Mycobacterium tuberculosis DNA sequences encoding immunostimulatory peptides

Abstract

Nucleotide sequences isolated from Mycobacterium tuberculosis are disclosed. These sequences are shown to encode immunostimulatory peptides. The invention encompasses, among other things, vaccine preparations formulated using these peptides.

Description

I. BACKGROUND

A. The Rise of Tuberculosis

Over the past few years the editors of the Morbidity and Mortality Weekly Report have chronicled the unexpected rise in tuberculosis cases. It has been estimated that worldwide there are one billion people infected with M. tuberculosis, with 7.5 million active cases of tuberculosis. Even in the United States, tuberculosis continues to be a major problem especially among the homeless, Native Americans, African-Americans, immigrants, and the elderly. HIV-infected individuals represent the newest group to be affected by tuberculosis. Of the 88 million new cases of tuberculosis expected in this decade approximately 10% will be attributable to HIV infection.

The emergence of multi-dug resistant strains of M. tuberculosis has complicated matters further and even raises the possibility of a new tuberculosis epidemic. In the U.S. about 14% of M. tuberculosis isolates are resistant to at least one drug, and approximately 3% are resistant to at least two drugs. M. tuberculosis strains have even been isolated that are resistant to all seven drugs in the repertoire of drugs commonly used to combat tuberculosis. Resistant strains make treatment of tuberculosis extremely difficult: for example, infection with M. tuberculosis strains resistant to isoniazid and rifampin leads to mortality rates of approximately 90% among HIV-infected individuals. The mean time to death after diagnosis in this population is 4-16 weeks. One study reported that of nine immunocompetent health care workers and prison guards infected with drug resistant M. tuberculosis, five died. The expected mortality rate for infection with drug sensitive M. tuberculosis is 0%.

The unrelenting persistence of mycobacterial disease worldwide, the emergence of a new, highly susceptible population, and the recent appearance of drug resistant strains point to the need for new and better prophylactic and therapeutic treatments of mycobacterial diseases.

B. Tuberculosis and the Immune System

Infection with M. tuberculosis can take on many manifestations. The growth in the body of M. tuberculosis and the pathology that it induces is largely dependent on the type and vigor of the immune response. From mouse genetic studies it is known that innate properties of the macrophage play a large role in containing disease (1). Initial control of M. tuberculosis may also be influenced by reactive γδ T cells. However, the major immune response responsible for containment of M. tuberculosis is via helper T cells (Th1) and to a lesser extent cytotoxic T cells (2). Evidence suggests that there is very little role for the humoral response. The ratio of responding Th1 to Th2 cells has been proposed to be involved in the phenomenon of suppression.

Th1 cells are thought to convey protection by responding to M. tuberculosis T cell epitopes and secreting cytokines, particularly interferon-γ, which stimulate macrophages to kill M. tuberculosis. While such an immune response normally clears infections by many facultative intracellular pathogens, such as Salmonella, Listeria or Francisella, it is only able to contain the growth of other pathogens such as M. tuberculosis and Toxoplasma. Hence, it is likely that M. tuberculosis has the ability to suppress a clearing immune response, and mycobacterial components such as lipoarabinomannan are thought to be potential agents of this suppression. Dormant M. tuberculosis can remain in the body for long periods of time and can emerge to cause disease when the immune system wanes due to age or other effects such as infection with HIV-1.

Historically it has been thought that one needs replicating Mycobacteria in order to effect a protective immunization. An hypothesis explaining the molecular basis for the effectiveness of replicating mycobacteria in inducing protective immunity has been proposed by Orme and co-workers (3). These scientists suggest that antigens are pinocytosed from the mycobacteria-laden phagosome and used in antigen presentation. This hypothesis also explains the basis for secreted proteins effecting a protective immune response.

Antigens that stimulate T cells from M. tuberculosis infected mice or from PPD-positive humans are found in both the whole mycobacterial cells and also in the culture supernatants (3, 4, 5-7, 34). Recently Pal and Horwitz (8) were able to induce partial protection in guinea pigs by vaccinating with M. tuberculosis supernatant fluids. Similar results were found by Andersen using a murine model of tuberculosis (9). Other studies include reference nos. 34, 12. Although these works are far from definitive they do strengthen the notion that protective epitopes can be found among secreted proteins and that a non-living vaccine can protect against tuberculosis.

For the purposes of vaccine development one needs to find epitopes that confer protection but do not contribute to pathology. An ideal vaccine would contain a cocktail of T-cell epitopes that preferentially stimulate Th1 cells and are bound by different MHC haplotypes. Although such vaccines have never been made there is at least one example of a synthetic T-cell epitope inducing protection against an intracellular pathogen (10). It is an object of this invention to provide M. tuberculosis DNA sequences that encode bacterial peptides having an immunostimulatory activity. Such immunostimulatory peptides will be useful in the treatment, diagnosis and prevention of tuberculosis.

II. SUMMARY OF THE INVENTION

The present invention provides DNA sequences isolated from Mycobacterium tuberculosis. Peptides encoded by these DNA sequences are shown to stimulate the production of the macrophage-stimulating cytokine, gamma interferon (“INF-γ”), in mice. Critically, the production of INF-γ by CD4 cells in mice has been shown to correlate with maximum expression of protective immunity against tuberculosis (11). Furthermore, in human patients with active “minimal” or “contained” tuberculosis, it appears that the containment of the disease may be attributable, at least in part, to the production of CD4 Th-1-like lymphocytes that release INF-γ (12).

Hence, the DNA sequences provided by this invention encode peptides that are capable of stimulating T-cells to produce INF-γ. That is, these peptides act as epitopes for CD4 T-cells in the immune system. Studies have demonstrated that peptides isolated from an infectious agent and which are shown to be T-cell epitopes can protect against the disease caused by that agent when administered as a vaccine (13, 10). For example, T-cell epitopes from the parasite Leishmania major have been shown to be effective when administered as a vaccine (10, 13-14). Therefore, the immunostimulatory peptides (T-cell epitopes) encoded by the disclosed DNA sequences may be used, in purified form, as a vaccine against tuberculosis.

As noted, the nucleotide sequences of the present invention encode immunostimulatory peptides. In a number of instances, these nucleotide sequences are only a part of a larger open reading frame (ORF) of an M. tuberculosis operon. The present invention enables the cloning of the complete ORF using standard molecular biology techniques, based on the nucleotide sequences provided herein. Thus, the present invention encompasses both the nucleotide sequences disclosed herein and the complete M. tuberculosis ORFs to which they correspond. However, it is noted that since each of the nucleotide sequences disclosed herein encodes an immunostimulatory peptide, the use of larger peptides encoded by the complete ORFs is not necessary for the practice of the invention. Indeed, it is anticipated that, in some instances, proteins encoded by the corresponding ORFs may be less immunostimulatory than the peptides encoded by the nucleotide sequences provided herein.

One aspect of the present invention is an immunostimulatory preparation comprising at least one peptide encoded by the DNA sequences presented herein. Such a preparation may include the purified peptide or peptides and one or more pharmaceutically acceptable adjuvants, diluents and/or excipients. Another aspect of the invention is a vaccine comprising one or more peptides encoded by nucleotide sequences provided herein. This vaccine may also include one or more pharmaceutically acceptable excipients, adjuvants and/or diluents.

Another aspect of the present invention is an antibody specific for an immunostimulatory peptide encoded by a nucleotide sequence of the present invention. Such antibodies may be used to detect the present of M. tuberculosis antigens in medical specimens, such as blood or sputum. Thus, these antigens may be used to diagnose tuberculosis infections.

The present invention also encompasses the diagnostic use of purified peptides encoded by the nucleotide sequences of the present invention. Thus, the peptides may be used in a diagnostic assay to detect the presence of antibodies in a medical specimen, which antibodies bind to the M. tuberculosis peptide and indicate that the subject from which the specimen was removed was previously exposed to M. tuberculosis.

The present invention also provides an improved method of performing the tuberculin skin test to diagnose exposure of an individual to M. tuberculosis. In this improved skin test, purified immunostimulatory peptides encoded by the nucleotide sequences of this invention are employed. Preferably, this skin test is performed with one set of the immunostimulatory peptides, while another set of the immunostimulatory peptides is used to formulate vaccine preparations. In this way, the tuberculin skin test will be useful in distinguishing between subjects infected with tuberculosis and subjects who have simply been vaccinated. In this manner, the present invention may overcome a serious limitation inherent in the present BCG vaccine/tuberculin skin test combination.

Other aspects of the present invention include the use of probes and primers derived from the nucleotide sequences disclosed herein to detect the presence of M. tuberculosis nucleic acids in medical specimens.

A further aspect of the present invention is the discovery that a significant proportion of the immunostimulatory peptides are homologous to proteins known to be located in bacterial cell surface membranes. This discovery suggests that membrane-bound peptides, particularly those from M. tuberculosis, may be a new source of antigens for use in vaccine preparations.

III. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the deduced amino acid sequence of the full length MTB2-92 protein. The nucleic acid sequence is contained within Seq. I.D. No. 67, the amino acid sequence is shown in Seq. I.D. No. 113.

IV. DESCRIPTION OF THE INVENTION

A. Definitions

Particular terms and phrases used herein have the meanings set forth below.

“Isolated”. An “isolated” nucleic acid has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA. The term “isolated” thus encompasses nucleic acids purified by standard nucleic acid purification methods. The term also embraces nucleic acids prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

The nucleic acids of the present invention comprise at least a minimum length able to hybridize specifically with a target nucleic acid (or a sequence complementary thereto) under stringent conditions as defined below. The length of a nucleic acid of the present invention is preferably 15 nucleotides or greater in length, although a shorter nucleic acid may be employed as a probe or primer if it is shown to specifically hybridize under stringent conditions with a target nucleic acid by methods well known in the art. The phrase a “peptide of the present invention” means a peptide encoded by a nucleic acid molecule as defined in this paragraph.

“Probes” and “primers”. Nucleic acid probes and primers may readily be prepared based on the nucleic acid sequences provided by this invention. A “probe” comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in reference nos. 15 and 16.

“Primers” are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.

As noted, probes and primers are preferably 15 nucleotides or more in length, but, to enhance specificity, probes and primers of 20 or more nucleotides may be preferred.

Methods for preparing and using probes and primers are described, for example, in reference nos. 15, 16 and 17. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).

“Substantial similarity”. A first nucleic acid is “substantially similar” to a second nucleic acid if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 75%-90% of the nucleotide bases, and preferably greater than 90% of the nucleotide bases. (“Substantial sequence complementarity” requires a similar degree of sequence complementarity.) Sequence similarity can be determined by comparing the nucleotide sequences of two nucleic acids using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wis.).

“Operably linked”. A first nucleic acid sequence is “operably” linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

“Recombinant”. A “recombinant” nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

“Stringent Conditions” and “Specific”. The nucleic acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence, e.g., to a full length Mycobacterium tuberculosis gene that encodes an immunostimulatory peptide.

The term “stringent conditions” is functionally defined with regard to the hybridization of a nucleic-acid probe to a target nucleic acid (i.e., to a particular nucleic acid sequence of interest) by the hybridization procedure discussed in Sambrook et al. (1989) (reference no. 15) at 9.52-9.55. See also, reference no. 15 at 9.47-9.52, 9.56-9.58; reference no. 18 and reference no. 19.

Nucleic-acid hybridization is affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide-base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.

In preferred embodiments of the present invention, stringent conditions are those under which DNA molecules with more than 25% sequence variation (also termed “mismatch”) will not hybridize. Such conditions are also referred to as conditions of 75 % stringency (since hybridization will occur only between molecules with 75% sequence identity or greater). In more preferred embodiments, stringent conditions are those under which DNA molecules with more than 15% mismatch will not hybridize (conditions of 85% stringency). In most preferred embodiments, stringent conditions are those under which DNA molecules with more that 10% mismatch will not hybridize (i.e. conditions of 90% stringency).

When referring to a probe or primer, the term “specific for (a target sequence)” indicates that the probe or primer hybridizes under stringent conditions substantially only to the target sequence in a given sample comprising the target sequence.

“Purified”—a “purified” peptide is a peptide that has been extracted from the cellular environment and separated from substantially all other cellular peptides. As used herein, the term peptide includes peptides, polypeptides and proteins. In preferred embodiments, a “purified” peptide is a preparation in which the subject peptide comprises 80% or more of the protein content of the preparation. For certain uses, such as vaccine preparations, even greater purity may be necessary.

“Immunostimulatory”—the phrase “immunostimulatory peptide” as used herein refers to a peptide that is capable of stimulating INF-γ production in the assay described in section B 5 below. In preferred embodiments, an immunostimulatory peptide is one capable of inducing greater than twice the background level of this assay determined using T-cells stimulated with no antigens or negative control antigens. Preferably, the immunostimulatory peptides are capable of inducing more than 0.01 ng/ml of INF-γ in this assay system. In more preferred embodiments, an immunostimulatory peptide is one capable of inducing greater than 10 ng/ml of INF-γ in this assay system.

B. Materials and Methods

1. Standard Methodologies

The present invention utilizes standard laboratory practices for the cloning, manipulation and sequencing of nucleic acids, purification and analysis of proteins and other molecular biological and biochemical techniques, unless otherwise stipulated. Such techniques are explained in detail in standard laboratory manuals such as Sambrook et al. (15); and Ausubel et al. (16).

Methods for chemical synthesis of nucleic acids are discussed, for example, in reference nos. 20 and 21. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers.

2. Isolation of Mycobacterium Tuberculosis DNA Sequences Encoding Immunostimulatory Proteins

Mycobacterium tuberculosis DNA was obtained by the method of Jacobs et al. (22). Samples of the isolated DNA were partially digested with one of the following restriction enzymes HinPI, HpaII, AciI, TaqI, BsaHI, NarI. Digested fragments of 0.2-5 kb were purified from agarose gels and then ligated into the BstBI site in front of the truncated phoA gene in one or more of the three phagemid vectors pJDT1, pJDT2, and JDT3.

A schematic representation of the phagemid vector pJDT2 is provided in Mdluli et al. (1995) (reference no. 31). The pJDT vectors were specifically designed for cloning and selecting genes encoding cell wall-associated, cytoplasmic membrane associated, periplasmic or secreted proteins (and especially for cloning such genes from GC rich genomes, such as the Mycobacterium tuberculosis genome). The vectors have a BstBI cloning site in frame with the bacterial alkaline phosphatase gene (phoA) such that cloning of an in-frame sequence into the cloning site will result in the production of a fusion protein. The phoA gene encodes a version of the alkaline phosphatase that lacks a signal sequence; hence, only if the DNA cloned into the BstBI site includes a signal sequence or a transmembrane sequence can the fusion protein be secreted to the medium or inserted into cytoplasmic membrane, periplasm or cell wall. Those clones encoding such fusion proteins may be detected by plating clones on agar plates containing the indicator 5-bromo-4-chloro-3-indolyl-phosphate. Alkaline phosphatase converts this indicator to a blue colored product. Hence, those clones containing secreted alkaline phosphatase fusion proteins will produce the blue color.

The three vectors in this series (pJDT1, 2 and 3) have the BstBI restriction sites located in different reading frames with respect to the phoA gene. This increases the likelihood of cloning any particular gene in the correct orientation and reading frame for expression by a factor of 3. Reference no. 31 describes pJDT vectors in detail.

3. Selection of Secreted Fusion Proteins

The recombinant clones described above were transformed into E. coli and plated on agar plates containing the indicator 5-bromo-4-chloro-3-indolyl-phosphate. Production of blue pigmentation, produced as a result of the action of alkaline phosphatase on the indicator, indicated the presence of secreted cytoplasmic membrane periplasmic, cell wall associated or outer membrane fusion proteins (because the bacterial alkaline phosphatase gene in the vector lacks a signal sequence and could not otherwise escape the bacterial cell). A similar technique has been used to identify M. tuberculosis genes encoding exported proteins by Lim et al. (32).

Those clones producing blue pigmentation were picked and grown in liquid culture to facilitate the purification of the alkaline phosphatase fusion proteins. These recombinant clones were designated according to the restriction enzyme used to digest the Mycobacterium tuberculosis DNA (thus, clones designated A#2-1, A#2-2 etc were produced using Mycobacterium tuberculosis DNA digested with AciI).

4. Purification of Secreted Fusion Proteins

PhoA fusion proteins were extracted from the selected E. coli clones by cell lysis and purified by SDS polyacrylamide gel electrophoresis. Essentially, individual E. coli clones are grown overnight at 30° C. with shaking in 2 ml LB broth containing ampicillin, kanamycin and IPTG. The cells are precipitated by centrifugation and resuspended in 100 μL Tris -EDTA buffer. 100 μL lysis buffer (1% SDS, 1 mMEDTA, 25 mM DTT, 10% glycerol and 50 mM tris-HCl, pH 7.5) is added to this mixture and DNA released from the cells is sheared by passing the mixture through a small gauge syringe needle. The sample is then heated for 5 minutes at 100° C. and loaded onto an SDS PAGE gel (12 cm×14 cm×1.5 mm, made with 4% (w/v) acrylamide in the stacking section and 10% (w/v) acrylamide in the separating section). Several samples from each clone are loaded onto each gel.

The samples are electrophoresed by application of 200 volts to the gel for 4 hours. Subsequently, the proteins are transferred to a nitrocellulose membrane by Western blotting. A strip of nitrocellulose is cut off to be processed with antibody, and the remainder of the nitrocellulose is set aside for eventual elution of the protein. The strip is incubated with blocking buffer and then with anti-alkaline phosphatase primary antibody, followed by incubation with anti-mouse antibody conjugated with horse radish peroxidase. Finally, the strip is developed with the NEN DuPont Renaissance kit to generate a luminescent signal. The migratory position of the PhoA fusion protein, as indicated by the luminescent label, is measured with a ruler, and the corresponding region of the undeveloped nitrocellulose blot is excised.

This region of nitrocellulose, which contains the PhoA fusion protein, is then incubated in 1 ml 20% acetronitrile at 37° C. for 3 hours. Subsequently, the mixture is centrifuged to remove the nitrocellulose and the liquid is transferred to a new test tube and lyophilized. The resulting protein pellet is dissolved in 100 μL of endotoxin-free, sterile water and precipitated with acetone at −20° C. After centrifugation the bulk of the acetone is removed and the residual acetone is allowed to evaporate. The protein pellet is re-dissolved in 100 μL of sterile phosphate buffered saline. This procedure can be scaled up by modification to include IPTG induction 2 hours prior to cell harvesting, washing nitrocellulose membranes with PBS prior to acetonitrile extraction and lyophilization of acetonitrile extracted and acetone precipitated protein samples.

