CA2346218A1 - Tuberculosis vaccine and diagnostic reagents based on antigens from the mycobacterium tuberculosis cell - Google Patents

Abstract

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

Description

TB vaccine and diagnostic based on antigens from the M. tuberculosis cell BACKGROUND OF THE INVENTION
Human tuberculosis (TB) caused by Mycobacterium tuberculosis is a serious global health problem responsible for approximately 3 million deaths annually, according to WHO. The world-wide incidence of new tuberculosis cases has been progressively falling for the last decade but the recent years have markedly changed this trend due to the advent of AIDS and the appearance of multidrug resistant strains of Mycobacterium tuberculosis.
The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. BCG generally induces a high level of acquired resistance in animal models of tuberculosis, but several human trials in developing countries have failed to demonstrate significant protection. Notably, BCG is not approved by the FDA for use in the United States because BCG vaccination impairs the specificity of the Tuberculin skin test for diagnosis of TB infection.
This makes the development of a new and improved vaccine against tuberculosis an urgent matter which has been given a very high priority by the WHO. Many attempts have been made to define the protective Mycobacterial substances and a series of experiments were conducted to compare the protective efficacy of vaccination with live versus killed preparations of M. tuberculosis (Orme IM. Infect.lmmun.1988;
56:3310-12).
The conclusion of these studies was that vaccination of mice with dead M.
tuberculosis administered without adjuvants only induced short term protection against TB, whereas live M.tuberculosis vaccines induced efficient immunological memory. This information was the background for the further search for protective substances focused on antigens actively secreted from the live Mycobacteria (Andersen P. Infect.lmmun.1994;
62:2536-44, Honivitz et al. Proc. Natl Acad. Sci. USA 1995; 92:1530-4, Pal PG et al.
infect.lmmun.
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

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 5.
By the term "virulent Mycobacterium" is understood a bacterium capable of causing the tuberculosis disease in a mammal including a human being. Examples of virulent Mycobacteria are M. tuberculosis, M. africanum, and M. bovis.
By "a TB patient" is understood an individual with culture or microscopically proven infection with virulent Mycobacteria, and/or an individual clinically diagnosed with TB and who is responsive to anti-TB chemotherapy. Culture, microscopy and clinical diagnosis of TB is well known by the person skilled in the art.
A significant decrease or increase is defined as a decrease or increase which is significant at the 95% level by comparison of immunised and placebo-treated groups using an appropriate statistical analysis such as a Student's two-tailed T
test.
By the term "PPD positive individual" is understood an individual with a positive Mantoux test or an individual where PPD induces an increase in in itro recall response determined by release of IFN-y of at least 1,000 pgiml from Peripheral Blood Mononuclear Cells (PBMC) or whole blood, the induction being performed by the addition

3 PCT/DK99/00538 of 2.5 to 5 pg PPD/ml to a suspension comprising about 1.0 to 2.5 x 105 PBMC, the release of IFN-y being assessable by determination of IFN-y in supernatant harvested 5 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 "a" means "at least one" unless explicitly indicated otherwise. The "polypeptide" is used to referrer to short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues), and longer peptides (the usual interpretation of "polypeptide", i.e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, 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, 20 amino acids long. However, also longer polypeptides having a length of e.g.
25, 50, 75, 100, 125, 150, 175 or 200 amino acids are within the scope of the present invention.

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 (tower percentages of other polypeptide material are preferred, e.g. at most 4%, at most 3%, at most 2%, at most 1%, and at most'/Z%). It is preferred that the substantially pure polypeptide is at least 96% pure, i.e. that the poiypeptide 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 (Nn,-N~~)J°° , wherein Nd;, is the total number of non-identical residues in the two sequences Nn~
when aligned and wherein Nre, is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Nd;,=2 and N~ef=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 (Nd;f-2 and N~ef=8). Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program or the BLASTN program

5 (Pearson W.R and D.J. Lipman (1988) PNAS USA 85:2444-2448)(www.ncbi.nlm.nih.govIBLAST). 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, 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 pofypeptides, 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);

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 m); and 0) 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 immunofogical 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;
0) blotting the gel of n) to a PVDF membrane;

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 r); and t) formulating the polypeptide of s) with an adjuvant substance in an immunological composition.
A third embodiment is a method of producing a polypeptide originating from the cell membrane in an immunological composition, said method comprising the steps of:
a) killing a sample of virulent Mycobacteria;
b) centrifugating the sample of a) c) resuspending the pellet of b) in PBS supplemented with EDTA and phenylmethylsulfonyl fluoride and sonicating for 15 min d) lysing the suspension of c) e) centrifugating the lysed suspension of d) f) ultracentrifugating the supernatant of e) g) resuspending the pellet of f) in PBS
h) subject the suspension of g) to a Triton X-114 extraction i) applying the resuspension of h) to 2 dimensional electrophoresis;
j) blotting the gel of i) to a PVDF membrane;
k) subjecting the spots on j) to N-terminal sequencing;
I) searching a database for homology with the sequence of k) to identify the nucleotide sequence;
m) cloning the nucleotide sequence of I) into an expression system; and n) isolating and purifying the polypeptide expressed in m);
o) formulating the polypeptide of n) with an adjuvant substance in an immunological composition.
A fourth embodiment is a method of producing a polypeptide originating from the cytosol in an immunological composition comprising the steps of:
a) killing a sample of virulent Mycobacteria;
b) centrifugating the sample of a) c) resuspending the pellet of b) in PBS supplemented with EDTA and phenylmethylsulfonyl fluoride and sonicating for 15 min

8 d) iysing 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 m); 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 105 cells isolated from the spleen of said mouse, the addition of the polypeptide resulting in a concentration of the polypeptide of not more than 20 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, 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° virulent Mycobacteria, the induction being performed by the addition of the polypeptide to a suspension comprising about 2 x 105 cells isolated from the spleen, the addition of the polypeptide resulting in a concentration of not more than 20 p.g per ml suspension, the release of IFNJy being assessable by determination of IFN-y in supernatant harvested 3 days after the addition of the polypeptide to the suspension,

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 10s virulent Mycobacteria/ml 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 2.5 x 105 PBMC or whole blood cells, 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, 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 ~glml 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 pgiml from Peripheral Blood Mononuclear Cells (PBMC) withdrawn from an individual who is clinically or subclinically infected with a virulent Mycobacterium, the induction being performed by the addition of the polypeptide to a suspension comprising about 1.0 to 2.5 x 105 PBMC, the addition of the polypeptide resulting in a concentration of not more than 20 p.g per ml suspension, the release of IFN-y being assessable by determi-nation 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 clinically or subclinically infected with a virulent Mycobacterium, the induction being performed by the addition of the polypeptide to a suspension comprising about 1.0 to 2.5 x 105 PBMC, the addition of the polypeptide resulting in a concentration of not more than pg per ml suspension, the release of IFN~y being assessable by determination of IFN-y 5 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

10 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 15 application patch of at most 100 ~.g 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 20 p.g per ml suspension, such as 15 ug, 10 p.g, 5 pg, 3 pg, 2 pg, or 1 p,g polypeptide per ml suspension.

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, C3H/HeJ-Lps or BALE 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 1FN-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, 1. et S.H.E.
Kaufmann ( 1987) J Immunol.138(12):4408-13). However other cytokines could be relevant when monitoring the immunological response to the polypeptide, such as IL-12, TNF-a, IL-4, IL-5, IL-10, IL-6, TGF-(3. 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, Gaseous 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

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.Immunol.154:3359).
The property described in ii) will also be satisfied if the release of IFN-y from T-lymphocytes withdrawn during primary infection is 2,000 pg/ml, such as 3,000 pglml. 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, 10 weeks, 11 weeks, 12 weeks or 15 weeks. In another embodiment of the invention, the last vaccination challenge is given subcutaneously with 5x104 virulent Mycobacteria. As will be known by the person skilled in the art, the number of viable bacteria in the lung is presently considered to be relevant to the degree of bacterial infection of the animal. An equally important measure is the determination of the number of viable bacteria in the spleen, lymph node, or blood.
The amount of polypeptide and adjuvant used for vaccinating will depend on the animal model used, e.g. the mouse strain. When a mouse model is used it is preferred that the amount of polypeptide used for vaccinating the mouse is between 2 and 20 fig, such as between 5 and 15 pg, preferably 10 p,g. For larger animals such as guinea pigs, Beers, cows, primates, badgers, and possums higher doses such as 5 to 50 ~g 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

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 ~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 (e.g.
peroxidase labeled) secondary antibody and reaction is thereafter visualized by the use of OPD and H202 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 ~g 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).

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 fig, such as 80~g, 70 p.g, 60 pg, 50 pg, 40 fig, or 30 pg. 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 mare 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.

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.
5 These polypeptides in purified form, or non-naturally occurring, i.e.
recombinantly or synthetically produced, are considered part of the invention. It is understood that a polypeptide which has any of the properties i) - ix) and has a sequence identity of at least 80% with any of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 75, 77 and 79 or has a sequence identity of 10 at least 80% to any subsequence thereof is considered part of the invention. (n a preferred embodiment the sequence identity is at feast 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

15 considered part of the invention. Also, any B-cell epitope of the polypeptides disclosed in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 75, 77 and 79 is considered part of the invention.
Although the minimum length of a T-cell epitope has been shown to be at least 6 amino acids, it is normal that such epitopes are constituted of longer stretches of amino acids.
Hence it is preferred that the polypeptide fragment of the invention has a length of at least 7 amino acid residues, such as at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, or at least 30 amino acid residues.
In both immunodiagnostics and vaccine preparation, it is often possible and practical to prepare antigens from segments of a known immunogenic protein or polypeptide.
Certain epitopic regions may be used to produce responses similar to those produced by the entire antigenic polypeptide. Potential antigenic or immunogenic regions may be identified by any of a number of approaches, e.g., Jameson-Wolf or Kyte-Doolittle antigenicity analyses or Hopp and Woods (Hopp et Woods, (1981), Proc Natl Acad Sci USA 78/6:3824-8) hydrophobicity analysis (see, e.g., Jameson and Wolf, (1988) Comput Appl Biosci, 4(1):181-6; Kyte and Doolittle, (1982) J Mol Biol, 157(1):105-32;
or U.S.
Patent No. 4,554,101). Hydrophobicity analysis assigns average hydrophilicity values to each amino acid residue; from these values average hydrophilicities can be calculated

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 one-dimensional structure, are located near to each other in the folded protein.
Within the meaning of the present context, the expression epitope is intended to comprise the one-and three-dimensional structure as well as mimics thereof.. The term is further intended to include discontinuous B-cell epitopes. The linear B-cell epitopes can be identified in a similar manner as described for the T-cell epitopes above. However, when identifying B
celi epitopes the assay should be an ELISA using overlapping oligomers derived from the polypeptide as the coating layer on a microtiter plate as described elsewhere.
A non-naturally occurring polypeptide, an analogue, a subsequence, a T-cell epitope and/or a B-cell epitope of any of the described polypeptides are defined as any non-naturally occurring polypeptide, analogue, subsequence, T-cell epitope and/or 8-cell epitope of any of the polypeptides having any of the properties i)-ix).
Table 1 lists the antigens of the invention.

1?
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 AntigenN-Terminal sequenceNucleotide Amino acid sequence SEQ ID NO: sequence SEQ ID NO:

SEQ ID NO:

B

TB12.5 80 74 75 TB20.6 81 76 77 TB40.8 82 78 79 Each of the polypeptides may be characterised by specific amino acid and nucleic acid sequences. It will be understood that such sequences include analogues and variants produced by recombinant methods wherein such nucleic acid and polypeptide sequences have been modified by substitution, insertion, addition and/or deletion of one or more nucleotides in said nucleic acid sequences to cause the substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide. A
preferred nucleotide sequence encoding a polypeptide of the invention is a nucleotide sequence which 1) is a nucleotide sequence selected from the group consisting of SEQ ID NOs:
1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 74, 76 and 78 or an analogue of said sequence which hybridises with any of the nucleotide sequences shown in SEQ ID
NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 74, 76 or 78 or a nucleotide sequence complementary thereto, or a specific part thereof, preferably under stringent hybridisation conditions. By stringent conditions is understood, as defined in the art, 5-10°C under the melting point 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 I D 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 andlor subsequence. The analogous nucleotide fragment or nucleotide sequence may be derived from a bacterium, a mammal, or a human or may be partially or completely of synthetic origin. The analogue and/or subsequence may also be derived through the use of recombinant nucleotide techniques.
Furthermore, the terms "analogue" and "subsequence" are intended to allow for variations in the sequence such as substitution, insertion (including introns), addition, deletion and rearrangement of one or more nucleotides, which variations do not have any substantial effect on the polypeptide encoded by a nucleotide fragment or a subsequence thereof. The term "substitution" is intended to mean the replacement of one or more nucleotides in the full nucleotide sequence with one or more different nucleotides, "addition" is understood to mean the addition of one or more nucleotides at either end of the full nucleotide sequence, "insertion" is intended to mean the introduction of one or more nucleotides within the full nucleotide sequence, "deletion" is intended to indicate that one or more nucleotides have been deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it, and "re-arrangement" 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 ex-hibits the above hybridization pattern. Normally this will require a minimum sequence identity of at least 70% with a subsequence of the hybridization partner having SEQ ID
NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 74, 76 or 78. It is preferred that the nucleic acid fragment is longer than 10 nucleotides, such as at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, and at least 80 nucleotides long, and the sequence identity should preferable also be higher than 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, and at least 98%. It is most preferred that the sequence identity is 100%. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR
tech-nology of U.S. Patent 4,603,102, or by introducing selected sequences into recombinant vectors for recombinant production.

The nucleotide sequence to be modified may be of cDNA or genomic origin as discussed above, but may also be of synthetic origin. Furthermore, the sequence may be of mixed cDNA and genomic, mixed cDNA and synthetic or genomic and synthetic origin as discussed above. The sequence may have been modified, e.g. by site-directed mu-5 tagenesis, to result in the desired nucleic acid fragment encoding the desired polypep-tide.
The invention also relates to a replicable expression vector which comprises a nucleic acid fragment defined above, especially a vector which comprises a nucleic acid frag-10 ment encoding a polypeptide fragment of the invention. The vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
Thus, the vector may be an autonomously replicating vector, i.e, a vector which exists as an extrachromo-somal entity, the replication of which is independent of chromosomal replication;
15 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.
20 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 shorker DNA segments could be used, depending on the antigenic protein desired. Epitopic regions of the proteins expressed or encoded by the disclosed DNA could be included as relatively short segments of DNA. A wide variety of expression vectors is possible including, for example, DNA segments encoding reporter gene products useful for identification of heterologous gene products and/or resistance genes such as antibiotic resistance genes which may be useful in identifying transformed cells.
The vector of the invention may be used to transform cells so as to allow propagation of the nucleic acid fragments of the invention or so as to allow expression of the polypeptide fragments of the invention. Hence, the invention also pertains to a transformed cell harbouring at least one such vector according to the invention, said cell being one which does not natively harbour the vector and/or the nucleic acid fragment of the invention contained therein. Such a transformed cell (which is also a part of the invention) may be any suitable bacterial host cell or any other type of cell such as a unicellular eukaryotic organism, a fungus or yeast, or a cell derived from a multicellular organism, e.g. an ani-mal 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, 8. subtilis, or M. bovis BCG cell, and it is especially preferred that the transformed cell expresses a polypeptide according of the invention.
The latter opens for the possibility to produce the polypeptide of the invention by simply recovering it from the culture containing the transformed cell. In the most preferred embodiment of this part of the invention the transformed cell is Mycobacterium bovis BCG strain: Danish 1331, which is the Mycobacterium bovis strain Copenhagen from the Copenhagen BCG Laboratory, Statens Seruminstitut, Denmark.
The nucleic acid fragments of the invention allow for the recombinant production of the polypeptides fragments of the invention. However, also isolation from the natural source is a way of providing the polypeptide fragments as is peptide synthesis.
Therefore, the invention also pertains to a method for the preparation of a polypeptide fragment of the invention, said method comprising inserting a nucleic acid fragment as described in the present application into a vector which is able to replicate in a host cell, introducing the resulting recombinant vector into the host cell (transformed cells may be selected using various techniques, including screening by differential hybridization, identification of fused reporter gene products, resistance markers, anti-antigen antibodies and the like), culturing the host cell in a culture medium under conditions sufficient to effect expression of the polypeptide (of course the cell may be cultivated under conditions appropriate to the circumstances, and if DNA is desired, replication conditions are used), and recovering the polypeptide from the host cell or culture medium; or isolating the polypeptide from a short-term culture filtrate; or isolating the polypeptide from whole mycobacteria of the tuberculosis complex or from lysates or fractions thereof, e.g. cell wall containing fractions, or synthesizing the polypeptide by solid or liquid phase peptide synthesis.

The medium used to grow the transformed cells may be any conventional medium suitable for the purpose. A suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above. The methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purposes within the field of recombinant DNA. In the follow-ing a more detailed description of the possibilities will be given:
In general, of course, prokaryotes are preferred for the initial cloning of nucleic se quences of the invention and constructing the vectors useful in the invention.
For ex ample, 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 (ATCC No. 31446), E. coli B, and E. coli X 1776 (ATCC No. 31537). These examples are, of course, intended to be illustrative and not limiting.
Prokaryotes are also preferred for expression. The aforementioned strains, as well as E.
coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia mar-cesans, and various Pseudomonas species may be used. Especially interesting are rapid-growing mycobacteria, e.g. M. smegmatis, as these bacteria have a high degree of resemblance with mycobacteria of the tuberculosis complex and therefore stand a good chance of reducing the need of performing post-translational modifications of the expression product.
In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coliis typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al., 1977, Gene 2: 95). The pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
The pBR plasmid, or other microbial plasmids or phages must also contain, or be modified to contain, promoters which can be used by the microorganism for expression.

Those promoters most commonly used in recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., (1978), Nature, 35:515; Itakura et al., (1977), Science 198:1056; Goeddel et al., (1979), Nature 281:544) and a tryptophan (trp) promoter system (Goeddel et al., (1979) Nature 281:544;
EPO
Appl. Publ. No. 0036776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (Siebwenlist et al., (1980), Cell, 20:269). Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another promoter by artificial means.
After the recombinant preparation of the polypeptide according to the invention, the isolation of the polypeptide may for instance be carried out by affinity chromatography (or other conventional biochemical procedures based on chromatography), using a monoclonal antibody which substantially specifically binds the polypeptide according to the invention. Another possibility is to employ the simultaneous electroelution technique described by Andersen et al. in J. Immunol. Methods 161: 29-39.
According to the invention the post-translational modifications involves lipidation, gly-cosylation, cleavage, or elongation of the polypeptide.
In certain aspects, the DNA sequence information provided by this invention allows for the preparation of relatively short DNA (or RNA or PNA) sequences having the ability to specifically hybridize to mycobacterial gene sequences. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of the relevant sequence. The ability of such nucleic acid probes to specifically hybridize to the mycobacterial gene sequences lend them particular utility in a variety of embodiments.
Most importantly, the probes can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample. However, either uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructs.
Apart from their use as starting points for the synthesis of polypeptides of the invention and for hybridization probes (useful for direct hybridization assays or as primers in e.g.
PCR or other molecular amplification methods) the nucleic acid fragments of the WO 00/219$3 PCT/DK99/00538 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 func-tioning 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 ac-cording to the invention, the vaccine effecting in vivo expression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to infec-tions with mycobacteria of the tuberculosis complex in an animal, including a human being.
The efficacy of such a "DNA vaccine" can possibly be enhanced by administering the gene encoding the expression product together with a DNA fragment encoding a poly-peptide which has the capability of modulating an immune response. For instance, a gene encoding lymphokine precursors or lymphokines (e.g. IFN-y, IL-2, or IL-12) could be administered together with the gene encoding the immunogenic protein, either by ad-ministering two separate DNA fragments or by administering both DNA fragments included in the same vector. It also is a possibility to administer DNA
fragments compri-sing a multitude of nucleotide sequences which each encode relevant epitopes of the poiypeptides disclosed herein so as to effect a continuous sensitization of the immune system with a broad spectrum of these epitopes.
In one embodiment of the invention, any of the above mentioned polypeptides is used in the manufacture of an immunogenic composition to be used for induction of an immune response in a mammal against an infection with a virulent Mycobacterium.
Preferably, the immunogenic composition is used as a vaccine.
The preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251;

4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in liquid or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified. The active 5 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 10 the effectiveness of the vaccines.
In one embodiment the composition used for vaccination comprises at least one, but preferably at least 2, such as at least 3, 4, 5, 10, 15 or at least 20 different polypeptides of the invention.
In another embodiment the composition to be used for vaccine comprises, together with at least one polypeptide of the invention, at least one, but preferably at least 2, such as at least 3, 4, 5, 10, 15 or at least 20 polypeptides which are not polypeptides of the present invention but are derived from a virulent Mycobacterium such as a polypeptide belonging to the group of ST-CF (Elhay MJ and Andersen P, Immunology and cell Biology (1997) 75, 595-603). ESAT-6, CFP7, CFP10 (EMBL accession number: AL022120), CFP17, CFP21, CFP25, CFP29, MPB59, MPT59, MPB64, and MPT64.
The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.

