CHIMERIC PROTEIN, METHOD OF PRODUCTION AND USE THEREOF, AND ALSO A NUCLEIC ACID MOLECULE, EXPRESSION CASSETTE, EXPRESSION VECTOR, HOST CELL, COMPOSITION FOR THE DIAGNOSIS OF LEISHMANIASIS, KIT FOR THE DIAGNOSIS OF LEISHMANIASIS AND METHOD OF DIAGNOSIS OF LEISHMANIASIS IN VITRO

Abstract

The present invention relates to chimeric proteins, their uses and production method comprising native protein fractions from Leishmania infantum for the Visceral Leishmaniasis diagnosis. The invention also relates to nucleic acid, expression cassette, expression vector, host cell, visceral leishmaniasis diagnostic kit, visceral leishmaniasis diagnostic kit, visceral leishmaniasis diagnostic method, and vaccine composition.

Claims

1. Chimeric protein, characterized by comprising fractions of native Leishmania infantum proteins.

2. Chimeric protein according to claim 1, characterized by further comprising: spacers between the antigenic regions, optimized sequences at the 5′ end of the synthetic genes as well as in the region encoding the protein ends, such as: pRBS-SD1+6AA, ribosome binding site and Shine-Dalgarno sequence, pSS-gIII, t7 tag peptide, ET-6His; or translation termination codon; or a combination of these.

3. Chimeric protein according to claim 1 or 2, characterized by comprising an amino acid sequence having at least 90% sequence identity with 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. Nucleic acid molecule, characterized by comprising a nucleotide sequence with at least 90% sequence identity with 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 their degenerate sequences encoding the same amino acid sequences as defined by SEQ ID NO: 2, 4, 6 or 8, respectively.

5. Expression cassette, characterized by comprising the nucleic acid molecule as defined in claim 4 operably linked to a promoter and a transcription terminator.

6. Expression vector, characterized by comprising the nucleic acid molecule as defined in claim 4 or the expression cassette as defined in claim 5.

7. Host cell, characterized by comprising the nucleic acid molecule as defined in claim 4 or the expression cassette as defined in claim 5 or the expression vector as defined in claim 6

8. Method for the chimeric protein production, characterized by comprising the following steps: (a) transforming a host cell with the expression vector comprising said nucleic acid molecule as defined in claim 4, (b) culturing the said host cell for the production of the chimeric proteins; and (c) isolating said chimeric proteins from said cell or from the culture medium surrounding said cell.

9. Composition for diagnosis of Leishmaniasis, characterized by comprising one or more chimeric proteins as defined in any one of claims 1 to 3.

10. Composition according to claim 9, characterized by the fact that the diagnosis can still be performed on samples from dogs or humans.

11. Leishmaniasis diagnostic kit, characterized by comprising one or more chimeric proteins as defined in any one of claims 1 to 3 or a composition as defined in claim 9.

12. Kit according to claim 11, characterized by further comprising instructions for use.

13. Kit according to claim 11 or 12, characterized by further comprising a means for detecting the antigen/antibody complex, which may comprise a signal generator, capable of generating a detectable signal.

14. Kit according to any one of claims 11 to 13, characterized in that the diagnosis can be performed on samples from dogs or humans.

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

16. In vitro diagnostic method for Leishmaniasis, characterized by comprising the following steps: (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 with said human or dog serum sample, (b) contacting said one or more chimeric proteins or said composition with the biological sample to be tested for a sufficient time and under sufficient conditions for the formation of antibody/antigen complexes; and (c) detecting the chimeric protein/antibody complex formed in the previous step by means of a detection technique capable of generating a detectable signal in the presence of this antigen/antibody complex.

17. Method according to claim 16, characterized in that the biological sample is selected from the group comprising saliva, urine, serum or blood.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0069] FIG. 1 refers to the schematic representation of the chimeric proteins Q1 and Q5. The complete chimera is represented, showing, in the (Q1) in Q4, the Lci12-2-3-13 regions and, in Q5, the Lci12-2-3 regions flanked by XbaI/HindIII enzyme sites and the SalI/XhoI sites for truncations and the pSS-gIII optimization tag flanked by the NcoI site.

