阅读说明：本技术 嵌合蛋白、其生产方法和用途，以及核酸分子、表达盒、表达载体、宿主细胞、用于诊断利什曼病的组合物、用于诊断利什曼病的试剂盒和体外诊断利什曼病的方法 (Chimeric proteins, methods for their production and use, as well as nucleic acid molecules, expression cassettes, expression vectors, host cells, compositions for diagnosing leishmaniasis, kits for di) 是由 O·P·D·M·内托 A·M·雷森德 D·D·H·C·塔瓦雷斯 W·J·T·多斯桑托斯 A· 于 2020-02-14 设计创作，主要内容包括：本发明涉及嵌合蛋白、以及其用途和生产方法,所述嵌合蛋白包括用于诊断内脏利什曼病的天然婴儿利什曼原虫的蛋白级分。本发明还涉及核酸、表达盒、表达载体、宿主细胞、用于诊断内脏利什曼病的组合物、用于诊断内脏利什曼病的试剂盒、诊断内脏利什曼病的方法和疫苗组合物。(The present invention relates to chimeric proteins, including protein fractions of natural infant leishmania for diagnosis of visceral leishmaniasis, as well as uses and methods of production thereof. The invention also relates to nucleic acids, expression cassettes, expression vectors, host cells, compositions for diagnosing visceral leishmaniasis, kits for diagnosing visceral leishmaniasis, methods of diagnosing visceral leishmaniasis, and vaccine compositions.)

1. Chimeric protein, characterized in that it comprises a fraction of native infant leishmania proteins.

2. The chimeric protein according to claim 1, further comprising:

a spacer between the antigenic regions,

optimized sequences at the 5' end of the synthetic gene and in the region encoding the protein end, such as: pRBS-SD1+6AA,

a ribosome binding site and Shine-Dalgarno sequence,

pSS-gIII，

the t7 tag peptide is a peptide,

ET-6 His; or

A translation stop codon;

or a combination of these.

3. The chimeric protein according to claim 1 or 2, characterized in comprising an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 or SEQ ID No. 8.

4. A nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO 1, 3, 5 or 7 and degenerate sequences thereof encoding identical amino acid sequences as defined by SEQ ID NO 2, 4, 6 or 8, respectively.

5. An expression cassette comprising a nucleic acid molecule as defined in claim 4 operably linked to a promoter and a transcription terminator.

6. An expression vector comprising a nucleic acid molecule as defined in claim 4 or an expression cassette as defined in claim 5.

7. Host cell, characterized in that it comprises a nucleic acid molecule as defined in claim 4 or an expression cassette as defined in claim 5 or an expression vector as defined in claim 6.

8. A method for producing a chimeric protein, comprising the steps of:

(a) transforming a host cell with an expression vector comprising said nucleic acid molecule as defined in claim 4,

(b) culturing the host cell to produce the chimeric protein; and

(c) isolating the chimeric protein from the cell or from the medium surrounding the cell.

9. Composition for the diagnosis of leishmaniasis, characterized in that it comprises one or more chimeric proteins as defined in any one of claims 1 to 3.

10. The composition of claim 9, wherein the diagnosis is still performed on a sample from a dog or human.

11. Kit for the diagnosis of visceral leishmaniasis, characterized in that it comprises one or more chimeric proteins as defined in any one of claims 1 to 3 or a composition as defined in claim 9.

12. The kit of claim 11, further comprising instructions for use.

13. The kit of claim 11 or 12, further comprising a device for detecting antigen/antibody complexes, which may comprise a signal generator capable of generating a detectable signal.

14. The kit of any one of claims 11 to 13, wherein the diagnosis is performed on a sample from a dog or a human.

15. Use of one or more chimeric proteins as defined in any one of claims 1 to 3, of a composition as defined in claim 9 or 10 or of a kit as defined in any one of claims 11 to 14, characterized in that it is for the diagnosis of leishmaniasis.

16. A method for the in vitro diagnosis of leishmaniasis, comprising the steps of:

(a) providing one or more chimeric proteins as defined in any one of claims 1 to 3 or a composition as defined in claim 9 or 10 and a human or dog serum sample,

(b) contacting the one or more chimeric proteins or the composition with a biological sample to be tested for a sufficient time and under conditions sufficient for antibody/antigen complexes to form; and

(c) the chimeric protein/antibody complex formed in the previous step is detected by a detection technique capable of producing a detectable signal in the presence of the antigen/antibody complex.

17. The method of claim 16, wherein the biological sample is selected from the group consisting of saliva, urine, serum, or blood.

Technical Field

The present invention relates to the field of diagnostic medicine and biotechnology. In particular, the invention relates to polypeptides, more particularly chimeric proteins, for diagnostic applications of leishmania infantis in human, dog and other vertebrate hosts.

Background

Leishmaniasis (Leishmaniasis) is an infectious parasitic disease caused by flagellar protozoa belonging to the families Trypanosomatidae (Trypanosomatidae) and the genus Leishmania (Leishmania).

These protozoa have heterogeneous life cycles, alternately living in vertebrate hosts (including humans and other wild and/or domestic mammals, such as dogs) and vector dipteran insects (vector dipteran insects), the latter belonging to the genera phlebotomis (phlebotomis) and phlebotomia (lutzomia) (PACE, 2014).

Currently, there are about 30 known leishmania species, 10 of which are present in the Old continent (the Old World) and 20 of which are present in the New continent (the New World), of which about 20 are capable of causing cutaneous and/or visceral leishmaniasis.

Protozoa of the genus leishmania have two very different evolutionary forms, both in function and morphology, namely promastigotes (promastigotes) and amastigotes (amastigotes).

Amastigote forms exist in vertebrate hosts and take on a spherical to ovoid shape. They are fixed, obligate intracellular forms because they have short flagella located within the flagellar follicles, primarily as sites of endocytosis and exocytosis. They develop in the vacuole of cells of the mononuclear phagocytic system, and are therefore eosinophils of low pH adapted to this environment.

Promastigote forms exist in the gut of the vector (invertebrate host) and are elongated with length. They are extracellular in the form of flagellates, which may be expressed as promastigotes (circulating promastigotes), defined as more ovoid, mobile and replicating cells, or as infectious forms of late promastigotes (meta promastigotes), longer and without proliferative capacity. (KAYE; SCOTT, 2011; MURRAY et al, 2005).

Leishmaniasis has as a vector invertebrates belonging to the phylum Arthropoda, Insecta, Diptera, Arachnocampaceae, phlebotoideae. Two genera are known, the sandfly (present in the old continent) and the sand fly (present in the new continent) (AKHOUNDI et al, 2016; READY, 2013).

The life cycle of leishmania parasites is divided into two stages, the first in a vertebrate host (mammal) and the second in an invertebrate host (sand fly). The cycle begins when an infected female sandfly mosquito ingests blood from a vertebrate host (dog or human). At the same time as the blood is taken up, the insect ruminates the late promastigotes of the parasite (the infectious form, located in the valve flap of the vector) together with its salivary components.

The late promastigotes are then phagocytosed by different types of immune system cells found at the site of inoculation, where they differentiate into amastigote forms. This cycle is completed when the amastigote macrophages are engulfed by another sand fly, where they will undergo different stages of development until they return to a later form. Each of these phases is characterized by morphological and functional changes to ensure survival within the medium.

After the differentiation step, the late promastigotes will migrate to the valve for inoculation into another vertebrate host and continue the cycle (KAMHAWI, 2006; KAYE; SCOTT, 2011).