The proteins may be formulated into the vaccine as neutral or salt forms.
Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micrograms of active ingredient per vaccination with a preferred range from about 0.1 pg to 1000 p.g, such as in the range from about 1 ~g to 300 pg, and especially in the range from about 10 ug to 50 wg. 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 physiologi-cally acceptable dispersion), buccal, sublingual, nasal, rectal or transdermal routes. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the weight of the person to be vaccinated.
Some of the polypeptides of the vaccine are sufficiently immunogenic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant substance.
Various methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), commonly used as a 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as a 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101 °C
for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated {Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oif vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. According to the invention DDA (dimethyldioctadecylammonium bromide) is an interesting candidate for an adjuvant, but also Freund's complete and incomplete adjuvants as well as QuilA and RIBI adjuvants are interesting possibilities.
Other possibilities to enhance the immunogenic effect involve the use of immune modulating substances such as lymphokines (e.g. IFN-y, IL-2 and It_-12) or synthetic IFN-y 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 10 years, more preferably 1-5 years usually three years, will be desirable to maintain the desired levels of protective immunity.
In one embodiment of the invention a composition is produced comprising as the effective component a micro-organism, the micro-organism is a bacterium such as Mycobacterium, Salmonella, Pseudomonas and Escherichia, preferably Mycobacterium bovis BCG wherein at least one, such as at least 2 copies, such as at least 5 copies of a nucleotide fragment comprising a nucleotide sequence encoding a polypeptide of the invention has been incorporated into the genome of the micro-organism or introduced as a part of an expression vector in a manner allowing the micro-organism to express and optionally secrete the polypeptide. In a preferred embodiment, the composition comprises at least 2 different nucleotide sequences encoding at least 2 different polypeptides of the invention. In a much preferred embodiment, the composition comprises at least different nucleotide sequences encoding at least one polypeptide of the invention and at 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, CFP10, CFP17, CFP21, CFP25, CFP29, MPB59, MPT59, MPB64, and MPT64.
Individuals infected with virulent Mycobacteria can generally be divided into two groups.
The first group has an infection with a virulent Mycobacterium e.g. contacts of TB
patients. The virulent Mycobacterium may have established colonies in the lungs, but the individual has, as yet, no symptoms of TB. The second group has clinical symptoms of TB, as a TB patient.
In one embodiment of the invention, any of the above mentioned polypeptides are used for the manufacture of a diagnostic reagent that preferably distinguishes a subclinically or clinically infected individual (group I and group II) from an individual who has been BCG
vaccinated or infected with Mycobacterium avium or sensitised by non-tuberculosis Mycobacterium (NTM), and may distinguish a subclinically or clinically infected individual from an individual who has cleared a previous infection with a virulent Mycobacterium. It is most likely that specific polypeptides derived from SPE will identify group I and/or group II from individuals not infected with virulent Mycobacteria in the same way as ESAT-f and CFP10 (P.Ravn et al., (1998), J. Infectious Disease 179:637-45).
In one embodiment of the invention, any of the above discussed polypeptides are used for the manufacture of a diagnostic reagent for the diagnosis of an infection with a virulent Mycobacterium. One embodiment of the invention provides a diagnostic reagent for differentiating an individual who is clinically or subclinically infected with a virulent Mycobacterium from an individual not infected with virulent Mycobacterium, i.e. an individual who has been BCG vaccinated or infected with Mycobacterium avium or sensitised by non-tuberculosis Mycobacterium (NTM). Such a diagnostic reagent will distinguish between an individual in group I and/or II of the infection stages above, from an individual who has been vaccinated against TB. Another embodiment of the invention provides a diagnostic reagent for differentiating an individual who is clinically or subclinically infected with a virulent Mycobacterium from an individual who has a cleared infection with a virulent Mycobacterium. Such a diagnostic reagent will distinguish between an individual in group I and/or II of the infection stages above, from an individual who has cleared the infection.

Determination of an infection with virulent Mycobacterium will be instrumental in the, still very laborious, diagnostic process of tuberculosis. A number of possible diagnostic assays and methods can be envisaged (some more specifically described in the examples and the list of properties): a sample comprising whole blood or mononuclear cells {i.a. T-lymphocytes) from a patient could be contacted with a sample of one or more polypeptides of the invention. This contacting can be performed in vitro and a positive reaction could e.g. be proliferation of the T-cells or release of cytokines such as IFN-y into the extracellular phase (e.g. into a culture supernatant).
Alternatively, a sample of a possibly infected organ may be contacted with an antibody raised against a polypeptide of the invention. The demonstration of the reaction by means of methods well-known in the art between the sample and the antibody will be indicative of ongoing infection and could be used to monitor treatment effect by reduction in responses. It is of course also a possibility to demonstrate the presence of anti-Mycobacterial antibodies in serum by contacting a serum sample from a subject with at least one of the polypeptide fragments of the invention and using well-known methods for visualising the reaction between the antibody and antigen such as ELISA, Western blot, precipitation assays.
Also a method of determining the presence of virulent Mycobacterium nucleic acids in a mammal, including a human being, or in a sample, comprising incubating the sample with a nucleic acid sequence of the invention or a nucleic acid sequence complementary thereto, and detecting the presence of hybridised nucleic acids resulting from the incubation (by using the hybridisation assays which are well-known in the art), is included in the invention. Such a method of diagnosing TB might involve the use of a composition comprising at least a part of a nucleotide sequence as defined above and detecting the presence of nucleotide sequences in a sample from the animal or human being to be tested which hybridises with the nucleic acid sequence (or a complementary sequence) by the use of PCR techniques.
The invention also relates to a method of diagnosing infection caused by a virulent Mycobacterium in a mammal, including a human being, comprising locally applying (patch test) or intradermally injecting (Mantoux test) a polypeptide of the invention. These tests are both called a delayed hypersensitivity reaction (DTH). A positive skin response at the location of injection or application is indicative of the mammal including a human being, being infected with a virulent Mycobacterium, and a negative skin response at the location of injection or application is indicative of the mammal including a human being not having TB. A positive response is a skin reaction having a diameter of at least 5 mm larger than background, but larger reactions are preferred, such as at least 1 cm, 1.5 cm, 5 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 10 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 15 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 20 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.
25 The polypeptides according to the invention may be potential drug targets.
Once a particular interesting polypeptide has been identified, the biological function of that polypeptide may be tested. The polypeptides may constitute receptor molecules or toxins which facilitates the infection by the Mycobacterium and if such functionality is blocked, the infectivity of the virulent Mycobacterium will be diminished.
The biological function of particular interesting polypeptides may be tested by studying the effect of inhibiting the expression of the polypeptides on the virulence of the virulent Mycobacterium. This inhibition may be performed at the gene level such as by blocking the expression using antisense nucleic acid, PNA or LNA or by interfering with regulatory sequences or the inhibition may be at the level of translation or post-translational processing of the polypeptide.
Once a particular polypeptide according to the invention is identified as critical for virulence, an anti-mycobacterial agent might be designed to inhibit the expression of that polypeptide. Such anti-mycobacterial agent might be used as a prophylactic or therapeutic agent. For instance, antibodies or fragments thereof, such as Fab and (Fab')2 fragments, can be prepared against such critical polypeptides by methods known in the art and thereafter used as prophylactic or therapeutic agents A presently preferred embodiment is an extract of polypeptides obtainable by a method comprising the steps of a) killing a sample of virulent Mycobacteria;
b) centrifugating the sample of a) at 2,OOOg 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,OOOg 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 polycional antibody, which is specifically reacting with a poly-peptide of the invention in an immuno assay, or a specific binding fragment of said anti-body, 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 anti-bodies are subsequently isolated, suitably by immune affinity chromatography.
The production of monoclonals can be effected by methods well-known in the art, since the present invention provides for adequate amounts of antigen for both immunization and screening of positive hybridomas.

Examples EXAMPLE 1: Total extraction of proteins from dead M.tuberculosis bacteria.
1.5 x 109 bacteria/ml M.tuberculosis was heat treated at 55°C for 1.5 hours and checked for sterility. 10 ml of these heat killed bacteria was centrifuged at 2000 g for 40 min; the supernatant was discharged and the pellet resuspended in PBS containing 0.5%
Tween 20 and used as the antigen source. The pellet was sonicated with 20 rounds of 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 C5781/6J mice (Bomholtgaard, Denmark) were immunised subcutaneously at the base of the tail with vaccines of the following composition:
Group 1: BCG
Group 2: 1x 10' heat killed M.tuberculosislDDA (250 ~.g DDA) Group 3: 50 p.g ST-CF/DDA (250 pg) Group 4: 50 pg SPE/DDA (250 wg}
Group 5: Adjuvant control: DDA (250 p,g) 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 2). 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 t 203 11079 ~ 178 BCG <200 469 t 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 ali 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 1.5 x 109 colony forming units (CFU/ml) of M. tuberculosis H37Rv were inactivated by heat-killing at 60°C for 1.5 hour. The heat-killed Mycobacteria was centrifuged at 3,000 x g for 20 min; the supernatant was discarded and the pellet was resuspended in cold PBS.
This step was repeated twice. After the final wash, the pellet was resuspended in a homogenising buffer consisting of PBS supplemented with 10 mM EDTA and 1 mM of phenylmethylsulfonyl fluoride in a ratio of 1 ml buffer per 0.5 g of heat-killed Mycobacteria. The sample was sonicated on ice for 15 min (1-min-pulser-on110-sec-pulser off) and subsequently lysed three times with a French Pressure Cell at 12,000 Iblin2. 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.

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, 5 and twice with PBS. Soluble proteins were extracted from the cell wall by a 2 hour incubation with 2% SDS at 6°C. The insoluble cell wall core was removed by a centrifugation at 27,000 x g for 20 min and the SDS-extraction was repeated.
Finally, the pooled supernatants were precipitated with 6 volumes of chilled acetone and resuspended in PBS.
10 Preparation of cytosol and membrane:
To separate the cytosol and the membrane fraction, the pooled supernatants were ultracentrifugated at 100,000 x g for 2 hours at 5°C. The cytosol proteins in the supernatant were precipitated with acetone and resuspended in PBS. The pellet, representing the membrane fraction, was washed in PBS, ultracentrifugated, and finally 15 resuspended in PBS.
Triton X-114 extraction of cell wall and membrane:
To prepare protein fractions largely devoid of lipoarabinomannan, the cell wall and the membrane fraction were subjected to extraction with precondensed Triton X-114.
Triton X-114 was added to the protein sample at a final concentration of 4%. The solution was 20 mixed on ice for 60 min and centrifuged at 20,000 x g for 15 min at 4°C. The pellet containing residual insoluble material was extracted once more (membrane) or twice (cell wall), while the supernatant was warmed to 37°C to condense the Triton X-114. After centrifugation of the supernatant at 12,000 x g for 15 min, the aqueous phase and detergent phase were separated. The aqueous phase and detergent phase were washed 25 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 30 stimulating PBMCs with panels of narrow molecular mass fractions from membrane, cell wall, and cytosol obtained by the mufti-elution technique described by Andersen et al.
(1993) J. Immunol. Methods 161:29-39. The technique resulted in 30 sharply defined fractions and allowed an identification of immunological active regions, of potential as either diagnostic reagents or as vaccine components.

The study demonstrated that multiple targets within the cell 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 non-precipitated proteins were removed by centrifugation and precipitated proteins were resuspended in 20 mM imidazole pH 7Ø The protein solution was applied to a DEAE
Sepharose 6B column, equilibrated with 20 mM imidazole pH 7Ø Bound protein was eluted from the column using a salt gradient from 0 to 1 M NaCI, in 20 mM
imidazole pH
7Ø 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-IgG {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-terrninai sequencing and amino acid analysis Proteins of the fractions containing bands reactive with serum from TB
patients in Western blot were separated by 2D electrophoresis. Gels were blotted to PVDF

membranes and spots subjected to N-terminal sequencing on a Procise sequencer (Applied Biosystems).
The following N-terminal sequences were obtained ForTB15 :TERTAVLIKPDGIER
(SEQ ID NO: 39) ForTB18 :TDTQVTWLTQESHDR
(SEQ ID NO: 40) ForTB21 :MIDEALFDAEEKMEK
(SEQ ID NO: 41) ForTB33 :PLPADPSTDLSAYAQ
(SEQ ID NO: 42) ForTB38 :MLISQRPTLSEDVLT
(SEQ ID NO: 43) ForTB54 :TGNLVTKNSLTPDVR
(SEQ ID NO: 44) Sequence identity searches The N-terminal sequences obtained were used for an identity search using the blast program of the Sanger M. tuberculosis database http://www.sanger.ac.uk/Projects/M tuberculosis/blast server.shtml In addition, the GenEMBL database was searched using the BLASTP program (Altschul, Stephen F., Warren Gish, Webb Miller, Eugene W. Myers, and David J. Lipman (1990).
Basic local alignment search tool. J. Mol. Biol. 215:403-10.), to reveal proteins with homology to the full amino acid sequences obtained from the Sanger database.
Thereby, the following information was obtained For the 15 determined N-terminal amino acids for TB15 a 93% identical sequence was found in MTV008.01 c. Amino acid 5 of the determined N-terminal sequence (A) is an L in the sequence MTV008.01c.

Within the open reading frame the translated protein is 136 amino acids long.
The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 136 amino acids, which corresponds to a theoretical molecular mass of 14 509 Da and a theoretical pl of 5.36. The observed mass in SDS-PAGE
is 14 kDa.
TB15 has 80% sequence identity in a 139 amino acid overlap to a protein of M.
smegmatis. It is homologous to putative nucleoside diphosphate kinases from several species, e.g. 59% sequence identity to a 151 amino acid protein of Archaeoglobus fulgidus and 57% sequence identity to a 149 amino acid protein of Bacillus subtilis.

For the 15 determined N-terminal amino acids for TB18 a 100% identical sequence was found in MTCY017.33c.
Within the open reading frame the translated protein is 164 amino acids long.
The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 164 amino acids, which corresponds to a theoretical molecular mass of 17 855 Da and a theoretical pl of 4.81. The observed mass in SDS-PAGE
is 20 kDa.
TB18 has 94% sequence identity, in a 164 amino acid overlap, to a protein from M.
leprae. In addition, it is homologous to transcription elongation factors from several species, e.g. 32% sequence identity in a 114 amino acid overlap, to a protein from Zymomonas mobilis.

For the 15 determined N-terminal amino acids for TB21 a 100% identical sequence was found in MTCY274.13c.
Within the open reading frame the translated protein is 185 amino acids long.
The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 1.
This corresponds to a theoretical molecular mass of 20 829 Da and a theoretical 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.

For the 15 determined N-terminal amino acids for TB33 a 85% identical sequence was found in MTCY71.23. Amino acids 8 and 9 of the determined N-terminal sequence (T and D) are a P and a T in MTCY71.23, respectively.
Within the open reading frame the translated protein is 297 amino acids long.
The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 297 amino acids, which corresponds to a theoretical molecular mass of 33 323 Da and a theoretical pl of 4.91. The observed mass in SDS-PAGE
is 35 kDa.
TB33 has 83% sequence identity in a 296 amino acid overlap to a protein from M. leprae.
In addition, it is homologous to thiosulphate sulfurtransferases (rhodanese) from several species, e.g. 48% in a 131 amino acid overlap to rhodanese from Saccharopolyspora erythraea.

For the 15 determined N-terminal amino acids for TB38 a 100% identical sequence was found in MTCY13E12.10c.
Within the open reading frame the translated protein is 347 amino acids long.
The N-terminal sequence of the protein identified in the cytosoi 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 Sfreptomyces coelicolor.

For the 15 determined N-terminal amino acids for TB54 a 100% identical sequence was found in MTCY20B11.23c.
Within the open reading frame the translated protein is 495 amino acids long.
The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 495 amino acids, which corresponds to a theoretical molecular mass of 54 329 Da and a theoretical pl of 5.00. The observed mass in SDS-PAGE
is 60 kDa.

TB54 is homologous to adanosyl homocysteinases from several species, e.g. 73%
in a 90 amino acid overlap to S-adenosyl-L-homocysteine hydrolase from Triticum aestivum.
It contains a S-adenosyl-L-homocysteine hydrolase signature (PS00739).
Example 3a: Use of patient sera to identify M. tuberculosis cytosol antigens.
5 Anion exchange chromatography of the cytosol proteins and Western blot experiments with a pool of sera from TB patients were performed as described in Example 3.
N-terminal sequencing Proteins of the fractions containing TB12.5, TB20.6, and TB40.8 were separated by 2D
electrophoresis. Gels were blotted to PVDF membranes and spots subjected to N-10 terminal sequencing on a Procise sequencer (Applied Biosystems).
The following N-terminal sequences were obtained For TB12.5 :ALKVEMVTFDXSDPA
(SEQ ID NO: 80) 15 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.
20 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.
25 Within the open reading frame the translated protein is 115 amino acids long. The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 115 amino acids, which corresponds to a theoretical molecular mass of 12 512 Da and a theoretical pl of 4.91. The observed mass in SDS-PAGE
is 14 30 kDa.
No homology was found to TB12.5.
TB20.6 For the 15 determined N-terminal amino acids of TB20.6 a 100 % identical sequence was found in Rv3920c.
Within the open reading frame the translated protein is 187 amino acids long.
The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 1.
This gives a protein of 187 amino acids, which corresponds to a theoretical molecular mass of 20.559 Da and a theoretical pl of 4.14. The observed mass in SDS-PAGE
is 24 kDa.
TB20.6 has 73 % homology to a 193 amino acid protein of M. leprae. It has 59%
homology in a 184 amino acid overlap to a Jag-like protein from Streptomyces coelicolor.
TB40.8 For the 15 determined N-terminal amino acids of TB40.8 a 100 % identical sequence was found in Rv0331.
Within the open reading frame the translated protein is 388 amino acids long.
The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 388 amino acids, which corresponds to a theoretical molecular mass of 40 792 Da and a theoretical 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 non-precipitated proteins were removed by centrifugation and precipitated proteins were resuspended in 20 mM imidazole, pH 7Ø 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 ForT810C :MEVKIGITDSPRELV
(SEQ ID NO: 45) ForTBI5A : SAYKTVVVGTDDXSX
(SEQ ID NO: 46) ForTBl7 :MEQRAELVVGRALVV
(SEQ ID NO: 47) ForTB24 :ADIDGVTGSAGL(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 (M) is a V in the sequence found, corresponding to GTG being used as a start codon, instead of ATG.
Within the open reading frame the translated protein is 90 amino acids. The N-terminal sequence of the protein identified in the cytosol starts at amino acid 1.
This corresponds to a theoretical molecular mass of 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 CY0182.28. The X at position 13 of the determined N-terminal sequence corresponds to a G in MTCY0182.28 and the X at position 15 to a D.
Within the open reading frame the translated protein is 146 amino acids long.
The N
terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 146 amino acids, which corresponds to a theoretical molecular mass of 15 313 Da and a theoretical pl of 5.60. The observed mass in SDS-PAGE
is 16 kDa.

The highest sequence identity, 32% in a 34 amino acid overlap, was found to a conserved protein of Methanobacterium thermoautotrophicum.

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.

For the 15 determined N-terminal amino acids for TB24 a 92% identical sequence was found in MTCY07D11.03. The tentative N in position 13 of the determined amino acid sequence is a Q in MTCY07D11.03, and the A at position 15 is a G.
Within the open reading frame the translated protein is 216 amino acids long.
The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 216 amino acids, which corresponds to a theoretical molecular mass of 24 227 Da and a theoretical 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.
55% in a 72 amino acid overlap to ECF sigma factor RpoE1 from Myxococcus xanthus.

For the 15 determined N-terminal amino acids for TB27B a 100% identical sequence was found in MTCY017.23c.
Within the open reading frame the translated protein is 257 amino acids long.
The N-terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 257 amino acids, which corresponds to a theoretical molecular mass of 27 276 Da and a theoretical pl of 4.82. The observed mass in SDS-PAGE
is 28 kDa.

WO 00121983 PCTlDK99/00538 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: 50) Sequence identity search on the TB13A N-terminal sequence gave the following results:

For the 15 determined N-terminal amino acids for TB13A a 100% identical sequence was found in MTCY427.25.
Within the open reading frame the translated protein is 115 amino acids long.
The N
terminal sequence of the protein identified in the cytosol starts at amino acid no 2, with the N-terminal Met cleaved off.
This gives a protein of 115 amino acids, which corresponds to a theoretical molecular mass of 12 524 Da and a theoretical pl of 3.87. The observed mass in SDS-PAGE
is 10 kDa.
TB13A has 94% sequence identity to a 115 amino acid protein of M. leprae. It is homologous to putative acyl carrier proteins from several species, e.g. 59%
sequence identity to a 78 amino acid protein of Myxococcus xanthus and 56% to a 82 amino acid protein from Streptomyces coelicolor.
Identification of TB64 Biotinylated proteins were purified from the cytosol fraction in the following way: 12 mg of the cytosol fraction was added to 100 p.l of TetraLink Tetrameric Avidin Resin (Promega) in PBS, pH 7.4 in an eppendort tube. After incubation over night at 4°C, centrifugation (1000 g for 5 min) was performed and the resin was washed five times with PBS, pH 7.4, each time followed by centrifugation and collection of the supernatant.
Thereafter, 100 NI
of 4 times concentrated SDS-PAGE sample buffer (0.08 M Tris-HCI, 8% SDS, 16%

glycerol, 24 mM EDTA , pH 8.0) was added to the resin and it was boiled for 20 minutes.
After centrifugation the supernatant was collected and analysed for the presence of biotinylated proteins: The sample was analysed on SDS-PAGE followed by semi-dry blotting to nitrocellulose. The nitrocellulose membranes were incubated with alkaline 5 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 10 blotted to Problott PVDF membrane (Applied Biosystems, Foster City, CA) by semidry electroblotting in 10 mM CAPS, 10% methanol, pH 11. The PVDF membrane was stained with 0.1 % Coomassie R-250 in 40% methanol, 1 % acetid acid, and destained in 50% methanol. A band of 10 kDa which was identified as a biotinylated protein as described above was excised and subjected to N-terminal sequence analysis by 15 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) 20 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 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 25 the protein: GDLVVVLEAMKMENPVTA (residues 556-573, PROSITE pattern PS00188).
EXAMPLE 4: Identification of proteins from the cell wall.
Identification of TB11 B, TB16, TB16A, TB32, TB32A, and TB51.
Proteins contained in the cell wall fraction were separated by 2-D
electrophoresis. A
30 sample containing 120 mg protein was subjected to isoelectric focusing in a pH gradient from 4 to 7. The second dimension separation (SDS-PAGE) was carried out in a 10-20%
acrylamide gradient. After blotting onto a PVDF membrane, proteins could be visualised by Coomassie blue staining.

N-terminal sequencing.
The relevant spots were excised from the PVDF membrane and subjected to N-terminal sequencing using a Procise sequences (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: 55) TB51:MKSTVEQLSPTRVRI (SEQ ID NO: 56) N-terminal sequence identity searching and identification of the corresponding genes.
The N-terminal amino acid sequence from each of the proteins identified was used for a sequence identity search using the tblastn program at NCBI:
http:/lwww. ncbi. nl m. nih.govlcgi-bin/BLAST/nph-blast?Jform=0 The following information was obtained:
TB11 B:
The 14 as 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 as 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.

The amino acid sequence shows some similarity to other hypothetical Mycobacterial proteins.
TB16A:
The 15 as 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 pl of 4.44.
The apparent molecular mass in an SDS-PAGE gel is 14 kDa.
TB32:
The 14 as 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 as N-terminal sequence was found to be 100% identical to a sequence found within the Mycobacterium tuberculosis sequence MTV012.
20 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 far various dehydrogenases. Equivalent to Mycobacterium leprae FixB.
TB51:
The 15 as 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 pl of 4.3. The apparent molecular mass in an SDS-PAGE gel is 56 kDa.
The amino acid sequence shows similarities to trigger factor from several organisms.
Possible chaperone protein.

