BR102014011613A2 - vaccine compositions containing the recombinant antigens li1040, fc and cyclo, their applications and protective effects against leishmaniasis - Google Patents

Abstract

The present invention relates to the production of vaccine formulations containing recombinant proteins obtained by genetic engineering techniques (recombinant ona), in particular proteins li1040 (seq id No. 1; linj.32.1 040, gene 10: 5071843), fc (seq id No. 2 linj.36.0640, gene lo: 5073646) and cyclo (seq id No. 3; linj.08.0560, gene 10: 5066731), and their applications, given the protective effect obtained for li1 040, fce cyclo proteins in animals vaccinated and later challenged with leishmania infantum. The invention also relates to vaccine compositions containing epitope peptides obtained from these proteins: cyclo 10 (seq 10 no. 4; ekysvkvtg), fc 10: 1 o (seq lo no. 5; vollnvkam), l1 040 lo: 8 (seq lo # 6: rgwrpysl), l1 040 10: 7 (seq lo # 7; oyvknvsov), l1 040 lo: 34 (seq 10 no 8; spvaasaityaisnflpyannf), l1040 io: no 10 (seq 10 no 9; haaootlall ), a2 10: n11 (seq 10 no. 10; vgpqsvgpls), a2 10: n13 (seq 10 no. 11; sasaephkaavovgpls), a2 10: n14 (seq 10 no. 12; wgpqsvgpls) and diagnostic kits for leishmaniasis.

Description

(54) Title: VACCINE COMPOSITIONS CONTAINING THE RECOMBINANT ANTIGENS LI1040, FC AND CYCLO,

ITS APPLICATIONS AND PROTECTIVE EFFECTS AGAINST LEISHMANIOSIS (51) Int. Cl .: A61K 39/008; A61P 33/02; C07K 14/44; C12N 15/30 (73) Holder (s): FEDERAL UNIVERSITY OF MINAS GERAIS (72) Inventor (s): ANA PAULA SALLES MOURA FERNANDES; ADRIANA MONTE CASSIANO CANAVACI MARTINS; VICENTE DE PAULO MARTINS; LEOPOLDO FERREIRA MARQUES MACHADO; ANGELA VIEIRA SERUFO; DANIEL HENRIQUE DORO PEREIRA; DANIELLA CASTANHEIRA BARTHOLOMEU; TIAGO ANTÔNIO DE OLIVEIRA MENDES (85) Start date of the National Phase:

05/14/2014 (57) Abstract: The present invention reports the obtaining of vaccine formulations containing recombinant proteins obtained by genetic engineering techniques (recombinant ONA), in particular the proteins L1040 (Seq ID N ° 1; LinJ.32.1 040, Gene 10: 5071843), Fc (Seq ID No. 2 LinJ.36.0640, Gene IO: 5073646) and Cyclo (Seq ID No. 3; LinJ.08.0560, Gene 10: 5066731), and their applications, given the protective effect obtained for proteins Lil 040, F c and Cyclo in animals vaccinated and subsequently challenged with Leishmania infantum. The invention also deals with vaccine compositions containing epitope peptides obtained from these proteins: Cyclo 10 (Seq 10 N ° 4; EKYSVKVTG), F c 10: 1 O (Seq IO N ° 5; VOLLNVKAM), Lil 040 IO: 8 ( Seq IO No. 6: RGWRPYSL), Lil 040 10: 7 (Seq IO No. 7; OYVKNVSOV), Lil 040 IO: 34 (Seq 10 No. 8;

SPVAASAITYAISNFLPYANNF), LI1040 IO: N10 (Seq 10 N ° 9; HAAOOTLALL), A2 10: Nll (Seq 10 N ° 10; VGPQSVGPLS), A2 10: N13 (Seq 10 N ° ll; SASAEPHKAAVOVGPLS), A2 10: N14 ( Seq 10 N ° 12; WGPQSVGPLS) and diagnostic kits for Leishmaniasis.

1/22 “VACCINE COMPOSITIONS CONTAINING THE RECOMBINANT ANTIGENS LI1040, FC AND CYCLO, THEIR APPLICATIONS AND PROTECTIVE EFFECTS AGAINST LEISHMANIOSIS.” [001] The present invention relates to obtaining vaccine formulations containing recombinant proteins obtained by genetic engineering techniques (recombinant DNA), in particular proteins L1040 (Seq ID N ° 1; LinJ.32.1040, Gene ID: 5071843), Fc ( Seq ID N ° 2 LinJ.36.0640, Gene ID: 5073646) and Cyclo (Seq ID N ° 3; LinJ.08.0560, Gene ID: 5066731), and their applications, considering the protective effect obtained for proteins L1010, Fc and Cyclo in vaccinated animals and subsequently challenged with Leishmania infantum. The invention also deals with vaccine compositions containing epitope peptides obtained from these proteins: Cyclo ID: 11 (Seq ID N ° 4; EKYSVKVTG), Fc ID: 10 (Seq ID N ° 5; VDLLNVKAM), LÍ1040 ID: 8 (Seq ID N ° 6: RGWRPYSL), LÍ1040 ID: 7 (Seq ID N ° 7; DYVKNVSDV), LÍ1040 ID: 34 (Seq ID N ° 8; SPVAASAITYAISNFLPYANNF), LÍ1040 ID: N10 (Seq ID N ° 9; HAADDTLALL), A2 ID: N11 (Seq ID N ° 10; VGPQSVGPLS), A2 ID: N13 (Seq ID N ° 11; SASAEPHKAAVDVGPLS), A2 ID: N14 (Seq ID N ° 12; WGPQSVGPLS) and diagnostic kits for Leishmaniasis.

[002] Leishmaniasis is an infectious disease caused by parasites of the genus Leishmania, transmitted by insects, Phlebotomus pemiciosus and Lutzomyia longipalpis, main vectors. In its different clinical forms, an estimated 1.3 million new cases are estimated, with 20 to 30 thousand deaths per year (ALVAR, J „VELEZ, ID, BERN, C., HERRERO, M„ DESJEUX, P., CANO , J., JANNIN, J., DEN, BM Leishmaniasis Worldwide and Global Estimates of Its Incidence. PLoS ONE, v. 7, n. 5, p. E35671,2012).

