BR102021000794A2 - CHIMERIC PROTEIN, KIT, METHOD FOR DIAGNOSING LEISHMANIASIS, USE OF A CHIMERIC PROTEIN, VACCINE COMPOSITION AGAINST VISCERAL LEISHMANIASIS, AND, USE OF A VACCINE COMPOSITION - Google Patents

Abstract

CHIMERIC PROTEIN, KIT, METHOD FOR DIAGNOSIS OF LEISHMANIASIS, USE OF A CHIMERIC PROTEIN, VACCINE COMPOSITION AGAINST VISCERAL LEISHMANIASIS, AND, USE OF A VACCINE COMPOSITION The present invention relates to the field of diagnostic medicine, vaccinology and biotechnology. More specifically, the present invention relates to a chimeric protein for diagnostic application of visceral leishmaniasis in humans and dogs, including human immunodeficiency virus (HIV) coinfected individuals, and a vaccine containing said chimeric protein for prophylactic or therapeutic use.

Description

CHIMERIC PROTEIN, KIT, METHOD FOR DIAGNOSING LEISHMANIASIS, USE OF A CHIMERIC PROTEIN, VACCINE COMPOSITION AGAINST VISCERAL LEISHMANIASIS, AND, USE OF A VACCINE COMPOSITION FIELD OF THE INVENTION

[001] The present invention relates to the field of diagnostic medicine, vaccinology and biotechnology. More specifically, the present invention relates to a chimeric protein for diagnostic application of visceral leishmaniasis in humans and dogs, including individuals co-infected with the human immunodeficiency virus (HIV) and a vaccine containing said chimeric protein for prophylactic or therapeutic.

FUNDAMENTALS OF THE INVENTION

[002] Leishmaniasis are diseases caused by parasites of the genus Leishmania, with more than 20 species being known. Transmission occurs through the bite of an infected insect of the genera Phlebotomus and Lutzomyia. Three main types of leishmaniasis are described: visceral leishmaniasis, the most severe form; cutaneous; and mucocutaneous. The disease, if left untreated, can lead to death within two years in 95% of cases.

[003] Currently, leishmaniasis constitute an important global public health problem. Epidemiological data from the World Health Organization show that 900,000 to 1.3 million new cases and 20 to 30 thousand deaths occur annually. Countries in Africa, Asia and Latin America are the most affected, and in 2017, more than 90% of visceral leishmaniasis cases were reported by seven countries: Brazil, Ethiopia, India, Kenya, Somalia, Sudan and South Sudan. The occurrence of leishmaniasis is more frequent in low-income populations and is associated with poor nutrition, poverty and low functionality of the immune system, with children and young adults being the most affected.

[004] According to data from the Ministry of Health available on DATASUS, almost four thousand cases of visceral leishmaniasis were confirmed in Brazil in 2018, with the highest incidence in the northeast and north, followed by the Southeast. In that same year, almost three hundred deaths caused by visceral leishmaniasis were reported in the country.

[005] The current strategies of the Ministry of Health of Brazil for the prevention of visceral leishmaniasis are focused on the diagnosis and early treatment of human cases, in addition to the prevention and control of human cases, vectors and reservoirs, that is, dogs with positive serology and / or positive parasitological.

[006] The effectiveness of treatment is low, being influenced by both host-related factors and the species of parasite causing the disease. The drugs used in the treatment include: pentavalent antimonies, such as sodium stibogluconate and meglumine antimoniate, amphotericin B and its liposomal formulations, miltefosine, paramomycin, allopurinol and pentamidine, being used in monotherapy or in combination. All have limitations, such as long duration of treatment, resistance, acquired, high toxicity, side effects and high cost.

[007] Dogs play an important role in the maintenance of the disease, since an increasing number of infections in dogs can lead to an increase in the incidence of the disease in humans. Therefore, early detection of infected dogs is an essential factor in controlling the spread of the disease in humans. Prevention measures recommended by the Brazilian Ministry of Health include care for dogs, such as performing serological tests before adoption, application of insecticides, use of screens in kennels and the use of collars with 4% deltamethrin. Diagnostic failures, resulting for example from the low sensitivity of the tests applied and the prolonged stay of asymptomatic and infected dogs in endemic areas, negatively impact the control of leishmaniasis.

[008] Early diagnosis is also of paramount importance in human cases, since there is a high lethality rate in Brazil, being especially important in patients with HIV (Human Immunodeficiency Viruses), since co-infection with visceral leishmaniasis accelerates the appearance of first symptoms of AIDS (acquired immunodeficiency syndrome) and reduces the life expectancy of patients infected with the virus (Thakur S, Joshi J, Kaur S. Leishmaniasis diagnosis: an update on the use of parasitological, immunological and molecular methods [published online ahead of print, 2020 Mar 16] . J Parasit Dis. 2020;44(2):1‐20).

[009] The clinical diagnosis of human visceral leishmaniasis is not very useful, as there are asymptomatic cases, and mild or even severe cases have signs and symptoms common to other diseases, such as malaria, Chagas disease, leukemia, toxoplasmosis and schistosomiasis. . Thus, laboratory methods are the only way to confirm the diagnosis.

[0010] The laboratory diagnosis of canine visceral leishmaniasis is usually based on parasitological and serological techniques. The serological diagnostic tests recommended by the Ministry of Health are the dual-path immunochromatographic tests (DPP® CVL rapid test - manufactured by Bio-Manguinhos/Fiocruz, Rio de Janeiro), as a screening, and the ELISA with total extract, as a confirmatory test. . The tests currently available in the Brazilian public health network for the diagnosis of human visceral leishmaniasis are the indirect immunofluorescence reaction (IFAR) and the immunochromatographic OnSiteTM Leishmania – Beijing Genese Biotech Inc, which uses rK39 and K28 as antigens.

[0011] Molecular techniques are useful tools in the diagnosis of visceral leishmaniasis in cases with inconclusive results, being possible to perform in reference laboratories in order to have the diagnosis confirmation. It is possible to obtain highly sensitive and specific results using the real-time PCR technique. However, the use of PCR is a valuable tool for molecular and epidemiological studies, being little used in laboratory routine because it is a difficult method to perform, high cost, unfeasible in a laboratory with low infrastructure and difficult to perform in the field.

[0012] In this context, serological techniques are extremely relevant. The development of a Leishmania species-specific crude or recombinant antigen is a valuable step for serological diagnosis, as the sensitivity and specificity of the test depend on the antigen used.

[0013] Misdiagnosis can lead to death in patients with visceral leishmaniasis. A reliable and efficient diagnostic method for visceral leishmaniasis requires the detection of symptomatic and asymptomatic cases, high sensitivity and specificity, being easy to perform, low cost, feasible in the laboratory with low infrastructure and easy to perform in the field. The diagnostic tests currently available do not meet all these requirements and it is still possible to achieve higher sensitivity and specificity by improving the antigens used. In addition, it is necessary to develop a more sensitive method for the diagnosis of visceral leishmaniasis in cases of co-infection with HIV.

[0014] Regarding the prevention of the disease, it is important to note that so far there is no licensed vaccine in the world for human visceral leishmaniasis, but only against infection in dogs. Leish-Tec®, which uses the A2 protein as a recombinant antigen, is the only commercially available vaccine for use in dogs in Brazil. Leish-Tec® has an efficacy of 72%-80%, like the other vaccines available in Europe (Canileish and Letifend). Canileish and Leish-Tec vaccines are administered in 3 doses, which is still an inconvenience to achieve maximum protection quickly and, therefore, there is still room to improve vaccine efficacy and the way of administering vaccines in dogs.

[0015] The chimeric protein of the present invention, called DTL4, comprises repetitive immunogenic fragments of the A2 and K39 proteins. A2 is mainly expressed in amastigote forms of viscerotropic species, being predominantly composed of repetitive sequences of 10 amino acids. K39 contains repeating sequences of 39 amino acids and is related to Leishmania kinesin kinesin.

