BR102016019380A2 - VACCINE COMPOSITIONS FOR PROTECTION AGAINST VISCERAL LEISHMANIASIS, SYNTHETIC PEPTIDES AND USES - Google Patents

Abstract

vaccine compositions for protection against visceral leishmaniasis, synthetic peptides and uses. The present technology addresses vaccine compositions for the treatment and / or prevention of visceral leishmaniasis in mammals, comprised of six free or surface-expressed synthetic peptides of non-infectious bacteriophages, which have been selected by means of a phage display proteomic approach. applied alone or in possible combinations between them.

Description

(54) Title: VACCINE COMPOSITIONS FOR PROTECTION AGAINST VISCERAL LEISHMANIOSIS, SYNTHETIC PEPTIDES AND USES (51) Int. Cl .: C07K 7/06; A61K 39/008; A61P 33/02 (73) Holder (s): FEDERAL UNIVERSITY OF MINAS GERAIS (72) Inventor (s): EDUARDO ANTONIO FERRAZ COELHO; CARLOS ALBERTO PEREIRA TAVARES; LOURENA EMANUELE COSTA; BEATRIZ CRISTINA SILVEIRA SALLES; DANIEL MENEZES SOUZA;

MARIANA COSTA DUARTE; BRUNO MENDES ROATT (57) Abstract: Vaccine compositions for protection against visceral leishmaniasis, synthetic peptides and uses. This technology deals with vaccine compositions for the treatment and / or prevention of visceral leishmaniasis in mammals, comprised of six synthetic peptides free or expressed on the surface of non-infectious bacteriophages, which were selected using a proteomic phage display approach, for be applied alone or in possible combinations between them.

1/17 “Vaccine compositions for protection against visceral leishmaniasis, synthetic peptides and uses” [001] This technology deals with vaccine compositions for the treatment and / or prevention of visceral leishmaniasis (VL) in mammals, comprised of six synthetic peptides free or expressed on the surface of non-infectious bacteriophages, which were selected using a proteomic phage display approach, to be applied alone or in possible combinations between them.

[002] Leishmaniasis are neglected diseases caused by protozoan parasites of the genus Leishmania. It is estimated that 380 million people are exposed to the risk of contracting the disease and that an approximate incidence of 0.5 to 1.0 and 1.5 to 2.0 million new cases occurs each year due to the LV and cutaneous leishmaniasis (LT), respectively. In the Americas, approximately 90% of the cases of VL occur in Brazil, which makes this disease an important public health problem and therefore requires special attention by our competent authorities (World Health Organization. Control of the leishmaniasis: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, 22-26 March 2010. WHO Technical Report Series. (949).

http://whqlibdoc.who.int/trs/WHO_TRS_949_eng.pdf. 2010; Alvar J, et al. WHO Leishmaniasis Control Team. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 7: e35671,2012).

[003] The severity of the disease in the mammalian host, in which the man and the dog fall, can vary from a single cutaneous lesion, to the visceral form of the disease, which, if left untreated, can be fatal. The dog is the main domestic reservoir of the parasite and is considered an important source of transmission between the phlebotomine vector and the mammalian host (Grimaldi, G. and Tesh, RB Leishmaniasis of the New World: current concepts and implications for future research. Clin Microbiol Rev. 6; 230-50, 1993). Due to the ineffectiveness of the control measures now used, the difficulty in making a correct diagnosis and also the toxicity problems caused by conventional drugs; O

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2/17 development of vaccines that are capable of inducing protective immunity against VL becomes desirable (Tesh RB. Control of zoonotic visceral leishmaniasis: is it time to change strategies? Am J Trop Med Hyg.52; 287-92, 1995 ).

[004] The diagnosis of the disease is based on the evaluation of the clinical manifestations of the disease in conjunction with epidemiological surveys and with the detection of the intracellular stages of the parasites by examining the aspirates of patients' internal organs, in addition to other laboratory tests. Rapid diagnosis is essential to avoid death, however, for clinical diagnosis, the symptoms of the disease are similar to those of other more common diseases in our country, such as Chagas disease, malaria, among others. However, the parasitological methods, despite having high specificity, have limitations in their sensitivity, due to the non-homogeneous distribution of the parasites in the aspirates, in addition to the risks of internal hemorrhage when performing such technique, as it is invasive. In addition, such methods take a long time to make the slides, a fact that also hinders their use in routine medical practice (Tavares CA, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn.3 (5): 657 -67, 2003). In addition, the similarity to clinical symptoms with other diseases also makes it difficult to diagnose VL.