EXAMPLE 5: Cloning of the genes encoding TB10C, TB13A, TB17, TB11 B, TB16, TB16A, TB32, TB51 The genes encoding TB10C, TB13A, TB17, TB11 B, TB16, TB16A, TB32, TB51 were all cloned into the E. coli expression vector pMCT3, by PCR amplification with gene specific primers.
Each PCR reaction contained 10 ng of M. tuberculosis chromosomal DNA in 1x low salt Taq+ buffer (Stratagene) supplemented with 250 pM 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 pl reaction volume.
Reactions were initially heated to 94°C for 25 sec. and run for 30 cycles according to the following program; 94°C for 10 sec., 55°C for 10 sec., and 72°C for 90 sec., using thermocycler equipment from Idaho Technology.
The PCR fragment was ligated with TA cloning vector pCR~ 2.1 (Invitrogen) and transformed into E. coli. Plasmid DNA was thereafter prepared from clones harbouring the desired fragment, digested with suitable restriction enzymes and subcloned into the expression vector pMCT3 in frame with 6 histidine residues which are added to the N-terminal of the expressed proteins. The resulting clones were hereafter sequenced by cycle sequencing using the Dye Terminator system in combination with an automated gel reader (model 373A; Applied Biosystems) according to the instructions provided. Both strands of the DNA were sequenced.
Expression and metal affinity purification of recombinant proteins was undertaken essentially as described by the manufacturers. For each protein, 1 1 LB-media containing 100 pg/ml ampicillin, was inoculated with 10 ml of an overnight culture of XL1-Blue cells harbouring recombinant pMCT3 plasmids. Cultures were shaken at 37°C
until they reached a density of ODsoo= 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 OD28o gym. Fractions containing recombinant protein 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 OD28onm.
Fractions containing protein were pooled and dialysed against 25 mM Hepes buffer, pH
8.5.
Finally, the protein concentration and the LPS content were determined by the BCA
(Pierce, Holland) and LAL (Endosafe, Charleston, USA) tests, respectively.
For cloning of the individual proteins, the following gene specific primers were used TB10C : Primers used for cloning of TB10C
TB10C-F : CTG AGA TCT GTG GAG GTC AAG ATC GGT
(SEQ ID NO: 58) TB10C-R : CTC CCA TGG CTAC TTA CCC GCT CGT AGC AAC (SEQ ID NO: 59) TB10C-F and TB10C-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.
TB13A : Primers used for cloning of TB13A
TB13A-F : CTG AGA TCT CCT GTC ACT CAG GAA GAA
(SEQ ID NO: 60) TB13A-R : CTC CCA TGG GAA ACC GCC ATT AGC GGT
(SEQ ID NO: 61) TB13A-F and TB13A-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.
TB17 : Primers used for cloning of TB17 TB17-F : CCC AAG CTT ATG GAA CAG CGT GCG GAG
(SEQ ID NO: 62) TB17-R : CTC CCA TGG CGA CAC TCG ATC CGG ATT (SEQ ID NO: 63) TB17-F and TB17-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.
TB11 B : Primers used for cloning of TB11 B

TB11 B-F : CTG AGA TCT ATG CCA GTG GTG AAG ATC
{SEQ ID NO: 64) TB11 B-R : CTC CCA TGG TTA TGC AGT CTT GCC GGT (SEQ ID NO: 65) TB11B-F and TB11B-R create BG/II and Ncol sites, respectively, used for the cloning in 5 pMCT3.
TB16 : Primers used for cloning OF TB16 TB16-F : CTG AGA TCT GCG GAC AAG ACG ACA CAG
(SEQ ID NO: 66) TB16-R : CTC CCA TGG TAC CGG AAT CAC TCA GCC {SEQ ID NO: 67) TB16-F and TB16-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.
TB16A : Primers used for cloning of TB16A
TB16A-F : CTG AGA TCT CCA GTT TTG AGC AAG ACC {SEQ ID NO: 68) TB16A-R : CTC CCA TGG GCA CAT GCC TTA GCT GGC
(SEQ ID NO: 69) TB16A-F and TB16A-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.
TB32 : Primers used for cloning of TB32 TB32-F : CTG AGA TCT ATG TCA TCG GGC AAT TCA (SEQ ID NO: 70) TB32-R : CTC CCA TGG CTAC CTA AGT CAG CGA CTC GCG (SEQ ID NO: 71) TB32-F and TB32-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.
TB51 : Primers used for cloning of TB51 TB51-F : CTG AGA TCT GTG AAG AGC ACC GTC GAG
(SEQ ID NO: 72) TB51-R : CTC CCA TGG GTC ATA CGG TCA CGT TGT (SEQ ID NO: 73) TB51-F and TB51-R create BG/II and Ncol sites, respectively, used for the cloning in pMCT3.
TB15A: Primers used for cloning of TB15A:
TB15A-F: CTG CCA TGG CTA GGT GGT GTG CAC GAT C
(SEQ ID NO: 89) TB15A-R: CTG AAG CTT ATG AGC GCC TAT AAG ACC
{SEQ ID NO: 90) TB15-F and TB15-R create Ncol and Hindlll sites, respectively, used for the cloning in pMCT3.
TB21: Primers used for cloning of TB21:
TB21-F: CTG AGA TCT ATG ATT GAT GAGGCT CTC
(SEQ ID NO: 91 ) TB21-R: CTC CCA TGG AGC GGC CGC TAG ACC TCC (SEQ ID NO: 92) TB21-F and TB21-R create Bglll and Ncol sites, respectively, used for the cloning in pMCT3.
TB24: Primers used for cloning of TB24:
TB24-F: GGCTGAGACTC ATG GCC GAC ATC GAT GGT G
(SEQ ID NO: 93) TB24-R: CGTACCATGG TCA TGA CGA CAC CCC CTC GTG (SEQ ID NO: 94) TB24-F and TB24-R create Bglll and Ncol sites, respectively, used for the cloning in pMCT3.
TB32A: Primers used for cloning of TB32A:
TB32A-F: GGCTGAGACTC ATG GCT GAA GTA CTG GTG C (SEQ ID NO: 95) TB32A-R: CGTACCATGGCTA GCC GGC GAC CGC CGG TTC (SEQ ID NO: 96) TB32A-F and TB32A-R create Bgll I and Ncol sites, respectively, used for the cloning in pMCT3.

TB14: Primers used for cloning of TB14:
TB14-F: 5'-GTG ACC GAA CGG ACT CTG GT-3' (SEQ ID NO: 97) TB14-R: 5'-CTA GGC GCC GGG AAA CCA GAG-3' (SEQ ID NO: 98) TB18: Primers used for cloning of TB18:
TB18-F: 5'-ATG ACG GAT ACT CAA GTC ACC TG-3"
(SEQ ID NO: 99) TB18-R: 5'-GGA GTG GTA CGG CTC GGC GC-3' (SEQ ID NO: 100) T827: Primers used for cloning of TB27:
TB27-F: 5'-ATG ACG TAC GAA ACC ATC CT-3' (SEQ ID NO: 101) TB27-R: 5'-TCA TCG GTG GGT GAA CTG GGG-3' (SEQ ID NO: 102) TB33: Primers used for cloning of TB33:
TB33-F: 5'-ATG CCG CTT CCC GCA GAC CCT AG-3' (SEQ ID NO: 103) TB33-R: 5'-TAC GAC GGG TAC CAC TCC TGG-3' (SEQ ID NO: 104) TB38: Primers used for cloning of TB38:
TB38-F: 5'-ATG CTG ATC TCA CAG CGC CCC A-3' (SEQ ID NO: 105) TB38-R: 5'-AAG CTG TTC GGT TTC GGC GTA G-3' (SEQ ID NO: 106) TB54: Primers used for cloning of TB54:
TB54-F: 5' -ATG ACC GGA AAT TTG GTG AC-3' (SEQ ID NO: 107) TB54-R: 5'-TCA GTA GCG GTA GTG GTC CGG-3' (SEQ ID NO: 108) TB14,TB18,TB27,TB33,TB38 and TB54 will be cloned in ex-pressions vector pBAD-TOPO (Invitrogen).
Example 5a: Cloning of the genes encoding TB12.5, TB20.6, and TB40.8 The genes encoding TB12.5, TB20.6, and TB40.8 were all cloned into the E. coli expression vector pMCT3 as described in Example 5.

For cloning of the individual genes, the following gene specific primers were used:
TB12.5: Primers used for cloning of TB12.5:
5 TB12.5-F: CTG AGA TCT ATG GCA CTC AAG GTA GAG (SEQ ID NO: 83) TB12.5-R: CTC CCA TGG TTA TTG ACC CGC CAC GCA
(SEQ ID NO: 84) TB12.5-F and TB12.5-R create Bglll 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
(SEQ ID NO: 85) TB20.6-R: CTC CCA TGG CTA GTC GCG GAG CAC AAC
(SEQ ID NO: 86) TB20.6-F and TB20.6-R create 8glll 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 (SEQ ID NO: 87) TB40.8-R: CTC CCA TGG TCA CGT CTT CCA GCG GGT
(SEQ ID NO: 88) TB40.8-F and TB40.8-R create Bglll and Ncol sites, respectively, used for the cloning in pMCT3.
Expressionlpurification of recombinant proteins was performed as described in Example 5.
EXAMPLE 6: Evaluation of immunological activity of identified somatic proteins.
Each of the proteins identified in either the cell wall, cytosol or the cell membrane derived from M.tuberculosis will be evaluated for the immunological recognition in M.
tuberculosis infected animals or in TB patients.

IFN-~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 (H-2b), CBA/J (H-2k), DBA.2 (H-2d) and A.SW (H-2g) mice (Bomholtgaard, Ry, Denmark) will be infected i.v. via the lateral tail vein with an inoculum of 5 x 104 M.tuberculosis suspended in PBS in a vof. 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 104 M. tuberculosis i.v. After 30 days of infection the mice will be subjected to 60 days of antibiotic treatment with isoniazid (Merck and Co., Rahway, NJ) and rifabutin (Farmatalia Carlo Erba, Milano, Italy) then left for 200-240 days to ensure the establishment of resting long-term memory immunity. Such memory immune mice are very efficient protected against a secondary infection (Orme; Andersen, Boom 1993, J. Infect.Dis. 167:

1497). Long lasting immunity in this model is mediated by a population of highly reactive CD4 cells recruited to the site of infection and triggered to produce large amounts of IFN-y 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 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~ in response to mycobacterial antigens 5 (unpublished, manus in prep) indicative of early, subclinical infection.
Together these findings indicate that those individuals who are relatively protected (i.e.
minimal TB
patients) respond with high levels of IFN-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 10 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 15 >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 105 PBMC in 2001 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.
20 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 25 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 30 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 ELISAIVIIestern 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 35 material.

The diagnostic potential of polypeptides will initially be evaluated in 10 individuals with TB
infection and 10 individuals with no known exposure to virulent Mycobacteria.
High specificity, >80% ,will be the most important selection criteria for these polypeptides and a sensitivity >80% is desirable but sensitivity >30% is acceptable as combinations of several specific antigens may be preferred in a cocktail of diagnostic reagent recognised by different individuals.
Skin test reaction in TB infected guinea pigs To identify polypeptides as antigens with the potential as TB diagnostic reagents the ability of the proteins to induce a skin test response will be evaluated in the guinea pig model where groups of guinea pigs have been infected with either M.
tuberculosis or M.avium or vaccinated with BCG.
To evaluate the response in M.tuberculosis infected guinea pigs, female outbred guinea pigs will be infected via an ear vein with 1 x 104 CFU of M. tuberculosis H37Rv in 0.2 ml of PBS or aerosol infected (in an exposure chamber of a Middlebrook Aerosol Generation device) with 1x 105 CFU/ml of M.tuberculosis Erdman given rise to 10-15 granulomas per animal in the lung. After 4 weeks skin test will be performed with the polypeptides diluted in 0.1 ml of PBS and 24 hours after the injection reaction diameter is measured.
To evaluate the response in M.avium infected guinea pigs, female outbred guinea pigs will be infected intradermally with 2 x 106 CFU of a clinical isolate of M.avium (Atyp.1443;
Statens Serum Institut, Denmark). Skin test are performed 4 weeks after with the polypeptides diluted in 0.1 ml of PBS and 24 hours after the injection reaction diameter is measured.
To evaluate the response in BCG vaccinated guinea pigs, female outbred guinea pigs will be sensitized intradermally with 2 x 106 CFU of BCG (BCG Danish 1331; Statens Serum Institut). Skin test are performed 4 weeks after with the polypeptides diluted in 0.1 ml of PBS and 24 hours after the injection reaction diameter is measured.
If a polypeptide induces a significant reaction in animal infected with M.tuberculosis but not in BCG vaccinated guinea pigs this polypeptide may have a potential as a diagnostic reagent to differentiate between BCG vaccinated and M.tuberculosis infected individuals, which will hereafter be evaluated in the human population.

If a polypeptide induces a reaction in M.tuberculosis infected guinea pigs but not in guinea pigs infected with M.avium, this polypeptide may have a potential as a diagnostic reagent with respect to differentiate between an individual infected with M.
tuberculosis and an individual infected with Mycobacteria not belonging to the tuberculosis complex.
The polypeptide may also have a potential as a diagnostic reagent to differentiate between a M.avium and a M.tuberculosis infected individual.
Induction of protective immunity by the recombinant proteins in the mice model.
The recombinant polypeptides will be evaluated as immunological compositions in mice.
Female C57BLI6j 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-fi 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, N.Y.) supplemented with 1 % penicillinlstreptomycin (Gibco BRL, Life Technologies), 1 % non-essential-amino acids (FLOW, ICN Biomedicals, CA, USA), and 10% normal human ABO serum (NHS) from the local blood bank. The number and the viability of the cells were determined by Nigrosin staining. Cultures were established with 1.25 x 105 PBMCs in 100 pl in microtitre plates (Nunc, Roskilde, Denmark) and stimulated with ST-CF (5pg/ml), TB13A, TB15A, TB17, TB18, TB33, TB11 B, TB16A, TB16, TB32, and TB51 in a final concentration of 10 pg/ml. No antigen and phytohaemagglutinin (PHA) were used as negative and positive control, respectively.
Supernatants for the detection of cytokines were harvested after 5 days of culture, pooled, and stored at -80°C until used.
Cytokine analysis: Interferon-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 10, 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 pglml). 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.

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 PHA ST-CF TB13A
ag (1 ~g/ml) (5 ~g/ml) (10 ~g/ml) TB patients DonorNo PHA ST-CF TB13A
ag (1 ~glml) (5 ug/ml) (10 ~g/ml) 2 51 10058 64$9 0 Table 4 Stimulation of PBMCs from 6 healthy BCG vaccinated and 5 TB patients with recombinant TB15A. Responses to ST-CF and shown for comparison.
PHA are Results are given as pg IFN-y/ml.

BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB15A

(1 ug/ml) (5 ~g/ml) (10 ~g/ml) TB patients Donor No ag PHA ST-CF TB15A

(1 ~g/ml) (5 ~g/ml) (10 ~g/ml) Table 5 Stimulation of PBMCs from 6 healthy BCG vaccinated with recombinant TB17.

Responses to ST-CF and PHA are shown . Results are given for comparison as pg IFN-y/ml BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB17 {1 ~g/ml) {5 ~g/ml) (10 ~g/ml) 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-ylml BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB18 (1 ~g/ml) (5 uglml) (10 ~g/ml) TB patients Donor No ag PHA (1 ST-CF(5 TB18 (10 u9lml) pglml) ~.glml) 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.
5 BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB33 {1 ~g/ml) (5 ~g/ml)(10 ~g/ml) TB patients Donor No ag PHA ST-CF TB33 (1 ~g/ml) (5 ~g/ml) (10 ~g/ml) s3 Table 8 Stimulation of PBMCs from 3 healthy BCG vaccinated and 3 TB patients with recombinant TB11 B. Responses to ST-CF and PHA are shown for comparison.
Results are given as pg IFN-y/ml.
BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB11 B
(1 ~glml) (5 ~g/ml) (10 ~glml) TB patients Donor No ag PHA ST-CF TB11 B
(1 ~g/ml) (5 ~glml) (10 ~g/ml) 10 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 ~glml) (5 ~g/ml) (10 ~glml) TB patients Donor No ag PHA ST-CF TB16A

(1 ~glml) (5 ~glml) (10 ~glml) 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 oa IFN-Donor No ag PHA ST-CF TB16 (1 ~g/ml) (5 ~.g/ml) (10 ~g/ml) 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.
Donor No ag PHA ST-CF TB32 (1 ~glml) (5 ~g/ml) (10 ~g/ml) TB patients Donor No ag PHA ST-CF TB32 (1 ~g/ml) (5 ~g/ml) (10 ~g/ml) Table 12, Stimulation of PBMCs from 6 healthy BCG vaccinated with recombinant TB51.
Responses to ST-CF and PHA are shown for comparison. Results are given as pg IFN-y/ml.
5 BCG vaccinated control donors, no known TB exposure Donor No ag PHA ST-CF TB51 (1 ~g/ml) (5 ~glml) {10 ~g/ml) ,. " . """. . . -."., . ...

Figure legends:
Figure 1:
Long term protection against TB can be induced by immunisation with dead M. tuberculosis.
5 Mice received either: three immunisations with 1x10' CFU of dead M.tuberculosis H37Rv (squares); three immunisations with 50 pg of ST-CF (triangles); one immunisation with 5 x 104 CFU of live M.tuberculosis H37Rv (circle) and was hereafter cleared for the infection by administration of isoniazid in the drinking water. At 3, 6 and 12 month after the last immunisation the mice received an infection with M. tuberculosis H37Rv and two 10 weeks later the bacterial load and the resistance against TB in the spleens were determined.
Figure 2:
Mice received three immunisations with 50~,g of either of the three vaccines:
heat killed H37Rv, SPE or ST-CF or received a vaccination with BCG. Two weeks after a primary 15 infection the bacterial load in the spleen was used to determined the resistance against TB.

SEQ(JENCE 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 3.0 <210> 1 <211> 273 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(270) <400> 1 gtggag gtcaagatcggt atcacg gacagtccg cgcgagctg gtgttc 48 ValGlu ValLysIleGly IleThr AspSerPro ArgGiuLeu ValPhe tccagt gcgcagacgccc agtgag gtagaagaa ctcgtcagc aacgcg 96 SerSer AlaGlnThrPro SerGlu ValGluGlu LeuValSer AsnAla ctgcgc gacgactctggt ttgctg accctgacc gacgagcgg ggccgt 149 LeuArg AspAspSerGly LeuLeu ThrLeuThr AspGluArg GlyArg cgcttc ctaattcacacc gccagg atcgcctat gtcgagatc ggtgtc 192 ArgPhe LeuIleHisThr AlaArg IleAlaTyr ValGluIle GlyVal gcagac gcccgccgggtg ggcttc ggcgtcggg gtggacgcc gcaget 240 AlaAsp AlaArgArgVal GlyPhe GlyValGly ValAspAla AlaAla gggtcc gecggaaaggtt getacg agcgggtaa 273 GlySer AlaGlyLysVal AlaThr SerGly <210> 2 <211> 90 <212> PRT
<213> M.Tuberculosis <400> 2 Met Glu Val Lys Ile Gly Ile Thr Asp Ser Pro Arg Glu Leu Val Phe Ser Ser Ala Gln Thr Pro Ser Glu Val Glu Glu heu Val Ser Asn Ala Leu Arg Asp Asp Ser Gly Leu Leu Thr Leu Thr Asp Glu Arg Gly Arg ArgPheLeuIle Thr AlaArgIle Tyr Val Glu Ile Gly Val His Ala AiaAspAlaArg Val GlyPheGly Gly Val Asp Ala Ala Ala Arg Val GlySerAlaGly Val AlaThrSer Lys Gly <210> 3 <211> 348 <212> DNA
<213> M.Tuberculosis <220>

<221> CDS

<222> (1)...(345) <400> 3 gtgcct actcaggaa gaaatcatt gccggtatc gccgag atcatc 48 gtc ValPro ThrGlnGlu GluIleIle AlaGlyIle AlaGlu IleIle Val gaagag accggtatc gagccgtcc gagatcacc ccggag aagtcg 96 gta GluGlu ThrGlyIle GluProSer GluIleThr ProGlu LysSer Val ttcgtc gacctggac atcgactcg ctgtcgatg gtcgag atcgcc 144 gac PheVal AspLeuAsp IleAspSer LeuSerMet ValGlu IleAla Asp gtgcag gaggacaag tacggcgtc aagatcccc gacgag gacctc 192 acc ValGln GluAspLys TyrGlyVal LysIlePro AspGlu AspLeu Thr gccggt cgtaccgtc ggtgacgtt gtcgcctac atccag aagctc 240 ctg AlaGly ArgThrVal GlyAspVal ValAlaTyr IleGln LysLeu Leu gaggaa aacccggag gcggetcag gcgttgcgc gcgaag attgag 288 gaa GluGlu AsnProGlu AlaAlaGln AlaLeuArg AlaLys IleGlu Glu tcggag cccgatgcc gttgccaac gttcaggcg aggctt gaggcc 336 aac SerGlu ProAspAla ValAlaAsn ValGlnAla ArgLeu GluAla Asn gagtcc tga aag GluSer Lys <210> 9 <211> 115 <212> PRT

<213> M.Tuberculo sis <400> 4 MetPro ThrGlnGlu GluIleIle AlaGlyIle AlaGlu IleIle Val GluGlu ThrGlyIle GluProSer Glu Thr ProGlu LysSer Val Ile Phe Val Asp Asp Leu Asp Ile Asp Ser Leu Ser Met Val Glu Ile Ala Val Gln Thr Glu Asp Lys Tyr Gly Val Lys Ile Pro Asp Glu Asp Leu Ala Gly Leu Arg Thr Val Gly Asp Val Val Ala Tyr Ile Gln Lys Leu Glu Glu Glu Asn Pro Glu Ala A1a Gln Ala Leu Arg Ala Lys Ile Glu Ser Glu Asn Pro Asp Ala Val Ala Asn Val Gln Ala Arg Leu Glu Ala Glu Ser Lys <210>5 <211>411 <212>DNA

