BRPI1106308A2 - immunogenic composition of leishmania (leishmania), method, immunodiagnostic kit and vaccine for leishmaniasis - Google Patents

Abstract

immunogenic composition of leishmania (leishmania), method, immunodiagnostic kit and vaccine for leishmaniasis. The present invention describes a composition comprising proteins isolated from the parasite leishmania (leishmania) chagasi syn. l. (l.) infantum for application in the serological diagnosis of cases of asymptomatic and / or symptomatic canine visceral leishmaniasis (lvc), in addition to its application as vaccine antigens against leishmaniasis. These proteins were recognized by antibodies present in the sera of dogs with asymptomatic and / or symptomatic visceral leishmaniasis (lv), indicating that they are expressed by the parasite during active infection. Its identification was performed by a two-dimensional electrophoresis technique performed with the two stages of the parasite (promastigote and amastigote) that cause lv in Brazil, l. chagasi, followed by western-blot experiments employing serum samples from dogs with asymptomatic and / or symptomatic lv.

Description

"IMMUNOGENIC COMPOSITION OF Leishmania (Leishmania), METHOD, IMMUNODIAGNOSTIC KIT AND LEISHMANIASIS VACCINE" The present invention describes a composition comprising proteins identified in the parasite Leishmania (Leishmania) chagasi syn. L. (L.) infantum for application in the serological diagnosis of asymptomatic and symptomatic canine visceral leishmaniasis (CVL), in addition to its application as vaccine antigens against leishmaniasis. These proteins were recognized by antibodies present in the sera of dogs with asymptomatic and / or symptomatic visceral leishmaniasis (VL), indicating that they are expressed by the parasite during active infection. Their identification was performed by two-dimensional electrophoresis technique with the two stages of the parasite (promastigote and amastigote) that causes VL in Brazil, L. chagasi, followed by western-blot experiments using samples from dogs with asymptomatic VL and / or symptomatic.

Leishmaniasis are diseases present in about 88 countries located in tropical and subtropical regions of the world (http://www.who.int). They are caused by infection by protozoan parasites of the genus Leishmania. Many regions of the world are endemic to the different species of the parasite [E. Handman, Leishmaniasis: current status of vaccine development, Clin Microbiol Rev 14 (2001) 229-243]. This is the case in South America, where at least eight different parasite species co-exist and are capable of causing different clinical types of the disease in the mammalian host [A. Barrai, D. Pedral-Sampaio, G. Junior Grimaldi, H. Momen, D. McMahon-Pratt, A. Ribeiro de Jesus, R. Almeida, R. Badaro, M. Barral-Netto, EM Carvalho, Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease, Am J Trop Med Hyg 44 (1991) 536-546]. In the Americas, leishmaniasis are classified into two broad clinical categories: VL and cutaneous leishmaniasis (LT). This includes the following clinical manifestations: cutaneous leishmaniasis (LC), mucocutaneous leishmaniasis (CML) and diffuse cutaneous leishmaniasis (LCD). VL is the most severe form of the disease, fatal if left untreated and is mainly caused by the L. chagasi species. LC and CML are mainly caused by L. braziliensis species. However, it is important to highlight the infection caused by L. amazonensis that can cause the clinical forms of LC, LCD or VL [M. Barrai et al, 1991; G. Grimaldi, Jr., R.B. Tesh, Leishmaniasis of the New World: Current Concepts and Implications for Future Research, Clin Microbiol Rev 6 (1993) 230-250]. In the Old World, LC is caused by L. major, while VL is caused by L. infantum in the Mediterranean coastal countries and L. donovani in Asian countries. It is noteworthy that the species L. infantum and L. chagasi are currently considered identical [I.L. Mauritius, JR Stothard, MA Miles, The strange case of Leishmania chagasi, Parasitol Today 16 (2000) 188-189], The outcome of Leishmania infection is mainly determined by the interactions established between the parasite infecting strain and the immune system. of the host. In general, a protective immunity is associated with the development of a Th1-type cellular immune response capable of stimulating macrophages to destroy phagocytic parasites, since such cells are considered the main host cells of Leishmania. Such response can be detected in man by late type hypersensitivity reaction (DTH), which is based on intradermal inoculation of parasite extract, followed by local inflammatory reaction. Susceptibility to the disease, in turn, seems to be associated with the generation of a Th2-type cellular immune response and, in many cases, by the induction of a high humoral response to the parasite [L. Gradoni, An update on antileishmanial vaccine candidates and prospects for a canine Leishmania vaccine, Vet Parasitol 100 (2001) 87-103; D. McMahon-Pratt, J. Alexander, Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or visceral disease ?, Immunol Rev 201 (2004) 206-224.]. The presence of Leishmania parasite-specific antibodies produced after recognition of parasite proteins has been associated with the clinical manifestation of the disease [S.A. Miles, S.M. Conrad, R.G. Alves, S.M. Jeronimo, D.M. Mosser, A role for IgG immune complexes during infection with the intracellular pathogen Leishmania, J Exp Med 201 (2005) 747-754]. When asymptomatic, human patients develop a positive DTH response associated with a negative serology against parasitic antigens, reversing the parameters in patients with overt clinical symptoms [J.A. Crescent, F.T. Silveira, R. Lainson, CM Gomes, MD Laurenti, CE Corbett, A cross-sectional study on the clinical and immunological spectrum of human L. (L.) infantile chagasi infection in the Brazilian Amazon region, Trans Royal Soc Trop Med Hyg 103 (2009) 1250-1256]. There are patients with intermediate values of such parameters, who tend to have intermediate symptomatology of the disease, thus being called oligosymptomatic [Crescente et al, 2009].