[0016] Several studies investigate the use of A2 and its epitopes for vaccine use (Resende D.M., Caetano B.C., Dutra M.S., et al (2008) Epitope mapping and protective immunity elicited by adenovirus expressing the Leishmania amastigote specific A2 antigen: correlation with IFN -gamma and cytolytic activity by CD8+ T cells. Vaccine 26, 4585-4593; Coelho E.A., Tavares C.A., Carvalho F.A., et al (2003) 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, 3988-3994; Zanin F.H., Coelho E.A., Tavares C.A., et al (2007) 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, 1070-1077; Fernandes A.P., Costa M.M., Coelho E.A., et al (2008) Protective immunity against challenge with Leishmania ( Leishmania) chagasi in beagle dogs vaccinated with recombinant A2 protein. Vaccine 26, 5888-5895; Fernandes AP, Coelho EA, Machado-Coelho GL, et al. 2012 Making an antiamastigote vaccine for visceral leishmaniasis: rational, update and perspectives. Curr Opin Microbiol.;15(4):476-85). A2 has also been used as a target antigen in several assays for the detection of visceral leishmaniasis, being the only amastigote-specific protein marker in L. donovani (Coura-Vital W, Ker HG, Roatt BM, et al. Evaluation of change in canine diagnosis protocol adopted by the visceral leishmaniasis control program in Brazil and new proposal for diagnosis. PloS One. 2014; 9 (3): e91009; Technical note, N.11/2016/CPV/DFIP/DAS/GM/MAPA. [Internet] ] ; 2018. Available from: http://www.sbmt.org.br/portal/wp-content/uploads/2016/09/nota-tecnica.pdf; Sundar S, Rai M. Laboratory diagnosis of visceral Leishmaniasis. Clin Diagn Lab Immunol. 2002; 9(5): 951-958 ). Anti-A2 antibodies can be detected by ELISA in both human and canine serum samples. Detection varies between 60 and 82% of samples, being higher in symptomatic individuals (Ghedin et al. Antibody response against Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis. Clin. Diagn. Lab. immunol. 4:530- 535; Porrozzi et al. Comparative Evaluation of Enzyme-Linked Immunosorbent Assays Based on Crude and Recombinant Leishmanial Antigens for Serodiagnosis of Symptomatic and Asymptomatic Leishmania infantum Visceral Infections in Dogs. Clin Vaccine Immunol. 2007;14(5):544–548).

[0017] The K39 protein is the recombinant antigen used in the rapid test available in the Brazilian public health network. This test consists of an immunochromatographic assay and has a sensitivity and specificity of 91.2% and 94.5%, respectively. In ELISA tests, diagnosis with rK39 has sensitivity and specificity of up to 98% and 100%, respectively.

[0018] However, the sensitivity of tests using this antigen is significantly reduced in patients with HIV coinfection. Bangert et al., for example, report that the sensitivity of the rK39-based rapid immunochromatographic test was 83.1% in HIV-negative patients and 67.3% in HIV-positive patients (Bangert M, Flores-Chávez MD, Llanes-Acevedo IP et al. Validation of rK39 immunochromatographic test and direct agglutination test for the diagnosis of Mediterranean visceral leishmaniasis in Spain. PLoS Negl Trop Dis. 2018 Mar; 12(3):e0006277). Cota et al. reported in HIV positive patients a sensitivity of 60.9% and 45.6% for immunochromatographic and indirect immunofluorescence (IFA) tests based on K39, respectively (Cota GF, de Sousa MR, de Freitas Nogueira BM, et al. Comparison of parasitological, serological, and molecular tests for visceral leishmaniasis in HIV-infected patients: a cross-sectional delayed-type study. Am J Trop Med Hyg. 2013 Sep; 89(3):570-7.).

[0019] In addition, when used for diagnosing visceral leishmaniasis in asymptomatic dogs, rK39 ELISA assays also have reduced sensitivity and specificity, with values around 69.4% and 96%, respectively (Reithinger R, Quinnell RJ , Alexander B, et al. Rapid detection of Leishmania infantum infection in dogs: a comparative study using an immunochromatographic dipstick test, enzyme-linked immunosorbent assay, and PCR. J Clin Microbiol. 2002; 40 (7): 2352-2356; Mettler M , Grimm F, Capelli G, et al.Evaluation of enzyme-linked immunosorbent assays, an immunofluorescent-antibody test, and two rapid tests (immunochromatographicdipstick and gel tests) for serological diagnosis of symptomatic and asymptomatic Leishmania infection in dogs.J Clin Microbiol. 2005; 43 (11): 5515-5519; Grimaldi GJr, Teva A, Ferreira AL, et al. Evaluation of a novel chromatographic immunoassay based on Dual-Path Platform technology (DPP® CVL rapid test) for the serodiagnosis of can ine visceral leishmaniasis. Trans R Soc Trop Med Hyg. 2012; 106 (1): 54-59; Laurenti MD, de Santana LMVJr, Tomokane TY, et al. Comparative evaluation of the DPP®CVL rapid test for canine serodiagnosis in the area of visceral leishmaniasis. Vet Parasitol. 2014; 205 (3-4): 444-450.).

[0020] Although K39 is used in diagnostic tests, this antigen is not normally used in vaccine formulations. According to Reed et al., 2018, most of the immunodominant components of Leishmania, such as the repetitive antigens represented by K39, are not able to induce protection in animal models, regardless of the adjuvant formulation or inoculation method (Reed et al, Leishmania Vaccine Development : Exploiting the Host-Vector-Parasite Interface. 2018).

[0021] Proteins A2 and K39 have previously been used in combination for diagnostic use, but not in the chimeric protein form as in the present invention. Below are cited studies that make use of these two antigens together.

[0022] Patent document PI1013447-6 - Recombinant peptides, method and kit for immunodiagnostic test of leishmaniasis, for example, refers to a composition and kit for diagnosing visceral leishmaniasis based on the association of recombinant antigens A2 and rK39. More specifically, epitopes of these antigens, called peptides 47 and 13, respectively, are used. These peptides were synthesized alone and tested in association (adsorbed in the same well of the microtiter plate)

[0023] Costa et al. evaluated combinations of peptides derived from A2, K39, NH and LACK seeking to optimize the immunodiagnosis of human and canine visceral leishmaniasis (Costa et al. Improved canine and human visceral leishmaniasis immunodiagnosis using combinations of synthetic peptides in enzyme-linked immunosorbent assay PLoS neglected tropical diseases vol 6,5 (2012): e1622). Synthetic peptides were used, alone or combined in the same reaction. The combination of peptides 13 (derived from A2) and 47 (derived from K39) resulted in an increase in sensitivity and specificity in the ELISA test using canine sera with low antibody titers. It is worth noting that the sensitivity of peptide 47 was not affected by the combination with peptide 13 in canine sera with intermediate levels of antibodies, remaining at 85%. In humans, the combination of peptides 13 and 47 resulted in increased sensitivity and specificity in patients in the acute phase of infection, reaching 100% and 96%, respectively.

[0024] There are also studies in the state of the art dealing with the vaccine or diagnostic use of chimeric proteins obtained from the fusion of A2 or K39 (or their epitopes) to other peptides. It is worth mentioning that it is known in the state of the art that the combination of two or more antigens, obtained in isolation, can increase the immune response induced by vaccine compositions or the detection capacity of diagnostic kits. However, the combination of two or more antigens in the form of a chimeric protein does not follow the same premise, since the chimera obtained has a unique and unpredictable three-dimensional structure, which can completely alter its ability to bind antibodies. increase or decrease the antigenicity and/or serological detectability of antibodies in a biological sample (Hollingshead et al. Structure-based design of chimeric antigens for multivalent protein vaccines. Nat Commun 9, 1051 (2018)).

