WO2018051291A1 - Vaccine composition for protection against tegumentary leishmaniasis - Google Patents

Vaccine composition for protection against tegumentary leishmaniasis Download PDF

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WO2018051291A1
WO2018051291A1 PCT/IB2017/055614 IB2017055614W WO2018051291A1 WO 2018051291 A1 WO2018051291 A1 WO 2018051291A1 IB 2017055614 W IB2017055614 W IB 2017055614W WO 2018051291 A1 WO2018051291 A1 WO 2018051291A1
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leishmaniasis
leishmania
ifn
peptides
vaccine
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PCT/IB2017/055614
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French (fr)
Portuguese (pt)
Inventor
Eduardo Antônio FERRAZ COELHO
Carlos Alberto Pereira Tavares
Lourena Emanuele COSTA
Antônio Lúcio TEIXEIRA JÚNIOR
Beatriz Cristina SILVEIRA SALLES
Denise UTSCH GONÇALVES
Daniel MENEZES SOUZA
Mariana COSTA DUARTE
Bruno MENDES ROATT
Luiz Ricardo Goulart Filho
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Universidade Federal De Minas Gerais
Universidade Federal De Uberlândia - Ufu
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present technology deals with vaccine compositions comprising at least one of the nine peptides defined by SEQ ID NOs 1 to 9, capable of stimulating the development of a T helper 1 (Th1) -type cellular immune response.
  • Th1 T helper 1
  • gamma IFN- ⁇
  • IL-4 interleukin-4
  • Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and is endemic in 98 countries worldwide, with an estimated incidence of 1.0 to 2.0 million new cases each year. Of this amount, 0.5 to 1.5 million correspond to cases of cutaneous leishmaniasis (LT) (Desjeux, P. The increase in risk factors for leishmaniasis worldwide. Trans R Soe Trop Med Hyg. 95 (3): 239-43,2001), while About 500,000 cases of visceral leishmaniasis (VL) are reported annually (Alvar, J. et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 7 (5): 35671, 2012).
  • LT can manifest itself in three main clinical forms: cutaneous leishmaniasis (LC), the most common clinical form of the disease, mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (LCD) (WHO. Leishmaniasis. Fact sheet 375, 2014).
  • LC is characterized by the presence of a primary skin lesion that develops at the site of the sand fly bite, being unique or may lead to other lesions.
  • the disease tends to evolve to spontaneous cure, however, there are cases in which the lesion may remain active for several years and coexist with mucosal lesions that may appear later on as the disease progresses (Gontijo, B., Carvalho MDE, L.
  • L. braziliensis species is the main etiological agent of LC in Brazil (Marsden, PD Mucosal leishmaniasis (Escomel espundia, 1911). Trans R Soe Trop Med Hyg.80 (6): 859-76, 1986).
  • LM and LCD the most severe clinical forms of LT, are also caused by the L. braziliensis species, although L. amazonensis is responsible for the clinical forms with the worst treatment response for affected patients (Garcez, LM, et al.
  • Leishmania (Leishmania ) amazonensis-induced cutaneous leishmaniasis in the primate Cebus apella: A model for vaccine trials International Journal for Parasitology 32: 1755-1764, 2002).
  • the diagnosis of LT is based on the assessment of clinical manifestations of the disease in conjunction with epidemiological investigations and the detection of intracellular stages of the parasites through smears and / or biopsies of skin lesions and specimen culture. ; in addition to other laboratory tests. Rapid and accurate diagnosis is critical to prevent permanent damage and severe functional sequelae to patients; However, for clinical diagnosis, the symptomatology of LT is similar to that of other diseases common in our country, such as tuberculosis, paracoccidioidomycosis and leprosy.
  • Noninfectious diseases such as granulomas and neoplasms may also cause lesions that resemble ulcers caused by LT (Junqueira-Pedras, M. et al., Antibody subclass profile against Leishmania braziliensis and Leishmania amazonensis in the diagnosis and follow-up of mucosal leishmaniasis. Diagn Microbiol Infect Dis. 47 (3): 477-85, 2003).
  • Parasitological methods although presenting high specificity, have limitations in their sensitivity, due to the non-homogeneous distribution of parasites in the lesions, and the collection of samples is considered invasive by biopsies.
  • the ELISA technique has been used for presenting better sensitivity and specificity values (Goto, H .; Lindoso, JA.) Expert Rev Anti Infect Ther. 8 (4): 419- 33, 2010). This technique is considered an important tool for the diagnosis of LT and monitoring the exposure of individuals in endemic areas of the disease. Generally, these tests use antigens obtained from crude fractions and / or semi-purified molecules of the parasite, as well as recombinant proteins and / or synthetic peptides derived from them (Junqueira-Pedras, M. et al., Antibody subclass profile against Leishmania braziliensis and Leishmania.
  • LT is a major public health problem. To date, the diagnosis is poorly made and many patients end up receiving treatment late and when the disease has been established for many years. Thus, other measures to control the spread of the disease are necessary, such as the development of vaccines.
  • Vaccination with the Leishmania infantum ribosomal protein induces protection in BALB / c mice against Leishmania chagasi and Leishmania amazonensis challenge Microbes Infect 12: 967-77, 2010).
  • an effective vaccine for the population is not yet available on the market.
  • the Th1 response profile is suppressed by IL-4 production.
  • this cytokine thought to induce a Th2 response, is related to macrophage deactivation and disease establishment in animals, whereas cytokines such as IFN- ⁇ and IL-12 are related to protection against disease (Coelho, EAF , et al.lmmune responses induced by Leishmania (Leishmania) donovani A2 antigen, but not by LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection Infect Immun.
  • protected animals are those with a low parasitic burden associated with high levels of IFN- ⁇ and IL-12, as well as low levels of IL-4 and IL-10 secreted by cultured splenocytes.
  • antigens that are capable of stimulating the development of a Th1-type response, primared by IFN- ⁇ production in splenocytes of Leishmania-infected mice, could be considered promising vaccine candidates for use as leishmaniasis vaccines.
  • Antigens currently tested as vaccines against leishmaniasis are usually complete proteins, which contain regions common to proteins of other pathogens, which can lead to cross reactions in vaccinated animals. Also, such proteins may contain epitopes (peptides) that have a beneficial effect, but others that have a harmful effect on immunized animals, thereby compromising the efficacy of the vaccine.
  • the use of small antigens in the form of peptides tends to allow the development of a more specific vaccine so that the immunized dog or man can develop a beneficial immune response to only that antigen specific to him and the disease for which he is immunized. which it has been selected.
  • Such antigens also have a simpler, faster, higher yield and lower cost technical production compared to recombinant protein production; They also have good stability after production and thus can be made more easily available to the population.
  • Phage display technology is based on the expression of protein-associated peptides, proteins and / or antibody fragments present on the outer surface of recombinant bacteriophages.
  • the nucleotide sequence encoding the inserted peptides is genetically fused to a sequence encoding some bacteriophage proteins, resulting in a hybrid product, which is then exposed on the outer surface of viral particles (Smith GP Filamentous fusion phage: novel expression vectors que display cloned antigens on the virion surface (Science, 288: 1315-17, 1985).
  • Bacteriophage libraries expressing exogenous peptides have been used to identify various cell receptors, facilitating the identification of small molecules that bind with high affinity and mimic interaction with their natural ligands; as well as in the identification of peptides that interact with antibodies, without prior knowledge of the antigenic region recognized by the antibody.
  • Selection of high affinity molecules with respect to a particular target receptor, adhered to a solid surface, is performed by consecutive selection steps called "bio-panning cycles". The number of phage display bio-panning cycles determines the degree of enrichment of the phages that bind to the target receptor.
  • a population of high affinity phage clones with respect to 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).
  • bio-panning cycles can be tailored according to the research group that mastered such technology. For example, in our case, immunoglobulin molecules (IgGs) from sera from patients to be investigated were fused to the surface of microspheres (called "beads”) and such a hybrid was subjected to negative and positive uptake (selection) processes. to phage clones present in the recombinant library used.
  • IgGs immunoglobulin molecules
  • Patent document PI09030891 entitled "Recombinant peptides and Leishmania antigen mimetic protein motifs and their applications” deals with the characterization and use of recombinant peptides, their reverse sequences and their protein motifs in the treatment of leishmaniasis.
  • the technology differs from the present application because the experimental strategy used is different and does not contemplate the use of human cells or the dosage of human cytokines.
  • the peptides expressed in the selected clones are different from those described in the present application, since they were used as synthetic peptides, whereas in the present technology the bacteriophage clones themselves expressing the peptides of interest were used, that is, as being an immunogenic composition. already done.
  • Patent document BR102012033552 entitled “Polymeric Peptides, Process for Obtaining and Use for Leishmaniasis Immunodiagnosis”, deals with the production of polymeric peptides by the phage display technique for canine and human leishmaniasis immunodiagnosis.
  • the technology differs from that contained in the present application in that it comprises different types of peptide sequences and the method of obtaining them is different.
  • the polymeric peptides contained in said document are used for diagnosis, and not as a vaccine composition against leishmaniasis.
  • Patent document BR102013027542 entitled "Canine Visceral Leishmaniasis Vaccine Composition, Synthetic Peptides and Use", is a vaccine composition for the prevention and / or treatment of canine visceral leishmaniasis comprised of peptides expressed in bacteriophages. Said technology differs from the present application in that it comprises different sequences and canine disease alone, and does not contemplate the use of human cells or the dosage of human cytokines.
  • the present technology deals with a vaccine composition comprising at least one of nine peptides selected by a phage display proteomic approach as capable of inducing high IFN- ⁇ production and low levels of IL-4.
  • PBMCs peripheral blood mononuclear cells
  • the present technology deals with vaccine compositions comprising at least one of the nine peptides defined by SEQ ID NOs 1 to 9, capable of stimulating the development of a T helper 1 (Th1) -type cellular immune response with high production of gamma (IFN- ⁇ ) and low levels of interleukin-4 (IL-4) for the prevention of cutaneous leishmaniasis in mammals.
  • Th1 T helper 1
  • IFN- ⁇ gamma
  • IL-4 interleukin-4
  • the present technology comprises nine peptides defined by SEQ ID NOs. 1 to 9 and / or phage display selected bacteriophage clones expressing such peptides, and pharmaceutically and pharmacologically acceptable adjuvants, and their use in the treatment and / or prevention of cutaneous leishmaniasis in mammals.
  • compositions or products according to the present invention may be selected from molecules or products which are capable of stimulating the further development of a Th1-type cellular immune response that is dominated by the production of IFN- ⁇ and IL-12 cytokines.
  • the proposed vaccine composition may be administered by the intradermal, intramuscular, oral, nasal, intravenous, subcutaneous and / or injectable device.
  • the present invention may be better understood by the following non-limiting examples of the technology.
  • the project was approved by COEP / UFMG (protocol CAAE-323431.14.9.0000.5149) and serum pools were prepared using equal amounts of each sample for use in the bio-panning cycles.
  • IgG antibodies Purification of IgG antibodies. Purification of IgG-class immunoglobulins from the sera pools listed above was performed by their coupling to G protein-conjugated magnetic beads (Dynabeads, Invitrogen). To this end, 2x10 9 microsphere particles were washed 3 times in 0.1 M MES pH 5.0 buffer and added to them: (a) 300 ⁇ l of the LM patient sera pool to a final volume of 500 ⁇ ; (b) 300 from the LC patient sera pool for a final volume of 500 ⁇ ; (c) 300 ⁇ of the CD patient sera pool for a final volume of 500 ⁇ and (d) 300 ⁇ of the healthy patient serum pool for a final volume of 500 ⁇ .
  • the ratio of antibodies to the amount of microspheres in all cases was 1: 1. Then, an incubation was performed for 40 minutes. under constant stirring and at room temperature (RT). Antibody adsorbed microspheres were washed 3 times in 0.1 M MES pH 5.0 buffer to remove unbound IgG antibodies.
