CHIMERIC MOLECULE USEFUL IN IMMUNOTHERAPY FOR LEISHMANIASIS, WHICH INCLUDES A FRAGMENT OF THE PFR1 PROTEIN OF LEISHMANIA INFANTUM WITH SPECIFIC IMMUNODOMINANT EPITOPES

Abstract

The present invention claims an isolated nucleotide sequence characterized by encoding the PFR1 protein of Leishmania infantum or a fragment thereof. This PFR1 protein or a fragment thereof comprises at least a selected immunodominant epitope between the following group: SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8, where the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response in an animal, against the kinetoplastids causing the leishmaniasis disease. The immunodominant epitopes are cytotoxic T-lymphocyte activators and they present a high binding affinity for A2 type MHC Class I molecule.

Claims

1. A nucleotide sequence coding for a PFR1 protein of Leishmania infantum, or a fragment thereof, comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is capable of inducing an antigen-specific T cell cytotoxic immune response in an animal against kinetoplastids causing leishmaniasis disease.

2. The nucleotide sequence according to claim 1 coding for the 1-595 amino acids of the Leishmania infantum PFR1 protein, determined by SEQ ID NO: 12.

3. The nucleotide sequence according to claim 1 coding for the 160-595 amino acids of the Leishmania infantum PFR1 protein, determined by SEQ ID NO: 9.

4. The nucleotide sequence according to claim 1 coding for the 160-548 amino acids of the Leishmania infantum PFR1 protein, determined by SEQ ID NO: 10.

5. The nucleotide sequence according to claim 1 coding for the 160-385 amino acids of the Leishmania infantum PFR1 protein, determined by SEQ ID NO: 11.

6. A chimeric molecule comprising: (a) at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal, wherein the at least one nucleotide sequence is fused to (b) a nucleotide sequence coding for a Hsp70 protein of Trypanosoma cruzi, or for a fragments thereof, wherein the chimeric molecule is capable of inducing an antigen-specific T cell cytotoxic immune response in an animal against the kinetoplastids causing the leishmaniasis disease.

7. The chimeric molecule according to claim 6, wherein the nucleotide sequence coding for the Hsp70 protein of Trypanosoma cruzi, or for the fragment thereof, is determined by SEQ ID NO: 13 or fragments thereof.

8. A recombinant vector comprising: (a) at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal, or (b) a chimeric molecule, comprising: at least one nucleotide sequence from (a) fused to a nucleotide sequence coding for the HsD70 protein of Trypanosoma cruzi, or for a fragment thereof, wherein the chimeric molecule is capable of inducing an antigen-specific T cell cytotoxic immune response in an animal against the kinetoplastids causing the leishmaniasis disease; or comprising at least one nucleotide sequence from (a) fused to a nucleotide sequence coding for the Hsp70 protein of Trypanosoma cruzi determined by the SEQ ID NO: 13 or a fragment thereof.

9. The recombinant vector of claim 8, as part of a recombinant plasmid comprising the vector.

10. A pharmaceutical composition comprising: (a) an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal, or (b) a chimeric molecule, comprising an expression product of at least one nucleotide sequence from (a) fused to an expression product of a nucleotide sequence coding for the Hsp70 protein of Trypanosome cruzi, or for a fragment thereof, wherein the chimeric molecule is capable of inducing an antigen-specific T cell cytotoxic immune response in an animal against the kinetoplastids causing the leishmaniasis disease; or comprising an expression product of at least one nucleotide sequence from (a) fused to a nucleotide sequence coding for the Hsp70 protein of Trypanosoma cruzi determined by the SEQ ID No: 13 or a fragment thereof.

11. A method for treatment and/or prevention of kinetoplastid infections that cause leishmaniasis disease in an animal, comprising, use of an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum or a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.

12. The method of claim 11, wherein the animal is a human or a dog.

13. A method for the manufacture of a therapeutic or preventive vaccine, comprising use of an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.

