# Patent

## Title
Method for the screening of conserved secreted proteins

## Abstract

Conserved polypeptides from protozoan parasitic species which are secreted through the endoplasmic reticulum/Golgi dependent secretory pathway, their identification and their use.

## Background
The present invention relates to conserved secreted protein in protozoan parasites, to a method for the screening said proteins and to therapeutical applications thereof. More particularly, the present invention relates to excreted/secreted polypeptides and polynucleotides encoding same, compositions comprising the same, and methods of vaccination against protozoan parasites causing important diseases in humans.
The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. The three model organisms that have been most extensively studied are Trypanosoma brucei, the causative agent of African sleeping sickness, T. cruzi, responsible for Chagas disease in South America, and Leishmania, which causes Leishmaniasis in Asia, South America and Mediterranean countries. Half a billion people, primarily in tropical and subtropical areas of the world, are at risk of contracting these diseases. It is estimated that more than 20 million individuals are infected, resulting in extensive suffering and more than 100,000 deaths per year.
Chagas disease is one of the most important parasitic diseases and affects 15 million people in Central and South America. The annual worldwide incidence of new cases is estimated at around 50,000-200,000.
The diagnosis of T. cruzi infection is difficult because the symptoms of the infection are often absent or non-specific, and because the parasites themselves are usually below the level of detection in the infected subjects (Tarleton et al., 2007). Therefore, diagnosis generally depends on the measurement of T. cruzi-specific antibodies produced in response to the infection.
Conventional serological tests, such as the indirect hemagglutination assay, indirect immunofluorescence assay, and enzyme-linked immunosorbent assay (ELISA), are used widely in the countries where the infection is endemic. Most are based on the use of whole or semi-purified antigenic fractions from T. cruzi epimastigotes grown in axenic culture. A persistent problem with the conventional assays is the occurrence of inconclusive and false-positive results. Thus, there is no consensus on which parasite antigen preparation is best for detecting antibodies to T. cruzi. The World Health Organization and other expert groups recommend using at least two tests in parallel to confirm T. cruzi infection. Due to the lack of a true gold standard for the serologic diagnosis of T. cruzi infection, development of new diagnostic tools remains a challenge for Chagas disease.
High-sensitivity and high specificity ELISA methods using recombinant or synthetic peptides as antigens have been reported. The inclusion of recombinant antigens and synthetic peptides for the serological diagnosis of T. cruzi infection has been an advantage in terms of specificity increase. Nevertheless, single recombinant antigens display lower sensitivity when compared with conventional test using whole parasite extracts. The use of cocktails of recombinant antigens, mixtures of synthetic peptides or multi-epitope antigens have shown to increase sensitivity.
It is widely assumed that secreted/excreted factors in T. cruzi are highly immunogenic. Indeed, trypomastigote forms release several antigens into the supernatant of infected cell cultures. This complex mixture of antigens, termed TESA (trypomastigote excretory-secretory antigens), is highly immunogenic and has been used for the diagnosis of both acute and chronic Chagas. Remarkably, the components of the TESA mixture are currently unknown.
In order to complete their life cycle, these parasites have to adapt and develop in an insect vector (a tsetse fly, a triatomine bug, or a sandfly, respectively), and in a vertebrate host. These single-celled organisms have developed several strategies to modify their surrounding environment, modulate host immune responses, or interfere with the host's anti-microbial activity. Materials secreted by the parasite play pivotal roles in these processes. Secreted proteases belonging to the family of cystein- or metallo-proteases are generally thought to be involved in the manipulation of host immune responses in insect and vertebrate hosts. Biochemical analysis of the extra-cellular gp63 of Leishmania has revealed two forms of the protein, one released from the cell surface and another that is apparently directly secreted. The secreted form provides protection for Leishmania against insect-derived antimicrobial peptides. Parasites can also secrete enzymes involved in nutritional processes. Leishmania, like other trypanosomatids, are purine auxotrophs, and therefore are entirely dependent upon salvaging these essential nutrients from their hosts. To satisfy its essential purine requirements, Leishmania secretes a nuclease that may function externally of the parasite to hydrolyze and access host-derived nucleic acids. Secreted materials can also be directly involved in the invasion of target cells. Tc-TOX, a pore-forming protein of T. cruzi, allows the parasite to escape the endosome and reach the cytoplasm, its natural cellular environment. Experimental evidence suggests that Leishmania also possess a pore-forming protein that contributes to their release from host macrophages.
Together, these findings demonstrate that secreted materials are involved in processes that help the parasite survive in an environment more favorable for its own development. However, all of the factors in these processes are currently not clearly identified.
In eukaryotes, soluble secreted proteins typically contain N-terminal signal peptides that direct them to the translocation apparatus of the endoplasmic reticulum (ER). Following vesicular transport from the ER via the Golgi apparatus to the cell surface, lumenal proteins are released into the extracellular space by fusion of Golgi-derived secretory vesicles with the plasma membrane.
In trypanosomatids, it is presumed that molecules destined for the cell surface and secretion follow a typical eukaryotic pathway traveling from the ER to the Golgi apparatus, then to the cell surface via a flagellar reservoir membrane called the flagellar pocket. Nevertheless, a recent proteomic approach applied to the Leishmania secretome suggested that this parasite predominantly uses non-classical secretion pathways to directly export proteins, including the release of microvesicles (Silverman J M, Chan S K, Robinson D P, Dwyer D M, Nandan D, Foster L J, Reiner N E: Proteomic analysis of the secretome of Leishmania donovani. Genome Biol 2008, 9 (2):R35).
Comparative analyses have revealed that genomes of the trypanosomatid parasites causing disease in humans, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei are highly conserved. In addition, about 50% of predicted proteins in the genome were annotated as “hypothetical proteins even if for many of them some evidence of protein expression exists (El-Sayed N M, Myler P J, Blandin G, Berriman M, Crabtree J, Aggarwal G, Caler E, Renauld H, Worthey E A, Hertz-Fowler C et al: Comparative genomics of trypanosomatid parasitic protozoa. Science 2005, 309 (5733):404-409; Atwood J A, 3rd, Weatherly D B, Minning T A, Bundy B, Cavola C, Opperdoes F R, Orlando R, Tarleton R L: The Trypanosoma cruzi proteome. Science 2005, 309 (5733):473-476; Jones A, Faldas A, Foucher A, Hunt E, Tait A, Wastling J M, Turner C M: Visualisation and analysis of proteomic data from the procyclic form of Trypanosoma brucei. Proteomics 2006, 6 (1):259-267). Progress in controlling infections caused by these pathogens requires an improved understanding of the biology of these parasites so as to design novel treatment strategies. It is widely assumed that excreted/secreted factors play crucial roles in the biology or virulence of trypanosomatid parasites and may therefore represent targets for vaccines or rational drug design. In trypanosomatids the secretion process is not fully understood and various pathways may contribute to the formation of the “extracellular proteome”. Identification of secreted materials would enhance efforts towards understanding mechanisms of protein secretion in these medically important parasites
The availability of three draft trypanosomatid genome sequences provides valuable data for protein-mining using bioinformatic tools, especially for the localization or prediction of function for hypothetical proteins. Given that a significant number of trypanosomatid protein-coding genes are annotated as hypothetical, additional studies are needed to ascertain their function.
Various approaches aimed at characterizing such extracellular material were developed. Even if collecting a culture's supernatant is relatively easy to perform, identification of the different factors can be difficult because of the relatively low abundance of the constituents. Additionally, the in vitro growth of mammalian stages for trypanosomatids is impossible or laborious. Moreover, even if parasites are grown in cell-free and serum-free media, the culture's supernatant can be contaminated with materials that are not primarily “secreted” by the parasite. These materials may be shed from the parasite surface or can originate from dead organisms. Thus to avoid such pitfalls it is best to limit the parasite's incubation time in a serum-free media to a few hours. Further characterization by screening cDNA libraries with sera raised against culture medium supernatants has also been performed. However, using this approach proteins with a low abundance or that are poorly immunogenic are likely to be missed. Recently, the secretome of L. donovani promastigotes was analyzed by using a quantitative proteomic approach based on the SILAC methodology (Silverman et al., Genome Biology (2008), 9, R35). Although the authors identified a total of 358 proteins secreted into a promastigote conditioned medium, the SILAC methodology was unable to detect proteins that are well known to be secreted, e.g., chitinase and SAcP (secreted acid phosphatase). This may be explained by the relative ratios (extracellular versus cell-associated protein) used by the SILAC approach to identify extracellular proteins. For example, this methodology is limited in its ability to detect proteins for which the large majority is secreted (extracellular) and almost nothing remains within the cell. Consequently, no reliable ratio can be calculated. The methodology developed in WO 2006/108720 is totally different from the one of the instant invention. It is based on immunogenicity properties of materials released in the culture supernatants by the promastigote forms of L. major, and a cDNA library screening. By using this method only the immunogenic proteins released by the parasite in the culture supernatant may be identified. A subset of these proteins is potentially secreted by the classical secretory pathway, based on the predictions of the signal peptide in the polypeptide sequence. Another subset might be exported by other ways, since they do not carry a predicted signal peptide. Another difference with the instant invention is the stage-dependent identification: their starting point is the materials released by the stationary-phase promastigotes, whereas the instant invention is stage-independent, since the starting point is genomic-based (the sequences might be expressed by any stage of the parasite life-cycle) and transgenic parasites are used for overexpression of the target proteins. Van Ooij et a.l (Ploss Pathogens, 2008, 4 (6), 1-15) use a bioinformatic analysis for prediction of signal peptide, but specific for Plasmodium species, and their functional validation is only performed on Plasmodium. This methodology aimed to detect and study the function of secreted proteins that bear ER retention motif and the PEXEL (Plasmodium Export Element) motif, not described in trypanosomatids.
In view of this, the inventors designed a new experimental approach based on bioinformatics genome screening to identify new proteins conserved among protozoan parasites, particularly those conserved among the three main trypanosomatid pathogens and that are secreted via the classical pathway. Said methodology possesses two main advantages Firstly, detection of secreted proteins is not limited to a particular parasite stage since the approach relies on the use of transgenic Leishmania parasites to detect extracellular proteins. Secondly, it allows the detection of small quantities of extracellular proteins that otherwise may be missed by other methodologies, such as, quantitative mass spectrometry.
Consequently the invention relates to an isolated or purified conserved secreted polypeptide said polypeptide comprising an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS: 86 and 98 and functional derivatives thereof selected from the group comprising SEQ ID NOS: 88, 90, 92, 94, 96, 100, 102 and 173.
More specifically, the invention relates to an isolated or purified conserved secreted polypeptide according to claim 1 consisting of SEQ ID NOS: 86 or 98 and functional derivatives thereof selected from the group comprising SEQ ID NOS: 88, 90, 92, 94, 96, 100, 102 and 173.
According to the invention, said polypeptides may be identified by a method for identifying conserved polypeptides from protozoan parasitic species which are secreted through the endoplasmic reticulum/Golgi dependent secretory pathway, said method comprising the following steps:

    
    
        a. analysing a putative conserved polypeptide from protozoan parasitic species to determine if said polypeptide has a secretion signal peptide at the N-terminus and a cleavage site just behind said signal peptide,
        b. searching for orthologue, in related family members, of said polypeptide, said orthologue having a secretion signal peptide and a cleavage site just behind said signal peptide,
        c. analyzing said orthologue to determine if said orthologue has a secretion signal peptide at the N-terminus and a cleavage site just behind said signal peptide,
        d. selecting the conserved polypeptide and its corresponding orthologue having a secretion signal peptide and a cleavage site just behind said signal peptide,
        e. cloning the encoding genes for the polypeptide selected in step d) by mean of an expression vector,
        f. transfecting replicative protozoan parasitic cells,
        g. in vitro cultivating said cells under pH and temperature conditions naturally found in a host infected by a protozoan parasitic strain,
        h. analysing the presence of secreted polypeptides in the proliferative protozoan parasitic cells and
        i. identifying said secreted polypeptides by a protein identification method.
    
    


According to the invention, “conserved proteins” are defined by the presence of orthologous genes in the family members. For example for the three trypanosomatids, Leishmania, Trypanosoma cruzi and Trypanosoma brucei, (tri Tryp) the orthologous genes are defined by “Jaccard COG Clustering”. The orthology between the ‘tri Tryp’ organisms is predicted using a method known as Jaccard COG clustering. Briefly, this firstly involves the proteins within each organism's data set being grouped into clusters using reciprocal BLASTP searches, with an assigned cut off score. Representatives of each cluster for each organism are then chosen and three way reciprocal BLASTP searches are run again on these data sets. A clustered orthologous group (COG) is a group of proteins which contains reciprocal best hits between the genomes. (Aslett M et al, Int J Parasitol. 2005 35:481-93).
According to the invention “family members” is defined as organisms belonging to the same genera.
According to the invention, “protozoan parasites” generally refers to any protozoan organisms that are eukaryotic, unicellular, parasitic and characterized by at least one developmental stage within its vertebrate host. Parasites used in the methods of the invention include, but are not limited to, protozoan parasites that are members of the genera Toxoplasma, Neospora, Eimeria, Theileria, Sarcocystis, Cryptosporidium and Trypanosomatidae family (Trypanosoma and Leishmania).
In an advantageous embodiment of the invention, the putative conserved polypeptide has been identified by screening a database of information.
More specifically the instant invention concerns a method for identifying conserved secreted polypeptides from trypanosomatid parasites.
In an advantageous embodiment said trypanosomatid parasites are selected from the group comprising Leishmania major, Leishmania infantum, Leishmania tropica, Leishmania braziliensis, Leishmania donovani, Leishmania amazonensis, Leishmania chagasi, Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma vivax, Trypanosoma congolense, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. 
Other objects of the invention concern a method for identifying conserved polypeptides from trypanosomatid parasites which are secreted through the endoplasmic reticulum/Golgi dependent secretory pathway, said method comprising the following steps:

    
    
        a. analyzing a putative conserved polypeptide from a trypanosomatid parasite to determine if said polypeptide has a secretion signal peptide at the N-terminus and a cleavage site just behind said signal peptide,
        b. searching for orthologue, in related family members, of said identified polypeptide having a secretion signal peptide and a cleavage site just behind said signal peptide
        c. analyzing said orthologue to determine if said orthologue has a secretion signal peptide at the N-terminus and a cleavage site just behind said signal peptide,
        d. selecting the conserved polypeptide and its corresponding orthologue having a secretion signal peptide and a cleavage site just behind said signal peptide,
        e. cloning the encoding genes for the polypeptide selected in step d) by mean of an expression vector,
        f. transfecting replicative trypanosomatid cells at the promastigotes stage,
        g. in vitro cultivating said cells under pH and temperature conditions naturally found in a host cell infected by a trypanosomatid strain,
        h. collecting replicative transfected cells obtained in step g),
        i. incubating said cells in a serum free medium during 1 to 10 hours, preferably during 5 to 6 hours, at a temperature comprised between 25-27° C.,
        j. analysing the presence of secreted polypeptides by said cells and
        k. identifying said secreted polypeptides by a protein identification method.
    