  • the beads-antibody system was washed 2 times with 1 mL of 0.2 M triethanolamine buffer pH 8.2 and resuspended in 1 mL of covalent binding buffer (20 mM dimethylpimelinidate / HCI in triethanolamine buffer) for 30 minutes under constant stirring and at RT The reaction was neutralized by incubating the system with 1 ml 50 mM Tris buffer pH 7.5 for 15 minutes and at RT Then the incorporated microspheres were washed.
  • Bio-panning cycles phage display.
  • 1x10 12 viral particles from a recombinant library containing filamentous phage fused peptides (Ph.D.-C7C commercial library from New England, BioLabs ® , USA) were used. They were diluted in 290 ⁇ l of 0.1% TBS-Tween for ligand selection in the microspheres incorporated by IgG antibodies.
  • TBS-Tween 0.1%
  • the recombinant bacteriophage library was incubated for 30 minutes and RT with the IgG-coupled microspheres of healthy individuals and then the microspheres were precipitated by magnetic attraction to the Dynal Biotech support (No. 12020), the supernatant containing clones not adhering to IgGs; transferred to a new tube containing the IgG-coupled microspheres of the Chagas Disease patient group. Only phage clones that did not adhere to such antibodies were recovered.
  • the supernatant collected and recovered during negative selection was transferred to a new tube containing the IgG-coupled microspheres of patients with ML, and incubated for 30 minutes at RT.
  • LTM patient antibody bio-panning
  • phages of interest adhered to such IgGs were washed 10 times with 0.1% TBS-Tween 20 and eluted in 500 ⁇ _ 0.2 M glycine buffer pH 2.0. such a solution is neutralized by the addition of 75 ⁇ _ Tris-base pH 9.0.
  • Phage clones were recovered to perform the second step of the positive selection process. These recovered and eluted clones were passed into a new tube containing the IgG-coupled microspheres of LC patients.
  • microspheres containing the beads-antibody system precipitated in the antibody patient bio-panning tubes plus the phages of interest adhered to such IgGs were washed 10 times with 0.1% TBS-Tween 20 and eluted in 500 ⁇ l buffer. 0.2 M glycine pH 2.0, such solution being neutralized by the addition of 75 ⁇ l Tris-base pH 9.0. The process was repeated twice more, totaling 3 cycles of bioselection. After such procedure, the clones were titrated, individualized. [032] Titration and amplification of selected clones. At the end of the third cycle of positive selection, clones of recovered phages were titrated. For each titration, a culture plate was made.
  • the plates were incubated at 37 ° C for 16 hours. Then, 96 blue colonies were individualized, manually collected for DNA extraction and subsequent sequencing.
  • the sequencing reaction was performed using the extracted DNA (500 ng of DNA from each phage) from the 5 pmol clones of Primer 96 glll (5'-OH CCC TCA TAG TTA GCG TAA CG-3'-Biolabs) and mix (DYEnamic ET Dye Terminator Cycle Kit-Amersham Biosciences).
  • mice 8-week-old female BALB / c mice were used.
  • the animals were acquired from the ICB / UFMG Vivarium and kept in the Vibrary of the Department of Clinical Pathology of COLTEC, under appropriate management conditions.
  • the project was also approved by the UFMG Animal Experimentation Ethics Committee (protocol 043/201 1).
  • the IFLA / BR / 1967 / PH-8 sample from L. amazonensis was used.
  • the parasites were grown in complete Schneider's medium, which consisted of Scheneider's medium (SIGMA) plus 20% inactivated fetal bovine serum (SIGMA), 20 mM L-glutamine, 50 ⁇ g / mL gentamicin, 200 U / mL and 100 ⁇ g / mL streptomycin at pH 7.4 and 24 ° C.
  • SIGMA Scheneider's medium
  • SIGMA inactivated fetal bovine serum
  • 20 mM L-glutamine 20 mM L-glutamine
  • 50 ⁇ g / mL gentamicin 200 U / mL
  • 100 ⁇ g / mL streptomycin at pH 7.4 and 24 ° C.
  • mice were euthanized and had their spleen removed and macerated in complete DMEM (Sigma) medium, which was composed of 4.5 g / L glucose, 20 ⁇ g / mL gentamicin sulfate, 100 U / mL penicillin and 50 ⁇ g / mL streptomycin, pH 7.4.
  • the macerate was centrifuged at 1,200 xg for 10 minutes and the supernatant discarded.
  • Red blood cells were lysed with 3 ml lysis buffer (17 mM Tris-HCl pH 7.4 and 144 mM ammonium chloride) for 4 minutes, when DMEM medium was added to a final volume of 10 ml.
  • the material was centrifuged at 1,200 xg for 10 minutes and then the supernatant was discarded.
  • the pellet was resuspended in 1 ml complete DMEM, which was added with 20% inactivated fetal bovine serum.
  • Splenocytes were quantified in Newbauer chamber and 1 x 10 6 cells per ml were plated in 24-well plates (Nunc) in complete DMEM medium. Splenocytes were stimulated either with individual phage clones (1 x 10 10 phages), with wild phage (1 x 10 10 ) as a control or with the parasite soluble protein extract (SLA L. amazonensis, 50 ⁇ g), or incubated. without stimulus. Incubation occurred at 37 ° C in a 5% CO 2 oven and for 48 hours.
  • IFN- ⁇ and IL-4 cytokines were assayed by capture ELISA using commercial kits (BD OptEIA TM IFN- ⁇ and IL-4 mouse, Pharmingen, San Diego, CA, USA).
  • 25 clones (A4, A8, A9, A10, A12, B2, B3, B4, B7, B8, B10, C1, C6, C8, C12, E3, E4, E5, E8, E9, G1, G2, G4, H1 and H4) were selected to be used for in vitro stimulation of PBMCs derived from healthy individuals.
  • a positive control cells were incubated with phorbol myristate acetate (PMA, 25 ng / mL) and ionomycin (1 Mg / mL) in DMEM medium. Cultures were incubated for 48 hours at 37 ° C and 5% CO 2 . Afterwards, brefeldin A (Sigma) was added (10 Mg / mL concentration) over a period of 4 hours. Afterwards, 200 ⁇ l 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 ⁇ l).
  • FACS buffer 1x PBS with 0.5% fetal bovine serum albumin and 0 ⁇ l.
  • 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 prior to data acquisition and cytometric analysis. Measurements were performed on a FACScalibur ⁇ instrument (Becton Dickson - BD, USA) and analyzed with Cell-quest TM software (Franklin Lakes, NJ, USA) on the basis of 30,000 events per sample. Results are shown in Table 3. The ratio of IFN- ⁇ and IL-4 levels were calculated for each clone used and data are also presented.
  • immunogenic clones A4, A8, A9, A10, B2, B10, E3, E5 and E8 pose as vaccine candidates to be tested for LT prevention in different mammalian models.

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Abstract

The present technology relates to vaccine compositions comprising at least one of the nine peptides defined by SEQ ID NO 1-9, capable of stimulating the development of a T helper 1 (Th1)-type cell immune response with high production of interferon-gamma (IFN- ) and low levels of interleukin-4 (IL-4) for preventing tegumentary leishmaniasis in mammals.

Description

"COMPOSIÇÃO VACINAL PARA PROTEÇÃO CONTRA A  "VACINAL COMPOSITION FOR PROTECTION AGAINST
LEISHMANIOSE TEGUMENTAR"  TEGUMENT LEISHMANIASIS "
[01] A presente tecnologia trata de composições vacinais compreendendo pelo menos um dos nove peptídeos definidos pelas SEQ ID No 1 a 9, capazes de estimular o desenvolvimento de uma resposta imune celular do tipo T helper 1 (Th1 ) com elevada produção de Interferon-gamma (IFN-γ) e baixos níveis de lnterleucina-4 (IL-4), para prevenção da leishmaniose tegumentar em mamíferos. [01] The present technology deals with vaccine compositions comprising at least one of the nine peptides defined by SEQ ID NOs 1 to 9, capable of stimulating the development of a T helper 1 (Th1) -type cellular immune response. gamma (IFN-γ) and low levels of interleukin-4 (IL-4) for the prevention of cutaneous leishmaniasis in mammals.
[02] As leishmanioses são doenças causadas por parasitas protozoários do género Leishmania, sendo endémicas em 98 países no mundo, com uma incidência estimada de 1.0 a 2.0 milhões de novos casos a cada ano. Deste montante, 0.5 a 1.5 milhões correspondem a casos de leishmaniose tegumentar (LT) (Desjeux, P. The increase in risk factors for leishmaniasis worldwide. Trans R Soe Trop Med Hyg. 95 (3): 239-43,2001 ), enquanto cerca de 500 mil casos de leishmaniose visceral (LV) são registrados anualmente (Alvar, J. et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 7(5):35671 , 2012).  [02] Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and is endemic in 98 countries worldwide, with an estimated incidence of 1.0 to 2.0 million new cases each year. Of this amount, 0.5 to 1.5 million correspond to cases of cutaneous leishmaniasis (LT) (Desjeux, P. The increase in risk factors for leishmaniasis worldwide. Trans R Soe Trop Med Hyg. 95 (3): 239-43,2001), while About 500,000 cases of visceral leishmaniasis (VL) are reported annually (Alvar, J. et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 7 (5): 35671, 2012).
[03] Atualmente, cerca de 20 espécies do parasita são encontradas nas Américas, das quais 14 estão associadas a casos de LT. No Brasil, sete espécies foram identificadas como agentes etiológicos da doença, das quais seis pertencem ao subgênero Viannia: Leishmania (Viannia) braziliensis, Leishmania (V.) guyanensis, Leishmania (V.) lainsoni, Leishmania (V.) shawi e Leishmania (V.) lindenbergi (Lainson, R.; Shaw, J. J. Evolution, classification and geographical distribution. Academic Press:London, 1-120, 1987; Silveira, F. T. et al. An outbreak of cutaneous leishmaniasis among soldiers in Belém, Para State, Brazil, caused by Leishmania (Viannia) lindenbergi n. sp. A new leishmanial parasite of man in the Amazon region. Parasite.9(1 ):43-50, 2002). A sétima espécie pertence ao subgênero Leishmania, sendo a espécie Leishmania (Leishmania) amazonensis (Shaw, J. J.; Lainson, R. Leishmaniasis in Brazil. VI. Observations on the seasonal variations of Lutzomyia flaviscutellata in different types of forest and its relationship to enzootic rodent leishmaniasis (Leishmania mexicana amazonensis). Trans R Soe Trop Med Hyg. 66(5):709-17, 1972; Silveira, F. T.; Lainson, R.; Corbett, C. E. Clinicai and immunopathological spectrum of American cutaneous leishmaniasis with special reference to the disease in Amazonian Brazil: a review. Mem Inst Oswaldo Cruz. 99(3):239-51 , 2004). [03] Currently, about 20 parasite species are found in the Americas, of which 14 are associated with LT cases. In Brazil, seven species were identified as etiological agents of the disease, six of which belong to the subgenus Viannia: Leishmania (Viannia) braziliensis, Leishmania (V.) guyanensis, Leishmania (V.) lainsoni, Leishmania (V.) shawi and Leishmania ( V.) lindenbergi (Lainson, R.; Shaw, JJ Evolution, classification and geographical distribution. Academic Press: London, 1-120, 1987; Silveira, FT et al. An outbreak of cutaneous leishmaniasis among soldiers in Bethlehem, Para State, Brazil, caused by Leishmania (Viannia) lindenbergi sp The New Leishmanial Parasite of Man in the Amazon Region Parasite.9 (1): 43-50, 2002). The seventh species belongs to the subgenus Leishmania, being the species Leishmania (Leishmania) amazonensis (Shaw, JJ; Lainson, R. Leishmaniasis in Brazil. VI. Observations on seasonal variations of Lutzomyia flaviscutellata in different types of forest and its relationship to enzootic rodent leishmaniasis (Mexican Leishmania amazonensis) Trans R Soe Trop Med Hyg. 66 (5): 709-17, 1972; Silveira, F; Lainson, R.; Corbett, CE Clinical and immunopathological spectrum of American cutaneous leishmaniasis with special reference to the disease in Amazonian Brazil : a review Mem Inst Oswaldo Cruz 99 (3): 239-51, 2004).
