EP0972045A1 - NUCLEIC ACID FRAGMENTS AND POLYPEPTIDE FRAGMENTS DERIVED FROM $i(M. TUBERCULOSIS) - Google Patents

NUCLEIC ACID FRAGMENTS AND POLYPEPTIDE FRAGMENTS DERIVED FROM $i(M. TUBERCULOSIS)

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Publication number
EP0972045A1
EP0972045A1 EP98913536A EP98913536A EP0972045A1 EP 0972045 A1 EP0972045 A1 EP 0972045A1 EP 98913536 A EP98913536 A EP 98913536A EP 98913536 A EP98913536 A EP 98913536A EP 0972045 A1 EP0972045 A1 EP 0972045A1
Authority
EP
European Patent Office
Prior art keywords
seq
polypeptide
sequence
ala
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98913536A
Other languages
German (de)
English (en)
French (fr)
Inventor
Peter Andersen
Rikke Skjot
Ida Rosenkrands
Karin Weldingh
Peter Birk Rasmussen
Thomas Oettinger
Walter Florio
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Statens Serum Institut SSI
Original Assignee
Statens Serum Institut SSI
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Filing date
Publication date
Application filed by Statens Serum Institut SSI filed Critical Statens Serum Institut SSI
Priority to EP04076605A priority Critical patent/EP1449922B1/en
Publication of EP0972045A1 publication Critical patent/EP0972045A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a number of immunologically active, novel polypeptide fragments derived from the Mycobac- terium tuberculosis, vaccines and other immunologic compositions containing the fragments as immunogenic components, and methods of production and use of the polypeptides .
  • the invention also relates to novel nucleic acid fragments derived from M. tuberculosis which are useful in the preparation of the polypeptide fragments of the invention or in the diagnosis of infection with M. tuberculosis .
  • the invention further relates to certain fusion polypeptides, notably fusions between ESAT-6 and MPT59.
  • TB Human tuberculosis caused by Mycobacterium tuberculosis is a severe global health problem responsible for approximately 3 million deaths annually, according to the WHO.
  • the worldwide incidence of new TB cases has been progressively falling for the last decade but the recent years has markedly changed this trend due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis .
  • BCG The only vaccine presently available for clinical use is BCG, a vaccine which efficacy remains a matter of controversy. BCG generally induces a high level of acquired resistance in animal models of TB, but several human trials in developing countries have failed to demonstrate significant protection. Notably, BCG is not approved by the FDA for use in the United States.
  • Immunity to AT. tuberculosis is characterized by three basic features; i) Living bacilli efficiently induces a protective immune response in contrast to killed preparations; ii) Specifically sensitized T lymphocytes mediate this protection; iii) The most important mediator molecule seems to be interferon gamma (INF-7) .
  • ST-CF Short term-culture filtrate
  • the novel antigens may also be important drug targets .
  • the present invention is i . a . based on the identification and characterization of a number of previously uncharacterized culture filtrate antigens from AT. tuberculosis .
  • T cells mediating immunity are focused predomi- nantly to antigens in the regions 6-12 and 17-30 kDa of ST- CF.
  • 8 antigens in the low molecular weight region (CFP7, CFP7A, CFP7B, CFP8A, CFP8B, CFP9 , CFP10A, and CFP11) and 18 antigens (CFP16, CFP17, CFP19, CFP19B, CFP20, CFP21, CFP22, CFP22A, CFP23, CFP23A, CFP23B, CFP25, CFP26, CFP27, CFP28, CFP29, CFP30A, and CFP30B) in the 17-30 kDa region have been identified.
  • CFP19A and CFP23 have been selected because they exhibit relatively high homologies with CFP21 and CFP25, respectively, in so far that a nucleotide homology sequence search in the Sanger Database (cf. below) with the genes encoding CFP21 and CFP25, (cfp25 and cfp21 respectively) , shows homology to two AT. tuberculosis DNA sequences, orfl9A and orf23.
  • the two sequences, orfl9a and orf23 encode to putative proteins CFP19A and CFP23 with the molecular weights of approx. 19 and 23 kDa respectively.
  • the identity, at amino acid level, to CFP21 and CFP25 is 46% and 50%, respectively, for both proteins.
  • CFP21 and CFP25 have been shown to be dominant T-cell antigens, and it is therefore believed that CFP19A and CFP23 are possible new T-cell antigens. Furthermore, a 50 kDa antigen (CFP50) has been isolated from culture filtrate and so has also an antigen (CWP32) isolated from the cell wall in the 30 kDa region.
  • CFP50 50 kDa antigen
  • CWP32 antigen isolated from the cell wall in the 30 kDa region.
  • the present invention is also based on the identification of a number of putative antigens from AT. tuberculosis which are not present in ATyccbacterium bovis BCG strains.
  • the nucleotide sequences encoding these putative antigens are: rdl -orf2 , rdl -orf3 . rdl -orf4 , rdl -orf5 , rdl -orf8 , rdl -orf9a . and rdl - orf 9b.
  • the invention is based on the surprising discovery that fusions between ESAT-6 and MPT59 are superior immunogens compared to the unfused proteins, respectively.
  • the encoding genes for 33 of the antigens have been determined, the distribution of a number of the antigens in vari- ous ycobacterial strains investigated and the biological activity of the products characterized.
  • the panel hold antigens with potential for vaccine purposes as well as for diagnostic purposes, since the antigens are all secreted by metabolizing mycobacteria.
  • T-cell epitopes are responsible for the elicitation of the acquired immunity against TB, whereas B-cell epitopes are without any significant influence on acquired immunity and recognition of mycobacte- ria in vivo . Since such T-cell epitopes are linear and are known to have a minimum length of 6 amino acid residues, the present invention is especially concerned with the identification and utilisation of such T-cell epitopes.
  • the invention relates to a substantially pure polypeptide fragment which
  • a) comprises an amino acid sequence selected from the sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, and any one of 168- 171,
  • b) comprises a subsequence of the polypeptide fragment defined in a) which has a length of at least 6 amino acid residues, said subsequence being immunologically equivalent to the polypeptide defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensiti- zation with antigens derived from mycobacteria belonging to the tuberculosis complex, or
  • c) comprises an amino acid sequence having a sequence identity with the polypeptide defined in a) or the subsequence defined in b) of at least 70% and at the same time being immunologically equivalent to the polypeptide defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensitization with antigens derived from mycobacteria belonging to the tuberculosis complex, with the proviso that
  • polypeptide fragment is in essentially pure form when consisting of the amino acid sequence 1-96 of SEQ ID NO: 2 or when consisting of the amino acid sequence 87-108 of SEQ ID NO: 4 fused to -galactosidase,
  • the degree of sequence identity in c) is at least 95% when the polypeptide comprises a homologue of a polypeptide which has the amino acid sequence SEQ ID NO: 12 or a subsequence thereof as defined in b) , and
  • the polypeptide fragment contains a threonine residue corresponding to position 213 in SEQ ID NO: 42 when comprising an amino acid sequence of at least 6 amino acids in SEQ ID NO: 42.
  • polypeptide fragment denotes both short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues) , and longer peptides (the usual interpretation of "polypeptide", i.e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, oligopeptide, or polypeptide which may be chemically modified by being glycosylated, by being lipi- dated, or by comprising prosthetic groups) .
  • polypeptides also comprises native forms of peptides/proteins in mycobacteria as well as recombinant proteins or peptides in any type of expression vectors transforming any kind of host, and also chemically synthesized peptides.
  • substantially pure polypeptide fragment means a polypeptide preparation which contains at most 5% by weight of other polypeptide material with which it is natively associated (lower percentages of other polypeptide material are preferred, e.g. at most 4%, at most 3%, at most 2%, at most 1%, and at most ⁇ _.%) . It is preferred that the substantially pure polypeptide is at least 96% pure, i.e.
  • polypeptide constitutes at least 96% by weight of total polypeptide material present in the pre- paration, and higher percentages are preferred, such as at least 97%, at least 98%, at least 99%, at least 99,25%, at least 99,5%, and at least 99,75%. It is especially preferred that the polypeptide fragment is in "essentially pure form", i.e. that the polypeptide fragment is essentially free of any other antigen with which it is natively associated, i.e. free of any other antigen from bacteria belonging to the tuberculosis complex.
  • sequence when used in connection with a polypeptide of the invention having a SEQ ID NO selected from 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, and any one of 168-171 denotes any continuous stretch of at least 6 amino acid residues taken from the AT.
  • a polypeptide from different sources such as other bacteria or even from eukaryotic cells.
  • polypeptide when formulated in a vaccine or a diagnostic agent (i.e. together with a pharmaceutically acceptable carrier or vehicle and optionally an adjuvant) , will
  • I) confer, upon administration (either alone or as an immunologically active constituent together with other antigens) , an acquired increased specific resistance in a mouse and/or in a guinea pig and/or in a primate such as a human being against infections with bacteria belonging to the tuberculosis complex which is at least 20% of the acquired increased resistance conferred by Mycobacterium bovis BCG and also at least 20% of the acquired increased resistance conferred by the parent polypeptide comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of 168-171 (said parent polypeptide having substantially the same relative location and pattern in a 2DE gel prepared as the 2DE gel shown in Fig.
  • the acquired increased resistance being assessed by the observed reduction in mycobacterial counts from spleen, lung or other organ homogenates isolated from the mouse or guinea pig receiving a challenge infection with a virulent strain of M. tuberculo sis, or, in a primate such as a human being, being assessed by determining the protection against development of clinical tuberculosis in a vaccinated group versus that observed in a control group receiving a placebo or BCG (preferably the increased resistance is higher and corresponds to at least 50% of the protec- tive immune response elicited by AT. jovis BCG, such as at least 60%, or even more preferred to at least 80% of the protective immune response elicited by AT.
  • Jbovis BCG such as at least 90%; in some cases it is expected that the increased resistance will supersede that conferred by M. bovis BCG, and hence it is preferred that the resistance will be at least 100%, such as at least 110% of said increased resistance) ; and/or
  • this diagnostically significant immune response can be in the form of a delayed type hypersensitivity reaction which can e.g. be determined by a skin test, or can be in the form of IFN-7 release determined e.g. by an IFN- ⁇ assay as described in detail below.
  • a diagnostically significant response in a skin test setup will be a reaction which gives rise to a skin reaction which is at least 5 mm in diameter and which is at least 65% (preferably at least 75% such as at the least 85%) of the skin reaction (assessed as the skin reaction diameter) elicited by the parent polypeptide comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of 168-171.
  • the ability of the polypeptide fragment to confer increased immunity may thus be assessed by measuring in an experimental animal, e.g. a mouse or a guinea pig, the reduction in myco- bacterial counts from the spleen, lung or other organ homoge- nates isolated from the experimental animal which have received a challenge infection with a virulent strain of mycobacteria belonging to the tuberculosis complex after previously having been immunized with the polypeptide, as compared to the mycobacterial counts in a control group of experimental animals infected with the same virulent strain, which experimental animals have not previously been immunized against tuberculosis.
  • an experimental animal e.g. a mouse or a guinea pig
  • the comparison of the mycobacterial counts may also be carried out with mycobacterial counts from a group of experimental animals receiving a challenge infection with the same virulent strain after having been immu- nized with ATycojbacteriuiT. Jbovis BCG.
  • the mycobacterial counts in homogenates from the experimental animals immunized with a polypeptide fragment according to the present invention must at the most be 5 times the counts in the mice or guinea pigs immunized with Mycobacterium bovis BCG, such as at the most 3 times the counts, and preferably at the most 2 times the counts.