[003] The infection begins with the bite of the infected insect, with the injection of promastigote forms of the parasite in the skin of the vertebrate host that are phagocyted and replicate in the macrophage phagolysosome. Once in the phagolysosome, the promastigote forms differ from amastigotes that can then infect other macrophages or be ingested during insect repast. In the saliva of the vector, amastigotes again differentiate into promastigotes, completing the life cycle of the parasite (MURRAY, H.W .;

2/22

BERMAN, J.D .; DAVIES, C.R .; SARAVIA, N.G. Advances in leishmaniasis. Lancet 366: 1561-77, 2005).

[004] The clinical symptoms of the disease range from skin lesions (caused by the species L. (L.) major, L. (L.) tropica and L. (L.) Mexican) to mucocutaneous (caused mainly by L. (L.) braziliensis) and severe head infections (caused by L. (L.) infantum, L. (L.) donovani) (KEDZIERSKI, 2010). Currently, L. (L.) infantum and L. (L.) chagasi, prevalent in the old and new world, respectively, are considered a single species (MAURÍCIO, IL; STOTHARD, JR; MILES, MA Leishmania donovani complex: genotyping with the ribosomal internai transcribed spacer and the mini-exon. Parasitology (2004) 128, 263-267; KEDZIERSKI, L.

Leishmaniasis Vaccine: Where are We Today? J Glob Infect Dis. 2 (2): 17785, 2010.).

[005] In some geographic regions, leishmaniasis are mainly zoonoses, with several wild mammalian animals acting as reservoirs of Leishmania species, particularly rodents, edentates and marsupials in cutaneous Leishmaniasis (LC), and wild and domestic dogs in visceral Leishmaniasis (LV) ).

[006] VL is the systemic and most severe form, among the three main categories of leishmaniasis, with 200 to 400 thousand new cases estimated per year. An increase in the number of cases may occur due to a higher incidence or exposure to risk factors such as malnutrition, poverty, population mobility and environmental and climate changes; or due to the failure of vector and reservoir control - which are among the main prophylactic practices recommended by the World Health Organization (GRIMALDI JR G, TESH RB. Leishmaniasis of the New World: current concepts and implications for future research. Clin Microbiol Rev 6: 230-250, 1993; PALATNIK-DE-SOUSA, CB; SANTOS, WR; FRANCE-SILVA, JC; DA COSTA, RT; REIS, AB; PALATNIK, M .; MAYRINK, W .; GENARO, O. Impact of canine control on the epidemiology of canine and visceral human leishmaniasis in Brazil. Am. J.Trop. Med. Hyg. 65: 510-517, 2001).

3/22 [007] Dogs are susceptible to infection and considered the main reservoir for L. (L.) infantum (= L. (L.) chagasi) in different geographical regions of the globe (TESH, 1995). The parasite is transmitted from an infected dog to an uninfected one by the insect's bite, although transmission by blood transfusion (OWENS et al., 2001) has been reported. Humans become accidentally infected and do not act as reservoir hosts for L. (L.) infantum, except in cases where contaminated syringes are shared among drug users (CRUZ, I. et al. Leishmania in discarded syringes from intravenous drug users Lancet 359: 1124-1125, 2002).

[008] The control of canine Leishmaniasis is based on the treatment or sacrifice of infected animals. The treatment of canine Leishmaniasis with successful drugs used in the therapy of human visceral Leishmaniasis is of low efficacy and high cost, while the sacrifice of infected dogs is not often accepted for ethical and social reasons. In addition, the elimination of infected dogs has shown contradictory results in Brazil. Over 5-6 years, more than 2 million dogs have been examined and more than 160,000 seropositive dogs have been euthanized, but the incidence of human VL has not reduced to acceptable levels. Thus, immunoprophylactic measures have been considered as alternative promises in the prevention of canine VL. For this reason, considerable effort has been applied to studies of the immune response in canine VL, and several Leishmania antigens involved in this response have been characterized (ASHFORD, DA; DAVID, JR; FREIRE, M. et al. Studies on control of visceral leishmaniasis: impact of dog control on canine and human visceral leishmaniasis in Jacobina, Bahia, Brazil.Am J Trop Med Hyg 59: 53-57, 1998; BRAGA, MD; COELHO, IC; POMPEU, MM; EVANS, TG; MACAULLIFE , IT; TEIXEIRA, MJ; LIMA, JW Control of canine visceral leishmaniasis: comparison of results from a rapid elimination program of serum-reactive dogs using an immunoenzyme assay and slower elimination of serum-reactive dogs using filter paper elution indirect immunofluorescence. Rev. Rev. Soc. Bras. Med. Trop. 31: 419424, 1998).

4/22 [009] Symptomatic visceral leishmaniasis in dogs has been associated with immunological changes involving T cells. These changes include the absence of late type hypersensitivity to Leishmania antigens, a decrease in the number of T cells in the peripheral blood and a lack of IFN production. -γ and IL-2 by peripheral blood mononuclear cells (PBMC - peripheral blood mononuclear cells) in vitro. In symptomatic dogs, IL-4 expression was observed in mitogen-stimulated PBMC and detectable in bone marrow aspirate. Data for the cytokine IL-10 are still controversial in dogs. In addition, high titers of anti-Leishmania antibodies, which are not immunoprotective, are detected in symptomatic animals. In general, resistance to leishmaniasis results from the development of the cell-mediated immune response (CMI) and has been associated with the activation of IFN-γ, IL-2 and TNF-α producing Th1 cells (PINELLI, E .; GONZALO, RM; BOOG, CJP et al. Leishmania infantum specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex restricted manner. Eur J Immunol 25: 1594-1600, 1995; PINELLI, E .; GEBHARD, D .; MOMMAAS, AM et al. Infection of a canine macrophage cell line with Leishmania infantum: determination of nitric oxide production and anti-leishmanial activity.Vet Parasitol 92: 181-189, 2000; QUINNELL, RJ; COURTENAY, O .; SHAW, MA et al. Tissue cytokine responses in canine visceral leishmaniasis. J Infect Dis 183: 1421-1424, 2001).

