The present invention relates to new proteins of Leishmania major and to therapeutical and diagnostic applications thereof. More particularly, the present invention relates to excreted/secreted polypeptides and polynucleotides encoding same, compositions comprising the same, and methods of diagnosis, vaccination and treatment of Leishmaniasis.

Description of the Invention

SUMMARY

The present invention satisfies at least one of the above-mentioned needs.

More specifically, an object of the invention concerns an isolated polynucleotide comprising a sequence encoding an excreted/secreted polypeptide of Leishmania major, said sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 1 to 34 and functional fragments thereof.

Other objects of the invention concern an isolated or purified excreted/secreted polypeptide of Leishmania major, said polypeptide comprising an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS: 35 to 68 and functional derivatives thereof.

Still another object of the invention is to provide an immunogenic composition generating an immune response against a leishmaniasis, comprising a polynucleotide of the invention or a polypeptide of the invention, and an acceptable carrier.

Yet another object of the invention concerns a vaccine composition generating a protecting response against a leishmaniasis, comprising a polynucleotide of the invention or a polypeptide of the invention, and an acceptable carrier.

Yet another object of the invention concerns an antibody obtainable by the immunization of an animal with a polypeptide of the invention.

Yet another object of the invention concerns an expression or a cloning vector containing a polynucleotide of the invention.

Yet another object of the invention concerns a method for preventing and/or treating a patient against an infection with a Leishmania major strain, the method comprising the step of administering to the patient a therapeutically effective amount of a composition of the invention or of an antibody of the invention

Yet another object of the invention concerns a method for identifying an excreted/secreted polypeptide of a Leishmania major strain, comprising in vitro cultivating Leishmania promastigotes under pH and temperature conditions naturally found in a host cell infected by a Leishmania major strain.

Yet another object of the invention concerns an in vitro diagnostic method for the detection of the presence or absence of antibodies indicative of a Leishmania major strain, which bind to a polypeptide of the invention to form an immune complex, comprising the steps of a) contacting said polypeptide with a biological sample for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).

A further object of the invention concerns a diagnostic kit for the detection of the presence or absence of antibodies indicative of a Leishmania major strain, comprising: a polypeptide of the invention; a reagent to detect polypeptide-antibody immune complex; optionally a biological reference sample lacking antibodies that immunologically bind with said peptide; and optionally a comparison sample comprising antibodies which can specifically bind to said peptide; wherein said polypeptide, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.

A further object of the invention concerns an in vitro diagnostic method for the detection of the presence or absence of polypeptides indicative of a Leishmania major strain, which bind to an antibody of the invention to form an immune complex, comprising the steps of: a) contacting said antibody with a biological sample for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).

A further object of the invention concerns a diagnostic kit for the detection of the presence or absence of polypeptides indicative of a Leishmania major strain, comprising: an antibody of the invention; a reagent to detect polypeptide-antibody immune complex; optionally a biological reference sample lacking polypeptides that immunologically bind with said antibody; and optionally a comparison sample comprising polypeptides which can specifically bind to said antibody; wherein said antibody, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.

A further object of the invention concerns a genetically modified Leishmania strain comprising at least one gene having a sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 1 to 34, and wherein said at least one gene is underexpressed compared to a corresponding gene of a wild-type strain of Leishmania.

A further object of the invention concerns a genetically modified Leishmania strain comprising at least one gene having a sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 1 to 34, and wherein said at least one gene is inactivated.

A further object of the invention concerns a method for detecting the presence or absence of lymphocytic stimulation in a subject suspected of Leishmaniasis, comprising the steps of: a) obtaining a sample containing T Lymphocytes from said subject; b) contacting the T lymphocytes with a polypeptide of the invention; and c) detecting the presence or absence of a proliferative response of said T lymphocyte to the polypeptide.

A further object of the invention concerns a method for detecting the presence or absence of lymphocytic stimulation in a subject suspected of Leishmaniasis, comprising the steps of: a) obtaining a sample containing T Lymphocytes from said subject; b) contacting the T lymphocytes with a polypeptide of the invention; and c) detecting the presence or absence of cytokines indicative of lymphocytic stimulation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to excreted/secreted polypeptides of Leishmania major and polynucleotide encoding same and their use in the preparation of compositions and vaccines. More specifically, the present invention is concerned with compositions, vaccines and methods for providing an immune response and/or a protective immunity to mammals against a Leishmania major strain as well as methods for the diagnosis of a Leishmaniasis. The term "leishmaniasis" means an infection caused by any of the flagellate protozoans of the genus Leishmania, such as Leishmania major.