5. Determination of Immunostimulatory Capacity in Mice

The purified alkaline phosphatase— Mycobacterium tuberculosis fusion peptides encoded by the recombinant clones were then tested for their ability to stimulate INF-γ production in mice. The test used to determine INF-γ stimulation is as essentially that described by Orme et al. (11).

Essentially, the assay method is as follows: The virulent strain M. tuberculosis Erdman is grown in Proskauer Beck medium to mid-log phase, then aliquoted and frozen at −70° C. for use as an inoculant. Cultures of this bacterium are grown and harvested and nice are inoculated with 1×10 5 viable bacteria suspended in 200 μl sterile saline via a lateral tail vein on day one of the test.

Bone marrow-derived macrophages are used in the test to present the bacterial alkaline phosphatase- Mycobacterium tuberculosis fusion protein antigens. These macrophages are obtained by harvesting cells from mouse femurs and culturing the cells in Dulbecco's modified Eagle medium as described by Orme et al. (11). Eight to ten days later, up to ten μg of the fusion peptide to be tested is added to the macrophages and the cells are incubated for 24 hours.

The CD4 cells are obtained by harvesting spleen cells from the infected mice and then pooling and enriching for CD4 cells by removal of adherent cells by incubation on plastic Petri dishes, followed by incubation for 60 minutes at 37° C. with a mixture of J11d.2, Lyt-2.43, and GL4 monoclonal antibody (mAb) in the presence of rabbit complement to deplete B cells and immature T cells, CD8 cells, and γδ cells, respectively. The macrophages are overlaid with 10 6 of these CD4 cells and the medium is supplemented with 5 U IL-2 to promote continued T cell proliferation and cytokine secretion. After 72 hours, cell supernatants are harvested from sets of triplicate wells and assayed for cytokine content.

Cytokine levels in harvested supernatants are assayed by sandwich ELISA as described by Orme et al. (11).

6. Determination of Immunostimulatory Capacity in Humans

The purified alkaline phosphatase— Mycobacterium tuberculosis fusion peptides encoded by the recombinant clones or by synthetic peptides are tested for their ability to induce INF-γ production by human T cells in the following manner.

Blood from tuberculin positive people (producing a tuberculin positive skin test) is collected in EDTA coated tubes, to prevent clotting. Mononuclear cells are isolated using a modified version of the separation procedure provided with the NycoPrep™ 1.077 solution (Nycomed Pharma AS, Oslo, Norway). Briefly, the blood is diluted in an equal volume of a physiologic solution, such as Hanks Balanced Salt solution (HBSS), and then gently layered over top of the Nycoprep solution in a 2 to 1 ratio in 50 ml tubes. The tubes are centrifuged at 800× g for 20 minutes and the mononuclear cells are then removed from the interface between the Nycoprep solution and the sample layer. The plasma is removed from the top of the tube and filtered through a 0.2 micron filter and is then added to the tissue culture media. The mononuclear cells are washed twice: the cells are diluted in a physiologic solution, such as HBSS or RPMI 1640, and centrifuged at 400× g for 10 minutes. The mononuclear cells are then resuspended to the desired concentration in tissue culture media (RPMI 1640 containing 10% autologous serum, Hepes, non-essential amino acids, antibiotics and polymixin B). The mononuclear cells are then cultured in 96 well microtitre plates.

Peptides or PhoA fusion proteins are then added to individual wells in the 96 well plate, and cells are then placed in an incubator (37° C., 5% CO 2 ). Samples of the supernatants (tissue culture media from the wells containing the cells) are collected at various time points (from 3 to 8 days) after the addition of the peptides or PhoA fusion proteins. The immune responsiveness of T cells to the peptides and PhoA fusion proteins is assessed by measuring the production of cytokines (including gamma-interferon).

Cytokines are measured using an Enzyme Linked Immunosorbent Assay (ELISA), the details of which are described in the Cytokine ELISA Protocol in the PharMingen catalogue (PharMingen, San Diego, Calif.). For measuring for the presence of human gamma-interferon, wells of a 96 well microtitre plate are coated with a capture antibody (anti-human gamma-interferon antibody). The sample supernatants are then added to individual wells. Any gamma-interferon present in the sample will bind to the capture antibody. The wells are then washed. A detection antibody (anti-human gamma-interferon antibody), conjugated to biotin, is added to each well, and will bind to any gamma-interferon that is bound to the capture antibody. Any unbound detection antibody is washed away. An avidin peroxidase enzyme is added to each well (avidin binds tightly to the biotin on the detection antibody). Any excess unbound enzyme is washed away. Finally, a chromogenic substrate for the enzyme is added and the intensity of the colour reaction that occurs is quantitated using an ELISA plate reader. The quantity of the gamma-interferon in the sample supernatants is determined by comparison with a standard curve using known quantities of human gamma-interferon.

Measurement of other cytokines, such as Interleukin-2 and Interleukin-4, can be determined using the same protocol, with the appropriate substitution of reagents (monoclonal antibodies and standards).

7. DNA Sequencing

The sequencing of the alkaline phosphatase fusion clones was undertaken using the AmpliCycle thermal sequencing kit (Perkin Elmer, Applied Biosystems Division, 850 Lincoln Centre Drive, Foster City, Calif. 94404, U.S.A.), using a primer designed to read out of the alkaline phosphatase gene into the Mycobacterium tuberculosis DNA insert, or primers specific to the cloned sequences.

C. Results

1. Immunostimulatory Capacity

More than 300 fusion clones were tested for their ability to stimulate INF-γ production. Of these, 80 clones were initially designated to have some ability to stimulate INF-γ production. Tables 1 and 2 show the data obtained for these 80 clones. Clones placed in Table 1 showed the greatest ability to stimulate INF-γ production (greater than 10 ng/ml of INF-γ) while clones placed in Table 2 stimulated the production of between 2 ng/ml and 10 ng/ml of INF-γ. Background levels of INF-γ production (i.e., levels produced without any added M. tuberculosis antigen) were subtracted from the levels produced by the fusions to obtain the figures shown in these tables.

TABLE 1
Immunostimulatory AP-fusion clones
Seq			Sanger/TIGR ID of	Functional
ID	Name	INT	Mtb gene	Identification
2	AciI#1-152	>40,000	MTCY16By.09	glycerol-3-
				phosphate bind-
				ing periplasmic
				protein precursor
4	AciI#1-247	>40,000	MTCI364.18	fatty acid trans-
				port protein
65,66	AciI#1-264	>40,000	MTCY78.03c	unknown
62	AciI#1-435	>40,000	MTCY13D12.28	EmbA
75	HinP#1-27	>20,000	unfin. TIGR/gmt	unknown
			7458
67	HinP#2-92	>20,000	MTCY190.11c	cytochrome c oxi-
				dase subunit II
110	HinP#2-145	>20,000	unfin. TIGR/gmt	unknown
			7541
52	HinP#2-150	>20,000
48	HinP#1-200	>20,000	unfin. TIGR/gmt
			7491
54	HinP#3-30	>20,000	MTCY19H5.30c
6	AciI#2-2	>20,000	MTV003.10c	lipoprotein, peni-
				cillin binding
				protein
7	AciI#2-23	>20,000	MTCY 13E10/unfin.
			TIGR/gmt 7508
11	AciI#2-506	>20,000	MTCY253.27c	γ-glutamyl
				transpeptidase
				precursor
13	AciI#2-511	>20,000	MTCY50.08c	unknown
15	AciI#2-639	>20,000	MTCY02B12.02	unknown
16	AciI#2-822	>20,000	U27357 cds3/	unknown
			unfin. TIGR gmt
			7503
68	AciI#2-823	>20,000	MTCY77.20	unknown mem-
				brane protein
61	AciI#2-825	>20,000	MTCY31.03c
71	AciI#2-827	>20,000	MTCY01B2.15c	cytochrome d
				(ubiquinol) oxi-
				dase (appC)
22	AciI#2-898	>20,000	unfin. TIGR/gmt
			7554
27	AciI#2-1084	>20,000	unfin. TIGR/gmt
			7458
34	AciI#3-47	>20,000	MTCY50.02	oppA-like
36	AciI#3-133	>20,000	MTCY22G8	complement of
				ORF designated
38	AciI#3-166	>20,000	MTCY20H10.03	unknown/contains
			(19H5)	potential mem-
				brane spanning
				region
39	AciI#3-167	>20,000	MTCI28.14	unknown
41	AciI#3-206	>20,000	MTCY270.17	ftsQ
69	HinP#1-31	14,638		19kDa Antigen
47	HinP#1-144	13,546		unknown
70	HinP#1-3	11,550
111	AciI#1-486	11,416	MTCY13D12.26	embC (LysR
				family)
5	AciI#1-426	11,135
23	AciI#2-916	10,865	MTCY21D4.03c	unknown (signal
				peptide)
Abbreviations:
INF: pg/ml of INF-γ produced using fusion to stimulate immune T-cells. Sanger/TGIR ID of M. tuberculosis gene: matches produced from BLAST search of TIGR and Sanger Center databases. For Sanger matches, the information prior to the decimal point (e.g., MTCY21D4) identifies the cosmid clone and the numbers after the decimal point (e.g., .03) indicate the matching ORF within that cosmid; “c” indicates that the clone matched with the complement of that cosmid ORF sequence.

TABLE 2
Immunostimulatory AP-fusion clones
Seq	Clone		Sanger/TIGR ID of	Functional
ID	Name	INT	Mtb gene	Identification
1	AciI#1-62	3,126
—	AciI#2-14	6,907
8	AciI#2-26	3,089	unfin. TIGR/gmt	unknown
			7458
9	AciI#2-35	3,907	unfin. TIGR/gmt	unknown
			7458
76	AciI#2-147	5,464
12	AciI#2-508	7,052	MTCY20G9.23
—	AciI#2-510	2,445
14	AciI#2-523	2,479	MTCY427.10c	unknown
—	AciI#2-676	3,651
72	AciI#2-834	5,942
17	AciI#2-854	5,560	MTCY339.08c	unknown
18	AciI#2-872	2,361	MTCY22D7.18c	cstA-like
73	AciI#2-874	2,171	MTCY190.20	membrane protein
19	AciI#2-884D	2,729
21	AciI#2-894	3,396		unknown
24	AciI#2-1014	6,302
74	AciI#2-1018	4,642	MTCY270.11	UDP-N-
				acetylmuramooylalan
				yl-D-glutamyl-
				2,6,diaminopimelate-
				D-alanyl-D-alanyl
				ligase (murF)
25	AciI#2-1025	3,582	MTCY359.10	unknown membrane
				protein
—	AciI#2-1034	2,736
26	AciI#2-1035	3,454	MTCY04D9.11c	similar to penicillin
				binding proteins
28	AciI#2-1089	8,974	MTCY39.39	mpt 64
29	AciI#2-1090	7,449	MTCY04C12.18c	unknown membrane
				protein
30	AciI#2-1104	5,148	MTCY359.13	Precursor of Apa
				wag43 locus
31	AciI#3-9	3,160	MTCY164.01	unknown
32	AciI#3-12	3,891		penicillin binding
				protein
33	AciI#3-15	4,019
—	AciI#3-21	2,301
35	AciI3-78	2,905
37	AciI#3-134	3,895
40	AciI#3-204	4,774
42	AciI#3-214	7,333		unknown
112	AciI#3-243	2,857
43	AciI#3-281	2,943	MTCY19H5.32c
44	Bsa HI#1-21	8,122
45	HinP#1-12	2,905	MTCY49.31c	unknown
49	HinP#2-23	2,339
46	HinP#1-142	6,258	MTCY02B10.27c	unknown
—	HinP#2-4	6,567
50	HinP#2-143	3,689	MTCY274,09c	unknown,
				thioredoxin-like
51	HinP#2-145A	2,314
—	HinP#2-147	7,021	unfin. TIGR/gmt
			7491
53	HinP#3-28	2,980
55	HinP#3-34	2,564	MTCY25D10.07	unknown
56	HinP#3-41	3,296	P31953	Antigen 85c, 85b &
			P31952	85a precursor
			P17944
57	HpaII#1-3	2,360
58	HpaII#1-8	2,048	MTCY432	unknown
59	HpaII#1-10	4,178
60	HpaII#1-13	3,714	MTCY16B7.47	unknown partial
				ORF
Abbreviations:
INF: pg/ml of INF-γ produced using fusion to stimulate immune T-cells: Sanger/TGIR ID of M. tuberculosis gene: matches prodcuced from BLAST search of TIGR and Sanger Center databases. For Sanger matches, the information prior to the decimal point (e.g., MTCY21D4) identifies the cosmid clone and the numbers after the decimal point (e.g., .03) indicate the matching ORF within that cosmid; “c” indicates that the clone matched with the complement of that cosmid ORF sequence.

2. DNA Sequencing and Determination of Open Reading Frames

DNA sequence data for the sequences of the Mycobacterium tuberculosis DNA present in the clones shown in Tables 1 and 2 are shown in the accompanying Sequence Listing. The sequences are believed to represent the coding strand of the Mycobacterium DNA. In most instances, these sequences represent only partial sequences of the immunostimulatory peptides and, in turn, only partial sequences of Mycobacterium tuberculosis genes. However, each of the clones from which these sequences were derived encodes, by itself, at least one immunostimulatory T-cell epitope. As discussed in part V below, one of ordinary skill in the art will, given the information provided herein, readily be able to obtain the immunostimulatory peptides and corresponding fill length M. tuberculosis genes using standard techniques. Accordingly, the nucleotide sequences of the present invention encompass not only those sequences presented in the sequence listings, but also the complete nucleotide sequence encoding the immunostimulatory peptides as well as the corresponding M. tuberculosis genes. The nucleotide abbreviations employed in the sequence listings are as follows in Table 3:

TABLE 3
Symbol Meaning
A      A; adenine
C      C; cytosine
G      G; guanine
T      T; thymine
U      U; uracil
M      A or C
R      A or G
W      A or T/U
S      C or G
Y      C or T/U
K      G or T/U
V      A or C or G; not T/U
H      A or C or T/U; not G
D      A or G or T/U; not C
B      C or G or T/U; not A
N      (A or C or G or T/U) or (unknown or other
       or no base)
—      indeterminate*
*indicates an unreadable sequence compression.

The DNA sequences obtained were then analyzed with respect to the G+C content as a function of codon position over a window of 120 codons using the ‘FRAME’ computer program (Bibb, M. J.; Findlay, P. R.; and Johnson, M. W.; Gene 30: 157-166 (1984)). This program uses the bias of these nucleotides for each of the codon positions to enable the correct reading frame to be identified. As shown in tables 1 and 2, the sequences were also analyzed using the BLAST program on the TIGR database at the NCBI website (http://www.ncbi.gov/cgi-bin/BLAST/nph-tigrb1) and the Sanger Center website database (http://www/sanger.ac.uk/Projects/M_tuberculosis/blast_server.shtml). These sequence comparisons permitted matches with reported sequences to be identified and, for matches on the Sanger database, the identification of the open reading frame.

The sequence information revealed that a number of the clones contained overlapping sequence, as noted below:

Clone       Overlapping clone(s)
HinP#1-27   HinP#1-3
HinP#2-92   HinP#2-150, Aci#1-62, HpaII#1-3
HinP#1-200  AciI#2-147
AciI#2-639  AciI#2-676
AciI#3-47   AciI#3-204, AciI#3-243
AciI#3-133  AciI#3-134
AciI#3-166  AciI#3-15
AciI#3-167  AciI#3-78
AciI#2-916  AciI#2-1014
AciI#2-1089 HpaII#1-10
AciI#3-243  AciI#3-47

3. Identification of T Cell Epitopes in the Immunostimulatory Peptides

The T-Site program, by Feller, D. C. and de la Cruz, V. F., MedImmune Inc., 19 Firstfield Rd., Gaithersburg, Md. 20878, U.S.A., was used to predict T-cell epitopes from the determined coding sequences. It uses a series of four predictive algorithms. In particular, peptides were designed against regions indicated by the algorithm “A” motif which predicted alpha-helical periodicity (Margalit, H.; Spouge, J. L.; Cornette, J. L.; Cease, K. B.; DeLisi, C.; and Berzofsky, J. A., J. Immunol., 138:2213 (1987)) and amphipathicity and those indicated by the algorithm “R” motif which identifies segments which display similarity to motifs known to be recognized by MHC class I and class II molecules (Rothbard, J. B. and Taylor, W. R., EMBO J. 7:93 (1988)). The other two algorithms identify classes of T-cell epitopes recognized in mice.

4. Synthesis of Synthetic Peptides Containing T Cell Epitopes in Identified Immunostimulatory Peptides

A series of staggered peptides were designed to overlap regions indicated by the T-site analysis. These were synthesized by Chiron Mimotopes Pty. Ltd. (11055 Roselle St., San Diego, Calif. 92121, U.S.A.).

Peptides designed from sequences described in this application include:

		Seq
Peptide Sequence	Peptide Name	I.D. No.
Hin P#1-200 (6 peptides)
VHLATGMAETVASFSPS	HPI1-200/2	77
REVVHLATGMAETVASF	HPI1-200/3	78
RDSREVVHLATGMAETV	HPI1-200/4	79
DFNRDSREVVHLATGMA	HPI1-200/5	80
ISAAVVTGYLRWTTPDR	HPI1-200/6	81
AVVFLCAAAISAAVVTG	HPI1-200/7	82
AciI#2-827 (14 peptides)
VTDNPAWYRLTKFFGKL	CD-2/1/96/1	83
AWYRLTKFFGKLFLINF	CD-2/1/96/2	84
KFFGKLFLINFAIGVAT	CD-2/1/96/3	85
FLINFAIGVATGIVQEF	CD-2/1/96/4	86
AIGVATGIVQEFQFGMN	CD-2/1/96/5	87
TGIVQEFEFGMNWSEYS	CD-2/1/96/6	88
EFQFGMNWSEYSRFVGD	CD-2/1/96/7	89
MNWSEYSRFVGDVFGAP	CD-2/1/96/8	90
WSEYSRFVGDVFGAPLA	CD-2/1/96/9	91
EYSRFVGDVFGAPLAME	CD-2/1/96/10	92
SRFVGDVFGAPLAMESL	CD-2/1/96/11	93
WIFQWNRLPRLVHLACI	CD-2/1/96/12	94
WNRLPRLVHLACIWIVA	CD-2/1/96/13	95
GRAELSSIVVLLTNNTA	CD-2/1/96/14	96
HinP#1-3 (2 peptides)
GKTYDAYFTDAGGITPG	HPI1-3/2	97
YDAYFTDAGGITPGNSV	HPI1-3/3	98
HinP#1-3/HinP#1-200 combined peptides
WPQGKTYDAYFTDAGGI (HinP#1-3)	HPI1-3/1	99
	(combined)
ATGMAETVASFSPSEGS (HinP#1-200)		100
AciI#2-823 (1 peptide)
GWERRLRHAVSPKDPAQ	AI2-823/1	101
HinP#1-31 (4 peptides)
TGSGETTTAAGTTASPG	HPI1-31/1	102
GAAILVAGLSGCSSNKS	HPI1-31/2	103
AVAGAAILVAGLSGCSS	HPI1-31/3	104
LTVAVAGAAILVAGLSG	HPI1-31/4	105

These synthetic peptides were resuspended in phosphate buffered saline to be tested to confirm their ability to function as T cell epitopes using the procedure described in part IV(B)(6) above.

