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  Pharmaceutical Patents  

 

Title:  Identification of specific differentially expressed antigens
United States Patent: 
7,772,386
Issued: 
August 10, 2010

Inventors:
 Jungblut; Peter (Berlin, DE), Kaufmann; Stefan H. E. (Berlin, DE), Schaible; Ulrich (Berlin, DE), Mollenkopf; Hans (Berlin, DE), Raupach; Barbel (Berlin, DE), Zimny-Arndt; Ursula (Berlin, DE), Lamer; Stephanie (Berlin, DE), Mattow; Jens (Berlin, DE)
Assignee:
  Max-Planck-Gesellschaft zur Forderung der Wissenschaften E.V. (Berlin, DE)
Appl. No.:
 09/890,339
Filed:
 January 28, 2000
PCT Filed:
 January 28, 2000
PCT No.:
 PCT/EP00/00690
371(c)(1),(2),(4) Date:
 March 12, 2002
PCT Pub. No.:
 WO00/44392
PCT Pub. Date:
 August 03, 2000


 

Training Courses -- Pharm/Biotech/etc.


Abstract

The present invention relates to compositions useful in immunization against pathogenic organisms of the genus Mycobacterium and for diagnostic purposes. In particular, the present invention relates to a composition comprising at least one protein which is differentially expressed in a virulent strain as compared to an avirulent strain of Mycobacteria. Furthermore, the invention relates to compositions comprising fusion proteins, antigenic fragments, nucleic acid molecules encoding the aforementioned proteinaceous compounds and/or antibodies thereto. Additionally, the invention relates to pharmaceutical and diagnostic compositions comprising or employing compounds of the invention. In addition, the present invention relates to the use of the compounds of the invention for the treatment of Mycobacterium induced diseases and/or for the preparation of a vaccine for vaccination against Mycobacterium induced diseases.

Description of the Invention

This application is a continuation-of PCT Application No. PCT/EP00/00690, filed Jan. 28, 2000, which claims the benefit of EP99/101590.0, filed Jan. 29, 1999.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in both a paper copy and a computer readable copy, and which Sequence Listing is hereby incorporated by reference in its entirety.

The present invention relates to compositions useful in immunization against pathogenic organisms of the genus Mycobacterium and for diagnostic purposes. In particular, the present invention relates to a composition comprising at least one protein which is differentially expressed in a virulent strain as compared to an avirulent strain of a pathogenic Mycobacterium. Furthermore, the invention relates to compositions comprising fusion proteins, antigenic fragments, nucleic acid molecules encoding the aforementioned proteinaceous compounds and/or antibodies thereto. Additionally, the invention relates to pharmaceutical and diagnostic compositions comprising or employing compounds of the invention. In addition, the present invention relates to the use of the compounds of the invention for the treatment of Mycobacterium induced diseases and/or for the preparation of a vaccine for vaccination against Mycobacterium induced diseases.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.

Since the beginning of the 1980s, a new trend has been observed in the industrialized countries. On the one hand, resistances to antibiotics have increased, which make it difficult or even impossible to treat many of the disease-causing agents. On the other hand, new infectious diseases, which had been unknown up to now, arise, and old diseases return. For example, malaria and tuberculosis are old epidemics and increasingly surmounting in many different parts of the world. Especially tuberculosis (TB), a chronic infectious disease that is generally caused by infection with Mycobacterium tuberculosis, is a disease of major concern. Each year, 8 to 10 million new cases of TB are described, and, causing more than three million deaths per year, TB is a major disease in developing countries as well as an increasing problem in developed areas of the world due to, for example, antibiotic resistance.

Inhibiting the spread of TB will require effective vaccination and accurate, early diagnosis of the disease. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common Mycobacterium for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacterium bovis.

However, the safety and efficacy of BCG is a source of controversy, and some countries, such as the United States and the Netherlands, do not vaccinate the general public.

Additionally, it has been shown that BCG vaccination affords greater protection against leprosy than against tuberculosis (Ponninghaus, Lancet 339 (1992), 639). Furthermore, M. bovis BCG has failed to protect against TB in several trials (WHO, Tech. Rep. Ser. (1980), 651, 1-15) for reasons that are not entirely clear (Fine, Tubercle 65 (1984), 137-153). Additionally, it has been shown that the vaccine strain of M. bovis BCG only confers protection against the severe form of miliary tuberculosis in children (Fine, Lancet 346 (1995), 1339-1345). In contrast, its protective capacity against the most common form, pulmonary tuberculosis in adults, is low and highly variable (Colditz (1994), JAMA 271, 698).

Diagnosis of TB is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, which indicates exposure to Mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.

Therefore, it is of major concern that effective and safe vaccines and therapies for the immunization and the treatment of TB as well as useful, reliable diagnostics be developed.

The technical problem of the present invention was thus to provide compositions useful for effective immunization against pathogenic organisms, for effective therapy of infected humans and animals that can be reliably used in low doses and with substantially no side effects and/or for detection/diagnosis of pathogenic organisms in biological/medical samples.

The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to a composition comprising at least one protein which is differentially expressed in a virulent strain as compared to an avirulent strain of the genus Mycobacterium.

The term "composition", as used in accordance with the present invention, comprises at least one protein, an antigenic fragment of said protein, a fusion protein, a nucleic acid molecule and/or an antibody of this invention and, optionally, further molecules, either alone or in combination, like e.g. molecules which are capable of optimizing antigen processing, cytokines, immunoglobulins, lymphokines or CpG-containing DNA stretches or, optionally, adjuvants. The composition may be in solid, liquid or gaseous form and may be, inter alia, in form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). In a preferred embodiment, said composition comprises at least two, preferably three, more preferably four, most preferably five differentially expressed proteins.

