WO2000021983A2

WO2000021983A2 - Tuberculosis vaccine and diagnostic reagents based on antigens from the mycobacterium tuberculosis cell

Info

Publication number: WO2000021983A2
Application number: PCT/DK1999/000538
Authority: WO
Inventors: Peter Andersen; Karin Weldingh; Christina Veggerby Hansen; Walter Florio; Li Mei Meng Okkels; Rikke Louise Vinther SKJØT; Ida Rosenkrands
Original assignee: Statens Serum Institut
Priority date: 1998-10-08
Filing date: 1999-10-08
Publication date: 2000-04-20
Also published as: CA2346218A1; EP1117683A2; WO2000021983A3; AU766093B2; AU6078499A

Abstract

The present invention relates to substantially pure polypeptides, which has a sequence identity of at least 80 % to an amino acid sequence disclosed, or which is a subsequence of at least 6 amino acids thereof, preferably a B- or T-cell epitope of the polypeptides disclosed. The polypeptide or the subsequence thereof has at least one of nine properties. The use of the disclosed polypeptides in medicine is disclosed, preferably as vaccine or diagnostic agents relating to virulent Mycobacterium. The invention further relates to the nucleotide sequences disclosed and the nucleotide sequences encoding the disclosed polypeptides. Medical and non-medical use of the nucleotide sequences is disclosed.

Description

TB vaccine and diagnostic based on antigens from the M. tuberculosis cell

BACKGROUND OF THE INVENTION

Human tuberculosis (TB) caused by Mycobacterium tuberculosis is a serious global health problem responsible for approximately 3 million deaths annually, according to WHO. The world-wide incidence of new tuberculosis cases has been progressively falling for the last decade but the recent years have markedly changed this trend due to the advent of AIDS and the appearance of multidrug resistant strains of Mycobacterium tuberculosis.

The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. BCG generally induces a high level of acquired resistance in animal models of tuberculosis, 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 because BCG vaccination impairs the specificity of the Tuberculin skin test for diagnosis of TB infection.

This makes the development of a new and improved vaccine against tuberculosis an urgent matter which has been given a very high priority by the WHO. Many attempts have been made to define the protective Mycobacterial substances and a series of experiments were conducted to compare the protective efficacy of vaccination with live versus killed preparations of M. tuberculosis (Orme IM. Infect.lmmun.1988; 56:3310-12). The conclusion of these studies was that vaccination of mice with dead M. tuberculosis administered without adjuvants only induced short term protection against TB, whereas live M. tuberculosis vaccines induced efficient immunological memory. This information was the background for the further search for protective substances focused on antigens actively secreted from the live Mycobacteria (Andersen P. Infect.lmmun.1994; 62:2536- 44, Horwitz et al. Proc. Natl Acad. Sci. USA 1995; 92:1530-4, Pal PG et al. Infect. Immun. 1992; 60: 4781-92).

DETAILED DISCLOSURE OF THE INVENTION

The present inventors conducted a study comparing the long term protection against TB after vaccination three times with killed M. tuberculosis administered with DDA as an adjuvant with the long term protection obtained with ST-CF, and surprisingly similar levels of long term protection induced in the group receiving killed bacteria were found as in the group vaccinated with ST-CF/DDA (figure 1).

This leads to the conclusion that protective components can be found also among the components of the cell wall, cell membrane or cytosol derived from a preparation of dead virulent Mycobacteria.

It is thus an object of the present invention to provide a composition for the generation or determination of an immune response against a virulent Mycobacterium such as a vaccine for immunising a mammal, including a human being, against disease caused by a virulent Mycobacterium and a diagnostic reagent for the diagnosis of an infection with a virulent Mycobacterium.

By the terms "somatic protein" or "protein derived from the cell wall, the cell membrane or the cytosol", or by the abbreviation "SPE" is understood a polypeptide or a protein extract obtainable from a cell or a part. A preferred method to obtain a somatic protein is described in the examples, especially examples 2, 3, 4, and 5.

By the term "virulent Mycobacterium" is understood a bacterium capable of causing the tuberculosis disease in a mammal including a human being. Examples of virulent Mycobacteria are M. tuberculosis, M. africanum, and M. bovis.

By "a TB patient" is understood an individual with culture or microscopically proven infection with virulent Mycobacteria, and/or an individual clinically diagnosed with TB and who is responsive to anti-TB chemotherapy. Culture, microscopy and clinical diagnosis of TB is well known by the person skilled in the art.

A significant decrease or increase is defined as a decrease or increase which is significant at the 95% level by comparison of immunised and placebo-treated groups using an appropriate statistical analysis such as a Student's two-tailed T test.

By the term "PPD positive individual" is understood an individual with a positive Mantoux test or an individual where PPD induces an increase in in vitro recall response determined by release of IFN-γ of at least 1 ,000 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) or whole blood, the induction being performed by the addition of 2.5 to 5 μg PPD/ml to a suspension comprising about 1.0 to 2.5 x 10⁵ PBMC, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 5 days after the addition of PPD to the suspension compared to the release of IFN-γ without the addition of PPD.

By the term "delayed type hypersensitivity reaction" is understood a T-cell mediated inflammatory response elicited after the injection of a polypeptide into or application to the skin, said inflammatory response appearing 72-96 hours after the polypeptide injection or application.

By the term "IFN-γ" is understood interferon-gamma.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations thereof such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

By the term "a polypeptide" in the present application is generally understood a polypeptide of the invention, as will be described later. It is also within the meaning of "a polypeptide" that several polypeptides can be used, i.e. in the present context "a" means "at least one" unless explicitly indicated otherwise. The "polypeptide" is used to referrer to 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, oiigopeptide, or polypeptide which may be chemically modified by being phosphorylated, glycosylated, by being lipidated, or by comprising prosthetic groups). The definition of polypeptides 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 synthesised polypeptides. Within the scope of the invention is a polypeptide which is at least 6 amino acids long, preferably 7, such as 8, 9, 10, 11, 12 , 13, 14 amino acids long, preferably at least 15 amino acids, such as 15, 16, 17, 18, 19, 20 amino acids long. However, also longer polypeptides having a length of e.g. 25, 50, 75, 100, 125, 150, 175 or 200 amino acids are within the scope of the present invention. In the present context the term "purified polypeptide" 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 Y₂%). It is preferred that the substantially pure polypeptide is at least 96% pure, i.e. that the polypeptide constitutes at least 96% by weight of total polypeptide material present in the preparation, 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 is in "essentially pure form", i.e. that the polypeptide 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. This can be accomplished by preparing the polypeptide by means of recombinant methods in a non-mycobacterial host cell as will be described in detail below, or by synthesising the polypeptide by the well-known methods of solid or liquid phase peptide synthesis, e.g. by the method described by Merrifieid or variations thereof.

By the term "non-naturally occurring polypeptide" is understood a polypeptide that does not occur naturally. This means that the polypeptide is substantially pure, and/or that the polypeptide has been synthesised in the laboratory, and/or that the polypeptide has been produced by means of recombinant technology.

By the terms "analogue" and "subsequence" when used in connection with polypeptides is meant any polypeptide having the same immunological characteristics as the polypeptides of the invention described above with respect to the ability to confer increased resistance to infection with virulent Mycobacteria. Thus, included is also a polypeptide from a different source, such as from another bacterium or even from a eukaryotic cell.

The term "sequence identity" indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. If the two sequences to be compared are not of equal length, they must be aligned to best possible fit. The sequence identity can be calculated as

^\N„r^Nώf)ι o _{^ wnere}j_n pg_dlf is the total number of non-identical residues in the two sequences

Nref when aligned and wherein N_ref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (N_dlf=2 and N_re =8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (N_dlf=2 and N_ref=8). Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program or the BLASTN program (Pearson W.R and D.J. Lipman (1988) PNAS USA 85:2444-

2448)(www.ncbi. nlm.nih.gov/BLAST). In one aspect of the invention, alignment is performed with the global align algorithm with default parameters as described by X. Huang and W. Miller. Adv. Appl. Math. (1991) 12:337-357, available at http://www.ch.embnet.org/software/LALIGN_form.html.

When the term nucleotide is used in the following, it should be understood in the broadest sense. That is, most often the nucleotide should be considered as DNA. However, when DNA can be substituted with RNA, the term nucleotide should be read to include RNA embodiments which will be apparent for the person skilled in the art. For the purposes of hybridisation, PNA or LNA may be used instead of DNA. PNA has been shown to exhibit a very dynamic hybridisation profile and is described in Nielsen P E et al, 1991 , Science 254: 1497-1500). LNA (Locked Nucleic Acids) is a recently introduced ohgonucleotide analogue containing bicyclo nucleoside monomers (Koshkin et al., 1998, 54, 3607- 3630;Nielsen, N.K. et al. J.Am.Chem.Soc 1998, 120, 5458-5463).

It is surprisingly demonstrated herein that the SPE comprising polypeptides isolated from the cell wall, cell membrane and cytosol induces protective immunity against infection with M. tuberculosis in an animal model, when injected with an adjuvant. It is contemplated that these polypeptides, either alone or in combination, can be used as vaccine components.

It is further demonstrated that several polypeptides isolated from the cell wall, cell membrane or cytosol are recognised by human tuberculosis antisera. Therefore it is considered likely that these polypeptides, either alone or in combination, can be useful as diagnostic reagents in the diagnosis of tuberculosis.

One embodiment of the invention relates to a method for producing a polypeptide in an immunological composition comprising the steps of: a) killing a sample of virulent Mycobacteria; b) centrifugating the sample of a); c) resuspending the pellet of b) in PBS; d) centrifugating the suspension of c); e) extracting soluble proteins from the cytosol as well as cell wall and cell membrane from the supernatant of d) with SDS; f) centrifugating the extract of e); g) precipitating the supernatant of f) in cold acetone; h) resuspending the precipitate of g) in PBS; i) applying the resuspension of h) to 2 dimensional electrophoresis; j) blotting the gel of i) to a PVDF membrane; k) subjecting the spots on j) to N-terminal sequencing;

I) searching a database for homology with the sequence of k) to identify the nucleotide sequence; m) cloning the nucleotide sequence of I) into an expression system; n) isolating and purifying the polypeptide expressed in m); and o) formulating the polypeptide of n) with an adjuvant substance in an immunological composition.

Another embodiment is a method of producing a polypeptide originating from the cell wall in an immunological composition, said method comprising the steps of: a) killing a sample of virulent Mycobacteria; b) centrifugating the sample of a) c) resuspending the pellet of b) in PBS supplemented with EDTA and phenylmethylsulfonyl fluoride and sonicating for 15 min d) lysing the suspension of c) e) centrifugating the lysed suspension of d) f) resuspending the pellet of e) in homogenising buffer g) incubating the suspension of f) with RNase and DNase overnight h) incubating the suspension of g) with SDS i) centrifugating the incubated suspension of h) j) incubating the supernatant of i) with SDS k) precipitating the incubated supernatant of j) with acetone

I) resuspending the precipitate of k) in PBS m) subjecting the suspension of I) to a Triton X-114 extraction n) applying the resuspension of m) to 2 dimensional electrophoresis; o) blotting the gel of n) to a PVDF membrane; p) subjecting the spots on o) to N-terminal sequencing; q) searching a database for homology with the sequence of p) to identify the nucleotide sequence; r) cloning the nucleotide sequence of q) into an expression system; s) isolating and purifying the polypeptide expressed in r); and t) formulating the polypeptide of s) with an adjuvant substance in an immunological composition.

A third embodiment is a method of producing a polypeptide originating from the cell membrane in an immunological composition, said method comprising the steps of: a) killing a sample of virulent Mycobacteria; b) centrifugating the sample of a) c) resuspending the pellet of b) in PBS supplemented with EDTA and phenylmethylsulfonyl fluoride and sonicating for 15 min d) lysing the suspension of c) e) centrifugating the lysed suspension of d) f) ultracentrifugating the supernatant of e) g) resuspending the pellet of f) in PBS h) subject the suspension of g) to a Triton X-114 extraction i) applying the resuspension of h) to 2 dimensional electrophoresis; j) blotting the gel of i) to a PVDF membrane; k) subjecting the spots on j) to N-terminal sequencing;

I) searching a database for homology with the sequence of k) to identify the nucleotide sequence; m) cloning the nucleotide sequence of I) into an expression system; and n) isolating and purifying the polypeptide expressed in m); o) formulating the polypeptide of n) with an adjuvant substance in an immunological composition.