[0070] FIG. 2 refers to the confirmation of the subcloning of the chimeric genes. One can observe a 1% Agarose gel showing the genes Q1COM, Q1NN, Q1SX and Q5, by digestion with the enzyme duo XbaI/HindIII, the release of the complete chimeric genes (shown in red) in the expected sizes shown in table 1. Thus confirming the subcloning. The marker used was Ladder 1 Kb plus. Notes: Q1COM, was digested by the enzymes ScaI/XbaI/HindIII, because the insert is the same size as the vector, so additional digestion was performed with ScaI that leads to cleavage of the plasmid into two fragments and allows visualization of the insert as indicated.

[0071] FIG. 3 refers to the expression of the chimeric proteins. An SDS-PAGE gel showing the expression of the chimeric proteins in Escherichia coli Rosetta cells at the sizes described in Table 2 can be seen.

[0072] FIG. 4 refers to the western blot of the chimeric proteins. Recognition of the chimeric proteins by the Anti-Histidine monoclonal antibody can be observed. The reaction of the proteins is visualized in red in their predicted sizes.

[0073] FIG. 5 refers to the purification of the recombinant chimeric proteins Q1SX and Q5 by affinity chromatography. The panel shows the purification profile of the Q1SX and Q5 protein by affinity chromatography, highlighted in red.

[0074] FIG. 6 refers to the indirect ELISA of the Q1SX chimeric protein with the VL sera. The scatterplots and bar graphs show the sensitivity and specificity of the 50 human and 40 dog sera positive for VL against the Q1 chimeric protein. The horizontal line represents the cutoff of the reaction.

[0075] FIG. 7 refers to the indirect ELISA of the Q5 chimeric protein with the VL sera. The scatterplots and bar graphs show the sensitivity and specificity of the 50 human and 40 dog sera positive for VL against the Q5 chimeric protein. The scatter plot shows the human and dog sera against Q5. The horizontal line represents the cutoff of the reaction.

[0076] FIG. 8 refers to the indirect ELISA of the Q5 chimeric protein with LTA sera. The scatter plots show the cross-reaction of the 50 human sera positive for ATL against the Q5 chimeric protein. The horizontal line represents the cutoff of the reaction.

[0077] FIG. 9 refers to the indirect ELISA of the Q5 chimeric protein with HIV and HIV+/VL+ Coinfected sera. Scatter plots show the cross-reactivity of HIV-positive human sera and the sensitivity and specificity and of co-infected (VL+HIV) against the chimeric Q5 protein. The scatter plot shows the positive and negative human sera against Q5. The horizontal line represents the cutoff of the reaction.

DETAILED DESCRIPTION OF THE INVENTION

[0078] Unless differently defined, all technical and scientific terms used herein have the same meaning as understood by a person skilled in the subject matter to which the invention pertains. The terminology used in describing the invention is intended to describe particular embodiments only, and does not intend to limit the scope of the teachings. Unless otherwise stated, all numbers expressing quantities, percentages and proportions, and other numerical values used in the descriptive report and claims, should be understood as being modified in all cases by the term “about”. Thus, unless otherwise stated, the numerical parameters shown in the descriptive report and in the claims are approximations that may vary, depending on the properties to be obtained.

[0079] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques, and immunology, within the skill of the art. Such techniques are explained fully in the literature. Take a look, e.g. Fundamental Virology, 2nd Edition, vols. I & II (B. N. Fields and D. M. Knipe, eds.) Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989) Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0080] The polypeptides of the present invention exhibited reproducibility with respect to sensitivity and specificity. This suggests that the developed proteins can remain stable for long periods of time, maintaining their reactive capacity. It is possible that the composition of the stock buffer may have favored its stability, the use of protease inhibitors, the presence of a denaturing agent in the buffer, or even due to their amino acid sequence. Stable proteins are considered when there is interest for diagnostic application.