1. Polynucleotides and Polypeptides

In a first embodiment, the present invention concerns an isolated polynucleotide comprising a sequence encoding an excreted/secreted polypeptide of Leishmania major, said sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 1 to 34 and functional fragments thereof.

As used herein, the term "functional fragment" refers to a polypeptide which possesses biological function or activity that is identified through a defined functional assay and which is associated with a particular biologic, morphologic, or phenotypic alteration in a cell or cell mechanism.

By the term "substantially identical", it is meant that the polynucleotide of the invention has a nucleic acid sequence which is at least 65% identical, more particularly 80% identical and even more particularly 95% identical to any one of SEQ ID NO: 1 to 34.

Preferably, the polynucleotide of the invention comprises a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 1 to 13 (FIG. 2; Table 1: Group 1 (see Original Patent)) and functional fragments thereof, or from the group consisting of SEQ ID NOS 14 to 23 (FIG. 3; Table 1: Group 2 (see Original Patent)) and functional fragments thereof, or from the group consisting of SEQ ID NOS 24 to 26 (FIG. 4; Table 1: Group 3 (see Original Patent)) and functional fragments thereof, or from the group consisting of SEQ ID NOS 27 to 34 (FIG. 5; Table 1: Group 4 (see Original Patent)) and functional fragments thereof.

As used herein, the terms "Isolated or Purified" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a protein/peptide naturally present in a living organism is neither "isolated" nor purified, the same polynucleotide separated from the coexisting materials of its natural state, obtained by cloning, amplification and/or chemical synthesis is "isolated" as the term is employed herein. Moreover, a polynucleotide or a protein/peptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said organism.

Amino acid or nucleotide sequence "identity" and "similarity" are determined from an optimal global alignment between the two sequences being compared. An optimal global alignment is achieved using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453). "Identity" means that an amino acid or nucleotide at a particular position in a first polypeptide or polynucleotide is identical to a corresponding amino acid or nucleotide in a second polypeptide or polynucleotide that is in an optimal global alignment with the first polypeptide or polynucleotide. In contrast to identity, "similarity" encompasses amino acids that are conservative substitutions. A "conservative" substitution is any substitution that has a positive score in the blosum62 substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919). By the statement "sequence A is n % similar to sequence B" is meant that n % of the positions of an optimal global alignment between sequences A and B consists of identical residues or nucleotides and conservative substitutions. By the statement "sequence A is n % identical to sequence B" is meant that n % of the positions of an optimal global alignment between sequences A and B consists of identical residues or nucleotides.

As used herein, the term "polynucleotide(s)" generally refers to any polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. This definition includes, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. "Polynucleotide(s)" embraces short polynucleotides or fragments often referred to as oligonucleotide(s). The term "polynucleotide(s)" as it is employed herein thus embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells which exhibits the same biological function as the polypeptide encoded by SEQ ID NO.1 to 34. The term "polynucleotide(s)" also embraces short nucleotides or fragments, often referred to as "oligonucleotides", that due to mutagenesis are not 100% identical but nevertheless code for the same amino acid sequence.

In another embodiment, the present invention concerns an isolated or purified excreted/secreted polypeptide of Leishmania major comprising an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS: 35 to 68 and functional derivatives thereof. By the term "substantially identical", it is meant that the polypeptide of the present invention preferably has an amino sequence having at least 80% homology, or even preferably 85% homology to part or all of SEQ ID NO: 35 to 68.

Yet, more preferably, the polypeptide comprises an amino acid sequence substantially the same or having 100% identity with SEQ ID NO: 35 to 68.

According to a preferred embodiment, the polypeptide of the present invention comprises an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS: 35 to 47 (Annex A; Table 1: Group 1 (see Original Patent)) and functional derivatives thereof, or from the group consisting of SEQ ID NOS: 48 to 57 (Annex B; Table 1: Group 2) and functional derivatives thereof, or from the group consisting of SEQ ID NOS: 58 to 60 (Annex C; Table 1: Group3 (see Original Patent)) and functional derivatives thereof, or from the group consisting of SEQ ID NOS: 61 to 68 (Annex D; Table 1: Group 4) and functional derivatives thereof.

A "functional derivative", as is generally understood and used herein, refers to a protein/peptide sequence that possesses a functional biological activity that is substantially similar to the biological activity of the whole protein/peptide sequence. A functional derivative of a protein/peptide may or may not contain post-translational modifications such as covalently linked carbohydrate, if such modification is not necessary for the performance of a specific function. The term "functional derivative" is intended to the "fragments", "segments", "variants", "analogs" or "chemical derivatives" of a protein/peptide.