5. Confirmation of Immunostimulatory Capacity using T Cells from Tuberculosis Patients

The synthetic peptides described above, along with a number of the PhoA fusion proteins shown to be immunostimulatory in mice were tested for their ability to stimulate gamma interferon production in T-cells from tuberculin positive people using the methods described in part IV(B)(6) above. For each assay, 5×10 5 mononuclear cells were stimulated with up to 1 μg/ml M. tuberculosis peptide or up to 50 ng/ml Pho A fusion protein. M. tuberculosis filtrate proteins, Con A and PHA were employed as positive controls. An assay was run with media alone to determine background levels, and Pho A protein was employed as a negative control.

The results, shown in Table 4 below, indicate that all of the peptides tested stimulated gamma interferon production from T-cells of a particular subject.

TABLE 4
		Concentration of
	Concentration of	Interferon-gamma
Peptide or Pho A	Interferon-gamma	minus background
Fusion Protein Name	(pg/ml)	(pg/ml)
CD-2/1/96/1	256.6	153.3
CD-2/1/96/9	187.6	84.3
CD-2/1/96/10	134.0	30.7
CD-2/1/96/11	141.6	38.3
CD-2/1/96/14	310.2	206.9
HPI1-3/2	136.3	23.0
HPI1-3/3	264.2	160.9
AciI 2-898	134.0	30.7
AciI 3-47	386.8	283.5
M. tuberculosis filtrate	256.6	153.3
proteins (10 μg/ml)
M. tuberculosis filtrate	134.0	30.7
proteins (5 μg/ml)
Con A (10 μg/ml)	2839	2735.7
PHA (1%)	10378	10274.7
Pho A control	26.7	0
(10 μg/ml)
Background	103.3	0

V. CLONING OF FULL LENGTH Mycobacterium Tuberculosis T-Cell Epitope ORFs

Most the sequences presented represent only part of a larger M. tuberculosis ORF. If desired, the full length M. tuberculosis ORFs that include these provided nucleotide sequences can be readily obtained by one of ordinary skill in the art, based on the sequence data provided herein.

A. General Methodologies

Methods for obtaining full length genes based on partial sequence information are standard in the art and are particularly simple for prokaryotic genomes. By way of example, the full length ORFs corresponding to the DNA sequences presented herein may be obtained by creating a library of Mycobacterium tuberculosis DNA in a plasmid, bacteriophage or phagemid vector and screening this library with a hybridization probe using standard colony hybridization techniques. The hybridization probe consists of an oligonucleotide derived from a DNA sequence according to the present invention labelled with a suitable marker to enable detection of hybridizing clones. Suitable markers include radionuclides, such as P-32 and non-radioactive markers, such as biotin. Methods for constructing suitable libraries, production and labelling of oligonucleotide probes and colony hybridization are standard laboratory procedures and are described in standard laboratory manuals such as in reference nos. 15 and 16.

Having identified a clone that hybridizes with the oligonucleotide, the clone is identified and sequenced using standard methods such as described in Chapter 13 of reference no. 15. Determination of the translation initiation point of the DNA sequence enables the ORF to be located.

An alternative approach to cloning the full length ORFs corresponding to the DNA sequences provided herein is the use of the polymerase chain reaction (PCR). In particular, the inverse polymerase chain reaction (IPCR) is useful to isolate DNA sequences flanking a known sequence. Methods for amplification of flanking sequences by IPCR are described in Chapter 27 of reference no. 17 and in reference no. 23.

Accordingly, one aspect of the present invention is small oligonucleotides encompassed by the DNA sequences presented in the Sequence Listing. These small oligonucleotides are useful as hybridization probes and PCR primers that can be employed to clone the corresponding full length Mycobacterium tuberculosis ORFs. In preferred embodiments, these oligonucleotides will comprise at least 15 contiguous nucleotides of a DNA sequence set forth in the Sequence Listing, and in more preferred embodiments, such oligonucleotides will comprise at least 20 contiguous nucleotides of a DNA sequence set forth in the Sequence Listing.

One skilled in the art will appreciate that hybridization probes and PCR primers are not required to exactly match the target gene sequence to which they anneal. Therefore, in another embodiment, the oligonucleotides will comprise a sequence of at least 15 nucleotides and preferably at least 20 nucleotides, the oligonucleotide sequence being substantially similar to a DNA sequence set forth in the Sequence Listing. Preferably, such oligonucleotides will share at least about 75%-90% sequence identity with a DNA sequence set forth in the Sequence Listing and more preferably the shared sequence identity will be greater than 90%.

B. Example—Cloning of the Full Length ORF Corresponding to Clone HinP #2-92

Using the techniques described below, the full length gene corresponding to the clone HinP #2-92 was obtained. This gene, herein termed mtb2-92 includes an open-reading frame of 1089 bp (identified based on the G+C content relating to codon position). The alternative ‘GTG’ start codon was used, and this was preceded (8 bps upstream) by a Shine-Dalgarno motif. The gene mtb2-92 encoded a protein (termed MTB2-92) containing 363 amino acid residues with a predicted molecular weight of 40,436.4 Da.

Sequence homology comparisons of the predicted amino acid sequence of MTB2-92 with known proteins in the database indicated similarity to the cytochrome c oxidase subunit II of many different organisms. This integral membrane protein is part of the electron transport chain, subunits I and II forming the functional core of the enzyme complex.

1. Cloning the Full Length Gene Corresponding to HinP #2-92

The plasmid pHin2-92 was restricted with either BamH1 or EcoRI and then subcloned into the vector M13. The insert DNA fragments were sequenced under the direction of M13 universal sequencing primers (Yanisch-Perron, C. et al., 1985) using the AmpliCycle thermal sequencing kit (Perkin Elmer, Applied Biosystems Division, 850 Lincoln Centre Drive, Foster City, Calif. 94404, U.S.A.). The 5′-partial MTB2-92 DNA sequence was aligned using a GeneWorks (Intelligenetics, Mountain View, Calif., U.S.A.) program. Based on the sequence data obtained, two oligomers were synthesized. These oligonucleotides (5′ CCCAGCTTGTGATACAGGAGG 3′ (Seq. I.D. No. 106) and 5′ GGCCTCAGCGCGGCTCCGGAGG 3′ (Seq. I.D. No. 107)) represented sequences upstream and downstream, over an 0.8 kb distance, of the sequence encoding the partial MTB2-92 protein in the alkaline phosphatase fusion.

A Mycobacterium tuberculosis genomic cosmid DNA library was screened using PCR (Sambrook, J. et al., 1989) in order to obtain the full-length gene encoding the MTB2-92 protein. Two hundred and ninety-four bacterial colonies containing the cosmid library were pooled into 10 groups in 100 μl distilled water aliquots and boiled for 5 min. The samples were spun in a microfuge at maximal speed for 5 min. The supernatants were decanted and stored on ice prior to PCR analysis. The 100 μl-PCR reaction contained: 10 μl supernatant containing cosmid DNA, 10 μl of 10X PCR buffer, 250 μM dNTP's, 300 nM downstream and upstream primers, 1 unit Taq DNA polymerase.

The reactions were heated at 95° C. for 2 min and then 40 cycles of DNA synthesis were performed (95° C. for 30 s, 65° C. for 1 min, 72° C. for 2 min). The PCR products were loaded into a 1% agarose gel in TAE buffer (Sambrook, J. et al., 1989) for analysis.

The supernatant, which produced 800 bp PCR products, was then further divided into 10 samples and the PCR reactions were performed again. The colony which had resulted in the correctly sized PCR product was then picked. The cosmid DNA from the positive clone (pG3) was prepared using the Wizard Mini-Prep Kit (Promega Corp, Madison, Wis., U.S.A.). The cosmid DNA was further sequenced using specific oligonucleotide primers. The deduced amino acid sequence encoded by the MTB2-92 protein is shown in FIG. 1 .

2. Expression of the Full Length Gene

To conveniently purify the recombinant protein, a histidine tag coding sequence was engineered immediately upstream of the start codon of mtb2-92 using PCR. Two unique restriction enzyme sites for XbaI and HindIII were added to both ends of the PCR product for convenient subcloning. Two oligomers were used to direct the PCR reaction: (5′ TCTAGACACCACCACCACCACCACGTGACAC CTCGCGGGCCAGGTC 3′ (Seq. I.D. No. 108) and 5′ AAGCTTCGCCATGCCGC CGGTAAGCGCC 3′ (Seq. I.D. No. 109)).

The 100 μl PCR reaction contained: 1 μg pG3 template DNA, 250 μM dNTP's, 300 nM of each primer, 10 μl of 10X PCR buffer, 1 unit Taq DNA polymerase. The PCR DNA synthesis cycle was performed as above.

The 1.4 kb PCR products were purified and ligated into the cloning vector pGEM-T (Promega). Inserts were removed by digestion using both the XbaI and HindIII and the 1.4 kb fragment was directionally subcloned into the XbaI and HindIII sites of pMAL-c2 vector (New England Bio-Labs Ltd., 3397 American Drive, Unit 12, Mississauga, Ontario, L4V 1T8, Canada). The gene encoding MTB2-92 was fused, in frame, downstream of the maltose binding protein (MBP). This expression vector was named pMAL-MTB2-92.

3. Purification of the Encoded Protein

The plasmid pMAL-MTB2-92 was transformed into competent E. coli JM109 cells and a 1 liter culture was grown up in LB broth at 37° C. to an OD 550 of 0.5 to 0.6. The expression of the gene was induced by the addition of IPTG (0.5 mM) to the culture medium, after which the culture was grown for another 3 hours at 37° C. with vigorous shaking. Cultures were spun in the centrifuge at 10,000 g for 30 min and the cell pellet was harvested. This was re-suspended in 50 ml of 20 mM Tris-HCl, pH 7.2, 200 mM NaCl, 1 mM EDTA supplemented with 10 mM β mercaptoethanol and stored at −20° C.

The frozen bacterial suspension was thawed in cold water (0° C.), placed in an ice bath, and sonicated. The resulting cell lysate was then centrifuged at 10,000 g and 4° C. for 30 min, the supernatant retained, diluted with 5 volumes of buffer A and applied to an amylose-resin column (New England Bio-Labs Ltd., 3397 American Drive, Unit 12, Mississauga, Ontario, L4V 1T8, Canada) which had been pre-equilibrated with buffer A. The column was then washed with buffer A until the eluate reached an A 280 of 0.001 at which point, the bound MBP-MTB2-92 fusion protein was eluted with buffer A containing 10 mM maltose. The protein purified by the amylose-resin affinity column was about 84 kDa which corresponded to the expected size of the fusion protein (MBP: 42 kDa, MTB2-92 plus the histidine tag: 42 kDa).

The eluted MBP-MTB2-92 fusion protein was then cleaved with factor Xa to remove the MBP from the MTB2-92 protein. One ml of fusion protein (1 mg/ml) was mixed with 100 μl of factor Xa (200 μg/ml) and kept at room temperature overnight. The mixture was diluted with 10 ml of buffer B (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 6 M urea) and urea was added to the sample to a final concentration of 6 M urea. The sample was loaded onto the Ni-NTA column (QIAGEN, 9600 De Soto Ave., Chatsworth, Calif. 91311, U.S.A.) pre-equilibrated with buffer B. The column was washed with 10 volumes of buffer B and 6 volumes of buffer C (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 6 M urea). The bound protein was eluted with 6 volumes of buffer D (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 6 M urea).

At each stage of the protein purification, a sample was analysed by SDS polyacylamide gel electrophoresis (Laemmli, U.S. (1970) Nature ( London ), 227:680-685).

C. Correction of Sequence Errors

It is noted that some of the sequences presented in the Sequence Listing contain sequence ambiguities. Naturally, in order to ensure that the immunostimulatory function is maintained, one would utilize a sequence without such ambiguities. For those sequences containing ambiguities, one would therefore utilize the sequence data provided in the Sequence Listing to design primers corresponding to each terminal of the provided sequence and, using these primers in conjunction with the polymerase chain reaction, synthesize the desired DNA molecule using M. tuberculosis genomic DNA as a template. Standard PCR methodologies, such as those described above, may be used to accomplish this.

VI. EXPRESSION AND PURIFICATION OF THE CLONED PEPTIDES

Having provided herein DNA sequences encoding Mycobacterium tuberculosis peptides having an immunostimulatory activity, as well as the corresponding full length Mycobacterium tuberculosis genes, one of skill in the art will be able to express and purify the peptides encoded by these sequences. Methods for expressing proteins by recombinant means in compatible prokaryotic or eukaryotic host cells are well known in the art and are discussed, for example, in reference nos. 15 and 16. Peptides expressed by the nucleotide sequences disclosed herein are useful for preparing vaccines effective against M. tuberculosis infection, for use in diagnostic assays and for raising antibodies that specifically recognize M. tuberculosis proteins. One method of purifying the peptides is that presented in part V(B) above.

The most commonly used prokaryotic host cells for expressing prokaryotic peptides are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis Streptomyces or Pseudomonas may also be used, as is well known in the art. Partial or full-length DNA sequences, encoding an immunostimulatory peptide according to the present invention, may be ligated into bacterial expression vectors. One aspect of the present invention is thus a recombinant DNA vector including a nucleic acid molecule provided by the present invention. Another aspect is a transformed cell containing such a vector.

Methods for expressing large amounts of protein from a cloned gene introduced into Escherichia coli ( E. coli ) may be utilized for the purification of the Mycobacterium tuberculosis peptides. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described in reference no. 15 (ch. 17). Such fusion proteins may be made in large amounts, are relatively simple to purify, and can be used to produce antibodies. Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome binding site upstream of the cloned gene. If low levels of protein are produced, additional steps may be taken to increase protein production; if high levels of protein are produced, purification is relatively easy.

Often, proteins expressed at high levels are found in insoluble inclusion bodies. Methods for extracting proteins from these aggregates are described in ch. 17 of reference no. 15. Vector systems suitable for the expression of lacZ fusion genes include the pUR series of vectors (24), pEX1-3 (25) and pMR100 (26). Vectors suitable for the production of intact native proteins include pKC30 (27), pKK177-3 (28) and pET-3 (29). Fusion proteins may be isolated from protein gels, lyophilized, ground into a powder and used as antigen preparations.

Mammalian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, amphibian or avian species, may also be used for protein expression, as is well known in the art. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other prokaryotic and eukaryotic cells and cell lines may be appropriate for a variety of purposes, e.g., to provide higher expression, desirable glycosylation patterns, or other features.

Additionally, peptides, particularly shorter peptides, may be chemically synthesized, avoiding the need for purification from cells or culture media. It is known that peptides as short as 5 amino acids can act as an antigenic determinant and stimulate an immune response. Such peptides may be administered as vaccines in ISCOMs (Immune Stimulatory Complexes) as described by Janeway & Travers, Immunobiology: The Immune System In Health and Disease, 13.21 (Garland Publishing, Inc. New York, 1997). Accordingly, one aspect of the present invention includes small peptides encoded by the nucleic acid molecules disclosed herein. Such peptides include at least 5, and preferably 10 or more, contiguous amino acids of the peptides encoded by the disclosed nucleic acid molecules.

VII. SEQUENCE VARIANTS

It will be apparent to one skilled in the art that the immunostimulatory activity of the peptides encoded by the DNA sequences disclosed herein lies not in the precise nucleotide sequence of the DNA sequences, but rather in the epitopes inherent in the amino acid sequences encoded by the DNA sequences. It will therefore also be apparent that it is possible to recreate the immunostimulatory activity of one of these peptides by recreating the epitope, without necessarily recreating the exact DNA sequence. This could be achieved either by directly synthesizing the peptide (thereby circumventing the need to use the DNA sequences) or, alternatively, by designing a nucleic acid sequence that encodes for the epitope, but which differs, by reason of the redundancy of the genetic code, from the sequences disclosed herein.

Accordingly, the degeneracy of the genetic code further widens the scope of the present invention as it enables major variations in the nucleotide sequence of a DNA molecule while maintaining the amino acid sequence of the encoded protein. The genetic code and variations in nucleotide codons for particular amino acids is presented in Tables 5 and 6. Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from the DNA sequences disclosed herein using standard DNA mutagenesis techniques, or by synthesis of DNA sequences.

TABLE 5
The Genetic Code
	First	Second Pos'n	Third
	Pos'n	T	C	A	G	Pos'n
	T	Phe	Ser	Tyr	Cys	T
		Phe	Ser	Tyr	Cys	C
		Leu	Ser	Stop (och)	Stop	A
		Leu	Ser	Stop (amb)	Trp	G
	C	Leu	Pro	His	Arg	T
		Leu	Pro	His	Arg	C
		Leu	Pro	Gln	Arg	A
		Leu	Pro	Gln	Arg	G
	A	Ile	Thr	Asn	Ser	T
		Ile	Thr	Asn	Ser	C
		Ile	Thr	Lys	Arg	A
		Met	Thr	Lys	Arg	G
	G	Val	Ala	Asp	Gly	T
		Val	Ala	Asp	Gly	C
		Val	Ala	Glu	Gly	A
		Val (Met)	Ala	Glu	Gly	G
	“Stop (och)” stands for the ocre termination triplet, and “Stop (amb)” for the amber. ATG is the most common initiator codon; GTG usually codes for valine, but it can also code for methionine to initiate an mRNA chain.

TABLE 6
The Degeneracy of the Genetic Code
	Number of		Total
	Synontmous		Number of
	Codons	Amino Acid	Codons
	6	Leu, Ser, Arg	18
	4	Gly, Pro, Ala, Val, Thr	20
	3	Ile	3
	2	Phe, Tyr, Cys, His, Gln,	18
		Glu, Asn, Asp, Lys
	1	Met, Trp	2
	Total number of codons for amino acids	61
	Number of codons for termination	3
	Total number of codons in genetic code	64

Additionally, standard mutagenesis techniques may be used to produce peptides which vary in amino acid sequence from the peptides encoded by the DNA molecules disclosed herein. However, such peptides will retain the essential characteristic of the peptides encoded by the DNA molecules disclosed herein, i.e. the ability to stimulate INF-γ production. This characteristic can readily be determined by the assay technique described above. Such variant peptides include those with variations in amino acid sequence including minor deletions, additions and substitutions.