    


More specifically the instant invention concerns a method for identifying conserved polypeptides wherein the putative conserved polypeptide is identified by screening a genome selected from the accessible genome from of Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma vivax, Leishmania major, Leishmania braziliensis, in particular by screening the integrated Trypanosoma cruzi, genome resource TcruziDB.
In yet another embodiment of the invention, the replicative trypanosomatid cells used in step f) are promastigotes of Leishmania infantum. 
In yet another embodiment of the invention, the primers used in step e) are selected from the group comprising SEQ ID NO 124 to 165.
A further object of the invention is an isolated or purified conserved secreted polypeptide identified by the method of the invention, said polypeptide comprising an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS: 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 173 and functional derivatives or fragments 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.K 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 posttranslational natural processes and may be made by entirely synthetic methods, as well.
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: 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 173.
Yet an other object of the invention concerns an isolated polynucleotide comprising a sequence encoding a secreted polypeptide according to the invention, said sequence comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 85, 89, 91, 93, 97, 99, 101, 103, 107 or 109 and functional fragments thereof selected from SEQ ID NOS 87, 89, 91, 93, 95, 99, 101 and 172
According to the invention “functional derivatives or fragments” refers to polypeptides which possess 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: 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 172.
Amino acid or nucleotide sequence “identity” and “similarity” are determined from an optimal global alignment between the two sequences being compared. “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.
According to the invention, 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.
According to the invention, 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 NOS 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 173. 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 or sequence with high similarity.
Yet another object of the invention concerns an immunogenic composition generating an immune response against a leishmaniasis, comprising a polynucleotide corresponding to SEQ ID NOS 89, 97 or 107 or a polypeptide corresponding to SEQ ID NOS 90, 98 or 108 and an acceptable carrier.
Yet another object of the present invention concerns an immunogenic composition generating an immune response against a Chagas disease, comprising a polynucleotide corresponding to SEQ ID NOS 85, 93 or 103 or a polypeptide corresponding to SEQ ID NOS 86, 94 or 104 and an acceptable carrier.
Yet another object of the instant invention concerns an immunogenic composition generating an immune response against a sleeping sickness comprising a polynucleotide corresponding to SEQ ID NOS 91, 99, 101 or 109 or a polypeptide corresponding to SEQ ID NOS 92, 100, 102 or 110 and an acceptable carrier.
Thanks to the method of the invention it is possible to select a conserved protein from one organism selected from Trypanosoma cruzi, Trypanosoma brucei and Leishamia major and to use said protein or its corresponding nucleotide to treat one or more diseases selected from leishmaniasis, Chagas disease and sleeping sickness, i.e. the method according to the invention allows to treat two of them or the three together.
Consequently another object of the instant invention concerns an immunogenic composition generating an immune response against at least one disease selected from leishmaniasis, Chagas disease and sleeping sickness, comprising a polynucleotide corresponding to SEQ ID NOS 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 172 or a polypeptide corresponding to SEQ ID NOS 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 173.
Advantageously, the immunogenic composition according to the invention leads to an immune response which generates a cellular and/or humoral response, preferably a cellular response.
Yet another object of the invention is a vaccine composition generating a protecting response against a leishmaniasis, comprising a polynucleotide corresponding to SEQ ID NOS 89, 97 or 107 or a polypeptide corresponding to SEQ ID NOS 90, 98 or 108 and an acceptable carrier.
Yet another object of the invention is a vaccine composition generating a protecting response against a Chagas disease, comprising a polynucleotide corresponding to SEQ ID NOS 85, 93 or 103 or a polypeptide corresponding to SEQ ID NOS 86, 94 or 104 and an acceptable carrier.
Yet another object of the invention is a vaccine composition generating a protecting response against a sleeping sickness comprising a polynucleotide corresponding to SEQ ID NOS 91, 99, 101 or 109 or a polypeptide corresponding to SEQ ID NOS 92, 100, 102 or 110 and an acceptable carrier.
Yet another object of the invention is a vaccine composition generating a protecting response against a leishmaniasis, a Chagas disease or a sleeping sickness comprising a polynucleotide corresponding to SEQ ID NOS 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 172 or a polypeptide corresponding to SEQ ID NOS 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 173 and an acceptable carrier.
Yet another object of the instant invention is an antibody obtainable by the immunization of an animal with a polypeptide according to the invention.
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. As mentioned above, the present invention is preferably directed to antibodies that specifically bind to trypanosomatids 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 trypanosomatids 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 trypanosomatids 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 106 M−1, and preferably of at least about 107 M−1 and even more preferably 108 M−1 to 1010 M−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. They include complete antibodies, F(ab′)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′)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 trypanosomatids secreted polypeptides or derivative thereof. The term “immunological derivative” therefore encompasses “fragments”, “segments”, “variants”, or “analogs” of a polypeptide.
Yet another object of the invention is an expression or a cloning vector containing a polynucleotide of the invention and more preferably a host capable of expressing the polypeptide encoded by this polynucleotide.
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.
Yet another object of the invention concerns a method for preventing and/or treating a patient suffering from one or more diseases selected from leishmaniasis, Chagas disease and sleeping sickness, said method comprising the step of administering to the patient a therapeutically effective amount of a composition as defined above or of an antibody as defined above.
In another embodiment, the invention features the use of a polynucleotide according to the invention and/or a polypeptide according to the invention as a target for identifying a molecule capable of preventing a disease selected from leishmaniasis, Chagas disease and sleeping sickness or two of them or the three together.
In another embodiment, the invention proposes an in vitro diagnostic method for the detection of the presence or absence of antibodies indicative of one or more diseases selected from leishmaniasis, Chagas disease and sleeping sickness which bind to a polypeptide according to 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).
    
    


In another embodiment a diagnostic kit for the detection of the presence or absence of antibodies indicative of one or more diseases selected from leishmaniasis, Chagas disease and sleeping sickness is provided. Accordingly said kit comprises:

    
    
        a. a polypeptide according to the invention;
        b. a reagent to detect polypeptide-antibody immune complex; optionally a biological reference sample lacking antibodies that immunologically bind with said peptide; and
        c. 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.

    
    


The invention also proposes an in vitro diagnostic method for the detection of the presence or absence of polypeptides indicative of one or more diseases selected from leishmaniasis, Chagas disease and sleeping sickness 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).
    