To complete the complex spectrum of clinical manifestations of leishmaniasis, it should be noted that the variety of forms of the disease in the vertebrate host is not limited to humans alone. In urban and peri-urban areas of medium and large cities of Brazil, domestic dogs may be infected by viscerotropic species, such as L. chagasi, being sensitive to the development of VL, which may still be accompanied by cutaneous lesions. canine visceral-cutaneous leishmaniasis (CVL). CVL can be classified as symptomatic when three or more of the following clinical symptoms are found: weight loss and hair loss, adenopathy, onychogryphosis, hepatomegaly, conjunctivitis, spurious dermatitis in the nose, tail and ear tips; and are considered asymptomatic when no clinical manifestation is evident in the animal. It is also noteworthy that this clinical manifestation is responsible for about 60 to 80% of CVL cases in Brazil [L. Solano-Gallego, A. Koutinas, G. Miro, L. Cardoso, M.G. Pennisi, L. Ferrer, P. Bourdeau, G. Oliva, G. Baneth. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniasis, Vet Parasitol (2009)]. The disease is considered a serious veterinary and human health problem, as infected dogs, regardless of whether they are symptomatic or asymptomatic, behave as parasite reservoirs and can infect humans through the transmitting phlebotomine vectors [C.L. Barbieri, Immunology of canine leishmaniasis, Parasite Immunol 28 (2006) 329-337; R. Molina, C. Nicea, J. Nieto, M. San-Andres, F. Gonzalez, JA Castillo, J. Lucientes, J. Alvar, Infectivity of dogs naturally infected with Leishmania infantum to colonized Phlebotomus perniciosus, Trans Royal Soc Trop Med Hyg 88 (1994) 491-493].

In this context, the development of efficient diagnostic systems and vaccines based on parasite proteins that are antigenic and / or immunogenic, respectively, against the different clinical forms of leishmaniasis, becomes a complex but important task. The existence of high titers of parasite protein-specific antibodies is an important feature of human LVH and LVC, and the generation of a high humoral response has been related to the development of the disease. In LT, high antibody titers are found in symptomatic cases of the disease, but they are not homogeneous in cases of viscerotropic infections. Even in the absence of correlation between protective immunity and high antibody production, detection of such antibodies makes it possible to develop tools for serological diagnosis of the disease. The techniques employed in the diagnosis of LVH are well developed, however, the same does not apply to cases of CVL. The clinical and laboratory diagnosis of leishmaniasis is very difficult. Diagnosis of symptomatic forms is used to confirm the disease, while diagnosis of asymptomatic forms is important for disease control and reduction of infection rates. Additionally, an early diagnosis can improve the effectiveness of treatment as it can be done early in the disease. Traditionally, for a definitive diagnosis of the disease, it is necessary to find the parasite in lesions and / or aspirates performed in internal organs, such as samples obtained from punctures in the spleen, bone marrow and lymph nodes [B.L. Herwaldt, Leishmaniasis, Lancet 354 (1999) 1191-1199]. Traditional methods are based on the use of hematological staining of samples, in vitro cultures performed on infected tissue fragments and amplification of the parasite DNA by the polymerase chain reaction (PCR) technique [G. Spanakos, E.T. Piperaki, P.G. Menounos, N. Tegos, A. Flemetakis, N.C. Vakalis, Detection and species Identification of Old World Leishmania in clinical samples using a PCR-based method, Trans Royal Soc Trop Med Hyg 102 (2008) 46-53; G. Miro, L. Cardoso, M.G. Pennisi, G. Oliva, G. Baneth, Canine Leishmaniases — New Concepts and Insights on Expanding Zoonosis: Part Two, Trends Parasitol 24 (2008) 371-377]. Specimen collection to perform such techniques, however, is invasive. As in symptomatic cases there is a high production of specific antibodies specific to certain parasite proteins, the development of tools that enable the serodiagnosis of the disease presents an attractive alternative [Barbieri et al, 2006, K. Kar, Serodiagnosis of leishmaniasis, Crit Rev Microbiol 21 (1995) 123-152; L. Cardoso, H.D. Schallig, F. Neto, N. Kroon, M. Rodrigues, Serological survey of Leishmania infection in dogs from the municipality of Peso da Regua (Alto Douro, Portugal) using the direct agglutination test (DAT) and fast agglutination screening test (FAST) , Acta Tropica 91 (2004) 95-100; A.B. Reis, A. Teixeira-Carvalho, A.M. Vale, M.J. Marques, R.C. Giunchetti, W. Mayrink, L.L. Guerra, R.A. Andrade, R. Correa-Oliveira, O.A. Martins-Filho, Isotype patterns of immunoglobulins: hallmarks for clinical status and tissue parasite density in Brazilian dogs naturally infected by Leishmania (Leishmania) chagasi, Vet Immunol Immunopathol 112 (2006) 102-116]. The collection of serum samples for serological tests is considered less invasive compared to traditional parasitological methods and can be easily performed in endemic areas. However, such tests depend on the use of specific antigens and the clinical form of leishmaniasis to be diagnosed. The use of parasite proteins in the form of crude or semipurified extracts allows for high sensitivity values, especially in symptomatic visceral forms. However, in the cutaneous and mucocutaneous forms, and especially in the asymptomatic form of LVC and LVH, the sensitivity of the tests with the extracts is variable [Herwaldt et al 1999; I.A. Maalej, M. Chenik, H. Louzir, A. Ben Salah, C. Bahloul, F. Amri, K. Dellagi, Comparative evaluation of ELISAs based on ten recombinant or purified Leishmania antigens for the serodiagnosis of Mediterranean visceral leishmaniasis, Am J Trop Med Hyg 68 (2003) 312-320; A.J. Caldas, J. Costa, D.

