AU766093B2 - Tuberculosis vaccine and diagnostic reagents based on antigens from the mycobacterium tuberculosis cell - Google Patents

Description

WO 00/21983 PCT/DK99/00538 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. NatI 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 WO 00/21983 PCT/DK99/00538 2 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 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-y of at least 1,000 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) or whole blood, the induction being performed by the addition WO 00/21983 PCT/DK99/00538 3 of 2.5 to 5 .g PPD/ml to a suspension comprising about 1.0 to 2.5 x 10 5 PBMC, the release of IFN-y being assessable by determination of IFN-y in supernatant harvested days after the addition of PPD to the suspension compared to the release of IFN-y 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-y" 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 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, oligopeptide, 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, amino acids long. However, also longer polypeptides having a length of e.g. 25, 100, 125, 150, 175 or 200 amino acids are within the scope of the present invention.

WO 00/21983 PCT/DK99/00538 4 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 at most at most at most and at most 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 Merrifield 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 wherein Ndif is the total number of non-identical residues in the two sequences N,,f when aligned and wherein Nref 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 WO 00/21983 PCT/DK99/00538 AATCAATC (Ndif= 2 and Nref=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 (Ndi=2 and Nref= 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 oligonucleotide 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); WO 00/21983 PCT/DK99/00538 6 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 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; WO 00/21983 PCT/DK99/00538 7 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 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 WO 00/21983 PCT/DK99/00538 8 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; 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 and o) formulating the polypeptide of n) with an adjuvant substance in an immunological composition.

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-y 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 10 5 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 lag per ml suspension, the release of IFN-y being assessable by determination of IFN-y 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-y 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 10 4 virulent Mycobacteria, the induction being performed by the addition of the polypeptide to a suspension comprising about 2 x 10 5 cells isolated from the spleen, the addition of the polypeptide resulting in a concentration of not more than 20 p.g per ml suspension, the release of IFN-y being assessable by determination of IFN-y in supernatant harvested 3 days after the addition of the polypeptide to the suspension, WO 00/21983 PCT/DK99/00538 9 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 Mycobacterialml 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-y 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 x 10 5 PBMC or whole blood cells, the addition of the polypeptide resulting in a concentration of not more than 20 gg per ml suspension, the release of IFN-y being assessable by determination of IFN-y 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 lg/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-y 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 to 2.5 x 10 5 PBMC, the addition of the polypeptide resulting in a concentration of not more than 20 jg per ml suspension, the release of IFN-y being assessable by determination of IFN-y in supernatant harvested 5 days after the addition of the polypeptide to the suspension, and preferably does not induce such an IFN-y release in an individual not infected with a virulent Mycobacterium, vii) it induces a positive in vitro response determined by release of IFN-y of at least 500 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) withdrawn from an individual WO 00/21983 PCT/DK99/00538 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 x 105 PBMC, the addition of the polypeptide resulting in a concentration of not more than pg per ml suspension, the release of IFN-y being assessable by determination of IFN-y in supernatant harvested 5 days after the addition of the polypeptide to the suspension, and preferably does not induce such an IFN-y 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 100 pg 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 pg 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-y 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-y release as described with the IFN-y 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 ptg per ml suspension, such as 15 pg, 10 pg, 5 pg, 3 pig, 2 pg, or 1 pg polypeptide per ml suspension.

WO 00/21983 PCT/DK99/00538 11 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/1J, FVB/N, SJL/N, 129/SvJ, C3HIHeJ-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-y 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-y (Rook, G.A.W. (1990) Res. Microbiol. 141:253-256; Flesch, I. et S.H.E. Kaufmann (1987) J lmmunol.138(12):4408-13). However other cytokines could be relevant when monitoring the immunological response to the polypeptide, such as IL-12, TNF-a, IL-4, IL- IL-10, IL-6, TGF-P. 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-y response is presently believed to be the most relevant correlate of protective immunity as mice with a disruption of the gene coding for IFN-y 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 WO 00/21983 PCT/DK99/00538 12 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.lmmunol.154:3359).

The property described in ii) will also be satisfied if the release of IFN-y from Tlymphocytes 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-y, concentration of polypeptide, animal model, and other cytokines are equally relevant to property ii), and vice 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, 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 pg, such as between 5 and 15 lg, preferably 10 pg. For larger animals such as guinea pigs, deers, cows, primates, badgers, and possums higher doses such as 5 to 50 pg 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 WO 00/21983 PCT/DK99/00538 13 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 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-y from PBMC is determined in PBMC withdrawn from TB patients or PPD positive individuals more than 6 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-y, concentration of polypeptide, and other cytokines are equally relevant to property iv).

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 p.g/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 NaCI and 0.5% Tween-20 the serum or plasma from a TB 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 peroxidase labeled) secondary antibody and reaction is thereafter visualized by the use of OPD and

H

2 0 2 as described by the manufacturer (DAKO, Denmark). The OD value in each well is determined using an appropriate ELISA reader.

In a preferred embodiment the western blot is performed as follows: The polypeptide is applied in concentrations from 1-40 jug 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 NaCI and 0.5% Tween- 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 NaCI. 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).

WO 00/21983 PCT/DK99/00538 14 The property described in vi) will in particular be satisfied if the polypeptide does not induce such an IFN-y 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) regarding significant increase in IFN-y, 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-y release in an individual cleared of an infection with a virulent 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-y, concentration of polypeptide, and other cytokines are equally relevant to property vii).

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 pig, such as 80ig, 70 jig, 60 lg, 50 gg, 40 pg, or 30 g. 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.

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 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 identified polypeptides in the examples include a signal sequence.

WO 00/21983 PCT/DK99/00538 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 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, 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, 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 WO 00/21983 PCT/DK99/00538 16 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-y 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-y 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 onedimensional 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 oneand 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 Bcell 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 nonnaturally 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.

WO 00/21983 PCT/DK99/00538 17 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: 45 1 2 TB13A 50 3 4 39 5 6 46 7 8 TB17 47 9 TB18 40 11 12 TB21 41 13 14 TB24 48 15 16 TB27B 49 17 18 TB33 42 19 TB38 43 21 22 TB54 44 23 24 TB64 57 25 26 TB11B 51 27 28 TB16 52 29 TB16A 53 31 32 TB32 54 33 34 TB32A 55 35 36 TB51 56 37 38 TB12.5 80 74 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 WO 00/21983 PCT/DK99/00538 18 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 Tm, 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 WO 00/21983 PCT/DK99/00538 19 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 at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 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.

WO 00/21983 PCT/DK99/00538 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 mutagenesis, 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 fragment encoding a polypeptide fragment of the invention. The vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal 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 WO 00/21983 PCT/DK99/00538 21 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.

WO 00/21983 PCT/DK99/00538 22 The medium used to grow the transformed cells may be any conventional medium suitable for the purpose. A suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above. The methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purposes within the field of recombinant DNA. In the following a more detailed description of the possibilities will be given: In general, of course, prokaryotes are preferred for the initial cloning of nucleic sequences 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.

coliW3110 lambda-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may be used. 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. co/iis 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.

WO 00/21983 PCT/DK99/00538 23 Those promoters most commonly used in recombinant DNA construction include the Blactamase (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 a. in J. Immunol. Methods 161: 29-39.

According to the invention the post-translational modifications involves lipidation, glycosylation, 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 WO 00/21983 PCT/DK99/00538 24 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 infections 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 polypeptide which has the capability of modulating an immune response. For instance, a gene encoding lymphokine precursors or lymphokines IFN-y, 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 comprising a multitude of nucleotide sequences which each encode relevant epitopes of the polypeptides disclosed herein so as to effect a continuous sensitization of the immune system with a broad spectrum of these epitopes.

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; WO 00/21983 PCT/DK99/00538 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) 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 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%.

WO 00/2!983 PCT/DK99/00538 26 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, 2ethylamino 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, 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 4g to 1000 Ig, such as in the range from about 1 pg to 300 lag, and especially in the range from about 10 4g to jig. 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, intraperitoneal, intramuscular, subcutaneous or intradermal routes; the oral (on a solid physiologically acceptable base or in a physiologically 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 WO 00/21983 PCT/DK99/00538 27 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 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 IFN-y, IL-2 and IL-12) or synthetic IFNy inducers such as poly I: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 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 WO 00/21983 PCT/DK99/00538 28 least one polypeptide belonging to the group of ST-CF (Elhay MJ and Andersen P, Immunology and cell Biology (1997) 75, 595-603) such as ESAT-6, CFP7, 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.

WO 00/21983 PCT/DK99/00538 29 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 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-y into the extracellular phase 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 WO 00/21983 PCT/DK99/00538 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 WO 00/21983 PCT/DK99/00538 31 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 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 polypeptide of the invention in an immuno assay, or a specific binding fragment of said antibody, is also a part of the invention. The production of such polyclonal antibodies requires that a suitable animal be immunized with the polypeptide and that these antibodies are subsequently isolated, suitably by immune affinity chromatography. The production of monoclonals can be effected by methods well-known in the art, since the WO 00/21983 PCT/DK99/00538 present invention provides for adequate amounts of antigen for both immunization and screening of positive hybridomas.

WO 00/21983 PCT/DK99/00538 33 Examples EXAMPLE 1: Total extraction of proteins from dead M.tuberculosis bacteria.

x 109 bacteria/ml M.tuberculosis was heat treated at 55 0 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 and used as the antigen source. The pellet was sonicated with 20 rounds of 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: lx 10 7 heat killed M.tuberculosislDDA (250 gg DDA) Group 3: 50 gg ST-CF/DDA (250 lag) Group 4: 50 pg SPE/DDA (250 ig) Group 5: Adjuvant control: DDA (250 ig) in NaCI 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-y into the culture supernatants when stimulated in vitro with relevant antigens (Table WO 00/21983 PCT/DK99/00538 34 ST-CF and SPE induced a similar immune response while only a very low IFN-y release was observed after immunisation with BCG and stimulation with ST-CF.

Table 2 Recognition of protein preparations after immunisation presented as IFN-y 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 5x105 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: Subcellular fractionation of Mycobacterium tuberculosis x 10 9 colony forming units (CFU/ml) of M. tuberculosis H37Rv were inactivated by heat-killing at 60 0 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-secpulser off) and subsequently lysed three times with a French Pressure Cell at 12,000 Ib/in 2 The lysate was centrifuged at 27,000 x g for 20 min; the pellet was washed in homogenising buffer and recentrifuged. The pooled supernatants contained a mixture of cytosol and membrane components, while the pellet represented the crude cell wall.

WO 00/21983 PCT/DK99/00538 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 KCI, and twice with PBS. Soluble proteins were extracted from the cell wall by a 2 hour incubation with 2% SDS at 6 0 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 50C. 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 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 370C 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.

WO 00/21983 PCT/DK99/00538 36 The study demonstrated that multiple targets within the cell wall, membrane, and cytosol were recognised by the donors and initiated IFN-y 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 nonprecipitated 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 NaCI, 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 NaCI and 0.5% Tween-20, for 30 min. The serum pool was diluted in phosphate buffer pH 7.3 containing 0.37 M NaCI. 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 WO 00/21983 PCT/DK99/00538 37 membranes and spots subjected to N-terminal sequencing on a Procise sequencer (Applied Biosystems).

The following N-terminal sequences were obtained For TB15 ForTB18 ForTB21

:TERTAVLIKPDGIER

(SEQ ID NO: 39)

:TDTQVTWLTQESHDR

(SEQ ID NO:

:MIDEALFDAEEKMEK

(SEQ ID NO: 41)

:PLPADPSTDLSAYAQ

(SEQ ID NO: 42) :M LISQRPTLSEDVLT (SEQ ID NO: 43)

:TGNLVTKNSLTPDVR

(SEQ ID NO: 44) For TB33 For TB38 For TB54 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 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 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 is an L in the sequence MTV008.01c.

WO 00/21983 PCT/DK99/00538 38 Within the open reading frame the translated protein is 136 amino acids long. The Nterminal 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 pl of 5.36. The observed mass in SDS-PAGE is 14 kDa.

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 Nterminal 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 pl of 4.81. The observed mass in SDS-PAGE is 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 Nterminal 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 pl 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.

WO 00/21983 PCT/DK99/00538 39 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 Nterminal 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 pl of 4.91. The observed mass in SDS-PAGE is 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 Nterminal 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 pl 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 Nterminal 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 pl of 5.00. The observed mass in SDS-PAGE is kDa.

WO 00/21983 PCT/DK99/00538 TB54 is homologous to adanosyl homocysteinases from several species, e.g. 73% in a 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 Nterminal sequencing on a Procise sequencer (Applied Biosystems).

The following N-terminal sequences were obtained For TB12.5 :ALKVEMVTFDXSDPA (SEQ ID NO: 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 Nterminal 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 WO 00/21983 PCT/DK99/00538 41 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 Nterminal 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 Nterminal 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 pl 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 nonprecipitated 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 NaCI, 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 WO 00/21983 PCT/DK99/00538 42

:MEVKIGITDSPRELV

(SEQ ID NO:

:SAYKTVVVGTDDXSX

(SEQ ID NO: 46) For TB17 MEQRAELVVGRALVV (SEQ ID NO: 47) For TB24 A D I DGVTG SAG L(N)PA (SEQ ID NO: 48) ForTB27B :TYETILVERDQRVGI (SEQ ID NO: 49) 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 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 9 433 Da and a theoretical pl of 4.93. The observed mass in SDS-PAGE is 10 kDa.

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 Nterminal 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 15 313 Da and a theoretical pl of 5.60. The observed mass in SDS-PAGE is 16 kDa.

WO 00/21983 PCT/DK99/00538 43 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 pl 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 Nterminal 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 pl 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.

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 Nterminal 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 pl of 4.82. The observed mass in SDS-PAGE is 28 kDa.

WO 00/21983 PCT/DK99/00538 44 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 ForTB13A :PVTQEEIIAGIAEII (SEQ ID NO: 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 Nterminal 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 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 p.l of TetraLink Tetrameric Avidin Resin (Promega) in PBS, pH 7.4 in an eppendorf tube. After incubation over night at 4 0 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 pl of 4 times concentrated SDS-PAGE sample buffer (0.08 M Tris-HCI, 8% SDS, 16% WO 00/21983 PCT/DK99/00538 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 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: GDLVVVLEAMKMENPVTA (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.

WO 00/21983 PCT/DK99/00538 46 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:SGNSSLGIIVGIDD (SEQ ID NO: 54) TB32A:AEVLVLVEHAEGALK (SEQ ID NO: 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 pl 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 pl of 4.64. The apparent molecular mass in an SDS-PAGE gel is 17 kDa.

WO 00/21983 PCT/DK99/00538 47 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 128.

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 pl 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 pl 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.

WO 00/21983 PCT/DK99/00538 48 EXAMPLE 5: Cloning of the genes encoding TB10C, TB13A, TB17, TB11B, TB16, TB16A, TB32, TB51 The genes encoding TB10C, TB13A, TB17, TB11B, TB16, TB16A, TB32, TB51 were all cloned into the E. coliexpression vector pMCT3, by PCR amplification with gene specific primers.

Each PCR reaction contained 10 ng of M. tuberculosis chromosomal DNA in lx low salt Taq+ buffer (Stratagene) supplemented with 250 IM 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 C.1 reaction volume.

Reactions were initially heated to 94 0 C for 25 sec. and run for 30 cycles according to the following program; 94 0 C for 10 sec., 55 0 C for 10 sec., and 72 0 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. coil. 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 Nterminal 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 lag/ml ampicillin, was inoculated with 10 ml of an overnight culture of XL1-Blue cells harbouring recombinant pMCT3 plasmids. Cultures were shaken at 37 0 C until they reached a density of ODo 00 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 28 0 nm. Fractions containing recombinant protein WO 00/21983 PCT/DK99/00538 49 were pooled and dialysed against 3 M urea in 10 mM Tris-HCI, 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 NaCI. Fractions were analysed by SDS-PAGE and protein concentrations were estimated at OD280nm. Fractions containing protein were pooled and dialysed against 25 mM Hepes buffer, pH 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 Primers used for cloning of TB10C CTG AGA TCT GTG GAG GTC AAG ATC GGT CTC CCA TGG CTAC TTA CCC GCT CGT AGC AAC (SEQ ID NO: 58) (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 TB13A-R CTC CCA TGG GAA ACC GCC ATT AGC GGT (SEQ ID NO: (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 TB17-R CTC CCA TGG CGA CAC TCG ATC CGG ATT (SEQ ID NO: 62) (SEQ ID NO: 63) TB17-F and TB17-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

TB11B Primers used for cloning of TB11B WO 00/21983 PCT/DK99/00538 TB11B-F CTG AGA TCT ATG CCA GTG GTG AAG ATC TB11 B-R CTC CCA TGG TTA TGC AGT CTT GCC GGT (SEQ ID NO: 64) (SEQ ID NO: TB11B-F and TB11B-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 TB16-R CTC CCA TGG TAC CGG AAT CAC TCA GCC (SEQ ID NO: 66) (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 TB16A-R CTC CCA TGG GCA CAT GCC TTA GCT GGC (SEQ ID NO: 68) (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 TB32-R: CTC CCA TGG CTAC CTA AGT CAG CGA CTC GCG (SEQ ID NO: (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 TB51-R CTC CCA TGG GTC ATA CGG TCA CGT TGT (SEQ ID NO: 72) (SEQ ID NO: 73) WO 00/21983 PCT/DK99/00538 51 TB51-F and TB51-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.

Primers used for cloning of CTG CCA TGG CTA GGT GGT GTG CAC GAT C (SEQ ID NO: 89) CTG AAG CTT ATG AGC GCC TAT AAG ACC (SEQ ID NO: 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: 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.

WO 00/21983 WO 0021983PCT/DK99/00538 52 TB14: Primers used for cloning of TB14: TBI4-F: 5'-GTG ACC GMA OGG ACT CTG GT-3' TB14-R: 5'-CTA GGC GCC GGG AMA CCA GAG-3' (SEQ ID NO: 97) (SEQ ID NO: 98) T118: Primers used for cloning of TB1 8: TB 18-F 5'-ATG ACG GAT ACT CMA GTC ACC TG-3' TB18-R: 5'-GGA GTG GTA CGG CTC GGC GC-3' (SEQ ID NO: 99) (SEQ ID NO: 100) TB27: Primers used for cloning of TB27: TB27-F: 5'-ATG ACG TAC GMA ACC ATC CT-3' TB27-R: 5'-TCA TOG GTG GGT GMA CTG GGG-3' (SEQ ID NO: 101) (SEQ ID NO: 102) TB33: Primers used for cloning of TB33: TB33-F: 5'-ATG COG OTT CCC GCA GAO COT AG-3' TB33-R: 5'-TAC GAO GGG TAO CAC TOO TGG-3' (SEQ ID NO: 103) (SEQ ID NO: 104) TB38: Primers used for cloning of TB38: TB38-F: 5'-ATG CTG, ATC TOA CAG OGO COO A-3' TB38-R: 5'-AAG CTG TTO GGT HOC GGO GTA G-3' (SEQ ID NO: 105) (SEQ ID NO: 106) TB54: Primers used for cloning of TB54: TB54-F: 5' -ATG ACC GGA MAT HTG GTG AO-3' TB54-R: 5'-TCA GTA GOG GTA GTG GTO CGG-3' (SEQ ID NO: 107) (SEQ ID NO: 108) TB14,TBI 8,TB27,TB33,TB38 and TB54 will be cloned in ex-pressions vector pBAD- TOPO (Invitrogen).

Example 5a: Cloning of the genes encoding TBI 2.5, T1320.6, and TB4O.8 The genes encoding T1312.5, T620.6, and T1340.8 were all cloned into the E. coli expression vector pMCT3 as described in Example WO 00/21983 PCT/DK99/00538 53 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 TB12.5-R: CTC CCA TGG TTA TTG ACC CGC CAC GCA (SEQ ID NO: 83) (SEQ ID NO: 84) TB12.5-F and TB12.5-R create Bgll and Ncol 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 TB20.6-R: CTC CCA TGG CTA GTC GCG GAG CAC AAC (SEQ ID NO: (SEQ ID NO: 86) TB20.6-F and TB20.6-R create Bgll and Ncol 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 TB40.8-R: CTC CCA TGG TCA CGT CTT CCA GCG GGT (SEQ ID NO: 87) (SEQ ID NO: 88) TB40.8-F and TB40.8-R create Bg/ll and Ncol sites, respectively, used for the cloning in pMCT3.

