproliferation and IFN-γ and IL-5 production assays.

 Figure 10 shows the evaluation of the immune response of mice injected with saline, saline / saponin, pBK-C V plasmid without insert, rLci2B / saponin, pBK-C V-Lci2B or pBK-CMV-.Lci.2B followed by rLci2B / saponin. Figure 10 further shows the lymphoproliferation and IFN-γ and IL-5 production assays.

 Figure 11 shows the evaluation of the specific immune response of mice injected with saline, pBK-CMV plasmid without insert, pBK-CMV-ici2B or pBK-CMV-Lei3A.

Figure 12 shows the evaluation of the immune response of mice injected with saline, plasmid pBK-CMV without insert or a mixture of plasmids pBK-CMV-LcilA (pLcilA), pBK-CMV-Lci2B (pLci2B), pBK-CMV-2.C13 (pLci3A) and pBK-CMV-Lci4A (pLci4A).

 Figure 13 shows the immune response of dogs that were injected with saline / saponin, rLcilA / rLci2B / saponin or rLcilA / rLci2B / saponin in combination with pcDNA3.1-scca-IL-12.

 Detailed Description of the Invention

 The invention is based on the identification of L. chagasi I L. infantum proteins which were selected due to the presence of high titer antibodies against L. chagasi / L. infantum in human blood samples, which showed clinical manifestations of LV, or dogs that exhibited natural L. chagasi / L. infantum infection. The invention thus consists of the use of different antigens, Lcil, Lci2, Lci3, Lci4, Lci5, Lci6, Lci7, Lci8, Lci9, LcilO, Lcill, Lcil2, and part of these antigens, identified from one another. Leishmania chagasi / Leishmania infantum gene libraries useful for identifying, detecting and quantifying specific antibodies in biological material obtained from humans, dogs and other vertebrate hosts of Leishmania. These antigens or their genes or part of the genes encoding them can then be used for the diagnosis of leishmaniasis, either infection and / or disease.

The invention further proposes to use the antigens Lcil, Lci2, Lci3, Lci4, Lci5, Lci6, Lci8, Lci8, LcilO, Lcill, Lcil2, and Lcil3, or part of such antigens or part of the genes encoding them or formulations. containing these antigens in the treatment and / or vaccines against humans, dogs and other vertebrate hosts against leishmaniasis.

 Several prior art methods can be employed herein to obtain the polynucleotide sequences encoding the Lcil, Lci2, Lci3, Lci4, Lci5, Lci6, Lci7, Lci8, Lci9, LcilO, Lcill, Lcil2, and Lcil3 polypeptides. part of these polypeptides. Such processes include, but are not limited to: (i) DNA isolation technique using cDNA hybridization or probe genomic libraries for detection of homologous nucleotide sequences; (ii) antibody selection of expression libraries to detect cloned DNA fragments with shared structural aspects; (iii) polymerase chain reaction (PCR) on cDNA or genomic DNA using primers capable of amplifying DNA sequences of interest; (iv) computerized database searches of sequences similar to those of polynucleotides encoding Lcil, rLci2B, Lci3, Lci4, Lci5, Lci6, Lci7, Lci8, LcilO, Lcill, Lcil2, and Lcil3; and (v) chemical synthesis of polynucleotides.

In the present invention antigen identification was based on the knowledge that there is differential expression of Leishmania antigens during culture (in vitro) and during host infection (in vivo). Differential expression of Leishmania antigens is believed to be important in parasite adaptation during infection. The present invention used a strategy to identify immunoreactive antigens and thus antigens that are expressed during host infection.

In order to obtain new recombinant antigens to be evaluated in immunodiagnostic assays and / or as components of vaccine and / or immunotherapeutic method, a cDNA library and another genome of Leishmania chagasi / infantum were made. The cDNA library was made using RNA from purified hamster spleen amastigote forms while the genomic library was made using DNA from promastigote forms from Leishmania chagasi / ^' infantum ^' MHOM / BR2000 / Merivaldo2 strain. Using these libraries, and a second independently obtained second cDNA library, a set of recombinant clones encoding antigens with properties that enable them to be used for both diagnosis of visceral leishmaniasis as vaccine and / or immunotherapeutic constituents were selected.

Parasites

The Leishmania chagasi / infantum isolate used was obtained by aspiration of the human spleen with clinical manifestations of visceral leishmaniasis (VL). The patient, from whom Leishmania was isolated, resided in Jequié-Bahia, Brazil, which is an endemic area for visceral leishmaniasis. From isolation, Leishmania was maintained in the laboratory by successive passage in culture medium and liquid nitrogen freezing or successive passage in hamsters, where the infection exhibits a lethal outcome. This isolate (MHOM / BR2000 / Merivaldo2) was defined as Leishmania chagasi / ^' infantum by the isoenzyme profile.

To obtain promastigote forms, the parasites were cultured in Schneider's medium (Sigma-Aldrich, Saint Louis, MO, USA) with 20% fetal bovine serum (SBF, Invitrogen Corporation). Amastigote forms of this isolate were obtained from hamster spleen and liver, previously inoculated with 1x10 ⁸ promastigote forms of culture, submitted to a small number of successive passages intraperitoneally. For this, macerated liver and spleen tissue were centrifuged on a Percoll gradient according to the method described by Chang, KP (1980). Human cutaneous leishmania in a mouse macrophage line: propagation and isolation of intracellular parasites [Scienqe 209, 1240-1242].

Sera used

During the conduct of epidemiological surveillance activities in an endemic area of visceral leishmaniasis in Jequié-Bahia (BR), approximately one hundred domiciled or stray dogs were studied. Blood samples were collected and serum aliquots were obtained and stored at -20 ° C. In addition, Leishmania-specific humoral and cellular immune responses were evaluated in most of these dogs. Some of these animals were submitted to necropsy, and immediately after the sacrifice of each animal, aspiration of the spleen was performed. Part of the aspirated material from the spleen was transferred to tubes containing biphasic culture medium for detection of Leishmania, as previously described by Barrouin-Melo et al., 2006 [Barrouin-Melo, SM, Larangeira, DF, Andrade Filho, FA, Wheat, J., Juliao, FS, Franke, CR, Palis Aguiar, PH, Conrado dos Santos, WL, and Pontes de Carvalho, L. (2006). Can spleen aspirations be safely used for the parasitological diagnosis of canine visceral leishmaniasis? A study on asymptomatic and polysymptomatic animals. Vet J 171, 331-339].

 For selection of recombinant antigens in the Leishmania cDNA library (see description below), a four-dog serum mixture showing positive culture results for Leishmania detection and the late cutaneous hypersensitivity test (Montenegro reaction) was prepared with equal volumes of serum from each animal. For selection of recombinant Leishmania antigens in the genomic library (see description below), human serum samples with clinical manifestations of visceral leishmaniasis and infection confirmed by Leishmania finding in bone marrow aspirate were provided by Dr. Aldina Barral. , Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Salvador-Bahia. After Western blotting, a mixture of six sera with equal volume of each serum, selected by reacting separately to various antigens of promastigote forms of Leishmania chagasi, which was used to select clones from the genomic library, was prepared.

Making cDNA libraries and Leishmania genomics. chagasi / infantum. The cDNA library was made using RNA from Leishmania chagasi / infantum amastigote forms and reagents from Stratagene Corporation (Stratagene Corporation, La Jolla, USA) following the manufacturer's instructions. Briefly, total RNA extracted from amastigote forms using Trizol (Invitrogen) was used for reverse transcription. Subsequently, the synthesis of the second cDNA tape was performed. Restriction sites for EcoR I and Xho I endonucleases. were placed at the 5 'and 3' ends of Leishmania cDNA molecules. The Leishmania cDNA molecules were then ligated to two DNA fragments (right arm and left arm) of the bacteriophage λ ZAP Express, previously digested by the EcoR I and Xho I endonucleases, and the ligation product was incubated with the packaging proteins. to generate viral particles.

 The genomic library was also made using Stratagene reagents. Briefly, about 100 ng of Lemamania promastigote genomic DNA, partially digested by the Tsp509I endonuclease (New England Biolabs Inc., Ipswich, MA, USA) were ligated into the EcoR I-digested bacteriophage λ ZAP Express arms. thereafter, the ligation product was packaged to generate viral particles. Libraries were later titered before and after amplification of clones using XL1-Blue Escherichia coli.

Selection of Recombinant Antigens For selection of cDNA library antigens, petri dishes were prepared with solid NZY-1.5% Agar [0.5% (w / v) NaCl - 0.2% MgS0 ₄ 7H ₂ 0 ₂ ( m / v) - 0.5% yeast extract (m / v) - 1% hydrolyzed casein (m / v) - 1.5% agar (m / v)]. Over NZY-1.5% Agar, a layer formed from the mixture of NZY-0.7% Agar, E. coli XL1-Blue and about 1 x 10 ³ viral particles from the library was placed. Petri dishes were incubated at 42 ° C. About four hours later, nitrocellulose membranes, previously incubated with isopropyl thio-galactopyranosidium (IPTG), were placed on the top layer of culture medium and the petri dishes were incubated at 37 ° C for an additional eight hours. Nitrocellulose membranes were removed, blocked with 5% skimmed milk in 0.15 M phosphate buffered saline, pH 7.4 (PBS), with 0.05% Tween 20 (PBS-T) at 4 ° C for about 14 hours The membranes were then incubated with a dog serum mixture diluted 1: 800 in 5% skimmed milk - PBS-T for 1 h at room temperature (20 ° C - 23 ° C) under gentle agitation. Thereafter, the membranes were washed with PBS-T and incubated with peroxity-conjugated goat immunoglobulins diluted 1: 1000 in 5% skim milk - PBS-T, specific for dog IgG (Sigma-Aldrich) for more. at room temperature under gentle agitation. The membranes were washed and then developed with tetramethylbenzidine (TMB) and hydrogen peroxide in phosphate-citrate buffer. Selection of antigens from the genomic library was performed with human antibodies essentially according to the method described above. For this, 5% skimmed milk blocked nitrocellulose membranes - PBS-T were successively incubated with a 6: 1 diluted human sera mixture, 1: 3000 diluted peroxidase-conjugated rabbit immunoglobulins (Sigma-Aldrich) and peroxidase substrate. In nitrocellulose membranes, the areas corresponding to bacterial lysis plaques caused by bacteriophage λ ZAP Express clones were identified. Petri dish cylinders corresponding to bacteriophage clones inducing the production of specific antibody-reactive Leishmania recombinant polypeptides were collected. Each of these cylinders was separately transferred into an Eppendorf-type tube containing 200-500 μΐι SM buffer (0.1 M NaCl, 8 mM MgSO ₄ , 50 mM Tris-HCl, pH 7.5, and 0.01% gelatin). One to two drops of chloroform were added to each tube. To obtain isolated clones, two more cycles of lysis plate generation, nitrocellulose membrane impregnation and reaction with specific antibodies were performed. For use as a negative control in some experiments, a bacteriophage clone λ ZAP Express without Leishmania insert was obtained, following the manufacturer's guidelines (Stratagene).