Aquino, A.A. Silva, M. Barral-Netto, A. Barrai, Are there differences in clinical and laboratory parameters between children and adults with American visceral leishmaniasis ?, Acta Tropica 97 (2006) 252-258; S.A. Grevelink, E.A. Lerner, Leishmaniasis. J Am Acad Dermatol 34 (1996) 257-272]. Another important problem is the specificity of the tests, as there is a frequent cross-reaction of anti-Leishmania antibodies with proteins also found in other pathogens, such as trypanosomatids, Schistosoma, and a high number of false results. -positives in animals that reside in endemic areas of the disease but are not infected with Leishmania [Kar et al, 1995; C. Ferreira Ede, M. de Lana, M. Carneiro, A.B. Reis, D.V. Paes, E.S. da Silva, H. Schallig, C.M. Gontijo, Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations, Vet Parasitol 146 (2007) 235-241; R. Porrozzi, M.V. Santos da Costa, A. Teva, A. Falqueto, A.L. Ferreira, C.D. of Santos, A.P. Fernandes, R.T. Gazzinelli, A. Campos-Neto, G. Grimaldi, Jr., Comparative evaluation of enzyme-linked immunosorbent assays based on crude and recombinant leishmanial antigens for serodiagnosis of symptomatic and asymptomatic Leishmania infantum visceral infections in dogs, Clin Vaccine Immunol 14 (2007) 544-548; S.G. Reed, WG Shreffler, JM Burns, Jr., JM Scott, G. Orge Mda, HW Ghalib, M. Siddig, R. Badaro, An improved serodiagnostic procedure for visceral leishmaniasis, Am J Trop Med Hyg 43 (1990) 632-639 ]. The techniques employed in the serological diagnosis of CVL using parasite extracts also have limitations in that, in the early stages of the disease, infected animals may be sero-negative and others, clinically cured, may remain serologically reactive for many years [L. Ferrer, M.J. Aisa, X. Roura, M. Portus. Due to the low specificity of the serological tests currently used by the competent Health Bodies, many dogs that present positive results in the serological screening, however, absence of serological diagnosis and treatment of canine leishmaniasis, Vet Rec 136 (1995) 514-516]. Obvious clinical symptoms are most often sacrificed without the need for such a measure, however, as another measure of disease control.

For all the reasons mentioned, it is considered urgent to develop a serological diagnosis for CVL that presents high sensitivity and specificity and that allows, especially, the diagnosis of animals that are in the asymptomatic phase of the disease.