[0025] There has also been reported the application of different recombinant proteins for the serological diagnosis of Visceral Leishmaniasis in humans and dogs, more specifically, recombinant proteins developed from the recombinant proteins rkE16, rk26, rK39 and rA2 have been reported (Farahmand M, Nahrevanian, H. (2016). Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs. Iranian Biomedical Journal 20(3): 128-134 July 2016. doi: 10.7508/ibj.2016.03.001 ). There is still in the state of the art the development of enzyme immunoassay (ELISA) and immunochromatographic tests (ICT) for the serological diagnosis of human VL (LVH) and canine VL (LVC), using three recombinant proteins of Leishmania infantum (rFc , rC9 and rA2) selected from proteomic and genomic analyzes (dos Santos ARR, Serufo AV, Figueiredo MM, Godoi LC, Vitorio JG, Marcelino AP, de Avelar DM, Rodrigues FTG, Machado-Coelho GLL, Medeiros FAC, Jerônimo SMB , de Oliveira EJ, Nascimento FC, Texeira SMR, Gazzinelli RT, Nagem RAP, Fernandes AP. (2019). Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis. Memória Instituto Oswaldo Cruz, Rio de Janeiro , Vol. 114: e180405, 2019).

[0026] Patent US8911746 - Recombinant polyprotein vaccines for the treatment and diagnosis of leishmaniasis, for example, describes fusion polypeptides comprising combinations of the A2, KMP11, SMT and CPB antigens (or immunogenic portions or variants thereof) for use in the prevention, treatment and detection of leishmaniasis. These same antigens are used in the construction of the chimeric protein disclosed in patent US10364274 - Expression of chimeric KSAC protein and method of producing soluble proteins by high pressure, which describes a vaccine composition comprising such a chimeric protein and a method of producing the same using high pressure.

[0027] Patent US9909114 - Vaccines comprising leishmania polypeptides for the treatment and diagnosis of leishmaniasis, describes a fusion peptide for vaccine use comprising polypeptides or variants of NH and SMT combined with polypeptides or variants of CPB, mtHSP70, H2BN, A2, p21 or LeiF.

[0028] Patent US8771710 - Fusion proteins and their use in the diagnosis and treatment of leishmaniasis, describes a method for diagnosing canine or human visceral leishmaniasis based on a chimeric protein called rK28. rK28 is formed by immunogenic portions of peptides rK39, rK9 and rK26. The portion of rK39 used comprises residues 2110-2343 of K39 from L. donovani. The results obtained suggest that rK28 detects asymptomatic or subclinical cases of leishmaniasis with greater sensitivity than the respective antigens that form the chimeric protein alone. The patent also describes a possible vaccine use of this protein. It is worth mentioning that rK28 is used in the Dual-Path Platform (DPP®) CVL rapid test.

[0029] Boarino and collaborators also developed a multiepitope chimeric protein formed by the K26, K9 and K39 antigens. However, a fragment corresponding to a single subunit of 39 amino acids of K39 was used in this construction. These authors evaluated the use of this chimeric protein in the serological diagnosis of visceral leishmaniasis (Boarino et al. Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes of Leishmania infantum for Serodiagnosis of Visceral Leishmaniasis, 12(5), 647–653. 2005).

[0030] In view of the need for an assay for the diagnosis of human and canine visceral leishmaniasis that is fast, and has good precision and accuracy, the present technology consists of a method and a kit for the serological diagnosis of visceral leishmaniasis, based on a protein chimeric, which allows the qualitative determination of anti-Leishmania IgG antibodies in canine and human serum or plasma. Antibodies specific to Leishmania infantum, when present in the sample, bind to recombinant antigens immobilized on the plate, forming antigen-antibody complexes that are later identified by enzyme and substrate. The diagnostic method of the present invention is a reliable and efficient method, easy to perform, low cost, feasible in the laboratory with low infrastructure and easy to perform in the field.

[0031] In view of the absence of a vaccine available for human visceral leishmaniasis and the need for vaccine improvement for the canine vaccine (vaccines available for dogs show 70-80% efficacy in clinical trials), the fusion protein of the present invention can to compose vaccine formulations against human and canine visceral leishmaniasis. Due to the growing evidence of the emergence of resistance to treatment for leishmaniasis, which limits the number of already scarce drugs available, an effective prophylactic vaccine would be the best way to reduce infection and disease. A therapeutic vaccine would also be of great help in developing a more effective treatment.

[0032] The chimeric protein DTL4, which the present invention deals with, was constructed after extensive experimentation and is the result of the fusion of repetitive immunogenic fragments of the A2 and K39 proteins. Among the advantages of using a chimeric protein instead of several synthetic peptides or two recombinant proteins separately are greater ease, traceability and lower production cost, in addition to greater homogeneity between different batches.

[0033] Regarding diagnostic use, the secondary structure formed by several aligned and covalently linked peptides led to increased sensitivity and specificity of the test using DTL4 compared to A2 and K39 antigens, allowing better detection of anti-Leishmania infantum IgG even in patients HIV positive, without interference from other diseases such as cutaneous leishmaniasis, Chagas disease, increased rheumatoid factor, lupus, malaria, paracoccidioidomycosis, syphilis, tuberculosis and toxoplasmosis.

[0034] Regarding vaccine use, immunization with DTL4 resulted in a significant reduction in the parasite load in the spleen and liver of immunized animals, evidencing the ability of this chimeric protein to induce protection against visceral leishmaniasis. In addition, higher IgG2a production was observed after immunization with DTL4 compared to immunization with A2, suggesting that the protein of the present invention has a greater potential to trigger a Th1 response profile in mice, a valuable feature in a vaccine candidate against to leishmaniasis. The vaccine of the present invention can also be used in the treatment of leishmaniasis.

SUMMARY OF THE INVENTION

[0035] The present invention aims to provide a protein, defined by SEQ ID NO 1, for the diagnosis, prophylaxis and treatment of visceral leishmaniasis in humans and dogs.

[0036] Said protein is in the form of a chimeric protein construct comprising efficient antigens for the diagnosis, prophylaxis and treatment of visceral leishmaniasis.

[0037] Particularly, the present invention relates to the provision and use of a chimeric protein for the diagnosis, prophylaxis and treatment of visceral leishmaniasis in humans and dogs.

[0038] In a first embodiment, the present invention relates to a chimeric protein comprising antigenic regions of proteins efficient for the diagnosis of visceral leishmaniasis, including in patients coinfected with HIV.

• a) a proteína quimérica da presente invenção;
• b) um anticorpo secundário ou uma proteína, conjugados ou não a uma enzima ou a um marcador; e
• c) um reagente para detectar o anticorpo secundário ou a proteína.

[0039] In a second embodiment, the present invention provides a kit for the diagnosis of visceral leishmaniasis, the kit comprising:

• a) the chimeric protein of the present invention;
• b) a secondary antibody or a protein, whether or not conjugated to an enzyme or a label; and
• c) a reagent to detect the secondary antibody or protein.

• a) expor uma amostra à proteína quimérica da presente invenção;
• b) identificar as amostras contendo anticorpos reativos à proteína quimérica apontada acima utilizando reagentes apropriados.

[0040] In a third embodiment, the present invention provides a method for diagnosing visceral leishmaniasis comprising the steps of:

• a) exposing a sample to the chimeric protein of the present invention;
• b) identifying the samples containing antibodies reactive to the chimeric protein noted above using appropriate reagents.

[0041] In a third embodiment, the present invention provides the use of said chimeric protein in the preparation of a composition for the prophylaxis and treatment of human and canine visceral leishmaniasis.

[0042] In a fourth embodiment, the present invention provides a vaccine composition comprising the protein defined by SEQ ID NO 1 for the prophylaxis and treatment of human and canine visceral leishmaniasis.

[0043] In the descriptions below, the protein defined by SEQ ID NO 1 is referred to as DTL4.