 Obtaining pBK-C V phagemids

The phagemid (phagemid) were obtained according to the manufacturer's instructions (Stratagene). Briefly, E. coli XLl-Blue samples were infected with each bacteriophage clone λ separately, including the negative control clone, and the filamentous bacteriophage. helper ExAssist and then grown in NZY broth for 3 h. Following heating of the bacterial suspension for bacteriophage λ ZAP Express lysis, stranded bacteriophage samples packaged with single stranded DNA pBK-CMV displaying Leishmania insert or without insect (negative control) were used to infect E. coli XLOLR, to generate double stranded pBK-CMV DNA (dsDNA pBK-CMV). Thereafter, pBK-CMV dsDNAs were engineered as standard plasmids. Alternatively, in the case of the second cDNA library, the excision process generated clones with plasmid pBluescript SK (-) (Stratagene).

Characterization of recombinant antigens

The DNA sequences of the 5 'and 3' ends of the inserts present in the clones derived from the Leishmania chagasí / infantum genomic and cDNA libraries were obtained from sequencing reactions with deoxyribonucleotides using plasmid DNA samples. pBK-CMV obtained from bacteriophages isolated in the selection process. This sequencing was performed using universal primer oligonucleotides, such as M13F and M13R, which anneal to regions flanking the Leishmania inserts, and Applied Biosystems reagents (Applied Biosystems, Foster City, CA, USA), followed by if the recommendations the manufacturer. Reaction products were fractionated on ABI3100 sequencer (Applied Biosystems, Foster City, CA, USA). Sequencing of the ends of the pBluescript® SK (-) plasmid containing the Lcil3A insert was performed in the same manner. The identification of the inserts was performed using the sequences obtained in the preliminary sequencing in Basic Local Alignment Search Tool (BLAST) searches against trypanosomatid protein sequences available on the GeneDb database pages (www.genedb.org - "The Wellcome Trust Institute, Pathogen Sequencing Unit") and also from GenBank at NCBI (www.ncbi.nlm.nih.gov - "National Center for Biotechnology Information"). In subsequent steps, and in order to avoid redundancy, where multiple clones coding for the same protein, one or at most two clones coding for segments of each protein were arbitrarily chosen, and named with the name of the protein followed by one. capital letter. Most of the inserts contained in the selected cDNA library clones (Lci2A and Lci2B, Lci3A and Lci3B, LciâA and Lcil3A), as well as the insert of clone Lci9A from the genomic library, were then sequenced in their entirety using primer oligonucleotides. specifically annealing the internal sequences of the inserts and or deletion of internal segments by restriction enzyme digestion followed by new sequencing reaction. This sequencing was performed in part using the Alfll sequencer from Amersham Biosciences and in part the Applied Biosystems sequencer ABI3100. In the case of Lcil3A, part of the sequencing was performed by subcloning smaller internal fragments into a second cloning vector (pTZ18R, PL-Pharmacia, GenBank accession number: L08956) and sequencing with primers. M13F and M13R. For clones derived from the genomic library (Lci6A, LcilA, Lci8A, LcilOA, LclllA and Lcil2A), as well as the clones LcilA and Lci5A from the cDNA libraries, the partial sequences obtained from the ends of each insert were used in new searches with the BLAST program against L. chagasi / infantum nucleotide genomic sequences available on the GeneDb website. In the case of the 5 'end sequences, equivalent sequences (identical in practically 100% of the nucleotides) were found within regions predicted as protein coding whereas the corresponding 3' sequences aligned with nearby sequences within the same coding region or regions. neighboring intergenic / coding In all cases the 5 'and 3' sequences of the same fragment were compatible with the presence of a single chromosomal segment (or cDNA for LcilA and Lci5A) and thus delimited their ends. In most subsequent analyzes the coding regions identified by the 5 'sequences of each insert were then used.

 Subcloning in pRSET expression vector

In order to express their recombinant proteins fused to a 6-histidine tail at their amino-terminal end, which permits purification of these proteins by affinity chromatography, the inserts encoding segments or all of the proteins rLcil, rLci2, rLci3, rLci4, rLci5, rLci6, rLci7, rLci8, rLci9, rLcilO, rLcill, rLcil2 and rLcil3, contained in the selected clones, were subcloned into plasmids of the pRSET (Invitrogen) series using standard molecular biology methods. Briefly, for subcloning, each of the plasmids of interest and samples from one of the versions (A, B or C) of plasmid pRSET were subjected to double restriction endonuclease digestion. Afterwards, the products of the digestion reactions were purified after agarose gel fractionation and subjected to the binding reaction. Binding products were used to transform competent E. coli samples (DH5-alpha or TOP10) for subsequent isolation of recombinant clones containing the new plasmids. For each insert a distinct subcloning strategy was used, defined according to the occurrence of restriction sites compatible with the subcloning and the appropriate reading phase for the production of recombinant polypeptides. In some situations it was possible to subclone the inserts in their entirety while in other situations only smaller fragments were selected. In all cases, the resulting constructs encoded a fusion protein with a 32 amino acid segment (derived from the pRSET vector, MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKD, which includes the 6-stranded histidine sequence) at the amino-terminal end followed by the protein segment encoded by the recombinant DNA. Leishmania and, in between, a few amino acids encoded by the multiple cloning sites of the vector. Depending on the cloning sites used in the genomic library inserts without translation termination sites, some amino acids encoded by the vector were also included at the C-terminal end of the proteins.

 Following is an individual description of each of the subclones performed.

LcllA

 The ~ 3 kb fragment obtained by digestion with restriction enzymes BamE I and Xho I of plasmid pBK-CMV-LcilA, comprising the entire protein coding sequence plus 162 nt from the 5 'non-translatable end of its cDNA, was subcloned between the same sites as the pRSET-B vector. The resulting recombinant protein contains at its amino-terminal end, in addition to the segment encoded by the pRSET vector, an extra 54 amino acid segment encoded by the cDNA 5 'non-translatable end fragment (sequence:

 DPKHALALKLSEENTYAHRHTSLSLCALLRNPITTLLPPPPIPHAHTHTTTAAE

with a final predicted molecular weight of about 88 kDa. Lc ± 2A and Lc ± 2B

 Two distinct pBK-CMV clones with 1902 (Lci2A) and 1203 (Lci2B) nt cDNA inserts were identified. For expression of their recombinant proteins, the Lci2B insert was recovered by digestion with restriction enzymes Ba R I and Kpn I and subcloned at the same sites as plasmid pRSET-B.

Lc ± 3A and Lc ± 3B

Two distinct pBK-CMV clones with cDNA inserts with 2265 (Lci3A) and 1890 (Lci3B) nt cDNA inserts were identified. The original 2265 nt cDNA fragment (Lci3A) was subcloned as 2 fragments leading to the production of 2 fusion proteins with different antigenic properties. At first, a -1300 nt fragment was obtained by digestion with the EcoR I and Hind III enzymes and subcloned at the same sites as the pRSET-C vector (construct called pRSET-Lci3A-R22). A second ~ 1500 nt fragment was recovered by Sac I / Kpn I digestion and subcloned between the same sites as the pRSET-A vector (Lci3A-R3).

Lc ± 4A

 For expression of the respective recombinant protein the ~ 2180 bp fragment of the original cDNA was recovered by digestion with restriction enzymes BamE I and Kpn I and subcloned at the same sites as plasmid pRSET-B.

Lc ± 5A

 Two fragments of 915bp (Lci5A-I) and 468bp {c155-

II) were amplified from clone Lci5A with the same pair of oligonucleotides (5'-5'CGA GGT ACC GGC GCA GCA GCG TGA GGA GCA GGC3 'oligonucleotide; '). These fragments were cloned into the pGEM-T Easy vector (Promega) and subcloned into the Kpn I and EcoR I sites of the pRSET-C vector using restriction enzyme sites included in the oligonucleotides (underlined). Expression of the resulting plasmids in E. coli leads to the production of recombinant proteins and / or degradation products of ~ 33kDa and ~ 16kDa, respectively, for Lci5A-I and Lci5A-II.

Lei6A

A 582 bp fragment of the insert was recovered by digestion with restriction enzymes BamE 1 / Hind III and subcloned between the same sites as the pRSET-C vector. The resulting recombinant protein migrates on SDS-PAGE gel with an apparent molecular weight of ~ 30 kDa. A second fragment of about 3.8 Kb was obtained by digestion with BamE 1 / Kpn I and subcloned between the same sites as pRSET-C. This plasmid leads to the production of a ~ 40kDa recombinant protein probably generated by proteolytic cleavage. while retaining its N-terminal end, which includes the polyhistidine sequence, intact.

Lc ± 7A

 A 2429 bp fragment (which includes 1437 bp of the coding sequence plus 982 of the subsequent intergenic region) was recovered by digestion with BamE I / Sal I restriction enzymes and subcloned into the BamE I / Xho I sites of plasmid pRSET-B ( DNA end generated by Sal I digestion may bind to the DNA end generated by Xho I digestion). The resulting recombinant protein migrates on SDS-PAGE with a molecular weight of about 65 kDa.

Lc ± 8A

A 3145 bp fragment was recovered by digestion with BamH I / Xho I and subcloned at the same sites as the pRSET-B vector. The resulting recombinant protein migrates on SDS-PAGE with a molecular weight greater than 100 kDa. Variants of this protein were obtained by deletion in the pRSET-B cloned gene of multiple DNA fragments encoding the repetitive domains by partial digestions with the enzyme Bgl I. These variants were those used in the ELISA assays.

 Lc ± 9A

 A 2400 bp fragment was recovered by digestion with BamH I / Xho I and subcloned at the same sites as the pRSET-B vector generating the plasmid with the Lci9A-I insert. To obtain Lci9A-II the resulting plasmid was digested with Sal I and the DNA fragment corresponding to the vector plus part of the purified and rewired insert. In this ~ 1100 bp construct, the C-terminal half of the insert was removed. Expression of the resulting plasmids in E. coli leads to the production of recombinant proteins and / or degradation products of> 100kDa and ~ 40kDa, respectively, for LCIA9A-I and LCIA9A-II.

LcllOA

 For expression of the respective protein (rLcil0A-I) the LcilOA insert was fully recovered (~ 2600 bp) by digestion with restriction enzymes BamH I and Xho I, but with partial digestion with Xho I, and subcloned between the same sites of the plasmid pRSET-C. This protein migrates on SDS-PAGE gel with a molecular weight greater than 100kDa but during its purification is cleaved into a smaller polypeptide with an estimated ~ 70kDa weight. For expression of the second recombinant protein (rLcilOA-II), a 928 bp fragment recovered by full digestion with the same restriction enzymes was also subcloned into pRSET-C. The resulting recombinant protein migrates on SDS-PAGE gel with an apparent molecular weight of ~ 55 kDa.

