WO2018037343A1 - Vaccine compositions for protecting against visceral leishmaniasis, synthetic peptides and uses - Google Patents

Vaccine compositions for protecting against visceral leishmaniasis, synthetic peptides and uses Download PDF

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WO2018037343A1
WO2018037343A1 PCT/IB2017/055064 IB2017055064W WO2018037343A1 WO 2018037343 A1 WO2018037343 A1 WO 2018037343A1 IB 2017055064 W IB2017055064 W IB 2017055064W WO 2018037343 A1 WO2018037343 A1 WO 2018037343A1
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leishmaniasis
synthetic peptides
ifn
peptides
clones
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French (fr)
Portuguese (pt)
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Eduardo Antônio FERRAZ COELHO
Carlos Alberto Pereira Tavares
Lourena EMANUELE COSTA
Beatriz Cristina SILVEIRA SALLES
Daniel MENEZES SOUZA
Mariana COSTA DUARTE
Bruno MENDES ROATT
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Universidade Federal De Minas Gerais
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    • 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
    • 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
    • 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 addresses vaccine compositions for the treatment and / or prevention of visceral leishmaniasis (VL) in mammals, comprised of six free or surface-expressed synthetic peptides of non-infectious bacteriophages, which have been selected by an approach. phage display proteomics, to be applied alone or in possible combinations between them.
  • VL visceral leishmaniasis
  • Leishmaniasis are neglected diseases caused by protozoan parasites of the genus Leishmania. It is estimated that 380 million people are at risk of contracting the disease, and an approximate incidence of 0.5 to 1, 0 and 1, 5 to 2.0 million new cases occur each year as a result of the disease. LV and cutaneous leishmaniasis (LT), respectively. In the Americas, approximately 90% of VL cases occur in Brazil, which makes this disease a major public health problem and thus requires special attention by our competent authorities (World Health Organization. Control of the leishmaniasis: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, 22-26 March 2010.
  • the severity of the disease in the mammalian host in which both man and dog fall, can range from a single skin lesion to the visceral form of the disease which, if left untreated, can be fatal.
  • the dog is the main domestic reservoir of the parasite and is regarded as an important source of transmission between the sandfly vector and the mammalian host (Grimaldi, G. and Tesh, RB Leishmaniasis of the New World: current concepts and implications for future research. Clin Microbiol Rev. 6; 230-50, 1993).
  • the diagnosis of the disease is based on the assessment of clinical manifestations of the disease in conjunction with epidemiological investigations and the detection of intracellular stages of parasites by examination of patients' internal organ aspirates, as well as other laboratory tests. Rapid diagnosis is essential to prevent death, however, for clinical diagnosis, the symptomatology of the disease is similar to other common diseases in our country, such as Chagas disease, malaria, among others.
  • the parasitological methods although presenting high specificity, have limitations in their sensitivity, due to the inhomogeneous distribution of parasites in the aspirates, besides the risks of internal hemorrhage when performing this technique, as it is invasive.
  • ELISA Enzyme-Linked Immunosorbent Analysis
  • IFAT Indirect Immunofluorescence Reaction
  • DAT Direct Agglutination Test
  • Th1 response may be inhibited by production of cytokines such as IL-10.
  • these molecules are related to macrophage deactivation and disease establishment in animals, whereas cytokines such as IFN- ⁇ are associated with the disease protection phenotype (Zanin FH, et al.
  • immunogens are administered in mice associated with Th1 immune response inducing adjuvants. After immunizations, for evaluation of vaccine immunogenicity, cytokines such as IFN- ⁇ 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 these levels obtained with the parasitic loads found in animals is performed (Martins VT, et al. Antigenicity and protective efficacy of Leishmania). amastigote-specific protein, member of the superoxygenase family, against visceral leishmaniasis (PLoS Negl Trop Dis. 7; e2148, 2013). Thus, it is sought the association that, in animals considered as protected against VL, they have low parasitic load and that their splenocytes are capable of producing high levels of IFN- ⁇ and low levels of IL-10 after in vitro stimulation with immunogens.
  • cytokines such as IFN- ⁇ and IL-12 as Th1 response markers
  • Antigens currently used as leishmaniasis vaccines are usually complete proteins, which contain regions common to proteins of other diseases, which can lead to cross reactions in vaccinated animals. Also, such proteins may contain epitopes (or peptides) that have a beneficial effect, but others that have a detrimental effect on immunized hosts, thereby compromising vaccine efficacy.
  • the use of small antigens tends to allow the development of a more specific vaccine so that the immunized dog or man can develop a beneficial immune response to only that specific antigen and the disease for which it was selected.
  • Such antigens also have a simpler technical production, higher yield and lower cost compared to the production of recombinant proteins; They also have good stability and thus can be made available under better conditions for the population.
  • Phage display technology is based on the expression of peptides, proteins or antibody fragments associated with proteins 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 the viral particles (Smith GP Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228; 1315-1317, 1 985).
  • 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 the 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 given target receptor, adhered to a solid surface, is performed by consecutive selection steps called "bio-panning cycles". The number of biohazard cycles performed on the phage display determines the degree of enrichment of the phages that bind to a 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).
  • the bio-panning cycles can be tailored according to the group that dominates such technology.
  • IgG molecules from patient sera to be investigated are fused to the surface of microspheres (called "beads") and such a hybrid is subjected to negative or positive uptake (selection) processes in relation to clones.
  • Phage display technology has been used in several works to research 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 Immun.
  • Patent document BR102013027542 entitled “Canine Visceral Leishmaniasis Vaccine Composition, Synthetic Peptides and Use", is a vaccine composition for 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 of selected peptides, the methodology adopted was also different and that such patent document is only the study of canine disease.
  • 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 against 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 identified immunogens are different from those described in the present invention.
  • Patent application PI08004854 deals with the use of recombinant viral vectors expressing the Leishmania A2 antigen as a method of vaccination against leishmaniasis.
  • the technology differs from the present application in that it contemplates antigen different from those comprised in the present application and the experimental strategy is distinct.
  • the present technology deals with six bacteriophage peptides and / or clones expressing peptides of interest which were selected by the phage display technique.
  • Such clones expressing the peptides were immunogenic in two mammalian models, mouse and man, being able to induce the development of a Th1 cellular immune response profile, since both splenocytes of chronically L. infantum-infected mice and mononuclear cells of the mouse.
  • peripheral blood (PBMCs) from healthy individuals stimulated with individual clones were able to induce production of high levels of IFN- ⁇ and low levels of IL-10, thus demonstrating the ability of such immunogens to induce the development of an immune response related to protection against VL, in this case the Th1 response.
  • PBMCs peripheral blood
  • the present technology relates to vaccine compositions for the treatment and / or prevention of visceral leishmaniasis (VL) in mammals, comprised of six free or surface-expressed synthetic peptides of non-infectious bacteriophages, which have been selected by an approach.
  • phage display proteomics to be applied alone or in possible combinations between them.
  • Such clones are immunogenic in human and mouse cells and are capable of inducing the development of a Th1 cellular immune response profile.
  • the present technology comprises the peptides defined by SEQ ID NQ 1 to 6, free or surface-expressed non-infectious bacteriophages, as well as their use in the treatment and / or prevention of visceral leishmaniasis in different mammalian species. .
  • 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 primed for the production of IFN- ⁇ cytokines. and IL-12.
  • 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.
  • EXAMPLE 1 OBTAINING BACTERIOPHAGES EXPRESSING PROTEIN OF INTEREST Serum samples.
  • the project was approved by the UFMG COEP (CAAE-323431 .14.9.0000.5149).
  • IgG class immunoglobulins from the sera pools listed above were purified by coupling them to G protein conjugated magnetic beads (Dynabeads, Invitrogen). To this end, 1 x 10 11 microspheres were washed 3 times in 0.1 M MES pH 5.0 buffer and added to them: (a) 400 ⁇ _ of the LV patient serum pool to a final volume of 700 ⁇ ; (b) 400 ⁇ from the serum pool of patients with Chagas disease for a final volume of 700 ⁇ and (c) 400 ⁇ from the serum pool of healthy individuals for a final volume of 700 ⁇ . The ratio of antibodies to the amount of microspheres in all cases was 1: 1. Thereafter, incubation was performed for 40 minutes under constant agitation 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 / HCl in triethanolamine buffer) for 30 minutes under constant stirring and at RT The reaction was neutralized by incubating the system with 1 ml of 50 mM Tris buffer pH 7.5 for 15 min and at RT.
  • the incorporated samples 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 h at 37 ° C, then resuspended at 200 ⁇ L of TBS buffer.
