# Patent

## Title
Identification of specific differentially expressed antigens

## 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.

## Background
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; Blüggel, 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; Blüggel (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 20th 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 1α-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, Köhler 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′)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 μg 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®” 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-sn-glycero-3-hydroxphaosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), MF59 and RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80® 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, β-galactosidase, alkaline phosphatase), radioactive isotopes (like 32P or 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.



The figures show:
FIG. 1: 2-DE gel of total cell protein of (A) M. bovis BCG, (B) M. tuberculosis H37Rv and (C) culture supernatant of H37Rv.
FIG. 2: 2-DE pattern of M. bovis BCG Chicago cell proteins in 6 sectors (2a-2f). Identified proteins are marked with accession numbers corresponding to the accession numbers in Table 1.
FIG. 3: 2-DE pattern of M. tuberculosis H37Rv culture supernatant in 6 sectors (3a-3f). Identified proteins are marked with accession numbers corresponding to the accession numbers in Table 1.
FIG. 4: Pattern sectors showing differences in intensity or position between cell proteins of different mycobacterial strains.

    a) Comparison between A, C, E, M. bovis BCG Chicago and B, D, F, M. tuberculosis H37Rv. C645 is a mobility variant of C527. Both spots were identified as succinyl-CoA synthase α- chain (Rv 0952). C126 and C125 are mobility variants, both identified as oxidoreductases of aldo/keto reductase family (Rv2971). C31 is increased in intensity in BCG Chicago as compared with C53 of H37Rv. This protein was identified as alkyl hydroperoxide reductase chain C(Rv2428). C71 is absent in BCG Chicago and was identified as MPT64 (Rv1980c).
    b) Comparison of A and C, M. tuberculosis H37Rv with B and D, Erdman. Proteins of the glutamate family are increased in intensity in the Erdman pattern: A511 and A195 and their corresponding spots in H37Rv A386 and B17 are acetylornithine amino transferases ArgD (Rv1655) and D20 is N-acetyl-glutamylphosphate reductase (Rv1652). Two spots in A and B are shifted to a more acidic position in the Erdman pattern. A473 and A267 were identified as transcriptional regulator MoxR(Rv1479). The region shown in C and D reveals 3 intensity differences: D59 was identified as Rv 3213c; D153 as Rv1996; and D10 as haloalkane dehalogenase Rv2296.


FIG. 5: Pattern areas showing +/− differences or mobility variants between cell proteins of different mycobacterial strains. A, M. bovis BCG Chicago; B, M. bovis BCG Copenhagen; C, M. tuberculosis H37Rv; D, M. tuberculosis Erdman. The spots indicated by arrows were only detected on the patterns of the virulent strains Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis Erdman.

    a) The proteins A186 of M. tuberculosis H37Rv and A312 of M. tuberculosis Erdman were identified as 2-isopropylmalate synthase (LeuA) expressed from the gene Rv3710.
    b) The proteins A264 of M. tuberculosis H37Rv and A226 of M. tuberculosis Erdman were identified as s-adenosylmethionine synthase (MetK) expressed from the gene Rv1392.
    c) The proteins C527 of M. tuberculosis H37Rv and C336 of M. tuberculosis Erdman were identified as succinyl CoA synthase alpha chain (SucD) expressed from the gene Rv0952.
    d) The proteins C125 of M. tuberculosis H37Rv and C143 of M. tuberculosis Erdman were identified as oxidoreductase of aldo/keto reductase family expressed from the gene Rv2971.
    e) The protein D92 of M. tuberculosis H37Rv was identified as oxireductase expressed from the gene Rv0068.
    f) The proteins A187 of M. tuberculosis H37Rv and A509 of M. tuberculosis Erdman were identified as elongation factor G (FusA2) expressed from the gene Rv0120c.
    g) The proteins C236 of M. tuberculosis H37Rv and C271 of M. tuberculosis Erdman were identified as uridylate kinase (PyrH) expressed from the gene Rv2883c.
    h) The proteins C608 of M. tuberculosis H37Rv and C523 of M. tuberculosis Erdman were identified as ABC-type transporter expressed from the gene Rv1463.
    i) The proteins C416 of M. tuberculosis H37Rv and C487 of M. tuberculosis Erdman were identified as short-chain dehydrogenase/reductase family expressed from the gene Rv1856c.
    j) The proteins C278 of M. tuberculosis H37Rv and C315 of M. tuberculosis Erdman were identified as 1,3,4,6-tetrachloro-1,4-cyclohexadiene hydrolase (LinB) expressed from the gene Rv2579.
    k) The proteins C407 (lower part) of M. tuberculosis H37Rv and C474 (lower part) of M. tuberculosis Erdman were identified as phosphoribosylaminoimidazole carboxylase catalytic subunit (PurE) expressed from the gene Rv3275c.
    l) The proteins C144 of M. tuberculosis H37Rv and C2 of M. tuberculosis Erdman were identified as hypothetical protein expressed from the gene Rv2557.
    m) The proteins F52 of M. tuberculosis H37Rv and F44 of M. tuberculosis Erdman were identified as hypothetical protein expressed from the gene Rv3407.
    n) The proteins A607 of M. tuberculosis H37Rv and A148 of M. tuberculosis Erdman were identified as hypothetical protein expressed from the gene Rv3881c.
    o) The proteins B69 of M. tuberculosis H37Rv and B54 of M. tuberculosis Erdman were identified as hypothetical protein expressed from the gene Rv2449c.
    p) The proteins C176 of M. tuberculosis H37Rv and C404 of M. tuberculosis Erdman were identified as hypothetical protein expressed from the gene Rv0036c.
    q) The proteins C434 of M. tuberculosis H37Rv and C508 of M. tuberculosis Erdman were identified as hypothetical protein expressed from the gene Rv2005c.
    r) The proteins D12 of M. tuberculosis H37Rv, D115 of M. tuberculosis H37Rv, D115 of M. tuberculosis Erdman and D130 of M. tuberculosis Erdman were identified as transcriptional regulator (Crp/Fnr family) expressed from the gene Rv3676.