As used herein, the term "polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, selenoylation, sulfation and transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance: PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62(1992). Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.

2. Vectors and Cells

In a third embodiment, the invention is also directed to a host, such as a genetically modified cell, comprising any of the polynucleotide sequence according to the invention and more preferably, a host capable of expressing the polypeptide encoded by this polynucleotide.

Transformed or transfected cells preferably contemplated by the present invention contain a polynucleotide having a sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 1 to 13 and functional fragments thereof. Examples of such cells are those consisting of an Escherichia coli bacterium selected from the group consisting of Escherichia coli bacteria filed at the CNCM. under accession numbers I-3394, I-3393, I-3395, I-3396, I-3377, I-3371, I-3376, I-3373, I-3379, I-3397, I-3384, I-3383 and I-3382 on Feb. 24, 2005.

Other transformed or transfected cells preferably contemplated by the present invention contain a polynucleotide having a sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 14 to 23 and functional fragments thereof. Examples of such cells are those consisting of an Escherichia coli bacterium selected from the group consisting of Escherichia coli bacteria filed at the CNCM. under accession numbers I-3386, I-3378, I-3385, I-3381, I-3372, I-3392, I-3380, I-3367, I-3370, and I-3366 on Feb. 24, 2005.

Other transformed or transfected cells preferably contemplated by the present invention contain a polynucleotide having a sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 24 to 26 and functional fragments thereof. Examples of such cells are those consisting of an Escherichia coli bacterium selected from the group consisting of Escherichia coli bacteria filed at the CNCM. under accession numbers I-3365, I-3369 and I-3368 on Feb. 24, 2005.

Other transformed or transfected cells preferably contemplated by the present invention contain a polynucleotide having a sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 27 to 34 and functional fragments thereof. Examples of such cells are those consisting of an Escherichia coli bacterium selected from the group consisting of Escherichia coli bacteria filed at the CNCM. under accession numbers I-3364, I-3387, I-3391, I-3389, I-3390, I-3388, I-3374, and I-3375 on Feb. 24, 2005.

In another embodiment, the invention is further directed to cloning or expression vector comprising a polynucleotide sequence as defined above, and more particularly directed to a cloning or expression vector which is capable of directing expression of the polypeptide encoded by the polynucleotide sequence in a vector-containing cell.

As used herein, the term "vector" refers to a polynucleotide construct designed for transduction/transfection of one or more cell types. Vectors may be, for example, "cloning vectors" which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors" which are designed for expression of a nucleotide sequence in a host cell, or a "viral vector" which is designed to result in the production of a recombinant virus or virus-like particle, or "shuttle vectors", which comprise the attributes of more than one type of vector.

A number of vectors suitable for stable transfection of cells and bacteria are available to the public (e.g. plasmids, adenoviruses, baculoviruses, yeast baculoviruses, plant viruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses, Alphaviruses, Lentiviruses), as are methods for constructing such cell lines. It will be understood that the present invention encompasses any type of vector comprising any of the polynucleotide molecule of the invention.

In another embodiment, the invention is concerned with genetically modified Leishmania strains. A first preferred genetically modified Leishmania strain comprises at least one gene having a sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 1 to 34, and wherein said at least one gene is inactivated, preferably by knock-out. A second preferred genetically modified Leishmania strain contemplated by the present invention comprises at least one gene having a sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 1 to 34, and wherein said at least one gene is underexpressed compared to a corresponding gene of a wild-type strain of Leishmania. Methods by which such strains are genetically modified are known to one skilled in the art and will not be further discussed.

3. Antibodies

In another embodiment, the invention features purified antibodies that specifically bind to the isolated or purified polypeptide as defined above or fragments thereof. The antibodies of the invention may be prepared by a variety of methods using the polypeptides described above. For example, the polypeptide, or antigenic fragments thereof, may be administered to an animal in order to induce the production of polyclonal antibodies. Alternatively, antibodies used as described herein may be monoclonal antibodies, which are prepared using hybridoma technology (see, e.g., Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., 1981).

As mentioned above, the present invention is preferably directed to antibodies that specifically bind to Leishmanina major excreted/secreted polypeptides, or fragments thereof as defined above. In particular, the invention features "neutralizing" antibodies. By "neutralizing" antibodies is meant antibodies that interfere with any of the biological activities of any of the Leishmanina major excreted/secreted polypeptides. Any standard assay known to one skilled in the art may be used to assess potentially neutralizing antibodies. Once produced, monoclonal and polyclonal antibodies are preferably tested for specific Leishmanina major excreted/secreted polypeptides recognition by Western blot, immunoprecipitation analysis or any other suitable method.