While the site for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed protein variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence as described above are well known.

In order to maintain the functional epitope, preferred peptide variants will differ by only a small number of amino acids from the peptides encoded by the DNA sequences disclosed herein. Preferably, such variants will be amino acid substitutions of single residues. Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 7 when it is desired to finely modulate the characteristics of the protein. Table 7 shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions. As noted, all such peptide variants are tested to confirm that they retain the ability to stimulate INF-γ production.

TABLE 7
Original Residue Conservative Substitutions
Ala              ser
Arg              lys
Asn              gln, his
Asp              glu
Cys              ser
Gln              asn
Glu              asp
Gly              pro
His              asn; gln
Ile              leu, val
Leu              ile; val
Lys              arg; gln; glu
Met              leu; ile
Phe              met; leu; tyr
Ser              thr
Thr              ser
Trp              tyr
Tyr              trp; phe
Val              ile; leu

Substantial changes in immunological identity are made by selecting substitutions that are less conservative than those in Table 7, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. However, such variants must retain the ability to stimulate INF-γ production.

VIII. USE OF CLONED MYCOBACTERIUM SEQUENCES TO PRODUCE VACCINES

The purified peptides encoded by the nucleotide sequences of the present invention may be used directly as immunogens for vaccination. The conventional tuberculosis vaccine is the BCG (bacille Calmette-Guerin) vaccine, which is a live vaccine comprising attenuated Mycobacterium bovis bacteria. However, the use of this vaccine in a number of countries, including the U.S., has been limited because administration of the vaccine interferes with the use of the tuberculin skin test to detect infected individuals (see Cecil Textbook of Medicine (Ref. 33), pages 1733-1742 and section VIII (2) below).

The present invention provides a possible solution to the problems inherent in the use of the BCG vaccine in conjunction with the tuberculin skin test. The solution proposed is based upon the use of one or more of the immunostimulatory M. tuberculosis peptides disclosed herein as a vaccine and one or more different immunostimulatory M. tuberculosis peptides disclosed herein in the tuberculosis skin test (see section IX (2) below). If the immune system is primed with such a vaccine, it will be able to resist an infection by M. tuberculosis. However, exposure to the vaccine peptides alone will not induce an immune response to those peptides that are reserved for use in the tuberculin skin test. Thus, the present invention would allow the clinician to distinguish between a vaccinated individual and an infected individual.

Methods for using purified peptides as vaccines are well known in the art and are described in the following publications: Pal and Horwitz (1992) (reference no. 8) (describing immunization with extra-cellular proteins of Mycobacterium tuberculosis ); Yang et al. (1991) (reference no. 30) (vaccination with synthetic peptides corresponding to the amino acid sequence of a surface glycoprotein from Leishmania major ); Andersen (1994) (reference no. 9) (vaccination using short-term culture filtrate containing proteins secreted by Mycobacterium tuberculosis ); and Jardim et al. (1990) (reference no. 10) (vaccination with synthetic T-cell epitopes derived from Leishmania parasite). Methods for preparing vaccines which contain immunogenic peptide sequences are also disclosed in U.S. Pat. Nos. 4,608,251, 4,601,903, 4,599,231, 4,5995230, 4,596,792 and 4,578,770. The formulation of peptide-based vaccines employing M. tuberculosis peptides is also discussed extensively in International Patent application WO 95/01441.

As is well known in the art, adjuvants such as Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may be used in formulations of purified peptides as vaccines. Accordingly, one embodiment of the present invention is a vaccine comprising one or more immunostimulatory M. tuberculosis peptides encoded by genes including a sequence shown in the attached sequence listing, together with a pharmaceutically acceptable adjuvant.

Additionally, the vaccines may be formulated using a peptide according to the present invention together with a pharmaceutically acceptable excipient such as water, saline, dextrose and glycerol. The vaccines may also include auxiliary substances such as emulsifying agents and pH buffers.

It will be appreciated by one of skill in the art that vaccines formulated as described above may be administered in a number of ways including subcutaneous, intramuscular and intra-venous injection. Doses of the vaccine administered will vary depending on the antigenicity of the particular peptide or peptide combination employed in the vaccine, and characteristics of the animal or human patient to be vaccinated. While the determination of individual doses will be within the skill of the administering physician, it is anticipated that doses of between 1 microgram and 1 milligram will be employed.

As with many vaccines, the vaccines of the present invention may routinely be administered several times over the course of a number of weeks to ensure that an effective immune response is triggered. As described in International Patent Application WO 95/01441, up to six doses of the vaccine may be administered over a course of several weeks, but more typically between one and four doses are administered. Where such multiple doses are administered, they will normally be administered at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain the desired levels of protective immunity.

As described in WO 95/01441, the course of the immunization may be followed by in vitro proliferation assays of PBL (peripheral blood lymphocytes) co-cultured with ESAT6 or ST-CF, and especially by measuring the levels of IFN-γ released from the primed lymphocytes. The assays are well known and are widely described in the literature, including in U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064.

To ensure an effective immune response against tuberculosis infection, vaccines according to the present invention may be formulated with more than one immunostimulatory peptide encoded by the nucleotide sequences disclosed herein. In such cases, the amount of each purified peptide incorporated into the vaccine will be adjusted accordingly.

Alternatively, multiple immunostimulatory peptides may also be administered by expressing the nucleic acids encoding the peptides in a nonpathogenic microorganism, and using this transformed nonpathogenic microorganism as a vaccine. As described in International Patent Application WO 95/01441, Mycobacterium bovis BCG may be employed for this purpose, although this approach would destroy the advantage outlined above to be gained from using separate classes of the peptides as vaccines and in the skin test. As disclosed in WO 95/01441, an immunostimulatory peptide of M. tuberculosis can be expressed in the BCG bacterium by transforming the BCG bacterium with a nucleotide sequence encoding the M. tuberculosis peptide. Thereafter, the BCG bacteria can be administered in the same manner as a conventional BCG vaccine. In particular embodiments, multiple copies of the M. tuberculosis sequence are transformed into the BCG bacteria to enhance the amount of M. tuberculosis peptide produced in the vaccine strain.

Finally, a recent development in the field of vaccines is the direct injection of nucleic acid molecules encoding peptide antigens, as described in Janeway & Travers, Immunobiology: The Immune System In Health and Disease, 13.25 (Garland Publishing, Inc. New York, 1997); McDonnell & Askari, N. Engl. J. Med. 334:42-45 (1996). Thus, plasmids which include nucleic acid molecules described herein, or which include nucleic acid sequences encoding peptides according to the present invention may be utilized in such DNA vaccination methods.

IX. USE OF CLONED MYCOBACTERIUM SEQUENCES IN DIAGNOSTIC ASSAYS

Another aspect of the present invention is a composition for diagnosing tuberculosis infection wherein the composition includes peptides encoded by the nucleotide sequences of the present invention. The invention also encompasses methods and compositions for detecting the presence of anti-tuberculosis antibodies, tuberculosis peptides and tuberculosis nucleic acid sequences in body samples. Three examples typify the various techniques that may be used to diagnose tuberculosis infection using the present invention: an in vitro ELISA assay, an in vivo skin test assay and a nucleic acid amplification assay.

A. In Vitro ELISA Assay

One aspect of the invention is an ELISA that detects anti-tuberculosis mycobacterial antibodies in a medical specimen. An immunostimulatory peptide encoded by a nucleotide sequence of the present invention is employed as an antigen and is preferably bound to a solid matrix such as a crosslinked dextran such as SEPHADEX (Pharmacia, Piscataway, N.J.), agarose, polystyrene, or the wells of a microtiter plate. The polypeptide is admixed with the specimen, such as human sputum, and the admixture is incubated for a sufficient time to allow antimycobacterial antibodies present in the sample to immunoreact with the polypeptide. The presence of the immunopositive immunoreaction is then determined using an ELISA assay.

In a preferred embodiment, the solid support to which the polypeptide is attached is the wall of a microtiter assay plate. After attachment of the polypeptide, any nonspecific binding sites on the microtiter well walls are blocked with a protein such as bovine serum albumin. Excess bovine serum albumin is removed by rinsing and the medical specimen is admixed with the polypeptide in the microtiter wells. After a sufficient incubation time, the microtiter wells are rinsed to remove excess sample and then a solution of a second antibody, capable of detecting human antibodies is added to the wells. This second antibody is typically linked to an enzyme such as peroxidase, alkaline phosphatase or glucose oxidase. For example, the second antibody may be a peroxidase-labeled goat anti-human antibody. After further incubation, excess amounts of the second antibody are removed by rinsing and a solution containing a substrate for the enzyme label (such as hydrogen peroxide for the peroxidase enzyme) and a color-forming dye precursor, such as o-phenylenediamine is added. The combination of mycobacterium peptide (bound to the wall of the well), the human antimycobacterial antibodies (from the specimen), the enzyme-conjugated anti-human antibody and the color substrate will produce a color than can be read using an instrument that determines optical density, such as a spectrophotometer. These readings can be compared to a control incubated with water in place of the human body sample, or, preferably, a human body sample known to be free of antimycobacterial antibodies. Positive readings indicate the presence of anti-mycobacterial antibodies in the specimen, which in turn indicate a prior exposure of the patient to tuberculosis.

B. Skin Test Assay

Alternatively, the presence of tuberculosis antibodies in a patient's body may be detected using an improved form of the tuberculin skin test, employing immunostimulatory peptides of the present invention. Conventionally, this test produces a positive result to one of the following conditions: the current presence of M. tuberculosis in the patient's body; past exposure of the patient to M. tuberculosis; and prior BCG vaccination. As noted above, if one group of immunostimulatory peptides is reserved for use in vaccine preparations, and another group reserved for use in the improved skin test, then the skin test will not produce a positive response in individuals whose only exposure to tuberculosis antigens was via the vaccine. Accordingly, the improved skin test would be able to properly distinguish between infected individuals and vaccinated individuals.

The tuberculin skin test consists of an injection of proteins from M. tuberculosis that are injected intradermally. The test is described in detail in Cecil Textbook of Medicine (Ref. 33), pages 1733-1742. If the subject has reactive T-cells to the injected protein, the cells will migrate to the site of injection and cause a local inflammation. This inflammation, which is generally known as delayed type hypersensitivity (DTH) is indicative of M. tuberculosis antibodies in the patient's blood stream. Purified immunostimulatory peptides according to the present invention may be employed in the tuberculin skin test using the methods described in reference 33.

C. Nucleic Acid Amplification

One aspect of the invention includes nucleic acid primers and probes derived from the sequences set forth in the attached sequence listing, as well as primers and probes derived from the full length genes that can be obtained using these sequences. These primers and probes can be used to detect the presence of M. tuberculosis nucleic acids in body samples and thus to diagnose infection. Methods for making primers and probes based on these sequences are well known and are described in section V above.

The detection of specific pathogen nucleic acid sequences in human body samples by polymerase chain reaction amplification (PCR) is discussed in detail in reference 17, in particular, part four of that reference. To detect M. tuberculosis sequences, primers based on the sequences disclosed herein would be synthesized, such that PCR amplification of a sample containing M. tuberculosis DNA would result in an amplified fragment of a predicted size. If necessary, the presence of this fragment following amplification of the sample nucleic acid could be detected by dot blot analysis (see chapter 48 of reference 17). PCR amplification employing primers based on the sequences disclosed herein may also be employed to quantify the amounts of M. tuberculosis nucleic acid present in a particular sample (see chapters 8 and 9 of reference 17). Reverse-transcription PCR using these primers may also be utilized to detect the presence of M. tuberculosis RNA, indicative of an active infection.

Alternatively, probes based on the nucleic acid sequences described herein may be labelled with suitable labels (such a P 32 or biotin) and used in hybridization assays to detect the presence of M. tuberculosis nucleic acid in provided samples.

X. USE OF CLONED MYCOBACTERIUM SEQUENCES TO RAISE ANTIBODIES

Monoclonal antibodies may be produced to the purified M. tuberculosis peptides for diagnostic purposes. Substantially pure M. tuberculosis peptide suitable for use as an immunogen is isolated from the transfected or transformed cells as described above. The concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few milligrams per milliliter. Monoclonal antibody to the protein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of the M. tuberculosis peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected purified protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (1988).

B. Antibodies Raised Against Synthetic Peptides

An alternative approach to raising antibodies against the M. tuberculosis peptides is to use synthetic peptides synthesized on a commercially available peptide synthesizer based upon the amino acid sequence of the peptides predicted from nucleotide sequence data.

In a preferred embodiment of the present invention, monoclonal antibodies that recognize a specific M. tuberculosis peptide are produced. Optimally, monoclonal antibodies will be specific to each peptide, i.e. such antibodies recognize and bind one M. tuberculosis peptide and do not substantially recognize or bind to other proteins, including those found in healthy human cells.

The determination that an antibody specifically detects a particular M. tuberculosis peptide is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al., 1989). To determine that a given antibody preparation (such as one produced in a mouse) specifically detects one M. tuberculosis peptide by Western blotting, total cellular protein is extracted from a sample of human sputum from a healthy patient and from sputum from a patient suffering from tuberculosis. As a positive control, total cellular protein is also extracted from M. tuberculosis cells grown in vitro. These protein preparations are then electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. Thereafter, the proteins are transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove non-specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase; application of the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immuno-localized alkaline phosphatase. Antibodies which specifically detect the M. tuberculosis protein will, by this technique, be shown to bind to the M. tuberculosis -extracted sample at a particular protein band (which will be localized at a given position on the gel determined by its molecular weight) and to the proteins extracted from the sputum from the tuberculosis patient. No significant binding will be detected to proteins from the healthy patient sputum. Non-specific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-tuberculosis protein binding. Preferably, no antibody would be found to bind to proteins extracted from healthy donor sputum.

Antibodies that specifically recognize a M. tuberculosis peptide encoded by the nucleotide sequences disclosed herein are useful in diagnosing the presence of tuberculosis antigens in patients.

All publications and published patent documents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

XI. REFERENCES

1. Skamene, E. (1989). Genetic control of susceptibility to Mycobacterial infections. Ref. Infect. Dis. 11:S394-S399.

2. Kaufmann, S. H. E. (1991). Role of T-Cell Subsets in Bacterial Infections. Current Opinion in Immunology 3:465-470.

3. Orme, I. M., et al. (1992). T Lymphocytes Mediating Protection and Cellular Cytolysis During the Course of Mycobacterium-Tuberculosis Infection—Evidence for Different Kinetics and Recognition of a Wide Spectrum of Protein Antigens. Journal of Immunology 148:189-196.

4. Daugelat, S., et al. (1992). Secreted Antigens of Mycobacterium tuberculosis: characterization with T Lymphocytes from Patients and Contacts after Two-Dimensional Separation. J. Infect. Dis. 166:186-190.

5. Barnes et al. (1989). Characterization of T Cell Antigens Associated with the Cell Wall Protein-Peptidoglycan Complex of Mycobacterium tuberculosis. J. Immunol. 143:2656-2662.

6. Collins et al. (1988). Biological activity of protein antigens isolated from Mycobacterium tuberculosis culture filtrate. Infect. Immun. 56:1260-1266.

7. Lamb et al. (1989). Identification of Mycobacterial Antigens Recognized by T Lymphocytes, Rev. Infect. Dis. 11:S443-S447.

8. Pal, P. G., et al. (1992). Immunization with Extracellular Proteins of Mycobacterium tuberculosis Induces Cell-Mediated Immune Responses and Substantial Protective Immunity in a Guinea Pig Model of Pulmonary Tuberculosis. Infect. Immun. 60:4781-4792.

9. Andersen (1994). Infection & Immunity 62:2536.

10. Jardim et al. (1990). Immunoprotective Leishmania major Synthetic T Cell Epitopes. J. Exp. Med. 172:645-648.

11. Orme et al. (1993). Cytokine Secretion by CD4 T Lymphocytes Acquired in Response to Mycobacterium tuberculosis Infection. J. Immunology 151:518-525.

12. Boesen et al. (1995). Human T-Cell Responses to Secreted Antigen Fractions of Mycobacterium tuberculosis. Infection and Immunity 63:1491-1497.

13. Mougneau et al. (1995). Expression Cloning of a Protective Leishmania Antigen. Science 268:536-566.

14. Yang et al. (1990). Oral Salmonella typhimurium (AroA − ) Vaccine Expressing a Major Leishmanial Surface Protein (gp63) Preferentially Induces T Helper 1 Cells and Protective Immunity Against Leishmaniasis. J. Immunology 145:2281-2285.

15. Sambrook et al. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.

16. Ausubel et al., (1987). Current Protocols in Molecular Biology, ed. Greene Publishing and Wiley-Interscience: New York (with periodic updates).

17. Innis et al., (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego.

18. Kanehisa (1984). Nuc. Acids Res. 12:203-213, 1984.

19. Wetmur et al. (1968). J. Mol. Biol. 31:349-370.

20. Beaucage et al. (1981) Tetra. Letts. 22:1859-1862.

21. Matteucci et al. (1981). J. Am. Chem. Soc. 103:3185.

22. Jacobs et al. (1991) METHODS IN ENZYMOLOGY 204:537-555.

23. Earp et al. (1990). Nucleic Acids Research 18:3721-3729.

24. Ruther et al. (1983). EMBO J. 2:1791.

25. Stanley and Luzio (1984). EMBO J. 3:1429.

26. Gray et al. (1982). Proc. Natl. Acad. Sci. USA 79:6598.

27. Shimatake and Rosenberg (1981). Nature 292:128.

28. Amann and Brosius (1985). Gene 40:183.

29. Studiar and Moffatt (1986). J. Mol. Biol. 189:113.

30. Yang et al. (1991). Identification and Characterization of Host-Protective T-Cell Epitopes of a Major Surface Glycoprotein (gp63) from Leishmania major. Immunology 72:3-9.

31. Mdluli et al. (1995). New vectors for the in vitro generation of alkaline phosphatase fusions to proteins encoded by G+C-rich DNA. Gene 155:133-134.

32. Lim et al. (1995). Identification of Mycobacterium tuberculosis DNA Sequences Encoding Exported Proteins by Using phoA Gene Fusions. J. Bact. 177:59-65).