Lei1IA For the expression of LcillA protein three different strategies were used. In the first of these an 1874 bp fragment was recovered by digestion with the restriction enzymes BamR I / Not I and subcloned at the same sites as the pET21-A vector (Novagen). This same fragment, plus the Xho I site at its 3 'end, was then recovered from the resulting plasmid by digestion with the BamR I / Xho I enzymes and subcloned at the same sites as pRSET-A (LcillA-I). The 1167 bp gene fragment encoding LcillA-II was site flanked amplification for restriction enzymes BamR 1 / Xho I (5'-CGA GGA TCC oligonucleotide CCC AAC GGT GGT GGC AAC AGC; 3'-CGA CTC GAG oligonucleotide AGT TTG GGG CGG CGT GTG AGG), cloned into the pGEM-T-Easy vector and subcloned into the pRSET-A BamR 1 / Xho I sites. For LcillA-III, a 933 bp DNA fragment was also site flanked for the restriction enzymes BamR 1 / Xho I (5'-GCA oligonucleotide GGA TCC CCT CAC ACG CCG CCC CAA AC; 3'-GTC oligonucleotide CTC GAG CTG CAT AAA CAC AGT CCC GTC) and cloned into pGEM-T-Easy. Then part of this (679 bp) fragment was recovered by digestion with BamR I / Not I and subcloned at the same sites as pET21-A. The same fragment, added from the Xho I site at its 3 'end, was recovered from the resulting plasmid by digestion with the BamR I / Xho I enzymes and subcloned at the same sites as pRSET-A. All recombinant fragments have at their end additional amino acids derived from the inclusion of the restriction sites required to perform the cloning steps. Lc ± 12A

 For protein expression two distinct strategies were performed. In the first, a 2800 bp fragment obtained by digestion with BamH I / Pst I was subcloned between the same sites as the pRSET-B vector (Lcil2A-I, recombinant protein apparent molecular weight> 100 kDa). In a second strategy a smaller 1600 bp fragment was recovered by digestion with BamH 1 / Nco I and subcloned at the same sites as pRSET-B (Lcil2A-II, -90 kDa molecular weight).

Lcil3A

 A 1005 bp internal fragment recovered by digestion with Pst I was initially subcloned into the Pst I site of the pTZ18R plasmid vector, oriented such that the end coding for the initial protein stretch was positioned next to the T7 promoter. The resulting plasmid was then digested with the restriction enzymes BamH I and Hind III (sites flanking the original Pst I site) and the resulting fragment subcloned between pRSET-A BamH I / Hind III sites. The resulting plasmid encodes a recombinant protein whose amino-terminal end includes the amino acids encoded by the pRSET vector plus amino acids encoded by the pTZ18R cloning sites (totaling 40 amino acids). Another 24 amino acids are included at the carboxy terminal end of the recombinant protein, encoded by the cloning sites of plasmids pTZ18R and pRSET-A.

Production and purification of recombinant antigens Assays for recombinant Leishmania protein production were performed using E. coli samples from strain BL21 (DE3) LysS (Invitrogen) transformed with one of the plasmid constructs: pRSET-icilA, pRSET-Lci2B _r pRSE -Lci3A-R3, pRSET -Lci4A, pRSET-Lci5A-I, pRSET-Lci6A, pRSET-Lci7A, pRSET-LciSA-T, pRSET-Lei9A-1, pRSET-LcilOA-I, pRSET-LcillA-I, pRSET-Lcil2A-II, and pRSET- Lcil3A following the manufacturer's instructions (Invitrogen). In addition, assays were performed for production of recombinant protein rLcilA using E. coli strain TOP10F '(Invitrogen) transformed with plasmid construct pBK-CMV-LcilA, following the recommendations of the host bacterium manufacturers. The bacterial sediments resulting from the above tests were subjected to conventional processing using lysozyme, sodium deoxycholate acid and / or ultrasonication. Urea-soluble or solubilized recombinant proteins produced by BL21 (DE3) pLysS transformed by plasmid constructs were purified by affinity chromatography using sepharose-nickel and following the manufacturer's recommendations (Sepharose Chelating Fast Flow; Amersham Biosciences, Uppsalla, Sweden). The produced recombinant TOPIOF 'protein transformed with pBK-CMV was purified by ion exchange chromatography or electroelution. Purification by electroelution was performed on sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) using a Prep Celi model 491 apparatus (Bio-Rad Laboratories, Hercules, USA) following the manufacturer's recommendations. After purification, SDS was removed from the protein by essentially as described by Tuszynski and Warren, 1975 [Tuszynski, GP and L. Warren. (1975) "Removal of sodium dodecyl sulfate from proteins." Anal Biochem 67 (1): 55-65]. Protein concentration was determined by the method described by Bradford [Bradford, Marion M. (1976). The rapid and sensitive method for quantifying microgram quantities of protein using the principle of protein-dye binding. Anal Biochem. 72, 248-254]. Protein purity was assessed by SDS-PAGE.

 Plasmid DNA preparation for immunization

Escherichia coli colonies of the TOP10F '(Invitrogen) strain transformed with one of the plasmid constructs pBK-C V-LciA, pBK-CMV-Lci2B, pBK-CMV-Lci3A, or pBK-Cci plasmid without insert were separately used to inoculate about 5 to 10 ml of Luria-Bertani broth [LB, 1% (w / v) tripotone, 0.5% (w / v) yeast extract and 1% (w / w) NaCl / v), pH 7.0] with ampliciline at a concentration of 50 mg / ml. Bacterial suspensions were incubated for eight hours at 37 ° C under constant shaking at about 300 rpm. After this period, a sample from each culture was used to inoculate a larger volume of LB broth with amplicillin. After an incubation period of about 12 to 16 h at 37 ° C under constant shaking at 300 rpm, bacterial sediments were obtained by centrifugation at 6,000 xg and 4 ° C for 15 minutes and then stored at room temperature. -20 ° C until time of use. Bacterial sediments were processed and plasmids purified by ion exchange chromatographic columns using reagents and following Qiagen recommendations (Qiagen, GmbH, Hilden, Germany). After elution of the columns, washing each DNA sample with 70% ethanol, incubation at room temperature for about 20 minutes for residual ethanol evaporation, plasmid DNA corresponding to each different construct was resuspended in deionized water (Milli-Q, Millipore , Billerica, MA, USA) to achieve a concentration of about 1.5 to 2.0 mg / ml, and stored at -20 ° C. Prior to storage, an aliquot of each sample was used to determine concentration by spectrophotometry and integrity by agarose gel electrophoresis. At the time of use, each plasmid sample was precipitated with 0.1 M NaCl and 70% ethanol. Thereafter, each sample was resuspended at the concentration of 1 mg / mL DNA in sterile 0.85% NaCl solution (Isofar, Duque de Caxias, Brazil).

Animals

 Mice of the isogenic strain BALB / c, female or of both sexes, aged six to 12 weeks, produced by the vivarium of the Gonçalo Moniz Research Center (CPqGM-FIOCRUZ) in Salvador-Bahia (BR), were used. The mice were kept throughout the experiment in the CPqGM vivarium. The animals received food and water ad libitum.

Adult crossbreed dogs, aged between 4 and 9 years, of both sexes, were obtained from the Zoonosis Control Center of Dias D'Ávila, Lauro. de Freitas and Salvador (Bahia, BR). The animals were housed in the experimental kennels of the Central Laboratory of the State of Bahia (LACEN) and the Gonçalo Moniz Research Center (CPqGM) and handled according to Good Animal Experimentation Practices. Aiming at eliminating possible pre-existing exo and endoparasitosis, about three months before the start of the experiment, the dogs were treated with three doses of praziquantel 5 mg / kg, pirantel 14.4 mg / kg and 15 mg / kg febantel (Vedbrands Saúde Animal, Jacareí, Brazil), with a seven-day interval between consecutive doses, and with amitraz (Schering-Plow Veterinary, São Paulo, Brazil) at 12.5%, in the form of baths of varying duration and intervals, determined in accordance with veterinary assessment. Animals were immunized for prevention of leptospirosis caused by two strains, distemper, adenovirus, parainfluenza, parvovirus, viral hepatitis, coronavirus (eightfold vaccine, Fort Dodge Laboratories, USA) according to the manufacturer's recommendation, approximately 40 days prior to beginning of the experiment. Male dogs underwent orchiepididectomy to facilitate handling and reduce stress resulting from aggression in female heat periods (http://www.pasteur.saude.sp.gov.br/cao/cao_03.htm) around 190 days before the start of the experiment. Initially, each animal underwent immunological and parasitological evaluation to reduce the possibility of using a naturally infected animal by L. ch.aga.si in the experiment described here. Only animals were used with negative results in the two tests mentioned above,

 Lymphoproliferation Evaluation

In assays for evaluating immune response in mice, the spleen of each animal was removed after sacrifice. Suspension of isolated splenic cells was obtained. Splenic cells were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum [(v / v), SFB, Gibco BRL Life Technologies, USA], 2 m L-glutamine (Sigma-Aldrich) and 0.01 mM 2 mercaptoethanol (Sigma-Aldrich). Cells were distributed in triplicates (3 x 10 ⁵ / well in 200 μΐ _* ) in flat-bottomed 96-well microtiter plates (Costar Corning Inc., New York, USA) and incubated for three days in the absence of additional stimulation or concanavalin A (Con A, 2 μg / mL) or for five days with total extract of Leishmania chagasi / infantum antigens (10 μg / mL) or recombinant antigen (rLcilA, rLci2B, rLci3A or rLci4A at concentrations of 0.1 μg / mL, 1 μg / mL or 10 μg / mL) at 37 ° C in a humid atmosphere with 5% (v / v) CO ₂ . Twenty μΐ supplemented RPMI 1640 containing 1 μΟ ± thymidine [H] ³⁺ (Amersham Biosciences, Uppsalla, Sweden) were added to each well. After 18 hours, the plates were stored at -20 ° C. Cells were collected on fiberglass filter paper (Packard Canberra Company, Meriden, USA) and the evaluation of emission of beta particles emitted per minute (cpm) was performed on Matrix 9600 beta counter (Packard Canberra Company). Results were expressed by proliferation indices [arithmetic mean of particle count per minute (cpm) of cells stimulated with Con A or arithmetic mean divided antigen of cpm from cells cultured with supplemented medium only].

To measure cytokine production (interferon-gamma and IL-5), splenocytes (9 x 10 ⁵ / well at 600 μΐι) were cultured in 24-well flat-bottom polystyrene plates (Costar Corning Inc.) in medium. culture without additional stimulation or with Con A at 2 μg / mL _/ crude extract of L. chagasi antigens (10 μg / mL) or recombinant antigen (rLcilA, rLci2B, rLci3A or rLci4A, at 10 μg / mL for 48 hours at 37 ° C in a humid atmosphere with 5% CO2 After this period, the supernatant was collected stored at -20 ° C until the time of use.

For measurement of dog interferon gamma production, peripheral blood mononuclear cells (CMNSP) were obtained by Ficoll-Paque Plus gradient (Amersham Biosciences, Sweden) and following the manufacturer's recommendations. CMNSP were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum [(v / v), SFB, Gibco BRL Life Technologies, USA], 2 mM L-glutamine (Sigma-Aldrich) and 0.01 mM 2 mercaptoethanol (Sigma-Aldrich). CMNSP were then distributed (1 x 10 ⁷ / well in 2 mL) into 6-well polystyrene plates (Costar Corning Inc., USA) and then cultured for 48 h without further stimulation with Con A (10 pg / mL) or crude extract of. chagasi / infantum (20 g / mL) at 37 ° C with 5% CO ₂ . Supernatants were collected and stored at -20 ° C until time of use.