The invention will now be illustrated by reference to the following examples which are merely illustrative and are not to be construed as being a limitation of the scope of the present invention.
EXAMPLE 1
Mycobacterial Strains and their Culturing Conditions
M. tuberculosis H37Rv and Erdman as well as M. bovis BCG Chicago and Copenhagen (M. tub. H37Rv and Erdman, BCG Chicago from: Stammsammlung MPI für Infektionsbiologie, Berlin, BCG Copenhagen from: Statensernen Instittutet, Kopenhagen) were grown in Middlebrook medium (900 ml Difco 0713-01-7+100 ml ADC enrichment 0714-64-0) for 6-8 days at 37° C.; to a cell density of 108 cells per ml. For the preparation of culture supernatant proteins (CSN), mycobacterial strains were grown in Sauton medium (per 4 l of Sauton medium enriched with pyruvic acid sodium salt glucose: 16.00 g asparagine, 2.00 g magnesiumsuiphate-heptahydrate p.A., 8.00 g citric acid monohydrate, 2.00 g dipotassium hydrogenphosphate, 0.20 g ferri-ammoniumcitrate, 19.28 g D(+)-glucose monohydrate, 19.28 g pyruvic acid sodium salt, 240 ml glycerol (86-88%)) under permanent shaking for 10 to 15 days at 37° C. or without shaking for 30 days at 37° C. until a cell density of 1−2×108 cells per ml was reached.
EXAMPLE 2
Protein Separation and Identification Strategy for Differentially Expressed Proteins (Proteome Analysis)
Proteome analysis of a biological entity depends on separation methods appropriate for the complexity of the system. Whereas proteomes of ribosomes containing about 50-100 protein species can be investigated by small 2-DE systems (Kaltschmidt (1970), Anal. Biochem. 36: 401) or high-performance liquid chromatography (Kamp (1984), J. Chromatogr. 317: 181), proteome analysis of bacterial and higher organisms requires high-resolution techniques. The combination of isoelectric focusing and SDS-PAGE, both per se high-resolution methods (Vesterberg(1966), Acta Chem. Scand. 20: 820; Laemmli (1970), Nature 227: 680), and the use of large-sized gels (at least 20 cm×30 cm) results in a resolution power of 5,000-10,000 protein species with sufficient quality to allow the comparison of gels between different laboratories (Jungblut (1994), Electrophoresis 15: 685; Klose (1995), Electrophoresis 16: 1034).
Two virulent strains of M. tuberculosis, H37Rv and Erdman, and two vaccine strains, M. bovis BCG Chicago and Copenhagen, were analyzed. In order to prepare a cell protein fraction (CP), mycobacteria were as described in Example 1. The cells were washed in PBS and sonicated in the presence of proteinase inhibitors (TLCK: 100 μg/ml, E64: 25 μg/ml, Leupeptin: 50 μg/ml, Pepstatin A: 50 μg/ml), and the proteins were treated with 9 M urea, 70 mM DTT 2% ampholytes pH 2-4 (Serva Biochemicals, Germany) and 2% Triton X-100 to obtain completely denatured and reduced proteins. Culture supernatant proteins (CSN) were prepared from mycobacterial cultures grown in Sauton medium as described in Example 1. CSNs were collected by filtration and precipitation in 10% trichloroacetic acid. Samples were prepared according to standard methods and applied onto 2-DE gel systems (Klose, (1995), loc. cit., Jungblut (1999), loc. cit.).
For the resolution of the mycobacterial proteome, a 2-DE gel system in a 23 cm×30 cm version was applied and a resolution power of about 5,000 protein species was achieved. For subtractive analyses (as described in Aebersold (1990), Electrophoresis 11: 517) and database construction, 0.75 mm thick gels in the second dimension were used and silver staining was applied on these gels (Jungblut (1990), J. Biochem. Biophys. Meth. 21: 47). In order to identify proteins 1.5 mm thick gels were produced and the proteins were detected by Coomassie Brilliant Blue R250 (Eckerskorn (1988), Electrophoresis 9: 830) or G250 (Doherty (1998), Electrophoresis 19: 355), or negative staining (Fernandez-Patron (1995), Anal. Biochem. 224: 203).
The 2-DE pattern of all strains investigated are highly similar and since many landmark spots are known, these patterns are easily comparable. Only obvious differences readily recognizable by visual evaluation were employed to detect protein species from different mycobacterial strains with regard to intensity or position. Each comparison was repeated at least three times with different sample preparations of the same strains. Only differences confirmed in all preparations were accepted as strain specific.
Identification of proteins separated by 2-DE has been reviewed (Patterson (1995), Electrophoresis 16: 1791; P. Jungblut (1996), Electrophoresis 17: 839; Jungblut (1997), Mass Spectrometry Reviews 16: 145) 2-DE combines isoelectric focusing in the first dimension with SDS-PAGE (Sodiumdodecyl sulfate polyacrylamide gel electrophoresis) in the second dimension. The proteins are separated by two independent parameters, charge and molecular mass. Single amino acid changes may be detected. The resolution power of the used technique (gel size 23 cm×30 cm) is about 5000 protein species, which should be sufficient for a microorganism with about 3700 genes like Mycobacterium tuberculosis or bovis. The term protein species is defined as the smallest unit of a protein classification, defined by its chemical structure. In-gel tryptic digestion (Otto (1996), Electrophoresis 17: 1643) and MALDI-MS peptide mass fingerprinting (Henzel (1993), Proc. Natl. Acad. Sci. U.S.A. 90: 5011; Pappin (1993), Current Biology 3: 327; Mann (1993), Biol. Mass Spectrom. 22: 338; James (1993), Biochem. Biophys. Res. Commun. 195: 58) with the possibility of sequencing by post-source decay MALDI-MS (Spengler (1992), Rapid Commun. Mass Spectrom. 6: 105) were chosen in order to identify the first 263 proteins, with a priority for high intensity proteins and for variants between the investigated mycobacteriai strains. Peptide mass fingerprints were searched using the program MS-FIT (prospector.ucsfedu/ucsfhtml/msfit.htm) reducing the proteins of the NCBI database to the mycobacteriai proteins and to a molecular mass range estimated from 2-DE+/−20%, allowing a mass accuracy of 0.1 Da for the peptide mass. In the absence of matches the MOLECULAR mass window was extended. Partial enzymatic cleavages leaving two cleavage sites, oxidation of methionine, pyro-glutamic acid formation at N-terminal glutamine and modification of cysteine by acrylamide were considered in these searches.
The employed 2-DE methodology led to a resolution of the mycobacterial proteome into 1,800 distinct protein species. The composition of cellular as well as culture filtrate proteins of two strains of M. tuberculosis and of M. bovis BCG was compared. Hereby, 263 proteins have already been identified, 157 and 53 in the cell protein (CP) fraction of M. bovis BCG Chicago and M. tuberculosis (H37Rv and Erdman), respectively, as well as 53 proteins from H37Rv culture filtrate (CSN). From the CP patterns 8 proteins were unique for BCG, and 13 for M. tuberculosis H37Rv. Identification was performed by peptide mass fingerprinting (PMF) using matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry and if necessary by confirmation with post-source decay (PSD) sequencing.
EXAMPLE 3
Format of Mycobacterial 2-DE Databases for Electronic Access
Data obtained as described in Example 2 and 8 are shown in FIGS. 1 to 5 and illustrated in Tables 1 to 4. Further information is available via internet (www.mpiib-berlin.mpg.de/2DPAGE/). The 2D-PAGE database complies with all rules according to the World 2D-PAGE guidelines for building a federated database (Appel (1996), Electrophoresis 17: 540). To navigate through the database, a Java compatible browser is required (e.g. Netscape 4.0 or internet Explorer 4.0). The program consists of common gateway interface (CGI) scripts written in PERL. One set of data comprises three files. The link between the image file, the map file and the rational data file is built by their names. The image file is a high density scan of the 2-D gel. The map file describes the location and the size of the spots as polygons. The rational data file is a document in Microsoft Access format that is connected to the WWW server by an Open Database Connectivity (ODBC) driver from MySQL. This connection ensures that after a single transfer of all data, no further maintenance and administration work is required. The rational data file is located on a micro-computer with IP address at any location of choice. The Hyper Text Markup Language (HTML) documents displayed via internet are dynamically generated on the basis of the available data for each individual session. Properties of proteins are presented in annotation spot windows. An example for such an annotation is: Spot ID: C191, Mr (2-DE): 27100, Mr (theoretical) 28160, pi (2-DE) 4.7, Identification Method PMF/PSD, Sequence coverage 35%, Protein name electron transfer flavoprotein beta subunit, short name fixA, Rv-No Rv3029c, EMBL: Z99263, NCBI: 2414529, Ident. No MLCB637, Gene No MLCB637.03. The EMBL and NCBI Nos have hyperlinks to obtain easily more information.
EXAMPLE 4
Analysis of the Mycobacterial Protein Composition by Detailed Proteome Analysis
Whole cell preparations of mycobacteria resulted in 2-DE patterns containing 1,500-2,000 distinct protein spots depending on silver-staining conditions and the amount of sample applied to the gels. Standard patterns of M. bovis BCG Chicago and M. tuberculosis H37Rv chosen for the construction of the mycobacterial 2-DE database are shown in FIGS. 1a and b. Molecular mass and isoelectric point calibrations were obtained by internal mycobacterial marker proteins identified during this approach. Some marker proteins for calibration are: Spot A540, tuf, Rv0685, pl 5.3, Mr 43594; Spot A543, acn, Rv1475c, pl 4.9, Mr 102500; Spot A10, tig, Rv2462c, pl 4.4, Mr 50616; Spot B5, probable fatty acid-acyl CoA reductase, Rv1543, pl 9.1, Mr 36821, Spot C342, nuoC, pl 5.4, Mr 26932; Spot E54, rplL, Rv0652, pl 4.6, Mr 13441; Spot F58, probable heat-shock protein, pl 6.8, Mr 10269. Both mycobacterial species comprise patterns with a high density of spots in the acidic range, whereas in the basic range, spot density is clearly reduced. The patterns of the 4 strains investigated are highly similar and can be compared easily. They were divided into 6 sectors to promote data handling for visual inspection and personal computer evaluation (FIG. 2).
Selected proteins from the 6 sectors were identified by peptide mass fingerprinting (Pappin, Curr. Biology 3 (1993), 327) using MALDI-MS. Selected proteins from the 6 sectors were identified by peptide mass fingerprinting using MALDI-MS. Starting with the procedure as described in Otto (Electrophoresis 17 (1996), 1643) sensitivity was improved during the course of identification of 270 protein species by minimization. Identification starting from 1 spot per protein species was successful. Gel spots were washed in 500 μl 100 mM Tris/HCl pH 8.5 in 50% acetonitrile for 20 min at 30° C. Further pH stabilization and reduction of the concentration of acetonitrile was obtained by a following equilibration in 500 μl 100 mM Tris/HCl pH 8.1 in 10% acetonitrile. The gel was now shrunken by evaporation in an Eppendorf concentrator 5301 (Eppendorf, Hamburg, Germany) to about 20% of the starting volume. Depending on the gel spot size 20 to 100 μl of a buffer containing 100 mM Tris/HCl pH 8.1, 1 mM CaCl2 in 10% acetonitrile together with 0.5 pg trypsin/100 μl buffer were added. Trypsination was performed overnight at 37° C. Enzymatic digestion was stopped by 2% TFA solution. A minimized peptide collecting device, reducing the amount of reversed phase material (Octadecyl-functionalized silica gel, Aldrich, Steinheim, Germany) to about one fifth (Otto, (1996) loc. cit.) was used to wash and concentrate the sample. The salt-free bound peptides were then eluted from the column by 50 μl 60% acetonitrile in 0.1% TFA. A further improvement in sensitivity was obtained by the use of 50 mM ammoniumbicarbonat pH 7.8 in 10% acetonitrile as the digestion buffer, a volatile buffer allowing to omit the peptide collecting device and therefore reducing drastically surface contacts and therefore loss of the peptides. A protein was accepted as identified if peptides covering at least 30% of the complete sequence were detected. An assignment with a sequence coverage below 30% was only accepted, if (i) at least the 3 main peaks of the mass spectrum matched with a database sequence, (ii) the number of low intensity peaks was clearly reduced and the mass of the uncleaved protein fitted within 20%, or (iii) PSD confirmed a proposed protein. In particular, the method is characterized by the capability of analyzing whole pathogenic organisms (like mycobacteria) and/or fractions thereof due to the possibility of identification of differentially expressed protein(s)/protein species by peptide mass fingerprinting without confirmation by a further method. Most proteins matched with 1 database entry with a clearly higher number of common peptides as compared to the second candidate. Only 3 spots in BCG contained 2 proteins: BCG Chicago spot C100 includes a protein homologous to a conserved hypothetical M. tuberculosis H37Rv protein, Rv3075c, and, in addition, the transcription antitermination protein NusG, Rv0639. BCG Chicago C241 contains a probable adenylate kinase, Rv0733, and a probable transposase, Rv1041c; and C600a thioredoxin reductase, Rv3913, and 3-hydroxyacyl-CoA dehydrogenase, Rv0468. In some cases peptides of neighboring spots were detected in reduced intensity in addition to the peptides of the main protein.
Starting from Coomassie Brilliant Blue R-250 or G-250 or in some cases negatively stained gels, 312 mycobacterial protein spots were analyzed. From these spots peptide mass fingerprinting identified 263 proteins. Starting with the identification of M. bovis BCG strain Chicago CP, 157 proteins were identified. From M. tuberculosis strains H37Rv and Erdman 53 and 12 proteins were identified by PMF (peptide mass fingerprinting), respectively. Additional sequence information confirmed the PMF assignments for 34 proteins. Because all PSD results confirmed the PMF assignments, it could be shown that 30% sequence coverage is sufficient for protein identification. PSD had to be used only if the sequence coverage was <30%. As determined by PMF, all 23 H37Rv spots had the same identity as their counterparts at the same position in the BCG pattern. Proteins were identified by comparison of the spot position of these two mycobacterial species. This resulted in a total of 162 identified proteins in BCG Chicago and a total of 626 identified proteins in CP of all strains.
Identified proteins of the mycobacterial species investigated were classified according to the M. tuberculosis H37Rv gene classification of Cole (1998; loc. cit.) and assigned to the corresponding Rv-numbers (Tab. 1). After identification of about 3% of all predicted gene products, starting with the most common proteins, species of many categories were found. However, only within two categories, i.e. protein translation/modification and chaperones/heat shock, more than 40% of the predicted gene products were identified in the obtained 2-DE patterns. To date expression of 30 conserved hypotheticals and 6 unknowns, not described previously at the protein level, was revealed.
In the CSN of M. tuberculosis H37Rv approximately 300 proteins were resolved by 2-DE (FIGS. 1c and 3). So far, 53 protein spots were identified within the CSN of M. tuberculosis H37Rv (Table 1). Similar to the CP patterns, CSN patterns were highly comparable. As compared with CP, CSN proteins occurred relative to the total number of spots in more spot series (FIG. 1c). Of the 164 identified proteins in CP, 20 gene products and of 53 from CSN 12 appeared as more than 1 spot in the 2-DE patterns, suggesting their existence as different protein species, probably due to posttranslational modification, such as phosphorylation, glycosylation or acylation. The higher portion of spot series in CSN could be additionally caused by the higher load per protein on the gel, by a higher degree of posttranslational modifications of secreted proteins, or by degradation of proteins outside of the bacterial cell. For instance, in CSN three adjacent series containing 8 spots were stained. Four of these spots were identified by PMF as elongation factor Tu (tuf), Rv0685. The 14 kDa antigen (Rv2031c) and the 10 kDa chaperonin (Rv3418c) appeared as 6 and 5 spots, respectively. An example from CP, steroid dehydrogenase of BCG Chicago corresponding to Rv0148, occurred in 6 spots randomly distributed within one sector of the 2-DE pattern.
EXAMPLE 5
Comparison of Protein Patterns from Different M. Tuberculosis and M. bovis BCG Strains