A fourth embodiment is a method of producing a polypeptide originating from the cytosol in an immunological composition comprising the steps of: a) killing a sample of virulent Mycobacteria; b) centrifugating the sample of a) c) resuspending the pellet of b) in PBS supplemented with EDTA and phenylmethylsulfonyl fluoride and sonicating for 15 min d) lysing the suspension of c) e) centrifugating the lysed suspension of d) f) ultracentrifugating the supernatant of e) g) precipitating the supernatant of f) with acetone h) resuspending the precipitate of g) in PBS i) applying the resuspension of h) to 2 dimensional electrophoresis; j) plotting the gel of i) to a PVDF membrane; k) subjecting the spots on j) to N-terminal sequencing;

In particular, the invention relates to a polypeptide obtainable by a method as described above which polypeptide has at least one of the following properties:

i) it induces an in vitro recall response determined by a release of IFN-γ of at least 1 ,500 pg/ml from reactivated memory T-lymphocytes withdrawn from a C57BI/6J mouse within 4 days after the mouse has been rechallenged with 1 x 10⁶ virulent Mycobacteria, the induction being performed by the addition of the polypeptide to a suspension comprising about 2 x 10⁵ cells isolated from the spleen of said mouse, the addition of the polypeptide resulting in a concentration of the polypeptide of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 3 days after the addition of the polypeptide to the suspension,

ii) it induces an in vitro response during primary infection with virulent Mycobacteria, determined by release of IFN-γ of at least 1 ,500 pg/ml from T-lymphocytes withdrawn from a mouse within 28 days after the mouse has been infected with 5 x 10⁴ virulent Mycobacteria, the induction being performed by the addition of the polypeptide to a suspension comprising about 2 x 10⁵ cells isolated from the spleen, the addition of the polypeptide resulting in a concentration of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 3 days after the addition of the polypeptide to the suspension, iii) it induces a protective immunity determined by vaccinating an animal model with the polypeptide and an adjuvant in a total of three times with two weeks interval starting at 6- 8 weeks of age, 6 weeks after the last vaccination challenging with 5 x 10⁶ virulent Mycobacterialxx by aerosol and determining a significant decrease in the number of bacteria recoverable from the lung 6 weeks after the animal has been challenged, compared to the number recovered from the same organ in a mammal given placebo treatment,

iv) it induces in vitro recall response determined by release of IFN-γ of at least 1 ,000 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) or whole blood withdrawn from TB patients 0-6 months after diagnosis, or PPD positive individual, the induction being performed by the addition of the polypeptide to a suspension comprising about 1.0 to 2.5 x 10⁵ PBMC or whole blood cells, the addition of the polypeptide resulting in a concentration of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 5 days after the addition of the polypeptide to the suspension,

v) it induces a specific antibody response in a TB patient as determined by an ELISA technique or a western blot when the whole blood is diluted 1 :20 in PBS and stimulated with the polypeptide in a concentration of at the most 20 μg/ml and induces an OD of at least 0.1 in ELISA, or a visual response in western blot.

vi) it induces a positive in vitro response determined by release of IFN-γ of at least 500 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) withdrawn from an individual who is clinically or subclinically infected with a virulent Mycobacterium, the induction being performed by the addition of the polypeptide to a suspension comprising about 1.0 to 2.5 x 10⁵ PBMC, the addition of the polypeptide resulting in a concentration of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determi- nation of IFN-γ in supernatant harvested 5 days after the addition of the polypeptide to the suspension, and preferably does not induce such an IFN-γ release in an individual not infected with a virulent Mycobacterium,

vii) it induces a positive in vitro response determined by release of IFN-γ of at least 500 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) withdrawn from an individual clinically or subclinically infected with a virulent Mycobacterium, the induction being performed by the addition of the polypeptide to a suspension comprising about 1.0 to 2.5 x 10⁵ PBMC, the addition of the polypeptide resulting in a concentration of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 5 days after the addition of the polypeptide to the suspension, and preferably does not induce such an IFN-γ release in an individual not infected with a virulent Mycobacterium,

viii) it induces a positive DTH response determined by intradermal injection or local application patch of at most 100μg of the polypeptide to an individual who is clinically or subclinically infected with a virulent Mycobacterium, a positive response having a diameter of at least 10 mm 72-96 hours after the injection or application,

ix) it induces a positive DTH response determined by intradermal injection or local application patch of at most 100 μg of the polypeptide to an individual who is clinically or subclinically infected with a virulent Mycobacterium, a positive response having a diameter of at least 10 mm 72-96 hours after the injection, and preferably does not induce a such response in an individual who has a cleared infection with a virulent Mycobacterium. >

Any polypeptide fulfilling one or more of the above properties and which is obtainable from either the cell wall, cell membrane or the cytosol is within the scope of the present invention.

The property described in i) will also be satisfied if the release of IFN-γ from reactivated memory T-lymphocytes is 2,000 pg/ml, such as 3,000 pg/ml. In an alternative embodiment of the invention, the immunological effect of the polypeptide could be determined by comparing the IFN-γ release as described with the IFN-γ release from a similar assay, wherein the polypeptide is not added, a significant increase being indicative of an immunologically effective polypeptide. In a preferred embodiment of the invention, the addition of the polypeptide results in a concentration of not more than 20 μg per ml suspension, such as 15 μg, 10 μg, 5 μg, 3 μg, 2 μg, or 1 μg polypeptide per ml suspension. The property mentions as an example the mouse strain C57BI/6J as the animal model. As will be known by a person skilled in the art, due to genetic variation, different strains may react with immune responses of varying strength to the same polypeptide. It is presently unknown which strains of mice will give the best predictability of immunogenic reactivity in which human population. Therefore, it is important to test other mouse strains, such as C3H/HeN, CBA (preferably CBA/J), DBA (preferably DBA/2J), A/J, AKR/N, DBA 1 J, FVB/N, SJL/N, 129/SvJ, C3H/HeJ-Lps or BALB mice (preferably BALB/cA, BALB/cJ). It is presently contemplated that also a similar test performed in another animal model such as a guinea pig or a rat will have clinical predictability. In order to obtain good clinical predictability to humans, it is contemplated that any farm animal, such as a cow, pig, or deer, or any primate will have clinical predictability and thus serve as an animal model.

It should be noted, moreover, that tuberculosis disease also affects a number of different animal species such as cows, primates, guinea pigs, badgers, possums, and deers. A polypeptide which has proven effective in any of the models mentioned above may be of interest for animal treatment even if it is not effective in a human being.

It is proposed to measure the release of IFN-γ from reactivated T lymphocytes withdrawn from a C57BI/6J mouse within 4 days after the mouse has been rechallenged with virulent Mycobacteria. This is due to the fact that when an immune host mounts a protective immune response, the specific T-cells responsible for the early recognition of the infected macrophage stimulate a powerful bactericidal activity through their production of IFN-γ (Rook, G.A.W. (1990) Res. Microbiol. 141 :253-256; Flesch, I. et S.H.E. Kaufmann ( 1987) J Immunol.138(12):4408-13). However other cytokines could be relevant when monitoring the immunological response to the polypeptide, such as IL-12, TNF-α, IL-4, IL- 5, IL-10, IL-6, TGF-β. Usually one or more cytokines will be measured utilising for example the PCR technique or ELISA. It will be appreciated by the person skilled in the art that a significant increase or decrease in the amount of any of these cytokines induced by a specific polypeptide can be used in evaluation of the immunological efficacy of the polypeptide. The ability of a polypeptide to induce a IFN-γ response is presently believed to be the most relevant correlate of protective immunity as mice with a disruption of the gene coding for IFN-γ are unable to control a mycobacterial infection and die very rapidly with widespread dissemination, caseous necrosis and large abscesses (Flynn et al (1993) J.Exp.Med 178: 2249-2254, Cooper et al (1993) J.Exp.Med. 178:2243-2248). A specific model for obtaining information regarding the antigenic targets of a protective immunity in the memory model was originally developed by Lefford (Lefford et al (1973) Immunology 25:703) and has been used extensively in the recent years (Orme et al (1988). Infect.lmmun. 140:3589, P.Andersen and I. Heron (1993) J. Immunol.154:3359).

The property described in ii) will also be satisfied if the release of IFN-γ from T- lymphocytes withdrawn during primary infection is 2,000 pg/ml, such as 3,000 pg/ml. The comments on property i) regarding a significant increase in IFN-γ, concentration of polypeptide, animal model, and other cytokines are equally relevant to property ii), and wee versa.

The property described in iii) will also be satisfied if the protective immunity is determined by challenging the mouse more than 6 weeks after the last vaccination challenge such as 7 weeks, preferably 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or 15 weeks. In one embodiment of the invention the bacteria are recovered from the spleen more than 6 weeks after the last vaccination challenge such as 7 weeks, preferably 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or 15 weeks. In another embodiment of the invention, the last vaccination challenge is given subcutaneously with 5x10⁴ virulent Mycobacteria. As will be known by the person skilled in the art, the number of viable bacteria in the lung is presently considered to be relevant to the degree of bacterial infection of the animal. An equally important measure is the determination of the number of viable bacteria in the spleen, lymph node, or blood.

The amount of polypeptide and adjuvant used for vaccinating will depend on the animal model used, e.g. the mouse strain. When a mouse model is used it is preferred that the amount of polypeptide used for vaccinating the mouse is between 2 and 20 μg, such as between 5 and 15 μg, preferably 10 μg. For larger animals such as guinea pigs, deers, cows, primates, badgers, and possums higher doses such as 5 to 50 μg of a single polypeptide are preferred.

The comments on property i) regarding concentration of polypeptide and animal model are equally relevant to property iii), and vice versa.

In another aspect of property iii), the mice, or other animal model, are given the standard lethal dose of virulent Mycobacteria. The standard lethal dose varies from around 3x10⁵ to around 5x10⁶ virulent Mycobacteria depending on the specific strain of virulent Mycobacteria and strain of mice. The mortality in the mice is then monitored and compared to a placebo vaccinated control group. A significant decrease in mortality, measured as the mean survival time, will be indicative of an immunologically effective polypeptide. In a very recent paper it is shown that there is good correlation between 5 mortality of the individual animals and the bacterial counts in the same animals. (S.Baldwin (1998) Infect.lmmun 66:2951-2959).

The property described in iv) will also be satisfied if the release of IFN-γ from PBMC is determined in PBMC withdrawn from TB patients or PPD positive individuals more than 6 10 months after diagnosis such as 9 months, 1 year, 2 years, 5 years, or 10 years after diagnosis.

The comments on property i) regarding significant increase in IFN-γ, concentration of polypeptide, and other cytokines are equally relevant to property iv).

15

The property described in v) will in particular be satisfied, if the ELISA is performed as follows: the polypeptide of interest in the concentration of 1 to 10 μg/ml is coated on a 96 wells polystyrene plate (NUNC, Denmark) and after a washing step with phosphate buffer pH 7.3, containing 0.37 M NaCl and 0.5% Tween-20 the serum or plasma from a TB

20 patient is applied in dilution's from 1 :10 to 1 :1000 in PBS with 1% Tween-20. Binding of an antibody to the polypeptide is determined by addition of a labeled (e.g. peroxidase labeled) secondary antibody and reaction is thereafter visualized by the use of OPD and H₂O₂ as described by the manufacturer (DAKO, Denmark). The OD value in each well is determined using an appropriate ELISA reader.

25

In a preferred embodiment the western blot is performed as follows: The polypeptide is applied in concentrations from 1-40 μg to a SDS-PAGE and after electrophoresis the polypeptide is transferred to a membrane e.g. nitrocellulose or PVDF. The membrane is thereafter washed in phosphate buffer, pH 7.3, containing 0.37 M NaCl and 0.5% Tween-

30 20 for 30 min. The sera obtained from one or more TB patients were diluted 1 : 10 to 1 :1000 in phosphate buffer pH 7.3 containing 0.37 M NaCl. The membrane is hereafter washed four times five minutes in binding buffer and incubated with peroxidase- or phosphates-labeled secondary antibody. Reaction is then visualized using the staining method recommended by the manufacture (DAKO, Denmark).

35 The property described in vi) will in particular be satisfied if the polypeptide does not induce such an IFN-γ release in an individual not infected with a virulent Mycobacterium, i.e. an individual who has been BCG vaccinated or infected with Mycobacterium avium or sensitised by non-tuberculosis Mycobacterium (NTM). The comments on property i) 5 regarding significant increase in IFN-γ, concentration of polypeptide, and other cytokines are equally relevant to property vi).

The property described in vii) will in particular be satisfied if the polypeptide does not induce such an IFN-γ release in an individual cleared of an infection with a virulent 10 Mycobacterium, i.e. which does not have any positive culture, microscopically or clinically proven ongoing infection with virulent Mycobacterium. The comments on property i) regarding significant increase in IFN-γ, concentration of polypeptide, and other cytokines are equally relevant to property vii).

15 The property described in viii) will in particular be satisfied if the polypeptide does not induce such a response in an individual not infected with a virulent Mycobacterium, i.e. an individual who has been BCG vaccinated or infected with Mycobacterium avium or sensitised by non-tuberculosis Mycobacterium. In a preferred embodiment the amount of polypeptide intradermally injected or applied is 90 μg, such as 80 μg, 70 μg, 60 μg,

20 50 μg, 40 μg, or 30 μg. In another embodiment of the invention, the diameter of the positive response is at least 11 mm, such as 12 mm, 13 mm, 14 mm, or 15 mm. In a preferred embodiment the induration of erythema or both could be determined after administration of the polypeptide by intradermal injection, patch test or multipuncture. The reaction diameter could be positive after more than 48, such as 72 or 96 hours.

25

The property described in ix) will in particular be satisfied if the polypeptide does not induce such a response in an individual cleared of an infection with a virulent Mycobacterium, i.e. which does not have any positive culture or microscopically proven ongoing infection with virulent Mycobacterium. The comments on property viii) regarding

30 the amount of polypeptide intradermally injected or applied and the diameter of the positive response are equally relevant to property ix).

Preferred embodiments of the invention are the specific polypeptides which have been identified and analogues and subsequences thereof. It has been noted that none of the 35 identified polypeptides in the examples include a signal sequence. Until the present invention was made, it was unknown that the polypeptides with the amino acid sequences disclosed in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 75, 77 and 79 are expressed in live virulent Mycobacterium. These polypeptides in purified form, or non-naturally occurring, i.e. recombinantly or synthetically produced, are considered part of the invention. It is understood that a polypeptide which has any of the properties i) - ix) and has a sequence identity of at least 80% with any of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 75, 77 and 79 or has a sequence identity of at least 80% to any subsequence thereof is considered part of the invention. In a preferred embodiment the sequence identity is at least 80%, such as 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%. Furthermore, any T cell epitope of the polypeptides disclosed in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 75, 77 and 79 is considered part of the invention. Also, any B-cell epitope of the polypeptides disclosed in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 75, 77 and 79 is considered part of the invention.

Although 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. Hence it is preferred that 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, or at least 30 amino acid residues.