Cytokine Quantification ELISAs were performed to measure the concentration of murine IFN-γ and IL-5 present in splenocyte supernatants using 96-well High Binding (Corning Incorporated Life Sciences) reagents and reagents. Pharmigen following the manufacturer's instructions. Samples were evaluated in duplicates. The cytokine concentration of each sample was determined using a calibration curve, Graph Pad computer program, PRISM, version 4.0. and making correction by the dilution factor.

 Measurement of canine IFN-γ concentration in CMNSP supernatants was performed by capture ELISA using reagents from R&D Systems (R&D Systems Incorporation, Minneapolis, USA), following manufacturer's recommendations. Samples were evaluated in duplicate. Optical density reading at 450 nm was performed on a spectrophotometer (Emax Precison Microplate Reader, Molecular Devices Corporation, Sunnyvale, CA, USA). Canine IFN-γ concentration was determined using the arithmetic mean of optical density values of duplicates with the Softmax 3.0 computer program, corrected for the dilution factor, where appropriate.

Statistical analysis

Data were analyzed using parametric analysis of variance (ANOVA) tests. When ANOVA showed a statistically significant difference, the comparison between groups, two by two, was performed by Tukey post-test. THE The value defined for statistical significance was p <0.05.

 The following Examples are to be considered as illustrative of the present invention.

Isolation of recombinant antigens

 Screening of lambda phage clones from an L. chagas! CDNA library With a set of four sera from dogs naturally infected with Leishmania chagasi / infantum, with parasitological confirmation, led to the identification of 30 positive clones. Subsequent excision of pBK-CMV phagemids from each clone and sequencing of the 5 'and 3' ends of each insert allowed the identification of four distinct genes (Lcil, Lci2, Lci3 and Lci4). The phagemid clones had inserts encoding part or all of the polypeptides corresponding to those genes. Of these clones, 24 were found for the Lcil genes (including one with the LcilA insert), 2 for Lci2 (with the Lci2A and Lci2B inserts), 2 for Lci3 (with the Lci3A and Lci3B inserts) and 1 for the Lci4 ( with insert Lci4A). Further screening of the library with a mixture of three sera from human patients with visceral leishmaniasis led to the identification of a fifth gene (Lci5) by isolating a single phagemid clone containing the LciSA insert encoding part of the Lci5 corresponding polypeptide .

A second set of recombinant clones was selected from the screening of about 30,000 lambda phage clones from an L. genomic library. chagasi with a set of six sera from people diagnosed with visceral leishmaniasis (parasitologically confirmed diagnosis). This screening led to the identification of 60 positive clones, of which 43 were used for excision of the respective phagemids and sequencing of the 5 'and 3' ends. The analysis of the sequences obtained led to the identification of 7 other genes, including Lei 6 (33 clones, including one with the Lci6A insert), Lcil (4 clones, one with the Lci7A insert), Lci8 (2 clones, one with insert Lei8A), Lci9 (1 clone with Lci9A), LcilO (1 clone with LcilOA), Lcill (1 clone with LcillA) and Lcill (1 clone with Lcil2A).

 Finally, a third screening was performed on another L. chagasi cDNA library, this time using polyclonal rabbit serum produced against proteins in the L. braziliensis 67 and 94 kDa molecular weight range. This screening led to the identification of two distinct cDNAs that code for variants of the same protein, Lcil3. One of these was arbitrarily selected and the protein encoded by it is called rLcil3A.

Most of the identified genes code for proteins that have in common a predicted high molecular weight and a series of multiple repetitive motifs, which vary between proteins in amino acid length and composition. This is true for the genes Lci2, Lci3, Lcié, Lci5, Lci6, Lci8, Lci9, LcilO and Lcil2. The proteins encoded by the other three genes mentioned above (Lcil, Lci7 and Lcil3) have their associated function. to cellular stress events. Only Lelli codes for a hypothetical protein that does not fit into these two situations. Table 1 provides a summary of the most relevant results associated with each insert. More detailed data on such inserts will be described below. Figures 1 and 2 show an insert scheme containing repetitive segments. In these figures these segments are indicated by arrows of varying length according to their size. Figure 1 shows the inserts from the cDNA library, while Figure 2 shows those derived from the genomic library. In Figure 1, for the inserts corresponding to the Lcl2 and Lcl3 genes (A and B), the scheme shows only the length of the longest isolated cDNAs that were isolated (Lci2A and Lci3A), including the C-end-encoding sections. respective proteins (in black) and the 3 'untranslated regions. For Lci4 (C) is shown the complete coding region obtained from the genomic sequence as described in the text, as well as the complete Lcl4A cDNA with its 3 'untranslated region. In the case of Lcl5 (D) the N-terminal half of its coding region, also derived from the genomic sequence, as well as the full length of the LciSA cDNA is shown. In A, B, C and D the sections present in the respective recombinant proteins are marked in gray. In the case of Lci2, Lcl3 and Lci4, amino acid sequences of isolated repeatable segments are represented, which does not mean that all segments of the same protein are identical. Figure 2 shows the genes Lci6 (A), Lci8 (B), Lci9 (C), LcilO (D) and Lci22 (E) and / or the inserts present in the recombinant clones Lci6A, Lci8A, Lci9A, LcilOA, and Lcil2A, where the representation of repetitive segments and those present in the respective recombinant proteins is the same as that shown in Figure 1. Likewise, amino acid sequences of isolated segments are shown by way of illustration only. Characterization of recombinant antigens

The Lcil protein corresponds to the L. chagasi cytoplasmic homologue of the 70 kDa heat shock protein, better known as HSP70. This protein has been reported in numerous assays aimed at identifying immunogenic proteins from different Leishmania species. The term HSP comprises different families of proteins classified according to their molecular weight and acting as molecular chaperones, that is, helping other proteins to maintain their structure / function under stress conditions (such as heat shock) or shortly thereafter. synthesis. The HSP70 family comprises abundant proteins, extremely conserved throughout evolution, which can be found in different cell compartments. The recombinant protein produced after subcloning the cilA insert into the pRSET expression vector includes the entire protein coding region plus a 52 amino acid segment at its amino-terminal end encoded by the 5 'untranslatable region of its cDNA. In the L. infantum genome (accessed through http://www.genedb.org), four distinct genes have been mapped coding for this At least three of these appear to be identical to the one encoding the Lcil protein, identified with the accession numbers LinJ28_V3.3000, LinJ28_V3.2960 and LinJ28_V3.3060. The complete nucleotide sequence of the gene fragment coding for rLc1 protein, including the 5'UTR segment present in the recombinant clone, is shown in SEQ ID NO: 1 while the respective amino acid sequence is shown in SEQ ID NO: 2.

Clones containing the Lci2A and Lci2B inserts encode the carboxy terminal end of a protein classified as an N-3 family N-kinesin (according to classification proposed by Miki, H., Setou, M., Kaneshiro, K., and Hirokawa). (2001), Ali kinesin superfamily protein, KIF, genes in mouse and human (Proc. Atl. Acad. Sci. U.SA 98, 7004-7011), previously described in L. chagasi and called LcKin. . This protein has an estimated complete molecular weight of about 230 kDa and consists basically of a motor domain at its N-terminal end, followed by a region composed of a large number of 39 amino acid repeats. The region corresponding to the first 956 amino acids of this protein, which includes the N-terminal motor domain, has already been cloned and sequenced. Of this, the segment comprising the last 46 amino acids from the non-repetitive region near the motor domain plus 242 amino acids from the repetitive segments constitute the rK39 antigen, already used in diagnostic tests against visceral leishmaniasis. The use of rK39 in the diagnosis of visceral leishmaniasis is covered by US5,411,865 and US5,719,263 and Brazilian patent PI1101114-9 (application filed May 14, 1997). The rLci2A and rLci2B polypeptides are basically comprised of 39 amino acid repeating segments with a non-repeating 76 amino acid region at their C-terminal end. Figure IA schematically depicts the cDNA nucleotide sequence encoding the rLci2A polypeptide, illustrating the various repetitive 39 amino acid segments (including the amino acid sequence of one of these), the non-repetitive C-terminal region and the 3 'non-sequence. translated. A portion of the coding region contained in the Lci2B insert that has been subcloned into the pRSET vector can also be visualized to produce a recombinant protein fused to an N-terminal polyhistidine sequence. What differentiates the Lci2A-encoded polypeptide from that of the Lci2B-encoded polypeptide is the presence of a greater number of repeating domains present in the rLci2A polypeptide (11 repeats while in rLci2B only 5 completions are found) and a DNA mentoring segment in the 3 'region not translatable into Lc ± 2B (not shown in the figure), which does not interfere with the protein sequence. The protein fragments constituting the rLci2A / rLci2B polypeptides and that comprised by the rK39 protein then appear to be distinct fragments of a single L. chagasi / infantum protein, whose partial sequence in the genome is available under accession number LinJ14_V3.1180. However, as they are distinct fragments of the same protein there is a significant variation in the sequence of the repetitive domains of so that none of the domain sequences present in those encoded by the Lci2A and Lci2B inserts fit completely within the patented consensus sequence along with rK39 and most diverge significantly at different positions. Figure 3 shows the consensus sequence of the rK3 repeat domains (Fig. 3A) and the repeat domains present in the rLci2A and rLci2B proteins, highlighting the amino acids that differ from the rK39 consensus (Fig. 3B). The consensus generated by the repetitive domains of the rLci2B polypeptide is illustrated illustratively in Figure 3C, noting that a consensus generated from the sequences of the rLci2A polypeptide domains would be even more complex. The complete nucleotide sequence of the cDNA fragment encoding the rLci2A protein is shown in SEQ ID NO: 3 and the corresponding reading matrix amino acid sequence is shown in SEQ ID NO: 4. The protein nucleotide and amino acid sequences are shown. rLci2B are represented in SEQ IDs 5 and 6, respectively.