  • a more relevant assessment of the ability of the polypeptide fragment of the invention to confer increased resistance is to compare the incidence of clinical tuberculosis in two groups of individuals (e.g. humans or other primates) where one group receives a vaccine as described herein which contains an antigen of the invention and the other group receives either a placebo or an other known TB vaccine (e.g. BCG) .
  • the antigen of the invention should give rise to a protective immunity which is significantly higher than the one provided by the administration of the placebo (as determined by statistical methods known to the skilled artisan) .
  • the "tuberculosis- complex” has its usual meaning, i.e. the complex of mycobacteria causing TB which are Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, and ATycobacterium africanum.
  • metabolic mycobacteria means live mycobacteria that are multiplying logarithmically and releasing polypeptides into the culture medium wherein they are cultured.
  • sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences or between two nucleotide sequences of equal length:
  • sequence identity can be calculated as zef crawl — > wherein
  • N dif is the total number of non- identical residues in the two sequences when aligned and wherein N ref is the number of residues in one of the sequences.
  • sequence identity is used here to illustrate the degree of identity between the amino acid sequence of a given polypeptide and the amino acid sequence shown in SEQ ID NO:
  • amino acid sequence to be compared with the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of 168-171 may be deduced from a DNA sequence, e.g. obtained by hybridization as defined below, or may be obtained by conventional amino acid sequencing methods.
  • the sequence identity is preferably determined on the amino acid sequence of a mature polypeptide, i.e. without taking any leader sequence into consideration.
  • polypeptides which are not identical to the polypeptides having SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of 168-171 are embraced by the present invention.
  • the invention allows for minor variations which do not have an adverse effect on immunogenicity compared to the parent sequences and which may give interesting and useful novel binding properties or biological functions and immunogenicities etc.
  • Each polypeptide fragment may thus be characterized by specific amino acid and nucleic acid sequences.
  • sequences include analogues and variants produced by recombinant methods wherein such nucleic acid and polypeptide sequences have been modified by substitution, insertion, addition and/or deletion of one or more nucleotides in said nucleic acid sequences to cause the substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide.
  • DNA is used in the following, it should be understood that for the number of purposes where DNA can be substituted with RNA, the term DNA should be read to include RNA embodiments which will be apparent for the man skilled in the art.
  • PNA may be used instead of DNA, as PNA has been shown to exhibit a very dynamic hybridization profile (PNA is described in Nielsen P E et al., 1991, Science 254: 1497-1500) .
  • antigens from seg- ments of a known immunogenic protein or polypeptide.
  • Certain epitopic regions may be used to produce responses similar to those produced by the entire antigenic polypeptide.
  • Potential antigenic or immunogenic regions may be identified by any of a number of approaches, e.g., Jameson-Wolf or Kyte-Doolittle antigenicity analyses or Hopp and Woods (1981) hydrophobicity analysis (see, e.g., Jameson and Wolf, 1988; Kyte and Doo- little, 1982; or U.S. Patent No. 4,554,101).
  • Hydrophobicity analysis assigns average hydrophilicity values to each amino acid residue from these values average hydrophilicities can be calculated and regions of greatest hydrophilicity determined. Using one or more of these methods, regions of predicted antigenicity may be derived from the amino acid sequence assigned to the polypeptides of the invention.
  • T-cell epitopes are linear, deletion mutants of polypeptides having SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of 168-171 will, if constructed systematically, reveal what regions of the polypeptides are essential in immune recognition, e.g.
  • Another method utilises overlapping oligomers (preferably synthetic having a length of e.g. 20 amino acid residues) derived from polypeptides having SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of 168-171. Some of these will give a positive response in the IFN-7 assay whereas others will not.
  • the polypeptide fragment of the invention comprises an epitope for a T-helper cell .
  • the minimum length of a T-cell epitope has been shown to be at least 6 amino acids, it is normal that such epitopes are constituted of longer stretches of amino acids.
  • the polypeptide fragment of the invention has a length of at least 7 amino acid residues, such as at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, and at least 30 amino acid residues.
  • polypeptides of the invention are natively translation products which include a leader sequence (or other short peptide sequences) , whereas the product which can be isolated from short-term culture filtrates from bacteria belonging to the tuberculosis complex are free of these sequences.
  • a leader sequence or other short peptide sequences
  • the product which can be isolated from short-term culture filtrates from bacteria belonging to the tuberculosis complex are free of these sequences.
  • a preferred embodiment of the invention is a polypeptide which is free from amino acid residues -30 to -1 in SEQ ID NO: 6 and/or -32 to -1 in SEQ ID NO: 10 and/or -8 to -1 in SEQ ID NO: 12 and/or -32 to -1 in SEQ ID NO: 14 and/or -33 to -1 in SEQ ID NO: 42 and/or -38 to -1 in SEQ ID NO: 52 and/or -33 to -1 in SEQ ID NO: 56 and/or -56 to -1 in SEQ ID NO: 58 and/or -28 to -1 in SEQ ID NO: 151.
  • the polypeptide fragment of the invention is free from any signal sequence; this is especially interesting when the polypeptide fragment is produced synthetically but even when the polypeptide fragments are produced recombinantly it is normally acceptable that they are not exported by the host cell to the periplasm or the extracellular space; the polypeptide fragments can be recovered by traditional methods (cf. the discussion below) from the cytoplasm after disruption of the host cells, and if there is need for refolding of the polypeptide fragments, general refolding schemes can be employed, cf. e.g. the disclosure in WO 94/18227 where such a general applicable refolding method is described.
  • a suitable assay for the potential utility of a given polypeptide fragment derived from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of 168-171 is to assess the ability of the polypeptide fragment to effect IFN- 7 release from primed memory -lymphocytes .
  • Polypeptide fragments which have this capability are according to the invention especially interesting embodiments of the invention: It is contemplated that polypeptide fragments which stimulate T lymphocyte immune response shortly after the onset of the infection are important in the control of the mycobacteria causing the infection before the mycobacteria have succeeded in multiplying up to the number of bacteria that would have resulted in fulminant infection.
  • an important embodiment of the invention is a polypeptide fragment defined above which
  • IFN-7 induces a release of IFN-7 of at least 1,500 pg/ml above background level from about 1,000,000 human PBMC (peripheral blood mononuclear cells) per ml isolated from TB patients in the first phase of infection, or from healthy BCG vaccinated donors, or from healthy contacts to TB patients, the induction being performed by the addition of the polypeptide to a suspension comprising the about 1,000,000 PBMC per ml, the addition of the polypeptide resulting in a concentration of 1-4 ⁇ g polypeptide per ml suspension, the release of IFN-7 being assessable by determination of IFN-7 in supernatant harvested 2 days after the addition of the polypeptide to the suspension; and/or
  • the release effected by the polypeptide fragment gives rise to at least 1,500 pg/ml IFN-7 in the supernatant but higher concentrations are preferred, e.g. at least 2,000 pg/ml and even at least 3,000 pg/ml IFN-7 in the supernatant.
  • the IFN-7 release from bovine PBMC can e.g. be measured as the optical density (OD) index over background in a standard cytokine ELISA and should thus be at least two, but higher numbers such as at least 3, 5, 8, and 10 are preferred.
  • polypeptide fragments of the invention preferably com- prises an amino acid sequence of at least 6 amino acid residues in length which has a higher sequence identity than 70 percent with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one Of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of 168-171.
  • a preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.
  • the invention also pertains to a fusion polypeptide comprising at least one LO LO t t H H
  • first and second T-cell epitopes each have a sequence identity of at least 70% with the natively occurring sequence in the proteins from which they are derived and it is even further preferred that the first and/or second amino acid sequence has a sequence identity of at least 70% with the protein from which they are derived.
  • a most preferred embodiment of this fusion polypeptide is one wherein the first amino acid sequence is the amino acid sequence of ESAT-6 or MPT59 and/or the second amino acid sequence is the full-length amino acid sequence of the possible "parent" polypeptides listed above.
  • the fusion polypeptide fragment comprises ESAT-6 fused to MPT59 (advantageously, ESAT-6 is fused to the C- terminus of MPT59) and in one special embodiment, there are no linkers introduced between the two amino acid sequences constituting the two parent polypeptide fragments.
  • Another part of the invention pertains to a nucleic acid fragment in isolated form which
  • nucleic acid sequence which encodes a polypeptide or fusion polypeptide as defined above, or comprises a nucleic acid sequence complementary there- to, and/or
  • SEQ ID NO: 3 or a sequence complementary thereto
  • SEQ ID NO: 5 or a sequence complementary thereto
  • SEQ ID NO: 13 or a sequence complementary thereto
  • SEQ ID NO: 15 or a sequence complementary thereto
  • SEQ ID NO: 140 or a sequence complementary thereto SEQ ID NO: 142 or a sequence complementary thereto,
  • SEQ ID NO: 150 or a sequence complementary thereto
  • SEQ ID NO: 152 or a sequence complementary thereto
  • nucleic acid fragment when the nucleic acid fragment comprises a subsequence of SEQ ID NO: 41, then the nucleic acid fragment contains an A corresponding to position 781 in SEQ ID NO: 41 and when the nucleic acid fragment comprises a subsequence of a nucleotide sequence exactly complementary to SEQ ID NO: 41, then the nucleic acid fragment comprises a T corresponding to position 781 in SEQ ID NO: 41.
  • the nucleic acid fragment is a DNA fragment .
  • the preferred nucleic acid sequence when employed for hybridization studies or assays includes sequences that are complementary to at least a 10 to 40, or so, nucleotide stretch of the selected sequence.
  • a size of at least 10 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective.
  • Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • sequence when used in connection with the nucleic acid fragments of the invention is intended to indicate a continuous stretch of at least 10 nucleotides exhibits the above hybridization pattern. Normally this will require a minimum sequence identity of at least 70% with a subsequence of the hybridization partner having SEQ ID NO: 1, 3, 5, 7, 9, 11, 12, 15, 21, 41, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 87, 89, 91, 93, 140, 142, 144, 146, 148, 150, or 152.
  • the nucleic acid fragment is longer than 10 nucleotides, such as at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, and at least 80 nucleotides long, and the sequence identity should preferable also be higher than 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, and at least 98%. It is most preferred that the sequence identity is 100%.
  • Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Patent 4,603,102, or by introducing selected sequences into recombinant vectors for recombinant production.
  • nucleic acid fragment of the invention may be exchanged by others which, when expressed, result in a polypeptide identical or substantially identical to the polypeptide encoded by the nucleic acid fragment in question.
  • the invention thus allows for variations in the sequence such as substitution, insertion (including introns) , addition, deletion and rearrangement of one or more nucleotides, which variations do not have any substantial effect on the poly- peptide encoded by the nucleic acid fragment or a subsequence thereof.
  • substitution is intended to mean the replacement of one or more nucleotides in the full nucleotide sequence with one or more different nucleotides
  • addition is understood to mean the addition of one or more nucleotides at either end of the full nucleotide sequence
  • insertion is intended to mean the introduction of one or more nucleotides within the full nucleotide sequence
  • deletion is intended to indicate that one or more nucleotides have been deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it
  • “rearrangement” is intended to mean that two or more nucleotide residues have been exchanged with each other.
  • the nucleotide sequence to be modified may be of cDNA or genomic origin as discussed above, but may also be of syn- thetic origin. Furthermore, the sequence may be of mixed cDNA and genomic, mixed cDNA and synthetic or genomic and syn- LO t to F> H o L ⁇ o L ⁇ O L ⁇ tr Hi TJ CQ P CO Lq CD ⁇ PJ TJ P P. tr F- P > rt CQ PJ hi Hi rt ⁇ PJ o ft P ft TJ P. 3 rt
  • CD tr 3 LQ P.
