BR122023022690A2 - LEISHMANIA MIMETIC PEPTIDE SEQ. NO.ID.2: PEPTIDE AND USE - Google Patents

Abstract

leishmania mimetic peptide seq. no.id.2: peptide and use. The present invention deals with the characterization of mimetic peptides from Leishmania spp. used to induce immunogenic response and for diagnostic tests. the present invention comprises the identification of mimotopes of Leishmania spp. antigens. The peptides described in the present invention are useful in methods, diagnostic kits and biosensors, as well as immunological compositions that contain them as an active ingredient, such as vaccines.

Description

Field of Invention

[001]. The present invention deals with obtaining new antigens for use in diagnosis, prognosis (diagnostic method and kit), vaccines (immunological compositions) and in biosensors as a control measure for leishmaniasis from Leishmania spp. mimetic peptides. used alone and/or in associations.

History of the Invention

[002]. Leishmaniases are among the main neglected, re-emerging parasitic diseases and affect around 350 million individuals worldwide (Leishmaniasis: epidemiology and access to medicines. WHO. 2012). The disease is caused by protozoa of the genus Leishmania and infects humans in the Americas, Africa, Asia and Europe. In Latin America, cutaneous leishmaniasis (CL) is known as American cutaneous leishmaniasis (ATL) and is caused by different etiological agents: Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) naiffii, L. (V.) lainsoni, L. (V.) shawi, L. (Leishmania) amazonensis and L. (L.) americana. Visceral leishmaniasis (VL) is caused by Leishmania infantum (synonymous with L. chagasi), which is more severe and can cause death in the absence of correct diagnosis and early treatment (LAINSON, R. The American leishmaniases: some observations on their ecology and epidemiology. Royal Society of Tropical Medicine and Hygiene, v. 77, n. 5, p. 569-596, 1983; THOMAZ-SOCCOL, V. et al. Monophyletic origin of the genus Leishmania Ross, 1903. Annales de Parasitologie Humaine et Comparee, v. 68, n. 2, p. 107-108, 1993; THOMAZ-SOCCOL, V. et al. A new focus of cutaneous leishmaniasis in the central area of Paraná State, southern Brazil. Acta Tropica, v. 111, n 3, pp. 308-315. sep. 2009).

[003]. The clinical form of the disease develops after inoculation of the parasite in humans and is closely associated with the host's immune response, the species of Leishmania involved and the relationship between the parasite and its host (SARAVIA, N. G. et al. Mucocutaneous leishmaniasis in Colombia: Leishmania braziliensis. American Journal of Tropical Medicine and Hygiene, v. 34, n. 4, p. 714-720, Jul. 1985).

[004]. In humans, the disease occurs in four main clinical forms: localized cutaneous, mucocutaneous, diffuse cutaneous and visceral. The visceral form is the most serious of the disease and can cause fever, weight loss, hepatosplenomegaly, hypergammaglobulinemia, and can be fatal if left untreated (DESJEUX, P. Leishmaniasis: current situation and new perspectives. Microbiology and Infectious Diseases, v. 27, n. 5, p. 305-318, sept. 2004).

[005]. The main species of Leishmania that affect humans, as well as the clinical form related to each species, are listed in TABLE 1. TABLE 1 - CLINICAL FORMS AND MAIN SPECIES OF Leishmania THAT AFFECT HUMANS .

[006]. The induction of a host immune response to an antigen requires a series of complex interactions between different cell types (and their products), with the orchestrated secretion of proteins, surface receptors and signaling pathways and intracellular processes all occurring in a highly specialized manner. (GUERMONPREZ, P. et al. Antigen presentation and T cell stimulation by dendritic cells. Annual Review of Immunology, v. 20, p. 621-667, oct. 2002). After the parasite enters the host, several defense mechanisms are activated. Depending on the species of the parasite and the immune response developed by the host, the infection can cause a spectrum of disease that varies from self-healing lesions to disseminated and fatal infections (MOSSER, D. M. et al. Leishmania species: mechanisms of complement activation by five strains of promastigotes . experimental Parasitology, v. 62, n. 3, p. 394-404, Dec. 1986). The host's inability to control the infection is apparently related to two main factors: the ability of some Leishmania strains to resist the microbicidal effects of the host's innate and adaptive immunity events, as well as the establishment of a cellular and humoral immunity profile by the host. vertebrate host that favors an appropriate microenvironment for the installation of the parasite in the mononuclear phagocytic system (GRIMALDI, G. R.; TESH, R. B. Leishmaniases of the new world: current concepts and implications for future research. Clinical Microbiology Reviews, v. 6, p. 230- 250, Jul. 1993).

[007]. The diagnosis of leishmaniasis is based on clinical characteristics, epidemiological criteria and laboratory test results (GOTO, H.; LINDOSO, A. L. Current diagnosis and treatment of cutaneous and mucocutaneous leishmaniasis. Expert Review of Anti-infective Therapy, v. 8 , n. 4, p. 419-433, apr. 2010). However, there is no single method that can be adopted as the gold standard for diagnosing Leishmania spp. infections. (ANDRADE, A. S. R. et al. Use of DNA-based diagnostic methods for human leishmaniasis in Minas Gerais, Brazil. Acta Tropica, v. 78, n. 3, p. 261-262, mar. 2001; SZARGIKI, R. et al . Comparison of serological and parasitological methods for cutaneous leishmaniasis diagnosis in the state of Paraná, Brazil. Brazilian Journal of Infectious Diseases, v. 13, n. 1, p. 47-52, Feb. 2009).

[008]. During the laboratory diagnostic stage, several methods are applied to detect the parasite and identify the Leishmania species. Direct or parasitological methods isolate the parasite from skin ulcers (for the cutaneous form) or from bone marrow or lymph nodes (when it comes to VL). The material can be examined fresh (under an optical microscope) or inoculated into culture medium for parasite multiplication. The success in finding the parasite is inversely proportional to the time of evolution of the skin lesion, being rare after one year of infection. Lesions heavily contaminated with bacteria or fungi also contribute to reducing the sensitivity of the method. It is recommended to collect the material after local asepsis with soap and water. Leishmania species have variable growth capacity in culture and this method requires a series of laboratory conditions, trained personnel and a few days to acquire a sufficient number of promastigotes for investigations. These characteristics make the method of isolating the parasite in cultures unsuitable for large-scale epidemiological investigation (MONTENEGRO, J. Cutaneous reaction in leishmaniasis. Archives of dermatology and syphilology, v. 13, p. 187, 1926; FURTADO, T. Critérios for diagnosis of ATL. Brazilian Annals of Dermatology, v. 65, p. 51-86, Sept. 1980; NATIONAL HEALTH FOUNDATION. American Tegumentary Leishmaniasis Control Manual, Brasília, 2000).

[009]. The sensitivity of parasitological culture can be reduced by up to 80% when the infection is more than 12 months old. Chronic cases, such as those with a low parasite load in the lesion, result in an even more reduced sensitivity of this method (MONTENEGRO, J. Cutaneous reaction in leishmaniasis. Archives of dermatology and syphilology, v. 13, p. 187, 1926; FURTADO, T. Criteria for diagnosing ATL. Brazilian Annals of Dermatology, v. 65, p. 51-86, Sept. 1980; FOUNDATION NACIONAL DE SAÚDE. Manual for the Control of American Tegumentary Leishmaniasis, Brasília, 2000; LACHAUD, L. et al . Optimized PCR using patient blood samples for diagnosis and follow-up of visceral leishmaniasis, with special reference to AIDS patients. Journal of Clinical Microbiology, v.38, n. 1, p. 236-240, Jan. 2000).

[010]. Another possibility for diagnosing this parasite is the use of molecular techniques that search for fragments of the parasite's DNA. The PCR assay for L. infantum using peripheral blood as a clinical sample proved to be a highly efficient alternative for diagnosing the infection, exhibiting sensitivities of 82% to 100% and specificity of 100% (NUZUM, E. et al. Diagnosis of symptomatic visceral leishmaniasis by use of the polymerase chain reaction on patient blood. Journal of Infectious Diseases, v. 171, n. 3, p. 751-754, mar. 1995; LACHAUD, L. et al. Optimized PCR using patient blood samples for diagnosis and follow-up of visceral leishmaniasis, with special reference to AIDS patients. Journal of Clinical Microbiology, v.38, n. 1, p. 236-240, Jan. 2000). However, parasitemia can be episodic and may result in a low number of parasites in the blood at the time of collection and, consequently, non-detection by PCR. Furthermore, molecular techniques require specific apparatus and not all laboratories have highly complex equipment to carry out these analyzes (FICHOUX, Y. L. et al. Occurrence of Leishmania infantum parasitemia in asymptomatic blood donors living in an area of endemicity in southern France. Journal of Clinical Microbiology, v. 37, n. 6, p. 1953-1957, Jun. 1999).

[011]. The delayed skin hypersensitivity test (also called Montenegro) is used for the cutaneous form. This test can detect infection within a few weeks and evaluates cellular immunity. However, it is known that tests that use promastigotes or soluble parasite proteins as a source of sensitizing antigen can limit the specificity of the test and/or increase problems with the production of antigenic preparations (MONTENEGRO, J. Cutaneous reaction in leishmaniasis. Archives of dermatology and syphilology, v. 13, p. 187, 1926; GONTIJO, C. M. F. and MELO, M. N. Visceral leishmaniasis in Brazil: current situation, challenges and perspectives. Revista Brasileira de Epidemiologia, v. 7 n. 3, p. 338 - 349, sep. 2004).

[012]. Blood tests that detect antibodies (humoral immune response) are also useful. Many studies have been carried out to evaluate diagnostic methods for leishmaniasis. These studies emphasized the need to combine two or more indirect techniques for diagnosing the aforementioned parasitosis in order to make an accurate diagnosis (FABER, W. R. et al. Value diagnostic techniques for cutaneous leishmaniasis. Journal of the American Academy of Dermatology, n. 49, pp. 70-74, Jul. 2003; MARQUES-DA-SILVA, E. A. et al. Intramuscular immunization with P-36 (LACK) DNA vaccine induces a type 1 response but does not protect BALB/c mice against intravenous Leishmania chagasi challenge. Parasitology Research, v. 98, n. 1, p. 67-74, nov. 2005; SZARGIKI, R. et al. Comparison of serological and parasitological methods for cutaneous leishmaniasis diagnosis in the state of Paraná, Brazil. Brazilian Journal of Infectious Diseases, v. 13, n. 1, p. 47-52, Feb. 2009).

[013]. The enzyme-linked immunosorbent assay (ELISA) test for diagnosing leishmaniasis is considered a rapid, sensitive test for detecting antibodies and easily automatable. Studies aimed at improving this methodology and using purified antigens have been developed so that a quick, safe and effective way to diagnose this disease can be obtained (PORROZZI, R. et al. 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. Encoded by Genes from Five Families with Distinct Capacities for Serodiagnosis of Canine and Human visceral Leishmaniasis. The American Journal of Tropical Medicine and Hygiene, v. 85, n. 6, p. 1025-1034, Dec. 2011; COSTA, M. M. et al . Improved Canine and Human visceral Leishmaniasis Immunodiagnosis Using Combinations of Synthetic Peptides in Enzyme-Linked Immunosorbent Assay. PLOS Neglected Tropical Diseases, v. 6, no. 5, p. 1622, may. 2012; FUMAGALLI, M. A. C. Protection against Leishmania (Leishmania) amazonensis infection by immunization of BALB/c mice with synthetic peptides selected by Phage display and spot synthesis. 85 f. Dissertation (Postgraduate in Biochemical and Molecular Pharmacology) - Biological Sciences Sector, Federal University of Minas Gerais, Belo Horizonte, 2008).