  • 5 ⁇ of the IgG-coated beads were incubated for 1 h 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 substrate. tetramethylbenzidine. 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).
  • the recombinant bacteriophage library was incubated for 30 minutes and at 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).
  • supernatant containing clones that did not adhere 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. This process was repeated twice more, totaling 3 cycles of negative selection. The supernatant containing phages that did not bind to IgGs from sera from healthy individuals and those with Chagas disease was collected.
  • the collected supernatant was transferred to a new tube containing the IgG-coupled microspheres of patients with symptomatic and asymptomatic VL, and incubated at RT for 30 minutes.
  • the individualized blue colored colonies present in the culture plates obtained from the 3rd Q- cycle of biopannings were collected for DNA extraction.
  • 1.2 mL of the early-log phase E. coli ER2738 cell culture (OD 6 oonm ⁇ 0.3) was distributed into each well of a deepwell plate.
  • the plate was centrifuged for 1 hour, the supernatant was removed and the pellet was resuspended in iodide buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA and 4 M Nal). The plates were shaken vigorously and then absolute ethanol was added. After a 10 minute incubation, the plates were centrifuged (2,250 xg at 4 Q C and for 10 minutes) and the supernatant discarded. The precipitate containing the DNA was washed with 500 ⁇ 70% ethanol and centrifuged again. Finally, the DNA of each clone was diluted in 20 ⁇ _ of ultrapure water and its quality was evaluated after an electrophoretic run performed on 0.8% agarose gel stained with an ethidium bromide solution.
  • Sequencing of selected clones The 96 clones from the 3 Q positive selection cycle had their DNA extracted and sequenced. The reaction was processed on the MegaBace 1000 automatic sequencer (Amersham Biosciences) and in silico deduction of DNA amino acid sequences from phage of interest for identification of exogenous peptides was performed by the DNA2PRO12 program, which is designed for deduction of insert sequences from New England Biolabs libraries (Ph. D.-12TM or Ph.D.-C7CTM), as from other libraries of interest containing the initial and final vector sequences. The program automatically finds the position of the insert, translates it and indicates possible translation errors (such as unexpected codons or errors in the next sequence).
  • Infection challenge For experimental infection in animals, 1 x 10 7 promastigote stationary growth forms of L. infantum were inoculated into the right plantar cushion of 8 animals. Mice were monitored for 10 weeks after challenge, when animals were euthanized for immunogenicity experiments and determination of parasitic load.
  • Parasitic loading was performed to certify that the animals were indeed infected by the parasite.
  • Parasite loading in the spleen, liver, bone marrow and draining lymph nodes was assessed by a limiting dilution technique.
  • the results correspond to the mean ⁇ standard deviation of the groups, in two experiments performed and that presented similar results.
  • L. infantum infected BALB / c mice were euthanized 10 weeks after challenge 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 sulphate, 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 of complete DMEM, which was added with 20% inactivated fetal bovine serum. Splenocytes were quantified in a Newbauer chamber and 1 x 10 6 cells per ml were plated in 24 well plates (Nunc) in complete DMEM medium.
  • lysis buffer 17 mM Tris-HCl pH 7.4 and 144 mM ammonium chloride
  • 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. infantum, 50 ⁇ ), or incubated. without stimulus. Incubation occurred at 37 Q C in incubator with 5% CO 2 and 48 hours. Subsequently, the culture supernatant was collected and IFN- ⁇ and IL-10 cytokines were measured by capture ELISA using commercial kits (BD OptEIA TM mouse set kits, Pharmingen, San Diego, CA, USA). Results are shown in Table 2.
  • the ratio between IFN- ⁇ and IL-10 levels was calculated for each clone evaluated, in order to select only those phages capable of inducing higher IFN- ⁇ production and lower levels of IFN- ⁇ .
  • IL-10 and the results are also shown. The values presented are representative of two independently performed experiments that presented similar results. A wild phage clone, similar to the target phage but not expressing exogenous peptide and SLA L. infantum were used as controls.
  • 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 PBMCs stimulation, 10 6 cells per well were incubated in DMEM with 10 10 of each phage evaluated, either with wild phage (same concentration) or with L.
  • infantum SLA 50 ⁇ g / 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 ⁇ g / mL) in DMEM medium. Cultures were incubated for 48 hours at 37 ° C and 5% CO 2 . Afterwards, brefeldin A (Sigma) was added (concentration of 10 ⁇ g / mL) during a period of 4 hours.
  • PMA phorbol myristate acetate
  • ionomycin 1 ⁇ g / mL
  • PBMCs were permeabilized with FACS Perm-buffer (FACS buffer plus 0.5% saponin), incubated for 30 minutes in the dark and at room temperature; in the presence of 20 ⁇ _ of PE-labeled anti-cytokine mAbs antibody (IFN- ⁇ and IL-10, Serotec ® , USA).
  • FACS Perm-buffer FACS buffer plus 0.5% saponin
  • 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. The results are shown in table 3. The ratio between IFN- ⁇ and IL-10 levels was calculated for each clone and data are also presented.
  • the immunogens B1, D11, D12, F5, F6 and H2 pose as vaccine candidates to be tested for prevention of VL in different mammalian models.

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Abstract

The present technology relates to vaccine compositions for the treatment and/or prevention of visceral leishmaniasis in mammals, comprising six synthetic peptides that are free or expressed in the surface of non-infectious bacteriophages selected by means of a proteomic phase display approach for use in isolation or in any possible combinations.

Description

"Composições vacinais para proteção contra a leishmaniose visceral, peptídeos sintéticos e usos"  "Vaccine compositions for protection against visceral leishmaniasis, synthetic peptides and uses"
[001 ] A presente tecnologia trata de composições vacinais para o tratamento e/ou a prevenção de leishmaniose visceral (LV) em mamíferos, compreendida por seis peptídeos sintéticos livres ou expressos na superfície de bacteriófagos não infecciosos, que foram selecionados por meio de uma abordagem proteômica de phage display, para serem aplicados isoladamente ou em possíveis combinações entre os mesmos. [001] The present technology addresses vaccine compositions for the treatment and / or prevention of visceral leishmaniasis (VL) in mammals, comprised of six free or surface-expressed synthetic peptides of non-infectious bacteriophages, which have been selected by an approach. phage display proteomics, to be applied alone or in possible combinations between them.
[002] As leishmanioses são doenças negligenciadas causadas por parasitas protozoários do género Leishmania. Estima-se que 380 milhões de pessoas encontram-se expostas ao risco de contrair a doença e que uma incidência aproximada de 0,5 a 1 ,0 e 1 ,5 a 2,0 milhões de novos casos ocorra a cada ano em decorrência da LV e leishmaniose tegumentar (LT), respectivamente. Nas Américas, aproximadamente 90% dos casos de LV ocorre no Brasil, o que torna essa doença um importante problema de Saúde Pública e que requer, dessa forma, uma atenção especial por nossas autoridades competentes (World Health Organization. Control of the leishmaniasis: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, 22-26 March 2010. WHO Technical Report Series. (949). http://whqlibdoc.who.int/trs/WHO_TRS_949_eng.pdf. 2010; Alvar J, et al. WHO Leishmaniasis Control Team. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 7:e35671 , 2012).  Leishmaniasis are neglected diseases caused by protozoan parasites of the genus Leishmania. It is estimated that 380 million people are at risk of contracting the disease, and an approximate incidence of 0.5 to 1, 0 and 1, 5 to 2.0 million new cases occur each year as a result of the disease. LV and cutaneous leishmaniasis (LT), respectively. In the Americas, approximately 90% of VL cases occur in Brazil, which makes this disease a major public health problem and thus requires special attention by our competent authorities (World Health Organization. Control of the leishmaniasis: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, 22-26 March 2010. WHO Technical Report Series (949) http://whqlibdoc.who.int/trs/WHO_TRS_949_eng.pdf 2010; Alvar J, et al., WHO Leishmaniasis Control Team, Leishmaniasis worldwide and global estimates of its incidence (PLoS One. 7: e35671, 2012).