[04] A LT pode se manifestar sobre três formas clínicas principais: a leishmaniose cutânea (LC), forma clínica mais comum da doença, a leishmaniose mucosa (LM) e a leishmaniose cutâneo-difusa (LCD) (WHO. Leishmaniasis. Fact sheet. 375, 2014). A LC é caracterizada pela presença de uma lesão cutânea primária que se desenvolve no local da picada do flebotomíneo, sendo única ou podendo originar outras lesões. A doença tende a evoluir à cura espontânea, porém, há casos em que a lesão pode permanecer ativa por vários anos e coexistir com lesões mucosas que podem surgir mais adiante com o avançar da doença (Gontijo, B., Carvalho MDE, L. American cutaneous leishmaniasis. Rev Soe Bras Med Trop. 36(1 ): 71-80, 2003). A espécie L. braziliensis é o principal agente etiológico de LC no Brasil (Marsden, P. D. Mucosal leishmaniasis ("espundia" Escomel, 191 1 ). Trans R Soe Trop Med Hyg.80(6):859-76, 1986). A LM e LCD, formas clínicas mais graves de LT, são também causadas pela espécie L. braziliensis, embora L. amazonensis seja responsável pelas formas clínicas com pior resposta de tratamento para os pacientes acometidos (Garcez, L. M., et al. Leishmania (Leishmania) amazonensis-induced cutaneous leishmaniasis in the primate Cebus apella: A model for vaccine trials. International Journal for Parasitology. 32: 1755-1764, 2002). [05] O diagnóstico da LT baseia-se na avaliação das manifestações clínicas da doença em conjunto com inquéritos epidemiológicos e com a detecção dos estágios intracelulares dos parasitas, por meio de exames de esfregaços e/ou biópsias de lesões da pele e cultura de espécimes; além de outros exames laboratoriais. O diagnóstico rápido e preciso é fundamental para evitar danos permanentes e graves sequelas funcionais aos pacientes; porém, para o diagnóstico clínico, a sintomatologia da LT é semelhante à de outras doenças, comuns em nosso meio, tais como a tuberculose, paracoccidioidomicose e hanseníase. Doenças não infecciosas, como granulomas e neoplasias podem também causar lesões que se assemelham às úlceras causadas na LT (Junqueira-Pedras, M. et al. Antibody subclass profile against Leishmania braziliensis and Leishmania amazonensis in the diagnosis and follow-up of mucosal leishmaniasis. Diagn Microbiol Infect Dis. 47(3):477-85, 2003). Os métodos parasitológicos, apesar de apresentarem especificidade elevada, possuem limitações em sua sensibilidade, pela distribuição não homogénea dos parasitas nas lesões, além da coleta das amostras ser considerada invasiva por meio das biópsias. Além disso, tais métodos consomem grande tempo para a confecção das lâminas, fato que também dificulta sua utilização na prática médica rotineira (Weigle, K. A. et al. Diagnosis of cutaneous and mucocutaneous leishmaniasis in Colômbia: a comparison of seven methods. Am J Trop Med Hyg.36(3):489-96, 1987). [04] LT can manifest itself in three main clinical forms: cutaneous leishmaniasis (LC), the most common clinical form of the disease, mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (LCD) (WHO. Leishmaniasis. Fact sheet 375, 2014). LC is characterized by the presence of a primary skin lesion that develops at the site of the sand fly bite, being unique or may lead to other lesions. The disease tends to evolve to spontaneous cure, however, there are cases in which the lesion may remain active for several years and coexist with mucosal lesions that may appear later on as the disease progresses (Gontijo, B., Carvalho MDE, L. American cutaneous leishmaniasis Rev Soe Bras Med Trop 36 (1): 71-80, 2003). The L. braziliensis species is the main etiological agent of LC in Brazil (Marsden, PD Mucosal leishmaniasis (Escomel espundia, 1911). Trans R Soe Trop Med Hyg.80 (6): 859-76, 1986). LM and LCD, the most severe clinical forms of LT, are also caused by the L. braziliensis species, although L. amazonensis is responsible for the clinical forms with the worst treatment response for affected patients (Garcez, LM, et al. Leishmania (Leishmania ) amazonensis-induced cutaneous leishmaniasis in the primate Cebus apella: A model for vaccine trials International Journal for Parasitology 32: 1755-1764, 2002). [05] The diagnosis of LT is based on the assessment of clinical manifestations of the disease in conjunction with epidemiological investigations and the detection of intracellular stages of the parasites through smears and / or biopsies of skin lesions and specimen culture. ; in addition to other laboratory tests. Rapid and accurate diagnosis is critical to prevent permanent damage and severe functional sequelae to patients; However, for clinical diagnosis, the symptomatology of LT is similar to that of other diseases common in our country, such as tuberculosis, paracoccidioidomycosis and leprosy. Noninfectious diseases such as granulomas and neoplasms may also cause lesions that resemble ulcers caused by LT (Junqueira-Pedras, M. et al., Antibody subclass profile against Leishmania braziliensis and Leishmania amazonensis in the diagnosis and follow-up of mucosal leishmaniasis. Diagn Microbiol Infect Dis. 47 (3): 477-85, 2003). Parasitological methods, although presenting high specificity, have limitations in their sensitivity, due to the non-homogeneous distribution of parasites in the lesions, and the collection of samples is considered invasive by biopsies. Moreover, such methods consume a great deal of time for making the slides, a fact that also hinders their use in routine medical practice (Weigle, KA et al. Diagnosis of cutaneous and mucocutaneous leishmaniasis in Colombia: a comparison of seven methods. Am J Trop Med Hyg. 36 (3): 489-96, 1987).
[06] O diagnóstico laboratorial baseado na presença de anticorpos específicos por parte do hospedeiro infectado constitui-se em uma alternativa, com a sensibilidade variando em torno de 62% a 100% dos casos, dependendo da preparação antigênica utilizada. Uma das desvantagens de testes de sorodiagnóstico tem sido a baixa sensibilidade e/ou especificidade dos mesmos, provocada pelos baixos títulos de anticorpos específicos e pela reatividade cruzada com antígenos presentes em agentes infecciosos causando doenças relacionadas à LT, respectivamente (Guimarães, M. C ; Celeste, B. J.; Franco, E. L. Evaluation of dot enzyme-linked immunosorbent assay for mucocutaneous leishmaniasis and comparison with microplate enzyme immunoassay. J Clin Microbiol.24(3):364-7, 1986; Valli, L. C. et al. Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis. J Parasitol. 85(6): 1076-83, 1999). Dessa forma, muitos pacientes são considerados falso-negativos nas triagens sorológicas realizadas, embora estejam infectados pelo parasita Leishmania (Passos, V. M. et al. American cutaneous leishmaniasis: use of a skin test as a predictor of relapse after treatment. Buli World HeaLT Organ. 78(8):968-74, 2000; Brito, M. E. et al. Dynamics of the antibody response in patients with therapeutic or spontaneous cure of American cutaneous leishmaniasis. Trans R Soe Trop Med Hyg.95(2):203-6, 2001 ). [06] Laboratory diagnosis based on the presence of specific antibodies by the infected host is an alternative, with sensitivity ranging from about 62% to 100% of cases, depending on the antigen preparation used. One of the disadvantages of serodiagnostic tests has been their low sensitivity and / or specificity, caused by low specific antibody titers and cross-reactivity with antigens present. in infectious agents causing LT-related diseases, respectively (Guimarães, M. C; Celeste, BJ; Franco; EL; Evaluation of dot-immunosorbent assay for mucocutaneous leishmaniasis; and comparison with microplate enzyme immunoassay. J Clin Microbiol.24 (3) : 364-7, 1986; Valli, LC et al., Humoral immune responses between mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis J Parasitol 85 (6): 1076-83, 1999). Thus, many patients are considered false-negative in the serological screening performed, although they are infected by the Leishmania parasite (Passos, VM et al. American cutaneous leishmaniasis: use of a skin test as a predictor of relapse after treatment.) Bulli World HeaLT Organ. 78 (8): 968-74, 2000; Brito, ME Dynamics of the antibody response in patients with therapeutic or spontaneous cure of American cutaneous leishmaniasis Trans R Sound Trop Med Hyg.95 (2): 203-6, 2001).
[07] A técnica de ELISA vem sendo utilizada por apresentar melhores valores de sensibilidade e especificidade (Goto, H.; Lindoso, J. A. Current diagnosis and treatment of cutaneous and mucocutaneous leishmaniasis. Expert Rev Anti Infect Ther. 8(4):419-33, 2010). Tal técnica é considerada uma ferramenta importante para o diagnóstico da LT e o monitoramento da exposição dos indivíduos em áreas endémicas da doença. Geralmente, esses testes utilizam antígenos obtidos de frações brutas e/ou moléculas semi-purificadas do parasito, bem como proteínas recombinantes e/ou peptídeos sintéticos derivados dos mesmos (Junqueira-Pedras, M. et al. Antibody subclass profile against Leishmania braziliensis and Leishmania amazonensis in the diagnosis and follow-up of mucosal leishmaniasis. Diagn Microbiol Infect Dis.47(3):477-85, 2003). Entretanto, a heterogeneidade da resposta humoral observada em muitos pacientes com LT indica que, para um sorodiagnóstico efetivo da doença, uma combinação de antígenos em um mesmo ensaio seria desejável, quando comparado à utilização de antígenos isolados dos parasitas. Uma combinação de diferentes antígenos com valor diagnóstico também poderia detectar infecções assintomáticas sendo, dessa forma, altamente desejável para o diagnóstico da doença. [07] The ELISA technique has been used for presenting better sensitivity and specificity values (Goto, H .; Lindoso, JA.) Expert Rev Anti Infect Ther. 8 (4): 419- 33, 2010). This technique is considered an important tool for the diagnosis of LT and monitoring the exposure of individuals in endemic areas of the disease. Generally, these tests use antigens obtained from crude fractions and / or semi-purified molecules of the parasite, as well as recombinant proteins and / or synthetic peptides derived from them (Junqueira-Pedras, M. et al., Antibody subclass profile against Leishmania braziliensis and Leishmania. amazonensis in the diagnosis and follow-up of mucosal leishmaniasis (Diagn Microbiol Infect Dis.47 (3): 477-85, 2003). However, the heterogeneity of the humoral response observed in many LT patients indicates that for an effective serodiagnosis of the disease, a combination of antigens in the same assay would be desirable when compared to the use of antigens isolated from parasites. A combination of different antigens with diagnostic value could also detect asymptomatic infections and is thus highly desirable for the diagnosis of the disease.
[08] Apesar dos avanços científicos, a LT constitui-se em um importante problema de Saúde Pública. Até o presente momento, o diagnóstico é realizado de forma precária e muitos pacientes acabam por receber o tratamento de forma tardia e quando a doença já se estabeleceu por muitos anos. Dessa forma, outras medidas de controle de disseminação da doença fazem-se necessárias, tais como o desenvolvimento de vacinas.  [08] Despite scientific advances, LT is a major public health problem. To date, the diagnosis is poorly made and many patients end up receiving treatment late and when the disease has been established for many years. Thus, other measures to control the spread of the disease are necessary, such as the development of vaccines.