BRIEF DESCRIPTION OF THE FIGURES

[0044] Figure 1 refers to the expression and solubility test for the purification step of the chimeric protein DTL4. The asterisk and arrow signal the expression of soluble DTL4 in Escherichia coli BL21 (DE3) cells.

[0045] Figure 2 refers to the affinity chromatography test on a 5ml HisTrap HP (GE) column in the Akta system. The asterisk indicates the elution of DTL4 in the 40 kDa range.

[0046] Figure 3 refers to the DTL4 purification step. The 40kDa band can be seen indicating purified DTL4.

[0047] Figure 4 refers to ELISA assays for detection of anti-DTL4 total IgG in patient and dog sera. (A) Detection of anti-DTL4 IgG in a pool of patient sera samples positive (n=10) and negative (n=10) for visceral leishmaniasis, using different concentrations of the DTL4 protein. (B) Comparison of the performance of DTL4, A2 and K39 in detecting anti-DTL4 IgG in a pool of positive (Pos) (n=10) and negative (Neg) (n=10) patient sera samples for visceral leishmaniasis , using different protein dilutions (C) ELISA-DTL4 evaluation using 469 patient samples (154 positive and 315 negative). The dotted line represents the cut point. All positive samples were diagnosed by indirect immunofluorescence reaction (IFAT), in-house ELISA with rK39 and/or immunochromatography - It Leish® (rK39). (D) Detection of anti-DTL4 IgG in a pool of sera samples from positive (Pos) (n=10) and negative (Neg) (n=10) dogs for visceral leishmaniasis, using different concentrations of the DTL4 protein (E) Comparison of the performance of DTL4, A2 and K39 in the detection of anti-DTL4 IgG in a pool of sera samples from positive (Pos) (n=10) and negative (Neg) (n=10) dogs for visceral leishmaniasis, using different protein dilutions.

[0048] Figure 5 refers to an ELISA assay for detection of anti-DTL4 total IgG in sera from HVL+/HIV- (n=167), HVL+/HIV+ (n=62) and healthy (negative) donors. (n=169). All positive samples were diagnosed by parasitological and HIV test. The dotted line represents the cut point. HVL = human visceral leishmaniasis. HIV = human immunodeficiency virus.

[0049] Figure 6 refers to an ELISA assay for detection of anti-DTL4 IgG in a serum sample from patients with Visceral Leishmaniasis (positive control), Cutaneous Leishmaniasis, Chagas Disease, Rheumatoid Factor, Lupus, Malaria, Paracoccidioidomycosis, Syphilis, Tuberculosis, Toxoplasmosis. Samples from healthy donors were used as a negative control.

[0050] Figure 7 corresponds to the interpretation scheme of the results of the rapid test to detect IgG anti-Leishmania infantum. In the left panel (A) a rapid test scheme for detecting IgG anti-L. infantum. A positive sample and a negative sample are represented in B and C, respectively.

[0051] Figure 8 corresponds to the specific immunoglobulin total IgG and IgG2a response induced to (A) DTL4 and (B) A2 30 days after the last immunization. Statistical differences p<0.05, analyzed by ANOVA: (a) Saline (b) Poly-IC (c) A2+CPG+Alumen (d) DTL4+Poly-IC (e) DTL4+CPG+Alumen.

[0052] Figure 9 corresponds to the levels of (A) INF-γ and (B) IL-10 in splenocyte supernatants from Balb/c mice 30 days after the last immunization. Cultures were stimulated for 48 hours with DTL4 or RPMI. Statistical differences p<0.05, analyzed by ANOVA: (a) Saline (b) A2+CPG+Alumen (c) DTL4+CPG+Alumen.

[0053] Figure 10 evaluates the parasite load in (A) spleen and (B) liver by the limiting dilution assay, 30 days after the challenge of L. infantum infection. The results were expressed as the negative logarithm of the number of parasites and statistical differences p<0.05 were analyzed by ANOVA: (a) Saline (b) Poly-IC (c) A2+CPG+ALUMEN (d) DTL4+ POLY-IC

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention relates to a chimeric protein for the diagnosis, prophylaxis and treatment of canine or human visceral leishmaniasis. The invention further relates to a kit for the diagnosis of visceral leishmaniasis containing this protein and other reagents necessary for detection. Furthermore, the invention relates to a method for diagnosing visceral leishmaniasis based on the chimeric protein and/or the diagnostic kit of the present invention. Finally, the invention relates to a vaccine composition for the prophylaxis and treatment of visceral leishmaniasis containing the chimeric protein.

[0055] The chimeric protein of the present invention is characterized by comprising an amino acid sequence with at least 85% identity to the sequence defined by SEQ ID NO 1.

[0056] In a preferred embodiment, the chimeric protein of the present invention is characterized in that it comprises an amino acid sequence as defined by SEQ ID NO 1, referred to herein as DTL4.

• a) Uma proteína quimérica cuja sequência compreende a sequência de aminoácidos com pelo menos 85% de identidade em relação à sequência definida pela SEQ ID N° 1;
• b) Um anticorpo secundário ou uma proteína, conjugados ou não a uma enzima ou a um marcador;
• c) Um reagente para detectar o anticorpo secundário ou a proteína.

[0057] The Leishmaniasis Diagnostic Kit of the present invention comprises:

• a) A chimeric protein whose sequence comprises the amino acid sequence with at least 85% identity to the sequence defined by SEQ ID NO: 1;
• b) A secondary antibody or a protein, whether or not conjugated to an enzyme or a label;
• c) A reagent to detect the secondary antibody or protein.

[0058] In the Leishmaniasis Diagnosis Kit of the present invention, the chimeric protein can be immobilized on a solid support. This solid support can be composed of a material selected from the group comprising cellulose fiber, nitrocellulose, fiberglass, plastic, nylon, latex, polypropylene and/or polystyrene, the solid support consisting of microplates, microtiter plates, tubes, spheres or membranes.

[0059] The kit for diagnosis of leishmaniasis of the present invention may also contain a carrier selected from the group comprising nanomaterials and/or micromaterials of gold, silver and/or carbon, and/or microparticles (beads) simple or coated with different materials, preferably gold nanomaterials and/or micromaterials.

[0060] The secondary antibody of the leishmaniasis diagnostic kit of the present invention can be selected from the group comprising IgG, IgM, IgA, IgE and their subclasses; the protein may be selected from the group comprising Protein A, Protein G and/or lectins; the enzyme may be selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase, preferably peroxidase; and the label may be selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents.

[0061] The reagent for detecting the secondary antibody or the protein, when they are conjugated to an enzyme or a marker, of the Visceral Leishmaniasis Diagnostic Kit of the present invention can be selected from the group comprising chromogenic substrates, preferably solution containing hydrogen peroxide and tetramethylbenzidine.

[0062] The Leishmaniasis Diagnostic Kit of the present invention may be for enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay, or rapid immunochromatographic assay.

[0063] In a preferred embodiment, the Leishmaniasis Diagnosis Kit of the present invention is a visceral leishmaniasis diagnosis kit.

• a) Expor amostra a uma proteína quimérica cuja sequência compreende uma sequência de aminoácidos com pelo menos 85% de identidade em relação à sequência definida pela SEQ ID N°1;
• b) Identificar as amostras contendo anticorpos reativos à proteína quimérica da etapa “a” utilizando reagentes apropriados.

[0064] The Leishmaniasis diagnosis method of the present invention contains the following steps:

• a) Exposing sample to a chimeric protein whose sequence comprises an amino acid sequence with at least 85% identity to the sequence defined by SEQ ID NO:1;
• b) Identify the samples containing antibodies reactive to the chimeric protein from step “a” using appropriate reagents.