The genomes of the M. tuberculosis complex, comprising all 4 strains investigated, are highly conserved (Sreevatsan (1997), Proc. Natl. Acad. Sci. U.S.A. 94: 9869). The 2-DE patterns confirm the prediction that the vast majority of proteins have their counterparts in all strains investigated. However, clear differences in spot intensity, presence or absence, and position of the spots between these strains could be detected. Evaluation was concentrated on readily detectable spot variations, which were consistent in all obtained 2-DE patterns. The investigation was primarily aimed at the elucidation of proteins occurring exclusively in the virulent strains to detect potential virulence factors and candidate vaccine antigens (Table 2). Between BCG Chicago and H37Rv, 31 variants were detected. In comparison to BCG, H37Rv comprised 13 additional spots and lacked 8 spots; 9 spots were decreased in intensity and 1 spot was increased. Table 3 illustrates protein species which were either decreased or increased (1 spot) in intensity and denotes “intensity differences” between BCG Chicago and M. tuberculosis H37Rv. From the 31 variants, 25 were identified by PMF. Six identified proteins in H37Rv were without any counterpart in BCG: L-alanine dehydrogenase (40 kDa antigen, Rv 2780), isopropyl malate synthase (Rv 3710), nicotinate-nucleotide pyrophosphatase (Rv1596), MPT64 (Rv1980c), and 2 conserved hypotheticals (Rv2449c and Rv0036c). The absence of L-alanine dehydrogenase in BCG confirms a previous observation (Andersen (1992), Infect. Immun. 60: 2317) and shows that differentially expressed proteins can be detected by the methods described in the examples described herein. Eight of the +/−variants were shown to be mobility variants, possibly caused by amino acid exchanges or posttranslational modifications. Two obvious positional variations, 1 intensity and 1+/−variant are shown in FIG. 4a. Succinyl-CoA synthase alpha chain (Rv0952) shifted from a higher Mr variant in BCG to a lower one in H37Rv. An oxidoreductase of the aldo/keto reductase family (Rv2971) was shifted diagonally from a more basic, lower Mr form in BCG to a more acidic, higher Mr, form in H37Rv. Alkyl hydroxyperoxide reductase chain C(Rv2428) was decreased in H37Rv, and MPT64 (Rv1980c) occurred as an additional spot in H37Rv.
Comparison between M. tuberculosis Erdman and M. bovis BCG Chicago revealed 4 mobility variants, belonging to an oxidoreductase of the aldo/ketoreductase family described as Rv2971 in H37Rv, succinyl-CoA synthase α chain (Rv0952), S-adenosylmethionine synthase (Rv1392), and oxireductase (Rv0068).
Positional variants are interesting vaccine candidates, too, if the positional variation is caused by amino acid exchanges within the amino acid sequence relevant to T-cell recognition. Furthermore, if this is not the case, enzymes mediating a posttranslational modification are of interest for vaccine development or for diagnostic purposes.
Comparison of 2-DE patterns from M. tuberculosis H37Rv versus Erdman revealed 18 variant proteins, 16 of which were identified. In the M. tuberculosis Erdman proteome 6 protein species appeared to be increased in intensity; 2 protein species newly appeared; 6 were absent; and two represented mobility variants. Some examples are shown in FIG. 4b. Two spots of the acetylornithine aminotransferase ArgD (Rv1655) were present both in H37Rv and in Erdman, but both with clearly higher intensities in Erdman. The transcriptional regulator MoxR(Rv1479) was shifted to a more acidic position in the Erdman 2-DE pattern. The haloalkane dehalogenase (Rv2296), 2 spots containing L-alanine dehydrogenase (Rv2780), and protease IV (Rv0724) were absent from the Erdman proteome, whereas the unknown protein Rv3213c, sharing similarity with a Soj protein of possible relevance to chromosome segregation, and the conserved hypothetical protein Rv2641 were absent in the H37Rv proteome.
BCG Chicago and Copenhagen expressed highly similar 2-DE patterns. Only 3 obvious variants were identified. The conserved hypothetical protein Rv0968 was absent in the Copenhagen proteome, and 2 spots of a probable neuraminidase (Rv3463) were increased in intensity in the Chicago strain.
EXAMPLE 6
Classification of Identified Proteins
Of the 263 proteins identified by 2-DE in total CP and CSN of both M. tuberculosis H37Rv/Erdman and M. bovis BCG, about one third corresponded to housekeeping proteins involved in gene regulation, biosynthesis, degradation or metabolism. Amongst housekeeping proteins involved in transcription/translation, 4 polypeptides play a role in transcription control such as the RNA polymerase A (Rv3457c) and the transcription termination protein rho (Rv1297). Four proteins are ribosomal proteins such as the 50S L7/L12 (Rv0652), and 7 proteins are involved in protein translation and modification such as the elongation factors Tu (Rv0685) and Ts (Rv2889c) and the homolog to the transcription elongation factor greA of M. leprae (Rv1080). The EF-Tu was present in the CP as well as the CSN. This factor has been localized to the cell wall of M. leprae and is associated with the membrane and periplasmic space of other bacteria such as E. coli and Neisseria gonorrhoeae but its function remains uncertain (Marques (1998), Infect. Immun. 66: 2625; Jacobson (1976), Nature 261: 23; Porcella (1987), Microbiol. 142: 2481).
There are 2 two-component response regulators (Rv1626, Rv3133c) present in the proteome. One of these proteins, Rv1626, shows strong similarities to two-component systems of Methanobacterium thermoautotrophicum, Azetobacter vinelandii and Streptomyces coelicolor indicating the usage of environmental sensor and regulation systems by mycobacteria similar to other prokaryotes (Smith (1997), J. Bacteriol. 179: 7135; Gutierrez (1995), Mol. Microbiol. 18: 579; Brian (1996), J. Bacteriol. 178: 3221). In A. vinelandii, this protein is involved in negative regulation of the nitrite-nitrate reductase system. In S. coelicolor, a member of the Actinomycetaceae closely related to Mycobacteriaceae, it is a negative regulatory element in the synthesis of antibiotics. MoxR (Rv1479), which was apparently modified in H37Rv when compared to Erdman is a putative regulatory molecule probably involved in the formation of an active methanol dehydrogenase as shown for Paracoccus denitrificans (Van Spanning (1991), J. Bacteriol. 173: 6948). Similarly, the 40 kDa antigen (Rv2780), an alanine dehydrogenase, which is unique for M. tuberculosis and M. marinum (Andersen (1992), Infect. Immun. 60: 2317), was upregulated in H37Rv when compared to Erdman. It is unclear yet, whether this polypeptide is exclusively expressed in virulent mycobacteria. However, it could contribute to virulence because it has been implicated as part of the cell wall synthesis machinery since L-alanine is an important constituent of the peptidoglycan layer. Consistent with this notion, this protein is also present in the mycobacterial cell wall and even the outer-most capsule (Ortalo-Magne (1995), Microbiol. 141: 1609).
Twenty-five protein spots were identified as putative heat shock proteins including Hsp60 (groEL2; Rv0440), Hsp70 (dnaK; Rv0350), Hsp10 (groES; Rv3418), and ClpB (38; Rv0384c). Due to the high sequence homology between mycobacterial and human Hsp60 it has been suggested that this protein is involved in infection triggered autoimmune responses. DNA vaccination experiments also indicate that Hsp60 is a potential vaccine candidate (Tascon (1996), Nature Med. 888). A 14 kDa protein (hspX; Rv2031c) related to the heat shock protein alpha-crystalline, is a strong inducer of antibodies in patients with pulmonary tuberculosis (Verbon (1992), J. Bacteriol. 174: 1352). Interestingly, both M. bovis BCG and M. tuberculosis contain a putative rotamase (peptidyl-prolyl cis trans isomerase; Rv0009) homologous to cyclophilins, the specific receptors for the immunosuppressive drug cyclosporin A.
A number of proteins identified within the mycobacterial proteome are involved in biosynthesis/degradation of fatty acids and glycolipids which are essential components of the complex acid fast cell wall. Examples are the methoxy mycolic acid synthase 4 (Rv0642c), and the three molecular targets for the commonly used drugs against tuberculosis, isoniazid and ethambutol: The enoyl (ACP) reductase (Rv1484) and β-ketoacyl (ACP) synthase (Rv2246) are central to the biosynthesis of mycolic acids, and have recently been identified as targets for isoniazid (Mdluli (1998), Science 280: 1607; Rozwarski (1998), Science 279: 98; Sacchettini (1996), Res. Microbiol. 147: 36). The target for ethambutol, arabinosyl transferase (Rv0020c), participates in arabinogalactan synthesis and is specific for acid fast bacteria including mycobacteria (Lety (1997), Antimicrob. Agents Chemother. 41: 2629). Members of the antigen 85 complex (Rv1886c, Rv3803c, Rv3804c) are also part of the enzymatic cascade of the cell wall synthesis, i.e. mycolyl transferases, but apparently have also the potential to mediate mycobacterial binding to fibronectin (Belisle (1997), Science 276: 1420; Abou-Zeid (1988), Infect. Immun. 56: 3046). In addition, they are considered as vaccine candidates (Kaufmann and Andersen (1998), in “Chemical Immunology: Immunology of Intracellular Parasitism” (Ed. F. Y. Liew): 21-59).
Amongst the proteins identified within the mycobacterial proteome, several have been suggested as mycobacterial antigens of putative value for vaccine development and/or for diagnosis: These include the alanine dehydrogenase (Rv2780), Hsp60 (Rv0440), Hsp70 (Rv0350), members of the antigen 85 complex (Rv1886c, Rv3803c, Rv3804c), α crystalline (Rv2031) and the 35 kDa antigen (Rv2744c) (Kaufmann and Andersen (1998) loc. cit.; O′Connor (1990), Res. Microbiol. 141, 407). The mycobacteria specific 34 kDa protein, termed antigen 84 (Rv2145c), has been identified in M. kansasii, M. bovis BCG, M. leprae and M. tuberculosis and is recognized by antibodies in 60% of lepromatous leprosy patients (Hermans (1995), Infect. Immun. 63: 954). MPT64 (Rv1980c) and MPT51 (Rv3803c), a homolog to Antigen 85, are both CSN proteins and MPT64 is a known inducer of delayed type hypersensitivity responses in guinea pigs (Kaufmann and Andersen (1998) loc. cit.).
Although the acid fast cell wall and its enzymatic machinery contribute to mycobacterial survival in the host and resistance to host defense mechanisms, other factors must contribute to virulence of M. tuberculosis although they are far from being elucidated. As yet, only 5 potential virulence genes have been described: Catalase-peroxidase and superoxide dismutase which protect against reactive oxygen intermediates (ROI); noxR1 which confers resistance against reactive nitrogen intermediates (RNI); mce and sigA which encode macrophage-colonizing factor and sigma factor, respectively (Collins (1996), Trends Microbiol. 4: 426; Ehrt (1997), J. Exp. Med. 186: 1885; Arruda (1993), Science 261: 1454). In addition, the M. tuberculosis genome contains a homolog of smpB, a gene of Salmonella typhimurium involved in intracellular survival (Cole (1998) loc. cit.). Interestingly, none of these proteins were identified in this analysis. Furthermore, the genome sequence revealed several genes for lipases, phospholipases C, esterases and proteases potentially contributing to mycobacterial virulence (Cole (1998) loc. cit.). So far, only two alkyl hydroperoxide reductases (ahpC Rv2428, ahpD Rv2429) have been identified within the proteome.
Pathogenic mycobacteria survive intraphagosomally in host macrophages and interfere with phagosome maturation through mechanisms virtually unknown thus far (Russell (1997), Philos. Trans. R. Soc. Lond. B. Biol. Sci. 352: 1303). The HspX (α-crystalline; Rv2031c) has recently been shown to be important for intracellular survival of mycobacteria in macrophages (Harth (1994), Proc. Nat. Acad. Sci. U.S.A. 91: 9342; Clemens (1995), J. Bacteriol. 177: 5644). The urease and glutamine synthase of M. tuberculosis have been suggested to buffer the intraphagosomal pH and therefore block fusion with lysosomes (Sturgill-Koszycki (1996), EMBO J. 15: 6960; Schaible (1998), J. Immunol. 160: 1290). The mycobacterial phagosome represents an early endosomal compartment which intersects with the iron transport pathway (Dussurget (1998), Trends Microbiol. 6: 354; Gobin (1995), Proc. Nat. Acad. Sci. U.S.A. 92: 5189). There, proteins with high iron binding affinity such as exochelins, mycobactins and ferritin-like proteins (bfrA, bfrB) compete with the host cell iron handling system (Cole (1998) loc. cit.; Dussurget (1998) loc. cit.). Under conditions where iron is limited, these proteins have been detected by 2-DE (Dussurget (1998) loc. cit.).
In summary, of all proteins analyzed 39 polypeptides are conserved hypothetical proteins and 6 are unknown proteins using the information contained in the M. tuberculosis genome sequence. Furthermore, six identified proteins were detected in M. tuberculosis H37Rv, but could not be identified in M. bovis BCG. These proteins comprise: L-alanine dehydrogenase (40 kDa antigen, Rv 2780), isopropyl malate synthase (Rv 3710), nicotinate-nucleotide pyrophosphatase (Rv1596), MPT64 (Rv1980c), and 2 conserved hypotheticals (Rv2449c and Rv0036c).
EXAMPLE 7
Proteome Analysis Identifies Known Differences in Virulent and Avirulent Strains
As described herein above (see Example 5) two proteins could be identified which are expressed in M. tuberculosis H37Rv, but not in M. bovis BCG: L-alanine dehydrogenase (40 kDa antigen; Rv 2780) and MPT64 (Rv 1980c). The absence of alanine dehydrogenase in BCG has been described earlier (Andersen et al. Infect. Immun. 60, 2317 (1992)) and was confirmed by this approach. MPT64 (Rv1980c) is a CSN protein and is a known inducer of delayed type hypersensitivity responses in guinea pigs (S. H. K. Kaufmann and P. Andersen, in “Chemical Immunology: Immunology of Intracellular Parasitism” (Ed. F. Y. Liew), 1998: 21-59.). This protein was absent in the 2-DE patterns of BCG. This example illustrates the potential of the here described method for proteome analysis on strains of pathogenic organisms.
Furthermore, the example shows that differentially expressed proteins can be identified by this method.
EXAMPLE 8
Further Comparisons of Protein Patterns from Different M. tuberculosis and M. bovis BCG Strains
The 2-DE patterns of all four strains investigated (H37Rv, Erdman, Chicago and Copenhagen) are very conservative. The evaluation of 2-DE patterns comparing four strains of microorganism is difficult and time-consuming. In a second approach, therefore, the further analysis concentrated on +/− differences between the virulent strains as compared with the non-virulent strains. This investigation confirmed the results described in the examples described herein above. However, additional proteins Rv1511 (RD6), Rv1980c (RD2), Rv0222 (RD4), Rv1512 (RD6), Rv1978 (RD2), Rv2658c (RD13), Rv3875 (RD1), and Rv2074 (RD12) were found to be differentially expressed, confirming results from a comparison of the genome of M. tuberculosis with M. bovis by DNA Microarray (Science 284 (1999), 1520), where the loss of 16 regions (RD) in M. bovis BCG as compared to M. tuberculosis was described. Additionally, proteins occurring only in M. tuberculosis H37Rv and M. tuberculosis Erdman, but absent in Mycobacterium bovis BCG Chicago and Mycobacterium bovis BCG Copenhagen could be defined. These proteins COULD not be predicted by genomic investigations and comprised elongation factor G (Rv0120c), uridylate kinase (Rv2883c), ABC-type transporter (Rv1463), short chain dehydrogenase/reductase family protein (Rv 1856c), 1,3,4,6-tetrachloro-1,4,-cyclohexadiene hydrolase (Rv2579), phosphoribosylaminoimidazole carboxylase catalytic subunit (Rv3275c), hypothetical protein (Rv2557), and hypothetical protein (Rv3407). The sectors where these proteins occur in the virulent strains are shown in FIG. 5. The assignment of these protein species to their spot numbers and the link to the NCBI sequence database (www.ncbi.nlm.nih.gov/) by their accession No. are shown in Table 4.
Table 1: Proteins identified in 2-DE patterns of mycobacterial species. Proteins of M. tuberculosis H37Rv (H37Rv), Erdman (Erdman) and M. bovis BCG Chicago (Chic) and Copenhagen (Cop) were separated by 2-DE. The most intensive protein spots were identified by PMF using MALDI-mass spectrometry. The proteins were grouped according to the protein classification described in Cole et al. (Nature 393 (1998), 537), which is deduced from the E. coli gene classification of Riley (Microbiol. Rev. 57 (1993), 862). The numbers in brackets after each category refer to the total number of genes of this category (3). n.d., spot was not investigated; -, spot is absent; *, identified by MALDI-MS






