[005] Laboratory tests are based on the detection of parasite-specific antigens and / or antibodies in patient serum samples. In such cases, the Enzyme Binding Immunoadsorption Analysis (ELISA), the Indirect Immunofluorescence Reaction (IFAT) and the Direct Agglutination Test (DAT) are used, however, among some problems observed, they do not have the ability to differentiate patients with subclinical disease, with active disease or with the disease already cured (Tavares CA, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn. 3; 657-67, 2003).

[006] In the case of canine LV (LVC), serodiagnosis is hampered by factors related to the specificity and / or sensitivity of the antigens used. In this case, the occurrence of a high number of false positive results in healthy animals, however, living in endemic areas of the disease, as well as

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3/17 in animals with other diseases related to VL, such as Chagas disease, babesiosis, toxoplasmosis, rickettsiosis and ehrlichiosis; as well as the occurrence of false negative results, as in infected animals, however, with low titers of anti-Leishmania antibodies; have created great difficulties for the correct interpretation of serological tests in dogs (Scalone A, et al. Evaluation of the Leishmanía recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay. Vet. Parasitol. 104 ; 275-85, 2002; Mettler M, et al. Evaluation of enzyme-linked immunosorbent assays, an immunofluorescent-P. Antibody test, and two rapid tests (immunochromatographic-dipstick and gel tests) for serological diagnosis of symptomatic and asymptomatic Leishmania infections in dogs. J. Clin. Microbiol. 43; 5515-19, 2005). Such problems have also been observed in the serodiagnosis of human VL.

[007] In murine models, after the cure of the disease caused by the species Leishmanía major, the animals acquire lasting immunity against reinfection (Afonso LC and Scott P. Immune responses associated with susceptibility of C57BL / 10 mice to Leishmania amazonensis. Infect. & Immun 61; 2952-59, 1993). This fact has stimulated the development of research aimed at obtaining vaccine antigens that can be used as an effective measure to control the disease. There are antigens that are being evaluated and that partially meet the requirements to be used as candidates for the vaccine (Stager S, Smith DF, Kaye PM. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol 165; 7064-71, 2000; Dondji B, et al. Heterologous prime boost vaccination with the LACK antigen protects against visceral murine leishmaniasis. Infect Immun 73; 5286-89, 2005).

[008] Most studies performed use unique antigens, which are expressed in only one of the morphological forms of the parasite, be it promastigote or amastigote (Fernandes AP, et al. Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives. Curr Opin Microbiol.; 15: 1-10, 2012). Furthermore, such antigens are usually capable of inducing protection of the species-specific type and in a type of mammalian host,

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4 / vr normally, in murine models. Thus, there is no vaccine available on the market that is effective in combating leishmaniasis and that can be used in both dogs and humans.

[009] In the immune response against infection by Leishmania infantum, resistance to infection by the parasite is normally associated with the development of a Th1-type cellular immune response, excelled in the production of cytokines such as IFN-gamma (IFN-γ) , interleukin 12 (IL-12), among others, by the mammalian host. The immune effector mechanism responsible for controlling parasitism appears to be due to the activation of macrophages, via the production of high levels of nitric oxide and other molecules that attack internalized parasites (Scott P. Development and regulation of cell-mediated immunity in experimental leishmaniasis. Immunol Res. 27; 489-98, 2003). Studies based on the murine model of L. major infection in BALB / c mice, proposed by Sacks and Noben-Trauth (2002) defined the Th1 / Th2 paradigm of resistance and susceptibility, respectively, to infection and the role of cytokines such as IFN-gamma and interleukin-4 (IL-4), respectively, in the development of Th1 and Th2 cell lines (Sacks D and Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol.2; 845-58, 2002).

[0010] The development of a Th1 response can be inhibited by the production of cytokines such as IL-10. In this sense, these molecules are related to the deactivation of macrophages and the establishment of the disease in animals, while cytokines such as IFN-γ are associated with the disease protection phenotype (Zanin FH, et al. Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections. Microbes Infect 9; 1070-77, 2007; Chávez-Fumagalli MA, et al. Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB / c mice against Leishmania chagasi and Leishmania amazonensis challenge. Microbes Infect 12; 967-77, 2010; Martins VT, et al. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis PLoS Negl Trop Dis. 7; e2148, 2013).