33. Cecil Textbook of Medicine, (1992, 19th edition), Wyngaarden et al, eds. W. B. Saunders, Philadelphia, Pa.

34. Hubbard et al. (1992). Immunization of mice with mycobacterial culture filtrate culture proteins. Clin. exp. Immunol. 87: 94-98.

SEQUENCE LISTING
NUMBER OF SEQ ID NOS: 113
SEQ ID NO: 1
LENGTH: 267
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 1
acgcggacct cgaagttcat catcgagtga tacgtgccac acatctcggc gc agtggccc     60
acgaatgctc cggtcttggt gatttcttcg atctggaaga cgttgaccga gt tgtttgcc    120
accgggttag gcatcacgtc acgcttgaac aagaactccg gcacccagaa tg cgtgtatc    180
acatcggctg aggccatttg gaattcgata cgcttgccgg acggcagcac ca gcaccgga    240
atttcggtgc tggtgcccaa cgtctcg                                        267
SEQ ID NO: 2
LENGTH: 487
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 2
ctgatacgac gccggcaagg actacgacga ggtggcacag aattcaatgc gg cgctcatc     60
ggaaccgacg tgcccgacgt cgttttgctc gacgacngat ggtggttcca tt tcgccntc    120
agcggtgttc tgactgccct tgacgacctg ttcggccaag ttggggtgga ca caacggat    180
tacgtcgatt cgctgctggc cgactatgag ttcaacggcc gccattacgc tg tgccgtat    240
gctcgctcga cgccgctgtt ctactacaac aaggcggcgt ggcaacaggc cg gcctaccc    300
gaccgcggac cgcaatcctg gtcagagttc gacgagtggg gtccggagtt ac agcgcgtg    360
gtcggcgccg gtcgatcggc gcacggctgc gntaacgccg acctcatctc gt ggacgttt    420
cagggaccga actgggcatt cggcggtgcc tactccgaca agtggacatt ga cattgacc    480
gagcccg                                                              487
SEQ ID NO: 3
LENGTH: 511
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 3
ggcggccaga cngtcnggaa ctcgcnggcc attggtgtgg tgggaaccgc ga tcctcgac     60
gcaccgcttc gcggtcttgc agtgttcgat gccaatctgc cggccgggac gc tgccggat    120
ggcggcccgt tcaccgaggc tggtgacaag acctggcgtg tcgttccggg ca ctactccc    180
caggtcggtc aaggcaccgt caaagtgttc aggtataccg tcgagatcga ga acggtctt    240
gatcccacaa tgtacggcgg tgacaacgca ttcgcccaga tggtcgacca ga cgttgacc    300
aatcccaagg gctggaccca caatccgcaa ttcgcgttcg tgcggatcga ca gcggaaaa    360
cccgacttcc ggatttcgct ggtgtcgccg acgacagtgc gcggggggtg tg gctacgaa    420
ttccggctcg agacgtcctg ctacaacccg tcgttcggcg gcatggatcg cc aatcgcgg    480
gtgttcatca acgaggcgcg ctgggtacgc g                                   511
SEQ ID NO: 4
LENGTH: 512
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 4
gtgtgcaacc agtgtgtgtn cgtgtgcgaa ccagtgtgta gtggtaacca gg accacgtt     60
gcaaaccagt gttggagtgc agtgttgcgt gcnagtgttg cncgttgcag tg ttngncga    120
gccgagattg gaagttnccg acattaccgt tgccgacgtt gccctcgccg ac gttcgcca    180
agcccaggtt gcggacacgc cggtgattgt gcgtggggca atgancgggc tg ctggcccg    240
gccgaattcc aaggcgtcga tcggcacggt gttccaggac cgggccgctc gc tacggtga    300
ccgagtcttc ctgaaattcg gcgatcagca gctgacctac cgcgacgcta ac gccaccgc    360
caaccggtac gccgcggtgt tggccgcccg cggcgtcggc cccggcgacg tc gttggcat    420
catgttgcgt aactcaccca gcacagtctt ggcgatgctg gccacggtca ag tgcggcgc    480
tatcgccggc atgctcaact accaccagcg cg                                  512
SEQ ID NO: 5
LENGTH: 456
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 5
gcaacggaga ggtggactat gccggaccgg caccgcgaag gggttggtgc cg gcccgggt     60
ggtgacggtg cacattctgc gcaattcgct gagttccggt ggtgaccttc ct gggcgcgg    120
agtctgggcg cgctgatggc ggagcgaktg tgaccgaagg aantcngttc aa catccacg    180
gcgtcggggg cgtgctgtat caagcggtca ccgtcaggag acgccgacgg tg gtgtcgat    240
cgtgacggtg ctggtgctga tctacctgat caccaatctg ttggtggatc tg ctgtatgc    300
ggccctggac gccgnngatn cgctatggct gagcacacgg ggttctggct cg atgcctng    360
cgcgggttgc gccggcgtcc taaantcgtg atcgcgcggc gctgakcctg ct gattcttg    420
tcgtggcggc gtttccgtcg ttgtttaccg cagccg                              456
SEQ ID NO: 6
LENGTH: 172
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 6
tcncttatcg cttcagctgg catctgccca aggaccgaat ctggacctat ga cggccagc     60
tgaagatggc ccgcgacgaa gggcgttggc acgttcgctg gaccaccagc gg gttgcatc    120
ccaagctagg cgaacatcaa aggttcgcgc tacgagccga cccgccgcgg cg             172
SEQ ID NO: 7
LENGTH: 232
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 7
cttctcgcgc cagcncgtcc cgctgtccgg gatgncgcta ccggtcgtca gc gccaagac     60
ggtgcagctc aacgacggcg ggttggtgcg cacggtgcac ttgccggccc cc aatgtcgc    120
ggggctgctg agtgcggccn gcgtgccgct gttgcaaagc gaccacgtgg tg cccgccgc    180
gacggccccg atcgtcgaag gcatgcagat ccaggtgacc cgcaaatcgg at             232
SEQ ID NO: 8
LENGTH: 173
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 8
gttcgncgcg ctcaaaaggt tgacgatggt cacgtcgcac gtgctggccg ag accaaggt     60
ggatttcggt gaagacctca aaganctcta ctcgnatcgt caaggccctc aa cgacgacc    120
gaaaggattt cgtcacctcg ctgcagctgt tgctgacgtt cccatttccc aa c           173
SEQ ID NO: 9
LENGTH: 223
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 9
cctgttncaa cggtncnttc ncggaacgga cgacttctga tncgnnctcg gn cgttccct     60
cgcaccggtc gatggtgatc aaggtcagcg tcttcgcggt ggtcatgctg ct ggtggccg    120
ccggtctggt ggtggtattc ggggacttcc ggtttggtcc cacaaccgtc ta ccacgcca    180
ccttcaccga cncgtngcgg ctgaangcag gccagaaggt tcg                      223
SEQ ID NO: 10
LENGTH: 120
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 10
caacgagatc gcacccgtga ttaggaggtg acggtggcag cgccgacccc gt cgaatcgg     60
atcgaagtaa cgctccgtag acgccagctc gtccgcgccg atgccgacct gc cacccgtg    120
SEQ ID NO: 11
LENGTH: 162
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 11
cggcttccag cgggtgcgcc aagcacggcc ggtccgtgcg agatcgtccc ca atggcacg     60
ccggcgccca agacaccccc ggntaccgtg ccttcgtcgc gcaacctcgc ga ccaacccc    120
gagatcgcca ccnnctacng ccgggacatg accgtggtgc gg                       162
SEQ ID NO: 12
LENGTH: 133
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 12
gactggnccc gaygytgtgn ccgghncgth ggncghgchg cantcgaycc tg gccgttgc     60
ttcggtgccg ggttgttcat cgccttcgac cagttgtggc gctggaacag ca tagtggcg    120
ctagtgctat cgg                                                       133
SEQ ID NO: 13
LENGTH: 395
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 13
gcgcacactg cgcatgctgc cgtacccgcg ccaggcatga gtcttaggcc ga aatgcctg     60
gttaactggc gtgtcgtggt tgacccgcgg gcgtgcggct acagtgcatg ct gtgatcgg    120
cagtgggaga ggtagcggtg cggcgtaagg tgcggaggtt gactctggcg gt gtcggcgt    180
tggtggcttt gttcccggcg gtcgcggggt gctccgattc cggcgacaac aa accgggag    240
cgacgatccc gtcgacaccg gcaaacgctg agggccggca cggacccttc tt cccgcaat    300
gtggcggcgt cagcgatcag acggtgaccg agctgacaag ggtgaccggg ct ggtcaaca    360
ccgccaagaa gtcggtgggc tgccaatggc tggcg                               395
SEQ ID NO: 14
LENGTH: 175
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 14
ccagnccncc naacntgtyn cgntctcayy tcgccgtcgc tgccggtncg tg tgtgcacc     60
atctgcaccg acccgtgkaa cytcgatcac ganactggna gagntcaggc at naaagccg    120
gagtggcaca gcaacggtcg ctactggaat tggcgaagct ggatgctgag ct gac         175
SEQ ID NO: 15
LENGTH: 265
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 15
gggctggatt cgaggctcng tgcatgccgt acgactaggg gtagcgccca gc tgctcaat     60
accatcggtt ggataacaaa ggctgaacat gaatggcttg atctcacaag cg tgcggctc    120
ccaccgaccc cggcgcccct cgagcctggg ggctgtcgcg atcctgatcg cg gcgacact    180
tttcgcgact gtcgttgcgg ggtgcgggaa aaaaccgacc acggcgagct cc ccgagtcc    240
cgggtcgccg tcgccggaag cccac                                          265
SEQ ID NO: 16
LENGTH: 170
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 16
cgccatgncg aagcgmaccc cggtccggaa ggcctgcaca gttctagccg tg ctcgccgc     60
gacgctactc ctcgcctgcg gcggtcccac gcagccacgc agcatcacct tg acctttat    120
ncgcaacgcg caatcccagg ccaacgccga cgggatcatc gacaccgaca               170
SEQ ID NO: 17
LENGTH: 181
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 17
accngttccc gccggnctna cncncggtgc cgttgcaccg gccanctgca gc ctgccccg     60
acgccgaagt ggtgttcgcn ccgcggccgc ttcgaaccgc ccgggattgg ca cggtcggc    120
aabgcattcg tcagcnntgc gctcgaaggt caacaagaat gtcggggtct ac gcggtgaa    180
a                                                                    181
SEQ ID NO: 18
LENGTH: 95
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 18
aggtkacggt ggcagcgccg accccgtcga atcggwtcga agaaygctcc gk acacgcca     60
gctgcgtccg ygccgatgcc gacctgccac ccgtg                                95
SEQ ID NO: 19
LENGTH: 283
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 19
gcgcatgcgc aaccacttgg aatccgttga caatcgcatc ggtggccggc cc gtcggtga     60
ccgcntgcaa cagcgcggtg gtcaccnaca gcgaaaccag gtncttgtcg gc tccggagg    120
tggcgatgac gtggcgccgg gaggtgttga gggtcatgtc gttttcgcgn ta ggtgccct    180
cgatgattga tgacggaaag cnncgtngaa anttggcnat agcggcgttt gt ggtctgcn    240
atncgagcra ttnctgnctg tcagtgtagn cgtgtgtgat ggc                      283
SEQ ID NO: 20
LENGTH: 156
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 20
tcttctacaa ggacgccttc gccaagcacc aggagctgtt cgacgacttg gn cgtcaacg     60
tcaacaatgg cttgtccgat ctgtacragc aagwtcgagt cgctgccgnb cg caacgcga    120
cgagatcatc gaggacctac accgttgcca cgaaca                              156
SEQ ID NO: 21
LENGTH: 123
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 21
atnccgttcc actnccgcgg cagcagctgg ntttgcgcac acggtgaccc ag tggcgntt     60
ggtggggcct cgctgacggc gagtntggnc gagcgtcctc ggtcggtgnc ct ntcntccc    120
gcc                                                                  123
SEQ ID NO: 22
LENGTH: 823
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 22
cggtcacgca attgatggcc gcgcgcaagg scgcatggtg gagatgncca ac cacaccac     60
cggctgggtc cgcatggact tcgtggttcc cagtcgcggc ctgattgggt gg cgcaccga    120
cttcctcacc gagacccgtg gctccggtgt cgggcatgcg gtgttcgacg ga tnaccggc    180
catgggcggg ggagkccggg cccgnccaca ccggttctct ggtatcggac cg ggccggcg    240
ccatcacacc gttcgcgttg ctgcaactcg ccgatcgggg gcagttcttc gt cgagcccg    300
gccaacagan ccntacgagg ncantggctg ctgggatcaa cccccgtccg ga ggacctcg    360
acatcaatgt cacccggagn agnangctga ccnaacatgc gctcatcgac cg cggatgtc    420
atcgagacgg tngccaagcc gctgcagctg gatctcgagc gcgccatgga gt tatgtgcg    480
cccgacgaat gcgtcgaggt gaccccggag atcgtgcgga tccgcaaagt cg agctggcc    540
gccgccgccc gggctcgcag ccgggcgcgc accaaggcgc gtggctagca ac ttggcgcg    600
ctggccgcgc gagcgtaacg ccactgcgaa atccagcccg gcttttcgca gc cgggttac    660
gctcgtgggg gtactggata gcctgatggg cgtgcccagc ccagtccgcc gc gtctgtgt    720
gacggtcggc gcgttggtcg cgctggcgtg tatggtgttg gccgggtgca cg gtcagccc    780
gccgccggca ccccagagca ctgatacgcc gcgcagcaca ccg                      823
SEQ ID NO: 23
LENGTH: 103
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 23
cttccggcgg gacaacaaca ggtctcaccg gcgccacacc ctgacacctg at cgcgtctg     60
ccgatcccgg tcggagcacc cgggttccac cgctgtgccc ccc                      103
SEQ ID NO: 24
LENGTH: 207
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 24
gccaccggtt catcgcgtgg tgctggtcac cgccnggaan gcctcagcgg at cccctgct     60
gccaccgccg cctatccctg ccccagtctc ggcgccggca acagtcccgy cc gtgcagaa    120
cctcacggct ncthccgggc gggagcagca acaggttctc accggygccw ng yacccgca    180
ccgatcgcgt cgccgattcc ggtcgga                                        207
SEQ ID NO: 25
LENGTH: 204
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 25
ttncgcannc gttcatccag gtccactggt gtcgcanctc tcnntgatgc ac cggttccg     60
gatatatgtc nacatcnccs tcstcgtcct ggtgctggta ctnacgaacc tg atcgcgca    120
tttcaccaca ccgtgngcga gcatcgccac cgtcccggcc gccygcggtc gg actggtga    180
tcttggtkcg gagtagaggc ctgg                                           204
SEQ ID NO: 26
LENGTH: 207
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 26
ataccngtca tccngcacat ngtcaacctn gagtcggtnc tcacctacga gg cacgcccg     60
agatgcatca ctggtgctcg rtcagncctt cacggcttgg ccgccttccg gt aggaccgt    120
hgcatgcccg tcttcggcgc ctcgggtgtt cggtcctggc tctcgggctg ct ggccnctg    180
cgccccaccc cgcaccgggc cggcttc                                        207
SEQ ID NO: 27
LENGTH: 289
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 27
ccgtgatngg ccggnncgnc atgttacggg nagccgggna ttgcgntacg cc acggtgat     60
cgcgctggtg gccgcgctgg tggncngcgt gtacgtgctc tcgtccaccg gt aataagcg    120
caccatcgtg ggctacttca cctctgctgt cgggctctat cccggtgacc ag gtccgcgt    180
cctgggcgtc ccggtgggtg agatcgacat gatcgagccg cggtcgtccg ac gtcaagat    240
cactatgtcg gtgtccaagg acgtcaaggt gcccgtgsac gtgcaggcc                289
SEQ ID NO: 28
LENGTH: 198
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 28
ttgnaccang cctatcgcaa gccaatcacc tatgacacgc tgtggcaggc tg acaccgat     60
ccgctgccag tcgtcttccc cattgtgcaa ggtgaactga gcaangcaga cc ggacaaca    120
ggtatcgata gcgccgaatg ccggcttgga cccggtgaat tatcagaact ty gcagtcac    180
gaacgacggg gtgatttt                                                  198
SEQ ID NO: 29
LENGTH: 149
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 29
tcacganggt rynacmgcaa cwcgaccgcc acgtcasgcc gccgcgcacg aa gatcaccg     60
tgcctgcncg atgggtcgtg aacggaatag aaygcagcgg tgaggtcaan yg cgaagccg    120
ggaaccaaat ccggtgaccg cgtcggcat                                      149
SEQ ID NO: 30
LENGTH: 210
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 30
ggacccgcca agcatcagcc ggtcaacagc cgccgccggt ggccaaagtt cg agcagccg     60
ccggtatcgt gctcggcccg gctagaccaa aaactttacg ccagcgcccg aa gccacccg    120
actccaaggc ctcggcccgg ttgggttcgc acatgggtga gttctatatg cc ctacccgg    180
gcacccggtt caaccaggaa accgtctcgc                                     210
SEQ ID NO: 31
LENGTH: 255
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 31
cagnccgctg ncccggaact gttccagcag ctacaagacc ttcgacaacg tn gcgcgtca     60
acctgcantc gagcgcaacc tctcggtggc gctcaacgag tgttcgccgg ct tcaacccg    120
ctggacccgc gaaacctcga cgtgtccccg ctgccttcgc tggccaagcg cg ccgccgac    180
atcctgcgcc aggacgtggg cgggcaggtc gacattttcg atgtcaatgt gc ccaccatc    240
cagtacgacc agagc                                                     255
SEQ ID NO: 32
LENGTH: 164
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 32
aaynccnggc crtcgacggt nccggttcnc rccaccggtc tatatccacc cg ggtcnrca     60
ttmanantga ntmnccgccg gtgcggccgt cgagcgtgac ctggcatccc ct gagacgct    120
gctgggttgc cccggggagn tcgamantcg ggcatcgcac catc                     164
SEQ ID NO: 33
LENGTH: 237
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 33
acggacggca acgggatgcg acccgatccc accggtcgcc acgagggacg ct acttcgtc     60
gccgggcagc cganccgacc gtcngttcng cganggcgac ngccgaagcc gt tgacccac    120
nttggtcagc agcagctgga tsagtcaggt gccgttggtg tttcgccgtc ag cggtgtcg    180
gggtgggtgc gttctgggca ccgtcgactg tggtgggcgc tngcgggcgn tg gtggc       237
SEQ ID NO: 34
LENGTH: 371
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 34
cggatgctcg gcctccggta cccaactcga actcgcgccc acngcggacn gc agggccgc     60
ggttggcacc accagcgaca tcaatcangc aggatcccgc cacgttgcaa ga cggcggca    120
atcttcgcct gtcgctcacc gactttccgc ccaacttcaa catcttgcac at cgacggca    180
acaacgccga ggtcgcggcg atgatgaaag ccaccttgcc gcgcgcgttc at catcggac    240
cggacggctc gacgacggtc gacaccaact acttcaccag catcgagctg ac caggaccg    300
ccccgcaggt ggtcacctac accatcaatc ccgaggcggt gtggtccgac gg gaccccga    360
tcacctggcc g                                                         371
SEQ ID NO: 35
LENGTH: 26
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 35
gagaactccg ggccganttt tggaca                                          26
SEQ ID NO: 36
LENGTH: 202
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 36
tgtcggtagc gttcgcgtcc atgattgctc ttgcaacgct gttgacgctt at caatcaag     60
tcgtcggcac tccgtatatt cccggtggcg attctcccgc cgggaccgac tg ctcggagc    120
tggcttcgtg ggtatcgaat gcggcgacgg ccaggccggt tttcggagat ag gttcaaca    180
ccggcaacga ggaagcgcct tg                                             202
SEQ ID NO: 37
LENGTH: 319
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 37
ctanttttag aytnngtcgt gacatatccg ctgtacgcgt gggacggncc at tattggat     60
aatgcgtgat aagcaccaca agaantgatt ncctatggat attgtcggta nc gttcgcgt    120
ccatgattgc tcttgcaacg ctgttgacgc ttatcaatca agtcgtcggc ac tccgtata    180
ttcccggtgg cgattctccc gccgggaccg actgctcgga gctggcttcg tg ggtatcga    240
atgcggcgac ggccaggccg gttttcggag ataggttcaa caccggcaac ga ggaagcng    300
ccttggcggc tcggggctt                                                 319
SEQ ID NO: 38
LENGTH: 263
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 38
ggtacttgtc gtcgatggac tcccggtctc gattcaggaa cagcgtcccg ac gacaccgg     60
ctcccaccag cccgagaaac gccaccacgc cgcgagcgcc caccacagtc ga cggtgcca    120
gaacgcacca cccgacacgt gacggcgaaa caccaacggc acctgactga tg ccagctgc    180
tgctgaccaa gtgggcacgc tcggcgcgcc tcggaacgag tcgtcgctgc cg cgacgaag    240
acgcctcggc gacgtggatc ggg                                            263
SEQ ID NO: 39
LENGTH: 841
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 39
gcgttgcgcg ccctcgagca gtcnnttggc ggcgatcccg agacaatgat tc ccgacatc     60
cggtacacac cgaaccccaa cgatgcgccg ggcggcccgc tggtagaaag gg gaaatcgc    120
cagtgctgac tcgcttcatc cgacgccagt tgatcctttt tgcgatcgtc tc cgtagtgg    180
caatcgtcgt attgggctgg tactacctgc gaattccgag tctggtgggt at cgggcagt    240
acaccttgaa ggccgacttg cccgcatcgg gtggcctgta tccgacggcc aa tgtgacct    300
accgcggtat caccattggc aaggttactg ccgtcgagcc caccgaccag gg cgcacgag    360
tgacgatgag catcgccagc aactacaaaa tccccgtcga tgcctcggcg aa cgtgcatt    420
cggtgtcagc ggtgggcgag cagtacatcg acctggtgtc caccggtgct cc gggtaaat    480
acttctcctc cggacagacc atcaccaagg gcaccgttcc cagtgagatc gg gccggcgc    540
tggacaattc caatcgcggg ttggccgcat tgcccacgga gaagatcggc tt gctgctcg    600
acgagaccgc gcaagcggtg ggtgggctgg gacccgcgtt gcaacggttg gt cgattcca    660
ctcaagcgat cgtcggtgac ttcaaaacca acattggcga cgtcaacgac at catcgaga    720
actccgggcc gattttggac agccaggtca acacgggtga tcagatcgac ng ctgggcgc    780
gcaaattgaa caatctggcc gcacagaccg cgaccaggga tcagaacgtg cg aagcatcc    840
t                                                                    841
SEQ ID NO: 40
LENGTH: 209
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 40
gcggttggca ccaccagcga naatcagcag gndcccgcca cgttgcaaga cg gcggcaat     60
cttcgcctgt cgctcaccga ctttccgccc aacttcaaca tcttgcacat cg acggcaab    120
aabgccgagg tcgcggcgat gatgaaagcc accttgccgc gcgcgttcat ca tcggaccg    180
gacggctcga cgacggtcga caccaacta                                      209
SEQ ID NO: 41
LENGTH: 167
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 41
agatcgtcag tgagcagaac cccgccaaac cggccgcccg aggtgttgtt cg agggctga     60
aggcgctgct cgcgacggtc gntgctggcc gtcgtcggga tcgggcttng gc tcgcgctg    120
tacttcacgc cggcgatgtc ggcccgcgag atcgtgnatc atcggga                  167
SEQ ID NO: 42
LENGTH: 221
