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US20020172684A1 - Mycobacterium tuberculosis DNA sequences encoding immunostimulatory peptides and methods for using same - Google Patents

Mycobacterium tuberculosis DNA sequences encoding immunostimulatory peptides and methods for using same Download PDF

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US20020172684A1
US20020172684A1 US09/996,634 US99663401A US2002172684A1 US 20020172684 A1 US20020172684 A1 US 20020172684A1 US 99663401 A US99663401 A US 99663401A US 2002172684 A1 US2002172684 A1 US 2002172684A1
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Francis Nano
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Victoria University of Innovation and Development Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • 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 one billion people are infected with M. tuberculosis worldwide, 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.
  • M. tuberculosis multi-drug resistant strains of M. tuberculosis has complicated matters further and even raises the possibility of a new tuberculosis epidemic.
  • 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.
  • the expected mortality rate for infection with drug-sensitive M tuberculosis is 0%.
  • 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, Skamene, Ref. Infect. Dis. 11:S394-S399, 1989.
  • Initial control of M. tuberculosis may also be influenced by reactive T ⁇ cells.
  • the major immune response responsible for containment of M. tuberculosis is via helper T cells (Th1) and to a lesser extent cytotoxic T cells, Kaufmann, Current Opinion in Immunology 3:465-470, 1991.
  • Th1 helper T cells
  • Th2 helper T cells
  • Th1 cells are thought to convey protection by responding to M. tuberculosis T cell epitopes and secreting cytokines, particularly INF- ⁇ , that 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.
  • cytokines particularly INF- ⁇
  • Antigens that stimulate T cells from mice infected with M. tuberculosis or from PPD-positive humans are found in both the whole mycobacterial cells and also in the culture supernatants, Orme et al., Journal of Immunology 148:189-196, 1992; Daugelat et al., J. Infect. Dis. 166:186-190, 1992; Barnes et al., J. Immunol. 143:2656-2662, 1989; Collins et al., Infect. Immun. 56:1260-1266, 1988; Lamb et al., Rev. Infect. Dis. 11:S443-S447, 1989; and Hubbard et al., Clin. exp. Immunol.
  • the present invention provides inter alia, DNA sequences isolated from Mycobacterium tuberculosis . Peptides encoded by these DNA sequences stimulate the production of the macrophage-stimulating cytokine, gamma interferon (“INF- ⁇ ”), in mice. Critically, the production of INF- ⁇ by CD4 cells in mice correlates with maximum expression of protective immunity against tuberculosis, Orme et al., J. Immunology 151:518-525, 1993.
  • INF- ⁇ gamma interferon
  • the DNA sequences provided by this invention encode peptides that can of stimulate T-cells to produce INF- ⁇ . That is, these peptides act as epitopes for CD4 T-cells in the immune system.
  • 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, Mougneau et al., Science 268:536-566, 1995 and Jardim et al., J. Exp. Med. 172:645-648, 1990.
  • T-cell epitopes from the parasite Leishmania major have been shown to be effective when administered as a vaccine, Jardim et al., J.
  • the immunostimulatory peptides (T-cell epitopes) encoded by the DNA sequences according to the invention may be used, in purified form, as a vaccine against tuberculosis.
  • nucleotide sequences of the present invention encode immunostimulatory peptides.
  • these nucleotide sequences are only a part of a larger open reading frame (ORF) of an M. tuberculosis operon.
  • ORF open reading frame
  • the present invention enables the cloning of the complete ORF using standard molecular biology techniques, based on the nucleotide sequences provided herein.
  • the present invention encompasses both the nucleotide sequences disclosed herein and the complete M. tuberculosis ORFs to which they correspond.
  • 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.
  • immunostimulatory preparations comprising at least one peptide encoded by the DNA sequences presented herein.
  • a preparation may include the purified peptide or peptides and one or more pharmaceutically acceptable adjuvants, diluents, and/or excipients.
  • vaccines comprising one or more peptides encoded by nucleotide sequences provided herein.
  • a vaccine may include one or more pharmaceutically acceptable excipients, adjuvants, and/or diluents.
  • antibodies are provided that are specific for immunostimulatory peptides encoded by a nucleotide sequence according to the present invention. Such antibodies may be used to detect the presence 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 nucleotide sequences according to the present invention.
  • 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 improved methods of performing the tuberculin skin test to diagnose exposure of an individual to M. tuberculosis .
  • this improved skin test purified immunostimulatory peptides encoded by the nucleotide sequences of this invention are employed.
  • 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.
  • the tuberculin skin test will be useful in distinguishing between subjects infected with tuberculosis and subjects who have simply been vaccinated.
  • 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 is 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.
  • FIG. 1 shows the deduced amino acid sequence of the full-length MTB2-92 protein.
  • the nucleic acid sequence is contained within SEQ ID NO: 67, the amino acid sequence is shown in SEQ ID NO: 113.
  • a Mycobacterium tuberculosis specific binding agent binds substantially only cellular components derived from Mycobacterium tuberculosis . These cellular components include both extracellular and intracellular, proteins, glycoproteins, sugars, and lipids, that are found in Mycobacterium tuberculosis isolates.
  • Mycobacterium tuberculosis specific binding agent can be an anti- Mycobacterium tuberculosis antibody or other agent that binds substantially only to Mycobacterium tuberculosis.
  • anti- Mycobacterium tuberculosis antibodies encompasses monoclonal and polyclonal antibodies that are specific for Mycobacterium tuberculosis , i.e., which bind substantially only to Mycobacterium tuberculosis when assessed using the methods described below, as well as immunologically effective portions (“fragments”) of such antibodies. Immunologically effective portions of the antibodies include Fab, Fab′, F(ab′) 2 , Fabc, and Fv portions (for a review, see Better and Horowitz, Methods Enzymol., 178:476-496, 1989). Anti- Mycobacterium tuberculosis antibodies may also be produced using standard procedures described in a number of texts, including Harlow and Lane, Antibodies, A Laboratory Manual , Cold Spring Harbor Laboratory, 1988.
  • Sequence Identity The similarity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences are.
  • NCBI Basic Local Alignment Search Tool (BLASTTM Altschul et al. J. Mol. Biol., 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at http//www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
  • the “Blast 2 sequences” function in the BLAST program is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per-residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 45%, at least 50%, at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity.
  • 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.
  • 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 be readily 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, Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al. (ed.); Current Protocols in Molecular Biology , Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987.
  • Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length, that 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.
  • PCR polymerase chain reaction
  • 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.
  • 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.).
  • a first nucleic acid is “substantially similar” to a second nucleic acid if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the second 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.
  • a first nucleic acid sequence is “operably” linked with a second nucleic acid sequence whenever the first nucleic acid sequence is placed in a functional relationship with the nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • a “recombinant” nucleic acid 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.
  • 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.
  • 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. (ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, at pages 9.52-9.55, 9.47-9.52 and 9.56-9.58; Kanehisa, Nuc. Acids Res. 12:203-213, 1984; and Wetmur et al., J. Mol. Biol. 31:349-370, 1968.
  • 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 appreciated readily by those skilled in the art.
  • 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).
  • 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.
  • a “purified” peptide is a peptide that has been extracted from the cellular environment and separated from substantially all other cellular peptides.
  • the term “peptide” includes peptides, polypeptides and proteins.
  • 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 peptide refers to a peptide that is capable of stimulating INF- ⁇ production in the assay described in section B.5. below.
  • an immunostimulatory peptide is capable of inducing greater than twice the background level of this assay determined using T-cells stimulated with no antigens or negative control antigens.
  • the immunostimulatory peptides are capable of inducing more than 0.01 ng/mL of INF- ⁇ in this assay system.
  • an immunostimulatory peptide is one capable of inducing greater than 10 ng/mL of INF- ⁇ in this assay system.
  • 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. (ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al. (ed.), Current Protocols in Molecular Biology , Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987.
  • nucleic acids Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage et al., Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers.
  • Mycobacterium tuberculosis DNA was obtained by the method of Jacobs et al., Methods In Enzymology 204:537-555, 1991. Samples of the isolated DNA were partially digested with one of the following restriction enzymes HinPI, HpaII, AciI, TaqI, BsaHI, and NarI. Digested fragments of 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 pJDT3.
  • a schematic representation of the phagemid vector pJDT2 is provided in Mdluli et al., Gene 155:133-134, 1995.
  • 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.
  • phoA bacterial alkaline phosphatase gene
  • 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 (XP). Alkaline phosphatase cleaves XP to release a blue-colored product. Hence, those clones containing alkaline phosphatase fusion proteins with the enzymatic portion lying outside the cytoplasmic membrane will produce the blue color.
  • XP 5-bromo-4-chloro-3-indolyl phosphate
  • the three vectors in this series (pJDT1, pJDT2, and pJDT3) 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 three. Mdluli et al., Gene 155:133-134, 1995, describes pJDT vectors in detail.
  • 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).
  • 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 were grown overnight at 30° C. with shaking in 2 mL LB broth containing ampicillin, kanamycin, and IPTG. The cells were precipitated by centrifugation and resuspended in 100 ⁇ L Tris-EDTA buffer. To this mixture was added 100 ⁇ L lysis buffer (1% SDS, 1 mMEDTA, 25 mM DTT, 10% glycerol and 50 mM tris-HCl, pH 7.5). DNA released from the cells was sheared by passing the mixture through a small-gauge syringe needle.
  • the samples were electrophoresed by application of 200 volts to the gel for 4 hours. Subsequently, the proteins were transferred to a nitrocellulose membrane by Western blotting. A strip of nitrocellulose was cut off to be processed with antibody, and the remainder of the nitrocellulose was set aside for eventual elution of the protein. The strip was incubated with blocking buffer and then with anti-alkaline phosphatase primary antibody, followed by incubation with anti-mouse antibody conjugated with horseradish peroxidase. Finally, the strip was developed with the NEN DuPont RenaissanceTM kit to generate a luminescent signal. The migratory position of the PhoA fusion protein, as indicated by the luminescent label, was measured with a ruler, and the corresponding region of the undeveloped nitrocellulose blot was excised.
  • This region of nitrocellulose containing the PhoA fusion protein was then incubated in 1 mL 20% acetronitrile at 37° C. for 3 hours. Subsequently, the mixture was centrifuged to remove the nitrocellulose, and the liquid was transferred to a new test tube and lyophilized. The resulting protein pellet was dissolved in 100 ⁇ L of endotoxin-free, sterile water and precipitated with acetone at ⁇ 20° C. After centrifugation the bulk of the acetone was removed and the residual acetone was allowed to evaporate. The protein pellet was re-dissolved in 100 ⁇ L 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.
  • 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 mice are inoculated with 1 ⁇ 10 5 viable bacteria suspended in 200 ⁇ L sterile saline via a lateral tail vein on the first day 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., J. Immunology 151:518-525, 1993. Eight to ten days later, up to ten ⁇ g 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 ⁇ cells, respectively.
  • the macrophages are overlaid with 10 6 of these CD4 cells, and the medium is supplemented with 5 U interleukin-2 (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.
  • IL-2 interleukin-2
  • Cytokine levels in harvested supernatants are assayed by sandwich ELISA as described by Orme et al., J. Immunology 151:518-525, 1993.
  • 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- ⁇ production by human T cells in the following manner.
  • Blood from tuberculin-positive people is collected in EDTA-coated tubes to prevent clotting.
  • Mononuclear cells are isolated using a modified version of the separation procedure provided with the NycoPrepTM 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 atop 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.
  • a physiologic solution such as Hanks Balanced Salt solution (HBSS)
  • the plasma is removed from the top of the tube and filtered through a 0.2-micron filter.
  • the plasma is then added to the tissue culture media.
  • the mononuclear cells are washed twice by a procedure in which 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 INF- ⁇ ).
  • cytokines including INF- ⁇
  • Cytokines are measured using an Enzyme Linked Immunosorbent Assay (ELISA), the details of which are described in the Cytokine ELISA Protocol in the PharMingen catalog (PharMingen, San Diego, Calif.).
  • ELISA Enzyme Linked Immunosorbent Assay
  • To measure the presence of human INF- ⁇ wells of a 96-well microtitre plate are coated with a “capture antibody” (e.g., anti-human INF- ⁇ antibody). The sample supernatants are then added to individual wells. Any INF- ⁇ present in the sample binds to the capture antibody. The wells are then washed.
  • ELISA Enzyme Linked Immunosorbent Assay
  • a “detection antibody” (e.g., anti-human INF- ⁇ antibody), conjugated to biotin, is added to each well, and binds to any INF- ⁇ bound to the capture antibody. Any unbound detection antibody is washed away. An avidin-linked horseradish 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 quantified using an ELISA plate reader. The amount of the INF- ⁇ in the sample supernatants is determined by comparison with a standard curve using known amounts of human INF- ⁇ .
  • Measurement of other cytokines can be determined using the same protocols with the appropriate substitution of reagents (monoclonal antibodies and standards).
  • alkaline phosphatase fusion clones were undertaken using the AmpliCycleTM 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.
  • AmpliCycleTM thermal sequencing kit Perkin Elmer, Applied Biosystems Division, 850 Lincoln Centre Drive, Foster City, Calif. 94404, U.S.A.
  • fusion clones were tested for their ability to stimulate INF- ⁇ production. Of these, 80 clones initially were designated to have some ability to stimulate INF- ⁇ production. Tables 1 and 2 show the data obtained for these 80 clones. Clones listed in Table 1 showed the greatest ability to stimulate INF- ⁇ production (greater than 10 ng/mL of INF- ⁇ ), while clones listed in Table 2 stimulated the production of between 2 ng/mL and 10 ng/mL of INF- ⁇ . Background levels of INF- ⁇ 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.
  • nucleotide sequences of the present invention encompass not only those respective sequences presented in the sequence listings, but also the respective complete nucleotide sequence encoding the respective immunostimulatory peptides as well as the corresponding M. tuberculosis genes.
  • 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)
  • sequences were also analyzed using the BLASTTM program on the TIGRTM 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 identification of matches with reported sequences to be identified and, for matches on the Sanger database, the identification of the open reading frame.
  • T-Site 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.
  • the program uses a series of four predictive algorithms. In particular, peptides were designed against regions indicated by the algorithm “A” motif which predicts alpha-helical periodicity, Margalit et al., J. Immunol. 138:2213, 1987, and amphipathicity.
  • 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: Peptide Sequence Peptide Name SEQ ID NO:.
  • HinP#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 AciI2-827 (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 TGIVQEFEFGM
  • tuberculosis filtrate 256.6 153.3 proteins (10 ⁇ g/mL) M. tuberculosis filtrate proteins (5 ⁇ g/mL) 134.0 30.7 Con A(10 ⁇ g/mL) 2839 2735.7 PHA(1%) 10378 10274.7 PhoA control 26.7 0 (10 ⁇ g/mL) Background 103.3 0
  • 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 labeled with a suitable marker to enable detection of hybridizing clones.
  • Suitable markers include radio nucleotides, such as 32 P and non-radioactive markers such as biotin-avidin enzyme linked systems.
  • Methods for constructing suitable libraries, production and labeling of oligonucleotide probes, and colony hybridization are standard laboratory procedures and are described in standard laboratory manuals such as in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al. (ed.), Current Protocols in Molecular Biology , Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987.
  • the clone is identified and sequenced using standard methods such as described in Chapter 13 of reference Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Determination of the translation-initiation point of the DNA sequence enables the ORF to be located.
  • PCR polymerase chain reaction
  • IPCR inverse polymerase chain reaction
  • the present invention encompasses small oligonucleotides included in the DNA sequences presented in the respective Sequence Listings. 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 as set forth in the respective Sequence Listing.
  • the oligonucleotides can comprise a sequence of at least 15 nucleotides and preferably at least 20 nucleotides, the oligonucleotide sequence being substantially similar to a respective DNA sequence set forth in the respective Sequence Listing.
  • such oligonucleotides will share at least about 75%-90% sequence identity with a respective DNA sequence set forth in the respective Sequence Listing and more preferably the shared sequence identity will be greater than 90%.
  • 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 base pairs upstream) by a Shine-Dalgarno motif.
  • the gene mtb2-92 encodes a protein (termed MTB2-92) containing 363 amino acid residues with a predicted molecular weight of 40,436 Da.
  • the plasmid pHin2-92 was restricted with either BamH1 or EcoRI and then subcloned into the vector M13.
  • the inserted DNA fragments were sequenced under the direction of M13 universal sequencing primers, Yanisch-Perron et al., Gene 33:103-119, 1985, using the AmpliCycleTM 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 GeneWorksTM (Intelligenetics, Mountain View, Calif., U.S.A.) program. Based on the sequence data obtained, two oligomers were synthesized.
  • oligonucleotides (5′ CCCAGCTTGTGATACAGGAGG 3′ (SEQ ID NO: 106) and 5′ GGCCTCAGCGCGGCTCCGGAGG 3′ (SEQ ID 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 et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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- ⁇ L aliqots of distilled water 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- ⁇ L PCR reaction contained: 10 ⁇ L supernatant containing cosmid DNA, 10 ⁇ L of 10 ⁇ PCR buffer, 250 ⁇ M dNTPs, 300 nM downstream and upstream primers, and 1 unit Taq DNA polymerase.
  • 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′TCTAGACACCACCACCACCACCACCACGTGACACCT CGCGGGCCAGGTC 3′ (SEQ ID NO: 108) and 5′AAGCTTCGCCATGC CGCCGGTAAGCGCC 3′ (SEQ ID NO: 109)).
  • the 100- ⁇ L PCR reaction contained: 1 ⁇ g pG3 template DNA, 250 nM dNTP's 300 nM of each primer, 10 ⁇ L of 10 ⁇ PCR buffer, and 1 unit Taq DNA polymerase.
  • the PCR DNA synthesis cycle was performed as described 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 Xba I and HindIII and the 1.4-kb fragment was directionally subcloned into the Xba I and Hind III 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.
  • 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.
  • the cell pellet was re-suspended in 50 mL of 20 mM Tris-HCl, pH 7.2, 200 mM NaCl, 1 mM EDTA supplemented with 10 mM ⁇ -mercaptoethanol and stored at ⁇ 20° C.
  • 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 ⁇ L of Factor Xa (200 ⁇ g/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.
  • sequence ambiguities occur when the results from the sequencing reaction do not clearly distinguish between the individual base pairs. Therefore, substitute abbreviations denoting multiple base pairs are provided in Table 3, supra. These abbreviations denote which of the four bases could possibly be at a position that was found not to give a clear experimental result.
  • substituent abbreviations denote which of the four bases could possibly be at a position that was found not to give a clear experimental result.
  • 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.
  • PCR was used to amplify the complete predicted ORFs encoding the proteins identified in the immunogenicity study. Oligodeoxynucleotide primers were designed with restriction sites in order to clone the amplified fragments into expression vectors. Table 5 provides a description the primer sequences used. All PCR reactions were conducted in 20 ⁇ L using a 6:1 Taq polymerase: Pfu polymerase enzyme combination. The reaction mixes contained either 1 ⁇ L DMSO or 4 ⁇ L of Q solution (proprietary solution available from Qiagen, Dusseldorf, Germany, for denaturation in PCR) as a denaturant.
  • Q solution proprietary solution available from Qiagen, Dusseldorf, Germany, for denaturation in PCR
  • MBP maltose binding protein
  • GST glutathione S-transferase
  • PET 260 amino acid T7 gene 10 product
  • the different expression vectors used included pMAL-c2 (New England Biolabs), pGEX-4T3 (Pharmacia, Piscataway, N.J.), and pET-17xb (Novagen, Madison, Wis.). These expression vectors enabled the N-terminal fusion of the maltose binding protein (MBP), glutathione S-transferase (GST), or the 260 amino acid T7 gene 10 product (PET) containing the T7 ⁇ Tag® (Novagen, Madison, Wis.) to be added to the products of the cloned DNA.
  • MBP maltose binding protein
  • GST glutathione S-transferase
  • PET 260 amino acid T7 gene 10 product
  • Table 6 provides a summary of the results from cloning the PCR products into the various vectors described above.
  • SDS-PAGE and Western Blotting were used to identify the novel antigens expressed by the clones.
  • Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using 10% slab gels in a continuous buffer system, Laemmli, Nature (London) 227:680-685, 1970. Proteins were electrophoretically transferred from the gel to a nitrocellulose membrane using standard protocols, Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • E. coli BL21 plysS MBP-264 (SEQ ID NO: 114), PET-23 (SEQ ID NO: 117), MBP-506 (SEQ ID NO: 115), MPB-825 (SEQ ID NO: 116), PET-639 (SEQ ID NO: 119), PET-916 (SEQ ID NO: 120), PET-1084 (SEQ ID NO: 121), PET-47 (SEQ ID NO: 118); in E. coli BL21: GST-152 (SEQ ID NO: 122), GST-822 (SEQ ID NO: 124); and in E. coli SURE: GST-206 (SEQ ID NO: 125).
  • the recombinant fusion proteins MBP-506 (SEQ ID NO: 115), MBP-825 (SEQ ID NO: 116), GST-152 (SEQ ID NO: 122), GST-822 (SEQ ID NO: 124) GST-827 (SEQ ID NO: 126), PET-639 (SEQ ID NO: 119), PET-1084 (SEQ ID NO: 121) formed inclusion bodies that were harvested from the pellet following centrifugation of the bacterial sonicate.
  • the fusion proteins PET-916 and GST-206 were found primarily in the supernatant and underwent considerable breakdown in culture. Protein fractions were checked by SDS-PAGE using Coomassie Blue staining and approximate concentrations were determined by Western blotting.
  • amino acid sequences encoded by nucleic acid sequences described above are also shown in the sequence listing. These amino acid sequences are SEQ ID NOS: 128-141, which correspond to the nucleic acid sequences shown in SEQ ID NOS: 114-127, respectively.
  • 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.
  • 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.
  • E. coli Escherichia coli
  • 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 Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • 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.
  • 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.
  • 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 for stimulating 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.
  • 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 are presented in Tables 7 and 8, respectively.
  • variant DNA molecules may be derived from the DNA sequences disclosed herein using standard DNA mutagenesis techniques, or by synthesis of DNA sequences.
  • peptides that vary in amino acid sequence from the peptides encoded by the DNA molecules disclosed herein.
  • peptides will retain the essential characteristic of the peptides encoded by the DNA molecules disclosed herein, i.e., the ability to stimulate INF- ⁇ production. This characteristic can be readily determined by the assay technique described above.
  • variant peptides include those with variations in amino acid sequence including minor deletions, additions, and substitutions.
  • the site for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined.
  • 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.
  • preferred peptide variants will differ by only a small number of amino acids from the peptides encoded by the DNA sequences disclosed herein.
  • 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.
  • substitutions generally are made in accordance with the following Table 9 when it is desired to finely modulate the characteristics of the protein.
  • Table 9 shows amino acids that may be substituted for an original amino acid in a protein and that are regarded as conservative substitutions. As noted, all such peptide variants are tested to confirm that they retain the ability to stimulate INF- ⁇ production.
  • Substantial changes in immunological identity are made by selecting substitutions that are less conservative than those in Table 9, 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.
  • substitutions that, in general, are expected to produce the greatest changes in protein properties are 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.
  • a hydrophilic residue e
  • 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 (Bacillus Calmette-Guérin) vaccine, which is a live vaccine comprising attenuated Mycobacterium bovis bacteria.
  • BCG Bacillus Calmette-Guérin
  • 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, Wyngaarden et al. (eds.), Cecil Textbook of Medicine, 19 th ed., W. B. Saunders, Philadelphia, Pa., pages 1733-1742, 1992, 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 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, the system 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.
  • 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.
  • the vaccines may be formulated using a peptide according to the present invention together with a pharmaceutically acceptable excipient such as water, saline, dextrose, or glycerol.
  • a pharmaceutically acceptable excipient such as water, saline, dextrose, or glycerol.
  • the vaccines may also include auxiliary substances such as emulsifying agents and pH buffers.
  • vaccines formulated as described above may be administered in a number of ways including subcutaneously, intramuscularly, and by 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.
  • 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.
  • 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.
  • 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-released from the primed lymphocytes.
  • PBL peripheral blood lymphocytes
  • ESAT6 or ST-CF peripheral blood 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.
  • 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.
  • multiple immunostimulatory peptides may also be administered by expressing the nucleic acids encoding the peptides in a nonpathogenic microorganism, and using the transformed nonpathogenic microorganism as a vaccine.
  • 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.
  • 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 The 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.
  • a guinea pig protection study was undertaken to compare three candidate vaccine preparations with BCG. These included the Antigen 85 complex with IL-2 (Ag 85), a fusion-protein pool of twelve M. tuberculosis proteins in combination with IL-2 (FPP), and a control containing the adjuvant monophosphoryl lipid A (MPL).
  • Ag 85 the Antigen 85 complex with IL-2
  • FPP fusion-protein pool of twelve M. tuberculosis proteins in combination with IL-2
  • MPL adjuvant monophosphoryl lipid A
  • E. coli BL21 DE3 plysS (Novagen, Madison, Wis.): MBP-264 (SEQ ID NO: 114), MBP-506 (SEQ ID NO: 115), MBP-825 (SEQ ID NO: 116), PET-23 (SEQ ID NO: 117), PET-47 (SEQ ID NO: 118), PET-639 (SEQ ID NO: 119), PET-916 (SEQ ID NO: 120), PET-1084 (SEQ ID NO: 121); in E.
  • MBP-264 SEQ ID NO: 114
  • MBP-506 SEQ ID NO: 115
  • MBP-825 SEQ ID NO: 116
  • PET-23 SEQ ID NO: 117
  • PET-47 SEQ ID NO: 118
  • PET-639 SEQ ID NO: 119
  • PET-916 SEQ ID NO: 120
  • PET-1084 SEQ ID NO: 121
  • coli BL21 GST-152 (SEQ ID NO: 122), GST-511 (SEQ ID NO: 123), GST-822 (SEQ ID NO: 124); and in E. coli SURE (Stratagene, La Jolla, Calif.): GST-206 (SEQ ID NO: 125).
  • the Antigen 85 complex was kindly provided by Dr. John Belisle (Colorado State University, Fort Collins, Co.) through the TB research materials and vaccine testing contract (NIH, NIAID NOI AI-75320) (Belisle Science 276:1420-1422, 1997).
  • Vaccinations Guinea pigs were immunized subcutaneously two times at a three-week interval using 100 ⁇ g of AG85 complex with 20 ⁇ g Proleukin-PEG IL-2 (Chiron, Emeryville, Calif.) and emulsified in 100 ⁇ g Monophosphoryl Lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) adjuvant that had been solubilized in 0.02% triethanolamine and 0.4% dextrose by sonication (MPL-TeoA); and 100 ⁇ g of fusion proteins that had been pooled in equivalent concentrations with 20 ⁇ g PEG IL-2 (Chiron) in 100 ⁇ g MPL-TeoA adjuvant.
  • the positive control, BCG Copenhagen was injected once intradermally (10 3 bacilli/guinea pig), corresponding to the second set of injections.
  • Necropsy Guinea pigs were euthanized by the intraperitoneal injection of 1-3 mL of sodium pentobarbital (Sleepaway, Ft. Dodge, Iowa). The abdominal and thoracic cavities were opened aseptically and the spleen and right lower lung lobe were homogenised separately in sterile Teflon-glass homogenisers in 4.5 mL of sterile physiological saline. The number of viable M. tuberculosis organisms was determined by inoculating appropriate dilutions onto MiddlebrookTM 7H10 agar plates (Hardy Diagnostics, Santa Maria, Calif.). The colonies were counted after three weeks' incubation at 37° C. Data were expressed as mean log 10 number of viable organisms per portion of tissue.
  • compositions for diagnosing tuberculosis infection includes peptides encoded by one or more of 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.
  • 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.
  • SEPHADEX® Pharmaacia, Piscataway, N.J.
  • 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 ELISA.
  • the solid support to which the polypeptide is attached is the wall of a microtiter assay plate.
  • a protein such as bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • Excess BSA is removed by rinsing and the medical specimen is admixed with the polypeptide in the microtiter wells.
  • 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.
  • 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.
  • a substrate for the enzyme label such as hydrogen peroxide for the peroxidase enzyme
  • a color-forming dye precursor such as o-phenylenediamine
  • 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.
  • E. coli BL21 plysS MBP-506 (SEQ ID NO: 115), MPB-825 (SEQ ID NO: 116), PET-639 (SEQ ID NO: 119), PET-916 (SEQ ID NO: 120), PET-1084 (SEQ ID NO: 121); in E. coli BL21: GST-152 (SEQ ID NO: 122), GST-822 (SEQ ID NO: 124); and in E. coli SURE: GST-206 (SEQ ID NO: 125).
  • the recombinant fusion proteins MBP-506 (SEQ ID NO: 115), MBP-825 (SEQ ID NO: 116), GST-152 (SEQ ID NO: 122), GST-822 (SEQ ID NO: 124), PET-639 (SEQ ID NO: 119), PET-1084 (SEQ ID NO: 121) formed inclusion bodies that were harvested from the pellet following centrifugation of the bacterial sonicate.
  • the fusion proteins PET-916 and GST-206 were found primarily in the supernatant and underwent considerable breakdown in culture. Protein fractions were checked by SDS-PAGE using Coomassie Blue staining and approximate concentrations determined by Western blotting.
  • ELISA sera were obtained from 38 Brazilian individuals with pulmonary tuberculosis and that were HIV positive (TBH), from 20 individuals with extrapulmonary tuberculosis and that were HIV negative (EP-TB), and from 17 healthy volunteers.
  • Wells were coated with 200 ng of antigen in 50 ⁇ L of coating buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 adjusted to pH 9.6) and incubated for 1 hour. Plates were then aspirated, 250 ⁇ L of blocking buffer (0.5% BSA and 0.1% Thimerosal (Aldrich, Milwaukee, Wis.) in phosphate-buffered saline at pH 7.4) were added to each well, and the plates were incubated for a further 2 hours.
  • Plates were washed six times with 350 ⁇ L/well of washing solution (2 mL/L Tween 20 in PBS at pH 7.4) and serum was added at a 1:100 dilution in serum diluting buffer (blocking buffer with 2 mL/L Tween 20). Plates were incubated for 30 minutes and washed as before. 50 ⁇ L of a 1:50,000 dilution of HRP-Protein A (ZYMED, VWR) were added to each well. The plates were then incubated for 30 minutes and washed as before. 100 ⁇ L/well of TMB Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories) was added and the plates were incubated for 15 minutes in the dark.
  • washing solution 2 mL/L Tween 20 in PBS at pH 7.4
  • serum was added at a 1:100 dilution in serum diluting buffer (blocking buffer with 2 mL/L Tween 20). Plates were incubated for 30 minutes and
  • the reaction was stopped with 100 ⁇ L of 0.5 M H 2 SO 4 and the plates were read immediately at 450 nm.
  • Table 11 shows the overall seropositivity results for the nine full-length fusion proteins.
  • 71% of sera contained antibodies against at least one antigen (or 82% if the TB lysate individuals are included) and in the TBH group, 66% of sera contained antibodies against at least one antigen (or 84% if the TB lysate individuals are included).
  • specific antibody responses can be identified in the majority of the individual sera.
  • 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.
  • this test produces a positive result in one of the following conditions: the current presence of M. tuberculosis in the patient's body after exposure of the patient to M. tuberculosis and prior BCG vaccination.