[010] Some studies have demonstrated the involvement of CD8 + T lymphocytes in resistance to canine LV. A greater number of these lymphocytes were detected in asymptomatic dogs, experimentally infected by L. (L.) infantum, a fact not observed in symptomatic animals, suggesting that the direct lysis of macrophages infected by the parasite, by cytotoxic T lymphocytes, represents an additional effector mechanism resistance to LV. In dogs naturally infected with L. (L.) infantum, a reduction in the CD4 + and CD8 + populations was observed and the restoration of these cells was seen after drug treatment. The humoral response, in the absence of CMI, contributes to the susceptibility to the disease (PINELLI, E .; GONZALO, R.M .; BOOG, C.J.P.

5/22 et al. Leishmania infantum specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex restricted manner. Eur J Immunol 25: 1594-1600, 1995;

BOURDOISEAU, G .; BONNEFONT, C .; HOAREAU, E .; BOEHRINGER, C .; STOLLE, T .; CHABANNE, L. Specific lgG1 and lgG2 antibody and lymphocyte subset levels in naturally Leishmania infantum infected treated and untreated dogs. Vet Immunol Immunopathol 59: 21-30, 1997; MCMAHON-PRATT, D .; ALEXANDER, J. Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease? Immunol.Rev 201: 206-224, 2004). [011] Vaccines protect against the development of diseases, by making the immune system able to respond quickly to the pathogen. Historically, most vaccines have been focused on inducing high antibody titers; however, in many cases, cellular immunity involving CD4 + T cells, CD8 + T cells or both, as well as their IFN-γ cytokine, are essential. In such cases, an ideal immunization protocol should induce the generation of Tmc cells. In general, the first dose of a vaccine induces the expansion of specific T cells, which determines the frequency and the final number of CD8 ⁺ T cells that are maintained . Due to the differentiation kinetics of memory cells, a time interval between vaccine doses is usually necessary for effective induction of immune memory. The second dose of the vaccine, in addition to stimulating memory cells, may promote additional activation of specific virgin CD8 ⁺ T cells, which may not have been activated after the first dose (ESSER, MT; MARCHESE, RD; KIERSTEAD, LS; TUSSEY , LG; WANG, F .; CHIRMULE, N .; WASHABAUGH, MW Memory T cells and vaccines.Vaccine 21: 419-430, 2003; BADOVINAC, VP & HARTY, JT Programming, demarcating, and manipulating CD8 + T-cell memory. Immunol.Rev 211: 67-80, 2006).

[012] Experimental evidence shows that the initial dose of the antigen, and not its persistence in the host organism, is the main determinant of the clonal expansion of CD8 ⁺ T cells, thus being reflected in the population of memory cells. Other factors, such as the presence of molecules

6/22 co-stimulators, cytokines and CD4 + T cells also influence the quantity, quality and location of memory T cells. Thus, the manipulation of the initial phase of the response, by changing the time of exposure to the antigen and the microenvironmental conditions, allows changing the generation of memory cells, which can be of great practical use in immunization protocols. In addition, the nature of the antigen, the route of immunization, the dose and the presence of adjuvants are among the factors that influence the magnitude and duration of the cellular immune response after vaccination ESSER, M.T .; MARCHESE, R.D .; KIERSTEAD, L.S .; TUSSEY, L.G .; WANG, F .; CHIRMULE, N .; WASHABAUGH, M.W. Memory T cells and vaccines. Vaccine 21: 419-430, 2003; BADOVINAC, V.P. & HARTY, J.T. Programming, demarcating, and manipulating CD8 + T-cell memory. Immunol.Rev 211: 67-80, 2006).

[013] Several vaccination strategies have been tested against experimental leishmaniasis, focusing especially on efficacy against cutaneous leishmaniasis, more than visceral leishmaniasis. Protein A2 was first identified in L. (L.) donovani, being expressed only in amastigotes, and has been associated with the visceralization process. Vaccine containing the recombinant A2 protein, Leish-tec®, passed a phase III test and a protective efficacy of 71% was observed in vaccinated animals, based on the recovery of parasites from bone marrow aspirates. Among the dogs that showed anti-A2 antibodies in response to Leish-tec®, 82% protection was observed. In addition, Leish-tec® has been shown to be safe for a heterogeneous population of dogs, being the first vaccine based on recombinant protein licensed to dogs in the world, marketed in Brazil. The A2 antigen was able to induce protection in different hosts against infection, contains epitopes for CD8 ⁺ T cells and is associated with the ability of parasites to migrate to the spleen and liver of infected hosts, being specific to species with such characteristic. Considering these characteristics, the A2 antigen can be used as a model in the search and characterization of new vaccine antigens against species such as L. (L.) chagasi, with visceralization capacity (CHAREST, H .; MATLASHEWSKI,

G. Developmental gene expression in Leishmania donovani: differential

7/22 cloning and analysis of an amastigote stage-specific gene. Mol Cell Biol 14: 2975-84, 1994; HANDMAN, E. Leishmaniasis: current status of vaccine development. Clin Microbiol Rev 14: 229-43, 2001; COELHO, E.A .; TAVARES, C.A .; CARVALHO, F.A .; CHAVES, K.F .; TEIXEIRA, K.N .; RODRIGUES, R.C .; CHAREST, H .; MATLASHEWSKI, G .; GAZZINELLI, R.T .; FERNANDES A.P. 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.lmmun. 71.3988-3994, 2003).

[014] One way to select possible genes is by comparing the genomic sequences of these species with other species of Leishmania that do not cause visceral Leishmaniasis. The detailed comparison of three genomic sequences of Leishmania (L. (L.) infantum, L. (V.) braziliensis, L. (L.) major) has been described. Genes that are distributed differently between species, encode related proteins in parasitic host interactions and parasite survival in macrophages. There are still demonstrations that levels of expression and / or variation in the sequence of conserved genes among Leishmania species can contribute significantly to virulence and tissue tropism. Examples of this are some genes from L. (L.) donovani previously cloned and expressed in L. (L.) major, which gave this parasite the ability to visceralize, not seen in wild parasites that have the same genes (ZHANG, WW .; MATLASHEWSKI, G. Characterization of the A2-A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection. Mol Microbiol 39: 935-48, 2001. ZHANG, WW .; MATLASHEWSKI, G. Screening Leishmania donovani-specific genes required for visceral infection.Mol Microbiol 77 (2), 505-517, 2010. ZHANG, WW; MENDEZ, S .; GHOSH, A .; MYLER, P .; IVENS, A .; MATLASHEWSKI, G. Comparison of the A2 locus gene in Leishmania donovani and Leishmania 162 major and its control over cutaneous infection. J Biol Chem 278: 35508—35515, 2003; PEACOCK, CS et al. Comparative genomic analysis of three Leishmania

8/22 species that cause diverse human disease. Nat Genet. 39 (7): 839, 2007).