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 42
ccagntcctc nnatatcgac accctcnacn aagaccgctt cgcgagatca ac nctcagat     60
atncnnacta tcnccnntnc acgcacacct caacatnana naatngaact at ngncttcg    120
cctcaccacc aaggttcagg ttancggctg ncgtttkctc tkcgccggct cg aacacgcc    180
atcgtgcgcc ggkacacccg gatgtttgac gacccgctgc a                        221
SEQ ID NO: 43
LENGTH: 117
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 43
cggyccgnnc aayyygncgc gchncggygy agaggtcgny aaggtcgcca ag gtaacgct     60
gatcgayggg nacangcaag tattggtgna cttcaccgtg ghthgcthgc tg tyagc       117
SEQ ID NO: 44
LENGTH: 385
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 44
gaacctcctc gcccgcgctt ggcctagcat taatcgactg gcacgacagt tg cccgactg     60
ggtacacggc atggacgcaa cgcgaatgaa tgtgagttag ctcactcatt ag gcacccca    120
ggcgttgaca ctttatgctt ccggctcgtg tagttgtgtg ggaattgtgg ag cggataac    180
aatttcgacg acgaggaaac agctgtagac atggattgac gaatttgaat ac gactcact    240
ataggaattc gagctcggta cccggggatc ctctagagtc cttcgccgcg gg tcgccacc    300
atcagggcca gtgcgatcgc aagcgcgggg taccgggcgc catagtcttc ag catcggcg    360
tgttgaccgc agagaccgga cgggg                                          385
SEQ ID NO: 45
LENGTH: 285
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 45
cccgcagcag tacccgcagn cccacacccg ctatncgcag cccgaacagt tc ggtgcaca     60
gcccacccna gctcggcgtg cccggtcagt acggccaata ccagcagccg gg ccaatatg    120
nccagccggn acagtnacgn ccagcccggc cagtacgcna ccgcccggtc ag taccccgg    180
gcaatacggc ccgtatgncc agtcgggtca ggggtcgaag cgttcggttg cg gtgatcgg    240
cggcgtgatc gccgtgatgg ccgtgctgtt catcggcgcg gttct                    285
SEQ ID NO: 46
LENGTH: 186
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 46
gcncgtgncc gtgccgcccg gttgaacgtg agcngctgnc natngcccca gc cgagacga     60
gaacgtcccc gaggagtatg cagactggga agacgccgaa gactatgacg ac tatgacga    120
ctatgaggcc gcagaccagg aggccgcacg gtcggcatcc tggcgacggc gg ttgcgggt    180
ncggtt                                                               186
SEQ ID NO: 47
LENGTH: 409
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 47
gtcgctgaat gtgttgtcgg agaccgtnga tcagacctat ccgcacctga gc gccgccnt     60
cgacgggntg gctaagttct ccgacaccat cggcaagcgc gacgagcaga nt cacgcacc    120
tactagccca ggccaaccag gtggccagca tcctgggtga tcgcagtgag ca ggtcgacc    180
gcctattggt caacgctaag accctgatcg ccgcgttcaa cgagcgcggc cg cgcggtcg    240
acgccctgct ggggaacatc tccgctttct cgncccaggt gcaaaacctt na tcaacgac    300
aacccgaacc tgaaccatgt gctcgnnnag ctgcgcatcc tcancgacct gt tggtcgac    360
cgcaaggagg atttggctga aaccctgacg atcttgggca gattcagcg                409
SEQ ID NO: 48
LENGTH: 464
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 48
agnccgtgca ctggaacttc ggctcgatgt ctccgatgtg gacggcaagc tg atgatctc     60
ccggttggan gtcgattcga tgasaaatgn cttggcggct ggtggtgttc ga tgncctgg    120
caccactggc cacgatcgcc gccntggccg cgatcggcgn cttngctcgg ct ggcccctg    180
tggtgggttt cgacgtgctc ggtgttggtg ctgctggtgg tcgaaggtgt gg caatcaac    240
nttctggctg ttgcgtcgtg attcggtaac cgtcggtacc gacgacgatg cg cccgggct    300
gcgactggcc gttgtcttcc tgtgcgccgc cgcgatctcg gcggcggtgg tg actgggta    360
cctgcgctgg acgacaccgg accgcgactt caatcgggat tcccgggaag tg gtgcatct    420
tgccacgggg atggccgaga cggtcgcgtc attctccccg agcg                     464
SEQ ID NO: 49
LENGTH: 423
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 49
gtccaaggcc gtagcccacc tcctggaagt cgtaccacgt cgactcgacc ag gacggctg     60
cantcagcna cttcgtcaac ccggcgatca tcaacntgca cctacggcag tg tgnacgca    120
ccccggacca tcgcactggc cggggnttca cacgccgaac actgnctgac cg cactggat    180
ctgctnggtc gcatgcacca cttcaaggtg gtgacgtacc tcaaaatggg tt kcccgttg    240
tccaccgagg aagtcccgct gatncatggg caataacgct ccctatccgc ag tgtcacca    300
gtgggtgcaa gcggcgatgg ccaagttggt cgctgaccac cccgactacg tt ttcacaac    360
ctcgactcga ccgtggaaca tcaaacccgg cgatgtgatg ccagcaacct at gtcgggat    420
ctg                                                                  423
SEQ ID NO: 50
LENGTH: 279
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 50
cggtcgagcc gatgaacgtc tgcagttcac cgcaaccacg ctcagcggtg ct cccttcga     60
tgcgcaagcc tgcaaggcaa tgccgcggtg ttgtggttct ggacgccgtg gt gcccgttc    120
tgcaactgtc agaagccccc agccgcagcc aggtagcggc cgctaatccg gc ggtcacct    180
tcgtcggaat cgccacccgc gccgacgtcg gggcgatgca gagctttgtc tc gaagtaca    240
acctgaattt caccaacctc aatgacgccg atggtgtga                           279
SEQ ID NO: 51
LENGTH: 331
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 51
cggcccgscg gcgccntggt gaagcttggm gmmtgggtgn agcgcagctg cc caccacac     60
ggraccnngg tgcggacgcg gntgacgcgc ctggtggtca gcatcgtggc cg gtctgctg    120
ttgtatgcca gcttcccgcc gcgcaactgc tggtnggcgg cggtggttgs gc tncgcatt    180
gctggcctgg gtgctgaccc accgcgcgac gacaccggtg ggtgggctgg gc tacggcct    240
gctattcggc ctggtgttct acgtctcgtt gttgccgtgg atcggcgagc tg gtgnnccc    300
cgggccctgg ttggcactgg cgacgacgtg c                                   331
SEQ ID NO: 52
LENGTH: 507
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 52
tgtattcccg tcgatgcgtt gctgcaggta ggccttgaaa tcgttggggg tc acgacgcg     60
gacctcgaag ttcatcatcg agtgatacgt gccacacatc tcggcgcagt gg cccacgaa    120
tgctccggtc ttggtgattt cttcgatctg gaagacgttg accgagttgt tt gccaccgg    180
gttaggcatc acgtcacgct tgaacaagaa ctccggcacc cagaatgcgt gt atcacatc    240
ggctgaggcc atttggaatt cgatacgctt gccggacggc agcaccagca cc ggaatttc    300
ggtgctggtg cccaacgtct cgaccttgtc gaaattcagg taggtccggt cc tcggtgtt    360
gagcccgcgc accggcccga ccagctcttc gccgtacttg tccttgccct ct ggcttgga    420
aaccatggcg cgcttgcgct ccggatcggc accatcatag gtcagtgtgc cg tctttgaa    480
gttcaccctt tgatagccaa acttcca                                        507
SEQ ID NO: 53
LENGTH: 293
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 53
ccacacaaca caaatctacg tcgtaatgca gtcgtaagtc catccgacgt cg atggcaag     60
gacagcaccc gacggccaac ggcatataca tcgtcggctc gccggtcaca ag cacatcat    120
catggactcg tccactacgg cgtacccgtc aactcgccca acggatatcg ca ccgatgtc    180
gactggccac ccagatctcc tacagcggtg tcttcgtgca ctcagcgccg tg gtcggtgg    240
gggctcaggg ccacaccaac accagccatg gctgcctgaa cgtcagcccg ag c           293
SEQ ID NO: 54
LENGTH: 820
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 54
cgccgccggc gngcgctacc ggtgcgggag ggtacaccca agcantccgg ga ccggccgt     60
cycgccggga acgccgtgct cctacacacc ggcggcgggc gcgttgccac gg cccgacac    120
cccactaccc tgncgcgggc gccaccgttg gcccgttcgg tggacccgac tt cccggcac    180
cgctcgatgt ccagccgtcg ccgcctaatc ccgatgggcc gccgccgacg cc gggcatcc    240
taagtgctgg gcggccgggc gagccggctc cggctgttcc ggncataccg at gccsctgc    300
cgccgaacnn nnnnnnnnnt gcacgcaccc aaccgcttga gccgtttcct ga cgggacgg    360
gaggtagcaa ccaatgagca ccatcttcga catccgsagc ctgcgactgy cg aaactgtc    420
tgcaaaggta gtggtcgtcg gcgggttggt ggtggtcttg gcggtcgtgg cc gctgcggc    480
cggcgcgcgg ctctaccgga aactgactac cactaccgtg gtcgcrtatt tn ctstgagg    540
cgctcgcgct gtacccagga gacaaagtcc agatcatggg tgtgcgggtc gg ttctatcg    600
acaagatcga gccggccggc gacaagatgc gagtcacgtt gcactacagc aa caaatacc    660
aggtgccggc cacgnctacc gcgtcgatcc tcaaccccag cctggtggcc tc gcgcacca    720
tccagctgtc accgccgtac accggcggcc cggtcttgca agacggcgcg gt gatcccaa    780
tcgagcgcac ccaggtgccc gtcgagtggg atcagttgcg                          820
SEQ ID NO: 55
LENGTH: 117
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 55
cagccacctc gttcgccgcc gacatcgact atcagccgac ccggccactg ct gacctgat     60
cgccaacagc tggaggccct accggctgca gttcaattca cccgctgcgg gt cggcg       117
SEQ ID NO: 56
LENGTH: 242
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 56
aggtgtcgtg cttcatgcct ggcgcccaat ccagtttcta caccgactgg ta tcaccctt     60
cgcagacaaa cggccagaac tacacctaca agtgggagac cttccttacc ac acagatgc    120
ccgcctggct acaggccaac aaggcgtgtc ccccacaggc aacgcggcgg tg ggtctttc    180
gatctcgggc ggttccgcgc tgaccctggc cgcgtactac ccgcagcagt tc ccgtacgc    240
cg                                                                   242
SEQ ID NO: 57
LENGTH: 345
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 57
tgctgcagat agccaaggat cccgaggtcg tgattgatat cacgtctttc ca gtggaatt     60
ggaagtttgg ctatcaaagg gtgaacttca aagacggcac actgacctat ga tggtgccg    120
atccggagcg caagcgcgcc atggtttcca agccagaggg caaggacaag ta cggcgaag    180
agctggtcgg gccggtgcgc gggctcaaca ccgaggaccg gacctacctg aa tttcgaca    240
aggtcgagac gttgggcacc agcaccgaaa ttccggtgct ggtgctgccg tc cggcaagc    300
gtatcgaatt ccaaatggcc tcagccgatg tgatacacgc attct                    345
SEQ ID NO: 58
LENGTH: 262
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 58
cngactccaa cnagtgcgnt caancngntg tnccngacaa gaaggttcct ac atccgcaa     60
ntcggtgnaa ngccactgtg gatgcctacg acggaacggt cacgctgtac ca acaggacg    120
naaaaggatc cggtgctcaa ggcctggatg caggtcttcc ccggcacggt aa agcctaag    180
agcgacattg cgccggagct tgccgagcan ctgcggtatc ccgaggacct gt tcaaggtg    240
cagcgcatgt tgttggccaa at                                             262
SEQ ID NO: 59
LENGTH: 241
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 59
ccaccannna acrrcacagc tccggccrrc cgtncgcagg ccacccgcan cg tagtgctc     60
aaattcttcc aggacctcgg tggggyacat ccgtccacct ggtacaaggc ct tcaactac    120
aacctcgcga cctcgcagcc catcaccttc gacacgttgt tcgtgcccgg ca ccacgcca    180
ctggacagca tctaccccat cgttcagcgc gagctggcac gtcagaccgg tt tcggtgcc    240
g                                                                    241
SEQ ID NO: 60
LENGTH: 243
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 60
ccggcggatc tgcgtgacga ntgtatncca cggnactacc cgcggtcctt cc tcnantnc     60
cgccggncca gncgcagnct ncngatgtcc ngctataacc tgcgcgatcg cc gccgggct    120
gcccgacaac acggtgngcg ccgccgctgc ttccgccaat tctgggtgnc gg catnccgg    180
cagcgcccgg cccagcactg agagggggac gttgatgcgg tggccgacgg cg tggctgct    240
ggc                                                                  243
SEQ ID NO: 61
LENGTH: 2348
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 61
gcgctgtcat tcggacttcg gaccgcgttg gcggtggtgc tgatcatgaa nc tacgacgg     60
cgccaccggc agcttcccgt catgggtgct ctatccctgt gcgctggcca tg atggtgtt    120
ctcgaagtcg ttcagcgtgc tgcgcagcgc agtgacaccg agggtgatgc cg ccaaccat    180
cgacttggtc cgggtcaact cacggctgac cgtgttcggc ctgctcggcg gc accatcgc    240
tggtggcgcg attgcggccg gagtcgaatt cgtctgcacc cacctgttcc ag ctgccggg    300
cgcgttgttc gtcgtcgtcg cgatcaccat cgctggcgct tcgctgtcga tg cgcattcc    360
gcgctgggtc gaggtgacca gcggtgaggt cccggccaca ttgagctacc ac cgggatag    420
gggcagacta cggcgacngc tggccggagg aagtcaagaa cctcggcgga ac actccgac    480
aaccgttggg ccgcaacatc attacctccc tgtggggtaa ctgcaccatc aa ggtgatgg    540
tcggctttct gttcttgtat ccggcgtttg tcgccaaggc gcacgaagcc aa cgggtggg    600
tgcaattggg catgctgggc ctgatcggcg cggcggccgc ggtcggcaac tt cgccggca    660
atttcaccag cgcacgcctg cagctaggca ggccagctgt gctggtngtg cg ctgcaccg    720
tgctagttac cgtgttagcc atcgcggccg cggtggccgg cagcctggca gc gacagcga    780
ttgccaccct gatcacggca gggtccagtg ccattgctaa agcctcgctg ga cgcctcgt    840
tgcagcacga cctgcccgag gagtcgcggg catcggggtt tgggcgttcc ga gtcgactc    900
ttcagctggc ctgggtgctg ggcggcgcgg tgggcgtgtt ggtgtacacc ga gctgtggg    960
tgggcttcac tgcggtgagc gcgctgctga tcctgggtct ggctcagacc at cgtcagct   1020
tccgcggcga ttcgctgatc cctggcctgg gcggtaatcg gcccgtgatg gc cgagcaag   1080
aaaccacccg tcgtggtgcg gcggtggcgc cgnagtgaag cgcggtgtcg ca acgctgcc   1140
ggtgatcctg gtgattctgc tctcggtggc ggccggggcc ggtgcatggc tg ctagtacg   1200
cggacacggt ccgcagcaac ccgagatcag cgcttactcg cacgggcacc tg acccgcgt   1260
ggggccctat ttgtactgca acgtggtcga cctcgacgac tgtcagaccc cg cangcgca   1320
gggcgaattg ccggtaagcg aacgctatcc cgtgcagctc tcggtacccg aa gtcatttc   1380
ccgggcgccg tggcgtttgc tgcaggtata ccaggacccc gccaacacca cc agcacctt   1440
gtttcggccg gacacccggt tggcggtcac catccccact gtcgacccgc ag cgcgggcg   1500
gctgaccggg attgtcgtgc agttgctgac gttggtggtc gaccactcgg gt gaactacg   1560
cgacgntccg cacgcggaat ggtcggtgcg ccttatcttt tgacgaggcc gc ggctcgac   1620
gggacgctta agcgcggtcg gcgccaacgg tccgaagagc cgccgacacc cg gggcacat   1680
cggcgcatca tggaactgtg cggatcggag tcggggtttg caccacgccc ga cgcgcggc   1740
aggccgcggt ggaggctgcg ggccaggcgc gcgacgagct ggcgggtgag gc gccgtcgc   1800
tggcggtgtt gcttggatcg cgtgcacaca ccgaccgggc tgccgacgtc ct gagcgcgg   1860
tgctgcagat gatcgayccg cccgcgcttg tcggttgcat cgcccaggcc at cgtcgccg   1920
gccgccacga gatcgaggac gagcccgcgg tggtggtgtg gctggcgtcc gg cttggccg   1980
ccgagacatt ccagctggac tttgtccgta ccggctcggg tgccctgatc ac cggttatc   2040
ggttcgaccg caccgcccgg gatctgcatc tgctgctgcc ggacccgtac ac attcccgt   2100
cgaacctgct catcgagcac cccaacaccg acctgccggg caccgccgtc gt gggcggcg   2160
ntggtgagcg gcgggcgccg gcggggcgac acccggctgt tccgcgatca cg acgtgctc   2220
acctccggcg tcgtcggcgt gcgcctgccc gggatgcgcg gtgtmccggt cg tgtcgcag   2280
ggttgccggc cgatcggcta cccatacatc gtcaccggcg cggacggcat ac tgatcacc   2340
gagctcgg                                                            2348
SEQ ID NO: 62
LENGTH: 821
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 62
cgttacccgc tttacaccac cgccaaggcc aacctgaccg cgctcagcac cg ggctgtcc     60
agctgtgcga tggccgacga cgtgctggcc gagcccgacc ccaatgccgg ca tgctgcaa    120
ccggttccgg gccaggcgtt cggaccggac ggacgctggg cggtatcagt cc cgtcggct    180
tcaaacccga gggcgtgggc gaggacctca agtccgancc cggtggtctc ca aacccggg    240
ctggtcaact ccgatgcgtc gcccaacaaa cccaacgccg ccatcaccga ct ccgcgggc    300
accgccggag ggaagggccc ggntcgggat ncaacgggtt gcnacgcggc gc tgccgttc    360
nggattggac ccggcacgta ccccggtgat gggcagctac ggggagaaca ac ctggccgc    420
cacggccacc tcggcctggt accagttacc gccccgcagc ccggaccggc cg ctggtggt    480
ggtttccgcg gccggcgcca tctggtccta caaggaggac ggcgatttca tc tacggcca    540
gtccctgaaa ctgcagtggg gcgtcaccgg cccggacggc cgcatccagc ca ctggggca    600
ggtatttccg atcgacatcg gaccgcaacc cgcgtggcgc aatctgcggt tt ccgctggc    660
ctgggcgccg ccggaggccg acgtggcgcg cattgtcgcc tatgacccga ac ctgagccc    720
tgagcaatgg ttcgccttca ccccgccccg ggttccggtg ctggaatctc tg cagcggtt    780
gatcgggtca gcgacaccgg tgttgatgga catcgcgacc g                        821
SEQ ID NO: 63
LENGTH: 479
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 63
gccagccgtg atcggctgay cggncagntg atcaccaacc tcaacgtggt gc tgggcntc     60
gctggncgct cacacngatc ggttggacca gscggtgacg tcgctatcag cg ttgattca    120
ccggctcgcg caacgcaaga ccgacatctc caacgccgtg gcctacacca ac gcgccgcc    180
ggctcggtcg ccgatctnct gtcgcaggct cgcgcnncgt tggcgaangt gg ttcgcgag    240
accgatcggg tggccggcat cgcggccgcc gaccacgact acctcgacaa tc tgctcaac    300
acgctgccgg acaaatacca ggcgctggtc cgccagggta tgtacggcga ct tcttcgcc    360
ttctacctgt gcgacgtcgt gctcaaggtc aacggcaagg gcggccagcc gg tgtacatc    420
aagctggccg gtcaggacan gcnggcggtg cgcgccgaaa tgaaatcctt cg ccgaacg     479
SEQ ID NO: 64
LENGTH: 481
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 64
kgtctcgcgn ccttaacatc cggtcgcccc ancggtaatc tgcctgtgga tg ccgtccgg     60
aantataagc aaatggccag gagtgcgtga cgcagttatg gctcggtata gt tccgttnt    120
tgccccggac tgggggcgtg aggtggaact aatggcggtg tcgggtgata tt tccgacgg    180
caagcgacca tataggtgga tcgacggcaa taaasacacg ctctggccac gt ttcttggc    240
ggggaaaggg gtgatgctat cggagccaat ggtatcgcga caacacttgc ag atgccgcc    300
aaggccgatc acgctaatga cggattcggg gccacaaacg ttccccgttc tg gcggtttt    360
ctctgactac acctcagatc aaggtgtgat tttgatggat cgcgccagtt at cgggccca    420
ttggcaggat gatgacgtga cgaccatgtt tctttttttg gcnatncggg tg cgaatagc    480
g                                                                    481
SEQ ID NO: 65
LENGTH: 469
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 65
ggcgaggtca gtgaagccga ggaagcggaa aggagcgccc aatacggaac cg cctctccc     60
cgcgcgttgg ccgattcatt aaatgcagct ggcacgacag gtttcccgac tg gaamgcgg    120
gcagtgagcg caasgcaatt aatgtgagtt agctcactca ttaggcaccc ca ggctttac    180
actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tt tcacacag    240
gaaacagcta tgacatgatt acgaatttaa tacgactcac tatagggaat tc gagctcgg    300
tacccgggga tcctctagag tcgcttcggt tggcggcgac cagcagtgga tc cacggtgg    360
ccgcccgcgc ggcdtcatac accgccgcgg cctccttggc ctgtgcggcc sg cttagcgc    420
gcgtgttgct gccgtgctta gccanctggc atagggggct gccgcgcgc                469
SEQ ID NO: 66
LENGTH: 291
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 66
caggttcgac tgatctagct gnrrrccara ccggcacnag ncgacantta cc antacctg     60
acanacagnc cgntcnagcc aanccgnann naggangcag nagnaacagg ca gatgcatc    120
taatgatacc cgcggagtat atctccaacg tgatatatga aggtccgcgt gc tgactcat    180
tgtatgccgc cgaccagcga ttgcgacaat tagctgactc agttagaacg ac tgccgagt    240
cgctcaacac cacgctcgac gagctgcacg agaactggaa aggtagtttc a             291
SEQ ID NO: 67
LENGTH: 1306
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 67
gtgatacagg aggcgccaac agtgacacct cgcgggccag gtcgtttgca ac gcttgtcg     60
cagtgcaggc ctcagcgcgg ctccggaggg cctgcccgtg gtcttcgaca gc tggcgctc    120
gcagcaatgc tgggggcatt ggccgtcacc gtcagtggat gcagctggtc gg aagccctg    180
ggcatcggtt ggccggaggg cattaccccg gaggcacacc tcaatcgaga ac tgtggatc    240
ggggcggtga tcgcctccct ggcggttggg gtaatcgtgt ggggtctcat ct tctggtcc    300
gcggtatttc accggaagaa gaacaccgac actgagttgc cccgccagtt cg gctacaac    360
atgccgctag agctggttct caccgtcata ccgttcctca tcatctcggt gc tgttttat    420
ttcaccgtcg tggtgcagga gaagatgctg cagatagcca aggatcccga gg tcgtgatt    480
gatatcacgt ctttccagtg gaattggaag tttggctatc aaagggtgaa ct tcaaagac    540
ggcacactga cctatgatgg tgccgatccg gagcgcaagc gcgccatggt tt ccaagcca    600
gagggcaagg acaagtacgg cgaagagctg gtcgggccgg tgcgcgggct ca acaccgag    660
gaccggacct acctgaattt cgacaaggtc gagacgttgg gcaccagcac cg aaattccg    720
gtgctggtgc tgccgtccgg caagcgtatc gaattccaaa tggcctcagc cg atgtgata    780
cacgcattct gggtgccgga gttcttgttc aagcgtgacg tgatgcctaa cc cggtggca    840
aacaactcgg tcaacgtctt ccagatcgaa gaaatcacca agaccggagc at tcgtgggc    900
cactgcgccg agatgtgtgg cacgtatcac tcgatgatga acttcgaggt cc gcgtcgtg    960
acccccaacg atttcaaggc ctacctgcag caacgcatcg acgggaakac aa acgccgag   1020
gccctgcggg cgatcaacca gccgcccctt gcggtgacca cccacccgtt tg atactcgc   1080
cgcggtgaat tggccccgca gcccgtaggt taggacgctc atgcatatcg aa gcccgact   1140
gtttgagttt gtcgccgcgt tcttcgtggt gacggcggtg ctgtacggcg tg ttgacctc   1200
gatgttcgcc accggtggtg tcgagtgggc tggcaccact gcgctggcgc tt accggcgg   1260
catggcgttg atcgtcgcca ccttcttccg gtttgtggcc gcggat                  1306
SEQ ID NO: 68
LENGTH: 728
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 68
ggtgcctgcc atcggttcgc tggccacgct ggcatctttg gtctgttaga gg tatccgcg     60