[014]. Advances have been made with regard to the diagnosis and prevention of leishmaniasis during the last decade. However, there is still no single method that can be adopted as a standard, whether for diagnosis or prevention of the disease (ANDRADE, A. S. R. et al. Use of DNA-based diagnostic methods for human leishmaniasis in Minas Gerais, Brazil. Acta Tropica, v. 78, n. 3, p. 261-262, Mar. 2001). Within this context, it is necessary to search for purified antigens, with high sensitivity and safety for the diagnosis and prevention of the disease.

[015]. One technique that allows the search for antigenic targets is the Phage display technique, which stands out in the selection of mimotopes (protein sequences that have similarities with the original epitope) or continuous and discontinuous epitopes based on the antigen-antibody reaction. Once bacteriophages from a known library are selected by antibodies present in the serum of patients with a certain disease, their DNA can be extracted and sequenced. The identified peptide can be chemically synthesized for use in immunizations and immunodiagnostics (SOMPURAM, S. R. et al. Synthetic peptides identified from phage- displayed combinatorial libraries as immunodiagnostic assay surrogate quality-control targets. Clinical Chemistry, v. 48, n. 3, p 410-420, Mar. 2002; COELHO, E. A. F. et al. Immune responses induced by Leishmania (Leishmania) donovani A2, but not by LACK antigen, are protective against experimental L. (L.) amazonensis infection. Infection Immunology, v. 71, n. 7, p. 3988-3994, Jul. 2003; HAMBY P. G. et al. Use of peptide library screening to detect a previously unknown linear diagnostic epitope: Proof of Principle by Use of Lyme Disease Sera. Clinical and Vaccine Immunology, v. 12, n. 7, p. 801-807, Jul. 2005; SHARMA, A. et al. Specific and randomly derived immunoactive peptide mimotopes of mycobacterial antigens. Clinical and Vaccine Immunology, v. 13, n. 10, p. 1143-1154, Oct. 2006; GAUCI, C. et al. Protection of pigs against Taenia solium cysticercosis by immunization with novel recombinant antigens. Vaccine, vol. 30, no. 26, p. 3824-3828, jun. 2012).

[016]. Considering the multiplicity of factors that involve the transmission of leishmaniasis, it is difficult to formulate efficient strategies to control this disease. In this sense, progress is being made in the studies of new candidate antigens for vaccines against leishmaniasis, as immunoprophylaxis is recognized worldwide for being a safe and effective way of leading the immune system to develop defense against certain pathogenic agents.

[017]. The first-choice treatment for American cutaneous leishmaniasis is pentavalent antimony. Although this treatment is most often effective and indicated, disadvantages such as side effects, long duration of treatment and contraindication for heart disease, nephropathy, the elderly and pregnant women must be considered (MAYRINK, W. et al. Immunotherapy, immunochemotherapy and chemotherapy for American cutaneous leishmaniasis treatment. Revista da Sociedade Brasileira de Medicina Tropical, v. 39, n. 1, p. 14-21, feb. 2006).

[018]. The first studies looking for vaccines for the treatment of leishmaniasis date back to the beginning of the century, when strains of Leishmania tropica (originating from experimental infections in monkeys) were cultivated and sterilized and made up a vaccine applied to individuals with lesions characteristic of leishmaniasis. Analysis of the results demonstrated that the treatment was successful, as it provided a cure for the cases (ROW, R. Curative value of Leishmania culture vaccine in Oriental sore. British medical journal, v. 9, p. 540-541, 1912).

[019]. A protocol for immunotherapy was tested by Mayrink and colleagues by administering increasing doses of a vaccine composed of dead promastigotes to individuals with ATL. Series of 10 days of treatment were applied until the individuals were completely cured. Of the 122 participating patients, 60 received the vaccine and the rest were treated with conventional chemotherapy. The results of this trial confirm that 95% of patients treated with the vaccine were cured after a maximum of 20 series (400 days). Once again, immunotherapy has proven to be an effective treatment alternative for ATL, especially when conventional chemotherapy is not recommended (MAYRINK, W. et al. Treatment of American cutaneous leishmaniasis using vaccine. Anais Brasileiros de Dermatologia. v. 66, p. 55-59, 1991).

[020]. More recently, it was demonstrated that the association of antimony with the vaccine (immunochemotherapy) presented a high cure rate (100%). It was also found that the application of the vaccine reduced the salt volume by 17.9% and the curing time from 87 to 62 days; consequently, reducing side effects (MAYRINK, W. et al. Immunotherapy, immunochemotherapy and chemotherapy for American cutaneous leishmaniasis treatment. Revista da Sociedade Brasileira de Medicina Tropical, v. 39, n. 1, p. 14-21, feb. 2006 ).

[021]. The use of anti-Leishmania vaccine should also be considered in patients who have co-morbidities. The occurrence of leishmaniasis in patients with HIV or leprosy has increased, and these patients are generally resistant to conventional chemotherapy and have a poor prognosis in most cases. HIV-positive patients with ATL were treated with immunotherapy and in all cases regression of the lesion was achieved successfully (HERMETO, M. V., et al. Antimony-resistant American Cutaneous Leishmaniasis treated with immunotherapy. Memórias do Instituto Oswaldo Cruz; v. 87 , n.1, p.187, 1992; CUNHA, R. M. C. et al. Disseminated leishmaniasis in a patient infected with the HIV virus. Atypical clinical-morphological evolution. Revista da Sociedade Brasileira de Medicina Tropical, v. 27, n. 1, p. 389, 1994). In patients with an association of leishmaniasis and leprosy (lepromatous and indeterminate forms), three doses of vaccine combined with BCG over a period of three months were necessary to induce complete healing of the lesions (SILVEIRA, F. T., MAYRINK, W. diffuse anergic cutaneous leishmaniasis in the State of Pará, Brazil: Report of the cure of a case after 24 years of illness, after combined treatment of chemotherapy with immunotherapy. XXXIII Congress of the Brazilian Society of Tropical Medicine, Belo Horizonte, MG, Brazil, poster 42, p.129 , 1997).

[022]. The therapeutic arsenal of LTA is very restricted as the available drugs have high toxicity and none of them are effective. Relapse, therapeutic failure in immunocompromised patients and resistance to treatment are factors that motivate the search for an ideal drug. In this sense, it is desirable to carry out research aimed at curing the disease more quickly and with few side effects.

[023]. Therefore, the need to identify new biomolecules capable of generating an immunogenic response is evident not only in humans who already have the disease, but also in people who are in endemic areas, but who do not yet show symptoms. With this, it is possible to control the disease in three ways: early diagnosis, prophylaxis and treatment.

State of art

[024]. Several strategies have been used to identify antigens for use in the diagnosis of leishmaniasis.

[025]. In FARGEAS, C. et al. Synthetic Peptide-Based Enzyme-Linked Immunosorbent Assay for Serodiagnosis of visceral Leishmaniasis. Journal of Clinical Microbiology, vol. 34, no. 2, p. 241-248, Feb. In 1996, an ELISA test with antigens for VL, by cloning, was described, which achieved a sensitivity of 71% and a specificity of 93%. In PASSOS, S. et al. Recombinant Leishmania Antigens for Serodiagnosis of visceral Leishmaniasis. Clinical and diagnostic laboratory immunology, v. 12, no. 10, p. 1164-1167, oct. 2005, an antigen (KPM11) was described that has 100% sensitivity and specificity for the same disease. In KUMAR, S. et al. Identification and Characterization of a Novel Leishmania donovani Antigen for Serodiagnosis of visceral Leishmaniasis. The American Journal of Tropical Medicine and Hygiene, vol. 86, no. 4, p. 601 605, apr. In 2012, an antigen was obtained using SDS PAGE, which gave the test sensitivity and specificity of 95 and 100%, respectively.

[026]. Both in OLIVEIRA, S. G. G. et al. Characterization of Novel Leishmania infantum Recombinant Proteins Encoded by Genes from Five Families with Distinct Capacities for Serodiagnosis of Canine and Human Visceral Leishmaniasis. The American Journal of Tropical Medicine and Hygiene, vol. 85, no. 6, p. 1025-1034, dec. 2011, through cloning, as well as in COSTA, M. M. et al. Improved Canine and Human visceral Leishmaniasis Immunodiagnosis Using Combinations of Synthetic Peptides in Enzyme-Linked Immunosorbent Assay. PLOS Neglected Tropical Diseases, v. 6, no. 5, p. 1622, may. 2012, through B cell epitope prediction, sensitive tests for VL and canine leishmaniasis were studied. Of these two studies, the best result was observed by Costa, reaching 71% and 81% sensitivity and 100% specificity for VL and canine leishmaniasis, respectively.

[027]. In PORROZZI, R. et al. 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. Clinical and vaccine immunity, v. 14, no. 5, p. 544-548, May 2007, through cloning, and in FARIA, A. L. et al. High-Throughput Analysis of Synthetic Peptides for the Immunodiagnosis of Canine Visceral Leishmaniasis. PLOS Neglected Tropical Diseases, v. 5, no. 9, p.1310, sept. 2011, and FUMAGALLI, M. A. C. et al. Sensitive and specific serodiagnosis of Leishmania infantum infection in dogs by using peptides selected from hypothetical proteins identified by an immunoproteomic approach. Clinical and Vaccine Immunology, vol. 20, no. 6, p. 835-841, jun. 2013, through B cell epitope prediction, antigens for canine leishmaniasis were studied. None of them were able to present a test with sensitivity greater than 88% and specificity greater than 98%.

[028]. Regarding the development of vaccines for leishmaniasis, it can be said that vaccination against human leishmaniasis has been practiced for centuries. However, the first published vaccine studies were carried out in Asian countries, where live virulent strains of L. major were inoculated into the population, a practice known as leishmanization, whose objective would be to achieve lasting immunity after healing of the lesion, which was effective in Cutaneous leishmaniasis in the Old World. In inoculations carried out on people, the appearance of minor lesions was observed, followed by the patients' recovery. However, several problems prevent the widespread use of this procedure, including logistics, difficulty in standardizing the virulence of strains and occasional severe persistent lesions resulting from vaccine inoculation (NADIM, A. Immunity to Cutaneous Leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, v. 78, n. 6, p. 848, 1984; KHAMESIPOUR, A. et al. Use of an old method for evaluation of candidate vaccines against leishmaniasis. Vaccine, v. 28, n. 23, pp. 3642-3248, May 2005).

[029]. Many of the vaccines currently tested use killed parasites (first generation vaccines). The first clinical trials of immunization with dead Leishmania promastigotes against leishmaniasis in Brazil date back to 1939, when Salles-Gomes and Pessoa immunized a certain population with this type of preparation. The results suggested a protective effect of the vaccination schedule with a reduction in the size of the lesions in immunized individuals. Encouraged by the results, new tests against LTA in humans were developed. A suspension of dermotropic promastigote forms of Leishmania sp. prepared in phenolated solution was administered intramuscularly to volunteers. After 20 months of observation, a disease development rate of around 18% was found in negative control individuals, while among those vaccinated only 3.2% became ill (PESSOA, S. B, PESTANA, B. R. Essay on preventive vaccination in American cutaneous leishmaniasis with dead germs. Revista de Biologia e Higiene, v. 10, p. 112-118, 1941).

[030]. The studies by Pessoa and collaborators were followed much later by a series of tests carried out by Mayrink and collaborators (1979). Some modifications were introduced in the vaccination protocol (including reducing the number of Leishmania strains) and also in the preparation of the suspension. In this way, only five dermotropic strains (isolated from different areas in Brazil) were used, sonication of the parasites was also introduced and the total nitrogen content was used as a way to normalize the preparations.

[031]. Mayrink's group and collaborators used this vaccine on military personnel in the Amazon (a high-risk area for leishmaniasis). There was a reduction of 67.3% in 1980 and 7% in 1983 in the incidence of the disease among vaccinated individuals who started to present a positive Montenegro Test (IDR). It was also suggested that the intradermal test (IDR), in humans, would be an important indicator of the immune response which, in a way, would reflect a stage of protection. No disease exacerbation effect was observed (ANTUNES, C. M. F. et al. Controlled field trials of a vaccine against new world cutaneous leishmaniasis. The International Journal of Epidemiology, v.15, n.4, p. 572-580, dec.1986 ).