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

WO 00/21983 PCT/DK99/00538 54 IFN-y induction in the mouse model of TB infection The recognition of an antigen by IFN-y 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-y production in mice of four different haplotypes during a primary infection: 8-12 weeks old female mice C57BL/6j CBA/J DBA.2 (H-2d) and A.SW (H-2s) mice (Bomholtgaard, Ry, Denmark) will be infected i.v. via the lateral tail vein with an inoculum of 5 x 10 4 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-y 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 10 4 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.Infect.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 IFNy 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-y produced in response to any of the recombinantly produced proteins will be evaluated.

IFN-y induction in humans during infection with virulent Mycobacteria.

IFN-y is currently believed to be the best marker of protective immunity in humans. In patients with limited tuberculosis, high levels of IFN-y can be induced, in contrast to patients with severe TB who often respond with low levels of IFN-y (Boesen et al (1995), Human T-cell response to secreted antigen fractions of M.tuberculosis. Infection and Immunity 63(4):1491-1497). Furthermore, IFN-y release has been shown to correlate WO 00/21983 PCT/DK99/00538 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-y 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 minimal TB patients) respond with high levels of IFN-y. The ability of the polypeptides to induce IFN-y 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 y >30% of that induced by a crude antigen preparation like ST-CF, PPD and SPE.

Cultures will be established with 1 to 2 x 10 s PBMC in 200pl in microtiter plates (Nunc, Roskilde, Denmark) or with 1 ml of serum or plasma stimulated with the identified polypeptide and the IFN-y 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-y 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/Vestern 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.

WO 00/21983 PCT/DK99/00538 56 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 10 5 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.

WO 00/21983 PCT/DK99/00538 57 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-y 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.tuberculosislml. 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-y 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 RPMI 1640 medium (Gibco, Grand Island, 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 WO 00/21983 PCT/DK99/00538 58 the viability of the cells were determined by Nigrosin staining. Cultures were established with 1.25 x 105 PBMCs in 100 .l in microtitre plates (Nunc, Roskilde, Denmark) and stimulated with ST-CF (5gg/ml), TB13A, TB15A, TB17, TB18, TB33, TB11B, TB16A, TB16, TB32, and TB51 in a final concentration of 10 gg/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 0 C until used.

Cytokine analysis: Interferon-y (IFN-y) 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-y (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 Table 11, and Table 12 a marked release of IFN-y 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-y responses 1,000 pg/ml). Less than 1/3 of the donors recognised TB15A and TB11B at this level. Between 30 and 70% of the donors show intermediate IFN-y 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.

WO 00/21983 PCT/DK99/00538 59 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-y/ml.

BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB13A (1 pg/ml) (5 pg/ml) (10 pg/ml) 1 12 11572 10860 41 2 0 14257 11536 0 3 7 13270 8844 493 4 0 13193 2828 0 4 14239 14275 332 6 0 16278 12623 0 TB patients Donor No ag PHA ST-CF TB13A (1 pg/ml) (5 pg/ml) (10 pg/ml) 1 0 9914 3297 0 2 51 10058 6489 0 3 0 10587 9155 0 4 0 9458 5236 18 WO 00/21983 PCT/DK99/00538 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-y/ml.

BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF (1 pg/ml) (5 pg/ml) (10 pg/ml) 1 0 18860 3733 1478 2 0 16218 2856 0 3 94 18427 13998 0 4 0 17815 4255 0 0 15981 10830 441 6 81 16961 11165 8009 TB patients Donor No ag PHA ST-CF (1 pg/ml) (5 pg/ml) (10 pg/ml) 1 231 18854 6443 57 2 0 17213 2196 0 3 0 17880 1049 0 4 0 17777 2865 0 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 IFNy/ml BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB17 (1 pg/ml) (5 pg/ml) (10 pg/ml) 1 33 16696 7304 66 2 102 16878 6427 3 49 12161 11055 0 4 0 12949 2284 73 81 12129 6669 1029 6 0 12706 11762 656 WO 00/21983 PCT/DK99/00538 61 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-y/ml BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB18 (1 pg/ml) (5 gg/ml) (10 pg/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 mlg/mi) gg/mi) gg/ml) 1 60 12301 11057 265 2 7 10390 6123 167 3 34 11678 8136 1629 WO 00/21983 PCT/DK99/00538 62 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-y/ml.

BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB33 (1 tpg/ml) (5 pg/ml) (10 pg/ml) 1 589 10068 4426 721 2 1953 10817 6316 662 3 702 11837 1640 0 4 605 9463 2694 0 2471 7990 5979 0 TB patients Donor No ag PHA ST-CF TB33 (1 pg/ml) (5 pg/ml) (10 pg/ml) 1 0 3647 812 0 2 0 12266 920 0 3 0 12899 4388 0 4 0 10233 7989 0 WO 00/21983 PCT/DK99/00538 63 Table 8 Stimulation of PBMCs from 3 healthy BCG vaccinated and 3 TB patients with recombinant TB11B. Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN-y/ml.

BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB11B (1 Pg/ml) (5 pg/ml) (10 Pg/ml) 1 0 13682 9067 1379 2 0 13705 10169 2092 3 0 13231 7740 0 TB patients Donor Noag PHA ST-CF TB11B (1 g/ml) (5 pg/ml) (10 pg/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-y/ml.

BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB16A (1 pg/ml) (5 pg/ml) (10 pg/ml) 1 0 12816 1831 645 2 0 14530 10293 1404 TB patients Donor No ag PHA ST-CF TB16A (1 pg/ml) (5 pg/ml) (10 pg/ml) 1 0 11606 5460 42 2 0 11836 5837 977 3 388 12353 8401 958 4 0 9587 3169 499 43 10820 4869 593 WO 00/21983 PCTIDK99/00538 64 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 IFNy/ml in BCG vaccinated control donors, no known TB exposure.

Donor No ag PHA ST-CF TB16 (1 pg/ml) (5 pg/ml) (10 pg/ml) 1 33 16696 7304 0 2 102 16878 6427 292 3 49 12161 11055 514 4 0 12949 2284 24 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-y/ml.

BCG vaccinated control donors, no known TB exposure Donor Noag PHA ST-CF TB32 (1 pg/ml) (5 pg/ml) (10 pg/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 pg/ml) (5 pg/ml) (10 pg/ml) 1 60 12301 11057 562 2 7 10390 6123 206 3 34 11678 8136 83 WO 00/21983 PCT/DK99/00538 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 IFNy/ml.

BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB51 (1 ig/ml) (5 pg/ml) (10 pg/ml) 1 33 16696 7304 596 2 102 16878 6427 1155 3 49 12161 11055 2247 4 0 12949 2284 777 81 12129 6669 140 6 0 12706 11762 1123 66 Figure legends: Figure 1: Long term protection against TB can be induced by immunisation with dead M.

tuberculosis.

Mice received either: three immunisations with 1x10 7 CFU of dead M.tuberculosis H37Rv (squares); three immunisations with 50 jLg of ST-CF (triangles); one immunisation with 5 x 10 4 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 50 ig 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.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in S the field relevant to the present invention as it existed before the priority date of each 25 claim of this application.

t «e~e EDITORIAL NOTE APPLICATION NUMBER 60874/99 The following Sequence Listing pages 1 to 50 are part of the description. The claims pages follow on pages 67 to 73.

PCT/DK99/00538 WO 00/21983 1 SEQUENCE LISTING <110> Statens Serum Institute <120> TB vaccine and diagnostic based antigens from the M.tuberculosis cell <130> 21868PC1 <160> 108 <170> FastSEQ for Windows Version <210> 1 <211> 273 <212> DNA <213> M.Tuberculosis <220> <221> CDS <222> gtg Val 1 <400> gag gtc Glu Val 1 aag atc ggt atc acg gac Lys Ile Gly Ile Thr Asp 5 ccg cgc gag ctg Pro Arg Glu Leu gtg ttc 48 Val Phe tec agt gcg Ser Ser Ala ctg cgc gac Leu Arg Asp cag Gln acg ccc agt gag Thr Pro Ser Glu gaa gaa ctc gtc Glu Glu Leu Val agc aac gcg Ser Asn Ala cgg ggc cgt Arg Gly Arg gac tct ggt ttg Asp Ser Gly Leu ctg Leu acc ctg acc gac Thr Leu Thr Asp cgc ttc Arg Phe cta att cac acc Leu Ile His Thr gcc cgc cgg gtg Ala Arg Arg Val gcc Ala 55 agg atc gcc tat Arg Ile Ala Tyr gtc Val gag atc ggt gtc Glu Ile Gly Val gca Ala gac Asp ggc ttc ggc gtc Gly Phe Gly Val gtg gac gcc gca Val Asp Ala Ala 273 ggg tcc gcc gga Gly Ser Ala Gly <210> 2 <211> <212> PRT <213> M.Ti aag Lys gtt gct acg age Val Ala Thr Ser ggg taa Gly uberculosis <400> 2 Met Glu Val Lys Ile Gly Ile 1 5 Ser Ser Ala Gin Thr Pro Ser Leu Arg Asp Asp Ser Gly Leu Thr Asp Ser Pro Arg Glu Leu Val Phe 10 Glu Val Glu Glu Leu Val Ser Asn Ala 25 Leu Thr Leu Thr Asp Glu Arg Gly Arg PCT/DK99/00538 WO 00/2 1983 2 Arg Phe Leu Ala Asp Ala Ile His Thr Arg Arg Val 70 Ala 55 Gly 40 Arg Phe 45 Giu Ile Gly Val Ile Ala Tyr Val Gly Val Gly Val Asp Ala Ala Gi y Ser Ala Gly Lys Val Ala <210> 3 <211> 348 <212> DNA <213> M.Tubercuiosis <220> <221> CDS <222> (345) Thr Ser Gly gt g Val 1 <400> 3 cct gtc act cag Pro Val Thr Gin 5 gaa gaa atc att Giu Gu Ile Ile gcc Ala 10 gqt atc goc gag Gly Ile Ala Giu atc atc 48 Ile Ile gaa gag gta Giu Giu Val ttc gtc gac Phe Val Asp ggt atc gag ccg Gly Ile Giu Pro tcc Ser 25 gag atc aco ccg Giu Ile Thr Pro gag aag tcg Giu Lys Ser gag atC gCC Giu Ile Ala gac ctg gac atc Asp Leu Asp Ile gac Asp tcg ctg tcg atg Ser Leu Ser Met gtg cag Val Gin acc gag gac aag Thr Giu Asp Lys tac Tyr 55 ggc gtc aag atc Gly Val Lys Ile ccc Pro gac gag gac ctc Asp Giu Asp Leu 192 240 ggt ctg cgt acc Giy Leu Arg Thr ggt gac gtt gtc Gly Asp Val Val gcc Al a tac atc cag aag Tyr Ile Gin Lys gag gaa gaa aac Giu Giu Giu Asn gag gcg gct cag Giu Ala Ala Gin gcg Ala 90 ttg cgc gcg aag Leu Arg Ala Lys att. gag Ile Giu tcg gag aac ccc gat gcc gtt. gcc aac gtt cag gcg agg Ser Giu Asn Pro Asp Ala Val Ala Asn Val Gin Ala Arg ctt gag gcc Leu Glu Ala 110 100 105 gag tcc aag tga Glu Ser Lys 115 <210> 4 <211> 115 <212> PRT <213> M.Tubercuiosis <400> 4 Met Pro Val T 1 Glu Glu Val T 2 hr Gin Giu Glu Ile Ile Ala Gly Ile Ala hr Gly Ile Giu Pro 0 Ser Giu Ile Thr Pro 25 Gu Ile Ile Giu Lys Ser PCTIDK99/00538 WO 00/21983 Phe Val Asp Val Gin Thr Ala Gly Leu Asp Leu Asp Ile Asp Gly Ser Leu Ser Met Glu Ile Ala Glu Asp Leu Glu Asp Lys Arg Thr Val 70 Val Lys Ile Asp Val Val Glu Ala 75 Leu Ile Gin Lys Leu Glu Glu Asn Pro Glu Ala Ala iln Pro Asp Ala Vai Ala Asn 100 105 Ala 90 Val Arg Ala Lys Ile Glu Glu Ala Ser Giu Asn Glu Ser Lys 115 Gin Ala Arg Leu 110 <210> <211> 411 <212> DNA <213> M.Tuberculosis <220> <221> CDS <222> (408) <400> 5 acc gaa cgg act Thr Glu Arg Thr ctg gta ctg atc Leu Val Leu Ile ccg gat ggc atc Pro Asp Gly Ile gaa agg 48 Glu Arg cag ctg atc Gin Leu Ile atc gct gcg Ile Ala Ala ggc Gly gag atc atc agc Glu Ile Ile Ser cgc Arg 25 atc gag cgc aaa Ile Giu Arg Lys ggc ctc acc Gly Leu Thr qcc agc cag Ala Ser Gin ctg cag ctc agg Leu Gin Leu Arg acc Thr gtc agc gcg gag Val Ser Ala Glu cac tao His Tyr gcc gaa cat gaa Ala Giu His Glu ggc Gly 55 aaa cca ttc ttt Lys Pro Phe Phe gga Gly tcg ttg ctg gag Ser Leu Leu Glu atc acg tog ggt Ile Thr Ser Gly gtg gta gcg gcg Val Val Ala Ala ato Ile gtg gag gga acc Val Giu Gly Thr cga Arg gcc atc gcg gcg Ala Ile Ala Ala gtt Val cgc caa ctc gcc Arg Gin Leu Ala ggc Gly 90 ggc acc gac ccg Gly Thr Asp Pro gtg cag Val Gin 240 288 336 gcg gcg gcg Ala Ala Ala ttc aac ctg Phe Asn Leu 115 ccc Pro 100 ggc aca atc cgg Gly Thr Ile Arg gac ttc gct cta gag acg cag Asp Phe Aia Leu Giu Thr Gin 110 gtg cac ggg tct Val His Gly Ser gat Asp 120 tcg gcc gaa tcc Ser Ala Giu Ser cag cgc gaa 384 Gin Arg Glu atc gcg Ile Ala 130 ctc tgg ttt ccc Leu Trp Phe Pro ggc gcc tag 411 Gly Ala 135 <210> 6 PCT/DK99/00538 WO 00/21983 <211> <212> <213> 136

PRT

M.Tuberculosis <400> Thr Glu Met 1 Gin Ti Arg Thr Leu Val Leu Ile Lys 10 Ile Pro Asp Gly Ile Glu Arg Leu Ile Gly Ala Ala Leu Glu Ile Ile Ser Arg 25 Val Glu Arg Lys Gin Leu Arg Thr 40 Ser Ala Glu Leu Ser Gly Leu Thr Ala Ser Gin Leu Leu Glu His Tyr Phe Ile Ala Glu His Glu Lys Pro Phe Phe Gly Val Thr Ser Gly Val Val Ala Ala Ala Ile 75 Gly Glu Gly Thr Arg Ile Ala Ala Val Gly Gin Leu Ala Gly 90 Asp Thr Asp Pro Val Gin Ala Ala Ala Pro Thr Ile Arg Gly 105 Ser Phe Ala Leu Glu Thr Gin 110 Gin Arg Glu Phe Asn Leu 115 Ile Ala Leu 130 His Gly Ser Asp 120 Ala Ala Glu Ser Ala 125 Trp Phe Pro Gly 135 <210> 7 <211> 441 <212> DNA <213> M.Tuberculosis <220> <221> CDS <222> atg Met 1 <400> 7 agc gcc tat Ser Ala Tyr acc gtg gtg gta Thr Val Val Val gga Gly 10 acc gac ggt tcg Thr Asp Gly Ser gac tcg 48 Asp Ser tcg atg cga Ser Met Arg aag ttg atc Lys Leu Ile gta gat cgc gct Val Asp Arg Ala gcc Ala 25 cag atc gcc ggc Gin Ile Ala Gly gca gac gcc 96 Ala Asp Ala atc gcc tcg gca Ile Ala Ser Ala tac Tyr 40 cta cct cag cac gag gac gct cgc Leu Pro Gin His Glu Asp Ala Arg gcc gcc Ala Ala gac att ctg aag Asp Ile Leu Lys gac Asp 55 gaa ago tac aag Glu Ser Tyr Lys gtg Val acg ggc acc gcc Thr Gly Thr Ala ccg Pro atc tac gag atc Ile Tyr Glu Ile cac gac gcc aag His Asp Ala Lys gaa Glu 75 cga gcg cac aac Arg Ala His Asn gcc Ala ggt gcg aaa aac gtc gag gaa cgg ccg atc gtc ggc gcc ccg Gly Ala Lys Asn Val Glu Glu Arg Pro Ile Val Gly Ala Pro gtc gac Val Asp 288 gcg ttg gtg Ala Leu Val aac Asn 100 ctg gcc gat gag Leu Ala Asp Glu gag Glu 105 aag gcg gac ctg ctg gtc gtc 336 Lys Ala Asp Leu Leu Val Val PCT/DK99/00538 WO 00/21983 ggc aat gtc Gly Asn Val 115 ggt ctg agc acg Gly Leu Ser Thr gcg ggt cgg ctg Ala Gly Arg Leu gga tcg gta Gly Ser Val ccg gcc Pro Ala 130 aat gtg tca cgc Asn Val Ser Arg gcc aag gtc gac Ala Lys Val Asp ctg atc gtg cac Leu Ile Val His 432 acc acc tag Thr Thr 145 <210> 8 <211> 146 <212> PRT <213> M.Tuberculosis <400> Ser Ala Met 1 Ser 8 Tyr Lys 5 Thr Val Val Val Gly 10 Gin Thr Asp Gly Ser Asp Ser Met Arg Ala Val Asp Arg Ala Ala 25 Leu Ile Ala Gly Lys Leu Ile Ala Ala Asp Ile Ala Ser Ala Tyr Glu Pro Gin His Ala Asp Ala Asp Ala Arg Gly Thr Ala Ile Leu Lys Asp Ser Tyr Lys lie Val Arg Pro Gly Tyr Glu Ile Leu 70 Glu Asp Ala Lys Glu Val Ala His Asn Ala Lys Asn Val Leu Glu Arg Pro Ile 90 Lys Gly Ala Pro Val Asp Ala Leu Val Gly Asn Val 115 Pro Ala Asn 130 Thr Thr 145 Asn 100 Gly Ala Asp Glu Glu 105 Ala Ala Asp Leu Leu Ser Thr Ile 120 Ala Gly Arg Leu Leu Val Val 110 Gly Ser Val Ile Val His Val Ser Arg Arg 135 Lys Val Asp <210> 9 <211> 498 <212> DNA <213> M.Tuberculosis <220> <221> <222>

CDS

(495) atg Met 1 <400> 9 gaa cag cgt gcg gag ttg gtg gtt ggc Glu Gin Arg Ala Glu Leu Val Val Gly 5 10 cgg gca ctt gtc gtc gtc 48 Arg Ala Leu Val Val Val gtt gac gat Val Asp Asp cgc acg gcg Arg Thr Ala cac ggc gat His Gly Asp 25 gaa gac cac agc ggg ccg ctt 96 Glu Asp His Ser Gly Pro Leu gtc acc gag ctg ctc acc gag gcc ggg ttt gtt gtc gac ggc gtg gtg Val Thr Glu Leu Leu Thr Glu Ala Gly Phe Val Val Asp Gly Val Val WO 00/21983 PCTIDK99/00538 otg aac aca gcg Leu Asn Thr Ala gcg gtg tcg gc gac gag Ala Val Ser Ala Asp Glu gag atc oga aat Glu Ile Arg Asn gcg Ala gtg Val atc gqc ggg gtg Ile Gly Giy Val ctg gtg gtg tcg Leu Vai Val Ser ccg gaa gcc acc Pro Glu Ala Thr 90 gtc Val 75 ggc ggg acc ggg Gly Gly Thr Gly gtg Va1 192 240 288 acg cct cgc gat gto aco Thr Pro Arg Asp Val Thr cgc gao att ctg Arg Asp Ile Leu gac cgc Asp Arg gag atc otc Glu Ile Leu gga atc gtc Gly Ile Val 115 ggt Gly 100 ato goc gag gc Ile Ala Glu Ala cgo gcg too ggg Arg Ala Ser Gly otg too gcg Leu Ser Ala 110 gto too ggo Vai Ser Gly gao goo ggg ttg Asp Ala Gly Leu cgo ggo ctg gcg Arg Giy Leu Ala ggt Gly 125 ago acg Ser Thr 130 otg gtg gto aaO Leu Val Val Asn gcg ggt tog ogt Ala Gly Ser Arg tat Tyr 140 gcg gtg cgo gat Ala Val Arg Asp 432 480 gga atg gog aog otg aat Gly Met Ala Thr Leu Asn 145 150 ccg ota gog gca Pro Leu Ala Ala oag Gin 155 ato ato ggg oag Ile Ile Gly Gin ttg Leu 160 498 tcg ago ttg gag ato tga Ser Ser Leu Glu Ile 165 <210> <211> 165 <212> PRT <213> M.Tuberculosis <400> 10 Glu Gin Arg Ala Giu Leu Vai Val Met 1 Vai Val Gly 10 Glu Arg Ala Leu Val Vai Val Asp Asp Arg Thr Giu Leu Thr Ala His Gly Asp 25 Gly Asp His Ser Leu Thr Glu Ala Val Ser Ala 40 Glu Phe Val Val Asp Leu Gly Pro Leu Gly Val Val Asn Thr Ala Ala Asp Glu Ile Arg Asn Val Ile Ala Gly Gly Gly Val Thr Asp 70 Val Val Ser Val 75 Gly Thr Gly Val Pro Arg Asp Val Thr Pro Glu Ala Thr Arg Arg Asp Ile Leu Asp Arg Glu Ile Leu Gly Ile Val 115 Ser Thr Leu Gly 100 Ile Ala Giu Ala Ile 105 Ala Ser Gly Asp Ala Gly Leu Ser Arg Gly Leu Ala Gly Leu Ser Ala 110 Val Ser Gly Val Arg Asp Val Val Asn Ala Gly Ser Arg Tyr 130 Gly Met 145 Ala Thr Leu Asn Pro Leu Ala Ala Gin Ile Ile Gly Gin 150 155 Leu 160 WO 00/21983 Ser Ser Leu <210> <211> <212> <213> <220> <221> <222> PCTIDK99/00538 Glu Ile 165 495