Regarding LVH serodiagnosis, the use of a recombinant protein that contains the carboxy-terminal portion of the L. chagasi kinesin protein (rK39) currently forms the basis for the commercial serodiagnosis test and has high sensitivity and specificity [R. Badaro, D. Benson, M.C. Eulalio, M. Freire, S.Cunha, E.M. Netto, D. Pedral-Sampaio, C. Madureira, J.M. Burns, R.L. Houghton, J.R. David, S.G. Reed, rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis, J infect Dis 173 (1996) 758-761; S. Singh, V. Kumari, N. Singh, Predicting kala-azar disease manifestations in asymptomatic patients with latent Leishmania donovani infection by detection of antibody against recombinant K39 antigen, Clin Diagn Lab Immunol 9 (2002) 568-572; AND IS. Zijlstra, Y. Nur, P. Desjeux, E.A. Khalil, A.M. El-Hassan, J. Groen, Diagnosing visceral leishmaniasis with the recombinant K39 strip test. experience from the Sudan, Trop Med Int Health 6 (2001) 108-113; J.M. Burns, Jr., W.G. Shreffler, D.R. Benson, H.W.Ghalib, R. Badaro, S.G. Reed, Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis, Proc Natl Acad Sci USA 90 (1993) 775-779]. However, the serodiagnosis of asymptomatic forms of LVH and LVC, as well as the serodiagnosis of LC and CML in men, also presents sensitivity and / or specificity problems, such as the cross-reactivity with sera from patients with autoimmune diseases [P. Salotra, R. Singh, Rapid and reliable diagnostic tests for visceral leishmaniasis, Indian J Med Res 122 (2005) 464-467], Regarding the development of vaccines against leishmaniasis, research has currently aimed at identifying proteins from which immunization is capable of inducing a protective immune response [Handman et al 2001; R.N. Coler, S.G. Reed, Second-generation vaccines against leishmaniasis, Trends Parasitol 21 (2005) 244-249]. At first, a protein is considered a candidate for a class of vaccines when it is capable of inducing the generation of a Th1-type cellular immune response to parasite infection [Handman et al 2001]. Several parasitic antigens tested as purified, semipurified extracts, recombinant proteins and DNA vaccines have been evaluated in several experimental models (reviewed in [J. Kubar, K. Fragaki, Recombinant DNA-derived proteins. the field, Lancet Infect Dis 5 (2005) 107-114; M. Soto, L. Ramirez, M. Pineda, V. Gonzalez, P. Entringer, C. Indiani de Oliveira, I. Nascimento, A. Souza, L. Crow, C. Alonso, P. Bonay, C. Brodskyn, A. Barrai, M. Barral-Netto, S. Iborra, Searching for genes encoding Leishmania antigens for diagnosis and protection, Scholarly Research Exchange 2009 (2009)]). The protection induced by vaccine antigens depends to a large extent on their joint administration with immune response adjuvants. These include the use of unmethylated CpG motifs (CpG ODN) and saponins oligodeoxyribonucleotides or, alternatively, the use of DNA vaccines based on eukaryotic expression plasmids containing genes encoding the antigens of interest [S. Gurunathan, D.M. Klinman, R.A. Seder, DNA vaccines: immunology, application, and optimization, Annual Rev Immunol 18 (2000) 927-974; E. Dumonteil, DNA Vaccines against protozoan parasites, advances and challenges, J Biomed Biotechnol 2007 (2007) 905-920; J. Champsi, D. McMahon-Pratt, Membrane glycoprotein M-2 protects against Leishmania amazonensis infection, Infect Immun 56 (1988) 3272-3279; W.R. Santos, V.M. de Lima, E.P. de Souza, R.R. Bernardo, M. Palatnik, C.B. Palatnik de Sousa, Saponins, IL-12 and BCG adjuvant in the FML-vaccine formulation against murine visceral leishmaniasis, Vaccine 21 (2002) 30-43; E. Oliveira-Freitas, C.P. Casas, G.P. Borja-Cabrera, F.N. Santos, D. Nico, L.O. Souza, L.W. Tinoco, B.P. da Silva, M. Palatnik, J.P. Relative, C.B. Palatnik-de-Sousa, Acylated and deacylated saponins of Quiliaja saponaria mixture as adjuvants for the FML-vaccine against visceral leishmaniasis, Vaccine 24 (2006) 3909-3920; R.C. Giunchetti, R. Correa-Oliveira, O.A. Martins-Filho, A. Teixeira-Carvalho, B.M. Roatt, R. D. Oliveira Aguiar-Soares, W. Coura-Vital, R.T. de Abreu, L.C. Malaquias, N.F. Gontijo, C. Brodskyn, C.I. de Oliveira, DJ. Costa, M. de Lana, A.B. Reis, A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs, Vaccine 26 (2008) 623-638; A.P. Fernandes, M.M. Costa, E.A. Coelho, M.S. Michalick, E. Freitas, M.N. Melo, W.L. Tafuri, M. Resende, V. Hermont, C.F. Abrantes, R.T. Gazzinelli, Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle dogs vaccinated with recombinant A2 protein, Vaccine 26 (2008) 5888-5895; Gurunathan et al 2000]. Vaccine formulations have been employed mainly in experimental murine models which, although not considered similar to human and canine leishmaniasis, are initially taken into account to predict their success in mammalian hosts. The success of vaccine candidates has been linked to the generation of specific immune responses to vaccine antigens, as described above, mediated by the production of cytokines such as IFN-gamma, IL-12 and TNF-alpha, and the control of cytokine-mediated responses. associated with Th2-type disease susceptibility such as IL-4, IL-10 and TGF-beta [M. Barral-Netto, A. Barrai, C.E. Brownell, Y.A. Skeiky, L.R. Ellingsworth, D.R. Twardzik, S.G. Reed, Transforming growth factor-beta in leishmanial infection: a parasite escape mechanism, Science 257 (1992) 545-548; A. Gumy, JA Louis, P. Launois, The murine model of infection with L. major and its importance for the deciphering of mechanisms underlying differences in Th cell differentiation in mice from different genetic backgrounds, Int J Parasitol 34 (2004) 433- 444; N. Noben-Trauth, Susceptibility to L. major infection in the absence of IL-4, Immunol Lett 75 (2000) 41-44; D. Sacks, N. Noben-Trauth, The immunology of susceptibility and resistance to Leishmania major in mice, Nat Rev Immunol 2 (2002) 845-858], There are several limitations regarding the application of different vaccine candidates against leishmaniasis in experiments conducted in research laboratories for employment in the canine and / or human population.

Formulations successfully administered in murine models may not induce protection in dogs and humans, and many candidate molecules have been tested against a limited number of parasite species, usually against one species, making them specific only to that particular species. Leishmania strain (reviewed in [CB Palatnik-de-Sousa, Vaccines for leishmaniasis in the fore coming 25 years, Vaccine 26 (2008) 1709-1724]). The present invention describes the identification of 59 (fifty-nine) proteins isolated from the parasite Leishmania (Leishmania) chagasi syn. L. (L.) infantum for application in the serological diagnosis of asymptomatic and symptomatic canine visceral leishmaniasis (CVL), in addition to its application as vaccine antigens against leishmaniasis. Of these proteins, thirty-one (31) are proteins considered hypothetical in the parasite and were selected in the promastigote (15) and like-amastigote (16) stages of L. chagasi. The other proteins (28) had not been previously described for Leishmania, but had not yet been used for immunodiganosis or vaccine against leishmaniasis. The immunogenic composition containing the proteins and / or their epitopes (peptides) specific for B lymphocytes (LB) and CD8 + T lymphocytes, allows the development of a sensitive and specific serological diagnosis for the identification of dogs with symptomatic and asymptomatic VL. These proteins and / or peptides also constitute antigens for leishmaniasis vaccines, allowing the development of an effective multigenic vaccine against different strains of the parasite.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Two-dimensional electrophoresis (2DE) of total protein extracts of promastigote stationary growth and like-amastigote forms of Leishmania chagasi. The 2DE gels were obtained after separating the extracts of promastigote (in A) and like-amastigote (in B) forms (150 pg from each extract), with the first dimension in IEF pH 4-7 and the second dimension in SDS. -PAGE 12%, followed by staining with Coomassie Brilliant Blue G-250 colloidal. The figures are representative of three independent gels, which showed similar results.