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5/17 [0011] In studies evaluating LV vaccine candidates, immunogens are administered to mice associated with adjuvants that induce the Th1 immune response. After immunizations, to assess the immunogenicity of the vaccine, cytokines such as IFN-γ and IL-12, as markers of Th1 response; and IL-4 and IL-10, as markers of Th2 response, have their levels determined, and then a comparison is made between such levels obtained with the parasitic loads found in animals (Martins VT, et al. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis. PLoS Negl Trop Dis. 7; e2148, 2013). Thus, it is sought the association that, in animals considered to be protected against VL, that they have low parasitic load and that their splenocytes are capable of producing high levels of IFN-γ and low levels of IL-10 after in vitro stimulation of them with immunogens.

[0012] The antigens currently used as vaccines against leishmaniasis are usually complete proteins, which contain regions common to proteins from other diseases, which can generate cross reactions in vaccinated animals. Also, such proteins may contain epitopes (or peptides) that have a beneficial effect, but others that have a harmful effect on immunized hosts, thereby compromising the effectiveness of the vaccine. The use of small antigens tends to allow the development of a more specific vaccine, so that the immunized dog or man can develop a beneficial immune response against only that antigen, specific to him and the disease for which he was selected. Such antigens also present a simpler technical production, with higher yield and lower cost in relation to the production of recombinant proteins; besides having good stability and, thus, they can be made available in better conditions for the population. [0013] The phage display technology is based on the expression of peptides, proteins or fragments of antibodies associated with proteins present on the external surface of recombinant bacteriophages. The nucleotide sequence encoding the inserted peptides is genetically fused to a sequence encoding some bacteriophage proteins, giving rise to a hybrid product, which is then exposed on the outer surface of the viral particles (Smith G.P.

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6 / n

Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 228; 1315-1317, 1985). Bacteriophage libraries expressing exogenous peptides have been used to identify several cell receptors, facilitating the identification of small molecules that bind with high affinity and mimic the interaction with their natural ligands; as well as in the identification of peptides that interact with antibodies, without the need for prior knowledge of the antigenic region recognized by the antibody. The selection of molecules of high affinity in relation to a specific target receptor, adhered to a solid surface, is carried out by consecutive selection steps called “bio-panning cycles”. The number of biopanning cycles performed on a phage display determines the degree of enrichment of the phages that bind to a target receptor. A population of phage clones with high affinity for a given receptor can be obtained by performing 3 to 5 cycles of bio-panning (Crameri R, Suter M. Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production (Gene.137; 69-75, 1993).

[0014] Methodologically, the bio-panning cycles can be adapted according to the group that dominates such technology. For example, in our case, IgG molecules from the sera of patients to be investigated are fused to the surface of microspheres (called “beads”) and this hybrid is subjected to negative or positive uptake (selection) processes in relation to clones of phage present in the used recombinant library.

[0015] The phage display technology has been used in several studies for the research of new antigens, so that in the form of bacteriophage clones expressing the peptides of interest, or in the form of individual peptides produced synthetically; can be tested as vaccines against various diseases (Manoutcharian K, et al. Phage-displayed T-cell epitope grafted into immunoglobulin heavy-chain complementarity-determining regions: an effective vaccine design tested in murine cysticercosis. Infect Immun. 67: 4764-4770 , 1999; Manoutcharian K, et al. Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis. Vet Immunol Immunopatho. 99: 11-24,

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2004; Sartorius R, et al. The use of filamentous bacteriophage fd to deliver MAGEA10 or MAGE-A3 HLA-A2-restricted peptides and to induce strong antitumor CTL responses. J Immunol. 15; 180 (6): 3719-28, 2008).

[0016] Patent document BR1020130275425, entitled "Vaccine composition against canine visceral leishmaniasis, synthetic peptides and use", deals with a vaccine composition for the prevention and / or treatment of canine visceral leishmaniasis comprised by peptides expressed in bacteriophages. This technology differs from the present application in that it comprises different sequences of selected peptides, because the methodology adopted was also different and because such a patent document deals only with the study of canine disease.