Spot
Chic
Cop
H37Rv
Erdman
Cop
H37Rv
NCBI
Protein
Short-
Rv-


No
CP
CP
CP
CP
CSN
CSN
AccNo.
name
name
name









I Small-molecule metabolism

A Degradation (163)

1. Carbon compounds (22)






















65
A382*
A171
A436
A243
n.d.
n.d.
1871608
Succinate-semialdehyde
gabD2
Rv0234c










dehydrogenase


222
B14*
B41+
B47
B30
n.d.
n.d.
1654033
Succinate semialdehyde
gabD1
Rv1731



B26*
B65





dehydrogenase









2. Amino acids and amines (18)






















134
A260*
A91
A301
A415
n.d.
n.d.
2911027
Methylmalonate
mmsA
Rv0753c










semialdehyde










dehydrogenase


117
—
—
A132*
—
n.d.
A134*
231985
L-Alanine dehydrogenase
ald
Rv2780


255






2624302
(40kD Antigen)


196
—
—
A481*
—
n.d.
A124*
231985
L-Alanine dehydrogenase
ald
Rv2780


254






2624302
(40kD Antigen)









3. Fatty acids (119)






















146
B119*
B28
B34
B3
n.d.
n.d.
1850115
Acyl CoA synthase;
fadD2
Rv0270










similar to LCFA_ECOLI










P29212 long-chain-fatty-










acid-CoA ligase


208a
C600*
C337
C523
C384
n.d.
n.d.
2909544
3-Hydroxyacyl-CoA
fadB2
Rv0468










dehydrogenase


39
C360*
C377
C385
C452
n.d.
n.d.
1877369
Enoyl-CoA hydratase
echA3
Rv0632c


111
C456*
C488
C576
C552
n.d.
n.d.
1706568
Enoyl-CoA hydratase/
echA6
Rv0905










isomerase superfamily


125
A432*
A155
A627*
A310
n.d.
n.d.
2896711
Acetyl-CoA C-
fadA3
Rv1074c


169







acetyltransferase


124
A424*
A569
A397
A237
n.d.
n.d.
1729939
Acetyl-CoA
fadA4
Rvl323










acetyltransferase


219
A124*
A498
A170
A412
n.d.
n.d.
2916977
Acyl-CoA dehydrogenase/
fadE16
Rv1679










hypothetical protein










MTV047.14


128
A547*
A487
A566
A635
n.d.
n.d.
1877329
Acyl-CoA dehydrogenase
fadE25
Rv3274c


73
A518*
A100
A182
A409
n.d
n.d.
2916919
β oxidation complex, α
fadB
Rv0860










subunit (multiple










activities)


74
A272*
A328
A319
A534
n.d
n.d.
2916919
β oxidation complex, α
fadB
Rv0860





A392




subunit (multiple










activities)









4. Phosphorous compounds (4)






















18
C654*
C528
C230
C171
n.d.
C42*
2105066
Probable inorganic
ppa
Rv3628


264







pyrophosphatase









B Energy Metabolism (292)

1. Glycolysis (12)






















49
C627*
C416
C59*
C615
n.d.
n.d.
2094844
Fructose bisphosphate
fba
Rv0363c


182







aldolase


206
A353*
A517
A626
A50
n.d.
n.d.
3122120
Glyceraldehyde 3-
gap
Rv1436










phosphate dehydrogenase


131
A218*
A278
A489
A636
n.d.
n.d.
2131060
Glyceraldehyde 3-
gap
Rv1436










phosphate dehydrogenase


223
C511*
C500
C67
C482
n.d.
n.d.
2131058
Triosephosphate isomerase
tpi
Rv1438









2. Pyruvate dehydrogenase (6)






















151
A305*
A255+
A243
A532
n.d.
n.d.
2909538
Probable dihydrolipoamide
—
Rv0462




A301





dehydrogenase


152
A549*
A468
A325
A45
n.d.
n.d.
2909538
Probable dihydrolipoamide
—
Rv0462










dehydrogenase









3. TCA cycle (19)






















118
—
C501
C527*
C336
n.d.
n.d.
1524210
Succinyl-CoA synthase
sucD
Rv0952










alpha chain


100
C597*
C402
C404
C473
n.d.
n.d.
1524210
Succinyl-CoA synthase
sucD
Rv0952










alpha chain


209
C645*
C501
—
—
n.d.
n.d.
1524210
Succinyl-CoA synthase
sucD
Rv0952


47







alpha chain


127
A542*
A516
A117*
A57
n.d.
n.d.
2896735
Fumarase
fum
Rv1098c


170


63
C333*
C604
A500
C142
n.d.
n.d.
2695826
Malate dehydrogenase
mdh
Rv1240


2


147
A106*
A176
—
—
n.d.
n.d.
2791409
Aconitate hydratase
acn
Rv1475c


148
A543*
A423
—
—
n.d.
n.d.
2791409
Aconitate hydratase
acn
Rv1475c









4. Glyoxylate bypass (5)






















136
A357*
A426
A406
A316
n.d.
n.d.
1483535
Malate synthase
glcB
Rv1873c









5. Pentose phosphate pathway (11)
6. Respiration (60)

    
    
        a. Aerobic (30)
    
    
























101
C342*
C361
C356
C416
n.d.
n.d.
1781221
NADH dehydrogenase
nuoC
Rv3147










chain c









    
    
        b. Anaerobic (15)
        c. Electron transport (15)
    
    
