[0065] The identification of samples containing antibodies reactive to the chimeric protein, performed in step "b" of the Method for diagnosing visceral leishmaniasis of the present invention can be done by means of secondary antibodies or a protein that will be later identified using appropriate reagents. Such secondary antibodies or proteins may be conjugated to an enzyme or a label, and the identification of secondary antibodies or proteins may be by means of reagents capable of detecting the enzyme or label conjugated to the antibodies or proteins. Reagents capable of detecting the enzyme or the label conjugated to the secondary antibodies or proteins of the method for diagnosing visceral leishmaniasis of the present invention can be selected from the group comprising chromogenic substrates, preferably solution containing hydrogen peroxide and tetramethylbenzidine. Secondary antibodies may comprise IgG, IgM, IgA, IgE or subclasses thereof; the protein may comprise Protein A and/or Protein G and/or lectins; the enzyme may be selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase, preferably peroxidase; and the label may be selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents.

[0066] The sample of step "a" of the method for diagnosis of leishmaniasis of the present invention can be blood, serum, plasma and/or other body fluid, and the sample can be human or canine.

[0067] The method for diagnosing leishmaniasis of the present invention can be by enzyme immunosorbent assay, enzyme immunoassay or rapid immunochromatographic test.

[0068] In a preferred embodiment, the method for diagnosing leishmaniasis of the present invention is a method for diagnosing visceral leishmaniasis.

[0069] In another preferred embodiment, the Leishmaniasis Diagnosis Kit and the Leishmaniasis Diagnosis Method of the present invention are a kit and a method for the diagnosis of visceral leishmaniasis caused by parasitic infection of L. infantum, L. donovani, L. chagasi and/or L. amazonensis

[0070] The present invention further relates to a vaccine composition containing the chimeric protein defined by SEQ ID NO: 1.

[0071] In a preferred embodiment, the vaccine composition comprises a protein whose sequence comprises the amino acid sequence with at least 85% identity to the sequence defined by SEQ ID No. 1.

[0072] In another embodiment, the vaccine composition further comprises adjuvants.

[0073] Among the non-limiting examples of adjuvants that can be used in the present vaccine composition are Poly-IC and derivatives, water and oil emulsion or alum with unmethylated CpG oligonucleotide, water and oil emulsion or alum with lipid A; squalene; synthetic TLR4 agonist; liposomes associated with unmethylated CpG TLR or TLR4 agonist, Montanide and Monophosporyl Lipid A (MPL), or another adjuvant that induces Th1-type immune response.

[0074] According to this embodiment, the adjuvants are preferably selected from the group comprising Poly-IC and derivatives, CpG and aluminum hydroxide.

[0075] Preferably, the compositions of the present invention can be administered intravenously, intramuscularly or subcutaneously. More preferably, the compositions of the present invention are administered parenterally.

[0076] More specifically, examples of a preparation suitable for parenteral administration include injection, intravenous fluid, solution or suspension for infusion, lyophilized powder for suspension or solution, inhalant powder, and the like.

[0077] Furthermore, formulation according to the present invention may be in a form for oral administration.

[0078] More specifically, examples of a preparation suitable for oral administration include tablet, tablet, capsule, powder, fine granule, granule, solution, suspension, syrup and the like.

[0079] However, the form of preparation should not be limited to these alone.

[0080] In general, the appropriate dose can be administered in one to several portions, or can be administered every few days according to a defined prime-boost protocol.

[0081] In a preferred form of the invention, the composition comprises 10 to 30 µg of DTL4 per dose, a range of 1µg to 1mg of said protein may be used. Furthermore, 10µg to 1mg of Poly:IC adjuvant can be used per dose, preferably 50µg of said adjuvant per dose is used. In the formulation with the adjuvants CPG and Alum, 18µg of CpG and 30% (v/v) of Alum are preferably used, and the range from 1ug to 10 mg of CpG can be adopted, as well as the range from 10% to 90% of Alum.

[0082] The present invention also relates to a method for preventing and treating leishmaniasis wherein a protein whose sequence comprises the amino acid sequence having at least 85% identity to the sequence defined by SEQ ID NO: 1, or a vaccine composition according to the present invention, administered to a subject.

[0083] The present invention can be better understood by the following non-limiting examples.

Example 1 - Production of the chimeric protein DTL4

[0084] The chimeric protein of the present invention has a size of 40 kDa with histidine tag. The synthetic gene that encodes this protein was commercially acquired and cloned into an expression vector in E. coli. The vector used to produce the protein of the present invention was pET24a (+) in an E. coli BL21 (DE3) expression system, in LB (Lysogeny broth) culture medium with 50µg/ml of the antibiotic Kanamycin and inoculum dilution. of 1:100. The culture volume used in the expression protocol was 2 liters. Figure 1 shows the expression test result. After expression for 3 h at 37°C, the culture was centrifuged for 10 minutes at 8000 rpm. In the lysis step, the generated cell pellet was resuspended in 100 ml of the buffer consisting of 20 mM Tris-HCl, 100 mM NaCl pH 8, 5 mM DTT, 5 mM benzamidine and 1 mM PMSF. After resuspension, the resulting suspension was incubated in the homogenizer and then centrifuged at 40,000 g for 30 minutes. Finally, the supernatant was collected for protein purification by Fast Protein Liquid Chromatography (FPLC). In this process, a 5 ml HisTrap HP (GE) affinity column was used in an Akta Prime (GE) system. Purification steps were performed following the column manufacturer's instructions in binding buffer containing 20 mM Tris-HCl, 100 mM NaCl pH8 and elution buffer containing 20 mM Tris-HCl, 100 mM NaCl, 500 mM Imidazole, pH 8. Initially the column was equilibrated with 25 ml of binding buffer at a flow rate of 5 ml per minute, followed by passage of the sample at a flow rate of 3 ml per minute. After this step, the column was washed with 25 ml of binding buffer at a flow rate of 5 ml per minute and finally the protein was eluted in a gradient from 0 to 100% of elution buffer made in a final volume of 50 ml to 3 ml per minute in 2 ml fractions.

[0085] The result of the elution steps is shown in Figure 2 and the purified 40 kDa protein in Figure 3.

Example 2 – Diagnosis of visceral leishmaniasis using the DTL4 protein in an ELISA assay

[0086] The kit and method for diagnosis by ELISA have been validated using human and canine samples. Samples were collected from patients with suspected infection or healthy patients. The samples acquired were classified as being from the general population, without distinction of sex, age group or ethnicity. Suspected patients presented clinical symptoms of human visceral leishmaniasis and underwent clinical and laboratory diagnosis using the following methods: indirect immunofluorescence reaction (IFAT), in-house ELISA with rK39 and/or immunochromatography - It Leish® (rK39). Dog samples were submitted to clinical and laboratory diagnosis, using the following methods: indirect immunofluorescence reaction (IFAT), in-house ELISA with rK39, Kalasar Detect™, Bone marrow parasitology and polymerase chain reaction (PCR).