[09] As medidas de controle empregadas para reduzir o número de casos de leishmanioses visam, principalmente, à interrupção do ciclo biológico do parasita. Entretanto, o número de espécies de Leishmania, o caráter zoonótico da doença e a manutenção do parasita no ciclo silvestre dificultam a adoção de medidas efetivas (Tesh RB. Control of zoonotic visceral leishmaniasis: is it time to change strategies? Am J Trop Med Hyg.52:287-92, 1995). O controle do vetor pode ser realizado pela aplicação de inseticidas no ambiente doméstico e peridoméstico; entretanto, sua utilização apresenta relativa eficácia (Tesh RB. Control of zoonotic visceral leishmaniasis: is it time to change strategies? Am J Trop Med Hyg.52:287- 92,1995). A eliminação dos reservatórios silvestres, como marsupiais e roedores, não é considerada uma medida executável e ecologicamente correta, pois existe a possibilidade de adaptação do parasita a outros reservatórios existentes no ambiente (Grimaldi, G. e Tesh, R.B. Leishmaniasis of the New World: current concepts and implications for future research. Clin. Microbiol. Rev. 6:230-50, 1993; Gramiccia M, Gradoni L. The current status of zoonotic leishmaniases and approaches to disease control. Int J Parasitol. 35(1 1-12): 1 169-80. 2005). [010] Em modelos murinos, após a cura da LC causada pela espécie Leishmania major, os animais adquirem imunidade duradoura contra a re- infecção (Afonso LC e Scott P. Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis. Infect. & Immun. 61 :2952-59, 1993). Tal fato tem estimulado o desenvolvimento de pesquisas visando à obtenção de antígenos vacinais que possam ser utilizados como uma medida efetiva de controle da doença. Existem alguns antígenos que vêm sendo avaliados e que preenchem, parcialmente, requisitos para serem empregados como candidatos à vacina contra as leishmanioses (Stáger 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, Perez-Jimenez E, Goldsmith-Pestana K, Esteban M, McMahon-Pratt D. Heterologous prime - boost vaccination with the LACK antigen protects against murine visceral leishmaniasis. Infect Immun 73:5286-89,2005; 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). Entretanto, devido às dificuldades relacionadas à obtenção de um antígeno que seja de fácil produção e alto rendimento, baixo custo, que não estimule a produção de anticorpos específicos ao parasita nos animais imunizados, mas ao mesmo tempo em que seja imunogênico e capaz de induzir ao desenvolvimento de uma resposta imune celular específica; ainda não há disponível no mercado uma vacina eficaz para a população. [09] The control measures employed to reduce the number of leishmaniasis cases are aimed primarily at disrupting the parasite's biological cycle. However, the number of Leishmania species, the zoonotic character of the disease and the maintenance of the parasite in the wild cycle make it difficult to adopt effective measures (Tesh RB. Control of zoonotic visceral leishmaniasis: Am J Trop Med Hyg 52: 287-92, 1995). Vector control can be performed by applying insecticides in the domestic and peridomestic environment; however, its use is relatively effective (Tesh RB. Control of zoonotic visceral leishmaniasis: is it time to change strategies? Am J Trop Med Hyg.52: 287-92,1995). The elimination of wild reservoirs, such as marsupials and rodents, is not considered an executable and ecologically correct measure, as there is a possibility of adaptation of the parasite to other reservoirs in the environment (Grimaldi, G. and Tesh, RB Leishmaniasis of the New World: Current concepts and implications for future research Clinical Microbiol Rev. 6: 230-50, 1993; Gramiccia M, Gradoni L. The current status of zoonotic leishmaniases and approaches to disease control Int J Parasitol 35 (1 1-12 ): 1169-80. [010] In murine models, after LC healing caused by Leishmania major species, animals acquire lasting immunity against re-infection (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 aiming at obtaining vaccine antigens that can be used as an effective disease control measure. There are some antigens that have been evaluated and partially meet the requirements to be employed as candidates for the leishmaniasis vaccine (Stag S, Smith DF, Kaye PM. Immunization with a recombinant stage-regulated protein from Leishmania donovani induces protection against visceral leishmaniasis J Immunol 165: 7064-71, 2000; Dondji B, Perez-Jimenez E, Goldsmith-Pestana K, Esteban M, McMahon-Pratt D. Heterologous prime - boost vaccination with the LACK antigen protects against visceral murine leishmaniasis Infect Immun 73: 5286-89,2005; Chávez-Fumagalli MA, et al. Vaccination with the Leishmania infantum ribosomal protein induces protection in BALB / c mice against Leishmania chagasi and Leishmania amazonensis challenge Microbes Infect 12: 967-77, 2010). However, due to the difficulties related to obtaining an antigen that is easy to produce and high yield, low cost, that does not stimulate the production of parasite specific antibodies in immunized animals, but at the same time it is immunogenic and able to induce development of a specific cellular immune response; An effective vaccine for the population is not yet available on the market.
[01 1 ] Na resposta imune contra a LT, a resistência à infecção é relacionada com o desenvolvimento de uma resposta imune específica celular do tipo Th1 , primada pela produção de níveis elevados de citocinas tais como o IFN-γ por parte do hospedeiro mamífero. O mecanismo imunológico efetor responsável pelo controle dos parasitas é decorrente da ativação de macrófagos, via produção de níveis elevados de óxido nítrico, estimulado por essa citocina (Scott P. Development and regulation of cell-mediated immunity in experimental leishmaniasis. Immunol Res. 27:489-98, 2003). [01 1] In the immune response against LT, resistance to infection is related to the development of a Th1-type cellular specific immune response, which is dominated by the production of high levels of cytokines such as IFN-γ by the mammalian host. The effector immune mechanism responsible for the control of parasites is due to macrophage activation via production of high levels of nitric oxide stimulated by this cytokine (Scott P. Immunol Res. 27: 489-98, 2003).
[012] A utilização de modelos murinos em estudos de vacinação nas leishmanioses permitiu a identificação de dois subtipos distintos de linfócitos T que produzem e secretam citocinas capazes de induzir funções efetoras diferentes. Os estudos que utilizaram como base o modelo murino de infecção por L. major em camundongos BALB/c, proposto por Sacks & Noben-Trauth (2002), definiram o paradigma Th1/Th2 de resistência e susceptibilidade, respectivamente, à infecção e o papel de citocinas como o IFN-γ e a IL-4, respectivamente, no desenvolvimento de linhagens de células Th1 e Th2 (Sacks D and Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania majo In mice. Nat Rev Immunol. 2:845-58, 2002). [012] The use of murine models in leishmaniasis vaccination studies allowed the identification of two distinct T lymphocyte subtypes that produce and secrete cytokines capable of inducing different effector functions. Studies based on the murine model of L. major infection in BALB / c mice, proposed by Sacks & Noben-Trauth (2002), defined the Th1 / Th2 paradigm of resistance and susceptibility, respectively, to the role of infection. of cytokines such as IFN-γ and 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 majo In mice. Nat Rev Immunol. 2 : 845-58, 2002).
[013] Na LT, o perfil de resposta Th1 é reprimido pela produção de IL-4. Neste sentido, essa citocina, tida como indutora de uma resposta Th2, é relacionada à desativação de macrófagos e ao estabelecimento da doença nos animais, enquanto citocinas como IFN-γ e IL-12 estão relacionadas com a proteção contra a doença (Coelho, E. A. F., et al.lmmune 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 :3988-3994, 2003; Chávez-Fumagalli, M. A., et al. Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge. Microbes and Infection. 12:967-977, 2010;Nico, D., et al. Cross-protective immunity to Leishmania amazonensis is mediated by CD4+ and CD8+ epitopes of Leishmania donovani nucleoside hydrolase terminal domains. Frontiers in Immunology. 5:1-10, 2014). [013] In LT, the Th1 response profile is suppressed by IL-4 production. In this sense, this cytokine, thought to induce a Th2 response, is related to macrophage deactivation and disease establishment in animals, whereas cytokines such as IFN-γ and IL-12 are related to protection against disease (Coelho, EAF , et al.lmmune responses induced by Leishmania (Leishmania) donovani A2 antigen, but not by LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection Infect Immun. 71: 3988-3994, 2003; Chávez-Fumagalli, MA, et al Vaccination with the Leishmania infantum ribosomal protein induces protection in BALB / c mice against Leishmania chagasi and Leishmania amazonensis challenge Microbes and Infection 12: 967-977, 2010; Nico, D., et al. immunity to Leishmania amazonensis is mediated by CD4 + and CD8 + epitopes of Leishmania donovani nucleoside hydrolase terminal domains. Frontiers in Immunology. 5: 1-10, 2014).
[014] Em estudos de avaliação de candidatos vacinais na LT, os imunógenos são administrados em camundongos, associados aos adjuvantes de resposta Th1. Após algumas semanas, os animais vacinados são experimentalmente infectados e, após alguns meses, células do baço (chamados "esplenócitos") dos animais vacinados e infectados são retiradas, cultivadas e estimuladas in vitro com os antígenos utilizados nas imunizações e com o extrato proteico dos parasitas, a fim de se verificar o perfil de resposta imune gerada por meio da dosagem de citocinas desenvolvida nesses animais. Nesse caso, IFN-γ e IL-12, como marcadores de resposta Th1 , e IL-4 e IL-10, como marcadores de resposta Th2, têm seus níveis determinados, sendo então realizada uma comparação entre tais níveis obtidos com as cargas parasitárias encontradas nos animais (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). Sendo assim, os animais ditos como protegidos são aqueles que apresentam uma baixa carga parasitária associada com níveis elevados de IFN-γ e IL-12, bem como baixos níveis de IL-4 e IL-10, secretados pelos esplenócitos cultivados. Desta forma, antígenos que são capazes de estimular o desenvolvimento de uma resposta tipo Th1 , primada pela produção de IFN- γ em esplenócitos de camundongos infectados com Leishmania, poderiam ser considerados candidatos vacinais promissores para utilização como vacinas contra as leishmanioses.  [014] In studies evaluating vaccine candidates in LT, immunogens are administered in mice associated with Th1 response adjuvants. After a few weeks, the vaccinated animals are experimentally infected, and after a few months, spleen cells (called "splenocytes") from the vaccinated and infected animals are removed, cultured and stimulated in vitro with the immunization antigens and the protein extract of the animals. parasites, in order to verify the immune response profile generated by the cytokine dosage developed in these animals. In this case, IFN-γ and IL-12, as Th1 response markers, and IL-4 and IL-10, as Th2 response markers, have their levels determined, and then a comparison between such levels obtained with parasitic loads is performed. found in animals (Martins VT, et al. Antigenicity and protective efficacy of an amastigote-specific Leishmania protein, member of the superoxygenase family, against visceral leishmaniasis. PLoS Negl Trop Dis. 7: e2148, 2013). Thus, protected animals are those with a low parasitic burden associated with high levels of IFN-γ and IL-12, as well as low levels of IL-4 and IL-10 secreted by cultured splenocytes. Thus, antigens that are capable of stimulating the development of a Th1-type response, primared by IFN-γ production in splenocytes of Leishmania-infected mice, could be considered promising vaccine candidates for use as leishmaniasis vaccines.
[015] Os antígenos atualmente testados como vacinas contra as leishmanioses são usualmente proteínas completas, as quais contêm regiões comuns às proteínas de outros patógenos, o que pode gerar reações cruzadas nos animais vacinados. Também, tais proteínas podem conter epitopos (peptídeos) que apresentam efeito benéfico, mas outros que apresentam efeito nocivo nos animais imunizados, comprometendo, dessa forma, a eficácia da vacina. O uso de pequenos antígenos, sob a forma de peptídeos, tende a permitir o desenvolvimento de uma vacina mais específica, de modo que o cão ou homem imunizado possam desenvolver uma resposta imune benéfica frente apenas a tal antígeno, específica a ele e à doença para a qual o mesmo foi selecionado. Tais antígenos apresentam também uma produção técnica mais simples, rápida, de maior rendimento e menor custo, em relação à produção de proteínas recombinantes; além de terem boa estabilidade após sua produção e, dessa forma, poderem ser disponibilizados mais facilmente para a população. [015] Antigens currently tested as vaccines against leishmaniasis are usually complete proteins, which contain regions common to proteins of other pathogens, which can lead to cross reactions in vaccinated animals. Also, such proteins may contain epitopes (peptides) that have a beneficial effect, but others that have a harmful effect on immunized animals, thereby compromising the efficacy of the vaccine. The use of small antigens in the form of peptides tends to allow the development of a more specific vaccine so that the immunized dog or man can develop a beneficial immune response to only that antigen specific to him and the disease for which he is immunized. which it has been selected. Such antigens also have a simpler, faster, higher yield and lower cost technical production compared to recombinant protein production; They also have good stability after production and thus can be made more easily available to the population.