[0017] Patent document PI09030891, entitled "Recombinant peptides and protein motifs mimetic to Leishmania antigens and their applications", deals with the characterization and use of recombinant peptides, their reverse sequences and their protein motifs in the treatment against leishmaniasis. The technology differs from the present application because the experimental strategy used is different and does not include the use of human cells or the measurement of human cytokines. Also, the immunogens identified are different from those described in the present invention.

[0018] Patent document PI08004854 deals with the use of recombinant viral vectors expressing A2 Leishmania antigen as a method of vaccination against leishmaniasis. The technology differs from the present application in that it contemplates an antigen different from those included in the present application and the experimental strategy is different.

[0019] This technology deals with six peptides and / or clones of bacteriophages expressing peptides of interest and which were selected using the phage display technique. Such clones expressing the peptides were shown to be immunogenic in two mammalian models, mouse and man, being able to induce the development of a Th1 cellular immune response profile, since both splenocytes of mice chronically infected with L. infantum and mononuclear cells of the peripheral blood (PBMCs) from healthy individuals stimulated with individual clones were able to induce

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8/17 to the production of high levels of IFN-γ and low levels of IL-10, thus demonstrating the ability of such immunogens to induce the development of an immune response related to protection against LV, in this case, the Th1 response. In addition, such immunogens, represented by bacteriophage clones expressing the peptides of interest, have no similar amino acid sequence identity with others already described, so they are considered unpublished.

DETAILED TECHNOLOGY DESCRIPTION [0020] This technology deals with vaccine compositions for the treatment and / or prevention of visceral leishmaniasis (VL) in mammals, comprised of six synthetic peptides free or expressed on the surface of non-infectious bacteriophages, which were selected by through a proteomic approach of phage display, to be applied alone or in possible combinations between them. Such clones are immunogenic in human and mouse cells, being able to induce the development of a Th1 cellular immune response profile.

[0021] More specifically, the present technology comprises the peptides defined by SEQ ID N ° 1 to 6, free or expressed on the surface of non-infectious bacteriophages, as well as their use in the treatment and / or prevention of visceral leishmaniasis in different species of mammals .

[0022] The pharmaceutically and pharmacologically acceptable adjuvants in accordance with the present invention can be selected from among molecules or products that are capable of stimulating the further development of a Th1-type cellular immune response, which is excelled in the production of IFN-γ cytokines and IL-12. [0023] The proposed vaccine composition can be administered via intradermal, intramuscular, oral, nasal, intravenous, subcutaneous and / or as a device that can be injected.

[0024] The present invention can be better understood through the following examples, not limiting the technology.

[0025] EXAMPLE 1. OBTAINING BACTERIOPHAGES EXPRESSING PROTEINS OF INTEREST

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9/17 [0026] Serum samples. The following serum samples were used to perform the bio-panning cycles using the phage display technique: sera from patients with active VL (n = 40), sera from uninfected individuals residing in an endemic area of leishmaniasis (n = 50) and sera from patients with Chagas disease (n = 20). The project was approved by the UFMG COEP (CAAE323431.14.9.0000.5149).

[0027] Purification of IgG antibodies by coupling to G-protein microspheres. The immunoglobulins of the IgG class of pools from the sera listed above were purified by coupling them to magnetic beads conjugated to protein G (Dynabeads, Invitrogen). For this purpose, 1 x 10 ¹¹ microspheres were washed 3 times in MES buffer 0.1 M pH 5.0 and added to them: (a) 400 qL of the serum pool of patients with LV, for a final volume of 700 μL; (b) 400 qL of the serum pool of patients with Chagas disease, for a final volume of 700 qL and (c) 400 qL of the serum pool of healthy individuals, for a final volume of 700 qL. The ratio of antibodies to the amount of microspheres in all cases was 1: 1. Then, an incubation for 40 minutes was performed under constant agitation and at room temperature (RT). The microspheres adsorbed with the antibodies were washed 3 times in MES 0.1 M pH 5.0 buffer, in order to remove the non-adhered IgG antibodies.