[032]. The study by the aforementioned research group also revealed that vaccination increases lymphocyte proliferation and peripheral blood mononuclear cell responses, when compared to unvaccinated control individuals. They also observed a 90% correlation of these results with the positive skin test. The assays demonstrated that the serum of vaccinated individuals recognized eight main antigens with molecular masses estimated at 13.5, 25, 40, 63, 73, 85, 97 and 160 kDa (NASCIMENTO, E. et al. Vaccination of Humans against Cutaneous Leishmaniasis : Cellular and Humoral Immune Responses. Infection and immunity, v. 58, n. 7, p. 2198-2203, Jul. 1990).

[033]. In 1985, results of a vaccination campaign during an ATL epidemic in the city of Viana in Espírito Santo revealed statistically significant differences in infection levels between vaccinated and unvaccinated groups, with 87.6% of vaccinated individuals presenting a positive Montenegro Test after vaccination (MAYRINK, W. et al. An experimental vaccine against American dermal leishmaniasis: experience in the State of Espírito Santo, Brazil. Annals of Tropical Medicine and Parasitology, v. 79, n. 3 p. 259-269. jun. 1985). Despite the excellent results obtained with the campaign, criticism was made regarding its composition and identification of the strains that made up the vaccine suspension, as the various Leishmanian strains were not well characterized, which led to difficulties in manufacturing and standardizing the vaccine.

[034]. Scientific meetings were organized to analyze the appropriate vaccine strategy for research and vaccine formulation using only one strain of L. amazonensis. It was also decided that Biobrás SA (a pharmaceutical company interested in producing and marketing the product) would produce the vaccine, including good manufacturing practices in the process. Phase I trials were carried out in Rio de Janeiro. According to clinical and laboratory evaluations carried out, despite the high concentration of total protein per immunization, it was concluded that the vaccine was harmless if applied, and Phase II trials were initiated (MARZOCHI, K. B. F. et al. Phase I studies for the Leishvacin (Killed Leishmaniasis vaccine) in Brazil. Workshop on vaccine efficacy trial against Leishmaniasis, 1995). This vaccine was tested using adjuvants in 56 healthy volunteers who received vaccine and placebo or rhGM-CSF (Recombinant human granulocyte-macrophage colony-stimulating factor). The levels of IFN-gamma and IL-5 were significantly increased in the group that received the vaccine. The group that received only adjuvant had a significant response to vaccination compared to the placebo group (86% of all volunteers were IDR positive on day 42). The use of rhGM-CSF improved the immune response, indicating that it could optimize the immune response to the vaccine (FOLLADOR, I. et al. Immune responses to an inactive vaccine against American cutaneous leishmaniasis together with granulocyte-macrophage colony-stimulating factor. Vaccine, v. 31, p. 1365-1368, Jan. 2002). Despite all efforts, there is still no vaccine for human leishmaniasis in use.

[035]. The use of attenuated organisms or parasitic products is an approach that aims to avoid the dangers and inconveniences of using infectious parasites. Regarding second generation immunobiologicals, protocols have been developed and/or tested in several countries, but to date, none have been proven to be satisfactorily effective or viable (VELEZ, I. D. et al. Safety and immunogenicity of a defined vaccine for the prevention of cutaneous. Vaccine, v. 28, n. 1, p. 329-337, Dec. 2010; FUMAGALLI, M. A. C. et al. Sensitive and specific serodiagnosis of Leishmania infantum infection in dogs by using peptides selected from hypothetical proteins identified by an immunoproteomic approach. Clinical and Vaccine Immunology, v. 20, n. 6, p. 835-841, Jun. 2013).

[036]. Despite the existence of numerous studies aimed at the prophylaxis of human leishmaniasis, many gaps still need to be filled. Among the second generation vaccines tested for leishmaniasis, the ones that stand out are described below.

[037]. In SPITZER, N. et al. Long-term protection of mice against Leishmania major with a synthetic peptide vaccine. Vaccine, vol. 17, no. 11, p. 1298-1300, Mar. 1999, the GP63 antigen was used, obtained through cloning of L. major antigens in Balb/c mice. With this antigen, protection (absence of lesions) was achieved for 10 months after the challenge.

[038]. The study COELHO, E. A. F. et al. Immune responses induced by Leishmania (Leishmania) donovani A2, but not by LACK antigen, are protective against experimental L. (L.) amazonensis infection. Infection Immunology, vol. 71, no. 7, p. 3988-3994, Jul. 2003 uses the LACK and A2 antigens, obtained through cloning of L. donovani antigens in BALB/c mice. In this study, protection was observed with high levels of IFN-γ and low levels of IL-4, in addition to specific antibodies and decreased parasite quantification. However, only protection was observed with A2 and not with LACK, when the challenge was performed with other Leishmania strains.

[039]. In SALAY, G. et al. Testing of four Leishmania vaccine candidates in a mouse model of infection with Leishmania (Viannia) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World. Clinical Vaccine Immunology, v. 14, no. 9, p. 1173-1181, sep. 2007, 4 different proteins are described, obtained through cloning of L. braziliensis in Balb/c mice. No protection or reduction in the size and number of characteristic lesions was observed, nor was there any stimulation in the production of IFN-y.

[040]. In FUMAGALLI, M. A. C. Protection against Leishmania (Leishmania) amazonensis infection by immunization of BALB/c mice with synthetic peptides selected by Phage display and spot synthesis. 85 f. Dissertation (Postgraduate in Biochemical and Molecular Pharmacology) - Biological Sciences Sector, Federal University of Minas Gerais, Belo Horizonte, 2008 peptides are synthesized using the Phage Display technique from L. amazonensis in Balb/c mice. The tested targets were protected, with high production of IFN-y, but with low levels of IL-4, IL-10 and specific antibodies in the vaccinated animals.

[041]. Patent US5674503, filed on 09/21/1994, describes immunogenic peptides for leishmaniasis, with the gp64 protein being cited as one of them. However, the amino acid sequences described are different from those presented in the present patent.

[042]. Patent WO2006058243, filed on 11/23/2005, cites a useful formulation for increasing the immune response to Leishmana antigens, comprising antigens such as GP46, P4, GP63, p36-LACK and P8. However, the epitodes of these antigens are different from those presented in the present patent.

[043]. The leishmaniasis control measures implemented so far have been unable to eliminate or reduce transmission and prevent the occurrence of new epidemics. The methods available for diagnosis and research aimed at immunoprophylaxis of leishmaniasis still do not present the desired efficacy and applicability, although promising advances have been achieved with research into new tests. Considering the above, the need to produce purified antigens, with high sensitivity and safety for the prevention of leishmaniasis, is evident.

Brief Description of the Invention

[044]. The present invention patent aims to protect synthetic peptides, obtained through the Phage Display technique, containing mimotopes or epitopes that have the potential for serological diagnosis and also to activate lymphocytes. This last property is of particular importance, since synthetic peptides can also be used in vaccine protocols. The term “mimotope” refers to a peptide selected from random libraries expressed in phage that mimics the structure of an epitope. While, the term “antigen” refers to any substance that induces an immune response involving T or B cells.

[045]. In this way, the invention provides amino acid sequences in the form of peptides that can be used in different presentations, such as in diagnostic methods and kits or in immunological compositions.

[046]. The invention provides a diagnostic method for infectious diseases related to Leishmanias, this method comprises the following steps: a) Sample collection; b) Place the sample, after processing, in contact with at least one of the identified peptides; c) Detection of the bond between peptides and sample components.

[047]. The present invention also aims to develop a diagnostic kit for infectious diseases caused by Leishmania. This diagnostic kit comprises at least one of the identified peptides and reagents for detecting the reaction between antigen and components of the sample obtained from individuals.

[048]. The present invention also refers to immunological compositions that have as an active ingredient at least one of the identified peptides associated with one or more physiologically acceptable vehicles or adjuvants.

[049]. Another aspect of the present invention comprises the use of the peptides mentioned herein in biosensors, for detecting infection with Leishmania.

Citation of Figures

[050]. The attached figures will serve to provide a better understanding of the process of identifying peptides derived from peptide libraries presented in phages and their uses. It should be noted that the process is not limited to the species L. braziliensis, but is extended to other Leishmania species.

[051]. FIGURE 1 illustrates the bands obtained by SDS-PAGE of soluble antigens from L. braziliensis and L. amazonensis. Columns: 1 - Molecular mass standard; 2 - L. braziliensis; 3 - L. amazonensis. Twenty micrograms of protein were applied to each channel of a 15% polyacrylamide gel, then serum from patients with ATL was added and finally the gel was stained with silver nitrate.

[052]. FIGURE 2 illustrates the bands obtained by SDS-PAGE of total IgGs from patients with cutaneous leishmaniasis purified by ammonium sulfate precipitation and affinity chromatography. Columns: 1 - Molecular mass standard; 2 - Total IgGs precipitated by ammonium sulfate under non-reducing conditions; 3 - Total IgGs purified by affinity chromatography under non-reducing conditions; 4 - Total IgGs purified by affinity chromatography under reducing conditions. Three micrograms of protein were applied to each channel of a 10% polyacrylamide gel stained with Coomassie blue.

[053]. FIGURE 3 illustrates the reactivity of total IgGs from patients with ATL against L. braziliensis and L. amazonensis antigens - reaction observed in western blotting. Molecular mass standard (column 1). Forty micrograms of antigen from L. braziliensis (columns 2 and 3) and L. amazonensis (columns 4 and 5) were separated by 15% SDS-PAGE. Western blotting was performed with positive human serum for cutaneous leishmaniasis diluted 1:50 (columns 2 and 4) and 40 µg/mL of total IgGs (columns 3 and 5). Detection of the reaction was carried out with anti-human IgG antibody conjugated to peroxidase and using DAB as chromogen.

[054]. FIGURE 4 illustrates the reactivity of antigen-specific immunoglobulins by ELISA. A plate was sensitized with 5 µg/mL of L. braziliensis antigen and incubated with immunoglobulins starting from 100 µg/mL for total (light bars) and 10 µg/mL for antigen-specific immunoglobulins (dark bars). The detection of the reaction was carried out with an anti-human IgG Fc-specific antibody conjugated to peroxidase and OPD as chromogen.

[055]. FIGURE 5 illustrates (A) plate presenting the antigen-antibody reaction using the ELISA technique, demonstrating the increase in reactivity with each selection cycle (B) - absorbance obtained from each selection cycle using the ELISA test. An ELISA plate was sensitized with 5 µg/mL of anti-phage and incubated with phage (109/well) from cycles I, II, II, IV. Anti-L. braziliensis antibodies were subsequently added. Detection of the reaction was carried out with Fc-specific anti-human IgG antibody, conjugated to peroxidase and with OPD as chromogen. C: control (Wild phage).

[056]. FIGURE 6 illustrates the production of IgG in the serum of hamsters immunized with peptide 1, 2, 3 or a mix of the three peptides by ELISA. Non-immune: Serum was added from the group of animals that did not receive immunizations, Adj: Serum was added from the group of animals that received immunizations only with adjuvant, P1: Serum was added from the group of animals immunized with peptide 1 and adjuvant, P2: Serum from the group of animals immunized with peptide 2 and adjuvant, P3 was added: Serum from the group of animals immunized with peptide 3 and adjuvant, Pep 1, 2, 3 (MIX) was added: Serum from the group of immunized animals was added with peptides 1, 2, 3 and adjuvant. Sensitization of the ELISA plates was performed with 0.2 µg/ml of peptides 1, 2, 3 or with the MIX of the three. Serum samples from hamsters from different experimental groups were applied, at a dilution of 1:50 (poll of ten animals from each group). Detection of the reaction was performed with peroxidase-linked anti-hamster total IgG antibody (200 ng/mL) and OPD as chromogen.