  • 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 Wyngaarden et al. (eds.), Cecil Textbook of Medicine , W. B. Saunders, Philadelphia, Pa., 1992. If the subject has reactive T-cells to the injected protein, then 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 circulating M. tuberculosis antibodies in the patient.
  • DTH delayed type hypersensitivity
  • Purified immunostimulatory peptides according to the present invention may be employed in the tuberculin skin test using the methods described in Wyngaarden et al. (eds.), Cecil Textbook of Medicine , W. B. Saunders, Philadelphia, Pa., 1992.
  • 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 described in section V, above.
  • PCR polymerase chain reaction
  • PCR amplification employing primers based on the sequences disclosed herein may also be employed to quantify the amount of M. tuberculosis nucleic acid present in a particular sample (see chapters 8 and 9 of Innis et al., PCR Protocols: A Guide to Methods and Applications , Academic Press: San Diego, 1990). Reverse-transcription PCR using these primers may also be utilized to detect the presence of M. tuberculosis RNA, indicative of an active infection.
  • probes based on the nucleic acid sequences described herein may be labeled with suitable labels (such as 32 P or biotin-avidin enzyme linked systems) and used in hybridization assays to detect the presence of M. tuberculosis nucleic acid in provided samples.
  • suitable labels such as 32 P or biotin-avidin enzyme linked systems
  • 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 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 described below.
  • Monoclonal antibody to epitopes of the M. tuberculosis peptides identified and isolated as described herein can be prepared from murine hybridomas according to the classical method of Kohler and Milstein, Nature, 256:495, 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 with mouse myeloma cells by means of polyethylene glycol, and the excess unfused cells are destroyed by growth of the system on selective media comprising aminopterin (HAT media).
  • HAT media aminopterin
  • 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, Enzymol,. 70:419, 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, Antibodies, A Laboratory Manual , Cold Spring Harbor Laboratory, New York, 1988.
  • 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.
  • monoclonal antibodies that recognize a specific M. tuberculosis peptide are produced.
  • 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. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • a given antibody preparation such as one produced in a mouse
  • total cellular protein is extracted from a sample of human sputum from a healthy patient and from sputum from a patient suffering from tuberculosis.
  • 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.
  • an enzyme such as alkaline phosphatase.
  • 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.
  • 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.
  • n is a, c, g, or t/u.
  • n is a, c, g, or t/u.
  • n is a, c, g, or t/u.
  • n is a, c, g, or t/u.
  • n is a, c, g, or t/u; r is g or a.
  • n is a, c, g, or t/u.
  • n is a, c, g, or t/u; k is g or t/u.
  • n is a, c, g, or t/u.
  • n is a, c, g,
  • n is a, c, g, or t/u.
  • n is a, c, g, or t/u; k is g or t/u.
  • n is a, c, g, or t/u.
  • n is a, c, g, or t/u.
  • n is a, c, g, g, g, g, g, g, g, g, gtatncca cggnactacc cgcggtcctt cctcnantnc 60 cgccggncca gncgcagnct ncngatgtcc ngctataacc tgcgcgatcg ccgccgggct 120 gcccgacaac acggtgngcg ccgccgctgc tccgccaat tctgggtgnc ggcatnccgg 180 cagcgcccgg cccagcactg agagggggac gttgatgcgg tggccgacgg ctggctgct 240 ggc 243 61 2348 DNA Mycobacterium tuberculosis misc_feature (1)..(2348)

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Abstract

Nucleotide sequences isolated from Mycobacterium tuberculosis are disclosed. These sequences encode immunostimulatory peptides. Also disclosed are vaccine preparations formulated using these peptides.

Description

    CROSS REFERENCE TO RELATED CASES
  • This application is a continuation-in-part of co-pending U.S. application Ser. No. 08/990,823, filed Dec. 15, 1997, which is incorporated herein by reference. The Ser. No. 08/990,823 application claims priority from PCT Application No. U.S. 96/10375, filed Jun. 14, 1996, which claims priority from U.S. Provisional Application No. 60/000,254, filed Jun. 15, 1995, all of which are incorporated herein by reference.[0001]
    • I. BACKGROUND
  • A. The Rise of Tuberculosis [0002]
  • Over the past few years the editors of the [0003] Morbidity and Mortality Weekly Report have chronicled the unexpected rise in tuberculosis cases. It has been estimated that one billion people are infected with M. tuberculosis worldwide, 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-drug resistant strains of [0004] 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. [0005]
  • B. Tuberculosis and the Immune System [0006]
  • Infection with [0007] 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, Skamene, Ref. Infect. Dis. 11:S394-S399, 1989. Initial control of M. tuberculosis may also be influenced by reactive T γδ 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, Kaufmann, Current Opinion in Immunology 3:465-470, 1991. 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 [0008] M. tuberculosis T cell epitopes and secreting cytokines, particularly INF-γ, that 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, Orme et al., [0009] Journal of Immunology 148:189-196, 1992. These scientists suggest that antigens are pinocytosed from the mycobacterial-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 mice infected with [0010] M. tuberculosis or from PPD-positive humans are found in both the whole mycobacterial cells and also in the culture supernatants, Orme et al., Journal of Immunology 148:189-196, 1992; Daugelat et al., J. Infect. Dis. 166:186-190, 1992; Barnes et al., J. Immunol. 143:2656-2662, 1989; Collins et al., Infect. Immun. 56:1260-1266, 1988; Lamb et al., Rev. Infect. Dis. 11:S443-S447, 1989; and Hubbard et al., Clin. exp. Immunol. 87: 94-98, 1992. Recently Pal and Horwitz, Infect. Immun. 60:4781-4792, 1992, induced partial protection in guinea pigs by vaccinating with M. tuberculosis supernatant fluids. Similar results were found by Andersen using a murine model of tuberculosis, Andersen, Infection & Immunity 62:2536, 1994. Other studies include Hubbard et al., Clin. exp. Immunol. 87: 94-98, 1992, and Boesen et al., Infection and Immunity 63:1491-1497, 1995. 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.
    • II. SUMMARY OF THE INVENTION
  • 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, Jardim et al., [0011] J. Exp. Med. 172:645-648, 1990.
  • It is an object of this invention to provide [0012] 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.
  • The present invention provides inter alia, DNA sequences isolated from [0013] Mycobacterium tuberculosis. Peptides encoded by these DNA sequences 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 correlates with maximum expression of protective immunity against tuberculosis, Orme et al., J. Immunology 151:518-525, 1993. 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, Boesen et al., Infection and Immunity 63:1491-1497, 1995.
  • Hence, the DNA sequences provided by this invention encode peptides that can of stimulate 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, Mougneau et al., [0014] Science 268:536-566, 1995 and Jardim et al., J. Exp. Med. 172:645-648, 1990. For example, T-cell epitopes from the parasite Leishmania major have been shown to be effective when administered as a vaccine, Jardim et al., J. Exp. Med. 172:645-648, 1990; Mougneau et al., Science 268:536-566, 1995; and Yang et al., J. Immunology 145:2281-2285, 1990. Therefore, the immunostimulatory peptides (T-cell epitopes) encoded by the DNA sequences according to the invention 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 [0015] 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.
  • According to one aspect of the present invention, immunostimulatory preparations are provided 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. [0016]
  • According to another aspect of the invention, vaccines are provided comprising one or more peptides encoded by nucleotide sequences provided herein. Such a vaccine may include one or more pharmaceutically acceptable excipients, adjuvants, and/or diluents. [0017]
  • According to another aspect of the present invention, antibodies are provided that are specific for immunostimulatory peptides encoded by a nucleotide sequence according to the present invention. Such antibodies may be used to detect the presence of [0018] 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 nucleotide sequences according to 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 [0019] 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 improved methods of performing the tuberculin skin test to diagnose exposure of an individual to [0020] 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 [0021] 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 is homologous to proteins known to be located in bacterial cell-surface membranes. This discovery suggests that membrane-bound peptides, particularly those from [0022] 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 ID NO: 67, the amino acid sequence is shown in SEQ ID NO: 113.[0023]
    • IV. DESCRIPTION OF THE INVENTION
  • A. Definitions [0024]
  • Particular terms and phrases used herein have the meanings set forth below. [0025]
  • “Specific binding agent.” An agent that binds substantially only to a defined target. Thus, a [0026] Mycobacterium tuberculosis specific binding agent binds substantially only cellular components derived from Mycobacterium tuberculosis. These cellular components include both extracellular and intracellular, proteins, glycoproteins, sugars, and lipids, that are found in Mycobacterium tuberculosis isolates. As used herein, the term “Mycobacterium tuberculosis specific binding agent” can be an anti-Mycobacterium tuberculosis antibody or other agent that binds substantially only to Mycobacterium tuberculosis.
  • The term “anti-[0027] Mycobacterium tuberculosis antibodies” encompasses monoclonal and polyclonal antibodies that are specific for Mycobacterium tuberculosis, i.e., which bind substantially only to Mycobacterium tuberculosis when assessed using the methods described below, as well as immunologically effective portions (“fragments”) of such antibodies. Immunologically effective portions of the antibodies include Fab, Fab′, F(ab′)2, Fabc, and Fv portions (for a review, see Better and Horowitz, Methods Enzymol., 178:476-496, 1989). Anti-Mycobacterium tuberculosis antibodies may also be produced using standard procedures described in a number of texts, including Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • “Sequence Identity.” The similarity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences are. [0028]
  • Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, [0029] Adv. Appl. Math., 2:482, 1981; Needleman & Wunsch, J. Mol. Biol., 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins & Sharp, Gene, 73:237-244, 1988; Higgins & Sharp, CABIOS, 5:151-153, 1989; Corpet et al., Nucleic Acids Research, 16:10881-10890, 1988; Huang, et al., Computer Applications in the Biosciences, 8:155-165, 1992; and Pearson et al., Methods in Molecular Biology, 24:307-331, 1994. Altschul et al., J. Mol. Biol., 215:403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
  • The NCBI Basic Local Alignment Search Tool (BLAST™ Altschul et al. [0030] J. Mol. Biol., 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at http//www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
  • For comparisons of amino acid sequences of greater than about 30 amino acids, the “Blast 2 sequences” function in the BLAST program is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per-residue gap cost of 1). When aligning short peptides (fewer than about 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 45%, at least 50%, at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity. [0031]
  • “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. [0032]
  • 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. [0033]
  • “Probes” and “primers.” Nucleic acid probes and primers may be readily 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, Sambrook et al. (ed.), [0034] Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al. (ed.); Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987.
  • “Primers.” Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length, that 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. [0035]
  • 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. [0036]
  • Methods for preparing and using probes and primers are described, for example, in Sambrook et al. (ed.), [0037] Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al. (ed.), Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987; and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. 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 second 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. [0038]
  • “Operably linked.” A first nucleic acid sequence is “operably” linked with a second nucleic acid sequence whenever 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. [0039]
  • “Recombinant.” A “recombinant” nucleic acid 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. [0040]
  • “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 [0041] 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. (ed.), [0042] Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, at pages 9.52-9.55, 9.47-9.52 and 9.56-9.58; Kanehisa, Nuc. Acids Res. 12:203-213, 1984; and Wetmur et al., J. Mol. Biol. 31:349-370, 1968.
  • 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 appreciated readily by those skilled in the art. [0043]
  • 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). [0044]
  • 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. [0045]
  • “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. [0046]
  • “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 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. [0047]
  • B. Materials and Methods [0048]
  • 1. Standard Methodologies [0049]
  • 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. (ed.), [0050] Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al. (ed.), Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987.
  • Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage et al., [0051] Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers.
  • 2. Isolation of [0052] Mycobacterium tuberculosis DNA Sequences Encoding Immunostimulatory Proteins
  • [0053] Mycobacterium tuberculosis DNA was obtained by the method of Jacobs et al., Methods In Enzymology 204:537-555, 1991. Samples of the isolated DNA were partially digested with one of the following restriction enzymes HinPI, HpaII, AciI, TaqI, BsaHI, and NarI. Digested fragments of 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 pJDT3.
  • A schematic representation of the phagemid vector pJDT2 is provided in Mdluli et al., [0054] Gene 155:133-134, 1995. 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 (XP). Alkaline phosphatase cleaves XP to release a blue-colored product. Hence, those clones containing alkaline phosphatase fusion proteins with the enzymatic portion lying outside the cytoplasmic membrane will produce the blue color.
  • The three vectors in this series (pJDT1, pJDT2, and pJDT3) 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 three. Mdluli et al., [0055] Gene 155:133-134, 1995, describes pJDT vectors in detail.
  • 3. Selection of Secreted Fusion Proteins [0056]
  • The recombinant clones described above were transformed into [0057] 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., J. Bact. 177:59-65, 1995.
  • 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 [0058] 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 [0059]
  • PhoA fusion proteins were extracted from the selected [0060] E. coli clones by cell lysis and purified by SDS polyacrylamide gel electrophoresis. Essentially, individual E. coli clones were grown overnight at 30° C. with shaking in 2 mL LB broth containing ampicillin, kanamycin, and IPTG. The cells were precipitated by centrifugation and resuspended in 100 μL Tris-EDTA buffer. To this mixture was added 100 μL lysis buffer (1% SDS, 1 mMEDTA, 25 mM DTT, 10% glycerol and 50 mM tris-HCl, pH 7.5). DNA released from the cells was sheared by passing the mixture through a small-gauge syringe needle. The sample was 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 were loaded onto each gel.
  • The samples were electrophoresed by application of 200 volts to the gel for 4 hours. Subsequently, the proteins were transferred to a nitrocellulose membrane by Western blotting. A strip of nitrocellulose was cut off to be processed with antibody, and the remainder of the nitrocellulose was set aside for eventual elution of the protein. The strip was incubated with blocking buffer and then with anti-alkaline phosphatase primary antibody, followed by incubation with anti-mouse antibody conjugated with horseradish peroxidase. Finally, the strip was 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, was measured with a ruler, and the corresponding region of the undeveloped nitrocellulose blot was excised. [0061]
  • This region of nitrocellulose containing the PhoA fusion protein was then incubated in 1 mL 20% acetronitrile at 37° C. for 3 hours. Subsequently, the mixture was centrifuged to remove the nitrocellulose, and the liquid was transferred to a new test tube and lyophilized. The resulting protein pellet was dissolved in 100 μL of endotoxin-free, sterile water and precipitated with acetone at −20° C. After centrifugation the bulk of the acetone was removed and the residual acetone was allowed to evaporate. The protein pellet was re-dissolved in 100 μL of sterile phosphate buffered saline. [0062]
  • 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. [0063]
  • 5. Determination of Immunostimulatory Capacity in Mice [0064]
  • The purified alkaline phosphatase-[0065] 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 was essentially as described by Orme et al., J. Immunology 151:518-525, 1993.
  • Essentially, the assay method is as follows: The virulent strain [0066] 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 mice are inoculated with 1×105 viable bacteria suspended in 200 μL sterile saline via a lateral tail vein on the first day of the test.
  • Bone marrow-derived macrophages are used in the test to present the bacterial alkaline phosphatase-[0067] 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., J. Immunology 151:518-525, 1993. 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[0068] 6 of these CD4 cells, and the medium is supplemented with 5 U interleukin-2 (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., [0069] J. Immunology 151:518-525, 1993.
  • 6. Determination of Immunostimulatory Capacity in Humans [0070]
  • The purified alkaline phosphatase-[0071] 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 atop 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. The plasma is then added to the tissue culture media. The mononuclear cells are washed twice by a procedure in which 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. [0072]
  • 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[0073] 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 INF-γ).
  • Cytokines are measured using an Enzyme Linked Immunosorbent Assay (ELISA), the details of which are described in the Cytokine ELISA Protocol in the PharMingen catalog (PharMingen, San Diego, Calif.). To measure the presence of human INF-γ, wells of a 96-well microtitre plate are coated with a “capture antibody” (e.g., anti-human INF-γ antibody). The sample supernatants are then added to individual wells. Any INF-γ present in the sample binds to the capture antibody. The wells are then washed. A “detection antibody” (e.g., anti-human INF-γ antibody), conjugated to biotin, is added to each well, and binds to any INF-γ bound to the capture antibody. Any unbound detection antibody is washed away. An avidin-linked horseradish 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 quantified using an ELISA plate reader. The amount of the INF-γ in the sample supernatants is determined by comparison with a standard curve using known amounts of human INF-γ. [0074]
  • Measurement of other cytokines, such as IL-2 and interleukin-4 (IL-4), can be determined using the same protocols with the appropriate substitution of reagents (monoclonal antibodies and standards). [0075]
  • 7. DNA Sequencing [0076]
  • 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 [0077] Mycobacterium tuberculosis DNA insert, or primers specific to the cloned sequences.
  • C. Results [0078]
  • 1. Immunostimulatory Capacity [0079]
  • More than 300 fusion clones were tested for their ability to stimulate INF-γ production. Of these, 80 clones initially were designated to have some ability to stimulate INF-γ production. Tables 1 and 2 show the data obtained for these 80 clones. Clones listed in Table 1 showed the greatest ability to stimulate INF-γ production (greater than 10 ng/mL of INF-γ), while clones listed 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 [0080] 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 ID NO: Name INF Sanger ID of Mtb gene Functional Identification
    2 AciI#1-152 >40,000 MTCY16By.09 glycerol-3-phosphate binding periplasmic
    protein precursor
    4 AciI#1-247 >40,000 MTCI364.18 fatty acid transport 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 MTV023.04c
    67 HinP#2-92 >20,000 MTCY190.11c cytochrome c oxidase subunit II
    110 HinP#2-145 >20,000 MTV018.38c
    52 HinP#2-150 >20,000 MTCY190.11c COXII (same as 2-92)
    48 HinP#1-200 >20,000 MTV003.08
    54 HinP#3-30 >20,000 MTCY19H5.30c
    6 AciI#2-2 >20,000 MTV003.10c lipoprotein, penicillin binding protein
    7 AciI#2-23 >20,000 MTCY13E10.15c
    11 AciI#2-506 >20,000 MTCY253.27c 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 MTV004.48 unknown
    68 AciI#2-823 >20,000 MTCY77.20 unknown membrane protein
    61 AciI#2-825 >20,000 MTCY31.03c
    71 AciI#2-827 >20,000 MTCY01B2.15c cytochrome d (ubiquinol) oxidase (appC)
    22 AciI#2-898 >20,000 MTV005.02
    27 AciI#2-1084 >20,000 MTV023.03c
    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 potential membrane
    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 MTV025.111 19kDa Antigen
    47 HinP#1-144 13,546 MTCI28.11 unknown
    70 HinP#1-3 11,550 MTV023.04c same as HinP1-27
    111 AciI#1-486 11,416 MTCY13D12.26 embC (LysR family)
    5 AciI#1-426 11,135 MTV025.013c dppB (peptide transport permease)
    23 AciI#2-916 10,865 MTCY21D4.03c unknown (signal peptide)
  • [0081]
    TABLE 2
    Immunostimulatory AP-fusion clones
    SEQ ID NO: Clone Name INF Sanger/TIGR ID of Mtb gene Functional Identification
     1 AciI#1-62 3,126 MTCY190.11c COXII(same as 2-92)
     8 AciI#2-26 3,089 MTV023.02c
     9 AciI#2-35 3,907 MTV023.05c
    76 AciI#2-147 5,464 same as H2-147 or H1-200
    12 AciI#2-508 7,052 MTCY20G9.23
    14 AciI#2-523 2,479 MTCY427.10c unknown
    72 AciI#2-834 5,942 MTV016.33c
    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 MTCY21D4.03c
    21 AciI#2-894 3,396 MTV002.33c PPE family
    24 AciI#2-1014 6,302 MTCY21D4.03C same as 2-916
    74 AciI#2-1018 4,642 MTCY270.11 MURF
    25 AciI#2-1025 3,582 MTCY359.10 unknown membrane protein
    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 MTV003.10c penicillin binding protein
    33 AciI#3-15 4,019 MTCY20H10.03
    35 AciI#3-78 2,905 MTCI28.14 same as A3-167
    37 AciI#3-134 3,895 MTCY22G8.04 same as A3-133
    40 AciI#3-204 4,774 MTCY50.02 same as A3-47
    42 AciI#3-214 7,333 MTCY33.38 unknown
    112  AciI#3-243 2,857 MTCY50.02
    43 AciI#3-281 2,943 MTCY19H5.32c
    44 Bsa HI#1-21 8,122 M. bovis clone
    45 HinP#1-12 2,905 MTCY49.31c unknown
    49 HinP#2-23 2,339 MTCY0033.38 same as A30214
    46 HinP#1-142 6,258 MTCY02B10.27c unknown
    50 HinP#2-143 3,689 MTCY274.09c unknown, thioredoxin-like
    51 HinP#2-145A 2,314
    53 HinP#3-28 2,980 MTV009.03c LppS
    55 HinP#3-34 2,564 MTCY25D10.07 unknown
    56 HinP#3-41 3,296 P31953 Antigen 85c, 85b & 85a precursor
    P31952
    P17944
    57 HpaII#1-3 2,360 MTCY190.11c COXII
    58 HpaII#1-8 2,048 MTCY432 unknown
    59 HpaII#1-10 4,178 MTCY39.39 same as A2-1089
    60 HpaII#1-13 3,714 MTCY16B7.47 unknown partial ORF
  • 2. DNA Sequencing and Determination of Open Reading Frames [0082]
  • DNA sequence data for the sequences of the [0083] 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 respective coding strands of the Mycobacterium DNA. In most instances, these sequences represent only partial sequences of the respective immunostimulatory peptides and, in turn, only partial sequences of respective 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, given the information provided herein, readily can obtain the immunostimulatory peptides and corresponding full-length M. tuberculosis genes using standard techniques. Accordingly, the nucleotide sequences of the present invention encompass not only those respective sequences presented in the sequence listings, but also the respective complete nucleotide sequence encoding the respective 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 et al., [0084] Gene 30: 157-166, 1984. This program uses the bias of these nucleotides for each of the codon positions to identify the correct reading frame. 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 identification of 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 an number of potentially overlapping sequences or sequences from the same gene, as noted below: [0085]
    Clone Overlapping Sequence(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, H#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
    AeiI#3-167 AciI#3-78
    AciI#2-916 AciI#2-1014
    AciI#2-1089 HpaII#1-10
    AciI#3-243 AciI#3-47, AciI#3-204
    Hinp#2-23 AciI #3-214
  • 3. Identification of T Cell Epitopes in the Immunostimulatory Peptides [0086]
  • 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. The program uses a series of four predictive algorithms. In particular, peptides were designed against regions indicated by the algorithm “A” motif which predicts alpha-helical periodicity, Margalit et al., [0087] J. Immunol. 138:2213, 1987, and amphipathicity. Peptides were also designed against regions indicated by the algorithm “R” motif which identifies segments that display a similarity to motifs known to be recognized by MHC class I and class II molecules, Rothbard and Taylor, 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 [0088]
  • 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.). [0089]
  • Peptides designed from sequences described in this application include: [0090]
    Peptide Sequence Peptide Name SEQ ID NO:.
    HinP#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
    AciI2-827 (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
    WIFGWNRLPRLVHLACI 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 (combined) 99
    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. [0091]
  • 5. Confirmation of Immunostimulatory Capacity Using T Cells from Tuberculosis Patients [0092]
  • 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 production of INF-γ in T-cells from tuberculin-positive people using the methods described in part IV(B)(6), above. For each assay, 5×10[0093] 5 mononuclear cells were stimulated with up to 1 μg/mL M. tuberculosis peptide or up to 50 ng/mL PhoA fusion protein. M. tuberculosis filtrate proteins, Con A and PHA, were employed as positive controls. An assay was run with medium alone to determine background levels, and PhoA protein was employed as a negative control.
  • The results, shown in Table 4 below, indicate that all of the peptides tested stimulated INF-γ production from T-cells of a particular subject. [0094]
    TABLE 4
    Concentration of
    Concentration of INF-γ
    Peptide or PhoA INF-γ 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
    proteins (5 μg/mL) 134.0 30.7
    Con A(10 μg/mL) 2839 2735.7
    PHA(1%) 10378 10274.7
    PhoA control 26.7 0
    (10 μg/mL)
    Background 103.3 0
    • V. CLONING OF FULL-LENGTH MYCOBACTERIUM TUBERCULOSIS ORFs CONTAINING T-CELL EPITOPES
  • Most the sequences presented represent only part of a larger [0095] 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 [0096]
  • 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 [0097] 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 labeled with a suitable marker to enable detection of hybridizing clones. Suitable markers include radio nucleotides, such as 32P and non-radioactive markers such as biotin-avidin enzyme linked systems. Methods for constructing suitable libraries, production and labeling of oligonucleotide probes, and colony hybridization are standard laboratory procedures and are described in standard laboratory manuals such as in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al. (ed.), Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987.
  • 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 Sambrook et al. (ed.), [0098] Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. 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 amplifying of flanking sequences by IPCR are described in Chapter 27 of Innis et al., [0099] PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, 1990, and in Earp et al., Nucleic Acids Research 18:3721-3729, 1990.
  • Accordingly, the present invention encompasses small oligonucleotides included in the DNA sequences presented in the respective Sequence Listings. These small oligonucleotides are useful as hybridization probes and PCR primers that can be employed to clone the corresponding full-length [0100] 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 as set forth in the respective 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 can comprise a sequence of at least 15 nucleotides and preferably at least 20 nucleotides, the oligonucleotide sequence being substantially similar to a respective DNA sequence set forth in the respective Sequence Listing. Preferably, such oligonucleotides will share at least about 75%-90% sequence identity with a respective DNA sequence set forth in the respective Sequence Listing and more preferably the shared sequence identity will be greater than 90%. [0101]
  • B. Example—Closing of the Full-Length Orf Corresponding to Clone HinP #2-92 [0102]
  • 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 base pairs upstream) by a Shine-Dalgarno motif. The gene mtb2-92 encodes a protein (termed MTB2-92) containing 363 amino acid residues with a predicted molecular weight of 40,436 Da. [0103]
  • 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. Cytochrome c oxidase is part of the electron transport chain, in which the subunits I and II form the functional core of the enzyme complex. [0104]
  • 1. Cloning the Full-Length Gene Corresponding to HinP #2-92 [0105]
  • The plasmid pHin2-92 was restricted with either BamH1 or EcoRI and then subcloned into the vector M13. The inserted DNA fragments were sequenced under the direction of M13 universal sequencing primers, Yanisch-Perron et al., [0106] Gene 33:103-119, 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 ID NO: 106) and 5′ GGCCTCAGCGCGGCTCCGGAGG 3′ (SEQ ID 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 [0107] Mycobacterium tuberculosis genomic cosmid DNA library was screened using PCR, Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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 aliqots of distilled water 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 10×PCR buffer, 250 μM dNTPs, 300 nM downstream and upstream primers, and 1 unit Taq DNA polymerase.
  • The reactions were heated at 95° C. for 2 min, and 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 et al. (ed.), [0108] Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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 that 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. [0109]
  • 2. Expression of the Full-Length Gene [0110]
  • 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′TCTAGACACCACCACCACCACCACGTGACACCT CGCGGGCCAGGTC 3′ (SEQ ID NO: 108) and 5′AAGCTTCGCCATGC CGCCGGTAAGCGCC 3′ (SEQ ID NO: 109)). [0111]
  • The 100-μL PCR reaction contained: 1 μg pG3 template DNA, 250 nM dNTP's 300 nM of each primer, 10 μL of 10×PCR buffer, and 1 unit Taq DNA polymerase. The PCR DNA synthesis cycle was performed as described above. [0112]
  • The 1.4-kb PCR products were purified and ligated into the cloning vector pGEM-T (Promega). Inserts were removed by digestion using both Xba I and HindIII and the 1.4-kb fragment was directionally subcloned into the Xba I and Hind III 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. [0113]
  • 3. Purification of the Encoded Protein [0114]
  • The plasmid pMAL-MTB2-92 was transformed into competent [0115] E. coli JM109 cells and a 1-liter culture was grown up in LB broth at 37° C. to an OD550 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. The cell pellet 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 (20 mM Tris-HCl, pH 7.2, 200 mM NaCl, and 1 mM EDTA) and applied to an amylose-resin column (New England Bio-Labs Ltd., 3397 American Drive, Unit 12, Mississauga, Ontario, L4V 1T8, Canada) that had been pre-equilibrated with buffer A. The column was then washed with buffer A until the eluate reached an A[0116] 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). [0117]
  • At each stage of the protein purification, a sample was analyzed by SDS polyacylamide gel electrophoresis, Laemmli, [0118] Nature (London) 227:680-685, 1970.
  • C. Correction of Sequence Errors [0119]
  • Some of the sequences presented in the Sequence Listing may contain sequence ambiguities. Sequence ambiguities occur when the results from the sequencing reaction do not clearly distinguish between the individual base pairs. Therefore, substitute abbreviations denoting multiple base pairs are provided in Table 3, supra. These abbreviations denote which of the four bases could possibly be at a position that was found not to give a clear experimental result. 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 [0120] M. tuberculosis genomic DNA as a template. Standard PCR methodologies, such as those described above, may be used to accomplish this.
  • D. Additional Examples of Cloning of Full-Length Mtb-PhoA Fusion Proteins [0121]
  • Selected mtb-phoA fusions were sequenced using the Taq-Track™ sequencing system (Promega Corp.), and sequencing was directed from a primer located 48 bp upstream of the junction between the [0122] M. tuberculosis and phoA DNA. Sequences were compared to the databases of the M. tuberculosis genome projects, Cole et al., Nature 393:537-544, 1998, and the National Centre of Biotechnology Information at the National Library of Medicine (Bethesda, Md.) using the “BLASTX”, “BLASTN”, and “TBLAST” programs, Atschul et al., Nucleic Acids 25:3389-3402, 1997. A determination of signal peptide determination was made using the SignalP neural network trained on Gram-positive data, Neilson et al., Protein Eng. 10:1-6, 1997.
  • Whenever the upstream DNA sequence matched the raw sequence from the database of the [0123] M. tuberculosis genome projects, the extent of the reading frame and direction of translation were ascertained using a G+C analysis package, Bibb et al., Gene 30:157-166, 1984. A verification was made of whether the mtb-phoA fusion was in the same reading frame as the predicted ORF. In some cases, the extent of the ORF had already been assigned, and the assessment of the genome project was used for the fusion construction.
  • PCR was used to amplify the complete predicted ORFs encoding the proteins identified in the immunogenicity study. Oligodeoxynucleotide primers were designed with restriction sites in order to clone the amplified fragments into expression vectors. Table 5 provides a description the primer sequences used. All PCR reactions were conducted in 20 μL using a 6:1 Taq polymerase: Pfu polymerase enzyme combination. The reaction mixes contained either 1 μL DMSO or 4 μL of Q solution (proprietary solution available from Qiagen, Dusseldorf, Germany, for denaturation in PCR) as a denaturant. A manual hot start was used for all PCR reactions which consisted of an initial denaturization (95° C., 4 min.) followed by a final extension (72° C., 4 min.). Standard protocols were followed for cloning, Sambrook et al. (ed.), [0124] Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and expression of the Mycobacterium tuberculosis proteins as fusions in commercial expression vectors. The different expression vectors used included pMAL-c2 (New England Biolabs, Beverly, Mass.), pGEX-4T3 (Pharmacia, Piscataway, N.J.), and pET-17xb (Novagen, Madison, Wis.). Respectively, these expression vectors enabled the N-terminal fusion of the maltose binding protein (MBP), glutathione S-transferase (GST) or the 260 amino acid T7 gene 10 product (PET) containing the T7Tag® (Novagen, Madison, Wis.) to the products of the cloned DNA.