14. The method of claim 13, comprising the steps of: (a) identifying at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response in the animal against the kinetoplastids causing the leishmaniasis disease; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in the animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in the animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in the animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in the animal, (b) amplifying the at least one nucleotide sequence identified in (a) by PCR using genomic DNA from the kinetoplastid that causes the leishmaniasis disease, and oligonucleotides containing restriction enzymes sites that allow direct cloning of the amplicon into prokaryotic and/or eukaryotic expression vectors after its digestion with these restriction enzymes, (c) cloning that least one nucleotide sequence amplified in (b) for the production of the protein encoded by the at least one nucleotide sequence, (d) purifying the at least one protein(s) obtained in (c) by affinity chromatography, and (e) producing the vaccine for a safe inoculation.

15. A method for measuring a degree of infection of kinetoplastids that cause leishmaniasis disease in an animal, comprising use of a marker comprising an expression product of a nucleic acid sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.

16. The method of claim 15, wherein the animal is a human or a dog.

17. A method for generating protective immunological memory against infection of kinetoplastids causing leishmaniasis disease in an uninfected animal, comprising use of an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum or a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.

18. The method of claim 17, wherein the animal is a human or a dog.

19. A method for clarifying or generating partial or total clearance of kinetoplastids causing leishmaniasis disease of the tissues in an infected animal, comprising use of an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum or a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.

20. The method of claim 19, wherein the animal is a human or a dog.

Description

FIGURE LEGENDS

[0057] FIG. 1. Purified PFR-1 recombinant protein was electrophoresed in a 10% SDS-PAGE gel and visualized by coomasie blue staining. MW. Protein molecular weight marker.

[0058] FIG. 2. Measurement of Nitric Oxide (NO) concentration in the supernatant of macrophages culture stimulated with lipopolysaccharide (LPS); lipopolysaccharide+polymyxin B (LPS+PolB); purified PFR-1 protein (PFR-1); purified PFR-1 protein+polymyxin B (PFR1+PolB); L-NG-monomethyl arginine citrate (LNMMA); purified PFR-1 protein+L-NG-monomethyl arginine citrate (PFR1+LNMMA). Concentration is expressed in pmol/litre.

[0059] FIGS. 3A to 3C. Western blot analysis of PFR-1 and PFR1-Hsp70 protein expression in COS-7 cells transfected with the pCMV4 empty vector (lane 2) and pCMV4 PFR1 (lane 3); pCMV4 PFR1-Hsp70 (lane 4) and pCMV4 PFR1-Th70 (lane 5) constructs. No transfected cells are used as control (lane 1). Proteinmolecular weight marker (Lane 6). Poly clonal antibody against LiPFR1 (FIG. 3A), TcHsp70 (FIGS. 3B and 3C).

[0060] FIGS. 4A to 4B. FIG. 4A. IgG antibody level against PFR-1 recombinant protein in sera from mice inoculated with saline solution and immunized with pCMV4 PFR-1 and pCMV4 PFR1Hsp70 vectors, 14 (grey Bars) and 40 days (black bars) after the fourth immunization. FIG. 4B. Level of IgG1 (grey bars) and IgG2a (black bars) antibodies specific of the PFR-1 recombinant protein detected in the immunized mice referred in FIG. 4A at 14 and 40 days post fourth immunization. Bars represent the mean value of optical density of each group.

[0061] FIGS. 5A to 5D. Antibody level against Leishmania soluble antigens (SLA, FIGS. 5A, 5B and 5C) and PFR1 recombinant protein (FIG. 5D) in sera from C57BL/6 mice inoculated with saline solution (SS) and, pCMV4 plasmid (pCMV4) or immunized with pCMV4 PFR-1 (PFR1), pCMV4 PFR-1-Hsp70 (70c) and pCMV4 PFR-1 truncated Hsp70 (70t) recombinant vectors, 14 and 21 days after challenge with Leishmania infantum infective promastigotes. Bars represent the mean of optical density of each group.