[016] A tecnologia de phage display é baseada na expressão de peptídeos, proteínas e/ou fragmentos de anticorpos associados a proteínas presentes na superfície externa de bacteriófagos recombinantes. A sequência de nucleotídeos codificadora dos peptídeos inseridos é geneticamente fundida a uma sequência codificadora de algumas proteínas do bacteriófago, originando um produto híbrido, que é então exposto na superfície externa das partículas virais (Smith G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science.228: 1315-17, 1985).  [016] Phage display technology is based on the expression of protein-associated peptides, proteins and / or antibody fragments present on the outer surface of recombinant bacteriophages. The nucleotide sequence encoding the inserted peptides is genetically fused to a sequence encoding some bacteriophage proteins, resulting in a hybrid product, which is then exposed on the outer surface of viral particles (Smith GP Filamentous fusion phage: novel expression vectors que display cloned antigens on the virion surface (Science, 288: 1315-17, 1985).
[017] Bibliotecas de bacteriófagos expressando peptídeos exógenos têm sido usadas na identificação de diversos receptores celulares, facilitando a identificação de pequenas moléculas que se ligam com alta afinidade e mimetizam a interação com seus ligantes naturais; bem como na identificação de peptídeos que interagem com anticorpos, sem a necessidade do conhecimento prévio da região antigênica reconhecida pelo anticorpo. A seleção de moléculas de alta afinidade em relação a um determinado receptor-alvo, aderido a uma superfície sólida, é realizada por etapas consecutivas de seleção denominadas de "ciclos de bio-pannings". O número de ciclos de bio-pannings realizados em phage display determina o grau de enriquecimento dos fagos que se ligam ao receptor-alvo. Uma população de clones de fagos com elevada afinidade em relação a um dado receptor pode ser obtida com a realização de 3 a 5 ciclos de bio- pannings (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). Metodologicamente, os ciclos de bio-pannings podem ser adaptados de acordo com o grupo de pesquisa que domina tal tecnologia. Exemplificamente, em nosso caso, moléculas de Imunoglobulinas (IgGs) provenientes de soros de pacientes a serem investigados foram fundidas à superfície de microesferas (denominadas de "beads") e tal híbrido foi submetido a processos de captação (seleção) negativa e positiva em relação aos clones de fagos presentes na biblioteca recombinante utilizada. [017] Bacteriophage libraries expressing exogenous peptides have been used to identify various cell receptors, facilitating the identification of small molecules that bind with high affinity and mimic interaction with their natural ligands; as well as in the identification of peptides that interact with antibodies, without prior knowledge of the antigenic region recognized by the antibody. Selection of high affinity molecules with respect to a particular target receptor, adhered to a solid surface, is performed by consecutive selection steps called "bio-panning cycles". The number of phage display bio-panning cycles determines the degree of enrichment of the phages that bind to the target receptor. A population of high affinity phage clones with respect to 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). Methodologically, bio-panning cycles can be tailored according to the research group that mastered such technology. For example, in our case, immunoglobulin molecules (IgGs) from sera from patients to be investigated were fused to the surface of microspheres (called "beads") and such a hybrid was subjected to negative and positive uptake (selection) processes. to phage clones present in the recombinant library used.
[018] A tecnologia de phage display vem sendo utilizada em diversos trabalhos para a pesquisa de novos antígenos, para que na forma de clones de bacteriófagos expressando os peptídeos de interesse, ou na forma de peptídeos individuais produzidos sinteticamente; possam ser testados como vacinas contra diversas doenças (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 lmmun.67:4764-4770,1999; Manoutcharian K., et al. Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis. Vet Immunol Immunopathol.99:1 1-24, 2004;. Noya, O., et al. Immunodiagnosis of parasitic diseases with synthetic peptides. Curr. Prot. Peptide Sei.4:299-308, 2003). [019] O documento de patente PI09030891 , intitulado "Peptídeos recombinantes e motivos proteicos miméticos a antígenos de Leishmania e suas aplicações", trata da caracterização e utilização de peptídeos recombinantes, suas sequencias reversas e seus motivos proteicos no tratamento das leishmanioses. A tecnologia se difere do presente pedido devido à estratégia experimental utilizada ser diferente e não contemplar o uso de células humanas nem a dosagem de citocinas humanas. Também, os peptídeos expressos nos clones selecionados são diferentes dos descritos no presente pedido, uma vez que foram usados como peptídeos sintéticos, enquanto na presente tecnologia foram usados os próprios clones de bacteriófagos expressando os peptídeos de interesse, ou seja, como sendo uma composição imunogênica já pronta. [018] The technology of phage display has been used in several works for the research of new antigens, so that in the form of bacteriophage clones expressing the peptides of interest, or in the form of synthetically produced individual peptides; 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 lmmun.67: 4764- 4770,1999; Manoutcharian K., et al., Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis. Vet Immunol Immunopathol.99: 1 1-24, 2004; Noya, O., et al., Immunodiagnosis of parasitic diseases with synthetic peptides, Curr. Prot. Peptide Sci.4: 299-308, 2003). [019] Patent document PI09030891 entitled "Recombinant peptides and Leishmania antigen mimetic protein motifs and their applications" deals with the characterization and use of recombinant peptides, their reverse sequences and their protein motifs in the treatment of leishmaniasis. The technology differs from the present application because the experimental strategy used is different and does not contemplate the use of human cells or the dosage of human cytokines. Also, the peptides expressed in the selected clones are different from those described in the present application, since they were used as synthetic peptides, whereas in the present technology the bacteriophage clones themselves expressing the peptides of interest were used, that is, as being an immunogenic composition. already done.
[020] O documento de patente BR102012033552, intitulado "Peptídeos poliméricos, processo de obtenção e uso para imunodiagnóstico de leishmaniose", trata da obtenção de peptídeos poliméricos através da técnica de phage display para imunodiagnóstico da leishmaniose canina e humana. A tecnologia se difere da contida no presente pedido por compreender tipos diferentes de sequencias de peptídeos e pelo método de obtenção dos mesmos ser diferente. Além do mais, os peptídeos poliméricos contidos no citado documento são utilizados para o diagnóstico, e não como composição vacinai contra as leishmanioses. [020] Patent document BR102012033552, entitled "Polymeric Peptides, Process for Obtaining and Use for Leishmaniasis Immunodiagnosis", deals with the production of polymeric peptides by the phage display technique for canine and human leishmaniasis immunodiagnosis. The technology differs from that contained in the present application in that it comprises different types of peptide sequences and the method of obtaining them is different. In addition, the polymeric peptides contained in said document are used for diagnosis, and not as a vaccine composition against leishmaniasis.
[021 ] O documento de patente BR1020130275425, intitulado "Composição vacinai contra leishmaniose visceral canina, peptídeos sintéticos e uso", trata de uma composição vacinai para prevenção e/ou tratamento de leishmaniose visceral canina compreendida por peptídeos expressos em bacteriófagos. A referida tecnologia se diferencia do presente pedido por compreender sequências diferentes e por se tratar apenas de doença canina, além da não contemplar o uso de células humanas nem a dosagem de citocinas humanas. [022] A presente tecnologia trata de uma composição vacinai compreendendo pelo menos um dos de nove peptídeos selecionados por meio de uma abordagem proteômica de phage display, como sendo capazes de induzir uma produção elevada de IFN-γ e baixos níveis de IL-4 tanto em esplenócitos de camundongos cronicamente infectados com L. amazonensis quanto em células mononucleares do sangue periférico (PBMCs) de indivíduos sadios que foram estimulados com os clones individuais demonstrando, dessa forma, a capacidade de tais imunógenos em induzir o desenvolvimento de uma resposta imune relacionada à proteção contra a leishmaniose tegumentar (formas cutânea, cutâneo- difusa e mucosa da doença), no caso, de uma resposta imune Th1 , e em dois modelos distintos de mamíferos: camundongo e homem. [021] Patent document BR1020130275425, entitled "Canine Visceral Leishmaniasis Vaccine Composition, Synthetic Peptides and Use", is a vaccine composition for the prevention and / or treatment of canine visceral leishmaniasis comprised of peptides expressed in bacteriophages. Said technology differs from the present application in that it comprises different sequences and canine disease alone, and does not contemplate the use of human cells or the dosage of human cytokines. [022] The present technology deals with a vaccine composition comprising at least one of nine peptides selected by a phage display proteomic approach as capable of inducing high IFN-γ production and low levels of IL-4. splenocytes of mice chronically infected with L. amazonensis and peripheral blood mononuclear cells (PBMCs) from healthy individuals that were stimulated with individual clones, thereby demonstrating the ability of such immunogens to induce the development of an immune-related immune response. protection against cutaneous leishmaniasis (cutaneous, diffuse and mucosal forms of the disease), in the case of a Th1 immune response, and in two distinct mammalian models: mouse and man.
[023] No estado da técnica não foi encontrada tecnologia similar utilizando os peptídeos contidos no presente pedido. [023] In the prior art no similar technology has been found using the peptides contained in the present application.
DESCRIÇÃO DETALHADA DA TECNOLOGIA DETAILED DESCRIPTION OF TECHNOLOGY
[024] A presente tecnologia trata de composições vacinais compreendendo pelo menos um dos nove peptídeos definidos pelas SEQ ID No 1 a 9, capazes de estimular o desenvolvimento de uma resposta imune celular do tipo T helper 1 (Th1 ) com elevada produção de Interferon-gamma (IFN-γ) e baixos níveis de lnterleucina-4 (IL-4), para prevenção da leishmaniose tegumentar em mamíferos.  [024] The present technology deals with vaccine compositions comprising at least one of the nine peptides defined by SEQ ID NOs 1 to 9, capable of stimulating the development of a T helper 1 (Th1) -type cellular immune response with high production of gamma (IFN-γ) and low levels of interleukin-4 (IL-4) for the prevention of cutaneous leishmaniasis in mammals.
[025] A, a presente tecnologia compreende nove peptídeos definidos pelas SEQ ID N°1 a 9 e/ou clones de bacteriófagos selecionados através de phage display que expressam tais peptídeos, e adjuvantes farmacêutica e farmacologicamente aceitáveis, e seu uso.no tratamento e/ou prevenção da leishmaniose tegumentar em mamíferos.  A, the present technology comprises nine peptides defined by SEQ ID NOs. 1 to 9 and / or phage display selected bacteriophage clones expressing such peptides, and pharmaceutically and pharmacologically acceptable adjuvants, and their use in the treatment and / or prevention of cutaneous leishmaniasis in mammals.
[026] Os adjuvantes farmacêutica e farmacologicamente aceitáveis, de acordo com a presente invenção podem ser selecionados dentre moléculas ou produtos que sejam capazes de estimular ao maior desenvolvimento de uma resposta imune celular do tipo Th1 , que seja primada pela produção das citocinas IFN-γ e IL-12. Pharmaceutically and pharmacologically acceptable adjuvants according to the present invention may be selected from molecules or products which are capable of stimulating the further development of a Th1-type cellular immune response that is dominated by the production of IFN-γ and IL-12 cytokines.
[027] A composição vacinai proposta pode ser administrada pelas vias intradérmica, intramuscular, oral, nasal, intravenosa, subcutânea e/ou como dispositivo que possa ser injetado.  [027] The proposed vaccine composition may be administered by the intradermal, intramuscular, oral, nasal, intravenous, subcutaneous and / or injectable device.
[028] A presente invenção pode ser mais bem compreendida através dos seguintes exemplos, não limitantes da tecnologia.  The present invention may be better understood by the following non-limiting examples of the technology.
EXEMPLO 1. OBTENÇÃO DOS CLONES DE BACTERIÓFAGOS EXPRESSANDO AS PROTEÍNAS DE INTERESSE.  EXAMPLE 1. OBTAINING BACTERIOPHAGE CLONES EXPRESSING PROTEIN OF INTEREST.