With respect to antibodies of the invention, the term "specifically binds to" refers to antibodies that bind with a relatively high affinity to one or more epitopes of a protein of interest, but which do not substantially recognize and bind molecules other than the one(s) of interest. As used herein, the term "relatively high affinity" means a binding affinity between the antibody and the protein of interest of at least 10.sup.6 M.sup.-1, and preferably of at least about 10.sup.7 M.sup.-1 and even more preferably 10.sup.8 M.sup.-1 to 10.sup.10 M.sup.-1. Determination of such affinity is preferably conducted under standard competitive binding immunoassay conditions which is common knowledge to one skilled in the art. As used herein, "antibody" and "antibodies" include all of the possibilities mentioned hereinafter: antibodies or fragments thereof obtained by purification, proteolytic treatment or by genetic engineering, artificial constructs comprising antibodies or fragments thereof and artificial constructs designed to mimic the binding of antibodies or fragments thereof. Such antibodies are discussed in Colcher et al. (Q J Nucl Med 1998; 42: 225-241). They include complete antibodies, F(ab').sub.2 fragments, Fab fragments, Fv fragments, scFv fragments, other fragments, CDR peptides and mimetics. These can easily be obtained and prepared by those skilled in the art. For example, enzyme digestion can be used to obtain F(ab').sub.2 and Fab fragments by subjecting an IgG molecule to pepsin or papain cleavage respectively. Recombinant antibodies are also covered by the present invention.

Preferably, the antibody of the invention is a human or animal immunoglobulin such as IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgD carrying rat or mouse variable regions (chimeric) or CDRs (humanized or "animalized"). Furthermore, the antibody of the invention may also be conjugated to any suitable carrier known to one skilled in the art in order to provide, for instance, a specific delivery and prolonged retention of the antibody, either in a targeted local area or for a systemic application.

The term "humanized antibody" refers to an antibody derived from a non-human antibody, typically murine, that retains or substantially retains the antigen-binding properties of the parent antibody but which is less immunogenic in humans. This may be achieved by various methods including (a) grafting only the non-human CDRs onto human framework and constant regions with or without retention of critical framework residues, or (b) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Such methods are well known to one skilled in the art.

As mentioned above, the antibody of the invention is immunologically specific to the polypeptide of the present invention and immunological derivatives thereof. As used herein, the term "immunological derivative" refers to a polypeptide that possesses an immunological activity that is substantially similar to the immunological activity of the whole polypeptide, and such immunological activity refers to the capacity of stimulating the production of antibodies immunologically specific to the Leishmanina major excreted/secreted polypeptides or derivative thereof. The term "immunological derivative" therefore encompass "fragments", "segments", "variants", or "analogs" of a polypeptide.

4. Compositions and Vaccines

The polypeptides of the present invention, the polynucleotides coding the same, and antibodies produced according to the invention, may be used in many ways for the diagnosis, the treatment or the prevention of Leishmaniasis.

In another embodiment, the present invention relates to an immunogenic composition generating an immune response against a leishmaniasis, comprising a polynucleotide as defined above or a polypeptide as defined above, and an acceptable carrier. According to a related aspect, the present invention relates to a vaccine composition generating a protecting response against a leishmaniasis, comprising a polynucleotide as defined above or a polypeptide as defined above, and an acceptable carrier. As used herein, the term "treating" refers to a process by which the symptoms of Leishmaniasis are alleviated or completely eliminated. As used herein, the term "preventing" refers to a process by which a Leishmaniasis is obstructed or delayed. The composition of the vaccine of the invention comprises a polynucleotide and/or a polypeptide as defined above and an acceptable carrier.

As used herein, the expression "an acceptable carrier" means a vehicle for containing the polynucleotide and/or a polypeptide that can be injected into a mammalian host without adverse effects. Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i. e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.

Further agents can be added to the composition and vaccine of the invention. For instance, the composition of the invention may also comprise agents such as drugs, immunostimulants (such as .alpha.-interferon, .beta.-interferon, .gamma.-interferon, granulocyte macrophage colony stimulator factor (GM-CSF), macrophage colony stimulator factor (M-CSF), interleukin 2 (IL2), interleukin 12 (IL12), and CpG oligonucleotides), antioxidants, surfactants, flavoring agents, volatile oils, buffering agents, dispersants, propellants, and preservatives. For preparing such compositions, methods well known in the art may be used.