Figure 2. Immunoproteomic analysis of total protein extract of promastigote forms in steady-state growth of Leishmania chagasi. 2DE gels (in A) were obtained as shown in the legend of Figure 1. Immunoblots were identified after incubation of the cellulose membrane with asymptomatic (B) or symptomatic (C) dog serum samples. Membrane-bound antibodies were detected with dog anti-IgG goat conjugate at a dilution of 1: 5000. The x axis represents the isoelectric point (mp) of the proteins and the y axis represents the approximate molecular weight (kDa) of the proteins, determined by using a commercial protein standard for 2DE gels. The most reactive spots of the hypothetical or previously described proteins were numbered and their identities were determined and are shown in Tables 1 and 2. The figures are representative of three independent experiments, which showed similar results.

Figure 3. Immunoproteomic analysis of total protein extract of Leishmania chagasi like-amastigote forms. 2DE gels (in A) were obtained as shown in the legend of Figure 1. Immunoblots were identified after incubation of the cellulose membrane with asymptomatic (B) or symptomatic (C) dog serum samples. Membrane-bound antibodies were detected with dog anti-IgG goat conjugate at a dilution of 1: 5000. The x axis represents the isoelectric point (mp) of the proteins and the y axis represents the approximate molecular weight (kDa) of the proteins, determined by using a commercial protein standard for 2DE gels. The most reactive spots of the hypothetical or previously described proteins were numbered and their identities were determined and are shown in Tables 3 and 4. The figures are representative of three independent experiments, which yielded similar results.

Figure 4. Comparison of the expression profile of proteins of the promastigote and like-amastigote forms of Leishmania chagasi that were recognized by sera from dogs with symptomatic and / or asymptomatic VL. The diagrams show the number of spots recognized at each stage of the parasite, individually, or both. In (A), the total number of proteins recognized by asymptomatic (19 - 18%), symptomatic (64 -62%) and total (21 - 20%) dog sera. (B) Proteins expressed in the promastigote form of the parasite that have been recognized by asymptomatic (10-20%), symptomatic (25-49%) and total (16-31%) dog sera. C) Proteins expressed in the parasite-like-amastigote form that were recognized by asymptomatic (9 - 17%), symptomatic (39 - 74%) and total (5 - 9%) dog sera.

DETAILED DESCRIPTION OF TECHNOLOGY

The proteins described in the present invention were isolated from Leishmania chagasi strain MHOM / BR / 1970 / BH46. Promastigote growth-stage parasites were prepared after in vitro incubation at 24 ° C in complete Schneider's (Sigma) medium, which was composed of the addition of 20% inactivated fetal bovine serum (FBS, Sigma), 20 mM L-glutamine, 200 U / mL penicillin, 100 pg / mL streptomycin and 50 pg / mL gentamicin, at pH 7.2 [Rabbit EAF. Evaluation of protection levels and immune response induced by immunization with A2 and LACK antigens in experimental infection with Leishmania (Leishmania) major and Leishmania (Leishmania) amazonensis. Doctoral Thesis. Department of Biochemistry and Immunology, ICB, UFMG. 2004], Parasites at the like-amastigote stage were obtained by converting the promastigote forms into a steady-state growth phase (1010 parasites), which were incubated at 34 ° C with 5% CO2 for 48 h at pH 5.0 as described [ Doyle PS, Engel JC, Pepper PFP, Silva PP, Dwyer DM. Leishmania donovani: long-term culture of axenic amastigotes at 37 ° C. Exp Parasitol 1991; 73: 326-34]. The preparation of the extracts, the extraction of proteins from the L. chagasi promastigote and like-amastigote forms and the preparation of two-dimensional gels (2DE) were performed according to the protocol described by Lewis et al. (2000), with modifications [Lewis TS, Hunt JB, Aveline LD, Jonscher KR, Louie DF, Yeh JM, Nahreini TS, Resing KA, Ahn NG.

Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Mol Cell. 2000; 6: 1343-54]. Briefly, cells (1010 parasites) were washed 3 times in 40 mM Tris-HCl, pH 7.5, centrifuged at 5000 x g for 10 min and at 4 ° C. The precipitate was resuspended in lysis buffer [7 M urea, 2 M thiourea, 4% chol-amidopropyl dimethylammonio-1-propanesulfonate (CHAPS), 40 mM dithiothreitol (DTT), 2% IPG (pH 4 7) and 40mM Tris base] by adding a protease inhibitor cocktail (GE Healthcare, Upsala, Sweden). Samples were incubated for 1 h at room temperature under constant agitation. Protein purification was performed by protein precipitation by the 2D Clean Up Kit (GE Healthcare) according to the manufacturer's instructions. Extracts were quantified with the 2-D Quant-Kit (GE Healthcare). Aliquots were kept at -80 ° C until use.