[029] Amostras de soros. As seguintes amostras de soro foram utilizadas para a realização dos ciclos de bio-pannings na técnica de phage display: soros de indivíduos sadios residentes em área endémica da doença (n=20), soros de pacientes com leishmaniose cutânea (LC, n=20) ou mucosa (LM, n=20) e soros de pacientes com Doença de Chagas (DC, n=20). O projeto foi aprovado pelo COEP/UFMG (protocolo CAAE-323431.14.9.0000.5149) e pools de soros foram preparados usando quantidades iguais de cada amostra, para sua utilização nos ciclos de bio-pannings. Serum samples. The following serum samples were used for phage display bio-panning cycles: sera from healthy individuals living in an endemic area of the disease (n = 20), sera from patients with cutaneous leishmaniasis (LC, n = 20 ) or mucosa (ML, n = 20) and sera from patients with Chagas Disease (CD, n = 20). The project was approved by COEP / UFMG (protocol CAAE-323431.14.9.0000.5149) and serum pools were prepared using equal amounts of each sample for use in the bio-panning cycles.
[030] Purificação de anticorpos IgGs. A purificação das imunoglobulinas da classe IgG a partir dos pools de soros listados acima foi realizada por meio de seu acoplamento em microesferas magnéticas (beads magnéticos) conjugadas à proteína G (Dynabeads, Invitrogen). Para tal, 2x109 partículas de microesferas foram lavadas por 3 vezes em tampão MES 0.1 M pH 5.0 e foi adicionado às mesmas: (a) 300 do pool de soros de pacientes com LM, para um volume final de 500 μί; (b) 300 do pool de soros de pacientes com LC, para um volume final de 500 μί; (c) 300 μί do pool de soros de pacientes com DC, para um volume final de 500 μί e (d) 300 μί do pool de soros de indivíduos sadios, para um volume final de 500 μί. A proporção de anticorpos para a quantidade das microesferas em todos os casos foi de 1 :1. Em seguida, foi realizada uma incubação por 40 minutos sob agitação constante e à temperatura ambiente (T.A). As microesferas adsorvidas com os anticorpos foram lavadas por 3 vezes em tampão MES 0.1 M pH 5.0, com a finalidade de retirar os anticorpos IgGs não-aderidos. Para a finalização da ligação covalente entre as microesferas e os anticorpos, o sistema "beads-anticorpo" foi lavado 2 vezes com 1 mL de tampão trietanolamina 0.2M pH 8.2 e ressuspendido em 1 mL de tampão de ligação covalente (20 mM de dimetilpimelinidato/HCI em tampão trietanolamina) por 30 minutos, sob agitação constante e à T.A. A neutralização da reação foi feita pela incubação do sistema com 1 mL de tampão Tris 50 mM pH 7.5, por 15 minutos e à T.A. Em seguida, as microesferas incorporadas foram lavadas por 3 vezes em tampão TBS-T 0.1 % de Tween 20 e bloqueadas com solução de bloqueio (5% de BSA em TBS-T 0.05% de Tween 20) por 1 hora e a 37°C, sendo então ressuspendidas em 200 de tampão TBS. Para certificação do acoplamento, 5 dos beads cobertos pelas IgGs foram incubados por 1 hora e a 37°C com o anticorpo anti-lgG de humano (diluição de 1 :5.000). Após a incubação, os beads foram lavados por 3 vezes com TBS-Tween 0.1 % e a reação foi revelada pela adição do substrato tetrametilbenzidina. A reação foi então interrompida pela adição de ácido sulfúrico 2 N e a leitura da absorbância foi efetuada a 450 nm, em leitor de microplacas (Titertek Multiskan Plus, Flow Laboratories, USA). [030] Purification of IgG antibodies. Purification of IgG-class immunoglobulins from the sera pools listed above was performed by their coupling to G protein-conjugated magnetic beads (Dynabeads, Invitrogen). To this end, 2x10 9 microsphere particles were washed 3 times in 0.1 M MES pH 5.0 buffer and added to them: (a) 300 µl of the LM patient sera pool to a final volume of 500 μί; (b) 300 from the LC patient sera pool for a final volume of 500 μί; (c) 300 μί of the CD patient sera pool for a final volume of 500 μί and (d) 300 μία of the healthy patient serum pool for a final volume of 500 μί. The ratio of antibodies to the amount of microspheres in all cases was 1: 1. Then, an incubation was performed for 40 minutes. under constant stirring and at room temperature (RT). Antibody adsorbed microspheres were washed 3 times in 0.1 M MES pH 5.0 buffer to remove unbound IgG antibodies. For completion of covalent binding between microspheres and antibodies, the beads-antibody system was washed 2 times with 1 mL of 0.2 M triethanolamine buffer pH 8.2 and resuspended in 1 mL of covalent binding buffer (20 mM dimethylpimelinidate / HCI in triethanolamine buffer) for 30 minutes under constant stirring and at RT The reaction was neutralized by incubating the system with 1 ml 50 mM Tris buffer pH 7.5 for 15 minutes and at RT Then the incorporated microspheres were washed. 3 times in 0.1% Tween 20 TBS-T buffer and blocked with blocking solution (5% BSA in 0.05% Tween 20 TBS-T) for 1 hour at 37 ° C, then resuspended in 200 µl buffer TBS For coupling certification, 5 of the IgG-coated beads were incubated for 1 hour at 37 ° C with the human anti-IgG antibody (1: 5,000 dilution). After incubation, the beads were washed 3 times with 0.1% TBS-Tween and the reaction was revealed by the addition of the tetramethylbenzidine substrate. The reaction was then stopped by the addition of 2 N sulfuric acid and the absorbance reading was taken at 450 nm in a microplate reader (Titertek Multiskan Plus, Flow Laboratories, USA).
[031 ] Ciclos de bio-pannings: phage display. Para a realização dos ciclos de seleção negativa e positiva nos bio-pannings, 1x1012 partículas virais de uma biblioteca recombinante contendo peptídeos fusionados em fagos filamentosos (biblioteca comercial Ph.D.-C7C obtida da empresa New England, BioLabs®, USA) foram utilizadas. As mesmas foram diluídas em 290 de TBS-Tween 0.1 % para a seleção dos ligantes nas microesferas incorporadas pelos anticorpos IgGs. No 1 o ciclo de bio-panning, iniciou-se com a seleção negativa. Para tal, a seleção foi realizada com 150 das microesferas de proteína G acopladas com as IgGs purificadas, dos diferentes grupos de soros. Para a seleção negativa, a biblioteca de bacteriófagos recombinantes foi incubada por 30 minutos e a T.A. com as microesferas acopladas com as IgGs de indivíduos sadios e, em seguida, as microesferas foram precipitadas por atração magnética ao suporte Dynal Biotech (no. 12020), sendo o sobrenadante, contendo os clones que não se aderiram às IgGs; transferido para novo tubo contendo as microesferas acopladas com as IgGs do grupo de pacientes com Doença de Chagas. Apenas os clones de fagos que não se aderiram a tais anticorpos foram recuperados. Para a seleção positiva, o sobrenadante coletado e recuperado durante a seleção negativa foi transferido para um novo tubo contendo as microesferas acopladas com as IgGs de pacientes com LM, sendo incubadas por 30 minutos e à T.A. As microesferas contendo o sistema beads-anticorpos precipitado nos tubos dos bio-panning dos anticorpos de pacientes com (LTM), mais os fagos de interesse aderidos a tais IgGs, foram lavadas por 10 vezes com TBS-Tween 20 0.1 % e eluídos em 500 μΙ_ de tampão glicina 0.2 M pH 2.0, sendo tal solução neutralizada pela adição de 75 μΙ_ de Tris-base pH 9.0. Os clones de fagos foram recuperados para realizar a segunda etapa do processo de seleção positiva. Estes clones recuperados e eluídos foram passados para um novo tubo contendo as microesferas acopladas com as IgGs de pacientes com LC. As microesferas contendo o sistema beads-anticorpos precipitado nos tubos dos bio-panning dos anticorpos de pacientes com LC, mais os fagos de interesse aderidos a tais IgGs, foram lavadas por 10 vezes com TBS- Tween 20 0.1 % e eluídos em 500 de tampão glicina 0.2 M pH 2.0, sendo tal solução neutralizada pela adição de 75 de Tris-base pH 9.0. O processo foi repetido mais duas vezes, totalizando 3 ciclos de bio seleção. Após tal procedimento, os clones foram titulados, individualizados. [032] Titulação e amplificação dos clones selecionados. Ao fim do 3o ciclo de seleção positiva, os clones de fagos recuperados foram titulados. Para cada titulação, uma placa de cultura foi confeccionada. As placas foram incubadas à 37°C por 16 horas. Após, 96 colónias azuis foram individualizadas, coletadas manualmente para a extração do DNA e posterior sequenciamento. A reação de sequenciamento foi realizada utilizando o DNA extraído (500 ng do DNA de cada fago) dos clones com 5 pmol do Primer 96 glll (5'-OH CCC TCA TAG TTA GCG TAA CG-3'- Biolabs) e o pré-mix (DYEnamic ET Dye Terminator Cycle Kit-Amersham Biosciences). A dedução in silico das sequências de aminoácidos, para a identificação dos peptídeos exógenos, foi realizada pelo programa DNA2PR012, que é designado para a dedução de sequências de insertos das bibliotecas da New England Biolabs (Ph.D.-12™ ou Ph.D.-C7C™), como de outras bibliotecas de interesse que contenham as sequências inicial e final do vetor. [031] Bio-panning cycles: phage display. To perform the negative and positive selection cycles on bio-pannings, 1x10 12 viral particles from a recombinant library containing filamentous phage fused peptides (Ph.D.-C7C commercial library from New England, BioLabs ® , USA) were used. They were diluted in 290 µl of 0.1% TBS-Tween for ligand selection in the microspheres incorporated by IgG antibodies. In the 1st bio-panning cycle, it started with negative selection. For this, the selection was made with 150 of the G protein microspheres coupled with purified IgGs from different groups of sera. For negative selection, the recombinant bacteriophage library was incubated for 30 minutes and RT with the IgG-coupled microspheres of healthy individuals and then the microspheres were precipitated by magnetic attraction to the Dynal Biotech support (No. 12020), the supernatant containing clones not adhering to IgGs; transferred to a new tube containing the IgG-coupled microspheres of the Chagas Disease patient group. Only phage clones that did not adhere to such antibodies were recovered. For positive selection, the supernatant collected and recovered during negative selection was transferred to a new tube containing the IgG-coupled microspheres of patients with ML, and incubated for 30 minutes at RT. The patient antibody bio-panning (LTM) tubes, plus the phages of interest adhered to such IgGs, were washed 10 times with 0.1% TBS-Tween 20 and eluted in 500 μΙ_ 0.2 M glycine buffer pH 2.0. such a solution is neutralized by the addition of 75 μΙ_ Tris-base pH 9.0. Phage clones were recovered to perform the second step of the positive selection process. These recovered and eluted clones were passed into a new tube containing the IgG-coupled microspheres of LC patients. The microspheres containing the beads-antibody system precipitated in the antibody patient bio-panning tubes plus the phages of interest adhered to such IgGs were washed 10 times with 0.1% TBS-Tween 20 and eluted in 500 µl buffer. 0.2 M glycine pH 2.0, such solution being neutralized by the addition of 75 µl Tris-base pH 9.0. The process was repeated twice more, totaling 3 cycles of bioselection. After such procedure, the clones were titrated, individualized. [032] Titration and amplification of selected clones. At the end of the third cycle of positive selection, clones of recovered phages were titrated. For each titration, a culture plate was made. The plates were incubated at 37 ° C for 16 hours. Then, 96 blue colonies were individualized, manually collected for DNA extraction and subsequent sequencing. The sequencing reaction was performed using the extracted DNA (500 ng of DNA from each phage) from the 5 pmol clones of Primer 96 glll (5'-OH CCC TCA TAG TTA GCG TAA CG-3'-Biolabs) and mix (DYEnamic ET Dye Terminator Cycle Kit-Amersham Biosciences). In silico deduction of amino acid sequences for identification of exogenous peptides was performed by the DNA2PR012 program, which is designed for deduction of insert sequences from New England Biolabs libraries (Ph.D.-12 ™ or Ph.D .-C7C ™), as from other libraries of interest that contain the start and end sequences of the vector.