DNA

M. Tuberculosis

CUS

(492) atg Met 1 <400> 11 aog gat act Thr Asp Thr caa Gin 5 gtc acc tgg ttg Vai Thr Trp Leu caa gag tca cat Gin Giu Ser His gac cqa 48 Asp Arg oto aaa gca Leu Lys Ala gcc gaa atc Ala Glu Ile ctc gaO cag ctg Leu Asp Gin Leu gcg aat ogc ccg Ala Asn Arg Pro gto atc gcc Val Ile Ala gag aao ggc Glu Asn Gly aac gac cgc cgo Asn Asp Arg Arg ga a Giu 40 gaa ggc gac ctg Giu Gly Asp Leu cgc Arg gga tac Gly Tyr cac gcc gcc cgc His Ala Ala Arg gag cag ggc cag Giu Gin Gly Gin cag Gin gag gcc cgc att Giu Ala Arg Ile ogo Arg cag ctg cag gac Gin Leu Gin Asp ttg Leu 70 ctc agc aao gca Leu Ser Asn Ala a ag Lys gtt ggc gag gca Val Gly Giu Ala 192 240 288 aag caa tcc gqo Lys Gln Ser Gly gtc Val1 gca tta ccc ggt Ala Leu Pro Gly gtg gtc aag gtg Val Val Lys Val tac tac Tyr Tyr aac ggc gao Asn Gly Asp gag ggc gtc Giu Gly Val 115 aag Lys 100 tog gao agc gaa Ser Asp Ser Giu acg Thr 105 ttc ctc atc gcc Phe Leu Ile Ala acc cgc oag Thr Arg Gln 110 aat tca ccg Asn Ser Pro agc gac ggc aag Ser Asp Gly Lys gag gtc tao tog Giu Val Tyr Ser oto ggt Leu Gly 130 ggg goc ctg atc Giy Ala Leu Ile gao Asp 135 gcc aag gto ggc Ala Lys Val Gly gag Glu 140 acc cgc agc tac Thr Arg Ser Tyr acg Thr 145 gtg ccc aac ggc Val Pro Asn Giy acc gtg tog gtg Thr Val Ser Val acc Thr 155 ota gtc agc gcc Leu Vai Ser Ala gag Giu 160 ocg tao cac too tag 495 Pro Tyr His Ser <210> 12 <211> 164 <212> PRT WO 00/21983 PCT/DK99/00538 <213> M.Tuberculosis Met 1 Leu Ala <400> 12 Thr Asp Thr Lys Ala Glu Glu Ile Asn Gin Val Thr Trp Le 5 Le Asp Gin Leu Ile Thr Gin Glu 10 Ala Asn Arg Gly Asp Le Ser His Asp Arg Pro 25 Glu Asp Arg Arg Glu Val Ile Ala Glu Asn Gly Ala Arg Ile Glv Tvr His Ala Ala Arg Glu Glu Gin Gly Gin Arg Gin 55 Leu Gin Va1 Le Gin Asp Lys Le 70 Ala Ser Asn Ala Lys 75 Val Gly Glu Ala Pro Gin Ser Gly Le Pro Gly Val Lys Val Tyr Tyr Asn Gly Asp Glu Gly Val 115 Le Gly Gly Lys 100 Asp Ser Glu Leu Ile Ala Ser Asp Gly Lys Val Tyr Ser Thr Arg Gin 110 Asn Ser Pro Arg Ser Tyr Ala Leu Ile Asp Ala Lys Val Gly 130 Thr Val Glu 140 Leu Pro Asn Gly Ser 150 Val Ser Val Thr 155 Val Ser Ala Glu 160 Tyr His Ser <210> <211> <212> <213> <220> <221> <222> 13 558

DNA

M.Tuberculosis

CDS

.(555) atg Met 1 <400> 13 att gat gag Ile Asp Glu got Ala 5 ctc ttc gac gcc Le Phe Asp Ala gag aaa atq gag Glu Lys Met Glu aag gct Lys Ala gtg gog gtg Val Ala Val aac cot ggc Asn Pro Gly gca Ala cgt gac gac ctg Arg Asp Asp Le act ato cgt aco Thr Ile Arg Thr ggc cgo gc Gly Arg Ala ggt gog goo Gly Ala Ala atg tto tot ogg Met Phe Ser Arg aco ato gao tao Thr Ile Asp Tyr aco cog Thr Pro ato aog caa ctg Ile Thr Gin Lev ago ato aat gto Ser Ile Asn Val gag gog cgg ota Glu Ala Arg Le gtc Val gtg ata aag cog Val Ile Lys Pro gaa gc aat cag Glu Ala Asn Gin ttg Lev 75 cgo got ato gag Arg Ala Ile Glu act Thr gca att cgc aac Ala Ile Arg Asn gac ctt gga gtg Asp Lev Gly Val cc aco aac gao Pro Thr Asn Asp ggc gc Gly Ala ott att cgo gtg gc gta cog cag otc aco gaa gaa cgt cgg oga gag 336 WO 00/2 1983 Leu Ilie Arg ctg gtc aaa Leu Val Lys 115 cgt aat atc Arg Asn Ile PCT/DK99/00538 9 Thr Val1 100 cag Gin Ala Val Pro Gin Len 105 ggg Gly Giu Glu Arg gca aag cat Ala Lys His aag Lys 120 atg Met gag gag gcc Giu Giu Ala aag Lys 125 cgc Arg Arg Arg Glu 110 gtt teg gtg Val Ser Val atc cgt aag Ile Arg Lys cgt cgc aaa Arg Arg Lys 130 gaa ggc Giu Gly gcg Al a 135 gat Asp gag gaa etc Gin Giu Leu gag gcc ggc Gin Ala Gly 145 gac Asp gag Giu 150 caa Gin gag gte ggt Giu Val Gly gaa aag gat Gin Lys Asp etc Len 160 aag acc aeg Lys Thr Thr tac gte ace Tyr Val Thr eaa Gin 170 tag att gat gag ctg Ile Asp Giu Leu gtt aaa Val Lys 175 cac aaa gaa His Lys Giu ggC Gly 180 ctg ctg gag Leu Len Giu gte Val 185 Met Val1 Asn Thr Val1 Ala Leu Leu Arg Gin 145 Asp His <210> <211> <212> <213> <400> Ilie Asp Ala Vai Pro Giy Pro Ile Val Ile Ile Arg Ilie Arg Vai Lys 115 Asn Ile 130 Gly Giu Lys Thr Lys Giu <210> <211> <212> 14 Gin Al a Met Thr Lys Asn Val1 100 Gin Arg Al a Thr Gly 180 651

DNA

Al a Arg Phe Gin Pro Ser Ala Al a Arg Giy His 165 Glu Len Asp Ser Len Tyr 70 Asp Val Lys Lys Gin 150 Gin Leu Phe Asp Ar g Al a 55 Glu Leu Pro His Al a 135 Asp Tyr Leu Asp Len Ile 40 Ser Al a Giy Gin Lys 120 Met Gin Val1 Gin Ala Ser 25 Thr Ile Asn Val Leu 105 Gly Giu Val Thr Val 185 Gin 10 Thr Ile As n Gin As n 90 Thr Gin Giu Gly Gin 170 Glu Ilie Asp Val1 Len 75 Pro Giu Gin Len Arg 155 Ie Lys Arg T yr Pro Arg Thr Gin Ala His 140 Ala Asp Met Thr Tyr Gin Ala As n Arg Lys 125 Arg Gin Gin Gin Gi y Gly Ala Ile Asp Arg 110 Val1 Ile Lys Len Lys Arg Ala Arg Gin Gly Arg, Ser Arg Asp Val 175 Ala Ala Ala Len Thr Ala Gin Val1 Lys Len 160 Lys 14 185

PRT

M. Tubereniosis <213> M.Tnbereulosis <220> WO 00/21983 WO 0021983PCT[DK99/00538 <221> CDS <222> (648) atg Met 1 <400> gcc gac atc Ala Asp Ilie gqt gta acC ggt Gly Val Thr Gly tcg Ser 10 qcg ggt ctg cag Ala Gly Leu Gin cct ggg Pro Gly ccg tct gag Pro Ser Gin gcg att ccc Ala Ile Pro gag Glu aca gac gag gag ttq acc gcg cgt ttc Thr Asp Giu Gin Len Thr Ala Arg Phe 25 gag cgc gac Gin Arg Asp cgg atg acg Arg Met Thr ctg ttg gac cag Len Leu Asp Gin tac ggc ggt gcg Tyr Gly Gly Ala cgc aat Arg Asn ccg gcc gac gcc Pro Ala Asp Ala gag Giu 55 gac ttg ctc cag Asp Len Len Gin gag Gin acg atg gtg aag Thr Met Val Lys gcc Al a tat gcg gga ttt Tyr Ala Gly Phe cgt Arg 70 tcg ttc cgg cac Ser Phe Arg His acc aat ctc aag Thr Asn Len Lys gcc Al a tgg ctc tac cgg Trp Len Tyr Arg ctg acc aac acc Len- Thr Asn Thr tac Tyr atc aac agc tat Ilie Asn Ser Tyr cgc aag Arg Lys aaa cag cgg Lys Gin Arg caa ctg gcg Gin Len Ala 115 caa Gin 100 ccg gcg gag tat Pro Ala Gin Tyr acc gag cag atc Thr Gin Gin Ile acc gat tgg Thr Asp Trp 110 cgc tcg gct Arg Ser Ala tcc aac gcc gag Ser Asn Ala Gin cat His1 120 tcc tcg acc ggg Ser Ser Thr Giy ct g Len 125 gaa gtc Gin Val 130 gaa gcg tta gaa Gin Ala Len Gin gcg Al a 135 ttg ccg gac acc Len Pro Asp Thr atc aaa gag gcg Ile Lys Gin Ala ct g Len 145 cag gca ttg ccg Gin Ala Len Pro gaa Gin 150 gag ttc cgg atg Gin Phe Arg Met gtc tac tac gcc Val Tyr Tyr Ala gat Asp 160 gtc gaa ggt ttc Val Gin Gly Phe tac aag gag atc Tyr Lys Gin Ile gcc Ala 170 gag atc atg gat Gin Ile Met Asp act ccg Thr Pro 175 atc ggc acc Ile Giy Thr ggt ctt tta Gly Leu Len 195 gtg Val1 180 atg tcg agg ctt Met Ser Arg Len cgc ggc cga cgt Arg Giy Arg Arg cag ttq cgc Gin Len Arg 190 agg ggc gag Arg Gly Gin gcc gat gtg gcc Ala Asp Val Ala agg Arg 200 gat cgg ggg ttt Asp Arg Gly Phe gcc Al a 205 cag gcg Gin Ala 210 cac gag ggg gtg His Giu Gly Val tcg tca tga Ser Ser 215 WO 00/21983 WO 00/ 1983PCT/DK99/00538 Met Pro Al a Arg Al a Trp Lys Gin Glu Leu 145 Val Ile Gly Gin at g Met 1 atc Ile at g Met <210> 16 <211> 21 <212> PR <213> M <400> 16 Ala Asp Ii Ser Giu Gi Ile Pro Le Asn Pro Al Tyr Ala Gi Leu Tyr Ar Gin Arg Gi Leu Ala Se 115 Val Glu Al 130 Gin Ala Le' Giu Gly Ph Gly Thr Va 18 Leu Leu Al 195 Ala His G1 210 <210> 17 <211> 77 <212> DN.

<213> M.

<220> <221> CD <222> (1 <400> 17 acg tac ga Thr Tyr Gi e

U

U

a y g n 0 r a

U

e 1 0 a

U

Asp Thr Leu Asp Phe Ile Pro Asn Leu Pro Pro 165 Met Asp Gly Gly Asp Asp Ala Arg 70 Leu Ala Ala Giu Glu 150 Tyr Ser Val Val Val Giu Gin Giu.

Ser Thr Giu Giu Al a 135 Giu Lys Arq Al a Ser 215 Thr Glu Leu Asp Phe Asn Tyr His 120 Leu Phe Glu Leu Arq 200 Ser Gly Leu 25 Tyr Leu Arg Thr Pro 105 Ser Pro Ar g Ile His 185 Asp Ser 10 Thr Gi y Leu His Tyr 90 Thr Ser Asp Met Ala 170 Arg Arg Ala Al a Gly Gin Gly 75 Ile Giu Thr Thr Al a 155 Glu Gi y Gly 6

T

Tuberculosis Gin Glu Arg Met Leu Tyr Thr 110 Arg Lys Tyr Asp Gin 190 Arg Pro Arg Met Val1 Lys Arg Asp Ser Glu Al a Thr 175 Leu Gly Gi y Asp Thr Lys Ala Lys Trp Ala Ala Asp 160 Pro Arg Giu 4

A

Tuberculosis

S

(771) a acc atc u Thr Ile 5 acg ctg aac cgt ccc Thr Leu Asn Arg Pro aac gag gtc acc agc Asn Glu Val Thr Ser ctg gtc gag Leu Val Glu cag gca ctg Gin Ala Leu 25 gct gca acc Ala Ala Thr gat cag cga gtt Asp Gin Arg Val ggc att Gly Ile gcg ctc aac Ala Leu Asn gaa ctg gac Glu Leu Asp agc cag gtg Ser Gin Val gac ccg gac Asp Pro Asp gcc gcc gga Ala Ala Gly att ggg gcg Ile Gly Ala atc atc atc Ile Ile Ile acc ggt Thr Gly tcg gcc aaa Ser Ala Lys WO 00/21983 WO 0021983PCT/DK99/00538 gcc Al a gao atc aaa gaa Asp Ile Lys Glu gcc gac otg acg Ala Asp Leu Thr goc gac gog ttc Ala Asp Ala Phe aco Thr qcC gao ttc ttc Ala Asp Phe Phe acc tgg ggc aag Thr Trp Gly Lys goc gcc gtg cgc Ala Ala Val Arg aco Oog Thr Pro 288 acg ato gc Thr Ile Ala gcg atg atg Ala Met Met 115 gtg gog gga tao Val Ala Gly Tyr gog Ala 105 otc gqc gqt ggc Leu Gly Gly Gly tgo gag ctg Cys Glu Leu 110 aag tto gga Lys Phe Gly 336 384 tgo gao gtg otg Cys Asp Val Leu goo qco gao aoo Ala Ala Asp Thr oag 000 Gin Pro 130 gag ata aag otg Giu Ile Lys Leu ggo Gly 135 gtg ctg oca ggo atg ggo ggo too oag Val Leu Pro Giy Met Gly Gly Ser Gin 140 ogg Ar g 145 otg aoo cgg got Leu Thr Arg Ala ato Ile 150 ggo aag got aag Gly Lys Ala Lys gog Al a 155 atg gao oto ato Met Asp Leu Ile otg Leu 160 480 528 aoo ggg cgo aoo Thr Gly Arg Thr gao goo goc gag Asp Ala Ala Glu gag ogc ago ggt Giu Arg Ser Gly otg gtt Leu Val 175 toa ogg gtg Ser Arg Val goo aog aoo Ala Thr Thr 195 ocg goc gao gao Pro Ala Asp Asp ttg Leu 185 ctg aoo gaa goo Leu Thr Glu Ala agg goc aot Arg Ala Thr 190 atg goo aag Met Ala Lys 576 624 att tog oag atg Ile Ser Gin Met tog Ser 200 gco tog gog goo Ala Ser Ala Ala ogg Arg 205 gag goo Glu Ala 210 gto aao ogg got Val Asn Arg Ala tto Phe 215 gaa too agt ttg Glu Ser Ser Leu tco Ser 220 gag ggg otg oto Giu Gly Leu Leu tao Tyr 225 gaa ogo ogg ott Glu Arg Arg Leu tto Phe 230 oat tog got tto His Ser Ala Phe g og Ala 235 aoo gaa gao oaa Thr Glu Asp Gin too Ser 240 672 720 768 gaa ggt atg goa Glu Gly Met Ala tto ato gag aaa Phe Ile Glu Lys got ooo oag tto Ala Pro Gin Phe aoo oao Thr His 255 oga tga Arg <210> <211> <212> <213> 18 257

PRT

M. Tuberoulosis <400> 18 Met Thr Tyr Giu Thr Ile Leu Val Glu Arg Asp Gin Arg Val Giy Ile 1 5 10 WO 00/21983 PCT/DK99/00538 Ile Met Ile Ala Ala Thr Ala Gin Arg 145 Thr Ser Ala Glu Tyr 225 Glu Arg Thr Asn Gly Asp Asp Ile Met Pro 130 Leu Gly Arg Thr Ala 210 Glu Gly Leu Glu Ala Ile Phe Ala Met 115 Glu Thr Arg Val Thr 195 Val Arg Met Asn Val Ile Lys Phe Ala 100 Cys Ile Arg Thr Val 180 Ile Asn Arg Ala Pro Ser Ile Met 70 Thr Ala Val Leu Ile 150 Asp Ala Gin Ala Phe 230 Gin Ala Thr 55 Ala Trp Gly Leu Gly 135 Gly Ala Asp Met Phe 215 His Ala Ala 40 Gly Asp Gly Tyr Ile 120 Val Lys Ala Asp Ser 200 Glu Ser Leu 25 Thr Ser Leu Lys Ala 105 Ala Leu Ala Glu Leu 185 Ala Ser Ala 13 Asn Glu Ala Thr Leu Leu Ala Pro Lys Ala 170 Leu Ser Ser Phe Arg 250 Ala Leu Lys Phe 75 Ala Gly Asp Gly Ala 155 Glu Thr Ala Leu Ala 235 Leu Asp Ala Ala Ala Gly Thr Met 140 Met Arg Glu Ala Ser 220 Thr Asn Asp Phe Asp Val Gly Ala 125 Gly Asp Ser Ala Arg 205 Glu Glu Ser Asp Ala Ala Arg Cys 110 Lys Gly Leu Gly Arg 190 Met Gly Asp Gin Pro Ala Phe Thr Glu Phe Ser Ile Leu 175 Ala Ala Leu Gin Val Asp Gly Thr Pro Leu Gly Gin Leu 160 Val Thr Lys Leu Ser 240 Ala Phe Ile Glu Lys 245 Ala Pro Gin Phe Thr His 255 <210> <211> <212> <213> <220> <221> <222> <400> ccg ctt Pro Leu 19 894