[0081] As employed throughout the present application, the term “amino acid” denotes the ?-amino group that directly or in the form of a precursor can be encoded by a nucleic acid. The individual amino acids are encoded by nucleic acids consisting of three nucleotides, known as codons or 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 known as “degeneracy of the genetic code”. The term “amino acid”, as used in the present application, denotes the naturally occurring ?-amino acids, comprising alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0082] The terms “peptide”, “polypeptide” or “protein” can be used interchangeably, and refer to a polymer of amino acids connected by peptide bonds, regardless of the number of amino acid residues constituting this chain. Polypeptides, as used here, include “variants” or “derivatives” thereof, which refer to a polypeptide that includes variations or modifications, for example, substitution, deletion, addition, or chemical modifications in its amino acid sequence with respect to the reference polypeptide. Examples of chemical modifications are glycosylation, PEGlation, PEG alkylation, alkylation, phosphorylation, acetylation, amidation, etc. The polypeptide can be produced artificially from cloned nucleotide sequences using the recombinant DNA technique, or it can be prepared using a known chemical synthesis reaction.

[0083] More specifically, the term polypeptide of the present invention may also be understood as antigen, polyantigen or multieptope antigen, which consist of a junction of different epitopes that may or may not be linked by ligands (linkers) flexible or rigid, specific for a single pathogen or for different pathogens.

[0084] In a first embodiment, the present invention provides chimeric proteins comprising fractions of native Leishmania infantum proteins, which have been generated from antigenic combinations of three of the 13 so-called Lci proteins already described (Lci1 to Lci13), selected for presenting the best results for the human VL diagnosis (Lci2) or canine (Lci3 and Lci12).

[0085] Such proteins were generated from chemically synthesized genes that underwent multiple subcloning steps and consisted of the splicing of the same fragments of these three antigenic proteins.

[0086] The three proteins selected for the chimeric constructs (Lci2, Lci3 and Lci12) have no sequence homology with each other, but have similar structures, consisting of multiple copies of small motifs (Lci2-39 amino acids; Lci3-14 amino acids; Lci 12-8 amino acids) repeated numerous times and flanked by non-repeating regions, as described (MAGALHÃES et al., 2017; OLIVEIRA et al., 2011).

[0087] The selection of the antigenic regions of each Lci included in the chimeric constructs was based on size, solubility, and immunogenic potential criteria. The respective coding sequences were then optimized for expression in Escherichia coli and spliced into a sequence, taking into consideration the following parameters: reading phase, codon frequency, secondary mRNA structure, GC content distribution, and restriction sites.

[0088] Small spacers were included between the antigenic regions in the chimeric protein aiming to facilitate the folding conditions of the resulting recombinant protein. Optimized sequences at the 5′ end of the synthetic genes were also included, as well as in the region encoding the ends of proteins, such as: pRBS-SD1+6AA-ribosome binding site and Shine-Dalgarno sequence; pSS-gIII-N-terminal sequence of a bacteriophage envelope protein to optimize expression; t7 tag peptide-N-terminal sequence to stabilize and optimize expression; ET-6His-polyhistidine sequence (six) introduced at the C-terminal end to allow purification of the protein; and translation termination codon.

[0089] Restriction enzyme sites were also added to the synthetic genes at strategic points along the constructs, in order to allow easy modification of these genes, if necessary, by including or excluding some sequences and allowing further manipulation of their structure. These genes gave rise to the chimeric constructs Q1 (SEQ ID NO: 1, 3, and 5) and Q5 (SEQ ID NO: 7) with the predicted sizes of 2845 and 1986 bp (FIG. 1).

[0090] These differ in that the Q5 construct includes an additional repetitive segment of the Lci3 protein, absent from the Q1 construct, whereas the original Q1 construct features at the 3′ end of the gene a fragment encoding for the C-terminal region of a fourth antigenic protein, Lci13. In possession of the commercially synthesized gene constructs, they were then subcloned into the bacterial expression vector pRSETa, with confirmation of subcloning performed by digestion by the restriction enzyme pair XbaI/HindIII, releasing the DNA fragments of the expected size (FIG. 2) and by sequencing analysis.

[0091] For the Q1 construction only (SEQ ID NO: 1) Digests were performed on the fragment cloned into the plasmid pRSETa, using internal restriction sites present only in the synthetic gene, in order to generate truncated variants of this gene where different fragments encoding selected portions of the protein were removed.