[0062] FIG. 6. Lymphoproliferative response to PFR-1 protein in mice inoculated with saline solution (SS) and pCMV4 empty vector (pCMV4) and immunized with pCMV4-PFR1 (PFR1), pCMV4 PFR1-HSP70 (70c) and pCMV4 PFR1-Truncated Hsp70 (70t). Stimulation index was calculated as [(arithmetic mean of cpm (stimulated culture)arithmetic mean of cpm (control culture))/arithmetic mean of cpm]. The results represent the mean and standard deviation of three independent immunization experiments.

[0063] FIG. 7. Nitric Oxide production (NO) by peritoneal macrophages from immunized C57BL/6 mice inoculated with saline solution (SS) and pCMV4 empty vector (pCMV4) or immunized with pCMV4 PFR1 (PFR1), pCMV4 PFR1-Hsp70 (70c) and pCMV4 PFR1-truncated Hsp70 (70t) constructs after Leishmania infantum challenge in response to lipopolysaccharide (LPS), culture media (Medium), Leishmania soluble antigens (SLA) and PFR1 recombinant protein.

[0064] FIG. 8. Parasite burden in spleen, liver and bone marrow from mice inoculated with saline solution (SS) and empty vector pCMV4 (pCMV4) or immunized with pCMV4 PFR1 (PFR1), pCMV4 PFR1-Hsp70 (70c) and pCMV4 PFR1-truncated Hsp70 (70t) recombinant vectors at 14 and 21 days post challenged with Leishmania infantum infective promastigotes.

[0065] FIG. 9. Binding assay of HLA*02:01-restricted PFR1-derived peptides to TAP-deficient T2 cells. Percentage of maximal complex stabilization was calculated with the HB-ENV.sub.334-342 peptide fluorescence index as a reference. The binding of each peptide was determined at different concentrations of each peptide.

[0066] FIG. 10. Cytotoxic activity of the CD8.sup.+ T lymphocytes specific for the eight selected PFR1 peptides evaluated by the secretion of GzB through ELISPOT in splenocytes from B6-A2/K.sup.b mice inoculated with saline solution (SS) or immunized with pCMV4 PFR1 (PFR1) or pCMV4 PFR1-Hsp70 (PFR1-HSP70) recombinant vectors. Spots were visualized using a KS ELISPOT device (Zeiss). Only large spots with fuzzy borders were scored as spot-forming cells (SFC). Responses were considered significant if (i) a minimum of 150 SFC/106 splenocytes were detected after subtraction of the negative control (splenocytes without peptide), and additionally, (ii) the response was at least over two fold the negative control.

[0067] FIG. 11. Cytotoxic activity of the CD8.sup.+ T lymphocytes specific for the eight selected PFR1 peptides evaluated by the secretion of GzB through ELISPOT in splenocytes from B6-A2/Kb mice infected with Leishmania infantum infective promastigotes. Non-inoculated animals were used as control. Spots were visualized using a KS ELISPOT device (Zeiss). Only large spots with fuzzy borders were scored as spot-forming cells (SFC). Responses were considered significant if (i) a minimum of 250 SFC/106 splenocytes were detected after subtraction of the negative control (splenocytes without peptide), and additionally, (ii) the response was at least over two fold the negative control.

[0068] FIG. 12. Cytotoxic activity of the CD8+T lymphocytes specific for the eight selected PFR1 peptides evaluated by the secretion of GzB through ELISPOT in hepatocytes from B6-A2/Kb mice infected with Leishmania infantum infective promastigotes. Non-inoculated animals were used as control. Spots were visualized using a KS ELISPOT device (Zeiss). Only large spots with fuzzy borders were scored as spot-forming cells (SFC). Responses were considered significant if (i) a minimum of 75 SFC/106 hepatocytes detected after subtraction of the negative control (hepatocytes without peptide), and additionally, (ii) the response was at least over two fold the negative control.

[0069] FIG. 13. The prediction of potential HLA-A*02:01 ligands contained in L. Infantum PFR1 protein was carried out through the screening of the deduced amino acid sequence of PFR1 gene using three HLA-A2-binding affinity algorithms: SYPFEITHI (www.syfpeithi.de), RANKPEP (immunax.dfci.harvard.edu/Tools/rankpep.html) and BIMAS (theoretical half-time dissociation, www.bimas.cit.nih.gov/molbio/hla_bind/).