DNA

M.Tuberculosis

CDS

19 ccc gca gac cct age ccc Pro Ala Asp Pro Ser Pro gtg Val 1 acc Thr 10 ttg Leu ttg tcg gcc tac Leu Ser Ala Tyr gcc cat Ala His ccc gaa cgg Pro Glu Arg ccg ggc ctg Pro Gly Leu gtc ggc cat Val Gly His aac gac cca Asn Asp Pro ctc Leu gtg acc gcc gac Val Thr Ala Asp tgg Trp 25 gac Asp tcg gca cac Ser Ala His atg ggc gcg Met Gly Ala ctc tac gac Leu Tyr Asp gcg ate gtc gaa Ala Ile Val Glu tcc Ser 40 gtc Val gag gac gtc Glu Asp Val ttg Leu 96 144 192 240 att ccc ggc Ile Pro Gly cgg gtg cgc Arg Val Arg aag atc gac Lys Ile Asp cac acc gac ctc His Thr Asp Leu tac ate aac ggc gag cag ttc gcc gaa Tyr Ile Asn Gly Glu Gln Phe Ala Glu WO 00/21983 PCT/DK99/00538 ttg atg gac cgc Leu Met Asp Arg ggc atc gec cgc Gly Ile Ala Arg gat gac Asp Asp acc gtg gtg atc tat Thr Val Val Ile Tyr ggc gac aag Gly Asp Lys ctg ttc ggt Leu Phe Gly 115 agc Ser 100 aat tgg tgg gcc Asn Trp Trp Ala gcc Ala 105 tat gcg ttq tgg Tyr Ala Leu Trp gtg ttc acq Val Phe Thr 110 cgt gac ctc Arg Asp Leu cac gcc gac gtg His Ala Asp Val cga Arg 120 ctc ctc aac gqc Leu Leu Asn Gly ggc Gly 125 tgg ctc Trp Leu 130 gcc gag cgc cgg Ala Giu Arg Arg acc acc ttg gac Thr Thr Leu Asp ccg acc aag acc Pro Thr Lys Thr tgc Cys 145 acc ggt tat ccc Thr Gly Tyr Pro gtc Va1 150 gtg cag cgc aac Val Gin Arg Asn gca ccc atc cgc Ala Pro Ile Arg ttc aga gac gac Phe Arg Asp Asp ctg gcc atc ctg Leu Ala Ile Leu ggc Gly 170 gct cag ccg ctg Ala Gin Pro Leu atc gac Ile Asp 175 gta cgc tct Val Arg Ser tac ccc gag Tyr Pro Glu 195 ccc Pro 180 gag gag tac acc Glu Giu Tyr Thr aag cgc acc cat Lys Arg Thr His atg ccc gat Met Pro Asp 190 acg gcg gtg Thr Ala Val 576 624 gaa ggg gcg ctg Glu Gly Ala Leu gcc ggt cac atc Ala Giy His Ile cac att His Ile 210 ccg tgg ggg aag Pro Trp Gly Lys gcc Ala 215 gcc gac gaa agt Ala Asp Glu Ser cgg ttt cgc agc Arg Phe Arg Ser cgc Arg 225 gag gaa ttg gaa Glu Glu Leu Glu ctc tat gac ttc Leu Tyr Asp Phe aac ccg gac gac Asn Pro Asp Asp caa Gin 240 acc gtc gtc tat Thr Val Val Tyr cgc atc ggt gaa Arg Ile Gly Glu tcc agc cat acc Ser Ser His Thr tgg ttc Trp Phe 255 gtg ctc aca Val Leu Thr tcg tgg acc Ser Trp Thr 275 cac His 260 ctg ctg ggc aag Leu Leu Gly Lys gca Ala 265 gat gta cgg aac Asp Val Arg Asn tac gac ggc Tyr Asp Gly 270 gtc gcg ggc Val Ala Gly gag tgg ggc aac Glu Trp Gly Asn gtg cga gtg ccg Val Arg Val Pro atc Ile 285 gaa gaa Glu Glu 290 cca gga gtg gta Pro Gly Val Val ccc gtc gta tga Pro Val Val 295 <210> <211> 297 WO 00/21983 PCT/DK99/00538 Met 1 Pro Pro Val Asn Leu Gly Leu Trp Cys 145 Phe Val Tyr His Arg 225 Thr Val Ser Glu atg Met 1 <212> PRT <213> M.Tu <400> Pro Leu Pro Glu Arg Leu Gly Leu Ala Gly His Ile Asp Pro Arg Met Asp Arg Asp Lys Ser 100 Phe Gly His 115 Leu Ala Glu 130 Thr Gly Tyr Arg Asp Asp Arg Ser Pro 180 Pro Glu Glu 195 Ile Pro Trp 210 Glu Glu Leu Val Val Tyr Leu Thr His 260 Trp Thr Glu 275 Glu Pro Gly 290 <210> 21 <211> 1044 <212> DNA <213> M.Tu <220> <221> CDS <222> <400> 21 ctg atc tca Leu Ile Ser berculosis Ala 5 Val Ile Pro Val Lys Asn Ala Arg Pro Val 165 Glu Gly Gly Glu Cys 245 Leu Trp Val Asp Thr Val Gly Arg 70 Gly Trp Asp Arg Val 150 Leu Glu Ala Lys Arg 230 Arg Leu Gly Val Pro Ala Glu Ala 55 Asp Ile Trp Val Glu 135 Val Ala Tyr Leu Ala 215 Leu Ile Gly Asn Pro 295 Ser Asp Ser 40 Val Tyr Ala Ala Arg 120 Thr Gin Ile Thr Arg 200 Ala Tyr Gly Lys Ala 280 Val Pro Trp 25 Asp Lys Ile Arg Ala 105 Leu Thr Arg Leu Gly 185 Ala Asp Asp Glu Ala 265 Val Val Thr 10 Leu Glu Ile Asn Asp 90 Tyr Leu Leu Asn Gly 170 Lys Gly Glu Phe Arg 250 Asp Arg Leu Ser Asp Asp Gly 75 Asp Ala Asn Asp Asp 155 Ala Arg His Ser Ile 235 Ser Val Val Ser Ala Val Trp Glu Thr Leu Gly Val 140 Ala Gin Thr Ile Gly 220 Asn Ser Arg Pro Ala His Leu His Gin Val Trp Gly 125 Pro Pro Pro His Pro 205 Arg Pro His Asn Ile 285 Tyr Met Leu Thr Phe Val Val 110 Arg Thr Ile Leu Met 190 Thr Phe Asp Thr Tyr 270 Val Ala Gly Tyr Asp Ala Ile Phe Asp Lys Arg Ile 175 Pro Ala Arg Asp Trp 255 Asp Ala His Ala Asp Leu Glu Tyr Thr Leu Thr Ala 160 Asp Asp Val Ser Gin 240 Phe Gly Gly berculosis (1041) aac cga tcc Asn Arg Ser cag Gin cag cgc ccc Gln Arg Pro 5 ttc gtg atc Phe Val Ile acc ctg tcc gag gac gtc ctc acc gac Thr Leu Ser Glu Asp Val Leu Thr Asp 10 gaa ccg ctg gag ccg gga ttc ggc tac Glu Pro Leu Glu Pro Gly Phe Gly Tyr 25 WO 00/21983 PCT/DK99/00538 ace ctg ggc Thr Leu Gly aat tog ctg cgt Asn Ser Leu Arg acc ctg otg tcg Thr Leu Leu Ser tcg att ccc Ser Ile Pro gcg gcc Ala Ala gte acc age att Val Thr Ser Ile cge Arg 55 atc gat ggt gta Ile Asp Gly Val ctg Leu cac gaa ttc ace His Glu Phe Thr acg Thr gtg ccc ggg gtc Val Pro Gly Val aaa Lys 70 gaa gat gte ace Glu Asp Val Thr gag Glu ato atc ctg aat Ile Ile Leu Asn aag ago ctg gtg Lys Ser Leu Val tcc tog gag gag Ser Ser Glu Glu gao Asp gag cog gte ace Glu Pro Val Thr atg tao Met Tyr 288 eta cgc aag Leu Arg Lys ccg gcc ggc Pro Ala Gly 115 cag Gin 100 ggt ccg ggt gag Gly Pro Gly Glu ace gcc gge gac Thr Ala Gly Asp atc gtg cog Ile Val Pro 110 gcc acg ctg Ala Thr Leu 336 384 gte acc gtg cac Val Thr Val His aac Asn 120 ccc gge atg cac Pro Gly Met His aac gat Asn Asp 130 aag gqc aag ctg Lys Gly Lys Leu gtc gag etc gte Val Glu Leu Val gag egt ggc cge Glu Arg Gly Arg 432 480 ggc Gly 145 tat gte ccg gcg Tyr Val Pro Ala gtg Va1 150 caa aac cgg gct Gin Asn Arg Ala tcg Ser 155 ggt gcc gaa att Gly Ala Glu Ile ggg Gly 160 cgc att oca gte Arg Ile Pro Val gat Asp 165 tcc ate tac tea Ser Ile Tyr Ser ccg Pro 170 gtg ctc aaa gtg Val Leu Lys Val acc tao Thr Tyr 175 528 aag gtg gac Lys Val Asp ate etg gac Ile Leu Asp 195 gee Ala 180 ace egg gte gag Thr Arg Val Glu cgo ace gao tte Arg Thr Asp Phe gao aag otg Asp Lys Leu 190 gao gog etg Asp Ala Leu 576 624 gtg gag ace aag Val Glu Thr Lys aat Asn 200 tea ate age ccg Ser Ile Ser Pro gcg tcg Ala Ser 210 get gge aag acg Ala Gly Lys Thr gte gag ttg ttc Val Glu Leu Phe gge Gly 220 etg gea egg gaa Leu Ala Arg Glu etc Leu 225 aac gte gag gee Asn Val Giu Ala gaa Glu 230 ggo ate gag ate Gly Ile Glu Ile ggg Gly 235 ccg tog cog gee Pro Ser Pro Ala gee gat eac att Ala Asp His Ile gcg Ala 245 tea tto gee otg Ser Phe Ala Leu cog Pro 250 ate gao gac ctg Ile Asp Asp Leu gat otg Asp Leu 255 acg gtg egg Thr Val Arg te Ser 260 tao aac tgc etc Tyr Asn Cys Leu cgo gag ggg gtg Arg Giu Gly Val cac ace gtg His Thr Val 270 816 gge gaa otg gtg gog cgo ace gaa too gac ctg ott gao ato cgo aac WO 00/21983 WO 0021983PCT/DK99/00538 Giy Giu Leu 275 tte ggt cag Phe Gly Gin Vai Ala Arg Thr Giu 280 gag Glu Ser Asp Leu Leu Asp 285 ctg Leu Ile Arq Asn eac eag etg His Gin Leu aag tcc ate Lys Ser Ile gtg aag ate Val Lys Ile 290 ggc ctg Gly Leu aag Lys 300 gac Asp tea etc aag Ser Leu Lys ccg ccg age Pro Pro Ser ccc tcg gag Pro Ser Giu 305 geg Ala gtc Val1 320 tac Tyr 912 960 1008 ggc tac gac Gly Tyr Asp acc gge ace Thr Giy Thr tgg teg ace gag gge Trp Ser Thr Giu Giy 330 eag ett tag Gin Leu geg Ala 335 gac gag eag Asp Giu Gin qac Asp 340 gee gaa ace Ala Giu Thr 1044 Met As n Thr Al a Thr Lys Leu Pro As n Giy 145 Arg Lys Ile Ala Leu 225 Ala Thr Gly <210> <211> <212> <213> <400> Leu Ile Arg Ser Leu Gly Ala Val Val Pro Ser Leu Arg Lys Ala Gly 115 Asp Lys 130 Tyr Val Ile Pro Val Asp Leu Asp 195 Ser Ala 210 Asn Val Asp His Val Arg Giu Leu 22 347

PRT

M. Tuberculosis 22 Ser Gin Arg Pro Gin Phe Val Ile Asn Ser Leu Arg Thr Ser Ile Arg 55 Giy Val Lys Glu 70 Vai Val Ser Ser Gin Gly Pro Gly 100 Val Thr Val His Gly Lys Leu Giu 135 Pro Ala Val Gin 150 Val Asp Ser Ile 165 Ala Thr Arg Val 180 Val Glu Thr Lys Gly Lys Thr Leu 215 Giu Ala Giu Gly 230 Ile Ala Ser Phe 245 Ser Tyr Asn Cys 260 Val Ala Arg Thr Thr Giu Arg 40 Ile Asp Glu Giu Asn 120 Val1 Asn Tyr Giu Asn 200 Val1 Ile Ala Leu Glu Ser 10 Leu Leu Gly Thr Asp 90 Thr Gly Leu Ala Pro 170 Arg Ile Leu Ile Pro 250 Arg Asp Glu Glu Leu Val1 Giu 75 Gi u Ala Met Val Ser 155 Val Thr Ser Phe Gly 235 Ile Giu Leu Asp Pro Ser Leu Ile Pro Gly His Val 140 Gly Leu Asp Pro Gly 220 Pro Asp Gly Leu Val Gly Ser His Ile Val1 Asp Ile 125 Glu Al a Lys Phe Arg 205 Leu Ser Asp Val1 Asp Leu Phe Ile Giu Leu Thr Ile 110 Al a Arg Giu Val Asp 190 Asp Al a Pro Leu His 270 Ile Thr Gly Pro Phe Asn Met Val Thr Gly Ile Thr 175 Lys Ala Arg Ala Asp 255 Thr Arg Asp Tyr Gly Thr Leu Tyr Pro Leu Arg Gly 160 Tyr Leu Leu Giu Glu 240 Leu Val1 Asn WO 00/21983 PCT/DK99/00538 Phe Gly 290 Gly Leu 275 Gin Ser 280 Glu Lys Ser Ile Asp 295 Leu Ls Asp Ser Vai Lys Ile Lys 300 Asp 285 Leu His Gin Leu Pro Pro Ser Pro Ser Glu 305 Ala Asp 310 Ala Gly Tyr Asp Vai 325 Glu Gin Asp Tyr 340 Thr Giy Thr Trp Ser 330 Gin Leu Thr Giu Gly Ala Giu Thr Glu 345 <210> <211> <212> <213> <220> <221> <222> 23 1488

DNA

M.Tuberculosis

CDS

(1)..(1485) atg Met 1 <400> 23 acc gga aat Thr Giy Asn gtg acc aaa aat Vai Thr Lys Asn tcg Ser 10 otg ace oct gac gtt cgt Leu Thr Pro Asp Vai Arg aac ggc atc Asn Giy Ile cgc aaa gaa Arg Lys Glu gac Asp ttt aag atc gee Phe Lys Ile Ala gao Asp 25 otg tea eta gcg Leu Ser Leu Ala gat tto ggo Asp Phe Gly ctg atg tcg Leu Met Ser etc egg ate gee Leu Arg Ile Ala eac gag atg c00 His Giu Met Pro etg egg Leu Arg cgo gag tat gee Arg Giu Tyr Ala gag Glu 55 gtg oaa cc ctg Val Gin Pro Leu aag Lys ggg gee egg ate Gly Ala Arg Ile tcg Ser ggt tog etg eac Gly Ser Leu His atg Met acg gtg cag ace Thr Val Gin Thr gtg ttg ate gaa Val Leu Ile Glu etc ace gog ctg Leu Thr Aia Leu ggc Gly gee gaa gte cgo Ala Giu Vai Arg tgg Trp 90 gee tog tgo aac Ala Ser Cys Asn ate ttc Ile Phe toe ace cag Ser Thr Gin ace ccc gao Thr Pro Asp 115 gat Asp 100 cac gee gee gee His Ala Ala Ala gee Ala 105 gte gtg gte ggo Vai Vai Val Gly cog cac gge Pro His Gly 110 aag ggo gag Lys Giy Glu 336 384 gag coo aag ggt Glu Pro Lys Gly gte Val 120 cog gtq ttc gcg Pro Val Phe Ala acg etc Thr Leu 130 gaa gag tao tgg Glu Giu Tyr Trp gee gec gag eag Ala Ala Glu Gin atg Met 140 etc ace tgg ccg Leu Thr Trp Pro 432 480 gao Asp 145 ccc gao aag ccg Pro Asp Lys Pro gc Ala 150 aac atg ate etc Asn Met Ile Leu gat Asp 155 gac gge ggt gao Asp Gly Giy Asp ace atg ttg gtg ctg cgo ggc atg eag tat gag aaq gee ggc gtg gtg 528 WO 00/21983 PCT/DK99/00538 Thr ccg Pro otg Leu gcc Ala cgg Arg 225 aac Asn act Thr atc Ile ggc Gly gag Glu 305 gtg Va1 gcg Ala aag Lys gac Asp coct Pro 385 Met coo Pro cta Leu gag Glu 210 ctc Leu gto Val cgg Arg gg 0 Gly tgt Cys 290 ato Ile gto Val aco Thr gao Asp atg Met 370 cag Gin Leu gcc Ala cgg Arg 195 tcg Ser tao Tyr aao Asn cac His ggt Gly 275 gcg Ala gao Asp ac Thr ggo Gly cac His 355 goc Ala gto Val Val Leu Arg Gly Met Gin Tyr Glu Lys Ala Gly Val Val gag Glu 180 ac Thr gto Va1 oaa Gin gao Asp too Ser 260 aag Lys gag Glu oog Pro gto Va1 aac Asn 340 gcg Ala ggg Gly gao Asp 165 gag gac Glu Asp cgo ttc Arg Phe aag ggc Lys Gly tto gc Phe Ala 230 tcg gtg Ser Val 245 otg ato Leu Ile aag gto Lys Val gcg atg Ala Met ato aac Ile Asn 310 gag gag Glu Glu 325 aaa gao Lys Asp ato ctg Ile Leu otg gag Leu Glu otg tgg Leu Trp 390 gao Asp gag Glu gtc Va1 215 gcg Ala ac Thr gao Asp otc Leu aag Lys 295 gcg Ala goo Ala ato Ile gga Gly cgc Arg 375 aco Thr cc gc Pro Ala 185 aco gao Thr Asp 200 ac gag Thr Glu goo ggg Ala Gly aag too Lys Ser ggc ato Gly Ile 265 ato tgo Ile Cys 280 ggo cag Gly Gin ctg oag Leu Gin ato ggg Ile Gly ato atg Ile Met 345 aat ato Asn Ile 360 too ggg Ser Gly ttt ggo Phe Gly 170 gag Glu aag Lys gag Glu gat Asp aaa Lys 250 aao Asn ggo Gly gga Gly goo Ala gao Asp 330 otc Leu ggo Gly gcg Ala gao Asp tgg Trp gao Asp ac Thr ctg Leu 235 tto Phe cgo Arg tao Tyr gcg Ala atg Met 315 gc Ala gag Glu cac His aca Thr acg Thr 395 aag Lys aag Lys ac Thr 220 gc Ala gao Asp ggo Gly ggo Gly ogg Arg 300 atg Met gao Asp cac His tto Phe ogg Arg 380 ggo Gly gto tto Val Phe 190 tgg aoo Trp Thr 205 ac ggo Thr Gly tt cog Phe Pro aao aag Asn Lys ac gao Thr Asp 270 gao gto Asp Val 285 gto too Val Ser gag ggo Glu Gly ato gto Ile Val att aag Ile Lys 350 gao aao Asp Asn 365 gto aao Val Asn cgo tcg Arg Ser 175 otg aao Leu Asn aag ata Lys lie gtg otg Vai Leu gcg ato Ala Ile 240 tao ggo Tyr Gly 255 gcg ctg Ala Leu ggt aag Gly Lys gto aoo Val Thr tto gao Phe Asp 320 gta aco Val Thr 335 gcg atg Ala Met gag ato Glu Ile gto aag Val Lys ato ato Ile Ile 400 ggg cac Gly His 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 gtg ctg too gag ggg ogg otg otg aac ctg ggo aat gc aoo Val Leu Ser Giu Gly Arg Leu Leu Asn Leu Gly Asn Ala Thr 1248 WO 00/21983 WO 8021983PCT/DK99/00538 000 tcg ttc Pro Ser Phe atc gag otg Ile Glu Leu 435 ctg 000 aag Leu Pro Lys gt g Val 420 tgg Trp agc aac agc Ser Asn Ser tto Phe 425 gag Glu aac cag aog Asn Gin Thr 415 atc gco cag Ile Aia Gin 430 gtg tao cgg Val Tyr Arg gto gag goc Val Giu Ala acc aag aao Thr Lys Asn gao Asp 440 aag Lys tao gao aac Tyr Asp Asn gag Giu 445 oat His 1296 1344 1392 1440 oao oto gao His Leu Asp gtg got oga Vai Ala Arg 450 ott ggo Leu Gly ggt oao otg Gly His Leu 465 ggo Gly aoo Thr 470 ggt Giy otq aoo aag Leu Thr Lys gag Glu 475 gao Asp goo gaa tao Ala Giu Tyr ot 0 Leu 480 gto gao gto Val Asp Val gag Glu 485 ooo tao aag Pro Tyr Lys oao tao ogo His Tyr Arg 1485 1488 Met Asn Arg Leu Ser Leu Ser Thr Thr Asp 145 Thr Pro Leu Ala Arg <210> <211> <212> <213> <400> Thr Gly Gly Ilie Lys Glu Arg Arg Gly Ser Thr Ala Thr Gin Pro Asp 115 Leu Giu 130 Pro Asp Met Leu Pro Ala Leu Arg 195 Glu Ser 210 Leu Tyr 24 Asn Asp Leu Giu Leu Leu Asp 100 Glu Giu Lys Val Giu 180 Thr Val1 Gin 24 495