cggatggcca gtcctgttgg cggggnttgt cgccacgatt gccgcccgcg ct gaancccg    120
acgacgccga tgccctgccc accacggatc ggctgaccac ccgagcgaac cg tgcagatg    180
cttggttgac gagcctgctg gcgnccttcg cggcctcggc gaccatcggt gc catcggaa    240
ccgccgtcgc aacccacggc atccacagst ccagcatngg cggtatcgcg tt ggccgncg    300
tcaccggtgc gctgctgctg ctacgagcac gttcagcaga caccagaagg tc actggtgt    360
ttgccatctg tggaatcacc accgttgcaa cggcattnta ccgtcgccgc gg atcgggct    420
ctggaacacg ggccgtggat tgccgcgctg accgccatgc tggnccgccg tg gcaatgtt    480
tttgggcttc gtcgctcccg cgttgtcgct ctcgcccgtc acgtaccgca cc atcgaatt    540
gctggagtgt ctggcgctga tcgcaatggt tccattgacc gcttggstat gc ggcgccta    600
caggcgcgtt cgccacctcg acctgacatg gacatgacca cngtcccgta cc ctgcgcct    660
gctggtggta tcagcgctcg cgacgctgtc tgggttggga acgccggttg cg ccacgcgg    720
tttcgccg                                                             728
SEQ ID NO: 69
LENGTH: 1028
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 69
gktcncggtg atgtcgaccg tcggcacgac gagcgaaacc tcaccggtcg ac agtgtctg     60
cccgaggccg cagccgacgt gcccccggag accgcgcgcc aacacggtgc cg tacatgta    120
gcccgcacgg cgcatcatcg ccgagccggc gtagatgttt tcctgcacgg cg tgcgcggt    180
gaacccntcc ggcgccagca ccgccacctt tcccgcgtcc acgtcggcct gg gtggtgac    240
gccgagcacc ccaccgaaat gatcgacatg gctgtgggtg tagatgaccg sc gaccacgg    300
ggcggtcggc tccgcggtgg gcgcgataca agtccagcgc ggcggcggcc ac ctcggtgg    360
acaccaacgg gtcgatgacg atcagcccag tgtcaccctc aacgaagctg at attggaga    420
tatcgaatcc gcggacctga tagatgcccg gcaccacctg gtagaggccc tg tttcgcgg    480
tcagctggga ttgccgccac aggctgggat gcaccgatgt cggcgcggca cc gtcgagaa    540
acgagtacgc gtcgttgtcc cacaccacgc gaccatcggc agccttgatc ac acacgggg    600
acagcgcggc aatgaatccg cgatcggcgt cgtcgaaatc cgttgtgtca tg caacggta    660
acgagtgttc accgtgtgcc gcctggatga cggcagtngg gaggtttgtg tt ccatcggc    720
actacattgc cactactacg gtgcacgccg gtagatgccg ttggcgaacc ac gctaccga    780
ccagaaagag agaattttcc gccgcaccta gacctcgggc cctcntaacg cg catactgc    840
cgaagcggtc ctcaatgccg atggaccgct acgacaggca aaggagcaca gg gtgaagcg    900
tggactgacg gtcgcggtag ccggagccgc cattctggtc gcaggtcttt cc ggatgttc    960
aagcaacaag tcgactacag gaagcggtga gaccacgacc gcgngcaggc ac gacgcaag   1020
ccccggcg                                                            1028
SEQ ID NO: 70
LENGTH: 780
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 70
agatcaacac catcaccagt gcggtcatcg agttgctgca gggcnagggt gg tccgttgg     60
cgaacgtgct cgccgcyacc ggtgccttct cggcggcgct gggcgcacgc ga ccagctga    120
tcggcgaggt aatcaccaac ctcaacgcgg tgctggcgac cgtcgatgca aa gagcgcgc    180
aatttntcgg ccagtgtcga ccagctgcag cagctggtca gcggcctggc ca agaaccgg    240
gatccgatcg cgggcgccat ttcgccgctg gcgtcgacga cgacggatct ta cggaactg    300
ttgcggaatt cgcgccggcc gctgcaaggc atcctggaaa acgcccggcc gc tggctacc    360
gagctggaca accgaaaggc cgaggtcaas aacgacatcg agcagctcgg cg aggactac    420
ctgcgcctgt ccgcgctggg cagttacgga gcattcnttc aacatctact tc tgctcggt    480
gacgatcaag atcaacggac cggccggcag cgacatcctg ctgccgatcg gc ggccagcc    540
ggatcccagc aaggggaggt gcgcctttgc taaataggaa gccaagtagc aa acacgaac    600
gcgacccgnt ccgcaccggc atcttcggcc tggtgctggt gatctgcgtc gt cctgatcg    660
cattcggcta cagcgggttg cctttctggc cacagggcaa aacctacgac gc gtatttca    720
ccgacgccgg tgggatcacc cccggtaact cggtttatgt ctcgggcctc aa ggtgggcg    780
SEQ ID NO: 71
LENGTH: 689
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 71
ctatccgcaa ggcttcgcag acgctcggct gnaccgcaga atcgcggtgc ac ccacgatt     60
gccagtagcg cgggcccact cgtgcctact acacttcgtc gtagccaaat ca tcggcccc    120
gtagtatctc cggagatgac agatgaatgt cgtcgacatt tcgcggtggc ag ttcggtat    180
caccaccgtc tatcacttca ttttcgtnac cgctgaccat cggcctggcc cc ngctgatc    240
gcggtcatgc aaactgctgt nggtcgtcac cgataacccc gcctggtatc gc ctcaccaa    300
attcttcggc aaattgttcc tgatcaactt tgccatcggc gtggcgaccg ga atcgtgca    360
ggaatttcag ttcggcatga actggagcga gtactcccga ttcgtcggcg at gtcttcgg    420
cgccccgctg gccatggagg gcctggcggc cttcttcttc gaatccacct tc atcgggtt    480
gtggatcttc ggctggaaca ggctgccccg gctggtgcat ctggcctgca tc tggatcgt    540
cgcaatcgcg gtcaacgtgt ccgcgttctt catcatcgng gcaaactcct tc atgcagca    600
tccggtcggc gcgcactaca acccgaccac cgggcgtgcc gagttgagca gc atcgtcgt    660
gcctgctgac caacaacacc gcacaggcg                                      689
SEQ ID NO: 72
LENGTH: 274
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 72
ccgcagcacc gaggcaagca tcgcacccgt cgattcccgc catcccggcg ac atgatggt     60
catgtccgac accgacgccc gcacctcgct tcccgagttg accgcgctgc gc gtggacgc    120
cgcaacggat gcgtcggttc attcgatccc ggctcgaaat tggccatggc ga acgcatct    180
tgctgtgatg gttcgggcag tagatctcca ctgccgcact gataaactcg gg tcatggtc    240
gtcgtgaggc ggacagggta gaggcgcatg accg                                274
SEQ ID NO: 73
LENGTH: 252
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 73
gtgatgcctt ccagcattgg attggtcgtc ggttcgatgc tgtggcgaca ga taaaccgc     60
ctgttcgggg tgcgtggcct ctgctgggca gcgcactgct caacgccgct ct gcgctgct    120
gtgcatggtg gccgagtcgt gtgggcagtg ggttcacgcc tgggcgtact tc acggcgtt    180
cctgctggct acggtggccg ctcaaacggt ggtcgccgca tcgatatcgt gg atcagcgt    240
cctcgcgccc ga                                                        252
SEQ ID NO: 74
LENGTH: 160
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 74
ggcgccgccg tcgtgctggc cgcccggccc ggtgggggtg ccggccagcg tg gttccgcc     60
agtggccgcg ccgaacgtat tggccggcgt cctcgagcac gacaacgacg gg tcgggggc    120
ggcggtgctg gccgcgctgg ccaagctgcc acccggtggt                          160
SEQ ID NO: 75
LENGTH: 401
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 75
atcagccgcg ggtcgacgcc gccgatgacc tcgacgtcgt cgtcgtcgct gc cggtactc     60
aatccaatca ccatcctctt acgcaccttc taggagtgtg ttgctgcggc ag tgccgngc    120
cattcgtaga ttcgggcctc gccgttgtcg tagatcttcg cccacgacct cg atgtctct    180
aacgacacta gtccgtccgg cacngcaaan ccccgcaccg tcggagtgct gg tcaggnta    240
tagncggtac aggnggactt ggwwggcctc gagtanccga ggwwcgntct nc ccgttgcg    300
gncataggcc agaagatgaa ccggtgtaga ccgggcctgt tgcgagggtc gt agtcgtag    360
gtcccagagg tgtcggacgc ccaggttaat acacagcgtg c                        401
SEQ ID NO: 76
LENGTH: 248
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 76
gcagacctct ggccgctggt ggtgctgggt acctgcgctg gcgacaccgg ac cgcagacc     60
gtcaatcggg actcccggga acgtggtgcc atcttgccac ggggatggcc ga cgcggctc    120
gtcattctcc ccgagcgcac cggccgccgc tgttgaccgg gccgcggcga ct gatggtgc    180
ccgcacacgc gggcgggttc aaggagcaat acgccaagtc cagcgccgct ct cgcacggc    240
gcggtgtt                                                             248
SEQ ID NO: 77
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 77
Val His Leu Ala Thr Gly Met Ala Glu Thr Va l Ala Ser Phe Ser Pro
1               5                  10                  15
Ser
SEQ ID NO: 78
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 78
Arg Glu Val Val His Leu Ala Thr Gly Met Al a Glu Thr Val Ala Ser
1               5                  10                  15
Phe
SEQ ID NO: 79
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 79
Arg Asp Ser Arg Glu Val Val His Leu Ala Th r Gly Met Ala Glu Thr
1               5                  10                  15
Val
SEQ ID NO: 80
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 80
Asp Phe Asn Arg Asp Ser Arg Glu Val Val Hi s Leu Ala Thr Gly Met
1               5                  10                  15
Ala
SEQ ID NO: 81
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 81
Ile Ser Ala Ala Val Val Thr Gly Tyr Leu Ar g Trp Thr Thr Pro Asp
1               5                  10                  15
Arg
SEQ ID NO: 82
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 82
Ala Val Val Phe Leu Cys Ala Ala Ala Ile Se r Ala Ala Val Val Thr
1               5                  10                  15
Gly
SEQ ID NO: 83
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 83
Val Thr Asp Asn Pro Ala Trp Tyr Arg Leu Th r Lys Phe Phe Gly Lys
1               5                  10                  15
Leu
SEQ ID NO: 84
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 84
Ala Trp Tyr Arg Leu Thr Lys Phe Phe Gly Ly s Leu Phe Leu Ile Asn
1               5                  10                  15
Phe
SEQ ID NO: 85
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 85
Lys Phe Phe Gly Lys Leu Phe Leu Ile Asn Ph e Ala Ile Gly Val Ala
1               5                  10                  15
Thr
SEQ ID NO: 86
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 86
Phe Leu Ile Asn Phe Ala Ile Gly Val Ala Th r Gly Ile Val Gln Glu
1               5                  10                  15
Phe
SEQ ID NO: 87
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 87
Ala Ile Gly Val Ala Thr Gly Ile Val Gln Gl u Phe Gln Phe Gly Met
1               5                  10                  15
Asn
SEQ ID NO: 88
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 88
Thr Gly Ile Val Gln Glu Phe Glu Phe Gly Me t Asn Trp Ser Glu Tyr
1               5                  10                  15
Ser
SEQ ID NO: 89
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 89
Glu Phe Gln Phe Gly Met Asn Trp Ser Glu Ty r Ser Arg Phe Val Gly
1               5                  10                  15
Asp
SEQ ID NO: 90
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 90
Met Asn Trp Ser Glu Tyr Ser Arg Phe Val Gl y Asp Val Phe Gly Ala
1               5                  10                  15
Pro
SEQ ID NO: 91
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 91
Trp Ser Glu Tyr Ser Arg Phe Val Gly Asp Va l Phe Gly Ala Pro Leu
1               5                  10                  15
Ala
SEQ ID NO: 92
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 92
Glu Tyr Ser Arg Phe Val Gly Asp Val Phe Gl y Ala Pro Leu Ala Met
1               5                  10                  15
Glu
SEQ ID NO: 93
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 93
Ser Arg Phe Val Gly Asp Val Phe Gly Ala Pr o Leu Ala Met Glu Ser
1               5                  10                  15
Leu
SEQ ID NO: 94
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 94
Trp Ile Phe Gly Trp Asn Arg Leu Pro Arg Le u Val His Leu Ala Cys
1               5                  10                  15
Ile
SEQ ID NO: 95
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 95
Trp Asn Arg Leu Pro Arg Leu Val His Leu Al a Cys Ile Trp Ile Val
1               5                  10                  15
Ala
SEQ ID NO: 96
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 96
Gly Arg Ala Glu Leu Ser Ser Ile Val Val Le u Leu Thr Asn Asn Thr
1               5                  10                  15
Ala
SEQ ID NO: 97
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 97
Gly Lys Thr Tyr Asp Ala Tyr Phe Thr Asp Al a Gly Gly Ile Thr Pro
1               5                  10                  15
Gly
SEQ ID NO: 98
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 98
Tyr Asp Ala Tyr Phe Thr Asp Ala Gly Gly Il e Thr Pro Gly Asn Ser
1               5                  10                  15
Val
SEQ ID NO: 99
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 99
Trp Pro Gln Gly Lys Thr Tyr Asp Ala Tyr Ph e Thr Asp Ala Gly Gly
1               5                  10                  15
Ile
SEQ ID NO: 100
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 100
Ala Thr Gly Met Ala Glu Thr Val Ala Ser Ph e Ser Pro Ser Glu Gly
1               5                  10                  15
Ser
SEQ ID NO: 101
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 101
Gly Trp Glu Arg Arg Leu Arg His Ala Val Se r Pro Lys Asp Pro Ala
1               5                  10                  15
Gln
SEQ ID NO: 102
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 102
Thr Gly Ser Gly Glu Thr Thr Thr Ala Ala Gl y Thr Thr Ala Ser Pro
1               5                  10                  15
Gly
SEQ ID NO: 103
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 103
Gly Ala Ala Ile Leu Val Ala Gly Leu Ser Gl y Cys Ser Ser Asn Lys
1               5                  10                  15
Ser
SEQ ID NO: 104
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 104
Ala Val Ala Gly Ala Ala Ile Leu Val Ala Gl y Leu Ser Gly Cys Ser
1               5                  10                  15
Ser
SEQ ID NO: 105
LENGTH: 17
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 105
Leu Thr Val Ala Val Ala Gly Ala Ala Ile Le u Val Ala Gly Leu Ser
1               5                  10                  15
Gly
SEQ ID NO: 106
LENGTH: 21
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
OTHER INFORMATION: Description of Artificial Sequence:PCR Primer
SEQUENCE: 106
cccagcttgt gatacaggag g                                               21
SEQ ID NO: 107
LENGTH: 22
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
OTHER INFORMATION: Description of Artificial Sequence:PCR Primer
SEQUENCE: 107
ggcctcagcg cggctccgga gg                                              22
SEQ ID NO: 108
LENGTH: 46
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
OTHER INFORMATION: Description of Artificial Sequence:PCR Primer
SEQUENCE: 108
tctagacacc accaccacca ccacgtgaca cctcgcgggc caggtc                    46
SEQ ID NO: 109
LENGTH: 28
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
OTHER INFORMATION: Description of Artificial Sequence:PCR Primer
SEQUENCE: 109
aagcttcgcc atgccgccgg taagcgcc                                        28
SEQ ID NO: 110
LENGTH: 330
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 110
gcacgtcgtc gccagtgcca accagggccc ggggcnnacc agctcgccga tc cacggcaa     60
caacgagacg tagaacacca ggccgaatag caggccgtag cccagcccac cc accggtgt    120
cgtcgcgcgg tgggtcagca cccaggccag caatgcgnag cccaaccacc gc cgnccacc    180
agcagttgcg cggcgggaag ctggcataca acagcagacc ggccacgatg ct gaccacca    240
ggcgcgtcan ccgcgtccgc accgngtccc gtgtggtggg cagctgcgct nc acccakkc    300
kccaagcttc accanggcgc cgscgggccg                                     330
SEQ ID NO: 111
LENGTH: 431
TYPE: DNA
ORGANISM: Mycobacterium tuberculosis
FEATURE:
NAME/KEY: Modified base
OTHER INFORMATION: n represents a or g or c or t/u
SEQUENCE: 111
tgtcccgcat ggtagtcggg ctggnccngg tgatcgcttg cagctttngc cg tggatgtg     60
agaaaggaat atgttggtga tcaccatgtt tcgtgtactc gtggcgcgga tg acggcgct    120
ggcggtcgac gangtcgggc atgtccaccg tggaatacgc catcggtacc at cgcggcgg    180
ctgcnttcgg tgcgatcctc tacacggtcg tcaccgggga ttccattgtg tc ggcgctca    240
accgcatcat cggtcgcgcg ctcagcacca aggtttagcg tcgtgtgcgg gt gcgagcac    300
cgtggaagcg gcgttggcga tcgccaccct ggtgctggtg ctggtgctgt gc ctggcggg    360
cgtcaccgcg gtatcaatgc aggtgcgctg tatcgacgcg gcccgcgagg cc gctcgatt    420
ggccgcgcgc g                                                         431
SEQ ID NO: 112
LENGTH: 143
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 112
Ala Gly Gly Cys Gly Ala Cys Gly Gly Thr Th r Gly Gly Cys Ala Cys
1               5                  10                  15
Cys Ala Cys Cys Ala Cys Gly Ala Gly Thr Gl y Ala Gly Thr Cys Ala
20                  25                  30
Gly Gly Cys Ala Gly Gly Ala Gly Cys Cys Cy s Cys Gly Cys Cys Ala
35                  40                  45
Cys Gly Thr Thr Gly Cys Gly Gly Ala Cys Gl y Gly Cys Gly Cys Gly
50                  55                  60
Ala Ala Thr Cys Thr Thr Cys Gly Cys Cys Th r Gly Thr Gly Gly Cys
65                  70                  75                  80
Thr Cys Ala Cys Cys Gly Ala Cys Thr Thr Th r Cys Cys Gly Cys Cys
85                  90                  95
Cys Ala Ala Cys Thr Thr Cys Ala Ala Cys Gl y Ala Thr Cys Thr Thr
100                 105                 110
Gly Cys Ala Cys Ala Thr Gly Gly Ala Cys Gl y Gly Cys Ala Ala Gly
115                 120                 125
Ala Ala Cys Gly Cys Cys Gly Ala Gly Gly Th r Cys Gly Cys Gly
130                 135                 140
SEQ ID NO: 113
LENGTH: 363
TYPE: PRT
ORGANISM: Mycobacterium tuberculosis
SEQUENCE: 113
Met Thr Pro Arg Gly Pro Gly Arg Leu Gln Ar g Leu Ser Gln Cys Arg
1               5                  10                  15
Pro Gln Arg Gly Ser Gly Gly Pro Ala Arg Gl y Leu Arg Gln Leu Ala
20                  25                  30
Leu Ala Ala Met Leu Gly Ala Leu Ala Val Th r Val Ser Gly Cys Ser
35                  40                  45
Trp Ser Glu Ala Leu Gly Ile Gly Trp Pro Gl u Gly Ile Thr Pro Glu
50                  55                  60
Ala His Leu Asn Arg Glu Leu Trp Ile Gly Al a Val Ile Ala Ser Leu
65                  70                  75                  80
Ala Val Gly Val Ile Val Trp Gly Leu Ile Ph e Trp Ser Ala Val Phe
85                  90                  95
His Arg Lys Lys Asn Thr Asp Thr Glu Leu Pr o Arg Gln Phe Gly Tyr
100                 105                 110
Asn Met Pro Leu Glu Leu Val Leu Thr Val Il e Pro Phe Leu Ile Ile
115                 120                 125
Ser Val Leu Phe Tyr Phe Thr Val Val Val Gl n Glu Lys Met Leu Gln
130                 135                 140
Ile Ala Lys Asp Pro Glu Val Val Ile Asp Il e Thr Ser Phe Gln Trp
145                 1 50                 1 55                 1 60
Asn Trp Lys Phe Gly Tyr Gln Arg Val Asn Ph e Lys Asp Gly Thr Leu
165                 170                 175
Thr Tyr Asp Gly Ala Asp Pro Glu Arg Lys Ar g Ala Met Val Ser Lys
180                 185                 190
Pro Glu Gly Lys Asp Lys Tyr Gly Glu Glu Le u Val Gly Pro Val Arg
195                 200                 205
Gly Leu Asn Thr Glu Asp Arg Thr Tyr Leu As n Phe Asp Lys Val Glu
210                 215                 220
Thr Leu Gly Thr Ser Thr Glu Ile Pro Val Le u Val Leu Pro Ser Gly
225                 2 30                 2 35                 2 40
Lys Arg Ile Glu Phe Gln Met Ala Ser Ala As p Val Ile His Ala Phe
245                 250                 255
Trp Val Pro Glu Phe Leu Phe Lys Arg Asp Va l Met Pro Asn Pro Val
260                 265                 270
Ala Asn Asn Ser Val Asn Val Phe Gln Ile Gl u Glu Ile Thr Lys Thr
275                 280                 285
Gly Ala Phe Val Gly His Cys Ala Glu Met Cy s Gly Thr Tyr His Ser
290                 295                 300
Met Met Asn Phe Glu Val Arg Val Val Thr Pr o Asn Asp Phe Lys Ala
305                 3 10                 3 15                 3 20
Tyr Leu Gln Gln Arg Ile Asp Gly Asn Thr As n Ala Glu Ala Leu Arg
325                 330                 335
Ala Ile Asn Gln Pro Pro Leu Ala Val Thr Th r His Pro Phe Asp Thr
340                 345                 350
Arg Arg Gly Glu Leu Ala Pro Gln Pro Val Gl y
355                 360