[0028] For the completion of the covalent bond between the microspheres and the antibodies, the beads-antibody system was washed twice with 1 ml of 0.2M triethanolamine buffer pH 8.2 and resuspended in 1 ml of covalent bond buffer (20 mM of dimethylpimelinidate / HCl in triethanolamine buffer) for 30 minutes, under constant agitation and at RT The reaction was neutralized by incubating the system with 1 ml of 50 mM Tris buffer pH 7.5, for 15 min and at TA Then, the microspheres incorporated were washed 3 times in TBS-T 0.1% Tween 20 buffer and blocked with blocking solution (5% BSA in TBS-T 0.05% Tween 20) for 1 h at 37 ° C, then resuspended in 200 □ L of TBS buffer. To certify the coupling, 5 qL of beads covered by IgGs were incubated for 1 h and at 37 ° C with anti-human IgG antibody (dilution of 1: 5,000). After incubation, the beads were washed 3 times with 0.1% TBS-Tween and the reaction was revealed by adding the substrate

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10/17 tetramethylbenzidine. The reaction was then interrupted by the addition of 2 N sulfuric acid and the absorbance reading was performed at 450 nm in a microplate reader (Titertek Multiskan Plus, Flow Laboratories, USA).

[0029] Bio-panning cycles. To perform the bio-panning cycles using the phage display technique, negative and positive selection processes were performed using 1 x 10 viral particles from a recombinant library containing peptides fused in filamentous phages (commercial library Ph.D.C7C obtained from the company New England, BioLabs®, USA). It was diluted in 190 pL of 0.1% TBS-Tween for the selection of ligands in the microspheres incorporated by the IgG antibodies. In the first cycle of bio-panning, began with the negative selection. For this, the library of recombinant bacteriophages was incubated for 30 minutes and the TA with the microspheres coupled with the IgGs of healthy individuals, and then the microspheres were precipitated by magnetic attraction to the Dynal Biotech support (no. 12020), being the supernatant, containing clones that did not adhere to IgGs; transferred to a new tube containing the microspheres coupled with the IgGs of the group of patients with Chagas disease. Only those phage clones that did not adhere to such antibodies were recovered. This process was repeated two more times, totaling 3 negative selection cycles. The supernatant, containing phage that did not bind to IgGs from sera from healthy individuals and from those with Chagas' disease, was collected. For positive selection, the collected supernatant was transferred to a new tube containing the microspheres coupled with the IgGs of patients with symptomatic and asymptomatic VL, being incubated at RT and for 30 minutes. The microspheres containing the beads-antibody system precipitated in the biopannings tubes of the antibodies of the patients' sera, plus the phages of interest adhered to such IgGs, were washed 10 times with TBS-Tween 20 0.1% and eluted in 500 pL of glycine buffer 0.2 M pH 2.0, this solution being neutralized by the addition of 75 pL of Tris-base pH 9.0. The phage clones were recovered for later titration. The process was repeated two more times, totaling 3 positive selection cycles. After this procedure, the clones were individualized. [0030] Titrations, amplification and extraction of DNA from clones of interest. At the end of the 3rd positive selection cycle, the recovered phage clones were

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11/17 titles. For this, they were submitted to exponential serial dilutions

1Π 14 (log ¹⁰ ) in liquid LB medium with ampicillin, with dilutions of 10 to 10 for

Q dd the non-amplified eluates and 10 to 10 for the amplified eluates. For each titration, a culture plate was made. The plates were incubated at 37 ° C for 16 hours. Afterwards, the colonies that were blue and individualized were manually quantified. To estimate the values of the titers, the total number of colonies counted was multiplied by the dilution factor of each plate. The individualized and blue colored colonies present in the culture plates obtained from the 3rd cycle of bio-pannings were collected for DNA extraction. For phage amplification, 1.2 mL of the E. coli ER2738 cell culture in early-log phase (OD _600nm ~ 0.3) was distributed into each well of a deepwell plate.

[0031] With sterile toothpicks, 96 blue colonies were removed from the Petri dish and transferred to the culture dish. To each well, only one phage colony was added, totaling 96 colonies on the plate. It was sealed and incubated for 5 hours, under constant agitation and at 37 ^o C. After incubation, the plate was centrifuged for 20 minutes at 2,250 xg, the supernatant being transferred to another plate. To that 2 ^the plate, PEG / NaCl (1/6 of the total volume of the supernatant) was added, which was incubated for 14 hours and at 4 ^o C. After, the plate was centrifuged for 1 hour, the supernatant was removed and the pellet was resuspended in iodide buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA and 4 M NaI). The plates were shaken vigorously and then absolute ethanol was added. After a 10-minute incubation, the plates were centrifuged (2,250 xga at 4 ° C and for 10 minutes), with the supernatant discarded. The precipitate containing the DNA was washed with 500 pL of 70% ethanol and again centrifuged. Finally, the DNA of each clone was diluted in 20 pL of ultrapure water and its quality was assessed after an electrophoretic run performed on 0.8% agarose gel, stained with an ethidium bromide solution.