[0092] The goal of this step was to expand the number of chimeric proteins synthesized in order to see which of the possible alternatives and antigenic combinations enhances protein expression in the prokaryotic system.

[0093] A first digestion was performed with the enzyme NcoI that releases a 75 bp fragment in the N-terminal region of the gene encoding the expression optimization peptide pSS-gIII.

[0094] After purification and reconnection of the plasmid construct this fragment is lost and the resulting, slightly smaller chimeric gene encodes for a truncated Q1 protein, where this peptide is absent (Q1NN—SEQ ID NO: 4).

[0095] Using an equivalent strategy, a second digest was performed from the complete gene (SEQ ID NO: 1) using the SalI/XhoI enzymes. Since the restriction sites for these enzymes are complementary, ligation of the major fragment product of this digestion leads to the production of a chimeric gene (SEQ ID NO: 5) where the 1155 bp fragment encoding for Lci13 (Q1SX—SEQ ID NO: 6).

[0096] FIG. 2 also illustrates the fragments generated after digestion of the plasmids containing the two truncated constructs generated from the chimeric Q1 gene (SEQ ID NO: 1) and the sizes of each are summarized in Table 1.

TABLE-US-00001 TABLE 1 PRIMARY NAME Insert size (in pb) Protein size (kDa) Q1 2845 102  Q1NN 2734 99 Q1SX 1690 60 Q5 1960 98

[0097] In a second aspect, the invention provides nucleic acid molecules encoding the disclosed chimeric proteins.

[0098] The nucleic acid molecules of the present invention are represented, non-limiting, by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or sequences with at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or their degenerate sequences that encode the same polypeptide.

[0099] 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 (for example, GAU and GAC both encode Asp).

[0100] A subject matter expert would recognize that the degenerations are fully supported based on the information provided in the application and common knowledge of the prior art. For example, the degeneration of the genetic code (that is, different codons can encode the same amino acids) is common knowledge in the technique and the identity of the amino acid encoded by each codon is well established.

[0101] Based on information well known and established in the prior art, the subject matter expert is able to identify nucleotide substitutions that do not alter the resulting amino acid sequence. Thus, when in possession of both the nucleotide sequence of a gene and the amino acid sequence of the encoded protein, the subject matter expert will easily identify degenerates that encode the same protein, with the same amino acid sequence.

[0102] In a third aspect, the present invention provides an expression cassette comprising the nucleic acid according to the invention. The said cassette is placed under conditions that lead to the expression of the polypeptides of the present invention.

[0103] The expression cassette may further comprise sequences necessary for its expression, such as, promoters, enhancer and terminator sequences compatible with the expression system. Furthermore, the expression cassette can comprise spacer sequences, ligand sequences and suitable restriction sites. In addition, the cassette may also comprise a sequence coding for histidine tails.

[0104] In a fourth aspect, the present invention provides an expression vector comprising a nucleic acid molecule or an expression cassette according to the invention. Said vector can be used to transform a host cell and allow the expression of the nucleic acid according to the invention in said cell.

[0105] Advantageously, the expression vector comprises regulatory elements that enable the expression of a nucleic acid molecule and elements that enable its selection in the host cell according to the invention. The methods for selecting these elements depending on the host cell in which expression is desired, are well known to those versed in the technique and widely described in the literature.

[0106] The vectors can be constructed by classical molecular biology techniques, well known to the tech-savvy. Non-limiting examples of expression vectors suitable for expression in host cells are plasmids and viral or bacterial vectors.

[0107] In a fifth aspect, the present invention provides a host cell transiently or stably transformed/transfected with the nucleic acid, cassette or vector of the invention. The nucleic acid molecule, cassette or vector may be contained in the cell in the form of an episome or in chromosomal form.

[0108] The host cell can be a bacterial cell, yeast, filamentous fungi, protozoa, insects, animal and plant cells.