    TABLE 5
    Oligonucleotide Primers Used for PCR Amplification
    SEQ ID
    Clone Primer Sequence 5′ to 3′ NO:
    GST- 1-152F GTCAAGGATCCGGCATGGACCCGCTGAACCGCCGAC 142
    152 1-152R ATGTCGGGATCCAAGCTTTCGACGGTCGGCGCGTCGGCGCCGGG 143
    MBP- 1-264F GCAGATGCATCTAATGGGATCCGCGGAGTATATCTCC 144
    264 1-264R GGCGCCGTGGGTGTCAGCGAAGCTTACCTGGTTGTTG 145
    PET- 2-23F GGTGCCGAATTCGCGCCGATGCTGGACGCGG 146
    23 2-23R ACCCGAATTCCCAAGCTTGCTGCTCAAACCACTGTTCC 147
    MBP- 2-506F GCGCCCAAGGGATCCCCGGCTACCATGCCTTCG 148
    506 2-506R CTCGAAGGGATCCGCGTTCGTTTGGCCGCCCGC 149
    GST- 2-511F GGCAGTGGGATCCGTAGCGGTGCGGCGTAAGGTGCGG 150
    511 2-511R GACTTCGTGGATCCGGTCAAGACAAGCTTTGCGGTGATCAAGGCGGC 151
    PET- 2-639F CATGAATGAAGTTCATCTCACAAGCGTGCGGCTCCCACCGACCC 152
    639 2-639R CCTTGGCGAAGTTCTCAAAGGAAAGCTTCGAAGGCGG 153
    GST- 2-822F GGAGTTCGGATCCATCGCCATGCAACTCTCCTCCCGG 154
    822 2-822R GGGCAGTGGATCCGTGGTCAGCAAGCTTTCCCTAGAGTTTCGTGCG 155
    MBP- 2-825F GTGGCGCCGAAGTTCAAGCGCGGTGTCGCAACGCTG 156
    825 2-825R CGCTTAAGCGCGAAGCTTCGTCGAGCCGCG 157
    PET- 2-916F GACCGGAATTCATGATCCAGATCGCGCGCACCTGGCGG 158
    916 2-916R AACATGAATTCAAGCTTCGAGGCCGCCGACGAATCCGCTCACCG 159
    PET- 2-1084F CGGGTCGCCGAATTCACGCGGAGCCGGGGATTGCGC 160
    1084 2-1084R GGCGGAATTCAAGCTTCGGTTCATCCGCCGCCCCCATGC 161
    GST- 3-206F CCCCGGGGATCCGGGGGTGCTGGGATGACGG 162
    206 3-206R ACGACGGATCCTAAGCTTGCAGGCGCGCCGATACGCGGC 163
    GST- 2-827F TCTCCGGGGATCCCAGATGAATGTCGTCGACATTTC 164
    827 2-827R GGGTCTCCGGATCCCCCATACCGACATG 165
    GST- 1-247F CCGACTCGAGCGGCGGCGCACACACAACGGTC 166
    247 1-247R AATCCTCGAGCCCTGCGGTCGCCTTCCGAGCG 167
    PET- 3-47F ATCCGGCCCGAATTCGCTGACCGTGGCCAGCGACGA 168
    47 3-47R GATCGGGGAGAATTCCGCCGACTTAAGCTTCAGCTGAGCTGG 169
  • The different expression vectors used included pMAL-c2 (New England Biolabs), pGEX-4T3 (Pharmacia, Piscataway, N.J.), and pET-17xb (Novagen, Madison, Wis.). These expression vectors enabled the N-terminal fusion of the maltose binding protein (MBP), glutathione S-transferase (GST), or the 260 amino acid T7 gene 10 product (PET) containing the T7Tag® (Novagen, Madison, Wis.) to be added to the products of the cloned DNA. Table 6 provides a summary of the results from cloning the PCR products into the various vectors described above. [0125]
    TABLE 6
    Recombinant Plasmids for Cloning and Expression of the Full-length Proteins
    Plasmid Expression Cloning Fusion Predicted Mr
    Construct Vector Sites Sanger ID Product (kDa)
    pAM23E pET-17xb E MTCY13E10.15c PET-23 124 
    pAM47E pET-17xb E MTCY50.02 PET-47 94
    pAM152E pGEX-4T3 B MTCY16B7.09 GST-152 70
    pAM206E pGEX-4T3 B MTCY270.17 GST-206 60
    pAM247E pGEX-4T3 E MTC1364.18 GST-247 78
    pAM264E pMAL-c2 B, H MTCY78.03c MBP-264 68
    pAM506E pMAL-c2 B MTCY253.27c MBP-506 105 
    pAM511E pGEX-4T3 B MTCY50.08c GST-511 46
    pAM639E pET-17xb E MTCY02B12.02 PET-639 56
    pAM822E pGEX-4T3 B MTV004.48 GST-822 50
    pAM825E pMAL-c2 E, H MTCY31.03c MBP-825 60
    pAM827E pGEX-4T3 B MTCY01B2.15c GST-827 80
    pAM916E pET-17xb E MTCY21D4.03c PET-916 61
    pAM1084E pET-17xb E MTV023.03c PET-1084 76
  • SDS-PAGE and Western Blotting were used to identify the novel antigens expressed by the clones. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using 10% slab gels in a continuous buffer system, Laemmli, [0126] Nature (London) 227:680-685, 1970. Proteins were electrophoretically transferred from the gel to a nitrocellulose membrane using standard protocols, Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Western blots for the GST (Pharmacia), T7 gene 10 (Invitrogen), and MBP (NEB) tagged fusion proteins were conducted as per the suppliers' instructions. The chemiluminescent Renaissance™ system (DuPont NEN Renaissance, NEL-201) was used to image bound antibody.
  • Subsequently, the following fusion proteins were over-expressed in [0127] E. coli BL21 plysS: MBP-264 (SEQ ID NO: 114), PET-23 (SEQ ID NO: 117), MBP-506 (SEQ ID NO: 115), MPB-825 (SEQ ID NO: 116), PET-639 (SEQ ID NO: 119), PET-916 (SEQ ID NO: 120), PET-1084 (SEQ ID NO: 121), PET-47 (SEQ ID NO: 118); in E. coli BL21: GST-152 (SEQ ID NO: 122), GST-822 (SEQ ID NO: 124); and in E. coli SURE: GST-206 (SEQ ID NO: 125). The recombinant fusion proteins MBP-506 (SEQ ID NO: 115), MBP-825 (SEQ ID NO: 116), GST-152 (SEQ ID NO: 122), GST-822 (SEQ ID NO: 124) GST-827 (SEQ ID NO: 126), PET-639 (SEQ ID NO: 119), PET-1084 (SEQ ID NO: 121) formed inclusion bodies that were harvested from the pellet following centrifugation of the bacterial sonicate. The fusion proteins PET-916 and GST-206 were found primarily in the supernatant and underwent considerable breakdown in culture. Protein fractions were checked by SDS-PAGE using Coomassie Blue staining and approximate concentrations were determined by Western blotting.
  • An additional fusion protein, GST-247 (SEQ ID NO: 127), was constructed using a different cloning strategy. The PCR product resulting from a reaction using the primers described in Table 5 and the entire MTCI364.18 cds was digested with EcoR1. The resulting fragment was then cloned into pGEX-4T3, with the C-terminus adjacent to the GST sequence. The resulting protein fragment included amino acid 43 to amino acid 514 of the 597 amino acid protein predicted by the genome project. [0128]
  • The amino acid sequences encoded by nucleic acid sequences described above are also shown in the sequence listing. These amino acid sequences are SEQ ID NOS: 128-141, which correspond to the nucleic acid sequences shown in SEQ ID NOS: 114-127, respectively. [0129]
    • VI. EXPRESSION AND PURIFICATION OF THE CLONED PEPTIDES
  • The DNA sequences disclosed herein that encode [0130] Mycobacterium tuberculosis peptides having an immunostimulatory activity, as well as the corresponding full-length Mycobacterium tuberculosis genes, enable one of ordinary skill in the art 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 Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al. (ed.), Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987. 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 [0131] 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 [0132] 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 Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. 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 Sambrook et al. (ed.), [0133] Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Vector systems suitable for the expression of lacZ fusion genes include the pUR series of vectors, Ruther et al., EMBO J. 2:1791, 1983; pEX1-3, Stanley and Luzio, EMBO J. 3:1429, 1984; and pMR100, Gray et al., Proc. Natl. Acad. Sci. USA 79:6598, 1982. Vectors suitable for the production of intact native proteins include pKC30, Shimatake and Rosenberg, Nature 292:128, 1981; pKK177-3, Amann and Brosius, Gene 40:183, 1985; and pET-3, Studiar and Moffatt, J. Mol. Biol. 189:113, 1986. 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. [0134]
  • 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 for stimulating an immune response. Such peptides may be administered as vaccines in ISCOMs (Immune Stimulatory Complexes) as described by Janeway & Travers, [0135] 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 can 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 the epitope, but which differs, by reason of the redundancy of the genetic code, from the sequences disclosed herein. [0136]
  • 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 are presented in Tables 7 and 8, respectively. 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. [0137]
    TABLE 7
    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
  • [0138]
    TABLE 8
    The Degeneracy of the Genetic Code
    Number of Total
    Synonymous 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 that 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 be readily determined by the assay technique described above. Such variant peptides include those with variations in amino acid sequence including minor deletions, additions, and substitutions. [0139]
  • 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. [0140]
  • 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 9 when it is desired to finely modulate the characteristics of the protein. Table 9 shows amino acids that may be substituted for an original amino acid in a protein and that 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. [0141]
    TABLE 9
    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 9, 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 that, in general, are expected to produce the greatest changes in protein properties are 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. [0142]
    • VIII. USE OF CLONED MYCOBACTERIUM SEQUENCES TO PRODUCE VACCINES
  • A. Overview [0143]
  • 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 (Bacillus Calmette-Guérin) vaccine, which is a live vaccine comprising attenuated [0144] 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, Wyngaarden et al. (eds.), Cecil Textbook of Medicine, 19th ed., W. B. Saunders, Philadelphia, Pa., pages 1733-1742, 1992, 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 is based upon the use of one or more of the immunostimulatory [0145] 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, the system 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 et al., [0146] Infect. Immun. 60:4781-4792, 1992 (describing immunization with extra-cellular proteins of Mycobacterium tuberculosis); Yang et al., Immunology 72:3-9, 1991 (vaccination with synthetic peptides corresponding to the amino acid sequence of a surface glycoprotein from Leishmania major); Andersen, Infection & Immunity 62:2536, 1994 (vaccination using short-term culture filtrate containing proteins secreted by Mycobacterium tuberculosis); and Jardim et al., J. Exp. Med. 172:645-648, 1990 (vaccination with synthetic T-cell epitopes derived from Leishmania parasite). Methods for preparing vaccines that 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 No. 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 [0147] 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, or glycerol. The vaccines may also include auxiliary substances such as emulsifying agents and pH buffers. [0148]
  • It will be appreciated by one of ordinary skill in the art that vaccines formulated as described above may be administered in a number of ways including subcutaneously, intramuscularly, and by 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. [0149]
  • 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 No. 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. [0150]
  • 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-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. [0151]
  • 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. [0152]
  • Alternatively, multiple immunostimulatory peptides may also be administered by expressing the nucleic acids encoding the peptides in a nonpathogenic microorganism, and using the transformed nonpathogenic microorganism as a vaccine. As described in International Patent Application No. WO 95/01441, [0153] 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, [0154] Immunobiology: The Immune System In Health and Disease, page 13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari, N. Engl. J. Med. 334:42-45, 1996. Thus, plasmids that include nucleic acid molecules described herein, or that include nucleic acid sequences encoding peptides according to the present invention, may be utilized in such DNA vaccination methods.
  • B. Pool of 12 [0155] M. tuberculosis Proteins Confers Immunity
  • A guinea pig protection study was undertaken to compare three candidate vaccine preparations with BCG. These included the Antigen 85 complex with IL-2 (Ag 85), a fusion-protein pool of twelve [0156] M. tuberculosis proteins in combination with IL-2 (FPP), and a control containing the adjuvant monophosphoryl lipid A (MPL).
  • 1. Materials and Methods [0157]
  • The following fusion proteins were identified and cloned into expression vectors as described supra and then were over-expressed in [0158] E. coli BL21 (DE3) plysS (Novagen, Madison, Wis.): MBP-264 (SEQ ID NO: 114), MBP-506 (SEQ ID NO: 115), MBP-825 (SEQ ID NO: 116), PET-23 (SEQ ID NO: 117), PET-47 (SEQ ID NO: 118), PET-639 (SEQ ID NO: 119), PET-916 (SEQ ID NO: 120), PET-1084 (SEQ ID NO: 121); in E. coli BL21: GST-152 (SEQ ID NO: 122), GST-511 (SEQ ID NO: 123), GST-822 (SEQ ID NO: 124); and in E. coli SURE (Stratagene, La Jolla, Calif.): GST-206 (SEQ ID NO: 125).
  • The Antigen 85 complex was kindly provided by Dr. John Belisle (Colorado State University, Fort Collins, Co.) through the TB research materials and vaccine testing contract (NIH, NIAID NOI AI-75320) (Belisle [0159] Science 276:1420-1422, 1997).
  • Animals. Outbred female Hartley guinea pigs, that were specifically pathogen-free (Charles River Laboratories, North Willmington, Mass.) were held under barrier conditions in an ANL-3 biohazard laboratory. Owing to expense, experimental groups were limited to between three and five animals. They were housed one to a cage and given free access to water and guinea pig chow. Following aerogenic infection with [0160] M. tuberculosis H37Rv, the guinea pigs were monitored over a period of 27 weeks. After the first four weeks, the animals were weighed weekly, with the exception of a two-week period, and any animals demonstrating sudden significant weight loss were euthanised.
  • Bacterial infection. Guinea pigs were aerogenically infected with between 20 and 50 bacilli of [0161] M. tuberculosis H37Rv using a calibrated aerosol generation device (Glas-Col, Terre Haute, Ind.) that delivered the inoculum to each lung.
  • Vaccinations. Guinea pigs were immunized subcutaneously two times at a three-week interval using 100 μg of AG85 complex with 20 μg Proleukin-PEG IL-2 (Chiron, Emeryville, Calif.) and emulsified in 100 μg Monophosphoryl Lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) adjuvant that had been solubilized in 0.02% triethanolamine and 0.4% dextrose by sonication (MPL-TeoA); and 100 μg of fusion proteins that had been pooled in equivalent concentrations with 20 μg PEG IL-2 (Chiron) in 100 μg MPL-TeoA adjuvant. The positive control, BCG Copenhagen, was injected once intradermally (10[0162] 3 bacilli/guinea pig), corresponding to the second set of injections.
  • Necropsy. Guinea pigs were euthanized by the intraperitoneal injection of 1-3 mL of sodium pentobarbital (Sleepaway, Ft. Dodge, Iowa). The abdominal and thoracic cavities were opened aseptically and the spleen and right lower lung lobe were homogenised separately in sterile Teflon-glass homogenisers in 4.5 mL of sterile physiological saline. The number of viable [0163] M. tuberculosis organisms was determined by inoculating appropriate dilutions onto Middlebrook™ 7H10 agar plates (Hardy Diagnostics, Santa Maria, Calif.). The colonies were counted after three weeks' incubation at 37° C. Data were expressed as mean log10 number of viable organisms per portion of tissue.
  • Histological analysis. Sagital tissue sections were made through the middle of the left lower lobe. The tissue sections were fixed in 10% neutral buffered formalin and stained with hematoxylin and eosin. Prepared tissues were coded prior to evaluation by a board-certified pathologist. [0164]
  • 2. Results [0165]
  • Long-term survival assay. The survival of test groups after aerosol infection and their respective weight gain or loss are summarized in Table 10. [0166]
    TABLE 10
    Total Weight Change, Survival Length and Bacterial Loads in The
    Lung and Spleen For Individual Guinea Pigs within Different
    Vaccination Groups, After Aerogenic Infection with M. tuberculosis.
    Wt. Bacterial Bacterial
    Survival Change Load Load
    Group No (wks) (g) Log10 Lung Log10 Spleen
    BCG 117 27   159 3.65 4.93
    121 27    99 4.13 3.95
    130 27   151 4.13 3.65
    mean  27.0 ± 0.0136   136 ± 3.97 ± 0.16 4.18 ± 0.39
       33
    Ag85 155 25  −25
    156 20  −48
    158 27    86 5.91 3.65
    162 27    75 5.19 4.19
    167 27    86 5.94 3.83
    mean 25.2 ± 1.4     35 ± 5.68 ± 0.24 3.89 ± 0.16
       66
    MPL 170 27    81 5.31 4.95
    171 12 −205 >7.0 >7.0
    172 9 −231 6.51 6.49
    mean 16.0 ± 5.6  −118 ± 6.27 ± 0.87 6.15 ± 1.07
      173
    FPP 157 15    17 6.58 5.23
    166 27    82 5.48 2.65
    169 27   133 5.26 0.0*
    mean 23.0 ± 4.0     77 ± 5.77 ± 0.41 2.63 ± 1.51
       58
  • All positive-control animals vaccinated with BCG exhibited consistent weight gain and were healthy when the experiment was curtailed after 27 weeks. [0167]
  • Three out of five guinea pigs immunized with Ag85 survived to 27 weeks, as did 2 out of 3 guinea pigs vaccinated with the fusion protein mixture. All of these surviving animals showed reasonable weight gain. In contrast, 2 of 3 negative controls exhibited precipitous weight loss and died within the first 17 weeks of the experiment. That animal experienced dramatic weight loss throughout the latter few weeks of the experiment. [0168]
  • Bacterial Loads. Table 9 shows the individual bacterial loads found in the lung and spleen. Subsequent assessment of bacterial loads indicated that only BCG dramatically reduced bacterial numbers in the lungs. Approximately one-half log reduction in counts were observed in mice administered Ag85 of the fusion protein mixture. [0169]
  • Dissemination of bacteria to the spleen was reduced in all groups relative to the negative control and the fusion protein pool effected the greatest control on dissemination. [0170]
  • Comparative Histology. Guinea pigs immunized with BCG exhibited a few discrete granulomas in the lungs with a diffuse interstitial mononuclear cell infiltrate affecting approximately 70% of the lung parenchyma, with no evidence of necrosis, caseation or mineralization. In contrast, guinea pigs in the negative control group had a moderate to severe, multi-focal granulomatous pneumonia with extensive caseation and necrosis throughout the lung parenchyma. [0171]
  • A mixed response was seen in guinea pigs administered Ag85. In two animals that died before 27 weeks (at 20 and 25 weeks, respectively) a moderate, multi-focal, necrosuppurative granulomatous pneumonia was seen, with scattered aggregates of lymphocytes and areas of mineralization and fibrosis. In the three surviving animals the pathology was less severe, with increased numbers of aggregations of lymphocytes being evident and the granulomatous pneumonia scored as mild to moderate. A similar histological appearance was seen in the lungs of two guinea pigs immunized with fusion proteins that were still alive at 27 weeks. [0172]
    • IX. USE OF CLONED MYCOBACTERIUM SEQUENCES IN DIAGNOSTIC ASSAYS
  • Another aspect of the present invention is a composition for diagnosing tuberculosis infection. The composition includes peptides encoded by one or more of 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. [0173]
  • A. In Vitro ELISA Assay [0174]
  • 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 ELISA. [0175]
  • 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 (BSA). Excess BSA 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 anti-mycobacterial antibodies (from the specimen), the enzyme-conjugated anti-human antibody, and the color substrate produces 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. [0176]
  • B. Example of ELISA Using Eight Full-Length Clones [0177]
  • The following fusion proteins were over-expressed in [0178] E. coli BL21 plysS: MBP-506 (SEQ ID NO: 115), MPB-825 (SEQ ID NO: 116), PET-639 (SEQ ID NO: 119), PET-916 (SEQ ID NO: 120), PET-1084 (SEQ ID NO: 121); in E. coli BL21: GST-152 (SEQ ID NO: 122), GST-822 (SEQ ID NO: 124); and in E. coli SURE: GST-206 (SEQ ID NO: 125). The recombinant fusion proteins MBP-506 (SEQ ID NO: 115), MBP-825 (SEQ ID NO: 116), GST-152 (SEQ ID NO: 122), GST-822 (SEQ ID NO: 124), PET-639 (SEQ ID NO: 119), PET-1084 (SEQ ID NO: 121) formed inclusion bodies that were harvested from the pellet following centrifugation of the bacterial sonicate. The fusion proteins PET-916 and GST-206 were found primarily in the supernatant and underwent considerable breakdown in culture. Protein fractions were checked by SDS-PAGE using Coomassie Blue staining and approximate concentrations determined by Western blotting.
  • ELISA sera were obtained from 38 Brazilian individuals with pulmonary tuberculosis and that were HIV positive (TBH), from 20 individuals with extrapulmonary tuberculosis and that were HIV negative (EP-TB), and from 17 healthy volunteers. Wells were coated with 200 ng of antigen in 50 μL of coating buffer (15 mM Na[0179] 2CO3, 35 mM NaHCO3 adjusted to pH 9.6) and incubated for 1 hour. Plates were then aspirated, 250 μL of blocking buffer (0.5% BSA and 0.1% Thimerosal (Aldrich, Milwaukee, Wis.) in phosphate-buffered saline at pH 7.4) were added to each well, and the plates were incubated for a further 2 hours. Plates were washed six times with 350 μL/well of washing solution (2 mL/L Tween 20 in PBS at pH 7.4) and serum was added at a 1:100 dilution in serum diluting buffer (blocking buffer with 2 mL/L Tween 20). Plates were incubated for 30 minutes and washed as before. 50 μL of a 1:50,000 dilution of HRP-Protein A (ZYMED, VWR) were added to each well. The plates were then incubated for 30 minutes and washed as before. 100 μL/well of TMB Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories) was added and the plates were incubated for 15 minutes in the dark. The reaction was stopped with 100 μL of 0.5 M H2SO4 and the plates were read immediately at 450 nm. The mean and standard deviations (SD) were calculated from the sera of uninfected control subjects (n=17) and the cut-off for positive results was calculated as greater than the mean plus 3 SD, and for high level responses, as the mean plus 6 SD.
  • Table 11 shows the overall seropositivity results for the nine full-length fusion proteins. When individual sera were considered, in the EP-TB group, 71% of sera contained antibodies against at least one antigen (or 82% if the TB lysate individuals are included) and in the TBH group, 66% of sera contained antibodies against at least one antigen (or 84% if the TB lysate individuals are included). Thus, specific antibody responses can be identified in the majority of the individual sera. [0180]
  • Measurement of the serum antibodies provides a way to determine the antigen reactivity. For the two groups, the number of serum samples that reacted positively to each antigen, and those which reacted at a high level, are presented in Table 11. For patients with EP-TB, antibodies against GST-822 were found in 60% of individual sera and a third of these were high-level responses. Specific antibody responses to three other antigens (PET-639, MBP-825, and MBP-506) of between 35% and 45% were also found in the EP-TB group. The other five antigens, as well as the [0181] M. tuberculosis lysate, elicited responses in fewer sera from EP-TB patients (35% or less).
  • For patients with TBH, antibodies against MBP-506 were found in 61% of individual sera and over two thirds of these were high-level responses. GST-822 was recognized in 42% of sera and almost two-thirds of the specific antibody responses were at a high level. The other six antigens, as well as the [0182] M. tuberculosis lysate, elicited responses in fewer sera from EP-TB patients (35% or less).
  • This study demonstrated that most of the patients infected with [0183] M. tuberculosis produced serum antibodies to a variety of antigens. As has been seen for individuals with pulmonary TB, Lyashchenko et al., Infect. Immun. 66:3936-3940, 1998, sera responses confirm that antigen recognition and strength of response were heterogeneous in both EP-TB and TBH groups. Encouragingly, the majority of sera contained specific antibodies to the small set of antigens tested. This finding suggests that, for these two groups previously considered refractory to serodiagnosis, the combination of only a few well-recognized antigens might greatly improve diagnostic success. MBP-506 and GST-822, the two highly reactive and most frequently recognized antigens identified in this study, are potentially valuable candidates for inclusion in a serodiagnostic test.
    TABLE 11
    Antigen Recognition by Serum Antibodies in TB Patients
    Number (%) of responders
    EP-TB HIV +, TB+
    Antigen Totala High levelb Totala High levelb
    TB lysate  5 (25%) 4 20%) 13 34%)  6 (30%)
    PET-1084  0 (0%) 0 (0%)  4 11%)  1 (3%)
    PET-47  5 (25%) 1 (5%)  8 21%)  3 (8%)
    PET-916  3 (15%) 3 15%) 11 29%)  4 (11%)
    PET-639  8 (40%) 2 10%)  5 13%)  4 (11%)
    MBP-825  7 (35%) 1 (5%) 10 26%)  5 (13%)
    MBP-506  9 (45%) 3 15%) 23 61%) 16 (42%)
    GST-822 12 (60%) 4 20%) 16 42%) 10 (26%)
    GST-206  3 (15%) 2 10%)  5 13%)  3 (8%)
    GST-152  2 (10%) 1 (5%)  2 (5%)  0 (0%)
  • C. Skin Test Assay [0184]
  • 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 in one of the following conditions: the current presence of [0185] M. tuberculosis in the patient's body after 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 [0186] M. tuberculosis that are injected intradermally. The test is described in detail in Wyngaarden et al. (eds.), Cecil Textbook of Medicine, W. B. Saunders, Philadelphia, Pa., 1992. If the subject has reactive T-cells to the injected protein, then 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 circulating M. tuberculosis antibodies in the patient. Purified immunostimulatory peptides according to the present invention may be employed in the tuberculin skin test using the methods described in Wyngaarden et al. (eds.), Cecil Textbook of Medicine, W. B. Saunders, Philadelphia, Pa., 1992.
  • D. Nucleic Acid Amplification [0187]
  • 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 [0188] M. tuberculosis nucleic acids in body samples and thus to diagnose infection. Methods for making primers and probes based on these sequences are described in section V, above.
  • The detection of specific nucleic acid sequences of a pathogen in human body samples by polymerase chain reaction (PCR) amplification is discussed in detail in Innis et al., [0189] PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990, 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 can be detected by dot blot analysis (see chapter 48 of Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990). PCR amplification employing primers based on the sequences disclosed herein may also be employed to quantify the amount of M. tuberculosis nucleic acid present in a particular sample (see chapters 8 and 9 of Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990). 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 labeled with suitable labels (such as [0190] 32P or biotin-avidin enzyme linked systems) 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 [0191] M. tuberculosis peptides for diagnostic purposes. Substantially pure M. tuberculosis peptide suitable for use as an immunogen is isolated from 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 described below.
  • A. Monoclonal Antibody Production by Hybridoma Fusion [0192]
  • Monoclonal antibody to epitopes of the [0193] M. tuberculosis peptides identified and isolated as described herein can be prepared from murine hybridomas according to the classical method of Kohler and Milstein, Nature, 256:495, 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 with mouse myeloma cells by means of polyethylene glycol, and the excess unfused cells are 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, Enzymol,. 70:419, 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, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988.