  • the invention also relates to a replicable expression vector which comprises a nucleic acid fragment defined above, espe- cially a vector which comprises a nucleic acid fragment encoding a polypeptide fragment of the invention.
  • the vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachro o- somal entity, the replication of which is independent of chromosomal replication; examples of such a vector are a plasmid, phage, cosmid, mini-chromosome or virus.
  • the vector may be one which, when introduced in a host cell, is integrated in the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
  • Expression vectors may be constructed to include any of the DNA segments disclosed herein. Such DNA might encode an antigenic protein specific for virulent strains of mycobacteria or even hybridization probes for detecting mycobacteria nucleic acids in samples . Longer or shorter DNA segments could be used, depending on the antigenic protein desired. Epitopic regions of the proteins expressed or encoded by the disclosed DNA could be included as relatively short segments of DNA.
  • a wide variety of expression vectors is possible including, for example, DNA segments encoding reporter gene products useful for identification of heterologous gene products and/or resistance genes such as antibiotic resistance genes which may be useful in identifying transformed cells.
  • the vector of the invention may be used to transform cells so as to allow propagation of the nucleic acid fragments of the invention or so as to allow expression of the polypeptide fragments of the invention.
  • the invention also pertains to a transformed cell harbouring at least one such vector according to the invention, said cell being one which does not natively harbour the vector and/or the nucleic acid fragment of the invention contained therein.
  • a transformed cell (which is also a part of the invention) may be any suitable bacterial host cell or any other type of cell such as a unicellular eukaryotic organism, a fungus or yeast, or a cell derived from a multicellular organism, e.g. an animal or a plant.
  • a mammalian cell is used, although glycosylation of proteins is a rare event in proka- ryotes.
  • a prokaryotic cell is preferred such as a bacterium belonging to the genera ATycojbacterium, Salmonella, Pseudomonas, Bacillus and Eschericia .
  • the transformed cell is an E. coli , B . subti - lis, or AT. bovis BCG cell, and it is especially preferred that the transformed cell expresses a polypeptide according of the invention. The latter opens for the possibility to produce the polypeptide of the invention by simply recovering it from the culture containing the transformed cell .
  • the transformed cell is AfycoJbacterium jbovis BCG strain: Danish 1331, which is the Mycobacterium bovis strain Copenhagen from the Copenhagen BCG Laboratory, Statens Seruminstitut , Denmark.
  • nucleic acid fragments of the invention allow for the recombinant production of the polypeptides fragments of the invention. However, also isolation from the natural source is a way of providing the polypeptide fragments as is peptide synthesis.
  • the invention also pertains to a method for the preparation of a polypeptide fragment of the invention, said method comprising inserting a nucleic acid fragment as defined above into a vector which is able to replicate in a host cell, introducing the resulting recombinant vector into the host cell (transformed cells may be selected using various techniques, including screening by differential hybridization, identification of fused reporter gene products, resistance markers, anti-antigen antibodies and the like) , culturing the host cell in a culture medium under conditions sufficient to effect expression of the polypeptide (of course the cell may be cultivated under conditions appropriate to the circumstances, and if DNA is desired, replication condi- tions are used) , and recovering the polypeptide from the host cell or culture medium; or
  • polypeptide from whole mycobacteria of the tuberculosis complex or from lysates or fractions thereof, e.g. cell wall containing fractions, or
  • the medium used to grow the transformed cells may be any conventional medium suitable for the purpose.
  • a suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above.
  • the methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purposes within the field of recombinant DNA. In the following a more detailed description of the possibilities will be given:
  • prokaryotes are preferred for the initial cloning of nucleic sequences of the invention and constructing the vectors useful in the invention.
  • strains such as E. coli K12 strain 294 (ATCC No. 31446) , E. coli B, and E. coli X 1776 (ATCC No. 31537) .
  • E. coli K12 strain 294 ATCC No. 31446
  • E. coli B E. coli B
  • E. coli X 1776 ATCC No. 31557
  • Prokaryotes are also preferred for expression.
  • the aforementioned strains, as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may be used.
  • rapid-growing mycobacteria e.g. AT. smegmatis, as these bacteria have a high degree of resemblance with mycobacteria of the tuberculosis complex and therefore stand a good chance of reducing the need of performing post-translational modifications of the expression product.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al . , 1977, Gene 2: 95).
  • the pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microorganism for expression.
  • promoters most commonly used in recombinant DNA con- struction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al . , 1978; Itakura et al . , 1977; Goeddel et al., 1979) and a tryptophan (trp) promoter system (Goeddel et al . , 1979; EPO Appl . Publ . No. 0036776).
  • the isolation of the polypeptide may for instance be carried out by affinity chromatography (or other conventional biochemical procedures based on chromatography) , using a monoclonal antibody which substantially specifically binds the polypeptide according to the invention.
  • affinity chromatography or other conventional biochemical procedures based on chromatography
  • monoclonal antibody which substantially specifically binds the polypeptide according to the invention.
  • Another possibility is to employ the simultaneous electroelution technique described by Andersen et al . in J. Immunol. Methods 161: 29-39.
  • the post- translational modifications involves lipidation, glycosylation, cleavage, or elongation of the polypeptide.
  • the DNA sequence information provided by this invention allows for the preparation of relatively short DNA (or RNA or PNA) sequences having the ability to specifically hybridize to mycobacterial gene sequences.
  • nucleic acid probes of an appropriate length are prepared based on a consideration of the relevant sequence. The ability of such nucleic acid probes to specifically hybridize to the mycobacterial gene sequences lend them particular utility in a variety of embodiments.
  • the probes can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample. However, either uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructs.
  • nucleic acid fragments of the invention may be used for effecting in vivo expression of antigens, i . e . the nucleic acid fragments may be used in so-called DNA vaccines.
  • DNA vaccines a DNA fragment cloned in a vector which is non- replicative in eukaryotic cells may be introduced into an animal (including a human being) by e.g. intramuscular injection or percutaneous administration (the so-called "gene gun” approach) .
  • the DNA is taken up by e.g.
  • muscle cells and the gene of interest is expressed by a promoter which is functioning in eukaryotes, e.g. a viral promoter, and the gene product thereafter stimulates the immune system.
  • a promoter which is functioning in eukaryotes, e.g. a viral promoter, and the gene product thereafter stimulates the immune system.
  • the invention also relates to a vaccine comprising a nucleic acid fragment according to the invention, the vaccine effecting in vivo expression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to infections with mycobacteria of the tuberculosis complex in an animal, inclu- ding a human being.
  • the efficacy of such a "DNA vaccine” can possibly be enhanced by administering the gene encoding the expression product together with a DNA fragment encoding a polypeptide which has the capability of modulating an immune response.
  • a gene encoding lymphokine precursors or lympho- kines e.g. IFN-7, IL-2, or IL-12
  • lympho- kines e.g. IFN-7, IL-2, or IL-12
  • DNA fragments comprising a multitude of nucleotide sequences which each encode relevant epitopes of the polypeptides disclosed herein so as to effect a continuous sensitization of the immune system with a broad spectrum of these epitopes.
  • the polypeptide fragments of the inven- tion are excellent candidates for vaccine constituents or for constituents in an immune diagnostic agent due to their extracellular presence in culture media containing metabolizing virulent mycobacteria belonging to the tuberculosis complex, or because of their high homologies with such extra- cellular antigens, or because of their absence in M. bovis BCG.
  • an immunologic composition comprising a polypeptide or fusion polypeptide according to the invention.
  • it is preferred that it comprises an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant.
  • Suitable carriers are selected from the group consisting of a polymer to which the polypeptide (s) is/are bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the polypeptide (s) is/are covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet haemocyanin.
  • Suitable vehicles are selected from the group consisting of a diluent and a suspending agent.
  • the adjuvant is preferably selected from the group consisting of dimethyl- dioctadecylammonium bromide (DDA) , Quil A, poly I:C, Freund's incomplete adjuvant, IFN-7, IL-2, IL-12, monophosphoryl lipid A (MPL) , and muramyl dipeptide (MDP) .
  • DDA dimethyl- dioctadecylammonium bromide
  • Quil A Quil A
  • poly I:C poly I:C
  • Freund's incomplete adjuvant IFN-7
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • MPL monophosphoryl lipid A
  • MDP muramyl dipeptide
  • a preferred immunologic composition according to the present invention comprising at least two different polypeptide fragments, each different polypeptide fragment being a polypeptide or a fusion polypeptide defined above. It is preferred that the immunologic composition comprises between 3-20 different polypeptide fragments or fusion polypeptides.
  • Such an immunologic composition may preferably be in the form of a vaccine or in the form of a skin test reagent.
  • the invention therefore also pertain to a method for producing an immunologic composition according to the invention, the method comprising preparing, synthesizing or isolating a polypeptide according to the invention, and solubilizing or dispersing the polypeptide in a medium for a vaccine, and optionally adding other AT. tuberculosis antigens and/or a carrier, vehicle and/or adjuvant substance.
  • Such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations 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 take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
  • the proteins may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeuti- cally effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual ' s immune system to mount an immune response, and the degree of protection desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 ⁇ g to 1000 ⁇ g, such as in the range from about 1 ⁇ g to 300 ⁇ g, and especially in the range from about 10 ⁇ g to 50 ⁇ g.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • the tion may comprise several different polypeptides in order to increase the immune response .
  • the vaccine may comprise two or more polypeptides, where all of the polypeptides are as defined above, or some but not all of the peptides may be derived from a bacterium belonging to the AT. tuberculosis complex. In the latter example the polypeptides not necessarily fulfilling the criteria set forth above for polypeptides may either act due to their own immunogenicity or merely act as adjuvants. Examples of such interesting polypeptides are MPB64, MPT64, and MPB59, but any other substance which can be isolated from mycobacteria are possible candidates.
  • the vaccine may comprise 3-20 different polypeptides, such as 3-10 different polypeptides.
  • Another important aspect of the present invention is an improvement of the living BCG vaccine presently available, which is a vaccine for immunizing an animal, including a human being, against TB caused by mycobacteria belonging to the tuberculosis-complex, comprising as the effective compo- nent a microorganism, wherein one or more copies of a DNA sequence encoding a polypeptide as defined above has been incorporated into the genome of the microorganism in a manner allowing the microorganism to express and secrete the polypeptide.
  • the term "genome” refers to the chromosome of the microorganisms as well as extrachromosomally DNA or RNA, such as plasmids. It is, however, preferred that the DNA sequence of the present invention has been introduced into the chromosome of the non-pathogenic microorganism, since this will prevent loss of the genetic material introduced.
  • the non-pathogenic microorganism is a bacterium, e.g. selected from the group consisting of the genera Mycobacterium, Salmonella, Pseudomonas and Eschericia . It is especially preferred that the non-pathogenic microorganism is ATycoJbacteriui ⁇ bovis BCG, such as Mycobacterium bovis BCG strain: Danish 1331.
  • incorporation of one or more copies of a nucleotide sequence encoding the polypeptide according to the invention in a mycobacterium from a AT. jbovis BCG strain will enhance the immunogenic effect of the BCG strain.
  • the incorporation of more than one copy of a nucleotide sequence of the invention is contemplated to enhance the immune response even more, and consequently an aspect of the invention is a vaccine wherein at least 2 copies of a DNA sequence encoding a polypeptide is incorporated in the genome of the microorganism, such as at least 5 copies.