    


The invention further provides a diagnostic kit for the detection of the presence or absence of polypeptides indicative of one or more diseases selected from leishmaniasis, Chagas disease and sleeping sickness, comprising:

    
    
        a. an antibody according to the invention;
        b. a reagent to detect polypeptide-antibody immune complex; and
        c. optionally a biological reference sample lacking polypeptides that immunologically bind with said antibody; and
        d. 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 transformed or transfected cell that contains a vector as defined above, more preferably a transformed or transfected cell that contains a polynucleotide according to the invention, preferably a polynucleotide as defined above.
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 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 172 and functional fragments thereof. Examples of such cells are those consisting of replicative protozoan parasitic cells (egg Leishmania promastigotes, T. cruzi epimastigotes) transfected with a recombinant expression vector such as pTEX, widely used to express heterologous DNA sequences in the trypanosome genetic environment, containing sequence selected from the group consisting of SEQ ID NOS 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 172 and functional fragments thereof.
Another further object of the invention is a method for detecting the presence or absence of lymphocytic stimulation in a subject suspected of one or more diseases selected from leishmaniasis, Chagas disease and sleeping sickness, comprising the steps of:

    
    
        a. obtaining a sample containing T Lymphocytes from said subject;
        b. contacting the T lymphocytes with a polypeptide; and
        c. detecting the presence or absence of a proliferative response of said T lymphocyte to the polypeptide.
    
    


The instant invention also provides a method for detecting the presence or absence of lymphocytic stimulation in a subject suspected of one or more diseases selected from leishmaniasis, Chagas disease and sleeping sickness, comprising the steps of:

    
    
        a. obtaining a sample containing T Lymphocytes from said subject;
        b. contacting the T lymphocytes with a polypeptide as defined above; and
        c. detecting the presence or absence of cytokines indicative of lymphocytic stimulation.
    
    





The present invention will be more readily understood by referring to the following examples 1 to 3 and to FIGS. 1 to 11.
FIG. 1 illustrates the T. cruzi genes selected by in silico analysis. The 13 proteins bears the highest probability for the presence of the signal peptide and were selected for functional confirmation of bona fide secretion. The beta-tubulin T. cruzi gene (GeneID Tc00.1047053506563) was added to our sample as a potential negative control for protein.
SPP Signal peptide probability predicted by SignalP 3.0; CSP Maximal cleavage site probability predicted by SignalP.
FIG. 2 illustrates the primer design of the 13 T. cruzi putative secreted proteins conserved in trypanosomatids and T. cruzi. Tubulin is taken as negative control. F: Forward primer including the start codon; Rint: Internal reverse primer for RT-PCR; R: Reverse primer used for the amplification of the full-length ORF. In the column “Primer sequences” restriction sites used for cloning in the pTEX vector are in italics and the His-Tag sequence is in bold. Column “MW” corresponds to the expected molecular weight of the proteins. In the first column, last line Tc00.1047053506563.40 corresponds to Beta-tubulin gene.
FIG. 3 illustrates the ID of L. infantum orthologous genes and primers used for cloning. F: Forward primer including the start codon; R: Reverse primer used for the amplification of the full-length ORF. In the column “Primer sequences” restriction sites used for cloning in the pTEX vector are in italics and the His-Tag sequence is in bold. Column “MW” corresponds to the expected molecular weight of the proteins.
FIG. 4 illustrates the expression analysis of potentially secreted proteins during the life cycle of T. cruzi. RT-PCR analysis of total RNA from T. cruzi (clone derived from the Y strain) epimastigotes (E), trypomastigotes (T) and amastigotes (A), respectively, using cDNA obtained by gene-specific PCR primers listed in FIG. 2. Gene ID and expected lengths of cDNA are listed in order in FIG. 2. B: Blank. M: Molecular marker: Smart Ladder SF.
FIG. 5 illustrates PCR analyses in episomally-transfected L. infantum promastigotes (A) Amplification of NEO gene fragment in L. infantum episomally transfected promastigotes. (B) Amplification of full length transfected genes in L. infantum promastigotes. Specific forward and Reverse PCR primers and gene lengths are listed in order in FIG. 2. WT: Wild Type Parasites. M: Molecular marker: Smart Ladder SL.
FIG. 6 illustrates the protein expression in L. infantum episomally transfected promastigotes during the exponential phase of development (A) Western blot analysis of His-tagged proteins detected in whole cell lysate. Equal amounts of total protein (35 μg) were resolved by electrophoresis in 4-12% gradient gels (Invitrogen), blotted, and developed with anti-HisTag antibody followed by ECL (Amersham). Gene ID and theoretical molecular weight of detected proteins are listed in order in FIG. 2. (B) Identification of secreted proteins in whole cell lysate (Lys) and concentrated cell-free culture supernatant (CCFS) obtained from promastigotes incubated for 6 h in serum-free medium. Note the absence of β Tubulin in the concentrated supernatant of Line 8. Non-transfected L. infantum promastigotes (Wild Type) were used as a negative control. Protein molecular mass standards in kDa are shown on the left of each panel.
FIG. 7 illustrates the homologous expression of secreted proteins in L. infantum episomally transfected promastigotes according to the example. L. infantum promastigotes were transfected with genes: LinJ19.0410 and LinJ36.5780 corresponding to secreted proteins Tc00.1047053505789.10 (SEQ ID NO 93) and Tc00.1047053506155.99 (SEQ ID NO 103), respectively. Cell whole lysate (Lys) and concentrated cell-free culture supernatant (CCFS) were obtained as in FIG. 6. Tagged proteins were detected only in recombinant parasites transfected with LinJ19.0410 (58 kDa) (Line 1 and 2) and LinJ36.5780 (28 kDa) (Line 3 and 4). Non-transfected L. infantum promastigotes (Wild Type) were used as negative controls (Line 5 and 6). Protein molecular mass standards in kDa are shown on the left. Cell whole lysate (Lys), concentrated cell-free culture supernatant (CCFS) and electrophoresis procedure were as mentioned in FIG. 6. Non-transfected L. infantum promastigotes (Wild Type) were used as negative controls (Line 5 and 6). Protein molecular mass standards in kDa are shown on the left.
FIG. 8 illustrates the bioluminescence activity of intracellular Leishmania expressing episomal luciferase in infected macrophages in vitro. Recombinant L. infantum promastigotes over-expressing the secreted proteins pTEX734 LinJ19.0410 (▾) or pTEX-LinJ36.5780 (●) were co-transfected with the pSP735 YαHYGROαLUC carrying the firefly-luciferase gene. Survival of luciferase-expressing parasites was monitored in infected human monocyte cell line THP-1 differentiated into macrophages as indicated in the Methods section. Promastigotes transfected with the pTEX vector alone and the pSP-αHYGαLUC (□) were used as control for infection experiments. RLU (Relative Luminescence Units) were measured at various time points post-infection using the Steady Glo reagent. Results are expressed as the mean of three independent experiments, each carried out in triplicate.
FIG. 9 illustrates the bioluminescence activity of intracellular Leishmania expressing episomal luciferase in infected macrophages in vitro. Transgenic L. infantum promastigotes over-expressing the secreted proteins pTEX-LinJ19.0410 (▾) or pTEX-LinJ36.5780 (▴) were co-transfected with the pSP-YαHYGROαLUC carrying the firefly-luciferase gene. Survival of luciferase-expressing parasites was monitored in infected canine monocyte-macrophage cell line DH-82 (A) and murine macrophage cell line J774 (B). Promastigotes transfected with the pTEX vector alone and pSP-αHYGαLUC (▪) were used as controls for infection experiments. RLU (Relative Luminescence Units) were measured at various time points post-infection using the Steady Glo reagent. Results are expressed as the mean of three independent experiments, each carried out in triplicate.
FIG. 10 illustrates the purification of the recombinant protein rTc00.1047053509999.10 ((SEQ ID NO 85). Lane 1: cleared bacterial lysate; lane 2: flow-through; lanes 3-5: washes; lanes 6-8: eluates at pH 5,9; lanes 9-11: eluates at pH 4,5. MW: Molecular marker (BenchMark Protein Ladder, Invitrogen). The proteins were resolved by electrophoresis in a 4-12% gradient gel and visualized by Coomassie staining. The purified protein appeared in the pH 4.5 eluates as a band of 45 kDa corresponding to the theoretical molecular weight of the recombinant protein.
FIG. 11 represents the Western blot of the recombinant protein rTc00.1047053509999.10 (SEQ ID NO 85) with sera from non chagasic individuals (lane 1) and sera from chagasic patients (lanes 2-13)