[0087] The ELISA assay was performed in a sensitized microplate (Corning® 1x8 StripwellTM, 96 well plates, high binding surface, clear #2592) by applying to each well 100µL of carbonate buffer (15mM sodium carbonate, sodium bicarbonate 85mM, pH 9.5) containing 10ng/100µL of the DTL4 recombinant protein. After adding the sensitizing solution, the microplate was incubated overnight at a temperature between 2-8ºC and at the end of the incubation time the solution was discarded and 280µL of the blocking solution (10%w/v sucrose, 150mM sodium chloride and 1%w/v bovine albumin) was added to each well of the microplate. The blocking solution remained in the microplate for two hours at room temperature (between 15 and 30ºC), when it was discarded and the microplate was dried in a room with low humidity and at room temperature. The sensitized and dried microplate was stored at a temperature between 2-8ºC, in a properly sealed aluminum envelope. When performing the test, the wells to be used were separated considering: controls and samples (which could be tested in duplicate). The first cavity was separated for the Blank. 1000µL of sample diluent was pipetted (100mM anhydrous dibasic sodium phosphate, 17mM anhydrous potassium phosphate monobasic, 150mM sodium chloride, 28mM potassium chloride, 1%w/v bovine albumin, 2%w/v sucrose, D-mannitol 1%w/v, 50%w/v BND solution in 0.04%v/v dimethyl sulfoxide and 0.05%v/v tween 80) in a test tube (considering the number of samples and controls to be assayed). 10µL of the sample and controls were diluted in the previously pipetted sample diluent. 100µL of the sample and controls previously diluted and homogenized was added to each well of the plate. The wells were covered with the plate sealer and incubated for 45 minutes at 37°C ± 2°C. After the incubation time, the plate sealer was removed from the wells, and the contents of the wells were discarded by aspiration (using the microplate washer) or by decanting (by hand). Each well was filled with 350µL of washing solution (100mM anhydrous dibasic sodium phosphate, 18mM anhydrous potassium phosphate monobasic, 1500mM sodium chloride, 28mM potassium chloride, 0.1%v/v proclin and 1%v tween 20 /v) and then discarded (approximately 30 seconds later). A total of five such wash cycles were performed. Then, 100µL of conjugate (peroxidase-conjugated goat anti-human IgG antibody - FAPON Biotech, Inc BEEIGGI201, diluted 1:5000 in MOSS peroxidase diluent, Inc) was pipetted into each well. The wells were covered with the plate sealer and incubated for 30 minutes at 37°C ± 2°C. After incubation, the washing procedure was repeated, filling each well with 350µL of wash solution, then discarding it (approximately 30 seconds later) for a total of five wash cycles. In the next step, 100 µL of substrate (TMB Ultra Sensitive Substrate 2.08mMol - MOSS, Inc #TMBUS) were pipetted into all wells. The wells were covered with the plate sealer and incubated for exactly 15 minutes at room temperature. To stop the reaction, after 15 minutes, 100µL of stop solution (0.5M sulfuric acid solution) was added to all wells, and gently homogenized for ± 30 seconds. The absorbance reading was taken at 450 nm within 30 minutes after addition of the stop solution.

[0088] The reactivity of specific anti-DTL4 antibodies present in human and canine samples was tested by ELISA using a pool of ten positive sera samples and a pool of ten negative samples for human (Figure 4A) and canine visceral leishmaniasis (Figure 4D ), to determine the best protein concentration and which sera dilution could be applied in the evaluation of individual sera. Both assays (human serum and canine serum) demonstrated that the DTL4 protein is able to discriminate with high precision between positive and negative samples of visceral leishmaniasis, even when low protein concentration was used. Figures 4B and 4E provide a comparison of the performance of DTL4 with recombinant proteins A2 and K39, using human and canine sera, respectively. These assays demonstrate that DTL4 is more effective than A2 and K39 in discriminating human and canine samples positive for visceral leishmaniasis from negative samples. Furthermore, it should be noted that the reactivity of positive human sera with DTL4 remains high even at high dilutions of sera, while the same is not true for A2 or K39. In Figure 4C, 469 human samples (154 positive 315 negative) were evaluated. As seen in the figure, DTL4 discriminates negative and positive samples in a high proportion.

[0089] The sensitivity and specificity of the DTL4 protein was evaluated using a panel of human samples and compared to A2 and K39. DTL4 showed sensitivity and specificity of 89.5% and 93.7%, respectively, using a panel of 100 negative samples and 245 positive samples, tested by the It-Leish kit. The A2 protein showed a sensitivity of 53.5% and a specificity of 67.3% (100 negative samples and 141 positive samples, tested by the It-Leish kit), while the K39 protein showed a sensitivity of 71.4% and a specificity of 79 .1% (100 negative samples and 159 positive samples, tested by the It Leish kit).

Example 3 - Diagnosis of visceral leishmaniasis in HIV patients using the DTL4 protein in an ELISA assay.

[0090] Anti-DTL4 antibody levels were evaluated in sera from patients previously diagnosed with co-infection with visceral leishmaniasis and HIV. These samples were tested by DAT and parasitological test.

[0091] For the reaction, all reagents were placed to stabilize at room temperature (between 15 and 30ºC) for at least 20 minutes. Then, the wells to be used were separated and tested in duplicate, according to the ELISA assay described in example 2.

[0092] The sensitivity and specificity of patients co-infected with leishmaniasis and HIV (LVH+/HIV+) were 92.31% and 82.25%, respectively, whereas in patients with leishmaniasis but without HIV (LVH+/HIV -) the sensitivity was 94.61% and the specificity was 99.41% (Figure 5). This analysis was performed with 62 co-infected patients and 167 patients with visceral leishmaniasis whose infection was detected by the parasitological method, considered the gold standard, and 169 healthy donors.

Example 4 - Evaluation of interferents in the ELISA assay

[0093] For the evaluation of interferents, samples classified into 11 different groups were submitted to the ELISA assay following the methodology described in example 2. The following were evaluated: (i) positive samples for visceral leishmaniasis (Leishmania chagasi) - Infected individual; samples negative for visceral leishmaniasis and (ii) positive for cutaneous leishmaniasis, (iii) positive for Chagas disease (Trypanosoma cruzi), (iv) with increased levels of rheumatoid factor, (v) positive for lupus, (vi) positive for malaria (Plasmodium vivax and/or Plasmodium falciparum), (vii) positive for paracoccidioidomycosis, (viii) positive for syphilis (Treponema pallidum), (ix) positive for tuberculosis (Mycobacterium tuberculosis), (x) positive for toxoplasmosis (Toxoplasma gondii); and (xii) negative samples for visceral leishmaniasis (Leishmania chagasi) - Healthy donor;

[0094] The average absorbance obtained for each of the groups is represented in Figure 6. Considering the cut-off value for the test (approximately 0.2670, it is concluded that the clinical conditions tested (cutaneous leishmaniasis, Chagas disease, increased rheumatoid factor, lupus, malaria, paracoccidioidomycosis, syphilis, tuberculosis and toxoplasmosis) do not interfere with the visceral leishmaniasis diagnostic kit and method of the present invention.

Example 5 - Evaluation of the rapid test using the DTL4 protein

[0095] This assay used protein A conjugated to colloidal gold, antigen (DTL4) immobilized in the test line (LT) and protein A in the control line (LC) (Figure 7). The purpose of the test is to detect anti-DTL4 antibodies present in serum positive for visceral leishmaniasis, by means of the initial capture of antibodies by protein A conjugated to colloidal gold and later by the capture of specific antibodies (anti-DTL4) by the previously fixed antigen, on the test line.

[0096] To obtain the kit, first, gold nanoparticles with a diameter of 15 nm were prepared. For this, 10 ml of 1% tetrachloroauric acid (HAuCl4) and 10 ml of 4% sodium citrate were separately solubilized in 480 ml of distilled water. These solutions were added to boiling water under constant stirring. Heating and stirring were continued until the solution showed a red-orange color and an absorbance between 1.8 and 2 (520 nm). Subsequently, the pH 9 of the colloid was adjusted with 0.1M K2CO3 and the conjugation of protein A with the gold nanoparticles obtained was performed. After determining the ideal amount of antigen for conjugation, the container with the colloidal gold was manually shaken, the determined amount of protein was added, already resuspended, and the agitation was maintained for 20 minutes. Finally, 1% of the final volume of albumin was added (using a 10% BSA solution) and stirring was maintained for 30 minutes. At the end, 3% sucrose and 1.5 mL of conjugate were added to 30 cm of glass fiber for drying in an oven at 37°C for 2 hours, and then the conjugate was kept in a room with humidity control <40%.

[0097] For quick test assembly, membranes capable of absorbing the sample from the beginning to the end of the tape, fixing the test and control lines and allowing the flow of the sample and the conjugate to react with the lines can be used. Membranes of absorbent cellulose fiber, nitrocellulose, glass fiber or plastic backing can be used, even overlapping layers of these membranes can be used to group all necessary functions (Table 2).