[0032] Sequencing of the selected clones. The 96 clones from the 3rd cycle of positive selection had their DNA extracted and sequenced. The reaction was processed in the MegaBace 1000 automatic sequencer (Amersham

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Biosciences) and the in silico deduction of the DNA amino acid sequences of the phage of interest, for the identification of exogenous peptides, was carried out by the program DNA2PRO12, which is designated for the deduction of insert sequences from the New England Biolabs (Ph. D.-12TM or Ph.D.-C7CTM), as well as other libraries of interest that contain the initial and final vector sequences. The program automatically locates the position of the insert, translates it and indicates possible errors in the translation (such as unexpected codons or errors in the next sequence).

EXAMPLE 2. EVALUATION OF ANIMAL INFECTION.

[0033] Parasites. The L. infantum sample MHOM / BR / 1970 / BH46 was used. The parasites were grown in complete Schneider's medium, which consisted of Schneider's medium (SIGMA) plus 20% inactivated fetal bovine serum (SIGMA), 20 mM L-glutamine, 50 pg / mL gentamicin, 200 U / mL penicillin and 100 pg / ml streptomycin, at pH 7.4 and at 24 ° C, as described by Coelho et al., 2003 (Coelho EAF, et al. Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen, but not by the LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection. Infect Immun. 71: 3988-94, 2003).

[0034] Challenge infection. In order to carry out the experimental infection in the animals, 1 x 10 ⁷ promastigote forms in the stationary phase of growth of L. infantum were inoculated in the right footpad of 8 animals. The mice were monitored for 10 weeks after the challenge, when the animals were euthanized for the experiments of immunogenicity and determination of the parasitic load.

[0035] The parasitic load was carried out in order to certify that the animals were actually infected by the parasite. the parasitic load on the spleen, liver, bone marrow and draining lymph nodes was assessed using a limiting dilution technique. The animals (n = 8) were infected subcutaneously with this species of the parasite and, 10 weeks after infection, they were euthanized for organ collection and parasitological evaluation. In the analysis of the results, it was observed that all the evaluated organs had a high parasite load (represented by the parasite log), as shown in Table 1.

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13/17 way, it was found that the animals were infected with L. infantum. The results correspond to the mean ± standard deviation of the groups, in two experiments carried out and which presented similar results.

Table 1. Parasitic load in mice infected with L. infantum.

Evaluated organ Mean ± standard deviation of the log of the number of parasites Liver 5.5 ± 0.3 Spleen 6.5 ± 0.2 Bone marrow 4.0 ± 0.3 Lymph nodes 7.0 ± 0.5 draining

EXAMPLE 3. DETERMINATION OF CYTOKINES AND CALCULATION OF THE REASON BETWEEN LEVELS OBTAINED FROM IFN-γ AND IL-10 [0036] BALB / c mice infected with L. infantum were euthanized 10 weeks after the challenge and had their spleen removed, the same being removed macerated in complete DMEM (Sigma) medium, which was composed of 4.5 g / L of glucose, 20 pg / mL of gentamicin sulfate, 100 U / mL of penicillin and 50 pg / mL of streptomycin, pH 7.4. The macerate was centrifuged at 1,200 xg for 10 minutes and the supernatant was discarded. The red blood cells were lysed with 3 ml of lysis buffer (17 mM Tris-HCl pH 7.4 and 144 mM ammonium chloride) for 4 minutes, when the DMEM medium was added, to a final volume of 10 ml. The material was centrifuged at 1,200 xg for 10 minutes and, afterwards, the supernatant was discarded. The pellet was resuspended in 1 mL of complete DMEM, which was added with 20% inactivated fetal bovine serum. Splenocytes were quantified in a Newbauer chamber and 1 x 106 cells per mL were plated in 24-well plates (Nunc) in complete DMEM medium. Splenocytes were stimulated with individual phage clones (1 x 10 phages), with wild phage (1 x 10) as a control or with the parasitic soluble protein extract (SLA L. infantum, 50 pg), or incubated without stimulation . The incubation took place at 37 ° C in an oven with 5% CO ₂ and for 48 hours. Afterwards, the cultures supernatant was collected and the measurement of the cytokines IFN-γ and IL-10 was performed by capture ELISA using commercial kits (BD OptEIA TM set mouse kits, Pharmingen, San Diego, CA, USA). The results are shown in Table 2.