[0109] In a sixth aspect, the present invention provides a method for producing chimeric proteins of the present invention comprising the following steps transforming a host cell with the expression vector comprising said nucleic acid molecule or expression cassette, culturing the said host cell for the production of the chimeric proteins (in vivo expression systems); and isolating said chimeric proteins from said cell or from the culture medium surrounding said cell.

[0110] Particularly suitable expression systems include microorganisms, such as bacteria transformed with recombinant DNA expression vectors of bacteriophage, plasmid or cosmid; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus 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) or animal cell systems. It is also possible to employ cell-free translation systems to produce the polypeptides of the invention.

[0111] The introduction of the nucleic acid molecule, expression cassette, or the vector encoding a recombinant or synthetic protein of the present invention into host cells can be accomplished by methods described in many standard laboratory manuals, such as Davis et al. Basic Methods in Molecular Biology (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989).

[0112] In a preferential aspect, the expression of the different recombinant proteins of the present invention was carried out using the plasmid DNA of the generated chimeric constructs, which was transformed into E. coli cells and, after the period of growth and expression of the recombinant proteins, Total bacterial extracts from cells expressing the different proteins were fractionated on SDS-PAGE gel (see FIG. 3).

[0113] The transformed or transfected host cell described above is then grown in a suitable nutrient medium under conducive conditions that allow the expression of the recombinant proteins of the invention. The medium used to grow the cells can be any conventional medium suitable for growing host cells, such as minimal or complex medium containing appropriate supplements. Suitable media are available from commercial suppliers or can be prepared according to published prescriptions (for example, in the catalogs of the American Type Culture Collection). The proteins of the invention produced by the cells can then be recovered from the cell or culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the aqueous protein components from the supernatant or filtrate using a salt, e.g. ammonium sulfate, purification by a variety of chromatographic procedures, for example ion exchange chromatography, exclusion chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, or similar, depending on the type of polypeptide in question.

[0114] The obtained chimeric protein is then purified and biochemically characterized using, for example, methods common to the field of biochemistry, such as HPLC, SDS-PAGE, Western Blotting, pH gradient isoelectric focusing, circular dichroism. By means of these methods, it is possible to determine characteristics such as, for example, the yield of expression of the chimeric protein; the determination of the characteristics of secondary structures, as well as other characteristics whose determination is important for the development of a diagnostic composition for Visceral Leishmaniasis.

[0115] In one mode, the transformation of the host cell with the nucleic acid molecule of the present invention is accomplished by means of an expression vector. In a specific mode, the vector is pRSETa and the transformed host cell is E. coli Rosetta™ 2. Table 1 illustrates the expected molecular weight for each of the recombinant proteins encoded by the different chimeric genes obtained. Among the four evaluated constructions, only Q1SX (SEQ ID NO: 5) and the Q5 construction (SEQ ID NO: 7) allowed the expression of clearly identifiable bands in the total bacterial extracts.

[0116] The host cell culture conditions indicated in step (b) are known to a person skilled in the art. In one embodiment, culturing is performed in LB medium in the presence of an antibiotic, under stirring. In a specific culturing, the antibiotic is ampicillin and chloramphenicol. Culturing can take place at different temperatures for different periods of time. In a specific mode, culturing can be performed for about 4 to about 6 hours at a temperature of about 35° C. to about 37° C. In a preferred embodiment, the culturing is done at 37° C. for 6 hours under stirring.

[0117] The production of the chimeric protein referred to in step (b) can be performed with any technique known in the state of the art. In one embodiment, the induction of the chimeric protein expression of the invention is accomplished by adding IPTG to the culture medium, after obtaining adequate optical density.

[0118] To confirm the expression of the various truncations of the Q1 protein, the respective bacterial extracts were then subjected to Western Blot assays where the recognition of these proteins by a commercial monoclonal antibody directed against the polyhistidine sequence present in each of the proteins was evaluated.

[0119] As shown in FIG. 4, all three Q1 protein constructs were recognized by the monoclonal antibody, confirming their expression, while Q1SX (SEQ ID NO: 5) was expressed at higher levels than the others compatible with its visualization on SDS-PAGE gels.