[003] A gravidade da doença no hospedeiro mamífero, no qual se enquadram o homem e o cão, pode variar desde uma lesão cutânea única, até a forma visceral da doença que, se não tratada, pode ser fatal. O cão é o principal reservatório doméstico do parasita e é tido como uma importante fonte de transmissão entre o vetor flebotomíneo e o hospedeiro mamífero (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). Devido à ineficácia das medidas de controle ora utilizadas, pela dificuldade para um diagnóstico correto e também pelos problemas de toxicidade causados pelos fármacos convencionais; o desenvolvimento de vacinas que sejam capazes de induzir imunidade protetora contra a LV torna-se desejável (Tesh RB. Control of zoonotic visceral leishmaniasis: is it time to change strategies? Am J Trop Med Hyg.52;287-92, 1995). [003] The severity of the disease in the mammalian host, in which both man and dog fall, can range from a single skin lesion to the visceral form of the disease which, if left untreated, can be fatal. The dog is the main domestic reservoir of the parasite and is regarded as an important source of transmission between the sandfly vector and the mammalian host (Grimaldi, G. and Tesh, RB Leishmaniasis of the New World: current concepts and implications for future research. Clin Microbiol Rev. 6; 230-50, 1993). Due to the ineffectiveness of the control measures now used, the difficulty for a correct diagnosis and also the toxicity problems caused by conventional drugs; The Development of vaccines that are capable of inducing protective immunity against VL becomes desirable (Tesh RB. Control of zoonotic visceral leishmaniasis: is it time to change strategies? Am J Trop Med Hyg.52; 287-92, 1995).
[004] O diagnóstico da doença é baseado na avaliação das manifestações clínicas da doença em conjunto com inquéritos epidemiológicos e com a detecção dos estágios intracelulares dos parasitas por meio de exame de aspirados de órgãos internos dos pacientes, além de outros exames laboratoriais. O diagnóstico rápido é fundamental para evitar o óbito, porém, para o diagnóstico clínico, a sintomatologia da doença é semelhante à de outras doenças mais comuns em nosso meio, como a doença de Chagas, malária, entre outras. Porém, os métodos parasitológicos, apesar de apresentarem especificidade elevada, possuem limitações em sua sensibilidade, pela distribuição não homogénea dos parasitas nos aspirados, além dos riscos de hemorragia interna quando da realização de tal técnica, por ser invasiva. Além disso, tais métodos consomem grande tempo para a confecção das lâminas, fato que também dificulta sua utilização na prática médica rotineira (Tavares CA, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn.3(5):657-67, 2003). Em adição, a semelhança com os sintomas clínicos com outras doenças também dificulta o diagnóstico da LV.  [004] The diagnosis of the disease is based on the assessment of clinical manifestations of the disease in conjunction with epidemiological investigations and the detection of intracellular stages of parasites by examination of patients' internal organ aspirates, as well as other laboratory tests. Rapid diagnosis is essential to prevent death, however, for clinical diagnosis, the symptomatology of the disease is similar to other common diseases in our country, such as Chagas disease, malaria, among others. However, the parasitological methods, although presenting high specificity, have limitations in their sensitivity, due to the inhomogeneous distribution of parasites in the aspirates, besides the risks of internal hemorrhage when performing this technique, as it is invasive. Moreover, such methods consume a great deal of time for making the slides, a fact that also makes their use difficult in routine medical practice (Tavares CA, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn.3 (5): 657 -67, 2003). In addition, the similarity with clinical symptoms with other diseases also makes the diagnosis of VL difficult.
[005] Testes laboratoriais baseiam-se na detecção de antígenos e/ou anticorpos específicos aos parasitas em amostras de soros dos pacientes. Em tais casos, a Análise de Imunoadsorção por Ligação Enzimática (ELISA), a Reação de Imunofluorescência Indireta (IFAT) e o Teste de Aglutinação Direta (DAT) são utilizados, porém, dentre alguns problemas observados, os mesmos não apresentam a capacidade de diferenciar pacientes com a doença subclínica, com a doença ativa ou com a mesma já curada (Tavares CA, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn. 3;657-67, 2003).  Laboratory tests are based on the detection of parasite-specific antigens and / or antibodies in patient serum samples. In such cases, Enzyme-Linked Immunosorbent Analysis (ELISA), Indirect Immunofluorescence Reaction (IFAT) and Direct Agglutination Test (DAT) are used, but among some observed problems, they do not have the ability to differentiate. patients with the subclinical disease, with the active disease or with it already cured (Tavares CA, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn. 3; 657-67, 2003).
[006] No caso da LV canina (LVC), o sorodiagnostico apresenta-se dificultado por fatores relacionados à especificidade e/ou sensibilidade dos antígenos utilizados. Nesse caso, a ocorrência de um elevado número de resultados falso-positivos em animais saudáveis, porém, residentes em áreas endémicas da doença, bem como em animais portando outras doenças relacionadas à LV, tais como doença de Chagas, babesiose, toxoplasmose, rickettsiose e erlichiose; bem como a ocorrência de resultados falso-negativos, como em animais infectados, porém, com baixos títulos de anticorpos anti- Leishmania; têm criado grandes dificuldades para a interpretação correta dos testes sorológicos nos cães (Scalone A, et al. Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay. Vet. Parasitol. 104;275-85, 2002; Mettler M, et al. Evaluation of enzyme-linked immunosorbent assays, an immunofluorescent-P. antibody test, and two rapid tests (immunochromatographic-dipstick and gel tests) for serological diagnosis of symptomatic and asymptomatic Leishmania infections in dogs. J. Clin. Microbiol. 43;5515-19, 2005). Tais problemas têm sido também observados no sorodiagnóstico da LV humana. [006] In the case of canine VL (CVL), serodiagnosis is hampered by factors related to the specificity and / or sensitivity of the antigens used. In this case, the occurrence of a high number of false positive results in healthy animals, however, residing in endemic areas of the disease, as well as in animals carrying other VL-related diseases such as Chagas disease, babesiosis, toxoplasmosis, rickettsiosis and erlichiosis; as well as the occurrence of false negative results, as in infected animals, but with low anti-Leishmania antibody titers; have created major difficulties for the correct interpretation of serological tests in dogs (Scalone A, et al. Evaluation of the recombinant Leishmania K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay. Vet. Parasitol. 104 ; 275-85, 2002; Mettler M, et al Evaluation of enzyme-linked immunosorbent assays, an immunofluorescent-P. antibody test, and two rapid tests (immunochromatographic-dipstick and gel tests) for serological diagnosis of symptomatic and asymptomatic Leishmania infections in dogs J. Clin Microbiol 43; 5515-19, 2005). Such problems have also been observed in the serodiagnosis of human VL.
[007] Em modelos murinos, após a cura da doença causada pela espécie Leishmania major, os animais adquirem imunidade duradoura contra a reinfecção (Afonso LC and 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 antígenos que vêm sendo avaliados e que preenchem, parcialmente, requisitos para serem empregados como candidatos à vacina (Stãger S, Smith DF, Kaye PM. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol 165;7064-71 , 2000; Dondji B, et al. Heterologous prime - boost vaccination with the LACK antigen protects against murine visceral leishmaniasis. Infect Immun 73;5286-89, 2005).  [007] In murine models, upon cure of the disease caused by the Leishmania major species, animals acquire lasting immunity against reinfection (Afonso LC and Scott P. Immune responses associated with susceptibility of C57BL / 10 mice to Leishmania amazonensis. Infect. & Immun. 61; 2952-59, 1993). This fact has stimulated the development of research aiming at obtaining vaccine antigens that can be used as an effective disease control measure. There are antigens that have been evaluated and partially meet requirements to be used as vaccine candidates (Stager S, Smith DF, Kaye PM. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol 165; 7064-71, 2000; Dondji B, et al., Heterologous prime - boost vaccination with the LACK antigen protects against murine visceral leishmaniasis (Infect Immun 73; 5286-89, 2005).
[008] A maioria dos estudos realizados utiliza antígenos únicos, que são expressos em apenas uma das formas morfológicas do parasita, quer seja, promastigota ou amastigota (Fernandes AP, et al. Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives. Curr Opin Microbiol.;15:1 -10, 2012). Além disso, tais antígenos são usualmente capazes de induzir proteção do tipo espécie-específica e em um tipo de hospedeiro mamífero, normalmente, em modelos murinos. Dessa forma, não há disponível no mercado uma vacina eficaz no combate às leishmanioses e que possa ser usada tanto no cão quanto no homem. [008] Most studies performed use single antigens, which are expressed in only one of the morphological forms of the parasite, either promastigote or amastigote (Fernandes AP, et al. Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives Curr Opin Microbiol; 15: 1-10, 2012). In addition, such antigens are usually capable of inducing species-specific protection and a mammalian host type, usually in murine models. Thus, an effective leishmaniasis vaccine that can be used in both dogs and humans is not available on the market.