EXEMPLO 2. AVALIAÇÃO DA INFECÇÃO EM ANIMAIS  EXAMPLE 2. EVALUATION OF INFECTION IN ANIMALS
[033] Camundongos BALB/c fêmeas, de 8 semanas de idade, foram utilizados. Os animais foram adquiridos junto ao Biotério do ICB/UFMG e mantidos no Biotério do Departamento de Patologia Clinica do COLTEC, sob as condições apropriadas de manejo. O projeto foi também aprovado pelo Comité de Ética em Experimentação animal da UFMG (protocolo 043/201 1 ). A amostra IFLA/BR/1967/PH-8 de L. amazonensis foi utilizada. Os parasitas foram cultivados em meio Schneider's completo, o qual foi constituído pelo meio Scheneider's (SIGMA) acrescido com 20% de soro fetal bovino (SIGMA) inativado, 20 mM de L-glutamina, 50 μg/mL de gentamicina, 200 U/mL e 100 μg/mL de estreptomicina em pH 7,4 e a 24°C.  8-week-old female BALB / c mice were used. The animals were acquired from the ICB / UFMG Vivarium and kept in the Vibrary of the Department of Clinical Pathology of COLTEC, under appropriate management conditions. The project was also approved by the UFMG Animal Experimentation Ethics Committee (protocol 043/201 1). The IFLA / BR / 1967 / PH-8 sample from L. amazonensis was used. The parasites were grown in complete Schneider's medium, which consisted of Scheneider's medium (SIGMA) plus 20% inactivated fetal bovine serum (SIGMA), 20 mM L-glutamine, 50 μg / mL gentamicin, 200 U / mL and 100 µg / mL streptomycin at pH 7.4 and 24 ° C.
[034] A infecção nos animais (n=8) foi realizada com 1 x 106 formas promastigotas em fase estacionaria de crescimento de L. amazonensis por via subcutânea e no coxim plantar esquerdo. Dez semanas após, a carga parasitária foi avaliada pela técnica da diluição limitante na pata infectada, baço, fígado e linfonodos drenantes da pata infectada dos animais infectados (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). Para tal, os mesmos foram retirados após a eutanásia dos animais, pesados e macerados em meio de Schneider's completo. O macerado foi submetido a uma diluição seriada em placas de 96 poços, quando as mesmas foram incubadas durante 7 dias e a 24°C, sendo que a maior diluição na qual parasitas viáveis foram visualizados utilizando um microscópio trinocular invertido. [034] Infection in animals (n = 8) was performed with 1 x 10 6 promastigote stationary growth stages of L. amazonensis subcutaneously and on the left plantar cushion. Ten weeks later, the charge The parasitic parasite was evaluated by the technique of limiting dilution in the infected paw, spleen, liver and draining lymph nodes of the infected paw of infected animals (Rabbit 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). For this, they were removed after euthanasia of the animals, weighed and macerated in complete Schneider ' s medium. The macerate was serially diluted in 96-well plates when incubated for 7 days at 24 ° C, with the highest dilution at which viable parasites were viewed using an inverted trinocular microscope.
[035] Após as dez semanas, os animais infectados foram eutanasiados para a coleta de tecidos e órgãos para a avaliação parasitológica. Na análise dos resultados, observou-se que tanto a pata quanto os órgãos dos animais apresentavam elevada carga de parasitas (representada pelo log de parasitas), conforme mostrado na Tabela 1. Dessa forma, constatou-se que os animais estavam cronicamente infectados por L. amazonensis. Os resultados correspondem à média ± desvio-padrão dos grupos, em dois experimentos realizados e que apresentaram resultados similares.  [035] After ten weeks, infected animals were euthanized for tissue and organ collection for parasitological evaluation. In the analysis of the results, it was observed that both the paw and the organs had high parasite load (represented by the log of parasites), as shown in Table 1. Thus, it was found that the animals were chronically infected with L amazonensis. The results correspond to the mean ± standard deviation of the groups, in two experiments performed and that presented similar results.
Tabela 1. Carga parasitária em camundongos BALB/c infectados com L. amazonensis. Table 1. Parasitic loading in L. amazonensis infected BALB / c mice.
Figure imgf000018_0001
[036] Dessa forma, comprovando-se a infecção sistémica pelo parasita, o baço dos animais foi coletado e utilizado para a cultura e estimulação dos esplenócitos com os fagos individuais selecionados após o 3o ciclo de seleção positiva, e que tiveram suas sequências válidas determinadas; com vistas a permitir a identificação daqueles mais imunogênicos e com suas identidades relevadas. Para tal, camundongos foram eutanasiados e tiveram seu baço retirado, sendo o mesmo macerado em meio DMEM (Sigma) completo, o qual foi composto por 4.5 g/L de glicose, 20 μg/mL de sulfato de gentamicina, 100 U/mL de penicilina e 50 μg/mL de estreptomicina, pH 7.4. O macerado foi centrifugado a 1.200 x g por 10 minutos e o sobrenadante foi descartado. As hemácias foram lisadas com 3 ml_ de tampão de lise (17 mM Tris-HCI pH 7.4 e 144 mM cloreto de amónio) por 4 minutos, quando foi acrescentado o meio DMEM, para um volume final de 10 ml_. O material foi centrifugado a 1.200 x g por 10 minutos e, posteriormente, o sobrenadante foi descartado. O pellet foi ressuspendido em 1 ml_ de DMEM completo, o qual foi acrescentado com 20% de soro fetal bovino inativado.
Figure imgf000018_0001
[036] Thus, confirming the systemic parasite infection, the spleen of the animals was collected and used for cultivation and stimulation of the splenocytes with the selected individual phages after the 3rd positive selection cycle, and had their valid sequences determined; in order to allow the identification of those most immunogenic and their identities To this end, mice were euthanized and had their spleen removed and macerated in complete DMEM (Sigma) medium, which was composed of 4.5 g / L glucose, 20 μg / mL gentamicin sulfate, 100 U / mL penicillin and 50 μg / mL streptomycin, pH 7.4. The macerate was centrifuged at 1,200 xg for 10 minutes and the supernatant discarded. Red blood cells were lysed with 3 ml lysis buffer (17 mM Tris-HCl pH 7.4 and 144 mM ammonium chloride) for 4 minutes, when DMEM medium was added to a final volume of 10 ml. The material was centrifuged at 1,200 xg for 10 minutes and then the supernatant was discarded. The pellet was resuspended in 1 ml complete DMEM, which was added with 20% inactivated fetal bovine serum.
[037] Os esplenócitos foram quantificados em câmara de Newbauer e 1 x 106 células por ml_ foram plaqueadas em placas de 24 poços (Nunc), em meio DMEM completo. Os esplenócitos foram estimulados com os clones de fagos individuais (1 x 1010 fagos), com o fago selvagem (1 x 1010) como controle ou com o extrato proteico solúvel dos parasitas (SLA L. amazonensis, 50 μg), ou incubadas sem estímulo. A incubação ocorreu a 37°C em estufa com 5% de CO2 e por 48 horas. Após, o sobrenadante das culturas foi coletado e a dosagem das citocinas IFN-γ e IL-4 foi realizada por ELISA de captura utilizando kits comerciais (BD OptEIA TM set mouse IFN-y e IL-4, Pharmingen, San Diego, CA, USA). Splenocytes were quantified in Newbauer chamber and 1 x 10 6 cells per ml were plated in 24-well plates (Nunc) in complete DMEM medium. Splenocytes were stimulated either with individual phage clones (1 x 10 10 phages), with wild phage (1 x 10 10 ) as a control or with the parasite soluble protein extract (SLA L. amazonensis, 50 μg), or incubated. without stimulus. Incubation occurred at 37 ° C in a 5% CO 2 oven and for 48 hours. Subsequently, the culture supernatant was collected and IFN-γ and IL-4 cytokines were assayed by capture ELISA using commercial kits (BD OptEIA ™ IFN-γ and IL-4 mouse, Pharmingen, San Diego, CA, USA).
[038] Dos 96 clones isolados após o 3o ciclo de seleção positiva, 35 tiveram suas sequências identificadas e foram utilizados para a estimulação dos esplenócitos dos camundongos infectados com L. amazonensis e posterior dosagem das citocinas IFN-γ e IL-4. Os resultados são mostrados na tabela 2. [038] Of the 96 clones isolated after the third cycle of positive selection, 35 have their sequences identified and were used for stimulation of splenocytes of mice infected with L. amazonensis and subsequent dosage of cytokines IFN-γ and IL-4. The results are shown in table 2.
[039] Os valores apresentados são representativos de dois experimentos realizados de maneira independente e que apresentaram resultados similares. Um clone de fago selvagem, similar ao fago usado na técnica, mas que não expressa peptídeo exógeno e o SLA L. amazonensis foram utilizados como controles. Com os valores das concentrações das citocinas, a razão entre os níveis de IFN-γ e IL-4 foram calculados, a fim de selecionarmos apenas aqueles fagos que tenham sido capazes de induzir à produção dos maiores níveis de IFN-γ e dos menores níveis de IL-4.  [039] The values shown are representative of two independently performed experiments that yielded similar results. A wild phage clone, similar to the phage used in the art but not expressing exogenous peptide and SLA L. amazonensis were used as controls. With cytokine concentration values, the ratio between IFN-γ and IL-4 levels was calculated in order to select only those phages that were able to induce the production of the highest IFN-γ levels and the lowest levels. of IL-4.
Tabela 2. Dosagem das citocinas IFN-γ e IL-4 após estímulo in vitro dos esplenócitos de camundongos BALB/c infectados com L. amazonensis e cálculo da razão entre os valores. Table 2. Dosage of IFN-γ and IL-4 cytokines after in vitro stimulation of splenocytes of L. amazonensis infected BALB / c mice and calculation of the ratio between values.
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000020_0001
Figure imgf000021_0001
[040] Na análise dos resultados, observou-se que houve uma grande variação na produção das citocinas entre os clones avaliados. Os controles (fago selvagem e SLA L. amazonensis) apresentaram razão entre nos níveis de IFN-γ e IL-4 de 0,5 e 0,4, respectivamente. Aqueles clones cuja produção de IFN-γ foi, pelo menos, 2,0 vezes maior que a produção de IL-4 foram selecionados, haja vista que, após o estímulo de esplenócitos de camundongos infectados com L. amazonensis, os mesmos induziram a uma maior produção de citocinas do tipo Th1 , em relação à produção das citocinas Th2. Desta forma, 25 clones (A4, A8, A9, A10, A12, B2, B3, B4, B7, B8, B10, C1 , C6, C8, C12, E3, E4, E5, E8, E9, G1 , G2, G4, H1 e H4) foram selecionados para serem usados na estimulação in vitro de PBMCs derivados de indivíduos sadios. [040] In the analysis of the results, it was observed that there was a large variation in cytokine production among the evaluated clones. Controls (wild phage and SLA L. amazonensis) showed a ratio between IFN-γ and IL-4 levels of 0.5 and 0.4, respectively. Those clones whose IFN-γ production was at least 2.0 times higher than IL-4 production were selected, since, after stimulation of splenocytes from L. amazonensis-infected mice, they induced a higher production of Th1 cytokines than Th2 cytokines. Thus, 25 clones (A4, A8, A9, A10, A12, B2, B3, B4, B7, B8, B10, C1, C6, C8, C12, E3, E4, E5, E8, E9, G1, G2, G4, H1 and H4) were selected to be used for in vitro stimulation of PBMCs derived from healthy individuals.