  • B. Antibodies Raised Against Synthetic Peptides [0194]
  • An alternative approach to raising antibodies against the [0195] 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 [0196] 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 [0197] M. tuberculosis peptide is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique, Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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 that 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 [0198] 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. [0199]
  • Having illustrated and described the principles of the invention in multiple embodiments and examples, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. Therefore, the invention encompasses all modifications coming within the spirit and scope of the following claims. [0200]
  • 1 169 1 267 DNA Mycobacterium tuberculosis 1 acgcggacct cgaagttcat catcgagtga tacgtgccac acatctcggc gcagtggccc 60 acgaatgctc cggtcttggt gatttcttcg atctggaaga cgttgaccga gttgtttgcc 120 accgggttag gcatcacgtc acgcttgaac aagaactccg gcacccagaa tgcgtgtatc 180 acatcggctg aggccatttg gaattcgata cgcttgccgg acggcagcac cagcaccgga 240 atttcggtgc tggtgcccaa cgtctcg 267 2 487 DNA Mycobacterium tuberculosis misc_feature (1)..(487) n is a, c, g, or t/u. 2 ctgatacgac gccggcaagg actacgacga ggtggcacag aattcaatgc ggcgctcatc 60 ggaaccgacg tgcccgacgt cgttttgctc gacgacngat ggtggttcca tttcgccntc 120 agcggtgttc tgactgccct tgacgacctg ttcggccaag ttggggtgga cacaacggat 180 tacgtcgatt cgctgctggc cgactatgag ttcaacggcc gccattacgc tgtgccgtat 240 gctcgctcga cgccgctgtt ctactacaac aaggcggcgt ggcaacaggc cggcctaccc 300 gaccgcggac cgcaatcctg gtcagagttc gacgagtggg gtccggagtt acagcgcgtg 360 gtcggcgccg gtcgatcggc gcacggctgc gntaacgccg acctcatctc gtggacgttt 420 cagggaccga actgggcatt cggcggtgcc tactccgaca agtggacatt gacattgacc 480 gagcccg 487 3 511 DNA Mycobacterium tuberculosis misc_feature (1)..(511) n is a, c, g, or t/u. 3 ggcggccaga cngtcnggaa ctcgcnggcc attggtgtgg tgggaaccgc gatcctcgac 60 gcaccgcttc gcggtcttgc agtgttcgat gccaatctgc cggccgggac gctgccggat 120 ggcggcccgt tcaccgaggc tggtgacaag acctggcgtg tcgttccggg cactactccc 180 caggtcggtc aaggcaccgt caaagtgttc aggtataccg tcgagatcga gaacggtctt 240 gatcccacaa tgtacggcgg tgacaacgca ttcgcccaga tggtcgacca gacgttgacc 300 aatcccaagg gctggaccca caatccgcaa ttcgcgttcg tgcggatcga cagcggaaaa 360 cccgacttcc ggatttcgct ggtgtcgccg acgacagtgc gcggggggtg tggctacgaa 420 ttccggctcg agacgtcctg ctacaacccg tcgttcggcg gcatggatcg ccaatcgcgg 480 gtgttcatca acgaggcgcg ctgggtacgc g 511 4 512 DNA Mycobacterium tuberculosis misc_feature (1)..(512) n is a, c, g, or t/u. 4 gtgtgcaacc agtgtgtgtn cgtgtgcgaa ccagtgtgta gtggtaacca ggaccacgtt 60 gcaaaccagt gttggagtgc agtgttgcgt gcnagtgttg cncgttgcag tgttngncga 120 gccgagattg gaagttnccg acattaccgt tgccgacgtt gccctcgccg acgttcgcca 180 agcccaggtt gcggacacgc cggtgattgt gcgtggggca atgancgggc tgctggcccg 240 gccgaattcc aaggcgtcga tcggcacggt gttccaggac cgggccgctc gctacggtga 300 ccgagtcttc ctgaaattcg gcgatcagca gctgacctac cgcgacgcta acgccaccgc 360 caaccggtac gccgcggtgt tggccgcccg cggcgtcggc cccggcgacg tcgttggcat 420 catgttgcgt aactcaccca gcacagtctt ggcgatgctg gccacggtca agtgcggcgc 480 tatcgccggc atgctcaact accaccagcg cg 512 5 456 DNA Mycobacterium tuberculosis misc_feature (1)..(456) n is a, c, g, or t/u. 5 gcaacggaga ggtggactat gccggaccgg caccgcgaag gggttggtgc cggcccgggt 60 ggtgacggtg cacattctgc gcaattcgct gagttccggt ggtgaccttc ctgggcgcgg 120 agtctgggcg cgctgatggc ggagcgaktg tgaccgaagg aantcngttc aacatccacg 180 gcgtcggggg cgtgctgtat caagcggtca ccgtcaggag acgccgacgg tggtgtcgat 240 cgtgacggtg ctggtgctga tctacctgat caccaatctg ttggtggatc tgctgtatgc 300 ggccctggac gccgnngatn cgctatggct gagcacacgg ggttctggct cgatgcctng 360 cgcgggttgc gccggcgtcc taaantcgtg atcgcgcggc gctgakcctg ctgattcttg 420 tcgtggcggc gtttccgtcg ttgtttaccg cagccg 456 6 172 DNA Mycobacterium tuberculosis misc_feature (1)..(172) n is a, c, g, or t/u. 6 tcncttatcg cttcagctgg catctgccca aggaccgaat ctggacctat gacggccagc 60 tgaagatggc ccgcgacgaa gggcgttggc acgttcgctg gaccaccagc gggttgcatc 120 ccaagctagg cgaacatcaa aggttcgcgc tacgagccga cccgccgcgg cg 172 7 232 DNA Mycobacterium tuberculosis misc_feature (1)..(232) n is a, c, g, or t/u. 7 cttctcgcgc cagcncgtcc cgctgtccgg gatgncgcta ccggtcgtca gcgccaagac 60 ggtgcagctc aacgacggcg ggttggtgcg cacggtgcac ttgccggccc ccaatgtcgc 120 ggggctgctg agtgcggccn gcgtgccgct gttgcaaagc gaccacgtgg tgcccgccgc 180 gacggccccg atcgtcgaag gcatgcagat ccaggtgacc cgcaaatcgg at 232 8 173 DNA Mycobacterium tuberculosis misc_feature (1)..(173) n is a, c, g, or t/u. 8 gttcgncgcg ctcaaaaggt tgacgatggt cacgtcgcac gtgctggccg agaccaaggt 60 ggatttcggt gaagacctca aaganctcta ctcgnatcgt caaggccctc aacgacgacc 120 gaaaggattt cgtcacctcg ctgcagctgt tgctgacgtt cccatttccc aac 173 9 223 DNA Mycobacterium tuberculosis misc_feature (1)..(223) n is a, c, g, or t/u. 9 cctgttncaa cggtncnttc ncggaacgga cgacttctga tncgnnctcg gncgttccct 60 cgcaccggtc gatggtgatc aaggtcagcg tcttcgcggt ggtcatgctg ctggtggccg 120 ccggtctggt ggtggtattc ggggacttcc ggtttggtcc cacaaccgtc taccacgcca 180 ccttcaccga cncgtngcgg ctgaangcag gccagaaggt tcg 223 10 120 DNA Mycobacterium tuberculosis 10 caacgagatc gcacccgtga ttaggaggtg acggtggcag cgccgacccc gtcgaatcgg 60 atcgaagtaa cgctccgtag acgccagctc gtccgcgccg atgccgacct gccacccgtg 120 11 162 DNA Mycobacterium tuberculosis misc_feature (1)..(162) n is a, c, g, or t/u. 11 cggcttccag cgggtgcgcc aagcacggcc ggtccgtgcg agatcgtccc caatggcacg 60 ccggcgccca agacaccccc ggntaccgtg ccttcgtcgc gcaacctcgc gaccaacccc 120 gagatcgcca ccnnctacng ccgggacatg accgtggtgc gg 162 12 133 DNA Mycobacterium tuberculosis misc_feature (1)..(133) n is a, c, g, or t; y is t/u or c; h is a, c or t/u. 12 gactggnccc gaygytgtgn ccgghncgth ggncghgchg cantcgaycc tggccgttgc 60 ttcggtgccg ggttgttcat cgccttcgac cagttgtggc gctggaacag catagtggcg 120 ctagtgctat cgg 133 13 395 DNA Mycobacterium tuberculosis 13 gcgcacactg cgcatgctgc cgtacccgcg ccaggcatga gtcttaggcc gaaatgcctg 60 gttaactggc gtgtcgtggt tgacccgcgg gcgtgcggct acagtgcatg ctgtgatcgg 120 cagtgggaga ggtagcggtg cggcgtaagg tgcggaggtt gactctggcg gtgtcggcgt 180 tggtggcttt gttcccggcg gtcgcggggt gctccgattc cggcgacaac aaaccgggag 240 cgacgatccc gtcgacaccg gcaaacgctg agggccggca cggacccttc ttcccgcaat 300 gtggcggcgt cagcgatcag acggtgaccg agctgacaag ggtgaccggg ctggtcaaca 360 ccgccaagaa gtcggtgggc tgccaatggc tggcg 395 14 175 DNA Mycobacterium tuberculosis misc_feature (1)..(175) n is a, c, g, or t/u; y is t/u or c; k is g or t/u. 14 ccagnccncc naacntgtyn cgntctcayy tcgccgtcgc tgccggtncg tgtgtgcacc 60 atctgcaccg acccgtgkaa cytcgatcac ganactggna gagntcaggc atnaaagccg 120 gagtggcaca gcaacggtcg ctactggaat tggcgaagct ggatgctgag ctgac 175 15 265 DNA Mycobacterium tuberculosis misc_feature (1)..(265) n is a, c, g, or t/u. 15 gggctggatt cgaggctcng tgcatgccgt acgactaggg gtagcgccca gctgctcaat 60 accatcggtt ggataacaaa ggctgaacat gaatggcttg atctcacaag cgtgcggctc 120 ccaccgaccc cggcgcccct cgagcctggg ggctgtcgcg atcctgatcg cggcgacact 180 tttcgcgact gtcgttgcgg ggtgcgggaa aaaaccgacc acggcgagct ccccgagtcc 240 cgggtcgccg tcgccggaag cccac 265 16 170 DNA Mycobacterium tuberculosis misc_feature (1)..(170) n is a, c, g, or t/u; m is a or c. 16 cgccatgncg aagcgmaccc cggtccggaa ggcctgcaca gttctagccg tgctcgccgc 60 gacgctactc ctcgcctgcg gcggtcccac gcagccacgc agcatcacct tgacctttat 120 ncgcaacgcg caatcccagg ccaacgccga cgggatcatc gacaccgaca 170 17 181 DNA Mycobacterium tuberculosis misc_feature (1)..(181) n is a, c, g, or t/u; b is g, c or t/u. 17 accngttccc gccggnctna cncncggtgc cgttgcaccg gccanctgca gcctgccccg 60 acgccgaagt ggtgttcgcn ccgcggccgc ttcgaaccgc ccgggattgg cacggtcggc 120 aabgcattcg tcagcnntgc gctcgaaggt caacaagaat gtcggggtct acgcggtgaa 180 a 181 18 95 DNA Mycobacterium tuberculosis misc_feature (1)..(95) w is a or t/u; k is g or t/u; y is t/u or c. 18 aggtkacggt ggcagcgccg accccgtcga atcggwtcga agaaygctcc gkacacgcca 60 gctgcgtccg ygccgatgcc gacctgccac ccgtg 95 19 283 DNA Mycobacterium tuberculosis misc_feature (1)..(283) n is a, c, g, or t/u; r is g or a. 19 gcgcatgcgc aaccacttgg aatccgttga caatcgcatc ggtggccggc ccgtcggtga 60 ccgcntgcaa cagcgcggtg gtcaccnaca gcgaaaccag gtncttgtcg gctccggagg 120 tggcgatgac gtggcgccgg gaggtgttga gggtcatgtc gttttcgcgn taggtgccct 180 cgatgattga tgacggaaag cnncgtngaa anttggcnat agcggcgttt gtggtctgcn 240 atncgagcra ttnctgnctg tcagtgtagn cgtgtgtgat ggc 283 20 156 DNA Mycobacterium tuberculosis misc_feature (1)..(156) n is a, c, g, or t/u; w is a or t/u; b is g, c or t/u; r is g or a. 20 tcttctacaa ggacgccttc gccaagcacc aggagctgtt cgacgacttg gncgtcaacg 60 tcaacaatgg cttgtccgat ctgtacragc aagwtcgagt cgctgccgnb cgcaacgcga 120 cgagatcatc gaggacctac accgttgcca cgaaca 156 21 123 DNA Mycobacterium tuberculosis misc_feature (1)..(123) n is a, c, g, or t/u. 21 atnccgttcc actnccgcgg cagcagctgg ntttgcgcac acggtgaccc agtggcgntt 60 ggtggggcct cgctgacggc gagtntggnc gagcgtcctc ggtcggtgnc ctntcntccc 120 gcc 123 22 823 DNA Mycobacterium tuberculosis misc_feature (1)..(823) n is a, c, g, or t/u; k is g or t/u. 22 cggtcacgca attgatggcc gcgcgcaagg scgcatggtg gagatgncca accacaccac 60 cggctgggtc cgcatggact tcgtggttcc cagtcgcggc ctgattgggt ggcgcaccga 120 cttcctcacc gagacccgtg gctccggtgt cgggcatgcg gtgttcgacg gatnaccggc 180 catgggcggg ggagkccggg cccgnccaca ccggttctct ggtatcggac cgggccggcg 240 ccatcacacc gttcgcgttg ctgcaactcg ccgatcgggg gcagttcttc gtcgagcccg 300 gccaacagan ccntacgagg ncantggctg ctgggatcaa cccccgtccg gaggacctcg 360 acatcaatgt cacccggagn agnangctga ccnaacatgc gctcatcgac cgcggatgtc 420 atcgagacgg tngccaagcc gctgcagctg gatctcgagc gcgccatgga gttatgtgcg 480 cccgacgaat gcgtcgaggt gaccccggag atcgtgcgga tccgcaaagt cgagctggcc 540 gccgccgccc gggctcgcag ccgggcgcgc accaaggcgc gtggctagca acttggcgcg 600 ctggccgcgc gagcgtaacg ccactgcgaa atccagcccg gcttttcgca gccgggttac 660 gctcgtgggg gtactggata gcctgatggg cgtgcccagc ccagtccgcc gcgtctgtgt 720 gacggtcggc gcgttggtcg cgctggcgtg tatggtgttg gccgggtgca cggtcagccc 780 gccgccggca ccccagagca ctgatacgcc gcgcagcaca ccg 823 23 103 DNA Mycobacterium tuberculosis 23 cttccggcgg gacaacaaca ggtctcaccg gcgccacacc ctgacacctg atcgcgtctg 60 ccgatcccgg tcggagcacc cgggttccac cgctgtgccc ccc 103 24 207 DNA Mycobacterium tuberculosis misc_feature (1)..(207) n is a, c, g, or t/u; w is a or t/u; y is c or t/u; h is a, c or t/u. 24 gccaccggtt catcgcgtgg tgctggtcac cgccnggaan gcctcagcgg atcccctgct 60 gccaccgccg cctatccctg ccccagtctc ggcgccggca acagtcccgy ccgtgcagaa 120 cctcacggct ncthccgggc gggagcagca acaggttctc accggygccw ngyacccgca 180 ccgatcgcgt cgccgattcc ggtcgga 207 25 204 DNA Mycobacterium tuberculosis misc_feature (1)..(204) n is a, c, g, or t/u; y is c or t/u; s is g or c; k is g or t/u. 25 ttncgcannc gttcatccag gtccactggt gtcgcanctc tcnntgatgc accggttccg 60 gatatatgtc nacatcnccs tcstcgtcct ggtgctggta ctnacgaacc tgatcgcgca 120 tttcaccaca ccgtgngcga gcatcgccac cgtcccggcc gccygcggtc ggactggtga 180 tcttggtkcg gagtagaggc ctgg 204 26 207 DNA Mycobacterium tuberculosis misc_feature (1)..(207) n is a, c, g, or t/u; h is a, c or t/u; r is g or a. 26 ataccngtca tccngcacat ngtcaacctn gagtcggtnc tcacctacga ggcacgcccg 60 agatgcatca ctggtgctcg rtcagncctt cacggcttgg ccgccttccg gtaggaccgt 120 hgcatgcccg tcttcggcgc ctcgggtgtt cggtcctggc tctcgggctg ctggccnctg 180 cgccccaccc cgcaccgggc cggcttc 207 27 289 DNA Mycobacterium tuberculosis misc_feature (1)..(289) n is a, c, g, or t/u; s is g or c. 27 ccgtgatngg ccggnncgnc atgttacggg nagccgggna ttgcgntacg ccacggtgat 60 cgcgctggtg gccgcgctgg tggncngcgt gtacgtgctc tcgtccaccg gtaataagcg 120 caccatcgtg ggctacttca cctctgctgt cgggctctat cccggtgacc aggtccgcgt 180 cctgggcgtc ccggtgggtg agatcgacat gatcgagccg cggtcgtccg acgtcaagat 240 cactatgtcg gtgtccaagg acgtcaaggt gcccgtgsac gtgcaggcc 289 28 198 DNA Mycobacterium tuberculosis misc_feature (1)..(198) n is a, c, g, or t/u; y is c or t/u. 28 ttgnaccang cctatcgcaa gccaatcacc tatgacacgc tgtggcaggc tgacaccgat 60 ccgctgccag tcgtcttccc cattgtgcaa ggtgaactga gcaangcaga ccggacaaca 120 ggtatcgata gcgccgaatg ccggcttgga cccggtgaat tatcagaact tygcagtcac 180 gaacgacggg gtgatttt 198 29 149 DNA Mycobacterium tuberculosis misc_feature (1)..(149) n is a, c, g, or t/u; w is a or t/u; m is c or a; r is g or a. 29 tcacganggt rynacmgcaa cwcgaccgcc acgtcasgcc gccgcgcacg aagatcaccg 60 tgcctgcncg atgggtcgtg aacggaatag aaygcagcgg tgaggtcaan ygcgaagccg 120 ggaaccaaat ccggtgaccg cgtcggcat 149 30 210 DNA Mycobacterium tuberculosis 30 ggacccgcca agcatcagcc ggtcaacagc cgccgccggt ggccaaagtt cgagcagccg 60 ccggtatcgt gctcggcccg gctagaccaa aaactttacg ccagcgcccg aagccacccg 120 actccaaggc ctcggcccgg ttgggttcgc acatgggtga gttctatatg ccctacccgg 180 gcacccggtt caaccaggaa accgtctcgc 210 31 255 DNA Mycobacterium tuberculosis misc_feature (1)..(255) n is a, c, g, or t/u. 31 cagnccgctg ncccggaact gttccagcag ctacaagacc ttcgacaacg tngcgcgtca 60 acctgcantc gagcgcaacc tctcggtggc gctcaacgag tgttcgccgg cttcaacccg 120 ctggacccgc gaaacctcga cgtgtccccg ctgccttcgc tggccaagcg cgccgccgac 180 atcctgcgcc aggacgtggg cgggcaggtc gacattttcg atgtcaatgt gcccaccatc 240 cagtacgacc agagc 255 32 164 DNA Mycobacterium tuberculosis misc_feature (1)..(164) n is a, c, g, or t/u; m is a or c; r is g or a, y is t/u or c. 32 aaynccnggc crtcgacggt nccggttcnc rccaccggtc tatatccacc cgggtcnrca 60 ttmanantga ntmnccgccg gtgcggccgt cgagcgtgac ctggcatccc ctgagacgct 120 gctgggttgc cccggggagn tcgamantcg ggcatcgcac catc 164 33 237 DNA Mycobacterium tuberculosis misc_feature (1)..(237) n is a, c, g, or t/u; s is g or c. 33 acggacggca acgggatgcg acccgatccc accggtcgcc acgagggacg ctacttcgtc 60 gccgggcagc cganccgacc gtcngttcng cganggcgac ngccgaagcc gttgacccac 120 nttggtcagc agcagctgga tsagtcaggt gccgttggtg tttcgccgtc agcggtgtcg 180 gggtgggtgc gttctgggca ccgtcgactg tggtgggcgc tngcgggcgn tggtggc 237 34 371 DNA Mycobacterium tuberculosis misc_feature (1)..(371) n is a, c, g, or t/u. 34 cggatgctcg gcctccggta cccaactcga actcgcgccc acngcggacn gcagggccgc 60 ggttggcacc accagcgaca tcaatcangc aggatcccgc cacgttgcaa gacggcggca 120 atcttcgcct gtcgctcacc gactttccgc ccaacttcaa catcttgcac atcgacggca 180 acaacgccga ggtcgcggcg atgatgaaag ccaccttgcc gcgcgcgttc atcatcggac 240 cggacggctc gacgacggtc gacaccaact acttcaccag catcgagctg accaggaccg 300 ccccgcaggt ggtcacctac accatcaatc ccgaggcggt gtggtccgac gggaccccga 360 tcacctggcc g 371 35 26 DNA Mycobacterium tuberculosis misc_feature (1)..(26) n is a, c, g, or t/u. 35 gagaactccg ggccganttt tggaca 26 36 202 DNA Mycobacterium tuberculosis 36 tgtcggtagc gttcgcgtcc atgattgctc ttgcaacgct gttgacgctt atcaatcaag 60 tcgtcggcac tccgtatatt cccggtggcg attctcccgc cgggaccgac tgctcggagc 120 tggcttcgtg ggtatcgaat gcggcgacgg ccaggccggt tttcggagat aggttcaaca 180 ccggcaacga ggaagcgcct tg 202 37 319 DNA Mycobacterium tuberculosis misc_feature (1)..(319) n is a, c, g, or t/u; y is t/u or c. 37 ctanttttag aytnngtcgt gacatatccg ctgtacgcgt gggacggncc attattggat 60 aatgcgtgat aagcaccaca agaantgatt ncctatggat attgtcggta ncgttcgcgt 120 ccatgattgc tcttgcaacg ctgttgacgc ttatcaatca agtcgtcggc actccgtata 180 ttcccggtgg cgattctccc gccgggaccg actgctcgga gctggcttcg tgggtatcga 240 atgcggcgac ggccaggccg gttttcggag ataggttcaa caccggcaac gaggaagcng 300 ccttggcggc tcggggctt 319 38 263 DNA Mycobacterium tuberculosis 38 ggtacttgtc gtcgatggac tcccggtctc gattcaggaa cagcgtcccg acgacaccgg 60 ctcccaccag cccgagaaac gccaccacgc cgcgagcgcc caccacagtc gacggtgcca 120 gaacgcacca cccgacacgt gacggcgaaa caccaacggc acctgactga tgccagctgc 180 tgctgaccaa gtgggcacgc tcggcgcgcc tcggaacgag tcgtcgctgc cgcgacgaag 240 acgcctcggc gacgtggatc ggg 263 39 841 DNA Mycobacterium tuberculosis misc_feature (1)..(841) n is a, c, g, or t/u. 39 gcgttgcgcg ccctcgagca gtcnnttggc ggcgatcccg agacaatgat tcccgacatc 60 cggtacacac cgaaccccaa cgatgcgccg ggcggcccgc tggtagaaag gggaaatcgc 120 cagtgctgac tcgcttcatc cgacgccagt tgatcctttt tgcgatcgtc tccgtagtgg 180 caatcgtcgt attgggctgg tactacctgc gaattccgag tctggtgggt atcgggcagt 240 acaccttgaa ggccgacttg cccgcatcgg gtggcctgta tccgacggcc aatgtgacct 300 accgcggtat caccattggc aaggttactg ccgtcgagcc caccgaccag ggcgcacgag 360 tgacgatgag catcgccagc aactacaaaa tccccgtcga tgcctcggcg aacgtgcatt 420 cggtgtcagc ggtgggcgag cagtacatcg acctggtgtc caccggtgct ccgggtaaat 480 acttctcctc cggacagacc atcaccaagg gcaccgttcc cagtgagatc gggccggcgc 540 tggacaattc caatcgcggg ttggccgcat tgcccacgga gaagatcggc ttgctgctcg 600 acgagaccgc gcaagcggtg ggtgggctgg gacccgcgtt gcaacggttg gtcgattcca 660 ctcaagcgat cgtcggtgac ttcaaaacca acattggcga cgtcaacgac atcatcgaga 720 actccgggcc gattttggac agccaggtca acacgggtga tcagatcgac ngctgggcgc 780 gcaaattgaa caatctggcc gcacagaccg cgaccaggga tcagaacgtg cgaagcatcc 840 t 841 40 209 DNA Mycobacterium tuberculosis misc_feature (1)..(209) n is a, c, g, or t/u; b is g, c or t/u; d is g, t/u or a. 40 gcggttggca ccaccagcga naatcagcag gndcccgcca cgttgcaaga cggcggcaat 60 cttcgcctgt cgctcaccga ctttccgccc aacttcaaca tcttgcacat cgacggcaab 120 aabgccgagg tcgcggcgat gatgaaagcc accttgccgc gcgcgttcat catcggaccg 180 gacggctcga cgacggtcga caccaacta 209 41 167 DNA Mycobacterium tuberculosis misc_feature (1)..(167) n is a, c, g, or t/u. 41 agatcgtcag tgagcagaac cccgccaaac cggccgcccg aggtgttgtt cgagggctga 60 aggcgctgct cgcgacggtc gntgctggcc gtcgtcggga tcgggcttng gctcgcgctg 120 tacttcacgc cggcgatgtc ggcccgcgag atcgtgnatc atcggga 167 42 221 DNA Mycobacterium tuberculosis misc_feature (1)..(221) n is a, c, g, or t/u; k is g or t/u. 42 ccagntcctc nnatatcgac accctcnacn aagaccgctt cgcgagatca acnctcagat 60 atncnnacta tcnccnntnc acgcacacct caacatnana naatngaact atngncttcg 120 cctcaccacc aaggttcagg ttancggctg ncgtttkctc tkcgccggct cgaacacgcc 180 atcgtgcgcc ggkacacccg gatgtttgac gacccgctgc a 221 43 117 DNA Mycobacterium tuberculosis misc_feature (1)..(117) n is a, c, g, or t/u; y is t/u or c; h is a, c or t/u. 43 cggyccgnnc aayyygncgc gchncggygy agaggtcgny aaggtcgcca aggtaacgct 60 gatcgayggg nacangcaag tattggtgna cttcaccgtg ghthgcthgc tgtyagc 117 44 385 DNA Mycobacterium tuberculosis 44 gaacctcctc gcccgcgctt ggcctagcat taatcgactg gcacgacagt tgcccgactg 60 ggtacacggc atggacgcaa cgcgaatgaa tgtgagttag ctcactcatt aggcacccca 120 ggcgttgaca ctttatgctt ccggctcgtg tagttgtgtg ggaattgtgg agcggataac 180 aatttcgacg acgaggaaac agctgtagac atggattgac gaatttgaat acgactcact 240 ataggaattc gagctcggta cccggggatc ctctagagtc cttcgccgcg ggtcgccacc 300 atcagggcca gtgcgatcgc aagcgcgggg taccgggcgc catagtcttc agcatcggcg 360 tgttgaccgc agagaccgga cgggg 385 45 285 DNA Mycobacterium tuberculosis misc_feature (1)..(285) n is a, c, g, or t/u. 45 cccgcagcag tacccgcagn cccacacccg ctatncgcag cccgaacagt tcggtgcaca 60 gcccacccna gctcggcgtg cccggtcagt acggccaata ccagcagccg ggccaatatg 120 nccagccggn acagtnacgn ccagcccggc cagtacgcna ccgcccggtc agtaccccgg 180 gcaatacggc ccgtatgncc agtcgggtca ggggtcgaag cgttcggttg cggtgatcgg 240 cggcgtgatc gccgtgatgg ccgtgctgtt catcggcgcg gttct 285 46 186 DNA Mycobacterium tuberculosis misc_feature (1)..(186) n is a, c, g, or t/u. 46 gcncgtgncc gtgccgcccg gttgaacgtg agcngctgnc natngcccca gccgagacga 60 gaacgtcccc gaggagtatg cagactggga agacgccgaa gactatgacg actatgacga 120 ctatgaggcc gcagaccagg aggccgcacg gtcggcatcc tggcgacggc ggttgcgggt 180 ncggtt 186 47 409 DNA Mycobacterium tuberculosis misc_feature (1)..(409) n is a, c, g, or t/u. 47 gtcgctgaat gtgttgtcgg agaccgtnga tcagacctat ccgcacctga gcgccgccnt 60 cgacgggntg gctaagttct ccgacaccat cggcaagcgc gacgagcaga ntcacgcacc 120 tactagccca ggccaaccag gtggccagca tcctgggtga tcgcagtgag caggtcgacc 180 gcctattggt caacgctaag accctgatcg ccgcgttcaa cgagcgcggc cgcgcggtcg 240 acgccctgct ggggaacatc tccgctttct cgncccaggt gcaaaacctt natcaacgac 300 aacccgaacc tgaaccatgt gctcgnnnag ctgcgcatcc tcancgacct gttggtcgac 360 cgcaaggagg atttggctga aaccctgacg atcttgggca gattcagcg 409 48 464 DNA Mycobacterium tuberculosis misc_feature (1)..(464) n is a, c, g, or t/u. 48 agnccgtgca ctggaacttc ggctcgatgt ctccgatgtg gacggcaagc tgatgatctc 60 ccggttggan gtcgattcga tgasaaatgn cttggcggct ggtggtgttc gatgncctgg 120 caccactggc cacgatcgcc gccntggccg cgatcggcgn cttngctcgg ctggcccctg 180 tggtgggttt cgacgtgctc ggtgttggtg ctgctggtgg tcgaaggtgt ggcaatcaac 240 nttctggctg ttgcgtcgtg attcggtaac cgtcggtacc gacgacgatg cgcccgggct 300 gcgactggcc gttgtcttcc tgtgcgccgc cgcgatctcg gcggcggtgg tgactgggta 360 cctgcgctgg acgacaccgg accgcgactt caatcgggat tcccgggaag tggtgcatct 420 tgccacgggg atggccgaga cggtcgcgtc attctccccg agcg 464 49 423 DNA Mycobacterium tuberculosis misc_feature (1)..