[009] Na resposta imune contra a infecção por Leishmania infantum, normalmente, a resistência à infecção pelo parasita é associada com o desenvolvimento de uma resposta imune celular do tipo Th1 , primada pela produção de citocinas como o IFN-gamma (IFN-γ), a interleucina 12 (IL-12), entre outras, por parte do hospedeiro mamífero. O mecanismo imunológico efetor responsável pelo controle do parasitismo parece ser decorrente da ativação de macrófagos, via produção de níveis elevados de óxido nítrico e de outras moléculas que atacam os parasitas internalizados (Scott P. Development and regulation of cell-mediated immunity in experimental leishmaniasis. Immunol Res. 27;489-98, 2003). Estudos que utilizam como base o modelo murino de infecção por L. major em camundongos BALB/c, proposto por Sacks e Noben-Trauth (2002) definiram o paradigma Th1 /Th2 de resistência e susceptibilidade, respectivamente, à infecção e o papel de citocinas como o IFN-gama e a interleucina-4 (IL-4), respectivamente, no desenvolvimento de linhagens de células Th1 e Th2 (Sacks D e Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2;845-58, 2002).  In the immune response against Leishmania infantum infection, resistance to parasite infection is usually associated with the development of a Th1-type cellular immune response, dominated by the production of cytokines such as IFN-gamma (IFN-γ). , interleukin 12 (IL-12), among others, from the mammalian host. The effector immune mechanism responsible for controlling parasitism seems to be due to macrophage activation via production of high levels of nitric oxide and other molecules that attack internalized parasites (Scott P. Development and regulation of cell-mediated immunity in experimental leishmaniasis). Immunol Res. 27; 489-98, 2003). Studies based on the murine model of L. major infection in BALB / c mice proposed by Sacks and Noben-Trauth (2002) defined the Th1 / Th2 paradigm of infection resistance and susceptibility, respectively, and the role of cytokines. as IFN-gamma and interleukin-4 (IL-4), respectively, in the development of Th1 and Th2 cell lines (Sacks D and Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2: 845-58, 2002).
[0010] O desenvolvimento de uma resposta Th1 pode ser inibido pela produção de citocinas tais como a IL-10. Neste sentido, essas moléculas estão relacionadas com a desativação de macrófagos e com o estabelecimento da doença nos animais, enquanto que citocinas como IFN-γ estão associados com o fenótipo de proteção contra a doença (Zanin FH, et al. Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections. Microbes Infect 9;1070-77, 2007; Chávez-Fumagalli MA, et al. Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge. Microbes Infect 12;967-77, 2010; Martins VT, et al. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis. PLoS Negl Trop Dis. 7;e2148, 2013). [001 1 ] Em estudos avaliando candidatos vacinais contra a LV, os imunógenos são administrados em camundongos associados a adjuvantes indutores de resposta imune Th1 . Após as imunizações, para a avaliação da imunogenicidade da vacina, citocinas como 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). Dessa forma, procura-se a associação de que, nos animais tidos como protegidos contra a LV, que os mesmos apresentem baixa carga parasitária e que seus esplenócitos sejam capazes de produzir níveis elevados de IFN-γ e baixos níveis de IL-10 após a estimulação in vitro dos mesmos com os imunógenos. The development of a Th1 response may be inhibited by production of cytokines such as IL-10. In this sense, these molecules are related to macrophage deactivation and disease establishment in animals, whereas cytokines such as IFN-γ are associated with the disease protection phenotype (Zanin FH, et al. protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections Microbes Infect 9; 1070-77, 2007; Chávez-Fumagalli MA, et al Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB / c mice against Leishmania chagasi and Leishmania amazonensis challenge Microbes Infect 12; 967-77, 2010; Martins VT, et al Antigenicity and protective efficacy of a amastigote-specific protein, member of the superoxygenase family, against visceral leishmaniasis PLoS Negl Trop Dis. 7; e2148, 2013). In studies evaluating VL vaccine candidates, immunogens are administered in mice associated with Th1 immune response inducing adjuvants. After immunizations, for evaluation of vaccine immunogenicity, cytokines such as 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 these levels obtained with the parasitic loads found in animals is performed (Martins VT, et al. Antigenicity and protective efficacy of Leishmania). amastigote-specific protein, member of the superoxygenase family, against visceral leishmaniasis (PLoS Negl Trop Dis. 7; e2148, 2013). Thus, it is sought the association that, in animals considered as protected against VL, they have low parasitic load and that their splenocytes are capable of producing high levels of IFN-γ and low levels of IL-10 after in vitro stimulation with immunogens.
[0012] Os antígenos atualmente utilizados como vacinas contra as leishmanioses são usualmente proteínas completas, as quais contêm regiões comuns a proteínas de outras doenças, o que pode gerar reações cruzadas nos animais vacinados. Também, tais proteínas podem conter epitopos (ou peptídeos) que apresentam efeito benéfico, mas outros que apresentam efeito nocivo nos hospedeiros imunizados comprometendo, dessa forma, a eficácia da vacina. O uso de pequenos antígenos 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, de maior rendimento e menor custo em relação à produção de proteínas recombinantes; além de terem boa estabilidade e, dessa forma, poderem ser disponibilizados em melhores condições para a população.  Antigens currently used as leishmaniasis vaccines are usually complete proteins, which contain regions common to proteins of other diseases, which can lead to cross reactions in vaccinated animals. Also, such proteins may contain epitopes (or peptides) that have a beneficial effect, but others that have a detrimental effect on immunized hosts, thereby compromising vaccine efficacy. The use of small antigens tends to allow the development of a more specific vaccine so that the immunized dog or man can develop a beneficial immune response to only that specific antigen and the disease for which it was selected. Such antigens also have a simpler technical production, higher yield and lower cost compared to the production of recombinant proteins; They also have good stability and thus can be made available under better conditions for the population.
[0013] A tecnologia de phage display é baseada na expressão de peptídeos, proteínas 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; 1315-1317, 1 985). 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-panning". O número de ciclos de bio- pannings realizados em phage display determina o grau de enriquecimento dos fagos que se ligam a um 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). Phage display technology is based on the expression of peptides, proteins or antibody fragments associated with proteins 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 the viral particles (Smith GP Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228; 1315-1317, 1 985). 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 the 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 given target receptor, adhered to a solid surface, is performed by consecutive selection steps called "bio-panning cycles". The number of biohazard cycles performed on the phage display determines the degree of enrichment of the phages that bind to a 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).
[0014] Metodologicamente, os ciclos de bio-pannings podem ser adaptados de acordo com o grupo que domina tal tecnologia. Exemplificadamente, em nosso caso, moléculas de IgGs provenientes de soros de pacientes a serem investigados são fundidas à superfície de microesferas (denominadas de "beads") e tal híbrido é submetido a processos de captação (seleção) negativa ou positiva em relação aos clones de fagos presentes na biblioteca recombinante utilizada.  Methodologically, the bio-panning cycles can be tailored according to the group that dominates such technology. For example, in our case, IgG molecules from patient sera to be investigated are fused to the surface of microspheres (called "beads") and such a hybrid is subjected to negative or positive uptake (selection) processes in relation to clones. phages present in the recombinant library used.
[0015] 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 Immun. 67:4764-4770, 1999; Manoutcharian K, et al. Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis. Vet Immunol Immunopatho. 99:1 1 -24, 2004; Sartorius R, et al. The use of filamentous bacteriophage fd to deliver MAGE- A10 or MAGE-A3 HLA-A2-restricted peptides and to induce strong antitumor CTL responses. J Immunol. 15;180(6):3719-28, 2008). [0015] Phage display technology has been used in several works to research 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 Immun. 67: 4764-4770 Manoutcharian K et al Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis Vet Immunol Immunopatho 99: 11-1-24, 2004; Sartorius R, et al. The use of filamentous bacteriophage fd to deliver MAGE-A10 or MAGE-A3 HLA-A2-restricted peptides and to induce strong antitumor CTL responses. J Immunol. 15; 180 (6): 3719-28, 2008).
[0016] 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 de peptídeos selecionados, pela metodologia adotada ter sido também diferente e por tal documento de patente se tratar apenas do estudo da doença canina.  Patent document BR1020130275425, entitled "Canine Visceral Leishmaniasis Vaccine Composition, Synthetic Peptides and Use", is a vaccine composition for 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 of selected peptides, the methodology adopted was also different and that such patent document is only the study of canine disease.
[0017] 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 contra 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 imunógenos identificados são diferentes dos descritos na presente invenção.  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 against 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 identified immunogens are different from those described in the present invention.
[0018] O documento de patente PI08004854 trata da utilização de vetores virais recombinantes expressando o antígeno de A2 Leishmania como método de vacinação contra leishmaniose. A tecnologia se difere do presente pedido por contemplar antígeno diferente daqueles compreendidos pelo presente pedido e a estratégia experimental ser distinta.  Patent application PI08004854 deals with the use of recombinant viral vectors expressing the Leishmania A2 antigen as a method of vaccination against leishmaniasis. The technology differs from the present application in that it contemplates antigen different from those comprised in the present application and the experimental strategy is distinct.