  • the copies of DNA sequences may either be identical encoding identical polypeptides or be variants of the same DNA sequence encoding identical or homologues of a polypeptide, or in another embodiment be different DNA sequences encoding different polypeptides where at least one of the polypeptides is according to the present invention.
  • the living vaccine of the invention can be prepared by cultivating a transformed non-pathogenic cell according to the invention, and transferring these cells to a medium for a vaccine, and optionally adding a carrier, vehicle and/or adjuvant substance.
  • the invention also relates to a method of diagnosing TB caused by Mycobacterium tuberculosis, Mycobacterium africanum or AfycoJbacteriiun Jbovis in an animal, including a human being, comprising intradermally injecting, in the animal, a polypeptide according to the invention or a skin test reagent LO LO to to H F>
  • a blood sample comprising mononuclear cells (i . a . T-lymphocytes) from a patient could be contacted with a sample of one or more polypeptides of the invention.
  • This contacting can be performed in vi tro and a positive reaction could e.g. be proliferation of the T-cells or release cytokines such as 7-interferon into the extracellular phase (e.g. into a culture supernatant) ; a suitable in vivo test would be a skin test as described above.
  • a serum sample from a subject to contact with a polypeptide of the invention the demonstration of a binding between antibodies in the serum sample and the polypeptide being indicative of previous or ongoing infection.
  • the invention therefore also relates to an in vi tro method for diagnosing ongoing or previous sensitization in an animal or a human being with bacteria belonging to the tuberculosis complex, the method comprising providing a blood sample from the animal or human being, and contacting the sample from the animal with the polypeptide of the invention, a significant release into the extracellular phase of at least one cytokine by mononuclear cells in the blood sample being indicative of the animal being sensitized.
  • a significant release is herein meant that the release of the cytokine is significantly higher than the cytokine release from a blood sample derived from a non-tuberculous subject (e.g. a subject which does not react in a traditional skin test for TB) .
  • a significant release is at least two times the release observed from such a sample.
  • a sample of a possibly infected organ may be contacted with an antibody raised against a polypeptide of the invention.
  • the demonstration of the reaction by means of methods well-known in the art between the sample and the antibody will be indicative of ongoing infection. It is of course also a possibility to demonstrate the presence of anti-mycobacterial antibodies in serum by contacting a serum sample from a subject with at least one of the polypeptide fragments of the invention and using well-known methods for visualizing the reaction between the antibody and antigen.
  • a method of determining the presence of mycobacterial nucleic acids in an animal comprising administering a nucleic acid fragment of the invention to the animal or incubating the sample with the nucleic acid fragment of the invention or a nucleic acid fragment complementary thereto, and detecting the presence of hybridized nucleic acids resulting from the incubation (by using the hybridization assays which are well-known in the art) , is also included in the invention.
  • Such a method of diagnosing TB might involve the use of a composition comprising at least a part of a nucleotide sequence as defined above and detecting the presence of nucleotide sequences in a sample from the animal or human being to be tested which hybridize with the nucleic acid fragment (or a complementary fragment) by the use of PCR technique.
  • antigens are not present in AT. Jbovis BCG but are present in virulent mycobacteria point them out as interesting drug targets; the antigens may constitute receptor molecules or toxins which facilitate the infection by the mycobacterium, and if such functionalities are blocked the infectivity of the mycobacterium will be diminshed.
  • the gene encoding at least one of the polypeptides of the invention and the necessary control sequences can be introduced into avirulent strains of mycobacteria (e.g. BCG) so as to determine which of the polypeptides are critical for virulence.
  • mycobacteria e.g. BCG
  • anti-mycobacterial agents can be designed rationally to inhibit expression of the critical genes or to attack the critical gene products.
  • antibodies or fragments thereof such as Fab and (Fab') 2 fragments can be prepared against such critical polypeptides by methods known in the art and thereafter used as prophylactic or therapeutic agents.
  • small molecules can be screened for their ability to selectively inhibit expression of the critical gene products, e . g. using recombinant expression systems which include the gene's endogenous promoter, or for their ability to directly interfere with the action of the target. These small molecules are then used as therapeutics or as prophylactic agents to inhibit mycobacterial virulence.
  • anti-mycobacterial agents which render a virulent mycobacterium avirulent can be operably linked to expression control sequences and used to transform a virulent mycobacterium.
  • Such anti-mycobacterial agents inhibit the replication of a specified mycobacterium upon transcription or translation of the agent in the mycobacterium.
  • Such a "newly avirulent" mycobacterium would constitute a superb alternative to the above described modified BCG for vaccine purposes since it would be immunologically very similar to a virulent mycobacterium compared to e.g. BCG.
  • a monoclonal or polyclonal antibody which is spe- cifically reacting with a polypeptide of the invention in an immuno assay, or a specific binding fragment of said antibody, is also a part of the invention.
  • the production of such polyclonal antibodies requires that a suitable animal be immunized with the polypeptide and that these antibodies are subsequently isolated, suitably by immune affinity chromatography.
  • the production of monoclonals can be effected by methods well-known in the art, since the present invention provides for adequate amounts of antigen for both immunization and screening of positive hybridomas .
  • Fig. 1 Long term memory immune mice are very efficiently protected towards an infection with AT. tuberculosis. Mice were given a challenge of AT. tuberculosis and spleens were isolated at different time points. Spleen lymphocytes were stimulated in vi tro with ST-CF and the release of IFN-7 investigated (panel A) . The counts of CFU in the spleens of the two groups of mice are indicated in panel B. The memory immune mice control infection within the first week and produce large quantities of IFN-7 in response to antigens in ST-CF.
  • Fig. 2 T cells involved in protective immunity are predominantly directed to molecules from 6-12 and 17-38 kDa. Splenic T cells were isolated four days after the challenge with AT. tuberculosis and stimulated in vi tro with narrow molecular mass fractions of ST-CF. The release of IFN-7 was sake- gated
  • Fig. 3 Nucleotide sequence (SEQ ID NO: l) of cfp7.
  • the deduced amino acid sequence (SEQ ID NO: 2) of CFP7 is given in conventional one-letter code below the nucleotide sequence.
  • the putative ribosome-binding site is written in underlined italics as are the putative -10 and -35 regions. Nucleotides written in bold are those encoding CFP7.
  • Fig. 4 Nucleotide sequence (SEQ ID NO: 3) of cfp9 .
  • the deduced amino acid sequence (SEQ ID NO: 4) of CFP9 is given in conventional one- letter code below the nucleotide sequence.
  • the putative ribosome-binding site Shine Delgarno sequence is written in underlined italics as are the putative -10 and -35 regions. Nucleotides in bold writing are those encoding CFP9.
  • the nucleotide sequence obtained from the lambda 226 phage is double underlined.
  • Fig. 5 Nucleotide sequence of mpt ⁇ l .
  • the deduced amino acid sequence of MPT51 is given in a one-letter code below the nucleotide sequence.
  • the signal is indicated in italics, the putative potential ribosome-binding site is underlined.
  • the nucleotide difference and amino acid difference compared to the nucleotide sequence of MPB51 (Ohara et al . , 1995) are underlined at position 780.
  • the nucleotides given in italics are not present in AT. tuberculosis H37Rv.
  • Fig. 6 the position of the purified antigens in the 2DE system have been determined and mapped in a reference gel. The newly purified antigens are encircled and the position of well-known proteins are also indicated.
  • mice A group of efficiently protected mice was generated by infecting 8-12 weeks old female C57B1/6J mice with 5 x 10 4 AT. tuberculosis i.v. After 30 days of infection the mice were subjected to 60 days of antibiotic treatment with isoniazid and were then left for 200-240 days to ensure the establishment of resting long-term memory immunity. Such memory immune mice are very efficiently protected against a secondary infection (Fig. 1) . Long lasting immunity in this model is mediated by a population of highly reactive CD4 cells recruited to the site of infection and triggered to produce large amounts of IFN-7 in response to ST-CF (Fig. 1) (Andersen et al . 1995).
  • the plates were overlaid with sheets of nitrocellulose satu- rated with isopropyl -3-D- thiogalactopyranoside and incubation was continued for 2,5 hours at 37°C.
  • the nitrocellulose was removed and incubated with samples of the monoclonal antibodies in PBS with Tween 20 added to a final concentration of 0.05%.
  • Bound monoclonal antibodies were visualized by horse- radish peroxidase- conjugated rabbit anti-mouse immunoglobulins (P260, Dako, Glostrup, DK) and a staining reaction involving 5 , 5 ' , 3 , 3 ' - tetramethylbenzidine and H 2 0 2 .
  • AA226 (expressing ST- 3 reactive polypeptide CFP9) which has been deposited 28 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM 8377 and in accordance with the provisions of the Budapest Treaty
  • AA242 (expressing PV-2 reactive polypeptide CFP7) which has been deposited 28 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM 8379 and in accordance with the provisions of the Budapest Treaty.
  • the st-3 binding protein is expressed as a protein fused to jS-galactosidase, whereas the pv-2 binding protein appears to be expressed in an unfused version.
  • the approximately 3 kb M. tuberculosis derived EcoRI - EcoRI fragment from AA242 was subcloned in the EcoRI site in the pBluescriptSK + (Stratagene) and used to transform E. coli XL-lBlue (Stratagene) .
  • the approximately 5 kb AT. tuberculosis derived EcoRI - EcoRI fragment from AA226 was subcloned in the EcoRI site in the pBluescriptSK + (Stratagene) and used to transform E. coli XL-lBlue (Stratagene) .
  • the complete DNA sequence of both genes were obtained by the dideoxy chain termination method adapted for supercoiled DNA by use of the Sequenase DNA sequencing kit version 1.0 (United States Biochemical Corp., Cleveland, OH) and by cycle sequencing using the Dye Terminator system in combination with an automated gel reader (model 373A; Applied Biosystems) according to the instructions provided.
  • the sequences DNA are shown in SEQ ID NO: 1 (CFP7) and in SEQ ID NO: 3 (CFP9) as well as in Figs. 3 and 4, respectively. Both strands of the DNA were sequenced.
  • ORF open reading frame encoding a sequence of 96 amino acid residues was identified from an ATG start codon at position 91-93 extending to a TAG stop codon at position 379- 381.
  • the deduced amino acid sequence is shown in SEQ ID NO: 2 (and in Fig. 3 where conventional one-letter amino acid codes are used) .
  • CFP7 appear to be expressed in E. coli as an unfused version.
  • the nucleotide sequence at position 78-84 is expected to be the Shine Delgarno sequence and the sequences from position 47-50 and 14-19 are expected to be the -10 and -35 regions, respectively:
  • the protein recognised by ST- 3 was produced as a -galactosi- dase fusion protein, when expressed from the AA226 lambda phage.
  • the fusion protein had an approx. size of 116 - 117kDa
  • the nucleotide sequence at position 123 - 130 is expected to be the Shine Delgarno sequence and the sequences from posi- tion 73 - 78 and 4 - 9 are expected to be the -10 and -35 region respectively (Fig. 4) .
  • the ORF overlapping with the 5 '-end of the sequence of AA229 is shown in Fig. 4 by double underlining.
  • the two ORFs encoding CFP7 and CFP9 were PCR cloned into the pMST24 (Theisen et al., 1995) expression vector pRVNOl or the pQE-32 (QIAGEN) expression vector pRVN02, respectively.