(423) n is a, c, g, or t/u; k is t/u or g. 49 gtccaaggcc gtagcccacc tcctggaagt cgtaccacgt cgactcgacc aggacggctg 60 cantcagcna cttcgtcaac ccggcgatca tcaacntgca cctacggcag tgtgnacgca 120 ccccggacca tcgcactggc cggggnttca cacgccgaac actgnctgac cgcactggat 180 ctgctnggtc gcatgcacca cttcaaggtg gtgacgtacc tcaaaatggg ttkcccgttg 240 tccaccgagg aagtcccgct gatncatggg caataacgct ccctatccgc agtgtcacca 300 gtgggtgcaa gcggcgatgg ccaagttggt cgctgaccac cccgactacg ttttcacaac 360 ctcgactcga ccgtggaaca tcaaacccgg cgatgtgatg ccagcaacct atgtcgggat 420 ctg 423 50 279 DNA Mycobacterium tuberculosis 50 cggtcgagcc gatgaacgtc tgcagttcac cgcaaccacg ctcagcggtg ctcccttcga 60 tgcgcaagcc tgcaaggcaa tgccgcggtg ttgtggttct ggacgccgtg gtgcccgttc 120 tgcaactgtc agaagccccc agccgcagcc aggtagcggc cgctaatccg gcggtcacct 180 tcgtcggaat cgccacccgc gccgacgtcg gggcgatgca gagctttgtc tcgaagtaca 240 acctgaattt caccaacctc aatgacgccg atggtgtga 279 51 331 DNA Mycobacterium tuberculosis misc_feature (1)..(331) n is a, c, g, or t/u; m is a or c. 51 cggcccgscg gcgccntggt gaagcttggm gmmtgggtgn agcgcagctg cccaccacac 60 ggraccnngg tgcggacgcg gntgacgcgc ctggtggtca gcatcgtggc cggtctgctg 120 ttgtatgcca gcttcccgcc gcgcaactgc tggtnggcgg cggtggttgs gctncgcatt 180 gctggcctgg gtgctgaccc accgcgcgac gacaccggtg ggtgggctgg gctacggcct 240 gctattcggc ctggtgttct acgtctcgtt gttgccgtgg atcggcgagc tggtgnnccc 300 cgggccctgg ttggcactgg cgacgacgtg c 331 52 507 DNA Mycobacterium tuberculosis 52 tgtattcccg tcgatgcgtt gctgcaggta ggccttgaaa tcgttggggg tcacgacgcg 60 gacctcgaag ttcatcatcg agtgatacgt gccacacatc tcggcgcagt ggcccacgaa 120 tgctccggtc ttggtgattt cttcgatctg gaagacgttg accgagttgt ttgccaccgg 180 gttaggcatc acgtcacgct tgaacaagaa ctccggcacc cagaatgcgt gtatcacatc 240 ggctgaggcc atttggaatt cgatacgctt gccggacggc agcaccagca ccggaatttc 300 ggtgctggtg cccaacgtct cgaccttgtc gaaattcagg taggtccggt cctcggtgtt 360 gagcccgcgc accggcccga ccagctcttc gccgtacttg tccttgccct ctggcttgga 420 aaccatggcg cgcttgcgct ccggatcggc accatcatag gtcagtgtgc cgtctttgaa 480 gttcaccctt tgatagccaa acttcca 507 53 293 DNA Mycobacterium tuberculosis 53 ccacacaaca caaatctacg tcgtaatgca gtcgtaagtc catccgacgt cgatggcaag 60 gacagcaccc gacggccaac ggcatataca tcgtcggctc gccggtcaca agcacatcat 120 catggactcg tccactacgg cgtacccgtc aactcgccca acggatatcg caccgatgtc 180 gactggccac ccagatctcc tacagcggtg tcttcgtgca ctcagcgccg tggtcggtgg 240 gggctcaggg ccacaccaac accagccatg gctgcctgaa cgtcagcccg agc 293 54 820 DNA Mycobacterium tuberculosis misc_feature (1)..(820) n is a, c, g, or t/u. 54 cgccgccggc gngcgctacc ggtgcgggag ggtacaccca agcantccgg gaccggccgt 60 cycgccggga acgccgtgct cctacacacc ggcggcgggc gcgttgccac ggcccgacac 120 cccactaccc tgncgcgggc gccaccgttg gcccgttcgg tggacccgac ttcccggcac 180 cgctcgatgt ccagccgtcg ccgcctaatc ccgatgggcc gccgccgacg ccgggcatcc 240 taagtgctgg gcggccgggc gagccggctc cggctgttcc ggncataccg atgccsctgc 300 cgccgaacnn nnnnnnnnnt gcacgcaccc aaccgcttga gccgtttcct gacgggacgg 360 gaggtagcaa ccaatgagca ccatcttcga catccgsagc ctgcgactgy cgaaactgtc 420 tgcaaaggta gtggtcgtcg gcgggttggt ggtggtcttg gcggtcgtgg ccgctgcggc 480 cggcgcgcgg ctctaccgga aactgactac cactaccgtg gtcgcrtatt tnctstgagg 540 cgctcgcgct gtacccagga gacaaagtcc agatcatggg tgtgcgggtc ggttctatcg 600 acaagatcga gccggccggc gacaagatgc gagtcacgtt gcactacagc aacaaatacc 660 aggtgccggc cacgnctacc gcgtcgatcc tcaaccccag cctggtggcc tcgcgcacca 720 tccagctgtc accgccgtac accggcggcc cggtcttgca agacggcgcg gtgatcccaa 780 tcgagcgcac ccaggtgccc gtcgagtggg atcagttgcg 820 55 117 DNA Mycobacterium tuberculosis 55 cagccacctc gttcgccgcc gacatcgact atcagccgac ccggccactg ctgacctgat 60 cgccaacagc tggaggccct accggctgca gttcaattca cccgctgcgg gtcggcg 117 56 242 DNA Mycobacterium tuberculosis 56 aggtgtcgtg cttcatgcct ggcgcccaat ccagtttcta caccgactgg tatcaccctt 60 cgcagacaaa cggccagaac tacacctaca agtgggagac cttccttacc acacagatgc 120 ccgcctggct acaggccaac aaggcgtgtc ccccacaggc aacgcggcgg tgggtctttc 180 gatctcgggc ggttccgcgc tgaccctggc cgcgtactac ccgcagcagt tcccgtacgc 240 cg 242 57 345 DNA Mycobacterium tuberculosis 57 tgctgcagat agccaaggat cccgaggtcg tgattgatat cacgtctttc cagtggaatt 60 ggaagtttgg ctatcaaagg gtgaacttca aagacggcac actgacctat gatggtgccg 120 atccggagcg caagcgcgcc atggtttcca agccagaggg caaggacaag tacggcgaag 180 agctggtcgg gccggtgcgc gggctcaaca ccgaggaccg gacctacctg aatttcgaca 240 aggtcgagac gttgggcacc agcaccgaaa ttccggtgct ggtgctgccg tccggcaagc 300 gtatcgaatt ccaaatggcc tcagccgatg tgatacacgc attct 345 58 262 DNA Mycobacterium tuberculosis misc_feature (1)..(262) n is a, c, g, or t/u. 58 cngactccaa cnagtgcgnt caancngntg tnccngacaa gaaggttcct acatccgcaa 60 ntcggtgnaa ngccactgtg gatgcctacg acggaacggt cacgctgtac caacaggacg 120 naaaaggatc cggtgctcaa ggcctggatg caggtcttcc ccggcacggt aaagcctaag 180 agcgacattg cgccggagct tgccgagcan ctgcggtatc ccgaggacct gttcaaggtg 240 cagcgcatgt tgttggccaa at 262 59 241 DNA Mycobacterium tuberculosis misc_feature (1)..(241) n is a, c, g, or t/u; r is g or a. 59 ccaccannna acrrcacagc tccggccrrc cgtncgcagg ccacccgcan cgtagtgctc 60 aaattcttcc aggacctcgg tggggyacat ccgtccacct ggtacaaggc cttcaactac 120 aacctcgcga cctcgcagcc catcaccttc gacacgttgt tcgtgcccgg caccacgcca 180 ctggacagca tctaccccat cgttcagcgc gagctggcac gtcagaccgg tttcggtgcc 240 g 241 60 243 DNA Mycobacterium tuberculosis misc_feature (1)..(243) n is a, c, g, or t/u. 60 ccggcggatc tgcgtgacga ntgtatncca cggnactacc cgcggtcctt cctcnantnc 60 cgccggncca gncgcagnct ncngatgtcc ngctataacc tgcgcgatcg ccgccgggct 120 gcccgacaac acggtgngcg ccgccgctgc ttccgccaat tctgggtgnc ggcatnccgg 180 cagcgcccgg cccagcactg agagggggac gttgatgcgg tggccgacgg cgtggctgct 240 ggc 243 61 2348 DNA Mycobacterium tuberculosis misc_feature (1)..(2348) n is a, c, g, or t/u; m is a or c. 61 gcgctgtcat tcggacttcg gaccgcgttg gcggtggtgc tgatcatgaa nctacgacgg 60 cgccaccggc agcttcccgt catgggtgct ctatccctgt gcgctggcca tgatggtgtt 120 ctcgaagtcg ttcagcgtgc tgcgcagcgc agtgacaccg agggtgatgc cgccaaccat 180 cgacttggtc cgggtcaact cacggctgac cgtgttcggc ctgctcggcg gcaccatcgc 240 tggtggcgcg attgcggccg gagtcgaatt cgtctgcacc cacctgttcc agctgccggg 300 cgcgttgttc gtcgtcgtcg cgatcaccat cgctggcgct tcgctgtcga tgcgcattcc 360 gcgctgggtc gaggtgacca gcggtgaggt cccggccaca ttgagctacc accgggatag 420 gggcagacta cggcgacngc tggccggagg aagtcaagaa cctcggcgga acactccgac 480 aaccgttggg ccgcaacatc attacctccc tgtggggtaa ctgcaccatc aaggtgatgg 540 tcggctttct gttcttgtat ccggcgtttg tcgccaaggc gcacgaagcc aacgggtggg 600 tgcaattggg catgctgggc ctgatcggcg cggcggccgc ggtcggcaac ttcgccggca 660 atttcaccag cgcacgcctg cagctaggca ggccagctgt gctggtngtg cgctgcaccg 720 tgctagttac cgtgttagcc atcgcggccg cggtggccgg cagcctggca gcgacagcga 780 ttgccaccct gatcacggca gggtccagtg ccattgctaa agcctcgctg gacgcctcgt 840 tgcagcacga cctgcccgag gagtcgcggg catcggggtt tgggcgttcc gagtcgactc 900 ttcagctggc ctgggtgctg ggcggcgcgg tgggcgtgtt ggtgtacacc gagctgtggg 960 tgggcttcac tgcggtgagc gcgctgctga tcctgggtct ggctcagacc atcgtcagct 1020 tccgcggcga ttcgctgatc cctggcctgg gcggtaatcg gcccgtgatg gccgagcaag 1080 aaaccacccg tcgtggtgcg gcggtggcgc cgnagtgaag cgcggtgtcg caacgctgcc 1140 ggtgatcctg gtgattctgc tctcggtggc ggccggggcc ggtgcatggc tgctagtacg 1200 cggacacggt ccgcagcaac ccgagatcag cgcttactcg cacgggcacc tgacccgcgt 1260 ggggccctat ttgtactgca acgtggtcga cctcgacgac tgtcagaccc cgcangcgca 1320 gggcgaattg ccggtaagcg aacgctatcc cgtgcagctc tcggtacccg aagtcatttc 1380 ccgggcgccg tggcgtttgc tgcaggtata ccaggacccc gccaacacca ccagcacctt 1440 gtttcggccg gacacccggt tggcggtcac catccccact gtcgacccgc agcgcgggcg 1500 gctgaccggg attgtcgtgc agttgctgac gttggtggtc gaccactcgg gtgaactacg 1560 cgacgntccg cacgcggaat ggtcggtgcg ccttatcttt tgacgaggcc gcggctcgac 1620 gggacgctta agcgcggtcg gcgccaacgg tccgaagagc cgccgacacc cggggcacat 1680 cggcgcatca tggaactgtg cggatcggag tcggggtttg caccacgccc gacgcgcggc 1740 aggccgcggt ggaggctgcg ggccaggcgc gcgacgagct ggcgggtgag gcgccgtcgc 1800 tggcggtgtt gcttggatcg cgtgcacaca ccgaccgggc tgccgacgtc ctgagcgcgg 1860 tgctgcagat gatcgayccg cccgcgcttg tcggttgcat cgcccaggcc atcgtcgccg 1920 gccgccacga gatcgaggac gagcccgcgg tggtggtgtg gctggcgtcc ggcttggccg 1980 ccgagacatt ccagctggac tttgtccgta ccggctcggg tgccctgatc accggttatc 2040 ggttcgaccg caccgcccgg gatctgcatc tgctgctgcc ggacccgtac acattcccgt 2100 cgaacctgct catcgagcac cccaacaccg acctgccggg caccgccgtc gtgggcggcg 2160 ntggtgagcg gcgggcgccg gcggggcgac acccggctgt tccgcgatca cgacgtgctc 2220 acctccggcg tcgtcggcgt gcgcctgccc gggatgcgcg gtgtmccggt cgtgtcgcag 2280 ggttgccggc cgatcggcta cccatacatc gtcaccggcg cggacggcat actgatcacc 2340 gagctcgg 2348 62 821 DNA Mycobacterium tuberculosis misc_feature (1)..(821) n is a, c, g, or t/u. 62 cgttacccgc tttacaccac cgccaaggcc aacctgaccg cgctcagcac cgggctgtcc 60 agctgtgcga tggccgacga cgtgctggcc gagcccgacc ccaatgccgg catgctgcaa 120 ccggttccgg gccaggcgtt cggaccggac ggacgctggg cggtatcagt cccgtcggct 180 tcaaacccga gggcgtgggc gaggacctca agtccgancc cggtggtctc caaacccggg 240 ctggtcaact ccgatgcgtc gcccaacaaa cccaacgccg ccatcaccga ctccgcgggc 300 accgccggag ggaagggccc ggntcgggat ncaacgggtt gcnacgcggc gctgccgttc 360 nggattggac ccggcacgta ccccggtgat gggcagctac ggggagaaca acctggccgc 420 cacggccacc tcggcctggt accagttacc gccccgcagc ccggaccggc cgctggtggt 480 ggtttccgcg gccggcgcca tctggtccta caaggaggac ggcgatttca tctacggcca 540 gtccctgaaa ctgcagtggg gcgtcaccgg cccggacggc cgcatccagc cactggggca 600 ggtatttccg atcgacatcg gaccgcaacc cgcgtggcgc aatctgcggt ttccgctggc 660 ctgggcgccg ccggaggccg acgtggcgcg cattgtcgcc tatgacccga acctgagccc 720 tgagcaatgg ttcgccttca ccccgccccg ggttccggtg ctggaatctc tgcagcggtt 780 gatcgggtca gcgacaccgg tgttgatgga catcgcgacc g 821 63 479 DNA Mycobacterium tuberculosis misc_feature (1)..(479) n is a, c, g, or t/u; s is g or c. 63 gccagccgtg atcggctgay cggncagntg atcaccaacc tcaacgtggt gctgggcntc 60 gctggncgct cacacngatc ggttggacca gscggtgacg tcgctatcag cgttgattca 120 ccggctcgcg caacgcaaga ccgacatctc caacgccgtg gcctacacca acgcgccgcc 180 ggctcggtcg ccgatctnct gtcgcaggct cgcgcnncgt tggcgaangt ggttcgcgag 240 accgatcggg tggccggcat cgcggccgcc gaccacgact acctcgacaa tctgctcaac 300 acgctgccgg acaaatacca ggcgctggtc cgccagggta tgtacggcga cttcttcgcc 360 ttctacctgt gcgacgtcgt gctcaaggtc aacggcaagg gcggccagcc ggtgtacatc 420 aagctggccg gtcaggacan gcnggcggtg cgcgccgaaa tgaaatcctt cgccgaacg 479 64 481 DNA Mycobacterium tuberculosis misc_feature (1)..(481) n is a, c, g, or t/u; k is t/u or g. 64 kgtctcgcgn ccttaacatc cggtcgcccc ancggtaatc tgcctgtgga tgccgtccgg 60 aantataagc aaatggccag gagtgcgtga cgcagttatg gctcggtata gttccgttnt 120 tgccccggac tgggggcgtg aggtggaact aatggcggtg tcgggtgata tttccgacgg 180 caagcgacca tataggtgga tcgacggcaa taaasacacg ctctggccac gtttcttggc 240 ggggaaaggg gtgatgctat cggagccaat ggtatcgcga caacacttgc agatgccgcc 300 aaggccgatc acgctaatga cggattcggg gccacaaacg ttccccgttc tggcggtttt 360 ctctgactac acctcagatc aaggtgtgat tttgatggat cgcgccagtt atcgggccca 420 ttggcaggat gatgacgtga cgaccatgtt tctttttttg gcnatncggg tgcgaatagc 480 g 481 65 469 DNA Mycobacterium tuberculosis misc_feature (1)..(469) n is a, c, g, or t/u. 65 ggcgaggtca gtgaagccga ggaagcggaa aggagcgccc aatacggaac cgcctctccc 60 cgcgcgttgg ccgattcatt aaatgcagct ggcacgacag gtttcccgac tggaamgcgg 120 gcagtgagcg caasgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac 180 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag 240 gaaacagcta tgacatgatt acgaatttaa tacgactcac tatagggaat tcgagctcgg 300 tacccgggga tcctctagag tcgcttcggt tggcggcgac cagcagtgga tccacggtgg 360 ccgcccgcgc ggcdtcatac accgccgcgg cctccttggc ctgtgcggcc sgcttagcgc 420 gcgtgttgct gccgtgctta gccanctggc atagggggct gccgcgcgc 469 66 291 DNA Mycobacterium tuberculosis misc_feature (1)..(291) n is a, c, g, or t/u; r is g or a. 66 caggttcgac tgatctagct gnrrrccara ccggcacnag ncgacantta ccantacctg 60 acanacagnc cgntcnagcc aanccgnann naggangcag nagnaacagg cagatgcatc 120 taatgatacc cgcggagtat atctccaacg tgatatatga aggtccgcgt gctgactcat 180 tgtatgccgc cgaccagcga ttgcgacaat tagctgactc agttagaacg actgccgagt 240 cgctcaacac cacgctcgac gagctgcacg agaactggaa aggtagtttc a 291 67 1306 DNA Mycobacterium tuberculosis 67 gtgatacagg aggcgccaac agtgacacct cgcgggccag gtcgtttgca acgcttgtcg 60 cagtgcaggc ctcagcgcgg ctccggaggg cctgcccgtg gtcttcgaca gctggcgctc 120 gcagcaatgc tgggggcatt ggccgtcacc gtcagtggat gcagctggtc ggaagccctg 180 ggcatcggtt ggccggaggg cattaccccg gaggcacacc tcaatcgaga actgtggatc 240 ggggcggtga tcgcctccct ggcggttggg gtaatcgtgt ggggtctcat cttctggtcc 300 gcggtatttc accggaagaa gaacaccgac actgagttgc cccgccagtt cggctacaac 360 atgccgctag agctggttct caccgtcata ccgttcctca tcatctcggt gctgttttat 420 ttcaccgtcg tggtgcagga gaagatgctg cagatagcca aggatcccga ggtcgtgatt 480 gatatcacgt ctttccagtg gaattggaag tttggctatc aaagggtgaa cttcaaagac 540 ggcacactga cctatgatgg tgccgatccg gagcgcaagc gcgccatggt ttccaagcca 600 gagggcaagg acaagtacgg cgaagagctg gtcgggccgg tgcgcgggct caacaccgag 660 gaccggacct acctgaattt cgacaaggtc gagacgttgg gcaccagcac cgaaattccg 720 gtgctggtgc tgccgtccgg caagcgtatc gaattccaaa tggcctcagc cgatgtgata 780 cacgcattct gggtgccgga gttcttgttc aagcgtgacg tgatgcctaa cccggtggca 840 aacaactcgg tcaacgtctt ccagatcgaa gaaatcacca agaccggagc attcgtgggc 900 cactgcgccg agatgtgtgg cacgtatcac tcgatgatga acttcgaggt ccgcgtcgtg 960 acccccaacg atttcaaggc ctacctgcag caacgcatcg acgggaakac aaacgccgag 1020 gccctgcggg cgatcaacca gccgcccctt gcggtgacca cccacccgtt tgatactcgc 1080 cgcggtgaat tggccccgca gcccgtaggt taggacgctc atgcatatcg aagcccgact 1140 gtttgagttt gtcgccgcgt tcttcgtggt gacggcggtg ctgtacggcg tgttgacctc 1200 gatgttcgcc accggtggtg tcgagtgggc tggcaccact gcgctggcgc ttaccggcgg 1260 catggcgttg atcgtcgcca ccttcttccg gtttgtggcc gcggat 1306 68 728 DNA Mycobacterium tuberculosis misc_feature (1)..(728) n is a, c, g, or t/u. 68 ggtgcctgcc atcggttcgc tggccacgct ggcatctttg gtctgttaga ggtatccgcg 60 cggatggcca gtcctgttgg cggggnttgt cgccacgatt gccgcccgcg ctgaancccg 120 acgacgccga tgccctgccc accacggatc ggctgaccac ccgagcgaac cgtgcagatg 180 cttggttgac gagcctgctg gcgnccttcg cggcctcggc gaccatcggt gccatcggaa 240 ccgccgtcgc aacccacggc atccacagst ccagcatngg cggtatcgcg ttggccgncg 300 tcaccggtgc gctgctgctg ctacgagcac gttcagcaga caccagaagg tcactggtgt 360 ttgccatctg tggaatcacc accgttgcaa cggcattnta ccgtcgccgc ggatcgggct 420 ctggaacacg ggccgtggat tgccgcgctg accgccatgc tggnccgccg tggcaatgtt 480 tttgggcttc gtcgctcccg cgttgtcgct ctcgcccgtc acgtaccgca ccatcgaatt 540 gctggagtgt ctggcgctga tcgcaatggt tccattgacc gcttggstat gcggcgccta 600 caggcgcgtt cgccacctcg acctgacatg gacatgacca cngtcccgta ccctgcgcct 660 gctggtggta tcagcgctcg cgacgctgtc tgggttggga acgccggttg cgccacgcgg 720 tttcgccg 728 69 1028 DNA Mycobacterium tuberculosis misc_feature (1)..(1028) n is a, c, g, or t/u; k is t/u or g; s is g or c. 69 gktcncggtg atgtcgaccg tcggcacgac gagcgaaacc tcaccggtcg acagtgtctg 60 cccgaggccg cagccgacgt gcccccggag accgcgcgcc aacacggtgc cgtacatgta 120 gcccgcacgg cgcatcatcg ccgagccggc gtagatgttt tcctgcacgg cgtgcgcggt 180 gaacccntcc ggcgccagca ccgccacctt tcccgcgtcc acgtcggcct gggtggtgac 240 gccgagcacc ccaccgaaat gatcgacatg gctgtgggtg tagatgaccg scgaccacgg 300 ggcggtcggc tccgcggtgg gcgcgataca agtccagcgc ggcggcggcc acctcggtgg 360 acaccaacgg gtcgatgacg atcagcccag tgtcaccctc aacgaagctg atattggaga 420 tatcgaatcc gcggacctga tagatgcccg gcaccacctg gtagaggccc tgtttcgcgg 480 tcagctggga ttgccgccac aggctgggat gcaccgatgt cggcgcggca ccgtcgagaa 540 acgagtacgc gtcgttgtcc cacaccacgc gaccatcggc agccttgatc acacacgggg 600 acagcgcggc aatgaatccg cgatcggcgt cgtcgaaatc cgttgtgtca tgcaacggta 660 acgagtgttc accgtgtgcc gcctggatga cggcagtngg gaggtttgtg ttccatcggc 720 actacattgc cactactacg gtgcacgccg gtagatgccg ttggcgaacc acgctaccga 780 ccagaaagag agaattttcc gccgcaccta gacctcgggc cctcntaacg cgcatactgc 840 cgaagcggtc ctcaatgccg atggaccgct acgacaggca aaggagcaca gggtgaagcg 900 tggactgacg gtcgcggtag ccggagccgc cattctggtc gcaggtcttt ccggatgttc 960 aagcaacaag tcgactacag gaagcggtga gaccacgacc gcgngcaggc acgacgcaag 1020 ccccggcg 1028 70 780 DNA Mycobacterium tuberculosis misc_feature (1)..(780) n is a, c, g, or t/u; y is t/u or c. 70 agatcaacac catcaccagt gcggtcatcg agttgctgca gggcnagggt ggtccgttgg 60 cgaacgtgct cgccgcyacc ggtgccttct cggcggcgct gggcgcacgc gaccagctga 120 tcggcgaggt aatcaccaac ctcaacgcgg tgctggcgac cgtcgatgca aagagcgcgc 180 aatttntcgg ccagtgtcga ccagctgcag cagctggtca gcggcctggc caagaaccgg 240 gatccgatcg cgggcgccat ttcgccgctg gcgtcgacga cgacggatct tacggaactg 300 ttgcggaatt cgcgccggcc gctgcaaggc atcctggaaa acgcccggcc gctggctacc 360 gagctggaca accgaaaggc cgaggtcaas aacgacatcg agcagctcgg cgaggactac 420 ctgcgcctgt ccgcgctggg cagttacgga gcattcnttc aacatctact tctgctcggt 480 gacgatcaag atcaacggac cggccggcag cgacatcctg ctgccgatcg gcggccagcc 540 ggatcccagc aaggggaggt gcgcctttgc taaataggaa gccaagtagc aaacacgaac 600 gcgacccgnt ccgcaccggc atcttcggcc tggtgctggt gatctgcgtc gtcctgatcg 660 cattcggcta cagcgggttg cctttctggc cacagggcaa aacctacgac gcgtatttca 720 ccgacgccgg tgggatcacc cccggtaact cggtttatgt ctcgggcctc aaggtgggcg 780 71 689 DNA Mycobacterium tuberculosis misc_feature (1)..(689) n is a, c, g, or t/u. 71 ctatccgcaa ggcttcgcag acgctcggct gnaccgcaga atcgcggtgc acccacgatt 60 gccagtagcg cgggcccact cgtgcctact acacttcgtc gtagccaaat catcggcccc 120 gtagtatctc cggagatgac agatgaatgt cgtcgacatt tcgcggtggc agttcggtat 180 caccaccgtc tatcacttca ttttcgtnac cgctgaccat cggcctggcc ccngctgatc 240 gcggtcatgc aaactgctgt nggtcgtcac cgataacccc gcctggtatc gcctcaccaa 300 attcttcggc aaattgttcc tgatcaactt tgccatcggc gtggcgaccg gaatcgtgca 360 ggaatttcag ttcggcatga actggagcga gtactcccga ttcgtcggcg atgtcttcgg 420 cgccccgctg gccatggagg gcctggcggc cttcttcttc gaatccacct tcatcgggtt 480 gtggatcttc ggctggaaca ggctgccccg gctggtgcat ctggcctgca tctggatcgt 540 cgcaatcgcg gtcaacgtgt ccgcgttctt catcatcgng gcaaactcct tcatgcagca 600 tccggtcggc gcgcactaca acccgaccac cgggcgtgcc gagttgagca gcatcgtcgt 660 gcctgctgac caacaacacc gcacaggcg 689 72 274 DNA Mycobacterium tuberculosis 72 ccgcagcacc gaggcaagca tcgcacccgt cgattcccgc catcccggcg acatgatggt 60 catgtccgac accgacgccc gcacctcgct tcccgagttg accgcgctgc gcgtggacgc 120 cgcaacggat gcgtcggttc attcgatccc ggctcgaaat tggccatggc gaacgcatct 180 tgctgtgatg gttcgggcag tagatctcca ctgccgcact gataaactcg ggtcatggtc 240 gtcgtgaggc ggacagggta gaggcgcatg accg 274 73 252 DNA Mycobacterium tuberculosis 73 gtgatgcctt ccagcattgg attggtcgtc ggttcgatgc tgtggcgaca gataaaccgc 60 ctgttcgggg tgcgtggcct ctgctgggca gcgcactgct caacgccgct ctgcgctgct 120 gtgcatggtg gccgagtcgt gtgggcagtg ggttcacgcc tgggcgtact tcacggcgtt 180 cctgctggct acggtggccg ctcaaacggt ggtcgccgca tcgatatcgt ggatcagcgt 240 cctcgcgccc ga 252 74 160 DNA Mycobacterium tuberculosis 74 ggcgccgccg tcgtgctggc cgcccggccc ggtgggggtg ccggccagcg tggttccgcc 60 agtggccgcg ccgaacgtat tggccggcgt cctcgagcac gacaacgacg ggtcgggggc 120 ggcggtgctg gccgcgctgg ccaagctgcc acccggtggt 160 75 401 DNA Mycobacterium tuberculosis misc_feature (1)..