<213>M.Tuberculosis <220>

<221>CDS

<222>(1)...(408) <400> 5 gtgaccgaa cggactctg gtactgatc aagccggat ggcatcgaa agg 48 ValThrGlu ArgThrLeu ValLeuIle LysProAsp GlyIleGlu Arg cagctgatc ggcgagatc atcagccgc atcgagcgc aaaggcctc acc 96 GlnLeuIle GlyGluIle IleSerArg IleGluArg LysGlyLeu Thr atcgetgcg ctgcagctc aggaccgtc agcgcggag ttggccagc cag 144 IleAlaAla LeuGlnLeu ArgThrVal SerAlaGlu LeuAlaSer Gln cactacgcc gaacatgaa ggcaaacca ttctttgga tcgttgctg gag 192 HisTyrAla GluHisGlu GlyLysPro PhePheGly SerLeuLeu Glu 50 55 fi0 ttcatcacg tcgggtccg gtggtagcg gcgatcgt:ggagggaacc cga 240 PheIleThr SerGlyPro ValValAla AlaIleVal GluGlyThr Arg gccatcgcg gcggttcgc caactcgcc ggcggcacc gacccggtg cag 288 AlaIleAla AlaValArg GlnLeuAla GlyGlyThr AspProVal Gln gcggcggcg cccggcaca atccggggc gacttcget ctagagacg cag 336 AlaAlaAla ProGlyThr IleArgGly AspPheA:LaLeuGluThr Gln ttcaacctg gtgcacggg tctgattcg gccgaatcc gcgcagcgc gaa 384 PheAsnLeu ValHisGly SerAspSer AlaGluSer AlaGlnArg Glu atcgcgctc tggtttccc ggcgcctag 411 IleAlaLeu TrpPhePro GlyAla <210> 6 <211> 136 <212> PRT
<213> M.Tuberculosis <400> 6 Met Thr Glu Arg Thr Leu Val Leu Ile Lys Pro Asp Gly Ile Glu Arg Gln Leu Ile Gly Glu Ile Ile Ser Arg Ile Glu Arg Lys Gly Leu Thr Ile Ala Ala Leu Gln Leu Arg Thr Val Ser Ala Glu Leu Ala Ser Gln His Tyr Ala Glu His Glu Gly Lys Pro Phe Phe Gly Ser Leu Leu Glu Phe Ile Thr Ser Gly Pro Val Val Ala Ala Ile Val Glu Gly Thr Arg Ala Ile Ala Ala Val Arg Gln Leu Ala Gly Gly Thr Asp Pro Val Gln Ala Ala Ala Pro Gly Thr Ile Arg Gly Asp Phe Ala Leu Glu Thr Gln Phe Asn Leu Val His Gly Ser Asp Ser Ala Glu Ser Ala Gln Arg Glu Ile Ala Leu Trp Phe Pro Gly Ala <210> 7 <211> 941 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(438) <400> 7 atgagcgcc tataagacc gtggtggta ggaaccgac ggttcggac tcg 48 MetSerAla TyrLysThr ValValVal GlyThrAsp GlySerAsp Ser tcgatgcga gcggtagat cgcgetgcc cagatcgcc ggcgcagac gcc 96 SerMetArg AlaValAsp ArgAlaAla GlnIleAla GlyAlaAsp Ala aagttgatc atcgcctcg gcataccta cctcagcac gaggacget cgc 144 LysLeuIle IleAlaSer AlaTyrLeu ProGlnHis GluAspAla Arg gccgccgac attctgaag gacgaaagc tacaaggtg acgggcacc gcc 192 AlaAlaAsp IleLeuLys AspGluSer TyrLysVal ThrGlyThr Ala ccgatctac gagatcttg cacgacgcc aaggaacga gcgcacaac gcc 240 ProIleTyr GluIleLeu HisAspAla LysGluArg AlaHisAsn Ala ggtgcgaaa aacgtcgag gaacggccg atcgtcggc gccccggtc gac 288 GlyAlaLys AsnValGlu GluArgPro IleValGly AlaProVal Asp gcgttggtg aacctggcc gatgaggag aaggcggac ctgctggtc gtc 336 AlaLeuVal AsnLeuAla AspGluGlu LysAlaAsp LeuLeuVal Val ggc aat gtc ggt ctg agc acg atc gcg ggt cgg ctg ctc gga tcg gta 384 Gly Asn Val Gly Leu Ser Thr Ile Ala Gly Arg Leu Leu Gly Ser Val ccg gcc aat gtg tca cgc cgg gcc aag gtc gac gtg ctg atc gtg cac 432 Pro Ala Asn Val Ser Arg Arg Ala Lys Val Asp Val Leu Ile Val His acc acc tag 441 Thr Thr <210> 8 <211> 146 <212> PRT
<213> M.Tuberculosis <900> 8 Met Ser Ala Tyr Lys Thr Val Val Val Gly Thr Asp Gly Ser Asp Ser Ser Met Arg Ala Val Asp Arg Ala Ala Gln Ile Ala Gly Ala Asp Ala Lys Leu Ile Ile Ala Ser Ala Tyr Leu Pro Gln His Glu Asp Ala Arg Ala Ala Asp Ile Leu Lys Asp Glu Ser Tyr Lys Val Thr Gly Thr Ala Pro Ile Tyr Glu Ile Leu His Asp AIa Lys Glu Arg Ala His Asn Ala Gly Ala Lys Asn Val Glu Glu Arg Pro Ile Val Gly Ala Pro Val Asp Ala Leu Val Asn Leu Ala Asp Glu Glu Lys Ala Asp Leu Leu Val Val Gly Asn Val Gly Leu Ser Thr Ile Ala Gly Arg Leu Leu Gly Ser Val Pro Ala Asn Val Ser Arg Arg Ala Lys Val Asp Val Leu Ile Val His Thr Thr <210> 9 <211> 998 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(495) <400> 9 atg gaa cag cgt gcg gag ttg gtg gtt ggc cgg gca ctt gtc gtc gtc 48 Met Glu Gln Arg Ala Glu Leu Val Val Gly Arg Ala Leu Val Val Val gtt gac gat cgc acg gcg cac ggc gat gaa gac cac agc ggg ccg ctt 96 Val Asp Asp Arg Thr Ala His Gly Asp Glu Asp His Ser Gly Pro Leu gtc acc gag ctg ctc acc gag gcc ggg ttt gtt gtc gac ggc gtg gtg 149 Val Thr Glu Leu Leu Thr Glu Ala Gly Phe Val Val Asp Gly Val Val gcg gtg tcg gcc gac gag gtc gag atc cga aat gcg ctg aac aca gcg 192 Ala Val Ser Ala Asp Glu Val Glu Ile Arg Asn Ala Leu Asn Thr Ala gtg atc ggc ggg gtg gac ctg gtg gtg tcg gtc ggc ggg acc ggg gtg 240 Val Ile Gly Gly Val Asp Leu Val Val Ser Val Gly Gly Thr Gly Val acg cctcgcgat gtcaccccg gaagccacc cgcgac attctggaccgc 288 Thr ProArgAsp ValThrPro GluAlaThr ArgAsp IleLeuAspArg gag atcctcggt atcgccgag gccatccgc gcgtcc gggctgtccgcg 336 Glu IleLeuGly IleAlaGlu AlaIleArg AlaSer GlyLeuSerAla gga atcgtcgac gccgggttg tcgcgcggc ctggcg ggtgtctccggc 384 Gly IleValAsp AlaGlyLeu SerArgGly LeuAl.aGlyValSerGly agc acgctggtg gtcaacctc gcgggttcg cgttat gcggtgcgcgat 432 Ser ThrLeuVal ValAsnLeu AlaGlySer ArgTyr AlaValArgAsp gga atggcgacg ctgaatccg ctagcggca cagat:catcgggcagttg 480 Gly MetAlaThr LeuAsnPro LeuAlaAla GlnIle IleGlyGlnLeu tcg agcttggag atctga 498 Ser SerLeuGlu Ile <210> 10 <211> 165 <212> PRT
<213> M.Tuberculosis <400> 10 Met Glu Gln Arg Ala Glu Leu Val Val Gly Arg Ala Leu Val Val Val Val Asp Asp Arg Thr Ala His Gly Asp Glu Asp H:is Ser Gly Pro Leu Val Thr Glu Leu Leu Thr Glu Ala Gly Phe Val Val Asp Gly Val Val Ala Val Ser Ala Asp Glu Val Glu Ile Arg Asn Ala Leu Asn Thr Ala Val Ile Gly Gly Val Asp Leu Val Val Ser Val Gly Gly Thr Gly Val Thr Pro Arg Asp Val Thr Pro Glu Ala Thr Arg Asp Ile Leu Asp Arg Glu Ile Leu Gly Ile Ala Glu Ala Ile Arg Ala Ser Gly Leu Ser Ala Gly Ile Val Asp Ala Gly Leu Ser Arg Gly Leu Ala Gly Val Ser Gly Ser Thr Leu Val Val Asn Leu Ala Gly Ser Arg Tyr Ala Val Arg Asp Gly Met Ala Thr Leu Asn Pro Leu Ala Ala Gln Ile Ile Gly Gln Leu SerSer Glu Ile Leu <210> 11 <211> 495 <212> DNA

<213> M.Tuberculosis <220>

<221> CDS

<222> (1)...(992) <400> 11 atgacg act caagtcacc tggttgacc caagagtca catgac cga 48 gat MetThr Thr GlnValThr TrpLeuThr GlnGluSer HisAsp Arg Asp ctcaaa gag ctcgaccag ctgattgcg aatcgcccg gtcatc gcc 96 gca LeuLys Glu LeuAspGln LeuIleAla AsnArgPro ValIle Ala Ala gccgaa aac gaccgccgc gaagaaggc gacctgcgc gagaac ggc 199 atc AlaGlu Asn AspArgArg GluGluGly AspLeuArg GluAsn Gly Ile ggatac gcc gcccgcgag gagcagggc cagcaggag gcccgc att 192 cac GlyTyr Ala AlaArgGlu GluGlnGly GlnGl.nGlu AlaArg Ile His cgccag cag gacttgctc agcaacgca aaggttggc gaggca ccc 240 ctg ArgGln Gln AspLeuLeu SerAsnAla LysValGly GluAla Pro Leu aagcaa ggc gtcgcatta cccggttct gtggtcaag gtgtac tac 288 tcc LysGln Gly ValAlaLeu ProGlySer ValValLys ValTyr Tyr Ser aacggc aag tcggacagc gaaacgttc ctcat:cgcc acccgc cag 336 gac AsnGly Lys SerAspSer GluThrPhe LeuIleAla ThrArg Gln Asp gagggc agc gacggcaag ctcgaggtc tactcgccg aattca ccg 384 gtc GluGly Ser AspGlyLys LeuGluVal TyrSerPro AsnSer Pro Val ctcggt gcc ctgatcgac gccaaggtc ggcgagacc cgcagc tac 932 ggg LeuGly Ala LeuIleAsp AlaLysVal GlyGluThr ArgSer Tyr Gly acggtg aac ggcagcacc gtgtcggtg accctagtc agcgcc gag 480 ccc ThrVal Asn GlySerThr ValSerVal ThrLeuVal SerAla Glu Pro ccgtac tcc tag 495 cac ProTyr Ser His <210> 12 <211> 164 <212> PRT

<213> M.Tuberculosis <400> 12 MetThr AspThrGln ValThrTrp LeuThrGln GluSerHis AspArg LeuLys AlaGluLeu AspGlnLeu IleAlaAsn ArgProVal IleAla AlaGlu IleAsnAsp ArgArgGlu GluGlyAsp LeuArgGlu AsnGly GlyTyr HisAlaAla ArgGluGlu GlnGlyGln GlnGluAla ArgIle ArgGln LeuGlnAsp LeuLeuSer AsnAlaLys ValGlyGlu AlaPro LysGln SerGlyVal AlaLeuPro Gly5erVal ValLysVal TyrTyr AsnGly AspLysSer AspSerGlu ThrPheLeu IleAlaThr ArgGln GluGly ValSerAsp GlyLysLeu GluValTyr SerProAsn SerPro LeuGly GlyAlaLeu IleAspAla LysValGly GluThrArg SerTyr ThrVal ProAsnGly SerThrVal SerValThr LeuValSer AlaGlu ProTyr HisSer <210> 13 <211> 558 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(555) <400> 13 atgatt gatgagget ctcttcgac gccgaa gagaaaatg gagaagget 48 MetIle AspGluAla LeuPheAsp AlaGlu GluLysMet GluLysAla gtggcg gtggcacgt gacgacctg tcaact atccgtacc ggccgcgcc 96 ValAla ValAlaArg AspAspLeu SerThr IleArgThr GlyArgAla aaccct ggcatgttc tctcggatc accatc gactactac ggtgcggcc 144 AsnPro GlyMetPhe SerArgIle ThrIle AspTyrTyr GlyAlaAla accccg atcacgcaa ctggccagc atcaat gtccccgag gcgcggcta 192 ThrPro IleThrGln LeuAlaSer IleAsn ValProGlu AlaArgLeu gtcgtg ataaagccg tatgaagcc aatcag ttgcgcget atcgagact 240 ValVal IleLysFro TyrGluAla AsnGln LeuArgAla IleGluThr gcaatt cgcaactcc gaccttgga gtgaat cccaccaac gacggcgcc 288 AlaIle ArgAsnSer AspLeuGly ValAsn ProThrAsn AspGlyAla cttatt cgcgtggcc gtaccgcag ctcacc gaagaacgt cggcgagag 336 Leu Ile Arg Val Ala Val Pro Gln Leu Thr Glu Glu Arg Arg Arg Glu ctggtcaaacag gcaaagcat aagggggag gaggccaag gtttcg gtg 384 LeuValLysGln AlaLysHis LysGlyGlu GluAlaLys ValSer Val cgtaatatccgt cgcaaagcg atggaggaa ctccatcgc atccgt aag 432 ArgAsnIleArg ArgLysAla MetGluGlu LeuHisArg IleArg Lys gaaggcgaggcc ggcgaggat gaggtcggt cgcgcagaa aaggat ctc 980 GluGlyGluAla GlyGluAsp GluValGly ArgAlaGlu LysAsp Leu gacaagaccacg caccaatac gtcacccaa attgatgag ctggtt aaa 528 AspLysThrThr HisGlnTyr ValThrGln IleAspGlu LeuVal Lys cacaaagaaggc gagctgctg gaggtctag 558 HisLysGluGly GluLeuLeu GluVal <210> 14 <211> 185 <212> PRT
<213> M.Tuberculosis <400> 19 Met Ile Asp Glu A1a Leu Phe Asp Ala Glu Glu Lys Met Glu Lys Ala Val Ala Val Ala Arg Asp Asp Leu Ser Thr Ile Arg Thr Gly Arg Ala Asn Pro Gly Met Phe Ser Arg Ile Thr Ile Asp Tyr Tyr Gly Ala Ala Thr Pro Ile Thr Gln Leu Ala Ser Ile Asn Val Pro Glu Ala Arg Leu Val Val Ile Lys Pro Tyr Glu Ala Asn Gln Leu Arg Ala Ile Glu Thr Ala Ile Arg Asn Ser Asp Leu Gly Val Asn Pro Thr Asn Asp Gly Ala Leu Ile Arg Val Ala Val Pro Gln Leu Thr Glu Glu Arg Arg Arg Glu Leu Val Lys Gln Ala Lys His Lys Gly Glu Glu Ala Lys Val Ser Val Arg Asn Ile Arg Arg Lys Ala Met Glu Glu Leu His Arg Ile Arg Lys Glu Gly Glu Ala Gly Glu Asp Glu Val Gly Arg Ala Glu Lys Asp Leu Asp Lys Thr Thr His Gln Tyr Val Thr Gln Ile Asp Glu Leu Val Lys His Lys Glu Gly Glu Leu Leu Glu Val <210> 15 <211> 651 <212> DNA
<213> M.Tuberculosis <220>

<221> CDS

<222> (1)...(648) <400> 15 atggccgac atcgatggt gtaaccggt tcggcgggt ctgcag cctggg 98 MetAlaAsp IleAspGly ValThrGly SerAlaGly LeuGln ProGly ccgtctgag gagacagac gaggagttg accgcgcgt ttcgag cgcgac 96 ProSerGlu GluThrAsp GluGluLeu ThrAlaArg PheGlu ArgAsp gcgattccc ctgttggac cagctgtac ggcggtgcg ctgcgg atgacg 144 AlaIlePro LeuLeuAsp GlnLeuTyr GlyGlyA1<~LeuArg MetThr cgcaatccg gccgacgcc gaggacttg ctccaggag acgatg gtgaag 192 ArgAsnPro AlaAspAla GluAspLeu LeuGlnGlu ThrMet ValLys gcctatgcg ggatttcgt tcgttccgg cacggtacc aatctc aaggcc 240 AlaTyrAla GlyPheArg SerPheArg HisGlyThr_AsnLeu LysAla tggctctac cggatactg accaacacc tacatcaac agctat cgcaag 288 TrpLeuTyr ArgIleLeu ThrAsnThr TyrIleAsn SerTyr ArgLys aaacagcgg caaccggcg gagtatccg accgagcag atcacc gattgg 336 LysGlnArg GlnProAla GluTyrPro ThrGluG1I1IleThr AspTrp caactggcg tccaacgcc gagcattcc tcgaccggc3ctgcgc tcgget 389 GlnLeuAla SerAsnAla GluHisSer SerThrGly LeuArg SerAla gaagtcgaa gcgttagaa gcgttgccg gacaccgag atcaaa gaggcg 432 GluValGlu AlaLeuGlu AlaLeuPro AspThrGlu IleLys GluAla ctgcaggca ttgccggaa gagttccgg atggcggtc tactac gccgat 480 LeuGlnAla LeuProGlu GluPheArg MetAlaVal TyrTyr AlaAsp gtcgaaggt ttcccctac aaggagatc gccgagatc atggat actccg 528 ValGluGly PheProTyr LysGluIle AlaGluIllsMetAsp ThrPro atcggcacc gtgatgtcg aggcttcat cgcggccga cgtcag ttgcgc 576 IleGlyThr ValMetSer ArgLeuHis ArgGlyArg ArgGln LeuArg ggtctttta gccgatgtg gccagggat cgggggttt gccagg ggcgag 624 GlyLeuLeu AlaAspVal AlaArgAsp ArgGlyPhe AlaArg GlyGlu caggcgcac gagggggtg tcgtcatga 651 GlnAlaHis GluGlyVal SerSer <210> 16 <211> 216 <212> PRT
<213> M.Tuberculosis <900> 16 Met Ala Asp Ile Asp Gly Val Thr Gly Ser Ala Gly Leu Gln Pro Giy Pro Ser Glu Glu Thr Asp Glu Glu Leu Thr Ala Arg Phe Glu Arg Asp Ala Ile Pro Leu Leu Asp Gln Leu Tyr Gly Gly Ala Leu Arg Met Thr Arg Asn Pro Ala Asp Ala Glu Asp Leu Leu Gln Glu Thr Met Val Lys Ala Tyr Ala Gly Phe Arg Ser Phe Arg His Gly Thr Asn Leu Lys Ala Trp Leu Tyr Arg Ile Leu Thr Asn Thr Tyr Ile Asn Ser Tyr Arg Lys Lys Gln Arg Gln Pro Ala Glu Tyr Pro Thr Glu Gln Ile Thr Asp Trp Gln Leu Ala Ser Asn Ala Glu His Ser Ser Thr Gly Leu Arg Ser Ala Glu Val Glu Ala Leu Glu Ala Leu Pro Asp Thr Glu Ile Lys Glu Ala Leu Gln Ala Leu Pro Glu Glu Phe Arg Met Ala Val Tyr Tyr Ala Asp Val Glu Gly Phe Pro Tyr Lys Glu Ile Ala Glu Ile Met Asp Thr Pro Ile Gly Thr Val Met Ser Arg Leu His Arg Gly Arg Arg Gln Leu Arg Gly Leu Leu Ala Asp Val Ala Arg Asp Arg Gly Phe Ala Arg Gly Glu Gln Ala His Glu Gly Val Ser Ser <210> 17 <211> 779 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(771) <900> 17 atg acg tac gaa acc atc ctg gtc gag cgc gat cag cga gtt ggc att 48 Met Thr Tyr Glu Thr Ile Leu Val Glu Arg Asp Gln Arg Val Gly Ile atc acg ctg aac cgt ccc cag gca ctg aac gcg ctc aac agc cag gtg 96 Ile Thr Leu Asn Arg Pro Gln Ala Leu Asn Ala Leu Asn Ser Gln Val atg aac gag gtc acc agc get gca acc gaa ctg gac gat gac ccg gac 144 Met Asn Glu Val Thr Ser Ala Ala Thr Glu Leu Asp Asp Asp Pro Asp att ggg gcg atc atc atc acc ggt tcg gcc aaa gcg ttt gcc gcc gga 192 Ile Gly Ala Ile Ile Ile Thr Gly Ser Ala Lys Ala Phe Ala Ala Gly gccgacatc aaagaaatg gccgacctg acgttcgcc gacgcgttc acc 290 AlaAspIle LysGluMet AlaAspLeu ThrPheAla AspAlaPhe Thr gccgacttc ttcgccacc tggggcaag ctggccgcc gtgcgcacc ccg 288 AlaAspPhe PheAlaThr TrpGlyLys LeuAlaAla ValArgThr Pro acgatcgcc gcggtggcg ggatacgcg ctcggcggt ggctgcgag ctg 336 ThrIleAla AlaValAla GlyTyrAla LeuGlyGly GlyCysGlu Leu gcgatgatg tgcgacgtg ctgatcgcc gccgacacc gcgaagttc gga 384 AlaMetMet CysAspVal LeuIleAla AlaAspThr AlaLysPhe Gly cagcccgag ataaagctg ggcgtgctg ccaggcatg ggcggctcc cag 432 GlnProGlu IleLysLeu GlyValLeu ProGlyMet GlyGlySer Gln cggctgacccgg getatc ggcaagget aaggcgatg gacctcatc ctg 480 ArgLeuThrArg AlaIle GlyLysAla LysAlaMet AspLeuIle Leu accgggcgcacc atggac gccgccgag gccgagcgc:agcggtctg gtt 528 ThrGlyArgThr MetAsp AlaAlaGlu AlaGluArg SerGlyLeu Val tcacgggtggtg ccggcc gacgacttg ctgaccgaa gccagggcc act 576 SerArgValVal ProAla AspAspLeu LeuThrGlu AlaArgAla Thr gccacgaccatt tcgcag atgtcggcc tcggcggcc:cggatggcc aag 629 AlaThrThrIle SerGln MetSerAla SerAlaAla ArgMetAla Lys gaggcc aaccggget ttcgaatcc agtttgtcc:gaggggctg ctc 672 gtc GluAla AsnArgAla PheGluSer SerLeuSer GluGlyLeu Leu Val tacgaa cggcttttc cattcgget ttcgcgacc gaagaccaa tcc 720 cgc TyrGlu ArgLeuPhe HisSerAla PheAlaThr GluAspGln Ser Arg gaaggt gcagcgttc atcgagaaa cgcgetccc cagttcacc cac 768 atg GluGly AlaAlaPhe IleGluLys ArgAlaPro GlnPheThr His Met cgatga 774 Arg <210> 18 <211> 257 <212> PRT