The term "protein" means, in accordance with the present invention, a peptide(s) or (a) (poly)peptide(s) which encompass amino acid chains of any length, wherein the amino acid residues are linked by covalent peptide bonds. However, peptidomimetics of such proteins wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention. In accordance with this invention, a protein may comprise different protein species. A protein species is defined by its chemical composition and modifications of said peptide(s)/(poly)peptide(s) by, inter alia, glycosylations, acetylations, phosphorylations, lipidations or by amino acid exchanges, the term describes a chemically clearly-defined molecule and corresponds, inter alia, to one spot on a high-performace 2-DE pattern (Jungblut, Electorphoresis 17 (1996), 839-847). The term protein species is therefore defined as the smallest unit of a protein classification, defined by its chemical structure.

The term "differentially expressed" denotes in the context of the present invention proteins/protein species which are distinctly expressed, regulated and/or modified. Therefore, the term "differentially expressed" includes protein(s)/protein species that are absent in, that occur in different amounts in and/or that comprise different post-translating modifications in a "virulent" strain compared to an "avirulent" strain of a pathogenic organism. The term "differentially expressed" as used in accordance with the invention denotes therefore not only proteins/protein species which are missing in one strain as compared to another (+/- variants), but also comprises mobility variants and/or intensity variants. Intensity variants are protein species occurring in comperative protein 2DE-patterns which differ in amount. A +/- variant can be considered as an extreme intensity variant, where the protein species occurs in one pattern and is absent in the other. If the protein occurs in two different compared patterns at different positions, these two positions can be considered as indication for two different protein species of this protein (inter alia, due to secondary modifications as explained herein above) which are defined as mobility variants. These variants (+/-, intensity or mobility) can be detected by proteome analysis.

Previously, the determination of immunogenic antigenic and/or pathogenic determinants of pathogenic organisms had been hampered by the fact that it was not possible to analyze the whole proteome of such organisms, like Mycobacteria, by conventional means. However, the previously employed analysis of cellular fractions and/or fragments (like bacterial membranes) can only reflect a limited number of differentially expressed protein(s)/protein species, if any, due to the loss of proteinaceous material during fractionation and isolation of such fragments. In accordance with the present invention, a new method (as examplified in the examples) has been employed that allows the analysis of whole pathogenic organisms and it was surprisingly found, that a great number of differentially expressed proteins in a virulent strain as compared to an avirulent strain of Mycobacteria can be identified.

Differentially expressed proteins (protein species) may be identified, detected and/or brought into a biological correlation, inter alia, by proteome analysis of whole organisms (like mycobacteria) or, less preferred, of biochemically defined fractions (like, inter alia, lipoproteins, glycoproteins, phosphoproteins) or of biologically defined fractions (like, inter alia, membranes, cytosol, structural elements of a pathogenic organism); see, e.g. Wilkins (1997), "Proteome Research: New Frontiers in Functional Genomics, Springer-Publishers Berlin; Kahn, Science 270 (1995), 369-370; Jungblut, J. Biotech. 41 (1995), 111-120; Bluggel, Biospektrum 5 (1998), 39-44; Lohaus, Biospekturm 5 (1998), 32-39; Jungblut Electrophoresis 17 (1996), 839-847; Scheler, Electrophoresis 19 (1998), 918-927.

As known to the person skilled in the art, analysis of proteomes of lower complexity, e.g. ribosomes with 60 protein species, can be performed, inter alia, by protein/protein species separation and identification strategies, comprising, for example, 2-dimensional gel electrophoresis (2-DE; Kaltschmidt, Anal. Biochem. 36 (1970), 401) or HPLC (Kamp, J. Chromatogr. 317 (1984), 181). However, analysis of proteomes of higher complexity can be carried out, inter alia, by a combination of isoelectric focusing and SDS-PAGE (Vesterburg, Acta Chem. Scand. 20 (1966), 820; Laemmli, Nature 227 (1970), 680) and the use of large-sized gels (Jungblut, Electrophoresis 15 (1994), 685; Klose, Electrophoresis 16 (1995), 1034). Comparison of individual, specific 2-DE gels allows for the identification of differentially expressed proteins and the identification of proteins separated by 2-DE is known to the skilled artisan (see, e.g. Patterson, Electrophoresis 16 (1995), 1791; Jungblut, Electrophoresis 17 (1996), 839; Jungblut, Mass Spectrometry Reviews 16 (1997), 145; Kaufmann, Jahrbuch der MPG (1998), 42-57; Bluggel (1998), loc. cit., Schaible, DGHM-Kongress (1998), Einhoon-Resse Verlag (ISSN 1433-3988), 20).