Leishmaniasis is considered to be a major neglected disease, affecting mainly individuals from developing countries. Epidemics occur in africa, asia, the mediterranean, southwest europe, and tropical and subtropical regions in south and central america. The disease is prevalent in 98 countries, 72 of which are developing countries, with over 100 million new cases registered per year (ALVAR et al, 2012).

The disease can be divided into two major forms, which differ according to symptoms and clinical form and are associated with different species of leishmania. Cutaneous Leishmaniasis (LC) is characterized by the spread of skin lesions to the mucosa, whereas in Visceral Leishmaniasis (LV) the parasites reside in internal organs such as the spleen, lymph nodes, bone marrow and liver. VL caused by l.donovani and l.infantum has a lower incidence than LC, but is a more severe and fatal form if untreated (chappius et al, 2007).

Domestic dogs play a major role in the epidemiology of VL in the epidemiological region, as it is the main domestic host for the disease (reservoir). This importance comes from the fact that: calazar (the common name for the disease) is more prevalent in dogs than in humans, and human cases usually precede canine cases. (QUINNELL; CURTENAY, 2009).

The immunological and pathogenic mechanisms of leishmaniasis are complex and are associated with a large number of genetic and cellular factors involved in host resistance and susceptibility. For some time, it has been known that control of parasitic infections appears to be dependent on CD4+ T lymphocytes associated with a Th1 response and on the immune responses associated with Interleukins (IL) IL-12, IL-18, IL-27 that activate macrophages. Such as IL-12, IFN-gamma also produce an effective response by increasing the production of Th1 responses.

On the other hand, Th2 responses associated with cytokines such as IL-10 and IL-4 maintain the persistence of the parasite, and thus the progression of VL infection will depend on the response produced by the vertebrate host. Inactivation of macrophages can lead to failure of the host to control infection if the Th2 response is increased compared to the Th1 response.

It has recently been observed that other types of immune system cells, such as those known as Th17, appear to also play a key role in defining how a vertebrate host responds to infection by the causative agent of leishmaniasis (SRIVASTAVA et al, 2016).

Knowledge of the immune response to leishmaniasis has been enhanced with the use of experimental models, such as mice, and studies on human cells, but little is known about how it occurs in dogs.

Clinical symptoms of VL are associated with systemic infections, including long-lasting fever, loss of appetite, weight loss, fatigue, cough, abdominal pain, edema, and diarrhea, as well as spleen, liver, and lymph node enlargement, pancytopenia, anemia, and hyperglobulinemia.

In their early stages, these symptoms are easily confused with those of other diseases, resulting in delayed diagnosis of the disease and complicating its treatment (SINGH; SUNDAR, 2015; SRIVASTAVA et al, 2011; SRIVIDYA et al, 2012).

Canine VL diagnosis is also a point of great relevance for the control of the overall disease, as clinically healthy dogs can be infected, and even where certain clinical symptoms are evident, these can lead to misidentification of pathology (GOMES et al, 2008; NOLI; SARIDOMICHELAKIS, 2014).

In dogs, VL evolves slowly and is insidious, and is a systemic, severe disease whose clinical manifestations depend on the type of immune response expressed by the infected animal.

Clinical manifestations in infected dogs exhibit a range of clinical features ranging from a clear state of health to severe end-stages. Initially, the parasite is present at the site of the bite, but later visceral infection and spread through the dermis occurs. Alopecia caused by infection exposes a large area of extensive parasitic skin (PALTRINIERI et al, 2016; saridomichael, 2009).

Gold markers confirming VL diagnosis in both humans and dogs identify leishmania parasites in material obtained by biopsy of internal organs such as bone marrow and spleen. However, this approach is insensitive, labor intensive, and requires an invasive procedure (CHAPPUIS et al, 2007; SRIVASTAVA et al, 2011; SUNDAR; RAI,2002)

Molecular detection has recently been developed based on variations in PCR technology, and although they may show different sensitivity and specificity results depending on the method used, they are in general quite satisfactory with minimal invasiveness, but all require equipment that is not suitable for field use (DE RUITER et al, 2014).

However, VL is characterized by hypergammaglobulinemia or the high production of anti-leishmanial antibodies, which facilitates the detection of these antibodies using immunological techniques. Among these techniques, the following techniques stand out: IIF (indirect immunofluorescence), DAT (direct agglutination assay), Western immunoblot (Western-Blot), ELISA (enzyme immunoassay) and rapid detection.

IIF has considerable sensitivity but low specificity, where cross-reactivity with other diseases such as chagas disease, malaria and schistosomiasis is observed at low titers. Its application requires a high level of skill, experience, and specialized, high cost equipment. In addition, serial dilution of serum makes screening of large numbers of specimens laborious, a reaction that is less suitable for large-scale epidemiological studies.

DAT is based on the detection of direct agglutination of leishmania promastigotes that react with anti-leishmania antibodies in serum, leading to agglutination of promastigotes, and constitutes an easy-to-perform technique. However, there are difficulties in standardization and quality control of antigens.

One of the limitations of DAT is the long incubation time required (18 hours) and the necessity of multiple dilutions of serum or plasma, which makes the assay laborious and unsuitable for screening large numbers of samples.

ELISA assay is a modern method, allowing large numbers of assays to be performed in a short time, being the most widely used method for VL immunodiagnosis. This assay is highly sensitive, allowing the detection of low antibody titers, but can be inaccurate in the detection of subclinical or asymptomatic cases, since the performance of an ELISA assay in the diagnosis of VL, particularly canine VL, is not only related to the type of antigen used, but also to the clinical status of the dog being tested.

A rapid alternative to ELISA and using minimal infrastructure is the rapid immunochromatographic assay, which is based on nitrocellulose membrane immunochromatography, using a recombinant antigen immobilized on paper (rK39) (BOELAERT et al, 2004;et al, 2011; chappius et al, 2007; METTLER et al, 2005; SRIVASTAVA et al, 2011; SUNDAR; RAI, 2002).

The recombinant rK39 antigen is a 39 amino acid peptide from leishmania infantis and has been identified as highly attractive for diagnosis of human VL (SINGH et al, 1995). Rapid assays based on rK39 showed high sensitivity and specificity in human VL recognition assays (SUNDAR et al, 1998).

Therefore, this detection was initially considered as a promising tool in VL control procedures, as it requires a small amount of peripheral blood and is fast to perform and read (between 10 and 20 minutes) and can be used under field conditions (CHAPPUIS et al, 2006, 2007; GOMES et al, 2008).

However, there are differences between studies using rK39 in different countries of the world, for example in the continental africa, and show markedly different results. These data indicate that the sensitivity of an antigen may vary depending on the region in which it is used (BOELAERT et al, 2014; SRIVASTAVA et al, 2011).

In brazil, the diagnostic performance of K39 was considered reasonable only for confirmation of infection in suspected canine VL cases (QUINNELL et al, 2013). Recently, brazil used another immunochromatographic rapid assay based on a recombinant rK28 antigen, a synthetic gene, generated by fusion of several repeated sequences of leishmania donovani haspb1 and rK39, and used ELISA as a validation method (patahbi et al, 2010).

The assay was performed with sera from dogs infected with the disease from three states, Bahia (Bahia), Rio Grande dot north, and minax giras (Minas Gerais), and showed high sensitivity in symptomatic dogs but lower sensitivity in asymptomatic dogs (FRAGA et al, 2016).