[015] The ability of the immune system to respond to a particular antigen differs between individuals, as they have different patterns of MHC (major histocompatibility complex) genes. Antigen variations in pathogens and the extensive polymorphism of HLA molecules (human leukocyte antigens) increase the number of test targets for vaccines. In order to increase the scope of the immune response, vaccines with multiple antigens can be used, guaranteeing a better response, with a more varied pool of effector cells, even though the individual has a lower polymorphism in HLA molecules. It seems unlikely that a single antigen can induce a complete protective immune response against a complex parasite like Leishmania. Although the LinJ32_V3.1040 (Li1040), LinJ.36.0640 (Fc) and LinJ.08.0560 (Cyclo) genes, have already been reported in the state of the art, no material was found in the literature describing the use of the respective proteins encoded by them , in formulations for immunization against Leishmaniasis (PEACOCK, CS et al. Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet. 39 (7): 839-847, 2007; ZHANG, WW .; PEACOCK, CS; MATLASHEWSKI, G. A Genomic-Based Approach Combining In Vivo Selection in Mice to Identify a Novel Virulence Gene in Leishmania. PLoS Negl Trop Dis 2 (6): e248, 2008).

[016] After years of research and advances in genome sequencing techniques, vaccine development today employs new tools and does not necessarily begin with empirical tests based on the growth of microorganisms, as described by Louis Pasteur, but with information stored in It is called reverse vaccinology (VR) (PIZZA M, SCARLATO V, MASIGNANI V, GIULIANI MM, ARICÒ B, COMANDUCCI M, [017] et al. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287: 1816—20, 2000).

9/22 [018] When proposed for the first time, VR would be a definitive solution to problems in the discovery of antigens. Since all the genes encoded in the genome of a pathogenic species are known for sequencing, the list of vaccine candidates is finite and, in principle, all can be tested in animal models. In this way, protective antigens would not be lost or forgotten, despite the time and effort required to find them and define the most effective vaccine formulations (DONATI, C .; RAPPUOLI, R. Reverse vaccinology in the 21st century: improvements over the original design. Ann. NY Acad. Sci. 1285 115-132, 2013).

[019] However, for each VR-based vaccine project, the original analysis model must be modified to adapt to the peculiarities of the target species. Information such as biology, host-pathogen interactions and strain variability are important. Thanks to these large-scale sequencing technologies, hundreds of sequences of complete genomes can be used at the initial point of vaccine target selection. Thus, the epidemiological characterization of the pathogen has become an integral part of VR (DONATI, C .; RAPPUOLI, R. Reverse vaccinology in the 21st century: improvements over the original design. Ann. NY Acad. Sci. 1285 115-132, 2013).

[020] The testing of vaccine candidates in animal models is the final step in the entire VR process, which proves the induction or not of protective immunity. In this way, a great effort has been made towards a set of scanning procedures that can be integrated with the original selection of bioinformatics to significantly reduce the number of candidates that need to be tested on animals. New technologies, including microarrays, RNA-seq and proteomics, are generating data that, integrated with the VR process, can make it more efficient. In addition, technological advances in the program tools used in the selection process can be tested in new animal experiments DONATI, C .; RAPPUOLI, R. Reverse vaccinology in the 21st century: improvements over the original design. Ann. N.Y. Acad. Know. 1285 115-132,2013).

10/22 [021] Computational methods of epitope mapping now play an instrumental role in bench experiments because they facilitate the selection of immunogenic targets and the modeling of the immune response. Several programs have been developed in an attempt to predict, before in vitro or in vivo assays, information about possible regions of a protein capable of being recognized by cells of the immune system. The use of these programs helps in choosing possible vaccine candidates, and can effectively reduce the number of biological experiments, important for confirming the predicted data.

[022] The design of potential synthetic vaccines is an important application of epitope identification and prediction methods. Prediction methods for immunogenic peptides can lead to the rational development of vaccines. However, this information is not sufficient to predict how the immune system will respond and, therefore, candidates for synthetic vaccines should be tested experimentally to demonstrate their ability to generate a protective immune response.

[023] Therefore, the theoretical prediction of epitopes is a challenge, although significant progress has been made in the discovery and construction of synthetic peptides useful in research and diagnosis. Efforts are being made to develop peptide vaccines against HIV, HTLV-1, Streptococcus pyogenes and malaria. Also, synthetic peptides are being considered in the development of therapeutic vaccines to cure cancer, Alzheimer's and autoimmune diseases. Knowledge of bioinformatics in the development of epitope prediction methods is desirable and has an important contribution to the development of synthetic vaccines (QUAKKELAAR ED, MELIEF CJ. Experience with synthetic vaccines for cancer and persistent virus infections in nonhuman primates and patients. Adv Immunol. 114: 77-106, 2012; LYNCH, MP; KAUMAYA, PT Advances in HTLV-1 peptide vaccines and therapeutics. Curr Protein Pept Sei. 7 (2): 137-45, 2006; PANDEY M, SEKULOSKI S, BATZLOFF MR. Novel strategies for controlling Streptococcus pyogenes infection and associated diseases: from potential peptide vaccines to antibody

11/22 immunotherapy. Immunol Cell Biol. 87 (5): 391-9, 2009; MENÉNDEZGONZÁLEZ M, PÉREZ-PINERA P, MARTÍNEZ-RIVERA M, MUISIIZ AL, VEGA JA. Immunotherapy for Alzheimer's disease: rational basis in ongoing clinical trials. Curr Pharm Des. 17 (5): 508-20, 2011; MILLER MJ, FOY KC, KAUMAYA PT. Cancer immunotherapy: present status, future perspective, and a new paradigm of peptide immunotherapeutics. Discov Med. 15 (82): 166-76, 2013.).