[0070] FIGS. 14A to 14B. FIG. 14A. IgG antibody level against PFR1 recombinant protein in sera from mice inoculated with saline solution and immunized with pCMV4 436aaPFR1 and pCMV4 436aaPFR1Hsp70 vectors, 14 (black Bars) and 40 days (grey bars) after the fourth immunization. FIG. 14B. Level of IgG1 (black bars) and IgG2a (grey bars) antibodies specific of the PFR-1 recombinant protein detected in the immunized mice referred in FIG. 14A at 14 and 40 days post fourth immunization. Bars represent the mean value of optical density of each group.

[0071] FIG. 15. Lymphoproliferative response to PFR-1 protein in mice inoculated with saline solution (SS) or immunized with pCMV4 436aaPFR1 (436aaPFR1) and pCMV4 436aa PFR1-HSP70 (436aa PFR1 HSP70). Stimulation index was calculated as [(arithmetic mean of cpm (stimulated culture)arithmetic mean of cpm (control culture))/arithmetic mean of cpm]. The results represent the mean and standard deviation of three independent immunization experiments.

[0072] FIG. 16. Cytotoxic activity of the CD8+ T lymphocytes specific for the eight selected PFR1 peptides evaluated by the secretion of GzB through ELISPOT in splenocytes from B6-A2/Kb mice inoculated with saline solution or immunized with pCMV4 436aaPFR1 or pCMV4 436aa PFR1-Hsp70 recombinant vectors. Spots were visualized using a KS ELISPOT device (Zeiss). Only large spots with fuzzy borders were scored as spot-forming cells (SFC). Responses were considered significant if (i) a minimum of 150 SFC/106 splenocytes were detected after subtraction of the negative control (splenocytes without peptide), and additionally, (ii) the response was at least over two fold the negative control.

BIBLIOGRAPHY

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EXAMPLES

[0106] The following specific examples that are provided in this patent document are intended to illustrate the nature of the present invention. These examples are only for illustrative purposes and should not be interpreted as limitations to the invention that is claimed here. Therefore, the examples described below illustrate the invention without limiting the field of application of the same.

Example 1

[0107] 1.1. The PFR1 Protein Induces the Expression of Nitric Oxide (NO).

[0108] PFR1 protein of Leishmania infantum is encompassed within the family of characteristic proteins of paraflagellar rod proteins from kinetoplastids. In FIG. 1, we show the PFR1 recombinant protein of L. infantum expressed in a prokaryotic expression system and purified by affinity chromatography. For this protein we have demonstrated that it has the ability to activate the production of nitric oxide (NO), one of the main mechanisms that the macrophages have to eliminate pathogens that have phagocytosed into alveolar macrophages of nave rat, i.e. without any treatment or previous infection. This activation of nitric oxide production confers to the aforementioned protein a relevant immunological feature because NO favors the clearance of the amastigotes of Leishmania that multiply within the host macrophages. In FIG. 2, the values of NO production by these alveolar macrophages are collected by stimulating them with LPS (control) and the PFR1 protein. Interestingly, unlike the one detected for PFR1 protein, the activation observed for LPS is inhibited by the presence of polymyxin B (LPS activity-induced inhibitor), which discards any contamination with LPS from the PFR1 recombinant protein. On the other hand, the addition of L-N.sup.6-monomethyl Arginine (LNMMA), an inhibitor of the enzyme nitric oxide synthase, inhibits the NO activation induced by the PFR1 protein, indicating the mechanism of action of this protein. Similar results are obtained by the fusion PFR1-HSP70 protein.