The amount of polynucleotide and/or a polypeptide present in the compositions of the present invention is preferably a therapeutically effective amount. A therapeutically effective amount of polynucleotide and/or a polypeptide is that amount necessary to allow the same to perform their immunological role without causing, overly negative effects in the host to which the composition is administered. The exact amount of polynucleotide and/or a polypeptide to be used and the composition/vaccine to be administered will vary according to factors such as the type of condition being treated, the mode of administration, as well as the other ingredients in the composition.

5. Method for Identifying a Polypeptide of the Invention

In another object, the present invention provides a method for identifying an excreted/secreted polypeptide of a Leishmania major strain. The method comprises in vitro cultivating Leishmania promastigotes under pH and temperature conditions naturally found in a host cell infected by a Leishmania major strain. Preferably, the pH is about 5.5 and the temperature is about 35.degree. C. By "about", it is meant that the value of said pH or temperature can vary within a certain range depending on the margin of error of the method used to evaluate such pH or temperature.

In a related aspect, the excreted/secreted polypeptides identified by the method as defined above finds a particular use as drug target for identifying a molecule capable of preventing a Leishmaniasis.

6. Methods of Use

In another embodiment, the present invention relates to a method for preventing and/or treating a patient against an infection with a Leishmania major strain, the method comprising the step of administering to the patient a therapeutically effective amount of a immunogenic and/or a vaccine composition as defined above and/or an antibody as defined above.

The vaccine, antibody and immunogenic composition of the invention may be given to a patient through various routes of administration. For instance, the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os. The vaccine and the composition of the invention may also be formulated as creams, ointments, lotions, gels, drops, suppositories, sprays, liquids or powders for topical administration. They may also be administered into the airways of a subject by way of a pressurized aerosol dispenser, a nasal sprayer, a nebulizer, a metered dose inhaler, a dry powder inhaler, or a capsule. Suitable dosages will vary, depending upon factors such as the amount of each of the components in the composition, the desired effect (short or long term), the route of administration, the age and the weight of the mammal to be treated. Any other methods well known in the art may be used for administering the vaccine, antibody and the composition of the invention.

The present invention is also directed to an in vitro diagnostic method for the detection of the presence or absence of antibodies indicative of a Leishmania major strain, which bind to a polypeptide as defined above to form an immune complex, comprising the steps of a) contacting said polypeptide with a biological sample for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).

In a further embodiment, a diagnostic kit for the detection of the presence or absence of antibodies indicative of of a Leishmania major strain is provided. Accordingly, the kit comprises: a polypeptide as defined above; a reagent to detect polypeptide-antibody immune complex; optionally a biological reference sample lacking antibodies that immunologically bind with the polypeptide; and optionally a comparison sample comprising antibodies which can specifically bind to the polypeptide; wherein the polypeptide, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform the detection.

The present invention also proposes an in vitro diagnostic method for the detection of the presence or absence of polypeptides indicative a Leishmania major strain, which bind to the antibody of the present invention to form an immune complex, comprising the steps of: a) contacting the antibody of the invention with a biological sample for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).

In a further embodiment, a diagnostic kit for the detection of the presence or absence of polypeptides indicative of Leishmania major strain is provided. Accordingly, the kit comprises: an antibody as defined above; a reagent to detect polypeptide-antibody immune complex; optionally a biological reference sample lacking polypeptides that immunologically bind with the antibody; and optionally a comparison sample comprising polypeptides which can specifically bind to the antibody; wherein said antibody, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform the detection.

A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.

A further object of the invention concerns a method for detecting the presence or absence of lymphocytic stimulation in a subject suspected of Leishmaniasis, comprising the steps of: a) obtaining a sample containing T Lymphocytes from said subject; b) contacting the T lymphocytes with a polypeptide of the invention; and c) detecting the presence or absence of a proliferative response of said T lymphocyte to the polypeptide.

A further object of the invention concerns a method for detecting the presence or absence of lymphocytic stimulation in a subject suspected of Leishmaniasis, comprising the steps of: a) obtaining a sample containing T Lymphocytes from said subject; b) contacting the T lymphocytes with a polypeptide of the invention; and c) detecting the presence or absence of cytokines indicative of lymphocytic stimulation.
 

Claim 1 of 10 Claims

1. An isolated polynucleotide consisting of a sequence encoding an excreted/secreted polypeptide of Leishmania major, said sequence having SEQ ID NO:1 or said sequence having at least 95% identity to SEQ ID NO:1.
 

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