[057]. FIGURE 7 illustrates the reactivity of IgGs from hamsters inoculated with peptides against L. braziliensis antigens by western blotting. Thirty micrograms of L. braziliensis antigen were separated by 15% SDS-PAGE and after Western Blotting reacted with hamster serum diluted 1:100 (pool of hamster serum that received peptide 1 in channel 1, peptide 2 in channel 2, peptides 3 in channel 3, MIX of the three peptides in channel 4, in channel 5 the serum from the hamsters that received only adjuvant was added and in channel 6 the serum from the hamsters that did not receive any inoculation). The reaction was detected with an anti-hamster IgG antibody conjugated to peroxidase (100 µg/mL).

Detailed Description of the Invention

[058]. The present invention consists of peptides whose application includes methods of diagnosis and prophylaxis of Leishmaniasis. Such peptides were obtained from the identification of specific mimotopes of Leishmania antigens using the Phage Display technique, a methodology for presenting peptides or proteins, including antibodies, on the surface of filamentous phages. This technique allows the study of protein-ligand interactions, can be used in mapping monoclonal and polyclonal antibody epitopes, as well as identifying ligands for receptors and selecting substrates for enzymes (Azzazy H. M., Highsmith W.E Jr., Clin Biochem., 35: 425-445, 2002).

[059]. The Phage Display technique presents itself as an alternative for identifying Leishmania antigens, whether for use in diagnosis, immunotherapy, vaccines, or immunizations, since the methods previously used, as described in the state of the art, are not comprehensive for all Leishmania species, especially L. braziliensis.

[060]. Furthermore, the methods used to date have been based on prior knowledge of antigens for the characterization of new diagnostic reagents. An advantage of the Phage Display technique is that it does not require prior identification of the natural antigen against which an immune response is directed (Azzazy H. M., Highsmith W.E Jr., Clin. Biochem., 35: 425-445, 2002).

[061]. In this way, phage-presented peptide libraries were screened using antibodies and more specifically, antigen-specific antibodies.

[062]. Antigen-specific antibodies can be obtained not exclusively from samples from infected individuals, but can be obtained from samples from experimentally infected animals.

[063]. The term “sample” refers to a variety of sample types obtained from individuals such as blood, serum, plasma and other samples of biological origin.

[064]. Blood samples of human or animal origin can be processed to separate antibodies by procedures already known in the art such as centrifugation or further processed by additional procedures such as precipitation and/or affinity chromatography to separate specific subclasses of immunoglobulins. Furthermore, antibodies can be separated by binding to a column immobilized with antigen or antibodies can be separated from immunoblots (Harlow E., Lane D. Antibodies: a laboratory manual. New York: Cold Spring Harbor Laboratory, 1988. 726 p. ).

[065]. The antibodies used in the present invention for the selection of peptides from phage libraries came from serum samples from different individuals with ATL.

[066]. Antigen-specific antibodies denote, in the present invention, antibodies obtained from patient serum that are subjected to ammonium sulfate precipitation with subsequent purification by G-protein affinity chromatography and further purified against Leishmania sppa antigens after immunoblotting. However, obtaining antigen-specific antibodies is not limited to this example, but may involve the use of one or a combination of two or more procedures known in the art. Also, the antibodies obtained are not limited to the IgG class, but can include other classes and their subclasses.

[067]. The use of such antigen-specific antibodies to select peptides in phage libraries provides a greater opportunity to find mimetic peptides (mimotopes) of Leishmania antigens.

[068]. Phage libraries were screened using Leishmania spp. antigen-specific antibodies. as well as the same libraries were screened using Leishmania spp. antigen-specific antibodies. In the latter case, phages not linked to antibodies from patients with Leishmaniasis were subsequently used with Leishmania spp. antigen-specific antibodies, in order to prevent selection of nonspecific mimotopes.

[069]. Using Phage Display technology, phage/peptide clones were selected to recognize antibodies from patients with Leishmaniasis.

[070]. The identified peptides comprise mimotopes or also called mimotypes, as they are not natural epitopes, but are epitopes that mimic part of the natural epitope. Mimicry is due to similarity in sequence or similarity in physicochemical properties and special organization (Moreau V. et al., Bioinformatics, 22: 1088-1095, 2006).

[071]. One of the objectives of the present invention was to evaluate whether the identified peptides, which were, in turn, selected from peptide libraries expressed in phage, had the ability to react with antibodies from the serum of patients with Leishmaniasis.

[072]. The ability of peptides to react with antibodies from these patients was demonstrated in the present invention. The analysis involves incubating the sample, preferably serum, from individuals with leishmaniasis in the presence of one or more peptides. Binding of the sample antibody to the peptide(s) is then detected. It is understood that in addition to serum, other samples of biological origin that contain antibodies can be used in the analysis.

[073]. Peptides can be used individually or in combination. The term “combined” refers to a composition formed by two or more sequences listed in table 3. The term “combined” also refers to a peptide polymer, which in turn is a longer peptide formed by repeating of a peptide sequence which may or may not be separated by spacers or linkers. When present, spacers or linkers can be located between some sequences or between all peptide sequences. The polymer can be made up of any peptide sequence and the polymer can also comprise different peptide sequence combinations. Therefore, a polymer can be formed by one or more repeats of the first sequence, followed by one or more repeats of the second peptide sequence.

[074]. The peptide sequences covered by the present invention may undergo modifications resulting in variants, as long as these variant forms maintain the antigenic and/or immunogenic characteristics of the original sequences from which they were derived.

[075]. The term antigenic refers to the ability of the antigen to recognize the antibody or T cell receptor, while the term immunogenic refers to the ability to induce a humoral and/or cellular immune response.

[076]. Modifications include, but are not limited to, acetylation, amidation, formylation, biotinylation, phosphorylation, succinylation, sulfurylation, addition of one or more fatty acids, addition of one or more dyes such as AMC (7-amino-4-methyl-coumarin ), Cy3, Cy5, Dabcyl, Dansyl, DNP (2,4-dinitrophenyl), DNP-lysine, EDANS (5-2((2-aminoethyl)amino)naphthalene-1-sulfonic acid), fluorescein, NBD (7- nitrobenzene-2-oxa-1,3-diazol), p-nitro-aniline, rhodamine B and Tmra, replacement of one or more amino acids with a naturally occurring one, replacement of one or more amino acids with unnatural or special amino acids such as, citrulline, ornithine, a-acetyl-lysine, a-alanine, aminobenzoic acid, 6-aminocaproic acid, aminobutyric acid, dimethyl-lysine, hydroxyproline, mercaptopropionic acid, methyl-lysine, 3-nitro-tyrosine, norleucine, pyroglutamic acid, carbobenzoxyl and many others, addition of one or more naturally occurring amino acids, addition of one or more unnatural or special amino acids, substitution of one or more amino acids for heavy amino acids, addition of one or more heavy amino acids, substitution of one or more amino acids for D- amino acids, addition of one or more D-amino acids, cyclization, addition of spacers or linkers, addition of one or more disulfide bridges, addition of polyethylene glycol (PEG), labeling with radioactive isotopes, replacement of one or more amino acids by N- methyl amino acids, addition of one or more N-methyl amino acids and deletion of one or more amino acids.

[077]. Another modification is the conjugation of peptides to carrier proteins such as bovine serum albumin (BSA), ovalbumin, KLH (keyhole limpet hemocyanin), diphtheria toxoid, tetanus toxoid, etc. Several methods are available for conjugation of peptides to proteins, such as conjugation via EDC, conjugation via MBS and conjugation via glutaraldehyde.

[078]. Another modification that can be found in peptides is the formation of MAPs (Multiple Antigen Peptides), which are characterized by using ? and Q-amino lysine groups to form a backbone to which multiple peptide chains can be attached. MAPs are an alternative to conjugating antigens to carrier proteins.

[079]. It should be noted that when peptide sequences are used individually or in combination, this refers to the use of both original sequences and variant sequences.

[080]. Peptides (original sequences or variants) can be produced by recombinant DNA technology or by chemical synthesis and both methods are known in the art.

[081]. An objective of the present invention is to provide a method for diagnosing infection by Leishmania spp.. To this end, at least one of the peptides (isolated or combined) is immobilized on a solid support and after placing in contact the sample containing antibodies obtained from individuals with the peptide(s) the binding between antibody and peptide(s) is detected.

[082]. The antigen, which corresponds to at least one of the peptides, is placed in contact with a solid support so that it can bind and become adsorbed to that support. Solid supports include polystyrene or polyvinyl chloride (PVC) 96-well microplates, nitrocellulose membranes, polyvinylidene fluoride (PVDF) or nylon, polystyrene or agarose beads, glass, among others. The immobilized antigen is placed in contact with the sample containing antibodies for interaction. Molecules that have not specifically interacted with the antigen bound to the solid support are removed by washing with appropriate buffer solutions. To detect the interaction between antigen and antibody, a second labeled antibody (secondary antibody) is added. The solid support can be washed again to remove unbound antibodies. The reaction between antigen and antibody is revealed by a method, which depends on the label used, in order to produce a detectable or measurable signal. Signal intensity indicates the amount of antibody in the sample.

[083]. The antibody that recognizes the antigen is called the primary antibody. If this antibody is labeled, direct detection of the antigen is possible. Generally, the secondary antibody is labeled, so the antigen is detected indirectly.

[084]. The secondary antibody can be a polyclonal or monoclonal antibody which in turn is produced in host animals such as mice, rabbits, goats or sheep. The secondary antibody is a species-specific antibody to the primary antibody, that is, if the primary antibody is a mouse monoclonal antibody, the secondary antibody must be an anti-mouse antibody produced in a host animal other than mice. Secondary antibodies are available with specificity for the entire antibody molecule or for antibody fragments such as Fc or Fab regions as well as for different classes and subclasses.

[085]. The labels used in secondary antibodies can be radioactive isotopes, enzymes, or fluorophores. Generally, labels with enzymes or fluorophores are used. Enzymes provide a detectable signal by reacting with a specific substrate that generates a colored or fluorescent product or release of light. The most commonly used enzymes are alkaline phosphatase and peroxidase (HRP). The most commonly used fluorophores for labeling are fluorescein and rhodamine.

[086]. Secondary antibodies can also be labeled with biotin. The presence of biotin allows efficient detection with avidin or streptavidin labeled with enzymes or fluorophores. Systems with biotin have the characteristic of signal amplification, thus increasing sensitivity. Secondary antibodies can also be conjugated to agarose or colloidal gold.

[087]. Antibody binding proteins such as Protein A, Protein G, Protein A/G and Protein L can also be useful in detecting antibodies.

[088]. The detection and quantification of substances such as peptides, proteins and antibodies can be carried out using the ELISA (Enzyme-linked Immunosorbent Assays) technique, also called EIA (Enzyme Immunoassay). The ELISA technique is performed in 96- or 384-well polystyrene or PVC plates in which proteins, peptides or antibodies are passively bound. Small molecules such as peptides can be more efficiently immobilized on plates through peptide biotinylation and the use of streptavidin-coated plates. The format of the technique can be direct, indirect, sandwich or competition. Most commonly, detection of the reaction between antigen and antibody is done using fluorescent or enzymatic labels. The signal emitted after the addition of enzyme substrates can be colorimetric, chemiluminescent or fluorescent. Colorimetric substrates include PNPP, ABST, OPD and TMB.

[089]. The ELISA technique also allows the detection of cytokines such as interferon gamma (IFN-N). IFN-a is a cytokine produced mainly by T cells in response to antigens and its detection has shown usefulness in the diagnosis of Leishmaniasis (Geluk A. et al., Clin Vaccine Immunol., 15: 522-533, 2008). The analysis of IFN- is based on the principle that T cells from individuals sensitized with Leishmania spp. antigens. they produce IFN- when they encounter these antigens again. Therefore, increased levels of IFN- are indicative of infection. In the analysis of IFN-ü, whole blood (diluted or undiluted) or peripheral blood mononuclear cells, separated by a density gradient in FicollTM, are cultured in the presence of antigens, such as peptides and when present, sensitized T cells secrete IFN-ü . Cell culture supernatant containing the cytokine can then be detected by sandwich ELISA testing. Quantification of IFN- can also be done using ELISPOT (Enzyme-Linked Immunospot Assay).