In previous work carried out by some authors of the present invention and directly related to the present invention, attempts were made to identify new polypeptides of infant leishmania with potential for VL serodiagnosis (Oliveira et al, 2011; Magalhaes et al, 2017).

Neoantigens are then identified by screening infant leishmania cDNA expression libraries with sera from animals and human patients affected by the disease. As a result of this screening, and after sequence homology analysis of clones obtained with sequences from the genomic databases of leishmania infantis and leishmania major (l.major), clones encoding five different proteins were identified, designated Lci1 to Lci 5.

These fragments were then expressed in E.coli (Escherichia coli) and ELISA assays using sera from animals and humans affected by the disease were then used to evaluate the potential of the derived recombinant proteins in VL diagnosis.

One important result from these assays is that the most effective antigen to detect human VL is not the optimal antigen for canine VL and vice versa. (OLIVEIRA et al, 2011).

The two produced recombinant proteins (Lci1A and Lci2B) were then evaluated in a rapid immunochromatographic assay for diagnosis of dogs infected with leishmania infantum, and it could be verified that these two antigens, in cooperation with an assay based on rK28 protein, increased sensitivity from 88% to 93.5%, indicating that the combination of antigens is an excellent alternative for VL diagnosis (FRAGA et al, 2014).

Et al (2017) then identified a second new group of infant leishmania antigens in a new expression library screen, this time using a genomic library from patients infected with the pathogen and a set of sera. Seven novel antigens (Lci6 to Lci12) were identified, five of which were not yet correctly characterized in leishmania.

Six of these fragments were expressed in E.coli and the potential of the respective recombinant proteins in human and canine VL diagnosis was also evaluated. These are all considered to be unsatisfactory for human VL diagnosis: (Et al, 2017).

Et al further evaluated another protein identified in an early independently performed screening of infant leishmania cDNA library (CAMPOS et al, 2008) and then named Lci13, confirming that this protein is not considered satisfactory for human VL diagnosis.

Patent application PI 0900961-2 relates to 13 different leishmania antigens (Lci1 to Lci13) for the diagnosis of leishmaniasis. However, since none of the newly identified antigens proved to be effective in diagnosing VL in both human and canine forms, and the available serological tests were found to be less effective in diagnosing VL in dogs, we sought alternatives to develop a unique system for diagnosing VL in humans and dogs.

Furthermore, a simple diagnostic method for early and rapid identification of Visceral Leishmaniasis (VL) and applicable to both human and canine host samples would be an important tool to support combating and controlling this disease.

Currently, there is no good serological test available for canine VL diagnosis. In a meta-analysis study (PEIXOTO; DE OLIVERA; ROMERO,2015) carried out, it was determined that the antigens used in the main serological methods (ELISA and DPP) for diagnosing CVL were not highly accurate. This study also underscores the need for improved, improved quality and implementation of new assays to diagnose this disease.

The lack of reliable diagnostic methods has created a large gap in monitoring endemic areas, as most of the need for detection is for dogs in these areas. Because of this problem, reliable diagnosis of CVL requires a set of serological and molecular tests (ELISA, IFAT, PCR and qPCR), which is a very expensive practice (BOURDEAU et al, 2014). If the disease in dogs is not diagnosed and controlled, it may not be expected to be successfully controlled in humans.

In this regard, it should be clarified that the development of recombinant proteins (chimeric proteins) combining multiple antigenic determinants in a single molecule may lead to excellent proposals, as it facilitates the standardization of diagnostic tests, and studies have shown that a single molecule comprising multiple antigens may allow better distribution of antigenic determinants on ELISA plates than if multiple molecules were used in the same assay (CAMUSSONE et al, 2009).

Pioneering work and recent studies in the 1990's have highlighted the potential of chimeric proteins in diagnosing leishmania infection in infants (BOARINO et al, 2005; FARIA et al, 2015; SOTO et al, 1998). However, to date, the use of these molecules in the clinic, particularly for canine VL diagnosis, has been limited and/or has not resulted in significant improvements in the quality of disease diagnosis.

As can be seen, none of the prior art documents teach a single tool for VL diagnosis in humans or dogs. In response to the above-mentioned problems of prior art VL diagnostics, the present invention provides a selection of three recombinant proteins, selecting for optimal performance in human or canine VL diagnostics and the subsequent development of chimeric proteins comprising multiple antigenic determinants with high sensitivity and specificity.

The advantages of the present invention will be apparent from the description of the invention provided herein.

Disclosure of Invention

The present invention is directed to polypeptides for VL diagnosis in humans and dogs.

The polypeptide is in the form of a chimeric polypeptide construct comprising an antigenic region of an effective protein for VL diagnosis.

In particular, the invention relates to the production of synthetic chimeric proteins for use in VL diagnosis in humans and dogs.

In a first embodiment, the present invention provides a chimeric protein comprising an effective protein antigenic region for VL diagnosis.

In a second embodiment, the invention provides nucleic acid molecules encoding said chimeric proteins and degenerate sequences thereof.

In a third embodiment, the invention provides an expression cassette comprising said nucleic acid molecule.

In a fourth embodiment, the present invention provides an expression vector comprising said nucleic acid molecule or said expression cassette.

In a fifth embodiment, the invention provides a host cell comprising said nucleic acid molecule, said expression cassette or said expression vector.

In a sixth embodiment, the present invention provides a method of producing said chimeric protein, comprising the steps of:

transforming a host cell with an expression vector comprising the aforementioned nucleic acid molecule as defined above,

culturing the host cell to produce the chimeric protein; and

isolating the chimeric protein from the cell or from the medium surrounding the cell.

In a seventh embodiment, the present invention provides a diagnostic composition for visceral leishmaniasis comprising one or more chimeric proteins as defined above.

In an eighth embodiment, the present invention provides a diagnostic kit for visceral leishmaniasis comprising one or more chimeric proteins or a composition as defined above.

In a ninth embodiment, the present invention provides the use of one or more chimeric proteins in the diagnosis of visceral leishmaniasis.

In a tenth embodiment, the present invention provides a method of diagnosing visceral leishmaniasis, comprising the steps of:

contacting one or more chimeric proteins from a human or dog serum sample with a human or dog serum sample,

the chimeric protein/antibody complex formed in the previous step is detected using immunological detection techniques.

Drawings

FIG. 1 is a schematic representation of the chimeric proteins Q1 and Q5. The complete chimera is shown. The Lci12-2-3-13 region is shown in (Q1), the Lci12-2-3 region is shown in Q5, flanked by XbaI/HindIII enzyme sites and SalI/XhoI sites for truncation, and pSS-gIII optimized tags flanked by NcoI sites.

FIG. 2 indicates the confirmation of subcloning of the chimeric gene. It can be observed that 1% agarose gels showing the genes Q1COM, Q1NN, Q1SX and Q5, the complete chimeric gene (shown in red) was released in the expected size shown in Table 1 by digestion with the double enzyme XbaI/HindIII. Thus confirming subcloning. The label used was Ladder1Kb plus. Note that: q1COM was digested with the enzymes ScaI/XbaI/HindIII, and since the insert was the same size as the vector, additional digestion with ScaI resulted in cleavage of the plasmid into two fragments and allowed visualization of the insert as shown.

FIG. 3 refers to the expression of the chimeric protein. SDS-PAGE gels showing expression of the chimeric proteins in E.coli Rosetta cells of the sizes described in Table 2 can be seen.