  • the PCR amplification was carried out in a thermal reactor (Rapid cycler, Idaho Technology, Idaho) by mixing 10 ng plasmid DNA with the mastermix (0.5 ⁇ M of each oligonucleotide primer, 0.25 ⁇ M BSA (Stratagene), low salt buffer (20 mM Tris-HCl, pH 8.8, 10 mM KC1 , 10 mM (NH 4 ) 2 S0 4 , 2 mM MgS0 4 and 0,1% Triton X-100) (Stratagene), 0.25 mM of each deoxynucleo- side triphosphate and 0.5 U Taq Plus Long DNA polymerase (Stratagene) ) .
  • the mastermix 0.5 ⁇ M of each oligonucleotide primer, 0.25 ⁇ M BSA (Stratagene), low salt buffer (20 mM Tris-HCl, pH 8.8, 10 mM KC1 , 10 mM (NH 4 ) 2 S0 4 , 2 m
  • Final volume was 10 ⁇ l (all concentrations given are concentrations in the final volume) .
  • Predenatura- tion was carried out at 94°C for 30 s. 30 cycles of the following was performed; Denaturation at 94°C for 30 s, annealing at 55°C for 30 s and elongation at 72°C for 1 min.
  • the oligonucleotide primers were synthesised automatically on a DNA synthesizer (Applied Biosystems, Forster City, Ca, ABI- 391, PCR-mode) , deblocked, and purified by ethanol precipitation.
  • the cfp7 oligonucleotides (TABLE 1) were synthesised on the basis of the nucleotide sequence from the CFP7 sequence (Fig. 3) .
  • the oligonucleotides were engineered to include an Smal restriction enzyme site at the 5' end and a BamHl restriction enzyme site at the 3' end for directed subcloning.
  • the cfp9 oligonucleotides (TABLE 1) were synthesized partly on the basis of the nucleotide sequence from the sequence of the AA229 clone and partly from the identical sequence found in the Sanger database cosmid MTCY48 (Fig. 4) .
  • the oligo- nucleotides were engineered to include a Sma restriction enzyme site at the 5' end and a Hindlll restriction enzyme site at the 3' end for directed subcloning.
  • the pellet was resuspended in BC 40/100 buffer (20 mM Tris-HCl pH 7.9, 20% glycerol, 100 mM KCl, 40 mM Imidazole) and cells were broken by sonication (5 times for 30 s with intervals of 30 s) at 4°C. followed by centrifugation at 12,000 x g for 30 min at 4°C, the supernatant (crude extract) was used for purification of the recombinant antigens.
  • the two Histidine fusion proteins (His-rCFP7 and His-rCFP9) were purified from the crude extract by affinity chromatography on a Ni 2+ -NTA column from QIAGEN with a volume of 100 ml. His-rCFP7 and His-rCFP9 binds to Ni 2+ . After extensive washes of the column in BC 40/100 buffer, the fusion protein was eluted with a BC 1000/100 buffer containing 100 mM imidazole, 20 mM Tris pH 7.9, 20% glycerol and 1 M KCl. subsequently, the purified products were dialysed extensively against 10 mM Tris pH 8.0.
  • His-rCFP7 and His-rCFP9 were then separated from contaminants by fast protein liquid chromato- graphy (FPLC) over an anion- exchange column (Mono Q, Pharmacia, Sweden) . in 10 mM Tris pH 8.0 with a linear gradient of NaCl from 0 to 1 M. Aliquots of the fractions were analyzed by 10% -20% gradient sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS -PAGE) . Fractions containing purified either purified His-rCFP7 or His-rCFP9 were pooled. TABLE 1. Sequence of the cfp7 and cfp9 oligonucleotides 8 .
  • StF2 CTACTAAGCTTCCATGGTCAGGTCTTTTCGATGCTTAC 467 - 447 (SEQ ID NO : 46) (SEQ ID NO : 3 ) a
  • the cfp7 oligonucleotides were based on the nucleotide sequence shown in Fig . 3 (SEQ ID NO : 1) .
  • the cfp9 oligonucleotides were based on the nucleotide sequence shown in Fig . 4 (SEQ ID NO : 3 ) .
  • Nucleotides underlined are not contained in the nucleotide sequence of cfp7 and cfp9 .
  • the positions referred to are of the non -underlined part of the primers and correspond to the nucleotide sequence shown in Fig . 3 and Fig . 4 , re spe ct i e ly .
  • BCG Bacillus Calmette-Guerin
  • the first mutations described are the loss of the gene encoding MPB64 in some BCG strains (Li et al . , 1993 , Oettinger and Andersen, 1994) and the gene encoding ESAT- 6 in all BCG strain tested (Harboe et al . , 1996) , later 3 large deletions in BCG have been identified (Mahairas et al . , 1996 ) .
  • the region named RD1 includes the gene encoding ESAT- 6 and an other (RD2) the gene encoding MPT64.
  • ORFs with a minimum length of 96 bp have been predicted using the algorithm described by Borodovsky and Mclninch (1993) , in total 27 ORFs have been predicted, 20 of these have possible diagnostic and/or vaccine potential, as they are deleted from all known BCG strains.
  • the predicted ORFs include ESAT-6 (RD1-0RF7) and CFP10 (RD1-ORF6) described previously (S ⁇ rensen et al . , 1995), as a positive control for the ability of the algorithm. In the present is described the potential of 7 of the predicted antigens for diagnosis of TB as well as potential as candidates for a new vaccine against TB.
  • the nucleotide sequence of rdl -orf 2 from AT. tuberculosis H37Rv is set forth in SEQ ID NO: 71.
  • the deduced amino acid sequence of RD1-0RF2 is set forth in SEQ ID NO: 72.
  • the nucleotide sequence of rdl -orf 3 from M. tuberculosis H37Rv is set forth in SEQ ID NO: 87.
  • the deduced amino acid sequence of RD1-0RF2 is set forth in SEQ ID NO: 88.
  • H37Rv is set forth in SEQ ID NO: 89.
  • the deduced amino acid sequence of RD1-0RF2 is set forth in SEQ ID NO: 90.
  • the nucleotide sequence of rdl -orf 5 from AT. tuberculosis H37Rv is set forth in SEQ ID NO: 91.
  • the deduced amino acid sequence of RD1-0RF2 is set forth in SEQ ID NO: 92.
  • the nucleotide sequence of rdl -orf 8 from AT. tuberculosis H37Rv is set forth in SEQ ID NO: 67.
  • the deduced amino acid sequence of RD1-0RF2 is set forth in SEQ ID NO: 68.
  • the nucleotide sequence of rdl -orf 9a from M. tuberculosis H37Rv is set forth in SEQ ID NO: 93.
  • the deduced amino acid sequence of RD1-0RF2 is set forth in SEQ ID NO: 94.
  • the nucleotide sequence of rdl -orf9b from M. tuberculosis H37Rv is set forth in SEQ ID NO: 69.
  • the deduced amino acid sequence of RD1-ORF2 is set forth in SEQ ID NO: 70.
  • the DNA sequence rdl -orf 2 (SEQ ID NO: 71) contained an open reading frame starting with an ATG codon at position 889 - 891 and ending with a termination codon (TAA) at position 2662 - 2664 (position numbers referring to the location in RD1) .
  • the deduced amino acid sequence (SEQ ID NO: 72) contains 591 residues corresponding to a molecular weight of 64,525.
  • the DNA sequence rdl -orf 3 (SEQ ID NO: 87) contained an open reading frame starting with an ATG codon at position 2807 - 2809 and ending with a termination codon (TAA) at position 3101 - 3103 (position numbers referring to the location in RD1) .
  • the deduced amino acid sequence (SEQ ID NO: 88) con- tains 98 residues corresponding to a molecular weight of 9,799.
  • the DNA sequence rdl -orf 4 (SEQ ID NO: 89) contained an open reading frame starting with a GTG codon at position 4014 - 4012 and ending with a termination codon (TAG) at position 3597 - 3595 (position numbers referring to the location in RD1) .
  • the deduced amino acid sequence (SEQ ID NO: 90) con- tains 139 residues corresponding to a molecular weight of 14,210.
  • the DNA sequence rdl - orf 5 (SEQ ID NO: 91) contained an open reading frame starting with a GTG codon at position 3128 - 3130 and ending with a termination codon (TGA) at position 4241 - 4243 (position numbers referring to the location in RD1) .
  • the deduced amino acid sequence (SEQ ID NO: 92) contains 371 residues corresponding to a molecular weight of 37,647.
  • the DNA sequence rdl -orf 8 (SEQ ID NO: 67) contained an open reading frame starting with a GTG codon at position 5502 - 5500 and ending with a termination codon (TAG) at position 5084 - 5082 (position numbers referring to the location in RD1) , and the deduced amino acid sequence (SEQ ID NO: 68) contains 139 residues with a molecular weight of 11,737.
  • the DNA sequence rdl -orf 9a (SEQ ID NO: 93) contained an open reading frame starting with a GTG codon at position 6146 - 6148 and ending with a termination codon (TAA) at position 7070 - 7072 (position numbers referring to the location in RD1) .
  • the deduced amino acid sequence (SEQ ID NO: 94) contains 308 residues corresponding to a molecular weight of 33,453.
  • the DNA sequence rdl -orf 9b (SEQ ID NO: 69) contained an open reading frame starting with an ATG codon at position 5072 - 5074 and ending with a termination codon (TAA) at position 7070 - 7072 (position numbers referring to the location in RD1) .
  • the deduced amino acid sequence (SEQ ID NO: 70) contains 666 residues corresponding to a molecular weight of 70,650. Cloning of the ORF's rdl -orf 2. rdl -orf 3 , rdl -orf 4 , rdl -orf 5. rdl -orf 8 , rdl -orf 9a . and rdl -orf 9b.
  • ORF' s rdl - orf 2, rdl - orf 3 , rdl - orf 4 , rdl - orf 5 , rdl - orf 8 , rdl -orf 9a and rdl -orf 9b were PCR cloned in the pMST24 (Thei- sen et al .
  • Oligonucleotides were synthesized on the basis of the nucleotide sequence from the RD1 region (Accession no. U34848) .
  • the oligonucleotide primers were engineered to include an restriction enzyme site at the 5' end and at the 3' end by which a later subcloning was possible. Primers are listed in TABLE 2.
  • rdl -orf 2 A BamHl site was engineered immediately 5' of the first codon of rdl -orf2, and a Hindlll site was incorporated right after the stop codon at the 3' end.
  • the gene rdl -orf2 was subcloned in pQE32, giving pT096.
  • rdl -orf 3 A Smal site was engineered immediately 5' of the first codon of rdl -orf 3 , and a Ncol site was incorporated right after the stop codon at the 3' end.
  • the gene rdl -orf 3 was subcloned in pMST24, giving pT087.
  • rdl -orf 4 A Ba Hl site was engineered immediately 5' of the first codon of rdl -orf 4 , and a Hindlll site was incorporated right after the stop codon at the 3' end.
  • the gene rdl -orf4 was subcloned in pQE32, giving pT089.
  • rdl -orf 5 A BamHl site was engineered immediately 5' of the first codon of rdl -orf 5, and a Hindlll site was incorporated right after the stop codon at the 3' end.
  • the gene rdl -orf 5 was subcloned in pQE32, giving pT088.
  • rdl -orf 8 A BamHl site was engineered immediately 5' of the first codon of rdl -orf8, and a Ncol site was incorporated right after the stop codon at the 3' end.
  • the gene rdl -orf8 was subcloned in pMST24, giving pT098.
  • rdl -orf9a A BamHl site was engineered immediately 5' of the first codon of rdl -orf9a, and a Hindlll site was incorporated right after the stop codon at the 3' end.
  • the gene rdl -orf 9a was subcloned in pQE32, giving pT091.