In both immunodiagnostics and vaccine preparation, it is often possible and practical to prepare antigens from segments 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 (Hopp et Woods, (1981), Proc Natl Acad Sci USA 78/6:3824-8) hydrophobicity analysis (see, e.g., Jameson and Wolf, (1988) Comput Appl Biosci, 4(1): 181 -6; Kyte and Doolittle, (1982) J Mol Biol, 157(1): 105-32; 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. Alternatively, in order to identify relevant T-cell epitopes which are recognised during an immune response, it is also possible to use a "brute force" method: Since T-cell epitopes are linear, deletion mutants of polypeptides will, if constructed systematically, reveal what regions of the polypeptide are essential in immune recognition, e.g. by subjecting these deletion mutants to the IFN-γ assay described herein. A presently preferred method utilises overlapping oligomers (preferably synthetic ones having a length of e.g. 20 amino acid residues) derived from the polypeptide. Some of these will give a positive response in the IFN-γ assay whereas others will not. A preferred T-cell epitope is a T-helper cell epitope or a cytotoxic T-cell epitope.

B-cell epitopes may be linear or spatial. The three-dimensional structure of a protein is often such that amino acids, which are located distant from each other in the one- dimensional structure, are located near to each other in the folded protein. Within the meaning of the present context, the expression epitope is intended to comprise the one- and three-dimensional structure as well as mimics thereof. The term is further intended to include discontinuous B-cell epitopes. The linear B-cell epitopes can be identified in a similar manner as described for the T-cell epitopes above. However, when identifying B- cell epitopes the assay should be an ELISA using overlapping oligomers derived from the polypeptide as the coating layer on a microtiter plate as described elsewhere.

A non-naturally occurring polypeptide, an analogue, a subsequence, a T-cell epitope and/or a B-cell epitope of any of the described polypeptides are defined as any non- naturally occurring polypeptide, analogue, subsequence, T-cell epitope and/or B-cell epitope of any of the polypeptides having any of the properties i)-ix).

Table 1 lists the antigens of the invention. Table 1 The antigens of the invention by the names used herein as well as by reference to relevant SEQ ID NOs of N-terminal sequences, full amino acid sequences and sequences of nucleotides encoding the antigens

Antigen N-Terminal sequence Nucleotide Amino acid sequence SEQ ID NO: sequence SEQ ID NO: SEQ ID NO:

TB10C 45 1 2

TB13A 50 3 4

TB15 39 5 6

TB15A 46 7 8

TB17 47 9 10

TB18 40 11 12

TB21 41 13 14

TB24 48 15 16

TB27B 49 17 18

TB33 42 19 20

TB38 43 21 22

TB54 44 23 24

TB64 57 25 26

TB11 B 51 27 28

TB16 52 29 30

TB16A 53 31 32

TB32 54 33 34

TB32A 55 35 36

TB51 56 37 38

TB12.5 80 74 75

TB20.6 81 76 77

TB40.8 82 78 79

Each of the polypeptides may be characterised by specific amino acid and nucleic acid sequences. It will be understood that such 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. A preferred nucleotide sequence encoding a polypeptide of the invention is a nucleotide sequence which 1) is a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 74, 76 and 78 or an analogue of said sequence which hybridises with any of the nucleotide sequences shown in SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 74, 76 or 78 or a nucleotide sequence complementary thereto, or a specific part thereof, preferably under stringent hybridisation conditions. By stringent conditions is understood, as defined in the art, 5-10°C under the melting point T_m, cf. Sambrook et al, 1989, pages 11.45-11.49, and/or

2) encodes a polypeptide, the amino acid sequence of which has a 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 75, 77 and 79 and/or

3) constitutes a subsequence of any of the above mentioned nucleotide sequences, and/or

4) constitutes a subsequence of any of the above mentioned polypeptide sequences.

The terms "analogue" or "subsequence" when used in connection with the nucleotide fragments of the invention are thus intended to indicate a nucleotide sequence which encodes a polypeptide exhibiting identical or substantially identical immunological properties to a polypeptide encoded by the nucleotide fragment of the invention shown in any of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 74, 76 or 78, allowing for minor variations which do not have an adverse effect on the ligand binding properties and/or biological function and/or immunogenicity as compared to any of the polypeptides of the invention or which give interesting and useful novel binding properties or biological functions and immunogenicities etc. of the analogue and/or subsequence. The analogous nucleotide fragment or nucleotide sequence may be derived from a bacterium, a mammal, or a human or may be partially or completely of synthetic origin. The analogue and/or subsequence may also be derived through the use of recombinant nucleotide techniques.

Furthermore, the terms "analogue" and "subsequence" are intended to allow 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 polypeptide encoded by a nucleotide fragment or a subsequence thereof. The term "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, and "rearrangement" is intended to mean that two or more nucleotide residues have been exchanged with each other.

It is well known that the same amino acid may be encoded by various codons, the codon usage being related, inter alia, to the preference of the organisms in question expressing the nucleotide sequence. Thus, at least one nucleotide or codon of a nucleotide fragment of the invention may be exchanged by others which, when expressed, results in a polypeptide identical or substantially identical to the polypeptide encoded by the nucleotide fragment in question.

The term "subsequence" when used in connection with the nucleic acid fragments of the invention is intended to indicate a continuous stretch of at least 10 nucleotides which 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 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 74, 76 or 78. It is preferred that 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. The nucleotide sequence to be modified may be of cDNA or genomic origin as discussed above, but may also be of synthetic origin. Furthermore, the sequence may be of mixed cDNA and genomic, mixed cDNA and synthetic or genomic and synthetic origin as discussed above. The sequence may have been modified, e.g. by site-directed mu- tagenesis, to result in the desired nucleic acid fragment encoding the desired polypeptide.

The invention also relates to a replicable expression vector which comprises a nucleic acid fragment defined above, especially a vector which comprises a nucleic acid frag- ment 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. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromo- somal entity, the replication of which is independent of chromosomal replication; examples of such a vector are a plasmid, phage, cosmid, mini-chromosome and virus. Alternatively, 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. Hence, 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. Such 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. It is especially in cases where glycosylation is desired that a mammalian cell is used, although glycosylation of proteins is a rare event in prokaryotes. Normally, however, a prokaryotic cell is preferred such as a bacterium belonging to the genera Mycobacterium, Salmonella, Pseudomonas, Bacillus and Eschericia. It is preferred that the transformed cell is an E. coli, B. subtilis, or M. 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. In the most preferred embodiment of this part of the invention the transformed cell is Mycobacterium bovis BCG strain: Danish 1331 , which is the Mycobacterium bovis strain Copenhagen from the Copenhagen BCG Laboratory, Statens Seruminstitut, Denmark.

The 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.

Therefore, 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 described in the present application 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 conditions are used), and recovering the polypeptide from the host cell or culture medium; or

isolating the polypeptide from a short-term culture filtrate; or

isolating the polypeptide from whole mycobacteria of the tuberculosis complex or from lysates or fractions thereof, e.g. cell wall containing fractions, or

synthesizing the polypeptide by solid or liquid phase peptide synthesis. 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 ceil 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:

In general, of course, prokaryotes are preferred for the initial cloning of nucleic se- quences of the invention and constructing the vectors useful in the invention. For example, in addition to the particular strains mentioned in the more specific disclosure below, one may mention by way of example, strains such as E. coli K12 strain 294 (ATCC No. 31446), E. coli B, and E. coli X 1776 (ATCC No. 31537). These examples are, of course, intended to be illustrative and not limiting.

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 mar- cesans, and various Pseudomonas species may be used. Especially interesting are rapid-growing mycobacteria, e.g. M. 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.

In general, 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. For example, E. coins 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 plasmids or phages must also contain, or be modified to contain, promoters which can be used by the microorganism for expression. Those promoters most commonly used in recombinant DNA construction include the B- lactamase (penicillinase) and lactose promoter systems (Chang et al., (1978), Nature, 35:515; Itakura et al., (1977), Science 198:1056; Goeddel et al., (1979), Nature 281 :544) and a tryptophan (trp) promoter system (Goeddel et al., (1979) Nature 281 :544; EPO Appl. Publ. No. 0036776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (Siebwenlist et al., (1980), Cell, 20:269). Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another promoter by artificial means.

After the recombinant preparation of the polypeptide according to the invention, 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. Another possibility is to employ the simultaneous electroelution technique described by Andersen et al. in J. Immunol. Methods 161 : 29-39.

According to the invention the post-translational modifications involves lipidation, gly- cosylation, cleavage, or elongation of the polypeptide.

In certain aspects, 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. In these aspects, 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. Most importantly, 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.

Apart from their use as starting points for the synthesis of polypeptides of the invention and for hybridization probes (useful for direct hybridization assays or as primers in e.g. PCR or other molecular amplification methods) the 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. Recent research have revealed that 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. These newly discovered methods are reviewed in Ulmer et al., (1993), Curr. Opin. Invest. Drugs, 2:983-989 which hereby is included by reference.

Hence, 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 infec- tions with mycobacteria of the tuberculosis complex in an animal, including 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 poly- peptide which has the capability of modulating an immune response. For instance, a gene encoding lymphokine precursors or lymphokines (e.g. IFN-γ, IL-2, or IL-12) could be administered together with the gene encoding the immunogenic protein, either by administering two separate DNA fragments or by administering both DNA fragments included in the same vector. It also is a possibility to administer DNA fragments compri- sing 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.

In one embodiment of the invention, any of the above mentioned polypeptides is used in the manufacture of an immunogenic composition to be used for induction of an immune response in a mammal against an infection with a virulent Mycobacterium. Preferably, the immunogenic composition is used as a vaccine.

The preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251 ; 4,601 ,903; 4,599,231 ; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in liquid 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. In addition, if desired, 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.

In one embodiment the composition used for vaccination comprises at least one, but preferably at least 2, such as at least 3, 4, 5, 10, 15 or at least 20 different polypeptides of the invention.

In another embodiment the composition to be used for vaccine comprises, together with at least one polypeptide of the invention, at least one, but preferably at least 2, such as at least 3, 4, 5, 10, 15 or at least 20 polypeptides which are not polypeptides of the present invention but are derived from a virulent Mycobacterium such as a polypeptide belonging to the group of ST-CF (Elhay MJ and Andersen P, Immunology and cell Biology (1997) 75, 595-603). ESAT-6, CFP7, CFP10 (EMBL accession number: AL022120), CFP17, CFP21 , CFP25, CFP29, MPB59, MPT59, MPB64, and MPT64.

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. For 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 therapeutically 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 of active ingredient per vaccination with a preferred range from about 0.1 μg to 1000 μg, such as in the range from about 1 μg to 300 μg, and especially in the range from about 10 μg to 50 μg. Suitable regimes for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. Preferred routes of administration are the parenteral route such as the intravenous, intrapehtoneal, intramuscular, subcutaneous or intradermal routes; the oral (on a solid physiologically acceptable base or in a physiologi- cally acceptable dispersion), buccal, sublingual, nasal, rectal or transdermal routes. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the weight of the person to be vaccinated.

Some of the polypeptides of the vaccine are sufficiently immunogenic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant substance.

Various methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), commonly used as a 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as a 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. According to the invention DDA (dimethyldioctadecylammonium bromide) is an interesting candidate for an adjuvant, but also Freund's complete and incomplete adjuvants as well as QuilA and RIBI adjuvants are interesting possibilities.

Other possibilities to enhance the immunogenic effect involve the use of immune modulating substances such as lymphokines (e.g. IFN-γ, IL-2 and IL-12) or synthetic IFN- γ inducers such as poly l:C in combination with the above-mentioned adjuvants.

In many instances, it will be necessary to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations. The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-25 years, such as 20 years, preferably 15 or 10 years, more preferably 1-5 years usually three years, will be desirable to maintain the desired levels of protective immunity.

In one embodiment of the invention a composition is produced comprising as the effective component a micro-organism, the micro-organism is a bacterium such as Mycobacterium, Salmonella, Pseudomonas and Escherichia, preferably Mycobacterium bovis BCG wherein at least one, such as at least 2 copies, such as at least 5 copies of a nucleotide fragment comprising a nucleotide sequence encoding a polypeptide of the invention has been incorporated into the genome of the micro-organism or introduced as a part of an expression vector in a manner allowing the micro-organism to express and optionally secrete the polypeptide. In a preferred embodiment, the composition comprises at least 2 different nucleotide sequences encoding at least 2 different polypeptides of the invention. In a much preferred embodiment, the composition comprises at least 2 different nucleotide sequences encoding at least one polypeptide of the invention and at least one polypeptide belonging to the group of ST-CF (Elhay M J and Andersen P, Immunology and cell Biology (1997) 75, 595-603) such as ESAT-6, CFP7, CFP10, CFP17, CFP21 , CFP25, CFP29, MPB59, MPT59, MPB64, and MPT64.

Individuals infected with virulent Mycobacteria can generally be divided into two groups. The first group has an infection with a virulent Mycobacterium e.g. contacts of TB patients. The virulent Mycobacterium may have established colonies in the lungs, but the individual has, as yet, no symptoms of TB. The second group has clinical symptoms of TB, as a TB patient.

In one embodiment of the invention, any of the above mentioned polypeptides are used for the manufacture of a diagnostic reagent that preferably distinguishes a subclinically or clinically infected individual (group I and group II) from an individual who has been BCG vaccinated or infected with Mycobacterium avium or sensitised by non-tuberculosis Mycobacterium (NTM), and may distinguish a subclinically or clinically infected individual from an individual who has cleared a previous infection with a virulent Mycobacterium. It is most likely that specific polypeptides derived from SPE will identify group I and/or group II from individuals not infected with virulent Mycobacteria in the same way as ESAT-6 and CFP10 (P.Ravn et al., (1998), J. Infectious Disease 179:637-45).