(401) n is a, c, g, or t/u; w is t/u or a. 75 atcagccgcg ggtcgacgcc gccgatgacc tcgacgtcgt cgtcgtcgct gccggtactc 60 aatccaatca ccatcctctt acgcaccttc taggagtgtg ttgctgcggc agtgccgngc 120 cattcgtaga ttcgggcctc gccgttgtcg tagatcttcg cccacgacct cgatgtctct 180 aacgacacta gtccgtccgg cacngcaaan ccccgcaccg tcggagtgct ggtcaggnta 240 tagncggtac aggnggactt ggwwggcctc gagtanccga ggwwcgntct ncccgttgcg 300 gncataggcc agaagatgaa ccggtgtaga ccgggcctgt tgcgagggtc gtagtcgtag 360 gtcccagagg tgtcggacgc ccaggttaat acacagcgtg c 401 76 248 DNA Mycobacterium tuberculosis 76 gcagacctct ggccgctggt ggtgctgggt acctgcgctg gcgacaccgg accgcagacc 60 gtcaatcggg actcccggga acgtggtgcc atcttgccac ggggatggcc gacgcggctc 120 gtcattctcc ccgagcgcac cggccgccgc tgttgaccgg gccgcggcga ctgatggtgc 180 ccgcacacgc gggcgggttc aaggagcaat acgccaagtc cagcgccgct ctcgcacggc 240 gcggtgtt 248 77 17 PRT Mycobacterium tuberculosis 77 Val His Leu Ala Thr Gly Met Ala Glu Thr Val Ala Ser Phe Ser Pro 1 5 10 15 Ser 78 17 PRT Mycobacterium tuberculosis 78 Arg Glu Val Val His Leu Ala Thr Gly Met Ala Glu Thr Val Ala Ser 1 5 10 15 Phe 79 17 PRT Mycobacterium tuberculosis 79 Arg Asp Ser Arg Glu Val Val His Leu Ala Thr Gly Met Ala Glu Thr 1 5 10 15 Val 80 17 PRT Mycobacterium tuberculosis 80 Asp Phe Asn Arg Asp Ser Arg Glu Val Val His Leu Ala Thr Gly Met 1 5 10 15 Ala 81 17 PRT Mycobacterium tuberculosis 81 Ile Ser Ala Ala Val Val Thr Gly Tyr Leu Arg Trp Thr Thr Pro Asp 1 5 10 15 Arg 82 17 PRT Mycobacterium tuberculosis 82 Ala Val Val Phe Leu Cys Ala Ala Ala Ile Ser Ala Ala Val Val Thr 1 5 10 15 Gly 83 17 PRT Mycobacterium tuberculosis 83 Val Thr Asp Asn Pro Ala Trp Tyr Arg Leu Thr Lys Phe Phe Gly Lys 1 5 10 15 Leu 84 17 PRT Mycobacterium tuberculosis 84 Ala Trp Tyr Arg Leu Thr Lys Phe Phe Gly Lys Leu Phe Leu Ile Asn 1 5 10 15 Phe 85 17 PRT Mycobacterium tuberculosis 85 Lys Phe Phe Gly Lys Leu Phe Leu Ile Asn Phe Ala Ile Gly Val Ala 1 5 10 15 Thr 86 17 PRT Mycobacterium tuberculosis 86 Phe Leu Ile Asn Phe Ala Ile Gly Val Ala Thr Gly Ile Val Gln Glu 1 5 10 15 Phe 87 17 PRT Mycobacterium tuberculosis 87 Ala Ile Gly Val Ala Thr Gly Ile Val Gln Glu Phe Gln Phe Gly Met 1 5 10 15 Asn 88 17 PRT Mycobacterium tuberculosis 88 Thr Gly Ile Val Gln Glu Phe Glu Phe Gly Met Asn Trp Ser Glu Tyr 1 5 10 15 Ser 89 17 PRT Mycobacterium tuberculosis 89 Glu Phe Gln Phe Gly Met Asn Trp Ser Glu Tyr Ser Arg Phe Val Gly 1 5 10 15 Asp 90 17 PRT Mycobacterium tuberculosis 90 Met Asn Trp Ser Glu Tyr Ser Arg Phe Val Gly Asp Val Phe Gly Ala 1 5 10 15 Pro 91 17 PRT Mycobacterium tuberculosis 91 Trp Ser Glu Tyr Ser Arg Phe Val Gly Asp Val Phe Gly Ala Pro Leu 1 5 10 15 Ala 92 17 PRT Mycobacterium tuberculosis 92 Glu Tyr Ser Arg Phe Val Gly Asp Val Phe Gly Ala Pro Leu Ala Met 1 5 10 15 Glu 93 17 PRT Mycobacterium tuberculosis 93 Ser Arg Phe Val Gly Asp Val Phe Gly Ala Pro Leu Ala Met Glu Ser 1 5 10 15 Leu 94 17 PRT Mycobacterium tuberculosis 94 Trp Ile Phe Gly Trp Asn Arg Leu Pro Arg Leu Val His Leu Ala Cys 1 5 10 15 Ile 95 17 PRT Mycobacterium tuberculosis 95 Trp Asn Arg Leu Pro Arg Leu Val His Leu Ala Cys Ile Trp Ile Val 1 5 10 15 Ala 96 17 PRT Mycobacterium tuberculosis 96 Gly Arg Ala Glu Leu Ser Ser Ile Val Val Leu Leu Thr Asn Asn Thr 1 5 10 15 Ala 97 17 PRT Mycobacterium tuberculosis 97 Gly Lys Thr Tyr Asp Ala Tyr Phe Thr Asp Ala Gly Gly Ile Thr Pro 1 5 10 15 Gly 98 17 PRT Mycobacterium tuberculosis 98 Tyr Asp Ala Tyr Phe Thr Asp Ala Gly Gly Ile Thr Pro Gly Asn Ser 1 5 10 15 Val 99 17 PRT Mycobacterium tuberculosis 99 Trp Pro Gln Gly Lys Thr Tyr Asp Ala Tyr Phe Thr Asp Ala Gly Gly 1 5 10 15 Ile 100 17 PRT Mycobacterium tuberculosis 100 Ala Thr Gly Met Ala Glu Thr Val Ala Ser Phe Ser Pro Ser Glu Gly 1 5 10 15 Ser 101 17 PRT Mycobacterium tuberculosis 101 Gly Trp Glu Arg Arg Leu Arg His Ala Val Ser Pro Lys Asp Pro Ala 1 5 10 15 Gln 102 17 PRT Mycobacterium tuberculosis 102 Thr Gly Ser Gly Glu Thr Thr Thr Ala Ala Gly Thr Thr Ala Ser Pro 1 5 10 15 Gly 103 17 PRT Mycobacterium tuberculosis 103 Gly Ala Ala Ile Leu Val Ala Gly Leu Ser Gly Cys Ser Ser Asn Lys 1 5 10 15 Ser 104 17 PRT Mycobacterium tuberculosis 104 Ala Val Ala Gly Ala Ala Ile Leu Val Ala Gly Leu Ser Gly Cys Ser 1 5 10 15 Ser 105 17 PRT Mycobacterium tuberculosis 105 Leu Thr Val Ala Val Ala Gly Ala Ala Ile Leu Val Ala Gly Leu Ser 1 5 10 15 Gly 106 21 DNA Mycobacterium tuberculosis Description of Artificial SequencePCR Primer 106 cccagcttgt gatacaggag g 21 107 22 DNA Mycobacterium tuberculosis Description of Artificial SequencePCR Primer 107 ggcctcagcg cggctccgga gg 22 108 46 DNA Mycobacterium tuberculosis Description of Artificial SequencePCR Primer 108 tctagacacc accaccacca ccacgtgaca cctcgcgggc caggtc 46 109 28 DNA Mycobacterium tuberculosis Description of Artificial SequencePCR Primer 109 aagcttcgcc atgccgccgg taagcgcc 28 110 330 DNA Mycobacterium tuberculosis misc_feature (1)..(330) n is a, c, g, or t/u; k is t/u or g; s is c or g. 110 gcacgtcgtc gccagtgcca accagggccc ggggcnnacc agctcgccga tccacggcaa 60 caacgagacg tagaacacca ggccgaatag caggccgtag cccagcccac ccaccggtgt 120 cgtcgcgcgg tgggtcagca cccaggccag caatgcgnag cccaaccacc gccgnccacc 180 agcagttgcg cggcgggaag ctggcataca acagcagacc ggccacgatg ctgaccacca 240 ggcgcgtcan ccgcgtccgc accgngtccc gtgtggtggg cagctgcgct ncacccakkc 300 kccaagcttc accanggcgc cgscgggccg 330 111 431 DNA Mycobacterium tuberculosis misc_feature (1)..(431) n is a, c, g, or t/u. 111 tgtcccgcat ggtagtcggg ctggnccngg tgatcgcttg cagctttngc cgtggatgtg 60 agaaaggaat atgttggtga tcaccatgtt tcgtgtactc gtggcgcgga tgacggcgct 120 ggcggtcgac gangtcgggc atgtccaccg tggaatacgc catcggtacc atcgcggcgg 180 ctgcnttcgg tgcgatcctc tacacggtcg tcaccgggga ttccattgtg tcggcgctca 240 accgcatcat cggtcgcgcg ctcagcacca aggtttagcg tcgtgtgcgg gtgcgagcac 300 cgtggaagcg gcgttggcga tcgccaccct ggtgctggtg ctggtgctgt gcctggcggg 360 cgtcaccgcg gtatcaatgc aggtgcgctg tatcgacgcg gcccgcgagg ccgctcgatt 420 ggccgcgcgc g 431 112 143 PRT Mycobacterium tuberculosis 112 Ala Gly Gly Cys Gly Ala Cys Gly Gly Thr Thr Gly Gly Cys Ala Cys 1 5 10 15 Cys Ala Cys Cys Ala Cys Gly Ala Gly Thr Gly Ala Gly Thr Cys Ala 20 25 30 Gly Gly Cys Ala Gly Gly Ala Gly Cys Cys Cys Cys Gly Cys Cys Ala 35 40 45 Cys Gly Thr Thr Gly Cys Gly Gly Ala Cys Gly Gly Cys Gly Cys Gly 50 55 60 Ala Ala Thr Cys Thr Thr Cys Gly Cys Cys Thr Gly Thr Gly Gly Cys 65 70 75 80 Thr Cys Ala Cys Cys Gly Ala Cys Thr Thr Thr Cys Cys Gly Cys Cys 85 90 95 Cys Ala Ala Cys Thr Thr Cys Ala Ala Cys Gly Ala Thr Cys Thr Thr 100 105 110 Gly Cys Ala Cys Ala Thr Gly Gly Ala Cys Gly Gly Cys Ala Ala Gly 115 120 125 Ala Ala Cys Gly Cys Cys Gly Ala Gly Gly Thr Cys Gly Cys Gly 130 135 140 113 363 PRT Mycobacterium tuberculosis 113 Met Thr Pro Arg Gly Pro Gly Arg Leu Gln Arg Leu Ser Gln Cys Arg 1 5 10 15 Pro Gln Arg Gly Ser Gly Gly Pro Ala Arg Gly Leu Arg Gln Leu Ala 20 25 30 Leu Ala Ala Met Leu Gly Ala Leu Ala Val Thr Val Ser Gly Cys Ser 35 40 45 Trp Ser Glu Ala Leu Gly Ile Gly Trp Pro Glu Gly Ile Thr Pro Glu 50 55 60 Ala His Leu Asn Arg Glu Leu Trp Ile Gly Ala Val Ile Ala Ser Leu 65 70 75 80 Ala Val Gly Val Ile Val Trp Gly Leu Ile Phe Trp Ser Ala Val Phe 85 90 95 His Arg Lys Lys Asn Thr Asp Thr Glu Leu Pro Arg Gln Phe Gly Tyr 100 105 110 Asn Met Pro Leu Glu Leu Val Leu Thr Val Ile Pro Phe Leu Ile Ile 115 120 125 Ser Val Leu Phe Tyr Phe Thr Val Val Val Gln Glu Lys Met Leu Gln 130 135 140 Ile Ala Lys Asp Pro Glu Val Val Ile Asp Ile Thr Ser Phe Gln Trp 145 150 155 160 Asn Trp Lys Phe Gly Tyr Gln Arg Val Asn Phe Lys Asp Gly Thr Leu 165 170 175 Thr Tyr Asp Gly Ala Asp Pro Glu Arg Lys Arg Ala Met Val Ser Lys 180 185 190 Pro Glu Gly Lys Asp Lys Tyr Gly Glu Glu Leu Val Gly Pro Val Arg 195 200 205 Gly Leu Asn Thr Glu Asp Arg Thr Tyr Leu Asn Phe Asp Lys Val Glu 210 215 220 Thr Leu Gly Thr Ser Thr Glu Ile Pro Val Leu Val Leu Pro Ser Gly 225 230 235 240 Lys Arg Ile Glu Phe Gln Met Ala Ser Ala Asp Val Ile His Ala Phe 245 250 255 Trp Val Pro Glu Phe Leu Phe Lys Arg Asp Val Met Pro Asn Pro Val 260 265 270 Ala Asn Asn Ser Val Asn Val Phe Gln Ile Glu Glu Ile Thr Lys Thr 275 280 285 Gly Ala Phe Val Gly His Cys Ala Glu Met Cys Gly Thr Tyr His Ser 290 295 300 Met Met Asn Phe Glu Val Arg Val Val Thr Pro Asn Asp Phe Lys Ala 305 310 315 320 Tyr Leu Gln Gln Arg Ile Asp Gly Asn Thr Asn Ala Glu Ala Leu Arg 325 330 335 Ala Ile Asn Gln Pro Pro Leu Ala Val Thr Thr His Pro Phe Asp Thr 340 345 350 Arg Arg Gly Glu Leu Ala Pro Gln Pro Val Gly 355 360 114 709 DNA Mycobacterium tuberculosis 114 ggatccgcgg agtatatctc caacgtaata tatgaaggtc cgcgtgctga ctcattgtat 60 gccgccgacc agcgattgcg acaattagct gactcagtta gaacgactgc cgagtcgctc 120 aacaccacgc tcgacgagct gcacgagaac tggaaaggta gttcatcgga atggatggcc 180 gacgcggctt tgcggtatct cgactggctg tctaaacact cccgtcagat tttgcgaacc 240 gcccgcgtga tcgaatccct cgtaatggcc tatgaggaga cacttctgag ggtggtaccc 300 ccggcgacta tcgccaacaa ccgcgaggag gtgcgcaggc tgatcgcgag caacgtggcc 360 gggggtaaac actccagcaa tcgcagacct cgaggcacaa tacgagcagt accgggccga 420 aaatatccaa gcaatggacc gctatctaag ttggacccga tttgcgctat cgaagctgcc 480 ccgatggcgg gagccgccgc agatccacag gagcgggtag gtccaagagg ccggcgcggt 540 cttgcaggcc agcaacaatg ccgcggtcga ccaggcccat cgcttcgctg ctcgcacgac 600 acaccgcggt ttcagatgaa tcaggcgttt cacaccatgg tgaacatgtt gctgacgtgt 660 tttgcatgtc aggagaaacc gagatgacga tcaacaacca ggtaagctt 709 115 1831 DNA Mycobacterium tuberculosis 115 ggatccccgg ctaccatgcc ttcgtcgcgc aacctcgcga ccaaccccga gatcgccacc 60 ggctaccgcc gggacatgac cgtggtgcgg accgcccact atgcggcagc caccgccaat 120 ccgctggcca ctcaggtggc ctgccgagta ttgcgcgacg gtggtaccgc cgccgatgcc 180 gtcgtggccg cccaggcggt gctggggttg gtcgaaccgc aatcctccgg gatcggcggc 240 ggcggatatc tggtgtactt cgacgcccgc acgggctcag tgcaggccta cgacggccgt 300 gaggtggccc cagcggccgc caccgagaac taccttcgct gggtcagcga cgtcgaccgc 360 agcgcgccca ggcccaacgc ccgagcctcg ggacggtcga tcggagtacc gggcatcctg 420 cgaatgctgg agatggtgca caacgagcac gggcgcacac cctggcgcga cctcttcggc 480 cccgcggtaa cgctggccga tggcggtttt gacatcagcg ccaggatggg cgcggccatc 540 tccgacgctg cgccgcaact gcgagacgac ccggaggctc gcaagtattt cctcaatccc 600 gacggcagcc cgaaacccgc gggaacccgg ctgacgaacc ccgcgtactc aaaaaccctg 660 tccgccatcg cctccgccgg cgccaacgcc ttctattccg gcgacattgc ccacgacatc 720 gtggcggcgg cgagcgacac atcgaatggc cgcacgccgg gcctgttgac cattgaggac 780 ctggcgggtt acctcgccaa gagacgccaa ccgttgtgca cgacctatcg cggccgggag 840 atctgcggca tgccatcgtc gggtggcgtc gccgtggccg caaccttggg catcctcgag 900 cacttcccga tgagcgacta cgcgcccagc aaggtcgacc tcaacggcgg tcgcccgacc 960 gtgatggggg ttcacctgat agcggaggcc gaacggctgg cctatgccga ccgcgaccaa 1020 tatatcgctg acgtcgattt tgtccggctg cccggcggct cgctcaccac gctggttgac 1080 ccgggctact tggcagcacg cgccgcgcta atctcgccgc aacacagcat gggcagcgcc 1140 agaccggggg acttcggcgc accgacggcc gtcgccccgc cagtgcctga gcatggcacc 1200 agccacctca gcgtcgtcga ttcgtacggc aatgcggcca cgttgacgac gacggtggaa 1260 tcttcgttcg gctcctacca cctggtggac ggattcatcc tcaacaacca gctgagcgat 1320 ttcagcgccg agccacacgc tactgacgga tcaccggtgg ctaaccgggt cgagcctggg 1380 aagcgaccgc gcagttcgat ggcaccgacg ttggtgttcg atcactcgtc ggcggggcgc 1440 ggtgcgctgt acgcggtgct cggttctccg ggcggctcca tgatcatcca gttcgtcgtg 1500 aaaacacttg tggcgatgct ggattggggt ctgaatccgc agcaggcggt ttccctggtc 1560 gatttcggcg ccgcgaactc gccgcacact aacctcggcg gtgagaatcc cgagatcaac 1620 acttccgacg atggtgatca tgacccgctg gtgcaaggcc tgcgcgcgct ggggcatcga 1680 gttaatcttg ccgagcaatc cagtgggctc tcggcgatca cccgcagcga ggcgggttgg 1740 gccggcggcg ccgacccacg ccgcgaaggc gcggtcatgg gcgacgatgc ctgagccgtt 1800 cgccggcggg cggccaaacg aacgcggatc c 1831 116 516 DNA Mycobacterium tuberculosis 116 gaattcaagc gcggtgtcgc aacgctgccg gtgatcctgg tgattctgct ctcggtggcg 60 gccggggccg gtgcatggct gctagtacgc ggacacggtc cgcagcaacc cgagatcagc 120 gcttactcgc acgggcacct gacccgcgtg gggccctatt tgtactgcaa cgtggtcgac 180 ctcgacgact gtcagacccc gcaggcgcag ggcgaattgc cggtaagcga acgctatccc 240 gtgcagctct cggtacccga agtcatttcc cgggcgccgt ggcgtttgct gcaggtatac 300 caggaccccg ccaacaccac cagcaccttg tttcggccgg acacccggtt ggcggtcacc 360 atccccactg tcgacccgca gcgcgggcgg ctgaccggga ttgtcgtgca gttgctgacg 420 ttggtggtcg accactcggg tgaactacgc gacgttccgc acgcggaatg gtcggtgcgc 480 cttatctttt gacgaggccg cggctcgacg aagctt 516 117 2831 DNA Mycobacterium tuberculosis 117 gaattcgcgc cgatgctgga cgcggcggcc gcttgggatg gactggccga cgaattgggt 60 tcggccgcgg cctcgttttc ggcggtgacg gcggggctgg caggttcctc gtggctgggc 120 gcggcgtcga cggcgatgac gggagcggcc gccccctatc tgggctggtt gagcgcggcg 180 gcggcgcagg cccagcaggc ggccacccaa acccggctgg cggcggccgc cttcgaggca 240 gccctggcgg cgacggtaca tccggcgatc atctcggcca accgggcact gttcgtgtcg 300 ctggtggtct cgaacctgct gggccaaaac gccccggcga tcgcggccac cgaggccgcc 360 tacgagcaga tgtgggccca ggacgtggcg gcgatgtttg gctaccatgc cggggcttcg 420 gcggccgtct cggcgttgac accgttcggc caggcgctgc cgaccgtggc gggcggcggt 480 gcgctggtca gcgcggccgc ggctcaggtg accacgcggg tcttccgcaa cctgggcttg 540 gcgaacgtcg gcgagggcaa cgtcggcaac ggtaatgtcg ggaacttcaa tctcggctcg 600 gccaacatcg gcaacggcaa catcggcagc ggcaacatcg gcagctccaa catcgggttt 660 ggcaacgtgg gtcctgggtt gaccgcagcg ctgaacaaca tcggtttcgg caacaccggc 720 agcaacaaca tcgggtttgg caacaccggc agcaacaaca tcggcttcgg caataccgga 780 gacggcaacc gaggtatcgg gctcacgggt agcggtttgt tggggttcgg cggcctgaac 840 tcgggcaccg gcaacatcgg tctgttcaac tcgggcaccg gaaacgtcgg catcggcaac 900 tcgggtaccg ggaactgggg cattggcaac tcgggcaaca gctacaacac cggttttggc 960 aactccggcg acgccaacac gggcttcttc aactccggaa tagccaacac cggcgtcggc 1020 aacgccggca actacaacac cggtagctac aacccgggca acagcaatac cggcggcttc 1080 aacatgggcc agtacaacac gggctacctg aacagcggca actacaacac cggcttggca 1140 aactccggca atgtcaacac cggcgccttc attactggca acttcaacaa cggcttcttg 1200 tggcgcggcg accaccaagg cctgattttc gggagccccg gcttcttcaa ctcgaccagt 1260 gcgccgtcgt cgggattctt caacagcggt gccggtagcg cgtccggctt cctgaactcc 1320 ggtgccaaca attctggctt cttcaactct tcgtcggggg ccatcggtaa ctccggcctg 1380 gcaaacgcgg gcgtgctggt atcgggcgtg atcaactcgg gcaacaccgt atcgggtttg 1440 ttcaacatga gcctggtggc catcacaacg ccggccttga tctcgggctt cttcaacacc 1500 ggaagcaaca tgtcgggatt tttcggtggc ccaccggtct tcaatctcgg cctggcaaac 1560 cggggcgtcg tgaacattct cggcaacgcc aacatcggca attacaacat tctcggcagc 1620 ggaaacgtcg gtgacttcaa catccttggc agcggcaacc tcggcagcca aaacatcttg 1680 ggcagcggca acgtcggcag cttcaatatc ggcagtggaa acatcggagt attcaatgtc 1740 ggttccggaa gcctgggaaa ctacaacatc ggatccggaa acctcgggat ctacaacatc 1800 ggttttggaa acgtcggcga ctacaacgtc ggcttcggga acgcgggcga cttcaaccaa 1860 ggctttgcca acaccggcaa caacaacatc gggttcgcca acaccggcaa caacaacatc 1920 ggcatcgggc tgtccggcga caaccagcag ggcttcaata ttgctagcgg ctggaactcg 1980 ggcaccggca acagcggcct gttcaattcg ggcaccaata acgttggcat cttcaacgcg 2040 ggcaccggaa acgtcggcat cgcaaactcg ggcaccggga actggggtat cgggaacccg 2100 ggtaccgaca ataccggcat cctcaatgct ggcagctaca acacgggcat cctcaacgcc 2160 ggcgacttca acacgggctt ctacaacacg ggcagctaca acaccggcgg cttcaacgtc 2220 ggtaacacca acaccggcaa cttcaacgtg ggtgacacca ataccggcag ctataacccg 2280 ggtgacacca acaccggctt cttcaatccc ggcaacgtca ataccggcgc tttcgacacg 2340 ggcgacttca acaatggctt cttggtggcg ggcgataacc agggccagat tgccatcgat 2400 ctctcggtca ccactccatt catccccata aacgagcaga tggtcattga cgtacacaac 2460 gtaatgacct tcggcggcaa catgatcacg gtcaccgagg cctcgaccgt tttcccccaa 2520 accttctatc tgagcggttt gttcttcttc ggcccggtca atctcagcgc atccacgctg 2580 accgttccga cgatcaccct caccatcggc ggaccgacgg tgaccgtccc catcagcatt 2640 gtcggtgctc tggagagccg cacgattacc ttcctcaaga tcgatccggc gccgggcatc 2700 ggaaattcga ccaccaaccc ctcgtccggc ttcttcaact cgggcaccgg tggcacatct 2760 ggcttccaaa acgtcggcgg cggcagttca ggcgtctgga acagtggttt gagcagcaag 2820 cttgggaatt c 2831 118 1823 DNA Mycobacterium tuberculosis 118 gaattcgctg accgtggcca gcgacgaggc tgcgccccgg gcatcgcgtc tgcgctcagg 60 gcgtcgtttc aggggaaatc cagaccctgg acgcagactc gatattgggc tttcgcgtta 120 ttaacaccgc tcgtcgtggc tatggtgctc accggatgct cggcctccgg tacccaactc 180 gaactcgcgc ccactgcgga ccgcagggcc gcggttggca ccaccagcga catcaatcag 240 caggatcccg ccacgttgca agacggcggc aatcttcgcc tgtcgctcac cgactttccg 300 cccaacttca acatcttgca catcgacggc aacaacgccg aggtcgcggc gatgatgaaa 360 gccaccttgc cgcgcgcgtt catcatcgga ccggacggct cgacgacggt cgacaccaac 420 tacttcacca gcatcgagct gaccaggacc gccccgcagg tggtcaccta caccatcaat 480 cccgaggcgg tgtggtccga cgggaccccg atcacctggc gggacatcgc cagccagatt 540 catgcgatca gcggcgccga caaggcattc gagatcgctt ctagcagcgg cgccgagcgt 600 gtggcgtcgg taaccagagg ggtcgacgac cggcaggccg tggtgacgtt cgccaagccg 660 tacgcggagt ggcgcggtat gttcgcgggc aacggcatgc tgctgccggc cagtatgacc 720 gccacacccg aggcattcaa taagggtcaa ctcgatgggc ccggtccgtc ggcgggtccg 780 ttcgtcgtgt ctgccctgga ccgcaccgcg cagcgaatcg tgttgacccg taacccgaga 840 tggtgggggg cacggccacg cctggacagc atcacatacc tggtgctcga tgatgccgcc 900 cggctgccgg cgctgcagaa caacacaatc gacgccaccg gcgtcggcac actggaccag 960 ctgaccatcg cggcgcgcac caagggcatc tcgatccggc gcgcccccgg gcccagctgg 1020 tatcacttca ccctcaacgg tgcgcctggg tcgatcctcg ccgacaaggc gctgcgcctg 1080 gcgatcgcca agggcatcga ccgatacacc atcgccaggg tcgcccaata cggcctcacc 1140 agcgacccgg tgccactgaa caaccacgtc ttcgtcgccg gccaagacgg ctaccaggac 1200 aacagcggcg ttgtcgccta caacccggaa caagcgaaac gggagctgga cgccctgggc 1260 tggaggcgaa gcggcgcgtt ccgggagaag gacggtcgcc agctcgtcat ccgcgatctg 1320 ttctacgacg cacaaagcac ccggcagttc gcccagatcg cccaacacac cctggcgcag 1380 atcggcgtca aactcgaact tcaggccaag tccggcagcg gtttcttcag cgactacgtc 1440 aacgtggggg ctttcgacat cgcacagttc ggctgggtgg gcgacgcgtt tccgctgtca 1500 tcgctcaccc agatctacgc ttcggacggg gaaagcaact tcggcaagat cggtagcccg 1560 caaatcgacg ccgcgatcga gcgaacgctg gcagaactcg atcccggcaa ggcgagggcc 1620 ttggccaacc aggtcgacga gctgatctgg gccgaaggat tcagcctgcc gcttacccag 1680 tcgcccggca ccgttgcggt ccgcagcacg ctggccaact tcggcgcgac gggtctggca 1740 gacctggact acaccgccat cgggttcatg cgacgctgag ccggcggcga ccagctcagc 1800 tgaagcttaa gtcggcggaa ttc 1823 119 886 DNA Mycobacterium tuberculosis 119 gaattcatct cacaagcgtg cggctcccac cgaccccggc gcccctcgag cctgggggct 60 gtcgcgatcc tgatcgcggc gacacttttc gcgactgtcg ttgcggggtg cgggaaaaaa 120 ccgaccacgg cgagctcccc gagtcccggg tcgccgtcgc cggaagccca gcagatcctg 180 caagacagtt ccaaggcgac gaagggcctg cattccgtcc acgtggtggt gacggtaaac 240 aatctctcga ccctcccgtt tgagagcgtc gatgccgacg tgaccaacca accgcagggc 300 aatggccagg cggtgggcaa cgccaaggtc agaatgaagc ccaacacccc ggtggtggcc 360 accgagttcc tggtcacgaa caagaccatg tacacgaagc ggggcggcga ctatgtctcg 420 gtgggtccgg cggagaagat ctatgacccg ggcatcatcc tggacaagga ccgggggctg 480 ggcgcggtcg tcgggcaagt gcaaaacccg acaatccagg gacgtgacgc catcgacggc 540 ctggccaccg tcaaggtgtc cgggaccatc gacgccgcgg tgatcgatcc gatcgtgcct 600 cagctaggta agggtggggg caggctcccg ataaccttgt ggatcgtcga caccaacgcc 660 tcaacgccgg cacccgccgc gaacctggtg cggatggtca ttgacaagga ccaaggcaac 720 gtcgacatca cgctgtccaa ttggggtgcg ccggtcacca tcccgaaccc ggcgggataa 780 caggcgcgaa ccggcccggt ccagccccat cgctggtcga tggcctggcc ggtccggtac 840 tcgtccgcgg gcggaggccg ccttcgaaga aatcctttga gaattc 886 120 1020 DNA Mycobacterium tuberculosis 120 gaattcatga tccagatcgc gcgcacctgg cgggtcttcg caggcggcat ggccaccggt 60 ttcatcggcg tggtgctggt caccgccggg aaggcctcag cggatcccct gctgccaccg 120 ccgcctatcc ctgccccagt ctcggcgccg gcaacagtcc cgcccgtgca gaacctcacg 180 gcgcttccgg gcgggagcag caacaggttc tcaccggcgc cagcacccgc accgatcgcg 240 tcgccgattc cggtcggagc acccgggtcc accgctgtgc ccccgctgcc gccgccagtg 300 actcccgcga tcagcggcac acttcgggac cacctccggg agaagggcgt caagctggag 360 gcacagcgac cgcacggatt caaggcgctc gacatcacac tgcccatgcc gccgcgctgg 420 actcaggtgc ccgaccccaa cgtgcccgac gcgttcgtgg tgatcgccga ccggttgggc 480 aacagcgtct acacgtcgaa tgcgcagctg gtggtgtata ggctgatcgg tgacttcgat 540 cccgctgagg ccatcacaca cggctacatt gacagccaga aattgctcgc atggcagacc 600 acaaacgcct cgatggccaa tttcgacggc tttccgtcat caatcatcga gggcacctac 660 cgcgaaaacg acatgaccct caacacctcc cggcgccacg tcatcgccac ctccggagcc 720 gacaagtacc tggtttcgct gtcggtgacc accgcgctgt cgcaggcggt caccgacggg 780 ccggccaccg atgcgattgt caacggattc caagtggttg cgcatgcggc gcccgctcag 840 gcgcctgccc cggcacccgg ttcggcaccg gtgggactac ccgggcaggc gcctgggtat 900 ccgcccgcgg gcaccctgac accagtcccg ccgcgctagg tcgcgatgag gccgagcaga 960 aacacgggcc cgcatggagc tcggtgagcg gattcgtcgg cggcctcgaa gcttgaattc 1020 121 1354 DNA Mycobacterium tuberculosis 121 gaattcacgc ggagccgggg attgcgctac gccacggtga tcgcgctggt ggccgcgctg 60 gtgggcggcg tgtacgtgct ctcgtccacc ggtaataagc gcaccatcgt gggctacttc 120 acctctgctg tcgggctcta tcccggtgac caggtccgcg tcctgggcgt cccggtgggt 180 gagatcgaca tgatcgagcc gcggtcgtcc gacgtcaaga tcactatgtc ggtgtccaag 240 gacgtcaagg tgcccgtgga cgtgcaggcc gtgatcatgt cgccgaattt ggtggcggcg 300 cgcttcattc agctcacccc ggtgtatacc ggcggggcgg tactgcccga caacggtcgg 360 atcgatctgg atcgcaccgc ggtgccggtg gaatgggacg aggtgaaaga ggggctcacc 420 cggttggccg ccgacctgag tccggcggcg ggcgagctgc aggggccgct gggcgcggcg 480 atcaaccagg ccgcggacac ccttgacggc aacggagact cgttacacaa cgcgttgcgc 540 gagcttgcgc aggtcgccgg gcggctgggg gattcgcgcg gcgacatctt cggcaccgtc 600 aagaacctgc aggtactggt cgacgcgcta tcggagagcg acgagcagat tgtgcagttc 660 gccggccacg tggcatcggt gtcgcaggtg ctcgccgaca gctcggccaa tctggaccag 720 accctgggca cgctcaacca ggcgctgtcc gacatcaggg ggttcttgcg cgagaacaac 780 tcgacgctga tcgaaacggt gaatcagctc aacgactttg cgcagacgtt gagtgaccag 840 agcgagaaca tcgagcaagt gctgcacgtg gctgggccgg ggatcaccaa cttctacaac 900 atctatgacc ctgcgcaagg caccctcaac ggtctgttgt cgatacccaa cttcgctaac 960 ccggtgcagt tcatctgcgg cggttccttc gataccgccg cgggcccgtc ggcgccggac 1020 tactaccggc gcgccgagat ctgccgtgag cggctggggc cggtgctgcg ccggctcacg 1080 gtgaattacc cgccgatcat gttccacccg cttaacacga tcacggcgta caagggccag 1140 atcatctacg acaccccggc caccgaggcc aagtcggaga cgccggttcc ggaattgact 1200 tgggtacccg cgggaggagg agcgcctgtg ggcaaccccg cggatctgca gagcctactc 1260 gtcccaccgg cgcccggtcc ggcaccggcc ccgccggcgc cgggggcagg accgggcgag 1320 catgggggcg gcggatgaac cgaagcttga attc 1354 122 1326 DNA Mycobacterium tuberculosis 122 ggcatggacc cgctgaaccg ccgacaattc ctcgcgctgg ccgctgccgc cgccggcgtg 60 accgccggct gcgccgggat gggcggcggc ggttcggtga agtccggttc cggcccaatc 120 gacttctggt ccagtcatcc cggccaatcc agcgcggcgg aacgggagct gatcggtcgt 180 ttccaggacc gattccccac tctgtcggtc aagctgatcg acgccggcaa ggactacgac 240 gaggtggcac agaaattcaa tgcggcgctc atcggaaccg acgtgcccga cgtcgttttg 300 ctcgacgacc gatggtggtt ccatttcgcc ctcagcggtg ttctcactgc ccttgacgac 360 ctgttcggcc aagttggggt ggacacaacg gattacgtcg attcgctgct ggccgactat 420 gagttcaacg gccgccatta cgctgtgccg tatgctcgct cgacgccgct gttctactac 480 aacaaggcgg cgtggcaaca ggccggccta cccgaccgcg gaccgcaatc ctggtcagag 540 ttcgacgagt ggggtccgga gttacagcgc gtggtcggcg ccggtcgatc ggcgcacggc 600 tgggctaacg ccgacctcat ctcgtggacg tttcagggac cgaactgggc attcggcggt 660 gcctactccg acaagtggac attgacattg accgagcccg ccacgatcgc ggccggcaac 