7
C507*
C540
C598*
C222
n.d.
C40*
2791626
Electron transfer
fixB
Rv3028c


203







flavoprotein α subunit


280


281
n.d.
n.d
n.d.
n.d.
n.d.
C33*
2791626
Electron transfer
fixB
Rv3028c










flavoprotein α subunit


20
C191*
C113
C559*
C145
n.d.
n.d.
2414529
Electron transfer
fixA
Rv3029c


186







flavoprotein β subunit









7. Miscellaneous oxidoreductases and oxygenases (171)






















179
—
—
D92*
—
n.d.
n.d.
2808725
Probable oxidoreductase
—
Rv0068


305
D138
—
—
D100*
n.d.
n.d.
2808725
Probable oxidoreductase
—
Rv0068


22
C305*
C496
C577
C445
n.d.
n.d.
1877273
Steroid dehydrogenase
—
Rv0148


23
C321*
C340
C338
C394
n.d.
n.d.
1877273
Steroid dehydrogenase
—
Rv0148


23
C557*
C471
C339
C388
n.d.
n.d.
1877273
Steroid dehydrogenase
—
Rv0148


38
C379*
C388
C392
C465
n.d.
n.d.
1877273
Steroid dehydrogenase

Rv0148


38
C594*
C492
C394
C466
n.d.
n.d.
1877273
Steroid dehydrogenase
—
Rv0148


230
C653*
C308
C303
C353
n.d.
n.d.
1877273
Steroid dehydrogenase
—
Rv0148


96
C129*
D86
D81
D88
n.d.
n.d.
2695831
Putative dehydrogenase
—
Rv1245c


228
C350*
C359
C352
C410
n.d.
n.d.
2791388
Quinone oxidoreductase
qor
Rv1454c


105
D230*
D115
B2
B23
n.d.
n.d.
1694883
Putative oxidoreductase
—
Rv2951c


180
—
—
C125*
C143*
n.d.
n.d.
1694860
Oxidoreductase of
—
Rv2971


236







aldo/keto reductase family


210
C126*
C134
—
—
n.d.
n.d.
1694860
Oxidoreductase of
—
Rv2971










aldo/keto reductase family


129
A180*
C585
A490
A309
n.d.
n.d.
399009
NADP-dependent alcohol
adhC
Rv3045










dehydrogenase


51
C522*
C2
C41*
C310
n.d.
n.d.
2072661
Putative oxidoreductase
—
Rv3224


160


221
C274*
C286
C293
C334
n.d.
n.d
886104
Putative dehydrogenase
—
Rv3389c


95
C462*
C451
C482
C560
n.d.
n.d.
2104408
Short-chain alcohol
—
Rv3485c










dehydrogenase family









8. ATP-proton motive force (8)






















112
A6*
A247
A425
A116
n.d.
n.d.
1703652
ATP synthetase
atpA
Rv1308










alpha chain









C Central Intermediary Metabolism (45)

1. General (13)






















10
C500*
C578
A496
C235
n.d.
n.d.
1877280
Pyridine transhydrogenase
pntAA
Rv0155










subunit α1









2. Gluconeogenesis (2)






















153
A114*
A427
A48
A512
n.d.
n.d.
1871584
Phosphoenolpyruvate
pckA
Rv0211










carboxykinase









3. Sugar nucleotides (14)






















311
C314*
C331
C330
C381
n.d.
n.d.
2496483
Probable β-phosphoglucomutase/28.2 kD
—
Rv3400










protein CY78.28C









4. Amino sugars (1)
5. Sulphur metabolism (15)






















194
A220*
A228
—
—
n.d.
n.d.
2143298
Probable arylsulphatase
atsD
Rv0663









D Amino Acid Biosynthesis (95)

1. Glutamate family (19)






















155
D31*
D26
D28
D20*
n.d.
n.d.
3023331
N-acetyl-γ-glutamyl-
argC
Rv1652


304







phosphate reductase


227
C661*
C389
C393
C458*
n.d.
n.d.
1839006
Acetylglutamatekinase
argB
Rv1654


302


156
A344*
A395
B17*
A195*
n.d.
n.d.
1839007
Acetylornithine
argD
Rv1655


198






2829813
aminotransferase


235


226
A332*
A386
A386*
A511*
n.d.
n.d.
1839007
Acetylomithine
argD
Rv1655


197






2829813
aminotransferase


234









2. Aspartate family (21)






















215
C386*
C405
C409
C480
n.d.
n.d.
1729955
Homoserine synthase
thrC
Rv1295


81
A91*
A534
—
—
n.d.
n.d.
1542900
S-adenosylmethionine
metK
Rv1392










synthase


115
—
—
A264*
A226
n.d.
n.d.
1542900
S-adenosylmethionine
metK
Rv1392










synthase


225
C398*
C410
C417
C486
n.d.
n.d.
2498290
Dihydrodipicolinate
dapB
Rv2773c










reductase









3. Serine family (15)






















229
C539*
C287
C286
C332
n.d.
n.d.
2076692
Thiosulfate
cysA2
Rv0815c










sulfurtransferase


154
A193*
A241
A224
A328
n.d.
n.d.
2896714
Cystathionine β-synthase
cysM2
Rv1077


12
A545*
A391
B13
A520
n.d.
n.d.
2896730
Serine
glyA
Rv1093










hydroxymethyltransferase


13
B84*
B15
B60
A530
n.d.
n.d.
2896730
Serine
glyA
Rv1093










hydroxymethyltransferase









4. Aromatic amino acid family (15)
5. Histidine (11)
6. Pyruvate family (1)
7. Branched amino acid family (13)






















212
A51*
A542
A608
A141
n.d.
n.d.
2924446
Probable acetohydroxyacid
ilvX
Rv3509c










synthase I large subunit


174
—
—
A186*
A312
n.d.
n.d.
2960134
2-Isopropylmalate synthase
leuA
Rv3710









E Polyamine synthesis (1)

F Purines, Pyrimidines, Nucleosides and Nucleotides (60)

1. Purine ribonucleotide biosynthesis (20)






















94
C458*
C445
C472
C549
n.d.
n.d.
1870011
Ribose-phosphate-
prsA
Rv1017c










pyrophosphokinase


137
A186*
A237
A233
A320
n.d.
n.d.
1449391
GMP synthase
guaA
Rv3396c


138
A500*
A463
A247
A352
n.d.
n.d.
1449391
GMP synthase
guaA
Rv3396c









2. Pyrimidine ribonucleotide biosynthesis (9)
3. 2′-deoxyribonucleotide metabolism (12)
4. Salvage of nucleosides and nucleotides (10)
5. Miscellaneous nucleoside/nucleotide reactions (9)






















21a
C241*
C248
C254
C289
n.d.
C61*
2911007
Adenylate kinase
adk
Rv0733


265









G Biosynthesis of cofactors, prosthetic groups and carriers (117)

1. Biotin (8)
2. folic acid (11)
3. Lipoate (2)
4. Molybdopterin (20)
5. Panthotenate (4)
6. Pyridoxine (1)
7. Pyridine nucleotide (4)






















116
—
—
C266*
C298
n.d.
n.d.
2117241
Nicotinate-nucleotide
nadC
Rv1596










pyrophosphatase









8. Thiamine (4)
9. Riboflavine (8)
10.Thioredoxin, glutaredoxin and mycothiol (8)






















208b
C600*
C337
C523
C384
n.d.
n.d.
2808698
Thioredoxin reductase
trxB2
Rv3913


213
C584*
C564
—
C338
n.d.
n.d.
2808698
Thioredoxin reductase
trxB2
Rv3913


 80
E95*
E124
E82
E143
n.d.
n.d.
1729947
Thioredoxin
trxC
Rv3914









11. Menaquinone, PQQ, ubiquinone, and other terpenoids (15)
12. Heme and porphyrin (9)
13. Cobalamin (17)
14. Iron utilization (6)
H Lipid Biosynthesis (65)
1. Synthesis of fatty and mycolic acids (26)






















217
A476*
A387
A610*
A503
n.d.
n.d.
2909446
3-Oxoacyl-[ACP]
fabG4
Rv0242c


224







reductase


59
B116*
—
—
—
n.d.
n.d.
2909446
3-Oxoacyl-[ACP]
fabG4
Rv0242c










reductase


59
B46*
—
—
—
n.d.
n.d.
2909446
3-Oxoacyl-[ACP]
fabG4
Rv0242c










reductase


25
C414*
C429
C443
C515
n.d.
n.d.
1170564
Enoyl[ACP]reductase
inhA
Rv1484


107
D145*
D103
D100
D107
n.d.
n.d.
1155269
Enoyl[ACP]reductase
inhA
Rv1484


132
A222*
A485
A266
A371
n.d.
n.d.
1706747
β-ketoacyl-ACP synthase
kasB
Rv2246


141
A207*
A675
A199
A345
n.d.
n.d.
1877335
Acetyl/propionyl CoA
accD5
Rv3280










carboxylase β subunit









2. Modification of fatty and mycolic acids (14)






















214
C585*
C502
C50
C340
n.d.
n.d.
1575549
Methoxy mycolic acid
mmaA4
Rv0642c










synthase 4









3. Acyltransferases, mycoltransferases and phospholipid synthesis (25)






















104
B5*
D113
B14
B2
n.d.
n.d.
1723008
Probable fattyacid-acyl
—
Rv1543










CoA reductase


251
n.d.
n.d.
n.d.
n.d.
n.d.
C45*
393879
Antigen 85B precursor
fbpB
Rv1886c


37
C335*
C372
C363
C425
n.d.
C125*
804884
Antigen MPT51, mycolyl
fbpD
Rv3803c


267






2578420
transferase, MPB51










precursor


102
C540*
C319
C159*
C361
n.d.
n.d.
112764
Antigen 85A precursor
fbpA
Rv3804c


184


249
n.d.
n.d.
n.d.
n.d.
n.d.
C58*
112765
Antigen 85A precursor
fbpA
Rv3804c


250
n.d.
n.d.
n.d.
n.d.
n.d.
C14*
112765
Antigen 85A precursor
fbpA
Rv3804c









I Polyketide and Non-Ribosomal Peptide Synthesis (41)























98
D180*
D118
—
—
n.d.
n.d.
1403498
Probable ketoacyl
—
Rv1544










reductase









J Broad regulatory functions (187)

1. Repressors/activators (143)






















11
A8*
A726
A267*
—
n.d.
n.d.
2791413
Transcriptional regulator
moxR
Rv1479


199







MoxR homologue


232
—
—
—
A473*
n.d.
n.d.
2791413
Transcriptional regulator,
moxR
Rv1479










MoxR homologue


120
—
—
D12*
D115
n.d.
n.d.
2960100
Transcriptional regulator
—
Rv3676










(Crp/Fnr family)


121
D174
D111*
D115
D130
n.d.
n.d.
2960100
Transcriptional regulator
—
Rv3676










(Crp/Fnr family)









2. Two component systems (30)






















24
C561*
C227
C222
C266
n.d.
n.d.
2113910
Two-component response
—
Rv1626










regulator; similar also to










chemotaxis proteins


36
C659*
C415
C214
C493
n.d.
n.d.
1781234
Two-component response
—
Rv3133c










regulator









3. Serine-threonine protein kinases and phosphoprotein phosphatases (14)
II Macromolecule Metabolism
A Synthesis and Modification of Macromolecules (215)
1. Ribosomal protein synthesis and modification (58)






















70
F52*
F28
F45
F47
n.d.
F9*
1568592
30 S Ribosomal protein S6
rpsF
Rv0053


294






2829551


309
D131*
D154
D84
D93
n.d.
n.d.
1877389
50 S Ribosomal protein
rplJ
Rv0651










L10


28
E54*
E42
E42*
E77
n.d.
n.d.
585892
50 S Ribosomal protein
rplL
Rv0652


16







L7/L12


164


82
E173*
—
E138
E206
n.d.
n.d.
1806177
50 S Ribosomal protein
rpmC
Rv0709










L29









2. Ribosome modification and maturation (3)
3. Aminoacyl tRNA synthases and their modification (26)
4. Nucleoproteins (4)






