In one embodiment of the invention, any of the above discussed polypeptides are used for the manufacture of a diagnostic reagent for the diagnosis of an infection with a virulent Mycobacterium. One embodiment of the invention provides a diagnostic reagent for differentiating an individual who is clinically or subclinically infected with a virulent Mycobacterium from an individual not infected with virulent Mycobacterium, i.e. an individual who has been BCG vaccinated or infected with Mycobacterium avium or sensitised by non-tuberculosis Mycobacterium (NTM). Such a diagnostic reagent will distinguish between an individual in group I and/or II of the infection stages above, from an individual who has been vaccinated against TB. Another embodiment of the invention provides a diagnostic reagent for differentiating an individual who is clinically or subclinically infected with a virulent Mycobacterium from an individual who has a cleared infection with a virulent Mycobacterium. Such a diagnostic reagent will distinguish between an individual in group I and/or II of the infection stages above, from an individual who has cleared the infection. Determination of an infection with virulent Mycobacterium will be instrumental in the, still very laborious, diagnostic process of tuberculosis. A number of possible diagnostic assays and methods can be envisaged (some more specifically described in the examples and the list of properties): a sample comprising whole blood or 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 vitro and a positive reaction could e.g. be proliferation of the T-cells or release of cytokines such as IFN-γ into the extracellular phase (e.g. into a culture supernatant).

Alternatively, 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 and could be used to monitor treatment effect by reduction in responses. 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 visualising the reaction between the antibody and antigen such as ELISA, Western blot, precipitation assays.

Also a method of determining the presence of virulent Mycobacterium nucleic acids in a mammal, including a human being, or in a sample, comprising incubating the sample with a nucleic acid sequence of the invention or a nucleic acid sequence complementary thereto, and detecting the presence of hybridised nucleic acids resulting from the incubation (by using the hybridisation assays which are well-known in the art), is 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 hybridises with the nucleic acid sequence (or a complementary sequence) by the use of PCR techniques.

The invention also relates to a method of diagnosing infection caused by a virulent Mycobacterium in a mammal, including a human being, comprising locally applying (patch test) or intradermally injecting (Mantoux test) a polypeptide of the invention. These tests are both called a delayed hypersensitivity reaction (DTH). A positive skin response at the location of injection or application is indicative of the mammal including a human being, being infected with a virulent Mycobacterium, and a negative skin response at the location of injection or application is indicative of the mammal including a human being not having TB. A positive response is a skin reaction having a diameter of at least 5 mm larger than background, but larger reactions are preferred, such as at least 1 cm, 1.5 cm, and at least 2 cm in diameter. A skin reaction is here to mean erythema or induration of the skin, as directly measured. The composition used as the skin test reagent can be prepared in the same manner as described for the vaccines above.

In human volunteers, the generation of a significant immune response can alternatively be defined as the ability of the reagent being tested to stimulate an in vitro recall response by peripheral blood cells from at least 30% of PPD positive individuals previously vaccinated with that reagent or infected with a virulent Mycobacterium, said recall response being defined as proliferation of T cells or the production of cytokine(s) which is higher than the responses generated by cells from unimmunised or uninfected control individuals, with a 95% confidence interval as defined by an appropriate statistical analysis such as a Student's two-tailed T test.

Alternatively, a significant immune response could be detected in vivo by a test such as the generation of delayed type hypersensitivity in the skin in response to exposure to the immunising reagent, such response being significantly larger (with a 95% confidence interval as defined by appropriate statistical analysis such as a Student's two-tailed T test) in at least 30% of vaccinated or infected individuals than in placebo-treated or uninfected individuals.

The polypeptides according to the invention may be potential drug targets. Once a particular interesting polypeptide has been identified, the biological function of that polypeptide may be tested. The polypeptides may constitute receptor molecules or toxins which facilitates the infection by the Mycobacterium and if such functionality is blocked, the infectivity of the virulent Mycobacterium will be diminished.

The biological function of particular interesting polypeptides may be tested by studying the effect of inhibiting the expression of the polypeptides on the virulence of the virulent Mycobacterium. This inhibition may be performed at the gene level such as by blocking the expression using antisense nucleic acid, PNA or LNA or by interfering with regulatory sequences or the inhibition may be at the level of translation or post-translational processing of the polypeptide.

Once a particular polypeptide according to the invention is identified as critical for virulence, an anti-mycobacterial agent might be designed to inhibit the expression of that polypeptide. Such anti-mycobacterial agent might be used as a prophylactic or therapeutic agent. For instance, 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

A presently preferred embodiment is an extract of polypeptides obtainable by a method comprising the steps of a) killing a sample of virulent Mycobacteria; b) centrifugating the sample of a) at 2,000g for 40 minutes; c) resuspending the pellet of b) in PBS and 0.5% Tween 20 and sonicating with 20 rounds of 90 seconds; d) centrifugating the suspension of c) at 5,000g for 30 minutes; e) extracting soluble proteins from the cytosol as well as cell wall and cell membrane components from the supernatant of d) with 10% SDS; f) centrifugating the extract of e) at 20,000g for 30 minutes; g) precipitating the supernatant of f) with 8 volumes of cold acetone;

with an adjuvant substance.

In other words, the invention relates to use of an extract of polypeptides with an adjuvant substance for the preparation of a composition for the generation or determination of an immune response against a virulent Mycobacterium.

Finally, a monoclonal or polyclonal antibody, which is specifically reacting with a poly- peptide 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.

Examples

EXAMPLE 1 : Total extraction of proteins from dead M.tuberculosis bacteria.

1.5 x 10⁹ bacteria/ml M.tuberculosis was heat treated at 55°C for 1.5 hours and checked for sterility. 10 ml of these heat killed bacteria was centrifuged at 2000 g for 40 min; the supernatant was discharged and the pellet resuspended in PBS containing 0.5% Tween 20 and used as the antigen source. The pellet was sonicated with 20 rounds of 90 seconds and centrifuged 30 min at 5000 g to remove unbroken cells. The supernatant containing soluble proteins as well as cell wall and cell membrane components was extracted twice with 10% SDS to release proteins inserted in the cell wall and membrane compartments. After a centrifugation at 20.000 g for 30 min the supernatant was precipitated with 8 volume of cold acetone and resuspended in PBS at a protein concentration of 5 mg/ml and named: Somatic Proteins Extract (SPE).

Analysis of protective immune response for tuberculosis after immunisation with different M.tuberculosis protein preparations. The protective efficacy of SPE was evaluated in a vaccination experiment and compared to the two vaccines ST-CF and BCG, known to induce protection against TB.

Five groups of 6-8 weeks old, female C57BI/6J mice (Bomholtgaard, Denmark) were immunised subcutaneously at the base of the tail with vaccines of the following composition:

Group 1 : BCG

Group 2: 1x 10⁷ heat killed M.tuberculosislDDA (250 μg DDA) Group 3: 50 μg ST-CF/DDA (250 μg) Group 4: 50 μg SPE/DDA (250 μg)

Group 5: Adjuvant control: DDA (250 μg) in NaCl

The animals were injected with a volume of 0.2 ml. The mice of groups2, 3 and 4 were boosted twice at two weeks interval. Four weeks after the last immunisation three mice/group were sacrificed and the spleens removed. The immune response induced in the spleen cells was monitored by release of IFN-γ into the culture supernatants when stimulated in vitro with relevant antigens (Table 2). ST-CF and SPE induced a similar immune response while only a very low IFN-γ release was observed after immunisation with BCG and stimulation with ST-CF.

Table 2 Recognition of protein preparations after immunisation presented as IFN-γ release (pg/ml) after restimulation.

Immunogen No antigen ST-CF SPE

ST-CF <200 6752 ± 591 8431 ± 459

SPE <200 6621 ± 203 11079 ± 178

BCG <200 469 ± 32 ND

Seven weeks after the final immunisation the mice received a primary infection with 5x10 5 H37Rv in 0.1 ml iv. and two weeks later the mice were sacrificed and the spleens were isolated for bacterial enumeration (figure 2). BCG induced a high level of protection in the spleen as expected but so did the killed H37Rv, ST-CF and SPE and all preparations induced protection at almost the same level, with SPE as the most potent of these preparations.

These data demonstrate that there are components to be found among the somatic proteins of H37Rv which in an animal model protect against tuberculosis at the same level as BCG.

EXAMPLE 2: Subcelluiar fractionation of Mycobacterium tuberculosis

1.5 x 10⁹ colony forming units (CFU/ml) of M. tuberculosis H37Rv were inactivated by heat-killing at 60°C for 1.5 hour. The heat-killed Mycobacteria was centrifuged at 3,000 x g for 20 min; the supernatant was discarded and the pellet was resuspended in cold PBS. This step was repeated twice. After the final wash, the pellet was resuspended in a homogenising buffer consisting of PBS supplemented with 10 mM EDTA and 1 mM of phenylmethylsulfonyl fluoride in a ratio of 1 ml buffer per 0.5 g of heat-killed Mycobacteria. The sample was sonicated on ice for 15 min (1-min-pulser-on/10-sec- pulser off) and subsequently lysed three times with a French Pressure Cell at 12,000 lb/in². The lysate was centrifuged at 27,000 x g for 20 min; the pellet was washed in homogenising buffer and recent fuged. The pooled supernatants contained a mixture of cytosol and membrane components, while the pellet represented the crude ceil wall. Preparation of cell wall

The cell wall pellet, resuspended in homogenising buffer, was added RNase and DNase to a final concentration of 1 mg/ml and incubated overnight at 4°C. The cell wall was washed twice in homogenising buffer, twice in homogenising buffer saturated with KCl, and twice with PBS. Soluble proteins were extracted from the cell wall by a 2 hour incubation with 2% SDS at 6°C. The insoluble cell wall core was removed by a centrifugation at 27,000 x g for 20 min and the SDS-extraction was repeated. Finally, the pooled supernatants were precipitated with 6 volumes of chilled acetone and resuspended in PBS.

Preparation of cytosol and membrane:

To separate the cytosol and the membrane fraction, the pooled supernatants were ultracentrifugated at 100,000 x g for 2 hours at 5°C. The cytosol proteins in the supernatant were precipitated with acetone and resuspended in PBS. The pellet, representing the membrane fraction, was washed in PBS, ultracentrifugated, and finally resuspended in PBS.

Triton X-114 extraction of cell wall and membrane:

To prepare protein fractions largely devoid of lipoarabinomannan, the cell wall and the membrane fraction were subjected to extraction with precondensed Triton X-114. Triton X-114 was added to the protein sample at a final concentration of 4%. The solution was mixed on ice for 60 min and centrifuged at 20,000 x g for 15 min at 4°C. The pellet containing residual insoluble material was extracted once more (membrane) or twice (cell wall), while the supernatant was warmed to 37°C to condense the Triton X-114. After centrifugation of the supernatant at 12,000 x g for 15 min, the aqueous phase and detergent phase were separated. The aqueous phase and detergent phase were washed twice with Triton X-114 and PBS, respectively. The combined aqueous phases and residual insoluble material containing the majority of proteins were pooled, precipitated with acetone, and resupended in PBS.

The specificity of the human T-cell response in TB patients was investigated by stimulating PBMCs with panels of narrow molecular mass fractions from membrane, cell wall, and cytosol obtained by the multi-elution technique described by Andersen et al.

(1993) J. Immunol. Methods 161 :29-39. The technique resulted in 30 sharply defined fractions and allowed an identification of immunological active regions, of potential as either diagnostic reagents or as vaccine components. The study demonstrated that multiple targets within the cell wail, membrane, and cytosol were recognised by the donors and initiated IFN-γ release as well as cellular proliferation (unpublished results). The broad cellular response were directed towards both the low molecular mass as well as the some of the higher molecular mass fractions. These experiments suggest the existence of numerous target antigens among the cell wall, membrane, and cytosol fractions and it is therefore likely that some of these will have a potential as a protective or diagnostic reagent.

EXAMPLE 3: Identification of proteins from the cytosolic fraction

Use of patient sera to identify M. tuberculosis antigens This example illustrates the identification of antigens from the cytosol fraction by screening with serum from M. tuberculosis infected individuals in western blot. The reaction with serum was used as an indication that the proteins are recognised immunologically.

The cytosol was precipitated with ammonium sulphate at 80% saturation. The non- precipitated proteins were removed by centrifugation and precipitated proteins were resuspended in 20 mM imidazole pH 7.0. The protein solution was applied to a DEAE Sepharose 6B column, equilibrated with 20 mM imidazole pH 7.0. Bound protein was eluted from the column using a salt gradient from 0 to 1 M NaCl, in 20 mM imidazole pH 7.0. Fractions collected during elution was analysed on a silver stained 10-20% SDS- PAGE and on 2 dimensional electrophoresis.

For use in western blot a pool of serum from 5 TB patients was made. These patients ranged from minimal to severe TB. Nitrocellulose membranes were blocked with phosphate buffer, pH 7.3, containing 0.37 M NaCl and 0.5% Tween-20, for 30 min. The serum pool was diluted in phosphate buffer pH 7.3 containing 0.37 M NaCl. The blots incubated in serum dilution overnight at room temperature on a shaker. Membranes were washed for four times five minutes in the dilution buffer, and incubated with 1 :1 ,000 diluted peroxidase-labelled swine anti human-lgG (P214, Dako) for 1 hour at room temperature on a shaker. Blots were then washed for four times 5 min. in the dilution buffer and stained with DONS/TMB.

N-terminal sequencing and amino acid analysis

Proteins of the fractions containing bands reactive with serum from TB patients in

Western blot were separated by 2D electrophoresis. Gels were blotted to PVDF membranes and spots subjected to N-terminal sequencing on a Procise sequencer (Applied Biosystems).