PRT

M. Tuberoulosis Leu Phe Arg Tyr His Gly His Pro T yr Pro Leu 165 Glu Arg Lys Phe Val1 Lys Ile Ala Met 70 Al a Ala Lys Trp Al a 150 Arg Asp Phe Gly Al a Thr Ile Ala Glu Thr Glu Ala Gly Trp 135 Asn Gly Asp Glu Val1 215 Al a Lys Al a Glu 40 Val1 Val Val1 Al a Val1 120 Al a Met Met Pro Thr 200 Thr Ala Asn Asp 25 His Gin Gin Arq Ala 105 Pro Al a Ile Gin Al a 185 Asp Glu Gi y Ser 10 Leu Glu Pro Thr Trp 90 Val1 Val Glu Leu Tyr 170 Glu Lys Glu Asp Leu Ser Met Leu Ala 75 Al a Val Phe Gln Asp 155 Glu T rp Asp Thr Leu Pro Al a Gly Gi y Leu Cys Gly Trp 125 Leu Gi y Al a Val1 Trp 205 Thr Phe Asp Asp Leu Al a Ile Asn Pro 110 Lys Thr Gly Gly Phe 190 Thr Gly Pro Val1 Phe Met Arg Glu Ile His Gly T rp Asp Val 175 Leu Lys Vai Al a Arg Gly Ser Ile Thr Phe Gly Glu Pro Al a 160 Val Asn Ile Leu Ile 240 225 230 235 Asn Val Asn Asp Ser Val Thr Lys Ser Lys Phe Asp Asn Lys Tyr Gly WO 00/21983 PCT/DK99/00538 245 Ser Leu Ile Asp Gly Ile Thr Ile Gly Glu 305 Val Ala Lys Asp Pro 385 Val Pro Ile Leu Leu 465 Gly Arg Gly Cys 290 Ile Val Thr Asp Met 370 Gin Leu Ser Glu Pro 450 Gly Val His Gly 275 Ala Asp Thr Gly His 355 Ala Val Ser Phe Leu 435 Lys Gly Asp 260 Lys Glu Pro Val Asn 340 Ala Gly Asp Glu Val 420 Trp His His Val Lys Ala Ile Glu 325 Lys Ile Leu Leu Gly 405 Met Thr Leu Leu Glu 485 Val Met Asn 310 Glu Asp Leu Glu Trp 390 Arg Ser Lys Asp Thr 470 Gly Leu Lys 295 Ala Ala Ile Gly Arg 375 Thr Leu Asn Asn Glu 455 Lys Pro Ile 280 Gly Leu Ile Ile Asn 360 Ser Phe Leu Ser Asp 440 Lys Leu Tyr 265 Cys Gin Gin Gly Met 345 Ile Gly Gly Asn Phe 425 Glu Val Thr Lys Asn Gly Gly Ala Asp 330 Leu Gly Ala Asp Leu 410 Ala Tyr Ala Lys Pro 490 Arg Tyr Ala Met 315 Ala Glu His Thr Thr 395 Gly Asn Asp Arg Glu 475 Asp Gly Gly Arg 300 Met Asp His Phe Arg 380 Gly Asn Gin Asn Ile 460 Gin His Thr Asp 285 Val Glu Ile Ile Asp 365 Val Arg Ala Thr Glu 445 His Ala Tyr Asp 270 Val Ser Gly Val Lys 350 Asn Asn Ser Thr Ile 430 Val Val Glu Arg 255 Ala Gly Val Phe Val 335 Ala Glu Val Ile Gly 415 Ala Tyr Glu Tyr Tyr 495 Leu Lys Thr Asp 320 Thr Met Ile Lys Ile 400 His Gin Arg Ala Leu 480 <210> <211> 1803 <212> DNA <213> M.Tu <220> <221> CDS <222> <400> gct agt cac Ala Ser His berculosis (1800) gcc ggc Ala Gly gtg Val 1 gtc Val tcg agg atc Ser Arg Ile cgg atc tct aag Arg Ile Ser Lys 5 ggc Gly gtt ctc Val Leu gcc aat cgc Ala Asn Arg gag atc gca Glu Ile Ala gtg atc cgg Val Ile Arg gac gcc ggc Asp Ala Gly tcc ccg cat Ser Pro His acc tcg gcg Thr Ser Ala ctg Leu ccc age gtg Pro Ser Val tac gcc gaa Tyr Ala Glu gcg gcc cgc Ala Ala Arg gac gcc gag Asp Ala Glu ggc ggc cag Gly Gly Gin 48 96 144 192 gtt cgg ctg Val Arg Leu gag tec tat Glu Ser Tyr 70 gcc Ala ctg Leu gag gcg ttc Glu Ala Phe gac ttc gcc Asp Phe Ala aag Lys 75 ctc gac gcg Leu Asp Ala gca 240 Ala WO 00/21983 PCT/DK99/00538 22 gcc aag tec ggg gcc aac gcc atc cac ccc ggc tac ggc ttc cta gcg 288 Ala Lys Ser Gly Ala Asn Ala Ile His Pro Gly Tyr Gly Phe Leu Ala 90 gaa aat gcc gac ttc gcc cag gcg gtg atc gac gcc ggc ctg atc tgg 336 Glu Asn Ala Asp Phe Ala Gin Ala Val Ile Asp Ala Gly Leu Ile Trp 100 105 110 atc ggc ccc agc ccg cag tcg atc cgc gac ctg ggc gac aag gtc acg 384 Ile Gly Pro Ser Pro Gin Ser Ile Arg Asp Leu Gly Asp Lys Val Thr 115 120 125 gcc cgt cac atc gcg gcc cgc gct cag gcg ccc ctg gtg ccg ggt acc 432 Ala Arg His Ile Ala Ala Arg Ala Gin Ala Pro Leu Val Pro Gly Thr 130 135 140 ccc gat ccg gtc aaa ggc gcc gac gag gtg gtg gca ttc gcc gag gag 480 Pro Asp Pro Val Lys Gly Ala Asp Glu Val Val Ala Phe Ala Glu Glu 145 150 155 160 tac ggc ctg ccg ate gcg atc aag gcc gcc cac ggc ggc ggc ggc aag 528 Tyr Gly Leu Pro Ile Ala Ile Lys Ala Ala His Gly Gly Gly Gly Lys 165 170 175 ggc atg aag gtg gcc cgc acc atc gac gag att ccg gag ctg tac gag 576 Gly Met Lys Val Ala Arg Thr Ile Asp Glu Ile Pro Glu Leu Tyr Glu 180 185 190 tcg gcg gtg cgc gag gcc acg gcc gcg ttc ggc cgc ggt gag tgc tac 624 Ser Ala Val Arg Glu Ala Thr Ala Ala Phe Gly Arg Gly Glu Cys Tyr 195 200 205 gtg gag cgc tat ctc gac aag ccg cgc cac gtc gaa gca cag gtg atc 672 Val Glu Arg Tyr Leu Asp Lys Pro Arg His Val Glu Ala Gin Val Ile 210 215 220 gcc gac cag cac ggc aac gtc gtc gtc gcc ggc acc cgg gac tgc tcg 720 Ala Asp Gin His Gly Asn Val Val Val Ala Gly Thr Arg Asp Cys Ser 225 230 235 240 ctg cag cgc cgc tac cag aag ctg gtc gag gag gcg ccc gca ccg ttc 768 Leu Gin Arg Arg Tyr Gin Lys Leu Val Glu Glu Ala Pro Ala Pro Phe 245 250 255 ctg acc gac ttt caa cgc aaa gag ate cac gac tcg gcc aaa cgg att 816 Leu Thr Asp Phe Gin Arg Lys Glu Ile His Asp Ser Ala Lys Arg Ile 260 265 270 tgc aaa gag gcc cat tac cac ggc gcc ggc acc gtc gaa tac ctg gtc 864 Cys Lys Glu Ala His Tyr His Gly Ala Gly Thr Val Glu Tyr Leu Val 275 280 285 ggt cag gac ggc ttg atc tcg ttc ttg gag gtc aac acg cgc ctt cag 912 Gly Gin Asp Gly Leu Ile Ser Phe Leu Glu Val Asn Thr Arg Leu Gin 290 295 300 gta gaa cac ccg gtc acc gag gaa acc gcg ggc atc gac ttg gtg ctg 960 Val Glu His Pro Val Thr Glu Glu Thr Ala Gly Ile Asp Leu Val Leu 305 310 315 320 WO 00/21983 WO 0021983PCT/DK99/00538 cag caa ttc cgg Gin Gin Phe Arq ato Ile 325 gcc aac ggc gaa Ala Asn Gly Giu ctg gac atc acc Leu Asp Ile Thr gag gat Glu Asp 335 1008 coo acc ocg Pro Thr Pro gcg gqg cgt Ala Giy Arg 355 cgc Arg 340 ggg cac goc atc: Gly His Ala Ile ttc cgg ato aac Phe Arg Ile Asn ggc gag gac Gly Giu Asp 350 aag tto cac Lys Phe His 1056 1104 aac ttc cta ccg Asn Phe Leu Pro ccc ggg ccq gtg Pro Gly Pro Val cog ccg Pro Pro 370 tcc ggc ccc ggt Ser Gly Pro Gly gtg Val 375 cgg gtg gac tcc Arg Val Asp Ser gtc gag aco ggc Vai Giu Thr Gly tcg Ser 385 gtg ato ggc ggc Val Ilie Gly Giy cag Gin 390 ttc gac tcg atg The Asp Ser Met gcc aag ctg atc Ala Lys Leu Ile gtg Val1 400 1152 1200 1248 cac ggt goc gao His Gly Ala Asp gcc gag gcg ctg Ala Glu Ala Leu cgg goc ogg cgc Arg Ala Arg Arg gog Otg Ala Leu 415 aac gag ttc Asn Glu Phe gcc gtg gtg Ala Val Val 435 ggt Gly 420 gtc gaa ggc ctg Val Giu Gly Leu gcg Al a 425 acg gtc atc ccg Thr Val Ile Pro ttt cac cgc Phe His Arg 430 ggo ttt tog Gly The Ser 1296 1344 too gac ccg gca Ser Asp Pro Ala tto Phe 440 atc ggc gac gog Ile Gly Asp Ala gta oat Val His 450 aoo ogc tgg atc Thr Arg Trp Ile aoo gag tgg aat Thr Giu Trp Asn acc ato gag cc Thr le Glu Pro ttt Phe 465 aco gao ggc gaa Thr Asp Gly Giu oct Pro 470 oto gao gag gao Leu Asp Giu Asp cgg cog cgt cag Arg Pro Arg Gin aag Lys 480 1392 1440 1488 gtg gtc gtc gaa Val Val Val Giu gac ggt ogo ogo Asp Gly Arg Arg gaa gtc tog ctg Glu Val Ser Leu cog got Pro Ala 495 gat oto gog Asp Leu Ala ogg ogo aag Arg Arg Lys 515 ot g Leu 500 too aat ggc ggc Ser Asn Gly Gly ggt Gly 505 tgo gao cog gto Cys Asp Pro Val ggt gto ato Gly Val Ile 510 ggo gcg gog Gly Ala Ala 1536 1584 coo aag ocg cgc Pro Lys Pro Arg aag Lys 520 cgg ggt gog cac Arg Gly Ala His goc too Ala Ser 530 ggt gao gcg gtg Gly Asp Ala Val ac Thr 535 gog oct atg cag Ala Pro Met Gin ggc Gly 540 aco gta gtt aag Thr Val Val Lys 1632 tto Phe 545 gog gtc gaa gaa Ala Val Giu Glu ggg Gly 550 oaa gag gtc gtg goc ggc gao ota gtg Gin Giu Val Val Ala Gly Asp Leu Val 555 gt g Val 560 1680 gto oto gag gcg atg aag atg gaa. aac cog gtc acc gog cat aag gat 12 1728 WO 00/21983 Val Leu Glu ggc acc atc Gly Thr Ile ggc acg gtg Gly Thr Val 595 PCT/DK99/00538 Ala Met Lys Met 565 acc ggg ctg gcg Thr Gly Leu Ala 580 ctc gcc gag atc Leu Ala Glu Ile 24 Glu Asn Pro Val Thr Ala His Lys Asp 570 575 gtc gag gcg ggc gcg gcc atc acc cag Val Glu Ala Gly Ala Ala Ile Thr Gin 585 590 aag taa Lys 600 1776 1803 Val 1 Val Asp Ser Thr Ala Glu Ile Ala Pro 145 Tyr Gly Ser Val Ala 225 Leu Leu Cys Gly Val 305 Gin Pro <210> 26 <211> 60 <212> PR <213> M.

<400> 26 Ala Ser Hi Ala Asn Ar Ala Gly Lei Pro His Va.

Ser Ala Gli Lys Ser Gl Asn Ala As Gly Pro Se: 115 Arg His Il 130 Asp Pro Va Gly Leu Pr Met Lys Va 18( Ala Val Ar 195 Glu Arg Ty] 210 Asp Gin Hi Gin Arg Ar Thr Asp Ph( 26( Lys Glu Al 275 Gin Asp Gl 290 Glu His Pr Gin Phe Arc Thr Pro Arc 0

T

Tuberculosis s g u 1 u y p 3 r e 1 I 1 3 9 r 3 9 e 0 y o3 g g Ala 5 Gly Pro Arg Ser Ala Phe Pro Ala Lys Ile 165 Ala Glu Leu Gly Tyr 245 Gln His Leu Val Ile 325 Gly Gly Glu Ser Leu Tyr 70 Asn Ala Gin Ala Gly 150 Ala Arg Ala Asp Asn 230 Gin Arg Tyr Ile Thr 310 Ala His Ser Ile Val Ala 55 Leu Ala Gin Ser Arg 135 Ala Ile Thr Thr Lys 215 Val Lys Lys His Ser 295 Glu Asn Ala Arg Ala Ala 40 Asp Asp Ile Ala lie 120 Ala Asp Lys Ile Ala 200 Pro Val Leu Glu Gly 280 Phe Glu Gly Ile Ile Val 25 Val Glu Phe His Val 105 Arg Gin Glu Ala Asp 185 Ala Arg Val Val Ile 265 Ala Leu Thr Glu Glu Ala 10 Arg Tyr Ala Ala Pro 90 Ile Asp Ala Val Ala 170 Glu Phe His Ala Glu 250 His Gly Glu Ala Lys 330 Phe Arg Val Ala Phe Lys 75 Gly Asp Leu Pro Val 155 His Ile Gly Val Gly 235 Glu Asp Thr Val Gly 315 Leu Arg Ile Ile Glu Ala Ile Tyr Ala Gly Leu 140 Ala Gly Pro Arg Glu 220 Thr Ala Ser Val Asn 300 Ile Asp Ile Ser Arg Pro Leu Leu Gly Gly Asp 125 Val Phe Gly Glu Gly 205 Ala Arg Pro Ala Glu 285 Thr Asp Ile Asn Lys Ala Asp Gly Asp Phe Leu 110 Lys Pro Ala Gly Leu 190 Glu Gin Asp Ala Lys 270 Tyr Arg Leu Thr Gly Val Ala Ala Gly Ala Leu Ile Val Gly Glu Gly 175 Tyr Cys Val Cys Pro 255 Arg Leu Leu Val Glu 335 Glu Leu Arg Glu Gin Ala Ala Trp Thr Thr Glu 160 Lys Glu Tyr Ile Ser 240 Phe Ile Val Gin Leu 320 Asp Asp WO 00/21983 WO 00/ 1983PCT/DK99/00538 Ala Pro Ser 385 His Asn Ala ValI Phe 465 Val1 Asp Arg Ala Phe 545 Val1 Gly Gly atg Met 1 Gly Arg 355 Pro Ser 370 Val Ile Gly Ala Glu Phe Val Val 435 His Thr 450 Thr Asp Val Val Leu Ala Arg Lys 515 Ser Gly 530 Ala Val Leu Giu Thr Ile Thr Val 595 <210> <211> <212> <213> <220> <221> <222> <400> cca gtg Pro Val 340 As n Gly Gly Asp Gly 420 Ser Arg Gly Glu Leu 500 Pro Asp Glu Ala Thr 580 Leu Phe Pro Gly Arg 405 Val1 Asp Trp Giu Ilie 485 Ser Lys Al a Giu Met 565 Gi y Al a Pro Val1 375 Phe Giu Gly Ala Glu 455 Leu Gly Gly Arg Thr 535 Gin Met Al a Ile Al a 360 Arg Asp Al a Leu Phe 440 Thr Asp Arg Gly Lys 520 Ala Giu Glu Val Lys 600 345 Pro Val1 Ser Leu Al a 425 Ile Giu Glu Arg Giy 505 Arg Pro Val1 As n Giu 585 Gly Asp Met Al a 410 Thr Gly T rp Asp Val1 490 Cys Gi y Met Val1 Pro 570 Ala Pro Ser Le u 395 Arg Val1 Asp As n Ala 475 Giu Asp Ala Gin Al a 555 Val1 Gly Val1 Gly 380 Al a Ala Ile Ala Asn 460 Arg Val1 Pro His Gly 540 Gly Thr Ala 350 Lys Giu Leu Arg Phe 430 Gly Ile Arg Leu Gly 510 Gly Val Leu His Ile 590 Phe Thr Ile Al a 415 His Ph e Giu Gin Pro 495 Val Al a Val Val Lys 575 Thr His Gly Val 400 Leu Arg Ser Pro Lys 480 Ala Ile Al a Lys Val 560 Asp Gin 27 318

DNA

M. Tuberculosis

CDS

(315) 27 gtg aag atc Val Lys Ile 5 aac gca atc Asn Ala Ile gtg ccc gcc ggc Val Pro Ala Gly gct ggc Ala Gly ccc gag ctg Pro Giu Leu tcc ccg ggt Ser Pro Gly gaa cgc tac Giu Arg Tyr gag tgg gca aag cgg ttc gct Lys Arg Phe Ala cgc gcg cac gcg Arg Ala His Ala ctc ggc ttt Leu Gly Phe cag Gin cac His tta cgt ccg Leu Arg Pro gtc gag aac Val Giu Asn aag ggt gaa Lys Gly Glu gca ttc cag Ala Phe Gin ttc gtg gtg Phe Val Val tgg gag tc Trp Glu Ser aac ggg ccc gcc atc gca gcc cat gcc gga cac cgg gcc WO 00/21983 PCT/DK99/00538 Trp Ala Asn Gly Pro Ala Ile Ala Ala Ala Gly His Arg aac ccc gtg Asn Pro Val gac gtc ggt Asp Val Gly <210> <211> <212> <213> gcg acc ggt gct tcg ctg ctg Ala Thr Gly Ala Ser Leu Leu 90 ggg acc ggc aag act gca taa Gly Thr Gly Lys Thr Ala 100 105 28 105

PRT

M.Tuberculosis gaa ttc gag gtc Glu Phe Glu Val gtg ctt Val Leu <400> 28 Pro Val Val Glu Leu Glu Pro Gly Phe Ile Asn Ala Ile Glu Arg Val Pro Ala Gly Ala Gly Arg Phe Ala Ala His Ala Ser Glu Arg Tyr Ala Trp Ala Phe Leu Gly Phe Val Val Thr Gin 40 His Leu Arg Pro Val Glu Val Glu Asn Lys Gly Glu Ala Phe Gin Trp Glu Ser 55 Ala Asp Ala Asn Asn Gly Pro 70 Ala Thr Gly Ile Ala Ala His Glu Gly His Arg Pro Val Ala Ser Leu Leu Phe Glu Val Val Asp Val Gly Gly Lys Thr Ala 105 <210> 29 <211> 435 <212> DNA <213> M.Tuberculosis <220> <221> <222>