FIG. 4 refers to a Western immunoblot of the chimeric protein. Recognition of the chimeric protein by anti-histidine monoclonal antibodies was observed. The response of the protein is shown in red in its predicted size.

FIG. 5 refers to the purification of recombinant chimeric proteins Q1SX and Q5 by affinity chromatography. The figure shows the purification pattern of the Q1SX and Q5 proteins by affinity chromatography, highlighted in red.

FIG. 6 refers to an indirect ELISA of the Q1SX chimeric protein with VL serum. Scatter plots and bar graphs show the sensitivity and specificity of VL positive 50 human and 40 dog sera against the Q1 chimeric protein. The horizontal line represents the cut-off of the reaction.

FIG. 7 refers to an indirect ELISA of the Q5 chimeric protein with VL serum. Scatter plots and bar graphs show the sensitivity and specificity of VL positive 50 human and 40 dog sera against the Q5 chimeric protein. Scatter plots show sera from humans and dogs against Q5. The horizontal line represents the cut-off of the reaction.

FIG. 8 refers to an indirect ELISA of the Q5 chimeric protein with LTA serum. Scatter plots show cross-reactivity of ATL positive 50 human sera against the Q5 chimeric protein. The horizontal line represents the cut-off of the reaction.

FIG. 9 refers to an indirect ELISA of the Q5 chimeric protein with HIV and HIV +/VL + co-infected sera. Scatter plots show cross-reactivity of HIV positive human serum and sensitivity and specificity of co-infection (VL + HIV) against the chimeric Q5 protein. Scatter plots show positive and negative human sera against Q5. The horizontal line represents the cut-off of the reaction.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used to describe the invention is intended to describe particular embodiments only and is not intended to limit the scope of the teachings. Unless otherwise indicated, all numbers expressing quantities, percentages and proportions, and other numerical values, used in the descriptive report and claims, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the descriptive report and in the claims are approximations that may vary depending upon the properties sought to be obtained.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, which are within the skill of the art. These techniques are explained fully in the literature. See, e.g., Fundamental Virology, 2 nd edition, vols.i & II (b.n.fields and d.m.knipe, ed.) Handbook of Experimental Immunology, vols.i-IV (d.m.well and c.c.blackwell, ed., Blackwell Scientific Publications); T.E.Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); l. lehninger, Biochemistry (Worth Publishers, inc., current edition); sambrook, et al Molecular Cloning: A Laboratory Manual (2 nd edition, 1989) Methods In Enzymology (S.Colowick and N.Kaplan, ed., Academic Press, Inc.).

The polypeptides of the invention exhibit reproducibility in terms of sensitivity and specificity. This indicates that the developed protein can be kept stable for a long period of time, maintaining its reactivity. The composition of the stock buffer may be advantageous for its stability, the use of protease inhibitors, the presence of denaturants in the buffer, or even their amino acid sequence. Stable proteins are considered when of interest for diagnostic applications.

As used throughout this application, the term "amino acid" denotes an alpha-amino group that may be directly encoded by a nucleic acid or encoded in precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides, called codons or sets of bases (suit of bases). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code". The term "amino acid" as used herein denotes naturally occurring alpha-amino acids, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

The terms "peptide", "polypeptide" or "protein" are used interchangeably and refer to a polymer of amino acids linked by peptide bonds, regardless of the number of amino acid residues making up the chain. As used herein, a polypeptide, including "variants" or "derivatives" thereof, refers to a polypeptide that includes variations or modifications, such as substitutions, deletions, additions, or chemical modifications, in its amino acid sequence relative to a reference polypeptide. Examples of chemical modifications are glycosylation, pegylation, PEG alkylation, phosphorylation, acetylation, amidation, and the like. The polypeptides may be artificially produced from cloned nucleotide sequences using recombinant DNA techniques, or may be prepared using known chemical synthesis reactions.

More specifically, the term polypeptide according to the invention is also understood to mean an antigen, a multiple antigen or a multiple epitope antigen, which consists of a linkage of different epitopes, which may or may not be linked by flexible or rigid ligands (linkers), specific for a single pathogen or different pathogens.

In a first embodiment, the invention provides chimeric proteins comprising fractions of natural infant leishmania proteins (fractions) produced from the 13 antigen combinations already described, called three of the Lci proteins (Lci1 to Lci13), selected for presenting the best results of human VL diagnosis (Lci2) or canines (Lci3 and Lci 12).

These proteins are produced from chemically synthesized genes that undergo multiple subcloning steps and consist of splicing of identical fragments of the three antigenic proteins.

The three proteins selected for the chimeric constructs (Lci2, Lci3 and Lci12) have no sequence homology to each other, but have a similar structure, repeated several times by multiple copies of a small motif (Lci2-39 amino acids; Lci3-14 amino acids; Lci12-8 amino acids), flanked by non-repetitive regions, such as (A), (B), (C) and C) 3)Et al, 2017; OLIVEIRA et al, 2011).

The selection of the antigenic region of each Lci included in the chimeric construct is based on size, solubility and immunogenic potential criteria. The individual coding sequences were then optimized for expression in e.coli and spliced into sequences, taking into account the following parameters: reading frame (reading phase), codon frequency, mRNA secondary structure, GC content distribution and restriction sites.

Small spacers are included between the antigenic regions of the chimeric protein, intended to promote folding conditions of the resulting recombinant protein. Also included are optimized sequences for the 5' end of the synthetic gene and the region encoding the protein end, such as: pRBS-SD1+6 AA-ribosome binding site and Shine-Dalgarno sequence; pSS-gIII-N-terminal sequence of phage envelope protein for optimized expression; t7 tag peptide-N-terminal sequence for stable and optimized expression; ET-6 His-polyhistidine sequence (six) introduced at the C-terminus to allow purification of the protein; and a translation stop codon.

Restriction enzyme sites are also added to the synthetic genes at key points along the construct to allow easy modification of these genes by including or excluding some sequences and allowing further manipulation of their structure, if necessary. These genes produced chimeric constructs Q1(SEQ ID NOS: 1, 3, and 5) and Q5(SEQ ID NO:7) with predicted sizes of 2845 and 1986bp (FIG. 1).

These differences are that the Q5 construct included an additional repeated fragment of the Lci3 protein, absent from the Q1 construct, whereas the original Q1 construct had at the 3' end of the gene the characteristic of a fragment encoding the C-terminal region of the fourth antigenic protein Lci 13. Possessing commercially synthesized gene constructs, they were then subcloned into the bacterial expression vector pRSETa, digested with XbaI/HindIII by restriction enzymes, releasing DNA fragments of the expected size (FIG. 2) and subcloned by sequencing analysis.

For the Q1 construct (SEQ ID NO:1) only, the fragment cloned into plasmid pRSETa was digested with internal restriction sites present only in the synthetic gene to generate truncated variants of the gene with the different fragments encoding selected portions of the protein removed.

The goal of this step is to expand the number of chimeric proteins synthesized to see which possible surrogate and antigen combinations enhance protein expression in prokaryotic systems.

The first digestion was performed with the enzyme NcoI, which releases a 75bp fragment in the N-terminal region of the gene encoding the expression optimization peptide pSS-gIII.

After purification and religation of the plasmid construct, this fragment was lost and the resulting chimeric gene was slightly smaller, encoding a truncated Q1 protein in which the peptide was not present (Q1NN-SEQ ID NO: 4).