720 ttctatcgga actccatcca tggcaagggt tatgcggcgg tcgccaacga tattgccaac 780 gagttcgcca ccggaatcct ggcctcggcc gtggcatcca ccggctcgct ggccggcatc 840 accgcatctg cccgattcga cttcggcgcc gcaccgctgc ccacgggccc ggacgcagcg 900 cccgcctgtc cgacgggcgg tgcggggctg gcgataccgg ccaagctctc cgaggagcga 960 aaagtcaacg cgctcaagtt catcgcattc gtcaccaacc cgacgaacac cgcctacttc 1020 agccagcaaa ccggctatct gccggtgcgc aagtccgccg tcgacgatgc cagcgaacgg 1080 cactatctgg cggacaatcc ccgtgcgcgg gtggcgctcg accagctgcc acacacccgg 1140 acacaagact acgcacgggt tttcctgccc ggtggtgacc ggatcatctc cgccggcctg 1200 gaatccatcg ggctgcgcgg agccgacgtg accaagacct tcacgaacat ccaaaaacgg 1260 ttgcaggtca tcctggatcg gcagatcatg cggaagctgg cggggcatgg ctaacgttca 1320 ggatcc 1326 123 629 DNA Mycobacterium tuberculosis 123 ggatccgtag cggtgcggcg taaggtgcgg aggttgactc tggcggtgtc ggcgttggtg 60 gctttgttcc cggcggtcgc ggggtgctcc gattccggcg acaacaaacc gggagcgacg 120 atcccgtcga caccggcaaa cgctgagggc cggcacggac ccttcttccc gcaatgtggc 180 ggcgtcagcg atcagacggt gaccgagctg acaagggtga ccgggctggt caacaccgcc 240 aagaattcgg tgggctgcca atggctggcg ggcggcggta tcttgggccc gcacttctcc 300 ttctcctggt accgcggcag cccgatcggg cgggaacgca agaccgagga gttgtcgcgc 360 gcgagtgtcg aggacatcaa catcgacggc cacagcggtt tcatcgccat cggtaacgag 420 cccagtttgg gtgactcact gtgtgaagtc ggaatccagt tctccgacga cttcatcgaa 480 tggtcggtga gtttcagcca gaagccgttc ccgctgccgt gcgacatcgc caaagaactg 540 acccgccaat cgattgcgaa ttcgaaatga gacgtgtcct ggtcggtgcg gccgccttga 600 tcaccgcaaa gcttgtcttg accggatcc 629 124 692 DNA Mycobacterium tuberculosis 124 ggatccatcg ccatgcaact ctcctcccgg ttggaaaatc atcgcaagcc cttcccccgg 60 acggtatcga cagggcaggc tatcgccatg gcgaagcgca ccccggtccg gaaggcctgc 120 acagttctag ccgtgctcgc cgcgacgcta ctcctcggcg cctgcggcgg tcccacgcag 180 ccacgcagca tcaccttgac ctttatccgc aacgcgcaat cccaggccaa cgccgacggg 240 atcatcgaca ccgacatgcc cggttccggc ctcagcgccg acggcaaagc agaggcgcag 300 caggtcgcgc accaggtttc ccgcagagat gtcgacagca tctattcctc ccccatggcg 360 gccgaccagc agaccgccgg gccgttggcc ggcgaacttg gcaagcaagt cgagattctt 420 ccgggcctgc aagcgatcaa cgccggctgg ttcaacggca aacccgaatc aatggccaac 480 tcaacatata tgctggcacc ggcagactgg ctggccggcg atgttcacaa cactattccg 540 gggtcgatca gcggcaccga attcaattcc cagttcagcg ccgccgtccg caagatctac 600 gacagcggcc acaatacgcc ggtcgtgttc tcgcaggggg tagcgatcat gatctggacg 660 ctgatgaacg cacgaaactc tagggaaagc tt 692 125 1004 DNA Mycobacterium tuberculosis 125 ggatccgggg gtgctgggat gacggaacac aacgaggacc cacagatcga gcgcgtggcc 60 gacgacgccg ccgacgagga ggcggttacg gagccgttgg ccaccgaatc gaaggacgaa 120 ccggccgagc acccagaatt cgaagggccg cgtcggcgcg cccgccgcga acgtgccgaa 180 cgtcgcgccg cgcaggctcg agctaccgcg atcgagcagg ctcgccgcgc ggccaaacgg 240 cgagcccgcg ggcagatcgt cagtgagcag aaccccgcca aaccggccgc ccgaggtgtt 300 gttcgagggc tgaaggcgct gctcgcgacg gtcgtgctgg ccgtcgtcgg gatcgggctt 360 gggctcgcgc tgtacttcac gccggcgatg tcggcccgcg agatcgtgat catcgggatc 420 ggggcggtga gccgcgagga ggttctcgac gccgccagag tgcggccggc aacgccgttg 480 ctgcagatcg acacccaaca ggttgctgac cgagtggcca cgatccggcg ggtggccagt 540 gcgcgggtgc agcggcagta cccgtcggcc ttgcggatca ccatcgtcga gcgggtcccg 600 gtggtggtca aggatttttc ggacggcccg cacctttttg accgcgacgg cgtcgacttc 660 gcgaccgatc cgccaccgcc ggcgttgcct tatttcgatg tggacaatcc cggtcctagc 720 gatccgacga ccaaggcggc gctgcaggtg ttgaccgcgc tgcatcctga agttgcaagc 780 caggtggggc ggatcgcggc cccgtcggtg gcctcgatca ccctgacgtt ggccgatggc 840 cgcgtggtga tctggggaac caccgaccgc tgcgaagaga aggccgaaaa gctggcggcg 900 ctgttgaccc agccaggcag aacgtacgac gtgtccagcc ccgacctgcc gaccgtgaaa 960 tagccgaaaa aatgcccgcc gcgtatcggc gcgcctgcaa gctt 1004 126 1624 DNA Mycobacterium tuberculosis 126 ggatcccaga tgaatgtcgt cgacatttcg cggtggcagt tcggtatcac caccgtctat 60 cacttcattt tcgtaccgct gaccatcggc ctggccccgc tgatcgcggt catgcaaacg 120 ctgtgggtcg tcaccgataa ccccgcctgg tatcgcctca ccaaattctt cggcaaattg 180 ttcctgatca actttgccat cggcgtggcg accggaatcg tgcaggaatt tcagttcggc 240 atgaactgga gcgagtactc ccgattcgtc ggcgatgtct tcggcgcccc gctggccatg 300 gagggcctgg cggccttctt cttcgaatcc accttcatcg ggttgtggat cttcggctgg 360 aacaggctgc cccggctggt gcatctggcc tgcatctgga tcgtcgcaat cgcggtcaac 420 gtgtccgcgt tcttcatcat cgcggcaaac tccttcatgc agcatccggt cggcgcgcac 480 tacaacccga ccaccgggcg tgccgagttg agcagcatcg tcgtgctgct gaccaacaac 540 accgcacagg cggcgtttac ccacactgtc agcggtgcgc tgctgaccgc cgggaccttc 600 gtcgccgcgg tgagcgcctg gtggctggtc cgttcgagca ccacgcacgc cgactcagat 660 acccaagcca tgtatcgtcc cgcgaccatc ctggggtgtt gggttgcgtt ggccgccacg 720 gccgggttgt tgttcaccgg cgaccaccaa ggcaagctga tgttccagca gcagccgatg 780 aagatggcgt cggccgaatc gttgtgcgat acccagacag atccaaactt ctctgtcctg 840 acggtcggcc ggcaaaacaa ctgcgacagc ctcacccgtg tcatcgaagt gccctatgtg 900 ttgccgttcc tcgccgaggg ccggatcagc ggtgtgacgt tgcagggtat ccgcgatctg 960 cagcaggaat accagcagcg cttcggacca aacgactacc ggcccaacct cttcgtcacc 1020 tactggtcat ttcgcatgat gatcgggttg atggcgatcc cggtgctgtt cgcactgatt 1080 gcgctctggc tcacccgtgg cggccagatc cccaatcaac gctggttctc ctggctggcg 1140 ctgctaacca tgcccgcccc gttcctggcc aacagcgccg gatgggtgtt caccgagatg 1200 gggcgccagc cctgggtcgt cgtccctaac ccgaccggtg atcagctggt tcgactcacc 1260 gtcaaagcag gcgtctcgga tcactccgcc accgtggtcg ccacgtcttt gctgatgttc 1320 accttggtct acgcggtact tgcggtcatc tggtgctggc tgctcaagcg ttacatcgtc 1380 gaaggccccc tggaacacga cgcggaaccg gctgcgcacg gggcaccccg cgacgacgag 1440 gtagcaccat tgtcgtttgc ttactgaggc caactgaccc cggaaaggag cagccggtgg 1500 tactccaaga attgtggttc ggtgtcatcg cagcgctgtt cctcggtttc ttcatcctag 1560 aagggttcga cttcggcgtg ggcatgctga tggcgccgtt cgctcatgtc ggtatggggg 1620 atcc 1624 127 1414 DNA Mycobacterium tuberculosis 127 gaattccaag gcgtcgatcg gcacggtgtt ccaggaccgg gccgctcgct acggtgaccg 60 agtcttcctg aaattcggcg atcagcagct gacctaccgc gacgctaacg ccaccgccaa 120 ccggtacgcc gcggtgttgg ccgcccgcgg cgtcggcccc ggcgacgtcg ttggcatcat 180 gttgcgtaac tcacccagca cagtcttggc gatgctggcc acggtcaagt gcggcgctat 240 cgccggcatg ctcaactacc accagcgcgg cgaggtgttg gcgcacagcc tgggtctgct 300 ggacgcgaag gtactgatcg cagagtccga cttggtcagc gccgtcgccg aatgcggcgc 360 ctcgcgcggc cgggtagcgg gcgacgtgct gaccgtcgag gacgtggagc gattcgccac 420 aacggcgccc gccaccaacc cggcgtcggc gtcggcggtg caagccaaag acaccgcgtt 480 ctacatcttc acctcgggca ccaccggatt tcccaaggcc agtgtcatga cgcatcatcg 540 gtggctgcgg gcgctggccg tcttcggagg gatggggctg cggctgaagg gttccgacac 600 gctctacagc tgcctgccgc tgtaccacaa caacgcgtta acggtcgcgg tgtcgtcggt 660 gatcaattct ggggcgaccc tggcgctggg taagtcgttt tcggcgtcgc ggttctggga 720 tgaggtgatt gccaaccggg cgacggcgtt cgtctacatc ggcgaaatct gccgttatct 780 gctcaaccag ccggccaagc cgaccgaccg tgcccaccag gtgcgggtga tctgcggtaa 840 cgggctgcgg ccggagatct gggatgagtt caccacccgc ttcggggtcg cgcgggtgtg 900 cgagttctac gccgccagcg aaggcaactc ggcctttatc aacatcttca acgtgcccag 960 gaccgccggg gtatcgccga tgccgcttgc ctttgtggaa tacgacctgg acaccggcga 1020 tccgctgcgg gatgcgagcg ggcgagtgcg tcgggtaccc gacggtgaac ccggcctgtt 1080 gcttagccgg gtcaaccggc tgcagccgtt cgacggctac accgacccgg ttgccagcga 1140 aaagaagttg gtgcgcaacg cttttcgaga tggcgactgt tggttcaaca ccggtgacgt 1200 gatgagcccg cagggcatgg gccatgccgc cttcgtcgat cggctgggcg acaccttccg 1260 ctggaagggc gagaatgtcg ccaccactca ggtcgaagcg gcactggcct ccgaccagac 1320 cgtcgaggag tgcacggtct acggcgtcca gattccgcgc accggcgggc gcgccggaat 1380 ggccgcgatc acactgcgcg ctggcgccga attc 1414 128 228 PRT Mycobacterium tuberculosis 128 Gly Ser Ala Glu Tyr Ile Ser Asn Val Ile Tyr Glu Gly Pro Arg Ala 1 5 10 15 Asp Ser Leu Tyr Ala Ala Asp Gln Arg Leu Arg Gln Leu Ala Asp Ser 20 25 30 Val Arg Thr Thr Ala Glu Ser Leu Asn Thr Thr Leu Asp Glu Leu His 35 40 45 Glu Asn Trp Lys Gly Ser Ser Ser Glu Trp Met Ala Asp Ala Ala Leu 50 55 60 Arg Tyr Leu Asp Trp Leu Ser Lys His Ser Arg Gln Ile Leu Arg Thr 65 70 75 80 Ala Arg Val Ile Glu Ser Leu Val Met Ala Tyr Glu Glu Thr Leu Leu 85 90 95 Arg Val Val Pro Pro Ala Thr Ile Ala Asn Asn Arg Glu Glu Val Arg 100 105 110 Arg Leu Ile Ala Ser Asn Val Ala Gly Gly Lys His Ser Ser Asn Arg 115 120 125 Arg Pro Arg Gly Thr Ile Arg Ala Val Pro Gly Arg Lys Tyr Pro Ser 130 135 140 Asn Gly Pro Leu Ser Lys Leu Asp Pro Ile Cys Ala Ile Glu Ala Ala 145 150 155 160 Pro Met Ala Gly Ala Ala Ala Asp Pro Gln Glu Arg Val Gly Pro Arg 165 170 175 Gly Arg Arg Gly Leu Ala Gly Gln Gln Gln Cys Arg Gly Arg Pro Gly 180 185 190 Pro Ser Leu Arg Cys Ser His Asp Thr Pro Arg Phe Gln Met Asn Gln 195 200 205 Ala Phe His Thr Met Val Asn Met Leu Leu Thr Cys Phe Ala Cys Gln 210 215 220 Glu Lys Pro Arg 225 129 597 PRT Mycobacterium tuberculosis 129 Gly Ser Pro Ala Thr Met Pro Ser Ser Arg Asn Leu Ala Thr Asn Pro 1 5 10 15 Glu Ile Ala Thr Gly Tyr Arg Arg Asp Met Thr Val Val Arg Thr Ala 20 25 30 His Tyr Ala Ala Ala Thr Ala Asn Pro Leu Ala Thr Gln Val Ala Cys 35 40 45 Arg Val Leu Arg Asp Gly Gly Thr Ala Ala Asp Ala Val Val Ala Ala 50 55 60 Gln Ala Val Leu Gly Leu Val Glu Pro Gln Ser Ser Gly Ile Gly Gly 65 70 75 80 Gly Gly Tyr Leu Val Tyr Phe Asp Ala Arg Thr Gly Ser Val Gln Ala 85 90 95 Tyr Asp Gly Arg Glu Val Ala Pro Ala Ala Ala Thr Glu Asn Tyr Leu 100 105 110 Arg Trp Val Ser Asp Val Asp Arg Ser Ala Pro Arg Pro Asn Ala Arg 115 120 125 Ala Ser Gly Arg Ser Ile Gly Val Pro Gly Ile Leu Arg Met Leu Glu 130 135 140 Met Val His Asn Glu His Gly Arg Thr Pro Trp Arg Asp Leu Phe Gly 145 150 155 160 Pro Ala Val Thr Leu Ala Asp Gly Gly Phe Asp Ile Ser Ala Arg Met 165 170 175 Gly Ala Ala Ile Ser Asp Ala Ala Pro Gln Leu Arg Asp Asp Pro Glu 180 185 190 Ala Arg Lys Tyr Phe Leu Asn Pro Asp Gly Ser Pro Lys Pro Ala Gly 195 200 205 Thr Arg Leu Thr Asn Pro Ala Tyr Ser Lys Thr Leu Ser Ala Ile Ala 210 215 220 Ser Ala Gly Ala Asn Ala Phe Tyr Ser Gly Asp Ile Ala His Asp Ile 225 230 235 240 Val Ala Ala Ala Ser Asp Thr Ser Asn Gly Arg Thr Pro Gly Leu Leu 245 250 255 Thr Ile Glu Asp Leu Ala Gly Tyr Leu Ala Lys Arg Arg Gln Pro Leu 260 265 270 Cys Thr Thr Tyr Arg Gly Arg Glu Ile Cys Gly Met Pro Ser Ser Gly 275 280 285 Gly Val Ala Val Ala Ala Thr Leu Gly Ile Leu Glu His Phe Pro Met 290 295 300 Ser Asp Tyr Ala Pro Ser Lys Val Asp Leu Asn Gly Gly Arg Pro Thr 305 310 315 320 Val Met Gly Val His Leu Ile Ala Glu Ala Glu Arg Leu Ala Tyr Ala 325 330 335 Asp Arg Asp Gln Tyr Ile Ala Asp Val Asp Phe Val Arg Leu Pro Gly 340 345 350 Gly Ser Leu Thr Thr Leu Val Asp Pro Gly Tyr Leu Ala Ala Arg Ala 355 360 365 Ala Leu Ile Ser Pro Gln His Ser Met Gly Ser Ala Arg Pro Gly Asp 370 375 380 Phe Gly Ala Pro Thr Ala Val Ala Pro Pro Val Pro Glu His Gly Thr 385 390 395 400 Ser His Leu Ser Val Val Asp Ser Tyr Gly Asn Ala Ala Thr Leu Thr 405 410 415 Thr Thr Val Glu Ser Ser Phe Gly Ser Tyr His Leu Val Asp Gly Phe 420 425 430 Ile Leu Asn Asn Gln Leu Ser Asp Phe Ser Ala Glu Pro His Ala Thr 435 440 445 Asp Gly Ser Pro Val Ala Asn Arg Val Glu Pro Gly Lys Arg Pro Arg 450 455 460 Ser Ser Met Ala Pro Thr Leu Val Phe Asp His Ser Ser Ala Gly Arg 465 470 475 480 Gly Ala Leu Tyr Ala Val Leu Gly Ser Pro Gly Gly Ser Met Ile Ile 485 490 495 Gln Phe Val Val Lys Thr Leu Val Ala Met Leu Asp Trp Gly Leu Asn 500 505 510 Pro Gln Gln Ala Val Ser Leu Val Asp Phe Gly Ala Ala Asn Ser Pro 515 520 525 His Thr Asn Leu Gly Gly Glu Asn Pro Glu Ile Asn Thr Ser Asp Asp 530 535 540 Gly Asp His Asp Pro Leu Val Gln Gly Leu Arg Ala Leu Gly His Arg 545 550 555 560 Val Asn Leu Ala Glu Gln Ser Ser Gly Leu Ser Ala Ile Thr Arg Ser 565 570 575 Glu Ala Gly Trp Ala Gly Gly Ala Asp Pro Arg Arg Glu Gly Ala Val 580 585 590 Met Gly Asp Asp Ala 595 130 163 PRT Mycobacterium tuberculosis 130 Glu Phe Lys Arg Gly Val Ala Thr Leu Pro Val Ile Leu Val Ile Leu 1 5 10 15 Leu Ser Val Ala Ala Gly Ala Gly Ala Trp Leu Leu Val Arg Gly His 20 25 30 Gly Pro Gln Gln Pro Glu Ile Ser Ala Tyr Ser His Gly His Leu Thr 35 40 45 Arg Val Gly Pro Tyr Leu Tyr Cys Asn Val Val Asp Leu Asp Asp Cys 50 55 60 Gln Thr Pro Gln Ala Gln Gly Glu Leu Pro Val Ser Glu Arg Tyr Pro 65 70 75 80 Val Gln Leu Ser Val Pro Glu Val Ile Ser Arg Ala Pro Trp Arg Leu 85 90 95 Leu Gln Val Tyr Gln Asp Pro Ala Asn Thr Thr Ser Thr Leu Phe Arg 100 105 110 Pro Asp Thr Arg Leu Ala Val Thr Ile Pro Thr Val Asp Pro Gln Arg 115 120 125 Gly Arg Leu Thr Gly Ile Val Val Gln Leu Leu Thr Leu Val Val Asp 130 135 140 His Ser Gly Glu Leu Arg Asp Val Pro His Ala Glu Trp Ser Val Arg 145 150 155 160 Leu Ile Phe 131 943 PRT Mycobacterium tuberculosis 131 Glu Phe Ala Pro Met Leu Asp Ala Ala Ala Ala Trp Asp Gly Leu Ala 1 5 10 15 Asp Glu Leu Gly Ser Ala Ala Ala Ser Phe Ser Ala Val Thr Ala Gly 20 25 30 Leu Ala Gly Ser Ser Trp Leu Gly Ala Ala Ser Thr Ala Met Thr Gly 35 40 45 Ala Ala Ala Pro Tyr Leu Gly Trp Leu Ser Ala Ala Ala Ala Gln Ala 50 55 60 Gln Gln Ala Ala Thr Gln Thr Arg Leu Ala Ala Ala Ala Phe Glu Ala 65 70 75 80 Ala Leu Ala Ala Thr Val His Pro Ala Ile Ile Ser Ala Asn Arg Ala 85 90 95 Leu Phe Val Ser Leu Val Val Ser Asn Leu Leu Gly Gln Asn Ala Pro 100 105 110 Ala Ile Ala Ala Thr Glu Ala Ala Tyr Glu Gln Met Trp Ala Gln Asp 115 120 125 Val Ala Ala Met Phe Gly Tyr His Ala Gly Ala Ser Ala Ala Val Ser 130 135 140 Ala Leu Thr Pro Phe Gly Gln Ala Leu Pro Thr Val Ala Gly Gly Gly 145 150 155 160 Ala Leu Val Ser Ala Ala Ala Ala Gln Val Thr Thr Arg Val Phe Arg 165 170 175 Asn Leu Gly Leu Ala Asn Val Gly Glu Gly Asn Val Gly Asn Gly Asn 180 185 190 Val Gly Asn Phe Asn Leu Gly Ser Ala Asn Ile Gly Asn Gly Asn Ile 195 200 205 Gly Ser Gly Asn Ile Gly Ser Ser Asn Ile Gly Phe Gly Asn Val Gly 210 215 220 Pro Gly Leu Thr Ala Ala Leu Asn Asn Ile Gly Phe Gly Asn Thr Gly 225 230 235 240 Ser Asn Asn Ile Gly Phe Gly Asn Thr Gly Ser Asn Asn Ile Gly Phe 245 250 255 Gly Asn Thr Gly Asp Gly Asn Arg Gly Ile Gly Leu Thr Gly Ser Gly 260 265 270 Leu Leu Gly Phe Gly Gly Leu Asn Ser Gly Thr Gly Asn Ile Gly Leu 275 280 285 Phe Asn Ser Gly Thr Gly Asn Val Gly Ile Gly Asn Ser Gly Thr Gly 290 295 300 Asn Trp Gly Ile Gly Asn Ser Gly Asn Ser Tyr Asn Thr Gly Phe Gly 305 310 315 320 Asn Ser Gly Asp Ala Asn Thr Gly Phe Phe Asn Ser Gly Ile Ala Asn 325 330 335 Thr Gly Val Gly Asn Ala Gly Asn Tyr Asn Thr Gly Ser Tyr Asn Pro 340 345 350 Gly Asn Ser Asn Thr Gly Gly Phe Asn Met Gly Gln Tyr Asn Thr Gly 355 360 365 Tyr Leu Asn Ser Gly Asn Tyr Asn Thr Gly Leu Ala Asn Ser Gly Asn 370 375 380 Val Asn Thr Gly Ala Phe Ile Thr Gly Asn Phe Asn Asn Gly Phe Leu 385 390 395 400 Trp Arg Gly Asp His Gln Gly Leu Ile Phe Gly Ser Pro Gly Phe Phe 405 410 415 Asn Ser Thr Ser Ala Pro Ser Ser Gly Phe Phe Asn Ser Gly Ala Gly 420 425 430 Ser Ala Ser Gly Phe Leu Asn Ser Gly Ala Asn Asn Ser Gly Phe Phe 435 440 445 Asn Ser Ser Ser Gly Ala Ile Gly Asn Ser Gly Leu Ala Asn Ala Gly 450 455 460 Val Leu Val Ser Gly Val Ile Asn Ser Gly Asn Thr Val Ser Gly Leu 465 470 475 480 Phe Asn Met Ser Leu Val Ala Ile Thr Thr Pro Ala Leu Ile Ser Gly 485 490 495 Phe Phe Asn Thr Gly Ser Asn Met Ser Gly Phe Phe Gly Gly Pro Pro 500 505 510 Val Phe Asn Leu Gly Leu Ala Asn Arg Gly Val Val Asn Ile Leu Gly 515 520 525 Asn Ala Asn Ile Gly Asn Tyr Asn Ile Leu Gly Ser Gly Asn Val Gly 530 535 540 Asp Phe Asn Ile Leu Gly Ser Gly Asn Leu Gly Ser Gln Asn Ile Leu 545 550 555 560 Gly Ser Gly Asn Val Gly Ser Phe Asn Ile Gly Ser Gly Asn Ile Gly 565 570 575 Val Phe Asn Val Gly Ser Gly Ser Leu Gly Asn Tyr Asn Ile Gly Ser 580 585 590 Gly Asn Leu Gly Ile Tyr Asn Ile Gly Phe Gly Asn Val Gly Asp Tyr 595 600 605 Asn Val Gly Phe Gly Asn Ala Gly Asp Phe Asn Gln Gly Phe Ala Asn 610 615 620 Thr Gly Asn Asn Asn Ile Gly Phe Ala Asn Thr Gly Asn Asn Asn Ile 625 630 635 640 Gly Ile Gly Leu Ser Gly Asp Asn Gln Gln Gly Phe Asn Ile Ala Ser 645 650 655 Gly Trp Asn Ser Gly Thr Gly Asn Ser Gly Leu Phe Asn Ser Gly Thr 660 665 670 Asn Asn Val Gly Ile Phe Asn Ala Gly Thr Gly Asn Val Gly Ile Ala 675 680 685 Asn Ser Gly Thr Gly Asn Trp Gly Ile Gly Asn Pro Gly Thr Asp Asn 690 695 700 Thr Gly Ile Leu Asn Ala Gly Ser Tyr Asn Thr Gly Ile Leu Asn Ala 705 710 715 720 Gly Asp Phe Asn Thr Gly Phe Tyr Asn Thr Gly Ser Tyr Asn Thr Gly 725 730 735 Gly Phe Asn Val Gly Asn Thr Asn Thr Gly Asn Phe Asn Val Gly Asp 740 745 750 Thr Asn Thr Gly Ser Tyr Asn Pro Gly Asp Thr Asn Thr Gly Phe Phe 755 760 765 Asn Pro Gly Asn Val Asn Thr Gly Ala Phe Asp Thr Gly Asp Phe Asn 770 775 780 Asn Gly Phe Leu Val Ala Gly Asp Asn Gln Gly Gln Ile Ala Ile Asp 785 790 795 800 Leu Ser Val Thr Thr Pro Phe Ile Pro Ile Asn Glu Gln Met Val Ile 805 810 815 Asp Val His Asn Val Met Thr Phe Gly Gly Asn Met Ile Thr Val Thr 820 825 830 Glu Ala Ser Thr Val Phe Pro Gln Thr Phe Tyr Leu Ser Gly Leu Phe 835 840 845 Phe Phe Gly Pro Val Asn Leu Ser Ala Ser Thr Leu Thr Val Pro Thr 850 855 860 Ile Thr Leu Thr Ile Gly Gly Pro Thr Val Thr Val Pro Ile Ser Ile 865 870 875 880 Val Gly Ala Leu Glu Ser Arg Thr Ile Thr Phe Leu Lys Ile Asp Pro 885 890 895 Ala Pro Gly Ile Gly Asn Ser Thr Thr Asn Pro Ser Ser Gly Phe Phe 900 905 910 Asn Ser Gly Thr Gly Gly Thr Ser Gly Phe Gln Asn Val Gly Gly Gly 915 920 925 Ser Ser Gly Val Trp Asn Ser Gly Leu Ser Ser Lys Leu Gly Asn 930 935 940 132 592 PRT Mycobacterium tuberculosis 132 Glu Phe Ala Asp Arg Gly Gln Arg Arg Gly Cys Ala Pro Gly Ile Ala 1 5 10 15 Ser Ala Leu Arg Ala Ser Phe Gln Gly Lys Ser Arg Pro Trp Thr Gln 20 25 30 Thr Arg Tyr Trp Ala Phe Ala Leu Leu Thr Pro Leu Val Val Ala Met 35 40 45 Val Leu Thr Gly Cys Ser Ala Ser Gly Thr Gln Leu Glu Leu Ala Pro 50 55 60 Thr Ala Asp Arg Arg Ala Ala Val Gly Thr Thr Ser Asp Ile Asn Gln 65 70 75 80 Gln Asp Pro Ala Thr Leu Gln Asp Gly Gly Asn Leu Arg Leu Ser Leu 85 90 95 Thr Asp Phe Pro Pro Asn Phe Asn Ile Leu His Ile Asp Gly Asn Asn 100 105 110 Ala Glu Val Ala Ala Met Met Lys Ala Thr Leu Pro Arg Ala Phe Ile 115 120 125 Ile Gly Pro Asp Gly Ser Thr Thr Val Asp Thr Asn Tyr Phe Thr Ser 130 135 140 Ile Glu Leu Thr Arg Thr Ala Pro Gln Val Val Thr Tyr Thr Ile Asn 145 150 155 160 Pro Glu Ala Val Trp Ser Asp Gly Thr Pro Ile Thr Trp Arg Asp Ile 165 170 175 Ala Ser Gln Ile His Ala Ile Ser Gly Ala Asp Lys Ala Phe Glu Ile 180 185 190 Ala Ser Ser Ser Gly Ala Glu Arg Val Ala Ser Val Thr Arg Gly Val 195 200 205 Asp Asp Arg Gln Ala Val Val Thr Phe Ala Lys Pro Tyr Ala Glu Trp 210 215 220 Arg Gly Met Phe Ala Gly Asn Gly Met Leu Leu Pro Ala Ser Met Thr 225 230 235 240 Ala Thr Pro Glu Ala Phe Asn Lys Gly Gln Leu Asp Gly Pro Gly Pro 245 250 255 Ser Ala Gly Pro Phe Val Val Ser Ala Leu Asp Arg Thr Ala Gln Arg 260 265 270 Ile Val Leu Thr Arg Asn Pro Arg Trp Trp Gly Ala Arg Pro Arg Leu 275 280 285 Asp Ser Ile Thr Tyr Leu Val Leu Asp Asp Ala Ala Arg Leu Pro Ala 290 295 300 Leu Gln Asn Asn Thr Ile Asp Ala Thr Gly Val Gly Thr Leu Asp Gln 305 310 315 320 Leu Thr Ile Ala Ala Arg Thr Lys Gly Ile Ser Ile Arg Arg Ala Pro 325 330 335 Gly Pro Ser Trp Tyr His Phe Thr Leu Asn Gly Ala Pro Gly Ser Ile 340 345 350 Leu Ala Asp Lys Ala Leu Arg Leu Ala Ile Ala Lys Gly Ile Asp Arg 355 360 365 Tyr Thr Ile Ala Arg Val Ala Gln Tyr Gly Leu Thr Ser Asp Pro Val 370 375 380 Pro Leu Asn Asn His Val Phe Val Ala Gly Gln Asp Gly Tyr Gln Asp 385 390 395 400 Asn Ser Gly Val Val Ala Tyr Asn Pro Glu Gln Ala Lys Arg Glu Leu 405 410 415 Asp Ala Leu Gly Trp Arg Arg Ser Gly Ala Phe Arg Glu Lys Asp Gly 420 425 430 Arg Gln Leu Val Ile Arg Asp Leu Phe Tyr Asp Ala Gln Ser Thr Arg 435 440 445 Gln Phe Ala Gln Ile Ala Gln His Thr Leu Ala Gln Ile Gly Val Lys 450 455 460 Leu Glu Leu Gln Ala Lys Ser Gly Ser Gly Phe Phe Ser Asp Tyr Val 465 470 475 480 Asn Val Gly Ala Phe Asp Ile Ala Gln Phe Gly Trp Val Gly Asp Ala 485 490 495 Phe Pro Leu Ser Ser Leu Thr Gln Ile Tyr Ala Ser Asp Gly Glu Ser 500 505 510 Asn Phe Gly Lys Ile Gly Ser Pro Gln Ile Asp Ala Ala Ile Glu Arg 515 520 525 Thr Leu Ala Glu Leu Asp Pro Gly Lys Ala Arg Ala Leu Ala Asn Gln 530 535 540 Val Asp Glu Leu Ile Trp Ala Glu Gly Phe Ser Leu Pro Leu Thr Gln 545 550 555 560 Ser Pro Gly Thr Val Ala Val Arg Ser Thr Leu Ala Asn Phe Gly Ala 565 570 575 Thr Gly Leu Ala Asp Leu Asp Tyr Thr Ala Ile Gly Phe Met Arg Arg 580 585 590 133 259 PRT Mycobacterium tuberculosis 133 Glu Phe Ile Ser Gln Ala Cys Gly Ser His Arg Pro Arg Arg Pro Ser 1 5 10 15 Ser Leu Gly Ala Val Ala Ile Leu Ile Ala Ala Thr Leu Phe Ala Thr 20 25 30 Val Val Ala Gly Cys Gly Lys Lys Pro Thr Thr Ala Ser Ser Pro Ser 35 40 45 Pro Gly Ser Pro Ser Pro Glu Ala Gln Gln Ile Leu Gln Asp Ser Ser 50 55 60 Lys Ala Thr Lys Gly Leu His Ser Val His Val Val Val Thr Val Asn 65 70 75 80 Asn Leu Ser Thr Leu Pro Phe Glu Ser Val Asp Ala Asp Val Thr Asn 85 90 95 Gln Pro Gln Gly Asn Gly Gln Ala Val Gly Asn Ala Lys Val Arg Met 100 105 110 Lys Pro Asn Thr Pro Val Val Ala Thr Glu Phe Leu Val Thr Asn Lys 115 120 125 Thr Met Tyr Thr Lys Arg Gly Gly Asp Tyr Val Ser Val Gly Pro Ala 130 135 140 Glu Lys Ile Tyr Asp Pro Gly Ile Ile Leu Asp Lys Asp Arg Gly Leu 145 150 155 160 Gly Ala Val Val Gly Gln Val Gln Asn Pro Thr Ile Gln Gly Arg Asp 165 170 175 Ala Ile Asp Gly Leu Ala Thr Val Lys Val Ser Gly Thr Ile Asp Ala 180 185 190 Ala Val Ile Asp Pro Ile Val