  • rdl -orf 9b A Seal site was engineered immediately 5' of the first codon of rdl -orf 9b, and a Hind III site was incorporated right after the stop codon at the 3' end.
  • PCR fragments were digested with the suitable restriction enzymes, purified from an agarose gel and cloned into either pMST24 or pQE-32.
  • the seven constructs were used to transform the E. coli XLl-Blue. Endpoints of the gene fusions were determined by the dideoxy chain termination method. Both strands of the DNA were sequenced.
  • RD1-0RF8 Purification of recombinant RD1-0RF2, RD1-0RF3, RD1-ORF4, RD1-ORF5.
  • RD1-0RF8 RDl-0RF9a and RDl-0RF9b.
  • Recombinant antigen was prepared as described in example 2 (with the exception that pT091 was expressed at 30 °C and not at 37°C) , using a single colony of E. coli harbouring either the pT087, pT088, pT089, pTO90, pT091, pT096 or pT098 for inoculation. Purification of recombinant antigen by Ni 2+ affinity chromatography was also carried out as described in example 2.
  • His-rRDl-ORF ' s were extensively dialysed against 10 mM Tris/HCl, pH 8.5, 3 M urea followed by an additional purification step performed on an anion exchange column (Mono Q) using fast protein liquid chromatography (FPLC) (Pharmacia, Uppsala, Sweden) .
  • FPLC fast protein liquid chromatography
  • the purification was carried out in 10 mM Tris/HCl, pH 8.5, 3 M urea and protein was eluted by a linear gradient of NaCl from 0 to 1 M. Fractions containing the His-rRDl-ORF' s were pooled and subsequently dialysed extensively against 25 mM Hepes, pH 8.0 before use.
  • RDl-ORF2f CTGGGGATCCGCATGACTGCTGAACCG 886 - 903 RDl-ORF3f CTTCCCGGGATGGAAAAAATGTCAC 2807 - 2822 RDl-ORF4f GTAGGATCCTAGGAGACATCAGCGGC 4028 - 4015 RDl-ORF5f CTGGGGATCCGCGTGATCACCATGCTGTGG 3028 - 3045 RDl-ORF8f CTCGGATCCTGTGGGTGCAGGTCCGGCGATGGGC 5502 - 5479 RDl-ORF9af GTGATGTGAGCTCAGGTGAAGAAGGTGAAG 6144 - 6160 RDl-ORF9bf GTGATGTGAGCTCCTATGGCGGCCGACTACGAC 5072 - 5089 Antisense RDl-ORF2r TGCAAGCTTTTAACCGGCTTGGGGGTGC 2664 - 2644 RDl-ORF3r GATGCCATGGTTAGGCGAAG
  • oligonucleotides were constructed from the Accession number U34484 nucleotide sequence (Mahairas et al . , 1996) . Nucleotides (nt) underlined are not contained in the nucleotide sequence of RD1-ORF' s. The positions correspond to the nucleotide sequence of Accession number U34484.
  • nucleotide sequences of rdl-orf2, rdl-orf3 , rdl-orf4, rdl-orf5, rdl-orf8, rdl-orf9a, and rdl-orf9b from AT. tuberculosis H37Rv are set forth in SEQ ID NO: 71, 87, 89, 91, 67, 93, and 69, respectively.
  • deduced amino acid sequences of rdl -orf 2, rdl -orf 3, rdl -orf 4 rdl -orf 5, rdl -orf 8, rdl -orf 9a, and rdl -orf 9b are set forth in SEQ ID NO: 72, 88, 90, 92, 68, 94, and 70, respectively.
  • CFP29 containing fractions were blotted to PVDF membrane after tricine SDS-PAGE (Ploug et al., 1989).
  • the relevant bands were excised and subjected to amino acid analysis (Barkholt and Jensen, 1989) and N-terminal sequence analysis on a Procise sequencer (Applied Biosystems) .
  • X denotes an amino acid which could not be determined by the sequencing method used, whereas a "/" between two amino acids denotes that the sequencing method could not determine which of the two amino acids is the one actually present.
  • the N-terminal sequence of CFP29 was used for a homology search in the EMBL database using the TFASTA program of the Genetics Computer Group sequence analysis software package.
  • the search identified a protein, Linocin M18, from Brevibac- terium linens that shares 74% identity with the 19 N-terminal amino acids of CFP29.
  • PCR reactions were containing 10 ng of AT. tuberculosis chromosomal DNA in 1 x low salt Taq+ buffer from Stratagene supplemented with 250 ⁇ M of each of the four nucleotides (Boehringer Mannheim), 0,5 mg/ml BSA (IgG technology), 1% DMSO (Merck), 5 pmoles of each primer and 0.5 unit Tag+ DNA polymerase (Stratagene) in 10 ⁇ l reaction volume. Reactions were initially heated to 94 °C for 25 sec. and run for 30 cycles of the program; 94°C for 15 sec, 55°C for 15 sec. and 72 °C for 90 sec, using thermocycler equipment from Idaho Technology.
  • the fragment was excised from a 1% agarose gel, purified by Spin-X spinn columns (Costar) , cloned into pBluescript SK 11+ - T vector (Stratagene) and finally sequenced with the Seque- nase kit from United States Biochemical.
  • the first 150 bp of this sequence was used for a homology search using the Blast program of the Sanger Afycojbacterium tuberculosis database:
  • This program identified a Mycobacterium tuberculosis sequence on cosmid cy444 in the database that is nearly 100% identical to the 150 bp sequence of the CFP29 protein.
  • the sequence is contained within a 795 bp open reading frame of which the 5 ' end translates into a sequence that is 100% identical to the N-terminally sequenced 19 amino acids of the purified CFP29 protein.
  • CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28 the N-terminal amino acid sequence from each of the proteins were used for a homology search using the blast program of the Sanger Mycobacterium tuberculosis database:
  • CFP29 the first 150 bp of the DNA sequence was used for the search. Furthermore, the EMBL database was searched for proteins with homology to CFP29.
  • the translated protein is 162 amino acids long.
  • the N-terminal of the protein purified from culture filtrate starts at amino acid 31 in agreement with the presence of a signal sequence that has been cleaved off. This gives a length of the mature protein of 132 amino acids, which corresponds to a theoretical molecular mass of 13833 Da and a theoretical pi of 4.4.
  • the observed mass in SDS -PAGE is 17 kDa.
  • a sequence 100% identical to the 15 determined amino acids of CFP20 was found on the translated cosmid cscy09F9.
  • a stop codon is found at amino acid 166 from the amino acid M at position 1. This gives a predicted length of 165 amino acids, which corresponds to a theoretical molecular mass of 16897 Da and a pi of 4.2.
  • the observed molecular weight in a SDS -PAGE is 20 kDa.
  • the translated protein is 217 amino acids long.
  • the N- terminally determined sequence from the protein purified from culture filtrate starts at amino acid 33 in agreement with the presence of a signal sequence that has been cleaved off. This gives a length of the mature protein of 185 amino acids, which corresponds to a theoretical molecular weigh at 18657 Da, and a theoretical pi at 4,6.
  • the observed weight in a SDS-PAGE is 21 kDa.
  • the protein has 32,6% identity to a cutinase precursor with a length of 209 amino acids (CUTI_ALTBR P41744) .
  • the N- terminally determined sequence from the protein purified from culture filtrate begins at amino acid 33 in agreement with the presence of a signal sequence that has been cleaved off. This gives a length of the mature protein of 187 amino acids, which corresponds to a theoretical molecular weigh at 19665 Da, and a theoretical pi at 4.9. The observed weight in a SDS -PAGE is 25 kDa.
  • amino acid sequences of the purified antigens as picked from the Sanger database are shown in the following list.
  • the amino acids determined by N-terminal sequencing are marked with bold.
  • CFP20 (SEQ ID NO: 8) :
  • PCR reactions contained 10 ng of AT. tuberculosis chromosomal DNA in lx low salt Taq+ buffer from Stratagene supplemented with 250 mM of each of the four nucleotides (Boehringer Mannheim), 0,5 mg/ml BSA (IgG technology), 1% DMSO (Merck), 5 pmoles of each primer and 0.5 unit Tag-t- DNA polymerase (Stratagene) in 10 ⁇ l reaction volume. Reactions were initially heated to 94°C for 25 sec. and run for 30 cycles according to the following program; 94°C for 10 sec, 55°C for 10 sec. and 72°C for 90 sec, using thermocycler equipment from Idaho Technology.
  • DNA fragments were subsequently run on 1% agarose gels, the bands were excised and purified by Spin-X spin columns (Costar) and cloned into pBluescript SK 11+ - T vector (Stratagene) . Plasmid DNA was thereafter prepared from clones harbouring the desired fragments, digested with suitable restriction enzymes and subcloned into the expression vector pMCT6 in frame with 8 histidine residues which are added to the N-terminal of the expressed proteins. The resulting clones were hereafter sequenced by use of the dideoxy chain termination method adapted for supercoiled DNA using the
  • OPBR-51 ACAGATCTGTGACGGACATGAACCCG (SEQ ID NO: 117)
  • OPBR-52 TTTTCCATGGTCACGGGCCCCCGGTACT (SEQ ID NO: 118)
  • OPBR-51 and OPBR-52 create Bglll and Ncol sites, respectively, used for the cloning in pMCT6.
  • CFP20 Primers used for cloning of cfp20:
  • OPBR-53 ACAGATCTGTGCCCATGGCACAGATA (SEQ ID NO: 119)
  • OPBR-54 TTTAAGCTTCTAGGCGCCCAGCGCGGC (SEQ ID NO: 120)
  • OPBR-53 and OPBR-54 create Bglll and HinDIII sites, respect- ively, used for the cloning in pMCT6.
  • OPBR-55 ACAGATCTGCGCATGCGGATCCGTGT (SEQ ID NO: 121)
  • OPBR-56 TTTTCCATGGTCATCCGGCGTGATCGAG (SEQ ID NO: 122)
  • OPBR-55 and OPBR-56 create Bglll and Ncol sites, respective- ly, used for the cloning in pMCT6.
  • OPBR-57 ACAGATCTGTAATGGCAGACTGTGAT (SEQ ID NO: 123)
  • OPBR-58 TTTTCCATGGTCAGGAGATGGTGATCGA (SEQ ID NO: 124)
  • OPBR-57 and OPBR-58 create Bglll and Ncol sites, respective- ly, used for the cloning in pMCT6.
  • OPBR-59 ACAGATCTGCCGGCTACCCCGGTGCC (SEQ ID NO : 125)
  • OPBR-60 TTTTCCATGGCTATTGCAGCTTTCCGGC (SEQ ID NO: 126)
  • OPBR-59 and OPBR-60 create Bglll and Ncol sites, respective- ly, used for the cloning in pMCT6.
  • the lysate was applied to a column containing 25 ml of resuspended Talon resin (Clontech, Palo Alto, USA) .
  • the column was washed and eluted as described by the manufacturers .
  • ST-CF was precipitated with ammonium sulphate at 80% saturation.
  • the precipitated proteins were removed by centrifugation and after resuspension washed with 8 M urea.
  • CHAPS and glycerol were added to a final concentration of 0.5 % (w/v) and 5 % (v/v) respectively and the protein solution was applied to a Rotofor isoelectrical Cell (BioRad) .
  • the Rotofor Cell had been equilibrated with a 8M urea buffer containing 0.5 % (w/v) CHAPS, 5% (v/v) glycerol, 3% (v/v) Biolyt 3/5 and 1% (v/v) Biolyt 4/6 (BioRad) .