In order to further identify differentially expressed proteins, several techniques which are known in the art can be used. These techniques comprise, but are not limited to, in-gel digestions, electroelution procedures, microsequencing, amino acid analysis, Edman-sequencing or mass spectroscopy. For example, some techniques start directly from gel(s), others need a transfer to membranes by blotting. To the first group belong, inter alia, coelectrophoresis, internet comparison of position, peptide mapping by SDS-PAGE (Cleveland, J. Biol. Chem. 252 (1977), 1102), protein elution and MALDI-MS or N-terminal sequencing by Edman degradation (Edman, Acta Chem. Scand. 4 (1950), 283), enzymatic in-gel digestion, analysis of peptides directly in the mixture by mass spectrometry, peptide mass fingerprinting (Pappin, Curr. Biol. 3, (1993), 327), PSD-MALDI-MS (Spengler, Rapid Commun. Mass Spectrom. 6, (1992), 105), ESI-MS (electrospray-ionization-MS) and/or (after separation) by micro-HPLC. HPLC separated peptides may be further analysed, inter alia, by Edman degradation, PSD-MALDI-MS, MS/MS (Wilm, Nature 379, (1996), 466) or ladder sequencing (Thiede, FEBS Lett. 357, (1995), 65) in order to obtain a peptide sequence. Proteins immobilized on membranes allow the identification by immunostaining (Towbin, Proc. Natl. Acad. Sci. USA 76, (1979), 4350), N-terminal sequencing (either directly or after deblocking) (Hirano, Electrophoresis 14, (1993), 839), determination of the protein mass (Eckerskorn, Electrophoresis 13, (1992), 664), amino acid analysis (Jungblut, J. Prot. Chem. 11, (1992), 603) and/or enzymatic digestion with the same proteinchemical techniques as described for in-gel digestions. Results of such analysis are mass fingerprints.

The resulting peptide masses are searched by search programs (e.g. prospector.ucsf.edu/ucsfhtm13.2/msfit.htm; www.expasy.ch/tools/peptident.html) in sequence databases (EMBL, PIR, NCBI, MIPS, Swiss-Prot, OWL). By use of such mass fingerprints amino acid sequences can be deduced and sequenced. From these sequenced amino acid fragments degenerative oligonucleotides may be deduced and synthesized that may be used to screen, for example, genomic or cDNA libraries to identify and clone the corresponding GENE/cDNA.

Identified proteins may be produced by, for example, recombinant techniques or by biochemical or synthetic techniques which are known to the skilled artisan (Sambrook et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (1989); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989)).

Other methods for the elucidation of differentially expressed proteins include, but are not limited to, enzyme activity, receptor activity measurements, immunostainings, immunohistochemical methods.

As shown in the appended examples, differential protein expression can be detected by preparation of microorganisms or, less preferred, compartment/fragments thereof, 2-DE, subtractive analysis and identification of proteins by peptide mass fingerprinting (PMF) with or without confirmation by further methods.

Identification of protein species from 2-DE patterns by only one of the above-described methods, peptide mass fingerprinting or amino acid analysis, was described to lead to false identification (Cordwell, Electrophoresis 16 (1995), 438; Mortz, Biol. Mass. Spec. 23 (1993), 249). However, the present invention, surprisingly showed that differentially expressed proteins may be identified by peptide mass fingerprinting without confirmation by a further method. As examplified in the appended examples, improvements in the sample preparation, e.g. reduction of volumes and surface contacts, use of volatile buffers and improvements in mass spectrometry, introduction of delayed extraction, results in improved mass accuracy, resolution, and sensitivity, leading to high sequence coverage of at least 30%. This sequence coverage is sufficient for identification and needs no further confirmation. Thus, the present invention also concerns a method for identification of differentially expressed proteins as discussed above and illustrated in examples 2, 4 and 8.

The term "virulent strain", in accordance with the present invention, denotes the capacity of a pathogenic strain of the genus Mycobacterium to infect a host and/or to cause disease--defined broadly in terms of severity of symptoms in a host. Thus, a "virulent strain" might cause symptoms in a susceptible host, whereas another host might be unaffected by this strain, which can be therefore considered as being an "avirulent strain" in this second host. As used in accordance with the present invention, the term "avirulent strain" denotes strains of a Mycobacteria which is not capable of inducing infection and/or causing disease in a specific host or in a host species. The term "avirulent strains" denotes furthermore attenuated strains of microorganisms.

The terms "virulent" and "avirulent" strains not only relate to laboratory strains but also comprise wildtype strains. The virulency of a strain is known in the art and described, inter alia, in Brandis et al., "Lehrbuch der medizinischen Mikrobiologie", Gustav Fischer Verlag, 7. Auflage (1994), Zinsser Microbiology, ed Joklik, Willett, Amos, Wilten 20.sup.th edition, Appleton & Lange, 1992.

In a preferred embodiment of the composition of the present invention said strains are selected from the group consisting of M. tuberculosis, M. bovis, M. avium, M. africanum, M. kanasasii, M. intracellulare, M. ulcerans, M. paratuberculosis, M. simiae, M. scrofulaceam, M. szulgai, M. xenopi, M. fortuitum, M. chelonei M. leprae and M. marinum.

In a more preferred embodiment of the composition of the present invention said protein is differentially expressed in M. tuberculosis and in M. bovis.

In a particularly preferred embodiment the present invention relates to a composition wherein said virulent strain is M. tuberculosis H37Rv or M. tuberculosis Erdman and said avirulent strain is M. bovis BCG. Furthermore, the present invention relates to a composition wherein said protein is differentially expressed in M. tuberculosis H37Rv and M. tuberculosis Erdman as compared to M. bovis BCG.