Pro Gln Leu Gly Lys Gly Gly Gly Arg 195 200 205 Leu Pro Ile Thr Leu Trp Ile Val Asp Thr Asn Ala Ser Thr Pro Ala 210 215 220 Pro Ala Ala Asn Leu Val Arg Met Val Ile Asp Lys Asp Gln Gly Asn 225 230 235 240 Val Asp Ile Thr Leu Ser Asn Trp Gly Ala Pro Val Thr Ile Pro Asn 245 250 255 Pro Ala Gly 134 312 PRT Mycobacterium tuberculosis 134 Glu Phe Met Ile Gln Ile Ala Arg Thr Trp Arg Val Phe Ala Gly Gly 1 5 10 15 Met Ala Thr Gly Phe Ile Gly Val Val Leu Val Thr Ala Gly Lys Ala 20 25 30 Ser Ala Asp Pro Leu Leu Pro Pro Pro Pro Ile Pro Ala Pro Val Ser 35 40 45 Ala Pro Ala Thr Val Pro Pro Val Gln Asn Leu Thr Ala Leu Pro Gly 50 55 60 Gly Ser Ser Asn Arg Phe Ser Pro Ala Pro Ala Pro Ala Pro Ile Ala 65 70 75 80 Ser Pro Ile Pro Val Gly Ala Pro Gly Ser Thr Ala Val Pro Pro Leu 85 90 95 Pro Pro Pro Val Thr Pro Ala Ile Ser Gly Thr Leu Arg Asp His Leu 100 105 110 Arg Glu Lys Gly Val Lys Leu Glu Ala Gln Arg Pro His Gly Phe Lys 115 120 125 Ala Leu Asp Ile Thr Leu Pro Met Pro Pro Arg Trp Thr Gln Val Pro 130 135 140 Asp Pro Asn Val Pro Asp Ala Phe Val Val Ile Ala Asp Arg Leu Gly 145 150 155 160 Asn Ser Val Tyr Thr Ser Asn Ala Gln Leu Val Val Tyr Arg Leu Ile 165 170 175 Gly Asp Phe Asp Pro Ala Glu Ala Ile Thr His Gly Tyr Ile Asp Ser 180 185 190 Gln Lys Leu Leu Ala Trp Gln Thr Thr Asn Ala Ser Met Ala Asn Phe 195 200 205 Asp Gly Phe Pro Ser Ser Ile Ile Glu Gly Thr Tyr Arg Glu Asn Asp 210 215 220 Met Thr Leu Asn Thr Ser Arg Arg His Val Ile Ala Thr Ser Gly Ala 225 230 235 240 Asp Lys Tyr Leu Val Ser Leu Ser Val Thr Thr Ala Leu Ser Gln Ala 245 250 255 Val Thr Asp Gly Pro Ala Thr Asp Ala Ile Val Asn Gly Phe Gln Val 260 265 270 Val Ala His Ala Ala Pro Ala Gln Ala Pro Ala Pro Ala Pro Gly Ser 275 280 285 Ala Pro Val Gly Leu Pro Gly Gln Ala Pro Gly Tyr Pro Pro Ala Gly 290 295 300 Thr Leu Thr Pro Val Pro Pro Arg 305 310 135 445 PRT Mycobacterium tuberculosis 135 Glu Phe Thr Arg Ser Arg Gly Leu Arg Tyr Ala Thr Val Ile Ala Leu 1 5 10 15 Val Ala Ala Leu Val Gly Gly Val Tyr Val Leu Ser Ser Thr Gly Asn 20 25 30 Lys Arg Thr Ile Val Gly Tyr Phe Thr Ser Ala Val Gly Leu Tyr Pro 35 40 45 Gly Asp Gln Val Arg Val Leu Gly Val Pro Val Gly Glu Ile Asp Met 50 55 60 Ile Glu Pro Arg Ser Ser Asp Val Lys Ile Thr Met Ser Val Ser Lys 65 70 75 80 Asp Val Lys Val Pro Val Asp Val Gln Ala Val Ile Met Ser Pro Asn 85 90 95 Leu Val Ala Ala Arg Phe Ile Gln Leu Thr Pro Val Tyr Thr Gly Gly 100 105 110 Ala Val Leu Pro Asp Asn Gly Arg Ile Asp Leu Asp Arg Thr Ala Val 115 120 125 Pro Val Glu Trp Asp Glu Val Lys Glu Gly Leu Thr Arg Leu Ala Ala 130 135 140 Asp Leu Ser Pro Ala Ala Gly Glu Leu Gln Gly Pro Leu Gly Ala Ala 145 150 155 160 Ile Asn Gln Ala Ala Asp Thr Leu Asp Gly Asn Gly Asp Ser Leu His 165 170 175 Asn Ala Leu Arg Glu Leu Ala Gln Val Ala Gly Arg Leu Gly Asp Ser 180 185 190 Arg Gly Asp Ile Phe Gly Thr Val Lys Asn Leu Gln Val Leu Val Asp 195 200 205 Ala Leu Ser Glu Ser Asp Glu Gln Ile Val Gln Phe Ala Gly His Val 210 215 220 Ala Ser Val Ser Gln Val Leu Ala Asp Ser Ser Ala Asn Leu Asp Gln 225 230 235 240 Thr Leu Gly Thr Leu Asn Gln Ala Leu Ser Asp Ile Arg Gly Phe Leu 245 250 255 Arg Glu Asn Asn Ser Thr Leu Ile Glu Thr Val Asn Gln Leu Asn Asp 260 265 270 Phe Ala Gln Thr Leu Ser Asp Gln Ser Glu Asn Ile Glu Gln Val Leu 275 280 285 His Val Ala Gly Pro Gly Ile Thr Asn Phe Tyr Asn Ile Tyr Asp Pro 290 295 300 Ala Gln Gly Thr Leu Asn Gly Leu Leu Ser Ile Pro Asn Phe Ala Asn 305 310 315 320 Pro Val Gln Phe Ile Cys Gly Gly Ser Phe Asp Thr Ala Ala Gly Pro 325 330 335 Ser Ala Pro Asp Tyr Tyr Arg Arg Ala Glu Ile Cys Arg Glu Arg Leu 340 345 350 Gly Pro Val Leu Arg Arg Leu Thr Val Asn Tyr Pro Pro Ile Met Phe 355 360 365 His Pro Leu Asn Thr Ile Thr Ala Tyr Lys Gly Gln Ile Ile Tyr Asp 370 375 380 Thr Pro Ala Thr Glu Ala Lys Ser Glu Thr Pro Val Pro Glu Leu Thr 385 390 395 400 Trp Val Pro Ala Gly Gly Gly Ala Pro Val Gly Asn Pro Ala Asp Leu 405 410 415 Gln Ser Leu Leu Val Pro Pro Ala Pro Gly Pro Ala Pro Ala Pro Pro 420 425 430 Ala Pro Gly Ala Gly Pro Gly Glu His Gly Gly Gly Gly 435 440 445 136 437 PRT Mycobacterium tuberculosis 136 Gly Met Asp Pro Leu Asn Arg Arg Gln Phe Leu Ala Leu Ala Ala Ala 1 5 10 15 Ala Ala Gly Val Thr Ala Gly Cys Ala Gly Met Gly Gly Gly Gly Ser 20 25 30 Val Lys Ser Gly Ser Gly Pro Ile Asp Phe Trp Ser Ser His Pro Gly 35 40 45 Gln Ser Ser Ala Ala Glu Arg Glu Leu Ile Gly Arg Phe Gln Asp Arg 50 55 60 Phe Pro Thr Leu Ser Val Lys Leu Ile Asp Ala Gly Lys Asp Tyr Asp 65 70 75 80 Glu Val Ala Gln Lys Phe Asn Ala Ala Leu Ile Gly Thr Asp Val Pro 85 90 95 Asp Val Val Leu Leu Asp Asp Arg Trp Trp Phe His Phe Ala Leu Ser 100 105 110 Gly Val Leu Thr Ala Leu Asp Asp Leu Phe Gly Gln Val Gly Val Asp 115 120 125 Thr Thr Asp Tyr Val Asp Ser Leu Leu Ala Asp Tyr Glu Phe Asn Gly 130 135 140 Arg His Tyr Ala Val Pro Tyr Ala Arg Ser Thr Pro Leu Phe Tyr Tyr 145 150 155 160 Asn Lys Ala Ala Trp Gln Gln Ala Gly Leu Pro Asp Arg Gly Pro Gln 165 170 175 Ser Trp Ser Glu Phe Asp Glu Trp Gly Pro Glu Leu Gln Arg Val Val 180 185 190 Gly Ala Gly Arg Ser Ala His Gly Trp Ala Asn Ala Asp Leu Ile Ser 195 200 205 Trp Thr Phe Gln Gly Pro Asn Trp Ala Phe Gly Gly Ala Tyr Ser Asp 210 215 220 Lys Trp Thr Leu Thr Leu Thr Glu Pro Ala Thr Ile Ala Ala Gly Asn 225 230 235 240 Phe Tyr Arg Asn Ser Ile His Gly Lys Gly Tyr Ala Ala Val Ala Asn 245 250 255 Asp Ile Ala Asn Glu Phe Ala Thr Gly Ile Leu Ala Ser Ala Val Ala 260 265 270 Ser Thr Gly Ser Leu Ala Gly Ile Thr Ala Ser Ala Arg Phe Asp Phe 275 280 285 Gly Ala Ala Pro Leu Pro Thr Gly Pro Asp Ala Ala Pro Ala Cys Pro 290 295 300 Thr Gly Gly Ala Gly Leu Ala Ile Pro Ala Lys Leu Ser Glu Glu Arg 305 310 315 320 Lys Val Asn Ala Leu Lys Phe Ile Ala Phe Val Thr Asn Pro Thr Asn 325 330 335 Thr Ala Tyr Phe Ser Gln Gln Thr Gly Tyr Leu Pro Val Arg Lys Ser 340 345 350 Ala Val Asp Asp Ala Ser Glu Arg His Tyr Leu Ala Asp Asn Pro Arg 355 360 365 Ala Arg Val Ala Leu Asp Gln Leu Pro His Thr Arg Thr Gln Asp Tyr 370 375 380 Ala Arg Val Phe Leu Pro Gly Gly Asp Arg Ile Ile Ser Ala Gly Leu 385 390 395 400 Glu Ser Ile Gly Leu Arg Gly Ala Asp Val Thr Lys Thr Phe Thr Asn 405 410 415 Ile Gln Lys Arg Leu Gln Val Ile Leu Asp Arg Gln Ile Met Arg Lys 420 425 430 Leu Ala Gly His Gly 435 137 189 PRT Mycobacterium tuberculosis 137 Gly Ser Val Ala Val Arg Arg Lys Val Arg Arg Leu Thr Leu Ala Val 1 5 10 15 Ser Ala Leu Val Ala Leu Phe Pro Ala Val Ala Gly Cys Ser Asp Ser 20 25 30 Gly Asp Asn Lys Pro Gly Ala Thr Ile Pro Ser Thr Pro Ala Asn Ala 35 40 45 Glu Gly Arg His Gly Pro Phe Phe Pro Gln Cys Gly Gly Val Ser Asp 50 55 60 Gln Thr Val Thr Glu Leu Thr Arg Val Thr Gly Leu Val Asn Thr Ala 65 70 75 80 Lys Asn Ser Val Gly Cys Gln Trp Leu Ala Gly Gly Gly Ile Leu Gly 85 90 95 Pro His Phe Ser Phe Ser Trp Tyr Arg Gly Ser Pro Ile Gly Arg Glu 100 105 110 Arg Lys Thr Glu Glu Leu Ser Arg Ala Ser Val Glu Asp Ile Asn Ile 115 120 125 Asp Gly His Ser Gly Phe Ile Ala Ile Gly Asn Glu Pro Ser Leu Gly 130 135 140 Asp Ser Leu Cys Glu Val Gly Ile Gln Phe Ser Asp Asp Phe Ile Glu 145 150 155 160 Trp Ser Val Ser Phe Ser Gln Lys Pro Phe Pro Leu Pro Cys Asp Ile 165 170 175 Ala Lys Glu Leu Thr Arg Gln Ser Ile Ala Asn Ser Lys 180 185 138 230 PRT Mycobacterium tuberculosis 138 Gly Ser Ile Ala Met Gln Leu Ser Ser Arg Leu Glu Asn His Arg Lys 1 5 10 15 Pro Phe Pro Arg Thr Val Ser Thr Gly Gln Ala Ile Ala Met Ala Lys 20 25 30 Arg Thr Pro Val Arg Lys Ala Cys Thr Val Leu Ala Val Leu Ala Ala 35 40 45 Thr Leu Leu Leu Gly Ala Cys Gly Gly Pro Thr Gln Pro Arg Ser Ile 50 55 60 Thr Leu Thr Phe Ile Arg Asn Ala Gln Ser Gln Ala Asn Ala Asp Gly 65 70 75 80 Ile Ile Asp Thr Asp Met Pro Gly Ser Gly Leu Ser Ala Asp Gly Lys 85 90 95 Ala Glu Ala Gln Gln Val Ala His Gln Val Ser Arg Arg Asp Val Asp 100 105 110 Ser Ile Tyr Ser Ser Pro Met Ala Ala Asp Gln Gln Thr Ala Gly Pro 115 120 125 Leu Ala Gly Glu Leu Gly Lys Gln Val Glu Ile Leu Pro Gly Leu Gln 130 135 140 Ala Ile Asn Ala Gly Trp Phe Asn Gly Lys Pro Glu Ser Met Ala Asn 145 150 155 160 Ser Thr Tyr Met Leu Ala Pro Ala Asp Trp Leu Ala Gly Asp Val His 165 170 175 Asn Thr Ile Pro Gly Ser Ile Ser Gly Thr Glu Phe Asn Ser Gln Phe 180 185 190 Ser Ala Ala Val Arg Lys Ile Tyr Asp Ser Gly His Asn Thr Pro Val 195 200 205 Val Phe Ser Gln Gly Val Ala Ile Met Ile Trp Thr Leu Met Asn Ala 210 215 220 Arg Asn Ser Arg Glu Ser 225 230 139 320 PRT Mycobacterium tuberculosis 139 Gly Ser Gly Gly Ala Gly Met Thr Glu His Asn Glu Asp Pro Gln Ile 1 5 10 15 Glu Arg Val Ala Asp Asp Ala Ala Asp Glu Glu Ala Val Thr Glu Pro 20 25 30 Leu Ala Thr Glu Ser Lys Asp Glu Pro Ala Glu His Pro Glu Phe Glu 35 40 45 Gly Pro Arg Arg Arg Ala Arg Arg Glu Arg Ala Glu Arg Arg Ala Ala 50 55 60 Gln Ala Arg Ala Thr Ala Ile Glu Gln Ala Arg Arg Ala Ala Lys Arg 65 70 75 80 Arg Ala Arg Gly Gln Ile Val Ser Glu Gln Asn Pro Ala Lys Pro Ala 85 90 95 Ala Arg Gly Val Val Arg Gly Leu Lys Ala Leu Leu Ala Thr Val Val 100 105 110 Leu Ala Val Val Gly Ile Gly Leu Gly Leu Ala Leu Tyr Phe Thr Pro 115 120 125 Ala Met Ser Ala Arg Glu Ile Val Ile Ile Gly Ile Gly Ala Val Ser 130 135 140 Arg Glu Glu Val Leu Asp Ala Ala Arg Val Arg Pro Ala Thr Pro Leu 145 150 155 160 Leu Gln Ile Asp Thr Gln Gln Val Ala Asp Arg Val Ala Thr Ile Arg 165 170 175 Arg Val Ala Ser Ala Arg Val Gln Arg Gln Tyr Pro Ser Ala Leu Arg 180 185 190 Ile Thr Ile Val Glu Arg Val Pro Val Val Val Lys Asp Phe Ser Asp 195 200 205 Gly Pro His Leu Phe Asp Arg Asp Gly Val Asp Phe Ala Thr Asp Pro 210 215 220 Pro Pro Pro Ala Leu Pro Tyr Phe Asp Val Asp Asn Pro Gly Pro Ser 225 230 235 240 Asp Pro Thr Thr Lys Ala Ala Leu Gln Val Leu Thr Ala Leu His Pro 245 250 255 Glu Val Ala Ser Gln Val Gly Arg Ile Ala Ala Pro Ser Val Ala Ser 260 265 270 Ile Thr Leu Thr Leu Ala Asp Gly Arg Val Val Ile Trp Gly Thr Thr 275 280 285 Asp Arg Cys Glu Glu Lys Ala Glu Lys Leu Ala Ala Leu Leu Thr Gln 290 295 300 Pro Gly Arg Thr Tyr Asp Val Ser Ser Pro Asp Leu Pro Thr Val Lys 305 310 315 320 140 488 PRT Mycobacterium tuberculosis 140 Gly Ser Gln Met Asn Val Val Asp Ile Ser Arg Trp Gln Phe Gly Ile 1 5 10 15 Thr Thr Val Tyr His Phe Ile Phe Val Pro Leu Thr Ile Gly Leu Ala 20 25 30 Pro Leu Ile Ala Val Met Gln Thr Leu Trp Val Val Thr Asp Asn Pro 35 40 45 Ala Trp Tyr Arg Leu Thr Lys Phe Phe Gly Lys Leu Phe Leu Ile Asn 50 55 60 Phe Ala Ile Gly Val Ala Thr Gly Ile Val Gln Glu Phe Gln Phe Gly 65 70 75 80 Met Asn Trp Ser Glu Tyr Ser Arg Phe Val Gly Asp Val Phe Gly Ala 85 90 95 Pro Leu Ala Met Glu Gly Leu Ala Ala Phe Phe Phe Glu Ser Thr Phe 100 105 110 Ile Gly Leu Trp Ile Phe Gly Trp Asn Arg Leu Pro Arg Leu Val His 115 120 125 Leu Ala Cys Ile Trp Ile Val Ala Ile Ala Val Asn Val Ser Ala Phe 130 135 140 Phe Ile Ile Ala Ala Asn Ser Phe Met Gln His Pro Val Gly Ala His 145 150 155 160 Tyr Asn Pro Thr Thr Gly Arg Ala Glu Leu Ser Ser Ile Val Val Leu 165 170 175 Leu Thr Asn Asn Thr Ala Gln Ala Ala Phe Thr His Thr Val Ser Gly 180 185 190 Ala Leu Leu Thr Ala Gly Thr Phe Val Ala Ala Val Ser Ala Trp Trp 195 200 205 Leu Val Arg Ser Ser Thr Thr His Ala Asp Ser Asp Thr Gln Ala Met 210 215 220 Tyr Arg Pro Ala Thr Ile Leu Gly Cys Trp Val Ala Leu Ala Ala Thr 225 230 235 240 Ala Gly Leu Leu Phe Thr Gly Asp His Gln Gly Lys Leu Met Phe Gln 245 250 255 Gln Gln Pro Met Lys Met Ala Ser Ala Glu Ser Leu Cys Asp Thr Gln 260 265 270 Thr Asp Pro Asn Phe Ser Val Leu Thr Val Gly Arg Gln Asn Asn Cys 275 280 285 Asp Ser Leu Thr Arg Val Ile Glu Val Pro Tyr Val Leu Pro Phe Leu 290 295 300 Ala Glu Gly Arg Ile Ser Gly Val Thr Leu Gln Gly Ile Arg Asp Leu 305 310 315 320 Gln Gln Glu Tyr Gln Gln Arg Phe Gly Pro Asn Asp Tyr Arg Pro Asn 325 330 335 Leu Phe Val Thr Tyr Trp Ser Phe Arg Met Met Ile Gly Leu Met Ala 340 345 350 Ile Pro Val Leu Phe Ala Leu Ile Ala Leu Trp Leu Thr Arg Gly Gly 355 360 365 Gln Ile Pro Asn Gln Arg Trp Phe Ser Trp Leu Ala Leu Leu Thr Met 370 375 380 Pro Ala Pro Phe Leu Ala Asn Ser Ala Gly Trp Val Phe Thr Glu Met 385 390 395 400 Gly Arg Gln Pro Trp Val Val Val Pro Asn Pro Thr Gly Asp Gln Leu 405 410 415 Val Arg Leu Thr Val Lys Ala Gly Val Ser Asp His Ser Ala Thr Val 420 425 430 Val Ala Thr Ser Leu Leu Met Phe Thr Leu Val Tyr Ala Val Leu Ala 435 440 445 Val Ile Trp Cys Trp Leu Leu Lys Arg Tyr Ile Val Glu Gly Pro Leu 450 455 460 Glu His Asp Ala Glu Pro Ala Ala His Gly Ala Pro Arg Asp Asp Glu 465 470 475 480 Val Ala Pro Leu Ser Phe Ala Tyr 485 141 471 PRT Mycobacterium tuberculosis 141 Asn Ser Lys Ala Ser Ile Gly Thr Val Phe Gln Asp Arg Ala Ala Arg 1 5 10 15 Tyr Gly Asp Arg Val Phe Leu Lys Phe Gly Asp Gln Gln Leu Thr Tyr 20 25 30 Arg Asp Ala Asn Ala Thr Ala Asn Arg Tyr Ala Ala Val Leu Ala Ala 35 40 45 Arg Gly Val Gly Pro Gly Asp Val Val Gly Ile Met Leu Arg Asn Ser 50 55 60 Pro Ser Thr Val Leu Ala Met Leu Ala Thr Val Lys Cys Gly Ala Ile 65 70 75 80 Ala Gly Met Leu Asn Tyr His Gln Arg Gly Glu Val Leu Ala His Ser 85 90 95 Leu Gly Leu Leu Asp Ala Lys Val Leu Ile Ala Glu Ser Asp Leu Val 100 105 110 Ser Ala Val Ala Glu Cys Gly Ala Ser Arg Gly Arg Val Ala Gly Asp 115 120 125 Val Leu Thr Val Glu Asp Val Glu Arg Phe Ala Thr Thr Ala Pro Ala 130 135 140 Thr Asn Pro Ala Ser Ala Ser Ala Val Gln Ala Lys Asp Thr Ala Phe 145 150 155 160 Tyr Ile Phe Thr Ser Gly Thr Thr Gly Phe Pro Lys Ala Ser Val Met 165 170 175 Thr His His Arg Trp Leu Arg Ala Leu Ala Val Phe Gly Gly Met Gly 180 185 190 Leu Arg Leu Lys Gly Ser Asp Thr Leu Tyr Ser Cys Leu Pro Leu Tyr 195 200 205 His Asn Asn Ala Leu Thr Val Ala Val Ser Ser Val Ile Asn Ser Gly 210 215 220 Ala Thr Leu Ala Leu Gly Lys Ser Phe Ser Ala Ser Arg Phe Trp Asp 225 230 235 240 Glu Val Ile Ala Asn Arg Ala Thr Ala Phe Val Tyr Ile Gly Glu Ile 245 250 255 Cys Arg Tyr Leu Leu Asn Gln Pro Ala Lys Pro Thr Asp Arg Ala His 260 265 270 Gln Val Arg Val Ile Cys Gly Asn Gly Leu Arg Pro Glu Ile Trp Asp 275 280 285 Glu Phe Thr Thr Arg Phe Gly Val Ala Arg Val Cys Glu Phe Tyr Ala 290 295 300 Ala Ser Glu Gly Asn Ser Ala Phe Ile Asn Ile Phe Asn Val Pro Arg 305 310 315 320 Thr Ala Gly Val Ser Pro Met Pro Leu Ala Phe Val Glu Tyr Asp Leu 325 330 335 Asp Thr Gly Asp Pro Leu Arg Asp Ala Ser Gly Arg Val Arg Arg Val 340 345 350 Pro Asp Gly Glu Pro Gly Leu Leu Leu Ser Arg Val Asn Arg Leu Gln 355 360 365 Pro Phe Asp Gly Tyr Thr Asp Pro Val Ala Ser Glu Lys Lys Leu Val 370 375 380 Arg Asn Ala Phe Arg Asp Gly Asp Cys Trp Phe Asn Thr Gly Asp Val 385 390 395 400 Met Ser Pro Gln Gly Met Gly His Ala Ala Phe Val Asp Arg Leu Gly 405 410 415 Asp Thr Phe Arg Trp Lys Gly Glu Asn Val Ala Thr Thr Gln Val Glu 420 425 430 Ala Ala Leu Ala Ser Asp Gln Thr Val Glu Glu Cys Thr Val Tyr Gly 435 440 445 Val Gln Ile Pro Arg Thr Gly Gly Arg Ala Gly Met Ala Ala Ile Thr 450 455 460 Leu Arg Ala Gly Ala Glu Phe 465 470 142 36 DNA Artificial Sequence Description of Artificial SequencePCR Primer 142 gtcaaggatc cggcatggac ccgctgaacc gccgac 36 143 44 DNA Artificial Sequence Description of Artificial SequencePCR Primer 143 atgtcgggat ccaagctttc gacggtcggc gcgtcggcgc cggg 44 144 37 DNA Artificial Sequence Description of Artificial SequencePCR Primer 144 gcagatgcat ctaatgggat ccgcggagta tatctcc 37 145 37 DNA Artificial Sequence Description of Artificial SequencePCR Primer 145 ggcgccgtgg gtgtcagcga agcttacctg gttgttg 37 146 31 DNA Artificial Sequence Description of Artificial SequencePCR Primer 146 ggtgccgaat tcgcgccgat gctggacgcg g 31 147 38 DNA Artificial Sequence Description of Artificial SequencePCR Primer 147 acccgaattc ccaagcttgc tgctcaaacc actgttcc 38 148 33 DNA Artificial Sequence Description of Artificial SequencePCR Primer 148 gcgcccaagg gatccccggc taccatgcct tcg 33 149 33 DNA Artificial Sequence Description of Artificial SequencePCR Primer 149 ctcgaaggga tccgcgttcg tttggccgcc cgc 33 150 37 DNA Artificial Sequence Description of Artificial SequencePCR Primer 150 ggcagtggga tccgtagcgg tgcggcgtaa ggtgcgg 37 151 48 DNA Artificial Sequence Description of Artificial SequencePCR Primer 151 gacttcgtgg atccggtcaa gacaagcttt gcggtgatca aggcggcc 48 152 43 DNA Artificial Sequence Description of Artificial SequencePCR Primer 152 catgaatgaa ttcatctcac aagcgtgcgg ctcccaccga ccc 43 153 36 DNA Artificial Sequence Description of Artificial SequencePCR Primer 153 ccttggcgaa ttctcaaagg aaagcttcga aggcgg 36 154 37 DNA Artificial Sequence Description of Artificial SequencePCR Primer 154 ggagttcgga tccatcgcca tgcaactctc ctcccgg 37 155 46 DNA Artificial Sequence Description of Artificial SequencePCR Primer 155 gggcagtgga tccgtggtca gcaagctttc cctagagttt cgtgcg 46 156 35 DNA Artificial Sequence Description of Artificial SequencePCR Primer 156 gtggcgccga attcaagcgc ggtgtcgcaa cgctg 35 157 30 DNA Artificial Sequence Description of Artificial SequencePCR Primer 157 cgcttaagcg cgaagcttcg tcgagccgcg 30 158 38 DNA Artificial Sequence Description of Artificial SequencePCR Primer 158 gaccggaatt catgatccag atcgcgcgca cctggcgg 38 159 44 DNA Artificial Sequence Description of Artificial SequencePCR Primer 159 aacatgaatt caagcttcga ggccgccgac gaatccgctc accg 44 160 36 DNA Artificial Sequence Description of Artificial SequencePCR Primer 160 cgggtcgccg aattcacgcg gagccgggga ttgcgc 36 161 39 DNA Artificial Sequence Description of Artificial SequencePCR Primer 161 ggcggaattc aagcttcggt tcatccgccg cccccatgc 39 162 31 DNA Artificial Sequence Description of Artificial Sequence PCR primer 162 ccccggggat ccgggggtgc tgggatgacg g 31 163 39 DNA Artificial Sequence Description of Artificial Sequence PCR primer 163 acgacggatc ctaagcttgc aggcgcgccg atacgcggc 39 164 36 DNA Artificial Sequence Description of Artificial Sequence PCR primer 164 tctccgggga tcccagatga atgtcgtcga catttc 36 165 28 DNA Artificial Sequence Description of Artificial Sequence PCR primer 165 gggtctccgg atcccccata ccgacatg 28 166 32 DNA Artificial Sequence Description of Artificial Sequence PCR primer 166 ccgactcgag cggcggcgca cacacaacgg tc 32 167 32 DNA Artificial Sequence Description of Artificial Sequence PCR primer 167 aatcctcgag ccctgcggtc gccttccgag cg 32 168 36 DNA Artificial Sequence Description of Artificial Sequence PCR primer 168 atccggcccg aattcgctga ccgtggccag cgacga 36 169 42 DNA Artificial Sequence Description of Artificial Sequence PCR primer 169 gatcggggag aattccgccg acttaagctt cagctgagct gg 42

Claims (12)

1. An isolated immunostimulatory peptide selected from the group consisting of:
(a) an amino acid sequence selected from the group consisting of SEQ ID NOS: 126-138 and fragments thereof;
(b) amino acid sequences that differ from those specified in (a) by one or more conservative amino acid substitutions; and
(c) amino acid sequence having at least 60% sequence identity to the sequences specified in (a) or (b).
2. An isolated nucleic acid molecule, encoding an immunostimulatory peptide according to claim 1.
3. A method of stimulating an immune response, comprising administering to a subject one or more of the immunostimulatory peptides of claim 1.
4. A method of detecting a Mycobacterium tuberculosis specific binding agent, comprising:
(a) contacting a sample, suspected of containing a Mycobacterium tuberculosis specific binding agent, with one or more of the immunostimulatory peptides of claim 1;
(b) allowing a complex comprising the immunostimulatory peptide and the specific binding agent to form; and
(c) detecting the presence of the complex.
5. The method of claim 4, wherein the immunostimulatory peptide is selected from the group consisting of SEQ ID NOS: 126-138, and fragments thereof.
6. An immunostimulatory preparation, comprising one or more of the immunostimulatory peptides of claim 1.
7. The immunostimulatory preparation of claim 6, further comprising a pharmaceutically acceptable excipient, diluent, adjuvant, or a mixture thereof.
8. An isolated immunostimulatory peptide, comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 126-138.
9. A composition, comprising a Mycobacterium tuberculosis specific binding agent that specifically binds to the isolated immunostimulatory peptide of claim 1.
10. The composition of claim 9, wherein the Mycobacterium tuberculosis specific binding agent further comprises at least one polyclonal antiserum.
11. The composition of claim 10, wherein the Mycobacterium tuberculosis specific binding agent further comprises a monoclonal antibody.
12. A method for determining whether a subject has been previously infected with Mycobacterium tuberculosis, the method comprising:
(a) administering to a subject one or more of the immunostimulatory peptides of claim 1; and
(b) detecting an immune response in the subject.
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US7393540B2 (en) 2001-07-04 2008-07-01 Health Protection Agency Mycobacterial antigens expressed during latency
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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