1.2. Expression of PFR1 Protein in Eukaryotic Cells.

[0109] The determination of the correct expression of the proteins to study in eukaryotic cells is determined by the analysis of COS-7 transfected cells with genes encoding for PFR1 protein and chimeric fusion proteins HSP70-PFR1 and PFR1-H70T cloned into pCMV4 plasmid. The visualization of different proteins is made by Western blots analysis of cells extracts transfected with the respective abovementioned plasmids and induced PFR1-protein polyclonal antibodies. Thus, in FIG. 3 is observed, respectively, the presence of recognition bands with sizes of 70 kDa (corresponding to the PFR1 protein in the cells lane with the pCMV4 PFR1), 140 kDa in the lane of COS-7 cells transfected with the pCMV4 PFR1-Hsp70 plasmid and 96 kDa in the cells lane with the pCMV4 PFR1-Hsp70T plasmid. Cells containing the empty pCMV4 plasmid are not detected.

1.3 Antigen-Specific Humoral Response Induced by the Tested Molecules.

[0110] As example we show the results obtained after intramuscularly immunization in groups of 12 mice of the C57BL/6 strain, with 100 g of different plasmids under study, as well as negative controls (empty plasmid and saline solution). Each mouse was immunized 4 times every two weeks. Six weeks after the fourth immunization, six mice in each group were challenged intravenously with 105 infective promastigotes of the JPCM5 (MCAN/s/98/LLM-724) strain of L. infantum.

[0111] To analyze the humoral response generated in different groups of mice, blood samples were collected two weeks after each immunization and specific antibody measured. The obtained results indicate the presence of anti-PFR1 IgG antibodies 15 days post-second immunization in the group of mice immunized by the isolated PFR1 gene and two weeks after the third immunization in those immunized by the fused HSP70-PFR1 or PFR1-HSP70T genes. FIG. 4 shows the results for each molecule at 14 and 40 days post-fourth immunization. IgG levels registered in sera from mice immunized by the PFR1 gene fused to the HSP70 gene were higher than those detected in mice immunized with the isolated PFR1 gene, peaking within two weeks post-fourth immunization. In all cases the level of anti-PFR1 antibodies slightly descends after six weeks post-fourth immunization. The isotype analysis reflects that generated antibodies show a clear polarization of the response towards the IgG2a isotype (Th1-type immune response). Similar results were obtained for the PPFR1-H70T molecule.

[0112] The results obtained in PFR1 protein humoral response assays and total antigens of Leishmania (SLA) occurring in mice immunized by the molecules under study and challenged with L. infantum are shown in FIG. 5. Analysis of these results indicates that infection with Leishmania does not induce significant variation in IgG levels against PFR1 and the same polarization towards the IgG2a isotype is observed prior to the infection.

1.4 Antigen-Specific Cell Response Induced by the Molecules Under Study.

[0113] Lymphoproliferation tests were carried out three weeks after the fourth immunization. The spleens were extracted in sterile conditions and the obtained splenocytes were cultivated in vitro, with an increasing concentration of the recombinant PFR1 protein (0.4, 2 and 10 g/ml). In addition, in this assay a mitogen (ConA) and unstimulated splenocytes were included as a positive and negative control, respectively. From the results obtained, shown in FIG. 6, a significant cellular proliferation rate is observed as the recombinant PFR1 is present and, in addition, this stimulation is dose-dependent in the splenocytes from mice immunized by the recombinant testing molecules. Interestingly, this index was significantly higher in the groups receiving the PFR1 gene fused to the HSP70 and HSP70T. The proliferation rate (IE) for the splenocytes from mice immunized with these fusion molecules are approximately 25, while the measured index for splenocytes from mice immunized with the plasmid containing the isolated PFR1 gene is about 20. In both cases, above the controls; mice inoculated with the empty plasmid, IE=13 and saline solution, IE=4. These proliferation rates of splenic cells of mice immunized with the testing molecules remain with similar values after eight weeks post-fourth immunization. In addition, stimulation capacity is maintained after the challenge, noting a maximum of proliferation rate (cellular response) in mice immunized by the isolated PFR1 gene and after stimulating with 2 g/ml of the PFR1 recombinant protein.