Claims

1. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61, and 67.

2. A purified immunostimulatory peptide encoded by a nucleic acid molecule according to claim 1.

3. An antibody that specifically binds to a peptide according to claim 2.

4. A method of making an immunostimulatory preparation comprising:providing at least one purified immunostimulatory peptide according to claim 2; and combining the peptide with a pharmaceutically acceptable excipient.

5. A purified immunostimulatory peptide comprising at least 5 contiguous amino acids encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61, and 67.

6. An immunostimulatory preparation comprising at least one peptide according to claim 5 and a pharmaceutically acceptable excipient.

7. A peptide according to claim 5 wherein the peptide includes at least 10 contiguous amino acids encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61, and 67.

8. An immunostimulatory preparation comprising at least one peptide according to claim 7 and a pharmaceutically acceptable excipient.

9. An isolated nucleic acid molecule consisting of a nucleotide sequence selected form the group consisting of:(a) SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61 and 67; (b) nucleotide sequences complementary to a sequence defined in (a); (c) nucleic acid molecules of at least 15 nucleotides in length which have at least 75% sequence identity with molecules defined in (a) or (b), wherein the nucleic acid molecule encodes an immunostimulatory peptide; and (d) nucleic acid molecules comprising at least 15 consecutive nucleotides of a molecule defined in (a).

10. A recombinant vector including a nucleic acid molecule according to claim 9.

11. A nucleic acid probe comprising a nucleic acid molecule according to claim 9 conjugated with a detectable label.

12. A transformed cell containing a vector according to claim 10.

13. A method of detecting the presence of Mycobacterium tuberculosis DNA in a sample comprising contacting the sample with a nucleic acid probe according to claim 11 and detecting hybridization products that include the nucleic acid probe.

14. A method of isolating a Mycobacterium tuberculosis gene which gene encodes an immunostimulatory peptide, the method comprising the steps of:providing nucleic acids of Mycobacterium tuberculosis; contacting said nucleic acids with a probe or primer, the probe or primer comprising at least 15 consecutive nucleotides of a sequence selected form the group consisting of SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61, and 67 and sequences complementary thereto; identifying a gene hybridized to the probe or primer; and isolating the Mycobacterium tuberculosis gene, wherein the gene encodes an immunostimulatory peptide.

15. An isolated Mycobacterium tuberculosis nucleic acid molecule, said molecule encoding an immunostimulatory peptide and having at least 75% sequence identity with a nucleic acid probe comprising at least 15 contiguous bases of a sequence selected from SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61, and 67.

16. A purified immunostimulatory peptide encoded by the nucleic acid molecule of claim 15.

17. An immunostimulatory preparation comprising:a purified peptide according to claim 16; and a pharmaceutically acceptable excipient.

18. An improved tuberculin skin test, the improvement comprising the use of one or more immunostimulatory peptides according to claim 16.

19. An immunostimulatory peptide encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61, and 67, wherein the immunostimulatory peptide further comprises at least one conservative amino acid substitution.

20. An isolated Mycobacterium tuberculosis nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61, and 67.

21. A vector comprising the isolated Mycobacterium tuberculosis nucleic acid molecule as recited in claim 20.

22. A purified immunostimulatory peptide comprising at least 5 contiguous amino acids encoded by a Mycobacterium tuberculosis nucleic acid sequence consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61, and 67, wherein the nucleic acid molecule encodes a peptide that is expressed externally by Mycobacterium tuberculosis.

23. An isolated nucleic acid molecule consisting of a Mycobacterium tuberculosis nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 11, 15, 23, 34, 41, 61, and 67, wherein the nucleic acid molecule encodes a peptide that is expressed externally by Mycobacterium tuberculosis.