Inserts of two clones encode Lci3 protein segments, rLci3A and zLci3B. The Lci3 protein basically consists of a 14-fold repeated amino acid sequence (hundreds of times in the most similar L. major counterpart, which has more than 3,000 amino acids), followed by an unrepeated carboxy terminal end of about 240 amino acids. . ^' The gene encoding this protein appears to be present in the L. infantum genome, but its sequencing has not yet been completed (two incomplete sequences related accession numbers LinJ34_V3.0700 and LinJ34_V3.0710). A related T. cruzi protein with similar repetitive motifs has been described [Hoft, DF, Kim, KS, Otsu, K., Moser, DR, Yost, WJ, Blumin, JH, Donelson, JE, and Kirchhoff, LV (1989). ). Trypanosoma cruzi expressions diverse repetitive protein antigens. Infect Immun. 57, 1959-1967]. The protein encoded by clone Lci3A consists of 22 14-amino acid repeats with a consensus sequence essentially identical to that of L. major and a very large sequence conservation in the non-repetitive Carboxy-terminal region (outlined in Figure 1B). The only difference between the Lci3A and Lci3B inserts is that in the latter the number of repetitions is smaller (15 in all - data not shown). The complete nucleotide sequence of the cDNA fragment Lci3A is shown in SEQ ID NO: 7 and the amino acid sequence of the corresponding reading matrix is shown in SEQ ID NO: 8. The nucleotide sequence of the first subcloned fragment in the pRSET vector {Lci3A -R22) and the sequence of the respective protein fragment (rLci3A-R22) are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. Similarly, the nucleotide and amino acid sequences of the second subcloned fragment in the expression vector, Lci3A-R3, are described in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. As shown in Figure 1B, Lci3A-R22 and Lci3A-R3 vary in the number of 14 amino acid segment repeats and the length of the non-repetitive C-terminal sequence contained in each recombinant protein. The Lci4 protein is encoded by a gene identified in the L. infantum genome sequence bank with accession number LinJ36_V3.3690. This gene codes for a highly conserved eukaryotic protein called poly-ubiquitin and previously described in other Leishmania species. This protein contains multiple repeats (nine in all in the L. infantum protein) of the 76 amino acid ubiquitin coding sequence which in eukaryotic cells is covalently linked to different proteins. Binding to ubiquitin may serve as a signal for protein degradation by the proteasome complex or for transport to different subcellular compartments. The Lci4 protein fragment encoded by the clone we have isolated, rLci4A, comprises the last two complete copies of ubiquitin plus one third of the previous copy, totaling 173 amino acids. The ubiquitin sequence is highly conserved and there are no significant differences in its amino acid sequence when compared to other eukaryotic sequences. The complete nucleotide sequence of the Lci4 gene (LinJ36_V3.3690) is shown in SEQ ID NO: 13 and the corresponding amino acid sequence is shown in SEQ ID NO: 14. The nucleotide sequence of the insert Lci4A _{f is} subcloned into the pRSET vector and used in the production of the recombinant protein as well as the corresponding amino acid sequence are represented in SEQ ID NO: 15 and SEQ ID NO: 16, respectively. Schematic representations of the complete protein sequence and the rLci4A recombinant polypeptide can be seen in Figure 1C.

The Lci5 gene is the same as represented in the L. infantum GeneDb with accession number LinJ33_V3.3230. This gene codes for a hypothetical protein whose sequence has yet to be revised, as its reading phase is interrupted by translation termination codons, although it begins with a continuous sequence of 7665 bp. A schematic representation of the first 4.5Kb of this coding sequence can be seen in Figure 1D and demonstrates its organization into repetitive segments of two distinct classes. The Lci5A cDNA encodes a portion of this region encompassing 4 of the 148 amino acid repeat domains, totaling 2025 bp. The full sequence of the ~ 7.6Kb of the first full reading frame is represented by SEQ ID NO 17 and the corresponding amino acid sequence shown in SEQ ID NO 18. The nucleotide sequence of the insert Lci5A and the amino acid sequence encoded by it are represented in SEQ ID NO: 19 and SEQ ID NO: 20, respectively. The nucleotide sequence of the first cloned fragment in the pRSET vector (Lci5A-I) and the sequence of the respective protein fragment (rLci5A-I) are shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. Similarly, the nucleotide and amino acid sequences of the second subcloned fragment in the expression vector, LC1A-II, are described in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. The two recombinant fragments totaling 305 and 156 amino acids consist of one and two copies, respectively, of the repetitive segment of the protein and have the same N-terminal end. The schematic representation of these recombinant polypeptides is also illustrated in Figure 1D.

The Lci6 protein is encoded by the same gene identified in the L. infantum genome sequence bank with accession number LinJ26_V3.1950. This gene codes for the Leishmania ortholog of the protein called GB4, a protein previously described in Trypanosoma brucei that associates with cytoskeleton microtubule proteins. In T. brucei this protein is encoded by a very large gene composed of numerous 600 bp repeats, but its mature form is 28 kDa (Rindisbacher, L., Hemphill, A., and Seebeck, T. (1993). A repetitive protein from Trypanosoma brucei which caps the microtubules to the posterior end of the cytoskeleton (Mol. Bihem. Paras tol. 58, 83-96). In L. chagasi / infantum its counterpart is also encoded by a very large gene (7599 bp), but the repeated sequences are not as conserved as in T. brucei. The complete nucleotide sequence of the Lci6 gene is shown in SEQ ID NO: 25 and the corresponding amino acid sequence is shown in SEQ ID NO: 26. The nucleotide sequence of the Lci6A fragment subcloned into the pRSET vector is used in the production of recombinant protein as well as the corresponding amino acid sequence are represented in SEQ ID NO: 27 and SEQ ID NO: 28, respectively. The schematic representation of the rLci6A polypeptide illustrating the various repetitive motifs can be seen in Figure 2A. The Lci7 protein is encoded by the same gene identified with accession LinJ08_V3.1020 in the L. infantum genome. This gene codes for the stile stress-induced protein originally described in L. major. The use of this protein for immunization purposes as a component of a Leishmaniasis vaccine is provided for in US6,365,165, US6,613,337 and US6,709,661. Its use for diagnosis, however, is not explicit in the claims of said patents. This protein will be discussed in this patent then only as regards its use for diagnostic purposes. The complete nucleotide sequence of the Lei 7 gene is shown in SEQ ID NO: 29 and the corresponding amino acid sequence is shown in SEQ ID NO: 30. The nucleotide sequence of the Lci7A _r fragment subcloned into the pRSET vector is used in production. of the recombinant protein as well as the corresponding amino acid sequence are represented in SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

The Lci8 protein is encoded by the same gene identified in the L. infantum genome sequence bank with access code LinJ32_V3.2420. The main feature of this protein is its central segment consisting of 61 repeats of 10 amino acids encoded by identical DNA fragments flanked by sites for the Bgl I restriction enzyme. This central segment is flanked by N- and C-terminal regions of 331 and 242 amino acids in extension, respectively. Lci8 has orthologs identified in the genomes of L. major and Trypanosoma species. Your T. brucei orthologs is a intracellular membrane protein possibly associated with vesicular transport (Lee, MG, Russell, DG, D'Alesandro, PA and Van der Ploeg, LH (1994). Identification of membrane-associated proteins in Trypanosoma brucei encoding an internai, EARLRAEE amino acid repeat J Biol Chem 269, 8408-15). The nucleotide sequence of the ci8 gene is shown in SEQ ID NO: 33 and the corresponding amino acid sequence is shown in SEQ ID NO: 34. The nucleotide sequence of the cloned Lci8A clone segment in pRSET and the sequence of the respective recombinant protein fragment (Lci8A-I and rLci8A-I) are represented in SEQ ID NO: 35 and SEQ ID NO: 36, respectively. The schematic representation of the Lci8 gene and clone Lc8A, illustrating the central segment and repetitive motifs can be seen in Figure 2B.

The Lci9 gene is not represented by a single coding region duly annotated in the L. infantum genomic sequences, although segments identical to the Lci9A clone end sequences are found in two distinct portions of chromosome 28. Already in L. major a single homologous gene was found encoding a protein with short N- and C-terminal ends (169 and 97 amino acids, respectively) and a central region consisting of 14 repeats of a 25 amino acid motif followed by 11 other repeats with a related 34 amino acid motif ( (F28.3010). This protein has orthologs with low homology in Trypanosoma species and in the case of T. cr zi its ortholog was evaluated for its use in the diagnosis of patients with Chagas disease (Gruber, A. and Zingales, B. (1993) Trypanosoma cruzi: characterization of two recombinant antigens with potential application in the diagnosis of Chagas ¹ disease. J. Exp. Parasitol. 76, 1-12). The nucleotide sequence of the clone Lci9A insert with 13 repeats of 25 amino acids followed by 11 repeats with 34 amino acids flanked by non-repetitive end sections is shown in SEQ ID NO: 37 and the corresponding amino acid sequence is shown in SEQ ID NO: 38. This sequence was completely subcloned into pRSET generating clone Lci9A-I. The minor segment nucleotide sequence cloned into pRSET and the sequence of the respective recombinant protein fragment (Lci9A-II and rLci9A-II) are represented in SEQ ID NO: 39 and SEQ ID NO: 40, respectively. The schematic representation of L. infantum clone Lci9A, illustrating the central segment, repetitive motifs and recombinant proteins produced can be seen in Figure 2C.

LcilO is encoded by the same gene identified in the L. infantum genome sequence bank under accession number LinJ34_V3.2360, the sequencing of which has not yet been completed. This gene codes for a hypothetical L. infantum protein and an ortholog can be identified in Leishmania genomic sequences but not T. brucei or T. cruzi. Recently the same gene (identified by its previous GeneDb accession number - LinJ34.2140) was identified in another L. infantum expression library screening with serum from humans or hamsters infected with this protozoan (Goto, Y., Coler , RN, Guderian, J., Mohamath, R., and Reed, SG (2006). Cloning, characterization, and serodiagnostic evaluatiorx of Leishmania infantum tandem repeat proteins. Infect Immun. 74, 3939-3945), however, nothing further has been described for this protein. So far nothing conclusive can be inferred from this protein from its amino acid sequence, except that it is a protein that contains multiple repetitive domains of different sizes (ranging from 68 to 198 amino acids) following a non-repetitive N-terminal region. Figure 2D shows a schematic representation of the LcilOA clone with the different repetitive domains. In the same figure we can see the segment that was included in the rLcilOA-II protein and that includes only a small fraction of the segment containing the repetitive domains. The nucleotide sequence of the LcilO coding region is shown in SEQ ID NO: 41 and the corresponding amino acid sequence is shown in SEQ ID NO: 42. The nucleotide sequence of clone LcilOA (the same as in the Lcil0A-I insert) and the amino acid sequence of the respective recombinant protein fragment are represented in SEQ ID NO: 43 and SEQ ID NO: 44, respectively. Similarly, the nucleotide and amino acid sequences of the Lcil0A-II fragment are depicted in SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

The hypothetical Lei 11 protein is encoded by the same gene identified with the accession LinJ35_V3.4030 in the L. infantum genome. This is a hydrophilic protein conserved in different Leishmania species but with possible homologues in very poorly conserved Trypanosoma species. This protein contains a probable signal peptide at its N-terminal end suggesting extracellular membrane or secreted function. It has a high isoelectric point and is rich in the amino acids proline, glutamine, alanine and serine, which make up 50% of the protein and are often organized into 3 or 4 residue repeats. The nucleotide sequence of the Lcill gene is shown in SEQ ID NO: 47 and the corresponding amino acid sequence is shown in SEQ ID NO: 48. The nucleotide sequence of the first subcloned fragment in the pRSET vector (LcillA-I) and the sequence of respective protein fragment (rLcillA-I) are shown in SEQ ID NO: 49 and SEQ ID NO: 50, respectively. Similarly, the nucleotide and amino acid sequences of the second subcloned fragment in the expression vector, LcillA-II, are described in SEQ ID NO: 51 and SEQ ID NO: 52, respectively, and their respective LcillA-III sequences are shown. in SEQ IDs NOs: 53 and 54. The resulting three recombinant fragments start after the signal peptide and comprise distinct portions of the protein. RLcillA-I comprises amino acids 47-667 of the native protein, while rLcillA-II comprises amino acids 68-450 and rLcillA-III comprises amino acids 444-667.