EXAMPLE 1
Identification of Conserved Secreted Proteins in Trypanosomatids
1.1. Methods
1.1.1. In Silico Sequence Analysis
Release V 5.0 of the T. cruzi genome was extracted from the integrated T. cruzi genome resource TcruziDB. Protein sequences that do not bear an initial methionine amino acid were removed manually. Proteins belonging to large families of surface molecules, which include trans-sialidases, mucins, gp63s and mucin-associated surface proteins were also discarded. Finally ORFs encoding proteins bearing a molecular weight (MW) above 90 kDa were also eliminated. The software SignalP 3.0 was used to predict the presence of a secretion signal peptide and a cleavage site in amino acid sequences. Protein sequences having a secretion signal peptide probability greater than 0.8 associated with a cleavage site probability greater than 0.7 were analyzed for the presence of orthologs in the related Trypanosoma brucei and Leishmania major parasite databases.
1.1.2 Parasite Strains and Cultures
The T. cruzi TcY7 (or Y c17) clone derived from the Y strain (Allaoui A, Francois C, Zemzoumi K, Guilvard E, Ouaissi A: Intracellular growth and metacyclogenesis defects in Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele. Mol Microbiol 1999, 32 (6):1273-1286; Garzon E, Borges M C, Cordeiro-da-Silva A, Nacife V, Meirelles Mde N, Guilvard E, Bosseno M F, Guevara A G, Breniere S F, Ouaissi A: Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele elicits attenuated Chagas' disease in mice. Immunol Lett 2003, 89 (1):67-80) was used throughout this study. Epimastigotes were grown in liver infusion tryptose (LIT) medium supplemented with 10% FCS at 28° C. in standard conditions as described by Camargo E P (Growth And Differentiation In Trypanosoma Cruzi. I. Origin Of Metacyclic Trypanosomes In Liquid Media. Rev Inst Med Trop Sao Paulo 1964, 12:93-100) and harvested during the logarithmic growth phase. Metacyclic trypomastigotes, obtained from the differentiation of late stationary phase epimastigotes, were used to initiate infection of mouse fibroblasts (L929). Trypomastigotes and amastigotes were produced and harvested as previously described by the inventors (Mathieu-Daude F, Bosseno M F, Garzon E, Lelievre J, Sereno D, Ouaissi A, Breniere S F: Sequence diversity and differential expression of Tc52 immuno-regulatory protein in Trypanosoma cruzi: potential implications in the biological variability of strains. Parasitol Res 2007, 101 (5):1355-1363). Pellets for RNA purification were processed immediately in lysis buffer. The wild-type (WT) promastigote clone from L. infantum (MHOM/MA/67/ITMAP-263) was maintained at 26° C. by weekly sub-passages in SDM 79 medium supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin and 100 μg/ml streptomycin according to Brun R, Schonenberger (Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop 1979, 36 (3):289-292).
1.1.3. Reverse Transcription and PCR Amplifications
Total RNA was extracted from epimastigotes, amastigotes and trypomastigotes with the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturers' instructions, and treated with DNase I (DNA-free kit, Ambion Inc., Austin, Tex.). Reverse transcription was performed for 1 μg of total RNA using random hexamers and Superscript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) according to the manufacturers' instructions. The cDNA (4 μl of 1/10 dilutions) from each stage was amplified by PCR in a 20 μl reaction volume using 10 μl of Master Mix 1× (Promega, Madison, Wis.), 0.5 μM gene-specific forward and internal reverse primers (listed in FIG. 2) using the following cycling conditions: 94° C. for 3 min followed by 30 cycles of 94° C. for 30 s, 55° C. to 58° C. (according to the primer pair) for 30 s, 72° C. for 45 s and a final elongation at 72° C. for 5 min. Amplicons were electrophoresed on 2% agarose gels stained with ethidium bromide.
1.1.4. Cloning and Sequencing
The encoding genes selected through the in silico analysis were cloned into the pTEX expression vector, carrying the Neomycin resistance gene (NEO). Full length ORFs were amplified from genomic DNA with specific reverse and forward primers including different restriction sites and a 6-Histidine-Tag in the C-terminal region (see FIGS. 2 and 3). PCR reactions were carried out in 20 μl using 0.5 μM of each primer, 0.2 mM dNTP, 0.4 U of Phusion high-fidelity polymerase (Finnzymes, Espoo, Finland) and the following cycling conditions: 98° C. for 30 s followed by 25 cycles of 98° C. for 10 s, 64° C. to 68° C. for 15 s, 72° C. for 25 to 60 s (according to gene size), and a 72° C. elongation for 5 min. Digested and purified fragments were inserted into the dephosphorylated pTEX vector digested with the corresponding restriction enzymes. Cloned sequences were confirmed by restriction digestion and sequencing. Large scale preparations of the different constructs were performed using the plasmid midi kit (Promega).
1.1.5. Transfection Procedures
Promastigotes of the Leishmania infantum clone were electroporated as described elsewhere (Sereno D, Roy G, Lemesre J L, Papadopoulou B, Ouellette M: DNA transformation of Leishmania infantum axenic amastigotes and their use in drug screening. Antimicrob Agents Chemother 2001, 45 (4):1168-1173). Briefly, promastigotes were washed twice with HEPES-NaCl buffer saline (21 mM HEPES, 5 mM KCl, 0.7 mM NaH2PO4, 137 mM NaCl), resuspended at 108 cells/ml in HEPES-NaCl electroporation buffer (pH 7.2) supplemented with 6 mM glucose and cooled on ice for 10 min. Cells (108) were combined with 15 μg of vector, left on ice for 10 additional min, and electroporated using an Easyject Plus (Eurogentec, Liege, Belgium) apparatus set at 450 V and 450 μF, for one pulse. The cells were left on ice for a further 10 min and transferred to 4 ml of growth medium. The antibiotic G-418 (20 μg/ml) was added 24 h later, and parasites were sub-cultured at a dilution of 1/10 in 5 ml SDM in the presence of 20 μg/ml G418. Drug-resistant cells were observed 15-20 days later. Parasites were grown in the presence of gradually increasing concentrations of G418 and were routinely maintained in SDM containing 150 μg/ml of G418.
1.1.6. PCR Amplifications in Transfected Parasites
PCR amplifications were carried out to check for the presence of the NEO gene and the corresponding gene in transfected parasites. A fragment of 800 bp corresponding to the NEO gene was amplified with specific forward and reverse primers (SEQ ID NO 170 F5′ATGATTGAACAAGATGGATTGCACGCAGG3′ and SEQ ID NO 171 R5′ TCAGAAGAACTCGTCAAGAA 3′). Full length ORFs of the specific genes were amplified with primers listed in FIG. 2. PCR reactions were carried out in a 20 μl reaction volume using 10 μl of Master Mix 1× (Promega, Madison, Wis.), 0.5 μM NEO and gene-specific forward and reverse primers using the following cycling conditions: 94° C. for 3 min followed by 30 cycles of 94° C. for 30 s, 55° C. to 58° C. (according to the primer pair) for 30 s, 72° C. for 45 s to 2 min (according to gene size) and a final elongation at 72° C. for 5 min.
1.1.7. Production of Cell Free Culture Supernatants
To analyze the presence of secreted proteins in the supernatant, 1×109 L. infantum promastigotes from log-phase culture were collected by centrifugation, washed twice in HEPES-NaCl buffer, re-suspended in 40 ml of HEPES-NaCl (pH 7.2), 11 mM glucose, 200 μg/ml G-418 and incubated for 6 h at 27° C. Parasite viability was then assessed as previously described (Vergnes B, Sereno D, Madjidian-Sereno N, Lemesre J L, Ouaissi A: Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death. Gene 2002, 296 (1-2):139-150) and harvested by centrifugation at 2,100×g for 10 min at 4° C. The parasite pellet was stored at −80° C. for subsequent SDS-PAGE analysis and the supernatant filtered through a low retention 0.45 μm PVDF filter membrane (Millipore, Boston, Mass.). After addition of protease inhibitor cocktail (Sigma-Aldrich) the filtrate was concentrated up to 80-fold using an Amicon Ultra-Centrifugal Filter device, according to manufacturers' instructions (Amicon Bioseparations, MilliporeCorp). The concentrated cell-free culture supernatant was frozen and stored at −80° C.