72
F95*
—
—
—
n.d.
n.d.
1857251
Integration host factor
mlHF
Rv1388









1542896









5. DNA replication, repair, recombination and restriction/modification (69)






















19
C272*
C277
C226
C318
n.d.
C6*
1568593
Single strand binding
ssb
Rv0054


256







protein









6. Protein translation and modification (15)






















34
D12*
D41
D39*
D35
n.d.
D7*
1552563
Peptidyl-prolyl cis-trans
ppiA
Rv0009


167






2829514
isomerase (rotamase)


237


238
n.d.
n.d.
n.d.
n.d.
n.d.
D5*
2829514
Peptidyl-prolyl cis-trans
ppiA
Rv0009










isomerase


239
n.d.
n.d.
n.d.
n.d.
n.d.
C112*
2829514
Peptidyl-prolyl cis-trans
ppiA
Rv0009










isomerase


299
n.d.
n.d.
n.d.
n.d.
n.d.
C119*
2829514
Peptidyl-prolyl cis-trans
ppiA
Rv0009










isomerase


135
A572*
A452
A349*
A339
n.d.
n.d.
3261535
Elongation factor G
fusA
Rv0684


172


173
A148
—
A187*
A509
n.d.
n.d.
2181962
Elongation factor G
fusA2
Rv0120c


3
A540*
A579
A587*
A601
n.d.
A106*
399422
Elongation Factor EF-Tu
tuf
Rv0685


159






1333784


268


269
n.d.
n.d.
n.d.
n.d.
n.d.
A91*
399422
Elongation Factor EF-Tu
tuf
Rv0685









1333784


279
n.d.
n.d.
n.d.
n.d.
n.d.
A9*
399422
Elongation Factor EF-Tu
tuf
Rv0685









1333784


296
n.d.
n.d.
n.d.
n.d.
n.d.
A82*
399422
Elongation Factor EF-Tu
tuf
Rv0685









1333784


33
E86*
E65
E61
E110
n.d.
n.d.
2896717
Transcription elongation
greA
Rv1080c










factor G


240
n.d.
n.d.
n.d.
n.d.
n.d.
C108*
1710712
Ribosome recycling factor
frr
Rv2882c


241
n.d.
n.d.
n.d.
n.d.
n.d.
C91*
1710712
Ribosome recycling factor
frr
Rv2882c


103
C309*
C329
C324*
C377
n.d.
C71*
1706595
Elongation factor Ts (EF-
tsf
Rv2889c


185







Ts)


263









7. RNA synthesis, RNA modification and DNA transciption (32)






















1b
C100*
C601
C199
C522
n.d.
n.d.
1877377
Transcription
nusG
Rv0639










antitermination protein


56
A235*
A298
A285
A527
n.d.
n.d.
1710260
Transcription termination
rho
Rv1297


144







factor Rho


57
A246*
A309
A297
A236
n.d.
n.d.
1710260
Transcription termination
rho
Rv1297


145







factor Rho


58
A259*
A317
A312
A238
n.d.
n.d.
1710260
Transcription termination
rho
Rv1297










factor Rho


4
A435*
A324
A616
A621
n.d.
n.d.
2104380
α Subunit of RNA
rpoA
Rv3457c










polymerase


61
A40*
A330
A615
A622
n.d.
n.d.
2104380
α Subunit of RNA
rpoA
Rv3457c










polymerase









8. Polysaccharides (8)
B Degradation of Macromolecules (87)
1. RNA (6)
2. DNA (3)
3. Proteins, peptides and glycopeptides (34)






















195
A268
A332
A320*
—
n.d.
n.d.
1806192
Protein IV, signal peptide
sppA
Rv0724










peptidase









4. Polysaccharides, lipopolysaccharides and phospholipids (8)






















97
D136*
D82
—
D96
n.d.
n.d.
2104386
Probable neuraminidase
—
Rv3463


191
D99*
D3
D64
D67
n.d.
n.d.
2104386
Probable neuraminidase
—
Rv3463


192
D122*
D82
D78
D84
n.d.
n.d.
2104386
Probable neuraminidase
—
Rv3463









5. Esterases and lipases (27)
6. Aromatic hydrocarbons (9)
C Cell Envelope (366)
1. Lipoproteins (65)
2. Surface polysaccharides (39)






















270
—
—
C71
C84*
n.d.
C24*
127271
Secreted immunogenic
mpt64
Rv1980c


566







protein MPB64/MPT64


9
C648*
C156
C221
C187
n.d.
n.d.
1168374
Antigen 84
wag31
Rv2145c









3. Murein sacculus and peptidoglucan (28)
4. Conserved membrane proteins (23)
5. Other membrane proteins (211)






















89
D46*
D116 +
D9
D7
n.d.
n.d.
1731190
possible exported protein
—
Rv0475




D156









III Cell processes

A Transport/Binding Proteins (124)

1. Amino acids (18)
2. Cations (31)
3. Carbohydrates, organic acids and alcohols (19)
4. Anions (34)
5. Fatty acid transport (2)
6. Efflux proteins (20)
B Chaperones/Heat shock (16)






















6
A16*
A132
A368*
A212
n.d.
n.d.
416908
70 kD Heat shock protein
dnaK
Rv0350


157






2094929


261
n.d.
n.d.
n.d.
n.d.
n.d.
A31*
2094829
70 kD Heat shock protein
dnaK
Rv0350


66
C69*
C526
C132
C4
n.d.
n.d.
417087
Stimulates DnaK ATPase
grpE
Rv0351









2094830
activity


8
A524*
A5
A600*
A158
n.d.
n.d.
2909505
Heat shock protein
clpB
Rv0384c


168


284
n.d.
n.d.
n.d.
n.d.
n.d.
A1*
2909505
ClpB heat shock protein
clpB
Rv0384c


284
n.d.
n.d.
n.d.
n.d.
n.d.
A69*
2909505
ClpB heat shock protein
clpB
Rv0384c


284
n.d.
n.d.
n.d.
n.d.
n.d.
A77*
2909505
ClpB heat shock protein
clpB
Rv0384c


5
A67*
A743
A431*
A556
n.d.
A4
116244
60 kD Chaperonin 2
groEL2
Rv0440


158







(Protein CPN60 2) (Groel


262







Protein 2) (65 kD Antigen)










(Heat shock protein 65)










(Cell wall protein A)










(Antigen A)


75
A452*
A712
A295
A22 +
n.d.
n.d.
116244
60 kD Chaperonin 2
groEL2
Rv0440






A72


123
A418*
A144
A613
A246
n.d.
n.d.
116244
60 kD Chaperonin 2
groEL2
Rv0440


292
n.d.
n.d.
n.d.
n.d.
n.d.
A24*
116244
60 kD Chaperonin 2
groEL2
Rv0440


27
E103*
E84
E166*
E148
n.d.
E18*
231343
14 kD Antigen
hspX
Rv2031c


15


166


275


271
n.d.
n.d.
n.d.
n.d.
n.d.
E54*
231343
14 kD Antigen (16 kD
hspX
Rv2031c










Antigen) (Hsp 16.3)


272
n.d.
n.d.
n.d.
n.d.
n.d.
E11*
231343
14 kD Antigen (16 kD
hspX
Rv2031c










Antigen) (Hsp 16.3)


273
n.d.
n.d.
n.d.
n.d.
n.d.
E53*
231343
14 kD Antigen (16 kD
hspX
Rv2031c










Antigen) (Hsp 16.3)


274
n.d.
n.d.
n.d
n.d.
n.d.
E38*
231343
14 kD Antigen (16 kD
hspX
Rv2031c










Antigen) (Hsp 16.3)


285
n.d.
n.d.
n.d.
n.d.
n.d.
E51*
231343
14 kD Antigen (16 kD
hspX
Rv2031c










Antigen) (Hsp 16.3)


69
F58*
F29
F47
F51
n.d.
n.d.
1877324
Probable heat shock
—
Rv3269










protein, similar to










YW26_MYCTU Q10865










hypothetical 10.5 kd










protein


64
A14*
A133
A432
A267
n.d.
n.d.
421608
Heat shock protein groEL
groEL1
Rv3417c









1449370
Gpn60-1/60 kD chaperonin










1


17
E14*
E44
E100*
E231
n.d.
n.d.
116198
10 kD Chaperonin (Protein
groES
Rv3418c


165







CPN10) (Protein GroES)










(immunogenic Protein










MPB57)


242
n.d.
n.d.
n.d.
n.d.
n.d.
E45*
116200
10 kD Chaperonin
groES
Rv3418c


243
n.d.
n.d.
n.d.
n.d.
n.d.
E44*
116200
10 kD Chaperonin
groES
Rv3418c


244
n.d.
n.d.
n.d.
n.d.
n.d.
E46*
116200
10 kD Chaperonin
groES
Rv3418c


245
n.d.
n.d.
n.d.
n.d.
n.d.
E42*
116200
10 kD Chaperonin
groES
Rv3418c


246
n.d.
n.d.
n.d.
n.d.
n.d.
E41*
116200
10 kD Chaperonin
groES
Rv3418c









C Cell division (19)























190
D92*
D158
—
D59*
n.d.
n.d.
2072672
Very similar to Soj protein
—
Rv3213c


231







possible role in










chromosome segregation









D Protein and peptide secretion (14)























52
A10*
A407
A92
A651
n.d.
n.d.
2791502
Putative chaperone protein
tig
Rv2462c









E Adaptions and atypical conditions (12)























68
E135*
E41 +
E101
E168 +
n.d.
E23*
2105046
Cold shock protein,
cspA
Rv3648c


277

E88

E174


2811046
transcriptional regulator









F Detoxification (22)























108
E32*
E21
C44
C36
n.d.
n.d.
2501346
Thiol peroxidase
tpx
Rv1932


43
C31*
C527
C53
C437
n.d.
n.d.
2127453
Alkyl hydroperoxide
ahpC
Rv2428










reductase chain C


42
D91*
D57
D54
—
n.d.
n.d.
2127455
Member of AhpC/TSA
ahpD
Rv2429










family









IV Other

A Virulence (38)

B IS elements, Repeated sequences, and Phage (135)

1. IS elements (90)
a IS6110 (32)
b IS1081 (6)
c others (52)






















21b
C241*
C248
C254
C289
n.d.
n.d.
1869987
Probable transposase
—
Rv1041c









2. REP13E12 family (10)
3. Phage-related functions (35)
C PE and PPE families (167)
1. PE family (99)

    
    
        a. PE subfamily (38)
        b. PE_PGRS subfamily (61)
    
    


2. PPE family (68)
D Antibiotic Production and Resistance (14)
E Bacteriocin-Like Proteins (3)
F Cytochrome P450 Enzymes (22)
G Coenzyme F420-dependent enzymes (3)






















189
C368*
C387
—
C459
n.d.
n.d.
1817673
Probable coenzyme
—
Rv0407










F420-










dependent










enzyme









H Miscellaneous transferases (61)























133
A302*
A2
A482
A462
n.d.
n.d.
2791398
Nifs-like protein
—
Rv1464


93
D28*
D23
D24
C590
n.d.
n.d.
2326746
o-Methyltransferase
—
Rv1703c









I Miscellaneous phosphatases, lyases, and hydrolases (18)























200
D98
D65
D10*
—
n.d.
n.d.
2494371
Haloalkane dehalogenase
—
Rv2296









J Cyclases (6)

K Chelatases (2)