[024] Thus, the present technology is based on the cloning of the genes LinJ32_V3.1040 (LÍ1040), LinJ.36.0640 (Fc) and LinJ.08.0560 (Cyclo), expression and purification of recombinant proteins LÍ1040, Fc and Cyclo, for used in vaccine formulations against Leishmaniasis. As well as the Cyclo epitope peptides: Seq ID No. 4 (EKYSVKVTG); Fc (Seq ID N ° 5; VDLLNVKAM): Li1040: (Seq ID N ° 6: RGWRPYSL), Seq ID N ° 7 (DYVKNVSDV), Seq ID N ° 8 (SPVAASAITYAISNFLPYANNF), Seq ID N ° 9 (HAADDTLALL); A2: Seq ID No. 10 (VGPQSVGPLS), Seq ID No. 11 (SASAEPHKAAVDVGPLS), Seq ID No. 12 (WGPQSVGPLS), to be used for the same purpose.

[025] Through biological tests it was verified the ability of the proteins and peptides mentioned above to induce protection against Leismaniasis in experimental models.

BRIEF DESCRIPTION OF THE FIGURES [026] Figure 1 - Figure 1 shows how the gene sequences of L. infantum proteins were subcloned in E. coli XL1 blue. In A, Li1040, Fc and Cyclo gene amplification products are shown by PCR visualized on 1% agarose gel after electrophoretic run. In B the plasmid pGEM map is represented and in C the electrophoretic analysis of the PCR reactions is observed for different clones of each gene (Li1040, Fc and Cyclo).

[027] Figure 2: After obtaining Miniprep of the clones containing the insert with confirmed sequence, digestions of plasmids pGEMFc, pGEMLi1040 and pGEMCyclo were performed, as well as digestion of plasmid pET15b

12/22 with the corresponding restriction enzymes. After electrophoretic running, gel A corresponding to pGEMFc digestion, gel B1 to pGEMLi1040 digestion and gel C to pGEMCyclo digestion, the fragments corresponding to the inserts (sizes <1500 bp), as well as the digested pET15b (fragment of approximately 6000 bp , gel B2) were collected and purified from the gel, so that they could be bound and transformed into E. coli C41 for protein expression. D. Map of plasmid pET15b.

[028] Figure 3: Figure 3 shows the electrophoretic analysis of PCR reactions for different E. coli C41 clones after transformation with the binding product of each gene to plasmid pET15b, respectively, and selection with antibiotic ampicillin.

[029] Figure 4: Figure 4 shows the evaluation of the expression of proteins L1040, Fc and Cyclo at different induction times (T0, T1, T2 and T3), using the techniques of Western blot (L1010 and Fc) and polyacrylamide gel after SDS-PAGE (Cyclo), using the molecular weight marker (M) (PageRuler Plus Prestained Protein ladder - Thermo Scientific). [030] Figure 5: Figure 5 shows the evaluation of the L101040 protein solubility using the SDS-PAGE (A) and Western blot (B) methods after bacterial lysis. Each item listed in the image corresponds, respectively, to: 1. purified rLi1040; 2. First pellet treatment after bacterial lysis; 3. Second pellet treatment after bacterial lysis; 4. Supernatant after lysis and centrifugation; M. Molecular weight marker (PageRuler Plus Prestained Protein ladder - Thermo Scientific).

[031] Figure 6: Figure 6 refers to the real-time PCR evaluation of parasitism in the spleen (A), liver (B) and lymph nodes (C) of animals immunized with proteins rA2, L1010 and both, later challenged with L. infantum.

[032] Figure 7: Figure 7 refers to the real-time PCR evaluation of parasitism in the spleen of animals immunized with the proteins rLi1040, rFc or rCyclo, later challenged with L. infantum.

[033] Figure 8: Figure 8 shows the ELISA dosing graphs of IFNg and IL-10 in supernatants of culture of splenocytes restimulated by 48

13/22 hours. Each graph corresponds respectively to: A. IFNg levels; B. Levels of IL-10; C. 1FNg / IL-10 ratio.

[034] Figure 9: Figure 9 shows the ELISA dosage of antiL1-1040 antibodies during the immunization and challenge protocol. Each graph corresponds, respectively, to: A. IgG1 dosage; B. Dosage of IgG2a; C. lgG2a / lgG1 ratio; D. Total IgG measurement.

[035] Figure 10: Figure 10 shows the levels of cytokines obtained by CBA in splenocyte culture supernatants. A. IL-2 levels. B. IL-4 levels. C. IL-5 levels. D. Levels of TNF-α. E. Levels of IFN-γ.

DETAILED DESCRIPTION OF THE TECHNOLOGY [036] The present invention relates to obtaining vaccine formulations containing recombinant proteins obtained by genetic engineering techniques (recombinant DNA), in particular proteins L1040 (LinJ.32.1040, Gene ID: 5071843), Fc (LinJ. 36.0640, Gene ID: 5073646) and Cyclo (LinJ.08.0560, Gene ID: 5066731), and their applications, considering the protective effect obtained for proteins LI1040, Fc and Cyclo in vaccinated animals and subsequently challenged with Leishmania infantum. This technology is based on the cloning of the LinJ32_V3.104, LinJ.36.0640 and LinJ.08.0560 genes, expression of the recombinant proteins Li1040, Fc and Cyclo, respectively, and their purification, to be used concomitantly in vaccine formulations against Leishmaniasis.

[037] From the amino acid sequence of the Li1040, Fc and Cyclo proteins of L. infantum, obtained by the TriTrypDB database (http://tritrypdb.org/tritrypdb/), predictions were made for CD8 ⁺ T cell epitopes murines using the BIMAS program; NetCTL 1.2 in the prediction of human CD8 + T cell epitopes; NetMHC 3.4 in the prediction of murine CD8 T cell epitopes; NetMHCII 2.2 in the prediction of human and murine TCD4 cell epitopes; and BepiPred 1.0 in the prediction of linear B cell epitopes.

14/22 [038] The ability of these proteins and the epitope peptides derived from them to induce protection against the disease was verified in experimental models.