The following N-terminal sequences were obtai

ForTB15 :TERTAVLIKPDGIER (SEQ ID NO: 39) ForTB18 :TDTQVTWLTQESHDR (SEQ ID NO: 40)

For TB21 :M IDEALFDAEEKM EK (SEQ ID NO: 41) For TB33 : PLPADPSTDLSAYAQ (SEQ ID NO: 42) For TB38 :M LISQRPTLSEDVLT (SEQ ID NO: 43)

For TB54 :TGNLVTKNSLTPDVR (SEQ ID NO: 44)

Sequence identity searches

The N-terminal sequences obtained were used for an identity search using the blast program of the Sanger M. tuberculosis database : http://www.sanger.ac.uk/Projects/M_tuberculosis/blast_server.shtml

In addition, the GenEMBL database was searched using the BLASTP program (Altschul, Stephen F., Warren Gish, Webb Miller, Eugene W. Myers, and David J. Lipman (1990). Basic local alignment search tool. J. Mol. Biol.215:403-10.), to reveal proteins with homology to the full amino acid sequences obtained from the Sanger database.

Thereby, the following information was obtained :

TB15 For the 15 determined N-terminal amino acids for TB15 a 93% identical sequence was found in MTV008.01c. Amino acid 5 of the determined N-terminal sequence (A) is an L in the sequence MTV008.01c. Within the open reading frame the translated protein is 136 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.

This gives a protein of 136 amino acids, which corresponds to a theoretical molecular mass of 14 509 Da and a theoretical pi of 5.36. The observed mass in SDS-PAGE is 14 kDa.

TB15 has 80% sequence identity in a 139 amino acid overlap to a protein of M. smegmatis. It is homologous to putative nucleoside diphosphate kinases from several species, e.g. 59% sequence identity to a 151 amino acid protein of Archaeoglobus fulgidus and 57% sequence identity to a 149 amino acid protein of Bacillus subtilis.

TB18

For the 15 determined N-terminal amino acids for TB18 a 100% identical sequence was found in MTCY017.33c. Within the open reading frame the translated protein is 164 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.

This gives a protein of 164 amino acids, which corresponds to a theoretical molecular mass of 17 855 Da and a theoretical pi of 4.81. The observed mass in SDS-PAGE is 20 kDa.

TB18 has 94% sequence identity, in a 164 amino acid overlap, to a protein from M. leprae. In addition, it is homologous to transcription elongation factors from several species, e.g. 32% sequence identity in a 114 amino acid overlap, to a protein from

Zymomonas mobilis.

TB21

For the 15 determined N-terminal amino acids for TB21 a 100% identical sequence was found in MTCY274.13c.

Within the open reading frame the translated protein is 185 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 1.

This corresponds to a theoretical molecular mass of 20 829 Da and a theoretical pi of

5.81. The observed mass in SDS-PAGE is 22 kDa.

TB21 has 90% sequence identity in a 185 amino acid overlap to a protein from M. leprae.

In addition, it is homologous to ribosome recycling factors from several species, e.g. 63% in a 185 amino acid overlap to a protein from Streptomyces coelicolor. TB33

For the 15 determined N-terminal amino acids for TB33 a 85% identical sequence was found in MTCY71.23. Amino acids 8 and 9 of the determined N-terminal sequence (T and D) are a P and a T in MTCY71.23, respectively.

Within the open reading frame the translated protein is 297 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.

This gives a protein of 297 amino acids, which corresponds to a theoretical molecular mass of 33 323 Da and a theoretical pi of 4.91. The observed mass in SDS-PAGE is 35 kDa.

TB33 has 83% sequence identity in a 296 amino acid overlap to a protein from M. leprae.

In addition, it is homologous to thiosulphate sulfurtransferases (rhodanese) from several species, e.g. 48% in a 131 amino acid overlap to rhodanese from Saccharopolyspora erythraea.

TB38

For the 15 determined N-terminal amino acids for TB38 a 100% identical sequence was found in MTCY13E12.10c.

Within the open reading frame the translated protein is 347 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 1.

This corresponds to a theoretical molecular mass of 37 710 Da and a theoretical pi of

4.53. The observed mass in SDS-PAGE is 38 kDa.

TB38 is homologous to DNA-directed RNA polymerase alpha-chains from several species, e.g. 79% in a 321 amino acid overlap to a protein from Streptomyces coelicolor.

TB54

For the 15 determined N-terminal amino acids for TB54 a 100% identical sequence was found in MTCY20B11.23c.

Within the open reading frame the translated protein is 495 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.

This gives a protein of 495 amino acids, which corresponds to a theoretical molecular mass of 54 329 Da and a theoretical pi of 5.00. The observed mass in SDS-PAGE is 60 kDa. TB54 is homologous to adanosyl homocysteinases from several species, e.g. 73% in a 90 amino acid overlap to S-adenosyl-L-homocysteine hydrolase from Triticum aestivum. It contains a S-adenosyl-L-homocysteine hydrolase signature (PS00739).

Example 3a: Use of patient sera to identify M. tuberculosis cytosol antigens. Anion exchange chromatography of the cytosol proteins and Western blot experiments with a pool of sera from TB patients were performed as described in Example 3.

N-terminal sequencing

Proteins of the fractions containing TB12.5, TB20.6, and TB40.8 were separated by 2D electrophoresis. Gels were blotted to PVDF membranes and spots subjected to N- terminal sequencing on a Procise sequencer (Applied Biosystems).

The following N-terminal sequences were obtained :

For TB12.5 :ALKVEMVTFDXSDPA (SEQ ID NO: 80) For TB20.6 :ADADTTDFDVDAEAP (SEQ ID NO: 81)

For TB40.8 :SKTVLILGAGVGGLT (SEQ ID NO: 82)

Sequence identity searches was performed as described in Example 3.

Thereby, the following information was obtained :

TB12.5

For the 15 determined N-terminal amino acids of TB12.5 a 93 % identical sequence was found in Rv0801. The x in position 11 is a cysteine. Within the open reading frame the translated protein is 115 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.

This gives a protein of 115 amino acids, which corresponds to a theoretical molecular mass of 12 512 Da and a theoretical pi of 4.91. The observed mass in SDS-PAGE is 14 kDa.

No homology was found to TB12.5.

TB20.6 For the 15 determined N-terminal amino acids of TB20.6 a 100 % identical sequence was found in Rv3920c.

Within the open reading frame the translated protein is 187 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 1. This gives a protein of 187 amino acids, which corresponds to a theoretical molecular mass of 20.559 Da and a theoretical pi of 4.14. The observed mass in SDS-PAGE is 24 kDa.

TB20.6 has 73 % homology to a 193 amino acid protein of M. leprae. It has 59% homology in a 184 amino acid overlap to a Jag-like protein from Streptomyces coelicolor.

TB40.8

For the 15 determined N-terminal amino acids of TB40.8 a 100 % identical sequence was found in Rv0331.

Within the open reading frame the translated protein is 388 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.

This gives a protein of 388 amino acids, which corresponds to a theoretical molecular mass of 40 792 Da and a theoretical pi of 5.06. The observed mass in SDS-PAGE is 44 kDa. No homology was found to TB40.8.

Identification of abundant proteins

As immunity to tuberculosis is not B-cell but T-cell mediated, reactivity with serum from

TB patients was not the only selection criterion used to identify proteins from the cytosol. Further proteins were selected by virtue of their abundance in the cytosol. The cytosol was precipitated with ammonium sulphate at 80% saturation. The non- precipitated proteins were removed by centrifugation and precipitated proteins were resuspended in 20 mM imidazole, pH 7.0. The protein solution was applied to a DEAE Sepharose 6B column, equilibrated with 20 mM imidazole. Bound protein was eluted from the column using a salt gradient from 0 to 1 M NaCl, in 20 mM imidazole. Fractions collected during elution was analyzed on a silver stained 10-20% SDS-PAGE and on 2 dimensional electrophoresis. Fractions containing well separated bands were selected for 2D electrophoresis and blotted to PVDF, after which spots, visualised by staining with Coomassie Blue, were selected for N-terminal sequencing.

The following N-terminal sequences were obtained : ForTBIOC :MEVKIGITDSPRELV

(SEQ ID NO: 45)

ForTB15A :SAYKTVVVGTDDXSX

(SEQ ID NO: 46)

ForTB17 :MEQRAELVVGRALVV

(SEQ ID NO: 47)

For TB24 :ADIDGVTGSAGL(N)PA

(SEQ ID NO: 48)

For TB27B :TYETILVERDQRVGI

(SEQ ID NO: 49)

TB10C

No sequence identity was found, when searching the Sanger database using the blast program. However, when the blast program at Swiss-blast was used, a sequence was obtained.

For the 15 determined N-terminal amino acids for TB10C a 93% identical sequence was obtained. The first amino acid of the N-terminal sequence (M) is a V in the sequence found, corresponding to GTG being used as a start codon, instead of ATG. Within the open reading frame the translated protein is 90 amino acids. The N-terminal sequence of the protein identified in the cytosol starts at amino acid 1.

This corresponds to a theoretical molecular mass of 9433 Da and a theoretical pi of

4.93. The observed mass in SDS-PAGE is 10 kDa.

TB15A For the determined N-terminal sequence of TB15 a 78% identical sequence was found in CY01B2.28. The X at position 13 of the determined N-terminal sequence corresponds to a G in MTCY01B2.28 and the X at position 15 to a D.

Within the open reading frame the translated protein is 146 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.

This gives a protein of 146 amino acids, which corresponds to a theoretical molecular mass of 15313 Da and a theoretical pi of 5.60. The observed mass in SDS-PAGE is 16 kDa. The highest sequence identity, 32% in a 34 amino acid overlap, was found to a conserved protein of Methanobacterium thermoautotrophicum.

TB17

For the 15 determined N-terminal amino acids for TB17 a 100% identical sequence was found in MTV044.12.

Within the open reading frame the translated protein is 165 amino acids. The N-terminal sequence of the protein identified in the cytosol starts at amino acid 1. This gives a protein of 165 aa. Theoretical molecular mass 16 793 Da and a theoretical pi of 4.22. The observed mass in SDS-PAGE is 18 kDa. TB17 is homologous to putative molybdenum cofactor biosynthesis proteins from several species, e.g. 34% in a 103 amino acid overlap to moaCB from Synechococcus spp.

TB24

For the 15 determined N-terminal amino acids for TB24 a 92% identical sequence was found in MTCY07D11.03. The tentative N in position 13 of the determined amino acid sequence is a Q in MTCY07D11.03, and the A at position 15 is a G.

Within the open reading frame the translated protein is 216 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.

This gives a protein of 216 amino acids, which corresponds to a theoretical molecular mass of 24 227 Da and a theoretical pi of 4.91. The observed mass in SDS-PAGE is 28 kDa.

TB24 is homologous to a RNA polymerase sigma-E factors from several species, e.g.

55% in a 72 amino acid overlap to ECF sigma factor RpoE1 from Myxococcus xanthus.

TB27B For the 15 determined N-terminal amino acids for TB27B a 100% identical sequence was found in MTCY017.23c.

Within the open reading frame the translated protein is 257 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off. This gives a protein of 257 amino acids, which corresponds to a theoretical molecular mass of 27 276 Da and a theoretical pi of 4.82. The observed mass in SDS-PAGE is 28 kDa. TB27B has 86% sequence identity in a 257 amino acid overlap, to a protein from M. leprae. In addition, it is homologous to enoyl-CoA hydratases from several species, e.g. 66% in a 257 amino acid overlap to a protein from Rhizobium meliloti.

Identification of TB13A :

One protein spot was selected by its reaction with the monoclonal antibody ST-3 in western blot. N-terminal sequencing of the spot on the PVDF membrane corresponding to the ST-3 spot yielded the following results :

For TB13A : P V T Q E E I I A G I A E I I

(SEQ ID NO: 50)

Sequence identity search on the TB13A N-terminal sequence gave the following results:

TB13A

For the 15 determined N-terminal amino acids for TB13A a 100% identical sequence was found in MTCY427.25.

Within the open reading frame the translated protein is 115 amino acids long. The N- terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.

This gives a protein of 115 amino acids, which corresponds to a theoretical molecular mass of 12 524 Da and a theoretical pi of 3.87. The observed mass in SDS-PAGE is 10 kDa.

TB13A has 94% sequence identity to a 115 amino acid protein of M. leprae. It is homologous to putative acyl carrier proteins from several species, e.g. 59% sequence identity to a 78 amino acid protein of Myxococcus xanthus and 56% to a 82 amino acid protein from Streptomyces coelicolor.

Identification of TB64

Biotinylated proteins were purified from the cytosol fraction in the following way: 12 mg of the cytosol fraction was added to 100 μl of TetraLink Tetrameric Avidin Resin (Promega) in PBS, pH 7.4 in an eppendorf tube. After incubation over night at 4°C, centrifugation (1000 g for 5 min) was performed and the resin was washed five times with PBS, pH 7.4, each time followed by centrifugation and collection of the supernatant. Thereafter, 100 μl of 4 times concentrated SDS-PAGE sample buffer (0.08 M Tris-HCl, 8% SDS, 16% glycerol, 24 mM EDTA , pH 8.0) was added to the resin and it was boiled for 20 minutes. After centrifugation the supernatant was collected and analysed for the presence of biotinylated proteins: The sample was analysed on SDS-PAGE followed by semi-dry blotting to nitrocellulose. The nitrocellulose membranes were incubated with alkaline phosphatase labeled streptavidin (D396, DAKO, Glostrup, Denmark). Nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate was used as substrate.

N-terminal sequencing

The eluate from the TetraLink Tetrameric Avidin Resin was loaded on a precast 10-20% Tricine SDS-PAGE gel (Novex, San Diego, USA). After electrophoresis the gel was blotted to Problott PVDF membrane (Applied Biosystems, Foster City, CA) by semidry electroblotting in 10 mM CAPS, 10% methanol, pH 11. The PVDF membrane was stained with 0.1% Coomassie R-250 in 40% methanol, 1% acetid acid, and destained in 50% methanol. A band of 10 kDa which was identified as a biotinylated protein as described above was excised and subjected to N-terminal sequence analysis by automated Edman degradation using a Procise 494 sequencer (Applied Biosystems) as described by the manufacturer.