Using an equivalent strategy, a second digestion was performed from the entire gene (SEQ ID NO:1) using the SalI/XhoI enzyme. Since the restriction sites of these enzymes are complementary, ligation of the main fragment products of this digestion results in the generation of a chimeric gene (SEQ ID NO:5) in which the 1155bp fragment encoding Lci13 (Q1SX-SEQ ID NO:6) was removed.

FIG. 2 also shows the fragments generated after digestion of the plasmid containing the two truncated constructs generated from the chimeric Q1 gene (SEQ ID NO:1), the size of each fragment being summarized in Table 1.

TABLE 1

Main name	Inset size (in pb)	Protein size (kDa)
			Q1	2845	102
Q1NN	2734	99
			Q1SX	1690	60
Q5	1960	98

In a second aspect, the invention provides nucleic acid molecules encoding the disclosed chimeric proteins.

The nucleic acid molecules of the invention are represented, without limitation, by SEQ ID NO 1, 3, 5 or 7, or sequences having at least 90% identity to SEQ ID NO 1, 3, 5 or 7, or degenerate sequences thereof encoding the same polypeptides.

The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons when compared to a reference nucleic acid molecule encoding a given polypeptide. Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (e.g., GAU and GAC both encode Asp).

One skilled in the art will recognize that degeneracy is fully supported based on the information provided in the present application and common general knowledge in the art. For example, the degeneracy of the genetic code (i.e., different codons can encode the same amino acid) is well known in the art, and the identity of the amino acid encoded by each codon is well established.

Based on information well known and established in the art, one skilled in the art is able to identify nucleotide substitutions that do not alter the resulting amino acid sequence. Thus, when possessing both the nucleotide sequence of a gene and the amino acid sequence encoding a protein, one of skill in the art will readily recognize degeneracy encoding the same protein having the same amino acid sequence.

In a third aspect, the invention provides an expression cassette comprising a nucleic acid according to the invention. The expression cassette is placed under conditions which result in the expression of the polypeptide of the invention.

The expression cassette may also comprise sequences necessary for its expression, such as promoter, enhancer and terminator sequences compatible with the expression system. In addition, the expression cassette may comprise a spacer sequence, a ligand sequence and suitable restriction sites. In addition, the expression cassette may further comprise a sequence encoding a histidine tail.

In a fourth aspect, the present invention provides an expression vector comprising a nucleic acid molecule or expression cassette according to the invention. The vector may be used to transform a host cell and allow expression of the nucleic acid according to the invention in said cell.

Advantageously, the expression vector comprises regulatory elements capable of expressing the nucleic acid molecule and elements enabling its selection in the host cell according to the invention. Methods for selecting these elements according to the host cell desired for expression are well known to those skilled in the art and are widely described in the literature.

Vectors can be constructed by classical molecular biology techniques well known to those skilled in the art. Non-limiting examples of expression vectors suitable for expression in a host cell are plasmids and viral or bacterial vectors.

In a fifth aspect, the invention provides host cells transiently or stably transformed/transfected with a nucleic acid, expression cassette or vector of the invention. The nucleic acid molecule, expression cassette or vector may be comprised in the cell in episomal or chromosomal form.

The host cell may be a bacterial cell, yeast, filamentous fungus, protozoan, insect, animal and plant cell.

In a sixth aspect, the present invention provides a method for producing a chimeric protein of the invention, comprising the steps of: transforming a host cell with an expression vector comprising said nucleic acid molecule or expression cassette, culturing said host cell for production of the chimeric protein (in vivo expression system); isolating the chimeric protein from the cell or from the medium surrounding the cell.

Particularly suitable expression systems include microorganisms, such as bacteria transformed with recombinant DNA expression vectors of phage, plasmid or cosmid; yeast transformed with a yeast expression vector; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems or animal cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV [ cauliflower mosaic virus ]; tobacco mosaic virus, TMV [ tobacco mosaic virus ]) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids). Cell-free translation systems may also be used to produce the polypeptides of the invention.

Introduction of a nucleic acid molecule, expression cassette or vector encoding a recombinant or synthetic protein of the invention into a host cell can be accomplished by a number of standard Laboratory manuals such as those described in Davis et al Basic Methods in Molecular Biology (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989).

In a preferred aspect, the expression of the different recombinant proteins of the invention is carried out using the plasmid DNA of the resulting chimeric construct, transformed into E.coli cells, after the growth and expression of the recombinant proteins, the total bacterial extracts from the cells expressing the different proteins are fractionated on SDS-PAGE gels (see FIG. 3).

The transformed or transfected host cells described above are then grown in a suitable nutrient medium under conditions which are advantageous to allow expression of the recombinant protein of the invention. The medium used to culture the cells may be any conventional medium suitable for culturing host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (descriptions), for example, in catalogues of the American type culture Collection. The protein of the invention produced by the cells may then be recovered from the cells or culture medium by conventional procedures including separation of the host cells from the culture medium by centrifugation or filtration, precipitation of the aqueous protein fraction from the supernatant or filtrate using a salt such as ammonium sulfate, purification by various chromatographic methods such as ion exchange chromatography, exclusion chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography or similar methods, depending on the type of polypeptide in question.

The resulting chimeric proteins are then purified and biochemically characterized using, for example, methods commonly used in the field of biochemistry, such as HPLC, SDS-PAGE, western blotting, pH gradient isoelectric focusing, circular dichroism. By these methods, characteristics such as the expression yield of the chimeric protein can be determined; determination of secondary structural features, and other features of which determination of features is important for the development of diagnostic compositions for visceral leishmaniasis, among others.

In one mode, transformation of a host cell with a nucleic acid molecule of the invention is accomplished by an expression vector. In a specific mode, the vector is pRSETa and the transformed host cell is E^TM2. Table 1 shows the expected molecular weights of the individual recombinant proteins encoded by the different chimeric genes obtained. Constructs evaluated in four waysOf these, only the Q1SX (SEQ ID NO:5) and Q5 constructs (SEQ ID NO:7) allowed the expression of clearly distinguishable bands in the total bacterial extract.

The host cell culture conditions indicated in step (b) are known to the skilled person. In one embodiment, the culturing is performed in LB medium in the presence of antibiotics with stirring. In a specific culture, the antibiotics are ampicillin and chloramphenicol. The incubation may be performed at different temperatures for different times. In a specific mode, the culturing may be performed at a temperature of about 35 ℃ to about 37 ℃ for about 4 to about 6 hours. In a preferred embodiment, the incubation is carried out at 37 ℃ for 6 hours with stirring.

The production of the chimeric protein mentioned in step (b) may be carried out by any technique known in the art. In one embodiment, induction of expression of the chimeric protein of the invention is achieved by adding IPTG to the culture medium after sufficient optical density is obtained.

To confirm the expression of the various truncations of the Q1 protein, the respective bacterial extracts were then subjected to western immunoblot analysis in which the recognition of these proteins by commercial monoclonal antibodies directed against the polyhistidine sequences present in each protein was evaluated.

As shown in FIG. 4, all three Q1 protein constructs were recognized by monoclonal antibodies confirming their expression, while Q1SX (SEQ ID NO:5) was expressed at a level higher than the others, compatible with its visualization on SDS-PAGE gels.

Likewise, commercial antibodies directed against poly-histidine sequences also recognized the Q5 protein (SEQ ID NO: 8). Based on expression analysis, E.coli cultures expressing the Q1SX and Q5 proteins were amplified for large scale expression of these proteins and purified by affinity chromatography on nickel resin. FIG. 5 shows representative SDS-PAGE gels of Q1SX and Q5 protein purification.