The invention can be better understood from the following non-limiting examples:

Example 1- Subcloning of the gene sequences of L. infantum proteins in E. coli XL1 blue [039] The DNA fragment corresponding to the sequence of each studied protein was amplified by the PCR technique, using a set of specific primers. The amplification product was visualized after electrophoretic running on 1% agarose gel (Figure 1A) and, as noted, the PCR amplification was specific, as a single amplification product was obtained for each set of primers. The fragments obtained were of the expected size, being 1230 bp for Li'1040, 1281 bp for Fc and 1467 bp for Cyclo. Each fragment was ligated to the pGEM plasmid (Figure 1B) and transformed into competent E. coli XL1 blue bacteria. The selected colonies were grown in a liquid medium and subsequently evaluated by PCR to confirm the subcloning (Figure 1C). Several positive clones were obtained (Figure 1C). A sample of the extracted DNA was sequenced to confirm the insert and the reading phase (frame). The sequencing showed that the inserts showed no changes in the sequence and that the reading phase was correct. The pGEM plasmid containing the insert was digested with appropriate restriction enzymes, allowing the evaluation of the size of the released insert (Figure 2) and creating cohesive ends in the insert for plasmid transfer. The bacterial expression vector pET15b (Figure 2D), which allows the expression of the 6-amino acid histidine tail protein, was digested with the same restriction enzymes (Figure 2). The digested and purified insert and pET15b from the gel were ligated and transformed into E. coli XL1 blue. The selected colonies were evaluated by PCR to confirm cloning (Figure 3).

[040] The pET15b plasmid containing the insert was then transformed into competent E. coli C41 bacteria for protein expression.

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Example 2- Verification of expression and Purification of rLi1040, rFc and rCyclo [041] Protein expression was induced in recombinant E. coli C41 cultures, as described in the previous example, and was evaluated after separation of proteins by SDS-PAGE and Western blot detection using anti-histidine antibodies. The purification of recombinant Li1040, Fc and Cyclo was done in a six-histidine tail affinity system using nickel affinity chromatography (Qiagen) under denaturing condition, and any other purification system capable of purifying the recombinant Li1040 could be used.

[042] The expression and the best induction time for each protein were verified, collecting samples of the culture at times 0 (TO = before the addition of IPTG), 1 (T1 = after 1 h of induction), 2 (T2 = after 2 h of induction) and 3 (T3 = after 3 h of induction). The collected samples were lysed and the protein extract was subjected to separation by SDS-PAGE. Then, the Western blot technique was used to detect the expression of histidine-tailed proteins (Figure 4). It was observed that the best induction time for protein expression was 3 hours (T3) (Figure 4).

[043] The correct expression of proteins by the clones was observed, confirming that the binding of the insert in pET15b remained in the correct reading phase, as observed in the sequencing of plasmid pGEM containing the same insert. The weight of each recombinant protein seen on the Western blot membrane was close to that expected, with Li1040 (45.1 kD), Fc (48.5 kD) and Cyclo (55.5 kD) receiving 6 histidines and a plasmid fragment, and molecular weights estimated at 49.8 kD (rLi1040), 54.4 kD (rFc) and 62.2 kD (rCyclo), according to the ProtParam tool on the ExPASy Bioinformatics Resource Portal (http://web.expasy.org/protparam/).

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Example 3- Evaluation of protein solubility after bacterial lysis using the SDS-PAGE and Western blot techniques [044] To determine how the sample would be treated, after the bacterial lysis and centrifugation, the analysis of the supernatant and pellet by SDS -PAGE and Western blot, checking in which portion the proteins were in greater quantity. It was found that all proteins were present in greater amount in the pellet, although it is possible to visualize a small amount of L101040 protein in the supernatant (Figure 5) after lysis. These tests confirmed the hydrophobicity and solubility predictions by the ExPASy - ProtScale and Recombinant Protein Solubility Prediction programs.

Example 4 - Analysis of the purity of the rLi1040, rFc and rCyclo proteins after elution in a nickel column [045] After elution of the proteins in the nickel column, the samples were dialyzed and the dialyzed product was analyzed by SDS-PAGE and Western blot. As can be seen in Figure 6, a good degree of protein purity was obtained, which can be used in vaccine formulations for animal immunization.

Example 5 - Applications and protective effects of recombinant proteins rLi1040, rFc and rCyclo [046] Groups of BALB / c mice were immunized subcutaneously in the lower right dorsal region, with an initial injection (prime) and two booster injections (boost) , at intervals of 15 days each. Each group was immunized, respectively, with the recombinant proteins rLi1040, rFc, rCyclo, rLi1040 concomitant with rA2, and animals immunized only with the rA2 protein were used as a vaccine control group. It was observed that the subcutaneous vaccine and challenge protocols worked, since the group vaccinated with rA2 (vaccine control) showed a significant result of

17/22 protection, with less tissue parasitism compared to the saline group (Figure 7).

[047] With the results of real-time PCR, it was found that groups immunized with the protein rA2, Li1040 or both showed a significant decrease in spleen parasitism, compared to the group that received saline during immunizations (Figure 7A). The same decrease was observed in the lymph nodes. However, due to the variation presented by the saline group, there were no statistically significant differences between groups for parasitism of this organ (Figure 7C).

[048] Splenocytes from animals vaccinated with Li1040, Fc and Cyclo were restimulated with the respective proteins for 48 h. Supernatants were used to measure IFN-γ and IL-10 by ELISA. Splenocytes from animals vaccinated with rFc or rCyclo, when restimulated with the respective proteins, produced increased levels of IFN-γ compared to cells from the restimulated saline and MPLA groups (Figure 8A). Furthermore, IFN-y levels for the Fc and Cyclo groups were higher than those observed in the L1040 group. Regarding the cells of the control groups, there was no statistically significant difference between them when stimulated with each protein, although higher levels of IFN-γ were observed when the stimulus was with rFc or rCyclo (Figure 8A). '[049] Regarding the production of IL-10, the Fc group presented levels similar to those of its control groups, or even to those produced by the L1010 group (Figure 8B). Regarding the results for the rFc protein, it can be said that vaccination with this protein generated a preferential Th1 response (Figure 8C), with low production of IL-10, important in establishing a protective immunity against the parasite.