[0120] Similarly, the Q5 protein (SEQ ID NO: 8) was also recognized by the commercial antibody directed at the polyhistidine sequence. From the expression analysis, E. coli cultures expressing Q1SX and Q5 proteins were expanded for large scale expression of these proteins and purification by affinity chromatography on Nickel resin. FIG. 5 shows representative SDS-PAGE gels of the purification of the Q1SX and Q5 proteins.

[0121] The purified Q1SX and Q5 proteins were quantified by comparison with defined amounts of BSA on SDS-PAGE and used in ELISA assays aiming to evaluate their potential in the diagnosis of human and canine VL. Each protein was then evaluated with human sera from individuals proven to be infected with L. infantum, sera from healthy controls, sera from proven VL infected dogs and their respective healthy controls.

[0122] The isolation of the chimeric protein referred to in step (c) can be performed with any technique known in the state of the art. In an embodiment, purification is achieved by chromatography techniques. In a specific embodiment, purification is performed by nickel resin affinity chromatography. Non-limiting examples include the affinity chromatography method, ion exchange, other affinity or adsorption methods, ion pair, reversed phase, and molecular exclusion.

[0123] In a seventh aspect, the invention provides a composition, comprising one or more of the chimeric proteins according to the present invention.

[0124] In a particular aspect, the composition is used as a reagent for the Visceral Leishmaniasis diagnosis.

[0125] In an eighth aspect, the invention provides a Visceral Leishmaniasis diagnostic kit comprising one or more chimeric proteins or a composition as defined in the present invention.

[0126] Optionally, the kit also includes instructions for use. [00124] Additionally, 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.

[0127] The detection means can be those known in the technique. A non-limiting example of the detection means may be a conjugate comprised of an antibody coupled to a signal generating compound capable of generating a detectable signal.

[0128] In a ninth aspect, the invention provides the use of said chimeric proteins in the Visceral Leishmaniasis diagnosis.

[0129] In a tenth aspect, the invention provides a method for diagnosing Visceral Leishmaniasis comprising detecting the chimeric protein/antibody complex by using an immunological detection technique from a human or dog serum sample.

[0130] The evaluation of the chimeric proteins of the present invention has demonstrated that, from the results of the ELISA of the chimeric protein Q1 with sera from humans and dogs, it presented a sensitivity for human sera of (76%) and specificity of (100%), however, results with sera from dogs showed a sensitivity of (99%) and specificity of (100%) shown in FIG. 6.

[0131] The results of the Q5 chimeric protein ELISA showed a sensitivity for human sera of 81%, while the results with dog sera maintained a sensitivity of 99% (FIG. 7).

[0132] Based on the best results obtained for the Q5 protein, said protein was used in new ELISA assays to assess its cross recognition with sera from patients with tegumentary leishmaniasis. The results with these sera showed a nonspecific reaction of 8% (FIG. 8).

[0133] Sera from patients co-infected with VL and HIV were then evaluated, showing 98% sensitivity and 100% specificity (shown in FIG. 9) and no reaction with HIV-positive patients only. Table 2 shows the results of all analyzes with sera from the groups described above with Q5 and Q1SX protein, showing the sensitivity, specificity and amount of sera evaluated.

TABLE-US-00002 TABLE 2 Serum Type Sensitivity Specificity Quantity Serum Pos Human Piauí (Q5) 82% 100% 50 Human Piauí (Q1) 72%  96% 50 Coinfected Lv+/HIV+ 86% 100%  7 (Q5) Human LTA (Q5)  2% 100% 50 Humans HIV+/VL−  0% 100%  7 (Q5) Bahia Dogs (Q5) 99% 100% 37 Bahia Dogs (Q1) 99% 100% 37

[0134] Results of the Q1 and Q5 protein ELISA showing the amount of sera used in each test group and the sensitivity and specificity shown in each group as well. Divided into Dogs and Humans. The negative sera were also tested to calculate the values.