EXEMPLO 3. CULTURA DE PBMCS E DOSAGEM DE CITOCINAS.  EXAMPLE 3. PBMC CULTURE AND CYTOKINE DOSAGE.
[041 ] Após a verificação da imunogenicidade dos clones citados acima como capazes de induzir à produção elevada de IFN-γ e baixos níveis de IL-4 (no mínimo, cerca de 4 vezes maior que o fago selvagem e o SLA L. amazonensis) em esplenócitos de camundongos BALB/c cronicamente infectados com essa espécie de Leishmania, tais imunógenos foram avaliados quanto à capacidade de induzir a produção de níveis elevados de IFN-g e baixos níveis de IL-4 em PBMCs de indivíduos sadios. Para tal, células mononucleares do sangue periférico foram isoladas conforme descrito (Khamesipour A, et al. Phenotyping of circulating CD8+ T cell subsets in human cutaneous leishmaniasis. Microbes Infect. 9:702-1 1 , 2012). A cultura das células foi realizada conforme descrito (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(1 1 ):e49780, 2012). Para o estímulo dos PBMCs in vitro, 1 x 106 células por poço foram incubadas em meio DMEM com 1 x 1010 de cada fago avaliado, ou com o fago selvagem (mesma concentração) ou com SLA de L. amazonensis (50 Mg/mL). A incubação foi realizada a 37°C com 5% de C02 durante 5 dias. Como controle positivo, as células foram incubadas com acetato de miristato de forbol (PMA, 25 ng/mL) e ionomicina (1 Mg/mL) em meio DMEM. As culturas foram incubadas durante 48 horas a 37°C e em 5% de C02. Após, a brefeldina A (Sigma) foi adicionada (concentração de 10 Mg/mL), durante um período de 4 horas. Após, 200 de EDTA (Sigma) foram adicionados a cada tubo de cultura (para uma concentração final de 2 mM), e as células foram lavadas com tampão FACS (PBS 1x com 0,5% de albumina de soro fetal bovino e 0, 1 % de azida sódica), ressuspensas e imunocoradas com mAb marcado com FITC anti- CD4 ou anti-CD8 (Caltag, Burlingame, CA, USA), durante 30 minutos no escuro e à temperatura ambiente. Após a coloração da membrana, os procedimentos de lise e fixação foram realizados e os PBMCs foram permeabilizadas com FACS Perm-tampão (tampão do FACS mais 0,5% de saponina), com incubação durante 30 minutos no escuro e à temperatura ambiente; na presença de 20 μΙ_ de anticorpo mAbs anti-citocina PE- marcados (IFN-γ e IL-4, Serotec®, EUA). Após a coloração das citocinas intracitoplasmáticas, PBMCs foram lavadas com tampão FACS e fixadas em solução de fixação (10 g/L de paraformaldeído, 10,2 g/L de cacodilato de sódio e 6,63 g/L de cloreto de sódio, pH 7,2), para armazenamento a 4°C antes da aquisição dos dados e análise por citometria. As medições foram realizadas em um instrumento FACScalibur© (Becton Dickson - BD, EUA) e analisadas com o software Cell-quest™ (Franklin Lakes, NJ, EUA), na base de 30.000 eventos por amostra. Os resultados são mostrados na tabela 3. A razão entre os níveis de IFN-γ e IL-4 foram calculados para cada clone utilizado e os dados são também apresentados. Following verification of the immunogenicity of the above clones as capable of inducing high IFN-γ production and low levels of IL-4 (at least about 4-fold higher than wild phage and SLA L. amazonensis) in splenocytes of BALB / c mice chronically infected with this Leishmania species, such immunogens were evaluated for their ability to induce the production of high IFN-g levels and low IL-4 levels in healthy PBMCs. For this, 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: 702-1 1, 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 ). For in vitro stimulation of PBMCs, 1 x 10 6 cells per well were incubated in DMEM medium with 1 x 10 10 of each evaluated phage, either wild phage (same concentration) or L. amazonensis SLA (50 Mg / mL). Incubation was performed at 37 ° C with 5% CO 2 for 5 days. As a positive control, cells were incubated with phorbol myristate acetate (PMA, 25 ng / mL) and ionomycin (1 Mg / mL) in DMEM medium. Cultures were incubated for 48 hours at 37 ° C and 5% CO 2 . Afterwards, brefeldin A (Sigma) was added (10 Mg / mL concentration) over a period of 4 hours. Afterwards, 200 µl 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 µl). 1% sodium azide), resuspended and immunostained with anti-CD4 or anti-CD8 FITC-labeled mAb (Caltag, Burlingame, CA, USA) for 30 minutes in the dark and at room temperature. After membrane staining, lysis and fixation procedures were performed and PBMCs were permeabilized with FACS Perm-buffer (FACS buffer plus 0.5% saponin), incubated for 30 minutes in the dark and at room temperature. environment; in the presence of 20 μΙ_ of PE-labeled anti-cytokine mAbs antibody (IFN-γ and IL-4, Serotec ® , USA). After staining of 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 prior to data acquisition and cytometric analysis. Measurements were performed on a FACScalibur © instrument (Becton Dickson - BD, USA) and analyzed with Cell-quest ™ software (Franklin Lakes, NJ, USA) on the basis of 30,000 events per sample. Results are shown in Table 3. The ratio of IFN-γ and IL-4 levels were calculated for each clone used and data are also presented.
Tabela 3. Dosagem das citocinas IFN-γ e IL-4 após o estímulo in vitro de PBMCs obtidos de indivíduos sadios e cálculo da razão entre os valores. Table 3. Dosage of IFN-γ and IL-4 cytokines after in vitro stimulation of PBMCs obtained from healthy individuals and calculation of the ratio between values.
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000023_0001
Figure imgf000024_0001
[042] Na análise dos resultados, observou-se que houve uma grande diferença na produção de IFN-γ e IL-4 entre os clones selecionados. Os controles (fago selvagem e SLA L. infantum) apresentaram razão entre nos níveis de IFN-γ e IL-4 de 0,7 e 0,5, respectivamente. Dessa forma, aqueles clones cuja produção de IFN-γ foi, pelo menos, 3 vezes maior que a produção de IL-4, colocam-se como imunogênicos nos dois modelos mamíferos avaliados (camundongo e homem) e, dessa forma, como candidatos para serem empregados como vacinas contra a LT, haja vista que, após o estímulo de esplenócitos de camundongos infectados com L. amazonensis e de PBMCs de indivíduos sadios, tais clones tenham sido capazes de induzir uma maior produção de IFN-γ e baixos níveis de IL-4. As sequências de aminoácidos dos clones selecionados são mostradas na tabela 4. [042] In the analysis of the results, it was observed that there was a large difference in IFN-γ and IL-4 production between the selected clones. Controls (wild phage and SLA L. infantum) showed a ratio between IFN-γ and IL-4 levels of 0.7 and 0.5, respectively. Thus, those clones whose IFN-γ production was at least 3 times higher than IL-4 production, are immunogenic in both mammalian models evaluated (mouse and man) and thus as candidates for TL vaccines, since after stimulation of splenocytes from L. amazonensis-infected mice and PBMCs from healthy individuals, such clones were able to induce higher IFN-γ production and low IL levels. -4. The amino acid sequences of the selected clones are shown in table 4.
Tabela 4. Sequências de aminoácidos dos clones selecionados após avaliação de sua imunogenicidade em esplenócitos de camundongos infectados e PBMCs de indivíduos sadios  Table 4. Amino acid sequences of selected clones after evaluation of their immunogenicity in splenocytes of infected mice and PBMCs from healthy individuals.
Clone Sequências de Clone Sequences of
aminoácidos  amino acids
A4 Y L L C I S P  A4 Y L L C I S P
A8 G S R C Y P R  A8 G S R C Y P R
A9 D S A F R L K  A9 D Y F F L K
A10 A S F L K N R B2 AETVESC A10 ASFLKNR B2 AETVESC
B10 R S M E I D R  B10 R S M E I D R
E3 S F I N S H G  E3 Y F Y N Y H G
E5 G Y S A L V E  E5 G Y S A L V E
E8 DRRGYGL  E8 DRRGYGL
[043] Dessa forma, tais clones imunogenos A4, A8, A9, A10, B2, B10, E3, E5 e E8 colocam-se como candidatos a vacina para serem testados na prevenção contra a LT em diferentes modelos de mamíferos. Thus, such immunogenic clones A4, A8, A9, A10, B2, B10, E3, E5 and E8 pose as vaccine candidates to be tested for LT prevention in different mammalian models.

Claims

REIVINDICAÇÕES
1 - COMPOSIÇÃO VACINAL CONTRA LEISHMANIOSE TEGUMENTAR, caracterizada por compreender pelo menos um dos antígenos proteicos definidos pelas SEQ ID N° 1 a 9 ou qualquer combinação possível entre eles e excipientes farmacologicamente e farmaceuticamente aceitáveis.VACINAL COMPOSITION AGAINST TEGUMENTARY LEISHMANIASIS, characterized in that it comprises at least one of the protein antigens defined by SEQ ID NOS. 1 to 9 or any possible combination between them and pharmaceutically and pharmaceutically acceptable excipients.
2- COMPOSIÇÃO VACINAL CONTRA LEISHMANIOSE TEGUMENTAR, de acordo com a reivindicação 1 , caracterizada pelos antígenos serem sintéticos ou expressos na superfície externa de bacteriófagos. VACINAL COMPOSITION AGAINST TEGUMENTARY LEISHMANIASIS, according to claim 1, characterized in that the antigens are synthetic or expressed on the external surface of bacteriophages.
3- COMPOSIÇÃO VACINAL CONTRA LEISHMANIOSE TEGUMENTAR, de acordo com as reivindicações 1 e 2, caracterizada por ser administrada pelas vias intradérmica, intramuscular, oral, nasal, intravenosa, subcutânea e/ou como dispositivo que possa ser injetado.  Vaccine composition against cutaneous leishmaniasis according to claims 1 and 2, characterized in that it is administered by the intradermal, intramuscular, oral, nasal, intravenous, subcutaneous and / or injectable device.
4- COMPOSIÇÃO VACINAL CONTRA LEISHMANIOSE TEGUMENTAR, de acordo com as reivindicações 1 a 3, caracterizada por compreender adjuvantes farmacêutica e farmacologicamente aceitáveis, a fim de potencializar o desenvolvimento de uma resposta imune celular do tipo Th1. Vaccine composition against cutaneous leishmaniasis according to any one of claims 1 to 3, characterized in that it comprises pharmaceutically and pharmacologically acceptable adjuvants in order to enhance the development of a Th1-type cellular immune response.
5- PEPTÍDEOS SINTÉTICOS, caracterizados por compreenderem as sequências definidas pelas SEQ ID N°1 a 9. 5. SYNTHETIC PEPTIDES, characterized in that they comprise the sequences defined by SEQ ID NOS. 1 to 9.
6- USO dos peptídeos sintéticos, de acordo com a reivindicação 5, caracterizado por ser na preparação de composições vacinais para o tratamento e/ou prevenção da leishmaniose tegumentar em mamíferos. Use of synthetic peptides according to claim 5, characterized in that they are in the preparation of vaccine compositions for the treatment and / or prevention of cutaneous leishmaniasis in mammals.
7- USO da composição vacinai, de acordo com as reivindicações 1 a 4, caracterizado por ser no tratamento e/ou tratamento da leishmaniose tegumentar em mamíferos. Use of the vaccine composition according to claims 1 to 4, characterized in that it is in the treatment and / or treatment of cutaneous leishmaniasis in mammals.
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DATABASE Protein [O] 27 February 2015 (2015-02-27), "conserved hypothetical protein [Leishmania braziliensis MHOM/BR/75/M2904]", XP055481914, Database accession no. CAM45080 *
MIGUEL A. CHAVEZ-FUMAGALLI ET AL.: "Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach", CLIN VACCINE IMMUNOL, vol. 20, no. 6, 1 June 2013 (2013-06-01), pages 835 - 841, XP055467551 *

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