[090]. Therefore, in addition to samples containing antibodies, cell culture supernatants obtained from individuals can also be used as samples.

[091]. In addition to the ELISA technique, other techniques can be used to detect leishmania antigens or antibodies specific to them. Techniques include Western Blotting/immunoblotting, immunoprecipitation, agglutination, immunofluorescence, radioimmunoassay, Spot Synthesis, microarray, dipstick (immunochromatographic analysis), MAPIA (Multiantigen Print Immunoassay).

[092]. In Western Blotting or also called immunoblotting, antigens, such as peptides, for example in the form of conjugates, polymers or MAPs, could be transferred to membranes, generally nitrocellulose or polyvinylidene fluoride, after polyacrylamide gel electrophoresis. After blocking the membrane to prevent nonspecific binding, the membrane is incubated with antibodies from individual samples. Anti-Leishmania spp. antibodies. if present in the sample, they will bind to the antigen fixed on the membrane and can be detected by adding a labeled secondary antibody. A suitable substrate is then added to produce a detectable signal such as a chromogenic precipitate, chemiluminescence, or fluorescence. Several methods can be used to transfer antigens to membranes, such as simple diffusion, vacuum transfer or electrotransfer. Commonly, electrotransfer, also called electrophoretic elution, is the most used method and can be carried out using a wet or semi-dry system. Generally, secondary antibodies are labeled to enzymes or fluorophores. The signal obtained from chemiluminescent substrates can be detected using X-ray film or digital imaging equipment.

[093]. Other techniques such as immunodiffusion and agglutination could be used to detect leishmania antigens or anti-leishmania antibodies. These are low sensitivity techniques, but they are presented as alternatives for field applications and in less developed countries. Immunodiffusion is based on the precipitation that occurs when antigen and antibody diffuse in agar and the antigen-antibody complex formed appears in the form of a precipitation line or arc. While, the agglutination reaction is characterized by the formation of visible aggregates as a result of the interaction of specific antibodies and insoluble particles that contain antigenic determinants on their surface. Agglutination can occur with inert particles (latex particles, polystyrene, betonite, sepharose, etc.) or with unrelated antigenic cells such as red blood cells, which adsorb or fix soluble antigens.

[094]. The immunofluorescence technique is used to search for antibodies and antigens. In direct immunofluorescence, a specific antibody labeled with a fluorophore binds to the antigen in cells or tissues, forming an immunocomplex that is observed under a fluorescence microscope. In indirect immunofluorescence, antigens in cells or tissues are initially detected by incubation with a specific antibody directed against the antigen to be investigated followed by incubation with an anti-primary antibody labeled with fluorophores. Indirect immunofluorescence, for the search for antibodies, antigens fixed to glass slides are incubated with antibodies from samples to form the antigen-antibody complex. Afterwards, the secondary antibody labeled with a fluorophore is added to the preparation and if there is antibody in the sample, fluorescence will form. For amplification of the reaction, fluorophores can be conjugated to streptadine. Therefore, biotin labeled with the secondary antibody reacts with streptavidin conjugated to a fluorophore.

[095]. In radioimmunoassay reagents, such as antigen or antibody, are labeled with radioisotopes to detect unlabeled antigen or antibody in the sample. An unlabeled antigen (or antibody) is attached to a solid support, such as a plastic surface or particulate matrices, and the fraction of the antibody (or antigen) retained on the surface is then determined. The assay can be competitive or with excess reagent. Various radioisotopes can be used, among them the most used are 125I and 131I. In addition to the solid phase system, other methods such as immunoprecipitation, chemical separation and adsorbent techniques can be used to separate the free and bound fractions of the labeled substance.

[096]. In the Spot Synthesis technique, peptides are synthesized in defined positions (spot) on a cellulose membrane using the Fmoc/tBu chemical strategy. The cellulose membrane with bound peptides can be used to detect antibodies in samples following the same steps already described for Western Blotting.

[097]. Protein or peptide microarray is another technology that is useful in detecting antibodies in samples. In this technique, suitable glass slides containing antigens are incubated with samples containing antibodies. Specific antibodies bound to the antigen are detected by addition of fluorophore-labeled secondary antibody.

[098]. The immunochromatography method is also a tool for detecting antibodies in samples. In this test, the sample containing antibodies is deposited on a strip of nitrocellulose membrane. Antibodies in the sample bind to the detection antibody labeled with latex beads or colloidal gold attached to the membrane. The complex formed - sample antibodies and detection antibody - moves until it finds the antigen immobilized on the nitrocellulose membrane and if there are specific antibodies for the antigen in the sample, the strip will change color. The analysis can be qualitative or quantitative and in this case the method is combined with a detection system capable of quantifying the binding reaction.

[099]. MAPIA (Multiantigen Printing Immunoassay) is another antibody detection method and is based on the direct application of antigens by printing on nitrocellulose membranes followed by classical antibody detection methods, such as antibodies conjugated to enzymes and chromogenic substrates. MAPIA allows the detection of antibodies to several antigens in a single analysis.

[100] . In addition to methods for detecting anti-leishmanial antibodies in samples, other methods such as lymphoproliferation analysis also allow the assessment of exposure to infectious agents. The lymphoproliferation test evaluates, in cell culture, the responses of lymphocytes in contact with specific antigens and mitogens (generally, concanavalin A or phytohemagglutinin). Cell cultures include mononuclear cells separated from peripheral blood or whole blood cultures. The activation of the immune response promotes cell proliferation in the culture and this activation can be identified by cell counting under microscopy, by the incorporation of radioactive material into the DNA (for example, tritiated thymidine), by the reduction of tetrazolium blue (MTT), by the incorporation lysosomal neutral red dye and flow cytometry.

[101] . The detection of a humoral or cellular response to antigens, such as proteins and peptides, can be carried out using different methods that were presented here above, but are not limited to these.

[102] . The present invention also aims to develop a diagnostic kit for leishmaniasis. The kit may include any configuration and composition to perform the various analysis methods described here. The kit will be presented in a commercially packaged form containing the essential elements required to conduct an analysis according to the methods presented. Therefore, the kit comprises one or more peptides (isolated or combined/variant forms), devices, reagents and written instructions for performing the assay.

[103] . For example, a kit to detect the presence of anti-Leishmania antibodies. may contain one or more peptides for immobilization or already immobilized on a solid support and reagents for detecting the binding between sample antibodies and peptides such as anti-human IgG antibody conjugated to biotin and streptavidin conjugated to peroxidase. Or, another kit could consist of a device made up of immunochromatographic strips impregnated with peptides and detection antibodies and could also contain capillary pipettes and buffer solution.

[104] . The examples below demonstrate that peptides are capable of inducing the production of antibodies and, furthermore, these anti-peptide antibodies are recognized by natural antigens of Leishmania braziliensis, although such peptides can be used for any species of Leishmania. In addition to inciting a humoral response, peptides have the potential to induce a cellular response as shown in the example of a study of delayed-type hypersensitivity.

[105] . The delayed-type hypersensitivity reaction (DTH) allows the evaluation of the individual's previous exposure to the antigen as well as evaluating the cellular immune response of individuals to an antigen. DTH tests using specific reagents are extremely desired due to their simplicity of execution and reading, thus allowing their application in the field. The DTH test consists of intradermal application of the antigen, generally on the anterior surface of the forearm and after 48-72 hours the size of the induration is read. The amount of antigen applied will depend mainly on the composition of the composition and should be determined after extensive experimentation.

[106] . Immunological compositions for use in DTH testing comprise one or more peptides (isolated or combined/variant forms) associated with one or more physiologically acceptable carriers. An example of physiologically acceptable vehicles is saline solution plus preserving agents such as phenol.

[107] . Since peptides are capable of inducing a humoral response and/or cellular response, the present invention includes immunological compositions for use as vaccines, provided that vaccines are designed to enhance immunity and prevent infection in individuals.

[108] . Immunological compositions for use as vaccines comprise one or more peptides (isolated or combined/variant forms), preservatives, physiologically acceptable carriers and additional agents such as adjuvants, which can stimulate the individual's immune response to the immunogenic component. Examples of adjuvants include complete or incomplete Freund's adjuvant, aluminum hydroxide, liposomes, immunostimulating complexes (ISCOMS), etc. The composition of the formulation as well as the route of immunization and the number of doses to be administered to induce an effective humoral and/or cellular response must be determined experimentally.

Example 1: Biosealing of Phage-Displayed Libraries Patients and controls

[109] . Blood samples were collected from patients with ATL to obtain sera, selection of specific immunoglobulins against L. braziliensis and to evaluate the antigens produced in the present work. The volunteers came from the Hospital de Clínicas of the Federal University of Paraná (UFPR) and several Health Regions in the State of Paraná. Blood samples were also collected from healthy individuals who had no history of leishmaniasis or Chagas disease, lived in an area free from the disease and were not in contact with patients infected with these diseases. Sera were collected and stored in a freezer at -20°C.

Production of Leishmania braziliensis and L. amazonensis antigens

[110] . To perform the ELISA and Western blotting tests, antigens were produced from cultures of promastigotes of L. braziliensis (MHOM/BR/94/Cur22) and L. amazonensis (MHOM/BR/73/M2269). The strains used were previously characterized by isoenzymes and are stored in the cryobank of the UFPR Molecular Parasitology Laboratory.

[111] . Promastigotes were cultured in biphasic Novy-McNeal-Nicolle (NNN) medium, with the liquid phase being 0.9% sterilized, non-pyrogenic physiological saline at 24°C. The strains were cultured between five and seven days into new tubes containing culture medium. After obtaining six tubes, rich in promastigote forms, 0.5 mL of the supernatant from each tube was transferred to sterile culture bottles (100 mL) containing RPMI culture medium (Sigma-Aldrich, Saint Louis, Missouri, USA). enriched with 10% fetal bovine serum (Gibco, Rockville, Maryland, USA). After the parasites multiplied, the promastigote forms were recovered by filtration through sterilized gauze. The content obtained was subjected to centrifugation at 3,500 g for 10 minutes at 4°C. Washes were carried out with physiological solution 0.9%, 0.3%, 0.9% and PBS (phosphate-buffered saline) pH 7.2, respectively, to eliminate the remaining culture medium, lyse the red blood cells and remove the rabbit hemoglobin. Cell recovery was performed by centrifugation at 3,500 g for 10 minutes at 4°C.

[112] . After the last centrifugation, the parasites were disrupted by resuspending the sediment with sterilized distilled water in a volume equal to half the volume of that sediment. For complete lysis of the protozoa, six cycles of sudden freezing and thawing were also carried out, immersing the tubes in liquid nitrogen until freezing and immediately thawing in a water bath at 37°C. Keeping the antigens preserved in an ice bath, the Sonoplus HD 2070 Ultrasound (Bandelin, Berlin, Germany) was applied at 150 W for six series of 30 seconds at one-minute intervals.

[113] . The resulting product was centrifuged for 30 minutes in an ultra-centrifuge at 4°C and 14,000 g. The supernatants were recovered and filtered in a sterilizing system (0.22 µm membrane), which constituted the soluble antigen (AS) of L. braziliensis and L. amazonensis. Then, ASs proteins were measured using the Lowry method (LOWRY, O. H. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, v. 193, p. 265-275, nov. 1951; SZARGIKI, R. et al. Comparison of serological and parasitological methods for cutaneous leishmaniasis diagnosis in the state of Paraná, Brazil. Brazilian Journal of Infectious Diseases, v. 13, n. 1, p. 47 52, Feb. 2009).