The hypothetical Lcil2 protein is encoded by a gene whose sequence in the L. infantum genome has the accession number LinJ29_V3.0110. This protein has orthologs identified in the genomes of L. major and Trypanosoma species. In T. brucei his ortholog appears to be the protein called Tb-292, a membrane protein that appears to associate with regions near the nucleus and the parasite's flagellar pocket. Recently this protein was identified in a bioinformatics analysis that sought to identify proteins containing repetitive domains in the L. infantum genome and the region containing the repetitive domain of this protein was recognized by sera from individuals with visceral leishmaniasis [Goto, Y. , Coler, RN, and Reed, SG (2007). Bioinformatic identification of tandem repeat antigens of the Leishmania donovani complex. Infect Immun. 75, 846-851]. The L. infantum protein is composed of an N-terminal region of about 160 amino acids followed by a portion containing 30 repetitive 8 amino acid motifs and, at the end, a much larger C-terminal region containing possible transmembrane motifs and totaling about 2,000 amino acids. The complete nucleotide sequence of Lcil2 is shown in SEQ ID NO: 55 and the corresponding amino acid sequence is shown in SEQ ID NO: 56. The nucleotide sequence of the first subcloned fragment in the pRSET vector (Lcil2A-I) and the sequence. of the respective protein fragment (rLcil2A-I) are shown in SEQ ID NO: 57 and SEQ ID NO: 58, respectively. Similarly, the nucleotide and amino acid sequences of the second subcloned fragment in the expression vector, Lcil2A-II, are described in SEQ ID NO: 59 and SEQ ID NO: 60, respectively. The two recombinant fragments start at the first of 30 repetitive motifs, include the entire repetitive segment and part of the C-terminal region. The two proteins vary at their C-terminal end and total 928 and 534 amino acids for the rLcil2A-I and rLcil2A-Il polypeptides respectively. Figure 2E shows a schematic representation of the rLcil2A protein and the two recombinant proteins generated from the Lcil2A-I and Lcil2A-II inserts.

The cil3A insert codes for part of a protein that corresponds to a mitochondrial homologue of the L. chagasi / infantum HSP70 heat shock protein. The full coding sequence for Lcil3 is identified in the L. infantum genome with accession number LinJ30_V3.2530. Although the respective Lcil3 protein also belongs to the HSP70s family, the identity between the amino acid sequence of Lcil3 and Lcil (another HSP70 family protein) is less than 50%. ^' The Lcil3 protein is encoded by one of four L. chagasi / infantum mitochondrial HSP70 genes which, while not identical, encode proteins with a homology level of 94% or greater (Campos, RM, Nascimento, M., Ferraz, JC, Pereira, MM, Rocham PO, Thompson, GM, Cysne-Finkelstein, L., Figueiredo, RC, by Melo Neto, OP (2008). Distinct mitochondrial HSP70 homologues conserved in various Leishmania species suggest novel biological functions. ol. Biochem. Parasitol 160, 157-162]. The complete nucleotide sequence of the Lcil3 gene is shown in SEQ ID NO: 61 and the corresponding amino acid sequence is shown in SEQ ID NO: 62. The nucleotide sequence of the subcloned fragment in the pRSET vector. (Lcil3A) and the sequence of the respective recombinant protein fragment (rLcil3A) are shown in SEQ ID NO: 63 and SEQ ID NO: 64, respectively.

 Dipstick assays for anti-elshmanla antibody detection

Nitrocellulose paper discs were individually sensitized to the rLcilA, rLci2B, rLci3A, rLci3B and rLci5A polypeptides as described below. Sample of each Lambda ZAP bacteriophage clone encoding information for each of the rLcilA, rLci2B, rLci3A, rLci3B and rLci5A polypeptides was mixed with E. coli suspension, agar-containing culture medium and isopropyl-pD-thiogalactoside (IPTG) and then , was placed in a petri dish for production of each encoded recombinant protein. The amount of bacteriophages was previously determined by titration to cause confluent lysis plaques in the bacterial layer at the end of an incubation period of 4 hours at 42 ° C and 12 hours at 37 ° C. After a four hour incubation period of the bacteriophage-bacterial mixture, nitrocellulose paper discs were juxtaposed to the agar surface and incubated for 12 hours at 37 ° C to be sensitized with the recombinant proteins produced. As a negative control of the solid phase bound antigen, nitrocellulose discs were sensitized with agar plate lysates in which E. coli was infected by non-recombinant lambda ZAP bacteriophage. Promastigotes of L. chagasi / L. infantum, obtained from stationary phase cultures in Schneider's medium for insect cells containing 10% serum fetal cattle were lysed at 4 ° C (as a source of Leishmania antigen) and used to sensitize nitrocellulose paper at the concentration of 20 μg.mL ^-'L PBS in a 16 hour incubation at 4 ° C. Possibly protein binding sites remaining on nitrocellulose discs sensitized with recombinant or control antigens were blocked by incubation with 5% (w / v) PBS skimmed milk powder (PBS-L) for at least one hour at room temperature. Small (8 x 2 mm) rectangular pieces of nitrocellulose discs sensitized with the different antigens were cut and glued transversely on a single flexible plastic tape (8 x 700 mm dipstick) to form a matrix of insolubilized antigens arranged side by side, to be simultaneously tested in the dipstick test. The dipstick matrix was formed by: (i) rLcilA, rLci2B, rLci3A, rLci3B and rLci5A polypeptides in E. coli lysate; (ii) negative control of E. coli lysate; and (iii) positive control of Leishmania lysate. Sixty-one sera from parasitologically confirmed visceral leishmaniasis patients, and 14 sera from healthy non-endemic individuals were tested on the matrix of antigen-sensitized nitrocellulose scraps described above. This was done as described below. The antigenic matrix was incubated with 1: 400 serum dilutions in PBS-L containing 0.05% Tween 20 (PBS-LT) for two hours at room temperature. After four washes with PBS-T, antibody binding to antigens was revealed by successive incubations with antigen antibodies. Peroxidase conjugated human IgG Fc (specific for IgG) and a mixture of hydrogen peroxide and diaminobenzidine chromogen (Sigma-Aldrich) as described above. Staining of one of the nitrocellulose pieces of paper that formed the antigenic matrix was considered a positive reaction as shown in Figure 4.

 The test results of 61 sera from visceral leishmaniasis patients and 14 non-endemic healthy individuals in the dipstick assay showed sensitivity, calculated from these results, to 73.8% and 63 rLci2B and rLci5A antigens. , 9%, respectively, and specificity for each of the antigens was 100%.

Expression and purification of recombinant antigens

 Electrophoretic analysis of recombinant antigens produced and purified as described above, performed to assess the purity of these antigens, is illustrated in Figure 5. The results shown in this figure are representative of those observed for the other antigens. In some cases more than one band is observed which probably represents degradation products of the synthesized polypeptide, since they are not found with the other recombinant proteins.

 Use of recombinant antigens in ELISA immunodiagnostic assays

Wells of 96-well polystyrene microtiter plates (Cliniplate, Thermo Labsystems, Helsinki, Finland) were sensitized with antigen diluted in 100 μΐ of 0.1 M sodium carbonate-bicarbonate buffer (15 mM Na ₂ HCl ₃ and 28 mM NaHCO ₃ , pH 9.6) for 16 hours at 4 ° C. Total extract of Leishmania chagasi / 'infantum antigens or purified Leishmania recombinant antigens were used at concentrations of 10 μg / τL and 2 to 5 μg / mL, respectively. Non-antigen occupied protein binding sites were blocked with 200 L / well 5% skimmed milk in PBS (m / v) for 1 h at 37 ° C. The wells were washed three times with PBS-T. One hundred microliters of diluted sera [human 1: 1,600, canines 1: 400 (Figure 13), 1: 400 or 1: 200, and mouse 1: 200, Figures 10 and 11, 1: 5,000, Figure 9] in PBS-T containing 10% skim milk, were added to the wells in duplicates. The plates were incubated for 1h at 37 ° C. Negative control and positive control serum samples were used in each assay. The microtiter plate wells were washed three times with PBS-T. 100 μΐι per well of human IgG-class (diluted 1: 15,000), canine [diluted 1: 1,000 (Figure 13) or 1: 1,200 (Figure 6) or murine (diluted 1: 400 or 1: 4,000, were different lots of peroxidase-conjugated conjugate (Sigma-Aldrich or Jackson Immunoresearch) as appropriate for the assay. The plates were incubated for 1 h at 37 ° C. The wells were washed 3 times with PBS-T and the reaction was revealed by the addition of 100 µl tetramethylbenzidine (TMB; Sigma-Aldrich) in citrate / acetate buffer for 30 minutes at room temperature and in the dark. The enzymatic reaction was stopped by the addition of 50 µl of 4 M sulfuric acid. Optical density reading was performed 3 with 450 nm filter. Results were expressed as reciprocal and geometric means of reciprocal serum titers.

 ELISA results with sera from dogs naturally infected with Leishmania chagasi and sera from control dogs, initially with rLcilA, rLci2B, rLci4A, rLci6A, rLcilOA-II, rLci7A, rLcil2A-II and rLcil3A antigens, and then with the rLci5- antigens. I, rLci8A-I, rLci9A-I and rLcillA-I are shown in Figure 6. The sensitivities and specificities of these ELISAs for each recombinant antigen in the sera groups tested are shown in Table 2.

 The ELISA results for 43 sera from visceral leishmaniasis patients and 50 from healthy individuals with the antigens rLcilA, rLci2B, rLci6A, rLci7A, rLcil0A-I and rLcil2A-II are shown in Figure 7.

 Side flow assays for detection of anti-Lelshmanla antibodies

To further illustrate the use of the invention for detection of antibodies against recombinant polypeptides, a patented lateral-flow assay platform (Chembio, USA) was used to develop a kit that allowed detection of canine antibodies against recombinant polypeptides. . An equal parts mixture of rLcilA and rLci2B antigens was used to sensitize the bottom of a receptacle on the kit platform. 5 L volumes of undiluted serum from 23 dogs with parasitologically confirmed visceral leishmaniasis, and from 10 control dogs from a non-endemic area, were applied to a receptacle on the kit platform, followed by 40 μΐι of a wash solution. After an incubation period of 5 minutes, the formation of visible lines on the kit platform indicated positive reactions.

 An example of the results obtained with the

"lateral flow" produced against control dog sera and dogs naturally infected with Leishmania chagas! (formation of one and two violet bands, respectively), are illustrated in Figure 8. Table 3 presents a summary of the results obtained with the set of sera analyzed.