<213> M.Tuberculosis <400> 18 MetThr GluThrIle LeuValGlu ArgAspGln ArgValGly Ile Tyr Ile Thr Leu Asn Arg Pro Gln Ala Leu Asn Ala Leu Asn Ser Gln Val Met Asn Glu Val Thr Ser Ala Ala Thr Glu Leu Asp Asp Asp Pro Asp Ile Gly Ala Ile Ile Ile Thr Gly Ser Ala Lys Ala Phe Ala Ala Gly Ala Asp Ile Lys Glu Met Ala Asp Leu Thr Phe Ala Asp Ala Phe Thr 65 70 75 g0 Ala Asp Phe Phe Ala Thr Trp Gly Lys Leu Ala Ala Val Arg Thr Pro Thr Ile Ala Ala Val Ala Gly Tyr Ala Leu Gly Gly Gly Cys Glu Leu Ala Met Met Cys Asp Val Leu Ile Ala Ala Asp Thr Ala Lys Phe Gly Gln Pro Glu Ile Lys Leu Gly Val Leu Pro Gly Met: Gly Gly Ser Gln Arg Leu Thr Arg Ala Ile Gly Lys Ala Lys Ala Met Asp Leu Ile Leu Thr Gly Arg Thr Met Asp Ala Ala Glu Ala Glu Arg Ser Gly Leu Val Ser Arg Val Val Pro Ala Asp Asp Leu Leu Thr Glu Ala Arg Ala Thr Ala Thr Thr Ile Ser Gln Met Ser Ala Ser Ala Ala Arg Met Ala Lys Glu Ala Val Asn Arg Ala Phe Glu Ser Ser Leu Ser Glu Gly Leu Leu Tyr Glu Arg Arg Leu Phe His Ser Ala Phe Ala Thr Glu Asp Gln Ser Glu Gly Met Ala Ala Phe Ile Glu Lys Arg Ala Pro Gln Phe Thr His Arg <210> 19 <211> 894 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(891) <400> 19 gtgccgcttccc gcagaccct agccccacc ttgtcggcc tacgcc cat 48 ValProLeuPro AlaAspPro SerProThr LeuSerAla TyrAla His cccgaacggctc gtgaccgcc gactggttg tcggcacac atgggc gcg 96 ProGluArgLeu ValThrA1a AspTrpLeu SerAlaHis MetGly Ala ccgggcctggcg atcgtcgaa tccgacgag gacgtc_ttg ctctac gac 144 ProGlyLeuAla IleValG1u SerAspGlu AspValLeu LeuTyr Asp gtcggccatatt cccggcgcc gtcaagatc gactggcac accgac ctc 192 ValGlyHisIle ProGlyAla ValLysIle AspTrpHis ThrAsp Leu aac gac cca cgg gtg cgc gac tac atc aac ggc gag cag ttc gcc gaa 290 Asn Asp Pro Arg Val Arg Asp Tyr Ile Asn Gly Glu Gln Phe Ala Glu 65 70 75 g0 ttgatggac cgcaagggc atcgcccgc gatgacacc gtggtg atctat 288 LeuMetAsp ArgLysGly :CleAlaArg AspAspThr ValVal IleTyr ggcgacaag agcaattgg tgggccgcc tatgcgttg tgggtg ttcacg 336 GlyAspLys SerAsnTrp TrpAlaAla TyrAlaLeu TrpVal PheThr ctgttcggt cacgccgac gtgcgactc ctcaacggc ggccgt gacctc 389 LeuPheGly HisAlaAsp ValArgLeu LeuAsnGly GlyArg AspLeu tggctcgcc gagcgccgg gaaaccacc ttggacgtc ccgacc aagacc 432 TrpLeuAla GluArgArg GluThrThr LeuAspVal ProThr LysThr tgcaccggt tatcccgtc gtgcagcgc aacgatgca cccatc cgcgca 980 CysThrGly TyrProVal ValGlnArg AsnAspAla ProIle ArgAla ttcagagac gacgtgctg gccatcctg ggcgetcag ccgctg atcgac 528 PheArgAsp AspValLeu AlaIleLeu GlyAlaGln ProLeu IleAsp gtacgctct cccgaggag tacaccggc aagcgcacc catatg cccgat 576 ValArgSer ProGluGlu T'yrThrGly LysArgThr HisMet ProAsp taccccgag gaaggggcg ctgcgggcc ggtcacatc cccacg gcggtg 629 TyrProGlu GluGlyAla heuArgAla GlyHisIleaProThr AlaVal cacattccg tgggggaag gccgccgac gaaagtgga cggttt cgcagc 672 HisIlePro TrpGlyLys AlaAlaAsp GluSerGly ArgPhe ArgSer cgcgaggaa ttggaacgg ctctatgac ttcataaac ccggacgac caa 720 ArgGluGlu LeuGluArg LeuTyrAsp PheIleAsn ProAspAsp Gln accgtcgtc tattgccgc atcggtgaa cgctccagc:catacctgg ttc 768 ThrValVal TyrCysArg IleGlyGlu ArgSerSer HisThrTrp Phe gtgctcaca cacctgctg ggcaaggca gatgtacgg aactacgac ggc 816 ValLeuThr HisLeuLeu GlyLysAla AspValArg AsnTyrAsp Gly tcgtggacc gagtggggc aacgccgtg cgagtgccg atcgtcgcg ggc 864 SerTrpThr GluTrpGly AsnAlaVal ArgValPro IleValAla Gly gaagaacca ggagtggta cccgtcgta tga g9q GluGluPro GlyValVal ProValVal <210> 20 <211> 297 <212> PRT
<213> M.Tuberculosis <400> 20 Met Pro Leu Pro Ala Asp Pro Ser Pro Thr Leu Ser Ala Tyr Ala His Pro Glu Arg Leu Val Thr Ala Asp Trp Leu 5er Ala His Met Gly Ala Pro Gly Leu Ala Ile Val Glu Ser Asp Glu Asp Val Leu Leu Tyr Asp Val Gly His Ile Pro Gly Ala Val Lys Ile Asp Trp His Thr Asp Leu Asn Asp Pro Arg Val Arg Asp Tyr Ile Asn Gly Glu Gln Phe Ala Glu Leu Met Asp Arg Lys Gly Ile Ala Arg Asp Asp Th:r Val Val Ile Tyr Gly Asp Lys Ser Asn Trp Trp Ala Ala Tyr Ala Leu Trp Val Phe Thr Leu Phe Gly His Ala Asp Val Arg Leu Leu Asn Gly Gly Arg Asp Leu Trp Leu Ala Glu Arg Arg Glu Thr Thr Leu Asp Val Pro Thr Lys Thr Cys Thr Gly Tyr Pro Val Val Gln Arg Asn Asp Ala Pro Ile Arg Ala Phe Arg Asp Asp Val Leu Ala Ile Leu Gly Ala Gln Pro Leu Ile Asp Val Arg Ser Pro Glu Glu Tyr Thr Gly Lys Arg Thr His Met Pro Asp Tyr Pro Glu Glu Gly Ala Leu Arg Ala Gly His Ile: Pro Thr Ala Val His Ile Pro Trp Gly Lys Ala Ala Asp Glu Ser Gly Arg Phe Arg Ser Arg Glu Glu Leu Glu Arg Leu Tyr Asp Phe Ile Asn Pro Asp Asp Gln Thr Val Val Tyr Cys Arg Ile Gly Glu Arg Ser Ser His Thr Trp Phe Val Leu Thr His Leu Leu Gly Lys Ala Asp Val Arg Asn Tyr Asp Gly Ser Trp Thr Glu Trp Gly Asn Ala Val Arg Val Pro Ile Val Ala Gly Glu Glu Pro Gly Val Val Pro Val Val <210> 21 <211> 1094 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(1041) <400> 21 atg ctg atc tca cag cgc ccc acc ctg tcc gag gac gtc ctc acc gac 48 Met Leu Ile Ser Gln Arg Pro Thr Leu Ser Glu Asp Val Leu Thr Asp aac cga tcc cag ttc gtg atc gaa ccg ctg gag ccg gga ttc ggc tac 96 Asn Arg Ser Gln Phe Val I1e Glu Pro Leu Glu Pro Gly Phe Gly Tyr acc ctg ggc aat tcg ctg cgt cgc acc ctg ctg tcg tcg att ccc gga 199 Thr Leu Gly Asn Ser Leu Arg Arg Thr Leu Leu Ser Ser Ile Pro Gly gcg gcc gtc acc agc att cgc atc gat ggt gta ctg cac gaa ttc acc 192 Ala Ala Val Thr Ser Ile Arg Ile Asp Gly Val Leu His Glu Phe Thr acggtgcccggg gtcaaagaa gatgtcacc gagatc atcctgaat ctc 240 ThrValProGly ValLysGlu AspValThr GluIle IleLeuAsn Leu aagagcctggtg gtgtcctcg gaggaggac gagccg gtcaccatg tac 288 LysSerLeuVal ValSerSer GluGluAsp GluPro ValThrMet Tyr ctacgcaagcag ggtccgggt gaggttacc gccggc gacatcgtg ccg 336 LeuArgLysGln GlyProGly GluValThr AlaGly AspIleVal Pro ccggccggcgtc accgtgcac aaccccggc atgcac atcgccacg ctg 384 ProAlaGlyVal ThrValHis AsnProGly MetHis IleAlaThr Leu aacgataag ggcaagctggaa gtcgag ctcgtcgtc gagcgtggc cgc 432 AsnAspLys GlyLysLeuGlu ValGlu LeuValVal GluArgGly Arg ggctatgtc ccggcggtgcaa aaccgg gettcgggt gccgaaatt ggg 480 GlyTyrVal ProAlaValGln AsnArg AlaSerGly AlaGluIle Gly cgcattcca gtcgattccatc tactca ccggtgctc aaagtgacc tac 528 ArgIlePro ValAspSerIle TyrSer ProValLeu LysValThr Tyr aaggtggac gccacccgggtc gagcag cgcaccgac ttcgacaag ctg 576 LysValAsp AlaThrArgVal GluGln ArgThrAsp PheAspLys Leu atcctggac gtggagaccaag aattca atcagcccg cgcgacgcg ctg 624 IleLeuAsp ValGluThrLys AsnSer IleSerPro ArgAspAla Leu gcgtcgget ggcaagacgctg gtcgag ttgttcggc:ctggcacgg gaa 672 AlaSerAla GlyLysThrLeu ValGlu LeuPheGly LeuAlaArg Glu ctcaacgtc gaggccgaaggc atcgag atcgggccg tcgccggcc gag 720 LeuAsnVal GluAlaGluGly IleGlu IleGlyPro SerProAla Glu gccgatcac attgcgtcattc gccctg ccgatcgac gacctggat ctg 768 AlaAspHis IleAlaSerPhe AlaLeu ProIleAsp AspLeuAsp Leu acggtgcgg tcctacaactgc ctcaag cgcgagggg gtgcacacc gtg 816 ThrValArg SerTyrAsnCys LeuLys ArgGluGly ValHisThr Val ggc gaa ctg gtg gcg cgc acc gaa tcc gac ctg ctt gac atc cgc aac 864

17 Gly Glu Leu Val Ala Arg Thr Glu Ser Asp Leu Leu Asp Ile Arg Asn ttc ggt cag aag tcc atc gac gag gtg aag atc aag ctg cac cag ctg 912 Phe Gly Gln Lys Ser Ile Asp Glu Val Lys Ile Lys Leu His Gln Leu ggc ctg tca ctc aag gac agc ccg ccg agc ttc gac ccc tcg gag gtc 960 Gly Leu Ser Leu Lys Asp Ser Pro Pro Ser Phe Asp Pro Ser Glu Val gcg ggc tac gac gtc gcc acc ggc acc tgg tcg acc gag ggc gcg tac 1008 Ala Gly Tyr Asp Val Ala Thr Gly Thr Trp Ser Thr Glu Gly Ala Tyr gac gag cag gac tac gcc gaa acc gaa cag ctt tag 1044 Asp Glu Gln Asp Tyr Ala Glu Thr Glu Gln Leu <210> 22 <211> 347 <212> PRT
<213> M.Tuberculosis <900> 22 Met Leu Ile Ser Gln Arg Pro Thr Leu Ser Glu Asp Val Leu Thr Asp Asn Arg Ser Gln Phe Val Ile Glu Pro Leu Glu Pro Gly Phe Gly Tyr Thr Leu Gly Asn Ser Leu Arg Arg Thr Leu Leu Ser Ser Ile Pro Gly Ala Ala Val Thr Ser Ile Arg Ile Asp Gly Val Leu His Glu Phe Thr Thr Val Pro Gly Val Lys Glu Asp Val Thr Glu Ile Ile Leu Asn Leu Lys Ser Leu Val Val Ser Ser Glu Glu Asp Glu Pro Val Thr Met Tyr Leu Arg Lys Gln Gly Pro Gly Glu Val Thr Ala Gly Asp Ile Val Pro Pro Ala Gly Val Thr Val His Asn Pro Gly Met His Ile Ala Thr Leu Asn Asp Lys Gly Lys Leu Glu Val Glu Leu Val Va.1 Glu Arg Gly Arg Gly Tyr Val Pro Ala Val Gln Asn Arg Ala Ser Gly Ala Glu Ile Gly Arg Ile Pro Val Asp Ser Ile Tyr Ser Pro Val Leu Lys Val Thr Tyr Lys Val Asp Ala Thr Arg Val Glu Gln Arg Thr Asp Phe Asp Lys Leu Ile Leu Asp Val Glu Thr hys Asn Ser Ile Ser Pro Arg Asp Ala Leu Ala Ser Ala Gly Lys Thr Leu Val Glu Leu Phe Gl:y Leu Ala Arg Glu Leu Asn Val Glu Ala Glu Gly Ile Glu Ile Gly Pro Ser Pro Ala Glu Ala Asp His Ile Ala Ser Phe Ala Leu Pro Ile Asp Asp Leu Asp Leu Thr Val Arg Ser Tyr Asn Cys Leu Lys Arg Glu Gly Val His Thr Val Gly Glu Leu Val Ala Arg Thr Glu Ser Asp Leu Leu Asp Ile Arg Asn

18 PheGly LysSerIle AspGluVal LysIleLys LeuHisGln Leu Gln GlyLeu LeuLysAsp SerProPro SerPheAsp ProSerGlu Val Ser AlaGly AspValAla ThrGlyThr TrpSerThr GluGlyAla Tyr Tyr AspGlu AspTyrAla GluThrGlu GlnLeu Gln <210> 23 <211> 1488 <212> DNA

<213> M.Tuberculosis <220>

<221> CDS

<222> (1)...(1485) <400> 23 atgacc aatttggtg accaaaaat tcgctgacc cctgacgtt cgt 48 gga MetThr AsnLeuVal ThrLysAsn SerLeuThr ProAspVal Arg Gly aacggc gactttaag atcgccgac ctgtcacta gcggatttc ggc 96 atc AsnGly AspPheLys IleAlaAsp LeuSerLeu AlaAspPhe Gly Ile cgcaaa ctccggatc gccgagcac gagatgccc ggcctgatg tcg 144 gaa ArgLys LeuArgIle AlaGluHis GluMetPro GlyLeuMet Ser Glu ctgcgg gagtatgcc gaggtgcaa cccctgaag ggggcccgg atc 192 cgc LeuArg GluTyrAla GluValGln ProLeuLys GlyAlaArg Ile Arg tcgggttcg ctgcacatg acggtgcag accgcggtg ttgatcgaa acc 240 SerGlySer LeuHisMet ThrValGln ThrAlaVal LeuIleGlu Thr ctcaccgcg ctgggcgcc gaagtccgc tgggcctcg tgcaacatc ttc 288 LeuThrAla LeuGlyAla GluValArg TrpAlaSer CysAsnIle Phe tccacccag gatcacgcc gccgccgcc gtcgtggtc:ggcccgcac ggc 336 SerThrGln AspHisAla AlaAlaAla ValValVal GlyProHis Gly acccccgac gagcccaag ggtgtcccg gtgttcgcg tggaagggc gag 389 ThrProAsp GluProLys GlyValPro ValPheAla TrpLysGly Glu acgctcgaa gagtactgg tgggccgcc gagcagatg ctcacctgg ccg 932 ThrLeuGlu GluTyrTrp TrpAlaAla GluGlnMet:LeuThrTrp Pro gaccccgac aagccggcc aacatgatc ctcgatgac ggcggtgac gcc 480 AspProAsp LysProAla AsnMetIle LeuAspAsp GlyGlyAsp Ala acc atg ttg gtg ctg cgc ggc atg cag tat gag aag gcc ggc gtg gtg 528

19 ThrMet LeuValLeu ArgGly MetGlnTyrGlu LysAla GlyValVal ccgccc gccgaggag gacgac cccgccgagtgg aaggtc ttcctgaac 576 ProPro AlaGluGlu AspAsp ProAlaGluTrp LysVal PheLeuAsn ctgcta cggacccgc ttcgag accgacaaggac aagtgg accaagata 624 LeuLeu ArgThrArg PheGlu ThrAspLysAsp LysTrp ThrLysIle gccgag tcggtcaag ggcgtc accgaggagacc accacc ggcgtgctg 672 AlaGlu SerValLys GlyVal ThrGluGluThr ThrThr GlyValLeu cggctc taccaattc gccgcg gccggggatctg gccttc ccggcgatc 720 ArgLeu TyrGlnPhe AlaAla AlaGlyAspLeu AlaPhe ProAlaIle aacgtc aacgactcg gtgacc aagtccaaattc gacaac aagtacggc 768 AsnVal AsnAspSer ValThr LysSerLysPhe AspAsn LysTyrGly actcgg cactccctg atcgac ggcatcaaccgc ggcacc gacgcgctg 816 ThrArg HisSerLeu IleAsp GlyIleAsnArg GlyThr AspAlaLeu atcggc ggtaagaag gtcctc atctgcggctac ggcgac gtcggtaag 864 IleGly GlyLysLys ValLeu IleCysGlyTyr GlyAsp ValGlyLys ggctgt gcggaggcg atgaag ggccagggagcg cgggtc tccgtcacc 912 GlyCys AlaGluAla MetLys GlyGlnGlyAla ArgVal SerValThr gagatc gacccgatc aacgcg ctgcaggccatg atggag ggcttcgac 960 GluIle AspProIle AsnAla LeuGlnAlaMet MetGlu GlyPheAsp gtggtc accgtcgag gaggcc atcggggacgcc gacatc gtcgtaacc 1008 ValVal ThrValGlu GluAla IleGlyAspAla AspIle ValValThr gcgacc ggcaacaaa gacatc atcatgctcgag cacatt aaggcgatg 1056 AlaThr GlyAsnLys AspIle IleMetLeuGlu HisIle LysAlaMet aaggac cacgcgatc ctggga aatatcggccac ttcgac aacgagatc 1109 LysAsp HisAlaIle LeuGly AsnIleGlyHis PheAsp AsnGluIle gacatg gccgggctg gagcgc tccggggcgaca cgggtc aacgtcaag 1152 AspMet AlaGlyLeu GluArg SerGlyAlaThr ArgVal AsnValLys cctcag gtcgacctg tggacc tttggcgacacg ggccgc tcgatcatc 1200 ProGln ValAspLeu TrpThr PheGlyAspThr GlyArg SerIleIle gtgctg tccgagggg cggctg ctgaacctgggc aatgcc accgggcac 1298 ValLeu SerGluGly ArgLeu LeuAsnLeuGly AsnAla ThrGlyHis ccc tcg ttc gtg atg agc aac agc ttc get aac cag acg atc gcc cag 1296 Pro Ser Phe Val Met Ser Asn Ser Phe Ala Asn Gln Thr Ile Ala Gln atc gag ctg tgg acc aag aac gac gag tac gac aac gag gtg tac cgg 1349 Ile Glu Leu Trp Thr Lys Asn Asp Glu Tyr Asp Asn Glu Val Tyr Arg ctg ccc aag cac ctc gac gag aag gtg get cga atc cat gtc gag gcc 1392 Leu Pro Lys His Leu Asp Glu Lys Val Ala Arg Ile His Val Glu Ala ctt ggc ggt cac ctg acc aag ctg acc aag gag cag gcc gaa tac ctc 1490 Leu Gly Gly His Leu Thr Lys Leu Thr Lys Glu Gln Ala Glu Tyr Leu ggc gtc gac gtc gag ggt ccc tac aag ccg gac cac tac cgc tac 1985 Gly Val Asp Val Glu Gly Pro Tyr Lys Pro Asp His Tyr Arg Tyr tga 1488 <210> 29 <211> 495 <212> PRT
<213> M.Tuberculosis <400> 24 Met Thr Gly Asn Leu Val Thr Lys Asn Ser Leu Thr Pro Asp Val Arg Asn Gly Ile Asp Phe Lys Ile Ala Asp Leu Ser Leu Ala Asp Phe Gly

20 25 30 Arg Lys Glu Leu Arg Ile Ala Glu His Glu Met Pro Gly Leu Met Ser Leu Arg Arg Glu Tyr Ala Glu Val Gln Pro Leu Lys Gly Ala Arg Ile Ser Gly Ser Leu His Met Thr Val Gln Thr Ala Val Leu Ile Glu Thr Leu Thr Ala Leu Gly Ala Glu Val Arg Trp Ala Sex Cys Asn Ile Phe Ser Thr Gln Asp His Ala Ala Ala Ala Val Val Val Gly Pro His Gly Thr Pro Asp Glu Pro Lys Gly Val Pro Val Phe Ala Trp Lys Gly Glu Thr Leu Glu Glu Tyr Trp Trp Ala Ala Glu Gln Met Leu Thr Trp Pro Asp Pro Asp Lys Pro Ala Asn Met Ile Leu Asp Asp Gly Gly Asp Ala Thr Met Leu Val Leu Arg Gly Met Gln Tyr Glu Lys Ala Gly Val Val Pro Pro Ala Glu Glu Asp Asp Pro Ala Glu Trp Lys Val Phe Leu Asn Leu Leu Arg Thr Arg Phe Glu Thr Asp Lys Asp Lys Trp Thr Lys Ile Ala Glu Ser Val Lys Gly Val Thr Glu Glu Thr Thr Thr Gly Val Leu Arg Leu Tyr Gln Phe Ala Ala Ala Gly Asp Leu Ala Phe Pro Ala Ile Asn Val Asn Asp Ser Val Thr Lys Ser Lys Phe Asp Asn Lys Tyr Gly

21 Thr Arg His Ser Leu Ile Asp Gly Ile Asn Arg Gly Thr Asp Ala Leu Ile Gly Gly Lys Lys Val Leu Ile Cys Gly Tyr Gly Asp Val Gly Lys Gly Cys Ala Glu Ala Met Lys Gly Gln Gly Ala Arg Val Ser Val Thr Glu Ile Asp Pro Ile Asn Ala Leu Gln Ala Met Met Glu Gly Phe Asp Val Val Thr Val Glu Glu Ala Ile Gly Asp Ala Asp Ile Val Val Thr Ala Thr Gly Asn Lys Asp Ile Ile Met Leu Glu His Ile Lys Ala Met Lys Asp His Ala Ile Leu Gly Asn Ile Gly His Phe Asp Asn Glu Ile Asp Met Ala Gly Leu Glu Arg Ser Gly Ala Thr Arg Val Asn Val Lys Pro Gln Val Asp Leu Trp Thr Phe Gly Asp Thr Gly Arg Ser Ile Ile Val Leu Ser Glu Gly Arg Leu Leu Asn Leu Gly Asn Ala Thr Gly His Pro Ser Phe Val Met Ser Asn Ser Phe Ala Asn Gln Thr Ile Ala Gln Ile Glu Leu Trp Thr Lys Asn Asp Glu Tyr Asp Asn Glu Val Tyr Arg Leu Pro Lys His Leu Asp Glu Lys Val Ala Arg Ile His Val Glu Ala Leu Gly Gly His Leu Thr Lys Leu Thr Lys Glu Gln Ala Glu Tyr Leu Gly Val Asp Val Glu Gly Pro Tyr Lys Pro Asp His Tyr Arg Tyr <210> 25 <211> 1803 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(1800) <400> 25 gtgget agtcacgcc ggctcgagg atcgetcgg atctctaag gttctc 48 ValAla SerHisAla GlySerArg IleAlaArg IleSerLys ValLeu gtcgcc aatcgcggc gagatcgca gtgcgggtg atccgggcg gcccgc 96 ValAla AsnArgGly GluIleAla ValArgVal IleArgAla AlaArg gacgcc ggcctgccc agcgtggcg gtgtacgcc gaacccgac gccgag 194 AspAla GlyLeuPro SerValAla ValTyrAla GluProAsp AlaGlu tccccg catgttcgg ctggccgac gaggcgttc gcgctgggc ggccag 192 SerPro HisValArg LeuAlaAsp GluAlaPhe AlaLeuGly GlyGln acc tcg gcg gag tcc tat ctg gac ttc gcc aag atc ctc gac gcg gca 240 Thr Ser Ala Glu Ser Tyr Leu Asp Phe Ala Lys Ile Leu Asp Ala Ala