[0019] A presente tecnologia trata de seis peptídeos e/ou clones de bacteriófagos expressando peptídeos de interesse e que foram selecionados por meio da técnica de phage display. Tais clones expressando os peptídeos mostraram-se imunogênicos em dois modelos de mamíferos, camundongo e homem, sendo capazes de induzir ao desenvolvimento de um perfil de resposta imune celular Th1 , visto que tanto esplenócitos de camundongos cronicamente infectados com L. infantum quanto células mononucleares do sangue periférico (PBMCs) de indivíduos sadios estimulados com os clones individuais foram capazes de induzir à produção de níveis elevados de IFN-γ e baixos níveis de IL-10, demonstrando dessa forma a capacidade de tais imunógenos em induzir ao desenvolvimento de uma resposta imune relacionada à proteção contra a LV, no caso, da resposta Th1 . Em adição, tais imunógenos, representados pelos clones de bacteriofagos expressando os peptídeos de interesse, não têm identidade de sequência de aminoácidos similares com outros já descritos, de forma que são considerados inéditos. [0019] The present technology deals with six bacteriophage peptides and / or clones expressing peptides of interest which were selected by the phage display technique. Such clones expressing the peptides were immunogenic in two mammalian models, mouse and man, being able to induce the development of a Th1 cellular immune response profile, since both splenocytes of chronically L. infantum-infected mice and mononuclear cells of the mouse. peripheral blood (PBMCs) from healthy individuals stimulated with individual clones were able to induce production of high levels of IFN-γ and low levels of IL-10, thus demonstrating the ability of such immunogens to induce the development of an immune response related to protection against VL, in this case the Th1 response. In addition, such immunogens, represented by bacteriophage clones expressing the peptides of interest, do not have similar amino acid sequence identity to others already described, so they are considered unpublished.
DESCRIÇÃO DETALHADA DA TECNOLOGIA  DETAILED DESCRIPTION OF TECHNOLOGY
[0020] A presente tecnologia trata de composições vacinais para o tratamento e/ou a prevenção de leishmaniose visceral (LV) em mamíferos, compreendida por seis peptídeos sintéticos livres ou expressos na superfície de bacteriofagos não infecciosos, que foram selecionados por meio de uma abordagem proteômica de phage display, para serem aplicados isoladamente ou em possíveis combinações entre os mesmos. Tais clones são imunogênicos em células do homem e de camundongo, sendo capazes de induzir ao desenvolvimento de um perfil de resposta imune celular Th1 .  The present technology relates to vaccine compositions for the treatment and / or prevention of visceral leishmaniasis (VL) in mammals, comprised of six free or surface-expressed synthetic peptides of non-infectious bacteriophages, which have been selected by an approach. phage display proteomics, to be applied alone or in possible combinations between them. Such clones are immunogenic in human and mouse cells and are capable of inducing the development of a Th1 cellular immune response profile.
[0021 ] Mais especificamente, a presente tecnologia compreende os peptídeos definidos pelas SEQ ID NQ 1 a 6, livres ou expressos na superfície de bacteriofagos não infecciosos, assim como seu uso no tratamento e/ou prevenção da leishmaniose visceral em diferentes espécies de mamíferos. More specifically, the present technology comprises the peptides defined by SEQ ID NQ 1 to 6, free or surface-expressed non-infectious bacteriophages, as well as their use in the treatment and / or prevention of visceral leishmaniasis in different mammalian species. .
[0022] 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 pharmaceutically 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 primed for the production of IFN-γ cytokines. and IL-12.
[0023] A composição vacinai proposta pode ser administrada pelas vias intradérmica, intramuscular, oral, nasal, intravenosa, subcutânea e/ou como dispositivo que possa ser injetado. The proposed vaccine composition may be administered by the intradermal, intramuscular, oral, nasal, intravenous, subcutaneous and / or injectable device.
[0024] 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.
[0025] EXEMPLO 1. OBTENÇÃO DOS BACTERIOFAGOS EXPRESSANDO AS PROTEÍNAS DE INTERESSE [0026] Amostras de soro. As seguintes amostras de soro foram utilizadas para a realização dos ciclos de bio-pannings na técnica de phage display: soros de pacientes com LV ativa (n=40), soros de indivíduos não infectados residentes em área endémica de leishmanioses (n=50) e soros de pacientes com doença de Chagas (n=20). O projeto foi aprovado pelo COEP da UFMG (CAAE- 323431 .14.9.0000.5149). EXAMPLE 1. OBTAINING BACTERIOPHAGES EXPRESSING PROTEIN OF INTEREST Serum samples. The following serum samples were used for phage display bio-panning cycles: sera from patients with active VL (n = 40), sera from uninfected individuals living in an endemic area of leishmaniasis (n = 50) and sera from patients with Chagas disease (n = 20). The project was approved by the UFMG COEP (CAAE-323431 .14.9.0000.5149).
[0027] Purificação de anticorpos IgGs por acoplamento em microesferas de proteína-G. As imunoglobulinas da classe IgG de pools dos soros listados acima foram purificadas por meio de seu acoplamento em beads magnéticos conjugados à proteína G (Dynabeads, Invitrogen). Para tal, 1 x 1011 microesferas foram lavadas por 3 vezes em tampão MES 0.1 M pH 5.0 e foi adicionado às mesmas: (a) 400 μΙ_ do pool de soros de pacientes com LV, para um volume final de 700 μΐ; (b) 400 μΐ do pool de soros de pacientes com doença de Chagas, para um volume final de 700 μΐ e (c) 400 μΐ do pool de soros de indivíduos sadios, para um volume final de 700 μΐ. 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. Purification of IgG antibodies by coupling on G-protein microspheres. The IgG class immunoglobulins from the sera pools listed above were purified by coupling them to G protein conjugated magnetic beads (Dynabeads, Invitrogen). To this end, 1 x 10 11 microspheres were washed 3 times in 0.1 M MES pH 5.0 buffer and added to them: (a) 400 μΙ_ of the LV patient serum pool to a final volume of 700 μΐ; (b) 400 μΐ from the serum pool of patients with Chagas disease for a final volume of 700 μΐ and (c) 400 μΐ from the serum pool of healthy individuals for a final volume of 700 μΐ. The ratio of antibodies to the amount of microspheres in all cases was 1: 1. Thereafter, incubation was performed for 40 minutes under constant agitation 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.
[0028] 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 min e a 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 h e a 37°C, sendo então ressuspendidas em 200 □ L de tampão TBS. Para certificação do acoplamento, 5 μί dos beads cobertos pelas IgGs foram incubados por 1 h 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). 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 / HCl in triethanolamine buffer) for 30 minutes under constant stirring and at RT The reaction was neutralized by incubating the system with 1 ml of 50 mM Tris buffer pH 7.5 for 15 min and at RT. The incorporated samples 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 h at 37 ° C, then resuspended at 200 □ L of TBS buffer. For coupling certification, 5 μί of the IgG-coated beads were incubated for 1 h 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 substrate. tetramethylbenzidine. 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).
[0029] Ciclos de bio-pannings. Para a realização dos ciclos de bio-pannings pela técnica de phage display, processos de seleção negativa e positiva foram realizadas usando 1 x 1012 partículas virais de uma biblioteca recombinante contendo peptídeos fusionados em fagos filamentosos (biblioteca comercial Ph.D.- C7C obtida da empresa New England, BioLabs®, USA). A mesma foi diluída em 190 μΙ_ 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 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. Esse processo foi repetido mais duas vezes, totalizando 3 ciclos de seleção negativa. O sobrenadante, contendo fagos que não se ligaram às IgGs de soros de indivíduos sadios e daqueles com Doença de Chagas foi coletado. Para a seleção positiva, o sobrenadante coletado foi transferido para um novo tubo contendo as microesferas acopladas com as IgGs de pacientes com LV sintomática e assintomática, sendo incubadas à T.A. e por 30 minutos. As microesferas contendo o sistema beads-anticorpos precipitado nos tubos dos bio- pannings dos anticorpos dos soros dos pacientes, 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 posterior titulação. O processo foi repetido mais duas vezes, totalizando 3 ciclos de seleção positiva. Após tal procedimento, os clones foram individualizados. Cycles of bio-pannings. For phage display bio-panning cycles, negative and positive selection procedures were performed using 1 x 10 12 viral particles from a recombinant library containing filamentous phage fused peptides (commercial library Ph.D.-C7C obtained from New England, BioLabs®, USA). It was diluted in 190 μΙ_ 0.1% TBS-Tween for ligand selection in the microspheres incorporated by IgG antibodies. In the first cycle of bio-panning, began with the negative selection. For this, the recombinant bacteriophage library was incubated for 30 minutes and at 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). supernatant containing clones that did not adhere 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. This process was repeated twice more, totaling 3 cycles of negative selection. The supernatant containing phages that did not bind to IgGs from sera from healthy individuals and those with Chagas disease was collected. For positive selection, the collected supernatant was transferred to a new tube containing the IgG-coupled microspheres of patients with symptomatic and asymptomatic VL, and incubated at RT for 30 minutes. The microspheres containing the beads-antibody system precipitated in the patient sera antibody bio-panning tubes, plus the phages of interest adhered to such IgGs, were washed 10 times with TBS-Tween 20 0.1% and eluted in 500 μΙ_. 0.2 M glycine buffer pH 2.0, such solution being neutralized by the addition of 75 μΙ_ Tris-base pH 9.0. Phage clones were recovered for further titration. The process was repeated twice more, totaling 3 positive selection cycles. After such procedure, the clones were individualized.