 Induction of immune response in mice by immunization with recombinant plasmids and / or antigens

 Groups of six to 18 female or male and female mice aged 6-12 weeks were injected three times, with 21 days between each series of consecutive injections, with:

 (i) 200 µL of saline or 200 µL of 500 µg / ml rLci2B polypeptide in 500 µg / ml saline or saponin, 100 µL of each flank subcutaneously;

ii) 200 pL of saline, 200 pL of 500 pg / ml saponin saline, 200 pL of 500 pg / ml rLci2B polypeptide and 500 pg / ml saponin, 50 pg of pBK-CMV plasmid without insert or 50 pg pBK -CMV-Lci2B. In addition, one group was injected twice with 50 pg pBK-CMV-Lci2B and once with 200 pL of 500 pg / ml rLci2B polypeptide and 500 pg / ml saponin, with 21 days between each two consecutive injections. Saline, saline / saponin and rLci2B / saponin injections were performed subcutaneously whereas plasmid injections were performed by muscle followed by local electroporation (Mir, LM, MF Bureau, et al. (1999) "High-efficiency gene transfer into skeletal muscle mediated by electric pulses." Proc Natl Acad Sci USA 96 (8): 4262-7; Lucas, ML and R. Heller. (2001) "Immunomodulation by electrically enhanced delivery of IL-12 encoding DNA to murine skeletal muscle." Mol Ther 3 (1): 47-53] ;

 iii) 50 L of saline, 50 µl of pBK-CMV without insert or pBK-CMV-ic3A. Injections were performed by muscle, followed by local electroporation;

 (iv) 50 μL of saline, 200 g of pBK-CMV plasmid without insert or 200 pg of a mixture of equal amounts of plasmids pBK-CMV-LciA, pBK-CMV-Lci2B, pBK-CMV-Lci-M and pBK -CMV- c14A. Injections were performed by muscle followed or not by local electroporation.

 ELISAs for measurement of IgG1 and IgG2a subclass antibody titer against Leishmania antigens were performed essentially as described above, however using mouse IgG1 or IgG2a specific mouse antibodies (Sigma-Aldrich) and a conjugate avidin with peroxidase (Sigma-Aldrich).

Mice injected with the rLci2B polypeptide produce antibodies specific for the IgG class and IgG2a and IgG1 subclasses, as shown in Figure 9. Additionally, these animals develop lymphoproliferation and production of interferon gamma as well as interleukin-5 (IL-5), after specific stimulation in vitro. These results were obtained by ELISA assays performed with rLci2B polypeptide, mouse sera and peroxidase-conjugated anti-IgG (IgG), anti-IgG1 (IgG1) or anti-IgG2a (IgG2a) antibodies. Results of splenocyte interferon gamma (IFN-γ) and interleukin-5 (IL-5) lymphoproliferation assays were obtained after in vitro stimulation with the rLci2B polypeptide. Still in Figure 9 and following figures, the columns and bars correspond to mean and standard deviation (X + SD), respectively, of values obtained with at least five mice per group. IL-5 concentration was evaluated in splenocyte supernatant pool (* p <0.05).

Mice injected with plasmid encoding rLci2B (pBK-CMV-Lci2B) or pBK-CMV-Lci2B (administered by electroporation) and subsequently injected with the rLci2B polypeptide, as described herein, produced, respectively, a small and moderate amount of specific antibodies. of the IgG class (Figure 10). Animals injected with pBK-CMV-Lci2B or pBK-CMV-Lci2B and rLci2B alone developed antibodies specific exclusively and predominantly of the IgG2a subclass, respectively (Figure 10). Moreover, these animals have a tendency to develop lymphoproliferation and interferon gamma production, but not IL-5, following specific in vitro stimulation. In these results, ELISAs were performed with total antigen extract of. chagasi / L. infantum. Mice injected with plasmid encoding rLci3A (pBK-C V-Lci3.¾), as described above, developed IgG class specific antibodies, with a moderate amount of IgG2a subclass antibodies and a small amount of IgGl subclass antibodies (Figure 11). These results were also obtained by ELISA assays performed with total antigen extract of. nasturtiums! / L. infantum.

Mice injected with a mixture of plasmids encoding rLcilA, rLci2B, rLci3A and rLci4A (followed by electroporation - EL - or not) produced IgG class and IgG2a subclass antibodies (Figure 12A, Figure 12B _f Figure 12C). In these assays the ELISAS results were also performed with total extract of L. chagasi / L. infantum antigens.

 Induction of immune response in dogs by recombinant rLcilA and rLci2B in association with saponin

Groups of dogs were formed with one male and two females (Gl), one male and three females (G2) and one male and two females (G3). These animals were injected three times, on days 0, 21 and 42 of the experiment, subcutaneously, with 1 mL of saline with 1 mg of saponin (Gl) or 200 g of rLcilA, 200 μg of rLci2B and 1 mg saponin (G2). and G3), and with 0.5 ml of saline (Gl and G2) or 800 μς of single-stranded canine interleukin-12 plasmid (pcDNA3.1-scca-IL12, from Santos, LR, SM Barrouin-Melo, Chang , Y.F., Olsen, J.; McDonough, SP; Quimby, F.; dos Santos, WL Carvalho, L.C; Oliveira, GG (2004) Recombinant single-chain canine interleukin 12 induces gamma mRNA interferon expression in peripheral blood mononuclear cells of dogs with visceral leishmaniasis. Vet Immvmol Immunopathol 98 (1-2): 43-8] directly into the draining popliteal lymph node to promote intense biological activity.

 Dogs injected with LcllA and c ± 2B associated or not with single-chain canine IL-12 encoding plasmid produced reactive IgG class antibodies with crude Leishmania antigen extract (Figure 13). In these assays the ELISAS results were performed with total extract of L. chagasi antigens. infantum (crude extract), rLcilA or rLci2B, dog sera and peroxidase-conjugated anti-IgG antibodies. In the figure, each symbol and bars correspond to the value obtained for each dog and the arithmetic means of the groups, respectively (* p <0.05).

Thus, it is found that the present invention involves thirteen individual DNA molecules encoding Lcil, Lci2B, Lci3, Lci4, Lci5, Lci6, Lci7, Lci8, Lci9, LcilO, Lcill, Lcil2 and Lcil3 proteins in the Leishmania chagasi / Leishmania protozoan infantum and their use, or the use of recombinant proteins encoded by them, for diagnostic, therapeutic and vaccine applications. In the present invention, individual DNA molecules, as well as the proteins they encode, are used to prevent infection by Leishmania chagasi / infantum and other Leishmania species capable of causing disease in man or other mammals, including those of major importance. to treat diseases resulting from these infections. Recombinant proteins encoded by the DNA molecules are used for the detection of specific antibodies in biological samples, including serum, plasma, urine and saliva, obtained from humans and other mammals, particularly dogs, for the purpose of determining the diagnosis of visceral leishmaniasis or for the detection of infection. caused by Leishmania.

Table 1

 Protein Preliminary Identification GeneDB Accession Number Polypeptide Accession Number

 V3 protein on GenBank. recombinant

 Deposit available on 30/04/2007 available

Thermal shock protein LinJ28_V3.3060 XP 001470324 rLcilA HSP70 LinJ28_V3.2960

 LinJ28 V3.3000

 Lci2 N-Kinesin LinJ14_V3.1180 XP_001464298 rLci2B

 Partial Sequence Incomplete Sequence

 homology <100%.

 Hypothetical protein Sequence similar to: XP_001468457 rLcí3A-R22 repetitive motifs Li J34_V3.0700 Incomplete sequence rLci3A-R3

 LinJ34 V3.0710 homology <100%.

 Lci4 Poly ubiquitin LinJ36 V3.3690 Not Available rLci4A

Lci5 I Hypothetical protein with LinJ33_V3.3230. Contains Not Available rLci5A-I,

 repetitive motifs other out-of-phase CDS rLci5A-II

Lei6 I LinJ26-associated protein V3.1950 XP 001470534 rLci6A-I cytoskeleton GB4 rLci6A-II

Lci7 I LinJ08 V3.1020 XP Related Protein 001463435 rLci7A stress Stil

 Lci8 I Hypothetical Protein with LinJ32 V3.2420 XP 001467919 rLci8A

 repetitive motives

 Lci9 I Hypothetical protein with Not noted in L infantum Not available rLci9A-I, repetitive motifs Ortholog of LmjF28.3010. rLci9A-II

Hypothetical protein LinJ34_V3.2360 XP_001468598 rLcil0A-I, repetitive motifs Incomplete Sequence Incomplete Sequence rLcilOA-II

Lcill I Hypothetical protein LinJ35 V3.4030 XP 001469205 rLcillA-I, rLcillA-II signal peptide and

Lcil2 I Hypothetical Protein with LinJ29 V3.0110 XP 001466517 rLcil2A-I

 repetitive motifs rLcil2A-II

Thermal Shock Protein LinJ30 V3.2530 XP 001467098 rLcil3A HSP70 Mitochondrial

Table 2

a The specificity of anti-Leishmania antibody detection was determined by the use of serum samples from dogs with Demodexis (Demodex canis), Babesiose (Babesia canis) and Ehrlichiose (Anaplasma platys) diagnosed by parasitological examination. Values in parentheses correspond to number of positive results / number of samples tested. Serum samples from 14, 15 ^c or ^d 5 healthy dogs and non - endemic area for leishmaniasis were used to define the "cut off". Table 3

Proportion of sera from dogs infected with Leishmania chagasi (n = 23) and uninfected dogs (n = 10) reacting with an equal parts (mass / mass) mixture of rLcilA and rLci2B antigens in the lateral flow test.

To facilitate information about the sequences of the invention, Table 4 shows the sequence identifier numbers with additional information about them.

 Table 4

SEQ ID NO Additional Information Location

1 5'UTR Nucleotide Sequence (1) ... (162)

(LcilA) cDNA fragment including CDS (163) ... (2127)

 part of 5'UTR and CDS.

 2 Amino acid sequence of

 (rLcilA) recombinant fragment.

 3 CDS Nucleotide Sequence (1) ... (1575)

 (Lci2A) cDNA fragment including 3'UTR (1576) ... (1881) part of CDS and 3'UTR.

 4 Amino acid sequence of

 (rLci2A) recombinant fragment.

 5 CDS Nucleotide Sequence (1) ... (882)

 (Lci2B) cDNA fragment including 3'UTR (883) ... (1188) part of CDS and 3'UTR.

 6 Amino acid sequence of

 (rLci2B) recombinant fragment.

 7 CDS Nucleotide Sequence (1) ... (1633)

 (Lci3A) cDNA fragment including Lc ± 3A-R22 (2) ... (1282) part of CDS and 3'UTR. c ± 4A-R3 (800) ... (1630)

 3'UTR (1634) ... (2242)

8 Amino Acid Sequence of

 (rLci3A) recombinant fragment.

 9 Nucleotide Sequence of

 (Lci3A-R22) gene fragment encoding

 the recombinant protein

 10 Amino Acid Sequence of

 (rLci3A-R22) recombinant fragment

 11 Nucleotide Sequence of

 (ci3A-R3) gene fragment encoding

 the recombinant protein

 12 Amino Acid Sequence of

 (rLci3A-R3) recombinant fragment

 13 CDS Nucleotide Sequence (1) ... (2055)

 [cie] complete gene including CDS and 3'UTR (2056) ... (3696)

 3'UTR.

 14 Amino Acid Sequence

 (Lci4) complete.

 15 CDS Nucleotide Sequence (1) .. - (522)

 (Lci A) cDNA fragment including 3'UTR (523) ... (2163) part of CDS and 3'UTR.