1.5 Expression of Nitric Oxide (NO) in Mice Immunized and Infected by L. infantum.

[0114] As shown in FIG. 7, it can be seen a significant greater ability to produce nitric oxide (NO) in peritoneal macrophages of mice immunized with the testing molecules, especially with the fused ones (PFR1-HSP70 or PFR1-HSP70T) comparing to the control mice, both stimulated and unstimulated. In addition, this NO releasing ability of the mentioned immunized groups of mice increases significantly after stimulation with the recombinant PFR1 protein. On the other hand, such production was significantly higher in the infected mice versus the unchallenged ones.

1.6 Determination of the Capability of Inducing Protection Against L. infantum Infection.

[0115] The parasite load was analyzed by limiting dilution (Buffet et al., 1995, Antimicrob. Agents Chemother. 39 (9): 2167-2168), in the liver, spleen and bone marrow (target tissues of the parasite), after 14 and 28 days post-infection, showing a summary of the results in FIG. 8. Thus, after 14 days post-infection all groups of mice had parasites in the liver, however, in control groups (ss and pCMV4) the parasite load was significantly higher than the detected in the groups of animals immunized with different testing molecules, showing higher values in at least one order of magnitude. In addition, unlike the control groups, vaccinated mice with the testing molecules do not show an increase in hepatic parasitic load at 28 days post-infection. In fact, mice immunized with PFR1 gene fused to the full HSP70 gene (pPFR1-HSP70), showed and important parasite clearance in the liver. The analysis of the parasite load in spleen shows a similar pattern. Thus, after 14 days post-infection it is observed a significant higher parasite load (at least one order of magnitude) in splenic tissue of control mice (ss and pCMV4) versus vaccinated mice. In fact, mice vaccinated with PFR1 gene fused to the HSP70T gene (pPFR1-H70T) show no parasites in spleen after two weeks post-infection. Regarding bone marrow, only control groups (ss and pCMV4) account parasites, detected just after 28 days post-infection. Interestingly, none of the vaccinated mice presents parasites in this tissue. In summary, all vaccinated groups showed in all analyzed tissues during the infection a significantly lower parasitic load than the control groups (decrease between two to four orders of magnitude), pointing that these molecules confer a high level of protection against L. infantum infection, as intravenously administered.

[0116] The analysis of the expression pattern of cytokines of splenocytes of vaccinated mice versus control groups (cytometry measures in the supernatant of the cell culture using the Mouse Th1/Th2 Cytokine CBA-BD Biosciences kit) shows the existence of higher levels of TNF- and IFN- and with statistical significance in vaccinated mice with PFR1-HSP70 and PFR1-H70T chimeric constructs, stimulated or unstimulated with rPFR1 and/or SLA, versus the control groups (P<0.01). However, there is not a statistically significant variation among groups regarding IL-2 or IL-4 levels. Likewise, the analysis, after the challenge with Leishmania, of the level of the splenic macrophage activation of all the mice groups, problem and control, is measured by the expression pattern of CD80, CD86 and CD40 surface molecules. The results show that after 21 days post-infection the expression of CD86 and CD40 was significantly higher in the vaccinated group with chimeric constructions PFR1-HSP70 and PFR1-H70T versus the control (P<0.01).

1.7 Identification of T CD8+ Epitopes T in the PFR1 Protein of L. infantum.

[0117] The identification of epitopes in the sequence of the PFR1 protein of L. infantum able to be recognized by CD8+ T cells and, as a result of activating a cytotoxic antigen-specific response in the host, was carried out trying to identify epitopes capable of binding to MHC-dass I HLA. To do this, it was selected, by in silico analyses, different peptides capable of binding to HLA-A*0201 (expressed in the half of the human population), using three programs: SYFPEITHI, RANKPEP, and BIMAS. The deduced sequence of the PFR1 protein of Leishmania infantum (gene number AY702344) was introduced in each of the programs and it was selected the algorithm of binding to HLA-A*0201. The first two programs deliver a score to each peptide based on its theoretical affinity with the HLA molecule. On the other hand BIMAS scores the stability of binding with the HLA molecule, focusing on the theoretical binging time. Combining the results, eight theoretical epitopes of high binding affinity to class I HLA molecule were selected and their corresponding peptides were synthesized: SEQ ID No: 1-1864 (FMDIIGVKKV), SEQ ID No: 2-1865 (QLDATQLAQV), SEQ ID No: 3-1866 (KLLELTVYNC), SEQ ID No: 4-1868 (KMMEDIMNA), SEQ ID No: 5-1869 (AMHDGETQV), SEQ ID No: 6-1871 (QLQERLIEL), SEQ ID No: 7-1872 (MLYLTLGSL) and SEQ ID No: 8-1873 (KMVEYKSHL). The FIG. 13, tables 1 and 2, includes scores for the different peptides in the mentioned software.