CDS

(435) gtg Val 1 <400> 29 gcg gac aag acg Ala Asp Lys Thr 5 aca cag acg att Thr Gin Thr Ile tac Tyr 10 atc gac gcg gat Ile Asp Ala Asp cca ggc 48 Pro Gly gag gtg atg Glu Val Met tcg gag tat Ser Glu Tyr gcg atc gcc gac Ala Ile Ala Asp ate Ile gaa gcc tac ccg Glu Ala Tyr Pro caa tgg att Gin Trp Ile gag ggc tac Glu Gly Tyr aag gaa gtc gag Lys Glu Val Glu cta gag gcc gac Leu Glu Ala Asp gac Asp ccg aaa Pro Lys cga gcg cga atg Arg Ala Arg Met atg gac gca gcc Met Asp Ala Ala ate Ile ttc aaa gac acc Phe Lys Asp Thr ttg atc atg tcc tac gag tgg ccg gaa gac cgc caa tcg ctt ago tgg Leu Ile Met Ser Tyr Glu Trp Pro Glu Asp Arg Gin Ser Leu Ser Trp 240 WO 00/21983 PCT/DK99/00538 act ctc gaa tcc Thr Leu Glu Ser age Ser tcg ctg cta aag Ser Leu Leu Lys ctc gaa ggc acg Leu Glu Gly Thr tat cgc Tyr Arg 288 ttg gcg ccc Leu Ala Pro gac ctt gct Asp Leu Ala 115 aag Lys 100 ggt tct ggc act Gly Ser Gly Thr gag Glu 105 gtc acc tac gag Val Thr Tyr Glu ctt gcc gtc Leu Ala Val 110 gcg gaa cgc Ala Glu Arg 336 384 gtc ccc atg atc Val Pro Met Ile atg ctc aag cgt Met Leu Lys Arg agg ttg Arg Leu 130 tga *r ata gac ggc gcg Ile Asp Gly Ala ttg Leu 135 aag gat ctg aag Lys Asp Leu Lys aaa Lys 140 cga gtc gag ggc Arg Val Glu Gly <210> <211> 144 <212> PRT <213> M.Tuberculosis Met 1 Glu Ser <400> Ala Asp Lys Val Met Lys Glu Tyr Lys Thr Thr Gin Thr Ile Tyr Ile Asp Ala Asp Pro Gly 5 Ala Ile Ala Asp Ile 25 Leu Ala Tyr Pro Gin Trp Ile Glu Gly Tyr Lys Asp Thr Glu Val Glu Glu Ala Asp Pro Lys Arg Leu Ile Met Ala Arg Met Leu 55 Trp Asp Ala Ala Ile Gin Ser Tyr Pro Glu Asp Thr Arg Leu Ser Leu Ser Leu Glu Ser Ser Gly Leu Leu Lys Glu Gly Thr Tyr Arg Leu Ala Pro Asp Leu Ala 115 Arg Leu Ile 130 Lys 100 Val Ser Gly Thr Glu 105 Met Thr Tyr Glu Pro Met Ile Leu Lys Arg Lys Leu Ala Val 110 Ala Glu Arg Val Glu Gly Asp Gly Ala Leu 135 Asp Leu Lys Lys 140 <210> <211> <212> <213> <220> <221> <222> 31 441

DNA

M.Tuberculosis

CDS

<400> atg cca gtt Met Pro Val 31 ttg agc aag acc gtc gag gtc acc gcc gac gcc gca tcg Leu Ser Lys Thr Val Glu Val Thr Ala Asp Ala Ala Ser 10 WO 00/21983 atc atg gcc Ile Met Ala PCT/DK99/00538 gtt gcc gat atc Val Ala Asp Ile cgc tac cca gag Arg Tyr Pro Giu tgg aat. gaa Trp, Asn Giu ggg gtc aag Giy Val Lys ggc gca tgg gtg ctc gct cgc tac gat gac ggg cgt ccc Gly Ala Trp Val Leu Ala Arg Tyr Asp Asp Gly Arq Pro agc cag Ser Gin gtg cgg ctc gac Vai Arg Leu Asp gct gtt caa ggc Ala Vai Gin Giy atc Ile gag ggc acc tat Glu Giy Thr Tyr at c Ile cac gcc gtg tac His Ala Vai Tyr cca ggc gaa aac Pro Giy Giu Asn cag Gin 75 att caa acc gtc Ile Gin Thr Val atg Met cag cag ggt gaa Gin Gin Gly Giu ttt gcc aag cag Phe Ala Lys Gin cag ctg ttc agt Gin Leu Phe Ser gtg gtg Vai Val 288 gca acc ggc Ala Thr Gly acc atg ccg Thr Met Pro 115 gcc Aila 100 gcg agc ttg ctc Ala Ser Leu Leu gtg gac atg gac Vai Asp Met Asp gtc cag gtc Vai Gin Val 110 aac aac gtc Asn Asn Val 336 384 gtg ccc gag ccg Val Pro Giu Pro gtg aag atg ctg Vai Lys Met Leu ctc Leu 125 ctg gag Leu Giu 130 cat ctc gcc gaa His Leu Ala Giu ctc aag cag cgc Leu Lys Gin Arg qcc Al a 140 gag cag ctg gcg Giu Gin Leu Ala gcc agc taa Ala Ser 145 <210> <211> <212> <213> 32 146

PRT

M. Tuberculosis Met 1 Ile Gi y <400> 32 Pro Val Leu Met Ala Ile Val Lys Gly Lys Thr Val Giu Thr Ala Asp Ala Ala Ser Ala Asp Ile Giu 25 Ala Tyr Pro Giu Ala Trp Val Arg Tyr Asp Ser Gin Val Ile His Ala Asp Glu Trp Asn Giu Gly Arg Pro Gly Thr Tyr Arg Leu Asp Val Tyr Tyr 70 Giu Leu Phe Ala Val Gin Gly Ile Ile Gly Giu Asn Gin Gin Gin Gin Thr Val Gin Gly Ala Lys Gin Leu Phe Ser Val Ala Thr Gly Thr Met Pro 115 Leu Glu His Ala 100 Val1 Ser Leu Leu Thr 105 Val Asp Met Asp Pro Giu Pro Met 120 Leu Lys Met Leu Leu 125 Glu Val Gin Val 110 Asn Asn Val Gin Leu Ala Leu Ala Giu Asn Lys Gin Arg Ala WO 00/21983 130 Ala Ser 145 PCT/DK99/00538 <210> 33 <211> 894 <212> DNA atg Met 1 t ca Ser ttg Leu gco Al a cag Gin cag Gin cog Pro ctg Leu ct C Leu gtg Val1 145 Ccg Pro <213> <220> <221> <222> <400> tca tog Ser Ser cog goc Pro Ala cga aaa Arg Lys aco tgg Thr Trp gat cac Asp His got tog Ala Ser gcg goa Ala Ala atg gtc Met Val 115 ggt tog Gly Ser 130 ato ato Ile Ile gtg ota Val Leu M. Ti

CDS

33 ggo aat toa tot otg gga Gly Asn Ser Ser Leu Gly 5 goa cag gtt gog gtg ogg Ala Gin Val Ala Val Arg 25 ato oct ctg acg oto gtg Ile Pro Leu Thr Leu Val 40 otg gag gtg oca ctg ocg Leu Giu Val Pro Leu Pro 55 ggg ogo oac otg ato gao Gly Arg His Leu Ile Asp 70 Ctg ogo got. ggt ccc coo Leu Arq Ala Gly Pro Pro goc gtt coo aca ttg gtc Ala Val Pro Thr Leu Vai 100 105 gtg ggt tgt oto gga agt Val Gly Cys Leu Gly Ser 120 gto agt too ggc ctg oto Val Ser Ser Gly Leu Leu 135 cac gao gaa gat tog gtg His Asp Glu Asp Ser Val 150 gtt ggc gtt gao ggc tog Val Gly Val Asp Gly Ser 165 uberculosis att ato gto Ile Ile Val ggg ato gao gat Gly Ile Asp Asp t gg Trp cac His cg Pro gao Asp aog Thr 90 gao Asp ggg Gly ogo Arg atg Met tog Ser 170 gca Al a gcg Ala ggc Gly gca Al a 75 gt c Val atg Met cgg Arg cac His cog Pro 155 goc Ala got Ala gtg Val gtg Val 60 oto Leu cac His too Ser t gg T rp 9gg Al a 140 cat His too Ser cgg Arg tog Ser 45 otq Leu aag Lys agt Ser aaa Lys cog Pro 125 ca c His ccc Pro gag Giu gat Asp 30 cog Pro cga Arg gt g Val1 gaa Glu gao Asp 110 ggo Gly tgt Cys cag Gin ctg Leu gcg Al a gaa Giu t gg Trp gt t Val1 ato Ile gca Al a cgg Arg cg Pro ca a Gin gog Al a 175 gag Giu gt a Val1 oag Gin gaa Glu gt t Val gtg Val ctg Leu gtC Val1 gog Ala 160 ac Thr 96 144 192 240 288 336 384 432 480 528 gca ato goa Ala Ile Ala ttc gao gaa gog tog cgg oga aac gtg Phe Asp Glu Ala Ser Arg Arg Asn Vai gac ctg gtg gog Asp Leu Val Ala WO 00/21983 ctg cac gca Leu His Ala 195 tgg ccg gca Trp Pro Ala PCT/DK99/00538 tgg agc gac gtc Trp Ser Asp Val gtg tcg gag tgg Val Ser Glu Trp ccc Pro 205 gcc Ala gga ate gat Gly Ile Asp gag cgg ttg Glu Arg Leu act cag tcg Thr Gin Ser 210 gcg ggt Ala Gly atg Met 215 tat Tyr gag cag gtg Glu Gin Val ctg Leu 220 ata Ile tgg cag gag Trp Gin Glu ccc aac gta Pro Asn Val acc cgc gtg Thr Arg Val 225 gtg Val gtg Val 240 720 768 cgc gat cag Arg Asp Gin ccg Pro 245 gtc Val cgc cag ctc Arg Gin Leu gtc Val 250 cgc tcc gag Arg Ser Glu gaa gcc Glu Ala 255 cag ctg gtc Gin Leu Val ctg gtg ggg Leu Val Gly 275 gtc atc gtg Val Ile Val 290 gtg Val 260 tcg Ser ggc agc cgg Gly Ser Arg ggc cgc ggc ggc tac Gly Arg Gly Gly Tyr 265 gtt get cag ctg gcg Val Ala Gin Leu Ala gcc gga atg Ala Gly Met 270 cgg acg ccg Arg Thr Pro 816 864 gta ggc gaa Val Gly Glu acc Thr 280 ctg act tag Leu Thr 285 gca cgc gag Ala Arg Glu Met 1 Ser Leu Ala Gin Gin Pro Leu Leu Val 145 Pro Ala Leu <210> <211> <212> <213> <400> Ser Ser Pro Ala Arg Lys Thr Trp Asp His Ala Ser Ala Ala Met Val 115 Gly Ser 130 Ile Ile Val Leu Ile Ala His Ala 34 297

PRT

M.Tuberculosis 34 Gly Asn Ser Ser 5 Ala Gin Val Ala Ile Pro Leu Thr Leu Glu Val Pro 55 Gly Arg His Leu 70 Leu Arg Ala Gly Ala Val Pro Thr 100 Val Gly Cys Leu Val Ser Ser Gly 135 His Asp Glu Asp 150 Val Gly Val Asp 165 Phe Asp Glu Ala 180 Trp Ser Asp Val Leu Val Leu 40 Leu Ile Pro Leu Gly 120 Leu Ser Gly Ser Asp Gly Arg 25 Val Pro Asp Pro Val 105 Ser Leu Val Ser Arg 185 Val Ile 10 Trp His Pro Asp Thr 90 Asp Gly Arg Met Ser 170 Arg Ser Val Ala Val Val Leu His Ser Trp Ala 140 His Ser Val Trp Gly Arg Ser Leu Lys Ser Lys Pro 125 His Pro Glu Asp Pro Ile Asp Pro Arg Val Glu Asp 110 Gly Cys Gin Leu Leu 190 Gly Asp Ala Glu Trp Val Ile Ala Arg Pro Gin Ala 175 Val Ile Asp Glu Val Gin Glu Val Val Leu Val Ala 160 Thr Ala Asp WO 00/21983 WO 0021983PCT/DK99/00538 205 Ala Trp Pro 210 Ala Gly Thr Gin Ser Met 215 Tyr Ala Glu Gin Val Leu 220 Ile Giu Arg Leu Trp Gin Glu Pro Asn Val Thr Arg Val Arg Asp Gin Pro 245 Val Gin Leu Val Leu Val Gly 275 Val Ile Val 290 Val1 260 Ser Gi y Arg Gin Leu Val 250 Ser Arg Gly Arg 265 Arg Ser Giu Giu Ala 255 Gly Gly Tyr Ala Giy Met 270 Arg Thr Pro Val Gly Glu Thr Val 280 Leu Thr Ala Gin Leu Ala Arg Glu Ser 295 <210> <211> <212> <213> <220> <221> <222> 957

DNA

M. Tuberculosis

CDS

.(954) at g Met 1 <400> got gaa qta Ala Giu Val ctg Leu 5 gtg ctc gtt gag Val Leu Val Giu cac His 10 got gaa ggo gog Ala Giu Gly Ala tta aag Leu Lys aag gto ago Lys Val Ser goc goc gto Ala Ala Val gco Al a gaa ttg atc ac Glu Leu Ile Thr goc ogo goc ttg Ala Arg Ala Leu ggo gaa oca Gly Giu Pro ctg gtg gao Leu Val Asp gtc gto ggt gtg Val Val Gly Val ggg acg goc gog Gly Thr Ala Ala cog Pro ggg ott Gly Leu aag gog got ggt Lys Ala Ala Gly gco Ala 55 goc aag ato tao Ala Lys Ile Tyr gto Val goo gag too gao Ala Giu Ser Asp gtc gao aaa tao Val Asp Lys Tyr atc aco cog gog Ile Thr Pro Ala gto Val gao gtg ctg gc Asp Val Leu Ala ggg Gi y ctg goc gag too Leu Ala Glu Ser tcg Ser goc cot gcc ggc Ala Pro Ala Gly ota ato goc gc Leu Ile Ala Ala aco gog Thr Ala gao ggc aag Asp Gly Lys ctg ctg gto Leu Leu Val 115 gag Glu 100 ato goc ggc oga Ile Ala Gly Arg ott Leu 105 gog got cgg ato Ala Ala Arg Ile ggc tog ggt Gly Ser Gly 110 ggt gtc cac Gly Val His1 gao gtg gto gao Asp Val Val Asp gt g Val 120 aga gaa ggt gga Arg Glu Gly Gly gtg Val1 125 ago ato Ser Ile 130 ttc ggt ggg gog Phe Gly Gly Ala tto Phe 135 aco gtc gaa gog Thr Val Glu Ala cag Gin 140 goc aac ggc gao Ala Asn Gly Asp aco cog gtg atc aco gtg ogo gca gga goc gtg gag gcg gag oog gc WO 00/21983 PCT/DK99/00538 Thr 145 Pro Val Ile Thr Val Arg Ala Gly Ala 150 Glu Ala Glu Pro Ala 160 gcc ggc gcc ggt Ala Gly Ala Gly cag gtc agc gtg Gln Val Ser Val gaa Glu 170 gtg ccg got gcg Val Pro Ala Ala gcg gag Ala Glu 175 528 aac gcc gcc Asn Ala Ala ccg gag ctg Pro Glu Leu 195 agg Arg 180 atc acc gcg cgc Ile Thr Ala Arg ccg gcg gtc gcc Pro Ala Val Ala ggc gac cgg Gly Asp Arg 190 cgt ggt gtc Arg Gly Val 576 624 acc gag gcg acc Thr Glu Ala Thr gtg gtg gcc ggt Val Val Ala Gly ggc Gly 205 ggc agc Gly Ser 210 gcg gag aac ttc Ala Glu Asn Phe agc Ser 215 gtg gtc gag gcg Val Val Glu Ala ctg Leu 220 gcc gac tcg ctg Ala Asp Ser Leu ggc Gly 225 gcc gcg gtc ggg Ala Ala Val Gly gcc Ala 230 tcg cgt gcc gca Ser Arg Ala Ala gac tcc ggc tac Asp Ser Gly Tyr tac Tyr 240 ccg ggc cag ttc Pro Gly Gln Phe cag Gin 245 gtc ggc cag acc Val Gly Gin Thr ggc Gly 250 aag acg gtg tcg Lys Thr Val Ser ccc cag Pro Gin 255 ctc tac att Leu Tyr Ile atg cag acg Met Gin Thr 275 gcc Ala 260 ctg ggc atc tec Leu Gly Ile Ser ggg Gly 265 gcg atc cag cac Ala Ile Gin His cgc gct ggc Arg Ala Gly 270 gaa gag gcg Glu Glu Ala 816 864 tcc aag acc atc Ser Lys Thr Ile gcg gtc aac aag Ala Val Asn Lys gac Asp 285 ccg atc Pro Ile 290 ttt gag atc gcc Phe Glu Ile Ala gac Asp 295 tac ggg gtg gtg Tyr Gly Val Val gga Gly 300 gac ctg ttc aag Asp Leu Phe Lys 912 gtc Val 305 gct ccg cag ctg Ala Pro Gin Leu acc Thr 310 gag gcc ate aag Glu Ala Ile Lys cgc aag ggc Arg Lys Gly <210> <211> <212> <213> 36 318

PRT

M.Tuberculosis <400> 36 Ala Glu Val Leu Val Leu Met 1 Lys Val Ser Ala 5 Glu Leu Ile Val Gly Val Val Glu His 10 Thr Ala Ala Ala Glu Gly Ala Leu Lys Arg Ala Leu 25 Pro Gly Ala Ala Val Gly Leu Lys Leu Val Asp Val Thr Ala Ala Pro Ala Gly Glu Pro Leu Val Asp Glu Ser Asp Ala Ala Gly Ala 55 Lys Tyr Leu Ile Lys Ile Tyr Val Asp Thr Pro Ala Val Leu Ala WO 00/21983 WO 00/ 1983PCT/DK99/00538 Leu Asp Leu Ser Thr 145 Al a Asn Pro Gly Gly 225 Pro Leu Met Pro Val1 305 Al a Gly Leu Ile 130 Pro Gly Ala Giu Ser 210 Al a Gly T yr Gin Ile 290 Al a Gi u Lys Vai 115 Phe Val1 Al a Ala Leu 195 Al a Ala Gin Ile Thr 275 Phe Pro Ser Glu 100 Asp Giy Ile Gly Arg 180 Thr Glu Val1 Phe Al a 260 Ser Glu Ser Ile Val Gly Thr Glu 165 Ile Giu Asn Gly Gin 245 Leu Lys Ile Al a Al a Val1 Ala Val 150 Gin Thr Ala Phe Al a 230 Val Giy Thr Al a Pro Gly Asp Phe 135 Arg Val1 Ala Thr Ser 215 Ser Gly Ile Ile Asp 295 Ala Arg Val1 120 Thr Al a Ser Arg Ile 200 Val Arg Gin Ser Val 280 Tyr Gly Leu 105 Arg Val Gly Val1 Giu 185 Val1 Val Al a Thr Gly 265 Ala Gly Val1 90 Al a Giu Giu Ala Glu 170 Pro Val Giu Ala Gly 250 Ala Val1 Val Leu Ala Gly Ala Val 155 Val Ala Al a Al a Val 235 Lys Ile Asn Val Ile Arg Gly Gin 140 Glu Pro Val Gly Leu 220 Asp Thr Gin Lys Gly 300 Al a Ile Val 125 Al a Ala Al a Ala Gly 205 Ala Ser Val His Asp 285 Asp Al a Gly 110 Gly Asn Giu Al a Gly 190 Arg Asp Gly Ser Arg 270 Gi u Leu Thr Ser Val1 Gly Pro Al a 175 Asp Gly Ser Tyr Pro 255 Al a Glu Phe Ala Gly His Asp Ala 160 Giu Arg Val Leu Tyr 240 Gin Gly Ala Lys <210> <211> <212> <213> <220> <221> <222> <400> aag ago Lys Ser gag gtg Giu Val Gin Leu Thr Glu Ala Ile Lys Ala Arg Lys Gly 310 315 37 1401