22 gcc aag tcc 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 gaa aat gcc gac ttc gcc cag gcg gtg atc gac gcc ggc ctg atc tgg 336 Glu Asn Ala Asp Phe Ala Gln Ala Val Ile Asp Ala Gly Leu Ile Trp atcggcccc agcccgcag tcgatc cgcgacctgggc gacaag gtcacg 389 IleGlyPro SerProGln SerIle ArgAspLeuGly AspLys ValThr gcccgtcac atcgcggcc cgcget caggcgcccctg gtgccg ggtacc 932 AlaArgHis IleAlaAla ArgAla GlnAlaProLeu ValPro GlyThr cccgatccg gtcaaaggc gccgac gaggtggtggca ttcgcc gaggag 480 ProAspPro ValLysGly AlaAsp GluValValAla PheAla GluGlu tacggcctg ccgatcgcg atcaag gccgcccacggc ggcggc ggcaag 528 TyrGlyLeu ProIleAla IleLys AlaAlaHisGly GlyGly GlyLys ggcatgaag gtggcccgc accatc gacgagattccg gagctg tacgag 576 GlyMetLys ValAlaArg ThrIle AspGluIlePro GluLeu TyrGlu tcggcggtg cgcgaggcc acggcc gcgttcggccgc ggtgag tgctac 629 SerAlaVal ArgGluAla ThrAla AlaPheGlyArg GlyGlu CysTyr gtggagcgc tatctcgac aagccg cgccacgtcgaa gcacag gtgatc 672 ValGluArg TyrLeuAsp LysPro ArgHisValGlu AlaGln ValIle gccgaccag cacggcaac gtcgtc gtcgccggcacc cgggac tgctcg 720 AlaAspGln HisGlyAsn ValVal ValAlaGlyThr ArgAsp CysSer ctgcagcgc cgctaccag aagctg gtcgaggaggcg cccgca ccgttc 768 LeuGlnArg ArgTyrGln LysLeu ValGluGluA1<~ProAla ProPhe ctgaccgac tttcaacgc aaagag atccacgactcg gccaaa cggatt 816 LeuThrAsp PheGlnArg LysGlu IleHisAspSer AlaLys ArgIle tgcaaagag gcccattac cacggc gccggcaccgtc gaatac ctggtc 864 CysLysGlu AlaHisTyr HisGly AlaGlyThrVa1 GluTyr LeuVal ggtcaggac ggcttgatc tcgttc ttggaggtcaac acgcgc cttcag 912 GlyGlnAsp GlyLeuIle SerPhe LeuGluValAsn ThrArg LeuGln gtagaacac ccggtcacc gaggaa accgcgggcatc gacttg gtgctg 960 ValGluHis ProValThr GluGlu ThrAlaGlyIle AspLeu ValLeu

23 cag caa ttc cgg atc gcc aac ggc gaa aag ctg gac atc acc gag gat 1008 Gln Gln Phe Arg Ile Ala Asn Gly Glu Lys Leu Asp Ile Thr Glu Asp ccc acc ccg cgc ggg cac gcc atc gaa ttc cgg atc aac ggc gag gac 1056 Pro Thr Pro Arg Gly His Ala Ile Glu Phe Arg Ile Asn Gly Glu Asp gcg ggg cgt aac ttc cta ccg gcg ccc ggg ccg gtg aca aag ttc cac 1109 Ala Gly Arg Asn Phe Leu Pro Ala Pro Gly Pro Val Thr Lys Phe His ccg ccg tcc ggc ccc ggt gtg cgg gtg gac tcc ggt gtc gag acc ggc 1152 Pro Pro Ser Gly Pro Gly Val Arg Val Asp Ser Gly Val Glu Thr Gly tcg gtg atc ggc ggc cag ttc gac tcg atg ctg gcc aag ctg atc gtg 1200 Ser Val Ile Gly Gly Gln Phe Asp Ser Met Leu Ala Lys Leu Ile Val cac ggt gcc gac cgc gcc gag gcg ctg gcg cgg gcc cgg cgc gcg ctg 1298 His Gly Ala Asp Arg Ala Glu Ala Leu Ala Arg Ala Arg Arg Ala Leu aac gag ttc ggt gtc gaa ggc ctg gcg acg gtc atc ccg ttt cac cgc 1296 Asn Glu Phe Gly Val Glu Gly Leu Ala Thr Val Ile Pro Phe His Arg gcc gtg gtg tcc gac ccg gca ttc atc ggc gac gcg aac ggc ttt tcg 1344 Ala Val Val Ser Asp Pro Ala Phe Ile Gly Asp Ala Asn Gly Phe Ser gta cat acc cgc tgg atc gag acc gag tgg aat aac acc atc gag ccc 1392 Val His Thr Arg Trp Ile Glu Thr Glu Trp Asn Asn Thr Ile Glu Pro ttt acc gac ggc gaa cct ctc gac gag gac gcc cgg ccg cgt cag aag 1940 Phe Thr Asp Gly Glu Pro Leu Asp Glu Asp Ala Arg Pro Arg Gln Lys gtg gtc gtc gaa atc gac ggt cgc cgc gtc gaa gtc tcg ctg ccg get 1988 Val Val Val Glu Ile Asp Gly Arg Arg Val Glu Va.l Ser Leu Pro Ala gat ctc gcg ctg tcc aat ggc ggc ggt tgc gac ccg gtc ggt gtc atc 1536 Asp Leu Ala Leu Ser Asn Gly Gly Gly Cys Asp Pro Val Gly Val Ile cgg cgc aag ccc aag ccg c:gc aag cgg ggt gcg cac acc ggc gcg gcg 1584 Arg Arg Lys Pro Lys Pro Arg Lys Arg Gly Ala His Thr Gl.y Ala Ala gcc tcc ggt gac gcg gtg acc gcg cct atg cag ggc acc gta gtt aag 1632 Ala Ser Gly Asp Ala Val Thr Ala Pro Met Gln Gly Thr Val Val Lys ttc gcg gtc gaa gaa ggg caa gag gtc gtg gcc ggc gac cta gtg gtg 1680 Phe Ala Val Glu Glu Gly Gln Glu Val Val Ala Gly Asp Leu Val Val gtc ctc gag gcg atg aag atg gaa aac ccg gtc acc gcg cat aag gat 1728