[0118] To determine the binding capacity to the HLA-A*0201 molecule, it was performed binding tests to T2 cells, having a low expression capacity of transport-antigen molecules. The results, shown in FIG. 9, indicate that all the testing peptides performed a good or very good binding affinity to the HLA-A*0201 molecule, being in some cases superior to the HB-ENV.sub.334-242 peptide affinity used as control, for which a percentage of 100% binding is expected.

[0119] The analysis of the effective presentation capacity of these epitopes in the context of an experimental immunization with the testing molecules, took place in C57BL/6-A2/K.sup.b transgenic mice (they were modified to express the product of the chimeric gene HLA-A2.1/Kb, where alpha1 and alpha2 domains are the same as the HLA-A*0201 human molecule and the alpha3 domains, both transmembrane and cytoplasmic corresponding to the H-2K.sup.b murine molecule) immunized intramuscularly with pPFR1 and pPFR1-HSP70 molecules. Mice injected with saline solution (Sigma) were used as negative control. Six weeks after the fourth immunization the splenocytes of the different mice groups were stimulated with the testing peptides in the context of an ELISPOT assay using an anti-granzyme B antibody as probe. The results obtained (FIG. 10) show that five of the peptides gave a positive response to granzyme B (activation of antigen-specific CTLs) in vaccinated mice with the chimeric molecule pPFR1-HSP70 and one in those immunized with the molecule containing the isolated PFR1 gene. These results demonstrate that the above testing molecules are efficiently processed and presented in the context of the MHC-Class I. Furthermore it points that in particular those epitopes for which positive values are obtained are recognized by cytotoxic T lymphocytes (CTLs) of the immunized mice.

[0120] The analysis of the capacity to generate cytotoxic response in the course of an experimental infection with L. infantum was evaluated in C57BL/6-A2/Kb transgenic mice infected via i.v. with 10.sup.6 infective promastigotes of L. infantum (strain JPCM5). 170 Days after infection mice were sacrificed and their splenocytes or their not-parenchymal liver cells were exposed to the texting peptides in the context of an ELISPOT assay using Granzima B as detector antibody. The results show (FIGS. 11 and 12) that four of the tested peptides gave a positive response to Granzyme B in splenocytes and three of them also in not-parenchymal liver cells. Therefore, these peptides are presented to the CTLs in the course of experimental infection by the parasite. Interestingly, only one out of these four peptides also gave positive response in immunized mice, showing that the antigen presentation differs between immunization with plasmids and the course of the experimental infection.

Concluding Remarks

[0121] Example 1 conclusion: the results show that immunized mice with the testing molecules and subsequently challenged, do not present parasites in bone marrow, while those groups inoculated with the pCMV4 empty plasmid or saline control (control) present a high parasite load in the same tissue after 28 days post-infection. In addition, the parasite load in spleen and liver of immunized mice are between two to four orders of magnitude lower than the detected in the control groups (empty pCMV4 plasmid or saline solution). Immunized mice with the pPFR1-H70T molecule and Leishmania-infected, do not show parasites in spleen after 14 days post-infection.