V Conserved Hypotheticals (912)























54
A32
A62
A66*
A115
n.d.
A34*
1552575
MLB1770.15c
—
Rv0020c


301







similar to E235827/










hypothetical 38.4 kD










protein


53
A29*
A38
A56
—
n.d.
n.d.
1552575
MLB1770.15c
—
Rv0020c





A576




similar to E235827/










hypothetical 38.4 kD










protein


202
—
C178
C176*
C404
n.d.
n.d.
1552591
Hypothetical 27.6 kDa
—
Rv0036c










protein


126
A150*
A134
A151
A219
n.d.
n.d.
1568585
Similar to M. leprae
—
Rv0046c










G466956B1620_F3_113


60
D248*
D159
—
—
n.d.
n.d.
1871589
Hypothetical protein
—
Rv0216










MTCY08D5.11


293
n.d.
n.d.
n.d.
n.d.
n.d.
D15*
2909625
Hypothetical protein
—
Rv0566c










MTV039.04c


86
E122*
—
E137
E32
n.d.
E28*
2909628
Hypothetical protein
—
Rv0569


286







MTV039.07


84
F19*
F12
F18
F18
n.d.
n.d.
1524195
Similar to MTV007.08,
—
Rv0967










similar to G1001429/










hypothetical 18.9 kd










protein


67
F12*
—
—
F13
n.d.
n.d.
1524194
Conserved hypothetical
—
Rv0968


207
A202*
A254
A249
A350
n.d.
n.d.
2896736
Hypothetical protein
—
Rv1099c










Rv1099c; similar to










YWJI_BACSU (52.1%)


99
C376*
C589
C389
C463
n.d.
n.d.
1929075
Similar to M. leprae
—
Rv1201c










Q49948 U1756F


201
E152
E109
C376*
E192*
n.d.
n.d.
1722951
Hypothetical 18.2 kD
—
Rv1284


233







Protein CY373.03










similar to carboanhydrase


295
n.d.
n.d.
n.d.
n.d.
n.d.
F12*
1723000
Hypothetical 16.4 kD
—
Rv1558










protein CY48.07C


14
E160*
E114
E127
E199
n.d.
n.d.
2113920
Conserved hypothetical
—
Rv1636


260
n.d.
n.d.
n.d.
n.d.
n.d.
E22*
2113920
Conserved hypothetical
—
Rv1636


290
n.d.
n.d.
n.d.
n.d.
n.d.
E52*
2113920
Conserved hypothetical
—
Rv1636


205
C184*
C330
C181
C671
n.d.
n.d.
2924475
Similar to MTCY15F10.23
—
Rv1794


83
F3*
—
FS
E222
n.d.
n.d.
2225985
Hypothetical protein
—
Rv1875










MTCY180.43c


303
D13
D11
D59
D153*
n.d.
n.d.
1731252
Hypothetical 33.9 kD
—
Rv1996










Protein CY39.23C


183
C406
C422
C521*
C502
n.d.
n.d.
1731241
Conserved hypothetical
—
Rv2005c










30.9 kDa protein


30
E143*
E104
C336
E177
n.d.
n.d.
2104338
Similar to hypothetical
—
Rv2140c










17.1 kD E coli protein










YbhB


258
n.d.
n.d.
n.d.
n.d.
n.d.
C78*
2104338
Similar to hypothetical
—
Rv2140c










17.1 kD E. coli protein










YbhB


32
E82*
E143
E57
E108
n.d.
n.d.
2911105
Hypothetical protein
—
Rv2185c


176
—
B59
B69*
B54
n.d.
n.d.
2791489
Protein MTV008.05c
—
Rv2449c


50
C587*
C504
C243*
C456
n.d.
n.d.
2104288
Similar to YW12_MYCTU
—
Rv2623


181







Q10851 hypothetical 30.9










kD protein cy39.12


90
E127*
—
—
—
n.d.
n.d.
2104285
Conserved hypothetical
—
Rv2626c


91
E126*
—
—
—
n.d.
n.d.
2104285
Conserved hypothetical
—
Rv2626c


76
E158*
E181
E162
E195
n.d.
n.d.
2104285
Conserved hypothetical
—
Rv2626c


79
E1*
E74
E75
E156
n.d.
n.d.
2104285
Conserved hypothetical
—
Rv2626c


306
E50 +
E48 +
—
E84*
n.d.
n.d.
2829592
Hypothetical 16.0 kD
—
Rv2641



E65
E103





Protein CY441.11










YQCK_BACSU P45945


48
C171*
C424
C30*
C303
n.d.
n.d.
398959
35 kD Antigen
35kd_ag
Rv2744c


161


193
C226*
n.d.
C234
C267
n.d.
n.d.
1781160
Similar to hypothetical
—
Rv3054c










bacterial proteins/contains










aminotransferase class-II










pyridoxal-phosphate










attachment site










YieF_ECOLI (3e-20)










P31465


1a
C100*
n.d.
C199
C522
n.d.
n.d.
1781138
Conserved hypothetical;
—
Rv3075c


140







similar to citrate lyase β-










chains


310
B100*
B51
B10*
B12
n.d.
n.d.
2076700
Similar to C-terminal part
—
Rv3127


178







of hypothetical M.










tuberculosis protein










Y07J_MYCU Q11025/










similar to C-terminal part










MTCY02B10.19C


29
E156*
E111
C387
E193
n.d.
n.d.
1877314
Conserved hypothetical
—
Rv3555c


87
F5*
F3
F6
E116
n.d.
n.d.
2113924
Conserved-hypothetical
—
Rv3592


247
n.d.
n.d.
n.d.
n.d.
n.d.
F3*
2113924
Conserved hypothetical
—
Rv3592


297
n.d.
n.d.
n.d.
n.d.
n.d.
E50*
2960226
Hypothetical protein
—
Rv3874










MTV02709










similar to TR: 033084










(EMBL: Y14967)










MLCB628.13)


298
n.d.
n.d.
n.d.
n.d.
n.d.
E5*
2960226
Hypothetical protein
—
Rv3874










MTV027.09










similar to TR: 033084










(EMBL: Y14967)










MLCB628.13)









VI Unknowns (606)























41
F9*
F5
F9
E230
n.d.
n.d.
1877374
Unknown
—
Rv0636


92
C443*
C442
C466
C542
n.d.
n.d.
2896746
Unknown
—
Rv1109c


62
C497*
C84
A68
C123
n.d.
n.d.
1722975
some similarity to
—
Rv1324










thioredoxins


287
n.d.
n.d.
n.d.
n.d.
n.d.
E9*
1806236
Unknown
—
Rv1926c


85
E177*
E179
E144
E213
n.d.
n.d.
2104293
Unknown
—
Rv2619c


31
E120*
E157
C275
E157
n.d.
n.d.
1552871
Unknown; similar to
—
Rv3788










GREA_MYGLE p46808










transcription elongation










factor grea




















Total
3924
















Found on 2-DE patterns (Rv Nos)
150


Investigated spots
312


Identified spots by PMF
268


Identified spots by PMF and PSD
33


Identified spots by pattern comparison
267


Several spots of one gene in one strain
36


Cells
26


Supernatants
12


Common spots identified in H37Rv and Chicago by PMF
23


Identified spots in BCG Chicago PMF
152


Pattern comparison
10


Identified spots in BCG Copenhagen PMF
0


Pattern comparison
154


Identified spots in M.tub H37Rv PMF
41


Pattern comparison
113


Identified spots in Mtub Erdman PMF
12


Pattern comparison
144


Identified spots in M.tub H37 Rv CSN PMF
44








Pattern comparison
—








Several genes in one spot (e.g. 1a and 1b, 21, 208)
3


in prep, not identified:
44



























new

Name
Short-
Rv-



Spot
Spot
NCBI
Sanger + 
name
name


No
No
No
AccNo
NCBI
Sanger
Sanger





















26
C272(X9)
C579







(= D15) +
C580







C270








(= D14)







35
C247
C395
1781068
unknown; 





(X16)


similarityto 








hypothetical 








20.4 kDa 








protein




40
F22
F13







(X21)







55
A66
A385







(X36)







77 
E62
G2 







(X58)







78
E89
E102







(X59)







88
E15
E186







(X69)








(= F2)







109
F54
F63






110
D19
D18






113
Tub1







114
Tub2







119
Tub7

2072672
unknown, 








similarity to








Soj protein




122
Tub10







130
A333
C521






139
A95
C87






142
A413
A226






143
A47
A519






149 
A307
G4






150
A521
A297






162
Tub16







163
Tub17







171
Tub25







175
Tub29








(neu6)







177 
Tub31








(neul9)







188
A467
A257






204
Tub54







216
B41/B22 
A342







(Doppe
A132







Ispot)







218
C75
C155






220
C102
C641







Tub
F4







CSN








12







252
Tub
C51
398980
Antigen 85-C 





CSN

1877254
Precursor 85c





16







253
Tub
A117







CSN








17







257
Tub
C13







CSN








21







259 
Tub
C67







CSN








23







266
Tub
E32







CSN








30







276
Tub
E29







CSN








56







282
Tub
A51
2896711
beta-ketoacyl 





CSN


CoA thiolase





47







283
Tub
A30







CSN








48







288
Tub
C20







CSN








55







289
Tub
C44







CSN








59







291
Tub
E25







CSN








70







300
Tub
???







CSN 
nicht







62
vorha








nden






307
E28
E42






308
A184
C197
2909470
hypothetical 





(= C84)


protein 








MTV035 .09











Table 2: Protein variability between cell proteins (CP) of different strains. Four comparisons were performed: a, M. bovis BCG Chicago CP versus M. tuberculosis H37Rv CP; b, M. tuberculosis H37Rv CP versus Erdman CP; c, M. bovis BCG Chicago CP versus Copenhagen CP; and d, M. bovis Chicago CP versus M. tuberculosis Erdman CP. Each strain was prepared at least 3 times and at least gels of 3 independently prepared samples were compared. Some obvious differences were checked for reproducibility and only variations occurring reproducibly in all gels of one strain were accepted. From these 59 variant spots we identified 50 proteins. [↑] spot intensity increased; [↓] spot intensity decreased; [-] spot not detected on 2-DE pattern; MV mobility variant, spot position shifted, the following spot No corresponds to the shifted spot.
a) Comparison M. bovis BCG Chicago CP  M. tuberculosis H37Rv CP

















BCG








Chic
H37Rv
NCBI


No
CP
CP
AccNo
Name
Short-name
Rv-name







28
E54
E42
585892
50 S Ribosomal protein
rplL
Rv0652



[↑]
[↓]

L7/L12


42
D91
D54
2127455
Member of AhpC/TSA family
ahpD
Rv2429



[↑]
[↓]


43
C31
C53
2127453
Alkyl hydroperoxide reductase
ahpC
Rv2428



[↑]
[↓]

chain C


81
A91
[—]
1542900
S-adenosylmethionine synthase
metK
Rv1392




MV A264


115
[—]
A264
1542900
S-adenosylmethionine synthase
metK
Rv1392



MV A91


86
E122
E137
2909628
Hypothetical protein
—
Rv0569



[↑]
[↓]

MTV039.07


89
D46
D9
1731190
Hypothetical 21.5 kD protein
—
Rv0475



[↑]
[↓]

CY20G9.01 precursor; possible






exported protein


130
C521
A228

Not identified



[↑]
[↓]