1.1.8. Production of Parasite Lysates
Cell pellets of wild-type and episomally-transfected L. infantum promastigotes were re-suspended in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% Sodium deoxycholate and 0.1% SDS), incubated on ice for 30 min and sonicated three times for 20 s. The soluble phase was recovered by centrifugation at 10,000 g for 30 min (4° C.) and the protein concentration was determined using a Bradford protein assay (Bio-Rad Laboratories, Hercules, Calif.).
1.1.9. Gel Electrophoresis and Western Blot Analysis
Proteins from parasite lysates (35 μg) or from 80× concentrated cell-free supernatants (2 μg) were separated on a NuPAGE Bis-Tris gel (4-12%) in MOPS-SDS running buffer (Invitrogen) under reducing conditions (50 mM DTT), and transferred to a PVDF membrane (Hybond-P, Amersham). The membrane was rinsed twice in TBS and incubated for 1 h in the anti-His HRP conjugate blocking buffer (Qiagen). The membrane was then incubated in 1/3000 anti-His HRP conjugate antibody (Qiagen) for 1 h and washed seven times for 5 min in TBS-T buffer (TBS-0.5% Tween 20). Signals were detected by chemiluminescence emission using the ECL Plus Western blotting detection system and ECL Hyperfilms (GE Healthcare, UK).
1.2. Results
1.2.1. Bioinformatic Selection for Secreted Proteins in Trypanosomatids
The preliminary screening of the T. cruzi gene bank was performed to discard; (i) uncompleted sequences, (ii) housekeeping genes and sequences belonging to large gene families like the trans-sialidases, mucins, Mucin Associated Surface Proteins (MASPs), (iii) sequences encoding proteins with a molecular weight above 90 kDa, in order to facilitate subsequent gene cloning. The remaining coding sequences were screened for the presence of both the signal peptide and a secretion signal peptide peptidase (SPP) cleavage site. Only 216 sequences bore a 0.8 and 0.7 probability of carrying both the secretion signal peptide and the peptidase cleavage site, respectively. Among them, 91 (42%) were annotated as “hypothetical proteins, conserved” in the data bank. The final criterion for selected proteins likely to be secreted by the classical eukaryotic pathway was the presence of the signal peptide and the signal peptidase site in orthologous members of the related parasites; Leishmania major, L. infantum and T. brucei. Among the 91 sequences, only 45 showed orthologous members with the signal peptide criteria. The 13 proteins bearing the highest probability for the presence of the signal peptide were selected (FIG. 1) for functional confirmation of bona fide secretion. The beta-tubulin T. cruzi gene (GeneID Tc00.1047053506563—SEQ ID NO 83) was added to the sample as a potential negative control for protein secretion.
1.2.2. Expression Analysis of Potentially Secreted Proteins During the Life Cycle of T. cruzi 
The expression of the 13 different encoding genes was analysed throughout the developmental life cycle of T. cruzi. The beta-tubulin T. cruzi gene (GeneID Tc00.1047053506563—SEQ ID NO 83), constitutively expressed in all the three stages of T. cruzi was used as a positive control. The expression of each gene was supported by positive RT-PCR amplification in the infective trypomastigote and amastigote forms. Nevertheless two genes (Gene ID Tc00.1047053511901.30 (SEQ ID NO 17) and Tc00.1047053509999.10 (SEQ ID NO 85)) were negative for the amplification of cDNA in the non-infective epimastigote form, suggesting a possible stage-specific expression of these genes (FIG. 4).
1.2.3. Experimental Approach for the Detection of Secreted Proteins
A functional test to confirm the presence of selected proteins in the extracellular environment by detection of target proteins in cell-free supernatants was set-up. Thus the 13 selected encoding genes of T. cruzi and the gene encoding the beta-tubulin negative control were amplified from genomic DNA. A sequence encoding a 6×His-Tag was added at the C-terminal end of each of the encoded genes. This would allow to detect the protein in total parasite protein extracts or in cell-free supernatants. Amplified PCR products were cloned into the pTEX shuttle vector that is widely used for expression in trypanosomatids (Kelly J M, Ward H M, Miles M A, Kendall G: A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania. Nucleic Acids Res 1992, 20 (15):3963-3969). Transformation and selection of T. cruzi is not as easy to perform as for Leishmania, mainly due to longer periods required for selecting drug-resistant parasites. Since the inventors were aimed to develop a fast and reliable approach to identify trypanosomatid conserved secreted proteins, they used Leishmania as the recipient organism for the experimental validation of their selected proteins. Thus, Leishmania infantum promastigotes were transformed with pTEX carrying the 14 selected T. cruzi genes (including the beta-tubulin gene), and recombinant parasites were selected for resistance to Geneticin G418. Each parasite population was checked for the presence of both the NEOR gene and the selected gene whose secretion was to be analyzed. A specific 800 bp fragment, indicative of the presence of the NEOR gene, was detected in the transfected promastigotes and not in wild-type parasites (FIG. 5A). Moreover, the presence of each candidate gene in recombinant parasites was confirmed using specific primers (FIG. 5B). PCR were negative in Wild type parasites, demonstrating that the amplification for each gene is specific although they are conserved in trypanosomatids (data not shown). The expression of these genes was screened using an antibody directed against the His-Tag carried by the recombinant proteins (FIG. 6A). Western blot analysis demonstrated that; (i) the inventors were able to easily and specifically detect the 6×His tag protein in the extract derived from recombinant parasites, (ii) recombinant Leishmania expressed a relatively high level of T. cruzi protein, and (iii) the molecular weight of the detected tagged protein corresponded to the expected MW (see FIG. 2). The inventors thus set up an approach to detect recombinant proteins in cell-free supernatants. In order to limit potential contamination by proteins derived from dying organisms, they restricted incubation in serum-free mediums to 6 hours, and they checked the viability of parasite populations before and after this incubation period. Parasites and cell-free supernatants were collected if the viability of the cell population was above 98%. Western Blot analyses of the concentrated cell-free supernatants revealed that among the 14 proteins only 3 were actively secreted (Tc00.1047053506155.99 (SEQ ID NO 103), Tc00.1047053505789.10 (SEQ ID NO 93) and Tc00.1047053509999.10 (SEQ ID NO 85)) (FIG. 6B). These proteins represent genuine secreted material, since; (i) the over-expression of the beta-tubulin gene does not induce translocation of the beta-tubulin protein into the extracellular space (difference between Lys and CCFS in FIG. 6B), and (ii) the detection of the tagged protein in the cell-free supernatant is not related to the level of its expression by Leishmania (low abundance of Tc00.1047053506155.99 (SEQ ID NO 103) in FIG. 6B). As expected, for the three proteins it was observed a slight molecular weight difference between the tagged protein detected into the whole soluble extract and that detected in the cell-free supernatant. This observation could be explained by a loss of the Signal Peptide (FIG. 6B). As anticipated, no band was detected in wild-type parasites (FIG. 6B). Furthermore, to verify that secretion is not related to the heterologous expression system, two Leishmania genes (GenID LinJ19.0410 (SEQ ID NO 77) and LinJ36.5780 (SEQ ID NO 107)) corresponding to the orthologous of Tc00.1047053506155.99 gene (SEQ ID N0103) and Tc00.1047053505789.10 gene (SEQ ID NO 93), were selected to validate said approach. By using the same protocol as above, parasites expressing the 6×His tagged proteins were produced (See FIG. 3). As expected, the presence of the tagged protein in the extracellular medium was detected only in the episomally transfected parasites (FIG. 7).