EXAMPLES

Example 1

Bioinformatics Methods, Chemical Synthesis and Subcloning of Chimeric Genes

[0135] The prediction of the presence of linear B-cell epitopes in the sequences of selected proteins (Lci2, Lci3 and Lci12) was performed by the program BCPred12 (EL?MANZALAWY; DOBBS; HONAVAR, 2008). The sequences of the chimeric genes optimized for the expression in Escherichia coli were designed using the Gendesigner program (WELCH et al., 2011) and their commercial synthesis carried out by GenScript companies (GenScript, Piscataway, N.J., USA) (Q1 protein) and Thermo (Life Tech, Sao Paulo, Brazil). (Q5 protein).

[0136] These genes were delivered already cloned into the commercial vector pUC57 flanked by restriction sites for the enzymes XbaI/HindIII. For subcloning into the bacterial expression vector, pRSETa (Thermo Life Tech, Sao Paulo, Brazil), the chimeric genes were recovered by double digestion with the restriction enzymes XbaI/HindIII and subcloned into the same sites of the pRSETa vector.

[0137] After the first subcloning of the Q1 gene into the pRSETa vector two truncation reactions of this gene were performed. The first, from digestion with the enzyme NcoI, which led to the excision of the fragment encoding the pSS-gIII peptide, and the second with the SalI/XhoI enzyme pair, excising the fragment encoding the C-terminal region of the Lci13 protein. All final constructs were confirmed by digestion with restriction enzymes and sequencing.

Example 2

Expression and Purification of Recombinant Proteins

[0138] For the expression of the chimeric protein, plasmid constructs derived from the pRSETa vector were transformed into competent E. coli Rosetta™ 2 cells (Merck Millipore) followed by selection on LB medium (Luria Bertani) ampicillin-supplemented solid (100 μg/ml) and chloramphenicol (34 μg/ml) at 37° C.

[0139] Clones of the transformed cells were grown in liquid LB medium with the same concentrations of antibiotics and induction of expression of the recombinant proteins performed by adding IPTG to a final concentration of 0.1 mM, at an optical density (D.O) from 0.6 to 0.8 at 600 nm.

[0140] The result was visualized using a polyacrylamide gel (SDS-PAGE 15%) after staining the gel with Comassie Blue R-250. To obtain recombinant proteins, cell pellets obtained after the induction were resuspended in 20 mL of lysis buffer and equilibrated (100 mM sodium phosphate, 10 mM Tris, 8 M Urea, 20 mM imidazole—pH 8.0) and solubilized by ultrasonication in 5 pulses of 30 seconds at intervals of 1 minute at 4° C.

[0141] After centrifugation at 5000 rpm for 10 minutes, protein purification was performed by incubation with Ni-NTA agarose resin (Qiagen) followed by washes in the same buffer and in denaturing buffer at pH 6.0 (100 mM sodium phosphate, 10 mM Tris, 8 M Urea, 30 mM imidazole) and elution in denaturing buffer at pH 4.5 at 1 M imidazole. Aliquots were evaluated on SDS-PAGE gels as described.

Example 3

Western Blot Assays

[0142] For Western-blot assays chimeric proteins were fractionated on 15% SDS-PAGE gels and transferred to PVDF membranes (Immobilon-P Millipore®) which were blocked in TBS buffer (20 mM Tris, 500 mM NaCl, pH7,5) supplemented with 5% skim milk and 1% Tween-20.

[0143] The membranes were then incubated with the antibodies/sera against the target proteins at final dilutions of 1:3000 and 1:1000 in TBS buffer with 5% milk and 1% Tween-20. After washing with 1% TBS/Tween-20, a new incubation with rabbit anti IgG was performed, labeled with peroxidase, (Jackson Immunoresearch Laboratories), diluted 1:10000 in TBS buffer with 5% milk and 1% Tween-20.

[0144] After further washing, the membranes were revealed in a solution of 1.2 mM luminol, 0.4 mM iodophenol and 0.03% hydrogen peroxide for 2 minutes. These membranes were then dried and exposed to autoradiography film for 1 and 5 minutes, followed by developing the film.

Example 4

Human Sera Used

[0145] Initially, a panel of human sera consisting of two distinct sera groups was assembled. The first group was composed of 50 sera obtained from the control group in the serum library of the Laboratory of Virology and Experimental Therapy (LAVITE) of the JAM, set up for the yellow fever vaccine project. These sera come from healthy individuals living in a non-endemic area for visceral leishmaniasis (urban area of the city of Recife) and were kindly provided by Dr. Rafael Dhália.