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14/17 [0037] With the quantified cytokines, the ratio between the levels of IFN-γ and IL-10 was calculated for each clone evaluated, in order to select only those phages capable of inducing a greater production of IFN-γ and lower levels of IL-

10, and the results are also shown. The displayed values are representative of two experiments carried out independently and that presented similar results. A wild phage clone, similar to wild phage that does not express exogenous peptide and SLA L. infantum were used as controls. Table 2. Dosage of IFN-γ and IL-10 cytokines after in vitro stimulation of splenocytes from BALB / c mice infected with L. infantum and calculus of the ratio between the values. Immunogen Cytokines (in pg / mL) IFN-gamma IL-10 Reason A10 129 78 1.65 B1 132 87 2.52 D2 136 90 1.51 D11 116 70 2.65 C12 154 85 1.81 D12 137 88 2.44 E2 66 75 0.88 E3 48 86 2.56 E4 72 80 0.9 E6 239 70 1.41 E12 317 75 1.23 F5 164 83 2.97 F6 495 88 5.62 F7 40 79 0.5 F8 123 78 2.58 F10 122 66 1.85 F11 195 70 2.79 F12 564 80 0.7

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G10 163 74 1.4 H2 143 69 2.07 H6 104 75 1.39 H10 63 90 2.7 H11 80 89 0.9 H12 46 76 0.6 Wild Phage 70 77 0.6 SLA 59 84 0.4

[0038] In the analysis of the results, it was observed that there was a great variation in the production of cytokines among the pre-selected clones. The controls (wild phage and SLA L. infantum) showed a ratio between the levels of IFN-γ and IL-10 of 0.6 and 0.4, respectively. Those clones whose production of IFN-γ was at least 2.0 times greater than the production of IL-10 were selected given that, after stimulation of splenocytes from mice infected with L. infantum, they induced a greater production of Th1 type cytokines, compared to the production of Th2 cytokines. Thus, 10 clones (B1, D11, D12, E3, F5, F6,

F8, F11, H2 and H10) were selected to be used in the in vitro stimulation of PBMCs from healthy individuals.

EXAMPLE 4. CULTURE OF PBMCS AND DETERMINATION OF CYTOKINES.

[0039] Peripheral blood mononuclear cells were isolated as described (Khamesipour A, et al. Phenotyping of circulating CD8 ⁺ T cell subsets in human cutaneous leishmaniasis. Microbes Infect. 9: 702-11, 2012). Cell culture was performed as described (Roatt BM, et al. Performance of LBSap vaccine after intradermal challenge with L. infantum and saliva of Lu. Longipalpis: immunogenicity and parasitological evaluation. PLoS One.7 (11): e49780, 2012) . To stimulate PBMCs, 10 ^{6 * * *} cells per well were incubated in DMEM with

10 ¹⁰ of each phage evaluated, either with wild phage (same concentration) or with L. infantum SLA (50 pg / ml). Incubation was carried out at 37 ° C with 5% CO ₂ for 5 days. As a positive control, the cells were incubated with phorbol myristate acetate (PMA, 25 ng / mL) and ionomycin (1 pg / mL) in DMEM medium. The cultures were incubated for 48 hours at 37 ° C and in 5% CO2. Afterwards, brefeldin A (Sigma) was added (10 pg / mL concentration) during