24 Val Leu Glu Ala Met Lys Met Glu Asn Pro Val Thr Ala His Lys Asp ggc acc atc acc ggg ctg gcg gtc gag gcg ggc gcg gcc atc acc cag 1776 Gly Thr Ile Thr Gly Leu Ala Val Glu Ala Gly Ala Ala Ile Thr Gln ggc acg gtg ctc gcc gag atc aag taa 1803 Gly Thr Val Leu Ala Glu Ile Lys <210> 26 <211> 600 <212> PRT
<213> M.Tuberculosis <400> 26 Val Ala Ser His Ala Gly Ser Arg Ile Ala Arg Ile Ser Lys Val Leu Val Ala Asn Arg Gly Glu Tle Ala Val Arg Val Ile Arg Ala Ala Arg Asp Ala Gly Leu Pro Ser Val Ala Val Tyr Ala Glu Pro Asp Ala Glu Ser Pro His Val Arg Leu Ala Asp Glu Ala Phe Ala Leu Gly Gly Gln Thr Ser Ala Glu Ser Tyr Leu Asp Phe Ala Lys Ile Leu Asp Ala Ala Ala Lys Ser Gly Ala Asn Ala Ile His Pro Gly Ty:r Gly Phe Leu Ala Glu Asn Ala Asp Phe Ala Gln Ala Val Ile Asp Ala Gly Leu Ile Trp Ile Gly Pro Ser Pro Gln Ser Ile Arg Asp Leu Gly Asp Lys Val Thr Ala Arg His Ile Ala Ala Arg Ala Gln Ala Pro Leu Val Pro Gly Thr Pro Asp Pro Val Lys Gly Ala Asp Glu Val Val Ala Phe Ala Glu Glu Tyr Gly Leu Pro Ile Ala Lle Lys Ala Ala His Gly Gly Gly Gly Lys Gly Met Lys Val Ala Arg Thr Ile Asp Glu Ile Pro Glu Leu Tyr Glu Ser Ala Val Arg Glu Ala Thr Ala Ala Phe Gly Arg Gly Glu Cys Tyr Val Glu Arg Tyr Leu Asp Lys Pro Arg His Val Glu Ala Gln Val Ile Ala Asp Gln His Gly Asn Val Val Val Ala Gly Thr Arg Asp Cys Ser Leu Gln Arg Arg Tyr Gln Lys Leu Val Glu Glu Ala Pro Ala Pro Phe Leu Thr Asp Phe Gln Arg Lys Glu Ile His Asp Ser Ala Lys Arg Ile Cys Lys Glu Ala His Tyr His Gly Ala Gly Thr Va_L Glu Tyr Leu Val Gly Gln Asp Gly Leu Ile Ser Phe Leu Glu Val Asn Thr Arg Leu Gln Val Glu His Pro Val Thr Glu Glu Thr Ala Gly Ile Asp Leu Val Leu Gln Gln Phe Arg Ile Ala Asn Gly Glu Lys Leu Asp Ile Thr Glu Asp Pro Thr Pro Arg Gly His Ala Ile Glu Phe Arg Ile Asn Gly Glu Asp Ala Gly Arg Asn Phe Leu Pro Ala Pro Gly Pro Val Thr Lys Phe His Pro Pro Ser Gly Pro Gly Val Arg Val Asp Ser Gly Val Glu Thr Gly Ser Val Ile Gly Gly Gln Phe Asp Ser Met Leu Ala Lys Leu Ile Val His Gly Ala Asp Arg Ala Glu Ala Leu Ala Arg Ala Arg Arg Ala Leu Asn Glu Phe Gly Val Glu Gly Leu Ala Thr Val Ile Pro Phe His Arg Ala Val Val Ser Asp Pro Ala Phe Ile Gly Asp Ala Asn Gly Phe Ser Val His Thr Arg Trp Ile Glu Thr Glu Trp Asn Asn Thr Ile Glu Pro Phe Thr Asp Gly Glu Pro Leu Asp Glu Asp Ala Arg Pro Arg Gln Lys 465 ~ 470 475 480 Val Val Val Glu Ile Asp Gly Arg Arg Val Glu Val Ser Leu Pro Ala Asp Leu Ala Leu Ser Asn Gly Gly Gly Cys Asp Pro Val Gly Val Ile Arg Arg Lys Pro Lys Pro Arg Lys Arg Gly Ala His Thr Gly Ala Ala Ala Ser Gly Asp Ala Val Thr Ala Pro Met Gln Gly Thr Val Val Lys Phe Ala Val Glu Glu Gly Gln Glu Val Val Ala Gly Asp Leu Val Val Val Leu Glu Ala Met Lys Met Glu Asn Pro Val Thr Ala His Lys Asp Gly Thr Ile Thr Gly Leu Ala Val Glu Ala Gly Ala Ala Ile Thr Gln Gly Thr Val Leu Ala Glu Ile Lys <210> 27 <211> 318 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(315) <400> 27 atg cca gtg gtg aag atc aac gca atc gag gtg cc<: gcc ggc get ggc 48 Met Pro Val Val Lys Ile Asn Ala Ile Glu Val Pro Ala Gly Ala Gly ccc gag ctg gag aag cgg ttc get cac cgc gcg cac gcg gtc gag aac 96 Pro Glu Leu Glu Lys Arg Phe Ala His Arg Ala His Ala Val Glu Asn tcc ccg ggt ttc ctc ggc ttt cag ctg tta cgt ccg gtc aag ggt gaa 144 Ser Pro Gly Phe Leu Gly Phe Gln Leu Leu Arg Pro Val Lys Gly Glu gaa cgc tac ttc gtg gtg aca cac tgg gag tcc gat gaa gca ttc cag 192 Glu Arg Tyr Phe Val Val Thr His Trp Glu Ser Asp Glu Ala Phe Gln gcg tgg gca aac ggg ccc gcc atc gca gcc cat gcc gga cac cgg gcc 240 Ala Trp Ala Asn Gly Pro Ala Ile Ala Ala His Ala Gly His Arg Ala aac ccc gtg gcg acc ggt get tcg ctg ctg gaa ttc gag gtc gtg ctt 288 Asn Pro Val Ala Thr Gly Ala Ser Leu Leu Glu Phe Glu Val Val Leu gac gtc ggt ggg acc ggc aag act gca taa 318 Asp Val Gly Gly Thr Gly Lys Thr Ala <210> 28 <211> 105 <212> PRT
<213> M.Tuberculosis <400> 28 Met Pro Val Val Lys Ile Asn Ala Ile Glu Val Pro Ala Gly Ala Gly Pro Glu Leu Glu Lys Arg Phe Ala His Arg Ala His Ala Val Glu Asn Ser Pro Gly Phe Leu Gly Phe Gln Leu Leu Arg Pro Val Lys Gly Glu ' Glu Arg Tyr Phe Val Val Thr His Trp Glu Ser Asp Glu Ala Phe Gln Ala Trp Ala Asn Gly Pro Ala Ile Ala Ala His Ala Gly His Arg Ala Asn Pro Val Ala Thr Gly Ala Ser Leu Leu Glu Phe Glu Val Val Leu Asp Val Gly Gly Thr Gly Lys Thr Ala <210> 29 <211> 935 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(935) <900> 29 gtggcg gacaagacg acacagacg atttacatc gacgcg gatccaggc 98 ValAla AspLysThr ThrGlnThr IleTyrIle AspAla AspProGly gaggtg atgaaggcg atcgccgac atcgaagcc tacccg caatggatt 96 GluVal MetLysAla IleAlaAsp IleGluAla TyrPro GlnTrpIle tcggag tataaggaa gtcgagatc ctagaggcc gacgac gagggctac 144 SerGiu TyrLysGlu ValGluIle LeuGluAla AspAsp GluGlyTyr ccgaaa cgagcgcga atgttgatg gacgcagcc atcttc aaagacacc 192 ProLys ArgAlaArg MetLeuMet AspAlaAla IlePhe LysAspThr ttg atc atg tcc tac gag tgg ccg gaa gac cgc caa tcg ctt agc tgg 240 Leu Ile Met Ser Tyr Glu Trp Pro Glu Asp Arg Gln Ser Leu Ser Trp WO 00/219$3 PCT/DK99/00538 act ctc gaa tcc.agc tcg ctg cta aag tcc ctc gaa ggc acg tat cgc 288 Thr Leu Glu Ser Ser Ser Leu Leu Lys Ser Leu Glu Gly Thr Tyr Arg ttg gcg ccc aag ggt tct ggc act gag gtc acc tac gag ctt gcc gtc 336 Leu Ala Pro Lys Gly Ser Gly Thr Glu Val Thr Tyr Glu Leu Ala Val gac ctt get gtc ccc atg atc ggg atg ctc aag cgt aag gcg gaa cgc 384 Asp Leu Ala Val Pro Met Ile Gly Met Leu Lys Arg Lys Ala Glu Arg agg ttg ata gac ggc gcg ttg aag gat ctg aag aaa cga gtc gag ggc 932 Arg Leu Ile Asp Gly Ala Leu Lys Asp Leu Lys Lys Arg Val Glu Gly tga 435 <210> 30 <211> 149 <212> PRT
<213> M.Tuberculosis <400> 30 Met Ala Asp Lys Thr Thr Gln Thr Ile Tyr Ile Asp Ala Asp Pro Gly Glu Val Met Lys Ala Ile Ala Asp Ile Glu Ala Tyr Pro Gln Trp Ile Ser Glu Tyr Lys Glu Val Glu Ile Leu Glu Ala Asp Asp Glu Gly Tyr Pro Lys Arg Ala Arg Met Leu Met Asp Ala Ala Ile Phe Lys Asp Thr Leu Ile Met Ser Tyr Glu Trp Pro Glu Asp Arg Gln Ser Leu Ser Trp Thr Leu Glu Ser Ser Ser Leu Leu Lys Ser Leu Glu Gly Thr Tyr Arg Leu Ala Pro Lys Gly Ser Gly Thr Glu Val Thr Tyr Glu Leu Ala Val Asp Leu Ala Val Pro Met Ile Gly Met Leu Lys Arg Lys Ala Glu Arg Arg Leu Ile Asp Gly Ala Leu Lys Asp Leu Lys Lys Arg Val Glu Gly <210> 31 <211> 441 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(938) <400> 31 atg cca gtt ttg agc aag acc gtc gag gtc acc gcc gac gcc gca tcg 48 Met Pro Val Leu Ser Lys Thr Val Glu Val Thr Ala Asp Ala Ala Ser atc atg gcc atc gtt gcc gat atc gag cgc tac cca gag tgg aat gaa 96 Ile Met Ala Ile Val Ala Asp Ile Glu Arg Tyr Pro Glu Trp Asn Glu ggg gtc aag ggc gca tgg gtg ctc get cgc tac gat gac ggg cgt ccc 144 Gly Val Lys Gly Ala Trp Val Leu Ala Arg Tyr Asp Asp Gly Arg Pro agc cag gtg cgg ctc gac acc get gtt caa ggc atc gag ggc acc tat 192 Ser Gln Val Arg Leu Asp Thr Ala Val Gln Gly Ile Glu Gly Thr Tyr atc cac gcc gtg tac tac cca ggc gaa aac cag att caa acc gtc atg 290 Ile His Ala Val Tyr Tyr Pro Gly Glu Asn Gln Ile Gln Thr Val Met cag cag ggt gaa ctg ttt gcc aag cag gag cag ctg ttc agt gtg gtg 288 Gln Gln Gly Glu Leu Phe Ala Lys Gln Glu Gln Leu Phe Ser Val Val gca acc ggc gcc gcg agc ttg ctc acg gtg gac atg gac gtc cag gtc 336 Ala Thr Gly Ala Ala Ser Leu Leu Thr Val Asp Met Asp Val Gln Val acc atg ccg gtg ccc gag ccg atg gtg aag atg ctg ctc aac aac gtc 384 Thr Met Pro Val Pro Glu Pro Met Val Lys Met Leu Leu Asn Asn Val ctg gag cat ctc gcc gaa aat ctc aag cag cgc gcc: gag cag ctg gcg 432 Leu Glu His Leu Ala Glu Asn Leu Lys Gln Arg Ala Glu Gln Leu Ala gcc agc taa 441 Ala Ser <210> 32 <211> 196 <212> PRT
<213> M.Tuberculosis <400> 32 Met Pro Val Leu Ser Lys Thr Val Glu Val Thr Ala Asp Ala Ala Ser Ile Met Ala Ile Val Ala Asp Ile Glu Arg Tyr Pro Glu Trp Asn Glu Gly Val Lys Gly Ala Trp Val Leu Ala Arg Tyr Asp Asp Gly Arg Pro Ser Gln Val Arg Leu Asp Thr Ala Val Gln Gly Ile Glu Gly Thr Tyr Ile His Ala Val Tyr Tyr Pro Gly Glu Asn Gln Ile Gln Thr Val Met Gln Gln Gly Glu Leu Phe A:La Lys Gln Glu Gln Leu Phe Ser Val Val Ala Thr Gly Ala Ala Ser Leu Leu Thr Val Asp Met Asp Val Gln Val Thr Met Pro Val Pro Glu Pro Met Val Lys Met Leu Leu Asn Asn Val Leu Glu His Leu Ala Glu Asn Leu Lys Gln Arg Ala Glu Gln Leu Ala Ala Ser <210> 33 <211> 894 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(891) <400> 33 atg tca tcg ggc aat tca tct ctg gga att atc gtc ggg atc gac gat 98 Met Ser Ser Gly Asn Ser Ser Leu Gly Ile Ile Val Gly Ile Asp Asp tca ccg gcc gca cag gtt gcg gtg cgg tgg gca get cgg gat gcg gag 96 Ser Pro Ala Ala Gln Val Ala Val Arg Trp Ala Ala Arg Asp Ala Glu ttg cga aaa atc cct ctg acg ctc gtg cac gcg gtg tcg ccg gaa gta 144 Leu Arg Lys Ile Pro Leu Thr Leu Val His Ala Val Ser Pro Glu Val gcc acc tgg ctg gag gtg cca ctg ccg ccg ggc gtg ctg cga tgg cag 192 Ala Thr Trp Leu Glu Val Pro Leu Pro Pro Gly Val Leu Arg Trp Gln cag gat cac ggg cgc cac ctg atc gac gac gca ctc aag gtg gtt gaa 240 Gln Asp His Gly Arg His Leu Ile Asp Asp Ala Leu Lys Val Val Glu cag get tcg ctg cgc get ggt ccc ccc acg gtc cac agt gaa atc gtt 288 Gln Ala Ser Leu Arg Ala Gly Pro Pro Thr Val His Ser Glu Ile Val ccg gcg gca gcc gtt ccc aca ttg gtc gac atg tcc aaa gac gca gtg 336 Pro Ala Ala Ala Val Pro Thr Leu Val Asp Met Se:r Lys Asp Ala Val ctg atg gtc gtg ggt tgt ctc gga agt ggg cgg tgg ccg ggc cgg ctg 384 Leu Met Val Val Gly Cys Leu Gly Ser Gly Arg Trp Pro Gly Arg Leu ctc ggt tcg gtc agt tcc ggc ctg ctc cgc cac gcg cac tgt ccg gtc 432 Leu Gly Ser Val Ser Ser Gly Leu Leu Arg His Ala His Cys Pro Val gtg atc atc cac gac gaa gat tcg gtg atg ccg cat ccc cag caa gcg 480 Val Ile Ile His Asp Glu Asp Ser Val Met Pro His Pro Gln Gln Ala ccg gtg cta gtt ggc gtt gac ggc tcg tcg gcc tcc gag ctg gcg acc 528 Pro Val Leu Val Gly Val Asp Gly Ser Ser Ala Ser Glu Leu Ala Thr gca atc gca ttc gac gaa gcg tcg cgg cga aac gtg gac ctg gtg gcg 576 Ala Ile Ala Phe Asp Glu Ala Ser Arg Arg Asn Va:L Asp Leu Val Ala ctg cac gca tgg agc gac gtc gat gtg tcg gag tgg ccc gga atc gat 624 Leu His Ala Trp Ser Asp Val Asp Val Ser Glu Trp Pro Gly Ile Asp tgg ccg gca act cag tcg atg gcc gag cag gtg ctg gcc gag cgg ttg 672 Trp Pro Ala Thr Gln Ser Met Ala Glu Gln Val Leu Ala Glu Arg Leu gcg ggt tgg cag gag cgg tat ccc aac gta gcc ata acc cgc gtg gtg 720 Ala Gly Trp Gln Glu Arg Tyr Pro Asn Val Ala Ile Thr Arg Val Val gtg cgc gat cag ccg gcc cgc cag ctc gtc caa cgc tcc gag gaa gcc 768 Va1 Arg Asp Gln Pro Ala Arg Gln Leu Val Gln Arg Ser Glu Glu Ala cag ctg gtc gtg gtc ggc agc cgg ggc cgc ggc ggc tac gcc gga atg 816 Gln Leu Val Val Val Gly Ser Arg Gly Arg Gly Gly Tyr Ala Gly Met ctg gtg ggg tcg gta ggc gaa acc gtt get cag ctg gcg cgg acg ccg 869 Leu Val Gly Ser Val Gly Glu Thr Val Ala Gln Leu Ala Arg Thr Pro gtc atc gtg gca cgc gag tcg ctg act tag 894 Val Ile Val Ala Arg Glu Ser Leu Thr <210> 39 <211> 297 <212> PRT
<213> M.Tuberculosis <400> 39 Met Ser Ser Gly Asn Ser Ser Leu Gly Ile Ile Val Gly Ile Asp Asp Ser Pro Ala Ala Gln Val Ala Val Arg Trp Ala Ala Arg Asp Ala Glu Leu Arg Lys Ile Pro Leu Thr Leu Val His Ala Val. Ser Pro Glu Val Ala Thr Trp Leu Glu Val Pro Leu Pro Pro Gly Val Leu Arg Trp Gln Gln Asp His Gly Arg His Leu Ile Asp Asp Ala Leu Lys Val Val Glu Gln Ala Ser Leu Arg Ala Gly Pro Pro Thr Val His Ser Glu Ile Val Pro Ala Ala Ala Val Pro Thr Leu Val Asp Met Ser Lys Asp Ala Val Leu Met Val Val Gly Cys Leu Gly Ser Gly Arg Trp Pro Gly Arg Leu Leu Gly Ser Val Ser Ser Gly Leu Leu Arg His Ala His Cys Pro Val Val Ile Ile His Asp Glu Asp Ser Val Met Pro His Pro Gln Gln Ala Pro Val Leu Val Gly Val Asp Gly Ser Ser Ala Ser Glu Leu Ala Thr Ala Ile Ala Phe Asp Glu Ala Ser Arg Arg Asn Val Asp Leu Val Ala Leu His Ala Trp Ser Asp Val Asp Val Ser Glu Trp Pro Gly Ile Asp Trp Pro Ala Thr Gln Ser Met Ala Glu Gln Val Leu Ala Glu Arg Leu Ala Gly Trp Gln Glu Arg Tyr Pro Asn Val Ala Ile Thr Arg Val Val Val Arg Asp Gln Pro Ala Arg Gln Leu Val Gln Arg Ser Glu Glu Ala Gln Leu Val Val Val Gly Ser Arg Gly Arg Gly Gly Tyr Ala Gly Met Leu Val Gly Ser Val Gly Glu Thr Val Ala Gln Leu Ala Arg Thr Pro Val Ile Val Ala Arg Glu Ser Leu Thr <210> 35 <211> 957 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(954) <900> 35 atg get gaa gta etg gtg cte gtt gag cac get gaa ggc gcg tta aag 48 Met Ala Glu Val Leu Val Leu Val Glu His Ala Glu Gly Ala Leu Lys aag gtc agc gcc gaa ttg atc acc gcc gcc cgc gcc: ttg ggc gaa cca 96 Lys Val Ser Ala Glu Leu Ile Thr Ala Ala Arg Ala Leu Gly Glu Pro gcc gcc gtc gtc gtc ggt gtg ccg ggg acg gcc gcg ccg ctg gtg gac 144 Ala Ala Val Val Val Gly Val Pro Gly Thr Ala Ala Pro Leu Val Asp ggg ctt aag gcg get ggt gcc gcc aag atc tac gtc: gcc gag tcc gac 192 Gly Leu Lys Ala Ala Gly Ala Ala Lys Ile Tyr Val. Ala Glu Ser Asp ctt gtc gac aaa tac ctg atc acc ccg gcg gtc gac: gtg ctg gcc ggg 290 Leu Val Asp Lys Tyr Leu Ile Thr Pro Ala Val Asp Val Leu Ala Gly ctg gcc gag tcc tcg gcc cct gcc ggc gta cta atc: gcc gcc acc gcg 288 Leu Ala Glu Ser Ser Ala Pro Ala Gly Val Leu Ile: Ala Ala Thr Ala gac gge aag gag atc gcc ggc cga ett gcg get cgg atc ggc tcg ggt 336 Asp Gly Lys Glu Ile Ala Gly Arg Leu Ala Ala Arg Ile Gly Ser Gly ctg ctg gtc gac gtg gtc gac gtg aga gaa ggt gga gtg ggt gtc cac 389 Leu Leu Val Asp Val Val Asp Val Arg Glu Gly Gly Val Gly Val His agc atc ttc ggt ggg gcg ttc acc gtc gaa gcg cag gcc aac ggc gac 432 Ser Ile Phe Gly Gly Ala Phe Thr Val Glu Ala Gln Ala Asn Gly Asp acc ccg gtg atc acc gtg cgc gca gga gcc gtg gag gcg gag ccg gcc 480 Thr Pro Val Ile Thr Val Arg Ala Gly Ala Val Glu Ala Glu Pro Ala gcc ggc gcc ggt gag cag gtc agc gtg gaa gtg ccg get gcg gcg gag 528 Ala Gly Ala Gly Glu Gln Val Ser Val Glu Val Pro Ala Ala Ala Glu aac gcc gcc agg atc acc gcg cgc gaa ccg gcg gtc gcc ggc gac cgg 576 Asn Ala Ala Arg Ile Thr Ala Arg Glu Pro Ala Va.1 Ala Gly Asp Arg ccg gag ctg acc gag gcg acc att gtg gtg gcc ggt ggc cgt ggt gtc 624 Pro Glu Leu Thr Glu Ala Thr Ile Val Val Ala Gly Gly Arg Gly Val ggc agc gcg gag aac ttc agc gtg gtc gag gcg ctg gcc gac tcg ctg 672 Gly Ser Ala Glu Asn Phe Ser Val Val Glu Ala Leu Ala Asp Sex Leu ggc gcc gcg gtc ggg gcc tcg cgt gcc gca gtc gac tcc ggc tac tac 720 Gly Ala Ala Val Gly Ala Ser Arg Ala Ala Val Asp Ser Gly Tyr Tyr ccg ggc cag ttc cag gtc ggc cag acc ggc aag acg gtg tcg ccc cag 768 Pro Gly Gln Phe Gln Val Gly Gln Thr Gly Lys Thr Val Ser Pro Gln ctc tac att gcc ctg ggc atc tcc ggg gcg atc cag cac cgc get ggc 816 Leu Tyr Ile Ala Leu Gly Ile Ser Gly Ala Ile Gln His Arg Ala Gly atg cag acg tcc aag acc atc gtc gcg gtc aac aag gac gaa gag gcg 864 Met Gln Thr Ser Lys Thr Ile Val Ala Val Asn Lys Asp Glu Glu Ala ccg atc ttt gag atc gcc gac tac ggg gtg gtg gga gac ctg ttc aag 912 Pro Ile Phe Glu Ile Ala Asp Tyr Gly Val Val Gly Asp Leu Phe Lys gtc get ccg cag ctg acc gag gcc atc aag gcc cgc aag ggc 954 Val Ala Pro Gln Leu Thr Glu Ala Ile Lys Ala Arg Lys Gly tag 957 <210> 36 <211> 318 <212> PRT
<213> M.Tuberculosis <400> 36 Met Ala Glu Val Leu Val Leu Val Glu His Ala Glu Gly Ala Leu Lys Lys Val Ser Ala Glu Leu Ile Thr Ala Ala Arg Ala Leu Gly Glu Pro Ala Ala Val Val Val Gly Val Pro Gly Thr Ala Ala Pro Leu Val Asp Gly Leu Lys Ala Ala Gly Ala Ala Lys Ile Tyr Val. Ala Glu Ser Asp Leu Val Asp Lys Tyr Leu Ile Thr Pro Ala Val Asp Val Leu Ala Gly Leu Ala Glu Ser Ser Ala Pro Ala Gly Val Leu Ile Ala Ala Thr Ala Asp Gly Lys Glu Ile Ala Gly Arg Leu Ala Ala Arg Ile Gly Ser Gly Leu Leu Val Asp Val Val Asp Val Arg Glu Gly Gly Val Gly Val His Ser Ile Phe Gly Gly Ala Phe Thr Val Glu Ala Gln Ala Asn Gly Asp Thr Pro Val Ile Thr Val Arg Ala Gly Ala Val Glu Ala Glu Pro Ala Ala Gly Ala Gly Glu Gln Val Ser Val Glu Val Pro Ala Ala Ala Glu Asn Ala Ala Arg Ile Thr Ala Arg Glu Pro Ala Val Ala Gly Asp Arg Pro Glu Leu Thr Glu Ala Thr Ile Val Val Ala Gly Gly Arg Gly Val Gly Ser Ala Glu Asn Phe Ser Val Val Glu Ala Leu Ala Asp Ser Leu Gly Ala Ala Val Gly Ala Ser Arg Ala Ala Val Asp Ser Gly Tyr Tyr Pro Gly Gln Phe Gln Val Gly Gln Thr Gly Lys Thr Val Ser Pro Gln Leu Tyr Ile Ala Leu Gly Ile Ser Gly Ala Ile Gln His Arg Ala Gly Met Gln Thr Ser Lys Thr Ile Val Ala Val Asn Lys Asp Glu Glu Ala Pro Ile Phe Glu Ile Ala Asp Tyr Gly Val Val Gly Asp Leu Phe Lys Val Ala Pro Gln Leu Thr Glu Ala Ile Lys Ala Arg Lys Gly <210> 37 <211> 1401 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(1398) <400> 37 gtg aag agc acc gtc gag cag ttg agc ccc acc cgg gtt cgt atc aac 98 Val Lys Ser Thr Val Glu Gln Leu Ser Pro Thr Arg Val Arg Ile Asn gtg gag gtg cca ttc gcc gag ctt gag ccg gat ttc cag cgg gcc tac 96 Val Glu Val Pro Phe Ala Glu Leu Glu Pro Asp Phe Gln Arg Ala Tyr aaa gag ctg gcc aaa cag gtg cgg ctg ccc ggc ttc cgg ccc ggg aag 194 Lys Glu Leu Ala Lys Gln Val Arg Leu Pro Gly Phe Arg Pro Gly Lys gcg ccg gcc aaa cta ctc gaa gcc cgc atc ggc cgg gag gcc atg ctg 192 Ala Pro Ala Lys Leu Leu Glu Ala Arg Ile Gly Arg Glu Ala Met Leu gat caa atc gtc aac gat gcg ctg ccc agc cgg tac gga cag gcg gtg 240 Asp Gln Ile Val Asn Asp A1_a Leu Pro Ser Arg Tyr Gly Gln Ala Val gcc gag tcg gat gtc caa ccg ctc ggc cgg ccc aac atc gag gtg acc 288 Ala Glu Ser Asp Val Gln Pro Leu Gly Arg Pro Asn Ile Glu Val Thr aag aag gag tac ggc cag gac ctg caa ttc acc gcc gag gtc gac atc 336 Lys Lys Glu Tyr Gly Gln Asp Leu Gln Phe Thr Ala Glu Val Asp Ile cgc ccg aag atc agt ccc ccg gac ctg agc gcg ctg acg gtc tcg gtg 384 Arg Pro Lys Ile Ser Pro Pro Asp Leu Ser Ala Leu Thr Val Ser Val gat ccg atc gaa atc ggt gag gac gac gtc gac gcc gaa ctg cag tcg 432 Asp Pro Ile Glu Ile Gly Glu Asp Asp Val Asp Ala Glu Leu Gln Ser tta cgt acc cgg ttc ggc acc ctg acc gcg gtg gac cgg ccg gtg gcc 480 Leu Arg Thr Arg Phe Gly Thr Leu Thr Ala Val Asp Arg Pro Val Ala gtc ggc gac gtc gtc tcg atc gac ttg tct gcc acg gtc gac gga gag 528 Val Gly Asp Val Val Ser Ile Asp Leu Ser Ala Thr Val Asp Gly Glu gac ata ccg aac gca gcc get gag gga ctc tcc cac gag gtc ggc tcc 576 Asp Ile Pro Asn Ala Ala Ala Glu Gly Leu Ser His Glu Val Gly Ser ggc cgg ctc atc gca ggt ctc gac gac gcg gtt gtt ggt ctg tcc gcc 624 Gly Arg Leu Ile Ala Gly Leu Asp Asp Ala Val Val Gly Leu Ser Ala gac gag tcc cgg gtc ttc acc gcc aag ctg gca gcc ggc gag cac gcc 672 Asp Glu Ser Arg Val Phe Thr Ala Lys Leu Ala Ala Gly Glu His Ala ggg cag gaa get cag gtt acc gtc acg gtc agg tcg gtt aag gag cgc 720 Gly Gln Glu Ala Gln Val Thr Val Thr Val Arg Ser Val Lys Glu Arg gaa cta cca gag ccc gac gac gaa ttc gcg cag tta gcc agc gag ttc 768 Glu Leu Pro Glu Pro Asp Asp Glu Phe Ala Gln Leu Ala Sex Glu Phe gac agc atc gac gaa ttg cgg gcc agc ctc agc gac cag gtg cgc cag 816 Asp Ser Ile Asp Glu Leu Arg Ala Ser Leu Ser Asp Gln Val Arg Gln gcc aag cgc gcc cag cag gcc gag cag att cga aac gcc acc atc gat 864 Ala Lys Arg Ala Gln Gln Ala Glu Gln Ile Arg Asn Ala Thr Ile Asp gcg cta ctc gaa cag gtc gac gtg ccg ttg ccg gag tcg tat gtg cag 912 Ala Leu Leu Glu Gln Val Asp Val Pro Leu Pro Glu Ser Tyr Val Gln gcc caa ttc gac agc gtg ctg cac agc gcg ctc agc ggt ctt aat cac 960 Ala Gln Phe Asp Ser Val Leu His Ser Ala Leu Ser Gly Leu Asn His gac gaa gcc cgg ttc aat gag ttg ctc gtc gag caa ggc tcg tca cgc 1008 Asp Glu Ala Arg Phe Asn Glu Leu Leu Val Glu Gln Gly Ser Ser Arg gcg gcg ttc gat gcc gag gcg cgc acc gcc tca gaa aag gac gtc aag 1056 Ala Ala Phe Asp Ala Glu Ala Arg Thr Ala Ser Glu Lys Asp Val Lys agg cag ctg ttg cta gac gcc ctg gcc gat gag ctg cag gtc caa gtt 1104 Arg Gln Leu Leu Leu Asp Ala Leu Ala Asp Glu Leu Gln Val Gln Val ggc cag gat gat ctg acc gaa cga ctg gtg acg acg tct cgg caa tac 1152 Gly Gln Asp Asp Leu Thr Glu Arg Leu Val Thr Thr Ser Arg Gln Tyr ggc atc gag ccg cag cag ctg ttc ggc tac ctc caa gag cgc aac cag 1200 Gly Ile Glu Pro Gln Gln Leu Phe Gly Tyr Leu Gln Glu Arg Asn Gln ctg ceg acc atg ttc get gac gtg egg cgc gag etg gcg atc agg gcc 1248 Leu Pro Thr Met Phe Ala Asp Val Arg Arg Glu Leu Ala Ile Arg Ala gca gtg gag gcg gcg acg gtc acc gac agt gac gga aac acg atc gat 1296 Ala Val Glu Ala Ala Thr Val Thr Asp Ser Asp Gly Asn Thr Ile Asp acc agt gag ttc ttc ggc aag cgt gtg teg gcc ggt: gag get gag gag 1344 Thr Ser Glu Phe Phe Gly Lys Arg Vai Ser Ala Gly Glu Ala Glu Glu gcc gaa ccg gca gac gag ggt gcc gcg cgg gcg gcq tcc gac gaa gcg 1392 Ala Glu Pro Ala Asp Glu Gly Ala Ala Arg Ala Ala Ser Asp Glu Ala aca acg tga 1401 Thr Thr <210> 38 <211> 466 <212> PRT
<213> M.Tuberculosis <400> 38 Met Lys Ser Thr Val Glu Gln Leu Ser Pro Thr Arg Val Arg Ile Asn Val Glu Val Pro Phe Ala Glu Leu Glu Pro Asp Phe Gln Arg Ala Tyr Lys Glu Leu Ala Lys Gln Val Arg Leu Pro Gly Phe Arg Pro Gly Lys Ala Pro Ala Lys Leu Leu Glu Ala Arg Ile Gly Arg Glu Ala Met Leu Asp Gln Ile Val Asn Asp Ala Leu Pro Ser Arg Tyr Gly Gln Ala Val Ala Glu Ser Asp Val Gln Pro Leu Gly Arg Pro Asn Ile Glu Val Thr Lys Lys Glu Tyr Gly Gln Asp Leu Gln Phe Thr Ala Glu Val Asp Ile Arg Pro Lys Ile Ser Pro Pro Asp Leu Ser Ala Leu Thr Val Ser Val Asp Pro Ile Glu Ile Gly Glu Asp Asp Val Asp Ala Glu Leu Gln Ser Leu Arg Thr Arg Phe Gly 'rhr Leu Thr Ala Val Asp Arg Pro Val Ala Val Gly Asp Val Val Ser Ile Asp Leu Ser Ala Thr Val Asp Gly Glu Asp Ile Pro Asn Ala Ala Ala Glu Gly Leu Ser His Glu Val Gly Ser Gly Arg Leu Ile Ala Gly Leu Asp Asp Ala Val Val Gly Leu Ser Ala Asp Glu Ser Arg Val Phe Thr Ala Lys Leu Ala Ala Gly Glu His Ala Gly Gln Glu Ala Gln Val Thr Val Thr Val Arg Ser Val Lys Glu Arg Glu Leu Pro Glu Pro Asp Asp Glu Phe Ala Gln Leu Ala Ser Glu Phe Asp Ser Ile Asp Glu Leu Arg Ala Ser Leu Ser Asp Gln Val Arg Gln Ala Lys Arg Ala Gln Gln Ala Glu Gln Ile Arg Asn Ala Thr Ile Asp Ala Leu Leu Glu Gln Val Asp Val Pro Leu Pro Glu Ser Tyr Val Gln Ala Gln Phe Asp Ser Val Leu His Ser Ala Leu Ser Gly Leu Asn His Asp Glu Ala Arg Phe Asn Glu Leu Leu Val Glu Gln Gly Ser Ser Arg Ala Ala Phe Asp Ala Glu Ala Arg Thr Ala Ser Glu Lys Asp Val Lys Arg Gln Leu Leu Leu Asp Ala Leu Ala Asp Glu Leu Gln Val Gln Val Gly Gln Asp Asp Leu Thr Glu Arg Leu Val Thr Thr Ser Arg Gln Tyr Gly Ile Glu Pro Gln Gln Leu Phe Gly Tyr Leu Gln Glu Arg Asn Gln Leu Pro Thr Met Phe Ala Asp Val Arg Arg Glu Leu Ala Ile Arg Ala Ala Val Glu Ala Ala Thr Val Thr Asp Ser Asp Gly Asn Thr Ile Asp Thr Ser Glu Phe Phe Gly Lys Arg Val Ser Ala Gly Glu Ala Glu Glu Ala Glu Pro Ala Asp Glu Gly Ala Ala Arg Ala Ala Ser Asp Glu Ala Thr Thr <210> 39 <211> 15 <212> PRT
<213> M.Tuberculosis <900> 39 Thr Glu Arg Thr Ala Val Leu Ile Lys Pro Asp Gly Ile Glu Arg <210> 90 <211> 15 <212> PRT
<213> M.Tuberculosis <900> 40 Thr Asp Thr Gln Val Thr Trp Leu Thr Gln Glu Ser His Asp Arg <210> 41 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 41 Met Ile Asp Glu Ala Leu Phe Asp Ala Glu Glu Lys Met Glu Lys <210> 42 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 42 Pro Leu Pro Ala Asp Pro Ser Thr Asp Leu Ser Ala Tyr Ala Gln <210> 43 <211> 15 <212> PRT
<213> M.Tuberculosis <900> 43 Met Leu Ile Ser Gln Arg Pro Thr Leu Ser Glu Asp Val Leu Thr <210> 44 <211> 15 <212> PRT
<213> M.Tuberculosis <900> 44 Thr Gly Asn Leu Val Thr hys Asn Ser Leu Thr Pro Asp Val Arg <210> 45 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 95 Met Glu Val Lys Ile Gly Ile Thr Asp Ser Pro Arg Glu Leu Val <210> 46 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 46 Ser Ala Tyr Lys Thr Val Val Val Gly Thr Asp Asp Xaa Ser Xaa <210> 47 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 47 Met Glu Gln Arg Ala Glu Leu Val Val Gly Arg Ala Leu Val Val <210> 98 <211> 15 <212> PRT
<213> M.Tuberculosis <900> 48 Ala Asp Ile Asp Gly Val Thr Gly Ser Ala Gly Leu Asn Pro Ala <210> 49 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 49 Thr Tyr Glu Thr Ile Leu Val Glu Arg Asp Gln Arg Val Gly Ile <210> 50 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 50 Pro Val Thr Gln Glu Glu Ile Ile Ala Gly Ile Ala Glu Ile Ile <210> 51 <211> 19 <212> PRT
<213> M.Tuberculosis <900> 51 Pro Val Val Lys Ile Asn Ala Ile Glu Val Pro Ala Gly Ala <210> 52 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 52 Ala Asp Lys Thr Thr Gln Thr Ile Tyr Ile Asp Ala Asp Pro Gly <210> 53 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 53 Pro Val Leu Ser Lys Thr Val Glu Val Thr Ala Asp Ala Ala Ser <210> 54 <211> 14 <212> PRT
<213> M.Tuberculosis <900> 54 Ser Gly Asn Ser Ser Leu Gly Ile Ile Val Gly Ile Asp Asp <210> 55 <211> 15 <212> PRT
<213> M.Tuberculosis <900> 55 Ala Glu Val Leu Val Leu Val Glu His Ala Glu Gly Ala Leu Lys <210> 56 <211> 15 <212> PRT
<213> M.Tuberculosis <900> 56 Met Lys Ser Thr Val Glu Gln Leu Ser Pro Thr Arg Val Arg Ile <210> 57 <211> 11 <212> PRT
<213> M.Tuberculosis <400> 57 Val Ile Arg Arg Lys Pro Lys Pro Arg Xaa Arg <210> 58 <211> 27 <212> DNA
<213> M.Tuberculosis <400> 58 ctgagatctg tggaggtcaa gatcggt 27 <210> 59 <211> 31 <212> DNA
<213> M.Tuberculosis <400> 59 ctcccatggc tacttacccg ctcgtagcaa c 31 <210> 60 <211> 27 <212> DNA
<213> M.Tuberculosis <400> 60 ctgagatctc ctgtcactca ggaagaa 27 <210> 61 <211> 27 <212> DNA