For the first electrophoresis dimension, 150 pg of proteins were dissolved in a volume of 250 μΙ with rehydration solution [7 M urea, 2 M thiourea, 2% CHAPS, 40 mM DTT, 2% immobilized pH gradient ( IPG-buffer, pH 4-7, trace bromophenol blue)]. The samples were then applied to IPG strips (13 cm, pH 4-7; GE Healthcare) for passive overhydration at room temperature. After rehydration of the gel, isoelectric focusing was performed by running at 500 V for 1 h, 1,000 V for 1 h, and 8,000 V for 8 h using Multiphor II electrophoresis unit and EPS 3500 XL power supply equipment (Amersham, Piscataway). , NJ, USA).

After isoelectric focusing (IEF), each strip was incubated for 15 min in 10 ml of 50 mM Tris-HCI buffer solution at pH 8.8.6 M urea, 30% (v / v) glycerol, 2% (w / v ) SDS, 0.002% bromophenol blue and 125 mM DTT, followed by a second incubation step in the same buffer, without DTT, which was replaced by 125 mM iodacetamide. The IPG strips were transferred to a 12% polyacrylamide gel and sealed with agarose solution (agarose and bromophenol blue in Tris-glycine cathode buffer). Invitrogen's protein standard (BenchMark ™ Protein Ladder) was used. The electrophoretic run was performed in a Mini-Protean II system (BioRad) connected to the MultiTemp II cooling bath (Amersham Biosciences). Electrophoresis was performed at 200 V for 2 h in Tris / Glycine / SDS buffer.

The gels were stained with coomassie Brilliant Blue G-250 eolloidal, according to Neuhoff et al. (1988) [Neuhoff V, Arold N, Taube D, Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988; 9: 255-62]. The images were documented with the Typhoon scanner (GE Healthcare) and the analyzes were performed with the PDQuest ™ software (BioRad). Comparison of 2DE gels maps derived from three independent preparations. A representative preparation of each stage of the parasite was used. The theoretical pi of proteins was identified by de novo sequencing and calculated by the program ScanSite pl / Mw (http://scansite.mit.edu/calc_mw_pl.html).

Approximately 350 protein spots were visualized in the promastigote forms (Figure 1A) and 200 spots in the like-amastigote forms (Figure 1B) of Leishmania chagasi. Most spots in the promastigote forms were concentrated between 15 and 50 kDa, while in the like-amastigote forms they were concentrated between 25 and 70 kDa.

Proteins were transferred from 2DE gel to cellulose membranes by western blot technique and detection of antigenic proteins was performed using 60 samples of sera from dogs infected with L. chagasi (40 with symptomatic disease and 20 asymptomatic). ). In the following examples the western blot test was used. However, other techniques may also be employed to develop a laboratory diagnosis of CVL, such as: ELISA, dot-blot, radioimmunodiffusion and immunochromatography. The present invention may be better understood, without limitation, by the following examples: Example 1- Detection of Antigenic Proteins Detection of antigenic proteins was performed using sera samples from 60 dogs infected with L. chagasi ( 40 symptomatic and 20 asymptomatic), obtained in the metropolitan region of Belo Horizonte, Minas Gerais, Brazil. The animals were considered symptomatic when three or more of the following symptoms were present: weight and / or hair loss, adenopathy, onychogryphosis, hepatomegaly, ejunctivitis, spolemic dermatitis in the nose, tail or ear tips. The asymptomatic animals showed no clinical signs and symptoms. All serum samples from symptomatic and asymptomatic dogs were positive when tested by indirect immunofluorescence (IFAT) and the presence of amastigote forms of Leishmania was confirmed by microscopic observation and in vitro culture of popliteal and / or pre-scapular lymph node aspirates. bone marrow and / or tissue fragments. Serum samples from 20 dogs residing in non-endemic VL areas, without signs or clinical suspicion of VL and negative on serological and parasitological tests were the control group.

Total extracts of the promastigote and like-amastigote forms were electrophoretically separated as described above and transferred to cellulose membranes by western blot technique (Schleicher & Schull, Dassel, Germany) for 2 h at 400 mA. Membranes were blocked in 5% (w / v) skimmed milk powder in 1x PBS (pH 7.4) and 0.05% Tween 20 for 2 h at room temperature. Subsequently, the membranes were washed 6 times (10 min each) with blocking solution and preincubated with the serum pools of symptomatic and asymptomatic VL (diluted 1: 200 each) for 2 h at room temperature. Membranes were incubated with goat anti-dog IgG secondary antibody peroxidase conjugate (1: 5000 dilution) for 2 h. After 3 washes with 1x PBS and 0.5% Tween 20, the immunoblots were revealed by the addition of a solution composed of chloronafitol (12.5 mg), diaminobenzidine (25 mg) and 30 vol H2O2. (20 µl).

In the analysis of antibody immunoreactivity against proteins expressed in the promastigote form of parasites (Figure 2), and based on the comparison with the 2DE gel obtained previously (Figure 2A), it was observed that antibodies present in asymptomatic dog sera recognized approximately , 40 protein spots in the promastigote forms extract, (Figure 2B), whereas the symptomatic dog sera recognized approximately 80 proteins (Figure 2C).