Together, these results demonstrate that the approach used by the inventors allowed them to identify new and genuinely secreted proteins which are conserved between different species and are involved in the endoplasmic reticulum/Golgi-dependent secretory pathway.
EXAMPLE 2
Generation of Bioluminescent L. infantum Promastigotes and In Vitro Infection of Human, Canine and Murine Macrophages
2.1. Material and Methods
A homologous episomal expression system was devised to further examine the infection in vitro of two secreted proteins from L. infantum. The vector pSP-αHYGαLUC disclosed by El Fadili et al. (Mol Biochem Parasitol, 2002, 124:63-71) carrying the firefly-luciferase gene was used to co-transfect L. infantum promastigotes over-expressing the secreted proteins LinJ19.0410 (SEQ ID NO 97) ortholog of Tc00.1047053505789.10 (SEQ ID NO 93 or LinJ36.5780 (ortholog of. Tc00.1047053506155.99=SEQ ID NO 103). Recombinant parasites were selected for their growth in increasing concentrations of Hygromycin (up to 300 μg/ml) over a period of several weeks. Promastigotes transfected with the pTEX vector alone and the pSP-αHYGαLUC were used as controls for infection experiments. The survival of transfected parasites was evaluated within human leukemia monocyte cell line (THP-1 cells). THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml. THP-1 cells in the log phase of growth were differentiated into macrophages by incubation for 2 days in a medium containing 20 ng/ml of phorbol myristate acetate (Sigma-Aldrich). THP-1 cells treated with PMA were washed with prewarmed medium and then infected with stationary-phase promastigotes of transfected-Leishmania in a 24-well plate at a parasite/macrophage ratio of 10:1 for 4 h at 37° C. with 5% CO2. Non internalized parasites were removed. After different incubation periods (24 h to 96 h) Luciferase activity was determined using the Steady Glo reagent (Promega, Madison, Wis.), according to the manufacturers' instructions. After 5 min, the plate was read using a Multilabel Counter VICTOR2258 model 1420 (Perkin Elmer). Results are expressed as the mean of RLU (Relative Luminescence Units) activity of three independent experiments, each performed in triplicate. Statistical significance was analyzed by the Mann-Whitney U-test.
To further evaluate these secreted proteins (LinJ19.0410 and LinJ36.5780), the infectivity of the transgenic parasites in different cell cultures was assessed. By using the same infection protocol with slight modifications, the survival of transfected parasites within murine macrophage cell line J774 and the canine monocyte-macrophage cell line DH82 were evaluated.
Briefly, both lines were cultured in RPMI 1640 complete medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 2 mM sodium pyruvate, 100 units/ml gentamycin). Cell suspensions in the log phase of growth (107 cells/ml RPMI 1640) were treated with 25 ug/ml mitomycin for 20 min at 37° C., washed with PBS, and distributed into 24-well microplates at 2×105 cells/well in 500 μl of culture medium. The cells were infected with stationary-phase promastigotes (10 promastigotes/cell) and infection was determined by measuring luciferase activity as described before.
2.2. Results
Results are given in FIGS. 8 and 9.
We attempted to determine whether the expression of Leishmania secreted proteins could interfere with the capacity of recombinant parasites to replicate within human macrophages in vitro. Both confirmed secreted proteins (LinJ19.0410 and LinJ36.5780) from L. infantum were tested by using the luciferase reporter system in transfected parasites over-expressing these proteins. We used bioluminescence as a quantitative indicator of the viability and multiplication of parasites. The number of promastigotes cells and luciferase activity were linearly correlated for the different recombinant parasites before macrophage infection (data not shown). Results of in vitro infection showed that over-expression of secreted protein LinJ19.0410 (ortholog of Tc00.1047053505789.10) increases the capacity of Leishmania to survive in THP-1 differentiated macrophages as early as 24 h post-infection (FIG. 8). Furthermore, a statistically significant increase in luciferase activity of recombinant parasites expressing LinJ19.0410 was maintained throughout the experiments (P<0.05). This effect was not observed in parasites over-expressing LinJ36.5780 (ortholog of Tc00.1047053506155.99) where infectivity levels were similar to the control parasites transfected with the pTEX vector alone and the pSP-αHYGαLUC (FIG. 8). Results of these assays showed a significant survival advantage to Leishmania parasites over-expressing the gene LinJ19.0410 (Ortholog gene of Tc00.1047053505789.10) suggesting that this protein is involved in a process increasing survival and replication of the parasite inside its target cell.
As shown in FIG. 9, parasites expressing the protein LinJ19.0410 displayed higher luciferase activity in canine macrophages at 48 h post-infection, indicating enhanced virulence of transgenic parasites expressing this protein. Nevertheless, this effect was not observed in infected murine macrophages where luciferase activity was similar to control parasites and the transgenic parasites expressing LinJ36.5780. Taken together, these results suggest that the secreted protein LinJ19.0410 from L. infantum increases its infectivity in cell lines derived from both human and dogs, the main natural mammalian hosts of Leishmania parasites.
EXAMPLE 3
Immunogenic Properties of Conserved Secreted Proteins from Trypanosoma cruzi Identified by a Genomic Based Approach
3.1. Material and Methods
3.1.1. Bacterial Strains and Plasmids
Escherichia coli TOP10 (Invitrogen) was used for primary cloning, and E. coli BL21 (DE3) for protein expression. Encoding sequence for the secreted protein Tc00.1047053509999.10 was cloned under the control of the T7lac promoter. The plasmid vector used was pET-21b (Novagen, Madison, Wis.) conferring resistance to ampicillin and allowing expression of the target protein fused to a C-terminal tail of six Histidine residues.
3.1.2. Cloning of T. cruzi Genes into the Expression Vector
T. cruzi gene encoding the secreted protein was amplified from genomic DNA (TcY7clone derived from the Y strain) by PCR using specific forward and reverse primers (table 1). Tc00.1047053509999.10 gene (SEQ ID NO 85) was cloned without the amino-acid sequence corresponding to the predicted N-terminal signal peptide. PCR reactions were carried out in 20 μl using 0.5 μM of each primer, 0.2 mM dNTP, 0.4 U of Phusion high-fidelity polymerase (Finnzymes, Espoo, Finland) and the following cycling conditions: 98° C. for 30 s followed by 25 cycles of 98° C. for 10 s, 65° C. for 15 s, 72° C. for 45 s, and a 72° C. elongation for 5 min. Digested and purified fragments were inserted into the dephosphorylated pET-21b vector digested with the corresponding restriction enzymes. Cloned sequences were confirmed by restriction digestion and sequencing. Large scale preparations of the constructs were performed using the plasmid midi kit (Promega).