Example 2

2.1 Antigen-Specific Humoral Response.

[0122] As an example, it is shown the results obtained after intramuscularly immunization of 12-mice-groups of the C57BL16 strain, with 100 g of the different testing plasmids, as well as the negative controls (mice inoculated with saline solution). In this case, the tested plasmids contain the sequence corresponding to the fragment of the PFR1 protein of Leishmania infantum (436 amino acids length) comprised between amino acids 160 and 595 of the PFR1 protein. Each mouse was immunized 4 times every other week.

[0123] To analyze the generated humoral response in different groups of mice, blood collection was made two weeks after each immunization and antibody level detection in animal sera was measured. The results indicate that the presence of anti-PFR1 IgG antibodies appears 15 days post-fourth immunization in the immunized group with the isolated 436aaPFR1 gen and two weeks after the third immunization in those which carry fused 436aaPFR1-HSP70 genes. FIG. 14 (A) shows the results for each testing molecule at 14 and 40 days post-fourth immunization. IgG levels in sera from immunized mice with the 436aaPFR1 gen fused to the HSP70 gene were higher than those detected in the group immunized with the isolated 436aaPFR1 gene, with a maximum of antibodies within two weeks post-fourth immunization. In all cases, the level of anti-PFR1 antibodies slightly descends at six weeks post-fourth immunization. Analysis of isotypes (B) shows a clear polarization of the response towards the IgG2a isotype (Th1 type immune response) in released antibodies.

2.2 Antigen-Specific Cell Response

[0124] Lymphoproliferation tests were carried out three weeks after the fourth immunization. The spleens were extracted in sterile conditions and the obtained splenocytes were in vitro cultivated in the presence of an increasing concentration of the rPFR1 protein (0.4, 2 and 10 g/ml). In addition, a mitogen (ConA) and unstimulated splenocytes were included in this assay as a positive and negative control, respectively. From the results obtained, shown in FIG. 15, a significant cellular proliferation rate is observed as the recombinant PFR1 (rPFR1) is present and, in addition, this stimulation is dose-dependent in the splenocytes of mice immunized by the recombinant testing molecules. Interestingly, this index was significantly higher in the groups receiving the PFR1 gene fused to the HSP70 and HSP70T. The proliferation rate (IE) for the splenocytes of mice inoculated with these fusion molecules are approximately 22, while the measured index for splenocytes from mice immunized with the plasmid containing the isolated PFR1 gene is about 18. In both cases, above the controls; saline solution, IE=4. These proliferation rates of splenic cells of mice immunized with the testing molecules remain with similar values after eight weeks post-fourth immunization.

[0125] The analysis of the effective presentation capacity of these epitopes in the context of an experimental immunization with the testing molecules, took place in C57BL/6-A2/K.sup.b transgenic mice (they were modified to express the product of the chimeric gene HLA-A2.1/Kb, where alpha1 and alpha2 domains are the same as the HLA-A*0201 human molecule and the alpha3 domains, both transmembrane and cytoplasmic corresponding to the H-2K.sup.b murine molecule) immunized intramuscularly with 436aaPFR1 and 436aaPFR1-HSP70 molecules. Mice injected with saline solution (Sigma) were used as negative control. Six weeks after the fourth immunization the splenocytes of the different mice groups were stimulated with the testing peptides in the context of an ELISPOT assay using an anti-granzyme B antibody as probe. The results obtained (FIG. 16) show that five of the peptides gave a positive response to granzyme B (activation of antigen-specific CTLs) in vaccinated mice with the chimeric molecule 436aaPFR1-HSP70 and one in those immunized with the molecule containing the isolated 436aaPFR1 gene. These results demonstrate that the above testing molecules are efficiently processed and presented in the context of the MHC-Class I. Furthermore it points that in particular those epitopes for which positive values are obtained are recognized by cytotoxic T lymphocytes (CTLs) of the immunized mice.

Conclusion Example 2

[0126] the results show that the 436 amino acids fragment of the PFR1 protein performs very similarly to the complete protein, getting a similar pattern of immune response (humoral, cellular and cytotoxic). Therefore, since these parameters are those involved in protection against the parasite, we can conclude that this fragment behaves in all terms to study as the complete protein.