In the analysis of antibody immunoreactivity against proteins expressed in the parasite-like-amastigote form (Figure 3), and based on the comparison with the previously obtained 2DE gel (Figure 3A), - it was observed that antibodies present in asymptomatic dog sera recognized approximately 30 protein spots (Figure 3B), whereas symptomatic dog sera recognized approximately 70 proteins (Figure 3C).

As a control, total protein extracts from the L. chagasi promastigote and like-amastigote stages were submitted to two-dimensional electrophoresis, transferred to nitrocellulose membranes and incubated with control dog serum samples, and no recognition of any protein from the control group was found. Leishmania parasite (data not shown).

In the comparative analysis of the proteins identified in the promastigote and like-amastigote forms of L. chagasi with the symptomatic and asymptomatic dog sera, it was observed that most proteins of both stages were recognized by antibodies of dogs with symptomatic disease ( Figure 4).

Example 2- Identification of Antigenic Proteins Identification of proteins from antibodies in asymptomatic dog sera is of clinical relevance, as a large number of infected dogs are not detected in routine serological analyzes. In addition, proteins recognized in symptomatic and asymptomatic sera and in both stages of the parasite become attractive tools for the sensitive serological diagnosis of the disease, which is capable of detecting pathology alone or in association with the various clinical stages. introduces himself.

In order to establish a reference map of proteins in promastigote L. chagasi forms that were recognized in western blots with asymptomatic and symptomatic dog sera, 51 well-defined spots (25, 10 and 16 recognized by symptomatic, asymptomatic and by both, respectively) were selected and extracted from the 2DE gels. For proteins expressed as amastigote forms, 53 spots were selected (39, 9 and 5 recognized by symptomatic, asymptomatic sera and both, respectively) that were selected and extracted from 2DE gels. Protein extraction from 2DE gels was performed as described: the spots were washed with 25 mM ammonium bicarbonate and 50% acetonitrile until complete discoloration of the dye used. After drying, the gel fragments were placed in an ice bath with 50 μΙ protease solution and 20 ng / ml sequence grade-modified trypsin (Promega) in 25 mM ammonium bicarbonate (Promega Biosciences, CA). for 30 min. Excess protease solution was removed and replaced with 25 mM ammonium bicarbonate. Digestion was performed for 18 h at 37 ° C. Peptides were extracted twice for 15 min with 30 μΙ of 50% acetonitrile and 5% formic acid. The trypsin digestion product was concentrated in speed-vac (Savant, USA) to approximately 10 μΙ and desalted on a Zip-Tip column (C18 resin; P10, Millipore Corporation, Bedford, MA). The samples were mixed in a matrix (5 mg / ml recrystallized α-cyano-4-hydroxycinnamic acid) in a final volume of 1 ml (1: 1 ratio) and subjected to analysis.

MS and MS tandem analyzes were performed using the MALDI-TOF-TOF AutoFIex III ™ program in positive-reflective mode controlled by FlexControl ™ software for protein analysis and identification. A peptide calibration standard II (Bruker Daltonics) was used as a reference for instrument calibration and α-cyano-4-hydroxycinnamic acid as a matrix. Samples were placed on AnchorChip ™ 600/384 MTP targets (Bruker Daltonics) using the standard protocol for the dry drop method. Each spectrum was produced by data accumulation of 200 consecutive laser shots. The mass spectrum was calibrated using a standard calibration peptide mixture (Bruker Daltonics) consisting of angiotensin I & II, substance P, bombesin, ACTH clip 1-17, ACTH clip 18-39 and somatostatin 28 as internal standard, and peptide masses were determined as monoisotopic masses. The resulting spectrum was processed by Flex analysis software (Bruker Daltonics).

The peptides obtained from the MS / MS spectrum were evaluated manually. Sequenced peptides were searched in the database (http://blast.ncbi.nlm7nih.gov) searching in (Leishmania -taxid: 5658 organism). Low quality MS / MS spectra were not considered for sequencing or selection in a protein sequence database. Sequences of the sequenced peptides were extended to the nearest tryptic dividing point and the coincidence between experimental and theoretical molecular mass, together with the quality of alignment, was considered as protein identification criterion.

Among the identified proteins, some proteins are already described and were previously used for diagnosis, vaccine and / or treatment of leishmaniasis.

Hypothetical proteins that had not yet defined function in Leishmania are presented in Table 1. Proteins already known in Leishmania, but not yet used in diagnosis and vaccines in leishmaniasis are presented in Table 2. In both cases, the proteins were identified in promastigote stage of L. chagasi.

In the case of proteins recognized in the L. chagasi like-amastigote stage, hypothetical proteins that did not yet have a defined function in Leishmania are presented in Table 3. Proteins already known in Leishmania, but not yet employed in diagnosis and vaccines in Leishmaniasis. presented in Table 4.

Thus, such protein groups have the potential to make a serologically specific diagnosis for leishmaniasis and can be used as vaccines against leishmaniasis.

Table 1. List of fifteen (15) hypothetical promastigote proteins of Leishmania that were recognized by antibodies present in the sera of dogs with asymptomatic and / or symptomatic visceral leishmaniasis caused by Leishmania chagasi after identification by immunoproteomics.