[114] . Aliquots of the produced antigenic contents were made and stored in cryogenic tubes at -20°C for use in subsequent experiments.

[115] . After carrying out the protein dosage, 5.19 mg/mL was obtained for L. braziliensis and 1.58 mg/mL for L. amazonensis. Proteins were aliquoted and stored for all subsequent experiments.

Assessment of the electrophoretic profile of soluble antigens from L. braziliensis and L. amazonensis

[116] . To analyze the electrophoretic profile of AS from L. braziliensis and L. amazonensis produced, the Polyacrylamide Gel Protein Electrophoresis (SDS-PAGE) technique (LAEMMLI, U.K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage) was performed. T4. Nature. v. 227, p. 680-685, Aug. 1970). For this, 20 µg of each fraction of parasite antigens were diluted in 4X sample buffer, in the presence of the reducing agent beta-mercaptoethanol and heated at 100 °C for five minutes. This mixture was distributed in channels of 15% polyacrylamide gel. The electrophoretic run was performed at 100 V in 0.025M Tris, 0.2M glycine and 0.5% SDS buffer, pH 8.3.

[117] . After the run was finished, the gel was stained with silver nitrate. To do this, the gel was washed with distilled water for five minutes, fixed with 50% methanol for 20 minutes and washed for another 10 minutes with distilled water. Subsequently, the gel was fixed with 10% glutaraldehyde for 30 minutes, and washed with water four times for 10 minutes. To stain the proteins, a 1% silver nitrate solution was prepared by adding 53% 0.36% sodium hydroxide and 3.5% ammonium hydroxide. The gel was stained for 20 minutes in the dark and washed twice with distilled water for five minutes. For development, the gel was incubated with a solution containing 0.25% acetic acid and 0.1% formaldehyde. To stop the reaction, a stop solution containing 1% acetic acid was added for 20 minutes.

[118] . The electrophoretic profile of the soluble antigens of L. braziliensis and L. amazonensis was obtained on a 15% polyacrylamide gel under reducing conditions (FIGURE 1). Both antigenic extracts presented protein masses between 15 and 100 KDa. In column 2, approximately 12 bands can be observed present in the L. braziliensis antigens, while in column 3, a total of approximately 11 proteins can be observed for the L. amazonensis antigen. This result demonstrates the sharing of proteins between the two species studied.

[119] . This step was important to confirm the extraction of proteins to be used in the present study, as well as to evaluate the similarity of total proteins from the two Leishmania species available.

Selection of anti-L immunoglobulins. braziliensis

[120] . Sera from patients with ATL and individuals without ATL were tested using the indirect ELISA method to search for antibodies against Leishmania. To standardize this assay, 96-well plates (Costar, Cambridge, Massachusetts, USA), specific anti-human immunoglobulin G Fc conjugated to peroxidase (Sigma-Aldrich, Saint Louis, Missouri, USA) and the chromogen OPD (Orthophenylenediamine - Sigma-Aldrich, Saint Louis, Missouri, USA).

[121] . Four concentrations of L. braziliensis and L. amazonensis antigens were analyzed (125, 250, 500 and 1,000 ng/well), compared to three dilutions (1:25, 1:50 and 1:100) of the sera (positive controls and negatives and test samples) and three dilutions of the conjugate (1:5000, 1:7500 and 1:10,000). In all ELISA tests carried out during this work, sera were evaluated in duplicates and the results presented represent the average of each sample. The dilutions of the conjugate, serum and antigens capable of better discriminating between positive and negative controls were selected. The tests now have the following standardization: antigens 500 ng per well, serum diluted 1:100 and conjugate diluted 1:10,000.

[122] . To evaluate all patient sera and select immunoglobulins, the test was performed by sensitizing the plates with 100 µL of the antigen produced (500 ng) in coating buffer (Na2CO3 0.16%, NaHCO3 0.29%, pH 9.6) per well and overnight incubation at 4°C. After this step, the antigen solution was discarded and the plates were washed twice with washing solution (0.9% NaCl - 0.05% Tween 20) to remove unfixed antigen. The spaces in the wells not occupied by the antigen were blocked for one hour at 37°C, adding 120 µL of blocking solution (2% casein in PBS pH 7.4) per well. After incubation, the plate was washed again. Sera diluted (1:100) in incubation buffer (0.25% casein in PBS pH 7.4 - 0.05% Tween 20) were added to the plate and incubated in an oven at 37°C for one hour. The plates were washed again four times, 100 µL per well of anti-human conjugate diluted 1:10000 in incubation buffer were added and the plates were incubated for one hour at 37°C. The plates were washed four times and the substrate, consisting of an OPD tablet (2 mg) diluted in 10 mL of 0.15M citrate buffer, pH 5.0, and 2 µL of 30% hydrogen peroxide were applied. After 15 minutes, protected from light, the reaction was stopped by adding 20 µL of 1:20 sulfuric acid solution. The reading was carried out using a spectrophotometer (microplate reader) at a wavelength of 492 nm.

[123] . To determine the cut-off level of the test, the 37 human serum samples from a leishmaniasis-free zone in the State of Paraná were analyzed against AS. The average absorbance obtained from these 37 samples was added to twice the standard deviation obtained to calculate the Cut-off.

Precipitation of anti-Leishmania immunoglobulins with ammonium sulfate

[124] . Total immunoglobulins were selected for subsequent purification of IgGs against - L. braziliensis. For this, patient sera (positive in the indirect ELISA test described in item 2.6) were used, which made up a pool of human sera. This content was centrifuged for 30 minutes at 3,000 g at 4°C. In an ice bath and under agitation, the supernatant obtained had its immunoglobulins precipitated by slow addition of an equal volume of saturated ammonium sulfate solution (HARLOW, E.; LANE, D. Antibodies: a laboratory manual. New York: Cold Spring Harbor Laboratory, p. 726, 1988). The mixture was left to rest at 4°C overnight and then transferred to 50 mL tubes and centrifuged for 30 minutes at 3,000 g at 4°C. The supernatant was discarded and the immunoglobulins present in the precipitate were reconstituted in PBS pH 7.4. The contents were added to dialyzer membranes and dialyzed against PBS, pH 7.4, to remove ammonium sulfate.

[125] . The purity and molecular mass of the immunoglobulins were evaluated by SDS-Page.

Obtaining subclass G immunoglobulins (IgG)

[126] . To subsequently carry out the selection cycles (pannings) of the Phage display technique, the purification of anti-L. braziliensis IgGs from patients with ATL occurred in two stages. Initially, IgGs were purified by affinity chromatography on protein G-agarose, followed by purification of immunoglobulins from antigens transferred to polyvinylidene fluoride membrane (PVDF - Millipore Corporation, Billerica, Maryland, USA).

[127] . Class G immunoglobulins were obtained by affinity chromatography on protein G - agarose (Sigma-Aldrich, Saint Louis, Missouri, USA) with the aim that the Fc portion of the IgGs recognized the G protein molecule fixed on the resin and thus were possible to recover this specific type of antibody.

[128] . The binding between the antigen (Protein G - agarose) and the antibodies (immunoglobulins G) was carried out by applying a fraction of the total immunoglobulins to the protein G - agarose column, which remained under agitation for one hour at room temperature. The column was washed with 20 mM sodium phosphate buffer, pH 7.0 until no more proteins were detected by the Bradford test (BRADFORD, M. M. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, v. 72, p. 248-254, 1976). IgGs were eluted by pH change with 100 mM glycine buffer, 150 mM NaCl, pH 2.7, collected and neutralized with 1 M Tris-HCl buffer, pH 9.0. Then, the column was re-equilibrated with 20 mM sodium phosphate buffer, pH 7.0. The fractions containing the obtained immunoglobulins G were pooled in a single sample, dialyzed against PBS 7.4 (to remove excess salts that could hinder subsequent protein characterization steps) and concentrated using the Amicon Ultra-15 10 kDa device (Millipore Corporation, Billerica, Maryland, USA). The purity and molecular mass of the immunoglobulin solution were evaluated by SDS-PAGE.

Obtaining specific immunoglobulins G for L. braziliensis

[129] . To obtain anti-L. immunoglobulins G. braziliensis, two milligrams of L. braziliensis antigens were resolved by polyacrylamide gel electrophoresis and transferred to a PVDF membrane in a buffer composed of 25 mM Tris, 192 nM glycine and 20% methanol overnight at 24 V, followed by a hour at 48 V. After transfer, the membrane was blocked with 0.3% Tween 20 in PBS pH 7.4 for one hour, under agitation. Then, the membrane was washed with PBS Tween 20 0.05% (PBST-0.05%) for three times of five minutes and incubated with 3 mg of IgGs diluted in PBST-0.05%. After incubation for two hours, the membrane was washed with PBST-0.05% for five times of five minutes and the antibodies were eluted by addition of 0.1 M glycine buffer, 0.15 M NaCl, pH 2.7 under shaking. for 30 minutes. The eluted antibody solution was recovered and immediately neutralized with Tris-HCl, 1 M, pH 9.0. The antibodies obtained were concentrated using the Amicon Ultra 15 10kDa device (Millipore Corporation, Billerica, Maryland, USA) and protein measurement was performed using the Bradford method (BRADFORD, M. M. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, v. 72, p. 248-254, 1976).

[130] . To evaluate the purification and reactivity of anti-L. immunoglobulins. braziliensis, ELISA and Western blotting techniques were performed. For Western blotting, the SDS-PAGE technique was first performed with L. braziliensis and L. amazonensis antigens (40 µg of antigens per well) and then the gel containing the antigens was electrotransferred onto a PVDF membrane (Millipore Corporation, Billerica, Maryland, USA). The transfer took place under a current of 24 V overnight and 48 V for one hour in transfer buffer with pH 8.3 composed of 25 mM Tris, 192 nM glycine and 20% methanol.

[131] . After completing these steps, the membrane was blocked with 0.3% of 20% Tween in PBS pH 7.4 for one hour under agitation, in order to avoid non-specific interactions. The membrane was then washed four times for five minutes each with 0.05% Tween 20 in PBS pH 7.4 and then incubated for two hours, under agitation with the specific anti-L. immunoglobulins. braziliensis (40 µg/mL in PBS pH 7.4). After incubation, the membrane was washed four times for five minutes each. It was subsequently incubated for one hour, at room temperature, under agitation with the specific anti-human IgG Fc antibody conjugated to peroxidase (Sigma-Aldrich, Saint Louis, Missouri, USA) at a dilution of 1:5,000 for one hour. Finally, after washing again, development was carried out with a solution of 1 mg DAB (3,3'-Diaminobenzidine Tetrahydrochloride) in 1.5 mL of 50 mM Tris, 0.15 M, NaCl pH 7.6 and 1.2 µL of 30% H2O2 per strip (each strip was incubated with 1.5 mL). The developing solution was maintained until the appearance of bands and the reaction was stopped by washing the strips with PBS pH 7.4.

[132] . Of the 198 patient sera analyzed by the ELISA test, 57 sera were considered positive for LTA, as they presented absorbance greater than 0.139 (established Cut-off value). A pool of ± 50 mL was formed from the serum samples of these patients for immunoglobulin precipitation. SDS-PAGE electrophoresis, under reducing and non-reducing conditions, demonstrates the presence of the albumin band present in total human serum IgGs precipitated by ammonium sulfate (column two). Albumin has a molecular weight of approximately 60 kDa and column three shows the removal of this molecule, representing the success of the affinity chromatography stage to obtain subclass G immunoglobulins (FIGURE 2).

[133] . Immunoglobulins G, obtained after affinity chromatography, were evaluated for their reactivity against Leishmania sp antigens. by Western Blotting (FIGURE 3). Reactivity was observed against serum from a known positive patient (columns two and four) and against IgGs purified by affinity chromatography (columns three and five).

[134] . Reactive bands were visualized both with total IgG and in the presence of purified IgGs. It is noted that there is a cross-reaction between the different Leishmania species analyzed. An example can be seen in the reactivity between the 10 and 15 kDa and 20 kDa and 50 kDa bands.