DNA

M. Tuberculosis

CDS

(1398) 37 acc gto gag cag ttg ago ccc aco ogg gtt cgt Thr Val Giu Gin Leu Ser Pro Thr Arg Val Arg 5 10 oca ttc goc gag ott gag cog gat ttc cag cgg Pro Phe Ala Giu Leu Glu Pro Asp Phe Gin Arq gtg Val1 1 gtg Val1 ato aac Ile Asn goc tao Ala Tyr 25 ctg Leu ccc ggg aag Pro Gly Lys aaa gag otg Lys Glu Leu goc aaa cag gtg Ala Lys Gin Val cgg Ar g coo qgc tto Pro Gly Phe gog cog Ala Pro goc aaa ota ctc Ala Lys Leu Leu gaa Glu 55 9gg goc ogo ato ggc Ala Arg Ile Gly ogg Arg gag goc atg otg Glu Ala Met Leu 192 240 gat caa ato gtc aac Ctg ccc ago ogg tao qga cag gog gtg Asp Gin Ile Val Asn Ala Leu Pro Ser Arg 75 Tyr Gly Gin Ala Val WO 00/21983 WO 0021983PCTIDK99/00538 gcc gag Ala Glu aag aag Lys Lys ogo ocg Arg Pro gat ccg Asp Pro 130 tta cgt Leu Arg 145 gtc ggc Vai Gly gao ata Asp Ile Gly Arg gac gag Asp Giu 210 ggg cag Gly Gin 225 gaa cta Giu Leu gac agc Asp Ser gcc aag Ala Lys gog cta Ala Leu 290 gcc caa Ala Gin tog gat Ser Asp gag tac Glu Tyr 100 aag ato Lys Ile 115 atc gaa Ile Giu aoc ogg Thr Arg gao gto Asp Val ocg aao Pro Asn 180 cto ato Leu Ile 195 tcc cgg Ser Arg gaa got Glu Ala cca gag Pro Glu ato gao Ile Asp 260 ogo goo Arg Ala 275 oto gaa Leu Giu tto gao Phe Asp gtc caa ocg cto ggc cgg ccc aac atC gag Val1 ggC Gi y agt Ser atc Ile ttc Phe gt 0 Val 165 gca Ala goa Al a gt 0 Vai oag Gin coo Pro 245 gaa Glu oa g Gin oag Gin ago Ser Gin Pro oag gac Gin Asp Pro Pro ggt gag Giy Glu 135 ggo ac Giy Thr 150 tog ato Ser Ile gcc gct Aia Ala ggt otc Gly Leu tto aoo Phe Thr 215 gtt aoo Val Thr 230 gao gao Asp Asp ttg ogg Leu Arg oag goo Gin Ala gto gac Val Asp 295 gtg otg Val Leu Leu ctg Leu gao Asp 120 gao Asp otg Leu gao Asp gag Glu gao Asp 200 gc Ala gt 0 Val1 gaa Glu gco Al a gag Glu 280 gtg Val1 oa 0 His Gly oaa Gin 105 ot g Leu gac Asp ac Thr ttg Leu gga Gly 185 gao Asp aag Lys a cg Thr tto Phe agc Ser 265 oag Gin ocg Pro agc Ser Arg Pro Asn Ile Glu 90 ttc Phe a go Ser gt c Val gog Ala t ct Ser 170 ot c Leu gcg Aila ot q Leu gtc Val gog Ala 250 Otc Leu att Ile ttg Leu gog Ala acc Thr gcg Al a ga c Asp gtg Val 155 gcc Ala tc Ser gtt Val gca Al a ag Arg 235 ca g Gin a gc Ser cga Arg o cg Pro ctc Leu gcc Ala otg Leu goc Al a 140 gac Asp aoq Thr oac His gtt Val gc Al a 220 tog Ser tta Leu gac Asp aao As n gag Giu 300 ago Ser gag Giu aog Thr 125 gaa Glu ogg Arg gto Val1 gag Giu ggt Gly 205 ggc Gly gt t Val1 goc Al a oag Gin goo Ala 285 tog Ser ggt Gly gt 0 Val1 110 gt c Val1 ct g Leu cog Pro gao Asp gt c Val 190 ct g Leu gag Glu aag Lys agc Ser gt g Val1 270 acc Thr tat Tyr ctt Leu gtg aoo Val Thr gao ato Asp Ile tcg gtg Ser Val cag tog Gin Ser gtg goo Val Ala 160 gga gag Gly Giu 175 ggc too Gly Ser too goo Ser Ala oao goo His Ala gag ogo Giu Arg 240 gag tto Glu Phe 255 ogo cag Arg Gin ato gat Ile Asp gtg oag Val Gin aat oao Asn His 288 336 384 432 480 528 576 624 672 720 768 816 864 912 960 305 310 315 gao gaa gcc ogg tto aat gag ttg otc gtc gag oaa ggc tcg toa ogo 00 1008 WO 00/21983 PCT/DK99/00538 Asp Glu Ala Arg Phe Asn Glu Leu Leu 325 Val 330 Glu Gin Gly Ser Ser Arg 335 gcg gcg ttc Ala Ala Phe agg cag ctg Arg Gin Leu 355 gcc gag gcg cgc Ala Glu Ala Arg acc Thr 345 gcc tca gaa aag Ala Ser Glu Lys gac gtc aag Asp Val Lys 350 gtc caa gtt Val Gin Val 1056 1104 ttg cta gac gcc Leu Leu Asp Ala ctg Leu 360 gcc gat gag ctg Ala Asp Glu Leu cag Gin 365 ggc cag Gly Gin 370 gat gat ctg acc Asp Asp Leu Thr cga ctg gtg acg Arg Leu Val Thr acg Thr 380 tct cgg caa tac Ser Arg Gin Tyr atc gag ccg cag Ile Glu Pro Gin cag Gin 390 ctg ttc ggc tac Leu Phe Gly Tyr ctc Leu 395 caa gag cgc aac Gin Glu Arg Asn 1152 1200 1248 ctg ccg acc atg Leu Pro Thr Met gct gac gtg cgg Ala Asp Val Arg cgc Arg 410 gag ctg gcg ate Glu Leu Ala Ile agg gcc Arg Ala 415 gca gtg gag Ala Val Glu acc agt gag Thr Ser Glu 435 gcg Ala 420 gcg acg gtc acc Ala Thr Val Thr gac Asp 425 agt gac gga aac Ser Asp Gly Asn acg atc gat Thr Ile Asp 430 get gag gag Ala Glu Glu 1296 1344 ttc ttc ggc aag Phe Phe Gly Lys gtg tcg gcc ggt Val Ser Ala Gly gag Glu 445 gcc gaa Ala Glu 450 ccg gca gac gag Pro Ala Asp Glu ggt Gly 455 gcc gcg cgg gcg Ala Ala Arg Ala gcg Ala 460 tec gac gaa gcg Ser Asp Glu Ala 1392 aca acg tga Thr Thr 465 1401 <210> <211> <212> <213> 38 466

PRT

M.Tuberculosis <400> 38 Lys Ser Thr Val Glu Gin Leu Ser Pro 10 Glu Val Pro Phe Ala Glu Leu Glu Pro 25 Glu Leu Ala Lys Gin Val Arg Leu Pro Thr Arg Val Arg Ile Asn Ala Tyr Asp Phe Gin Arg Lys Gly Phe Ala Pro Ala Asp Gin Ile Arg Glu Pro Gly Lys Ala Met Leu Lys Leu Leu Glu 55 Val Asn Asp Ala 70 Arg Ile Gly Pro Ser Arg Arg Tyr Leu Gly Gin Ala Val Ala Lys Arg Glu Ser Asp Val Gin Pro Leu Gly Arg Lys Glu Tyr Gly Gin Asp Leu Gin Phe 100 105 Pro Lys Ile Ser Pro Pro Asp Leu Ser Pro Asn Ile Glu Thr Ala Glu Ala Leu Thr Val Asp Ile 110 Val Ser Val WO 00/21983 PCT/DK99/00538 36 115 120 125 Asp Pro Ile Glu Ile Gly Glu Asp Asp Val Asp Ala Glu Leu Gin Ser 130 135 140 Leu Arg Thr Arg Phe Gly Thr Leu Thr Ala Val Asp Arg Pro Val Ala 145 150 155 160 Val Gly Asp Val Val Ser Ile Asp Leu Ser Ala Thr Val Asp Gly Glu 165 170 175 Asp Ile Pro Asn Ala Ala Ala Glu Gly Leu Ser His Glu Val Gly Ser 180 185 190 Gly Arg Leu Ile Ala Gly Leu Asp Asp Ala Val Val Gly Leu Ser Ala 195 200 205 Asp Glu Ser Arg Val Phe Thr Ala Lys Leu Ala Ala Gly Glu His Ala 210 215 220 Gly Gin Glu Ala Gin Val Thr Val Thr Val Arg Ser Val Lys Glu Arg 225 230 235 240 Glu Leu Pro Glu Pro Asp Asp Glu Phe Ala Gin Leu Ala Ser Glu Phe 245 250 255 Asp Ser Ile Asp Glu Leu Arg Ala Ser Leu Ser Asp Gin Val Arg Gin 260 265 270 Ala Lys Arg Ala Gin Gin Ala Glu Gin Ile Arg Asn Ala Thr Ile Asp 275 280 285 Ala Leu Leu Glu Gin Val Asp Val Pro Leu Pro Glu Ser Tyr Val Gin 290 295 300 Ala Gin Phe Asp Ser Val Leu His Ser Ala Leu Ser Gly Leu Asn His 305 310 315 320 Asp Glu Ala Arg Phe Asn Glu Leu Leu Val Glu Gin Gly Ser Ser Arg 325 330 335 Ala Ala Phe Asp Ala Glu Ala Arg Thr Ala Ser Glu Lys Asp Val Lys 340 345 350 Arg Gin Leu Leu Leu Asp Ala Leu Ala Asp Glu Leu Gin Val Gin Val 355 360 365 Gly Gin Asp Asp Leu Thr Glu Arg Leu Val Thr Thr Ser Arg Gin Tyr 370 375 380 Gly Ile Glu Pro Gin Gin Leu Phe Gly Tyr Leu Gin Glu Arg Asn Gin 385 390 395 400 Leu Pro Thr Met Phe Ala Asp Val Arg Arg Glu Leu Ala Ile Arg Ala 405 410 415 Ala Val Glu Ala Ala Thr Val Thr Asp Ser Asp Gly Asn Thr Ile Asp 420 425 430 Thr Ser Glu Phe Phe Gly Lys Arg Val Ser Ala Gly Glu Ala Glu Glu 435 440 445 Ala Glu Pro Ala Asp Glu Gly Ala Ala Arg Ala Ala Ser Asp Glu Ala 450 455 460 Thr Thr 465 <210> 39 <211> <212> PRT <213> M.Tuberculosis <400> 39 Thr Glu Arg Thr Ala Val Leu Ile Lys Pro Asp Gly Ile Glu Arg 1 5 10 <210> <211> <212> PRT <213> M.Tuberculosis <400> WO 00/21983 37 Thr Asp Thr Gin Val Thr Trp Leu Thr Gin Glu Ser His Asp Arg 1 5 10 <210> 41 <211> <212> PRT <213> M.Tuberculosis <400> 41 Met Ile Asp Glu Ala Leu Phe Asp Ala Glu Glu Lys Met Glu Lys 1 5 10 <210> 42 <211> <212> PRT <213> M.Tuberculosis <400> 42 Pro Leu Pro Ala Asp Pro Ser Thr Asp Leu Ser Ala Tyr Ala Gin 1 5 10 <210> 43 <211> <212> PRT <213> M.Tuberculosis <400> 43 Met Leu Ile Ser Gin Arg Pro Thr Leu Ser Glu Asp Val Leu Thr 1 5 10 <210> 44 <211> <212> PRT <213> M.Tuberculosis <400> 44 Thr Gly Asn Leu Val Thr Lys Asn Ser Leu Thr Pro Asp Val Arg 1 5 10 <210> <211> <212> PRT <213> M.Tuberculosis <400> Met Glu Val Lys Ile Gly Ile Thr Asp Ser Pro Arg Glu Leu Val 1 5 10 <210> 46 <211> <212> PRT <213> M.Tuberculosis <400> 46 Ser Ala Tyr Lys Thr Val Val Val Gly Thr Asp Asp Xaa Ser Xaa 1 5 10 <210> 47 <211> <212> PRT <213> M.Tuberculosis PCT/DK99/00538 WO 00/21983 38 <400> 47 Met Glu Gin Arg Ala Glu Leu Val Val Gly Arg Ala Leu Val Val 1 5 10 <210> 48 <211> <212> PRT <213> M.Tuberculosis <400> 48 Ala Asp Ilie Asp Gly Val Thr Gly Ser Ala Gly Leu Asn Pro Ala 1 5 10 <210> 49 <211> <212> PRT <213> M.Tuberculosis <400> 49 Thr Tyr Glu Thr Ile Leu Val Giu Arg Asp Gin Arg Val Gly Ile 1 5 10 <210> <211> <212> PRT <213> M.Tuberculosis <400> Pro Val Thr Gin Giu Glu Ile Ile Ala Gly Ile Ala Glu Ile Ilie 1 5 10 <210> 51 <211> 14 <212> PRT <213> M.Tubercuiosis <400> 51 Pro Val Val Lys Ile Asn Ala Ile Giu Val Pro Ala Gly Ala <210> 52 <211> <212> PRT <213> M.Tubercuiosis <400> 52 Ala Asp Lys Thr Thr Gin Thr Ile Tyr Ile Asp Ala Asp Pro Giy 1 5 10 <210> 53 <211> <212> PRT <213> M.Tubercuiosis <400> 53 Pro Val Leu Ser Lys Thr Vai Giu Val Thr Ala Asp Ala Ala Ser 1 5 10 PCT/DK99/00538 <210> 54 <211> 14 WO 00/21983 WO 0021983PCTIDK99/00538 <212> PRT <213> M.Tubercuiosis Ser 1 <400> 54 Gly Asn Ser Ser Leu Gly Ile Ile Val Gly Ile Asp Asp 5 <210> <211> <212> PRT <213> M.Tubercuiosis <400> Ala Glu Val Leu Val Leu Val Glu His Ala Glu Gly Ala Leu Lys 1 5 10 <210> 56 <211> <212> PRT <213> M.Tuberculosis Met 1 <400> 56 Lys Ser Thr Val Glu Gin Leu Ser Pro Thr Arg Val Arg Ile 5 .10 <210> <211> <212> <213> 57 11

PRT

M. Tuberculosis 57 Arg Lys Pro Lys Pro Arg Xaa Arg 5 <400> Ile Arg Val1 1 <210> <211> <212> <213> 58 27

DNA

M. Tuberculosis <400> 58 ctgagatctg tggaggtcaa gatcggt <210> 59 <211> <212> <213> 31

DNA

M. Tuberculosis <400> 59 ctcccatggc tacttacccg ctcgtagcaa c <210> <211> 27 <212> DNA <213> M.Tuberculosis <400> ctgagatctc ctgtcactca ggaaqaa <210> 61 <211> 27 <212> DNA WO 00/21983 PCT/DK99/00538 <213> M.Tuberculosis <400> 61 ctcccatggg aaaccgccat tagcggt 27 <210> 62 <211> 27 <212> DNA <213> M.Tuberculosis <400> 62 cccaaqctta tggaacaqcg tgcggag 27 <210> 63 <211> 27 <212> DNA <213> M.Tuberculosis <400> 63 ctcccatggc qacactcgat ccggatt 27 <210> 64 <211> 27 <212> DNA <213> M.Tuberculosis <400> 64 ctgagatcta tgccagtgqt qaagatc 27 <210> <211> 27 <212> DNA <213> M.Tuberculosis <400> ctcccatggt tatqcagtct tgccggt 27 <210> 66 <211> 27 <212> DNA <213> M.Tuberculosis <400> 66 ctgagatctg cggacaagac gacacaq 27 <210> 67 <211> 27 <212> DNA <213> M.Tuberculosis <400> 67 ctcccatggt accggaatca ctcagcc 27 <210> 68 <211> 27 <212> DNA <213> M.Tuberculosis <400> 68 ctgagatctc cagttttqag caagacc 27 WO 00/21983 PCT/DK99/00538 41 <210> 69 <211> 27 <212> DNA <213> M.Tuberculosis <400> 69 ctcccatggg cacatgcctt agctggc 27 <210> <211> 27 <212> DNA <213> M.Tuberculosis <400> ctgagatcta tgtcatcggg caattca 27 <210> 71 <211> 31 <212> DNA <213> M.Tuberculosis <400> 71 ctcccatgqc tacctaagtc agcgactcgc g 31 <210> 72 <211> 27 <212> DNA <213> M.Tuberculosis <400> 72 ctgagatctg tgaagagcac cgtcgag 27 <210> 73 <211> 27 <212> DNA <213> M.Tuberculosis <400> 73 ctcccatggg tcatacggtc acgttgt 27 <210> 74 <211> 348 <212> DNA <213> M.Tuberculosis <220> <221> CDS <222> (348) <400> 74 atg qca ctc aag gta gag atg gtc act ttc gac tgc agc gac cct gcg 48 Met Ala Leu Lys Val Glu Met Val Thr Phe Asp Cys Ser Asp Pro Ala 1 5 10 aag ctt gcc ggc tgg tgg gcc gag cag ttc gat ggc acg acg cgt gaa 96 Lys Leu Ala Gly Trp Trp Ala Glu Gin Phe Asp Gly Thr Thr Arg Glu 25 ctg ctq ccc ggc gaa ttc qtc gtg gtc gcc cgg acc gat gga ccg cgg 144 Leu Leu Pro Gly Glu Phe Val Val Val Ala Arg Thr Asp Gly Pro Arg WO 00/21983 PCT/DK99/00538 ttg gga Leu Gly ttc cag aag gtg Phe Gin Lys Val gat ccc gcc cct Asp Pro Ala Pro ggg Gly aaa aac cgc gtg Lys Asn Arg Val cac His ctc gac ttc acg Leu Asp Phe Thr acc Thr 70 aag gac ctg gat Lys Asp Leu Asp gag gtg ttg cgc Glu Val Leu Arg ctg Leu 192 240 288 gtc gcc gcc gga Val Ala Ala Gly gcc Ala agt gag gtc ggg Ser Glu Val Gly cgg Arg 90 cat cag gtc ggc His Gin Val Gly gag agc Glu Ser ttt cgc tgg Phe Arg Trp gtg Val 100 gtg ctg gct gac Val Leu Ala Asp ccc Pro 105 gaa ggc aac gct Glu Gly Asn Ala ttt tgc gtg Phe Cys Val 110 gcg ggt caa taa 348 Ala Gly Gin 115 <210> <211> <212> <213> 115

PRT

M.Tuberculosis Met 1 Lys <400> Ala Leu Lys Leu Ala Gly Val 5 Trp Glu Met Val Thr Asp Cys Ser Asp Pro Ala Trp Ala Glu Gln 25 Val Asp Gly Thr Leu Leu Pro Leu Gly Phe His Leu Asp Gly Glu Phe Val Val 40 Asp Ala Arg Thr Asp Lys Thr Arg Glu Gly Pro Arg Asn Arg Val Gin Lys Val Phe Thr Thr 70 Pro Lys Pro Ala Pro Asp Leu Asp Val Ala His Val Leu Arg Ala Ala Gly Ala Val Ser Glu Val Gly Gln Val Gly Glu Ser Cys Val Phe Arg Trp Ala Gly Gln 115 Val 100 Leu Ala Asp Pro 105 Gly Asn Ala <210> <211> <212> <213> <220> <221> <222> 76 564

DNA

M.Tuberculosis

CDS

(564) <400> 76 atg gcc gac gct gac acc acc gac ttc Met Ala Asp Ala Asp Thr Thr Asp Phe 1 5 gtc gac gca gaa Val Asp Ala Glu gca ccg 48 Ala Pro ggt gga ggc gtc cgg gag gac acg gcg acg gat got gac gag gcc gac WO 00/21983 WO 0021983PCT/DK99/00538 Gly Gly Gly gat caa gaa Asp Gin Giu Arg Glu Asp Thr Ala Thr Asp Ala Asp Glu Ala Asp gag aga ttg gte Glu Arg Leu Val gag ggc gag att gca ggc gac tac Giu Gly Giu Ile Ala Gly Asp Tyr 144 ctg gaa Leu Glu gag tta ttg gac Giu Leu Leu Asp ttg gac ttc gat Leu Asp Phe Asp ggc Gly gac atc gac etc Asp Ile Asp Leu 192 240 gat Asp gtc gaa. ggc aat Val Giu Gly Asn cgt Arg 70 gcg gtg gtg age Ala Val Val Ser gac ggc agt gac Asp Gly Ser Asp gac Asp etg aae aag ttg Leu Asn Lys Leu ggg egc ggg gge Gly Arg Gly Giy gtg etc gac gct Val Leu Asp Ala etg cag Leu Gin 288 gaa ctc acc Giu Leu Thr ttg atg cta.