The following sequence was obtained:

VIRRKPKPRXR (SEQ ID NO: 57) Submission of this sequence to the Sanger Centre M. tuberculosis blast server identified the open reading frame Rv3285 (91% identity in 11 amino acids) encoding a protein of 600 amino acids. The determined sequence showed identity to amino acids 511 to 521 suggesting that the identified peptide is a C-terminal fragment of the protein. As expected, the pattern for biotinylation of a lysine was identified in the C-terminal part of the protein: GDLVWLEAMKMENPVTA (residues 556-573, PROSITE pattern PS00188).

EXAMPLE 4: Identification of proteins from the cell wall.

Identification of TB11B, TB16, TB16A, TB32, TB32A, and TB51.

Proteins contained in the cell wall fraction were separated by 2-D electrophoresis. A sample containing 120 mg protein was subjected to isoelectric focusing in a pH gradient from 4 to 7. The second dimension separation (SDS-PAGE) was carried out in a 10-20% acrylamide gradient. After blotting onto a PVDF membrane, proteins could be visualised by Coomassie blue staining. N-terminal sequencing.

The relevant spots were excised from the PVDF membrane and subjected to N-terminal sequencing using a Procise sequencer (Applied Biosystems). The following N-terminal sequences were obtained:

TB11B:PWKINAIEVPAGA (SEQ ID NO: 51)

TB16:ADKTTQTIYIDADPG (SEQ ID NO: 52)

TB16A:PVLSKTVEVTADAAS (SEQ ID NO: 53)

TB32:SGNSSLGllVGIDD (SEQ ID NO: 54) TB32A:AEVLVLVEHAEGALK (SEQ ID NO: 55)

TB51 -.MKSTVEQLSPTRVRI (SEQ ID NO: 56)

N-terminal sequence identity searching and identification of the corresponding genes.

The N-terminal amino acid sequence from each of the proteins identified was used for a sequence identity search using the tblastn program at NCBI: http://www.ncbi.nlm. nih.gov/cgi-bin/BLAST/nph-blast? Jform=0

The following information was obtained:

TB11B:

The 14 aa N-terminal sequence was found to be 100% identical to a sequence found on cosmid SCY06F7.

The identity is found within an open reading frame of 105 amino acids lenght corresponding to a theoretical molecular mass of 11 185 Da and a pi of 6.18. The apparent molecular mass in an SDS-PAGE gel is 12 kDa.

The amino acid sequence shows some low level similarity to oxygenases and hypothetical proteins.

TB16:

The 15 aa N-terminal sequence was found to be 100% identical to a sequence found within the Mycobacterium tuberculosis sequence MTV021.

The identity is found within an open reading frame of 144 amino acids length corresponding to a theoretical molecular mass of 16294 Da and a pi of 4.64. The apparent molecular mass in an SDS-PAGE gel is 17 kDa. The amino acid sequence shows some similarity to other hypothetical Mycobacterial proteins.

TB16A:

The 15 aa N-terminal sequence was found to be 100% identical to a sequence found on cosmid I28.

The identity is found within an open reading frame of 146 amino acids length corresponding to a theoretical molecular mass of 16 060 Da and a pi of 4.44. The apparent molecular mass in an SDS-PAGE gel is 14 kDa.

TB32: The 14 aa N-terminal sequence was found to be 100% identical to a sequence found within the Mycobacterium tuberculosis sequence MTCY1A10.

The identity is found within an open reading frame of 297 amino acids length corresponding to a theoretical molecular mass of 31654 Da and a pi of 5.55. The apparent molecular mass in an SDS-PAGE gel is 33 kDa. The amino acid sequence shows some similarity to other hypothetical Mycobacterial proteins.

TB32A:

The 15 aa N-terminal sequence was found to be 100% identical to a sequence found within the Mycobacterium tuberculosis sequence MTV012. The identity is found within an open reading frame of 318 amino acids length corresponding to a theoretical molecular mass of 31694 Da and a pi of 4.61. The apparent molecular mass in an SDS-PAGE gel is 32 kDa.

The amino acid sequence reveals high sequence identity to the fixB gene product from several organisms. Probable electron transfer flavoprotein alpha subunit for various dehydrogenases. Equivalent to Mycobacterium leprae FixB.

TB51 :

The 15 aa N-terminal sequence was found to be 100% identical to a sequence found within the Mycobacterium tuberculosis sequence MTV008. The identity is found within an open reading frame of 466 amino acids length corresponding to a theoretical molecular mass of 50587 Da and a pi of 4.3. The apparent molecular mass in an SDS-PAGE gel is 56 kDa.

The amino acid sequence shows similarities to trigger factor from several organisms. Possible chaperone protein. EXAMPLE 5: Cloning of the genes encoding TB10C, TB13A, TB17, TB11 B, TB16, TB16A, TB32, TB51

The genes encoding TB10C, TB13A, TB17, TB11 B, TB16, TB16A, TB32, TB51 were all cloned into the E. coli expression vector pMCT3, by PCR amplification with gene specific primers.

Each PCR reaction contained 10 ng of M. tuberculosis chromosomal DNA in 1x low salt Taq+ buffer (Stratagene) supplemented with 250 μM 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 Taq+ DNA polymerase (Stratagene) in 10 μl 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.

The PCR fragment was ligated with TA cloning vector pCR^® 2.1 (Invitrogen) and transformed into E. coli. Plasmid DNA was thereafter prepared from clones harbouring the desired fragment, digested with suitable restriction enzymes and subcloned into the expression vector pMCT3 in frame with 6 histidine residues which are added to the N- terminal of the expressed proteins. The resulting clones were hereafter sequenced 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.

Expression and metal affinity purification of recombinant proteins was undertaken essentially as described by the manufacturers. For each protein, 1 I LB-media containing 100 μg/ml ampicillin, was inoculated with 10 ml of an overnight culture of XL1-Blue cells harbouring recombinant pMCT3 plasmids. Cultures were shaken at 37°C until they reached a density of OD₆oo⁼ 0.4 - 0.6. IPTG was hereafter added to a final concentration of 1 mM and the cultures were further incubated 4 - 16 hours. Cells were harvested, resuspended in 1x sonication buffer + 8 M urea and sonicated 5 x 30 sec. with 30 sec. pausing between the pulses. After centrifugation, the lysate was applied to a column containing 10 ml of resuspended Talon resin (Clontec, Palo Alto, USA). The column was washed and eluted as described by the manufacturers.

After elution, all fractions (1.5 ml each) were subjected to analysis by SDS-PAGE using the Mighty Small (Hoefer Scientific Instruments, USA) system and the protein concentrations were estimated at OD_{280 nm}- Fractions containing recombinant protein were pooled and dialysed against 3 M urea in 10 mM Tris-HCl, pH 8.5. The dialysed protein was further purified by FPLC (Pharmacia, Sweden) using 1 ml HiTrap columns (Pharmacia, Sweden) eluted with a linear salt gradient from 0 - 1 M NaCl. Fractions were analysed by SDS-PAGE and protein concentrations were estimated at OD_280nm- Fractions containing protein were pooled and dialysed against 25 mM Hepes buffer, pH 8.5. Finally, the protein concentration and the LPS content were determined by the BCA (Pierce, Holland) and LAL (Endosafe, Charleston, USA) tests, respectively.

For cloning of the individual proteins, the following gene specific primers were used :

TB10C : Primers used for cloning of TB10C :

TB10C-F : CTG AGA TCT GTG GAG GTC AAG ATC GGT (SEQ ID NO: 58)

TB1 OC-R : CTC CCA TGG CTAC TTA CCC GCT CGT AGC AAC (SEQ ID NO: 59)

TB10C-F and TB10C-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

TB13A : Primers used for cloning of TB13A : TB13A-F : CTG AGA TCT CCT GTC ACT CAG GAA GAA (SEQ ID NO: 60)

TB13A-R : CTC CCA TGG GAA ACC GCC ATT AGC GGT (SEQ ID NO: 61)

TB13A-F and TB13A-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

TB17 : Primers used for cloning of TB17 :

TB17-F : CCC AAG CTT ATG GAA CAG CGT GCG GAG (SEQ ID NO: 62)

TB17-R : CTC CCA TGG CGA CAC TCG ATC CGG ATT (SEQ ID NO: 63)

TB17-F and TB17-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

TB11 B : Primers used for cloning of TB11 B TB11B-F : CTG AGA TCT ATG CCA GTG GTG AAG ATC (SEQ ID NO: 64) TB11B-R : CTC CCA TGG TTA TGC AGT CTT GCC GGT (SEQ ID NO: 65)

TB11 B-F and TB11 B-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

TB16 : Primers used for cloning OF TB16 :

TB16-F : CTG AGA TCT GCG GAC AAG ACG ACA CAG (SEQ ID NO: 66)

TB16-R : CTC CCA TGG TAC CGG AAT CAC TCA GCC (SEQ ID NO: 67)

TB16-F and TB16-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

TB16A : Primers used for cloning of TB16A :

TB16A-F : CTG AGA TCT CCA GTT TTG AGC AAG ACC (SEQ ID NO: 68)

TB16A-R : CTC CCA TGG GCA CAT GCC TTA GCT GGC (SEQ ID NO: 69)

TB16A-F and TB16A-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

TB32 : Primers used for cloning of TB32 :

TB32-F : CTG AGA TCT ATG TCA TCG GGC AAT TCA (SEQ ID NO: 70) TB32-R : CTC CCA TGG CTAC CTA AGT CAG CGA CTC GCG (SEQ ID NO: 71 )

TB32-F and TB32-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

TB51 : Primers used for cloning of TB51 :

TB51-F : CTG AGA TCT GTG AAG AGC ACC GTC GAG (SEQ ID NO: 72)

TB51-R : CTC CCA TGG GTC ATA CGG TCA CGT TGT (SEQ ID NO: 73) TB51-F and TB51-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

TB15A: Primers used for cloning of TB15A:

TB15A-F: CTG CCA TGG CTA GGT GGT GTG CAC GAT C (SEQ ID NO: 89) TB15A-R: CTG AAG CTT ATG AGC GCC TAT AAG ACC (SEQ ID NO: 90)

TB15-F and TB15-R create Ncol and Hindlll sites, respectively, used for the cloning in pMCT3.

TB21 : Primers used for cloning of TB21 :

TB21-F: CTG AGA TCT ATG ATT GAT GAGGCT CTC (SEQ ID NO: 91) TB21-R: CTC CCA TGG AGC GGC CGC TAG ACC TCC (SEQ ID NO: 92)

TB21-F and TB21-R create Bglll and Ncol sites, respectively, used for the cloning in pMCT3.

TB24: Primers used for cloning of TB24:

TB24-F: GGCTGAGACTC ATG GCC GAC ATC GAT GGT G (SEQ ID NO: 93)

TB24-R: CGTACCATGG TCA TGA CGA CAC CCC CTC GTG (SEQ ID NO: 94)

TB24-F and TB24-R create Bglll and Ncol sites, respectively, used for the cloning in pMCT3.

TB32A: Primers used for cloning of TB32A:

TB32A-F: GGCTGAGACTC ATG GCT GAA GTA CTG GTG C (SEQ ID NO: 95)

TB32A-R: CGTACCATGGCTA GCC GGC GAC CGC CGG TTC (SEQ ID NO: 96)

TB32A-F and TB32A-R create Bglll and Ncol sites, respectively, used for the cloning in pMCT3. TB14: Primers used for cloning of TB14:

TB14-F: 5^'-GTG ACC GAA CGG ACT CTG GT-3^' (SEQ ID NO: 97)

TB14-R: 5^'-CTA GGC GCC GGG AAA CCA GAG-3^' (SEQ ID NO: 98)

TB18: Primers used for cloning of TB18:

TB18-F: 5^'-ATG ACG GAT ACT CAA GTC ACC TG-3' (SEQ ID NO: 99)

TB18-R: 5^'-GGA GTG GTA CGG CTC GGC GC-3^' (SEQ ID NO: 100)

TB27: Primers used for cloning of TB27:

TB27-F: 5^'-ATG ACG TAC GAA ACC ATC CT-3^' (SEQ ID NO: 101)

TB27-R: 5^'-TCA TCG GTG GGT GAA CTG GGG-3^' (SEQ ID NO: 102)

TB33: Primers used for cloning of TB33:

TB33-F: 5^'-ATG CCG CTT CCC GCA GAC CCT AG-3^' (SEQ ID NO: 103)

TB33-R: 5^'-TAC GAC GGG TAC CAC TCC TGG-3^' (SEQ ID NO: 104)

TB38: Primers used for cloning of TB38:

TB38-F: 5^'-ATG CTG ATC TCA CAG CGC CCC A-3^' (SEQ ID NO: 105)

TB38-R: 5^'-AAG CTG TTC GGT TTC GGC GTA G-3^' (SEQ ID NO: 106)

TB54: Primers used for cloning of TB54:

TB54-F: 5^' -ATG ACC GGA AAT TTG GTG AC-3^' (SEQ ID NO: 107)

TB54-R: 5^'-TCA GTA GCG GTA GTG GTC CGG-3^' (SEQ ID NO: 108)

TB14,TB18,TB27,TB33,TB38 and TB54 will be cloned in ex-pressions vector pBAD- TOPO (Invitrogen).