  • Isoelectric focusing was performed in a pH gradient from 3-6. The fractions were analyzed on silver-stained 10-20% SDS-PAGE. Fractions with similar band patterns were pooled and washed three times with PBS on a Centriprep concentrator (Amicon) with a 3 kDa cut off membrane to a final volume of 1-3 ml. An equal volume of SDS containing sample buffer was added and the protein solution boiled for 5 min before further separation on a Prep Cell (BioRad) in a matrix of 16% polyacrylamide under an electrical gradient. Fractions containing well separated bands in SDS-PAGE were selected for N-terminal sequencing after transfer to PVDF membrane.
  • ST-CF was precipitated with ammonium sulphate at 80% saturation and redissolved in PBS, pH 7.4, and dialysed 3 times against 25mM Piperazin-HCl , pH 5.5, and subjected to chroma- tofocusing on a matrix of PBE 94 (Pharmacia) in a column connected to an FPLC system (Pharmacia) .
  • the column was equilibrated with 25 mM Piperazin-HCl, pH 5.5, and the elution was performed with 10% PB74-HC1, pH 4.0 (Pharmacia) .
  • ST-CF was concentrated approximately 10 fold by ultrafiltration and proteins were precipitated at 80 % saturation, redissolved in PBS, pH 7.4, and dialysed 3 times against PBS, pH 7.4.
  • 5.1 ml of the dialysed ST-CF was treated with RNase (0.2 mg/ml, QUIAGEN) and DNase (0.2 mg/ml, Boeh- ringer Mannheim) for 6 h and placed on top of 6.4 ml of 48 % (w/v) sucrose in PBS, pH 7.4 , in Sorvall tubes (Ultracrimp 03987, DuPont Medical Products) and ultracentrifuged for 20 h at 257,300 x g max , 10°C. The pellet was redissolved in 200 ⁇ l of 25 mM Tris-192 mM glycine, 0.1 % SDS, pH 8.3.
  • CFP27, CFP30A and CFP50 ST-CF was concentrated approximately 10 fold by ultrafiltration and ammonium sulphate precipitation in the 45 to 55 % saturation range was per- formed.
  • Proteins were redissolved in 50 mM sodium phosphate, 1.5 M ammonium sulphate, pH 8.5, and subjected to thiophilic adsorption chromatography on an Affi-T gel column (Kern-En- Tec) . Proteins were eluted by a 1.5 to 0 M decreasing gradient of ammonium sulphate.
  • Fractions with similar band patterns in SDS-PAGE were pooled and anion exchange chromatography was performed on a Mono Q HR 5/5 column connected to an FPLC system (Pharmacia) . The column was equilibrated with 10 mM Tris-HCl, pH 8.5, and the elution was performed with a gradient of NaCl from 0 to 1 M. Fractions containing well separated bands in SDS-P
  • CFP7A and CFP25A were obtained as described above except for the following modification: ST-CF was concentrated approximately 10 fold by ultrafiltration and proteins were precipi- tated at 80 % saturation, redissolved in PBS, pH 7.4, and dialysed 3 times against PBS, pH 7.4. Ammonium sulphate was added to a concentration of 1.5 M, and ST-CF proteins were loaded on an Affi T-gel column. Elution from the Affi T-gel column and anion exchange were performed as described above.
  • Heat treated H37Rv was subtractionated into subcellular fractions as described in S ⁇ rensen et al 1995.
  • the Cell wall fraction was resuspended in 8 M urea, 0.2 % (w/v) N-octyl ⁇ - O glucopyranoside (Sigma) and 5 % (v/v) glycerol and the protein solution was applied to a Rotofor isoelectrical Cell (BioRad) which was equilibrated with the same buffer. Isoelectric focusing was performed in a pH gradient from 3-6. The fractions were analyzed by SDS-PAGE and fractions con- taining well separated bands were polled and subjected to N- terminal sequencing after transfer to PVDF membrane.
  • Fractions containing CFP7A, CFP8A, CFP8B, CFP16, CFP19 , CFP19B, CFP22A, CFP23A, CFP23B, CFP27, CFP30A, CWP32, and CFP50A were blotted to PVDF membrane after Tricine SDS-PAGE (Ploug et al, 1989) .
  • the relevant bands were excised and subjected to N-terminal amino acid sequence analysis on a Procise 494 sequencer (Applied Biosystems) .
  • the fraction containing CFP25A was blotted to PVDF membrane after 2 -DE PAGE (isoelectric focusing in the first dimension and Tricin SDS-PAGE in the second dimension) .
  • the relevant spot was excised and sequenced as described above.
  • CFP7A AEDVRAEIVA SVLEVWNEG DQIDKGDVW LLESMYMEIP VLAEAAGTVS (SEQ ID NO : 81)
  • CFP8A DPVDDAFIAKLNTAG (SEQ ID NO: 73)
  • CFP8B DPVDAIINLDNYGX (SEQ ID NO: 74)
  • CFP16 AKLSTDELLDAFKEM (SEQ ID NO: 79)
  • CFP19 TTSPDPYAALPKLPS (SEQ ID NO: 82)
  • CFP19B DPAXAPDVPTAAQLT (SEQ ID NO: 80)
  • CFP22A TEYEGPKTKF HALMQ (SEQ ID NO: 83)
  • CFP23A VIQ/AGMVT/GHIHXVAG (SEQ ID NO: 76)
  • CFP23B AEMKXFKNAIVQEID (SEQ ID NO: 75)
  • CFP25A AIEVSVLRVF TDSDG (SEQ ID NO: 78)
  • CWP32 TNIWLIKQVPDTWS (SEQ ID NO: 77)
  • CFP27 TTIVALKYPG GWMA (SEQ ID NO: 84)
  • CFP30A SFPYFISPEX AMRE (SEQ ID NO: 85)
  • CFP50 THYDWVLGA GPGGY (SEQ ID NO: 86)
  • N-terminal amino acid sequence from each of the proteins was used for a homology search using the blast program of the Sanger Mycobacterium tuberculosis database:
  • CFP7A, CFP8A, CFP8B, CFP16, CFP19, CFP19B , CFP22A, CFP25A, CFP27, CFP30A, CWP32, and CFP50 the following information was obtained:
  • the identity is found within an open reading frame of 71 amino acids length corresponding to a theoretical MW of CFP7A of 7305.9 Da and a pi of 3.762.
  • the observed molecular weight in an SDS-PAGE gel is 7 kDa.
  • CFP8A A sequence 80% identical to the 15 N-terminal amino acids was found on contig TB_1884. The N-terminally determined sequence from the protein purified from culture filtrate starts at amino acid 32. This gives a length of the mature protein of 98 amino acids corresponding to a theoretical MW of 9700 Da and a pi of 3.72 This is in good agreement with the observed MW on SDS-PAGE at approximately 8 kDa. The full length protein has a theoretical MW of 12989 Da and a pi of 4.38.
  • CFP8B A sequence 71% identical to the 14 N-terminal amino acids was found on contig TB_653. However, careful re-eva- luation of the original N-terminal sequence data confirmed the identification of the protein.
  • the N-terminally determined sequence from the protein purified from culture filtrate starts at amino acid 29. This gives a length of the mature protein of 82 amino acids corresponding to a theoreti- cal MW of 8337 Da and a pi of 4.23. This is in good agreement with the observed MW on SDS-PAGE at approximately 8 kDa. Analysis of the amino acid sequence predicts the presence of a signal peptide which has been cleaved of the mature protein found in culture filtrate.
  • CFP16 The 15 aa N-terminal sequence was found to be 100% identical to a sequence found on cosmid MTCY20H1.
  • the identity is found within an open reading frame of 130 amino acids length corresponding to a theoretical MW of CFP16 of 13440.4 Da and a pi of 4.59.
  • the observed molecular weight in an SDS-PAGE gel is 16 kDa.
  • CFP19 The 15 aa N-terminal sequence was found to be 100% identical to a sequence found on cosmid MTCY270.
  • the identity is found within an open reading frame of 176 amino acids length corresponding to a theoretical MW of CFP19 of 18633.9 Da and a pi of 5.41.
  • the observed molecular weight in an SDS-PAGE gel is 19 kDa.
  • CFP22A The 15 aa N-terminal sequence was found to be 100% identical to a sequence found on cosmid MTCY1A6.
  • the identity is found within an open reading frame of 181 amino acids length corresponding to a theoretical MW of CFP22A of 20441.9 Da and a pi of 4.73.
  • the observed molecular weight in an SDS-PAGE gel is 22 kDa.
  • CFP25A The 15 aa N-terminal sequence was found to be 100% identical to a sequence found on contig 255.
  • the identity is found within an open reading frame of 228 amino acids length corresponding to a theoretical MW of CFP25A of 24574.3 Da and a pi of 4.95.
  • the observed molecular weight in an SDS-PAGE gel is 25 kDa.
  • CFP27 The 15 aa N-terminal sequence was found to be 100% identical to a sequence found on cosmid MTCY261.
  • CFP30A Of the 13 determined amino acids in CFP30A, a 100% identical sequence was found on cosmid MTCY261.
  • the identity is found within an open reading frame of 248 amino acids length corresponding to a theoretical MW of CFP30A of 26881.0 Da and a pi of 5.41.
  • the observed molecular weight in an SDS-PAGE gel is 30 kDa.
  • CWP32 The 15 amino acid N-terminal sequence was found to be 100% identical to a sequence found on contig 281. The identity was found within an open reading frame of 266 amino acids length, corresponding to a theoretical MW of CWP32 of 28083 Da and a pi of 4.563. The observed molecular weight in an SDS-PAGE gel is 32 kDa.
  • CFP50 The 15 aa N-terminal sequence was found to be 100% identical to a sequence found in MTVO38.06. The identity is found within an open reading frame of 464 amino acids length corresponding to a theoretical MW of CFP50 of 49244 Da and a pi of 5.66. The observed molecular weight in an SDS-PAGE gel is 50 kDa.
  • the first reading frame encodes a 254 amino acid protein of which the first 26 aa constitute a putative leader peptide that strongly indicates an extracellular location of the protein.
  • the mature protein is thus 228 aa in length corresponding to a theoretical MW of 23149.0 Da and a Pi of 5.80.
  • the protein is named CFP23.
  • the second reading frame encodes an 231 aa protein of which the first 44 aa constitute a putative leader peptide that strongly indicates an extracellular location of the protein.
  • the mature protein is thus 187 aa in length corresponding to a theoretical MW of 19020.3 Da and a Pi of 7.03.
  • the protein is named CFP19A.
  • CFP19B CFP22A.
  • CFP23 CFP25A.
  • CFP27 CFP30A, CWP32 and CFP50 in the EMBL database.
  • amino acid sequences derived from the translated genes of the individual antigens were used for homology searching in the EMBL and Genbank databases using the TFASTA algorithm, in order to find homologous proteins and to address eventual functional roles of the antigens.
  • CFP7A has 44% identity and 70% similarity to hypothetical Methanococcus jannaschii protein (AT. jannaschii from base 1162199-1175341), as well as 43% and 38% identity and 68 and 64% similarity to the C-terminal part of B. stearofc ermo- philus pyruvate carboxylase and Streptococcus mutans biotin carboxyl carrier protein.
  • CFP7A contains a consensus sequence EAMKM for a biotin binding site motif which in this case was slightly modified (ESMKM in amino acid residues 34 to 38) .
  • CFP16 RplL gene, 130 aa. Identical to the AT. jbovis 50s ribosomal protein L7/L12 (ace. No P37381) .
  • CFP19 has 47% identity and 55% similarity to E. coli pectinesterase homolog (ybhC gene) in a 150 aa overlap.