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16/17 a period of 4 hours. Afterwards, 200 pL of EDTA (Sigma) was added to each culture tube (to a final concentration of 2 mM), and the cells were washed with FACS buffer (1x PBS with 0.5% fetal bovine serum albumin and 0 , 1% sodium azide), resuspended and immunostained with anti-CD4 or anti-CD8 labeled mAb (Caltag, Burlingame, CA, USA), for 30 minutes in the dark and at room temperature. After staining the membrane, lysis and fixation procedures were performed and PBMCs were permeabilized with FACS Permtampão (FACS buffer plus 0.5% saponin), with incubation for 30 minutes in the dark and at room temperature; in the presence of 20 pL of PE-labeled anti-cytokine mAbs antibody (IFN-γ and IL-10, Serotec®, USA). After staining the intracytoplasmic cytokines, PBMCs were washed with FACS buffer and fixed in fixation solution (10 g / L paraformaldehyde, 10.2 g / L sodium cacodylate and 6.63 g / L sodium chloride, pH 7.2), for storage at 4 ° C before data acquisition and analysis by cytometry. Measurements were performed on a FACScalibur® instrument (Becton Dickson - BD, USA) and analyzed with Cell-quest ™ software (Franklin Lakes, NJ, USA), based on 30,000 events per sample. The results are shown in Table 3. The ratio between the levels of IFNγ and IL-10 was calculated for each clone and the data are also presented.

Table 3. Dosage of IFN-γ and IL-10 cytokines after in vitro stimulation of PBMCs obtained from healthy individuals and calculation of the ratio between values.

Immunogen Cytokines (in pg / mL) IFN-gamma IL-10 Reason B1 205 89 2.3 D11 244 95 2.56 D12 233 100 2.33 E3 101 112 0.9 F5 241 87 2.77 F6 316 102 3.1 F8 106 88 1.2 F11 104 69 1.5 H2 255 92 2.77

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H10 106 76 1.4 Wild Phage 68 97 0.7 SLA 57 113 0.5

[0040] In the analysis of the results, it was observed that there was a big difference in the production of IFN-γ and IL-10 among the selected clones. The controls (wild phage and SLA L. infantum) showed a ratio between the levels of IFN-γ and IL-10 of 0.7 and 0.5, respectively. Thus, those clones whose production of IFNγ was at least 2.0 times greater than the production of IL-10, place themselves as immunogenic in two mammal models evaluated (mouse and man) and, thus, as candidates to be used as vaccines against VL, given that, after stimulation of splenocytes from mice infected with L. infantum and PBMCs from healthy individuals, such clones have been able to induce a greater production of IFN-γ and low levels of IL-10. The amino acid sequences of the selected clones are shown in Table 4.

Table 4. Strings amino acids of the selected clones after evaluation of its immunogenicity in mouse splenocytes infected and PBMCs from healthy individuals. Clone Amino acid sequences B1 V Q S W S V R D11 G P N L L L V D12 V S V Met R G R F5 T L W P F K L F6 D L C T Q I T H2 G S R S Y P R

[0041] Thus, the immunogens B1, D11, D12, F5, F6 and H2 are placed as candidates for vaccine to be tested in the prevention against VL in different mammalian models.

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Claims (7)

1- VACCINE COMPOSITIONS AGAINST VISCERAL LEISHMANIOSIS, characterized by comprising at least one of the protein antigens defined by SEQ ID N ° 1 to 6 or any possible combination between them, and pharmacologically and pharmaceutically acceptable excipients. 2- VACCINE COMPOSITIONS AGAINST VISCERAL LEISHMANIOSIS, according to claim 1, characterized by the antigens being expressed on the external surface of bacteriophages. 3- VACCINE COMPOSITIONS AGAINST VISCERAL LEISHMANIOSIS, according to claim 1, characterized by being administered intradermally, intramuscularly, orally, nasally, intravenously, subcutaneously and / or as a device that can be injected. 4- VACCINE COMPOSITIONS AGAINST VISCERAL LEISHMANIOSIS, according to claims 1 to 3, characterized by the association of pharmaceutically and pharmacologically acceptable adjuvants, in order to potentiate the development of a Th1-type cellular immune response. 5- SYNTHETIC PEPTIDES, characterized by comprising the sequences defined by SEQ ID N ° 1 to 6. 6- USE of synthetic peptides, as defined by claim 5, characterized by being in the preparation of vaccine compositions for the treatment and / or prevention of visceral leishmaniasis. 7- USE of vaccine compositions, as defined by claims 1 to 4, characterized by being in the treatment and / or prevention of visceral leishmaniasis in mammals. Petition 870160045682, of 23/08/2016, p. 26/28 1/1