[0098] For impregnation of nitrocellulose membranes, the DTL4 protein was diluted in dialysis buffer (phosphate buffer, pH 7.4) in order to reach a known final concentration. In the same way, the protein A to be impregnated in the control line was diluted in phosphate buffer (10mM, pH 7.4) in order to reach a known concentration.

[0099] Subsequently, the nitrocellulose membranes were impregnated with DTL4 protein [320 ng/strip (1mg/mL)] in the test line and protein A (0.3 mg/mL) in the control line.

[00100] After the impregnation process, the membranes were dried and overlapped, the test strips were cut and packaged in cassettes, and then the kits were ready for use.

[00101] After assembly, cutting and packaging, the strips were tested by applying 10 µL of human serum (positive or negative for visceral leishmaniasis) and 60 µL of running buffer. Visual reading was performed after 10 minutes. The test is considered positive when the test and control lines appear; and considered negative when only the control line is visible. The test is considered invalid when the control line is not visible (Figure 7).

[00102] To evaluate the performance of the kit, different parameters were evaluated, among them the definition of the conjugation method, the volume of conjugate used (5 µL to 20 µL), the volume of the applied sample (5 and 10 µL), the antigen concentration in the test line, different dilutions of protein A in the control line, the type of nitrocellulose membrane, the running buffer and the reading time.

[00103] Different running buffers were tested, being: (1) 20 mM sodium tetraborate, 0.1% Triton X-100, 0.1% sodium azide and 1% BSA, pH 8.5; and (2) 0.01 M Sodium Tetraborate, 0.5% Tween 20, pH 8.2.

[00104] The buffer that showed the best performance was 2, 0.01M sodium tetraborate, 0.5% Tween 20, pH 8.2.

[00105] For the standardization of the immunochromatographic test, several parameters were tested and they were optimized to have the best performance of the assay.

[00106] The parameters defined were: the method of conjugation of protein A with the particles of colloidal gold (15 nm) in suspension, pore size of the nitrocellulose membrane, antigen concentration in the test line, composition of the control line, volume of the conjugate, sample volume, running buffer type, running buffer volume and read time.

[00107] The parameters that presented the highest color intensity in the test and control lines were selected, without compromising the specificity of the assay. Therefore, the best combination of parameters that resulted in positivity with positive human serum for visceral leishmaniasis and negativity with negative human serum for leishmaniasis was defined.

[00108] The tests showed the best results using the standardized parameters, described in Table 3.

[00109] To evaluate the performance of the rapid test kit for the diagnosis of human visceral leishmaniasis, biological samples were applied (100 positive for human visceral leishmaniasis and 100 negative from healthy donors). The sensitivity and specificity achieved with these samples were 95 and 100%, respectively. In order to assess the specificity of the diagnosis, cross-reactivity was evaluated by applying samples of diseases such as: Cutaneous Leishmaniasis, Chagas' disease, malaria, toxoplasmosis and rheumatoid factor. There was no cross-reactivity in any case, proving the high specificity of this product in diagnosing human visceral leishmaniasis.

Example 6 – Immunization of mice using the chimeric protein DTL4

[00110] In order to explore the potential of the chimeric protein DTL4 as a candidate antigen for a vaccine formulation against human visceral leishmaniasis, tests in mice were performed in combination with two immunological adjuvants already approved for human vaccination, Polynosinic-polycytidyl acid ( Poly-IC) and CPG associated with Alumen. The immunization assay is described below.

[00111] Female BALB/c mice, aged 6 to 8 weeks, were divided into five groups and immunized with three subcutaneous doses of DTL4 (with Poly-IC or CPG+Alumen adjuvants), saline, Poly-IC or A2: Group 1 (n=10) - immunization with a formulation composed of 10µg of DTL4 and 50µg of Poly-IC; Group 2 (n=10) - immunization with doses consisting of 10µg of DTL4 associated with 18µg of CpG and 30% (v/v) of Alumen; Groups 3 (n=10) and 4 (n=10) - considered negative control groups - were submitted to sterile saline at 0.9% and 50µg of Poly-IC, respectively; Group 5 (n=10) - positive control group, received 10µg of recombinant protein A2 (A2) associated with 18µg of CpG and 30% (v/v) of Alumen. The total volume of 100μL/mouse was adopted as the dose and the interval of 21 days was established between each of the three doses.

[00112] To evaluate the immunogenicity of DTL4, four animals from each group were euthanized thirty days after the last vaccination dose. Serum and spleen samples from all animals were collected and used to examine, respectively, the immune responses of B and T cells. At the same time, six immunized mice (n=6) were then challenged with inoculation of 1x107 L. infantum promastigotes (strain PP75) in sterile saline, subcutaneously. Finally, thirty days after the challenge, the animals were euthanized, and serum, spleen and liver samples were collected for analysis. The number of viable parasites per milligram of tissue (liver and spleen) was determined using limiting dilution (LDA) assays.

Humoral response - Evaluation of immunoglobulin levels total IgG, IgG2a in mice immunized with DTL4

[00113] In order to predict the response profile triggered by immunization with the DTL4 protein, the levels of total IgG and IgG2a immunoglobulins were evaluated in the immunized animals. It is known that IgG2 isotype immunoglobulin levels imply a T-helper 1 (Th1) type response pattern, with high levels of Interferon-gamma (IFN-γ). The induction of this type of response is a desirable feature for vaccine candidates against leishmaniasis, given that IFN-γ leads to macrophage activation, which is necessary for the elimination of parasites. Poly-IC and its derivatives, CPG, saponin, MPLA (monophosphoryl lipid A) and GLA (Glucopyranosyl Lipid A) are examples of adjuvants capable of inducing Th1-type immune response.

[00114] Total IgG and IgG2a immunoglobulin levels were determined by the enzyme-linked immunosorbent assay (ELISA-Enzyme-Linked Immunosorbent Assay). Briefly, 96-well plates were sensitized with DTL4 protein (2.5ng/100ul/well) or A2 protein (50ng/100ul/well) for 12 hours, diluted in carbonate buffer, under refrigeration. After washing the wells, the block (5% skim milk diluted in 1x phosphate buffer + 0.005% Tween 20) was added and the plate was incubated for 2 h at room temperature. The mouse serum, diluted 1:500 (in 2% skimmed milk diluted in 1x phosphate buffer + 0.005% Tween 20) was incubated for 1 hour at 37°C, as established by the titration curve suggested by the manufacturer and analyzed in duplicate. Total IgG and IgG2a anti-mouse antibodies, conjugated with peroxidase (Southern Biotech), were used in a 1:40,000 dilution after washing the wells, and added to the plates for 1 hour at 37°C. In the next step, after washing the wells, 100 µL of substrate (TMB Ultra Sensitive Substrate 2.08mMol – MOSS) were pipetted and the plate was incubated for 15 minutes in the dark. To stop the reaction, 100µL of stop solution (0.5M sulfuric acid solution) was added. Finally, optical densities were read at 450nm on the Multiskan-Go microplate reader.

[00115] The results demonstrated that mice immunized with DTL4 had higher levels of IgG and IgG2a than the group of mice immunized with protein A2 and controls (Figure 8A and 8B). Furthermore, it is worth noting that the DTL4 protein, when associated with the Poly-IC adjuvant, induced higher IgG2a secretion compared to the other experimental groups, even when the antigen used in the ELISA was A2 (Figure 8 B).

[00116] Therefore, surprisingly, the results demonstrated that immunization with the DTL4 protein was more effective than immunization with A2 for inducing IgG2a production. This data suggests that immunization with DTL4 triggers a Th1 response profile in mice, a valuable feature in a vaccine candidate against leishmaniasis.

Cellular response - Evaluation of Interferon-gamma (IFN-γ) and Interleukin-10 (IL-10) Production in immunized mice

[00117] Interferon-gamma (IFN-γ) and Interleukin-10 (IL-10) play an essential role in cellular immunity against Leishmania infection. The cytokine IFN-γ is the main Th1 marker associated with host protection in L. infantum infection, inducing macrophage activation and causing the death of intracellular pathogens. On the other hand, IL-10 is a cytokine related to the persistence of the parasite in the host, inhibiting macrophage activation and, consequently, modulating the cellular response and causing the appearance of clinical forms of the disease.