[0030] Titulações, amplificação e extração do DNA dos clones de interesse. Ao final do 3Q ciclo de seleção positiva, os clones de fagos recuperados foram titulados. Para tal, os mesmos foram submetidos a diluições seriadas exponenciais (log10) em meio LB líquido com ampicilina, sendo as diluições de 10"1 até 10"4 para os eluatos não amplificados e de 10"8 a 10"11 para os eluatos amplificados. Para cada titulação, uma placa de cultura foi confeccionada. As placas foram incubadas à 37QC por 16 horas. Após, as colónias que se apresentavam azuis e individualizadas foram quantificadas manualmente. Para estimar os valores dos títulos, o número total de colónias contadas foi multiplicado pelo fator de diluição de cada placa. As colónias individualizadas e de cor azul presentes nas placas de cultura obtidas do 3Q ciclo de bio-pannings foram coletadas para a extração do DNA. Para a amplificação dos fagos, 1 ,2 mL da cultura de células E. coli ER2738 em fase early-log (OD6oonm~0,3) foram distribuídos em cada poço de uma placa deepwell. DNA titrations, amplification and extraction of clones of interest. At the end of the 3 Q positive selection cycle, the recovered phage clones were Titled. For this, they were submitted to exponential serial dilutions (log 10 ) in liquid LB medium with ampicillin, with dilutions of 10 "1 to 10 " 4 for non-amplified eluates and 10 "8 to 10 " 11 for eluates. amplified. For each titration, a culture plate was made. The plates were incubated at 37 Q C for 16 hours. Afterwards, the blue and individualized colonies were manually quantified. To estimate the titre values, the total number of counted colonies was multiplied by the dilution factor of each plate. The individualized blue colored colonies present in the culture plates obtained from the 3rd Q- cycle of biopannings were collected for DNA extraction. For phage amplification, 1.2 mL of the early-log phase E. coli ER2738 cell culture (OD 6 oonm ~ 0.3) was distributed into each well of a deepwell plate.
[0031 ] Com palitos de dente esterilizados, 96 colónias azuis foram retiradas da placa de Petri e transferidas para a placa de cultura. A cada poço, apenas uma colónia de fago foi adicionada, totalizando 96 colónias na placa. A mesma foi vedada e incubada por 5 horas, sob agitação constante e a 37°C. Após a incubação, a placa foi centrifugada por 20 minutos a 2.250 x g, sendo o sobrenadante transferindo para outra placa. A essa 2- placa, foi adicionado PEG/NaCI (1 /6 do volume total do sobrenadante), sendo a mesma incubada por 14 horas e a 4°C. Após, a placa foi centrifugada por 1 hora, o sobrenadante foi retirado e o pellet foi ressuspendido em tampão iodeto (10 mM de Tris HCI pH 8.0, 1 mM de EDTA e 4 M de Nal). As placas foram agitadas vigorosamente e, em seguida, adicionou-se etanol absoluto. Após uma incubação de 10 minutos, as placas foram centrifugadas (2.250 x g a 4QC e por 10 minutos), sendo o sobrenadante descartado. O precipitado contendo o DNA foi lavado com 500 μΙ_ de etanol 70% e novamente centrifugado. Finalmente, o DNA de cada clone foi diluído em 20 μΙ_ de água ultrapura e sua qualidade foi avaliada após uma corrida eletroforética realizada em gel de agarose 0.8%, corado com uma solução de brometo de etídio. With sterile toothpicks, 96 blue colonies were removed from the Petri dish and transferred to the culture plate. To each well, only one phage colony was added, totaling 96 colonies on the plate. It was sealed and incubated for 5 hours under constant agitation at 37 ° C. After incubation, the plate was centrifuged for 20 minutes at 2,250 xg and the supernatant transferred to another plate. To this plate was added PEG / NaCl (1/6 of the total volume of the supernatant) and incubated for 14 hours at 4 ° C. Afterwards, the plate was centrifuged for 1 hour, the supernatant was removed and the pellet was resuspended in iodide buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA and 4 M Nal). The plates were shaken vigorously and then absolute ethanol was added. After a 10 minute incubation, the plates were centrifuged (2,250 xg at 4 Q C and for 10 minutes) and the supernatant discarded. The precipitate containing the DNA was washed with 500 μΙ 70% ethanol and centrifuged again. Finally, the DNA of each clone was diluted in 20 μΙ_ of ultrapure water and its quality was evaluated after an electrophoretic run performed on 0.8% agarose gel stained with an ethidium bromide solution.
[0032] Sequenciamento dos clones selecionados. Os 96 clones provenientes do 3Q ciclo de seleção positiva tiveram seu DNA extraído e foram sequenciados. A reação foi processada no sequenciador automático MegaBace 1000 (Amersham Biosciences) e a dedução in silico das sequências de aminoácidos do DNA dos fagos de interesse, para a identificação dos peptídeos exógenos, foi realizada pelo programa DNA2PRO12, que é designado para a dedução de sequências de insertos das bibliotecas da New England Biolabs (Ph.D.-12TM ou Ph.D.-C7CTM), como de outras bibliotecas de interesse que contenham as sequências inicial e final do vetor. O programa automaticamente localiza a posição do inserto, traduz o mesmo e indica os possíveis erros na tradução (tais como códons inesperados ou erros na sequência próxima). Sequencing of selected clones. The 96 clones from the 3 Q positive selection cycle had their DNA extracted and sequenced. The reaction was processed on the MegaBace 1000 automatic sequencer (Amersham Biosciences) and in silico deduction of DNA amino acid sequences from phage of interest for identification of exogenous peptides was performed by the DNA2PRO12 program, which is designed for deduction of insert sequences from New England Biolabs libraries (Ph. D.-12TM or Ph.D.-C7CTM), as from other libraries of interest containing the initial and final vector sequences. The program automatically finds the position of the insert, translates it and indicates possible translation errors (such as unexpected codons or errors in the next sequence).
EXEMPLO 2. AVALIAÇÃO DA INFECÇÃO EM ANIMAIS.  EXAMPLE 2. EVALUATION OF INFECTION IN ANIMALS.
[0033] Parasitas. A amostra MHOM/BR/1970/BH46 de L infantum foi utilizada. Os parasitas foram cultivados em meio Schneider's completo, o qual foi constituído pelo meio Schneider's (SIGMA) acrescido com 20 % de soro fetal bovino (SIGMA) inativado, 20 mM de L-glutamina, 50 μg/mL de gentamicina, 200 U/mL de penicilina e 100 μg/mL de estreptomicina, em pH 7,4 e a 24QC, conforme descrito por Coelho et al., 2003 (Coelho EAF, et al. Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen, but not by the LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection. Infect Immun. 71 :3988-94, 2003). Parasites. Sample MHOM / BR / 1970 / BH46 from L infantum was used. The parasites were cultured in complete Schneider's medium, which consisted of Schneider's medium (SIGMA) plus 20% inactivated fetal bovine serum (SIGMA), 20 mM L-glutamine, 50 μg / mL gentamicin, 200 U / mL penicillin and 100 μg / mL streptomycin, at pH 7.4 and 24 Q C, as described by Coelho et al., 2003 (EAF rabbit, et al. Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen, but not by the LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection (Infect Immun. 71: 3988-94, 2003).