Example 5a: Cloning of the genes encoding TB12.5, TB20.6, and TB40.8

The genes encoding TB12.5, TB20.6, and TB40.8 were all cloned into the E. coli expression vector pMCT3 as described in Example 5. For cloning of the individual genes, the following gene specific primers were used:

TB12.5: Primers used for cloning of TB12.5: TB12.5-F: CTG AGA TCT ATG GCA CTC AAG GTA GAG (SEQ ID NO: 83)

TB12.5-R: CTC CCA TGG TTA TTG ACC CGC CAC GCA (SEQ ID NO: 84)

TB12.5-F and TB12.5-R create BglW and Λ/col sites, respectively, used for the cloning in pMCT3.

TB20.6: Primers used for cloning of TB20.6:

TB20.6-F: CTG AGA TCT ATG GCC GAC GCT GAC ACC (SEQ ID NO: 85)

TB20.6-R: CTC CCA TGG CTA GTC GCG GAG CAC AAC (SEQ ID NO: 86)

TB20.6-F and TB20.6-R create BglW and Λ/col sites, respectively, used for the cloning in pMCT3.

TB40.8: Primers used for cloning of TB40.8:

TB40.8-F: CTG AGA TCT ATG AGC AAG ACG GTT CTC (SEQ ID NO: 87) TB40.8-R: CTC CCA TGG TCA CGT CTT CCA GCG GGT (SEQ ID NO: 88)

TB40.8-F and TB40.8-R create BglW and Λ/col sites, respectively, used for the cloning in pMCT3.

Expression/purification of recombinant proteins was performed as described in Example 5.

EXAMPLE 6: Evaluation of immunological activity of identified somatic proteins.

Each of the proteins identified in either the cell wall, cytosol or the cell membrane derived from M.tuberculosis will be evaluated for the immunological recognition in M.tuberculosis infected animals or in TB patients. IFN-γ induction in the mouse model of TB infection

The recognition of an antigen by IFN-γ producing T cells in M.tuberculosis infected animals or in TB patients is presently believed to be the most relevant correlate of protective immunity. We will therefore evaluate the ability of the polypeptides of the invention to induce an IFN-γ production in mice of four different haplotypes during a primary infection: 8-12 weeks old female mice C57BL/6J (H-2^b), CBA/J (H-2^k), DBA.2 (H-2^d) and A.SW (H-2^S) mice (Bomholtgaard, Ry, Denmark) will be infected i.v. via the lateral tail vein with an inoculum of 5 x 10⁴ M.tuberculosis suspended in PBS in a vol. of 0.1 ml. 14 days postinfection the animals will be sacrificed and spleen cells isolated and tested for proliferation and the IFN-γ release in response to stimulation with the recombinantly produced proteins.

As a specific model we will analyse the recognition of the purified polypeptides of the invention the mouse model of memory immunity to TB: A group of efficiently protected mice will be generated by infecting 8-12 weeks old female C57BI/6J mice with 5 x 10⁴ M.tuberculosis i.v. After 30 days of infection the mice will be subjected to 60 days of antibiotic treatment with isoniazid (Merck and Co., Rahway, NJ) and rifabutin (Farmatalia Carlo Erba, Milano, Italy) then left for 200-240 days to ensure the establishment of resting long-term memory immunity. Such memory immune mice are very efficient protected against a secondary infection (Orme; Andersen, Boom 1993, J.lnfect.Dis. 167: 1481- 1497). 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- γ in response to M.tuberculosis antigens. This model will be used to identify single antigens recognised by protectiveT cells. Memory immune mice will be reinfected with 1 x 10⁶ M.tuberculosis i.v and splenic lymphocytes harvested at day 4-6 of reinfection and proliferation and the amount of IFN-γ produced in response to any of the recombinantly produced proteins will be evaluated.

IFN-γ induction in humans during infection with virulent Mycobacteria. IFN-γ is currently believed to be the best marker of protective immunity in humans. In patients with limited tuberculosis, high levels of IFN-γ can be induced, in contrast to patients with severe TB who often respond with low levels of IFN-γ (Boesen et al (1995), Human T-cell response to secreted antigen fractions of M.tuberculosis. Infection and Immunity 63(4):1491-1497). Furthermore, IFN-γ release has been shown to correlate inversely with the severity of disease as determined by X-ray findings (Sodhi A, et al (1997) Clinical correlates of IFN-gamma production in patients with Tuberculosis, Clinical Infectious disease. 25; 617-620). Healthy exposed contacts of sputum positive TB patients also produce very high levels of IFN-γ in response to mycobacterial antigens (unpublished, manus in prep) indicative of early, subclinical infection. Together these findings indicate that those individuals who are relatively protected (i.e. minimal TB patients) respond with high levels of IFN-γ. The ability of the polypeptides to induce IFN-γ release in cultures of PBMC or whole blood from 20 PPD responsive patients with microscopy or culture proven TB (0-6 month after diagnosis), exposed household contacts, or BCG vaccinated individuals from different geographical regions will be evaluated. Evaluation of donors from different geographical regions will enable us to take into account the influence of i.e. exposure to virulent Mycobacterium or NTM (Non- Tuberculous Mycobacteria) and different genetic background. The most important selection criteria for vaccine candidates are the polypeptides which are recognised by >30% of the donors with a level of IFN γ >30% of that induced by a crude antigen preparation like ST-CF, PPD and SPE.

Cultures will be established with 1 to 2 x 10⁵ PBMC in 200μl in microtiter plates (Nunc, Roskilde, Denmark) or with 1 ml of serum or plasma stimulated with the identified polypeptide and the IFN-γ release measured by ELISA. Polypeptides of the invention frequently recognised will be preferred.

The use of polypeptides as diagnostic reagents:

A polypeptide has diagnostic potential in humans when it is inducing significantly higher responses in patients with microscopy or culture positive tuberculosis compared to PPD positive or PPD negative individuals with no known history of TB infection or exposure to M.tuberculosis but who may or may not have received a prior BCG vaccination, have been exposed to non-tuberculous mycobacteria(NTM), or be actively infected with M. avium. To identify polypeptides capable of discriminating between the above mentioned groups, the level of response and the frequency of positive responders to the polypeptide is compared. By positive responders are meant i) in vitro IFN-γ release by PBMC or whole blood stimulated with the polypeptide of at least 3-500 pg/ml above background or another cut off relating to the specific test kit used, ii) reactivity by human serum or plasma from TB patients with the polypeptide using conventional antibody ELISA/Western blot or iii) in vivo delayed type hypersensitivity response to the polypeptide which is at least 5 mm higher than the response induced by a control material. The diagnostic potential of polypeptides will initially be evaluated in 10 individuals with TB infection and 10 individuals with no known exposure to virulent Mycobacteria. High specificity, >80% ,will be the most important selection criteria for these polypeptides and a sensitivity >80% is desirable but sensitivity >30% is acceptable as combinations of several specific antigens may be preferred in a cocktail of diagnostic reagent recognised by different individuals.

Skin test reaction in TB infected guinea pigs

To identify polypeptides as antigens with the potential as TB diagnostic reagents the ability of the proteins to induce a skin test response will be evaluated in the guinea pig model where groups of guinea pigs have been infected with either M.tuberculosis or M. avium or vaccinated with BCG.

To evaluate the response in M.tuberculosis infected guinea pigs, female outbred guinea pigs will be infected via an ear vein with 1 x 10⁴ CFU of M.tuberculosis H37Rv in 0.2 ml of PBS or aerosol infected (in an exposure chamber of a Middlebrook Aerosol Generation device) with 1x 10⁵ CFU/ml of M.tuberculosis Erdman given rise to 10-15 granulomas per animal in the lung. After 4 weeks skin test will be performed with the polypeptides diluted in 0.1 ml of PBS and 24 hours after the injection reaction diameter is measured.

To evaluate the response in M.avium infected guinea pigs, female outbred guinea pigs will be infected intradermally with 2 x 10⁶ CFU of a clinical isolate of M.avium (Atyp.1443; Statens Serum Institut, Denmark). Skin test are performed 4 weeks after with the polypeptides diluted in 0.1 ml of PBS and 24 hours after the injection reaction diameter is measured.

To evaluate the response in BCG vaccinated guinea pigs, female outbred guinea pigs will be sensitized intradermally with 2 x 10⁶ CFU of BCG (BCG Danish 1331 ; Statens Serum Institut). Skin test are performed 4 weeks after with the polypeptides diluted in 0.1 ml of PBS and 24 hours after the injection reaction diameter is measured.

If a polypeptide induces a significant reaction in animal infected with M.tuberculosis but not in BCG vaccinated guinea pigs this polypeptide may have a potential as a diagnostic reagent to differentiate between BCG vaccinated and M.tuberculosis infected individuals, which will hereafter be evaluated in the human population. If a polypeptide induces a reaction in M.tuberculosis infected guinea pigs but not in guinea pigs infected with M.avium, this polypeptide may have a potential as a diagnostic reagent with respect to differentiate between an individual infected with M.tuberculosis and an individual infected with Mycobacteria not belonging to the tuberculosis complex. The polypeptide may also have a potential as a diagnostic reagent to differentiate between a M.avium and a M.tuberculosis infected individual.

Induction of protective immunity by the recombinant proteins in the mice model.

The recombinant polypeptides will be evaluated as immunological compositions in mice. Female C57BL/6J mice of 6-8 weeks old (Bomholtgaard, Denmark) will be immunised subcutaneously at the base of the tail with the recombinantly produced polypeptides with DDA as adjuvant. The mice will be vaccinated with a volume of 0.2 ml in total of three times with two weeks interval between each immunisation. One week after last immunisation the mice will be bled and the blood cells isolated. The immune response induced will be monitored by release of IFN-γ into the culture supernatant when stimulated in vitro with the homologous proteins.

6 weeks after the last immunisation the mice will be aerosol challenged with 5.5 ml of 5 x 10⁶ viable M.tuberculosis/m\. After 6 weeks of infection the mice will be killed and the number of viable bacteria in lung and spleen determined by plating serial 3-fold dilution of organ homogenates on 7H11 plates. Colonies will be counted after 2-3 weeks of incubation and the levels of protection induced by each of the single polypeptide will be determined.

Example 6a: Interferon-γ induction in human TB patients and BCG vaccinated

Human donors: PBMC were obtained from healthy BCG vaccinated donors with no known exposure to M. tuberculosis and from patients with culture or microscopy proven infection with TB. Blood samples were drawn from the TB patients 0-6 months after diagnosis of tuberculosis, and 20 months to 40 years after BCG vaccination.

Lymphocyte preparations and cell culture: PBMC were freshly isolated by gradient centrifugation of heparinized blood on Lymphoprep (Nycomed, Oslo, Norway) and stored in liquid nitrogene until use. The cells were resuspended in complete RPM I 1640 medium (Gibco, Grand Island, N.Y.) supplemented with 1% penicillin/streptomycin (Gibco BRL, Life Technologies), 1% non-essential-amino acids (FLOW, ICN Biomedicals, CA, USA), and 10% normal human ABO serum (NHS) from the local blood bank. The number and the viability of the cells were determined by Nigrosin staining. Cultures were established with 1.25 x 10⁵ PBMCs in 100 μl in microtitre plates (Nunc, Roskilde, Denmark) and stimulated with ST-CF (δμg/ml), TB13A, TB15A, TB17, TB18, TB33, TB11 B, TB16A, TB16, TB32, and TB51 in a final concentration of 10 μg/ml. No antigen and phytohaemagglutinin (PHA) were used as negative and positive control, respectively. Supernatants for the detection of cytokines were harvested after 5 days of culture, pooled, and stored at -80°C until used.

Cytokine analysis: Interferon-γ (IFN-γ) was detected with a standard sandwich ELISA technique using a commercially available pair of monoclonal antibodies (Endogen) and used according to the manufacturers instruction. Recombinant IFN-γ (Endogen) was used as a standard. All data are means of duplicate wells and the variation between wells did not exceed 10 % of the mean. Cytokine levels below 50 pg/ml were considered negative. Responses of 10 individual donors are shown in TABLE 3.

As shown in Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11 , and Table 12 a marked release of IFN-γ is observed after stimulation with some of the recombinant proteins. For 50% of the donors, stimulation with TB18, TB32, and TB51 give rise to high IFN-γ responses (> 1 ,000 pg/ml). Less than 1/3 of the donors recognised TB15A and TB11 B at this level. Between 30 and 70% of the donors show intermediate IFN-γ response (> 500 pg/ml) when stimulated with TB17 and TB16A whereas only limited response was obtained by TB13A, TB33, and TB16. However, TB13A, TB33 and TB16 may still be of immunological importance and meet some of the other properties of the present invention. E.g. as demonstrated for TB33 which is recognised by a pool of sera from human TB-patients.

Table 3 Stimulation of PBMCs from 6 healthy BCG vaccinated and 4 TB patients with recombinant TB13A. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN-γ/ml.

BCG vaccinated control donors, no known TB exposure

Donor No ag PHA ST-CF TB13A

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 12 11572 10860 41

2 0 14257 11536 0

3 7 13270 8844 493

4 0 13193 2828 0

5 4 14239 14275 332

6 0 16278 12623 0

TB patients

Donor No ag PHA ST-CF TB13A

(1 μg l) (5 μg/ml) (10 μg/ml)

1 0 9914 3297 0

2 51 10058 6489 0

3 0 10587 9155 0

4 0 9458 5236 18

Table 4 Stimulation of PBMCs from 6 healthy BCG vaccinated and 5 TB patients with recombinant TB15A. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN-γ/ml.