The purified Q1SX and Q5 proteins were quantified by comparison with the amount of BSA determined on SDS-PAGE and used in ELISA assays to assess their potential in human and canine VL diagnosis. Each protein was then evaluated with human serum from individuals who demonstrated infection with leishmania infantum, serum from healthy controls, serum from dogs who demonstrated VL infection, and their respective healthy controls.

The isolation of the chimeric proteins mentioned in step (c) can be carried out by any technique known in the art. In one embodiment, purification is achieved by chromatographic techniques. In one embodiment, the purification is performed by nickel resin affinity chromatography. Non-limiting examples include affinity chromatography, ion exchange, other affinity or adsorption methods, ion pairing, reverse phase, and size exclusion.

In a seventh aspect, the present invention provides a composition comprising one or more chimeric proteins according to the invention.

In a particular aspect, the composition is used as an agent for the diagnosis of visceral leishmaniasis.

In an eighth aspect, the present invention provides a diagnostic kit for visceral leishmaniasis comprising one or more chimeric proteins or a composition as defined herein.

Optionally, the kit further comprises instructions for use.

In addition, the kit may further comprise a means for detecting the antigen/antibody complex, which may comprise a signal generator capable of generating a detectable signal.

The detection means may be those known in the art. A non-limiting example of a detection device may be a conjugate consisting of an antibody coupled to a signal generating compound capable of generating a detectable signal.

In a ninth aspect, the invention provides the use of said chimeric protein in the diagnosis of visceral leishmaniasis.

In a tenth aspect, the present invention provides a method of diagnosing visceral leishmaniasis, comprising detecting chimeric protein/antibody complexes from a human or dog serum sample by using an immunological detection technique.

Evaluation of the chimeric protein of the present invention has demonstrated that it has sensitivity (76%) and specificity (100%) to human serum as shown in fig. 6 from the ELISA results of the chimeric protein Q1 using human and dog sera, whereas the results of the dog sera showed sensitivity (99%) and specificity (100%).

The ELISA results for the Q5 chimeric protein showed 81% sensitivity to human serum, while the results for the dog serum maintained 99% sensitivity (fig. 7).

Based on the best results obtained for the Q5 protein, the protein was used in a new ELISA assay to evaluate its cross-recognition with sera from patients with cutaneous leishmaniasis. The results for these sera showed 8% non-specific reaction (fig. 8).

Sera from patients co-infected with VL and HIV were then evaluated, showing 98% sensitivity and 100% specificity (as shown in figure 9) and no response only to HIV positive patients. Table 2 shows the results of analysis of all sera of the above group using Q5 and Q1SX proteins, showing the sensitivity, specificity and serum amount of the sera evaluated.

TABLE 2

The ELISA results for the Q1 and Q5 proteins showed the amount of serum used in each test group and the sensitivity and specificity shown in each group. Divided into dogs and humans. Negative sera were also tested to calculate each value.

Examples

Example 1

Bioinformatics method, chemical synthesis and subcloning of chimeric genes

Prediction of the presence of linear B-cell epitopes in the sequences of selected proteins (Lci2, Lci3 and Lci12) was carried out by the program BCPred12 (EL-MANZALAWY; DOBBS; HONAVAR, 2008). Chimeric gene sequences optimized for expression in E.coli were designed using the Gendesigner program (WELCH et al, 2011) and were generated by GenScript (GenScript, Piscataway, New Jersey, USA) (Q1 protein) and Thermo (Life Tech,paulo, Brazil) (protein Q5) were synthesized commercially.

These genes have been cloned into the commercial vector pUC57 flanked by enzymesRestriction sites for XbaI/HindIII. For subcloning into the bacterial expression vector pRSETa (Thermo Life Tech,paulo, Brazil), the chimeric gene was recovered by double digestion with restriction enzymes XbaI/HindIII and subcloned into the same site of the pRSETa vector.

After the Q1 gene was first subcloned into the pRSETa vector, two truncation reactions of the gene were performed. The first reaction was a digestion with the enzyme NcoI, resulting in the excision of the fragment encoding the pSS-gIII peptide, and the second reaction was the excision of the fragment encoding the C-terminal region of the Lci13 protein with the SalI/XhoI enzyme pair. All final constructs were confirmed by restriction enzyme digestion and sequencing.

Example 2

Expression and purification of recombinant proteins

To express the chimeric protein, plasmid constructs derived from pRSETa vector were transformed into competent E^TM2 cells (Merck Millipore) were then selected at 37 ℃ on solid LB medium (Luria Bertani) supplemented with ampicillin (100. mu.g/ml) and chloramphenicol (34. mu.g/ml).

Clones of the transformed cells were grown in liquid LB medium with the same concentration of antibiotic and expression of the recombinant protein was induced by adding IPTG to a final concentration of 0.1mM, and an optical density at 600nm (D.O) of 0.6 to 0.8.

After staining the gel with Coomassie blue R-250, the results were visualized using polyacrylamide gel (SDS-PAGE 15%). To obtain recombinant proteins, the cell pellet obtained after induction was resuspended in 20mL lysis buffer and equilibrated (100mM sodium phosphate, 10mM Tris, 8M urea, 20mM imidazole-ph 8.0) and sonicated by 5 pulses for 30 seconds at 1 minute intervals at 4 ℃.

After centrifugation at 5000rpm for 10 minutes, the cells were washed by incubation with Ni-NTA agarose resin (Qiagen) and then in the same buffer and in a denaturing buffer at pH6.0 (100mM sodium phosphate, 10mM Tris, 8M Urea, 30mM imidazole) and eluted with 1M imidazole in a denaturing buffer at pH 4.5. Aliquots were evaluated on SDS-PAGE gels as described.

Example 3

Western blot analysis

For Western blot analysis, chimeric proteins were fractionated on 15% SDS-PAGE gels and transferred to PVDF membrane (Immobilon-P)) Blocking was performed in TBS buffer (20mM Tris, 500mM NaCl, pH7.5) supplemented with 5% skim milk and 1% Tween-20.

The membrane was then incubated with antibodies/serum against the target protein at final dilutions of 1:3000 and 1:1000 in TBS buffer containing 5% milk and 1% Tween-20. After washing with 1% TBS/Tween-20, a new incubation with peroxidase-labeled rabbit anti-IgG (Jackson Immunoresearch laboratories) was performed, diluted 1:10000 in TBS buffer containing 5% milk and 1% Tween-20.

After further washing, the membrane was exposed to a solution of 1.2mM luminol, 0.4mM iodophenol and 0.03% hydrogen peroxide for 2 minutes. These films were then dried and exposed to autoradiography film for 1 and 5 minutes, and the film was then developed.

Example 4

Human serum for use

Initially, a set of human sera consisting of two different serogroups was assembled. The first group consisted of 50 sera from a control group in the IAM virology and laboratory of experimental treatment (LAVITE) serum bank established for the yellow fever vaccine program. These sera were obtained from healthy individuals living in the non-endemic region of visceral leishmaniasis (urban region of leishmania) and were provided by doctor Rafael Dh a lia fries.

The second group consisted of 50 sera from visceral leishmaniasis patients with clinical and laboratory diagnosis (confirmed by parasitological examination). This information was provided by the fries of caros Henrique Costa from the Federal University of piauyi.

All human sera were collected by the appropriate ethics committee after approved use as follows: the ethical committee (0116/2005) from the University of the peouee Federal approved the use of serum from VL patients; the negative control sera were included in the study approved by the ethics committee of the minimum of Health (25000.119007/2002-03).