In an even more preferred embodiment of the composition of the present invention said differentially expressed protein is 2-isopropyl malate synthase (Rv3710), s-adenosylmethionine synthase (metK, RV1392), succinyl-CoA synthase a-chain (sucD, RV0952), oxidoreductase of aldo/keto reductase family (Rv2971), oxidoreductase (Rv0068), elongation factor G (FusA2, Rv0120c), uridylate kinase (PyrH, Rv2883c), ABC-type transporter (Rv1463), short chain dehydrogenase/reductase family (RV1856C), hydrolase (LinB, Rv2579), phosphoribosylamino-imidazole carboxylase catalytic subunit (PurE, Rv3275c), hypothetical protein (Rv2557), hypothetical protein (Rv3407), hypothetical protein (Rv3881c), hypothetical protein (Rv2449c), hypothetical protein (Rv0036c), hypothetical protein (Rv2005c) or transcriptional regulator (Crp/Fr family) (Rv 3676). As shown in the appended examples, whereas 2-isopropyl malate synthase (Rv3710) is expressed in M. tuberculosis H37Rv, it is not detected and identified in M. bovis BCG. Furthermore, s-adenosylmethionine synthase (metK, RV1392), succinyl-CoA synthase a-chain (SUCD, Rv0952), oxidoreductase of aldo/keto reductase family (Rv2971) or oxidoreductase (Rv0068), represent protein species which are differentially expressed in M. tuberculosis H37Rv and M. bovis BCG and represent mobility variants. As intensity variants may be considered proteins corresponding to the Rv numbers Rv0652, Rv2429, Rv2428, RV0569, Rv0475, Rv3463, Rv3054c. As +/--variants may be considered Rv2883c, Rv0120c, Rv1463, Rv2579, Rv3275c, Rv3407, Rv3881c, Rv2449c, Rv0036c, Rv2005c or Rv3676. As shown in the appended examples, whereas elongation factor G (Rv0120c), uridylate kinase (Rv2883c), ABC-type transporter (Rv1463), short chain dehydrogenase/reductase family protein (Rv 1856c), 1,3,4,6-tetracholoro-1,4,-cyclohexadiene hydrolase (Rv2579), phosphoribosylaminoimidazole carboxylase catalytic subunit (Rv3275c), hypothetical protein (Rv2557), and hypothetical protein (Rv3407) are expressed in M. tuberculosis H37Rv and M. tuberculosis Erdman, they are not detected in M. bovis BCG Chicago and M. bovis BCG Copenhagen. Furthermore, protein spot A607 in M. tuberculosis H37Rv and the corresponding spot A148 in M. tuberculosis Erdman have no counterparts in M. bovis BCG Chicago and M. bovis BCG Copenhagen. This protein was identified herein as the hypothetical protein Rv3881c. Furthermore, spots C434 from M. tuberculosis H37Rv and the corresponding spot C508 from M. tuberculosis Erdman have no counterparts in M. bovis BCG Chicago and M. bovis Copenhagen. They were identified as a hypothetical protein (Rv2005c). Rv2005c occurs at the 2-DE pattern in another form at a different position in all four strains. Additionally, the spots B69, C176, D12 and D115 of M. tuberculosis H37Rv with their counterparts in M. tuberculosis Erdman, B54, C404, D115 and D130, respectively, have no counterparts in M. bovis BCG Chicago and M. bovis BCG Copenhagen. B69 was identified as a hypothetical protein (Rv2449c). C176 was identified as a hypothetical protein (Rv0036c). D12 and D115 of M. tuberculosis H37Rv were identified as transcriptional regulator (Crp/Fnr family) (Rv3676). As will be described herein below these proteins/protein species might serve, inter alia, in pharmaceutical and diagnostic compositions. Cole (Nature 393 (1998), 537) published the complete sequence of the M. tuberculosis H37Rv genome and identified a total of 3924 individual genes which were classified according to the classification of Riley (Microbiol. Rev. 57 (1993), 862). Identifications of this putative genes were performed by homology searches of deduced open reading frames from other microorganisms. Therefore, the term "Rv numbers" as employed herein corresponds to clearly defined nucleic acid sequences (deduced open reading frames) as describes in Cole et al., (loc. cit.). However, for most of the identified putative genes of M. tuberculosis, it is not clearly shown that they are actually expressed additional sequence information on mycobacterial genes is also available from the Sanger Centre, U. K. Under www.sanger.ac.uk/Projects/M_tuberculosis/ information on the genomic sequence of M. tuberculosis is available. Therefore, the "Rv-numbers" not only refer to nucleic acid sequences but also to protein sequences as deposited in the Sanger database. Further information on M. tuberculosis sequence is available from the Institut Pasteur, Paris under bioweb.pasteur.fr/GenoList/TubercuList/.

The invention also relates to a composition comprising an antigenic fragment of the protein as defined herein.

The term "antigenic fragment", as used herein, refers to the ability of said fragment to elicit an immune response (e.g. humoral or cellular) in a subject, such as a human, and/or in a biological sample. These fragments may consist entirely of the antigenic and/or immunogenic portion of the protein or may contain additional sequences. The additional sequences may be derived from said protein or may be heterologous, and such additional sequences may (but need not) be antigenic and/or immunogenic. The antigenicity of an amino acid sequence can be deduced/predicted by methods known to the person skilled in the art as for example described in Parker, J. Immunol. 152 (1994), 163 (bimas.dcrt.nih.gov:80/molbio/hla_bind/), Meister, Vaccine 13 (1995), 581-591 or Bull, Biochem. Biophys. 161 (1974), 665-670. Furthermore, computer predictions may be employed to elucidate hydrophilicity and/or antigenicity of amino acid sequences and stretches. Such computer programs may be Garnier analysis of the on the plot v. 2.5e package, the GCG-software derived from HGMP resource Center Cambridge (Rice (1995) Programme Manual for the EGCG package, Cambridge (B10 IKQ, England) or the programme based on Kyte/Dolittle, J. Mol. Biol. 157 (1982), 105-132 (see also www.expasy.ch/cgi-bin/protscale.pl).