[0034] Infecção desafio. Para a realização da infecção experimental nos animais, 1 x 107 formas promastigotas em fase estacionária de crescimento de L. infantum foram inoculadas no coxim plantar direito de 8 animais. Os camundongos foram monitorados durante 10 semanas após o desafio, quando foi realizada a eutanásia dos animais para os experimentos de imunogenicidade e determinação da carga parasitária. [0034] Infection challenge. For experimental infection in animals, 1 x 10 7 promastigote stationary growth forms of L. infantum were inoculated into the right plantar cushion of 8 animals. Mice were monitored for 10 weeks after challenge, when animals were euthanized for immunogenicity experiments and determination of parasitic load.
[0035] A carga parasitária foi realizada a fim de atestar que os animais estavam realmente infectados pelo parasita, A carga parasitária no baço, fígado, medula óssea e linfonodos drenantes foi avaliada por meio de uma técnica de diluição limitante. Os animais (n=8) foram infectados subcutaneamente com essa espécie do parasita e, 10 semanas após a infecção, os mesmos foram eutanasiados para a coleta dos órgãos e avaliação parasitológica. Na análise dos resultados, observou- se que todos os órgãos avaliados apresentaram elevada carga de parasitas (representada pelo log de parasitas), conforme mostrado na Tabela 1 . Dessa forma, constatou-se que os animais estavam infectados por L. infantum. Os resultados correspondem à média ± desvio-padrão dos grupos, em dois experimentos realizados e que apresentaram resultados similares. Parasitic loading was performed to certify that the animals were indeed infected by the parasite. Parasite loading in the spleen, liver, bone marrow and draining lymph nodes was assessed by a limiting dilution technique. The animals (n = 8) were subcutaneously infected with this parasite species and, 10 weeks after infection, they were euthanized for organ collection and parasitological evaluation. In the analysis of the results, it was observed that all organs evaluated presented high parasite load (represented by the log of parasites), as shown in Table 1. Of that Thus, the animals were found to be infected with L. infantum. 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 infectados por L. infantum. Table 1. Parasitic burden in L. infantum infected mice.
Órgão avaliado Média ± desvio-padrão do log do número de parasitas Evaluated organ Mean ± standard deviation of log number of parasites
Fígado 5,5±0,3 Liver 5.5 ± 0.3
Baço 6,5±0,2  Spleen 6.5 ± 0.2
Medula óssea 4.0±0,3  Bone Marrow 4.0 ± 0.3
Linfonodos  Lymph Nodes
7.0±0,5  7.0 ± 0.5
drenantes  draining
EXEMPLO 3. DOSAGEM DE CITOCINAS E CALCULO DA RAZÃO ENTRE OS NÍVEIS OBTIDOS DE IFN^ E IL-10  EXAMPLE 3. DOSAGE OF CYTOKINES AND RATIO CALCULATION BETWEEN IFN ^ AND IL-10 LEVELS
[0036] Camundongos BALB/c infectados com L. infantum foram eutanasiados 10 semanas após o desafio 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. 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. infantum, 50 μς), ou incubadas sem estímulo. A incubação ocorreu a 37QC 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-10 foi realizada por ELISA de captura utilizando kits comerciais (BD OptEIA TM set mouse kits, Pharmingen, San Diego, CA, USA). Os resultados são mostrados na Tabela 2. [0037] Com as citocinas quantificadas, a razão entre os níveis de IFN-γ e IL-10 foi calculada para cada clone avaliado, com vistas a selecionarmos apenas aqueles fagos capazes de induzir a uma maior produção de IFN-γ e menores níveis de IL- 10, e os resultados são também mostrados. Os valores apresentados são representativos de dois experimentos realizados de maneira independente e que apresentaram resultados similares. Um clone de fago selvagem, similar aos fagos- alvo, mas que não expressa peptídeo exógeno e o SLA L. infantum foram utilizados como controles. L. infantum infected BALB / c mice were euthanized 10 weeks after challenge 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 sulphate, 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 of complete DMEM, which was added with 20% inactivated fetal bovine serum. Splenocytes were quantified in a 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. infantum, 50 μς), or incubated. without stimulus. Incubation occurred at 37 Q C in incubator with 5% CO 2 and 48 hours. Subsequently, the culture supernatant was collected and IFN-γ and IL-10 cytokines were measured by capture ELISA using commercial kits (BD OptEIA ™ mouse set kits, Pharmingen, San Diego, CA, USA). Results are shown in Table 2. With the quantified cytokines, the ratio between IFN-γ and IL-10 levels was calculated for each clone evaluated, in order to select only those phages capable of inducing higher IFN-γ production and lower levels of IFN-γ. IL-10, and the results are also shown. The values presented are representative of two independently performed experiments that presented similar results. A wild phage clone, similar to the target phage but not expressing exogenous peptide and SLA L. infantum were used as controls.
Tabela 2. Dosagem das citocinas IFN-γ e IL-10 após estímulo in vitro dos esplenocitos de camundongos BALB/c infectados com L. infantum e cálculo da razão entre os valores.  Table 2. Dosage of IFN-γ and IL-10 cytokines after in vitro stimulation of splenocytes of L. infantum infected BALB / c mice and calculation of the ratio between values.
Citocinas (em pg/mL)  Cytokines (in pg / mL)
Imunógeno IFN-gamma IL-10 Razão  IFN-gamma immunogen IL-10 Ratio
A10 129 78 1 ,65 A10 129 78 1, 65
B1 132 87 2,52B1 132 87 2.52
D2 136 90 1 ,51D2 136 90 1, 51
D1 1 1 16 70 2,65D1 1 1 16 70 2.65
C12 154 85 1 ,81C12 154 85 1.81
D12 137 88 2,44D12 137 88 2.44
E2 66 75 0,88E2 66 75 0.88
E3 48 86 2,56E3 48 86 2.56
E4 72 80 0,9E4 72 80 0.9
E6 239 70 1 ,41E6 239 70 1, 41
E12 317 75 1 ,23E12 317 75 1, 23
F5 164 83 2,97F5 164 83 2.97
F6 495 88 5,62F6 495 88 5.62
F7 40 79 0,5F7 40 79 0.5
F8 123 78 2,58F8 123 78 2.58
F10 122 66 1 ,85F10 122 66 1.85
F1 1 195 70 2,79F1 1 195 70 2.79
F12 564 80 0,7 G10 163 74 1 ,4F12 564 80 0.7 G10 163 74 1, 4
H2 143 69 2,07H2 143 69 2.07
H6 104 75 1 ,39H6 104 75 1.39
H10 63 90 2,7H10 63 90 2.7
H1 1 80 89 0,9H1 1 80 89 0.9
H12 46 76 0,6H12 46 76 0.6
Fago selvagem 70 77 0,6 Wild Phage 70 77 0.6
SLA 59 84 0,4  SLA 59 84 0.4
[0038] Na análise dos resultados, observou-se que houve uma grande variação na produção das citocinas entre os clones pré-selecionados. Os controles (fago selvagem e SLA L. infantum) apresentaram razão entre nos níveis de IFN-γ e IL-10 de 0,6 e 0,4, respectivamente. Aqueles clones cuja produção de IFN-γ foi, pelo menos, 2,0 vezes maior que a produção de IL-10 foram selecionados haja vista que, após o estímulo de esplenócitos de camundongos infectados com L. infantum, os mesmos induziram a uma maior produção de citocinas do tipo Th1 , em relação à produção das citocinas Th2. Desta forma, 10 clones (B1 , D1 1 , D12, E3, F5, F6, F8, F1 1 , H2 e H10) foram selecionados para serem usados na estimulação in vitro de PBMCs provenientes de indivíduos sadios.  In the analysis of the results, it was observed that there was a large variation in cytokine production among the preselected clones. Controls (wild phage and SLA L. infantum) showed a ratio between the IFN-γ and IL-10 levels of 0.6 and 0.4, respectively. Those clones whose IFN-γ production was at least 2.0 times higher than IL-10 production were selected since, after stimulation of splenocytes from L. infantum-infected mice, they induced a higher production of Th1 cytokines in relation to the production of Th2 cytokines. Thus, 10 clones (B1, D1 1, D12, E3, F5, F6, F8, F1 1, H2 and H10) were selected for in vitro stimulation of PBMCs from healthy individuals.
EXEMPLO 4. CULTURA DE PBMCS E DOSAGEM DE CITOCINAS.  EXAMPLE 4. PBMCS CULTURE AND CYTOKINE DOSAGE.