188
A257
[—]

Not identified


189
C368
[—]
1817673
Probable coenzmyme
—
Rv0407






F420-dependent






enzyme


190
D92
[—]
2072672
Very similar to Soj protein,
—
Rv3213c






possible role in chromosome






segregation


191
D99
D64
2104386
Probable neuraminidase
—
Rv3463



[↑]
[↓]


192
D122
D78
2104386
Probable neuraminidase
—
Rv3463



[↑]
[↓]


193
C226
C234
1781160
Contains aminotransferase class
—
Rv3054c



[↑]
[↓]

II pyridoxal-phosphate






attachment site


194
A220
[—]
2143298
Probable arylsulphatase
atsD
Rv0663


209
C645
[—]
1524210
Succinyl-CoA synthase α chain
sucD
RV0952




MV C527


118
[—]
C527
1524210
Succinyl-CoA synthase α chain
sucD
Rv0952



MV C645


210
C126
[—]
1694860
Oxidoreductase of aldo/keto
—
Rv2971




MV C125

reductase family


180
[—]
C125
1694860
Oxidoreductase of ado/keto
—
Rv2971



MV C126


reductase family


113
[—]
A607

Not identified


114
[—]
A115

Not identified


116
[—]
C266
2117241
Nicotinate-nucleotide
nadC
Rv1596






pyrophosphatase


117
[—]
A132
231985
L-Alanine dehydrogenase (40 kD
ald
Rv2780






Antigen)


162
C155
C151

Not identified



[↓]
[↑]


174
[—]
A186
2960134
2-Isopropylmalate synthase
leuA
Rv3710


176
[—]
B69
2791489
Conserved hypothetical protein
—
Rv2449c






MTV008.05c


177
[—]
B3

Not identified


179
[—]
D92
2808725
Oxidoreductase
—
Rv0068



MV D138


423
D138
[—]
2808725
Oxidoreductase
—
Rv0068




MV D92


202
[—]
C176
1552591
Conserved hypothetical
—
Rv0036c






hypothetical 27.6 kDa protein


566
[—]
C71
127271
Immunogenic MPB64/MPT64
mpt64
Rv1980c






(antigen MPB64/MPT64)









b) Comparison M. tuberculosis H37Rv CP  M. tuberculosis Erdman CP


















H37Rv
Erdman
NCBI





No
CP
CP
AccNo
Name
Short-name
Rv-name







117
A132
[—]
231985
L-Alanine dehydrogenase (40 kD
ald
Rv2780






antigen)


119
D96
[—]

Not identified


122
E151
[—]

Not identified


179
D92
[—]
2808725
Oxidoreductase
—
Rv0068




MV D100


305
[—]
D100
2808725
Oxidoreductase
—
Rv0068



MV D92


195
A320
[—]
1806192
Protease IV, signal peptide
sppA
Rv0724






peptidase


196
A481
[—]
231985
L-Alanine dehydrogenase (40 kD
ald
Rv2780






Antigen)


234
A386
A511
2829813
Acetylomithine aminotransferase
argD
Rv1655



[↓]
[↑]

(ACOAT)


235
B17
A195
2829813
Acetylornithine aminotransferase
argD
Rv1655



[↓]
[↑]

(ACOAT)


199
A267
[—]
2791413
Transcriptional regulator, MoxR
moxR
Rv1479




MV A473

homologue


232
[—]
A473
2791413
Transcriptional regulator, MoxR
moxR
Rv1479



MV A267


homologue


200
D10
[—]
2494371
Haloalkane dehalogenase
—
Rv2296


233
C376
E192
1722951
Hypothetical 18.2 kD protein
—
Rv1284



[↓]
[↑]

CY373.03


231
[—]
D59
2072672
Similar to Soj protein possible
—
Rv3213c






role in chromosome segregation


302
C393
C458
1839006
Acetylglutamate kinase
argB
Rv1654



[↓]
[↑]


303
D59
D153
1731252
Hypothetical 33.9 kD protein
—
Rv1996



[↓]
[↑]

CY39.23C


304
D28
D20
3023331
N-acetyl-γ-glutamyl-phosphate
argC
Rv1652



[↓]
[↑]

reductase


306
[—]
E84
2829592
Hypothetical 16.0 kD protein
—
Rv2641






CY441.11









c) Comparison M. bovis BCG Chicago CP  M. bovis BCG Copenhagen CP


















Chicago
Copenhagen
NCBI





No
CP
CP
AccNo
Name
Short-name
Rv-name







67
F12
[—]
1524194
Conserved hypothetical
—
Rv0968


191
D99
D3
2104386
Probable neuraminidase
—
Rv3463



[↑]
[↓]


192
D122
D82
2104386
Probable neuraminidase
—
Rv3463



[↑]
[↓]









d) Comparison M. bovis BCG Chicago CP  M. tuberculosis Erdman CP


















Chicago
Erdman
NCBI





No
CP
CP
AccNo
Name
Short-name
Rv-name







11
A8
[—]
2791413
Transcriptional regulator MoxR
moxR
Rv1479




MV A473

homologue


232
[—]
A473
2791413
Transcriptional regulator, MoxR
moxR
Rv1479



MV A8


homologue


81
A91
[—]
1542900
S-adenosylmethionine synthase
metK
Rv1392




MV A226


209
C645
[—]
1524210
Succinyl-CoA synthase α chain
sucD
RV0952




MV C336


210
C126
[—]
1694860
Oxidoreductase of aldo/keto
—
Rv2971




MV C143

reductase family


236
[—]
C143
1694860
Oxidoreductase of aldo/keto
—
Rv2971



MV C126


reductase family









Table 3: Intensity Variants Identified on 2-DE Patterns of M. bovis BCG Chicago and M. tuberculosis H37Rv.



















BCG Chic
H37Rv
NCBI

Short-




No
CP
CP
AccNo
Name
name
Rv-name
Status







28
E54
E42
585892
50 S Ribosomal protein L7/L12
rplL
Rv0652
Compare Copenhagen-



[↑]
[↓]




H37Rv


42
D91
D54
2127455
Member of AhpC/TSA family
ahpD
Rv2429
Compare Copenhagen-



[↑]
[↓]




H37Rv


43
C31
C53
2127453
Alkyl hydroperoxide reductase
ahpC
Rv2428
Compare Copenhagen-



[↑]
[↓]

chain C


H37Rv


86
E122
E137
2909628
Hypothetical protein
—
Rv0569
Compare Copenhagen-



[↑]
[↓]

MTV039.07


H37Rv


89
D46
D9
1731190
Hypothetical 21.5 kD protein
—
Rv0475
Compare Copenhagen-



[↑]
[↓]

CY20G9.01 precursor;


H37Rv






possible exported protein


130
C521
A228

Not identified


Compare Copenhagen



[↑]
[↓]




H37Rv


191
D99
D64
2104386
Probable neuraminidase
—
Rv3463
Compare Copenhagen-



[↑]
[↓]




H37Rv


192
D122
D78
2104386
Probable neuraminidase
—
Rv3463
Compare Copenhagen-



[↑]
[↓]




H37Rv


193
C226
C234
1781160
Contains aminotransferase
—
Rv3054c
Compare Copenhagen-



[↑]
[↓]

class-II pyridoxal-phosphate


H37Rv






attachment site


162
C155
C151

Not identified


Compare Copenhagen-



[↑]
[↓]




H37Rv
















TABLE 4







Differentially expressed proteins between the virulent strains of M. tuberculosis and


M. bovis BCG (+/− variants):


Comparison M. bovis BCG Chicago CP ←→ M. tuberculosis H37Rv CP


←→ M. bovis BCG Copenhagen CP ←→ M. tuberculosis Erdman CP
















BCG
BCG









Chicago
Copenhagen
H37Rv
Erdman
NCBI

Short-


No
CP
CP
CP
CP
AccNo
Protein name
name
Rv-name





Xx2
—
—
A187
A509
2181962
Elongation factor G
FusA2
Rv0120c


Xx4
—
—
C236
C271
1709963
Uridylate kinase
PyrH
Rv2883c


Xx6
—
—
C608
C523
2791397
ABC-type transporter
—
Rv1463


Xx7
—
—
C416
C487
1781191
Short chain dehydro-
—
Rv1856c








genase/reductase family


Xx9
—
—
C278
C315
1478233
1,3,4,6-tetrachloro-1,4,-
LinB
Rv2579








cyclohexadiene








hydrolase


Xx10
—
—
C407
C474
2500011
Phosphoribosylamino-
PurE
Rv3275c





Lower
Lower

imidazole carboxylase





part
part

catalytic subunit


Xx11
—
—
C144
C2
2496493
Hypothetical protein
—
Rv2557


Xx13
—
—
F52
F44
2496475
Hypothetical protein
—
Rv3407


Xx1
—
—
A607
A148
2960233
Hypothetical protein
—
Rv3881c


Xx3
—
—
B69
B54
2791489
Hypothetical protein
—
Rv2449c


Xx5
—
—
C176
C404
1552591
Hypothetical protein
—
Rv9936c


Xx8
—
—
C434
C508
1731241
Hypothetical protein
—
Rv2005c


Xx12
—
—
D12
D115
2960100
Transcriptional
—
Rv3676



—
—
D115
D130

regulator(Crp/Fnr








family)

## 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.

2. A composition comprising at least one nucleic acid molecule of claim 1.

3. A composition comprising a nucleic acid molecule of claim 1, wherein said composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

4. The composition of claim 3, wherein said composition is a vaccine.

5. A composition comprising a nucleic acid molecule of claim 1, wherein said composition is a diagnostic composition further comprising suitable means for detection.

6. A method for the production of a vaccine against a virulent strain of the M. tuberculosis comprising the steps of:
(a) recombinantly expressing a differentially expressed 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, or a fusion protein comprising said protein, and
(b) combining said recombinantly expressed protein, or said fusion protein with a pharmaceutically acceptable carrier.

7. A method for the production of a vaccine against a virulent strain of M. tuberculosis comprising combining a vector comprising a nucleic acid molecule of claim 1 with a biologically acceptable carrier, wherein said nucleic acid molecule in said vector is placed under the control of an expression control sequence.

8. A method of preventing, ameliorating or treating tuberculosis induced by M. tuberculosis in a subject in need thereof comprising administering an effective amount of the vaccine of claim 4 to the subject.

9. A method of detecting the presence of M. tuberculosis in a sample, comprising contacting the composition of claim 5 with a sample suspected of containing M. tuberculosis, and detecting the presence of a polynucleotide 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 Rv0068, Rv3407, or a combination of Rv0068 and Rv3407 in the sample.

10. The method of claim 9, wherein said detection is indicative of tuberculosis.

11. An isolated or purified nucleic acid molecule coding for hypothetical protein (Rv3407) (SEQ ID NO: 2) from M. tuberculosis, or a fusion protein comprising said protein.

12. A composition comprising a nucleic acid molecule of claim 11, wherein said composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

13. The composition of claim 12, wherein said composition is a vaccine.

14. An isolated or purified nucleic acid molecule coding for oxidoreductase (Rv0068) (SEQ ID NO: 1) from M. tuberculosis, or a fusion protein comprising said protein.

15. A composition comprising a nucleic acid molecule of claim 14, wherein said composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

16. The composition of claim 15, wherein said composition is a vaccine.