Antigenic fragment may be produced recombinantly using a polynucleotide sequence that encodes the antigenic fragment or may be produced by biochemical or synthetic techniques. Those methods are known to those of ordinary skill in the art (see, e.g. Sambrook et al., loc. cit.; Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, N.Y. (1988); Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2146; Stewart, "Solid Phase Peptide Synthesis", WH Freeman Co, San Francisco (1969); Scopes, "Protein Purification", Springer Verlag, New York, Heidelberg, Berlin (1987); Janson, "Protein Purification, Principles, High Resolution Methods and Applications", VCH Publishers, New York, Weinheim, Cambridge (1989); Wrede, "Concepts in Protein Engineering and Design", Walter de Gruyter, Berlin, N.Y. (1994); Wittmann-Liebold, Jungblut "Analysis and Characterization of Proteins", 47-107).

Additionally, the invention relates to a fusion protein comprising a protein and/or an antigenic fragment as defined in the above.

The protein and/or the antigenic fragment of the present invention can comprise a further domain, said domain being linked by covalent or non-covalent bonds. The linkage can be based on genetic fusion according to the methods known in the art (Sambrook et al., loc. cit.; Ausubel, loc. cit.) or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686. The additional domain present in the fusion protein comprising the protein of the invention may be joined directly (i.e. with no intervening amino acids) or may be linked by a flexible linker, advantageously a polypeptide linker, wherein said polypeptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N-terminal end of the protein or vice versa. The above described fusion protein may further comprise a cleavable linker or cleavage site, which, for example, is specifically recognized and cleaved by proteinases or chemical agents. Cleavable linker sequences include, but are not limited to, Factor XA or enterokinase (Invitrogen, San Diego, Calif., USA).

Additionally, said further domain may be of a predefined specificity or function. In this context, it is understood that the protein of the invention may be further modified by conventional methods known in the art. This allows for the construction of fusion proteins comprising the protein of the invention and other functional amino acid sequences, e.g., immunologically relevant proteins like cytokines, lymphocytes, interferones, or protein tags (GST, GFP, h-myc peptide, FLAG, HA peptide) which may be derived from heterologous proteins.

In yet another preferred embodiment the present invention relates to a composition comprising at least one differentially expressed protein as defined herein above wherein said differentially expressed protein is biochemically, biophysically and/or recombinantly modified. Such modifications may comprise amino acid substitutions, deletions, insertions, additions and/or duplications wherein said modified differentially expressed protein should still comprise at least one antigenic fragment or epitope which is specifically recognized by an antibody directed to, raised to and/or engineered to detect the non-modified differentially expressed protein as defined herein above. The non-modified amino acid sequence of a differentially expressed protein is deducible for the person skilled in the art as described herein above, inter alia, by employing biochemical and recombinant methods and sequence databases. Additionally, the non-modified amino acid sequence of a differentially expressed protein as defined herein above may be deduced from nucleic acid sequences and/or proposed open reading frames as known to the person skilled in the art. For example, the complete genome sequence of M. tuberculosis H37Rv is published in Cole et al. (1998, loc. cit.).

In addition, the invention relates to a fusion protein comprising at least two proteins as defined herein and/or (an) antigenic fragment(s) as defined herein.

In a further embodiment the fusion protein of the present invention comprises an immunostimulatory molecule.

The term "immunostimulatory molecule" denotes in accordance with the present invention molecules or fragments thereof which, inter alia, activate and/or stimulate the humoral and cellular response of an immune system. They might, e.g. activate antigen-presenting cells, stimulate natural killer cells, enhance the production of antibodies directed against an antigen and/or a pathogen or induce the proliferation of cells of the immune system. These molecules are known in the art and comprise, inter alia, cytokines, lymphokines, immunoglobulins, interleukins and/or complement factors (see, e.g. Paul, "Fundamental Immunology", Raven Press (1989); Schaible, Adv. In Immunology 71 (1999), 261-377).

In one further preferred embodiment of the fusion protein of the present invention said fusion protein comprises a molecule capable of optimizing antigen processing.

Cellular immune recognition is mediated by a special class of lymphoid cells, T-cells. These cells do not recognize whole antigens but instead they respond to degraded peptide fragments thereof which appear on the surface of the target cell bound to proteins called major histocompatibility complex (MHC) molecules (antigen processing). Essentially all nucleated cells have MHC class I molecules, whereas MHC II are restricted to immune cells with special presenting qualities. Molecules which are capable of optimizing antigen processing are known in the art and comprise, inter alia, listeriolysin, which improves MHC class I restricted immune responses (see, e.g., Hess, PNAS 95 (1998), 5299-5304).

The term "fusion protein" as employed hereinabove also relates to chimeric proteins wherein said chimeric protein comprises at least one differentially expressed protein and/or (a), preferably antigenic, fragment(s) thereof in combination with at least one other protein, peptide or fragment(s) thereof. Furthermore, said chimeric protein may comprise at least two modified differentially expressed proteins as defined herein above.

The invention also relates to a composition comprising at least one fusion protein as defined hereinabove.

The invention further relates to a nucleic acid molecule coding for a modified differentially expressed protein as defined herein, the antigenic fragment as defined herein and/or a fusion protein as defined herein.

The nucleic acid molecule of the invention or employed in methods or compositions of the invention may be DNA such as cDNA or RNA such as mRNA. Additionally, the nucleic acid molecule of the invention may be PNA. Its origin may be natural, synthetic or semisynthetic or it may be a derivative, such as said peptide nucleic acid (Nielsen, Science 254 (1991), 1497-1500). Furthermore, said nucleic acid molecule may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination. Preferably, said nucleic acid molecule is part of a vector.

Such vectors may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.

Furthermore, the vectors may, in addition to the nucleic acid sequences of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, translation initiation codon, translation and insertion site for introducing an insert into the vector. Preferably, the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells.

Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV-HSV thymiakine kinase promoter, SV40, RSV-promoter (Rous sarcome virus), human elongaticn factor 1.alpha.-promoter, CMV enhancer or SV40-enhancer. For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter or the trp promoter, has been described. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORTI (GIBCO BRL), or prokaryotic expression vectors, such as lambda gt11. Beside the nucleic acid molecules of the present invention, the vector may further comprise nucleic acid sequences encoding for secretion signals. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used leader sequences capable of directing the protein/(poly)peptide to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a protein thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the proteins, antigenic fragments or fusion proteins of the invention may follow. Of course, the vector can also comprise regulatory regions from pathogenic organisms.

Furthermore, said vector may also be a gene transfer or targeting vector. Gene therapy, which is based on introducing therapeutic genes (for example for vaccination) into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, vector systems and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, Schaper, Current Opinion in Biotechnology 7 (1996), 635-640 or Verma, Nature 389 (1997), 239-242 and references cited therein. The nucleic acid molecules of the invention and vectors as described herein above may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention. In addition to recombinant production, fragments of the protein, the fusion protein or antigenic fragments of the invention may be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al. (1969) Solid Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2154). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City Calif.) in accordance with the instructions provided by the manufacturer. Various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

The invention in addition relates to a composition comprising at least one nucleic acid molecule as defined herein and/or at least one nucleic acid molecule coding for any of the differentially expressed proteins as defined herein. Said nucleic acid molecule coding for a differentially expressed protein, codes preferably for Rv3710, Rv1392, Rv0952, Rv2971, Rv0068, Rv0120c, Rv2883c, Rv1463, Rv1856c, Rv2579, Rv3275c, Rv2557, Rv3407, Rv3881c, Rv2449c, Rv0036c, Rv2005c or Rv3676.

Most preferably said nucleic acid molecule is the nucleic acid molecule as disclosed under said Rv-number under www.sanger.ac.uk/Projects/M_tuberculosis or bioweb.pasteur.fr/GenoList/TubercuList. However, the present invention relates also to compositions comprising at least one Nucliec acid molecule which hybridizes under stringent conditions to the complementary strand of the nucleic acid molecule of any of the above cited Rv-numbers. "Stringent conditions" are preferably conditions as described in Sambrook (Molecular Cloning, A Laboratory Manual, 2nd edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Such hybridizing sequences show preferably an identity of at least 50%, more preferably of at least 70% and most preferably of at least 90% on the nucleic acid level to the sequences described above. The molecules hybridizing to the nucleic acid molecules as disclosed under the above cited Rv-numbers or to the nucleic acid molecules of the invention thus also comprise fragments, derivatives and allelic variants of the above-described nucleic acid molecules which encode a differentially expressed protein (or a fragment thereof) as described in the present invention. In this regard, fragments are defined as parts of the nucleic acid molecules, which are long enough in order to encode the at least one epitope/antigenic fragment which is specifically recognized by an antibody directed to, raised to and/or engineered to detect a differentially expressed protein as defined herein. The term derivatives means that the sequences of these hybridizing molecules differ from the sequences of the above-mentioned nucleic acid molecules at one or more positions and that they exhibit a high degree of homology to these sequences. Hereby, homology means a sequence identity of at least 50%, in particular an identity of at least 60%, preferably of more than 70% and still more preferably a sequence identity of more than 90%. The deviations occurring when comparing with the above-described nucleic acid molecules might have been caused by deletion, substitution, insertion or recombination.

Said composition is useful, inter alia, for medical and diagnostic purposes, in particular, for pharmaceutic and vaccination purposes.

Moreover, the invention relates to an antibody or a fragment or a derivative thereof directed against the protein as defined herein, the antigenic fragment of the invention, the nucleic acid molecule of the invention or the fusion protein as defined herein. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric or single chain antibodies or fragments or derivatives of such antibodies.

The general methodology for producing antibodies is well-known and has been described in, for example, Kohler and Milstein, Nature 256 (1975), 494 and reviewed in J. G. R. Hurrel, ed., "Monoclonal Hybridoma Antibodies: Techniques and Applications", CRC Press Inc., Boco Raron, Fla. (1982), as well as that taught by L. T. Mimms et al., Virology 176 (1990), 604-619. As stated above, in accordance with the present invention the term "antibody" relates to monoclonal or polyclonal antibodies. Antibody fragments or derivatives comprise F(ab').sub.2, Fab, Fv or scFv fragments; see, for example, Har'ow and Lane, "Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor, N.Y. Preferably the antibody of the invention is a monoclonal antibody. Furthermore, in accordance with the present invention, the derivatives can be produced by peptidomimetics. Such production methods are well known in the art and can be applied by the person skilled in the art without further ado.

Furthermore, the invention relates to a composition comprising at least one antibody, a fragment or a derivative thereof as defined above. Such antibodies, fragments or derivatives can be used for diagnostic or for pharmaceutical purposes, i.e. for the treatment of Mycobacteria-induced diseases or the vaccination against these pathogens.

The invention also relates to a composition as defined above which is a pharmaceutical composition further comprising, optionally, a pharmaceutically acceptable carrier.

The pharmaceutical composition may comprise the proteins of the present invention, the fusion proteins of the present invention, antigenic fragments of the invention and/or antibodies (or their fragments or derivatives) of the invention, either alone or in combination. The pharmaceutical composition of the present invention may be used for effective therapy of infected humans and animals and/or for vaccination purposes.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient and/or diluent. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. If the regimen is a continuous infusion, it should also be in the range of 1 .mu.g to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins, interferons and/or CpG-containing DNA stretches, depending on the intended use of the pharmaceutical composition.