[135] . The reactivity of the specific IgGs obtained against L. braziliensis was evaluated by ELISA (FIGURE 4). The assay demonstrates that specific immunoglobulins, even at lower concentrations, present tests with higher absorbances than those found in the test with total immunoglobulins. Thus, it is concluded that the purification method allowed the concentration of antigen-specific antibodies to be used in the Phage display technique. The purification of the immunoglobulins specific to L. braziliensis was successful because, taking the first pair of bars on the graph as an example, it was observed that the IgGs specific to L. braziliensis, at a concentration 10 times lower than that of Total IgG showed higher absorbance (FIGURE 4).

Expression of peptides on the surface of phages (Phage Display)

[136] . After obtaining IgG anti-L. braziliensis from patient serum, the Phage display technique was performed as described by Villard et al. (2003), with modifications described below.

[137] . Initially, two immunotubes (Nunc, Roskilde, Denmark) were coated with 10 µg of protein G (Sigma-Aldrich, Saint Louis, Missouri, USA) diluted in TBS (Tris-buffered saline - 50 mmol/L, NaCl 150 mmol/L L, pH 7.5) and were incubated, under shaking, at 4°C, overnight. Subsequently, the immunotubes were washed twice with wash buffer (0.05% TBS-T), blocked with 3% BSA in 0.05% TBS-T for two hours at 37 °C and washed again. The two immunotubes had different stages in the Phage display technique. In tube one, 5x 1012 phages from four different peptide libraries (LX4, LX8, X8CX8, X15) obtained from J. Scott (Simon Fraser University, Canada) were added, which remained incubated overnight at 4°C. In immunotube two, 5 µg of specific IgGs diluted in 1.5 mL of TBS were added where they remained overnight at 4°C. After the incubation period, the supernatant from immunotube two was discarded and the supernatant from immunotube one (containing phages that do not bind to protein G) was added to immunotube two where it remained, under incubation, overnight at 4°C. Four pannings were performed and, in the first, 5 µg/mL of the specific anti-L. antibody was used. braziliensis, and for the second, third and fourth pannings, 2.5 µg/mL, 1 µg/mL and 1 µg/mL respectively.

[138] . The immunotube that remained during the development of this methodology (tube two) had its supernatant discarded and was then washed 10 times with TBST-0.5% and five times with TBS-T 0.05% to remove unbound phage. Subsequently, the phages whose peptides were recognized by the patient's antibodies were eluted for 30 minutes by changing the pH of the solution with the aid of glycine buffer, 0.1 mol/L, pH 2.2, BSA 1 mg/mL. The eluate was transferred to a microtube and immediately neutralized with Tris-HCl buffer, 2 mol/L, pH 9.0.

[139] . For amplification of the phages, to 10 mL of the solution of bacteria Escherichia coli (E. coli), strain K91 (optical density at 550 nm = 1.9) previously prepared in Luria Bertani (LB) medium, 1.5 mL were added of the eluate containing the phages. The content was incubated for 10 minutes at 37°C without shaking and subsequently added to 100 mL of LB medium pre-heated to 37°C containing 0.2 µg/mL of tetracycline (the tetracycline resistance gene present in the genome of Phage is what enables the growth and selection of bacteria infected with phages). After 30 minutes of stirring at 225 rpm, the tetracycline concentration was adjusted to 20 µg/mL. Bacterial growth occurred overnight at 37°C and under agitation at 225 rpm.

[140] . The amplified phages were purified with precipitation in 20% polyethylene glycol 8000 (PEG 8,000), 2.5 M NaCl. For this, the bacterial culture was first placed in 250 mL tubes and centrifuged at 4°C, at 5,000 g for 20 minutes. The pellet formed (mainly composed of bacteria) was resuspended in LB medium and stored in a -80°C freezer with 15% glycerol. The supernatant containing the phages was transferred to a new tube, where 15% of 20% PEG 8,000, 2.5 M NaCl was added. The content was mixed by inversion and precipitated overnight on ice at 4°C. The solution was centrifuged at 4°C, at 12,000 g for 40 minutes. The supernatant was discarded and the precipitate was resuspended in TBS buffer. Subsequently, the solution was transferred to microcentrifuge tubes and centrifuged at 14,500 g for 15 minutes at 4°C to remove possible cell debris. The phages obtained were used in the next selection cycle or stored at -20°C.

[141] . The phage pool was titrated by adding 10 µL of each of the phage dilutions 10-1, 10-2 and 10-4 for the partial titration and 10-6, 10-8 and 10-10 for the final titration. ), in 200 µL of previously cultured E. coli K91 bacteria. Partial titration means the title of eluted and non-amplified phages and total titration means the title of phages after amplification. The microtubes were incubated for 15 minutes at 37°C without shaking and for another 15 minutes at 37°C under shaking at 225 rpm.

[142] . The cells were plated on LB agar containing 20 µg/mL of tetracycline, for subsequent colony counting. The plates were incubated overnight in an oven at 37°C. The titer was determined by multiplying the number of colonies by the plate dilution factor and by 100 to obtain the number of phages per mL.

Example 2: Phage reactivity

[143] . After the fourth selection cycle, the reactivity of the panning phages recognized by patient antibodies was evaluated by ELISA testing.

[144] . A 96-well plate (Costar, Cambridge, Massachusetts, USA) was sensitized with 0.5 µg/mL of commercial anti-phage antibodies (Sigma-Aldrich, Saint Louis, Missouri, USA) in coating buffer (100 mM NaHCO3, pH 8.6), overnight at 4°C. The wells were washed twice with 0.9% NaCl Tween 20 - 0.05%. Free sites were blocked using a 2% casein solution in PBS pH 7.4 for 1 hour at 37°C. To each well, 109 phages eluted from each selection cycle were added along with incubation buffer. The plate was incubated for one hour at 37°C. Afterwards, the wells were washed four times and incubated at 37°C for one hour with anti-L IgGs. braziliensis (10 µg/well), and subsequently, with the anti-human IgG antibody specific Fc portion conjugated to peroxidase (Sigma-Aldrich, Saint Louis, Missouri, USA) diluted 1:10,000. The wells were washed again four times. To reveal the reaction, the substrate was added, consisting of a tablet of 2 mg of OPD diluted in 10 mL of citrate buffer pH 5.0 and 2 µL of 30% hydrogen peroxide. After 15 minutes, protected from light, the reaction was stopped by adding 20 µL of 1:20 sulfuric acid solution to the entire plate. The reading was carried out by taking the plate immediately to the microplate reader at a wavelength of 492 nm.

[145] . To identify phage clones capable of binding to antibodies from patients with leishmaniasis, the most immunoreactive panning was chosen. Isolated colonies were obtained after cultivation on LB agar in 140 x 15mm Petri dishes. After growth, each isolated bacterial colony was pinched with sterilized toothpicks and transferred to a well of a 96-well U-bottom culture plate containing 200 µL of LB medium and 20 µg/mL tetracycline. After overnight cultivation in a shaker with rotation of 225 rpm at 37°C, the plate was centrifuged at 1,600 g for 20 minutes at 4°C. The supernatant from the culture plate containing the phages was used directly to perform the ELISA test.

[146] . Each phage was tested by ELISA test with patient serum. Patient serum diluted 1:100 was used. The clones were selected with a pool of high absorbance sera (above 0.5) in an ELISA test with L. braziliensis AS. The phages were also tested with pooled sera with absorbances of 0.2 and 0.3.

[147] . When evaluating the reactivity of each of the four pannings by ELISA, it was possible to observe a progressive increase in the reaction intensity going from the first to the fourth selection cycle (FIGURE 5). The number of phages eluted was always lower than the amount incubated, as the phages with greater affinity to the antibodies immobilized in the tube were bound to them by the specific interaction of these molecules (TABLE 2). An increase in output phages can be observed with each selection cycle, demonstrating the enrichment of specific phages. These results indicate that the selection of phages reactive with anti-Leishmania antibodies occurred. TABLE 2 - ENTRY AND EXIT PHAGES OBTAINED FROM BIOSELECTION BY PHAGE DISPLAY WITH ANTIGEN-SPECIFIC ANTIBODIES

Selection of clones

[148] . The fourth selection cycle was chosen to infect an E. coli culture and obtain isolated colonies on a plate containing LB medium. Each clone was tested by ELISA using culture supernatant and patient serum with high absorbance (above 0.5). In this way, 428 clones were tested. Of these, 36 were reactive against sera from patients with ATL. The phages were also tested with a pool of sera with absorbances of 0.2 and 0.3 in the ELISA test with the AS, but the results did not allow the differentiation of positive and negative tests and therefore studies with these sera were interrupted.

Example 3: Obtaining peptides Obtaining amino acid sequences

[149] . The most reactive phage clones (absorbance above 0.3) were selected for DNA sequencing and subsequent identification of the amino acid sequence inserted into the phages. For this, the positive phage clones amplified in E. coli were subjected to DNA extraction using the QIAprep spin M13 kit (Qiagen, Hilden, Germany), carried out according to the manufacturer's instructions. The concentration of nucleic acids was measured using a Nanodrop spectrophotometer (Thermo Scientific - Wilmington, Delaware, USA), the DNAs were aliquoted and stored at -20°C until use. Sequencing reactions were performed with the Big Dye Terminator v3.1 Kit (Applied Biosystems, California, USA). For each reaction, 50 ng of DNA was used in a final volume of 10 µL, containing 1 µL of Big Dye Terminator v3.1 Ready Reaction MIX, 1 µL of 5X sequencing buffer and 1.6 pmol of the reverse primer (5'-TCG GCA AGC TCT TTT AGC - 3'). The sequencing conditions were: one minute at 96°C for initial DNA denaturation followed by 35 cycles of denaturation at 96°C for 10 seconds, annealing at 50°C for five seconds and extension at 60°C for four minutes. For DNA purification, isopropanol was added to the microtubes to a final concentration of 60%. After homogenization and incubation at room temperature for 20 minutes, the tubes were centrifuged at 15,000 g for 25 minutes. Isopropanol was removed by inversion on absorbent paper and 70% ethanol was added to the tubes. After centrifugation at 15,000 g for 10 minutes, the ethanol was removed, the tubes were placed in an oven at 37°C to dry the DNA pellet and stored at -20°C.

[150] . The analysis of DNA sequences from the ABI PRISM 377 automatic sequencer (Applied biosystems, Foster City, California, USA) was carried out using the equipment's own software.

[151] . After identifying the viral DNA, the Proteomics Tools program available at http://www.expasy.ch was used (Tools option - DNA-Proteins - Translate, protein translation reading 3' ->5', using the first reading window - frame 1) to obtain the amino acid sequence encoded by the DNA segment inserted into the phage, as well as to obtain the estimated weight of each peptide. The peptide sequences obtained were analyzed for their similarity with other sequences already registered in GenBank.

[152] . Three different peptide sequences were obtained and are presented in TABLE 3. The sequences were compared with others deposited in GenBank to verify homology with proteins from Leishmania spp. It was observed that peptides 1, 2 and 3 aligned with proteins from Leishmania sp. as shown in TABLES 4, 5 and 6. TABLE 3 - SEQUENCE OF PEPTIDES OBTAINED AFTER PERFORMING THE PHAGE DISPLAY TECHNIQUE STARTING FROM G IMMUNOGLOBULINS SPECIFIC FOR Leishmania sp.

Peptide synthesis

[153] . Once selected, the peptides were chemically synthesized using the Fmoc (fluorenylmethyloxycarbonyl) strategy, using a solid phase peptide synthesis protocol (resin as an insoluble solid support), which uses special amino acids for in vitro synthesis. These amino acids have the amine group protected by the Fmoc group and are coupled to the resin. The protocol was carried out as described in MERRIFIELD, R. B. Solid-phase peptide synthesis. Advances in Enzymology and Related Areas of Molecular Biology, v.32, p. 221-96, 1965 and using the MultiPep RS automatic peptide synthesizer (Intavis Bioanalytical Instruments, Nattermannallee, Germany).