BCG vaccinated control donors, no known TB exposure

Donor Noag PHA ST-CF TB15A

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 0 18860 3733 1478

2 0 16218 2856 0

3 94 18427 13998 0

4 0 17815 4255 0

5 0 15981 10830 441

6 81 16961 11165 8009

TB patients

Donor Noag PHA ST-CF TB15A

(1 μg/mi) (5 μg/ml) (10 μg/ml)

1 231 18854 6443 57

2 0 17213 2196 0

3 0 17880 1049 0

4 0 17777 2865 0

5 0 17487 5321 0

Table 5 Stimulation of PBMCs from 6 healthy BCG vaccinated with recombinant TB17. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN- γ/ml

BCG vaccinated control donors, no known TB exposure

Donor Noag PHA ST-CF TB17

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 33 16696 7304 66

2 102 16878 6427 50

3 49 12161 11055 0

4 0 12949 2284 73

5 81 12129 6669 1029

6 0 12706 11762 656 Table 6 Stimulation of PBMCs from 3 healthy BCG vaccinated and 3 TB patients with recombinant TB18. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN-γ/ml

BCG vaccinated control donors, no known TB exposure

Donor No ag PHA ST-CF TB18

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 82 20862 15759 842

2 7 17785 10088 1855

3 912 16198 11350 6838

TB patients

Donor No ag PHA (1 ST-CF(5 TB18 (10 μg/ml) μg/ml) μg/ml)

1 60 12301 1 1057 265

2 7 10390 6123 167

3 34 11678 8136 1629

Table 7 Stimulation of PBMCs from 5 healthy BCG vaccinated and 6 TB patients with recombinant TB33. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN-γ/ml.

BCG vaccinated control donors, no known TB exposure

Donor No ag PHA ST-CF TB33

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 589 10068 4426 721

2 1953 10817 6316 662

3 702 11837 1640 0

4 605 9463 2694 0

5 2471 7990 5979 0

TB patients

Donor No ag PHA ST-CF TB33

(1 μg l) (5 μg/ml) (10 μg/ml)

1 0 3647 812 0

2 0 12266 920 0

3 0 12899 4388 0

4 0 10233 7989 0

Table 8 Stimulation of PBMCs from 3 healthy BCG vaccinated and 3 TB patients with recombinant TB11 B. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN-γ/ml.

BCG vaccinated control donors, no known TB exposure

Donor No ag PHA ST-CF TB11 B

(1 μg/mi) (5 μg/ml) (10 μg/ml)

1 0 13682 9067 1379 2 0 13705 10169 2092 3 0 13231 7740 0

TB patients

Donor No ag PHA ST-CF TB11 B

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 0 13285 8025 0 2 0 13157 3945 0 3 0 13207 4485 0

Table 9. Stimulation of PBMCs from 2 healthy BCG vaccinated and 5 TB patients with recombinant TB16A. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN-γ/ml.

BCG vaccinated control donors, no known TB exposure

Donor No ag PHA ST-CF TB16A

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 0 12816 1831 645

2 0 14530 10293 1404

TB patients

Donor No ag PHA ST-CF TB16A

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 0 11606 5460 42

2 0 11836 5837 977

3 388 12353 8401 958

4 0 9587 3169 499

5 43 10820 4869 593 Table 10. Stimulation of PBMCs from 6 healthy BCG vaccinated with recombinant TB16. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN- γ/ml in BCG vaccinated control donors, no known TB exposure.

Donor No ag PHA ST-CF TB16

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 33 16696 7304 0

2 102 16878 6427 292

3 49 12161 11055 514

4 0 12949 2284 24

5 81 12129 6669 58

6 0 12706 11762 36

Table 11. Stimulation of PBMCs from 3 healthy BCG vaccinated and 3 TB patients with recombinant TB32. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN-γ/ml.

BCG vaccinated control donors, no known TB exposure

Donor No ag PHA ST-CF TB32 (1 μg/ml) (5 μg/ml) (10 μg/ml)

1 82 20862 15759 1614 2 7 17785 10088 3385 3 912 16198 11350 9863

TB patients

Donor No ag PHA ST-CF TB32 (1 μg/ml) (5 μg/ml) (10 μg/ml)

1 60 12301 11057 562 2 7 10390 6123 206 3 34 11678 8136 83 Table 12. Stimulation of PBMCs from 6 healthy BCG vaccinated with recombinant TB51. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN- γ/ml.

BCG vaccinated control donors, no known TB exposure

Donor No ag PHA ST-CF TB51

(1 μg/ml) (5 μg/ml) (10 μg/ml)

1 33 16696 7304 596

2 102 16878 6427 1155

3 49 12161 11055 2247

4 0 12949 2284 777

5 81 12129 6669 140

6 0 12706 11762 1123

Figure legends:

Figure 1 :

Long term protection against TB can be induced by immunisation with dead M.tuberculosis. Mice received either: three immunisations with 1x10⁷ CFU of dead M.tuberculosis H37Rv (squares); three immunisations with 50 μg of ST-CF (triangles); one immunisation with 5 x 10⁴ CFU of live M.tuberculosis H37Rv (circle) and was hereafter cleared for the infection by administration of isoniazid in the drinking water. At 3, 6 and 12 month after the last immunisation the mice received an infection with M.tuberculosis H37Rv and two weeks later the bacterial load and the resistance against TB in the spleens were determined.

Figure 2:

Mice received three immunisations with 50μg of either of the three vaccines: heat killed H37Rv, SPE or ST-CF or received a vaccination with BCG. Two weeks after a primary infection the bacterial load in the spleen was used to determined the resistance against TB.

Claims

1. A substantially pure polypeptide, which has a sequence identity of at least 80% to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 75, 77 and 79 or a subsequence of at least 6 amino acids thereof, wherein the polypeptide or the subsequence thereof has at least one of the following properties:

i) the polypeptide induces an in vitro recall response determined by a release of IFN-γ of at least 1 ,500 pg/ml from reactivated memory T-lymphocytes withdrawn from a mouse within 4 days after the mouse has been rechallenged with 1 x 10⁶ virulent Mycobacteria, the induction being performed by the addition of the polypeptide to a suspension comprising about 2 x 10⁵ cells isolated from the spleen of said mouse, the addition of the polypeptide resulting in a concentration of the polypeptide of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 3 days after the addition of the polypeptide to the suspension,

ii) the polypeptide induces an in vitro response during primary infection with virulent Mycobacteria, determined by release of IFN-γ of at least 1 ,500 pg/ml from T-lymphocytes withdrawn from a mouse within 28 days after the mouse has been infected with 5 x 10⁴ virulent Mycobacteria, the induction being performed by the addition of the polypeptide to a suspension comprising about 2 x 10⁵ cells isolated from the spleen, the addition of the polypeptide resulting in a concentration of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 3 days after the addition of the polypeptide to the suspension,

iii) the polypeptide induces a protective immunity determined by vaccinating an animal with the polypeptide and an adjuvant in a total of three times with two weeks interval starting at 6-8 weeks of age, 6 weeks after the last vaccination challenging with 5 x 10⁶ virulent /Wycooacter/a/ml by aerosol and determining a significant decrease in the number of bacteria recoverable from the spleen 6 weeks after the animal has been challenged, compared to the number recovered from the same organ in an animal given placebo treatment,

iv) the polypeptide induces in vitro recall response determined by release of IFN-γ of at least 1 ,000 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) withdrawn from TB patients or PPD positive individuals 0-6 months after diagnosis, the induction being performed by the addition of the polypeptide to a suspension comprising about 1.0 to 2.5 x 10⁵ PBMC, the addition of the polypeptide resulting in a concentration of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 5 days after the addition of the polypeptide to the suspension,

v) the polypeptide induces a specific antibody response in a TB patient as determined by an ELISA technique or a western blot when the whole blood is diluted 1 :20 in PBS and stimulated with the polypeptide in a concentration of not more than 20 μg/ml.

vi) the polypeptide induces a positive in vitro response determined by release of IFN-γ of at least 500 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) withdrawn from an individual who is clinically or subclinically infected with a virulent Mycobacterium, the induction being performed by the addition of the polypeptide to a suspension comprising about 1.0 to 2.5 x 10⁵ PBMC, the addition of the polypeptide resulting in a concentration of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 5 days after the addition of the polypeptide to the suspension, and does not induce such an IFN-γ release in an individual not infected with a virulent Mycobacterium,

vii) the polypeptide induces a positive in vitro response determined by release of IFN-γ of at least 500 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) withdrawn from an individual clinically or subclinically infected with a virulent Mycobacterium, the induction being performed by the addition of the polypeptide to a suspension comprising about 1.0 to 2.5 x 10⁵ PBMC, the addition of the polypeptide resulting in a concentration of not more than 20 μg per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 5 days after the addition of the polypeptide to the suspension, and does not induce such a IFN-γ release in an individual who has a cleared infection with a virulent Mycobacterium,

viii) the polypeptide induces a positive DTH response determined by intradermal injection of at most 100 μg of the polypeptide to an individual who is clinically or subclinically infected with a virulent Mycobacterium, a positive response having a diameter of at least 10 mm 72 hours after the injection, and does not induce such a response in an individual not infected with a virulent Mycobacterium, ix) the polypeptide induces a positive DTH response determined by intradermal injection of at most 100 μg of the polypeptide to an individual who is clinically or subclinically infected with a virulent Mycobacterium, a positive response having a diameter of at least 5 10 mm 72 hours after the injection, and does not induce such a response in an individual who has a cleared infection with a virulent Mycobacterium.

2. A substantially pure polypeptide which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,

10 26, 28, 30, 32, 36, 38, 75, 77 and 79.

3. A polypeptide according to any of claims 1 or 2, which comprises an amino acid sequence which has a sequence identity of at least 80% to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,

15 22, 24, 26, 28, 30, 32, 36, 38, 75, 77 and 79 and/or is a subsequence thereof.

4. A purified or non-naturally occurring polypeptide as defined in any of claims 1-3 which comprises a T cell epitope.

20 5. A purified or non-naturally occurring polypeptide as defined in any of claims 1-4 which comprises a B cell epitope.

6. A polypeptide according to any of claims 1-5, wherein the polypeptide is encodable by a nucleic acid sequence, which sequence 25

1) is the DNA sequence selected from the group consisting of SEQ ID NOs: 33, 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 35, 37, 74, 76, and 78 or an analogue of said sequence which hybridises with any of the DNA sequences shown in SEQ ID NOs: 33, 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 35, 37, 74, 76, or 78 or a DNA

30 sequence complementary thereto, or a specific part thereof, preferably under stringent hybridisation conditions, and/or

2) encodes a polypeptide, the amino acid sequence of which has a 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:

35 34, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 75, 77 and 79 and/or 3) constitutes a subsequence of any of the above mentioned DNA sequences, and/or

7. A polypeptide as defined in any of claims 1-6 for use in medicine.

8. Use of a polypeptide as defined in any of claims 1-6 for the manufacture of a diagnostic reagent for the diagnosis of an infection with a virulent Mycobacterium.

9. Use of a polypeptide as defined in any of claims 1-6 for the manufacture of a composition for induction of a protective immune response in a mammal against infection with a virulent Mycobacterium.

10. A composition comprising a polypeptide as defined in any of claims 1-7, further comprising at least one other polypeptide derived from a virulent Mycobacterium.

11. A composition comprising, as the effective component, a micro-organism, wherein at least one copy of a DNA sequence comprising a DNA sequence encoding a polypeptide as defined in any of claims 1-6 has been incorporated into the genome of the microorganism in a manner allowing the micro-organism to express and optionally secrete the polypeptide.

12. A diagnostic reagent for diagnosing an infection with a virulent Mycobacterium comprising a polypeptide as defined in any of claims 1-7, optionally in combination with a pharmaceutically acceptable carrier or vehicle.

13. A diagnostic reagent according to claim 12 for differentiating an individual who is clinically or subclinically infected with a virulent Mycobacterium from an individual not infected with virulent Mycobacterium.

14. A diagnostic reagent according to any of claims 12 for differentiating an individual who is clinically or subclinically infected with a virulent Mycobacterium from an individual who has a cleared infection with a virulent Mycobacterium.

15. A diagnostic reagent according to any of claims 12 for diagnosing an infection with Mycobacterium tuberculosis.

16. An extract of polypeptides obtainable by a method comprising the steps of a) killing a sample of virulent Mycobacteria; b) centrifugating the sample of a) at 2,000g for 40 minutes; c) resuspending the pellet of b) in PBS and 0.5% Tween 20 and sonicating with 20 rounds of 90 seconds; d) centrifugating the suspension of c) at 5,000g for 30 minutes; e) extracting soluble proteins from the cytosol as well as cell wall and cell membrane components from the supernatant of d) with 10% SDS; f) centrifugating the extract of e) at 20,000g for 30 minutes; g) precipitating the supernatant of f) with 8 volumes of cold acetone; with an adjuvant substance.

17. Use of an extract of polypeptides with an adjuvant substance according to claim 16 for the preparation of a composition for the generation of an immune response against a virulent Mycobacterium.

18. A method of screening for inhibition of the infectivity of a virulent Mycobacterium belonging to the tuberculosis complex, said method comprising

a) inhibiting the expression of one or more of the polypeptides according to the invention, and

b) observing the effect, if any, on the infectivity of the bacteria.

19. A method according to claim 18 wherein the expression is inhibited by blocking the transcription of the polypeptides or by interfering with regulatory sequences.

20. A method according to claim 19, wherein the inhibition is at the level of translation or post-translational processing of the polypeptides or by direct interaction with the polypeptides.

21. A method of using the polypeptides having a significant effect on the infectivity of a virulent Mycobacterium as tested in any of claims 18-20 for designing a prophylactic or therapeutic agent.

22. A nucleotide sequence which is a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3,5,7,9,11,13, 15, 17, 19,21,23,25,27,29,31,33,35, 37, 74, 76 and 78 or an analogue of said sequence which hybridises with any of the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, 74, 76 or 78 or a nucleotide sequence complementary thereto, or a specific part or subsequence thereof, preferably under stringent hybridisation conditions.

23. A monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide according to any of claims 1-7 in an immuno assay, or a specific binding fragment of said antibody.