TABLE 1







Primers used for cloning into the pET21b expression vector












Amplification





product size
MW


Gene ID
Primer sequencesa
(bp)
(kDa)b





Tc00.1047053509999.10
F CATCGCAGCATATGGCAGAAGAGGAGGACGTGAGG
1182
45.8



R GCACTGACTCTCGAGGCCGCACCAGCGCTCCAGAA







F, Forward primer and R, Reverse primer used for the amplification of the encoding


sequence without the predicted secretion signal peptide.


aRestriction sites used for cloning in the pET21b vector are indicated in italics.


bExpected molecular weight of the protein.






3.1.3. Expression and Purification of T. cruzi Recombinant Proteins

The plasmid carrying the target protein was used to transform E. coli BL21(DE3) strain. Colonies were grown overnight at 37° C. with shaking in auto-inducing media ZYP-5052 (Studier F W, 2005) containing 100 μg/ml of ampicillin. Cleared E. coli lysates were obtained from cell pellets (50-ml cultures) re-suspended in 5 ml of buffer containing 100 mM NaH2PO4, 10 mM Tris-Cl and 8 M urea (pH 8.0). The clarified supernatant was applied to a Ni-NTA agarose column (Qiagen) and the recombinant protein was eluted in 8M Urea buffer at pH 4.5. To assess the purity of the proteins, eluted fractions were analyzed in SDS-PAGE gel by stained with Coomassie blue and/or by immunoblotting with an Anti-His (C-Term)-HRP antibody as detailed below.
3.1.4. SDS-PAGE
SDS-PAGE analysis was performed on a NuPAGE Bis-Tris gel (4-12%) in MOPS-SDS running buffer (Invitrogen) under reducing conditions (50 mM DTT). The gels were either stained with Coomassie blue or electroblotted onto PVDF membranes (Hybond-P, Amersham) for western blot analyses.
3.1.5. Western Blot Analysis
The PVDF membranes were rinsed twice in TBS and used for immuno-detection of the recombinant proteins with an Anti-His (C-Term)-HRP antibody (Invitrogen) according to the manufacturer's instructions. Membranes used for detection of anti-T. cruzi antibodies in patients' sera were rinsed twice in TBS and blocked overnight in TBS-Tween 0.1% (TBS-T) and 5% non-fat milk. Strips of paper (5 mm) were then cut and incubated separately in a 1:2,500 dilution of human serum for 2 h at room temperature. Strips were then washed five times (5 min) in TBS-T and incubated for 1 h at room temperature with HRP-conjugated anti-human IgG (heavy and light chain specific) (Nordic Immunology) diluted 20,000 times in TBS-T and 5% non-fat milk. The strips were washed as before, and the immune complexes were revealed by chemiluminescence emission using the ECL Plus Western blotting detection system and ECL Hyperfilms (GE Healthcare, UK).
3.1.6. Patients' Sera
Positive sera were obtained from infected chagasic individuals from an endemic region located in northeast Argentina (Chaco Province), a free-vector city located in north-western Argentina (Salta-city) and from Bolivian children treated with Benznidazole (5 mg/kg/day) in the community of Tupiza (Potosi department), an area under controlled vector transmission. Negative sera were obtained from healthy blood donors of the same region respectively. The T. cruzi infection status was determined by using two conventional tests: commercial ELISA and IHA based on parasite homogenate antigens. Positive sera for both reactions were considered as true positives. Negative sera for both reactions were considered as true negatives.
3.2. Results
They are given in FIGS. 10 and 11.
3.2.1. Expression and Purification of Recombinant Protein rTc00.1047053509999.10 (SEQ ID NO 85)
In order to determine the antigenic properties of this T. cruzi secreted protein, the recombinant protein in E. coli was produced to subsequently analyse its reactivity with chagasic patient sera. Protein expression was assessed in transformed bacteria carrying the recombinant plasmid. The recombinant protein was expressed and purified under denaturing conditions in the E. coli BL21 (DE3) (FIG. 10).
3.2.2. Reactivity of Sera from Chagasic Patients with rTc00.1047053509999.10 (SEQ ID NO 85)
The ability of the recombinant protein rTc00.1047053509999.10 to detect chagasic infections was evaluated by Western Blot. As shown in FIG. 11 the recombinant protein rTc00.1047053509999.10 was recognized by sera from chagasic patients as a single band. Sera from healthy donors did not recognize the recombinant protein. Anti-Tc00.1047053509999.10 antibodies were detected in 80% (18/22) of sera from chagasic patients. The analysed sera belong to patients originating from different regions of Latin America. Six sera are from patients living in North-western Argentina (Salta-city) and presenting heart failure as detected by electrocardiogram and echocardiogram. Five out of these sera were positive by Western blot analyses. Among six sera of children from Northeast Argentina (Chaco-province), only 5 recognized the recombinant protein. The remaining sera correspond to five Bolivian children and five Mexican patients that recognized the T. cruzi protein in four out the five sera tested.
Furthermore, potential cross-reactivity with antibodies from five patients infected with L. infantum was evaluated. The recombinant protein was not recognized by these sera suggesting that the antibodies detected by chagasic patients are specific for T. cruzi infection.
In view of the antigenic properties of T. cruzi Tc00.1047053509999.10 (SEQ ID NO 85) said recombinant protein may represent an interesting antigen to specifically identify anti-T. cruzi antibodies in sera and may represent a new potential diagnostic tool for Chagas disease.

## Claims

1. A method of diagnosing Chagas disease caused by Trypanosoma cruzi in an in vitro assay comprising the steps of:
a. contacting a biological sample with a polypeptide comprising an amino acid sequence SEQ ID NO: 86 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), where in the presence of an immune complex is indicative of Chagas disease.