 16 Amino Acid Sequence of

(rLci4A) recombinant fragment. 17 LclSA Nucleotide Sequence (1456) ... (3480)

(Lcl5) I is ^the complete CDS. Lcl5A-I (2482) ... (3396)

 LCIA-II (2,882) ... (2941)

R 18 amino acid sequence

 (Lci5) complete CDS.

 19 Nucleotide Sequence of

 (Lci5A) cDNA

 20 Amino Acid Sequence of

 (rLci5A) fragment encoded by

 cDNA.

 21 Nucleotide Sequence of

 (Lci5A-I) 1st recombinant fragment.

 22 1st amino acid sequence

 (rLci5A-I) recombinant fragment.

 23 Nucleotide Sequence of

 (LC5A-II) 2nd recombinant fragment.

24 of ^the second amino acid sequence

 (rLci5A-II) recombinant fragment.

 25 c ± 6A Nucleotide Sequence (802) ... (4641)

(c16) complete gene.

 26 Amino Acid Sequence

 (Lci6) complete.

 27 Nucleotide Sequence of

 (Lci6A) gene fragment encoding

 for recombinant protein.

 28 Amino Acid Sequence of

 (rLci6A) recombinant protein.

 29 Lc ± 7A Nucleotide Sequence (303) ... (1736)

(Law 7) complete gene.

 30 Amino Acid Sequence

 (Lci7) complete.

 Nucleotide Sequence of

 (Lci7A) gene fragment encoding

 for recombinant protein.

 32 Amino Acid Sequence of

 (rLci7A) recombinant protein.

 33 Lci8 LC18A-I Nucleotide Sequence (580) ... (3723)

(Lci8) of the complete gene

 34 Lci8 Amino Acid Sequence

 (Lci8) complete

 35 Nucleotide Sequence of

 (Lci8A-I) gene fragment encoding

 the recombinant protein.

 36 Amino Acid Sequence of

 (rLci8A-I) recombinant protein.

 37 Lc ± 9A-I Nucleotide Sequence (2) ... (2398)

(Lci9A) complete gene fragment. Lc ± 9A-II (2) ... (1099)

38 Amino Acid Sequence of

 (Lci9A) protein encoded by

 gene fragment.

 39 Nucleotide Sequence of

 (Lci9A-II) fragment encoding the

recombinant protein. 40 Amino Acid Sequence of

 (rLci9A-II) recombinant protein.

 41 Cl10A-I Nucleotide Sequence (1210) ... (3816)

(LcilO) CDS incomplete. cl10A-II (1210) ... (2148)

42 Amino Acid Sequence of

 (LcilO) CDS incomplete.

 43 Nucleotide Sequence of

 {LcilOA-1) gene fragment encoding

 for recombinant protein.

 44 Amino Acid Sequence of

 (rLcilOA-I) recombinant protein.

 45 Nucleotide Sequence of

 {LcilOA-II) gene fragment encoding

 for recombinant protein.

 46 Amino Acid Sequence of

 (rLcilOA-II) recombinant protein.

 47 IcillA-I Nucleotide Sequence (357) ... (2220)

(Lcill) CDS. LcillA-II (420) ... (1568)

 czllA-III (1548) ... (2220)

48 Amino Acid Sequence of

 (Lcill) complete protein.

 49 Nucleotide Sequence of

 {LcillA-I) gene fragment encoding

 for recombinant protein.

 50 Amino Acid Sequence of

 (rLcillA-I) recombinant protein.

 51 Nucleotide Sequence of

 (LcillA-II) gene fragment encoding

 for recombinant protein.

 52 Amino Acid Sequence of

 (rLcillA-II) recombinant protein.

 53 Nucleotide Sequence of

 (LcillA-III) gene fragment encoding

 for recombinant protein.

 54 Amino Acid Sequence of

 (rLcillA-III) recombinant protein.

 55 Lcx12A-I Nucleotide Sequence (457) ... (3240)

(Lk 2) CDS. Lc ± 12A-II (457) ... (2058)

56 Amino Acid Sequence of

 (rLcil2) CDS.

 57 Nucleotide Sequence of

 {Lcil2A-I) gene fragment encoding

 for recombinant protein

 58 Amino Acid Sequence of

 (rLcil2A-I) recombinant protein

 59 Nucleotide Sequence of

 (LCÍ12A-II) gene fragment encoding

 for recombinant protein

 60 Amino Acid Sequence of

 (rLcil2A-II) recombinant protein

 61 c ± 13A Nucleotide Sequence (835) ... (1839)

(Lcil3) CDS complete.

 62 Amino Acid Sequence of

(Lcil3) CDS. 63 Nucleotide Sequence of

 (Lcil3A) cDNA fragment encoding

 for recombinant protein.

 64 Amino Acid Sequence of

 (rLcil3A) recombinant protein

 Thus, the description of the present invention demonstrates that use of different recombinant antigens obtained from Leishmania chagasi / Leishmania infantum genes identify, detect and quantify specific antibodies in biological material obtained from humans, dogs and other vertebrate hosts of Leishmania.

 It should be clear that the present invention is not limited to the embodiments described herein. Those skilled in the art will appreciate that any particular feature introduced should be understood only as something that has been described for ease of understanding.

Claims

 Substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

 An isolated polynucleotide encoding the polypeptide of SEQ ID NO: 4, with the nucleotide sequence represented by SEQ ID NO: 3.

 Substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12.

 4. Isolated polynucleotide encoding the polypeptide of SEQ ID NO: 8, with the nucleotide sequence represented by SEQ ID NO: 7.

 5. A substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.

 6. Isolated polynucleotide encoding the polypeptide of SEQ ID NO: 14, with the nucleotide sequence represented by SEQ ID NO: 13.

 7. cDNA fragment characterized by having SEQ ID NO: 15.

A substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises a amino acid sequence represented by SEQ ID NO: 18 or SEQ ID NO: 20 or SEQ ID NO: 22 or SEQ ID NO: 24.

 An isolated polynucleotide encoding the polypeptide of SEQ ID NO: 18, with the nucleotide sequence represented by SEQ ID NO: 17.

 CDNA fragment characterized by having SEQ ID NO: 19 or SEQ ID NO: 21 or SEQ ID NO: 23.

 11. A substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises an amino acid sequence represented by SEQ ID NO: 26 or SEQ ID NO: 28.

 An isolated polynucleotide encoding the polypeptide of SEQ ID NO: 26, with the nucleotide sequence represented by SEQ ID NO: 25.

 13. Gene fragment characterized by having SEQ ID NO: 27.

 14. A substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises an amino acid sequence represented by SEQ ID NO: 34 or SEQ ID NO: 36.

 An isolated polynucleotide encoding the polypeptide of SEQ ID NO: 34, with the nucleotide sequence represented by SEQ ID NO: 33.

 16. Gene fragment characterized by SEQ ID NO: 35.

17. Substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises an amino acid sequence represented by SEQ ID NO: 38 or SEQ ID NO: 40.

 18. Isolated polynucleotide encoding the polypeptide of SEQ ID NO: 38, with the nucleotide sequence represented by SEQ ID NO: 37.

 Substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises an amino acid sequence represented by SEQ ID NO: 42 or SEQ ID NO: 44 or SEQ ID NO: 46.

 20. Isolated polynucleotide encoding the polypeptide of SEQ ID NO: 42, with the nucleotide sequence represented by SEQ ID NO: 41.

 21. Gene fragment characterized by having SEQ

ID NO: 43 or SEQ ID NO: 45.

 22. A substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises an amino acid sequence represented by SEQ ID NO: 48 or SEQ ID NO: 50 or SEQ ID NO: 52 or SEQ ID NO: 54

 23. Isolated polynucleotide encoding the polypeptide of SEQ ID NO: 48, with the nucleotide sequence represented by SEQ ID NO: 47.

 24. Gene fragment characterized by having SEQ

ID NO: 49 or SEQ ID NO: 51 or SEQ ID NO: 53.

25. A substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises a amino acid sequence represented by SEQ ID NO: 56 or SEQ ID NO: 58 or SEQ ID NO: 60.

 26. Isolated polynucleotide encoding the polypeptide of SEQ ID NO: 56, with the nucleotide sequence represented by SEQ ID NO: 55.

 27. Gene fragment characterized by having SEQ ID NO: 57 or SEQ ID NO: 59.

 28. A substantially purified polypeptide having an immunogenic portion of a Leishmania antigen, wherein said antigen comprises an amino acid sequence represented by SEQ ID NO: 62 or SEQ ID NO: 64.

 29. Isolated polynucleotide encoding the polypeptide of SEQ ID NO: 62, with the nucleotide sequence represented by SEQ ID NO: 61.

 30. cDNA fragment characterized by having SEQ ID NO: 63.

 31. Antibodies characterized in that they bind to polypeptides of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO: 64.

32. Pharmaceutical compositions characterized in that they are used to induce an immune response against Leishmania in mammals, said compositions consisting of a immunogenically effective amount of one or more of the polypeptides represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO : 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 36 , SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO: 64 and a pharmaceutically acceptable carrier and / or adjuvant capable of eliciting an immune response predominantly cellular and can promote (i) protection against infection and / or disease development, (ii) fully developed disease regression, and (iii) reduction of parasitism, including skin, and / or parasite transmission to insect vector, consequently providing disease control.

 A method for inducing an immune response against Leishmania infection in mammals which comprises administering the pharmaceutical compositions described in claim.

34

 3 Method for detecting Leishmania in a sample characterized by the steps of:

(a) contacting a suspected sample containing Leishmania with a reagent capable of identifying one or more of the polypeptides represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO. : 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 , SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO: 64, which will bind to a component of a pathogen; and,

 (b) revealing, directly or indirectly, the reagent binding to the pathogen component.

 A method according to claim 34 wherein the reagent capable of binding to the pathogen component is an oligonucleotide for the identification of one or more of the polynucleotides represented by SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55, SEQ ID NO: 61.

A method according to claim 34 wherein the reagent capable of binding to the pathogen component is an oligonucleotide or polynucleotide encoding any of the polypeptides represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, 'SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO : 58, SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO: 64.

37. Method for detecting antibodies characterized by detecting antibodies to polypeptides represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ^'ID NO : 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 , SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO : 64, using the following steps:

 (a) contacting a sample of biological material from a mammal suspected of containing Leishmania into one or more of the polypeptides represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO : 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 SEQ ID NO: 62 or SEQ ID NO: 64 under conditions that allow antibodies to bind to such polypeptides; and,

 (b) detecting binding of the reagent to oligopeptides or peptides.

38. Method for detecting polypeptides in a sample suspected of containing Leishmania characterized in that the method detects one or more of the polypeptides represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO: 64 by following these steps:

 (a) contacting the sample with a reagent capable of identifying the polypeptides under conditions that allow the reagent to bind to the polypeptides; and,

 (b) detecting binding to oligopeptides or peptides.

 39. Method for detecting nucleic acids in a sample suspected of containing Leishmania characterized in that the method detects nucleic acids encoding one or more of the polypeptides represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO: 64, which uses oligonucleotides encoding said polypeptides or peptides.

40. Diagnostic Leishmania kit for detecting antibodies which bind to one or more of the polypeptides represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO-16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO : 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 , SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO: 64, alone, or in association with each other or with other antigens.