In a preferred embodiment of the present invention the pharmaceutical composition as defined herein is a vaccine.

Vaccines may be prepared, inter alia, from one or more proteins, derivatives of the proteins, nucleic acid molecules, fusion proteins, antigenic fragments or antibodies, fragments of said antibodies or derivatives of the antibodies of the invention.

For example, nucleic acid molecules of the invention may be used for gene vaccination or as DNA vaccines. Routes for administration of gene/DNA vaccines are well known in the art and DNA vaccination has been successfully used to elicit alloimmune, anti-tumor and antiidiotype immune responses (Tighe M. et al., Immunology Today 19 (1998), 89-97). Moreover, inoculation with nucleic acid molecules/DNA has been found to be protective in different modes of disease (Fynan, Proc. Natl. Acad. Sci. U.S.A. 90 (1993), 11478-11482; Boyer, Nat. Med. 3 (1997), 526-532; Webster, Vaccine 12 (1994), 1495-1498; Montgomery et al., DNA Cell Biol. 12 (1993), 777-783; Barry, Nature 311 (1995), 632-635; Xu and Liew, Immunology 84 (1995), 173-176; Zhoug, Eur. J. Immunol. 26 (1996), 2749-2757; Luke, J. Inf. Dis. 175 (1997), 91-97; Mor, Biochem. Pharmacology 55 (1998), 1151-1153; Donelly, Annu. Rev. Immun. 15 (1997), 617-648; MacGregor, J. Infect. Dis. 178 (1998), 92-100).

The proteins, nucleic acid molecules, fusion proteins, antigenic fragments or antibodies, fragments or derivatives of said antibodies of the invention used in a pharmaceutical composition as a vaccine may be formulated e.g. as neutral or salt forms. Pharmaceutically acceptable salts, such as acid addition salts, and others, are known in the art. Vaccines can be, inter alia, used for the treatment and/or the prevention of an infection with pathogens and are administered in dosages compatible with the method of formulation, and in such amounts that will be pharmacologically effective for prophylactic or therapeutic treatments.

Proteins, protein fragments and/or protein derivatives used as vaccines are well known in the art (see, e.g. Cryz, "Immunotherapy and Vaccines", VCH Weinheim (1991); Paul (1989), loc. cit.). Furthermore, it has been shown that even intracellular enzymes of bacterial pathogens can act as antigenic entities which provide immunological protection (Michetti, Gastroenterology 107 (1994), 1002; Radcliff, Infect. Immun. 65 (1997), 4668; Lowrie, Springer Semin. Immunopathol. 19 (1997), 161)

A vaccination protocol can comprise active or passive immunization, whereby active immunization entails the administration of an antigen or antigens (like the compositions of the present invention or proteins, nucleic acid molecules, fusion proteins, antigenic fragments or antibodies, fragments of said antibodies or derivatives of the antibodies of the present invention) to the host/patient in an attempt to elicit a protective immune response. Passive immunization entails the transfer of preformed immunoglobulins or derivatives or fragments thereof (e.g., the antibodies, the derivatives or fragments thereof of the present invention) to a host/patient. Principles and practice of vaccination and vaccines are known to the skilled artisan, see, for example, in Paul, "Fundamental Immunology" Raven Press, New York (1989) or Morein, "Concepts in Vaccine Development", ed: S. H. E. Kaufmann, Walter de Gruyter, Berlin, N.Y. (1996), 243-264. Typically, vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection also may be prepared. The preparation may be emulsified or the protein may be encapsulated in liposomes. The active immunogenic ingredients often are mixed with pharmacologically acceptable excipients which are compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol and the like; combinations of these excipients in various amounts also may be used. The vaccine also may contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. For example, such adjuvants can include aluminum compositions, like aluminumhydroxide, aluminumphosphate or aluminumphospho-hydroxide (as used in "Gen H-B-Vax.RTM." or "DPT-Impfstoff Behring"), N-acetyl-muramyl-L-th reonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-s- n-glycero-3-hydroxphaosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), MF59 and RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80.RTM. emulsion. Further adjuvants may comprise DNA or oligonucleotides, like, inter alia, CpG-containing motifs (CpG-oligonucleotides; Krieg, Nature 374 (1995), 546-549; Pisetsky, An. Internal. Med. 126 (1997), 169-171).

The vaccines usually are administered by intravenous or intramuscular injection. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include but are not limited to polyalkylene glycols or triglycerides. Oral formulation include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tables, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.

Vaccines are administered in a way compatible with the dosage formulation, and in such amounts as will be prophylactically and/or therapeutically effective. The quantity to be adminstered generally is in the range of about 5 micrograms to about 250 micrograms of antigen per dose, and depends upon the subject to be dosed, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection sought. Precise amounts of active ingredient required to be administered also may depend upon the judgment of the practitioner and may be unique to each subject. The vaccine may be given in a single or multiple dose schedule. A multiple dose is one in which a primary course of vaccination may be with one to ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reinforce the immune response, for example, at one to four months for a second dose, and if required by the individual, a subsequent dose(s) after several months. The dosage regimen also will be determined, at least in part, by the need of the individual, and be dependent upon the practitioner's judgment. It is contemplated that the vaccine containing the immunogenic compounds of the invention may be administered in conjunction with other immunoregulatory agents, for example, with immunoglobulins, with cytokines or with molecules which optimize antigen processing, like listeriolysin.

In a preferred embodiment, the composition of the present invention is a diagnostic composition further comprising, optionally, suitable means for detection.