[050] The Cyclo group had higher levels of IL-10 than its controls and other vaccine groups (Figure 8B). In addition, cells from the control groups also produced more IL-10 when restimulated with rCyclo, than with rLi1040; and levels similar to those restored with rFc (Figure 8B). When analyzing the data for the rCyclo protein, it can be said that there was a predominance of the Th1 response pattern (Figure 8C), despite the high

18/22 levels of IL-10 found (Figure 8B). When comparing the ratio between the levels of IFN-γ and IL-10 in the vaccinated groups, a higher value was observed for the Fc group compared to the Cyclo group, a difference that was not statistically significant when the values of the Fc group were compared to the L1040 group (Figure 7C). The production of high levels of IFN-γ (Figure 8A) showed that the Fc and Cyclo proteins are immunogenic.

[051] Splenocytes from animals vaccinated with the Li1040 protein produced increased levels of IFN-γ compared to cells in the saline and MPLA groups (Figure 8A); in addition to low levels of IL-10, similar to those found in their control groups (Figure 8B). With these data, it can be said that vaccination with the L101040 protein generated a preferential Th1 response (Figure 8C), with low IL-10 production, important in the establishment of a protective immunity against the parasite. This information also shows the immunogenicity of the Li1040 protein, showing its potential as a vaccine.

Example 6- Evaluation of the humoral response generated by the L101040 protein by ELISA [052] The humoral response generated by the rLi1040 protein was also evaluated in serum samples. The results showed that immunization with protein Li1040, or A2 + Li'1040, was able to generate specific antibodies, with high IgG production and its subclasses lgG1 and lgG2a (Figure 9A, 9B and 9D). Through the ratio of lgG2a to lgG1 (Figure 9C), it can be noted that, during immunization and after the challenge, the production of lgG2a was greater than lgG1 in animals in the Li1040 group, or A2 + L101040. It is known that the high production of lgG2a is directly related to the production of IFN-γ. Thus, the results found in the groups that received LI1040 alone or associated with A2, are in accordance with the literature, since high levels of IFN-γ were produced by these groups (Figures 8A).

[053] The protection observed in the groups that received the LI1040 protein (Figure 7A) can be related not only to the high production of IFN-γ (Figures 8A), associated with the activation of TCD4 (Th1) and TCD8 cells, but also the greater production of lgG2a (Figure 9C). This antibody is

19/22 related to opsonization activities, activation of the complement system and death of pathogens; whereas lgG1 is generally associated with neutralization. Resistance to Leishmania infection has been attributed not only to a cellular response preferentially of the Th1 type, but also to the increased production of IgG2a by the hosts. BALB / c mice, susceptible to infection by L. major, mount a predominantly Th2 response and greater production of IgG1; while C57BL / 6 mice have a predominant Th1 response with main production of lgG2a.

Example 7- Prediction of epitopes for human and murine TCD8, TCD4 and B cells, using different prediction programs [054] The sequences of the selected proteins were initially subjected to the prediction of epitopes for TCD8, TCD4 and B cells, human and murines, using the prediction programs BIMAS, NetCTL 1.2, NetMHCII 2.2 and BepiPred 1.0, which allowed to obtain a diverse set of epitopes with recognition by different hosts.

[055] For NetCTL 1.2, a conservative approach was used to minimize the probability of identifying false-positive epitopes, using a cut-off greater than 1, which means 70% sensitivity and 98.5% specificity. For NetMHCII 2.2, 26 HLA class II supertypes were considered, being 14 HLADR, 6 HLA-DQ and 6 HLA-DP. Peptides of 9-15 amino acids that bind with an affinity of less than 50 nM to at least 30% of different alleles were considered potential epitopes. The program was also used in the prediction of epitopes for 2 murine MHC II alleles (Η-2-IAb and Η-2-IAd), considering as a cut-off a binding affinity less than 50 nM. For BepiPred 1.0, a cut-off equal to 1.3 was used for each amino acid, guaranteeing a specificity of 96% and 13% sensitivity.

[056] From the BIMAS program, peptides were obtained containing 9 amino acids, and others made up of larger regions (chimeras), generated by the overlapping of predicted epitopes for the same haplotype, or

20/22 among different MHC I haplotypes. The predicted epitopes were included in table 1.

[057] When using the other prediction programs, the predicted epitopes for each cell type and host were pointed out within the amino acid sequence through the coordinates containing the first and last amino acids, by computational analysis. The choice of new peptides was based on the following criteria: i) recognition by BALB / c (adopted experimental model); ii) recognition by a greater number of cell types and hosts; iii) TCD8 recognition, if possible, in more than one host. The predicted and selected epitopes were also included in table 1.

Table 1: Epitopes predicted for human and murine TCD8, TCD4 and B cells

Protein Sequence Peptide coordinates Biologically tested Cyclo EKYSVKVTG 282-290 yea Fc VDLLNVKAM 227-135 yea Li 1040 RGWRPYSL 127-135 yea Li 1040 DYVKNVSDV 310-318 yea Li 1040 SPVAASAITYAISNFLPYANNF 19-40 Yes Li 1040 HAADDTLALL 288-297 yea A2 VGPQSVGPLS 47-56 Yes (control, already described in the literature) A2 SASAEPHKAAVDVGPLS 23-39 Yes (control, already described in the literature) A2 WGPQ.SVGPLS 40-49 Yes

Example 8- Evaluation of immunogenicity in biological assay for predicted osepitopes [058] BALB / c animals infected with promastigotes were used in the stationary phase of Leishmania amazonensis, subcutaneously, in the footpad of the right hind leg of each animal. The infection was followed up weekly with a caliper measurement of the lesion. When the diameter of the lesion reached a certain size, after approximately 3 months, the animals were sacrificed by cervical dislocation, and the spleens

21/22 collected and kept refrigerated. The organs were divided into RPMI medium supplemented with fetal bovine serum, penicillin and streptomycin. The cells were centrifuged and the supernatant was discarded after centrifugation. Splenocytes were treated with erythrocyte lysis solution. Sterile PBS was added to the tubes, which were then subjected to further centrifugation. The supernatant was discarded and the pellet resuspended in PBS / 1% SFB. The samples were diluted in Tripan blue in a proportion of 1: 100 and counted in a Neubauer chamber. The cells were adjusted to a concentration of 2 x 10 ⁶ cells / ml. The final volume was 1.5 mL per well, in a 48-well plate. As a positive control of proliferation, cells were stimulated with Concanavalin A (ConA); and as basal control of proliferation, wells were prepared only with complete RPMI medium. The plates were incubated in suitable conditions for 72 h. After incubation, the contents of each well were homogenized and transferred to a microcentrifuge tube. The tubes were centrifuged and the supernatant frozen for further analysis of cytokine production by CBA.