[0039] 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, 106 células por poço foram incubadas em DMEM com 1010 de cada fago avaliado, ou com o fago selvagem (mesma concentração) ou com SLA de L. infantum (50 μg/mL). A incubação foi realizada a 37°C com 5% de CO2 durante 5 dias. Como controle positivo, as células foram incubadas com acetato de miristato de forbol (PMA, 25 ng/mL) e ionomicina (1 μg/mL) em meio DMEM. As culturas foram incubadas durante 48 horas a 37°C e em 5% de CO2. Após, a brefeldina A (Sigma) foi adicionada (concentração de 10 μg/mL) durante um período de 4 horas. Após, 200 μΙ_ 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 1 x 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-10, Serotec®, USA). 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-10 foi calculada para cada clone e os dados são também apresentados. [0039] Peripheral blood mononuclear cells were isolated as described (Khamesipour A, et al. Phenotyping of circulating CD8 + T cell subsets in human cutaneous leishmaniasis. Microbes Infect. 9: 702-1 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 PBMCs stimulation, 10 6 cells per well were incubated in DMEM with 10 10 of each phage evaluated, either with wild phage (same concentration) or with L. infantum SLA (50 μg / 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 μg / mL) in DMEM medium. Cultures were incubated for 48 hours at 37 ° C and 5% CO 2 . Afterwards, brefeldin A (Sigma) was added (concentration of 10 μg / mL) during a period of 4 hours. Afterwards, 200 μΙ_ EDTA (Sigma) was added to each culture tube (to a final concentration of 2 mM), and the cells were washed with FACS buffer (1 x PBS with 0.5% fetal bovine serum albumin and 0.1% 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; in the presence of 20 μΙ_ of PE-labeled anti-cytokine mAbs antibody (IFN-γ and IL-10, 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. The results are shown in table 3. The ratio between IFN-γ and IL-10 levels was calculated for each clone and data are also presented.
Tabela 3. Dosagem das citocinas IFN-γ e IL-10 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-10 cytokines after in vitro stimulation of PBMCs obtained from healthy individuals and calculation of the ratio between values.
Citocinas (em pg/mL)  Cytokines (in pg / mL)
Imunógeno IFN-gamma IL-10 Razão IFN-gamma immunogen IL-10 Ratio
B1 205 89 2,3B1 205 89 2.3
D1 1 244 95 2,56D1 1 244 95 2.56
D12 233 100 2,33D12 233 100 2.33
E3 101 1 12 0,9E3 101 1 12 0.9
F5 241 87 2,77F5 241 87 2.77
F6 316 102 3,1F6 316 102 3.1
F8 106 88 1 ,2F8 106 88 1, 2
F1 1 104 69 1 ,5F1 1 104 69 1, 5
H2 255 92 2,77 H10 106 76 1,4H2 255 92 2.77 H10 106 76 1.4
Fago selvagem 68 97 0,7 Wild Phage 68 97 0.7
SLA 57 113 0,5  SLA 57 113 0.5
[0040] Na análise dos resultados, observou-se que houve uma grande diferença na produção de IFN-γ e IL-10 entre os clones selecionados. Os controles (fago selvagem e SLA L. infantum) apresentaram razão entre nos níveis de IFN-γ e IL-10 de 0,7 e 0,5, respectivamente. Dessa forma, aqueles clones cuja produção de IFN- γ foi, pelo menos, 2,0 vezes maior que a produção de IL-10, colocam-se como imunogênicos em dois modelos de mamíferos avaliados (camundongo e homem) e, dessa forma, como candidatos para serem empregados como vacinas contra a LV, haja vista que, após o estímulo de esplenócitos de camundongos infectados com L. infantum e de PBMCs de indivíduos sadios, tais clones tenham sido capazes de induzir uma maior produção de IFN-γ e baixos níveis de IL-10. As sequências de aminoácidos dos clones selecionados são mostradas na Tabela 4. Tabela 4. Sequências de aminoácidos dos clones selecionados após avaliação de sua imunogenicidade em esplenócitos de camundongos infectados e PBMCs de indivíduos sadios.  In the analysis of the results, it was observed that there was a large difference in IFN-γ and IL-10 production between the selected clones. Controls (wild phage and SLA L. infantum) showed a ratio between IFN-γ and IL-10 levels of 0.7 and 0.5, respectively. Thus, those clones whose IFN-γ production was at least 2.0 times higher than IL-10 production are immunogenic in two mammalian models evaluated (mouse and man), and thus As candidates for use as VL vaccines, after the stimulation of splenocytes from L. infantum-infected mice and from PBMCs from healthy individuals, such clones were able to induce a higher production of IFN-γ and low levels. IL-10 levels. The amino acid sequences of the selected clones are shown in Table 4. Table 4. Amino acid sequences of the selected clones after evaluation of their immunogenicity in splenocytes from infected mice and PBMCs from healthy individuals.
Clone Sequências de aminoácidos  Clone Amino Acid Sequences
B1 VQSWSVR  B1 VQSWSVR
D11 G PN LLLV  D11 G PN LLLV
D12 VSVMetRG R  D12 VSVMetRG R
F5 TLWPFKL  F5 TLWPFKL
F6 D L C T Q I T  F6 D L C T Q I T
H2 GS RSYP R  H2 GS RSYP R
[0041] Assim, os imunógenos B1, D11, D12, F5, F6 e H2 colocam-se como candidatos a vacina para serem testados na prevenção contra a LV em diferentes modelos de mamíferos.  Thus, the immunogens B1, D11, D12, F5, F6 and H2 pose as vaccine candidates to be tested for prevention of VL in different mammalian models.

Claims

REIVINDICAÇÕES
1 - COMPOSIÇÕES VACINAIS CONTRA LEISHMANIOSE VISCERAL, caracterizadas por compreenderem pelo menos um dos antígenos proteicos definidos pelas SEQ ID N° 1 a 6 ou qualquer combinação possível entre eles, e excipientes farmacologicamente e farmaceuticamente aceitáveis. VACCINARY COMPOSITIONS AGAINST VISCERAL LEISHMANIOSIS, characterized in that they comprise at least one of the protein antigens defined by SEQ ID NOS. 1 to 6 or any possible combination thereof, and pharmaceutically and pharmaceutically acceptable excipients.
2- COMPOSIÇÕES VACINAIS CONTRA LEISHMANIOSE VISCERAL, de acordo com a reivindicação 1 , caracterizadas pelos antígenos serem expressos na superfície externa de bacteriófagos. VACCINAL COMPOSITIONS AGAINST VISCERAL LEISHMANIOSIS according to claim 1, characterized in that the antigens are expressed on the external surface of bacteriophages.
3- COMPOSIÇÕES VACINAIS CONTRA LEISHMANIOSE VISCERAL, de acordo com a reivindicação 1 , caracterizadas por serem administradas pelas vias intradérmica, intramuscular, oral, nasal, intravenosa, subcutânea e/ou como dispositivo que possa ser injetado.  VACCINARY COMPOSITIONS AGAINST VISCERAL LEISHMANIOSIS according to claim 1, characterized in that they are administered by the intradermal, intramuscular, oral, nasal, intravenous, subcutaneous and / or injectable device.
4- COMPOSIÇÕES VACINAIS CONTRA LEISHMANIOSE VISCERAL, de acordo com a reivindicação 1 a 3, caracterizadas pela associação de adjuvantes farmacêutica e farmacologicamente aceitáveis, a fim de potencializar o desenvolvimento de uma resposta imune celular do tipo Th1 . VACCINAL COMPOSITIONS AGAINST VISCERAL LEISHMANIOSIS according to claims 1 to 3, characterized by the combination of 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 NQ1 a 6. SYNTHETIC PEPTIDES 5, characterized in that they comprise the sequences defined by SEQ ID NO : Q 1 to 6.
6- USO dos peptídeos sintéticos, conforme definidos pela reivindicação 5, caracterizado por ser na preparação de composições vacinais para o tratamento e/ou prevenção da leishmaniose visceral.  Use of synthetic peptides as defined in claim 5, characterized in that they are in the preparation of vaccine compositions for the treatment and / or prevention of visceral leishmaniasis.
7- USO das composições vacinais, conforme definido pelas reivindicações 1 a 4, caracterizado por ser no tratamento e/ou prevenção da leishmaniose visceral em mamíferos.  Use of the vaccine compositions as defined by claims 1 to 4, characterized in that they are in the treatment and / or prevention of visceral leishmaniasis in mammals.
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BRPI0903089A2 (en) * 2009-08-12 2011-04-12 Univ Fed De Uberlandia recombinant peptides and leishmania antigens mimetic protein motifs and their applications

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BRPI0903089A2 (en) * 2009-08-12 2011-04-12 Univ Fed De Uberlandia recombinant peptides and leishmania antigens mimetic protein motifs and their applications

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Title
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, June 2013 (2013-06-01), pages 835 - 841, XP055467551 *

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