For diagnosis and quantification of pathogens like Mycobacteria, pathogenic fragments, their derivatives, their (poly)peptides (proteins), their polynucleotides, etc. in clinical and/or scientific specimens, a variety of immunological methods, as well as molecular biological methods, like nucleic acid hybridization assays, PCR assays or DNA Enzyme Immuno Assays (DEIA; Mantero et al., Clinical Chemistry 37 (1991), 422-429) have been developed and are well known in the art. In this context, it should be noted that the nucleic acid molecules of the invention may also comprise PNAs, modified DNA analogs containing amide backbone linkages. Such PNAs are useful, inter alia, as probes for DNA/RNA hybridization. The proteins of the invention may be, inter alia, useful for the detection of anti-pathogenic (like, e.g., anti-bacterial or anti-viral) antibodies in biological test samples of infected individuals. It is also contemplated that antibodies and compositions comprising such antibodies of the invention may be useful in discriminating acute from non-acute infections.

The diagnostic composition optionally comprises suitable means for detection. The proteins, antigenic fragments, fusion proteins and antibodies or fragments or derivatives thereof described above are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, animal red blood cells, or red blood cell ghosts, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing nucleic acids, (poly)peptides, proteins, antibodies, microorganisms etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions and the like. Examples of immunoassays which can utilize said proteins, antigenic fragments, fusion proteins, antibodies or fragments or derivatives of said antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western blot assay. Furthermore, these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzym Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno. Assay), and CLIA (Chemioluminescent Immune Assay). Other detection methods that are used in the art are those that do not utilize tracer molecules. One prototype of these methods is the agglutination assay, based on the property of a given molecule to bridge at least two particles.

The proteins, antigenic fragments, antibodies, nucleic acid molecules and/or fusion proteins of the invention can be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention.

Appropriate labels and methods for labeling are known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, .beta.-galactosidase, alkaline phosphatase), radioactive isotopes (like .sup.32P or .sup.125I), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums).

A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention and comprise, inter alia, covalent coupling of enzymes or biotinyl groups, iodinations, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases). Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immuno assays", Burden, RH and von Knippenburg (Eds), Volume 15 (1985), "Basic methods in molecular biology"; Davis L G, Dibmer M D; Battey Elsevier (1990), Mayer et al., (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987), or in the series "Methods in Enzymology", Academic Press, Inc.

Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.

Said diagnostic composition may be used for methods for detecting a pathogenic organism in a biological and/or medical sample and/or for detecting expression of a protein or a nucleic acid molecule of the invention by detecting the presence of mRNA coding for a protein of the invention which comprises, for example, obtaining mRNA from pathogen preparations (like bacterial or viral preparations) and contacting the mRNA so obtained with a probe/primer comprising a nucleic acid molecule capable of specifically hybridizing with a nucleic acid molecule of the invention under suitable conditions and detecting the presence of mRNA hybridized to the probe/primer. Further diagnostic methods leading to the detection of nucleic acid molecules in a sample comprise, e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR), Southern blotting in combination with nucleic acid hybridization, comparative genome hybridization (CGH) or representative difference analysis (RDA). These methods for assaying for the presence of nucleic acid molecules are known in the art and can be carried out without any undue experimentation.

The invention relates further to a method for the production of a vaccine against a virulent strain of the genus Mycobacterium comprising the steps of (a) recombinant expression of a differentially expressed protein as defined above, an antigenic fragment as defined above or the fusion protein of the invention, and (b) combining said recombinantly expressed differentially expressed protein, antigenic fragment or fusion protein with a pharmaceutically acceptable carrier.

Furthermore, the invention relates to a method for the production of a vaccine against a virulent strain of the genus Mycobacterium by combining a vector comprising a nucleic acid molecule encoding a differentially expressed protein, an antigenic fragment or the fusion protein of the invention with a biologically acceptable carrier, wherein said nucleic acid molecule in said vector is placed under the control of an expression control sequence.

Moreover, the invention relates to the use of a nucleic acid molecule encoding a differentially expressed protein, an antigenic fragment as defined above or the fusion protein of the invention for the methods as described herein.

The invention further relates to the use of at least one of the proteins, an antigenic fragment, a nucleic acid molecule, a fusion protein or the antibody or fragments or derivatives thereof as defined herein for the preparation of a composition for the treatment of a Mycobacteria-induced disease.

The invention further relates to the use of at least one of the proteins, an antigenic fragment, a nucleic acid molecule, a fusion protein or the antibody or fragments or derivatives thereof as defined herein for the preparation of a vaccine for vaccination against a Mycobacteria-induced disease.

In a preferred embodiment of the use of the present invention said Mycobacteria induced disease is selected from the group consisting of tuberculosis, leprosy, tropical skin ulcer, ulceration, abscess, pulmonary disease, granulomatous (skin) disease, opportunistic infections with non-tuberculous mycobacteria as well as from diseases elicited by atypical mycobacteria such as M. avium including pulmonary disease, lymphadenitis, cutaneous and disseminated diseases, e.g. in immunocompromised patients. The use is not restricted to Mycobacteria-induced diseases in humans but comprises also the use of the present invention in animal diseases, like bovine tuberculosis.
 

Claim 1 of 16 Claims

1. An isolated or purified nucleic acid molecule coding for a protein selected from the group consisting of oxidoreductase (Rv0068) (SEQ ID NO: 1) from M. tuberculosis, hypothetical protein (Rv3407) (SEQ ID NO: 2) from M. tuberculosis, and a fusion protein comprising said Rv0068 or said Rv3407 protein or a combination of Rv0068 and Rv3407.

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