Leu Met Leu 115 ttg gcg gtg cat Leu Ala Val His cag Gin 105 aag ace ggt gtg Lys Thr Gly Val cgg age cgg Arg Ser Arg 110 gag gaa. ttg Giu Glu Leu 336 384 gae ate gcg agg Asp Ile Aia Arg ega cgg cgg egc Arg Arg Arg Arg egg Arg 125 gcg gcg Ala Aia 130 etg gec gac gag Leu Ala Asp Giu gtg Val1 135 gcg egg cga gtg Ala Arg Arg Val gee Ala 140 gaa aee ggt gae Giu Thr Gly Asp 432 480 ege Arg 145 gag gaa. etc gtt Giu Giu Leu Val atg aeg ceg tte Met Thr Pro Phe egg aag ate gte Arg Lys Ile Vai eac His 160 gat geg gtt gea Asp Ala Val Ala geg Ala 165 gtg eca ggt gtg Val Pro Gly Val age gaa age gaa.

Ser Giu Ser Giu gge gtg Gly Val 175 gag eca, gaa Glu Pro Giu egc Arg 180 ega gte gtt gtg Arg Val Val Val ege gac tag Arg Asp <210> <211> <212> <213> 77 187

PRT

M. Tuberculosis Met 1 Gi y Asp Leu Asp Leu <400> 77 Ala Asp Ala Asp Thr Thr Asp Phe Asp 5 10 Gly Gly Val Arg Giu Asp Thr Ala Thr 25 Gin Giu Giu Arg Leu Val Ala Giu Gly 40 Val Asp Ala Giu Ala Pro Asp Ala Asp Giu Ile Ala Asp Giu Ala Asp Gly Asp Tyr Ile Asp Leu Glu Giu Leu Leu Asp Val Val Glu Gly Asn Arg Ala 70 Asn Lys Leu Val Gly Arg Leu Asp Phe Asp Val Val Ser Gly Ser Asp Asp Gin Gly Gly Giu Val Leu Asp Ala Leu WO 00/2 1983 Giu Leu Thr Leu Met Leu 115 Ala Ala Leu PCT/DK99/00538 Leu 90 Lys Ala Val His Gin 105 Arg Thr Gly Val Ile Ala Arg Arg Ser Arg 110 Giu Glu Leu Thr Gly Asp T rp 120 Al a Arg Arg Arg Ar g 125 Glu Ala Asp Giu 130 Arg Glu Val 135 Met Arg Arq Vai Al a 140 Arg Glu Leu Val 145 Asp Pro 150 Val Thr Pro Phe Glu 155 Lys Ile Val His 160 Val1 Ala Val Ala Al a 165 Arg Pro Gly Val His Ser 170 Arg Asp Giu Ser Glu Glu Pro Glu Arg 180 Val Val Val Leu 185 <210> <211> <212> <213> <220> <221> <222> 78 1167

DNA

M. Tuberculosis

CDS

(1167) <400> 78 agc aag acg Ser Lys Thr gt t Val ctc atc ctt ggc Leu Ile Leu Gly gcg Al a 10 ggt gtc ggc qgc Gly Val Gly Gly ctg acc Leu Thr acc gcc gac Thr Ala Asp ttg gtg gac Leu Vai Asp ctc cgt caa ctg Leu Arg Gin Leu cta Leu cca cct gag gat Pro Pro Giu Asp cga atc ata Arg Ile Ile ttg cta tgg Leu Leu Trp agg agc ttt qac Arg Ser Phe Asp ggg Gly 40 acg ctg ggc ttg Thr Leu Gly Leu t cg Ser gtg ttg Val Leu cqg gqc tgg cgq Arg Giy Trp Arg cgg Arg 55 cct gac gac gtc Pro Asp Asp Val cgc Arg gtc cgc ccc acc Val Arg Pro Thr gcg Ala gcg tcg ctq ccc Ala Ser Leu Pro ggt Gly 70 gtg gaa atg gtt Val Giu Met Val act Thr 75 gca acc gtc gcc Ala Thr Val Ala cac His att gac atc gcg Ile Asp Ile Ala cag gta gtg cac Gin Val Val His acc Thr gac aac agc gtc Asp Asn Ser Vai atc ggc Ile Gly tat gac gcg Tyr Asp Ala gtt ccc gga Val Pro Gly 115 ttg Leu 100 gtg atc gca tta Val Ile Ala Leu ggt Gly 105 gcg gcg ctg aac Ala Ala Leu Asn acc gac gcc Thr Asp Ala 110 ggc cag ttc Gly Gin Phe 336 384 ctg tcg gac gcg Leu Ser Asp Ala ctc Leu 120 gac gcc gac gtc Asp Ala Asp Val gcg Aia 125 tac acc Tyr Thr 130 ctg gac ggc gcg Leu Asp Gly Ala gag ctg cgt gcg aag gtc gag gcg ctc Giu Leu Arg Ala Lys Vai Glu Ala Leu 140 gag cat ggc cgg atc gct gtg gct atc gcc ggg gtg ccg ttc aaa tgc WO 00/21983 WO 00/ 1983PCT/DK99/00538 Giu 145 coa Pro gao Asp ccg Pro tcg Ser got Ala 225 too Ser gc Al a gao Asp gat Asp goo Al a 305 ogo Arg goo Al a gat Asp ogg Arg His gco Ala ogo Arg ot g Leu atg Met 210 ogo Arg gaa Glu gog Ala oog Pro gog Ala 290 gt g Val cat His tgo Cys tto Phe gag Glu 370 Gly gca Al a tac T yr cog Pro 195 ot 0 Le u gto Val cog Pro 9gg Al a cgc Arg 275 acc Thr tto Phe ot 0 Leu tao Tyr tto Phe 355 tt~t Phe Arg Ile Ala Val Ala ccg Pro gc Al a 180 atg Met aag Lys ga t Asp tto Phe cgg Arg 260 aco Thr gt g Val1 gc Al a ggt Gly gt o Val1 340 got Ala ca c His 150 ttc gaa Phe Glu 165 acc gga Thr Gly coo gtt Pro Val gat cac Asp His gag gcc Glu Ala 230 gat ctg Asp Leu 245 toa gcg Ser Ala otg too Leu Ser otg acg Leu Thr gaa gc Giu Ala 310 tac gao Tyr Asp 325 gag aco Giu Thr cog tog Pro Ser gag gag Glu Giu gog Al a aco Thr goa Ala ggt Gly 215 gca Al a ott Leu ggt Gly act Thr ctg Leu 295 cag Gln gt a Val ggt Gly gog Ala gog Ala gt a Val ggt Giy 200 gt 0 Val1 agg Arg gco Al a ot 0 Leu ago Ser 280 cog Pro gc Al a got Al a gat Asp ccc Pro 360 Ile Ala Gly 155 ttt ctg ato Phe Leu Ile 170 cag atc gao Gin Ile Asp 185 ccc gag gtc Pro Glu Val ggc ttc oat Gly Phe His aog atg cac Thr Met His 235 gtg gto ccc Val Val Pro 250 ago gaa too Ser Glu Ser 265 goc gao aao Ala Asp Asn aat ggc aaa Asn Gly Lys gca gtt gtc Ala Val Val 315 gag ogo cac Glu Arg His 330 cac cag gca His Gin Ala 345 tog gtg acg Ser Val Thr gc Ala acg Thr ggc Gly ct Pro 220 tto Phe cog Pro ggg Gly gt g Val cog Pro 300 gc la tto Phe gcc Ula ct g Leu Val Pro Phe Lys Cys gc Al a tto Phe gag Gi u 205 ogo Arg ggt Gly cac His t gg Trp tgg Trp 285 ot g Leu cac His aco Thr aag Lys tao Tyr 365 caa Gin acg Thr 190 got Ala aag Lys gao Asp gt g Val ata Ile 270 gc Al a coo Pro ggc Gly ggc Gly ggc Gi y 350 cog Pro otc Leu 175 cot Pro ttg Leu gc Al a ggc Gly coo Pro 255 coo Pro ato Ile aag Lys gt 0 Val1 aog Thr 335 gao Asp cog Pro 160 ggt *Gly gao Asp gt c Val1 ota Leu a cg Thr 240 too Ser gt g Val ggc Gly got Al a goc Ala 320 ggc Gly ggo Gly tog Ser 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 aag gto gca oaa gaa otg goc tgg otg aco Lys Val Ala Gin Glu Leu Ala Trp Leu Thr cgo tgg aag acg tga Arg Trp Lys Thr 1167 WO 00/21983 PCT/DK99/00538 385 Met 1 Thr Leu Val Ala Ile Tyr Val Tyr Glu 145 Pro Asp Pro Ser Ala 225 Ser Ala Asp Asp Ala 305 Arg Ala Asp Arg Arg 385 <210> 79 <211> 388 <212> PRT <213> M.T <400> 79 Ser Lys Thr Ala Asp Thr Val Asp Arg Leu Arg Gly Ala Ser Leu Asp Ile Ala uberculosis Asp Pro Thr 130 His Ala Arg Leu Met 210 Arg Glu Ala Pro Ala 290 Val His Cys Phe Glu 370 Trp Ala Gly 115 Leu Gly Ala Tyr Pro 195 Leu Val Pro Ala Arg 275 Thr Phe Leu Tyr Phe 355 Phe Lys Leu 100 Leu Asp Arg Pro Ala 180 Met Lys Asp Phe Arg 260 Thr Val Ala Gly Val 340 Ala His Thr Val Leu 5 Leu Arg Ser Phe Trp Arg Pro Gly 70 Ala Gin Val Ile Ser Asp Gly Ala Ile Ala 150 Phe Glu 165 Thr Gly Pro Val Asp His Glu Ala 230 Asp Leu 245 Ser Ala Leu Ser Leu Thr Glu Ala 310 Tyr Asp 325 Glu Thr Pro Ser Glu Glu Ile Leu Gly Gin Leu Leu 25 Asp Gly Thr 40 Arg Pro Asp 55 Val Glu Met Val Val His Ala Leu Gly 105 Ala Leu Asp 120 Ala Glu Leu 135 Val Ala Ile Ala Ala Phe Thr Val Gin 185 Ala Gly Pro 200 Gly Val Gly 215 Ala Arg Thr Leu Ala Val Gly Leu Ser 265 Thr Ser Ala 280 Leu Pro Asn 295 Gln Ala Ala Val Ala Glu Gly Asp'His 345 Ala Pro Ser 360 Lys Val Ala 375 Ala 10 Pro Leu Asp Val Thr 90 Ala Ala Arg Ala Leu 170 Ile Glu Phe Met Val 250 Glu Asp Gly Val Arg 330 Gin Val Gln Gly Pro Gly Val Thr 75 Asp Ala Asp Ala Gly 155 Ile Asp Val His His 235 Pro Ser Asn Lys Val 315 His Ala Thr Glu Val Glu Leu Arg Ala Asn Leu Val Lys 140 Val Ala Thr Gly Pro 220 Phe Pro Gly Val Pro 300 Ala Phe Ala Leu Leu 380 Gly Asp Ser Val Thr Ser Asn Ala 125 Val Pro Ala Phe Glu 205 Arg Gly His Trp Trp 285 Leu His Thr Lys Tyr 365 Ala Gly Arg Leu Arg Val Val Thr 110 Gly Glu Phe Gin Thr 190 Ala Lys Asp Val Ile 270 Ala Pro Gly Gly Gly 350 Pro Trp Leu Ile Leu Pro Ala Ile Asp Gin Ala Lys Leu 175 Pro Leu Ala Gly Pro 255 Pro Ile Lys Val Thr 335 Asp Pro Leu Thr Ile Trp Thr His Gly Ala Phe Leu Cys 160 Gly Asp Val Leu Thr 240 Ser Val Gly Ala Ala 320 Gly Gly Ser Thr <210> WO 00/21983 PCT/DK99/00538 47 <211> <212> PRT <213> M.Tuberculosis <400> Ala Leu Lys Val Glu Met Val Thr Phe Asp Xaa Ser Asp Pro Ala 1 5 10 <210> 81 <211> <212> PRT <213> M.Tuberculosis <400> 81 Ala Asp Ala Asp Thr Thr Asp Phe Asp Val Asp Ala Glu Ala Pro 1 5 10 <210> 82 <211> <212> PRT <213> M.Tuberculosis <400> 82 Ser Lys Thr Val Leu Ilie Leu Gly Ala Gly Val Gly Gly Leu Thr 1 5 10 <210> 83 <211> 27 <212> DNA <213> M.Tuberculosis <400> 83 ctgagatcta tggcactcaa ggtagag 27 <210> 84 <211> 27 <212> DNA <213> M.Tuberculosis <400> 84 ctcccatggt tattgacccg ccacgca 27 <210> <211> 27 <212> DNA <213> M.Tuberculosis <400> ctgagatcta tggccgacgc tgacacc 27 <210> 86 <211> 27 <212> DNA <213> M.Tuberculosis <400> 86 ctcccatggc tagtcgcgqa gcacaac 27 <210> 87 <211> 27 <212> DNA WO 00/21983 PCT/DK99/00538 48 <213> M.Tuberculosis <400> 87 ctgagatcta tgaqcaagac ggttctc 27 <210> 88 <211> 27 <212> DNA <213> M.Tuberculosis <400> 88 ctcccatqgt cacgtcttcc agcgggt 27 <210> 89 <211> 28 <212> DNA <213> M.Tuberculosis <400> 89 ctgccatggc taggtqgtgt gcacgatc 28 <210> <211> 27 <212> DNA <213> M.Tuberculosis <400> ctgaaqctta tgagcgccta taagacc 27 <210> 91 <211> 27 <212> DNA <213> M.Tuberculosis <400> 91 ctgagatcta tgattqatqa ggctctc 27 <210> 92 <211> 27 <212> DNA <213> M.Tuberculosis <400> 92 ctcccatgga gcggccgcta gacctcc 27 <210> 93 <211> <212> DNA <213> M.Tuberculosis <400> 93 ggctgagact catggccqac atcgatggtg <210> 94 <211> 31 <212> DNA <213> M.Tuberculosis <400> 94 cgtaccatgg tcatqacgac accccctcgt g 31 WO 00/21983 PCT/DK99/00538 49 <210> <211> <212> DNA <213> M.Tuberculosis <400> ggctgagact catggctgaa gtactggtgc <210> 96 <211> 31 <212> DNA <213> M.Tuberculosis <400> 96 cqtaccatqg ctagccggcg accgccggtt c 31 <210> 97 <211> <212> DNA <213> M.Tuberculosis <400> 97 gtgaccgaac ggactctggt <210> 98 <211> 21 <212> DNA <213> M.Tuberculosis <400> 98 ctaggcgccg ggaaaccaga g 21 <210> 99 <211> 23 <212> DNA <213> M.Tuberculosis <400> 99 atqacggata ctcaagtcac ctq 23 <210> 100 <211> <212> DNA <213> M. Tuberculosis <400> 100 ggagtggtac ggctcggcgc <210> 101 <211> <212> DNA <213> M.Tuberculosis <400> 101 atqacgtacg aaaccatcct <210> 102 <211> 21 <212> DNA <213> M.Tuberculosis WO 00/21983 PCT/DK99/00538 <400> 102 tcatcggtgq gtgaactggg g 21 <210> 103 <211> 23 <212> DNA <213> M.Tuberculosis <400> 103 atqccgcttc ccgcaqaccc tag 23 <210> 104 <211> 21 <212> DNA <213> M.Tuberculosis <400> 104 tacgacgggt accactcctg g 21 <210> 105 <211> 22 <212> DNA <213> M.Tuberculosis <400> 105 atgctgatct cacagcgccc ca 22 <210> 106 <211> 22 <212> DNA <213> M.Tuberculosis <400> 106 aagctgttcg qtttcggcgt ag 22 <210> 107 <211> <212> DNA <213> M.Tuberculosis <400> 107 atgaccqgaa atttggtgac <210> 108 <211> 21 <212> DNA <213> M.Tuberculosis <400> 108 tcagtagcgg tagtggtccg g 21

Claims (25)

1. A substantially pure polypeptide, which has a sequence identity of at least to an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 34, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 36 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-y 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 10 5 cells isolated from the spleen of said mouse, the i addition of the polypeptide resulting in a concentration of the polypeptide of not more 15 than 20 jpg per ml suspension, the release of IFN-y being assessable by determination of IFN-y 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-y 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 10 5 cells isolated from the spleen, the addition of the polypeptide resulting in a concentration of not more 25 than 20 pg per ml suspension, the release of IFN-y being assessable by determination of IFN-y 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 Mycobacterialml 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, 68 iv) the polypeptide induces in vitro recall response determined by release of IFN-y 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 ig per ml suspension, the release of IFN-y being assessable by determination of IFN-y 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 tg/ml. vi) the polypeptide induces a positive in vitro response determined by release of IFN-y o. 15 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 10 5 PBMC, the addition of the polypeptide resulting in a concentration of not more than 20 utg per ml suspension, the release of IFN-y being assessable by determination of IFN-y in supernatant harvested 5 days after the addition of the polypeptide to the suspension, and does not induce such an IFN-y release in an individual not infected with a virulent Mycobacterium, vii) the polypeptide induces a positive in vitro response determined by release of IFN-y 25 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 10 5 PBMC, the addition of the polypeptide resulting in a concentration of not more than 20 pg per ml suspension, the release of IFN-y being assessable by determination of IFN-y in supernatant harvested 5 days after the addition of the polypeptide to the suspension, and does not induce such a IFN-y release in an individual who has a cleared infection with a virulent Mycobacterium, 69 viii) the polypeptide induces a positive DTH response determined by intradermal injection of at most 100 ptg 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 pig 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 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: 38, 34, 2, 4, 6, 8, 10, 12, 14, 16, 15 18, 20, 22, 24, 26, 28, 30, 32 and 36.

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: 38, 34, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 36 and/or is a subsequence thereof.

4. An isolated or recombinant polypeptide as defined in any of claims 1-3 which comprises a T cell epitope. 25 5. An isolated or recombinant polypeptide as defined in any of claims 1-4 which I comprises a B cell epitope.

6. A polypeptide according to any of claims 1-5, wherein the polypeptide is encoded by a nucleic acid sequence, said sequence comprising i) is the DNA sequence selected from the group consisting of SEQ ID NOs: 37, 33, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and 35 or an analogue of said sequence which hybridises with any of the DNA sequences shown in SEQ ID NOs: 37, 33, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and 35 or a DNA sequence complementary thereto, or a specific part thereof, preferably under stringent hybridisation conditions, and/or ii) 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: 38, 34, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 36 and/or iii) constitutes a subsequence of any of the above mentioned DNA sequences, and/or iv) constitutes a subsequence of any of the above mentioned polypeptide sequences.

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 :i.o 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 S 15 composition for induction of a protective immune response in a mammal against infection with a virulent Mycobacterium. 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 micro-organism in a manner allowing the micro-organism to express and 25 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. 71

14. A diagnostic reagent according to claims 12 or claim 13 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 to 14 for diagnosing an infection with Mycobacterium tuberculosis.

16. An extract of polypeptides obtained 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 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 15 membrane components from the supernatant of d) with 10% SIDS; 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 25 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. 72 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. An isolated nucleotide sequence which is a nucleotide sequence selected from the group consisting of SEQ ID NOs: 37, 33, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 29, 31 and 35 or an analogue of said sequence which hybridises with any of the nucleotide sequences shown in SEQ ID NOs: 37, 33, 1, 3, 5, 7, 9, 11, 13, 15, 17, -19, 21, 23, 25, 27, 29, 31 and 35 or a nucleotide sequence complementary thereto, or i a specific part or subsequence thereof, preferably under stringent hybridisation 15 conditions.

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

24. A polypeptide as defined by any one of claims 1 to 7 as hereinbefore described with reference to the figures and/or examples. o*e 2 25. Use of a polypeptide as defined by claim 8 or claim 9 as hereinbefore described 25 with reference to the figures and/or examples.

26. A composition as defined by claim 10 or claim 11 as hereinbefore described with reference to the figures and/or examples.

27. A diagnostic reagent as defined by any one of claims 12 to 15 as hereinbefore described with reference to the figures and/or examples.

28. An extract of polypeptides as defined by claim 16 as hereinbefore described with reference to the figures and/or examples. 73

29. Use of an extract of polypeptides as defined by claim 17 as hereinbefore described with reference to the figures and/or examples. A nucleic acid as defined by claim 22 as hereinbefore described with reference to the figures and/or examples. DATED THIS SIXTEENTH DAY OF JULY 2003 STATENS SERUM INSTITUT Patent Attorneys for the Applicant F.B.RICE CO see.