[059] The obtained supernatants were used in the detection of cytokines by CBA (Cytometric Bead Array), by the Mouse Th1 / Th2 cytokine kit (BD). A mixture of beads and Reagent B (PE-labeled detection reagent) was added to each sample. The standard curve was also prepared, and Reagent B was also added to each dilution. The 96-well plates were incubated at room temperature and protected from light. The tubes were prepared for compensation, tube A (compensation beads + buffer), tube B (compensation beads + FITC-conjugated antibody control + buffer) and tube C (compensation beads + PE-conjugated antibody control + buffer), which were ready for later reading.

[060] After incubation, buffer (Reagent F) was added to each sample and the plates were centrifuged at. The supernatant from each well was aspirated with the aid of a vacuum pump, leaving a minimum volume to avoid aspiration of the pellet. The entire content was transferred to tubes and the reading was performed on a flow cytometer. Data analysis was performed by FCAP

22/22 mL per well, in a 48-well plate. As a positive control of proliferation, cells were stimulated with Concanavalin A (ConA); and as basal control of proliferation, wells were prepared only with complete RPMI medium. The plates were incubated in suitable conditions for 72 h. After incubation, the contents of each well were homogenized and transferred to a microcentrifuge tube. The tubes were centrifuged and the supernatant frozen for further analysis of cytokine production by CBA.

[060] The obtained supernatants were used in the detection of cytokines by CBA (Cytometric Bead Array), by the Mouse Th1 / Th2 cytokine kit (BD). A mixture of beads and Reagent B (PE-labeled detection reagent) was added to each sample. The standard curve was also prepared, and Reagent B was also added to each dilution. The 96-well plates were incubated at room temperature and protected from light. The tubes were prepared for compensation, tube A (compensation beads + buffer), tube B (compensation beads + FITC-conjugated antibody control + buffer) and tube C (compensation beads + PE-conjugated antibody control + buffer), which were ready for later reading.

[061] After incubation, buffer (Reagent F) was added to each sample and the plates were centrifuged at. The supernatant from each well was aspirated with the aid of a vacuum pump, leaving a minimum volume to avoid aspiration of the pellet. The entire content was transferred to tubes and the reading was performed in a flow cytometer. Data analysis was performed using the FCAP Array software, relating the average fluorescence intensity of the samples to the values obtained on the calibration curve constructed in the test. [062] All peptides tested were shown to be immunogenic by the increased production of IL-4 and / or IL-5 and / or IFN-γ (Figure 10).

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Claims (8)

1- Leishmaniasis vaccine composition characterized by comprising at least one of the three recombinant proteins of Seq ID N ° 1 to 3 or at least one of the peptides of Seq ID N ° 4 to 12, and pharmacologically and pharmaceutically acceptable excipients. 2- Leishmaniasis vaccine composition, according to claim 1, characterized by being administered by dermal, intramuscular, intravenous, intraperitoneal, subcutaneous, transdermal routes or as devices that can be implanted or injected. 3- Use of recombinant proteins and peptides defined in claim 1, characterized by being in the preparation of vaccine compositions against leishmaniasis. 4- Use of the vaccine composition defined in claim 1, characterized by being in the treatment and prevention of canine and human leishmaniasis. 1/8 FIGURES FIGURE 1 2/8, ECO «H7C8: bcsaiw / i ^ HáTtf t1lr29 / Sca Jtiiwj s. rMl ii5fS3i PSI) 4 & S «.! _z 8ixmc2 II2e7) _z 8af »Hlp» 9} _z xC.XhOil324) 1 (331) h'- 1 ^ co toesl · Xba Itf-Kb BçHiwwj SçrA Líí »| ^ • Spb içe «») \ - ScoN Ιί’ΐΗ X AS * Tí IpZK) j pET-15b $ 7GW! ? 2. \ l <-ScJl <lim33> 2 | i f jSstc fim®?) !! h <& | i pAf * 3 ίί.'Ί4ΓΓί 5 Ül ‘/ e * sw? <£ h 7¾ SsoUrt 1 Líwa? <\ Sap / \ BSW07 ίίΐΜμ X ACC Íf35 * c> 8S & A Ifísri »7 Ttfttll! («*« · 'ΛΉρα l (x? “TP ^k 8sm toro *) 'Mm ltC7Ci> / P? $ HA 3Γ2β = β · ϊ> pieces and £ 2 »M3 Mu Íí231« i ·, Βερλί li | 23M) FIGURE 2 FIGURE 3 3/8 FIGURE 4 A. Cyclo B. Fc C. Li1040 D.A2 FIGURE 5 4/8 A. Spleen B. Liver Number of parasites / 10 ⁶ cells Number of parasites / 10 ⁶ cells 0.00860.00660.00460.00260.00060.0004-r 0.00030.00020.00010.0000IOílO o _ II —-— I - ^ r O ° T 0 Oo 4® -A® τ-1-1-r :. Lymph nodes 6-, 54321- ¹ · 0.75040.50040.25040.0004- ^ 0.0-r -0.5-1.0Animal group I Animal group Animal group FIGURE 6 5/8 Spleen FIGURE 7 6/8 Stimuli Stimuli Saline ES3 MPLA and Liio4o Fc niii Cyclo Stimuli FIGURE 8 7/8 A. lgG1 B. lgG2a lgG2a / lgG1 ratio O Abs _O o «2nm Saline U1040 A2 + LÍ1Q40 - * MPLA. lgG2a / lgG1 D. Total IgG Days after first dose Li 1040 A2 + LI1040 Saline LI1040 A2 + LI1040 MPLA FIGURE 9 8/8 Stimuli Stimuli Stimuli Stimuli 3000η 2500- 3 £ 2000- Ç" Q. 1500- 50-r O z 40- Ll_ 30- 20- 10- n ° _o ã n%.> O Stimuli FIGURE 10 1/1