[0146] The second group consisted of 50 sera from patients with visceral leishmaniasis with clinical and laboratory diagnosis (confirmed by parasitological examination) This information was kindly provided by Dr. Carlos Henrique Costa, from the Federal University of Piauí.

[0147] All human sera were collected after approval for use, by appropriate ethics committees, as described below: the use of serum from VL patients was approved by the ethics committee of the Federal University of Piauí (0116/2005); the sera from the negative controls were included in a study approved by the ethics committee of the Ministry of Health (25000.119007/2002-03).

[0148] Sera from patients with cutaneous leishmaniasis, included in project CAEE 0014.0.095.000-05, approved by the ethics committee of CPqAM-FIOCRUZ, were also used (Mar. 8, 2008).

[0149] Finally sera from patients with coinfection (HIV/VL) and the sera from patients with HIV only, also evaluated in this proposal, were provided by Dr. Zulma Medeiros, from a project approved by the ethics committee of the Centro de Pesquisas Aggeu Magalhães—FIOCRUZ (CAEE: 53495816.0.0000.5190).

Example 5

Dog Serums Used

[0150] About one hundred domesticated or stray dogs were studied from an endemic area of visceral leishmaniasis in Jequié-BA.

[0151] Blood samples were collected, and from there, aliquots of serum were obtained and stored at −20° C. For some of these animals an aspiration puncture of the spleen was performed and the aspirate used for culture and detection of Leishmania, according to the method previously described (BARROUIN-MELO et al., 2006).

[0152] We also used 90 sera from dogs with VL provided in partnership with Dr. Valeria Pereira. All dogs were treated according to the guidelines for animal experimentation of the Oswaldo Cruz Foundation. [00151] The use of the dog sera in this study was approved by the Ethics Committee on Animal Use (CPqGM-FIOCRUZ, Ceua, protocol N.040/2005 and CPqAM-FIOCRUZ, Ceua, protocol 27/2016).

Example 6

Indirect Enzyme Immunoassay

[0153] After quantification, the recombinant proteins were used for sensitization of microtiter plates, diluted in carbonate-bicarbonate buffer (NaCO3, 0.05 M; pH 9.6), for a concentration of 400 ng (1 ?g for the total extract of the Leishmania infantum parasite, used as a reaction control) per well with a volume of 100 ?1 per well, and kept for 16 hours at 4° C. in a humid chamber.

[0154] After washes with PBS (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 1.8 mM) blocking was performed with PBS plus 10% skim milk, followed by further washing with PBS plus 0.05% Tween-20. Incubation with the human and canine sera was then carried out, diluted in PBS Tween-20 with 10% milk at dilutions of 1:2500 and 1:900, respectively.

[0155] Washing and incubation steps were again followed with the conjugates, anti-Human IgG or anti-Canine IgG linked to peroxidase, for 1 hour at room temperature in a humid chamber, in dilutions of 1:10000 and 1:1200, respectively.

[0156] After further washing, development was carried out in citrate-phosphate buffer (Na2HPO4, C6H8O7, 0.1 M, pH:5.0) in the presence of the chromogen OPD (Orthophenylenediamine) in a 0.01% concentration (300 μl) and hydrogen peroxide (in a 4% concentration, 3 μl) in a darkroom for 30 minutes, and the reaction was stopped with sulfuric acid (H2SO4 at 2.5 M). The reading was performed in a 490 nm filter in the Benchmark Plus Microplate Manager 5.2 (BIO-RAD).

Example 7

Statistical Analysis of the Indirect ELISA Results

[0157] 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).

[0158] For the association between the variables and their determinants, the chi-square test was used in a double-entry table (2×2) relating the diagnosis of the disease and the test result.

[0159] Sensitivity is given by the percentage of positives detected by the test among individuals known to be ill (positive parasitological), and specificity, by the percentage of negatives among non-diseased individuals.

[0160] The scatter plot was obtained using the GraphPad Prism software (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla Calif. USA).

REFERENCES

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