<213> M.Tuberculosis <400> 61 ctcccatggg aaaccgccat tagcggt 27 <210> 62 <211> 27 <212> DNA
<213> M.Tuberculosis <400> 62 cccaagctta tggaacagcg tgcggag <210> 63 <211> 27 <212> DNA
<213> M.Tuberculosis <900> 63 ctcccatggc gacactcgat ccggatt 27 <210> 64 <211> 27 <212> DNA
<213> M.Tuberculosis <400> 69 ctgagatcta tgccagtggt gaagatc 27 <210> 65 <211> 27 <212> DNA
<213> M.Tuberculosis <400> 65 ctcccatggt tatgcagtct tgccggt 27 <210> 66 <211> 27 <212> DNA
<213> M.Tuberculosis <400> 66 ctgagatctg cggacaagac gacacag 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 cagttttgag caagacc 27 <210> 69 <211> 27 <212> DNA
<213> M.Tuberculosis <900> 69 ctcccatggg cacatgcctt agctggc 27 <210> 70 <211> 27 <212> DNA

<213> M.Tuberculosis <900> 70 ctgagatcta tgtcatcggg caattca 27 <210> 71 <211> 31 <212> DNA

<213> M.Tuberculosis <400> 71 ctcccatggc 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> 79 <211> 398 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(398) <400> 79 atg gca ctc aag gta gag atg gtc act ttc gac tgc agc gac cct gcg 98 Met Ala Leu Lys Val Glu Met Val Thr Phe Asp Cys Ser Asp Pro Ala 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 Gln Phe Asp Gly Thr Thr Arg Glu ctg ctg ccc ggc gaa ttc gtc 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 ttg gga ttc cag aag gtg ccc gat ccc gcc cct ggg aaa aac cgc gtg 192 Leu Gly Phe Gln Lys Val Pro Asp Pro Ala Pro Gly Lys Asn Arg Val cac ctc gac ttc acg acc aag gac ctg gat gcc gag gtg ttg cgc ctg 290 His Leu Asp Phe Thr Thr Lys Asp Leu Asp Ala Glu Val Leu Arg Leu gtc gcc gcc gga gcc agt gag gtc ggg cgg cat cag gtc ggc gag agc 288 Val Ala Ala Gly Ala Ser Glu Val Gly Arg His Gln Val Gly Glu Ser ttt cgc tgg gtg gtg ctg get gac ccc gaa ggc aac get ttt tgc gtg 336 Phe Arg Trp Val Val Leu Ala Asp Pro Glu Gly Asn Ala Phe Cys Val gcg ggt caa taa 348 Ala Gly Gln <210> 75 <211> 115 <212> PRT
<213> M.Tuberculosis <400> 75 Met Ala Leu Lys Val Glu Met Val Thr Phe Asp Cys Ser Asp Pro Ala Lys Leu Ala Gly Trp Trp Ala Glu Gln Phe Asp Gly Thr Thr Arg Glu Leu Leu Pro Gly Glu Phe Val Val Val Ala Arg Thr Asp Gly Pro Arg Leu Gly Phe Gln Lys Val Pro Asp Pro Ala Pro Gly Lys Asn Arg Val His Leu Asp Phe Thr Thr Lys Asp Leu Asp Ala Glu Val Leu Arg Leu Val Ala Ala Gly Ala Ser Glu Val Gly Arg His Gln Val Gly Glu Ser Phe Arg Trp Val Val Leu Ala Asp Pro Glu Gly Asn Ala Phe Cys Val Ala Gly Gln <210> 76 <211> 569 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(564) <900> 76 atg gcc gac get gac acc acc gac ttc gac gtc gac gca gaa gca ccg 48 Met Ala Asp Ala Asp Thr Thr Asp Phe Asp Val Asp Ala Glu Ala Pro ggt gga ggc gtc cgg gag gac acg gcg acg gat get gac gag gcc gac 96 Gly Gly Gly Val Arg Glu Asp Thr Ala Thr Asp Ala Asp Glu Ala Asp gat caa gaa gag aga ttg gtc gcc gag ggc gag att gca ggc gac tac 149 Asp Gln Glu Glu Arg Leu Val Ala Glu Gly Glu Ile Ala Gly Asp Tyr ctg gaa gag tta ttg gac gtg ttg gac ttc gat ggc gac atc gac ctc 192 Leu Glu Glu Leu Leu Asp Val Leu Asp Phe Asp Gly Asp Ile Asp Leu gat gtc gaa ggc aat cgt gcg gtg gtg agc atc gac ggc agt gac gac 240 Asp Val Glu Gly Asn Arg Ala Val Val Ser Ile Asp Gly Ser Asp Asp ctg aac aag ttg gtc ggg cgc ggg ggc gag gtg ctc gac get ctg cag 288 Leu Asn Lys Leu Val Gly Arg Gly Gly Glu Val Leu Asp Ala Leu Gln gaa ctc acc cgg ttg gcg gtg cat cag aag acc ggt gtg cgg agc cgg 336 Glu Leu Thr Arg Leu Ala Val His Gln Lys Thr Gly Val Arg Ser Arg ttg atg cta gac atc gcg agg tgg cga cgg cgg cgc cgg gag gaa ttg 384 Leu Met Leu Asp Ile Ala Arg Trp Arg Arg Arg Arg Arg Glu Glu Leu gcg gcg ctg gcc gac gag gtg gcg cgg cga gtg gcc gaa acc ggt gac 432 Ala Ala Leu Ala Asp Glu Val Ala Arg Arg Val Ala Glu Thr Gly Asp cgc gag gaa ctc gtt cca atg acg ccg ttc gaa cgg aag atc gtc cac 480 Arg Glu Glu Leu Val Pro Met Thr Pro Phe Glu Arg Lys Ile Val His gat gcg gtt gca gcg gtg cca ggt gtg cac agc gaa agc gaa ggc gtg 528 Asp Ala Val Ala Ala Val Pro Gly Val His Ser Glu Ser Glu Gly Val gag cca gaa cgc cga gtc gtt gtg ctc cgc gac tag 564 Glu Pro Glu Arg Arg Val Val Val Leu Arg Asp <210> 77 <211> 187 <212> PRT
<213> M.Tuberculosis <400> 77 Met Ala Asp Ala Asp Thr Thr Asp Phe Asp Val Asp Ala Glu Ala Pro Gly Gly Gly Val Arg Glu Asp Thr Ala Thr Asp Ala Asp Glu Ala Asp Asp Gln Glu Glu Arg Leu Val Ala Glu Gly Glu Ile Ala Gly Asp Tyr Leu Glu Glu Leu Leu Asp Val Leu Asp Phe Asp Gly Asp Ile Asp Leu Asp Val Glu Gly Asn Arg Ala Val Val Ser Ile Asp Gly Ser Asp Asp Leu Asn Lys Leu Val Gly Arg Gly Gly Glu Val Leu Asp Ala Leu Gln Glu Leu Thr Arg Leu Ala Val His Gln Lys Thr Gly Val Arg Ser Arg Leu Met Leu Asp Ile Ala Arg Trp Arg Arg Arg Arg Arg Glu Glu Leu Ala Ala Leu Ala Asp Glu Val Ala Arg Arg Val Ala Glu Thr Gly Asp Arg Glu Glu Leu Val Pro Met Thr Pro Phe Glu Arg Lys Ile Val His Asp Ala Val Ala Ala Val Pro Gly Val His Ser Glu Ser Glu Gly Val Glu Pro Glu Arg Arg Val Val Val Leu Arg Asp <210> 78 <211> 1167 <212> DNA
<213> M.Tuberculosis <220>
<221> CDS
<222> (1)...(1167) <400> 78 atg agc aag acg gtt ctc atc ctt ggc gcg ggt gtc ggc ggc ctg acc 48 Met Ser Lys Thr Val Leu Ile Leu Gly Ala Gly Val Gly Gly Leu Thr acc gcc gac acc ctc cgt caa ctg cta cca cct gag gat cga atc ata 96 Thr Ala Asp Thr Leu Arg Gln Leu Leu Pro Pro Glu Asp Arg Ile Ile ttg gtg gac agg agc ttt gac ggg acg ctg ggc ttg tcg ttg cta tgg 149 Leu Val Asp Arg Ser Phe Asp Gly Thr Leu Gly Leu Ser Leu Leu Trp gtg ttg cgg ggc tgg cgg cgg cct gac gac gtc cgc: gtc cgc ccc acc 192 Val Leu Arg Gly Trp Arg Arg Pro Asp Asp Val Arg Val Arg Pro Thr gcggcgtcgctg cccggtgtg gaaatg gttactgca accgtcgcc cac 240 AlaAlaSerLeu ProGlyVal GluMet ValThrAla ThrValAla His attgacatcgcg gcccaggta gtgcac accgacaac agcgtcatc ggc 288 IleAspIleAla AlaGlnVal ValHis ThrAspAsn SerValIle Gly tatgacgcgttg gtgatcgca ttaggt gcggcgctg aacaccgac gcc 336 TyrAspAlaLeu ValIleAla LeuGly AlaAlaLeu AsnThrAsp Ala gttcccggactg tcggacgcg ctcgac gccgacgtc:gcgggccag ttc 384 ValProGlyLeu SerAspAla LeuAsp AlaAspVal.AlaGlyGln Phe tacaccctggac ggcgcgget gagctg cgtgcgaag gtcgaggcg etc 432 TyrThrLeuAsp GlyAlaAla GluLeu ArgAlaLys ValGluAla Leu gag cat ggc egg atc get gtg get ate gec ggg gtg ccg ttc aaa tgc 480 Glu His Gly Arg Ile Ala Val Ala Ile Ala Gly Val Pro Phe Lys Cys cca gcc gca ccg ttc gaa gcg gcg ttt ctg atc gcc gcc caa ctc ggt 528 Pro Ala Ala Pro Phe Glu Ala Ala Phe Leu Ile Ala Ala Gln Leu Gly gac cgc tac gcc acc gga acc gta cag atc gac acg ttc acg cct gac 576 Asp Arg Tyr Ala Thr Gly Thr Val Gln Ile Asp Thr Phe Thr Pro Asp ccg ctg ccg atg ecc gtt gca ggt cce gag gtc ggc gag get ttg gtc 624 Pro Leu Pro Met Pro Val Ala Gly Pro Glu Val Gly Glu Ala Leu Val tcgatgctc aaggatcac ggtgtc ggcttccat cctcgcaag gcccta 672 SerMetLeu LysAspHis GlyVal GlyPheHis ProArgLys AlaLeu 210 2.15 220 getcgcgtc gatgaggcc gcaagg acgatgcac ttcggtgac ggeacg 720 AlaArgVal AspGluAla AlaArg ThrMetHis PheGlyAsp GlyThr tccgaaccg ttcgatctg cttgcc gtggtcccc ccgcacgtg ccctcc 768 SerGluPro PheAspLeu LeuAla ValValPro ProHisVal ProSer gccgcggcg cggtcagcg ggtctc agcgaatcc gggtggata cccgtg 816 AlaAlaAla ArgSerAla GlyLeu SerGluSer GlyTrpIle ProVal gacccgcgc accctgtcc actagc gccgacaac gtgtgggcc atcggc 864 AspProArg ThrLeuSer ThrSer AlaAspAsn ValTrpAla IleGly gatgegacc gtgctgacg ctgeeg aatggcaaa ccgctgecc aagget 912 AspAlaThr ValLeuThr LeuPro AsnGlyLys ProLeuPro LysAla gccgtgttc gccgaagcc caggccgca gttgtcgcc cacggcgtc gcc 960 AlaValPhe AlaGluAla GlnAlaAla ValValAla HisGlyVal Ala cgccatctc ggttacgac gtagetgag cgccacttc accggcacg ggc 1008 ArgHisLeu GlyTyrAsp ValAlaGlu ArgHisPhe:ThrGlyThr Gly gcctgctac gtcgagacc ggtgatcac caggcagcc aagggcgac ggc 1056 AlaCysTyr ValGluThr GlyAspHis GlnAlaAla LysGlyAsp Gly gatttcttc getccgtcg gcgccctcg gtgacgctg taeccgccg tcg 1104 AspPhePhe AlaProSer AlaProSer ValThrLeu TyrProPro Ser cgggagttt cacgaggag aaggtcgca caagaactg gcctggctg acc 1152 ArgGluPhe HisGluGlu LysValAla GlnGluLeu AlaTrpLeu Thr cgctggaag acgtga 1167 ArgTrpLys Thr <210> 79 <211> 388 <212> PRT
<213> M.Tuberculosis <400> 79 Met Ser Lys Thr Val Leu Ile Leu Gly Ala Gly Val Gly Gly Leu Thr Thr Ala Asp Thr Leu Arg Gln Leu Leu Pro Pro Glu Asp Arg Ile Ile Leu Val Asp Arg Ser Phe Asp Gly Thr Leu Gly Leu Ser Leu Leu Trp Val Leu Arg Gly Trp Arg Arg Pro Asp Asp Val Arg Val Arg Pro Thr Ala Ala Ser Leu Pro Gly Val Glu Met Val Thr Ala Thr Val Ala His Ile Asp Ile Ala Ala Gln Val Val His Thr Asp Asn Ser Val Ile Gly Tyr Asp Ala Leu Val Ile Ala Leu Gly Ala Ala Leu Asn Thr Asp Ala Val Pro Gly Leu Ser Asp Ala Leu Asp Ala Asp Val Ala Gly Gln Phe Tyr Thr Leu Asp Gly Ala Ala Glu Leu Arg Ala Lys Val Glu Ala Leu Glu His Gly Arg Ile Ala Val Ala Ile Ala Gly Val Pro Phe Lys Cys Pro Ala Ala Pro Phe Glu Ala Ala Phe Leu Ile Ala Ala Gln Leu Gly Asp Arg Tyr Ala Thr Gly Thr Val Gln Ile Asp Thr Phe Thr Pro Asp Pro Leu Pro Met Pro Val Ala Gly Pro Glu Val Gly Glu Ala Leu Val Ser Met Leu Lys Asp His G.Ly Val Gly Phe His Pro Arg Lys Ala Leu Ala Arg Val Asp Glu Ala Ala Arg Thr Met His Phe Gly Asp Gly Thr Ser Glu Pro Phe Asp Leu Leu Ala Val Val Pro Pro His Val Pro Ser Ala Ala Ala Arg Ser Ala Gly Leu Ser Glu Ser Gly Trp Ile Pro Val Asp Pro Arg Thr Leu Ser Thr Ser Ala Asp Asn Val Trp Ala Ile Gly Asp Ala Thr Val Leu Thr Leu Pro Asn Gly Lys Pro Leu Pro Lys Ala Ala Val Phe Ala Glu Ala Gln Ala Ala Val Val Ala His Gly Val Ala Arg His Leu Gly Tyr Asp Val Ala Glu Arg His Phe Thr Gly Thr Gly Ala Cys Tyr Val Glu Thr Gly Asp His Gln Ala Ala Lys Gly Asp Gly Asp Phe Phe Ala Pro Ser Ala Pro Ser Val Thr Leu Tyr Pro Pro Ser Arg Glu Phe His Glu Glu Lys Val Ala Gln Glu Leu Ala Trp Leu Thr Arg Trp Lys Thr <210> 80 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 80 Ala Leu Lys Val Glu Met Val Thr Phe Asp Xaa Ser Asp Pro Ala <210> 81 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 81 Ala Asp Ala Asp Thr Thr Asp Phe Asp Val Asp Ala Glu Ala Pro <210> 82 <211> 15 <212> PRT
<213> M.Tuberculosis <400> 82 Ser Lys Thr Val Leu Ile Leu Gly Ala Gly Val Gly Gly Leu Thr <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> 85 <211> 27 <212> DNA
<213> M.Tuberculosis <400> 85 ctgagatcta tggccgacgc tgacacc 27 <210> 86 <211> 27 <212> DNA
<213> M.Tuberculosis <400> $6 ctcccatggc tagtcgcgga gcacaac 27 <210> 87 <211> 27 <212> DNA

<213> M.Tuberculosis <400> 87 ctgagatcta tgagcaagac ggttctc 27 <210> 88 <211> 27 ' <212> DNA

<213> M.Tuberculosis <400> 88 ctcccatggt cacgtcttcc agcgggt 27 <210> 89 <211> 28 <212> DNA

<213> M.Tuberculosis <400> 89 ctgccatggc taggtggtgt gcacgatc 28 <210> 90 <211> 27 <212> DNA

<213> M.Tuberculosis <900> 90 ctgaagctta tgagcgccta taagacc 27 <210> 91 <211> 27 <212> DNA

<213> M.Tuberculosis <400> 91 ctgagatcta tgattgatga ggctctc 27 <210> 92 <211> 27 <212> DNA

<213> M.Tuberculosis <400> 92 ctcccatgga gcggccgcta gacctcc 27 <210> 93 <211> 30 <212> DNA

<213> M.Tuberculosis <400> 93 ggctgagact catggccgac atcgatggtg 30 <210> 94 <211> 31 <212> DNA
<213> M.Tuberculosis <900> 94 cgtaccatgg tcatgacgac accccctcgt g 31 <210> 95 <211> 30 <212> DNA
<213> M.Tuberculosis <400> 95 ggctgagact catggctgaa gtactggtgc 30 <210> 96 <211> 31 <212> DNA
<213> M.Tuberculosis <400> 96 cgtaccatgg ctagccggcg accgccggtt c 31 <210> 97 <211> 20 <212> DNA
<213> M.Tuberculosis <900> 97 gtgaccgaac ggactctggt 20 <210> 98 <211> 21 <212> DNA
<213> M.Tuberculosis <900> 98 ctaggcgccg ggaaaccaga g 21 <210> 99 <211> 23 <212> DNA
<213> M.Tuberculosis <400> 99 atgacggata ctcaagtcac ctg 23 <210> 100 <211> 20 <212> DNA
<213> M.Tuberculosis <400> 100 ggagtggtac ggctcggcgc 20 <210> 101 <211> 20 <212> DNA
<213> M.Tubercuiosis <400> 101 atgacgtacg aaaccatcct 20 <210> 102 <211> 21 <212> DNA
<213> M.Tuberculosis <400> 102 tcatcggtgg gtgaactggg g 21 <210> 103 <211> 23 <212> DNA

<213> M.Tuberculosis <400> 103 atgccgcttc ccgcagaccc tag 23 <210> 104 <211> 21 <212> DNA

<213> M.Tuberculosis <900> 109 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 gtttcggcgt ag 22 <210> 107 <211> 20 <212> DNA
<213> M.Tuberculosis <400> 107 atgaccggaa atttggtgac 20 <210> 108 <211> 21 <212> DNA
<213> M.Tuberculosis <400> 108 tcagtagcgg tagtggtccg g 21

Claims (23)

1. A substantially pure polypeptide, which has a sequence identity of at least 80% to SEQ
ID NOs 34 or a subsequence of at least 8 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-.gamma. 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 10 6 virulent Mycobacteria, the induc-tion being performed by the addition of the polypeptide to a suspension comprising about 2 x 10 6 cells isolated from the spleen of said mouse, the addition of the polypeptide resulting in a concentration of the polypeptide of not more than 20 µg per ml suspension, the release of IFN-.gamma. being assessable by determination of IFN-.gamma. in supermatant 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 Myco-badana, determined by release of IFN-.gamma. of at least 1,500 pg/ml from T-lymphocytes with-drawn 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 sus-pension comprising about 2 x 10 5 cells isolated from the spleen, the addition of the poly-peptide resulting in a concentration of not more than 20 µg per ml suspension, the release of IFN-.gamma. being assessable by determination of IFN-.gamma. 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 10 6 virulent Myco-bacteria/ml by aerosol and determining a significant decrease in the number of bacteria re-coverable 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, 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 stimu-lated with the polypeptide in a concentration of not more than 20 µg/ml vi) the polypeptide reduces a positive in vitro response determined by release of IFN-.gamma. of at least 500 pg/ml from Peripheral Blood Mononuclear Cells (PBMC) withdrawn from an indi-vidual 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 µg per ml suspension, the release of IFN-.gamma. being assessable by determination of IFN-.gamma. in supernatant harvested 5 days after the addition of the polypeptide to the suspension, and does not induce such an IFN-.gamma. release in an individual not infected with a virulent Mycobac-ferium, viii) the polypeptide induces a positive DTH response determined by intradermal injection of at most 100 µg of the polypeptide to an individual who is clinically or subclinically infected with a virulent Mycobacterium, a positive response having a diameter of at feast 10 mm 72 hours after the injection, and does not induce such a response in an individual not infected with a virulent Mycobacterium.

2. A substantially pure polypeptide which comprises an amino acid sequence consisting of SEQ ID NO: 34.

3. A polypeptide according to any of claims 1 or 2, which comprises an amino add sequence which has a sequence identity of at least 80% to an amino acid sequence consisting of SEQ
ID NO: 34 and/or is a subsequence thereof.

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

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

6. A polypeptide according to any of claims 1-5, wherein the polypeptide is encodable by a nucleic acid sequence, which sequence 1) is the DNA sequence consisting of SEQ ID NO: 33 or an analogue of said sequence which hybridises with any of the DNA sequences shown in SEQ ID NO: 33 or a DNA
se-quence complementary thereto, or a specific part thereof, under stringent hybridization con-ditions, and/or 2) encodes a polypeptide, the amino acid sequence of which has a 8096 sequence identity with an amino acid sequence consisting of SEQ ID NO: 34 and/or 3) constitutes a subsequence of any of the above mentioned DNA sequences, and/or 4) constitutes a subsequence of any of the above mentioned polypeptide sequences.

7. A polypeptide as defined in any of claims 1-8 for use in medicine.

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

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

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

11. A composition comprising, as the effective component, a recombinant micro-organism, wherein at least one copy of a DNA sequence comprising a DNA sequence encoding a poly-peptide 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 secrete the polypeptide.

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

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

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

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

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

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

18. A method of screening for inhibition of the infectivity of a virulent Mycobacterium be-longing to the tuberculosis complex, said method comprising a) inhibiting the expression of one ar 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 tran-scription of the polypeptides or by interfering with regulatory sequences.

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

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

22. A nucleotide sequence which is a nucleotide sequence consisting of SEG ID
NO: 33 or an analogue of said sequence which hybridises with any of the nucleotide sequences shown in SEQ IQ NO: 33 or a nucleotide sequence complementary thereto, or a specific part or subsequence thereof, under stringent hybridisation conditions.

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