Serum from cutaneous leishmaniasis patients was also used, including in item CAEE0014.0.095.000-05, approved by the CPqAM-FIOCRUZ ethics committee (03/08/2008).

Finally, doctor Zulma Medeeros supplied Aggeu from Centro de PesquisasSerum from patients co-infected with pooled (HIV/VL) and HIV-only infected patients from the ethical Committee-FIOCRUZ approved project (CAEE:53495816.0.0000.5190) were also evaluated in this protocol.

Example 5

Use of dog serum

Approximately one hundred domesticated or wandering dogs in the visceral leishmaniasis endemic area from jequie-BA were studied.

Blood samples were collected and aliquots of serum were obtained therefrom and stored at-20 ℃. For some of these animals, aspiration puncture of the spleen was performed and aspirates were used to culture and detect Leishmania according to the method described previously (BARROUIN-MELO et al, 2006).

We also used 90 sera from VL-bearing dogs offered in collaboration with doctor Val-era Pereira. All dogs were treated according to the animal's laboratory guidelines of the Oswaldo Cruz Foundation.

The Use of dog sera in this study was approved by the Animal Use Ethics Committee on Animal Use (CPqGM-FIOCRUZ, Ceua, protocol N.040/2005 and CPqAM-FIOCRUZ, Ceua, protocol 27/2016).

Example 6

Indirect enzyme immunoassay

After quantification, the recombinant protein was used for sensitization of microtiter plates in carbonate-bicarbonate buffer (NaCO)₃0.05M; pH9.6) to a concentration of 400ng (1. mu.g of total extract of Leishmania infantis parasite, used as reaction control) per well, in a volume of 100. mu.l per well, and kept in a humidified chamber at 4 ℃ for 16 hours.

In PBS (NaCl 137mM, KCl 2.7mM, Na)₂HPO₄ 10mM，KH₂PO₄1.8mM), blocked with PBS plus 10% skim milk, and further washed with PBS plus 0.05% Tween-20. Human and canine sera were then incubated at dilutions 1:2500 and 1:900 respectively in PBS Tween-20 containing 10% milk.

After washing and incubation steps with conjugate-peroxidase-linked anti-human IgG or anti-canine IgG were performed again, they were diluted 1:10000 and 1:1200 respectively in a humid chamber at room temperature for 1 hour.

After further washing, in the dark room in the presence of 0.01% concentration (300. mu.l) of chromogen OPD (o-phenylenediamine) and hydrogen peroxide (at 4% concentration, 3. mu.l) in citrate-phosphate buffer (Na)₂HPO₄、C₆H₈O₇0.1M, pH:5.0) for 30 minutes, followed by development with sulfuric acid (2.5M H)₂SO₄) The reaction was terminated. The readings were performed on a 490nm filter in Benchmark Plus Microplate Manager 5.2 (BIO-RAD).

Example 7

Statistical analysis of indirect ELISA results

The parameters of sensitivity, specificity, positive and negative predictive value and confidence interval were estimated using MedCalc Software (version 12.3) (MedCalc Software, Ostend, Belgium).

For the association between variables and their determinants, chi-square tests were used in a duplex table (2x2) relating disease diagnosis and test results.

Sensitivity is given by the percentage of positive detected by the test in known diseased individuals (positive parasites) and specificity is given by the percentage of negative in non-diseased individuals.

Scatter plots were obtained using GraphPad Prism Software (GraphPad Prism version 6.00for Windows, GraphPad Software, La Jolla California USA).

Reference to the literature

AKHOUNDI,M.et al.A Historical Overview of the Classification,Evolution,and Dispersion of Leishmania Parasites and Sandflies.PLoS Neglected Tropical Diseases,v.10,n.3,p.1–40,2016。

AVLAR,J.et al.Leishmaniasis worldwide and global estimates of its incidence.PLoS ONE,v.7,n.5,2012。

BARROUIN-MELO,S.M.et al.Can spleen aspirations be safely used for the parasitological diagnosis of canine visceral leishmaniosisA study on asymptomatic and polysymptomatic animals.Veterinary Journal,v.171,n.2,p.331–339,2006。

BOARINO,A.et al.Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes of Leishmania infantum for SeroVisceral Leishmaniasis diagnosis.Clinical and Vaccine Immunology,v.12,n.5,p.647–653,2005。

BOELAERT,M.et al.A comparative study of the effectiveness of diagnostic tests for visceral leishmaniasis.Am.J.Trop.Med.Hyg.,v.70,n.1,p.72-77,Jan.2004。

BOELAERT,M.et al.Rapid tests for the diagnosis of visceral leishmaniasis in patients with suspected disease.Cochrane.Database.Syst.Rev.,v.6,n.1469–493X(Electronic),p.CD009135,2014。

BOURDEAU,P.et al.Management of canine leishmaniosis in endemic SWEuropean regions:a questionnaire-based multinational survey.Parasites&Vectors,v.7,n.1,p.110,2014。

CAMPOS,R.M.et al.Distinct mitochondrial HSP70 homologues conserved in various Leishmania species suggest novel biological functions.Molecular and Biochemical Parasitology,v.160,n.2,p.157–162,2008。

CAMUSSONE,C.et al.Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis.Clinical and Vaccine Immunology,v.16,n.6,p.899–905,2009。

C.et al.Evaluation of two rK39 dipstick tests,direct agglutination test,and indirect fluorescent antibody test for diagnosis of visceral leishmaniasis in a new epidemic site in highland Ethiopia.American Journal of Tropical Medicine and Hygiene,v.84,n.1,p.102-106,2011。

CHAPPUIS,F.et al.A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis.BMJ(Clinical research ed.)v.333,n.7571,p.723,2006。

CHAPPUIS,F.et al.Visceral leishmaniasis:what are the needs for diagnosis,treatment and controlNat.Rev.Microbiol.,v.5,n.1740–1534(Electronic)p.873-882,nov.2007。

DE RUITER,C.M.et al.Molecular tools for diagnosis of visceral leishmaniasis:systematic review and meta-analysis of diagnostic test accuracy.J.Clin.Microbiol.,v.52,n.1098–660X(Electronic),p.3147-3155,Sept.2014。

EL-MANZALAWY,Y.；DOBBS,D.；HONAVAR,V.Predicting linear B-cell epitopes using string kernels.Journal of Molecular Recognition,v.21,n.4,p.243-255,jul.2008。

FARIA,A.R.et al.Novel Recombinant Multiepitope Proteins for the Diagnosis of Asymptomatic Leishmania infantum-Infected Dogs.PLoS Neglected Tropical Diseases,v.9,n.1,p.13–16,2015。

FRAGA,D.B.M.et al.A multicentric evaluation of the recombinant Leishmania infantum antigen-based immunochromatographic assay for the serodiagnosis of canine visceral leishmaniasis.Parasit.Vectors,v.7,n.1756–3305(Electronic),p.136,2014。

FRAGA,D.B.M.et al.The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs.PLoS Neglected Tropical Diseases,v.10,n.1,p.1–11,2016。

GOMES,Y.M.et al.Diagnosis of canine visceral leishmaniasis:Biotechnological advances.Veterinary Journal,v.175,n.1,p.45–52,2008。

KAMHAWI,S.Phlebotomine sand flies and Leishmania parasites:friends or foesTrends in Parasitology,v.22,n.9,p.439–445,2006。