[154] . This technique consists of fixing the C-terminal amino acid of the peptide on an insoluble solid support and then lengthening the peptide chain by successive additions of residues from the C-terminal to the N-terminal portion. These amino acids have the amine group protected by the Fmoc group, their side chain is also protected by a protecting group to avoid unwanted reactions.

[155] . Cycles of coupling (of amino acids with their activators) and deprotection (removal of the Fmoc group by the presence of 20% piperidine) are carried out until all amino acids of the peptide to be synthesized are coupled. After the end of the last cycle, the peptide without the Fmoc group of the last amino acid is removed from the resin by a step called cleavage. At this stage, the protective groups of the side chain are also eliminated. This solution is then filtered and precipitated with ice-cold ethyl ether, thus obtaining the peptide. After centrifugation, the ether is eliminated by evaporation. The peptides were lyophilized, weighed, dissolved in milli-Q water and stored at -20°C at a concentration of 1 mg/mL.

[156] . The identity of the peptides was confirmed by mass spectrometry on an Autoflex spectrometer (Bruker Daltonics, Bremen, Germany) and FlexAnalysis software (Bruker Daltonics, Bremen, Germany). The technique was carried out at the Center for Molecular and Structural Biology - Federal University of Santa Catarina (UFSC).

Conjugation of peptides to carrier protein

[157] . In the animal immunization stage, in order for the peptides to stop being haptens, it was necessary to couple these molecules with the KLH protein (hemocyanin from the Keyhole limpet mollusk) with a specific kit (Pierce, Rockford, Illinois, USA) for this purpose.

[158] . The KLH protein is isolated from the hemolymph of a mollusk and due to its high molecular weight it is an important immunogen. Also, because it is a mollusc protein, it has a large phylogenetic distance from mammals, a fact that favors the immunogenicity of this molecule. The large number of available lysine residues makes KLH one of the main peptide coupling molecules (HERMANSON, G.T. Bioconjugate techniques. USA:Academic, 785p., 1996).

[159] . The activated carrier protein was reconstituted in ultrapure water and two milligrams of the peptides were weighed and dissolved in conjugation buffer containing DMF (N,N-Dimethylmethanamide). An aliquot of the peptide was separated to calculate the free sulfhydryl groups with Ellmann's reagent. The peptide and activated protein were mixed and incubated under shaking for 2 hours at room temperature. Subsequently, the content was centrifuged at 2000 g for 5 minutes and the supernatant was recovered and subjected to a filtration column to remove EDTA and sodium azide. Protein measurement was performed using the Micro BCATM Protein Assay kit (Pierce, Rockford, Illinois, USA).

Production of anti-peptide antibodies

[160] . The KLH-peptides (1, 2, 3 alone and the MIX of the three peptides) diluted in sterile 0.9% NaCl were used to immunize golden hamsters (Mesocricetus auratus), as described in GAMBOA, D, K. et al. Evaluation of an in vitro and in vivo model for experimental infection with Leishmania (Viannia) braziliensis and L. (V.) peruviana. Parasitology, vol. 135, p. 319-326, Mar. 2008. Four-week-old female hamsters were purchased from the supplier Anilab de Paulínea - SP. After arriving at the vivarium, the animals had an adaptation period of one week.

[161] . Throughout the experiment, the animals were maintained with food and water ad libitum. To carry out this experiment, six experimental groups were constituted (group 1, peptide 1 + adjuvant; group 2, peptide 2 + adjuvant; group 3, peptide 3 + adjuvant; group 4 peptides 1, 2 and 3 + adjuvant; group 5, adjuvant; group 6, control) composed of ten animals per group. Animals were immunized with individual or combined peptides (MIX). A negative control group did not receive any type of immunization. Another group received only the adjuvant in an immunization vehicle.

[162] . The synthetic peptides were emulsified in the first immunization, in complete Freund's adjuvant (Sigma-Aldrich, Saint Louis, Missouri, USA) and, in the remaining immunizations, in incomplete Freund's adjuvant (Sigma-Aldrich, Saint Louis, Missouri, USA). 20 µg of KLH-peptide was inoculated per animal, on the back and subcutaneously, in a total volume of 100 µL. Four doses of this inoculum were applied at 30-day intervals (TABLE 7). Non-immune serum was used as a negative control and immune serum was obtained seven days after the last immunization. Blood collections were performed by puncturing the saphenous vein of previously anesthetized animals. TABLE 7 - HAMSTER IMMUNIZATION PROTOCOL WITH KLH-PEPTIDES

[163]. After collecting blood from the hamsters immunized with the peptides, an ELISA test was performed to evaluate the presence of specific anti-peptide antibodies.

[164] . Plate sensitization with antigen was performed by applying 0.2 µg/well of each peptide, diluted in 0.05 M carbonate buffer solution pH 9.6, and incubated overnight at 4°C. After this step, the plates were washed twice with washing solution (0.9% NaCl - Tween 20 - 0.05%) and blocked by adding 120 µL of blocking buffer (2% casein in PBS pH 7.4) per well. After incubation for one hour in an oven at 37°C, the plate was washed twice. The sera, diluted 1:50 in incubation buffer (0.25% casein in PBS pH 7.4 - 0.05% Tween 20 - pool of ten animals from each group), were applied to the wells and the plate was incubated for a hour in an oven at 37°C. Subsequently, the plate was subjected to four washes to remove immunoglobulins not bound to the antigen. Peroxidase-linked anti-hamster total IgG conjugate (Sigma-Aldrich, Saint Louis, Missouri, USA) was added at a concentration of 200 ng/mL. The plate was incubated for another hour in an oven at 37°C.

[165] . After incubation, the plate was washed four times and the substrate, consisting of an OPD tablet (2 mg) diluted in 10 mL of citrate buffer pH 5.0 and 2 µL of 30% hydrogen peroxide, was applied. After incubation for 15 minutes, protected from light, the reaction was stopped by adding 20 µL of 1:20 sulfuric acid solution to the entire plate. The reading was performed by taking the plate immediately to the microplate reader and the absorbance was determined at a wavelength of 492 nm.

[166] . To evaluate the presence of specific anti-peptide antibodies, serum from hamsters immunized with peptides were evaluated by ELISA (FIGURE 6).

[167] . All animals that were immunized with peptides showed reactivity against peptides 1, 2 and 3 alone and against the peptide pool. Peptides 1 and 2 alone induced reactivity equal to or close to that of the peptide MIX. Regarding the hamsters immunized with peptide 3, it was observed that they had greater reactivity towards the MIX of peptides present in the ELISA plate. Non-immunized animals and those that received only adjuvant did not produce antibodies against the peptides.

Assessment of IgG reactivity

[168] . To evaluate whether anti-peptide antibodies produced in hamsters would recognize L. braziliensis antigens, the Western blotting technique was used.

[169] . Firstly, the L. braziliensis antigens were subjected to the SDS-PAGE electrophoresis technique according to item 2.5. After electrotransfer, the membrane was blocked with 3% BSA, 20-0.05% Tween in PBS pH 7.4 for one hour under agitation. The membrane was then washed four times for five minutes and was incubated with the pool of serum from the immunized animals (diluted 1:100 in incubation buffer - 3% BSA, 20-0.05% Tween in PBS pH 7, 4). After washing, peroxidase-linked hamster anti-total IgG conjugate was added at 100 ng/mL. After incubation, four washes were performed and development was carried out with a solution containing 1 mg of DAB in 1.5 mL of 50 mM Tris, 0.15 M NaCl, pH 7.6 and 1.2 µL of 30% H2O2 per strip. (each strip was incubated with 1.5 mL). The developing solution was incubated until bands appeared and the reaction was stopped by washing the strips with PBS pH 7.4.

[170] . After performing the Western Blotting technique, using the AS of L. braziliensis and serum from hamsters immunized with peptides as antigens, it was possible to verify the reactivity of the antigens against antibodies from animals immunized with peptides 1, 2, 3 and MIX ( FIGURE 6). For animals that received only the adjuvant, there was no reactivity. Hamsters that received peptide 1 produced antibodies that recognized proteins between 76 and 150 kDa. Sera from hamsters that received peptides 2 and 3 produced antibodies that reacted with proteins with a mass between 52 and 76 kDa from L. braziliensis, as did the MIX of peptides 1, 2 and 3. Sera from animals that did not receive the peptides (negative controls) did not react to L. braziliensis antigens.

[171] . Analysis of the results obtained demonstrates that the selected methodology (Phage display technique) was appropriate, since peptide sequences with homology to the main virulence factors of Leishmania spp. they were obtained. Furthermore, these sequences demonstrated to be immunogenic in experimental models for leishmaniasis and anti-peptide antibodies recognized L. braziliensis antigens. Therefore, these peptides can be used to detect antibodies in diagnostic tests and vaccines.

Claims (13)

1. MIMETIC PEPTIDE FROM Leishmania spp. with antigenic properties, characterized by comprising SEQ. ID. No:2. 2. MIMETIC PEPTIDE FROM Leishmania spp., according to claim 1, characterized in that it comprises the peptide in its original form and with possible amino acid substitutions from 1 to 15 and can be replaced by a single Alanine residue, provided that this substitution is, singular. 3. MIMETIC PEPTIDE according to claims 1 to 2, characterized in that it is presented as an isolated sequence or is linked to a carrier such as hemocyanin (KLH) or bovine serum albumin (BSA). 4. MIMETIC PEPTIDE FROM Leishmania spp., according to claims 1, 2, or 3, characterized in that it is produced by chemical synthesis. 5. MIMETIC PEPTIDE FROM Leishmania spp., according to claims 1, 2, or 3, characterized in that it is produced by recombinant DNA technology. 6. IMMUNOGENIC COMPOSITION USING THE MIMETIC PEPTIDE (SEQ. ID. No. 2) defined in claims 1, 2 or 3, characterized in that it comprises the preparation of an immunogenic composition containing the peptide seq. ID. No. 2 at a concentration of 20 to 100 ng, to stimulate or discourage the human or animal immune system against human or animal leishmaniasis. 7. IMMUNOGENIC COMPOSITION defined in claim 6, characterized in that it comprises at least one physiologically acceptable adjuvant. 8. METHOD FOR IMMUNODIAGNOSIS OF LEISHMANIASIS using PEPTIDE SEQ as antigen. ID. AT THE. 2 defined in claim 1,2,3, 4 or 5 characterized by comprising the steps of: a) binding antibodies against Leishmania spp. from a sample of biological fluid such as blood, serum, plasma, saliva, urine or tears, to the antigen (Seq. ID. No. 1), attached to a solid support; b) contact of the antibodies from step (a) with a secondary antibody or a protein, conjugated to an enzyme or a marker and which bind to the antibodies from step (a); c) detection of antibodies against leishmaniasis in the aforementioned sample by detection of the secondary antibody or protein specifically linked to the antibody against leishmaniasis. 9. METHOD according to claim 8, characterized by carrying out step (a) using the peptide (Seq. ID. No. 2) as defined in claims 1 or 2 at a concentration of 0.2 to 0.4 pg /pit. 10. METHOD according to claim 8, characterized by carrying out step (b) by diluting the primary antibody 1:50 in the incubation buffer (0.25% casein in PBS pH 7.4 - Tween 20 at 0.05 %) and the secondary antibody (species-specific) at a concentration of 200 ng/mL. 11. METHOD according to claim 8, characterized in that the protein used in step (b) is preferably protein A or protein G. 12. METHOD according to claim 8, characterized in that the enzyme used in step (b) is selected from the group comprising peroxidase, alkaline phosphatase, xanthine oxidase and penicillinase. 13. METHOD according to claim 8, characterized in that the marker used in step (b) is selected from enzymes, radioisotopes, biotin, avidin, neutrovidin, chromophores, fluorophores and chemiluminescents.