Table 2. List of the nine (9) proteins already described in promastigote forms of Leishmania that were recognized by antibodies present in the sera of dogs with asymptomatic and / or symptomatic visceral leishmaniasis caused by Leishmania chagasi after identification by immunoproteomics.

Table 3- List of sixteen (16) hypothetical proteins of Leishmania-like-amastigote forms that were recognized by antibodies present in the sera of dogs with asymptomatic and / or symptomatic visceral leishmaniasis caused by Leishmania chagasi after identification by immunoproteomics.

Table 4- List of the nineteen (19) proteins already described in Leishmania-like-amastigote forms that were recognized by antibodies present in the sera of dogs with asymptomatic and / or symptomatic visceral leishmaniasis caused by Leishmania chagasi after identification by immunoproteomics.

Claims (21)

Immunogenic composition of Leishmania, characterized in that it comprises at least one protein selected from the group consisting of the sequences SEQ ID NOS. 1 to 59 or their corresponding epitopes (peptides) more specific for B lymphocytes (LB) and CD8 + T lymphocytes and pharmacologically acceptable excipients. . Immunogenic composition of Leishmania according to claim 1, characterized in that it preferably comprises at least one of the Leishmania-specific proteins selected from the group consisting of the sequences SEQ ID NOs. 1 to 11, or their corresponding more specific epitopes (peptides) for B lymphocytes (LB) and CD8 + T lymphocytes and pharmacologically acceptable excipients. Method for immunoassay of Leishmaniasis characterized by comprising: a) binding of Leishmania-specific antibodies from a sample to at least one protein selected from the group described consisting of the sequences SEQ ID NOs. 1 to 59 and / or its epitopes (peptides) more specific correspondents for B lymphocytes (LB) and CD8 T lymphocytes attached to a solid support or carrier; b) contacting the antibodies of step (a) with a secondary antibody or protein, conjugated to an enzyme or label and binding to the antibodies of step (a); c) detecting Leishmania specific antibodies in the above sample by detecting the secondary antibody or protein specifically bound to said artà-Leishmania antibody. Method for immunoassay testing of leishmaniasis according to claim 3, characterized in that the samples are selected from the group consisting of blood, serum, plasma and other body fluid. Method for immunoassay testing of leishmaniasis according to claim 3, characterized in that the solid support is selected from a group of materials consisting of nitrocellulose, nylon, latex, polypropylene, polystyrene or other materials used for the same purpose as those described above. Method for immunoassay of leishmaniasis according to claim 3, characterized in that the carrier is a gold particle. Method for immunoassay of leishmaniasis according to claim 3, characterized in that the secondary antibody is selected from a group consisting of IgG, IgM, IgA, IgE and their subclasses. A method for immunoassaying leishmaniasis testing according to claim 3, wherein the protein of step (b) is selected from a group consisting of Protein A and / or Protein G. A method for immunoassay of leishmaniasis according to claim 3, characterized in that the enzyme is selected from the group consisting of peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. The method for immunoassay of leishmaniasis according to claim 3, wherein the marker is selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent. The method for immunoassay testing of Leishmaniasis according to claim 3, wherein the Leishmania-specific antibody detection step is selected from the group consisting of fluorescence, immunofluorescence, immunoluminescence, absorbance or radioisotope detection. 12. Leishmaniasis immunoassay test kit, comprising: - at least one of the selected protein group consisting of the sequences SEQ ID Nos. 1 to 59; or their corresponding more specific epitopes (peptides) for B lymphocytes (LB) and CD8 + T lymphocytes; a secondary antibody or protein, wherein the secondary antibody or protein is conjugated to an enzyme or a marker, and a reagent to detect the enzyme or marker used; Leishmaniasis immunoassay test kit according to claim 12, characterized in that the proteins or epitopes are attached to a solid support or carrier. Leishmaniasis immunoassay test kit according to claim 13, characterized in that the solid support is selected from the material group consisting of nitrocellulose, nylon, latex, polypropylene and polystyrene. Leishmaniasis immunoassay test kit according to claim 13, characterized in that the carrier consists of gold particles. Leishmaniasis immunoassay test kit according to claim 12, characterized in that the secondary antibody is selected from the group consisting of IgG, IgM, IgA, IgE and / or their respective subclasses. Leishmaniasis immunoassay test kit according to claim 12, characterized in that the protein conjugated to an enzyme or marker is selected from the group consisting of protein A and / or protein G. An immunodiagnostic leishmaniasis test kit according to claim 12, wherein said marker is selected from the group consisting of enzymes, redioisotopes, biotin, chromophores, fluorophores and chemiluminescent. Leishmaniasis immunoassay test kit according to claim 12, characterized in that the enzyme is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, ureaase, xanthine oxidase, glucose oxidase and penicillinase. 20. LEISHMANIOSIS VACCIN, characterized in that it comprises at least one protein selected from a group consisting of the sequences SEQ ID NOS. 1 to 59 or their corresponding epitopes (peptides) more specific for B lymphocytes (LB) and CD8 + T lymphocytes and pharmacologically excipients. acceptable. A Leishmaniasis vaccine according to claim 20, preferably comprising at least one of the Leishmania-specific proteins selected from a group consisting of the sequences SEQ ID NOs: 1 to 11, or their corresponding more specific epitopes (peptides) for B lymphocytes (LB) and CD8 + T lymphocytes and pharmacologically acceptable excipients.