[00118] To evaluate the production of Interferon-gamma (IFN-γ) and Interleukin-10 (IL-10), 1x106 splenocytes from vaccinated mice were incubated at 37°C with 5% CO2 for 48 hours in the presence of 10ug /mL of DTL4, or Roswell Park Memorial Institute (RPMI) culture medium (unstimulated control). Duoset® ELISA kits (R&D Systems) were used to quantify IL-10 and IFN-γ secretion in cell culture supernatants, according to the manufacturer's instructions.

[00119] The results showed that both immunization with DTL4 antigen and immunization with A2 are able to induce IFN-γ production after in vitro stimulation with DTL4 for 48 hours (Figure 2-A). IL10 levels detected after stimulation with both antigens were low, with no statistical difference between them (Figure 2-B).

Reduction of parasite load

[00120] To evaluate the effect of immunization with DTL4 on Leishmania infection, the animals were euthanized thirty days after the challenge with L. infantum, and the organs (liver and spleen) were harvested for quantification of viable parasites per milligram of tissue, by limiting dilution assay (LDA). The liver and spleen of the mice were ground and homogenized with 5 mL of Schneider medium. Tissue debris was removed by centrifugation at 50xg and 4°C for 1 minute. The supernatant was collected and centrifuged at 1540xg and 4°C for 10 minutes. The obtained pellet was resuspended in 500μL of Schneider medium and subjected to a 1:10 dilution (in duplicate) in 96-well microplates with a final volume of 200 μL. The microplates were incubated at 26°C for 14 days, and the presence of parasites was evaluated weekly. The last dilution in which at least one parasite could be identified in the well was considered as the final titer. The results were expressed as the negative logarithm of the number of parasites.

[00121] The results demonstrated that immunization with the DTL4 antigen, as well as the A2 antigen, resulted in a significant decrease in the parasite load after challenge compared to the control groups (saline and poly-IC) (Figure 3). It is worth mentioning that, based on the analysis of the average of the results found in the limiting dilution assay, it is possible to perceive a greater reduction of parasitism in the spleen and liver by immunization with DTL4 compared to protein A2 (Table 1).

[00122] The results obtained demonstrate, therefore, the success of formulations containing the DTL4 antigen for the development of new vaccine formulations for human visceral leishmaniasis, with superior results in relation to A2 with regard to the potential for inducing a Th1 immune response and reduction of parasites in the liver and spleen.

therapeutic potential

[00123] To evaluate the therapeutic potential of DTL4, female BALB/c mice aged 6 to 8 weeks were infected with 107 L.infantum promastigotes (strain PP75) in sterile saline, subcutaneously. Thirty days after infection, animals were treated with DTL4 + Poly:IC Poly:IC (between 10 and 100 µg of DTL4 and 25 and 50 µg of Poly:IC adjuvant, per dose). Amphotericin B and saline were used as positive and negative controls, respectively. The treatment was carried out for 1 month. After treatment, the animals were euthanized, and serum, spleen and liver samples were collected for analysis. The number of viable parasites per milligram of tissue (liver and spleen) was determined using limiting dilution (LDA) assays.

Claims (19)

Chimeric protein, characterized in that it comprises an amino acid sequence with at least 85% identity with respect to SEQ ID NO 1. Chimeric protein according to claim 1, characterized in that it comprises an amino acid sequence as defined by SEQ ID NO 1. Kit for the diagnosis of leishmaniasis, characterized by the fact that it comprises:

• a) a chimeric protein as defined in claim 1 or 2;
• b) a secondary antibody or a protein, whether or not conjugated to an enzyme or a label;
• c) a reagent to detect the secondary antibody or protein.

Kit, according to claim 3, characterized in that the chimeric protein is immobilized on a solid support. Kit, according to claim 4, characterized in that said solid support is composed of a material selected from the group comprising cellulose fiber, nitrocellulose, fiberglass, plastic, nylon, latex, polypropylene and/or polystyrene, consisting of solid support on microplates, microtiter plates, tubes, beads or membranes. Kit, according to claim 3, characterized in that it also contains a carrier selected from the group comprising nanomaterials and/or micromaterials of gold, silver and/or carbon, and/or microparticles (beads) simple or covered with different materials, preferably nanomaterials and/or gold micromaterials. Kit, according to claim 3, characterized in that said secondary antibody is selected from the group comprising IgG, IgM, IgA, IgE and their respective subclasses; said protein is selected from the group comprising Protein A, Protein G and/or lectins; the enzyme is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase, preferably peroxidase; and the label is selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents. Kit, according to claim 3, characterized in that the reagent for detecting the secondary antibody or the protein, when they are conjugated to an enzyme or a label, is selected from the group comprising chromogenic substrates, preferably a solution containing hydrogen peroxide and tetramethylbenzidine. Method for the diagnosis of leishmaniasis, characterized by the fact that it contains the following steps:

• a) exposing a sample to a chimeric protein as defined in claim 1 or 2;
• b) identifying the samples containing antibodies reactive to the chimeric protein of step “a” using appropriate reagents.

Method, according to claim 9, characterized in that step "b" for identification of samples containing antibodies reactive to said chimeric protein is carried out by means of secondary antibodies or a protein that will later be identified using appropriate reagents, in which said secondary antibodies or proteins are conjugated to an enzyme or a label. Method, according to claims 9 or 10, characterized in that the identification of secondary antibodies or proteins is carried out by means of reagents capable of detecting the enzyme or the marker conjugated to said antibodies or proteins,

• a) wherein the reagents capable of detecting the enzyme or the label conjugated to secondary antibodies or proteins are selected from the group comprising chromogenic substrates, preferably solution containing hydrogen peroxide and tetramethylbenzidine,
• b) wherein said secondary antibody is IgG, IgM, IgA, IgE or subclasses thereof; the protein is Protein A and/or Protein G and/or lectins; the enzyme is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase, preferably peroxidase; and the label is selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents.

Method, according to claim 9, characterized in that said sample of step "a" sample is blood, serum, plasma and/or other body fluid. Method, according to claims 9 or 10, characterized in that said sample is human or canine. Method according to any one of claims 9 to 11, characterized in that it is carried out by enzyme immunosorbent assay, enzyme immunoassay or rapid immunochromatographic test. Use of a chimeric protein as defined in claim 1 or 2, characterized in that it is in the preparation of a composition for the prophylaxis and/or treatment of leishmaniasis. Vaccine composition, characterized in that it comprises a chimeric protein as defined in claim 1 or 2, and adjuvants. Vaccine composition, according to claim 16, characterized in that the adjuvants are selected from the group comprising Poly-IC and derivatives, water and oil emulsion or alum with unmethylated CpG oligonucleotide, water and oil emulsion or alum with lipid A; squalene; synthetic TLR4 agonist; liposomes associated with unmethylated CpG TLR or TLR4 agonist, Montanide and Monophosporyl Lipid A, or another adjuvant that induces a Th1-type immune response. Vaccine composition according to claims 16 and 17, characterized in that it comprises 1 µg to 1 mg of the protein as defined in claim 1 or 2, more preferably 10-100 µg of said protein per dose, and
10 µg to 1 mg of the polyinosinic-polycytidyl acid adjuvant, more preferably 50 µg of said adjuvant per dose, or
1 µg to 10 mg CPG and 10% to 90% Alum, more preferably 18 µg CpG and 30% (v/v) Alum. Use of the composition defined in claim 18, characterized in that it is for the preparation of a vaccine that induces an immune response for the prevention and/or treatment of leishmaniasis in a human or dog, preferably visceral leishmaniasis, caused by parasitic infection of L. infantum, L. donovani and/or L. chagasi

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