KAYE,P.；SCOTT,P.Leishmaniasis:complexity at the host-pathogen interface.Nature reviews.Microbiology,v.9,n.8,p.604–615,2011。

MAGELLAN,F.B.et al.Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis.PLoS ONE,v.12,n.9,p.e0184867,2017。

METTLER,M.et al.Evaluation of enzyme-linked immunosorbent assays,an immunofluorescent-antibody test,and two rapid tests (immunochromatographic-dipstick and gel tests)for serological diagnosis of symptomatic and asymptomatic Leishmania infections in dogs.J.Clin.Microbiol.,v.43,n.0095–1137(Print)p.5515-5519,nov.2005。

MURRAY,H.W.et al.Advances in leishmaniasis.Lancet,v.366,n.1474–547X(Electronic)p.1561-1577,Oct.2005。

NOLI,C.；SARIDOMICHELAKIS,M.N.An update on the diagnosis and treatment of canine leishmaniosis caused by Leishmania infantum(syn.L.chagasi).The Veterinary Journal,v.202,n.3,p.425–435,2014。

OLIVEIRA,G.G.et al.Characterization of novel Leishmania infantum recombinant proteins encoded by genes from five families with distinct capacities for serodiagnosis of canine and human visceral leishmaniasis.Am.J.Trop.Med.Hyg.,v.85,n.1476–1645(Electronic),p.1025-1034,Dec.2011。

PACE,D.Leishmaniasis.Journal of Infection,v.69,n.S1,p.S10-S18,2014。

PALTRINIERI,S.et al.Laboratory tests for diagnosing and monitoring canine leishmaniasis.Veterinary Clinical Pathology,v.45,n.4,p.552–578,2016。

PATTABHI,S.et al.Design,development and evaluation of rK28-based point-of-care tests for improving rapid diagnosis of visceral leishmaniasis.PLoS Neglected Tropical Diseases,v.4,n.9,2010。

PEIXOTO,H.M.；DE OLIVEIRA,M.R.F.；ROMERO,G.A.S.Serological diagnosis of canine visceral leishmaniasis in Brazil:systematic review and meta-analysis.Tropical Medicine&International Health,v.20,n.3,p.334–352,2015。

QUINNELL,R.J.et al.Evaluation of rK39 rapid diagnostic tests for canine visceral leishmaniasis:longitudinal study and meta-analysis.PLoS.Negl.Trop.Dis.,v.7,n.1935–2735(Electronic)

QUINNELL,R.J.；COURTENAY,O.Transmission,reservoir hosts and control of zoonotic visceral leishmaniasis.Parasitology,v.136,n.14,p.1915,2009。

READY,P.D.Biology of Phlebotomine Sand Flies as Vectors of Disease Agents.Annual Review of Entomology,v.58,n.1,p.227–250,2013。

SARIDOMICHELAKIS,M.N.Advances in the pathogenesis of canine leishmaniosis:Epidemiologic and diagnostic implications.Veterinary Dermatology,v.20,n.5–6,p.471–489,2009。

SINGH,O.P.；SUNDAR,S.Developments in Diagnosis of Visceral Leishmaniasis in the Elimination Era.v.2015,2015。

SINGH,S.et al.Diagnostic and prognostic value of K39 recombinant antigen in Indian leishmaniasis.J.Parasitol.,v.81,n.0022–3395(Print)p.1000-1003,dec.1995。

SOTO,M.et al.Multicomponent chimeric antigen for serodiagnosis of canine visceral leishmaniasis.Journal of Clinical Microbiology,v.36,n.1,p.58–63,1998。

SRIVASTAVA,P.et al.Diagnosis of visceral leishmaniasis.Trans.R.Soc.Trop.Med.Hyg.,v.105,n.1878–3503(Electronic),p.1-6,jan.2011。

SRIVASTAVA,S.et al.Possibilities and challenges for developing a successful vaccine for leishmaniasis.Parasites&Vectors,v.9,n.1,p.277,2016。

SRIVIDYA,G.et al.Diagnosis of visceral leishmaniasis:developments over the last decade.Parasitol.Res.,v.110,n.1432–1955(Electronic),p.1065-1078,Mar.2012。

SUNDAR,S.et al.Rapid accurate field diagnosis of Indian visceral leishmaniasis.Lancet,v.351,n.9102,p.563–565,1998。

SUNDAR,S.；RAI,M.Laboratory diagnosis of visceral leishmaniasis.Clin.Diagn.Lab Immunol.,v.9,n.1071–412X(Print)p.951-958,Sep.2002。

WELCH,M.et al.Designing genes for successful protein expression.1.ed.[s.l.]Elsevier Inc.,2011.v.498。

The claims (modification according to treaty clause 19)

1. A chimeric protein comprising an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, or SEQ ID No. 8.

2. A nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO 1, 3, 5 or 7 and degenerate sequences thereof encoding identical amino acid sequences as defined by SEQ ID NO 2, 4, 6 or 8, respectively.

3. The nucleic acid molecule of claim 2, further comprising:

a spacer between the antigenic regions,

optimized sequences at the 5' end of the synthetic gene and in the region encoding the protein end, such as: pRBS-SD1+6AA,

a ribosome binding site and Shine-Dalgarno sequence,

pSS-gIII，

the t7 tag peptide is a peptide,

ET-6 His; or

A translation stop codon;

or a combination of these.

4. An expression cassette comprising a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7 and degenerate sequences thereof, operably linked to a promoter and a transcription terminator.

5. Expression vector comprising a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7 and degenerate sequences thereof, or an expression cassette as defined in claim 4.

6. Host cell, characterized in that it comprises a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7 and degenerate sequences thereof, or an expression cassette as defined in claim 4, or an expression vector as defined in claim 5.

7. A method for producing a chimeric protein, comprising the steps of:

(a) transforming a host cell with an expression vector comprising a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7 and degenerate sequences thereof,

(b) culturing the host cell to produce the chimeric protein; and

(c) isolating the chimeric protein from the cell or from the medium surrounding the cell.

8. Composition for the diagnosis of leishmaniasis, characterized in that it comprises one or more chimeric proteins as defined in claim 1.

9. The composition of claim 8, wherein the diagnosis is still performed on a sample from a dog or human.

10. Kit for the diagnosis of visceral leishmaniasis, characterized in that it comprises one or more chimeric proteins as defined in claim 1 or a composition as defined in claim 8.

11. The kit of claim 10, further comprising instructions for use.

12. The kit of claim 10 or 11, further comprising a device for detecting antigen/antibody complexes, which may comprise a signal generator capable of generating a detectable signal.

13. The kit of any one of claims 10 to 12, wherein the diagnosis is performed on a sample from a dog or a human.

14. Use of one or more chimeric proteins as defined in claim 1, of a composition as defined in claim 8 or 9, or of a kit as defined in any one of claims 10 to 13, for the diagnosis of leishmaniasis.

15. A method for the in vitro diagnosis of leishmaniasis, comprising the steps of:

(a) providing one or more chimeric proteins as defined in claim 1 or a composition as defined in claim 8 or 9 and a human or dog serum sample,

(b) contacting the one or more chimeric proteins or the composition with a biological sample to be tested for a sufficient time and under conditions sufficient for antibody/antigen complexes to form; and

(c) the chimeric protein/antibody complex formed in the previous step is detected by a detection technique capable of producing a detectable signal in the presence of the antigen/antibody complex.

16. The method of claim 15, wherein the biological sample is selected from the group consisting of saliva, urine, serum, or blood.