  • CFP19A has between 38% and 45% identity to several cutinases from different fungal sp.
  • CFP19A has 46% identity and 61% similarity to CFP25 as well as 50% identity and 64% similarity to CFP21 (both proteins are earlier isolated from the ST-CF) .
  • CFP23 has between 38% and 46% identity to several cutinases from different fungal sp.
  • CFP23 has 46% identity and 61% similarity to CFP25 as well as 50% identity and 63% similarity to CFP21 (both proteins are earlier isolated from the ST-CF) .
  • CFP25A CFP25A has 95% identity in a 241 aa overlap to a putative AT. tuberculosis thymidylate synthase (450 aa accession No p28176) .
  • CFP27 CFP27 has 81% identity to a hypothetical AT. leprae protein and 64% identity and 78% similarity to Rhodococcus sp. proteasome beta-type subunit 2 (prcB(2) gene).
  • CFP30A has 67% identity to Rhodococcus proteasome alfa-type 1 subunit.
  • CWP32 The CWP32 N-terminal sequence is 100% identical to the Mycobacterium leprae sequence MLCB637.03.
  • CFP50 The CFP50 N-terminal sequence is 100% identical to a putative lipoamide dehydrogenase from AT. leprae (Accession 415183)
  • CFP7A, CFP8A, CFP8B Cloning of the genes encoding CFP7A, CFP8A, CFP8B. CFP16. CFP19. CFP19A. CFP22A. CFP23 , CFP25A, CFP27, CFP30A, CWP32. and CFP50.
  • the genes encoding CFP7A, CFP8A, CFP8B, CFP16, CFP19, CFP19A, CFP22A, CFP23, CFP25A, CFP27, CFP30A, CWP32 and CFP50 were all cloned into the expression vector pMCT6, by PCR amplification with gene specific primers, for recombinant expression in E. coli of the proteins.
  • PCR reactions contained 10 ng of AT. tuberculosis chromosomal DNA in IX low salt Taq+ buffer from Stratagene supplemented with 250 mM of each of the four nucleotides (Boehringer Mannheim), 0,5 mg/ml BSA (IgG technology), 1% DMSO (Merck), 5 pmoles of each primer and 0.5 unit Tag+ DNA polymerase (Stratagene) in 10 ml reaction volume. Reactions were initially heated to 94°C for 25 sec. and run for 30 cycles of the program; 94°C for 10 sec, 55°C for 10 sec. and 72°C for 90 sec, using thermocycler equipment from Idaho Technology.
  • Plasmid DNA was hereafter prepared from clones harbouring the desired fragments, digested with suitable restriction enzymes and subcloned into the expression vector pMCT6 in frame with 8 histidines which are added to the N- terminal of the expressed proteins.
  • the resulting clones were hereafter sequenced by use of the dideoxy chain termination method adapted for supercoiled DNA using the Sequenase DNA sequencing kit version 1.0 (United States Biochemical Corp., USA) and by cycle sequencing using the Dye Terminator system in combination with an automated gel reader (model 373A; Applied Biosystems) according to the instructions provided. Both strands of the DNA were sequenced.
  • CFP7A Primers used for cloning of cfplA:
  • OPBR-79 AAGAGTAGATCTATGATGGCCGAGGATGTTCGCG (SEQ ID NO: 95)
  • OPBR-80 CGGCGACGACGGATCCTACCGCGTCGG (SEQ ID NO: 96)
  • OPBR-79 and OPBR-80 create Bgrlll and BamHl sites, respectively, used for the cloning in pMCT6.
  • CFP8A Primers used for cloning of cfp8A:
  • CFP8A-F CTGAGATCTATGAACCTACGGCGCC (SEQ ID NO: 154)
  • CFP8A-R CTCCCATGGTACCCTAGGACCCGGGCAGCCCCGGC (SEQ ID NO: 155)
  • CFP8A-F and CFP8A-R create Bglll and Ncol sites, respectively, used for the cloning in pMCT6.
  • CFP8B Primers used for cloning of cfp8B:
  • CFP8B-F CTGAGATCTATGAGGCTGTCGTTGACCGC (SEQ ID NO: 156)
  • CFP8B-R CTCCCCGGGCTTAATAGTTGTTGCAGGAGC (SEQ ID NO: 157)
  • CFP8B-F and CFP8B-R create Bglll and Smal sites, respectively, used for the cloning in pMCT6.
  • CFP16 Primers used for cloning of cfpl6:
  • OPBR-104 CCGGGAGATCTATGGCAAAGCTCTCCACCGACG (SEQ ID NOs : 111 and 130)
  • OPBR-105 CGCTGGGCAGAGCTACTTGACGGTGACGGTGG (SEQ ID NOs: 112 and 131)
  • OPBR-104 and OPBR-105 create Bgrlii and Ncol sites, respect- ively, used for the cloning in pMCT6.
  • OPBR-96 GAGGAAGATCTATGACAACTTCACCCGACCCG (SEQ ID NO: 107)
  • OPBR-96 and OPBR-97 create Bgrlii and Ncol sites, respective- ly, used for the cloning in pMCT6.
  • CFP19A Primers used for cloning of cfpl9A:
  • OPBR-88 CCCCCCAGATCTGCACCACCGGCATCGGCGGGC (SEQ ID NO: 99)
  • OPBR-88 and OPBR-89 create Bgrlii and BamHl sites, respective- ly, used for the cloning in pMCT6.
  • CFP22A Primers used for cloning of cfp22A:
  • OPBR-90 CCGGCTGAGATCTATGACAGAATACGAAGGGC (SEQ ID NO : 101)
  • OPBR-91 CCCCGCCAGGGAACTAGAGGCGGC (SEQ ID NO: 102)
  • OPBR-90 and OPBR-91 create Bgili and Ncol sites, respective- ly, used for the cloning in pMCT6.
  • OPBR-86 CCTTGGGAGATCTTTGGACCCCGGTTGC (SEQ ID NO: 97)
  • OPBR-87 GACGAGATCTTATGGGCTTACTGAC (SEQ ID NO: 98)
  • OPBR-86 and OPBR-87 both create a B lll site used for the cloning in pMCT6.
  • CFP25A Primers used for cloning of cfp25A:
  • OPBR-106 GGCCCAGATCTATGGCCATTGAGGTTTCGGTGTTGC (SEQ ID NO: 113)
  • OPBR-107 CGCCGTGTTGCATGGCAGCGCTGAGC (SEQ ID NO: 114)
  • OPBR-106 and OPBR-107 create Bgrlii and Ncol sites, respect- ively, used for the cloning in pMCT6.
  • OPBR-92 CTGCCGAGATCTACCACCATTGTCGCGCTGAAATACCC (SEQ ID NO: 103)
  • OPBR-93 CGCCATGGCCTTACGCGCCAACTCG (SEQ ID NO: 104)
  • OPBR-92 and OPBR-93 create Bgrlii and Ncol sites, respective- ly, used for the cloning in pMCT6.
  • CFP30A Primers used for cloning of cfp30A:
  • OPBR-94 GGCGGAGATCTGTGAGTTTTCCGTATTTCATC (SEQ ID NO: 105)
  • OPBR-95 CGCGTCGAGCCATGGTTAGGCGCAG (SEQ ID NO: 106)
  • OPBR-94 and OPBR-95 create Bgrlii and Ncol sites, respective- ly, used for the cloning in pMCT6.
  • C P32-F GCTTAGATCTATGATTTTCTGGGCAACCAGGTA (SEQ ID NO: 158)
  • CWP32-F and CWP32-R create Bglll and Nco sites, respective- ly, used for the cloning in pMCT6.
  • CFP50 Primers used for cloning of cfp50:
  • OPBR-100 GGCCGAGATCTGTGACCCACTATGACGTCGTCG (SEQ ID NO: 109)
  • OPBR-101 GGCGCCCATGGTCAGAAATTGATCATGTGGCCAA (SEQ ID NO: 110)
  • OPBR-100 and OPBR-101 create Bglll and Ncol sites, respect- ively, used for the cloning in pMCT6.
  • CFP27, CFP30A, CWP32, and CFP50 proteins are examples of CFP7A, CFP8A.
  • CFP8B CFP16, CFP19. CFP19A, CFP22A.
  • CFP25A CFP27, CFP30A, CWP32, and CFP50 proteins.
  • the lysate was applied to a column containing 25 ml of resuspended Talon resin (Clontech, Palo Alto, USA) .
  • the column was washed and eluted as described by the manufacturers .
  • ST-CF was concentrated approximately 10 -fold by ultrafiltration and ammonium sulphate precipitation at 80 % saturation. Proteins were redissolved in 50 mM sodium phosphate, 1.5 M ammonium sulphate, pH 8.5, and subjected to thiophilic adsorption chromatography on an Affi-T gel column (Kem-En-Tec) . Proteins were eluted by a 1.5 to 0 M decreasing gradient of ammonium sulphate. Fractions with similar band patterns in SDS-PAGE were pooled and anion exchange chromatography was performed on a Mono Q HR 5/5 column connected to an FPLC system (Pharmacia) .
  • the column was equilibrated with 10 mM Tris -HCl, pH 8.5, and the elution was performed with a gradient of NaCl from 0 to 1 M. Fractions containing well separated bands in SDS-PAGE were selected.
  • N-terminal amino acid sequence analysis was performed on a Procise 494 sequencer (applied Biosystems) .
  • CFP7B PQGTVKWFNAEKGFG (SEQ ID NO: 168)
  • CFPIOA NVTVSIPTILRPXXX (SEQ ID NO: 169)
  • CFP11 TRFMTDPHAMRDMAG (SEQ ID NO: 170)
  • CFP30B PKRSEYRQGTPNWVD (SEQ ID NO: 171) "X" denotes an amino acid which could not be determined by the sequencing method used.
  • N-terminal amino acid sequence from each of the proteins was used for a homology search using the blast program of the Sanger Mycobacterium tuberculosis genome database:
  • CFP7B For CFP7B a sequence 100% identical to 15 N-terminal amino acids was found on contig TB_2044 and on locus MTY15C10.04 with EMBL accession number: z95436. The identity was found within an open reading frame of 67 amino acids length corresponding to a theoretical MW of 7240 Da and a pi of 5.18.
  • amino acid sequences of the purified antigens as picked from the Sanger database are shown in the following list.
  • PCR reactions contained 10 ng of AT. tuberculosis chromosomal DNA in IX low salt Taq+ buffer from Stratagene supplemented with 250 mM of each of the four nucleotides (Boehringer
  • Plasmid DNA was hereafter prepared from clones harbouring the desired fragments, digested with suitable restriction enzymes and subcloned into the expression vector pMCT6 in frame with 8 histidines which are added to the N- terminal of the expressed proteins.
  • the resulting clones were hereafter sequenced by use of the dideoxy chain termination method adapted for supercoiled DNA using the Sequenase DNA sequencing kit version 1.0 (United States Biochemical Corp., USA) and by cycle sequencing using the Dye Terminator system in combination with an automated gel reader (model 373A; Applied Biosystems) according to the instructions provided. Both strands of the DNA were sequenced.
  • CFP7B Primers used for cloning of cfp7B:
  • CFP7B-F CTGAGATCTAGAATGCCACAGGGAACTGTG (SEQ ID NO : 160)
  • CFP7B-R TCTCCCGGGGGTAACTCAGAGCGAGCGGAC (SEQ ID NO: 161)
  • CFP7B-F and CFP7B-R create Bglll and Smal sites, respectively, used for the cloning in pMCT6.

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